Quality assessment of South African herbal medicines by means of HPLC fingerprinting

Journal of Ethnopharmacology 101 (2005) 75–83

Quality assessment of South African herbal medicines by means of HPLC fingerprinting E.P. Springfield a,∗ , P.K.F. Eagles a , G. Scott b a

South African Traditional Medicines Research Group, School of Pharmacy, University of the Western Cape, Bellville, South Africa b Department of Botany, University of Cape Town, Rondebosch, Western Cape, South Africa Received 12 November 2004; received in revised form 1 December 2004; accepted 26 March 2005 Available online 24 May 2005

Abstract An estimated 70% of South Africans regularly use traditional plant medicines. Incorporation of these medicines within the formal health care system, which is the stated intention of the Health Ministry, requires the establishment of standards for quality control. Except in the case of a handful of South African plant species, such standards are lacking. Of central importance with respect to quality control is correct identification of the species concerned, whether in the fresh, dried or powdered state. In cases where botanical identification is impossible, high performance liquid chromatography (HPLC) with diode array detection (DAD), offers an alternative qualitative profile and is being increasingly used for the authentication of crude drugs or their extracts. As a contribution to establishing quality standards for South African plant species used as traditional medicines, HPLC-DAD “fingerprints” of 60 commonly-used species have been generated in our laboratory. One of these species is presented here, together with UV spectra of individual components represented by major peaks in the HPLC profiles. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: HPLC-DAD; Medicinal plants; Chironia baccifera; Quality control; South Africa

1. Introduction In man’s quest for food during the early nomadic period of his existence he would most certainly have encountered some plants that were poisonous, others that would serve as adequate foods, and still others that produced bizarre and unusual effects by altering his consciousness. Among this latter group were those that would simultaneously relieve pain and counteract disease (Emboden, 1997). These experiences, passed on from generation to generation, are today recognized for their vital role in global health care and the application of scientific method to ancient philosophical systems has led to Natural Medicine and Ethnopharmacology ∗ Corresponding author at: Programme on Mycotoxins and Experimental Carcinogenesis (PROMEC), Medical Research Council, PO Box 19070, Tygerberg 7505, South Africa. Tel.: +27 21 938 0376; fax: +27 21 938 0260. E-mail address: [email protected] (E.P. Springfield).

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.03.012

becoming important areas of enquiry; these considerations may contribute to the ongoing discussions between naturaland modern medicine (Vogel, 1991). Despite the use of herbal medicines over many centuries, only a relatively small number of plant species has been scientifically validated in South Africa. Safety and efficacy data are available for an even smaller number of plants, their extracts and active ingredients and preparations containing them. The quantity and quality of safety and efficacy data on traditional medicines are far from sufficient to meet the criteria needed to support their use worldwide. The reasons for the lack of such data include not only national health care policies, but also a lack of adequate or accepted research methodology for evaluating traditional medicines (WHO, 1978). The World Health Assembly over the last decade, has adopted a number of resolutions drawing attention to the fact that 80% of the population in many developing countries relies on traditional plant medicines for its health care needs,

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and that the work force represented by traditional medical practitioners is a potentially important resource for primary health care. The World Health Organization (WHO), in pursuance of its goal of providing affordable, accessible and culturally acceptable health care to the global population, has encouraged the rational use of traditional, plant based medicines by member states of the World Health Assembly (WHO, 1998) and, to this purpose, developed technical guidelines for the assessment of herbal medicines (WHO, 2000). The trade in and use of crude indigenous herbal products is at present largely unregulated in South Africa. In many countries education, training and research in ethnomedicine have not been accorded due attention and support. In order to meet an urgent need for data relating to the quality, safety and efficacy of South African herbal medicines, pharmaceutical monographs for 60 indigenous plant species used in traditional practice were recently drawn up in accordance with WHO guidelines. This work, carried out by the South African Traditional Medicines Research Group (SATMERG, 1999), in the laboratories of the School of Pharmacy, University of the Western Cape (UWC), focused principally on quality control of South African plant medicines. Except in the case of a handful of plant species, quality standards do not exist for South African herbal medicines. Of central importance with respect to quality control is correct identification of the species concerned, whether in the fresh, dried or powdered state. Consequently, careful attention was given to the establishment of identity standards and quality control profiles, using a combination of taxonomy, classical microscopy, thin layer chromatography (TLC) and HPLC-DAD. HPLC-DAD is being increasingly utilized for the screening of drugs, vitamins and natural products and has also been applied to the identification of poisoning by traditional medicines (Foukaridis et al., 1994). In HPLC, qualitative solute identification can be achieved via comparison of retention data. However, the results can be considered unequivocal only if additional independent physical/chemical methods are applied. For this reason the coupling of HPLC to DAD, yielding on-line UV spectra, is of paramount interest (Engelhardt and Konig, 1989). Some of the advantages of DAD application are highlighted in the work of Fell et al. (1984), Hill et al. (1987) and Hayashida et al. (1990). When reporting the results of pharmacological research on herbal medicines (Amabeoku et al., 2001; Springfield, 2002; Bienvenu et al., 2002), often based on whole plant or partially fractionated extracts, an HPLC-UV profile of the active extract/s is useful, where the identity of individual active(s) is not known or not finally clarified (Bauer and Tittel, 1996). In this paper, Chironia baccifera L. (Gentianaceae) serves as an example of the approach to assess quality control, adopted in compiling South African medicinal plant monographs. The overground parts of Chironia baccifera are used traditionally to treat haemorrhoids, sores, acne, eczema and boils. Microchemical tests in our laboratories indicated pos-

itive for the presence of tannins and saponins but negative for alkaloids and cardiac-, cyanogenic- and anthraquinone glycosides. Although some active compounds have been isolated from Chironia species (Wolfender et al., 1993), individual compound identification is not the aim of the present study. HPLC-UV-DAD results are presented to show that this method can aid in the assessment of quality control of raw herbal material, especially in laboratories of developing countries.

2. Experimental 2.1. Materials Chironia baccifera is a shrub with widespread distribution in the Western- and Eastern Cape Provinces of South Africa. To allow for the effects of infraspecific variation on secondary chemistry, three collections of this species were collected from different geographical locations within its range, as follows: Cedarberg Mountains, Tygerberg Nature Reserve- and Kirstenbosch Botanic Garden, South Africa. Authentication was done by one of the co-authors and voucher specimens (Trd 14 for Cedarberg Mountain collection; Trd 36 for Kirstenbosch Botanic Garden collection and Trd 118 for Tygerberg Nature Reserve collection) were prepared and deposited in the UWC herbarium. All material was rinsed in distilled water to remove dust or soil and dried at 40 ◦ C in a ventilated oven and stored in dried, amber glass containers. The dried material was milled to shorten the extraction period (Eloff, 1998) and passed through a sieve of mesh size 850 ␮m. 2.2. Preparation of extracts 2.2.1. Dichloromethane (DCM) extraction The milled plant powder (1 g) was shaken (Labcon shaker) for 20 min with 10 ml DCM. The extract was filtered through glass wool and evaporated to dryness, on a Buchi Rotavapor (Labortechnik, Switzerland) at a temperature not exceeding 40 ◦ C. The dried residue was re-dissolved to 5 ml with 100% MeOH. 2.2.2. Methanol (MeOH) extraction The plant residue remaining after DCM extraction was shaken for 20 min with 10 ml 100% MeOH and the resultant extract treated as for Section 2.2.1. 2.3. Instrument and chromatographic procedure The MeOH and DCM extracts were filtered through a 0.45 ␮m syringe prior to analysis. Spectra were generated on a Beckman System Gold HPLC, consisting of a double pump Programmable Solvent Module 126, Diode Array Detector Module model 168, with 32 Karat Gold software supplied by Beckman; Column C18 Bondapak 5 ␮m and dimensions

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250 mm × 4.6 mm (Luna-Separations). Ambient temperature was maintained at 23 ◦ C. 2.3.1. Methanol extract Mobile phase, solvent A: MeOH; solvent B: 5% glacial acetic acid (CH3 COOH); Mode: gradient, increasing the organic phase MeOH from 20 to 90% over 18 min; flow rate: 1 ml/min; reference standard: rutin (2.5 g dissolved in 100 ml MeOH); injected volume: 10 ␮l. The run time was 25 min. Detection wavelength of 270 nm on Channel A and 360 nm on Channel B. The wavelength scanning range was 200–600 nm at 2 nm/step. 2.3.2. Dichloromethane extracts Mobile phase, solvent A: acetonitrile (CH3 CN); solvent B: HPLC grade water (H2 O); Mode: gradient, increasing the organic phase (CH3 CN) from 60 to 90% over 12 min, flow rate: 1.5 ml/min; reference standard: thymol (2.5 g dissolved in 100 ml MeOH), injected volume: 10 ␮l. Detection wavelength of 254 nm on Channel A and 270 nm on Channel B. The wavelength scanning range was 200–600 nm at 2 nm/step. The room temperature was controlled by an LG air-conditioning system at 23 ◦ C. 2.4. Selection of spectra The DCM and MeOH spectra generated following analysis of the three separate collections were compared and major common peaks identified using super-imposable features of the software. In order to establish whether the peaks selected represented true identity and purity (Bauer and Tittel, 1996) in all three spectra, their UV spectra were compared by making use of overlay features of the diode array detector model 168 (Fig. 1). A correlation coefficient >0.95 indicated co-identity. In cases where both MeOH and DCM extracts

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yielded characteristic fingerprint spectra, both were included in the monograph.

3. Discussion As can be seen from Fig. 2, the MeOH spectra (270 nm) representing three different collections of Chironia baccifera are qualitatively sufficiently similar to represent a chemical fingerprint for this species. Although quantitative differences are evident, major peaks (1–4) are common to all three spectra. As mentioned above, qualitative identification can be made using retention data. In the present case, comparison of the retention data of the selected peaks indicated co-identity, as evidenced by a very low standard deviation (Table 1). The co-identity of these peaks was validated by means of their UV spectra (Fig. 3), which indicated the purity of each selected peak in the chromatogram, relative to its retention time and Λmax . Fig. 4 represents chemical fingerprints of the dichloromethane extracts of three collections of Chironia baccifera at 254 nm. Qualitatively these chromatograms are identical with respect to peaks 1–4, although a slight quantitative difference is present between the selected compounds. The same procedure for peak identification as used for the MeOH extracts was followed in the case of DCM extracts. The retention data of the selected peaks in the DCM extracts also showed a very low standard deviation (Table 2). The co-identity of these peaks was validated by means of their UV spectra (Fig. 5) relative to retention time and Λmax . As in the case of the MeOH spectra, one of these spectra can therefore be considered to be representative of the species concerned. It is important however that decisions as to the representivity of a particular spectrum take into account the effects of extrinsic (environmental) and intrinsic (genetic) factors on plant secondary chemistry (Harborne, 1998). The three collections analysed in the present study displayed remarkable qualitative similarity as regards their Table 1 Retention times (min) of the representative compounds in the MeOH extracts of Chironia baccifera Peak

Trd 14

Trd 36

Trd 118

Standard deviation

1 2 3 4

12.12 12.72 21.00 22.07

12.21 12.78 21.02 22.15

12.23 12.76 21.05 22.03

0.06 0.03 0.03 0.06

Table 2 Retention times (min) of the representative compounds in the DCM extracts of Chironia baccifera

Fig. 1. Overlay of UV spectra of one peak, with same retention time, from the three different collections.

Peak

Trd 14

Trd 36

Trd 118

Standard deviation

1 2 3 4 5

7.74 8.40 8.96 9.45 9.72

7.65 8.31 8.88 9.37 9.63

7.62 8.44 8.87 9.36 9.62

0.06 0.07 0.05 0.05 0.06

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Fig. 2. Chromatograms of MeOH extracts of three collections of Chironia baccifera.

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Fig. 3. UV spectra of the representative compounds in the MeOH extracts of Chironia baccifera.

HPLC profiles, despite the fact that the plant material had been gathered in its natural habitat at different seasons, from different geographical locations and in different soil types. Indeed, qualitative similarity in HPLC profiles was noted for almost all of the 60 plant species examined in our laboratories, permitting the inclusion of a fingerprint spectrum in each monograph. Harborne (personal communication) noted that infraspecific variation in plant secondary chemistry was in his experience mainly quantitative, which is relevant to the potency of medicinal herbs but less so to their correct identification. The peak for the reference standard, rutin (Fig. 6), appeared at retention time (min) of 19.66, and is used to monitor the MeOH extracts. The peak for the reference standard, thymol (Fig. 7), appeared at retention time (min) of 6.29, and is used to monitor the DCM extracts. These references standards are run during a sequence of analyses as an external standard. Because their retention times are known, a correction factor can be applied to the unknown plant chromatogram should any significant shift occur in the retention time of the reference standard.

The isolation from their respective parent species of such individual “actives” as digoxin, morphine and quinine has led to a pharmacological preference for using single chemical entities rather than whole plant extracts. This is based on the supposition that any plant possessing clinical effectiveness must contain one (or at most a few) active principle(s), which can therefore replace a total extract (Phillipson, 1995). Advantages of this approach include accurate dosage and avoidance of natural variation in bioactivity. Sufficient examples now exist however of bioactivity being the product of synergism between all compounds present in a particular plant extract. In yet other cases, individual “actives” have proved to be either too toxic for human use or of limited solubility. In the case of traditional medicines, isolation and identification of individual “actives” may be beyond the capability or budget of many developing countries. Health ministries may also simply prefer a traditional approach, on the grounds that many prescription drugs can cause nutrition depletion and may give rise, inter alia, to osteoporosis, heart and blood pressure problems, tooth decay or birth defects (Von Geusau, 2001) as well as other adverse effects.

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Fig. 4. Chromatograms of DCM extracts of three collections of Chironia baccifera.

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Fig. 5. UV spectra of the representative compounds (peaks 1–5) in the DCM extracts of Chironia baccifera.

The results of the present study demonstrate that it is possible, using HPLC-DAD to generate a species – specific spectrum confirming the “chemical fingerprint” of a medicinal plant species, provided that a standard species – specific spectrum already exists, and can be validated by hyphenated chromatographic and spectrometric techniques. For further exploration of the secondary chemistry of an unknown plant

species, it is evident that HPLC-DAD can provide some information about the chemical classes present in a herbal plant extract (Bauer and Tittel, 1996), and may be used optimally together with mass spectroscopy (Gong et al., 2003). In the present study, the different fingerprint spectra obtained for MeOH and DCM extracts demonstrated the effect of extracting solvent on the fingerprint spectrum (Bauer and

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Fig. 6. Chromatogram of reference standard, rutin, for the MeOH extracts with UV spectra. λmax : 256–345–359 nm.

Fig. 7. Chromatogram of reference standard, thymol, for the DCM extracts with UV spectra. λmax : 276 nm.

Tittel, 1996) and underscored the need for strict adherence to a standard method of extraction in order to provide reproducible spectra. In the case of herbal medicines whose active constituents are unknown (the situation in most developing countries), the use of reference compounds (e.g. rutin or other flavonoids for MeOH spectra; thymol or cineole for DCM spectra) exemplifies the principles of one approach for rapid-scanning detection using HPLC.

4. Conclusion In developing countries, including South Africa and most African Union (AU) states, traditional medicines are an important but often unacknowledged component of the health care system. Pharmaceutical chemists in these countries are well equipped to bridge the gap between traditional and Western allopathic medical systems. The application of standard pharmaceutical methods to the quality assurance, safety as-

sessment and efficacy testing of traditional medicines constitutes the first step in the process of bringing them from the field into the clinic, dispensary and hospital. In developing countries seeking to promote the rational use of herbal medicines, correct species identification is of paramount importance to quality assurance, as very few traditional herbs are cultivated and almost all raw material is obtained from natural stands of vegetation. Consequently mis-identification or adulteration can easily occur. A combination of HPLC “fingerprinting” and online UV spectrum detection via a diode array configuration add value to conventional botanical methods used in the quality control of herbal medicines. HPLC-DAD, a sensitive, rapid, and economical technique, can be used in establishing a code of practice for the quality control of herbal extracts. This methodology, which serves as a chemical marker together with a combination of taxonomy, classical microscopy and TLC is applied to the preparation of pharmaceutical monographs for plant species used as tra-

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ditional medicines (Scott et al., 2004), and ultimately to the compilation of a national herbal pharmacopoeia for South Africa. The implementation of quality control processes for herbal medicines is continually faced with new challenges and therefore subject to ongoing modification and adjustment (Bauer and Tittel, 1996; Anderson and Burney, 1998; Bast et al., 2002; Choi et al., 2002; Ang et al., 2002; Koll, 2003; Sherma, 2003; Gong et al., 2003, 2004; Le Gall et al., 2004).

Acknowledgements The authors are grateful to the South African Medical Research Council for financial support for this project and Mr. Dick Loubser for his valuable technical assistance.

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