An Annual Research Publication of Southwestern UniversityMatias H. Aznar Memorial College of Medicine

Redemptorist Plaza, Camputhaw, Cebu City 6000Telephone Nos.: +63 412 2942 • +63 412 2501

SWU-MHAM College of MedicineMedical Research JournalBasic&ClinicalSciences

Volume 1 Number 1 ISSN - 2449-3333 April 2015

NATIONAL EDITORIAL BOARD

Dr. Jose M. Oclarit, Ph.D.Biochemistry/Physiology

Editor-in-Chief

Dr. Arlene M. Diaz Dr. Ma. Socorro G. Manaloto Pharmacology/Family Medicine Obstetrics & Gynecology

Dr. Donny Jay Yu Dr. Ma. Luisita Caturza Internal Medicine-Pulmonology Pediatrics

Dr. Virgette Mollaneda Dr. Fe Cabugao Internal Medicine-Cardiology Microbiology

Dr. Axel Elises Dr. Arvizminda Ladiao Surgery Internal Medicine

Dr. Dale Pasco Dr. Moamar Tingkahan Surgery Physiology

Dr. Luditha L Pe Dr. Anette Salillas Biochemistry/Pediatrics Pathology/Cytology

Dr. Vanessa Neri Fajardo Allergologist/ Internal Medicine

SWU-MHAM COLLEGE OF MEDICINE MEDICAL RESEARCH JOURNAL FOR BASIC AND CLINICAL SCIENCES

Copyright@2015. Swu-Mham College of Medicine Medical Research Journal for Basic and Clinical Sciences

SWU-MHAM College of MedicineResearch Department

Redemptorist Plaza, Camputhaw, Cebu City 6000Telephone No. (032) 412-2942 • www.swumhamcollegeofmedicine.com

MHAM MEDICAL RESEARCH JOURNAL is a national journal publishing articles that emphasize the molecular, cellular, and functional basis of therapy. All articles are rigorously reviewed. The Journal favors publication of full-length papers covering basic medical sciences: pharmacology, physiology, biochemistry, pathology, parasitology, and microbiology. Articles that report profound observations are also accepted. Articles should be written at a level accessible to readers who are non-specialists in the topic of the article themselves, but who are interested in the research. The Journal welcomes reviews on topics of wide interest to investigators in the medical sciences. We particularly encourage submission of focused reviews.

Contents in this Volume

Title Pages

1. HOBNAIL VARIANT: REPORT OF A RARE AND AGGRESSIVE PAPILLARY THYROID CARCINOMA 1-6 Salillas,Annette L. MD, FPSP,FASCP, MIAC Nadela, Lev V. MD

2. PERIPHERAL NERVE-BLOCKING EFFECT FROM AN ALKYLAMIDE OBTAINED FROM TOOTHACHE PLANT FLOWER: A PROSPECT LOCAL ANESTHETIC AGENT 7-14 John Paul L. de la Cruz, Lica Jamane L. Carabaña, Randulf B. Copino, Maria Theresa M. Celestino, Kim Gregorio E. Cuizon III, Roey C. Yee, Dr. Arlene M. Diaz and Jose M. Oclarit, Ph.D

3. SUPPRESSION OF CARRAGEENAN-INDUCED ACUTE INFLAMMATION BY ANNONA SQUAMOSA (ATIS) ETHANOL SEED EXTRACT ON MALE ALBINO MICE 15-21

JF Perocho, SP Mollaneda, MJ Montederamos, G Montesclaros and A Muslimen and Arlene M. Diaz M.D.

4. THE NEPHROTOXIC EFFECT OF EXOGENOUS TRIMERIC GLUTATHIONE ON MALE ALBINO RATS 23-31 Philipp Vincent B. Lim,IoCamille D. Garcia, Janine H. Fuderanan, Joanna Rose B. Ipong, Ruffaidah M. Guro, Sudarat Klongrua Advisers: Arlene M. Diaz M.D. and Jose M. Oclarit Ph.D.

5. ANTI-PLATELET AGGREGATION ACTIVITY OF ATUNA RACEMOSA (TABON-TABON) FRUIT EXTRACT ON AGGREGATE INDUCED HUMAN PLATELET SUSPENSION 33-38

LC Liao, K Lasdoce, JW Lim, GK Macaya, R Subbaand Jose M.Oclarit,Ph.D

6. ANTIBACTERIAL ACTIVITY OF THE MORINGA OLEIFERA LAM (MALUNGGAY) PLANT PARTS ON PENICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS IN VITRO. 39-44 Arlene Maceren DiazM.D, FPSECP, Ed.D.

7. ANTI-DIABETIC POLYSACCHARIDE FROM A MANGROVE PLANT, SONNERATIA ALBA OBTAINED BY A REVERSE-PHASE (C-18) CHROMATOGRAPHY 45-46

Jose M. Oclarit and Nancy J. Morada

8. GUIDE TO AUTHORS 47-50

9. GUIDELINES ON WRITING A RESEARCH PROPOSAL 51-55

HOBNAIL VARIANT: REPORT OF A RARE AND AGGRESSIVE PAPILLARY THYROID CARCINOMA.

Salillas,Annette L. MD, FPSP,FASCP, MIAC1, Nadela, Lev V. MD2

1 Professor and Chairman of Pathology, SWU-MHAM College of Medicine, Cebu City, Philippines. Pathology Department Chairman, Gov. Celestino Gallares Memorial Hospital.

2 Pathology Resident, Gov. Celestino Gallares Memorial Hospital, Tagbilaran City, Bohol, Philippines.

Abstract

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, as well as the most common endocrine malignancy. PTC with prominent hobnail features is a recently recognized PTC variant. Its histological hallmark is represented by elongated cells showing a high nuclear/cytoplasmic ratio and a hobnail appearance. Immunohistochemistry and molecular studies have been performed showing aggressive clinical behaviour. Therefore, the correct recognition of Hobnail Variant of Papillary Thyroid Carcinoma features in preoperative FNA, in particular, the “comet-like” cell appearance representing the cytological counterpart of the histological hobnailing is extremely important. Thus, a better patient management might benefit from its early diagnosis on fine needle aspiration (FNA) samples. Introduction: Fine Needle Aspiration Biopsy(FNAB) is very accurate in diagnosing the most common thyroid malignancy, papillary thyroid carcinomas (PTC). Hobnail PTC (HPTC) is rare and a newly recognized variant with aggressive clinical behaviour which was first described by Asioli et al in 2010 [1]. These tumors commonly occur in women and are associated with significant mortality; 50% of the eight reported cases [1]. The aim of this study is to emphasize the important role of FNA in recognizing pre-operatively a rare and clinically aggressive variant of Papillary Thyroid Carcinoma (PTC) useful for suggesting a more aggressive neck surgery. Case report: A 54 year old, female with a 10 year history of a left anterior neck mass. Ultrasound was donewhich showed an enlarged left thyroid with a hypoechoic solid mass. FNAB was requested and read as Follicular Neoplasm in a background of colloid goiter with subsequent total thyroidectomy . Histopathology findings: The thyroidectomy specimen weighed 60 grams. The left lobe was enlarged to 8.5 cm x 4 cm x 2.7 cm and section shows a well delineated, white tan solid mass with a greatest dimension of 3.7 cm (fig. 1). The right

thyroid measures 3.7 cm x 3.2 cm x 0.7 cm and section reveals brown areas with one focal cream tan solid mass measuring 0.3 cm in widest dimension. The left thyroid mass microscopically showed tumor with papillary growth pattern and variably sized follicles (fig. 2a); some of the tumor cell nuclei were eccentrically placed causing bulging of the nuclei at the tip of the cell imparting the so called “hobnail” appearance to the cells (fig. 2b). Loss of cellular cohesiveness was evident leading to cell detachment (fig. 2c).On higher magnification, both the papillae and neoplastic follicles were mostly lined by cuboidal to elongated cells with abundant, well defined cytoplasmic borders. There are few nuclei showing multiple “soap bubble like” intranuclear inclusions (fig 2d). Many clusters of malignant cells with hobnail features form micropapillary structures without fibrovascular core comprising >30%. A concomitant tall cell/oncocytic cells were also identified (fig. 2d). A diagnosis of Hobnail Variant of Papillary Thyroid Carcinoma was rendered. The right thyroid mass revealed Papillary Thyroid Microcarcinoma (fig. 3). Cytological review: FNA was reviewed on the basis of the histological evidence of tumor cell nuclei which are eccentrically placed causing bulging of the nuclei at the tip of the

HOBNAIL VARIANT: REPORT OF A RARE AND AGGRESSIVE PAPILLARY THYROID CARCINOMA.

Salillas,Annette L. MD, FPSP,FASCP, MIAC1, Nadela, Lev V. MD2

1 Professor and Chairman of Pathology, SWU-MHAM College of Medicine, Cebu City, Philippines. Pathology Department Chairman, Gov. Celestino Gallares Memorial Hospital.

2 Pathology Resident, Gov. Celestino Gallares Memorial Hospital, Tagbilaran City, Bohol, Philippines.

Abstract

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, as well as the most common endocrine malignancy. PTC with prominent hobnail features is a recently recognized PTC variant. Its histological hallmark is represented by elongated cells showing a high nuclear/cytoplasmic ratio and a hobnail appearance. Immunohistochemistry and molecular studies have been performed showing aggressive clinical behaviour. Therefore, the correct recognition of Hobnail Variant of Papillary Thyroid Carcinoma features in preoperative FNA, in particular, the “comet-like” cell appearance representing the cytological counterpart of the histological hobnailing is extremely important. Thus, a better patient management might benefit from its early diagnosis on fine needle aspiration (FNA) samples. Introduction: Fine Needle Aspiration Biopsy(FNAB) is very accurate in diagnosing the most common thyroid malignancy, papillary thyroid carcinomas (PTC). Hobnail PTC (HPTC) is rare and a newly recognized variant with aggressive clinical behaviour which was first described by Asioli et al in 2010 [1]. These tumors commonly occur in women and are associated with significant mortality; 50% of the eight reported cases [1]. The aim of this study is to emphasize the important role of FNA in recognizing pre-operatively a rare and clinically aggressive variant of Papillary Thyroid Carcinoma (PTC) useful for suggesting a more aggressive neck surgery. Case report: A 54 year old, female with a 10 year history of a left anterior neck mass. Ultrasound was donewhich showed an enlarged left thyroid with a hypoechoic solid mass. FNAB was requested and read as Follicular Neoplasm in a background of colloid goiter with subsequent total thyroidectomy . Histopathology findings: The thyroidectomy specimen weighed 60 grams. The left lobe was enlarged to 8.5 cm x 4 cm x 2.7 cm and section shows a well delineated, white tan solid mass with a greatest dimension of 3.7 cm (fig. 1). The right

thyroid measures 3.7 cm x 3.2 cm x 0.7 cm and section reveals brown areas with one focal cream tan solid mass measuring 0.3 cm in widest dimension. The left thyroid mass microscopically showed tumor with papillary growth pattern and variably sized follicles (fig. 2a); some of the tumor cell nuclei were eccentrically placed causing bulging of the nuclei at the tip of the cell imparting the so called “hobnail” appearance to the cells (fig. 2b). Loss of cellular cohesiveness was evident leading to cell detachment (fig. 2c).On higher magnification, both the papillae and neoplastic follicles were mostly lined by cuboidal to elongated cells with abundant, well defined cytoplasmic borders. There are few nuclei showing multiple “soap bubble like” intranuclear inclusions (fig 2d). Many clusters of malignant cells with hobnail features form micropapillary structures without fibrovascular core comprising >30%. A concomitant tall cell/oncocytic cells were also identified (fig. 2d). A diagnosis of Hobnail Variant of Papillary Thyroid Carcinoma was rendered. The right thyroid mass revealed Papillary Thyroid Microcarcinoma (fig. 3). Cytological review: FNA was reviewed on the basis of the histological evidence of tumor cell nuclei which are eccentrically placed causing bulging of the nuclei at the tip of the

Date Received: March 2015 Date Accepted: April 10, 2015

1

cell imparting the so-called 'hobnail' appearance to the cells. The FNA smears are cellular composed of medium-sized neoplastic follicular cells arranged in branching cell groups, some in micropapillary and microfollicular structures,others are dispersed single cells with distinct “tear-drop” cytoplasm, apically placed nuclei that produced a surface bulge leading to a “comet-cell” like appearance (fig. 4a-c). At higher magnification, nuclei showed variable degrees of atypia with nuclear grooves and inclusions readily seen (fig. 4b-e). Other areas show single cells with abundant cytoplasm and sharp borders and aggregated in tile-like manner (fig. 4f-g) Discussion: Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, as well as the most common endocrine malignancy. It has an excellent clinical prognosis, even with local lymph node involvement at initial surgery. Hobnail Papillary Thyroid Carcinoma (HPTC) is a rare and newly recognized variant with highly aggressive behaviour , high incidence of invasion, and metastasis [1- 3]. These aggressive subtypes of PTC have been associated with higher rate of extrathyroidal extension, multifocality,nodal and distant metastasis, recurrence and resistance to radioactive iodine therapy [4]. This case was a female in her 5th decade of life with a 3.7cm in size solid left anterior neck mass . Studies have shown that these tumors commonly occur in women, with average age of 28 to 78 years old [5] and patients usually presented with a neck mass and cervical lymphadenopathy [1]. Tumor size ranged from 1 cm to 5.8 cm (mean 3.0cm) [5]. They are usually multifocal with variably sized complex papillary structures without fibrovascular core lined by cells with increased nuclear/cytoplasmic ratios and apically placed nuclei that produced a surface bulge (hobnail appearance) [1] . Typical features of HPTC according to Bellivicine et al [6] includes the presence of hobnail and papillary/micropapillary cyto-architectural patterns, single “tear-drop” cells similar to “comet-like cells”,high N/C ratio and severe crowding which were also seen in this case. Recently, the cytological features of HPTC have been reported as “comet-like” cells, suggesting that this feature represents the cytological counterpart of the histological hobnailing [6]. Thus, the presence on FNA of “comet-like “ cells may lead to the preoperative suspicion of HPTC [6]

There is no specific immunohistochemical or molecular markers in HPTC [7]. Thyroglobulin, TTF-1, HBME-1, and p53 were positive in all cases, and there was membrane staining for β-catenin and E-cadherin [1]. Despite high mitotic rate and the presence of necrosis, HPTC should not be included in the group of poorly differentiated carcinomas, because of the presence of conventional nuclear features of PTC, absence of convoluted nuclei [7], diffuse cytoplasmic staining for thyroglobulin [8] and presence of BRAF mutation [9]. To date, only a few cases of PTC with prominent hobnail features have been described and most of these had an aggressive behavior [6]. This patient exhibited >30% of malignant cells with hobnail features forming micropapillary structures with concomitant tall cell/oncocytic cells also noted. According to the study of Baloch et al [4], tumors with more than 30% hobnail/micropapillary features were often very aggressive, although 2 patients with tumors with 10% hobnail/micropapillary features also had poor outcomes [4]. Concomitant tall cell features (TCF) (40% of cases), and others with small foci of undifferentiated (anaplastic) thyroid carcinoma (ATC) were also seen in some patients [2]. Seven of eleven patients were Stage III or IV at presentation [2]. At median follow- up of 14 months, three of seven patients had recurred: one with coexistent ATC and TCF, one with TCF, and one without ATC or TCF. One patient died from their disease one year after surgery [2] Most PTC is managed by standard total thyroidectomy. However, in some PTC variants with a more aggressive behaviour such as the tall cell, columnar, solid , diffuse sclerosing and hobnail variants , the correct recognition on pre-op FNA is useful for suggesting more aggressive neck surgery [10]. Conclusion: Hobnail Variant of Papillary Thyroid Carcinoma features show distinct cytological features whereby its recognition in preoperative FNA,in particular, the “comet-like” cell feature representing the cytological counterpart of the histological hobnailing is extremely important. Reporting these features in a cytopathology report is very useful which may lead to additional screening to rule out lymph node and /or distant metastases. Thereby allowing early aggressive neck surgery with adjuvant radioactive iodine management and close observation for recurrence.

References:

1. Asioli S, Erickson L, Sebo T et al: Papillary Thyroid Carcinoma with prominent hobnail features: A new aggressive variant of moderately differentiated papillary carcinoma. A clinicopathologic, Immunohistochemical, and Molecular Study of Eight cases. Am J of Surg Pathol: January 2010 Volume 34 -Issue 1- pp 44-52.

2. Lubitz C, Parangi S, Faquin W, Sadow P: Hobnail variant of papillary thyroid carcinoma: an institutional case series and molecular profile. Endocrine Surgery Unit, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston , Massachusettes. 2014 June; 24 (6): 958-65. EPub 2014 March 6.

3. Motosugi U, Murata S, Nagata K et al: Thyroid papillary carcinoma with micropapillary and hobnail growth pattern: a histological variant with intermediate malignancy. Thyroid 2009; 19:535-537.

4. Baloch Z, LiVolsi V, Tondon R: Aggressive Variants of Follicular Cell Derived Thyroid Carcinoma The So-Called “Real Thyroid Carcinomas”. J ClinPathol. 2013; 66(9): 733-743.

5. Asioli S, Erickson LA, Righi A, Lloyd RV: Papillary Thyroid Carcinoma with Hobnail Features :Histopathologic criteria to predict aggressive behavior. Hum Pathol 2013; 44(3): 320-8.

6. Bellevicine C, Cozzolino I, Umberto M et al: Cytological and Molecular Features of Papillary Thyroid Carcinoma with Prominent Hobnail Features: A Case Report. ActaCytol 2012;56:560–564

7. Asioli S, Maletta F, Pagni F et al: Cytomorphologic and Molecular Features of Hobnail Variant of Papillary Thyroid Carcinoma: Case series and Literature Review. Diag Cytol.2013; Vol 42, No.1: 78-84.

8. Asioli S, Erickson LA, Righi A et al: Poorly

differentiated carcinoma of the thyroid: Validation of the Turin proposal and analysis of IMP3 expression. Mod Pathol 2010; 23:1269-1278.

9. Volante M, Rapa I, Gandhi M et al: RAS mutation are the predominant molecular alteration in poorly differentiated thyroid carcinomas and bear prognostic impact. J ClinEndocrinolMetab 2009; 94: 4735-4741.

10. Ohori NP , Schoedel KE: Cytopathology of high grade papillary thyroid carcinomas: tall-cell variant, diffuse sclerosing variant, and poorly differentiated papillary thyroid carcinoma. Diagn Cytopathol 1999; 20: 19-23

2

References:

1. Asioli S, Erickson L, Sebo T et al: Papillary Thyroid Carcinoma with prominent hobnail features: A new aggressive variant of moderately differentiated papillary carcinoma. A clinicopathologic, Immunohistochemical, and Molecular Study of Eight cases. Am J of Surg Pathol: January 2010 Volume 34 -Issue 1- pp 44-52.

2. Lubitz C, Parangi S, Faquin W, Sadow P: Hobnail variant of papillary thyroid carcinoma: an institutional case series and molecular profile. Endocrine Surgery Unit, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston , Massachusettes. 2014 June; 24 (6): 958-65. EPub 2014 March 6.

3. Motosugi U, Murata S, Nagata K et al: Thyroid papillary carcinoma with micropapillary and hobnail growth pattern: a histological variant with intermediate malignancy. Thyroid 2009; 19:535-537.

4. Baloch Z, LiVolsi V, Tondon R: Aggressive Variants of Follicular Cell Derived Thyroid Carcinoma The So-Called “Real Thyroid Carcinomas”. J ClinPathol. 2013; 66(9): 733-743.

5. Asioli S, Erickson LA, Righi A, Lloyd RV: Papillary Thyroid Carcinoma with Hobnail Features :Histopathologic criteria to predict aggressive behavior. Hum Pathol 2013; 44(3): 320-8.

6. Bellevicine C, Cozzolino I, Umberto M et al: Cytological and Molecular Features of Papillary Thyroid Carcinoma with Prominent Hobnail Features: A Case Report. ActaCytol 2012;56:560–564

7. Asioli S, Maletta F, Pagni F et al: Cytomorphologic and Molecular Features of Hobnail Variant of Papillary Thyroid Carcinoma: Case series and Literature Review. Diag Cytol.2013; Vol 42, No.1: 78-84.

8. Asioli S, Erickson LA, Righi A et al: Poorly

differentiated carcinoma of the thyroid: Validation of the Turin proposal and analysis of IMP3 expression. Mod Pathol 2010; 23:1269-1278.

9. Volante M, Rapa I, Gandhi M et al: RAS mutation are the predominant molecular alteration in poorly differentiated thyroid carcinomas and bear prognostic impact. J ClinEndocrinolMetab 2009; 94: 4735-4741.

10. Ohori NP , Schoedel KE: Cytopathology of high grade papillary thyroid carcinomas: tall-cell variant, diffuse sclerosing variant, and poorly differentiated papillary thyroid carcinoma. Diagn Cytopathol 1999; 20: 19-23

3

Fig. 1. Left lobe Fig 2. Left thyroid mass. a. Thin arborizing papillae with prominent hobnail cells, 10x. b. Hobnail cells,

20x. c. Loss of cellular cohesiveness resulting to tufts of detaching cells, 10x. d. Tall and oncocytic cells lining micropapillae without fibrovascular core, 10x .

Fig 3. Papillary microcarcinoma in the right lobe, 4x.

4

Fig 3. Papillary microcarcinoma in the right lobe, 4x.

5

Fig 4. FNA smears. a,b,c. The comet-like cells are shown with “tear-drop” cytoplasm. 10x,40,x100x. d,e. Soap-bubble - like nuclear inclusions are recognizable, 40x. f,g. Single cells with abundant cytoplasm and sharp borders aggregated in a tile-like manner, 40x.

Peripheral Nerve-blocking Effect from an Alkylamide Obtained from Toothache Plant flower: A

Prospect Local Anesthetic Agent

John Paul L. de la Cruz, Lica Jamane L. Carabaña, Randulf B. Copino, Maria Theresa M. Celestino, Kim Gregorio E. Cuizon III, Roey C. Yee, Dr. Arlene M. Diaz and Jose M. Oclarit, Ph.D

1Southwestern University – Matias H. Aznar Memorial College of Medicine Redemptorist Plaza, Camputhaw, Cebu City 6000

Philippines

Abstract

The fast-growing popularity of folk medicine had conceived the objective of investigating the Toothache Plant’s

local anesthetic property. The study qualitatively-isolated the active compound of the plant extract, evaluated its peripheral nerve-blocking effect using a quasi-experimental design, and investigated its toxicological properties. The extract was phytochemically analysed. The isolation of the active compound was done using thin layer chromatography and high performance liquid chromatography. The peripheral nerve-blocking activity was evaluated using muscle-nerve module of Rhinella marinus and the toxicity test was assessed using Artemia salina assay. Phytochemically, the extract contains alkaloids, glycosides, flavanoids, tannins, anthraquinone, saponins and cardiac glycosides. Chromatographically, it displayed an active compound with an Rf value of 0.5 while in HPLC qualitative-analysis, four prominent peaks were seen. The peak with a retention time of 6 minutes and 40 seconds is identified as an alkylamide – spilanthol which causes the anesthetic activity. The arrests of muscular contractions were evaluated on the parameters: onset, duration, and anesthetic effect quality. There were significant differences seen between the plant extract-treated groups and the 2% lidocaine hydrochloride group on the duration parameter, while none on the onset and the anesthetic effect quality. Toxicologically, the LC50 increased when the mean extract concentration and duration of exposure were decreased. The forgoing findings implied a substantial peripheral nerve-blocking effect of the extract comparable to 2% lidocaine hydrochloride. Introduction

The use of medicinal plants as a source of relief

from illness is as old as humankind1. The World Health Organization has estimated that 80% of the population in developing countries2 use traditional medicine for primary health care and for treating diseases3.

Plants continue to be a major source of drugs for

the world populations2 and substances derived from plants alone constitute 25% of prescribed medicines1. Approximately half of about 250,000 flowering plant species occurring globally are found in the tropical forests4 which continue to produce natural products with valuable compounds for the development of new drugs5.

The increasing demand on herbal medicines and their acceptance in international market is due to its potent pharmacological potential and high therapeutic value for human populace2. With the advent of scientific breakthroughs and discovery of many drugs and other innovations in the field of medicine, a vast array of human conditions can now be remedied with ease.

Despite the availability of commercially

available medicines and other health care commodities, cost remains high which prevents easy access to safe and effective health care. It is beyond denial that the high cost of drugs, particularly branded medications, constitutes as the prime reason for most of patient’s non-compliance to the pharmacologic regimen6. With this, some people resort to alternative treatment modalities, of which herbal therapy is of foremost popularity.

In sum, the continued rise in drug cost as well as

the fast-growing popularity and deepened confidence in the use of herbal therapy are the principal motivators of conceiving the objective of investigating Toothache Plant’s local anesthetic property.

Toothache Plant, a traditionally used folk medicine4 that grows throughout the tropics7, has

*Corresponding author: John Paul L. De la Cruz Southwestern University – Matias H. Aznar Memorial College of Medicine Cebu city 6000 Corresponding author email address: jp.delacruz88@yahoo.com

6

Peripheral Nerve-blocking Effect from an Alkylamide Obtained from Toothache Plant flower: A

Prospect Local Anesthetic Agent

John Paul L. de la Cruz, Lica Jamane L. Carabaña, Randulf B. Copino, Maria Theresa M. Celestino, Kim Gregorio E. Cuizon III, Roey C. Yee, Dr. Arlene M. Diaz and Jose M. Oclarit, Ph.D

1Southwestern University – Matias H. Aznar Memorial College of Medicine Redemptorist Plaza, Camputhaw, Cebu City 6000

Philippines

Abstract

The fast-growing popularity of folk medicine had conceived the objective of investigating the Toothache Plant’s

local anesthetic property. The study qualitatively-isolated the active compound of the plant extract, evaluated its peripheral nerve-blocking effect using a quasi-experimental design, and investigated its toxicological properties. The extract was phytochemically analysed. The isolation of the active compound was done using thin layer chromatography and high performance liquid chromatography. The peripheral nerve-blocking activity was evaluated using muscle-nerve module of Rhinella marinus and the toxicity test was assessed using Artemia salina assay. Phytochemically, the extract contains alkaloids, glycosides, flavanoids, tannins, anthraquinone, saponins and cardiac glycosides. Chromatographically, it displayed an active compound with an Rf value of 0.5 while in HPLC qualitative-analysis, four prominent peaks were seen. The peak with a retention time of 6 minutes and 40 seconds is identified as an alkylamide – spilanthol which causes the anesthetic activity. The arrests of muscular contractions were evaluated on the parameters: onset, duration, and anesthetic effect quality. There were significant differences seen between the plant extract-treated groups and the 2% lidocaine hydrochloride group on the duration parameter, while none on the onset and the anesthetic effect quality. Toxicologically, the LC50 increased when the mean extract concentration and duration of exposure were decreased. The forgoing findings implied a substantial peripheral nerve-blocking effect of the extract comparable to 2% lidocaine hydrochloride. Introduction

The use of medicinal plants as a source of relief

from illness is as old as humankind1. The World Health Organization has estimated that 80% of the population in developing countries2 use traditional medicine for primary health care and for treating diseases3.

Plants continue to be a major source of drugs for

the world populations2 and substances derived from plants alone constitute 25% of prescribed medicines1. Approximately half of about 250,000 flowering plant species occurring globally are found in the tropical forests4 which continue to produce natural products with valuable compounds for the development of new drugs5.

The increasing demand on herbal medicines and their acceptance in international market is due to its potent pharmacological potential and high therapeutic value for human populace2. With the advent of scientific breakthroughs and discovery of many drugs and other innovations in the field of medicine, a vast array of human conditions can now be remedied with ease.

Despite the availability of commercially

available medicines and other health care commodities, cost remains high which prevents easy access to safe and effective health care. It is beyond denial that the high cost of drugs, particularly branded medications, constitutes as the prime reason for most of patient’s non-compliance to the pharmacologic regimen6. With this, some people resort to alternative treatment modalities, of which herbal therapy is of foremost popularity.

In sum, the continued rise in drug cost as well as

the fast-growing popularity and deepened confidence in the use of herbal therapy are the principal motivators of conceiving the objective of investigating Toothache Plant’s local anesthetic property.

Toothache Plant, a traditionally used folk medicine4 that grows throughout the tropics7, has

*Corresponding author: John Paul L. De la Cruz Southwestern University – Matias H. Aznar Memorial College of Medicine Cebu city 6000 Corresponding author email address: jp.delacruz88@yahoo.com

Date Received: Dec. 10, 2014 Date Accepted: Jan. 25, 2015

7

demonstrated a numbing effect for toothaches, stomatitis and other throat complaints4. The plant genus possesses phytochemicals: alkaloids, glycosides, flavonoids, tannins, anthraquinones, saponins, and cardiac glycosides8. The most of its biological activity is attributed to alkylamide9, 10 – spilanthol which causes the local anesthetic effect11, 12.

Hence, the present study isolated the active

compound of the extract using thin layer chromatography and high performance liquid chromatography. Its significant peripheral nerve-blocking activity was evaluated using muscle-nerve module of Rhinella marinus, and the toxicity test was assessed using Artemia salina assay. Methods

A quasi-experimental research design was used

in this study. This research design permitted the researchers to systematically manipulate independent variables and make observations. The independent variable of the study includes the modes of treatment namely: Toothache Plant flower extract, 2% lidocaine hydochloride, and Plain NSS. The dependent variable was the peripheral nerve-blocking effects of such treatments on the randomly assigned group samples. Preliminary Preparation

The protocol for experimentation was approved

by Dr. Jocelyn A. Tingson of Institutional Animal Care and Use Committee, College of Veterinary Medicine, Southwestern University, Cebu City, and was in accordance with international standard on the care and use of experimental animals8.

The appropriate sample size was determined by

power analysis done by Mr. Dexter S. Ontoy, Center for Research, Cebu Normal University. Thirty-two cane toads (Rhinella marinus) were randomly chosen as subjects of the experiment. It was made certain that the subjects were devoid of any ailment or physical aberration at the time of experimentation under the supervision of a veterinarian. Moreover, the researchers ensured that the samples were more or less identical in size and weight. The cane toad had a weight ranges from 40 to 50 grams.

The toothache plant flower were collected,

identified and authenticated by the Department of Agriculture, Experimental Station, Mandaue City. The collected flowers were cleaned with running water and were air-dried for 2 weeks to remove the minimum biological moisture of the samples. When completely dried, 90 grams of plant samples were crushed using mortar and pestle.

The crushed plant material was soaked in 80%

HPLC grade ethanol with a ratio of 2 part solute to 1 part solvent for 48 hours. After 48 hours of soaking, the flowers were passed through a filter paper, fed in a soxhlet apparatus until complete exhaustion. The extract was collected and dried under a vacuum using a rotary evaporator machine at a temperature of 78.37oC (the boiling point of ethanol) at a rate of 100 rpn for 30 minutes until most of the ethanol was remove. The extract was lit and the absence of fire confirmed the absence of ethanol. The final extract was placed in an amber prescription bottle, stored it at a temperature of 4oC and away from direct sunlight. Chemical Analysis

The freshly prepared extract was qualitatively

analysed chemically using phytochemical screening test, thin layer chromatography and high performance liquid chromatography. Phytochemically, the tests for alkaloids, glycosides, flavonoids, tannins, anthraquinones, saponins, and cardiac glycosides13 were carried.

In the thin layer chromatography, the silica gel

TLC plates (20 x 20 cm) were spotted with 10ul (10 mg/ml) of the extract at a height of 2 cm of from the bottom. It was eluted with 200 mL of hexane and ethyl acetate in proportion 2:1 (v/v) and stored in a glass tank for 3 hours. Afterwards, the plate was taken out and dried for 24 hours at room temperature under a stream of air to remove the eluent solvent system. Thereafter, the separated constituents were visualized under UV light at wavelengths of 254 nm to observe the bands. The Rf value (ratio between distance travelled by the sample and distance travelled by the solvent) was identified. 9

The silica with the compound was mixed in a

magnetic stirrer with 95 % ethanol and filtrated thrice to remove completely the solid. The clean and green solution was subjected to an evaporative step using a rotary evaporator machine.

In the reverse phase high performance liquid

chromatography, the C18 column (50mm x 2.1 mm, 3 μm particle size, 110 Å pore size) equipped with a 0.5 μm pre column filter was carried out. The separation was conducted with mobile phase composition of 1% acetic acid in nanopure water and HPLC grade acetonitrile in 50:50 solvent ratio. The HPLC machine was ran for 22 minutes with a flow rate of 0.2 mL/min and an injection volume of 10 μL. 11

Electrophysiological Assay In gastrocnemius muscle-sciatic nerve

preparation, the Rhinella marinus were double pithed though the foramen magnum then into the cranium. The gastrocnemius muscles were prepared, weighed in grams one at a time and were kept moist with Ringers solution. After acquiring the weight, the amount of solution of the positive control was identified using the formula 20mg/kg/ml14 (2% lidocaine hydrochloride’s strength per ml). The 0.01ml dose was administered after considering the weight of each thigh of the experimental animals which was 10 g each.

The dosing of the plant crude extract was

prepared according to the dose of the experimental control. In 100% concentration, 100mg of pure crude extract was diluted with 5ml triple distilled water (20mg/ml) and used 0.01ml in order to have the same amount of strength with the positive control. In the 50% concentration, 50mg of the extract was diluted to 5ml triple distilled water and used 0.01ml of the prepared solution.

The extract was evaluated for its peripheral

nerve-blocking property using the muscle-nerve module of Rhinella marinus on the parameters: onset, duration, and quality of local anesthetic effect. The 2% lidocaine hydrochloride and plain NSS were used as the positive and negative controls respectively. All records of muscular contractions were taken using the kymograph machine at a speed of 580 mm/sec.

In the onset and duration parameters, the

gastrocnemius muscle-sciatic nerve was stimulated with 5 mAmp with a frequency setting of 1 per 20 seconds to prevent muscular fatigue. The recording starts after an arrest of muscular contraction was seen (onset) and ends after first contraction from the arrest (duration) was noticed. In the evaluation the quality of local anesthetic parameter, however, the break shocks were used to determined threshold stimulus. After a lapse of 20 seconds, a slightly stronger stimulus was applied (1 mAmp, 3 mAmp and 5 mAmp) to the gastrocnemius muscle-sciatic nerve. The experiment was continued until there was no further increase in response of the muscles. Toxicological Assay

The 5 mg of toothache plant flower extract was

dissolved in 0.5 ml methanol and assigned as solution A while 0.5 ml of solution A was diluted to 10 ml methanol and was named as solution B.

100µL of solution B, 50µL of solution A and

500µL of solution A were pipetted and transferred into

separate test tubes and were labelled as 1, 2, 3 respectively. The control test tube was prepared as well with 1mL of methanol. 5 replicates were prepared and then solutions were dried after.

The sample vials were diluted to 5 ml artificial

sea water (3.8 g of rock salt per 100 ml distilled water) and a final concentration of 10, 100 and 1000 µg/ml were made respectively.

With a 9 inch pipette, 10 nauplii were transferred into each test tube labelled as 1, 2, 3 and control. A yeast suspension (3mg/5ml of artificial sea water) was dropped as food in each test tube. The test tubes were kept under illumination. The survivors were counted using 3x magnifying glass after 6 hours and after 24 hours. The percent death for each dose level and for the control vial were recorded and the LC50 from the sixth and twenty-four hour counts were determined with the Probit analysis method.13

Statistical Treatment

A descriptive statistics was used to determine the

profile of subjects in terms of the parameters used in local anesthetic measurement. After ascertaining homogeneity of the raw data, one-way analysis of variance (ANOVA) was used to determine the presence of a statistically significant difference in the mean onset and duration of peripheral nerve-blocking effect. The two-way analysis of variance (ANOVA) was used in the quality of local anaesthetic parameter in post-treatment groups. In conjunction to ANOVA, the post hoc analysis utilizing the Tukey-test (t-test) was used to compare the mean onset, duration of peripheral nerve-blocking effect and the quality of anesthesia between and among treatment groups: the experimental groups (50% and 100% concentrations), the positive control, the negative control.

Results Chemical Assay

The chemical investigation using phytochemical

screening test revealed that the extract contains alkaloids, glycosides, flavanoids, tannins, anthraquinone, saponins and cardiac glycosides while the thin layer chromatography presented one (1) active compound with an Rf value of 0.5 after subjecting it to 256 nm UV light that was isolated as seen in figure 1. It revealed in high performance liquid chromatographic qualitative analysis that the extract has 4 prominent peaks as seen in figure 2. The peak with a retention time of 6 minutes and 40 seconds suggests an alkylamide – spilanthol, a known constituent of the genus Spilanthes.

8

Electrophysiological Assay In gastrocnemius muscle-sciatic nerve

preparation, the Rhinella marinus were double pithed though the foramen magnum then into the cranium. The gastrocnemius muscles were prepared, weighed in grams one at a time and were kept moist with Ringers solution. After acquiring the weight, the amount of solution of the positive control was identified using the formula 20mg/kg/ml14 (2% lidocaine hydrochloride’s strength per ml). The 0.01ml dose was administered after considering the weight of each thigh of the experimental animals which was 10 g each.

The dosing of the plant crude extract was

prepared according to the dose of the experimental control. In 100% concentration, 100mg of pure crude extract was diluted with 5ml triple distilled water (20mg/ml) and used 0.01ml in order to have the same amount of strength with the positive control. In the 50% concentration, 50mg of the extract was diluted to 5ml triple distilled water and used 0.01ml of the prepared solution.

The extract was evaluated for its peripheral

nerve-blocking property using the muscle-nerve module of Rhinella marinus on the parameters: onset, duration, and quality of local anesthetic effect. The 2% lidocaine hydrochloride and plain NSS were used as the positive and negative controls respectively. All records of muscular contractions were taken using the kymograph machine at a speed of 580 mm/sec.

In the onset and duration parameters, the

gastrocnemius muscle-sciatic nerve was stimulated with 5 mAmp with a frequency setting of 1 per 20 seconds to prevent muscular fatigue. The recording starts after an arrest of muscular contraction was seen (onset) and ends after first contraction from the arrest (duration) was noticed. In the evaluation the quality of local anesthetic parameter, however, the break shocks were used to determined threshold stimulus. After a lapse of 20 seconds, a slightly stronger stimulus was applied (1 mAmp, 3 mAmp and 5 mAmp) to the gastrocnemius muscle-sciatic nerve. The experiment was continued until there was no further increase in response of the muscles. Toxicological Assay

The 5 mg of toothache plant flower extract was

dissolved in 0.5 ml methanol and assigned as solution A while 0.5 ml of solution A was diluted to 10 ml methanol and was named as solution B.

100µL of solution B, 50µL of solution A and

500µL of solution A were pipetted and transferred into

separate test tubes and were labelled as 1, 2, 3 respectively. The control test tube was prepared as well with 1mL of methanol. 5 replicates were prepared and then solutions were dried after.

The sample vials were diluted to 5 ml artificial

sea water (3.8 g of rock salt per 100 ml distilled water) and a final concentration of 10, 100 and 1000 µg/ml were made respectively.

With a 9 inch pipette, 10 nauplii were transferred into each test tube labelled as 1, 2, 3 and control. A yeast suspension (3mg/5ml of artificial sea water) was dropped as food in each test tube. The test tubes were kept under illumination. The survivors were counted using 3x magnifying glass after 6 hours and after 24 hours. The percent death for each dose level and for the control vial were recorded and the LC50 from the sixth and twenty-four hour counts were determined with the Probit analysis method.13

Statistical Treatment

A descriptive statistics was used to determine the

profile of subjects in terms of the parameters used in local anesthetic measurement. After ascertaining homogeneity of the raw data, one-way analysis of variance (ANOVA) was used to determine the presence of a statistically significant difference in the mean onset and duration of peripheral nerve-blocking effect. The two-way analysis of variance (ANOVA) was used in the quality of local anaesthetic parameter in post-treatment groups. In conjunction to ANOVA, the post hoc analysis utilizing the Tukey-test (t-test) was used to compare the mean onset, duration of peripheral nerve-blocking effect and the quality of anesthesia between and among treatment groups: the experimental groups (50% and 100% concentrations), the positive control, the negative control.

Results Chemical Assay

The chemical investigation using phytochemical

screening test revealed that the extract contains alkaloids, glycosides, flavanoids, tannins, anthraquinone, saponins and cardiac glycosides while the thin layer chromatography presented one (1) active compound with an Rf value of 0.5 after subjecting it to 256 nm UV light that was isolated as seen in figure 1. It revealed in high performance liquid chromatographic qualitative analysis that the extract has 4 prominent peaks as seen in figure 2. The peak with a retention time of 6 minutes and 40 seconds suggests an alkylamide – spilanthol, a known constituent of the genus Spilanthes.

9

Figure 1.(Left) TLC plate under 265nm of UV light. (Right) Determination of Rf value (0.5)

Figure 2.HPLC result of toothache plant flower showing 4

prominent peaks. The peak number 7 with a retention time of 6 minutes and forty seconds suggest alkylamide – spilanthol.

Electrophysiological Assay

The toothache plant flower extract in a

concentration of 50% and 100% exhibited a peripheral nerve-blocking effect comparable to anesthetic effect produced by 2% lidocaine hydrochloride as seenin figure 3. However, the increase from 50% to 100% concentration of Toothache Plant flower extract presented an earlier onset and longer duration of local anesthetic action.

Figure 3.The mean onset and duration of peripheral nerve blocking effect of (a) Plain NSS (b) 2% lidocaine HCl (c) 50% concentration (d) 100% concentration of the Toothache Plant flower extract The mean onset of local anesthetic effect of the

100% and 50% concentrations were 35 seconds and 276 seconds respectively while in the 2% lidocaine hydrochloride, a lapse of 138 seconds was seen before an anesthetic effect was produced. The mean duration of the 100% and 50% concentration were 4235 seconds and 2640 seconds respectively, while in standard drug, the duration was 3420 seconds.

Statistically, the one-way ANOVA revealed

significant differences in the mean onset as seen in table 1 and duration parameter as seen table in 2 in the post-treatment groups with a p-value of 0.000. In conjunction to one-way ANOVA, the post-hoc T-test revealed that the onset of local anesthetic activity of the 50% and 100% concentrations against the positive control were insignificantly different with a p value of 0.112 and 0.384 respectively while there is a significant difference seen among the experimental group with a p value that lies in a 0.05 level as seen in table 3.

Treatment Mean Difference Std. Error p-Value

1

(Neg.

control)

4 5365.00* 59.60 0.000

3 5190.00* 59.60 0.000

2 5295.00* 59.605 0.00

2

(Pos.

control)

4 70.00 59.605 0.384

3 105.00 59.60 0.112

1 105.00* 59.60 0.000

3

(50%

conc.)

4 175.00* 59.60 0.006

2 105.00 59.605 0.112

1 210.00* 59.60 0.000

4

(100%

conc.)

3 175.00* 59.605 0.006

2 70.00 59.605 0.384

1 35.00* 59.60 0.000

Treatment

Duration of the Anesthetic Effect (in Seconds)

F-ratio

p-Value

Mean (in sec) Standard deviation

1 (Control) 5400.00* 0.000

7868.82 0.000 2 (Positive) 105.00 30.000

3 (50% conc.) 210.00 114.891

4 (100% conc.) 35.00 None

*the onset for the control used the whole duration of the study as a proxy value for the purpose of testing the difference using ANOVA Table 1.Comparison of the mean scores of the onset of the

anesthetic effect between treatments (one-way ANOVA).

Treatment Duration of the Anesthetic

Effect (in Seconds) F-ratio p-

Value Mean (in sec)

Standard deviation

1 (Control) 0.00 0.000

133.733 0.000 2 (Positive) 3,720.00 492.341

3 (50% conc.) 2,940.00 388.844

4 (100% conc.) 4,465.00 253.180

Table 2. Comparison of the mean scores of the duration of the

anesthetic effect between treatments (One-way ANOVA)

*The mean difference is significant at the 0.05 level.

Table 3. Post-hoc (Tukey Test) multiple comparisons for onset

of peripheral nerve-blocking effect after the treatment

The duration parameter of 50% and 100%

concentration against the positive control and among the experimental groups revealed significant differences with a p value of 0.05 as seen in table 4.

Treatment Mean Difference Std. Error p-Value

1

(Neg. control)

2 3720.00* 239.191 0.000 3 745.00* 239.191 0.039 4 4465.00* 239.191 0.000

2

(Pos. control)

1 -1525.00* 239.191 0.000 3 -780.00* 239.191 0.030 4 2940.00* 239.191 0.000

3

(50% conc.)

1 -745.00* 239.191 0.039 2 780.00* 239.191 0.030 4 3720.00* 239.191 0.000

4

(100% conc.)

1 -4465.00* 239.191 0.000 2 -2940.00* 239.191 0.000 3 -3720.00* 239.191 0.000

* The mean difference is significant at the 0.05 level. Table 4.Post-hoc (Tukey Test) Multiple Comparisons for

duration of peripheral nerve-blocking effect after the treatment In the quality of local anesthetic effect

parameter, the plant-extract treated groups and the positive control group presented a substantially diminished contraction even if a stronger stimulus was induced from 1 mAmp to 3mAmp to 5mAmp as seen in figure 4. However, no significant differences were seen between the plant-extract treated groups and the positive control group as seen in table 5.

Figure 4. Estimated marginal means of contractionbetween

treatments and between varying strength of stimulus (a) Plain NSS, (b) 2% lidocaine HCl, (c) 50%, (d) 100% concentration of the Toothache Plant flower extract

10

Treatment

Duration of the Anesthetic Effect (in Seconds)

F-ratio

p-Value

Mean (in sec) Standard deviation

1 (Control) 5400.00* 0.000

7868.82 0.000 2 (Positive) 105.00 30.000

3 (50% conc.) 210.00 114.891

4 (100% conc.) 35.00 None

*the onset for the control used the whole duration of the study as a proxy value for the purpose of testing the difference using ANOVA Table 1.Comparison of the mean scores of the onset of the

anesthetic effect between treatments (one-way ANOVA).

Treatment Duration of the Anesthetic

Effect (in Seconds) F-ratio p-

Value Mean (in sec)

Standard deviation

1 (Control) 0.00 0.000

133.733 0.000 2 (Positive) 3,720.00 492.341

3 (50% conc.) 2,940.00 388.844

4 (100% conc.) 4,465.00 253.180

Table 2. Comparison of the mean scores of the duration of the

anesthetic effect between treatments (One-way ANOVA)

*The mean difference is significant at the 0.05 level.

Table 3. Post-hoc (Tukey Test) multiple comparisons for onset

of peripheral nerve-blocking effect after the treatment

The duration parameter of 50% and 100%

concentration against the positive control and among the experimental groups revealed significant differences with a p value of 0.05 as seen in table 4.

Treatment Mean Difference Std. Error p-Value

1

(Neg. control)

2 3720.00* 239.191 0.000 3 745.00* 239.191 0.039 4 4465.00* 239.191 0.000

2

(Pos. control)

1 -1525.00* 239.191 0.000 3 -780.00* 239.191 0.030 4 2940.00* 239.191 0.000

3

(50% conc.)

1 -745.00* 239.191 0.039 2 780.00* 239.191 0.030 4 3720.00* 239.191 0.000

4

(100% conc.)

1 -4465.00* 239.191 0.000 2 -2940.00* 239.191 0.000 3 -3720.00* 239.191 0.000

* The mean difference is significant at the 0.05 level. Table 4.Post-hoc (Tukey Test) Multiple Comparisons for

duration of peripheral nerve-blocking effect after the treatment In the quality of local anesthetic effect

parameter, the plant-extract treated groups and the positive control group presented a substantially diminished contraction even if a stronger stimulus was induced from 1 mAmp to 3mAmp to 5mAmp as seen in figure 4. However, no significant differences were seen between the plant-extract treated groups and the positive control group as seen in table 5.

Figure 4. Estimated marginal means of contractionbetween

treatments and between varying strength of stimulus (a) Plain NSS, (b) 2% lidocaine HCl, (c) 50%, (d) 100% concentration of the Toothache Plant flower extract

11

* The mean difference is significant at the 0.05 level. Table 5.Post-hoc (Tukey-test) Multiple Comparisons on

contraction between treatments. The comparison of mean scores of the

contraction response to the fixed factors: the treatment and the stimulus with the use of the two-way ANOVA presented that there is a strong interaction between the treatment and the strength of stimulus in producing a contraction with a significant difference of 0.000 as seen table 6.

Source Sum of Squares df Mean Square F-ratio p-Value

Corrected Model 183.729a 11 16.703 49.085 .000

Intercept 111.021 1 111.021 326.265 .000

Treatment 135.229 3 45.076 132.469 .000

Amplitude 30.542 2 15.271 44.878 .000

Treatment * Amplitude 17.958 6 2.993 8.796 .000

Error 12.250 36 .340

Total 307.000 48

Corrected Total 195.979 47

a. R Squared = .937 (Adjusted R Squared =0.918)

Table 6 .Comparison of the mean scores of the contraction

responses (Two-way ANOVA with the fixed factors: treatment and stimulus.

Toxicological Assay Toxicologically, the LC50 increased when the

mean extract concentration and duration of exposure were decreased from 303.07µg/ml on the sixth-hour as seen in figure 5.a, and 11.98µg/ml on the twenty-fourth-hour as seen in figure 5.b with a confidence of 95% and 92% respectively.

Figure 5. LC50 dose response curve of Toothache Plant

floweron different concentrations: 10, 100, 1000 μg/ml on Artemi salina assay (a) after 6 hours (b) after 24 hours

Discussion

In the chemical analyses, the extract of

Toothache Plant flower constitutes an alkylamide. This is based on the result of 0.5 Rf value in the thin layer chromatography which is similar to the result of the study done by Costa S. et al (2013) wherein an alkylamide was isolated. The presence of an alkylamide was further concretized through the high performance liquid chromatography where the peaks were compared to nomenclature of alkylamides and the study done Bae, S (2007) of which spilanthol was isolated through series of high performance liquid chromatography processes. It

Treatment Mean Difference Std. Error Sig.

1 (Neg. control)

2 3.67* 0.238 0.000

3 3.83* 0.238 0.000 4 4.08* 0.238 0.000

2 (Pos. control)

1 3.67* 0.238 0.000

3 0.17 0.238 0.896 4 0.42 0.238 0.314

3 (50% conc.)

1 3.83* 0.238 0.000

2 0.17 0.238 0.896 4 0.25 0.238 0.722

4 (100% conc.)

1 4.08* 0.238 0.000

2 0.42 0.238 0.314 3 0.25 0.238 0.722

a

b

revealed that at peak number 7 with a retention time of 6 minutes and 40 seconds suggest the presence of an alkylamide – spilanthol which causes numbness and tingle.

Electrophysiologically, the plant extract

exhibited a local anesthetic activity on the muscle-nerve module of Rhinella marinus. Based on the mean onset and duration of peripheral nerve-blocking effect, significant differences were seen among the treatment groups receiving the plant extact (50% and 100% concentration) and 2% lidocaine hydrochloride after subjecting the data to the one-way ANOVA. This reflects that the groups were varied from each other in producing an arrest of muscular contraction and with a substantial degree of duration of peripheral nerve-blocking.

The post-hoc t-test, in conjunction to one-way

ANOVA, revealed no significant differences among the experimental groups (50% and 100% concentration) and positive control group. This means that the both concentration of the experimental groups have the same effect as to the positive control in producing an onset of peripheral nerve-blocking.

Significant differences were seen among the

treatment groups in the duration parameter. This reveals that the groups were varied from each other in producing an effect. The 100% concentration produced a longer duration compared to the 50% concentration and positive control. However, comparing the 50% concentration to the positive control, the positive control produced a longer duration of action.

In the parameter of the quality of anesthesia, both the experimental (50% and 100% concentration) and the positive control groups statistically presented no significant difference. A substantial diminished to no muscular contraction were seen even the stimulus was increased. This expresses that the quality of local anesthetic effect of the positive control is similar to the 50% and 100% concentration of the plant extract.

Established by the brine shrimp toxicology assay

using Probit Analysis of LC50 revealed that at the end of sixth-hour, 50% of the population will be protected from death when an amount of 303.07µg/ml will be given. In the twenty-fourth-hour, 50% of the population will be rendered protected when an amount of 11.98µg/ml will be administered. Toxicologically, the LC50 has an inversely proportional association with the duration of exposure and the concentration of the extract. The variation of lethal concentration values were due to the sensitivity of Artemia salina, the concentration of the plant extract, and the duration of exposure of Artemia salina to the Toothache Plant flower extract. These results implies that

amount of the extract is inversely proportional to the length of exposure - the lesser length of exposure, the greater the amount of extract that can be given and vice versa. Toxicity would arise to the remainder of the other 50% of the population when there will be any discrepancies to the proportions of the extract to the time of exposure.

As supported by well-established literature and

related experiments, the peripheral nerve-blocking effect of Toothache Plant flower can be attributed to the alkylamide9, 10, spilanthol11, 12, which is present in the flower. As cited in previous studies, spilanthol isolated from genus Spilanthes exhibited local anesthetic effect11 by binding and blocking the Na+ channels in a complex time-dependent manner such as that of how lidocaine hydrochloride works9. Major functional effect of local anesthetics suppresses cellular excitability, particularly at high rates of stimulation15.

The effects have generally been interpreted using

modulated receptor theory, which postulates that local anesthetics bind preferentially to the inactivated state of the channel. High-affinity local anesthetic binding induces additional non-conducting states which further reduce the probability of channel opening16. Furthermore, majority of lidocaine hydrochloride block appears to develop gradually during depolarization with a time course parallel to channel inactivation, a significant fraction of local anesthetic block also develops very rapidly, suggesting an additional interaction between the local anesthetic and the open channel17. Either mechanism could enhance use dependence if the drug-bound state liberates drug slowly enough to delay channel repriming.

The data gathered consistently affirms the

potential peripheral nerve-blocking property of Toothache Plant flower extract on Rhinella marinus.

Conclusions and Recommendations

The foregoing findings highly implied a

substantial potential peripheral nerve-blocking effect of Toothache Plant flower comparable to the marketed local anesthetic drug, the 2% lidocaine hydrochloride.

In view of the findings obtained, the researchers

would suggest to subject the extract to Nuclear Magnetic Resonance (NMR) and obtain its molecular structure; determine the minimum concentration of the extract that would render a local anesthetic activity; use mammalian species as test organism; use other route of administration of the extract that would create an anesthetic effect; and design other pharmacological studies of Toothache Plant flower aside from its peripheral nerve-blocking activity.

12

revealed that at peak number 7 with a retention time of 6 minutes and 40 seconds suggest the presence of an alkylamide – spilanthol which causes numbness and tingle.

Electrophysiologically, the plant extract

exhibited a local anesthetic activity on the muscle-nerve module of Rhinella marinus. Based on the mean onset and duration of peripheral nerve-blocking effect, significant differences were seen among the treatment groups receiving the plant extact (50% and 100% concentration) and 2% lidocaine hydrochloride after subjecting the data to the one-way ANOVA. This reflects that the groups were varied from each other in producing an arrest of muscular contraction and with a substantial degree of duration of peripheral nerve-blocking.

The post-hoc t-test, in conjunction to one-way

ANOVA, revealed no significant differences among the experimental groups (50% and 100% concentration) and positive control group. This means that the both concentration of the experimental groups have the same effect as to the positive control in producing an onset of peripheral nerve-blocking.

Significant differences were seen among the

treatment groups in the duration parameter. This reveals that the groups were varied from each other in producing an effect. The 100% concentration produced a longer duration compared to the 50% concentration and positive control. However, comparing the 50% concentration to the positive control, the positive control produced a longer duration of action.

In the parameter of the quality of anesthesia, both the experimental (50% and 100% concentration) and the positive control groups statistically presented no significant difference. A substantial diminished to no muscular contraction were seen even the stimulus was increased. This expresses that the quality of local anesthetic effect of the positive control is similar to the 50% and 100% concentration of the plant extract.

Established by the brine shrimp toxicology assay

using Probit Analysis of LC50 revealed that at the end of sixth-hour, 50% of the population will be protected from death when an amount of 303.07µg/ml will be given. In the twenty-fourth-hour, 50% of the population will be rendered protected when an amount of 11.98µg/ml will be administered. Toxicologically, the LC50 has an inversely proportional association with the duration of exposure and the concentration of the extract. The variation of lethal concentration values were due to the sensitivity of Artemia salina, the concentration of the plant extract, and the duration of exposure of Artemia salina to the Toothache Plant flower extract. These results implies that

amount of the extract is inversely proportional to the length of exposure - the lesser length of exposure, the greater the amount of extract that can be given and vice versa. Toxicity would arise to the remainder of the other 50% of the population when there will be any discrepancies to the proportions of the extract to the time of exposure.

As supported by well-established literature and

related experiments, the peripheral nerve-blocking effect of Toothache Plant flower can be attributed to the alkylamide9, 10, spilanthol11, 12, which is present in the flower. As cited in previous studies, spilanthol isolated from genus Spilanthes exhibited local anesthetic effect11 by binding and blocking the Na+ channels in a complex time-dependent manner such as that of how lidocaine hydrochloride works9. Major functional effect of local anesthetics suppresses cellular excitability, particularly at high rates of stimulation15.

The effects have generally been interpreted using

modulated receptor theory, which postulates that local anesthetics bind preferentially to the inactivated state of the channel. High-affinity local anesthetic binding induces additional non-conducting states which further reduce the probability of channel opening16. Furthermore, majority of lidocaine hydrochloride block appears to develop gradually during depolarization with a time course parallel to channel inactivation, a significant fraction of local anesthetic block also develops very rapidly, suggesting an additional interaction between the local anesthetic and the open channel17. Either mechanism could enhance use dependence if the drug-bound state liberates drug slowly enough to delay channel repriming.

The data gathered consistently affirms the

potential peripheral nerve-blocking property of Toothache Plant flower extract on Rhinella marinus.

Conclusions and Recommendations

The foregoing findings highly implied a

substantial potential peripheral nerve-blocking effect of Toothache Plant flower comparable to the marketed local anesthetic drug, the 2% lidocaine hydrochloride.

In view of the findings obtained, the researchers

would suggest to subject the extract to Nuclear Magnetic Resonance (NMR) and obtain its molecular structure; determine the minimum concentration of the extract that would render a local anesthetic activity; use mammalian species as test organism; use other route of administration of the extract that would create an anesthetic effect; and design other pharmacological studies of Toothache Plant flower aside from its peripheral nerve-blocking activity.

13

Literature Cited:

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2. Elumalai A, Pendem N, Eswaraiah MC, Naresh V: An updated annual review on antipyretic medicinal plants. Int J Univers Pharm Life Sci 2012, 2:207-15.

3. Vanamala U, Elumalai A, Eswaraiah MC, Shaik A: An updated review on diuretic plants. Int J Pharm Biol Arch 2012, 3:29-31.

4. Tiwari KL, Jadhav SK, Joshi V. Journal of Chinese

Integrative Medicine 2011, 9:1170-1178.

5. Harbone JE: Phytochemical methods. London, Chapman and Hall Ltd 1973, 49-88.

6. Consult M: Complementary and alternative medicine.

Clinical topic tours. St. Louis, Elsevier 2003.

7. Prachayasittikuli S, Prachayasittikuli P: High therapeutic potential of Spilanthes acmella: a review .EXCLI Journal 2013, 12:291-312.

8. Tiwari H, Kakkar A: Phytochemical examination of

Spilanthes acmella Murr.J Indian Chem Soc 1990: 67:784-785.

9. Chakraborty A, Devi Bharti RK, Rita S, Singh IT:

Local anesthetic effect of Spilanthes acmella in experimental animal models. Indian J Pharmacol 2002, 34:144-145.

10. Chavez P, Chavez E, Salinas C, Torres J: Acmella

radicans var. radicans: in vitro culture establishment and alkylamide content. In vitro Cell Dev Biol Plant 2003, 39:37-41.

11. Bae SS, Ehrmann BM, Ettefagh KA, Cech NB: A

validated liquid chromatography-electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem Anal. 2010, 21: 438-443.

12. Rani SA, Murty SU: Antifungal potential of flower

head extract of Spilanthes acmella Linn. Afr J Biomed Res. 2006, 9: 67-68.

13. Guevara B: A Guidebook to Plant Screening:

Phytochemical and Biological. Philippines, UST-RCNS 2004.

14. Trease GE, Evans WC: Pharmacognosy, 14th ed. New

Delhi, India, Elsevier, 2005, 53: 431-512.

15. Hilde B: Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J. Gen. Physiol 1977, 69: 497-515.

16. Carterall WA: Molecular Mechanism of Gating and

Drug Block Sodium Channels. Sodium Channels and Neuronal Hyperexcitability. Novartis Foundation Symposia 2001: 206.

17. Chernoff DM, Strichartz, GR: Tonic and phasic

block of neuronal sodium currents by 5-hydroxyhexano-2',6'-xylide, a neutral lidocaine homologue. Journal of General Physiology 1989, 93:1075-90.

Suppression of Carrageenan-Induced Acute Inflammation by Annona squamosa (Atis) Ethanol Seed Extract on Male Albino Mice

JF Perocho*, SP Mollaneda, MJ Montederamos, G Montesclaros and A Muslimen

and Arlene M. Diaz M.D.

Southwestern University – Matias H. Aznar Memorial College of Medicine Redemptorist Plaza, Camputhaw Cebu City 6000

Philippines

Abstract

Philippines is greatly blessed to have an abundant supply of plants as one of its natural resources. The vision and mission of Philippine Institute of Traditional and Alternative Health Care (PITAHC) inspired the researchers to utilize plant products and explore their potential. The focus of this study is to find the influence of Annona squamosa ethanol seed extract on the inflammatory process, whether it mitigates or aggravates. Laboratory Test Tube Method was utilized for the preliminary and confirmatory test for alkaloids. Soxhlet extraction was used to obtain the crude extract and the latter was then formulated into an ointment. Fifteen male albino mice, distributed as Experimental (Annona squamosa), Positive (Betamethasone Betnovate) and Negative (PNSS) respectively, were used. 0.03 mL of carrageenan solution was injected into the dorso-sacral portion of the male albino mice intradermally. Mean Percentage reduction was obtained after four, eight, twelve, sixteen, twenty and twenty-four hours and skin-nip biopsy twenty-four hours after induction. Statistically, One-way Analysis of Variance indicated that there is significant difference in the mean percentage reduction of swelling between and within treatments. Post-Hoc Bonferroni –Holm (All-Pairwise) Test further delineated the significant difference in the mean percentage reduction of swelling between Positive and Negative group as well as between Experimental and Negative Group. Meaning, the ability of Positive group to reduce swelling is different from that of Negative group and likewise with the Experimental from that of Negative group. However, there is no significant difference in the mean percentage reduction of swelling between Experimental and Positive Group implicating their similar reduction of swelling ability. Histologically, greatest anti-inflammatory activity was seen in the experimental group, followed by positive group and the negative group. Findings were conclusive on the presence of alkaloid in the seeds of Annona squamosa making it responsible for its anti-inflammatory activity on the carrageenan-induced dorso-sacral swelling on male albino mice. Keywords: Carrageenan, Annonas squamosa, Inflammation, Suppression Introduction

Plants are great source of therapeutic and

phytochemical properties that can be very beneficial once these ingredients are properly extracted, isolated and processed.The congress implemented Republic Act No. 8423way back 1997 which creates the Philippine Institute of Traditional and Alternative Health Care (PITAHC) aiming to encourage scientific research and to develop traditional and alternative health care systems that have direct impact on public health care.

Inflammation is a complex reaction to injurious agents such as microbes and usually damaged necrotic cells that consist of vascular responses, migration and activation of leukocytes and systemic reactions. The unique feature of inflammatory process is the reaction of blood vessels, leading to the accumulation of leukocytes in extravascular tissues. Inflammation is fundamentally a protective

response and the ultimate goal is to get rid of the microorganisms of both the initial cause of cell injury and the consequence of injury. 1 Inflammation can be acute or chronic. Acute is rapid in onset, is of short duration, lasting for minutes, several hours or few days. Its main characteristics are the exudation of fluid and plasma (edema) and the emigration of leukocytes predominantly neutrophils. Neutrophil activation is an early event in the up-regulation of the inflammatory response. Its peak appearance in acute inflammation is within the 24th -48th hour.Chronic is of longer duration and is associated histologically with the presence of lymphocytes and macrophages, the proliferation of blood vessels, fibrosis and tissue necrosis. 1

Anti-inflammatory is a name given to drugs that act to stop the swelling and to minimize the pain, redness and heat

Betamethasone Betnovate ointment is a potent glucocorticosteroid with anti-inflammatory and

*Corresponding author/research presenter Corresponding author email address: iammeemay1@gmail.com

14

Suppression of Carrageenan-Induced Acute Inflammation by Annona squamosa (Atis) Ethanol Seed Extract on Male Albino Mice

JF Perocho*, SP Mollaneda, MJ Montederamos, G Montesclaros and A Muslimen

and Arlene M. Diaz M.D.

Southwestern University – Matias H. Aznar Memorial College of Medicine Redemptorist Plaza, Camputhaw Cebu City 6000

Philippines

Abstract

Philippines is greatly blessed to have an abundant supply of plants as one of its natural resources. The vision and mission of Philippine Institute of Traditional and Alternative Health Care (PITAHC) inspired the researchers to utilize plant products and explore their potential. The focus of this study is to find the influence of Annona squamosa ethanol seed extract on the inflammatory process, whether it mitigates or aggravates. Laboratory Test Tube Method was utilized for the preliminary and confirmatory test for alkaloids. Soxhlet extraction was used to obtain the crude extract and the latter was then formulated into an ointment. Fifteen male albino mice, distributed as Experimental (Annona squamosa), Positive (Betamethasone Betnovate) and Negative (PNSS) respectively, were used. 0.03 mL of carrageenan solution was injected into the dorso-sacral portion of the male albino mice intradermally. Mean Percentage reduction was obtained after four, eight, twelve, sixteen, twenty and twenty-four hours and skin-nip biopsy twenty-four hours after induction. Statistically, One-way Analysis of Variance indicated that there is significant difference in the mean percentage reduction of swelling between and within treatments. Post-Hoc Bonferroni –Holm (All-Pairwise) Test further delineated the significant difference in the mean percentage reduction of swelling between Positive and Negative group as well as between Experimental and Negative Group. Meaning, the ability of Positive group to reduce swelling is different from that of Negative group and likewise with the Experimental from that of Negative group. However, there is no significant difference in the mean percentage reduction of swelling between Experimental and Positive Group implicating their similar reduction of swelling ability. Histologically, greatest anti-inflammatory activity was seen in the experimental group, followed by positive group and the negative group. Findings were conclusive on the presence of alkaloid in the seeds of Annona squamosa making it responsible for its anti-inflammatory activity on the carrageenan-induced dorso-sacral swelling on male albino mice. Keywords: Carrageenan, Annonas squamosa, Inflammation, Suppression Introduction

Plants are great source of therapeutic and

phytochemical properties that can be very beneficial once these ingredients are properly extracted, isolated and processed.The congress implemented Republic Act No. 8423way back 1997 which creates the Philippine Institute of Traditional and Alternative Health Care (PITAHC) aiming to encourage scientific research and to develop traditional and alternative health care systems that have direct impact on public health care.

Inflammation is a complex reaction to injurious agents such as microbes and usually damaged necrotic cells that consist of vascular responses, migration and activation of leukocytes and systemic reactions. The unique feature of inflammatory process is the reaction of blood vessels, leading to the accumulation of leukocytes in extravascular tissues. Inflammation is fundamentally a protective

response and the ultimate goal is to get rid of the microorganisms of both the initial cause of cell injury and the consequence of injury. 1 Inflammation can be acute or chronic. Acute is rapid in onset, is of short duration, lasting for minutes, several hours or few days. Its main characteristics are the exudation of fluid and plasma (edema) and the emigration of leukocytes predominantly neutrophils. Neutrophil activation is an early event in the up-regulation of the inflammatory response. Its peak appearance in acute inflammation is within the 24th -48th hour.Chronic is of longer duration and is associated histologically with the presence of lymphocytes and macrophages, the proliferation of blood vessels, fibrosis and tissue necrosis. 1

Anti-inflammatory is a name given to drugs that act to stop the swelling and to minimize the pain, redness and heat

Betamethasone Betnovate ointment is a potent glucocorticosteroid with anti-inflammatory and

*Corresponding author/research presenter Corresponding author email address: iammeemay1@gmail.com

Date Received: Dec. 10, 2014 Date Accepted: April 10, 2015

15

immunosuppressive properties. They inhibit the production of arachidonic acid by phospholipases in the membrane. This effect is mediated by intracellular steroid receptor that, when activated by an appropriate steroid, increase expression of specific proteins capable of inhibiting phospholipase. Steroids also inhibit the synthesis of COX-2. They are prescribed to treat various inflammatory skin disorders, such as eczema and dermatitis,that have not responded to milder steroids. 2 The ointment contains betamethasone valerate 0.1%. Ointment preparation is more suitable for dry, scaly areas of skin and is applied over the affected area twice a day and is gently rubbed in the skin. Each gram of the 0.1% ointment contains 1.2 mg betamethasone valerate (equivalent to 1.0 mg betamethasone) in an ointment base consisting of mineral oil, white petrolatum andhydrogenated lanolin.3

A downfall regarding its use is that it can cause skin irritation, thinning of the skin, changes in skin pigmentation and excessive hair growth. Prolonged use on extensive areas of skin result in enough corticosteroid being absorbed to have side effects on other parts of the body by causing a decrease in the production of natural hormones by the adrenal glands.

To avoid these undesirable effects, we are laying a research endeavour to evaluate alternatives for steroidal anti-inflammatory drugs. Having known its negative effects, there is always room for improvement, a room for new studies that can help alleviate problems with great advantages and fewer side effects.

Annona squamosa (Figure 1) is a member of Annonaceae family. It is a small, semi-deciduous tree, 3-7 m in height, with a broad, open crown or irregularly spreading branches. Bark is light brown with visible leaf scars and smooth to slightly fissured into plates. Leaves occur singly, 6-17 x 3-6 cm, lanceolate or oblong lanceolate, pale green on both surfaces and are hairy when young. Flower is greenish-yellow, fragrant, on slender hairy stalks, produced singly or in short lateral clusters about 2.5 cm long. Fruit is round, heart shaped, ovate or conical, 5-10 cm in diameter, with many round protuberances and is greenish-yellow when ripe with a white, powdery bloom. The seeds are oblong, deep brownish-black, compressed, with a pale swelling at the hilum or albumen filled with numerous transverse, brown lines of clefts. The genus name, ‘Annona’ is from the Latin word ‘anon’, meaning ‘yearly produce’, referring to the production of fruits of the various species in this genus. Annona squamosa is cultivated throughout the Philippines. 4

Roots, leaves, fruits, seeds and bark of the plant has multiple uses and can be easily grown in dry climates as well. The fruits are very high in calorific value and are a rich source of minerals and vitamins. They are used by athletes for their high energy content. Leaves are shown to have anti-diabetic properties. It is also known for its hepato-protective powers and scientists have experimentally proven the efficacy of the alcoholic extract of the leaves and stem in malignant tumors. Its efficacy as anti-depressant and anti-oxidant has also been experimentally proven by various scientists. They are also used as anti-depressants, in epilepsy and in spinal cord disorders. Fruits are sweet, haematinic, act as a sedative, stimulant and function as expectorant and tonic. Roots are powerful purgatives and are also used in dysentery.5

The seeds are powerful insecticides and powdered seeds are used for removing head lice. It is also used as an effective pesticide in agriculture and horticulture. Folk people commonly grind seeds and apply them as poultice to reduce swellings. 4

Seeds contain isolation of Alkaloids Cyclosquamosin A to I, Squamtin A, Annosquamosin A, Annonastatin, Squamocin O1 and O2, Squamosinin-A, Reticulacin-1, Annotemoyin-1 and 2, Samoquasine A, Squamostatin C and D, (2,4-cis and trans)-bullatacinone, Annonin I and VI and Squamostene-A. 4

Cyclopeptide alkaloids generally crystallize easily. The melting points are mostly over 200 °C. Most of them are levorotatory. Cyclopeptide alkaloids are rather weak bases and sparingly soluble in water but readily so in alcohols, CHCl3, and some other organic solvents.6

Several authors evaluated and validated the anti-arthritic, anti-inflammatory and analgesic activity of combined extract of Annona squamosa and Nigella sativa in various animal models.7 Combined extract decreased the paw volume in carageenan treated rats. Combined extract shows moderate central and peripheral analgesic activities in hot plate method and acetic acid induced writhing method in mice. Combined extract of Annona squamosa and Nigella sativa, possesses anti-arthritic, anti-inflammatory and analgesic property.7

Parallel studies were conducted of two natural cyclopeptide alkaloids Cyclosquamosin D and Met-cherimolacyclopeptide B and their analogs in Annona squamosa seeds.8 Compounds were screened for anti-inflammatory activity by evaluating their inhibitory effects on the production of pro-inflammatory cytokines using lipopolysaccharide stimulated macrophages J7741.1 cell line, Compounds have significant anti-inflammatory activity in suppressing the secretion of IL-6 and TNF-α.8

Two new cyclic peptides cyclosquamosin H and I, together with known six cyclic peptide squamin A and B, cyclosquamosin A, D and E and cherimolacyclopeptide B from the seeds of Annona squamosal were isolated in 2008. Cherimolacyclopeptide was isolated from this plant for the first time and Cyclosquamosin D showed an inhibitory Fig. 1 – Annona squamosafruit and seed

effect on the production of inflammatory cytokines within lipopolysaccharide and Pam3Cys-stimulated J774A1 macrophages.9

Stems of Annona squamosa also exhibited immunomodulating effects in leukocytes. A cellular model using isolated human neutrophils, which are important in the pathogenesis of rheumatoid arthritis, ischemia-reperfusion injury, chronic obstructive pulmonary disease, asthma and other inflammatory diseases, was established in order to elucidate the anti-inflammatory functions of 16beta,17-dihydroxy-ent-kauran-19-oic acid In summary, the presented results indicate that the inhibitory effects of Annona squamosa on respiratory burst and degranulation of human neutrophils are through the inhibition of cytosolic calcium mobilization, but not via the cAMP-dependent pathways.10

Soxhlet Extraction Method was used to obtain the Annona squamosa seed extract for its adaptogenic and antidepressant activity. Carrageenans are high molecular weight sulphate polygactans derived from several species of red seaweeds (Rhodophyceae). Carrageenans are large, highly flexible molecules that curl forming helical structures. This gives them the ability to form a variety of different gels at room temperature. They are widely used in the food and other industries as thickening and stabilizing agents. 12

Carrageenan-induced edema has been commonly used as experimental model for acute inflammation and is believed to be biphasic. Histamine, serotonin and bradykinin are the first detectable mediators in the early phaseof carrageenan-induced inflammation and diminishes in two hours. Prostaglandins (PG’s) are involved in the increase vascular permeability and are detectable beginning at the end of the first phase to the late phase of inflammation. It is mediated by bradykinin, leukotrienes, polymorphonuclear cells and prostaglandins produced by tissue macrophages. Local and or systemic inflammation is associated with enhanced levels of pro-inflammatory cytokines.12

Materials and Methods

This study employed quasi-experimental method to evaluate the anti-inflammatory effect of Annona squamosa ethanol seed extract on the carrageenan-induced inflammation among male albino mice. Independent Variable includes the Annona squamosa ethanol seed extract formulated into an ointment with reduction of carrageenan-induced swelling as the dependent variable.

The research procedure was screened, reviewed and approved by the Institutional Animal Care and Use Committee (IACUC)of Southwestern University headed by Dr. Jocelyn A. Tingson.

The Annona squamosa seeds were gathered by handpicking, collected from its fruits and were dried. One hundred grams of ground seeds were placed in an Erlenmeyer flask wherein 80% ethanol enough to soak the seeds was added. The plant sample was soaked for 24-48

hours. After maceration, it was subjected into soxhlet extraction process for eight hours. Flame Test Method was then utilized to further eliminate the ethanol.

The obtained extract was subjected into Laboratory Test Tube Method for the preliminary and confirmatory test for alkaloids. Twenty grams of plant extract was evaporated to a syrupy consistency over a hot plate. Five mL of 2M hydrochloric acid (HCl) was added, heated with stirring for 5 minutes and was cooled. ½ gm of sodium chloride (NaCl) was added, stirred and filtered. The filtrate was washed with enough 2M HCl in order to bring the filtrate to a volume of about 5 mL. 1 mL of the filtrate was taken and was tested with 2 to 3 drops of Dragendorff’s reagent. Positive for alkaloid yielded orange precipitate. Another 1 ml of the filtrate was tested with 2 to 3 drops of Mayer’s reagent. Positive for alkaloid gave white precipitate. 13

To confirm the presence of alkaloid, dropwise of enough 28% Ammonia was added to the remaining 3 mL of the filtrate until the solution is alkaline to the litmus paper. 10 mL of chloroform was added to the alkaline solution. The chloroform extract was evaporated into dryness under the hot plate. The residue was taken up with 5 mL of 2M HCl and was stirred over a hot plate for about 2 minutes and was allowed to cool. It was filtered and the filtrate was divided into two portions. One portion was tested with Mayer’s reagent and to other with Dragendorff’s reagent. The results were recorded as (+) for slight turbidity, (++) for definite turbidity and (+++) for heavy precipitation. 13

To make a 30 gram ointment, 1.3 grams of white wax, 23.3 grams of white petrolatum and 5.4 gram of A. squamosa ethanol seed extract were prepared. The white wax was melted in a suitable dish on a hot plate with the addition of white petrolatum. Incorporation method is used in adding the effective concentration of A. squamosa extract to the prepared ointment base. It was stirred and was left to congeal. 14

Fifteen (15) Male Albino mice (20-30 grams) were maintained under good hygienic condition under standard environmental condition (22-28°C, 60-70% relative humidity, 12-hour light-dark cycle) and fed with standard feeds and water ad libitum. They were voided from disturbances that would likely excite them.

Subjects were grouped into three, designated as A, B, and C. Group A will receive Annona squamosa ointment, Group B will receive Betamethasone Betnovate ointment and Group C with Normal Saline Solution. The mice were placed in their respective cages and were acclimatized for seven days for them to adjust to their new environment. They were given food and water for sustenance. After the experiment, they were euthanized via cervical dislodgement method.

The hair of the dorso-sacral portion of the mice was removed using Veet hair removal cream. A carrageenan saline solution was prepared by dissolving one (1) gram powder added with 100 ml of hot distilled water (0.1 ml of 1% freshly prepared suspension of carrageenan

16

effect on the production of inflammatory cytokines within lipopolysaccharide and Pam3Cys-stimulated J774A1 macrophages.9

Stems of Annona squamosa also exhibited immunomodulating effects in leukocytes. A cellular model using isolated human neutrophils, which are important in the pathogenesis of rheumatoid arthritis, ischemia-reperfusion injury, chronic obstructive pulmonary disease, asthma and other inflammatory diseases, was established in order to elucidate the anti-inflammatory functions of 16beta,17-dihydroxy-ent-kauran-19-oic acid In summary, the presented results indicate that the inhibitory effects of Annona squamosa on respiratory burst and degranulation of human neutrophils are through the inhibition of cytosolic calcium mobilization, but not via the cAMP-dependent pathways.10

Soxhlet Extraction Method was used to obtain the Annona squamosa seed extract for its adaptogenic and antidepressant activity. Carrageenans are high molecular weight sulphate polygactans derived from several species of red seaweeds (Rhodophyceae). Carrageenans are large, highly flexible molecules that curl forming helical structures. This gives them the ability to form a variety of different gels at room temperature. They are widely used in the food and other industries as thickening and stabilizing agents. 12

Carrageenan-induced edema has been commonly used as experimental model for acute inflammation and is believed to be biphasic. Histamine, serotonin and bradykinin are the first detectable mediators in the early phaseof carrageenan-induced inflammation and diminishes in two hours. Prostaglandins (PG’s) are involved in the increase vascular permeability and are detectable beginning at the end of the first phase to the late phase of inflammation. It is mediated by bradykinin, leukotrienes, polymorphonuclear cells and prostaglandins produced by tissue macrophages. Local and or systemic inflammation is associated with enhanced levels of pro-inflammatory cytokines.12

Materials and Methods

This study employed quasi-experimental method to evaluate the anti-inflammatory effect of Annona squamosa ethanol seed extract on the carrageenan-induced inflammation among male albino mice. Independent Variable includes the Annona squamosa ethanol seed extract formulated into an ointment with reduction of carrageenan-induced swelling as the dependent variable.

The research procedure was screened, reviewed and approved by the Institutional Animal Care and Use Committee (IACUC)of Southwestern University headed by Dr. Jocelyn A. Tingson.

The Annona squamosa seeds were gathered by handpicking, collected from its fruits and were dried. One hundred grams of ground seeds were placed in an Erlenmeyer flask wherein 80% ethanol enough to soak the seeds was added. The plant sample was soaked for 24-48

hours. After maceration, it was subjected into soxhlet extraction process for eight hours. Flame Test Method was then utilized to further eliminate the ethanol.

The obtained extract was subjected into Laboratory Test Tube Method for the preliminary and confirmatory test for alkaloids. Twenty grams of plant extract was evaporated to a syrupy consistency over a hot plate. Five mL of 2M hydrochloric acid (HCl) was added, heated with stirring for 5 minutes and was cooled. ½ gm of sodium chloride (NaCl) was added, stirred and filtered. The filtrate was washed with enough 2M HCl in order to bring the filtrate to a volume of about 5 mL. 1 mL of the filtrate was taken and was tested with 2 to 3 drops of Dragendorff’s reagent. Positive for alkaloid yielded orange precipitate. Another 1 ml of the filtrate was tested with 2 to 3 drops of Mayer’s reagent. Positive for alkaloid gave white precipitate. 13

To confirm the presence of alkaloid, dropwise of enough 28% Ammonia was added to the remaining 3 mL of the filtrate until the solution is alkaline to the litmus paper. 10 mL of chloroform was added to the alkaline solution. The chloroform extract was evaporated into dryness under the hot plate. The residue was taken up with 5 mL of 2M HCl and was stirred over a hot plate for about 2 minutes and was allowed to cool. It was filtered and the filtrate was divided into two portions. One portion was tested with Mayer’s reagent and to other with Dragendorff’s reagent. The results were recorded as (+) for slight turbidity, (++) for definite turbidity and (+++) for heavy precipitation. 13

To make a 30 gram ointment, 1.3 grams of white wax, 23.3 grams of white petrolatum and 5.4 gram of A. squamosa ethanol seed extract were prepared. The white wax was melted in a suitable dish on a hot plate with the addition of white petrolatum. Incorporation method is used in adding the effective concentration of A. squamosa extract to the prepared ointment base. It was stirred and was left to congeal. 14

Fifteen (15) Male Albino mice (20-30 grams) were maintained under good hygienic condition under standard environmental condition (22-28°C, 60-70% relative humidity, 12-hour light-dark cycle) and fed with standard feeds and water ad libitum. They were voided from disturbances that would likely excite them.

Subjects were grouped into three, designated as A, B, and C. Group A will receive Annona squamosa ointment, Group B will receive Betamethasone Betnovate ointment and Group C with Normal Saline Solution. The mice were placed in their respective cages and were acclimatized for seven days for them to adjust to their new environment. They were given food and water for sustenance. After the experiment, they were euthanized via cervical dislodgement method.

The hair of the dorso-sacral portion of the mice was removed using Veet hair removal cream. A carrageenan saline solution was prepared by dissolving one (1) gram powder added with 100 ml of hot distilled water (0.1 ml of 1% freshly prepared suspension of carrageenan

17

in hot distilled water). 0.03 mL of carrageenan solution was injected into the dorso-sacral portion of the mice using a 25-gauge needle intradermally. The target area was allowed to swell for five minutes and the diameter was measured using vernier caliper and was recorded as baseline. The decrease in the mean diameter after the treatment of the test solutions was recorded after four (4), eight (8), twelve (12), sixteen (16), twenty (20) and twenty-four (24) hours.

The A. squamosa ethanol seed extract was administered as ointment and Betamethasone Betnovate ointment and the Normal Saline Solution were applied topically enough to cover the inflamed site. Treatment were given twice; first was an hour after the induction of swelling and the second was twelve hours thereafter.

Skin specimen was taken using skin nip biopsy on the 24th hour post-treatment. Specimens were taken from the dorso-sacral inflamed area of the test animals about 6 mm in length, 2 mm in width and 3 mm in thickness using sterile forceps and scalpel. The specimens were individually placed in a vial each containing enough to soak 10% formalin.

The properly labeled specimen was sent to the laboratory of Sacred Heart Hospital for processing and slide preparations. The prepared slides were examined microscopically by Dr. Abelardo J. Alera, certified pathologist, utilizing a Modified Likert’s Scalein the grading of the severity of inflammation and vascular congestion or edema.

The reading of slides was done in MHAM Southwestern University, 3rd floor, General Pathology Department Laboratory with the use of a binocular microscope. The parameters used in the study were numbers of polymorphonuclear cells and the presence of vascular congestion or edema. The distribution of inflammatory infiltrates was also noted.

All data was recorded and was further processed for analysis. Data was evaluated with the use of descriptive statistics in terms of mean percentage reduction of swelling among the experimental, positive control and negative control. One-way ANOVA was used as statistical test for measuring any significant difference in the mean percentage reduction of swelling after treatment at different time intervals between and among groups (positive, negative and experimental). Posthoc test: Bonferroni-Holmwas used in conjunction with ANOVA to find out which means were significantly different from the other. For histologic assessment, the comparison on degree of inflammation was measured using Modified Likert’s scale.

Results and Discussions

Preliminary and Confirmatory Test for Alkaloids. The presence of alkaloid responsible for the anti-inflammatory effect of Annona squamosa seed was investigated using Preliminary and Confirmatory test utilizing Laboratory Test Tube Method (Table 1). The extract yielded orange color with Dragendorff’s reagent and white color for Mayer’s reagent, both indicating the presence of alkaloid. Confirmatory test showed mark precipitation indicating strong alkaloidal presence. Table 1 – Laboratory Test Tube Method

Reagent Preliminary Test Confirmatory Test Interpretation

Theoretical Result

Actual Result

Theoretical Result

Actual Result

Dragendorff Orange Orange Precipitate Precipitate +

Mayer White White Precipitate Precipitate +

Results of Anti-inflammatory Activity

The mean baseline diameter measurement of the swelling (Figure 2) induced by Carrageenan in the dorso-sacral portion of male albino mice per treatment group was obtained 5 minutes after induction. The mean baseline diameter of induction of the experimental group is 7.7mm. The positive and negative group had baseline mean diameter of 7.5 mm and 8.2 mm, respectively.

Fig. 2 - Mean Baseline Measurement of swelling induced by Carrageenan on treatment groups

7

7.5

8

8.5

Experimental Positive Negative

Diameter (mm)

Treatment

ExperimentalPositive

20 39 45

61

87 96

25

48 62 67

93 97

10 20 27

36

58 75

4 8 12 16 20 24

Perc

enta

ge

Treatment

Experimental

Positive

Negative

Fig. 3 - Mean percentage reduction of swelling in treatment groups at different time intervals

Mean percentage reduction of swelling (Figure 3) after 4, 8, 12, 16, 20 and 24 hours of treatment administration was obtained. Experimental group has a mean baseline diameter of 7.7 mm. After 4 hours, swelling reduces by 20%, then 39%, 45%, 61%, 87% and 96% after 8, 12, 16, 20 and 24 hours post treatment with Annona squamosa ointment. For positive control, mean baseline diameter is 7.5 mm. Reduction of swelling is noted to be 25 %, 48%, 62%, 67%, 93% and 97% after 4, 8, 12, 16, 20 and 24 hour post treatment with Betamethasone Betnovate ointment. Mean baseline diameter of the Negative control group is 8.2 mm. Percentage reduction showed 10%, 20%, 27%, 36% 58% and 75% after 4,8,12,16, 20 and 24 hours post treatment with Normal Saline Solution. It is observed that experimental and positive group almost have 100 % reduction after the 24th hour of treatment.

One-way Analysis of Variance was performed to test if there is significant difference in the mean percentage reduction of swelling measurements after treatment at different time intervals between and within groups. Statistical significance was considered at p<0.05. Results revealed a p-value of 0.001 in the treatment variable, which means, there is a significant difference in the effect of different treatments.

Post-Hoc Bonferroni –Holm (All-Pairwise) Test was used in conjunction with One-way ANOVA to find out which means in the percentage reduction of swelling were significantly different from the other. Results revealed that there is significant difference in the mean percentage reduction of swelling between Positive and Negative group as well as between Experimental and Negative Group. There is no significant difference in the mean percentage reduction of swelling between Experimental and Positive Group.

Histopathology

The degree of inflammation at cellular level was graded by the use of Modified Likert’s Scale (Table 2). Mean frequency analysis (Table 3) is rounded to the nearest whole number. Based on the result, the Experimental group shows Grade 2 (Fig. 4) which is classified as MILD TO MODERATE INFLAMMATION with Polymorphonuclear cells of 26-49%, few to most vascular congestion and mild to moderate edema. Positive group shows Grade 3 (Fig. 5) classified as MODERATE INFLAMMATION with polymorphonuclear cells of 50%, most of the vascular congestion, moderate edema. For negative control, results shows Grade 5 (Fig.6) classified as SEVERE INFLAMMATION with Polymorphonuclear cells of >75%, all vascular congestion in area of inflammation including in the periphery and severe edema.

The above data supports the anti-inflammatory

effect of Annona squamosa ethanol seed extract on the carrageenan-induced inflammation at dorso-sacral area of male albino mice.

Table 2 – Modified Likert’s Scale for Histopathologic Severity of Inflammation

GRADE INTERPRETATION

0 NO INFLAMMATION

1 MILD INFLAMMATION

(PMN’S <25%, few vascular channels in areas of

inflammation, mild edema)

2 MILD TO MODERATE INFLAMMATION

(PMN’s 26-49%, few to most vascular congestion and

mild to moderate edema)

3 MODERATE INFLAMMATION

(PMN’s 50%, most of the vascular congestion, moderate

edema)

4 MODERATE TO SEVERE INFLAMMATION

(PMN’s 51-74%, most to all of vascular congestion,

moderate to severe edema)

5 SEVERE INFLAMMATION

(PMN’s >75%, all vascular congestion in area of

inflammation including in the periphery and severe

edema)

Fig. 4 Histologic Morphology of Experimental Control (Grade 2)

Fig. 5 Histologic Morphology of Positive Control (Grade 3)

18

Mean percentage reduction of swelling (Figure 3) after 4, 8, 12, 16, 20 and 24 hours of treatment administration was obtained. Experimental group has a mean baseline diameter of 7.7 mm. After 4 hours, swelling reduces by 20%, then 39%, 45%, 61%, 87% and 96% after 8, 12, 16, 20 and 24 hours post treatment with Annona squamosa ointment. For positive control, mean baseline diameter is 7.5 mm. Reduction of swelling is noted to be 25 %, 48%, 62%, 67%, 93% and 97% after 4, 8, 12, 16, 20 and 24 hour post treatment with Betamethasone Betnovate ointment. Mean baseline diameter of the Negative control group is 8.2 mm. Percentage reduction showed 10%, 20%, 27%, 36% 58% and 75% after 4,8,12,16, 20 and 24 hours post treatment with Normal Saline Solution. It is observed that experimental and positive group almost have 100 % reduction after the 24th hour of treatment.

One-way Analysis of Variance was performed to test if there is significant difference in the mean percentage reduction of swelling measurements after treatment at different time intervals between and within groups. Statistical significance was considered at p<0.05. Results revealed a p-value of 0.001 in the treatment variable, which means, there is a significant difference in the effect of different treatments.

Post-Hoc Bonferroni –Holm (All-Pairwise) Test was used in conjunction with One-way ANOVA to find out which means in the percentage reduction of swelling were significantly different from the other. Results revealed that there is significant difference in the mean percentage reduction of swelling between Positive and Negative group as well as between Experimental and Negative Group. There is no significant difference in the mean percentage reduction of swelling between Experimental and Positive Group.

Histopathology

The degree of inflammation at cellular level was graded by the use of Modified Likert’s Scale (Table 2). Mean frequency analysis (Table 3) is rounded to the nearest whole number. Based on the result, the Experimental group shows Grade 2 (Fig. 4) which is classified as MILD TO MODERATE INFLAMMATION with Polymorphonuclear cells of 26-49%, few to most vascular congestion and mild to moderate edema. Positive group shows Grade 3 (Fig. 5) classified as MODERATE INFLAMMATION with polymorphonuclear cells of 50%, most of the vascular congestion, moderate edema. For negative control, results shows Grade 5 (Fig.6) classified as SEVERE INFLAMMATION with Polymorphonuclear cells of >75%, all vascular congestion in area of inflammation including in the periphery and severe edema.

The above data supports the anti-inflammatory

effect of Annona squamosa ethanol seed extract on the carrageenan-induced inflammation at dorso-sacral area of male albino mice.

Table 2 – Modified Likert’s Scale for Histopathologic Severity of Inflammation

GRADE INTERPRETATION

0 NO INFLAMMATION

1 MILD INFLAMMATION

(PMN’S <25%, few vascular channels in areas of

inflammation, mild edema)

2 MILD TO MODERATE INFLAMMATION

(PMN’s 26-49%, few to most vascular congestion and

mild to moderate edema)

3 MODERATE INFLAMMATION

(PMN’s 50%, most of the vascular congestion, moderate

edema)

4 MODERATE TO SEVERE INFLAMMATION

(PMN’s 51-74%, most to all of vascular congestion,

moderate to severe edema)

5 SEVERE INFLAMMATION

(PMN’s >75%, all vascular congestion in area of

inflammation including in the periphery and severe

edema)

Fig. 4 Histologic Morphology of Experimental Control (Grade 2)

Fig. 5 Histologic Morphology of Positive Control (Grade 3)

19

Fig. 6 Histologic Morphology of Negative Control (Grade 5) Table 3 – Mean Frequency Analysis of Degree of Inflammation of

Treatment Groups

Laboratory Test Tube Method showed positive, thus, agreeing Dellai, A. et. al, 20108 and Yang Y.,et al, 20089 regarding the presence of alkaloids in Annona squamosa seeds. Mechanism is thought to be due to their inhibitory effects on the production of pro-inflammatory cytokines and in suppressing the secretion of IL-6 and TNF-

α as well as in the immunomodulation of the effects of leukocytes.10

The anti-inflammatory activity of Annona squamosa ethanol seed extract made into ointment was investigated using carrageenan-induced dorso-sacral model. Edema is based on the principle of release of various inflammatory mediators by the carrageenan.Same amount of carrageenan solution is induced (0.03mL) but different mean baseline measurement is produced; 7.7mm, 7.5mm and 8.2 mm for Experimental, Positive and Negative Control respectively. Carrageenan produced uneven swelling that diffuses from one area to another making this as a drawback with regards to its use as an inflammation inducer. Mean percentage reduction of swelling showed almost 100% complete reduction in both experimental and negative group.

Statistically, One-way Analysis of Variance indicated that there is a significant difference in the mean percentage reduction of swelling between and within treatments. Post-Hoc Bonferroni –Holm (All-Pairwise) Test further delineate the significant difference in the mean percentage reduction of swelling between Positive and Negative group as well as between Experimental and Negative Group. Meaning, the ability of Positive group to reduce swelling is different from that of Negative group and likewise with the Experimental from that of Negative group. However, there is no significant difference in the mean percentage reduction of swelling between Experimental and Positive Group implicating their similar reduction of swelling ability.

The histologic grading using the Modified Likert scale was done to confirm the suppression of inflammation at cellular level. Signs of acute inflammation like polymorphonuclear cell infiltration, vascular congestion reflecting edema were considered as parameters in the evaluation of inflammation. Experimental group got the greatest anti-inflammatory activity (Grade 2) followed by the Positive (Grade 3) and then the Negative ( Grade 5). Negative group had severe degree of inflammation despite treatment. This result supported the idea that the reduction of swelling in the negative control group is not due to the anti-inflammatory effect of the treatment (Normal Saline) but due to the diminishing effect of carrageenan as described by Bartosikova.12

Conclusions and Recommendations Findings were conclusive on the presence of alkaloid in the seeds of Annona squamosa making it responsible for its anti-inflammatory activity on the carrageenan-induced dorso-sacral swelling on male albino mice.

Based upon the findings, the researchers would like to recommend future researchers to use complete randomnization of research subjects, to determine the acute dermal toxicity, ascertain the peak effect and perform sensitivity test of Annona squamosa ointment, to formulate other dosage form like creams, gels and lotions, to speculate

Treatment Grade (x)

Frequency (f)

Experimental 0 0

1 2

2 3

3 0

4 0

5 0

Σ

5 Mean Frequency

1.6

Grade

2

Positive 0 0

1 0

2 2

3 3

4 0

5 0

Σ

5 Mean Frequency

2.6

Grade

3

Negative 0 0

1 0

2 0

3 0

4 2

5 3

Σ

5 Mean Frequency 4.6 Grade 5

other routes of administration on the anti-inflammatory effect of Annona squamosa and to find other inducers of inflammation. Literature Cited 1. Kumar,V, Mitchell R, Abbas, A and Fausto N.,Robbins

and Cotran Pathologic Basis of Disease8thedition.Philadelphia.PA.Saunders, Elsevier Inc. 2010

2. Katzung, Bertram, Trevor, Anthony, Masters, Susan. Katzung and Trevor’s Pharmacology 8th edition. Mc.Graw-Hill Companies, Inc. 2008

3. Fun, Leong Wai.et.al.MIMS Philippines PIMS 126thedition.UBMMedica. 2010

4. Rummel, Dietmar. Useful Plants of the Philippines; A Scientific Guide of Modern Botanical Medicine. Phytopharmacology. 2005

5. Quisumbing, Eduardo. Medicinal Plants of the Philippines. Quezon City: Katha Publishing. 1995

6. J.S. Davies: Amino Acids, Proteins and Peptides, Vol.32 Royal Society of Chemistry, Athenacum Press Ltd., Gateshead Tyne and Wear. 2001

7. Manvi FV, Nanjawade BK, ShingSanjiv: Pharmacological Screening of Combined Extract of Annona squamosal and Nigella sativa. Volume 2, issue 2, 520-529. 2011

8. Dellai A, Maricic I, Kumar V, Arutyunyan S, Bouraoui A, Nefzi A: Parallel synthesis and anti-inflammatory activity of cyclic peptides cyclosquamosin D and Met-cherimolacyclopeptide B and their analogs. 20(19):5653-5657. 2010

9. Yang YL, Hua KF, Chuang PH, Wu SH, Wu KY, Chang FR, Wu YC.: New cyclic peptides from the seeds of Annonasquamosa L. and their anti-inflammatory activities.56(2):386-392. 2008

10. Y Shang-Hsin, C. Fang-Rong, WU YangChang, Y. Yu-Liang, Z. Shi-Kai, H. Tsong-Long: An anti-inflammatory entkaurane from the stems of Annonasquamosa that inhibits various human neutrophil functions. Plantamedica. 71: 10, 904-909. 2005

11. Nagaraj Kolkur: Annona squamosa Seed Extractfor its Adaptogenic and Antidepressant Activity.71:1-19. 2010

12. Bartosikova, Necas J. Carrageenan: A review. VeterinarniMedicina, 58, 2013 (4): 187–205 Review Article. 2013

13. Aguinaldo, Alicia M., Espeso, Erlinda I., Guevarra Beatrice Q., Nonata, Maribel G. Phytochemical, Microbiological and Pharmacological Screening of Medicinal Plants. UST Manila Philippines, Publication of Research. 2004

20

other routes of administration on the anti-inflammatory effect of Annona squamosa and to find other inducers of inflammation. Literature Cited 1. Kumar,V, Mitchell R, Abbas, A and Fausto N.,Robbins

and Cotran Pathologic Basis of Disease8thedition.Philadelphia.PA.Saunders, Elsevier Inc. 2010

2. Katzung, Bertram, Trevor, Anthony, Masters, Susan. Katzung and Trevor’s Pharmacology 8th edition. Mc.Graw-Hill Companies, Inc. 2008

3. Fun, Leong Wai.et.al.MIMS Philippines PIMS 126thedition.UBMMedica. 2010

4. Rummel, Dietmar. Useful Plants of the Philippines; A Scientific Guide of Modern Botanical Medicine. Phytopharmacology. 2005

5. Quisumbing, Eduardo. Medicinal Plants of the Philippines. Quezon City: Katha Publishing. 1995

6. J.S. Davies: Amino Acids, Proteins and Peptides, Vol.32 Royal Society of Chemistry, Athenacum Press Ltd., Gateshead Tyne and Wear. 2001

7. Manvi FV, Nanjawade BK, ShingSanjiv: Pharmacological Screening of Combined Extract of Annona squamosal and Nigella sativa. Volume 2, issue 2, 520-529. 2011

8. Dellai A, Maricic I, Kumar V, Arutyunyan S, Bouraoui A, Nefzi A: Parallel synthesis and anti-inflammatory activity of cyclic peptides cyclosquamosin D and Met-cherimolacyclopeptide B and their analogs. 20(19):5653-5657. 2010

9. Yang YL, Hua KF, Chuang PH, Wu SH, Wu KY, Chang FR, Wu YC.: New cyclic peptides from the seeds of Annonasquamosa L. and their anti-inflammatory activities.56(2):386-392. 2008

10. Y Shang-Hsin, C. Fang-Rong, WU YangChang, Y. Yu-Liang, Z. Shi-Kai, H. Tsong-Long: An anti-inflammatory entkaurane from the stems of Annonasquamosa that inhibits various human neutrophil functions. Plantamedica. 71: 10, 904-909. 2005

11. Nagaraj Kolkur: Annona squamosa Seed Extractfor its Adaptogenic and Antidepressant Activity.71:1-19. 2010

12. Bartosikova, Necas J. Carrageenan: A review. VeterinarniMedicina, 58, 2013 (4): 187–205 Review Article. 2013

13. Aguinaldo, Alicia M., Espeso, Erlinda I., Guevarra Beatrice Q., Nonata, Maribel G. Phytochemical, Microbiological and Pharmacological Screening of Medicinal Plants. UST Manila Philippines, Publication of Research. 2004

21

The Nephrotoxic Effect Of Exogenous Trimeric Glutathione On

Male Albino Rats

Philipp Vincent B. Lim,IoCamille D. Garcia, Janine H. Fuderanan, Joanna Rose B. Ipong, Ruffaidah M. Guro, Sudarat Klongrua Advisers: Arlene M. Diaz M.D. and Jose M. Oclarit Ph.D.

Southwestern University – Matias H. Aznar Memorial College of Medicine, Inc.

Redemptorist Plaza, Camputhaw Cebu City 6000 Philippines

Abstract

Glutathione refers to a tripeptide L-gamma-glutamyl-L-cysteinyl-glycine. It is a powerful antioxidant that detoxifies harmful compounds. It helps maintain the integrity of red blood cells by preventing oxidative stress. It also helps trap free radicals that can damage DNA and RNA. Glutathione is mainly used for its antioxidant properties. It is only in Asia that it is used for its skin lightening promise. However the introduction of large doses of glutathione to the body was implicated to produce adverse effects, one of these is kidney failure. A commercially prepared glutathione was administered intravenously to male albino rats. Two experimental groups received ten doses of glutathione, one group received a higher dosage than the other. A control group was untreated as a negative control. The rats were sacrificed and their kidneys were subjected to histopathologic examination. Both groups receiving glutathione demonstrated histologic changes that resembled the occurrence of injury. It showed tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, and hemorrhage.

A significant difference was seen in the two experimental groups when compared to the control group, which demonstrated with mild histologic changes. Between the two experimental groups, the group with a higher dosage demonstrated an insignificantly higher degree of damage.

Keywords: Glutathione, tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, hemorrhage, L-gamma-glutamyl-L-cysteinyl-glycine

The Nephrotoxic Effect Of Exogenous Trimeric Glutathione On

Male Albino Rats

Philipp Vincent B. Lim,IoCamille D. Garcia, Janine H. Fuderanan, Joanna Rose B. Ipong, Ruffaidah M. Guro, Sudarat Klongrua Advisers: Arlene M. Diaz M.D. and Jose M. Oclarit Ph.D.

Southwestern University – Matias H. Aznar Memorial College of Medicine, Inc.

Redemptorist Plaza, Camputhaw Cebu City 6000 Philippines

Abstract

Glutathione refers to a tripeptide L-gamma-glutamyl-L-cysteinyl-glycine. It is a powerful antioxidant that detoxifies harmful compounds. It helps maintain the integrity of red blood cells by preventing oxidative stress. It also helps trap free radicals that can damage DNA and RNA. Glutathione is mainly used for its antioxidant properties. It is only in Asia that it is used for its skin lightening promise. However the introduction of large doses of glutathione to the body was implicated to produce adverse effects, one of these is kidney failure. A commercially prepared glutathione was administered intravenously to male albino rats. Two experimental groups received ten doses of glutathione, one group received a higher dosage than the other. A control group was untreated as a negative control. The rats were sacrificed and their kidneys were subjected to histopathologic examination. Both groups receiving glutathione demonstrated histologic changes that resembled the occurrence of injury. It showed tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, and hemorrhage.

A significant difference was seen in the two experimental groups when compared to the control group, which demonstrated with mild histologic changes. Between the two experimental groups, the group with a higher dosage demonstrated an insignificantly higher degree of damage.

Keywords: Glutathione, tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, hemorrhage, L-gamma-glutamyl-L-cysteinyl-glycine

22

The Nephrotoxic Effect Of Exogenous Trimeric Glutathione On

Male Albino Rats

Philipp Vincent B. Lim,IoCamille D. Garcia, Janine H. Fuderanan, Joanna Rose B. Ipong, Ruffaidah M. Guro, Sudarat Klongrua Advisers: Arlene M. Diaz M.D. and Jose M. Oclarit Ph.D.

Southwestern University – Matias H. Aznar Memorial College of Medicine, Inc.

Redemptorist Plaza, Camputhaw Cebu City 6000 Philippines

Abstract

Glutathione refers to a tripeptide L-gamma-glutamyl-L-cysteinyl-glycine. It is a powerful antioxidant that detoxifies harmful compounds. It helps maintain the integrity of red blood cells by preventing oxidative stress. It also helps trap free radicals that can damage DNA and RNA. Glutathione is mainly used for its antioxidant properties. It is only in Asia that it is used for its skin lightening promise. However the introduction of large doses of glutathione to the body was implicated to produce adverse effects, one of these is kidney failure. A commercially prepared glutathione was administered intravenously to male albino rats. Two experimental groups received ten doses of glutathione, one group received a higher dosage than the other. A control group was untreated as a negative control. The rats were sacrificed and their kidneys were subjected to histopathologic examination. Both groups receiving glutathione demonstrated histologic changes that resembled the occurrence of injury. It showed tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, and hemorrhage.

A significant difference was seen in the two experimental groups when compared to the control group, which demonstrated with mild histologic changes. Between the two experimental groups, the group with a higher dosage demonstrated an insignificantly higher degree of damage.

Keywords: Glutathione, tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, hemorrhage, L-gamma-glutamyl-L-cysteinyl-glycine

Date Received: Dec. 10, 2014 Date Accepted: April 6, 2015

*Corresponding author Philipp Vincent B. Lim,Matias H. Aznar Memorial College of Medicine, Inc.

Introduction

Society has often associated the concept of beauty with fair and white skin. To supply this bustling demand, the market exploded with a variety of whitening products, from herbal whitening soaps and cream, to the most popular now, glutathione. Glutathione in the past was mainly used for its anti-oxidant effects that fight against free radicals that destroy our cells. It prevents chronic inflammation by boosting antioxidants like vitamins C and E in our body. At present, glutathione has become a commercial success. The Philippines, and some countries in Asia, take glutathione for whitening purposes, though its property as a skin lightener and its safety are not yet established. When the intravenous glutathione became a trend, many immediately shifted from its oral form to the intravenous form. Intravenous injections, however, due to sudden increase of glutathione in the body was implicated to produce side effects like stomach ache, diarrhea, kidney failure, blood poisoning and Stevens-Johnson syndrome. There have been issues that tackled the upsurge of intravenous glutathione administration and its serious consequences. The rapid increase in the utilization of the intravenous form of the supplement has spurred

many questions about its efficacy and adverse effect. With this study, information can be generated to help elucidate the possible adverse effects of glutathione to the body, specifically the kidneys.

Glutathione is generally very good for health. Our body actually produces it, and we need it to fight off toxins and free radicals. Exogenous sources of these trimeric compounds have been proven to promote better health. In our country where a significant number of the population are currently using exogenous sources of glutathione, the need to employ research and development strategies for further discovery of chemotherapeutic effects of these substances is widely called for. However, similar attention and emphasis should also be directed on the adverse effects, if any, of the new emerging products such as glutathione. The adverse effects on health may outweigh the beneficial effects, thus this cannot be overlooked.

Therefore, the aim of this study is to evaluate the effects of exogenous trimeric glutathione on the kidneys of male Albino rats by determining the corresponding gross and microscopic pathologic changes that it may cause after administering glutathione intravenously.

The term glutathione is typically used as a collective term to refer to the tripeptide L-gamma-glutamyl-L-cysteinyl-glycine in both its reduced and dimeric forms. Glutathione is widely found in all forms of life and plays an essential role in the health of organisms, particularly

The Nephrotoxic Effect Of Exogenous Trimeric Glutathione On

Male Albino Rats

Philipp Vincent B. Lim,IoCamille D. Garcia, Janine H. Fuderanan, Joanna Rose B. Ipong, Ruffaidah M. Guro, Sudarat Klongrua Advisers: Arlene M. Diaz M.D. and Jose M. Oclarit Ph.D.

Southwestern University – Matias H. Aznar Memorial College of Medicine, Inc.

Redemptorist Plaza, Camputhaw Cebu City 6000 Philippines

Abstract

Glutathione refers to a tripeptide L-gamma-glutamyl-L-cysteinyl-glycine. It is a powerful antioxidant that detoxifies harmful compounds. It helps maintain the integrity of red blood cells by preventing oxidative stress. It also helps trap free radicals that can damage DNA and RNA. Glutathione is mainly used for its antioxidant properties. It is only in Asia that it is used for its skin lightening promise. However the introduction of large doses of glutathione to the body was implicated to produce adverse effects, one of these is kidney failure. A commercially prepared glutathione was administered intravenously to male albino rats. Two experimental groups received ten doses of glutathione, one group received a higher dosage than the other. A control group was untreated as a negative control. The rats were sacrificed and their kidneys were subjected to histopathologic examination. Both groups receiving glutathione demonstrated histologic changes that resembled the occurrence of injury. It showed tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, and hemorrhage.

A significant difference was seen in the two experimental groups when compared to the control group, which demonstrated with mild histologic changes. Between the two experimental groups, the group with a higher dosage demonstrated an insignificantly higher degree of damage.

Keywords: Glutathione, tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, hemorrhage, L-gamma-glutamyl-L-cysteinyl-glycine

23

aerobic organisms. It is found in all eukaryotic cells, including all mammalian tissue. Reduced L-glutathione (GSH) is available to the mammalian organism through cellular synthesis from constituent amino acids, and also through dietary intake (Jayyab, 1992).

Glutathione (reduced) is known chemically as N-L-gamma-glutamyl-L-cysteinyl-glycine and is abbreviated as GSH. Its molecular formula is C10H17N3O6S and its molecular weight is 307.33 daltons. Glutathione disulfide is also known as L-gamma-glutamyl-L-cysteinyl-glycine disulfide and is abbreviated as GSSG. Its molecular formula is C20H32N6O12S2. Nature has built the cysteine molecule into the glutathione tripeptide to make the amino acid more soluble and allow it to have redox buffering activity in a living tissue environment. Glutathione also plays roles in catalysis, metabolism, signal transduction, gene expression and apoptosis. It is a cofactor for glutathione S-transferases, enzymes which are involved in the detoxification of xenobiotics, including carcinogenic genotoxicants, and for the glutathione peroxidases, crucial selenium-containing antioxidant enzymes. It is also involved in the regeneration of ascorbate from its oxidized form, dehydroascorbate. There are undoubtedly roles of glutathione that are still to be discovered (Jayyab, 1992).

Beginning in our 20’s the levels of glutathione in our body begins to decline, and by the ages of 70 - 80, these levels can drop to as low as 30% - 40% of what we had available to us in early adulthood. The cause of this decline may be related to a decrease in the body’s ability to create glutathione as efficiently as when we were young, or it could be that as we age, our body’s requirement for glutathione may elevate significantly due to the natural decline of our overall health, and the oxidative stresses that may accompany this decline. It is interesting to point out that certain studies have discovered that individuals who have lived 100+ years have levels of glutathione in their bodies that are higher than any other age category (Keller, 2013).

Doses of 600 milligrams of aerosolized reduced glutathione twice daily have been used in idiopathic pulmonary fibrosis, chronic inflammatory upper respiratory tract disease,and HIV infection (Borok et al., 1991). Intramuscular glutathione is given for chemoprotection against cisplatin toxicity in patients with advanced gastric cancer, glutathione 1500 milligrams/square meter was given intravenously prior to cisplatin, followed by 600 mg/m2 intramuscularly on days 2 to 5. Patients in this study received a weekly regimen of cisplatin (40 mg/m2), epirubicin, 5-fluorouracil, 6S-leucovorin and granulocyte colony-stimulating factor (Cascinu et al., 1995).Intravenous doses of 1.5 grams/square meter have been given as chemoprotection against cisplatin toxicity in weekly cisplatin-based regimens for the treatment of gastric carcinoma. In ovarian carcinoma, doses of 2500 to 5000 mg or 1500 mg/m2 have been given immediately prior to cisplatin doses in cisplatin-alone or cisplatin-based regimens (Bohm et al., 1999).

The kidneys are highly dependent on an adequate supply of glutathione to maintain normal function. This is due, in part, to high rates of aerobic metabolism, particularly in the proximal tubules. Additionally, the kidneys are potentially exposed to high concentrations of oxidants and reactive electrophiles. Renal cellular concentrations of GSH are maintained by both intracellular synthesis and transport from outside the cell. Although function of specific carriers has not been definitively demonstrated, it is likely that multiple carriers are responsible for plasma membrane transport of GSH (Pressman and Buff, 1997).

Materials and methods The study used a true experimental research design to determine if exogenous trimetric glutathione has a nephrotoxic effect on male Albino rats. For a duration of fifteen (15) days, two experimental groups of Albino rats, each having six rats, was be given different doses of intravenous glutathione and a negative control group with six rats did not receive any substance or chemical. After the treatment period, the kidneys of the subjects were removed and prepared for microscopic examination. Research Subject.A total of eighteen (18) male Albino rats were used in the research. The rats were randomly assigned to three groups with six (6) rats each, designated as Experimental group 1, Experimental group 2, and Control group. Experimental group 1 received intravenous glutathione with a dose of 30mg/kg of body weight, Experimental group 2received 60mg/kg of body of glutathione intravenously, and the Control group was a negative control and received no treatment. Research Instrument.The research instrument that was used for this study is the binocular compound light microscope to examine the prepared kidney slides from the research subjects. A histopathologic grading system was utilized to quantify the degree of morphologic renal changes among the research subjects and data sheets were used to record the results. Exogenous Glutathione Utilized. The exogenous glutathione that was utilized was commercialized one in a powder form contained in a vial that contains 650mg/vial. It is mixed with a 5ml 0.9% NaCl saline placed in a nebule. It also comes with a 500 mg Vitamin C in a 2ml ampoule that is also mixed with the glutathione vial. Administration of Glutathione. The glutathione was administered for fifteen (15) days to both experimental groups through an injection into the vein on the tail. All injections were performed around 12:00 noon. For the first week, glutathione was administered every other day, then daily for the second week, with a total of 10 sessions. To have a successful injection of the tail vein it needed to be dilate the veins. This can be accomplished by placing the tail in warm water for 1 minute. The veins can be seen when the tip of the tail is lifted and rotated slightly in either direction. The tip of the needle can be followed visually as it penetrates the vein. The formation of a bleb at the site indicates improper placement of the needle. A second

attempt can be performed by removing the needle and trying a site on the same vessel in a more proximal location on the tail. Determination of Nephrotoxic Effect. The rats from each group were sacrificed after the treatment period through cervical dislocation and dissection for removal of the kidneys was performed in the MHAM, Physiology Laboratory. The specimens from each group were placed in glass vials containing 10% formalin and were properly labeled according to group, number and laterality (eg. Exp Grp 1, Rat 2, Left Kidney), there were a total of 36 specimens. The specimens obtained were submitted to Sacred Heart Hospital Laboratory for the slide preparation and processing. The slides were examined in Vicente Sotto Memorial Medical Center by a licensed pathologist, Dr. Chamberlain Agtuca Jr., for histopathologic changes and were graded according to the level of kidney tissue damage

using a grading system (Appendix I). Data recording and subsequent tabulation of results were done for data analysis and interpretation. Statistical Analysis. The Kruskal–Wallis one-way analysis of variance by ranks is a non-parametric method for testing whether samples originate from the same distribution. It is used for comparing more than two samples that are independent, or not related. The parametric equivalent of the Kruskal-Wallis test is the one-way analysis of variance (ANOVA). When the Kruskal-Wallis test leads to significant results, then at least one of the samples is different from the other samples. The test does not identify where the differences occur or how many differences actually occur. It is an extension of the Mann–Whitney U test to 3 or more groups. The Mann-Whitney U Test would help analyze the specific sample pairs for significant differences.

Acclimatization of subjects for 7 days

Administration of glutathione for 15 days for a total 10 sessions

Utilization of improvised tube to restrain rat Insertion of IV catheter into the vein in the tail

Injection of glutathione using tuberculin syringe (30mg/kg to Exp Group 1 and 60mg/kg to Exp Group 2)

Ensure entry into the vein by backflow of blood

Experimental flow chartAdministration of Glutathione

24

attempt can be performed by removing the needle and trying a site on the same vessel in a more proximal location on the tail. Determination of Nephrotoxic Effect. The rats from each group were sacrificed after the treatment period through cervical dislocation and dissection for removal of the kidneys was performed in the MHAM, Physiology Laboratory. The specimens from each group were placed in glass vials containing 10% formalin and were properly labeled according to group, number and laterality (eg. Exp Grp 1, Rat 2, Left Kidney), there were a total of 36 specimens. The specimens obtained were submitted to Sacred Heart Hospital Laboratory for the slide preparation and processing. The slides were examined in Vicente Sotto Memorial Medical Center by a licensed pathologist, Dr. Chamberlain Agtuca Jr., for histopathologic changes and were graded according to the level of kidney tissue damage

using a grading system (Appendix I). Data recording and subsequent tabulation of results were done for data analysis and interpretation. Statistical Analysis. The Kruskal–Wallis one-way analysis of variance by ranks is a non-parametric method for testing whether samples originate from the same distribution. It is used for comparing more than two samples that are independent, or not related. The parametric equivalent of the Kruskal-Wallis test is the one-way analysis of variance (ANOVA). When the Kruskal-Wallis test leads to significant results, then at least one of the samples is different from the other samples. The test does not identify where the differences occur or how many differences actually occur. It is an extension of the Mann–Whitney U test to 3 or more groups. The Mann-Whitney U Test would help analyze the specific sample pairs for significant differences.

Acclimatization of subjects for 7 days

Administration of glutathione for 15 days for a total 10 sessions

Utilization of improvised tube to restrain rat Insertion of IV catheter into the vein in the tail

Injection of glutathione using tuberculin syringe (30mg/kg to Exp Group 1 and 60mg/kg to Exp Group 2)

Ensure entry into the vein by backflow of blood

Experimental flow chartAdministration of Glutathione

25

Results and discussions Gross Observation.Figure 3 shows gross pictures of the kidneys. Grossly, the kidneys from the three groups appeared similar to each other although they varied in size. The kidneys were dark red to brown in color, firm in consistency and smooth in texture.

A B C Fig. 1 Gross Pictures of the kidneys of male Albino rats from

Experimental Group 1 (A), Experimental Group 2 (B) and the Control Group (C)

Microscopic Observation.The table on the next page shows the histopathologic grading system that used to quantify the nephrotoxic effect of glutathione on the kidneys. It was based on how much of the microscopicfield presents with tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, and hemorrhage. This method was applied by Liu et al. in grading cisplatin induced nephrotoxicity in 2003.

Figure 2 shows microscopic images from the 3 three groups with tubular cell swelling. Cellular swelling is a feature of reversible cell injury that can be recognized under the light microscope. It appears whenever cells are incapable of maintaining ionic and fluid homeostasis andis the result of failure of energy-dependent ion pumps in the plasma membrane. The cells affected look larger with abundant cytoplasm.

Fig. 2 Microscopic Images from Experimental Group 1 (A), Experimental Group 2 (B), and the Control Group (C) showing varying degrees of tubular cell swelling (black arrow).

Table 1 Histopathologic grading system utilized to determine the nephrotoxic effect of glutathione on the kidneys.

Grade 0 Normal

Grade 1 <25% of microscopic field with area of tubular epithelial cell

swelling, vacuolar degeneration, tubular cell necrosis and

hemorrhage.

Grade 2 25-50% of microscopic field with area of tubular epithelial cell

swelling, vacuolar degeneration, tubular cell necrosis and

hemorrhage.

Grade 3 50-75% of microscopic field with area of tubular epithelial cell

swelling, vacuolar degeneration, tubular cell necrosis and

hemorrhage.

Grade 4 >75% of microscopic field with area of tubular epithelial cell

swelling, vacuolar degeneration, tubular cell necrosis and

hemorrhage.

Figures 3 shows the grades of the three groups according to tubular cell swelling. Experimental Group 1 has a mean frequency of 2.92, while Experimental Group 2 got a mean frequency of 3.33. The two experimental groups have high grades when compared to the control group which has a mean frequency of 1.58. Figure 4 presents microscopic images of vacuolar degeneration. On microscopic examination, small clear vacuoles are seen within the cytoplasm; these represent distended and pinched-off segments of the endoplasmic reticulum. This pattern of nonlethal injury is sometimes called hydropic change. Figures 5 Shows the grade of the three groups according to vacuolar degeneration. Experimental Group 1 has a mean frequency of 2.5, while Experimental Group 2 got a mean frequency of 2.75. The control group has a mean frequency of 1.5. Figure 6 highlights tubular cell necroses that are seen in the slides. Tubular epithelial cells in the tubules are necrotic, and many have become detached from their basement membranes and been

sloughed into the tubular lumens. Necrotic cells show increased eosinophilia when stained. Necrotic cells lose their nuclei, appear fragmented with leakage of contents.Figures 7 shows the grade of the three groups according to tubular cell necrosis. Both Experimental Group 1 and Experimental Group 2 got a mean frequency of 3.5, while the control group has a mean frequency of 1.25. Figure 8 shows microscopic pictures of hemorrhage within the renal tissues. Red blood cells have escaped from the blood vessels and have accumulated in the tubules and interstitial spaces of the kidney. The cells stains darker compared to the surrounding parenchymal cells. Figures 9 shows the grade of the three groups according to hemorrhage. Experimental Group 1 has a mean frequency of 2.67, while Experimental Group 2 got a mean frequency of 2.75. The two experimental groups have a higher grade of hemorrhage when compared to the control group which has a mean frequency of 1.17.

Figure 3. Tubular Cell Swelling Grading

Fig. 4 Microscopic Images from Experimental Group 1 (A), Experimental Group 2 (B), and the Control Group (C) showing varying degrees of vacuolar degeneration (black arrow).

26

Figures 3 shows the grades of the three groups according to tubular cell swelling. Experimental Group 1 has a mean frequency of 2.92, while Experimental Group 2 got a mean frequency of 3.33. The two experimental groups have high grades when compared to the control group which has a mean frequency of 1.58. Figure 4 presents microscopic images of vacuolar degeneration. On microscopic examination, small clear vacuoles are seen within the cytoplasm; these represent distended and pinched-off segments of the endoplasmic reticulum. This pattern of nonlethal injury is sometimes called hydropic change. Figures 5 Shows the grade of the three groups according to vacuolar degeneration. Experimental Group 1 has a mean frequency of 2.5, while Experimental Group 2 got a mean frequency of 2.75. The control group has a mean frequency of 1.5. Figure 6 highlights tubular cell necroses that are seen in the slides. Tubular epithelial cells in the tubules are necrotic, and many have become detached from their basement membranes and been

sloughed into the tubular lumens. Necrotic cells show increased eosinophilia when stained. Necrotic cells lose their nuclei, appear fragmented with leakage of contents.Figures 7 shows the grade of the three groups according to tubular cell necrosis. Both Experimental Group 1 and Experimental Group 2 got a mean frequency of 3.5, while the control group has a mean frequency of 1.25. Figure 8 shows microscopic pictures of hemorrhage within the renal tissues. Red blood cells have escaped from the blood vessels and have accumulated in the tubules and interstitial spaces of the kidney. The cells stains darker compared to the surrounding parenchymal cells. Figures 9 shows the grade of the three groups according to hemorrhage. Experimental Group 1 has a mean frequency of 2.67, while Experimental Group 2 got a mean frequency of 2.75. The two experimental groups have a higher grade of hemorrhage when compared to the control group which has a mean frequency of 1.17.

Figure 3. Tubular Cell Swelling Grading

Fig. 4 Microscopic Images from Experimental Group 1 (A), Experimental Group 2 (B), and the Control Group (C) showing varying degrees of vacuolar degeneration (black arrow).

27

Fig. 5 Vacuolar Degeneration Grading

Fig. 6 Microscopic Images from Experimental Group 1 (A), Experimental Group 2 (B), and the Control Group (C) showing varying degrees of tubular cell necrosis (black arrow).

Figure 7 Tubular Cell Necrosis Grading

Figure 8 Microscopic Images from Experimental Group 1 (A), Experimental Group 2 (B), and the Control Group (C) showing varying degrees of hemorrhage (black arrow).

28

Figure 7 Tubular Cell Necrosis Grading

Figure 8 Microscopic Images from Experimental Group 1 (A), Experimental Group 2 (B), and the Control Group (C) showing varying degrees of hemorrhage (black arrow).

29

Figure 9 Hemorrhage Grading

microscopic inspection using the histopathologic grading system, a significant difference is observed in microscopic morphology between the experimental groups (receiving 30mg/kg and 60 mg/kg of exogenous trimeric glutathione) when compared to the control group.However, histologically, there is no significant difference in microscopic morphology between Experimental group 1 receiving 30mg/kg of exogenous trimeric glutathione and Experimental group 2 receiving 60mg/kg of exogenous trimeric glutathione.

Glutathione, when given in high dosage and in frequent doses was able to produce changes in the histology of the kidneys that resembles the nephrotoxic effects of certain drugs. The mechanism on how glutathione can produce a nephrotoxic effect is unclear, A probable cause can be attribute to it molecular structure, as a tripeptide, it is a large molecule that cannot be readily filtered by the glomerulus of the kidney and could results into its deposition into the different structures in the kidney that can alter its capacity to filter and reabsorb. Another possible mechanism is the precipitation of the cystine amino acids in glutathione that’s results into stone formation.

Literature cited

Alarif, S. (2012). Blood chemical changes and renal histological alterations induced by gentamicin in rats. Expert Opinion on Drug Safety.

Bohm S., et al. (1999). Dose intensification of platinum compounds with glutathione protection as induction chemotherapy for advanced ovarian carcinoma. Oncology.

Borok Z., et al. (1991). Effect of glutathione on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis. Lancet, Vol 338, pp 215-216.

Cascinu S., et al. (1995). Neuroprotective effect of reduced glutathione on cisplatin-based chemotherapy in advanced gastric cancer: a randomized double-blind placebo-controlled trial. J Clin Oncol, Vol 13, pp 26-32.

Chavez, L. (2011). White skin comes with a price. Foods for BetterHealth.

Eaton, D. and Pooler, J. (2009). Vander’s Renal Physiology 7th Edition. LANGE Physiology Series, McGraw – hill Medical.

Gartner, L. P. and Hiatt, J. L. (2009).Color Atlas and Text of Histology 5th Edition. Philadelphia, Pennsylvania. Lippincott Williams and Wilkins.

Guyton, A. C. and Hall, J. E. (2006).Textbook of Medical Physiology 11th Edition. Philadelphia, Pennsylvania. Saunders, Elsevier Inc.

Jones D. P., et al. (1992). Glutathione in Foods Listed in National Cancer Institute’s Health Habits and History Food Frequency Questionnaire. Nutrition and Cancer.

Kidd, P. M. (2001). Glutathione: Systematic Protectant Against Oxidative and FreeRadical Damage. CA – A Cancer Journal for Clinicians.

Kumar, V. et.al. (2010). Robbins and Cotran Pathologic Basis of Disease 8th Edition. Philadelphia, Pennsylvania. Saunders, Elsevier Inc.

Liu, H. and Baliga, R. (2003). Cytochrome P450 2E1 Null Mice Provide Novel Protection Against Cisplatin-induced Nephrotoxicity and Apoptosis. Kidney International, Vol. 63 pp. 1687–1696

Murray, R. K., et al. (2003). Harper’s Illustrated Biochemistry 26th Edition. United States of America, McGraw – Hill Companies.

Pressman, A. H. and Buff, S. (1997).GSH Phenomenon: Nature’s Most Powerful Antioxidant and Healing Agent. New York, St. Martin’s Press.

Okajima K., et al. (1998). Interorgan Cooperation in Enzymatic Processing and Membrane Transport of Glutathione S-Conjugates. American Journal of the Medical Sciences.

Ormstad K., Orrenius S. (1980). Metabolism of Extracellular Glutathione in Small Intestine and Kidney. Expert Opinion on Drug Metabolism and Toxicology.

Sies, H. (1985). Reduced and Oxidized Glutathione Efflux from Liver. The Medical Letter on Drugs and Therapeutics.

The Kruskal – Wallis Test was used to demonstrate that in all 4 microscopic parameters there was a significant difference in morphology between the 3 groups. The test does not identify where the differences occur or how many differences actually occur; therefore we need now to look where the difference lies between and among the three groups. In order to do this, a post hoc analysis is done. In this case we used Bonferroni-corrected Mann-Whitney U Test since this is a non-parametric test to compare two groups simultaneously. The Bonferroni-corrected Mann-Whitney U Test is utilized. In this post-hoc test, if the p-value is lesser than 0.05, there is a significant difference. In all 4 parameters when the Experimental Group 1 and Group 2 were compared to each other, it has a p-value greater than 0.05, so the two groups have the same or comparable effects or no significant difference is observed. While the Control Group when compared to both experimental groups, it has a p-value lesser than 0.05, therefore there is a significant difference when we compare the control group to the experimental groups.

As shown in the tables above, with the use of Bonferroni-corrected Mann-Whitney U Test, based on the 4 parameters of tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, and hemorrhage; there is a significant difference in the microscopic morphology in the kidneys of the subjects between Experimental Group 1 and the Control Group. As shown also by the tables above. There is a significant difference in the microscopic morphology in the kidneys of the subjects between Experimental Group 2 and the Control Group. Finally, when comparing Experimental Group 1 and Experimental Group 2,the microscopic morphology of the two experimental groups did not have a significant difference.

Conclusion

The administration on exogenous trimeric glutathione through an intravenous route caused kidney injury to male Albino rats in both experimental groups as manifested by tubular epithelial cell swelling, vacuolar degeneration, tubular cell necrosis, and hemorrhage.

On kidney dissection, there were no visible significant gross changes or difference noted on any of the specimens obtained from the three groups. Upon

30

microscopic inspection using the histopathologic grading system, a significant difference is observed in microscopic morphology between the experimental groups (receiving 30mg/kg and 60 mg/kg of exogenous trimeric glutathione) when compared to the control group.However, histologically, there is no significant difference in microscopic morphology between Experimental group 1 receiving 30mg/kg of exogenous trimeric glutathione and Experimental group 2 receiving 60mg/kg of exogenous trimeric glutathione.

Glutathione, when given in high dosage and in frequent doses was able to produce changes in the histology of the kidneys that resembles the nephrotoxic effects of certain drugs. The mechanism on how glutathione can produce a nephrotoxic effect is unclear, A probable cause can be attribute to it molecular structure, as a tripeptide, it is a large molecule that cannot be readily filtered by the glomerulus of the kidney and could results into its deposition into the different structures in the kidney that can alter its capacity to filter and reabsorb. Another possible mechanism is the precipitation of the cystine amino acids in glutathione that’s results into stone formation.

Literature cited

Alarif, S. (2012). Blood chemical changes and renal histological alterations induced by gentamicin in rats. Expert Opinion on Drug Safety.

Bohm S., et al. (1999). Dose intensification of platinum compounds with glutathione protection as induction chemotherapy for advanced ovarian carcinoma. Oncology.

Borok Z., et al. (1991). Effect of glutathione on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis. Lancet, Vol 338, pp 215-216.

Cascinu S., et al. (1995). Neuroprotective effect of reduced glutathione on cisplatin-based chemotherapy in advanced gastric cancer: a randomized double-blind placebo-controlled trial. J Clin Oncol, Vol 13, pp 26-32.

Chavez, L. (2011). White skin comes with a price. Foods for BetterHealth.

Eaton, D. and Pooler, J. (2009). Vander’s Renal Physiology 7th Edition. LANGE Physiology Series, McGraw – hill Medical.

Gartner, L. P. and Hiatt, J. L. (2009).Color Atlas and Text of Histology 5th Edition. Philadelphia, Pennsylvania. Lippincott Williams and Wilkins.

Guyton, A. C. and Hall, J. E. (2006).Textbook of Medical Physiology 11th Edition. Philadelphia, Pennsylvania. Saunders, Elsevier Inc.

Jones D. P., et al. (1992). Glutathione in Foods Listed in National Cancer Institute’s Health Habits and History Food Frequency Questionnaire. Nutrition and Cancer.

Kidd, P. M. (2001). Glutathione: Systematic Protectant Against Oxidative and FreeRadical Damage. CA – A Cancer Journal for Clinicians.

Kumar, V. et.al. (2010). Robbins and Cotran Pathologic Basis of Disease 8th Edition. Philadelphia, Pennsylvania. Saunders, Elsevier Inc.

Liu, H. and Baliga, R. (2003). Cytochrome P450 2E1 Null Mice Provide Novel Protection Against Cisplatin-induced Nephrotoxicity and Apoptosis. Kidney International, Vol. 63 pp. 1687–1696

Murray, R. K., et al. (2003). Harper’s Illustrated Biochemistry 26th Edition. United States of America, McGraw – Hill Companies.

Pressman, A. H. and Buff, S. (1997).GSH Phenomenon: Nature’s Most Powerful Antioxidant and Healing Agent. New York, St. Martin’s Press.

Okajima K., et al. (1998). Interorgan Cooperation in Enzymatic Processing and Membrane Transport of Glutathione S-Conjugates. American Journal of the Medical Sciences.

Ormstad K., Orrenius S. (1980). Metabolism of Extracellular Glutathione in Small Intestine and Kidney. Expert Opinion on Drug Metabolism and Toxicology.

Sies, H. (1985). Reduced and Oxidized Glutathione Efflux from Liver. The Medical Letter on Drugs and Therapeutics.

31

Anti-platelet Aggregation Activity Of Atuna racemosa (Tabon-tabon) Fruit Extract On Aggregate Induced Human Platelet Suspension

LC Liao, K Lasdoce, JW Lim, GK Macaya, R Subba and Jose M. Oclarit,Ph.DSouthwestern University – Matias H. Aznar Memorial College of Medicine

Redemptorist Plaza, Camputhaw, Cebu City 6000 Philippines

Abstract

Over the past decade, the platelet has emerged as a pivotal entity in thromboembolic related diseases and bleeding disorders. There is resurgent interest in exploring the antiplatelet activity of natural products from medicinal plant extracts because these are more economical and affordable. Studies showed that herbal plants such as Atuna racemosa have an anti-inflammatory and antiplatelet effect due to its various compounds, flavonoids and triterpene metabolite.

This randomized experimental study was conducted to determine the anti-platelet effect of Atuna racemosa(local name: tabon-tabon) fruit extract using the mesocarp part on aggregate induced human platelet suspension with blood type O+. The study used a Giemsa Dye for the Giemsa Microplate Assay where the platelet suspension, test extracts and controls are placed to assess for presence or absence indicated by presence of violet gels. The method detected at an all-or-none response at a set concentration selecting for potent inhibitors.

Out of the 12 test samples in the tabon-tabon with ethanol and chloroform free extract, ten test samples inhibited platelet aggregation at a concentration of 4µL. The extracts of Atuna racemosa possessed an antiplatelet aggregation activity relative to the positive control (clopidogrel) using the Giemsa Microplate Assay as exhibited in the absence of gel.

Keywords: Antiplatelet aggregation activity, Giemsa microplate assay, Atuna racemosa (tabon-tabon), Violet gels, Giemsa dye, Clopidogrel, Platelet concentrate

Introduction

The ‘preeminence of the platelet’ has spawned a variety of drugs that have been shown in large-scale randomized trials to improve patient outcomes in revascularization procedures. It is an excellent example ‘what is old is new’, the platelet had been observed as a potential source of inflammatory markers decades ago. These observations had been largely ignored until recently, and it is now evident that the platelet is in fact a key mediator of thrombosis as well as of inflammation. New insights at the cellular and genomic levels will probably generate novel drugs to inhibit platelet function more effectively and safely than previously possible.

Platelets play an essential role in the development of acute coronary and cerebrovascular ischemic events and participate in the formation of

atherosclerosis. Ischemic cardiovascular events represent the leading cause of mortality and morbidity worldwide, and platelet aggregation and thrombus formation are the main effectors of this (Gresele et al., 2008). There is a wide consensus that intensified antiplatelet therapy as contributors to the reduction of major atherothrombotic complications in cardiovascular disease (Geisler et al., 2010).

Cardiovascular (CV) disease is the largest single killer in the US and Europe. A significant factor in CV and related diseases is the formation of a blood clot (thrombus), which can occlude blood vessels or the heart. Arterial occlusion can result in myocardial infarction, stroke and peripheral ischemia, while venous occlusion can cause thrombosis and pulmonary embolism (Hillery, 2000).

Antiplatelet agents are therefore considered as a significant tool in the treatment and prevention of thromboembolic disorders. Despite well known role of synthetic blood thinning agents to manage efficient secondary deterrence of thromboembolic

*Address correspondence to: LC Liao, SWU- Matias H. Aznar Memorial College of Medicine

E-mail address:lindycherubliao@gmail.com; Cell No.: 09269803032

32

Anti-platelet Aggregation Activity Of Atuna racemosa (Tabon-tabon) Fruit Extract On Aggregate Induced Human Platelet Suspension

LC Liao, K Lasdoce, JW Lim, GK Macaya, R Subba and Jose M. Oclarit,Ph.DSouthwestern University – Matias H. Aznar Memorial College of Medicine

Redemptorist Plaza, Camputhaw, Cebu City 6000 Philippines

Abstract

Over the past decade, the platelet has emerged as a pivotal entity in thromboembolic related diseases and bleeding disorders. There is resurgent interest in exploring the antiplatelet activity of natural products from medicinal plant extracts because these are more economical and affordable. Studies showed that herbal plants such as Atuna racemosa have an anti-inflammatory and antiplatelet effect due to its various compounds, flavonoids and triterpene metabolite.

This randomized experimental study was conducted to determine the anti-platelet effect of Atuna racemosa(local name: tabon-tabon) fruit extract using the mesocarp part on aggregate induced human platelet suspension with blood type O+. The study used a Giemsa Dye for the Giemsa Microplate Assay where the platelet suspension, test extracts and controls are placed to assess for presence or absence indicated by presence of violet gels. The method detected at an all-or-none response at a set concentration selecting for potent inhibitors.

Out of the 12 test samples in the tabon-tabon with ethanol and chloroform free extract, ten test samples inhibited platelet aggregation at a concentration of 4µL. The extracts of Atuna racemosa possessed an antiplatelet aggregation activity relative to the positive control (clopidogrel) using the Giemsa Microplate Assay as exhibited in the absence of gel.

Keywords: Antiplatelet aggregation activity, Giemsa microplate assay, Atuna racemosa (tabon-tabon), Violet gels, Giemsa dye, Clopidogrel, Platelet concentrate

Introduction

The ‘preeminence of the platelet’ has spawned a variety of drugs that have been shown in large-scale randomized trials to improve patient outcomes in revascularization procedures. It is an excellent example ‘what is old is new’, the platelet had been observed as a potential source of inflammatory markers decades ago. These observations had been largely ignored until recently, and it is now evident that the platelet is in fact a key mediator of thrombosis as well as of inflammation. New insights at the cellular and genomic levels will probably generate novel drugs to inhibit platelet function more effectively and safely than previously possible.

Platelets play an essential role in the development of acute coronary and cerebrovascular ischemic events and participate in the formation of

atherosclerosis. Ischemic cardiovascular events represent the leading cause of mortality and morbidity worldwide, and platelet aggregation and thrombus formation are the main effectors of this (Gresele et al., 2008). There is a wide consensus that intensified antiplatelet therapy as contributors to the reduction of major atherothrombotic complications in cardiovascular disease (Geisler et al., 2010).

Cardiovascular (CV) disease is the largest single killer in the US and Europe. A significant factor in CV and related diseases is the formation of a blood clot (thrombus), which can occlude blood vessels or the heart. Arterial occlusion can result in myocardial infarction, stroke and peripheral ischemia, while venous occlusion can cause thrombosis and pulmonary embolism (Hillery, 2000).

Antiplatelet agents are therefore considered as a significant tool in the treatment and prevention of thromboembolic disorders. Despite well known role of synthetic blood thinning agents to manage efficient secondary deterrence of thromboembolic

*Address correspondence to: LC Liao, SWU- Matias H. Aznar Memorial College of Medicine

E-mail address:lindycherubliao@gmail.com; Cell No.: 09269803032

Date Received: December 15, 2014 Date Accepted: April 10, 2015

33

diseases complaints, these drugs may still lead to hemorrhagic events and upper gastrointestinal bleeding as main drawbacks. There have been interests in exploring the anti-platelet activity of natural products from medicinal plant extracts because these are cost effective, easily to synthetic blood thinners in this prospective. Thus available form of indigenous resources and are comparable native approaches of herbal treatment are part of the culture and the prevailing method of curative therapy. However, most of these traditional remedies have not been validated scientifically.

There is therefore a critical need for new anti-platelet therapies that do not increase risk of bleeding. In many developing countries, a large percentage of the populations still rely heavily on traditional practitioners and medicinal plants to meet primary health needs. Although modern medicine may be available in these countries, herbal medicines such as Atuna racemosa, have often maintained popularity for historical and cultural reasons.

For the past ten years, there are many reports of anti platelet effect of Atuna Racemosa(tabon-tabon), however it is not well established. Recent studies showed that Atuna racemosa has an antibacterial activity, where they identified a plant, the Atun tree or Atuna racemosa, with purported antibacterial properties in the kernel (Buenz et al., 2005). There are currently no other studies being made on the plant aside from its antibacterial activity therefore our goal is to determine whether the fruit extract from its mesocarp have an antiplatelet aggregating activity.

Material and Methods Collection Experimental Sample Grinding of Plant Material

Eight hundred (800) grams of mesocarp of Atuna racemosa (tabon-tabon) plant material, raw grind using an electric blender to improve the subsequent extraction by rendering the sample more homogenous, increasing the surface area, and facilitating the penetration of solvent into the cells.

Preparation of Test Controls

Volume preparation of 60% ethanol/40% chloroform solvents was used. Ethanol solution was prepared in 600mL, placed in a clean, empty erlenmeyer flask and 400mL chloroform solvent was

placed in a clear flask. The two solvents were mixed to obtain 1000mL.

Extraction Method: Maceration

In a closed glass container, pulverized plant material was placed in the mixed solution of ethanol and chloroform and was allowed to stand for 48 hours. The container was covered with a coil to prevent loss of solvent. After some time in contact, the solvent was decanted through a whatman filter paper and the sample is mixed through stirring or swirling using a stirring rod.

For water soluble solvent it is soaked with 60% ethanol (C2H6O) and 40% chloroform (CHCl3)for lipid soluble extraction.

Pure extraction: Evaporate to dryness using Rotary Evaporator

The flask with the filtrate solution was placed on the rotary evaporator. The used of a bump trap prevents the solution from accidentally splashing into the condenser (and being contaminated). A metal or Keck clip was used to secure the flask and the bump trap. The aspirator vacuum is turned on. The flask was lowered into the water bath (or the water bath was raised to immerse the flask in the warm water at about 40 degrees centigrade at reduced pressure. The solvent started collecting on the condenser and dripped into the receiving flask. To prevent evaporation in the receiving flask, a cooling bath on the receiver was used and a dry-ice condenser was used. Once all the solvent evaporate to dryness, the vacuum was released. The flask is raised out of the water bath and the spinning is discontinued. Some of the filtrate was in semi-liquid; a hair blower maintained at a room temperature was used.

Preparation of Positive control and Negative control

Clopidogrel was used as the positive control as it has been known for its antiplatelet activity. Plain normal saline solution or PNSS was used as negative control since it was used as a solvent for the preparation of test solutions. To promote platelet aggregation, 0.25 Calcium chloride was used.

Preparation of Platelet Concentrate (PRC Bloodbank Procedure)

This was conducted in Philippine Red Cross National Headquarters, Cebu City by a licensed technologist. A donor was bled using a needle attached to a quadruple Optipac bag. The blood type

Page | 3

taken was O+. The collected whole blood was made to stand until a certain amount of plasma could be visibly seen before it was centrifuged at 4000 rpm for 8-10 minutes at a temperature of 22 C.

A Fenwaloptipress II machine was used to separate the centrifuged blood into packed RBC and plasma by transferring the above mentioned components into different transference empty bag. This procedure would leave behind the platelets and a small amount of red blood cells in a mother bag.

The mother bag was then left to stand before spinning at 1,100 rpm for 6-7 minutes at a temperature of 22 C. The optipress machine was again used to separate the platelet concentrate from the remaining red blood cells. The former was stored at room temperature with constant rotation and could be transferred within five days from processing.

Platelet stored at room temperature causes activation of platelet aggregation so constant agitation and incubation at 37 C is needed to gradually decrease the aggregation-of-platelet-prior-to-rest-for-antiplatelet-function.

Preparation of Giemsa Stain

To make a 10% w/v concentration of Giemsa stain, one gram of Giemsa powder was dissolved in enough distilled water resulting to a volume of ten milliliters.

Giemsa Microplate Assay (Pimentel et al.,2003)

The Platelet suspension (200µL) was transferred to wells in a 96- well plate and was incubated at 37 C for five minutes. The test solutions tabon-tabon extract with ethanol and chloroform free extract were added in each well for about 4µL; the same was done with the positive control (Clopidogrel) 40µL was placed in the microplate well and 4µL negative control (PNSS). The plate was incubated at 37 C for 30 minutes, a temperature that mimics the human body temperature of a person. Platelets were induced to aggregate with the addition of 4µL 0.25 calcium chloride in each well (Born et al. 1963). The plate was incubated at 37 C for 30 minutes. Four microliters of 10% Giemsa dye were added to each well. The microplate was incubated further to five minutes. Giemsa dye was used to clearly visualize the gel formation. The microplate was inverted, or the absence of violet gels

indicates antiplatelet aggregation activity. Each well was observed for the presence or absence of violet gels on the microplate indicated platelet aggregation and the absence of such gels indicated inhibitory action.

Results and Discussion Description of Plant Material and Test Extracts

The plant material obtained after using an electric blender was solid, dark brown in color. The product of milling changed into juicy, yellow in color after using a rotatory evaporator. (See Figure 2 )

The 600mL ethanol solvent was transparent, clear in color. (See Figure 3) The 400mL choloroform solvent was opaque, clear in color. (See Figure 3)

The ethanol solvent mixed with the choloroform solvent yields a cloudy effect. (See Figure 4) The pure tabon-tabon extract with ethanol and choloroform solvent was dark orange in color. (See Figure 5)

Fresh platelet concentrate was dark yellow. Giemsa Microplate Assay Results Platelet concentrate from a human blood type O+ was used in the assay. The test solution (tabon-tabon with ethanol and chloroform free extract) at a concentration of 4µL showed absence of gel in well no. 1-5 and 8-10. Well no. 5 and 6 showed presence of gel in the test solution. The positive control, clopidogrel at a concentration of 40µLshowed no formation of violet gels except in well no. 3. Plain normal saline solution (PNSS) at a concentration of 4µL was used as a negative control and there were also formation of violet gels. (See Figure 1 and Table 2)

Fig. 1 : 96- Well Microplate showing the Giemsa Microplate Assay Results1-12 (+): Positive control (clopidogrel 40µL)1-12 (-): Negative control (PNSS 4µL)1-12 (E): Experimental group (tabon-tabon with ethanol and chloroformfree extract 4µL)

Positive Control

Negative Control

Experimental Group

1 2 3 4 5 6 7 8 9 10 11 12 Well No:

34

Page | 3

taken was O+. The collected whole blood was made to stand until a certain amount of plasma could be visibly seen before it was centrifuged at 4000 rpm for 8-10 minutes at a temperature of 22 C.

A Fenwaloptipress II machine was used to separate the centrifuged blood into packed RBC and plasma by transferring the above mentioned components into different transference empty bag. This procedure would leave behind the platelets and a small amount of red blood cells in a mother bag.

The mother bag was then left to stand before spinning at 1,100 rpm for 6-7 minutes at a temperature of 22 C. The optipress machine was again used to separate the platelet concentrate from the remaining red blood cells. The former was stored at room temperature with constant rotation and could be transferred within five days from processing.

Platelet stored at room temperature causes activation of platelet aggregation so constant agitation and incubation at 37 C is needed to gradually decrease the aggregation-of-platelet-prior-to-rest-for-antiplatelet-function.

Preparation of Giemsa Stain

To make a 10% w/v concentration of Giemsa stain, one gram of Giemsa powder was dissolved in enough distilled water resulting to a volume of ten milliliters.

Giemsa Microplate Assay (Pimentel et al.,2003)

The Platelet suspension (200µL) was transferred to wells in a 96- well plate and was incubated at 37 C for five minutes. The test solutions tabon-tabon extract with ethanol and chloroform free extract were added in each well for about 4µL; the same was done with the positive control (Clopidogrel) 40µL was placed in the microplate well and 4µL negative control (PNSS). The plate was incubated at 37 C for 30 minutes, a temperature that mimics the human body temperature of a person. Platelets were induced to aggregate with the addition of 4µL 0.25 calcium chloride in each well (Born et al. 1963). The plate was incubated at 37 C for 30 minutes. Four microliters of 10% Giemsa dye were added to each well. The microplate was incubated further to five minutes. Giemsa dye was used to clearly visualize the gel formation. The microplate was inverted, or the absence of violet gels

indicates antiplatelet aggregation activity. Each well was observed for the presence or absence of violet gels on the microplate indicated platelet aggregation and the absence of such gels indicated inhibitory action.

Results and Discussion Description of Plant Material and Test Extracts

The plant material obtained after using an electric blender was solid, dark brown in color. The product of milling changed into juicy, yellow in color after using a rotatory evaporator. (See Figure 2 )

The 600mL ethanol solvent was transparent, clear in color. (See Figure 3) The 400mL choloroform solvent was opaque, clear in color. (See Figure 3)

The ethanol solvent mixed with the choloroform solvent yields a cloudy effect. (See Figure 4) The pure tabon-tabon extract with ethanol and choloroform solvent was dark orange in color. (See Figure 5)

Fresh platelet concentrate was dark yellow. Giemsa Microplate Assay Results Platelet concentrate from a human blood type O+ was used in the assay. The test solution (tabon-tabon with ethanol and chloroform free extract) at a concentration of 4µL showed absence of gel in well no. 1-5 and 8-10. Well no. 5 and 6 showed presence of gel in the test solution. The positive control, clopidogrel at a concentration of 40µLshowed no formation of violet gels except in well no. 3. Plain normal saline solution (PNSS) at a concentration of 4µL was used as a negative control and there were also formation of violet gels. (See Figure 1 and Table 2)

Fig. 1 : 96- Well Microplate showing the Giemsa Microplate Assay Results1-12 (+): Positive control (clopidogrel 40µL)1-12 (-): Negative control (PNSS 4µL)1-12 (E): Experimental group (tabon-tabon with ethanol and chloroformfree extract 4µL)

Positive Control

Negative Control

Experimental Group

1 2 3 4 5 6 7 8 9 10 11 12 Well No:

35

Table 2 : Results and Interpretation of Antiplatelet Aggregation Activity using Giemsa Microplate Assay

Legend:

A- Absence of gel indicating inhibition of platelet aggregation (Positive result)

P- Presence of gel indicating platelet aggregation (Negative result)

Platelets have glycoprotein receptors which in the presence of calcium signal the platelet to aggregate. Appearance of violet gels in the wells indicated aggregation of platelets which was further visualized with the used of Giemsa dye. In the study, Calcium chloride (0.25M) was used to induce platelet aggregation.

Giemsa dye was used because it stains platelet well. Though giemsa dye is violet, possible factors that cause deviation or its blue coloration may be due to excessive staining time, inadequate washing and that the wash water was too alkaline (Allison et al. 1992)

Absence of gels exhibit antiplatelet aggregate activity and this was shown on the positive control, clopidogrel even in the presence of calcium chloride. It irreversibly inhibit the binding of ADP to its receptors which would then interfere with the signals that would promote aggregation of platelets.

The negative control (PNSS), did not exhibit antiplatelet activity as shown by presence of violet gel. This can reasonably show evidence that it did not contribute to the activity of the platelet aggregation even with the presence of calcium chloride. It did not

significantly contain active substances that may add to the inhibitory activity of the test solutions.

The experimental group, tabon-tabon extract were able to inhibit platelet aggregation as shown by the absence of violet gels in well no. 1-4 and 7-12. Based on studies, tabon-tabon contains flavonoids and tripertenoid. But this plant’s metabolite is more on the former.

Flavonoids are polyphenolic compounds that are ubiquitous in nature and its antiplatelet activity can be attributed to the increased production of prostacyclin by endothelian cells. Minimal concentrations of flavonoids can significantly lower the platelet aggregation. (Bojic et al., 2011).

Triterpenoid on the other hand, was said to have shown a potent ability to inhibit adenosine diphosphate and epinephrine-induced human platelet aggregation. (Alvarado-Castillo, 2012). These two constituents, flavonoids and tripertenoid are known to be polar and nonpolar constituents respectively. This explains that the constituents responsible for the activity from the mesocarp of A. racemosa using ethanol and chloroform as a solvent exhibit inhibition of platelet aggregation. The result does not quantify the extent of inhibition but will only provide qualitative data, specifically positive or no activity.

Conclusion

Out of the 12 test samples in experimental group the tabon-tabon with ethanol and chloroform free extract ten test samples in well numbers 1-4 and 7-12 as seen in Figure 1 inhibited platelet aggregation at a concentration of 4µL as shown by absence of violet gel in Giemsa Microplate Assay. The organic solvent that extracted the constituents from the tabon-tabon mesocarp was ethanol and chloroform, which has shown to promote antiplatelet aggregation relative to the positive control, clopidogrel.

The extracts of Atuna racemosa (tabon-tabon) extract possessed an antiplatelet aggregation activity using the Giemsa Microplate Assay as exhibited in the absence of gel.

Test solution and

controls

1 2 3 4 5 6 7 8 9 10 11 12

Positive

control(Clopidogrel

40µL)

A A P A A A A A A A A A

Negative

control(PNSS 4µL)

P P P P P P P P P P P P

Experimental group(A.racemosa)with ethanol and chloroform free extract 4µL

A A A A P P A A A A A A

1 2 3 4 5 6 7 8 9 10 11 12

Positive control

Negative control

Experimentalgroup

Legend:

= 200µL platelet +40µL (2mg/mL Clopidogrel in NSS) +4µL CaCl2 + 4µL Giemsa dye

= 200µL platelet +4µL (PNSS)

+4µL CaCl2 + 4µL Giemsa dye

= 200µL platelet +4µL (tabon-tabon with ethanol and chloroform free extract)

+4µL CaCl2 + 4µL Giemsa dye

36

1 2 3 4 5 6 7 8 9 10 11 12

Positive control

Negative control

Experimentalgroup

Legend:

= 200µL platelet +40µL (2mg/mL Clopidogrel in NSS) +4µL CaCl2 + 4µL Giemsa dye

= 200µL platelet +4µL (PNSS)

+4µL CaCl2 + 4µL Giemsa dye

= 200µL platelet +4µL (tabon-tabon with ethanol and chloroform free extract)

+4µL CaCl2 + 4µL Giemsa dye

37

Literature cited

Satyajit D. Sarker and Lutfun Nahar. Natural Products Isolation Methods and Protocols 3rd Edition, New York: Springer Science Business Media 2012 3:1, 75, 427

Saekar Halabi and Ashish Battah. Phytochemical and Antiplatelet Investigation of Gundelia tournifortii, Russia: Facts on File 2010 43:496-500 Lazarus, G. et al. Masters Dissertation of Platelet, India: Sheffield 2011

Bhatt, D. Scientific and Therapeutic Advances in Antiplatelet Therapy, Washington D.C., Life Science 2003 2:15-20

Bojic, M. et al. Evaluation of antiaggregatory activity of flavonoid aglycone series. Nutritional Journal 2011 10:1-8

Zia-Ul-Haq, M. et al. Anti-platelet activity of methanolic extract of Grewia asiatica L. leaves and Terminalla chebula Retz. Fruits. USA Today 2012 2:1-4

Pimentel, Sheila et al. Platelet Aggregation Inhibitors from Philippine Marine Invertebrate Samples Screened in a New Microplate Assay 2002 5:395-400

James Hamuel Doughari. Phytochemicals: Extraction methods, basic structures and mode of action as potential chemotherapeutic agents. 2008 2:57-65

Ashley, K. and Lincoff, M. Pharmacologic inhibition of platelet function: Correlation between in vitro tests of platelet function and clinical outcomes 2008 8:283

Singh, RB et al. Hypertension and stroke in Asia: prevalence, control and strategies in developing countries for prevention 2000 14:749-763

Imran, I. et al. Antiplatelet activity of methanolic extract of Acacia leucophloea bark 2012 6:4185-4188 Sanchez, Dulce Tabon-tabon sauce Philstarnews 2009

Antibacterial Activity of the Moringa oleifera Lam (Malunggay) Plant Parts on Penicillin-resistant Staphylococcus aureus in vitro.

Arlene Maceren Diaz M.D, FPSECP, Ed.D Department of Pharmacology

Matias H. Aznar Memorial College of Medicine Southwestern University, Cebu City, Phils

Abstract:

The aim of this study is to determine whether the antibacterial activity of Moringa oleifera lam (Malunggay) plant parts (Leaves, bark and roots) are effective against Penicillin-resistant Staphylococcus aureus in vitro. This is a true experimental study conducted at the Pharmacology and Microbiology Departments of the College of Medicine, Matias H.Aznar Memorial College of Medicine, Southwestern University. The study population, Penicillin-resistant Staphylococcus aureus was isolated from two sources: one from a hospital patient and one from a laboratory stock culture.

Seven hundred seventy-six (776) petri dishes were seeded with Penicillin-resistant Staphylococcus aureus.(338 plates with isolate from the hospital; 338 plates with the isolate from the laboratory stock culture). One hundred ninety-four (194) plates from the hospital source and hundred ninety-four (194) plates from the laboratory stock culture were treated with the alcoholic extract of the M.oleifera plant parts. The rest of the plants from both sources were treated with aqueous extract of the plants parts. Antibacterial activity was determined by measuring the zones of inhibition an area of clearing around the agar cup which signifies inhibition of bacterial growth. Using the Kirby Bauer interpretative chart for plant extract the results were then interpreted as follows: <6mm=negative; 7-11mm=weak; 12-16mm=moderately sensitive ; >17mm=strong or sensitive.

Both the hospital and laboratory stock culture isolates treated with the alcoholic and aqueous extracts of the M. oleifera leaves demonstrated 100% strong antibacterial activity. Majority of the root extracts demonstrated a moderate antibacterial while most of the alcohol bark extract showed a weak antibacterial activity. One hundred percent (100%) of the aqueous bark extract did not exhibit any antibacterial activity . There was a significance difference between the antibacterial activity of the alcoholic and aqueous extracts of the leaves, bark and roots in both hospital and laboratory cultures (p value = 0.00001). There was no difference in the antibacterial activity of the alcoholic and aqueous extract of M. oleifera leaves of both hospital and laboratory cultures (p value-1). The result of this study showed that it is Moringa oleifera leaves extract (alcoholic and aqueous) that has a strong anti-bacterial activity against Penicillin resistant Staphylococcus aureus. Future studies should include determination of the active substance responsible for the antibacterial activity of the leaves; testing of the stability of the extracts at room temperature and the Minimum inhibitory concentration of both the alcoholic and aqueous leave extract on Penicillin resistant S.aureus should be evaluated.

Key words: Antibacterial, Moringa Oleifera Lam (Malunggay) , Penicillin resistant Staphylococcus aureus

Introduction

The present thrust of our government today is geared towards the use of herbal medicine as an alternative to the ever rising cost of commercially available drugs. One plant of promising potential is the plant Moringa oleifera Lam. of the family Moringaceae.. It occurs widely in the Philippines and is commonly known as Malunggay in Tagalog; Marunggay in Ilocanos; Kamalongan, Mangit or

kalamunggay in the Visayas, Kalamunggai in the Bicol area, Kalungai for Kapampangans; and Horseraddish, Ben Tree or drumstick tree in English.6

The plant is a small tree, about eight meters or less in height with corky bark and soft white wood. The leaves are alternate usually thrice-pinnate about 25-50 cm long. These leaflets are thin ovate to elliptic and 1-2 cm long. The pod is 15-30 cm long pendulous, three angled and nine ribbed. It seeds are

38

Date Received: January 2015 Date Accepted: April 10, 2015

Antibacterial Activity of the Moringa oleifera Lam (Malunggay) Plant Parts on Penicillin-resistant Staphylococcus aureus in vitro.

Arlene Maceren Diaz M.D, FPSECP, Ed.D Department of Pharmacology

Matias H. Aznar Memorial College of Medicine Southwestern University, Cebu City, Phils

Abstract:

The aim of this study is to determine whether the antibacterial activity of Moringa oleifera lam (Malunggay) plant parts (Leaves, bark and roots) are effective against Penicillin-resistant Staphylococcus aureus in vitro. This is a true experimental study conducted at the Pharmacology and Microbiology Departments of the College of Medicine, Matias H.Aznar Memorial College of Medicine, Southwestern University. The study population, Penicillin-resistant Staphylococcus aureus was isolated from two sources: one from a hospital patient and one from a laboratory stock culture.

Seven hundred seventy-six (776) petri dishes were seeded with Penicillin-resistant Staphylococcus aureus.(338 plates with isolate from the hospital; 338 plates with the isolate from the laboratory stock culture). One hundred ninety-four (194) plates from the hospital source and hundred ninety-four (194) plates from the laboratory stock culture were treated with the alcoholic extract of the M.oleifera plant parts. The rest of the plants from both sources were treated with aqueous extract of the plants parts. Antibacterial activity was determined by measuring the zones of inhibition an area of clearing around the agar cup which signifies inhibition of bacterial growth. Using the Kirby Bauer interpretative chart for plant extract the results were then interpreted as follows: <6mm=negative; 7-11mm=weak; 12-16mm=moderately sensitive ; >17mm=strong or sensitive.

Both the hospital and laboratory stock culture isolates treated with the alcoholic and aqueous extracts of the M. oleifera leaves demonstrated 100% strong antibacterial activity. Majority of the root extracts demonstrated a moderate antibacterial while most of the alcohol bark extract showed a weak antibacterial activity. One hundred percent (100%) of the aqueous bark extract did not exhibit any antibacterial activity . There was a significance difference between the antibacterial activity of the alcoholic and aqueous extracts of the leaves, bark and roots in both hospital and laboratory cultures (p value = 0.00001). There was no difference in the antibacterial activity of the alcoholic and aqueous extract of M. oleifera leaves of both hospital and laboratory cultures (p value-1). The result of this study showed that it is Moringa oleifera leaves extract (alcoholic and aqueous) that has a strong anti-bacterial activity against Penicillin resistant Staphylococcus aureus. Future studies should include determination of the active substance responsible for the antibacterial activity of the leaves; testing of the stability of the extracts at room temperature and the Minimum inhibitory concentration of both the alcoholic and aqueous leave extract on Penicillin resistant S.aureus should be evaluated.

Key words: Antibacterial, Moringa Oleifera Lam (Malunggay) , Penicillin resistant Staphylococcus aureus

Introduction

The present thrust of our government today is geared towards the use of herbal medicine as an alternative to the ever rising cost of commercially available drugs. One plant of promising potential is the plant Moringa oleifera Lam. of the family Moringaceae.. It occurs widely in the Philippines and is commonly known as Malunggay in Tagalog; Marunggay in Ilocanos; Kamalongan, Mangit or

kalamunggay in the Visayas, Kalamunggai in the Bicol area, Kalungai for Kapampangans; and Horseraddish, Ben Tree or drumstick tree in English.6

The plant is a small tree, about eight meters or less in height with corky bark and soft white wood. The leaves are alternate usually thrice-pinnate about 25-50 cm long. These leaflets are thin ovate to elliptic and 1-2 cm long. The pod is 15-30 cm long pendulous, three angled and nine ribbed. It seeds are

*Corresponding author Arlene Maceren Diaz M.D.

39

three angled and winged on the angles and its flowers are white. 16

A substance from the roots called Pterygospermin was isolated which showed an antibiotic property. 6 Zopf. Eilert and coworkers isolated an antibacterial substance also from the seed and determined its structure to be 4-(-L-Rhamnosyloxy) benzyl isothiocyanate A, which is effective against gram positive bacteria and fungi. 4

Duero, Generosa and Puerto from MCU Manila, made a study of the antibacterial activity of alcoholic and aqueous extracts of Moringa Oleifera leaves, using Staphylococcus aureus and Escherichia coli. The results showed that both alcoholic and aqueous extracts of the leaves has very strong antibiotic property against Staphylococcus aureus. 5

Staphylococcus aureus belongs to the family of Micrococaceae. It is non motile, 0.8 to 1.0 mm in diameter that forms irregular grapelike clusters. They are gram positive. The most reliable property to diagnose Staphylococcus aureus is the ability to produce coagulase, and enzyme that causes coagulation of plasma. Most Staphylococcus aureus are members of the normal flora of the skin and mucous membrane but they can cause suppuration, abscesses, carbuncles, paronychia, impetigo contagiosa, and also systemic diseases such as pneumonia, empyema, endocarditis, puerperal fever, brain abscess, parotitis, phlebitis, pyelonephritis, toxic shock syndrome, food poisoning and even fatal septicema. 10

The inappropriate use and abuse of antibiotics that normally kill this organism leads to the emergence of a strain that produces beta lactamase or Penicillinase, which is an enzyme that breaks the beta lactam ring of Penicillin drugs and Cephalosporins (The beta Lactam antibiotics) and nullify the bacterial effect of these drugs. Such strain is referred to as Penicillin-resistant Staphylococcus aureus or Beta-Lactamase producing Staphylococcus aureus. 12, 4, 8

The emergence of Penicillin-resistant Staphylococcus aureus adds more to the burden of cost of treatment to those who will be having such an infection. It would be of great significance to determine if M. oleifera plant part extracts (leaves, bark, and roots,) would exhibit a strong antibacterial activity to such strain. This will be of great help to our people belonging to the lower socio-economic group. Despite the many traditional antimicrobial uses of M. oleifera, not much research has been conducted on the antibacterial activity of the plant parts on Penicillin resistant Staphylococcus aureus. Itis for this reason, that the researcher conducted this study in order to investigate whether the antibacterial

activities of M. oleifera plant parts (leaves, bark, and roots) are effective against Penicillin-resistant Staphylococcus aureus in vitro.

Methodology

The test organism, Staphylococcus aureus was taken from two sources: one from a hospital patient and another from a laboratory stock culture of the Microbiology Department of Matias H. Aznar Memorial College of Medicine, Southwestern University. An antibiogram was done using beta-lactam antibiotics: Penicillin, Ampicillin, and Cloxacillin. The isolate taken from the hospital was found resistant to all of the beta-lactam antibiotics, while the isolate from the laboratory was found resistant to both Penicillin and Ampicillin but sensitive to Cloxacillin. The Moringa oleifera plant parts was collected from Dumlog, Talisay City Cebu, Philippines.Two hundred (200) grams of each Moringa oleifera plant parts (leaves, bark, and roots) were mixed with three hundred (300) ml of 95% ethyl alcohol to produce the alcoholic extract. Another two hundred (200) grams of each part were mixed with the same amount of distilled water to produce the aqueous extract. Each mixture was blended separately. It was then placed in separate beakers, sealed tightly with aluminum foil and stored at refrigeration temperature (40 to 80 C). The mixture was filtered just before use and the plant residue discarded. The anti-microbial activity of each plant parts on the test organism was determined on the 14th

day of storage, since the preliminary testing of the fresh extracts did not exhibit any antibacterial activity. Using the agar cup method (Mueller-Hinton Agar), a total of seven-hundred-seven six (776) Petri dishes were seeded with Penicillin-resistant Staphylococcus aureus. Three hundred eighty-eight plates (388) were seeded with the isolate from the hospital and the rest of the plates with the isolate from the laboratory stock culture. Of the plates seeded with the hospital isolate, one hundred ninety-four (194) were used for the aqueous plant extracts and the remaining one hundred ninety-four (194) were used for the alcoholic extracts. For the plates using the aqueous extracts, three 6-mm holes were bored aseptically (holes were bored 25 mm apart to prevent overlapping of zone of inhibition.) The holes were designated to each of the Moringa oleifera plant parts (leaves, bark, and roots). Using a micropipette,0.05 ml of each plant extract was then placed into the designated holes. For the alcoholic extract a similar

procedure was carried out but an additional hole for 95%ethyl alcohol was placed to act as a negative control To the remaining three hundred eighty-eight (388) petri dishes seeded with the isolate from the laboratory stock culture as similar procedure was done in which 194 plates were used for the aqueous extract and 194 plates for the alcoholic extracts of the plant parts .The plates were incubated at 370 C for 18-24 hours. After incubation the antimicrobial activity was evaluated by measuring the diameter of clear zones of inhibition (an area of cleaning around the Agar cup which signifies inhibition of bacterial growth) to the nearest tenth of a millimeter using a celluloid millimeter ruler and a magnifying glass and then using the Kirby Bauer chart for interpretation of plant extracts the results were interpreted as follows: <6 mm=negative; 7-11mm=weak; 12-16mm=moderately sensitive ; >17mm=strong or sensitive.

Results:

The hospital isolate that was treated with the alcoholic extract of the plant showed that the leaves demonstrated 100% strong antibacterial activity. The root extract exhibited only 8.7% (17 plates) strong antibacterial activity and 56.3% moderate antibacterial activity; The bark extract did not show any strong sensitivity only 11.3% (22 plates exhibited moderately sensitive antibacterial activity. Majority,of the bark extract, 62.7% did not exhibited any antibacterial activity .All of the alcohol control did not exhibit any antibacterial activity. (Table 1)

Table 1: Antibacterial activity of alcoholic extract of M. oleifera plant parts on Penicillin resistant Staph. aureus (Hospital Source)

* Values expressed in percentage

The laboratory isolate treated with alcohol extract also showed that the leaves demonstrated 100% strong anti-bacterial activity. While the root extracts 20.10% showed strong antibacterial activity (39 plates). Only 0.5% (1 plate) of the bark extracts showed strong anti-bacterial activity. Majority of the roots extracts showed 64.95% (121 plates) moderately sensitive anti-bacterial activity. While majority of the bark extracts 42.78% (83 plates) exhibited weak anti-bacterial activity. The 95% ethyl alcohol did not exhibited any antibacterial activity (Table 2)

Table 2: Antibacterial activity of alcoholic extract of M. oleifera plant parts on Penicillin resistant Staph.aureus (Laboratory Stock Culture)

Treatment Negative Weak Moderate Sensitive TotalLeaves 100 100Bark 22 42.78 35 0.5 100Root 1 14 64.95 20.10 10095 ethyl alcohol 100 100

* Values expressed in percentage

Aqueous extracts of the leaves on the hospital organism still exhibited 100% strong anti-bacterial activity. Majority of the roots extract and bark extract showed negative anti-bacterial activity. (Table 3)

Table 3: Antibacterial activity of alcoholic extract of M. oleifera plant parts on Penicillin resistant Staph.aureus (Laboratory Stock)

Treatment Negative Weak Moderate Sensitive Total

Leaves 100 100Bark 100 100Root 81 14 5 100

* Values expressed in percentage

In the laboratory isolate, the aqueous extracts of the leaves demonstrated 100% strong antibacterial activity. Majority of the aqueous extract of the roots, 61% (118 plates) showed negative antibacterial activity, only 2.5% (5 plates) exhibited strong anti-bacterial activity, while 19.5 (38 plates) moderate anti-bacterial activity, and 17% (33 plates) showed weak anti-bacterial activity. The aqueous extract of the bark did not exhibit any anti-bacterial activity at all. (Table 4)

Treatment Negative Weak Moderate Sensitive Total

Leaves 100 100

Bark 62.7 26 11.3 100

Root 8.7 26% 56.3 9 100

95 ethyl alcohol 100 100

40

procedure was carried out but an additional hole for 95%ethyl alcohol was placed to act as a negative control To the remaining three hundred eighty-eight (388) petri dishes seeded with the isolate from the laboratory stock culture as similar procedure was done in which 194 plates were used for the aqueous extract and 194 plates for the alcoholic extracts of the plant parts .The plates were incubated at 370 C for 18-24 hours. After incubation the antimicrobial activity was evaluated by measuring the diameter of clear zones of inhibition (an area of cleaning around the Agar cup which signifies inhibition of bacterial growth) to the nearest tenth of a millimeter using a celluloid millimeter ruler and a magnifying glass and then using the Kirby Bauer chart for interpretation of plant extracts the results were interpreted as follows: <6 mm=negative; 7-11mm=weak; 12-16mm=moderately sensitive ; >17mm=strong or sensitive.

Results:

The hospital isolate that was treated with the alcoholic extract of the plant showed that the leaves demonstrated 100% strong antibacterial activity. The root extract exhibited only 8.7% (17 plates) strong antibacterial activity and 56.3% moderate antibacterial activity; The bark extract did not show any strong sensitivity only 11.3% (22 plates exhibited moderately sensitive antibacterial activity. Majority,of the bark extract, 62.7% did not exhibited any antibacterial activity .All of the alcohol control did not exhibit any antibacterial activity. (Table 1)

Table 1: Antibacterial activity of alcoholic extract of M. oleifera plant parts on Penicillin resistant Staph. aureus (Hospital Source)

* Values expressed in percentage

The laboratory isolate treated with alcohol extract also showed that the leaves demonstrated 100% strong anti-bacterial activity. While the root extracts 20.10% showed strong antibacterial activity (39 plates). Only 0.5% (1 plate) of the bark extracts showed strong anti-bacterial activity. Majority of the roots extracts showed 64.95% (121 plates) moderately sensitive anti-bacterial activity. While majority of the bark extracts 42.78% (83 plates) exhibited weak anti-bacterial activity. The 95% ethyl alcohol did not exhibited any antibacterial activity (Table 2)

Table 2: Antibacterial activity of alcoholic extract of M. oleifera plant parts on Penicillin resistant Staph.aureus (Laboratory Stock Culture)

Treatment Negative Weak Moderate Sensitive TotalLeaves 100 100Bark 22 42.78 35 0.5 100Root 1 14 64.95 20.10 10095 ethyl alcohol 100 100

* Values expressed in percentage

Aqueous extracts of the leaves on the hospital organism still exhibited 100% strong anti-bacterial activity. Majority of the roots extract and bark extract showed negative anti-bacterial activity. (Table 3)

Table 3: Antibacterial activity of alcoholic extract of M. oleifera plant parts on Penicillin resistant Staph.aureus (Laboratory Stock)

Treatment Negative Weak Moderate Sensitive Total

Leaves 100 100Bark 100 100Root 81 14 5 100

* Values expressed in percentage

In the laboratory isolate, the aqueous extracts of the leaves demonstrated 100% strong antibacterial activity. Majority of the aqueous extract of the roots, 61% (118 plates) showed negative antibacterial activity, only 2.5% (5 plates) exhibited strong anti-bacterial activity, while 19.5 (38 plates) moderate anti-bacterial activity, and 17% (33 plates) showed weak anti-bacterial activity. The aqueous extract of the bark did not exhibit any anti-bacterial activity at all. (Table 4)

Treatment Negative Weak Moderate Sensitive Total

Leaves 100 100

Bark 62.7 26 11.3 100

Root 8.7 26% 56.3 9 100

95 ethyl alcohol 100 100

41

Table 4: Antibacterial activity of alcoholic extract of M. oleifera parts on Penicillin resisitant Staph.aureus (Laboratory Stock Culture

Treatment Negative Weak Moderate Sensitive Total

Leaves 100 100Bark 100 100 Root 61 17 19.5 2.5 100

* Values expressed in percentage

The proportion of the plates that showed strong antibacterial activity of the different Malunggay plant parts extracts were compared using the Chi-square test. The test showed significant difference in the anti-bacterial activity between the alcohol and aqueous extracts of the leaves, bark and roots of malunggay plant parts both in the hospital and laboratory cultures. (With alpha set at 5%, the p value was 0.00001).

However, the alcohol and aqueous extracts of the Malunggay leaves showed no difference in the anti-bacterial activity both in hospital and laboratory stock culture. They both showed a strong anti-bacterial activity against Penicillin-resistant Staphylococcus aureus.

Discussion

The results of this study showed that both alcoholic and aqueous extract of Moringa oleifera has a strong anti-bacterial activity against Penicillin-resistant Staphylococcus aureus as compared to that of the bark and roots. Although there were no previous studies of the anti-bacterial activity of Moringa oleifera plant parts extracts on Penicillin-resistant Staphylococcus aureus, our study confirms the results of Duero, Generoso Pedro of MCU, Manila that both the alcoholic and aqueous extracts of Moringa oleifera leaves has strong anti-bacterial activity against Staphylococcus aureus. It also reaffirms the results of the study of Masilungan and Maranon et al. that the bacteria belonging to the Micrococaceae family such as Micrococcus aureus and staphylococcus aureus are sensitive to the antibacterial activity of both the alcoholic and aqueous M. oleifera leaves extract. The said study, however, also showed that the flowers and fruits had a strong anti-bacterial activity against Micrococcus aureus, but such plant parts were not used in this study, because of seasonal availability.

The findings of Olmedo Adolfo of MCU, Manila was different from our results. His study

concluded that Staphylococcus aureus were resistant to both the alcoholic and aqueous extracts of Moringa oleifera leaves. The difference in the results could be due to the extract that was used. The Olmedo study used fresh extracts of the plant, while our study showed that using the fresh extracts would not exhibit any antibacterial activity. Even when the stored extracts were used on the 5th day, only 60% of the leaves showed a strong anti-bacterial activity. It was not until the tenth to the fourteenth day that 100% of the leaves showed anti-bacterial activity.

The result of this study is also in agreement with the study of Sahar, Kafi and Haitham, in which the results showed that the alcoholic and aqueous (water) extract of M. oleifera leaves exhibited strong antimicrobial activity against Staphylococcus auerus at 500mg/ml concentration. Their study also showed that the alcoholic leaves extract exhibited a strong antibacterial activity against Methicillin Resistant S.auerus,22 however our study did not use such organism.

Although this study was not concerned with the determination of the active substance in the leaves that is responsible for the strong anti-bacterial activity against Penicillin-resistant Staphylococcus aureus. The antimicrobial activity of the leaves may be attributed to the presence of an array of phytochemicals. as identified by Bukar, Suarez, Entenza and other authors. Most importantly, the presence of a short polypeptide named 4 (ά– L –rhamnosyloxy) benzyl- isothiocyanate in M. oleifera. They postulated that the peptide may act directly on microorganisms that will result in growth inhibition by disrupting cell membrane synthesis or synthesis of essential enzymes 2, 11,21, 23

Conclusion

Based on the findings of this study, it is the Moringa oleifera leaves alcoholic and aqueous extract that demonstrated a strong anti-bacterial activity on Penicillin-resistant Staphylococcus aureus. However, using the fresh extracts of the plant would not show any anti-bacterial activity. It is the refrigerated stored leaves extract that showed strong inhibitory activity against Penicillin-resistant Staphylococcus aureus.

Recommendations

Future studies should include determination of the active substance responsible for the antibacterial activity of the leaves. Furthermore, the stability of the extracts at room temperature should be tested and the Minimum inhibitory concentration of both the alcoholic and aqueous leave extract on Penicillin resistant S.aureus should be evaluated.

Literature Cited

1. Bauer, A.W.; Kirby, W.M.M.; Sheriss J.C. et al. Antibiotics susceptibility testing by a single disc method. American Journal of Clinical Pathology. 1966; 45-493.

2. Bukar, A., Uba, A. and Oyeyi, T. I. (2010). Antimicrobial profile of Moringa oleifera lam. Extracts Against some food – borne microorganisms, Bayero Journal of Pure and Applied Sciences, 3(1): 43 – 48. [19] Suarez, M., Entenza, J.M. and Dorries, C. (2003): Expres

3. Cáceres A, Cabrera O, Morales O, Mollinedo P, Mendia P. Pharmacological Propertiesof Moringa oleifera. 1: Preliminary screening for antimicrobial activity. JEthnopharmacol, 1991; 33:213-6.

4. Clark, W.; Brater D. C., Johnson A. Goths Medical Pharmacology. Mosby year Book Inc.13th Ed.; 1992; 58-652.

5. Duero, Generosa Puerto, The Antibacterial Activity of the leaves of Moringa oleifera Lam. MCU Manila, 1984: Imprint, PCHRD, MRN 003063, HRN-P890835.

6. Eduardo Quisumbing. Medicinal Plants of the Philippines. Katha Publishing Co. Inc.1978; 346-349

7. Eilert, V. Walters, B; Nahrsted, A. The Antibiotics Principles of Seeds of Moringa oleifera Lam and Moringa stenopetata, Plants Medica. 1981 42: 55-61.

8. Forbes, B. A; Sahm, D.F; Weissfeld, A.S.: Scott and Bailey. Diagnostic Microbiology. (2007) 12TH Ed..

9. Harvey M. (2005). Moringa leaf powder - The world’s greatest unknown supplement. pp. 23-34.

10. Jawetz, Melnick, & Adelberg's (2012) Medical Microbiology 26th edition .Lange McGraw-Hill Publishing Co.

11. Karthika1,S., Ravishankar, M., Mariajancyrani, J., and Chandramohan, G. (2013): Study on phytoconstituents from Moringa oleifera leaves. Asian Journal of Plant Science and Research, 2013, 3(4):63-69

12. Katzung, Betram G, Masters Susan ,Trevor ,

Anthony (2012) Basic and Clinical Pharmacology. 12th Ed. Lange Medical Publications.

13. Khesorn, N. (2006). Antimicrobial activity of the capsules of Moringa oleifera (Moringaceae). CMU J. 5(3): 365-368.

14. Marcu M. G. (2004). Ideal food for obese and malnourishment. Pp 67-89.

15. Masilungan, V. Maranon, J. Valencia V. et al. Screening of the Philippines Higher Plants for Antibacterial Substance. The Philippines Journal of Science. Vol. 84, No. 3. September 1955; 275-299.

16. Merill E. Flora of Manila. Bureau of Printing Manila. Xl+498

17. Moreillon P. and Mermod N. (2005). Structure-function characterization and optimization of a plant-derived antibacterial peptide. Pp 3-6.

18. Nickon, F., Saud, Z.A., Rehman, M.H. and Haque, M.E. (2003) In vitro antimicrobial activity of the compound isolated from chloroform extract of M. oleifera Lam. Pak. J. Biol. Sci 22:1888 -1890

19. Ocampo, Leonila. Compilation of Phytochemical, Microbiological and Pharmacological Screening of Medicinal Plants. College of Pharmacy, Southwestern University. November 1995; p5; p34

20. Olmedo, Adolfo, Phytochemical and Microbiological Analysis of Moringa oliefera Lam (Malunggay). MCU, Manila 1981: Imprint, PCHRD, MRN-003285. HRN-P900037.

21. Rebecca, H.S.U., Sharon, M., Arbainsyah, A. and Lucienne, D. (2006): Moringa oleifera: medicinal and socio-economic uses.

42

Recommendations

Future studies should include determination of the active substance responsible for the antibacterial activity of the leaves. Furthermore, the stability of the extracts at room temperature should be tested and the Minimum inhibitory concentration of both the alcoholic and aqueous leave extract on Penicillin resistant S.aureus should be evaluated.

Literature Cited

1. Bauer, A.W.; Kirby, W.M.M.; Sheriss J.C. et al. Antibiotics susceptibility testing by a single disc method. American Journal of Clinical Pathology. 1966; 45-493.

2. Bukar, A., Uba, A. and Oyeyi, T. I. (2010). Antimicrobial profile of Moringa oleifera lam. Extracts Against some food – borne microorganisms, Bayero Journal of Pure and Applied Sciences, 3(1): 43 – 48. [19] Suarez, M., Entenza, J.M. and Dorries, C. (2003): Expres

3. Cáceres A, Cabrera O, Morales O, Mollinedo P, Mendia P. Pharmacological Propertiesof Moringa oleifera. 1: Preliminary screening for antimicrobial activity. JEthnopharmacol, 1991; 33:213-6.

4. Clark, W.; Brater D. C., Johnson A. Goths Medical Pharmacology. Mosby year Book Inc.13th Ed.; 1992; 58-652.

5. Duero, Generosa Puerto, The Antibacterial Activity of the leaves of Moringa oleifera Lam. MCU Manila, 1984: Imprint, PCHRD, MRN 003063, HRN-P890835.

6. Eduardo Quisumbing. Medicinal Plants of the Philippines. Katha Publishing Co. Inc.1978; 346-349

7. Eilert, V. Walters, B; Nahrsted, A. The Antibiotics Principles of Seeds of Moringa oleifera Lam and Moringa stenopetata, Plants Medica. 1981 42: 55-61.

8. Forbes, B. A; Sahm, D.F; Weissfeld, A.S.: Scott and Bailey. Diagnostic Microbiology. (2007) 12TH Ed..

9. Harvey M. (2005). Moringa leaf powder - The world’s greatest unknown supplement. pp. 23-34.

10. Jawetz, Melnick, & Adelberg's (2012) Medical Microbiology 26th edition .Lange McGraw-Hill Publishing Co.

11. Karthika1,S., Ravishankar, M., Mariajancyrani, J., and Chandramohan, G. (2013): Study on phytoconstituents from Moringa oleifera leaves. Asian Journal of Plant Science and Research, 2013, 3(4):63-69

12. Katzung, Betram G, Masters Susan ,Trevor ,

Anthony (2012) Basic and Clinical Pharmacology. 12th Ed. Lange Medical Publications.

13. Khesorn, N. (2006). Antimicrobial activity of the capsules of Moringa oleifera (Moringaceae). CMU J. 5(3): 365-368.

14. Marcu M. G. (2004). Ideal food for obese and malnourishment. Pp 67-89.

15. Masilungan, V. Maranon, J. Valencia V. et al. Screening of the Philippines Higher Plants for Antibacterial Substance. The Philippines Journal of Science. Vol. 84, No. 3. September 1955; 275-299.

16. Merill E. Flora of Manila. Bureau of Printing Manila. Xl+498

17. Moreillon P. and Mermod N. (2005). Structure-function characterization and optimization of a plant-derived antibacterial peptide. Pp 3-6.

18. Nickon, F., Saud, Z.A., Rehman, M.H. and Haque, M.E. (2003) In vitro antimicrobial activity of the compound isolated from chloroform extract of M. oleifera Lam. Pak. J. Biol. Sci 22:1888 -1890

19. Ocampo, Leonila. Compilation of Phytochemical, Microbiological and Pharmacological Screening of Medicinal Plants. College of Pharmacy, Southwestern University. November 1995; p5; p34

20. Olmedo, Adolfo, Phytochemical and Microbiological Analysis of Moringa oliefera Lam (Malunggay). MCU, Manila 1981: Imprint, PCHRD, MRN-003285. HRN-P900037.

21. Rebecca, H.S.U., Sharon, M., Arbainsyah, A. and Lucienne, D. (2006): Moringa oleifera: medicinal and socio-economic uses.

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International Course on Economic Botany. National Herbarium Leiden, Netherlands. pp.2 – 6.

22. Sahar, M. Khier; Kafi, S.K.; Haitham,Elbir: The antimicrobial activity and phyto chemical characteristics of Moringa oleifera Seeds,

Leaves and Flowers (2014) World Journal of Pharmaceutical Research Vol 4, pp. 258-271

23. Suarez, M., Entenza, J.M. and Dorries, C. (2003): Expression of a plant derived peptide harbouring water – cleaning and antimicrobial activities. Biotechnol. Bioeng. 81:13 – 20

Anti-diabetic Polysaccharide from a Mangrove plant, Sonneratia alba Obtained by a Reverse-Phase (C-18) Chromatography

Jose M. Oclarit1 and Nancy J. Morada2 1Department of Biochemistry, SWU-Matias H. Aznar Memorial College of Medicine

Redemptorist Plaza, Camputhaw, Cebu City 6000 Philippines

2Department of Biology, Mountain View College Valencia City, Bukidnon

Abstract

Mangrove trees have high potential in marine natural products being abundant in many shorelines all over the tropical regions of the globe. The study on anti-diabetic compound was conducted with intense interest using a mangrove species Sonneratia alba because its close relative terrestrial plant, Lagerstromia speciosa had previously shown many anti-diabetic properties. Molecular markers are said to be similar among closely related species belonging to the same family or taxon, hence, this concept was used as the framework of the study (Cohen et al., 2008). Isolation proceeds using several chromatographic techniques: (1) gravity column chromatography(GCC) both stepwise gradient and isocratic elution, and (2) high performance liquid chromatography (HPLC) with isocratic elution. First, the air dried leaf samples of S. alba (900 g) were obtained and were cut into smaller pieces and soaked with methanol (3.75 L) for a period of 4-7 days. Supernatant liquid were dried in vacuo using a Rotary Evaporator (BUCHI R110) and residues were then reconstituted to a small volume (ca. 100 mL) and loaded to silica column (9.7 cm x 15 cm). The column was eluted following step-wise gradient with the following concentrations: 50%, 70%, 90% and 100% methanol: water, respectively. Elution is 2x its column volume. Each fraction was collected separately and was chemically assayed for the presence of tannins using colorimetric method.

Majority of the tannins were detected on fractions that were eluted with 50:50 methanol:water (v/v) and so this was then evaporated to dryness and loaded again to silica column with isocratic elution of 60:40 (water:methanol v/v ratio). Fractions that were positive for the presence of tannins were collected and then dried for further purification. To remove non-polar components, liquid partitioning followed

using methanol: water:ethyl-acetate (5:25:70, respectively, v/v ratio). Tannins which were recovered in the polar fractions were loaded onto the High Performance Liquid Chromatograph (HPLC) apparatus detected at 254 nm using a Reverse Phase Column (Synerg 4u Hydro-RP80A, 250 x 10 mm 4u SN: 348934-4). There were about ten (10 major peaks that were observed (see Fig. 1).

Figure 1. HPLC Chromatogram of polar fractions obtained from isocratic aqueous sub-fraction of S. alba extract ran under silica gravity column developed with 50:50 (methanol: water) at GCC Column dimension 15 cm x 9. Peak 1 was singled out for analysis because majority of the residues were recovered in this fraction as evident in the chromatogram. It has a retention time of 3.05 minutes or a retention volume of 9.15 mL. Based on this chromatographic movement, the molecule is moving too fast along the C18 gel hence frontal in character and therefore doubtful about its purity. However, when it was subjected to LC Chromatograph it was found out to

Date Received: December 10, 2014 Date Accepted: January 12, 2015

44

Date Received: December 10, 2014 Date Accepted: January 12, 2015

Anti-diabetic Polysaccharide from a Mangrove plant, Sonneratia alba Obtained by a Reverse-Phase (C-18) Chromatography

Jose M. Oclarit1 and Nancy J. Morada2 1Department of Biochemistry, SWU-Matias H. Aznar Memorial College of Medicine

Redemptorist Plaza, Camputhaw, Cebu City 6000 Philippines

2Department of Biology, Mountain View College Valencia City, Bukidnon

Abstract

Mangrove trees have high potential in marine natural products being abundant in many shorelines all over the tropical regions of the globe. The study on anti-diabetic compound was conducted with intense interest using a mangrove species Sonneratia alba because its close relative terrestrial plant, Lagerstromia speciosa had previously shown many anti-diabetic properties. Molecular markers are said to be similar among closely related species belonging to the same family or taxon, hence, this concept was used as the framework of the study (Cohen et al., 2008). Isolation proceeds using several chromatographic techniques: (1) gravity column chromatography(GCC) both stepwise gradient and isocratic elution, and (2) high performance liquid chromatography (HPLC) with isocratic elution. First, the air dried leaf samples of S. alba (900 g) were obtained and were cut into smaller pieces and soaked with methanol (3.75 L) for a period of 4-7 days. Supernatant liquid were dried in vacuo using a Rotary Evaporator (BUCHI R110) and residues were then reconstituted to a small volume (ca. 100 mL) and loaded to silica column (9.7 cm x 15 cm). The column was eluted following step-wise gradient with the following concentrations: 50%, 70%, 90% and 100% methanol: water, respectively. Elution is 2x its column volume. Each fraction was collected separately and was chemically assayed for the presence of tannins using colorimetric method.

Majority of the tannins were detected on fractions that were eluted with 50:50 methanol:water (v/v) and so this was then evaporated to dryness and loaded again to silica column with isocratic elution of 60:40 (water:methanol v/v ratio). Fractions that were positive for the presence of tannins were collected and then dried for further purification. To remove non-polar components, liquid partitioning followed

using methanol: water:ethyl-acetate (5:25:70, respectively, v/v ratio). Tannins which were recovered in the polar fractions were loaded onto the High Performance Liquid Chromatograph (HPLC) apparatus detected at 254 nm using a Reverse Phase Column (Synerg 4u Hydro-RP80A, 250 x 10 mm 4u SN: 348934-4). There were about ten (10 major peaks that were observed (see Fig. 1).

Figure 1. HPLC Chromatogram of polar fractions obtained from isocratic aqueous sub-fraction of S. alba extract ran under silica gravity column developed with 50:50 (methanol: water) at GCC Column dimension 15 cm x 9. Peak 1 was singled out for analysis because majority of the residues were recovered in this fraction as evident in the chromatogram. It has a retention time of 3.05 minutes or a retention volume of 9.15 mL. Based on this chromatographic movement, the molecule is moving too fast along the C18 gel hence frontal in character and therefore doubtful about its purity. However, when it was subjected to LC Chromatograph it was found out to

Date Received: December 10, 2014 Date Accepted: January 12, 2015

45

be 99% pure as evidenced by a single peak shown in Fig. 2.

Fig. 2. LC- data of Peak No. 1 (active fraction) showing homogenous and pure fraction after running at Reverse Phase Column with Acetonitrile:Water (30:70 w/w).

Several elutions were tested ranging from 20% to 70% acetonitrile in water moving along the Reverse Phase (C18) gel with dimensions described above. After several trials, the best chromatographic separation was obtained at 30% acetonitrile in water, detected at 254 nm. This isolate was then subjected to proton-Nuclear Magnetic Resonance spectrometry (H-NMR) to identify the whole nuclear molecule. Based on the NMR data the molecule is a complex polysaccharide as shown in Fig. 3 with peaks observed at 3.4 to 4.5 ppm deuterated methanol as solvent matrix.

Fig. 3. Proton-NMR data showing peaks indicating that the pure substance from Peak No. 1 is a polysaccharide (peaks revealed from 3.4 tp 4.5 ppm) Peak 1 was subjected to anti-diabetic bioassay using standard Glucometer (SIMPDO). Data showed that it has tremendous blood glucose attenuating activity because it has reduced significantly the sugar level by about 19.2% during the first 6 hours and reduced further to 66.9% after 12 hours which is consistent with the results of Selvamani et al., (2008). Therefore, we conclude that this polysaccharide molecule obtained from S. alba leaf extracts has a very effective anti-diabetic property and it was effectively isolated using a reverse phase column C18 gel with highly polar eluent. Literature Cited Cohen, M.C.L.; J. T. F. Guimara; M. Fran; R. J. Lara

and H. Behling. 2008. Tannin as an indicator of paleomangrove in sediment cores from Amapa, Northern Brazil. Springer DOI 10.1007/s11273-008-9100.

Selvamani, P.; S. Latha; K. Elayaraja; P.S. Babu; J.K.

Gupta; T.K. Pal; L.K. Gosh and D. J. Sen. 2008. Anti-diabetic activity of the ethanol extract of Capparis sepiaria leaves. Indian J Pharm Sci 70: 378-80

Acknowledgment: This research is made possible through grants from the Commission on Higher Education Scholarship, Mountain View College, Center for Biomolecular Sciences and the Center for Research and the Crews Laboratory of Natural Products Chemistry of the University of California-Santa Cruz, California, U.S.A.

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MHAM MEDICAL RESEARCH JOURNAL: Basic and Clinical SciencesA Research Journal on Basic Medical and Clinical Sciences of the

Matias H Azanar Memorial College of MedicineSouthwestern University

Cebu City

GUIDE TO AUTHORS

IMPORTANT INFORMATION • Submission of a paper will be held to imply that the manuscript contains original unpublished work and is not being submitted for publication elsewhere. Manuscripts should present novel findings addressing significant medical questions. Studies that fail to do so may be rejected without review. Quantitative conclusions must be based on truly quantitative methods.

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the Journal Editors of any such requests and (2) publication of the accepted manuscript in an online issue is suspended until authorship has been agreed. After the accepted manuscript is published in an online issue: Any requests to add, delete, or rearrange author names in an article published in an online issue will follow the same policies as noted above and result in a corrigendum.

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Introduction State the objectives of the work and provide an adequate background, avoiding a detailed literature survey or a summary of the results.

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version. All instructions for proofing will be given in the e-mail we send to authors, including alternative methods to the online version and PDF. We will do everything possible to get your article published quickly and accurately - please upload all of your corrections within 48 hours. It is important to ensure that all corrections are sent back to us in one communication. Please check carefully before replying, as inclusion of any subsequent corrections cannot be guaranteed. Proofreading is solely your responsibility.

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Guidelines on Writing a Research Proposal

Introduction

This is a guide to writing research proposal in Pharmacology. The same principles apply to dissertation proposals and to proposals to most funding agencies

Proposal Writing

Proposal writing is important to your pursuit of a graduate degree. The proposal is, in effect, an intellectual scholastic (not legal) contract between you and your committee. It specifies what you will do, how you will do it, and how you will interpret the results. In specifying what will be done it also gives criteria for determining whether it is done. In approving the proposal, your committee gives their best judgment that the approach to the research is reasonable and likely to yield the anticipated results. They are implicitly agreeing that they will accept the result as adequate for the purpose of granting a degree. (Of course you will have to write the thesis in acceptable form, and you probably will discover things in the course of your research that were not anticipated but which should be addressed in your thesis, but the minimum core intellectual contribution of your thesis will be set by the proposal.) Both parties benefit from an agreed upon plan.

The objective in writing a proposal is to describe what you will do, why it should be done, how you will do it and what you expect will result. Being clear about these things from the beginning will help you complete your thesis in a timely fashion. A vague, weak or fuzzy proposal can lead to a long, painful, and often unsuccessful thesis writing exercise. A clean, well thought-out, proposal forms the backbone for the thesis itself. The structures are identical and through the miracle of word-processing, your proposal will probably become your thesis.

A good thesis proposal hinges on a good idea. Once you have a good idea, you can draft the proposal in an evening. Getting a good idea hinges on familiarity with the topic. This assumes a longer preparatory period of reading, observation, discussion, and incubation. Read everything that you can in your area of interest. Figure out what are the important and missing parts of our understanding. Figure out how to build/discover those pieces. Live and breath the topic. Talk about it with anyone who is interested. Then just write the important parts as the proposal. Filling in the things that we do not know and that will help us know more: that is what research is all about.

Proposals help you estimate the size of a project. Don’t make the project too big. Our pharmacology research program statement used to say that a thesis is equivalent to a published paper in scope. These days, sixty double spaced pages, with figures, tables and bibliography, would be a long paper. Your proposal will be shorter, perhaps five pages and certainly no more than fifteen pages. (For perspective, the NSF limits the

length of proposal narratives to 15 pages, even when the request might be for multiple hundreds of thousands of dollars.) The merit of the proposal counts, not the weight. Shoot for five pithy pages that indicate to a relatively well-informed audience that you know the topic and how its logic hangs together, rather than fifteen or twenty pages that indicate that you have read a lot of things but not yet boiled it down to a set of prioritized linked questions.

Different Theses, Similar Proposals

This guide includes an outline that looks like a “fill-in the blanks model” and, while in the abstract all proposals are similar, each proposal will have its own particular variation on the basic theme. Each research project is different and each needs a specifically tailored proposal to bring it into focus. Different advisors, committees and agencies have different expectations and you should find out what these are as early as possible; ask your advisor for advice on this. Further, different types of thesis require slightly different proposals. What style of work is published in your sub-discipline?

Characterizing theses is difficult. Some thesis are “straight science”. Some are essentially opinion pieces. Some are policy oriented. In the end, they may well all be interpretations of observations, and differentiated by the rules that constrain the interpretation. (Different advisors will have different preferences about the rules, the meta-discourse, in which we all work.)

In the abstract all proposals are very similar. They need to show a reasonably informed reader why a particular topic is important to address and how you will do it. To that end, a proposal needs to show how your work fits into what is already known about the topic and what new contribution your work will make. Specify the question that your research will answer, establish why it is a significant question, show how you are going to answer the question, and indicate what you expect we will learn. The proposal should situate the work in the literature, it should show why this is an (if not the most) important question to answer in the field, and convince your committee (the skeptical readers that they are) that your approach will in fact result in an answer to the question.

Theses which address research questions that can be answered by making plan-able observations (and hypothesis testing) are preferred and perhaps the easiest to write. Because they address well-bounded topics, they can be very tight, but they do require more planning on the front end. Theses which are largely based on synthesis of observations, rumination, speculation, and opinion formation are harder to write, and usually not as convincing, often because they address questions which are not well-bounded and essentially unanswerable. (One ‘old saw’ about research in the social sciences is that the finding

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is always: “some do and some don’t”. Try to to avoid such insight-less findings.) One problem with this type of project is that it is often impossible to tell when you are “done”. Another problem is that the nature of argument for a position rather than the reasoned rejection of alternatives to it encourages shepherding a favored notion rather than converging more directly toward a truth. (A good proposal is a boon here, too.)

Literature review-based theses involve collection of information from the literature, distillation of it, and coming up with new insight on an issue. One problem with this type of research is that you might find the perfect succinct answer to your question on the night before (or after) you turn in the final draft --- in someone else’s work. This certainly can knock the wind out of your sails. (But note that even a straight-ahead science thesis can have the problem of late in the game discovering that the work you have done or are doing has already been done, this is where familiarity with the relevant literature by both yourself and your committee members is important.)

A Couple of Models for Proposals

A Two Page (Preliminary Proposal) Model

Here is a model for a very brief (maybe five paragraph) proposal that you might use to interest faculty in sitting on your committee. People who are not yet hooked may especially appreciate its brevity.

In the first paragraph, the first sentence identifies the general topic area. The second sentence gives the research question, and the third sentence establishes its significance.

The next couple of paragraphs gives the larger historical perspective on the topic. Essentially list the major schools of thought on the topic and very briefly review the literature in the area with its major findings. Who has written on the topic and what have they found? Allocate about a sentence per important person or finding. Include any preliminary findings you have, and indicate what open questions are left. Restate your question in this context, showing how it fits into this larger picture.

The next paragraph describes your methodology. It tells how will you approach the question, what you will need to do it.

The final paragraph outlines your expected results, how you will interpret them, and how they will fit into the our larger understanding

The (Longer) Standard Model

The two outlines below are intended to show both what are the standard parts of a proposal and of a science paper. Notice that the only real difference is that you change “expected results” to “results” in the paper, and usually leave the budget out, of the paper.

A Basic Proposal Outline:

Introduction Topic area Research question Significance to knowledge Literature review Previous research others & yours Interlocking findings and Unanswered questions Your preliminary work on the topic The remaining questions and inter-locking logic Reprise of your research question(s) in this contextMethodology Approach Data needs Analytic techniques Plan for interpreting resultsExpected resultsBudgetBibliography (or References) -------------------------------------------------------------------------Another Model:The Basic Thesis Outline

Introduction Topic area Research question (finding?) Significance to knowledge Literature review Previous research others & yours Interlocking findings and Unanswered questions Your preliminary work on the topic The remaining questions and inter-locking logic Reprise of your research question(s) in this contextMethodology Approach Data needs Analytic techniques Plan for interpreting resultsResultsDiscussion and ConclusionsBibliography

Another Model of Outline:

Introduction Topic area Research Question and its significance to knowledge Literature review Previous research Your preliminary work on the topic The remaining questions and their inter-locking logic Reprise of your resulting question in this contextMethodology Approach to answering the question Data needs Analytic techniques Plan for interpreting resultsBudgetExpected resultsBibliography / References

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Each of these outlines is very similar. You probably see already that the proposal’s organization lends itself to word-processing right into the final thesis. It also makes it easy for readers to find relevant parts more easily. The section below goes into slightly more detail on what each of the points in the outline is and does.

The Sections of the Proposal

The Introduction

Topic Area

A good title will clue the reader into the topic but it can not tell the whole story. Follow the title with a strong introduction. The introduction provides a brief overview that tells a fairly well informed (but perhaps non-specialist) reader what the proposal is about. It might be as short as a single page, but it should be very clearly written, and it should let one assess whether the research is relevant to their own. With luck it will hook the reader’s interest.

What is your proposal about? Setting the topical area is a start but you need more, and quickly. Get specific about what your research will address.

Question

Once the topic is established, come right to the point. What are you doing? What specific issue or question will your work address? Very briefly (this is still the introduction) say how you will approach the work. What will we learn from your work?

Significance

Why is this work important? Show why this is it important to answer this question. What are the implications of doing it? How does it link to other knowledge? How does it stand to inform policy making? This should show how this project is significant to our body of knowledge. Why is it important to our understanding of the world? It should establish why I would want to read on. It should also tell me why I would want to support, or fund, the project.

Literature Review

State of our knowledge

The purpose of the literature review is to situate your research in the context of what is already known about a topic. It need not be exhaustive, it needs to show how your work will benefit the whole. It should provide the theoretical basis for your work, show what has been done in the area by others, and set the stage for your work.

In a literature review you should give the reader enough ties to the literature that they feel confident that you have found, read, and assimilated the literature in the field. It might do well to include a paragraph that summarizes each article’s

contribution, and a bit of ‘mortar’ to hold the edifice together, perhaps these come from your notes while reading the material. The flow should probably move from the more general to the more focused studies, or perhaps use historical progression to develop the story. It need not be exhaustive; relevance is ‘key’.

Outstanding questions

This is where you present the holes in the knowledge that need to be plugged, and by doing so, situate your work. It is the place where you establish that your work will fit in and be significant to the discipline. This can be made easier if there is literature that comes out and says “Hey, this is a topic that needs to be treated! What is the answer to this question?” and you will sometimes see this type of piece in the literature. Perhaps there is a reason to read old AAG presidential addresses.

Research Questions in Detail

Your work to date

Tell what you have done so far. It might report preliminary studies that you have conducted to establish the feasibility of your research. It should give a sense that you are in a position to add to the body of knowledge.

Methodology

Overview of approach

This section should make clear to the reader the way that you intend to approach the research question and the techniques and logic that you will use to address it.

Data Collection

This might include the field site description, a description of the instruments you will use, and particularly the data that you anticipate collecting. You may need to comment on site and resource accessibility in the time frame and budget that you have available, to demonstrate feasibility, but the emphasis in this section should be to fully describe specifically what data you will be using in your study. Part of the purpose of doing this is to detect flaws in the plan before they become problems in the research.

Data Analysis

This should explain in some detail how you will manipulate the data that you assembled to get at the information that you will use to answer your question. It will include the statistical or other techniques and the tools that you will use in processing the data. It probably should also include an indication of the range of outcomes that you could reasonably expect from your observations.

Interpretation

In this section you should indicate how the anticipated outcomes will be interpreted to answer the research question. It

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is extremely beneficial to anticipate the range of outcomes from your analysis, and for each know what it will mean in terms of the answer to your question.

Expected Results

This section should give a good indication of what you expect to get out of the research. It should join the data analysis and possible outcomes to the theory and questions that you have raised. It will be a good place to summarize the significance of the work.

It is often useful from the very beginning of formulating your work to write one page for this section to focus your reasoning as you build the rest of the proposal.

Bibliography

This is the list of the relevant works. Some advisors like exhaustive lists. I think that the Graduate Division specifies that you call it “Bibliography”. Others like to see only the literature which you actually cite. Most fall in between: there is no reason to cite irrelevant literature but it may be useful to keep track of it even if only to say that it was examined and found to be irrelevant.

Use a standard format. Order the references alphabetically, and use “flag” paragraphs as per the University’s Guidelines.

Tips and Tricks

Read. Read everything you can find in your area of interest. Read. Read. Read. Take notes, and talk to your advisor about the topic. If your advisor won’t talk to you, find another one or rely on ‘the net’ for intellectual interaction. Email has the advantage of forcing you to get your thoughts into written words that can be refined, edited and improved. It also gets time stamped records of when you submitted what to your advisor and how long it took to get a response.

Write about the topic a lot, and don’t be afraid to tear up (delete) passages that just don’t work. Often you can re-think and re-type faster than than you can edit your way out of a hopeless mess. The advantage is in the re-thinking.

Very early on, generate the research question, critical observation, interpretations of the possible outcomes, and the expected results. These are the core of the project and will help focus your reading and thinking. Modify them as needed as your understanding increases.

Use some systematic way of recording notes and bibliographic information from the very beginning. The classic approach is a deck of index cards. You can sort, regroup, layout spatial arrangements and work on the beach. Possibly a slight improvement is to use a word-processor file that contains bibliographic reference information and notes, quotes etc. that you take from the source. This can be sorted, searched, diced

and sliced in your familiar word-processor. You may even print the index cards from the word-processor if you like the ability to physically re-arrange things.

Even better for some, is to use specialized bibliographic database software. Papyrus, EndNote, and other packages are available for PCs and MacIntoshs. The bib-refer and bibTex software on UNIX computers are also very handy and have the advantage of working with plain ASCII text files (no need to worry about getting at your information when the wordprocessor is several generations along). All of these tools link to various word-processors to make constructing and formating your final bibliography easier, but you won’t do that many times anyway. If they help you organize your notes and thinking, that is the benefit.

Another pointer is to keep in mind from the outset that this project is neither the last nor the greatest thing you will do in your life. It is just one step along the way. Get it done and get on with the next one. The length to shoot for is “equivalent to a published paper”, sixty pages of double spaced text, plus figures tables, table of contents, references, etc. is probably all you need. In practice, most theses try to do too much and become too long. Cover your topic, but don’t confuse it with too many loosely relevant side lines.

This is not complete and needs a little rearranging.

The balance between Introduction and Literature Review needs to be thought out. The reader will want to be able to figure out whether to read the proposal. The literature review should be sufficiently inclusive that the reader can tell where the bounds of knowledge lie. It should also show that the proposer knows what has been done in the field (and the methods used).

The balance may change between the proposal and the thesis. It is common, although not really desirable, for theses to make reference to every slightly related piece of work that can be found. This is not necessary. Refer to the work that actually is linked to your study, don’t go too far afield (unless your committee is adamant that you do ;-).

Useful References:

Krathwohl, David R. 1988. How to Prepare a Research Proposal: Guidelines for Funding and Dissertations in the Social and Behavioral Sciences. Syracuse University Press.

Recent National Science Foundations Guidelines for Research Proposals can be found on the NSF website, www.nsf.gov.

Chamberlain, T.C. “The Method of Multiple Working Hypotheses”, reprinted in Science, Vol 148, pp754-759. 7 May 1965.

Platt, J. “Strong Inference” in Science, Number 3642, pp. 347-353, 16 October 1964.

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Strunk and White The Elements of Style

Turabian, Kate. 1955 (or a more recent edition) A Manual for Writers of Term Papers, Theses and Dissertations, University of Chicago Press.

Mortimer J. Adler and Charles Van Doren. 1940 (‘67, ‘72 etc). How to Read a Book. Simon and Schuster Publishers. New York City, NY.

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Officials of the Southwestern University MATIAS H AZNAR MEMORIAL COLLEGE OF MEDICINE

BOARD OF TRUSTEES

Madame Elma Aznar Sierra Chairman/President

Mr. Merelo B. Aznar

Member

Merlo B. Aznar Member

Dr. Carlito Astillero

Member

Mr. Daniel P. Lariba Jr. Member

MEDICAL SCHOOL OFFICIALS

Madame Elma Aznar Sierra President

Mr. Daniel P. Lariba Jr.

Assistant to the President/ Head, Human Resource Department

Mr. Merelo B. Aznar Treasurer

Ms. Rosario Angelica A. Sierra

Comptroller

Dr. Arlene M. Diaz Dean

Dr. Allan Librando

Associate Dean (Basic Sciences)

Dr. Ma. Socorro G. Manaloto Chief of Clinics/ Associate Dean (Clinical Sciences)

Dr. Luditha L. Pe College Registrar

Dr. Yvonne M. Lanuza

College Secretary

Dr. Jose Micabalo Oclarit Director, Department of Research

Mr. Matti H. Aznar Assistant Treasurer

Mr. Merelo H. Aznar

Administrator

Officials of the Southwestern University MATIAS H AZNAR MEMORIAL COLLEGE OF MEDICINE

BOARD OF TRUSTEES

Madame Elma Aznar Sierra Chairman/President

Mr. Merelo B. Aznar

Member

Merlo B. Aznar Member

Dr. Carlito Astillero

Member

Mr. Daniel P. Lariba Jr. Member

MEDICAL SCHOOL OFFICIALS

Madame Elma Aznar Sierra President

Mr. Daniel P. Lariba Jr.

Assistant to the President/ Head, Human Resource Department

Mr. Merelo B. Aznar Treasurer

Ms. Rosario Angelica A. Sierra

Comptroller

Dr. Arlene M. Diaz Dean

Dr. Allan Librando

Associate Dean (Basic Sciences)

Dr. Ma. Socorro G. Manaloto Chief of Clinics/ Associate Dean (Clinical Sciences)

Dr. Luditha L. Pe College Registrar

Dr. Yvonne M. Lanuza

College Secretary

Dr. Jose Micabalo Oclarit Director, Department of Research

Mr. Matti H. Aznar Assistant Treasurer

Mr. Merelo H. Aznar

Administrator