Research Journal of Recent Sciences ______ ________________________ ______ ______ ____ ___ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 41 - 46 (201 3 ) Res. J. Recent . Sci. International Science Congress Association 41 In Vitro Antioxidant and Antimicrobial Activity of Methanolic root Extracts of Hyptis suaveolens Javed Ahmadą, Iffat khan˛, Ashfaq Ahmad˛ and kaushar Imamą ąDepartment of biochemical Engineering and Food Technology, Harcourt butler technological Institut e, Kanpur, INDIA ˛Department of Bioscience, Integral University Lucknow, INDIA Available online at: www.isca.in Received 29 th November 2012, revised 27 th December 2012, accepted 24 th January 201 3 Abstract The plant Hyp tis is a potent medicinal herb and a well known medicinal plant in herbal world. Crude methanolic extract of Hyptis suaveolens were screened for their in vitro antimicrobial activity against pathogenic microorganisms; S.epidermidis; K.pneumoniae B.subtilis ;E.aerogens; B.cereus.In - vitro antioxidant and antimicrobial activity determined using 2, 2 - diphenyl - 1 - picrylhydrazyl (DPPH) and agar well diffusion method respectively. In addition,exract of Hyptis suaveolens prepared by soxlet apparatus and were partiall y purified by preparatory thin layer chromatography (TLC). Results indicated a potent antioxidant and antimicrobial activity of methanolic root extract of Hyptis suaveolens. Keywords: Hyptis suaveolens , methanolic extract , antioxidant , antimicrobial , DPPH , agar well diffusion assay . Introduction The plant, Hyptis suaveolens commonly known as “Wilayati tulsi” belongs to the family Lamiaceae and is an ethnobotanically important medicinal plant. Almost all parts of this plant are being used in traditional medicine to treat various diseases 1 . It is approximately 2 meters high, having branches and long, white piliferous stems. Its flowers are purple or white, its leaves oval, wrinkled and pointed. An antioxidant is a molecule that slows or prevents the oxida tion of the molecules. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. As a result,antioxidants are often considered as reducing agents such as thiols, ascorbic acid , polyphenols 2 . Antioxidants are widely used as ingredients in the dietary supplements in order to maintain health and to prevent diseases such as cancer and coronary heart disease 3 . These compounds may be synthesized in the body or obtained f rom the diet 4 . Many aromatic, medicinal and spice plants contain compounds that possess confirmed strong antioxidative components. One of the documented health promoting activities of many fruits and vegetables is their ability to scavenge naturally produc ed free radicals and hence acting as antioxidants 5 . The leaves of H. suaveolens have been utilized as a stimulant, carminative, galactogogue and as a cure for parasitic cutaneous diseases. Crude leaf extract is also used as a relief to colic and stomach a che 6,7 . Hyptis suaveolens has been reported to as tonics, emmenagogues, diaphoretics, antispasmodics, burns and wounds, antimicrobial, antibacterial, antispasmodic, analgesic, anti - inflammatory, headaches, anticatarrhal, anticutaneous, Insecticidal Effect , Acaricidal Effect, 12 Larvicidal Effect, Toxicity Concerns Hepatotoxicty, Chronic Toxicity Study 1,6 - 11. The efficacy of Hyptis suaveolens oil as a preservative of the cereals, pulses, nuts and spices against fungal spoilage Hyptis suaveolens (L.) and has been widely used as a stimulant, carminative, lactagogue and to treat colic disorders 1,6 . Figure - 1 Plant of Hyptis suaveolens A wide range of chemical compounds including terpenoids, alkaloids, acidic polysaccharide and 33 constituents were identified in the oil of Hyptis suaveolens isolated from its leaves. Extracts and metabolites from this plant have been found to possess pharmacological and insecticidal activities 1 . Hyptis suaveolens was targeted on the basis of folkloric uses which suggest its tox icity to microbes, coupled with its importance as Research Journal of Recent Sciences ______ _ _ _________________________ ______ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 41 - 46 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 42 food to humans 11 . Soluble solvent extracts of the plant were tested for phytochemicals which revealed the existence of alkaloids, flavonols, flavones, flavonones, terpenoids, tannins, aldehydes and ketones and the absence of steroids, saponins and anthraquinones 7 . Wound Healing Activity of Hyptis suaveolens is a traditional pubescent annual herb found throughout India. On the basis of traditional use and literature references, this plant was selected for the screening of wound healing property 11 . Toxic secondary metabolites from plants were extracted, tested and proved to affect insect nerve functions and behaviors. A considerable number of studies have emphasized the research and development of herbal subst ances for controlling mosquitoes 12,13,14 . These botanical extracts could also be used along with other insecticides under integrated vector control.Studies carried out so far have shown that some photochemicals acts as general toxicant (insecticide/ Larvic ide) both against adult as well as larval stage of mosquito while others interfere with reproduction 14 . In this communication bioactivity of essential oil extracted from Hyptis suaveolens was tested against Aspergillus flavus Link , Aspergillus niger 15 . An timicrobial Study of the volatile oil distilled from the overground parts of H. suaveolens showed activity against bacteria and fungi 6 . Material and Methods Chemicals: 2, 2 - diphenyl - 1 - picrylhydrazyl ( DPPH), dimethyl sulfoxide (DMSO), Methanol (MeOH), Thi n layer chromatography (TLC) plates, silica gel. All other chemicals were of AR grade. Test microorganism which were used in this experiment are : Klebsiella pneumoniae , B.cereus, E.aerogens, B.substilus, S.epidermidus, at different concentration (20 ,30, 40,50 µg/ml) concentration was used as a standard. Sample Preparation:Plant Material : Plant material Hyptis suaveolens was collected in flowering stage from local area of Lucknow near Integral University. The shade dried plant material was crushed into fi ne powder. Preparation of Methanolic Extract of Hyptis suaveolens : Around 15 gm fresh shade dried plant material was powdered and wrapped in muslin cloth. It was extracted by Soxhlet apparatus with 150 ml of methanol . The percolation process was continue d until the extraction process was completed (indicated by transparent colour). The extract was allowed to cool and then poured in to a petri plate, left for drying. The dried extract was scratched and was collected in eppendorf tube and weighed, used for further phytochemical screening. Figure shows the soxhlet apparatus In vitro antimicrobial assay: Agar well diffusion method: Cell suspensions to a final concentration of 10 to 10 cfu/ml were prepared from different subcultures. 0.1 ml of inoculums of t est plant extract was spread on Muller Hinton agar plates. 4 millimetres – diameter wells were punched into MHA. A 30 µl aliquot of each suspension was dispensed into the wells. The plates were incubated for 18hr at 37° C. Antimicrobial activity was evaluat ed by measuring the zone of inhibition against the test plant extract. Each experiment was carried out in triplicates. Determination of Minimum Inhibitory Concentration (MIC): MIC may be defined as the lowest concentration of the extract that prevent visi ble bacterial growth. Broth micro dilution assay was performed for determination of MIC , bacterial strain were cultured at 37° C and suspended in nutrient broth to obtain final inoculums density of 10 cfu/ml. The 0.5 ml of this suspension was added to 0.5 ml of susceptibility test broth containing serial two fold dilution of plant extract. All the t est tubes were incubated at 37° C for 20 hours. Antioxidant assay: Antioxidant assays: Each sample was dissolved in 95% methanol at a concentration 1µg/ml and th en diluted to prepare the series concentrations for antioxidant assays. Reference chemicals were used for comparison in all assays. DPPH radical scavenging activity assay . The DPPH assay was done according to the method of Brand - Williams, Cuvelier, and Ber set with some modifications. The stock solution was prepared by dissolving 24 mg DPPH with 100 ml methanol and then stored at 20 0 C until needed. The working solution was obtained by diluting DPPH solution with methanol to obtain an absorbance of about 0.98 0 (±0.02) at 517 nm using the spectrophotometer. A 3 ml aliquot of this solution was mixed with 100 ll of the fractions at varying concentrations (25 – 250 µg/ml). The solution in the test tubes were shaken well and incubated in the dark for 15 min at room temperature. Then the absorbance was taken at 517 nm. The scavenging activity was estimated based on the percentage of DPPH radical scavenged using the following equation: Scavenging effect (OD of control absorbance - OD of sample Research Journal of Recent Sciences ______ _ _ _________________________ ______ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 41 - 46 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 43 absorbance)/OD of control a bsorbance×100: EC50 value is the effective concentration that could scavenge 50% of the DPPH radicals. Ascorbic acid was used as standard. TLC Study of Flavonoids: 1 gm dried mass of plant was extracted with 10 ml methanol on rotary shaker for 24hrs. The flavonoid spots were separated using chloroform and methanol (4:1) solvent mixture. The colours of these spots were recorded under Ultraviolet light (254nm). Results and Discussion Crude methanolic extract of Hyptis suaveolens were screened for their in v itro antioxidant and antimicrobial properties. DPPH method was used to determine total antioxidant activity. Antimicrobial activity was determined by using agar well diffusion assay. Our results indicated a potent antioxidant and antimicrobial activity of methanolic root extract of Hyptis suaveolens . As shown in Table1 a significant decrease was observed in absorbance with increasing concentration, which showed better antioxidant power. In addition, our data also showed ( table 2 and fig ure 1) an increase in percent inhibition of free radicals by increasing concentration of plant extracts. The DPPH antioxidant assay was based on the ability of DPPH a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for visible deep purple color. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the changes in absorbance. T he an tioxidant capacity observed was not solely from the phenolic contents, but could possibly be due to the presence of some other phytochemicals such as ascorbic acid, tocopherol and pigments as well as the synergistic effects among them, which also contribut e to the total antioxidant capacity. Table - 1 Show the absorbance of Hyptis suaveolens methanolic root extract at various sample concentration Sample concentration(µg/ml) Ascorbic acid(OD) HsMe(root)(OD) 25 µg/ml 1.279±.001 1.234±.002 50 µg/ml 1.212±.0 05 1.219±.012 100 µg/ml 1.048±.008 1.208±.005 150 µg/ml 0.884±.009 1.169±0.004 250 µg/ml 0.571±.015 1.134±0.001 Table - 2 Percent Inhibition of DPPH mediated free radical generation by Hyptis suaveolens methanolic root ex tract at various concentrations Sample concentration(µg/ml) Ascorbic Acid (%scavenging inhibition) HsME(root) (% scavenging inhibition) 25 µg/ml 5.1 0.1615 50 µg/ml 10.3 1.373 100 µg/ml 22.3 2.323 150 µg/ml 34.4 5.411 250 µg/ml 57.4 8.481 Figure - 1 Graph representing Percentage inhibition of Hyptis (root) against Ascorbic acid Research Journal of Recent Sciences ______ _ _ _________________________ ______ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 41 - 46 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 44 Figure - 2(a) The TLC plate under ultraviolet light Figure - 2(b) Rf values of bands Emergence of multi - drug resistance in human and animal pathogenic bacteria as well as undes irable side effects of certain antibiotics has triggered immense interest in the search for new antimicrobial drugs from the plant source. In the present study methanolic extracts of plant have been tested against resistant bacteria. The antimicrobial acti vity of the extracts and their potency was quantitatively assessed by the presence or absence of inhibition zone and zone diameter. The results of antimicrobial activity of Hyptis suaveolens was encouraging and that the plant extract showed significant ant imicrobial activity against different bacterial strains. In the figure 3 - 6, the antimicrobial activity of methanolic extract of Hyptis suaveolens (root) against five bacterial strain B.cereus, E.aerogens, K.pneumoniae, B.substilus, S.epidermidus at differe nt concentration (20,30,40,50 µg/ml) was found in the following decreasing order B.cereus� S.epidermidus� E.aerogens� K.pneumoniae� B.subtilis Figure - 3 Zone of inhibition of Hyptis methanolic root extract against S.epidermidus Figure - 4 Zone of inhib ition of Hyptis methanolic root extract against K.pneumonia Research Journal of Recent Sciences ______ _ _ _________________________ ______ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 41 - 46 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 45 Figure - 5 Zone of inhibition of Hyptis methanolic root extract against B.cereus Figure - 6 Zone of inhibition of Hyptis methanolic root extract against B.subtilis Determination of antibacte rial activity by agar well diffusion assay showed that methanolic extract of Hyptis root exhibited the antibacterial effect against pathogenic as well as non - pathogenic test bacteria. Significant effect on growth inhibition of gram positive and gram negati ve bacteria was also noticed. It was noted that among all tested organisms: the gram positive bacterial strain S.epidermidus and B.cereus registered maximum susceptibility to the methanolic extract at 50 µg/ml of the entire Hyptis root used, with the maxim um inhibitory zone of 12 mm and 17 mm respectively . These differences may be attributed to the fact that while the cell wall in Gram - positive bacteria consist of a single layer that of Gram - negative is a multi - layered and quite complex structure. The resul ts provided evidence that the studied plant might indeed be potential sources of natural antioxidant and antimicrobial agents. Based on these results, it is possible to conclude that methanolic extracts of Hyptis suaveolens had different levels of antioxid ant and antimicrobial activity. The obtained results might be considered sufficient to further studies for the isolation and identification of the active principles and to evaluate of possible synergism among extract components for their antioxidant and an timicrobial activity. Conclusion Crude methanolic extract of Hyptis suaveolens were screened for their in vitro antioxidant and antimicrobial properties. DPPH method was used to determine total antioxidant activity. Antimicrobial activity was determined b y using agar well diffusion assay. Results indicated a potent antioxidant and antimicrobial activity of methanolic root extract of Hyptis suaveolens.Based on these results, it is possible to conclude that methanolic extracts of Hyptis suaveolens possesses potent antioxidant and antimicrobial activity. The obtained results might be considered sufficient to further studies for the isolation and identification of the active compounds, to evaluate possible synergism among extract components for their antioxidan t and antimicrobial activity. References 1. Asprey G.F., Medlcinal Plants of Jamaica, West Indian Medical Journal , 2(4) 2. Nayak Praveen S., Nayak Shweta, Shety Ranjan and Das P., Hyptis suaveolens Poit: A Review on Its Phytochemical and Pharmacological Profile , research journal of pharmacognosy and phytochemistry review, 2(1), (2010 ) 3. Sies. H., Oxidative stress: Oxidants andantioxidants, Exp - Physiol , 82(2), 291 - 295 ( 1997 ) 4. Vertuani S., Angust A. and Manfredini S., The antioxidants and proantioxidants network: an overview, CurrPharm Des , 10(4), 1677 - 1694 (2004 ) 5. Bauer, The Complete Idiot`s Guide to Total Nutrition , Alpha Books , 76 - 78 (2002) 6. Mandal S.M., Mondal K.C., Dey S. and Patil B.R., Antimicrobial activity of the leaf extracts of Hyptis suaveolens (L.) poit, In dian Journal of Pharmaceutical Sciences, 69(4) , 568 - 569 (2007) 7. Vermab Amita, Mukerjeea Alok, Vijayakumarc M. and Mishraa Shanti Bhushan, Anti - hyperglycemic activity of leaves extract of Hyptis suaveolens L. Poit in streptozotocin induced diabetic rats, Asi an Pacific Journal of Tropical Medicine , 4(9) , 689 – 693( 2011) 8. Sharma N., Verma U.K. and Tripathi Abhishek, Bioactivity Of Essential Oil From Hyptis Suaveolens Against Storage Mycoflora, Proc. Int. Conf . Israel, 99 - 116 ( 2007) 9. Barbara Conti, Giovanni Benelli, Guido Flamini, Pier Luigi Cioni, Raffaele Profeti, Lucia Ceccarini, Mario Research Journal of Recent Sciences ______ _ _ _________________________ ______ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 41 - 46 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 46 Macchia, Angelo Canale, Larvicidal and repellent activity of Hyptis suaveolens (Lamiaceae) essential oil against the mosquito Aedes albopictus Skuse (Diptera: Culicidae), Parasitol Res. , 110, 2013 – 2021,DOI 10.1007/s00436 - 011 - 2730 - 8 (2012) 10. Renisheya Joy Jeba Malar T., Sushna S.L., Johnson M., Janakiraman N. and Renola Joy Jeba Ethal T., Bio - Efficacy Of The Leaves Extracts Of Hyptis Suaveolens(L.) Poit Against The Fish Pathogens, Life Science Microbiology , 2(1), (2012) 11. Shenoy Chitra, Patil M.B. and Kumar Ravi, Wound Healing Activity of Hyptis suaveolens (L.) Poit (Lamiaceae), International Journal of Pharm Tech Research , 1(3) , 737 - 744 (2009) 12. Bielakovic G., Nikolova D., Gluud L.L. and Si monetti. R.G., Gluud C., Mortality in randomised trials of antioxidant supplements for primary and secondary prevention: systematic review and meta analysis, JAMA , 297(8) , 842 - 857 (2007) 13. Fabricant D.S. and Farnsworth N.R., The value of plants used in trad itional medicine for drug discovery, Environ. Health Perspect ., 109(1) , 69 – 75 (2001) 14. Okigbo R.N., Okeke J.J. and Madu N.C., Larvicidal effects of Azadirachta indica, Ocimum gratissimum and Hyptis suaveolens against mosquito larvae, Journal of Agricultural Technology, 6(4), 703 - 719 (2010) 15. Parsons W. and Cuthbertson E., Weeds of Natural Ecosystems: a field guide to environmental weeds of the Northern Territory., 490 – 492 ( 1992)