TABLE - 1 Physicochemical analysis and extractive values of (Percentage) leaf powder of Aegle marmelos Var. I and Var. III Sl.No Parameters Variant-I (%) 1 Organoleptic characteristics a. Appearance b. Colour c. Smell d. Taste Powder Greenish Brown Aromatic Pungent with astringent Variant-III (%) Powder Green Aromatic Pungent with astringent 2. Loss on drying 105 o C 6.2-8.5 6.0-8.8 3. Total Ash value 13.57 11.90 Acid insoluble ash 1.10 1.32 Sulphated ash 3.66 3.81 44 Solubility values Alcohol 7.09 7.18 5 Water 6.76 6.59 Extractive values in a) Petroleum ether 11.916 10.182 b) Benzene 2.020 2.136 c) Chloroform 3.238 2.110 d) Ethanol 14.217 14.731 143
e) Water 9.282 10.391 TABLE - 2 Fluorescent analysis of leaf powder of Aegle marmelos Var. I and Var.III Powder+ Reagent Variant-I Variant-III Sl.No used Day light UV-light Day light UV-light 1 Powder Pale green Light green Light green Light green 2 Powder+H 2 SO 4 Light Black Dark Black Black Dark Black 3 Powder+HNO 3 Brown Brownish yellow 4 Powder + HCl Light green Greenish yellow 5 Powder + Picric acid 6 Powder + Iodine Solution 7 Powder+FeCl 3 (5%) Solution Light green Yellowish green Dark yellow Light pink Light green Yellowish Brown Greenish pink Greenish yellow Green Green Green Green Green Dark green Pale green Light Green 8 Powder + NaOH Green Dark green Yellow Dark yellow 9 Powder +NH 4 OH Green Greenish yellow Green Greenish brown 144
145
TABLE - 3 Qualitatine Phytochemical screening of leaf powder of Aegle marmelos Var. I and Var. III S. No Compound Tested 1. Carbohydrate 2. Alkaloids Test applied/ Variant-I Variant-III Reagent used Pet.ether Benzene Chloroform Alcohol Water Pet.ether Benzene Chloroform Alcohol Water Fehilings - - - + + - - - + + Benedicts - - - + + - - - - + Mayer s - - - - + - - - - + Wagner s - - + + + - - + + + Hager s - - + + + - - + + + Dragendroff s - - + + + - - - - - 3. Phytosterols L.B. Test + + + + + + + + + + 4. Tannins & phenols 10% lead Acetate - - - + + - - - + + 5. Fixed oil & fats Spot test + + - - - + + - - - 6. Saponins Foam test - - - + + - - - + + 7. Gum & mucilage Alcoholic Precipitation - - - - + - - - - + constituents: + = present; - = Absent 146
TABLE - 4 Quantiative Phytochemical Estimation of leaf powder of Aegle marmelos Var. I and Var.III Compounds Variant-I Aegle marmelos Varian-III Total alkaloids 0.27 0.24 Total Terpenoids 0.85 0.68 Total Glycoside 2.23 1.89 Tannin 2.47 2.11 Ascorbic acid 0.92 0.87 Results are mean of 5 values and expressed in percentage 147
TABLE - 5 TLC profile of extracts on leaf powder of Aegle marmelos Var. I and Var.III S.No Samples Extracts Ethyl alcohol 1 Variant-I Water Ethyl alcohol 2 Variant-III Water Solute Distance (cm) R f Value 10.7 0.78 7.3 0.53 5.5 0.40 6.2 0.45 5.0 0.36 3.6 0.26 12.2 0.89 8.4 0.61 6.1 0.45 7.1 0.52 5.3 0.39 3.8 0.28 Distance of solvent front: 13.7(cm) 148
TABLE - 6 HPTLC profiles of leaf extracts of Aegle marmelos Variant-I and Variant-III. S.No Sample Total number of spots Number of major spots Retention factor (R f ) % of Area % Yield (100gm) 0.21 12.58 0.45 0.26 19.10 0.69 1 Var. I 10 5 0.41 10.68 0.38 0.50 12.77 0.46 0.62 18.56 0.67 0.02 13.17 1.50 2 Var. III 7 4 0.21 19.70 2.25 0.26 29.79 3.40 0.61 19.10 2.18 Solvent system used : Acetone: Alcohol (1:1) Amount applied : 10µl of 20 mg/ml solution. Spray reagent : Pankal D 149
TABLE - 7 Phytochemicals identified in the alcoholic extract of leaf of Aegle marmelos variant-i (GC-MS) No RT Name of the compound 1 9.22 2 9.31 Cyclohexane, 1-ethenyl-1- methyl-2,4-bis(1- methylethenyl)- Cyclohexane, 1-ethenyl-1- methyl-2,4-bis(1- methylethenyl)-, [1S- (1à,2á,4á)]- Molecular Formula C15H24 C15H24 MW Peak Area % 204 1.87 204 4.61 3 9.79 Caryophyllene C15H24 204 4.21 4 10.22 à-caryophyllene C15H24 204 1.05 5 10.60 Dodecanoic acid, methyl ester C13H26O2 214 41.09 6 13.09 Methyl tetradecanoate C15H30O2 242 6.21 7 14.83 3,7,11,15-Tetramethyl-2- hexadecen-1-ol C20H40O 296 0.61 8 15.92 Hexadecanoic acid, methyl ester C17H34O2 270 4.74 9 18.48 9,12-Octadecadienoic acid, methyl ester, (E,E)- C19H34O2 294 0.74 10 18.56 9-Octadecenoic acid (Z)-, methyl ester C19H36O2 296 4.09 11 18.93 Octadecanoic acid, methyl ester C19H38O2 298 2.97 12 25.27 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 25.76 13 29.65 Squalene C30H50 410 2.05 150
TABLE - 8 Phytochemicals identified in the alcoholic extract of leaf of Aegle marmelos variant-iii (GC-MS) No RT Name of the compound 1 9.20 2 8.40 Cyclohexane, 1-ethenyl-1- methyl-2,4-bis(1- methylethenyl)- Cyclohexane, 1-ethenyl-1- methyl-2,4-bis(1- methylethenyl)-, [1S- (1à,2á,4á)]- 3 10.40 à-caryophyllene Molecular Formula C15H24 C15H24 C15H24 MW Peak Area % 204 1.87 204 4.61 204 1.05 4 9.62 Dodecanoic acid, methyl ester C13H26O2 214 40.02 5 13.50 Methyl tetradecanoate C15H30O2 242 6.21 6 14.83 3,7,11,15-Tetramethyl-2- hexadecen-1-ol C20H40O 296 0.61 7 15.89 Hexadecanoic acid, methyl ester C17H34O2 270 4.74 8 18.48 9,12-Octadecadienoic acid, methyl ester, (E,E)- C19H34O2 294 0.74 9 18.56 9-Octadecenoic acid (Z)-, methyl ester C19H36O2 296 4.09 10 24.22 1,2-Benzenedicarboxylic C24H38O4 acid, diisooctyl ester 390 22.65 11 29.65 Squalene C30H50 410 2.05 151
TABLE 9 Biological activities of major compounds in leaf of Aegle marmelos No Name of the compound Compound Nature Activity Cyclohexane, 1-ethenyl-1- Anti-tumor, Analgesic 1 methyl-2,4-bis(1- Antibacterial, Antiinflammatory methylethenyl)- Sesquiterpene Sedative, Fungicide Cyclohexane, 1-ethenyl-1- Anti-tumor, Analgesic 2 methyl-2,4-bis Antibacterial, Antiinflammatory (1-methylethenyl)-, Sesquiterpene Sedative, Fungicide [1S-(1à,2á,4á)]- Anti-tumor, Analgesic 3 Caryophyllene Antibacterial, Antiinflammatory Sesquiterpene Sedative, Fungicide Anti-tumor, Analgesic 4 à-caryophyllene Antibacterial, Antiinflammatory 5 Dodecanoic acid, methyl ester 6 Methyl tetradecanoate 7 8 9 10 11 12 3,7,11,15-Tetramethyl-2- hexadecen-1-ol Hexadecanoic acid, methyl ester 9,12-Octadecadienoic acid, methyl ester, (E,E)- 9-Octadecenoic acid (Z)-, methyl ester Octadecanoic acid, methyl ester 1,2-Benzenedicarboxylic acid, diisooctyl ester 13 Squalene Sesquiterpene Lauric acid ester Myristic acid ester Terpene alcohol Palmitic acid ester Linoleic acid ester Oleic acid ester Stearic acid ester Plasticizer compound Sedative, Fungicide Antioxidant, Antibacterial, COX- 1 & COX-2 inhibitor, Antiviral, Hypocholesterolemic, Candidicide Antioxidant, Cancer preventive, Nematicide, Lubricant Hypocholesterolemic Antimicrobial Antiinflammatory Antioxidant, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Flavor,Hemolytic 5-Alpha reductase inhibitor Antiinflammatory, Hypocholesterolemic, Cancer preventive, Hepatoprotective,Nematicide, Insectifuge Antihistaminic, Antieczemic Antiacne,5-Alpha reductase inhibitor Antiandrogenic, Antiarthritic, Anticoronary, Insectifuge Antiinflammatory, Antiandrogenic, Cancer preventive, Dermatitigenic, Hypocholesterolemic, 5-Alpha reductase inhibitor, Anemiagenic, Insectifuge, Flavor No activity reported Antimicrobial, Anti fouling Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemo preventive, Lipoxygenase- 152
Triterpene inhibitor, Pesticide Courtesy:Dr.Duke s com. TABLE - 10 Acute Toxicity Studies Necropsy Findings on Aegle marmelos variant-i and Variant-III Group & Dose No Animal Mark Sex Body Weight Physical obserwvation Gross Pathology I control 1 HM M NAD NAD Solitary petechial lesion 2 BH M NAD NAD NAD 3 TM M NAD NAD NAD 4 LM M NAD NAD NAD 5 NM M NAD NAD Solitary blood spot in the lung (Var-I) 6 HM F NAD NAD NAD 7 BM F NAD NAD NAD 8 TM F NAD NAD NAD 9 LM F NAD NAD NAD 10 NM F NAD NAD NAD II 100mg/kg 11 HM M NAD NAD NAD bodyweight, 12 BH M NAD NAD NAD 13 TM M NAD NAD NAD 14 LM M NAD NAD NAD 15 NM M NAD NAD NAD 16 HM F NAD NAD NAD 17 BM F NAD NAD NAD 18 TM F NAD NAD Lung few petechiaes (Var-II) 19 LM F NAD NAD NAD 20 NM F NAD NAD NAD III200mg/kg 1 HM M NAD NAD NAD bodyweight, 2 BH M NAD NAD NAD 3 TM M NAD NAD Lung congested (Var-II) 4 LM M NAD NAD NAD 5 NM M NAD NAD NAD 6 HM F NAD NAD NAD 7 BM F NAD NAD NAD 8 TM F NAD NAD NAD 9 LM F NAD NAD Few blood sports lungs (Var-I) 10 NM F NAD NAD NAD IV 300 mg/kg 11 HM M NAD NAD NAD bodyweight, 12 BH M NAD NAD NAD 13 TM M NAD NAD NAD 14 LM M NAD NAD NAD 15 NM M NAD NAD NAD 16 HM F NAD NAD NAD 17 BM F NAD NAD NAD 18 TM F NAD NAD NAD 19 LM F NAD NAD NAD 20 NM F NAD NAD NAD Sign M-Male; F-Female; N-Normal; NAD-No Abnormality Detected; HM-Head Mark; BM-Body Mark; TM-Tail Mark; LM-Leg Mark; NM-No Mark. Note: Var. I = A.marmelos variant.i; Var.III = A.marmelos variant. III 153
154
TABLE - 11 Antiulcer property of leaf of A. marmelos var. I on ethanol-induced gastric ulcers in rats S.No. Group Animal weight Gastric volume (ml) Ulcer score ph Na + mg/l K + mg/l Free acidity (mg/l) Total acidity (mg/l) Pepsin (µg/ml) Total protein (µg/ml) Total hexose Total carbohydrates (µg/ml) Hexos Fucose Sialic acid Carbohydrate amine and protein ratio 1. Control 150.164 ± 3.35 2.071 ± 0.147 3.331 ± 0.080 2.234 ± 0.035 2.107 ± 0.080 0.852 ± 0.033 41.32 ± 1.501 70.31 ± 1.032 8.074 ± 0.074 616.7 ± 14.400 185.82 ± 3.971 282.35 ± 3.79 51.64 ± 1.031 34.35 ± 0.81 0.89± 0.01 2. Standard 151.3 ± 5.5*** 2.090 ± 0.126** 1.030 ± 0.135** 5.0834 ± 0.194*** 1.71 ± 0.053*** 0.7115 ± 0.016*** 31 ± 0.63*** 47.66 ± 1.032*** 5.054 ± 0.66*** 316.63 ± 4.130*** 310 ± 3.160*** 361.80 ± 1.15*** 30.31 ± 1.85*** 46 ± 0.0632*** 2.36 ± 0.07*** 3. 100mg/kg 153.167± 7.166*** 3.672± 0.315*** 3.031 ± 0.050*** 2.01 ± 0.134 NS 1.925 ± 0.030** 0.843 ± 36 ± 0.012 NS 0.631*** 62 ± 1.413*** 7.31 ± 0.172*** 500.82 ± 4.32*** 201.5 ± 4.413*** 285.165± 47.833 ± 34.64± 3.310 Ns 1.472 ns 0.981*** 1.36 ± 0.04*** 4. 200mg/kg 152.130± 5.520*** 3.062± 0.074*** 2.813 ± 0.073*** 3.16 ± 0.139*** 1.84 ± 0.037*** 0.803 ± 0.007** 32.81 ± 0.750*** 60.165 ± 0.751*** 7.790 ± 0.327*** 455.23 ± 6.375*** 250.165± 1.328*** 303.5 ± 6.74** 44.30 ± 1.54*** 41.831 ± 0.75*** 1.40 ± 0.16*** 5. 300mg/kg 151.830± 8.75*** 2.123± 2.563 ± 0.125 ns 0.050*** 3.9 ± 0.087*** 1.855 ± 0.041*** 0.792 ± 0.009** 27.66 ± 1.02*** 55.165 ± 1.72*** 6.73 ± 0.06*** 420.84 ± 2.481*** 260.831± 3.83*** 389.81 ± 1.95** 42.33 ± 2.060*** 43.830 ± 0.512*** 1.75 ± 0.20 Values are expressed as mean ± SD, n = 6 compared to control *** p < 0.001. ; ** p <0.01 ; * p < 0.05 ; NS Not significant 155
TABLE - 12 Antiulcer property of leaf of A. marmelos var. III on ethanol-induced gastric ulcers in rats S.No. Group Animal weight Gastric volume (ml) Ulcer score ph Na + mg/l K + mg/l Free acidity (mg/l) Total acidity (mg/l) Pepsin (µg/ml) Total protein (µg/ml) Total hexose Total carbohydrates (µg/ml) Hexos Fucose Sialic acid amine Carbohydrate and protein ratio 1. Control 150.164 2.071 ± 3.331 ± 2.234 ± 2.107 ± 0.852 ± 41.32 ± 70.31 ± 8.074 ± 616.7 ± 185.82 ± 282.35 ± 51.64 ± 34.35 ± 0.89 ± 0.01 ± 3.35 0.147 0.080 0.035 0.080 0.033 1.501 1.032 0.074 14.400 3.971 3.79 1.031 0.81 2. Standard 151.3 ± 2.090 ± 1.030 ± 5.0834 ± 1.71 ± 0.7115 ± 31 ± 47.66 ± 5.054 ± 316.63 ± 310 ± 361.80 ± 30.31 ± 46 ± 2.36 ± 5.5*** 0.126** 0.135** 0.194*** 0.053*** 0.016*** 0.63*** 1.032*** 0.66*** 4.130*** 3.160*** 1.15*** 1.85*** 0.0632*** 0.07*** 3. 100mg/kg 153.80± 6.532** 4.10± 0.174*** 2.831 ± 0.060*** 2.870 ± 1.804 ± 0.109 NS 0.030** 0.843 ± 41.830 ± 0.022 NS 1.631*** 61.32 ± 1.413*** 7.37 ± 0.256*** 473.52 ± 2.663*** 211.5 ± 4.313*** 285.9± 45.0 ± 41.69 ± 3.65 Ns 1.78 ns 0.781*** 1.23 ± 0.04*** 4. 200mg/kg 150.630± 3.242± 2.531 ± 3.3 ± 1.92 ± 0.83 ± 38.3 ± 54.65 ± 6.23 ± 383.83 ± 2.655± 316.15 ± 36.50 ± 39.031 ± 1.02 ± 4.260*** 0.131*** 0.083*** 0.41** 0.047*** 0.021** 0.832*** 1.031*** 0.37*** 3.61*** 3.545*** 4.52** 0.84*** 0.63*** 0.02*** 5. 300mg/kg 152.110± 4.910 2.731± 1.510 ± 0.517 ns 0.050*** 4.165 ± 0.381*** 1.844 ± 0.03*** 0.810 ± 0.0132** 37.4 ± 0.833** 47 ± 0.631** 6.394 ± 0.247** 345.0± 7.072*** 273.01± 3.65** 321.18 ± 3.760** 36.1 ± 1.867*** 37.54 ± 0.512*** 1.93 ± 0.05 Values are expressed as mean ± SD, n = 6 compared to control; *** p < 0.001. ; ** p <0.01 ; * p < 0.05 ; NS Not significant 156
TABLE 13 Effect of the alcoholic extracts of leaf of A. marmelos variant-i and variant-iii on Carrageenin Induced Rat-Paw Oedema in rats Group Dose Volume of paw oedema (ml) Inhibition (%) Normal saline 5 ml kg-1 1.24±0.09 --- A.marmelos variant-i A.marmelos variant-i A.marmelos variant-i A.marmelos variant-iii A.marmelos variant-iii A.marmelos variant-iii 100mg kg-1 0.74±0.04*** 40.32 200mg kg-1 0.54±0.04*** 56.45 300mg kg-1 0.40±0.09*** 67.74 100mg kg-1 0.90±0.07** 27.41 200mg kg-1 0.81±0.07** 34.67 300mg kg-1 0.74±0.07** 40.32 Ibuprofen 20mg kg-1 0.26±0.04** 79.03 Values are expressed in mean ± SEM, for six animals in each group. **P<0.01 ***P<0.001 157
TABLE 14 Effects of the alcoholic extracts of leaf of A. marmelos variant-i and variant-iii on Cotton-Pellet Granuloma in rats Group Dose Weight of the cottonpellet (mg) Normal saline 5 ml kg-1 49.40±1.18 --- A.marmelos variant-i A.marmelos variant-i A.marmelos variant-i A.marmelos variant-iii A.marmelos variant-iii A.marmelos variant-iii 100mg kg-1 36.04±1.28 27.04 200mg kg-1 21.45±0.81** 56.63 300mg kg-1 18.45±0.81*** 62.65 100mg kg-1 39±1.28 21.05 200mg kg-1 36±1.42 27.12 300mg kg-1 33.20±1.19 32.79 Ibuprofen 20mg kg-1 16.85±0.65*** 65.89 Values are expressed in mean ± SEM, for six animals in each group. **P<0.01 ***P<0.001 Inhibition(%) 158
TABLE 15 Effect of A.marmelos variant-i and variant-iii on yeast induced hyperpyrexia in rats Group Dose The reduced rectal temperature (C o ) post-drug at 1 hour Inhibition (%) Normal saline 5 ml kg -1 0.09±0.04 - A.marmelos variant-i 300 mg kg -1 0.72±0.07** 60.00 variant-iii 300mg kg -1 0.89±0.06 74.16 Paracetamol 100mg kg -1 1.20±0.06 *** 100.00 Values are expressed in mean ± SEM, for six animals in each group. **P<0.01 ***P<0.001 159
TABLE 16 Antioxidant activity of Ethanolic extracts of Aegle marmelos leaf (LPO Method) Aegle marmelos Group Dose O.D (Mean ± SD) Inhibition (%) Control 1.20±0.007 0 variant-i variant- III 100 mg /kg 0.40±0.006 66.6 200 mg /kg 0.33±0.005 72.5 300 mg /kg 0.28±0.008** 76.6 100 mg/ kg 0.16±0.005 86.6 200 mg /kg 0.14±0.006** 88.3 300 mg/kg 0.10±0.008*** 91.6 Values are expressed in mean ± SEM, for six animals in each group. **P<0.01 ***P 0.001 160
TABLE - 17 Antioxidant activity of Ethanolic extracts of Aegle marmelos leaf (Nitric Oxide method) Aegle marmelos Group Dose O.D (Mean ± SD) Inhibition (%) Control - 1.22±0.006 0 variant-i variant-iii 100 mg /kg 0.72±0.002 40.98 200 mg /kg 0.46±0.005 62.29 300 mg /kg 0.44±0.009** 63.93 100 mg/ kg 0.24±0.005** 80.32 200 mg /kg 0.13±0.004** 89.34 300 mg/kg 0.09±0.004*** 92.62 Values are expressed in mean ± SEM, for six animals in each group. **P<0.01 ***P 0.001 161
TABLE - 18 Antioxidant activity of Ethanolic extracts of leaf of Aegle marmelos (GSH method) Group Dose O.D (Mean ± SD) Inhibition (%) Control - 1.31±0.005 0 100 mg /kg 0.82±0.005 37.40 variant-i 200 mg /kg 0.69±0.002 47.32 Aegle marmelos 300 mg /kg 0.43±0.005** 67.17 100 mg/ kg 0.42±0.020** 67.93 variant-iii 200 mg /kg 0.38±0.008** 70.99 300 mg/kg 0.32±0.010** 77.09 Values are expressed in mean ± SEM, for six animals in each group. **P<0.01 ***P 0.001 162
163
TABLE - 19 Anti Microbial activity of leaf of Aegle marmelos variant-i and variant-iii Name of the Organism 10mg % Variant-I 20mg % Zone of inhibition (mm) 30mg % 10mg % Variant-III 20mg % 30mg % Amoxicillin / Clotrimazol e Escherichia coli - - - - - - 09 Streptococcus pyogenes - - - 15 18 21 12 Helicobacter pylori 7.1 9.2 10.1 8.2 10.8 12.5 06 Pseudomonas aeruginosa 6.1 8.2 9.2 7.4 8.7 9.9 25 Aspergillus niger - - - 9.2 10.1 14.2 23 Candida albicans - - - - - - 20 Trichoderma viride 3.1 7.1 9.4 5.2 7.3 10.1 24 Fussarium spp. - - - 9.1 9.4 13.1 12 164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188