RESEARCH ARTICLE Leaf extract of Momordica charantia

National Journal of Physiology, Pharmacy and Pharmacology 5342021 | Vol 11 | Issue 05

RESEARCH ARTICLE

Leaf extract of Momordica charantia: Effects on liver enzymes, duodenum antioxidant enzymes, and disaccharidases with liver and duodenum histology

Olusoji Adebusoye Oyesola, Ifabunmi Oduyemi Osonuga, Olagboye Emmanuel Taiwo, Ifedolapo Ibukunoluwa-Gloria Owoeye

Department of Physiology, Faculty of Basic Medical Sciences, Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria

Correspondence to: Olusoji Adebusoye Oyesola, E-mail: [email protected]

Received: February 20, 2021; Accepted: March 14, 2021

ABSTRACT

Background: Momordica charantia (MC) plant rich in phytochemicals, promoting sound health conditions, contains nutritional and nutraceutical components. Its popular in traditional and folk’s medicine, extract is used to treat and remedy diseases. Adverse effects produce toxic symptoms in children and laboratory animals. Aims and Objectives: The study investigated activities of liver enzymes, antioxidant enzymes, and disaccharidases in the duodenum and evaluated the histomorphological changes in the duodenum and liver of normal rats orally administered with aqueous leaf extract of MC. Materials and Methods: Fifteen male Wistar rats weight between 150 g and 200 g used for this experiment were divided into three groups equally. Group A received only water and Groups B and C received 100 and 400 mg/kg body weight of extract orally for 21 days. Liver enzymes, duodenum antioxidant enzymes, and disaccharidases were determined using standard methods. Histology of duodenum and liver was studied with hematoxylin and eosin staining. Results: Observation from statistically compared results showed that catalase, superoxide dismutase, and glutathione in the duodenum reduced significantly. Malondialdehyde only increases in Group C but not significant. Maltase, sucrose, and lactase in the duodenum reduced significantly. Total protein, albumin, alanine aminotransferase, and aspartate aminotransferase in the liver reduced significantly, alkaline phosphatase showed increase not significant for the two doses (P < 0.05). Histological study from duodenum and liver showed some degenerated villi, loss of epithelial cell, and goblet cells in the duodenum, central vein constriction with hepatocyte hypertrophy, degeneration, and sinusoid dilation. Conclusion: MC has health benefits with ability to neutralized free radicals, preserve functions of liver and if care is not taken, it can induce negative histomorphological changes in the duodenum and the liver.

KEY WORDS: Duodenum; Enzyme activities; Histomorphology; Liver; Momordica charantia

INTRODUCTION

About 80% of the population of many developing countries over the world continues to use traditional medicine as a primary source of treatment for many medical cases because

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DOI: 10.5455/njppp.2021.11.02069202114032021

National Journal of Physiology, Pharmacy and Pharmacology Online 2021. © 2021 Olusoji Adebusoye Oyesola, et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creative commons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.

of cost or/and unavailability of modern drugs, along with many side effects of prolong use. Therefore, researchers shift focus to scientific evaluation of traditional drugs of plants origin. Momordica charantia (MC), one of such plant, known as bitter melon or bitter gourd belonging to the family of Cucurbitaceae are found grown in the tropical and sub-tropical regions. The leaves and fruits of this plant were found rich in phytochemicals that promote sound health conditions. It contains both nutritional and nutraceutical components. Its popularity is most found in traditional and folk’s medicine.[1,2] The fruits of the plant are commonly used vegetable in different parts of the world. The whole plant

National Journal of Physiology, Pharmacy and Pharmacology

Oyesola et al. Effects of Momordica charantia effects on liver and duodenum

535 National Journal of Physiology, Pharmacy and Pharmacology 2021 | Vol 11 | Issue 05

(especially the seed, fruits, and the leaves) have significant pharmacological effects. Extracts from the plant have been used in treatment, prevention, and remedy of diabetes since ancient times in many developing countries.[3,4] Fraction of MC (fruits, vines, leaves, and roots) has been used in folk’s medicine as remedy of diseases, such as toothache, diarrhea, furuncle, diabetes, dysmenorrhea, eczema, gout, jaundice, leprosy, pile, pneumonia, and psoriasis.

Some pharmacological function of MC include antidiabetic, abortifacient, anthelmintic, contraceptive, antimalarial, laxative rheumatism, and scabies.[3,5,6] Studied medicinal properties of MC include hypoglycemic, antibacterial, antiviral, anti-tumor, immunomodulation, antioxidant, antidiabetes, anthelmintic, antimutagenic, antilipolytic, antifertility, hepatoprotective, and anti-inflammatory activities as well as antiulcerogenic, antioxidative, and immune modulatory activities.[7-9] MC proteins (α- and β-mormocharin) have been confirmed to have inhibitory effect against human immune deficiency virus. Its extract was used as a broad spectrum antibacterial agent to fight infections.[10] Listed beneficial effects are attributed to the various bioactive components of MC, which are important sources of phytoconstituents used to treat various diseases since ancient times.[11]

Many various pharmacological activities have been credited to MC but there are also some adverse effects that have been reported in the past years which may limit its wider application, therefore, there is need to study them. Some toxic symptoms reported in the previous studies include induction of hypoglycemic coma in children, abortion, or even death in laboratory animals.[12] Oyesola et al.[13] reported that prolonged uncontrolled administration of aqueous leaf extract of MC to rats will induce damages to gastric mucosal which was associated with decrease mucus concentration and acid production in the stomach as a result of distortion caused to cellular integrity of the gastric mucosa as well as reduced antioxidant enzymes level.

This study is conducted to evaluate the level of disaccharidases (maltase, lactase, and lactase) and antioxidant enzyme in the duodenum, the level of liver enzyme, and histology of liver and duodenum in normal rats exposed to oral administration of aqueous leaf extract of MC.

MATERIALS AND METHODS

Plant Material Preparation and Extraction

Fresh leaves of MC were obtained from an indigenous farm in Sagamu, Ogun state in the southwest part of Nigeria. The plant was authenticated by a botanist from the Department of Plant Science, Olabisi Onabanjo University, Ago Iwoye, Ogun state, Nigeria. Forestry Research Institute of Nigeria located in Ibadan confirmed it with herbarium number

109921. The plant materials were picked and sorted to eliminate all unwanted materials including the seeds. The dust-free leaves of MC were spread out on racks to dry in a ventilated room away from excessive heat and sunlight. The leaf dried to a constant weight, was grounded into fine powder, and weighed. Extraction procedure described by Mandal et al.[14] and Oben et al.,[15] was modified, 1000 g of the dried leaf was soaked in 5000 ml of distilled water for 3 days under refrigeration. A funnel plugged with glass wool was used to filter the resultant liquid. Further extraction was done with 5000 ml of distilled water 2–3 times until there was no more extractable material from the leaf, judging from the obtained water color. Filtrate aliquot was poured into beakers of known weight and dried in an oven at about 40°C. 4000 mg of the crude leaf extract was dissolved in 100 ml of distilled water to obtained 400 mg/kg dosage. One milliliter of the solution containing 40 mg extract was given to 100 g rat. 1000 mg of the crude leaf extract was dissolved in 100 ml of distilled water to obtained 100 mg/kg dosage. One milliliter of the solution containing 10 mg extract was given to 100 g rat. They were stored in the refrigerator until they were needed.

Animal and Grouping

Fifteen male Wistar rats used for this study were obtained from a reputable animal house in Ibadan, Oyo state, Nigeria. They weighed between 150 g and 200 g. They were housed in standard plastic cages under normal atmospheric condition in the Department of Physiology Animal House, Olabisi Onabanjo University, Ogun state, Nigeria. They acclimatized for 2 weeks before the commencement of the study, they were fed with commercial rat feed that was available. They were supplied with water ad libitum and were randomly distributed into three groups (n = 5). Group A, the control group, received only distilled water, Groups B and C received 100 and 400 mg/kg body weight of aqueous leaf extract of MC, respectively. The administration was done orally and lasted for 21 days. International, national, and institutional rules and guidelines with the use and care of laboratory animals were followed during the study. The ethics committee of the department gave approval for the study.

Determination of Catalase (CAT), Superoxide Dismutase (SOD), Glutathione (GSH), and Malondialdehyde (MDA) Activities in the Duodenum

CAT activity was determine from the duodenum homogenate after centrifugation with the method described by Sinha.[16] Dichromate was reduced in acetic acid to chromic acetate when heated in the presence of H2O2 to form perchromic acid. Absorbance measured colorimetrically at 530 nm. SOD activity was determined by the method of Misra and Fridovich.[17] The method is based on the ability of SOD to inhibit the autoxidation of epinephrine caused by O2 generated by xanthine oxidase reaction. GSH activity was determined



by the method describe by Beutler et al.,[18] gastric proteins were precipitated with 10% trichloroacetic acid, and the colored product was developed by 10 mM 5, 5’-dithiobis 2-nitrobenzoic acid solution in 0.3 M phosphate solution. Absorbance measured at 412 nm. MDA concentration in the duodenum was determined after centrifugation from the tissue homogenate following the modified method of Ohkawa et al.[19] Absorbance was measured at 412 nm.

Determination of Disaccharidases (Maltase, Sucrose, and Lactase) Activities in the Duodenum

Maltase, sucrose, and lactase activities were determined from the freshly prepared duodenum homogenate using the method of Dahlqvist[20] and expressed in units 9 µmol of substrate hydrolyzed per minute at 37°C per mg protein.

Determination of Liver Enzyme (Total Protein [TP], Albumin [Alb], Aspartate Aminotransferase [AST] Alanine Aminotransferase [ALT], and Alkaline Phosphatase [ALP])

The method of Lowry et al.[21] was used to determine protein content, used bovine serum Alb as a standard. Doumas et al.[22] method was used to determine serum Alb concentration. Liver enzymes (AST, ALT, and ALP) activities were determined by the procedure described Kind and King.[23] Ultraviolet–visible spectrophotometer model was used to monitor the absorbance.

Liver and Duodenum Histology

Tissue sample of the liver and duodenum was washed, dehydrated by alcohol, cleared in xylene, and embedded in paraffin. The paraffin blocks were cut into sections of 5 µm thickness and were stained with hematoxylin and eosin (H and E) and examined under the light microscope. This was performed according to the method described by Ogihara and Okabe.[24] Photomicrographs were also taken.

Statistical Analysis

Obtained result was statistically analyzed with the use of SPSS software version 16.0. Results were expressed as mean ± standard error of mean (SEM). Student’s t-test was used to compare differences between animal groups. Significant level was taken at a probability level of 5% (P < 0.05).

RESULTS

The results of the antioxidant enzymes activities (CAT, SOD, and GSH) and lipid peroxidation (MDA) are shown in Table 1 when rats were orally espoused to aqueous leaf extract of MC at dose 100 and 400 mg/kg bwt. Observed results when statistically compared showed decreases in the activities of CAT, SOD, and GSH which were significant (P < 0.05) in Groups B and C when compared with Group A. MDA statistically compared revealed an increase in Group C that was not significant and no difference was recorded in Group B when they were compared with Group A.

Table 2 shows the activities of disaccharidases (maltase, sucrose, and lactase) in the duodenum during oral administration of 100 and 400 mg/kg (Groups B and C) of aqueous leaf extract of MC. Observation across the table showed decreased activities that were significant (P < 0.005) in all the three measured enzymes for Groups B and C when compared with Group A.

Table 3 shows the level of liver enzymes (TP, Alb, AST, ALT, and ALP) in normal rats during oral administration of aqueous leaf extract of MC (100 and 400 mg/kg). Comparison of Group B and C with Group A showed decreases that were significant (P < 0.05) for protein, Alb, and AST. The decrease observed in ALT group was only significant in Group B. Observation from ALP showed increases that were significant for Groups B and C when compared with Group A.

Plates A and B [Figures 1 and 2] are the photomicrographs of duodenum and liver, respectively, with H and E staining ×400: Slide A from plate A is the normal control Group A with blue arrow showing normal villi with simple columnar epithelium and red arrow showed goblet cells, the cellular and extracellular core constituents are present. Slide B from plate A (100 mg/kg concentration of MC – Group B) blue arrow showed degenerating villi with loss of epithelial cell and green arrow showed goblet cells. The cellular and extracellular villous core constituents showed very slight degeneration. Slide C from plate A (400 mg/kg concentration of MC) blue arrow showed fairly degenerated villi with distorted histoarchitecture.

Observation from plate B (liver photomicrograph) showing slide A which is normal control Group A; red arrow showed

Table 1: Antioxidant enzymes activities in the duodenum during oral administration of aqueous extract of Momordica charantia rats

Groups CAT (unit/mg protein) SOD (unit/mg protein) GSH (unit/mg protein) MDA (unit/mg protein)Group A (Control) 23.41±0.67 5.23±0.19 0.75±0.04 0.02±0.003Group B (100 mg/kg MC) 18.91±0.47* 3.53±0.30* 0.12±0.01* 0.02±0.001Group B (400 mg/kg MC) 12.86±0.42* 2.87±0.10* 0.16±0.02* 0.03±0.003CAT: Catalase; SOD: Superoxide dismutase; GSH: Glutathione; MDA: Malondialdehyde; MC: Momordica charantia; Values are expressed as means±SEM (n=5); *values considered significant at P<0.05 when compared with Group A



hepatocytes, green arrow showed sinusoid which are apparently normal. Slide B (100 mg/kg concentration of MC) showed constricted central vein in red arrow, slightly hypertrophied hepatocytes were showed in green arrow and blue arrow showed mild dilated sinusoids. Slide C (400 mg/kg concentration of MC) dilated endothelium of the central vein is showed in red arrow, degenerated hepatocytes are showed in green arrow while blue arrow showed dilated sinusoids.

DISCUSSION

MC has medicinal properties and frequently been used in traditional medicine[25,26] is studied to evaluate its effects on duodenum and liver of normal rats. MC aqueous leaf extract was found to reduce the level of antioxidant enzymes in the duodenum, reduced the level of disaccharidases (maltase, sucrose, and lactase) in the duodenum and reduced the liver enzymes level. The histological study from the duodenum showed villi degeneration with loss of epithelial cell and goblet cell. The histoarchitecture of the duodenum was distorted. Liver histology also showed hepatocytes degeneration with dilated sinusoids.

The results of the antioxidant enzymes from this study are consistent with the known antioxidant properties of MC according to Semiz and Sen.[27] Antioxidant plays an important role to protect the body against damages caused by reactive oxygen species.[28] The endogenous antioxidant enzymes (e.g., SOD, CAT, GSH, and GPx) are responsible for the detoxification of deleterious oxygen radicals.[29] Plant extract and products were reported to contain significant antioxidant

activity,[30] which may be an important property of plant medicines associated with the treatment of many ill-fated diseases including diabetes.[31] Several evidences indicate that oxidative stress play a role in chronic inflammatory disease, which are closely related to pathophysiological processes that can activate each other.[32] MC beneficial properties were found to be dependent on its anti-inflammatory and antioxidant activities.[33,34] The reduced disaccharidases (maltase, sucrose, and lactase) activities in the duodenum were consistent with the report of Chipiti et al.,[35] that one of the major characteristics of drugs in the management of Type 2 diabetes inhibition of carbohydrate digesting enzymes[36] as recorded in this study. This procedure reduced the glucose uptake from the gastrointestinal tract to control hyperglycemic condition by modulating the activities of the disaccharidases.

The reduced liver enzymes (TP, Alb, AST, and ALT level significantly decrease in Groups B and C) activities were in agreement with the results of other researchers that demonstrated hepatoprotective effects of MC.[37,38] However, ALP increases in Groups B and C when compared with the control. However, trace of unsafety can be extrapolated for the liver because at the dose of 400 mg/kg concentration in Group C, TP, Alb, AST, and ALT increased slightly but they were not significant when compared with Group B (100 mg/kg concentration of MC). ALP also showed a slight decrease in Group C, over Group B, still lower than Group A the control group.

The risk of MC consumption was reported by Baldwa et al.,[39] Srivastava et al.,[31] Welihinda et al.,[40] and Fan[41] in association with acute gastric ulcer and intestinal bleeding, given speculation that the toxic bioactive substances producing such effect maybe alkanoids, lectin, or charatin.[42] This is consistent with the observed trend in the histology of duodenum and liver in this study (Plates A and B). Villi degeneration and loss of epithelial and goblet cells were noticed in plate A; slide B, there were degeneration of villi and distortion of histoarchitecture of duodenum in plate A; slide C. The observed trends can be extrapolated for the liver histology in plate B and slides B and C. Liver exhibits hypertrophied hepatocytes with constricted central vein and dilated sinusoid with degenerated hepatocytes, respectively. More recently, Oyesola et al.[13] reported gastric mucosal damage which was related to decrease in mucus concentration of the stomach and decreased acid production from the

Table 2: Disaccharidases activities in the duodenum during oral administration of aqueous extract of

Momordica charantia ratsGroups Maltase

unit/mg protein

Sucrose unit/mg protein

Lactase unit/mg protein

Group A (control)

1.65±0.05 1.87±0.006 1.71±0.03

Group B (100 mg/kg MC)

1.68±0.04* 1.15±0.25* 1.43±0.04*

Group C (400 mg/kg MC)

1.23±0.11* 0.42±0.0002* 1.36±0.02*

MC: Momordica charantia; values are expressed as means±SEM (n=5); *values considered significant at P<0.05 when compared with Group A

Table 3: Level of liver enzymes in normal rats during oral administered with aqueous leaf extract of Momordica charantia ratsGroups TP (mg/dl) Alb (mg/dl) AST (IU/L) ALT (IU/L) ALP (IU/L)Group A (control) 77.5±3.54 43.33 92.00±7.16 26.00±2.68 35.00±0.89Group B (100 mg/kg MC) 68.5±0.22* 28.67 64.00±5.37* 17.17±1.05* 41.33±0.95Group C (400 mg/kg MC) 70.5±0.89* 28.83 72.00±8.94* 22.50±4.25 39.00±1.10TP: Total protein; Alb: Albumin; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; MC: Momordica charantia; values are expressed as means±SEM (n=5); *values considered significant at P<0.05 when compared with Group A



stomach through distortion caused to the cellular integrity of the gastric mucosa.

CONCLUSION

Results from this study revealed that aqueous leaf extract of MC was able to reduce significantly the activities of antioxidant enzymes (CAT, SOD, and GSH) and disaccharidase (maltase, sucrose, and lactase) in the duodenum and liver enzymes (TP, Alb, AST, and ALT) of normal rats during oral administration. Histological study of the duodenum showed degenerated villi and loss of epithelial and goblet cell, in the liver tissue, there

was constriction of central vein and hypertrophied hepatocyte that degenerated with sinusoid dilation. MC has many health benefits, as reported by this study, but its administration will require proper guidance to avoid health complications.

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Figure 2: Photomicrograph of the liver in normal rats during oral administered of aqueous leaf extract of Momordica charantia rats (×400) hematoxylin and eosin

Figure 1: Photomicrograph of the duodenum in normal rats during oral administered of aqueous leaf extract of Momordica charantia rats (×400) hematoxylin and eosin



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How to cite this article: Oyesola OA, Osonuga IO, Taiwo OE, Owoeye IIG. Leaf extract of Momordica charantia: Effects on liver enzymes, duodenum antioxidant enzymes, and disaccharidases with liver and duodenum histology. Natl J Physiol Pharm Pharmacol 2021;11(05):534-539.

Source of Support: Nil, Conflicts of Interest: None declared.

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RESEARCH ARTICLE Leaf extract of Momordica charantia