Screening Antibacterial Activity of Various Extracts of Lawsonia inermis.

P. Arun^1*, K.G. Purushotham¹, Johnsy Jayarani¹, Vasantha Kumari¹ and D. Chamundeeswari²

¹Dr.M.G.R.Educational and Research Institute, Dr. M. G. R University, Maduravoyal. Chennai-600 095, Tamil Nadu, India.

²Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India.

ABSTRACT:

Lawsonia inermis known as Henna is a woody and flowering plant found in North Africa and South west Asia. Its leaves extensively in the treatmernt of urinary tract infection in Siddha system of medicine. Lawsonia inermis was subjected to antibacterial analysis. A battery of assays were performed on different extracts of Lawsonia inermis (Henna) for antibacterial activities. Antibacterial effects of n-hexane, choloroform and methanol extracts of the leaves extract of Lawsonia inermis exhibited various degree of inhibition activity. It was observed that leaves extract were promising against gram positive and gram negative bacteria viz. Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Proteus mirablis. This study showed that Lawsonia inermis could inhibit certain bacteria.

KEYWORDS: Anti-bacterial activity, Lawsonia inermis, Henna and Medicinal plants.

INTRODUCTION:

Medicinal plants are the most exclusive source of life saving drugs for the majority of the world’s population. Bioactive compounds currently extracted from plants are used as food additives, dyes, insecticides, cosmetics, perfumes and fine chemicals. These compounds belong to a group collectively known as secondary metabolites¹. A monotypic genus, represented by Lawsonia inermis, native of North Africa and South- west Asia, widely cultivated as an ornamental and dye plant. Lawsonia inermis, syn. Lawsonia alba (Henna) is a flowering plant the sole species in the genus in the family Lythraceae. A glabrous, much branched shrub or small tree with grayish brown bark. Leaves opposite, Sub–sessile, elliptic or broadly lanceolate, entire, acute or obtuse, often mucronulate, Flowers numerous, small, white or rose – colored, fragrant, in large terminal pyramidal panicle cymes; capsule globose, about the size of a pea, with numerous, pyramidal, smooth seeds².Henna has been found to exhibit Antibacterial, Antifungal and Dermatological properties. It is useful in coloring of skin, scalp and nails etc. Henna has also shown anti-diarrhoel, diuretic, emmanagogue and abortifacient prophetically and is found to be practically non-toxic³. Alcoholic extract of shade dried leaves of Lawsonia inermis intraperitonially injected to rats showed anti-inflammatory activity comparable to that of hydrocortisone⁴.Chloroform extract of leaves exhibit promising antibacterial activity against Shigella and Vibrio cholera⁵, Astringent properties⁶.

No antifungal activity was detected for henna aqueous solutions⁷.Most of the medicinal plants have identified and used for treatment of human diseases are well documented⁸.

Collection and Identification:

Lawsonia inermis (Henna) leaves were collected from Botanical garden, madras university campus, Maduravoyal, Chennai. The specimen thus obtained was identified and authenticated by- Dr. K. Balakrishnan, Research Officer, Central Research Institute for Ayurveda and Siddha (Central Council for Ayurveda and Siddha), Arumbakkam, Chennai. The voucher specimen has been also deposited in Herbarium of Department of Botany, University of Chennai.

Extraction and Isolation:

Lawsonia inermis leaves were taken in different aspirator bottles and successively extracted with solvents in the order n-hexane, chloroform and methanol after each extraction, the solvents were evaporated under reduced pressure using high vacuum conditions. These extracts were used for antibacterial assays.

Antibacterial Activity:

Sample preparation:

All the leaves extracts (viz., n-hexane, chloroform, and methanol) prepared in Dimethyl sulfoxide (Dmso) (5 mg/ ml) and aliquots were used to test the antibacterial activity. The antibacterial activity had been tested against five different species of gram positive and gram negative bacteria. Bacterial test cultures were maintained on the stock culture Brain heart infusion agar (BHA). From the stock culture a loop full of the fresh culture was inoculated in Brain heart infusion broth. The seed broths were incubated at 37°C ±1oC for 24 hours. Inoculate were prepared by diluting 24 hours old cultures in saline. A dilution of 1: 100 was used in all the tests.

MATERIALS AND METHODS:

Materials:

• Mueller Hinton Agar (Himedia)

• Brain heart infusion agar (Himedia)

• Culture plates

• Sterile cork borer

• Chloramphenicol (Himedia)

• Autoclave

• Incubators

• Wire loop

• Bacterial Test Cultures

Antibacterial Assay:

Antibacterial activity was performed by well diffusion technique⁹. Petri plates (10x10 cm) were prepared with Mueller Hinton agar. 0.1 ml of the diluted culture was spread evenly over plate with loop or sterile glass spreader. The plates were dried for 30 minutes at 37°C. Wells of 6 mm (approximate) diameter were cut with sterile cork borer in the inoculated agar. The wells were filled with the plant extract. Chloramphenicol (1 mg/ml) was used as control in the other well. The plates were incubated for 72 hours at 37°C. At the end of incubation period, the clear zone of inhibition around the wells was measured in millimeter (mm). The results are expressed in Table 1. Following the antibacterial assay, all the extracts were tested for their activity against the following bacterial culture.

Test microorganisms:

The bacteria used for antibacterial study were Staphylococcus aureus (MTCC 087), Escherichia coli (MTCC 729), Klebsiella pneumoniae (MTCC 432), Pseudomonas aeruginosa (MTCC 1688) and Proteus mirablis (MTCC 425).

Control:

Chloramphenicol (1mg/ml) – (MERCK)

RESULTS AND DISCUSSION:

Plants have been a source of medicinal compounds since pre-historic time. It is well established that all parts of plants were used in Unani systems of medicine for centuries. However, the discovery and use of synthetic drugs led to a dramatic decline in the popularity of herbal products used in the therapy. Nevertheless the realization of harmful toxic effect of a large number of synthetic drug led to for alternative sources which would be safe and effective in various ailments.

Antibacterial Activity of Leaves Extract:

The screening results of various leaves extract of Lawsonia inermis (Table 1) exhibited activity against the gram positive and gram negative organisms, whereas n-hexane and chloroform extracts were found active against Staphylococcus auereus, Klebsiella pneumoniae, Proteus mirablis and Pseudomonas aeruginosa (zone of inhibition range 16mm-24mm) and inactive against Escherichia coli.

Table-1: Evaluation of Antibacterial Activity of Lawsonia inermis

CRUDE EXTRACTS	Zone of inhibition(mm)
	Antibacterial Activity
	Staphylococcus aureus	Klebsiella pneumonia	E.coli	Proteus mirablis	Pseudomonas aeruginosa
n-Hexane (5mg/ml)	14	22	0	18	21
Chloroform (5mg/ml)	18	16	0	20	18
Methanol (5mg/ml)	22	24	21	22	20
Chloramphenicol (1mg/ml)	24	28	26	28	24

Methanol extracts have shown maximum activity against Staphylococcus auereus, Klebsiella pneumoniae, Proteus mirablis, Escherichia coli and Pseudomonos aeruginosa (zone inhibition 21mm-24mm). Consequently it can be suggested that the activity of Methanol extract is much higher as compared to the n-Hexane and Choloroform extract of Lawsonia inermis. As such the different chemical constituents elaborated by different spp. of Lawsonia inermis displayed are coumarin, Alkaloids, Flavonoids, Quinones and Tannins and the antibacterial activity of Lawsonia inermis may emencipate due to either of these constituents. In conclusion, the summary of the biological activity evaluation of the leaves extracts of Lawsonia inermis can be delineated as follows: The antibacterial activity of leaves extract were promising against Staphylococcus aureus, Klebsiella pneumoniae, E.coli, Proteus mirablis and Pseudomonas aeruginosa. Crude methanol extract has maximum activity against both gram positive and gram negative bacteria.

ACKNOWLEDGEMENTS:

We are grateful to our Prof. Dr. R. Vasantha Kumari for her constant support, encouragement and guidance throughout the study.

REFERENCES:

1. Ahmad H, Ghulam RB and Latif A (2004). Medicinal flora of the Thar Desert- an overview of problems and their feasible solutions. Zonas Áridas., 8: 1-11.

2. Wealth of India, (1992) CSIR Publication 47-49

3. Lemordant D, Foresteier(1983) Traditional medicinal uses and pharmacological properties of Lawsonia inermis L.Lythraceae J.Agric. Bot., 30(1): 69-89.

4. Singh S, Shrivastava NM, Modi NT, Safi AQ (1982) Anti-inflammatory activity of Lawsonia inermis. Current Science, 51(9): 470-471.

5. Ahmed I, Aquil F and Shafiullah R.A.K (2003) Antibacterial and antifungal properties of crude alcoholic extract and fractions of Lawsonia inermis, proceedings of first national interactive meet on medicinal and aromatic plants, IMAP, Lucknow, UP, India, 364-369

6. Kirtikar and Basu, II, (1947) J. Sci. industry 1078-1079.

7. Millet-Clerk J, Michel D, Levy F, Chaumont J.P (1989) Invitro antifungal activity of henna and of lawsone vis-a-v. Pityrosporum ovale. Plantes medicinales et Phytotherapie, 23(3): 162-168.

8. Iqbal Ahmed, Zafer Mehmood and Faiz Mohammad (1998). Screening of Indian medicinal for their antimicrobial properties. J. Ethnopharmacol, 62: 183-193

9. Saeed S and Tariq P (2006). Effects of some seasonal vegetables and fruits on the growth of bacteria. Pakistan Journal of Biological Sciences, 9(8): 1547-1551.

Received on 07.03.2010

Accepted on 12.04.2010

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 2(3): May-June 2010, 185-187