Testa: The study of the macroscopic characters of cashew nut testa reveal the following characteristics: Shape: kidney shaped Size: 3-5 mm thick Textu

Transcription

1 6.1 AUTHENTICATION OF PLANT MATERIAL The botanical identity of the plant specimen of cashew was confirmed by a taxonomist at Department of Botany, Botanical Survey of India, Pune; (M.S). It was authenticated to be Anacardium occidentale Linn. belonging to family Anacardiaceae. A Voucher specimen number YOGA1/No.BSI/WC/Tech/2008/69, was obtained. A copy of the authentication certificate is attached as Appendix - I. 6.2 STANDARDIZATION OF PLANT MATERIAL Identification Tests a) Organoleptic characters: Leaves: The powder of dried cashew leaves is green in color, with no characteristic odour and the taste is slightly astringent. Testa: The powder of dried cashew testa is dark brown in color, with an aromatic odour and slightly astringent taste. b) Macroscopic characteristics: In the macroscopic study of cashew leaves and testa, as depicted in Figure 6.1, the following characteristics were observed: Leaves: The study of the macroscopic characters of fresh leaves reveal the following characteristics: Type: petiolated Shape: elliptic obovate 4 to 22 cm long and 2 to 15 cm broad Base: cuneate Tip: obtuse Venation: reticulate Margin: entire and smooth Arrangement: spiral Texture: leathery P a g e 194

2 Testa: The study of the macroscopic characters of cashew nut testa reveal the following characteristics: Shape: kidney shaped Size: 3-5 mm thick Texture: irregular surface with fragile texture Color: dark brown (a) Leaves of cashew (b) Twig of cashew (c) Stem of cashew (d) Testa of cashew Figure 6.1: Macroscopic characters of cashew leaves and testa P a g e 195

3 c) Microscopic analysis and powder characteristics: The results of microscopic analyses and powder characteristics study of leaves of cashew are depicted in Figure Figure 6.6. Free hand sections of the leaves were taken. A drop of phloroglucinol and hydrochloric acid each was used to detect cellular arrangement in the sections of leaves and in the powdered drug. Photomicrographs of the sections were also recorded with the help of Motic software. Cuticle Epidermis Palisade Cells (a) T.S. of lamina of cashew leaf (b) T.S. of lamina of cashew leaf Figure 6.2: T.S of lamina of cashew leaf Parenchyma Collenchyma Upper Epidermis P a g e 196

4 Upper Epidermis Mesophyll Vascular Bundle (xylem and phloem) Lower Epidermis Figure 6.3: T.S. of cashew leaf through midrib Stomata (a) Stomata of cashew leaf Subsidiary cells Guard cells (b) Stomata of cashew leaf Figure 6.4: Microscopic images of stomata of cashew leaf P a g e 197

5 Epidermis Cortex Fibre (a) Petiole of cashew leaf Pericyclic fibres Xylem and Phloem (b) Petiole of cashew leaf Pith (c) Petiole of cashew leaf Figure 6.5: T.S. of petiole of cashew leaf P a g e 198

6 Trichomes (a) Trichomes of cashew leaf Epidermal cells Stomata (b) Epidermal cells of cashew leaf Lamina portion with palisade cells, parenchyma and epidermal cells Polygonal covering trichomes (c) Palisade cells of cashew leaf Figure 6.6: Powder characteristics of leaves of cashew P a g e 199

7 Study of transverse section of Leaf: As observed in Figure 6.2 and Figure 6.3, the upper epidermis consisted of a single layer of barrel-shaped cells. The epidermal cells were covered by a thick cuticle and stomata were found along the epidermis. The mesophyll consisted of two to three layers of compact cylindrical palisade cells and 4-5 layers of parenchyma. In the midrib region; the upper epidermis was distinct, followed by few layers of collenchymas and a wide region of different sizes of parenchyma cells. The vascular bundle region was covered by endodermis and most of the part of midrib was filled with corticle parenchyma and lignified xylem. Each vascular bundle protected by an upper and a lower patch of sclerenchyma cells. A wide nonlignified phloem region was found towards the lower epidermis protected by thick sclerenchyma cells. The xylem was formed of vessels arranged into 5-8 rows of vessels, in each row there were 2-6 vessels. The parenchyma cells below the vascular bundle were formed of 3-5 layers varying sizes of cells. As seen in Figure 6.4, the stomata were paracytic, rubiaceous celled with irregular subsidiary cells. Study of transverse section of petiole: The general structure of the transverse section of the petiole appeared circular. The outermost layer is formed of one layer of epidermis with no hairy structures. The vascular bundles are arranged in a circle, and each vascular bundle is preceded by pericyclic fibers. The phloem region is formed of primary and secondary phloem and they are followed by the xylem. The pith is a wide region of thickened parenchyma cells (Figure 6.5). Study of diagnostic characters (powder characteristics) of leaves: The diagnostic characters revealed in study of powder of cashew leaves were epidermal cells, stomata, palisade cells and trichomes as seen in Figure 6.6. The trichomes were single celled covering trichomes with sharp ends. Some collapsed trichomes were also observed. Epidermal cells with ranunculaceous stomata. Stomata were surrounded by subsidiary cells, resembling other epidermal cells. Epidermal cells are polygonal with irregular celled stomata. The palisade cells, parenchyma with epidermal cells resemble the lamina portion of the leaves. P a g e 200

8 6.2.2 Physicochemical analysis of cashew leaves and testa The physicochemical analysis of cashew leaves and testa was carried out as per the procedures and parameters mentioned in the Ayurvedic Pharmacopoeia of India, and the results obtained are mentioned in Table Table 6.1: Determination of ash values Type of ash Leaves of cashew (%) ± SEM Testa of cashew (%) ± SEM Total ash Acid insoluble ash Water soluble ash Sulphated ash 10.5 ± ± ± ± ± ± ± ± 0.5 n=3 determinations for values of each test mentioned above Table 6.2: Determination of loss on drying Leaves of cashew (% w/w) ± SEM Testa of cashew (%w/w) ± SEM Loss on drying 7.5 ± ± 0.3 n=3 determinations for values of the test mentioned above Table 6.3: Determination of various extractive values Extract Leaves of cashew (%) ± SEM Testa of cashew (%) ± SEM Alcohol soluble extractive Water soluble extractive Ether soluble extractive 20.9 ± ± ± ± ± ± 0.5 n=3 determinations for values mentioned above Table 6.4: Determination of ph values Extract of cashew leaves ± SEM Extract of cashew testa ± SEM ph values 5.5 ± ± 0.3 n=3 determinations for values mentioned above P a g e 201

9 Determination of physicochemical parameters has been introduced in Ayurvedic pharmacopoeia and in monographs of various herbal drugs. There are no reports found for the determination of physicochemical parameters of cashew leaves and testa and hence these investigations can serve as a reference for any further determinations. The total ash method measures the total amount of material remaining after ignition and the amount of heavy metals and inorganic compounds and includes physiological and non-physiological ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the plant surface. In the physiochemical analysis, it was found that the determination of ash values showed a higher value of ash present in cashew leaves as compared to testa (Table 6.1). The total ash values, and water soluble ash values of both testa and leaves were found to be higher than acid insoluble ash and sulphated ash values. An excess of water in medicinal plant materials will encourage microbial growth, the presence of fungi or insects, and deterioration of phytoconstituents following hydrolysis. Limits for water content should therefore be set for every given plant material. The test for loss on drying determines both water and volatile matter. The results shown in Table 6.2, indicate that the leaves of cashew have a higher moisture content as compared to testa. Determination of extractive values reveals the amount of active constituents extracted with solvents from a given amount of medicinal plant material. As indicated in Table 6.3, it was observed that testa of cashew exhibited higher extractive values as compared to leaves with alcohol, water and ether as extracting solvents. The alcohol and water soluble extractive values were found to be higher than ether soluble extractive values for leaves and testa of cashew. The alcohol soluble extractive value was found to be greater, as being a relatively non-polar solvent as compared to water, alcohol was able to extract polar as well as non polar components. In the determination of ph values of aqueous extracts of cashew leaves and testa the leaves of cashew were found to have a more acidic ph than testa probably due to the presence of higher amounts of constituents like anacardic acids (Table 6.4). P a g e 202

10 6.3 EXTRACTION OF PLANT MATERIAL The extraction of cashew leaves and testa were carried out by various techniques. The results of extractive values obtained for each of the methods are as mentioned in Table Table 6.5: Determination of extractive values (Soxhlet extraction) Extract Leaves of cashew (% w/w) ± SEM Testa of cashew (% w/w) ± SEM Ethanol extract Methanol extract ± ± ± ± 0.30 n=3 determinations for values mentioned above Table 6.6: Determination of extractive values (Decoction) Extract Leaves of cashew Testa of cashew nut (% w/w) ± SEM (% w/w) ± SEM Aqueous extract 8.64 ± ± 0.90 n=3 determinations for values mentioned above Table 6.7 (A): Determination of extractive values (Microwave assisted Time (sec.) extraction) Microwave Extraction (Low power 140 Watt) % Yield ± (SEM) Methanol extract of Microwave Extraction (Low power-140 Watt) % Yield ± (SEM) Aqueous extract of cashew leaves cashew leaves ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.80 Soxhlet extraction at ( C) for 18 hr 8.60 ± ± 0.80 P a g e 203

11 Figure 6.7: Effect of extraction time on extractive yields in microwave assisted extraction (cashew leaves) Table 6.7 (B): Extractive yields of leaves with optimised conditions for MAE Extraction for 120 secs. Leaves Of Cashew At Low Power (In Percentage W/W) ± SEM Aqueous extract 29.0 ± 0.7 Methanol extract 20.2 ± 1.2 n=3 determinations for values mentioned above Table 6.8: Extractive yields of Testa for MAE Extraction for 10 mins. Testa of cashew At Low Power (In percentage w/w) ± SEM Aqueous extract 35.0 ± 0.5 Methanol extract 44.2 ± 1.0 n=3 determinations for values mentioned above Table 6.9: Determination of various extractive values (polyphenol fractions) Extract Extractive value (In percentage w/w) ± SEM Polyphenols of leaves 3.2 ± 1.0 Polyphenols of testa 5.4 ± 0.2 n=3 determinations for values mentioned above P a g e 204

12 Extraction methods In literature, reports are found where Soxhlet has been used as control for comparison with other extraction techniques. The extraction of cashew leaves and testa with methanol and ethanol were carried out by Soxhlet extraction and with water by decoction process. Since cashew is reported to be a rich source of phenolic compounds and tannins, the extraction of testa and leaves was carried out with solvents viz. ethanol and methanol. These solvents extract polar as well as nonpolar phytoconstituents. Several studies have been reported on the comparison of MAE with other available conventional techniques. Recovery of phytoconstituents can be enhanced by study of the behavior of three variables, namely irradiation time, irradiation power and amount of extracting solvent. Extraction yields of plant materials depend on the various extraction conditions. In order to study the effect of Microwave assisted extraction on extractive yields of leaves of cashew, the method of extraction was optimized. Water and methanol, were used as extracting solvents. A threefold increase in the yield of aqueous extract was observed by use of MAE within 120 seconds as compared to Soxhlet extraction for 18 hrs extraction time. Microwaves give better extraction with polar solvents, especially with water which has a high dielectric constant. Thus, extractive yield of leaves with water was found to be higher than that of methanol. For MAE of testa, there was no significant change in the extractive yield even for an extraction period of 10 mins. The yield obtained by MAE in 10 mins. Extraction time was found similar to the yield obtained by soxhlet extraction carried out for 18 hours. However, a reduction in extraction time and solvent consumption was observed. Hence, it can be said that MAE can be a more effective technique compared to conventional extraction methods for faster and economical extraction of plant materials. Polyphenols are compounds of great interest to researchers worldwide for their varying beneficial effects in various diseases (Na, 2008 and Larrauri, 1997). The cashew nut testa is reported to be rich source of polyphenols and cashew nut shell liquid is well known for its tannin content and is widely used by the tanning industries (Subramanian, 1969). Hence an attempt was made to extract polyphenols from leaves and testa of cashew. P a g e 205

13 6.4 PRELIMINARY PHYTOCHEMICAL SCREENING OF EXTRACTS The results of qualitative chemical tests for various extracts of cashew are tabulated below Table 6.10: Qualitative chemical tests Sr.No Chemical Tests EL AL ET MT AT 1 Test for Carbohydrates Molisch's test Benedict's test Test for Non-reducing sugars Test for Gums Test for mucilage Tests for Proteins Millons test Tests for Amino Acids Ninhydrin test Test for Fats and Fixed Oil Stain test Saponification test Test for Sterols and Triterpenoids Libermann- Buchard test P a g e 206

14 7 Test for Glycosides Legal s test for Cardiac Glycosides Keller Killiani test [for Deoxy sugars] Froth Test for Saponin Glycosides Sodium picrate test (grignard reaction) for Cyanogenetic Glycosides Tests for Coumarin Glycosides Test for Flavonoids Shinoda test (Magnesium Hydrochloride reduction) Alkaline reagent test for Flavonoids Tests for Alkaloids Dragendorff s test Test for Tannins and Phenolic Compounds Ferric chloride test Tests for organic acids Calcium chloride test P a g e 207

15 In the table, the following abbreviations were used: EL: Ethanol extract of leaves AL: Aqueous extract of leaves ET: Ethanol extract of testa MT: Methanol extract of testa AT: Aqueous extract of testa The symbol (+) denotes presence and (-) denotes absence of phytoconstituents. From the qualitative chemical tests performed for estimation of various phytoconstituents in extracts of cashew leaves and testa the following results were obtained (Table 6.10): Ethanol extract of leaves: The ethanol extract of leaves was found to contain carbohydrates, proteins, saponin glycosides, flavonoids, alkaloids, tannins and phenolic compounds. Aqueous extract of leaves: The aqueous extract of leaves was found to contain carbohydrates, proteins, saponin glycosides, flavonoids, alkaloids, tannins and phenolic compounds. Ethanol extract of testa: The ethanol extract of testa was found to contain carbohydrates, proteins, flavonoids, alkaloids, tannins and phenolic compounds. Methanol extract of testa: The methanol extract of testa was found to contain carbohydrates, proteins, flavonoids, alkaloids, tannins and phenolic compounds. Aqueous extract of testa: The aqueous extract of testa was found to contain carbohydrates, proteins, flavonoids, alkaloids, tannins and phenolic compounds. Gums, mucilage, amino acids, and organic acids were found to be absent in leaves and testa of cashew. The extraction for leaves and testa were carried out with solvents of similar polarity. Polar solvents, viz. water, ethanol and methanol were used for extraction. Hence, phytoconstituents of similar nature were found in extracts of testa and leaves, except for saponin glycosides which were present in leaves and not in testa. P a g e 208

16 6.5 ISOLATION OF CATECHIN Isolation of catechin was carried out by Preparative Thin layer chromatography (P-TLC). The presence of catechin in various extracts of cashew was confirmed by co-chromatography with reference standard catechin. The spot with R f value identical to the marker catechin was isolated. The crude catechin thus obtained was recrystallised with hot water and the percentage yield of pure catechin from crude catechin was calculated. The weights of catechin obtained before and after recrystallisation are as below: Percentage yield of crude Catechin: 1g of the extract 90 mg of crude catechin Thus, %Yield of crude catechin = 9.0% w/w Percentage yield of catechin after recrystallisation: 1g of the extract 50 mg of Pure catechin Thus, %Yield of Pure catechin = 5.0 w/w Identification of isolated catechin The identity and purity of isolated catechin was further confirmed by chemical spectral and chromatographic studies standard catechin was used for comparison with the isolated catechin. Thus catechin was characterized for the following chemical characteristics: Physicochemical and spectral characteristics: Colour and shape: Catechin appeared to be a buff white colored powder. Melting point: Melting point of isolated catechin was found to be C. It was found to be identical to the melting point of reference catechin. ph: ph of the catechin solution was found to be 6.5. P a g e 209

17 Solubility: Catechin was found to be soluble in methanol, water, and ethanol. Chemical test for catechin: Catechin when added to vanillin and hydrochloric acid solution produced a pink colour. The test was found to be positive for isolated catechin. The UV λmax (nm) : Isolated and marker catechin were found to exhibit a similar λmax at 273nm. Table 6.11: Physiochemical and spectral studies of isolated Catechin Sr. No. Parameters Values for reference catechin Values for isolated catechin 1 Colour and crystal shape Buff white colored powder Buff white colored powder 2 Melting point( 0 C) C C C 3 Derivatization (Ethanolic FeCl 3 ) Bluish black color Bluish black color 4 HPLC Rt (min.) HPTLC Rf UV λmax (nm) 273 nm 273nm 7 ph of 1% solution in water 8 Solubility Soluble in methanol, water, chloroform and insoluble in benzene Soluble in methanol, water, chloroform and insoluble in benzene Figure 6.8: Structure of catechin P a g e 210

18 UV spectral analysis of catechin Figure 6.9: UV absorption spectrum of marker catechin Figure 6.10: UV absorption spectrum of isolated catechin Result: As indicated in Table 6.11 and Figure , it was observed that isolated catechin the spectral, chemical and chromatographic characteristics similar to reference catechin, thus indicating the identity of isolated catechin. P a g e 211

19 HPTLC profile of isolated catechin In order to ascertain the purity of isolated catechin, HPTLC studies were performed using marker catechin as the reference standard. Working solutions of 1mg/ml of isolated catechin and standard catechin were prepared in methanol and the HPTLC analysis was carried out using the following optimised conditions. Stationary phase : Silica gel 60 GF 254 (Merck) Mobile phase : Toluene: ethyl acetate: methanol: formic acid (6: 6:1:0.1) Saturation time : 30 min. Band width : 7 mm Detection wavelength : 273 nm Isolate Marker Isolate Marker Isolate Marker In White nm Figure 6.11: HPTLC video images of catechin P a g e 212

20 Figure 6.12: HPTLC chromatogram of marker catechin Figure 6.13: HPTLC chromatogram of isolated catechin Result: As shown in Figure , the isolated catechin gave a single isolated band at R f of 0.45 similar to that of marker catechin. The percentage purity of isolated catechin was found to be 99.82% when compared with standard catechin. P a g e 213

21 HPLC profile of Isolated catechin In order to ascertain the purity of isolated catechin, HPLC studies were carried out using marker catechin as the reference standard. Standard catechin and isolated catechin were analyzed by HPLC using the following conditions: System : TOSOH-CCPM HPLC method : Reverse Phase Column : C 18 (ODS)Phenomenex (250 x 4.60mm)-5µ Mobile phase : Methanol (100%) HPLC Grade Flow rate : 1ml/min Wavelength : 273nm Figure 6.14: HPLC chromatogram of reference catechin P a g e 214

22 Figure 6.15: HPLC chromatogram of isolated catechin Result: As seen in Figure 6.14 and Figure 6.15, isolated catechin gave a single, sharp and well resolved peak at R t of 2.6 min similar to that of reference catechin. The HPLC profile of the standard and isolated catechin was found to be identical at retention time (R t ) of 2.6 min. The HPLC profile of isolated catechin gave a, well isolated peak with purity greater than 99.65%. P a g e 215

23 6.6 CHROMATOGRAPHIC STUDIES The HPTLC analysis of various extracts of cashew leaves and testa were carried out by the optimized chromatographic conditions by HPLC and HPTLC techniques and various components of the extracts were analysed densitometrically. Catechin was used as a marker and the amount of it present in various extracts was quantified. HPTLC Analysis Optimized Chromatographic parameters Stationary Phase: Precoated, aluminum backed HPTLC plates (20cm 20 cm, 0.2mm thickness, 5 6 µm particle size. Mobile phase : Toluene:ethylacetate:MeOH:formic acid (6:6:1:0.1v/v/v/v) Saturation time : 15 mins. Development distance: 80 mm Derivatising agent: 5% alcoholic FeCl 3 solution Detection wavelength: 254 nm Calibration curve of catechin In order to establish a calibration curve for estimation of catechin, the limit of detection (LOD) and limit of quantitation (LOQ) were determined. The values obtained for LOD and LOQ were 0.1 and 0.3 µg / µl respectively. The calibration concentration range was between µg / µl. Figure 6.16: Calibration curve of catechin for HPTLC method P a g e 216

24 The chromatograms obtained for various extracts of cashew leaves and testa and polyphenol fractions are as depicted in Figure The amount of catechin in various extracts was estimated and is listed in Table It was found that aqueous extract of leaves and testa contained the maximum amount of catechin as compared to the other extracts. In the polyphenol fraction of cashew leaves and testa the maximum amount of catechin was found from the fraction prepared from aqueous extracts. The images of fingerprints and spectra of various extracts and fractions at different wavelengths are shown in Figure 6.25 and Table 6.12: Catechin content in various extracts of cashew by HPTLC Sr.No Sample % of Catechin Cashew leaf extracts 1 Ethanol extract 4.75% 2 Aqueous extract 5.70% Cashew testa extracts 3 Methanol extract 12.75% 4 Ethanol extract 13.09% 5 Aqueous extract 13.65% Polyphenol fraction of cashew testa 6 Aqueous extract 16.40% 7 Ethanol extract 15.44% Polyphenol fraction of cashew leaves 8 Aqueous extract 7.0% 9 Ethanol extract 5.50% P a g e 217

25 Figure 6.17: HPTLC chromatogram of ethanol extract of leaves Figure 6.18: HPTLC chromatogram of aqueous extract of leaves P a g e 218

26 Figure 6.19: HPTLC chromatogram of ethanol extract of testa Figure 6.20: HPTLC chromatogram of methanol extract of testa P a g e 219

27 Figure 6.21: HPTLC chromatogram of aqueous extract of testa Figure 6.22: HPTLC chromatogram of polyphenol fraction of testa P a g e 220

28 Figure 6.23: HPTLC chromatogram of polyphenol fraction of leaves Figure 6.24: HPTLC chromatogram of standard catechin P a g e 221

29 HPTLC Fingerprints of extracts of leaves and testa of cashew AL EL MT AT ET IC RC 366nm (a) AL EL MT AT ET IC RC 254nm (b) AL EL MT AT ET IC RC EL ET RC White Light white light after derivatization (c) (d) P a g e 222

30 HPTLC Fingerprints of Polyphenol fractions of leaves and testa of cashew RC PL PL PT RC PL PL PT RC PL PL PT nm 254 nm White Light (a) (b) (c) Figure 6.25: HPTLC video images (fingerprints of various extracts and polyphenol fractions of cashew) EL RC AT MT ET Figure 6.26: Spectra of catechin in various extracts The abbreviations denoted on the tracks are as follows: RC - Reference catechin IC - Isolated catechin PL - Polyphenols of cashew leaves MT - Methanol extract of cashew testa PT - Polyphenols of cashew testa AT - Aqueous extract of cashew testa AL - Aqueous extract of cashew leaves ET - Ethanol extract of cashew testa EL Ethanol extract of cashew leaves P a g e 223

31 HPLC Analysis The HPLC analysis of various extracts of cashew leaves and testa were carried out by the optimized chromatographic conditions. Catechin was used as a marker and the amount of catechin present in various polyphenol fractions, extracts of cashew prepared by conventional extraction process as well as by microwave extraction was quantified. Optimized Chromatographic parameters The optimized parameters for HPLC analysis were: Solvent system: Methanol:Water (90:10 v/v) Flow rate: 1ml/min Column: C18 column Detection wavelength: 254 nm Calibration curve of catechin In order to establish a calibration curve for estimation of catechin, the limit of detection (LOD) and limit of quantitation (LOQ) were determined. The values obtained for LOD and LOQ were 0.1 and 0.3 µg / µl respectively. The calibration concentration range was between µg / µl. Figure 6.27: Calibration curve of catechin by HPLC P a g e 224

32 The peaks in HPLC fingerprints were identified by comparing the retention times in the chromatograms of extracts with those of reference standard catechin peak. The Chromatograms obtained for various extracts of cashew leaves and testa and polyphenol fractions are as depicted below in Figure The catechin content in various extracts of testa and leaves prepared by conventional techniques was quantified and is listed in Table Aqueous extract of testa and leaves were found to contain maximum amount of catechin. The catechin content in methanol and aqueous extracts of leaves prepared by microwave assisted extraction (MAE) was quantified and is listed in Table It was observed that the extraction time of 120 secs. yielded the maximum amount of catechin in methanol and aqueous extract of leaves prepared by MAE. Table 6.13: Catechin content in various extracts of estimated by HPLC Sr.No. Extract % Catechin content Cashew leaf extracts 1 Ethanol extract 4.95 ± Aqueous extract 5.83 ± 0.9 Cashew testa extracts 3 Methanol extract ± Ethanol extract ± Aqueous extract ± 0.5 Table 6.14: Catechin content in various extracts prepared by MAE Sr. No. Time (sec.) Microwave Extraction (Low power 140 Watt) % Yield ± (SEM) Microwave Extraction (Low power-140 Watt) % Yield ± (SEM) Methanol extract of cashew leaves ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.7 Aqueous extract of cashew leaves P a g e 225

33 Figure 6.28: HPLC chromatogram of standard catechin P a g e 226

34 Figure 6.29: HPLC chromatogram of aqueous extract of cashew leaves P a g e 227

35 Figure 6.30: HPLC chromatogram of ethanol extract of cashew leaves P a g e 228

36 Figure 6.31: HPLC chromatogram of aqueous extract of cashew testa P a g e 229

37 Figure 6.32: HPLC chromatogram of ethanol extract of cashew testa P a g e 230

38 Figure 6.33: HPLC chromatogram of methanol extract of cashew testa P a g e 231

39 Figure 6.34: HPLC chromatogram of polyphenol fraction of cashew testa P a g e 232

40 Figure 6.35: HPLC chromatogram of polyphenol fraction of cashew leaves P a g e 233

41 Figure 6.36: HPLC chromatogram of aqueous extract prepared by microwave extraction (for 50 seconds) at low power P a g e 234

42 Figure 6.37: HPLC chromatogram of aqueous extract prepared by microwave extraction (for 70 seconds) at low power P a g e 235

43 Figure 6.38: HPLC chromatogram of aqueous extract prepared by microwave extraction (for 90 seconds) at low power P a g e 236

44 Figure 6.39: HPLC chromatogram of aqueous extract prepared by microwave extraction (for 120 seconds) at low power P a g e 237

45 Figure 6.40: HPLC chromatogram of aqueous extract prepared by microwave extraction (for 130 seconds) at low power P a g e 238

46 Figure 6.41: HPLC chromatogram of aqueous extract prepared by microwave extraction (for 150 seconds) at low power P a g e 239

47 Figure 6.42: HPLC chromatogram of aqueous extract prepared by microwave extraction (for 180 seconds) at low power P a g e 240

48 Figure 6.43: HPLC chromatogram of methanol extract prepared by microwave extraction (for 50 seconds) at low power P a g e 241

49 Figure 6.44: HPLC chromatogram of methanol extract prepared by microwave extraction (for 70 seconds) at low power P a g e 242

50 Figure 6.45: HPLC chromatogram of methanol extract prepared by microwave extraction (for 90 seconds) at low power P a g e 243

51 Figure 6.46: HPLC chromatogram of methanol extract prepared by microwave extraction (for 120 seconds) at low power P a g e 244

52 Figure 6.47: HPLC chromatogram of methanol extract prepared by microwave extraction (for 130 seconds) at low power P a g e 245

53 Figure 6.48: HPLC chromatogram of methanol extract prepared by microwave extraction (for 150 seconds) at low power P a g e 246

54 Figure 6.49: HPLC chromatogram of methanol extract prepared by microwave extraction (for 180 seconds) at low power P a g e 247

55 6.7 EFFECT OF VARIOUS DRYING METHODS ON THE POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF CASHEW LEAVES The leaves of cashew were subjected to various drying conditions in order to study the effect of varying temperatures on the polyphenol content and antioxidant activity of the extracts. The results of the antioxidant activity and total phenolic content estimation are detailed in section Quantitation of catechin content of various extracts of cashew The catechin content in extracts of leaves subjected to drying conditions was carried out by HPLC method. The conditions used for analysis are mentioned below: The optimized parameters for HPLC analysis were: Solvent system: Methanol:Water (90:10 v/v) Flow rate: 1ml/min Column: C 18 column Detection wavelength: 254 nm Table 6.15: Comparison of various drying techniques of cashew leaves based on catechin content Sr.No Extracts % of Catechin ± SEM 1 Oven dried leaves 6.11% ± Sun dried leaves 7.94 % ± Fresh leaves 5.70% ± Shade dried leaves 7.50 % ± 0.53 n=3 determinations for each of the values mentioned above P a g e 248

56 Figure 6.50: HPLC Chromatogram of extract of cashew leaves subjected to oven drying P a g e 249

57 Figure 6.51: HPLC chromatogram of extract of sun dried cashew leaves P a g e 250

58 Figure 6.52: HPLC chromatogram of extract of shade dried cashew leaves P a g e 251

59 Figure 6.53: HPLC chromatogram of extract of fresh cashew leaves P a g e 252

60 As observed in the Table 6.15 and Figure it is observed that the extracts prepared from cashew leaves exposed to sunlight for drying contained the maximum amount of catechin. Various temperatures significantly influence the extraction yield of phenolics from plants and the antioxidant activity of the phenolic compounds. In this study the cashew leaves were exposed to various drying conditions and upon chromatographic quantitation, a considerable difference was observed in the catechin content of various extracts. The results suggest that the order of catechin content in various extracts prepared was as Sun dried > Shade dried > Oven dried > Fresh leaves. The leaves of cashew are taken as food accompaniments in Malaysia (Abas, 2006) and finding a suitable method to preserve them along with maintaining their antioxidant effect would be of help to the consumers. The results showed that only oven drying brought about significant reduction in catechin content with fresh leaves as the comparison group. This might be due to some chemical transformations during the process of drying. Thermal processing of food is primarily intent to inactivate pathogens and other deteriorative microorganisms capable of making it unsuitable for human consumption. However, it is believed that thermal treatments are the main cause of the depletion in natural antioxidants (Mokbel, 2005; Mohan, 2008). In this study, increasing the temperature to about 80 C seemed to cause depletion in the antioxidant content. Since many plants/fruits have antioxidants, it is important to maintain this nutrient content for its benefit by controlling the extraction temperature or exposure of leaves to temperatures higher than 40 0 C. Sun dried leaves were found to contain the maximum amount of catechin, which suggests that if leaves dried in sunlight are consumed in some form instead of fresh leaves, the availability of antioxidants would be more as compared to fresh or shade dried leaves. In recent years polyphenols have received increasing attention from chemists and food technologists. Another phenomenon that might affect the Polyphenol content or antioxidant activity of the leaves subjected to various drying conditions is the browning effect. Such compounds present in food have been found to take part in both enzymic and nonenzymic browning reactions. The specific enzymes that take part in browning reactions involving polyphenols have been known by different P a g e 253

61 names but in general can be referred to as polyphenoloxidases. Enzyme chemists have been able to isolate, purify, and characterize polyphenoloxidase enzymes from several sources. The oxidation of polyphenolic substrates, secondary reactions, inhibition, and activation have also been investigated thoroughly during the last few decades. During the processing and storage of food products, especially fruits and vegetables, several nonenzymic changes leading to browning involving polyphenols have been noted. The common cause of darkening of many products is attributable to the interaction between polyphenols and heavy metals, especially iron. Formation of colored anthocyanidin pigments has been suspected in others. The inhibition of enzymic discoloration involving polyphenols is best effected by application of heat or by influence of certain chemicals. In addition to these the effect of other agents such as freezing (Negishi, 1964), moisture content (Draudt, 1966) and so on have also been investigated to a limited extent. Since browning of this nature involves an enzymic step, factors such as concentration of the substrate, ph of the medium, and availability of oxygen have an influence on the rate of the reaction. In practice, however, some of these factors are difficult to control during storage and processing of food materials. In a comparative study between fruits and vegetables, it was noted that polyphenoloxidases of fruits are more stable than those of vegetables (Yankov, 1963). Thus, a thorough detailed investigation of the enzymic reactions occurring in leaves of cashew would be required in order to arrive to mechanisms that lead to increase in catechin content of leaves exposed to sunlight. P a g e 254

62 6.8 EVALUATION OF ANTIOXIDANT ACTIVITY The extracts and fractions of cashew leaves and testa were subjected to various in vitro and cell line based antioxidant assays, in order to ascertain their antioxidant effect. The results obtained from various assays and tests are detailed below Assessment of Free Radical Scavenging Capacity in vitro A. DPPH. radical scavenging assay The radical scavenging activities of the extracts and fractions of leaves and Testa of cashew were estimated by comparing the percentage inhibition of formation of DPPH. radicals by the extracts and those of Ascorbic acid. The results of the assay are as indicated in Table Extracts of Cashew Leaves A steady increase in the percentage inhibitions of the absorbance of the DPPH radicals by the extracts up to a concentration of 16.0 µg/ml, and 18.0 µg/ml for aqueous and ethanolic extracts respectively, after which there was a leveling off with much slower increase in inhibition. This pattern of DPPH inhibition is commonly observed with plant extracts. Overall, the Aqueous and Ethanol extracts of leaves of cashew were able to inhibit the formation of DPPH. radicals. The aqueous extract and ethanol extracts had IC 50 values of µg/ml and 9.41µg/mL respectively which is inversely related to its antioxidant ability. The IC 50 value of Ascorbic acid (standard) was found to be 5.30µg. Based upon the IC 50 values of the extracts it can be concluded that, ethanol extract is more potent as an antioxidant than aqueous extract. Extracts of Cashew Testa Radical scavenging activities of the extracts of cashew testa, were estimated by comparing the percentage inhibition of formation of DPPH radicals by the extracts and those of Ascorbic acid depicting a steady increase in the percentage inhibitions of the absorbance of the DPPH radicals by the extracts up to a concentration of 14.0 µg/ml for aqueous and methanol extracts and 20.0 µg/ml P a g e 255

63 for ethanol extract, after which there was a slower increase in inhibition. Overall, the aqueous, ethanol and methanol extracts of cashew were able to inhibit the formation of DPPH. radicals and had IC 50 values of 7.62, 6.68 and 7.23µg/mL of dried extract which is inversely related to its antioxidant ability. Among the three extracts of testa that were tested for antioxidant activity, ethanol extract was found to be most potent with the least IC 50 value comparable with Ascorbic acid used as standard. This suggests that ethanol extract of testa can exhibit significant antioxidant activity at a much lower concentration. Polyphenol Fractions of Cashew Polyphenolic compounds are well known in literature for their antioxidant effects and hence an attempt was made to evaluate the efficacy of polyphenol rich fraction from cashew leaves and testa. The IC 50 values for Polyphenol fraction of test and leaves were found to be 7.51 and 7.42µg/mL respectively which is inversely related to its antioxidant ability. Thus, the results indicate that the polyphenol fractions may serve as potential antioxidant candidates. Extracts Prepared By Microwave Extraction Microwave assisted extraction has been known to increase the extractive yields of substances. In case of materials containing polyphenols, anthocyanins and flavanoids this behaviour of increase in yields can occur for two reasons: (i) at a high temperature, new compounds can be generated as a result of non-enzymatic browning or the Maillard reaction. These compounds, referred to as melanodins or Maillard reaction products (MRPs), possess antioxidant activity and function as an antioxidant via a chain-breaking mechanism. Several authors have noted that the antioxidant activity afforded by the generation of MRPs does not compensate for that lost by the phenolic compounds (Morales, 2001; Yilmaz, 2005), and (ii) during oxidation of polyphenolics, the oxidation products formed during the intermediate stages have shown to posses greater antioxidant activity than the endogenous polyphenolics; however, these intermediate compounds are only temporary (Manzocco, 2000). At the same time, constituents with moieties possessing antioxidant behaviour and bound to different components of the food/plant matrix can be released / cleaved from cell walls during thermal P a g e 256

64 operations thereby allowing them to exhibit antioxidant activity. With an increase in extraction time in microwave extraction an increase in the antioxidant behavior was observed. The extraction time of 180 seconds exhibited the least IC 50 values. The results thus indicate that microwave assisted extraction can effectively extract antioxidant compounds from cashew leaves with water as the extracting solvent. Extracts of Leaves Exposed to Various Drying Conditions Drying of plant material at high temperatures can result in significant degradation of the polyphenolics and also affect antioxidant and free-radical scavenging capacities (Larrauri, 1997). In the experiments carried out, it was observed that the order of antioxidant activity for extracts of leaves exposed to various drying conditions was Sun dried > Shade dried > Oven dried > Fresh leaves. Thus, it can be interpreted that sunlight temperature and browning of leaves in sun light might lead to some reactions that lead to increase in the antioxidant activity of the compounds. P a g e 257

65 Table 6.16: Results of DPPH. radical scavenging assay Sr.No. Sample IC 50 value (µg/ml) ± SD Extracts of Cashew Leaves 1 Aqueous Extract ± Ethanol Extract 9.41 ± 0.73 Extracts of Cashew Testa 3 Aqueous Extract 7.23 ± Ethanol Extract 6.68 ± Methanol Extract 7.62 ± 0.45 Polyphenol Fractions of Cashew 6 Polyphenols of leaves 7.51 ± Polyphenols of testa 7.42 ± 0.36 Extracts Prepared By Microwave Extraction 8 50 seconds 8.90 ± seconds 8.63 ± seconds 8.30 ± seconds 7.40 ± seconds 7.59 ± seconds 7.20 ± seconds 6.90 ± 0.67 Extracts of Leaves Exposed to Various Drying Conditions 15 Sun dried leaves 11.2 ± Oven dried leaves 13.5 ± Shade dried leaves 12.6 ± Fresh leaves 14.3 ± Ascorbic acid (Control) 5.3 ± 0.96 P a g e 258

66 B. Nitric oxide scavenging activity Sodium nitro-prusside in aqueous solution at physiological ph spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen, leading to reduced production of nitrite ions. Extracts of Cashew Leaves Overall, the ethanol extract of cashew leaves showed higher nitric oxide scavenging ability compared to the aqueous extract as indicated in Table The IC 50 values of ethanol and aqueous extracts were found to be µg and µg of dry extract respectively. The presence of high levels of phenolic compounds in the ethanol extract may have partly contributed to the observed antioxidant activities. This study provided evidence on the potential health benefits of cashew leaves. However, a detailed investigation of the molecular mechanisms responsible for this activity is further required to understand the mechanism of action of cashew leaves as antioxidant. Extracts of Cashew Testa Sodium nitroprusside in aqueous solution at physiological ph spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen, leading to reduced production of nitrite ions. Overall, the ethanol extract of cashew testa showed higher nitric oxide scavenging ability compared to the aqueous extract and methanol extract. The IC 50 values of aqueous, ethanol and methanol extracts were found to be 518.6, and µg/mL respectively. All three extracts exhibited comparable antioxidant activity. Ethanol extract was found to be most potent antioxidant with the least IC 50 value when compared with Ascorbic acid used as standard. Polyphenol Fractions of Cashew Based upon the results of Griess assay it was observed that polyphenols of cashew testa and leaves had considerable antioxidant activity as compared to Ascorbic acid used as a control. However, a considerable difference between the antioxidant activity of both the fractions was not observed. P a g e 259

67 Extracts Prepared By Microwave Extraction Microwave assisted extraction led to an increase in the extractive yield as observed in the previous experiments. The effect of this extraction process on the antioxidant compounds in cashew leaves was ascertained by evaluation of antioxidant activity. It was observed that the antioxidant effect increased with increase in extraction time period. However, after 130 seconds there was no considerable difference in the antioxidant activity of the extracts. Extracts of Leaves Exposed to Various Drying Conditions Among the extracts of leaves exposed to varying drying conditions, it was observed that leaves dried in sunlight exhibited the maximum antioxidant effect as compared to shade dried, fresh and oven dried leaves. Thus, it can be inferred that Sun drying may be the optimal drying condition amongst other conditions selected in the study which leads to increase in the antioxidant phytoconstituents. P a g e 260

68 Table 6.17: Results of nitric oxide scavenging activity Sr.No. Sample IC 50 value (µg/ml) ± SD Extracts of Cashew Leaves 1 Aqueous Extract ± Ethanol Extract ± 0.98 Extracts of Cashew Testa 3 Aqueous Extract ± Ethanol Extract ± Methanol Extract ± 0.47 Polyphenol Fractions of Cashew 6 Polyphenols of leaves ± Polyphenols of testa ± 1.3 Extracts Prepared By Microwave Extraction 8 50 seconds ± seconds ± seconds ± seconds ± seconds ± seconds ± seconds ± 0.40 Extracts of Leaves Exposed to Various Drying Conditions 15 Sun dried leaves ± Oven dried leaves ± Shade dried leaves ± Fresh leaves ± Ascorbic acid (Control) ± 0.83 P a g e 261

69 6.8.2 Determination of Antioxidant Capacity against Lipid Peroxidation A. Thiobarbituric acid Reacting substances (TBARS) test Lipid peroxidation (LPO) can inactivate cellular components and plays an important role in oxidative stress in biological systems. Furthermore, several toxic byproducts from the peroxidation can damage other bio-molecules (Box, 1997; Esterbauer, 1996). It is well established that transition of metal ions such as iron and copper stimulate lipid peroxidation through various mechanisms (Halliwell, 1984). These may either generate hydroxyl radicals to initiate the lipid peroxidation process or propagate the chain process via decomposition of lipid hydroperoxides (Braughler, 1987). In this study, the extracts inhibited the lipid peroxidation to a considerable extent as compared to standard i.e. Ascorbic acid. The effect of extracts against lipid peroxidation could be attributed to presence of phenolics, flavonoids, and glycosides. Ethanol extract of cashew testa and leaves exhibited good peroxidation inhibitory activity than methanol and aqueous extracts. As observed in the results shown in Table 6.18, the ethanol extract of leaves and aqueous extract of testa showed better anti-lipid Peroxidation activity as compared to the other extracts. Table 6.18: Results of anti-lipid peroxidation activity Sr.No Extract IC 50 Value (µg/ml) ± SD Extracts of Cashew Leaves 1 Ethanol extract ± Aqueous extract ± 0.47 Extracts of Cashew Testa 3 Ethanol extract 83.3 ± Methanol extract ± Aqueous extract ± 0.64 Control 6 Ascorbic acid ± 0.33 P a g e 262

70 6.8.3 Determination of Total Phenolics Content A. Folin - Ciocalteu method Phenolic compounds have been proved to be responsible for the antioxidant activity in plants. The amounts of total phenolics in cashew extracts were measured in this study. These extracts were found to have various phenolic levels as indicated in Table The ethanol extract of cashew test and leaves, Sun dried leaves extract and Microwave extracted leaves for 180 seconds had the highest content of total phenolics. The various levels of phenolics in these extracts could be partly due to the differences in growing conditions. Under field conditions, the phenolic compositions of plant tissues vary considerably with seasonal, genetic, and agronomic factors (Hilton, 1973). In addition, a large variability at various stages of maturation and growing conditions such as temperature and extraction conditions affect the contents of phenolic compounds (Zheng, 2001). Table 6.19: Results of total phenolics content estimation Sr.No. Sample Total Phenolic content (mggae/g of extract) ± SD Extracts of Cashew Leaves 1 Aqueous Extract ± Ethanol Extract ± 0.98 Extracts of Cashew Testa 3 Aqueous Extract ± Ethanol Extract ± Methanol Extract ± 0.56 Extracts Prepared By Microwave Extraction 6 50 seconds ± seconds ± seconds ± seconds ± seconds ± seconds ± seconds ± 0.63 Extracts of Leaves Exposed to Various Drying Conditions 13 Sun dried leaves ± Oven dried leaves ± Shade dried leaves ± Fresh leaves ± 0.89 P a g e 263

71 6.8.4 Antioxidant Capacity in Cultured Cells NF-E2-related factor (Nrf2) is responsible for regulation of antioxidant response element (ARE) driven expression of genes encoding the majority of phase II detoxification and antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase-1 (NQO1), glutathione S-transferases, glutamate cysteine ligase, and heme oxygenase-1 (HO-1). Basal and inducible antioxidant/phase II detoxifying enzyme expression was found to be abrogated in the Nrf2-deficient mice (Ramos-Gomez, 2001; Xu, 2006). The association of Nrf2 with ARE in the promotor regions of antioxidant genes is a key regulatory step in stress protein expression. Keap1 has been identified as a cytosolic binding protein for Nrf2 which associates with the Kelch domain of Keap1, and is sequestered in association with the actin cytoskeleton under normal physiological conditions, which in turn allows proteasomal degradation of Nrf2. Under oxidative stress or treatment with electrophilic reagents, Nrf2 is released through the oxidation of the cysteine residues on Keap1, allowing Nrf2 to translocate into the nucleus (Nguyen, 2004). A. ROS Assay : The cell-permeable dye 2',7'-dichlorofluoresceindiacetate (H 2 DCFDA) is oxidized by hydrogen peroxide, peroxinitrite (ONOO - ), and hydroxyl radicals (OH ) to yield the fluorescent molecule 2'7'-dichlorofluorescein. Thus, dye oxidation is an indirect measure of the presence of these reactive oxygen intermediates, calculated by difference in the mean fluorescence of a treated sample to that of the untreated one. Catechin, polyphenols of cashew testa, and aqueous extract of cashew testa were found to inhibit ROS production. The results of ROS assay for some selected extracts, showed a concentration dependent decrease in production ROS by oxidation of H 2 DCFDA dye after 3 hrs incubation period are shown in Figure Varying concentrations of tbh 2 O 2 (0, 75 and 150 µm) were used to induce oxidative stress conditions. With 0 µm tbh 2 O 2, pre-incubation with plant extracts exhibited slight variations in already low ROS levels. But with oxidative stress conditions induced by 75 or 150 µm tbh 2 O 2, increasing concentration of the extracts decreased the amount of ROS levels formed, as indicated by a decrease in fluorescence signal. P a g e 264

72 Figure 6.54: ROS assay of catechin, 3 hours after tbh 2 O 2 stimulation Figure 6.55: ROS assay of polyphenols of testa, 3 hours after tbh 2 O 2 stimulation P a g e 265

73 Figure 6.56: ROS assay of aqueous extract of cashew testa, 3 hours after tbh 2 O 2 stimulation B. Viability Assay: The Cell Proliferation Reagent WST-1 provides a colorimetric assay for the quantification of cell viability and proliferation. WST-1 is a tetrazolium salt that when in contact with metabolically active cells gets cleaved to formazan by mitochondrial dehydrogenases. The formazan dye is then measured using a scanning spectrophotometer at wavelengths nm. The effect of plant extracts on cell viability under oxidative stress conditions induced by tbh 2 O 2 after 3 hrs incubation period is shown in Figure In the graphs indicated below EBM2 is used to represent the culture medium used as a control to ascertain whether the media components or plant extracts affect WST-1 dye processing in the absence of cells. First of all, incubation pre-treated cells with vehicle (o µm tbh 2 O 2 ) showed that the WST-1 dye was processed to formazan and thus that the cells were viable. Incubation with tbh 2 O 2 decreased cell viability as indicated by decreased formazan production. Pre-incubation with increasing concentrations of catechin, ethanol extract of cashew leaves, ethanol extract of cashew testa and P a g e 266

74 polyphenols of cashew testa and leaves were found to rescue cell viability after H2O2 treatment to some extent, however, a complete rescue of cell viability was not observed for any of the extracts. Unexpectedly, the presence of formazan was detected in all EBM-2 wells, suggesting that cells were mistakenly added to the wells. Figure 6.57: Viability assay of ethanolic extract of cashew leaves, 3 hrs after stimulation with tbh 2 O 2 P a g e 267

75 Figure 6.58: Viability assay of ethanolic extract of cashew testa, 3 hrs after stimulation with tbh 2 O 2 Figure 6.59: Viability assay of polyphenols of cashew testa, 3 hrs after stimulation with tbh 2 O 2 P a g e 268

76 Figure 6.60: Viability assay of catechin, 3 hrs after stimulation with tbh 2 O 2 Figure 6.61: Viability assay of polyphenols of cashew leaves, 3 hrs after stimulation with tbh 2 O 2 P a g e 269

77 C. Angiogenesis assay: The method is based on the differentiation of ECs on a basement membrane matrix, Matrigel, derived from the Engelbreth-Holm-Swarm tumor. ECs from human umbilical cords as well as from other sources differentiate and form capillary-like structures on Matrigel in the presence of 10% bovine calf serum (BCS) and 0.1 mg/ml of endothelial cell growth supplement (ECGS), which is a mixture of both acidic and basic fibroblast growth factor (Croix, 2000). An inhibition of angiogenesis was observed by tbh 2 O 2. The ability of the extracts to rescue this inhibition of angiogenesis caused by tbh2o2 was assessed. HMEC cells were seeded on matrigel. Measurement of angiogenic capacity was based on the mean tube length observed after 24 hrs. Photographs of the tubes formed were taken with the help of Olympus DP71 Microscope and the mean tubule length was quantified with Angioquant software. The deleterious effect of oxidative stress on angiogenic capacity of HMEC cells was observed on untreated cells as well as cells pretreated with catechin. Catechin, showed no significant antiangiogenic effect nor any statistically significant inhibitory effect on the angiogenesis inhibiting activity of tbh 2 O 2. As observed in Figure 6.62, in-vitro investigations have indicated that catechin was not able to inhibit several key events of the angiogenic process. In our experiments we were unable to derive conclusions about the effect of extracts on the angiogenesis inhibiting activity of tbh 2 O 2. The data obtained from Matrigel assays needs to be reanalyzed. Reports have indicated that certain polyphenolic compounds inhibit certain angiogenesis processes such as proliferation and migration of endothelial cells and vascular smooth muscle cells and the expression of two major proangiogenic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2, by both redox-sensitive and redox-insensitive mechanisms (Kondo, 2002). P a g e 270

78 EGM 2 H 2 O Catechin 2.5 Catechin + H 2 O 2 Figure 6.62: Representative matrigel assay of catechin (2.5 micrg) after 24 hrs incubation D. Western blot analysis: The proteins were isolated from cells treated with varying concentrations of catechin, control, tbhq and tbh 2 O 2 for 3 hrs. The expression of Nrf2 and betaactin by proteins extracted from pretreated HMEC cells was measured by Western blot analysis with indicated specific antibodies. The experiments were repeated three times a representative blot is shown below in Figure Upon activation, Nrf2 protein is stabilized and translocates to the nucleus to heterodimerize with other leucine zipper transcription factors such as Nrf1, mafk, jund, and c-fos, and bind to ARE in target gene promoters. To study the effects of pretreatment with plant extracts on Nrf2 protein expression, Western blot analysis was performed. P a g e 271

79 The image depicted in Figure 6.63 represents the blot prepared to evaluate NrF2 expression. Multiple bands of low signals at varying positions were observed in the lane of standard NrF2 lysate. This casts a doubt on location of the exact band for NrF2 in the controls as well as in the lysates of cells treated with catechin. The protocol adopted needs to be optimized to obtain a higher signal of the protein of interest, so that the expression of the protein can be better visualized and quantified. Thus the experiments of Western Blot need to be repeated, in order to conclude about the effect of catechin on NrF2 protein expression in HMECs. Where, 1 o Antibody- Nrf2 H-300 and 2 o - Swine antibody Anti rabbit immunolglobulin/hrp. L Ladder, M- medium, CL-Control lysate, N- Nrf2 lysate, t- tbh 2 O C, 25 C and 125 c are varying concentrations of catechin in micrograms used for treatment. Figure 6.63 Western Blot for Nrf2 expression of HMEC cells treated with varying concentrations of catechin, tbhq and hydrogen peroxide. P a g e 272

80 E. RT-PCR analysis: Exposure of HMEC cells to catechin increased the NrF2 protein levels as observed in the western blot assay, whereas an increase was not found upon tbhq and tbh 2 O 2 stimulation of cells. tbhq was used as a positive control for NrF2 activation, however an optimal increase in the induction of NrF2 target genes was not observed even with tbhq treatment. Expression of phase II enzymes is important in protecting the cells against stress conditions. We evaluated mrna expression profiles of phase 2 enzymes in catechin, tbhq (positive control) and tbh 2 O 2 treated cells using real-time PCR. Treatment of HMEC cells with 2.5 and 25µM concentration of catechin resulted in an upregulation of the Nrf2 target gene HMOX. Upregulation of the Nrf2 target gene HMOX was observed compared to tbhq (positive control) and vehicle as the control. At a 25µM concentration of catechin for GCLC and NQO-1 a decrease was observed upto 1.5 and 0.6 fold respectively. The treatment with tbh 2 O 2 also exhibited a decrease in the responses for HMOX, GCLC, and GCLM, and NQO-1 upto 1.8, 0.9, 0.5 and 0.9 fold respectively. Hence, we may infer that a down regulation of HMOX, and GCLM, and NQO-1 genes was observed with tbh 2 O 2. The expression of Nrf2 genes GCLC and GCLM was decreased upto 1.3 and 0.8 fold by tbh 2 O 2. Hence, we observe a down regulation of 3 out of 4 Nrf2 genes by catechin as indicated in the results of our experiments depicted in Figure P a g e 273

81 Figure 6.64: Effect of catechin, tbhq and tbh 2 O 2 stimulation on expression of NrF2 genes in HMEC s ***p< and **p to Significant p - values were obtained for HMOX and GCLM gene expression by catechin. The objective of the present study was to evaluate the effect of oxidative stress on cultured endothelial cells (treated with plant extracts), specifically with reference to the intracellular protective mechanism that is governed by Nrf2. Several compounds, including known Nrf2 activators, bioactive plant extracts, phenolic and catechin fractions from cashew were tested for their potential to reduce oxidative stress and its detrimental effects on HMECs. Growing evidence indicates an important role for ROS in diabetes, hypertension, restenosis after balloon angioplasty and atherosclerosis. However, little and contradictory information exists about the mechanisms by which ROS elicit their effect on the structure and function of the cells of the blood vessels. Fewer data exist on the correlation between the ROS status and endothelial cell death. In the present work, plant extracts which exhibited direct antioxidant effect in in-vitro assays like DPPH radical Scavenging assay and Greiss assay were used as candidates for various indirect in-vitro assays on HMECs. ROS assay was P a g e 274

82 performed to establish the antioxidant potential of the plant extracts. For mimicking oxidative stress, we selected tert-butyl hydrogen peroxide (tbh 2 O 2 ). The cells pretreated with plant extracts showed a decrease in ROS levels after tbh 2 O 2 stimulation, indicated by a decrease in dye flurorescence. The effect may be attributed to the activity of plant extracts to reduce ROS formation, especially in the concentration range from 2.5 to 25µg. We questioned whether the oxygen radicals affect directly the HMECs, whether their effect is dependent on concentration and whether this insult may lead to cell death. To this purpose, cultured HMECs were exposed to oxidative stress for different time intervals and concentrations. The ability of plant extracts to reverse the effect of oxidative stress on cell viability was estimated by use of WST-1 dye assay. The results showed that exposure of cultured HMECs to tbh 2 O 2 led to an expected decrease in cell viability. The cell viability decreased with increasing concentrations of tbh 2 O 2, even in the cells treated with cashew extracts. Thus we may infer that, although the plant extracts exhibit significant antioxidant activity in the ROS assay, their mode of reduction of ROS species does not improve cell viability as measured by mitochondrial dehydrogenase activity. Several recent studies have indicated that polyphenols, flavanols and anthocyanins have in vitro and in vivo antiangiogenic properties by inhibiting the expression of two strong proangiogenic factors, VEGF and matrix metalloproteinase (MMP-2), and also by preventing the proliferation and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) (Diaz, 1997). The antiangiogenic properties of polyphenolic compounds could contribute to explain the reduced risk of coronary heart diseases (viz. Arteriosclerosis) and cancer mortality following chronic consumption of moderate amounts of red wine and green tea. Angiogenesis is a key process in the development of several pathologies, including cancer, and the inhibition of angiogenesis is regarded as a promising tool to combat cancer. To develop novel angiogenesis inhibiting agents, the phenotype of tumor endothelial cells is subject to intensive investigation to identify putative target molecules for interference (Croix, 2000). Proper validation P a g e 275

83 of target molecules and inhibitors requires reproducible experimental in vitro approaches. Tissue-specific micro-environmental factors have a pronounced influence on the phenotype of the endothelial cells within the tissue. HMEC have been extensively used in angiogenesis research, and we used HMEC cells to evaluate the effect of plant extracts on angiogenesis. In vitro investigations have indicated that polyphenolic compounds are able to inhibit several key events of the angiogenic process such as proliferation and migration of endothelial cells and vascular smooth muscle cells and the expression of two major proangiogenic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase- 2, by both redox-sensitive and redox-insensitive mechanisms (Kondo, 2002). However, in our experiments we were unable to derive conclusions about the effect of extracts on the angiogenesis inhibiting activity of tbh2o2. The data obtained from Matrigel assays needs to be reanalyzed. Nrf2 levels in cells are regulated by further phosphorylation, nuclear export, and degradation, which may be enhanced by ARE-linked expression of Keap1 (Jain, 2007). Nrf2 may also exhibit ARE-linked expression. We investigated the activation status of Nrf2 in human microvascular endothelial cells by assessing nuclear translocation of Nrf2 by immunoblotting in protein fractions. Quantitative Western blotting for Nrf2 revealed increased expression of a protein at 57-kDa in fractions of cells treated with 25 microgram concentration of catechin. The lysates from tbhq and tbh 2 O 2 stimulated cells did not increase the expression to a greater extent as compared to catechin. This may be due to inability of tbhq and tbh 2 O 2 to translocate Nrf2 to Antioxidant Response enzyme (ARE) binding sites in nucleus with stimulation period of 3 hours. The experiments need to be designed more appropriately in order to conclude about the Nrf2 expression activity of catechin, tbhq and tbh 2 O 2. A sharp distinct band was observed at 57-kDa in all the lanes. But as this band also appears to be in the negative control lanes of the lysates of cells treated with lysates alone, we are uncertain about locating the right band. P a g e 276

84 We next focused on the effect of the plant extracts on the expression of Nrf2 target genes. The results from q-pcr experiments suggest that in Human microvascular endothelial cells, catechin activates Nrf2 in a concentration dependent manner. This effect was observed as an increase in HMOX1 expression, a target gene of Nrf2. A down regulation of most of the selected NrF2 genes was observed in our experiments with tbhq and tbh 2 O 2, and catechin. Hence the experiments should be still investigated further with proper positive controls in order to infer about the effect of catechin on expression of the target genes of Nrf2. P a g e 277

85 6.9 PHARMACOLOGICAL INVESTIGATIONS OF CASHEW EXTRACTS FOR ANTIDIABETIC ACTVITY Acute Oral Toxicity Studies Acute Toxic Class Method Acute Oral Toxicity Studies were carried out in Albino mice following OECD 423 Guidelines, for extracts which showed a better antioxidant activity in vitro and the results obtained are indicated in Table Dose limit at 2000 mg/kg (single dose) was administered to mice and observed for 14 days. The crude extract/s and polyphenol fractions of leaves and testa of cashew did not produce toxic symptoms or changes in behavior or death and found to be safer in mice upto the dose of 2000 mg/kg body weight, except for Polyphenol fraction of leaves. Animals treated with ethanol extracts of leaves and testa of cashew exhibited normal body weight gain and food intake throughout the study. Animals treated with polyphenol fraction of leaves showed a slight abnormal contractions in the abdominal region, possibly due to high polyphenolic content which might have caused gastric irritation. Acute toxicity tests have shown that the LD 50 of the extract in mice was higher than 2000 mg/kg except for Polyphenol fraction of leaves for which the limit was 2000 mg/kg and it s categorized under category 5 and category 4 of GSH as per OECD guidelines 423 respectively. The data describing the toxicity of ethanol extracts and polyphenol fractions of ethanol extract of cashew leaves and testa indicates a moderate toxicity of polyphenol fraction of cashew leaves. Nevertheless, the folk medicine generally uses aqueous extracts of the cashew leaves (Konan, 2007). We may therefore conclude that the long history of the cashew leaves used in folk medicine without toxicity reports seems to be largely supported by the data shown here. P a g e 278

86 Table 6.20: Acute toxicity studies of extracts of cashew Test Substance Ethanol Extract Dose Level 2000 LD 50 Cut off value 5000 Mortality at selected doses LD 50 cutoff > 2000 mg/kg of Testa mg/kg mg/kg 0/6 Category 5 of b.w. b.w GSH Ethanol Extract > 2000 mg/kg of Leaves mg/kg mg/kg 0/6 Category 5 of b.w. b.w GSH Polyphenols of ethanol extract of Testa 2000 mg/kg b.w mg/kg b.w 0/6 > 2000 mg/kg Category 5 of GSH Polyphenols of > 300 mg/kg ethanol extract mg/kg mg/kg 1/6 Category 4 of of leaves b.w. b.w GSH P a g e 279

87 6.9.2 Evaluation of The Effect Of Cashew Leaves And Testa Extracts In Streptozotocin-Nicotinamide Induced Type-II Diabetic Rats From the eight extracts prepared from leaves and testa of cashew, three extracts were selected for checking the hypoglycemic activity based upon the acute toxicity study and antioxidant effects and IC50 values. The doses of the extracts were selected based upon the literature available for the cashew leaves and testa extracts. The extracts screened by STZ - Nicotinamide Induced Type 2 Diabetes Mellitus were then selected for evaluation of antidiabetic activity by Neonatal streptozotocin model. The study groups for interventional study of STZ-Nicotinamide model containing six animals each were as follows: Group 1: Normal control [treated with saline] Group 2: Positive control [treated with Glibenclamide 0.45 mg/kg] Group 3: Diabetic control [treated with streptozotocin (60 mg/kg i.p) 15 min after the administration of (100 mg/kg i.p) nicotinamide] Group 4: Treatment group [treated with ethanol extract of cashew testa 175 mg/kg] Group 5: Treatment group [treated with polyphenol fraction of cashew testa 50 mg/kg] Group 6: Treatment group [treated with ethanol extract of cashew leaves 100 mg/kg] Group 7: Treatment group [treated with ethanol extract of cashew testa 350 mg/kg in divided doses] Extracts, fractions and standard were administered in the form of oral solution and suspension once daily for 15 consecutive days to diabetic animals. Control animals received only vehicle. For blood glucose levels, the blood was withdrawn by tail snipping on day 0,7,15 and estimated using glucose strips (Accu check active, Roche diagnostics, Germany).On day 15,blood was collected and estimated for various biochemical parameters. The results obtained for each of the parameters are given in Table P a g e 280

88 Determination of Physical end points Table 6.21: Effect of extracts on body weight on rats Treatment Normal control Body weight (g) On day 1 Day 7 Day ± ± ± 5.33 Diabetic control Glibenclamide 0.45 mg/kg Ethanol extract of cashew testa (175 mg/kg) Polyphenols of cashew Testa (50 mg/kg) Ethanol Extract of cashew leaves (100 mg/kg) Ethanol extract of cashew testa (350 mg/kg) in divided doses ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.16 Values are expressed as mean ± SEM, n = 6 * Significantly different from diabetic control, p<0.05 As observed in Table 6.21, ethanol extract of leaves, ethanol extract of testa and polyphenols of cashew testa and ethanol extract of leaves (double dose i.e. 350 mg/kg) evaluated for their antidiabetic effects on STZ-Nicotinamide induced model. The results showed no significant difference in the body weights of diabetic animals treated with extracts as compared with diabetic control at p<0.05. P a g e 281

89 Determination of Biochemical End points Table 6.22: Effect of extracts on fasting blood glucose levels in rats Fasting blood glucose mg/dl ± SEM Treatment On day 0 Day 7 Day 15 Normal control ± ± ± 2.75 Diabetic control ± ± ± 8.09 Glibenclamide 0.45 mg/kg Ethanol extract of cashew testa (175 mg/kg) Polyphenols of cashew Testa (50 mg/kg) Ethanol Extract Of cashew leaves (100 mg/kg) Ethanol extract of cashew testa (350 mg/kg) in divided doses ± ± 9.15* ± 8.10* ± ± 7.45* ± 5.61* ± ± 6.2* ± 2.3* ± ± 7.1* ± 3.61* ± ± 5.9* ± 8.6* Values are expressed as mean ± SEM, n = 6; * Significantly different from Control, p<0.05 As observed in Table 6.22, ethanol extract of leaves, ethanol extract of testa and polyphenols of cashew testa and ethanol extract of leaves (double dose i.e. 350 mg/kg) tested for their antidiabetic effects on STZ-Nicotinamide induced model. The diabetic animals treated with extracts showed a significant decrease in the fasting blood glucose levels as compared with diabetic control at p<0.05. P a g e 282

90 Table 6.23: Effect of extracts on percent reduction in fasting blood glucose levels in rats Treatment % Reduction in FBG Day 7 Day 15 Normal control 0.73 ± ± 1.0 Diabetic control ± ± 1.3 Glibenclamide 0.45 mg/kg ± ± 1.6 Ethanol extract of cashew testa (175 mg/kg) 14.9 ± ± 2.5 Polyphenols of cashew Testa (50 mg/kg) ± ± 1.8 Ethanol Extract Of cashew leaves (100 mg/kg) ± ± 3.5 Ethanol extract of cashew testa (350 mg/kg) in divided doses ± ± 1.3 Percent reduction in glycemia was calculated with respect to the zero day level according to the following formula: Percent reduction in glycemia = [(G i -G t )/G i ] x 100 Where G i is initial glycemia values and G t is the glycemia value at 7 and 15 days. As seen in the results in Table 6.23, ethanol extract of testa, polyphenols of cashew testa and ethanol extract of leaves, at single dose levels and ethanol extract of testa at double dose caused a 14.9%, 15.31%, % and 26.14% reduction in the fasting blood glucose levels of diabetic animals treated with extracts on day 7. On day 15 a reduction of 38.02%, 46.49%, 40.51% and % in the fasting blood glucose levels of diabetic animals treated with extracts was observed. P a g e 283

91 Table 6.24: Effect of extracts on serum biochemical parameters in rats Serum parameters on day 15 Treatment TG (mg/dl) TC (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) VLDL-C (mg/dl) Normal control 72.81±3.27* 75.21±1.7* 72.3 ± ± ±0.6* Diabetic control ± ± ± ± ± 0.6 Glibenclamide (0.45 mg/kg) ± 1.2* ± 3.8* 38.2 ± ± ±0.2* Ethanol extract of cashew testa (175 mg/kg) 78.44±3.87* ±4.05* 39.7 ± ±4.2* 15.6±0.7* Polyphenols of cashew Testa (50 mg/kg) ± 2.7* 95.0 ± 3.0* 39.2 ± ± ±0.4* Ethanol Extract Of cashew leaves (100 mg/kg) ± 2.4* 90.3 ± 2.4* 45.1±5.8* 25.2 ± ±0.9* Ethanol extract of cashew testa (350 mg/kg) in divided doses ± 1.4* 93.3 ± 7.4* 42.1 ± ± ±0.6* P a g e 284

92 Values are expressed as mean ± SEM, n = 6,*Significantly different from diabetic control,p<0.01 TG Triglyceride; TC - Total cholesterol; HDL-C High density lipoprotein cholesterol LDL-C - Low density lipoprotein cholesterol VLDL-C Very low density lipoprotein cholesterol LDL-C, VLDL- C calculated using friedwald formula VLDL = TG/5; LDL=TC-(HDL+TG/5) As observed in the results stated in Table 6.24, ethanol extract of testa, polyphenols of cashew testa and ethanol extract of leaves, at single dose levels and ethanol extract of testa at double dose levels tested for their antidiabetic effects on STZ-Nicotinamide induced model showed statistically significant results as compared with diabetic control at p<0.01 for the lipid profile Viz, triglyceride, total cholesterol, and VLDL-c levels. The total cholesterol showed statistically significant results as compared with diabetic control at p<0.01. The fundamental mechanism underlying hyperglycemia involves over-production (excessive hepatic glycogenolysis and gluconeogenesis) and decreased utilization of glucose by the tissues (Latner, 1958). Persistent hyperglycemia, the common characteristic of diabetes can cause most diabetic complications. In all patients, treatment should aim to lower blood glucose to near-normal levels. The diabetic syndrome in rats administered STZ and partially protected with suitable dosages of nicotinamide is characterized by stable moderate hyperglycemia, glucose intolerance and altered but significant glucose stimulated insulin secretion (Masiello, 1998). In our investigation, the blood glucose level estimation studies revealed that the ethanolic extracts and Polyphenol fractions of cashew leaves and testa have the capacity to lower blood glucose levels. The marked increase in serum triglycerides and cholesterol observed in diabetic rats is in agreement with the findings of Nikkila and Kekki, The most common lipid abnormalities in diabetes are hypertriglyceridemia and hypercholesterolemia (Khan, 1995; Mitra, 1995). Hypertriglyceridemia is also associated with metabolic consequences of hypercoagulability, hyperinsulinemia, P a g e 285

93 insulin resistance and insulin intolerance (Gingsberg, 1994). In our study, administration of the extract to the STZ induced diabetic rats significantly (p < 0.05) improved these parameters. The observed hypolipidaemic effect may be because of decreased cholesterogenesis and fatty acid synthesis. Various studies on medicinal plants have reported a similar lipid lowering activity (Ram, 1997; Sharma, 1997; Jouad, 2003). The characteristic loss of body weight associated with STZ induced diabetes is due to increased muscle wasting in diabetes (Swanston-Flat, 1990). The animals treated with extracts of cashew testa and leaves showed a weight loss in our studies, which may be directly due to the lipid lowering activity of the extract or indirectly to the influence on various lipid regulation systems. The significant antidiabetic activity of the cashew extracts in our study may be attributed to its principle antioxidant constituents. Longer duration studies on chronic models may contribute towards the development of a potent antidiabetic drugs and help gain insights into molecular mechanisms of action of herbal drugs. P a g e 286

94 6.9.3 Evaluation of the effect of cashew leaves and testa extracts in neonatal Streptozotocin induced (n- STZ) rat model of Type 2 Diabetes Mellitus The aim of our study was to investigate the effects of bioactive extracts of leaves and testa of cashew in streptozotocin induced neonates. Various biochemical and physical parameters were estimated and the results are indicated in Table Determination of physical endpoints Table 6.25: Effect of extracts on body weight in rats Treatment Body weight (g) On day 1 Day 10 Day 20 Day 30 Normal control ± ± ± ±5.1 Diabetic control Pioglitazone 2 mg/kg Ethanol extract Of leaves (100 mg/kg) Ethanol extract of testa (175 mg/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±15.3 Values are expressed as mean ± SEM, n = 6 * Significantly different from diabetic control, p<0.05 As observed from the results shown in Table 6.25, ethanol extract and polyphenols of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2-stz) model of type 2 diabetes in rats at single dose levels. Upto a period of 15 days and 30 days, there was no significant difference observed in the body weights of the treatment group and the diabetic control group at p<0.05. P a g e 287

95 Determination of Biochemical End points Table 6.26: Effect of extracts on fasting blood glucose levels in rats Treatment Fasting blood glucose (mg/dl) ± SEM On day 0 Day 15 Day 30 Normal control ± ± ± 1.82 Diabetic control ± ± ± 5.40 Pioglitazone 2 mg/kg ± ± 5.47* ±3.30* Ethanol extract of cashew leaves( ± ± ±2.87* mg/kg) Ethanol extract of testa (175 mg/kg) ± ± ±4.36* Values are expressed as mean ± SEM, n = 6; * Significantly different from Control, p<0.05 As indicated in the results shown in Table 6.26, ethanol extract and polyphenols of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2- STZ) model of type 2 diabetes in rats at single dose levels. The extracts showed statistically significant reduction in the reduction of blood glucose levels at p<0.05 on day 30. P a g e 288

96 Table 6.27: Effect of extracts on serum insulin and glycated haemoglobin in rats Treatment Serum insulin on day 30 (Uiu/ml) Glycated haemoglobin on day 30 (%) FIRI on day 30 Normal control ± ± ±5.1 Diabetic control ± ± ±14.8 Pioglitazone 5 mg/kg ± ± 0.18* ±5.86* Ethanol extract Of cashew leaves (100 mg/kg) Ethanol extract of testa (175 mg/kg) ± ± 0.17* ±4.69 * ± ± ± 4.10* Values are expressed as mean ± SEM, n = 6; * Significantly different from Control, p<0.05 As observed from the results shown in Table 6.27, ethanol extract and polyphenols of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2-stz) model of type 2 diabetes in rats at single dose levels. A statistically significant decrease in the glycated haemoglobin levels was observed at p<0.05. Ethanol extract of leaves and ethanol extract of testa decreased the fasting insulin resistance index (FIRI) and it was comparable to standard pioglitazone. A decrease in serum insulin levels was also observed as compared with diabetic control but it was not found to be statistically significant at p<0.05. P a g e 289

97 Table 6.28: Effect of extracts on serum triglyceride levels in rats Treatment Serum triglyceride( mg/dl) ± SEM On day 0 Day 15 Day 30 Normal control ± ± ± 4.13 Diabetic control ± ± ± 7.55 Pioglitazone 2 mg/kg ± ± ± 2.52* Ethanol extract Of cashew leaves( ± ± ± 4.47* mg/kg) Ethanol extract of testa (175 mg/kg) ± ± ± 4.55 * Values are expressed as mean ± SEM, n = 6; * Significantly different from Control, p<0.05 Ethanol extract and polyphenols of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2-stz) model of type 2 diabetes in rats at single dose levels. The triglycerides levels on day 30 were found to be decreased and statistically significant as compared to diabetic control at p<0.05. P a g e 290

98 Table 6.29: Effect of extracts on serum total cholesterol levels in rats Treatment Serum total cholesterol (mg/dl) ± SEM On day 0 Day 15 Day 30 Normal control ± ± ± 3.87 Diabetic control ± ± ± 4.95 Pioglitazone 2 mg/kg ± ± 2.64 * ± 3.18 * Ethanol extract Of cashew leaves( ± ± ± 3.21 * mg/kg) Ethanol extract of testa (175 mg/kg) ± ± ± 2.81 * Values are expressed as mean ± SEM, n = 6; * Significantly different from Control, p>0.05 As shown in Table 6.29, ethanol extract and polyphenols of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2-stz) model of type 2 diabetes in rats at single dose levels. Decrease in total cholesterol levels were found as compared with diabetic control and the values were significant at p<0.05. P a g e 291

99 Table 6.30: Effect of extracts on lipid parameters in rats Treatment Serum Parameter on day 30 HDL-C (mg/dl) LDL-C (mg/dl) VLDL-C (mg/dl) Normal control ± ± ± 0.82 Diabetic control ± ± ± 1.51 Pioglitazone 5 mg/kg ± ± 3.80 * ± 0.78 * Ethanol extract of cashew leaves ± ± ± 0.89 * (100 mg/kg) Ethanol extract of testa (175 mg/kg) ± ± ± 0.91 * Values are expressed as mean ± SEM, n = 6 * Significantly different from diabetic control, p>0.05 TG Triglyceride; TC - Total cholesterol; HDL-c High density lipoprotein cholesterol LDL-C - Low density lipoprotein cholesterol VLDL-C Very low density lipoprotein cholesterol LDL-C, VLDL- c calculated using friedwald formula VLDL = TG/5;LDL=TC-(HDL+TG/5) As observed in the results shown in Table 6.30, ethanol extract and polyphenols of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2- STZ) model of type 2 diabetes in rats at single dose levels. The lipid profiles for VLDL-C levels showed statistically significant results as compared with diabetic control at p<0.05. However a significant reduction in LDL-C and HDL-C were not observed at p<0.05. P a g e 292

100 Table 6.31: Effect of extracts on renal function biomarkers in rats Treatment Normal control Diabetic control Pioglitazone 2 mg/kg Ethanol extract Of cashew leaves(100 mg/kg) Ethanol extract of testa (175 mg/kg) Serum biomarkers of Liver and Kidney on day 30 SGOT SGPT Urea Creatinine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01* ± ± ± ± 0.02* Values are expressed as mean ± SEM, n = 6 * Significantly different from diabetic control, p>0.05 As indicated in Table 6.31, Ethanol extract and polyphenols of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2-stz) model of type 2 diabetes in rats at single dose levels. The renal markers were accessed to ascertain the effect of drug treatment in diabetic rats. However, there was no significant difference observed between the treatment group and diabetic control at p<0.05. P a g e 293

101 The Histological and Histochemical Studies After blood sampling for the biochemical analysis, the animals were sacrificed, quickly dissected, and small slices of the liver and kidney were taken and fixed in 10% formalin. The specimens were dehydrated in ascending grades of ethanol, cleared in xylene, and embedded in paraffin wax. Sections of 6 µm in thickness were prepared and stained with Haematoxylin and Eosin to examine under microscopy at 100x magnification. Histopathological Results Liver: The liver of control rats appears to be divided into the classical hepatic lobules; each is formed of cords of hepatocytes radiating from the central vein to the periphery of the lobule. The cell cords were separated by narrow blood sinusoids (Figure a). The histopathological examination of diabetic rats showed periportal necrosis of the hepatocytes near the portal areas. The liver also, showed dilated and congested portal vessels as well as areas of inflammatory cell infiltration (Figure b). In diabetic rats treated with standard Pioglitazone liver of control rats appears to be divided into the classical hepatic lobules; each is formed of cords of hepatocytes radiating from the central vein to the periphery of the lobule. The cell cords were separated by narrow blood sinusoids as in normal control rat (Figure c). In diabetic rats treated with extracts of testa and leaves, the liver architecture appears more or less like control (Figure d and e). Kidney: Examination of the kidney of the normal control rats revealed normal glomeruli with thin glomerular basement membranes, normal cellularity and patent capsular space surrounding by proximal and distal were normal (Figure a). Light microscopy of the kidney sections from diabetic rats showed an increase in the mesangial cell and matrix of the glomeruli and hyalinization of the arterioles (Figure b). In diabetic rats treated with pioglitazone, the kidney architecture appears more or less like normal control (Figure 6.66 c). In diabetic rats treated with leaves and testa extract, the kidney architecture appears more or P a g e 294

102 less like normal control with the exception of some inflammatory infiltration that appeared in the interstitum (Figure 6.66 d and e). Pancreas: In the pancreas of normal control rats many round (Figure a) and elongated islets were evenly distributed throughout the cytoplasm, with their nucleus lightly stained than the surrounding acinar cells. In diabetic rats, (Figure b) the islets were damaged, shrunken in size and infiltration of lymphocytes was observed. In rats treated with plant extracts and standard Pioglitazone, islets were comparable to normal rats and there was not much shrinkage in size of the islet although slight damage was observed. (Figure 6.67 c, d and e). P a g e 295

103 a b c d e Figure 6.65: Photomicrographs of liver a) normal rat b) diabetic rat c) diabetic rats treated with standard drug d) diabetic rat treated with ethanol extract of leaves e) diabetic rats treated with ethanol extract of testa. (Sections treated with hematoxylin and eosin x 100) P a g e 296

104 a b c d e Figure 6.66: Photomicrographs of kidney a) normal rat b) diabetic rat c) diabetic rats treated with standard drug d) diabetic rat treated with ethanol extract of leaves e) diabetic rats treated with ethanol extract of testa. (Sections treated with hematoxylin and eosin x 100) P a g e 297

105 a b c d e Figure 6.67: Photomicrographs of pancreas a) normal rat b) diabetic rat c) diabetic rats treated with standard drug d) diabetic rat treated with ethanol extract of leaves e) diabetic rats treated with ethanol extract of testa. (Sections treated with hematoxylin and eosin x 100) P a g e 298

106 In further agreement with Portha et al. (2002),it can be suggested that this animal model is also suitable for measuring insulin secretion in comparison with a (nonglucose-dependent) insulin-secreting drug like tolbutamide. The data obtained from the experiments provided a clear evidence that an oral single dose of this plant extracts stimulates insulin secretion and this may partially explain the mechanism of the efficacy of cashew leaves and testa. A model of type 2 diabetes can be induced in rats by either i.v. (tail vein) or i.p. treatment with STZ in the first days of life. At 8 10 weeks of age and thereafter, rats neonatally treated with STZ manifest mild basal hyperglycemia, an impaired response to the glucose tolerance test, and a loss of pancreatic β-cell sensitivity to glucose (Pascoe and Storlien, 1990). The (n-stz) rat model exhibits a clear basal hyperglycemia with glucose intolerance, high HbA1c values, a strong reduction of pancreatic insulin stores, a decreased (50%) basal plasma insulin level, and a lack of plasma insulin response to glucose (Portha et al., 2002). It has been observed that STZ at first abolished the pancreatic β-cell response to glucose, but a temporary return of responsiveness then appears which is followed by its permanent loss (Mythili et al., 2004). It is necessary to reemphasize that natural products display several effects besides lowering blood glucose in these experimental models. In view of the lack of parallel studies of their toxicity, these models of diabetes induced by either alloxan or STZ are considered a screening step in the search for drugs for the treatment of diabetes. Experimental diabetes in animals has provided considerable insight into the physiologic and biochemical derangement of the diabetic state. Many of this derangement were in the form of significant changes in lipid metabolism and structure (Sochar, 1985). These structural changes are clearly oxidative in nature and are associated with development of vascular disease (Baynes, 1999). In diabetic rats, increased lipid peroxidation was also associated with hyperlipidaemia (Morel, 1989). During diabetes, a profound alteration in the concentration and composition of lipids occurs. Liver and kidney are important for glucose and lipid homeostasis, they participates in the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol, phospholipids P a g e 299

107 and triglycerides. Thus it is expected to have changes in liver and kidney during diabetes (Seifter, 1982). The results obtained indicate that the n-stz diabetic animal group developed a moderate type 2 diabetes; however the animals were in better conditions during the experiments (with lower blood-sugar concentrations); it confirms that the (n- STZ) model is suitable for investigations on type 2 diabetes. Thus, the results presented here suggest that that these extracts of cashew testa and leaves could be developed as a phytomedicine. P a g e 300

108 6.10 DEVELOPMENT OF FORMULATION OF ETHANOL EXTRACT OF CASHEW TESTA Pre-compression Parameters (Micrometric evaluation) 1. Determination of Water uptake characteristics (moisture sorption study in desiccators) 10 % of DCP 8 % of DCP 6% of DCP 4 % of DCP 2 % of DCP Figure 6.68: Physical appearance of the DEP s containing various percentages of DCP after 15 days in desiccators P a g e 301

109 Based on the results of the moisture content studies, the dry extract preparation containing 10% DCP was selected for tablet formulation (Table 6.32). Hence, density, flow property and compressibility of this dry extract blend were further investigated. Table 6.32: Water content of the dry extract preparations (DEP) Quantity of DCP in Dry Water content (%) ± SD extract powder (DEP) 2.0 % 5.5 ± % 5.1 ± % 4.7 ± % 4.3 ± % 3.5 ± Density Compressibility indices less than 15% are indicative of free-flowing powders; indices greater than 40% usually correspond to very poor flow (Carr, 1965). As shown in the Table 6.33, the compression index had decreased form 29.8 to 19.9 indicating the improvement of the flow properties due to the formulation of the crude extract in to dry extract preparation using DCP. Table 6.33: Porosity, compression index and Hausner ratio of the dry extract preparation (DEP) as well as the crude extract Sample Porosity (%) Compression (Carr s) index (%) Hausner ratio DEP Ethanol extract of testa P a g e 302

110 The HR is a simple test usually used to evaluate fluidity, where values less than 1.25 indicate good flow properties and values greater than 1.5 indicate poor flow properties (Fonner et al.,1966). As shown in the table, the dry extract preparation decreased the HR from 1.42 to 1.25, indicating the improvement of flowability after mixing with DCP. A. Powder flow properties Angle of repose The dry extract preparation showed an angle of repose 36.2 which is classified as passable (fair) flow according to Wells and Aulton (1988) and the flow rate was found to be 7.9 gm/sec. B. Formulation of Tablet Based on the preliminary investigation, the tablet formulation containing dry extract preparation was formulated with various proportions of different additives as mentioned below in Table Table 6.34: Optimised formula of tablet formulation Sr.No Ingredients Quantity for each tablet of 350 mg 1 Extract mg 2 Avicel -102 (directly compressible Micro crystalline Cellulose- MCC) 85.0 mg 3 Dibasic Calcium Phosphate (DCP) 35.0 mg 4 Croscarmellose 36.0 mg 5 Talc 7.0 mg P a g e 303

111 C. Post Compression parameters The results of post compression parameters and results of various pharmacopoeial tests of the tablets prepared from the optimized formula are shown in Table and in Figure Shape and color of tablets Tablets were brown in color and flat circular shape when observed under a lens by placing the tablets in light. Uniformity of thickness Three tablets were picked randomly from each batch of formulation and thickness was measured individually with dial caliper (Mitutoyo, Japan). The thickness of the tablet ranged from 4.9 ± 0.01 to 4.10 ± 0.05 mm. Uniformity in values indicates that the tablets were compressed without sticking to dies or punches. Hardness testing The hardness of the tablets was between 3.5 kg/cm kg/cm 2. The lower standard deviation value indicated that hardness of the tablets were almost uniform and possess good mechanical strength and sufficient hardness. Friability testing The friability of compressed tablets was within approved range (<1%) in the tablets. This indicates that that the tablets possess good mechanical strength. Weight Variation All the tablets passed the weight variation tests as the % weight variation was within the pharmacopoeial limits of ± 10%. The weight of all the tablets were found to be almost uniform. This can be attributed due to the good flow property and good compressibility of the tablets. Content uniformity testing The drug content of the tablets was ascertained spectrophotometrically for five times. The catechin content of the tablets were between 8.88 ± 0.12 to 8.10 ± 0.13 mg when determined spectrophotometrically at 273 nm. The Limit of detection (LOD) and Limit of quantitation (LOQ) were found to be 10 and 30 micrograms P a g e 304

112 respectively. The calibration range was established between micrograms. Disintegration Test The internal structure of tablet, i.e. pore size distribution, water penetration into tablets and swelling of disintegration substance are suggested to be the mechanism of disintegration. The disintegration time increases with increase in DCP and MCC content. The disintegration time for tablets were found to be less than 2.5 mins. Dissolution conditions The cumulative drug release was calculated based upon the amount of catechin present in each tablet. The drug releases at 10 mins, 15 mins and 30 mins were 28.9 %, 72.6% and 93.50% respectively. The rapid dissolution might be due to rapid breakdown of the tablet and faster absorption of the drug. Stability testing In the stability studies of the formulation the tablets were analysed for 6 months period for drug content uniformity, hardness, in vitro disintegration time and friability. From the results obtained we could conclude that the tablets were stable and they retained their original properties. The results of the post compression parameters are listed in Table Figure 6.69: Tablets prepared from ethanol extract of testa P a g e 305