Bangladesh J. Life Sci.

22(2): 65-73, 2010 (December)

Antibacterial activity of the ethanol extracts of Hibiscus rosa-sinensis leaves and flowers against clinical isolates of bacteria
Borhan Uddin, Tareq Hossan, Sudip Paul, Tanjir Ahmed, Taslima Nahar, and Sohel Ahmed* Department of Biochemistry and Molecular Biology, Jahangirnagar University Savar, Dhaka 1342, Bangladesh Abstract
Crude preparations of the different parts of Hibiscus rosa-sinensis have been traditionally used in folk medicine for various purposes. In the present study, we have evaluated the antibacterial activity of the extracts of H. rosa-sinensis leaves and flowers against some clinical isolates of bacteria by simple agar-well diffusion and bacteriological enumeration method. In the preliminary screening experiment, all of the bacterial isolates showed varying degrees of sensitivity to the flower extracts excluding Klebsiella pneumoniae. We found Staphylococcus aureus, a Gram-positive bacterium as the most sensitive to the extracts of flowers at the applied doses of 50 and 100 mg/well. On the other hand, most of the tested bacterial isolates were resistant to the extracts of leaves excluding S. aureus and Salmonella typhimurium. In bacteriological enumeration study, all of the bacterial isolates showed substantial extent of sensitivity to the different extracts used. Our findings clearly demonstrate that the flower extracts of H. rosa-sinensis had stronger antibacterial effects than that of leaves and raises the possibility of using the extracts as antibacterial agents in treating pathological conditions caused by S. aureus and S. typhimurium infection. Although the effect of the H. rosa-sinensis flowers and leaves against some pathogenic bacteria in vitro is promising, further microbiological and pharmacological studies will be required before starting clinical trials.

Key words: Hibiscus rosa-sinensis, antibacterial activity, infections INTRODUCTION The range of pathogenic bacteria is wide and so is the variety of diseases caused by them. Despite the existence of potent antimicrobial agents, resistant or multi-resistant strains are continuously emerging, imposing the need for a continuous search and development of new drugs (Barbour et al., 2004; Machado et al., 2003). Hence, many efforts have been exploited to discover new antimicrobial compounds from various kinds of sources such as soil, microorganisms, animals and plants. One such resource is medicinal plants, and their systematic screening may result in the discovery of novel effective compounds. In fact, plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines (Farombi, 2003). There has been a revival of interest in herbal medicines due to increased awareness of the limited ability of synthetic pharmaceutical products to control major diseases.
* Author to whom all correspondences should be made.
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Hibiscus rosa-sinensis L., popularly known as Joba in Bangladesh, is an evergreen shrub which is in leaf all the year. It is a sweet, astringent, cooling herb that checks bleeding, soothes irritated tissues and relaxes spasms. The leaves are anodyne, aperient, emollient and laxative. The flowers are hermaphrodite and are pollinated by insects. The flowers are aphrodisiac, demulcent, emmenagogue, emollient and refrigerant. They are used in the treatment of excessive and painful menstruation, cystitis, venereal diseases, feverish illnesses, carbuncles, mumps, sores, coughs. Mucilage prepared from the H. rosa-sinensis root has been used in the treatment of coughs (Duke and Ayensu, 1985; Chopra et al., 1986). The flowers of H. rosa-sinensis have been reported in the ancient Indian medicinal literature with beneficial effects in heart diseases (Nadkarni et al., 1976). In recent times, both experimental and clinical studies have shown that the dried flower powder of H. rosa-sinesis has significant protective effects in ischemic heart disease (Jonadet et al., 1990 and Yamasaki et al., 1996). Ethanolic extracts of H. rosa-sinesis flowers showed the most significant anticonvulsant and hypotensive activity among the crude extracts (Kasture et al., 2000, Siddiqui et al., 2006). In a previous study, Shivananda et al., (2007) reported on 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 (Gauthaman et al., 2006, Yamasaki et al., 1996). Bacterial infection play important role in many of the pathological conditions where extracts of H. rosa-sinensis extracts have been used as traditional medication. Considering the therapeutic potential of this plant, in the present study we aimed to evaluate the antibacterial activity of ethanol extracts of H. rosa-sinensis leaves and flowers against some clinically important bacteria. MATERIALS AND METHODS This study was conducted in Research Laboratories, Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka 1342. Preparation of the extracts of H. rosa-sinensis leaves and flowers: The leaves and flowers of H. rosa-sinensis were collected from the Botanical Garden at Jahangirnagar University, during the month of February 2009 and weighed individually (leaves: 55.0 g and flowers: 66.0 g) . The fresh leaves were first cut and grinded to produce paste. The paste materials were packed in a soxhlet apparatus and subjected to mild hot continuous percolation for 12 hour using absolute ethanol as solvent. The same procedure was followed for extraction from the fresh flowers. The extracts were then concentrated under reduced pressure using rotary evaporator. The concentrated extracts were further dried by placing them within desiccators and weighed again (leaves: 4.5 g and flowers: 1.4 g). Thus, the approximate yields of the extracts from leaves and flowers were 8% (w/w) and 2% (w/w) respectively. Formulation of plant extracts and standard antibiotic: The dried ethanol-free extracts were dissolved in sterile distilled water to a final concentration of 1.0 g/ml. Simultaneously, standard antibiotic solution of ampicillin was prepared and the concentration of ampicillin used was 1.0 mg/ml. Stock solutions of the extracts and standard antibiotic were then stored at -20 C in aliquots and used throughout the experiment.
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Bacterial isolates: A total of 7 clinical isolates belonging to different bacterial species were collected from the Department of Microbiology, Bangladesh Institute for Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM) Hospital, Dhaka. Among them only one was Gram-positive (Staphylococcus aureus) and others were Gramnegative (Salmonella typhimurium, Eschericia coli, Citrobacter sp., Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa). Pure cultures of bacteria were maintained at 4 C on nutrient agar slants. Morphological characteristics such as shape, size, form, opacity, and pigment production turbidity of the 24 hours bacterial cultures were observed. The identity of these bacterial isolates was confirmed through conventional biochemical tests (George, 1984; MacFaddin, 1980; Ryan and Ray, 2004).

In vitro antibacterial activity study by agar-well diffusion method: The antibacterial activity of ethanolic extracts of H. rosa-sinensis flowers and leaves were tested against clinical isolates by agar-well diffusion method as described by Perez et al., 1990 and Bauer et al., 1966 with minor modifications. Briefly, nutrient broth and agar media were prepared in a clean bench under laminar air flow. Each plate of nutrient agar media was prepared by pouring 25.0 ml of nutrient agar onto a 10.0 cm glass petri dish that gave rise to a uniform media thickness of 4.0 mm. First, a loop full of each test bacterial isolate was inoculated into 30 ml of nutrient broth in a 250 ml conical flask aseptically and incubated in an orbital shaker incubator at 100 rpm for 15-18 hours at 37 C. Cork borers of different diameters starting from 5.0 mm to 7.0 mm have been used to punch well in agar plates for studying antibacterial activity in vitro by agar-well diffusion method (Tippayatum and Chonhenchob, 2007; Nkere and Iroegbu, 2005 and Boyanova et al., 2005). In our study, we used a cork borer of 7.0 mm diameter. Then an aliquot of 100 l inocula (McFarland turbidity standard 2) for each bacterial isolate was evenly spread by a sterile glass spreader onto a previously bored nutrient agar plates and plates were allowed to dry at room temperature. Subsequently, 50 and 100 l of extracts (1.0 g/ml) of leaves and flowers were poured into the wells. Physiological saline was used as negative control and ampicillin (20.0 l) was used as positive control. Then plates were kept at 2-8 C in a refrigerator for 2 hours to allow diffusion of the extracts into the agar and further incubated at 37 C for 24 hours. The diameter of zone of inhibition was measured to the nearest millimeter (Okeke et al., 2001; Uddin et al., 2007). The tests were performed in duplicate for and the best results were taken. The whole experiment was performed under strict aseptic conditions according to the protocol shown in Figure 1.

In vitro antibacterial activity study by bacteriological enumeration method: The protocol of this method is shown in Figure 2. Each isolate of the test bacteria were allowed to grow in two different groups of vials at 37 C for 24 hours. One group of vials contained only nutrient broth and thus served as control, while the other group contained the extracts of H. rosasinensis leaves and flowers at a concentration of 100.0 mg/ml of nutrient broth. After
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overnight growth, the samples were diluted with nutrient broth and 10.0 l from different dilutions was spread onto the petri dishes with sterile glass rod spreader. Again, the plates were incubated at 37 C for 24 hours. The number of the total viable bacteria per milliliter was calculated for both the control and extracts-supplemented groups of vials (Harley and Harley, 2005). The percent of sensitive bacteria was calculated by the formula, percent of sensitive bacteria = {(C E)/C}100, where C indicates colony forming unit (CFU) per ml in control group and E represents CFU per ml in vials supplemented with the extracts. Antibacterial screening with pathogenic bacteria Streaking onto nutrient agar media Inoculation of pure colonies into nutrient broth
Incubation at 37 C for 15-18 hours

Spreading onto previously bored nutrient agar media Pouring of saline, plant extracts and ampicillin solution into respective wells Saline (100 l) Leaf extracts (50/100 mg/well) Flower extracts (50/100 mg/well) Ampicillin (20 g/well)

Incubation at 37 C for 24 hours Measurement of the diameter of zone of inhibition in the nearest mm Figure 1. Protocol for in vitro antibacterial activity study by agar-well diffusion method.

Stock culture of bacteria with pathogenic potential Inoculation of pure colonies into different groups of vials Control vials (Nutrient broth only) Vials with nutrient broth + flower extracts Vials with nutrient broth + leaf extracts

Incubation of all vials at 37 C for overnight growth (24 hours) Serial dilution with sterile nutrient broth (10 -2, 10-4, 10-6) Spreading onto nutrient agar media and incubation at 37 C for 24 hours Enumeration of the total viable bacterial count (CFU/ml)
Figure 2. Protocol for in vitro antibacterial activity study bacteriological enumeration method.
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RESULTS AND DISCUSSION Identities of the test bacterial isolates were confirmed through biochemical characterization for catalase, indole production, Voges-Proskaur, methyl red, citrate utilization, H2S production, oxidase, urease, phenylalanine deamination, triple sugar iron agar, ornithine decarboxylase and lysine decarboxylase (Table 1). Table 1. Identification of bacterial isolates by conventional biochemical tests
TSI test Bacteria S. aureus P. vulgaris P. aeruginosa Citrobacter spp E. coli S. typhimurium K. pneumoniae Cat + + + + IP + + VP MR CU HSP OX Urease PD OD LD Butt Slant Gas + + + + + + + + + + + + + + + + + + + + + + + + + + + A K K N A N K A A K N K N K N + N

Here, Cat = Catalase, IP= Indole Production, VP = Voges-Proskaur, MR = Methyl Red, CU = Citrate Utilization, HSP = H2 S production, OX = Oxidase, PD = Phenylalanine Deaminase, OD = Ornithine Decarboxylase, LD = Lysine decarboxylase, TSI = Triple sugar Iron Agar test, A = Acidic, K = Alkaline, N = Not done, + sign denotes positive and sign denotes negative.

We found that the extracts of H. rosa-sinensis flowers showed stronger antibacterial activity than that of leaves (Table 2). The maximum zone of inhibition (29 mm) was observed against S. aureus, followed by P. vulgaris (25 mm), P. aeruginosa (24 mm) and Citrobacter sp. (24 mm) and the lowest against S. typhimurium (13 mm) at the highest amount of flower extracts (100 mg/well) (Table 2). All the test bacteria responded to the extracts in a dose-dependent manner. However, K. pneumoniae was found to be resistant to the flower extracts at any of the applied doses (50 and 100 mg/well). Thus the extracts of H. rosa-sinensis flowers showed strong antibacterial activity against S. aureus and moderate activity against P. vulgaris, P. aeruginosa and Citrobacter sp. S. aureus was also the most sensitive bacterium to the extracts of H. rosa-sinensis leaves (Table 2). This was followed by S. typhimurium and E. coli. Rest bacterial isolates were found to be resistant to either of the applied doses of the extracts of leaves. In the bacteriological enumeration method, all the bacteria showed significant percentage of sensitivity to the plant extracts. S. typhimurium, E. coli and P. aeruginosa showed 100 % sensitivity to both the extracts of leaves and flowers at the applied dose of 100 mg/ml of nutrient broth (Table 3). Some of the bacterial isolates that showed resistance in agar-well diffusion method were found sensitive in bacteriological enumeration method. This might be due to the inferior diffusion of extracts in the agar-well diffusion method.
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Table 2. Antibacterial effects of the extracts of flowers and leaves Diameter of zone of inhibition (mm) Extracts of flowers Amount of extracts/well Staphylococcus aureus Proteus vulgaris Pseudomonas aeruginosa Citrobacter sp. Escherichia coli Salmonella typhimurium Klebsiella pneumoniae 50 mg 26 18 22 18 12 10 0 100 mg 29 25 24 24 15 13 0 Extracts of leaves 50 mg 14 0 0 0 0 11 0 100 mg 22 0 0 0 13 14 0 Ampicillin 20 g 22 17 12 15 20 10 19

Here, 0 denotes no zone of inhibition. The results are expressed as the radial extent of the annular zone of inhibition including the 7.0 mm diameter of the well.

Table 3. Percent of sensitive bacteria to the plant extracts Bacteria tested Salmonella typhimurium Extracts Control Leaves Flowers Control Leaves Flowers Control Leaves Flowers Control Leaves Flowers Control Leaves Flowers Citrobacter spp. Control Leaves Flowers CFU106/ml 780 0 0 2000 0 0 376 0 0 1632 0 2 748 475 7 588 584 51 % of sensitive bacteria 100.0 100.0 100.0 100.0 100.0 100.0 100.0 99.9 36.5 99.1 0.7 91.3

Escherichia coli

Pseudomonas aeruginosa

Staphylococcus aureus

Klebsiella pneumoniae

Here, CFU denotes colony forming unit indicating viable bacterial counts; 0 denotes no growth i.e., 100% sensitive.
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Although we have also tried smaller doses such as 10 and 25 mg/well, but did not find any notable inhibition of bacterial growth (data not shown). In the present study, we have shown only the susceptibilities of clinical bacterial isolates to 50 and 100 mg of the plant extracts of H. rosa-sinensis per well. Application of higher doses of the extracts prepared from selected medicinal plants in studying antibacterial activity by agar-well diffusion and disc-diffusion methods have also been reported (Adeshina et al., 2010; Cheruiyot et al., 2009 and Masoodi et al., 2008).

S. aureus is a leading cause of community-acquired and hospital-acquired infections. It is commonly found on the skin and in the nose of healthy people (Kluytmans et al., 1997). Occasionally, staphylococci cause infections that are minor, such as pimples, boils, and other skin conditions or sometimes serious and fatal, such as blood infections, carditis, meningitis, or pneumonia. It produces numerous toxins including super-antigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Over the past 50 years, S. aureus has undergone incremental changes in genetic complement that have resulted in the emergence of antibiotic-resistant strains and appear to be successful in transmitting and causing disease in the hospital setting (Curran and Al-Salihi, 1980). In the present study, S. aureus showed sensitivity to the extracts prepared from both leaves and flowers. This indicates that the extracts of leaves and flowers have immense potential to be used in treating infections caused by S. aureus.

The aim of the study was to investigate whether the folk uses of the herbal preparations derived from the plant H. rosa-sinensis in some diseases caused by bacterial infection had any scientific basis. It is clear from the findings of this study, that H. rosa-sinensis flowers and leaves contain important constituents that confer its antibacterial activity and may be used in treating pathological conditions caused particularly by S. aureus and S. typhimurium. A number of previous studies reported that H. rosa-sinensis contains flavonoids, cyanidin, querecetin, hentriacontane, calcium oxalate, thiamine, riboflavin, niacin, ascorbic, citric, tartaric and oxalic acid (Shukla and Mishra, 2001). Recently, four new phytoconstituents have been isolated from alcoholic extracts of H. rosa-sinensis leaf and flower (Siddiqui et al., 2006). These compounds may come into play (either individually or synergistically) to confer the antibacterial potential of this plant, particularly against S. aureus and S. typhimurium. Findings of the present study clearly demonstrates the scientific basis of traditional medication with the extracts of prepared from H. rosa-sinensis and reveals its potential in complementary alternative medicine. In the present study, only the susceptibilities of clinical bacterial isolates to the extracts of H. rosa-sinensis leaves and flowers have been examined. Therefore, further microbiological studies would be carried out to determine the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of the extracts prepared from H. rosa-sinensis flowers and leaves against S. aureus and S.
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typhimurium. Biochemical and pharmacological investigations would also be necessary to establish the exact antibacterial principles for use in complementary alternative medicine.

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