Minimal Inhibitory Concentration and Minimal Bactericidal Concentration of Peppermint (Mentha piperita) extracts against standard microorganisms
Ms. Hiral Vasavada1, Dr. Sailaja Inampudi2
1Ph.D Scholar, Department of Biotechnology, Parul University of Applied Sciences,
Parul University, Limda, Waghodia, Vadodara, Gujarat - 391769, India.
2Faculty of Department of Biotechnology, Parul University of Applied Sciences,
Parul University, Limda, Waghodia, Vadodara, Gujarat - 391769, India.
*Corresponding Author E-mail: vasavadahiral74@gmail.com, inampudisailaja@rediffmail.com
ABSTRACT:
Different plant extracts are considerably safe from infectious agents and may be used for medical purposes. The present research was conducted against the six standard microorganisms to quantify the antimicrobial activities of peppermint (Mentha piperita) extracts. The traditional approaches of minimum bactericidal concentration (MBC) and minimal inhibitory concentration (MIC) were used to approximate the antibacterial activities of ethanol, methanol, and chloroform peppermint extracts. The inhibitory function of the ethanol extract was comparable to that of chloroform (10 to 80mg/ml) and methanol (10 to 80mg/ml) against all gram-negative microorganisms. The minimum value of MIC was recorded for Streptococcus pyogenes (5mg/ml for extract of ethanol), followed by E. coli (10mg/ml for extract of ethanol) and then by Enterococcus faecalis (15mg/ml for extract of ethanol). With respect to the standard microorganisms, the MBC values were higher for both extracts than the corresponding MIC values. This work demonstrated the possible efficacy of antibacterial action on M. Piperita extracts from normal microorganisms (A. Baumenii, Escherichia coli, Streptococcus pyogenes, Enterococcus faecalis, Pseudomonas aeruginosa and Klebsiella pneumoniae), particularly ethanol extract. In summary, the peppermint ethanol extract had important growth-inhibiting effects on observed standard micro-organisms, followed by chloroform and methanol extracts. Further to in vitro and in vivo studies on a wide variety of natural microorganisms and therapeutic isolates are required to investigate and standardize the inhibitory activity of peppermint extracts against the most dangerous human pathogenic agents.
KEYWORDS: Mentha piperita, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC).
INTRODUCTION:
There are many grounds for AMR’s growth. Some of the more critical of these is antibiotic overuse/and/or excessive use of antimicrobials, which allows resistance production and dissemination far more possible3.
Inadequate professional treatment of an infection associated with unsuitable antimicrobial medication leads to selective strain and accelerates AMR. In addition to this, the practice of adding antibiotics to farm feed promotes drug resistance. More than half of US-produced antibiotics are used for agricultural purposes4-6. Leading infectious disease killers inequitably affect the developing countries. Not only can we blame new infectious disease pathogens, also aggressive species threatening communities where public health system is broken, vaccinations are not readily available; substandard and falsified medications occur, lack of exposure to clinical facilities, and inappropriate conduct accessing health treatment7.
Several reports have documented that bacteria have gained tolerance owing to extended therapy with modern antibiotics with wide-ranging effectiveness by growth-inhibitory effects on target species, making current antibiotic medication practically ineffective. Therefore, the demand for creating alternate approaches to traditional antibiotic therapy is increased.8 The antimicrobial activity of substances deriving from plant extracts has been recognized and widely studied for many years for medicinal purposes9,10. Mentha piperita L. is a hybrid of Mentha spicata L., a medicinally important plant belonging to the Lamiaceae family and commonly known as peppermint11. It was developed by the ancient Egyptians, and recorded in the 13th century Icelandic pharmacopoeia12. It is widely grown in temperate parts of the world, particularly in Europe, North America and North Africa, but is nowadays cultivated in all parts of the world13. It is grown primarily for its oil which is extracted from the flowering plant's leaves14. Peppermint oil is used for herbal flavouring and oral products such as toothpastes, dental creams, and mouth washes. Traditionally, higher and aromatic plants have been used in folk medicine as well as for extending food shelf life, showing inhibition against bacteria, fungi and yeast. The majority of their properties stem from essential oils produced as secondary metabolites15. Methanol extract have been used in the assessment of many plant extracts16. Similarly, ethanol extract have also been used in the assessment of many plant extracts17,18. Other than methanol and ethanol, ethylacetate extract is also employed to assess the antimicrobial properties of essential oils from plants.19 The current study was undertaken to estimate the antimicrobial activities of peppermint (Mentha piperita) extracts against the six standard microorganisms.
MATERIAL AND METHODS:
Study population:
This descriptive study was conducted at the Department of Biotechnology, Parul University of Applied sciences, Parul University, Vadodara, Gujarat, India during a 6-month period from June-2019 to December 2019. A total of six standard microorganisms were included in this study.
Standard microorganisms:
All standard microorganisms were obtained from National Chemical Laboratory, Pune, India. The selected organisms were Acinetobacter baumenii, Escherichia coli, Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae and Pseudomonas aeruginosa. Gram positive organisms included Enterococcus faecalis and Streptococcus pyogenes. While, gram negative organisms included Acinetobacter baumenii, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. The bacterial strains were grown in the sterile Muller Hinton Medium nutrient broth and maintained on nutrient agar slants at 4°C.
Peppermint collection:
The aerial parts of peppermint (M. piperita) were obtained from the plant grown in Vadodara city at full flowering stage in June–September 2019. The plant was identified by the Department of Pharmacy, Parul University, Vadodara, India. The samples were cleaned in the shade using sterile double distilled water to prevent volatility of the plant material constituents and to keep the natural colour of the sample fixed. They were then air-dried and powdered using a milling machine and kept in a cool dry place until ready for extraction with the selected solvents.
Peppermint extracts preparation:
Soxhlet Extraction or Hot continuous extraction was employed. The powdered plant material was soaked in ethanol, methanol and chloroform in individual beakers by keeping them in a shaker for 3 days. Soxhlet Extraction or Hot continuous extraction was employed. All the extracts were stored at 2°C to 8°C for antibacterial activity testing.
Inoculum preparation:
All bacterial isolates were grown to the exponential phase in tryptic soy broth (TSB) (Difco Laboratories, Detroit, USA) at 37°C for 18 h and adjusted to a final density of 108 cfu/ml.
Antimicrobial testing:
The antimicrobial activity of ethanol, methanol and chloroform extracts of peppermint was carried out using the standard minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) methods (CLSI, 2014).
The minimum inhibitory concentration:
The MIC of peppermint extracts was determined by the broth microdilution method following the guidelines of the CLSI (CLSI, 2014). All strains were cultured in sterile Muller Hinton (MH) agar (Oxoid, UK) and incubated at 37°C for 24 h prior to MIC determination. An inoculum density equivalent to 0.5 McFarland of each of the test organisms was prepared in sterile saline (0.84% NaCl). Double strength MH broth (100ml) containing 5% dimethyl sulfoxide (DMSO) was dispensed into wells of 96-well microtiter plates. Two row lines in each plate were used as controls (in a serial dilution of 32–0.015mg/ml): One row line with vancomycin (BioMerieux, Marcy-L’Etoile, France) as a positive control for Gram-positive isolates and another row line with tobramycin (BioMerieux) for Gram-negative strains. The stock extract solutions were diluted and transferred into the first well, and serial dilutions were performed so that concentrations in the range of 80–1.25mg/ml, (i.e. 80, 40, 20, 10, 5 and 2.5mg/ml) were obtained. To each well, 15 ml of each bacterial suspension (equivalent to 0.5 McFarland/100) was added. The set was allowed to incubate aerobically at 37°C for 24 h. The assay for each of the standard microorganisms was repeated three times to assess and attest reproducibility. MIC was defined as the lowest concentration of each extract that inhibited visible growth.
The minimal bactericidal concentration:
The wells with no visible growth were selected and samples were used to determine the MBC. Briefly, after homogenization, a loop of each suspension was cultured on sterile MH agar. This culture was incubated aerobically at 37°C overnight. The MBC of each was estimated from the culture medium in which no visible microbial growth was recorded upon examination. This experiment was repeated three times to ensure reproducibility.
RESULTS:
The MIC values of ethanol, methanol and chloroform extracts of peppermint against six standard microorganisms are listed in Table 1. Overall, the ethanol extract of peppermint had strong growth inhibitory effects on most of the standard microorganisms, followed by the chloroform and ethanol extracts. The ethanol extract exhibited a stronger antibacterial activity against Gram-positive (5–15 mg/ml) than Gram-negative (10–80mg/ml) bacteria. The lowest MIC value was seen for S. pyogenes (5 mg/ml for the ethanol extract), followed by E. coli (10 mg/ml for ethanol extract) and E. faecalis (15 mg/ml for the ethanol extract). The highest MIC value (80 mg/ml) was recorded for A. baumannii for all the three extracts and P.aeruginosa against methanol and ethanol extracts. The MBC values of ethanol, methanol and chloroform extracts of peppermint against six standard microorganisms are listed in Table 2. The MBC values of all extracts were higher than the corresponding MIC values for the majority of study bacteria. However the MBC values were equal to the MIC values for the chloroform extract against Acinetobacter baumenii and S. pyogenes.
Table 1: The minimal inhibitory concentration (MIC) values of three peppermint extracts against six standard microorganism
|
Standard microorganisms |
MIC values (mg/ml) Peppermint extracts |
MIC values (µg/ml) Positive control |
|||
|
Methanol Extract |
Ethanol Extract |
Chloroform extract |
Tobramycin |
Vancomycin |
|
|
Acinetobacter baumenii |
80 |
80 |
80 |
1.0 |
2.0 |
|
E. coli |
40 |
10 |
10 |
0.5 |
1.0 |
|
S. pyogenes |
15 |
5 |
10 |
0.5 |
0.5 |
|
E. faecalis |
15 |
15 |
15 |
0.25 |
0.25 |
|
K. pneumoniae |
40 |
20 |
20 |
1.0 |
1.0 |
|
P. aeruginosa |
80 |
80 |
40 |
0.5 |
0.5 |
Table 2: The minimal bactericidal concentration (MBC) values of three peppermint extracts against six standard microorganisms
|
Standard microorganisms |
MBC values (mg/ml) Peppermint extracts |
||
|
Methanol extract |
Ethanol extract |
Chloroform extract |
|
|
Acinetobacter baumenii |
80 |
80 |
80 |
|
E. coli |
20 |
20 |
40 |
|
S.pyogenes |
10 |
2.5 |
10 |
|
E.faecalis |
20 |
10 |
20 |
|
K.pneumoniae |
40 |
20 |
80 |
|
P.aeruginosa |
80 |
80 |
80 |
DISCUSSION:
AMR is growing worldwide at an alarming pace, compromising our ability to treat infectious diseases, as well as undermining many other advances in health and medicine. Awareness of the health and economic consequences of AMR constitutes a heavy and growing burden on high- income countries, middle- income countries and low-income countries, requiring urgent action at national, regional and global levels, particularly in view of the limited development of new antimicrobial agents8,9. Thus, there is a need for the discovery of new substances from natural sources, including plants to use their extracts as antimicrobial substances. A recent in-vitro study by Sharma and Karnwal (2018) stressed on the impact of herbal extracts in biocontroling of four human pathogenic bacteria20. Many microorganisms, which cause damage to human health, exhibit drug resistance due to inadequate use and misuse of antibiotics. So, the alternative strategies to fight antibiotic-resistant bacteria are the use of natural antimicrobial substances such as plant essential oils and their components21,22. Our study showed the effective antimicrobial action of M. piperita extracts, especially the ethanol peppermint extract against S. pyogenes, E. faecalis, E. coli and K. pneumonia. These results are consistent with some previous studies. One study reported that the MBC for E. coli, K. pneumonia, P. aeruginosa, S. aureus, and S. faecalis were >7.50, 3.50, >7.50, >7.50, and 3.50 mg/ml, respectively23. In one study, M. piperita antimicrobial activity was recorded only against S. aureus and not E. coli24. Another study reported higher value of the mean MBC for peppermint oil25. One study showed that the compounds from M. piperita possess potent antimicrobial activity, suggesting that M. piperita leaf extracts contain effective active constituents responsible for eliminating bacterial pathogens26. The differences in M. piperita antimicrobial activity could be due to a difference in chemical composition of the oils collected from different parts of the world. The weak antimicrobial action of different M. piperita extracts (other than the ethanol extract) was seen for P. aeruginosa, S. maltophilia and A. baumannii, as these Gram-negative pathogens express a variety of determinants that confer resistance to a broad array of antimicrobial agents. Mechanisms of resistance include impaired entry through the bacterial outer membrane, production of antibiotic-modifying enzymes, active efflux, and target mutations that reduce antimicrobial affinity. Similar findings were found by other studies who evaluated the antimicrobial activity of peppermint oil samples against pathogenic and potentially pathogenic bacteria. All of the tested peppermint oils demonstrated higher activity against gram-positive bacteria and weaker against gram-negative bacteria27,28.
To the best of our knowledge, our study is advantageous in investigating the antibacterial activity of M. piperita extracts against these standard microorganisms. Our finding is of considerable concern. The pathogenic forms of these standard microorganisms are notorious for their remarkable innate and acquired resistance to multiple antimicrobial classes, and to survive in nosocomial environments. This study has some limitations. The first limitation is that standard microorganisms were representative of its pathogenic clinical isolates. Second, the study investigated the antimicrobial activity against only standard microorganisms. It is also likely that extensive further research is needed to prove the available antibacterial effect from peppermint extracts against a wide collection of standard microorganisms having potential for pathogenecity.
This study highlights the potential antibacterial activity for M. piperita extracts, especially ethanol extract, against Streptococcus pyogenes, Escherichia coli, Enterococcus faecalis and Klebsiella pneumonia suggesting that, with further research and refinement, M. piperita leaf extracts could find a place in the treatment of some microbial infections in topical applications. Further in vitro and in vivo studies on a large number of standard microorganisms having potential for pathogenecity are necessary to further investigate and standardize the inhibitory effect of peppermint extracts. Nevertheless, the promising results in this study may open a window for potential new anti-bacterial agents.
ACKNOWLEDGEMENT:
The authors are grateful to the authorities of Parul University, Department of Biotechnology, Parul University of Applied Sciences, Waghodia, Vadodara for the facilities.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
REFERENCES:
1. Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF, Sumpradit N, Vlieghe E, Hara GL, Gould IM, Goossens H, Greko C. Antibiotic resistance—the need for global solutions. The Lancet infectious diseases. 2013; 13(12): 1057-98.
2. Allcock S, Young EH, Holmes M, Gurdasani D, Dougan G, Sandhu MS, Solomon L, Török ME. Antimicrobial resistance in human populations: challenges and opportunities. Global Health, Epidemiology and Genomics. 2017; 2.
3. Uchil RR, Kohli GS, Kate Khaye VM, Swami OC. Strategies to combat antimicrobial resistance. Journal of clinical and diagnostic research: JCDR. 2014; 8(7): ME01.
4. Goldberg AM, Hartung T. Protecting more than animals. Scientific American. 2006; 294(1) :84-91.
5. Laxminarayan R. How broad should the scope of antibiotics patents be?. American Journal of Agricultural Economics. 2002; 84(5): 1287-92.
6. Horrigan L, Lawrence RS, Walker P. How sustainable agriculture can address the environmental and human health harms of industrial agriculture. Environmental health perspectives. 2002; 110(5): 445-56.
7. Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology. 2010; 8(6): 423-35.
8. George J. Bioactive Screening and Antimicrobial Activity of Selected Three Medicinal Plants on Chosen Microbes. Research Journal of Pharmacy and Technology. 2014; 7(11): 1264-9.
9. Anup K, Mohan K, Suraj S, Sandip F, Bhushan F, Prashant W. Antimicrobial Activity of Some Important Medicinal Plants of India against Some Plant and Human Pathogens. Research Journal of Pharmacy and Technology. 2010; 3(3): 924-6.
10. Hindi NK, Abdul-Husin IF, Al-Mahdi ZK, Ewadh RM, Hossain AO, Kadhim MJ, Alnasraui AH, Al-Yaseri AA. Effectiveness of Aqueous extract of Green, Black and Red Tea Leaves against some types of Gram positive and negative bacteria. Research Journal of Pharmacy and Technology. 2017; 10(6): 1957-62.
11. Singh R, Shushni MA, Belkheir A. Antibacterial and antioxidant activities of Mentha piperita L. Arabian Journal of Chemistry. 2015; 8(3): 322-8.
12. Pramila DM, Xavier R, Marimuthu K, Kathiresan S, Khoo ML, Senthilkumar M, Sathya K, Sreeramanan S. Phytochemical analysis and antimicrobial potential of methanolic leaf extract of peppermint (Mentha piperita: Lamiaceae). Journal of Medicinal Plants Research. 2012 Jan 16; 6(2): 331-5.
13. Jeyakumar E, Lawrence R, Pal T. Comparative evaluation in the efficacy of peppermint (Mentha piperita) oil with standards antibiotics against selected bacterial pathogens. Asian Pacific Journal of Tropical Biomedicine. 2011; 1(2): S253-7.
14. Bupesh G, Amutha C, Nandagopal S, Ganeshkumar A, Sureshkumar P, Murali K. Antibacterial activity of Mentha piperita L. (peppermint) from leaf extracts-a medicinal plant. Acta Agriculturae Slovenica. 2007; 89(1): 73.
15. Sarbeen JI. Preliminary phytochemical analysis of Peppermint Oil and Tulsi Oil. Research Journal of Pharmacy and Technology. 2015; 8(7): 929-31.
16. Nuraskin CA, Idroes R, Soraya C. Study of Inhibition of Methanol Extract of Laban Leaf (Vitex Pinnata) against Sreptococcus mutans with Microdilution. Research Journal of Pharmacy and Technology. 2019; 12(12): 6037-40.
17. Nair DU, Saraswathy MP, Kishore N, Pully NR. Phytochemical and Antimicrobial Evaluation of Luffa cylindrica Linn. Leaf and Flower Extracts–An In-Vitro Study. Research Journal of Pharmacy and Technology. 2010; 3(2): 438-41.
18. Thilagavathi T, Kathiravan G. Phytochemical Analysis and Antimicrobial Activity of Ethanolic Leaf Extract of Ficus racemosa Linn. Research Journal of Pharmacy and Technology. 2017; 10(2): 537-40.
19. Anowi CF, Cardinal NC, Ike C, Ezeokafor E. Antimicrobial properties of the ethyl acetate extract of the stem bark of Nauclea latifolia. Int. J. Adv. Pharm. Biol. Chem. 2012; 1: 183-91.
20. Sharma H, Karnwal A. Impact of herbal extracts in biocontroling of four human pathogenic bacteria-an in-vitro study. Research Journal of Pharmacy and Technology. 2018; 11(7): 2895-900.
21. Swamy MK, Akhtar MS, Sinniah UR. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review. Evidence-Based Complementary and Alternative Medicine. 2016; 2016.
22. Harfouch RM, Darwish M, Al-Asadi W, Mohammad AF, Gharib NM, Haroun M. Antibacterial Activity of Essential Oils of Rosmarinus officinalis, Salvia officinalis and Anthemis nobilis Widespread in the Syrian Coast. Research Journal of Pharmacy and Technology. 2019; 12(7): 3410-2.
23. Andoğan BC, Baydar H, Kaya S, Demirci M, Özbaşar D, Mumcu E. Antimicrobial activity and chemical composition of some essential oils. Archives of Pharmacal research. 2002; 25(6): 860-4.
24. Thosar N, Basak S, Bahadure RN, Rajurkar M. Antimicrobial efficacy of five essential oils against oral Pathogens: An in vitro study. European Journal of Dentistry. 2013; 7(S 01): S071-7.
25. Castillo-Juárez I, González V, Jaime-Aguilar H, Martínez G, Linares E, Bye R, Romero I. Anti-Helicobacter pylori activity of plants used in Mexican traditional medicine for gastrointestinal disorders. Journal of Ethnopharmacology. 2009; 122(2): 402-5.
26. Zaidi S, Dahiya P. In vitro antimicrobial activity, phytochemical analysis and total phenolic content of essential oil from Mentha spicata and Mentha piperita. International Food Research Journal. 2015; 22(6): 2440.
27. Chao SC, Young DG, Oberg CJ. Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses. Journal of Essential Oil Research. 2000; 12(5): 639-49.
28. Prabuseenivasan S, Jayakumar M, Ignacimuthu S. In vitro antibacterial activity of some plant essential oils. BMC Complementary and Alternative Medicine. 2006; 6(1): 39.
Received on 11.07.2020 Modified on 17.09.2020
Accepted on 15.10.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(9):4662-4666.
DOI: 10.52711/0974-360X.2021.00810