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Abstract

The composition of root chemicals was studied for 7 samples of Ligularia tongolensis collected in Yunnan and Sichuan Provinces of China. The structures of 2 new 3β-angeloyloxy-6β-acyloxyfuranoeremophilan-15-oic acids were determined. It was found that the plant harbors chemical diversity in the acyloxy groups in 3,6-bis(acyloxy)eremophilan-15-oic acids. The presence of a 3-methylpentanoate moiety at C-3 appears geographically differentiated to a degree. Consistent with this low diversity, results of DNA analysis indicated little genetic differentiation, although introgression was inferred for one of the samples.

Keywords

furanoeremophilane internal transcribed spacer (ITS)diversity

The genus Ligularia is highly diversified in the Hengduan Mountains area of China. We have been studying the chemical diversity within each species while referring to DNA data as an index of genetic diversification. To date, we have found various degrees of intraspecific diversity in many species.^1,2 Sesquiterpenoids of the eremophilane type have been found in many species; furanoeremophilanes, in particular, are the major components in major Ligularia species. Ligularia tongolensis (Franch.) Hand.-Mazz. is found in northwestern Yunnan, western Sichuan, and southeastern Tibet.³ We previously reported that L. tongolensis in northwestern Yunnan Province (Shangrila County) and southwestern Sichuan Province (Daocheng County) produced 3,6-bis(acyloxy)furanoeremophilan-15-oic acids and 3-acyloxyfuranoeremophilan-15,6-olides.⁴ Related eremophilane-(12,8),(15,6)-diolides were isolated by Han et al.⁵ The chemical composition, judged from TLC patterns, was very similar among our L. tongolensis samples except for the presence of tetradymol in some samples collected in Shangrila County.⁴ Hybrids of L. tongolensis and L. cymbulifera were also analyzed and the ability to produce tetradymol in L. tongolensis was inferred to have been introgressed from L. cymbulifera.⁶

In the course of our subsequent search in the field, we collected 32 additional samples of L. tongolensis. Seven of the samples (samples 1-7), collected in 2015 to 2017 at locations shown in Table 1 and Figure 1, were analyzed in detail. Samples 1 and 2 (4 km apart from each other) were collected in Wenchuan County, Sichuan, which is about 500 km from the collection area of our previous L. tongolensis samples⁴ (Figure 1, red circle). Sample 3 was collected at Pachahai, Shangrila County, Yunnan, where the above hybrid samples were collected.⁶ Sample 4 was collected at Tianchi, Shangrila County, where we collected various Ligularia hybrids, for which the chemical outcomes of hybridization were described.^7
-9 Samples 5, 6 (4 km apart from each other), and 7 were collected in Muli County, Sichuan.

Figure 1

Collection locations of the present Ligularia tongolensis samples (squares). Circles indicate major cities. Solid and dotted lines indicate rivers and province boundaries, respectively. The red circle indicates the collection area of our previous 17 samples.⁴

Table 1

Collection Locales and Isolated Compounds of Ligularia tongolensis Samples.

Sample no.	Specimen no.	Locality^a	Elevation(m)	Isolated compounds
1	2015-04	Balangshan (Wenchuan, Sichuan)	2900	1a-1d, 3
2	2015-05	Balangshan (Wenchuan, Sichuan)	3100	1a, 1c, 1d, 2
3	2016-220	Pachahai (Shangrila, Yunnan)	3600	1b, 3, 5
4	2016-221	Tianchi (Shangrila, Yunnan)	3600	1a-1d, 4
5	2017-02	Liziping (Muli, Sichuan)	3600	1a-1d, 6, 7
6	2017-06	Liziping (Muli, Sichuan)	4000	5
7	2017-27	Wachang (Muli, Sichuan)	3900	1a-1d

^aCounty and province in parentheses.

The EtOH extract of each root sample was subjected to Ehrlich’s test on TLC, a facile method to detect furanoeremophilanes.^10,11 All the samples showed TLC spots similar to those of our previous samples, suggesting that the composition of furanoeremophilanes was similar. Sample 4 showed an additional strong Ehrlich-positive spot of tetradymol at R _f = 0.66 (hexane/EtOAc 7:3).

Compounds in each sample were separated with such standard methods as silica-gel column chromatography (CC) and HPLC, while air oxidation and decomposition were carefully avoided. From the Wenchuan samples (samples 1 and 2), 1a to 1 d were obtained together with 2 ^12
-14 and 3.¹² While compounds 1a ^12,15 and 1b ¹² were known, 1c and 1d were new. From sample 3, 1b, 3, and 5 ⁴ were isolated. From sample 4, tetradymol (4)¹⁶ was isolated in addition to a mixture of 1a to 1 d. From 2 of the Muli samples (samples 5 and 7), 1a to 1 d were obtained. β-Bisabolene (6) and β-eudesmol (7) were also isolated from sample 5. In contrast, 5 was isolated as the major component from sample 6 (Figure 2).

Figure 2

Isolated compounds from Ligularia tongolensis. The asterisks denote new compounds.

Attempts to isolate the new compounds 1c and 1d in pure form by chromatography were unsuccessful. Their structures were deduced from the MS and NMR spectra of a mixture of 1a, 1c, and 1d. ¹H and ¹³C NMR signals of the furanoeremophilane skeleton and those of an angelate at C-3 of 1a, 1c, and 1d almost overlapped, indicating that 1c and 1d were furanoeremophilanes with an angeloyloxy group at C-3β (Table 2). The difference among the compounds was in the acid part of the ester moiety at C-6. High-resolution LCMS (ESI) of the mixture showed 3 peaks of 1a, 1c, and 1d (1a: m/z 467.2022, C₂₅H₃₂O₇; 1c: m/z 469.2227, C₂₅H₃₄O₇; 1d: m/z 481.2176, C₂₆H₃₄O₇) ([M+Na]⁺). A fragment ion resulting from the elimination of ROH at C-6 was observed at m/z 367.1486 ([M+Na-ROH]⁺) for each of 1a, 1c, and 1d. The acyloxy group at C-6 (allylic) is eliminated more easily than that at C-3.¹² The molecular formula of 1c indicated that the acyloxy group at C-6 was -OCOC₄H₉ and its structure was determined to be 2-methylbutanoate from the correlations shown in Figure 3. The acyloxy group in 1d was similarly determined to be 3-methylpent-2-enoate (Figure 3). The geometry of the double bond was deduced to be the E-form from the δ value of H₃-6″ (δ 2.12). The 2 methyl groups of the senecioate (= 3-methylbut-2-enoate) moiety in 6-senecioyloxy-1,10-epoxyfuranoeremophilane was observed at δ 2.13 (Z-Me) and 1.43 (E-Me) in C₆D₆.¹⁷ HMBC correlation was observed between H-6 and C-1″ for both 1c and 1d, supporting that 1a, 1c, and 1d are variants of the acyloxy group at C-6.

Figure 3

Key COSY and HMBC correlations for the acid part of 1c and 1d at C-6.

Table 2

¹H and ¹³C NMR of New Compounds 1c and 1d (C₆D₆).^a

Carbon no.	1c		1d
	δ _H	δ _C	δ _H	δ _C
1	0.97-1.08 (2H, m)^b	26.4-26.5^b	0.97-1.08 (2H, m)^b	26.4-26.5^b
2	1.82-1.92 (m)^b,c 2.11-2.26 (m)^b,c	26.9	1.90-2.00 (m)^b,c 2.18-2.31 (m)^b,c	26.9
3	5.65-5.75 (m)^b,c	70.1	5.88 (br dt, 12.0, 5.8)	70.1
4	3.30-3.35 (m)^b,c	49.9	3.41-3.45 (m)	50.1
5		41.6		41.6
6	6.48 (br s)	68.1	6.61 (br s)	67.4
7		114.7		115.1
8		150.3		150.1
9	1.95-2.04 (m)^b,d 2.60-2.69 (m)^b,d	26.4-26.5^b	1.95-2.04 (m)^b,d 2.60-2.69 (m)^b,d	26.4-26.5^b
10	2.37-2.50 (m)^b,d	37.0	2.37-2.50 (m)^b,d	36.9
11		119.9		120.0
12	6.91 (s)	139.1	6.92 (s)	139.1
13	1.88 (d, 1.1)	9.1	1.93 (d, 1.2)	8.9
14	1.13 (br s)^b,d	19.4	1.13 (br s)^b,d	19.5
15		179.0		179.0
1′		166.3		166.3
2′		127.8-128.4^b		127.8-128.4^b
3′	5.67 (br q, 7.3)^b	138.5	5.67 (br q, 7.3)^b	138.5
4′	1.95-2.00 (m)^b,d	15.9	1.95-2.00 (m)^b,d	15.9
5′	1.82-1.85 (m)^b,d	20.7	1.82-1.85 (m)^b,d	20.7
1″		176.3		167.0
2″	2.30 (quint d, 7.2, 6.5)	41.8	5.75-5.78 (m)	113.9
3″	1.34 (dqd, 13.5, 7.3, 6.5)1.79-1.89 (m)^b,c	26.5	-	163.8
4″	0.86 (t, 7.3)	12.1	1.66 (qd, 7.4, 1.0)	33.8
5″	1.20 (d, 7.2)	17.6	0.66 (t, 7.4)	11.1
6″			2.12 (d, 0.9)	19.1

^aDetermined on a mixture of 1a, 1 c, and 1 d.

^bThe equivalent signals of 1a, 1 c, and 1 d overlapped.

^cDetermined from the COSY spectrum.

^dNo exact value could be determined.

The chemical composition was also analyzed for each sample by LCMS. The total ion chromatograms (TICs) of samples 1 to 7 are shown in Figure 4. In samples 1 and 2, 1a and 1c were the major components. Compound 2, detected as another major peak, is a lactonization product of 1a to 1d (vide infra). In sample 3, 1b and 5 were detected. In sample 4, 1a to 1d and tetradymol (4) were detected. In samples 5 and 7, another compound was detected at t _R = 19.4 minutes in addition to 1a to 1d (Figure 4, compound A). Its mass spectrum (m/z 345, 245) was very similar to those of 1a to 1d, suggesting that it was a related furanoeremophilan-15-oic acid; however, we could not identify the compound. In sample 6, 5 was detected as the most major component. Some additional peaks were observed in samples 6 (t _R about 18 minutes) and 7 (t _R 16-17 minutes); however, we were unable to identify the compounds of the peaks.

Figure 4

Total ion chromatograms of samples 1 to 7. The differences in the retention time among the samples are within experimental error.

The LCMS data indicate that the ratio of 3,6-bis(acyloxy)-eremophilan-15-oic acids, 1a to 1d and 5, was variable among the samples. These compounds are indistinguishable by TLC and the variation could not be uncovered in our previous study.⁴ This raised the possibility that there may have been the variation among our previous samples as well. Therefore, we analyzed some of our previous samples collected in 2003, with newly assigned sample numbers of 8 to 21 (Supplemental Table S1 for the correspondence between the new and old sample numbers), and the 25 additional samples collected in 2004 to 2011 (samples 22-46) by LCMS. The TICs of samples 8 to 11 are shown in Figure 5 (Supplemental Figures S1-S6 for the others). Four peaks were detected at t _R = 8.2 (m/z 245; 3), 13.1 (m/z 345, 245; 2), 13.3 (m/z 347, 245), and 15.1 (m/z 361, 245) minutes. The absence of the 3,6-bis(acyloxy) compounds 1a to 1d and the concomitant presence of 2 and 3 were presumably due to lactonization and elimination during storage of the extracts in EtOH: 1a to 1d were converted into 2 ¹² and then into 3 (Scheme 1). The structures of the compounds in the peak at t _R = 13.3 and 15.1 were identified as 8 ¹³ and 9,⁴ respectively, on the basis of the LCMS data obtained with previously purified materials. Compound 8 has been isolated from L. hookeri ¹³ but not from L. tongolensis; however, its lactone derivative, an eremophilane-(12,8),(15,6)-diolide with a 2-methylbutanoate moiety at C-3, was isolated from L. tongolensis by Han et al.⁵ Eremophilan-12,8-olides are likely to be derived from furanoeremophilanes.^18,19 Compound 9, presumably generated from 5 or related compound(s), was previously isolated from L. tongolensis.⁴ Compounds 8 and 9 also result in 3 through the elimination of the C-3 ester moiety (Scheme 1).

Figure 5

Total ion chromatograms of samples 8 to 11.

Scheme 1

Lactonization of 3,6-bis(acyloxy)furanoeremophilan-15-oic acids. See Figure 2 for A, B, and C.

Supposing that the rate of the conversion of 2, 8, and 9 into 3 is the same, the variable ratios of 2, 8, and 9 in samples 8 to 11, seen in Figure 5, indicates a variation in the acyloxy group at C-3. Among the furanoeremophilanes obtained from Ligularia, compounds with 3-methylpentanoyloxy moiety at C-3 (5 and/or 9) have been isolated only from L. tongolensis ^4,6 and L. hookeri.¹³ On the criterion of the presence/absence of these characteristic compounds, all the 46 samples could be grouped into a 5/9-producing and a nonproducing type. The producing type includes 16 samples from Yunnan (samples 3, 8-10, 13, 14, 16-19, 23-26, 31, and 46) and 3 samples from southern Sichuan (6, 35, and 37). The nonproducing type includes most of the other samples from Sichuan (samples 1, 2, 5, 7, 11, 20, 21, 28-30, 32-34, 36, 38-45) and the other 5 samples from Yunnan (samples 4, 12, 15, 22, and 27) (Figures 4, 5, S1-S6). This suggests some geographical differentiation in the presence of 3-methylpentanoic acid. Our previous study did not find 1a to 1d in our samples collected in 2002.⁴ Unfortunately, we could not reanalyze the 2002 samples due to decomposition; however, the samples may have been of the 5/9-producing type, because 5 and 9 were isolated as major components.

The base sequence of the ITS1-5.8S-ITS2 of the ribosomal RNA gene in the nuclear genome was determined for samples 1 to 11. The results are shown in Table 3. Sample 6 has an exceptionally large number (27) of multiple-base sites. As we discussed previously, it indicates hybridization with some other species.⁶ Since the morphology of the sample is of L. tongolensis, it must have undergone backcrossing; namely, it is introgressed. The bases at these sites cannot be accounted for by the superposition of L. tongolensis and L. cymbulifera sequences (Table 3). The hybridization partner is currently unknown because database search using a putative introgressive sequence found no clear candidate with a very good agreement. The sequences of the other samples are essentially the same, indicating that they are not differentiated genetically.

Table 3

Differences in the ITS1-5.8S-ITS2 Region Among Ligularia tongolensis Samples.^a

Sample no.	ITS1																													5.8S								ITS2
										1	1	1	1	1	1	1	1	1	1	1	1	1	2	2	2	2	2	2	2				1	1	1	1	1									1	1	1	1	1	1	1	2	2	2	2	2
		1	1	2	2	2	6	6	9	0	0	0	1	1	1	2	3	4	6	6	7	9	1	1	3	3	3	3	4			3	2	2	3	5	6		1	1	2	2	2	3	4	0	0	1	1	7	8	8	0	1	1	2	2
	6	1	2	1	5	6	2	8	2	1	3	4	0	2	6	7	2	4	6	7	8	8	0	5	1	7	8	9	6	1	5	4	2	4	6	7	4	3	5	9	0	7	8	6	5	1	7	1	5	4	2	8	9	3	5	0	1
1	A	C	C	C	C	C	C	C	G	C	T	C	T	G	T	A	C	T	G	C	G	K	C	T	T	A	C	A	C	C	C	C	Y	G	C	C	C	T	A	T	G	Y	G	A	G	C	C	C	C	G	Y	C	T	G	T	T	T
2	A	C	C	C	C	C	C	C	G	C	T	C	T	G	T	A	C	T	G	C	G	G	C	T	T	A	C	A	C	C	C	Y	Y	G	C	C	C	T	A	T	G	Y	G	A	G	C	C	Y	C	G	C	C	T	G	T	T	T
3	A	C	C	C	C	C	Y	C	G	C	T	C	T	G	T	A	C	T	R	C	G	G	C	T	T	A	C	A	Y	C	C	C	C	G	C	C	C	T	A	T	G	C	G	A	G	C	C	C	Y	G	C	C	T	R	T	T	T
4	A	T	C	Y	C	Y	C	C	T	C	T	C	T	G	T	A	T	T	G	C	T	G	C	T	T	A	C	A	C	C	C	C	C	A	C	Y	Y	C	R	T	G	C	G	T	G	C	Y	C	C	K	C	C	T	G	T	T	T
5^b	W	C	C	C	Y	C	C	C	G	C	T	C	T	G	T	A	C	T	G	C	G	G	C	T	T	A	C	A	C	C	Y	C	C	G	C	C	C	T	A	T	G	C	K	W	G	C	C	Y	C	G	C	C	T	G	T	T	T
6	W	C	C	C	C	C	C	M	G	Y	W	M	K	R	Y	A	C	K	G	C	G	G	C	T	Y	A	Y	R	C	Y	C	C	C	G	Y	Y	C	T	A	Y	R	Y	K	W	R	Y	C	Y	C	G	Y	Y	Y	G	Y	Y	T
7	T	C	C	C	C	C	C	C	G	C	T	C	T	G	T	A	C	T	G	C	G	G	C	T	T	A	C	A	C	C	C	C	C	G	C	C	C	T	A	T	G	C	T	T	G	C	C	T	Y	G	C	C	T	G	T	T	T
8^c	A	C	C	C	C	C	Y	C	G	C	T	C	T	G	T	A	C	T	R	C	G	G	C	T	T	A	C	A	Y	C	C	C	C	G	C	C	C	T	A	T	G	C	G	A	G	C	C	C	Y	G	C	C	T	R	T	T	T
9	A	C	C	C	C	C	Y	C	G	C	T	C	T	G	T	A	C	T	G	C	G	G	Y	T	T	A	C	A	C	C	C	C	C	G	C	C	C	T	A	T	G	C	G	A	G	C	C	C	C	G	C	C	T	G	T	T	T
10^c	A	C	C	C	C	C	Y	C	G	C	T	C	T	G	T	A	C	T	G	C	G	G	C	T	T	A	C	A	Y	C	C	C	C	G	C	C	C	T	A	T	G	C	G	A	G	C	C	C	Y	G	C	C	T	G	T	T	T
11	A	T	C	Y	C	C	C	C	T	C	T	C	T	G	T	A	T	T	G	C	T	G	C	T	T	A	C	A	C	C	C	C	C	A	C	C	C	C	R	T	G	C	G	T	G	C	Y	C	C	G	C	C	T	G	T	T	T
Ref^d	A	C	C	C	C	C	C	C	G	C	T	C	T	G	T	A	C	T	G	C	G	G	C	T	T	A	C	A	C	C	C	C	C	G	C	C	C	T	A	T	G	C	G	A	G	C	C	C	T	G	C	C	T	G	T	C	C
Ref^e	A	C	T	C	C	C	C	C	G	C	T	C	T	A	C	C	T	A	G	A	G	G	C	C	C	T	C	G	C	C	C	C	C	G	C	C	C	C	A	C	G	C	G	T	A	C	C	C	C	T	C	C	C	G	T	C	C

^aThe base numbering is according to the database entry DQ272337 of L. tongolensis. K = G + T; M = A + C; R = A + G; W = A + T; Y = C + T.

^bA sequence with C₆ in place of C₅ at 154 to 159 of ITS1 was also present.

^cA sequence with T₃ in place of T2 at 156 to 157 of ITS2 was also present.

^dA L. tongolensis sequence in the database (DQ272337).

^eA L. cymbulifera sequence in the database (DQ272330).

Tetradymol (4) was detected in sample 4 as a major component. Tetradymol is a major compound of L. cymbulifera,⁴ a species abundant in the Shangrila area, Yunnan, and it is considered ecologically useful as an allelopathic.²⁰ As described previously, the ability to produce tetradymol in L. tongolensis is likely to be acquired through introgression from L. cymbulifera.⁶ However, no evidence of hybridization is seen in the ITS1-5.8S-ITS2 sequence of sample 4 (Table 3). One possible explanation is that backcrossing eliminated the L. cymbulifera sequence from the rRNA gene locus while the gene(s) for tetradymol production remained in sample 4.

In conclusion, L. tongolensis harbors some chemical diversity in the acyloxy groups of 3,6-bis(acyloxy)eremophilan-15-oic acid. This range of variation within the same terpenoid framework suggests that the diversity in L. tongolensis is limited, which appears consistent with DNA sequence data.

Experimental

General

NMR, JEOL ECX-400 spectrometer (400 MHz for ¹H; 100 MHz for ¹³C) or Varian Unity Plus 500 spectrometer (500 MHz for ¹H; 125 MHz for ¹³C); IR, JASCO FT/IR-230 spectrometer; MS, JEOL JMS-700 MStation or CMATE II. CC was performed on silica gel (Wakosil C-200 or C-300). Analytical TLC was carried out on Merck Kieselgel 60 F254, 0.2 mm thickness, with either Ehrlich's reagent (p-dimethylaminobenzaldehyde and HCl)^10,11 or p-anisaldehyde/AcOH/H₂SO₄ as visualizing agent. HPLC was carried out by use of either a Shimadzu LC-20AT pump with an SPD-20A Prominence UV/VIS detector, a GL Sciences GL-7410 pump with a GL-7450 UV detector, or a JASCO 880-PU pump with an 875-UV detector, and a Hitachi D-2500 Chromato-Integrator, a Shimadzu C-R8A Chromatopac, or a SOMA OPTICS MDL-102 recorder, with either a GL Sciences Inertsil PREP-ODS column (20 × 250 mm), a Kanto Mightysil Si60 (10 × 250 mm) column, or a Nacalai Tesque Cosmosil 5SL-II (10 × 250 mm) column. LCMS was measured on an Agilent 1100 series LC/MSD mass spectrometer. See Reference ²¹ for details. High-resolution LCMS (ESI) was measured on a Waters SYNAPT G2-Si HDMS with a UPLC-I Class system and an electrospray ionization interface. UPLC was carried out by use of an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, Waters).

Plant Material

Samples were collected in August 2015, 2016, and 2017 at locations shown in Table 1 and Figure 1. Each sample was identified by X. G. (author). Voucher specimen numbers are 2015-04, 2015-05, 2016-220, 2016-221, 2017-02, 2017-05, and 2017-27 for samples 1 to 7, respectively (Kunming Institute of Botany).

Isolation of Compounds

EtOH extraction of dried root of sample 1 (23.7 g) afforded an extract (2.24 g). CC (n-hexane-EtOAc, gradient) of the extract resulted in 9 fractions. CC (n-hexane-EtOAc) of fraction 5 (eluted with n-hexane-EtOAc 90:10) afforded a mixture of 1a, 1b, 1c (121.5 mg, ratio 1:1:2). From fraction 6 (eluted with n-hexane-EtOAc 90:10 to 80:20), 3 mixtures of 1a, 1c, 1d (108.2 mg, ratio 10:5:2; 17.8 mg, ratio 6:2:1; 43.7 mg, 8:3:1) and 3 (0.3 mg) were obtained by CC (n-hexane-EtOAc) and HPLC (n-hexane-EtOAc). From fraction 7 (eluted with n-hexane-EtOAc 80:20), 2 mixtures of 1a, 1c, 1d (5.5 mg, ratio 7:4:1; 11.6 mg, ratio 5:1:1) were obtained by CC (n-hexane-EtOAc) and HPLC (n-hexane-EtOAc).

EtOH extraction of dried root of sample 2 (19.6 g) afforded an extract (1.25 g). CC (n-hexane-EtOAc, gradient) of the extract afforded 8 fractions. From fraction 4 (eluted with n-hexane-EtOAc 90:10), 2 (1.1 mg) was obtained by repeated HPLC (n-hexane-EtOAc). From fraction 5 (eluted with n-hexane-EtOAc 90:10 to 80:20), a mixture of 1a, 1c, 1d (351.0 mg, ratio 7:5:1) was afforded by CC (n-hexane-EtOAc) and HPLC (n-hexane-EtOAc). From fraction 6 (eluted with n-hexane-EtOAc 80:20 to 60:40), 1a (0.4 mg) was obtained by HPLC (n-hexane-EtOAc).

EtOAc extraction of dried root of sample 3 (1.9 g) afforded an extract (116.2 mg). CC (n-hexane-EtOAc, gradient) of the extract afforded 6 fractions. From fraction 3 (eluted with n-hexane-EtOAc 95:5 to 90:10), 1b (2.2 mg), 3 (1.1 mg), and 5 (0.1 mg) were obtained by repeated HPLC (n-hexane-EtOAc).

EtOH extraction of dried root of sample 4 (6.9 g) afforded an extract (630.5 mg). CC (n-hexane-EtOAc, gradient) of the extract afforded 9 fractions. From fraction 3 (eluted with n-hexane-EtOAc 96:4 to 94:6), 4 (0.8 mg) was obtained by CC (n-hexane-EtOAc) and HPLC (n-hexane-EtOAc). From fraction 4 (eluted with n-hexane-EtOAc 94:6 to 92:8), a mixture of 1a, 1b, 1c, 1d (69.9 mg, ratio 2:1:3:1) was obtained by HPLC (n-hexane-EtOAc). From fractions 5 (eluted with n-hexane-EtOAc 92:8 to 86:14) and 6 (eluted with n-hexane-EtOAc 86:14 to 80:20), mixtures of 1a, 1c, 1d (216.3 mg, ratio 2:2:1; 51.5 mg, ratio 3:1:2) were obtained.

EtOH extraction of dried root of sample 5 (13.0 g) afforded an extract (1021.0 mg). CC (n-hexane-EtOAc, gradient) of the extract afforded 13 fractions. From fraction 2 (eluted with n-hexane-EtOAc 96:4), 6 (24.4 mg) was obtained. From fraction 9 (eluted with n-hexane-EtOAc 70:30), 7 (4.8 mg) and 2 (0.6 mg) were isolated by repeated HPLC (n-hexane-EtOAc). From fraction 10 (eluted with n-hexane-EtOAc 60:40), 1c (16.1 mg), mixtures of 1a, 1b, 1c (22.0 mg, ratio 1:2:3), of 1a, 1c, 1d (18.3 mg, ratio 2:2:3), and of 1c, 1d (24.2 mg, ratio 1:1) were obtained by HPLC (n-hexane-EtOAc).

EtOH extraction of dried root of sample 6 (1.7 g) afforded an extract (54.1 mg). CC (n-hexane-EtOAc, gradient) of the extract afforded 2 fractions. From the polar fraction (eluted with n-hexane-EtOAc 80:20 to 50:50), 5 (43.4 mg) was obtained.

EtOH extraction of dried root of sample 7 (4.2 g) afforded an extract (122.8 mg). CC (n-hexane-EtOAc, gradient) of the extract afforded 4 fractions. From fraction 3 (eluted with n-hexane-EtOAc 91:9 to 86:14), a mixture of 1a, 1b, 1c, 1d (27.8 mg, ratio 7:2:6:2) was obtained by HPLC (n-hexane-EtOAc).

Compounds 1c and 1d

¹H and ¹³C NMR (C₆D₆): see Table 2.

HR-LCMS (ESI): 1c: m/z 469.2227 [M+Na⁺] (100) (calcd for C₂₅H₃₄O₇Na: 469.2203), 367.1529 [M-C₄H₉COOH+Na⁺] (32); 1d: m/z 481.2176 [M+Na⁺] (100) (calcd for C₂₆H₃₄O₇Na: 481.2203), 367.1529 [M-C₅H₉COOH+Na⁺] (28).

DNA Analysis

Purification of DNA from dried leaves, amplification of the ITS1-5.8S-ITS2 region with polymerase chain reaction, purification of the amplification product, and DNA sequencing were carried out as previously described.²²

Supplemental Material

Supplementary material - Supplemental material for Diversity of Furanoeremophilane Composition in Ligularia tongolensis

Supplemental material, Supplementary material, for Diversity of Furanoeremophilane Composition in Ligularia tongolensis by Yasuko Okamoto, Yumi Nakadozono, Kosuzu Shiojiri, Sayaka Suehiro, Yoshinori Saito, Yosuke Matsuo, Takashi Tanaka, Chiaki Kuroda, Motoo Tori, Xun Gong and Ryo Hanai in Natural Product Communications

Footnotes

Acknowledgments

The authors thank Dr Takayuki Kawahara, Forestry and Forest Products Research Institute, Dr Yoshinosuke Usuki, Osaka City University, and Dr Katsuyuki Nakashima, TokushimaBunri University, for their help in sample collection and valuable discussion.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was partly supported by a Grant-in-Aid for Scientific Research from JSPS (No. 25303010), the Japan-China Scientific Cooperation Program from JSPS and NSFC, and a Strategic Research Foundation Grant-Aided Project for Private Universities from the MEXT, Japan.

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