THIN LAYER CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY Thin layer chromatography is the best known technique of plant biochemistry. TLC is used for preliminary separation and determination of plant constituents. It is helpful for proper identification, authentication of phytoconstituents. In present study the TLC was performed to prepare qualitative TLC fingerprinting profile of methanol fruit and leaf extracts of studied plant parts. Attempt was made to separate flavonoids in studied plant parts. TLC fingerprinting is of key importance for standardization of herbal drugs. It is a powerful and relatively rapid technique to distinguish between chemical classes. A simple chromatographic technique of quantitative microscopy is an important analytical technique for powdered drugs, especially when chemical and other methods of evaluation of crude drug fails to accurate measure of quality. 6.1.1 Preparation of thin layer plates Thin layer glass plate (5cm 15cm) in size and.25 mm thickness was prepared by spreading aqueous slurry of finely grind solid silica gel. The plates were activated in an oven at 11 c -12 cfor 3 minutes before sample spotting. 6.1.2 Sample application and chromatogram development The collected methanol extract was applied as a spot 1cm from edge of the plate by using capillary tube. The plate was then placed in closed container saturated with vapors of developing solvents i.e. Ethyl acetate: formic acid: acetic acid: water in volume ratio 1:11:11:26 as a mobile phase and care was taken to avoid direct contact between the sample and developer. It shows good separation of components. After developing the plate up to two third of the length of plate it was removed from container and dried. After dryness they were spray with flavonoids reagent (1% ethanolic-2-aminoethyl diphenlyborinte solution) and examined in ultra violet chamber. Ph.D. Thesis: Miss Sandhya V. Rodge, 215 86

6.1.3 Observations TLC chromatogram of in the methanol extracts the spots were characterized by Rf values and colour obtained under UV light which are tabulated in Table No 46-57. The methanol extract was selected as it contains flavonoids as a major phytoconstituents and exhibits more fluorescence spots. Solvent system: Ethyl acetate: formic acid: acetic acid: water in volume ratio 1:11:11:26. Chamber saturation: 3 minutes. Temperature: 29-33. Detection: UV at 256nm. 6.2.1 Thin layer chromatography of Flavonoids of Coccinia grandis leaf The thin layer chromatography of methanol leaf extract of Coccinia grandis shows separation of two flavonoids i.e. rutin and quercetin with distinctive colours. Two spots were observed with different colour and Rf values determined by measuring the distance traveled by solvent and distance traveled by the solute. The spot 1with orange colour and spot 2 shows yellow colour (Fig No 48). The Rf values are.42 and.85 respectively. It was compared with standard Rf values for the confirmation of the rutin and quercetin (Table No 46). Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids 1 Orange.42.44 Orange Rutin 2 Yellow.85.87 yellow Quercetin Table No 46: TLC of Coccinia grandis leaf 6.2.2 Thin layer chromatography of Flavonoids of Coccinia grandis fruit The thin layer chromatography of methanol fruit extract of Coccinia grandis shows separation of two spots with different colours. The spot one with orange colour (Fig No 48) and Rf value is.4 which confirmed as rutin and spot two with greenish yellow colour and.84 Rf value confirmed as quercetin (Table No 47). 87 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Spot No. Colour of spot Rf value Standard Rf value Colour of Flavonoids Flavonoids 2 Orange yellowish.4.44 Orange Rutin 3 Greenish yellow.84.87 Yellow Quercetin Table No 47: TLC of Coccinia grandis fruit 6.3.1 Thin layer chromatography Lagenaria siceraria leaf Thin layer chromatography of methanol leaf extract of Lagenaria siceraria exhibit the separation of two flavonoids with distinctive colours developed after spray of 1% ethanol 2-aminoethyl and diphenlyborinte solution. The spot 1 with orange colour and its Rf value is.42 confirmed as rutin, spot second with yellowish green colour (Fig No 49) and Rf value is.86 confirmed as quercetin (Table No 48). Spot No. Colour of spot Rf value Standard Rf value Colour of Flavonoids Flavonoids 1 Orange.46.44 Orange Rutin 2 Yellowish green.86.87 Yellow green Quercetin Table No 48: TLC of Lagenaria siceraria leaf 6.3.2 Thin layer chromatography Lagenaria siceraria fruit The thin layer chromatography of Lagenaria siceraria fruit extract differentiated in to two spots with distinct colours. By measuring Rf values it is identified as flavonoids. The spot 1 with orange colour and Rf value is.42 which confirms the presence of rutin. The spot 2 with yellow colour (Fig No 49) and Rf value is.89 confirms quercetin (Table No 49). Ph.D. Thesis: Miss Sandhya V. Rodge, 215 88

Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids 1 Orange.42.44 Orange Rutin 3 yellow.89.87 Yellow Quercetin Table No 49: TLC of Lagenaria siceraria fruit 6.4.1 Thin layer chromatography of flavonoids of Trichosanthes tricuspidata leaf TLC of Trichosanthes tricuspidata leaf extract shows two spots with different colours and Rf values, the spot no 1 indicates orange colour with Rf value.43 and spot no. 2 was yellow coloured (Fig No 5) and its Rf is values.88. On the basis of Rf values and colour it is confirmed as rutin and quercetin (Table No 5). Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids 1 Orange.41.44 Orange Rutin 2 Yellow.88.87 Yellow Quercetin Table No 5: TLC of Trichosanthes tricuspidata leaf 6.4.2 Thin layer chromatography of flavonoids of Trichosanthes tricuspidata fruit The thin layer chromatography of methanol fruit extract of Trichosanthes tricuspidata shows separation of two flavonoids with distinctive colour. The first spot with orange brown colour and Rf value is.46 confirmed as rutin, spot no 2 with yellowish green colour (Fig No 5) and Rf value is.85 confirmed quercetin (Table No 51). Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids 2 Orange brown.47.44 Orange Rutin 3 Yellowish green.85.87 Yellow Quercetin Table No 51: TLC of Trichosanthes tricuspidata fruit 89 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

6.5.1 Thin layer chromatography of flavonoids of Diplocyclos palmatus leaf Thin layer chromatography of extract of Diplocyclos palmatus leaf extract shows separation of two flavonoids with distinctive colours developed after spray of 1% ethanol 2-Aminoethyl biphenyl borinate solution. The spot one with yellowish orange colour and Rf value is.43 confirmed as rutin. The spot no 2 with yellow colour (Fig No 51) and Rf value is.87 confirmed as quercetin (Table No 52). Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids 1 Yellowish orange.43.44 Orange Rutin 2 Yellow.87.87 Yellow Quercetin Table No 52: TLC of Diplocyclos palmatus leaf 6.5.2 Thin layer chromatography of Diplocyclos palmatus fruit The thin layer chromatography of Diplocyclos palmatus fruit extract exhibit two spots with distinct colours, by measuring Rf values the identification of flavonoids were confirmed. The spot no. 1 with greenish orange colour and Rf value is.42 confirms flavonoids rutin, spot no.2 with yellowish brown colour (Fig No 51) and Rf value is.88confirms the flavonoids quercetin (Table No 53). Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids 1 Greenish orange.42.44 Orange Rutin 2 Yellowish brown.82.86 Yellow Quercetin Table No 53: TLC of Diplocyclos palmatus fruit 9 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

6.6.1 Thin layer chromatography of Cucumis setosus leaf The thin layer chromatography of Cucumis setosus leaf extract exhibit the presence of two spots with different colours and Rf values. The spot no.1 with orange brown colour and Rf value is.46 confirmed as rutin. The spot no. 2 with yellow colour (Fig No 52) and Rf value.85 confirms as quercetin (Table No 54). Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids 1 Orange brown.46.44 Orange Rutin 2 Yellow.85.87 Yellow Quercetin Table No 54: TLC of Cucumis setosus leaf 6.6.2 Thin layer chromatography of Cucumis setosus fruit The thin layer chromatography of Cucumis setosus fruit extract shows two spots with distinct colours. The spot no. 1 with orange colour and Rf value is.48 confirms flavonoids rutin, spot no. 2 with yellowish green colour (Fig No 52) and Rf value is.86 confirms flavonoids quercetin (Table No. 55). Spot No. Colour of spot Rf value Standard Rf value Colour of Flavonoids Flavonoids 1 Orange.48.44 Orange Rutin 2 Yellowish green.85.87 Yellow Quercetin Table No 55: TLC of Cucumis setosus fruit 91 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

92 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Plant name Rutin Quercetin Plant parts Leaf Fruit Leaf Fruit Coccinia grandis.42.4.85.84 Lagenaria siceraria.46.42.86.89 Trichosanthes tricuspidata.41.47.88.85 Diplocyclos palmatus.43.42.87.82 Cucumis setosus.46.48.85.85 Table No 56: TLC of leaf and fruit extract of studied plants The TLC of present study noticed that the Rf values of leaf are.46 and.46 which concludes the presence of rutin was detected in Lagenaria siceraria and Cucumis setosus. Whereas, in fruit extract the Rf values are.47 and.48 in Trichosanthes tricuspidata and Cucumis setosus respectively. The leaf extract shows.88 and.87 are the Rf values in Trichosanthes tricuspidata and Diplocyclos palmatus respectively confirming the presence of quercetin. While the fruit extract exhibit.89 Rf value in Lagenaria siceraria indicating presence of quercetin (Table No 56). The TLC of reveals presence of rutin and quercetin in leaf and fruit extract further it was confirmed HPLC analysis. Ph.D. Thesis: Miss Sandhya V. Rodge, 215 93

6.7 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS The determination of rutin and quercetin the HPLC analysis was performed at Chemistry Research Laboratory, Deogiri College, Aurangabad. a) Materials and reagents Quercetin and rutin were purchased from Sigma-Aldrich, Bangalore, India. Methanol, water, acetic acid and Acetonitrile were of HPLC grade from SD fine chemicals, Mumbai, India. All the reagents and chemicals used were of analytical and HPLC grade. Water (HPLC grade) was obtained from Milli Q RO system. b) Plant Material The dried powdered leaf and fruit drug of selected medicinal plants were used for HPLC analysis. c) Preparation of sample solution About.1 g of dry plant powder was accurately weighed in a stopper tube, and then in the stopper tube, 1. ml of aqueous methanol (4%) was added and incubated in for about 24 hrs. After that the tube was sonicated in an ultrasonic bath (Model: USC 1, Frequency: 5 Hz, Amkette industries Pvt. Ltd, India), for 15 min. The contents of the tube were filtered through Whatman filter paper No 41 (Merck, India). The filtrate was used for further analysis. d) Preparation of standard solution The standard stock solution (1 mg/ml) of quercetin and rutin were prepared by dilution in methanol. These stock solutions were stored in light resistant containers and used for calibration curve of quercetin and rutin. e) Mobile Phase Preparation The solutions of Methanol (HPLC Grade), acetonitrile (HPLC grade) and Water (HPLC Grade) with 1 acetic acid in the ratio of 4:15:45 were prepared and mixed thereafter it is sonicated. f) Instrumentation and Chromatographic conditions HPLC analysis was performed with Aglient technology HPLC system. The analysis was performed by using Zorbax SB-C18, 25 X 4.6 mm internal diameter with 5 micron Ph.D. Thesis: Miss Sandhya V. Rodge, 215 94

particle size column and PDA detector set at 298 nm, in conjunction with a mobile phase of Methanol, Acetonitrile and Water with 1% acetic acid in the ratio of 4:15:45%(v/v) at a flow rate of 1. ml/min. The run time of samples was 1 minute and the operating pressure: 131 bar. The separation was performed at ambient temperature. The injection volume was 2μl. The methanolic leaf and fruit extracts of selected plants are used for HPLC analysis for the confirmation of flavonoids which are detected in TLC process. The HPLC chromatographic pattern corresponding to the methanol extract of selected plants were monitored at 298 nm is shown in Spectral Graph Nos 4-23. In the present study, characterization of chemical compounds was performed based on UV/visible spectrometry, as well as by the comparison of retention times with those of semi synthesized reference compound Rutin and Quercetin. The retention time of standard rutin and quercetin is 2.593 and 5.643 respectively. The compound was confirmed by HPLC with UV spectrometry provided peak information and retention time in minute. The retention time of rutin in Cucumis setosus fruit found to be 2.37 (Spectral Graph No. 5), by comparing with standard retention time it is observed that the presence amount of rutin in Cucumis setosus fruit having.78 percentage. The quercetin was not detected. The retention time of rutin in Lagenaria siceraria fruit found to be 2.413 (Spectral Graph No. 7), by comparing with standard retention time it is observed that the presence amount of rutin in L. siceraria fruit having.52 percentage. The quercetin was not detected. The retention time of rutin in Trichosanthes tricuspidata fruit found to be 2.657 (Spectral Graph No. 9), by comparing with standard retention time it is observed that the presence amount of rutin in T. tricuspidata fruit having.3 percentage. The quercetin was not detected. The retention time of rutin in Coccinia grandis fruit found to be 2.53 (Spectral Graph No. 11), by comparing with standard retention time it is observed that the presence Ph.D. Thesis: Miss Sandhya V. Rodge, 215 95

amount of rutin in C. grandis fruit is.78%. The retention time of quercetin is 5.613 (SpectralGraphNo1) showing.125% amount of quercetin. The retention time of rutin in Diplocyclos palmatus fruit found to be 2.52 (Spectral Graph No. 13), by comparing with standard retention time it is observed that the presence.85% amount of rutin. Whereas, the retention time of quercetin is 5.77 (Graph No 12). The amount of quercetin found in D. palmatus is about.24%. The retention time of rutin in Cucumis setosus leaf found to be 2.363 (Spectral Graph No. 15), by comparing with standard retention time it is observed that the presence amount of rutin in Cucumis setosus leaf having a.26 percentage. The quercetin was not detected. The retention time of rutin in Trichosanthes tricuspidata leaf found to be 2.443 (Spectral Graph No. 17), by comparing with standard retention time it is observed that the presence amount of rutin in T. tricuspidata leaf having.9 percentages. The quercetin was not detected. The retention time of rutin in Coccinia grandis leaf found to be 2.57 (Spectral Graph No. 19), by comparing with standard retention time it is observed that the presence amount of rutin in C. grandis leaf has.164 percentage but the quercetin was not found. The retention time of rutin in Diplocyclos palmatus leaf is 2.67 (Spectral Graph No. 21), by comparing with standard retention time it is observed that the presence.55 amount of rutin in D. palmatus leaf. The quercetin is absent. The retention time of rutin in Lagenaria siceraria leaf is 2.657 (Spectral Graph No.23), by comparing with standard retention time it is observed that the presence of.43 amount of rutin in L. siceraria leaf. The quercetin was absent. In present study quantification of rutin in methanol extract it is observed that maximum percentage of rutin is detected in Diplocyclos palmatus fruit and Cucumis setosus i.e. about.85% and.26% respectively. The notable percentage of rutin also detected in Coccinia grandis leaf and fruit i.e..164% and.121% respectively. Ph.D. Thesis: Miss Sandhya V. Rodge, 215 96

Trichosanthes tricuspidata leaf and Cucumis setosus fruit has rutin content.9% and.78% respectively. The rutin also detected in Diplocyclos palmatus leaf, Lagenaria siceraria fruit, L. siceraria leaf and Trichosanthes tricuspidata fruit i.e..55%,.52%,.43% and.3% respectively. The Coccinia grandis fruit has maximum percentage of quercetin i.e..125% and minimum percentage of quercetin.24% is detected in Diplocyclos palmatus. The flavonoids quercetin is not found in leaf and fruits of Lagenaria siceraria, Trichosanthes tricuspidata and Cucumis setosus (Table No 57). Sample Sample Name Flavonoid content in plant at 1mg/ml conc. Rutin (%) Quercetin (%) 1 C. setosus fruit.78-2 L. siceraria fruit.52-3 T. tricuspidata fruit.3-4 C. grandis fruit.121.125 5 D. palmatus fruit.85.24 6 C. setosus leaf.26-7 T. tricuspidata leaf.9-8 C. grandis leaf.164-9 D. palmatus leaf.55-1 L. siceraria leaf.43 - Table No 57: HPLC analysis of selected pants STD: Rutin and quercetin: RQ std. p1, RQ std. P2, RQ std. P3 Std. Concentration: 1 mg/ml (dissolve in 1 % HPLC grade methanol) Sample concentration: 1 mg/ml (dissolve in 1 % HPLC grade methanol) Rutin and Quercetin present in plant extract calculated as: Avg. of Sample area / avg. of std. area std.conc. / sample conc. 1 =. % Ph.D. Thesis: Miss Sandhya V. Rodge, 215 97

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\standard Rutin and Quercetin 1st. Method: Acquired: Printed: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met 7/31/214 12:5:7 PM 8/1/214 2:25:36 PM 2 2 2.423 1 1 3.13 5.443 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 1: HPLC chromatogram of standard Rutin and Quercetin 298 nm Results 2.423 19552697 23.5 1914963 35.8 3.13 5317694.63 4198.79 5.443 647442198 76.32 3367389 63.41 848286799 1. 53176 1. 98 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\standard Rutin and Quercetin 2nd Acquired: Printed: 7/31/214 1:3:45 PM 8/1/214 2:27:47 PM 2 5.393 2 1 1 2.36 2.947 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 2: HPLC chromatogram of standard Rutin and quercetin VWD: Signal A, 298 nm Results 2.36 433325943 45.65 433227 59.25 2.947 12812481 1.35 93844 1.38 5.393 5318933 53. 26782471 39.37 949157357 1. 682412 1. 99 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\standrd Rutin and Quercetin3rd Acquired: Printed: 7/31/214 1:16:18 PM 8/1/214 2:28:26 PM 15 5.643 15 1 1 5 1.66 2.127 2.593 3.18 5 1 2 3 4 5 6 7 8 9 1 Spectral Graph No: 3 HPLC chromatogram of standard Rutin and Quercetin 1.66 3359127.49 419115.85 2.127 111555.16 125397.25 2.593 296163833 43.16 27996884 56.85 3.18 285593.42 344868.7 5.643 38268369 55.77 2363553 41.35 686169472 1. 49249817 1. Avg. of std. rutin area = 383388943 and avg. of std. Quercetin area = 511471667 1 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis setosus fruit runs1st Acquired: 7/3/214 2:55:39 PM Printed: 8/1/214 1:34:56 PM 4 2.37 2.227 4 2 2 1.343 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 4: HPLC chromatogram of Cucumis setosus fruit 1.343 25943834 37.66 844139 48.85 2.37 88988 11.62 469249 27.15 2.227 34944984 5.72 414789 24. 6889786 1. 1728177 1. 11 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis setosus fruitruns 2nd Acquired: 7/3/214 3:8:5 PM Printed: 8/1/214 1:45:5 PM 75 75 5 25 2.53 3.12 5 25 1.773 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 5: HPLC of Cucumis setosus fruit 1.773 31458762 4.97 126449 58.3 2.53 1634119 21.28 511981 24.74 3.12 2898581 37.75 35899 16.96 76785681 1. 269329 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 243597/383388943 1/1 1 =.78 % For quercetin: Ph.D. Thesis: Miss Sandhya V. Rodge, 215 12

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria sicerariafruit runs 1st.Method: Acquired: Printed: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met 7/3/214 3:2:51 PM 8/6/214 2:6:55 PM 15 1 5 1.713 1.957 2.3 2.62 2.84 15 1 5 1.513 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 6: HPLC of Lagenaria siceraria fruit 1.513 34251977 14.3 22722 25.18 1.713 1591567 6.51 15816 17.52 1.957 32137625 13.16 178333 19.73 2.3 233769 9.57 1217595 13.49 2.62 7783822 3.19 98779 1.87 2.84 1372489 53.54 119229 13.21 24417571 1. 923816 1. Ph.D. Thesis: Miss Sandhya V. Rodge, 215 13

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria siscerariafruit runs2nd. Acquired: 7/3/214 3:34:5 PM Printed: 8/1/214 1:48:35 PM 15 1 1.53 2.9 2.413 2.627 3.93 15 1 5 5 1.297 1.743 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 7: HPLC of Lagenaria siceraria fruit 1.297 31716118 11.29 246485 22.3 1.53 238948 8.19 18986 16.29 1.743 3827399 13.63 287417 18.88 2.9 2441782 8.69 133599 12.9 2.413 876647 3.12 176681 9.74 2.627 3714822 13.18 1352446 12.23 3.93 117684455 41.9 93578 8.46 28881714 1. 115433 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 166858 /383388943 1/1 1 =.52 % For quercetin: 14 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Tricosanthes tricuspidata fruit run 1st Acquired: 7/3/214 3:47:11 PM Printed: 8/7/214 12:25:45 PM 75 5 25 1.59 1.93 2.483 75 5 25 1.183 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 8: HPLC of Trichosanthes tricuspidata fruit 1.183 27836694 39.36 12294 36.74 1.59 1491119 21.8 87341 26.1 1.93 1812337 25.59 657818 19.66 2.483 9878226 13.97 585827 17.51 7728366 1. 3346446 1. 15 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Tricosanthes tricuspidata fruit runs 2nd Acquired: 7/3/214 4:2:47 PM Printed: 8/7/214 12:35:56 PM 4 2 1.74 2.83 2.657 4 2 1.353 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 9: HPLC of Trichosanthes tricuspidata fruit 1.353 1977759 46.15 868253 38.76 1.74 9222436 21.52 549187 24.52 2.83 519338 12.11 449675 2.7 2.657 86653 2.22 37294 16.65 42855286 1. 22455 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 9271614.5 /383388943 1/1 1 =.3 % For quercetin: % Area % Report 16 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia grandis fruit runs 1st. P4 - A.dat Acquired: 7/31/214 1:28:26 PM Printed: 8/7/214 12:41:25 PM 2 2 1 1.713 2.1 4.247 1 2.637 5.733 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 1: HPLC of Coccinia grandis fruit 1.713 144196 8.35 61456 7.8 2.1 435931 2.59 456357 5.29 2.637 43853342 26.8 3613888 41.93 4.247 4486216 26.65 498718 5.79 5.733 611325 36.34 344212 39.91 16815191 1. 8619631 1. 17 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia grandis fruit runs 2nd. Acquired: 7/31/214 1:41:5 PM Printed: 8/7/214 12:42:56 PM 2 2.53 2 1 1.573 1.95 1 5.613 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 11: HPLC of Coccinia grandis fruit 1.573 11263453 9.77 495759 7.41 1.95 598238 5.19 373799 5.59 2.53 3775952 26.68 22921 34.25 5.613 67312814 58.36 3527731 52.75 115334527 1. 6687499 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 37314647/383388943 1/1 1 =.121 % For quercetin: 6426569.5/ 511471667 1/1 1 =.125 % 18 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos palmatus fruit runs first time Acquired: 7/31/214 1:54:59 PM Printed: 8/7/214 2:19:2 PM 6 2.477 5.75 6 4 4 2 2 1.683 2.7 2.627 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 12: HPLC of Diplocyclos palmatus fruit 1.683 2116678 27.13 12581 21.91 2.7 16138279 2.68 1124235 24.1 2.477 7277489 9.33 821866 17.55 2.627 19227856 24.64 13582 22.11 5.75 14221666 18.23 67551 14.43 7831998 1. 4682494 1. 19 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos palmatus fruit runs 2 nd Acquired: 7/31/214 2:7:26 PM Printed: 8/7/214 2:21:52 PM 1 1.7 2.52 1 5 5.77 5 2.27 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 13: HPLC of Diplocyclos palmatus fruit 1.7 25472319 28.69 125691 27.85 2.27 21615529 24.35 1652661 36.8 2.52 346771 34.32 163355 23.68 5.77 1123181 12.65 524 11.67 88787359 1. 44977 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 248477837/383388943 1/1 1 =.85 % For quercetin: 12726734 / 511471667 1/1 1 =.24 % 11 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis setosus leaf runs1st Acquired: 7/31/214 2:51:31 PM Printed: 8/7/214 2:3:12 PM 15 1 1.787 1.923 2.97 4.1 15 1 5 5 2.5 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 14: HPLC of Cucumis setosus leaf 1.787 227881 9.5 1685112 17.56 1.923 11489633 4.96 164915 17.18 2.97 183612 7.9 179965 18.75 2.5 5878341 21.97 29926 3.31 4.1 128916822 55.66 1554588 16.2 231598797 1. 9596941 1. 111 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis setosus leaf runs 2 nd. Acquired: 7/31/214 3:3:31 PM Printed: 8/7/214 2:34:9 PM 3 2 1 1.647 1.79 1.96 2.99 3.233 3.987 4.41 7.12 3 2 1 2.183 2.363 2.827 1 2 3 4 5 6 7 8 9 1 Results Spectral Graph No 15; HPLC of Cucumis setosus leaf 1.647 33694179 4.78 2499826 8.26 1.79 14728686 2.9 236442 7.62 1.96 325221 4.29 262491 8.67 2.183 444333 6.31 3911486 12.93 2.363 76543722 1.86 4565752 15.9 2.827 66136573 9.39 3857577 12.75 2.99 38974944 5.53 323675 1.68 3.233 9489727 13.46 2537728 8.39 3.987 61779962 8.77 2177838 7.2 4.41 195884 27.8 194676 6.41 7.12 474556 6.73 6924 2.1 7463227 1. 326215 Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 6371131.5 /383388943 1/1 1 =.26 %. For quercetin: 112 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANETrichosanthes tricuspidata leaf runs 1st Acquired: 7/31/214 3:16:27 PM Printed: 8/1/214 2:1:57 PM 298 nm Results 1.227 41248 6.88 3247524 13.12 1.37 15423991 2.65 2368533 9.57 1.51 26852531 4.61 2398456 9.69 1.76 39329546 6.75 3386874 13.68 1.95 55628351 9.55 3482788 14.7 2.393 5725322 9.82 299258 12.9 2.57 396423 5.3 2531218 1.23 2.817 68272912 11.71 248759 8.28 3.447 213152661 36.57 1787664 7.22 6.71 35875155 6.16 57967 2.5 Total 58279214 1. 24752291 1. 2 1.37 1.51 2.57 2.817 3.447 2 1 6.71 1 1.227 1.76 1.95 2.393 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 16: HPLC of Trichosanthes tricuspidata leaf Ph.D. Thesis: Miss Sandhya V. Rodge, 215 113

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Trichosanthes tricuspidata leaf runs 2 nd Acquired: 7/31/214 3:33:28 PM Printed: 8/1/214 2:12:51 PM 2 1 1.823 2.23 2.443 2.727 3.13 3.3 3.643 3.877 5.393 7.173 2 1 1.677 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 17: HPLC of Trichosanthes tricuspidata leaf 1.677 36955418 9.9 316522 17.76 1.823 33893431 8.34 25897 11.57 2.23 28378443 6.98 2192431 12.32 2.443 2538553 6.16 1863288 1.47 2.727 3863147 9.5 1953521 1.98 3.13 19587245 4.82 1492151 8.39 3.3 2771364 6.81 1346923 7.57 3.643 1764413 4.33 1297527 7.29 3.877 926758 22.79 1294159 7.27 5.393 5384381 13.24 731329 4.11 7.173 32255321 7.93 44661 2.27 4656525 1. 1779469 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 27972488 /383388943 1/1 1 =.9 % For quercetin: 114 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia grandis leaf runs 1st Acquired: 7/31/214 3:47:27 PM Printed: 8/1/214 2:14:32 PM 4 4 1.783 2 2.65 2.987 3.29 3.837 2 1.64 2.133 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 18: HPLC of Coccinia grandis leaf 1.64 841552 12.67 658354 25.2 1.783 49628786 7.47 415464 15.9 2.133 174458 16.17 6265 23.75 2.65 56283329 8.48 2595453 9.93 2.987 326343 4.56 22929 8.77 3.29 78273996 11.79 2244658 8.59 3.837 25828853 38.86 25219 7.85 66433485 1. 26127973 1. 115 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia grandis leaf runs 2nd time Acquired: 7/31/214 4::59 PM Printed: 8/7/214 2:42:44 PM 4 4 2 2.57 3.227 3.763 2 1.583 2.73 1 2 3 4 5 6 7 8 9 1 Spectral Graph No19: HPLC of Coccinia grandis leaf 1.583 167166698 21.99 8211671 33.92 2.73 13693344 17.9 799939 33.4 2.57 453117 5.92 2969822 12.27 3.227 873449 11.48 263285 1.86 3.763 32466719 42.7 239797 9.91 76262748 1. 2429138 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 5657218 /383388943 1/1 1 =.164 % For quercetin: 116 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos palmatus leaf runs 1 st time Method: Acquired: Printed: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met 7/31/214 4:12:57 PM 8/7/214 2:45:11 PM 4 1.58 1.72 4 2 2.563 3.247 3.763 2 2.63 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 2: HPLC of Diplocyclos palmatus leaf 1.58 6828179 12.83 4499184 22.67 1.72 3564724 7.52 3261457 16.44 2.63 1361614 21.86 777381 35.67 2.563 2246218 4.65 1817632 9.16 3.247 59849147 12.63 1674964 8.44 3.763 191994139 4.51 1511628 7.62 473975321 1. 19842246 1. 117 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos palmatus leaf runs 2 nd time Acquired: 7/31/214 4:24:51 PM Printed: 8/7/214 2:46:24 PM 4 4 1.623 2 2.11 2.67 3.293 3.82 7.443 2 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 21: HPLC of Diplocyclos palmatus leaf 1.623 7357235 19.68 342986 23.8 2.11 95915636 25.65 71879 48.7 2.67 12464413 3.33 146381 9.54 3.293 46345765 12.4 1296612 8.79 3.82 132685589 35.49 117447 7.96 7.443 1289158 3.45 28468 1.93 373875333 1. 14745766 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 17255315.5 /383388943 1/1 1 =.55 % For quercetin: 118 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Area % Report Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria siceraria leaf runs 1 st time Acquired: 7/31/214 4:35:17 PM Printed: 8/1/214 2:21:23 PM 3 2 1.62 3 2 1 2.6 2.82 3.28 7.87 7.45 1 2.1 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 22: HPLC of Lagenaria siceraria leaf 1.62 73195298 18.75 336584 26.38 2.1 72919419 18.68 4527518 35.49 2.6 14946128 3.83 1386496 1.87 2.82 36156324 9.26 1589194 12.46 3.28 166711976 42.71 131667 1.27 7.87 5678145 1.45 299177 2.35 7.45 279428 5.31 278689 2.18 39316718 1. 12756825 1. 119 Ph.D. Thesis: Miss Sandhya V. Rodge, 215

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria siceraria leaf runs 2 nd time Acquired: 7/31/214 4:47:7 PM Printed: 8/1/214 2:22:37 PM 15 1 5 2.163 2.657 2.88 3.333 5.123 7.513 15 1 5 1.683 1.813 1 2 3 4 5 6 7 8 9 1 Spectral Graph No 23: HPLC of Lagenaria siceraria leaf 1.683 323176 9.89 265387 22.51 1.813 316893 9.24 2229469 19.26 2.163 44154982 13.52 1845946 15.95 2.657 11969523 3.66 1195585 1.33 2.88 369253 11.5 1633136 14.11 3.333 9434245 28.89 1154516 9.98 5.123 57813137 17.7 6462 5.58 7.513 19759679 6.5 262577 2.27 326592938 1. 11572816 1. Avg. of Sample area / avg. of std. area std. conc. / sample conc. 1 For Rutin: 13457825.5/383388943 1/1 1 =.43 %. For quercetin: 12 Ph.D. Thesis: Miss Sandhya V. Rodge, 215