Phenolic Constituents of Elsholtzia blanda Benth

Abstract

Objectives: Elsholtzia blanda Benth. is a Vietnamese medicinal plant, used to treat inflammation, cough, and flu. Therefore, this current study focused on the isolation of the active ingredients from this plant and their NO production inhibitory activity. Methods: E. blanda aerial parts were dried, minced, and ultrasonically extracted with methanol and further separated by various chromatographic methods. The chemical structures of isolates were revealed by nuclear magnetic resonance, high resolution lectrospray ionization mass spectrometry, and circular dichroism spectra. The nitric oxide inhibition effects were evaluated in lipopolysaccharide-activated RAW264.7 cells. Results: Ten phenolic components [1 new (1) and 9 known (2-10)] were isolated and their chemical structures determined. Compound 5 (innovanoside, IC₅₀= 19.23 ± 1.43 µM) and 6 (maltol-O-β-D-glucopyranoside, IC₅₀ = 21.73 ± 1.27 µM) showed the highest activity, and 2-4 showed moderate inhibition (IC₅₀ = 47.36 ∼ 57.29 µM). The other compounds were inactive with IC₅₀ values >100 µM. Conclusions: One previously undescribed and 9 known phenolic components isolated from E. blanda have been screened for their nitric oxide inhibitory activity. Compounds 2-6 showed moderate to significant inhibition with IC₅₀ values of 19.23∼57.29 µM.

Introduction

The genus Elsholtzia Willd. (Lamiaceae) contains 44 species, which are widely distributed in East Asia, in Vietnam, China, and Korea.^1,2 In Vietnam, there are 7 species: Elsholtzia winitiana Craib., Elsholtzia ciliata (Thunb.) Hyl., Elsholtzia blanda Benth., Elsholtzia rugulosa Hemsl., Elsholtzia pilosa Benth., Elsholtziacommunis (Collett & Hemsl.) Diels, and Elsholtzia penduliflora W.W.Sm. ³ Acording to The World Flora Online, E. blanda has 2 synonyms, Aphanochilus blandus Benth. and Perilla elata D.Don. ³ In folk medicines, E. blanda has been used for treating nephritis, and has antiseptic and pain-relieving effects.^1,2 Previous papers reported that phenolics are the main chemical constituents of E. blanda and some of these exhibited significantly NO inhibition effects.^3–7 This article reports 10 phenolics (1-10) isolated from E. blanda (1 was a previously undescribed compound), and their effects on NO production in lipopolysaccharide (LPS)-activated RAW264.7 cells.

Results and Discussion

The methanol extract of dried E. blanda aerial parts was fractionated by successive chromatographic steps to yield 1 previously undescribed phenolic derivative (1) and 9 known compounds (2-10) (Figure 1). The infrared (IR) spectrum of 1 exhibited absorption bands at 1073 (C-O-C), 1652 (C = C), 1704 (C = O), and 3380 cm⁻¹ (OH). The high resolution electrospray ionization mass spectrometry (HR-ESI-MS) of 1 exhibited peaks at m/z 559.1588 [M + ³⁵Cl]⁻ (calcd. for [C₂₅H₃₂O₁₂³⁵Cl]⁻: 559.1587, Δ = +0.18 ppm) and m/z 561.1598 [M + ³⁷Cl]⁻ (calcd. for [C₂₅H₃₂O₁₂³⁷Cl]⁻: 561.1558, Δ = +7.0 ppm), indicating a molecular formula of C₂₅H₃₂O₁₂. Compound 1 had 25 carbons, including 12 of 2 aromatic rings, 6 of 1 glucose moiety, an acetyl, 1 methylene bearing oxygen atom, 2 methoxy, and 2 oxygenated methine groups, as identified by ¹³C nuclear magnetic resonance (NMR) and HSQC (Heteronuclear Single Quantum Coherence) spectra (Table 1). The ¹H NMR spectrum of 1 showed 2 ABX-type couplings of 2 aromatic rings [δ_H 7.13 (H-2, d, J = 1.8 Hz), 6.69 (H-5, d, J = 8.0 Hz), 6.90 (H-6, dd, J = 8.0, 1.8 Hz)] and [δ_H 7.46 (H-2ʹ, d, J = 1.8 Hz), 6.98 (H-5ʹ, d, J = 8.0 Hz), 7.52 (H-6ʹ, dd, J = 8.0, 1.8 Hz)], 1 anomeric proton (δ_H 4.16, d, J = 7.8 Hz), 2 methoxy groups (δ_H 3.80 and 3.81, each 3H, s), an acetyl (δ_H 2.53), 1 oxygenated methylene group (δ_H 3.93), and 2 oxygenated methine groups [δ_H 5.09 (H-7, d, J = 7.2 Hz) and 4.71 (H-8, m)]. The NMR data of 1 were very similar to those of lycocernuaside D, except for the additional signals of a glucose moiety. ⁸ In the Heteronuclear Multiple Bond Correlation (HMBC) spectrum of 1, an anomeric proton H-1″ (δ_H 4.16) correlated with C-7 (δ_C 77.9), H-7 (δ_H 5.09) correlated with C-1 (δ_C 129.7)/C-2 (δ_C 111.5)/C-3 (δ_C 148.8)/C-8 (δ_C 84.1)/C-9 (δ_C 60.7), and methoxy protons (δ_H 3.80) correlated with C-3 (δ_C 148.8). These indicated that the glucose moiety was linked to C-7 by an ether linkage and the methoxy group attached to C-3. In addition, HMBC correlation from H-8 (δ_H 4.71) to C-4ʹ (δ_C 154.3), from methoxy protons (δ_H 3.81) to C-3ʹ (δ_C 151.2), from H-2ʹ (δ_H 7.46) and H-6ʹ (δ_H 7.52) to C-7ʹ (δ_C 199.4), and from H₃-8ʹ (δ_H 2.53) to C-7ʹ were observed confirming a C8-O-C4ʹ linkage, and OCH₃ group at C-3ʹ, and a CH₃CO- group attached to C-1ʹ, similar to that of lycocernuaside D (Figure 2). ⁸ The large values of J_7,8 = 7.2 Hz suggested a threo-configuration for compound 1, as in the cases of 7-O-ethylguaiacylglycerol, J_7,8 = 6.6 Hz for threo and J_7,8 = 5.0 Hz for the erythro-isomer. ⁹ The absolute configuration of compound 1 was determined from the circular dichroism (CD) spectrum (Figure 3). Compound 1 showed a Cotton effect at 240 nm (negative) in its CD spectrum confirming the 8R configuration.^10,11 The above evidence indicated the (7R,8R) configuration for 1. The β-form of the glucosyl linkage was deduced from the large value of J_{H−1ʹʹ/H−2ʹʹ} = 7.8 Hz. The presence of a D-glucose sugar in 1 was determined by acid hydrolysis. ¹² Thus, 1 was concluded to be (7R,8R)-7-O-β-D-glucopyranosyllycocernuaside D, a previously undescribed compound (Supplemental Figures S1-12).

Figure 1.

Structures of the isolates from Elsholtzia blanda.

Figure 2.

Key HMBC and COSY interactions of 1. Abbreviations: COSY, COrrelation SpectroscopY; HMBC, Heteronuclear Multiple Bond Correlation.

Figure 3.

Cd spectrum of 1.

Table 1.

¹H and ¹³C NMR Spectroscopic Data for 1. ^a

Pos.	δ C	δ H b	Pos.	δ C	δ H b
1	129.7	–	4′	154.3	–
2	111.5	7.13 (d, 1.8)	5′	116.1	6.98 (d, 8.0)
3	148.8	–	6′	124.3	7.52 (dd, 8.0, 1.8)
4	147.6	–	7′	199.4	–
5	115.4	6.69 (d, 8.0)	8′	26.3	2.53 (s)
6	119.5	6.90 (dd, 8.0, 1.8)	3′-OCH3	56.6
7	77.9	5.09 (d, 7.2)	Glc
8	84.1	4.71 (m)	1″	100.6	4.16 (d, 7.8)
9	60.7	3.93 (d, 4.8)	2″	75.2	3.27 (dd, 9.0, 7.8)
3-OCH3	56.3	3.80 (s)	3″	78.2	3.26 (dd, 9.6, 9.0)
1′	131.9	–	4″	71.6	3.30 (dd, 9.6, 9.0)
2′	112.6	7.46 (d, 1.8)	5″	78.0	78.0 (ddd, 9.0, 5.4, 1.8)
3′	151.2	–	6″	62.8	3.69 (dd, 11.4, 5.4) 3.88 (dd, 11.4, 1.8)

^a

CD₃OD.

^b

(mult., J in Hz).

Abbreviation: NMR, nuclear magnetic resonance; Pos, position; s, singlet; d, doublet; m, multiplet.

The other isolates were identified as maltol-6′-O-(5-O-p-coumaroyl)-(1→6)-β-D-apiofuranosyl-β-D-glucopyranoside (2), ¹³ hedyotoside B (3), ¹⁴ shimobashiraside B (4), ¹⁵ innovanoside (5), ¹⁶ maltol-O-β-D-glucopyranoside (6), ¹⁶ methyl ester of rosmarinic acid (7), ¹⁷ 8-hydroxypinoresinol 4′-O-β-D-glucoside (8), ¹⁸ 47,9-trihydroxy-3,3′-dimethoxy-8-O-4′-neolignan-9′-O-β-D-glucopyranoside (9), ¹⁹ and cyclo-olivil-9-O-β-glucopyranoside (10). ²⁰

As shown in previous studies, some plant phenolic secondary metabolites show significant NO production inhibition.^2,21 The leaves of E. blanda are used in folk medicine to cure some inflammatory diseases. ¹ Therefore, the isolates were screened for their NO production effects in LPS-activated RAW264.7 cells. All the isolates showed noncytotoxic activity (at a concentration of 100 µM, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay). Their inhibition effects were then evaluated (concentrations of 0.8, 4.0, 20, and 100 µM). Compounds 2-6 showed moderate to significant inhibition with IC₅₀ values ranging from 19.23 to 57.29 µM (Supplemental Table S1). These results suggested that compounds which have a maltol moiety show stronger NO production inhibition than the others. ²²

Material and Methods

General

The equipment and experimental procedures are shown in Supplemental materials.

The Plant Materials

E. blanda Benth. (aerial parts) was obtained in Melinh-Vinhphuc Province (1.2021), and identified by Dr Le Tuan Anh at Vietnam National Museum of Nature. The specimen (NCCT-P175) was kept at IMBC, VAST.

Extraction and Purification

This section is shown in Supplemental materials.

(7R,8R)-7-O-β-D-Glucopyranosyllycocernuaside D (1)

Colorless amorphous powder; $[\alpha {]}_D^{25}$ : +13.4 (c 0.05, MeOH); IR (KBr) ν_max 3380 (OH), 1704 (C = O), 1652, 1603 (C = C Ar), 1168 and 1073 (C-O-C) cm⁻¹; CD (MeOH, c 0.2 mg/mL) λ (θ: mdeg): 214 (−1.79), 240 (−1.32); HR-ESI-MS m/z 559.1588 [M + ³⁵Cl]⁻, calcd. for [C₂₅H₃₂O₁₂³⁵Cl]⁻: 559.1587 (Δ = +0.18 ppm); m/z 561.1598 [M + ³⁷Cl]⁻, calcd. for [C₂₅H₃₂O₁₂³⁷Cl]⁻: 561.1558 (Δ = +7.0 ppm). ¹³C NMR (125 MHz) and ¹H NMR (500 MHz) data (see Table 1).

For the NMR data of 2-5 see Supplemental Table S2, for the other compounds: see Supplemental materials.

Conclusions

Ten phenolic compounds (1-10) including a new one (1), have been isolated from E. blanda. The chemical structures of these compounds were elucidated from IR, HR-ESI-MS, 1-dimensional and 2-dimensional NMR spectra. The absolute configuration of 1 was determined from its CD spectrum. Compounds 5 (IC₅₀ 19.23) and 6 (IC₅₀ 21.73 µM) showed the highest NO inhibition. Compounds 2-4 showed moderate inhibition with IC₅₀ values of 47.36∼57.29 µM. The other isolates were inactive with IC₅₀ values over 100 µM. The above results suggest that the phenolic compounds which have the maltol moiety play an important role in the NO production inhibitory activity of E. blanda and further studies on the anti-inflammatory effects of these maltol derivatives should be continued.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231192213 - Supplemental material for Phenolic Constituents of Elsholtzia blanda Benth

Supplemental material, sj-docx-1-npx-10.1177_1934578X231192213 for Phenolic Constituents of Elsholtzia blanda Benth by Duong Thi Dung, Pham Hai Yen, Do Thi Trang, Bui Huu Tai and Phan Van Kiem in Natural Product Communications