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

The plant Rhodiola 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.

1. Introduction

The plant Rhodiola 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 of Rh. 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 of Rh. 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 of Rh. rosea.

S. no.	Compounds	Structure
1	Chlorogenic acid: C16H18O9
2	Rosiridin: C16H28O7
3	Rosavin: C20H28O10
4	Salidroside: C14H20O7
5	Rhodiolin (rhodiolinin): C25H20O10

Information on the content of salidroside and rosavin in Rh. 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 of Rh. 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 of Rh. 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 and n-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 of Rh. 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 of Rh. 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 230 ηm 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 range m/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.

Figure 2 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 of Rh. 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 the m/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 group	Identification	Formula	Calculated mass	Observed mass [M-H]−	Observed mass [M+H]+	Observed mass [M+Na]+	MS/MS stage 1 fragmentation	MS/MS stage 2 fragmentation	References
Polyphenols
1	Flavonol	Acacetin [linarigenin; buddleoflavonol]	C16H12O5	284.2635	—	285	—	240	212; 183; 165	Mentha [51]; Ocimum [41]
2	Flavonol	Kaempferol	C15H10O6	286.2363	—	287.11	—	269; 189; 133	213; 119	Rhodiola sachalinensis [52, 53]; Rhodiola crenulata [35, 54]; Rhodiola sacra [55]; Impatiens glandulifera Royle [56]
3	Flavonol	Quercetin	C15H10O7	302.2357	—	303.09	—	123; 147; 201; 233; 256	135; 175; 201	Rhodiola rosea [57]; Rhodiola dumulosa [58]; Rhodiola crenulata [35, 59]; Impatiens glandulifera Royle [56]; Eucalyptus [42]; Triticum [43]
4	Flavonol	Herbacetin (3, 5, 7, 8-tetrahydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one)	C15H10O7	302.2357	—	303.08	—	285	212; 268	Rhodiola rosea [3, 60–62]; Rhodiola crenulata [35]; Ocimum [41]
5	Flavonol	Dihydroquercetin (taxifolin; taxifoliol)	C15H12O7	304.2516	—	305.1	—	287; 269; 249; 231; 217; 147	269; 227; 213; 173; 161	Larix dahurica [63]; Eucalyptus [42]; Vitis vinifera [37]
6	Flavonol	Herbacetin 8-methyl ether	C16H12O7	316.2623	—	317.06	—	298; 183; 112	279; 228; 129	Rhodiola crenulata [35]; Rhodiola dumulosa [64]
7	Flavonol	Gossypetin (articulatidin; equisporol; 8-methoxy-hydroxyquercetin)	C15H10O8	318.2351	—	319.03	—	300.97	228; 166; 110	Rhodiola rosea [3, 62]
8	Flavonol	Mearnsetin	C16H12O8	332.2617	—	333.1	—	317; 292; 195	221; 183	Eucalyptus [42]
9	Flavonol	Rhodalin (herbacetin-8-O-beta-D-xylopyranoside)	C20H18O11	434.3503	—	434.96	—	389.90; 266.93	308; 345; 267; 167	Rhodiola rosea [17]
10	Flavonol	Taxifolin-O-pentoside	C20H20O11	436.371	—	436.99	—	391; 285; 177	352; 269; 173	Vitis vinifera [37]
11	Flavonol	Quercitrin (quercetin 3-L-rhamnoside; quercetrin)	C21H20O11	448.3769	—	448.90	—	302.95	169; 303	Lotus japonicus [65]; Rhodiola rosea [62]; Rhodiola crenulata [35, 59]
12	Flavonol	Rhodiolatuntoside	C21H20O11	448.3769	—	450.92	—	332.90	200.89; 154.87	Rhodiola sachalinensis [66]; Rhodiola crenulata [67]
13	Flavonol	Rhodiolinin (rhodiolin)	C25H20O10	480.4203	—	480.95	—	401; 313; 233; 173	357; 313; 269; 233; 145	Rhodiola rosea [2, 16]; Rhodiola sachalinensis [52, 53, 68]; Rhodiola crenulata [69]
14	Flavonole glycoside	Kaempferol-3-xylosyl-glycoside	C26H28O15	580.4915	—	581.09	—	331; 509; 469; 375; 243	330.89; 287.99; 141.74	Rhodiola rosea [61]
15	Flavonole glycoside	Rhodiosin	C27H30O16	610.5175	—	610.82	—	303; 449	169	Rhodiola rosea [2, 16, 70, 71]; Rhodiola sachalinensis [52, 68]; Rhodiola crenulata [69]
16	Flavonole glycoside	Rhodiolgidin	C27H30O17	626.5179	—	627.30	—	344.78	344.7	Rhodiola rosea [3, 17]; Rhodiola crenulata [35]
17	Flavan-3-ol	Catechin	C15H14O6	290.2681	—	291.97	—	250	227	Rhodiola rosea [50]; Rhodiola crenulata [35]; strawberry, cherimoya [36]; pear [45]
18	Flavan-3-ol	Epicatechin ((2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-chromanetriol)	C15H14O6	290.2681	—	291.1	—	261; 273; 217; 173; 163	243; 191; 173; 143	Rhodiola rosea [50]; Rhodiola crenulata [35]; Rhodiola kirilowii [72]
19	Flavan-3-ol	Gallocatechin ((+)-gallocatechin)	C15H14O7	306.27	305.06	—	—	179; 168; 261	124	Red wine [73]; Licania ridigna [74]
20	Flavan-3-ol	(-)-Epicatechin gallate	C22H18O10	442.3723	—	443.01	—	363.12	319.16	Rhodiola rosea [39]; Rhodiola crenulata [35, 75]; Rhodiola kirilowii [50, 76]
21	Flavanone	Eriodictyol-7-O-glucoside (pyracanthoside; miscanthoside)	C21H22O11	450.3928	—	451.00	—	333; 433; 155	288; 201	Impatiens glandulifera Royle [56]
22	Flavone	Luteolin	C15H10O6	286.2363	285.02	—	—	241; 168; 124	124.02	Rhodiola crenulata [35, 54]; Rhodiola kirilowii [72]; Rhodiola sachalinensis [53, 77]
23	Flavone	Tricin	C17H14O7	330.2889	329.18	—	—	299; 311; 229; 171	211.04; 125.14	Triticum aestivum L. [77, 78]; Rhodiola rosea [61, 79]; Rhodiola sacra [55]; Rhodiola sachalinensis [53]; Rhodiola crenulata [59]
24	Flavone	Luteolin-7-O-α-L-rhamnoside	C21H20O10	432.3775	430.99	—	—	284.93	283.93	Rhodiola crenulata [35];
25	Flavone	Tricin 7-O-glucoside	C23H24O12	492.4295	—	493.11	—	401; 292; 201	383; 329; 280; 156	Rhodiola rosea [61, 70, 79]; Rhodiola crenulata [59]
26	Flavone	Apigenin-O-hexoside derivative	C26H25O12	529.4695	—	531.08	—	433; 485; 243; 177	399; 310	Strawberry [36]
27	Flavone	Tricetin trimethyl ether, 7-O-hexoside malonylated	C27H28O15	592.5022	591.23	—	—	533; 437; 323	197.01	Triticum aestivum L. [77]
28	Flavone	Tricin, 7-O-glucoronyl-O-hexoside	C29H32O18	668.5536	—	669.13	—	419; 375; 271	375; 243; 171	Triticum aestivum L. [77]
29	Flavone	Tricin trimethyl ether, 7-O-hexosyl-hexoside	C30H36O17	668.5966	—	669.01	—	419; 557; 331; 287	375; 331; 215	Triticum aestivum L. [77]
30	Flavone	Tricin, O-pentoside; O-dihexoside	C35H44O21	800.7113	—	801.24	—	409; 655; 509; 252	—	Triticum aestivum L. [77]
31	Hydroxycinammic acid	Ferulic acid	C10H10O4	194.184	—	195.07	—	176.8	—	Rhodiola crenulata [35]; Triticum [43]
32	Hydroxycinammic acid	Caffeoylmalic acid	C13H12O8	296.2296	—	297.09	—	279; 211; 163	265; 163; 135	Strawberry [36]
33	Cinnamate ester	4-O-p-Coumaroylquinic acid	C16H18O8	338.3098	—	338.94	—	189; 151	—	Pear [45]
34	Cinnamic alcohol glycoside)	Rosin (trans-cinnamyl O-beta-D-glycopyranoside)	C15H20O6	296.3157	—	297.06	—	255; 179; 115	215; 110	Rhodiola rosea [16, 49, 80]; Rhodiola crenulata [35]; Rhodiola sachalinensis [53]
35	Cinnamic alcohol glycoside	Triandrin	C15H20O7	312.3151	—	313.21	—	268.14	240; 211; 193	Rhodiola crenulata [35, 54]; Rhodiola rosea [10, 81]
36	Cinnamic alcohol glycoside	Sachaliside 1	C15H20O7	312.3151	311.13	—	—	309.08; 182.96	247.08; 119.01	Rhodiola rosea [9]
37	Cinnamic alcohol glycoside	p-Hydroxyphenacyl-β-D-glucopyranoside	C14H18O8	314.2879	—	314.97	—	294; 163	—	Rhodiola crenulata [35, 82];
38	Cinnamic alcohol glycoside	(2E)-3-(4-methoxyphenyl)-2-propen-1-yl-beta-D-glycopyranoside	C16H22O7	326.3417	325.09	—	—	182.99	119.09	Rhodiola rosea [9]
39	Cinnamic alcohol glycoside	Coniferin	C16H22O8	342.3411	—	343.01	—	240; 301; 129	240; 183	Rhodiola crenulata [35, 54]
40	Phenylpropanoid (cinnamicacid derivative glycoside)	Chlorogenic acid (3-O-caffeoylquinic acid)	C16H18O9	354.3087	—	355.04	—	335; 285; 203	200.0	Rhodiola rosea [2]; Eucalyptus [42]; Triticum [43];
41	Cinnamic alcohol glycoside	Rosavin (trans-cinnamil O-(6′-O-alpha-L-arabinopyranosyl-beta-D-glycopyranoside)	C20H28O10	428.4303	—	—	451.00	333; 155; 201	200.94	Rhodiola rosea [16, 49, 83]; Rhodiola crenulata [84]; Rhodiola sachalinensis [53]; Rhodiola quadrifida [2, 85]
42	Cinnamic alcohol glycoside	Rosarin (trans-cinnamyl O-(6′-O-alpha-L arabinofuranosyl-beta-D-glycopyranoside)	C20H28O10	428.4303	—	429.01	—	285; 199	384; 328; 230; 159	Rhodiola rosea [9, 16, 49, 83]; Rhodiola sachalinensis [53]
43	Phenylpropanoid (cinnamic acid derivative)	Di-O-caffeoylquinic acid	C25H24O12	516.4509	—	516.86	—	352; 431; 276	200; 135	Pear [45]
44	Gallic acid derivative	6-O-galloyl-salidroside	C21H24O11	452.4087	—	453.09	—	435; 209; 336	226; 336; 417	Rhodiola crenulata [35, 54]; Rhodiola rosea [39]
45	Gallic acid derivative	1,2,6-Tri-O-galloyl-beta-D-glucoside	C27H24O18	636.4687	—	637.28	—	507; 566; 620; 488; 366; 189	—	Rhodiola rosea [39]
46	Anthocyanidin	Pelargonidin-3-glucoside (callistephin)	C21H21ClO10	468.8444	—	469.88	—	357.05	247.00	Triticum [43]
47	Anthocyanidin	Pelargonidin (3-O-(6-O-malonyl-beta-D-glucoside))	C24H23O13	519.4388	—	520.10	—	433; 184	307; 163	Gentiana lutea [86]; wheat [87]
48	Proanthocyanidin	Proanthocyanidin B1 (procyanidin B1; procyanidin dimer B1)	C30H26O12	578.5202	577.21	579.07	—	197; 254; 351; 393; 407; 421	196.94; 133.04; 182.93	Pear [45]; Eucalyptus [42]
49	Anthocyanidin	Cyanidin-3-(3″,6″-dimalonylglucoside)	C27H24O17	620.4773	—	621.17	—	619; 432; 264	601; 518; 419	Wheat [87]
50	Anthocyanidin	Pelargonidin (3-O-(6-O-malonyl-beta-D-glucoside)-5-beta-D-glucoside	C30H33O18	681.5812	—	682.10	—	515.58; 353.14	351; 295; 173	Gentiana lutea [86]
51	Coumarin	Esculetin (cichorigenin; esculetin)	C9H6O4	178.1415	—	179.02	—	147.01	119.03	Ledum palustre [38]; Vitis vinifera [37]
52	Coumarin	Esculin (esculin; esculoside; polichrome)	C15H16O9	340.2821	—	340.91	—	133; 283; 322	175; 133	Dog plasma [38]; rat plasma [88]
53	Coumarin glucoside	Fraxin (Fraxetin-8-O-glucoside)	C16H18O10	370.3081	—	370.97	—	356; 193; 123	207.02	Dog plasma [38]; rat plasma [88]
54	Lignan	Hinokinin	C20H18O6	354.3533	—	355.01	—	337; 283; 203	239; 133	Triticum aestivum L. [89]; Bursera simaruba [90]
55	Lignan	Pinoresinol	C20H22O6	358.3851	—	359.02	—	341; 187	323; 187	Triticum aestivum L. [78]; Eucommia cortex [47]
56	Aryl-beta-glycoside	Arbutin	C12H16O7	272.2512	—	273.17	—	217; 163	161.09	Strawberry, blueberry, pear [91]; pear [45]
Others
57	Natural water-soluble vitamin	L-ascorbic acid	C6H8O6	176.1241	—	176.98	—	145.00	117.03	Strawberry, lemon, papaya [36]
58	Aldaric acid	Glucaric acid (D-glucaric acid)	C6H10O8	210.1388	—	211.01	—	192; 115	129.05	Cherimoya, papaya [36]
59	Monobasic saturated carboxylic acid	Palmitic acid (hexadecanoic acid; palmitate)	C16H32O2	256.4241	—	257.02	—	237; 137	221; 125	Salviae [44]
60	Acyclic alcohol nitrile glycoside	Heterodendrin ((2R)-2-(β-D-glucopyranosyloxy)-3-methylbutanenitrile)	C11H19O6N	261.2717	—	263.96	—	155; 228	—	Rhodiola crenulata [35]
61	Monobasic saturated carboxylic acid	Linolenic acid (alpha-linolenic acid; linolenate)	C18H30O2	278.4296	—	279.1	—	261; 243; 187; 123	173; 131	Salviae [44]; rice [48]
62	Phenylethane glycoside	Picein (ameliaroside; salicinerin; salinigrin; piceoside)	C14H18O7	298.2901	—	299	—	271; 211; 179	254; 225; 197	Rhodiola rose [9]; Rhodiola crenulata [82]
63	Phenylethane glycoside	Salidroside (2-(4-hydroxyphenyl) ethyl β-D-glucopyranoside)	C14H20O7	300.3044	—	301.15	—	240; 201	183; 110	Rhodiola crenulata [35, 54]; Rhodiola rosea [1, 92, 93]; Rhodiola sachalinensis [53]; Rhodiola kirilowii [2]
64	Phenylethane glycoside	Icariside D2	C14H20O7	300.3044	—	301.06	—	240; 201; 135	183; 113	Rhodiola rosea [39]; Rhodiola crenulata [54, 82]; Rhodiola sacra [55];
65	Acyclic alcohol glycoside	Creoside II	C14H26O7	306.352	—	307.99	—	199; 255	—	Rhodiola crenulata [35, 54]
66	Phenylethane glycoside	Viridoside	C15H22O7	314.331	—	315.04	337.11	319.13; 209.08	151; 207; 262; 301	Rhodiola viridula [94]; Rhodiola rosea [83]; Rhodiola crenulata [35]; Rhodiola sachalinensis [53]
67	Acyclic alcohol glycoside	Rosiridin (3,7-dimethylocta-2,6-diene-1,4-diol; 1-O-beta-D-glucopyranoside)	C16H28O7	332.3893	—	333.02	—	247; 175	181.93	Rhodiola crenulata [35]; Rhodiola rosea [2, 17, 49]; Rhodiola sachalinensis [95]
68	Acyclic alcohol glycoside	Rhodioloside A	C16H28O8	348.3887	—	349.02	371.03	271; 281; 305; 331; 257; 231; 219; 167; 141	268; 256; 243; 229; 215; 193; 143	Rhodiola rosea [1, 92]; Rhodiola crenulata [35]
69	Acyclic alcohol glycoside	Rhodioloside D	C16H30O8	350.4046	—	351.06	—	258; 220; 131	257; 141	Rhodiola rosea [1, 83, 92]; Rhodiola crenulata [35]
70	Tetracyclic diterpenoid	Grayanotoxin II	C20H32O5	352.4651	—	353.04	—	335; 282; 203	315; 245; 113	Grayanotoxins [96]
72	Benzidine glycoside	Phenylmethyl (6-O-alpha-L-arabinopyranosyl-beta-D-glycopyranoside)	C18H26O10	402.3930	—	402.86	—	343; 283; 175	283	Rhodiola rosea [83]; Rhodiola sachalinensis [53]
73	Acyclic alcohol glycoside	Rhodiooctanoside	C19H36O10	424.4831	—	424.94	—	290.96	173; 261	Rhodiola crenulata [35, 54]; Rhodiola kirilowii [97]; Rhodiola sacra [98]
74	Phenylethane glycoside	Mongrhoside	C20H30O11	446.4456	—	446.65	—	243; 379; 311	174.84	Rhodiola rosea [83]
75	Acyclic alcohol glycoside	Creoside V	C21H38O10	450.5204	—	473.15	—	471; 254; 401	463.61	Rhodiola crenulata [35];
76	Hydroxy acid	Ursolic acid	C30H48O3	456.7003	—	457.17	—	412; 307	368; 269	Ocimum [41]; pear [45]
77	Acyclic alcohol glycoside	Rhodioloside E	C21H38O11	466.5198	—	467.95	—	399.94; 265; 332	331.88	Rhodiola rosea [1, 92]; Rhodiola crenulata [35, 54]; Rhodiola sachalinensis [13]; Rhodiola sacra [55]
78	Acyclic alcohol glycoside	Rhodioloside B	C22H38O12	494.5299	493.22	—	517.97	447; 220	314.98	Rhodiola rosea [1, 92]; Rhodiola crenulata [35]

The CID spectrum (collision induced dissociation spectrum) in negative ion modes of Rhodioloside B from Rh. rosea is shown in Figure 3.

Figure 3

CID spectrum of the rhodioloside B from Rh. 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 from Rh. rosea, m/z 291.13.

It was identified in the bibliography in extracts from Rhodiola 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 from Rhodiola 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

The Rhodiola 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 the Rhodiola 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 containing Rh. 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.