ANTIMICROBIAL ACTIVITY OF THREE MEDICINAL PLANTS ( ARTEMISIA INDICA , MEDICAGO FALCATA AND TECOMA STANS )

Background: Artemisia indica , Medicago falcata and Tecoma stans are traditionally being use for medicinal purposes in Pakistan. Present study was designed to check in-vitro efficacy of these plants against selected bacterial and fungal strains. Methodology: Chloroform, butanol, ethyl acetate and n-hexane extracts of these plants were used for antimicrobial screening. Antibacterial activity was tested against four pathogenic bacterial strains i.e. Escherichia coli , Pseudomonas aeruginosa , Salmonella typhi and Staphylococcus aureus while antifungal activity was tested against four fungal strains i.e. Aspergillus flavus , Aspergillus niger , Aspergillus fumigatus and Fusarium solani . Results : Chloroform, butanol and ethyl acetate extracts of Artemisia indica , Medicago falcata and Tecoma stans showed high inhibitory activities (between 15-20 mm) against E. coli, P. aeruginosa and S. aureus. However, all extracts of Artemisia indica showed inhibitory activities (12-14 mm) against Salmonella typhi. As antifungal activities, the n-Hexane and chloroform extracts of Artemisia indica have completely inhibited the growth of Aspergillus flavus and Fusariun solani , respectively. Ethyl acetate and butanol extract of Medicago falcata completely inhibited Fusarium solani and Aspergillus fumigates , respectively. The n-hexane extract of Tecoma stan completely inhibited Fusarium solani , while its ethyl acetate extract shows excellent activity against Aspergillus niger . Conclusions: These findings provide scientific evidence of traditional use of medicinal plants and also indicate the potential of these plants for the development of antimicrobial agents.


Introduction
Medicinal plants are abundant source of antimicrobial molecules.A wide range of medicinal plants extracts are used to treat several infections as they have potential antimicrobial activity.Some of these bioactive molecules are screened and traded in market as raw material for many herbal industries (Renisheya et al., 2011).Experts turned their concentration back towards obtaining advantages from medicinal plants after observing more side effects of synthetic drugs compared to their benefits (Bushra et al., 2012).It is estimated that about 35,000 to 70,000 plants species are used as medicinal plants out of 422127 reported worldwide plant species (Bibi et al., 2011).In Pakistan 80% of the population belonging to the rural areas depends on the traditional medicines (Munir et al., 2013).Artemisia indica is a perennial plant species mostly found in Asia (India, China, Japan, Nepal and Pakistan) and Europe.It can grow to a height of 6 feet; contain a stalk of small reddish brown or yellow colour.In Pakistan, its infusion is traditionally used in the treatment of nervous and spasmodic effect, asthma and helpful in increasing appetite (Nadeem et al., 2013).In Nepal, the juice of Artemisia indica is used to treat stomachic, diarrhea, dysentery, abdominal pains and relieving burning sensation in conjunctives (Satyal et al., 2012).Moreover, the roots are antiseptic and used as tonic for the kidneys (Sarnim et al., 2013).Medicago falcata is also a perennial herb belonging to the family Fabaceae and is extremely cold tolerant and winter hardy.It is native to Mediterranean basin, but has worldwide distribution.It contains widely branched roots, a deep set crown, less upright stems, narrow leaves and yellow flower.
The leaves of Medicago falcata contain large source of vitamins and a number of proteins.Its seeds are commonly used salad, sandwiches and cooked in soups.An appetite enhancing tea is also made from the leaves.Its flavour is somewhat slightly laxative (Muthu et al., 2012).Tecoma stans known as yellow elder is an erect shrub or small tree belong to family Bignonaniceae.The plant has been used for a variety of purposes in phytomedicines, treating diabetes and digestive problems.Extracts from Tecoma stans leaves have been found to inhibit the growth of yeast infections (Jennie et al., 2003).A growing body of literature is available on the medicinal properties of these three medicinal plant species.
However, the literature mining revealed that Artemisia indica, Medicago falcata and Tecoma stans have not been studied for antimicrobial activities.Hence, the present study is first systematic attempt to analyse the antibacterial and antifungal potential of these three species on selected bacterial and fungal strains.The objectives of the study were to screen out selected medicinal plants of Kohat for their antimicrobial activities.

Collection of plant sample
Fresh samples of Artemisia indica, Medicago falcata and Tecoma stans were collected from district Kohat, Khyber Pakhtunkhwa (KP), Pakistan, and authenticated at department of Botany, Kohat University of Science and Technology, Kohat, KP.These plant samples were washed with tap water in order to remove the dust.Collected plant materials were then dried under the shade and mashed with the help of mortar and pestle.

Preparation of extract
About 30g powdered material of each plant species was soaked in 300 mL solvent of n-hexane, chloroform, Ethyl acetate and Butanol for 2 weeks, shaked well twice a day and then filtered.The filtrates were then evaporated under reduce pressure to obtain a gummy residue with the help of rotary evaporator.All extracts were stored in sterile glass bottle at room temperature until screened.

Antibacterial bioassay
The different solvent soluble fractions were subjected to antibacterial evaluation against four bacterial strains Escherichia coli (E.coli), Pseudomonas aeruginosa (P.aeruginosa), Salmonella typhi (S. typhi), Staphylococcus aureus (S. aureus) as described by Usman et al. (2013).Muller Hinton Agar (Oxoid UK) was prepared in conical flask in accordance to the directions provided by the manufacturer.The media along with petri dishes, pipette and metallic borer were sterilized in autoclave for 15 minutes at 121 o C and 15 psi pressure.The media was poured into Petri dishes under aseptic condition (Yogeshi et al., 2004).The stock solutions of corresponding fractions were prepared in dimethyl sulfoxide (DMSO) (Perez et al., 1990).
The modified method of Perez et al., (1990) was followed.All of the four bacterial strains were obtained from Department of Microbiology, Kohat University of Science and Technology, Kohat, KPK, Pakistan.Bacterial culture was inoculated on MHA (corresponding to 10 6 CFU/ml).Bacterial strains were spread on the solidified agar media, then 7 mm wells were punched in the agar media by using sterile metallic borer.Stock solutions of crude extract and fractions in DMSO at concentration of 20 mg/ml were prepared and 200 μl from each stock solution was added into respective wells (Rehman et al., 2001).The petri dishes were incubated at 37 o C for 24 hours and control wells containing antibiotic (Levofloxacin), which is a positive control, was also run side by side.After 24 hours antibacterial activities were measured as diameter of the zones of inhibition and compared with the zone of inhibition of control (Levofloxacin).

Antifungal bioassay
The antifungal bioassay was determined by agar tube dilution method (Islam et al., 2011).Four fungal strains i.e.Aspergillus flavus (A.flavus), Fusariun solani (F.solani), Aspergillus fumigatus (A.fumigatus) and Aspergillus niger (A.niger) were used for antifungal activities.To refresh fungal strains, 13g/L nutrient broth in distilled water was prepared.Sterilized in autoclave and four flasks of 250ml were filled from broth, to each flask fungal colonies were inoculated separately.These flasks were then placed in incubator at 30°C for 3 days for refreshing fungal strains.SDA was used for growth of fungal strains.The flask was sterilized in autoclave at 121°C for 15 minutes at 1.5 pounds pressure.Antibiotic, Clotrimazole (Canesten) an antifungal drug was taken as a positive control and dissolved in distilled water (30μg/ 6μL) while DMSO was incorporated as negative control.About 7ml of medium was added to clean, dry and sterilized test tubes.Solutions of crude extracts and sub fractions were prepared each of 2μg/μL.One ml of sample (2μg/μL) was also added to each test tube, the test tube was kept in inclined position to make a slant.The same process was repeated for all of test tubes.
After cooling and solidifying, the fungi inoculums suspension was spread over the SDA medium uniformly using sterile cotton swabs.After that the test tubes were kept in incubator for 3 days at 30°C.After 3 days the fungal growth was observed in each test tube by the absence and presence of fungal strains.

Results and Discussion
In the present study, the antibacterial and antifungal activities of A. indica, M. falcata and T. stans were recorded against different bacterial strains including E. coli, S. typhi, P. aeruginosa and S. aureus, and fungal strains like A. flavus, F. solani, A. fumigatus and A. niger.The extracts of these plants showed variable activities.

Antimicrobial Activities Antibacterial activity
The extract of A. indica was evaluated for in vitro antibacterial activity against, E. coli, S. typhi, P. aeruginosa and S. Aureus indicating different zones of inhibition (Table 1).Our results revealed that chloroform, butanol, ethyl acetate extract of A. indica showed significantly higher inhibitory activity (between 15-19 mm) against E. coli, P. aeruginosa and S. aureus.All extracts of A. indica showed activity (12-14 mm) against S. typhi (Table1).The present investigation has shown that the chloroform, butanol, ethyl acetate extract of A. indica have active phytochemical, which can inhibit the growth of pathogenic bacteria and fungi.Farhat et al., (2011) investigated that Artemisia spp.have great antibacterial and anti allergic potential due to the presence of large amounts of Flavonoids (Table 2).
It was found that chloroform, butanol, ethyl acetate and n-hexane extract of M. falcata showed significantly higher zone of inhibition (17-19 mm) against E. coli.While all extracts showed moderate activity (12-13 mm) against strains of P. aeruginosa, S. Typhi and S. aureus (Table 1).According to Baloch et al., (2013), the antibacterial activity of the chloroform, butanol, ethyl acetate and n-hexane extracts of Medicago spp.possess good antibacterial activity against Bacillus subtilis, E. coli, P. aeruginosa, S. typhi and S. aureus.The antimicrobial activity of the tested extracts and fractions are comparable with the standard drugs.These activities may be due to strong occurrence of different chemical compounds such as flavonoids, tannins, alkaloids, steroids, phenols and saponins (Table 2).
All four extract of T. stans showed significantly highest zone of inhibition (16-20 mm) against S. aureus and E. coli.Ethyl acetate and n-hexane extract showed no activity against P. aeruginosa and S. typhi, respectively (Table 1).Our results are supported by the study of Sing et al. (2011), in which he reported that ethanol, methanol and water extracts of T. stans can be effective against tested bacteria (S. aureus, E. coli, P. aeruginosa and S. typhi.Phytochemical analysis revealed the presence of alkaloids, flavonoids, saponins, phenols, steroids, anthraquinones and tannins (Table 2).The flavonoids from plant extracts have been found to possess antimicrobial and antioxidants properties in various studies (Amarlal et al., 2009;Lin et al., 2008).The alkaloids are also effective in their antimicrobial (Erdemoglu et al., 2007) and antioxidant (Benabdesselam et al., 2007) activities.

Antifungal activities
The ethyl acetate and butanol extracts of A. indica showed activity against all studied fungal strains.However, n-hexane and chloroform fractions did not show any activity against A. flavus and F. solani, respectively (Table 3).Similarly, the n-hexane and chloroform extracts of M. falcata showed activity against all studied fungal strains.Our results are also in line with the study of Juvatkar et al. (2012), who reported that the aqueous, chloroform and n-hexane extracts of Artemisia spp.Baloch et al. (2013) investigated that the antifungal activity of the different extract and different fractions from aerial parts of Medicago possess good antifungal activities against Microsporum, Candida albicans, Aspergillus flavus, Fusarium solani, Aspergillus fumigatus and Candida glaberata.shows significant antibacterial and antifungal activities.More significant antifungal activities of n-hexane and chloroform extract may be due to the combine effect of glycoside, saponins, alkaloid, tannin and flavonoids (Table 2).Furthermore, the phytochemical estimation of crude and its fractions showed the presence of alkaloids, flavonoids, phenols, tannins and diterpenes (Ireland, and Dziedzic, 1986) which contributes action against fungal strains.However, its ethyl acetate and butanol fractions did not show any activity against F. solani and A. fumigatus (Table 3).
Chloroform fraction of T. stans showed activity against all fungal strains, however its n-hexane and butanol fractions did not show activity against F. solani and A. fumigatus.Ethyl acetate fraction has only showed activity against A. flavus.The results are also supported by Farhat et al. (Farhat et al., 2011), who reported that T. stans is more active against F. solani and A. niger.It may be due to the presence of saponins in T. stans, which are naturally occurring surface active glycosides.Many pharmacological activities have been reported about saponins such as antibiotic, antifungal, antiviral (Ireland, and Dziedzic, 1986).Farhat et al. (2011) also confirmed the presence of significant saponins amount in T. stans.

Conclusions
It can be concluded that most fraction of medicinal plant A. indica, M. falcata and T. stans showed potential antimicrobial activities against the tested bacterial and fungal strains.The antimicrobial activities may be due to strong occurrence of active compounds i.e. saponins, tannins, alkaloids, steroids, phenols and flavonoids.Results of our findings confirmed the use of A. Indica, M. falcata and T. stans as traditional medicine.However, these medicinal plant species may be subjected to detailed phytochemical and pharmacological studies in order to find out new drugs against pathogenic bacterial and fungal strains.

Table 1 :
Zone of inhibition (mm) against four bacterial strains.

Table 2 :
Review on the ethnomedicinal uses and phytochemical of studied plant species.Above ground parts are grind to prepare paste with water and applied on wounds, (ii) Dry or fresh leaves are used as tonic, (iii) Plant is taken orally for treating anaemia, diabetes, asthma, ulcers, rheumatism, colitis, haemorrhage, menopausal complaints, pre-menstrual tension, fibroids etc, (iv) A poultice of heated leaves is used in earache

Table 3 :
Activities of different plant extracts against four fungal strains.