Microbiological analysis and total aflatoxins levels from shoot powder of Phyllanthus amarus (Schum. and from

Phyllanthus amarus (Schum. and Thonn) has been used traditionally for treating over 50 diseases globally including malaria in Tororo, Uganda. Although efficacy and acute toxicity studies of this plant have been determined, safety regarding microbiological analysis and total aflatoxins levels in this Ugandan shoot powder of P. amarus is still lacking. The microbiological analysis was determined according to the procedures recommended in WHO guidelines. Total aflatoxins were determined using Afla Test Kit, VICAM Series (South Africa). In terms of microbial analysis, the herbs were prepared based on indigenous knowledge according to the WHO criteria. Although this herbal shoot powder had microbial safety requirements with key microbial contaminants (i.e. mold fungi, Salmonella typhi , and Escherichia coli ) within acceptable ranges, however, aflatoxins in all the investigated samples were beyond acceptable levels. Current results are useful in developing and establishing public health standards for the production and safe handling of herbal products of the Ugandan P. amarus .


Introduction
Currently, the use of botanicals and traditional medicines is widespread around the world (Marcelo and Taís, 2012). The cost and poor access to allopathic medicines have served to a great extent as a stepping stone to the popularity of traditional medicines. According to the National Centre for Complementary and Alternative Medicine (NCCAM), the United States of America reported that 40% of its population depends on complementary and alternative medicines (NCCAM, 2013). Botanical dietary supplements represents distinctively one of the major contributors of complementary and alternative medicines in the USA (Kessler, 2001), with previous estimated returns of $5.1 billion annually towards the close of 1990s (Eisenberg, 1998).
According to Wu et al., (2014), about 18% of the US populations (over 38 million US citizens) tested were using herbal preparations and dietary supplements in 2007. It is estimated that the majority of people in developing countries as well as a vast number of the rural communities globally depend on botanicals as their primary sources of health care in controlling infectious diseases such as malaria (Birdi et al., 2006). Wu et al., (2014) added that a number of populations were also reported using high rates of herbs and supplements, including women (Schaffer et al., 2003;Kennedy, 2005), middle aged adults (Schaffer et al., 2003), Caucasians (Mackenzie et al., 2003;Kelly et al., 2006), Asians (Bair et al., 2002;Tanaka et al., 2008) and Latinos (Howell et al., 2006). Previously Waako, (2003 stated that in many communities, people were linked to culture and hence strongly believed that traditional medicines were readily accessible, safe and affordable. According to Oudhia, (2008), P. amarus and Plumbago zylenica were traditionally used globally for treating over 50 and 35 diseases and disease conditions, respectively.
Medicinal plants constituted very valuable aspect of pharmaceutical, pharmacological as well as medical developmental studies, which provided raw materials for drug synthesis and for pharmacologically active compounds used for treatment of infectious diseases such as malaria (Si-Yuan et al., 2013). A previous few examples reported by Sebisubi, (2006) supported this idea including; acetyl salicylic acid derived from the willow bark and morphine, which were sources of many active compounds exhibiting narcotic and analgesic properties. Moreover, procaine derived from cocaine and quinine from cinchona bark, both served as precursors of antimalarial compounds such as chloroquine and primaquine. MoH, (1987) in an earlier study stated that in African countries such as Uganda, it is recommended to include ethno medicine in local health teams and in Primary Health Care delivery.
Unfortunately, points of weakness against use of herbal medicines were lack of documented standard procedures for their collection, preparation, administration as well as storage of their products (OAU/STRC, 1991). Accordingly, there is an urgent need to evaluate the traditional procedures of processing these selected medicinal plants according to international recognized requirements (WHO. 1998(WHO. , 2003. Parts of the current study regarding the extent of knowledge, attitude and practices (KAP) of herbs of malaria, and standard use of P. amarus have already been published (Odda et al., 2013). Recently, efficacies of P. amarus, antimalarial activity and acute toxicity have also been published (Odda et al., 2018). However, safety aspects related to processing and storage practices based on indigenous knowledge (IK) of microbial analysis and aflatoxins levels of this important Ugandan herb have not yet been documented. Novel Research in Microbiology Journal, 2018 Current study is therefore a pioneer effort based on use of locally available antimalarial medicinal plants or their natural products with internationally acceptable levels of safety, efficacy, and good quality. This is the basis for herbal products that contain consistent levels of active ingredients, predictable pharmacological and physiological effects. Therefore, the aims of our study were to determine the microbiological analysis and total aflatoxins levels in this P. amarus antimalarial herb when used singly in Tororo, Uganda. According to the Uganda Bureau of Statistics at 2017, Tororo District has an approximate population of 517,080 with a sex ratio of 94.2 males per every100 females (UBOS, 2017). Kamuli is the village where the studied herb was used as mono-herb in the treatment of malaria fever based on indigenous knowledge (IK) of the herbalists (Fig. 1).

Microbiological studies
P. amarus shoots were collected from Kamuli Village, Pagoya LCI zone approximately 10 Km from Tororo town in Eastern Uganda, during December, 2012. Samples were identified at the Department of Biological Sciences Herbarium, Makerere University prior to air-drying at room temperature under shade, and then were ground into powder using an electric mill. Some of the powder was used for microbiological analysis, whereas the rest was stored for total aflatoxins levels determination. The microbiological analysis was carried out according to the procedures recommended in WHO. (1998) guidelines.
One gram (1g) proportion of each sample was aseptically taken after thorough mixing into a sterile flask containing 99 ml of sterile dist. water. These samples were soaked for 2-3 min. with occasional stirring. Each of these produced suspensions was shaken for 10 min. to obtain a homogenous suspension of microorganisms. These suspensions were diluted serially by taking 1 ml of the stock suspension and then adding it to 99 ml of sterile dist. water to obtain a dilution of 10 -1 ; this step was repeated serially to obtain 10 -2 -10 -6 dilutions. 0.1 ml of each of these dilutions was plated separately on Nutrient agar, Xylose Lysine Deoxycholate agar (XLD) and Potato dextrose agar (PDA) media using a glass spreader.
A pair of Nutrient agar plates were inoculated and incubated in 2 sets for each dilution; one set of plates used for the isolation of Coli forms was incubated in a Griffin incubator (UK) at 37°C for 24 h., whereas the second set of plates used to confirm if the coli form was E. coli incubated using a Memmert incubator (Germany) at 45°C for 24 h. The XLD plates enriched with Selenite F were also incubated at 37°C for 24 h., while PDA plates used to isolate mold fungi were incubated at 28°C for 7 days. After incubation, separate bacterial and fungal colonies were counted. On the other hand, E .coli (ATCC 25922) was used as a reference culture.

Determination of Aflatoxins levels in P. amarus shoot powder
The toxicological aspect of this study was carried out at the Department of Food Science, Makerere University (VICAM Afla Test® WBSR, 2008); the total aflatoxins of interest were AFB 1 , AFB 2 , AFG 1 and AFG 2 . Aflatoxins levels determination were carried out using VICAM Afla Test, because it has the advantage of measuring these key aflatoxins without using toxic solvents such as chloroform, and can be used in any laboratory with a High performance liquid chromatography (HPLC) system. Before screening of dried P. amarus shoot powder for estimation of aflatoxins levels, calibration were performed according to manufacturer's instructions i.e. performing the test using low, high calibrator and yellow mycotoxin standard, whose results are presented here with the test sample result (02-1C). Having satisfied the calibration requirements, samples for aflatoxins determination passed through the following steps:

Sample extraction
25 g of each ground P. amarus sample was mixed with 5 g NaCl and then placed in a blender to which 100 ml of methanol: water (80:20) was added. The mixture was blended at high speed for 1 min.; the extract was then filtered through filter paper. Novel Research in Microbiology Journal, 2018 Five milliliter of the filtrate was diluted with 20 ml of 10% Tween 20 solution and mixed well. The diluted extract was passed through glass micro-fiber filter into glass syringe barrel to measure 4 ml, thus become ready for extraction with column chromatography.

Column chromatography
4 ml of the diluted extract (equivalent to 0.2 g sample) was passed through Afla Test®-P affinity column at the rate of 1-2 drops/second until air comes through the column. This was followed by passing 10 ml of purified water through the column at a rate of about 2 drops/second, this process was repeated twice. Elution of the affinity column was carried out by passing 1.0 ml of HPLC grade methanol through the column at the rate of 1-2 drops/second, 1 ml of this elute was then collected in a glass cuvette. Finally, 1.0 ml of the Afla Test® Developer was added to collected elute in the cuvette and were mixed well, then the cuvette was placed in a calibrated fluorometer. The readout of total aflatoxins concentrations was carried out after 60 seconds.

Microbiological analysis of the P. amarus shoot powder
Results of the microbiological analysis of P. amarus herbal powder included E. coli, S. typhi, yeasts and mold fungi as the key microbes of herbal products specified by WHO. (1998). Among the coliforms, E. coli is responsible for most of herbal toxic effects and hence it has been cut off here. E. coli ATCC 25922 was used as a reference organism to demonstrate if the culture media was still capable of supporting E. coli growth or not, hence minimize false negative results regarding this bacterium. On day 7, one of the dilutions which was processed and stored according to indigenous knowledge (IK) coded (02-1C), had molds (greenish gray colonies) growing on PDA medium, whereas the samples processed and stored according to WHO guidelines coded (02-1D) had no such growth. The overall results of this microbiological analysis are demonstrated in Table 1.
Following 24 h. incubation at 37 o C, IK (02-1C) processed herbal powder had 620 cfu/g coli forms (pink colonies) indicating that poor unhygienic method of preparation was used. Whereas the second set of plates of the same sample incubated at 45 o C had no growth (absence of E. coli) indicating absence of fecal contamination. It was also noticed that IK (02-1C) processed samples had 120 cfu/g of yeasts/molds, whereas the samples who's processing met WHO criteria (02-1D) had 20 cfu\g only. In terms of microbial analysis, current results recorded that prepared herbs based on indigenous knowledge conformed to specifications just as those handled according to the WHO criteria.

Screening of aflatoxins levels of P. amarus powder
According to Bbosa et al., (2013), exposure to high levels of aflatoxins especially AFB 1 , AFB 2 , AFG 1 and AFG 2 , are associated with Hepatocellular carcinoma (HCC). However, the WHO and other regulatory bodies like European Medicines Agencies (EMEA) now demanded that raw materials for making drugs including herbal preparations should be screened for total aflatoxins to ensure that they are safe and of acceptable qualities (WHO, 2013;EMEA, 2005).
Results of total aflatoxins in herbal products presented in this work were only assessed at baseline level because aflatoxins would still be there for six months later. Unfortunately, after testing for these aflatoxins in IK processed samples, there was a technical problem with the Novel Research in Microbiology Journal, 2018 HPLC equipment before processing according to WHO guidelines; hence tests for the (02-1D) could not be carried out, accordingly were not described in Table (2). Note: To determine whether the preparations had acceptable or unacceptable levels (too contaminated for human use) according to United States Pharmacopeia (USP), Vol. 12 (Updated as of 2015) as follows: Salmonella should be absent in at least 25 g of the powder or preparations; yeasts and molds must have minimum acceptable level of 10 5 cfu/g; maximum is 10 6 cfu/g, whereas E. coli acceptable level is 10 2 /g; maximum limit is 10 3 /g of the sample.

Discussion
There is a growing concern about the quality and safety of herbal drugs due to microbial contamination caused by many environmental factors such as; temperature, humidity, extent of rainfall during pre-harvesting and post-harvesting periods, handling practices and the storage conditions of medicinal plant materials (Marcelo and Taís, 2012).
Results of the presence study on microbial analysis of shoot powder of P. amarus processed by IK showed the presence of total coliform count of 620 cfu/g sample which indicated poor handling processes. This was in accordance with previous studies of Kaume et al., (2012);Noor et al., (2013) who reported the presence of coliform from herbal medicines available in Kenya and Bangladesh, respectively. Although total coliform bacteria are unlikely to cause disease but their presence in this product indicates that the disease causing pathogens may also be present. There were no any common fecal contaminating isolates detected in the studied samples. This was in contrary to the findings of Abba et al., (2009) stated that fungal deterioration adversely affects the chemical composition of raw materials and thereby decreases the medicinal potency of herbal drugs. Marcelo and Taís, (2012) added that although high fungal loads may be acceptable due to the natural origin of those products, however, they indicated the possibility of their spoilage and mycotoxigenesis.
The shoot powder of P. amarus processed by IK coded (02-1C) underwent aflatoxins screening. The common molds notorious for aflatoxins production are A. flavus, A. parasticus, A. nomius, and A. bombyicis. Given that the studied herbs were collected in the way mimicking how collection is done based on IK, however, it is possible that the recommended good collection procedures were not followed. This is attributable to the fact that these molds can survive on plant debris, tree leaves, decaying wood, animal excreta, dead insects, stored grains, and even immunocompromised humans and animals. The other factors commonly linked to aflatoxins production are incomplete drying and poor storage practices which led to molds growth (Ogiehor, 2002). Subroto, (2011) reported that optimum conditions for aflatoxins production were temperatures in range of 24-35°C, and substrate moisture content exceeding 7% (10% with ventilation). Previous studies by Kitya et al., (2010) confirmed that traditional methods of food processing were contributing to risk factors of HCC if safety is not considered to these products. Novel Research in Microbiology Journal, 2018 Therefore, guidelines for proper processing procedures must be available and utilizable at this level.

Conclusion
Although antimalarial shoot powder of P. amarus subjected to microbial safety analysis had key herbal contaminants such as; yeasts, molds, S. typhi, and E. coli within acceptable ranges, however, aflatoxins in all the samples investigated were beyond these acceptable levels. Current results are useful in developing and establishing public health standards for the production and safe handling of herbal products from the Ugandan P. amarus.