In Europe and North America, Sedum roseum (L.) Scop. (commonly named under its synonym R. rosea L.) is the most well-known and widely used among the different species. It is also known as golden root, or artic root which reputedly demonstrates the economic importance and the geographical distribution of the plant. It has a rich history of traditional use in Russia, Europe and Asia with various uses according to the region (e.g., as shown in Table 1).

In Europe, the first documented medicinal use of R. rosea can be traced back to Dioscorides in 77 A.D. (Brown et al., 2002). In C. v. Linne's Materia Medica, the root of R. rosea was recommended for several conditions such as headaches, “hysteria,” hernias and discharges (C. v. Linne, 1749 in Panossian et al., 2010). Throughout the years, it has appeared in many pharmacopeias and medicinal books of different countries such as Sweden, France, Norway, Germany, Iceland, Estonia, and Russia (Brown et al., 2002; Alm, 2004; Panossian et al., 2010; Shikov et al., 2014).

In China, 73 different Rhodiola species have been reported, mainly in the northwest and southwest regions such as Tibet and the Sichuan province. The adaptogenic and tonic properties of the Rhodiola plants have been widely used in traditional Chinese and Tibetan medicine (Li and Zhang, 2008). They are generally referred to with the Pinyin name Hong Jing Tian [red (or glorious) view of heaven] with slight alterations for each species (Table 2).

R. crenulata can be traced back to Tibetan medicine books including “The Four Medical Tantras” (rgyud-bzhi in Tibetan, Si Bu Yi Dian in Chinese), Yue Wang's Classical Medicinal Book (Somaratsa in Tibetan, Yue Wang Yao Zhen in Chinese), and Jing Zhu Materia Medica [Shel Gong Shel Phreng in Tibetan, Jing Zhu Ben Cao in Chinese (Lu and Lan, 2013)]. It is used for treatment of cough, hemoptysis, pneumonia, and abnormal vaginal discharge. In Traditional Chinese Medicine (TCM), it has effects of nourishing qi as well as promoting blood circulation and is mainly prescribed for qi deficiency and blood stasis (QDBS), stroke, hemiplegia, and fatigue. It is commonly used in China and Tibet for treating altitude sickness.

Research on the phytochemistry and pharmacology of Rhodiola spp. was initiated in the 1960s in the Soviet Union and Scandinavia, mainly focusing on R. rosea (Brown et al., 2002). After the turn of the century the interest in this plant spread globally. Intensive phytochemical research led to the detection of known and novel compounds in R. rosea and related species (Ma et al., 2006; Yousef et al., 2006). Between 2000 and 2015 an increased number of publications stemming from Asian research groups have focused on the detection of novel compounds from Rhodiola species, usually in combination with their respective pharmacological assessments (Fan et al., 2001; Nakamura et al., 2007, 2008).

There are more than a few hundred pharmacological studies on medicinal Rhodiola species (mainly on R. rosea) that show a wide range of activities reflecting their diverse traditional use. They possess adaptogenic and stress-protective (neuro-cardio and hepato protective) and antioxidant effects, as well as stimulating effects on the central nervous system, including on cognitive functions such as attention, memory and learning; anti-fatigue effects; antidepressive and anxiolytic effects; endocrine activity normalizing; and life-span increasing effects (Aslanyan et al., 2010; Sarris et al., 2011; Panossian et al., 2013). The main active compounds are reputedly phenylpropanoids (rosavin, rosarin, rosin) and phenylethanoids (salidroside and tyrosol).

Rhodiola roots and rhizomes are highly valuable products traded at an international level. Since the majority of R. rosea and R. crenulata raw material supplied still comes from wild-collection, their intensive collection leads to scarcity (Galambosi, 2006; Lu and Lan, 2013).

Herbal preparations of Rhodiola species (mainly R. rosea) are extensively utilized around the globe. There is an increasing number of commercial products available on the American, Asian and European markets, either as food supplements or herbal medicines. R. rosea herbal monographs have been included in many Pharmacopeias worldwide. On the other hand, R. crenulata is the only species used medicinally in TCM (Table 3).

Due to this rapid increase of Rhodiola raw material demand, other Rhodiola species such as R. fastigiata, R. sachalinensis, R. quadrifida, Rhodiola sacra (Prain ex Hamet) S. H. Fu and Rhodiola serrata H. Ohba have been sold on the market (Xin et al., 2015). Since there is not any consistent worldwide quality control programme, inadequate quality assessment of Rhodiola spp. is a common issue. This raises concerns about possible adulteration and misidentification issues. The lack of genuine drug material, confusion over the Chinese Pin Yin name of the drug when sourcing from China and accidental or deliberate adulteration during the manufacturing stage may contribute to low quality of final products.

The analytical techniques currently available focus on identifying R. rosea or R. crenulata through chromatographic methods. Other species of Rhodiola have generally not been considered. R. sachalinensis presents a particular problem as it may contain similar marker compounds to R. rosea (and some sources suggest that it is the same species—see http://www.kew.org/mpns-portal).

Forty-two batches of Rhodiola market samples (i.e., not authenticated) were collected between October 2014 and January 2015 from different suppliers including retail outlets, The internet, pharmaceutical companies in seven different locations (Beijing, Guangdong, Qinghai, Anhui, Hebei, Jilin, and Hong Kong SAR) and in China, Germany and Russia. These raw-material samples were mainly labeled as R. rosea, R. crenulata, R. sachalinensis, and R. quadrifida. 18 batches of authenticated plant material were provided by Agroscope Institute (Switzerland). The samples were rhizomes of R. rosea plants propagated from different wild Swiss populations (Mattmark, Carrasino, and Nomnon) or botanical gardens (Switzerland and Germany). In addition, authenticated R. rosea samples which were grown from seeds or provided to the institute by Dr. Bertalan Galambosi were also included. Lastly, in June 2015, samples of R. crenulata and R. fastigiata roots and rhizomes were collected from Garze, Sichuan, China (altitude 4500 m). These samples were authenticated by Professor Shuyuan Li, (Guangdong Pharmaceutical University, Guangzhou, China). Botanical reference materials (BRMs) for R. rosea, R. crenulata, and R. sachalinensis were obtained from the National Institute of Food and Drug Control (NIFDC, China), Dr. William Schwabe (Germany) and Agroscope (Switzerland). BRMs for R. quadrifida and R. fastigiata were provided by Professor Alexander Shikov (Saint-Petersburg Institute of Pharmacy, Russia) and Dr. Anthony Booker (UCL School of Pharmacy). R. fastigiata was authenticated by Professor Shuyuan Li (Guangdong Pharmaceutical University, Guangzhou, China).

All the collected samples were deposited in the herbarium of the UCL School of Pharmacy (London, UK). A detailed description of the investigated samples including their origins and representative symbols are provided in Supplement (S1).

Crude root samples were ground to powder using a household grinder (EK1665ROFOB, Salter, UK) and sieved (0.70 mm mesh). All the powder samples were kept in 1.5 ml tubes (Eppendorf AG.) at 4°C until use.

Deuterium oxide (D₂O), methanol-d₄ (99.8% D, MeOD), dimethyl sulfoxide-d₆ (DMSO-d₆), and tetramethylsilane (TMS) were obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). Salidroside, gallic acid, rosarin, and rosavin were purchased from Sigma-Aldrich Chemicals (St Luis, USA). Tyrosol was purchased from Acros organics (New Jersey, US). Water used in this study was purified by using ULTRAPURE water system (Millipore, Germany). All other chemicals were of analytical grade.

By incorporating the whole region (0–10 ppm) and Pareto (Par) scaling, a significant clustering is observed in R. rosea samples (Figure 2). R. rosea can be differentiated distinctly from the rest of the species based on their principal component variability.

According to the spectra of the species (Figure 3), the aromatic region (6–8 ppm) is dominated by the main marker compounds (rosavin and salidroside). Hence, this region was analyzed independently using Par scaling (Figure 4). Based on the scores plot produced, Rhodiola species were separated more clearly compared to the scores plot of the whole region.

R. crenulata and R. quadrifida were also separated from the rest of the species. However, in this model they were clustered together. This suggests that there is no crucial metabolomic difference between them in the aromatic region. At this point it was considered important to visually inspect the spectra of the BRM's and detect any differences that might be lost with the integration of the data. R. crenulata BRM has an additional quartet at 6 ppm not detected in the rest of the species. This quartet can also be found in all the other R. crenulata samples investigated (figures not shown).

Therefore, an effective separation between R. crenulata and R. quadrifida samples can be accomplished by combining the PCA results with the detection of the additional peaks on the ¹H-NMR spectra only present in R. crenulata samples between 5 and 6 ppm.

We also studied the group-pair comparisons in PCA model with Par scaling (Figure 5). The score plots showed that Rhodiola species separated well (A: R. crenulata with other Rhodiola species; B: R. rosea with other Rhodiola species; C: R. crenulata with R. rosea).

The main differences were between δ 7.5–7.3 ppm (PC1) and δ 7.0–6.8 ppm (PC2). The chemical shift of the main variable metabolites were mainly rosavin, rosarin, and cinnamyl alcohol derivatives.

The metabolites detected were elucidated by the analyses of the ¹H-NMR spectra as well as the comparison with the reference compounds, together with the in-house NMR chemical shift database (Mudge et al., 2013; Luo et al., 2015). The summary of the assignment of ¹H-NMR spectral peaks obtained from the R. rosea, R. crenulata, and R. sachalinensis BRM extracts are provided in Supplement (S3).

The band position and visibility of the standards rosavin, rosarin, and salidroside (Figure 6) appear with characteristic colors and increasing retention factors (Rfs) 0.19, 0.26, and 0.31, respectively. Under UV light 254 nm, salidroside is not visible. Under 366 nm, after derivitisation with sulfuric acid, rosavin and rosarin appear as pale pink bands and salidroside as a green one.

Gallic acid shows good visibility under UV 254 nm, while it is not easily detected under UV 366 nm at a dark blue back-round. Tyrosol is visible in 254 nm but less clear in 366 nm.

The raw plant material obtained from the market was also studied by our HPTLC method (list of samples in Supplement S4). Under UV 254 nm (Figure 7), there were two obvious bands among these samples (Rf = 0.27 and 0.48). However, due to lack of reference standards, their identity remains unknown. Further studies need to be conducted using NMR and LC-MS. The majority of the samples investigated contained concentrations of tyrosol similar to the standard raw material used (R24, R30, and R31). Samples R1–R6 contained lower levels of this compound possibly due to their longer storage time. Therefore, tyrosol could be considered as a marker to study duration of Rhodiola storage. It was also found that only five samples (R9, R25, R58, R59, and R24) contained high levels of rosavin, which turned out to be the ones from R. rosea. Moreover, this result can also be verified by the NMR results (Figure 5). However, it is not evident whether there is adulteration of R. sachalinensis in R. rosea since their metabolites are similar.

Under UV 366 nm after derivatisation eight samples (R1, R5, R6, R15, R27, R32, R61, and R64) had a low concentration of salidroside (Rf = 0.31). These samples could have been kept for a long time after collection and the salidroside content could have decreased due to lack of a good storage environment.

Combining the results of HPTLC and ¹H-NMR multivariate statistical analysis, we also analyzed the adulteration rate among all the market samples (Supplement, S4).

Thirty percent of the Rhodiola samples collected from the market were not, as declared on the label, i.e., either R. rosea or R. crenulata. Some R. rosea samples were also being sold as R. crenulata. 47.7% of raw material samples were not labeled properly and their species information were not clearly illustrated to customers. This highlights a clear lack of proper local government policies and good quality control strategies.

According to our study, different Rhodiola species (including R. rosea and R. crenulata) can be found in the Chinese market. However, they are neither sold separately nor well-identified. Therefore, there is a high potential of adulteration and substitution among these species.

Since in the value chain, mixing of batches and, therefore, potentially also of species, is of major concern, the possibility of qualitatively and quantitatively detecting plant mixtures was also investigated. The additional species chosen for this study was R. crenulata which is considered to be the most common adulterant of R. rosea. The selected BRMs were weighed individually in different proportions and then added together in an Eppendorf reaction tube. The rest of the sample preparation was identical to the methodology for the ¹H-NMR spectroscopy. The samples were renamed as seen in Table 4. After the acquisition of the spectra, they were baseline and phase corrected and zeroed to the TMS peak in Topspin 3.2.

In all samples, the salidroside peak intensity remains almost the same since this constituent is present in both species. The peaks of rosavin are gradually decreasing with the addition of R. crenulata, whereas the characteristic quartet at 6 ppm due to the presence of an unknown compound is increasing with the addition of R. crenulata and it is not detected in R. rosea at all (Figure 8).

The acquired spectra were bucketed using Amix and only focused on this region (6 ppm). When the whole quartet was integrated into a single bucket, the observed increase of its intensity was not adequately represented. Therefore, the bucket size used changed to 0.002 ppm and only incorporated the first peak of the quartet (6.0028–6.0048 ppm). The buckets obtained from Amix were transferred into Excel, where the relationship between the bucket value and the percentage of R. crenulata in the mixture was expressed graphically as a calibration curve. The bucket value of the respective peak is increasing in a linear mode (Figure 9).

Similar results can also be obtained with HPTLC analysis. The HPTLC fingerprints produced consist of the over-spotted BRM extracts in different volumes as seen in Table 5. The final volume applied was 5 μl.

As seen in Figure 10, when the loading volume of the R. rosea decreases, the representative markers of this species (rosavin and rosarin) decrease as well. However, the band for salidroside, (since it occurs in both species) remains almost the same.

By gradually increasing the R. crenulata proportion, several bands gradually appear above salidroside that could potentially be used as markers for the qualitative and semi-quantitative HPTLC analysis of mixtures of these two Rhodiola species. Further work needs to be carried out to determine the identity and species-specificity of these compounds.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.