Sage Journals: Discover world-class research

Abstract

The chemical composition of 3 samples of Ligularia dictyoneura was studied. Nine furanoeremophilanes 1 to 9 and bakkenolide A (10) were isolated, among which compound 6 was a new furanoeremophilane-15,6-olide. These results, together with our previous results on 20 L. dictyoneura samples, show that furanoeremophilane is the major compound in the peripheries of the plant’s distribution area, while eremophilan-8-one, in the center. DNA analysis suggests that a furanoeremophilane producer in the center, an exception, is introgressed; namely, the ability to produce furanoeremophilane in the sample may have its origin in some other plant.

Keywords

diversity furanoeremophilane internal transcribed spacer (ITS)

We are studying the chemical and genic diversity in Ligularia (Asteraceae) in the Hengduan Mountains area of China using root chemicals and evolutionarily neutral base sequences as the indices.^1,2 Eremophilane-type sesquiterpenoids have been isolated from most of the major Ligularia species and many Ligularia species have been found to harbor intraspecific diversity in their root chemicals. On the basis of the isolated chemicals and our observation on the abundance of the plants in the field, we have proposed a hypothesis that furanoeremophilane-producing species or intraspecific groups are ecologically advantageous over those producing eremophilan-8-ones.¹

Ligularia dictyoneura (Franch.) Hand.-Mazz. grows in grassy slopes and scrubs in northwestern Yunnan and southwestern Sichuan Provinces.³ Some eremophilane sesquiterpenes have been isolated from this species.^4

-7 We isolated eremophilan-8-one derivatives from samples collected in Shangrila (formerly Zhongdian) area, Yunnan, and furanoeremophilanes from samples collected in its surrounding areas (Figure 1).⁸ On the basis of the TLC patterns of their root extracts, furanoeremophilane-producing L. dictyoneura samples were subgrouped into 6 chemotypes (types 1-6).⁸ Because furanoeremophilanes are believed to be generated from eremophilan-8-ones⁹ (Scheme 1), furanoeremophilane-producing populations of L. dictyoneura may have evolved from those producing eremophilan-8-ones.

Scheme 1

Biosynthesis of furanoeremophilanes.

Figure 1

Collection locations of L. dictyoneura (squares). Circles and triangles indicate major cities and peaks, respectively. Solid and dotted lines indicate rivers and province boundaries, respectively. The green circle indicates the area of 8 samples with eremophilan-8-one.⁸

Here we report the results of analyses on 3 samples of L. dictyoneura. They were collected further afield from our previous L. dictyoneura samples. Samples A to C were collected in Yanbian, Yanyuan, and Litang counties, Sichuan, respectively (Table 1 and Figure 1). One of the compounds isolated from sample A was new. The compounds from samples B and C were only listed previously¹ and here we describe the details of their analyses.

Table 1

Collection Localities and Isolated Compounds of Ligularia dictyoneura Samples.

Sample no.	Specimen no.	Locality^a	Elevation (m)	Isolated compounds	Chemotype
A	2012-52	Gesala (Yanbian)	2500	1-6	Type 2
B	2007-25	Gongmushan (Yanyuan)	2700	1-5, 7, 8	Type 7
C	2007-103	Jiawa (Litang)	3900	1-3, 5, 7- 10	Type 8

^aCounty in parentheses. All in Sichuan Province.

The EtOH extract of each root sample was subjected to Ehrlich’s test, which is a facile method to detect furanoeremophilanes.^10,11 The 3 samples were positive to the test. The TLC pattern of sample A was similar to that of the previous type-2 samples and the chemotype of this sample was judged to be type 2 (Table 1).⁸ Sample B showed 2 large pink spots at R _f = 0.59 and 0.65 (hexane-EtOAc 7:3). In addition, 3 small pink spots (R _f = 0.43, 0.76, and 0.88) and an orange spot of ligularone (R _f = 0.80) were observed. Sample C showed the same 2 large spots; however, of the 4 minor spots, the orange spot and the least polar spot were absent. These TLC patterns were different from that of any of the previously analyzed samples of types 1 to 6, and, therefore, new chemotypes 7 and 8 were assigned to samples B and C, respectively (Table 1).¹

Compounds in each sample were isolated by use of silica gel column chromatography (CC) and HPLC. From sample A, compounds 1,¹² 2 (ligularol),¹³ 3,^14,15 4,¹⁶ 5,^17,18 and a new compound 6 were isolated. From sample B, 1 to 5, 7,¹⁹ and 8 (ligularone)^13,20 were isolated. From sample C, 1 to 3, 5, 7, 8, 9 (tetradymol),²¹ and 10 (bakkenolide A)^22,23 were isolated. The structure of the new compound 6 was determined as follows.

The molecular formula of 6 was determined to be C₂₀H₂₄O₅ from the high-resolution CIMS (m/z 345.1697) and ¹³C NMR (4 CH₃, 3 CH₂, 6 CH, and 7 quaternary C) data. The IR spectrum showed the presence of a γ-lactone (1780 cm^-1) and a conjugated ester carbonyl group (1711 cm^-1). The ¹H and ¹³C NMR spectra showed the signals typical of furanoeremophilane [δ _H 6.86 (br s, H-12), 1.93 (d, H₃-13), 0.77 (s, H₃-14)] with an angeloyl moiety [δ _H 5.71 (qq, H-3′), 1.79 (quint, H₃-5′), and 1.93 (dq, H₃-4′); δ _C 166.4 (C-1′) and 128.0 (C-2′)] (Table 2). Two oxygenated methines [δ _H 5.11 (td, H-1) and 4.46 (s, H-6)] and another carbonyl [δ _C 174.0 (C-15)] were observed. These data, as well as the lack of a 15-Me signal in the ¹H NMR spectrum, suggested that 6 was a furanoeremophilan-15,6-olide with an angeloyloxy group. The position of the angeloyloxy group was determined to C-1α from the COSY spectrum (H-4/H-3/H-2/H-1/H-10/H-9) and the splitting pattern of H-1 (td, 5.3, 11.6 Hz). The planar structure was established by HMBC correlations (Figure 2). The configurations of the stereogenic centers were determined from the NOESY spectrum showing correlations between Me-14 and H-10 and between Me-14 and H-1 (Figure 2). Thus, the structure of compound 6 was established as depicted.

Table 2

¹H and ¹³C NMR Data of New Compound 6 (C₆D₆).

Carbon no.	δ _H	δ _C
1	5.11 (td, 5.3, 11.6)	70.3
2	1.00 (dq, 5.2, 12.5) 1.55 (m)	25.8
3	1.20 (dq, 4.2, 13.1) 1.52 (m)	17.6
4	1.65 (dd, 3.5, 12.6)	40.5
5	-	42.0
6	4.46 (s)	79.9
7	-	115.2
8	-	150.4
9	2.25 (dd, 10.0, 17.6) 2.37 (dd, 7.5, 17.5)	19.7
10	2.08 (ddd, 5.2, 7.8, 10.0)	40.2
11	-	120.4
12	6.86 (br s)	138.9
13	1.93 (d, 1.2)	8.3
14	0.77 (s)	19.5
15	-	174.0
1′	-	166.4
2′	-	128.0
3′	5.71 (qq, 1.3, 7.2)	138.5
4′	1.93 (qd, 1.3, 7.2)	15.9
5′	1.79 (quint, 1.3)	20.7

Figure 2

Selected COSY, HMBC, and NOESY correlations of compound 6.

The base sequence of the ITS1-5.8S-ITS2 region was determined for samples A to C. Samples 3 to 14 and 16 to 19 from our previous study on L. dictyoneura ⁸ were also reanalyzed. The adoption of a new set of primers²⁴ and a new sequencing reaction kit has improved the quality of data and allowed us to determine the sequences that had remained unknown in these samples. The results, as well as those previously determined for samples 1, 2, 15, and 20, are shown in Table 3. Subjecting the data to a phylogenetic analysis program MEGA X²⁵ returned no phylogenetic tree with a significant bootstrapping value, implying that the samples have not differentiated very much genetically. (See below for discussion on sample 9.)

Table 3

Differences in the ITS1-5.8S-ITS2 Region Among Ligularia dictyoneura Samples.^{a
b}

ITS1

5.8S

ITS2

Sample no.^c

B^e

C^e

9^f

17^g

^aK = G + T; R = A + G; S = C + G; W = A + T; Y = C + T; - = deletion.

^bThe base numbering based on the sequence of sample 15, which had been deposited in the database (AB299047).

^cSamples A to C, the present work; samples 1 to 20, our previous work.⁸

^dA sequence with TTTT was also present.

^eA sequence with AAA in place of AA at 252 to 253 of ITS1 was also present.

^fA sequence without AC at 163 to 164 of 5.8S was also present.

^gA sequence with T in place of GGA at 170 to 172 of ITS2 was also present.

Nine furanoeremophilanes were isolated from the 3 L. dictyoneura samples. Of them, 4 were 15-oxygenated derivatives (4-7) and 3 were ligularol derivatives (2, 3, and 8). Two of the compounds isolated from sample A, ligularol (2) and its lactone 5, were previously isolated from a type-2 sample (sample 3)⁸; however, the acid part of the 1α-acyloxyfuranoeremophilan-15,6α-olide was different between sample A and sample 3: angelate in 6 from sample A and acetate in 11 from sample 3.⁸ Thus, there is variation among type-2 samples. Although samples B and C were given different chemotypes,¹ the compounds in them were very similar (Table 1). Our chemotyping of L. dictyoneura has relied on TLC, which is facile and should be useful for diversity studies^1,8; however, the present results of compound isolation indicate that the chemical spectrum of L. dictyoneura is rather continuous.

The eremophilan-8-one type of L. dictyoneura is found in the Shangrila area, where the plant is most abundant (Figure 1, green circle); the furanoeremophilane type, in the surrounding areas. The only exception is sample 9 in our previous study: Although it was surrounded by eremophilan-8-one producers, furanoeremophilanes 1 and 2 were isolated from its extract. We surmised that the high diversity in L. dictyoneura may have arisen from hybridization of previously separate populations.^1,8 In this context, it is noteworthy that sample 9 contained 13 multiple-base sites and 2 indels in the ITS1-5.8S-ITS2 region; in no other sample, the former number was as large (Table 3). (Information of this kind cannot be handled by MEGA X.) This may suggest an introgression that resulted in the production of furano compounds in sample 9. The mechanism(s) which led L. dictyoneura in the peripheral areas to produce furano compounds is not known. However, an ultimate explanation for the phenomenon could be that the environments in these areas are not suitable to L. dictyoneura and the production of furano compounds helps the plant survive.

The characteristic components in 4 of the 8 furanoeremophilane-producing types (types 2, 4, 7, and 8) are 15-oxygenated derivatives. 15-Oxygenated furanoeremophilanes have been isolated from L. tongolensis,²⁶ L. cymbulifera,²⁶ L. vellerea (Shangrila-type),¹⁹ and L. hookeri.²⁷ Because the distribution areas of these plants and L. dictyoneura almost overlap (Shangrila County and the surrounding areas, see Figure 1), the production of similar compounds in these species may not be a coincidence.

Experimental

General

NMR, JEOL ECX-400 (400 MHz for ¹H; 100 MHz for ¹³C) or JEOL AL 400 spectrometer; 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 or Kanto silica gel 60 N [spherical neutral]). Analytical TLC was carried out on Merck Kieselgel 60 F254, 0.2 mm thickness, using either Ehrlich’s reagent (p-dimethylaminobenzaldehyde and HCl) or p-anisaldehyde/AcOH/H₂SO₄ as the visualizing agent. HPLC was carried out using either a Shimadzu LC-20AT pump with a SPD-20A Prominence UV/VIS detector or a GL Sciences GL-7410 pump with a GL-7450 UV detector, and a Hitachi D-2500 Chromato-Integrator or a Shimadzu C-R8A Chromatopac, with either a GL Sciences Inertsil PREP-ODS column (20 × 250 mm) or a Kanto Mightysil Si60 (10 × 250 mm) column. DNA was purified from dried leaves using DNeasy Plant Mini Kit (Qiagen). A 30-cycle amplification of the ITS1-5.8S-ITS2 region by polymerase chain reaction was carried out with LC5 and LC6 primers²⁴ and HotStarTaq plus Master Mix Kit (Qiagen). The amplified DNA was separated by agarose gel electrophoresis and purified with High Pure PCR Product Purification Kit (Roche Diagnostics). Sequencing reactions were carried out with LC1 to LC4 primers²⁴ and BigDye Terminator Cycle Sequencing Kit Ver. 3.1 (Applied Biosystems). Sequence determination was carried out on a 3130xl Genetic Analyzer (Applied Biosystems).

Plant Materials

Samples were collected at locations shown in Table 1 and Figure 1 in August 2012 (sample A) and 2007 (samples B and C) and identified by X. G. (author). Voucher specimen numbers are 2012-52, 2007-25, and 2007-103, respectively (Kunming Institute of Botany).

Isolation of Compounds

Extraction of dried root of sample A (22.4 g) with EtOH afforded an extract (2.08 g). Silica-gel CC (n-hexane-EtOAc, gradient) of the extract afforded 5 fractions. CC (n-hexane-EtOAc) of Fr. 1 (eluted with n-hexane) afforded 7 fractions (Fr. 1-1 to 1-7). From Frs. 1-2 and 1-3, 1 (17.1 mg) and 3 (63.4 mg) were obtained, respectively, without further purification. From Fr. 1-5, 4 (6.2 mg) was isolated after purification with HPLC (n-hexane-EtOAc 80:20). From Fr. 1-6, 2 (1.9 mg) was isolated with HPLC (n-hexane-EtOAc 90:10). CC (n-hexane-EtOAc) and HPLC (n-hexane-EtOAc 75:25) of Fr. 4 (eluted with n-hexane-EtOAc 80:20) afforded 5 (10.4 mg) and 6 (30.3 mg)..

CC (SiO₂, n-hexane-EtOAc, gradient) of a part (182.8 mg) of the EtOH extract of dried root of sample B (7.7 g) afforded 4 fractions. From Fr. 1 (eluted with n-hexane), 1 (0.4 mg), 3 (1.4 mg), 4 (5.1 mg), and 8 (6.7 mg) were isolated by repeated CC (n-hexane-EtOAc). Fr. 2 consisted of 2 (27 mg). From Frs. 3 and 4, 5 (5.0 mg) was obtained by repeated CC (n-hexane-EtOAc). Compound 7 (11 mg) was isolated from another part of the root extract (200.1 mg) by CC (n-hexane-EtOAc, 20:1).

CC (SiO₂, n-hexane-EtOAc, 20:1) of a part (166.7 mg) of the EtOH extract of dried root of sample C (7.7 g) afforded 4 fractions. From Fr. 1, 1 (0.8 mg), 3 (5.9 mg), and 8 (1.1 mg) were isolated by repeated CC (n-hexane/EtOAc 100:1 and 80:1). From Fr. 2, 10 (4.5 mg) was obtained by CC (n-hexane/EtOAc 40:1). From Fr. 3, 2 (6.1 mg), 5 (5.9 mg), and 9 (16 mg) were obtained by repeated CC (n-hexane/EtOAc 20:1 and n-hexane/Et₂O 80:1). From Fr. 4, 5 (7.2 mg) was obtained by CC (n-hexane/EtOAc 20:1). Compound 7 (12 mg) was obtained from another part of the root extract (194.5 mg) by repeated CC (n-hexane-EtOAc, 20:1).

Compound 6

An oil.

[α]_D ²⁵ +67.2 (c, 0.361, MeOH).

IR (neat/NaCl): 1780, 1711, 1645, 1234, 1157, 945, 756 cm^-1.

¹H and ¹³C NMR (C₆D₆): see Table 2.

MS (CI, CH₄): m/z (%) = 345 [M+H⁺] (95), 245 [M+H⁺-AngOH] (100), 199 (51).

HRMS-CI m/z [M+H⁺] calcd for C₂₀H₂₅O₅: 345.1702; found: 345.1697.

Footnotes

Acknowledgements

The authors thank Dr Takayuki Kawahara, Forestry and Forest Products Research Institute, and Dr Yoshinori Saito, Nagasaki University, for assistance in sample collection and 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: Grants-in-Aid for Scientific Research from JSPS (Nos. 16404008, 21404009, and 25303010) and Strategic Research Foundation Grant-Aided Projects for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

References

Kuroda

Hanai

Nagano

Tori

Gong

. Diversity of furanoeremophilanes in major Ligularia species in the Hengduan Mountains. Nat Prod Commun. 2012;7(4):539-548.doi:10.1177/1934578X1200700431

Kuroda

Hanai

Tori

et al. Diversity in furanoeremophilane composition produced by Ligularia species (Asteraceae) in the Hengduan Mountains area of China. J Synth Org Chem Jpn. 2014;72(6):717-725.doi:10.5059/yukigoseikyokaishi.72.717

Liu

Illarionova

. Ligularia. In: Wu

ZY.

Raven

PH.

Hong

DY.

, eds. Flora of China Vol. 20-21. Beijing and St. Louis: Science Press and Missouri Botanical Garden Press; 2011:376-415.

Tan

AM.

HP.

Yang

Zhang

Wang

ZT.

Hao

. Chemical constituents of Ligularia dictyoneura . Yao Xue Xue Bao. 2003;38(12):924-926.

Tan

AM.

Wang

ZT.

Hong

Hao

. Study on the chemical constituents of Ligularia dictyoneura . J China Pharm Univ. 2002;33(2):104-106.

Huang

Liu

et al. Two new sesquiterpenes from Ligularia dictyoneura . Phytochem Lett. 2013;6(3):315-317.doi:10.1016/j.phytol.2013.03.006

Wang

Zhan

Chen

. Two new eremophilane sesquiterpenoids from Ligularia dictyoneura . Nat Prod Res. 2019; Doi: 10.1080/14786419.2018.1556652.

Nagano

Iwazaki

Matsushima

et al. High diversity of Ligularia dictyoneura in chemical composition and DNA sequence. Chem Biodivers. 2007;4(12):2874-2888.doi:10.1002/cbdv.200790237

Gaikwad

NW.

Madyastha

. Biosynthesis of β-substituted furan skeleton in the lower furanoterpenoids: a model study. Biochem Biophys Res Commun. 2002;290(1):589-594.doi:10.1006/bbrc.2001.6232

10.

Kuroda

Ueshino

Nagano

. Ehrlich’s Reaction of furanoeremophilanes. Bull Chem Soc Jpn. 2004;77(9):1737-1740.doi:10.1246/bcsj.77.1737

11.

Kuroda

Nishio

. Substituent effect in color of Ehrlich's test of tetrahydrobenzofuran. Nat Prod Commun. 2007;2(5):581-585.doi:10.1177/1934578X0700200514

12.

Ishii

Tozyo

Minato

. Studies on sesquiterpenoids. Part XIII. Components of the root of Ligularia fischeri Turcz. J Chem Soc, C. 1966:1545-1548.doi:10.1039/j39660001545

13.

Ishii

Tozyo

Minato

. Studies on sesquiterpenoids – IX. Structure of ligularol and ligularone from Ligularia sibirica Cass. Tetrahedron. 1965;21:2605-2610.

14.

Nagano

Kuroda

Moriyama

Tsuyuki

Takahashi

. Mass spectra of furanoeremophilane derivatives. Bull Chem Soc Jpn. 1982;55(4):1221-1227.doi:10.1246/bcsj.55.1221

15.

Tori

Okamoto

Tachikawa

et al. Isolation of new eremophilane-type sesquiterpenoids, subspicatins A–D and subspicatolide from Ligularia subspicata, and chemical and genetic diversity of the species. Tetrahedron. 2008;64(38):9136-9142.doi:10.1016/j.tet.2008.06.077

16.

Bohlmann

Grenz

. Ein neues Furanoeremophilan-derivat aus Ligularia macrophylla . Phytochemistry. 1979;18(3):491-492.doi:10.1016/S0031-9422(00)81898-1

17.

Ishizaki

Tanahashi

Takahashi

Tori

. Furanoeremophilan-14β,6α-olide, a new furanosesquiterpene lactone from Ligularia h odgsoniiHook.f. The structure and nuclear Overhauser effects. J Chem Soc D. 1969(10):551-552.doi:10.1039/C29690000551

18.

Ishizaki

Tanahashi

Tsuyuki

Takahashi

Tori

. Furanoeremophilan-14β,6α-olide. A new furanosesquiterpene lactone of an eremophilane-type from Ligularia hodgsonii Hook.f. Bull Chem Soc Jpn. 1979;52(4):1182-1186.doi:10.1246/bcsj.52.1182

19.

Tori

Nakamizo

Mihara

et al. Chemical and genetic diversity of Ligularia vellerea in Yunnan, China. Phytochemistry. 2008;69(5):1158-1165.doi:10.1016/j.phytochem.2007.12.001

20.

Tada

Moriyama

Tanahashi

Takahashi

. Furanoeremophilane-6β,10β-diol and its derivatives. new furanosesquiterpenes from Ligularia japonica Less. Bull Chem Soc Jpn. 1974;47(8):1999-2002.doi:10.1246/bcsj.47.1999

21.

Jennings

PW.

Reeder

SK.

Hurley

JC.

Caughlan

CN.

Smith

. Isolation and structure determination of one of the toxic constituents from Tetradymia glabrata . J Org Chem. 1974;39(23):3392-3398.doi:10.1021/jo00937a019

22.

Abe

Onoda

Shirahata

Kato

Woods

MC.

Kitahara

. The structure of bakkenolide-A. Tetrahedron Lett. 1968;9(3):369-373.doi:10.1016/S0040-4039(01)98763-5

23.

Constantino

MG.

Oliveira

KTde.

Polo

EC.

Silva

GVJda.

Brocksom

. Core structure of eremophilanes and bakkanes through niobium catalyzed Diels-Alder reaction: synthesis of (+/-)-bakkenolide A. J Org Chem. 2006;71(26):9880-9883.doi:10.1021/jo061722x

24.

Nagano

Matsushima

Yamada

Hanai

Gong

Kuroda

. Two new furanoeremophilane sesquiterpenoids from: Ligularia oligonema . Nat Prod Commun. 2010;5(1):1-4.doi:10.1177/1934578X1000500101

25.

Kumar

Stecher

Knyaz

Tamura

. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547-1549.doi:10.1093/molbev/msy096

26.

Hanai

Gong

Tori

et al. Chemical and genetic study of Ligularia tongolensis , Ligularia cymbulifera , and Ligularia atroviolacea in the Hengduan Mountains of China. Bull Chem Soc Jpn. 2005;78(7):1302-1308.doi:10.1246/bcsj.78.1302

27.

Saito

Shiosaki

Fujiwara

et al. Eremophilanes from Ligularia hookeri collected in China and structural revision of 3β-acyloxyfuranoeremophilan-15,6-olide. Chem Pharm Bull. 2018;66(6):668-673.doi:10.1248/cpb.c18-00191

Diversity in Eremophilane Components of Ligularia dictyoneura in Yunnan and Sichuan Provinces of China

Abstract

Keywords

Experimental

General

Plant Materials

Isolation of Compounds

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References