Comparative Analysis of Far East Sikhotinsky Rhododendron (Rh. sichotense) and East Siberian Rhododendron (Rh. adamsii) Using Supercritical CO2-Extraction and HPLC-ESI-MS/MS Spectrometry.

Rhododendron sichotense Pojark. and Rhododendron adamsii Rheder have been actively used in ethnomedicine in Mongolia, China and Buryatia (Russia) for centuries, as an antioxidant, immunomodulating, anti-inflammatory, vitality-restoring agent. These plants contain various phenolic compounds and fatty acids with valuable biological activity. Among green and selective extraction methods, supercritical carbon dioxide (SC-CO2) extraction has been shown to be the method of choice for the recovery of these naturally occurring compounds. Operative parameters and working conditions have been optimized by experimenting with different pressures (300-400 bar), temperatures (50-60 °C) and CO2 flow rates (50 mL/min) with 1% ethanol as co-solvent. The extraction time varied from 60 to 70 min. A HPLC-UV-VIS-ESI-MS/MS technique was applied to detect target analytes. A total of 48 different biologically active components have been identified in the Rh. adamsii SC-CO2 extracts. A total of 31 different biologically active components have been identified in the Rh. sichotense SC-CO2 extracts.

1. Introduction

A total of 19 species of rhododendrons are growing in the territory of Russia, the main part of which (13 species) are found only in the flora of the Russian Far East and Eastern Siberia [1]. Russian researchers classify the genus Rhododendron somewhat differently than foreign ones [2]. At present, there is no unified classification scheme for a taxon, since the genus is very large—more than 800 species, as well as the presence of a large number of convergent characters among its representatives, complicating the construction of a natural classification [3].

The genus system, developed and adopted by Russian scientists, divides the genus Rhododendron into subgenera and series. In it, Rhododendron adamsii Rehder and Rhododendron parvifolium Adams are assigned to the subgenus Osmothamnus Maximowicz (Fragrantia E. Busch series and Parvifolia E. Busch series) (Table 1). Species of Rh. dauricum L., Rh. ledebourii Pojarkova, Rh. sichotense Pojarkova and Rh. micronulatum Turczaninowia constitute the series Daurica Pojarkova subgenus Rhodorastrum (Maxim.) Drude [4].

Table 1

Classification of genus Rhododendron L.

No.	Species or Variety	Subgenus	Row
1	Rh. sichotense Pojark.; Rh. micronulatum Pojark.; Rh. dauricum L.; Rh. ledebourii Pojark.	Rhodorastrum	Daurica Pojark.
2	Rh. parvifolium Adams [Rh. lapponicum (L.) Wahlenb.]	Osmothamnus Maximowicz	Parvifolia E. Busch
3	Rh. adamsii Rehd. [Rh. fragrans (Adams) Maxim.]		Fragrantia E. Busch

Rhododendron sichotense Pojark. is a plant from the genus of rhododendrons [5]. It grows in the Far East (Primorsky Krai) on the eastern slope of the Sikhote Alin ridge. This type of rhododendron is included in the Red Book of the Russian Federation [6]. From its closest relatives, Rhododendron dauricum and Rhododendron micronulatum is distinguished by larger, sometimes more than 7 cm wide, flowers and wider, green leaves from the underside, not falling leaves. Large flowers, lush foliage, winter hardiness suggest that Rh. sichotense is a very promising garden plant for areas with a harsh climate [7].

Rh. adamsii Rheder is a shrub found in Eastern Siberia and Baikal in the alpine and subalpine zones of the mountains, forming a shrub tundra, and at the upper border of the forest at an altitude of 1200–2500 m above sea level. This is a shrub from 1 to 3 m in height with falling leaves. The flowers are pink, large enough, and open before the leaves appear. The leaves are narrow, green above, grayish green below, and very fragrant. Rh. adamsii grows in pine and deciduous forests with grass and moss, on forest edges and especially on rocky mountains [8].

Traditional medicine uses different types of rhododendron to treat a number of diseases of the respiratory system, the gastrointestinal tract, chronic skin diseases, hypertension, rheumatism, helminthiases, etc. [9]. The stupefying smell formed during the burning of leaves and caused by the sharp evaporation of volatile terpenes has long been used by indigenous ethnic groups of Siberia and Far East as a psychoactive, analgesic, and narcotic drug [10,11].

Rh. adamsii Rehder is used as a stimulant and tonic by the populations of Buryatia, Mongolia and China. Decoctions and tinctures of it are used for cold diseases, as a diuretic agent for cardiac edema, as well as an adaptogen [12]. Pharmacological studies have shown that Rh. adamsii has antimicrobial, anti-inflammatory, immunomodulating, antioxidant effects [13,14].

In essential oil of Rh. adamsii, it is possible to isolate the components present both in the leaves and stems of the plant: α- and β-pinenes, β-myrcene, cis-β-ocimene, isoledene, aromadendrene, humulene, β-farnesol, γ-murolene, β-selinene, ledene, α-farnesol, δ-cadinene, trans-nerolidol, spathulenol, β-elemenone, germacrone [15]. The essential oil of the stems of the plant contains germacrene D and germacrene B, which are absent from the essential oil of the leaves. In almost all samples of essential oil of leaves and stems of Rh. adamsii there is found 4-phenyl-2-butanone, the content of which is from 3 to 13%, as well as its related 4-phenyl-2-butanol, the content of which is from 1.9 to 7.4% [16].

This study considers the possibility and effectiveness of supercritical carbon dioxide (SC-CO2) extraction of biologically active substances from stems and leaves of Rh. adamsii. Previously, the authors of this article successfully used SC-CO2 extraction to obtain biologically active substances from plants of the Far Eastern taiga Panax ginseng, Rhodiola rosea, and Schisandra chinensis, which are extremely popular in traditional medicines of Southeast Asia [17,18].

Supercritical fluid extraction (SFE) has been used since the late 1970s to analyze food products, isolate biologically active substances and determine lipid levels in food, as well as levels of toxic substances [19,20,21]. The use of SFE for fractionation and/or enrichment of certain components in products has been reported since the 1980s [22,23,24]. 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. Easy solvent removal from the final product, high selectivity and the use of moderate temperatures in the extraction process are the main attractive factors of supercritical technology, leading to a significant increase in research for use in the food and pharmaceutical industries [25,26,27].

An alternative to the use of co-solvents in the case of poorly soluble or practically insoluble compounds is to completely change the process scheme using the so-called supercritical solvent extraction (SAE). Industrial-scale devices with technological schemes containing CO2 processing plants have already been developed; therefore, most of the solvent/anti-solvent is recovered. SAE increases are associated with the same process conditions as the pressure, temperature and concentration of solutes in the slurry. However, the main parameter is the molar fraction of CO2. It depends on the relative flow rate of the CO2 and the solvent liquid to set the supercritical precipitant composition for the CO2/solvent mixture used [28].

Popova et al. [29] (2018) investigated the possibility of SC-CO2 extracting chlorophylls and carotenoids of Ledum palustre L. (Rhododendron tomentosum Harmaja) by supercritical fluid extraction using supercritical carbon dioxide and a co-solvent of ethyl alcohol as a solvent. It has been found that by varying the pressure and temperature of the fluid, the duration of processing and the moisture content of the raw material, extracts can be obtained enriched in one or both of the recoverable pigments. Furthermore, in this case, the amount of ethanol used as a co-solvent was 5% and was necessary and sufficient for efficient extraction of pigments with supercritical CO2.

The anti-inflammatory activity of two extracts from the aerial parts of Ledum palustre L. (Rh. tomentosum Harmaja) has been reported by [30]. The volatile oil was obtained by SC-CO2 extraction and the essential oil by hydrodistillation (HD). The anti-inflammatory activity was evaluated by the subcutaneous carrageenan injection-induced hind paw oedema. The results show that L. palustre essential oil enhanced a significant inhibition of oedema (50–73%) for HD oil and (52–80%) for SFE oil.

The results of SC-CO2-extraction of leaves and branches of rhododendrons, in particular, indicated that, when using this technology, the extract contained all biologically active components of the plant, as well as inert mixtures of extracted compositions. This study is devoted to comparative mass spectrometry of extracted biologically active substances from two closely related subgenus of rhododendron: Rh. sichotense Pojark. and Rh. adamsii Rehder.

2. Results and Discussion

In the first instance, the influence of the supercritical parameters (CO2 flow rate, temperature, % co-solvent, and pressure) on extraction yield was investigated. The cumulative quantitative extracts yield are summarized in the Table 2. Several experimental conditions were investigated working in a pressure range of 300–400 bar, with co-solvent EtOH and a temperature range of 50–60 °C.

Table 2

Extraction yield of Rh. adamsii presented depending on operational parameters (pressure, temperature, CO2 flow rate, % co-solvent).

No.	Temperature (°C)	Pressure (Bar)	CO2 Flow Rate (mL/min)	% Co-Solvent EtOH	Extraction Yield (mg/g)
1	50	300	30	1	1.23
2	50	350	50	2	3.27
3	50	370	30	1	3.25
4	50	400	50	1	4.15
5	60	300	30	2	5.81
6	60	350	30	2	7.12
7	60	370	50	1	10.86
8	60	400	30	2	8.13
9	70	300	30	1	7.15
10	70	350	50	1	7.28
11	70	370	30	1	8.10
12	70	400	30	1	7.90

Orthogonal projection representing the extraction yield at 300 to 400 Bar and 50–70 °C is shown in Figure 1. The best results were obtained at 370 Bar and 60 °C. An ion trap amaZon SL BRUKER DALTONIKS equipped with an electron spray ionization (ESI) source in the negative and positive ion modes and analysis of fragmented ions was used in this scientific work.

Figure 1

Orthogonal projection representing the extraction yield of target analytes at 300 to 400 Bar and 50–70 °C.

A screening of biologically active substances from Rh. adamsii sample and Rh. sichotense sample was obtained using this method. Typical base peak chromatograms (BPC) of analyzed target analytes are shown in the Supplementary Material. Identification of compounds was assigned by comparison of their UV-Vis spectra and mass spectrometric data obtained under both negative and positive electron spray ionization (ESI−/ESI+) conditions and with the scientific literature. Under these conditions a total of 800 peaks were detected in the ion chromatogram.

Series studies by HPLC–MS/MS of both samples of rhododendrons (Rh. sichotense and Rh. adamsii) were carried out, and the results of studies of the target compounds are presented below (Table 3).

Table 3

Compounds identified from SC-CO2 extracts by two varieties of rhododendron: Rh. sichotense and Rh. adamsii.

NO.	Identification	Formula	Calculated Mass	Rh. adamsii (104)	Rh. adamsii (105)	Rh. adamsii (109)	Rh. adamsii (110)	Rh. adamsii (108)	Rh. adamsii (112)	Rh. adamsii (116)	Rh. adamsii (117)	Rh. adamsii (118)	Rh. adamsii (122)	Rh. adamsii (123)	Rh. sicho (175)	Rh. sicho (176)	Rh. sicho (177)	Rh. sicho (178)	Rh. sicho (190)	Rh. sicho (192)	Rh. sicho (193)	References
1	Lepalol [5-(3-Furyl)-2-methyl-1-penten-3-ol	C10H14O2	166.217																			[31,45,48]
2	Caffeic acid [(2E)-3-(3,4-Dihydroxyphenyl)acrylic acid]	C9H8O4	180.1574																			[10,35,42,43,50]
3	Azelaic acid (Nonanedioic acid)	C9H16O4	188.2209																			[15,16]
4	Calamenene [Cis-Calamenene]	C15H22	202.3352																			[15,16,38]
5	Germacron	C15H22O	218.3346																			[4,5,10,42,44]
6	Myristic acid (Tetradecanoic acid; N-Tetradecanoic acid)	C14H28O2	228.3709																			[15,16]
7	Pentadecanoic acid (Pentadecylic acid)	C15H30O2	242.3975																			[15,16]
8	Palmitoleic acid	C16H30O2	254.4082																			[15,16]
9	Cis-cyclopropan-9,10-hexadecanoic acid	C17H32O2	268.4348																			[15,16]
10	Linoleic acid (Linolic acid; Telfairic acid)	C18H32O2	280.4455																			[15,16,49]
11	Stearic acid (Octadecanoic acid; Stearophanic acid)	C18H36O2	284.4772																			[15,16]
12	Kaempferol	C15H10O6	286.2363																			[36,39,40,43,50]
13	Cis-cyclopropan-9,10-octadecanoic acid	C19H32O2	292.4562																			[15,16]
14	Nonadecanoic acid (N-Nonadecanoic acid)	C19H38O2	298.5038																			[15,16]
15	Kaempferol 5-methyl ether	C16H12O6	300.2629																			[39]
16	Farrerol	C17H16O5	300.3059																			[34,39]
17	Quercetin	C15H10O7	302.2357																			[34,36,39,40,43,50]
18	Dihydroquercetin (Taxifolin; Taxifoliol)	C15H12O7	304.2516																			[35,39,40]
19	Cannabigerorcinic acid (Cannabigerorcinolic acid; Cannabiorcogerolic acid	C18H24O4	304.3808																			[15,16]
20	Docosane	C22H46	310.6006																			[48]
21	8-Demethyleucalyptin [5-Hydroxy-4′,7-dimetoxy-6-methylflavone; Pabalate; Sodium salicylate]	C18H16O5	312.3166																			[33]
22	Arachic acid (Arachidic acid; eicosanoic acid)	C20H40O2	312.5304																			[15,16]
23	Azaleatin [5-O-Methylquercetin]	C16H12O7	316.2623																			[10,39,41,42]
24	Myricetin	C15H10O8	318.2351																			[36,34,39,40,50]
25	Gossypetin [Articulatidin; Equisporol]	C15H10O8	318.2351																			[37]
26	Ampelopsin [Dihydromyricetin; Ampeloptin]	C15H12O8	320.251																			[39]
27	Heneicosanoic acid (Heneicosylic acid)	C21H42O2	326.557																			[15,16]
28	Myricetin 5-Methyl ether [5-O-Methylmyricetin]	C16H12O8	332.2617																			[39]
29	Esculin [Aesculin; Esculoside; Polichrome]	C15H16O9	340.2821																			[33,41]
30	Behenic acid (Docosanoic acid)	C22H44O2	340.5836																			[15,16]
31	Pentacosane (N-Pentacosane)	C25H52	352.6854																			[48]
32	Chlorogenic acid	C16H18O9	354.3087																			[10,35,42]
33	Scopolin [Scopoloside; Scopoletin-7-glucoside; Murrayin]	C16H18O9	354.3087																			[41]
34	Tricosanoic acid (N-Tricosanoic acid)	C23H46O2	354.6101																			[15,16]
35	Lignoceric acid (Tetracosanoic acid)	C24H48O2	368.6367																			[15,16]
36	Fraxin (Fraxetin-8-O-glucoside)	C16H18O10	370.3081																			[33]
37	Daurichromenic acid	C23H30O4	370.4819																			[15,16]
38	Pentacosanoic acid (N-Pentacosanoic acid)	C25H50O2	382.6633																			[15,16]
39	Fraxetin-7-O-beta-glucuronide	C16H16O11	384.2916																			[41]
40	Beta-Sitosterin [Beta-Sitosterol]	C29H50O	414.7067																			[10,30,42]
41	Cyanidin-3-alpfa-l-arabinoside	C20H19O10	419.3589																			[10,42]
42	Montanic acid (Amyrin; Beta-Amyrenol)	C28H56O2	424.743																			[15,16]
43	Alpha-Amyrin [Viminalol]	C30H50O	426.7174																			[30]
44	Lupeol [Fagarasterol; Clerodol; Monogynol B; Lupenol]	C30H50O	426.7174																			[30]
45	Dihydroquercetin-3-arabinofuranoside	C20H16O11	432.3344																			[10,42]
46	Afzelin [ Kaempferol-3-Rhamnoside; Kaempferin]	C21H20O10	432.3775																			[39,40]
47	Quercetin-3-O-beta-xyloside (Reynoutrin; Quercetin 3-O-Beta-d-Xylopyranoside)	C20H17O11	433.3424																			[34]
48	Avicularin (Quercetin 3-Alpha-l-Arabinofuranoside; Avicularoside)	C20H18O11	434.3503																			[10,33,39,40,42]
49	Pentoside dihydroquercetin		436																			[40]
50	Erithrodiol [3-beta-Erytrodiol]	C30H50O2	442.7168																			[30]
51	Uvaol	C30H50O2	442.7168																			[30]
52	Quercitrin [Quercetin 3-l- Rhamnoside; Quercetrin]	C21H20O11	448.3769																			[33,39,46]
53	Catechin-7-O-glucoside	C21H24O11	452.4087																			[34]
54	Micromeric acid	C30H46O3	454.6844																			[30]
55	Hyperoside (Quercetin 3-O-galactoside; Hyperin)	C21H20O12	464.3763																			[10,33,34,39,40,41,42]
56	Quercetin 3-O-glucoside [ Isoquercitrin]	C21H20O12	464.3763																			[33,46]
57	Alpha.-Tocopherol-Beta-d-Mannoside [Dihydro-2H-Chromen-6-YI Hexofuranoside]	C35H60O7	592.8467																			[48]

Colors are added for readability to avoid confusing columns. Green shades Rh. adamsii. blue Rh. sichotense.

Table 4 summarized all the molecular masses of the target analytes isolated from SC-CO2 of Rh. sichotense and Rh. adamsii. Among them, 57 biologically active substances were authenticated (m/z values and fragment ions) by comparison with the literature data [4,5,10,11,12,13,15,16,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53]. A total of 48 different biologically active components have been identified in the Rh. adamsii SC-CO2 extracts. A total of 31 different biologically active components have been identified in the Rh. sichotense SC-CO2 extracts.

Table 4

Components identified from the SC-CO2 extracts of Rh. sichotense and Rh. adamsii.

No.	Identification	Formula	Calculated Mass	Observed Mass [M − H]−	Observed Mass [M + H]+	Observed Mass [M + Na]+	MS/MS Stage 2 Fragmentation	MS/MS Stage 3 Fragmentation	MS/MS Stage 4 Fragmentation	Species of Rhododendron
1	Lepalol [5-(3-Furyl)-2-methyl-1-penten-3-ol	C10H14O2	166.217	165.06			147.01			Rh. adamsii
2	Caffeic acid [(2E)-3-(3,4-Dihydroxyphenyl) acrylic acid]	C9H8O4	180.1574		181.08		163.03; 135.11			Rh. adamsii
3	Azelaic acid [Nonanedioic acid]	C9H16O4	188.2209			210.09	192.12	175.06; 136.12		Rh. adamsii
4	Calamenene [Cis-Calamenene]	C15H22	202.3352		203.09		147.05	119.06		Rh. sichotense
5	Germacron	C15H22O	218.3346		219.06		201.07; 149.07	159.07		Rh. adamsii
6	Myristic acid (Tetradecanoic acid; N-Tetradecanoic acid)	C14H28O2	228.3709			251.09	150.48	149.08		Rh. adamsii
7	Pentadecanoic acid (Pentadecylic acid)	C15H30O2	242.3975		243.06		201.01; 137.05	181.05; 135.04		Rh. adamsii
8	Palmitoleic acid	C16H30O2	254.4082			277.09	275.04; 207.05	256.99	157.11	Rh. adamsii
9	Cis-cyclopropan-9,10-hexadecanoic acid	C17H32O2	268.4348		269.02		185.97; 121.08	176.96	154.98	Rh. adamsii
10	Linoleic acid [Linolic acid; Telfairic acid]	C18H32O2	280.4455			303.06	285.05; 163.00	180.95; 135.06	162.99	Rh. adamsii
11	Stearic acid [Octadecanoic acid; Stearophanic acid]	C18H36O2	284.4772		285.07		284.18; 229.07; 163.02	180.90; 135.05	163.03	Rh. adamsii; Rh. sichotense
12	Kaempferol [3,5,7-Trihydroxy-2-(4-hydro- xyphenyl)-4-H-chromen-4-one]	C15H10O6	286.2363		287.00		286.24; 204.96; 163.02	181.02	162.88	Rh. adamsii
13	Cis-cyclopropan-9,10-octadecanoic acid	C19H32O2	292.4562		293.05		274.98	256.99; 162.98	201.03	Rh. adamsii
14	Nonadecanoic acid [N-Nonadecanoic acid]	C19H38O2	298.5038		300.09		243.04	201.02		Rh. adamsii; Rh. sichotense
15	Kaempferol 5-methyl ether	C16H12O6	300.2629		300.98		283.01; 177.01	264.98	200.98	Rh. adamsii; Rh. sichotense
16	Farrerol [5,7-Dihydroxy-2-(4-hydroxyphenyl)-6,8-dimethylchroman-4-one]	C17H16O5	300.3059		301.05		283.04	241.01; 162.96		Rh. adamsii; Rh. sichotense
17	Quercetin [2-(3,4-Dihydroxyphenyl)-3,5,7-trihy- droxy-4-H-chromen-4-one]	C15H12O7	302.2357	301.09	303.08		285.01; 163.02	180.97; 145.00	162.98	Rh. adamsii; Rh. sichotense
18	Dihydroquercetin [Taxifolin; Taxifoliol]	C15H12O7	304.2516	303.09			285.04	266.96; 241.09; 215.05; 135.05	171.02	Rh. adamsii
19	Cannabigerorcinic acid [Cannabigerorcinolic acid; Cannabiorcogerolic acid]	C18H24O4	304.3808	303.08			285.05	241.07; 159.07	159.01	Rh. adamsii
20	Docosane	C22H46	310.6006		311.10		293.06; 167.01	259.03	240.97; 162.96	Rh. adamsii
21	8-Demethyleucalyptin [5-Hydroxy-4′,7-dimetoxy-6-methylflavone; Pabalate; Sodium salicylate]	C18H16O5	312.3166	311.14			311.10; 182.99			Rh. adamsii
22	Arachic acid [Arachidic acid; eicosanoic acid]	C20H40O2	312.5304	311.14		335.04	303.06; 195.01	284.99; 238.14; 163.00	180.89; 135.14	Rh. adamsii
23	Azaleatin [5-O-Methylquercetin]	C16H12O7	316.2623	315.08			297.01; 167.04	235.04; 149.00		Rh. adamsii
24	Myricetin [3,5,7-Trihydroxy-2-(3,4,5-Trihydroxyphenyl)-4H-Chromen-4-One]	C15H10O8	318.2351	317.08			299.01; 241.01	240.06; 197.09	238.99; 197.04	Rh. adamsii; Rh. sichotense
25	Gossypetin [Articulatidin; Equisporol]	C15H10O8	318.2351		319.07		287.09; 176.98	146.99		Rh. adamsii
26	Ampelopsin [Dihydromyricetin; Ampeloptin]	C15H12O8	320.251	319.08			317.01; 275.09	257.12; 217.11		Rh. adamsii
27	Heneicosanoic acid [Heneicosylic acid]	C21H42O2	326.557	325.11	327.08		271.01; 217.03; 177.06	149.10		Rh. adamsii; Rh. sichotense
28	Myricetin 5-Methyl ether [5-O-Methylmyricetin]	C16H12O8	332.2617	331.03			168.94	149.96		Rh. sichotense
29	Esculin [Aesculin; Esculoside; Polichrome]	C15H16O9	340.2821		341.09		281.01; 217.11; 151.06	174.96		Rh. adamsii; Rh. sichotense
30	Behenic acid [Docosanoic acid]	C22H44O2	340.5836		341.05		323.10; 243.11; 177.04	159.05		Rh. adamsii; Rh. sichotense
31	Pentacosane (N-Pentacosane)	C25H52	352.6854		353.12		270.97; 162.97	180.93	162.96	Rh. sichotense
32	Chlorogenic acid	C16H18O9	354.3087		355.09		287.05; 164.02	180.95	163.03	Rh. adamsii; Rh. sichotense
33	Scopolin [Scopoloside; Scopoletin-7-glucoside; Murrayin]	C16H18O9	354.3087		355.02		323.00	303.96; 184.89	162.86	Rh. adamsii; Rh. sichotense
34	Tricosanoic acid [N-Tricosanoic acid]	C23H46O2	354.6101		355.08		322.96; 163.00	180.96	162.96	Rh. adamsii
35	Lignoceric acid [Tetracosanoic acid]	C24H48O2	368.6367	367.12	369.08		351.08; 285.02; 218.92; 162.98	163.02	144.97	Rh. adamsii; Rh. sichotense
36	Fraxin (Fraxetin-8-O-glucoside)	C16H18O10	370.3081		371.08		338.99	320.96; 177.03	224.96	Rh. adamsii; Rh. sichotense
37	Daurichromenic acid	C23H30O4	370.4819		371.09		352.98; 287.08; 235.08; 179.02	231.04; 205.05; 162.99	180.93; 144.97	Rh. adamsii; Rh. sichotense
38	Pentacosanoic acid [N-Pentacosanoic acid]	C25H50O2	382.6633		383.07	405.08	351.04; 287.99	229.04	211.03	Rh. adamsii; Rh. sichotense
39	Fraxetin-7-O-beta-glucuronide	C16H16O11	384.2916	383.09			365.09; 190.96	266.97; 215.02	170.97	Rh. adamsii; Rh. sichotense
40	Beta-Sitosterin [Beta-Sitosterol]	C29H50O	414.7067		415.04		384.02	369.01	338.00	Rh. adamsii; Rh. sichotense
41	Cyanidin-3-alpfa-l-arabinoside	C20H19O10	419.3589	418.51			399.05; 319.02; 194.99	381.068 162.02	337.02; 253.08	Rh. adamsii
42	Montanic acid [Octacosanoic acid]	C28H56O2	424.743		425.02		407.00	389.00; 348.98; 298.99; 240.97	333.00; 280.97; 173.02	Rh. adamsii; Rh. sichotense
43	Alpha-Amyrin [Viminalol]	C30H50O	426.7174		427.05		408.27; 308.99; 202.91	389.02; 309.01	373.08; 229.10; 142.80	Rh. adamsii; Rh. sichotense
44	Lupeol [Fagarasterol; Clerodol; Monogynol B; Lupenol]	C30H50O	426.7174		427.04		409.01; 202.99	389.02; 247.99	370.96; 264.80	Rh. adamsii
45	Dihydroquercetin 3-arabinofuranoside	C20H16O11	432.3344		433.97		352.95	323.53; 271.96	241.95; 181.87	Rh. adamsii
46	Afzelin [ Kaempferol-3-Rhamnoside; Kaempferin]	C21H20O10	432.3775	431.04			413.00; 372.98; 216.94	354.95; 167.01	336.98; 148.91	Rh. sichotense
47	Quercetin-3-O-beta-xyloside (Reynoutrin; Quercetin 3-O-Beta-d-Xylopyranoside)	C20H17O11	433.3424		434.90		302.94	256.92; 164.96	228.91; 159.11	Rh. sichotense
48	Avicularin (Quercetin 3-Alpha-l-Arabinofuranoside; Avicularoside)	C20H18O11	434.3503	433.09			415.07; 335.01; 176.98	397.06; 190.99	353.07; 253.99	Rh. adamsii; Rh. sichotense
49	Pentoside dihydroquercetin		436	435.16			416.54; 300.99; 231.01	397.02; 205.96	361.11; 283.02; 188.80	Rh. adamsii; Rh. sichotense
50	Erithrodiol [3beta-Erytrodiol]	C30H50O2	442.7168	441.12			425.06; 381.05; 300.03; 217.04	363.06; 246.02	319.08; 201.02	Rh. adamsii
51	Uvaol	C30H50O2	442.7168		443.22		425.01; 233.07	407.02; 325.01	388.99; 231.11	Rh. adamsii; Rh. sichotense
52	Quercitrin [Quercetin 3-l-Rhamnoside; Quercetrin]	C21H20O11	448.3769		448.89		370.95; 282.93	352.95; 176.98	334.90; 222.92; 176.97	Rh. adamsii
53	Catechin-7-O-glucoside	C21H24O11	452.4087		453.17		435.15; 336.07; 209.06	417.16; 336.11; 226.12	209.09	Rh. sichotense
54	Micromeric acid	C30H46O3	454.6844		455.05		408.98; 246.98	391.05; 287.96	250.96	Rh. adamsii
55	Hyperoside (Quercetin 3-O- galactoside; Hyperin)	C21H20O12	464.3763		465.02		302.91	256.94; 190.87	228.96; 172.75	Rh. sichotense
56	Quercetin 3-O-glucoside [Isoquercitrin]	C21H20O12	464.3763		465.08		447.00	386.96	369.12; 172	Rh. sichotense
57	Alpha.-Tocopherol-Beta-d-Mannoside [Dihydro-2H-Chromen-6-YI Hexofuranoside]	C35H60O7	592.8467		593.11		533.08	461.10	433.11	Rh. sichotense

Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figure 11 shows examples of the decoding spectra (collision-induced dissociation (CID) spectrum) of the ion chromatogram obtained using tandem mass spectrometry. The CID spectrum in negative ion modes of fraxetin-7-O-beta-glucoronide from Rh. adamsii and Rh. sichotense are shown in Figure 2 and Figure 3.

Figure 2

Collision-induced dissociation (CID) spectrum of fraxetin-7-O-beta-glucoronide from Rh. adamsii, m/z 383.10.

Figure 3

CID spectrum of fraxetin-7-O-beta-glucoronide from Rh. sichotense, m/z 383.13.

Figure 4

CID spectrum of Beta-sitosterin from Rh. adamsii, m/z 415.04.

Figure 5

CID spectrum of Beta-sitosterin from Rh. sichotense, m/z 415.05.

Figure 6

CID spectrum of quercetin from Rh. adamsii, m/z 301.07.

Figure 7

CID spectrum of quercetin from Rh. sichotense, m/z 301.07.

Figure 8

CID spectrum of myricetin from Rh. adamsii, m/z 317.10.

Figure 9

CID spectrum of myricetin from Rh. sichotense, m/z 317.14.

Figure 10

CID spectrum of farrerol from Rh. adamsii, m/z 301.06.

Figure 11

CID spectrum of farrerol from Rh. sichotense, m/z 301.06.

The [M − H]− ion produced fragment ion with m/z 383.10 (Figure 2). The fragment ion with m/z 383.10 produced characteristic daughter ion with m/z 321.07, m/z 215.07 and m/z 149.09. The fragment ion with m/z 215.07 formed two daughter ions with m/z 171.02, m/z 212.86. It was identified in the bibliography in extract from rhododendron L. palustre [13,30,31,32,33,45].

The [M − H]− ion produced fragment ion with m/z 383.13 (Figure 3).The fragment ion with m/z 383.13 produced two fragments with m/z 365.07, m/z 191.00. The fragment ion with m/z 365.07 produced two characteristic daughter ions with m/z 267.06 and m/z 215.02. The fragment ion with m/z 215.02 formed a daughter ion with m/z 170.99. It was identified in the bibliography of the methanolic extract from rhododendron L. palustre [30,31,32,33,44].

The CID spectrum in positive ion modes of Beta-sitosterin from Rh. adamsii and Rh. sichotense is shown in Figure 4 and Figure 5.

The [M + H]+ ion produced one fragment with m/z 415.04 (Figure 4). The fragment ion with m/z 384.02 produced one daughter ion with m/z 369.01. The fragment ion with m/z 369.01 formed a daughter ion with m/z 338.00. It was identified in the bibliography in the extract from rhododendrons L. palustre [30,31,32,33,41,44,45] and Rh. adamsii [10,12,15,16,42,52].

The [M + H]+ ion produced one fragment with m/z 384.06 (Figure 5). The fragment ion with m/z 384.06 produced one daughter ion with m/z 369.03. The fragment ion with m/z 369.03 formed a daughter ion with m/z 338.05. It was identified in the bibliography in the extract from rhododendrons L. palustre [30,31,32,33,41,44,45] and Rh. adamsii [10,12,15,16,42,52].

The CID spectrum in negative ion modes of quercetin from Rh. adamsii and Rh. sichotense is shown in Figure 6 and Figure 7.

The [M − H]− ion produced one fragment ion with m/z 283.03 (Figure 6). The fragment ion with m/z 283.03 produced two daughter ions with m/z 255.11 and m/z 177.08. The fragment ion with m/z 255.11 formed two daughter ions with m/z 253.03 and m/z 173.05. It was identified in the bibliography in extracts from rhododendrons Rh. sichotense, Rh. micronulatum [38,39,40]; Rh. ungernii [34]; Rhodiola crenulata [36]; Rh. adamsii [10,12,15,16,42,52]; Rh. parvifolium [10]; Ocimum [43].

The [M − H]− ion produced one fragment ion with m/z 283.03 (Figure 7). The fragment ion with m/z 283.03 produced three daughter ions with m/z 255.09, m/z 177.06 and m/z 252.99. The fragment ion with m/z 252.99 formed two daughter ions with m/z 211.08 and m/z 173.05. It was identified in the bibliography in extracts from rhododendrons Rh. sichotense, Rh. micronulatum [38,39,40]; Rh. ungernii [34]; Rhodiola crenulata [36]; Rh. adamsii [10,12,15,16,42,52]; Rh. parvifolium [10]; Ocimum [43].

The CID spectrum in negative ion modes of myricetin from Rh. adamsii and Rh. sichotense is shown in Figure 8 and Figure 9.

The [M − H]− ion produced three fragment ions with m/z 299.03, m/z 259.05 and m/z 148.99 (Figure 8). The fragment ion with m/z 299.03 produced two daughter ions with m/z 241.04 and m/z 281.01. The fragment ion with m/z 241.04 formed a daughter ion with m/z 238.99. It was identified in the bibliography in extracts from rhododendrons Rh. sichotense, Rh. micronulatum [39,40,41]; Rh. ungernii [35]; Rhodiola crenulata [37]; Rh. adamsii [16,17,43,53]; Rh. parvifolium [41]; Ocimum [44].

The [M − H]− ion produced two fragment ions with m/z 299.06, m/z 241.01 (Figure 9). The fragment ion with m/z 299.06 produced two daughter ions with m/z 228.04 and m/z 129.92. The fragment ion with m/z 228.04 formed a daughter ion with m/z 227.10. It was identified in the bibliography in extracts from rhododendrons Rh. sichotense [4,5]; Rh. ungernii [34]; Rhodiola crenulata [36]; Rh. adamsii [10,15,16,42,52]; Rh. parvifolium [10,40].

The CID spectrum in positive ion modes of farrerol from Rh. adamsii and Rh. sichotense is shown in Figure 10 and Figure 11. The [M + H]+ ion produced three fragment ions with m/z 283.02, m/z 244.99 and m/z 162.98 (Figure 10). The fragment ion with m/z 283.02 produced two daughter ions with m/z 240.98 and m/z 163.01. The fragment ion with m/z 240.98 formed a daughter ion with m/z 170.96. It was identified in the bibliography in extracts from rhododendrons Rh. dauricum [38,39,40,41]; Rh. ungernii [34].

The [M + H]+ ion produced two fragment ions with m/z 282.95 and m/z 180.98 (Figure 11).

The fragment ion with m/z 180.98 formed a daughter ion with m/z 163.00. It was identified in the bibliography in extracts from rhododendrons Rh. dauricum [38,39,40]; Rh. ungernii [34].

3. Materials and Methods

3.1. Materials

The objects of study were purchased samples of Rh. sichotense (leaves and stems) from Primorsky Krai (the eastern slope of the Sikhote Alin ridge) and Rh. adamsii (leaves and stems) from the area near lake Baykal, Russia. All samples were morphologically authenticated according to the current standard of Russian Pharmacopeia [54]. All samples were immediately washed weighed by 10 g aliquot then frozen and kept until extraction.

3.2. Chemicals and Reagents

HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), MS-grade formic acid was from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was prepared from a SIEMENS ULTRA clear (SIEMENS water technologies, Munich, Germany), and all other chemicals were analytical grade.

3.3. Liquid Chromatography

HPLC was performed using a Shimadzu LC-20 Prominence HPLC (Kanda-Nishikicho 1-chrome, Shimadzu, Chiyoda-ku, Tokyo, Japan), equipped with a UV-sensor and a Shodex ODP-40 4E (250 × 4.6 mm, particle size: 4 μm) reverse phase C18 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; 60–75 min, 25–0% A; control washing 75–120 min 0% A. The entire HPLC analysis was performed using a UV-VIS detector SPD-20A (Kanda-Nishikicho 1-chrome, Shimadzu, Chiyoda-ku, Tokyo, Japan) at wavelengths of 230 and 330 nm, at 17 °C provided with a column oven CTO-20A (Kanda-Nishikicho 1-chrome, Shimadzu, Chiyoda-ku, Tokyo, Japan) with an injection volume of 20 μL.

3.4. SC-CO2 Extraction

SC-CO2 extraction was performed using the Supercritical fluid system -500 (Thar SCF Waters, Milford, MA, USA) supercritical pressure extraction apparatus. System options include: Co-solvent pump (Thar Waters P-50 High Pressure Pump), for extracting polar samples. CO2 flow meter (Siemens, Munich, Germany), to measure the amount of CO2 being supplied to the system, multiple extraction vessels, to extract different sample sizes or to increase the throughput of the system. Flow rate was 50 mL/min for liquid CO2 and 1.00 mL/min for EtOH. Samples for extraction of 10 g of frozen Rh. sichotense and Rh. adamsii pre-cut into pieces no more than 1 cm were used. Several experimental conditions were investigated, working in a pressure range of 300–400 bar, with 1% of C2H5OH as co-solvent and a temperature range of 50–60 °C. The extraction time was counted after reaching the working pressure and equilibrium flow, and it was 60–70 min for each sample.

3.5. Mass Spectrometry

MS analysis was performed on an ion trap amaZon SL (BRUKER DALTONIKS, Bremen, Germany) equipped with an ESI source in negative ion mode. The optimized parameters were obtained as follows: ionization source temperature: 70 °C, gas flow: 4 L/min, nebulizer gas (atomizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500 V, fragmentary: 280 V, collision energy: 60 eV MS/MS MS4 (four stages of separation). An ion trap was used in the scan range m/z 100–1700 for MS and the capture rate was one spectrum/s for MS and two spectrum/s for MS/MS. All experiments were repeated three times. A four-stage ion separation mode (MS/MS mode) was implemented.

4. Conclusions

Aiming to optimize the extraction of target analytes from the Rh. sichotense and Rh. adamsii leaves and stems, several experimental conditions were investigated working in a pressure range of 300–400 bar, with 1% of C2H5OH as co-solvent and a temperature range of 50–60 °C. Although this approach is not quantitative for evaluation of each analyte, it is semiquantitative when comparing a series of extractions and allows better comparison of the yield without loss of individual analytes during fractionation and sample preparation. The best results were obtained at 370 bar and 60 °C.

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 four-stage ion separation mode was implemented to perform correct identification. Under these conditions a total of 800 peaks were detected in the ion chromatogram. An optimized extraction process with SC-CO2 (co-solvent 1% ethanol) provided the samples for an accurate analytical study by HPLC–MS/MS technique. A total of 50 different biologically active components were identified in the Rh. adamsii SC-CO2 extracts. A total of 30 different biologically active components were identified in the Rh. sichotense SC-CO2 extracts.

An analysis of the similarity of the composition of biologically active components of Rh. sichotense and Rh. adamsii revealed their significant relationship. An analysis of morphometric parameters and composition of Rh. sichotense and Rh. adamsii flavonoids from populations of the Far East and Siberia indicated the presence of ecological and geographical variability. Inter-population differences in morphometric indicators are more significant than in chemical ones. Moreover, the former is more associated with climatic factors, while the latter are more associated with edaphic growth factors. The Rh. adamsii extract was more diverse in chemical composition than Rh. sichotense. A particularly large difference was observed in acidic components such as caffeic acid, azelaic acid, myristic acid, pentadecanoic acid, palmitoleic acid, linoleic acid. The extract of Rh. sichotense contained mainly stearic acid, nonadecanoic acid, montanic acid, behenic acid, tetracosanoic acid and chlorogenic acid. The flavonoid content of both rhododendrons was mainly the same.

These data could support future research for the production of a variety of pharmaceutical products containing ultra-pure SC-CO2 extracts of Rh. sichotense and Rh. adamsii. The richness of various biologically active compounds, including flavonoids: quercetin, kaempferol, dihydroquercetin, farrerol, myricetin, etc. provides great opportunities for the design of new drugs based on extracts from this species of rhododendron.