Available online at www.sciencedirect.com South African Journal of Botany 82 (2012) 60 – 66 www.elsevier.com/locate/sajb Review Ensuring quality in herbal medicines: Toxic phthalates in plastic-packaged commercial herbal products A.R. Ndhlala, B. Ncube, J. Van Staden ⁎ Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Available online 5 September 2012 Abstract There is a proliferation in the use of commercial herbal preparations, most notably liquid preparations of plant material packaged in plastic containers. The quality of these herbal preparations has always and still remains questionable. A number of research institutes and research groups in tertiary institutions throughout South Africa have embarked on the development of quality control programmes to test herbal medicines for safety and efficacy. However, very few of these groups have placed any emphasis on commercial herbal mixtures. This paper describes some effects of toxic phthalates such as bis(2-ethylhexyl) phthalates, (a common plasticiser) used in cheap soft plastics and its metabolites. Analytical methods for the determination, quantification and monitoring of DEHP in herbal remedies to ensure safety are also highlighted. © 2012 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Bis(2-ethylhexyl) phthalates; Herbal products; Phthalates; Quality control Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phthalate contaminants in herbal products . . . . . . . . . . . . . . . DEHP biomarkers of exposure and effects . . . . . . . . . . . . . . 3.1. Phthalates and the reproductive development in human infants 3.2. Phthalates and respiratory function, asthma, and allergy . . . . 4. The fate of DEHP: Its metabolites and their effects . . . . . . . . . . 5. Tolerable daily intake and minimal risk level . . . . . . . . . . . . . 6. Analytical methods for determining DEHP . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Despite its negative consequence on plant biodiversity and destabilisation of most ecosystems, the inter-relationship between ⁎ Corresponding author. Tel.: +27 33 2605130; fax: + 27 33 2605897. E-mail address: rcpgd@ukzn.ac.za (J. Van Staden). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 61 62 62 62 62 63 63 64 64 64 society and nature, and the importance of herbal medicine to human health, continues to dominate most medical traditions. Plants form an integral part of virtually every medicinal system in both modern and less civilised societies. South Africa is one of the countries that are richly bestowed with both floral and cultural diversity, and for this reason the population exploits phytomedicines as part of their medical 0254-6299/$ -see front matter © 2012 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2012.07.004 A.R. Ndhlala et al. / South African Journal of Botany 82 (2012) 60–66 tradition. A large number of medicinal plants are regularly sold as either crude, unprocessed material, semi-processed or processed as commercial herbal preparations (CHP) on various traditional markets across the country (Mander, 1998; Ndhlala et al., 2011b; Williams et al., 2000). As a result of urbanisation and the consequent commercialisation of traditional health care, the demand for herbal medicine has increased significantly in most South African cities. There exists an insatiable market for these products from most urban dwellers, on which traders capitalise. Among the herbal products sold in urban areas, CHPs dominate this market (Ndhlala et al., 2011b). This has become a modern avenue for traditional plant-derived therapeutics in South Africa. Although herbal medicines are widely used for the prevention, diagnosis, treatment and management of disease, quality control remains the biggest challenge. The widely held perception, by herbal medicinal users, that natural plant-derived products are safe, effective and non-toxic, has often left unsuspecting consumers exposed to a multitude of risks associated with the use and/or overuse of these products. It is important that the general public be warned of the range of possible dangers that emanate from using these plant-derived products. CHPs present some complications to herbal medicine with regard to efficacy, safety and quality. Chemical interactions between and among the constituents of the component plant species in each preparation could be diverse and result in synergism, antagonism, or toxicity (Ncube et al., 2012; Ndhlala et al., 2009) within the human body. Herbal preparations are produced and packaged by private entrepreneurs (Ndhlala et al., 2009), and their quality, efficacy and safety are determined by the manufacturing conditions, condition and quality of material used for packaging, quantity and type of component plant species used, as well as their storage and handling, all of which remain solely under the control of the manufacturer and distributor. Production and marketing of these products have become a lucrative business in most cities of South Africa, to such an extent that their quality and safety has often been overlooked in an effort to make a profit. As a result, consumers are offered products that are sub-standard and packaged using cheap recycled plastic materials (Nair et al., 2012). Could such herbal products deliver health benefits or are they a potential health hazard to consumers? Logically, one would be compelled to view such products as weapons of death. Herbal preparations in general, are probably characterised with impure, contaminated and sometimes lethally toxic components that pose risks to humankind. A solution to this lies in intensive screening of these products and the subsequent enacting of, and strict adherence to, quality and safety regulations. Considering the large number of people relying on these products on a day to day basis and the associated health concerns, an interest was aroused in their pharmacological and toxicological screening (Nair et al., 2012; Ndhlala et al., 2010a,b, 2011a), in which both positive and negative effects have been reported. Among the negative reports on the CHP, the most recent and potentially worrying discovery, is the isolation of a commercial plasticiser di(2-ethylhexyl) phthalate (DEHP) from one of the most popular preparations (Nair et al., 2012). This adds another dimension to the safety and quality concerns of CHPs sold in South African medicinal markets. An assessment of the possible 61 risks of phthalate contaminants and its metabolites in herbal products, as well as the minimum daily tolerable levels, are outlined in this review. Analytical methods for the determination, quantification and monitoring of DEHP in herbal remedies to ensure safety are also highlighted. 2. Phthalate contaminants in herbal products An increasing social concern has been aroused by the adverse effects on public health caused by phthalates. Phthalates have been shown to have endocrine disrupting properties resulting in carcinogenic effects, metabolic disorders, and developmental and reproductive defects (Hauser et al., 2006; Mahood et al., 2007; Willhite, 2001). DEHP is used as a plasticiser in polyvinyl chloride (PVC) plastics, rubber, cellulose and styrene production to impart flexibility, strength, broad-range temperature tolerance, and optical clarity (Chen et al., 2004). Phthalates are used in a wide variety of consumer products and applications, that include gelling agents, medical devices, cosmetics, adhesives, lubricants, dispersants, food wrappings, nail polish, plastic goods, kitchen plastic ware and emulsifying agents (Chen et al., 2004; Wormuth et al., 2006). Since phthalates are not chemically bound to these products, leaching and migration of these substances result in significant environmental contamination and human exposure is a common phenomenon (Clewell et al., 2008; Mahood et al., 2007; Wormuth et al., 2006). CHPs are, particularly those that are packed using cheap recycled plastic materials, no exception to this contamination. These contaminants could reach alarming levels in these products in South Africa, owing to the fact that there are no effective quality regulation mechanisms and/or policies with regard to the manufacturing and handling of these products (Ndhlala et al., 2011b). The highly toxic phthalate compound (DEHP) isolated from one of the popular herbal preparations (‘Sejeso’ herbal mixture) in South Africa, was found to be 43.3 mg/L (Nair et al., 2012). An important concern about DEHP relates to its potential to act as a non-genotoxic hepatocarcinogen, a reproductive and developmentally toxic substance and its neurodegenerative effects (Hauser et al., 2006; Koch et al., 2006; Willhite, 2001; Wittassek and Angerer, 2008). Due to its widespread use in a variety of consumer products and its associated health risks to humans, the World Health Organisation (WHO) have implemented strict guidelines with regard to the tolerable daily intake levels of DEHP (WHO, 2003). The recommended dose for patients using ‘Sejeso’ herbal mixture would translate to each individual consuming on average 7.8 mg DEHP per day. Compared to the daily tolerable levels of 8 μg/L and 1.5 mg/kg for drinking water and food consumed respectively, this figure (7.8 mg) is higher than the recommended values. These results indicate how exposed consumers of these herbal products are. In many cases, one patient might be using more than one CHP at any given time per day, resulting in the accumulation of these toxic contaminants to alarming levels. The risks could even be far more, considering the fact that consumers rarely stick to the recommended doses on the container labels, but tend to take higher doses, with the belief that the healing effects will be faster and more effective. 62 A.R. Ndhlala et al. / South African Journal of Botany 82 (2012) 60–66 It could be hypothesised that these contaminants are not only confined to one herbal mixture but could be a common characteristic of some plastic-packaged CHPs since most of them are packaged in similar plastic containers. Phthalate contaminants in herbal products could either be emanating from the plastic containers in which they are packaged (to a larger extent) and/or during the manufacturing process. The levels of these chemical substances could have far reaching effects on the health of current and future generations of the population relying on these herbal products. Against a background of widespread use, ubiquitous exposure and concerns about the ability of phthalates to cause adverse health effects in humans, quality assurance of botanicals and herbal preparations should be a prerequisite if these health concerns are to be addressed. Very little research has been done regarding the safety and quality of CHPs used in South Africa and hence the need for further studies in this regard, particularly on phthalates. The literature to this aspect (phthalates in CHPs used in South Africa) is indeed scarce and hence this review is to bring this to the fore to stimulate further research work on CHPs and other food products. 3. DEHP biomarkers of exposure and effects Through experimental and epidemiological studies, evidence has accumulated for the association of other detrimental health effects associated with phthalate exposure. Phthalates are rapidly hydrolyzed followed by oxidation and are mainly excreted via urine (Wittassek and Angerer, 2008). Thus, the content of phthalate metabolites in human urine acts as an indicator of recent internal exposure to the respective parent phthalate. Several studies have assessed phthalate exposure of the general population by measuring phthalate metabolites in human urine samples. 3.1. Phthalates and the reproductive development in human infants Exposure to environmental chemicals with antiandrogenic activity, such as phthalates, is considered one of the causes for the increased incidence of testicular dysgenesis syndrome (TDS). The foetus is considered to be the most sensitive stage of life to the potential developmental and reproductive toxicity of phthalates (Wittassek et al., 2009). Some phthalates have been found to be developmental and reproductive toxicants in rats, with pronounced effect on the male reproductive system (Borch et al., 2006; Foster, 2005; Gray et al., 2006). Recent research has focused on the effects of certain phthalate esters on reproduction, commonly on male reproductive development in experimental animals. There are indications that phthalates may be associated with abnormal sexual development and birth defects in humans (McKee et al., 2004; Sharpe, 2008; Swan, 2008). The pathways of androgen action are similar in both experimental animals and humans, suggesting that phthalates may cause comparable adverse effects on reproduction and development in humans compared to experimental animals. Several epidemiological studies suggest that environmental exposure to a number of phthalates may be associated with adverse reproductive outcomes, like alterations in semen parameters, DNA damage in sperm, reduced reproductive hormone levels in adult men and decreased anogenital distance in male infants (Duty et al., 2003; Swan, 2008). The relationship between phthalate exposure and adverse effects in human reproduction impacts negatively on their offspring. 3.2. Phthalates and respiratory function, asthma, and allergy The term allergy describes adverse health effects that might result from the stimulation of a specific immune response (Kimber and Dearman, 2010). One important aspect of allergic sensitisation is that some chemicals may have qualitative, as well as quantitative, effects on specific immune responses. The possibility that phthalates may present a risk factor for the development of allergies and asthma has been described in several case studies (Bornehag et al., 2004; Bornehag and Nanberg, 2009; Jaakkola et al., 2004; Kimber and Dearman, 2010). This has led to the conclusion that some cleaning products used in domestic environments are associated with an increased risk of asthma (Bornehag et al., 2004; Rumchev et al., 2004). In addition, occupational exposure to high concentrations of phthalate fumes has been linked to asthma and other respiratory symptoms (Andrasch et al., 1976; Markowitz, 1989; Nielsen et al., 1989). A variety of respiratory symptoms such as cough, work-related shortness of breath, wheezing and rhinitis, as well as a decline in forced expiratory volume (FEV1) were found to be increased in exposed workers compared to control groups (Kimber and Dearman, 2010). Persuasive as these investigations are to an association between household cleaning and clinical diseases such as respiratory reactions, they do not necessarily implicate cleaning materials as being responsible for an increase in the incidence of allergic sensitisation. The implication is that certain phthalates may act as adjuvants and thereby predispose to, and/or otherwise enhance, acquisition of allergic sensitisation (Kimber and Dearman, 2010). It is appropriate, therefore, in advance of reviewing published data on the association between phthalates and allergy, to consider the nature of phthalates and their function as adjuvants. In this regard, two independent studies were conducted in Sweden (Bornehag et al., 2004) and Bulgaria (Kolarik et al., 2008) in which the association between the phthalate content of house dust and allergic symptoms in children was investigated. Both studies reported a significant association between the concentration of DEHP in house dust and allergic symptoms (asthma or wheezing) in children. The implication that phthalates might play a role in the development of respiratory disorders, asthma and allergy can therefore not be overlooked. 4. The fate of DEHP: Its metabolites and their effects Humans can be exposed to phthalates through ingestion, inhalation and dermal contact when using phthalate-containing consumer products (Silva et al., 2007). Human exposure to phthalates is facilitated by nutrition, medical devices and personal care products. After absorption, phthalates are readily metabolised to hydrolytic monoesters which can be further metabolised to other oxidative products before excretion in urine or faeces either A.R. Ndhlala et al. / South African Journal of Botany 82 (2012) 60–66 63 Table 1 The effect of three DEHP metabolites used as markers of human exposure. Compound Toxic effects References MEHP • Anti-androgenic (disruption of testosterone action) • Inhibition of LH-stimulated steroid formation • May be toxic to the reproductive system • Confirmed animal carcinogen with unknown relevance to humans • Priority water pollutant • Probable endocrine disruptor • Prohibited in EU cosmetics • Gastrointestinal or liver toxicity hazards • Immune system toxicity • Respiratory system toxicity • Respiratory toxicity hazards • Persistent, bioaccumulative toxicant • Prohibited in EU cosmetics Grasso et al. (1993), Fan et al. (2010) and Piché et al. (2012) MEOHP MEHHP as free or conjugated species with elimination half-lives of less than 24 h (Herr et al., 2009). In the liver, phthalates are oxidised by xenobiotic metabolising enzymes such as cytochrome P450 (Dirven et al., 1993). There are however, differences in the excretion pattern of urinary DEHP metabolites between species. Rats, for example, excrete more of the oxidised urinary metabolites than humans and monkeys which excrete (glucuronide) conjugated metabolites (Albro, 1986). The toxic effects of phthalate metabolites are outlined in Table 1. The main known DEHP metabolites excreted by humans are mono (2-ethylhexyl) phthalate (MEHP), mono (5-carboxy2-ethylpentyl) phthalate, mono (2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) (Dirven et al., 1993) represented in Fig. 1 and Table 1. The other equally important phthalate metabolites include phthalic acid (PA), monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-3-carboxypropyl phthalate (MCPP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), monocyclohexyl phthalate (MCHP), monobenzyl phthalate (MBzP), mono-n-octyl phthalate (MOP), mono-isononyl phthalate (MNP), mono-isodecyl phthalate (MDP), mono-n-hexyl phthalate (MHxP), mono-n-heptyl phthalate (MHpP), mono-2ethyl-5-carboxypentyl phthalate (MECPP), mono-carboxy-nheptyl phthalate (MCHpP), monocarboxy-isooctyl phthalate (MCOP), mono-hydroxyisononyl phthalate (MHNP), monooxoisononyl phthalate (MONP), and mono-carboxyisononyl phthalate (MCNP) (Silva et al., 2007). These metabolites are excreted in urine predominantly as glucuronide conjugates (Kato et al., 2004). The differences in the rate of metabolism among phthalates could result in urinary excretion of higher levels of the monoester metabolites (MEHP, MEOHP and MEHHP) of the phthalates with short alkyl chains than for DEHP, making these metabolites suitable biomarkers for DEHP exposure (Kato et al., 2004). The metabolism of DEHP involves the hydrolysis of one ester bond, giving rise to MEHP, followed by oxidation to MEOHP and MEHHP. Apostolidis et al. (2002), OSPAR (2002) and Bornehag et al. (2004) Apostolidis et al. (2002), OSPAR (2002), and Bornehag et al. (2004) 5. Tolerable daily intake and minimal risk level Based on the experimental data derived from animal studies, the Scientific Committee on Toxicity, Ecotoxicity and the Environment obtained tolerable daily intakes (TDI) and minimal risk level (MRL) values for phthalates (Yen et al., 2011). The WHO has set the TDI for DEHP at 8 μg/L for drinking water and 1.5 mg/kg for food (WHO, 1996, 2003). It is therefore important to carry out quality checks on the levels of DEHP and its metabolites in commonly consumed products like CHPs. As mentioned before, Nair et al. (2012) calculated exposure levels of 7.8 mg DEHP per day in a commercial herbal mixture (Sejeso herbal mixture, Ingwe brand). The authors warned that the levels were alarming and reflected a lack of effective quality control in the traditional medicine sector in South Africa. 6. Analytical methods for determining DEHP Several analytical methods have been reported for the determination of DEHP in foods, biological fluids, and tissue samples. These include high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectroscopy (MS) and nuclear magnetic resonance techniques (NMR) (Aignasse et al., 1995; Dirven et al., 1993; Faouzi et al., 1999; Herr et al., 2009; Nair et al., 2012). These methods often differ in extraction and sample preparation procedures involved. Nevertheless, all the techniques are tedious and time consuming (Aignasse et al., 1995). The idea is however, to use a sensitive and specific analytical method to determine DEHP and its metabolites in any matrix. HPLC has been the most useful and sensitive method to determine concentrations of DEHP and its metabolites in food and liquid matrices (Kambia et al., 2001). However, as revealed by Torto et al. (2007), Africa as a continent has its unique challenges for analytical chemists in sample preparation for chromatographic 64 A.R. Ndhlala et al. / South African Journal of Botany 82 (2012) 60–66 O for clean-up as compared to silica gel adsorbents (Fatoki and Ogunfowokan, 1993). Given the recent surge and popularity of plastic packaged commercial herbal remedies, the South African government should provide adequate support for analytical studies and control of levels of DEHP in formulated products. WHO has already published several technical guidelines for appropriate approaches to assessing chemical toxins in herbal medicines (WHO, 1996, 2003). Despite the fact that a lot of research has been conducted on the effects of DEHP, its replacement with safer chemicals still remains a challenge. Providing adequate research facilities and experienced personnel, as well as setting up common accepted analytical standards through bilateral recognition and through international and regional regulatory cooperation for herbal medicines should be considered. CH3 O CH3 O CH3 O CH3 bis (2-ethylhexyl) phthalates (DEHP) O CH3 O OH CH3 O 7. Conclusions mono (2-ethylhexyl) phthalate (MEHP) In conclusion, DEHP is a liquid widely used to make plastics more flexible. A potential exists for DEHP contamination of food and CHPs during processing, handling, transportation and packaging. Most cases of exposure occur from the leaching of DEHP from plasticised products during their use. The level of exposures is affected by the thickness of the plastic item, the temperature, storage conditions and the specific nature of the product. Mandatory quality control mechanisms should therefore be implemented for the manufacturing and packaging of products in plastic containers intended for human consumption. OH O O CH3 OH CH3 O mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) O O Acknowledgements CH3 O OH CH3 O This work was supported by the Claude Leon Foundation, the University of KwaZulu-Natal (UKZN) College of Science and Agriculture and the UKZN Research Office. mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) References Fig. 1. DEHP and its metabolites as biomarkers in human exposure. analyses. This has led to the development of a protocol for the determination of phthalate esters and polycyclic aromatic hydrocarbons in both aquatic samples and sediments (Torto et al., 2007). In the investigation of simple methods for extraction of phthalates, two approaches were employed either using liquid– liquid extraction or solid-phase extraction. For liquid–liquid extraction, extraction efficiencies for chloroform, dichloromethane (DCM) and ethyl acetate were investigated for the determination of phthalates in recovery experiments (Torto et al., 2007). The authors concluded that DCM was the best extracting solvent for phthalates as the other solvents formed emulsions, which were difficult to separate from the aqueous medium and consequently gave low recovery. Dichloromethane was cited as suitable because of its low cost, availability in highly purified form, inertness and easy separation from water because of its higher density. Alumina was cited as the better suitable sorbent material Aignasse, M.F., Prognon, P., Stachowicz, M., Gheyouche, R., Pradeau, D., 1995. A new simple and rapid HPLC method for determination of DEHP in PVC packaging and releasing studies. International Journal of Pharmaceutics 113, 241–246. Albro, P.W., 1986. The biochemical toxicology of di-(2-ethylhexyl) and related phthalates: testicular atrophy and hepatocarcinogenesis. Review of Biochemistry and Toxicology 8, 73–119. Andrasch, R.H., Bardana Jr., E.J., Koster, F., Pirofsky, B., 1976. Clinical and bronchial provocation studies in patients with meatwrappers' asthma. The Journal of Allergy and Clinical Immunology 58, 291–298. Apostolidis, S., Chandra, T., Demirhan, I., Cinatl, J., Doerr, H.W., Chandra, A., 2002. Evaluation of carcinogenic potential of two nitro-musk derivatives, musk xylene and musk tibetene in a host-mediated in vivo/in vitro assay system. Anticancer Research 22, 2657–2662. Borch, J., Axelstad, M., Vinggaard, A.M., Dalgaard, M., 2006. Diisobutyl phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in foetal rat testis. Toxicology Letters 163, 183–190. Bornehag, C.G., Nanberg, E., 2009. Phthalate exposure and asthma in children. International Journal of Andrology 33, 1–13. Bornehag, C.G., Sundell, J., Weschler, C.J., Sigsgaard, T., Lundgren, B., Hasselgren, M., Hägerhed-Engman, L., 2004. The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case– control study. Environmental Health Perspectives 112, 1393–1397. A.R. Ndhlala et al. / South African Journal of Botany 82 (2012) 60–66 Chen, C.Y., Ghule, A.V., Chen, W.Y., Wang, C.C., Chiang, Y.S., Ling, Y.C., 2004. Rapid identification of phthalates in blood bags and food packaging using ToF-SIMS. Applied Surface Science 231–232, 447–451. Clewell, R.A., Kremer, J.J., Williams, C.C., Campbell Jr., J.L., Andersen, M.E., Borghoff, S.J., 2008. Tissue exposures to free and glucuronidated monobutylyphthalate in the pregnant and fetal rat following exposure to di-n-butylphthalate: evaluation with a PBPK model. Toxicological Sciences 103, 241–259. Dirven, H.A.A.M., Van den Broek, P.H.H., Jongeneelen, F.J., 1993. Determination of four metabolites of the plasticizer di(2-ethylhexyl)phthalate in human urine samples. International Archives of Occupational and Environmental Health 64, 555–560. Duty, S.M., Singh, N.P., Silva, M.J., Barr, D.B., Brock, J.W., Ryan, L., Herrick, R.F., Christiani, D.C., Hauser, R., 2003. The relationship between environmental exposure to phthalates and DNA damage in human sperm using the neutral comet assay. Environmental Health Perspectives 111, 1164–1169. Fan, J., Traore, K., Li, W., Amri, H., Huang, H., Wu, C., Chen, H., Zirkin, B., Papadopoulos, V., 2010. Molecular mechanisms mediating the effect of mono-(2-ethylhexyl) phthalate on hormone-stimulated steroidogenesis in MA-10 mouse tumor leydig cells. Endocrinology 151, 3348–3362. Faouzi, M.A., Khalfi, F., Dine, T., Luyck, M., Brunet, C., Gressier, B., Goudaliez, F., Cazin, M., Kablan, J., Belabed, A., Cazin, J.C., 1999. Stability, compatibility and plasticizer extraction of quinine injection added to infusion solutions and stored in polyvinyl chloride (PVC) containers. Journal of Pharmaceutical and Biomedical Analysis 21, 923–930. Fatoki, O.S., Ogunfowokan, A.O., 1993. Procedure cleanup technique for determination of phthalate esters in an aquatic environment. International Journal of Environmental Studies 44, 237–243. Foster, P.M., 2005. Mode of action: impaired foetal leydig cell function — effects on male reproductive development produced by certain phthalate esters. Critical Reviews in Toxicology 35, 713–719. Grasso, P., Heindel, J.J., Powell, C.J., Reichert Jr., L.E., 1993. Effects of mono (2-ethylhexyl) phthalate, a testicular toxicant, on follicle-stimulating hormone binding to membranes from cultured rat Sertoli cells. Biology of Reproduction 48, 454–459. Gray Jr., L.E., Wilson, V.S., Stoker, T., Lambright, C., Furr, J., Noriega, N., Howdeshell, K., Ankley, G.T., Guillette, L., 2006. Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals. International Journal of Andrology 29, 96–104. Hauser, R., Meeker, J.D., Duty, S., Silva, M.J., Calafat, A.M., 2006. Altered semen quality in relation to urinary concentrations of phthalate monoester and oxidative metabolites. Epidemiology 17, 682–691. Herr, C., Nieden, A., Koch, H.M., Schuppe, H.-C., Fieber, C., Angerer, J., Eikmann, T., Stilianakis, N.I., 2009. Urinary di(2-ethylhexyl)phthalate (DEHP) metabolites and male human markers of reproductive function. International Journal of Hygiene and Environmental Health 212, 648–653. Jaakkola, J.J., Parise, H., Kislitsin, V., Lebedeva, N.I., Spengler, J.D., 2004. Asthma, wheezing, and allergies in Russian schoolchildren in relation to new surface materials in the home. American Journal of Public Health 94, 560–562. Kambia, K., Dine, T., Gressier, B., Germe, A.-F., Luyckx, M., Bruneta, C., Michaud, L., Gottrand, F., 2001. High-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma. Journal of Chromatography B 755, 297–303. Kato, K., Silva, M.J., Reidy, J.A., Hurtz, D., Malek, N.A., Needham, L.L., Nakazawa, H., Barr, D.B., Calafat, A.M., 2004. Mono(2-ethyl-5-hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate as biomarkers for human exposure assessment to di-(2-ethylhexyl) phthalate. Environmental Health Perspectives 112, 327–330. Kimber, I., Dearman, R.J., 2010. An assessment of the ability of phthalates to influence immune and allergic responses. Toxicology 271, 73–82. Koch, H.M., Preuss, R., Angerer, J., 2006. Di(2-ethylhexyl) phthalate (DEHP): human metabolism and internal exposure-an update and latest results. International Journal of Andrology 29, 155–165. Kolarik, B., Naydenov, K., Larrson, M., Borenhag, C.G., Sundell, J., 2008. The association between phthalates in dust and allergic diseases among Bulgarian children. Environmental Health Perspectives 116, 98–103. 65 Mahood, I.K., Scott, H.M., Brown, R., Hallmark, N., Walker, M., Sharpe, R.M., 2007. In utero exposure to di(n-butyl) phthalate and testicular dysgenesis: comparison of fetal and adult end points and their dose sensitivity. Environmental Health Perspectives 115 (Suppl. 1), 55–61. Mander, M., 1998. The Marketing of Indigenous Medicinal Plants in Southern Africa: A Case Study in KwaZulu-Natal. FAO, Rome. Markowitz, J.S., 1989. Self-reported short- and long-term respiratory effects among PVC-exposed firefighters. Archives of Environmental Health 44, 30–33. McKee, R.H., Butala, J.H., David, R.M., Gans, G., 2004. NTP center for the evaluation of risks to human reproduction reports on phthalates: addressing the data gaps. Reproduction Toxicology 18, 1–22. Nair, J.J., Ndhlala, A.R., Chukwujekwu, J.C., Van Staden, J., 2012. Isolation of di(2-ethylhexyl) phthalate from a commercial South African cognate herbal mixture. South African Journal of Botany 80, 21–24. Ncube, B., Finnie, J.F., Van Staden, J., 2012. In vitro antimicrobial synergism within plant extract combinations from three South African medicinal bulbs. South African Journal of Botany 139, 81–89. Ndhlala, A.R., Stafford, G.I., Finnie, J.F., Van Staden, J., 2009. In vitro pharmacological effects of manufactured herbal concoctions used in KwaZulu-Natal South Africa. Journal of Ethnopharmacology 122, 117–122. Ndhlala, A.R., Anthonissen, R., Stafford, G.I., Finnie, J.F., Verschaeve, L., Van Staden, J., 2010a. In vitro cytotoxic and mutagenic evaluation of thirteen commercial herbal mixtures sold in KwaZulu-Natal, South Africa. South African Journal of Botany 76, 132–138. Ndhlala, A.R., Finnie, J.F., Van Staden, J., 2010b. In vitro antioxidant properties, HIV-1 reverse transcriptase and acetylcholinesterase inhibitory effects of traditional herbal preparations sold in South Africa. Molecules 15, 6888–6904. Ndhlala, A.R., Finnie, J.F., Van Staden, J., 2011a. Plant composition, pharmacological properties and mutagenic evaluation of a commercial Zulu herbal mixture: Imbiza ephuzwato. Journal of Ethnopharmacology 133, 663–674. Ndhlala, A.R., Stafford, G.I., Finnie, J.F., Van Staden, J., 2011b. Unregistered commercial herbal preparations in KwaZulu-Natal: the urban face of traditional medicine. South African Journal of Botany 77, 830–843. Nielsen, J., Fahraeus, C., Bensryd, I., Akesson, B., Welinder, H., Linden, K., Skerfving, S., 1989. Small airways function in workers processing polyvinylchloride. International Archives of Occupational and Environmental Health 61, 427–430. OSPAR, 2002. OSPAR List of Substances of Possible Concern. OSPAR Convention for the Protection of the Marine Environment of North-east Atlantic. Piché, C.D., Sauvageau, D., Vanlian, M., Erythropel, H.C., Robaire, B., Leask, R.L., 2012. Effects of di-(2-ethylhexyl) phthalate and four of its metabolites on steroidogenesis in MA-10 cells. Ecotoxicology and Environmental Safety 79, 108–115. Rumchev, K., Spickett, J., Bulsara, M., Phillips, M., Stick, S., 2004. Association of domestic exposure to volatile organic compounds with asthma in young children. Thorax 59, 746–751. Sharpe, R.M., 2008. Additional effects of phthalate mixtures on fetal testosterone production. Toxicological Science 105, 1–4. Silva, M.J., Samandar, E., Preau Jr., J.L., Reidy, J.A., Needham, L.L., Calafat, A.M., 2007. Quantification of 22 phthalate metabolites in human urine. Journal of Chromatography B 860, 106–112. Swan, S.H., 2008. Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans. Environmental Research 108, 177–184. Torto, N., Mmualefea, L.C., Mwatseteza, J.F., Nkoane, B., Chimuka, L., Nindi, M.M., Ogunfowokan, A.O., 2007. Sample preparation for chromatography: an African perspective. Journal of Chromatography. A 1153, 1–13. WHO, 1996. Guidelines for Drinking Water Quality. WHO Press, Geneva, Switzerland. WHO, 2003. Diethyl Phthalate. Concise International Chemical Assessment Document 52. WHO Press, Geneva, Switzerland. Willhite, C.C., 2001. Weight-of-evidence versus strength-of-evidence in toxicologic hazard identification: di(2-ethylhexyl)phthalate (DEHP). Toxicology 160, 219–226. Williams, V.L., Balkwill, K., Witkowski, E.T.F., 2000. Unraveling the commercial market for medicinal plants and plant parts on the Witwatersrand, South Africa. Economic Botany 54, 310–327. 66 A.R. Ndhlala et al. / South African Journal of Botany 82 (2012) 60–66 Wittassek, M., Angerer, J., 2008. Phthalates: metabolism and exposure. International Journal of Andrology 31, 131–138. Wittassek, M., Angerer, J., Kolossa-Gehring, M., Schäfer, S.D., Klockenbusch, W., Dobler, L., Günsel, A.K., Müller, A., Wiesmüller, G.A., 2009. Fetal exposure to phthalates-a pilot study. International Journal of Hygiene and Environmental Health 212, 492–498. Edited by AM Viljoen Wormuth, M., Scheringer, M., Vollenweider, M., Hungerbuhler, K., 2006. What are the sources of exposure to eight frequently used phthalic acid esters in Europeans? Risk Analysis 26, 803–824. Yen, T.-H., Lin-Tan, D.-T., Lin, J.-L., 2011. Food safety involving ingestion of foods and beverages prepared with phthalate-plasticizer containing clouding agents. Journal of the Formosan Medical Association 110, 671–684.