Abstract
The DCA (Drug Control Authority) of Malaysia implemented phase 3 registration of traditional medicines in January 1992 with special emphasis on the quality, efficacy, and safety of all dosage forms of these medicines. For this reason, a total of 100 herbal products containing Smilax myosotiflora were purchased in the Malaysian market and analyzed for mercury content, as mercury is a recognized reproductive toxicant. The products were analyzed using cold vapor atomic absorption spectrophotometry. It was found that 89% of the above products do not exceed 0.5 ppm of mercury. Heavy metal poisoning such as mercury has been associated with traditional medicines. Therefore, it is important that doctors and health care practitioners are aware of these risks and finding ways to minimize them, including questions pertaining to the use of these remedies during the routine taking of a patient’s history.
Keywords
There has been a resurgence of interest in traditional and alternative systems of medicine worldwide (Eisenberg et al. 1998; Ernst 2000a; Ernst and White 2000; MacLennan, Wilson, and Taylor 1996; Mohamad Taha 2002; Saras 2003; Zaidi 2002). The growing use of herbal medicines to treat a wide range of illnesses has been enhanced by the recognition that some diseases do not have any appropriate treatments, and by the belief that herbal remedies are innocuous. There is also the belief that what is natural can only be good and that herbal medicines are superior to synthetic drugs (Shultz, Hansel, and Tyler 1998; Tyler 1999). However, the risks contributing to the toxicity of herbal remedies, are among others, presence of contaminants such as microorganisms, heavy metals, microbial toxins, pesticides, fumigation agents, radioactivity, and synthetic and animal drug substances (Capasso et al. 2000).
Therefore, in Malaysia, the phase 3 registration for traditional medicines, which was implemented on 1 January 1992 under the Control of Drugs and Cosmetics Regulation 1984, emphasizes the quality, efficacy, and safety (including the detection of the presence of heavy metals) in all pharmaceutical dosage forms of traditional medicine preparations (Guidelines for application for registration of pharmaceutical products containing scheduled poisons and non-scheduled poisons 1993; Guidelines on Good Manufacturing Practice for Traditional Medicines 1999; Mohamad Taha 2002, Ramli 2001). Since then and until December 2003, DCA (Drug Control Authority) Malaysia managed to receive a total of 24,519 applications for registration, out of which only 49.4% have been approved whereas 37.3% have been rejected (Annual Report 2003).
In Malaysia, one of the herbal remedies is Smilax myosotiflora and commonly known as follow.
In Malaysia, S. myosotiflora is a slender climber, with the thin stem often clinging to adjacent objects for support, and has a great reputation as an aphrodisiac among the Malays and the jungle tribes alike. It is the rhizome that is eaten as an aphrodisiac, whereas the leaves and fruits are used internally for syphilis (Burkhill 1966; Compendium of Medicinal Plants Used in Malaysia 2002). Aphrodisiac is defined as any substance that arouses the sexual instinct, induces veneral desire, and/or increases sexual pleasure and performance (Choudhary and Rahman 1997).
A large number of studies in mercury determination have been published. The four most common and reliable techniques for mercury determination are spectrophotometry after chelation with dithizone, atomic absorption and emission spectrophotometry, neutron activation analysis, and gas chromatography. Of these, cold vapor atomic absorption spectrophotometer (CVAAS) has become the most popular technique because the mercury compounds found in the samples can be reduced to elemental mercury that occurs as vapor (Adeloju, Dhindsa, and Tandon 1994).
In the present study, we analyzed the mercury content using CVAAS in a total of 100 products (both registered and unregistered with the DCA Malaysia) in various pharmaceutical dosage forms of S. myosotiflora preparations, which were bought in the Malaysian market, including both night (products were sold to the public at night on a specific weekday) and weekend (products were sold to the public only on Saturdays and Sundays) markets, after a random sampling to reduce bias (Schefler 1984) and allow each sample an equal chance of being selected for analysis (Newman 1995).
MATERIALS AND METHODS
Treatment of Glasswares
All glassware was soaked with aqua regia (HCl:HNO3 = 3:2) for 2 h and then washed with deionized water (Deionizer Elga B113) prior to use.
Reagents
All reagents were analytical grade. Hydrochloride acid 37% (specific gradient 1.33; Merck, Darmstadt, Germany); nitric acid 65% (specific gradient 1.40; Merck); stannous chloride dihydrate (product no. 10270; BDH, Poole, England), and mercury stock solution, 1000 ppm (product no. 141454 K; BDH, Poole, England) were used in this study.
Wet Digestion of Samples
A total of 100 samples (see Tables 1 and 2) were digested using freshly prepared aqua regia wet digestion. Approximately 1.5 g of the sample was weighed and placed in a 100 ml Quickfit round bottom flask before 25.0 ml of freshly prepared aqua regia were added. The mixture was refluxed in a water bath for 6 to 8 h (or until dissolved), and allowed to cool before filtering. The residue was washed several times with deionized water. The combined aqueous extract was then made up to 50.0 ml with deionized water.
This extraction procedure was repeated for the same sample (replicate) and the blank (containing aqua regia only). Coarse particles, such as tablets, pills or powders, and capsules, were grounded to fine powder or particles prior to wet digestion.
CVAAS
One-milliliter extract, blank or standard solution, was added to 70 ml deionized water in a 150-ml Quickfit conical flask. The aqueous solution was added to 2 ml of 10% stannous chloride and then was aspirated by gas stream into the flameless pathway of the monochromatic light of GBC 906 AA model atomic absorption spectrophotometer (complete with inbuilt Window-based 906 program software). The absorbance was recorded when stable and the data were analyzed. The operating procedures used were slit width: 0.5 nm, current: 3.0 mA, and wavelength: 253.7 nm.
Preparation of Stannous Chloride
Ten grams stannous chloride dihydrate (SnCl2·2H2O) was dissolved in 20 ml of hot concentrated HCl and diluted to 100 ml with deionized water. The resulting solution was heated with a metallic tin until the precipitate disappeared. The solution was stored in a glass bottle containing tin powder.
Mercury Standard Solution
Mercury stock solution (1000 ppm) was diluted serially, using deionized water, to produce a standard solution of 0.1 μg/ml This was followed by adding 70 ml of deionized water into each of the seven 150 ml Quickfit conical flasks each. They were then added to 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml of 0.1 μg/ml mercury stock solution to produce 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.30 μg of mercury, respectively. Two milliliters of 10% stannous chloride were added to each flask and were aspirated by a gas stream into the flameless pathway of the monochromatic light. The absorbance was recorded when stable and the data were analyzed, thus producing a mercury standard curve.
RESULTS
Mercury Determination
The quality requirement for traditional medicines in Malaysia is not exceeding 0.5 ppm for mercury (http://www.bpfk.gov.my/default.asp; Jaafar 1995; Regulating supplements 2003). Results showed that 11 out of 100 of these products (11%) possessed 0.63 to 1.72 ppm of mercury and, therefore, do not comply with the quality requirement for traditional medicines in Malaysia (Tables 1 and 2).
Out of these 11 products, 6 Malaysian products were registered with the DCA Malaysia (Table 1). These include Radix Ubi Jaga (possessed 1.24 ± 0.37 ppm of mercury), M-Ubi Jaga (possessed 0.63 ± 0.21 ppm of mercury), Gold Box Ubi Jaga Plus (possessed 1.02 ± 0.56 ppm of mercury), Maajun Pak Tani (possessed 1.52 ± 0.21 ppm of mercury), Maajun Petani (possessed 1.26 ± 0.21 ppm of mercury), and Maajun Ubi Jaga (possessed 1.72 ± 0.34 ppm of mercury).
However, the rest of the products were manufactured in Indonesia but still have not registered with the DCA Malaysia (Table 2). These include Jiwa Super Pill Ubi Jaga Plus (possessed 0.63 ± 0.12 ppm of mercury), Jiwa Great Pill Ubi Jaga Plus (possessed 0.63 ± 0.21 ppm of mercury), Jiwa Tender Pill Ubi Jaga Plus (possessed 0.61 ± 0.20 ppm of mercury), Jiwa Extra Pill Ubi Jaga Plus (possessed 0.61 ± 0.21 ppm of mercury), and Jiwa Force Pill Ubi Jaga Plus (possessed 0.62 ± 0.12 ppm of mercury).
This study showed that 89% of the products complied with the quality requirement for traditional medicines in Malaysia pertaining to mercury content.
DISCUSSION
The use of aqua regia in the acid digestion could ensure total extraction of metals from both inorganic and organic samples and virtually eliminates loss of mercury compared to conventional wet digestion (Lee and Laufmann 1971). Stannous chloride was used as a reducing agent for the reduction of mercury (I or II) to atomic mercury (0). CVAAS is the predominant technique for mercury analysis due to its high selectivity and sensitivity (Adeloju. Dhindsa, and Tandon 1994; Beaty and Kerber 1993; Bruhn et al. 1994; EPA Test Methods for Evaluating Solid Waste 1986; Landi and Fagioli 1994). However, use of this method is limited to mercury because other metals are solid at normal room temperature (Beaty and Kerber 1993). Hence, the elemental mercury, which is liquid at normal room temperature, has a high vapor pressure and is readily converted into the vapor phase.
The sensitivity of the CV technique is far greater than can be achieved by the conventional AAS. This is mainly because of 100% sampling efficiency, because all the mercury in the sample solution placed in the reaction flask is chemically atomized and transported to the sample cell for measurement (Beaty and Kerber 1993).
Although this study showed that 89% of the tested products complied with the quality requirement for traditional medicines in Malaysia pertaining to mercury, these products cannot be assumed safe from mercury contamination because of batch-to-batch inconsistency (Ko 1998). This could be attributed to the various sources of raw materials, including the plant materials and also the different manufacturing process to obtain the various forms of products.
As such, the DCA Malaysia has specified that the validity of pharmaceutical products (nonpoisons) to be limited to a maximum of 5 years. Applicants will have to submit an application for re-registration for the reassessment of the quality, efficacy, and safety of their products. In addition, the DCA may reject, cancel or suspend the registration of any product if deemed necessary (Newsletter of the Drug Control Authority Malaysia 2001).
The traditional belief that the traditional medicines, particularly the natural herbal remedies, are of natural origin and, hence, inherently safe (Capasso et al. 2000; Ernst 1999; Friedman 1996) and without any adverse effects is sometimes unfounded (Ramli 2001).
There have been numerous reports of adverse effects associated with mercury poisoning through traditional Chinese (Chi et al. 1993; Ernst 2000b; Espinoza, Mann, and Bleasdell 1995; Kang-Yum and Oransky 1992; Li et al. 2000), Indian (Kew, Morris, and Aihic 1993; Sheerin et al. 1994), and Malaysian (Ahsan 2000) medicines.
Several possibilities could be responsible for the presence of mercury in these medicines. Among them were that these medicinal plants may have grown in seriously polluted soil (Schilcher 1983) or these products may also contain animal or mineral products that may also contaminated with heavy metal (Chuang et al. 2000).
Mercury has a spectrum of toxic effects, including being a reproductive toxicant. It has adverse effects on the male reproductive system and this is important to humans because this plant is traditionally eaten for its aphrodisiac property (Burkhill 1966; Compendium of Medicinal Plants Used in Malaysia 2002). Among the adverse effects caused by the presence of mercury are the induction of testicular deformation in seminiferous tubules and Leydig cells (Athar and Vohora 1995) and sperm abnormality in human (Ernst and Lauritsen 1991). Higher blood mercury concentration is associated with male infertility (Choy et al. 2002). Further studies showed that membranes of acrosomal cap, the midpiece, and the tail of human sperm are potential binding sites for mercury (Ernst, Christensen, and Lauritsen 1991). Subsequently, disruptions of sperm membrane permeability, mitochondrial function, DNA synthesis by the microtubules, and motion generation by the microtubule sliding assembly are possible mechanisms of mercury toxicity (Ernst, Christensen, and Lauritsen 1991; Liu et al. 1995; Vogel, Margolis, and Mottet 1985). Other than the sperm themselves, supporting cells in the testis (Ernst, Moller-Madsen, and Danscher 1991), epididymis (Working, Bus, and Hamm 1985), and the seminal vesicles (Li, Wu. and Yang 1995) are also possible targets of mercury toxicity. This will finally result in semen abnormality and clinical infertility.
Therefore, it is important that doctors and health care practitioners are aware of the potential risks associated with these products and, therefore, finding ways to minimize them (Ernst 2000c), including questions pertaining to the use of these remedies during the routine taking of a patient’s history (Ernst 1999). Such information will go beyond the possibility of heavy metal contaminations and include the possibility of detecting herbal toxicities and herbal-drug interactions (De Smet 1995; De Smet and D’Arcy 1996; Ernst 1999; Ernst et al. 2001).
Footnotes
Tables
This work was supported by the research grant of Universiti Sains Malaysia (304/PFARMASI/633118).