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Ravishankar Vamadevaiah Mathada, Praful Siddhalingappa Jevoor, Rajashree Ravishankar

Jawharlal Nehru Medical College, K.L.E. University, Belgaum, Karnataka State, INDIA �

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BACKGROUND: Encephalopathy is a major complication in juvenile diabetes mellitus which cripples the potential physiomorphological growth and development in early childhood. It is very essential to diagnose and initiate the treatment at the earliest to prevent the possible complications. The ancient medical science Ayurveda mentions number of remedies to treat cognitive dysfunctions, the herbal root of Clitoria ternatea plant is one among them. METHODS: The diabetes was induced in 22 days (post natal) wistar rats by giving intra peritoneal injection of Streptozotocin at a dose of 60mg/kg body weight (bw). After the confirmation of diabetic state, the treatment with oral administration of alcoholic root extract of �� at a dose of 100 mg/kg b.w. was started immediately and continued for one month of duration. At the end of 30 days treatment the animals were sacrified, brain and pancreatic tissue was collected and they were subjected to gross histological studies. RESULTS: On microscopy the brain tissue was showing homogenous architecture, the hippocampal CA 3 region neurons were showing gross viable changes in the cell morphology, and in the pancreatic tissue, there was a reduction in the cell hypertrophy along with relatively less inflammatory changes in the islet cells of Langerhans. CONCLUSSION: The alcoholic root extract of herb �� has shown significant gross impact in preventing the possible complications related to brain hippocampal area CA3 and pancreatic tissue in juvenile diabetic rat experimental models. These benefits could be due to interference of number of chemical compounds present in this extract. (��)� ��: Hippocampus, hyperglycemia, juvenile diabetes, piknosis, Streptozotocin�

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GİRİŞ: Ensefalopati, erken çocukluk dönemindeki potansiyel fizyomorfolojik büyüme ve gelişimi yavaşlatan juvenil diyabetes mellitustaki major komplikasyondur. Olası komplikasyonların önlenmesi için erken dönemde teşhis ve tedaviye başlanması çok önemlidir. Eski bir tıp bilimi olan Ayurveda, kognitif bozuklukların tedavisi için birçok şifadan bahsetmektedir; Clitoria ternatea bitkisinin kökü bunlardan birisidir. YÖNTEM: Diyabet, 60 mg/kg vücut ağırlığına dozda Streptozotosin’in periton içine enjeksiyonu ile doğum sonrası 22 günlük wistar sıçanlarda oluşturuldu. Diyabetik durumun onaylanmasından sonra, 100 mg/kg dozunda �� alkolik kök ekstresinin oral verilmesi ile tedaviye derhal başlandı ve bir ay süresince devam edildi. Otuz günlük tedavinin sonunda, hayvanlar öldürüldü; beyin ve pankreatik doku toplandı ve histolojik çalışmalar yapıldı. BULGULAR: Homojen yapıyı gösteren beyin dokusu mikroskopisinde, hipokampal CA3 bölgesi nöronları hücre morfolojisinde belirgin değişiklik gösterdi ve pankreas dokusunda, Langerhans hücrelerinde göreceli olarak daha az enflamatuar değişikliklerle beraber hücre hipertrofisinde bir azalma vardı. SONUÇ: �� bitkisinin alkolik kök ekstresi, juvenil diyabetik sıçan deneysel modelinde, beyin hipokampal bölgesi CA3 ve pankreatik doku ile ilişkili muhtemel komplikasyonların önlenmesinde belirgin etki gösterdi. Bu faydalar, ekstre içinde bulunan çok sayıda kimyasal bileşiğin katılımıyla olabilir. �� -�Hipokampüs, hiperglisemi, jüvenil diyabet, piknosiz, Streptozotosin

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Received May 10, 2011; accepted January 19, 2012 DOI 10.5455/spatula.20120119052120 Published online in ScopeMed (www.scopemed.org). Spatula DD. 2012; 2(1): 9-16.

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The human brain contains about 50 billion neurons and 500 billion neuroglia cells with a dense capillary bed. The general microscopic architecture of brain varies from one region to other. Phylogenetically the archycortex includes hippocampus, which is mainly concerned with cognitive functions like learning and memory; the hippocampal formation comprises sibiculum, hippocampus proper, and the dentate gyrus. The hippocampus is also known as Ammon’s horn, form the research point of view it is divided into CA1, CA2, CA3 and CA4 Cornu ammonis (CA) zones. Hippocampus lies in the floor of inferior horn of lateral ventricle of temporal lobe, which resembles the sea horse, hence the name. The largest afferent connections of hippocampal formation include prefrontal path fibers and the main efferent connections include the papez circuit [1].The brain shows rapid postnatal developments with great acceleration to achieve its effective functional status.�

In the early childhood brain shows several changes with respect to increase in its size, synaptic contacts, secretion of neurotransmitters, increased capillary networking, and the completion process of myelination of corticospinal tracts to achieve voluntary control on the micturition and defecation. The juvenile diabetes is considered as a life threatening condition, wherein it cripples the potential learning abilities by causing its impact on the developing brain tissue which results in cognitive dysfunction. These postnatal modifications are affected by excess of circulating unused glucose and its end metabolic products in the said Hyperglycemic condition, due to deficiency of hormone insulin.

The hyperglycemia in its long course affects the peripheral nerves and the central nervous system including the brain. There are number of supporting literature which is suggestive of impairment in the function of brain in different duration of hyperglycemia in the experimentally induced diabetic animal models. It is well known fact that hyperglycemia through its Advanced glycated end products(AGES) is causing multiple life threatening macro vascular and micro vascular complications like cardiovascular diseases, renal disease, cerebrovascular disorders, hypertension, neuropathy, retinopathy encephalopathy etc., in case of both Insulin Dependent and Non Insulin Dependent Diabetes Mellitus. Among the number of life threatening complications, the diabetic encephalopathy is least stressed in the existing literature [2]. The important areas of brain like

cerebral cortex and hippocampus plays a major role in long term potentiation of memory and there are experimental evidences of its electrophysiological impairment in different duration of diabetic condition suggesting its possible impact of hyperglycemia on cognitive functions. An early diagnosis and treatment of Juvenile diabetes can prevent the way long impact of hyperglycemia on the developing young brain [3].�

The incidence of childhood diabetes is increasing at the rate of 3-4% world wide and in India it is about 2% of over all incidence of diabetes [4].

Though at present an effective therapy is available in the field of modern medicine, however this rapidly growing ailment still needs a holistic, alternative, cost effective and easily approachable therapy. This ailment is not an invention of recent century but it has already documented in the pages of history and in the evolution of different systems of medical science around the world including Ayurveda, siddha, traditional Chinese medicine etc. These literatures indicate the awareness of the condition like diabetes mellitus. Even today almost 60% of the people in the world are still depending on conventional medicines for their sufferings. The oldest medical science Ayurveda has mentioned this condition as PRAMEHA,�its signs and symptoms are comparable with diabetes mellitus. The medical science Ayurveda has explained its definition, Etiological factors, classification, pathogenesis, treatment and prognosis of this disease condition, in the texts like Charaka Samhita and Sushrata Samhita, they are known to be the oldest treatises of medicine and surgery. There are more than 800 drugs are known to be showing its effect on diabetes.�

The herb shankapushpi (��) mentioned in the Texts of Ayurveda which belongs to Group of memory enhancing drugs called Madhya rasayanas, they were used as an excellent intellect promoter and they were also used to treat number of cognitive function disorders. This herb was called by different names like Shankapushpi, Aparagita etc [5].

It grows through out India in hedges, thick forests and also it can be cultivated in the gardens. This is a normal perennial twinning herb which has terete stem, branches and leaves. The leaves are compound, impairipinnate and the leaflets are 5-7 in number they are available in blue and white flowered variety. The roots, leaves and seeds are exhibiting their pharmacological activity. Its roots carry medicinal value as an intellect promoting, ophthalmic, laxative and aphrodisiac, and also it is used as tonic in vitiated conditions of body. According to Ayurvedic treatises like

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Bhavaprakasha, Dhanvantarinigantu, the name Clitoriaternatea is compared with the plant Shankapushpa. There are number of preparations mentioned in the classical texts containing this herb as an important ingradient [6].

As per modern classification �� Linn. belongs to the family Fabaceae, it contains compounds like Kaempferol-3-�-rhamnosyl,1-6 glycoside and kaempferol-3-�-rhamnosyl, 1-6 galactoside, kaempferol, 3-� glucoside, kamempferol-3-0 rutionoside, kaempferol-3-� neohesperidoside and clitorin [7]. There are number of herbs like �� wild, ��, ��, � �� etc. are used in cognitive function disorders, are mentioned in the Ayurvedic literature [8,9], out of which herb �� (Linn) extract was tested in number of experiments for its beneficial effects including diabetes mellitus and cognitive functions [10,11].

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�� Young Juvenile inbred 22 days old Wistar rats

(post natal) of either sex were procured as per Resolution code. JNMC/ Institutional animal ethical committee (IAEC)/2/1/2008� taken and they were housed in the central animal house maintained in 12 hours day and 12 night cycle in a well ventilated rooms by giving standard food pellets and water and proper care was taken to maintain hygienic environment by experts [12].

On the 22nd day overnight fasted rats were given intra peritoneal streptozotocin injection at a dose of 60 mg/kg body weight (bw) [13,14]. The steady state of diabetes was confirmed at the end of 5th day after the streptozotocin injection.

After a span of 20 minutes the 5% glucose solution was given for next 48 hours to prevent the hypoglycemic impact. On the fifth day the Fasting blood sugar of rats was assessed by using Optimum Exceed Glucometer and strips. The rats having the fasting blood sugar (FBS) between 200-400 mg/100 ml were selected for our experimental groups.�

�4 �� (Figure a) herb was grown in

our Jawaharlal Nehru medical college herbal garden, Belgaum. The plant was authenticated by the scientist and Botanist Dr. Harsha Hegde, from Indian Council for Medical Research (ICMR). Its roots were shade dried and were cut into small pieces were later made into coarse powder by putting into the

pulverizer. By using the soxhlet extraction procedure [15] first the powder was defatted by using the petroleum ether. Later, the same powder was used for extraction with Absolute alcohol. The extract was dried by using the water evaporation bath and finally the powder was collected in a clean container with lid [9].� The experimental groups were containing 6 rats each. The age matched normal control rats and diabetic rats were kept in a standard environment.

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5�� 6�Medicinal plant Clitoria ternatea (Linn).

The diabetic treatment group was started with the

extract administration (22days old rats+ 5 days diabetic attention period) immediately and it was continued for 30 days i. e. 27 days+30 days = 57 days.

The extract of �� was given orally at a dose of 100 mg/kg bw along with the distilled water as a preventive therapy, immediately after the confirmation of the diabetic state [11].The treatment was stopped at the end of the 30th day and the rats were sacrificed by using the overdose of anesthetic ether.

4� �� The rats were transcardially perfused with 0.9%

of normal saline was followed by 10% of formalin by inserting the needle into left ventricular chamber. The sufficiently perfused rats were dissected immediately and its skull was decapitated and brain was removed and later the abdomen was dissected to collect the pancreatic tissue. Brain and pancreatic tissue were put into 10% Formalin as a fixative, later it was subjected to standard procedure of tissue

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processing and paraffin embedding. By using the Rotary microtome sections with the thickness of 5 microns were taken and they were mounted on glass slides by using combination of distyrene, plasticizer and xylene (DPX).

After complete drying, the sections were cleared with xylene, rehydrated with different grades of alcohol and distill water, finally the brain sections were stained with Cresyl violet and pancreas was stained with hematoxylin and Eosin. The Hippocampal area CA3 and pancreas was analyzed for its qualitative gross morphological changes [16].

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At the end of the treatment, the pancreatic tissue was showing good integrity in a non homogenous pattern, the islets were showing reduced insulitis, the exocrine part of pancreas was showing reduction in the lining cell hypertrophy (Figure b). The cortical hippocampal CA 3 area was showing well appreciated cell integrity without any gross noticeable neuronal shrinkage, cytoplasm was clear (Figure c). The hippocampal pyramidal cells were showing increase in its size (Soma) in the CA 3 region with clear and homogenous architecture, when it was compared with age matched untreated diabetic control groups.

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5�� 6� Drug Treated group showing pancreas with reduce cell hypertrophy, Islet cells reappeared in many places. 10X magnification.

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.� �� 7��-�The pancreas was disrupted showing obvious tissue fragmentation. Integrity of pancreatic tissue was affected to the greater extent with the disruption

of capsule and binding connective tissue elements. The pancreatic acinus were widely spread, its cells were showing hypertrophy. Intact colonies of islet cells were few. The isolated islet cells were showing inflammatory changes [17], and there was infiltration of lymphocytes (Figure d). �

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5�� 6� Drug Treatment group Hippocampal CA 3 Region, densely packed viable neurons (cresyl violet stain). 40X magnification.

5�� 6� Diabetic group showing disrupted vessels, Islets with infiltration of lymphocytes along with exocrine pancreatic lining cell hypertrophy. 10X magnification.

The brain cortex was showing severe neurodegeneration [18,19] with vacuolization and cavitation in many places, shrunken cells of hippocampus area CA 3 was showing cytoplasm with chromatolysis indicating strong affinity towards cresyl violet stain. The neurons were showing shrinkage with irregular profile in a non homogenous pattern which was very well appreciated in the hippocampal CA3 region (Figure e).

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5�� 6� Diabetic group Hippocampus CA 3 region showing comparatively less cell Density, cell profile is irregular showing shrunken wall, many cell cytoplasms showing piknosis. 40X magnifications (cresyl violet stain).

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pancreas was intact showing its healthy exocrine acinus with intact epithelial lining, the endocrine part was showing numerous groups of islets without infiltration by lymphocytes, it was the indication of the absence of inflammatory changes. Tissue capsule was intact with clear appearance of lobular pattern (Figure f).The brain cerebral cortex was free from cavity, vacuolization and the Hippocampal CA 3 area cells were devoid of chromatolysis and the cell cytoplasm was homogenous and was showing well appreciable nucleus with nucleolus (Figure g).

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5�� 6�Normal Control arrows indicating many intact Islets, pancreatic tissue showing homogenous intact architecture. 10X magnification.

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The diabetic rats received treatment with �� root extract was showing its possible

involvement in preventing the juvenile diabetic hyperglycemia induced degenerative changes in the hippocampal area CA3 region. These changes maybe due to the number of compounds present in the plant extract, probably they are motivating the recovery in the endocrine part of the pancreas, and this in turn bringing the antihyperglycemic effect which is resulting in hippocampal cell recovery in the brain. By this we can tentatively come to a conclusion that the clitoria ternatea plant root extract can influence by showing some beneficial effects on the brain hippocampal cell recovery. Though this experiment needs further detailed evaluation but the present experimental data is supporting the basic rationality of using herbal drugs as an important ingredient in number of medicinal formulations.

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5�� 6 Normal Control Group Hippocampal CA 3 region cell cytoplasm is showing homogenous architecture showing normal viable neuron cell profile. 40X magnification (cresyl violet stain).

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Equilibrium in the metabolic state of an individual plays an important role to support the postnatal growth and development of an individual. Post natal development of body is highly influenced by number of factors including the genetic, environmental, nutrition, immunology etc., to achieve a disease free productive life span. Our body depends on the basic elements like carbohydrates, protein and lipids for its survival.

The glucose is the basic need of our body requirements, which is needed for the production of energy and it is a basic food for the brain cells to survive. The blood glucose is the one which is

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having wide range of fluctuations because of most of our food articles contains glucose as one of the main component. Homeostasis of glucose is mainly influenced by functional efficacy of endocrine part of the pancreas. Juvenile diabetes is a condition where the excess of unutilized circulating glucose load takes a different abnormal path to produce toxic metabolic end products which in tern affects the young susceptible developing brain.

Especially in the juvenile diabetes it was believed that the interplay between genetic and environmental factors are playing important role in the clinical manifestation of the disease [20].

The chemical induction of diabetes in young rats, which is causing the destruction of pancreatic islets resembles the early child hood diabetic model without autoimmune antibodies [21].

An early onset of Juvenile diabetes usually associated with mild central brain atrophy along with significant differences in the intellectual performance. This shows that the nervous system development may be adversely affected by this metabolic syndrome, which is hampering the cognitive functions in the early childhood [22].

Neuropathy is one of the major complications contributing to morbidity in patients with diabetes mellitus. It is well known fact that diabetes leads to a wide range of peripheral nerve deficits such as reduced motor nerve conduction velocity, impaired sciatic nerve regeneration, axonal shrinkage in association with reduced neurofilament delivery and deficient anterograde axonal transport etc., out of which the complications of central nervous system in juvenile diabetes are least stressed. The rats with experimentally induced diabetes by Streptozotocin (STZ) the nerve damage is similar in many ways which resembles human diabetic neuropathy. Quantitative analysis of the spine density of hippocampal neurons clearly revealed significant decrease in the number of dendritic spines in 30 and 60 days old diabetic rats, it is suggesting the sensitive response of dendritic morphology in diabetes [23].�

The impairment in learning and memory has been recognized as a complication of diabetes; both spatial learning and Long term potentiation (LTP) expression were impaired in severely hyperglycemic rats as compared with non diabetic controls. Both electrophysiological and morphological evidence are indicating the neurological changes in the diabetics and the experimental rats have shown obvious loss of neurons in association with micro vascular abnormalities [3].

The structural plasticity is an important phenomenon which takes place in the hippocampal neurons; they are capable to undergo modification when they are under the influence of metabolic effect of drugs or toxins, which intern influencing the cognitive functions. These neurons are sensitive to show its response to stress, glucose, drugs or chemicals in the form of a selective atrophy in number of disorders. However the reversible changes were observed especially in the CA3 hippocampal region by the process of rehabilitation of stressed rats [24].

In diabetic stress the worst affected ones are the insulin dependent tissues, constant hyperglycemia will not spare even the very sensitive brain tissue though which is not insulin dependent. The analysis of Golgi impregnated hippocampal sections of diabetic rats have reveled reduced normal course of apical dendritic arborizations of CA3 principal neurons as indicated by the decreased number of branching points and the reduction in total dendritic length. The apical dendritic atrophy is potentiated further in diabetic animals by the glycemic stress, a paradigm that does not affect the hippocampal morphology in non diabetic animals [25]. The untreated Streptozotocin induced diabetic rats have shown neuronal loss in the frontal cortex along with the white matter defects was associated with cognitive dysfunction [19, 26].

Brain cerebral cortical neurons are influenced by chemical components from the plant origin [18]. Since several thousands of years human being is using number of herbs for his sufferings and benefits, use of one such herb in the animal model suggesting the oral administration of extract of �� leaf and flower in diabetic rats showed significant decrease in the blood glucose and glycosylated hemoglobin levels, at the same time there was an increased quantity of serum insulin level [11].

The experiments have revealed the effects of aqueous root extract of �� (CT) on the acetylcholine content of the rat hippocampus clearly indicated its significant usage in impaired cognitive disorders. The administration of Clitoriaternatea root extract at a dose of 100 mg/kg bw was showing marked memory enhancing property in neonatal rats and a little impact on its general motor activity. Thus it appears that the Clitoria ternatea extract must have brought some permanent changes in the brain which is responsible for improved learning and memory[10], this could be due to the influence of phytochemicals on the neurotransmitters.

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In our study design we are able to achieve better recovery impact on the diabetic rat brain as well as the pancreatic tissue. The severe infiltrating lymphocytes [17] in the disrupted pancreatic islets in the diabetic rats showed the recovery through their improvement in the integrity of pancreatic tissue. The changes in the pancreas was also indicting the possible recovery of exocrine pancreatic acinus as well as the reduction in the severity of inflammation in the endocrine part of pancreas along with positive reversible hippocampal neuron changes are indicating the viable changes in the neurons without degenerative changes. In addition to this there was a significant increase in the size of the neuron body (soma) is indicating the boosting effect of plant extract. When compared with age matched normal and diabetic controls these changes are showing favoring the positive reversible impact of �� root extract on newly diagnosed juvenile diabetic rat brain as well as on the pancreatic tissue. It is a question routinely asked regarding the basic rationality and proof behind the usage of herbs in number of ailments, though the traditional medical science has its own version of scientific explanations which is not universally comprehensive. The man is dependent on number of naturally, easily and freely available cost effective herbs for his sufferings, since generations. This experimental work though needs further evaluation, but the present work can support the basic and rational utility of clitoria ternatia on juvenile diabetic rat animal model. In future, the advanced research on this herb can benefit the human to a greater extent. The interdependency between different specie of plants and animals, since the time of origin of life on the earth must be have influenced their survival. There may be a mutual molecular compromising influence of these diversified species on each other, might have resulted in the survival and existence of both the species. This could be the reason why the rational use of plant and plant products are benefiting the human since centuries.�

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Thus plants are reputed to have its anti ageing and memory enhancing properties, their experimental pharmacological activities indicating their potential efficacy for its clinical use. Here the clitoria ternatea root extract has shown its positive impact on the cerebral cortical hippocampal CA3 neurons and on the pancreatic tissue of juvenile diabetic rats when compared among different

experimental groups. The different compounds present in the whole crude plant extract may have some advantage when compared the trials with isolated compounds. Here we have taken the whole root extract; its detailed mechanism of action can be understood through trials by isolating the phytochemical constituents to prove the superiority of crude extracts from isolated compounds. This study gives more validity to already existing therapeutic use of this herb in the field of herbal medicine. Such early prevention oriented experimental study could be an effective therapy in preventing the complications of hyperglycemia in the early childhood diabetes.

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The author would like to acknowledge the Dr P.S. Jevoor, for his guidelines and support to complete this work. The author also would like to acknowledge the K.L.E University, Belgaum, INDIA, for their complete technical support and encouragement to carry out this experimental works.

This Ph.D. research work is purely self-funded, it has not utilized any financial support from any sources. Here the author is clearly declaring that he do not have any competing interests.

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�1.� Fitzgerald MJT, Folan-curran J. Olfactory and limbic system,

4th edition, W. B. Saunders; 2002.p.275-292. 2.� Moore DJ, Gregory JM, Kumah-crystal YA, Simmons JH,

Mitigating micro-and vascular complications of diabetes beginning in adolescence.www.dovepress.com/mitigating-micro-and [ Access date;12-11-2011]

3.� Biessels GJ, Kamaal A, Ramakers GM, Urban IJ, Spruijt BM, Erkelens DW et al. Place learning and hippocampal synaptic plasticity in streptozotocin-induced Diabetic rats, Diabetes.1996; 45(9):1259-66.

4.� Devendra D, Liu E, Eisenbarth GS, Type 1 diabetes; recent developments. www.bmj.com/content/328/7442/750.full.pdf. [Access date 18-11-2011]

5.� Warrier PK, Nambiar VPK, Kutty C. Indian Medicinal Plants. Kottakal, Orient Longman limited; 1994; 2:129-132.

6.� Kirtikar KR, Basu BD. Indian Medicinal plants, Second edition, International book distributors, Dehradun; 1999.p.802-803.

7.� Rastogi RP, Mehrotra BN. Compendium of Indian Medicinal plants, Drug research Perspectives; Publication and information, Directorate, New Delhi; 1980: 2;196.

8.� Howes MJR, Houghton PJ. Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function, Pharmacology Biochemistry and Behavior. 2003; 75(3):513-527.

9.� Madhyastha S, Somayaji SN, Bairy KL, Prakash, Madhyastha P. Neuroprotective effect of �� leaf extract

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��

treatment on cognition and hippocampal morphology against prenatal stress, Thai journal of Physiological sciences. 2007; 20(2):79-88.

10.� Daisy P, Rajathi M. Hypoglycemic effects of ��Linn. (Fabaceae) in alloxan-induced diabetes in rats. Tropical Journal of Pharmaceutical Research. 2009; 8(5):393-398.

11.� Rai KS, Murthy D, Karanth KS, Rao MS. ��(Linn) Root extracts treatment during growth spurt period enhances learning and memory in rats. Indian journal of pharmacology. 2001; 45(3):305-313.

12.� Lawlor MM, The proper care of laboratory Rodents, Psychological department, United kingdom.labanimals. awionline.org/pubs/cq/two.pdf. [Access date 11-1-2012]

13.� Chattopadhyay S, Ramanathan M, Das J, Bhattacharya SK. Animal Models in experimental diabetes mellitus. Indian Journal of Experimental Biology. 1999; 35:1141-1145.

14.� Szkudelski T, The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas, physiological research. 2001; 50:536-546.

15.� Rangari VD. Pharmocognacy and photochemistry, General methods of extraction, isolation and purification, 1st edition, career publications, Nasik; 2008.p.129-139.

16.� Carleton and Drury. Histological techniques, 3rdedition, university press London; 1957.p.51-76.

17.�Miyazaki A, Hanafusa T, Yamada K, Miyagawa J, Fujino-Kurihara H, Nakajima H, et al. Predominance of T-lymphocytes in pancreatic islets and spleen of prediabetic non-obese diabetic (NOD) mice: a longitudinal study. Clinical Exp. Immunology. 1985; 60:622-630.

18.� Nwaopara AO, Anibeze CIP, Akpuka FC. Histological signs of Neurodegeneratioan in the cerebrum of rats fed with diet containing yaji: The complex Nigerian soya meat sauce. Asian Journal of Medical Science. 2010; 2(1):16-21.

19.� Sima AAF, Zhang W, Muzik O, Kreipke CW, Rafols JA, Hoffman WH. Sequential abnormalities in type I diabetic encephalopathy and effects of C-peptide. Review of diabetic studies 2009; 6(3):211-222.

20.� Gillespie KM, Gale EAM, Bingley PJ, High familial risk and genetic susceptibility in early onset childhood diabetes,Journal of Diabetes. 2002; 51:210-214.

21.� Kumar P, Clark M. Kumar and Clark’s clinical medicine, Diabetes mellitus and other disorders of metabolism, Saunders Elsevier; 2009.p.1031.

22.� Ferguson SC, Blane A,Wardlaw J, Frier BM, Perros P, McCrimmon RJ , Deary IJ. Influence of an early onset age of type 1 diabetes on cerebral structure and cognitive function. Diabetic care. 2008; 5:1431-1437.

23.� Joghataie MT, Roughani M, Jalali M,Shrayeli M, Tourandokht B. Dendritic spine changes in medial prefrontal cortex of male diabetic rats using, Golgi impregnation method. Archives of Iranian medicine. 2007; 10(1):54-58.

24.� Rao BSS, Madhavi R, Sunanda, Raju TR. Complete reversal of dendritic atrophy in CA3 neurons of the hippocampus by rehabilitation in restraint stressed rats. Current science. 2001; 80(5):653-659.

25.�Mararinos AM, McEwen BS. Experimental diabetes in rats’ causes hippocampal dendritic and synaptic reorganization and increased glucocorticoid reactivity to stress. PNAS (Proceeding of the national academy of sciences USA). 2006; 97(20):11056-11061.

26.� Revsin Y, Saravia F, Roig P, Lima A, Kloet ERD, Homo-Delarche F et al. Neurological and astroglial alterations in the hippocampus of mouse model for type I diabetes. Brain research. 2005; 1038(1):22-31.

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Journal of Herbal Medicine and Toxicology 6 (1) 15-19 (2012)ISSN : 0973-4643 Review Article

CHEMICAL INDUCTION OF DIABETES MELLITUS IN RATS: ANEXPERIMENTAL CHALLENGE

M.V. Ravishankar, P. S. Jevoor, Parineetha PB*, Rajashree R**,V.S.Shirol.Department of Anatomy, 1 Department of Physiology, 2Department of Biochemistry, Jawaharlal Nehru Medical

College, KLE University, BELGAUM – 590010Corresponding author email: [email protected]

Received : 12 August, 2011 Revised : 3 October, 2011 Accepted : 4 January, 2012

ABSTRACT: Diabetes Mellitus is a syndrome clinically characterized by constanthyperglycemic state of an individual, irrespective of food intake, due to pancreatic orextra pancreatic causative factors. Research on diabetes has extended in leaps andbounds because of its increasing incidence throughout the world. Experiments inunderstanding the diabetes has reached from gross to molecular level. Various animalsare routinely chosen as diabetic experimental models, which resemble the clinicalsyndrome to some extent. Tackling with the distinctive experimental induction of insulin-dependent and non insulin-dependent diabetes mellitus in animals is still anexperimental challenge, having many methods, drawbacks and limitations especiallyin achieving the ultimate goal of inducing a relatively stable diabetic rat models. Oneof the methods which are regularly chosen in experimental induction of diabetes inrats is by using chemicals. In this article, the author would like to highlight some ofthe drawbacks and limitations of experimental induction of diabetes using chemicalsbased partly on his research experience in this field.

Key words- Alloxan, Diabetes mellitus (DM), Streptozotocin(STZ)

INTRODUCTION

Diabetes mellitus (DM) is a chronic disorder ofcarbohydrate, fat, and protein metabolism. DMrepresents a heterogeneous group of disorders thathave hyperglycemia as a common feature. A relativeor absolute deficiency in insulin secretory response,which translates into impaired carbohydrate (glucose)use, is a characteristic feature of DM, resulting inhyperglycemia. Traditionally, DM has been classifiedinto two major categories; primary which is the mostcommon form, arising from a defect in insulinproduction and/or its action in the peripheral tissues;and secondary which is arising from any diseasecausing the extensive destruction of pancreatic islets,such as pancreatitis, tumors, certain drugs, ironoverload (hemochromatosis), surgical removal ofpancreatic substance, or acquired or genetic factors1.The classification is named as primary and secondarydiabetes on the basis of the causative factors

irrespective of the age of the patient.Further, in primaryDM, the term Type I (type a) is used to denote DMassociated with auto immune antibodies andType Iwithout autoimmune antibodies( type b)[1,2]. The termType II DM usually associated with obesity and insulinresistance in adults. Proper understanding of thecomplex pathogenesis of diabetes mellitus hasprogressed greatly through the use of animal models.Generally used animal models include Rat, Mice,Chinese Hamster etc[3]. Diabetes can be induced inanimals in various ways, like by using differentchemical agents, using biological agents like viruseswhich target the islet cells in the body,manipulatingthe immune system by transferring the serum ofalready infected animal, using the hormones likecortisol or growth hormones etc, surgical methods likepartial/complete pancreatectomy and by usinggenetically susceptible diabetic model through genetic

Journal of Herbal Medicine & Toxicology

16

manipulation and selective breeding being anexpensive method to use generally[4-6].

The induction of experimental diabetes in the rat usingchemicals which selectively destroys the pancreaticbeta-cells is relatively convenient method, currentlybeing followed by many researchers in the developingcountries. In the recent few decades research on DMhas taken greater pace to achieve effective treatmentto benefit mankind. Even though presently cell linestudies, culturing etc. are in practice as an alternativeto animal experiments,screening of many drug needsbasically experimental evidence of its acute and toxicstudy, which can be understood only through the useof animal models. The induction of stable diabeticstate in the animal experiments is really a challengingone to achieve, because of its limitations in manyaspects.

MATERIALS AND METHODS

Chemical Induction of DM in rats-Our allexperimental trials are carried out under the Resolutioncode. JNMC/ Institutional Animal Ethical Committee(IAEC)/2/1/2008. The chemical induction of thediabetes is very widely practiced in the field of medicaland pharmaceutical research, chemicals likealloxan(2,4,5,6 Tetra-oxo-hexahydropyrimidine )andstreptozotocin ( 2-deoxy-23-methyl-3nitrosoureido-D-glucopyronanose) are widely used chemicals indiabetic induction. Alloxan is a highly toxic chemicalwas routinely used some times before the chemicalstreptozotocin usage was realized.Alloxancan inducethe diabetes at the cost of more death rates in animalsexcept in geunipigs.Streptozotocin is comparativelyless toxic and it can be used even on the neonatal ratexperimental studies. Currently, streptozotocin isconsidered to be more reliable and popular becauseof its effective induction and less death rate in injectedanimals.The literature is indicating the difference inchemical dose used in the experimental trials. Theroutinely used rat model i. p.(intra peritoneal) dose ofSTZ is 90 mg/kg body weight and that of alloxan I.Pdose ranges from 60 mg/kg body weight to 120 mg/kg body weight[7,8]. The streptozotocin and alloxancan be given in combination to achieve the effectivediabetic condition in the animal like dogs[9], or thestreptozotocin also can be given in split low dosepattern to achieve the diabetes, also can be followedin some selected animals . The gueni pigs are showing

more resistance towards alloxan in diabeticinduction[10]..

The first and fore most thing is that an effectivediabetogenic chemical is very expensive,it should beobtained in fresh form, not to be exposed to air ortemperatures and should be stored in very coldatmosphere.When they are dissolved with the bufferor saline they should be injected immediately otherwise they will be get oxidized even before we inject.In vivo the half life of alloxan is less than 1 minuteand that of streptozotocin is about 5 minutes duration.The root of administration is very important to achievethe targeted condition[7]. It is a must to inject thedrugs like streptozotocin by using very freshlyprepared citrate buffer preferably chilled to obtaineffective results, altered pH of these buffers can makeour drugs useless,where its effective pH range isfluctuating with a very narrow margin.There areexperiments indicating the usage of chemical alloxanwith[11] or without[12] using buffers, which can givethe effective results. Age of the animal is also animportant factor to achieve the good results. Whencompare the effective diabetic inductionbetweenadults, young and neonatal rats, they are showing thehigher resistant because of the difference in theircapacity of quick beta cell regeneration. It is observedin the neonates that they will show initial transienthyperglycemia followed by euglycemia with in fewdays after injection. Their plasma glucose and insulinlevels are not much significantly differ from the controlgroups. The glucose level exhibited in such rat modelswith the blood sugar level above 100mg% can beconsider as diabetic ones[13,14]. Often in our neonatalexperiments we feel difficulty in maintaining theuniformity in the animals on the basis of their age orweight, because it depends on the number of rat pupsdelivered and fed by mother rat, accordingly thenutrition status may not match with the age or withthe other rats in the same group.

Most likely the study on experimental diabetes targetsthe condition like IDDM or NIDDM.In spite offollowing the recommended instructions, there arefrequent failures in obtaining the good number ofeffective diabetic animals, because the diabtogenicdrugs can cause the complete loss of islets which willresemble the IDDM of type A where the autoimmuneantibodies are absent, they may die if the treatment isnot startedimmediately. Thedrug induced diabetes

17

Ravishankar et al.

targeting the induction ofNIDDM is a difficult taskbecause of its impartial effect on targeted islets, toget the control over the extent of damage which iscausing on the pancreas requires a meticulousassessment, because it generallymay resembles themost advanced state of NIDDM. Most of the type IIdiabetes are due to Extra pancreatic causes, whichplaysa major-role rather the defective insulinsecretions.If at all any such significant secretion defectexists it resembles much advanced NIDDM, in suchsituation only the animals arelikely to become moreinsulin dependent.

The juvenile diabetes most often conventionally impliesthe IDDMin child hood; in fact initially it resemblesNIDDM till the advanced derangement in insulinsecretion.Most often the chemical induction ofconventional NIDDM will bias the most commoncause of peripheral receptor defect in the animalmodel, but still the chemical induction of diabetestargeting the islet cells are very widelypracticedbecause of its resemblance with type I or type IIdiabetic model, at any one particular stage.

In case of NIDDM the defects in insulin secretion issubtle in the beginning, our chemical induction of DMresembles early sate of IDDM or late state ofNIDDM were the clinical symptoms are overlapping,on such situation we have to treat the animals asaccording to the degree of blood sugar levels withoutmaking an effort to label it as IDDM or NIDDM[15].Interestingly the streptozotocin is the chemical inducesboth the type of diabetes.

Most of theconventional diabetic research targetingonly the islets cells in pancreas where its extent ofdamage can’t be manipulated, there are overlappingpathophysiological events, this may misdirect the goalsand objectives of the researcher. The microscopicobservations shows the impact on whole pancreatictissue including its exocrine part [16], leads todisruption in enzyme activity, this may be resulting inthe acceleration of metabolic derangement.

When the chemical causes the damage to whole ofthe pancreas which is showing the involvement ofexocrine part of the pancreas may contribute inestablishing the high extent of glucose derangement.The chemical induction could be a better model ofdiabetes mellitus where the pancreatic causes aretargeted, in such circumstances the researcher

invariably has to ignore the genetic and immunefactors, which are the pre requisites for themanifestation of clinically resembling hyperglycemicstate of IDDM diabetes especially in the youngindividuals except the experiments which are involvingthe transgenic and immune modified animals.

The choice of rout of drug administration is also animportant factor to achieve the objective of theexperiments. The intraperitoneal root seems to be themost convenient root to inject the drugs, here the modeof administration of drug is also considered to achievethe targeted effectivediseasemanifestation. As analternative, the Intravenous root of drug delivery,though it looks simple but not convenient, becauseinyoung rat tail veins are very difficult to trace forparenteral injections,they are so small that often itruptures with needle pricks.The different root ofdiabetogenic drug delivery may not be equallyeffective in inducing thediabetes, the extent of damagethrough the intravenous administration is certainlydiffers from intaperitoneal administration. Theduration of diabetic condition depends once again onthe two factors mentioned above often resulting inthe reversibility. The type I can be achieved throughthe i. v. administration of the streptozotocin at a doseof 40mg/kg body weight and Type II diabetes at adose of 90 mg/kg body weight intraperitonially [17]

The half life of the drug and the rout of administrationof chemical having the great relevance, the drug likealloxan having 1 minute of half life insde the body,which is too short to target the damage required. Insuch circumstances the speed interavenous rout ofadministration is the ultimate choice, the administrationof the same drug through intraperitoneal rout maynot be effective[18]. One should be careful in choosingthe root of administration of the diabetogenic drugand the dose, it matters in establishing the type of theclinical diabetes targeting in our experiments.

Collection of Blood-Drawing the blood from therat tail vein is difficult in young rats,the overnight fastedrats are unlikely to bleed in spite of multiple tail veinpricks.Retroorbital venous punctureis anotherroutinely used site for blood collection, requiresanaesthesia preferably anesthetic ether, often it bleedsprofusely and animal may die.Here one should takecare that a slight high dose of anaesthesia can causedeath instantaneously, the anesthetic ether is the mostproffered one,but some of the investigators use

Journal of Herbal Medicine & Toxicology

18

chloroform for temporary sedation effect which ismost likely to cause the death instantaneously. Thechloroform can be used to sacrifice the rat as andwhen required rationally. Retroorbital prick is not idealin brain related research works because it can damagethe brain tissueif the person is not skilledor its processof recovery may lead to thrombus formation. Eventhough the investigator selects usually a particularbreed of animals for his experiments, sometimes theyare showing very high resistance against thediabetogenic drugs partially or completely, the animalsin the group may die or showing the difference in thedrug response.Even normal dose in the experimentcan lead to death of animals,investigator may have toloose many animals allotted through the ethicalclearance just to understand its diabetogenic drugdosage and its survival rate.

Drug injection response-After the drug injectionthe animal generally shows triphasic resonse i.e. firststage hyperglycemia, second stage hypoglycemia inthis stage during the next 48 hours following theinjection care should be taken to prevent the possibleadverse hypoglycemic impact especially on the braintissue by giving dextrose. Finally in the third stagestable hyperglycemia can be achieved after 5-6 days.But it may not sustain the same condition even forfew weeks due to reversibility in the course of itspathophysiological response, probably it may be dueto the recovery in the endocrine part of pancreas.Thehistological evidence is clearly indicating its capacityof regeneration in the pancreatic exocrine andendocrine pat of the pancreatic tissue, especially thisreversible phenomenon can be better appreciated inthe juvenile rats.Thesereversibility in diabetic animalscertainly hamper the research relatedparametersespeciallystructural relatedchangesplanned with in short duration. Because the impactof glucose derangement takes about 3-4weeks toshow appreciable structural changesin the body forexamplein the brain tissue the neuronal dendriticarborization , dendritic spine count, neuronalchromatolysis, shrinkage etc.

Tolerance response-The glucose tolerance test isconsidered to be one of the reliable investigations toconfirm the diabetes[19]. The animals often showingabsolute Fasting euglycemic response in spite ofdiabetogenic drug administration, When such animals(rat) are subjected to glucose tolerance test (GTT)

after sufficient overnight fasting surprisingly its resultsindicate that they are diabetic by showing theintolerance to glucose by sugar levels as high as400mg/dl, this confirmation practically may not bepossible to do in all groups of rats and it is more painful,time consuming and expensive.

CONCLUSION

There is a uncertainty prevails in every stage of thestable chemical diabetic induction in rat animal model,it even depends on the factors like age, of the ratswhich we incorporate in our experiments. There arewide variations in the induction through this methodwhich is including the animal, age, dose, root,etc.Though a lot of research is going on in the field ofIDDM and NIDDM by using such chemicals, theresearchers should contemplate deeply to correlatehis aims, objectivesand the cause involved in diseasemanifestation. All the above said problems could beovercomethrough proper understanding theirpharmacokinetic and pharmacodynamics profile of thechemicals should be taken into the consideration foran effective results. Subsequently the pilot studies canfacilitate the corrections, modifications or alterationin the experimental design on right time. Certainlyabove mentioned our experimental drawbacks maybe an eye opener to a new researcher in this field tounderstand, face or to overcome from the expectedor unexpected experimental out comes.

ACKNOWLEDGEMENT

This research work under the K.L.E. University,Belgaum, INDIA, has not used any financial supportfrom any sources. The author would like toacknowledge Dr P.S. Jevoor who has supported meto understand experimental drawbacks and DrV.S.Shirol, for their complete technical support tocomplete this work. Dr Rajashree and Dr.ParineethBhat, helped in literature review and documentation.The author also would like to acknowledge K.L.EUniversity, Belgaum, INDIA, for their encouragementand support to carry out the experimental works.

REFERENCES[1] Ramzi VK., Cotran S., Robbins SL.: Robbins basic

pathology 7th edition, Harcourt (India) New Delhi,chapter pp635-655 (1977).

19

[2] William E.: winter, Neil harris and desmondschatz.20(4):10-18(2002).

[3] Lawlor MM.: The proper care of laboratory Rodents,Psychological department, Comfortable quarter forLaboratory animals, Viktor Reinhardt, Editor. AnimalWelfare Institute, Washington, DC, Royal Holloway,Egham, Surrey, Tw 20 OEX, United kingdom (1997)..

[4] Etuk. EU.: Animals model for studying Diabetesmellitus, Agriculture and Biology journal of northAmerica, ISSN Print: 2151-7517,ISSN online: 2151-75252010, Science HU, Http/www.scihub.org/ABJNA

[5] J.W. Yoon,T.Morishima,P.R.McClintok,M.Austin andA.L.Notkins.: Journal of virology, 50: 684-690(1984).

[6] Kotnik K, Popova E, Todiras M, Mori MA, AleninaN, et al.: (2009) PLoS ONE 4(4): e5124. doi:10.1371/journal.pone.0005124

[7] Szkudelski T,.: Physiol. Res. 50:536-546 (2001).[8] Chattopadhyay S, Ramanathan M, Das J.,

Bhattacharya SK.: Ind Jr Exp Biol. 35: 1141-1145(1997)[9] Anderson HR., Stitt AW., Gardiner TA., Loyd SJ.,

Archer DB.: 27:281-285(1993).[10] Oguzhanyildiz, Erdalkaraoz, Semihsenoz,

Gonendeniz.: Acta pharmaceuticaturcica, XXXIX,2:.65-71 (1997).

[11] Singh PK, Baxi DB, Mukharjee R, Selvaraj J.,Ramachandran AV.: Jr herbal med toxicol. 45-54(2010)

[12] Daisy P., Rajathi M..: Tropical Jr Pharmaceutical Res.8:393-398(2009).

[13] Arulmazhi DK., Veeranjaneyulu A., Bodhankar SL.:Ind. Jr Pharmacol. 36:217-221(2000).

[14] Ayse can et al.: Biological pharmacol. 27:694-698(2004).

[15] Kumar P, Clark M.:, Kumar and clark’s clinicalmedicine Chapter 19, Diabetes mellitus and otherdisorders of metabolism, Saunders Elsevier., 7th

edition pp1029-1075(2009).[16] Eliakim-Ikechukwu CF, Obri AI.: Nigerian Jr Physiol.

Sci. 24:153-155(2008).[17] M.N.Ghosh, Fundamentals of experimental

pharmacology, Chapter 29., 3rd edition, Kolkata pageno. 184.

[18] Jain DK., Rajkumararya.: Ind. Jr Pharmacol. 43:91-92(2011).

[19] Verspohl EJ.: Planta Medica, 68:581-590(2000)..

Ravishankar et al.

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This article can be downloaded from http://www.ijlbpr.com/currentissue.php

Int. J. LifeSc. Bt & Pharm. Res. 2013 Mathada V Ravishankar and Praful S Jevoor, 2013

EFFECT OF INDIVIDUAL AND COMBINATIONOF HERBAL EXTRACTS ON GLUCOSE TOLERANCE,

IN EUGLYCEMIC RATSMathada V Ravishankar1* and Praful S Jevoor1

Research Paper

Objective: To assess the glucose tolerance of alcoholic plant root extract of Clitoria ternatea Linnand Salacia chinensis Wight, individually and in combination. Methods: The 20 wistar rats eachweighing around 150g were randomly divided in to 4 groups, each group containing 5 rats. Normalcontrol group was fed with 2g/kg body weight glucose alone and treatment groups were administeredwith 100mg/kg body weight extract of Clitoria ternatea Linn and Salacia chinensis Wight separatelyand in combination, all these administrations were done orally by using oral gastric tube which isfollowed by the administration of 2 g/kg body weight of sugar. Later blood sugar was measuredafter every 30min till the end of 90 min duration. The results were summarized by mean +/-standard deviation of blood sugar levels at regular intervals of time, one way Analysis of variance(ANOVA) was used to compare the mean blood sugar level of 4 groups which is followed bySchiff’s multiple comparison test. Results: Both the drugs have shown increased glucose tolerance,but when they were administered in combination the results have shown increased tolerance ofglucose in rats at the end of 90 min when compared with the extracts administered individually.Conclusion: The herbs Clitoria ternatea Linn and Salacia chinensis Wight have the capacity toincrease the glucose tolerance but their combined administration is showing relatively a higherdegree of tolerance against their administration individually, this effect could be due to cellular levelinteraction of number of phytochemicals to increase the post prandial glucose tolerance.

Keywords: Herbal extracts, Glucose tolerance, Clitoria ternatea Linn, Salacia chinensis Wight,Euglycemia, Prediabetic, Streptozotocin.

*Corresponding Author: Mathada V Ravishankar, [email protected]

INTRODUCTIONDiabetes Mellitus is a chronic disorder of

carbohydrate, fat, and protein metabolism, due

to this condition relative or absolute deficiency of

insulin secretory response. Which translates into

impaired carbohydrate (glucose) utility resulting

ISSN 2250-3137 www.ijlbpr.comVol. 2, No. 1, January 2013

© 2013 IJLBPR. All Rights Reserved

Int. J. LifeSc. Bt & Pharm. Res. 2013

1 Department of Anatomy, J.N. Medical College, K.L.E. University, Belgaum- 590010, Karnataka, India.

into hyperglycemia and glucose intolerance,

which represents a heterogeneous group of

disorders that have hyperglycemia as a common

feature.

It is well known fact that hyperglycemia is

causing multiple life threatening complications

218



like cardiovascular diseases, renal disease,

cerebrovascular disorders, hypertension,

neuropathy, etc. in both Insulin Dependent

Diabetes Mellitus (IDDM) and Non Insulin

Dependent Diabetes Mellitus (NIDDM). Among

the number of life threatening problems

hyperglycemia in its long course affects the

peripheral and the central nervous system. In

many regions of the world the disease prevention

through the holistic approach is highly

appreciated by the principles of Ayurveda where

it implies equally on the prevention and cure of

number of prevailing ailments (Etuk, 2012; Pulok

et al., 2011).

Ethnobotony is a field which deals with the

scientific study of relationship that exists between

the people and plant where the hundreds of herbs

have been used since centuries for the human

benefit (Sharma et al., 2010). The knowledge of

utility of herbs were realized through constant

practice, timely improved human civilization has

started their one way of documentation regarding

the understanding the disease etiology, pathology,

treatment and prognosis in a rational manner,

later which are mentioned in the classical

literature of Ayurvedic medicine.

The experimental trials generally recommend

to start with simple tests which should provide

healthy reproducible, affordable, applicable

results. The phytochemicals are recommended

to start with crude extracts given orally or

intraperitoneally (Verspohl , 2002). The herb

shankapushpi ( Clitoria ternatea Linn) belongs to

Group of Madhya rasayana (brain tonic) drugs

which were used to treat number of cognitive

functional disorders as mentioned in the Texts of

Ayurveda. This herb was called by different names

like Shankapushpi, Aparagita, etc. Its flower

resembles the conch shell hence the name

shankapushpi.

It grows through out India in hedges and thick

forest and also cultivated in gardens, this is a

normal perennial twinning herb which has terete

stems and branches, leaves are compound,

impairipinnate, leaflets are 5-7 in number, available

in blue and white flowered variety. According to

the scholars of Ayurvedic treatises like

Bhavaprakasha, Dhanvantari nigantu (dictionary)

the roots, leaves and seeds are having

pharmacological utility, particularly the roots carry

medicinal value as an intellect promoting

ophthalmic, laxative, aphrodisiac, and as tonic

used in vitiated conditions of body. Other different

parts of shankapushpi plant was used to prove

its properties like antihyperglycemic, cognitive

function supporter, antidiarrhoeal, etc. (Upwat et

al., 2010). There are number of preparations

mentioned in the classical texts containing the

herb shankapushpi as an important ingredient

(Kirtikar and Basu, 1999).

The Saptha chakra (Salacia chinensis Wight)

it is a woody shrub with blackish branches; stem

is pale yellow colored with yellowish flowers, it is

found in Southern Orissa, Kerala, Malabar, and

regions from Mumbai to Coorg , coastal areas

and near the river banks and forests. It is also

called by different names like Swarna moola,

Saptharangi, etc. Classical texts like Sahasrayoga,

Yogamrutam and Chikitsa manjari have quoted

extensively about the use of this herb in the

management of diabetes. Its root bark is light

brownish colored and on the cross section the

root shows seven circles, hence the name ‘Sapta

Chakra” (Sastry, 2005).

As per the modern botanical classification the

herb shankapushpi is identified as Clitoria

ternatea Linn. belongs to the family Fabaceae. It

contains compounds like kaempferol-3-O-

rhamnosyl,1-6 glycoside and kaempferol-3-O-

219



rhamnosyl,1-6 galactoside, kaempferol 3-O-

glucoside, kamempferol-3-O-rutionoside,

kaempferol-3-O neohesperidoside and clitorin. As

per the modern botanical classification the herb

Salacia chinensis Wight, belongs to family

Celastraceae, contains compounds like

triterpenes, Q,T,U assigned as friedelan-1,3,dione

bearing 24-al, and 24-ol and 24 oic groups

respectively (Rastogi and Mehrotra, 1980-1984).

MATERIALS AND METHODSHealthy 20 wistar rats each weighing around 150g

of either sex were procured as per the resolution

code JNMC/Institutional animal ethical committee

(IAEC) 2/1/2008 and divided them randomly into

4 groups, each group containing 5 rats (Xang and

Tan, 2000).

The well grown plant Clitoria ternatea Linn (white

flowered) roots were collected from the college

campus garden of J. N. Medical College, Belgaum

and the well grown Salacia chinensis Wight roots

were collected from Western Ghats forest near

Sirsi taluk of Uttarakannada district, Karnataka

state, India. Both the plants were authenticated

by the scientist and Botanist Dr. Harsha Hegde,

from Indian Council of Medical Research

(ICMR) Regional Centre Belgaum, India.

Clitoria ternatea Linn and Salacia chinensis Wight

roots were separately shade dried and cut into

small pieces, made into coarse powder by putting

into pulverizer, by using the soxhlet extraction

procedure. First the powder was defatted by using

the petroleum ether; later the same powder was

used for extraction with absolute alcohol. The

extract was dried by using the hot water

evaporation bath and later it was collected in a

clean container covered with lid.

Grouping and Treatment

The 20 rats each weighing 150 g were randomly

selected under 4 groups, each group was

containing 5 rats. After the confirmation of their

Fasting Blood Sugar in the overnight fasted rats,

the Group I normal control (NC) fed with glucose

alone, simultaneously the remaining groups II, III,

IV were fed with herbal extracts individually and

in combination which is followed by the

administration of glucose 2 gm/kg body weight at

the end of the 30 min. In all the groups these drugs

were administered once which is followed by

monitoring the glucose levels at regular intervals.

Group II was fed with 100mg/kg body weight

of alcoholic Extract of Clitoria ternatea Linn alone

(Lawlor, 1997). Group III was fed with 100mg/kg

body weight of alcoholic extract of Salacia

chinensis Wight alone, Group IV was fed with the

100mg/kg body weight of each above mentioned

both the herbal extracts in combination (Rai et

al., 2001; and Sahayam et al., 2011).

Observations

The blood sugar was checked regularly at 30 min

of interval, up to 90 min of duration by using

Glucometer device Optium Xceed belongs of

Abbott diabetes care Ltd. (Kumar et al., 2007).

Statistical Analysis

The results were summarized by mean ±

standard deviation of blood sugar levels at different

intervals of time, one way ANOVA was used to

compare the mean blood sugar level of 4 groups

(Table 1). For multiple comparison of mean

deference, Schiff’s test was used P<0.05 is

considered to be statistically significant.

There was significant difference in the mean FBS

of all the 4 groups, the difference was found in mean

FBS of all the 4 groups, a significant difference was

found between group 1 with group 2 and 3, other

groups did not differ significantly. At first 30 min

interval there were significant differences in the mean

220



blood sugar levels of all the 4 groups and the

significant difference in the mean value of group 1

with group 3 and 4 were found.

There was significant difference in mean sugar

level at 60 min of time with the group 1, which

differed significantly with group 4 but others did

not differ much. At the 90 min, group 4 differed

significantly with group 1, 2, 3 but others did not

differ much. In summary the major difference was

found between the groups 4 with other 1, 2, 3

groups in this study.

DISCUSSIONThe change in quality and quantity of food habits

of present generation has drastically changed

when compared to past. Sedentary work and lack

of physical exercise is adversely influenced by

our food habits. The routine use of adulterated,

modernized, highly refined food articles are

becoming the strong reasons for number of

ailments. Consumption of inevitable excess of

carbohydrates are leading to excess of circulating

blood sugar load on body in its long run showing

Table 1: Blood Sugar Level in mg/100 ml (Mean ± SD)

Groups FBS At 30 min At 30 min At 90 min

Mean ± SD Mean ± SD Mean ± SD Mean ± SD

1 92.6 ± 6.58 185.8 ± 9.57 148.8 ± 18.83 107 ± 12.04

2 74.2 ± 6.68 148 ± 26.3 111.4 ± 26.29 101.4 ± 13.27

3 70.6 ± 5.41 124.4 ± 15.34 115.2 ± 14.014 84.4 ± 16.89

4 81.4 ± 4.39 130.6 ± 24.35 79.8 ± 16.57 54.8 ± 14.66

F3,16 13.780 7.547 8.393 13.393

P < .001 <0.002 <.001 <.001

Mg/100 ml FBS 30 m 60 m 90 m (duration in min)

Figure 1: Blood Sugar Levels at Regular Intervalsof 30 min Duration in all Experimental Groups

Blo

od G

luco

se L

evel Normal Control (NC)

Clitoria ternatea Linn (CLT)

Salacia chinensis Wight (SLA)

Combination (COM)

221



its impact on the endocrine part of pancreatic

cells leading to gradual inefficiency in handling

the glucose load in the body by influencing

Prediabetic changes in the body. Childhood

obesity is also becoming an important

precipitating factor for diabetes in young

individuals and the same in adults can lead to

number of ailments from simple neuropathy to

nephropathy. It has been debated during the last

decades whether asymptomatic, unrecognized

diabetes or even a lesser degree of hyperglycemia

in random samples indicating the increased risk

of Cardiovascular Disease (CVD) and death;

however investigator who studied the association

between hyperglycemia and the development of

diabetic complications focus on fasting glucose

levels (Tuomilehto, 2002). In diabetic studies it is

generally recommended to start with simple tests

which should provide highly effective reproducible

results and it should not be much time consuming.

The euglycemic animals are used for testing

potential oral hypoglycemic agents; this is still a

valid screening method which is often used in

addition to diabetic animal models (Verspohl,

2002). Large interventional studies have shown

that achieving and maintaining near normal

glycemic levels can reduce the risk of micro

vascular and macro vascular complications in

type 2 diabetes. The impact of post prandial blood

sugar control is equally important to prevent its

long course complications (Parkin and Neil

Brooks, 2002). The strict control over the blood

sugar level possibly by changing our life style and

food habits, along with the rational use of cost

effective and easily available herbal supplement

is the essential need of time. The WHO (world

health organization) Expert committee

recommended and also high lightened the

investigation of antihyperglycemic agents from

plant origin which are used in traditional medicine

for the treatment of diabetes mellitus. The

numbers of anti hyperglycemic agents from the

plant origin are used in traditional medicine are

showing their beneficial effects. In this

experimental study on glucose tolerance

particularly we have chosen the drugs called

saptharangi (Salacia chinensis Wight) which is

used in the diabetes as an important

antihyperglycemic agent, and another drug

shankapushpi (Clitoria ternatea Linn) which

belong to Medhya rasayana, to support the brain

cells by counteracting the adverse impact of

glucose.

The above mentioned plant extracts used in

combination have shown much greater reduction

in the postprandial blood sugar at the end of 90

min of time when it was compared with individually

administered experimental groups (Kumar et al.,

2007). This effect could be due to molecular level

combined interaction of different phytochemicals

present in our two different herbal different

extracts. Herbs are well known, human friendly

which are easily available and cost effective

source to treat number of disorders with respect

to their effective prevention and cure. Even today

60% of world populations rely upon their traditional

medicine. The world’s oldest medical sciences

like Ayurveda and traditional Chinese medicine

have mentioned number of herbs and herbal

formulations to tackle this hyperglycemic

condition Salacia chinensis Wight is one among

them. The experimental drug trials with the

isolated compounds like Mangiferin from the

Salacia chinensis Wight in diabetic rats have

shown significant decrease in blood sugar levels

along with increased insulin content in the body,

which support the rationality of its ethnobotonical

usage (Sellamurthy et al., 2009; Melanie-Howes

222



and Peter J Houghton, 2003). The gross Study

trials by using the aqueous extracts of Clitoria

ternatea Linn leaf and flower in diabetic rats are

also significantly decreased the levels of blood

glucose and glycosylated hemoglobin. At the

same time there was an increased serum insulin

levels to nearly matching with normal control

groups, which is suggestive of its pancreatic

effect (Daisy and Rajathi, 2009). The probable

action of herbs may be through their stimulatory

effect on the endocrine part of pancreas which is

manifested as increased glucose tolerance

(Eliakim-Ikechukwu and Obri, 2009). Our main

interest of this study is to assess how best these

herbal extracts individually and in combination can

effect the glucose tolerance. Our study is

exclusively based on its ethnobotonical utility

where the people in the past were dependent only

on easily available, cheap and effective source

available around them for number of ailments.

This indicating that the conventional use of crude

herbs in diabetes contains some effective and

essential phytochemical constituents, which are

need to be identified, isolated and trialed in

advanced research set up to prove their

mechanism of anti hyperglycemia. The Ayurvedic

principles consider whole human body, mind and

spirit along with therapeutic preventive or curative

treatments. While dealing with the maintenance

of health in healthy individual and treating the

disease in a holistic way is well acceptable in the

present generation.

Hyperglycemia however is not a simple

medical issue, the blood glucose level tend to

show diurnal and seasonal variations. Thus we

have firm evidence that rapid load of post prandial

glucose can be delayed. In the current era of

evidence-based medicine this knowledge should

be the strongest argument to test the glucose

intolerance in the people especially those who

tend to face the risk in future.

Here we have chosen the whole root extracts,their mechanism of action can be understood byfurther trials by isolating their phytochemicalconstituents. Irrespective of any predisposingfactors for glucose intolerance, early adoptionpractice of herbal supplements as a part of ourlifestyle accompanied with good diet and exercisecertainly can reduce the long term consequenceson endocrine part of pancreas by preventing theearly exhaustion of islets, which is an ultimatechange taking place in the chronic diabeticsforcing the individual to become more dependenton the insulin therapy.

In western medicine we have a clear indicationof drug action site in the body, but the utility ofcrude extract which is a combination of manyknown and unknown compounds which mayinfluence the antihyperglycemic effect in acomplex pattern. The herbs may act by reducingthe glucose production from the liver tissue bypreventing the glycogenolysis or it may preventthe glucose absorption from the intestines so thatthe post prandial glucose load can be substantiallyreduced or it may act as stimulant for theproduction of insulin secretion by supporting thetissue recovery changes in the pancreatic isletcells or it may help many insulin dependent tissueslike skeletal muscles to utilize the glucose in aeffective manner. To prove the antihyperglycemiceffects which needs further trials to prove an

effective natural alternate therapy.

COMPETING INTERESTSAuthor declares that he do not have any

competing interest.

ACKNOWLEDGMENTThe author would like to acknowledge the K.L.E

University, Belgaum, India, for their complete

technical support to carry out this experimental

works.

223



REFERENCES1. Daisy P and Rajathi M (2009), “Research

Article: Hypoglycemic Effects of Clitoriater-

natea Linn. (Fabaceae) in Alloxan-induced

Diabetes in Rats”, Tropical Journal of

Pharmaceutical Research, Vol. 8, No. 5, pp.

393-398.

2. Eliakim-Ikechukwu C F and Obri A I (2009),

“Histological Changes in the Pancreas

Following Administration of Ethanolic

Extract of Alchorneacordifolia Leaf In Alloxan

– Induced Diabetic Wistar Rats”, Nigerian

Journal of Physiological Sciences, Vol. 24,

No. 2, pp. 153-155.

3. Etuk E U (2012), “Animals Model For

Studying Diabetes Mellitus, Agriculture and

Biology Journal of North America”, ISSN

Print: 2151-7517, ISSN Online: 2151-

75252010, Science HU, http/www.scihub.

org/ABJNA (Access date 13-4-2012).

4. Kirtikar K R and Basu B D (1999), “Indian

Medicinal Plants”, Second Edition, pp. 802-

803, International Book Distributors,

Dehradun.

5. Kumar G, Banu G S, Maheshwaran R, Rema

S, Pandian M R and Murugesan A G (2007),

“Effect of Plumbago zeylanica L. on Blood

Glucose And Plasma Antioxidant Status in

STZ Diabetic Rats”, Journal of Natural

Remedies,Vol. 7, No. 1, pp. 66-71.

6. Lawlor M M (1997), Comfortable Quarters

for Laboratory Animals, The Proper Care of

Laboratory Rodents, Psychological

Department, Editor Viktor Reinhardt, Animal

Welfare Institute, Washington, DC 1997,

Royal Holloway, Egham, Surrey, Tw 20

OEX, United Kingdom, labanimals.

awionline.org/pubs/cq/two.pdf (Access date

11-1-2012).

7. Melanie- Howes J R and Peter J Houghton

(2003), “Plants Used in Chinese and Indian

Traditional Medicine for Improvement of

Memory and Cognitive Function-review

Article”, Pharmacology Biochemistry and

Behavior, Vol. 75, No. 3, pp. 513-517.

8. Parkin C G, and Neil Brooks (2002), “Is Post

Prandial Glucose Control Important? Is It

Practical In Primary Care Settings?”,

Clinical Diabetes, Vol. 20, No. 2, pp. 71-76.

9. Pulok K M, Venkatesan N Sateesh Kumar

and Micheal Heinrich (2011), “Review, The

Ayurvedic Medicine Clitoriaternatea From

Traditional Use To Scientific Assessment”,

Doi ; 10.1016/jep.2008.09.009, cite of link

using DOI, copy right@2008Elsevier Ireland

Ltd. All rights reserved (Access date 22-7-

2011).

10. Rai K S, Murthy D, Karanth K S and Rao M

S (2001), “Clitoriaternatea (Linn) Root

Extracts Treatment During Growth Spurt

Period Enhances Learning and Memory in

Rats”, Indian Journal of Pharmacology, Vol.

45, No. 3, pp. 305-313.

11. Rangari V D2008), Pharmocognacy and

Photochemistry, General Methods of

Extraction, isolation and Purification, First

Edition, Part I, Chapter VIII, pp. 129-138,

Career Publications, Nasik Maharashtra.

12. Rastogi R P and Mehrotra B N (1980-1984),

“Compendium of Indian Medicinal Plants,

Drug research Perspectives”, A Central Drug

Research Institute, Lucknow, Publication

and information, Directorate, New Delhi, Vol.

2, pp. 96 and 564.

224



13. Sahayam C S, Duby G P, Rajamanickaam

G V and Brinda P (2011), “Comapritive Study

of Antidiabetic Potential of Salacia Species

In Hippocrateaceae Family”, Journal of

Pharmacy Research, 2Vol. 4, pp. 1113-1114.

14. Sastry J L N (2005), Dravyaguna Vignana

(A Study of Essential Medicinal Plants in

Ayurveda), Chaukhamba orientalia,

Varanasi, 2nd Edition, pp. 770-771.

15. Sellamurthy P S, Maniappan B P, Primal S

M and Murugesan Kandasamy (2009),

“Antihyperglycemic Effect of Mangiferin in

Streptozotocin induced Diabetic Rats”,

Journal of Health Sciences, Vol. 55, No. 2,

pp. 206-214.

16. Sharma S, Rathi N, Kamal B, Pundir D,

Kaur B and Arya S (2010), “Conservation of

Biodiversity of Highly Important Medicinal

Plants of India Through Tissue Culture

Technology-a Review”, Agriculture and

Biology Journal of North America, Vol. 1,

No. 5, pp. 827-833.

17. Tuomilehto J (2002), “Point: A Glucose

Tolerance Test is Important for Clinical

Practice”, Diabetes Care, Vol. 25, No. 10,

pp. 880-882.

18. Upwat N K, Roshanpatel, Naheedwaseem

and Naveen Kumarmahobia (2010),

“Research Article, Evaluation of Antidiarr-

hoel Activity of Root of Clitoriaternatialinn”,

International Journal of Pharmaceutical

Sciences Review and Research, Vol. 5, No.

1, pp. 131-134.

19. Verspohl E J (2002), “Recommended

testing in diabetes research”, Planta

medica, Vol. 68, pp. 581-590.

20. Xang X F and Tan B K H (2000), “Effects of

Ethnolic Extract of Gynura Procumbens on

Serum Glucose, Cholesterol and

Triglyceride Levels In Normal And

Streptozotocin Induced Diabetic Rats”,

Singapore Medical Journal, Vol. 41, No. 1,

www.sma.org .sg/smj/4101/art ic les/

4101a2.htm (Access date 7-7-2012).

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