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Abstract

Objective

Heliciopsis terminalis (Kurz.) Sleum (family Proteaceae) is distributed in India, Mianma, Thailand, Cambodia, Vietnam, and China. In Vietnamese traditional medicine, it is used as an antidote for detoxifying the liver and detoxifying alcohol, a contraceptive, and to treat infections. The purpose of this study is to determine the chemical constituents of the plant, as well as their in vitro hepatoprotective and antioxidant activities.

Methods

The dried powdered sample was ultrasonically extracted with MeOH, and the extract was fractionated by various chromatographic methods, including high-performance liquid chromatography. The structures of the isolated compounds were identified by NMR, high-resolution electrospray ionization mass spectrometry, and CD spectral analysis, and then screened for their in vitro hepatoprotective and antioxidant activities.

Results

One new phenolic glycoside, named helitermioside (1), and eight known compounds (2-9) were isolated from the trunk and branches of H terminalis. All of the isolates were reported from H terminalis for the first time. Compound 6 showed antioxidant activity in thiobarbituric acid reactive substances (TBARS) lipid peroxidation inhibition assays, with an IC₅₀ value of 61.16 ± 1.17 µg/mL, compared to that of Trolox (positive control), IC₅₀ = 7.06 ± 0.11 µg/mL. At a concentration of 100 µg/mL, none of the isolates exhibited hepatocellular protective activity.

Conclusion

Nine phenolic compounds (1–9) were recorded from H terminalis. Of these, compound 1 was new. Compound 6 exhibited moderate antioxidant activity in TBARS lipid peroxidation inhibition assays, but none of the compounds exhibited hepatocellular protective activity against CCl₄-induced toxicity.

Keywords

Proteaceae phenolic compound antioxidant activity hepatoprotective activity

Introduction

Heliciopsis terminalis (Kurz.) Sleum (Proteaceae) grows wild in Vietnam. The wood of this plant has the effect of detoxifying the liver and detoxifying alcohol, and is used as a contraceptive and to treat infections in traditional medicine in Vietnam.¹ In previous papers, several macrocyclics and phenolic glycosides have been reported for H terminalis and H lobata (Merr.) Sleum, along with their in vitro antioxidant and hepatoprotective activities.^2-5 The wood of H lobata and H terminalis are very similar, so according to folk experience, these materials are used interchangeably in the treatment of liver diseases without distinguishing them. Therefore, the wood of H terminalis was chosen for further study of its in vitro antioxidant and hepatoprotective activity. In this paper, we report one new (1) and eight known phenolics (2–9) from H terminalis, and their hepatoprotective and antioxidant effects.

Results and Discussion

Helitermioside (1) was isolated as a colorless powder. In the IR spectrum of 1, the functionalities were identified at 3343 cm⁻¹ (OH), 1701 (C = O), 1608 cm⁻¹ (olefinic), 1078 and 1038 cm⁻¹ (ether). Compound 1 (Figure 1) had the molecular formula of C₂₈H₃₆O₁₆, as determined from the ion peaks at m/z 629.2085 [M + H]⁺ (calcd for [C₂₈H₃₇O₁₆]⁺: 629.2085, Δ = + 1.4 ppm); and m/z 646.2348 [M + NH₄]⁺ (calcd for [C₂₈H₃₆O₁₆NH₄]⁺: 646.2342, Δ = + 0.9 ppm) in the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), indicating 11 degrees of unsaturation. The ¹H NMR spectrum of 1 showed a para-substituted aromatic ring [δ_H 7.09 (2H, d, J = 8.0 Hz, H-3ʹʹ and H-5ʹʹ) and 7.85 (2H, d, J = 8.0 Hz, H-2ʹʹ and H-6ʹʹ), a 1,3,4,5- substituted aromatic ring [δ_H 6.32 (2H, s)], three methoxy groups [δ_H 3.65 (6H, s) and 3.57 (3H, s)] and two anomeric protons at δ_H 4.97 (d, J = 7.5 Hz, H-1ʹ) and 5.22 (d, J = 7.5 Hz, H-1ʹʹʹ). The correlation spectroscopy (COSY) correlations of sugar molecules were observed and are shown in Figure 2. The heteronuclear single quantum coherence and ¹³C NMR spectra of 1 revealed two symmetric aromatic rings, a carbonyl (δ_H 165.2, C-7ʹʹ), three methoxy groups (δ_H 55.8 × 2 and 60.2), one glucose moiety [δ_C 100.2, 73.1, 76.3, 70.0, 73.8 (5 x CH), and 64.2 (CH₂)],⁶ and one galactose [δ_C 98.1, 70.2, 71.4, 67.0, 74.7 (5 x CH), and 60.8 (CH₂)] (Table 1).⁷ In addition, the presence of one galactose moiety was identified by J_{3ʹʹʹ,4ʹʹʹ} = 3.0 Hz and J_{4ʹʹʹ,5ʹʹʹ} ∼ 0 Hz, indicating equatorial orientation of H-4ʹʹʹ. The linking of the molecule parts was evidenced from the heteronuclear multiple bond correlation spectrum (Figure 2). Correlations of H-1ʹ and C-1, H₂-6ʹ and C-7ʹʹʹ, and H-1ʹʹʹ and C-4ʹʹ were observed. These confirmed that the glucose moiety was linked to C-1, the p-hydroxyphenoyl group to C-6ʹ,⁶ and the galactose moiety to C-4. In addition, the large J values (7.5 Hz) confirmed β-glycoside linkages.^6,7 The presence of D-glucose and D-galactose in the molecule of compound 1 was confirmed by acid hydrolysis.^8,9 Therefore, compound 1 was determined to be 34,5-trimethoxyphenyl 1-O-β-D-[6-O-(4-O-β-D-galactopyranosyl)-p-hydroxyphenoyl]-glucopyranoside and named as helitermioside (Supplemental Figures S1−S10).

Figure 1.

Chemical structures of 1–9.

Figure 2.

Key HMBC and COSY correlations of 1. Abbreviations: COSY, correlation spectroscopy; HMBC: heteronuclear multiple bond correlation.

Table 1.

¹H NMR and ¹³C NMR Spectroscopic Data for 1 in DMSO-d₆.

Pos.	^a δ _C	^bδ_H (mult, J in Hz)	Pos.	^a δ _C	^bδ_H (mult, J in Hz)
1	153.5	−	1ʹʹʹ	98.1	5.22 (d, 7.5)
2, 6	94.4	6.32 (s)	2ʹʹʹ	70.2	3.44 (dd, 6.0, 7.5)
3, 5	153.1	−	3ʹʹʹ	71.4	3.94 (dd, 6.0, 3.0)
4	132.7	−	4ʹʹʹ	67.0	3.42 (brd, 3.0)
1ʹ	100.2	4.97 (d, 7.5)	5ʹʹʹ	74.7	3.72 (m)
2ʹ	73.1	3.25 (dd, 9.0, 7.5)	6ʹʹʹ	60.8	3.47 (dd, 12.0, 5.0)
2ʹ	73.1	3.25 (dd, 9.0, 7.5)	6ʹʹʹ	60.8	3.69 (dd, 12.0, 2.0)
3ʹ	76.3	3.33 (t, 9.0)	3-OCH₃	55.8	3.65 (s)
4ʹ	70.0	3.26 (t, 9.0)	4-OCH₃	60.2	3.57 (s)
5ʹ	73.8	3.80 (ddd, 9.0, 5.0, 2.0)	5-OCH₃	55.8	3.65 (s)
6ʹ	64.2	4.57 (dd, 12.0, 2.0)	3ʹ-OH		5.35 (brs)
6ʹ	64.2	4.22 (dd, 12.0, 5.0)	3ʹ-OH		5.35 (brs)
1ʹʹ	122.9	−	2ʹʹʹ-OH		5.14 (brs)
2ʹʹ, 6ʹʹ	131.1	7.85 (d, 8.0)	3ʹʹʹ-OH		4.98 (brs)
3ʹʹ, 5ʹʹ	115.9	7.09 (d, 8.0)	4ʹʹʹ-OH		4.69 (brs)
4ʹʹ	161.4	−	6ʹʹʹ-OH		4.49 (brs)
7ʹʹ	165.2	−

^a125 MHz, ^b500 MHz.

The other compounds were identified as 34,5-trimethoxyphenyl 1-O-β-D-apiofuranosyl-(1ʹʹ→6ʹ)-β-D-glucopyranoside (2),¹⁰ rhyncoside C (3),¹¹ 34,5-trimethoxyphenyl 1-O-β-D-glucopyranoside (4),¹² ficuscarpanoside B (5),¹³ hovetrichoside A (6),¹⁴ threo-syringylglycerol (7),¹⁵ helicide (8),¹⁶ and (7R,8S)-dihydrodehydrodiconeferyl alcohol 4-O-β-D-glucopyranoside (9)¹⁷ by comparisons of their physical and NMR data (including CD spectral data for 9) with those of reported data. This is the first isolation of 2–9 from H terminalis.

Compounds 1–9 were tested for their hepatoprotective and antioxidant activities. Compound 6 showed moderate antioxidant activity in thiobarbituric acid reactive substances (TBARS) lipid peroxidation inhibition assays with an IC₅₀ value of 61.16 ± 1.17 µg/mL compared to the IC₅₀ value of 7.06 ± 0.11 µg/mL of Trolox (Supplemental Tables S1 and S2). None of the isolates showed hepatocellular activity against the toxic induction caused by CCl₄, as the viability of cells was lower than that of the blank compound, DMSO.

Materials and Methods

General

Refer to Supplemental Material.⁵

Plant Materials

The H terminalis (Kurz.) Sleum sample (coded: HTG0821) was harvested in December 2021 at BacKan, Vietnam and identified by Dr ND Trong (Hanoi University of Pharmacy).

Extraction and Isolation

H terminalis trunk and branches (10 kg, dried powder) were extracted ultrasonically by MeOH (3 times, each 30 L, 60 min) to obtain the extract (120 g). This was suspended in H₂O and partitioned successively with EtOAc to obtain the EtOAc extract (32.0 g) and water layer (HTW). The HTW was fractionated on a diaion column, eluted by MeOH/H₂O (1/4, 1/1, 4/1, and 1/0 each 3.0 L) to get four fractions, HTN1–HTN4. HTN2 (8.2 g) was separated on a silica gel column, eluting with CH₂Cl₂/MeOH (gradient 10/1–3/1) to obtain 3 fractions, HTN2A-HTN2C. HTN2A (430 mg) was fractionated by high-performance liquid chromatography (HPLC), eluting with MeOH in H₂O (1/6) to give 8 (10.6 mg, t_R 6.5 min) and 7 (10.6 mg, t_R 7.8 min). HTN2B (1.2 g) was purified by HPLC eluting with MeOH in H₂O (1/6) to give 5 (83.1 mg, t_R 13.5 min). HTN2C (220 mg) was purified by HPLC eluting with MeOH in H₂O (1/5) to yield 4 (7.5 mg, t_R 8.5 min). HTN3 (9.6 g) was fractionated on a silica gel column, and eluted with CH₂Cl₂/MeOH (gradient 20/1–4/1) to obtain 3 fractions, HTN3A-HTN3C. HTN3A (310 mg) was purified by HPLC, eluting with MeOH in H₂O (1/6) to give 3 (4.2 mg, t_R 16.2 min). HTN3B (250 mg) was treated by HPLC eluting with MeOH in H₂O (1/6) to yield 3 (7.5 mg, t_R 18.0 min). HTN3C (240 mg) was purified by HPLC eluting with MeOH in H₂O (1/5) to give 2 (7.5 mg, t_R 22.3 min). HTN4 (3.5 g) was isolated using a silica gel column by eluting with CH₂Cl₂/MeOH (10/1) to give 2 fractions, HTN4A and HTN4B. HTN4B (510 mg) was purified by HPLC, eluting with MeOH in H₂O (1/4) to give 1 (35.3 mg, t_R 24.6 min) and 9 (21.3 mg, t_R 28.4 min).

Helitermioside (1)

Colorless powder, $[α]_{D}^{25}$ : + 3.4° (c 0.1, MeOH). IR (KBr) ν_max: 3343, 2923, 1701, 1608, 1078, and 1038 cm⁻¹. HR-ESI-MS: m/z 629.2085 [M + H]⁺, calcd. for [C₂₈H₃₇O₁₆]⁺: 629.2085, Δ = + 1.4 ppm; m/z 646.2348 [M + NH₄]⁺, calcd for [C₂₈H₃₆O₁₆NH₄]⁺: 646.2342, Δ = + 0.9 ppm. ¹H and ¹³C NMR data See Table 1.

Antioxidant and Hepatoprotective Assay

See supporting information.

Acid Hydrolysis

See supporting information.

Conclusions

In this study, one new compound, named as helitermioside (1), and eight phenolics (2–9) were recorded from H terminalis. Compound 6 exhibited moderate antioxidant activity in TBARS lipid peroxidation inhibition assays, but compounds 4, 6, 8, and 9 exhibited moderate hepatocellular protective activity against CCl₄ toxic induction.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231174993 - Supplemental material for Hepatoprotective and Antioxidant Activities of Phenolic Compounds from Heliciopsis terminalis

Supplemental material, sj-docx-1-npx-10.1177_1934578X231174993 for Hepatoprotective and Antioxidant Activities of Phenolic Compounds from Heliciopsis terminalis by Bui V. Trung, Duong H. Anh, Pham H. Viet and Phan Van Kiem in Natural Product Communications

Footnotes

Author Contribution

Research idea was conceived by BVT and PHV; isolation, bioactivity assay were performed by BVT and DHA; structure elucidation and writing were handeled by BV Trung and PV Kiem.

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) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Phan Van Kiem

Supplemental Material

Supplemental material for this article is available online.

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