Introduction
Medicinal plants are used for treatment in almost all cultures. Ensuring the safety, quality and efficacy of these plants has recently become a fundamental issue in developing countries. By standardizing and evaluating the health of active plant compounds, herbal medicines can help usher in a new era of health care systems for the treatment of human diseases in the future. Knowledge of traditional medicine and medicinal plants can play an essential role in exploiting and discovering natural resources of plants [2]. According to the definition of the World Health Organization (WHO), a medicinal plant is a plant that contains substances in one or more of its organs that can be used for therapeutic purposes or precursors for pharmaceutical semi-synthesis [1]. Herbal products are often considered harmless, but studies have shown that they may contain pathogenic microorganisms [3]. Microbial contamination of medicinal plants can be related to a wide range of contaminants, including bacteria, fungi and viruses [4]. The pollutants that pose a serious health risk are pathogenic bacteria such as Salmonella, Escherichia coli, Staphylococcus aureus, Shigella species and other gram-positive and gram-negative bacteria [6-10]. The presence of bacteria such as E. coli and Salmonella in medicinal plants indicates fecal contamination and poor sanitary conditions in their preparation and storage [11]. Raw materials collected with non-scientific methods are usually exposed to many pathogenic pollutants, and most of them are contaminated by pathogenic microorganisms before harvesting and also during transportation and storage. Evidence shows that plant contamination can be transmitted to humans [11, 12]. This study aims to investigate the prevalence of microbial and fungal contamination in four plants from the Lamiaceae family in herb shops in Rasht, Iran.

Methods
For this study, a total of 61 samples were randomly collected from the herbal shops of Rasht City in 2020 (15 from each of mint, wild mint, and rosemary plants and 16 from lemon balm plant). The city was divided into 5 regions. The mentioned plants were randomly collected from 3 herbal shops in each region. The collected samples all included the leaves of the study plants. The collected data were coded and entered into SPSS software, version 16. Mean±SD, median (first quartile/third quartile) and minimum/maximum were used to describe quantitative variables. Also, qualitative variables were described based on frequency and percentage. The normal distribution of quantitative variables was measured using skewness and skewness values, Q-Q plot and Shapiro-Wilk test. According to the USP chapter 1111 related to non-sterile solid edible products (non-aqueous), the allowed TAMC and TYMC are equal to 103 and 102 CFU/g, respectively [26]. The Wilcoxon signed rank test was used to compare the results with the USP standard. The statistical significance level was set as P<0.05.

Results
The results of this study showed that about 73.7% of the collected samples were infected with bacteria, 86.6% with fungi and 44.2% with E. coli. Out of 16 samples of lemon balm, 13(81.2%) were infected with bacteria, 15(93.7%) were above the limit of fungal infection, and 6(37.5%) were infected with E. coli. Out of 15 rosemary plant samples, 5(33.3%) were infected with bacteria, 9(60%) were infected with fungi, and 11(73.3%) with E. coli. Out of 15 samples of mint plant, 14(93.3%) had bacterial contamination, all (100%) had fungal contamination, and 4(26.6%) were positive for E. coli contamination. Out of 15 wild mint plant samples, 13(86.6%) were infected with bacteria, 14(93.3%) with fungi, and 6(40%) with E. coli.
The results of E. coli tests were reported as positive if a colony was observed, and as negative if no colony was formed, according to the USP guideline No. 62 [27]. In this regard, a total of 27 samples were positive and brick-colored colonies were observed on the surface of the plate, while 34 samples were negative, out of which 26 had only color change and 8 did not have any color change or colony on them. The mean counts of bacteria and fungi in the plant samples were 3.39×105 and 6.3×105, respectively.
The median counts of bacteria isolated from lemon balm, mint, and wild mint plants were 5.42×103, 9.6×103, and 7.45×103, respectively, which were significantly different from the minimum standard count of 103 (P<0.001). The median counts of fungi isolated from lemon balm, mint, and wild mint plants were 3.8×103, 2.7×103, 9.5×102, respectively, which were significantly different from the minimum standard count of 102 (P<0.001). In this study, no significant difference was observed between the count of fungi isolated from rosemary plant and the minimum standard count of 102 (P=0.170).

Conclusion
Collection and use of medicinal plants are not always done in sanitary conditions. Most of the plants dry out in the air, which leads to the spread of contamination in the plants caused by bacteria and fungi in the air and soil. Microbial contamination of plants limits their use in food, pharmaceutical and cosmetic products [19]. The results of this research showed that almost all the examined samples of plants had bacterial and fungal contamination. Mint samples had the highest percentage of bacterial and fungal contamination, while the bacterial and fungal contamination of rosemary plant samples were at the lowest level. In addition to the conditions of cultivation, harvesting, drying and storage, the presence of some substances such as phenolic compounds in plants can prevent the growth of microorganisms [38]. There is a possibility of contamination of medicinal plants at any time in different stages of cultivation and preparation. To prevent microbial contamination, medicinal plants must be packed under strict regulations. Also, according to the reports of the absence of some strains such as staphylococcus, bacillus and clostridium in decoctions of plant samples compared to dry samples of plants, the use of boiling method can be a suitable method to reduce the microbial load of used medicinal plants. Also, storage in dry conditions and proper ventilation helps to maintain the quality of medicinal plants [39]. In improper storage conditions, the microbiota in plant products may increase and cause health risks for consumers, especially people with weak immune systems such as older adults and children. Therefore, it is very important to minimize the microbiota by washing or heating and maintaining hygiene in preparation. It is also recommended to keep the storage temperature at a low level and reduce the storage time as much as possible [40]. Considering the high microbial contamination of the examined plant in this study, It is necessary to consider more precise monitoring policies by the authorities supervising the health of plant products in herb shops of Rasht, Iran.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the Ethics Committee of Guilan University of Medical Sciences (Code: IR.GUMS.REC.1400.124).

Funding
This study was financially supported by Masoumeh Rohani.

Authors' contributions
Conceptualization, study design and initial draft preparation: Shirin Parvinroo; Data collection, analysis and interpretation: Elahe Rafiei and Masoumeh Rohani; Statistical analysis: Elahe Rafiei; Funding acquisition: Shirin Parvinroo and Masoumeh Rohani; Supervision: Shirin Parvinroo and Zahra Hesari; Critical revision: All authors.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors would like to thank the Faculty of Pharmacy, Guilan University of Medical Sciences for their corporation in this study.




References