Natural Product Research Formerly Natural Product Letters ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20 Authentication of Rhodiola rosea, Rhodiola quadrifida and Rhodiola rosea liquid extract from the Ukrainian market using HPTLC chromatographic profiles Kateryna Khokhlova & Oleksandr Zdoryk To cite this article: Kateryna Khokhlova & Oleksandr Zdoryk (2019): Authentication of Rhodiola�rosea, Rhodiola�quadrifida and Rhodiola�rosea liquid extract from the Ukrainian market using HPTLC chromatographic profiles, Natural Product Research, DOI: 10.1080/14786419.2019.1591398 To link to this article: https://doi.org/10.1080/14786419.2019.1591398 View supplementary material Published online: 28 Mar 2019. Submit your article to this journal Article views: 3 View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2019.1591398 SHORT COMMUNICATION Authentication of Rhodiola rosea, Rhodiola quadrifida and Rhodiola rosea liquid extract from the Ukrainian market using HPTLC chromatographic profiles Kateryna Khokhlovaa and Oleksandr Zdorykb a Department of Technology of Drugs, National University of Pharmacy, Kharkiv, Ukraine; Department of Quality, Standardization and Certification of Drugs, National University of Pharmacy, Kharkiv, Ukraine b ABSTRACT ARTICLE HISTORY Rhodiola rosea and Rhodiola quadrifida are widely distributed and sold in Eastern Europe. The purpose of this paper was to identify R.rosea, R.quadrifida and Rhodiola rosea liquid extract (RRLE) in the Ukrainian market and bring out adulteration cases using chromatographic characterisation by HPTLC. The multiple samples of R.rosea, R.quadrifida and RRLE were compared; the optimal chromatographic conditions for identification of R.rosea and RRLE based on the presence of rosavins and salidroside as well as for identification of R.quadrifida based on the presence of salidroside were proposed; the specific HPTLC fingerprints were obtained; the acceptance criteria for each product were set. The adulteration cases for R.rosea and RRLE samples were established. The dependence on handling R.rosea and presence of rosavins was determined. It was assumed that low-quality raw materials or inefficient technology process were used for RRLE. The consistency of HPTLC fingerprints for R.quadrifida samples was established. Received 19 December 2018 Accepted 2 March 2019 KEYWORDS Rhodiola rosea; Rhodiola quadrifida; golden root; rosavins; salidroside; authentication; highperformance thin-layer chromatography 1. Introduction The genus Rhodiola L. (Crassulaceae) is widely spread in high altitudes of Asia, Europe and North America. Traditionally, it is used in adaptogens, antidepressants, and anti-inflammatory remedies (Brown et al. 2002; Zhou et al. 2014; Chiang et al. 2015). CONTACT Kateryna Khokhlova [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1591398 ß 2019 Informa UK Limited, trading as Taylor & Francis Group 2 K. KHOKHLOVA AND O. ZDORYK There are mainly three products from Rhodiola species are present in the Ukrainian market. They are Sedum roseum (L.) Scop. (Syn. Rhodiola rosea L.) and Sedum quadrifidum Pall. (Syn. Rhodiola quadrifida (Pall.) Fisch. & C.A.Mey.) crude drugs, and Rhodiola rosea liquid extract (RRLE). Vernacular names for R. rosea and R.quadrifida in Russian and Ukrainian as well as their traditional usage and distribution are given in Table S1. RRLE is registered and sold as a herbal drug over-the-counter medicines in Ukraine and Russia (1:1, ethanol 40%). However, it has recently been reported (Kurkin 2015) that the RRLE technology is not optimal and its stability is under the question. Recently, a huge amount of adulterated R. rosea products has been discovered in the European market (Booker, Jalil, et al. 2016). The results showed that the amount of marker substances (rosavin or salidroside) were either insufficient or absent, and some of the analyzed products were adulterated with closely related Rhodiola species (Booker, Jalil, et al. 2016). However, the absence of rosavins may not always indicate adulteration. If not handled properly, rosavins may be subject to enzymatic degradation and, thus, be absent in a final product (Kurkin et al. 1985, Bejar et al. 2017). On the other hand, poor quality of R. rosea may be due to the growth region (Kurkin et al. 1985). In accordance with the paper (Wiedenfeld et al. 2007), a great variability in the composition and amount of pharmacologically active compounds was observed in plant materials of R. rosea and R. quadrifida acquired from different sources. A large number of Rhodiola species as well as morphological similarity of the herbal raw materials (HRM) for the major species, complicate proper authentication of the plant. Recent studies of Rhodiola species include the development of the quality control methods using such modern techniques as microscopy, high-performance thin layer chromatography (HPTLC), HPLC, DNA, MS and NMR, etc. (Li J et al. 2008; Li and Zhang 2008; Zhang et al. 2015; Booker, Zhai, et al. 2016). Among other pharmacopoeial tests, the HPTLC identification method is a powerful tool for authentication of herbal products and detection of adulteration (Reich and Schibli 2007; Toniolo et al. 2014; USP HMC 2015; Council of Europe 2016). The aim of this study was to compare and improve the existing TLC/HPTLC methods for identification of R. rosea, to develop the HPTLC identification method for RRLE and R. quadrifida, to prevent adulteration in the Ukrainian market. 2. Results and discussion The appropriate HPTLC chromatographic conditions for R.rosea, RRLE and R.quadrifida based on the existing TLC/HPTLC methods (Arzamastsev and Kosyireva 1990; USP HMC 2015; SRP 2016) for R.rosea were selected. Additional conditions were tested (Table S2). Different constituents of interest for R.rosea (such as phenylpropanoid glycosides (rosavin and rosarin), phenylethanoids (salidroside) and tyrosol) and R.quadrifida (such as salidroside, tyrosol and gallic acid) were tested as the reference standards. Other widely spread pharmacopoeial species (PPRC 2010) such as R. crenulata from China was compared (Figure S1). The optimal chromatographic conditions for identification of R.rosea and RRLE based on the presence of rosavins and salidroside as well as for identification of NATURAL PRODUCT RESEARCH 3 R.quadrifida based on the presence of salidroside in the mobile phase—ethyl acetate-methanol-water-formic acid (77:13:10:2) sourced from USP (USP HMC 2015) were proposed (Figure S2). For preparation of R.rosea and R.quadrifida samples, ethanol absolute was selected. The extraction technique was shaking for 10 min. For RRLE preparation, 40% ethanol was proposed. Derivatization with aniline-diphenylaminephosphoric acid was selected. Using the white light after derivatization, the proposed conditions for sample preparation allow obtaining the ‘clearest’ and the most consistent HPTLC fingerprints of the products (Figure S3). R.rosea and RRLE were detected under UV 254 nm (rosavin and rosarin presence) and white light after derivatization (characteristic fingerprints for five distinct grey zones of different intensity including the zones due to rosavins and salidroside). R. quadrifida was detected under white light (pinkish zone in the upper part of chromatogram) and UV 254 nm (quenching zone in the upper part of chromatogram) before derivatization as well as under white light after derivatization (zone due to salidroside and characteristic reddish zone in the upper part of chromatogram). The acceptance criteria that include the Table description and the examples of the typical chromatograms for each of the product were set (Figures S4–S6). The Table description shows the sequence of zones present in the chromatograms obtained with the standard solutions and the sample solution. This approach of interpretation of TLC/HPTLC results is used in European Pharmacopeia (Council of Europe 2016) for representation of the minimum quality criteria for acceptance of authenticity. The zones in the chromatogram are identified by letters which correspond to the descriptions in the table above (Figures S4–S6). Using the proposed HPTLC method, eleven samples of R. rosea and ten samples of R. quadrifida caudex with roots of different geographical origin were analyzed (Figures S6 and S7). The fingerprints of R. rosea samples (Figure S7, tracks 3–6, 8–10) were quite consistent with respect to the zone position, color and slight differences in intensity (black figured arrow). For the samples of R. rosea (Figure S7: tracks 2, 7, 11, 12), there were no characteristic chromatographic fingerprints (red frame). Also, no salidroside or rosavin selected as the specific marker for R. rosea was found. Three samples of R. rosea that failed the proposed HPTLC test were from Altai and one was from the Carpathians. Furthermore, all of the tested samples had the similar appearance, colour, the specific odour of rose (that could help easily differentiate R. rosea from other widely spread species such as R. crenulata) and taste. The samples were not damaged and all of them met the requirements of macroscopy and microscopy tests. The samples looked similar despite their sizes and shapes. Those eight samples that had similar chromatographic fingerprints and passed the proposed HPTLC test were comminuted roots and rhizomes (Figure S8). Among the four samples that failed the HPTLC test, three samples were the whole-piece (Figure S8B), and one was in the form of powder. Thus, despite the fact that chopped, whole-piece (Arzamastsev and Kosyireva 1990; SRP 2016) and powder HRM (SRP 2016) are allowed to be used by the monographs of USSR and the State Russian pharmacopoeias for R. rosea, the processing and handling procedures for R.rosea root and rhizoma could impact the quality and be the reason for the absence of rosavins in the finished product. The obtained data showed the dependence on the degree of comminution of chopped parts of R. rosea and the 4 K. KHOKHLOVA AND O. ZDORYK presence of rosavins and proved the assumption about the possible enzymatic degradation of the HRM (Kurkin 2015; Bejar et al. 2017). The obtained data showed the dependence on the degree of comminution for chopped parts of R. rosea and the presence of rosavins. Also, the assumption on possible enzymatic degradation of HRM (Kurkin 2015; Bejar et al. 2017) was confirmed. The HPTLC fingerprints for all the ten samples of R. quadrifida were consistent and passed the proposed HPTLC test (Figure S6). The HPTLC analysis for three RRLE samples from different manufacturers was conducted. To exclude the matrix influence of the liquid extract on HPTLC fingerprints, laboratory samples of RRLE were used as a reference. As shown in Figure S7, none of RRLE samples had all the marker zones, while the laboratory RRLE samples had characteristic HPTLC fingerprints for R. rosea. To prove that degradation/fermentation of drug was not responsible, the influence of environmental factors (such as the influence of light, temperature, acid, alkali, peroxide to change in pH ±10%) was studied. The results of stress tests shown that marker substances were stable to the action of stress factors. Thus, the failure of the proposed HPTLC test could be caused by the use of initial HRM of low quality due to inappropriate handling or botanical mistake, as well as by the use of other related species/adulteration or ineffective processing of the drug. 4. Conclusions The specific HPTLC method for authentication of the R.rosea, R.quadrifida and RRLE was proposed. It allows discriminating the falsified products of R.rosea and RRLE from the Ukrainian market. Acknowledgments Thanks are due to the Head of the CAMAG laboratory, Dr Eike Reich and MD Debora Frommenwiler personally for the inspiration and scientific support in conducting research. Disclosure statement No potential conflict of interest was reported by the authors. ORCID Kateryna Khokhlova http://orcid.org/0000-0002-7151-6763 Oleksandr Zdoryk http://orcid.org/0000-0002-2721-0281 References Arzamastsev AP, Kosyireva NS, editors. 1990. Gosudarstvennaya farmakopeya SSSR [State SSSR Pharmacopoeia]. 11th ed. 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