Evaluation of Anti bacterial Activity of Various Extract of Hibiscus syriacus

 

Punasiya R*, Joshi A, Sainkediya K, Tirole S, Joshi P, Das A and Yadav R

GRY Institute of Pharmacy Borawan Khargone (M.P.) 451228

*Corresponding Author E-mail: rakeshpunasiya@yahoo.com

 

ABSTRACT:

Bioassays for antimicrobial activities were carried out using leaves of Hibiscus syriacus plants. Petrolium ether, Dichloro-methane, Isopropyl alcohol extracts from leaves of Hibiscus syriacus was  prepared and tested against gram positive bacteria i.e. Bacillus cereus (MTCC 430) , Staphylococcus aureus(MTCC 3160 )gram negative bacteria Escherichia coli(MTCC433) and Klebsilla pneumoniae(MTCC432). Both the Petrolium ether and Isopropyl alcohol extracts showed considerable activity against all the test organisms. The Dichloro-methane extracts of Hibiscus syriacus which showed no activity against all the bacteria. The Minimum Inhibitory Concentration (MIC) of the plant extracts ranged from 0.01 mg/ml to 100 mg/ml. The antibacterial activities of both the Petroleum ether and Isopropyl alcohol plant extracts were comparable to those of selected chemical antibiotics suggesting their potential as alternatives to the antibiotics in the treatment of infections caused by these microorganisms.

 

KEYWORDS: Bioassays, Petrolium ether, Isopropyl alcohol extract, Antimicrobial activity, Agar diffusion, Minimum inhibition Concentration (MIC)

 

 


INTRODUCTION:

We know that everything in this world is changing day by day or time to time. Since many years ago Ayurveda or herbal drugs were very popular but last few decades it was replaced by allopathic system of medicine which was frequently accepted by world. But latter due to their undesirable or toxic effect can reflect interest of human being towards Ayurveda or herbal drugs. Human being has been using herbs (plants) for different purpose like food, medicine, beautifying with advancement of science and technology. Different parts of plant like leaves, flowers, stem, bark, roots, and seed are used in cosmetics. These have been used in both forms such as fresh form and dried form.1, 2

 

Medicinal plants are gifts of nature to cure limitless number of diseases among human beings.3 The abundance of plants on the earth’s surfaces has led to an increasing interest in the investigation of different extracts obtained from traditional medicinal plants as potential sources of new antimicrobial agents. Researches have shown that all different extracts of the plants leaves which possess antimicrobial property.4 The genus Hibiscus is widely distributed over Korea, China, India, and Siberia. The dried roots of Hibiscus syriacus are used as a folk medicine in the orient5.

 

Hibiscus is the common name and genus name for a group of about 250 species of shrubs, trees, and herbaceous plants in the Malvaceae family, characterized by alternate leaves and generally large and showy, often bell-shaped flowers with stamens united into a tubular structure surrounding the style. Also known by the common name of rosemallow, this genus includes such well-known members as rose of Sharon (Hibiscus syriacus) and China rose (Hibiscus rosa-sinensis), being the most commonly known species for medicinal purposes.6H. syriacus, is highly resistant to pests and diseases and is frost-tolerant.7 It is also being used for the production of fibre, medicinal product and beverage additives in some Asian countries. 8

 

 

Hibiscus syriacus is a widely cultivated ornamental shrub in the genus Hibiscus. Common names include Rose of Sharon Shrub Althea, and Rose of Althea. It is a flowering shrub in the plant family Malvaceae native to much of Asia. It is vase-shaped, reaching 2-4 m in height. It is widely planted in areas with hot summers for its very attractive white, pink, red, lavender, or purple edible flowers. Individual flowers are short lived, lasting only a day. However, numerous flower buds are produced on the shrub's new growth, which provide prolific flowering over a long summer blooming period (July-September).Up to now, research has focused mainly on the propagation of other ornamental Hibiscus species such as H. syriacus and H. rosasinensis. 10

 

Presently more attention is being paid to CNS active, cytoprotective, immunomodulators and chemotherapeutic plant products. Nutraceutics have opened up an entirely new field for exploration and in the near future, dietary modulations of disease immerge as an alternative mode of therapy. At the same time a decreasing trend has been noticed towards evaluation of plants for their effect on the autonomic nervous system and fertility control.11

 

MATERIALS AND METHODS:

Selection of Plant:

Drug discovery from medicinal plants has evolved to includes numerous fields of inquiry and various methods of analysis. The process typically begins with a botanist, ethanopharmacologist who identifies the plant of interest.Collection may involve species with known biological activity. On the basis of intensive literature survey Hibiscus Syriacus. was selected for present study.

 

Collection And Authentication of Plant:

The plant Hibiscus Syriacus was collected from Campus of Jawaharlal Institute of Technology and GRY Institute of pharmacy Borawan Khargone district, West Nimar region of Madhya Pradesh India in the month of Agust 2010.

The taxonomic identities of these plants were confirmed by Dr. S.K. Mahajan formerly professor of Govt. P.G. College Khargone (M.P.) 12

 

Preparation of Whole Plant Powder:

The plant of Hibiscus Syriacus was dried under shade and then powdered with a mechanical grinder. The powder was passed through sieve No. 20 and stored in an airtight container for further use.

 

Extraction of Plant Material:

Extraction is the term used pharmaceutically, involves the separation of medicinally active portion of plant or animal tissues from the inactive or inert components by using selective solvents in standard extraction procedure. These include decoction, infusion, percolation, maceration, Digestion.

 

Soxhlet Extraction (Continuous hot percolation):

Soxhlet extraction is a very old, clean up method, but it is still relatively, widely used in plant analysis. This method is convenient and widely used for extraction because of its continuous process, less time and solvent consumption than maceration and percolation. The main advantages of the soxhlet technique are that is an automatic and continuous method that does not require much manipulation. One disadvantage is that the extractives are heated during extraction at the boiling point of the solvent employed and thermally labile compounds may hydrolyze, decompose or produce artifacts.

 

The 85g powder of Hibiscus Syriacus. was subjected to defated to petroleum ether & soxhlet (Continuous hot percolation) extraction with Petroleum ether, Dichloro methane and isopropyl alcohal. The solvent was removed by evapouratig under low pressure and evaporation.The different extract thus obtain was subjected to further studies.13

 

Preliminary Phytochemical Screening14,15,16

The different extract of leaves of plant Hibiscus Syriacus was subjected for qualitative examination of Phytoconstituents and tests were performed:

 

Test for Glycosides:

Legal Test:  Dissolve the extract in pyridine and add sodium nitroprusside solution to make it alkaline. The formation of pink red to red color shows the presence of glycosides.

 

Baljet Test:  To 1ml of the test extract, add 1ml of sodium picrate solution and the yellow to orange color reveals the presence of glycosides.

 

Test for Steroids and triterpenoids:

Libermann-Burchard Test:  1gm of the test substance was dissolved in a few drops of chloroform, 3ml of acetic anhydride, 3ml of glacial acetic acid were added, warmed and cooled under the tap and drops of concentrated sulphuric acid were added along the sides of the test tube. Appearance of bluish-green color shows the presence of sterols.

 

Salkowski Test: 1 gm of test substance was treated with few drop of concentrated H2SO4, red colour at lower layer indicate the presence of steroid and yellow colour at lower layer indicate triterpenoids

 

Test for Flavonoid:

Alkaline reagent Test: Take 1 ml of extract and added few drops of NaoH solution, yellow colour is obtained. Then added few drops of dil. Acids, white colour indicate the presence of Flavonoids.

 

Test for Amino Acids:

Ninhydrins Test:  Take 1 ml of extract and added few drops of ninhydrins reagent, purple to violet colour indicate the presence of Amino acids.

 

Alkaloids, carbohydrates, tannins, phenolics compounds and saponin tests were performed but not given any positive results.

 

Antimicrobial assay:

The antimicrobial activity of three different extracts of plant Hibiscus syriacus was tested against four pathogenic bacteria, viz, gram positive, Bacilus Cereus (MTCC 430) and Staphylococcus aureus (MTCC 3160) and gram negative Escherichia Coli (MTCC 433), and Klebsiella pneumoniae (MTCC 432). The agar diffusion method of followed for the antimicrobial assay. Inoculums was prepared from the 24 hours old culture of bacterial isolates in nutrient broth. Nutrient agar plates were prepared and the inocula were seeded by spread plate method.

 

The extracts were applied to sterile nutrient agar plates. All the samples were done in duplicate. Both positive and negative controls were determined, for negative control the three solvents were also used to determine their effect on test organisms. Antibiotics were also used to compare the effectiveness of the plants extracts with that of the ciprofloxacin antibiotics. Nutrient agar plates incubated for next 24 hour at 37oC. After that the inhibition zone surrounding the wall by the diffusion of compounds was measured in mm diameter. The minimum inhibitory concentration (MIC) of the extracts against bacteria was also determined. This was done by soaking the paper disc in different concentration of the plant extracts using the same method of agar diffusion. Zones of inhibition in mm were also measured.17, 18

 

RESULT AND DISCUSSION:

Preliminary phytochemical screening

Preliminary phytochemical screening revealed the presence of various phytoconstituents which were identified by various testing methods (Table-1).

 

Table No. 1: Preliminary Phytochemical Screening

S. N.

Phytoconstituents

Petrolium ether

Dichloro-methane

Isopropyl alcohol

1.

Alkaloids

-

-

-

2.

Glycosides

-

-

+

3.

Flavonoids

+

+

+

4.

Tannins

-

-

-

5.

Carbohydrates

-

-

-

6.

Amino acids

+

+

+

7.

Steroids/ Triterpenoids

+

+

+

8.

Saonins

-

-

-

(+) indicate presence;  (–)indicate absence

 

Antibacterial Activity

Table No. 2:  Antibacterial activity of Petroleum ether, Dichloromethane and isopropyl alcohol extract of   Hibiscus Syriacus.

Sr. No.

 

Bacterial strain

Inhibition Zone (mm) of Ciprofloxacin

Inhibition Zone (mm) of

Petroleum Ether

Inhibition Zone(mm) of Dichloromethane

Inhibition Zone (mm) of isopropyl alcohol

1

Bacillus cereus (MTCC 430)

39

10

00

28

2

Staphylococcus aureus (MTCC 3160 )

32

10

00

25

3

Escherichia coli(MTCC433)

30

8

00

24

4

Klebsilla pneumonia (MTCC432)

24

9

00

22

 

All extract of plant Hibiscus syriacus  shown antibacterial activity (Table-2) from which petroleum ether isopropyl alcohol extract shown maximum Antibacterial activity but dichloro methane extract of plant Hibiscus syriacus was not given any Antibacterial activity against two gram positive and two gram negative bacterial strains. Petroleum ether, and isopropyl alcohol extract of plant Hibiscus syriacus shown Antibacterial activity against two gram positive and two gram negative bacterial strains. Isopropyl alcohol gives maximum antibacterial activity compare to other two extract.

 

 

Zone of inhibition of petroleum ether extract:

 

Bacillus cereus (MTCC 430)

Staphylococcus aureus (MTCC 3160)

 

Escherichia coli (MTCC433)

 

Klebsilla pneumoniae(MTCC432)

Zone of inhibition of Isopropyl alcohol

 

Bacillus cereus (MTCC 430)

 

Staphylococcus aureus (MTCC 3160)

 

Escherichia coli (MTCC433)

 

Klebsilla pneumoniae(MTCC432)

 

CONCLUSION:

The whole plant of Hibiscus syriacus, belonging to the family Malvaceae was taken up for the study to screen and give a report on the possible pharmacognostical, phytochemical studies. The exhaustive extraction of the plant material was done with petroleum Ether, Dichloromethane and Isopropyl alcohol the extract was screened for the presence of medicinally active Phytoconstituents.

 

The Test extract (Hibiscus syriacus) shows potent Antibacterial Activity. Thus these studies provide a scientific support to the selected medicinal plant which claims its use in folklore medicine. The antibacterial activity of different extracts of Hibiscus syriacus was compared to standard ciprofloxacin antibiotics. The Isopropyl alcohol and Petroleum ether extract of Hibiscus syriacus was shown the better and potent antibacterial activity respectively. The isopropyl alcohol extract shows greater inhibition of bacterial growth then other two extracts.

 

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4.        Bonjar, Glls and Farrokhi, P.R., 2004. Antibacillus activity of some plants used in traditional medicine of Iran.Niger. J. Nat. Prod. Med., 8: 34-39. Braude, A.I., 1982. Microbiology. W. B. Sauders Company, London.

5.        Sung Won Kwon, Soon Sun Hong, Jong In Kim, and Il Ho Ahn, 2003. Antioxidant Properties of Heat-treated Hibiscus syriacus. Biology Bulletin, Vol. 30, No. 1, pp. 15–16.

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7.        Yang L.J., Hidaka M. , and Masaka H. Agrobacterium-Mediated Transformation Of Hibiscus Syriacus Ans Regeneration of Transgenic Plants 1996 Plant Tissue Letters, 13 (2) 161-167.

8.        Yang L.J., Hidaka M., Masaka H., and Uozumi T. In Vitro Regeneration from leaf and petiole explant of Plants Hibiscus Syriacus L 1995 Plant Tissue Letters, 12 (2) 173-177.

9.        Govinden-S., Boodia N., Dussooa C., R. Gunowa R., S. Deensah S., S. Facknath S. and B. Rajkomar B. 2009. Vegetative Propagation and Tissue Culture Regeneration of Hibiscus sabdariffa L. (Roselle). World Journal of Agricultural Sciences 5 (5): 651-661.

10.     Plants for a Future: Hibiscus syriacus. Bailey, L. H. (2005). Manual of Gardening (Second Edition). Project Gutenberg Literary Archive Foundation.

11.     Pereira JV, Pereira MSV, Sampaio FC, Sampaio MCC, Alves PM, Araújo CRF, Higino JS. Efeito antibacteriano e antiaderente in vitro do extrato da Punica granatum Linn. sobre microrganismos do biofi lme dental. Rev Bras Farmacogn 16: (2006) pp: 88-93.

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13.     Nandkarni_K.M._“Indian_materia_medica”1954, Vol_I. Bombay_Popular Prakashan Mumbai pp 373.

14.     Kokate C.K. “Practical Pharmacognosy” 1997, Fourth edition. By Vallabh  prakashan, Delhi,. pp 107-111.

15.     Raman. N., 2006. Phytochemical techniques, Published by: new India publishing agency New Delhi. pp. 25.

16.     Khandelwal K.R., Practical Pharmacognosy Techniques and Experiments, Published By Nirali Prakashan, pp 1497. Mehta R.M. Text Book of Dispensing Pharmacy,.2nd ed. 2005. By Vallabh Prakashan pp247.

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Received on 09.02.2011          Modified on 10.03.2011

Accepted on 20.03.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 4(5): May 2011; Page 819-822