# Simultaneous Determination of 78 Compounds ofRhodiola rosea Extract by Supercritical CO2-Extraction and HPLC-ESI-MS/MS Spectrometry

Authors: Alexander M. Zakharenko; Mayya P. Razgonova; Konstantin S. Pikula; Kirill S. Golokhvast
Journal: Biochemistry Research International
 (2021)

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## Abstract

The plantRhodiola rosea L. of family Crassulaceae was extracted using the supercritical CO2-extraction method. Several experimental conditions were investigated in the pressure range of 200–500 bar, with the used volume of cosolvent ethanol in the amount of 1% in the liquid phase at a temperature in the range of 31–70°C. The most effective extraction conditions are pressure 350 bar and temperature 60°C. The extracts were analyzed by HPLC with MS/MS identification. 78 target analytes were isolated from Rhodiola rosea (Russia) using a series of column chromatography and mass spectrometry experiments. The results of the analysis showed a spectrum of the main active ingredients Rh. rosea: salidroside, rhodiolosides (B and C), rhodiosin, luteolin, catechin, quercetin, quercitrin, herbacetin, sacranoside A, vimalin, and others. In addition to the reported metabolites, 29 metabolites were newly annotated in Rh. rosea. There were flavonols: dihydroquercetin, acacetin, mearnsetin, and taxifolin-O-pentoside; flavones: apigenin-O-hexoside derivative, tricetin trimethyl ether 7-O-hexosyl-hexoside, tricin 7-O-glucoronyl-O-hexoside, tricin O-pentoside, and tricin-O-dihexoside; flavanones: eriodictyol-7-O-glucoside; flavan-3-ols: gallocatechin, hydroxycinnamic acid caffeoylmalic acid, and di-O-caffeoylquinic acid; coumarins: esculetin; esculin: fraxin; and lignans: hinokinin, pinoresinol, L-ascorbic acid, glucaric acid, palmitic acid, and linolenic acid. The results of supercritical CO2-extraction from roots and rhizomes of Rh. rosea, in particular, indicate that the extract contained all biologically active components of the plant, as well as inert mixtures of extracted compositions.


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## Body

## 1. Introduction

The plantRhodiola rosea L. of family Crassulaceae is widely used in traditional medicine and traditional medical systems (Tibetan, Chinese, and Korean). Rhizomes and plant roots are mainly used for the preparation of medicinal products [1, 2].The plant has an established popular name “golden root.” The name is determined not only by the color of the rhizome but also by its high price. The main medicinal raw material ofRh. rosea is rhizomes with roots, which are harvested from the end of flowering until the completion of the plant’s vegetation. Rh. rosea grows in the mountains in the north of the European part of Russia, Siberia, the Urals, the mountains of Altai, the Tien Shan and the Far East, the mountains of Western Europe, Scandinavia, Mongolia, and on the spurs of the Himalayas. Brush wood of Rh. rosea is located at an altitude of 1700–2200 m above sea level. Since about the 80s, Rh. rosea has been one of the main adaptogenic plants and competes with such well-known adaptogens such as Panax ginseng and Eleutherococcus. Adaptogens are a pharmacological group of drugs of natural or synthetic origin, which can increase the body’s resistance to various adverse environmental conditions [3–5].Rh. rosea roots and rhizomes contain organic acids (citric, malic, oxalic, and succinic acid) and sugars (fructose, sucrose, glucose, sedoheptulose, essential oil, phenolic compounds, monoterpenes, sterols, cinnamon alcohol, and manganese) [6–8].The active biologically active substances ofRh. rosea are tyrosol, salidroside, caffeic acid, gallic acid, methyl gallate, flavonoids (astragalin, kaempferol, rhodionine, rhodiosin, rhodiolinin, and rhodiolgin), and tannins of the pyrogallol group (Table 1). Monoterpenes are represented by rosiridol and its glycoside rosiridin, and sterols are represented by β-sitosterol and daucosterol. Cinnamon glycosides—rosin, rosarin, and rosavin—were isolated from the roots of Rh. rosea [9].Table 1
Some of the main active compounds ofRh. rosea.
S. no.CompoundsStructure1Chlorogenic acid: C16H18O92Rosiridin: C16H28O73Rosavin: C20H28O104Salidroside: C14H20O75Rhodiolin (rhodiolinin): C25H20O10Information on the content of salidroside and rosavin inRh. rosea is numerous and contradictory [10, 11]; Zang et al., 2019). Researchers still have not come to a consensus on the localization and activity of specialized biosyntheses, the nature of seasonal changes in glycoside content, and the variability in the accumulation of these substances in wild and cultivated plants [12–14].Detailed comparative studies of the content of salidroside and rosavin in the organs of wild-growing and cultivated plants were carried out. Performed using a unified determination method showed the presence of glycosides only in the roots and caudex. The presence of rosavin and salidroside in the aerial organs (stems, leaves, inflorescences, and seeds) was not detected in any case [15].Plants from different places of growth differed significantly in the accumulation of individual glycosides. The content of salidroside in the plant caudex varied from 9 to 20 mg/g dry weight. The largest accumulation of this glycoside was characterized by plants growing on rocks on the coast of the Barents Sea (Norway), as well as Ural plants growing on outcrops of bedrock with an insignificant soil layer. The minimum salidroside content was found in Altai plants. The highest content of rosavin (32 mg/g) was found in the caudex of plants of the subalpine ecotope in the Polar Urals, the lowest (10–12 mg/g) being in plants growing on the islands and the coast of the Barents Sea. Cultivated plants were not inferior for accumulation of rosavin to wild plants.Differences in the accumulation of glycosides by plants of various ecotopes were revealed. So, in the Subpolar Urals, in the caudex of plants growing in faults and on ledges of rocks, more salidroside accumulates, but these plants were characterized by a low content of rosavin, 1.5–2 times less than in plants of the subalpine ecotope [15].Cinnamic glycosides, and in particular rosavin, are believed to be the hallmark of the chemotaxonomic trait ofRh. rosea [16, 17]. Recently, however, literature has reported that this glycoside is present in other species of the genus Rhodiola L. The results confirmed the presence of rosavin in the caudex of Rh. iremelica Boriss. The concentration of salidroside and rosavin in the plant caudex was 7.1 ± 2.4 and 15.3 ± 2.9 mg/g, respectively. In the underground part of Rh. quadrifida (Pall.) Fisch. et Mey, rosavin was not detected, and the content of salidroside was about 10 mg/g dry weight [15].In official medical practice,Rh. rosea root extract is intended for oral administration as a tonic and immunomodulating therapeutic agent. In the study of alcoholic extracts of Rh. rosea, their hepatoprotective, nootropic, cardioprotective, and antiarrhythmic properties were clearly demonstrated [18–20].Cinnamic glycosides, also called cinnamyl glycosides and salidroside, are the main carriers of the biological activity ofRh. rosea, causing a positive pharmacological effect. With the presence of rosavin, rosin, and rosarin, many researchers attribute the increased biological activity of extracts of Rh. rosea, compared with drugs from other species of Rhodiola. Studies have shown the stimulating effect of drugs on the central nervous system. Of great interest is the ability of Rh. rosea to increase the body’s resistance to the effects of various stress factors [21, 22]. Rh. rosea extract has immune stimulating, hepatoprotective, and antimicrobial effects [23, 24]. Studies have also been conducted on the antitumor effect of Rh. rosea extract [25–27].This study considers the effectiveness of supercritical CO2-extraction of biologically active substances from roots and rhizomes of Rh. rosea. Previously, the authors of this article successfully used supercritical CO2 extraction to obtain biologically active substances from plants of the Far Eastern taiga Panax ginseng, Rhododendron adamsii, Schisandra chinensis, and sea cucumber which are extremely popular in traditional medicine of Southeast Asia [28, 29].Supercritical fluid extraction (SFE) has been used since 1960s to analyze food and pharmaceutical products, isolate biologically active substances, and determine lipid levels in food and levels of toxic substances. In addition, the products do not have residues of organic solvents, which occur with conventional extraction methods, and solvents can be toxic, for example, in the case of methanol andn-hexane. High selectivity, easy solvent removal from the final product, and the use of moderate temperatures in the extraction process are the main attractive factors of SFE, leading to a significant increase in research for use in the food and pharmaceutical sectors [30, 31].In Sweden, an article was published in 2009 that examined the extraction of rosavin from the roots and rhizomes ofRh. rosea using supercritical CO2-extraction. In this case, water was selected as a modifier of supercritical extraction, which gave a synergistic effect on the extraction yield of rosavin [32]. In China, researchers used supercritical CO2-extraction with ethanol modifier [33]. The purpose of this study was to extract the maximum amount of salidroside from the roots of Rh. rosea. The extraction conditions were chosen so that the yield of salidroside during supercritical extraction was much higher than the yield of the product when using classical extraction using a Soxhlet apparatus.The results of SC-CO2-extraction of from roots and rhizomes of Rh. rosea, in particular, indicate that when using this technology, the extract contained all biologically active components of the plant, as well as inert mixtures of extracted compositions.


## 2. Experimental

### 2.1. Materials

Ground, dried root ofRh. rosea was obtained from the area near Lake Baikal, Russia. All samples were morphologically authenticated according to the current standard of Russian Pharmacopeia [34]. The volume weighted mean diameter of the powder was found as 550 μm, as determined by dynamic light scattering (Hydro 2000MU Malvern Instruments Ltd.).


### 2.2. Chemicals and Reagents

HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), and MS-grade formic acid was purchased from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was prepared from Siemens Ultra-Clear water purification system (Siemens Water Technologies, Germany), and all other chemicals were analytical grade.


### 2.3. Supercritical Fluid Extraction

A supercritical fluid extraction system was Thar SFE-500F-2-FMC50 (Thar Technology Inc., Pittsburgh, PA, USA) which is used in supercritical extraction. CO2 was compressed to the required pressure using a supercritical extraction compressor (Thar SFC, USA). A hot casing string heated the extraction vessel; the temperature was regulated by a thermostat (±1°C). A metering valve controlled the pressure. Shredded Rhodiola roots (50 g) were wrapped in a filter paper, charged to a one-liter extractor, and extracted with supercritical CO2 compressed to a supercritical state at a liquid flow rate of 250 g/min. Seven SFE extracts were obtained under different pressure conditions (100–400 bar) and temperatures (31–70°C). Ethanol served as the cosolvent in all cases. The extracts were collected in a separator. The pressure and temperature of the supercritical CO2 were optimized experimentally to achieve the maximum yield of the product during extraction.


### 2.4. Liquid Chromatography

HPLC was performed using Shimadzu LC-20 Prominence HPLC (Shimadzu, Japan), equipped with an UV-sensor and a Shodex ODP-40 4E reverse phase column to perform the separation of multicomponent mixtures. The gradient elution program was as follows: 0.01–4 min, 100% A; 4–60 min, 100–25% A; and 60–75 min, 25–0% A; control washing 75–120 min 0% A. The entire HPLC analysis was done with a DAD detector at wavelengths of 230ηm and 330 ηm; the temperature corresponded to 17°C. The injection volume was 1 ml.


### 2.5. Mass Spectrometry

MS analysis was performed on an ion trap amaZon SL (Bruker Daltoniks, Germany) equipped with an ESI source in the negative ion mode. The optimized parameters were obtained as follows: ionization source temperature, 70°C; gas flow, 4l/min; nebulizer gas (atomizer), 7.3 psi; capillary voltage, 4500 V; end plate bend voltage, 1500 V; fragmentary, 280 V; and collision energy, 60 eV. An ion trap was used in the scan rangem/z 100–1.700 for MS and MS/MS. The capture rate was one spectrum/s for MS and two spectra/s for MS/MS. Data collection was controlled by Windows software for Bruker Daltoniks. All experiments were repeated three times. A two-stage ion separation mode (MS/MS mode) was implemented.


## 2.1. Materials

Ground, dried root ofRh. rosea was obtained from the area near Lake Baikal, Russia. All samples were morphologically authenticated according to the current standard of Russian Pharmacopeia [34]. The volume weighted mean diameter of the powder was found as 550 μm, as determined by dynamic light scattering (Hydro 2000MU Malvern Instruments Ltd.).


## 2.2. Chemicals and Reagents

HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), and MS-grade formic acid was purchased from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was prepared from Siemens Ultra-Clear water purification system (Siemens Water Technologies, Germany), and all other chemicals were analytical grade.


## 2.3. Supercritical Fluid Extraction

A supercritical fluid extraction system was Thar SFE-500F-2-FMC50 (Thar Technology Inc., Pittsburgh, PA, USA) which is used in supercritical extraction. CO2 was compressed to the required pressure using a supercritical extraction compressor (Thar SFC, USA). A hot casing string heated the extraction vessel; the temperature was regulated by a thermostat (±1°C). A metering valve controlled the pressure. Shredded Rhodiola roots (50 g) were wrapped in a filter paper, charged to a one-liter extractor, and extracted with supercritical CO2 compressed to a supercritical state at a liquid flow rate of 250 g/min. Seven SFE extracts were obtained under different pressure conditions (100–400 bar) and temperatures (31–70°C). Ethanol served as the cosolvent in all cases. The extracts were collected in a separator. The pressure and temperature of the supercritical CO2 were optimized experimentally to achieve the maximum yield of the product during extraction.


## 2.4. Liquid Chromatography

HPLC was performed using Shimadzu LC-20 Prominence HPLC (Shimadzu, Japan), equipped with an UV-sensor and a Shodex ODP-40 4E reverse phase column to perform the separation of multicomponent mixtures. The gradient elution program was as follows: 0.01–4 min, 100% A; 4–60 min, 100–25% A; and 60–75 min, 25–0% A; control washing 75–120 min 0% A. The entire HPLC analysis was done with a DAD detector at wavelengths of 230ηm and 330 ηm; the temperature corresponded to 17°C. The injection volume was 1 ml.


## 2.5. Mass Spectrometry

MS analysis was performed on an ion trap amaZon SL (Bruker Daltoniks, Germany) equipped with an ESI source in the negative ion mode. The optimized parameters were obtained as follows: ionization source temperature, 70°C; gas flow, 4l/min; nebulizer gas (atomizer), 7.3 psi; capillary voltage, 4500 V; end plate bend voltage, 1500 V; fragmentary, 280 V; and collision energy, 60 eV. An ion trap was used in the scan rangem/z 100–1.700 for MS and MS/MS. The capture rate was one spectrum/s for MS and two spectra/s for MS/MS. Data collection was controlled by Windows software for Bruker Daltoniks. All experiments were repeated three times. A two-stage ion separation mode (MS/MS mode) was implemented.


## 3. Results and Discussion

Several experimental conditions were investigated in the pressure range 200–500 bar, with the used volume of cosolvent ethanol in the amount of 1% in the liquid phase at a temperature ranging 31–70°C. Ethanol was used as the modifier due to its high solubility in CO2 and high polarity and ability to disturb solute-plant matrix bonding. As a result of using a wide range of pressures and temperatures empirically, the most efficient extraction conditions were found for extracting target analytes from the Rh. rosea roots. The most effective extraction conditions are pressure 350 bar and temperature 60°C (Figure 1).Figure 1
The effect of pressure and temperature on extraction efficiency of total yield of biologically active compounds (mg/g of extractable substance).Obtaining chemical profiles is an extremely important result in the biological analysis system. In this work, we used the HPLC-ESI-MS/MS method with additional ionization and analysis of fragmented ions. High accuracy mass spectrometric data were recorded on an ion trap amaZon SL (Bruker Daltoniks) equipped with an ESI source in the negative ion mode. The two-stage ion separation mode (MS/MS mode) was implemented.Figure2 shows the distribution density of the analyzed chemical profiles in the ion chromatogram of the Rh. rosea supercritical CO2-extract, realized by mass spectrometry in the two-stage ion separation mode (MS/MS mode).Figure 2
Distribution density of the analyzed chemical profiles in the ion chromatogram ofRh. rosea supercritical CO2-extract.Visually, a rather high-density distribution of the target analytes in the analyzed extract was observed. All the chemical profiles of the samples were obtained by the HPLC-ESI-MS/MS method. A total of 300 peaks were detected in the chromatogram. By comparing them/z values, the RT and the fragmentation patterns with the MS2 spectral data taken from the literature [2, 17, 35–50] or to search the data bases (MS2T, MassBank, HMDB). 78 metabolites were putatively identified as phenols, aromatic compounds, phenyl alkanoids, flavonoids, monoterpenoids, acyclic alcohol glycosides, anthocyanins etc. In addition to the reported metabolites, a number of metabolites were newly annotated in Rh. rosea.A unifying system table consists of the molecular masses of the target analytes isolated from the supercritical CO2-extract of Rh. rosea for ease of identification (Table 2).Table 2
Polyphenols and other substances identified from the SC-CO2 extracts of Rh. rosea.
No.Compound groupIdentificationFormulaCalculated massObserved mass [M-H]−Observed mass [M+H]+Observed mass [M+Na]+MS/MS stage 1 fragmentationMS/MS stage 2 fragmentationReferencesPolyphenols1FlavonolAcacetin [linarigenin; buddleoflavonol]C16H12O5284.2635—285—240212; 183; 165Mentha [51]; Ocimum [41]2FlavonolKaempferolC15H10O6286.2363—287.11—269; 189; 133213; 119Rhodiola sachalinensis [52, 53]; Rhodiola crenulata [35, 54]; Rhodiola sacra [55]; Impatiens glandulifera Royle [56]3FlavonolQuercetinC15H10O7302.2357—303.09—123; 147; 201; 233; 256135; 175; 201Rhodiola rosea [57]; Rhodiola dumulosa [58]; Rhodiola crenulata [35, 59]; Impatiens glandulifera Royle [56]; Eucalyptus [42]; Triticum [43]4FlavonolHerbacetin (3, 5, 7, 8-tetrahydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one)C15H10O7302.2357—303.08—285212; 268Rhodiola rosea [3, 60–62]; Rhodiola crenulata [35]; Ocimum [41]5FlavonolDihydroquercetin (taxifolin; taxifoliol)C15H12O7304.2516—305.1—287; 269; 249; 231; 217; 147269; 227; 213; 173; 161Larix dahurica [63]; Eucalyptus [42]; Vitis vinifera [37]6FlavonolHerbacetin 8-methyl etherC16H12O7316.2623—317.06—298; 183; 112279; 228; 129Rhodiola crenulata [35]; Rhodiola dumulosa [64]7FlavonolGossypetin (articulatidin; equisporol; 8-methoxy-hydroxyquercetin)C15H10O8318.2351—319.03—300.97228; 166; 110Rhodiola rosea [3, 62]8FlavonolMearnsetinC16H12O8332.2617—333.1—317; 292; 195221; 183Eucalyptus [42]9FlavonolRhodalin (herbacetin-8-O-beta-D-xylopyranoside)C20H18O11434.3503—434.96—389.90; 266.93308; 345; 267; 167Rhodiola rosea [17]10FlavonolTaxifolin-O-pentosideC20H20O11436.371—436.99—391; 285; 177352; 269; 173Vitis vinifera [37]11FlavonolQuercitrin (quercetin 3-L-rhamnoside; quercetrin)C21H20O11448.3769—448.90—302.95169; 303Lotus japonicus [65]; Rhodiola rosea [62]; Rhodiola crenulata [35, 59]12FlavonolRhodiolatuntosideC21H20O11448.3769—450.92—332.90200.89; 154.87Rhodiola sachalinensis [66]; Rhodiola crenulata [67]13FlavonolRhodiolinin (rhodiolin)C25H20O10480.4203—480.95—401; 313; 233; 173357; 313; 269; 233; 145Rhodiola rosea [2, 16]; Rhodiola sachalinensis [52, 53, 68]; Rhodiola crenulata [69]14Flavonole glycosideKaempferol-3-xylosyl-glycosideC26H28O15580.4915—581.09—331; 509; 469; 375; 243330.89; 287.99; 141.74Rhodiola rosea [61]15Flavonole glycosideRhodiosinC27H30O16610.5175—610.82—303; 449169Rhodiola rosea [2, 16, 70, 71]; Rhodiola sachalinensis [52, 68]; Rhodiola crenulata [69]16Flavonole glycosideRhodiolgidinC27H30O17626.5179—627.30—344.78344.7Rhodiola rosea [3, 17]; Rhodiola crenulata [35]17Flavan-3-olCatechinC15H14O6290.2681—291.97—250227Rhodiola rosea [50]; Rhodiola crenulata [35]; strawberry, cherimoya [36]; pear [45]18Flavan-3-olEpicatechin ((2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-chromanetriol)C15H14O6290.2681—291.1—261; 273; 217; 173; 163243; 191; 173; 143Rhodiola rosea [50]; Rhodiola crenulata [35]; Rhodiola kirilowii [72]19Flavan-3-olGallocatechin ((+)-gallocatechin)C15H14O7306.27305.06——179; 168; 261124Red wine [73]; Licania ridigna [74]20Flavan-3-ol(-)-Epicatechin gallateC22H18O10442.3723—443.01—363.12319.16Rhodiola rosea [39]; Rhodiola crenulata [35, 75]; Rhodiola kirilowii [50, 76]21FlavanoneEriodictyol-7-O-glucoside (pyracanthoside; miscanthoside)C21H22O11450.3928—451.00—333; 433; 155288; 201Impatiens glandulifera Royle [56]22FlavoneLuteolinC15H10O6286.2363285.02——241; 168; 124124.02Rhodiola crenulata [35, 54]; Rhodiola kirilowii [72]; Rhodiola sachalinensis [53, 77]23FlavoneTricinC17H14O7330.2889329.18——299; 311; 229; 171211.04; 125.14Triticum aestivum L. [77, 78]; Rhodiola rosea [61, 79]; Rhodiola sacra [55]; Rhodiola sachalinensis [53]; Rhodiola crenulata [59]24FlavoneLuteolin-7-O-α-L-rhamnosideC21H20O10432.3775430.99——284.93283.93Rhodiola crenulata [35];25FlavoneTricin 7-O-glucosideC23H24O12492.4295—493.11—401; 292; 201383; 329; 280; 156Rhodiola rosea [61, 70, 79]; Rhodiola crenulata [59]26FlavoneApigenin-O-hexoside derivativeC26H25O12529.4695—531.08—433; 485; 243; 177399; 310Strawberry [36]27FlavoneTricetin trimethyl ether, 7-O-hexoside malonylatedC27H28O15592.5022591.23——533; 437; 323197.01Triticum aestivum L. [77]28FlavoneTricin, 7-O-glucoronyl-O-hexosideC29H32O18668.5536—669.13—419; 375; 271375; 243; 171Triticum aestivum L. [77]29FlavoneTricin trimethyl ether, 7-O-hexosyl-hexosideC30H36O17668.5966—669.01—419; 557; 331; 287375; 331; 215Triticum aestivum L. [77]30FlavoneTricin, O-pentoside; O-dihexosideC35H44O21800.7113—801.24—409; 655; 509; 252—Triticum aestivum L. [77]31Hydroxycinammic acidFerulic acidC10H10O4194.184—195.07—176.8—Rhodiola crenulata [35]; Triticum [43]32Hydroxycinammic acidCaffeoylmalic acidC13H12O8296.2296—297.09—279; 211; 163265; 163; 135Strawberry [36]33Cinnamate ester4-O-p-Coumaroylquinic acidC16H18O8338.3098—338.94—189; 151—Pear [45]34Cinnamic alcohol glycoside)Rosin (trans-cinnamyl O-beta-D-glycopyranoside)C15H20O6296.3157—297.06—255; 179; 115215; 110Rhodiola rosea [16, 49, 80]; Rhodiola crenulata [35]; Rhodiola sachalinensis [53]35Cinnamic alcohol glycosideTriandrinC15H20O7312.3151—313.21—268.14240; 211; 193Rhodiola crenulata [35, 54]; Rhodiola rosea [10, 81]36Cinnamic alcohol glycosideSachaliside 1C15H20O7312.3151311.13——309.08; 182.96247.08; 119.01Rhodiola rosea [9]37Cinnamic alcohol glycosidep-Hydroxyphenacyl-β-D-glucopyranosideC14H18O8314.2879—314.97—294; 163—Rhodiola crenulata [35, 82];38Cinnamic alcohol glycoside(2E)-3-(4-methoxyphenyl)-2-propen-1-yl-beta-D-glycopyranosideC16H22O7326.3417325.09——182.99119.09Rhodiola rosea [9]39Cinnamic alcohol glycosideConiferinC16H22O8342.3411—343.01—240; 301; 129240; 183Rhodiola crenulata [35, 54]40Phenylpropanoid (cinnamicacid derivative glycoside)Chlorogenic acid (3-O-caffeoylquinic acid)C16H18O9354.3087—355.04—335; 285; 203200.0Rhodiola rosea [2]; Eucalyptus [42]; Triticum [43];41Cinnamic alcohol glycosideRosavin (trans-cinnamil O-(6′-O-alpha-L-arabinopyranosyl-beta-D-glycopyranoside)C20H28O10428.4303——451.00333; 155; 201200.94Rhodiola rosea [16, 49, 83]; Rhodiola crenulata [84]; Rhodiola sachalinensis [53]; Rhodiola quadrifida [2, 85]42Cinnamic alcohol glycosideRosarin (trans-cinnamyl O-(6′-O-alpha-L arabinofuranosyl-beta-D-glycopyranoside)C20H28O10428.4303—429.01—285; 199384; 328; 230; 159Rhodiola rosea [9, 16, 49, 83]; Rhodiola sachalinensis [53]43Phenylpropanoid (cinnamic acid derivative)Di-O-caffeoylquinic acidC25H24O12516.4509—516.86—352; 431; 276200; 135Pear [45]44Gallic acid derivative6-O-galloyl-salidrosideC21H24O11452.4087—453.09—435; 209; 336226; 336; 417Rhodiola crenulata [35, 54]; Rhodiola rosea [39]45Gallic acid derivative1,2,6-Tri-O-galloyl-beta-D-glucosideC27H24O18636.4687—637.28—507; 566; 620; 488; 366; 189—Rhodiola rosea [39]46AnthocyanidinPelargonidin-3-glucoside (callistephin)C21H21ClO10468.8444—469.88—357.05247.00Triticum [43]47AnthocyanidinPelargonidin (3-O-(6-O-malonyl-beta-D-glucoside))C24H23O13519.4388—520.10—433; 184307; 163Gentiana lutea [86]; wheat [87]48ProanthocyanidinProanthocyanidin B1 (procyanidin B1; procyanidin dimer B1)C30H26O12578.5202577.21579.07—197; 254; 351; 393; 407; 421196.94; 133.04; 182.93Pear [45]; Eucalyptus [42]49AnthocyanidinCyanidin-3-(3″,6″-dimalonylglucoside)C27H24O17620.4773—621.17—619; 432; 264601; 518; 419Wheat [87]50AnthocyanidinPelargonidin (3-O-(6-O-malonyl-beta-D-glucoside)-5-beta-D-glucosideC30H33O18681.5812—682.10—515.58; 353.14351; 295; 173Gentiana lutea [86]51CoumarinEsculetin (cichorigenin; esculetin)C9H6O4178.1415—179.02—147.01119.03Ledum palustre [38]; Vitis vinifera [37]52CoumarinEsculin (esculin; esculoside; polichrome)C15H16O9340.2821—340.91—133; 283; 322175; 133Dog plasma [38]; rat plasma [88]53Coumarin glucosideFraxin (Fraxetin-8-O-glucoside)C16H18O10370.3081—370.97—356; 193; 123207.02Dog plasma [38]; rat plasma [88]54LignanHinokininC20H18O6354.3533—355.01—337; 283; 203239; 133Triticum aestivum L. [89]; Bursera simaruba [90]55LignanPinoresinolC20H22O6358.3851—359.02—341; 187323; 187Triticum aestivum L. [78]; Eucommia cortex [47]56Aryl-beta-glycosideArbutinC12H16O7272.2512—273.17—217; 163161.09Strawberry, blueberry, pear [91]; pear [45]Others57Natural water-soluble vitaminL-ascorbic acidC6H8O6176.1241—176.98—145.00117.03Strawberry, lemon, papaya [36]58Aldaric acidGlucaric acid (D-glucaric acid)C6H10O8210.1388—211.01—192; 115129.05Cherimoya, papaya [36]59Monobasic saturated carboxylic acidPalmitic acid (hexadecanoic acid; palmitate)C16H32O2256.4241—257.02—237; 137221; 125Salviae [44]60Acyclic alcohol nitrile glycosideHeterodendrin ((2R)-2-(β-D-glucopyranosyloxy)-3-methylbutanenitrile)C11H19O6N261.2717—263.96—155; 228—Rhodiola crenulata [35]61Monobasic saturated carboxylic acidLinolenic acid (alpha-linolenic acid; linolenate)C18H30O2278.4296—279.1—261; 243; 187; 123173; 131Salviae [44]; rice [48]62Phenylethane glycosidePicein (ameliaroside; salicinerin; salinigrin; piceoside)C14H18O7298.2901—299—271; 211; 179254; 225; 197Rhodiola rose [9]; Rhodiola crenulata [82]63Phenylethane glycosideSalidroside (2-(4-hydroxyphenyl) ethylβ-D-glucopyranoside)C14H20O7300.3044—301.15—240; 201183; 110Rhodiola crenulata [35, 54]; Rhodiola rosea [1, 92, 93]; Rhodiola sachalinensis [53]; Rhodiola kirilowii [2]64Phenylethane glycosideIcariside D2C14H20O7300.3044—301.06—240; 201; 135183; 113Rhodiola rosea [39]; Rhodiola crenulata [54, 82]; Rhodiola sacra [55];65Acyclic alcohol glycosideCreoside IIC14H26O7306.352—307.99—199; 255—Rhodiola crenulata [35, 54]66Phenylethane glycosideViridosideC15H22O7314.331—315.04337.11319.13; 209.08151; 207; 262; 301Rhodiola viridula [94]; Rhodiola rosea [83]; Rhodiola crenulata [35]; Rhodiola sachalinensis [53]67Acyclic alcohol glycosideRosiridin (3,7-dimethylocta-2,6-diene-1,4-diol; 1-O-beta-D-glucopyranoside)C16H28O7332.3893—333.02—247; 175181.93Rhodiola crenulata [35]; Rhodiola rosea [2, 17, 49]; Rhodiola sachalinensis [95]68Acyclic alcohol glycosideRhodioloside AC16H28O8348.3887—349.02371.03271; 281; 305; 331; 257; 231; 219; 167; 141268; 256; 243; 229; 215; 193; 143Rhodiola rosea [1, 92]; Rhodiola crenulata [35]69Acyclic alcohol glycosideRhodioloside DC16H30O8350.4046—351.06—258; 220; 131257; 141Rhodiola rosea [1, 83, 92]; Rhodiola crenulata [35]70Tetracyclic diterpenoidGrayanotoxin IIC20H32O5352.4651—353.04—335; 282; 203315; 245; 113Grayanotoxins [96]72Benzidine glycosidePhenylmethyl (6-O-alpha-L-arabinopyranosyl-beta-D-glycopyranoside)C18H26O10402.3930—402.86—343; 283; 175283Rhodiola rosea [83]; Rhodiola sachalinensis [53]73Acyclic alcohol glycosideRhodiooctanosideC19H36O10424.4831—424.94—290.96173; 261Rhodiola crenulata [35, 54]; Rhodiola kirilowii [97]; Rhodiola sacra [98]74Phenylethane glycosideMongrhosideC20H30O11446.4456—446.65—243; 379; 311174.84Rhodiola rosea [83]75Acyclic alcohol glycosideCreoside VC21H38O10450.5204—473.15—471; 254; 401463.61Rhodiola crenulata [35];76Hydroxy acidUrsolic acidC30H48O3456.7003—457.17—412; 307368; 269Ocimum [41]; pear [45]77Acyclic alcohol glycosideRhodioloside EC21H38O11466.5198—467.95—399.94; 265; 332331.88Rhodiola rosea [1, 92]; Rhodiola crenulata [35, 54]; Rhodiola sachalinensis [13]; Rhodiola sacra [55]78Acyclic alcohol glycosideRhodioloside BC22H38O12494.5299493.22—517.97447; 220314.98Rhodiola rosea [1, 92]; Rhodiola crenulata [35]The CID spectrum (collision induced dissociation spectrum) in negative ion modes of Rhodioloside B fromRh. rosea is shown in Figure 3.Figure 3
CID spectrum of the rhodioloside B fromRh. rosea, m/z 493.05.The [M−H]− ion produced two fragments with m/z 447.00 and m/z 219.49 (Figure 3). The fragment ion with m/z 447.00 yields a daughter ion at m/z 314.98. The interpretation of the observed MS/MS spectra in comparison with those found in the literature was the main tool for putative identification of polyphenols. It was identified in the bibliography in extracts from Rh. rosea [50], from Rhodiola crenulata [35].The CID spectrum in the negative ion mode of luteolin-7-O-α-L-rhamnoside from Rh. rosea is shown in Figure 4.Figure 4
CID spectrum of luteolin-7-O-α-L-rhamnoside from Rh. rosea, m/z 430.99.The [M−H]− ion produced fragment with m/z 284.93 (Figure 5). The fragment ion with m/z 284.93 yields a daughter ion at m/z 283.93.Figure 5
CID spectrum of catechin fromRh. rosea, m/z 291.13.It was identified in the bibliography in extracts fromRhodiola crenulata [35]. The CID spectrum in the positive ion mode of catechin from Rh. rosea is shown in Figure 5. The [M+H]+ ion produced fragments with m/z 273.14 and m/z 217.09 (Figure 5). It was identified in the bibliography in extracts from Rh. rosea [50], from strawberry, cherimoya [36], and pear [45].We isolated 78 target analytes fromRhodiola rosea L. (Crassulaceae) using a series of column chromatography and mass spectrometry experiments. The structures were elucidated using the data of stepwise fragmentation of ions during MS/MS spectrometry and compared with spectroscopic data in the literature. It is accepted that glycosides of cinnamon alcohol, and in particular Rosavin, are a distinctive chemotaxonomic sign of Rh. rosea [17]. However, lately, information has appeared in the literature on the presence of this glycoside in other species of the genus Rhodiola L. [15]. Thus, we can summarize the research that the supercritical extraction of the roots of Rh. rosea gives an extract that is extremely effective in terms of the composition of biologically active substances, which should find further application in both pharmacological, medical, and perfumery developments. In this regard, research on the development of a technology for obtaining supercritical drugs from rhizomes and roots of Rh. rosea, containing a complex of biologically active substances of this plant, and the development of modern drugs on their basis, presented primarily in the form of solid dosage forms, are relevant.


## 4. Conclusions

TheRhodiola rosea L. family Crassulaceae contains a large number of polyphenolic compounds and other biologically active substances. In this work, we tried to conduct a comparative metabolomic study of biologically active substances of Rh. rosea obtained from the area near Lake Baikal, Russia. HPLC in combination with a Bruker Daltoniks ion trap (tandem mass spectrometry) was used to identify target analytes in extracts.The results showed the presence of 78 polyphenols and other compounds corresponding to theRhodiola rosea family Crassulaceae L. species. In addition to the reported metabolites, 29 metabolites were newly annotated in Rh. rosea. There were flavonols: dihydroquercetin, acacetin, mearnsetin, and taxifolin-O-pentoside; flavones: apigenin-O-hexoside derivative, tricetin trimethyl ether 7-O-hexosyl-hexoside, tricin 7-O-glucoronyl-O-hexoside, and tricin O-pentoside and O-dihexoside; flavanone: eriodictyol-7-O-glucoside; flavan-3-ol gallocatechin; hydroxycinnamic acid; caffeoylmalic acid; di-O-caffeoylquinic acid; coumarins: esculetin; esculin, fraxin; lignans: hinokinin, pinoresinol, L-ascorbic acid, glucaric acid, palmitic acid, linolenic acid, etc.The findings may support future research into the production of various pharmaceutical and dietary supplements containingRh. rosea extracts. A wide variety of biologically active compounds opens up rich opportunities for the creation of new drugs and biologically active additives based on extracts from family Crassulaceae.



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