MINIMUM INHIBITORY CONCENTRATION AND ANTIOXIDENT PROPERTIES OF ANDROGRAPHIS PANICULATA USING DIFFERENT SOLVENT EXTRACTS

Transcription

1 MINIMUM INHIBITORY CONCENTRATION AND ANTIOXIDENT PROPERTIES OF ANDROGRAPHIS PANICULATA USING DIFFERENT SOLVENT EXTRACTS S.Savitha 1*, C.Rathnavijaya 2 1. Faculty, Department of microbiology, 2.Department of biochemistry, Sri Sankara Arts and Science college, Asur, Kumbakonam *E. mail: gmail.com Abstract: This work mainly focus on the potential antibacterial value of various extracts of Andrographis paniculata against various pathogenic microorganisms (E.coli, Klebsilla and Pseudomonas) by employing minimal inhibitory concentration. In siddha system of medicine the herb Nilavembu (A. paniculata) used to treat various ailments including infection diseases even viral origin too according to its phamacodynamics. Minimal inhibitory concentration values were compared with control the result revealed that the methanol extract is potent in inhibiting bacterial growth of gram negative organism. Medicinal plants are a source for a wide variety of natural antioxidants. Free radical scavenging potential of various extract (methanol, ethanol, and aqueous) of the Andrographis paniculata, a medicinal plant was evaluated by using reducing power. The reducing power method ascorbic acid was used as a standard for determining reducing power. Significant results were observed in the methanol extract has estimated parameters. The methanol extracts of the plant exhibited appreciable activity as compared to the aqueous and ethanol extract. There by indicating that the Andrographis paniculata has promising free radical scavenging activity. Keywords: MIC, Antioxidant, A. paniculata, Free radical, 1. INTRODUCTION World is endowed with a rich wealth of medicinal plants. Man cannot survive on this earth for long life without the plant kingdom because the plant products and their active constituents played an important role (perumal samy et al. 1998). In response to the increased popularity and great demand for medicinal plants, a number of conservation groups are recommending that wild medicinal plants be brought in to cultivation. A large number of medicinal plants and their purified constituents have shown beneficial therapeutic potentials various herbs and spices have been reported to exhibit antioxidants activity and antibacterial activity including Woodfordia fructicosa, Catharanthus roseus, Lippie alba, Andrographis paniculata and etc. the majority of the MIC and antioxidant activity in due to the flavones, isoflavones, flavonoids, anthicyanin, catechins and isocatechins (Aquil.F, 2006). Braunter and Grein in 1994, natural plant products may offer a new source of antibacterial agents. In recent years antimicrobial properties of Indian medicinal plants have been increasingly reported the traditional treatment approach is of much significance. Especially in India due to the endemic presence of infective gastro intestinal diseases which are the major causes of infants and adult mortality (Miranda et al, 1993) Andrographis paniculata Andrographis paniculata is a herbaceous plant in the kingdom plantae, family acanthaceae, genus Andrographis native to India and srilanka. It is widely cultivated in southern asia. It grows erect to a height of cm in moist shady places with glabrous leaves and white flowers with rose-purple spots on the petals. Stem dark green, m in hight,2-6 mm in diameter.it does best in a sunny location. The seeds are sown during May-June. The seedlings are transplanted at a distance of 60 cm x 30 cm. village and ethnic communities in India have been using this herb for treating a verity of ailments Medicinal use of A.paniculata Mostly the leaves and roots were for medicinal purposes such as traditional siddha and ayurvedic systems of medicine in India and some other countries for multiple clinical applications.the plant extract exhibits antityphiod, antifungal activity, antioxidants, antihepatic, antithrombogenic, anti- 21

2 inflammatory, anti snake venom, antipyretic properties and also it is used as an immunostimulent agent. A recent study conducted at bastyr university, confirm the anti-hiv activity of an andrographolide. The andrographolide means the chief constituent extracted from the leaves of the plant.andrographis paniculata as it is called in tamil nilavembu is used in siddha medicine to treat fever like chikenguinea, swine flu,thyphiod etc. List of vernacular names of A.paniculata Language Common name Assamese Chiorta Marathi Oli-kiryata Bengali Kalmegh Oriya Bhuinimba English Green chirayta, creat, king of bitters, andrographis, India echinacea Gujarati Kariyatu Sanskrit Kalmegha, Bhunimba Hindi Kirayat Tamil Nilavembu Kannada Nelaberu Nelavemaa or Nelavepu ("Nela" means ground and Telugu "Vemaa" means neem. It indicates the bitter taste of neem much as does the Malaysian name which means "bile of the earth") Malayalam Nilavembu, Kiriyattu 1.3. Antioxidant Antioxidant may be defind as radical scavengers which protect the human body against free radical that may cause pathological condition. The supply of oxygen is absolutely essential for the existence of higher organisms. As the saying goes too much of even the best is bad. Very high concentrations of O2 are found to be toxic and can damage tissues. The present day concepts of toxicity are due to the involvement of oxygen free radicals or reactive oxygen species. Molecule or molecular species that contain one or more unpaired electron and is capable of independent existence are caused free radicals. Free radicals are highly reactive and are capable of damaging almost all types of biomolecules. Free radicals are caused many disease such as cardiovascular disease, diabetes, cataract, male infertility, aging process, Parkinson s disease, multiple sclerosis, liver cirrhosis, muscular dystrophy, toxemia of pregnancy etc., The antioxidant activities of extracts were determined by different invitro methods such as DPPH free radical scavenging assay, Reducing power method, assay of antioxidant enzymes, super oxide radical scavenging activity nitric oxide radical scavenging assay, ABTS radical scavenging assay (P.K. Jain et al,2008) 1.4. Minimum inhibitory concentration Minimal inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. MIC values can be determined by a number of standard test procedures. The most commonly employed methods are the tube dilution method and agar dilution method. 22

3 Test products that are not clear or precipitate the growth which is about same as tube dilution method except dilution are plated on agar. Serial dilutions are made of the products in bacterial growth media. The test organisms are then added to the dilutions of the products, incubated and scored for growth. This procedure is a standard assay for antimicrobial activity. MIC is important in diagnostic laboratories to confirm resistance of microorganism to antimicrobial agents. An MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent an organism. Clinically, the MIC are used not only to determine the amount of antibiotic that the patient win receive but the type of antibiotic used, which in true lowers the opportunity for microbial resistance to specific antimicrobial agents(mohammed Ibrahim). The present study has focused on the determination of MIC and antioxidant properties of the plant Andrographis paniculata of acanthaceae family. 2. MATERIALS AND METHODOLOGY 2.1. Plant material The Andrographis paniculata were collected from kumbakonam. The plant materials were washed with water, cut into pieces; sun dried for 5 days and then dried in an oven below 60º C. the dried plant materials were then pulverized into coarse powder in a grinding machine. (Md.Rajib Ahsan 2009) 2.2. Preparation of crude extraction The crude extract was obtained by using andrographis paniculata powder 10 g was dissolved in 100 ml of ethanol and methanol and kept on a rotary shaker for 24 h. the extract was filtered, then centrifuged at 5000 rpm for 15 min and was dried under reduced pressure. The yield obtained for crude methanol extract was 20.93% with respect to the initial dry materials. The crude extract was stored at 4ºC in airtight bottles.(chamnan Patarapanich etal, 2007) 2.3. Bacterial Strains The test organisms were supplied by the swasti lab, kumbakonam Tamil Nadu, India. Three intestinal organisms (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa) were used in this study. The organisms were sub-cultured on Mueller Hinton Agar medium, incubated at 37 C for 24 h and stored at 4 C in the refrigerator to maintain stock culture Minimal inhibitory concentration: For determination of minimum inhibitory concentration (MIC) and minimum microbicidial concentration (MMC), the broth twofold macro dilution method in Muller-Hinton broth was applied. Briefly, test strains were grown in a nutrition medium containing progressively lower dilutions of the test extract and incubated at 37 C. The last two tubes were free of test extract and served as growth control in broth and respective solvent. After incubation, approximately the 10 μl of content of each test-tube was transferred with a loop onto Muller Hinton agar. Agar plates were incubated for an appropriate time under aerobic conditions at 37 C. MIC was defined as the lowest concentration of extract that allows no more than 20% bacterial growth and MMC as the lowest extract concentration from which the microorganisms did not recover and grow when transferred to fresh medium.table-2, 3&4. 23

4 THE MICROORGANISMS ARE IDENTIFIED BY FOLLOWING TEST Name the test E.Coli Klebsiella Pseudomonas Gram stain negative negative negative Morphology Rods Rod Rod Motility Motile Motile Motile Biochemical test Indole Positive negative negative Methyl red Negative Positive Positive Vogus prouskuar Positive Negative Negative Citrate utilization Negative Positive positive A. paniculata A.paniculata Powder Anti oxidant property method Minimal inhibitory concentration method 24

5 2.5. Determination of Antioxidant activity Determination of Reducing power The reducing power of the extracts can be determined. Different concentration of methanol ethanol and water extract of Andrographis paniculata was prepared ranging from 20 to 100µg/ml Standard Solution preparation An ascorbic acid stock solution was prepared from which various concentrations like 20, 40, 60, 80, and 100 µg/ml were prepared Procedure 1ml of each extract was taken in different concentration (20, 40, 60, 80 and 100 mg/ml) mixed with 2.5 ml of phosphate buffer (0.2M, ph 6.6) and 2.5 ml of potassium ferricyanide. The mixture was incubated at 50 C for 20 minutes. 2.0 ml of TCA was added to the mixture and centrifuged at 3000 rpm for 10 minutes. 2.5 ml of upper layer of solution was taken and mixed with 2.5 ml of distilled water and 0.5 ml of ferric chloride i.e. in the ratio of 1:1:2 and the absorbance were measured at 700 nm in ultra violet visible spectrophotometer. Ascorbic acid was used as a standard solution. Phosphate buffer used as blank solution. Increased absorbance signified the increase in reducing power (Caia-2004; Chen-2007). Table-1 4. RESULT AND DISCUSSION In this present study revealed that the three crude extracts of Andrographis paniculata have the antibacterial and antioxidant properties. The antibacterial properties were carried out by using the assay of minimal inhibitory concentration (MIC). And the antioxidant properties are performed by using assay of reducing power. The reducing power of the aqueous, ethanol, methanol crud extract of the Andrographis paniculata. The reducing power of the methanolic extract of Andrographis paniculata was also determined and it was found significant but less as compared to the standard selected ascorbic acid. At 100 µg/ml concentration of extract highest reducing power was observed i.e as compare to of Standard ascorbic acid (Table-1). The next high reducing power was observed in aqueous extract of the Andrographis paniculata i.e But the ethanol extract have very low reducing power when compare to others. The reducing ability of a compound generally depends on the presence of reductants which have been exhibited anti oxidative potential by breaking the free radical chain, donating a hydrogen atom. The presence of reductants (i.e. antioxidants) in Andrographis paniculata extracts causes the reduction of the Fe 3+ ferricyanide complex to the ferrous form. Therefore, the Fe 2+ can be monitored by measuring the formation of Perl s Prussian blue at 700 nm. Extract of Andrographis paniculata possesses antioxidant and anti-inflammatory properties. It inhibits formation of oxygen derived free radicals such as superoxide, hydroxyl radicals, lipid peroxidation and nitric oxide (Sheeta, K.) Koshy Philip(2010) Decolouration due to reaction of antioxidants in samples with the stable free DPPH radical was measured spectrophotometerically. the free radical scavenging potential of A. paniculata fruit extract (88.13%) is significantly higher than both A. paniculata leaf extract (86.87%) and A. paniculata stem extract (80.48%). All samples had significantly lower DPPH free radical scavenging activity compared to the positive control (94.86%). 25

6 Table- 1 ASSAY OF REDUCING POWER T.NO concentration Std. Anrographis paniculata (extract) in µg/ml Ascorbic acid Absolute Methanol Ethanol T T T T T T.NO Table-2 MIC of the ethanol extracts against three pathogenic bacteria MH broth medium added (ml) Ethanol extract µg/ml Inoculums added (µl) Bacterial growth observed against E.coli Klebsiella Pseudomonas T T T T T All determinations were done in triplicates. + Growth; - no growth Table-3 MIC of the Methanol extracts against three pathogenic bacteria T.NO MH broth medium added (ml) Methanol extract µg/ml Inoculums added (µl) Bacterial growth observed against E.coli Klebsiella Pseudomanas T T T T T All determinations were done in triplicates. + Growth; - no growth T.NO Table-4 MIC of the Aqueous extracts against three pathogenic bacteria MH broth medium added (ml) aqueous extract µg/ml Inoculums added (µl) Bacterial growth observed against E.coli Klebsiella Pseudomanas T T T T T All determinations were done in triplicates. + Growth; - no growth 26

7 FIGURE: 1 ASSAY OF REDUCING POWER ascorbic acid aquesous methanol ethanol Roy Chaudhuri B, in 1984 conducted the microbiologycal profile of andrographis paniculata, in his study the plant act as high microbicidal agent. The results are Total bacterial count:not more than CFU/gm,Yeasts and moulds:not more than 100 CFU/gm, E.coli: Absent, S.aureus: Absent, Salmonella typhi: Absent, Pseudomonas aeruginose: Absent. Antimicrobial agents have been used in clinical practice for over 40 years ( Zhanel et al., 1991). In the present study, in vitro antibacterial activity of A. paniculata against Gram positive and Gram negative bacteria was measured by disk diffusion method. In the present work, methanol extract of A. paniculata has diaplayed maximum antibacterial activity against Klebsiella pneumoniae than chloroform and aqueous extract. Out of the three soxhlet extract, methanol extract of the A paniculata showed highest antibacterial activity against Enterobacter sp. A lowantimicrobial activity was observed in the extract of chloroform and aqueous extracts. Antibacterial studies revealed that the aqueous extract of the A. paniculata exhibit anti- salmonella profile. The results indicated that the aqueous extract could be used for the treatment of typhoid infections caused by Gram negative Salmonella typhi. The chloroform and methanol and aqueous extracts showed antibacterial sentivity against Staphylococcus aureus in impetigo (Martin and Ernst, 2003). Screening of extracts from several other medicinal plants shown activity against Staphylococcus aureus. It was inferred from the results that the A.paniculata could be used for treating skin infectious conditions produced by Gram positive strain Staphylococcus aureus. Some of the bacteria which is used in this study are the common microbes caused various infective ailments. Klebsiella pneumoniae is a commonest pathogen for respiratory infectious, uninary tract infections and wound infection (Schneiders et al., 2003). In the present work chloroform extract of A. paniculata displayed antibacterial activity against Klebsiella pneumoniae. Out of the three soxhlet extracts, methanol extract of A. paniculata showed maximum antibacterial sensitivity than chloroform and aqueous extracts. Antibacterial sensitivity studies revealed that aqueous extract of A.paniculata exhibited an interesting anti-salmonella profile. 27

8 REFERENCES [1] Aquil F, Ahmed I,Mehmood Z. Antioxidants and free radical scavenging properties of twel trasitionally used Indin medicinal plants.turk J Biol 30:177-83,2006. [2] Koshy Philip (February 2010): Journal of Medicinal Plants Research Vol. 4(3), pp , [3] Martin, K.W. and Ernst, E. Herbal Medicine for treatement of bacterial infections a review of controlled clinical traits. J.Antimicrobial chemotherapy : [4] Md.rajib Ahsan,Km Monirul Islam, and et al.,(2009),world journal of agricultural science 5(5): [5] Miranda,L., Peria sathyavathi,m., sirasal Antarkar,D.S. and vaidya,a.b.(1993).a In vitro action of selected medicinal plants against micro organismsinvolved in human gasro intestinal infections. J.Res.Ayurveda sidha. 8: [6] P.K.Jain and R.K Agarwl. Asian J.Exp.Sci.,Vol.22,No.3,2008; Antioxidant and free radical scavenging properties of developed Mono and Polyherbal formulation. [7] Perumalsomy,R., Ignacimuthu,S. and sem,a.(1998). Screening of 34 indian medicinal plants for antibacterial properties.j.ethanopharmaco.62: [8] Roy Chaudhuri B. and M.K.Podar: Methods Find.Exp.Clin. Pharmaacol.,6:9,(1984) [9] Schneiders, T.. Amyes, G.B. and Levy. Role of AcrR and Rama in fluproguinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore. Antimicrobial agents and chemotherapy (a): [10] Sheeta, K and et.al., Antioxidant and anti-inflammatory activities of the plant Andrographis paniculata Nees, Immunopharmacol Immunotoxicol., 28 (1): ) [11] Zhanel, G.G., Karlowsky, J.A., Bohan, D.J. and Davidson, R. Antimicrobial acitivity of sub inhibitory concentrations of Aminoglucosides against Pseudomonas aeruginosa as determined by the post antibiotic effect. Antimicrobial agents and chemotherapy : [12] Chamnan patarapanich,suwanna laungcholatan and Nuntakan Mahaverawat, HPLC determination of active diterpene lactones from Andrographis paniculata Nees planted in various seasons and regions in Thailand. Thai J. Pharm. Sci. 31 (2007)