Chemical Constituents from Memecylon Scutellatum with Nitric Oxide Inhibitory Activity

Abstract

Objectives: Memecylon scutellatum (Lour.) Hook. & Arn. (Melastomataceae) is a relatively lesser-known species within this genus. This study aims to explore its chemical structure profile and evaluate its ability to inhibit NO production. Methods: The chemical structures of MS were elucidated by comparing their NMR data, and all compounds were evaluated for NO inhibition using the Griess assay. Results: One new compound namely, memeloside (1) and fourteen known compounds, 1-O-(β-D-glucopyranosyl)-2-[2-methoxy-4-(ω-hydroxypropyl)-phenoxy]-propan-3-ol (2), (2R)-O-[4′-(3″-hydroxypropyl)-2′,6′dimethoxyphenyl]-3-O-β-D-glucopyranosyl-sn-glycerol (3), schizandriside (4), 2-butanone 4-glucopyranoside 6′-O-gallate (5), epicatechin (6), epigallocatechin (7), epigallocatechin gallate (8), myricitrin (9), benzyl O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (10), benzyl glucopyranoside (11), 3,4,5-trimethoxyphenyl-β-D-glucopyranoside (12), icariside B1 (13), gallic acid (14), and (7R,8R)-threo-7,9,9ʹ-trihydroxy-3,3ʹ-dimethoxy-8-O-4ʹ-neolignan 4-O-β-D-glucopyranoside (15) were isolated from the leaves of M. scutellatum. Compounds 4 and 5 significantly exhibited inhibition of NO production, with IC₅₀ values of 88.92 ± 1.42 µM and 17.14 ± 0.98 µM, respectively. Conclusions: One new compound, named memeloside (1) and fourteen known compounds isolated from the leaves of M. scutellatum. Compounds 4 and 5 demonstrated significant inhibition of NO production, with IC₅₀ values of 88.92 ± 1.42 µM and 17.14 ± 0.98 µM, respectively. These results suggested compound 5 could be a promising candidate for further anti-inflammatory research.

Introduction

Memecylon scutellatum (Lour.) Hook. & Arn. (Melastomataceae) is a lesser-known plant species indigenous to tropical and subtropical regions. This species has attracted scientific interest due to its potential medicinal properties, primarily driven by its traditional use in folk medicine across Southeast Asia. Ethnobotanical studies have documented its application in treating various ailments, including wounds, infections, and inflammation. ¹ Despite its extensive use in traditional medicine, there is a paucity of scientific literature that thoroughly examines the chemical composition and biological activities of M. scutellatum.

In recent years, the growing interest in natural remedies and the urgent need for new drugs to combat emerging diseases have intensified the search for plants with significant bioactive properties. Memecylon species reported to possess potential pharmacological effects. ² Previous studies on M. malabaricum, ³ M. umbellatum, ⁴ and M. edule ⁵ have identified compounds with antidiabetic, antimicrobial, and anti-inflammatory activities. However, chemical components and biological investigations of M. scutellatum have not been studied yet. Herein, we report the chemical constituents and anti-inflammatory activity of M. scutellatum.

Materials and Methods

General Experimental Procedures

All NMR spectra were recorded on a Bruker 600 MHz (Bruker, Massachusetts, United States). High-resolution mass data were obtained from Korea Basic Science Institute (KBSI, Chuncheon Center). Column chromatography (CC) was performed on silica-gel (Kieselgel 60, 230-400 mesh, Merck, Darmstadt, Germany), RP-18 resins (30-50 μm, Fuji Silysia Chemical Ltd, Kasugai, Japan) or HP-20 diaion (Mitsubishi Chemical Corporation, Tokyo, Japan). For thin-layer chromatography (TLC), pre-coated silica-gel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) and RP-18 F254S (0.25 mm, Merck, Darmstadt, Germany) plates were used. HPLC was carried out using an AGILENT 1100 HPLC system using J'sphere H-80 column (250 × 20 mm), a flow rate of 3.0 mL, and a DAD detector.

Plant Material

The leaves of M. scutellatum were collected at Phuc Yen, Vinh Phuc, Vietnam in September 2022 and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (USTH-MS01) was deposited at the Institute of Ecology and Biological Resources, VAST.

Extraction and Isolation

The dried powder of M. scutellatum leaves (9.5 kg) was sonicated with methanol (15 L MeOH at 45 °C, 2 times, each 2 h) and then filtered through filter paper, removed solvent under reduced pressure to yield 950 g of MeOH extract. The MeOH extract was suspended in water and successively partitioned with n-hexane and ethyl acetate, giving n-hexane (MS1, 45.1 g), ethyl acetate extracts (MS2, 66.6 g), and water layer (MS3).

The water layer (MS3) was chromatographed on a Diaion HP-20 column chromatography (CC) eluting with water to remove polar components, then increase the concentration of MeOH in water (25, 50, 75% and 100% MeOH, each 1 L) to obtain four fractions, MS3A (59.2 g), MS3B (25.3 g), MS3C (23.5 g), and MS3D (2.0 g). MS3A was chromatographed on a silica gel CC eluting with gradient solvents of dichloromethane/MeOH (20/1, 10/1, 5/1, 2.5/1, v/v) to give four fractions, MS3A1–MS3A4. MS3A1 was chromatographed on a silica gel CC eluting with CH₂Cl₂/MeOH (7/1, v/v) to give four fractions, MS3A1A-MS3A1D. MS3A1A was chromatographed on an RP-18 CC eluting with MeOH/water (1/3, v/v) to obtain two fractions, MS3A1A1 (80.0 mg) and MS3A1A2 (80.0 mg). MS3A1A1 was purified by an HPLC using 18% MeCN in water to yield 11 (5.0 mg, t _R 27.4 min). MS3A1A2 was purified by an HPLC using 15% MeCN in water to yield compounds 5 (7.0 mg, t _R 51.6 min) and 10 (25.0 mg, t _R 44.1 min). MS3A1C was purified by an HPLC using 15% MeCN in water to yield compound 1 (5.0 mg, t _R 36.4 min). MS3A1D was chromatographed on an RP-18 CC eluting with MeOH/water (1/3, v/v) to obtain compound 14 (15.0 mg) and two fractions, MS3A1D1 (30.0 mg) and MS3A1D2 (70.0 mg). MS3A1D1 was purified by an HPLC using 13% MeCN in water to yield 13 (8.0 mg, t _R 22.0 min). MS3A1D2 was purified by an HPLC using 13% MeCN in water to yield 4 (12.5 mg, t _R 29.2 min). MS3A2 was chromatographed on a silica gel CC eluting with CH₂Cl₂/acetone/water (1/2/0.1, v/v/v) to give five fractions, MS3A2A (320 mg), MS3A2B (900 mg), MS3A2C (910 mg), MS3A2D (1.5 g), and MS3A2E (2.3 g).MS3A2A was chromatographed on an RP-18 CC eluting with MeOH/water (1/2, v/v) to obtain two fractions, MS3A2A1 (180.0 mg) and MS3A2A2 (50.0 mg). MS3A1A1 and MS3A2A2 were chromatographed on an HPLC eluting with 20% MeCN in water to yield 6 (11.0 mg, t _R 19.6 min) and 7 (8.0 mg, t _R 30.1 min), respectively. MS3A2B was chromatographed on a Sephadex CC eluting with MeOH/water (9/1, v/v) and then on an HPLC eluting with 15% MeCN in water to yield 8 (18.0 mg, t _R 28.0 min). MS3A2C was chromatographed on an RP-18 CC eluting with MeOH/water (1/2, v/v) and then on an HPLC eluting with 20% MeCN in water to yield 12 (6.0 mg, t _R 30.1 min). MS3A2D was chromatographed on an RP-18 CC eluting with MeOH/water (1/2, v/v) and then on an HPLC eluting with 15% MeCN in water to yield 9 (13.0 mg, t _R 44.9 min). MS3A2E was chromatographed on an RP-18 CC eluting with MeOH/water (1/2, v/v) to obtain three fractions, MS3A2E1-MS3A2E3. MS3A2E1 was chromatographed on a silica gel CC eluting with CH₂Cl₂/MeOH/water (4.5/1/0.1, v/v/v), then on an HPLC eluting with 15% MeCN in water to yield 3 (5.2 mg, t _R 35.9 min). MS3A2E2 was purified by an HPLC using 13% MeCN in water to yield 15 (5.0 mg, t _R 39.1 min). MS3A2E3 was purified by an HPLC using 15% MeCN in water to yield 2 (3.0 mg, t _R 37.3 min).

2-Hydroxy-6-Methylhept-5-en-3-yl) 3-O-[β-D-Apiofuranosyl-(1′′→6′)]-β-D-Glucopyranoside (1)

White amorphous powder; $[\alpha {]}_D^{25}$ ‒46.0 (c 0.1, MeOH); C₂₇H₃₀O₁₂; HR-ESI-MS m/z: 461.1980 [M + Na]⁺ (Calcd. for [C₁₉H₃₄O₁₁Na]⁺, 461.1993); ¹H-NMR (CD₃OD, 600 MHz) δ_H: 1.19 (d, J = 6.6 Hz, H-1), 3.74 (m, H-2), 3.44 (m, H-3), 2.29 (m, H_a-4), 2.42 (m, H_b-4),5.30 (t, J = 7.2 Hz, H-5),1.72 (s, H-7),1.66 (s, H-8), 4.40 (d, J = 7.8 Hz, H-1ʹ), 3.25 (dd, J = 7.8, 9.0 Hz, H-2ʹ), 3.37 (t, J = 9.0 Hz, H-3ʹ), 3.32 (m, H-4ʹ), 3.39 (m, H-5ʹ), 3.63 (dd, J = 6.0, 12.0 Hz, H_a-6ʹ), 3.88 (dd, J = 1.8, 12.0 Hz, H_b-6ʹ), 5.01 (d, J = 2.4 Hz, H-1ʹʹ), 3.92 (d, J = 2.4 Hz, H-2ʹʹ), 3.78 (d, J = 9.6 Hz, H_a-4ʹʹ), 3.97 (d, J = 9.6 Hz, H_b-4ʹʹ), and 3.66 (s, H-5ʹʹ); ¹³C-NMR (CD₃OD, 150 MHz) δ_C: 19.3 (C-1), 69.9 (C-2), 86.5 (C-3), 31.8 (C-4), 121.1 (C-5), 134.1 (C-6), 26.0 (C-7), 18.2 (C-8), 105.7 C-1ʹ), 75.6 (C-2ʹ), 77.9 (C-3ʹ), 71.6 (C-4ʹ), 76.8 (C-5ʹ), 68.5 (C-6ʹ), 110.9 (C-1ʹʹ), 78.0 (C-2ʹʹ), 80.6 (C-3ʹʹ), 75.0 (C-4ʹʹ), and 65.8 (C-5ʹʹ).

Acid Hydrolysis

Compound 1 (2.0 mg) was dissolved in 1.0 N HCl (dioxane−H₂O, 1:1, v/v, 1.0 mL) and then heated to 80 °C in a water bath for 3 h. The acidic solution was neutralized with silver carbonate and the solvent thoroughly driven out under N₂ gas overnight. After extraction with CHCl₃, the aqueous layer was concentrated to dryness using N₂ gas. The residue was dissolved in 0.1 mL of dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL) was added to the solution. The reaction mixture was heated at 60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was added, followed by heating at 60 °C for 1.5 h. The dried product was partitioned with n-hexane and H₂O (0.1 mL, each), and the organic layer was analyzed by GC: Column: column of SPB-1 (0.25 mm × 30 m); detector FID, column temp 210 °C, injector temp 270 °C, detector temp 300 °C, carrier gas He (2.0 mL/min). The retention times of persilylated glucose and apiose were founded to be 14.11 and 6.70 min, respectively, when compared with the standard solutions prepared by the same reaction from the standard monosaccharides. The retention times of persilylated D-glucose, L-glucose, D-apiose, and L-apiose rhamnose were 14.11, 14.26, 6.70 and 6.95 min, respectively.

Cell Culture

Murine macrohage RAW 264.7 cells were provided by Professor Lee Jeong Hyung, Kangwon National University, Gangwo-do, Korea. Cells were cultured at 37 °C in a humidified 5% CO₂ incubator in DMEM (Gibco, Invitrogen, USA), 10% FBS, 1% penicillin/streptomycin solution.

Cell Viability

Cytotoxicity of compounds was determined by MTT assay.^6,7 Cells were grown in 96-well plates (50,000 cells/ 200 μL/ well). Cells were incubated in 24 h (37 °C, 5% CO₂) before treated with the MTT solution (0.5 mg/mL) in 4 h. The formazan crystals formed were dissolved with isopropanol. Absorbance was measured at 570 nm using a Biotek microplate reader (Biotek Instruments, USA).

Estimation of Nitric Oxide Level

Murine macrophage RAW 264.7 cells were grown in 96-well plates at a concentration of 50,000 cells/200 μl/well. After an incubation in a humidified incubator at 37 °C, 5% CO₂ for 24 h, cells were pre-activated for 30 min with compounds, then added 1 µg/mL lipopolysaccharide (Escherichia coli 0111: B4; Sigma Aldrich, USA). Cells were incubated for 24 h, and nitric oxide level was evaluated by Griess assay with a ratio of 1:1 (v/v) and calculated by standard curve of NaNO₂. Optical density was read at 540 nm wavelength by Biotek microplate reader (Biotek Instruments, USA).

Statistical Analysis

The results are presented as mean ± SD of three independent experiments. Statistical significance between multiple groups was evaluated using ANOVA, followed by the Dunnett method for comparisons. P value of < 0.05 was considered statistically significant.

Results and Discussion

Dried leaves of M. scutellatum were extracted with MeOH and fractionated with dichloromethane, ethyl acetate (EtOAc), and water. From the water layer and subsequently separation, one new and fourteen known compounds were isolated.

Compound 1 was obtained as a white amorphous powder and its molecular formula was determined to be C₁₉H₃₄O₁₁ by the high-resolution electrospray ionization mass spectrometry (HR EI MS) at m/z 461.1980 [M + Na]⁺ (Calcd. for [C₁₉H₃₄O₁₁Na]⁺, 461.1993). The ¹H-NMR spectrum of 1 (methanol-d₄) showed signals for two methyl groups at δ_H 1.66 (3H, s) and 1.72 (3H, s), suggesting the presence of a prenyl group, one secondary methyl group at δ_H 1.19 (d, J = 6.6 Hz), and two anomeric protons at δ_H 4.40 (1H, d, J = 7.8 Hz) and 5.01 (1H, d, J = 2.4 Hz) suggesting the presence of two sugar units. The ¹³C-NMR and HSQC spectra revealed signals of 19 carbons, 8 of which were assigned to isooctane-1-ene diol aglycone and 11 assigned to the two monosaccharide moieties. HMBC correlations between H-1 (δ_H 1.19) and C-2 (δ_C 69.9)/C-3 (δ_C 86.5), between H-7 (δ_H 1.72)/H-8 (δ_H 1.66) and C-5 (δ_C 121.1)/C-6 (δ_C 134.1), and between H-5 (δ_H 5.30) and C-3 (δ_C 86.5)/C-4 (δ_C 31.8)/C-6 (δ_C 134.1)/C-7 (δ_C 26.0)/C-8 (δ_C 18.2) and also COSY correlations of H-1 (δ_H 1.19)/H-2 (δ_H 3.74)/H-3 (δ_H 3.44)/H-4 (δ_H 2.29 and 2.42)/H-5 (δ_H 5.30) indicated the presence of 6-methylhept-5-ene-2,3-diol. The ¹³C-NMR spectral data of 1 showed the presence of one β-apiofuranosyl moiety and one β-glucopyranosyl moiety. ⁸ In addition, acid hydrolysis of 1 revealed D-apiose and D-glucose as sugar components (identified as trimethylsilyl (TMS) derivatives by GC method. The HMBC correlations between H-1′ (δ 4.40) and C-3 (δ_C 86.5), between H-1′′ (δ_C 5.01) and C-6′ (δ_C 68.5), between H-6′ (δ 3.63 and 3.88) and C-1′′ (δ 110.9) were observed (see Figure 1). These observations suggested the sequence of sugar linkages of 1 and the position of the sugar moiety at C-3. Compound 1 was obtained as small amount (5.0 mg). Thus, the absolute configuration for aglycone has been unidentified yet. Consequently, the structure of 1 was determined to be 2-hydroxy-6-methylhept-5-en-3-yl) 3-O-[β-D-apiofuranosyl-(1′′→6′)]-β-D-glucopyranoside, a new compound named memeloside.

Figure 1.

The key HMBC and COSY correlations of compound 1.

The known compounds were identified to be 1-O-(β-D-glucopyranosyl)-2-[2-methoxy-4-(ω-hydroxypropyl)-phenoxy]-propan-3-ol (2), ⁹ (2R)-O-[4′-(3″-hydroxypropyl)-2′,6′dimethoxyphenyl]-3-O-β-D-glucopyranosyl-sn-glycerol (3), ¹⁰ schizandriside (4), ¹¹ 2-butanone 4-glucopyranoside 6′-O-gallate (5), ¹² epicatechin (6), ¹³ epigallocatechin (7), ¹³ epigallocatechin gallate (8), ¹³ myricitrin (9), ¹⁴ benzyl O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (10), ¹⁵ benzyl glucopyranoside (11), ¹⁶ 3,4,5-trimethoxyphenyl-β-D-glucopyranoside (12), ¹⁷ icariside B1 (13), ¹⁸ gallic acid (14), ¹⁷ and (7R,8R)-threo-7,9,9ʹ-trihydroxy-3,3ʹ-dimethoxy-8-O-4ʹ-neolignan 4-O-β-D-glucopyranoside (15) ¹⁹ (Figure 2). Their chemical structures were elucidated by comparison of their NMR data with those reported in the literatures.

Figure 2.

The chemical structure of compound 1.

Nitric oxide (NO) is a signaling molecule that plays a crucial role in the development of inflammation and is recognized as a pro-inflammatory mediator. NO inhibitors represent a significant therapeutic advancement in the treatment of inflammatory diseases. ²⁰ To assess the cytotoxic effects of all isolated compounds, we conducted the MTT assay. The results showed that compounds 3, 4, 7, 8, 10, 12, 13, and 15 induced more than 30% cell death and significantly reduced cell viability compared to the control at a concentration of 100 µM. Moreover, Table 1 indicated that compounds 4 and 5 inhibited more than 50% NO production. As a result, compounds 4 and 5 were selected for further evaluation of inhibitory NO production in a dose-dependent manner. As depicted in Figure 3, the IC₅₀ values for compounds 4 and 5 were 88.92 ± 1.42 µM and 17.14 ± 0.98 µM, respectively. Notable, compound 5 exhibits a moderate effect on NO inhibition, in comparison to cardamonin, positive control, ²¹ which has an IC₅₀ value of 1.17 ± 0.15 µM. A study reported that a concentration of 528.61 ± 1.59 μg/mL of M. umbellatum Burm.f leaf methanolic extract scavenged 50% of the nitric oxide generated during incubation. ²² In comparison, the IC₅₀ value of M. scutellatum methanolic extract for NO production inhibition was found to be 66.99 ± 1.35 μg/mL (unpublish data), indicating its potential anti-inflammatory effects. The limitation of the study is the preliminary evaluation of anti-inflammatory activity via NO production, requiring further investigation in the future.

Figure 3.

Effect of compounds 4 and 5 on the NO production.

Table 1.

Inhibitory Effect of NO Production of Compounds 1-15 in LPS-Induced RAW 264.7 Macrophages.

	% NO inhibition		Cell viability
Compounds	30 µM	100 µM	30 µM	100 µM
1	4.6 ± 0.4	7.45 ± 0.9	98.6 ± 1.1	95.9 ± 1.7
2	20.8 ± 1.5	44.7 ± 0.6	88.6 ± 1.6	76.8 ± 1.2***
3	16.1 ± 0.7	38.0 ± 0.2	88.6 ± 1.6	66.4 ± 0.7***
4	7.8 ± 1.0	55.2 ± 0.2***	80.6 ± 1.1	60.2 ± 0.9***
5	66.4 ± 0.4***	91.9 ± 0.3***	94.5 ± 1.3	86.3 ± 1.2
6	15.3 ± 1.0	37.2 ± 0.7	92.7 ± 1.2	76.9 ± 1.5***
7	16.4 ± 0.6	38.2 ± 1.2	73.1 ± 0.9***	69.2 ± 0.6***
8	10.6 ± 0.5	12.5 ± 0.7	68.7 ± 0.5***	66.8 ± 0.2***
9	14.3 ± 1.9	16.1 ± 0.4	96.6 ± 1.6	88.9 ± 1.8
10	1.3 ± 1.7	35.4 ± 1.7	88.5 ± 1.5	66.6 ± 1.1***
11	15.6 ± 0.7	33.8 ± 1.0	84.7 ± 1.6	78.5 ± 1.9***
12	13.2 ± 0.6	40.1 ± 0.5	75.4 ± 0.3***	63.9 ± 1.1***
13	24.7 ± 0.2	37.5 ± 0.9	77.1 ± 1.5***	68.9 ± 1.5***
14	5.7 ± 0.4	28.9 ± 0.6	83.5 ± 0.5	72.1 ± 0.5***
15	17.7 ± 0.5	27.1 ± 0.3	78.0 ± 0.6***	69.6 ± 1.3***
Cardamonin a	52.0 ± 0.5***		80.0 ± 0.2

^a

Cardamonin was used as positive control, showed NO inhibition of 52.0 ± 0.5% at a concentration of 3 μM. Each bar show mean ± SD of three independent experiments performed in triplicate (***P < 0.001 compared LPS). Values are shown as means ± SD (n = 3).

Conclusions

In our study, we identified one new compound, named memeloside (1) along with fourteen known compounds isolated from the leaves of M. scutellatum. Among these, compounds 4 and 5 demonstrated significant inhibition of NO production, with IC₅₀ values of 88.92 ± 1.42 µM and 17.14 ± 0.98 µM, respectively. These results indicated that compound 5 may be a potential candidate for further anti-inflammatory investigation.