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Abstract

A new phenylpropane (1) and 9 known (2-10) compounds were isolated from the methanol extract of Pandanus tonkinensis roots. Their chemical structures were determined as (7S)-2,6-dimethoxyphenyl-7,9-propanediol-1-O-β-D-glucopyranoside (1), isorhapontigenin (2), pinoresinol-4,4′-di-O-β-D-glucoside (3), isoeucommin A (4), pinoresinol-4′-O-β-D-glucoside (5), acanthoside B (6), eucommin A (7), urolignoside (8), benzyl O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (9), and (6S,9S)-roseoside (10) by comprehensive analysis of high-resolution electron spray ionization mass spectrum and nuclear magnetic resonance spectral data, as well as by comparison of their spectral data with those reported in the literature. In addition, the stereochemistry of 1 was successfully determined by both theoretical and calculated CD spectra. All the isolates were tested for their lipid peroxidation inhibitory effects by in vitro assay. Compounds 2-7 exhibited significantly lipid peroxidation inhibitory effects with IC₅₀ values of 21.3 ± 1.7, 61.9 ± 3.9, 57.5 ± 5.5, 10.4 ± 0.7, 28.9 ± 0.3, 54.2 ± 3.5 µM, respectively, compared to that of the positive control, trolox (31.4 ± 2.2 µM).

Keywords

pandanaceae phenylpropanoid lignan lipid peroxidation inhibitory

Introduction

The plant Pandanus tonkinensis Mart. ex B. Stone belongs to Pandanaceae family, which is widely distributed in the northern provinces of Vietnam, such as Hoa Binh, Vinh Phuc, Bac Giang, and Ninh Binh, and is used in folk medicine to treat liver diseases very effectively.^1,2 However, up to now, there has been no publication on the chemical composition of this plant. In the program to search for antioxidant components from Vietnamese medicinal plants, the roots of P tonkinensis were selected for research. This paper reports the isolation and structure determination of 1 new and 9 known compounds from the methanol extract of the roots of this plant. In addition, the lipid peroxidation inhibitory effects of the isolated compounds were also evaluated by in vitro assay.

Results and Discussion

Compound 1 (Figure 1) was obtained as a white amorphous powder. Its molecular formula was determined to be C₁₇H₂₆O₉ from the pseudo-molecular ion peaks at m/z 397.1470 [M + Na]⁺ (calcd. for [C₁₇H₂₆O₉Na]⁺, 397.1469, Δ = + 0.2 ppm), and m/z 392.1910 [M + NH₄]⁺ (calcd. for [C₁₇H₃₀O₉N]⁺, 392.1915, Δ = −1.2 ppm) in the high-resolution electron spray ionization mass spectrum (HRESIMS), indicating 5 degrees of unsaturation. The ¹H nuclear magnetic resonance (NMR) spectrum of 1 exhibited 2 aromatic protons at δ_H 6.71 (2H, s) suggesting a 1,3,4,5-substituted symmetrical aromatic ring, one methyl doublet signal at δ_H 0.94 (3H, d, J = 7.2 Hz), 2 methoxy groups at δ_H 3.89 (6H, s), and an anomeric proton at δ_H 4.85 (1H, d, J = 7.8 Hz). The ¹³C NMR and HSQC spectra of 1 showed signals of 17 carbon atoms, including 4 nonprotonated, 8 methines, 2 methylenes, and 3 methyls. Of these, 6 carbon signals (δ_C 105.6, 75.8, 77.8, 71.4, 78.3, and 62.6) were assigned to a glucopyranosyl group. The above evidence suggested that 1 was a phenylpropane glycoside having 2 methoxy groups at C-3 and C-5 of the aromatic ring.³ In the propane side chain, the observed HSQC correlations of H-7 (δ_H 4.51)/C-7 (δ_C 76.6), H-8 (δ_H 1.76)/C-8 (δ_C 30.0), and H-9 (δ_H 0.94)/C-9 (δ_C 10.6), as well as the proton and carbon chemical shifts, the multiplets, and ¹H-¹H coupling constants indicated the presence of a -CH(OH)-CH₂-CH₃ moiety.³ In the HMBC spectrum of 1, H-8 correlated to C-1 (δ_C 143.4)/C-2 and C-5 (δ_C 105.0), methoxy protons (δ_H 3.89) correlated to C-3/C-5 (δ_C 154.1), and the anomeric proton (H-1′, δ_H 4.85) correlated to C-4 (δ_C 135.3) (Supplemental Figures S1). This evidence confirmed the propane moiety attached to C-1, 2 methoxy groups linked to C-3 and C-5, and the glucose attached to C-4. Furthermore, the coupling constant of the anomeric proton, J_{H-1′/H-2′} = 7.8 Hz, indicated a β-glucopyranosyl linkage. Later, the presence of a D-glucose moiety was identified by acid hydrolysis, treatment with cysteine methyl ester and O-tolyl isothiocyanate, followed by HPLC analysis and comparison with the t_R values of authentic D/L-glucose.⁴ All the above evidence corresponded to that of (7R)-2,6-dimethoxyphenyl-7,9-propanediol-1-O-β-D-glucopyranoside.⁴ However, the optical rotation of 1 ([α]_D²3: + 9.0) differed from that of (7R)-2,6-dimethoxyphenyl-7,9-propanediol-1-O-β-D-glucopyranoside ([α]_D²³: - 10.0)³ suggesting a (7S)-configuration of 1. This suggestion was confirmed based on both theoretical and calculated CD spectra. The 2 possible configurations 7S/7R of 1 were submitted for calculation of their theoretical ECD spectra and compared with experimental results (Figure 2). The experimental CD analysis of 1 was in good agreements with the 7S-configuration. Consequently, the complete structure of compound 1 was elucidated as (7S)-2,6-dimethoxyphenyl-7,9-propanediol-1-O-β-D-glucopyranoside (Supplemental Figures S2-S7).

Figure 1.

Chemical structures of compounds 1-10.

Figure 2.

Theoretical calculated ECD spectra of 2 possible stereoisomers and experimental CD for compound 1.

The other compounds were identified as isorhapontigenin (2),⁵ pinoresinol-4,4′-di-O-β-D-glucoside (3),⁶ isoeucommin A (4),⁷ pinoresinol-4′-O-β-D-glucoside (5),⁸ acanthoside B (6),⁹ eucommin A (7),¹⁰ urolignoside (8),^11,12 benzyl O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (9),¹³ and (6S,9S)-roseoside (10)¹⁴ by comparisons of their NMR spectroscopic data with those reported in the literature (Supplemental Figures S8-S43). Furthermore, the (6S,9S)-configuration of 10 was determined based on the positive Cotton effect at 240 nm in its CD spectrum and the lower carbon chemical shift of C-9 (δ_C 74.6 ppm).¹⁴

Phenolic compounds, including lignans, are a type of secondary plant metabolite exhibiting diverse structures, which exhibit potentially beneficial bioactive properties due to antioxidant activity.^15–18 Therefore, all the isolates were tested for their lipid peroxidation inhibitory effects by in vitro assay.^19,20 At a concentration as high as 100 µg/mL, compounds 2-7 exhibited significantly lipid peroxidation inhibitory effects with inhibition in the range from 72.7% to 88.4%, compared to the positive control, trolox, 87.1%, whereas compounds 1, and 8-10 showed no activity (Supplemental Table S1). Further evaluation of their lipid peroxidation inhibitory effects showed that compounds 2-7 exhibited significant lipid peroxidation inhibitory effects with IC₅₀ values of 21.3 ± 1.7, 61.9 ± 3.9, 57.5 ± 5.5, 10.4 ± 0.7, 28.9 ± 0.3, 54.2 ± 3.5 µM, respectively, compared to that of the positive control, trolox (31.4 ± 2.2 µM). Regarding structure activity relationship, our results suggested that the lignan glycoside compounds having a furofuran structure showed the most lipid peroxidation inhibitory effects (Table 1).

Table 1.

Lipid Peroxidation Inhibitory Activity of Compounds 1-10.

Compounds	IC₅₀ (µM)	Compounds	IC₅₀ (µM)
1	>100	6	28.9 ± 0.3
2	21.3 ± 1.7	7	54.2 ± 3.5
3	61.9 ± 3.9	8	>100
4	57.5 ± 5.5	9	>100
5	10.4 ± 0.7	10	>100
Trolox^a	31.4 ± 2.2

Positive control.

Materials and Methods

General Experimental Procedures

Optical rotation was measured on a Jasco P-2000 polarimeter, IR spectra on a Spectrum Two FT-IR spectrometer, CD spectra on a Chirascan spectrometer (Applied Photophysics), NMR spectra on a Bruker Avance NEO 600 MHz spectrometer, and HRESIMS on a SCIEX X500 QTOF LC/MS. Flash column chromatography was performed using either silica gel or reversed phase (RP-18) resins as adsorbent. The ratio between the amount of silica gel and fraction was 20/1 (w/w). A fraction collector was set by volume per tube (15 mL/tube). Fractionation was monitored by thin layer chromatography (TLC). Contents of test tubes showing a similar TLC pattern were combined. TLC was carried out on either precoated silica gel 60 F₂₅₄ and/or RP-18 F_254S plates. Compounds were visualized by UV irradiation (254 and 365 nm) and by spraying with H₂SO₄ solution (5%), followed by heating with a heat gun. HPLC was conducted on an Agilent 1100 system including quaternary pump, autosampler, DAD detector, and preparative HPLC column YMC J'sphere ODS-H80 (4 µm, 20 × 250 mm). An isocratic mobile phase with a flow rate of 3 mL/min was used in pre-HPLC.

Plant Material

The roots of Pandanus tonkinensis Mart. ex B. Stone were collected in Son Duong District, Hoa Binh Province, Vietnam, in April 2021, and identified by Dr Do Thi Xuyen, Head of Department of Botanic, Faculty of Biology, VNU University of Science. A voucher specimen (code HNU 024663) is kept at the Herbarium, Vietnam National University, Hanoi, Vietnam.

Extraction and Isolation

Dried powder of P tonkinensis roots (14 kg) was sonicated with methanol (3 times, each 25 L MeOH). After removal of solvent, the MeOH extract (40 g) was suspended in water and then partitioned with n-hexane, dichloromethane, and ethyl acetate to give the corresponding residues (PT1, 2.5 g, PT2, 3.7 g, PT3, 5.3 g), and water (PT4, 27.0 g). After checking by TLC, PT4 (26.5 g) was further chromatographed on a Diaion HP-20 column eluting with water to remove sugar, then with increasing concentration of methanol in water (25, 50, and 100%) to obtain 3 fractions, PT4A (2.4 g), PT4B (2.6 g), and PT4C (21.0 g). PT4C (20.0 g) was chromatographed on a silica gel column eluting with dichloromethane/methanol (v/v) 20:1, 5:1, 1:1, and 0:1 (each 2 L) to give 4 corresponding subfractions (PT4C1-PT4C4). PT4C1 (14.0 g) was further chromatographed on a YMC column eluting with acetone/water (1/3, v/v) to give 7 smaller fractions, PT5A-PT5G. PT5C (5.8 g) was fractionated by silica gel CC eluting with dichloromethane/methanol (7/1, v/v) to obtain 6 subfractions, PT6A-PT6F. PT6A (83.0 mg) was chromatographed by HPLC (J’sphere H-80 column, 250 mm length × 20 mm ID, eluting with 15% acetonitrile in water, a flow rate of 2.5 mL/min) to give compounds 1 (t_R 30.689, 8.7 mg) and 10 (t_R 35.024, 20.2 mg). PT6D (172.6 mg) was separated by HPLC (J’sphere H-80 column, 250 mm length × 20 mm ID, eluting with 13% acetonitrile in water, a flow rate of 2.5 mL/min) to give compound 9 (t_R 37.50, 36.0 mg). PT6E (151.3 mg) was chromatographed by HPLC (J’sphere H-80 column, 250 mm length × 20 mm ID, eluting with 15% acetonitrile in water, a flow rate of 2.5 mL/min) to give compounds 2 (t_R 35.240, 6.0 mg) and 3 (t_R 40.158, 16.4 mg). Fraction PT5F (1.4 g) was separated by silica gel CC eluting with dichloromethane/methanol (10/1, v/v) to give 6 smaller fractions, PT7A-PT7F. PT7A (93.9 mg) was chromatographed by HPLC (J’sphere H-80 column, 250 mm length × 20 mm ID, eluting with 20% acetonitrile in water, a flow rate of 2.5 mL/min) to give compounds 4 (t_R 48.319, 4.9 mg), 5 (t_R 50.933, 40.4 mg), 6 (t_R 53.606, 9.6 mg), and 7 (t_R 57.438, 4.5 mg). PT7E (17.8 mg) was chromatographed by HPLC using the same conditions to give compound 8 (t_R 24.60, 4.1 mg).

(7S)-2,6-Dimethoxyphenyl-7,9-Propanediol-1-O-β-D-Glucopyranoside (1)

Colorless amorphous powder, $[α]_{D}^{25}$ : + 9.0 (c 0.1, MeOH); IR (KBr) ν_max: 3428, 2935, 1650, 1078 cm⁻1. HRESIMS m/z 397.1470 [M + Na]⁺ (calcd. for [C₁₇H₂₆O₉Na]⁺, 397.1469, Δ = + 0.2 ppm) and m/z 392.1910 [M + NH₄]⁺ (calcd. for [C₁₇H₃₀O₉N]⁺, 392.1915, Δ = - 1.2 ppm).

¹H NMR (CD₃OD, 600 MHz) δ (ppm): 6.71 (2H, s, H-2, H-6), 4.51 (1H, t, J = 7.2 Hz, H-7), 1.76 (2H, m, H-8), 0.94 (3H, d, J = 7.2 Hz, H-9), 4.85 (1H, d, J = 7.8 Hz, H-1′), 3.50 (1H, dd, J = 9.0, 7.8 Hz, H-2′), 3.43 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 3.44 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.22 (1H, m, H-5′), 3.68 (1H, dd, J = 12.0, 5.0 Hz, H_a-6′), 3.79 (1H, dd, J = 12.0, 5.0 Hz, H_b-6′), 3.89 (6H, s, 2 × OCH₃). ¹³C NMR (CD₃OD, 150 MHz) δ (ppm): 143.4 (C-1), 105.0 (C-2, C-6), 154.1 (C-3, C-5), 135.3 (C-4), 76.6 (C-7), 30.0 (C-8), 10.6 (C-9), 105.6 (C-1′), 75.8 (C-2′), 77.8 (C-3′), 71.4 (C-4′), 78.3 (C-5′), 62.6 (C-6′), 57.0 (2 × OCH₃).

Acid Hydrolysis of Compound 1

Compound 1 (1.5 mg) was treated with 2 N aqueous HCl (2 mL) in sealed flask at 90 °C for 2 h. The acidic aqueous mixture was dried, CHCl₃ (1 mL) was added, and the CHCl₃ solution was extracted with H₂O (1 mL). The aqueous fraction was evaporated to dryness to obtain the liberated sugar. Sugar samples, including the saccharide hydrolysis product of 1, D-glucose and L-glucose (Sigma Aldrich) were separately dissolved in 1 mL pyridine and heated with 2 mg L-cysteine methyl ester at 60 °C for 1 h, and then 2.5 μL O-tolyl isothiocyanate was added to the reaction mixture and further reacted at 60 °C for 1 h. Then, the reaction mixture was analyzed on a 250 × 4.6 mm i.d. Ultimate™ XB-C18 column (Welch Material, Inc.) at 35 °C with isocratic elution with 25% CH₃CN in 0.5% formic acid for 40 min at a flow rate of 0.8 mL/min, and detection with an UV detector (at 250 nm). Under these conditions, the standard sugars gave peaks at t_R (min) 22.0 and 23.0 for L-glucose and D-glucose, respectively. A peak at t_R (min) 23.0 (D-glucose) for 1 was observed.

Antioxidant TBARS assay^19,20

Thiobarbituric acid reactive substances (TBARS) assay values are usually reported in MDA equivalents, a compound formed from the decomposition of polyunsaturated fatty acid lipid peroxides. Briefly, a 0.1 mL sample at difference doses was added to 1 mL of mice brain homogenate (10%) and 0.8 mL phosphate buffer in the presence of 0.1 mL Fenton reagent (FeSO₄ 0.1 mM: H₂O₂ 15 mM at a ratio of 1:1). After incubating the mixture at 37 °C for 15 min, 1 mL of trichloroacetic acid 10% was added to each tube and the tubes were centrifuged at 12000 rpm for 5 min. The supernatant was then mixed with 1 mL of 0.8% thiobarbituric acid at a ratio of 2:1 and heated at 100 °C for 15 min. After cooling, the absorbance of the mixture was determined at 532 nm by using a microplate reader (BioRad). The percentage inhibition was calculated by the following formula:

% Inhibition of lipid peroxidation = [\frac{(O D_{control} – O D_{sample})}{O D_{control}}] \times 100

Conclusions

Phytochemical study of the methanol extract of P tonkinensis roots led to the isolation of a new phenyl propane, (7S)-2,6-dimethoxyphenyl-7,9-propanediol-1-O-β-D-glucopyranoside (1), and 9 known (2-10) compounds. The chemical structure of these compounds were determined by comprehensive analysis of HRESIMS and NMR spectral data, as well as by comparison of their spectral data with those reported in the literature. The stereochemistry of 1 was successfully determined by both theoretical and calculated CD spectra. Compounds 2-7 exhibited significant lipid peroxidation inhibitory effects in an in vitro antioxidant TBARS assay with IC₅₀ values of 21.3 ± 1.7, 61.9 ± 3.9, 57.5 ± 5.5, 10.4 ± 0.7, 28.9 ± 0.3, 54.2 ± 3.5 µM, respectively, compared to that of the positive control, trolox, (31.4 ± 2.2 µM).

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221088372 - Supplemental material for Lignans and Other Compounds From the Roots of Pandanus tonkinensis and Their Lipid Peroxidation Inhibitory Activity

Supplemental material, sj-docx-1-npx-10.1177_1934578X221088372 for Lignans and Other Compounds From the Roots of Pandanus tonkinensis and Their Lipid Peroxidation Inhibitory Activity by Dinh Thi Huyen Trang, Pham Hung Viet, Duong Hong Anh, Bui Huu Tai, Ngo Quoc Anh, Nguyen Xuan Nhiem and Phan Van Kiem in Natural Product Communications

Footnotes

Acknowledgments

The authors would like to thank Dr Do Thi Xuyen, Department of Botanic, Faculty of Biology, VNU University of Science for the identification of the plant sample, and Dr Ngo Duc Phuong, Institute of Vietnamese Medicinal Plants, Ministry of Health for collecting the plant sample.

Author Contribution

Research idea: PH Viet, DH Anh, NX Nhiem, PV Kiem. Isolation: DTH Trang. Structure elucidation and writing: BH Tai, DTH Trang, DH Anh, PV Kiem.

Declaration of Conflicting Interests

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

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Department of Science and Technology of Ho Chi Minh City.

ORCID iD

Phan Van Kiem

Supplemental material

Supplemental material for this article is available online.

List of Abbreviations

References

Chi

. Dictionary of Medicinal Plants in Vietnam. Medical Publishing House; 2012: 820-840.

Bich

Chung

Chuong

, et al. The Medicinal Plants and Animals in Vietnam. vol 1. Sci. Technol. Publ. House Hanoi; 2004: 700-701.

Ishikawa

Kondo

Kitajima

. Water-soluble constituents of coriander. Chem Pharm Bull. 2003;51(1):32-39. Doi:10.1248/cpb.51.32

Tanaka

Nakashima

Ueda

Momii

Kouno

. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem Pharm Bull. 2007;55(6):899-901. Doi:10.1248/cpb.55.899

Fernández-Marín

Guerrero

García-Parrilla

, et al. Isorhapontigenin: a novel bioactive stilbene from wine grapes. Food Chem. 2012;135(3):1353-1359. Doi: 10.1016/j.foodchem.2012.05.086

Schumacher

Scholle

Holzl

Khudeir

Hess

Muller

. Lignans isolated from valerian: identification and characterization of a new olivil derivative with partial agonistic activity at A1 adenosine receptors. J Nat Prod. 2002;65(10):1479-1485. Doi: 10.1021/np010464q

Sugiyama

Kikuchi

. Studies on constituents of Osmanthus species. VII. Structures of lignan glycosides from the leaves of Osmanthus asiaticus Nakai. Chem Pharm Bull. 1991;39(2):483-485. Doi:10.1248/cpb.39.483

Cha

Kim

Lee

Subedi

Kim

Lee

. Phytochemical constituents of Capsella bursa-pastoris and their anti-inflammatory activity. Nat Prod Sci. 2018;24(2):132-138. Doi:10.20307/nps.2018.24.2.132

Kobayashi

Karasawa

Miyase

Fukushima

. Studies on the constituents of Cistanchis herba. V. Isolation and structures of two new phenylpropanoid glycosides, cistanosides E and F. Chem Pharm Bull. 1985;34(4):1452-1457. Doi: 10.1248/cpb.32.3009

10.

Deyama

Ikawa

Nishibe

. The constituents of Eucommia ulmoides Oliv. II. Isolation and structures of three new lignan glycosides. Chem Pharm Bull. 1985;33(9):3651-3657. Doi: 10.1248/cpb.33.3651

11.

Shen

Hsieht

Kuo

. Neolignan glycosides from Jasminum urophyllum. Phytochemistry. 1998;48(4):719-723. Doi:10.1016/s0031-9422(98)00032-6

12.

Kuang

Xia

Yang

Wang

. Lignan constituents from Chloranthus japonicus Sieb. Arch Pharm Res. 2009;32(3):329-334. doi:10.1007/s12272-009-1303-1

13.

Kawahara

Fujii

Kata

Ida

Akita

. Chemoenzymatic synthesis of naturally occurring benzyl 6-O-glycosyl-β-D-glucopyranosides. Chem Pharm Bull. 2005;53(8):1058-1061. doi: 10.1248/cpb.53.1058

14.

Yamano

Ito

. Synthesis of optically active vomifoliol and roseoside stereoisomers. Chem Pharm Bull. 2005;53(5):541-546. doi: 10.1248/cpb.53.541

15.

Maeda

Masuda

Tokoroyama

. Studies on the preparation of bioactive lignan by oxidative coupling reaction. I. Preparation and lipid peroxidation inhibitory effects of benzofuran lignans related to shizotenuins. Chem Pharm Bull. 1994;42(12):2500-2505. Doi: 10.1248/cpb.42.2500

16.

Rezende

Davino

Barros

SBM

Kato

. Antioxidant activity of aryltetralone lignans and derivatives from Virola sebifera (Aubl.). Nat Prod Res. 2005;19(7):661-666. Doi: 10.1080/14786410412331302118

17.

Barker

. Lignans. Molecules. 2019;24(7):1424. Doi: 10.3390/molecules24071424

18.

Ambriz-Pérez

Leyva-López

Gutierrez-Grijalva

Heredia

. Phenolic compounds: natural alternative in inflammation treatment. A review. Cogent Food Agric. 2016;2:1131412. Doi: 10.1080/23311932.2015.1131412

19.

Badmus

Adedosu

Fatoki

, et al. Lipid peroxidation inhibition and antitradical activities of some leaf fractions of Manggifera indica. Acta Polon Pharm. 2011;68(1):23-29.

20.

Giang

Thao

Nga

Trung

Anh

Viet

. Evaluation of the antioxidant, hepatoprotective, and anti-inflammatory activities of bisresorcinol isolated from the trunk of Heliciopsis terminalis. Pharm Chem J. 2019;53(7):628-634. Doi:10.1007/s11094-019-02051-7

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