IV EXPERIMENTAL RESULTS

Transcription

1 IV EXPERIMENTAL RESULTS Tinospora cordifolia was collected from various regions of South India, totally five accessions were collected, among these five accessions, based on their in vitro free radical scavenging activity the plant for further study was selected. The results of the analysis during the course of the experiments are presented below: 4.1. IN VITRO FREE RADICAL SCAVENGING ACTIVITY Free radical scavenging activity of Tinospora cordifolia wild plant was preliminarily analyzed from first accession in different extracts like methanol, aqueous and chloroform, the extracts were tested in in vitro modals via antiradical activity by using DPPH (1,1 diphenyl 2 picrylhydrazyl). The result shows that (Fig.4.1), methanol extract have a very greater antiradical activity in scavenging DPPH radical with maximum inhibition of 82.32%. The other extracts had inhibition of 64% (aqueous) and 47% (chloroform), when compared with standard ascorbic acid (85%). Likewise all the remaining accession s samples were analyzed further and only methanol extracts had high antiradical activity by using DPPH, from the accession plants analyzed (Fig.4.2). Results shows that the third accession sample have significantly high scavenging activity (91.3%) when compared to the other accession samples so, further analysis conducted with third accession place plant samples only. Fig.4.3. depicts the DPPH free radical scavenging activity of Tinospora cordifolia stem methanol extract in accession three. From the result, it was confirmed that the antiradical activity of methanol extract of Tinospora cordifolia stem exhibited free radical scavenging activity in dose dependant manner. The third accession place plant methanol extract were further tested two different in vitro models i.e., Superoxide radical scavenging activity in riboflavinlight NBT system and Reducing power assay by the transformation of Fe3+ to Fe2+. Fig.4.4. represents the superoxide radical scavenging activity exhibited by the methanol extract of Tinospora cordifolia stem extract of third accession place plant at different concentrations. The result shows that at 250µg of extract the maximum inhibition (98.63%) was observed at 590nm. 1

2 The reducing power assay of methanol extract of Tinospora cordifolia stem extract of third accession place plant was estimated by the transformation of Fe3+ to Fe2+ (Fig.4.5.). The reducing ability was found to increase with rising concentration in sample, a concentration of 250µg shows to have maximum reducing power which was compared to that of standard Gallic acid STANDARDIZATION OF TINOSPORA CORDIFOLIA WILD PLANT CRUDE DRUG Physico-chemical analysis The results of physico-chemical parameters analysis of powdered Tinospora cordifolia stem were shown in Table.4.1 to Table.4.3. It reveals that the organoleptic characterization of plant (Table.4.1), identification of extraneous material in crude drug (Table. 4.2.), physic-chemical analysis of crude drug (Table.4.3), heavy metal analysis (Table. 4.4), microbial analysis (Table. 4.5) were in the range and they were as per the recommendations of WHO, to proceed with the Tinospora cordifolia stem crude drug. The organoleptic characterization of Tinospora cordifolia crude drug was characterized as, the crude drug has the colour in creamy white to gray, odourless and bitter taste. The analysis of extraneous materials indicated that the foreign matters like sand, silico, insect infestation and rodent contaminations, were within the WHO limits. It was found that the total ash content (17.6 %, w/w), acid insoluble ash (1.16 %, w/w), water soluble extract (27.84%, w/w) and alcohol soluble extract (35.76%, w/w) were at normal level. And from the further analysis of Tinospora cordifolia stem methanol extract, further the extract has the following heavy metals, such as Lead ppm, Arsenic ppm, Cadmium ppm and, Mercury ppm, all the above metals were present within the WHO limits. Microbial analysis was performed for specific pathogens in methanol extract of Tinospora cordifolia, the results shows that E.coli and Salmonella, Shigella was absent and 10 2 CFU of entero bacteria, CFU of total Heterotrophic Bacteria and CFU yeast and mould were found in one gram of sample. It indicates that the levels of pathogens were present within the WHO limits. From the analysis of preliminary phytochemical testing (Table.4.6) the presence of high amount of alkaloids, glycosides, proteins, amino acids, tannins and phenolics 2

3 and all other secondary metabolites were confirmed in methanol extract of Tinospora cordifolia. It means that Tinospora cordifolia can be used as a source for drug and preceded further for the identification of active molecule from this plant material MICROPROPAGATION OF TINOSPORA CORDIFOLIA Table.4.7 and Table shows the standardization of surface sterilization in different types of explants as well as the influence of Cytokinins on shoot induction from different explants of Tinospora cordifolia in M.S medium at 20 days culture. Among the different sterilized substance and different explants like node, internodes and shoot tips, the better regeneration was found to 0.1% HgCl 2 solution for about 6 minutes followed by three to five wash with sterilized distilled water to remove the traces of HgCl 2 solution. Different explants were then cultured in M S supplemented with different concentrations of ( µm) BA, ( µm) KIN and ( µm) 2iP alone. The auxiliary buds on the nodal cuttings showed visible growth after five days in culture and most of them grow into shoots within 20 days. Shoots formation was affected by the concentration of hormones used in the medium. Among different Cytokinins (BA, KIN, and 2iP) used for shoot induction, the better result was produced only in nodal explants with KIN 4.36µM, produces 2.23 ± 0.1 cm length with 1.8 ± 0.1 numbers of shoots with 70% response when compared with other Cytokinins. After a month of shoot induced culture, creates a phenolic exudation problem in shoots formation, media had discoloration and explants were browning and the buds were break, so further analysis was needed to control the phenolic leaching out, the protocols were found by treating with different phenolic exudation controlling substances. The results were given in Table.4.9 These explants were allowed to stand for 3 to 4 hours in double distilled water to facilitate the phenolics and a characteristic gummy substance of polysaccharide leaching out of explants. Later single bud explants (1-2cm each) with upper portion were ished with 1% Bavistin (w/v) for 5min. followed by 3 times washed in sterile distilled water. The explants were surface sterilized with 0.1% (w/v) HgCl 2 for 6 minutes, and later rinsed 4 or 5 times with sterile distilled water. Later the edges of the 3

4 explants were trimmed and nodal explants (1cm each) containing axillary bud were used for the experiment. The different mediums used were polyvinyl pyrollidone (0.01%) (Fig.4.6- a and Fig.4.6- b), activated charcoal (1.5%) (Fig.4.7- a and Fig.4.7- b), silver nitrate (20%) (Fig.4.8 a and Fig.4.8 b), PVP (0.01%) + ascorbic acid (0.01%) (Fig.4.9- a; Fig b and Fig.4.9- c) and with 4.36 µm of KIN in M S medium. Among these combinations, silver nitrate (20%) with KIN (4.36 µm) gives 100% response with 3.01± 0.1 cm length of shoots with 2.01± 0.1 numbers of shoots (Fig.4.8 a and Fig.4.8 b) within 16 days of cultures. It indicates that Silver Nitrate at 20% along with KIN (4.36µM) will control the Phenolic exudation in shoot induction. After, the determination of best treatments for shoots induction. The effect of different concentration of Cytokinins (BA, KIN, and 2iP) alone or combination of BA+ NAA and BA + IAA on shoot proliferation in nodal explants of Tinospora cordifolia in M S medium showed in Table and Table Nodal explants of Tinospora cordifolia were cultured on MS medium supplemented with various concentrations of BA alone and in combination with NAA or IAA for multiple shoots proliferation. Among the different experiment, BA (8.82 µm) alone showed better growth response and produced 4.81± 0.2 numbers of shoots with an average length of 3.9± 0.1cm after a 20 days of culture (Fig E; F and G). Other experiments of KIN and 2iP different concentrations produced less number of shoots, whereas 8.82 µm BA with different concentration of NAA produced very less number of shoots and high concentration of NAA produced dark green cultures. The same results of dark green were obtained in BA 8.86 µm with different concentration of IAA. The effect of Cytokinins, BA ( µm), KIN ( µm) and 2iP ( µm) alone and combination of BA +KIN was analysed (Table and Table.4.13.) on elongation of shoot regenerated from nodal explants of Tinospora cordifolia on MS medium. 4

5 Among the above different experiment of shoot elongation with an average length of 4.82± 0.4 cm with 4.61± 0.2 numbers of shoots produced, the media containing 8.82 µm of BA alone with 76% of response when compared with other concentrations and the other experiments of Cytokinins treatment, the 6.88 µm BA µm KIN produce the average length of 6.8 ± 0.1 cm with 4.8 ± 0.4 shoots at 86% of response (Fig H; I & J) when compared with other experiments. The regeneration of shoots were excised and cultured on half strength of M S supplemented with 3% sucrose and different concentration of Auxins in IAA ( /13 µm), IBA ( µm) and NAA ( µm) were used in alone. The results were provided in Table Rootining was noticed in all the concentrations of Auxins used, however, maximum number of shoots rooted in 6.43 µm IBA at 85% of response (Fig K) followed by µm which produces 80% of response when compared with other concentration of Auxins at 27 days on medium. After 30 days of growth, lateral roots were produced from main root. The rooted plants were transplanted ex vitro and raised in pots (Fig L and Fig M) containing red soil, vermiculite and farmyard manure in 1:1:1 ratio, kept under green house conditions for one month followed by their field transfer. Approximately 80% of plantlets survived (Fig N) CALLUS CULTURE During the present set of experiments, different concentrations of Cytokinins BAP ( µm), BA ( µm) combined with different concentration of Auxins 2,4 - D ( µm), NAA ( µm) were tried for callus induction the results was represented in Table and Table BAP was combined with 2,4-D/NAA and BA combined with 2,4-D or NAA on MS medium using three experiments, including leaf, nodal segment and internodes. The callusing response was initiated in the form of swelling of the explants on the fifth to sixth day in cease of nodal explants and 2-3 weeks in use of leaf and internodes explants. However, callusing rate was markedly affected by type of primary explants using in the following order. Nodes > internodes > leaf. Nodes were used for the best source of explants for callogenesis and further secondary metabolites production experiments. 5

6 After 30 days the best callus was produced from the low concentration of Cytokinins and Auxin in nodal explants. The better primary callus was produced in the combination of BAP (0.56 µm) D (0.46 µm) concentration (Fig.4.11-A) and BA (0.44 µm) + 2,4 D (0.46 µm) also produce the healthier callus in nodal explants (Fig.4.11-B). The high concentration of BAP and 2,4-D/ NAA were not producing the callus in nodes, leaf and internodes. Table and Table shows the effect of plant growth regulators in proliferation of secondary callus formation from the best primary callus of Tinospora cordifolia nodal explants. The fresh pieces of callus tissue (25-30days) dissected from primary established culture. The callus were sub cultured with BAP ( µm) combined with 2,4 D ( µm)/ NAA ( µm) and BA ( µm) with 2,4 D ( µm)/ NAA ( µm). The out of four categories of experiments, the better significant amount of cell biomass ± 1.1 (mg dry wt.) was obtained after 32 days (Fig.4.11-C) from BAP (0.56 µm) + 2, 4-D (0.46 µm). At the same time BAP (0.56 µm) + NAA (0.54) combination was produce (Fig.4.11-D) ± 1.3 (mg dry wt.) biomass. And the experiment of (Table. 4.18) BA (0.44 µm) +2,4-D (0.46 µm) combined regulators produce ± 1.1 (mg dry wt.) cell biomass at 32 days culture (Fig.4.11-E). At the same time of experiments BA (0.44 µm) + NAA (0.54 µm) combination formed ± 1.2 (mg dry wt.) cell biomass (Fig.4.11-F) when compared to the other combinations of plant growth regulators. Table.4.19 and Table present the influence of plant growth regulators, sucrose and essential amino acids on proliferation of secondary callus from best primary callus. Freshly formed primary callus (25-30 days) was sub cultured on development medium. This development medium consists of full strength MS medium and 5% sucrose, essential amino acids along with BAP ( µm) combined with 2,4 D ( µm) or NAA ( µm) and BA ( µm) with 2,4 D ( µm) or NAA ( µm). 6

7 From the above treatments BAP (0.56 µm) + 2,4-D (0.46 µm) produces the highly significant amount of ± 1.3 (mg dry wt.) Cell biomass in 32 days culture (Fig.4.11-G). At the same time BAP (0.56 µm) + NAA (0.54) produced ± 1.1 (mg dry wt.) cell biomass (Fig.4.11-H). And the treatment of BA (0.44 µm) + 2,4- D (0.46 µm) produces ± 1.4(mg dry wt.) cell biomass at 32 days culture (Fig I), BA (0.44 µm) + NAA (0.54 µm) produce less amount of ± 1.3 (mg dry wt.) cell biomass (Fig J), when compared to other experimental treatments. Table and Table presents the effect of Cytokinins and Auxin for response, to production of biomass and percentage of Berberine in during secondary callus. Table and Table shows the effect of plant growth regulators, sucrose and essential amino acids for production of biomass and percentage of Berberine content in secondary callus. From the experimental reports (Table To Table. 4.20), the best cell biomass produced callus was weighed and dried, the secondary metabolite content (Berberine) was isolated and quantified. It was found that the BAP (0.56 µm) + 2,4-D (0.46 µm) plant growth regulators produces a callus biomass of ± 1.1 (mg dry wt.) contains 1.7± 0.3 % of Berberine. BAP (0.56 µm) + NAA (0.54) combination treatment produces ± 1.3(mg dry wt.) of biomass containing 1.2± 1.0 % of Berberine. The BA (0.44 µm) + 2,4- D (0.46 µm) combination produced ± 1.1 (mg dry wt.) having 0.6± 1.5 % of Berberine, at the same time BA (0.44 µm) + NAA (0.54 µm) produces ± 1.2 (mg dry wt.) biomass contains 0.4± 0.1 % of Berberine. The same type of experiments was conducted along with modified MS medium (add 5% sucrose + essential amino acids) to produce better biomass as well as high Berberine content also. Table: Shows that the combination of BAP (0.56 µm) + 2,4-D (0.46 µm) produces significant amount of cell biomass ± 1.3 (mg dry wt.) with 2.8 ± 0.2 % of Berberine. The combination of BAP (0.56 µm) + NAA (0.54) produces ± 1.1(mg dry wt.) biomass containing 1.6± 0.3% of Berberine. 7

8 Other combinations of different concentration of Cytokinins and Auxins (Table. 4.24) produces less weight of biomass and low content of Berberine. Table depicts the effect of growth regulators, sucrose and essential amino acids on proliferation of secondary callus biomass at different ages. The proliferation of secondary callus biomass weight was calculated at different ages (using log phase callus only), the fresh pieces primary callus was inoculated the modified improved MS medium (5% sucrose + essential amino acids) with treatment of BAP (0.56 µm) + 2, 4-D (0.46 µm) produces significant amount of biomass ( ± 1.3 mg dry wt.) at 32 days of culture. The proliferation was declaiming after 32 days (Fig.4.12). The optimum callus proliferation produced at 32 days in modified culture medium ISOLATION, IDENTIFICATION AND QUANTIFICATION OF BERBERINE FROM TINOSPORA CORDIFOLIA Isolation of Berberine from Tinospora cordifolia Wild plant stem, micropropagated plant stem and callus materials were powdered (100g) separately and extracted with methanol (400ml) in soxhlet extractor for 8hours. Solvent was removed by distillation and residue was dissolved in sufficient volume of hot water. At this stage the resin was separated out by filtration of hot solution. The solution was treated with excess of hydrochloric acid for the separation of Berberine as Berberine hydrochloride for few hours. Berberine hydrochloride was dissolved in hot water with few drops of 10 percent sodium hydroxide solution. Acetone was added to the solution and diluted with equal volume of water. The mixture was allowed to stand overnight at 5 o C. After separation it was ished with ice-cold water. The dry Berberine Acetone were dissolved in a mixture absolute alcohol and chloroform (10:1). The solution was heated to boiling and allowed for cooling at 5 o C. The crystals of pure Berberine separated out (Fig.4.13). The percentage of Berberine calculated by weighing TLC fingerprint profile of Berberine Thin layer chromatography of the Berberine containing wild plant stem, micropropagated plant stem and callus materials extracts and standard Berberine were 8

9 carried out on TLC. The extracted Berberine and standard Berberine was dissolved in methanol and subjected to TLC, using acetonitrile: water (60: 40) solvent system as mobile phase. The plates were treated with Iodine vapour (Fig a, Fig b & Fig c) and R f values were calculated. It was confirmed that from the R f values (0.57) of bands obtained in all the extracts and standard Berberine that the Berberine isolated from extract was pure and same can be used for further studies Analysis of Tinospora cordifolia Berberine content through HPLC The HPLC method was used to identify and quantify the Berberine from Tinospora cordifolia, the plant materials. The validation of quantification was performed for samples in five replicates which were collected from random samples. Satisfactory retention times and good resolution of Berberine was achieved using phenomenex -5µ (Luma) C18 column, clout with acetonitrile: water 60:40(V/V) at a flow rate of 0.5ml/min. A sharp and symmetric peak of Berberine was obtained with good baseline resolution and minimal tailing, thus facilitating the accurate measurement of peak area. The HPLC analysis was carried out in isocratic condition and a retention time of 5.15 min. and detected at a wavelength of 265nm. for obtaining Berberine. Co-chromatography was also performed with authentic samples (Fig. 4.15). The Berberine concentration of methanol extract of wild Tinospora cordifolia was found to be % (w/w) Fig.4.16 and aqueous (0.150%) Fig. 4.17, chloroform (0.020%) w/w Fig.4.18 and Fig The similar conditions was also applied for the micropropagated and callus materials to develop HPLC for them. The Berberine concentration of methanol extract of micropropagated and callus of Tinospora cordifolia was found to be 1.2 % (w/w) and 2.8 % (w/w) respectively (Fig.4.20; Fig.4.21 and Fig.4.22) STUDY OF ACUTE TOXICITY IN RATS TREATED WITH METHANOL EXTRACT OF TINOSPORA CORDIFOLIA The acute toxicity study was conducted in wistar albino rats weighting g. The whole plant methanol extract was given by oral route. Initially the animals were administrated with a single dose of 50 mg/kg of body weight and the dose was increased step by step to 250mg, 500mg, 1000mg, 2000mg and 3000mg/kg body weight. All the groups animals (n=3) almost continuously observation for 9

10 mortality and behavioural changes during first 2 hrs. and then at 6 th and 24 th hours. The observations of behavioural changes in body weight, food and water intake as well as cage side observations were reported (Table. 4.26). There was no abnormality observation in all groups, the whole plant methanol extract have no significant toxic effect in wistar albino rats and the plant materials was found to be nontoxic demonstrated by the following reports Respiration When administered 3000 mg/kg/oral (acute toxicity dose) of Tinospora cordifolia in rats individually, there was no alteration in the respiration up to the end of study, indicating the safety and efficacy of the extract at the above dose level Writhing When administered an acute toxicity doses of Tinospora cordifolia in rats there was no significant change in the writing reflux, even after 24 hours. This experiment proved that Tinospora cordifolia did not act on cholinergic receptor Tremor Tinospora cordifolia on acute toxicity doses did not produce any tremor even after 24 hours, thus it did not alter dopamine energy activity in the basal ganglia and also cholinergic receptors indicated that the safety of the extract in CNS activities Convulsion Certain plants extract at high doses may show GABA inhibitory and glutamate excitatory activities at post synaptic levels (Sodium influx) leading to electrical discharges which causes convulsion. Herbal extracts containing spinal stimulant compounds when administered at high does may produce convulsive disorders. The convulsive action may exhibit initial stimulation followed by depression (Rang H.P et al., 2005). Administration of Tinospora cordifolia at acute toxicity doses (3000mg/kg) and analysis shown in Table There was no cause of any convulsions up to the end of the study period. Hence, the plant extract did not have any action on CNS either on GABA inhibitory or glutamate excitatory receptors Hind limb paralysis 10

11 Plant extract containing higher doses of CNS stimulant compounds, activates excitatory amino acids, NMDA receptors and allowed excessive influx of calcium which leads to tetanus and finally cell death cytotoxicity by producing temporary hind limb paralysis (Sharma H.C and Sharma K.K. 2001). Tinospora cordifolia at acute toxicity doses (3000mg/kg) did not produce hind limb paralysis throughout the study period (Table ) Sense of touch and sound Plant extract and some anesthetic drugs effect sensation of touch and sound by acting on ascending reticular activating system ( proprioceptive) entero captive organs which receive stimuli from muscle and joints (Chatterjee.C.C.1994). Tinospora cordifolia at acute toxicity doses (3000mg/kg) in rats does not alter the sense of touch and sound responses up to 24 hours (Table ) Salivation Drugs acting directly on cholinergic or histaminic receptors may stimulate salivary secretions (Dandya P.C and Kulkarani S.K; 2004). Results (Table ) indicates that the Tinospora cordifolia extract at acute toxicity doses (3000mg/kg) does not alter the salivary secretion Diarrhoea On administration of acute toxicity doses (3000mg/kg) of Tinospora cordifolia extract in rats does not produce any electrogenic chloride secretion and hence the gastrointestinal mortality was not altered, thereby no sign of diarrhoea was observed Mortality From the experiment studies (Table ), it was observed that when Tinospora cordifolia was administrated at a dose of 3000mg/kg of body weight, there was no mortality. From the acute toxicity studies (as per OECD- 423 guidelines) gross behaviour studies in rats, the LD 50 value of Tinospora cordifolia was determined as 3000mg/kg/oral individually. At this dose level, no sign of writhing, tremor, convulsion, hind limb paralysis, sense of touch and sound, salivation, diarrhoea was observed in the rats, skin, fur, eyes, 11

12 mucous membrane, and behaviour pattern were normal. There was no mortality up to the dose level 3000mg/kg and the animals were alive up to the end of the study HYPOGLYCEMIC AND ANTIOXIDANT ACTIVITY OF TINOSPORA CORDIFOLIA WILD STEM, MICROPROPAGTED PLANTS AND CALLUS MATERIAL IN ALLOXAN INDUCED DIABETIC RATS During the experimental periods the changes in body weight were observed and tabulated in Table: The body weight found to be increased in normal and Tinospora cordifolia plant stem methanol extract of treated diabetic rats and in diabetic rats it was decreased when compared with normal (Fig: 4.23). Methanol extract of callus materials of treated diabetic rats significantly (P< 0.01) regain the body weight when compared to other micropropagated and wild plant extract treated groups. The changes in urine sugar during the experimental period was observed to find out the effectiveness of the methanol extract of wild, micropropagted plants stem and callus material (Table: 4.28.). The excretion of urine sugar was found to be increased in diabetic rats when compared with normal. Tinospora cordifolia wild, micropropagted plants stem and callus material of methanol extract treated diabetic rats was reverses back to the significantly (P< 0.01) normal condition, the absence of urine sugar in experimental rats. Table: represents the effect of Tinospora cordifolia stem methanol extract on blood glucose in normal, diabetic, micropropagated and callus material treated in alloxan induced diabetic rats. Significant reduction in the blood glucose level in Tinospora cordifolis stem methanol treated experimental rats was seen. If reverses back to the normal level in blood glucose after treatment of Tinospora cordifolis wild plant material (Fig: 4.24). The Tinospora cordifolia callus material was significantly (P< 0.01) reduced the blood glucose level when compared to the micropropagated and wild plant. The blood glucose level was elevated in diabetic rats when compared to normal. The Effect of Tinospora cordifolia extracts on animal liver weight in normal, diabetic and micropropagated Tinospora cordifolia treated and Tinospora cordifolia callus material treated diabetic rats was given as Table: The liver weight was decreased in diabetic rats when compared to normal. And significantly 12

13 (P< 0.01) increased in the liver weight in Tinospora cordifolia stem methanol treated experimental rats was noticed. It has normal liver weight after treatment of Tinospora cordifolia wild plant material. The Tinospora cordifolia callus material was significantly (P< 0.01) increased the animal liver weight level when compared to the micropropagated and wild plant (Fig: 4.25). Table: shows the effect of Tinospora cordifolia extract on serum protein and liver tissue protein in normal, diabetic, micropropagated Tinospora cordifolia plant treated and Tinospora cordifolia callus material treated diabetic rats. It was found that the level of serum protein and liver tissue protein were found to be decreased in alloxan induced diabetic rats when compared with normal. The decreased level of serum protein and liver tissue protein were bringing back to significantly (P< 0.01) near normal in Tinospora cordifolia methanol extract treated experimental diabetic rats. The callus material of Tinospora cordifolia methanol extract treated group was significantly (P< 0.01) increased the level of serum protein as well as liver tissue protein when compared to the micropropagated and wild plant treatments. The effect of Tinospora cordifolia extract on cholesterol and blood urea level in normal, diabetic, micropropagated Tinospora cordifolia treated and Tinospora cordifolia callus material treated diabetic rats was estimated and presented in Table: The levels of serum cholesterol and blood urea were found to be elevated in alloxan induced diabetic rats when compared to normal. After treatment of Tinospora cordifolia extract, it brings back to near normal levels of cholesterol and blood urea in experimental rats. The callus material of Tinospora cordifolia was significantly (P< 0.01) decreased in serum cholesterol and blood urea level when compared to the other materials. Effect of Tinospora cordifolia plant extract on liver glycogen and glycosylated haemoglobine levels shown in Table: 4.33, in normal, diabetic, micropropagated Tinospora cordifolia and Tinospora cordifolia callus material treated with diabetic rats. The level of liver glycogen was changed over the experimental period. The liver glycogen was decreased in diabetic condition, at the same time the glycosylated 13

14 hemoglobin level was highly increased in diabetic rats when compared with normal. After treatment of Tinospora cordifolia stem methanol extract, the liver glycogen level was gradually increased in alloxan induced diabetic rats. The glycosylated hemoglobin level also significantly (P<0.01) decreased in the treatment of Tinospora cordifolia extract. The callus material of Tinospora cordifolia was highly significantely (P< 0.01) effective in reversing back of liver glycogen and glycosylated hemoglobin when compared to the other treatment like micro propagated and callus plant materials. Table: indicates the activity of serum glutamate pyruvate transaminase (SGPT) and liver glutamate pyruvate transaminase (GPT) in normal, diabetic, wild, micropropagated Tinospora cordifolia and Tinospora cordifolia callus material treated with experimental diabetic rats. The levels of SGPT and GPT were increased in alloxan induced diabetic rats when compared with normal. After the oral treatment of Tinospora cordifolia (500 mg/kg of b/w) elevated levels of SGPT and GPT were coming to the normal levels (P< 0.01). The decrease in SGPT and GPT levels in the treatment of callus material, Tinospora cordifolia, micropropagated and wild stem methanol extracts were effective. Table: shows the changes in hexokinase, fructose 1,6 -bi- phosphatase, glucose -6- phosphatase and glycogen synthase levels in liver sample of normal, diabetic, wild, micropropagated Tinospora cordifolia extract treated and Tinospora cordifolia callus material extract treated diabetic rats. The levels hexokinase and glycogen synthase were found to be decreased whereas the levels of fructose 1,6-biphosphatase and glucose -6- phosphatase were significantly raised when compared with normal. These altered enzyme activities in diabetic rats found to be corrected to near normal after the treatment with Tinospora cordifolia stem methanol extracts. The methanol extract of Tinospora cordifolia callus material was highly active when compared to the other experimental treatments ANTIOXIDANT ACTIVITY OF TINOSPORA CORDIFOLIA WILD STEM, MICROPROPAGTED PLANTS AND CALLUS MATERIAL IN ALLOXAN INDUCED DIABETIC RATS The levels of erythrocytes membrane lipid peroxide, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase activities in normal and experimental rats 14

15 were given in Table: The levels of lipid peroxidase and catalase activity were significantly increased in alloxan induced diabetic rats. Whereas the activity of superoxide dismutase, glutathione peroxidase were found to be decreased significantly when compared with normal rats. After Tinospora cordifolia extract treatment the altered levels of erythrocytes membranes lipid peroxide, catalase and the activity of superoxide dismutase, glutathione peroxidase were reverted back to near normal. The callus culture material of Tinospora cordifolia methanol extract was effectively altered the near normal condition of enzyme when compared with micropropagated and wild plant stem methanol extract. The levels of liver lipid peroxide, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase activities in normal and experimental rats were given in Table: The level of lipid peroxide was significantly increased in alloxan induced diabetic rats. Whereas the activity of SOD, CAT, glutathinone peroxidase where found to be decreased significantly when compared with normal rats. After Tinospora cordifolia extract treatment the levels of liver lipid peroxide and the activity of catalase, superoxide dismutase, and glutathione peroxidase were altered back to the near normal. The callus cultured material of Tinospora cordifolia methanol extract treatement was significantly altered the SOD, CAT, glutathinone peroxidase levels when compared with other materials of Tinospora cordifolia treatments IN SILICO DOCKING ANALYSIS OF BERBERINE AT DI-PEPTIDYL PEPTIDASE 4 PROTEIN Induced Fit Docking Induced Fit Docking study indicates the possibility of ligand docking into binding site of the receptor where the receptor was rigid and the ligand was free to move, that has been filtered out from many compounds using virtual screening. Further, it shows the binding between the ligand into a rigid receptor, which assumes the better one with low energy values. The purpose of this method was to eliminate the steric clashes, and then the appropriate interaction will be resulted (Wang et al., 2009). The IFD was performed after obtaining the least energy, after series of filtration from 15

16 HTVS and standard precision using GLIDE docking. The compounds reported to have low energy value of Kcal were reported in Table And Table From the analysis of the inhibitor bound to DPP4, it clearly states that the quinoline derivative group fits tightly into the hydrophobic pocket and also the amino group has some interaction with glutamic acid 205 and 206. The amine also exhibited hydrogen interaction with tyrosine 662 (Fig.4.26; Fig.4.27 and Fig. 4.28). These key interactions anchor the molecule into the DPP4 binding site. With the observation of these compounds, this projects to a large solvent-exposed werea and can occupy, shows many conformation of different groups including with polar functionality. Unlike the existing inhibitors, the serine 630 from the catalytic triad plays a crucial role in binding and combine with the fact that most of the key contacts with subsequent binding energy from this inhibitor within the active site of DPP4 derives from residues in the β-propeller domain (Table. 4.39). 16

17 Fig Protein (Di-peptidyl peptidase 4) ligand (berberine) interaction Fig Representation of protein appearance in Ribbon form Fig Representation of Protein ligand interaction with distance Lipinski s Rule of 5 This screening methodology states that poor absorption or permeation were more likely when a ligand molecule violates Lipinski s rule of five i.e., has more than 5 hydrogen bond donors, the molecular weight was less 500, the log P was over 5 and the sum of N and O was over 10. The Ligand of the present study has well qualified in Lipinski s filter VALIDATION OF IN-SILICO STUDIES The hypoglycemic effect of methanol extract of Tinospora cordifolia wild, micropropagated, callus materials and isolated Berberine in normal and alloxan induced diabetic rats in different time intervals, up to 8 hours of observed results were shown in Table The level of blood glucose was significantly decreased in the treatment of single dose of Berberine (10mg/kg of b/w.), with the maximum of reduction 74% was unchanged for 8 hours, at the same time the other treatments such as Tinospora cordifolia stem methanol extract of wild, micropropagated, callus material (500mg/kg of b/w.) also significantly decreased the blood glucose levels, and the maximum reduction to the level of 48%, 51%, and 65% respectively seen when compared with normal rats. 17

18 18