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ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 1  |  Page : 15-18  

Antimicrobial effect of chloroform Hibiscus sabdariffa extract on pathogenic bacteria


1 Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
2 Medical Plants Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
3 Cellular and Molecular Research Center, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran
4 Department of Epidemiology and Biostatistics, School of Health, Shahrekord University of Medical Sciences, Shahrekord, Iran
5 Deputy of Research and Technology, Shahrekord, Iran
6 Student Research Committee, Shahrekord University of Medical Sciences, Shahrekord, Iran

Date of Submission28-Oct-2018
Date of Decision08-Dec-2018
Date of Acceptance03-Apr-2019
Date of Web Publication20-Jul-2020

Correspondence Address:
Prof. Mahmoud Rafieian-Kopaei
Medical Plants Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpnr.JPNR_19_18

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   Abstract 


Objective: The aim of this study was to determine the antimicrobial effect of Hibiscus sabdariffa extract on oral and dental bacterial pathogens. Materials and Methods: In this study, the extract of H. sabdariffa was isolated by maceration with chloroform and concentrated by a rotary evaporator. The antimicrobial effects were examined using broth microdilution, and minimum inhibitory concentration and minimum bactericidal concentration values were determined. Next, the effect of different concentrations of extract on the growth of Streptococcus pyogenes, Streptococcus mutans, and Streptococcus salivarius was investigated by disc diffusion method. Finally, the data were analyzed using SPSS 16 software (SPSS Inc., 233 South Wacker Drive, 11th Floor, Chicago, IL 60606-6412), and the results were presented as mean (± standard deviation). Results: The results of this study showed that the chloroform extract of H. sabdariffa had inhibitory and bactericidal effects on three bacteria in both broth microdilution and disc diffusion methods. In broth microdilution, S. mutans and S. salivarius were more resistant to H. sabdariffa extract than S. pyogenes. In disc diffusion method, the highest inhibitory effect of the extract was observed on S. pyogenes at 25 μg/ml with a growth inhibition zone diameter of 13.03 ± 0.15 mm. Conclusion: According to the results of this study, it can be argued that the chloroform extract of H. sabdariffa is effective in inhibiting S. pyogenes, S. mutans, and S. salivarius in both broth microdilution and disc diffusion methods.

Keywords: Antibiotic, antimicrobial effect, chloroform extract, Hibiscus sabdariffa, oral cavity and teeth


How to cite this article:
Aleebrahim-Dehkordy E, Rafieian-Kopaei M, Zamanzad B, Deris F, Sharifih A, Reyhanian A. Antimicrobial effect of chloroform Hibiscus sabdariffa extract on pathogenic bacteria. J Pharm Negative Results 2020;11:15-8

How to cite this URL:
Aleebrahim-Dehkordy E, Rafieian-Kopaei M, Zamanzad B, Deris F, Sharifih A, Reyhanian A. Antimicrobial effect of chloroform Hibiscus sabdariffa extract on pathogenic bacteria. J Pharm Negative Results [serial online] 2020 [cited 2020 Aug 11];11:15-8. Available from: http://www.pnrjournal.com/text.asp?2020/11/1/15/290201




   Introduction Top


In recent years, the tendency to the use of herbal drugs and the use of these drugs for treatment and prevention of illnesses has increased dramatically globally, especially in Iran. The side effects and the high cost of chemical drugs have led people to turn to phytomedicine. Convenient access and lack of pathogenic resistance are among the other factors that make medicinal plants become more and more important.[1] Iran has many medicinal plant species that can replace expensive chemical treatments that also lead to side effects. The antimicrobial and antifungal effects of Iranian medicinal plants have encouraged many researchers to study them, and the outcome of these studies can lead to self-sufficiency and independence of the country's pharmaceutical industries.[2] In light of dental treatments, antimicrobial agents, either oral or topical (mouthwash), are much frequently used to control oral bacterial plaques to decrease oral microbial flora during recovery on a daily basis.[3] On the other hand, patients with rare diseases or people at risk of microbial infection, such as those susceptible to endocarditis and immunodeficiency and patients with diabetes, are required to use antimicrobial agents to control dental plaque and prevent gum disease and tooth decay. There are over 500 different types of bacteria in the oral cavity that are associated with oral diseases. Oral infections such as periodontitis and other periodontal conditions are caused by aerobic and anaerobic pathogens.[4] One of the most common oral diseases is periodontal disease-dependent, chronic dental plaque, with a prevalence of 80%–90%. This inflammation leads to the destruction of soft and hard periodontal tissues and ultimately tooth decay and tooth loss.[5] In addition, periodontitis leads to an increase in systemic inflammatory responses associated with systemic diseases such as cardiovascular diseases, rheumatoid arthritis, and adverse outcomes of pregnancy such as preterm labor and preeclampsia.[6],[7],[8] Important risk factors for periodontitis include smoking, obesity, stress, cardiovascular disease, and diabetes, especially in people whose disease is not well controlled.[9] Medicinal plants and their secondary compounds play an important role in preventing various pathogens, and it is very important to use them as low-risk natural antibiotics to treat bacterial infections and other diseases.[10] Sour tea, botanically named Hibiscus sabdariffa, is a plant of the Malvaceae family, which is named differently in different countries. More than 300 species of this plant are found in tropical and subtropical regions of the world. This plant is not native to Iran, and its cultivation in Iran has been reported only in Sistan and Baluchestan provinces. H. sabdariffa is an annual, branched plant with reddish green color, alternate leaves; paw-shaped petioles; and serrated leaves. In the petals of this plant, there are various compounds and alkaloids such as ascorbic acid, anthocyanin, beta-carotene, beta-sitosterol, citric acid, galactose, hepcidin, saccharides, polysaccharides, and pectin. In traditional medicine, it is used for the treatment of kidney stones and as an antibacterial and antifungal agent.[11],[12] Because the oral cavity contains numerous bacterial species and the use of chemical drugs is associated with side effects in the oral cavity such as change in natural flora, an experimental study showed the antimicrobial effect of the chloroform extract of the plant Sour tea at different concentrations on common oral and dental microorganisms. Among these pathogenic bacteria, oral streptococci could be mentioned, out of which Streptococcus pyogenes, Streptococcus mutans, and Streptococcus salivarius will be studied in the current work. These bacteria have a great ability to convert carbohydrates to acids and play a major role in dental caries. Given the importance of oral and dental health in today's societies, the use of more body-compatible compounds as alternatives to antibiotics is necessary due to drug resistance and side effects. The aim of this study was to determine the antimicrobial effect of H. sabdariffa extract on bacterial pathogens affecting the oral cavity and teeth.


   Materials and Methods Top


Extraction

After obtaining H. sabdariffa and identifying it with reference to the botanical keys and Iranian flora, and also matching the samples with Herbarium samples, to prepare the chloroform extract, we pulverized 300 g of the plant using a mill and conducted extraction using chloroform. A rotary evaporator was used to concentrate the extract at 40°C.

Steps and procedures for performing microbial tests

To conduct microbial tests of standard bacteria, S. pyogenes PTCC1447, S. mutans PTCC1683, and S. salivarius PTCC1448 were obtained from the Iranian Research Organization for Science and Technology.

Evaluation of antimicrobial activity using well microplate method

After bacterial culture and preparation of microbial suspension, broth microdilution method was used to determine the antimicrobial effect of the extract in a sterile, 96-well plate with reference to the McFarland standard (105 colony-forming unit/ml). In this method, the first well was selected as negative control and the second well was selected as positive control (Clinical and Laboratory Standards Institute (CLSI), 2012).

Evaluation of the antimicrobial activity of the antibiotics using disc diffusion method

To determine the inhibitory effect of the extracts on the bacteria, a disc diffusion method known as the Kirby–Bauer test was used. For this purpose, after isolating the bacteria from all strains, 0.5 McFarland concentration was prepared in Mueller–Hinton broth and cultured on a Mueller–Hinton agar. The discs containing the extract were then placed at appropriate intervals and incubated for 24 h at 37°C until different concentrations of the extract were examined for antibacterial properties. Then, the diameter of the inhibition zone around the discs containing the extract was measured, and the results were examined based on the CLSI.[13] For each concentration of the extract, the rest was performed in triplicate.

Preparation of different dilutions of extract

To prepare various dilutions, 4.04 g of the extract weighed using a digital scale was mixed with 1 ml of 5% dimethyl sulfoxide solution, and then different dilutions (μg/ml) of the extract were prepared using Mueller–Hinton broth.

After adding the culture medium and extract to the microplate wells, 14 Landa bacteria were added and after dilution, the samples were incubated at 37°C for 24 h. The concentration of the last (most diluted) well without turbidity was considered to indicate minimum inhibitory concentration (MIC) and for determining the minimum bactericidal concentration (MBC), all the wells that did not have turbidity were cultured separately on the agar, and then incubated at 37°C for 24 h. The lowest concentration of the extract in which the bacteria could not grow was determined to indicate MBC. Experiments to determine the MIC and MBC for each concentration of extract were conducted in triplicate.

Data analysis

The mean values of MICs, MBCs, and growth inhibition zones of different concentrations of extract for bacteria were measured using ANOVA and Tukey's test, and the results were presented as mean (± standard deviation).


   Results Top


In this study, the antimicrobial effect of H. sabdariffa was investigated by broth microdilution method, and the results are shown in [Table 1] and [Table 2]. According to [Table 1] and [Table 2], the chloroform extract of the plant exhibited inhibitory and microbial effects on the bacteria studied. Among the studied bacteria, S. mutans and S. salivarius were more susceptible to the H. sabdariffa extract than S. pyogenes. The lowest (best) inhibitory effect was observed on S. pyogenes with 170.67 ± 73.9 μg/ml.
Table 1: The results on the minimum inhibitory concentration of the chloroform extract of Hibiscus sabdariffa on the bacteria studied (μg/ml)

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Table 2: The results on the minimum bactericidal concentration of the chloroform extract of Hibiscus sabdariffa on the bacteria studied (μg/ml)

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Various concentrations of the chloroform extract of H. sabdariffa also exhibited significant inhibitory effects on the bacteria studied. The results from the disc diffusion method regarding the effect of various concentrations of H. sabdariffa extract on the bacteria are shown in [Table 3].
Table 3: The mean diameter of growth inhibition zone for the strains studied against the chloroform extract of Hibiscus sabdariffa (mm)

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According to [Table 3], the highest inhibitory effect of H. sabdariffa extract was observed on S. pyogenes 25 μg/ml with a growth inhibition zone diameter of 13.03 ± 0.15 mm and its least effect on S. mutans at a concentration of 25 μg/ml with a nongrowth hole diameter of 97.5 ± 0.06 mm.


   Discussion Top


Increase in bacterial resistance to antibiotics and the side effects due to the use of these drugs are among the causes of the extensive research in the field of medicinal plants. Today, the use of various antibiotics has led to the emergence of excessive antibiotic-resistant species in patients due to lack of proper diagnosis.[14],[15],[16] According to the experiments and the results obtained from our study, it was found that the chloroform extract of H. sabdariffa in both broth microdilution and disc diffusion methods had inhibitory effects on the three bacteria. In broth microdilution method, S. mutans and S. salivarius were more susceptible to H. sabdariffa extract than S. pyogenes. The lowest (best) inhibitory effect was observed on S. pyogenes at 73.79 ± 170.66 μg/ml. This difference in the antibacterial properties of H. sabdariffa extract at various concentrations used in this study can be due to the solvent type and extraction method. Promising results of the present study on the inhibition of pathogenic bacteria can be an effective strategy to treat oral and dental infections. The antimicrobial effects of H. sabdariffa have also been investigated in various studies on other pathogens. In a research on the antibacterial effects of H. sabdariffa using two methods, plate cylinder and bioautography, on standard strains of Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, the methanol and ethyl acetate extracts of the plant were found to have antibacterial effect on the six microbial strains at 25 mg/ml with a minimum inhibition zone of 12 ± 3.0 μg/ml. In the bioautography method, the flavonoid extract caused a growth inhibition zone against four of the bacteria, namely, S. epidermidis, B. subtilis, E. coli, and K. pneumoniae.[17] In another study, the effect of H. sabdariffa extract at different concentrations on E. coli and S. aureus was investigated by dilution method, and the results showed that E. coli was highly resistant to penicillin (75.9%), followed by erythromycin (58.3%), tetracycline (56.9%), and cefixime (37%), and S. aureus was highly resistant to penicillin (83.5%), followed by cefixime (80%), erythromycin (55.6%), and tetracycline (26.1%). The results of this study indicated the beneficial effect of ethanolic extract of H. sabdariffa on E. coli and antibiotic-resistant S. aureus so that the MIC of ethanolic extract of the plant for these two bacteria was found to be 16 and 4 mg/ml, respectively.[18] In another study, ethanolic extract of H. sabdariffa was observed to be a strong inhibitor of E. coli and P. aeruginosa.[18] In other studies, the antimicrobial activity of H. sabdariffa against E. coli, P. aeruginosa, and Bacillus has been demonstrated.[19] In a study by Timothy et al., the aqueous extract of H. sabdariffa acted as an inhibitor of S. aureus, E. coli, K. pneumoniae, and P. aeruginosa at 10–160 mg/ml.[20]


   Conclusion Top


According to the results of this study, it can be argued that the chloroform extract of H. sabdariffa is effective in inhibiting the S. pyogenes, S. mutans, and S. salivarius in both broth microdilution and disc diffusion methods. As a result, the plant can be used as a natural compound to inhibit the growth of bacteria and also to produce various mouthwashes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Ghasemi A. Medicinal and Aromatic Plants (Recognizing and Evaluation of Their Effects). Iran: Islamic Azad University Publication; 2008. p. 500.  Back to cited text no. 1
    
2.
Omidbaigi R. Processing and Production of Medicinal Plants. 3rd ed. Iran: Astan Ghods Razavi Press; 2006. p. 347.  Back to cited text no. 2
    
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Chen F, Wang D. Novel technologies for the prevention and treatment of dental caries: A patent survey. Expert Opin Ther Pat 2010;20:681-94.  Back to cited text no. 3
    
4.
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 2005;43:5721-32.  Back to cited text no. 4
    
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Detert J, Pischon N, Burmester GR, Buttgereit F. The association between rheumatoid arthritis and periodontal disease. Arthritis Res Ther 2010;12:218.  Back to cited text no. 5
    
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Desvarieux M, Demmer RT, Rundek T, Boden-Albala B, Jacobs DR Jr., Sacco RL, et al. Periodontal microbiota and carotid intima-media thickness: The oral infections and vascular disease epidemiology study (INVEST). Circulation 2005;111:576-82.  Back to cited text no. 6
    
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de Pablo P, Chapple IL, Buckley CD, Dietrich T. Periodontitis in systemic rheumatic diseases. Nat Rev Rheumatol 2009;5:218-24.  Back to cited text no. 7
    
8.
Xiong X, Buekens P, Fraser WD, Beck J, Offenbacher S. Periodontal disease and adverse pregnancy outcomes: A systematic review. BJOG 2006;113:135-43.  Back to cited text no. 8
    
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AlJehani YA. Risk factors of periodontal disease: Review of the literature. Int J Dent 2014;2014:182513.  Back to cited text no. 9
    
10.
Aleebrahim-Dehkordy E, Rafieian-Kopaei M, Bahmani M. Study of antibacterial effect of total hydroalcoholic extract of Thymus daenensis and Lavandula officinalis on Listeria monocytogenes, an agent of food spoilage, using microdilution. Der Pharm Chem 2016;8:238-42.  Back to cited text no. 10
    
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Hirunpanich V, Utaipat A, Morales NP, Bunyapraphatsara N, Sato H, Herunsalee A, et al. Antioxidant effects of aqueous extracts from dried calyx of Hibiscus sabdariffa Linn. (Roselle)in vitro using rat low-density lipoprotein (LDL). Biol Pharm Bull 2005;28:481-4.  Back to cited text no. 11
    
12.
Mozaffari-Khosravi H, Jalali-Khanabadi BA, Afkhami-Ardekani M, Fatehi F. Effects of sour tea (Hibiscus sabdariffa) on lipid profile and lipoproteins in patients with type II diabetes. J Altern Complement Med 2009;15:899-903.  Back to cited text no. 12
    
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Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disks Susceptibility Tests; Approved Standards-Eleventh Edition, Mo2-A11. USA: Clinical and Laboratory Standards Institute; 2009.  Back to cited text no. 13
    
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Mohammadi-Sichani M, Amjad L, Mohammadi-Kamalabadi M. Antibacterial activity of methanol extract and essential oil of Achillea wilhelmsii against pathogenic bacteria. Zahedan J Res Med 2011;13:9-14.  Back to cited text no. 14
    
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Bayat A, Shahanipour K. The antibacterial effect of methanolic and aqueous extract of Stachys schegleevii (Poulk) Leave on bacteria causing urinary infection. Qom Univ Med Sci J 2015;9:33-9.  Back to cited text no. 15
    
16.
Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: A molecular, biological, and epidemiological study. Lancet Infect Dis 2010;10:597-602.  Back to cited text no. 16
    
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Tabatabai Yazdi F, Ali Zadeh Behbahani B, Vasi i AR, Mortazavi SA, Moradi S. Investigation of the extracts antibacterial effect of Hibiscus sabdariffa against strains of antibiotic resistance on pathogenic bacteria “in vitro”. FSCT 2015;13:23-31.  Back to cited text no. 17
    
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Hammad M, Sallal AK. Effect of date extract on growth and hemolytic activity of Streptococcus pyogenes. New Microbiol 2002;25:495-7.  Back to cited text no. 18
    
19.
Mounnissamy VM, Kavimani S, Gunasegarn R. Antibacterial from Hibiscus sabdariffa. Antiseptic 2002;99:81-2.  Back to cited text no. 19
    
20.
Moshafi MH, Forutan H, Mehrabani M. Investigation of antibacterial effects of Hibiscus sabdariffa L. dried calyx by agar diffusion and bioautographic methods. J Kermanshah Univ Med Sci 2006;13:103-10.  Back to cited text no. 20
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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