Evaluation of in Vitro Antimicrobial Effects of Aqueous Extract of Tribulus terrestris Against Oral Bacteria

Kiani, Zahra; Mohammad Parast Tabas, Pouria; khalilpour, khashayar; Goldani Moghadam, Mahjoube; Zare_Bidaki, Majid

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Kiani Z, Mohammad Parast Tabas P, khalilpour K, Goldani Moghadam M, Zare_Bidaki M. Evaluation of in Vitro Antimicrobial Effects of Aqueous Extract of Tribulus terrestris Against Oral Bacteria. mljgoums 2021; 15 (5) :7-12
URL: http://mlj.goums.ac.ir/article-1-1377-en.html

Evaluation of in Vitro Antimicrobial Effects of Aqueous Extract of Tribulus terrestris Against Oral Bacteria

Zahra Kiani¹

, Pouria Mohammad Parast Tabas²

, Khashayar Khalilpour³

, Mahjoube Goldani Moghadam⁴

, Majid Zare_Bidaki

⁵

1- Pharmacology department, Faculty of Pharmacy, Birjand University of Medical Sciences, Birjand, Iran
2- Student Research Committee, BSc Student in Medical Laboratory Science, Birjand University of Medical Sciences, Birjand, Iran
3- Faculty of Dentistry, Birjand University of Medical Science, Birjand, Iran
4- Orthodontic Department, Faculty of Dentistry, Birjand University of Medical Sciences, Birjand, Iran
5- Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran , m.zare@live.co.uk

Keywords: Tribulus [MeSH], Anti-Bacterial Agents [MeSH], bacteria [MeSH]

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Evaluation of in Vitro Antimicrobial Effects of Aqueous Extract of Tribulus terrestris Against Oral Bacteria

Zahra Kiani
(PhD) Pharmacology department, Faculty of Pharmacy, Birjand University of Medical Sciences, Birjand, Iran, kiani.za@gmail.com
Pouria Mohammad Parast Tabas
(BSc) Student Research Committee, Birjand University of Medical Sciences, Birjand, Iran, pouriamp1377@gmail.com
Khashayar Khalilpour
(PhD) Faculty of Dentistry, Birjand University of Medical Science, Birjand, Iran, khashayar.khalilpour@yahoo.com
Mahjoube Goldani Moghadam
(PhD) Orthodontic Department, Faculty of Dentistry, Birjand University of Medical Sciences, Birjand, Iran, Mahdjoube.gm@gmail.com
Majid Zare-Bidaki
(PhD) Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran, m.zare@live.co.uk

Corresponding author: Majid Zare-Bidaki
Tel: +9856 3238 1616
Email: m.zare@live.co.uk
Address: Medical Microbiology Department, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran

ABSTRACT

Background and objectives: Medicinal plants have long been considered as one of the most important pillars of traditional medicine. Existing challenges in the treatment of diseases, particularly infectious diseases, are major drivers for herbal medicine studies. Tribulus terrestris has been widely used in traditional medicine to treat various diseases. This study aimed to investigate in vitro antibacterial effect of the aqueous extract of T. terrestris on several oral bacteria.

Methods: In this experimental study, after preparing the aqueous extract of T. terrestris, minimum inhibitory and bactericidal concentrations (MIC and MBC) of the extract were determined against standard strains of Streptococcus mutans, Staphylococcus aureus, Klebsiella pneumoniae and Streptococcus pyogenes using the broth microdilution method. The experiments were repeated three times and the results were analyzed with SPSS 22 using the one-way analysis of variance (ANOVA) and LSD statistical tests with the significance level set at 0.05.

Results: The aqueous extract of T. terrestris had the highest inhibitory effect on S. pyogenes and S. mutans, and the difference between the MIC and MBC values was significant (P <0.05). However, no such effect was observed against S. aureus and K. pneumonia at concentrations below 50 mg/ml when compared to ampicillin and chlorhexidine.
Conclusion: The aqueous extract of T. terrestris has significant antibacterial effects against S. pyogenes and S. mutans. Therefore, it can be incorporated into topical formulations such as toothpaste and mouthwash products after further in vivo and toxicity experiments.
Keywords: Tribulus, Anti-Bacterial Agents, bacteria

INTRODUCTION
Nowadays, emergence of drug-resistant infectious diseases due to the inappropriate use of antibiotics is an important public health challenge. Alternative methods such as the use of bacteriocins, medicinal plants essential oils, antibodies, phage therapy, quorum-sensing inhibitors, and nanotherapy have been suggested to tackle this problem (1, 2). Medicinal plants have long been considered as one of the most important pillars of traditional medicine around the world. It is estimated that almost 80% of the world’s population depends on herbal products for healthcare needs, especially in developing countries (3). Medicinal plants have several bioactive compounds such as flavonoids, terpenes and terpinoids, which have shown antibacterial, anti-inflammatory, anticancer and antiviral effects (4-7). The mechanisms through which medicinal plants kill bacteria are different than antibiotics. This is a clinically significant advantage in the treatment of resistant infections (8, 9). Tribulus terrestris, commonly known as puncture vine, is a medicinal plant belonging to the Zygophyllaceae family. It is one of the native plants of the southeastern region of Iran that grows in tropical and arid regions. The plant contains bioactive compound such as triterpene glycosides (saponins), flavonoids, alkaloids and tannins that have been shown to have diuretic, aphrodisiac, anti-urolithic, immunomodulatory, hypolipidemic, cardioprotective, antidepressant, anxiolytic, hepatoprotective, anti-inflammatory, analgesic, antispasmodic, anticancer and antibacterial effects (10-12).
As the most prevalent chronic infection of oral cavity, periodontitis and dental caries have serious health and economic burdens. They are caused by deregulation of oral microbiota and subsequently not only affects oral health, but also correlates with some systemic diseases, such as diabetes, cancer and atherosclerosis, which indicates that the prevention and treatment of dental caries are important to attenuate this global health risk (13-15). Streptococcus mutans is a common resident microflora in dental plaque and also known as the main cause of dental caries. Staphylococcus aureus is a transient microflora of nasal mucosa but sometimes leads to purulent infections, systemic infections as well as nosocomial infections. Klebsiella pneumoniae is a transient normal microflora of the nasopharynx that may cause a type of pulmonary pneumonia. Streptococcus pyogenes is another transient normal flora of the upper respiratory tract and the main causative agent of purulent sore throat (16-18). Due to the presence of active compounds in T. terrestris and their potential antibacterial effects, in this study, we evaluated the in vitro effects of aqueous extract of the plant against the above mentioned oral bacteria to find scientific evidence for the production of a cheap and effective topical formulation of T. terrestris.

MATERIALS AND METHODS
The fruit of T. terrestris was purchased from a traditional market in Birjand, Iran, and authenticated by an expert botanist (Department of Botany, University of Birjand, Iran). It was washed, shade-dried and then powdered. To obtain an aqueous extract, 100 g of the powder were added to 1000 ml of boiling distilled water and boiled for 30 minutes. The solution was filtered through a filter paper (Whatman paper No. 41) and lyophilized using a vacuum freeze dryer (Dena Vacuum Industry, model FD-5005-BT, Iran). Then, the lyophilized extract was dissolved in autoclaved distilled water and passed through a 0.45 μm filter (19).
Four standard strains of different bacterial species including S. aureus (ATCC 29213), K. pneumoniae (ATCC 700603), S. mutans (ATCC 35668) and S. pyogenes (ATCC 10403) were purchased from the Pasteur Institute of Iran (Tehran, Iran). After preparation of bacterial suspensions, these suspensions were placed in cryotube and kept at -70 °C until use. To regenerate each strain of bacteria, we took a bullet impregnated with the desired bacteria from each cryotube and placed it in a test tube containing 3 ml of nutrient broth medium (Merck Co., Germany). Then, the tubes were incubated at 37 °C for 24 hours. After regenerating the bacterial strains, they were cultured on nutrient agar and blood agar and then isolated as pure colonies. These colonies were used to prepare microbial suspensions with a concentration of 0.5 McFarland (1.5 X 10⁸cfu/ml). To grow S. mutans and S. pyogenes, they were incubated in candle jars.
We used the latest edition of the Clinical and Laboratory Standards Institute(CLSI) guidelines to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the extract on the studied bacteria (20). To prepare the required concentrations of the extract, we dissolved 100 mg of the extract in 1 ml of sterile distilled water. The resulting solution was sterilized using a 0.45 μm filter paper and stored in a sterile, dark glass container at 3 °C until use. The MIC values were determined using the broth microdilution method (21).
Different dilutions were prepared from the T. terrestris extract. From each dilution, 100 μl were added to wells of a sterile 96-well microplate (Microteb Co., Iran) containing 100 μl of bacterial suspension in Mueller Hinton broth (Merck Co., Germany) with a concentration equal to half the McFarland standard. A well containing only medium and another containing only the extract were considered as negative control. Wells containing the bacterial suspension and the medium were considered as positive controls. The experiments were performed with chlorhexidine and ampicillin to compare data.
After inoculating the bacteria, the microplate was shaken for 30 seconds to make the mixture completely uniform. In the last step, the microplate was incubated at 37 °C for 24 hours. After incubation, turbidity of the wells was observed visually. The lowest concentration of extract that inhibited the growth of bacteria was determined as the MIC. The experiment was performed in triplicate.
To determine the MBC of the extract, 10 µl were taken from turbid wells (MIC concentration and above) in sterile condition and inoculated on Mueller Hinton agar (Merck Co., Germany). Streptococci were inoculated on blood agar (Merck Co., Germany). After 24 hours of incubation at 37 °C, the lowest dilution in which no colony appeared was determined as the MBC (22). All experiments were repeated three times.
Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed by SPSS Statistics 22.0 (IBM SPSS Statistics for Windows, IBM Corp, USA). Data were compared using the one-way analysis of variance (ANOVA) and LSD tests. P-values < 0.05 were considered statistically significant.

RESULTS
The aqueous extract of T. terrestris showed antibacterial effects against the selected bacteria. As shown in table 1, S. pyogenes was most sensitive to the extract with MIC and MBC values of 8.33

2.89 and 16.67

5.77, respectively. MIC and MBC values against S. mutans were 43.33

5.77 and 46.67

5.77, respectively. Bacterial growth was not observed in the wells containing the extract and culture medium as well as the culture medium alone (negative control). However, growth was observed in all wells containing the culture medium and the bacterial suspension (Positive control), which confirms the accuracy of the experiments.
Table 1. The MIC and MBC of the aqueous extract of T. terrestris, chlorhexidine and ampicillin on some oral pathogenic bacteria

	T. terrestris		Chlorhexidine		Ampicillin
Microorganism	MIC	MBC	MIC	MBC	MIC	MBC
S. pyogenes	8.33 2.89	16.67 5.77	500	500	0.25	0.25
S. mutans	43.33 5.77	46.67 5.77	500	500	3	3
S. aureus	>50	>50	8	8	32	32
K. pneumoniae	>50	>50	8	16	64	64

DISCUSSION
In this study, we examined the antibacterial effect of the aqueous extract of T. terrestris against several normal microflora and pathogenic bacteria of the oral cavity. Based on the findings, the aqueous extract of this plant had significant antibacterial effects on standard strains of the tested bacteria. The results indicated that the extract was more effective against S. pyogenes (MIC=8.33 mg/ml) and S. mutans (MIC=43.33 mg/ml). Al-Bayati et al. investigated the effects of aqueous, ethanolic and chloroform extracts of different parts of the Iraqis T. terrestris on some gram-positive and gram-negative bacteria. They reported that the aqueous extract of the plant had significant antibacterial effects on most of the bacteria tested, such as S. aureus (MIC=2.5 mg/ml) and K. pneumoniae (2.5 mg/ml). The lowest MIC values were observed for the ethanolic extract of fruit, leaf and root of the plant (23). The difference between the MIC value in our study and that in the Al-Bayati et al. may be due to the different geographical locations of the plant, as well as the different strains of bacteria in the two studies.
Soleimanpour et al. reported the antibacterial effects of ethanolic extract of T. terrestris against some bacteria, including of S. mutans (MIC and MBC=25 mg/ml) and S. aureus (MIC and MBC=50 mg/ml). They also reported the MIC and MBC values of chlorhexidine to be 0.0625 mg/ml and 0.125 mg/ml, respectively (24). The inconsistency between the results of their study and ours could be due to the use of different types of extracts.
Hakemi-Vala et al. examined the effects of the total aqueous and dimethyl sulfoxide extracts and phytophenol fraction (Benzoxazine derivative) of T. terrestris on some bacteria. The total extract showed a good antibacterial effect against P. aeruginosa (MIC=125 mg/ml), E. coli (MIC=62.5 mg/ml) and B. subtilis (MIC=500 mg/ml) but had no effect against S. aureus, S. epidermidis, P. aeruginosa, K. pneumonia, and Candida albicans. They also showed that the antibacterial effect was not due to the benzoxazine derivatives of the plant (25) .
Antibacterial effects of T. terrestris may be due to bioactive compounds such as frostanol, spirostanol, saponins, tigogenin, etc. By separating saponins from T. terrestris and performing antibacterial assays, Mohammed et al. showed that saponins can inhibit the growth of S. aureus and K. pneumonia by the disk diffusion method. They suggested that these compounds alter the surface tension of the bacterial outer membrane, causing changes in the membrane and killing the microorganism. In addition, due to their free carbonyl group, flavonoids can react with proteins on the surface of the bacterial membrane and kill the microorganism. The biological content of T. terrestris may vary in different geographical regions (10, 26, 27).
With the rapid emergence of antimicrobial-resistant microorganisms, there is an urgent need to implement some strategies including antibiotic stewardship programs and find alternatives for antibiotics. Indeed, medicinal plants have bioactive compounds with different mechanisms of action that have great potential to provide effective, biocompatible and economical solutions to accelerate the development of antimicrobial drugs. Therefore, more research is needed to provide a comprehensive knowledge of medicinal plants.

CONCLUSION
This study showed that T. terrestris has significant antibacterial effects against S. pyogenes and S. mutans. Therefore, it can be incorporated into topical formulations such as toothpaste and mouthwash products after further in vivo and toxicity experiments.

ACKNOWLEDGMENTS
This research was supported by the Birjand University of Medical Sciences, Birjand, Iran (grant number: 455366). The authors would like to thank the personnel of central research laboratory of Birjand University of Medical Sciences for helping us in the study.

ETHICS APPROVAL
This study was approved by the ethics committee of Birjand University of Medical Sciences, Iran (IR.BUMS.REC.1400.056)

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.

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Research Article: Research Article | Subject: Microbiology
Received: 2021/04/4 | Accepted: 2021/06/21 | Published: 2021/08/31 | ePublished: 2021/08/31

References

1. Zare Bidaki M, Arab M, Khazaei M, Afkar E, Zardast M. Anti-bacterial effect of zataria multiflora boiss. Essential oil on eight gastrointestinal pathogenic species. The Horizon of Medical Sciences. 2015;21(3):155-61. [View at Publisher] [DOI:10.18869/acadpub.hms.21.3.155] [Google Scholar]

2. Siadaty S, Siadati A, editors. New medicinal knowledge and consumption of medicinal plants. Conference Proceedings, Tonekabon Branch, Islamic Azad University; 2007. [View at Publisher] [Google Scholar]

3. Ekor M. The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Frontiers in pharmacology. 2014;4:177. [View at Publisher] [DOI:10.3389/fphar.2013.00177] [PubMed] [Google Scholar]

4. Tamokou Jde D, Chouna JR, Fischer-Fodor E, Chereches G, Barbos O, Damian G, et al. Anticancer and antimicrobial activities of some antioxidant-rich cameroonian medicinal plants. PLoS One. 2013;8(2):e55880 [View at Publisher] [DOI:10.1371/journal.pone.0055880] [PubMed] [Google Scholar]

5. Tetali SD. Terpenes and isoprenoids: a wealth of compounds for global use. Planta. 2019 ;249(1):1-8 [View at Publisher] [DOI:10.1007/s00425-018-3056-x] [PubMed] [Google Scholar]

6. Yonekura-Sakakibara K, Higashi Y, Nakabayashi R. The Origin and Evolution of Plant Flavonoid Metabolism. Front Plant Sci. 2019; 10: 943. [View at Publisher] [DOI:10.3389/fpls.2019.00943] [PubMed] [Google Scholar]

7. Mahizan NA, Yang SK, Moo CL, Song AA, Chong CM, Chong CW, Abushelaibi A, Lim SE, Lai KS. Terpene Derivatives as a Potential Agent against Antimicrobial Resistance (AMR) Pathogens. Molecules. 2019 19;24(14):2631. [View at Publisher] [DOI:10.3390/molecules24142631] [PubMed] [Google Scholar]

8. Vambe M, Aremu A, Chukwujekwu J, Finnie J, Van Staden J. Antibacterial screening, synergy studies and phenolic content of seven South African medicinal plants against drug-sensitive and-resistant microbial strains. South African Journal of Botany. 2018;114:250-9. [View at Publisher] [DOI:10.1016/j.sajb.2017.11.011] [Google Scholar]

9. Okwu MU, Olley M, Akpoka AO, Izevbuwa OE. Methicillin-resistant Staphylococcus aureus (MRSA) and anti-MRSA activities of extracts of some medicinal plants: A brief review. AIMS Microbiol. 2019 15;5(2):117-137. [View at Publisher] [DOI:10.3934/microbiol.2019.2.117] [PubMed] [Google Scholar]

10. Chhatre S, Nesari T, Somani G, Kanchan D, Sathaye S. Phytopharmacological overview of Tribulus terrestris. Pharmacogn Rev. 2014 ;8(15):45-51. [View at Publisher] [DOI:10.4103/0973-7847.125530] [PubMed] [Google Scholar]

11. Zhu W, Du Y, Meng H, Dong Y, Li L. A review of traditional pharmacological uses, phytochemistry, and pharmacological activities of Tribulus terrestris. Chem Cent J. 2017 11;11(1):60. [View at Publisher] [DOI:10.1186/s13065-017-0289-x] [PubMed] [Google Scholar]

12. Semerdjieva IB, Zheljazkov VD. Chemical constituents, biological properties, and uses of Tribulus terrestris: A review. Natural Product Communications. 2019;14(8):1934578X19868394. [View at Publisher] [DOI:10.1177/1934578X19868394] [Google Scholar]

13. Qiu W, Zhou Y, Li Z, Huang T, Xiao Y, Cheng L, et al. Application of Antibiotics/Antimicrobial Agents on Dental Caries. Biomed Res Int. 2020 31;2020:5658212. [View at Publisher] [DOI:10.1155/2020/5658212] [PubMed] [Google Scholar]

14. Liu XR, Xu Q, Xiao J, Deng YM, Tang ZH, Tang YL, et al. Role of oral microbiota in atherosclerosis. Clin Chim Acta. 2020 ;506:191-195. [View at Publisher] [DOI:10.1016/j.cca.2020.03.033] [PubMed] [Google Scholar]

15. Arweiler NB, Netuschil L. The Oral Microbiota. Adv Exp Med Biol. 2016;902:45-60. [View at Publisher] [DOI:10.1007/978-3-319-31248-4_4] [PubMed] [Google Scholar]

16. Yadav K, Prakash S. Dental caries: A microbiological approach. J Clin Infect Dis Pract. 2017;2(1):1-15. [View at Publisher] [DOI:10.4172/2476-213X.1000118] [Google Scholar]

17. Nomura R, Matayoshi S, Otsugu M, Kitamura T, Teramoto N, Nakano K. Contribution of Severe Dental Caries Induced by Streptococcus mutans to the Pathogenicity of Infective Endocarditis. Infect Immun. 2020 22;88(7):e00897-19. [DOI:10.1128/IAI.00897-19] [PubMed] [Google Scholar]

18. Al-Otaibi MK, Ahmed S, Al-Abdullah FA, Sabbagh OM, Al-Qahtani JM, Al-Mutairi FH, et al. Bacteriological correlation between dental plaque and chronic tonsillitis. Journal of Interdisciplinary Dentistry. 2019;9(3):119. [View at Publisher] [DOI:10.4103/jid.jid_5_19] [Google Scholar]

19. Alizadeh Behbahani B, Noshad M, Falah F. Cumin essential oil: Phytochemical analysis, antimicrobial activity and investigation of its mechanism of action through scanning electron microscopy. Microb Pathog. 2019 ;136:103716. [View at Publisher] [DOI:10.1016/j.micpath.2019.103716] [PubMed] [Google Scholar]

20. Clinical, Institute LS. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute Wayne, PA; 2017. [View at Publisher]

21. Cugnata NM, Guaspari E, Pellegrini MC, Fuselli SR, Alonso Salces RM. Optimal concentration of organic solvents to be used in the broth microdilution method to determine the antimicrobial activity of natural products against Paenibacillus larvae. Journal of Apicultural Science. 2017; 61(1): 37-53. [DOI:10.1515/jas-2017-0004] [Google Scholar]

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