Extraction and Pharmacological Evaluation of Some Extracts of Tridax procumbens and Capparis decidua

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1 International Journal of Applied Research in Natural Products Vol. 1(4), pp. 5-12, Dec 2008-Jan 2009 Available online Healthy Synergies Publications Original Article Extraction and Pharmacological Evaluation of Some Extracts of Tridax procumbens and Capparis decidua Sharma B*, Kumar P Laboratory of Tissue Culture and Secondary Metabolites, Department of Botany, University of Rajasthan, Jaipur, India Summary: Free and bound flavonoids of different parts of Tridax procumbens L. (Asteraceae) and Capparis decidua Forsk (Edgew) (Capparaceae) have been studied for their antimicrobial activities using disc diffusion assay, against two Gram negative bacteria (Escherichia coli MTCC 46 and Proteus mirabilis MTCC 425), one Gram positive bacteria (Staphylococcus aureus MTCC 87), and a fungi (Candida albicans MTCC 183). Minimum inhibitory concentration (MIC) of the extracts was evaluated by micro broth dilution method, while minimum bactericidal/ fungicidal concentration determined by subculturing the relevant samples. Both plants exhibited broad spectrum antimicrobial activity. Free and bound flavonoids of T. procumbens flowers and C. decidua stem were found to be more potent. C. albicans was found to be most susceptible organism followed by S. aureus, P. mirabilis, and E. coli. Among 14 extracts tested, 12 were found to be active, while 2 extracts showed no activity at tested concentration. Total activity (TA) was calculated for the extracts, to relate MIC of the extracts with its amount isolated from 1 g dried plant part. Results of the present study indicate that T. procumbens and C. decidua can be exploited for future antimicrobial drugs. Industrial relevance: Adverse effects of popular antibiotics and multidrug resistant strains of pathogens have lead rapid search for new antimicrobials. Because of the long history of plants in the treatment of different human ailments, most of the herbal drugs are believed to be safer than the synthetic drugs with no side effects; therefore medicinal plants have gained more importance as possible source of alternative and effective drugs. Plants and natural products remain as an untapped reservoir of potentially useful chemical compounds not only as drugs but also as unique templates that could serve as a starting point for synthetic analogues. Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in the drug development programmes in the pharmaceuticals industries. Keywords: Capparis decidua, MBC, MFC, MIC, Total activity, Tridax procumbens Introduction Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural resources. Traditional medicine is an important source of potentially useful new compounds for the development of chemotherapeutic agents (Racio et al., 1989). Emergence of pathogenic microorganisms that are resistant/ multi resistant to major class of antibiotics has increased in recent years due to indiscriminate use of synthetic antimicrobial drugs (Karaman et al., 2003). In addition, high cost and adverse side effects are commonly associated with popular synthetic antibiotics (such as hypersensitivity, allergic reactions, immjunosupression etc.) and are major burning global issues in treating infectious diseases (Schinor et al., 2007). Although pharmacological industries had produced considerable number of commercial antibiotics time to time but resistance in pathogens towards these drugs too has increased at high rate and multi drug resistant microorganisms have exacerbated the situation (Nino et al., 2006). In the present scenario, there is an urgent and continuous need of exploration and development of cheaper, effective new plant based drugs with better bioactive potential and least side effects. Hence, recent attention has been paid to biologically active extracts and compounds from plant species used in herbal medicines (Essawi and Srour, 2000). Antimicrobials of plant origin have enormous therapeutic potential and have been used since time immemorial. They have been proved effective in the treatment of infectious diseases simultaneously mitigating many of the side effects which are often associated with synthetic antibiotics (Iwu at al., 1999). Positive response of plant based drugs (less/ no side effects) might lies in the structure of the natural products which reacts with toxins and/or pathogens in such a way that less harm is done to other important molecules or physiology of host. It is because of this reason that drug designing studies nowadays have come up as new field of research. *Corresponding Author: Tel: bindushr111@gmail.com Accepted 27 August

2 Sharma, Kumar Tridax procumbens L. (Asteraceae) and Capparis decidua Forsk (Edgew) (Capparaceae) were selected in the present study for evaluation of their antimicrobial activities. Both the plants are well adapted to the harsh (xerophytic) climatic conditions and are well known for their medicinal properties among local natives. Tridax procumbens is known for its wound healing activities. Whole plants is made into paste and applied on fresh cuts (Dhar et al., 2003). In ayurvedic medicine C. decidua is recorded as a hepatic stimulant and protectant. Decoctions from C. decidua root bark have been traditionally used for dropsy, anaemia, arthritis, and gout. It is used for the treatment of asthma, ulcer, piles, and urinary problems (Warrier et al., 2003). Escherichia coli, Staphylococcus aureus, Proteus mirabilis, and Candida albicans have been proved to be major causal organisms of various human infections and have been selected for the present study. E. coli and P. mirabilis are the culprits for human urinary tract infections (Venier et al., 2007) and most of the human intestinal infections are due to the bacterium E. coli. S. aureus causes a variety of suppurative, wound infections and food poisoning in human beings. Major causative agent of nosocomial infections is S. aureus (Alvarez et al., 2006) along with E. coli and P. mirabilis. C. albicans is notorious for causing candidiasis and candida vaginitis. The present investigation evaluated the antibacterial and anticandidal effects of free and bound flavonoids of Tridax procumbens (root, stem, leaf, and flowers), and Capparis decidua (root, stem, and fruit).the study was carried out along with the standard drugs (terbinafine for fungus and streptomycin for bacteria). Materials and Methods Plant material and extraction procedure: Different parts of T. procumbens (root, stem, leaf, and flowers) and C. decidua (root, stem, and fruits) were collected in the month of August from the western parts of India (Jaipur, Rajasthan). Plants were identified by senior taxonomist Dr. Sudhakar Mishra, at Department of Botany, University of Rajasthan and sample vouchers were submitted to the herbarium, Botany Department, University of Rajasthan. Selected plant parts were separately shade dried, finely powdered using a blender, and subjected to extraction following the method of Subramanian and Nagarjan, Hundred grams of each finely powdered sample was Soxhlet extracted with 80% hot methanol (500 ml) on a water bath for 24 h and filtered. Filtrate was re-extracted successively with petroleum ether (fraction I), ethyl ether (fraction II), and ethyl acetate (fraction III) using separating funnel. Petroleum ether fractions were discarded as being rich in fatty substances, whereas ethyl ether and ethyl acetate fractions were analyzed for free and bound flavonoids, respectively. Ethyl acetate fraction of each of the samples was hydrolyzed by refluxing with 7% H2SO4 for 2 h (for removal of bounded sugars from the flavonoids). Resulting mixture was filtered and filtrate was extracted with ethyl acetate in separating funnel. Ethyl acetate extract thus obtained was washed with distilled water to neutrality. Ethyl ether (free flavonoids) and ethyl acetate fractions (bound flavonoids) were dried in vaccuo and weighed (Table 3). The extracts were stored at 4 C and were re-suspended in their respective solvents to get 10 mg/ml for antimicrobial assay. Free and bound flavonoids: Free and bound flavonoids were extracted by well established method of Subramanian and Nagarjan (1969) and the protocol has also been followed by several others workers (Kumar and Khanna, 1986; Kumar et al., 1989; Kumar and Khanna, 1994; Bhadauria and Kumar, 1999; Mewari and Kumar, 2008). The extraction protocol which has been carried out in the present investigation is exclusively meant for free and bound flavonoids. In the extraction procedure ethyl ether and ethyl acetate fractions are supposed to contain free and bound flavonoids, respectively. Here bound flavonoids imply that the flavonoids are bounded with sugar moiety. These bounded sugar moieties are removed (from ethyl acetate fraction) by acid hydrolysis during extraction. If sugar part of the flavonoids is not removed, extracts do not react with spraying reagents (i.e. 5% fehling solution and 1% AlCl 3 solution) during TLC analysis. On the other hand, after removal of sugar part, flavonoids become free from sugars bounded to it and now react with spraying reagent and give colour reactions during TLC analysis. Spraying reagents 5% fehling solution and 1% AlCl 3 solution are exclusively used to detect flavonoids (Harborne, 1984). Ethyl ether fraction contains free flavonoids i.e. flavonoids free from sugar moiety and during TLC analysis and thus shows positive colour reactions with the spraying reagents. In the ethyl ether fraction there is no need of acid hydrolysis as flavonoids in this fraction are not bound with sugar. Screening of antimicrobial activity: Test pathogenic microorganisms: E. coli (MTCC 46), S. aureus (MTCC 87), P. mirabilis (MTCC 425), and C. albicans (MTCC 183) were procured from Microbial Type Culture Collection, IMTECH, Chandigarh, India. Bacterial strains were grown and maintained on Muller-Hinton Agar medium, while yeast was maintained on Sabouraud Dextrose Agar medium. Disc diffusion assay (Andrews, 2001) was performed for screening. MH agar and SD agar base plates were seeded with the bacterial and fungal inoculum, respectively (inoculum size CFU/ml for bacteria and cell/ml for yeast). Sterile filters paper discs (Whatman no. 1, 6 mm in diameter) were impregnated with 100 µl of each of the extract (10 mg/ml) to give a final concentration of 1 mg/disc and left to dry in vaccuo so as to remove residual solvent, which might interfere with the determination. Extract discs were then placed on the seeded agar plates. Each extract was tested in triplicate with streptomycin (1 mg/disc) and terbinafine (1 mg/disc) as standard for bacteria and fungi, respectively. The plates were kept at 4 C for 1 h for diffusion of extract, thereafter were incubated at 37 C for bacteria (24 h) and 27 C for fungi (48 h). Activity index for each extract was calculated (Table 1). Determination of minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC): Minimum inhibitory concentration (MIC) was determined for each plant extract showing antimicrobial activity against test pathogens. Broth microdilution method (Basri and Fan, 2005) was followed for determination of MIC values. Plant extracts were resuspended in acetone (which has no activity against test microorganisms) to make 10 mg/ml final concentration and then two fold serially diluted; added to broth media of 96-wells of microtiter plates. Thereafter 100 µl inoculum (for bacteria CFU/ml and cell/ml for yeast) was added to each well. Bacterial 6

3 Pharmacological evaluation of T. procumbens and C. decidua and fungal suspensions were used as negative control, while broth containing standard drug was used as positive control. The microtiter plates were incubated at 37 C for 24 h for bacteria and 28 C for 48 h for yeast. Each extract was assayed in duplicate and each time two sets of microplates were prepared, one was kept for incubation while another set was kept at 4 C for comparing the turbidity in the wells of microplate. The MIC values were taken as the lowest concentration of the extracts in the well of the microtiter plate that showed no turbidity after incubation. The turbidity of the wells in the microtiter plate was interpreted as visible growth of microorganisms. The minimum bactericidal/fungicidal concentration (MBC/MFC) was determined by subculturing 50 µl from each well showing no apparent growth. Least concentration of extract showing no visible growth on subculturing was taken as MBC/MFC. Total activity (TA) determination: Total activity is the volume at which test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the amount of extract from 1 g plant material by the MIC of the same extract or compound isolated and is expressed in ml/g (Eloff, 2004). Results Antimicrobial activity (assessed in terms of inhibition zone and activity index) of the plant extracts, tested against selected microorganisms were recorded (Table 1). In the present study total 14 extracts of different parts of selected plants were tested for their bioactivity, among which 12 extracts showed significant antimicrobial potential against test microbes. However, two extracts showed no activity against any selected microorganism at tested concentration. Most susceptible organism in the investigation was C. albicans against which, most of the plant extracts showed inhibition zone (more than the standard) and best activity was observed by bound flavonoids of C. decidua root, where IZ of mm, 2.3 ± 0.03 AI and MIC value of mg/ml was recorded. E. coli was found to be the most resistant microbe, against which only four extracts showed antibacterial effect. Best antibacterial activity against E. coli (IZ mm, AI ± 0.003, MIC mg/ml) and S. aureus (IZ mm, AI ± 0.006, MIC mg/ml) was observed for bound flavonoids of T. procumbens flowers whereas, free flavonoids of C. decidua stem showed best activity against P. mirabilis (IZ mm, AI ± 0.002, MIC mg/ml). Bound flavonoids of C. decidua stem showed excellent bioactivity against S. aureus (IZ mm, AI ±0.006, MIC mg/ml) and P. mirabilis (IZ mm, AI 0.76 ± 0.016, MIC mg/ml). MIC and MBC/MFC values (Table 2) were evaluated for those plant extracts, which were showing activity in diffusion assay. The range of MIC and MBC/MFC of extracts recorded was mg/ml and mg/ml, respectively. In the present investigation lowest MIC value mg/ml was recorded against S. aureus, P. mirabilis, and C. albicans whereas, against E. coli mg/ml lowest MIC was observed, indicating significant antimicrobial potential of test extracts. MIC and MBC/MFC values were found equal for four extracts of T. procumbens and five extract of C. decidua. Amount of extract isolated from plant parts and total activity (TA) was calculated and recorded (Table 3). Total activity indicates the volume at which extract can be diluted with still having ability to kill microorganism. Most of the extracts showed high values of TA against S. aureus, P. mirabilis and C. albicans, which proves the potential to inhibit the growth of the test microorganisms, even at low concentration. Maximum TA values calculated were 12.82, , , and ml against E. coli, S. aureus, P. mirabilis, and C. albicans, respectively. Discussion Resistance in microorganisms to many antibiotics has resulted in morbidity and mortality from treatment failure and increased health care costs. Though a number of antibiotics are available but increasing capability of microbes to develop multidrug resistance has encouraged search for new, safe and effective bioactive agents of herbal origin. Results of the present study showed that 12/14 plant extracts tested inhibited the growth of selected bacteria and fungi, indicating broad spectrum bioactive nature of selected two plants. Free and bound flavonoids of T. procumbens flowers showed broad range of activity by suppressing the growth of all microbes under investigation. In the present study most of the extracts of C. decidua were found to be potent inhibitor of tested organisms except E. coli, against which only one extract of the plant showed activity. Excellent activity was shown by free and bound flavonoids of stem of C. decidua by showing low MIC and MBC/MFC values. MBC/MFC values were found higher than the MIC values of the extracts against microorganisms tested; indicate the bacteriostatic/fungistatic effects of the extracts. Three extracts of T. procumbens viz. bound flavonoids of root, leaf and flower were found to be bactericidal against P. mirabilis and bound flavonoids of leaf were found to be fungicidal against C. albicans. On the other hand, free and bound flavonoids of stem of C. decidua were recorded bactericidal against S. aureus whereas, bound flavonoids of root of C. decidua were bactericidal against E. coli and fungicidal against C. albicans. 7

4 Sharma, Kumar Table 1: Antimicrobial activity of Tridax procumbens and Capparis decidua Plant Plant part Extract Test microorganism E. coli S. aureus P. mirabilis C. albicans IZ (mm) AI IZ (mm) AI IZ (mm) AI IZ (mm) AI Tridax procumbens Root E ± ±0.006 E ± ± ±0.01 Stem E ± ±0.006 E ± ± ±0.017 Leaf E E ± ±0.006 Flower E ± ± ± ±0.003 E ± ± ± ±0.006 Capparis decidua Root E E ± ± ±0.03 Stem E ± ± ±0 E ± ± ±0.02 Fruit E ±0.03 E ± ± ±0.003 IZ = Inhibition zone in mm (mean value; including 6 mm diameter of disc), AI = Activity Index (IZ developed by extract/iz developed by standard), ± = SEM, ( ) = No activity, E 1 = Free flavonoids, E 2 = Bound flavonoids, Extracts assayed in triplicate, IZ of standard drug streptomycin against E. coli (20 mm), S. aureus (25 mm), P. mirabilis (24 mm) and IZ of terbinafine against C. albicans (10 mm). 8

5 Anti-nociceptive and anti-inflammatory activities of Newbouldia laevis Table 2: MIC and MBC/MFC values of Tridax procumbens and Capparis decidua against test pathogens Plant Plant part Extract Test microorganism E. coli S. aureus P. mirabilis C. albicans *MIC *MBC *MIC *MBC *MIC *MBC *MIC *MBC Root E E Tridax procumbens Stem E E Leaf E Flower E E Capparis decidua Root E Stem E E Fruit E E *in mg/ml E 1 = Free flavonoids; E 2 = Bound flavonoids MIC= Minimum Inhibitory Concentration, MBC/MFC= Minimum Bactericidal/Fungicidal Concentration 9

6 Sharma, Kumar Table 3: Quantity and Total Activity of free and bound flavonoids of Tridax procumbens and Capparis deciduas Plant Plant part Extract Amount of extract mg/g dried plant part Total activity (ml/g) E. coli S. aureus P. mirabilis C. albicans Tridax procumbens Root E E Stem E E Leaf E Flower E E Capparis decidua Root E Stem E E Fruit E E 1 = Free flavonoids E 2 = Bound flavonoids Total Activity = Extract per gram dried plant part MIC of extract E

7 Anti-nociceptive and anti-inflammatory activities of Newbouldia laevis Gram positive bacteria S. aureus was the second most susceptible organism after fungi C. albicans, which supported the finding that plant extracts are usually more active against Gram positive bacteria than Gram negative (Lin et al., 1999; Palombo and Semple, 2001). Susceptibility differences between Gram-positive and Gram-negative bacteria may be due to cell wall structural differences between these classes of bacteria. The Gram-negative bacterial cell wall outer membrane appears to act as a barrier to many substances including synthetic and natural antibiotics (Tortora et al., 2001). Bioactive potential of flavonoids have been proved by several workers (Ilic et al., 2004; Cushnei and Lamb, 2005; Mandalari et al., 2007). Though antimicrobial effects of C. decidua (Gaind et al., 1969; Ghadeib and Shatayeh, 1999; Almas, 2001) and T. procumbens (Samy and Ignacimuthu, 1999; Taddei and Rosas-Romero, 2000; Sanchez et al., 2005) have been demonstrated previously but still the literature available is meager with the flavonoids. Mostly the crude extracts have been screened, that too without MIC, MBC/MFC and TA determination. Such studies could only indicate their antimicrobial potential but are not helpful in establishing them as an antibiotic. In the present study IZ, AI, MIC, MBC/MFC and TA have been evaluated for each extract. For most of the extracts MIC values recorded were very low, indicating strong bioefficacy of the selected plants. Extracts under study not only inhibit the bacterial/fungal growth but the IZ developed, was more or less permanent when compared with the IZ developed by the standard drug used, as after sometime bacterial/fungal colonies could be easily seen in IZ developed by standard drugs. In the light of the fact that microorganism are becoming resistant against the drugs in use, present investigation is of great significance, as far as the future drugs are concerned and advocates uses of selected plants by the pharmaceutical industries for preparing plant based antimicrobials drugs. Acknowledgement Authors are thankful to the Head, Botany Department, University of Rajasthan, Jaipur, India for providing all the facilities to carry out the work. References Almas K The antimicrobial effects of seven different types of Asian Chewing sticks. 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