CHAPTER IV RESULTS 4.1: Survey:

Transcription

1 CHAPTER IV RESULTS This chapter includes the findings of the ethno-botanical survey of medicinal plants used to treat leucorrhoea by the herbal healers of Mayong, District Morigaon, Assam, India and in vivo and in vitro antifungal and anti inflammatory effect of the methanolic extract of Solanum torvum Sw. (berries), which was selected for study from the survey report due to its effectiveness and high popularity among the herbal healers of Mayong. Before incorporated the in vivo and in vitro antifungal and anti inflammatory effect of Solanum torvum Sw. (berries), preliminary chemical analysis (proximate analysis) of the plant material and qualitative Thin Layer Chromatographic findings was presented. 4.1: Survey: As a major part of the objective of the present study, and extensive and intensive ethnobotanical survey of medicinal plants which were used by different herbal healers of Mayong to treat leucorrhoea was conducted with the help of prepared questionnaire (see page no. 66). Information about 20 numbers of different plant species from different families were spotted during the survey period. The majority of herbal preparation for leucorrhoea were prepared from whole plant (25%), bark (20%), fruit (20%), leaf (15%), flower (15%) and root (10%) while seed was used much less frequently. In almost 80% of the cases fresh plant material was used to prepare remedies, while others dried powder was used. Over 65% of the remedies were applied orally while the remaining was applied topically. Many remedies were prepared as mixture of multiple ingredients by mixing with goat milk, sugar, turmeric juice and honey. A complete overview of all plants encountered is given in Table 4.1. On the basis of the ethnobotanical survey work, one plant species was selected for the study; the plant species was Solanum torvum Sw. According to many of the local herbal healers of Mayong, those who interviewed during the survey period, Solanum torvum 93

2 Sw. was the only plant species which was heavily prescribed as a medicine for leucorrhoea due to its effectiveness and its easy availability. The detailed observations are presented in a tabular form with their vernacular names in alphabetic order in Table 4.1. The scientific name, families, parts used, plant s local name, plant state, preparation, mode of use and the photographs are also given (Fig. 4.1). Table 4.1: List of medicinal plants documented in the present survey (January 2010 to December 2010). Botanical name Family Part/parts used Local name Plant state Mode of Preparation Route of administration Terminalia arjuna (Roxb.) Wight. &Arn. Combretaceae Bark Arjun Fresh Aqueous infusion Drink Sarca indica (Roxb.) Fabaceae Bark Asok Dried Aqueous Infusion Drink Emblica officinalis Gairtn. Euphorbiaceae Raw fruit Amlakhi Fresh Aqueous infusion with honey Drink Phyllostachys bambosuides Siebold& Zuce. Poeceae Leaf Bah Fresh Leaf paste with honey Drink Adhatoda vasica Nees L. Acanthaceae Root Bahoka Fresh Aqueous infusion with honey Drink Phyllanthas fraturnus L. Euphorbiaceae Whole plant Bhuiaml okhi Fresh Juice Drink Ficus bangalences L. Moraceae Bark Bat Dried Directly use Topical application 94

3 Acacia Arabica Lam. Fabaceae Bark Babool Dried Decoction Topical application Punica granatum L. Piniaceae Flower Dalim Fresh Fine paste with white sandal powder and hot milk Drink Cassia tora (L.) Roxb. Caesalpiniacea e Whole plant Dadigdi ga Fresh Directly use Topical application Solanum indicum Linn. Solanaceae Raw fruit Tita vecuri Fresh Directly use Topical application Ficus hlispida L. Moraceae Ripe fruit Dimoru Dried Fruit powder with sugar Drink Cynodon dactylon (L.) Pers. Poeceae Whole plant Dubori Fresh Juice with honey Drink Solanum torvum Sw. Solanaceae Fruit, flower Hati vekuri Fresh Directly use Topical application Sida rhombifolia L. Malvaceae Whole plant Hun boreal Fresh Aqueous infusion with goat milk Drink Ageratum conyzodes Lam. Asteraceae Whole plant Gendhal i bon Fresh Aqueous infusion with goat milk Drink Hibiscus rosa sinensis L. Malvaceae Flower Jaba Fresh Juice with honey Drink 95

4 Solanum xanthocarpu m Schrad & Wendl. Solanaceae Leaf Kota bengena Fresh Directly use Topical applicati On Gossypium arboretum L. Malvaceae Root Kopah Fresh Aqueous infusion with honey Drink Lawsonia inermis L. Lythraceae Leaf, Seed Jetuka Fresh Directly use Topical application The photographs of the respective survey plants are given in Fig

5 Terminalia arjuna (Roxb.)Wight. & Arn. Sarca indica Roxb. Emblica officinalis Gairtn. Phyllostachys bambosuides Siebold.& Zuce. 97

6 Adhatoda vasica Nees L. Phyllanthas fraturnus l. Ficus bangalences L. Acacia arabica Lam. 98

7 Punica granatum L. Cassia tora (L.) Roxb. Solanum indicum Linn. Ficus hlispida L. 99

8 Cynodon dactylon (L.) Pers. Solanum torvum Sw. Sida rhombifolia L. Ageratum conyzodes Lam. 100

9 Hibiscus rosa sinensis L. Solanum xanthocarpum Schrad. &Wendle. Gossypium arboreum L. Lawsonia inermis L. Fig Photographs showing 20 different plant species documented as an anti leucorrhoeal plant during the survey. (Photographs were taken by author at the homes of harbal healears and at the field during the studyperiod). 101

10 4.2: Preliminary chemical analysis: The preliminary chemical analysis or the proximate analysis of the plant material was done using the standard AOAC method (1990). According to this, the total moisture content was studied by completely drying the plant material (5 g) in a hot air oven ( C). Subtracting the content dry weight from the fresh weight gave the total moisture content of the plant sample. For estimation of the total ash, measured plant sample was heated in a low flame to a charred mass. The mass was broken and ignited in muffle furnace at C for 4-5 hrs. After cooling the weight of the burned sample was taken which gave the value of total ash. For estimation of the total protein the plant sample was digested with concentrated sulphuric acid in the presence of catalyst, sodium sulphate and copper sulphate in a kjeldahl flask. The organic nitrogen present in the plant sample was converted into ammonium sulphate. Ammonium liberated by making the solution alkaline was distilled in 10 ml of N/10 sulphuric acid which was then back titrated. The protein content was obtained by multiplying the nitrogen value with 6.12 for estimation of total fibre, the plant sample was successfully digested in 1.25% of sulphuric acid and 1.25% of sodium hydroxide. The digested plant sample was completely dried in a hot air oven following by ignition of muffle furnace at C for 2-3 hrs. It was then cooled and weighed. The loss of weight was the weight of the total fibre. Total fat was estimated by extracting plant sample in ether which was continuously volatized at C, condensed and allowed to pass through the thimble containing the sample in a soxhlet s apparatus. After completing total ash formation the ash was digested in concentrated HCl and water. The mixture was then filtered and the sample was made acid free by repeated washing with distilled water. The volume was finally made up to 250 ml now called as aliquot. This aliquot was used for the estimation of inorganic metals viz.ca, P, Zn, Cu, Fe, Mg and Mn. For Calcium estimation measured volume of aliquot was treated with ammonium oxalate which precipitated all Ca present in the aliquot taken. The precipitate on treatment with sulphuric acid dissolved, forming calcium sulphate liberating free oxalic acid which was quantitatively estimated by titration against standard N/10 KMnO4 solution to arrive at the Ca content present in the given solution. For quantitative estimation of phosphorus, the aliquot of known volume was treated with ammonium molybdate solution and 102

11 concentrated nitric acid which precipitated P as a yellow precipitate of ammonium phosphomolybdate. The precipitate was washed till free from acid and dissolved in a measured volume (10 ml) of N/10 NaOH. The excess alkali was back titrated against N/10 HCl to arrive at the exact quantity of N/10 NaOH required. The quantity of N/10 NaOH corresponds to the amount of P present in the sample. The other metals were estimated by Flame Atomic Absorption Spectrophotometer (Perkin Elmer 2380) with air acetylene flame. The result of the preliminary chemical analysis or proximate analysis of the plant sample is presented in the Table 4.2. The result of the preliminary chemical analysis shows good amount of moisture, Ca, Zn and Mg. 4.3: Preliminary phytochemical screening: Preliminary qualitative phytochemical screening of plant sample was done using standard methods of analysis (Sofowora, 1993; Evans, 1996; Haughton and Raman, 1998), (See page no ) where the abundance of the compound present was determined by observing the intensity of the colouration. The results of the preliminary phytochemical screening are presented in Table 4.3. The result shows strong indication for the presence of tannin, phenol, alkaloid, saponin and flavonoid, where very low indication for the presence of steroids, and glycosides and no indication for the presence of terpenoid and volatile oil. 103

12 Table- 4.2: Result of the preliminary chemical analysis of plant sample showing high concentration of moisture, Ca, Zn and Mg. Parameters Total moisture Results 80.5% (W/V) Total ash 12.33% (W/V) Total fibre 1.25% (W/V) Total fat 1.23% (W/V) Total protein 1.73% (W/V) Inorganic metal content (µg/g) Calcium (Ca) 2.3 Phosphorus (P) Zinc (Zn) Iron (Fe) Copper (Cu) Manganese (Mn) Magnesium (Mg)

13 Table-4.3: Results of preliminary phytochemical analysis of plant sample showing strong presence of Tannin, Phenol, Alkaloid, Flavanoid and Saponin. (Experiment was repeated for 4 times). Phytochemicals Indication Tannin +++ Phenol +++ Alkaloid +++ Flavonoid +++ Steroid + Saponin +++ Glycosides + Terpenoid _ Volatile oil _ +++: strong indication of presence, ++: medium indication of presence, +: very low indication of presence, -: absent. 4.4: Conformational qualitative phytochemical analysis: Flavonoid, phenol, alkaloid and saponin are those phytochemicals, which act as an antifungal and anti inflammatory agent. For separation of these four phytochemicals Thin Layer Chromatography (TLC) plates were used. Chloroform and methanol (19:1) solvent mixture was used as a mobile phase for flavonoid, chloroform and methanol (27:0.3) solvent mixture was used as mobile phase for Phenols, chloroform and methanol (15:1) solvent mixture as a mobile phase for alkaloid and chloroform, glacial acetic acid, methanol and water (64:34:12:8) solvent mixture as a mobile phase for 105

14 saponin. After the running of mobile phase over plant sample spot contained silica gel plate it was found that, the four phytochemicals were present in the plant sample. Different fractions of flavonoid, phenols, alkalois and saponin were detected as spot over the silica gel plate. Table 4.4: Results of conformational qualitative phytochemical analysis using TLC showing the presence of Flavonoid, Phenol, Alkaloid and Saponin with their fraction numbers. (Experiment was repeated for 4 times) Sl.no Phytochemicals Detection Fraction number 1 Flavonoid Phenol Alkaloid Saponin + 3 Photos of TLC plates were given in Fig A B C D Fig Photographs showing TLC plates for detection of flavonoid showing 3 fraction (A), phenols showing 4 fraction (B), alkaloids showing 5 faction (C) and finally saponin showing 5 fractions (D). All the TLC plates were visualised using Iodene vapour except Phenols. Phenols are detected using pholin-phenol reagent. 106

15 4.5: Acute toxicity study of the plant extracts (LD-50 study): A number of doses of the methanol extract of Solanum torvum Sw. (berries) were administered to rat in the (LD-50) study. Rat were subjected to different doses of MLE (150, 300, 500, 1000, 2000, 3000, 5000, 8000 and 10,000 mg/kg) (p.o.) for four consecutive days and their mortality, loss of body weight and general behaviour recorded from the first dose up to 72 hrs after the first administration. As in the Table 4.5 no mortality (0%) was recorded. Table-4.5: Treatment schedule of administration of plant extract for acute toxicity study in rat. Group Doses (mg/kg) Normal control Not significant Mortality 0% Solanum torvum Sw ,000 (berries) Mortality 0% 0% 0% 0% 0% 0% 0% 0% 0% n=6 4.6: In vivo antifungal study: The washed blastoconidia of Candida albicans were inoculated intravaginally in rat with 10 7 cells (in 150µl of sterile saline solution). 2 days after inoculation the animals were given different plant extract doses (300 mg/kg and 600 mg/kg body weight) intravaginally. Ketoconazole was used as a positive control and only vehicle as a negative control. Simultaneously the vaginal lavage that released by the vagina of rats due to Candida infection was collected by rolling a sterile cotton swab over the vaginal cavity in day 5 day 15 post- infection. Determination of the number of Candida organisms was conducted in duplicate after serial 10-fold dilution of washing fluid and plating on nutrient agar. All plates were incubated at 30 C for 24hr for each series of dilutions. The number of viable cells was determined using the drop count method. After the counting of CFU of Candida albicans present in the vaginal lavage collected from each 107

16 groups at day 5 and day 15, it was found that the vehicle treated group showed 0% reduction on CFU of Candida albicans in both day 5 and day 15. On the other hand the both extract treated group (group II and group III) displayed reduction in percentage of CFU at day 5 and day 15. At day 5 the group II showed a little bit lesser percentage of reduction of CFU then the group III, but at day 15 both the group showed same result by reducing the percentage of CFU up to 98.89%. The positive control group showed maximum reduction at day 15 (100%). The statistical data of this experiment and reduction in the percentage of CFU of Candida albicans were recorded in the Fig 4.3 and 4.4. Vehicle treated 300 mg/kg B.W. 600 mg/kg B.W. Ketoconazole extract treated extract treated treated Day 5th Day 15th Fig Figure showing log CFU±SD value of Candida albicans present in vaginal lavage collected from rats of treated groups. Stdent s t- test was done comparing with vahicle treated group where, *P<

17 Day 5th 300 mg/kg B.W. 600 mg/kg B.W Ketoconazole extract treated extract treated treated Day 15th Fig Figure showing percentage of CFU reduction in Day 5 and Day 15 in each treated groups. After completion of the treatment, animals were sacrificed under mild dose of anaesthesia (diethyl-ether) followed by cervical dislocation. Vaginas were removed from the sacrificed animals and then fixed in toto by immersion in Bouin s solution for at least 48hr. The histological slides were prepared by using PAS stain protocol (See page no. 88, 89). The changes in the histo-architecture of vaginal epithelium and presence of fungal spores and hyphae over the vaginal epithelial lining are presented in Fig. 3.3A-H. Striking changes in the presence of fungal hyphae and spores were observed in the vehicle treated ovariectomized rat (negative control) (Fig A, B) when compared with treated groups (Fig. 4.5 C-H). Presence of a lot of fungal hyphae (H) and spores (S) were found over the epithelium (E) was found in vehicle treated group (Fig. 3.3.A, B). Reductions of fungal hyphae (H) and spores (S) over epithelium (E) were also not seen in the 300 mg/kg concentration of extract treated group (Fig C, D). Complete reduction of fungal hyphae and spores were seen in both 600mg/kg concentration of extract preaped group as well as positive control group (Fig E-H) 109

18 B A (400X) (200X) C D (400X) (200X) F E (400X) (200X) H G (400X) (200X) Fig Histological photomicrograph of fungal blastoconidia inoculated vaginal tissues of ovariectomized female rat showing presence of fungal hyphae(h) and spores (S) in vehicle treated group (A,B). 300mg/kg extract treated group (C, D) showed again no reduction of fungal growth over the vaginal epithelium (E). While the both 600mg/kg extract treated group (E, F) as well as positive control group (G, H) showed complete reduction of fungal growth over vaginal epithelium (E) and vaginal cavity (VC). 110

19 4.7: In vivo anti inflammatory study: 4.7.1: Carrageenan induced rat paw edema model: Edema was induced in left hind paw on each rat by injecting 1% carrageenan solution in all the 4 groups. All the animals were pre-treated with vehicle (group I), 300 mg/kg body weight concentration of plant extract (group II), 600 mg/kg body weight concentration of plant extract (group III) and 10 mg/kg body weight concentration of indomethacene as a positive control (group IV) before giving the carrageenan injection. At the acute phase of experiment the result showed a significant increasing in the paw volume (P<0.01) at 1 hr after injection of carrageenan in group III animals (3.2±0.048). On the other hand the value taken in the 2 nd hr after carrageenan injection showed simultaneously decreasing in the paw volume in group III animals (3.13±0.009) which is significant at P< 0.01 again (Table: 4.6). Table 4.6: Effect of methanolic extract of Solanum torvum Sw. and reference drug on the swelling of rat hind paw induced by carrageenan (acute phase). No Treatment 0hr 1hr 2hr 3hr I Vehicle treated 3.05± ± ± ±0.048 II Extract treated (300 mg/kg body weight) 3.05±0.029 NS 3.34±0.071 NS 3.22±0.058 NS 3.16±0.041 NS III Extract treated (600 mg/kg body weight) 2.97±0.048NS 3.2±0.048** 3.13±0.009** 3.07±0.025 NS Indomethacene treated as IV positive control(10mg/ 3.13±0.048 NS 3.35±0.029 NS 3.25±0.029 NS 3.2±0.025 NS kg body weight) Mean± SEM. n=4, Student s t-test: *P< 0.05;**P<0.01; NS : Not Significant : Percentage of inhibition in paw volume (acute phase): The percentage of inhibition in paw volume of rat was calculated to ensure the effectiveness of plant extract treated groups of different concentration comparing with the positive control group. It was found that at this acute phase of experiment the group II does not show satisfactory result on the inhibiting inflammation but on the other hand 111

20 the group III animals displayed adequate result (16.66%) at the 3 rd hour of experiment, where the positive control group displayed 41.66% of inhibition in paw volume (Fig. 4.6). Fig Figure shows the % inhibition of rat paw volume induced by carrageenan (acute phase). At the chronic phase of experiment the result showed a significant decreasing in the paw volume (P<0.01) at second day after injection of carrageenan in group III (3.07±0.029) and group IV (3.18±0.009) animals. On the other hand the value taken in the day 15 after carrageenan injection showed strong result on decreasing of paw volume in group IV animals (3.13±0.029) which is significant at P< 0.05 ( Table: 4.7). 112

21 Table 4.7: Effect of methanolic extract of Solanum torvum Sw. and indomethacin on the swelling of rat hind paw induced by carrageenan. (chronic phase). No Treatment Day1 Day2 Day3 Day4 Day15 I Vehicle treated 3.17± ± ± ±0.011 NS 3.11±0.011 NS II Extract treated (300 mg/kg body weight) 3.16±0.041 NS 3.14±0.002 NS 3.08±0.018 NS 3.06±0.023 NS 3.06±0.023 NS III Extract treated (300 mg/kg body weight) 3.07±0.041 NS 3.03±0.029** 3±0.023 NS 2.99±0.024 NS 2.98±0.018 NS IV Indomethac ene treated as positive control(10 mg/kg body weight) 3.2±0.05 NS 3.18±0.009** 3.15±0.029 NS 3.13±0.041 NS 3.13±0.029* Mean± SEM. n=4, Student s t-test: *P<0.05;**P<0.01; NS : Not Significant : Percentage of inhibition in paw volume (chronic phase): Like the acute phase of experiment, the percentage of inhibition in chronic phase was also calculated and result displayed very low percentage of inhibition in 2 nd experimental group at day1 and day2 (8.33%, 10%) comparing to the positive control group (group IV). On the other hand group III was showing promising result for inhibiting paw volume at day 1 and day2 (16.66%, 40%). But at day3 and day4 the group II showed an amazing result by inhibiting the paw volume better then the 3 rd experimental group where higher concentration of plant extract was used. At day 15 the both experimental group showed same result (83.33%), where the positive control group showed 100% inhibition (Fig. 4.7). 113

22 Fig Figure showing percentage of inhibition against swelling of rat hind paw induced by carrageenan (chronic phase) : Formaldehyde induced rat paw edema model: Like carrageenan, edema was induced in left hind paw on each rat by injecting 1% formaldehyde solution in all the 4 groups. All the animals were pre-treated with vehicle (group I), 300mg/kg body weight concentration of plant extract (group II), 600 mg/kg body weight concentration of plant extract (group III) and 10 mg/kg body weight concentration of indomethacene as a positive control (group IV) before giving the formaldehyde injection. At the acute phase of experiment the result showed a significant decreasing in the paw volume (P<0.05) at first hour, second hour and third hour after injection of formaldehyde in positive control groups. But in the experimental groups (group II and group III) no significant result was seen at the acute phase of experiment (Table. 4.8). 114

23 Table 4.8: Effect of methanolic extract of Solanum torvum Sw. and reference drug on the swelling of rat hind paw induced by formaldehyde (acute phase) No Treatment 0hr 1hr 2hr 3hr I Vehicle treated 2.63± ± ± ±0.029 II Extract 2.67±0.025 NS 3.53±0.048 NS 3.4±0.04 NS 3.3±0.092 NS treated (300 mg/kg body weight) III Extract 2.63±0.048 NS 3.47±0.063 NS 3.35±0.065 NS 3.33±0.048 NS treated (600 mg/kg body weight) IV Indomethace ne treated as positive control(10mg /kg body weight) 2.6±0.041 NS 3.23±0.111* 3.23±0.095* 3.15±0.087* Student s t-test: *P< 0.05; NS : Not Significant. 115

24 : Percentage of inhibition in paw volume (acute phase): The result of the percentage of inhibition in paw volume of rat at formaldehyde induced rat paw edema model showed not a very pleasing result at acute phase. Both the experimental group displayed neck to neck result, where the positive control group showed higher percent of inhibition comparing to the both group II and group III (Fig. 4.8). Fig Figure showing percentage of inhibition against swelling of rat hind paw induced by formaldehyde (acute phase). At the chronic phase of experiment the result showed a significant decreasing in the paw volume (P<0.01) in both the experimental group from second to 15 th day after giving formaldehyde injection. The positive control group was showing also significant result from day 1(P<0.05) to day15 (P<0.01) (Table: 4.9). 116

25 Table 4.9: Effect of methanolic extract of Solanum torvum Sw. and reference drug on the swelling of rat hind paw induced by formaldehyde (chronic phase). No Treatment Day 1 Day2 Day 3 Day4 Day15 I II III Vehicle treated Extract treated (300 mg/kg body weight) Extract treated (600 mg/kg body weight) 3.45± ± ± ± ± ±0.092 NS 3.15±0.029** 2.97±0.058** 2.97±0.048** 2.93±0.025** 3.30±0.048 NS 3.07±0.029** 2.85±0.029** 2.83±0.025** 2.75±0.029** Indomethac ene treated IV as positive control( ±0.087* 3.03±0.025** 2.83±0.025** 2.77±0.063** 2.67±0.048** mg/kg body weight) Mean± SEM. n=4, Student s t-test: *P<0.05;**P<0.01; NS : Not Significant : Percentage of inhibition in paw volume (chronic phase): Calculation of percentage of inhibition in chronic phase of formaldehyde induced modal of rat revealed that the 3 rd experimental group showed very close result with each other in percentage of inhibition in paw volume with the positive control group, where the result of 2 nd experimental group displayed lower percentage of inhibition comparing to the 3 rd experimental group and the positive control group from 1 st day to 15 th day of experiment (Fig. 4.9). 117

26 Fig Figure showing percentage of inhibition against swelling of rat hind paw induced by formaldehyde (chronic phase). 4.8: In vitro antifungal study: Antifungal effect of Solanum torvum Sw. methanolic extract against Candida albicans was carried out In vitro by using disc diffusion assay and micro dilution broth assay : Disc diffusion assay: The disc diffusion assay was carried out by loading different concentration of Solanum torvum Sw. extract (12.5, 25, 50, 100, 150, 200, 250 and 300 mg/ml) on sterilized filter paper discs. Ketoconazole (30 mg/disc) was used as standard antifungal agent, whereas sterilized saline water was used as a negative control. The extract loaded discs were then positioned on the Candida albicans inoculated agar surface and incubated at 37 C for 24 hr. After incubation clear zone of inhibition of fungal growth was found surrounding the filter paper disc at 100, 150 and 200 mg/ml concentration of plant extract as well as ketoconazole loaded disc. 12.5, 25 and 50 mg/ml concentration of extract displayed no zone of inhibition (Fig 4.10). 118

27 Fig Figure showing increasing in the diameter of zone of inhibition with increasing concentration of Solanum torvum Sw. plant extract. (Mean± SEM. n=4, Student s t-test: **P< 0.01) : Micro dilution broth assay: Different concentration of plant extract was prepared (12.5, 25, 50, 100, 150, 200, 250 and 300 mg/ml) in sterile nutrient broth using sterilized test tubes for determining the MIC value. Ketoconazole which is a standard antifungal drug (30 mg/ml) was used as positive control; whereas sterilized saline water was used as negative control. The result of micro dilution broth method revealed that 200 mg/ml concentration of Solanum torvum Sw. extract is the Minimum Inhibitory Concentration (MIC) which showed clearly visible inhibition of fungal growth to 100 mg/ml concentration of extract showed no inhibition, where 150 mg/ml concentration showed a little inhibition of fungal growth (Table 4.10). Table 4.10: MIC study of Solanum torvum Sw. plant extract showing MIC value at 200 mg/ml concentration of plant extract. (Study was repeated for 4 times) Concentration of MLE and ketoconazole 12.5 mg/ml 25 mg/ml 50 mg/ml 100 mg/ml 150 mg/ml 200 mg/ml 30 mg/ml ketoconaz ole Observation NB: _ : No Inhibition +: very little inhibition, ++: little inhibition, +++: clearly visible inhibition, ++++ complete inhibition. 119

28 4.8.3: Determination of the minimum fungicidal concentration: 50 µl from each test tube which indicate no growth of all respective candidal yeast, were sub cultured onto fresh nutrient agar plates after overnight incubation. The plates were incubated at 35 C for 24 to 48 hrs until the visible growth was observed and it was found that 200 mg/ml concentration of plant extract showed no growth of fungal colonies in the agar plate while the 150 mg/ml concentration of plant extract showed more than 5 colonies of Candida albicans. That is why the Minimum Fungicidal Concentration (MFC) value was considered as 200 mg/ml concentration of Solanum torvum Sw. extract. Table 4.11: MFC study of Solanum torvum Sw. plant extract showing MFC value at 200 mg/ml concentration (Study was repeated for 4 times). Sl. No Concentration of plant extract showing growth inhibition in MIC test (mg/ml) Observation (colonies of Candida albicans found in agar plates) Remarks More than 5 colonies No colonies Could be considered as MFC value The photographs of the respective experiment on In vitro antifungal effect are given in Fig and

29 Fig Photo showing Minimum Inhibitory Concentration of Solanum torvum Sw. methanolic extract against Candida albicans. Fig Photo showing inhibition in growth of Candida albicans colony on the increasing order of Solanum torvum Sw. methanolic extract concentration. 4.9: In vitro anti inflammatory study: The inhibition of hypotonicity induced rat RBC membrane lysis i.e; stabilisation of RBC membrane was taken as a measure of the anti inflammatory activity. The percentage of membrane stabilisation for methanolic extracts and hydrocortisone sodium were done at 50, 100, 150, 200, 250, 500, 1000 µg/ml. Methanolic extracts of Solanum torvum Sw. are effective in inhibiting the heat induced haemolysis of rat RBC at different concentrations ( µg/ml) as shown in Table It showed the maximum inhibition 94.97% at 1000 µg/ml. With the increasing concentration the 121

30 membrane haemolysis is decreased as shown in Fig and membrane stabilisation / protection is increased as shown in Fig Table 4.12: Results of In vitro anti-inflammatory effect of Solanum torvum Sw. showing percentage of haemolysis and protection of rat RBC membrane when treated with plant extract and hydrocortisone sodium. Conc. % Hemolysis of % Protection % Hemolysis % Protection (µg/ml) S. Torvum Sw. MLE of S. Torvum Sw. MLE of Hydrocortisone sodium of Hydrocortisone sodium Fig Effect of Solanum torvum Sw. on rat RBC membrane hemolysis. The test sample showed decreased amount of membrane hemolysis with the increased amount of plant extract and hydrocortisone sodium. 122

31 Fig Effect of Solanum torvum Sw. on rat RBC membrane stabilisation. The test sample showed increased amount of membrane protection with the increased amount of plant extract and hydrocortisone sodium. 123