New Cadinane Sesquiterpenes from the Stems of Kadsura Heteroclita

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Article New Cadinane Sesquiterpenes from the Stems of Kadsura heteroclita

1, 1,2, 1,2 1 1 Liang Cao †, Nuzhat Shehla †, Shumaila Tasneem , Mengru Cao , Wenbing Sheng , Yuqing Jian 1, Bin Li 1, Caiyun Peng 1, M. Iqbal Choudhary 1,2,*, Atta-ur-Rahman 2, Duan-fang Liao 1 and Wei Wang 1,2,* 1 TCM and Ethnomedicine Innovation & Development International Laboratory, Innovative Materia Medica Research Institute, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China; [email protected] (L.C.); [email protected] (N.S.); [email protected] (S.T.); [email protected] (M.C.); [email protected] (W.S.); [email protected] (Y.J.); [email protected] (B.L.); [email protected] (C.P.); dﬂ[email protected] (D.-f.L.) 2 H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan; [email protected] * Correspondence: [email protected] (M.I.C.); [email protected] (W.W.); Tel.: +86-136-5743-8606 (M.I.C.); +92-21-4824924 or +92-21-4819010 (W.W.) They contributed equally to this work. † Received: 10 March 2019; Accepted: 26 April 2019; Published: 28 April 2019

Abstract: As part of our continual eﬀorts to exploit ‘Tujia Ethnomedicine’ for their pharmacophoric functionalities, we herein investigated Kadsura heteroclita collected from a deep Wulin mountain area in northern Hunan province. The current study resulted in the isolation of three new sesquiterpenes: 6α,9α,15-trihydroxycadinan-4-en-3-one (1), (+)-3,11,12-trihydroxycalamenene (2), (–)-3,10,11,12-tetrahydroxy-calamenene (3), along with four known sesquiterpenes (4–7), and a cytochalasin H (8). Their chemical structures were elucidated by 1D-, and 2D-NMR spectroscopy, and HRESI-MS, CD spectrometry. The antioxidant, and cytotoxic activities of the compounds were evaluated. Compound 8 exhibited a strong antioxidant eﬀect with an IC50 value of 3.67 µM on isolated human polymorphonuclear cells or neutrophils.

Keywords: Kadsura heteroclita; Schisandraceae; sesquiterpenes; antioxidant; Tujia ethnomedicine

1. Introduction In the last few decades, phytochemical work on the family Schisandraceae has resulted in a seminal contribution to novel structural frameworks for triterpenoidal skeletons [1–4]. Recently, some promising sesquiterpenes has also been reported from the plants of family Schisandraceae [5–10]. In our previous research, we reported the identification of several bioactive lignanoids from the genus kadsura [11] that highlight the immense diversity in the chemical constituents of these plants as well as in their important biological activities. Herein, we report the identification of three new sesquiterpenoids (1–3) from kadsura heteroclita, amongst compound 1 was a structural analogue of our previously reported compound 4, while compounds 2–3 possess calamenene core skeletons, which are identified for the first time from this species. With the major distribution in China, genus Kadsura (Schisandraceaea) is represented by sixteen species, half of which are mainly distributed in the southwest and southeast part of China [12]. Kadsura heteroclita is a vine plant primarily distributed in the southwest part of China, has been used in Traditional Chinese Medicine (TCM) as “Hai Feng Teng” or “Ji Xue Teng” for the treatment of menstrual irregularities and blood deficiencies [13], by activating the blood circulation, relieving pain, and eliminating dampness. In the “Hu Ping” and “Xue Feng” mountain areas of northwest Hunan

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Molecules 2019, 24, x 2 of 10 province, the stems of K. heteroclita are used to treat traumatic injuries and rheumatoid arthritis by Hunanone of theprovince, Chinese the ethnic stems minorities of K. heteroclita known are as the used Tujia to peopletreat traumati [14,15].c Traditionallyinjuries and theyrheumatoid named arthritisthe stems by of oneK. heteroclitaof the Chineseas “Xue ethni Tong”c minorities which meansknown“circulating as the Tujia thepeople blood [14,15]. system”, Traditionally which is usedthey namedto treat the blood stems related of K. diseases heteroclita [16 as]. “Xue These Tong” features which triggered means our “circulating research enthusiasmthe blood system”, to explore which the isbioactive used to metabolites treat blood fromrelated this diseases species that[16]. canThes bee relatedfeatures to triggered its clinical our application. research enthusiasm The previous to explorework implied the bioactive that the metabolites plant contains from triterpenoidsthis species that [17 –can19], be lignans related [20 to– its22 ]clinical and sesquiterpenes application. The [6]. previousIn the present work work, implied we reported that the three plant new contains sesquiterpenes triterpenoids (1–3) (Figure [17–19],1) along lignans with [20–22] four known and sesquiterpenes [6]. 6α ,9Inα the-dihydroxycadinan-4-en-3-one present work, we reported three (4)[ 6new], isoepicubenol sesquiterpenes (5 )[(123–3],) (Figure cryptomeridiol 1) along with(6)[24 four], 1β known,4β,7α-trihydroxyeudesmane sesquiterpenes 6α,9α-dihydroxycadinan-4-en-3-one (7)[25,26], and a cytochalasin H(4) ( 8[6],)[27 isoepicubenol,28] isolated from(5) [23], the cryptomeridiolstems and roots ( of6) K.[24], heteroclita 1β,4β,7αas-trihydroxyeudesmane shown in Figure1. (7) [25,26], and a cytochalasin H (8) [27,28] isolated from the stems and roots of K. heteroclita as shown in Figure 1.

Figure 1. Structures of compounds 1–8. Figure 1. Structures of compounds 1–8. 2. Results and Discussion 2. Results and Discussion 2.1. Structure Characterization of the Isolated Compounds (1–3) 2.1. Structure Characterization of the Isolated Compounds (1–3) 6α,9α,15-Trihydroxycadinan-4-en-3-one (1) (Figure2) was isolated as a white amorphous powder. + The molecular6α,9α,15-Trihydroxycadinan-4-en-3-one formula was established as C 15(1H) 22(FigureO4 from 2) itswas HRESI-MS isolated (mas/z a289.1419, white amorphous [M + Na] powder.calcd. 289.1410), The molecular indicating formula five degrees was established of unsaturation. as C15H The22O41 fromH-NMR its HRESI-MS spectrum (Table (m/z 1289.1419,) the revealed [M + + 1 Na]presence calcd. of 289.1410), one secondary indicating group fi (δveH 1.00,degrees d, J of= 6.9),unsaturation. a tertiary methylThe H-NMR group (spectrumδH 1.83, s), (Table one olefinic 1) the revealedmethine presence group (δH of6.85, one secondary s), and a terminal group (δ olefinicH 1.00, d, methylene J = 6.9), a tertiary group ( δmethylH 4.77, group 4.90, brs(δH each)1.83, s), in one the olefinicstructure. methine The 13 groupC-NMR (δH and 6.85, DEPT s), and NMR a terminal spectra olefinic of compound methylene1 indicated group (δ fifteenH 4.77, carbon4.90, brs signals each) 13 inattributed the structure. to one The carbonyl C-NMR carbon and (δ CDEPT200.5), NMR four olefinicspectra carbonsof compound (δC 155.2, 1 indicated 146.6, 137.0, fifteen 114.6), carbon and signalsthree oxygenated attributed to carbons one carbonyl (δC 73.3, carbon 71.9, 59.5). (δC 200.5), Apart four from olefinic these carbons signals, three(δC 155.2, methines 146.6, (137.0,δC 37.3, 114.6), 48.6, and50.2), three two methylenesoxygenated (δcarbonsC 36.8, 37.0),(δC 73.3, and 71.9, two methyl59.5). Apart groups from (δC these15.0, 23.0)signals, were three also methines observed. ( BesidesδC 37.3, 48.6,the two 50.2), double two methylenes bonds and one(δC 36.8, carbonyl, 37.0), theand remaining two methyl two groups unsaturation (δC 15.0, degrees23.0) were together also observed. with the Besideschemical the shift two patterns double suggestedbonds and that one the carbonyl, compound the isremaining a bicycle two sesquiterpene unsaturation based degrees on a cadinanetogether withskeleton. the chemical A downfield shift chemical patterns shiftsuggested of an olefinic that the proton compound (δH 6.85, is a s) bicycle conjugated sesquiterpene with carbonyl based carbon on a cadinane(δC 200.5) skeleton. showed the A presencedownfield of chemic an α-βalunsaturated shift of an ketone olefinic moiety proton in the(δH skeleton6.85, s) conjugated of 1. with carbonylBy carefully carbon (δ comparingC 200.5) showed the 1 H-the and presence13C-NMR of anchemical α-β unsaturated shifts data ketone (Table mo1iety) of in1 thewith skeleton that of ofknown 1. compound 6α,9α-dihydroxycadinan-4-en-3-one (4), which was also reported previously from K. heteroclitaBy carefully[6], we comparing revealed the major1H- and di ff13erenceC-NMR between chemical the shifts two structures.data (Table The 1) of chemical 1 with shiftthat of knownC-15 (δ Ccompound15.3, δ3H 1.81, 6α,9α 3H)-dihydroxycadina correspondingn-4-en-3-one to a methyl group(4), which in 4 changedwas also reported to oxygenated previously methylene from K.(δC heteroclita59.5, δ2H [6],4.16, we 2H) revealed in 1. This the deductionmajor difference was supported between bythe HMBC two structures. correlations The of chemical H2-15 with shift C-3 of C-15(δC 200.5), (δC 15.3, C-4 δ3H (δ C1.81,137.0), 3H) and corresponding C-5 (δC 155.2). to a An methyl isopropenyl group in unit 4 changed was also to identified oxygenated in the methylene skeleton (δC 59.5, δ2H 4.16, 2H) in 1. This deduction was supported by HMBC correlations of H2-15 with C-3 (δC 200.5), C-4 (δC 137.0), and C-5 (δC 155.2). An isopropenyl unit was also identified in the skeleton on the basis of the presence of terminal methylene (δC 114.6, δH 4.77, 4.90 brs each), a tertiary methyl

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Figure 3. 1H−1H COSY, HMBC and NOESY correlations of compound 2. A: Key 1H−1H COSY (bold ─), and HMBC (→) correlations. B: Key NOESY ( ) correlations.

Compound 3 (–)-3,10,11,12-tetrahydroxycalamenene was isolated as white amorphous powder and its molecular formula C15H22O4 was established based on its HRESIMS data (m/z 289.1396, [M + Molecules 2019, 24, x 3 of 10 Na]+ calcd. 289.1410). The IR spectra revealed the presence of hydroxyl (3379 cm−1) and phenolic (1623 cm−1) groups. The 1H-NMR spectrum (Table 1) indicated two aromatic (δH 6.68, 6.93, s each) hydrogen, signal (δC 23.0, δ3H 1.83, 3H) along with olefinic quaternary carbon (δC 146.6). The HMBC correlations and three methyl (δH 1.42, 1.57, 2.25, s each) groups. of H3-13 (δH 1.83) to C-7 (δC 48.6), C-11 (δC 146.6), and C-12 (δC 114.6), and of H2-12 (δH 4.77, 4.90 brs The 13C-NMR and DEPT spectra (Table 1) exhibited fifteen carbon signals, including three each) with C-7 (δC 48.6) and C-13 (δC 23.0), and HMBC correlation of H-7 (δH 2.54) with C-13 helped methyls (δC 15.6, 22.0, 22.5), three methylenes (δC 21.0, 32.5, 69.1), three methines (δC 38.9, 108.1, 126.7), us to assign the isopropenyl unit at the C-7 position. The HMBC correlation of H3-14 (δH 1.00) to C-1 and six quaternary carbons (δC 72.3, 75.9, 122.0, 133.6, 140.6, 152.2). The similarity of the 1H and 13C- (δC 50.2), C-9 (δC 71.9), and C-10 (δC 37.3), along with COSY cross peaks between H3-14 and H-10 (δH NMR spectroscopic data of 3 with those of 2 (Table 1) suggested that they were analogues. Detailed 2.14) revealed a hydroxyl group attached to C-9. The HMBC signals of H2-2 (δH 2.39, 2.46 dd each) comparison of the chemical shifts showed that C-10 in 3 was an oxygenated quaternary carbon (δC and H2-8 (δH 2.00, 1.65, m each) with C-6 (δC 73.3) supported the presence of another hydroxyl group 72.3)located rather at thanC-6, thusa methine the planar (δC 32.4, structure δH 2.64, of qd, compound J = 14.2, 7.0) 1 has in 2been. This determined inference was (Figure further 2). confirmed by the TheHMBC relative correlations configuration of H3 -14of 1 ( wasδH 1.57) determined and H-2 by (δ HNOESY 6.68) to spectra C-10 ( inδC a72.3). combination The whole of couplingplanar structure of 3 was determined (Figure 4) by referencing the compound 2, and by surveying the 2D constant analysis. The observed NOESY correlations between the spin systems H3-14 (δH 1.00)/H-9 NMR spectral data. (δH 3.47), H-9/Hβ-8 (δH 1.65), Hβ-8/H-7 (δH 2.54), and H-7/H-9 suggested that H-9, H-7, CH3-14, and Hβ- MoleculesThe relative2019, 24, 1664conﬁguration of 3 was assigned by detailed analysis of its NOESY spectra, in which3 of 10 8 were positioned in β-orientation, while correlations of H-1 (δH 2.21)/H-10 (δH 2.14), H-10/Hα-8 (δH H3-14 (δH 1.57) was correlated with H-7 (δH 2.79, t J = 2.7), illustrating the β-orientation of H-7, while 2.00), Hα-8/H3-13 revealed that H-1, Hα-8, H-10 and the isopropenyl unit were α-oriented. According the 1,2-isopropanediol group was1 α-oriented. The absence of correlations between H3-14, H3-13 and onto the the coupling basis of the constants presence in ofthe terminal H-NMR, methylene δH 2.39 (J (=δ C13.9,114.6, 17.3δH Hz)4.77, was 4.90 assigned brs each), as H-2 a tertiaryβ because methyl the H2-12 also suggested that H3-14 and the 1,2-isopropanediol group were in trans conformation [34– signaldihedral (δC angle23.0, δof3H H-21.83,β and 3H) H-1 along should with oleﬁnicbe close quaternary to 180, thus carbon δH 2.46 (δ (CJ =146.6). 5.1, 17.3 The Hz) HMBC was correlationsassigned as 37]. The 1H coupling constant between H2-8 and H-7 were 2.7 Hz in triplicate, indicating that H-7 and ofH-2 Hα3-13 [29]. (δ HThe1.83) observed to C-7 ( δNOESYC 48.6), C-11correlations (δC 146.6), between and C-12 H-2 (δβC/H-7114.6), further and ofproved H2-12 this. (δH 4.77,The 4.90relative brs H-8α, H-8β are in ee and ae formation. According to 3D molecular model and coupling constant of H- each)configuration with C-7 of (δ C6-OH48.6) has and been C-13 provisionally (δC 23.0), and determined HMBC correlation as α on of the H-7 basis (δH 2.54)of alike with chemical C-13 helped shift 7, the 1,2-isopropanediol group and 10-OH are in an axial position, which may be induced by the Van usvalues to assign of known the isopropenyl compound unit4, for at which the C-7 the position. configuration The HMBC had been correlation determined of H by3-14 X-ray (δH experiment1.00) to C-1 der Waals force between the 10-OH and 11-OH or 12-OH. The conformation change may also have ([6].δC 50.2), C-9 (δC 71.9), and C-10 (δC 37.3), along with COSY cross peaks between H3-14 and H-10 resulted in the up fields shift of H2-12 from (δH 3.57, 3.42) in 2 to (δ2H 2.85) in 3, and the positive cotton (δH 2.14)The revealedabsolute aconfiguration hydroxyl group could attached be determined to C-9. The by HMBCthe application signals ofof Hhelicity2-2 (δH of2.39, α-β 2.46 unsaturated dd each) effect at 263 nm in CD spectra (Figure S36) of 3. OR experiment approved the compound to be a andketone H2 -8as (describedδH 2.00, 1.65, previously m each) [30]. with The C-6 (observedδC 73.3) supported negative cotton the presence effect at of 248 another nm (Figure hydroxyl S12) group was laevoisomer,locatedconsistent at C-6,with thus thus the the helicity the compound planar rule, structure was hence named the of configuration compound as (–)-(7S, 110has Rof)-3,10,11,12-tetrah 1 been was determineddeduced toydroxycalamenene. be (Figure (–)-12S).,6R,7R,9R,10 R- 6,9,15-trihydoxycadinan-4-en-3-one.

1 1 1 1 FigureFigure 4. 2.1H1−H1H1 COSY,H COSY, HMBC HMBC and and NOESY NOESY correlations correlations of of compound compound 3.1 .AA: Key: Key 1H1H−1H1 HCOSY COSY (bold (bold Figure 2. H− H COSY, HMBC and NOESY correlations of compound 1. A: Key H−− H COSY (bold ─–),─), and HMBC ( →→)) correlations. correlations. BB:: Key Key NOESY NOESY ( ( )) correlations.correlations. ), and HMBC→ ( ) correlations. B: Key NOESY ( ) correlations. Table 1. 1H and 13C-NMR spectroscopic data for compound 1–3 (δ in ppm, J in Hz). 2.2. AntioxidantTable Activity 1. 1H of and Isolated 13C-NMR Compounds spectroscopic data for compound 1–3 (δ in ppm, J in Hz). Num. 1 a 2 b 3 b Num.The antioxidant bioassay1 a of compounds (1–8) 2 wasb performed on human whole 3 b blood δH δC δH δC δH δC neutrophils initially,δ Hin which 8 showedδC potential toδH inhibit the generationδC of reactiveδH oxygen speciesδC 1 2.21 (dt, 13.9, 4.9) 50.2 143.7 140.6 1 2α 2.212.46 (dt, (dd, 13.9, 5.1, 4.9) 17.3) 50.2 37.0 6.66 (s) 112.8 143.7 6.68 (s) 108.1 140.6 2α 2β 2.462.39 (dd, (dd, 5.1, 13.9, 17.3) 17.3) 37.0 6.66 (s) 112.8 6.68 (s) 108.1 2β 3 2.39 (dd, 13.9, 17.3) 200.5 152.3 152.2 3 4 200.5 137.0 120.7152.3 122.0152.2 5 6.85 (s) 155.2 7.12 (s) 133.1 6.93 (s) 126.7 4 6 137.0 73.3 127.8120.7 133.6122.0 5 7β 2.54 6.85 (dd, (s) 13.4 3.3) 155.2 48.6 3.09 7.12 (dd, (s) 8.6, 5.6) 133.144.0 2.79 6.93 (t, 2.7) (s) 38.9 126.7 6 8α 2.00 (m)73.3 36.8 2.00 (m)127.8 23.0 2.23 (dt,12.4 2.9) 21.0133.6 8β 1.65 (m) 1.81 (m) 1.35 (m) 7β 2.54 (dd, 13.4 3.3) 48.6 3.09 (dd, 8.6, 5.6) 44.0 2.79 (t, 2.7) 38.9 9α 71.9 1.23 (m) 31.2 1.39 (dd, 11.8, 2.2) 32.5 8α 9β 3. 2.00 47 (td, (m) 10.9, 4.4) 36.8 2.00 1.93 (m) (m) 23.0 2.23 2.00 (dt,12.4 (m) 2.9) 21.0 8β 10α 1.652.14 (m) (m) 37.3 2.64 1.81 (dq, (m) 14.2 7.0) 32.4 1.35 (m) 72.3 9α 11 71.9 146.6 1.23 (m) 76.5 31.2 1.39 (dd, 11.8, 2.2) 75.9 32.5 12a 4.77 (s) 114.6 3.57 (d 11.1) 67.7 2.85 (s) 69.1 9β 12b 3. 47 (td, 4.90 10.9, (s) 4.4) 1.93 3.42 (m) (d11.1) 2.85 2.00 (s) (m) 10α 13 2.14 1.83 (m) (s) 37.3 23.0 2.64 (dq,1.14 14.2 (s) 7.0) 22.7 32.4 1.42 (s) 22.5 72.3 11 14 1.00 (d 6.9)146.6 15.0 1.24 (d 7.1) 21.6 76.5 1.57 (s) 22.0 75.9 12a 15 4.77 4.16 (s) (d 1.14) 114.6 59.5 3.57 (d 2.20 11.1) (s) 15.567.7 2.25 2.85 (s) (s) 15.6 69.1 a b 12b 4.90 (s) Recorded at 600 MHz in CD 3.423OD. (d 11.1)Recorded at 600 MHz in CDCl3. 2.85 (s) 13 1.83 (s) 23.0 1.14 (s) 22.7 1.42 (s) 22.5

The relative configuration of 1 was determined by NOESY spectra in a combination of coupling constant analysis. The observed NOESY correlations between the spin systems H3-14 (δH 1.00)/H-9 (δH 3.47), H-9/Hβ-8 (δH 1.65), Hβ-8/H-7 (δH 2.54), and H-7/H-9 suggested that H-9, H-7, CH3-14, and Hβ-8 were positioned in β-orientation, while correlations of H-1 (δH 2.21)/H-10 (δH 2.14), H-10/Hα-8 (δH 2.00), Hα-8/H3-13 revealed that H-1, Hα-8, H-10 and the isopropenyl unit were α-oriented. 1 According to the coupling constants in the H-NMR, δH 2.39 (J = 13.9, 17.3 Hz) was assigned as H-2β because the dihedral angle of H-2β and H-1 should be close to 180, thus δH 2.46 (J = 5.1, 17.3 Hz) was assigned as H-2α [29]. The observed NOESY correlations between H-2β/H-7 further proved this. The relative configuration of 6-OH has been provisionally determined as α on the basis of alike chemical shift values of known compound 4, for which the configuration had been determined by X-ray experiment [6]. Molecules 2019, 24, x 5 of 10

Molecules 2019, 24, 1664 4 of 10

Figure 3. 1H−1H COSY, HMBC and NOESY correlations of compound 2. A: Key 1H−1H COSY (bold ─), andThe HMBC absolute (→) correlations. configuration B: Key could NOESY be ( determined) correlations. by the application of helicity of α-β unsaturated ketone as described previously [30]. The observed negative cotton effect at 248 nm (FigureCompound S12) 3 was (–)-3,10,11,12-tetrahydroxycalamenene consistent with the helicity rule, hence was theisolated configuration as white amorphous of 1 was deduced powder to be and (–)-1its molecularS,6R,7R,9 Rformula,10R-6,9,15-trihydoxycadinan-4-en-3-one. C15H22O4 was established based on its HRESIMS data (m/z 289.1396, [M + Na]+ calcd.(+ )-3,11,12-Trihydroxycalamenene289.1410). The IR spectra revealed (2 )the was presence prepared of ashydroxyl a white (3379 amorphous cm−1) and powder phenolic and possessed(1623 + cm−1a) groups. molecular The formula 1H-NMR of Cspectrum15H22O3 ,(Table which 1) was indicated determined two aromatic by HRESIMS (δH 6.68, (m/z 6.93,273.1471, s each) [M hydrogen,+ Na] calcd. 1 1 and 273.1461).three methyl IR spectra(δH 1.42, revealed 1.57, 2.25, the s presenceeach) groups. of hydroxyl (3385 cm− ) and phenolic (1620 cm− ) groups. 1 TheThe H-NMR13C-NMR spectrum and DEPT (Table spectra1) indicated (Table two1) exhibite aromaticd fifteen ( δH 6.66, carbon 7.12, signals, s each) hydrogens,including three and two methylssinglet (δC (15.6,δH 1.14, 22.0, 2.20, 22.5), s each)three andmethylenes one doublet (δC 21.0, methyl 32.5, ( δ69.1),H 1.24, three d J =methines7) groups. (δC 38.9, 108.1, 126.7), 13 and six quaternaryThe C-NMR carbons and DEPT(δC 72.3, spectra 75.9,(Table 122.0,1 )133.6, exhibited 140.6, fifteen 152.2). carbon The similarity signals, including of the 1H three and methyls13C- NMR(δ Cspectroscopic15.5, 21.6, 22.7), data three of 3 with methylenes those of ( δ2C (Table23.0, 31.2,1) suggested 67.7), four that methines they were (δC analogues.32.4, 44.0, 112.8,Detailed 133.1), comparisonand five of quaternary the chemical carbons shifts ( δshowedC 76.5, 120.7,that C-10 127.8, in 133.1,3 was 152.3).an oxygenated Two aromatic quaternary methine carbon signals (δC (δC 72.3)112.8, ratherδ Hthan6.66 a methine s), (δC 133.1, (δC 32.4,δH δ7.12H 2.64, s) qd, together J = 14.2, with 7.0) fourin 2. aromaticThis inference carbons was ( furtherδC 120.7, confirmed 127.8, 133.1, by the152.3) HMBC suggesting correlations a tetra-substituted of H3-14 (δH 1.57) aromatic and H-2 spin ( systemδH 6.68) with to C-10 a para (δCconfiguration. 72.3). The whole Consideration planar structureof the of unsaturation 3 was determined degrees (Figure and chemical 4) by referencing shift characteristics, the compound it was 2, unambiguously and by surveying proposed the 2D that NMRcompound spectral data.2 should be a bicyclic sesquiterpene with the calamenene skeleton [31–33]. The Therelative HMBC con correlationsfiguration of of 3 H-5was ( δassignedH 7.12) with by detailed C-15 (δc analysis 15.5), and of C-7its NOESY (δC 44.0), spectra, and of in H-2 which (δH 6.66) H3-14with (δH C-101.57) (wasδC 32.4), correlated and C-4 with (δC H-7120.7) (δH indicated2.79, t J = 2.7), that illustrating the two cycles the wereβ-orientation connected of H-7, from while C-7, and the 1,2-isopropanediolC-10, which allowed group us to was fully α determine-oriented. The the aromaticabsence of moiety correlations [31]. The between phenolic H3-14, hydroxyl H3-13 groupand at H2-12C-3 also was suggested assigned that by the H3-14 HMBC and correlationsthe 1,2-isopropanediol of H3-15 (δ Hgroup2.20) were to C-3 in ( δtransC 152.3) conformation and C-5 (δ [34–C 133.1). 37]. Meanwhile,The 1H coupling the correlationsconstant between of H2-12 H2-8 (δ Hand3.57, H-7 3.42, were d 2.7 J = Hz11.1) in withtriplicate, C-7 ( δindicatingC 44.0), C-11 that (δ H-7C 76.5), and and H-8αC-13, H-8 (δβC are22.7), in ee and and of ae H formation.3-13 (δH 1.14) According with C-7 to (δ 3DC 44.0), molecular C-11(δ modelC 76.5), and and coupling C-12 (δC constant67.7) in the of HMBCH- 7, thespectra 1,2-isopropanediol corroborated group the attachment and 10-OH of are 1,2-isopropanediol in an axial position, group which at positionmay be induced C-7. Furthermore, by the Van the der WaalsHMBC force correlations between of the H 310-OH-14 (δH and1.24, 11-OH d J = or7.1) 12 to-OH. C-1 The (δC conformation143.7), C-9 (δ Cchange31.2), andmayC-10 also (haveδC 32.4), resultedalong in with the up COSY field crosss shift peaks of H2-12 between from ( HδH3 3.57,-14(δ 3.42)H 1.24, in d2 Jto= (δ7.1)2H 2.85)/H-10 in ( δ3H, and2.64, the dq positiveJ = 14.2, cotton 7.0)/H 2-9 effect(δ Hat1.23, 263 nm 1.93) in/H CD2-8 (spectraδH 1.81, (Figure 2.00)/H-7 S36) (δ Hof3.09) 3. OR determined experiment the approved cyclohexane the ring.compound Hence to the be planar a laevoisomer,Moleculesstructure 2019 thus of, compound24 ,the x compound2 with was the named calamenene as (–)-(7 coreS,skeleton 10R)-3,10,11,12-tetrah [31] was establishedydroxycalamenene. (Figure3). 5 of 10

FigureFigure 4. 1H 3.−11HH COSY,1H COSY, HMBC HMBC and andNOESY NOESY correlations correlations of compound of compound 3. 2A.: A:Key Key 1H1−H1H1 COSYH COSY (bold (bold –), Figure 3. 1H−−1H COSY, HMBC and NOESY correlations of compound 2. A: Key 1−H−1H COSY (bold ─), andand HMBC HMBC ( (→)) correlations. correlations. B B:: Key Key NOESY NOESY ( ( )) correlations.correlations. ─), and HMBC→ (→) correlations. B: Key NOESY ( ) correlations. 2.2. AntioxidantComparison Activity of of theIsolated chemical Compounds shifts data of compound 2 with that of known compound 3,12-dihydroxycalameneneCompound 3 (–)-3,10,11,12-tetrahydroxycalamenene [31] suggesting that two compounds was isolated have as a white close resemblanceamorphous powder in their andThe its antioxidant molecular formulabioassay C 15ofH 22compoundsO4 was established (1–8) wasbased performed on its HRESIMS on human data ( m/zwhole 289.1396, blood [M + planar structure, except that C-11 (δC 76.5) in compound 2 was found to be an oxygenated quaternary neutrophilsNa]+ calcd. initially, 289.1410). in which The IR 8 showed spectra revealpotentialed the to inhibitpresence the of generation hydroxyl (3379of reactive cm−1) oxygenand phenolic species (1623 carbon instead of a methine, as indicated by the HMBC correlations of H-7 (δH 3.09), H2-12 (δH 3.42, cm−1) groups. The 1H-NMR spectrum (Table 1) indicated two aromatic (δH 6.68, 6.93, s each) hydrogen, 3.57), and H3-13 (δH 1.14) with C-11.

and threeThe relativemethyl conﬁguration(δH 1.42, 1.57, 2.25, of 2 wass each) assigned groups. by detailed analysis of its NOESY spectra and the 1 The 13C-NMR and DEPT spectra (Table 1) exhibited fifteen carbon signals, including three H-NMR coupling constants. The observed NOESY correlations between H3-14 (δH 1.24) and H-7 methyls (δC 15.6, 22.0, 22.5), three methylenes (δC 21.0, 32.5, 69.1), three methines (δC 38.9, 108.1, 126.7), (δH 3.09) indicated that H3-14 and H-7 were co-facial, while H3-14 and 1,2-isopropanediol group andwere sixtrans quaternaryto each othercarbons [34 (–δ37C 72.3,], unlike 75.9, the 122.0,cis conformations133.6, 140.6, 152.2). [31– 33The]. Thesimilarity above of deductions the 1H and were 13C- NMR spectroscopic data of 3 with those of 2 (Table 1) suggested that they were analogues. Detailed further proved by the NOESY cross peaks between H-10 (δH 2.64) and H3-13 (δH 1.14). The relative comparisonstereochemistry of the at chemical the cyclohexene shifts showed ring wasthat alsoC-10 deducedin 3 was froman oxygenated the 1H coupling quaternary constants carbon[ 34(δC]. 72.3)The couplingrather than constants a methine between (δC 32.4, H-7 δH 2.64, and H-8qd, αJ =and 14.2, between 7.0) in 2. H-10 This andinference H-9β waswere further 8.6 and confirmed 14.2 Hz, by the HMBC correlations of H3-14 (δH 1.57) and H-2 (δH 6.68) to C-10 (δC 72.3). The whole planar structure of 3 was determined (Figure 4) by referencing the compound 2, and by surveying the 2D NMR spectral data. The relative conﬁguration of 3 was assigned by detailed analysis of its NOESY spectra, in which H3-14 (δH 1.57) was correlated with H-7 (δH 2.79, t J = 2.7), illustrating the β-orientation of H-7, while the 1,2-isopropanediol group was α-oriented. The absence of correlations between H3-14, H3-13 and H2-12 also suggested that H3-14 and the 1,2-isopropanediol group were in trans conformation [34– 37]. The 1H coupling constant between H2-8 and H-7 were 2.7 Hz in triplicate, indicating that H-7 and H-8α, H-8β are in ee and ae formation. According to 3D molecular model and coupling constant of H- 7, the 1,2-isopropanediol group and 10-OH are in an axial position, which may be induced by the Van der Waals force between the 10-OH and 11-OH or 12-OH. The conformation change may also have resulted in the up fields shift of H2-12 from (δH 3.57, 3.42) in 2 to (δ2H 2.85) in 3, and the positive cotton effect at 263 nm in CD spectra (Figure S36) of 3. OR experiment approved the compound to be a laevoisomer, thus the compound was named as (–)-(7S, 10R)-3,10,11,12-tetrahydroxycalamenene.

Figure 4. 1H−1H COSY, HMBC and NOESY correlations of compound 3. A: Key 1H−1H COSY (bold ─), and HMBC (→) correlations. B: Key NOESY ( ) correlations.

Molecules 2019, 24, x 5 of 10 Molecules 2019, 24, x 5 of 10

Figure 3. 1H−1H COSY, HMBC and NOESY correlations of compound 2. A: Key 1H−1H COSY (bold Figure 3. 1H−1H COSY, HMBC and NOESY correlations of compound 2. A: Key 1H−1H COSY (bold ─), and HMBC (→) correlations. B: Key NOESY ( ) correlations. ─), and HMBC (→) correlations. B: Key NOESY ( ) correlations. Compound 3 (–)-3,10,11,12-tetrahydroxycalamenene was isolated as white amorphous powder Compound 3 (–)-3,10,11,12-tetrahydroxycalamenene was isolated as white amorphous powder and its molecular formula C15H22O4 was established based on its HRESIMS data (m/z 289.1396, [M + andMolecules its molecular2019, 24, 1664 formula C15H22O4 was established based on its HRESIMS data (m/z 289.1396, 5[M of 10+ Na]+ calcd. 289.1410). The IR spectra revealed the presence of hydroxyl (3379 cm−1) and phenolic (1623 Na]+ calcd. 289.1410). The IR spectra revealed the presence of hydroxyl (3379 cm−1) and phenolic (1623 cm−1) groups. The 1H-NMR spectrum (Table 1) indicated two aromatic (δH 6.68, 6.93, s each) hydrogen, cm−1) groups. The 1H-NMR spectrum (Table 1) indicated two aromatic (δH 6.68, 6.93, s each) hydrogen, and respectively,three methyl showing(δH 1.42, 1.57, that 2.25, these s each) hydrogens groups. have a trans-diaxial relationship, while the CH3-14 and three methyl (δH 1.42, 1.57, 2.25, s each) groups. andThe the13C-NMR 1,2-isopropanediol and DEPT spectra group must(Table both 1) exhibite be equatoriald fifteen and carbontrans -orientedsignals, including to each otherthree [34]. The 13C-NMR and DEPT spectra (Table 1) exhibited fifteen carbon signals, including three methylsOR experiment(δC 15.6, 22.0, approved 22.5), three the methylenes compound (δCto 21.0, be 32.5, dextrorotatory 69.1), three methines form (+12.1), (δC 38.9, very 108.1, similar 126.7), to the methyls (δC 15.6, 22.0, 22.5), three methylenes (δC 21.0, 32.5, 69.1), three methines (δC 38.9, 108.1, 126.7), and reportedsix quaternary compound carbons (+ )-(1(δC S72.3,,4R)-7-methoxycalamenene 75.9, 122.0, 133.6, 140.6, 152.2). [34,35]. The Hence similarity the compound of the 1H and was 13 namedC- and six quaternary carbons (δC 72.3, 75.9, 122.0, 133.6, 140.6, 152.2). The similarity of the 1H and 13C- NMRas spectroscopic (+)-(7S, 10S)-3,11,12-trihydroxycalamenene. data of 3 with those of 2 (Table 1) The suggested absolute that configuration they were analogues. of C-11 remained Detailed to be NMR spectroscopic data of 3 with those of 2 (Table 1) suggested that they were analogues. Detailed comparisondetermined. of the CD chemical spectra shifts of 2 (Figure showed S24) that was C-10 also in recorded3 was an tooxygenated give the information quaternary forcarbon the further(δC comparison of the chemical shifts showed that C-10 in 3 was an oxygenated quaternary carbon (δC 72.3)research, rather than which a methine displayed (δC 32.4, a negative δH 2.64, cotton qd, J = eff 14.2,ect at 7.0) 238 in nm 2. This and inference 276 nm. was further confirmed 72.3) rather than a methine (δC 32.4, δH 2.64, qd, J = 14.2, 7.0) in 2. This inference was further confirmed by the HMBCCompound correlations 3 (–)-3,10,11,12-tetrahydroxycalamenene of H3-14 (δH 1.57) and H-2 (δH 6.68) was to isolatedC-10 (δC as 72.3). white The amorphous whole planar powder by the HMBC correlations of H3-14 (δH 1.57) and H-2 (δH 6.68) to C-10 (δC 72.3). The whole planar structureand its of molecular3 was determined formula (Figure C H 4)O bywas referencing established the basedcompound on its 2, HRESIMS and by surveying data (m /zthe289.1396, 2D structure of 3 was determined15 (Figure22 4 4) by referencing the compound 2, and by surveying the 2D NMR[M spectral+ Na]+ data.calcd. 289.1410). The IR spectra revealed the presence of hydroxyl (3379 cm 1) and phenolic NMR spectral data. − (1623The relative cm 1) groups. configuration The 1H-NMR of 3 was spectrumassigned by (Table detailed1) indicated analysis two of its aromatic NOESY ( spectra,δ 6.68, in 6.93, which s each) The −relative configuration of 3 was assigned by detailed analysis of its NOESYH spectra, in which H3-14hydrogen, (δH 1.57) andwas threecorrelated methyl with (δH H-71.42, (δ 1.57,H 2.79, 2.25, t J s= each)2.7), illustrating groups. the β-orientation of H-7, while H3-14 (δH13 1.57) was correlated with H-7 (δH 2.79, t J = 2.7), illustrating the β-orientation of H-7, while the 1,2-isopropanediolThe C-NMR andgroup DEPT was spectra α-oriented. (Table The1) exhibited absence fifteenof correlations carbon signals, between including H3-14, H three3-13 methylsand the 1,2-isopropanediol group was α-oriented. The absence of correlations between H3-14, H3-13 and H2-12(δ Calso15.6, suggested 22.0, 22.5), that three H3-14 methylenes and the 1,2-isopropanediol (δC 21.0, 32.5, 69.1), group three were methines in trans (conformationδC 38.9, 108.1, [34– 126.7), H2-12 also suggested that H3-14 and the 1,2-isopropanediol group were in trans conformation [34– 1 1 37]. andThe sixH coupling quaternary constant carbons between (δC 72.3, H2-8 75.9, and 122.0,H-7 were 133.6, 2.7 140.6,Hz in triplicate, 152.2). The indicating similarity that of H-7 the andH and 37]. The 1H coupling constant between H2-8 and H-7 were 2.7 Hz in triplicate, indicating that H-7 and H-8α13,C-NMR H-8β are spectroscopic in ee and ae formation. data of 3 with According those of to2 (Table3D molecular1) suggested model that and they coupling were analogues. constant of Detailed H- H-8α, H-8β are in ee and ae formation. According to 3D molecular model and coupling constant of H- 7, thecomparison 1,2-isopropanediol of the chemical group shiftsand 10-OH showed are that in an C-10 axial in 3position,was an oxygenated which may quaternarybe induced carbonby the (Vanδ 72.3) 7, the 1,2-isopropanediol group and 10-OH are in an axial position, which may be induced by theC Van der ratherWaals thanforce abetween methine the (δ 10-OH32.4, δ and2.64, 11-OH qd, Jor= 1214.2,-OH. 7.0) The in 2conformation. This inference change was may further also confirmed have der Waals force betweenC the 10-OHH and 11-OH or 12-OH. The conformation change may also have resultedby the in HMBCthe up field correlationss shift of ofH2 H-123-14 from (δH (δ1.57)H 3.57, and 3.42) H-2 in (2δ Hto 6.68)(δ2H 2.85) to C-10 in 3 (,δ andC 72.3). the positive The whole cotton planar resulted in the up fields shift of H2-12 from (δH 3.57, 3.42) in 2 to (δ2H 2.85) in 3, and the positive cotton effectstructure at 263 nm of 3 inwas CD determined spectra (Figure (Figure S36)4) byof 3 referencing. OR experiment the compound approved2, theand compound by surveying to be the a 2D effect at 263 nm in CD spectra (Figure S36) of 3. OR experiment approved the compound to be a laevoisomer,NMR spectral thus the data. compound was named as (–)-(7S, 10R)-3,10,11,12-tetrahydroxycalamenene. laevoisomer, thus the compound was named as (–)-(7S, 10R)-3,10,11,12-tetrahydroxycalamenene.

FigureFigure 4. 1H 4.−11HH COSY,1H COSY, HMBC HMBC and andNOESY NOESY correlations correlations of compound of compound 3. 3A. :A Key: Key 1H1H−1H1 HCOSY COSY (bold (bold –), Figure 4. 1H−1H COSY, HMBC and NOESY correlations of compound 3. A: Key 1−H−1H COSY (bold ─), and HMBC ( →)) correlations. correlations. BB:: Key Key NOESY NOESY ( ( )) correlations.correlations. ─), and HMBC→ (→) correlations. B: Key NOESY ( ) correlations. 2.2. AntioxidantThe relative Activity conﬁguration of Isolated Compounds of 3 was assigned by detailed analysis of its NOESY spectra, in which 2.2. Antioxidant Activity of Isolated Compounds H3-14 (δH 1.57) was correlated with H-7 (δH 2.79, t, J = 2.7), illustrating the β-orientation of H-7, while The antioxidant bioassay of compounds (1–8) was performed on human whole blood the 1,2-isopropanediolThe antioxidant bioassay group was ofα -oriented.compounds The ( absence1–8) was of correlationsperformed betweenon human H3 -14,whole H3-13 blood and neutrophils initially, in which 8 showed potential to inhibit the generation of reactive oxygen species neutrophilsH2-12 also suggested initially, in that which H3-14 8 showed and the potential 1,2-isopropanediol to inhibit the group generation were in transof reactiveconformation oxygen [species34–37]. 1 The H coupling constant between H2-8 and H-7 were 2.7 Hz in triplicate, indicating that H-7 and

H-8α, H-8β are in ee and ae formation. According to 3D molecular model and coupling constant of H-7, the 1,2-isopropanediol group and 10-OH are in an axial position, which may be induced by the Van der Waals force between the 10-OH and 11-OH or 12-OH. The conformation change may also have resulted in the up ﬁelds shift of H2-12 from (δH 3.57, 3.42) in 2 to (δ2H 2.85) in 3, and the positive cotton eﬀect at 263 nm in CD spectra (Figure S36) of 3. OR experiment approved the compound to be a laevoisomer, thus the compound was named as (–)-(7S, 10R)-3,10,11,12-tetrahydroxycalamenene.

2.2. Antioxidant Activity of Isolated Compounds The antioxidant bioassay of compounds (1–8) was performed on human whole blood neutrophils initially, in which 8 showed potential to inhibit the generation of reactive oxygen species (ROS). Further study on isolated human polymorphonuclear cells or neutrophils exhibited that 8 possessed strong antioxidant eﬀects with an IC value = 3.68 0.22 µM, comparing to the positive control Trolox with 50 ± IC value = 77.23 1.62 µM. 50 ± Molecules 2019, 24, 1664 6 of 10

2.3. Cytotoxic Activity of Isolated Compounds Compounds 2–8 were also evaluated for their cytotoxicity against human tumor cells: HL-60 (acute leukemia), HepG-2 (liver hepatocellular carcinoma), HCT-16 (colorectal carcinoma), BGC-823 (gastric carcinoma cell) using MTT assay. Unfortunately, all the tested compounds showed the IC50 value at more than 100 µM. Since different biochemical, molecular and genetic mechanisms are involved in the development and progression of these cancers, the treatment also requires the specific agents. Ideally, the test compounds should be assayed for their cytotoxic potential on large number of cancer cell lines in the preliminary screening, with the aim of finding a lead compound to study the mechanism of action to treat such malicious tumors, which might be a limiting factor for any significant cytotoxicity that is lacking in our study [38].

3. Materials and Methods

3.1. Plant Material The stems of Kadsura heteroclita were collected in Shimen, Hunan, China, in September, 2014. The plant was identiﬁed by Prof. Wei Wang, and a voucher specimen (KH-shimen-201409) has been deposited in the TCM and Ethnomedicine Innovation & Development International Laboratory, Innovative Materia Medica Research Institute, School of Pharmacy, Hunan University of Chinese Medicine.

3.2. General and Solvents Optical rotations were measured on a Perking-Elmer 341-MC digital polarimeter. UV spectra were recorded on a TU-1900 spectrophotometer (Shimadzu Europa GmbH, Duisburg, Germany). Experimental CD spectra were recorded on a JASCO J-815 Circular Dichroism (CD) Spectropolarimeter (Jasco, Mary’s Court Easton, MD, USA). A Hitachi 260-30 spectrometer was used for scanning IR spectroscopy. ID and 2D NMR spectra were performed on Bruker ARX-600 spectrometers ((Bruker Technology Co., Ltd., Karlsruhe, Germany) using TMS as the internal standard, with chemical shifts (δ) expressed in ppm and referenced to the solvent signals. HRESIMS were performed on a UPLC/xevo G2 Qtof spectrometer (Waters Corporation, Milford, MA, USA). Column chromatography (CC) was performed with silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China), and Sephadex LH-20 (Pharmacia). Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) with an Alltima C18 (5µ ODS 10 mm 250 mm) column. Fractions were monitored by thin-layer chromatography (TLC) (Qingdao × Marine Chemical Inc., Qingdao, China), and spots were visualized by heating the TLC silica gel plates sprayed with 10% H2SO4 in EtOH (10:90, v/v). Petroleum ether (PE), ethyl acetate (EtOAc), n-butanol (n-BuOH), dichloromethane (CH2Cl2) and methanol (MeOH) were purchased from Shanghai Titan Scientiﬁc Co., Ltd, Shanghai, China. MeOH (HPLC grade) and Water (HPLC grade) were purchased from Merck KGaA, 64271 Darmstadt, Germany.

3.3. Extraction and Isolation The air-dried stems and roots of K. heteroclita (400 kg) were powdered and extracted with 95% aqueous ethanol (3 400 L, 3 days each) at room temperature and concentrated under reduced pressure × to afford a crude extraction (8 kg). 1 kg of crude extraction was suspended in H2O (10 L) and was partitioned between water and n-BuOH (10 L) to afford the n-BuOH fraction, repeated for 3 times, the n-BuOH layer was combined and evaporated to yield a residue of 251.5 g. Then 240.0 g of n-BuOH fraction was chromatographed on a silica gel column (3.0 kg, 100–200 mesh) using dichloromethane/methanol as elution solvents (from 99.8:0.2 to 0:100 gradient system), to afford fraction A–J. Fraction C (35.1 g) was chromatographed using silica gel CC (0.9 kg, 200–300 mesh), eluted with a DCM/MeOH gradient system (99.8:0.2 to 0:100) to obtain ten fractions (Fr. C-1 to Fr. C-10). Molecules 2019, 24, 1664 7 of 10

Fr. C-5 (3.1 g) was loaded to silica gel (0.1 kg, 200–300 mesh) column and eluted with petroleum ether/ethyl acetate (90:10 to 20:80) to produce eight fractions (Fr. C-5-a to Fr. C-5-h). Fr. C-5-b (227.0 mg) was isolated by silica gel column (9.0 g, 200–300 mesh), by eluting with PE/EtOAc (40:60) to afford compound 5 (12.0 mg). Fr. C-10 (2.0 g) was subjected to silica gel CC (0.15 kg, 200–300 mesh) eluted with PE/EtOAc (80:20 to 0:100) to give twelve fractions (Fr. C-10-1 to Fr. C-10-12). Fr. C-10-7 was purified by semipreparative HPLC, with a solvent of MeOH/H2O (3 ml/min, 75:25), to afford 8 (4.3 mg). Fraction D (20.6 g) was separated over silica gel CC (0.6 kg, 200–300 mesh) with a gradient elution of DCM/MeOH (99.5:0.5 to 0:100) to give ten fractions (Fr. D-1 to Fr. D-10). Fr. D-3 (1.5 g) was repeated eluting on silica gel CC (0.2 kg, 200–300 mesh) and produced six subfractions (D-3-a to D-3-f), D-3-d (350.0 mg) was purified by silica gel CC (50.0 g, 200–300 mesh) eluting with PE/EtOAc (90:10 to 0:100) to yield two crystals 4 (6.5 mg) and 6 (120.5 mg). Fraction D-5 (1.2 g) was chromatographed by silica gel CC (0.1 kg, 200–300 mesh) eluted with PE/EtOAc (90:10 to 0:100) to give six fractions (Fr. D-5-a to Fr. D-5-f). Fr. D-5-a (214.7 mg) was applied to another silica gel CC (20.0 g, 200–300 mesh) eluted with PE/EtOAc (80:20) to give six fractions (Fr. D-5-a-1 to Fr. D-5-a-6). Fr. D-5-a-5 (42.0 mg) was purified by semipreparative HPLC (2 mL/min, MeOH/H2O, 75:25) to yield 3 (1.8 mg) and 2 (7.6 mg) respectively. The Fr. D-6 (1.0 g) was repeated on silica gel (0.1 kg, 200–300 mesh) column eluting with PE/EtOAc (50:50 to 0:100) to yield eight fractions (Fr. D-6-a to Fr. D-6-h). Fr. C-6-f (201.3 mg) was separated by sephadex LH 20 (10.0 g) column with the washing solvent methanol/chloroform (1:1), to produce four fractions (Fr. C-6-f-1 to C-6-f-4). Fr. C-6-f-2 (21.7 mg) was purified by semi prepare HPLC (MeOH/H2O, 75: 25) to obtain compound 1 (2.1 mg). Fr. D-7 (0.5 g) was subjected to silica gel CC (50.0 g, 200–300 mesh) eluted with PE/EtOAc (80:20 to 0:100) to provide eight fractions (Fr. D-7-a to Fr. D-7-h). Fr. D-7-g (54.8 mg) was purified by semipreparative HPLC, with a solvent system of MeOH/H2O (3 mL/min, 70:30), to afford compounds 7 (10.2 mg).

3.4. Spectroscopic Data of New Compounds

6α,9α,15-Trihydroxycadinan-4-en-3-one (1): white solid (MeOH); [α]20 20.0 (c 0.25, MeOH); UV(MeOH) D − λmax (log ε) 230 (3.10) nm; CD (c 0.50, MeOH) λmax (∆ε) 248 ( 4.42), 270 (+3.66), 332 (+4.99) nm; IR 1 1 − 13 νmax 3410, 2930, 1713, 1668, 1606, 1283, 1255, 1098 cm− ; H and C-NMR data, see Table1; positive + HRESIMS m/z 289.1419 [M + Na] (calcd for C15H22O4Na, 289.1416).

20 (+)-3,11,12-Trihydroxycalamenene (2): white, amorphous solid; [α]D +12.1 (c 1.9, MeOH); UV(MeOH) λmax (log ε) 283 (3.41) nm; CD (c 0.33 MeOH) λmax (∆ε) 238 ( 5.49) nm, 276 ( 8.68); IR νmax 3385, 2960, − 1 1 13 − 2932, 2873, 1620, 1505, 1457, 1381, 1260, 1187, 1114, 1039 cm− ; H and C-NMR data, see Table1; + positive HRESIMS m/z 273.1471 [M + Na] (calcd for C15H22O3Na, 273.1461).

(–)-3,10,11,12-Tetrahydroxy-calamenene (3): white, amorphous solid; [α]20 2.2 (c 0.45, MeOH); D − UV(MeOH) λmax (log ε) 220 (3.90) nm, 280 (3.45) nm; CD (c 0.12, MeOH) λmax (∆ε) 215 (+11.85), 237 1 1 13 ( 2.42), 263 (+6.16) nm; IR νmax 3379, 2932, 1623, 1463, 1421, 1376, 1196, 1044 cm− ; H and C-NMR − + data, see Table1; positive HRESIMS m/z 289.1396 [M + Na] (calcd for C15H22O4Na, 289.1416).

3.5. Antioxidant Assay Chemiluminescence assay was utilized to measure the antioxidant activity. It is a sensitive method to measure the inhibition of ROS production [39]. Phorbol 12-myristate 13-acetate (PMA) is used as an inducer for the generation of various ROS by macrophages and neutrophils, and PMA is used as an activator of protein kinase C and activator of NADPH oxidase in our study [11]. Different concentrations of samples were incubated with either diluted whole blood 40 µL (1:25 dilution in sterile PBS, pH 7.4) or PMN 40 µL (Polymorphonuclear cells, 1 106/mL) suspended in × HBSS++. The cells were stimulated with 40 µL of opsonized zymosan followed by luminol as enhancer and then HBSS++ was added to adjust the final volume to 200 µL. The final concentrations of the samples in the mixture were 20, 10, 5, 2.5 and 0.625 µM. Microplates were then incubated at 22 °C for Molecules 2019, 24, 1664 8 of 10

30 min. The results were measured by Enspire Multimode Plate Reader (PerkinElmer, Inc., Winter Street Waltham, MA, USA), as counts per second (CPS). Inhibition percentage (%) calculated as:

Inhibition percentage (%) = 100 (CPStest/CPS ) 100 − control × 3.6. Cytotoxicity Assays The cytotoxicity assay was performed using an MTT assay [40], the experiment method referenced to the reported literature [11] and the following human tumor cell lines were used: HL-60 (acute leukemia), HepG-2 (liver hepatocellular carcinoma), HCT-16 (colorectal carcinoma), BGC-823 (gastric carcinoma cell). Each tumor cell line was exposed to test compounds in triplicate for 48 hr., with Taxol used as a positive control substance.

Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/9/1664/s1, Figures S1–S12: 1D and 2D NMR, HRESIMS, UV, IR, CD spectra of compound 1. Figures S13–S24: 1D and 2D NMR, HRESIMS, UV, IR, CD spectra of compound 2. Figures S25-S36: 1D and 2D NMR, HRESIMS, UV, IR, CD spectra of compound 3. Figures S37–S46: H and C-NMR spectra of compounds 4–8. Author Contributions: W.W. (corresponding author) and D.-f.L. conceived and designed the idea of the study; L.C., N.S. contributed to isolating work and paper writing; Y.J., B.L., C.P. performed the NMR spectra elucidating; M.C., W.S. contributed to the HRESI-MS, IR and other spectra experiment work; S.T. designed the content of bioassays; W.W., M.I.C., A.-u.-R. contributed to revision, funding acquisition, project administration and supervision. All of the authors read and approved the final manuscript. Funding: This work was supported in part by National Natural Science Foundation of China (81673579, 81874369 and 81374062), Ministry of Science and Technology of P.R.China(2018FY100703) and Hunan Science and Technology Department (2019JJ50315, 2018SK 2110 and 2081). Acknowledgments: The authors would like to thank Bin Liu in Hunan University for valuable suggestion in Bioassay. Conflicts of Interest: The authors declare no conflict of interest.

References

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Sample Availability: Samples of the compounds 1–8 are available from the authors.

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