International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(1), 46-51, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 46 Quality-Based Microbial Contamination Analysis of Nutraceuticals Baba-Moussa F1*, Adjanohoun A, Anihouvi V.B., Ahouandjnou H, Sanni S, Omansen T.F., Kotchoni S.O., Toukourou F. 1, Baba-Moussa L.Laboratoire de Microbiologie et de Technologie Alimentaire, Faculté des Sciences et Techniques/Université d’Abomey-Calavi, ISBA-Champ de foire Cotonou, BENIN Centre de Recherches Agricoles Sud / Institut National des Recherches Agricoles du Bénin, BENIN Laboratoire de Biotechnologie Alimentaire, Département de Nutrition et Sciences Alimentaires, Faculté des Sciences Agronomiques, Université d’Abomey-Calavi, 01 BP 526, Cotonou, BENIN University of Groningen, University Medical Center Groningen, Department of Internal Medicine/Infectious Diseases, P.O. Box 30.001, 9700 RB Groningen, NETHERLANDS Rutgers University, Department of Biology and Center for Computational and Integrative Biology, 315 Penn St., Camden, NJ 08102, USA Laboratoire de Biologie et de Typage Moléculaire en Microbiologie, Faculté des Sciences et Techniques/Université d’Abomey-Calavi, 05 BP 1604 Cotonou, BENIN Available online at: www.isca.in Received 3rd November 2012, revised 10th November 2012, accepted 29th November 2012Abstract The aim of this study was to evaluate the microbial contaminations of medicinal plants used as traditional herbal medicines sold in pharmacies in Cotonou in Benin. Twenty (20) medicinal plants involved in the preparation of improved herbal medicines, eleven (11)improved herbal medicines sold in pharmacies and fourteen (14) improved herbal medicines sold by herbalists were studies. The microbial contaminations (mesophilic bacteria, yeasts and moulds) of the samples were analyzed. Our results revealed that the samples were contaminated with a wide range of pathogenic microorganisms, including Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Samples sold by herbalists were the most highly contaminated. The herbal medicines sold in pharmacies were also contaminated by Escherichia coli (81.82%), Staphylococcus aureus 27.27% and Pseudomonas aeruginosa (18.18%). These products did not meet the European Pharmacopoeia standards. Our data suggest that nutraceutical processing as well as storage conditions among others must be critically improved in order to meet the standards required for any pharmaceutical drugs. Keywords: Phytomedicines improved, microbiological quality, specified germs, Benin. IntroductionEthnobotany has been the source of several drug discoveries to treat a wide range of human diseases. The development of drug resistance in synthetic anti-malarial drug therapies has led to the use of combined artemisinin-based (CTA) drug therapy. However, there has been lately several accounts of resistance to the ACT therapy. On the other hand, the Artemisia annua L (Asteraceae) nutraceutical has been proven to be very effective to treat the disease, without inducing drug type of resistance. Currently in Europe, there is a trend of return to nutraceuticals for human disease treatment. In Africa, the several socio-economical factors including poverty, lack of drug accessibility have forced people to embrace the use of nutraceuticals to treat a wide range of human diseases. The use of nutraceuticals has been further enhanced by the highly diverse and rich African flora and fauna, readily and cheaply available. About 75% of Africans rely on medicinal plants for a variety of ailments. In Benin, more than 85% of the population relies on traditional medicine for health care needs. This explains the rapid expansion of global market of medicinal plant derived nutraceuticals that represents currently over 60 billion U.S. dollars per year. The traditional medicine represents an economically sustainable alternative enabling the developing countries to cope with highly expensive modern medicine therapies. The declaration of Abuja (Nigeria) in April 2001 aimed at making traditional medicine research a priority for Africa. Since then considerable effort has been put on identifying, characterizing and promoting the use of medicinal plants and herbal medicines in Africa. The production of phytochemicals and nutraceuticals has been improved to minimize the cytotoxicity while increasing their efficacy in treating a wide range of ailments. These improved nutraceuticals have been approved to be sold in pharmacies and they have been adopted by the community at large. However, there has been no report on the microbial content analysis of these products, which might occur during packaging, storage and distribution. One of the stringent safety regulations of these products is the quality control before commercialization. Unfortunately, only a quarter of African countries have such safety regulation rules before commercialization of nutraceuticlas The WHA42.43 resolution of World Health Assembly urged the country members to implement the safety control regulation of neutraceuticals before their commercialization. In Benin, there is not safety regulation, and no microbial analytical control is performed before International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 46-51, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 47 commercialization of nutraceuticals widely embraced by the population. In this study, we aim to evaluate the microbial content of nutraceuticals as well as pharmaceuticals commercialized in different pharmacies in Cotonou, Benin. Material and MethodsMaterials: The samples used in this study are: medicinal plants purchased at market (table 1), improved and processed medicinal herbals purchased from herbalists, and the nutraceuticals purchased from different pharmacies (table 2). In total we considered 20 medicinal plants were purchased from two highly populated markets of Cotonou (Dantokpa and Gbegamey). In addition we purchased 11 nutraceuticals from pharmacies and 14 other nutraceuticals were purchased at the National Nutraceutical Fair (MTA) of 24 August 2011 at “Stade de l’amitie” in Cotonou. Survey of registered medicinal plants in form of nutraceuticals: The survey was conducted in ninety-three (93) pharmacies in Cotonou with the guidance of the pharmacists. The survey was performed according to the following criteria: Inclusion criteria: Available neutraceuticals made through infusion or maceration with no antibacterial and/or antifungal properties, administered orally Non-inclusion criteria: Unavailable nutraceuticals, with antibacterial and/or antifungal agents, administered by any (s) other (s) path (s) or as a decoction and alcoholic maceration. Determination of viable aerobic microbial germs: The both agar plate and Sabouraud containing chloramphenicol cell count approach was use to determine the number of bacteria and fungi present in the samples as described in the European Pharmacological guideline. A volume of 1 mL of inoculums of SM or its serial dilutions is deposited in the empty Petri dishes and covered by 15 to 20 mL of PCA agar. The SM solutions were seeded in duplicate. Plates were then homogenized and allowed to solidify under sterile hood. The Petri dishes were then incubated at 35 ° C for 48 hours. About 15 to 20 mL of Sabouraud Chloramphenicol Agar is distributed aseptically in Petri dishes. After cooling, the plates were seeded in duplicate by filing it with 0.1 mL of the SM or its serial dilutions. The microbial drop was on the Petri dish spread and incubated at 25 ° C for 96 hours. Research of specific microbial germs: Staphylococcus aureus (SA) the most pathogenic species, SA, responsible for food spoilage and food contamination was assessed on Baird Parker Agar containing potassium telluride. Pseudomonas aeruginosa: We assessed the highly resistant bacteria that can grow in antiseptic or antibiotic solution on cetrimide agar. Escherichia coli: To identify E. coli bacteria, we traced the marker of fecal contamination, an efficiency indicator from treatment on eosin methylene blue agar. MS previously prepared and used to prepared serial dilutions and kept at room temperature under the sterile hood for six hours (6 pm). 0.1 mL of MS was used to inoculate three selective media respectively. The control containing no bacterial was performed under the same conditions. The duration of incubation was 72 h for all selective media inoculated. Table-1 The medicinal plants, herbal drugs and the places of purchase N° Medicinal plants Herbal drugs Purchase locations 01 Carica papaya Leaf Market Dantokpa 02 Cassia alata Leaf Market Dantokpa 03 Citrus aurentifolia Leaf Market Dantokpa 04 Combretum glutinosum Leaf Market Dantokpa 05 Combretum micranthum Leaf Market Dantokpa 06 Crataeva religiosa Stem bark Market Dantokpa 07 Eucalyptus globulus Leaf Market Dantokpa 08 Ficus spp Leaf Market Dantokpa 09 Funtumia africana Leaf Market Dantokpa 10 Imperata cylindrica Leaf Market Dantokpa 11 Lippia chevalieri Leaf Market Gbégamey 12 Lippia rugosa Leaf Market Gbégamey 13 Lonchocarpus cyanescens Leaf Market Gbégamey 14 Morinda lucida Stem bark Market Gbégamey 15 Nauclea latifolia Root bark Market Gbégamey 16 Newbouldia laevis Leaf Market Gbégamey 17 Ocimum basilicum Leafy stem Market Gbégamey 18 Ocimum gratissimum Leaf Market Gbégamey 19 Panax ginseng Root Market Gbégamey 20 Phaseolus vulgaris Leaf Market Gbégamey International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 46-51, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 48 Table-2 Phytomedicinal forms and places of purchase N° Improved phytomedicines (PI) Forms of administration Places to buy 01 PI 01 Tea Pharmacy 02 PI 02 Tea Pharmacy 03 PI 03 Tea Pharmacy 04 PI 04 Powder Pharmacy 05 PI 05 Oral solution Pharmacy 06 PI 06 Oral solution Pharmacy 07 PI 07 Aqueous maceration Pharmacy 08 PI 08 Oral solution Pharmacy 09 PI 09 Aqueous maceration pharmacy 10 PI 10 Oral solution Pharmacy 11 PI 11 Oral solution Pharmacy 12 PI 12 Oral solution Stade de l’amitie 13 PI 13 Oral solution Stade de l’amitie 14 PI 14 Oral suspension Stade de l’amitie 15 PI 15 Oral suspension Stade de l’amitie 16 PI 16 Oral suspension Stade de l’amitie 17 PI 17 Oral solution Stade de l’amitie 18 PI 18 Infusion bag Stade de l’amitie 19 PI 19 Infusion bag Stade de l’amitie 20 PI 20 Infusion bag Stade de l’amitie 21 PI 21 Tea Stade de l’amitie 22 PI 22 Aqueous maceration Stade de l’amitie 23 PI 23 Infusion bag Stade de l’amitie 24 PI 24 Tea Stade de l’amitie 25 PI 25 Aqueous maceration Stade de l’amitie Results and Discussion Results of the field survey: Almost all pharmacies in Cotonou, Benin have embraced the selling of nutraceuticals. However, 66.67%of the pharmacies surveyed doubt the quality and/or the efficacy of the medicinal derived nutraceuticals, while 2.22% of pharmacies have not fully embraced the selling of nutraceuticals. Most of the nutraceuticals found in the pharmacies are not approved and account for 63.64% of the medicines preferred by the folk Marketing approval/license to sell plant based medicines/nutraceuticals is very complicated and generally not enforced. Among the well recognized licensing agencies, the Direction for Food and Applied Nutrition (DANA) is the most structured in the Country. As such, it has issued more than two out of the four marketing licenses of the recognized approved nutraceuticals in the pharmacies. No improved phytomedicine has been approved from the federal recognized agency, the National Program of the Pharmacopoeia and Traditional Medicine (PNPMT). Herbal medicines are mainly sold as liquid improved infusions (45.46%) either in water or alcoholic drink They are generally packaged in bottles (63.64%) and paper packaging (36.36%) devices For the bottle packaging they are generally in content of 100 mL, 250 mL and/or 1000 mL. In addition, most of these packaging medicines have no expiration date, although relatively very few contain expiration date. Specifically, more than half (54.55%) of herbal medicines sold in improved pharmacies have no expiration date. Results of counts of detected viable aerobic microorganisms: In this analysis, we only take into account plates containing 30-300 colonies for further characterizations. The results of different microorganisms identified are summarized in tables 3, 4 and 5. Identification and Characterization of specific microbial germs: The entire medicinal plants (100%) analyzed in this work have been contaminated by the following three microbial organisms: Staphylococcus aureus, identified in three samples out of the eleven samples analyzed. i.e. 27.27%; Pseudomonas aeruginosa, identified in two out of the eleven samples analyzed, i.e. 18.18% and Escherichia coli, identified in all the samples analyzed, i.e. 100%. Additional investigation showed that samples PI 05 and PI 08 were void of E. coli, indicating that 81.82% of all analyzed samples were contaminated by E. coli. In summary the nutraceuticals sold in pharmacies were most contaminated by Escherichia coli followed by Staphylococcus aureus. In addition of the microbial contamination data, no nutraceuticals considered in this work meets the European Pharmacopoeia standards. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 46-51, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 49 Among the phytomedicines purchased directly from herbalists Staphylococcus aureus was identified in twelve out of fourteen samples analyzed, i.e. 85.71%, Pseudomonas aeruginosa was identified in four out of fourteen samples, i.e. 28.57%, while Escherichia coli was present in all samples with exception of four samples, i.e. 92.86%. They are also not conforming to the European Pharmacopoeia standards. In this work, we surveyed eleven herbal medicines from registered pharmacies in the city of Cotonou, Benin (West Africa) and evaluated the microbial contamination profile of the medicines. The size of samples analyzed in this study was higher than that of Coulibaly which was six (06) herbal medicines in Mali. About88.17% of medicines sold in pharmacies in the city of Cotonou were herbal medicines, indicating that nutraceuticals are the most popular mode of medication in Benin. There are, however, pharmacies that do not sell these nutraceuticals due to their uncertain quality and low hygienic standard of processing. The pharmacies that refused to sell these nutraceuticals generally referred to the lack of marketing authorization and the lack of proper expiration date on the packaging of the medicines.Terminology /indications of improved phytomedicines are very arbitrary in this region of the world. They are not generally related to chemical properties of the plants, and most of the chemicals in the neutraceuticals were not properly studied and were generally unknown. This reflects the need to study for the validation of new indications (Phaseolus vulgaris) indicated in the removal of cholesterol, weight loss. The liquid forms of nutraceuticlas are more available, but also they were the most contaminated by the microorganisms. The form (maceration, infusion or decoction) of processing were not indicated in the packaging device. This is generally important because it can easily affect the storage conditions, the expiration date and the modification of the active ingredients of the nutraceuticals. According to Agassounon et al.10, at room temperature (26 ° C) macerations are not to be stored as long as decoctions. They generally can be kept more than three days. The shelf life is longer with the preparation that took into account a higher hygienic condition such as sterilization with filter through Millipore membrane 0.4 diameter10. The use of 1000 mL bottle packaging, which takes longer to be used are generally the most contaminated because the storage conditions were not generally followed both in the pharmacies and by the patients. Table-3 Microbial contamination counts in the medicinal plants S. No. Medicinal plants Number of colonies per ml of product (UFC/mL) Bacteria Yeasts and molds 01 Carica papaya 32.10 2 3.10 4 02 Cassia alata 59.10 2 6.10 4 03 Citrus aurentifolia 5.10 2 2.10 4 04 Combretum glutinosum 19.10 2 5.10 2 05 Combretum micranthum 40.10 2 5.10 4 06 Crataeva religiosa 99.10 2 12.10 3 07 Eucalyptus globulus 7.10 1 34.10 3 08 Ficus spp 4.10 3 52.10 2 09 Funtumia africana 27.10 2 8.10 3 10 Imperata cylindrica 36.10 2 14.10 4 11 Lippia chevalieri 41.10 2 18.10 4 12 Lippia rugosa 45.10 2 27.10 3 13 Lonchocarpus cyanescens 50.10 2 38.10 3 14 Morinda lucida 1.10 2 2.10 4 15 Nauclea latifolia 18.10 2 37.10 3 16 Newbouldia laevis 5.10 2 3.10 3 17 Ocimum basilicum 5.10 3 40.10 3 18 Ocimum gratissimum 6.10 3 24.10 3 19 Panax ginseng 18.10 1 36.10 3 20 Phaseolus vulgaris 25.10 3 25.10 3 Average number 4,4.10 3 3,9.10 4 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 46-51, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 50 Table-4 Microbial contamination counts in nutraceuticals sold in pharmacies S. No. Code of improved phytomedicines Number of colonies per ml of product (UFC/mL) Bacteria Yeasts and molds 01 PI 01 000 000 02 PI 02 8.10 2 000 03 PI 03 1.10 4 000 04 PI 04 1.10 2 000 05 PI 05 8.10 3 45.10 2 06 PI 06 16.10 4 000 07 PI 07 15.10 4 5.10 3 08 PI 08 1.10 4 4.10 3 09 PI 09 14.10 4 4.10 3 10 PI 10 3.10 4 5.10 2 11 PI 11 18.10 4 000 Average number 6,3.10 4 1,6.10 3 Table-5 The fungal versus bacterial contaminants in herbalmedicines purchased from herbalists S. No. Code of improved phytomedicines Number of colonies per ml of product (UFC/mL) Bacteria yeasts and molds 01 PI 12 000 000 02 PI 13 000 000 03 PI 14 4.10 4 49.10 3 04 PI 15 18.10 5 2.10 7 05 PI 16 000 000 06 PI 17 000 000 07 PI 18 5.10 2 000 08 PI 19 16.10 2 000 09 PI 20 3.10 2 000 10 PI 21 18.10 5 000 11 PI 22 31.10 2 000 12 PI 23 8.10 2 000 13 PI 24 18.10 5 1.10 7 14 PI 25 2.10 6 6.10 5 Average number 5,3.10 5 2,2.10 6 The total viable aerobic microorganisms were evaluated A total of twenty (20) medicinal plants were analyzed. Herbal drugs analyzed are mainly leaves (15/20) as they are commonly used in African traditional medicine. Yeasts and molds are the main contaminants; they generally affect the organoleptic characteristics of the herbal products. Places of harvest and drying conditions and storage are generally the main reasons for these contaminations. As above mentioned herbal medicines purchased from pharmacies were mainly in liquid forms (7 out of 11). They are also contaminated with bacteria at the rate of 14.32 times that of medicinal plants. This can be explained by the Proliferation of microbial contaminants of herbal medicine use and/or contamination occurred during the nutraceutical processing. To obtain improved herbal and medicinal plants that meet the approved standards, the plant materials used should always be disinfected. The compliance with good practice (GMP), packaging and appropriate storage conditions are (7 out of 11) not always observed. Compliance with good practice (GMP), appropriate packaging and control of intermediates in the production of herbal medicines need improvement. This study found that herbal medicines purchased from herbalists are mostly in liquid forms (8 of 14). They are slightly contaminated by yeasts and molds. For those contaminated by fungi, the fungal contamination count was very high (10 CFU/ml). The result is the same for bacterial contaminants. The most contaminated samples were the oldest products registered in the pharmacies compared to the newer products, which were less contaminated. The nutraceutical manufacturing process also plays a significant in the microbial contamination. The macerations are more easily contaminated than the decoctions.Medicinal plants purchased from different places (markets and Dantokpa Gbegamey) in Cotonou were all contaminated with Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. These results are similar with those obtained by Agassounon et al.11who found E. coli in all samples of International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 46-51, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 51 medicinal plants analyzed.In these markets, the mixture of herbal drugs, the absence of disinfectant during nutraceutical processing and good storage conditions could explain the high level of medicinal contamination. During storage, the conditions for cross-contamination increases the rate of microbial germ counts and complicate the issue of nutraceutical safety. The rate of herbal medicine contaminations reported in this study is similar with that reported by Baba-Moussa et al.12 (83%) in food sold by hawkers in the city of Cotonou. However, studies conducted by Coulibaly revealed an absence of Salmonella and Shigella in six (06) improved herbal analyzed.Herbal medicines purchased from herbalists are heavily contaminated with Staphylococcus aureus and Escherichia coli. Given the habitat of these two organisms (human digestive tract and genitals), contamination of human origin is suspected in these samples. This reflects a lack of strict hygienic measures. The strong presence of Staphylococcusaureus and Escherichia coli in the phytomedicines confirms the involvement of manipulations. ConclusionFollowing this study, eleven (11) improved herbal medicines sold in pharmacies in the city of Cotonou were analyzed. The results show that none of these herbal medicines meets the required European Pharmacopoeia standards. Improved herbal medicines sold in pharmacies and those sold by herbalists are highly contaminated with highly pathogenic microbial germs, including Staphylococcus aureus, Pseudomonas aeruginosa andEscherichia coli. References 1.Lutgen P., La tisane d’Artemisia annua, une puissante Polythérapie, 2ème congrès « Maladies tropicales, aspects humanitaires et scientifiques, Luxembourg, (2009)2.Pousset J.L., Plantes médicinales d’Afrique : Comment les reconnaître et les utiliser ? Edisud, 288 (2004)3.Ministère de la Santé, Manuel d’initiation des professionnels de la santé aux systèmes d’éducation et de transmission du savoir en Médecine Traditionnelle au Bénin, Cotonou: PMLS 2 / Banque Mondiale, 80 (2011)4.Organisation Mondiale de la Santé,Stratégie de l’OMS pour la médecine traditionnelle pour 2002-2005, Genève, 65 (2002)5.Pousset J.L., Place des médicaments traditionnels en Afrique, Med. Trop., 66, 606-609 (2006)6.Organisation Mondiale de la Santé. Réglementation des médicaments à base de plantes, la situation dans le monde, Genève, 59 (1998)7.Direction des Pharmacies du Médicaments et des Explorations Diagnostiques., Liste des Officines de Pharmacie du Bénin, DPMED, Cotonou, 14 (2011)8.Rodier J., L’analyse de l’eau. 8 eds. Paris: DUNOD, 1383 (2005)9.Coulibaly S.L., Contribution à l’évaluation de la qualité des médicaments traditionnels améliorés, Thèse de doctorat d’Etat en pharmacie, Université de Bamako, Mali, (2008)10.Agassounon Djikpo Tchibozo M.,Evaluation de la qualité hygiénique-Etudes phytochimique et pharmacologique de quelques plantes de la médecine traditionnelle béninoise, Thèse unique de Doctorat, Université de Lomé, Togo, (2004)11.Agassounon Djikpo Tchibozo M., Toukourou F., de Souza C. and Gbeassor M., Identification de la flore microbienne de six plantes médicinales utilisées en médecine traditionnelle béninoise, Microbiol. Hyg. Alim., 18, 24-29 (2006)12.Baba-Moussa L., Bokossa Y.I., Baba-Moussa F., Ahissou H., Adeoti Z., Yehouenou B., Mamadou A., Toukourou F. and Sanni A., Etude des possibilités de contamination des aliments de rues au Bénin : cas de la ville de Cotonou, J. Rech. Sci. Univ. Lomé, , 149-156 (2006)