The Ethnopharmacological Uses, Metabolite Diversity, and Bioactivity of Rhaponticum uniflorum (Leuzea uniflora): A Comprehensive Review

Rhaponticum uniflorum (L.) DC. (syn. Leuzea uniflora (L.) Holub) is a plant species of the Compositae (Asteraceae) family that is widely used in Asian traditional medicines in China, Siberia, and Mongolia as an anti-inflammatory and stimulant remedy. Currently, R. uniflorum is of scientific interest to chemists, biologists, and pharmacologists, and this review includes information from the scientific literature from 1991 to 2022. The study of the chemodiversity of R. uniflorum revealed the presence of 225 compounds, including sesquiterpenes, ecdysteroids, triterpenes, sterols, thiophenes, hydroxycinnamates, flavonoids, lignans, nucleosides and vitamins, alkanes, fatty acids, and carbohydrates. The most studied groups of substances are phenolics (76 compounds) and triterpenoids (69 compounds). Information on the methods of chromatographic analysis of selected compounds, as well as on the quantitative content of some components in various organs of R. uniflorum, is summarized in this work. It has been shown that the extracts and some compounds of R. uniflorum have a wide range of biological activities, including anti-inflammatory, antitumor, immunostimulatory, anxiolytic, stress-protective, actoprotective, antihypoxic, anabolic, hepatoprotective, inhibition of PPARγ receptors, anti-atherosclerotic, and hypolipidemic. Published research on the metabolites and bioactivity of R. uniflorum does not include clinical studies of extracts and pure compounds; therefore, an accurate study of this traditional medicinal plant is needed.


Introduction
Rhaponticum Vaill. is a small genus from the tribe Cynareae of the Asteraceae family that is distributed mainly in tropical and subtropical regions of Europe, Asia, and Africa. In total, more than 20 species belong to the genus and are distributed in a narrow strip in the Northern hemisphere from the Atlantic coast to the Pacific Ocean [1]. Close to Rhaponticum are the Mediterranean monotypic genus Leuzea and the small Asian genus Stemmacantha, which, combined, include approximately 10 species. Many species of Rhaponticum are of economic importance, and some have been introduced into cultivation as ornamental or medicinal plants. R. carthamoides (also known as Maral root) is widespread from Central Asia to Siberia and Xinjiang; it is a medicinal plant and a source of ecdysteroids; it is recommended as part of combination therapy for asthenia, physical and mental overwork, impotency, and during convalescence [2]. North African endemic species R. acaule is used as an aperitif, cholagogue, depurative, digestive, stomachic, and tonic in North and Central Tunisia [3]. Creeping knapweed or R. repens is a traditional medicine in Central Asia; it is applied as an emetic, antiepileptic, and anti-malaria remedy [4].

Review Strategy
To produce a relevant review, international databases (e.g., Scopus, Web of Science, PubMed, and Google Scholar) were used. Because most studies have been performed by Chinese and Russian scientists, national electronic resources (e.g., Chinese research databases (Wanfang and CNKI Journals) and the Russian scientific database (eLibrary)) were included in the search. These resources contain relevant articles that are not indexed by international databases. Only original papers written in English, Chinese, and Russian, and published in journals prior to October 2022, were considered. An exception was made for the ethnopharmacological data collected from books. The search keywords used included plant names (e.g., "Rhaponticum uniflorum", "Leuzea uniflora", "Stemmacantha uniflora", "Fornicium uniflorum") and metabolite names. The list of R. uniflorum compounds includes secondary metabolites mostly correlated with ethnopharmacological uses and bioactivities of the plant, and, for a more complete picture, information about primary metabolites is also mentioned in this manuscript.
Comparing the chemodiversity of the ecdysteroids in R. uniflorum with that of the more-studied species R. carthamoides (in which more than 50 compounds of this class have been identified so far [20]), it can be assumed that there are many more compounds in the composition of the steroid metabolome of R. uniflorum.
Different organs of R. uniflorum are the sources of 25 non-ecdysteroid triterpenoids (57-81), including 23 compounds isolated from the roots and five components detected in

Chromatographic Analysis of R. uniflorum
Despite the widespread use of R. uniflorum as a medicinal plant, only few methods for the quantitative analysis of this plant material using liquid chromatography are known (Table 3). To separate the main ecdysteroids of the herb and roots of R. uniflorum (28,25,41,50,53), six variants of high performance liquid chromatography analysis on reversedphase sorbents have been proposed, i.e., using the columns Ultrasphere ODS [7], Zorbax ODS [28], ProntoSIL 120-5 C18 [56], YMC-Pack C18 [57], GLC Mastro C18 [43], and Waters Acquity UPLC HSS T3 C18 [9] with 100-250-mm length [7,9,28] or 60-mm microcolumns [56]. Mixtures of methanol, acetonitrile, water, perchlorate buffer, and formic acid have been used as eluents to achieve separation in isocratic and gradient modes. The total duration of the analysis varied from 15 to 70 min. Analysis of the dominant components of R. uniflorum flowers has also been performed under reversed phase HPLC conditions using a mixture of phosphoric acid and acetonitrile [57]. The chosen analysis conditions allowed separation of six compounds, including 28, 109, 116, 128, 147, and 163.

Chromatographic Analysis of R. uniflorum
Despite the widespread use of R. uniflorum as a medicinal plant, only few methods for the quantitative analysis of this plant material using liquid chromatography are known (Table 3). To separate the main ecdysteroids of the herb and roots of R. uniflorum (28, 25, 41,  50, 53), six variants of high performance liquid chromatography analysis on reversed-phase sorbents have been proposed, i.e., using the columns Ultrasphere ODS [7], Zorbax ODS [28], ProntoSIL 120-5 C18 [56], YMC-Pack C18 [57], GLC Mastro C18 [43], and Waters Acquity UPLC HSS T3 C18 [9] with 100-250-mm length [7,9,28] or 60-mm microcolumns [56]. Mixtures of methanol, acetonitrile, water, perchlorate buffer, and formic acid have been used as eluents to achieve separation in isocratic and gradient modes. The total duration of the analysis varied from 15 to 70 min. Analysis of the dominant components of R. uniflorum flowers has also been performed under reversed phase HPLC conditions using a mixture of phosphoric acid and acetonitrile [57]. The chosen analysis conditions allowed separation of six compounds, including 28, 109, 116, 128, 147, and 163.
According to the quantitative analysis of R. uniflorum, the content of individual compounds in different organs may vary ( Table 4). The concentration of the dominant ecdysteroid 20-hydroxyecdysone (28) in raw materials collected in Russia was 0.02-1.06% [28,56]. Plants growing in China are characterized by a higher content of 28 in the leaves (up to 1.35%) than in the roots (0.45%) [7,57]. The level of other ecdysteroids (25, 41, 50, and 53) was characterized as trace. The concentration of the basic phenolic compounds in R. uniflorum flowers varied from 0.03-0.05% for 128 to 0.42-2.26% for 163 [57]. Table 3. HPLC analysis conditions used for the separation of selected R. uniflorum metabolites.   [7] 0.62 0.05 0.02 Russia [28] Tr. 0.03-0.47 Tr.

Anti-Inflammatory Activity
The study of the anti-inflammatory mechanisms of R. uniflorum roots and flowers demonstrated their effectiveness in in vitro and in vivo studies [8,9,16,19,58]. Ethanol extract of R. uniflorum roots significantly inhibited the secretion of nitric oxide (NO) and inflammatory cytokines in the culture of RAW 264.7 mouse macrophages and peritoneal macrophages without the manifestation of cytotoxicity [58]. The extract significantly suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 while simultaneously inducing the expression of heme oxygenase 1 [58]. The inhibition of phosphorylation and degradation of the IκBα factor led to the prevention of nuclear translocation of the NF-κB transcription factor, which, in turn, controls the expression of immune response, apoptosis, and cell cycle genes. A pronounced ability of the R. uniflorum root extract to suppress mitogen-activated protein kinases (MAPKs), such as ERK1/2, p38, and JNK, was revealed in a culture of lipopolysaccharide (LPS)-stimulated macrophages [8]. The lipophilic components of the hexane and chloroform fractions of R. uniflorum had a greater inhibitory effect on NO production in a culture of LPS-stimulated macrophages and suppressed the transcription of the iNOS messenger RNA [8]. The butanol and ethyl acetate fractions reduced the synthesis of prostaglandin PGE2, while the hexane and ethyl acetate fractions led to the suppression of interleukin-1β [8]. Overall, these facts demon-strate the effectiveness of the R. uniflorum root extract as an anti-inflammatory agent acting through the activation of NF-κB and MAPK signaling pathways. Investigation of the anti-inflammatory activity of the R. uniflorum flower extract demonstrated its facilitating potential after doxorubicin-initiated cardiotoxicity of embryonic rat cardiomyocytes H9c2 [16]. In in vivo experiments, R. uniflorum flower extract prevented LPS-induced pathological alterations of lung bronchoalveolar lavage fluid (BALF) [9]. Downregulation of F4/80 antigen expression in lungs and suppression of LPS-induced elevations in BALF and lung tissue levels of myeloperoxidase were observed with the simultaneous reduction of expression of proteins p-p38, p-JNK, p-ERK (mitogen-activated protein kinase signaling pathway), TLR4, Myd88, p-IκB, and p-p65 (Toll-like receptor 4 and NF-κB signaling pathway) [9]. The abovementioned results indicated that the R. uniflorum flower extract ameliorated LPS-induced acute lung injury by suppressing the inflammatory response and enhancing antioxidant capacity.

Antitumor Activity
The root extracts of R. uniflorum in in vitro studies reduced the proliferation of AGS human gastric adenocarcinoma cells [59], SCC 15 oral cancer cells [60], and human lung adenocarcinoma cells A549 and H1299 tumor cells [61]. The extracts inhibited messenger RNA (mRNA) and expressed transcription factors protein C-ets-1 (ETS1), and peroxiredoxin 1 (Prx1) resulted in the suppression the growth and proliferation of SCC 15 cells [60]. Animal experiments with H 22 hepatoma cells demonstrated reduction of transplanted tumor grow caused by reducing DNA fragmentation and microvascular density and worsening the expression of signaling proteins, such as vascular endothelial growth factors (VEGF) and hypoxia-inducible factor 1α (HIF-1α), indicating an antiangiogenic and proapoptotic effect on H 22 cells [62]. Root ethyl acetate extract affected the growth of SCC15 epidermoid carcinoma cells, reducing their viability and inducing their apoptosis. Treatment of cells with this fraction promoted the expression of messenger RNA and E-cadherin, while reducing the expression of peroxiredoxin 1, vimentin, and the SNAI1 protein influenced the program of the epithelial-mesenchymal transition, significantly reducing tumor growth [63]. The aqueous extract of R. uniflorum roots (100-400 mg/kg) slowed tumor growth by 27-38% in mice with transplanted H22 tumors, improving the immune system and antioxidant status of the organism [64].

Immune-Stimulating Activity
The immunostimulatory effect of the R. uniflorum root extract has been described for the experimental immune suppressions caused by azathioprine, owing to the increasing activity of the cellular, humoral, and macrophage components of the body s immune system [65]. The extract from the leaves of R. uniflorum is an effective immune stimulant in cyclophosphamide-induced immunodeficiency [66].

Nervous System Effects
A study on the anti-anxiety effect of R. uniflorum showed that animals treated with dry root extract (200-300 mg/kg) had higher overall locomotor activity compared to control animals. Administration of the R. uniflorum extract had a pronounced anti-anxiety effect under conditions of unpunished behavior. An increase in exploratory activity and a decrease in the feeling of fear and anxiety in animals was explained by a decrease in their level of emotionality [67]. The administration of the extract stimulated cognitive functions, accelerated the development of conditioned reflexes, and ensured the long-term preservation of memory. The use of the R. uniflorum root extract in mice with galactoseinduced aging contributed to the prevention of mitochondrial degeneration, increased the level of succinate dehydrogenase and superoxide dismutase in brain tissues, and decreased the level of MDA, monoamine oxidase, and lactate dehydrogenase activity [68]. Finally, it led to a decrease in the concentration of lipoperoxides and lipofuscin in brain tissues, positively affecting the learning and memory processes [69]. The leaf extract of R. uniflorum Biomolecules 2022, 12, 1720 23 of 28 (50-200 mg/kg) resulted in the adaptation of animals to unfamiliar conditions, an increase in orienting-exploratory activity, and the formation of a conditioned reflex with positive reinforcement, which has generally indicated a pronounced anti-anxiety effect [70]. After 30 min hypobaric hypoxia and 3 h reoxygenation, the use of R. uniflorum leaf extract (100 mg/kg) limited the formation of pyknotic neurons, sharply hypochromic neurons, and "shadow cells" in the cortex of cerebral hemispheres, indicating a neuroprotective effect during hypoxia/reoxygenation [71].

Stress-Protective Activity
In models of 18 h immobilization stress and psycho-emotional stress, it was found that extracts from the herb and roots of R. uniflorum (100 mg/kg) had a pronounced stressprotective effect, reducing the involution of immunocompetent organs (adrenals, thymus, spleen), delaying the development of deep destruction of the gastric mucosa, reducing the level of MDA, and increasing the concentration of reduced glutathione and the activity of catalase and superoxide dismutase [67]. After administration of R. uniflorum extracts, there was a decrease in blood concentration of adrenaline, norepinephrine, adrenocorticotropic hormone, corticosterone, and aldosterone [72]. The positive effect of extracts is due to the limitation of hyperactivation of sympathetic-adrenal and hypothalamic-pituitary-adrenal stress-realizing systems.

Actoprotective and Anabolic Activity
Administration of the R. uniflorum root extract (100 mg/kg) led to an increase in overall physical endurance in experimental animals, which affected the increase in working capacity, improved energy supply of working tissues, and increased ATP content in skeletal muscles [68]. A decrease in the severity of metabolic acidosis and the intensity of free radical processes also prolonged the possibility of performing physical work. An increase in the animal body weight, up to 16% compared with the control after application of the R. uniflorum root extract (100 mg/kg), occurred owing to an increase in the skeletal muscle mass [67]. An increase in the muscle protein synthesis and DNA and RNA concentrations was observed without a noticeable effect on blood glucose and somatotropic hormone levels, which indicated an anabolic effect of the R. uniflorum root extract.

Antihypoxic and Anti-Ischemic Activity
Dry extracts of R. uniflorum (50-200 mg/kg) demonstrated pronounced antihypoxic effect, while the effectiveness of root extract was higher in models of hypercapnic and hemic hypoxia, and the herb extract was more effective in histotoxic hypoxia [67]. Intragastric administration of R. uniflorum leaf extract (50-200 mg/kg, 14 days) before bilateral carotid artery occlusion led to a decrease in the total mortality of experimental animals, a decrease in neurological deficit, and a decrease in the severity of cerebral edema [73].

Hepatoprotective Activity
Root ethanol extract of R. uniflorum increased cell viability at H 2 O 2 -induced liver cell damage in in vitro models [74,75]. Pre-treatment of mice with an aqueous R. uniflorum root extract attenuated CCl 4 -induced liver damage, decreased the activity of alanine aminotransferase and aspartate aminotransferase in serum, reduced the concentration of hydroperoxides and malondialdehyde in the liver, increased the level of catalase, glutathione peroxidase, and superoxide dismutase, and reduced glutathione [76]. A decrease in the activity of Na + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase in liver mitochondria and a decrease in the hepatocyte DNA damage indicated a pronounced hepatoprotective effect of the extract on the function of the damaged organ.

Anti-Aterosclerotic and Hypolypidemic Activity
In a hypercholesterol diet model in birds, the R. uniflorum root extract was found to reduce the incidence and severity of atherosclerotic vascular lesions while protecting the ultra-microstructural integrity of cells [77]. The ethanol R. uniflorum root extract reduced the levels of triglycerides and the low-and high-density lipoproteins in the blood of mice with experimental hyperlipidemia and prevented lipid accumulation in hepatocytes [78].

Other Activities
Peroxisome activator-activated receptors (PPARs) are a group of nuclear receptors that play an essential role in the regulation of metabolism. Gamma-type receptors (PPARγ) are expressed in all tissues of the body and are a therapeutic target for the treatment of obesity, diabetes, cancer, and other diseases. The R. uniflorum root extract, as well as its component 7-chloroarctinone b (89), inhibited the rosiglitazone-induced transcriptional activity of PPARγ [79]. Plasmon resonance indicated that 89 binds to PPARγ receptors, blocking the ability of PPARγ agonists to interact with the ligand-binding domains of the receptors (PPARγ-LBD). The ability of 89 to inhibit hormonal and rosiglitazone-induced adipocyte differentiation was confirmed using the Gal4/UAS model and two hybrid yeast methods, indicating its potential efficacy for the treatment of metabolic diseases.

Toxicity
The study of acute toxicity of R. uniflorum dry extracts from the herb and roots at doses of 3.5-10 g/kg demonstrated no death of animals after intragastric administration [83]. After intraperitoneal administration, the LD 50 values were 5.8 (herb extract) and 9.5 g/kg (root extract). Long-term administration of the extracts had no negative effect on the morpho-functional parameters of the central nervous, cardiovascular, and urinary systems, organs of the gastrointestinal tract, metabolism, peripheral blood parameters, and the hemostasis system of laboratory animals [83]. Application of the extract as single injection at doses of 100 and 1000 mg/kg did not have local irritating or mutagenic effects. These results indicate that R. uniflorum extracts belong to the practically non-toxic group.

Conclusions
This review summarizes the scientific literature concerning the chemical composition, methods of analysis, and biological activity of traditional medicine Rhaponticum uniflorum. The presented data indicate a good degree of knowledge of the metabolites of the roots and herb of R. uniflorum. Of particular interest are the anti-inflammatory components of R. uniflorum, such as sesquiterpenes [84], ecdysteroids [85], triterpenes [86], thiophenes [87], and flavonoids [88]. Owing to the confirmed presence of these compounds in the plant, we understand its ethnopharmacological use as an anti-inflammatory agent. Despite promising information on the chemical and pharmacological composition of R. uniflorum and its extracts, biological studies of individual compounds are still insufficient. We note a lack of studies on metabolites (e.g., sesquiterpenes, triterpenes, and thiophenes) in aboveground organs. The composition of phenolic compounds of the whole plant has not been fully studied to date. Carbohydrates remain an unexplored class of compounds for R. uniflorum and the genus Rhaponticum in general. It is necessary to expand our knowledge about the organ-specific distribution of substances in the plant, as well as the influence of the environmental conditions of R. uniflorum growth on its chemical profile. Owing to the current level of scientific interest in R. uniflorum and its extracts, new data on the pharmacological efficacy of pure compounds in various pathologies should be expected in the near future. Therefore, we believe that this review is a starting point for future research on the health benefits of consuming products containing R. uniflorum, especially modern dosage forms (e.g., nanoformulations), which will contribute to a wider inclusion of this natural component in new pharmacological products.

Patents
Available patent information suggests that R. uniflorum extracts were registered as components of complex antihypoxic and adaptogenic remedy [89], cosmetic composition with a purpose of lipometabolism promoter [90], soy sauce [91], and granulated insecticide [92], as well as an independent medicine with stress-protective [93] or anxiolytic activity [94].