sign in sign up
<<
Home , Henna

Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349

ISSN: 2319-7706 Volume 2 Number 5 (2013) pp. 342-349 http://www.ijcmas.com

Original Research Article Antimicrobial activity of extracts against some human pathogens

M. Kannahi*and K.vinotha

P.G and Research Department of Microbiology S.T.E.T Women s college, Sundarakkottai, Mannargudi, Thiruvarur (District), Tamil Nadu, . *Corresponding author e-mail: [email protected]

A B S T R A C T

In this present study, henna were collected and selected for antimicrobial activity against some human pathogens. Among this study, K e y w o r d s henna leaves were extracted with methanol, ethanol and aqueous by solvent extraction method. By this study, the pathogens are isolated from wound Antimicrobial sample. The isolated organisms were identified based on cultural, activity; morphological and biochemical characteristics. Hence, the isolated bacterial inhibition zone; isolates were confirmed asS.aureus, S.mutans, P.aeruginosa and fungal solvent isolates such as A.niger, A.flavus and Fusarium. In this study, antimicrobial extracts; activity was performed by disc diffusion and well diffusion method. The henna maximum activity was showed in methanol extraction against all isolated leaves human pathogens. In this ethanol extraction also shows maximum activity (100%). There was no activity in aqueous extract. This result may open important perspectives alternative antimicrobial therapies.

Introduction

Medicinal plants are potential renewable In the past, medicinal plants were used natural resources and are generally intensively in folkloric medicine for considered to play a beneficial role in treatment of various disorders. Today, it is human health care. Since the dawn of estimated that about 80 % of people in civilization, medicinal plants are known as developing countries rely on traditional nature s best gift to cure a number of medicines for their primary health care. diseases of men and animals. There about Traditional medicines are becoming 121 clinically useful prescription drugs popular, due to high toxicity and adverse that is worldwide derived from higher effects of orthodox medicament. This has plants. About 70 percent of them came to led to sudden increase in the number of the attention of pharmaceutical houses herbal industries in the drug market. because of their use in traditional medicine Several plant species are used by various (Abelson, 1990). indigenous systems such as Siddha,

342 Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349

Ayurveda, Unani and Allopathy for the drugs for the treatment of variety of treatment of different ailments. This human ailments. India has about 18, 000 review emphasizes the traditional uses and species of angiosperms, of with about biological properties of Lawsonia inermis 2,500 species are considered as important (Farnsworth et al., 1985). source of medicinal and aromatic chemical components (Cown, 2009). Lawsonia inermis (Lythraceae) is a perennial plant commonly called as henna, In modern medicine also, plants occupy a having different vernacular names in India very significant place as raw material for viz., in Hindi, Mendika, some important drugs although synthetic Rakigarbha in Sanskrit, Mailanchi in drugs and antibiotics brought about a (Kirtikarkr and Basu, 1956). It Malayalam, revolution into controlling different Muruthani in Tamil, Benjati in Oriya, disease. But these synthetic drugs are out Mayilanchi in kannada and Mehedi in of reach to millions of people. Traditional Bengali [is native to and medicines are used by about 60 percent of South East Asia, and often cultivated as an the world s population. These are not only ornamental plant throughout India, Persia, used for primary health care not just in and along the African coast of the rural areas in developing countries, but (Malekzadeh, 1968). also in developed countries as well as Henna grows better in tropical savannah modern medicinal plants, minerals, and and tropical arid zones, in latitude between organic matter, the herbal drugs are 15° and 25° N and S, produces highest prepared from medicinal plants only. content in between temperature 35 to 45°C. The optimal soil temperature ranges ° The main interest in plants because of their for germination are 25 to 30 C. medicinally and pharmacologically important active ingredients rapidly. Medicinal plant products have been Henna leaves are very popular studied extensively and their role in to color hand, finger, nails and hair. The several useful biological activities, such as dye molecule, Lawson is the chief antibacterial, antifungal, insecticides and constituents of the plant; its highest herbicides have been well documented concentration is detected in the petioles number of plant products with the useful (0.5-1.5%). In folk medicines, henna has bioactive properties is increasing rapidly been used as astringent, antihemorrhagic, as on outcome of several on going intestinal antineoplastic, cardio-inhibitory, research programs on investigation of hypotensive, sedative and also as biological activities of a number of plants. therapeutic against amoebiasis, headache, These bioactive compounds have several jaundice and leprosy (Raja sekaran, 2001). as lead molecules for the development of many synthetic antibiotics. Hence , the present study was undertaken with the India is one of the pioneers in the preparation of different solvents extracts development and well documented of Lawsonia inermis and determination of indigenous systems at medicine, namely antimicrobial activity of its leaf extracts Ayurveda, Siddha and Unani. For by disc diffusion and well diffusion millennia, the India population has method. depended upon mostly plant based crude

343 Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349

Materials and Methods (4hrs) were uniformly spread on solidified nutrient agar medium. The filter paper Sample Collection discs prepared with plant extracts were carefully placed over the spreaded cultures Wound sample ( specimen) was collected by using sterile needle and incubated at with the help of specialist using sterile 370 C for 24hrs. After the incubation swabs. The swabs were immediately period, the plates were examined for immersed into normal saline and inhibitory zones (including the diameter of transported to the laboratory. The the disc) (Gupta et al., 1993). specimen should be collected before any antimicrobial agents were administrated. Well Diffusion Technique (Bauer et al. , 1966) Isolation and identification of pathogens 24 hrs old bacterial cultures were used for The swabs were immediately inoculated well diffusion method. One well of 5 mm on Mecconkey agar, Mannitol salt agar, size will be made in the help of sterile Kings B medium which were incubated at borer under aseptic condition in laminar 37 0 C for 18 24 hrs. air flow chamber. The wells were loaded with different concentration such as 25%, Collection of plant material 50%, 75%, and 100% of the leaf extracts (5g in 100ml of solvent). The plates were The plant materials used for this study incubated at 370C for 24 hours. The plates were collected from the rural area of were observed after 24 hrs for clearing Mahathevappatanam, Thiruvarur District, zone around the well. The zone of Tamil Nadu, South India. inhibition was calculated by measuring the diameter of the inhibition zone around the Plant Extract preparation well. The reading were taken in all the replicates the average values were The shade dried plant materials were tabulated (Baues et al., 1996). pulverized into coarse powder and extracted in soxhelt apparatus using Antifungal Activity methanol, ethanol and aqueous extracts Disc Diffusion method (Perz et al., 1990) were also prepared. The collected methanol, ethanol and aqueous extracts Potato-Dextrose Agar medium was were concentrated under vacuum (500C) prepared and transferred to sterile petri dried and weighed for methanol , ethanol dishes and allowed to solidify. A and aqueous extracts taken at different suspension of identified three fungal spp concentration 25%, 50%, 70% and 100% was added to media and swabs the entire against test pathogens. surface of the agar medium separately. The inoculums were equally distributed in Antibacterial Activity surface of the media by rotating the plate. Sterile 5mm disc in diameter dipped in Disc Diffusion method (Perz et al., 1990) solutions of the various solvent ethanol, Antibacterial activity of the extracts was methanol and aqueous plant extracts. The tested by Disc-diffusion method (Kirby plates were left for 1 hour at room and Bauer, 1966). 0.2 ml bacterial cultures temperature as a period of pre incubation

344

Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349 diffusion to minimize the effects of Standard deviation = variation in time between the applications 2 of the different solutions. Then the plates 0 N were incubated at 24 C for 2-3 days and Where, observed for antimicrobial activity. The diameters of the zone of inhibition was (X-X)2 = The sum of the square of the calculated and compared with the deviation of each value from the mean. standards (Gupta, 1997). N = Number of observation. Well Diffusion Technique (Bauer et al., 1966) Results and Discussion The fungal culture used for well diffusion method. The Sabouraud agar plates were In the previous studies has been found to prepared and the fungal spores were be a best solvent for extraction of the spreaded on the Sabouraud agar plates. active ingredient ( asarone ) from leaves. One well of 5mm, size made in the agar Though, solvents such as methnol, ethanol plates with the help of sterile cork borer and aqueous used the most of the previous under aseptic condition in laminar air flow studies studies were also found to be chamber. The well were loaded with suitable for extracrtion of active 0.2ml of leaf extracts. The plates were ingredients. It is well established that the incubated at 280C for 48hrs. - asarones found in leaf, and rhizomes tissues are responsible for almost The plate was observed after 48 hrs for all of the antimicrobial activities of the clearing zone around the well. The zone Lawsonia inermis (MacGaw et al., 2002). of inhibition was calculated by measuring the diameter of the inhibition zone around The ethanol and water extract of the back the well(Alexpolous and Mims, 1993). of Lawsonia inermis showed antimicrobial activity against gram Statistical Analysis positive and gram negative bacteria Diacyl heptenoid and its Fyrosdia crosyenin were Random sampling was used for the entire identified at the constituent responsible for test. The data of all the parameters were this activity (Saxena et al.,1990). In this statistically analyzed and expressed as work, aqueous extracts of Lawsonia Mean + SD by using the formula given by inermis inhibits the pathogens (Essawi and Srour, 2000). S.aureus, S.mutans and P.aeruginosa potentially at 100% concentration. this extracts control the bacteria and fungal pathogen which cause skin disease. Mean ( X ) = X

The antibacterial activity may be attributed N to not only a single active principle but to Where, a cocktain of a variety of active principles X = Sum of all values of variable or alkaloid (Britto, 2001; Eassawi and Srour, 2000; Bisignano et al., 2000. N = Number of observation Osawa et al., 2000)

345 Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349

Several recent papers reported that the of methanol extracts of Lawsonia inermis presences of antibacterial activity are due against Streptococcus mutans showed to flavonoides (Harborne, 1988). minimum activity (2.3±1.516mm) at 25% Alkaloids are important defence of the followed by 50% (3.6±3.23mm), 75% plats against pathogenic organisms and (5±5.12mm) and 100% 17±16.04mm). herbivores. It also toxin for insects which The activity of methanol extracts of further modify the alkaloids and Lawsonia inermis against pseudomonas incorporate them into their own defense aeruginosa, showed maximum activity secretion. was obtained at 75% (4.6±3.16mm) followed by 100% (3.3±2.16mm), 50% The present study has been under taken to (2.8±2.4mm) and 25% (2.6±2.1mm). The find out the effectiveness of the different ethanol extracts of Lawsonia inermis extracts of Lawsonia inermis leaves gainst leavesagainst Staphylococcus aureus some human bacterial and fungal showed minimum activity (3.1±3.21mm) pathogens. at 25% concentration and maximum activity (8.1±6.2mm) at 100% level. Isolation of organisms

Nutrient agar and PDA plates were The ethanol extracts of Lawsonia inermis prepared. Then the wound samples were leaves Streptococcus mutans showed inoculated in to the agar plates. The minimum activity (2.0±4.31mm) at 25 % bacterial colonies were identified by concentration maximum activity gram s staining and biochemical test (9.1±5.1mm) at 100%. The ethanol where as fungi by lactophenol cotton blue extracts of Lawsonia inermis leaves mounting technique. against pseudomonas aeruginosa showed minimum activity (9.1±8.61mm) an 25% Antimicrobial activity concentration and maximum activity (7.6±6.41mm) at 100%. There was no The antimicrobial potency of Lawsonia activity showed by aqueous extracts of inermis leaf extracts were tested against Lawsonia inermis against Staphylococcus some human pathogenic bacteria and fungi aureus, Streptococcus mutans, and was quantitatively assessed for the Psseudomonas aeruginosa. The present presence or absence of zone of inhibition. investigation has been undertaken to find The results relative to antibacterial activity out the effectiveness of the methanol, was observed by measuring the diameter ethanol and aqueous extracts of Lawsonia of the zone of inhibition. inermis against some human pathogenic microorganism such as S.aureus, Antibacterial activity of Lawsonia S.mutans, P.aerugenosa, A.niger, A.flavus inermis by Disc diffusion method and Fusarium.

The activity of methanol extracts of Antifungal activity of Lawsonia inermis Lawsonia inermis leaves against By Disc diffusion method Staphylococcus aureus showed minimum activity (2.3±2.01mm) at 25 % The methanol extracts of lawsonia inermis concentration and maximum activity leavesshowed maximum activity at 100% (9.3±8.9mm) at 100% level. The activity level (2.5±2.3mm) and minimum activity

346

Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349

Table.1 Antibacterial activity of methanolic extracts of Lawsonia inermis (mm)

Zone of inhibition(mm) S.No Organisms 25% 50% 75% 100% 1 S.aureus 2.3±2.01 2.3±3.0 2.8±2.7 2.5±2.3 2 S.mutans 2.3±2.0 2.4±3.1 5.1±4.0 6.6±4.4 3 P.aeruginosa 2.6±2.1 2.8±2.4 4.6±3.16 3.3±2.16 Value are triplicate and represented as mean ± standard deviation Table.2 Antibacterial activity of ethanolic extracts of Lawsonia inermis (mm) Zone of inhibition(mm) S.No Organisms 25% 50% 75% 100% 1 S.aureus 3.1±3.21 2.3±3.0 2.8±2.7 8.1±6.2 2 S.mutans 2.0±4.31 2.4±3.1 5.1±4.0 9.1±4.1 3 P.aeruginosa 2.6±2.1 2.8±2.4 7.6±6.4 9.1±8.61

Table.3 Antifungal activities of methanolic extracts of Lowsonia inermis (mm)

Zone of inhibition(mm) S.No Organisms 25% 50% 75% 100% 1 A.niger 0.3±1.1 2.3±3.0 2.8±2.7 2.5±2.3 2 A.flavus 8.3±9.1 4.4±3.1 5.1±4.0 2.5±8.1 3 Fusarium 2.6±2.1 2.8±2.4 4.6±3.16 4.7±4.4 Value are triplicate and represented as mean ± standard deviation Table.4 Antifungal activity of ethanolic extracts of Lawsonia inermis (mm) Zone of inhibition(mm) S.No Organisms 25% 50% 75% 100% 1 A.niger 3.5±1.4 2.3±3.0 2.8±2.7 2.03±3.03 2 A.flavus 8.3±9.1 3.04±5.5 5.1±4.0 7.4±6.5 3 Fusarium 2.6±2.1 6.1±9.5 4.6±3.16 9.1±8.9 Value are triplicate and represented as mean ± standard deviation at 25 % (0.3±1.1mm) against A.flavus. The activity at 100% (3.5±1.4mm) and methanol extracts of Lawsonia inermis minimum activity at 25% (2.03±3.03mm) leavesshowed maximum activity at 100% against A.flavus. Ethanol extracts of (2.5.0±8.1mm) minimum activity at 25% Lawsonia inermis showed maximum (8.3±9.1mm) against A.niger. For activity at 100% (7.4±6.5mm) and Fusarium also it showed maximum minimum activity at 50% (3.04±5.5mm) activity of methanol extracts at 100% against A.niger. The ethanol extracts of (4.7±4.4mm), followed by 25% inermis showed maximum activity at (1.3±1.8mm), 50% (2.2±2.1mm) and 75% 100% (9.1±8.9mm) and minimum activity (1.3±1.8mm). The ethanol extracts of 50% (6.1±9.5mm) against Fusarium. Lawsonia inermis showed maximum There was no activity showed by aqueous

347 Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349 extracts of Lawsonia inermis against this study, the pathogens are isolated from Staphylococcus aureus, Streptococcus soil. The isolated organisms were mutans, and Pseudomonas aeruginosa. identified based on cultural, morphological and biochemical characteristics. Hence the Well Diffusion method isolated bacterial colonies were confirmed as, S.aureus, S.mutans, P.aeruginosa and The same results were obtained by well fungal colonies such as A.niger, A.flaves diffusion method for different against all and Fusarium. By this study, antimicrobial organisms. Overall results of this study activity was performed by disc diffusion revealed that maximum activity of the leaf and well diffusion method. The maximum extracts of Lawsonia inermis was observed activity was showed in methanol at 100% level against both bacteria and extraction against all isolated human fungi. So usage of the extracts at a pathogens. In this ethanol extraction, also concentration of 100% might be effective shows maximum activity (100%). There to treat the some human pathogens was no activity in aqueous extract. This (Table.4) Aspergillus is pathogenic to result may open important perspectives humans. It causes Aspergillosis. Out of alternative antimicrobial therapies. In this eight species of Aspergillus, Aspergillus studies are required to study the effect and niger, fumigates account for most toxicity of these compounds in infections. experimental animals (in vivo) and to establish if they could be safety used as The antimicrobial activity potency of antibacterial agent against the pathogens. Lawsonia inermis leaves extracts were Thus we concluded that the medicinal tested against human pathogenic plants could be used as drug to treat the microorganism and the quantitatively infections caused by bacteria and fungi assessed for the presence or absence of without any side effects. zone of inhibition. References The hiumble Lawsonia inermis has value in the treatments of acne. The seeds are Abelson, P.H., 1990. Med.Plants Sci. 247; used, which contain proteins, lecithins, 513. carbohydrates and fats. It is nutritious and Alexpolous, C.J., and Mims, C.W . 1993. anti dermatitis agent. They have been used Introductory mycology wiley Eastern in cases of acne and on faded, wrinkled Ltd New Delhi. pp.555-556. [14]. In the present study Lawsonia Baues ,A.W., W.M. Kirby, H. Truck and inermis leaf extract was studied for their shreeies, J.C. 1996. Anragonistic antiseptic and vasodilator activities and susceptibility. Testing by standardized antimicrobial activity against skin disease well diffusion method . Am.. J. Clin. causing pathogens. Pathol. 45: 495-496. Cown, M.M., 2009. plant products as In this present study, henna leaves were antimicrobial agents. Clin.Microbiol collected and selected for antimicrobial Rev.2(4):564-582 activity against some human pathogens. Essawi, T., and M. Srour .2000. Screening Among this study, henna leaves were of some Palestinian medicinal plants extracted with methanol, ethanol and for antibacterial activity. J. aqueous by solvent extraction method. By Ethnopharmacol. 70: 343-349 (2000).

348 Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 342-349

Farnsworth, N.R., O. Akorele , A.S. Bingel and SoeJar to DD GUO 2.1985. Medicinal plants in therapy Bull.World. Health.Organization. 63(6):965-98%. Gupta, Ali, M and Alam, M.S. 1993. Anapthaquinoe from Lawsonia inermis stem park. Phytochem. 33(3):723-724 Gupta, S.P., 1997. Measures of Dispersion. In statistical methods.pp. 833-834. Kirtikarkr, and Basu, B.Q. 1956. India medicinal plants, vol.3.International book distributors, New Delhi,India. MacGaw, L. J., A.K. Jaguar and Van Staden. J. 2002. Isolation of B- asarones, an antibacterial and anthermintic compound, from Acores . South African J. Bot. 68:31-35. Malekzadeh, F.. 1968. Antimicrobial activity of Lawsonia inermis L. Am.Soc.Microbiol. 16(4):663-634. Raja sekaran, P.E., 2001. Medicinal Plant Sector in India.pp.23-31.

349