In Vitro Antibacterial Activity of Leaf Extracts of Hibiscus Syriacus (L)

 

Rakesh Punasiya1* and Sujit Pillai2

1Department of Pharmaceutical Biotechnology, GRY Institute of Pharmacy, Borawan, Khargone-451228, India

2Department of Pharmaceutical Chemistry, GRY Institute of Pharmacy, Borawan, Khargone, MP

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

 

ABSTRACT:

The antimicrobial activity of different crude extract like petroleum ether, benzene, chloroform, methanol, and aqueous of Hibiscus syriacus L. plants used in traditional Indian medicine was tested by well diffusion method against four bacterial pathogens: Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432), and Bacillus subtilis (MTCC 121) bacteria. The minimum inhibitory concentration (MIC) was determined for evaluating the potential leaves extract. The antibacterial activity of methanol extracts (0.5 g/ml) of Hibiscus syriacus L. showed maximum zone of inhibition (19.1 mm, 19.6 mm, 22.9 mm and 25.4 mm, respectively) against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432), and Bacillus subtilis (MTCC 121) bacteria. MIC was tested at various concentrations from 0.625 mg/ml to 100 mg/ml for all the leaves extracts. The Klebsiella spp was the most resistant strain and various concentrations of all the leaves extract benzene extract showed less activity against the tested microorganism. The present screening result demonstrated that the methanol leaves extract of plants Hibiscus syriacus L. has potent antibacterial activity and the studied plants may be new source for novel antibacterial.

 

KEYWORDS: Antibacterial activity, Well diffusion method, Zone of inhibition, Pathogens

 

 


INTRODUCTION:

In the recent times, focus on the research has all over the world and a large body of evidence has been collected to show the immense potential of medicinal plants used in traditional system. Various medicinal plants have been studied using modern scientific approaches, and the results have revealed the potential of medicinal plants. [1] According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs. Therefore, such plants should be investigated to better understand their properties, safety and efficacy. [2]

 

Now a day, there have been increases in antibiotic resistant strains of clinically important pathogens, which have led to the emergence of new bacterial strains that are multi-resistant. The non-availability and high cost of new generation antibiotics with limited effective span have resulted in increase in morbidity and mortality. Therefore, there is a need to look for substances from other sources with proven antimicrobial activity. [3] Plant compounds are of interest as a source of safer or more effective substitutes than synthetically produced antimicrobial agents. Phytochemical progress has been aided enormously by the development of rapid and accurate methods of screening plants for particular chemicals. [4] The treatment of infectious diseases is facing a major problem at present with many microbes developing resistance to widely used antibiotics and antiviral therapies. [5] One way to prevent antibiotic resistance of pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents. [6] Within the last few decades, there has been the emergence of about 30 threatening infectious diseases with the majority capable of affecting humans. [7]

The genus Hibiscus is widely distributed over Korea, china, India, and Siberia. The Hibiscus syriacus L. (Malveceae) are used as a fork medicine in the orient [8] for the cure of hematochezia, dysentery, obstruction due to wind phlegm, and vomiting of food. [9] Hibiscus syriacus L. (Rose-of-Sharon) is valued for large flowers produced in summer when few other shrubs bloom. It is useful as a garden accent due to its strict, upright habit. The open, loose branches and light green leaves make Rose-of-Sharon ideally suited to formal or informal plantings, and with a little pruning makes an attractive, small specimen tree. The plant grows in sun or partial shade and in any soil. Rose-of-Sharon grows 8 to 10 feet tall and spreads 4 to 10 feet. [10] In the present study, the antimicrobial activity of petroleum ether, benzene, chloroform, and methanol and water extract of Hibiscus syriacus L. leaves was investigated. Antifungal agents have been used for treatment of fungal infections. The root bark of Hibiscus syriacus (Malvaceae), which is widely distributed over East Asia, has been used as an antifungal agent for treatment of athlete’s foot. [11] 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. [12]

 

Hibiscus rosasinensis Linn. is commonly known as China rose belongs to family Malvaceae. The flowers are considered emollient and demulcent, soacked in coconut water are given to induce labor. Decoction of flower is given in bronchial catarrh. Leaves are emollient, aperient, anodyne and laxative. Roots are demulcent and used for coughs. Decoction of roots is used for venereal diseases and as antipyretic. [13] Hibiscus anthocyanins, groups of phenolic natural pigments present in the dried flower of Hibiscus sabdariffa and Hibiscus rosasinensis have been found to have cardioprotective, hypocholesterolemic, antioxidative and heaptoprotective effects in animals. [14] The antibacterial activity in vitro as well as the wound-healing activity of the ethanol extracts of H. rosa-sinensis flowers in vivo. Flowers contain anthocyanins, which may be responsible for its antioxidant effects. Bacterial infection play important role in many of the pathological conditions where extracts of H. rosa-sinensis extracts have been used as traditional medication. [15]

 

MATERIALS AND METHODS:

Selection of plant

Drug discovery from medicinal plants has evolved to include 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 were selecting for present study.

 

Collection of plant materials

The leaves of Hibiscus syriacus L. was collected from the month of august-September from the Garden of Jawaherlal Institute of Technology and G. R. Y. Institute of Pharmacy vidya vihar Borawan district Khargone western nimar region of Madhya Pradesh, India.

 

Authentication of plant 

The plant Hibiscus syriacus L. was identified and authenticated by Dr S.K. Mahajan, (Retd) Botanist from Government  College, Khargone Madhaya Pradesh. The herbarium of the plant specimens were prepared and deposited in the Department of Pharmacognosy, G. R. Y. Institute of Pharmacy vidya vihar Borawan district Khargone madhaya Pradesh, India, under voucher no. G.R.Y.I.P. 43.

 

Preparation of extract

Extraction of organic component

The leaves were initially separated from the main plants body and rinsed with distilled water and shade dried and then homogenized into fine powder and stored in air tight bottles. It was then passed through the 40 mesh sieve Dried and powered plant defatted firstly to remove fatty material for this purpose 1000 gram of weighed powered leaves of Hibiscus syriacus L. was packed in Soxhlet and extracted with petroleum ether at 60-80°c for 36 hours. The marc was removed and dried then it was subjected to continuous hot extraction with organic solvent like benzene, chloroform, and methanol respectively extracts in soxhlet for 36 hours according to their polarity index. After complete extraction the solvent was evaporated and concentrated to dry residue. The petroleum ether, benzene, chloroform, and methanolic extract of  Hibiscus syriacus L. leaves yielded  greenish brown and deep blue semi solid residue  then it were filtered with the help of muslin cloth. The supernatant was collected and the solvent was evaporated by distillation and the extract was concentrate under reduce pressure.

 

Extraction of aqueous component

Dried leaf material was extracted with 2000 ml of water by boiling the mixture. The sample was then filtered through Whatman No. 1 filter paper and concentrated to dryness under reduced pressure. Crude extracts were then stored in the dark at 10oC until use. The percentage yield for each extract was determined. [16]

 

Preliminary phytochemical screening

The methods described by Harborne (1978) with slight modifications were used to test for the presence of the active ingredients in the test sample. The plant materials were screened for the presence of different classes of secondary metabolites including alkaloids, flavonoids, phenols, saponins, tannins, anthocyanins, anthraquinones, sterols, and triterpenes using previously described   methods. [17]

 

Tested microorganisms

Two Gram-positive bacteria (Bacillus cereus (MTCC-430), Bacillus subtilis (MTCC 121), two Gram-Negative organisms (Staphylococcus epidermis (MTCC-435), Klebsiella pneumoniae (MTCC-432) were included. The microorganisms were kindly provided by the Microbial type culture collection Chandigard India. The microorganisms were cultivated on nutrient agar medium and stored at 4oC.

 

Antimicrobial activity by agar well-diffusion method

500 mg of each extract of Hibiscus syriacus L was dissolved in 1ml of each solvent. Suspension of micro-organisms was made in sterile normal saline and adjusted. Culture media was prepared using nutrient agar and were autoclaved at 121o, 15 psi for 15 minutes. A volume of 20 ml agar was transfer on Petridis and allowed to solidify. Each labelled medium plate was uniformly inoculated with a test organism by using a sterile cotton swab rolled in the suspension to streak the plate surface in a form that lawn growth can be observed. Each Petridis were divided into four sectors and in each sector 4 mm bore was made using sterile borer. 0.1ml of the various extract concentration were dropped into each, appropriate labeled well. Other solvents used for extraction apart from water were tested neat for each organism. The inoculated plates were kept in the refrigerator for 1 hour to allow the extracts to diffuse into the agar. [18] The Nutrient Agar plates were incubated at 37OC for 24 hours. Antimicrobial activity was determined by measuring the diameter of zones of inhibition (mm) produced after incubation. O.O5% of chloramphenicol was used as control. [19]

 

Minimum inhibitory concentration

Determination of MIC and MBC

The minimum inhibitory concentration (MIC) of the crude extracts was also determined by comparing the various concentrations of plant extracts, which have different inhibitory effect, and selecting the lowest concentration of extract showing inhibition. After incubating at 24 h at 37°C, the MIC of each sample was determined by measuring the optical density in the spectrophotometer (620 nm), and comparing the result with those of the non inoculated nutrient agar. All the samples were prepared in       triplicates. [20]

 

Data Analysis

Each experiment of the antimicrobial activity was assessed with six serial dilutions for each compound and then replicated three times. Results were expressed as mean ±SE and statistically analyzed using an analysis of variance (ANOVA). Differences are considered significant when P‹0.05.

 

RESULT:

Preliminary phytochemical screening

Our preliminary phytochemical screening of H.syriacus L. leaves (table 1) revealed the presence of carbohydrate, glycosides, steroids, triterpenes, flavonoids, alkaloid, tannins, and saponins.    

 


 

 

Table 1 Phytochemical screening of leaves extract of Hibiscus syriacus L.

S.No.

Phytochemical

Pet. Ether

Benzene

Chloroform

Methanol

Water

1

Carbohydrates

-

+

+

+

+

2

glycosides

-

-

-

+

+

3

Steroids

+

+

+

-

-

4

Proteins

-

-

+

+

-

5

Flavonoids

-

-

-

+

+

6

Tannins

+

+

+

+

+

7

Alkaloids

-

-

-

-

+

 

 

Table2. Antibacterial activity of different leaves extract by disc diffusion method.

Bacterial sp.

Zone of Inhibition in mm of leaves extract of Hibiscus syriacus

Petroleum ether extract

Benzene extract

Chloroform extract

Methanol extract

Water extract

Chloramphenicol (std Drug)

Bacillus cereus

(MTCC-430)

08.1

07.2

10.1

19.6

17.1

27.0

Bacillus subtilis

(MTCC-121)

10.3

9.8

10.6

25.4

21.8

35.2

Staphylococcus epidermis (MTCC-435)

09.3

8.6

10.4

22.9

20.9

31.5

Klebsiella pneumonia (MTCC-432)

08.0

7.4

9.2

19.1

16.4

22.4

 

 


Antibacterial Activity of the Plant Extracts

The antibacterial activity of the plant extracts are depicted in Table 2. The results indicated that the plants extracts showed antibacterial activities at variable degrees against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432) and Bacillus subtilis (MTCC 121) bacteria. Methanolic leaves extract of Hibiscus syriacus displayed the most important spectrum of activity, its inhibitory effects being observed against all the bacterial strains, followed by the all other leaves extract of Hibiscus syriacus. The methanolic leaves extract of Hibiscus syriacus showed the highest activity against Bacillus subtilis (MTCC-121). Antibacterial activity of leaves extract of Hibiscus syriacus L. was evaluated in vitro against four bacterial species. Among the five extract (petroleum ether, benzene, chloroform, methanol, and aqueous) used in the study.  Methanol extract displayed maximum antibacterial activity against all the bacterial species tested. Among four bacterial species screened Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432) and Bacillus subtilis (MTCC 121) bacteria were most susceptible to the methanol extract of leaves of Hibiscus syriacus L. (19.1 mm, 19.6 mm, 22.9mm, 25.4mm) respectively. Whereasv the water extract of Hibiscus syriacus L. exhibited significant antibacterial activity (16.5 mm, 17.1 mm, 20.9 mm, and 21.8 mm) against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432) and Bacillus subtilis (MTCC 121) bacteria respectively. Petroleum ether(8.1 mm, 8.0 mm, 9.0 mm, 10.3 mm), benzene (7.2 mm, 7.4 mm, 8.6 mm, 9.8 mm) and chloroform (9.2 mm, 10.1 mm, 10.4 mm, 10.6 mm) leaves extract of Hibiscus syriacus L. fails to elict significant antibacterial activity against tested bacterial species.

 

All The leaves extract of Hibiscus syriacus show antibacterial activity against the majority of the bacteria, their inhibitory effect being noted against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumoniae (MTCC-432), and Bacillus subtilis (MTCC 121) bacterial strains tested respectively. Chloramphenicol used as reference antibiotic showed variable inhibitory activity on different strains of bacteria. Result obtained revealed potential application of leaves extract of Hibiscus syriacus L. for the treatment of bacterial infection.

 

The MIC values (table 2) ranged 0.001 and 0.625 mg/ml. The lowest minimal value of 0.078 mg/ml was recorded with the methanol leaves extract of Hibiscus syriacus L against Bacillus subtilis (MTCC-121). 

 


 

Table3. Minimum inhibitory concentration of different leaves extract.

Bacterial sp.

Minimum inhibitory concentration (mg/ml)

Petroleum ether extract

Benzene extract

Chloroform extract

Methanol extract

Water extract

Chloramphenicol (std Drug)

Bacillus cereus

(MTCC-430)

0.312

0.625

0.625

0.312

0.312

0.002

Bacillus subtilis

(MTCC-121)

0.312

0.312

0.625

0.078

0.156

0.001

Staphylococcus epidermis (MTCC-435)

0.312

0.312

0.625

0.156

0.156

0.001

Klebsiella pneumonia (MTCC-432)

0.625

0.625

0.625

0.312

0.312

0.004

 

 

 


DISCUSSION:

The Medicinal plants have been main source for drugs over many countries in both developed and developing world. Traditional medicines products are not officially recognized in many countries, and the European Union presently developing regulatory laws for quality traditional medicines. [21] Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antibacterial activity assay. [22]

 

Hibiscus rosasinensis was screened for antibacterial activity against human pathogenic bacterial strains. Most of the extracts have shown antibacterial activity against these pathogens. E. coli are common member of the normal flora of large intestine. It is predominant facultative organism in the gastrointestinal tract and colonizes the tract within hours or few days. It is responsible for causing diarrhea, which is characterized by rapid onset of watery non-bloody

fluid. [23]

 

Each of the extract tested in the present study displayed antibacterial activity on Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432), Bacillus subtilis (MTCC 121) bacterial strains tested. However, differences were observed between antibacterial activities of the extracts.  Therefore, the inhibitory activity found herein against Gram-positive as well as Gram-negative bacteria. Phytochemical screening results of leaves extract of Hibiscus syriacus was in accordance with the results previously obtained. This latter suggested that the presence of flavonoids which interfere with cell division) in Hibiscus syriacus could account for its antimicrobial activity. They demonstrated that methanol extract of Hibiscus syriacus possess inhibitory activities against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432), Bacillus subtilis (MTCC 121). In this report, the antibacterial activity was observed against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432), Bacillus subtilis (MTCC 121). The results of the present work also bring additional data on the antibacterial activity of Hibiscus syriacus, since we report for the first time its activity against Bacillus cereus (MTCC-430), Klebsiella Staphylococcus epidermis (MTCC-435), Klebsiella pneumonia (MTCC-432), and Bacillus subtilis (MTCC 121). The different phytochemicals found here should then explain its antibacterial activity against different bacterial strains tested. The plants of the Hibiscus family were already found to possess flavonoids, terpenoids and glycosides that could explain their antibacterial        activity. [24-25]

 

The emergence of multiple drug resistant bacteria has become a major cause of failure of the treatment of infectious diseases. [26] The antibacterial activity of the methanol and aqueous extracts were additional information of the antibacterial activities of this plant Hibiscus syriacus. Chloramphenicol confirmed that an active efflux system expressed by tested bacteria is responsible for their resistance to chloramphenicol. The wide substrate specificity of these pumps could allow them to provoke extrusion of various active antibacterial compounds, preventing their inhibitory effects. Therefore, the low antibacterial activities of these plants shown in the present work should thus be due to the resistance of bacteria strains tested. The contrast between high numbers of secondary metabolite classes found in these extracts reinforces the idea that the detection of the classes of phytochemicals in plants is not a guarantee for a good antibacterial property. [27] This shows that the studied extract mostly exhibited bacteriostatic effects.

 

REFERENCES:

1.       Rosa RM, Melecchi MIS, Halmenschlager RDC, Abad FC, Simoni CR, Caramao EB et al. Antioxidant and mutagenic properties of Hibiscus tiliaceus methanolic extract. J agri food chem 2006; 54(19):7324-7330.

2.       Doughari JH, El-mahmood AM, Tyoyina I. Antimicrobial activity of leaf extracts of Senna obtusifolia (L). Afr J Pharma Pharmacol 2008; 2(1):7-13.

3.       Akinsulire O, Aibinu I, Adenipekun A, Adelowotan T, Odugbemi T. In vitro antimicrobial activity of crude extracts from plants bryophyllum pinnatum and kalanchoe crenata. Afr J Tradit CAM 2007; 4(3):338-344.

4.       Banso A. Phytochemical and antibacterial investigation of bark extracts of Acacia nilotica. J Med Plants Res 2009;3(2):82-85.

5.       Punasiya R, Verma R, Pillai S. In vitro hair growth promoting activity of various leaves extract of Hibiscus syriacus L. on albino rats. Int. J. of Pharm. Life Sci. 2014; 5(5): 3565-3569.

6.       Rojas J, Ochoa V, Ocampo SA, Munoz JF. Screening for antimicrobial activity of ten medicinal plants used in Colombian folkloric medicine: A possible alternative in the treatment of non-nosocomial infections. BMC Compl Alter Med 2006; 6(2):1-6.

7.       Li XZ, Nikaido H. Efflux-mediated drug resistance in bacteria. Drugs 2004; 64:159-204.

8.       Punasiya R, Yadav A, Gaurav K, Pillai S. Formulation and evaluation of herbal gel containing extract of Hibiscus syriacus. Research journal of pharmacy and technology.2014; 7:3296-3300.

9.       Ryoo IJ, Yun BY, Lee IK, Kim YH, Lee IS, Ahn JS et al.  Hydroxyhibiscone A, a Novel neutrophil elastase inhibitor from Hibiscus syriacus. J Microbiolo Biotechnol 2010; (8):1189-1191. 

10.     Gilman EF, Watson DG. Hibiscus syriacus  Rose-of-Sharon., a series Environmental Horticulture Department, Florida Cooperative Extension Service, Instit Food  Agril Sciences, Univr Florida. 1993.

11.     Punasiya R,  Devre K, Pillai S. Pharmacognostic and pharmacological overview on Hibiscus syriacus L. Int. J. of Pharm. Life Sci. 2014 ; 5(6): 3617-3621.

12.     Punasiya R, Joshi A, Sainkediya K, Tirole S, Joshi P, Das A et al. Evaluation of Anti bacterial Activity of Various Extract of Hibiscus syriacus. Res J Pharmacy Technol 2011; (4) 5: 819- 822.

13.     Baqir S. Shaikh M. Maleka FA, Sheikh D. Studies of antibacterial activity of ethanolic extracts from nericum indicum and habiscus rosasinensis. J Islamic acad of sciences1994; 7(3): 167-168.

14.     Tolulope O M. Cytotoxicity and antibacterial activity of Methanolic extract of Hibiscus sabdariffa. J Med Plants Res 2007; 1(1): 9-13.

15.     Xiaoli L, Mouming Z, Jinshui W, Bao Y, Yueming J. Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China. J Food Compo Anal 2008; 21:219–228.

16.     Ruben P. Gajalakshmi K. In vitro antibacterial activity of Hibiscus rosa-sinensis flower extract against human pathogens. Asian j tropi biomed 2012; 399-403. 

17.     Djeussi DE, Noumedem J, Seukep J, Fankam A, Voukeng I, Tankeo1 Bet al. Antibacterial activities of selected edible plants extracts against multidrug-resistant Gram-negative bacteria. BMC ComplAlter Med 2013; 13:164.

18.     Atata R, Sani A, Ajewole SM. Effect of stem back extracts of Enantia chloranta on some clinical isolates. Biokemistr 2003; 15(2):84–92.

19.     National committee for clinical laboratory standards, author. Methods for dilution, antimicrobial susceptibility tests for bacteria that grow aerobically. 5th ed. 2000. p. 30.

20.     Nascimento GGF, Lacatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts and Phytochemicals on antibiotic-resistant bacteria. Braz. J. Microbiol 200; 31(4): 886-891.

21.     Mahesh B, Satish. Antimicrobial activity of some important medicinalplant    against plant and human pathogens. Wor j agri sciences. 2008; 4 (s): 839-843.

22.     Prasannabalaji N, Muralitharan G, Sivanandan M, Kumaran S, Pugazhvendan S R. Antibacterial activities of some Indian traditional plant extracts. Asian pacific j trop disease. 2012; S291-S295.

23.     Tomoko N, Takashi A, Hiromu T, Yuka I, Hiroko M, Munekazu I et al. Antibacterial activity of extracts preparated from tropical and subtropical plants on methicillin-resistant Staphylococcus aureus. J health Sci 2000; 48: 273-276.

24.     Olaleye MT. Cytotoxicity and antibacterial activity of methanolic extract of Hibiscus sabdariffa. J Med Plants Res. 2007; 1:009–013.

25.     Tane P, Tatsimo S D, Ayimele G A, Conolly J D. Bioactive metabolites from Afromomum species. In 11th NAPRECA Symposium Book of Proceedings. Antananarivo, Madagascar. 2005; 214–223.

26.     Kumar S, Nancy, Singh D, Kumar V. Evaluating the antibacterial activity of plant extracts against bacterial pathogens. J drug deliv therapeu  2012; 2(4): 182-185

27.     Achenbach H, Renner C. The annonidines - a new class of prenylated bisindole alkaloids from Annonidium mannii. Constitu West Afr med plants. XVIII:  Heterocycles. 1985; 23:2075–2081.

 

 

 

Received on 21.11.2014                    Accepted on 20.12.2014  

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Res. J. Topical and Cosmetic Sci. 5(2): July –Dec. 2014 page 51-55