CHAPTER 3 PHYTOCHEMICAL SCREENING AND ANTI-MICROBIAL ACTIVITY OF THE EXTRACTS OF SELECTED MEDICINAL PLANTS

CHAPTER 3 PHYTOCHEMICAL SCREENING AND ANTI-MICROBIAL ACTIVITY OF THE EXTRACTS OF SELECTED MEDICINAL PLANTS 3.1. INTRODUCTION Medicinal plants are of important therapeutic aid for various ailments. Naturally occurring anti-microbials can be derived from plants, animal tissues, or microorganisms (Gordon and David 2001). The shortcomings of the drugs available today propel the discovery of new pharmacotherapeutic agents in medicinal plants (Cordell 1993). Secondary metabolites produced by plants constitute a major source of bioactive substances. The scientific interest in these metabolites has increased today with the search of new therapeutic agents from plant source, due to the increasing development of the resistance pattern of microorganisms to most currently used anti-microbial drugs. According to World Health Report of infectious diseases 2000, overcoming antibiotic resistance is one of the major issues of the present millennium. Hence the last decade witnessed an increase in the investigation of plants as a source of human disease management (Prashanth et al. 2001). Furthermore, it is estimated that twothirds of the world population rely on traditional remedies due to the limited availability and high prices of most pharmaceutical products (Tagboto and Townson 2001). In recent years, multiple drug resistance in human pathogenic microorganisms has developed due to indiscriminate use of commonly available commercial antimicrobial drugs in the treatment of infectious diseases. This situation forced scientists 52

in search of new novel anti-microbial chemotherapeutic agents from various sources, like medicinal plants and marine sources. 3.2. MATERIALS AND METHODS 3.2.1. Plant Material collection and authentication The plants chosen for the study were authenticated by Botanical Survey of India, Southern Regional Centre, Tamil Nadu Agricultural University campus, Coimbatore. Table 3.1 depicts the plant parts used, area of collection and authentication number. Table 3.1 Details of the plants chosen for the study Place Authentication S. Name of the Plant collected Parts Used Number assigned No from by BSI 1 Blepharis maderaspatensis (L.) Heyne ex Roth. Pannaipuram, Theni Dt. Tamil Nadu Leaf BSI/SRC/5/23/2012-13/Tech-1887 2 Pannaipuram, Acacia leucophloea BSI/SRC/5/23/2012- Theni Dt. Bark (Roxb.) Willd. 13/Tech-460 Tamil Nadu 3 Acacia caesia (L.) Willd. Pannaipuram, BSI/SRC/5/23/2012- Theni Dt. Bark 13/Tech-1887 Tamil Nadu 4 Kanyakumari Eleutherine palmifolia (L.) BSI/SC/5/23/10- Dt. Bulb Merr. 11/Tech-1697 Tamil Nadu 5 Solanum nigrum (L.) Pannaipuram, BSC/SC/5/23/10- Leaf Theni Dt. 11/Tech-45 53

Tamil Nadu 6 Acalypha indica (L.) Pannaipuram, Theni Dt. Tamil Nadu Leaf BSC/SC/5/23/10-11/Tech-47 7 Tridax procumbens (L.) Pannaipuram, Theni Dt. Tamil Nadu Flower BSC/SC/5/23/10-11/Tech46 3.2.2. Processing of collected plant materials The plant material (Leaves) were separated and were washed in running tap water thrice and once in distilled water once and processed. They were shade dried at room temperature 28 C until they become brittle and dry. Dried plant materials were powdered for extraction. The powdered plant materials were stored in air tight container at room temperature until further analysis. 3.2.3. Extraction of active drug principle from plant materials Organic solvent extraction is a process for separating the desired substance from plant materials. The two common extraction methods, hot continuous successive extraction (Soxhlet apparatus) and Maceration were carried out to extract the active drug principle from the plant materials. The solvents ranging from non-polar to polar solvents viz. ether (Boiling point-35 C), Benzene (Boiling point-80 C), Chloroform (Boiling point-61 C), Acetone (Boiling point-56 C), Ethanol (Boiling point-78 C) and Methanol (Boiling point-64 C) were used in extraction. 54

3.2.3.1. Hot continuous extraction In this method, the finely ground powder (25 gm) was placed in a porous bag or thimble made out of strong filter paper, which was placed in chamber of the Soxhlet apparatus (Figure 3.1). The extracting solvent (250 ml) in flask was heated at its boiling point, and its vapors condense in condenser. The condensed extractant drips into the thimble containing the plant powder and extracts it by contact. When the level of liquid in chamber rises to the top of siphon tube, the liquid contents of chamber siphon into flask. The advantage of this method was that large amounts of drug can be extracted with a much smaller quantity of solvent. This was economical in terms of time, energy and consequently financial inputs. Figure 3.1 Soxhlet apparatus 55

3.2.3.2. Maceration In this process, the coarsely powdered plant part (25 gm) was placed in a stoppered container with the solvent (250 ml) and allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved. The mixture then was clarified by filtration after standing for some time. The Extracts were filtered concentrated by evaporating the solvent on the rotary evaporator under reduced pressure at 40 C (Figure 3.2) which was used for further analysis. Figure 3.2 Rotary Vacuum Evaporator 56

3.2.4. Qualitative phytochemical screening of the plant extracts Preliminary phytochemical screening was conducted in order to observe the chemical nature of active extracts following the standard protocols (Trease and Evans 2002, Kokate 1994, Trease and Evans 1989, Mace 1963, Pew 1948, Shinoda 1928). 3.2.4.1. Ferric chloride test for phenols The extract (50 mg) was dissolved in 5 ml of distilled water. To this, a few drops of neutral 5% ferric chloride solution was added. A dark green colour formation indicates the presence of phenolic compounds. 3.2.4.2. Xanthoprotein test for tannins About 0.5 g of each of the extract was taken in a boiling tube and was boiled with 20 ml distilled water and then filtered. To the filtrate, a few drops of 0.1% ferric chloride were added and was mixed well and allowed to stand for some time, and was later observed for brownish green or a blue-black colouration. 3.2.4.3. Salkowski test for terpenoids To 0.5 g of the extract, 2 ml of chloroform and concentrated H2SO4 (3 ml) were carefully added to form a layer. A reddish brown colouration of the interface indicates the presence of terpenoids. 57

3.2.4.4. Test for flavonoids To a portion of dissolved extract 10 % ferric chloride solution was added. Green or blue colour formation indicates the presence of flavonoids. 3.2.4.5. Test for alkaloids To 0.5 g of the extract, 5 ml of 1 % aqueous hydrochloric acid was added. Few drops of Mayer s reagent, was treated with 1 ml of filterate and another 1 ml of filtrate was treated with few drops of dragendorff s reagent. Turbidity of precipitate with either of these reagents indicates the presence of alkaloids. 3.2.5. Anti-microbial activity 3.2.5.1. Anti-microbial activity of the plant extracts Anti-microbial potential of the plant extracts were examined by well diffusion technique against the test organisms. The test organisms (5 gram positive, 5 gram negative bacteria and one Fungus) used in this study was obtained from Microbial Type Culture Collection (MTCC), Chandigarh, India. The MTCC codes were as follows: Lactobacillus acidophilus (MTCC code- 447), Bacillus subtilis (MTCC code- 121), Salmonella paratyphi (MTCC code-3220), Shigella sonnei (MTCC code-2957), Klebsiella pneumonia (MTCC code-3384), Streptococcus sp. (MTCC code-497), Staphylococcus aureus (MTCC code-3381), Pseudomonas aeruginosa (MTCC code- 424) and Candida albicans (MTCC code-183). Methicillin resistant Staphylococcus aureus (MRSA) and multidrug resistant Acinetobacter baumannii were obtained from Microbiological laboratory, Coimbatore, Tamil Nadu, India. The strains were sub cultured and grown in nutrient broth at suitable temperatures. 58

Log phase cultures of test organisms in nutrient broth were seeded by spreadplate method on Mueller-Hinton agar. Approximately wells of uniform size (0.65 cm) were made with a cork-borer onto the plates inoculated with test organisms, each well being placed 3 cm apart. Crude plant extracts of 50 µl were respectively added into the well aseptically. Control wells with 50 µl of the pure solvents were also made on the respective plates and were incubated at 37 C for 24 h. 3.2.5.2. Anti-microbial activity of commercial antibiotics Anti-microbial potential of Amoxyclav, Ampicillin A, Ceftazidime, Chloramphenicol, Ciprofloxacin, Erythromycin, Gentamicin, Imipenem, Methicillin, Nalidixic acid, Nitrofurantonin, Norfloxacin, Tetracycline, Kanamycin, Vancomycin, Fusidic acid and Ketoconazole were examined by disc diffusion technique against the test organisms. Log phase cultures of test organism grown in nutrient broth were seeded by spread-plate method on Mueller-Hinton agar. Commercial antibiotic discs were placed on the plates inoculated with test organisms, each disc being placed 3 cm apart and were incubated at 37 C for 24 h. This was taken as positive control (Jebakumar et al. 2005). 3.2.6. Quantitative phytochemical screening of the plant extracts After the qualitative confirmation of phytoconstituents by preliminary phytochemical tests, the plant extracts were taken for quantitative estimation. 3.2.6.1. Estimation of total phenol content About 0.1 ml of the extract (10 µg/ml) was mixed with 0.5 ml of Folin- Ciocalteu reagent (diluted 1:10 ratio with distilled water) and 1.5 ml of sodium 59

carbonate. The resulting mixture was vortexed for 15 sec and incubated at 40 C for 30 min for colour development. The absorbance of total phenolics was measured at 765 nm. The experiment was conducted in triplicates and the results were expressed as Mean ± SEM values (Ponmari et al. 2012). 3.2.6.2. Estimation of total alkaloid content About 5 g of the extract was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added and covered and allowed to stand for 4 h. This was filtered and the extract was concentrated to one-quarter of the original volume on a water bath. Concentrated ammonium hydroxide was added dropwise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed. The experiment was conducted in triplicates and the results were expressed as Mean ± SEM values. 3.3. RESULTS 3.3.1. Qualitative phytochemical screening Phytochemicals present in the plant parts were extracted by hot continuous successive extraction and by maceration method using six different solvents of varying polarity. Table 3.2, 3.3, 3.4 and 3.5 depicts the presence of phytochemicals (Alkaloids, flavonoids, phenols, terpenoids and tannins) in the extracts of Blepharis maderaspatensis, Solanum nigrum, Acalypha indica and Tridax procumbens respectively. Table 3.6 depicts the presence of phytochemicals (Alkaloids, flavonoids, phenols, terpenoids and tannins) in the ethanol extract of Acacia leucophloea, Acacia caesia and Eleutherine palmifolia. Both the extraction methods showed almost similar results. Flavonoids were present only in methanolic extracts of all the plants. Polar 60

solvent extracts of Acalypha indica and Tridax procumbens showed presence of tannins which were absent in other plant extracts. Terpenoids were present in non polar solvent extracts of Acalypha indica. Alkaloids, phenols and terpenoids were present in all the polar solvent extracts. The results clearly showed that polar solvent extracts of all the plants had the presence of almost all the phytochemicals analyzed. Table 3.2 Qualitative phytochemical analysis of Blepharis maderaspatensis Leaf Alkaloids Flavonoids Phenols Terpenoids Tannins - - - - - Blepharis maderaspatensis Leaf Cold Extracts Hot Extracts Benzene - - - - - Chloroform + - - - - Acetone + - - + - Ethanol + - + + - Methanol + + + + - - - - - - Benzene - - - - - Chloroform + - - - - Acetone + - - + - Ethanol + - + + - Methanol + + + + - 61

Table 3.3 Qualitative phytochemical analysis of Solanum nigrum Leaf Alkaloids Flavonoids Phenols Terpenoids Tannins - - - - - Solanum nigrum Leaf Cold Extracts Hot Extracts Benzene - - - - - Chloroform - - + + - Acetone + - + + - Ethanol + - + + - Methanol + + + + - - - - - - Benzene - - - - - Chloroform - - + + - Acetone + - + + - Ethanol + - + + - Methanol + + + + - Table 3.4 Qualitative phytochemical analysis of Acalypha indica Leaf Acalypha indica Leaf Hot Extracts Alkaloids Flavonoids Phenols Terpenoids Tannins - - - + - Benzene - - - + - Chloroform + - - + - Acetone + - + + + Ethanol + - + + + Methanol + + + + + 62

Cold Extracts - - - + - Benzene - - - + - Chloroform + - - + - Acetone + - + + + Ethanol + - + + + Methanol + + + + + Table 3.5 Qualitative phytochemical analysis of Tridax procumbens Leaf Tridax procumbens Flower Hot Extracts Cold Extracts Alkaloids Flavonoids Phenols Terpenoids Tannins - - - - - Benzene - - - - - Chloroform + - + - - Acetone + - + + + Ethanol + - + + + Methanol + + + + + - - - - - Benzene - - - - - Chloroform + - + - - Acetone + - + + + Ethanol + - + + + Methanol + + + + + 63

Table 3.6 Qualitative phytochemical analysis of the plants ethanolic extracts Alkaloids Flavonoids Phenols Terpenoids Tannins Acacia leucophloea Bark ethanolic extract Acacia caesia Bark ethanolic extract Eleutherine palmifolia Bulb ethanolic extract + - + + - + - + + - + - + + - 3.3.2. Anti-microbial activity Antibiogram pattern of a wide range of commercial available antibiotics were tested against eleven test organisms (Table 3.7). When the results obtained were compared, both chloramphenicol and imipenem inhibited the growth of all test organisms to various level, hence they were taken as positive controls for the study. 64

Table 3.7 Antibiogram Pattern of Commercial antibiotic discs Zone of inhibition (+)=10-15 mm (++)=15-20 mm (+++)= 21mm (-)= No Zone NA- Not Applicable NT- Not Tested Lactobacillus acidophilus Bacillus subtilis Salmonella paratyphi Shigella sonnei Klebsiella pneumonia Streptococcus sp. Staphylococcus aureus MRSA Pseudomonas aeruginosa Acinetobacter baumannii Candida albicans Amoxyclav +++ +++ +++ + + +++ +++ NT + NT NA Ampicillin A ++ ++ ++ + + + +++ NT + NT NA Ceftazidime + ++ +++ + + - +++ NT + NT NA Chloramphenicol +++ +++ +++ +++ +++ ++ +++ NT ++ NT NA Ciprofloxacin ++ +++ +++ +++ +++ +++ ++ NT + NT NA Erythromycin +++ +++ ++ ++ +++ ++ + NT + NT NA Gentamicin ++ ++ +++ + +++ ++ + NT + ++ NA Imipenem +++ +++ +++ +++ +++ +++ +++ NT ++ NT NA Methicillin - ++ - + +++ - ++ NT + NT NA Nalidixic acid - +++ +++ ++ ++ + + NT + NT NA Nitrofurantonin ++ +++ ++ + ++ + ++ NT + NT NA Norfloxacin + ++ +++ +++ + ++ ++ NT + NT NA Tetracycline +++ +++ +++ ++ +++ +++ +++ NT + NT NA Kanamycin + ++ ++ ++ ++ ++ + NT + NT NA Vancomycin +++ +++ - + ++ + + NT + NT NA Fusidic acid NT NT NT NT NT NT NT +++ NT NT NA Ketoconazole NA NA NA NA NA NA NA NA NA NA +++ 65

Antibiogram pattern exhibited by various solvent extract of Blepharis maderaspatensis leaf was depicted in Table 3.8.Hot chloroform and ethanolic extracts exhibited good inhibition pattern against both the gram positive and gram negative bacterial test organisms including bacterial skin pathogen Staphylococcus aureus and the fungal skin pathogen Candida albicans. Table 3.8 Antibiogram Pattern of Blepharis maderaspatensis Leaf Zone of inhibition (+)=10-15 mm (++)=15-20 mm (+++)= 21mm (-)= No Zone Lactobacillus acidophilus Bacillus subtilis Salmonella paratyphi Shigella sonnei Klebsiella pneumonia Streptococcus sp. Staphylococcus aureus MRSA Pseudomonas aeruginosa Acinetobacter baumannii Candida albicans Blepharis maderaspatensis Leaf Hot Extracts Cold Extracts - - - - - - - - - - - Benzene - - - - - - - - - - - Chloroform ++ + - + + + ++ - - - ++ Acetone - - - - - - - - - - + Ethanol + - + + + + ++ + - + + Methanol - - - - - - - - - - - - - - - - - - - - - - Benzene - - - - - - - - - - Chloroform - - + - + + + - - - + Acetone - + ++ + ++ + + - - - +++ Ethanol - - + + + + + + - + ++ Methanol + + ++ + + + ++ - - - + 66

On the other hand cold polar solvent extracts showed better zones of inhibition against the test organisms. Cold and hot ethanol extracts controlled almost all the pathogens including Pseudomonas aeruginosa, Acinetobacter baumannii and Methicillin resistant Staphylococcus aureus. Acinetobacter baumannii and Pseudomonas aeruginosa were not inhibited by any solvent extracts. Antibiogram pattern of Solanum nigrum leaf is given in Table 3.9. Both hot and cold extracts of acetone, ethanol and methanol exhibits similar Antibiogram pattern. Both the gram positive and gram negative test organisms were controlled by these extracts especially skin pathogens Staphylococcus aureus and Candida albicans were controlled well by cold and hot polar solvent extracts. Methicillin resistant Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa were not inhibited by any solvent extracts. 67

Table 3.9 Antibiogram Pattern of Solanum nigrum Leaf Zone of inhibition (+)=10-15 mm (++)=15-20 mm (+++)= 21mm (-)= No Zone Lactobacillus acidophilus Bacillus subtilis Salmonella paratyphi Shigella sonnei Klebsiella pneumonia Streptococcus sp. Staphylococcus aureus MRSA Pseudomonas aeruginosa Acinetobacter baumannii Candida albicans Solanum nigrum Leaf Hot Extracts Cold Extracts - - - - - - - - - - - Benzene - - - - - - - - - - - Chloroform - - - - - - - - - - - Acetone - - - - + + + - - - ++ Ethanol + + + + + + + - - - + Methanol ++ + + - + + + - - - + - - - - - - - - - - - Benzene - - - - - - - - - - - Chloroform - - - - - - - - - - - Acetone - + ++ ++ ++ + ++ - - - +++ Ethanol - + + + + + + - - - + Methanol - + + + ++ + - - - - ++ Table 3.10 summarizes the Antibiogram pattern shown by Acalypha indica leaf. Benzene, chloroform, acetone, ethanol and methanol hot extracts showed inhibition over almost all the test organisms. The same pattern was observed in Benzene, chloroform, acetone, ethanol and methanol cold extracts respectively. Lactobacillus acidophilus and Bacillus subtilis were not inhibited by the cold extracts of all the solvents used. Cold extracts of all solvent and hot non polar extracts did not inhibit Lactobacillus acidophilus and Bacillus subtilis. Methicillin resistant Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa were not inhibited by any solvent extracts. 68

Table 3.10 Antibiogram Pattern of Acalypha indica Leaf Zone of inhibition (+)=10-15 mm (++)=15-20 mm (+++)= 21mm (-)= No Zone Lactobacillus acidophilus Bacillus subtilis Salmonella paratyphi Shigella sonnei Klebsiella pneumonia Streptococcus sp. Staphylococcus aureus MRSA Pseudomonas aeruginosa Acinetobacter baumannii Candida albicans Acalypha indica Leaf Hot Extracts Cold Extracts - - - - - - - - - - - Benzene - - - + - ++ ++ - - - + Chloroform ++ + - ++ ++ +++ ++ - - - + Acetone ++ + + ++ +++ ++ ++ - - - + Ethanol +++ ++ ++ ++ + +++ +++ - - - ++ Methanol + - + + ++ +++ +++ - - - ++ - - - - - - - - - - - Benzene - - - + + - - - - - + Chloroform - - + ++ + + + - - - + Acetone - - + ++ + + ++ - - - +++ Ethanol - - ++ ++ ++ ++ ++ - - - ++ Methanol - - ++ + + ++ + - - - ++ Antibiogram pattern of Tridax procumbens flower (Table 3.11) showed similar pattern of anti-microbial activity exhibited by Acalypha indica leaf. Benzene, chloroform, acetone, ethanol and methanol hot and cold extracts showed inhibition over almost all the test organisms. Methicillin resistant Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa were not inhibited by any solvent extracts. 69

Table 3.11 Antibiogram Pattern of Tridax procumbens Flower Zone of inhibition (+)=10-15 mm (++)=15-20 mm (+++)= 21mm (-)= No Zone Lactobacillus acidophilus Bacillus subtilis Salmonella paratyphi Shigella sonnei Klebsiella pneumonia Streptococcus sp. Staphylococcus aureus MRSA Pseudomonas aeruginosa Acinetobacter baumannii Candida albicans Tridax procumbens Flower Hot Extracts Cold Extracts - - - + - - - - - - + Benzene - - + + + + + - - - ++ Chloroform - - + + + + + - - - + Acetone - - + ++ + ++ + - - - + Ethanol - - + + + + + - - - + Methanol - - + + ++ ++ ++ - - - ++ - - - - - - - - - - - Benzene - - - + - - - - Chloroform ++ - + + + - - - + Acetone +++ + - + + ++ + - - - + Ethanol ++ - - + + + - - - - Methanol ++ - - ++ + - - - - When comparing the anti-microbial activity and phytochemical analysis of Blepharis maderaspatensis, Solanum nigrum, Acalypha indica and Tridax procumbens it was found that the solvent ethanol is good in extracting the phytochemicals and on the other hand ethanolic extracts of all the plants showed higher range of Antibiogram pattern, hence ethanolic extracts of Acacia leucophloea, Acacia caesia and Eleutherine palmifolia were alone checked for their anti-microbial activity against the test organisms. Table 3.12 shows the Antibiogram pattern for the ethanolic extract of Acacia leucophloea, Acacia caesia and Eleutherine palmifolia. Acacia leucophloea and 70

Acacia caesia showed same inhibition range against the test organisms. Eleutherine palmifolia exhibited higher level of inhibition rate over the test organisms than other plant extracts. Moreover Eleutherine palmifolia was good in controlling Methicillin resistant Staphylococcus aureus and Acinetobacter baumannii. These two pathogens are those which develop resistance to all the available antibiotics and also resistant to all other plant extract tested, causing a great menace to human health. Table 3.12 Antibiogram Pattern of the plant extracts Zone of inhibition (+)=10-15 mm (++)=15-20 mm (+++)= 21mm (-)= No Zone Lactobacillus acidophilus Bacillus subtilis Salmonella paratyphi Shigella sonnei Klebsiella pneumonia Streptococcus sp. Staphylococcus aureus MRSA Pseudomonas aeruginosa Acinetobacter baumannii Candida albicans Acacia leucophloea Bark ethanolic extract Acacia caesia Bark ethanolic extract Eleutherine palmifolia Bulb ethanolic extract + + + + + - + - + - + + + + + - - + - + - + ++ +++ ++ +++ +++ +++ +++ ++ + ++ +++ 3.3.3. Quantitative phytochemical screening Among the three plant extracts estimated for phenolic and alkaloid content, Blepharis maderaspatensis showed higher content of phenols and alkaloids followed by Acacia leucophloea and Acacia caesia (Table 3.13).Phenolic content was more than alkaloid content in all the plant extracts. 71

Table 3.13 Total phenolic and alkaloid content of different plant extracts Plant extracts Total phenol Alkaloid content (mg/g) content (mg/g) Blepharis maderaspatensis ethanolic extract. 48.43±0.15 11.11±0.17 Acacia leucophloea ethanolic extract. 37.17±1.13 9.69±0.88 Acacia caesia ethanolic extract. 22.72±0.30 7.46±1.94 Values are represented as Mean ± SEM with triplicate estimations 3.4. DISCUSSION It is observed in general that polar solvents are good in extracting the drug principle from all the plants parts taken in this study. The findings are the extraction of drug molecule can be considerably improved by raising the temperature. In addition, the composition of the herbal extracts depends on the type of the solvent system and the temperature employed in the extraction. Hence, chloroform is the least preferred solvent among the extreme polar and non-polar solvents. The spectrum of compounds present in the extracts vary considerably depending on the hydrophilic or hydrophobic nature of the drug principle. The polar solvents ethanol and acetone are more efficient in extracting the drug principle. From the experimental results, it was found that the non-polar solvents (petroleum ether and benzene) are not efficient as the polar solvents (chloroform, acetone, ethanol and methanol) in extracting the anti-microbial drug principle. It was noted in general that polar solvents are good in extracting the drug principle from all the plant parts taken in the study especially, from the aerial parts of 72

the plant. The mode of extraction and the temperature employed in extraction determines the extract s activity/efficiency. The authenticity of the anti-microbial activity exhibited by all the extracts in comparison to the commercially available broad spectrum antibiotics was checked, among imipenem that controls all the three Gram negative test pathogens, namely, Shigella sonnei, Salmonella paratyphi and Klebsiella pneumonia and it also controls the growth of the Gram positive pathogens Staphylococcus aureus and Lactobacillus acidophilus. Imipenem-resistant strains were rarely found in clinical practice and bacteria resistant to newer beta-lactams and aminoglycosides were generally very susceptible to this new carbapenem (Jones 1985). The chloramphenicol controls the Gram positive Lactobacillus acidophilus and Gram negative Salmonella paratyphi. Chloramphenicol is a broad-spectrum antibiotic exhibiting bacteriostatic activity against many Gram-negative and Gram-positive bacteria as well as chlamydiae, rickettsiae and mycoplasmas (Antonio et al. 2010). Initial analysis of the solvent system was done on the influence of temperature in extraction process. The phytochemical constituent in the best extract that gives good anti-microbial activity was also analyzed. Acetone is a good solvent that widely extracts the drug principles from almost all the folkloric plants (Gami and Parabia 2011). An interesting finding in the acetone solvent extraction is that; B. maderaspatensis leaf cold acetone extract exhibited a significant control over the test pathogens. The hot acetone leaf extract of the same plant showed no anti-microbial effect. In general, the cold extracts show more activity in comparison to their corresponding hot extracts. This gives an inference that some constituents of phytochemicals may be heat labile that may lose its activity upon hot extraction protocols. Otherwise, some other phytochemical components which produce 73

synergistic activity were found along with the drug principle. It may be lost upon hot extraction. Ethanolic extracts also exhibited a significant control of the test organisms in comparison with the commercial antibiotics. It is significant to note that while comparing anti-microbial activity of the A. indica leaf hot and cold extract of ethanol, hot extract exhibits higher zone of inhibition corresponding to the respective cold extract. This indicates that certain drug molecules in A. indica leaf are slowly extracted by ethanol cold extraction, while the same drug molecule is exhaustively extracted only by hot extraction. The finding here is that the extraction of drug molecule can be considerably improved by raising the temperature and the composition of the herbal extracts depends on the type of the solvent system and the temperature applied in the extraction. On observing the anti-microbial activity of the methanolic extracts, almost all showed good inhibition. While comparing the hot and cold extracts of B. maderaspatensis leaf, hot extracts completely lost its anti-microbial activity, whereas the cold extract had significant anti-microbial activity. This again supports the finding that, the temperature in the hot extraction may be responsible for the loss of the drug principle s activity that may be heat labile. Among the benzene extracts, A. indica leaf extract exhibited anti-microbial activity that controls Gram positive bacteria to a greater extent than the Gram negative bacteria. This may be due to the hydrophobic drug principle found in the leaf parts of A. indica that is efficiently extracted by the non-polar solvent benzene extract. Both hot and cold benzene extracts of A. indica leaf, T. procumbens flower exhibit some feeble anti-microbial activity. 74

On the other hand, polar solvent extracts of T. procumbens flower and A. indica leaf have increased the zone of inhibition than non-polar solvent extracts. This gives an inference that some hydrophilic drug principles present in the above aerial plant parts are efficiently extracted by polar solvents, namely, methanol and ethanol than the nonpolar solvent benzene. Comparing the chloroform extracts, both hot and cold extracts of A. indica leaf exhibited good anti-microbial activity, but comparatively lesser activity than the extracts of polar solvents (ethanol and methanol). This gives an indication that the drug principles in the A. indica leaf are hydrophilic in nature and they are eluted well using the polar solvents. At the same time, hydrophilic drug principle found in the plant parts are later eluted well upon with the successive polar solvent extracts, namely, methanol, ethanol and acetone. Hence, chloroform is a least preferred solvent than the extreme non-polar solvents. The anti-microbial pattern of each solvent extracts and their phytochemical constituent s analysis of the different solvent extracts consequently obtained in different stages of hot extraction revealed the compounds and elution strength of the respective solvents. The spectrum of compounds varies considerably depending on the hydrophilic or hydrophobic nature of the drug principle. Analysis of the best solvent system for extracting the drug molecule from each plant part was done by analyzing the anti-microbial activity of all the solvent extracts of the plant parts. The anti-microbial activity exhibited by the A. indica leaf polar solvent extracts is comparatively more than the non-polar solvent extracts. Hence, the ingredients are extracted well by the polar solvents. This finding is in correlation with the earlier finding that acetone and methanol extracts are efficient in extracting the drug principle that control fungal pathogen Candida albicans from A. indica leaf (Jebakumar et al. 2005). In the folkloric medicinal system, there is a practice of using this whole plant extract in water base by just squeezing this plant leaves over the 75

wounds and the microbial borne skin infections. From the experimental results, it is found that both the extreme polar (methanol) and the non-polar solvents (petroleum ether and benzene) are not good in extracting the drug principles, whereas the intermediate solvent extracts like chloroform and acetone are good in extracting the drug principle as observed from their anti-microbial activity. Polar solvent extracts of S. nigrum are exhibiting good anti-microbial activity, but there is no activity in the non-polar solvent extracts. Polar or the aqueous extracts may be the best solvent system in extracting drug principle from S. nigrum. The findings are in correlation with the reports that S. nigrum leaf aqueous extracts are good in controlling the internal ulcers in the enteric tract (Mallika and Chennam 2006). Acacia leucophloea are good in controlling both gram positive and gram negative test organism and also the fungal pathogen Candia albicans. Klebsiella pneumonia, MRSA and Acinetobacter baumannii showed resistance to Acacia caesia. Methicillin-resistant Staphylococcus aureus (MRSA) does not show resistance to Eleutherine palmifolia bulb ethanol extract or otherwise ethanol extract Eleutherine palmifolia bulb controlled Methicillin-resistant Staphylococcus aureus and this activity was higher than the standard antibiotic tested. Acinetobacter baumannii was more sensitive to Eleutherine palmifolia Linn. bulb ethanol extract than gentamycin. The activities exhibited by the 100 µl of the Eleutherine palmifolia bulb ethanol extract which is more or less equivalent to stronger and broader spectrum anti-microbial compound gentamycin. Moreover elucidation of the drug principle in this extract is subject of significance. 76

It is also generally noticed from the successive extraction experimental findings, that there is no universal solvent for extracting anti-microbial principles from all plant parts. In some plants polar solvents are good in extracting anti-microbial drug principles, whereas in some plants non-polar solvents are good in extracting the drug molecule. Surprisingly in some plants intermediate solvents (acetone and chloroform) are good in extracting the drug molecule from the plant part. However every plant that is reported in traditional medicine has to be initially screened/ extracted with all solvents to find the best solvent in extracting the drug molecule. An interesting finding is that both cold and hot extraction methods do have difference in extracting efficiency. Hence for each plant part the extraction efficiency has to be standardized by both hot and cold extraction separately to find the efficienct extraction process. 77