And East Siberian Rhododendron (Rh. Adamsii) Using Supercritical CO2-Extraction and HPLC-ESI-MS/MS Spectrometry

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Article 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

Mayya Razgonova 1,2,* , Alexander Zakharenko 1,2 , Sezai Ercisli 3 , Vasily Grudev 4 and Kirill Golokhvast 1,2,5

1 N.I. Vavilov All-Russian Institute of Plant Genetic Resources, 190000 Saint-Petersburg, Russia; [email protected] (A.Z.); [email protected] (K.G.) 2 SEC Nanotechnology, Far Eastern Federal University, 690950 Vladivostok, Russia 3 Agricultural Faculty, Department of Horticulture, Ataturk University, 25240 Erzurum, Turkey; [email protected] 4 Far Eastern Investment and Export Agency, 123112 Moscow, Russia; [email protected] 5 Paciﬁc Geographical Institute, Far Eastern Branch of the Russian Academy of Sciences, 690041 Vladivostok, Russia * Correspondence: [email protected]

Academic Editors: Seung Hwan Yang and Satyajit Sarker Received: 29 June 2020; Accepted: 12 August 2020; Published: 19 August 2020

Abstract: 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.

Keywords: Rhododendron sichotense; Rhododendron adamsii; supercritical fluid extraction; HPLC–MS/MS; phenolic compounds

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.

Molecules 2020, 25, 3774; doi:10.3390/molecules25173774 www.mdpi.com/journal/molecules Molecules 2020, 25, 3774 2 of 20

Busch series) (Table1). 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]. 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].

Table 1. Classiﬁcation of genus Rhododendron L.

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

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 [19–21]. The use of SFE for fractionation and/or enrichment of certain components in products has been reported since the 1980s [22–24]. In addition, the products do not have residues of organic solvents, which occur with conventional extraction methods, and solvents can be toxic, Molecules 2020, 25, 3774 3 of 20 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 [25–27]. 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 Table2. 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. Orthogonal projection representing the extraction yield at 300 to 400 Bar and 50–70 ◦C is shown in Figure1. 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. 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. Molecules 2020, 25, 3774 4 of 20

Table 2. Extraction yield of Rh. adamsii presented depending on operational parameters (pressure, temperature, CO2 ﬂow rate, % co-solvent).

Temperature CO Flow Rate % Co-Solvent Extraction No. Pressure (Bar) 2 (◦C) (mL/min) EtOH Yield (mg/g) 1 50 300 30 1 1.23 2 50 350 50 2 3.27 3 50 370 30 1 3.25 Molecules 42020, 25, x FOR 50 PEER REVIEW 400 50 1 4.15 4 of 21 5 60 300 30 2 5.81 9 70 300 30 1 7.15 6 60 350 30 2 7.12 10 70 350 50 1 7.28 7 60 370 50 1 10.86 11 70 370 30 1 8.10 12 870 60 400 400 30 30 1 2 8.13 7.90 9 70 300 30 1 7.15 Orthogonal10 projection 70 representing 350 the extraction 50 yield at 300 to 400 1 Bar and 50–70 7.28 °C is shown in Figure11 1. The best 70 results were obtained 370 at 370 Bar 30 and 60 °C. An ion 1 trap amaZon 8.10 SL BRUKER DALTONIKS equipped with an electron spray ionization (ESI) source in the negative and positive 12 70 400 30 1 7.90 ion modes and analysis of fragmented ions was used in this scientific work.

12 10 8 6 4 70 2 0 60 300 350 370 50 PRESSURE 400 0-2 2-4 4-6 6-8 8-10 10-12

Figure 1.1. Orthogonal projection representingrepresenting the extraction yield of target analytes at 300 to 400 Bar

and 50–70 ◦°C.C.

SeriesA screening studies of by biologically HPLC–MS /activeMS of bothsubstances samples from of rhododendrons Rh. adamsii sample (Rh. sichotenseand Rh. sichotenseand Rh. adamsii)sample werewas obtained carried out, using and this the method. results ofTypical studies base of thepeak target chromatograms compounds (BPC) are presented of analyzed below target (Table analytes3). are shownTable4 in summarized the supplementary all the molecularmaterial. Identifi massescation of the of targetcompounds analytes was isolated assigned from by comparison SC-CO 2 of Rh.of their sichotense UV-Visand spectraRh. adamsii. and Amongmass spectrometric them, 57 biologically data obtained active substancesunder both were negative authenticated and positive (m/z valueselectron and spray fragment ionization ions) by (ESI comparison−/ESI+) conditions with the literatureand with data the [ 4scientific,5,10–13,15 literature.,16,30–53]. Under A total these of 48 diconditionsfferent biologically a total of 800 active peaks components were detected have been in the identified ion chromatogram. in the Rh. adamsii SC-CO2 extracts. A total of 31Series different studies biologically by HPLC–MS/MS active components of both have samples been identified of rhododendrons in the Rh. sichotense (Rh. sichotenseSC-CO2 andextracts. Rh. adamsii) were carried out, and the results of studies of the target compounds are presented below (Table 3). 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 [4,5,10–13,15,16,30–53]. 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.

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Table 3. Compounds identiﬁed from SC-CO2 extracts by two varieties of rhododendron: Rh. sichotense and Rh. adamsii.

Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Calculated NO. Identiﬁcation Formula adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii sicho sicho sicho sicho sicho sicho sicho References Mass (104) (105) (109) (110) (108) (112) (116) (117) (118) (122) (123) (175) (176) (177) (178) (190) (192) (193) Lepalol [5-(3-Furyl)- 1 2-methyl-1- C10H14O2 166.217 [31,45,48] penten-3-ol Caﬀeic acid [(2E)-3-(3,4- [10,35,42,43, 2 C H O 180.1574 Dihydroxyphenyl) 9 8 4 50] acrylic acid] Azelaic acid 3 C H O 188.2209 [15,16] (Nonanedioic acid) 9 16 4 Calamenene 4 C H 202.3352 [15,16,38] [Cis-Calamenene] 15 22 5 Germacron C15H22O 218.3346 [4,5,10,42,44] Myristic acid (Tetradecanoic acid; 6 C H O 228.3709 [15,16] N-Tetradecanoic 14 28 2 acid) Pentadecanoic acid 7 C H O 242.3975 [15,16] (Pentadecylic acid) 15 30 2 8 Palmitoleic acid C16H30O2 254.4082 [15,16] Cis-cyclopropan- 9 9,10-hexadecanoic C17H32O2 268.4348 [15,16] acid Linoleic acid (Linolic 10 C H O 280.4455 [15,16,49] acid; Telfairic acid) 18 32 2 Stearic acid 11 (Octadecanoic acid; C18H36O2 284.4772 [15,16] Stearophanic acid) [36,39,40,43, 12 Kaempferol C H O 286.2363 15 10 6 50] Cis-cyclopropan- 13 9,10-octadecanoic C19H32O2 292.4562 [15,16] acid Nonadecanoic acid 14 (N-Nonadecanoic C19H38O2 298.5038 [15,16] acid) Kaempferol 5-methyl 15 C H O 300.2629 [39] ether 16 12 6 16 Farrerol C17H16O5 300.3059 [34,39] [34,36,39,40, 17 Quercetin C H O 302.2357 15 10 7 43,50] Dihydroquercetin 18 C H O 304.2516 [35,39,40] (Taxifolin; Taxifoliol) 15 12 7 Cannabigerorcinic acid (Cannabigerorcinolic 19 C H O 304.3808 [15,16] acid; 18 24 4 Cannabiorcogerolic acid Molecules 2020, 25, 3774 6 of 20

Table 3. Cont.

Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Calculated NO. Identiﬁcation Formula adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii sicho sicho sicho sicho sicho sicho sicho References Mass (104) (105) (109) (110) (108) (112) (116) (117) (118) (122) (123) (175) (176) (177) (178) (190) (192) (193) 20 Docosane C22H46 310.6006 [48] 8-Demethyleucalyptin [5-Hydroxy-40,7- dimetoxy-6- 21 C H O 312.3166 [33] methylﬂavone; 18 16 5 Pabalate; Sodium salicylate] Arachic acid 22 (Arachidic acid; C20H40O2 312.5304 [15,16] eicosanoic acid) Azaleatin 23 C H O 316.2623 [10,39,41,42] [5-O-Methylquercetin] 16 12 7 [34,36,39,40, 24 Myricetin C H O 318.2351 15 10 8 50] Gossypetin 25 [Articulatidin; C15H10O8 318.2351 [37] Equisporol] Ampelopsin 26 [Dihydromyricetin; C15H12O8 320.251 [39] Ampeloptin] Heneicosanoic acid 27 C H O 326.557 [15,16] (Heneicosylic acid) 21 42 2 Myricetin 5-Methyl 28 ether C16H12O8 332.2617 [39] [5-O-Methylmyricetin] Esculin [Aesculin; 29 Esculoside; C15H16O9 340.2821 [33,41] Polichrome] Behenic acid 30 C H O 340.5836 [15,16] (Docosanoic acid) 22 44 2 Pentacosane 31 C H 352.6854 [48] (N-Pentacosane) 25 52 32 Chlorogenic acid C16H18O9 354.3087 [10,35,42] Scopolin [Scopoloside; 33 Scopoletin-7-glucoside; C16H18O9 354.3087 [41] Murrayin] Tricosanoic acid 34 C H O 354.6101 [15,16] (N-Tricosanoic acid) 23 46 2 Lignoceric acid 35 C H O 368.6367 [15,16] (Tetracosanoic acid) 24 48 2 Fraxin 36 C H O 370.3081 [33] (Fraxetin-8-O-glucoside) 16 18 10 37 Daurichromenic acid C23H30O4 370.4819 [15,16] Pentacosanoic acid 38 C H O 382.6633 [15,16] (N-Pentacosanoic acid) 25 50 2 Fraxetin-7-O-beta- 39 C H O 384.2916 [41] glucuronide 16 16 11 Beta-Sitosterin 40 C H O 414.7067 [10,30,42] [Beta-Sitosterol] 29 50 Molecules 2020, 25, 3774 7 of 20

Table 3. Cont.

Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Rh. Calculated NO. Identiﬁcation Formula adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii adamsii sicho sicho sicho sicho sicho sicho sicho References Mass (104) (105) (109) (110) (108) (112) (116) (117) (118) (122) (123) (175) (176) (177) (178) (190) (192) (193) Cyanidin-3-alpfa-l- 41 C H O 419.3589 [10,42] arabinoside 20 19 10 Montanic acid 42 (Amyrin; C28H56O2 424.743 [15,16] Beta-Amyrenol) Alpha-Amyrin 43 C H O 426.7174 [30] [Viminalol] 30 50 Lupeol [Fagarasterol; 44 Clerodol; Monogynol C30H50O 426.7174 [30] B; Lupenol] Dihydroquercetin-3- 45 C H O 432.3344 [10,42] arabinofuranoside 20 16 11 Afzelin [ Kaempferol- 46 3-Rhamnoside; C21H20O10 432.3775 [39,40] Kaempferin] Quercetin-3-O-beta- xyloside (Reynoutrin; 47 C H O 433.3424 [34] Quercetin 3-O-Beta-d- 20 17 11 Xylopyranoside) Avicularin (Quercetin 3-Alpha-l- [10,33,39,40, 48 C H O 434.3503 Arabinofuranoside; 20 18 11 42] Avicularoside) Pentoside 49 436 [40] dihydroquercetin Erithrodiol 50 C H O 442.7168 [30] [3-beta-Erytrodiol] 30 50 2 51 Uvaol C30H50O2 442.7168 [30] Quercitrin [Quercetin 52 3-l- Rhamnoside; C21H20O11 448.3769 [33,39,46] Quercetrin] 53 Catechin-7-O-glucoside C21H24O11 452.4087 [34] 54 Micromeric acid C30H46O3 454.6844 [30] Hyperoside (Quercetin [10,33,34,39– 55 3-O-galactoside; C H O 464.3763 21 20 12 42] Hyperin) Quercetin 56 3-O-glucoside [ C21H20O12 464.3763 [33,46] Isoquercitrin] Alpha.-Tocopherol-Beta- d-Mannoside 57 C H O 592.8467 [48] [Dihydro-2H-Chromen- 35 60 7 6-YI Hexofuranoside] Colors are added for readability to avoid confusing columns. Green shades Rh. adamsii. blue Rh. sichotense. Molecules 2020, 25, 3774 8 of 20

Table 4. Components identiﬁed from the SC-CO2 extracts of Rh. sichotense and Rh. adamsii.

Calculated Observed Mass Observed Mass Observed Mass MS/MS Stage 2 MS/MS Stage 3 MS/MS Stage 4 Species of No. Identiﬁcation Formula Mass [M H] [M + H]+ [M + Na]+ Fragmentation Fragmentation Fragmentation Rhododendron − − Lepalol [5-(3-Furyl) 1 C H O 166.217 165.06 147.01 Rh. adamsii -2-methyl-1-penten-3-ol 10 14 2 Caﬀeic acid [(2E)-3- 2 (3,4-Dihydroxyphenyl) C9H8O4 180.1574 181.08 163.03; 135.11 Rh. adamsii acrylic acid] Azelaic acid 3 C H O 188.2209 210.09 192.12 175.06; 136.12 Rh. adamsii [Nonanedioic acid] 9 16 4 Calamenene 4 C H 202.3352 203.09 147.05 119.06 Rh. sichotense [Cis-Calamenene] 15 22

5 Germacron C15H22O 218.3346 219.06 201.07; 149.07 159.07 Rh. adamsii Myristic acid 6 (Tetradecanoic acid; C14H28O2 228.3709 251.09 150.48 149.08 Rh. adamsii N-Tetradecanoic acid) Pentadecanoic acid 7 C H O 242.3975 243.06 201.01; 137.05 181.05; 135.04 Rh. adamsii (Pentadecylic acid) 15 30 2

8 Palmitoleic acid C16H30O2 254.4082 277.09 275.04; 207.05 256.99 157.11 Rh. adamsii Cis-cyclopropan-9,10- 9 C H O 268.4348 269.02 185.97; 121.08 176.96 154.98 Rh. adamsii hexadecanoic acid 17 32 2 Linoleic acid [Linolic acid; 10 C H O 280.4455 303.06 285.05; 163.00 180.95; 135.06 162.99 Rh. adamsii Telfairic acid] 18 32 2 Stearic acid 284.18; 229.07; Rh. adamsii; 11 [Octadecanoic acid; C H O 284.4772 285.07 180.90; 135.05 163.03 18 36 2 163.02 Rh. sichotense Stearophanic acid] Kaempferol [3,5,7- Trihydroxy-2-(4-hydro- 286.24; 204.96; 12 C H O 286.2363 287.00 181.02 162.88 Rh. adamsii xyphenyl)-4-H-chromen- 15 10 6 163.02 4-one] Cis-cyclopropan-9,10- 13 C H O 292.4562 293.05 274.98 256.99; 162.98 201.03 Rh. adamsii octadecanoic acid 19 32 2 Nonadecanoic acid Rh. adamsii; 14 C H O 298.5038 300.09 243.04 201.02 [N-Nonadecanoic acid] 19 38 2 Rh. sichotense Kaempferol 5-methyl Rh. adamsii; 15 C H O 300.2629 300.98 283.01; 177.01 264.98 200.98 ether 16 12 6 Rh. sichotense Molecules 2020, 25, 3774 9 of 20

Table 4. Cont.

Calculated Observed Mass Observed Mass Observed Mass MS/MS Stage 2 MS/MS Stage 3 MS/MS Stage 4 Species of No. Identiﬁcation Formula Mass [M H] [M + H]+ [M + Na]+ Fragmentation Fragmentation Fragmentation Rhododendron − − Farrerol [5,7-Dihydroxy- Rh. adamsii; 16 2-(4-hydroxyphenyl)-6,8- C H O 300.3059 301.05 283.04 241.01; 162.96 17 16 5 Rh. sichotense dimethylchroman-4-one] Quercetin [2-(3,4- Dihydroxyphenyl)-3,5,7- Rh. adamsii; 17 C H O 302.2357 301.09 303.08 285.01; 163.02 180.97; 145.00 162.98 trihydroxy-4-H- 15 12 7 Rh. sichotense chromen-4-one] Dihydroquercetin 266.96; 241.09; 18 C H O 304.2516 303.09 285.04 171.02 Rh. adamsii [Taxifolin; Taxifoliol] 15 12 7 215.05; 135.05 Cannabigerorcinic acid 19 [Cannabigerorcinolic acid; C18H24O4 304.3808 303.08 285.05 241.07; 159.07 159.01 Rh. adamsii Cannabiorcogerolic acid]

20 Docosane C22H46 310.6006 311.10 293.06; 167.01 259.03 240.97; 162.96 Rh. adamsii 8-Demethyleucalyptin [5-Hydroxy-40,7-dimetoxy- 21 6-methylﬂavone; C18H16O5 312.3166 311.14 311.10; 182.99 Rh. adamsii Pabalate; Sodium salicylate] Arachic acid [Arachidic 284.99; 238.14; 22 C H O 312.5304 311.14 335.04 303.06; 195.01 180.89; 135.14 Rh. adamsii acid; eicosanoic acid] 20 40 2 163.00 Azaleatin 23 C H O 316.2623 315.08 297.01; 167.04 235.04; 149.00 Rh. adamsii [5-O-Methylquercetin] 16 12 7 Myricetin [3,5,7-Trihydroxy- Rh. adamsii; 24 C H O 318.2351 317.08 299.01; 241.01 240.06; 197.09 238.99; 197.04 2-(3,4,5-Trihydroxyphenyl)- 15 10 8 Rh. sichotense 4H-Chromen-4-One] Gossypetin [Articulatidin; 25 C H O 318.2351 319.07 287.09; 176.98 146.99 Rh. adamsii Equisporol] 15 10 8 Ampelopsin 26 [Dihydromyricetin; C15H12O8 320.251 319.08 317.01; 275.09 257.12; 217.11 Rh. adamsii Ampeloptin] Heneicosanoic acid 271.01; 217.03; Rh. adamsii; 27 C H O 326.557 325.11 327.08 149.10 [Heneicosylic acid] 21 42 2 177.06 Rh. sichotense Molecules 2020, 25, 3774 10 of 20

Table 4. Cont.

Calculated Observed Mass Observed Mass Observed Mass MS/MS Stage 2 MS/MS Stage 3 MS/MS Stage 4 Species of No. Identiﬁcation Formula Mass [M H] [M + H]+ [M + Na]+ Fragmentation Fragmentation Fragmentation Rhododendron − − Myricetin 5-Methyl ether 28 C H O 332.2617 331.03 168.94 149.96 Rh. sichotense [5-O-Methylmyricetin] 16 12 8 Esculin [Aesculin; 281.01; 217.11; Rh. adamsii; 29 C H O 340.2821 341.09 174.96 Esculoside; Polichrome] 15 16 9 151.06 Rh. sichotense Behenic acid 323.10; 243.11; Rh. adamsii; 30 C H O 340.5836 341.05 159.05 [Docosanoic acid] 22 44 2 177.04 Rh. sichotense Pentacosane 31 C H 352.6854 353.12 270.97; 162.97 180.93 162.96 Rh. sichotense (N-Pentacosane) 25 52 Rh. adamsii; 32 Chlorogenic acid C H O 354.3087 355.09 287.05; 164.02 180.95 163.03 16 18 9 Rh. sichotense Scopolin [Scopoloside; Rh. adamsii; 33 Scopoletin-7-glucoside; C H O 354.3087 355.02 323.00 303.96; 184.89 162.86 16 18 9 Rh. sichotense Murrayin] Tricosanoic acid 34 C H O 354.6101 355.08 322.96; 163.00 180.96 162.96 Rh. adamsii [N-Tricosanoic acid] 23 46 2 Lignoceric acid 351.08; 285.02; Rh. adamsii; 35 C H O 368.6367 367.12 369.08 163.02 144.97 [Tetracosanoic acid] 24 48 2 218.92; 162.98 Rh. sichotense Fraxin Rh. adamsii; 36 C H O 370.3081 371.08 338.99 320.96; 177.03 224.96 (Fraxetin-8-O-glucoside) 16 18 10 Rh. sichotense 352.98; 287.08; 231.04; 205.05; Rh. adamsii; 37 Daurichromenic acid C H O 370.4819 371.09 180.93; 144.97 23 30 4 235.08; 179.02 162.99 Rh. sichotense Pentacosanoic acid Rh. adamsii; 38 C H O 382.6633 383.07 405.08 351.04; 287.99 229.04 211.03 [N-Pentacosanoic acid] 25 50 2 Rh. sichotense Fraxetin-7-O-beta- Rh. adamsii; 39 C H O 384.2916 383.09 365.09; 190.96 266.97; 215.02 170.97 glucuronide 16 16 11 Rh. sichotense Beta-Sitosterin Rh. adamsii; 40 C H O 414.7067 415.04 384.02 369.01 338.00 [Beta-Sitosterol] 29 50 Rh. sichotense Cyanidin-3-alpfa-l- 399.05; 319.02; 41 C H O 419.3589 418.51 381.068 162.02 337.02; 253.08 Rh. adamsii arabinoside 20 19 10 194.99 Montanic acid 389.00; 348.98; 333.00; 280.97; Rh. adamsii; 42 C H O 424.743 425.02 407.00 [Octacosanoic acid] 28 56 2 298.99; 240.97 173.02 Rh. sichotense Alpha-Amyrin 408.27; 308.99; 373.08; 229.10; Rh. adamsii; 43 C H O 426.7174 427.05 389.02; 309.01 [Viminalol] 30 50 202.91 142.80 Rh. sichotense Molecules 2020, 25, 3774 11 of 20

Table 4. Cont.

Calculated Observed Mass Observed Mass Observed Mass MS/MS Stage 2 MS/MS Stage 3 MS/MS Stage 4 Species of No. Identiﬁcation Formula Mass [M H] [M + H]+ [M + Na]+ Fragmentation Fragmentation Fragmentation Rhododendron − − Lupeol [Fagarasterol; 44 Clerodol; Monogynol B; C30H50O 426.7174 427.04 409.01; 202.99 389.02; 247.99 370.96; 264.80 Rh. adamsii Lupenol] Dihydroquercetin 45 C H O 432.3344 433.97 352.95 323.53; 271.96 241.95; 181.87 Rh. adamsii 3-arabinofuranoside 20 16 11 Afzelin [ 413.00; 372.98; 46 Kaempferol-3-Rhamnoside; C H O 432.3775 431.04 354.95; 167.01 336.98; 148.91 Rh. sichotense 21 20 10 216.94 Kaempferin] Quercetin-3-O-beta-xyloside 47 (Reynoutrin; Quercetin C20H17O11 433.3424 434.90 302.94 256.92; 164.96 228.91; 159.11 Rh. sichotense 3-O-Beta-d-Xylopyranoside) Avicularin (Quercetin 3-Alpha-l- 415.07; 335.01; Rh. adamsii; 48 C H O 434.3503 433.09 397.06; 190.99 353.07; 253.99 Arabinofuranoside; 20 18 11 176.98 Rh. sichotense Avicularoside) Pentoside 416.54; 300.99; 361.11; 283.02; Rh. adamsii; 49 436 435.16 397.02; 205.96 dihydroquercetin 231.01 188.80 Rh. sichotense Erithrodiol 425.06; 381.05; 50 C H O 442.7168 441.12 363.06; 246.02 319.08; 201.02 Rh. adamsii [3beta-Erytrodiol] 30 50 2 300.03; 217.04 Rh. adamsii; 51 Uvaol C H O 442.7168 443.22 425.01; 233.07 407.02; 325.01 388.99; 231.11 30 50 2 Rh. sichotense Quercitrin [Quercetin 334.90; 222.92; 52 3-l-Rhamnoside; C H O 448.3769 448.89 370.95; 282.93 352.95; 176.98 Rh. adamsii 21 20 11 176.97 Quercetrin] 435.15; 336.07; 417.16; 336.11; 53 Catechin-7-O-glucoside C H O 452.4087 453.17 209.09 Rh. sichotense 21 24 11 209.06 226.12

54 Micromeric acid C30H46O3 454.6844 455.05 408.98; 246.98 391.05; 287.96 250.96 Rh. adamsii Hyperoside (Quercetin 55 3-O- galactoside; C21H20O12 464.3763 465.02 302.91 256.94; 190.87 228.96; 172.75 Rh. sichotense Hyperin) Quercetin 3-O-glucoside 56 C H O 464.3763 465.08 447.00 386.96 369.12; 172 Rh. sichotense [Isoquercitrin] 21 20 12 Alpha.-Tocopherol-Beta-d- Mannoside [Dihydro-2H- 57 C H O 592.8467 593.11 533.08 461.10 433.11 Rh. sichotense Chromen-6-YI 35 60 7 Hexofuranoside] Molecules 2020, 25, 3774 12 of 20 Molecules 2020,, 25,, xx FORFOR PEERPEER REVIEWREVIEW 13 of 21

FiguresFigures 22–11–11 showsshows examplesexamples ofof thethe decodingdecoding spectraspectra (collision-induced(collision-induced dissociationdissociation (CID)(CID) spectrum)spectrum) of of the the ion ion chromatogram chromatogram obtained obtained using using tandemtandem tandem massmass mass spectrometry.spectrometry. spectrometry. TheThe The CIDCID CID spectrumspectrum spectrum inin in negativenegative ion ion modes modes of of fraxetin-7- fraxetin-7-OO-beta-glucoronide-beta-glucoronide from from Rh.Rh. adamsii adamsii andandand Rh.Rh. sichotense sichotense areare shown shown in in FiguresFigures 2 and 33..

Intens.Intens. 183. Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: -MS, 65.8min #2635 183. 1- Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: -MS, 65.8min #2635 x10x1077 1- 443.10443.10 11 1-1- 383.10383.10 1-1- 3-3- 539.14539.14 773.50773.50 00 x10x1066 183.183. 1-1- RhododendronRhododendron AdamsiiAdamsii SFESFE 16.0416.04 #2#2 Vol2Vol2 MS4_-1_01_109.d:MS4_-1_01_109.d: -MS2(3-MS2(383.10),83.10), 65.9min65.9min #2637#2637 365.10365.10 66 44 22 00 x10x1055 183.183. 1-1- RhododendronRhododendron AdamsiiAdamsii SFESFE 16.0416.04 #2#2 Vol2Vol2 MS4_-1_01_109.d:MS4_-1_01_109.d: -MS3(3-MS3(383.10->365.10),83.10->365.10), 65.9min65.9min #2640#2640 215.07215.07 44 1-1- 321.07321.07 2 2 149.09149.09 00 x10x1055 183.183. RhododendronRhododendron AdamsiiAdamsii SFESFE 16.0416.04 #2#2 Vol2Vol2 MS4_-1_01_109.d:MS4_-1_01_109.d: -MS4(3-MS4(383.10->365.10->215.07),83.10->365.10->215.07), 66.0min66.0min #2643#2643 1-1- 171.02171.02 0.50.5 4-4- 212.86212.86 0.00.0 200200 400400 600600 800800 10001000 12001200 14001400 16001600 18001800 20002000 m/z m/z FigureFigure 2. 2. Collision-inducedCollision-induced dissociation dissociation ((CID) (CID) spectrum spectrum of fraxetin-7- of fraxetin-7-O-beta-glucoronide-beta-glucoronideO-beta-glucoronide fromfrom from Rh. adamsiiRh. adamsii,, m/z, 383.10.m/z 383.10.

The [M H]− ion produced fragment ion with m/z 383.10 (Figure2). The fragment ion with m/z The [M −− H]H]− −ionion producedproduced fragmentfragment ionion withwith m/z 383.10383.10 (Figure(Figure 2).2). TheThe fragmentfragment ionion withwith m/z 383.10383.10 produced produced characteristic characteristic daughter daughter ion ion with with m/zm/z 321.07,321.07,321.07, m/zm/z 215.07215.07215.07 andand and m/zm/z 149.09.149.09.149.09. TheThe The fragmentfragment fragment ionionion withwith with m/zm/z 215.07215.07215.07 formedformed formed twotwo two daughterdaughter daughter ionsions ions withwith with m/zm/z 171.02,171.02,171.02, m/zm/zm/z 212.86.212.86.212.86. ItIt It waswas was identifiedidentified identiﬁed inin in thethe the bibliographybibliography in in extract extract from from rhododendron rhododendron L.L. palustre palustre [13,30–33,45].[13,30–33,45].[13,30–33,45].

Intens.Intens. 14. Rh sichotense 01.06 MS4 #2_-1_01_176.d: -MS, 5.9min #198 14. 1- Rh sichotense 01.06 MS4 #2_-1_01_176.d: -MS, 5.9min #198 x10x1077 1- 369.15369.15 1.01.0 1-1- 0.50.5 383.13383.13 1-1- 210.99210.99 478.96478.96 0.00.0 x10x1066 14.14. 1-1- RhRh sichotensesichotense 01.0601.06 MS4MS4 #2_-1_01_176.d:#2_-1_01_176.d: -MS2(383.13),-MS2(383.13), 5.9min5.9min #2#20000 1.51.5 365.07365.07 1.01.0 0.50.5 191.00191.00 0.00.0 x10x1055 14.14. 1-1- RhRh sichotensesichotense 01.0601.06 MS4MS4 #2_-1_01_176.d:#2_-1_01_176.d: -MS3(383.13->365.07),-MS3(383.13->365.07), 66.0min.0min #204#204 215.02215.02 1.01.0 1-1- 0.50.5 267.06267.06

0.00.0 x10x1044 14.14. RhRh sichotensesichotense 01.0601.06 MS4MS4 #2_-1_01_176.d:#2_-1_01_176.d: -MS4(383.13->365.07->21-MS4(383.13->365.07->215.02),5.02), 6.1min6.1min #208#208 22 170.99170.99

00 200200 400400 600600 800800 10001000 12001200 14001400 16001600 18001800 m/z FigureFigure 3. 3. CIDCID spectrum spectrum of of fraxetin-7- fraxetin-7-OO-beta-glucoronide-beta-glucoronide-beta-glucoronide fromfrom from Rh.Rh. sichotense sichotense,, ,m/zm/z 383.13.383.13.

The [M H]−− ion produced fragment ion with m/z 383.13 (Figure3).The fragment ion with m/z The [M −− H]H] −ionion producedproduced fragmentfragment ionion withwith m/z 383.13383.13 (Figure(Figure 3).The3).The fragmentfragment ionion withwith m/z 383.13383.13 produced twotwo fragments fragments with withm/ zm/z365.07, 365.07,365.07,m/z 191.00.m/z 191.00.191.00. The fragment TheThe fragmentfragment ion with ionionm / zwithwith365.07 m/z produced 365.07365.07 producedtwo characteristic two characteristic daughter daughter ions with ionsm/z with267.06 m/z and 267.06267.06m/z andand215.02. m/z 215.02.215.02. The fragment TheThe fragmentfragment ion with ionionm withwith/z 215.02 m/z 215.02formed formed a daughter a daughter ion with ionm with/z 170.99. m/z 170.99.170.99. It was ItIt identiﬁed waswas identifiedidentified in the bibliographyinin thethe bibliographybibliography of the methanolicofof thethe methanolicmethanolic extract extractfrom rhododendron from rhododendronL. palustre L. palustre[30–33 , [30–33,44].44[30–33,44].]. Rh. adamsii Rh. sichotense The CID spectrum in positive ion modes of Beta-sitosterin from and is shown in Figures4 and5.

Molecules 2020,, 25,, xx FORFOR PEERPEER REVIEWREVIEW 14 of 21

MoleculesThe2020 CID, 25 spectrum, 3774 in positive ion modes of Beta-sitosterin from Rh. adamsii and Rh. sichotense13 of is 20 shown in Figures 4 and 5.

Intens.Intens. 91. Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: +MS, 52.2-52.7min #2076-2096 7 91. 1+ Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: +MS, 52.2-52.7min #2076-2096 x107 1+ 270.27 1+ 415.04 1+ 1 455.01 1+ 455.01 3+ 3+ 3+ 701.72 601.97 804.73 886.56 07 x107 91. 1+ Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: +MS2(415.04), 52.3-52.7min #2079-2098 384.02

0.5

0.06 x106 91. 1+ Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: +MS3(415.04->384.02), 52.3-52.8min #2082-2100 369.01 4

06 x106 91. Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: +MS4(415.04->384.02->369.01), 52.4-52.8min #2085-2102 1+ 338.00

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z FigureFigure 4. 4. CIDCID spectrum spectrum of of Beta-sitosterin Beta-sitosterin from from Rh.Rh. adamsii adamsii, ,m/zm/z 415.04.415.04.

The [M + H]+ ion produced one fragment with m/z 415.04 (Figure4). The fragment ion with The [M + H]+ ion produced one fragment with m/z 415.04 (Figure 4). The fragment ion with m/z m/z 384.02 produced one daughter ion with m/z 369.01. The fragment ion with m/z 369.01 formed a 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 daughter ion with m/z 338.00. It was identified in the bibliography in the extract from rhododendrons L. palustre [30–33,41,44,45] and Rh. adamsii [10,12,15,16,42,52]. L. palustre [30–33,41,44,45] and Rh. adamsii [10,12,15,16,42,52].

Intens.Intens. 237. Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS, 69.6min #2778 6 237. 1+ Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS, 69.6min #2778 x106 1+ 6 355.10 1+ 4 1+ 415.05 2+ 1+ 2 270.26 559.14 782.36 06 x106 237. 1+ Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS2(415.05), 69.7min #2782 384.06

06 x106 237. 1+ Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS3(415.05->384.06), 69.8min #2786 369.03

0.5

0.05 x105 237. Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS4(415.05->384.06->369.03), 69.9min #2790 1+ 338.05 4

0 200 400 600 800 1000 1200 1400 1600 1800 m/z FigureFigure 5. 5. CIDCID spectrum spectrum of of Beta-sitosterin Beta-sitosterin from from Rh.Rh. sichotense sichotense, ,m/zm/z 415.05.415.05.

+ The [M + H]+ ion produced one fragment with m/z 384.06 (Figure5). The fragment ion with The [M + H]+ ion produced one fragment with m/z 384.06 (Figure 5). The fragment ion with m/z m z 384.06 produced one daughter ion with m z 369.03. The fragment ion with m z 369.03 formed a 384.06/ produced one daughter ion with m/z 369.03./ The fragment ion with m/z/ 369.03 formed a m z daughterdaughter ion ion with with m/z/ 338.05.338.05. It It was was identified identiﬁed in in the the bibliography bibliography in in the the extract extract from from rhododendrons rhododendrons L. palustre Rh. adamsii L. palustre [30–33,41,44,45][30–33,41,44,45] and and Rh. adamsii [10,12,15,16,42,52].[10,12,15,16,42,52]. Rh. adamsii Rh. sichotense TheThe CID spectrumspectrum inin negative negative ion ion modes modes of quercetinof quercetin from from Rh. adamsiiand and Rh. sichotenseis shown is shownin Figures in Figures6 and7. 6 and 7.

IntensIntens . . 189. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS, 65.5min #2619 7 189. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS, 65.5min #2619 x107 301.07 1- 383.06 1 383.06

07 x107 189. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS2(301.07), 65.6min #2620 283.03

06 x106 189. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS3(301.07->283.03), 65.6min #2623 255.11 1- 177.08 1

05 x105 189. Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS4(301.07->283.03->255.11), 65.7min #2626 2 173.05

1 253.03 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z FigureFigure 6. 6. CIDCID spectrum spectrum of of quercetin quercetin from from Rh.Rh. adamsii adamsii, ,m/zm/z 301.07.301.07.

Molecules 2020, 25, 3774 14 of 20 Molecules 2020, 25, x FOR PEER REVIEW 15 of 21 Molecules 2020, 25, x FOR PEER REVIEW 15 of 21 The [M − H]− ion produced one fragment ion with m/z 283.03 (Figure 6). The fragment ion with The [M H]− ion produced one fragment ion with m/z 283.03 (Figure6). The fragment ion with − − m/zm/z 283.03283.03The [M produced produced − H] ion producedtwo two daughter daughter one fragment ions ions with with ion m/zm with/z 255.11255.11 m/z 283.03and and m/zm (Figure/z 177.08. 6). The The fragmentfragment fragment ion ion ion with with with m/zm/z m/z 283.03 produced two daughter ions with m/z 255.11 and m/z 177.08. The fragment ion with m/z 255.11255.11 formed formed two two daughter daughter ions ions with with m/zm/z 253.03253.03 and and m/zm/z 173.05.173.05. It It was was identified identiﬁed in in the the bibliography bibliography 255.11 formed two daughter ions with m/z 253.03 and m/z 173.05. It was identified in the bibliography inin extracts extracts from from rhododendrons Rh. sichotense, Rh. micronulatum micronulatum [38–40][38–40];; Rh.Rh. ungernii ungernii [34];[34]; RhodiolaRhodiola in extracts from rhododendrons Rh. sichotense, Rh. micronulatum [38–40]; Rh. ungernii [34]; Rhodiola crenulatacrenulata [36];[36]; Rh.Rh. adamsii adamsii [10,12,15,16,42,52];[10,12,15,16,42,52]; Rh.Rh. parvifolium parvifolium [10][10];; OcimumOcimum [43].[43]. crenulata [36]; Rh. adamsii [10,12,15,16,42,52]; Rh. parvifolium [10]; Ocimum [43]. Intens . 194. Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS, 63.6min #2532 x107 1- Intens . 194. 383.09 Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS, 63.6min #2532 x107 1- 4 383.09 1- 24 301.071- 02 301.07 x107 194. 1- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS2(301.07), 63.6min #2533 0 x107 194. 283.051- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS2(301.07), 63.6min #2533 283.05 0.5 0.5 0.0 x100.05 194. Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS3(301.07->283.05), 63.7min #2537 x105 194. 1- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS3(301.07->283.05), 63.7min #2537 177.061- 255.09 4 177.06 255.09 4 2 1- 2 252.991- 252.99 0 05 x10x105 194. Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS4(301.07->283.05->252.99), 63.8min #2541 194. 1-1- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS4(301.07->283.05->252.99), 63.8min #2541 173.05173.05 1

211.08211.08 0 200200 400400 600600 800 800 1000 1000 1200 1200 1400 1400 1600 1600 1800 m/z1800 m/z FigureFigureFigure 7. 7. 7.CID CIDCID spectrum spectrum spectrum of quercetinof of quercetin quercetin from from from Rh. Rh.sichotenseRh. sichotense sichotense, m/z ,301.07., m/zm/z 301.07.301.07.

The [M H]− ion produced one fragment ion with m/z 283.03 (Figure7). The fragment ion with The [M[M −−− H]H] −ion− ion produced produced one one fragment fragment ion ion with with m/z m/z 283.03 283.03 (Figure (Figure 7). The 7). fragmentThe fragment ion with 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 m/zm/z 283.03 producedproduced three three daughter daughter ions ions with with m/z m/z 255.09, 255.09, m/z m/z177.06 177.06 and andm/z 252.99.m/z 252.99. The fragmentThe 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 ionion with m/zm/z 252.99252.99 formed formed two two daughter daughter ions ions with with m/z m/z 211.08 211.08 and and m/z m/z173.05. 173.05. It was It identifiedwas identified in the in the bibliography in in extrac extractsts from rhododendrons Rh.Rh. sichotense, sichotense, Rh. Rh. micronulatum micronulatum [38–40][38–40; ];Rh.Rh. ungernii ungernii [34]; bibliography in extracts from rhododendrons Rh. sichotense, Rh. micronulatum [38–40]; Rh. ungernii [34];Rhodiola Rhodiola crenulata crenulata[36]; [36];Rh. Rh. adamsii adamsii[10 [10,12,15,16,42,52];,12,15,16,42,52]; Rh. Rh. parvifolium parvifolium [[10]10];; Ocimum [43].[43]. [34]; Rhodiola crenulata [36]; Rh. adamsii [10,12,15,16,42,52]; Rh. parvifolium [10]; Ocimum [43]. TheThe CID spectrum in negative ion modesmodes ofof myricetinmyricetin fromfromRh. Rh. adamsiiadamsiiand andRh. Rh. sichotensesichotenseis is shown The CID spectrum in negative ion modes of myricetin from Rh. adamsii and Rh. sichotense is shownin Figures in Figures8 and9 .8 and 9. shown in Figures 8 and 9.

Intens . 36. Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS, 39.8min #1570 6 1- x10 1- Intens . 36. 303.09 Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS, 39.8min #1570 6 1- 317.101- x10 1- 1 181.07 303.09 1- 317.10 01 181.07 x106 36. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS2(317.10), 39.8min #1571 299.03 1.00 x106 36. 1- 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS2(317.10), 39.8min #1571 299.03 0.51.0 259.05 148.99 1- 0.0 259.05 x100.55 36. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS3(317.10->299.03), 39.9min #1574 2 148.99 241.04 0.0 x105 36. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS3(317.10->299.03), 39.9min #1574 1 241.041- 2 281.01 0 x1014 36. 1- Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS4(317.10->299.03->241.04), 39.9min #1577 1- 281.01 4 238.99 0 x104 36. Rhododendron Adamsii SFE 14.04 #2 Vol1 MS4_-1_01_105.d: -MS4(317.10->299.03->241.04), 39.9min #1577 2 1- 4 238.99 0 2 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z 0 200 Figure400 8. CID600 spectrum800 of myricetin1000 from 1200Rh. adamsii1400, m/z 317.10.1600 1800 2000 m/z FigureFigure 8. 8. CIDCID spectrum spectrum of of myricetin myricetin from from Rh.Rh. adamsii adamsii,, m/zm/z 317.10.317.10. 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 The [M H]− ion produced three fragment ions with m/z 299.03, m/z 259.05 and m/z 148.99 The [M − −H] ion− produced three fragment ions with m/z 299.03, m/z 259.05 and m/z 148.99 (Figure fragment(Figure8). ion The with fragment m/z 241.04 ion withformedm/ za299.03 daughter produced ion with two m/z daughter 238.99. It ions was with identifiedm/z 241.04 in the and m/z 8).bibliography The fragment in extrac ion withts from m/z rhododendrons299.03 produced Rh. two sichotense, daughter Rh. ions micronulatum with m/z 241.04 [39–41] and; Rh. m/z ungernii 281.01. The 281.01. The fragment ion with m/z 241.04 formed a daughter ion with m/z 238.99. It was identiﬁed in the fragment[35]; Rhodiola ion crenulata with m/z [37]; 241.04 Rh. adamsii formed [16,17,43,53]; a daughter Rh. ionparvifolium with m/z [41] ;238.99. Ocimum It [44]. was identified in the bibliographybibliography in extractsextractsfrom from rhododendrons rhododendronsRh. Rh. sichotense, sichotense, Rh. Rh. micronulatum micronulatum[39 –[39–41]41]; Rh.; Rh. ungernii ungernii[35]; [35];Rhodiola Rhodiola crenulata crenulata[37]; [37];Rh.adamsii Rh. adamsii[16,17 [16,17,43,53];,43,53]; Rh. parvifoliumRh. parvifolium[41]; [41]Ocimum; Ocimum[44]. [44].

Molecules 2020, 25, 3774 15 of 20

Molecules 2020,, 25,, xx FORFOR PEERPEER REVIEWREVIEW 16 of 21

Intens . 12. 1- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS, 3.1min #121 Intens7 . 12. 1- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS, 3.1min #121 x107 317.14 x10 317.14 1- 1- 1 401.12 1 401.12 1- 1- 665.00 773.14 0 665.00 773.14 x1007 1- x107 12. 1- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS2(317.14), 3.1min #122 1.0 12. 299.06 Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS2(317.14), 3.1min #122 1.0 299.06 1- 0.5 1- 0.5 241.01 241.01 0.0 x100.05 1- x105 12. 1- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS3(317.14->299.06), 3.2min #126 12. 228.04 Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS3(317.14->299.06), 3.2min #126 228.04 6 6 4 4 3- 2 3- 2 129.92 129.92 0 x1004 4- x104 12. 4- Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS4(317.14->299.06->228.04), 3.3min #130 12. 227.10 Rh sichotense 01.06 MS4 #5_-1_01_190.d: -MS4(317.14->299.06->228.04), 3.3min #130 227.10

1 1

0 0 200 400 600 800 1000 1200 1400 1600 1800 m/z 200 400 600 800 1000 1200 1400 1600 1800 m/z Figure 9. CID spectrum of myricetin from Rh. sichotense, m/z 317.14. Figure 9. CID spectrum of myricetin from Rh. sichotense, m/z 317.14.

The [M H]− ion produced two fragment ions with m/z 299.06, m/z 241.01 (Figure9). The fragment The [M −− H]− ion produced two fragment ions with m/z 299.06, m/z 241.01 (Figure 9). The ion with m/z 299.06 produced two daughter ions with m/z 228.04 and m/z 129.92. The fragment ion with fragment ion with m/z 299.06 produced two daughter ions with m/z 228.04 and m/z 129.92. The m/z 228.04 formed a daughter ion with m/z 227.10. It was identiﬁed in the bibliography in extracts from fragment ion with m/z 228.04 formed a daughter ion with m/z 227.10. It was identified in the rhododendrons Rh. sichotense [4,5]; Rh. ungernii [34]; Rhodiola crenulata [36]; Rh. adamsii [10,15,16,42,52]; bibliography in extracts from rhododendrons Rh. sichotense [4,5]; Rh. ungernii [34]; Rhodiola crenulata Rh. parvifolium [10,40]. [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 The CID spectrum in positive ion modes of farrerol from Rh. adamsii and Rh. sichotense is shown in Figures 10 and 11. The [M + H]+ ion produced three fragment ions with m/z 283.02, m/z 244.99 in Figures 10 and 11. The [M + H]+ ion produced three fragment ions with m/z 283.02, m/z 244.99 and and m/z 162.98 (Figure 10). The fragment ion with m/z 283.02 produced two daughter ions with m/z m/z 162.98 (Figure 10). The fragment ion with m/z 283.02 produced two daughter ions with m/z 240.98 240.98 and m/z 163.01. The fragment ion with m/z 240.98 formed a daughter ion with m/z 170.96. It was 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 [38–41]; Rh. ungernii [34]. identified in the bibliography in extracts from rhododendrons Rh. dauricum [38–41]; Rh. ungernii [34].

Intens . 102. Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS, 53.8min #2142 Intens . 102. 1+ Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS, 53.8min #2142 x107 1+ 1+ x107 1+ 415.061+ 301.06 415.06 1 301.06 1+ 1 1+ 4+ 1+ 627.02 4+ 3+ 1+ 627.02 701.894+ 3+ 570.62 701.89 820.66 570.62 820.66 1053.14 1170.74 0 1053.14 1170.74 x1006 102. 1+ Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS2(301.06), 53.8min #2145 x106 102. 1+ Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS2(301.06), 53.8min #2145 283.02 1+ 283.02 1+ 1 162.98 1 162.98 244.99 1 244.99

05 x1005 102. 1+ Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS3(301.06->283.02), 53.9min #2148 x10 102. 240.981+ Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS3(301.06->283.02), 53.9min #2148 240.98

1 163.01 1 163.01

0 x1004 102. Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS4(301.06->283.02->240.98), 54.0min #2151 x104 102. Rhododendron Adamsii SFE 16.04 #2 Vol2 MS4_-1_01_109.d: +MS4(301.06->283.02->240.98), 54.0min #2151 1.0 170.96 1.0 170.96 0.5 0.5 0.0 0.0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z FigureFigure 10. 10. CIDCID spectrum spectrum of of farrerol farrerol from from Rh.Rh. adamsii adamsii,, m/zm/z 301.06.301.06.

+ TheThe [M [M ++ H]H]+ ionion produced produced two two fragment fragment ions ions with with m/zm/ z282.95282.95 and and m/zm/ z180.98180.98 (Figure (Figure 11). 11).

Intens . 9. 1+ Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS, 2.8min #109 Intens . 9. 1+ 1+ Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS, 2.8min #109 x106 301.04 1+ x106 301.04 425.01 4 425.01 4 1+ 2+ 1+ 2 1+ 2+ 1+ 2 192.96 651.95 751.69 831.92 192.96 651.95 751.69 831.92 1062.62 1258.80 1062.62 1258.80 1508.52 0 1508.52 x1006 1+ x106 9. 1+ Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS2(301.04), 2.8min #110 9. 180.98 Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS2(301.04), 2.8min #110 180.98 2 2 1 2+ 1 2+ 282.95 282.95 406.86 0 406.86 x1004 x104 9. Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS3(301.04->180.98), 2.9min #114 9. 163.00 Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS3(301.04->180.98), 2.9min #114 163.00 4 4 2 2 402.99 402.99 0 0 9. Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS4(301.04->180.98->163.00), 3.0min #118 9. Rh sichotense 01.06 MS4 #5_-1_01_190.d: +MS4(301.04->180.98->163.00), 3.0min #118 1 1 0 0 -1 -1-1 200 400 600 800 1000 1200 1400 1600 1800 m/z 200 400 600 800 1000 1200 1400 1600 1800 m/z FigureFigure 11.11. CIDCID spectrumspectrum ofof farrerolfarrerol fromfromRh. Rh. sichotenseadamsii, m/z, m/ z301.06.301.06.

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 [38–40]; Rh. ungernii [34].

Molecules 2020, 25, 3774 16 of 20

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 [38–40]; 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. Molecules 2020, 25, 3774 17 of 20

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.

Supplementary Materials: The following are available online, Figure S1: Chemical profiles of the Rh. adamsii sample represented ion chromatogram from SC-CO2 extract; Figure S2: Chemical profiles of the Rh. sichotense sample represented ion chromatogram from SC-CO2 extract. Author Contributions: Conceptualization, A.Z. and K.G.; methodology, A.Z., M.R.; software, M.R.; validation, M.R., K.G.; formal analysis, M.R., A.Z., V.G.; investigation, M.R. and A.Z.; resources, K.G. and A.Z.; data curation, K.G.; writing—original draft preparation—M.R., A.Z., S.E.; writing—review and editing A.Z., V.G., S.E. and K.G.; visualization, M.R., A.Z., S.E. and V.G.; supervision, K.G.; project administration, A.Z., K.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Council on Grants of the President of the Russian Federation (CΠ3156.2019.4). Conflicts of Interest: The authors declare no conflict of interest.

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