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2019 Authentication of Rhodiola rosea, Rhodiola quadrifida and Rhodiola rosea liquid extract using HPTLC

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:
To link to this article: https://doi.org/10.1080/14786419.2019.1591398
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Published online: 28 Mar 2019.
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Authentication of Rhodiola rosea, Rhodiola quadrifida and
Rhodiola rosea liquid extract from the Ukrainian market
using HPTLC chromatographic profiles
Kateryna Khokhlovaa
and Oleksandr Zdorykb
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
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
Rhodiola rosea; Rhodiola
quadrifida; golden root;
rosavins; salidroside;
authentication; highperformance thin-layer
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
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
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
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
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.
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.
Kateryna Khokhlova
Oleksandr Zdoryk
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