Chapter # 4 RESULTS AND DISCUSSION

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1 Chapter # 4 RESULTS AND DISCUSSION

2 Chapter # 4 RESULTS AND DISCUSSION The research work was initiated to evaluate the bioactivities of three plants namely Artocarpus altilis, Artocarpus heterophyllus and Piper betle in order to study their potential roles as antimicrobial, insecticidal, anti arthritic, anti inflammatory, antioxidant and antidiabetic agents. The plant kingdom holds many species of plants containing substances of medicinal interest which are yet to be investigated. Large numbers of plants are constantly being screened for their chemical and pharmacological properties. With the application of modern techniques of isolation and pharmacological evaluation many new herbal drugs find their way to medicine as purified substances 27. The pharmaceutical industries are developing many synthetic drugs that help to alleviate the chronic diseases. The adverse health effects due to the long term usage of synthetic drugs include severe side effects and resistance of microbes against these drugs. On the other hand synthetic drugs are expensive and their availability is also restricted to the affordable wealthier populations. In recent times, research on phytomedicines is happening at a faster pace all over the world. The herbal drugs are of tremendous importance in terms of safety, efficacy and cost effectiveness. The phytomedicines offer extraordinary benefits due to the combination of medicinal ingredients with vitamins, minerals and several other nutrients 85. Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen substituted derivatives. Polyphenols are widely distributed plant derived dietary constituents and have been implicated as the active components in a number of herbal and traditional medicines. More than five thousand plant polyphenols have been identified and several of them are known to possess a wide spectrum of pharmacological properties 93. Flavonoids are low molecular weight bioactive polyphenols and are widely distributed among the plant kingdom. Flavonoids have been reported to exert broad range of biological activities which includes vasodilatory, antibacterial, antiviral, anti allergic, cytotoxic, anti tumour, anti inflammatory action. In addition, flavonoids are known to inhibit lipid peroxidation, platelet aggregation, capillary permeability and fragility, 9

3 cyclo-oxygenase and lipoxygenase enzyme activities. They exert these effects as antioxidants, free radical scavengers, chelators of divalent cation. They are also reported to inhibit variety of enzymes like alkaline phosphatase, hydrolases, hyalouronidase, camp phosphodiesterase, lipase, alpha glucosidase, alpha amylase, kinase etc 56. The concentration of phytochemicals varies with different parts of the plant and hence the leaves of Artocarpus heterophyllus, fruit of Artocarpus altilis and roots of Piper betle were selected for the determination of flavonoids and their bioactivities. 4.1 EXTRACTION AND PHYTOCHEMICAL SCREENING OF PLANT MATERIAL Extraction is a very crucial step for the recovery of bioactive constituents. Suitable extraction of bioactive compounds was carried out by sequential extraction with solvents of different polarities. The solvents and the conditions used for extraction greatly influence the yield and bioactivities if the extracts 62. Aqueous, methanol, acetone and ethanol extracts were prepared and the yield of extract obtained from 1 g of dry plant material was measured for each extract. The percentage yield obtained using different solvents from all the three plants is represented in the Table The highest yield for all the selected plants was obtained from the solvent methanol. The methanolic extracts of Artocarpus altilis showed an yield of 15.5 %, Artocarpus heterophyllus (18.8 %) and Piper betle (17.6%) respectively. After obtaining the crude extract from plant material, phytochemical screening can be performed using the appropriate tests to get an idea regarding the type of phytochemicals existing in the extract mixture. Plant synthesizes a wide variety of chemical compounds which can be grouped according to their bio synthetic origin, chemical class and functional groups into primary and secondary metabolites 151. Knowledge of the chemical constituents of plants is essential for the discovery of therapeutic agents and such information will be valuable in disclosing new resources of such chemical substances. The phytochemical experiments performed on the three plant extracts showed the presence or absence of different constituents such as flavonoids, tannins, terpenoids, saponins, steroids and acid compounds in them (Table 4.1.2, and 4.1.4). These classes of compounds possess good antioxidant potential and have considerable effects on human nutrition and health. 91

4 Phytochemical screening of the different crude solvent extracts revealed that the methanolic extracts of Artocarpus altilis, Artocarpus heterophyllus and Piper betle showed presence of flavonoids in abundance. The polyphenolic extracts of plants are always a mixture of different classes of compounds which are selectively soluble in the solvents. The use of an alcoholic solution has shown to provide satisfactory results for the extraction process of polyphenols 142. Methanol was found to be the best solvent for extracting important polyphenolic compounds like flavonoids from Artocarpus altilis, Artocarpus heterophyllus and Piper betle. Variation in the polyphenolic contents is related to the biosynthesis in the plants which is influenced by a variety of agricultural and environmental factors including cultivar, climate, postharvest treatments etc. The variation of the polyphenolic contents between the plants may be due to phytochemical diversity as reported earlier by some workers. Phytochemicals mainly include polyphenolic compounds like saponins, tannins, catechins, isocatechins, alkaloids, coumarin, lignans, anthraquinones, anthocyanins, cardiac glycosides, cyanogenic glycosides and flavonoids, alkaloids and terpenoids etc. The solubility of each polyphenolic component depends on its physical property and chemical nature and varies from solvent to solvent. Majority of the components are soluble in organic solvents than in water and this might be the reason for extracting significantly higher amount of polyphenols in methanolic solvent than in water

5 Table 4.1.1: Percentage yield of the plant extracts with different solvents Methanolic Water Ethanolic Acetone Plants extract extract extract extract (%) (%) (%) (%) Artocarpus heterophyllus Artocarpus altilis Piper betle Table 4.1.2: Qualitative Phytochemical screening of different solvent extracts of Artocarpus heterophyllus Tests n- hexane Acetone Ethyl Methanol Water acetate Flavonoids Terpenoids Steroids Alkaloids Tannins Glycosides Saponins Carbohydrates Proteins : Not present; +: Present but low in abundance; ++: Present and moderate in abundance; +++: Present and high in abundance 93

6 Table 4.1.3: Qualitative Phytochemical screening of different solvent extracts Artocarpus altilis Tests n- hexane Acetone Ethyl Methanol Water acetate Flavonoids Terpenoids Steroids Alkaloids Tannins Glycosides Saponins Carbohydrates Proteins : Not present; +: Present but low in abundance; ++: Present and moderate in abundance; +++: Present and high in abundance Table 4.1.4: Qualitative Phytochemical screening of different solvent extracts Piper betle Tests n- hexane Acetone Ethyl Methanol Water acetate Flavonoids Terpenoids Steroids Alkaloids Tannins Glycosides Saponins Carbohydrates Proteins : Not present; +: Present but low in abundance; ++: Present and moderate in abundance; +++: Present and high in abundance 94

7 4.2 DETECTION OF LECTINS AND EVALUATION OF HAEMAGGLUTINATION ACTIVITY Lectins are a class of proteins of that bind carbohydrates without modifying them. Originally, the term lectin was restricted to soluble, multivalent proteins capable of agglutination and historically was limited to proteins of plant origin. However, today the term lectin is used in a broad sense to denote all types of carbohydrate-binding proteins that do not catalyze reactions with their ligands. There are numerous lectins available in the nature, and the fact that they can be easily prepared in the purified form and accessible for chemical manipulation makes them potential tools for biological research. The applications of lectins ranges from identification of microorganisms, cell surface biology studies to cancer research and they are serve as probes for the characterization and isolation of simple and complex sugars 118. The studies on detection of lectins revealed that in the methanolic extracts of Artocarpus heterophyllus, lectins were present in abundance; Artocarpus altilis extract had lectins in trace amounts whereas it was absent in the Piper betle extracts (Table 4.2.1). The haemagglutination activity tests done on the human blood groups A, B, AB and O showed that the methanolic extracts of Artocarpus heterophyllus exhibited strong haemagglutination activity, Artocarpus altilis extract showed weak agglutination and Piper betle extract showed no agglutination (Table 4.2.2). Various studies have shown that plants that are rich in anti nutritional compounds like lectins possess antimicrobial activity against a number of microorganisms 154. Antimicrobial activity of lectins is an interesting and important topic of study because of the abundant prevalence of pathogenic microorganisms in the environment. Plant sources can be used to reduce the pathogenic effect of such microorganisms as they are eco friendly and do not cause toxicity to the environment. The significance of lectins as biotechnological tools has been established by immunological studies conducted in order to determine the role of these proteins on the lymphocyte cell division. It was found that the lectin of Phaseolus vulgaris (red kidney bean), called as phytohemagglutinin (PHA), possessed the ability to stimulate lymphocytes to undergo mitosis 47. Studies have been performed to evaluate the role of lectins in immune response like the stimulation of cytokine secretion, activation of monocytes and 95

8 macrophages and reactive oxygen species production by splenocytes 113. Various investigators have reported the influence of lectins in the field of microbiology as valuable tools to verify the role of interaction between the pathogen and carbohydrates present in host cells and its importance in disease development. It has been documented that the pathogen Helicobcter pylori infects human cells through an interaction involving a lectin 19. Reports reveal that plant lectins can be used in the diagnosis and treatment of malignant tumors. In neoplastic cells the glycosylation of the proteins and lipids is changed which generates membrane signaling molecules capable of inducing several processes directly related to tumor progression such as cell adhesion, angiogenesis, cellular mitosis and metastasis 18. Neoplastic cells are associated with frequent changes in glycans and these may be considered specific to tumor cells. Some examples of these changes are the antigens of the ABH and Lewis system. These antigens are observed in several carcinomas and appear to be related to tumor progression 15. Due to the peculiar feature of specific binding to carbohydrates, lectins have been used as biomarkers to identify aberrant glycans expressed by neoplastic cells and therefore provide better possibility of accurate diagnosis and treatment. Plant lectins have been widely used to understand the pro inflammatory mechanisms and sources of new compounds with pro healing effect. de Oliveira Silva et al., demonstrated the immunomodulatory activity of ConBr, a lectin isolated from Canavalia brasiliensis seeds. The studies showed that ConBr was able to induce in vitro proliferation of spleen cells with minimal damage to the cell structure. These findings indicate the potential immunomodulatory effect of this lectin in relation with the intrinsic role of carbohydrates in intercellular communication related to the inflammatory process 47. The results of the present investigation clearly confirm the presence of lectins in abundance in the Artocarpus heterophyllus extracts therefore indicating its biological applications in medical diagnostics and therapeutics. 96

9 Table 4.2.1: Detection of lectins in methanolic plant extracts Artocarpus heterophyllus Artocarpus altilis Piper betle extract extract extract = Trace, +++ = Abundant, --- = Absent Table 4.2.2: Evaluation of haemagglutination activity of methanolic plant extracts Dilutions Artocarpus heterophyllus Artocarpus altilis Piper betle Blood Groups 1:2 1:4 1:8 1:16 1:2 1:4 1:8 1:16 1:2 1:4 1:8 1:16 A A B B AB AB O O : No agglutination, +: Weak agglutination, ++: Moderate agglutination, +++: Strong agglutination 97

10 4.3 DETERMINATION OF TOTAL PHENOLIC, FLAVONOID AND FLAVONOL CONTENTS AND IN VITRO ANTIOXIDANT ACTIVITY OF PLANT EXTRACTS Antioxidants are absolutely critical for maintaining optimal cellular and systemic health and well being. Antioxidant based drugs and formulations for the prevention and treatment of complex diseases like Alzheimer s disease and cancer have appeared during last three decades. Recent studies have shown that a number of phytochemicals including flavonoids, polyphenols, terpenes and various plant extracts exerted an antioxidant action 13. There is also a considerable amount of evidence revealing an association between individuals who have diet rich in fresh fruits and vegetables and the decreased risk of cardiovascular diseases and certain forms of cancer 167. There is currently immense interest in natural antioxidants and their role in human health and nutrition. Polyphenols are widely distributed secondary metabolites with antioxidant and antiradical properties. Results of the present study indicated that there was a close correlation between the antioxidant capacity and the amount of phenolics, flavonoids, and flavonols present in the plant. Total polyphenols play a vital role in anti oxidization as well as in the biological functions of the plant 32. Phenolic compounds are also very important plant constituents because their hydroxyl groups confer the scavenging ability and considerable in vitro antioxidant and antidiabetic activity 32. Phenolic compounds of plants are classified into several categories. The main among these are the flavonoids which have potent antioxidant activities 41. Flavonoids naturally occur in plants and are believed to have positive effects on human health and form important components of human and animal diet. Studies on flavonoid and its derivatives have shown a wide range of antibacterial, antiviral, anti inflammatory, anticancer, and antiallergic activities 41. Flavonoids have been shown to be very potent scavengers of most oxidizing molecules including singlet oxygen and various free radicals implicated in various diseases. So comparable with the findings in the literature for other plant extracts, results of this investigation suggested that phenolic acids, flavonoids and flavonols may be the major contributors for the antioxidant activity. It is known that different phenolic compounds show different responses in the Folin- 98

11 Ciocalteu method. Similarly the molecular antioxidant response of phenolic compounds also varies remarkably depending on their chemical structureand also due to the interference of other chemical components present in the extract such as sugars or vitamins like ascorbic acid 137. The concentration of phenolics in the methanolic extracts of Artocarpus heterophyllus, Piper betle and Artocarpus altilis was determined using Folin-Ciocalteu method and the flavonoid and flavonol contents were quantified by spectrophotometric method with aluminum chloride. The content of phenolics in extracts was expressed in terms of gallic acid equivalent (mg of GA/g of extract).the content of flavonoids was expressed in terms of quercetin equivalent (mg of QE/g of extract). The summary of quantities of total polyphenols identified in the three tested extracts is shown in Table The methanolic extract of Artocarpus heterophyllus showed the highest amounts of phenolics, flavonoids and flavonols followed by Piper betle and Artocarpus altilis extracts. The difference in the concentration of total polyphenols in the three plant extracts may be due to the difference in the parts of the plants used for the study. These data are in agreement with other studies reporting the total phenolic, flavonoid and flavonol concentration in different plant parts 145. The concentration of polyphenols in plant extracts depends on the polarity of solvents used in the extract preparation 174. Based on the obtained values of the concentration of total polyphenols in the examined extracts of Artocarpus heterophyllus, Piper betle and Artocarpus altilis, it was found that the highest concentration of these compounds in the extracts was obtained using alcoholic solvents like methanol. Several methods have been used to determine the in vitro antioxidant activity of plant extracts in order to allow rapid screening of substances with low and high antioxidant activity so that only the extracts with potent in vitro activity can be further confirmed using in vivo models 67. Free radicals are known to play an important role in a wide variety of pathological manifestations. Antioxidants fight against these free radicals and protect us from various diseases. They act either by scavenging the ROS or protecting the antioxidant defense mechanism 67. Metal chelating capacity is significant since it reduces the concentration of the transition metal that catalyzes lipid peroxidation. The electron donation ability of natural products can be measured by using methanol solution of 2,2 -diphenyl-1- picrylhydrazyl radical (DPPH). DPPH is a very stable free radical. The method relies on scavenging of DPPH through 99

12 the addition of a radical species or antioxidant that gives rise to the reduced form DPPH and decolourizes the DPPH solution with the loss of its violet colour. The degree of colour change is proportional to the concentration and potency of the antioxidants. A large decrease in the absorbance of the reaction mixture indicates significant free radical scavenging activity of the compound under investigation 144. Unlike in vitro generated free radicals such as the hydroxyl radical and superoxide anion DPPH has the advantage of being unaffected by certain side reactions such as metal ion chelation and enzyme inhibition. The antioxidant activity of the three extracts is expressed in terms of percentage of scavenging activity in Figure Parallel to examination of the antioxidant activity of plant extracts, the values of the standard compound BHT were obtained and compared to the values of the antioxidant activity. The examination of antioxidant activities of the three plant extracts showed different values. The obtained values varied from 12.5% to 8.2%. The largest capacity to neutralize DPPH radicals was found for A. heterophyllus extract, which neutralized 5% of free radicals at the concentration of μg/ml followed by Piper betle extract. A moderate activity was found for A. altilis extract. All the three methanolic plant extracts exhibited less capacity for neutralization of DPPH radicals compared to the standard BHT. In the present study among all the three methanolic plant extracts tested, A. heterophyllus extract showed significantly higher inhibition percentage and positively correlated with total phenolic content compared to Piper betle and Artocarpus altilis extracts. Results of this study strongly suggest that the plant extracts contain phytochemical constituents that are capable of donating hydrogen to a free radical to scavenge the potential damage. 1

13 Table 4.3.1: Total phenolic, flavonoid and flavonol contents in the methanolic plant extracts Crude methanolic plant extracts Total phenolics (mg GAE/g) Flavonoid (mg QE/g) Flavonol (mg QE/g) Artocarpus heterophyllus extract Artocarpus altilis extract ± ± ± ± ± ±.7 Piper betle extract ± ± ±1.5 Values are mean ± SD of triplicate analysis 9 8 Artocarpus heterophyllus Artocarpus altilis Piper betel BHT Scavenging activity (%) Concentration of plant extracts (µg/ml) Figure 4.3.1: DPPH radical scavenging activity of plant extracts (values are expressed as mean ± SD, n = 3) 11

14 4.4 ISOLATION AND PURIFICATION OF FLAVONOID FRACTIONS The methanolic extracts of Artocarpus heterophyllus, Artocarpus altilis and Piper betle were subjected to column chromatography and collected fractions were subjected to TLC analysis. The flavonoid fractions based on the R f values were pooled together (Figures ). The fractions were labelled as Jackfruit flavonoid fraction (JFF), Bread fruit flavonoid fraction (BFF) and Piper betle flavonoid fraction (PFF). JFF yielded five flavonoids: 1 (56 mg), 2 (98 mg), 3 (165 mg), 4 (8 mg), 5 (32 mg). PFF yielded eight flavonoids: 1 (152 mg), 2 (86 mg), 3 (23 mg), 4 (62 mg), 5 (6 mg), 6 (trace amounts), 7 (22 mg), 8 (trace amounts). BFF yielded five flavonoids: 1 (74 mg), 2 (12 mg), 3 (16 mg), 4 (29 mg), 5 (44 mg). The TLC plates which showed the presence of these compounds was examined under UV light (245 and 366 nm). 4.5 EVALUATION OF ANTIMICROBIAL ACTIVITY OF PLANT FRACTIONS There are many important reasons to screen for novel alternative antimicrobial substances from natural sources mainly plants. The toxicity and side effects of the drugs presently used in health care and medicine being a major area of concern. The generation of drugs in plenty from natural sources with more efficacy, low cost of production and low or negligible side effects has become a prime focus of the pharmacological industry. All over the world and also in the developing countries, most infectious bacterial diseases have resulted in the death of individuals. These organisms include Gram positive and Gram negative bacteria like different species of Staphylococcus, Bacillus, Pseudomonas and Salmonella which cause severe infections in humans 6. The development of synthetic antibiotics have been successful in eliminating these organisms to an extent but pose the limitations like development of drug resistance by microorganisms, high cost and adverse side effects on the host. In such scenarios, natural products which are a part of our daily diet serve as the best candidates for new antibacterial drug discovery. Phenolic compounds have various defensive functions in plants which include cell wall strengthening and repair and antimicrobial activities 1.The flavonoid fraction of Artocarpus heterophyllus (JFF) exhibited significantly higher growth inhibition 12

15 of microorganisms compared to Artocarpus altilis (BFF) and Piper betle (PFF) fractions at different concentrations and showed maximum inhibition zones at 1 μg /disc (Table 4.5.1). Zone of inhibition was found highest against B. subtilis (35.±1. mm) followed by S. aureus (3.±1. mm). Moderate antibacterial potential was observed against E. coli and P. aeruginosa (26.±1. mm). JFF significantly inhibited the growth of microorganisms at different concentrations and showed maximum inhibition zones at 1 μg /disc. Zone of inhibition was found highest against B. subtilis (31. ± 1. mm) followed by S. aureus (24.333±.577). Moderate antibacterial potential was observed against E. coli (2.±1. mm zone of inhibition) and P. aeruginosa (18.±1. mm). PFF significantly inhibited growth of microorganisms at different concentrations and showed maximum inhibition zones at 1 μg/disc. Zone of inhibition was found highest against B. subtilis (32.±1. mm) followed by S. aureus (28.±1. mm). Moderate antibacterial potential was observed against E.coli and P. aeruginosa (25.±1. mm). These results were compared with a standard antibiotic and it revealed that the antibacterial activities of the conventional antibiotics were higher than that of the plant fractions at the same concentration in accordance to literature.all the three plant fractions (JFF, BFF and PFF) showed considerable antifungal activity at different concentrations and showed maximum inhibition zones at 1 μg /disc (Table 4.5.2). JFF strongly inhibited the growth of tested fungal strains at all concentrations compared to BFF and PFF. JFF demonstrated excellent antifungal potential and inhibited the growth of Aspergillus niger (29. ± 1. mm zone of inhibition), Aspergillus flavus (26. ± 1. mm zone of inhibition), Penicillium sp. (24.± 1. mm zone of inhibition), Rhizopus sp. ( ±.577 mm zone of inhibition) at the concentration of 1 μg/disc. PFF was second most effective antifungal extract and showed zone of inhibition of 24.±1., 23.±1., 21.±1., 2.±1. mm against A.niger, A.flavus, Penicillium sp., Rhizopus sp. respectively. BFF demonstrated moderate activity and was found to be comparatively the least effective with a zone of inhibition of 2.± 1., 18.± 1., 17.± 1., 16. ±2. mm against A.niger, A.flavus, Penicillium sp., Rhizopus sp. respectively. These results were compared with a standard antifungal drug and it revealed that the antifungal activities of the conventional antifungal drugs were higher than 13

16 that of the plant fractions at the same concentration in accordance to reports from literature 12. The presence of antimicrobial substances in the higher plants is well known. Plants have served as the resources of novel drug compounds and plant derived medicines have made significant contribution towards human health. The results of present investigation clearly indicate that the antimicrobial activity vary with the species of the plants and plant material used. All the three plant fractions showed less activity in case of Gram negative bacteria E.coli and P. aeruginosa while the Gram positive bacteria B.sutilis were the most susceptible bacteria followed by S.aureus. It has been already reported by many workers that Gram positive bacteria are more susceptible towards plant extracts as compared to Gram negative bacteria.these differences may be attributed to fact that the Gram positive bacteria possess a single layer cell wall whereas the Gram negative cell wall is multilayered structure 135. Amongst the three plant species investigated, JFF showed the most remarkable activity against all the microorganisms. The antimicrobial activity of plant extracts has been attributed to the nature of biologically active components like carbohydrates, proteins, amino acids, alkaloids, flavonoids and phenolic compounds which have been reported to possess anti microbial activity 38. Flavonoids in plants have been classified under phenolic groups. The mechanism of action of antimicrobial flavonoids includes inhibition of cytoplasmic membrane function, energy metabolism and synthesis of nucleic acids. They also alter cell membrane permeability which could result in uncoupling of oxidative phosphorylation and inhibition of active transport. The preliminary phytochemical screening of the three plant extracts revealed the presence of flavonoids in abundance. Therefore the antimicrobial activity of the plant extracts may be due to the flavonoids present in them. The present investigation therefore ascertains the value of the plants understudy which could be of considerable interest for the development of new drugs to treat ailments caused by human clinical pathogens. 14

17 Table 4.5.1: Antibacterial activity of plant fractions Plant Fractions Concentration (μg/disc) Diameter of zone of inhibition (mm) S. aureus B.subtilis E. coli P. aeruginosa ±1. 16.± ± ±1. BFF 5 17.±1. 18.±1. 18.±1. 14.± ± ± ±1. 17.± ± ±1. 2.±1. 18.± ±1. 19.±1. 16.±1. 14.±1. JFF 5 27.±1. 22.±1. 19.±1. 19.± ±1. 28.±1. 24.±1. 21.± ±1. 35.±1. 26.±1. 26.± ±2. 17.±1. 17.±1. 17.±1. PFF 5 22.±2. 21.± ± ± ±1. 24.± ± ± ±1. 32.±1. 25.±1. 25.±1. Standard antibiotic ± ± ± ±2.517 Values are mean ± SD of triplicate analysis 15

18 Table 4.5.2: Antifungal activity of plant fractions Diameter of zone of inhibition (mm) Plant Fractions Concentration (μg/disc) A. niger A. flavus Penicillium sp. Rhizopus sp ± ± ± 2. 1.± 2. BFF ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1. JFF ± ± ± ± ± ± ± ± ± ± ± ± ± ±1. 1.±1. 12.±1. PFF ±.5 17.±1. 14.±1. 16.± ± ±1. 17.±1. 18.± ±1. 23.±1. 21.±1. 2.±1. Standard antifungal drug 5 35.±2. 3.±2. 28.±1. 38.±1. Values are mean ± SD of triplicate analysis 16

19 4.6 EVALUATION OF LARVICIDAL AND INSECTICIDAL ACTIVITY OF PLANT FRACTIONS Though larvicides play a vital role in controlling fruit flies (Drosophila melanogaster), these also show a negative impact in areas of beneficial and non target organisms. In view of an increasing interest in developing plant origin larvicides as an alternative to chemical larvicides, this study was undertaken to assess the larvicidal potential of the plant extracts against fruit fly larvae.in the present study, the effects of various extracts on the level of nucleic acids and protein were studied in D. melanogaster larvae in a dose dependent manner. The larvae of D. melanogaster (2 in each set) were treated with different concentrations of plant fractions for 24 and 48 hours. The control represents larval treatment with water. The effect of PFF and JFF on larval mortality is shown in figures and PFF and JFF possessed goog larvicidal activity whereas BFF extract did not exhibit any significant larvicidal activity. PFF and JFF had a reducing effect on the nucleic acid and protein content in the larvae in a dose dependent manner in correlation to their larvicidal activity. BFF did not show any significant reduction in the nucleic acid and protein content in the larvae (Figures ). The findings showed that PFF and JFF can be developed as ecofriendly larvicides. Nucleic acids and protein contents are regarded as important biomarkers of the metabolic potential of cells, as these play the main role in regulating the different activities of cells. Since insects have very little carbohydrate, protein is used to meet the increased energy demand. Proteins are mainly involved in the architecture of the cell which is the chief source of nitrogenous metabolism. The decreases in total protein level with the increasing dose of the plant fractions suggest the high protein hydrolytic activity due to elevation of protease activity. Inhibition of DNA synthesis, thus, might affect both protein as well as protein synthesis machinery 18. Screening of plant extracts or fractions for deleterious effect on insects is one of the important approaches in the search of novel biological insecticides. The use of synthetic compounds to control insect pests is associated with several adverse effects that include loss of efficacy, insect resistance, human and ecotoxicity, contamination of water and soil and toxicity to non target species 198. Therefore, there is an urgent need to develop safe, convenient, environmental and 17

20 low cost alternatives. Plant extracts are considered to be less toxic, non-pollutant and easily biodegradable. The insecticidal activity of fractions was studied against Bruchus pisorum, Tribolium castaneum, Sitophilus oryzae and is shown in Table and Figure PFF and JFF showed good insecticidal activity whereas BFF showed poor insecticidal activity against all the three tested insect species. JFF possessed excellent insecticidal activity against Sitophilus oryzae causing 8% mortality of this species. JFF also exhibited good (6%) activity against Tribolium castaneum but only a weak activity was observed towards Bruchus pisorum (4%). PFF exhibited a good mortality activity in case of Bruchus pisorum (8%) and Sitophilus oryzae (7%) and also caused lethality to 5% in case of Tribolium castaneum.bff showed weak activities against the tested insect species inhibiting the growth of Bruchus pisorum, Sitophilus oryzae and Tribolium castaneum respectively by 2, 1 and 1%. To overcome the increasing problems associated with the use of toxic synthetic products there has been an urgent need of developing safer, alternative crop protectants such as botanical insecticides, anti feedants and repellents. The insecticidal activity of a large number of polyphenolic compounds, essential oils and other plant extracts has been assessed against several major agricultural pests. The results of the present study indicate the antifeedant property of PFF and JFF which may be due to the different bioactive flavonoids present in them. The plant kingdom comprises a rich pool of phytochemicals that may be widely used instead of synthetic insecticides in pest control programmes. The efficacy of phytochemicals against mosquito larvae has been reviewed according to their chemical nature and the mosquito larvicidal potential of several plant derived secondary materials, such as lactones, alkanes, alkenes, alkynes, essential oils, fatty acids, terpenes, alkaloids, steroids, flavonoids and lignans. The isolation of several bioactive toxic principles from various plants and their toxicity against different mosquito species have been reported in literature 91. Generally the active toxic ingredients of plant extracts have been found to be secondary metabolites that are synthesized by the plants to protect them from herbivores. The insects feed on these secondary metabolites and are targeted by these toxic substances with relatively non specific effects on a wide range of molecular targets. These targets include nucleic acids, proteins (enzymes, receptors, signalling molecules, ion-channels and structural proteins), cell membranes and various other cellular components. This affects the insect 18

21 physiology in many different ways and at various receptor sites including the nervous system (neurotransmitter synthesis, storage, release, binding, and uptake, activation and function of receptor, enzymes associated with signal transduction pathway). The mechanism of action of plant secondary metabolites on insect system has been found to be due to several physiological disruptions such as inhibition of acetylecholinestrase, GABA-gated chloride channel, sodium and potassium ion pumps, cellular respiration, neuronal membrane action, blockage of calcium channels, disruption of hormonal balance, mitotic catastrophie, disruption of the molecular events of morphogenesis etc 151. The results obtained from the present study clearly indicate that PFF and JFF were quite effective as insecticides for providing a better and excellent alternate for the control of insects. 19

22 % Mortality % Mortality Concentration of PFF (ppm) Concentration of JFF (ppm) Figure 4.6.1: Mortality curve of Drosophila Figure 4.6.2: Mortality curve of Drosophila melanogaster larvae for the determination melanogaster larvae for the determination of of LC 5 of PFF (mean ± SD, n = 3) LC 5 of JFF (mean ± SD, n = 3) Protein (mg/ g wet tissue) control Concentration of PFF (ppm) Figure 4.6.3: Effect of PFF on the level of protein after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3) 11

23 2 1.8 Protein (mg/ g wet tissue) control Concentration of JFF (ppm) Figure 4.6.4: Effect of JFF on the level of protein after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3) Protein (mg/ g wet tissue) control Concentration of BFF (ppm) Figure 4.6.5: Effect of BFF on the level of protein after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3) 111

24 DNA (mg/ g wet tissue) control Concentration of PFF (ppm) Figure 4.6.6: Effect of PFF on the level of DNA after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3).25 DNA (mg/ g wet tissue) control Concentration of JFF (ppm) Figure 4.6.7: Effect of JFF on the level of DNA after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3) 112

25 .25 DNA (mg/ g wet tissue) control Concentration of BFF(ppm) Figure 4.6.8: Effect of BFF on the level of DNA after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3).3.25 RNA (mg/ g wet tissue) control Concentration of PFF (ppm) Figure 4.6.9: Effect of PFF on the level of RNA after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3) 113

26 .3.25 RNA (mg/ g wet tissue) Concentration of JFF(ppm) Figure 4.6.1: Effect of JFF on the level of RNA after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3) RNA (mg/ g wet tissue) control Concentration of BFF (ppm) Figure : Effect of BFF on the level of RNA after 48 hours exposure of Drosophila melanogaster larvae (values are expressed as mean ± SD, n = 3) 114

27 Table 4.6.1: Insecticidal activity of plant fractions against Bruchus pisorum, Tribolium castaneum and Sitophilus oryzae (values are expressed as mean ± SD, n = 3) Mortality (%) Plant Fractions Bruchus pisorum Tribolium Sitophilus oryzae castaneum JFF BFF PFF Control Permethrin (Reference insecticide) Bruchus pisorum Tribolium castaneum Sitophilus oryzae Figure : Insecticidal activity of plant fractions against Bruchus pisorum, Tribolium castaneum and Sitophilus oryzae (values are expressed as mean ± SD, n = 3) 115

28 4.7 BRINE SHRIMP CYTOTOXICITY AND PHYTOTOXICITY ASSAY OF PLANT FRACTIONS The brine shrimp lethality assay is a simple, rapid and inexpensive bioassay for testing the bioactivity of plant extracts and fractions which in most cases correlates reasonably well with cytotoxicity and anti tumor properties. The assay is considered a useful tool for preliminary assessment of toxicity and it has been used for the detection of plant extract toxicity, fungal toxins, heavy metals, pesticides etc 95. In the present study the brine shrimp lethality of JFF, BFF and PFF was determined using the procedure of McLaughlin 111. The fractions with LD 5 values higher than 1 µg/ml were considered to possess low toxicity whereas those with LD 5 values lower than 1 µg/ml were thought to possess significant toxicity 111. All the three fractions showed LD 5 values greater than 1 µg/ml thereby indicating that they do not possess cytotoxic activity (Table 4.7.1). All the three fractions exhibited no toxicity in pea seed bioassay at 1 µg/ml (Figures 4.7.1, and 4.7.3). Currently agents capable of inhibiting cell division, inducing apoptosis or modulating cell signalling have been used in cancer treatment. The evaluation of cytotoxic potential of plant extracts is necessary to ensure their antiproliferative property. Reports suggest that many active components in plants can act as apoptosis inducers and tumour suppressors in cancer cells. Allelopathy is the beneficial or harmful effect of one plant on another by release of inhibitory substances 145. In this mechanism plant release the phytototoxic chemicals into their surroundings and this exudation occurs from the roots that affects seeds and seedlings of other species located within a limited range. Allelopathic effect of crops and weeds has the aim of using naturally produced allelochemicals to reduce reliance on herbicides. There is evidence that many plant species have the potential to be used as excellent sources of natural chemicals that can be used in developing natural herbicides 147. The present study showed that there was no adverse effect on growth stimulation or inhibition indicating that the three plant fractions were non cytotoxic and did not possess anti tumor properties. There was no pronounced effect on the shoot growth and root growth by the fractions indicating that the three plant fractions were non-phytotoxic. 116

29 Table 4.7.1: Percentage mortality of brine shrimps at different concentrations of plant fractions and respective LC 5 values Plant fractions Mortality (%) 1 µg/ml 1 µg/ml 1 µg/ml LC 5 (µg/ml) JFF 26% 18.3% 1% >1 BFF 15.8% 12% 5.2% >1 PFF 32% 22% 14% > Root Length (cm) Control Fractions.4.2 JFF BFF PFF Figure 4.7.1: Effect of plant fractions on root elongation (Values are mean ± SD of triplicate analysis) 117

30 Shoot Length (cm) JFF BFF PFF Control Fractions Figure 4.7.2: Effect of plant fractions on shoot elongation (Values are mean ± SD of triplicate analysis) 12 1 Control 5 µg/ml 1 µg/ml Germination (%) JFF BFF PFF Figure 4.7.3: Effect of plant fractions on seed germination (Values are mean ± SD of triplicate analysis) 118

31 4.8 ENZYME INHIBITORY ASSAYS OF PLANT FRACTIONS Alpha amylase, Alpha glucosidase and Tyrosinase inhibitory activity For a long time natural products from plants have been used for the treatment of diabetes, mainly in developing countries where the resources are limited and affordability and access to modern treatment is a problem. Extensive research has been carried out to screen the bioactivity of these inhibitors because of their significant importance in health care and medicine. Plant food rich in polyphenols have been reported to cause effects similar to insulin in the utilization of glucose and act as good inhibitors of key enzymes like alpha amylase and alpha glucosidase associated with type 2 diabetes and lipid peroxidation in tissues 57. Studies have also shown that the bioactivity of polyphenols in plants is linked to their antioxidant activity and many of these plants also possess hypoglycaemic properties 9. Higher plants, animals and microorganisms are found to produce a large number of different protein inhibitors of alpha amylases and alpha glucosidases in order to regulate the activity of these enzymes. Some of these enzyme inhibitors act by directly blocking the active centre of the enzyme at various local sites. In animals alpha amylase inhibitors decrease the high glucose levels that can occur after a meal by slowing the speed with which alpha amylase can convert starch to simple sugars. This is of importance in diabetic people where low insulin levels prevent the fast clearing of extracellular glucose from the blood. Hence diabetics tend to have low alpha amylase levels in order to keep their glucose levels under control. Plants also use alpha amylase inhibitors as a defence mechanism as a protection from insects 12. These inhibitors alter the digestive action of alpha amylases and proteinases in the gut of insects and inhibit their normal feeding behaviour. Therefore alpha amylase inhibitors have potential roles in controlling blood sugar levels and crop protection. Alpha glucosidase inhibitors are used as oral antidiabetic drugs for treating type 2 diabetes mellitus. They act by preventing the digestion of carbohydrates such as starch. Carbohydrates are normally converted into simple sugars which can be absorbed through the intestine. Alpha glucosidase inhibitors act as competitive inhibitors of alpha glucosidase 119

32 enzyme needed to digest carbohydrates. The intestinal alpha glucosidases hydrolyze complex carbohydrates to glucose and other monosaccharides in the small intestine. Inhibition of these enzyme systems helps to reduce the rate of digestion of carbohydrates. Less amounts of glucose is absorbed because the carbohydrates are not broken down into glucose molecules. In diabetics the short term effect of these enzyme inhibitor drug therapies is to decrease high blood glucose levels. The presently used synthetic enzyme inhibitors cause gastrointestinal side effects such as diarrhea, flatulence, abdominal bloating etc. Therefore natural alpha amylase and glucosidase inhibitors from the dietary plants can be used as an effective therapy for treating post prandial hyperglycemia with minimal side effects 193. The inhibitory activity JFF, BFF and PFF on alpha amylase and alpha glucosidase was investigated in this study and the results are shown in Figures and JFF, BFF and PFF showed an IC 5 value of ±11.14, 7.58±9.66, ±13.9 µg/ml respectively in the alpha amylase inhibition assay and ±1.29, 76.9±9.55, 96.56±12.93 µg/ml respectively in the alpha glucosidase inhibition assay. JFF showed the greater percentage inhibition of the alpha amylase and alpha glucosidase enzymes compared to PFF and BFF. Percent alpha amylase and alpha glucosidase inhibition of the three fractions was plotted as a function of concentration in comparison with acarbose as shown in Figures and The results indicate that out of the three fractions, JFF exhibited good anti alpha amylase activity, PFF and BFF showed appreciable alpha amylase inhibitory activity. The findings also revealed that that the three fractions efficiently inhibited alpha glucosidase enzyme in vitro. There was a dose dependent increase in percentage inhibitory activity against alpha glucosidase by all the three plant fractions. In humans, increased activity of this enzyme leads to overproduction of melanin in epidermal layers causing hyper pigmentation of the skin. Hyper pigmentation can also be caused by excessive exposure to UV light, adverse drug reactions and melanoma. The compounds inhibiting melanin biosynthesis have proven to have application as whitening agents in the cosmetic industry. The tyrosinase inhibitor diminishes melanin production as the activity of this enzyme is the rate controlling step of melanin synthesis. Hence tyrosinase inhibitors can be clinically very useful for the treatment of some skin disorders associated with melanin hyper pigmentation and depigmentation after sunburns 187. The inhibitory activity 12

33 of JFF, BFF and PFF on tyrosinase was investigated in this study and the results are shown in Figure Percent tyrosinase inhibition of the three fractions was plotted as a function of concentration in comparison with L-ascorbic acid as shown in Figure It was found that out of the three plant extracts, PFF showed the maximum antityrosinase activity followed by BFF whereas JFF exhibited poor antityrosinase activity. The inhibition was increased with increasing concentration of fractions. JFF ( μg/ml), BFF (85.4 μg/ml) and PFF (67.6 μg/ml) showed 5% tyrosinase inhibition activity at the mentioned concentrations.the results of the present study indicate that out of the three plant fractions, PFF showed the maximum antityrosinase activity. PFF however exhibited lower antityrosinase activity than L-ascorbic acid, which is a potent tyrosinase inhibitor commonly used in skin whitening cosmetic products. With respect to the results of this investigation, PFF can probably serve as therapeutic or cosmetic agent and can be used as potential sources of novel tyrosinase inhibitor for treating variety of skin related disorders. 121

34 IC5 (µg/ml) IC5 (µg/ml) BFF PFF JFF Acarbose BFF PFF JFF Acarbose Figure 4.8.1: Effect of plant fractions on Figure 4.8.2: Effect of plant fractions on alpha alpha amylase inhibition (mean ± SD, n = 3) glucosidase inhibition (mean ± SD, n = 3) IC5 (µg/ml) JFF PFF BFF Ascorbic acid Figure 4.8.3: Effect of plant fractions on tyrosinase inhibition (Values are expressed as mean ± SD, n = 3) 122

35 4.9 GLUCOSE UPTAKE ASSAY BY YEAST CELLS AND EVALUATION OF EFFECT OF PLANT FRACTIONS ON HAEMOGLOBIN GLYCOSYLATION Regulation of glucose level in the blood of the diabetic patient in can prevent the various complications associated with the disease. The long term maintenance of plasma glucose concentration under a variety of nutritional conditions is one of the most important and closely regulated processes in the mammalian species. In Yeast (Saccharomyces cerevisiae), glucose transport takes place through facilitated diffusion 3. Type 2 Diabetes is characterised by the deficiency of insulin causing increased amount of glucose in blood. After the treatment of the yeast cells with the three plant fractions, the glucose uptake was found to increase in a dose dependent manner. Figures a, b, c depict the percentage increase in glucose uptake by the yeast cell at different glucose concentration i.e. 25 mm, 1 mm and 5 mm respectively. JFF exhibited significantly higher activity BFF and PFF at all glucose concentrations showing the maximum increase at 1 mm Glucose concentration i.e % increase at 2 µg/ml of the plant fraction (Figure b). Results also indicated that JFF and BFF are efficient in increasing the glucose uptake by yeast cells as compared to standard drug metronidazole. The in vitro glucose uptake assays indicated that all the three fractions have good antidiabetic activity. The amount of glucose that remains in the medium after a specific time is as an indicator of the glucose uptake by the yeast cells. The rate of uptake of glucose into yeast cells wasfound to be linear in all the three glucose concentrations. JFF exhibited significantly higher activity than PFF and BFF at all concentrations. However the highest percentage of glucose uptake was seen in 1 mm glucose concentration. Recently, the mechanism of glucose transport across the yeast cell membrane has been receiving attention as in vitro screening assay for evaluating the hypoglycaemic effect of various compounds and medicinal plants. The studies on the transport of non metabolizable sugars and certain metabolizable glycosides suggest that glucose transport across the yeast cell membrane is mediated by certain stereospecific membrane carriers. It is reported that in yeast cells sugar transport is an extremely complex process. The glucose is transported in yeast cells by a facilitated diffusion process. Facilitated diffusion is mediated by carrier proteins that transport solutes down the concentration 123

36 gradient. This suggests that effective transport is only attained if there is removal of intracellular glucose 58. An increase in the glycosylation of the haemoglobin was observed when haemoglobin was incubated with increasing concentration of the glucose (2 mg/ml, 6 mg/ml and 1 mg/ ml) over a period of 72 hours (Figure 4.9.2). However the plant fractions significantly inhibited the haemoglobin glycosylation which is indicated by the presence of increasing concentration of haemoglobin (Figure 4.9.3). JFF exhibited higher inhibition of glycosylation as compared with the standard gallic acid. JFF, BFF and PFF also displayed the inhibition of haemoglobin glycosylation at different concentrations of the glucose over the period of 72 hours, indicating that the plant fractions decreases the formation of the glucose- haemoglobin complex and thus free haemoglobin increases. Increased concentration of glucose in the blood causes its binding with the hemoglobin which may result in the formation of the reactive oxygen species.the plant fractions can therefore cause the inhibition of the glycosylation end products. The results suggest that the plant fractions inhibit the binding of glucose to hemoglobin, since at higher concentration of glucose the concentration of haemoglobin was found to be higher. Literature reports have shown that the formation of advanced glycated end products is now known to be the source of free radicals which eventually aggravates the state of an increased oxidative stress in diabetes mellitus 171. The inhibition of formation of glycated end products implies lower level of free radicals in diabetes and hence reduces diabetic complications. In the management of diabetes appropriate attention has to be been given to the reducing properties of monosaccharides. In addition to direct glycosylation reactions, monosaccharides can enolize and thereby reduce molecular oxygen under physiological conditions, yielding alpha ketoaldehydes, hydrogen peroxide and free radical intermediates 193. In vivo the occurrence of this process leads to the elevated plasma peroxides found in diabetics complication and may also contribute to protein modification reactions. Thus the high levels of glucose in the blood and its relation with erythrocytes has become important in studying the pathophysiological mechanisms of diabetes, as several abnormal features of red blood cells have been demonstrated in diabetes mellitus patients 157. It is evident from the results of the present study that the administration of JFF significantly inhibits glycosylation of haemoglobin compared to PFF and BFF and as such the formation of advanced glycated end products may be inhibited by the plant fraction. This observed effect might be attributed by the presence of unique bioactive flavonoids in the fraction. 124

37 % Increase in Glucose Uptake % Increase by STD (Metronidazole) % Increase by BFF % Increase by JFF % Increase by PFF Concentration of fractions (µg/ml) % Increase in Glucose uptake % Increase by STD (Metronidazole) % Increase by BFF % Increase by JFF % Increase by PFF Concentration of fractions (µg/ml) % Increase by STD (Metronidazole) % Increase by BFF % Increase in Glucose uptake % Increase by JFF % Increase by PFF Concentration of fractions (µg/ml) Figure 4.9.1: The comparative percentage (%) increase in glucose uptake by yeast cells due to the effect of plant fractions and reference Standard (Metronidazole) (values are expressed as mean ± SD, n = 3) a: 25 mm glucose concentration; b: 1 mm glucose concentration; c: 5 mm glucose concentration 125

38 1.2 1 Absorbance at 443nm mg/ml glucose concentration 6 mg/ml glucose concentration 1 mg/ml glucose concentration.2 24 hrs 48hrs 72hrs Incubation time period (hours) Figure 4.9.2: Estimation of haemoglobin glycosylation over a period of 72 hours (values are expressed as mean ± SD, n = 3) Absorbance at 443nm hrs 48hrs 72hrs Standard (Gallic acid) BFF JFF PFF Figure 4.9.3: Effect of plant fractions on haemoglobin glycosylation as compared with standard Gallic acid (values are expressed as mean ± SD, n = 3) 126

39 4.1 EVALUATION OF IN VITRO ANTI ARTHRITIC AND ANTI INFLAMMATORY ACTIVITY OF PLANT FRACTIONS Rheumatoid arthritis is a type of auto immune disease and constitutes a major cause of disability in individuals. Pathogenesis of rheumatoid arthritis is associated with auto antibodies, immune complexes, T-cell-mediated antigen-specific responses and T-cell-independent cytokine networks. Based on the pathophysiological mechanisms specific treatment interventions can be designed and developed to suppress joint destruction and synovial inflammation in rheumatoid arthritis. Modern medicine uses various drugs like Methotrexate, Dexamethasone, Phenylbutazone and Indomethacin to relive the pain and inflammation in Rheumatoid arthritis which includes characteristic side effects. In recent years there is an interest in the clinical usage of indigenous drugs in chronic arthritic disorders because of their efficacy and being free from serious toxic effects. Most of the investigators have reported that denaturation of protein, membrane lysis and proteinase action are the cause of rheumatoid arthritis. It has been reported the production of auto antigens in certain arthritic disease may be due to membrane lysis, denaturation of protein and proteinase action 23. Production of auto antigens in certain rheumatic diseases may be due to in vivo denaturation of proteins. Mechanism of denaturation probably involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding 23. The results for the inhibition of the protein denaturation are presented in Figure The maximum percentage inhibition of protein denaturation membrane stabilisation and proteinase inhibitory action were shown by JFF. It was observed that only JFF exhibited a considerable inhibition of protein denaturation as compared with the standard i.e % and 84.27% inhibition at 2 µg/ml respectively. PFF and BFF showed moderate protein denaturation inhibition activity as compared to the standard. All the three fractions at 2 µg/ml significantly protected the lysis of the human erythrocyte membrane induced by hypotonic solution. JFF and PFF showed the greater membrane stabilisation activity than BFF (Figure 4.1.2). In the screening for the proteinase inhibitory activity, JFF (73.36%) showed significant inhibition of the proteinase as compared with the standard Diclofenac sodium (83.74%) at 2 µg/ml concentration. BFF and PFF showed considerably less proteinase 127

40 inhibition activity than JFF (Figure 4.1.3). The results of the study reveals that JFF is capable of controlling the production of auto antigen and inhibits denaturation of protein, membrane lysis and proteinase action in rheumatic disease. JFF, BFF and PFF were subjected to erythrocyte membrane stabilization induced haemolysis by using a hypotonic solution. The cell vitality depends on the integrity of their membranes and exposure of erythrocytes to hypotonic medium results in lysis of its membrane due to osmotic imbalance and is accompanied by haemolysis and oxidation of haemoglobin. An injury to erythrocyte membrane will further render the cells more susceptible to secondary damage through free radical induced lipid peroxidation 19. It is therefore expected that phytochemicals and their synergistic action with membrane stabilizing properties should offer significant protection to the cell membrane against injurious substances. Plant bioactive compounds with membrane stabilizing properties are well known for their ability to interfere with release of phospholipases that trigger the formation of inflammatory mediators 48. Studies on the membrane stabilizing activity of Phyllanthus amarus have shown that an increase in the surface area to volume ratio of the cells could be brought about by an expansion of membrane or shrinkage of the cell and an interaction with membrane proteins 17. This mechanism may be applicable in our studies as it has also been shown that the deformability and cell volume of erythrocytes is found to be closely related to the intracellular content of calcium. A possible explanation for the stabilizing activity of JFF, BFF and PFF is due to an increase in surface area or volume ratio of the cells which could be brought about by an expansion of membrane or cell shrinkage and an interaction with membrane protein 48. Similar studies on in vitro anti inflammatory activity of leaf extracts of Basella also exhibited membrane stabilization effect by inhibiting hypotonicity induced lysis of erythrocyte membrane 97. Stabilization of cell membrane is important in limiting the inflammatory response by preventing the release of lysosomal constituents such as bactericidal enzymes and proteases of activated neutrophils which cause further tissue inflammation and damage. The membrane stabilizing activity of JFF, BFF and PFF may be due to the presence of active flavonoids. JFF, BFF and PFF were then subjected to the evaluation of prevention of protein denaturation effect on bovine serum albumin solution. Several anti inflammatory drugs have shown dose dependent ability to inhibit thermally induced protein denaturation 13. Denaturation of proteins is a well proved 128

41 cause of inflammation in conditions like rheumatoid arthritis 23.Thus protection against protein denaturation may play an important role in the antirheumatic activity of NSAIDS. Studies have been reported that methanolic extract of Murraya koenigi leaves produces significant antiinflammatory activities in dose dependent manner in membrane stabilization and inhibition of protein denaturation 17. The ability of plant extracts to bring down thermal denaturation of proteins is possibly contributed for its anti inflammatory activity. Therefore it was found that JFF, BFF and PFF eventually led to effective RBC membrane stabilization and inhibition of protein denaturation, both contributing to its in vitro anti arthritic and anti inflammatory activity. Many Ayurvedic practitioners in India are using various indigenous plants for the treatment of different types of arthritic conditions. The scientific research to elucidate the actual efficacy and other limitations of these herbal drugs would definitely widen their scope for future use if they are proved to be really effective. This is very important for the problem of rheumatism and arthritis due to the absence of the precise synthetic drugs for its treatment. The currently available drugs are able to provide only symptomatic relief and are also not free from side effects. Therefore the target strategy should be to discover newer drugs from plant kingdom which may provide appropriate therapeutic cure and would be free from undesirable side effects and economical. From the results of present study it is shown that out of the three plant fractions, JFF possessess significant anti arthritic and anti inflammatory activity and hence could be beneficial for further work as an active anti arthritic and anti inflammatory agent. 129

42 % Inhibition by STD (Diclofenac sodium) % Inhibition by Piper betle % Inhibition by A. heterophyllus % Inhibition by A. altilis 9 % protein denaturation inhibition Concentration of plant fractions (µg/ml) Figure 4.1.1: Effect of plant fractions and standard drug on inhibition of protein denaturation (mean ± SD, n = 3) % stabilisation by STD (Diclofenac sodium) % stabilisation by Piper betle % stabilisation by A. heterophyllus % stabilisation by A. altilis % membrane stabilisation Concentration of plant fractions (µg/ml) Figure 4.1.2: Effect of plant fractions and standard drug on membrane stabilization (mean ± SD, n = 3) 13

43 % Inhibition by STD (Diclofenac sodium) % Inhibition by Piper betle % Inhibition by A. heterophyllus % Inhibition by A. altilis 9 8 % proteinase inhibition Concentration of plant fractions (µg/ml) Figure 4.1.3: Effect of plant fractions and standard drug on proteinase inhibition (values are expressed as mean ± SD, n = 3) 131

44 4.11 EVALUATION OF IN VIVO ANTI DIABETIC AND ANTI OXIDANT ACTIVITY OF PLANT FRACTIONS Diabetes mellitus is a chronic disorder which results in increased concentration of glucose in the blood and in turn causes damage to many of the body s system mainly the blood, nerves, kidney, pancreas etc. It is associated with disturbances in carbohydrate, lipid, and protein metabolism and characterized by changes in oxidative stress biomarkers, including superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione levels, vitamins, lipid peroxidation, nitrite concentration, non enzymatic glycosylated proteins and hyperglycemia. Both experimental and clinical studies suggest that oxidative stress plays an important role in the pathogenesis of both types of diabetes mellitus 155. Free radicals are formed in diabetes by glucose oxidation, non enzymatic glycation of proteins and the subsequent oxidative degradation of glycated proteins. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to increased lipid peroxidation, damage of cellular organelles and enzymes and development of insulin resistance. These consequences of oxidative stress can increase the development of complications of diabetes mellitus 171. Under diabetic conditions, ROS are produced mainly through the glycation reaction which occurs in various tissues. In diabetes mellitus alterations in the endogenous free radical scavenging defense mechanisms may lead to ineffective scavenging of reactive oxygen species thereby resulting in oxidative damage and tissue injury. The mechanism of implication of oxidative stress in the pathogenesis of diabetes is suggested by oxygen free radical generation, non enzymatic protein glycosylation, auto oxidation of glucose, impaired glutathione metabolism, alteration in antioxidant enzymes, lipid peroxides formation and decreased ascorbic acid levels. GSH and other defense mechanisms against free radicals like the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) help to eliminate superoxide, hydrogen peroxide and hydroxyl radicals. The levels of these antioxidant enzymes greatly influence the susceptibility of various tissues to oxidative stress and are associated with the development of complications in diabetes. This is particularly relevant and dangerous for the pancreatic beta islet which is one of the tissues that has the lowest levels of intrinsic antioxidant defences

45 The flavonoid fractions of the three plants namely JFF, BFF and PFF demonstrated significant in vivo antioxidant and antidiabetic effects at 3 mg/kg on STZ induced diabetic rats. Acute toxicity studies revealed the non toxic nature of the methanolic extracts of the plants. There was no lethality or any toxic reactions found with the selected dose until the end of the study period. The diabetic rats were found to ingest greater quantity of diet compared to control rats (2 g / day in the first week to 4 g/day in the sixth week). It was observed that the rats treated with JFF showed a marked normalisation of food intake (2 g /day in the first week to 25 g/day in the sixth week, 2 g / day in the first week to 24 g/day in the sixth week for BFF and 2 g/day in the first week to 25 g/day in the sixth week for PFF. The rats treated with standard drug Glibenclamide (1 mg/kg) also showed a reduction in food uptake (2 g/day in the first week to 3 g/day in the sixth week). The results from the study clearly indicated that the flavonoid fractions of the three plants exhibited significant hypoglycaemic activity in STZ induced diabetic rats. At the end of 45 days treatment, the body weight of normal rats, in diabetic groups treated with JFF, BFF and PFF and in standard drug treated group increased significantly by 182, 174, 163, 177 mg/kg respectively whereas there was a decrease in the body weight in the diabetic control (135 mg/kg) as shown in figure The diabetic group treated with JFF and PFF showed considerable increase in body weight which was comparable to that of normal control group and higher that the diabetic rats treated with drug. The diabetic group treated with BFF showed less increase in body weight compared to the diabetic group treated with JFF and PFF. The blood glucose levels in the rats were analysed after fasting them overnight. The fasting blood glucose levels of normal rats on 45 th day were found to be mg/dl. There was a significant lowering of blood glucose levels in the rats treated with Glibenclamide in comparison to diabetic rats. The results shown in figure indicate that in comparison to the normal drug those rats which were treated with JFF and PFF showed normal levels of the sugar. Though BFF treated rats showed a reduction in blood glucose levels, it was still in the diabetic range. The results indicate that treatment with JFF and PFF has significant effects on normalisation of blood glucose levels. Treatment with all the three fractions significantly increased plasma insulin levels beyond those observed in diabetic control and drug treated group. (Figure ). Glycogen levels in the liver of all the 133

46 groups of rats were measured on the 45 th day. The glycogen levels were significantly reduced in diabetic rats in comparison to normal rats. Rats treated with Glibenclamide were found to have increased levels of glycogen levels compared to diabetic rats. A significant increase in the liver glycogen was seen in the rats treated with JFF and PFF whereas animals treated with BFF showed decreased levels of liver glycogen with respect to the control group. But this was not restored to the normal group glycogen level (Figure ). In the view of glycogen level, there may be three possible ways of antidiabetogenic action; one possible way may be increased insulin level. Other possible ways of antidiabetic action of fractions may be by preventing the inactivation of the glycogen synthetase and by synthesize the glycogen synthetase 197. Non enzymatic glycosylation is the deleterious binding of sugars to protein that is commonly observed in diabetes and aging. In diabetes mellitus, the non enzymatic glycosylation reaction has been stepped up due to elevated concentration of glucose in the blood. Some glycosylated proteins are digested by macrophages, lysosome containing cells or extracellular proteolytic systems. Similarly, non enzymatic glycosylation reaction occurs in the protein in plasma containing haemoglobin. After glycosylation, haemoglobin is digested by macrophages and total haemoglobin levels are decreased significantly. Haemoglobin carries oxygen from lungs and releases carbondioxide to lungs. The decrease in total haemoglobin level induces tissue hypoxia 3. The total haemoglobin levels and glycated haemoglobin (HbAIC) levels were measured on the first day and the 45 th day respectively. Diabetic rats showed a significant decrease in the level of total haemoglobin and significant increase in the level of glycated haemoglobin. The administration fraction and glibenclamide to diabetic rats restored the changes in the level of total hemoglobin and glycosylated haemoglobin to near normal levels (Figure a and b). This could be due to the result of improved glycemic control produced by the fractions. In diabetes the excess glucose present in blood reacts with haemoglobin. Therefore, the total haemoglobin level is decreased in diabetic rats. The glycated haemoglobin level was increased significantly in the diabetic group in comparison with the control group. After treatment with the fractions and glibenclamide, the levels of glycated haemoglobin were resettled towards the control group. Administration of JFF, BFF and PFF prevented a significant elevation in 134

47 glycosylated haemoglobin thereby increasing the level of total haemoglobin in diabetic rats (Figure b). In uncontrolled or poorly controlled diabetes, an increased glycosylation of a number of proteins including haemoglobin has been observed. Glycation of proteins is a frequent phenomenon, but in the case of hemoglobin a non-enzymatic reaction occurs between glucose and the N-end of the beta chain. The level of glycosylated haemoglobin level in diabetic patients is increased to approximately 16 %. In diabetes the excess glucose present in blood reacts with haemoglobin. When blood glucose levels are high, glucose molecules attach to the hemoglobin in erythrocytes. The longer hyperglycemia occurs in blood, more glucose will bind to erythrocytes and higher will be the the glycosylated hemoglobin levels. The rate of formation of glycated hemoglobin has been observed to be proportional to blood glucose level 9. It reveals the integral blood glucose concentration over a period of time. The assessment of HbAIC level is of interest as an important means of determining the efficacy of blood glucose clearance by antidiabetic agents. It is considered a reliable index in glycaemic control 92. In the diabetic animals, HbAIC level increased significantly suggesting glycosylation of hemoglobin in the presence of hyperglycaemia. Glycosylated hemoglobin shows reduced affinity to oxygen 24, a process that generates free radicals. A number of medicinal plants have been reported to reduce HbAIC formation 1. In the fraction and glibenclamide treated groups marked decrease in HbAIC concentration was observed when compared to that of diabetic animals indicating decrease in blood glucose level and recovery of hemoglobin. All the three fractions were shown to reduce the HbAIC levels, BFF however showed more effective decrease compared to JFF and PFF confirming its antidiabetic potency. 135

48 Body weight (g) a Body weight (g) b a: Changes in body weight on day 1 b: Changes in body weight on 27 th day 25 c Body weight (g) c: Changes in body weight on 45 th day Figure : Changes in body weight due to the treatment of JFF, BFF and PFF in normal and experimental animals (mean ± SEM) 136

49 Fasting blood glucose (mg/dl) Plasma insulin (µu/ml) Figure : Effect of fractions on fasting blood glucose in normal and and experimental animals on 45 th day Figure : Effect of fractions on plasma insulin levels in normal and experimental animals (mean ± SEM) post treatment (mean ± SEM) Glycogen levels (mg/g) Figure : Liver glycogen levels in normal and experimental animals (mean ± SEM) 137

50 Haemoglobin level (Day ) mg/dl Figure a: Level of total haemoglobin in normal and experimental animals (mean ± SEM) Glycated Haemoglobin level (Day 45) mg/dl Figure b: Level of glycated haemoglobin in normal and experimental animals (mean ± SEM) 138

51 The levels of serum lipids are usually elevated in diabetes mellitus and such an elevation represents a risk factor for coronary heart disease. This abnormal high level of serum lipids is mainly due to the uninhibited actions of lipolytic hormones on the fat depots mainly due to the action of insulin. Under normal circumstances, insulin activates the enzyme lipoprotein lipase, which hydrolyses triglycerides. However in diabetes lipoprotein lipase is not activated due to insulin deficiency resulting in hypertriglyceridaemia. Also insulin deficiency is associated with hypercholesterolaemia. Insulin deficiency may be responsible for dyslipidaemia, because insulin has an inhibitory action on HMG-CoA reductase, a key rate-limiting enzyme responsible for the metabolism of cholesterol rich LDL particles. The mechanisms responsible for the development of hypertriglyceridemia and hypercholesterolemia in uncontrolled diabetes in humans are due to a number of sequentially occurring metabolic abnormalities 9. In this study, diabetic rats showed hypercholesterolaemia and hypertriglyceridaemia and the treatment with plant fractions significantly decreased both cholesterol and triglyceride levels. Elevated levels of cholesterol and triglycerides were observed in the diabetic group. (Figures and ) Elevated levels of phospholipids were seen in diabetic rats. A significant reduction in the levels of phospholipid was seen in the diabetic rats treated with JFF. PFF treated diabetic rats also showed considerable lowering of phospholipid levels whereas an elevated phospholipid level was seen in BFF treated rats (Figure ). Phospholipids are components of cell membrane and are most abundant of the membrane lipids. These are highly susceptible to free radicals and their levels in blood increases when there in high free radical production. Studies have shown that during the progression of diabetes there is a marked increase in the free radical production 1. Therefore, reducing the blood lipid level is a target for delaying the complications of diabetes. The present study shows the efficacy of JFF and PFF in normalising the levels of phospholipids. Therefore it can be suggested that the fractions normalises the phospholipid levels by increasing the antioxidant status in the cell. 139

52 Total cholesterol (mg/dl) Triglycerides (mg/dl) Figure : Total blood cholesterol levels in normal and experimental animals Figure : Level of triglycerides in normal and experimental animals (mean ± SEM) (mean ± SEM) Phospholipids (mg/dl) Figure : Phospholipid levels in normal and experimental animals (mean ± SEM) 14

53 The three flavonoid fractions (JFF, BFF and PFF) were studied for its antioxidant effects by investigating hepatic tissue and pancreatic tissue peroxidation, hydroperoxide levels, TBARS and activities of antioxidant enzymes, glutathione S-transferases, glutathione peroxidase, SOD, catalase. Antioxidant actions of food may be through inhibitory actions on ROS or by direct scavenging of free radicals. The antioxidant enzymes (AOE) include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidise (GPx) and indirectly glutathione reductase (GR). Their role as protective enzymes are well known and have been extensively investigated both in in vivo and in vitro models 133. Lipid peroxide mediated tissue damage has been observed in the development of type I and type II Diabetes mellitus. The results of fraction induced changes on the antioxidant defense system has been evaluated by estimating TBARS and HPx levels in liver and pancreas of fraction treated rats. The results show statistically significant increases in levels of these lipid peroxidation biomarkers in the liver and pancreas of diabetic control rats (Figures , and ). Glucose auto oxidation generates these free radicals and can cause lipid peroxidation. In this study, liver and pancreas TBARS and HPx levels were significantly lowered in fraction treated groups compared to diabetic rats indicating reduced lipid peroxidation mediated tissue damage. The levels of endogenous antioxidants may also be upregulated by increasing expression of the genes encoding the antioxidant enzymes SOD, CAT and GPx. AOE and antioxidant molecules can inhibit free radical production by chelating the transition metal catalysts, breaking chain reactions, reducing concentrations of ROS and/or scavenging initiating radicals 193. In the present study, treatment of rats with fractions significantly increased rat liver, serum and pancreas GST, SOD, CAT and GPx activities (Figures , , and ). There were significant increase in all hepatic CAT, SOD, GST and GPx activities in rats administered with JFF and PFF suggesting that liver plays predominant role in protection against free radicals. Although similar significant levels of elevations were observed in GPx, CAT and SOD activities in different tissues of in rats administered with JFF and PFF. The maximum AOE activity was observed in JFF treated rats. SOD, CAT and GPx are major free radical scavenging enzymes that have shown to be reduced in a number of pathophysiological processes and diseases such as diabetes. Thus, activation of these AOE by the administration of fractions clearly shows their free radical 141

54 scavenging activity which could exert a beneficial action against pathophysiological alterations caused by the presence of superoxide and hydroxide radicals. In addition JFF caused significant elevations on serum, hepatic and pancreatic GSTs which are responsible for the metabolism of numerous xenobiotics and play a major cellular antioxidant role. GSTs also have peroxidase and isomerise activity. They bind covalently with reactive metabolites formed from carcinogens and non covalently with lipophilic compounds and offer protection against oxidative stress 85. The screening of antioxidant properties of plants have been performed increasingly in the last few decades with the hope of finding an effective remedy for several diseases related to oxidative stress like type 2 diabetes, neurodegenerative diseases, and some types of cancer. There is also a huge demand for natural antioxidants in food industries for replacing the synthetic preservatives. Bioactive compounds from plants function as small molecular weight antioxidants through direct antiradical, chain-breaking of the free radical generation and interaction with transition metals. Other mechanisms include the inhibition of ROS generating enzymes such as xanthine oxidase, inducing nitric oxide synthase, and improving the endogenous cellular antioxidant mechanisms such as the up-regulation of the activity of SOD 65. Furthermore phenolic compounds function as powerful antioxidants as they possess the ability to quench and neutralize free radicals and reactive oxygen species. Flavonoids are one of the most diverse and widespread groups of secondary metabolites which are probably the most natural phenolics capable of exhibiting in vitro and in vivo antioxidant activities. Plant flavonoids which show an antioxidant activity in vitro have been found to function as antioxidants in vivo as well. A strong corelation between the total phenolic content and antioxidant activity in fruits, vegetables, grain products, and plant subjects of ethnopharmacological treatments has also been reported 93. Therefore, the in vitro antioxidative effect of BFF, JFF and PFF may be due to the presence of important bioflavonoids. The enzymic antioxidant systems such as catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase exhibit a coordinated role in the prevention of oxidative damage by ROS and thereby help to maintain a balanced redox status. Hence, they can serve as a potential marker of susceptibility, early and reversible tissue damage, and of decrease in antioxidant defense. JFF, BFF and PFF restored the levels of antioxidant enzymes such as SOD and CAT almost back to the normal levels. SOD is one of the 142

55 chief cellular defence enzymes that convert superoxide radicals to water and oxygen. SOD plays an important role in the elimination of ROS and protects cells against the deleterious effects of super oxide anion derived from the peroxidative process in liver and kidney tissues 55 and the observed increase in SOD activity suggests that JFF, BFF and PFF have an efficient protective mechanism in response to ROS. Catalases are heme containing proteins that protect the cells from toxic effects of ROS by converting hydrogen peroxide to water and oxygen. Catalases are the most important hydrogen peroxide removing enzyme and also a key component of anti oxidative defense system 55. Here catalase activity was increased and then restored to normal levels on administration of JFF, BFF and PFF. Peroxidase is an enzyme that catalyzes the reduction of hydroperoxides and protects the cell from peroxidative damage 65. Therefore it can be proposed from the results of this investigation that additive and synergistic antioxidant activity of phytochemicals such as flavonoids present in JFF, BFF and PFF are responsible for its potent antioxidant activity. The levels of enzymatic antioxidants such as glutathione, SOD, catalase, and peroxidase were improved in fraction treated group as compared to control. This might be due to the action of phenolic compounds present in the fractions which facilitate the removal of the free radicals. 143

56 2.5 a mm/1 g tissue mm/1 g tissue b Figure : TBARS levels in normal and experimental animals (mean ± SEM) a: TBARS levels in liver; b: TBARS levels in pancreas 144

57 a b mm/1 g tissue mm/1 g tissue Figure : Hydroperoxide levels in normal and experimental animals (mean ± SEM) a: Hydroperoxide levels in liver; b: Hydroperoxide levels in pancreas nmol MDA/ g wet tissue weight a nmol MDA/g wet tissue weight b Figure : Lipid peroxidation in normal and experimental animals (mean ± SEM) a: Lipid peroxidation levels in liver; b: Lipid peroxidation levels in pancreas 145

58 µm NADPH decomposed/min/mg protein a µm NADPH decomposed/min/mg protein b µm NADPH decomposed/min/mg protein c Figure : Glutathione S Transferase activity in normal and experimental animals (mean ± SEM) a: Serum Glutathione S Transferase activity; b: Hepatic Glutathione S Transferase activity; c: Pancreatic Glutathione S Transferase activity 146

59 µm NADPH decomposed/min/mg protein a µm NADPH decomposed/min/mg protein b µm NADPH decomposed/min/mg protein c Figure : Glutathione peroxidase activity in normal and experimental animals (mean ± SEM) a: Serum Glutathione peroxidase activity; Hepatic Glutathione peroxidase activity; Pancreatic Glutathione peroxidase activity 147

60 U/mg protein a U/mg protein b U/mg protein c Figure : Superoxide dismutase activity in normal and experimental animals (mean ± SEM) a: Hepatic superoxide dismutase activity; b: Blood superoxide dismutase activity; c: Pancreatic superoxide dismutase activity 148

61 a b µm H2O2 decomposed /min/mg protein µm H2O2 decomposed /min/mg protein µm H2O2 decomposed /min/mg protein c Figure : Catalase activity in normal and experimental animals (mean ± SEM) a: Hepatic catalase activity; b: Blood catalase activity; c: Pancreatic catalase activity 149

62 Effect of JFF, BFF and PFF on activities of different enzymes of glucose metabolism was evaluated using pancreas and liver homogenate of normal animals as enzyme source. The activity of glucose-6-phosphate dehydrogenase, the first regulatory enzyme of pentose phosphate pathway was found to be decreased in diabetic animals and increased in fraction treated animals. The activity of glucose-6-phosphate dehydrogenase in fraction treated animals was higher in comparison to untreated diabetic animals indicating improvement in glucose utilization mediated by this enzyme (Figure ). Insulin decreases gluconeogenesis by decreasing the activity of key enzyme, glucose-6-phosphatase. In fraction treated rats, the activity of enzyme glucose-6-phosphatase was seen significantly reduced in liver (Figure ). Animals treated with JFF showed maximum decrease in the enzyme activity followed by PFF whereas BFF showed increased enzyme activity. This may be due to increased insulin secretion, which is responsible for the repression of glucose-6-phosphatase enzyme 175. Activity of glucokinase, the first regulatory enzyme of glycolytic pathway was also increased by the three flavonoid fractions (JFF, BFF and PFF) as shown in Figure Most of the glucokinase in a mammal is found in the liver, and it provides approximately 95% of hexokinase activity in hepatocytes. Glucokinase plays an important role in diabetes. It is involved in glucose uptake in the pancreas and liver, which are defective in type 2 diabetes mellitus. Hypoglycaemia or hyperglycaemia may also reduce or alter the functional efficiency of the glucokinase enzyme molecule resulting in increasing or decreasing sensitivity of β-cell insulin response to glucose 197. As insulin is one of the most important regulators of glucokinase synthesis, diabetes of all types diminishes the synthesis and activity of glucokinase by a variety of mechanisms. Furthermore, it has been shown that insulin has a direct stimulatory effect on mitochondrial protein synthesis in isolated rat hepatocytes 114. Treatment with the three flavonoid fractions (JFF, BFF and PFF) fractions elevated the activity of glucokinase in the liver. The experimental group of animals treated with JFF showed maximum glucokinase activity. It can be attributed from previously documented reports in the literature that the flavonoid fractions may stimulate insulin secretion which activates glucokinase and thereby enhances the utilization of glucose leading to profound decrease in blood glucose levels. The antidiabetic activity seems to be a result of increase in glucose utilization by the fractions. The results of 15

63 some liver function parameters like serum levels of alanine amino transferase and aspartate amino transferase were evaluated and are presented in the figure The fact that there was no significant change in serum levels of these enzymes in the flavonoid fraction treated rats strongly indicates the non hepatotoxic effect of the fractions. It is shown that ALT and AST always increase following hepatotoxicity and thus are reliable markers of liver integrity. The observed insignificant difference in the activities of ALT and AST of the experimental groups when compared to the control group supports it non hepatotoxicity. The results of the present investigation show that the JFF and PFF possess an antidiabetic effect in addition to antioxidant activity, which may be attributed to its protective action on lipid peroxidation and to the enhancing effect on cellular antioxidant defense contributing to the protection against oxidative damage in streptozotocin induced diabetes 124.Therefore detection of hypoglycemic activity in JFF, BFF and PFF along with protective effect against STZ challenge and preventive action on lipid peroxidation provides scientific rationale of use of A. heterophyllus, A. altilis and P. betle as antidiabetic plants. 151

64 Glucose -6.phosphate dehydrogenase activity (U/g/min) Figure : Hepatic Glucose -6-Phosphate dehydrogenase activity in normal and experimental animals (mean ± SEM) Glucose-6-phosphatase activity (U/g/min) Figure : Hepatic Glucose -6-Phosphatase activity in normal and experimental animals (mean ± SEM) 152

65 a b Glucokinase U/mg protein Glucokinase U/mg protein Figure : Glucokinase activity in normal and experimental animals (mean ± SEM) a: Hepatic glucokinase activity; b: Pancreatic glucokinase activity a b Serum Alanine amino transferase (U/L) Serum Aspartate amino transferase (U/ L) Figure : Serum ALT and AST activity in normal and experimental animals (mean ± SEM) a: Serum ALT activity; b: Serum AST activity 153

66 4.12 IDENTIFICATION OF COMPOUNDS IN THE FRACTIONS BY HPLC AND NMR SPECTROSCOPY The HPLC analysis of JFF, BFF and PFF was carried out and the HPLC chromatogram of these fractions have been shown in Figures a, b and c. The 1H NMR and 13C NMR data of the isolated compounds is shown in Table The chemical structures of the isolated compounds are represented in figure All the identified compounds are matched with the findings of the available literature 5. The NMR spectral data of these compounds were identical to those published in literature 5. HPLC analysis JFF showed the presence of flavonoids namely morusin, rutin and kaempferol. Literature reports show that as a result of cytotoxicity guided fractionation, nine flavonoids namely artocarpin, cudraflavone C, 6-prenylapigenin, kuwanon C, norartocarpin, albanin A, cudraflavone B, brosimone I and artocarpanone were identified from the methanol extract of the wood of Artocarpus heterophyllus. Two flavonoids characterized as 5, 2 -dihydroxy-7,4 -dimethyoxyflavanone and 8-(γ,γ-dimethylallyl)-5,2,4 - trihydroxy-7-methoxyflavone, respectively and have been isolated from the root of Artocarpus heterophyllus. 2, 4, 6 --trioxygenated flavanones, heteroflavanones A and B were isolated from the root bark of Artocarpus heterophyllus 2. Their structures were elucidated as 5 hydroxy-7,2,4,6 -tetramethoxyflavanone and 8-(γ,γdimethylallyl)5-hydroxy-7,2,4,6 tetramethoxyflavanone. Three phenolic compounds were characterized as artocarpesin [(5,7,2,4 -tetrahydroxy-6-β-methylbut-3-enyl) flavone], norartocarpetin (5,7,2,4 -tetrahydroxyflavone) and oxyresveratrol (trans- 2, 4, 3, 5 -tetrahydroxystilbene) by spectroscopic methods 24. BFF showed the presence of cyclomorusin, rutin and kaempferol. Spectral data of compounds were in good agreement with those in the literatures. Reports suggest the isolation of six prenylated flavones, viz. morusin, cudraflavone B, cycloartobiloxanthone, artonin E, cudraflavone C and artobiloxanthone from the root bark of A. altilis. Five geranyl dihydrochalcones have been isolated from the leaves of A. altilis 177. Three flavonoids ellagic acid, querecetin, calycosin and two Phenolic compounds Gallic acid and Catechin have been reported to be detected in the hydrochloric acid extracts and acetone extracts of Piper betle leaves

67 The method developed for HPLC fingerprinting provided a quick analysis of the methanolic extracts. The conditions used led to a good separation of the peaks which could be identified in the chromatogram (Figures a, b and c) by comparison with the chromatogram of the respective reference compounds. PFF showed the presence of catechin, morin and quercetin. JFF showed the presence of morusin, rutin and kaempferol. BFF showed the presence of cyclomorusin, rutin and kaempferol. Kaempferol was present in high levels in JFF and BFF while Quercetin was abundant in PFF. Flavonoids are found as secondary metabolites in plants and are important compounds due to their ability to serve as powerful antioxidants. Many phenolic compounds have been reported to possess effective antioxidant, anticarcinogenic, anti inflammatory, antidiabetic, antibacterial and antiviral activities. Plant flavonoids are an important part of the daily diet because of their beneficial effects on human nutrition. The most important biological activity of flavonoids is their antioxidants properties. It is seen that oxidative stress results from an imbalance between the generation of oxygen derived radicals and the body s antioxidant potential 132. Various studies have shown that diabetes mellitus is associated with increased generation of free radicals and decrease in antioxidant potential. Due to these events, there is a disturbance caused in the normal balance between radical formation and protection against them in cells. This eventually causes oxidative damage of cell components such as proteins, lipids, and nucleic acids. An increased oxidative stress has been reported in both insulin dependent (type 1) and non-insulin-dependent diabetes (type 2) 124. Multiple factors contribute to the increased in oxidative stress in diabetes. Predominant among these factors is glucose autoxidation leading to the production of free radicals. Other factors include cellular oxidation or reduction imbalances and reduction in antioxidant defenses which includes decreased cellular antioxidant levels and a reduction in the activity of antioxidant enzymes that scavenge the free radicals. In addition to this, levels of some prooxidants such as ferritin and homocysteine are also elevated in diabetes. Another important factor is the interaction of advanced glycation end products (AGEs) with specific cellular receptors called AGE receptors. Elevated levels of AGE are formed under hyperglycemic conditions. They are formed when glucose interacts with specific aminoacids on proteins forming a compound that then undergoes further chemical reactions. Glycation of 155

68 protein alters protein and cellular function, and binding ofages to their receptors can lead to modification in cell signaling and further production of free radicals 14. Currently, there has been a considerable interest in finding natural antioxidants from plant materials inorder to replace synthetic ones. Data from laboratory studies and scientific reports reveal that plants contain a large variety of substances that possess antioxidant activity 33. Phytochemicals with antioxidant effects include phenylpropanoids, cinnamic acids, coumarins, diterpenes, flavonoids, lignans, triterpenes, monoterpenes and tannins. Natural antioxidants occur in all higher plants and in all parts of the plants like wood, bark, stems, pods, leaves, fruit, roots, flowers, pollen, and seeds etc 99. The HPLC results of this investigation revealed the presence of important flavonoids like catechin, morin, quercetin, morusin, cyclomorusin, rutin and kaempferol in the three plants. Quercetin belongs to the group of flavonoids with powerful antioxidant activity. It is also a natural anti histamine and anti inflammatory agent. Previous studies showed that quercetin may help to prevent cancer, especially prostate cancer and also inhibited human breast cancer cells (MCF 7 and MDA MB 231) significantly 58. Regarding to the obtained results, quercetin was found as abundant flavonoid in PFF. In this study, catechin was detected in PFF and this flavonoid is reported for its antioxidant activity and ability to control postmenopausal cancer in woman. Kaempferol, quercetin and quercetin derivative rutin (3-O-rhamnosylglucoside) are flavonols that exist as a variety of glycosides or in aglycone form. The aglycone forms of kaempferol and quercetin are structurally similar, differing only by one hydroxyl group in the B-ring. Research on cell culture models has shown important biochemical effects of these compounds, which are relevant to carcinogenesis, metal chelation, antioxidant, anti inflammatory properties and the inhibition of hepatic enzymes involved in glucose metabolism etc 138. Morin is one of the naturally occurring bioflavonoids, originally isolated from members of the Moraceae family. It is found to be present mostly in different herbs and fruits including onion, seed weeds, mill, fig, almond, red wine and oranges. Morin is reported to exhibit several pharmacological properties such as antioxidant, anti inflammatory, chemoprotective, anticancer and antidiabetic. Sreedharan et al., reported that morin supplementation to cancerous rats significantly attenuated oxidative stress and decreased the activities of SOD, CAT, and GPx and brought them to normal levels 171. In addition, morin 156

69 has shown to protect the human hepatocytes, myocytes, endothelial cells and red blood cells against free radical induced oxidative damages. The chemical structure of morin and other bioflavonoids can be distinguished by the presence of two aromatic rings connected by c-pyrone ring where polar hydroxyl groups are bind at various positions. These hydroxyl groups have been proposed to be responsible for the free radical scavenging properties of morin and other naturally occurring bioflavonoids 176. One of the main advantages of morin is its very minimal toxicity even at higher dose usage in animal models. Literature reports revealed that another important bioflavonoid cyclomorusin exhibited antimicrobial, antioxidant, antitubercular and antiplasmodial activities and also showed moderate cytotoxicity against KB (human oral epidermoid carcinoma) and BC (human breast cancer) cell lines 171. Therefore it is seen that plants particularly those with high levels and strong antioxidant compounds have an important role in improvement of disorders involving oxidative stress such as diabetes mellitus. The findings of the present studies suggest that the selected plants possessed different combinations of bioflavonoid compounds that appear to be responsible for their bioactivities. A further detailed research on the specific bioactivity testing of each of the isolated compounds is recommended in order to correlate the relative effectiveness of these compounds in treating various diseases. 157

70 Table H-NMR and 13 C-NMR data of the isolated compounds Kaempferol: 1H NMR [3 MHz, solvent CDCl3, (ppm)]: 6.2 d (1H, J = 1.5 Hz, H-6), 6.44 d (1H, J = 1.5 Hz, H-8), 6.88 d (2H, J = 9. Hz, H- 3, 5 ), 8.11 d (2H, J = 8.8 Hz, H-2, 6 ) Rutin: 1 H NMR (chemical shift δ in ppm, coupling constant J in Hz) 6.2(1H, d, J=2, C6- H), 6.4 (1H, d, J=2, C8-H),7.55 (1H, d, J=2.1,C2-H),6.86 (1H, d J=9,C5-H),7.56 (1H, dd, J=9,2.1, C6-H),9.71 (1H, s, C4-OH), 9.21 (1H, s, C3-OH), (1H, s, C5-OH), 1.86 (1H, s, C7-OH), 5.35 (1H, d, J=7.4, H1-G),5.12 (1H, d, J=1.9, H1-R), 1. (3H, d, J=6.1,CH3-R); 13 C NMR (chemical shift δ in ppm) (C-2),134.1 (C-3), (C-4), (C-5), 99.5 (C-6), (C-7), 94.5 (C-8), (C-9), 14.8 (C-1), (C-1), 116.1(C-2), (C-3), (C-4), (C-5), 122. (C-6),11.6 (C1-G), 74.9 (C2-G), 77.3 (C3-G), 72.7 (C4-G), 76.7 (C5-G), 67.9 (C6-G), 12.2 (C1-R), 7.8 (C2-R), 71.2 (C3-R), 71.4 (C4-R), 69.1(C5-R), 18.6 (C6-R) Cyclomorusin: 1H NMR (CDCl3, 4 MHz) ä 1.47 (6H, s, Me-17 and Me-18), 1.7 (3H, s, Me- 12), 1.98 (3H, s, Me-13), 5.44 (1H, d, J ) 9.6 Hz, H-1), 5.61 (1H, d, J ) 1. Hz, H-15), 6.25 (1H, s, H-6), 6.26 (1H, d, J ) 9.6 Hz, H-9), 6.48 (1H, d, J ) 2.8 Hz, H-3 δ), 6.61 (1H, dd, J ) 8.4, 2.8 Hz, H-5 δ), 6.67 (1H, d, J ) 1. Hz, H-14), 7.66 (1H, d, J ) 8.4 Hz, H-6δ), (1H, s, OH-5) Catechin: 1H NMR spectra showed peaks at δtms 4.56 [H-2, d, J(H-2, H-3a) 7.8 Hz], 4. [H-3, ddd, J(H-3a, H-4e) 5.58 Hz, J(H-3a, H-4a) 8.5 Hz, J(H-3a, H-2a) 7.8 Hz], 2.54 [H- 4a, dd, J(H-4a, H-3a) 8.5 Hz, J(H-4a, H-4e) 16.1 Hz], 2.9 [H-4e, dd, J(H-4e, H-3a) 5.5 Hz, J(H-4e, H-4a) 16.1 Hz], 5.87 [H-6, d, J(H-6, H-8) 2.3 Hz], 6.1 [H-8, d, J(H-8, H-6) 2.3 Hz], 6.89 [H-2, d, J(H-2, H-6 ) 1.95 Hz], 6.79 [H-5, d, J(H-5, H-6 ) 8.7 Hz], 6.73 [H-6, dd, J(H-6, H-2 ) 1.94 Hz, J(H-6, H-5 ) 8.19 Hz] and 8. (phenolic protons, m). 13C-NMR (5 MHz DMSO): Carbon atoms showed peaks at δtms 27.7 (C-4), 66.3 (C-3), 8.9 (C-2), 93.9 (C-6), 95.1 (C-8), (C-2), (C-5 ), 18.4 (C-6 ) and other aromatic carbons showed peaks at δ of 99.1, 13.6, 144.6, 144.8, 155.3, and Morin: 1H NMR (4MHz, CD3OD): δ6.19 (1H, s, H-6), 6.39 (1H, s, H-8), 6.87 (1H, d, H-5 ), 7.63 (1H, d, H-6 ), 7.68 (1H, s, H-3 ). 13C-NMR (1MHz, CD3OD): δ (C- 8), (C-6), (C-1), (C-3 ), (C-5 ), (C-6 ), (C-1 ), (C-3), (C-2), (C-4 ), (C-9), (C-2 ),161.8 (C-5), (C-7), (C-4) Quercetin: 1H NMR [3 MHz, solvent CDCl3, δ (ppm)]: 6.22 d (1H, J = 2. Hz, H-6), 6.42 d (1H, J = 2. Hz, H-8), 7.1 d (1H, J = 9.1 Hz, H-5 ), 7.44 dd (1H, J = 9., 2.5 Hz, H-6 ), 7.72 d (1H, J = 2.1 Hz, H-2 );13C-NMR (1MHz, CD3OD): δ (C-8), (C-6), (C-1), (C-2 ), (C-5 ), (C-6 ), (C-1 ), (C-3), (C-3 ), (C-4 ), (C-2), (C-9), (C-5), (C-7), (C-4) Morusin: 1H NMR(CDCl3, 4 MHz) 1.45(3H,dq,J=1.;.4 Hz,H13), 1.44(6H, s, H17 dan 18), 1.61(3H,q,J=1.3Hz,H12), 3.13(2H,dd d, J=1.;6.8Hz, H9), 5.14(iH, td, J=6.9; 1.4 Hz, H1), 5.47(1H, d,j=1hz, H15 ), 6.21(1H,d,J=.7 Hz, H6), 6.45 (1H, dd, J=8.4;2.3 Hz, H5 ), 6.65 (1H, d, J=2.2 Hz, H3 ), 6.63 (1H, dd, J=1.;.7 Hz, H14), 7.11 (1H, d, J=8.4 Hz, H6 ) 158

71 Figure a Figure b Figure c Figure : HPLC chromatogram of fractions a: JFF - (1) morusin (2) rutin (3) kaempferol b: PFF- (1) catechin (2) morin (3)quercetin c: BFF- (1) cyclomorusin (2) rutin (3) kaempferol 159

72 (1) (2) (3) (4) (5) (6) (7) Figure : Chemical structures of isolated compounds (1) Quercetin (2) Kaempferol (3) Catechin (4) Morin (5) Morusin (6) Cyclomorusin (7) Rutin 16

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