Distinction between Stephania tetrandra and Aristolochia fangchi by Hyperspectral Imaging and UHPLC-MS

Reviewed: Tankeu S, Vermaak I, Chen W, Sandasi M, Viljoen A. Differentiation between two ‘‘fang ji” herbal medicines, Stephania tetrandra and the nephrotoxic Aristolochia fangchi, using hyperspectral imaging. Phytochemistry. November 26, 2015; [epub ahead of print]. doi: 10.1016/j.phytochem.2015.11.008.

The use of botanical ingredients containing aristolochic acids (AAs), which are nitrophenanthrene carboxylic acids with carcinogenic, mutagenic, and nephrotoxic properties, is at the origin of a number of endemic manifestations of kidney disease (aristolochic acid nephropathy [AAN]), notably, reports from Belgium in the 1990s with 128 cases of AAN due to the ingestion of an herbal weight loss product. The product, which was supposed to contain Stephania tetrandra (Menispermaceae), actually contained the AA-containing Aristolochia fangchi (Aristolochiaceae). It is not clear if the substitution was accidental, due to the similarity of the Chinese common names, han fang ji (S. tetrandra) and guang fang ji (A. fangchi), or if it was the unintentional consequence of the perceived legitimate interchangeability of the species. The two species have historically been considered allied drugs, share the pharmacopeial name Fangji Radix,¹ and are frequently sold interchangeably in trade, hence the possibility of substitution.² Once the causal relationship between AA ingestion and the development of AAN and increased occurrence of upper tract urothelial carcinomas was established, a large number of cases of kidney disease worldwide, including kidney transplants and fatalities, could be attributed to the consumption of botanical ingredients containing AAs, or – in the case of the Balkan-endemic nephropathy – to bread made from wheat (Triticum spp., Poaceae) flour contaminated with seeds of Aristolochia clematitis.³

Methods to detect substitution of S. tetrandra with A. fangchi are available, and include macroscopic analysis, botanical microscopy, high-performance thin-layer chromatography (HPTLC), and high-performance liquid chromatography with ultraviolet/visible detection (HPLC-UV/Vis).^2,4,5 Hyperspectral imaging (HSI) is proposed as an alternative method due to the ease in sample preparation, the short analysis time, and the affordability of the instrumentation. The aim of this study was to evaluate the performance of HSI as an accurate method to discriminate between the two botanical ingredients. One authenticated sample of each species was ground up, passed through a sieve, and six aliquots (100 mg each) were subjected to HSI analysis. Discrimination was achieved by principal component analysis (PCA) of the spectral data. Looking at the trends in the data at various wavelengths allowed explaining the chemical variation between the two species. Furthermore, the authors assessed the ability of this model to predict the level of adulteration by preparing samples of S. tetrandra that were adulterated with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% of A. fangchi. The method gave good results for predicting the amount of adulterant, in particular with adulteration levels above 50%, where the accuracy was within 6% of the actual result. A result of 6% for the lowest amount, the substitution with 10% A. fangchi, was determined by HSI. Ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) was used to validate the results of the HSI analysis.

Comment: The toxicity issues related to consumption of AAs have remained a concern, in particular in Asian countries where many AA-containing herbs are part of the traditional medicine system and are still used, although less frequently since some Asian countries – e.g., China, Taiwan, and Japan – have implemented a ban on the use of most herbs that contain AAs. Methods that allow the detection of AA-containing herbs are helpful, in particular if these methods are easy to use, affordable, and have a low limit of detection. HSI fulfills most of these requirements, and the approach has been shown to be accurate in the detection of substitution of S. tetrandra with A. fangchi. However, since the authors did not determine the limit of detection (LOD) of the method, it is unclear if this technique is sufficient for finding AAs in materials that may contain small admixtures of AA-containing plants. In contrast, some of the other available methods can detect even small amounts of adulteration of A. fangchi in S. tetrandra: microscopy (1% admixture), HPTLC (1% admixture), and HPLC (LOD: 0.5 mg/g for AAs).² Because most botanical material is comprised of multiple batches that are mixed into one larger batch, it is important that even small amounts of adulteration be detectable. In addition, the HPTLC and HPLC methods allow the detection of any AA-containing plant materials, not only A. fangchi.

A larger sample set (to account for the variability in the materials), and an extension to other frequently confused species, e.g., the replacement of mu tong (Akebia quinata, Lardizabalaceae) with guan mu tong (Aristolochia manshuriensis), would be helpful additions to the presented HSI data. Hopefully, better education about the toxicity of AA-containing plants may eventually eliminate the occurrence of AAN.

The American Botanical Council, the American Herbal Pharmacopoeia (AHP), and the National Center for Natural Products Research (NCNPR) at the University of Mississippi, the three partners in the Botanical Adulterants Program, have published on this subject.^2,4,6 The article in HerbalGram was published in 2000.⁶ A complete characterization of AA-containing plants and those with which they can be mixed was published by AHP in 2006 in collaboration with USFDA, State Food and Drug Administration (SFDA, China), Therapeutic Goods Administration (TGA, Australia), NCNPR (University of Mississippi), and the Royal Gardens at Kew (UK).²

References

1.     Yen K-Y. The Illustrated Chinese Materia Medica: Crude and Prepared. Taipei, Taiwan: SMC Publishing, Inc.; 1992.

2.     Upton R, ed. Aristolochic Acid Evaluation Monograph: Characterization of Selected Plants that Contain or May Be Adulterated with Aristolochic Acid. Scotts Valley, CA: American Herbal Pharmacopoeia; 2006.

3.     Debelle FD, Vanherweghem J-L, Nortier JL. Aristolochic acid nephropathy: a worldwide problem. Kidney Int. 2008;74(2):158-169.

4.     Joshi VC, Avula B, Khan IA. Authentication of Stephania tetrandra S. Moore (fang ji) and differentiation of its common adulterants using microscopy and HPLC analysis. J Nat Med. 2008;62(1):117-121.

5.     Chan W, Hui KM, Poon WT, Lee KC, Cai Z. Differentiation of herbs linked to “Chinese herb nephropathy” from the liquid chromatographic determination of aristolochic acids. Anal Chim Acta. 2006;576(1):112-116.

6.     Chen JK. Nephropathy associated with the use of Aristolochia. HerbalGram. 2000;(48):44-45.