Phytochemical analysis and antimicrobial evaluation of Andrographis lineata Nees leaves and stem extracts

The present investigation was designed to evaluate the antibacterial, antifungal properties and physiochemical screening of the various leaves and stem extracts of Andrographis lineata. Acetone, ethanol, methanol, petroleum ether and chloroform extracts of shade dried plant leaf and stem of A. lineata were tested for antibacterial, antifungal and phytochemical analysis. The antibacterial properties of various extracts of leaves and stem of A. lineata were assayed using the standard disc diffusion method, against five strains of bacterial species, namely, Proteus vulgaris, Escherichia coli, Klebsiela pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa. Among different solvent extracts studied, the methanol leaf extract showed highest antibacterial activity against staphylococcus aureus (19.41 mm); followed by petroleum ether extract (18.30 mm). The highest inhibition zone observed for acetone extract of A. lineata leaves against P. vulgaris was 17.40 mm followed by ethanol extract (16.10 mm). The highest activity of chloroform extract against K. pneumoniae was 14.07 mm. Phytochemical investigation confirmed the presence of flavonoids, saponins, gums and mucilages, triterpenoids, steroids, glycosides, phenolic compounds and tannins. The acetone leaf extracts of A. lineata showed highest antifungal activity against Penicillium pinophilum (13.40 mm). The methanol leaf extracts showed maximum activity against Aspergillus niger (12.15 mm). The petroleum ether leaf extracts showed highest activity against P. pinophilum (12.11 mm). The ethanol leaf extracts showed significant activity against Aspergillus flavus (11.24 mm). The chloroform leaf extracts showed higher activity against A. niger (9.75 mm). The leaf extract showed more inhibitory effect than the stem extracts. The present research justifies the claimed uses of this herb in the traditional system of medicine to treat different diseases.


INTRODUCTION
Medicinal plants are eminent natural sources for the treatment of various ailments since primitive times. Infectious ailments are the second leading effect of death worldwide. Treatment of infections continues to be problematic in recent time because of the severe side effects of some drugs and the growing resistance to antimicrobial properties. Hence, investigation for newer, safer and more potent antimicrobial is in pressing need. Herbal medications have received much attention as a source of new antibacterial drugs since they are considered as time-tested and comparatively safe both for human use and the environment. (Faziy-Bazzaz et al., 2005) World Health Organization (WHO) recognized that medicinal plants play an important role in the health care of about 80% of the world population in developing countries and depend largely on traditional medicine (Ikegami et al., 2003). The increase in prevalence of multiple drug resistance has slowed down the progress of new synthetic antimicrobial medicines and has necessitated the investigation for new antimicrobial sources for replacement. Herbs are supposed to be safe but numerous unsafe and fatal side effects have recently been reported (Fabricant and Fansworth, 2001). Hence, there is an critical need to study the evaluation of antimicrobial activity of herbs, which will be useful in the treatment of various diseases caused by microorganisms.
Phytoconstitiuents from medicinal plants showing antimicrobial properties have the potential of filling this demand, because their structures varies from those of the more studied microbial reasons, and therefore their mode of achievement may too very likely differ (Al-Bayati and Al-Mola, 2008). There is growing attention in correlating the phytochemical of a medicinal plant with its pharmacological activity (Chen et al., 2008;Costa et al., 2008;Kumar et al., 2004). Screening active compounds from plants regulate the discovery of modern drugs, which have efficient protection and treatment roles against many diseases including cancer (Sheeja and Kultan, 2007). The useful medicinal effects of plant materials typically effect from the combinations of secondary products present in the plant. In plants, these compounds are mostly secondary metabolites such as flavonoids, tannins, alkaloids, steroids, phenolic compound and resins fatty acids gums which are efficient of producing physiological action on body (Bishnu et al., 2009).
Species of Andrographis Wallich ex Nees (Acanthaceae) are used in the Indian systems of medicines such as Ayurveda, Homeopathy, Naturopathy, Amchi , Modern, Siddha and Unani and exhibit antipyretic properties (Kirtikar and Basu, 1975). This genus consists of 40 species distributed in Tropical Asia. Among them 24 species have been found mainly in the hill areas of Tamilnadu, India (Gamble, 1924), of which 18 species are reported to be endemic in India (Ahmedullah and Nayar, 1986). Andrographis lineata Nees is a medicinal herb (Alagesaboopathi, 1993) found wild in Shevaroy Hills of Salem District , Tamilnadu (11°45'N and 11 55' and 78°11'to 78°20'E) with elevation up to 1600 m. Various medicinal properties such as antipyretic  anti-inflammatory  antivenom (Balu and Alagesaboopathi, 1995) anti-diabetic, jaundice , diabetes, snake bite, skin diseases and also as veterinary medicine have been attributed to this plant in the traditional system of Indian medicine (Ayyanar et al., 2008;Karuppusamy, 2007;Kadhirvel et al., 2010;Sivaperumal et al., 2010;Sangameswaran and Ilango, 2010). It is used as hepatoprotective (Sangameswaran et al., 2008;Sharma Bhawna and Sharma Upendra Kumar, 2009) antibacterial (Perumalsamy and Ignacimuthu, 2000) and diuretic (Sangameswaran et al., 2007). Three flavonoids were isolated from the leaf extract (Hari Kishore et al., 2003), therefore, it is necessary to establish the scientific basis for therapeutic action of this plant. The present research is an attempt to evaluate the phytochemical, antibacterial and antifungal activities of the leaves and stem of A. lineata.

Collection and identification of plant materials
A. lineata leaves and stem were collected in July 2010 from Shevaroy Hills of Salem district of Tamilnadu, India and dried at 31°C for 15 days. The plants specimens were identified and confirmed with the Flora of Tamilnadu and voucher specimen (No.21/10.07.2010 CA) deposited in the department of Botany, Government Arts College (Autonomous), Salem for the future reference.

Preparation of plant extracts
The solvents used were acetone, ethanol, methanol, petroleum ether and chlororform. The powdered leaves and stem (25 g) were taken and extracts were prepared with Soxhlet using 150 ml of solvent. The extract was filtered through membrane filter (0.45 µm size) with the aid of a suction pump. The obtained filtrate was evaporated to dryness at 37°C. The extract was then weighed dissolved in the minimal volume of dimethyl sulphoxide (Silva et al., 1997) and used for phytochemical, antibacterical and antifungal activity.

Microorganisms used
In the experiment, we have used five bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Proteus vulgaris) and five fungi (Penicillium pinophilum, Fusarium solani, Asoergillus flavus, A.niger and Alternaria alternata). All the cultures were procured in pure form from the Biomedical Engineering Research Foundation, Salem, Tamilnadu, India.

Antibacterial assay
The agar well diffusion method was employed for the determination of antimicrobial activity of the extracts. The Petriplates containing 20 ml of Muller Hinton Agar medium were seeded with 24 h culture of the microorganisms. The wells (6 mm in diameter) were cut from the agar and the extract solution (5 mg/ml) were then added into it. The plates were incubated at 37°C for 24 h. The diameter of the inhibition zone were measured on millimeters (mm).Each experiment was performed in triplicates, repeated twice and were tabulated

Antifungal activities
The antifungal activities of A. lineata were proved in a radical growth inhibition properties. A fungal plug was placed in the centre of the Potato Dextrose Agar Plate. Extract (50 mg/ml) was applied into the wells .The Petriplates were incublated in the dark at 23°C. Antifungal activities were observed as a cresent shaped zone of inhibition at the mycelial form. The effect on fungal growth was expressed qualitatively. Ciprofloxacin and fluconazole were used as positive controls for bacteria and fungi, respectively.

Phytochemical screening
Phytochemical investigation was carried out on the acetone, ethanol, methanol, petroleum ether, chloroform extracts and on the powdered specimens using standard procedures to identify the phytoconstituents as described by Sofowara (1993) and Kokate et al. (2003). The leaf and stem extracts was assayed for the presence of tannins, flavonoids, triterpenoids, saponins, steroids and sterols, glycosides, phenolic compounds, gums and mucilages.

RESULTS AND DISCUSSION
The phytochemical screening of A. lineata investigated   phytochemicals suggests that the plant might be important in medical and pharmaceutical industry. Table 2 show qualitative evaluation of the percentage phytoconstituents of A. lineata. A. lineata has the maximum percentage yield of flavonoids (19.60%) in chloroform leaf extract. The content of gums and mucilages were found highest (15.77%) in methanol leaf extract. A. lineata contained the least yield of phenolic compounds (0.15%) but the maximum percentage yield of tannins (7.80%). Triterpenoids obtained from the plant were found to be minimal (0.54 to 0.15%). Saponin recorded high yield of 4.35% and least yield was found in 1.83%. Steroids were obtained in the plant but the yields recorded were 0.75 to 0.29%. The content of glycosides that was found in A. lineata was 0.70 to 0.29%. The results of antibacterial properties of acetone, ethanol, methanol, petroleum ether and chloroform extracts of the leaves and stem of A. lineata are given in Table 3. All the extracts exhibited broad spectrum of activity. When the five extracts were compared with each other and with that of standard antibiotic Ciprofloxacin, the methanol leaf extract showed the highest activity compared to that of the ethanol, acetone, chloroform and petroleum ether extracts. The extract obtained using methanol showed highest activity against S. aureus (19.41 mm), P. aeruginosa (16.45 mm) and K. pneumoniae (16.40 mm) and minimal inhibition zone was observed against P. aeruginosa (10.30 mm). No activity was observed against P. vulgaris and E. coli. The study made on acetone extract recorded highest activity against P. vulgaris (17.40 mm), S. aureus (15.65 mm) and P. aeruginosa (14.63 mm). Further, it showed least activity against K. pneumoniae (10.41 mm) in stem extract.
Ethanol extract showed maximum activity against P. vulgaris (16.10 mm), P. aeruginosa (15.30 mm) and S. aureus (14.36 mm) and the minimal activity was against K. pneumoniae (8.70 mm). Whereas it has no activity against P. aeruginosa in stem extract. The extracts using petroleum ether showed highest inhibition zone observed against S. aureus (18.30 mm), P. aeruginosa (16.50 mm), E.coli (15.10 mm) and P. vulgaris (14.16 mm). Least inhibition zone was observed against K. pneumoniae (6.40 mm) and E. coli (8.40 mm). It has no activity against pathogen like P. aeruginosa in stem extract. The chloroform extract antimicrobial activity results showed diameter of inhibition zones ranging from 14.07 to 9.36 mm, with the highest zone was inhibition shown towards K. pneumoniae (14.07 mm). Minimum inhibition zone was observed against E. coli (9.36 mm). The phytochemical investigation and quantitative estimation of the percentage yields of chemical constituents of the plant studied was that the leaf and stem were rich in flavonoids, gums and mucilages, saponins and tannins. The plant was known to show medicinal activity as well as exhibiting physiological properties (Sofowara, 1993). Tannin has been found to have antibacterial, antiulcer and antiviral activities (Moreman, 1998). Flavonoids on the other hand, are potent water soluble antioxidants and free radical scavengers, which prevent oxidative cell damage and have strong anti-cancer activity (Okwu, 2004;Okwu and Josaiah, 2006). Saponin possesses specific chemical, physical and biological properties that make them useful as medicines. Some of the biological activities include antimicrobial (Margineanu et al., 1976) and hemolytic effects (Abe et al., 1981).
The antibacterial activity of another species of this family (Acanthaceae) has been reported (Santhi et al., 2006;Mishra et al., 2009). Currently the attention has been directed toward extracts and biologically active compounds isolated from familiar plant species. The use of medicinal plants plays an essential role in convening the main health needs in developing countries and these plants may offer a modern source of antibacterial, antifungal and antiviral agents with significant activity against infective microorganisms (Shadomy et al., 1985;Odds, 1989). Oliveira et al. (2007) reported the antimicrobial activity of Syzygium cumini extract from leaves. Kumar and Vaidhyalingam (2010) reported the antibacterial and antifungal activity of Rubus racemosus extract from aerial parts. Alagesaboopathi (2011) reported the phytochemical screening and antimicrobial potential of Andrographis ovata extract from leaves.
The antifungal activity of acetone, ethanol, methanol, petroleum ether and chloroform extracts of leaf and stem of A. lineata were evaluated by measuring the diameters of zones of growth inhibition of the fungal colonies and the results are given in Table 4. Antifungal activity denoted that the tested fungal strains are most susceptible to acetone extract. Antifungal activity results of acetone extract recorded the diameter of inhibition zones ranging from 7.20 to 13.40 mm with the highest inhibition zone observed against P. pinophilum (13.40 mm). Minimum inhibition zone was noticed against F. solani (7.20 mm). Where it has no activity against A. niger in stem extract. The investigation made on ethanol extract showed a highest activity against P. pinophilum (11.48 mm), Alternaria alternata (10.31 mm), A. flavus (10.24 mm) and F.solani (10.18 mm) and the minimal activity against A. alternata (6.03 mm) in stem extract. It has no activity against A. niger in stem extract.
The extract using methanol showed maximum activity against A. niger (12.15 mm), P. pinophilum (10.70 mm) and A. alternata (10.70 mm). Least inhibition zone was showed against A. alternata (5.90 mm) in stem extract. Petroleum ether extract pointed out maximum activity against P. pinophilum (12.11 mm), A. niger (10.70 mm) and A. flavus (9.11 mm) and the minimum activity against F. solani (5.14 mm) in stem extract. No activity was noticed against P. pinophilum in stem extract. Observation made from chloroform extract showed a highest activity against A. flavus (11.38 mm), P. pinophilum (10.36 mm) and A. niger (9.75 mm) and the minimum activity against F. solani (4.70 mm).
Several workers have screened a large number of plants belonging to angiosperms and gymnosperms for their fungitoxic activities. Mainly the aqueous extracts of expressed juice of plants have been used to evaluate their fungitoxicity (Sawant, 1999;Thapliyal et al., 2000;Alagesaboopathi and Balu, 2000). However some of the researchers have used organic extracts.
The use of 50% ethanol ensures extraction of highest compounds as well as facilities further purification of active fractions (Dhar et al., 1973).
The results of the current study revealed that antibacterial efficacies of acetone, ethanol, methanol, petroleum ether and chloroform extracts varied in effectiveness which may be attributed to the presence of saponins and tannin. The presence of phenolic compounds in the plant indicates the antimicrobial properties. In the present research, the author also observed the antibacterial assay, which agrees with the findings of Ofokansi et al. (2005). The phytochemicals are Known to have antimicrobial properties (Gupta et al., 2010). Hence, these plants can be used to discover bioactive natural products that may lead to the development of new pharmaceuticals research activities.