Rapid Mass Spectrometric Study of a Supercritical CO2-Extract from Woody Liana Schisandra Chinensis by HPLC-SPD-ESI-MS/MS

molecules

Article Rapid Mass Spectrometric Study of a Supercritical CO2-extract from Woody Liana Schisandra chinensis by HPLC-SPD-ESI-MS/MS

Mayya Razgonova 1,2,* , Alexander Zakharenko 1,2 , Konstantin Pikula 1,2, Ekaterina Kim 1, Valery Chernyshev 1,2, Sezai Ercisli 3 , Giancarlo Cravotto 4 and Kirill Golokhvast 1,2,5

1 REC Nanotechnology, School of Engineering, Far Eastern Federal University, Sukhanova 8, 690950 Vladivostok, Russia; [email protected] (A.Z.); [email protected] (K.P.); [email protected] (E.K.); [email protected] (V.C.); [email protected] (K.G.) 2 N.I. Vavilov All-Russian Institute of Plant Genetic Resources, B. Morskaya 42-44, 190000 Saint-Petersburg, Russia 3 Agricultural Faculty, Department of Horticulture, Ataturk University, Erzurum 25240, Turkey; [email protected] 4 Dipartimento di Scienza e Technologia del Farmaco, University of Turin, Via P. Giuria 9, 10125 Turin, Italy; [email protected] 5 Paciﬁc Geographical Institute, Far Eastern Branch of the Russian Academy of Sciences, Radio 7, 690041 Vladivostok, Russia * Correspondence: [email protected]

Academic Editor: David Barker Received: 20 April 2020; Accepted: 8 June 2020; Published: 10 June 2020

Abstract: Woody liana Schisandra chinensis contains valuable lignans, which are phenylpropanoids with valuable biological activity. Among green and selective extraction methods, supercritical carbon dioxide (SC-CO2) was shown to be the method of choice for the recovery of these naturally occurring compounds. Carbon dioxide (CO ) was the solvent with the flow rate (10 25 g/min) with 2% ethanol 2 − as co-solvent. In this piece of work operative parameters and working conditions were optimized by experimenting with different pressures (200–400 bars) and temperatures (40–60 ◦C). The extraction time varied from 60 to 120 min. HPLC-SPD-ESI -MS/MS techniques were applied to detect target analytes. Twenty-six different lignans were identified in the S. chinensis SC-CO2 extracts.

Keywords: Schisandra chinensis; supercritical ﬂuid extraction; HPLC-SPD-ESI-MS/MS; lignans

1. Introduction Schisandra chinensis (Turczaninowia) Baillon, is a medicinal plant (of the Schisandraceae family), known for its ethnomedicinal applications [1]. Its use in Chinese medicine dates back about 15 centuries, second only to ginseng. S. chinensis is included in the traditional Chinese medicine formula Sheng-Mai San, which has been used in the treatment of cardiovascular diseases [2]. Schisandra-based drugs, with the common names Shengmai-injection, Shengi Wuweizi-Pan and Shengmai-Yin are also included in the Chinese Pharmacopoeia [3]. The genus Schisandra (Schisandraceae family) consists of 25 species, two of which, namely Schisandra chinensis and Schisandra repanda (Maximowiczia nigra (Maxim.) Nakai), have a history of medicinal use [4]. Schisandra chinensis (synonyms: Kadsura chinensis, Maximoviczia amurensis, Maximoviczia chinensis, Maximoviczia japonica, Sphaerostemma japonicum, Wu wei zi) is endemic in northwest China (Heilongjiang Province), Korea, Russia (Primorye and Amur regions as well as Khabarovsk territory), Shikotan, Kunashir, Iturup, and on the island of Sakhalin.

Molecules 2020, 25, 2689; doi:10.3390/molecules25112689 www.mdpi.com/journal/molecules Molecules 2020, 25, 2689 2 of 10 Molecules 2020, 25, x FOR PEER REVIEW 2 of 11

RussianRussian academician KomarovKomarov V.L.V.L. made made a a botanical botanical description description of ofS. S. chinensis chinensis(Turcz.) (Turcz.) Baill. Baill. for forthe the first first time time in in 1901 1901 and and gave gave the the first first information information about about its its healing healing eeffect,ffect, havinghaving brought it from from expeditionsexpeditions to the Far EastEast [[5,6].5,6]. ThisThis isis aa deciduousdeciduous liana, liana, climbing climbing up up neighboring neighboring trees, trees, up up to to 10 1015−15 m − mlong. long. The The stem stem is is covered covered with with wrinkled, wrinkled, flakyflaky dark dark brown brown bark. bark. TheThe leavesleaves areare ellipticalelliptical 5−1010 cm cm − long,long, 3 3−5cm5 cm wide, wide, dicotyledonous dicotyledonous flowers, flowers, up up to to 1.5 1.5 cm cm in in diameter, diameter, with with a a distinct distinct lemon lemon aroma, aroma, − multi-berrymulti-berry fruits, fruits, up up to to 10 10 cm cm long, long, juicy juicy red red s seedseeds with with a a smooth smooth shiny shiny surface, surface, yellowish-brown. yellowish-brown. PlantPlant grinding grinding develops develops an an intense intense characteristic characteristic smell smell while while the the taste taste is is spicy spicy and and bitter-burning. bitter-burning. TheThe whole whole plant plant has has a a specific specific lemon lemon smell. smell. The The modern modern use use of of S.S. chinensis chinensis startedstarted with with a a large large number number ofof pharmacological pharmacological and and clinical clinical studies studies conducted conducted in the former USSR in the period 1940 −19601960 [7,8]. [7,8]. − VariousVarious descriptions of of the the specific specific properties properties of of S.S. chinensis chinensis areare available available in in English English in in reviews reviews ofof Far Far Eastern Eastern medicinal medicinal plants. plants. [9]. [9]. However, However, a a large large amount amount of of information information that that was was reported reported in in RussianRussian journals journals [4,10–16] [4,10–14] is is practically practically no nott accessible accessible to to foreign foreign scientists [17]. [15]. MoreMore than than 40 40 individual individual lignans lignans have have been been reported reported in in the the literature, 11 11 of of them, them, namely namely schisandrin,schisandrin, gomisin gomisin J, J, gomisin gomisin A, A, gomisin gomisin G, G, angeloygomisin H, H, angeloygomisin O, O, schisantherin A,A, schisantherin B, B, γ-schisandrin-schisandrin (schisandrin (schisandrin B) B) and and schisandrin schisandrin C, C, characterize characterize the the S.S. chinensis chinensis (Turcz.)(Turcz.) Baill. Baill. present present in in several several pharmacopeias. pharmacopeias. It It has has a a chemical chemical composition composition that that differs differs from from the the non-pharmacopeianon-pharmacopeia species species SchizandraSchizandra sphenanthera sphenanthera Rehd. et et Wils Wils [18,19]. [16,17]. LignansLignans are are phenylpropane phenylpropane dimers cons consistingisting of two propane residues CC66–C3.. Lignans Lignans are are found found inin various various parts parts of of the the plant, plant, especially especially in in the the seeds, seeds, the the underground underground parts, parts, the the wood wood and and woody woody stems.stems. They They may may be be present present in in plants plants in in free free fo formrm and in the form of glycosidesglycosides [[20].18]. Schizandra lignanslignans are are called called schisandrins. schisandrins. The The chemical chemical skeleton skeleton of of S.S. chinensis chinensis lignanslignans is is depicted depicted in in Figure Figure 1 andand all all the the substituents substituents are are presented presented in Table 1 1..

FigureFigure 1. 1. StructureStructure of of S.S. chinensis chinensis lignans.lignans.

The lignans of S. chinensis have been typically extracted with ethanol or hazardous potentially The lignans of S. chinensis have been typically extracted with ethanol or hazardous potentially toxic organic solvents such as methanol, chloroform and n-hexane. A valid green alternative, in which toxic organic solvents such as methanol, chloroform and n-hexane. A valid green alternative, in which there is no need to work with a large number of organic solvents and the production does not need there is no need to work with a large number of organic solvents and the production does not need explosion-proof rooms, is represented by supercritical fluid extraction (SFE) with many advantages explosion-proof rooms, is represented by supercritical fluid extraction (SFE) with many advantages compared to common extraction methods (maceration, percolation Soxhlet extraction) [19,20]. compared to common extraction methods (maceration, percolation Soxhlet extraction) [21,22]. SFE is a green, mild and selective extraction process, one of the best processes to get rid of residual solvent in theTable extract. 1. Chemical Among structures the supercritical of S. chinensis solvents, lignans, carbon according dioxide to the is authors the most [20,23–26]. common, offering several advantages, because it is non-toxic, non-flammable, cost-effective, environmentally friendly №and renewableCompound [ 25–27Formula]. The SFER1 methodR2 is activelyR3 R studied4 R5 andR6applied R7 inR the8 processingR9 R10 of plantR11 Schisandrin A 1 C24H32O6 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H CH3 CH3 H materials(Deoxyschisandrin) [28,29]. TheSchisandrol lignans A of S. chinensis were extracted by supercritical CO2 (SC-CO2) using ethanol as 2 C24H32O7 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H CH3 OH H co-solvent(Schisandrin) [30–32 ]. Different parts of the plant were extracted by SFE, isolating 36 compounds from the leaves,Schisandrin 43 compounds B from lignified stems and 36 compounds from rhizomes and roots. S. chinensis 3 (Gomisin N, C23H28O6 CH3 CH3 CH3 CH3 CH2 H CH3 H CH3 H extractsIsokadsuranin) contain a volatile fraction rich in essential oils (terpenes: monoterpenes, sesquiterpenes; terpenoids:Schisandrol alcohols, B esters, ketones) and a non-volatile part (carboxylic acids and lignans). 4 C23H28O7 CH2 CH3 CH3 CH3 CH3 CH3 H CH3 OH H (Gomisin A) 5 Schisandrin C C22H24O6 CH2 CH3 CH3 CH3 CH3 H CH3 H CH3 H 6 Isoschisandrin C24H32O7 CH3 CH3 CH3 CH3 CH3 CH3 CH3 OH CH3 H H 7 Gomisin K1 C23H30O6 H CH3 CH3 CH3 CH3 CH3 H CH3 H CH3 H 8 Gomisin K2 C23H30O6 H CH3 CH3 CH3 CH3 CH3 CH3 H CH3 H H

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Table 1. Chemical structures of S. chinensis lignans, according to the authors [18,21–24].

№ Compound Formula R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 Schisandrin A 1 C H O CH CH CH CH CH CH CH H CH CH H (Deoxyschisandrin) 24 32 6 3 3 3 3 3 3 3 3 3 Schisandrol A 2 C H O CH CH CH CH CH CH CH H CH OH H (Schisandrin) 24 32 7 3 3 3 3 3 3 3 3 Schisandrin B 3 (Gomisin N, C23H28O6 CH3 CH3 CH3 CH3 CH2 H CH3 H CH3 H Isokadsuranin) Schisandrol B 4 C H O CH CH CH CH CH CH H CH OH H (Gomisin A) 23 28 7 2 3 3 3 3 3 3

5 Schisandrin C C22H24O6 CH2 CH3 CH3 CH3 CH3 H CH3 H CH3 H

6 Isoschisandrin C24H32O7 CH3 CH3 CH3 CH3 CH3 CH3 CH3 OH CH3 HH

7 Gomisin K1 C23H30O6 H CH3 CH3 CH3 CH3 CH3 H CH3 H CH3 H

8 Gomisin K2 C23H30O6 H CH3 CH3 CH3 CH3 CH3 CH3 H CH3 HH Schisanhenol 9 C H O CH CH H CH CH CH CH H CH HH (Gomisin K3) 23 30 6 3 3 3 3 3 3 3

10 Gomisin H C23H30O7 CH3 CH3 H CH3 CH3 CH3 CH3 H CH3 OH H

11 Tigloylgomisin H C28H36O8 CH3 CH3 Tigloyl CH3 CH3 CH3 CH3 H CH3 OH H

12 Angeloygomisin H C28H36O8 CH3 CH3 Angeloyl CH3 CH3 CH3 CH3 H CH3 OH H

13 Benzoylgomisin H C30H34O8 CH3 CH3 Benzoyl CH3 CH3 CH3 CH3 H CH3 OH H

4 Gomisin J C22H28O6 H CH3 CH3 CH3 CH3 H H CH3 H CH3 H

15 Schisanhenol B C22H26O6 CH3 CH3 H CH3 CH2 H CH3 H CH3 H

16 Gomisin N C23H28O6 CH3 CH3 CH3 CH3 CH2 CH3 H CH3 HH

17 Gomisin L1 C22H26O6 CH3 CH3 H CH3 CH2 H CH3 H CH3 H

18 Gomisin L2 C22H26O6 H CH3 CH3 CH3 CH2 H CH3 H CH3 H

19 Gomisin M1 C22H26O6 CH3 CH3 H CH3 CH2 CH3 H CH3 HH

20 Gomisin M2 C22H26O6 CH3 CH3 CH3 H CH2 CH3 H CH3 HH

21 Gomisin O C23H28O7 CH2 CH3 CH3 CH3 CH3 CH3 CH3 H H OH

22 Isogomisin O C23H28O7 CH3 CH3 CH3 CH3 CH2 H CH3 H CH3 OH

23 Angeloylsogomisin O C28H34O8 CH2 CH3 CH3 CH3 CH3 CH3 CH3 H H O-angeloyl

24 Gomisin P C23H28O8 CH2 CH3 CH3 CH3 CH3 H CH3 OH CH3 OH

25 Tigloylgomisin P C28H34O9 CH2 CH3 CH3 CH3 CH3 H CH3 OH CH3 O-tigloyl Angeloylgomisin P 26 C H O CH CH CH CH CH H CH OH CH O-angeloyl (Schisantherin C) 28 34 9 2 3 3 3 3 3 3 Schisantherin A 27 C H O CH CH CH CH CH CH CH OH H O-bensoyl (Gomisin C) 30 32 9 2 3 3 3 3 3 3 Schisantherin B 28 (Gomisin B, C28H34O9 CH2 CH3 CH3 CH3 CH3 CH3 CH3 OH H O-angeloyl Schisandrer B)

29 Gomisin S C23H30O7 CH3 CH3 CH3 CH3 CH3 CH3 H CH3 H CH3 OH Gomisin R 30 C H O CH CH CH CH CH H CH HH (6-Epi-gomisin) 22 24 7 2 3 3 2 3 3

31 Deangeloylgomisin B C23H28O8 CH2 CH3 CH3 CH3 CH3 CH3 CH3 OH H OH

32 Gomisin F C28H34O9 CH3 CH3 CH3 CH3 CH2 CH3 CH3 OH H O-angeloyl

33 Gomisin G C30H32O9 CH3 CH3 CH3 CH3 CH2 CH3 CH3 OH H O-bensoyl

34 Epigomisin O C23H28O7 CH2 CH3 CH3 CH3 CH3 CH3 CH3 CH3 HH

35 Angeloylgomisin Q C29H38O9 CH3 CH3 CH3 CH3 CH3 CH3 H CH3 CH3 OH O-angeloyl

2. Results and Discussion Aiming to optimize the extraction of target analytes from the S. chinensis woody liana, several experimental conditions were investigated. Carbon dioxide (CO2) was the solvent with the ﬂow rate (10 25 g/min) and 2% ethanol as co-solvent in the liquid phase. Extraction was performed in the − pressure range of 200–400 bar and the temperature range of 40–60 ◦C. The best results were obtained at 350 bar and 60 ◦C. Increasing the pressure from 350 to 400 bar practically gave no increase in yields. The temperature of 60 ◦C was chosen as the maximum allowable to avoid the decomposition of target analytes. In this work HPLC-SPD-ESI-MS/MS techniques were used with additional ionization and analysis of fragmented ions. High-accuracy mass spectrometric data were recorded on an ion trap amaZon SL BRUKER DALTONIKS equipped with an ESI source in the mode of negative ions. The three-stage ion separation mode was implemented. Under these conditions a total of 800 peaks were detected in the ion chromatogram (Figure2). Molecules 2020, 25, x FOR PEER REVIEW 4 of 11 Molecules 2020, 25, 2689 4 of 10 three-stage ion separation mode was implemented. Under these conditions a total of 800 peaks were detected in the ion chromatogram (Figure 2).

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Figure 2. Chemical profiles of S. chinensis (Russia), ion chromatogram from SC-CO2 extract. Figure 2. Chemical profiles of S. chinensis (Russia), ion chromatogram from SC-CO2 extract. Although this approach is not quantitative for evaluating each analyte, it is semiquantitative Althoughwhen comparing this approach a series of extractions is not quantitative and allows better for evaluating comparison of each the yield analyte, without it isloss semiquantitative of whenindividual comparing analytes a series during of fractionation extractions and and sample allows preparation. better comparison Only the total ofextraction the yield yields without loss of individualwere completely analytes duringquantified. fractionation and sample preparation. Only the total extraction yields were completelyTable quantified. 2 summarizes all the molecular masses of the target analytes isolated from SC-CO2 of S. chinensis. Among them, 26 biologically active substances were authenticated as lignans (m/z values Tableand fragment2 summarizes ions) by comparison all the molecular with literature masses data [2,24,25,35–38]. of the target analytes isolated from SC-CO 2 of S. chinensis. Among them, 26 biologically active substances were authenticated as lignans (m/z values and fragment ions) by comparisonTable 2. Lignans with literature from S. chinensis data SC-CO [2,222 extract.,23,33 –36]. Obser MS/MS MS/MS MS/MS Calcula- ved Observe Stage 1 Stage 2 Stage 3 № Identification Table 2. LignansFormula fromted S. chinensisMass dSC-CO Mass 2 extract. Fragme Fragmentat Fragment Mass [M + [M + Na]+ Observed Observed ntation i-on ation Calcula-ted H]+ MS/MS Stage 1 MS/MS Stage 2 MS/MS Stage 3 № Identification Formula Mass Mass Schisandrin C [(12S,13R)-3,22- Mass + + Fragmentation Fragmentati-on Fragmentation [M + H] [M + Na] 323.01; dimethoxy-12,13-dimethyl-5,7,18,20- Schisandrin C 355.01; 299.02; 307.98; 1 [(12S,13R)-3,22-dimethoxy-12,13-dimethyl-tetraoxapentacyclo C22H24O6 384.4224 385.02 323.02 269.03;323.01; 299.02; 235.05 1 [13.7.0.02,10.04,8.017,21]docosa-5,7,18,20-tetraoxapentacyclo C22H24O6 384.4224 385.02 355.01; 323.02 307.98; 235.05 234.98269.03; 234.98 [13.7.0.02,10.04,8.017,21]docosa-1(22),1(22),2,4(8),9,15,17(21)-hexaene] 2,4(8),9,15,17(21)-hexaene] Gomisin M1 (Gomisin L1) [(9S,10R)- Gomisin M1 (Gomisin L1) [(9S,10R)-4,5, 290.99; 4,5,19-trimethoxy-9,10-dimethyl-15,17-19-trimethoxy-9,10-dimethyl-15, 290.99; 394.03; 260.97;260.97; 242.89; 2 C22H26O6 386.4382 408.95 394.03; 2 17-dioxatetracyclodioxatetracyclo [10.7.0.02,7.014,18] C22H26O6 386.4382 408.95 326.08; 274.96 242.89;172.97 326.08; nonadeca-1(19),2,4,6,12,14(18)-hexaen-3-ol][10.7.0.02,7.014,18]nonadeca- 172.97 274.96 1(19),2,4,6,12,14(18)-hexaen-3-ol]Gomisin L2 356.98; 325.00; [(9S,10R)-3,4,19-trimethoxy-9,10-dimethyl-15,17 284.93; 259.03; 297.04; 226.98; 3 C H O 386.4382 386.98 -dioxatetracyclo[10.7.0.02,7.014,18] 22 26 6 226.99;356.98; 167.02; 182.97 nonadeca-1(19),2,4,6,12,14(18)-hexaen-5-ol] 325.00;137.17 Gomisin L2 [(9S,10R)-3,4,19-trimethoxy- 284.93; 297.04; 9,10-dimethyl-15,17-Gomisin M2 3 [(9S,10R)-3,4,5-trimethoxy-9,10-dimethyl-15,17 C22H26O6 386.4382 386.98 355.01;259.03; 324.01; 226.98;339.98; 324.02; 4 dioxatetracyclo[10.7.0.02,7.014,18]nonad C22H26O6 386.4382 387.01 324.94; 296.90 -dioxatetracyclo[10.7.0.02,7.014,18] 226.99;284.97 182.97284.97; 226.96 nonadeca-1(19),2,4,6,12,14(18)-hexaen-19-ol]eca-1(19),2,4,6,12,14(18)-hexaen-5-ol] 167.02; Gomisin J [(9S,10R)-3,4,15,16-tetramethoxy-9,10- 325.03;137.17 357.01; 227.01; 241.00; 5 C H O 388.4541 389.04 226.98; 198.98 Gomisindimethyltricyclo[10.4.0.02,7]hexadeca M2 [(9S,10R)-3,4,5-trimethoxy- 22 28 6 226.96; 286.97 339.98;269.03; 297.01 355.01; -1(16),2,4,6,12,14-hexaene-5,14-diol]9,10-dimethyl-15,17- 324.02; 324.94; 4 C22H26O6 386.4382 387.01 324.01; dioxatetracyclo[10.7.0.02,7.014,18]nonadPregomisin 237.07; 205.03; 284.97; 296.90 284.97 6 [5-[(2S,3R)-4-(3-hydroxy-4,5-dimethoxyphenyl)eca-1(19),2,4,6,12,14(18)-hexaen-19-ol] C22H30O6 390.47 391.00 288.91; 326.96; 226.96205.00; 173.00 -2,3-dimethylbutyl]-2,3-dimethoxyphenol] 359.00 Gomisin J [(9S,10R)-3,4,15,16- 325.03; 227.01; Schisandrin B (Gomisin N, Isokadsuranin) [3,4,5,19-tetramethoxy-9,10-dimethyl-15,17tetramethoxy-9,10- 357.01; 241.00; 226.98;322.97; 295.03; 57 C CH22HO 28O6 400.3648 388.4541 401.07 389.04 369.04 354.04; 338.00 -dioxatetracyclo[10.7.0.02,7.014,18]nonadeca-1dimethyltricyclo[10.4.0.02,7]hexadeca- 23 28 6 226.96; 269.03; 198.98 264.03 1(16),2,4,6,12,14-hexaene-5,14-diol](19),2,4,6,12,14(18)-hexaene] 286.97 297.01 Schisanhenol (Gomisin K3) Pregomisin [5-[(2S,3R)-4-(3-hydroxy- 237.07; 340.03; 315.01; [(9S,10R)-4,5,14,15,16-pentamethoxy 371.01; 340.03; 205.00; 324.99; 270.99; 8 C H O 402.4807 403.05 300.98; 286.01; 6 -9,10-dimethyltricyclo[10.4.0.02,7]4,5-dimethoxyphenyl)-2,3- 23C3022H630O6 390.47 391.00 301.01;205.03; 259.00 227.02 173.00233.07 dimethylbutyl]-2,3-dimethoxyphenol]hexadeca-1(16),2,4,6,12,14-hexaen-3-ol] 288.91; Gomisin O [(8R,9S,10S)-3,4,5,19-tetramethoxy-9,10-dimethyl 9 C H O 416.3642 417.01 356.97; 373.01 329.00 313.97; 270.00 -15,17-dioxatetracyclo[10.7.0.02,7.014,18] 23 28 7 nonadeca-1(19),2,4,6,12,14(18)-hexaen-8-ol] Erigomisin O [(8S,9S,10S)-3,4,5,19-tetramethoxy-9,10- 356.98; 340.98; 10 C H O 416.3642 416.96 328.95; 313.98 dimethyl-15,17-dioxatetracyclo[10.7.0.02,7.014, 23 28 7 308.97 18]nonadeca-1(19),2,4,6,12,14(18)-hexaen-8-ol] Schisandrin A (Deoxyschisandrin) [(9R,10S)-3,4,5,14,15,16-hexamethoxy-9,10- 316.00; 346.99; 300.96; 284.95; 11 C H O 416.5073 417.01 dimethyltricyclo[10.4.0.02,7] 24 32 6 402.01 242.02 hexadeca-1(16),2,4,6,12,14-hexaene] 279.00; 322.89; 259.94;220.86; 12 Demethylated metabolites of Schisandrol A 440.95 306.86; 258.89; 137.02 202.99 Molecules 2020, 25, 2689 5 of 10

Table 2. Cont.

Observed Observed Calcula-ted MS/MS Stage 1 MS/MS Stage 2 MS/MS Stage 3 № Identiﬁcation Formula Mass Mass Mass Fragmentation Fragmentati-on Fragmentation [M + H]+ [M + Na]+ Schisandrol A (Schisandrin) [(9R,10S)-3,4,5,14,15,16-hexamethoxy- 13 C H O 432.5067 433.50 415.03 384.04; 359.03 368.99; 353.05 9,10-dimethyltricyclo[10.4.0.02,7]hexadeca- 24 32 7 1(16),2,4,6,12,14-hexaen-9-ol] 332.90; 348.90; 332.89; 274.94; 14 7, 8-Dihydroxy-schisandrin C24H32O8 448.5061 470.95 200.84; 230.30; 244.93; 202.98; 274.74 155.17 Tigloylgomisin O [[(8R,9S,10S)-3,4,5,19-tetramethoxy- 208.01; 250.08; 15 9,10-dimethyl-15,17-dioxatetracyclo C28H34O8 498.5648 521.92 304.99; 359.99; 191.00; 375.89 [10.7.0.02,7.014,18]nonadeca-1(19),2,4,6,12,14(18) 402.85; 436.83 -hexaen-8-yl] (E)-2-methylbut-2-enoate] Angeloylsogomisin O [[(9S,10S,11R)-3,4,5,19-tetramethoxy-9,10 323.00; 341.00; -dimethyl-15,17-dioxatetracyclo 308.98; 262.97; 16 C H O 498.5648 387.16 355.12 295.02; 262.94; [10.7.0.02,7.014,18]nonadeca-1(19),2,4, 28 34 8 220.24 210.100 6,12,14(18)-hexaen-11-yl] (Z)-2-methylbut-2-enoate] Angeloygomisin H [[(9S,10S)-10-hydroxy-4,5,14,15,16- 368.93; 433.87; 368.92; 352.97; pentamethoxy-9,10-dimethyl-3 472.83; 334.94; 299.90; 244.80; 17 C H O 500.3806 500.95 -tricyclo[10.4.0.02,7]hexadeca-1(16) 28 36 8 288.84; 244.92; 208.95; 156.99; ,2,4,6,12,14-hexaenyl] 207.21; 169.02 125.89 (Z)-2-methylbut-2-enoate] 407.87; 392.92; 422.91; 328.94; 18 Micrantherin A C H O 500.5806 522.93 364.93; 350.88; 28 36 8 386.00; 476.94 320.91; 295.02 Gomisin E [(11R,12R,15R,24S,25S)-12-hydroxy-18,19,20- trimethoxy-11,12,24,25-tetramethyl- 384.98; 355.03; 19 C H O 514.3642 514.99 354.99; 322.97 4,6,9,14-tetraoxapentacyclo[13.7.3.03,7.08, 28 34 9 322.99 22.016,21]pentacosa-1,3(7),8(22), 16,18,20-hexaen-13-one] Schisantherin D [[(11S,12S,13S)-12-hydroxy-3,22-dimethoxy- 380.89; 408.36; 12,13-dimethyl-5,7,18,20- 20 C H O 520.5272 542.89 451.55; 334.99; tetraoxapentacyclo[13.7.0.02,10.04, 29 28 9 200.93 8.017,21]docosa-1(22),2,4(8),9,15, 17(21)-hexaen-11-yl] Benzoylgomisin O [[(8R,9S,10S)-3,4,5,19-tetramethoxy-9,10 364.66; 308.93; 21 -dimethyl-15,17-dioxatetracyclo C H O 520.5703 542.91 380.89 30 32 9 193.02 [10.7.0.02,7.014,18]nonadeca -1(19),2,4,6,12,14(18)- hexaen-8-yl] benzoate] Benzoylgomisin H 491.30; 448.09; [[(9S,10S)-10-hydroxy-4,5,14,15,16 421.07; 399.03; 22 -pentamethoxy-9,10-dimethyl C H O 522.5862 522.99 271.39; 213.02 30 34 8 377.05; 335.11; -3-tricyclo[10.4.0.02,7]hexadeca 302.95; 269.78 -1(16),2,4,6,12,14-hexaenyl] benzoate] Gomisin D 510.97; 478.98; 382.92; 354.95; [12,25-dihydroxy-18,19,20-trimethoxy-11,12,24, 400.97; 372.91; 339.03; 312.11; 23 25-tetramethyl-4,6,9,14- C H O 530.5636 553.97 28 34 10 334.94; 248.99; 277.00; 248.99; tetraoxapentacyclo[13.7.3.03,7.08,22.016,21] 202.87 189.03 pentacosa-1,3(7),8(22),16, 18,20-hexaen-13-one] Gomisin G [[(9S,10S,11S)-10-hydroxy-3,4,5, 19-tetramethoxy-9,10-dimethyl-15 436.92; 414.99; 422.80; 390.84; 24 ,17-dioxatetracyclo[10.7.0.02,7.014,18] C H O 536.3697 536.93 30 32 9 371.03; 341.04 360.99 nonadeca-1(19),2,4,6, 12,14(18)-hexaen-11-yl] benzoate] Schisantherin A (Gomisin C) [[(8S,9S,10S)-9-hydroxy-3,4,5,19-tetramethoxy-9, 414.99; 371.05; 341.01; 310.01; 25 10-dimethyl-15,17-dioxatetracyclo[10.7.0.02,7.014 C H O 536.5697 537.95 370.99; 341.02 30 32 9 340.98 282.06 ,18]nonadeca-1(19),2,4,6,12,14(18)- hexaen-8-yl] benzoate] Benzoylgomisin Q [[(8S,9S,10S)-9-hydroxy-3,4,5,14,15,16- 415.05; 436.98; 384.03; 400.01; 369.02; 352.99; 26 hexamethoxy-9,10-dimethyl-8-tricyclo C H O 552.3121 552.99 31 36 9 384.03 359.00 338.00 [10.4.0.02,7]hexadeca-1(16),2, 4,6,12,14-hexaenyl] benzoate]

Figure3 shows examples of the decoding spectra (collision-induced dissociation (CID) spectrum) of the ion chromatogram obtained using tandem mass spectrometry. The CID spectrum in positive ion modes of schisandrin B (gomisin N, isokadsuranin) from Russian S. chinensis. Molecules 2020, 25, 2689 6 of 10 Molecules 2020,, 25,, xx FORFOR PEERPEER REVIEWREVIEW 7 of 11

Intens.Intens. 34.34. SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS,+MS, 42.2min42.2min #16#162828 66 1+1+ x10x10 1+ 44 1+ 471.06471.06 401.07401.07 1+1+ 485.02485.02 1+1+ 1+1+ 2 1+1+ 1+1+ 1+ 1+1+ 603.02 2 1+1+ 1+1+ 1+1+ 1+ 553.02553.02 603.02 2+2+ 4+4+ 2+2+ 3+3+ 277.04277.04 385.03385.03 418.96 511.01511.01 128.10128.10 193.07193.07 351.01351.01 418.96 672.11672.11 708.86708.86 741.01741.01 786.74786.74 00 x10x1066 34.34. SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS2(401.07),+MS2(401.07), 4242.2min.2min #1630#1630

1.01.0 369.04369.04

0.50.5 191.11191.11 0.00.0 x10x1055 34.34. SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS3(401.07->369+MS3(401.07->369.04),.04), 42.3min42.3min #1633#1633 355.01 44 355.01 1+ 1+ 2+2+ 338.01 22 338.01354.04354.04 00 x10x1055 34.34. SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS4(401.07->369+MS4(401.07->369.04->354.04),.04->354.04), 42.4min42.4min #1636#1636 1+1+ 322.97322.97 11 295.03295.03 1+1+ 264.03264.03 00 200200 300300 400400 500500 600600 700700 800800 m/z FigureFigure 3.3. Collision-induced Collision-inducedCollision-induced dissociation dissociationdissociation (CID) (CID) spectrum spectrum of the schisandrinof the sch Bisandrinisandrin (gomisin N,BB isokadsuranin)(gomisin(gomisin N,N, isokadsuranin)isokadsuranin)from S. chinensis fromfrom, m/z S.401.07. chinensis,, m/z 401.07.401.07.

+ TheThe [M [M ++ H]H]+ ionionion producedproduced produced oneone one fragmentfragment fragment withwith with m/zm/z 369.04369.04369.04 (Figure(Figure (Figure 3).33).). TheThe The fragmentfragment ionion with with m/zm/z 369.04369.04 further further formed formed two two daughter daughter ions ions with with m/zm/z 354.04354.04354.04 andand and m/zm/zm/z 338.00.338.00.338.00. TheThe The fragmentfragment fragment ionion ion withwith with m/zm/z 354.04354.04 produced produced three three daughter daughter ions ions with with m/zm/z 322.97,322.97, m/zm/z 295.03,295.03, andand m/zm/zm/z 264.03.264.03. TheThe CID spectrumspectrum inin positive positive ion ion modes modes of schisantherinof schisantherin A (gomisin A (gomisin C) from C) fromS. chinensis S. chinensisis shown isis shownin Figure in 4Figure. 4.

Intens.Intens. 60.60. SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS,+MS, 53.9min53.9min #20#208383 x10x1077 1+1+ 401.01 401.01 1+1+ 1+1+ 441.04441.04 537.95537.95 0.50.5 1+ 1+ 1+1+ 2+2+ 1+ 1+1+ 1+ 1+1+ 275.02 468.97 684.97684.97 157.06157.06 203.13203.13 275.02 343.19343.19 468.97 597.04597.04 652.92652.92 722.42722.42 0.00.0 x10x1066 60.60. 1+1+ SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS2(537.95),+MS2(537.95), 5353.9min.9min #2084#2084 414.99414.99 44 2 1+1+ 2 1+1+ 371.05371.05 340.98340.98 00 x10x1066 60.60. 1+1+ SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS3(537.95->414+MS3(537.95->414.99),.99), 54.0min54.0min #2087#2087 370.99370.99 1.01.0 1+1+ 341.02 0.50.5 341.02

0.00.0 x10x1055 60.60. SchisandraSchisandra SFESFE 04.0204.02 #3#3 VolVol 11 MS4_-1_01_63.d:MS4_-1_01_63.d: +MS4(537.95->414+MS4(537.95->414.99->370.99),.99->370.99), 54.1min54.1min #2090#2090 1+1+ 44 341.01341.01 1+1+ 22 1+1+ 310.01310.01 282.06282.06 00 200200 300300 400400 500500 600600 700700 800800 m/z FigureFigure 4. 4. CIDCID spectrum spectrum of of the the schisanth schisantherinerin A A (gomisin (gomisin C) C) from from S.S. chinensis chinensis,, , m/zm/z 537.95.537.95.537.95.

+ TheThe [M [M ++ H]H]+ ionionion producedproduced produced threethree three fragmentsfragments withwithwith mm/z/z 414.99, 414.99,414.99,m m/z/z 371.05 371.05371.05 and andandm m/zm/z/z 340.98 340.98340.98 (Figure (Figure(Figure4). 4).The The fragment fragment ion ion with withm/ zm/z414.99 414.99414.99 produced producedproduced two twotwo characteristic characteristiccharacteristic daughter daughterdaughter ions ionsions with withwithm/z m/z370.99 370.99370.99 and andandm/z m/z341.02. 341.02.341.02. The TheThe fragment fragmentfragment ion ionion with withwithm m/z/z 370.99 370.99370.99 formed formedformed three threethreedaughter daughterdaughter ionsionsions withwith m/zm/z 341.01,341.01, m/zm/z 310.01,310.01,310.01, andand m/zm/zm/z 282.06.282.06.282.06. TheThe CID CID spectrum spectrum in in positive positive ion ion modes modes of of benzoylgomisin benzoylgomisin Q Q is is shown shown in in Figure Figure 55.. + The [M + H] ion produced three fragments with m/z 415.05, m/z 436.98 and m/z 384.03 (Figure5). The fragment ion with m/z 415.05 produced three daughter ions with m/z 400.0, m/z 384.03 and m/z 359.00. The fragment ion with m/z 384.03 yielded three daughter ions with m/z 369.02, m/z 352.99, and m/z 338.00.

Molecules 2020, 25, 2689 7 of 10 Molecules 2020, 25, x FOR PEER REVIEW 8 of 11

Intens. 185. Schisandra SFE 04.02 #3 Vol 1 MS4_-1_01_63.d: +MS, 80.4min #3100 x107 1+ 490.99 1+ 1 425.04 1+ 1+ 3+ 552.99 1+ 162.99 205.15 275.08 353.11 520.96 631.09 715.08 755.10 0 x106 185. 1+ Schisandra SFE 04.02 #3 Vol 1 MS4_-1_01_63.d: +MS2(552.99), 80.5min #3102 6 415.05 4 1+ 436.98 2 1+ 384.03 0 x106 185. 1+ Schisandra SFE 04.02 #3 Vol 1 MS4_-1_01_63.d: +MS3(552.99->415.05), 80.6min #3105 384.03

2 1+ 1+ 359.00 400.01 0 x106 185. Schisandra SFE 04.02 #3 Vol 1 MS4_-1_01_63.d: +MS4(552.99->415.05->384.03), 80.7min #3108 1+ 369.02 1 1+ 1+352.99 338.00 0 200 300 400 500 600 700 800 m/z FigureFigure 5 5.. CIDCID spectrum spectrum of of the the be benzoylgomisinnzoylgomisin Q Q from from S.S. chinensis chinensis, ,m/zm/z 522.99.522.99.

3. Materials and Methods The [M + H]+ ion produced three fragments with m/z 415.05, m/z 436.98 and m/z 384.03 (Figure 5).3.1. The Materials fragment ion with m/z 415.05 produced three daughter ions with m/z 400.0, m/z 384.03 and m/z 359.00. The fragment ion with m/z 384.03 yielded three daughter ions with m/z 369.02, m/z 352.99, and m/z 338.00.As the objects of the study, samples of S. chinensis (woody liana) were purchased from the area of the Peschanka river near Lazovsky district (Sikhote Alin), Primorsky Krai, located at 43◦320 N and 3.134 Materials◦330 E, Russia. and Methods All samples were morphologically authenticated according to the current standard of Russian Pharmacopeia [37]. 3.1. Materials 3.2. Chemicals and Reagents As the objects of the study, samples of S. chinensis (woody liana) were purchased from the area of theHPLC-grade Peschanka river acetonitrile near Lazovsky was purchased district (Sikhote from Fisher Alin), Scientific Primorsky (Southborough, Krai, located at UK), 43°32 MS-grade′ N and 134°33formic′ E, acid Russia. was fromAll samples Sigma-Aldrich were morphologically (Steinheim, Germany). authenticated Ultra-pure according water to the was current prepared standard from a ofSIEMENS Russian Pharmacopeia ULTRA clear (SIEMENS [39]. water technologies, Germany), and all the other chemicals were analytical grade. 3.2. Chemicals and Reagents 3.3. SC-CO2 Extraction HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), MS-grade SC-CO extraction was performed using the SFE-500 system (Thar SCF Waters, Milford, USA) formic acid was2 from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was prepared from a supercritical pressure extraction apparatus. System options included co-solvent pump (Thar Waters SIEMENS ULTRA clear (SIEMENS water technologies, Germany), and all the other chemicals were P-50 High Pressure Pump), for extracting polar samples. CO flow meter (Siemens, Germany), analytical grade. 2 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 3.3. SC-CO2 Extraction CO2 and 1.76 mL/min for EtOH. Extraction samples of 10 g Schisandra chinensis wood were used. TheSC-CO extraction2 extraction time was was counted performed after reaching using the the SFE-50 working0 system pressure (Thar and SCF equilibrium Waters, flow,Milford, and USA) it was supercritical6 h for each sample.pressure extraction apparatus. System options included co-solvent pump (Thar Waters P-50 High Pressure Pump), for extracting polar samples. CO2 flow meter (Siemens, Germany), to measure3.4. Liquid the Chromatography 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 HPLC was performed using Shimadzu LC-20 Prominence HPLC (Shimadzu, Japan), equipped CO2 and 1.76 mL/min for EtOH. Extraction samples of 10 g Schisandra chinensis wood were used. The with an UV VIS detector. The analytical reverse phase column used was a Shodex ODP-40 4E C18 extraction time− was counted after reaching the working pressure and equilibrium flow, and it was 6 (4.6 250 mm, particle size: 4 µm) to perform the separation of multicomponent mixtures. The gradient h for× each sample. 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 3.4. Liquid Chromatography− − SPD-20A (Shimadzu, Japan) at wavelengths of 230 and 330 nm, at 17 ◦C provided with column oven CTO-20AHPLC (Shimadzu, was performed Japan) using with Shim an injectionadzu LC-20 volume Prominence of 20 µL. HPLC (Shimadzu, Japan), equipped with an UV−VIS detector. The analytical reverse phase column used was a Shodex ODP-40 4E C18 (4.63.5. × Mass 250 Spectrometrymm, particle size: 4 μm) to perform the separation of multicomponent mixtures. The gradientMS elution analysis program was performed was as follows: on an ion0.01 trap−4 min, amaZon 100% SL A; (BRUKER4−60 min, DALTONIKS,100−25% A; 60 Germany)−75 min, 25equipped−0% A; control with an washing ESI source 75− in120 negative min 0% ion A. mode.The entire The HPLC optimized analysis parameters was performed were obtained using asa UV−VIS detector SPD-20A (Shimadzu, Japan) at wavelengths of 230 and 330 nm, at 17 °C provided with column oven CTO-20A (Shimadzu, Japan) with an injection volume of 20 μL.

Molecules 2020, 25, 2689 8 of 10

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: 1500V, fragmentary: 280 V, collision energy: 60 eV. An ion trap was used in the scan range m/z 100 1.700 for MS and MS/MS. The capture rate was one − spectrum for MS and two spectra for MS/MS. Data collection was controlled by Windows software for BRUKER DALTONIKS. All experiments were repeated three times. A two-stage ion separation mode (MS/MS mode) was implemented.

4. Conclusions

An optimized extraction process with SC-CO2 (and co-solvent 2% ethanol) of woody liana S. chinensis provided the samples for an accurate analytical study by HPLC-SPD-MS/MS techniques. Twenty-six different lignans typical of S. chinensis species were identified. This method allows one to get all the studied ligands in a single extract without using a series of approaches and solvents, different in polarity, which not only reduces the environmental pressure, but also simplifies the production process. These data could support future investigations on the quality of pharmaceutical preparations containing these S. chinensis extracts. This is because the biological activity is related to the presence of the identified lignans. Their excellent transcutaneous penetration may offer new therapeutic approaches with transdermal preparations based on SC-CO2 extracts of S. chinensis.

Author Contributions: Conceptualization, G.C. and K.G.; methodology, A.Z. and K.P.; software, M.R.; validation, M.R., E.K., and, S.E.; formal analysis, M.R. and K.P.; investigation, M.R. and A.Z.; resources, K.G. and A.Z.; data curation, G.C. and S.E.; writing—original draft preparation—M.R. and V.C.; writing—review and editing A.Z., S.E. and K.G.; visualization, M.R. and E.K.; supervision, K.G.; project administration, A.Z. 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 (CΠ-3156.2019.4). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples fractions are available from the authors.

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