Chemical Compositions of Eupatorium heterophyllum Leaf Samples from Yunnan and Sichuan Provinces of China—Isolation of 13 New Sesquiterpene Lactones

Eight samples of Eupatorium heterophyllum leaves were collected at different locations in Yunnan and Sichuan provinces in China, and their chemical constituents were investigated. Thirteen previously undescribed sesquiterpene lactones—seven germacranolides, three eudesmanolides, two guaianolides, and a 2-norelemanolide—were isolated, and their structures were elucidated based on extensive spectroscopic analyses. The major constituents in the six samples from northwestern Yunnan and Sichuan are hiyodorilactones A and B, whereas that in the two samples from the region near Kunming, Yunnan is eupatoriopicrin. These results and previously reported results suggest the presence of locality-dependent intra-specific diversity in the chemical constituents of E. heterophyllum leaves.


Introduction
The Hengduan Mountains and surrounding areas consist of many mountain ranges and deep river valleys with the elevation range from approximately 1500 to 6000 m, which separate these areas into various subdivisions. These areas are also climatically diverse and rich in plant resources, providing us with good plant materials for the study of diversity in secondary metabolites.
Eupatorium heterophyllum DC. (Asteraceae) is an endemic species in China. It is widely distributed in grasslands and forest areas at altitudes of 1700-3000 m in Southwest China (Sichuan, Yunnan, and Guizhou provinces and Xizang Autonomous Region) and has not been artificially cultivated [1]. We have been studying the intra-specific diversity in leaf and root chemicals of E. heterophyllum native to Yunnan and Sichuan provinces [2][3][4][5][6] as part of our continuing research on the chemical diversity of Asteraceae plants for obtaining unique secondary metabolites produced by limited populations within a species and for getting an insight into a chemical aspect of adaptation/differentiation. To date, diversity was observed in the minor constituents of the root chemicals of E. heterophyllum samples taken from different locations, affording various heterocyclic aromatic compounds, such as benzofuran/dihydrobenzofuran derivatives and propynyl thiophenes, in addition to some common major constituents [2][3][4]. In particular, seven oligomeric benzofurans were recently isolated for the first time from the sample collected in Lijiang City of Yunnan Province, which suggest an ongoing diversification of secondary metabolites in this species [4,5]. The chemical composition of the leaves, which is mainly composed of sesquiterpene lactones, is significantly different from that of the roots. Our previous study has suggested the presence of a higher degree of chemical diversity in the leaves than that in the roots: hiyodorilactones A (1) and B (3), which were marked major constituents, and several germacranolides with a hydroperoxy group at C-1β were found in the leaf samples from northwestern Yunnan and southwestern Sichuan (this chemotype is referred to as hiyodorilactone-type), whereas almost no hiyodorilactones and a small amount of eupatoriopicrin (26) were detected in the sample from a region near Kunming [6]. However, the overall chemical composition of the samples from the latter region has not been sufficiently clarified due to the presence of many unstable or conformationally flexible constituents. Therefore, further phytochemical studies using newly collected samples are required for understanding the chemical diversity of E. heterophyllum leaves. In this study, eight additional samples of E. heterophyllum leaves were collected in Yunnan (samples 1, 2, 7, and 8) and Sichuan (samples 3-6) provinces of China, and a detailed phytochemical study for each sample was performed. Herein, we report the isolation and structure elucidation of thirteen new sesquiterpene lactones from MeOH extracts of the collected samples as well as the differences in chemical compositions of them.

LC-MS Analysis
For an initial assessment of chemical diversity, a portion of each fresh leaf sample collected at the locations shown in Figure 1 was immediately extracted with EtOH, and the chemical composition of the extract was analyzed using LC-MS. The base peak ion (BPI) chromatograms of the samples are shown in Figure 2. Major peaks were identified using isolated compounds ( Figure 3). The samples from northwestern Yunnan (samples 1 and 2) and Sichuan (samples [3][4][5][6] showed major peaks corresponding to hiyodorilactone D (38) [7], hiyodorilactone A (1) [8], eupaformosanin (2) [9], hiyodorilactone B (3) [8], and 20-desoxyeupaformosanin (4) [10] at t R = 7.95, 8.58, 9.17, 9.68, and 10.05 min, respectively, indicating that these chemical compositions are of hiyodorilactone-type samples. In contrast, the BPI chromatograms of the samples taken from a region near Kunming (samples 7 and 8) showed a significant peak of eupatoriopicrin (26) [11] at t R = 9.54 min, suggesting the presence of two chemotypes in the samples. There were some differences in the minor peaks of samples 7 and 8. Several peaks derived from (E,E)-germacranolides (25 [12], 28 [13], 27 [14], and 30 [15]) were detected at t R = 10.66, 10.77, 10.90, and 12.59 min, respectively, for sample 7. Weak peaks characteristic of hyodorilactone-type samples were observed for sample 8 but not for sample 7. quiterpene lactones, is significantly different from that of the roots. Our previous stu has suggested the presence of a higher degree of chemical diversity in the leaves than t in the roots: hiyodorilactones A (1) and B (3), which were marked major constituents, a several germacranolides with a hydroperoxy group at C-1β were found in the leaf samp from northwestern Yunnan and southwestern Sichuan (this chemotype is referred to hiyodorilactone-type), whereas almost no hiyodorilactones and a small amount of eu toriopicrin (26) were detected in the sample from a region near Kunming [6]. Howev the overall chemical composition of the samples from the latter region has not been su ciently clarified due to the presence of many unstable or conformationally flexible cons uents. Therefore, further phytochemical studies using newly collected samples are quired for understanding the chemical diversity of E. heterophyllum leaves. In this stu eight additional samples of E. heterophyllum leaves were collected in Yunnan (sample 2, 7, and 8) and Sichuan (samples 3-6) provinces of China, and a detailed phytochem study for each sample was performed. Herein, we report the isolation and structure e cidation of thirteen new sesquiterpene lactones from MeOH extracts of the collected sa ples as well as the differences in chemical compositions of them.

LC-MS Analysis
For an initial assessment of chemical diversity, a portion of each fresh leaf sam collected at the locations shown in Figure 1 was immediately extracted with EtOH, a the chemical composition of the extract was analyzed using LC-MS. The base peak (BPI) chromatograms of the samples are shown in Figure 2. Major peaks were identifi using isolated compounds ( Figure 3). The samples from northwestern Yunnan (sample and 2) and Sichuan (samples 3-6) showed major peaks corresponding to hiyodorilacto D (38) [7], hiyodorilactone A (1) [8], eupaformosanin (2) [9], hiyodorilactone B (3) [8], a 20-desoxyeupaformosanin (4) [10] at tR = 7.95, 8.58, 9.17, 9.68, and 10.05 min, respective indicating that these chemical compositions are of hiyodorilactone-type samples. In c trast, the BPI chromatograms of the samples taken from a region near Kunming (samp 7 and 8) showed a significant peak of eupatoriopicrin (26) [11] at tR = 9.54 min, suggest the presence of two chemotypes in the samples. There were some differences in the mi peaks of samples 7 and 8. Several peaks derived from (E,E)-germacranolides (25 [12], [13], 27 [14], and 30 [15]) were detected at tR = 10.66, 10.77, 10.90, and 12.59 min, resp tively, for sample 7. Weak peaks characteristic of hyodorilactone-type samples were served for sample 8 but not for sample 7.  Locations of the collected samples of E. heterophyllum (green and purple squares). Samples 9-16 are samples 1-8 from our previous report [6]. Solid and dotted lines indicate rivers and boundaries of provinces, respectively.

Isolation and Structural Elucidation of Leaf Chemicals
Dried leaves of each sample were extracted with MeOH, and the compounds were separated using silica-gel column chromatography and normal phase HPLC to yield 63 compounds, 13 of which were previously unreported. The isolated compounds were categolized into six types: (E,Z)-germacranolides and their oxidative analogs, (E,E)-germacranolides and their oxidative analogs, eudesmanolides, guaianolides, flavonoids, and others, as listed in Figure 3 and Table 1.  Table 7 and Figure 1).
The molecular formula of 51 was determined to be C 24 H 30 O 10 using HRFABMS. Its 1 H and 13 C NMR data (Table 5) were closely related to those of eupakirunsin H [41], suggesting that 51 was also a eudesmanolide. The downfield shift of H-3 (δ H 5.20) and H-8 (δ H 5.83) in the 1 H NMR spectrum as well as the COSY and HMBC correlations shown in Figure 4 indicated that the hydroxy group at C-3 and tigloyloxy group at C-8 in eupakirunsin H were replaced by acetoxy and 4 -acetoxy-5 -hydroxytigloyl groups, respectively, in 51. The HRESIMS spectrum of compound 53 showed a [M + Na] + peak at m/z 415.1368, which suggests the molecular formula C 20 H 24 O 8 with nine degrees of unsaturation. The 1 H and 13 C NMR spectra revealed the presence of one methyl, two oxymethylenes, three oxymethines, two exocyclic double bonds, one trisubstituted double bond, one tetrasubstituted double bond, and two carbonyls ( Table 6). The above spectroscopic data accounted for six degrees of unsaturation, and therefore, 53 should be tricyclic. Compound 53 was deduced to be a guaianolide with oxygen-functionalities at C-3, C-6, and C-8, one of which is a 4 ,5 -dihydroxytigloyloxy group, as evidenced by the COSY and HMBC correlations shown in Figure 4. A significant downfield shift of H-8 (δ H 5.72) as well as the NOESY correlation between H-13b and H-8 suggested the presence of a 4 ,5 -dihydroxytigloyloxy moiety at C-8 and a γ-lactone between C-12 and C-6. Moreover, the molecular formula of 53 and the chemical shift of C-3 (δ 94.2) suggested the presence of a hydroperoxy group at this position. The elucidated planar structure of 53 is shown in Figure 4. The NOESY spectrum showed a cross-peak between H-7 and H-1, H-8, and H-9α, indicating that these hydrogens were in the same orientation ( Figure 5). H-3 showed NOE correlations with H-2a and H-2b, but not with H-1, indicating the α-orientation of hydroperoxy group. Finally, H-6 was assigned a β-orientation owing to its coupling constant (J 6,7 = 10.5 Hz). Therefore, 53 was identified as 8β-(4 ,5 -dihydroxytigloyloxy)-3α-hydroperoxyguaia-4,10(14),11(13)-trien-(12,6α)-olide. HRFABMS and 1D/2D NMR spectra of 54 revealed that it is also a guaianolide with the same molecular formula as that of 53 (Table 6 and Figures 4 and 5). A COSY correlation between two olefinic protons at C-2 (δ C 133.0; δ H 5.77) and C-3 (δ C 137.2; δ H 6.01) indicated a disubstituted double bond. The chemical shift of C-4 observed at δ C 95.2 in the 13 C NMR spectrum and NOE correlation between H 3 -15 and H-6 indicated the presence of a hydroperoxy group at C-4α. Thus, 54 was concluded to be a 4α-hydroperoxy-2-ene isomer of 53.
The experimental ECD spectra of 17, 18, 24, 45, 50, 51, 54, and 62 showed a similar trend to that of 10, especially the negative Cotton effect around 210 nm mainly owing to the α-methylene-γ-lactone moiety. This indicated that the absolute configurations at C-6 and C-7 of these compounds are the same as those of 10 while the other chromophore might have a weaker contribution to their experimental ECD spectra [18]. In addition, considering the biosynthesis of sesquiterpenoids in higher plants, the other new compounds, 31, 36, 40, and 53, would have the same stereochemistry.

Discussion
In this study, 63 compounds, including 13 new compounds, were isolated from 8 leaf samples of E. heterophyllum collected in Yunnan and Sichuan provinces (Figure 3). Among the isolated compounds, 57 compounds were sesquiterpene lactones, including germacranolides (1-40), eudesmanolides (41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52), guaianolides (53-56), and elemanolides (62). Most of the new compounds would be produced from major constituents vir oxidative metabolism, which often involve the introduction of a hydroperoxy group. The chemical composition of each sample is summarized in Table 1. The major peaks in the BPI chromatograms ( Figure 2) confirm the major constituents in each sample. Hiyodorilactones A (1) and B (3) are the major constituents in samples 1-6, confirming that hiyodorilactone-type samples are predominant in Sichuan and northwestern Yunnan regions. In contrast, eupatoriopicrin (26), which is not detected in samples 1-6, is the major constituent in samples 7 and 8 from the region near Kunming. A variety of (E,E)-germacranolides and eudesmanolides are also contained in these samples. Therefore, samples 7 and 8 should be classified as another chemotype, eupatoriopicrin-type. Considering these results and previously obtained results [6], we conclude the presence of locality-dependent intra-specific diversity in the leaf chemicals of E. heterophyllum.
Eupatoriopicrin (26) showed higher cytotoxicity against HL-60 cells than hyodorilactones A (1) and B (3) [42]. Moreover, Pan et al. recently reported genetic diversity in E. heterophyllum [43]. Notably, the geographical distribution of the different genotypes is in good agreement with the chemotypes observed in this study, indicating that the intraspecific diversity in the leaf chemicals of E. heterophyllum is related to its genetic background. These observations can provide us a new insight into a chemical aspect of adaptation and differentiation of Eupatorium plants. We plan to conduct further chemical studies on E. heterophyllum sampled from other regions to obtain secondary metabolites produced by

LC-MS Analysis
Parts of the fresh leaves (a few grams) of each sample were extracted with ethanol immediately after harvesting, and the extracted ethanol solutions were filtered and then separated using a Waters Acquity™ UPLC I-Class system coupled with a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm). The temperature of the column was held at 45 • C. The mobile phases, which consisted of two solvent systems (eluent A, 0.1% formic acid in the water, v/v; eluent B (0.1% formic acid in methanol, v/v)), were delivered at a flow rate of 0.25 mL/min using a linear gradient program. The linear elution gradient program was set as follows: 0 min (95:5)-14.75 min (2:98)-17.00 min (2:98)-17.20 min (95:5)-20.00 min (95:5).
A Waters SYNAPT G2-Si HDMS mass spectrometer was connected to the UPLC system via an ESI interface. The conditions of analysis were as follows: capillary voltage was set at 1.5 kV under positive ion and 2.0 kV under negative mode, sampling cone voltage at 40.0 V, source temperature at 120 • C, desolvation gas temperature at 500 • C. The cone gas flow was at 500 L/h, desolvation gas at 1200 L/h, and the nebulizer gas at 6.5 bar.

Calculation of ECD Spectra
A conformational search was performed using the Monte Carlo method and the MMFF94 force field with Spartan 20 (Wavefunction, Irvine, CA, USA). The obtained lowenergy conformers within 6 kcal/mol were optimized at the B3LYP/6-31G(d,p) level in MeOH (PCM). The vibrational frequencies were also calculated at the same level to confirm their stability, and no imaginary frequencies were found. The energies, oscillator strengths, and rotational strengths of the low-energy conformers were calculated using TDDFT at the CAM-B3LYP/6-31G(d,p) in MeOH (PCM) level and weight-averaged. The ECD spectra were simulated using GaussView [44] with the overlapping Gaussian function with 0.35 eV exponential half-width, and UV correction was performed (redshifted by 10 nm). All DFT calculations were performed using Gaussian 09 [45].  Table 4.  Table 4.  Table 5.  Table 5.  Table 5.  Table 6.