Abstract: The clinical usefulness of gentamicin is limited due to the development of nephrotoxicity. Several natural agents have been used to ameliorate drugs toxicity. The survey of literature reveals that the Mentha piperita Linn. is found to be used in the traditional system of medicine. In the course of an ongoing UOH-project evaluate the effects of M. piperita L. on nephrotoxicity in rat model. So, the present study was designed to determine the pharmacological dose (oral LD50) and antibacterial activity of M. piperita leaf extracts for nephrotoxicity study. Freshly prepared ethanolic and aqueous extracts of M. piperita (EMPet and AMPet) at the following concentrations, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 g kg–1 b.wt., were orally administered to rats to find out the LD50 values of them. The LD50 was calculated by both arithmetically and graphically according to the method of Ghosh. The antibiotic activities of both extracts were tested against a variety of Gram-positive and Gram-negative bacteria. The LD50 of EMPet was found to be 3.7 and 3.6 g kg–1 b.wt., by arithmetic and graphical method, respectively. Similarly, AMPet were 4.8 and 4.69 g kg–1 b.wt., by arithmetic and graphical method, respectively. The inhibition zone for both Gram-negative and Gram-positive bacteria range from 5.0-20 mm and the lowest minimum inhibitory concentrations values were found in Staphylococcus. hominis. In conclusion, this pilot study revealed that EMPet and AMPet administered at a dose of 300 and 400 mg kg–1 b.wt., were effective, respectively. The active chemical compounds present in M. piperita have potential antibacterial activity.
INTRODUCTION
Mentha piperita, the peppermint plant belongs to the Family Lamiaceae. It is an aromatic and carminative herb cultivated throughout all regions of the world (Saharkhiz et al., 2012) have traditionally been used in folk remedy or in complementary and alternative medical therapy. The peppermint is widely used as flavoring, additive in foods, the preparation of toothpaste, chewing gum, mouthwash, soaps, sweets, balms or creams and cough medicine (Iwu et al., 1999; Georgiev and Stoyanova, 2006; Cragg and Newman, 2001; Sharafi et al., 2010) and other hygienic products and in pharmaceutical formulations (Simoes and Spitzer, 2000). A literature study reveals that peppermint has been ascribed a variety of biological properties, viz., antiallergenic (Inoue et al., 2002), antibacterial (Shapiro et al., 1994), anti-inflammatory (Inoue et al., 2002), antimycotic (Pattnaik et al., 1996), antitumor (Ohara and Matsuhisa, 2002), antiviral (Yamasaki et al., 1998), gastrointestinal protective (Mahmood et al., 2003), hepatoprotective (Akdogan et al., 2003) and chemopreventive (Samman et al., 1998). Several other studies have shown that it has antioxidant, antiperoxidative properties (Krishnaswamy and Raghuramulu, 1998; Al-Sereiti et al., 1999; Dorman et al., 2003). It is also used for antimutagenic purpose (Hossain et al., 2012) and symptomatic relief of the common cold (Stojanova et al., 2000). The formulation products from peppermint are used to decrease symptoms of irritable bowel syndrome and decrease digestive symptoms such as dyspepsia, nausea (Sharafi et al., 2010; Hossain et al., 2009) and used as an analgesic and to treat headache (Samarth et al., 2006). Mentha piperita contains active ingredients, such as menthol, menthone and menthyl acetate flavonoids, polymerized polyphenols, carotenes, tocopherols, saponin and choline (Saharkhiz et al., 2012; Iwu et al., 1999; Georgiev and Stoyanova, 2006; Cragg and Newman, 2001; Sharafi et al., 2010) together with several other minor constituents, including pulegone, menthofuran and limonene (Nair, 2001) and some of its constituents may have immunomodulating properties (Juergens et al., 2004, 2003; Raphael and Kuttan, 2003; Hamada et al., 2002) and effective in conditions such as arthritis and rheumatism (Darshan and Doreswamy, 2004).
Gentamicin (GM) is widely applied in human clinical practices for treatment of life threatening Gram-negative infections (Nagai and Takano, 2004; Tavafi, 2012). The antibiotics also cause drug induced a dose-dependent nephrotoxicity in 10-20% of therapeutic courses. Therefore, the clinical usefulness of this drug is limited due to the development of nephrotoxicity (Cuzzocrea et al., 2002). Thus, a therapeutic approach to protect or reverse renal damage would have very important clinical consequences. Several natural agents have been used to ameliorate some toxic and carcinogenic and drugs toxicity. The survey of literature reveals that the Mentha piperita Linn. are found to be used in the traditional system of medicine as a liver tonic. Many studies shows that various oral dose of M. piperita extracts were used viz g kg–1 b.wt. (Sharma et al., 2007; Samarth and Samarth, 2009) and 100 mg kg–1 b.wt. (Thangapandiyan et al., 2013). However nephroprotective activity of M. piperita has not been scientifically investigated. In the course of an ongoing UOH-project (CM4 2013) to evaluate the effects of M. piperita L. on nephrotoxicity in rat model. So, the present study was design to determine the LD50 and antibacterial activity of M. piperita leaf extracts.
MATERIALS AND METHODS
Preparation of plant extracts: Separated leave of M. piperita (Fig. 1a) was washed with tap water to remove the dust and other foreign materials (Fig. 1b). Washed leaves were dried under shade for one week (Fig. 1c). Approximately about 500 g of air-dried whole leaves were pulverized into powdered form (Fig. 1d) by using heavy duty commercial blender.
Preparation of ethanolic Mentha piperita extracts (EMPet): The powder samples (50 g) were extracted with 95% ethanol (1:3 w/v) by using Soxhlet extractor at 37°C for two days. The total yield was 4.67 g (9.34% w/w) of dark greenish extract. The EMPet from M. piperita was reconstituted to a final concentration of 5% (w/v) using aqueous solution of gum acacia 5%, (Fig. 1e) for further treatments.
Preparation of aqueous Mentha piperita extracts (AMPet): The aqueous extracts of M. piperita leaves were prepared according to the method of Hossain et al. (1992). The M. piperita leaves yielded 13% light greenish semisolid which was stored at 0-4°C until used.
Acute toxicity studies: Male Wistar albino rats weighing 130-140 g (7-8 weeks of age) were used for acute toxicity studies. The animals were divided into number of experimental groups (lower doses and higher doses groups) 10 animals for each group. All animals were allowed to fast by withdrawing the food and water for 18 h. Freshly prepared EMPet and AMPet at the following concentrations, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 g kg–1 b.wt., were orally administered to rats to find out the LD50 values of them. The animals were provided with food and water immediately after the plant drugs administration.
| Fig. 1(a-f): | Various stages of extraction of M. piperita leaves, (a): Fresh M. piperita L, (b): Separated cleaned leaves, (c): Dried leaves under shadow, (d): Powdered leaves, (e): Gum acacia and (f): Final extracts of M. peperita (EMPet and AMPet) |
The LD50 value of the plant extracts was calculated by both arithmetically and graphically according to the method of Ghosh (1984). For the interpretation of the toxicity data, the observed percentage mortality was converted into probit by referring to Table 1 (Ghosh, 1984). The LD50 of the plant extracts was calculated by the following formula:
Determinations of antimicrobial activity: Antibiotic activity of EMPet and AMPet were tested against a variety of Gram-positive and Gram-negative clinical isolates according to Kirby-Bauer method as described by Hudzicki (2009). One plate of each test microorganism was taken and colonies were transferred into normal saline under aseptic conditions. Density of each microbial suspension was adjusted to be equal to that of 106 CFU mL–1 (standardized by 0.5 McFarland standard). The bacterial suspensions were then spread uniformly with sterile swab stick on Nutrient Agar (NA) plates. Sterile filter paper disks were then placed onto the bacterial culture thus spread on the NA plates maintaining uniform distance from each other with a sterile forceps. Different concentrations (5-20 μL) of the plant extract from a 1% (w/v) solution were then delivered onto the filter paper disks. The plates were then kept at room temperature for 15 min. Then the plates were incubated at 37°C for 24 h. The zones of inhibitions around the disks were measured and recorded.
RESULTS
The LD50 of the M. piperita leaves extracts was calculated by using the formula:
The LD50 of EMPet was found to be 3700 mg kg–1 b.wt., by arithmetic method (Table 2) and also it was found 3.6058 g kg–1 b.wt., by graphical method (Fig. 2). Similarly, the LD50 of AMPet was found to be 4800 mg kg–1 b.wt., by arithmetic method (Table 3) and also it was found 4.6989 g kg–1 b.wt., by graphical method (Fig. 3). Then 1/10th of the LD50 values of both EMPet and AMPet were fixed as pharmacological dose. From both arithmetic and graphical methods shows the EMPet administered at the dose of 300 mg kg–1 b.wt. and AMPet administered at the dose of 400 mg kg–1 b.wt., were effective than the rest of the doses (Table 4). The antibacterial activity of EMPet and AMPet were evaluated according to their zones of growth inhibition against various pathogens measured in mm (Fig. 4). The inhibition zone for both Gram-negative and Gram-positive bacteria range from 5.0-20 mm and the lowest minimum inhibitory concentrations values were found for the S. hominis. All the tested microorganisms EMPet showed more potential antibacterial activity compared with AMPet.
DISCUSSION
The aim of the present study was to calculating the LD50 values for the EMPet and AMPet, given orally in rats, because of wide differences in the reported results from other studies (Sharma et al., 2007; Thangapandiyan et al., 2013; Samarth and Samarth, 2009). The dose dependent studies were carried out to find out effective pharmacological dose of the plant extracts for further experimental studies. The LD50 of M. piperita leaves extracts were then fixed 1/10th as pharmacological doses.
| Table 1: | Transformation of percentage mortalities to probits |
| Table 2: | Results of the lethal doses determination after oral ingestion of EMPet (n = 10) |
| *: The data below 2.5 g kg–1 b.wt. and above 5.0 g kg–1 b.wt., were omitted for calculation, #: Corrected formula for 0% dead = 100×0.25/n for 100% dead = 100× (n-0.25)/n, where n is the number of animals in each group LD50 of EMPet = 5000-(13,000/10) = 3700 mg kg–1 b.wt. | |
| Table 3: | Results of the lethal doses determination after oral ingestion of AMPet (n = 10) |
| *: The data below 3.5 g kg–1 b.wt. and above 6.0 g kg–1 b.wt., were omitted for calculation, #: Corrected formula for 0% dead = 100×0.25/n for 100% dead = 100X (n-0.25/n), where, n is the number of animals in each group and LD50 of AMPet = 6000 - (12,000/ 10) = 4800 mg kg–1 b.wt. | |
| Table 4: | LD50 and pharmacological doses of EMPet and AMPet |
The EMPet administered at a dose of 300 mg kg–1 b.wt., were effective. Similarly, the AMPet administered at 400 mg kg–1 b.wt., were effective than the rest of the doses.
Phytochemicals derived from plant products serve as a prototype to develop less toxic and more effective medicines in controlling the growth of microorganism (Kelmanson et al., 2000; Ahmad and Beg, 2001). These plant products have significant therapeutic application against human pathogens including bacteria. Numerous studies have been conducted with the extracts of various plants, screening antimicrobial activity as well as for the discovery of new antimicrobial compounds (Guleria and Kumar, 2006; Zakaria et al., 2007). In the present investigation, different extracts of M. piperita was evaluated for exploration of their antibacterial activity against certain Gram-negative and Gram-positive bacteria which was regarded as human pathogenic microorganism.
| Fig. 2: | Graphical representation of LD50 of EMPet LD50 = antilog 0.557 = 3.6058 g kg–1 b.wt. |
| Fig. 3: | Graphical representation of LD50 of AMPet LD50 = antilog 0.672 = 4.6989 g kg–1 b.wt. |
| Fig. 4: | Antibacterial effects of EMPet and AMPet on Gram-negative and Gram-positive bacteria strains |
The alcoholic extract of M. piperita showed significant antibacterial activity against clinically isolated microorganisms than aqueous extract. It is clear indicates that the effectiveness of the extracts largely depends on the type of solvent used. This will support the synergistic efficacy to treat the Gram-negative bacteria with gentamicin with minimize nephrotoxicity.
CONCLUSION
In conclusion, this pilot study revealed that the ethanolic and aqueous extracts of Mentha piperita administered at a dose of 300 and 400 mg kg–1 b.wt., were effective respectively. Finally, it can be conclude the active chemical compounds present in Mentha piperita have potential antibacterial activity.
ACKNOWLEDGEMENTS
We are grateful to Dr. Ashfaque Hossain from Department of Microbiology, for his encouragement and helpful discussions. This project was funded by the Deanship of Scientific Research, University of Hail, Kingdom of Saudi Arabia and award number (CM4 2013).