Introduction
Medicinal plants are considered as vegetables containing secondary metabolites, also called active principles, which are substances that act as pharmacological beneficial or harmful molecules on the organism (Martins & S. 2018).
The use of plants with medicinal attributes were the first medicines to be empirically used for curing diseases affecting humans. This knowledge has been passed on for generations trough different cultures until our time (Devesa 2004; Marinoff 2006).
The World Health Organization estimates that the primary attention of health, up to 80 % of the population in developing countries, is based on traditional medicine, by culture tradition or because there are no other options. While, as for developed countries, many people turn to several types of natural remedies because they think “natural” is synonym of innocuous (WHO, 2004; da-Silva et al., 2012; Luitel et al., 2014; Ávila, 2017). That is why, the consumption of medicinal plants has been increasing, even though it is not entirely regulated. In Mexico, medicinal plants are poorly regulated, since according to legislation, their approval consists in “just demonstrating that these remedies are safe and under no circumstances, assert any effectivity or therapeutic power against a specific disease” (Gómez, 2009). In general, these treatments are guaranteed only by tradition but lack of scientific support (Enríquez et al., 2005; Marinoff et al., 2009). On the other hand, the Comisión Federal para la Protección contra Riesgos Sanitarios (COFEPRIS) has subscribed a cooperation at international level, with a perspective until 2025, of efficiently easing the availability of these innocuous and high quality products, with the purpose of protecting public health (Gómez, 2009).
Another problem related to consumption and commercialization of medicinal plants, is the degree of innocuousness due to the presence of chemical contaminants (heavy metals, and pesticides) (Marinoff et al., 2009; Kishan et al., 2014; Kumar et al., 2015; Rodrigues et al., 2017) and biological contaminants (fungi, bacteria, and toxins) (Arias et al., 1999; Sánchez et al., 2006), mainly due to bad management of these products in any of the phases of the production-commercialization chain (cultivation, harvest, transportation, storing and sale) (Kneifel et al., 2002; Codex Alimentarius, 2014). In Mexico, it has been documented that more than 85 % of the species commercialized in local markets and naturist stores are collected in the wild form; this method does not normally count on effective handling programs and lack of sufficient control by government (Bye & Linares, 1990; Juárez-Rosete et al., 2013).
Among the main agents responsible for biological contamination in medicinal plants, bacteria, fungi, and toxic metabolites stand out. With respect to bacteria, the group of fecal coliforms, aside from being an indicator of fecal contamination, represents a health risk for consumers, since some types of bacteria in this group are considered to be the cause of serious diseases, like Escherichia coli enterohemorrhagic strain (EHEC), bacteria species causing hemorrhagic colitis and hemolytic-uremic syndrome (Momoh et al., 2011; Pullés 2014). Moreover, filamentous fungi, are cosmopolitan microorganisms but under certain conditions, they can produce toxic metabolites, called mycotoxins, which have toxic severe effects that are related to hepatic, renal, digestive, reproductive, immunosuppressive diseases, and to the developing of tumors (Marroquín-Cardona et al., 2014; Kabak & Dobson 2015; Abrunhosa et al., 2016; Pemán & Quindos 2016). For the reasons abovementioned, the presence of this type of contaminants in medicinal plants and herbal products represent a serious risk to consumers’ health.
The aim of this research was to assess the presence of bacteria (total coliforms, fecal and Escherichia coli), fungi and total aflatoxins in medicinal plants commercialized in Tepic city, Nayarit, Mexico.
Material and Methods
This research work was carried out in the city of Tepic, Nayarit, Mexico, which is located in western Mexico (21º 30 59 and 104º 53 39) at an altitude of 920 masl, with a warm sub-humid climate with rains in the summer.
The sampling of medicinal plants was realized inside the city (Figure 1). A total of 28 establishments (naturist stores) were previously located. Then, the people in charge of those establishments were invited to participate; they were asked, using a structured questionnaire, what type of medicinal plant was the most commercialized during the period of autumn-winter and spring-summer.
According to the results, 17 establishments accepted to participate. In each establishment, a sample was taken from one of the three most commercialized medicinal plants for each one of the seasons. As for the most commercialized plants during the period of autumn-winter, the sampling was realized from January to February and as for the plants during the period of spring-summer, the sampling was realized from August to September, 2016. The samples, collected in situ, were placed in sterile plastic bags for their transportation to the laboratory.
Microbiological analysis
For the microbiological analysis, the samples were prepared according to Norma Oficial Mexicana NOM-110SSA1-1994. For this, 10 g of each sample were weighed in aseptic conditions and placed in plastic bags with special filters for peristaltic homogenizer (Stomacher, BagMixer® 400, St Nom France). Subsequently, 90 mL of phosphatebuffered saline (PBS) solution at pH 7.4 were added and mixed for three minutes in the Stomacher. Finally, the solution was recovered in a sterile flask (work sample).
Determination of total Coliforms bacteria (TC), fecal coliform bacteria (FC), and E. coli
The determination of these bacterial groups was realized according to the most probable number (MPN) method described in CCAYAC-M-004/11.
Presumptive test: For each sample, a series of 3 tubes with 10 mL of sterile lauryl sulfate broth (LSB) was used, at a 1.5X concentration. Ten mL of the sample were added to the tubes; the determination was carried out by duplicate, in addition a positive control (tube with E. coli) and a negative control (tube with no bacterial inoculum) were used, in asepsis conditions. Later on, the tubes were taken to incubation at 35 °C during a period of 24 to 48 h.
Confirmative test: From each presumptively positive tube (production of gas in Durham tube), Three inoculations with bacteriological loop were transferred to the same number of tubes containing 5 mL of Brilliant-Green Bile Lactose Broth (BD Bioxon® Mexico) at 2 %. Each determination was realized in duplicate; as well, a positive control (tube with E. coli) and a negative one (not inoculated tube) were used. Subsequently, a series of tubes was incubated at 35 ± 0.5 °C during 48 h for TC and at the same time, another series of tubes was incubated at 44.5 ± 0.2 °C in bain-marie (VWR® International, USA) for the determination of FC. In order to establish the MPN/g, the corresponding tables established by CCAYAC-M-004/11 were reviewed.
For the confirmative test of E. coli, from each one of the samples which were positive to the FC test (formation of gas in Durham tube), three inoculations with bacteriological loop were transferred in the EC-MUG4 medium (4-methylumbelliferyl-beta-D-glucuronide) (Difco™, France) and was incubated at 44.5 ± 0.2 °C in bain-marie (VWR® International, USA) during 24 h; a positive and negative controls were used. Concluding the period of incubation, the tubes were placed in an Ultra Violet light Chamber (365 nm) (ChemiDoc-It2 Imager, UK), in which the presence of fluorescence was observed.
Determination of fungi
The determination of fungi was realized under the Norma Oficial Mexicana NOM-111-SSA1-1994. For this, 1 mL of the work sample was deposited in Petri dishes using a sterile pipette for each one of them, then 20 mL of sterile acidified Potato Dextrose Agar (PDA) (BD Bioxon®, Mexico) with tartaric acid (Fermont, Mexico) were poured on them at 10 %. The dishes were homogenized; they were left to solidify by placing them on a cold horizontal surface. Candida spp. was used as a positive control and a not inoculated plate as negative control. Later on, the Petri dishes were incubated at room temperature for five days in darkness. Once the time passed, the morphology of the mycelium colonies was observed and described. The test was carried out by duplicate.
For the identification of potentially aflatoxigenic fungi, the colonies obtained in PDA agar were inoculated in the Czapeck Yeast Extract medium (CYA) (BD Bioxon®, Mexico) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) (Difco™, France) mediums, and were later taken to incubated at room temperature for five days in darkness. Once passed this time, the morphology of the obtained colonies was described and the microscopic structures were observed with lactophenol blue (Hycel, Mexico) at 40X. For the identification of the genera and species of the fungi, dichotomic criteria according to Koneman et al. (2008) were considered.
Quantification of total aflatoxins (AF)
Prior to the quantification of total aflatoxins (AF), a methanolic extraction was performed. Briefly, 5 g of the samples were weighed and mixed with 25 mL of methanol at 70 % in a blender (Osterizer®, USA) for 3 min. The suspension obtained was passed through a filter paper (Whatman™ No. 1, GE UK) and the resulting solution was diluted with distilled water (1:2).
The quantification was performed by following the manufacturers’ recommendations on the ELISA commercial kits for total aflatoxins (RIDASCREEN®FAST Aflatoxin R-Biopharm AG, Germany) with a detection limit of 1.7 ppb. Firstly, the samples to be analyzed and 50 µL of the standard solution were added to the wells of in the microplate, then, 50 µL of the conjugate aflatoxin-enzyme and 50 µL of the antibody antitoxin were added. Later, the samples were mixed and incubated for 10 min at room temperature. Next, the content was emptied on a clean absorbent paper and three washings were realized, adding 250 µL of the washing solution into each well. Then, 100 µL of the substrate (chromogen) were added into each well and were mixed and incubated for 5 min in darkness at room temperature. Finally, 100 µL of H2SO4 were added to stop the reaction. The absorbances of the samples were measured at 450 nm in a microplate reader (EPOCH®, BioTel Instruments, USA). In order to calculate the concentration of AF in the samples, RIDA®SOFT Win software (Germany) (Art. No. Z9999) was used.
Results and Discussion
The consumption of medicinal plants is largely rooted in countries influenced by ancestral cultures; moreover, the lack of access to these systems of institutional health and allopathic medicine, encourages the consumption of these types of products. In addition to this, nowadays the consumption of these types of plants has become popular in all social levels around the world, based on the argument that “natural means innocuous” (Devesa et al., 2004; Enríquez et al., 2005; Marinoff, 2006).
The presence of bacteria and fungi in medicinal plants was demonstrated in this paper, microorganisms that besides being potential pathogens, are indicators of an incorrect handling and level of innocuousness of the products. On the other hand, the presence of aflatoxins, metabolites produced by certain species of fungi with carcinogenic properties, was evidenced here. For this study, 28 establishments dedicated to the selling of medicinal plants and located in downtown Tepic, Nayarit were asked to participate; 17 of them accepted to provide information and participate on this research.
According to the data obtained from the surveys, the three most commercialized medicinal plants during autumnwinter period were “gordolobo,” also known as common mullein (Verbascum thapsus L.), “eucalyptus” (Eucalyptus mellidora L.) and “hierba del zorrillo” a.k.a. petiveria (Petiveria alliacea L.), which were collected in the period of January-February 2016, while in the period of springsummer, the most commercialized medicinal plants were “cola de caballo” (Equisetum arvense L.), “caña agria” (Costus spicatus L.) and “guámara” (Bromelia pinguin L.) collected in the period of August-September 2016 (Table 1). From the 17 participant stores, according to the existence of plants of the moment of the sampling, 83 samples were collected: 16 of gordolobo, 15 of eucalipto, 11 of hierba del zorrillo, 15 of cola de caballo, 15 of caña agria and 11 of guámara.
Season | Medicinal herbs | Part of the plant marketed and analyzed |
Plant presentation for sale |
Therapeutic use* |
---|---|---|---|---|
Autumn- Winter |
Gordolobo (V. Thapsus L.) |
Flowers and leaves | Dry | RD |
Eucalipto (E. melliodora L.) |
Leaves | Dry | RD | |
H. del Zorrillo (P. alliacea L.) |
Roots and leaves | Dry | RD | |
Spring-Summer | Cola de caballo (E. arvense L.) |
Full plant | Fresh | UD |
Caña ágria (C. spicatus L.) |
Stem | Fresh | UD | |
Guámara (E. penguin L.) | Fruit | Fresh | UD |
*RD= enfermedades del tracto respiratorio; UD= enfermedades del tracto urinario.
From the analyzed samples, 43/83 presented contamination by total coliforms, 10/83 by fecal coliforms and 5/83 by E. coli. The proportion of positive samples for TC was the following: “cola de caballo” (10/15), “gordolobo” (10/16), “hierba del zorrillo” (7/11), “guámara” (7/11), “eucalipto” 5/15) and “caña agria” (2/15); this last plant presenting less frequency of contamination in comparison to the rest (p<0.05).
As to the contamination by FC, the plant with higher frequency of contamination by these types of bacteria, was “hierba del zorrillo” (4/11), followed by “cola de caballo” (3/15), “gordolobo” (2/16) and “eucalipto” (1/15), while, as for “caña agria” and “guámara,” the presence of these bacteria was not detected. On the other hand, the presence of E. coli was only detected in samples of “hierba del zorrillo” (4/11) and “gordolobo” (1/16) (Table 2).
Season | Medicinal herbs | Total coliforms | Fecal coliforms |
E. coli | |
---|---|---|---|---|---|
PPS | >10 MPN/g* |
PPS | PPS | ||
Autumn- Winter | Gordolobo (V. thapsus L.) |
10/16 | 10/16 | 2/16 | 1/16 |
Eucalipto (E.melliodora L.) |
5/15 | 5/15 | 1/15 | ND | |
H. del zorrillo (P. alliacea L.) |
7/11 | 7/11 | 4/11 | 4/11 | |
Spring- Summer |
Cola de caballo (E. arvense L.) |
12/15 | 12/15 | 3/15 | ND |
Caña agria (C. spicatus L.). |
2/15 | 2/15 | 0/15 | ND | |
Guámara (B. penguin L.) |
7/11 | 4/11 | 0/11 | ND | |
Total of contaminated samples | 43/83 | 40/83 | 10/83 | 5/83 |
PPS= Proportion of positive samples, *proportion of samples with MPN/g>10, ND= Not Detectable.
Therefore, data obtained in the present work indicated that presence of TC was detected in 51.8 % of the analyzed samples, and FC in 12 % of them. The aforementioned indicates that analyzed medicinal plants were not properly handled or have been in contact with fecal matter. The biological contamination of this type of products can present at different stages throughout their production process (cultivation, harvest, transportation, storing, packaging and point of sales). According to Kneifel et al. (2002), the critical points where a major risk of bacterial contamination exists for this type of products, are during cultivation and harvest.
From the 43 samples with presence of TC, only 3 presented values ≤10 MPN/g, which is a limit value of microbiological specifications for tea and infusion of plants (NORMA Oficial Mexicana NOM-218-SSA1.2011; CODEX STAN 192-1995). Therefore, 40 samples were exempt of these specifications, aside from these samples, 9 presented FC and 5 presented contamination by E. coli. Therefore it is considered that this type of medicinal plants represents a potential risk for consumers’ health. As well, the presence of FC and E. coli in this type of samples evidenced the possibility that other etiological agents of infections for human beings, such as Shigella and Salmonella, among others, could be present.
With respect to the determination of fungi, from the 6 types of plants analyzed, 5 resulted multi-contaminated by more than one species of fungi. “Gordolobo” and “cola de caballo” were the plants with the most mycotic contamination (5 genera of fungi of each one), followed by “caña agria” (4 genera), “eucalipto” (3 genera) and “hierba del zorrillo” (2 genera), while “guámara” was only contaminated by one type of fungus. Fungus genus the most frequently found in the analyzed plants was Aspergillus spp (24/83), followed by Penicillum spp (19/83), Rhizopus spp (19/83), Mucor spp (4/83), Paecilomyces spp (3/83), Fusarium spp (2/83) and Alternaria spp (2/83) (Table 3). This way, the most frequent fungi in the analyzed samples were Aspergillus, Penicillium and Rhizopus (p<0.001), compared to the rest of the genera identified.
Season | Medicinal herbs |
Aspergillus spp | No Penicillum spp | Fusarium spp | of samples contaminated by fungi Alternaria spp | Rhizopus spp | Mucor spp | Paecilomyces spp |
---|---|---|---|---|---|---|---|---|
Autumn- Winter | Gordolobo (V. thapsus L.) |
8/16 | 6/16 | ND | ND | 5/16 | 2/16 | 1/16 |
Eucalipto (E.melliodora L.) |
3/15 | 1/15 | ND | ND | ND | 1/15 | ND | |
H. del zorrillo (P. alliacea L.) |
ND | ND | 1/11 | ND | ND | 1/11 | ND | |
Spring-Summer | Cola de caballo (E. arvense L.) |
7/15 | 7/15 | 1/15 | 1/15 | 6/15 | ND | 2/15 |
Caña agria (C. spicatus L.). |
6/15 | 5/15 | ND | 1/15 | 3/15 | ND | ND | |
Guámara (B. penguin L.) |
ND | ND | ND | ND | 5/11 | ND | ND |
*ND= Not detectable.
Due to the fact that Aspergillus genus is a potent producer of aflatoxins, its species were identified. From the 24 samples contaminated by this fungus, A. niger was isolated in 19 samples, A. fumigatus in 3 and A. terreus in 2 (Table 4).
Season | Medicinal herbs | A. niger | A. terreus | A. fumigatus |
---|---|---|---|---|
Autumn- Winter | Gordolobo (V. thapsus L.) |
6 | 1 | 1 |
Eucalipto (E. melliodora L.) |
3 | ND | ND | |
H. del zorrillo (P. alliacea L.) |
ND | ND | ND | |
Spring-Summer | C. de caballo (E. arvense L.) |
5 | 1 | 1 |
Caña agria (C. spicatus L.). |
5 | ND | 1 | |
Guámara (B. penguin L.) |
ND | ND | ND | |
Total of contaminated herbs | 19 | 2 | 3 |
*ND= Not detectable
In a report about medicinal plants realized by Bugno et al. (2006), in Sao Paulo, Brazil, they found that more than 50 % of the samples were contaminated by diverse fungi, exceeding the limit allowed by the pharmacopoeia of the United States. The dominant load was Aspergillus followed by Penicillum, which were similar results to those found in this study (Bugno et al., 2006). Other reports have also demonstrated that in medicinal plants, the genera Penicillum, Aspergillus niger and Fusarium can be found more frequently (Abou-Arab et al., 1999), being these genera considered as storage fungi, since during this stage, the possibility of its incidence increases, due to the fact that its growth mainly depends on temperature and relative humifity (Rodríguez-Tito et al., 2012).
Aspergillus is a potent producer of mycotoxins, therefore when identifying the species in the analyzed samples, and when finding A. niger (present in 19 of 24 samples), followed by A. fumigatus (present in 3 of 24 samples) and A. terreus (found in 2 of 24 samples), it suggested that the environmental conditions of storing of the medicinal plants were proper for the growth of these fungi and the production of its toxins. Similar studies demonstrated that the species of Aspergillus genus potentially mycotoxic were: flavus, parasiticus, ochraceus niger and fumigatus (Bugno et al., 2006). However, About-Arab et al. (1999), found only the species A. ochraceus and A. niger with the capacity of producing mycotoxins in medicinal plants.
With respect to the determination of aflatoxins, the results indicated that from the 83 analyzed samples, 37 were positive (44.6 %). From these 37 positive samples, 18 (21.7 %) presented levels above the allowed limits (>20ppb) (FAO, 2003). In this sense, the samples of “eucalipto” (100 %) and “gordolobo” (93.8 %) were the plants with the highest quantity of AF, followed by “cola de caballo” (26.6 %), “caña agria” (13.3 %) and “hierba del zorrillo” (9 %), while in the samples of “guámara,” no presence of AF was detected (Table 5). When analyzing the frequency of positive samples for this group of mycotoxins, the statistical analysis indicated higher frequency for this type of contaminant in “eucalipto” and “gordolobo” (p<0.001) in comparison to rest of analyzed plants.
Season | Medicinal herbs | Positive samples | Contaminated samples (>20 ppb) |
|
Range |
---|---|---|---|---|---|
Autumn- Winter | Gordolobo (V. thapsus L.) |
15/16 | 2/15 | 26.34 | 5.6-37.2 |
Eucalipto (E. melliodora L.) |
15/15 | 14/15 | 250.75 | 2.13-960.1 | |
H. del zorrillo (P. alliacea L.) |
1/11 | 0/1 | - | - | |
Spring-Summer | Cola de caballo (E. arvense L.) |
4/15 | 2/4 | 21 | 4.94-41.76 |
Caña agria (C. spicatus L.). |
2/15 | 0/2 | 2,61 | 2.45-2.78 | |
Guámara (B. penguin L.) |
ND | - | - | - | |
Total | 37 | 18 | - | - |
*ND= Not detectable
Studies carried out by Bugno et al. (2006) found specifically that 27.6 % of A. flavus has the capacity of producing AFB1 o AFB1 + B2, while 45.5 % of A. parasiticus has the capacity of producing AFB1, B2, G1 and G2, which demonstrated that the presence of fungi involved a potential risk for the production of mycotoxins, especially during prolonged storage in inadequate conditions and without temperature control. In this sense, obtained data in this work, pointed out that the number of samples of “gordolobo” and “eucalipto” contaminated by Aspergillus genus is lower with respect to the number of positive samples for total AF. These data suggested that the chemical components like the essential oils present in “gordolobo” and “eucalipto” can inhibit the growth of fungi. There are studies performed with the extract of essential oil of “eucalipto,” where the inhibition of growth of Aspergillus section flavi (A. parasiticus and A. flavus) was observed (Kneifel et al., 2002; Elshafie et al., 2002; Bluma et al., 2008).
Conclusion
The present research demonstrated the presence of biological contaminants (coliforms bacteria, fungi and mycotoxins) in medicinal plants with infectious, toxic and in extreme cases, carcinogenic potential. Therefore it is indispensable that competent authorities effectively and strictly regulate the selling process of these products, but also their processes of cultivation, harvest, and storage, which are critical points to ensure their innocuousness.