Atractylodes macrocephala Rhizomes Contain Anti-inflammatory Sesquiterpenes

Abstract

Two new compounds, named eudesm-4(15),7-diene-3α,9β,11-triol (1) and eudesm-4(15),7-diene-1β,3α,9β,11-tetraol (2) together with three known sesquiterpene lactones (1S,5R,7R,10R)-secoatractylolactone (3), (1S,5R,7R,10R)-secoatractylolactone-11-O-β-D-glucopyranoside (4) atractylenolide III (5) were isolated from the rhizomes of Atractylodes macrocephala. Their structures were elucidated by using one-dimensional (1D) and 2D-NMR spectra and high resolution electrospray ionization (HR-ESI)-MS data. Compound 5 exhibited the most active anti-inflammatory activity with IC₅₀ values of 27.5 µM in inhibiting of nitric oxide production. Compounds 1, 2, and 3 showed moderate effects while compound 4 was inactive.

Introduction

Atractylodes macrocephala Koidz (Asteraceae) is a valuable traditional medicinal plant with many pharmacological activities. In traditional medicine, the rhizome of this plant treats several diseases, such as diarrhea, constipation, and abdominal distension.¹⁾ Previous studies reported various bioactivities of A. macrocephala, including hepatoprotective,²⁾ anti-oxidative,³⁾ anti-tumor, anti-viral, and anti-inflammatory effects.⁴⁾ This plant’s chemical constituents include sesquiterpenoids, triterpenoids, polyacetylenes, coumarins, phenyl propanoids, flavonoids, steroids, benzoquinones, and polysaccharides, which showed significant bioactivities.^5–8) In this study, we isolated two new eudesman sesquiterpenes (1–2), and three known sesquiterpene lactones (3–5) (Fig. 1). In a study on bioactivity screening, we found the aqueous extract of A. macrocephala species exhibited remarkable inhibition activity on nitric oxide (NO) production (72% inhibition at 20 µg/mL). Two new compounds 1 and 2 were isolated based on bioassay-guided isolation.

Fig. 1. Structures of Sesquiterpenes 1–5 from Rhizomes of A. macrocephala

Results and Discussion

Compound 1 was obtained as a colorless solid. It molecular formula was identified as C₁₅H₂₄O₃, deduced from the molecular ion negative peak high resolution electrospray ionization (HR-ESI)-MS at m/z 251.1645 [M−H]⁻ (Calcd 251.1647, C₁₅H₂₃O₃). The ¹H-NMR spectrum manifested signals for an exomethylene with two triplets at δ_H 4.80 (1H, t, J = 1.5 Hz, H-15β), 5.08 (1H, t, J = 1.5 Hz, H-15α), an olefinic proton at δ_H 5.55 (1H, d, J = 1.5 Hz, H-8) which were characteristic signals for an eudesmane skeleton. In addition, two oxymethine protons as a broad singlet at δ_H 4.28 (1H, br s, H-3) and a doublet at δ_H 4.04 (1H, d, J = 2.0 Hz, H-9) together with three methyl singlets at δ_H 0.69 (3H, s, H-14), 1.34 (3H, s, H-13), 1.35 (3H, s, H-12) were observed. Combination of ¹³C-NMR and heteronuclear single quantum coherence (HSQC) spectra displayed fifteen carbons, including three methyls (3×CH₃), four methylenes (4×CH₂), four methines (4×CH), and four quaternary carbons (4×Cq). We determined the linkage position of methylene (CH₂-15), and one methyl and propan-2-ol-2-yl with the heteronuclear multiple bond connectivity (HMBC) spectrum. The correlations of H-15α (δ_H 5.08) together H-15β (δ_H 4.80) with C-3 (δ_C 74.0), C-4 (δ_C 152.1), C-5 (δ_C 40.1), and of H-12 (δ_H 1.35) together H-13 (δ_H 1.34) with C-7 (δ_C 145.5), C-11 (δ_C 72.9) were demonstrated an exomethylene (CH₂-15), and propan-2-ol-2-yl attached at C-4, C-7, respectively. Besides, the interactions of H-14 (δ_H 0.69) with C-1 (δ_C 32.4), C-5 (δ_C 40.1), C-9 (δ_C 79.2) confirmed methyl (CH₃-14) was linked at C-10 (Fig. 2). The ¹H–¹H correlation spectroscopy (COSY) spectrum appeared during spin–spin coupling between the protons of H-1 (δ_H 1.78) with H-2α (δ_H 1.83), of H-2β (δ_H 1.77) with H-3 (δ_H 4.28), of H-5 (δ_H 2.56) with H-6 (δ_H 2.08), and of H-8 (δ_H 5.55) with H-9 (δ_H 4.04), which confirmed this compound was a eudesmane-type sesquiterpene. The small coupling constant of H-3 (br s) due to its equatorial-axial and equatorial-equatorial relationships with H-2 indicated the equatorial orientation of H-3. In addition, the splitting pattern of H-5 (dd, J = 9.0, 2.0 Hz) demonstrated the axial-axial and axial-equatorial relationships with H-6. Moreover, the nuclear Overhauser effect spectroscopy (NOESY) spectrum showed the interactions of H-5 (δ_H 2.56) with H-9 (δ_H 4.04) but no correlation of H-5 and H-14, which suggested that the junction of the eudesmane rings was trans-fused. The chemical shift of proton in the methyl group (CH₃-14) at δ_H 0.69 revealed the β-orientation, which was the same as in cases of eudesmane-type sesquiterpenes from Atractylodes species.^3,8–10) The results deduced that proton H-9 was α-oriented and proton H-3 was β-oriented. Therefore, we considered compound 1 a new sesquiterpene named eudesm-4(15),7-diene-3α,9β,11-triol.

Fig. 2. Key HMBC (H→C), COSY (━) and NOESY (↔) Correlations of Compounds 1 and 2

The molecular formula of compound 2 was identified as C₁₅H₂₄O₄, also deduced from molecular ion negative peak HR-ESI-MS at m/z 267.1607 [M−H]⁻ (Calcd for C₁₅H₂₃O₄, 267.1596). Comparing ¹H, and ¹³C-NMR spectra of 2 with those of 1 revealed they were similar except for the appearance of the oxymethine proton signal at δ_H 4.18 (H-1) and chemical shift of C-1 changed from upfield in 1 (δ_C 32.4) to downfield in 2 (δ_C 76.1). This suggested that 2 was a hydroxylated product of 1 at C-1. The relative configurations of 2 were determined by the proton coupling constants and NOESY experiment. The equatorial orientation of H-3 was assigned by the small coupling constant of H-3 (J = 3.0 Hz). In constrast, the di-axial and axial-equatorial relationships between H-1 and H-2α, H-2β were recognized by the doublet of doublet signal of H-1 (J = 12.0, 4.5 Hz), which indicated the axial position of H-1. Extensive analysis of the NOESY spectrum revealed the interaction of H-1 (δ_H 4.18) with H-9 (δ_H 4.38), H-9 with H-5 (δ_H 2.51). Through its spectrometry analysis and comparison with previously published literature,⁹⁾ we determined compound 2 was a new sesquiterpene named eudesm-4(15),7-diene-1β,3α,9β,11-tetraol. Due to the paucity of compounds after biological assay, the absolute configurations of 1 and 2 could not be determined in this study.

Compounds 3, 4 and 5 were identified as (1S,5R,7R,10R)-secoatractylolactone (3) (1S,5R,7R,10R)-secoatractylolactone-11-O-β-D-glucopyranoside (4),¹¹⁾ and atractylenolide III (5).¹²⁾ Previously, (1S,5R,7R,10R)-secoatractylolactone (3) were reported as a product from enzymatic hydrolysis of 4.¹¹⁾ This is the first time the compound 3 was isolated from nature.

The isolated compound were evaluated for their inhibitory effect of NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in comparision with the effect of cardamonin as a positive control (IC₅₀ = 2.14 ± 0.14 µM). The results showed that atractylenolide III (5) was the most active, while compounds 1–3 expressed remarkable inhibitory activities with IC₅₀ values of 43.5, 60.1, and 32.7 µM, respectively. The reason bioactive compound 3 was stronger than 1 and 2 could be because 3 had two hydroxyls and an acetyl group at the same face of the cycloheptane ring. It could increase in bioactivities of 3 by hydrogen bonds created from these groups. Compound 4, the glucosyl product of 3, was inactive.

Conclusion

Five secquiterpenes including two new eudesmane-type sesquiterpenes as eudesm-4(15),7-diene-3α,9β,11-triol (1), eudesm-4(15),7-diene-1β,3α,9β,11-tetraol (2) and three known sesquiterpene lactones as (1S,5R,7R,10R)-secoatractylolactone (3), (1S,5R,7R,10R)-secoatractylolactone-11-O-β-D-glucopyranoside (4) and atractylenolide III (5) were isolated from the methanol extract of Atractylodes macrocephala. The inhibitory effect of those All the isolated counpounds showed significant inhibitory effect of NO production with the IC₅₀ range from 27.5 to 60.1 µM. Among them, compound 5 had the highest IC₅₀ value at 27.5 µM.

Experimental

Plant Material

The rhizomes of A. macrocephala were gathered in Quan Ba, Ha Giang province, Vietnam in Oct. 2019 and authenticated by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources (VAST). The voucher specimens (AM12) were deposited at the Agro-Pharmaceutical Department, Center for Research and Technology Transfer.

Extraction and Isolation

The dried and powdered rhizomes of A. macrocephala (2.5 kg) were extracted with methanol at room temperature (3 × 5 L, overnight). After removing methanol solvent under reduced pressure to obtain a crude residue (200.0 g, 8%), which was then dispersed in water (1000 mL), and sequentially partitioned with dichloromethane (DCM) to yield 45.0 g (1.8%) of DCM extract. The aqueous layer was passed through a Diaion HP-20 column, eluted with H₂O, MeOH:H₂O (25/75) and MeOH to yield 3 fractions AMW1, AMW2, and AMW3 (30 g, 1.2%). DCM extract (45 g) was seeded on a silica gel column eluted with a gradient of n-hexane:CH₃COCH₃ (1 : 100→100 : 1) to give fraction AML1.5. On a reversed-phase silica gel column, fraction AML1.5 underwent further purification and was eluted with CH₃COCH₃ : H₂O (1 : 6) to give 5 (79.0 mg, 0.00316%). Fraction AMW3 (30 g) was sequestered on a silica gel column eluted with a gradient of CH₂Cl₂ : MeOH (100 : 1→0 : 100) to yield two fractions as AMWB1.10 and AMWB1.11. Fraction AMWB1.10 purified through silica gel column eluted with CH₂Cl₂ : MeOH : H₂O (12 : 1 : 0.01) to afford two fractions, AMWB1.1 and AMWB1.2. Fraction AMWB1.1 was detached on a C-18 column and eluted with a solvent mix of CH₃COCH₃ : H₂O (1 : 4) to obtain 1 (11.6 mg, 0.000464%). Fraction AMWB1.2 was segregated on a C-18 column and eluted with CH₃COCH₃ : H₂O (1 : 4) to give 3 (2.0 mg, 0.00008%). Fraction AMWB1.11 was detached on a silica gel column and eluted with mobile phase as CH₂Cl₂ : MeOH : H₂O (10 : 1 : 0.01) to afford three fractions as AMWB2.4, AMWB2.7, AMWB2.9. Fraction AMWB2.4 was segregated with a silica gel column, eluted with CH₂Cl₂ : CH₃COCH₃ (1 : 1), to yield 2 (7.5 mg, 0.0003%). Compound 4 (7.9 mg, 0.000316%) was detached on a C18 reversed-phase column and eluted with mobile phase as CH₃COCH₃ : H₂O (1 : 6) from fraction AMWB2.9.

Eudesm-4(15),7-diene-3α,9β,11-triol (1): Colorless solid; [α]_D²⁴=−23.4 (c 0.02, CH₃OH); IR ν_max (KBr): 3332 (OH), 1708 (C=C), 1025 (C–O) cm⁻¹; ¹H-NMR (500 MHz, CD₃OD) and ¹³C-NMR (125 MHz, CD₃OD: see Table 1; HR-ESI-MS m/z: [M−H]⁻ m/z 251.1645 (Calcd for C₁₅H₂₃O₃, 251.1647).

Table 1. ¹H- and ¹³C-NMR Data of Compounds 1 and 2

No.	1		2
	δH	δC	δH	δC
1	1.78 (2H, m)	32.4	4.18 (1H, dd, 12.0, 4.5)	76.1
2	1.83 (1H, m, H-2α)	30.6	1.96 (1H, ddd, 13.5, 4.5, 3.0, H-2α)	38.5
	1.77 (1H, m, H-2β)		1.69 (1H, ddd, 13.5, 12.0, 3.0, H-2β)
3	4.28 (1H, br s)	74.0	4.36 (1H, t, 3.0)	74.3
4	—	152.1	—	150.4
5	2.56 (1H, dd, 9,0, 2.0)	40.1	2.51 (1H, dd, 9,0, 2.0)	38.6
6	2.08 (2H, m)	25.4	2.15 (2H, m)	25.0
7	—	145.5	—	145.4
8	5.55 (1H, d, 1.5)	123.3	5.52 (1H, dd, 3.0, 1.5)	122.1
9	4.04 (1H, d, 2.0)	79.2	4.38 (1H, dd, 4.5, 2.5)	80.4
10	—	40.4	—	43.2
11	—	72.9	—	72.9
12	1.35 (3H, s)	29.1	1.35 (3H, s)	29.0
13	1.34 (3H, s)	29.0	1.35 (3H, s)	28.9
14	0.69 (3H, s)	11.0	0.70 (3H, s)	4.9
15	5.08 (1H, t, 1.5, H-15α)	110.8	5.14 (1H, t, 1.5, H-15α)	112.2
	4.80 (1H, t, 1.5, H-15β)		4.88 (1H, t, 1.5, H-15α)

Eudesm-4(15),7-diene-1β,3α,9β,11-tetraol (2): Colorless solid; [α]_D²⁴=−15.2 (c 0.02, CH₃OH); IR ν_max (KBr): 3363 (OH), 1708 (C=C), 1029 (C–O) cm⁻¹; ¹H-NMR (500 MHz, CD₃OD) and ¹³C-NMR (125 MHz, CD₃OD: see Table 1; HR-ESI-MS m/z: [M−H]⁻ m/z 267.1607 (Calcd for C₁₅H₂₃O₄, 267.1596).

Acknowledgments

The Graduate University of Science and Technology funds this research (Grant GUST.STS.T2020-HH04).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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