molecules

Article Crepidatumines C and D, Two New Indolizidine Alkaloids from crepidatum Lindl. ex Paxt.

1, 2, 1 2 2 1 Xiaolin Xu †, Zesheng Li †, Runmei Yang , Houguang Zhou , Yanbin Bai , Meng Yu , Gang Ding 1,* and Biao Li 1,*

1 Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, 2 Dehong Institute of Tropical Agricultural Science, Dehong 678600, China * Correspondence: [email protected] or [email protected] (G.D.); [email protected] (B.L.); Tel.: +86-010-5783-3281 (G.D.) Theses authors contributed equally to this work. †  Received: 15 July 2019; Accepted: 20 August 2019; Published: 23 August 2019 

Abstract: Two new indolizidine alkaloids, crepidatumines C (1) and D (2), together with crepidine (3), isocrepidamine (4), and crepidamine (5) were isolated from the Dendrobium crepidatum Lindl. ex Paxt. X-ray diffraction experiments established the absolute configurations of known compounds 3 and 4. The planar structures and relative configurations of new compounds 1 and 2 were elucidated by extensive spectra analysis including HR-ESI-MS, NMR (1H, 13C, 1H-1H COSY, HSQC, HMBC, and NOESY spectra), and the absolute configurations of 1 and 2 were suggested on the basis of possible biosynthetic pathways. The biological results confirmed that isocrepidamine (4) displayed a potent hypoglycemic effect in vitro without cytotoxicity.

Keywords: Dendrobium crepidatum Lindl. ex Paxt.; indolizidine alkaloids; crepidatumines; structure elucidation; hypoglycemic effect

1. Introduction Dendrobium, a genus of , is distributed in south of China [1,2]. The stems of several Dendrobium species are used as precious traditional Chinese medicines with the effect of maintaining gastric tonicity, enhancing the production of body fluids, and relieving and curing symptoms of dryness and body heat [3]. Dendrobium crepidatum Lindl. ex Paxt. Is considered as one of the sources of “Shi-Hu”. It produces indolizine-type alkaloids, and so far, only several indolizine-type alkaloids and two stilbene derivatives have been isolated from this medicinal plant [4–9]. Two indolizidine alkaloids, crepidatumines a and B, with novel skeletons, together with the stereoisomer of dendrocrepidine B and dendrocrepine, were isolated during our previous chemical investigation of this medicinal plant [10]. Actually, the biosynthesis of indolizidine alkaloids from the Dendrobium crepidatum Lindl. ex Paxt. is still not clear. In order to obtain the potential intermediates or shunt products of the biosynthesis, five indolizidine alkaloids including the two new analogs crepidatumines C (1) and D (2) together with crepidine (3), isocrepidamine (4), and crepidamine (5) were isolated from the total alkaloid extract [5]. (Figure1) In this paper, the structure elucidation, biological evaluation, and possible biogenetic origin of compounds 1–5 arereported.

Molecules 2019, 24, 3071; doi:10.3390/molecules24173071 www.mdpi.com/journal/molecules Molecules 2019, 24, 3071 2 of 9 MoleculesMolecules 20192019,, 2424,, xx 2 2 ofof 99

FigureFigureFigure 1. 1.1. Structures StructuresStructures of of 11––55.. .

2. Results2.2. ResultsResults and andand Discussion DiscussionDiscussion TheTheThe structures structures structures of of of compounds compounds compounds3 33and andand 4 44 were werewere determineddetermineddetermined toto to bebe be crepidinecrepidine crepidine andand and isocrepidamine,isocrepidamine, isocrepidamine, whichwhichwhich were were were supported supported supported by X-ray by by X-raydi X-rayffraction diffraction diffraction experiments experiments experiments (Figure 2 (Figure (Figure), and compound 2), 2), and and compound compound5 was characterized 55 waswas to becharacterizedcharacterized crepidamine toto basedbebe crepidaminecrepidamine on the NMR basedbased data onon [thethe5]. NMRNMR data[5].data[5].

Figure 2. X-ray crystal structure of 3 and 4. FigureFigure 2.2. X-rayX-ray crystalcrystal structurestructure ofof 33 andand 44.. The molecular formula of 1 was determined to be C18H25NO2 based on the HRESIMS (m/z The molecular formula of 1 was determined to be C18H25NO2 based on the HRESIMS (m/z 288.1968The [M + molecularH]+, calcd formula 288.1964). of 1 The was1 H, determined13C and HMQC to be C spectra18H25NO of2 1 based(Table on1) revealed the HRESIMS the presence (m/z 288.1968 [M + H]++, calcd 288.1964). The 11H, 1313C and HMQC spectra of 1 (Table 1) revealed the of two288.1968 methyls, [M + five H] ,methylenes, calcd 288.1964). three The methines, H, C andan oxygenated HMQC spectra carbon of 1 with (Table chemical 1) revealed shift thevalue presencepresence ofof twotwo methyls,methyls, fivefive methylenes,methylenes, threethree methines,methines, anan oxygenatedoxygenated carboncarbon withwith chemicalchemical shiftshift δC = 76.5, a keto group with chemical shift value δC = 206.8, and a mono-substituted phenyl ring. valuevalue δδCC == 76.5,76.5, aa ketoketo groupgroup withwith chemicalchemical shiftshift valuevalue δδCC == 206.8,206.8, andand aa mono-substitutedmono-substituted phenylphenyl In addition, a singlet proton formed as a free hydroxyl or amino group.The 1H-1H COSY correlations ring.ring. In In addition, addition, a a singlet singlet proton proton formed formed as as a a freefree hydroxyl hydroxyl or or amino amino group.The group.The 11H-H-11HH COSY COSY established three isolated spin-systems including a mono-substituted phenyl unit, and a fragment: correlationscorrelations establishedestablished threethree isolatedisolated spin-systemsspin-systems includingincluding aa mono-substitutedmono-substituted phenylphenyl unit,unit, andand –C-12–C-7–C-8–C-9–C-10–C-11–C-1–C-2–. Analysis of HMBC correlations elucidated the structure aa fragment: fragment: –C-12–C-7–C-8–C-9–C-10–C-11–C-1–C-2–. –C-12–C-7–C-8–C-9–C-10–C-11–C-1–C-2–. Analysis Analysis of of HMBC HMBC correlations correlations elucidated elucidated of 1 (Figure3). The HMBC cross peaks from 6-OH to C-5, C-6, C-7, and C-1 determined the direct thethe structure structure of of 11 (Figure(Figure 3). 3). The The HMBC HMBC cross cross peaks peaks from from 6-OH 6-OH to to C-5, C-5,0 C-6, C-6, C-7, C-7, and and C-1’ C-1’ connectivitiesdetermineddetermined of thethe C-6 directdirect with connectivitiesconnectivities C-5, C-7, and ofof C-1 C-6C-60 with withwith a C-5,C-5, hydroxyl C-7,C-7, andand group C-1’C-1’ anchoredwithwith aa hydroxylhydroxyl at C-6; groupgroup the correlations anchoredanchored of CHat3at-4 C-6;C-6; with thethe C-2 correlationscorrelations and C-3 established ofof CHCH33-4-4 withwith the linkage C-2C-2 andand of C-3C-3 CH establishedestablished3-4 with C-2 thethe and linkagelinkage C-3; and ofof CHCH correlations33-4-4 with with C-2C-2 of and CH and 2 -5 withC-3;C-3; C-1 andand and correlationscorrelations C-9, and H-9 ofof CH withCH22-5-5 C-1, withwith established C-1C-1 andand C-9,C-9, an and indolizidineand H-9H-9 withwith ring C-1,C-1, system. establishedestablished Thus, anan the indolizidineindolizidine planar structure ringring of 1system.system.was characterized. Thus, Thus, the the planar planar The structurerelative structure configuration of of 11 was was characterized. characterized. of 1 was determined The The relative relative on configuration configuration the basis of analysis of of 11waswas of NOESYdetermineddetermined correlations. onon thethe basisbasis The NOESYofof analysisanalysis correlations ofof NOESYNOESY fromcorrelations.Thecorrelations.The H-5b, H-7 NOESY toNOESY H-20 (H-6correlationscorrelations0), and fromfrom H-5b, H-5bH-5b, H-7 toH-7 H-9 confirmedtoto H-2’H-2’ (H-6’),(H-6’), that these andand from protonsfrom H-5bH-5b or toto groups H-9H-9 confirmedconfirmed were on that thethat samethesethese protons sideprotons of the oror groupsgroups corresponding werewere onon the piperidinethe samesame sideside ring; of the corresponding piperidine ring; the correlations of –CH2-2 with H-5b, and of H-1 and 6-OH theof correlations the corresponding of –CH2 -2 piperidine with H-5b, ring; and the of correlations H-1 and 6-OH of –CH with2-2 H-5a with demonstrated H-5b, and of that H-1 theseand 6-OH protons werewithwith close H-5aH-5a to eachdemonstrateddemonstrated other in space. thatthat thesethese Thus, protonsprotons the relative werewere close configurationclose toto eacheach otherother of 1 was inin space.space. determined Thus, Thus, the (Figurethe relative relative3). configurationconfigurationThe HRESIMS ofof 11 ( waswasm/z determined286.1809determined [M (Figure(Figure+ H]+ 3).,3). calcd 286.1807) assigned the molecular formula of 2 as 1 13 C18H23NO2. The H, C and HMQC spectra of 2 (Table1) revealed the presence of one methyl, five methylenes, four methines, an oxygenated carbon with chemical shift value δC = 76.5, a keto group with chemical shift value δC = 208.5, and a mono-substituted phenyl ring. In addition, there are two singlet protons as free hydroxyl or amino groups. These data accounted for all the 1H and 13C-NMR resonances together considering the degrees of unsaturation, implying that 2 possessed a tricyclic system. The 1H-1H COSY correlations established three isolated proton spin-systems including 2 a mono-substituted phenyl unit, and two fragments: –C-12–C-7–C-8–C-9–C-10–C-11–C-1–C-2–2 and Molecules 2019, 24, 3071 3 of 9

–C-4–C-5–. Analysis of HMBC correlations elucidated the structure of 2 (Figure4). Correlations of H-5 with C-1 and C-9, and H-9 with C-1 established an indolizidine ring system. The HMBC cross peaks from 6-OH to C-5, C-6, C-7, and C-10 determined the direct connectivities of C-6 with C-5, C-7, and C-10 with a hydroxyl group anchored at C-6.The correlations of H-4 with C-2 and C-3 established the linkage of C-4 with C-2 and C-3. Thus, the planar structure of 2 was characterized. The relative configuration of 2 was determined on the basis of analysis of NOESY correlations. The correlations from H-5 with H-1, H-7, H-9 and 7-OH implied that these groups possessed β-configurations. The NOESY correlations from 12-Me to H-20 (H-60) confirmed that 12-Me and the mono-substituted phenyl group were on the same side of the corresponding furan ring. Thus, the relative configuration of 2 was determined (Figure4).

a,b Table 1. NMR spectroscopic data of 1 and 2 in (DMSO-d6)(δ in ppm and J in Hz) .

Pos 1 2 b a b a δC , Type δH , mult. (J in Hz) δC , Type δH , mult. (J in Hz) 1 74.8, CH 4.10, m 59.9, CH 3.37, m 2.33, dd (10.8, 13.8) 2 48.6, CH 2.46, m 44.1, CH 2 2 1.92, ddd (1.8, 4.2, 13.8) 3 206.8, qC 209.6, qC 1.13, ddd (1.8, 3.0, 13.2) 4 30.9, CH 2.07, s 38.6, CH 3 2 2.63, t (13.2) 5a 2.95, d (10.8) 67.1, CH 66.7, CH 3.00, dd (3.0, 13.2) 5b 2 2.69, d (10.8) 6 76.5, qC 76.2, qC 7 38.4, CH 1.96, m 31.7, CH 2.46, m 1.46, m 8 36.3, CH 1.49, m 35.5, CH 2 2 1.74, dt (3.0, 11.4) 9 63.9, CH 2.43, m 51.4, CH 3.15, m 1.65, m 1.40, m 10 30.2, CH 29.7, CH 2 1.46, m 2 1.97, m 1.67, m 1.39, m 11 30.8, CH 29.1, CH 2 1.25, m 2 2.04, m 12 14.4, CH3 0.48, d (6.6) 16.1, CH3 0.71, d (6.6) 10 146.1, qC 143.9, qC 20/60 128.2, CH 7.46, dd (1.2, 8.4) 126.9, CH 7.41, br d (7.2) 30/50 125.6, CH 7.31, br d (8.4) 128.2, CH 7.34, dd (7.2) 40 126.8, CH 7.21, br t (8.4) 127.0, CH 7.24, dd (7.2) 6-OH 4.76, s 4.53, s a Assignments were based on HSQC, HMBC, and 1H-1H COSY experiments. b NMR spectroscopic data were recorded at 600 MHz (1H NMR), 150 MHz (13C NMR). Molecules 2019, 24, x 3 of 9

1 1 FigureFigure 3. 3.1 H-H-1HH COSY, HMBC, and and NOESY NOESY correlations correlations of of1. 1.

The HRESIMS (m/z 286.1809 [M + H]+, calcd 286.1807) assigned the molecular formula of 2 as C18H23NO2. The 1H, 13C and HMQC spectra of 2 (Table 1) revealed the presence of one methyl, five methylenes, four methines, an oxygenated carbon with chemical shift value δC = 76.5, a keto group with chemical shift value δC = 208.5, and a mono-substituted phenyl ring. In addition, there are two singlet protons as free hydroxyl or amino groups. These data accounted for all the 1H and 13C-NMR resonances together considering the degrees of unsaturation, implying that 2 possessed a tricyclic system. The 1H-1H COSY correlations established three isolated proton spin-systems including a mono-substituted phenyl unit, and two fragments: –C-12–C-7–C-8–C-9–C-10–C-11–C-1–C-2– and –C-4–C-5–. Analysis of HMBC correlations elucidated the structure of 2 (Figure 4). Correlations of H-5 with C-1 and C-9, and H-9 with C-1 established an indolizidine ring system. The HMBC cross peaks from 6-OH to C-5, C-6, C-7, and C-1’ determined the direct connectivities of C-6 with C-5, C-7, and C-1’ with a hydroxyl group anchored at C-6.The correlations of H-4 with C-2 and C-3 established the linkage of C-4 with C-2 and C-3. Thus, the planar structure of 2 was characterized. The relative configuration of 2 was determined on the basis of analysis of NOESY correlations. The correlations from H-5 with H-1, H-7, H-9 and 7-OH implied that these groups possessed β-configurations. The NOESY correlations from 12-Me to H-2’ (H-6’) confirmed that 12-Me and the mono-substituted phenyl group were on the same side of the corresponding furan ring. Thus, the relative configuration of 2 was determined (Figure 4).

Figure 4.1H-1H COSY, HMBC, and NOESY correlations of 2.

The absolute configurations of 1 and 2 were suggested to be same as those of 3–5 on the basis of similar biosynthesis pathway (Figure 5). Structures of 3 and 4 were determined by the X-ray diffraction experiments, and showed that 4 is a racemate.

3 Molecules 2019, 24, x 3 of 9

Figure 3. 1H-1H COSY, HMBC, and NOESY correlations of 1.

The HRESIMS (m/z 286.1809 [M + H]+, calcd 286.1807) assigned the molecular formula of 2 as C18H23NO2. The 1H, 13C and HMQC spectra of 2 (Table 1) revealed the presence of one methyl, five methylenes, four methines, an oxygenated carbon with chemical shift value δC = 76.5, a keto group with chemical shift value δC = 208.5, and a mono-substituted phenyl ring. In addition, there are two singlet protons as free hydroxyl or amino groups. These data accounted for all the 1H and 13C-NMR resonances together considering the degrees of unsaturation, implying that 2 possessed a tricyclic system. The 1H-1H COSY correlations established three isolated proton spin-systems including a mono-substituted phenyl unit, and two fragments: –C-12–C-7–C-8–C-9–C-10–C-11–C-1–C-2– and –C-4–C-5–. Analysis of HMBC correlations elucidated the structure of 2 (Figure 4). Correlations of H-5 with C-1 and C-9, and H-9 with C-1 established an indolizidine ring system. The HMBC cross peaks from 6-OH to C-5, C-6, C-7, and C-1’ determined the direct connectivities of C-6 with C-5, C-7, and C-1’ with a hydroxyl group anchored at C-6.The correlations of H-4 with C-2 and C-3 established the linkage of C-4 with C-2 and C-3. Thus, the planar structure of 2 was characterized. The relative configuration of 2 was determined on the basis of analysis of NOESY correlations. The correlations from H-5 with H-1, H-7, H-9 and 7-OH implied that these groups possessed β-configurations. The NOESY correlations from 12-Me to H-2’ (H-6’) confirmed that 12-Me and the Moleculesmono-substituted2019, 24, 3071 phenyl group were on the same side of the corresponding furan ring. Thus, the4 of 9 relative configuration of 2 was determined (Figure 4).

1 1 FigureFigure 4. 4.H-1H-1HH COSY,COSY, HMBC, and and NOESY NOESY correlations correlations of of2. 2.

TheThe absolute absolute configurations configurations of of1 1and and 22 werewere suggested to to be be same same as as those those ofof 3–35– on5 on the the basis basis of similarof similar biosynthesis biosynthesis pathway pathway (Figure (Figure5 , 5). Supplementary Structures of 3 Materials). and 4 were Structures determined of by3 theand X-ray4 were determineddiffraction by experiments, the X-ray di andffraction showed experiments, that 4 is a racemate. and showed that 4 is a racemate. Molecules 2019, 24, x 4 of 9

3

FigureFigure 5. 5.TheThe possible possible biosyntheticbiosynthetic pathway pathway of of compounds compounds 1–15–. 5.

TheThe hypoglycemic hypoglycemic eff ect effect of compound of compound4 (isocrepidamine) 4 (isocrepidamine) was wasevaluated evaluated using using the high the glucose high modelglucose of HepG2 model cells. of HepG2 As a result, cells. As at thea result, concentrations at the concentrations of 200 µmol of/L, 200 this μmol/L, compound this compound significantly increasedsignificantly the glucose increased consumption the glucose by consumption 34% compared by with 34% the compared model group, with the which model hadnon-cytotoxicity group, which as perhadnon-cytotoxicity the cell counting as kit-8 per (CCK-8)the cell counting assay (Figure kit-8 (CCK-8)6). assay (Figure 6).

A B

Figure 6.Effect of compound 4 (C4) on cell viability (A) and glucose consumption (B) in the HepG2 cell. Data are shown as the mean ± SD (n = 6). ** p < 0.01, *** p < 0.001 versus model.

Table 1.NMR spectroscopic data of 1 and 2 in (DMSO-d6) (δ in ppm and J in Hz) a b.

Pos 1 2 δC b, type δH a, mult. (J in Hz) δC b, type δH a, mult. (J in Hz) 1 74.8, CH 4.10, m 59.9, CH 3.37, m 2.33, dd (10.8, 13.8) 2 48.6, CH2 2.46, m 44.1, CH2 1.92, ddd (1.8, 4.2, 13.8) 3 206.8, qC 209.6, qC 1.13, ddd (1.8, 3.0, 13.2) 4 30.9, CH3 2.07, s 38.6, CH2 2.63, t (13.2) 5a 67.1, CH2 2.95, d (10.8) 66.7, CH 3.00, dd (3.0, 13.2)

4 Molecules 2019, 24, x 4 of 9

H H O H CO2 O H H CO2H H H H Me Me HO C 2 N N OH OH 2

HO2C Me OH SCoA H H H CO H H 2 H O O O O O Me Me HO C 2 H N H N OH OH NH2 1 OH CO H CO H H N 2 2 2 O O NH Me 2 N OH SCoA O HO2C OH OH CoAS [O] O O O N H N H Me Me CO OH SCoA 2 5 H H O O O

HO Me H OH O H O HO C CO2H H 2 H H OH O OH O [O] N H H N H Me N Me Me Me HO2C CO HO2C 2 H H 4 H H 3 Figure 5.The possible biosynthetic pathway of compounds 1–5.

The hypoglycemic effect of compound 4 (isocrepidamine) was evaluated using the high glucose model of HepG2 cells. As a result, at the concentrations of 200 μmol/L, this compound Moleculessignificantly2019, 24 increased, 3071 the glucose consumption by 34% compared with the model group, which5 of 9 hadnon-cytotoxicity as per the cell counting kit-8 (CCK-8) assay (Figure 6).

(A) (B)

FigureFigure 6.6.EffectEffect of compound 4 (C4) onon cellcell viabilityviability ((AA)) andand glucoseglucose consumptionconsumption ((BB)) inin thethe HepG2HepG2 cell.cell. DataData areare shownshown asas thethe meanmean ± SD (n = 6). ** ** pp << 0.01,0.01, *** *** pp << 0.0010.001 versus versus model. model. ±

3. Experimental Section a b. Table 1.NMR spectroscopic data of 1 and 2 in (DMSO-d6) (δ in ppm and J in Hz)

3.1. General ExperimentalPos Procedures 1 2 δC b, type δH a, mult. (J in Hz) δC b, type δH a, mult. (J in Hz) Optical rotations were measured on a PerkinElmer 241 polarimeter (Perkin Elmer, Inc., Waltham, MA, USA), and1 UV 74.8,data CH were determined 4.10, m on a ThermoGenesys-10S 59.9, CH UV-vis 3.37, m spectrometer (Fisher 2.33, dd (10.8, 13.8) Scientific, Illkirch,2 France).48.6, CH2 IR data2.46, were m recorded 44.1, using CH a2Nicolet IS5FT-IR spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were obtained on a JASCO1.92, J-810 ddd spectrometer (1.8, 4.2, 13.8) (JASCO, Tokyo, Japan). 1H and3 13C-NMR206.8, dataqC were acquired with a Bruker209.6, qC 600 spectrometer (Bruker, Rheinstetten, 1.13, ddd (1.8, 3.0, 13.2) Germany) using4 solvent30.9, signalsCH3 (DMSO-2.07,d6 ;s δH 2.50/δ38.6,C 39.5) CH as2 references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS2.63, t (13.2) were obtained using a TOF-ESI-MS5a (Waters 67.1, Synapt CH2 G2, Milford, 2.95, d (10.8) MA, USA). 66.7,Semipreparative CH 3.00, HPLC dd (3.0, separation 13.2) was carried out using a Lumtech instrument packed with a YMC-Pack ODS-A column (YMC Co., Ltd., Kyoto, 4 Japan, 5 µm, 250 10 mm). Sephadex LH-20(Pharmacia Biotech AB, Uppsala, Sweden) and silica gel × (200–300 mesh) (Qingdao Marine Chemical Plant, Qingdao, China) were used.

3.2. Plant Materials The stems of Dendrobium crepidatum Lindl. ex Paxt. were collected from Ruili Resource Nursery of Dendrobium Germ Plasm and Resources, the Ministry of Agriculture and Rural Affairs of the People’s Republic of China (Yunnan, China) in August 2017. The sample was identified by one of the co-authors Ze-Sheng Li from Yunnan Dehong Institute of Tropical Agricultural Science (Yunnan, China). a voucher specimen was deposited in the herbarium of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences (Beijing, China).

3.3. Extraction and Isolation The dried stems of Dendrobium crepidatum Lindl. ex Paxt. (9.0 kg) were extracted under reflux with 95% ethanol (50 L 3 h, three times). The combined extract was suspended with water, and extracted × with petroleum ether and CH2Cl2 three times separately. The fraction of CH2Cl2 was concentrated into extracts, and dissolved in 5% hydrochloric acid filtered, then adjusted to pH 10 with ammonia water. Finally, it was extracted by CH2Cl2 three times at room temperature. The CH2Cl2 extract was obtained the total alkaloids 90 g of crude extract. The original extract was fractionated on a silica gel CC eluted with petroleum ether- acetone (50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 5:1, 2:1 and 0:1, v/v, each 6.6 L) to give five fractions (Fr.1 to Fr.5). Fr.2 (10 g) was fractionated on a silica gel column chromatography (CC) using petroleum ether-acetone isocratic elution (30:1) to afford six fractions (Fr.2.1–Fr.2.6). Fr.2.1 (10.0 g) was purified by semi-preparative HPLC (60–100% MeOH-H2O for 30.0 min, v/v, 2 mL/min) to obtain crepidine (3; 105 mg, tR 29.0 min), and isocrepidamine (4; 2.0 g, tR 32.7 min). Separation of Fr.2.4 (2.0 g) was performed over Sephadex LH-20 (CH2Cl2: MeOH/1:1) to give four fractions (Fr.2.4.1–Fr.2.4.4). Fr.2.4.2 (500 mg) was further purified by semi-preparative HPLC (60–100% MeOH-H2O for 30 min, v/v, 2 mL/min) to obtain crepidamine (5; 30.0 mg, tR 21.0 min). Fr.2.4.3 (1.0 g) was purified by Molecules 2019, 24, 3071 6 of 9

semi-preparative HPLC (60–100% MeOH-H2O for 30.0 min, v/v, 2 mL/min) to obtain crepidatumine C (1; 4.0 mg, tR 32.8 min), and crepidatumine D (2; 25.0 mg, tR 24.7 min). 25 Compound 1: white powder; [α]D 3.00 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.68); − 1 1 13 IR (neat) υmax 2930, 1714, 1036, 766, 704 cm− ; for H-NMR and C-NMR data see Table1; + Positive HR-ESI-MS: m/z 288.1964 (calcd. for C18H26NO2 [M + H] , 288.1968). 25 Compound 2: white powder; [α]D 4.00 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (3.81); − 1 1 13 IR (neat) υmax 3482, 2964, 1708, 999, 776, 709 cm− ; for H-NMR and C-NMR data see Table1; + Positive HR-ESI-MS: m/z 286.1807 (calcd. for C18H24NO2 [M + H] , 286.1809).

3.4. X-Ray Crystallographic Analysis of 3 and 4.

Upon crystallization from n-Hexane–CH2Cl2 (10:1) using the vapor diffusion method, colorless crystals were obtained for 3.C21H29NO3,M = 343.45, orthorhombic, a = 5.7476(3) Å, b = 17.5786(5) Å, c = 17.7942(6) Å, U = 1797.84(11) Å3, T = 109.1(3), space group P212121 (No. 19), Z = 4, µ(Cu Kα) = 0.666, 9652 reflections measured, 3383 unique (Rint = 0.0609), which were used in all calculations. The final wR (F2) was 0.1367 (all data). Crystallographic data for the structure of 3 has been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1936544) (Table2).

Table 2. Crystal data and structure refinement for 3.

Identification Code 3

Empirical formula C21H29NO3 Formula weight 343.45 Temperature/K 109.1(3) Crystal system orthorhombic

Space group P212121 a/Å, b/Å, c/Å 5.7476(3), 17.5786(5), 17.7942(6)

α/◦, β/◦, γ/◦ 90, 90, 90 Volume/Å3 1797.84(11) Z 4 3 %calc/mg mm− 1.269 1 µ/mm− 0.666 F (000) 744 Crystal size/mm3 0.35 0.12 0.02 × × 2θ range for data collection 7.06 to 142.74◦ Index ranges 6 h 7, 18 k 21, 21 l 19 − ≤ ≤ − ≤ ≤ − ≤ ≤ Reflections collected 9652 Independent reflections 3383[R(int) = 0.0609 (inf-0.9Å)] Data/restraints/parameters 3383/0/231 Goodness-of-fit on F2 1.030

Final R indexes [I > 2σ (I) i.e., Fo > 4σ (Fo)] R1 = 0.0510, wR2 = 0.1302

Final R indexes [all data] R1 = 0.0551, wR2 = 0.1367 Largest diff. peak/hole/e Å 3 0.275/ 0.288 − − Flack Parameters 0.2(2) Completeness 0.993 Molecules 2019, 24, 3071 7 of 9

Upon crystallization from n-Hexane–CH2Cl2 (10:1) using the vapor diffusion method, colorless crystals were obtained for 4.C20H27NO4,M = 345.42, orthorhombic, a = 6.6679(4) Å, b = 10.7681(4) Å, c = 24.2111(9) Å, U = 1738.38(13) Å3, T = 107.75(10), space group P212121 (no. 19), Z = 4, µ (Cu Kα) = 0.737, 5698 reflections measured, 3256 unique (Rint = 0.0271), which were used in all calculations. The final wR (F2) was 0.1106 (all data).Crystallographic data for the structure of 4 has been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1908235) (Table3).

Table 3. Crystal data and structure refinement for compound 4.

Identification Code 4

Empirical formula C20H27NO4 Formula weight 345.42 Temperature/K 107.75(10) Crystal system orthorhombic

Space group P212121 a/Å, b/Å, c/Å 6.6679(4), 10.7681(4), 24.2111(9)

α/◦, β/◦, γ/◦ 90, 90, 90 Volume/Å3 1738.38(13) Z 4 3 %calc/mg mm− 1.320 1 µ/mm− 0.737 F (000) 744 Crystal size/mm3 0.350 0.340 0.100 × × 2θ range for data collection 8.988 to 142.446◦ Index ranges 4 h 7, 11 k 13, 29 l 29 − ≤ ≤ − ≤ ≤ − ≤ ≤ Reflections collected 5698 Independent reflections 3256[R(int) = 0.0271 (inf-0.9Å)] Data/restraints/parameters 3256/0/230 Goodness-of-fit on F2 1.054

Final R indexes [I > 2σ (I) i.e., Fo > 4σ (Fo)] R1 = 0.0397, wR2 = 0.1072

Final R indexes [all data] R1 = 0.0421, wR2 = 0.1106 Largest diff. peak/hole/e Å 3 0.312/ 0.240 − − Flack Parameters 0.13(14) − Completeness 0.9984

3.5. In Vitro Evaluation of Compound 4 Cell culture: Human hepatoma cells HepG2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone). The medium was supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (HyClone) in a humidified atmosphere of 5% CO2 and 37 ◦C. Assay for cell viability: The assay for cell viability was determined with the cell counting kit-8 (CCK-8). HepG2 cells were seeded in 96-well plates as 2.5 103 cells each well. After culturing for 24 h, × the control group was added with serum-free medium, while the experimental groups were with the medium containing different concentrations (50, 100, and 200 µmol/L) of compound 4 or 200 µmol/L of metformin for another 24 h. Then the cells were treated with CCK-8 for 4 h. Finally, the absorbance was measured at 450 nm. The cell survival rate was calculated as the absorbance of each treated well divided by the control. Molecules 2019, 24, 3071 8 of 9

Assay for hypoglycemic activity: For the experiment, the cells were seeded in 96-well plates as 1 104 cells each well. After culturing for 24 h, the medium containing different concentrations × (50, 100 and 200 µmol/L) of compound 4 was added for 24 h. The cells with 200 µmol/L metformin treatment were taken as positive control and the cells with phenol red-free DMEM as control. After the drug treatment, the glucose concentrations of the medium were determined with the glucose oxidase method. The glucose consumption of each well was obtained by subtracting the glucose concentrations of the experimental medium from the control group.

4. Conclusions Two new indolizidine alkaloids crepidatumines C (1) and D (2) together with crepidine (3), isocrepidamine (4), and crepidamine (5) were isolated from the Dendrobium crepidatum Lindl. ex Paxt., and their structures were determined by HR-ESI-MS, NMR (1H, 13C, 1H-1H COSY, HSQC, HMBC, and NOESY spectra), and X-ray diffraction experiments. The results enrich the chemical diversity and further provide the key intermediates in the biosynthetic pathway of indolizidine alkaloids from Dendrobium crepidatum Lindl. ex Paxt., implying that more minor intermediates or shunt products might exist in the medicinal . In addition, the biological study showed a potent hypoglycemic effect of isocrepidamine (4) in vitro without cytotoxicity.

Supplementary Materials: The following including NMR, IR, UV and HR-ESI-MS spectra of compounds 1 and 2 are available online. Author Contributions: X.X. performed the isolation; Z.L., H.Z. and Y.B. provided and identified the medicinal plants; Y.Y. performed the biological experiments; M.Y. provided the mass data; G.D. analyzed the NMR spectra; G.D. and B.L. wrote the manuscript. Funding: We gratefully acknowledge financial support from the National Key R & D Program of China (grant number 2018YFC1706200), Species and Varieties Resource Protection (Tropical Crops) Program “Germ Plasm and Resources Protection of Dendrobium” from the Ministry of Agriculture and Rural Affairs of the People’s Republic of China (grant number 151821301354052710); CAMS Initiative for Innovative Medicine (grant number CAMS-2016-I2M-2-003), and the National Natural Science Foundation of China (grant number 81473331). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds (1 and 2) are available from the authors.

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