Chemical Constituents of Callicarpa giraldii and Their Anti-Inflammatory Activity

Abstract

Callicarpa giraldii (family Verbenaceae) has many medical properties and is commonly used in traditional Chinese medicine. However, its chemical constituents have not been investigated. In this study, nine compounds present in the leaves and stems were isolated and characterized. These were negundonorin B (1), lupeol (2), erythrodiol (3), isoacteoside (4), forsythoside B (5), dibutyl phthalate (6), verbascoside (7), poliumoside (8), and 3-methoxy-4-hydroxybenzaldehyde (9). The structures were identified by comparison of their NMR data with those reported in the literature. The anti-inflammatory effects of compounds 1-6 were tested on lipopolysaccharide-stimulated RAW264.7 macrophages. Several compounds showed significant (p < .05) inhibitory effects.

Introduction

Callicarpa giraldii Hesse e × Rehd., family Verbenaceae, is indigenous to the Chinese mainland, but found primarily in southern China. It is a shrub of 3 m height with broadly elliptic to oblong-lanceolate leaves and grows in sparse forests at an altitude of 200 to 3400 m.^1,2 The plant has been well documented to be effective in traditional medicine for the treatment of traumatic injuries, inflammation, and bleeding syndromes. ³ Callicarpa species are rich in terpenoids, volatile oils, flavonoids, and phenylethanoid glycosides, and hence are pharmacologically important.^4,5 However, to the best of our knowledge, the constituents of C giraldii have not been investigated. As a part of our ongoing research on anti-inflammatory compounds isolated from Callicarpa, ⁶ nine known compounds have been isolated from the methanol extracts of the leaves and stems of C giraldii by various chromatographic methods. The structures were identified by comparing their spectroscopic data with those reported in the literature. The anti-inflammatory activity of the compounds (1-6) was detected by measuring nitric oxide (NO) release in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages using the Griess test (Figure 1).

Figure 1.

Structures of compounds 1 to 9.

Results and Discussion

The leaves and stems of C giraldii were extracted with methanol; the dry extract was suspended in H₂O and fractionated successively with light petroleum (PE), ethyl acetate, and n-butanol. The residual H₂O and PE soluble fractions were further purified by silica gel, Sephadex LH-20, and MCI gel column chromatography, and by semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC). Nine known compounds were obtained, which were identified by comparing their spectroscopic data with those reported in the literature. The compounds were negundonorin B (1), ⁷ lupeol (2), ⁸ erythrodiol (3), ⁹ isoacteoside (4), ¹⁰ forsythoside B (5), ¹¹ dibutyl phthalate (6), ¹² verbascoside (7), ¹³ poliumoside (8), ¹⁴ and 3-methoxy-4-hydroxybenzaldehyde (9). ¹⁵

Effects of Compounds 1 to 6 on NO Level

Since C giraldii has been traditionally used as a hemostatic and anti-inflammatory medicinal plant, ³ compounds 1 to 6 were tested for their anti-inflammatory effect. In order to avoid false-positive results, CCK8 kits were used to measure the effect of these compounds on cell viability (Supplemental material Figure 19). The dimethyl sulfoxide content was less than 0.02%. Most compounds at concentrations less than 100 µmol·L⁻¹ showed no obvious cytotoxicity, except for compound 1. Compound 3 significantly (p < .05) inhibited the production of NO at a relatively low concentration (25 µmol·L⁻¹). The effect of compound 6 at 200 µmol·L⁻¹ was almost comparable to that of dexamethasone (DEX) at 0.5 µmol·L⁻¹, which was selected as the positive control (p = .8542). The inhibitory effects of the compounds are shown in Figure 2.

Figure 2.

Effect of compounds 1 − 6 on nitric oxide inhibition in LPS-stimulated RAW264.7 cells; the values are expressed as mean ± standard deviation, n = 5, **p < .01, ***p < .001 versus the group treated with LPS alone.

Of the compounds isolated, erythrodiol (3) has been shown to be a good candidate for the treatment and prevention of atherosclerosis. ¹⁶ Shen et al ¹⁷ investigated the biotransformation of this compound by various microorganisms, and most of the metabolites showed good anti-inflammatory effects with lower toxicity. This suggests that erythrodiol might be considered as a potential anti-inflammatory drug. Although the effect of compound 6 is similar to that of 0.5 µmol·L⁻¹ DEX (p = .8542), its effective concentration is as high as 200 µmol·L⁻¹. In recent years, reports on the anti-inflammatory effect of Callicarpa species have gradually increased. Wu et al ¹⁸ investigated the anti-inflammatory pharmacodynamic basis of several phenylethanoid glycosides isolated from Callicarpa kwangtungensis, which revealed obvious inhibitory effects on inflammatory factors tumor necrosis factor-α (TNF-α), IL-6, NO, and the generation of reactive oxygen species (ROS) in RAW 264.7 macrophages. Sun et al ¹⁹ also found that the total phenylethanoid glycosides of C kwangtungensis can suppress the Na⁺-K⁺-ATPase/Src/ERK1/2 pathway and inhibit apoptosis mediated by oxidative stress and inflammation to protect cardiomyocytes from myocardial injury. In general, the development of anti-inflammatory drugs from Callicarpa species is worthy of further exploration.

Experimental Section

General Experimental Procedures

Nuclear magnetic resonance (NMR) spectroscopy was performed using a Bruker AVANCE 500 spectrophotometer (Bruker Daltonics). High resolution mass spectrum (HR-MS) data were recorded using Bruker maXis impact liquid chromatograph mass spectrometer (LC-MS) (Bruker Daltonics). Silica gel (Qingdao Haiyang Chem. Co.), MCI gel (Mitsubishi Chemical Corp.), Sephadex LH-20 (Pharmacia), and octadecylsilane (ODS) (PrePAK-500/C18, YMC) were used for column chromatography. Semipreparative RP-HPLC separation was performed using SEP LC-52 (Separation Technology Co Ltd) and a Cosmosil Packed Column (10 × 250 mm, NacalaiTesque Inc.). Thin-layer chromatography (TLC) was performed using precoated silica gel GF254 plates (Qingdao Haiyang Chem. Co.). All the reagents were of analytical grade (Guangzhou Reagent Factory.).

Plant Material

The leaves and stems of C giraldii Hesse e × Rehd. were collected from Yalian Township, Yongde County, Lincang City, Yunnan Province, China, in September 2018. It was botanically identified by Prof. Gu-hua Ye (South China Botanical Garden). A voucher specimen (number: 201680915) was deposited in the Chinese Herb Medicine Museum, Guangdong Pharmaceutical University. After collection, the plant material was dried in the shade and then ground into a coarse powder.

Extraction and Isolation

The methanol extract of dried leaves and stems of C giraldii (28 kg) was obtained at room temperature. The total cold leached solution was concentrated under reduced pressure to form a dry extract (840 g), which was then suspended in water and extracted successively with PE (60 °C-90 °C), ethyl acetate, and n-butanol. The PE extract (165 g) was subjected to chromatography using a silica gel column (PE/EA 100:1-1:1 v/v), and monitored by TLC analysis, which yielded twelve combined fractions (Fr. A1-A12). Fr. A5 was further subjected to silica gel column chromatography using methanol as the mobile phase to yield compound 2. Fr. A7 was purified by Sephadex LH-20 column chromatography (CH₂Cl₂/MeOH = 1:1), silica gel column chromatography, and MCI column chromatography (MeOH/H₂O = 1:5-1:0) to yield compound 3 (7.1 mg). Fr. A10 was separated successively by MCI (MeOH/H₂O = 1:5-1:0), and Sephadex LH-20 (CH₂Cl₂/MeOH = 1:1) chromatography, and purified by semipreparative HPLC, to yield compounds 1 (98% methanol water, t_R = 18.408 min, 9.6 mg), 6 (98% methanol, t_R = 7.522 min, 11.2 mg), and 9 (35% methanol water, t_R = 17.7078 min, 17.8 mg).

The water extract (540 g) was fractionated on a MCI column with methanol water (10%-100%; each ratio was 2-3 column volumes) as the mobile phase. Six fractions were combined according to TLC analysis: Fr. C1 (10%), Fr. C2 (30%∼60%), Fr. C3 (70%), Fr. C4 (80%), Fr. C5 (90%), and Fr. C6 (100%). Fr. C2 was fractionated by silica gel chromatography with dichloromethane-methanol (50:1, 30:1, 15:1, 9:1, 7:1, 5:1, 3:1, 1:1) as the mobile phase. It yielded nineteen components, among which compounds 7 (43% methanol water, t_R = 13.869, 11.5 mg), 4 (43% methanol water, t_R = 21.568 min, 12.4 mg), 5 (43% methanol water, t_R = 17.078 min, 11.9 mg), and 8 (43% methanol water, t_R = 18.930 min, 10.2 mg) were isolated and purified by MCI, ODS (MeOH/H₂O = 1:5-1:0), and RP-HPLC.

Bioassays

RAW264.7 macrophages were obtained from the China Center for Type Culture Collection. In the cell viability test, RAW264.7 cells were seeded onto a 96-well plate (2 × 10⁴ cells/well) containing Dulbecco's Modified Eagle's medium with 10% fetal bovine serum, and incubated at 37 °C for 24 h. The cells were washed and then incubated with different concentrations of the compound for 18 h. CCK8 reagent (10 µL) (APExBIO Technology LLC.) was added to the above, and the mixture was further incubated for another 30 min. To measure cell viability, the absorbance was recorded at 450 nm using a full wavelength microplate reader (Thermo Fisher Scientific Inc., San Jose). DEX (Sigma, St. Louis) was used as a positive control. In the NO inhibitory activity test, RAW264.7 cells were seeded onto 96-well plates at a density of 8 × 10⁴ cells/well, cultured overnight, and then subsequently treated with 100 ng·mL⁻¹ LPS in either the absence or presence of various concentrations of compounds for 18 h. Nitrite release in the culture media was determined using the Griess test. After that, 50 µL of cell culture medium was mixed with 50 µL of Griess reagent A and B (Beyotime Biotechnology.) in turn. The NO concentration was measured at 540 nm using NaNO₂ as a standard.

Supplemental Material

sj-docx-2-npx-10.1177_1934578X211051801 - Supplemental material for Chemical Constituents of Callicarpa giraldii and Their Anti-Inflammatory Activity

Supplemental material, sj-docx-2-npx-10.1177_1934578X211051801 for Chemical Constituents of Callicarpa giraldii and Their Anti-Inflammatory Activity by Qiao-Qi Yi, Zhong-Han Wu, Fan Xu and Hong-Gang Wang in Natural Product Communications