Rhodiola rosea L., [Crassulaceae, syn. Sedum rhodiola Vill] is affiliated with a group of perennial shrubs commonly associated with subarctic and alpine areas of eastern Siberia. Currently, Rhodiola rosea is found in hard-to-reach rocky terrains, sea cliffs and mountains of the northern hemisphere, ranging from the low Arctic to the high temperate zones of Asia, Europe, and North America (Scandinavia, Iceland, Greenland, Alaska or Canada), making the world supply from wild populations on an industrial scale extremely limited (Blomkvist et al., 2009).

To date, 74 accepted species have been identified within the Rhodiola genus. Many of them are used in national systems of traditional medicine, including Rhodiola heterodonta (Hook.f. and Thomson) Boriss, Rhodiola quadrifida (Pall.) Fisch and Mey, Rhodiola semenovii (Regel and Herder) Boriss, Rhodiola kirilowii (Regel) Maxim., and Rhodiola crenulata (Hook.f. and Thomson) H.Ohba. Of all these species, R. rosea is the one most studied for its phytochemical properties as well as its use in medicine and therapy. R. rosea is one of the main representatives of the group of plant adaptogens used for medicinal purposes linked to stress and age-related impairments of cognitive functions as a tonic and immunomodulating therapeutic agent (European Food Safety Authority (EFSA), 2012). The most valuable parts of the plant used in therapy are R. rosea roots and rhizomes, of a golden metallic colour, harvested after at least a 5-year vegetative period (Galambosi, 2006).

R. rosea has a long history of use as a medicinal plant, appearing in reports from Asian and European countries. The therapeutic use of Rhodiola rosea roots traces back to Tibetan Traditional Medicine (TTM), which has influenced its more recent use in Traditional Chinese medicine (TCM), where it was employed to treat altitude sickness, fatigue, and mood disorders, as well as headaches, diarrhoea, skin conditions and swelling, typically in the form of infusions and tinctures (Tao et al., 2019).

The European Medicines Agency classifies the dry extract (DER 1.5–5:1) obtained from R. rosea roots and rhizomes as a traditional herbal medicinal product used for the prevention and temporary relief of fatigue and sensation of psycho-physical weakness (European Medicines Agency, 2012). Currently, in the European Union, R. rosea preparations are used as active ingredients in registered traditional herbal medicinal products (HMP) and as ingredients in dietary supplements, suggesting potential benefits on memory, learning, and attention. Supplements containing Rhodiola are recognised as beneficial for managing physical and mental stress in the European Food Safety Authority’s (EFSA) consolidated list of Article 13 health claims (European Food Safety Authority, 2012). Moreover, R. rosea root extracts can be found in “functional foods”, sports and energy drinks, alcoholic beverages, as well as the cosmetic industry (European Commission, 2020; Lamadrid et al., 2019).

The multidirectional properties of R. rosea - concerning its antibacterial, anxiolytic, antioxidant, immunomodulating, cardio and neuroprotective, as well as nootropic effects are strongly correlated with the rich phytochemical composition of its underground organs (Lee et al., 2013; Zhang et al., 2022). Chemical investigation on this plant revealed approximately 150 chemical entities, encompassing phenylethanoids (i.e., salidroside with its aglycone–tyrosol and its derivatives), phenylpropanoids (i.e., rosin, rosavin, rosarin), and flavonoids characteristic of this species (herbacetin, gossypetin and kaempferol derivatives), as well as cyanogenic glycosides, gallic acid derivatives and proanthocyanidin constituents among others (Alperth et al., 2019). Although it is still not entirely clear which compounds are responsible for the adaptogenic effects of the raw material, the observed effects have been considered to be mainly based on phenylethane and phenylpropane derivatives. The latest edition of the European Pharmacopoeia (11th edition, 2023) emphasises the quality assessment of R. rosea-based products by considering the content of marker compounds, salidroside and rosavins, in a ratio not lower than 1:3. In turn, the United States Pharmacopeia guidelines assess the quality mark for the raw material as containing not less than 0.3% of the phenylpropanoid glycosides rosarin, rosavin and rosin (these three compounds are also collectively referred to as ‘rosavins’) calculated as rosavin, and not less than 0.08% of salidroside, calculated on a dry weight basis (United States Pharmacopeial Convention, 2017; Ezra Bejar and John, 2017). Salidroside has been identified in several species of the Rhodiola genus, including R. crenulata, R. rosea, R. sachalinensis, and R. kirilowii, while rosavin has been isolated specifically from the roots of R. rosea and R. sachalinensis (in smaller amounts) (Booker et al., 2016; Nakamura et al., 2008). Despite the presence of salidroside in most species of the Rhodiola genus, its relatively low content in R. rosea does not allow for its unequivocal recognition as a marker compound for this species. Consequently, monitoring the presence of rosavin, as a distinctive chemotaxonomic marker, seems to be considered synonymous with the high quality of the tested preparations.

The rising demand for R. rosea has been paralleled by an increase in dietary supplements claiming its inclusion. Considering the economic advantage of producing dietary supplements over medicinal products, as well as the lack of precise guidelines for analysing their composition, proper quality control of R. rosea preparations poses a significant global challenge within the pharmaceutical industry. The plant’s limited native range, coupled with its difficult accessibility, prolonged growth period, and morphological similarities within the Rhodiola genus, has led to frequent adulteration with other species (Nikolaichuk et al., 2021). Furthermore, the adaptogenic properties attributed to R. rosea, together with the growing number of individuals seeking relief from excessive psycho-physical stress, fatigue, and exhaustion, have rendered R. rosea an endangered species in many regions. The global demand for adaptogenic medicinal plants such as R. rosea and its key compounds, salidroside and rosavins, has also driven an increase in preclinical and clinical studies aimed at uncovering new applications for R. rosea in modern phytotherapy, which has further depleted the natural resources of the raw material (Hung et al., 2011; Lu et al., 2022).

The aim of our study was to assess the phytochemical analysis of commercial products of the underground parts of R. rosea and/or their potential contaminants. Herein, HPTLC and LC-MS fingerprints describing the phytochemical composition of 13 preparations containing R. rosea unprocessed roots or their extracts were analysed, with a particular emphasis on assessing the rosavin presence within these products. The traditional use of R. rosea is well-documented for its adaptogenic and stimulating effects. As the search for natural substances that help the human body adapt to challenges from technological advancements and modern working conditions continues, we analysed the qualitative composition of the tested extracts, including rosavin, and correlated these findings with their anti-inflammatory effects in a BV2 microglial cell model. Incorporating the potential impact of the tested samples on inflammatory responses within a microglial model may provide a valuable tool for additional quality control of adaptogenic plant materials.

Neuroinflammation plays a crucial role in the pathogenesis of neuropsychiatric and neurodegenerative disorders. A growing body of evidence suggests that excessive microglial activation and the overproduction of pro–inflammatory mediators underlie the development of psychophysical exhaustion, depression, anxiety, and neurodegeneration. In addition to its role in synaptic plasticity, learning, and memory, microglia are essential for maintaining central nervous system (CNS) homeostasis. Due to their involvement in these processes, microglia may become an important target for therapeutic interventions aimed at modulating neuroinflammatory responses and mitigating disease progression (Gao et al., 2023; Li et al., 2022).

Rhodiola rosea L., a traditional medicinal plant widely used in Eastern Europe and Asia, is recognized for its neurostimulatory, antidepressant, and adaptogenic properties. Studies have demonstrated its efficacy in enhancing cognitive performance, reducing fatigue, and alleviating stress–related symptoms. In shift workers, short–term administration of R. rosea extract improved concentration, focus, and reaction speed under stress. Similarly, in a study on 80 healthy students, a 14–days regimen of 400 mg R rosea root extract significantly reduced anxiety, stress, anger, and confusion while improving mood (Cropley et al., 2015). Additionally, a phase II clinical trial comparing R. rosea with sertraline indicated a moderate antidepressant effect in mild to moderate depression, with fewer adverse effects than the conventional drug (Mao et al., 2015). The available literature provides partial insights into the pathomechanism underlying this effect. Available review reports systematize the available evidence indicating the inhibitory effects of the raw material on the secretion of inflammatory mediators, reactive oxygen species (ROS), and nitric oxide (NO) as well as its anti–apoptotic functions in in vitro models employing microglial and macrophage cells. Notably, the majority of studies focus on assessing the bioactivity of isolated single compounds, predominantly salidroside, a constituent found in multiple species within this genus and rosavins as leading anti–inflammatory components (Liu et al., 2024). Furthermore, there is a limited number of studies that establish a correlation between the biological activity of the extract and its chemical composition (Ma et al., 2018; Pu et al., 2020).

The growing global demand for Rhodiola rosea products, spurred by increasing scientific evidence supporting its efficacy, presents significant challenges to the conservation and sustainable use of this species. The expanding use of R. rosea in herbal medicines, dietary supplements, cosmetics, and food additives, combined with its limited cultivation areas, high agricultural costs, lengthy growing period, low dispersal ability, and low rate of germination and seedling survival is contributing to a gradual decline in the availability of this raw material worldwide (Galambosi, 2006). Depletion of natural populations and habitat destruction have led to the disappearance of Rhodiola species in many regions, as most of the raw material is collected from the wild, and the plant needs several years to regenerate (Howes et al., 2020).

The increasing demand for raw materials, coupled with their decreasing availability, has led to a significant rise in cases of intentional and accidental adulteration of R. rosea products. A major concern in this context is the admixture or substitution of R. rosea roots and rhizomes with those from other Rhodiola species, which are morphologically similar and difficult to distinguish, particularly in underground structures. Recent phylogenetic studies have highlighted significant evolutionary convergence among the 74 species of Rhodiola (Zhang et al., 2014). Although the color of the flowers can differentiate R. rosea from R. crenulata and other purple–flowering species, this convergence is particularly noticeable in the roots and rhizomes of Rhodiola, which lack distinct diagnostic features, making species identification based on these structures difficult (Cuerrier A and Rapinski, 2014; Zhu et al., 2017).

According to a 2016 market study, approximately 75% of the global Rhodiola extract production originates from Asia, with around 13% sourced from Europe, 5% from the United States, and 7% from other regions (Center, 2016). The high volume of imports from Asia, particularly from China, into the U.S. and European herbal supplement markets has led to frequent adulteration or substitution of R. rosea raw materials with other Asian Rhodiola species. This is compounded by the interchangeable use of various Rhodiola species names in different regions, with species like R. crenulata, R. heterodonta, and R. kirilowii being labeled under the common term “rhodiola” in China (Cunningham et al., 2020). Despite limited data, it is widely recognized that European sources of R. rosea are scarce. The commercial collection of R. rosea in Europe is of relatively low economic significance due to high labor costs and logistical challenges associated with harvesting in remote mountainous regions. Furthermore, many European populations of R. rosea are legally protected, including those in Bulgaria, the Czech Republic, Austria, and Germany (Fisheries, 2022).

The United States Herbal Medicines Compendium identifies several Rhodiola species as potentially confounding in the commercial supply of Rhodiola rosea, including R. crenulata, R. kirilowii, R. sacra, R. serrata, R. sachalinensis, and R. yunnanensis (United States Pharmacopeial Convention, 2014). Our analysis indicates the intentional or accidental substitution of R. rosea with R. crenulata in at least 5 of the tested products, as suggested by the presence of compounds characteristic of R. crenulata, such as creoside I, IV, and crenulatin. A typical example of a species involved in the adulteration of roseroot preparations is also R. sachalinensis, a species with a similar chemical composition to R. rosea but containing lower amounts of rosavins and salidroside. Some botanists even regard R. sachalinensis as the same species or a subspecies of R. rosea (Gontcharova et al., 2009). However, chemotaxonomic studies conducted by Booker et al. show differences between R. rosea and R. sachalinensis, which, based on general metabolomic methods and specific high–performance thin–layer chromatography (HPTLC) spectra and limited geographical range, allow treating them as separate species (Booker et al., 2016; Liu et al., 2013).

Although adulteration of preparations with other Rhodiola species seems to be a major challenge for the pharmaceutical market, meeting the requirements for good quality raw materials for the growing industrial demand is also a significant problem. The key aspect seems to be the requirements of the European Pharmacopoeia for R. rosea roots and rhizomes in terms of the content of marker compounds, i.e., salidroside and rosavins.

R. rosea is available on the market in various forms, including cut/dried, powdered rhizomes and roots, dry or liquid extracts, as well as dry extracts in solid dosage forms like capsules and tablets (Brinckmann et al., 2021). Market analysis indicates that the global demand for R. rosea products is constantly growing, and with it, the number of adulterated or poor–quality preparations (Bernard, 2016). The scale of the problem is illustrated by earlier studies assessing the quality of preparations declaring the content of R. rosea, available on the European market. Ma et al. found that approximately 33% of 18 commercial Rhodiola rosea rhizome powder extract samples did not display a consistent Rapid Resolution Liquid Chromatography (RRLC) profile and were missing the characteristic peaks of rosarin, rosavin, and rosin or did not meet the claimed contents standards for the marker compounds for authentic R. rosea rhizomes (Ma et al., 2011). Similarly, a study by Booker A. et al. (2016) assessing the composition of 40 commercial preparations available on the EU market, claiming to contain R. rosea, confirmed the absence of rosavin in approximately 20% of the cases. Furthermore, in the remaining 80%, based on NMR Spectroscopy and HPTLC methods, the rosavin content was lower than declared. Additionally, 25% of tested dietary supplements were found to be substituted with other Rhodiola species, and one product was adulterated with synthetic 5–hydroxytryptophan (5–HTP) (A. Booker et al., 2016). A study conducted by Ruhsam M et al., in 2018 found that only 5 of 13 roseroot supplements contained R. rosea, with no visible admixture of other species (Ruhsam and Hollingsworth, 2018). In a study presented by Marchev A. et al., of 30 commercial products purchased from various suppliers, only 23% had rosavin and salidroside levels consistent with the amounts specified on the label, as confirmed by HPLC analysis. Additionally, 37% of the products did not meet the typical standardisation (1% salidroside and 3% rosavins), which may suggest potential adulteration (Marchev et al., 2020). In the study by Cunningham A. et al., they also found the presence of additives of extracts from the roots of Astragalus and Lycium species in preparations, declaring the presence of species of the Rhodiola genus (Cunningham et al., 2020).

Despite a smaller research group, our study indicates that almost 60% of the tested preparations did not contain the declared content of a single raw material–R. rosea roots/rhizomes or their extracts. Moreover, we detected the presence of rosavin in only 9 out of 13 tested preparations (using the LC–MS method), with 4 of these showing only trace amounts of the marker compound. Notably, the misidentification or accidental use of other Rhodiola species was most frequently observed in the tablet/capsule group. This could be attributed to the ease of substituting powdered extracts and the challenges associated with their precise identification.

Numerous studies have evaluated the quality and composition of commercial R. rosea products, highlighting the potential substitution of R. rosea with other Rhodiola species in dietary supplements. Such substitutions are often driven by the greater availability and ease of large–scale cultivation of alternative species. Notably, while the substitution of R. rosea with R. crenulata is widely recognized as a primary authenticity issue in R. rosea products, R. crenulata roots and rhizomes are preferred in Traditional Chinese Medicine (TCM) due to their higher availability in Asia, consistent with previous reports. However, from a safety perspective, the consumption of R. rosea is endorsed by the European Medicines Agency, whereas other species within the Rhodiola genus lack similar regulatory support. Furthermore, prior research underscores the efficacy of R. rosea as an adaptogen, demonstrating its beneficial effects in managing stress–related conditions, enhancing both mental and physical performance, and supporting immune function. In addition, few reports indicate its potential effectiveness in the adjunctive therapy and prevention of microglia–generated neuroinflammation associated with the development of neurodegenerative diseases, e.g., Parkinson’s or Alzheimer’s disease (Morgan and Grundmann, 2017).

The interchangeable use of different species within the same genus may, therefore, create some variations in chemical composition, potentially affecting the quality, safety, and biological activity of the product. The assessment of the anti–inflammatory effect of the tested preparations in the model of LPS–stimulated microglia may allow for the indirect correlation of the quality of the tested extracts with their biological effect.

Among the tested samples, the highest anti–inflammatory activity was observed for Z3 and Z4 among the unmodified raw material samples, K4 among the capsule formulations, T1 and T3 among the tablet formulations, and P1 in the powdered form (Figures 7, 8). Notably, the most significant inhibition of both TNF–α and IL–6 secretion was recorded for the ethanolic extract of preparation Z3, which was confirmed to contain Rhodiola rosea. Specifically, the infusion of Z3 resulted in TNF–α secretion inhibition, reducing its level to 62.0% ± 9.5%. Furthermore, Z3 reduced IL–6 secretion to 61.4% ± 14.2% and 74.8% ± 7.7% for the ethanol extract and infusion, respectively, relative to the 100% release observed in the stimulated control (Figure 8). The anti–inflammatory effects of R. rosea crude extract and its primary constituents, as well as their concentration–dependent modulation of iNOS, TNF–α, IL–1β, IL–6, and other inflammatory mediators, have been previously documented in the studies of Lee et al. and Jiang et al., among others (Borgonetti et al., 2020; Jiang et al., 2024; Lee et al., 2013). These findings are particularly relevant in the context of neuroinflammation modulation, a mechanism proposed to underlie the anti–stress effects of adaptogenic formulations (Ahmed et al., 2016; Guo et al., 2021).

An analysis of the composition of biologically active preparations suggests that their anti–inflammatory effects may partially correlate with the concentration of the marker compound for Rhodiola rosea. A comparative assessment of rosavin peak intensities in the analyzed samples of unprocessed plant material (Supplementary Table S3A, Supplementary Material) revealed a significantly higher concentration of rosavin in the Z3 preparation compared to the other samples.

Previous research has emphasized the role of phenylpropanoid derivatives, such as rosavins, and phenylethanol derivatives in contributing to the adaptogenic properties of R. rosea, exerting pleiotropic pharmacological effects on the neuroendocrine and immune systems (Fan et al., 2022; Maadawi, 2017). However, most studies have primarily focused on the biological activity of salidroside, with limited investigations into the effects of other compounds, including rosavin, within this cellular model (Lee et al., 2013; Zhang et al., 2022).

Thus, the neuroinflammation model employed in the present study enabled the evaluation of rosavin activity, representing one of the first investigations of this compound in a microglial cell model. As demonstrated, rosavin significantly and dose–dependently inhibited IL–6 and TNF–α secretion in LPS–stimulated microglia. Specifically, at a concentration of 5 μM, rosavin reduced IL–6 secretion to 34.3% ± 3.3% compared to the stimulated control (100% secretion), while a 43.5% ± 4.9% inhibition was observed at a concentration of 2 µM (Figure 8). Although data on rosavin’s effects in microglial cell models remain scarce, the present findings align with previous reports on its anti–inflammatory properties in other models, such as murine macrophages (Liu et al., 2024).

Interestingly, some preparations that did not contain R. rosea or rosavin—specifically P1, T1, and T3—also exhibited notable anti-inflammatory activity. These findings indicate that, although the selected cellular model is effective in capturing the activity of rosavin and standardized extracts, it may be insufficient for evaluating the overall quality or authenticity of complex commercial formulations.

It is also important to note that, although the BV2 microglial cell line is an immortalized murine model that does not fully capture the complexity of human microglia, it remains a widely used and well-established system for the preliminary investigation of neuroinflammatory mechanisms. Owing to their reproducible and well-characterized response to pro-inflammatory stimuli such as LPS, BV2 cells offer a practical and informative platform for the initial evaluation of anti-inflammatory properties in natural products (Marrazzo et al., 2023). Nevertheless, limitations related to species origin and monoculture conditions highlight the need for future studies employing more physiologically relevant models, including human-derived systems or co-culture approaches, to more accurately assess the biological activity of such preparations.

Nevertheless, our study confirms the potential of Rhodiola rosea as an agent mitigating neuroinflammatory processes, with rosavin identified as a compound potentially responsible for the observed biological activity of the raw material. With regard to other species within the Rhodiola genus, a comprehensive analysis should be conducted to assess their biological activities and to identify the specific compounds responsible for the observed effects.

The growth of the dietary supplement market necessitates detailed analyses of product composition and quality control. The increasing number of companies producing dietary supplements, along with rising consumer demand increases the risk of intentional or accidental contamination or incorrect product composition. European regulations governing the content of medicinal products containing Rhodiola rosea roots, rhizomes or their extracts necessitate the production of high–quality preparations with confirmed efficacy. The questionable quality of available preparations, claiming to contain R. rosea roots or rhizomes, may stem from either intentional or unintentional substitution of raw materials, as well as improper storage, which can lead to the degradation of key marker compounds. The objectives of this study were achieved by using an interdisciplinary approach (primarily based on LC–MS analysis) to better understand the sources of potential adulteration of individual herbal samples and their impact on biological activity. The identification of R. rosea in only 40% of the preparations underlines that rigorous control and standardisation of herbal supplements are crucial to understanding their therapeutic activity and preventing adulteration.