Anti-inflammatory sesquiterpene and triterpene acids from Mesona procumbens Hemsley

Mesona procumbens Hemsley is a plant conventionally processed to provide popular food materials and herbal medicines in Asia. In this study, six triterpene acids, including five new ones (mesonaic acids D-H, 1–5), and one proximadiol-type sesquiterpene (7) were isolated from the methanolic extract of the air-dried M. procumbens. Chemical structures of 1‒7 were established by spectroscopic methods, especially 2D NMR techniques (¹H–¹H COSY, HSQC, HMBC, and NOESY) and HRESIMS. Concerning their biological activities, compounds 1, 2, 6, and 7 were examined manifesting high inhibition toward the pro-inflammatory NO production with EC₅₀ values ranging from 12.88 to 21.21 µM, outrunning the positive control quercetin (24.12 µM). The mesoeudesmol B (7) identified from M. procumbens is the very first example, which exhibited high anti-inflammatory activity diminishing the level of the lipopolysaccharide-induced NO in RAW264.7 macrophage cells, thereby suppressing the secretion of pro-inflammatory cytokines TNF-α and IL-6 and the level of two critical downstream inflammatory mediators iNOS and COX-2.

1 Introduction

Mesona procumbens Hemsley (Hsian-tsao), an annual herb belonging to the Lamiaceae family, is distributed in the tropical and subtropical regions of South Asia, such as Taiwan, Indonesia, Thailand, Vietnam, and southern China (Feng et al., 2012). This herb is conventionally used alone as a heat-clearing (Qingre) and detoxifying (Jiedu) agent or filled in a prescription of traditional Chinese medicine typically for the treatment of heat-shock, hypertension, diabetes, hepatic disease, and various inflammations, such as joint and muscle pains (Yen and Hung, 2000; Hung and Yen, 2002). Hsian-tsao (also known as grass jelly herb) is preferably consumed as herbal tea, herbal jelly dessert (grass jelly), or dessert soup given its unique smell, refreshing taste, and medicinal benefits from the major component, polysaccharide gum, which is the most attractive target in this edible plant (Lai and Liao, 2002; Lai et al., 2003; Zhuang et al., 2010). Recent studies further revealed that the Hsian-tsao aqueous extract can stop disease progression of liver fibrosis through its apoptotic effects on the activation of hepatic stellate cells (Yeh et al., 2019). The crude polysaccharides from Hsian-tsao demonstrated a wound healing activity in the streptozotocin-induced diabetic mouse model (Fan et al., 2021). Numerous pharmacological properties, such as anti-inflammatory (Huang et al., 2012), antioxidant (Lin et al., 2018), antihypertensive (Yeh et al., 2009), antimutagenic (Yen et al., 2001), DNA damage protection (Yen et al., 2000), liver fibrosis prevention (Shyu et al., 2008), and renal protective activities, from M. procumbens extracts have been independently reported from time to time (Yang et al., 2008). In addition, some such chronic inflammations such as cardiovascular, cancer, diabetes, arthritis, pulmonary, and autoimmune diseases reported (Singh et al., 2019) can be improved by natural products for they possess some yet-unknown chemo-preventive activities (Azab et al., 2016; Huang et al., 2021).

This study aimed at investigating untapped anti-inflammatory ingredients from Hsian-tsao rather than the known polysaccharide gum. Seven new chemical entities were identified herein, including five new triterpene acids (two 24-nor-oleanane-type triterpenes 1‒2, one 24-nor-ursane-type triterpene 3, one ursane-type triterpene acid 4, and one ursane-type seco-triterpene acid 5), one known 2α,3α,19α-trihydroxy-24-norursa-4 (23),12-dien-28-oic acid (6), and one new proximadiol (cryptomeridiol)-type sesquiterpene 7 isolated from the methanolic extract of M. procumbens. Having identified their chemical structures, these compounds were subjected to biochemical assay for evaluating their anti-inflammatory capacities against the lipopolysaccharide-induced NO production in RAW264.7 macrophage cells.

2 Materials and methods

2.1 General experimental procedures

Optical rotations were determined by a JASCO P-2000 polarimeter at 25°C. Infrared (IR) spectra were recorded on a Thermo Scientific Nicolet iS5 FTIR spectrometer. The ECD spectra were measured by a JASCO J-715 spectropolarimeter. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were established by a Thermo Scientific Ultimate 3000 UHPLC System with a Thermo Scientific Q Exactive™ Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer. NMR (nuclear magnetic resonance) spectra, including ¹H, ¹³C, DEPT, ¹H–¹H COSY, HMBC, HSQC, and NOESY, were recorded on Varian Unity Inova 500 MHz (5 mm SWPFG/TRPFG probe) or Varian VNMRS 600 MHz spectrometers (cold probe) and the chemical shifts were referenced by deuterated solvent methanol-d₄. Silica gel 60 (Merck, 70–230 and 230–400 meshes), C₁₈ gel (Chromatorex, 40–75 mesh), Diaion HP-20 (Mitsubishi Chemical Co.), and Sephadex LH-20 (GE) were used for column chromatography. Preparative HPLC (high-performance liquid chromatography) was performed using a Shimadzu LC-8A pump and an SPD-10A VP UV detector (210 and 254 nm wavelengths) with a Cosmosil 5C₁₈ AR-II column (250 × 20 mm, Nacalai Tesque). TLC (thin-layer chromatography) analyses were conducted on pre-coated silica gel plates (Merck, Kieselgel 60 F₂₅₄, 1 mm) and sprayed with anisaldehyde–sulfuric acid reagent and then heated at 100°C.

2.2 Plant material

The whole plants of the air-dried M. procumbens Hemsley (8.0 kg) were purchased from Starsci Biotech company in September 2019. A voucher specimen (No. NRICM-20190901) was deposited in the Herbarium of Division of Chinese Materia Medica Development, NRICM, Taipei, Taiwan.

2.3 Extraction, isolation, and purification

The air-dried plant of M. procumbens Hemsley (8.0 kg) was shredded into 5 mm and extracted with methanol (80 L) at 50°C thrice, and the combined extract was concentrated under reduced pressure removing methanol to obtain the methanolic crude extract. The crude extract (ca. 796.5 g) was dissolved in ddH₂O and then the aqueous solution was further sequentially partitioned with n-hexane and dichloromethane (CH₂Cl₂) to obtain the hexane, CH₂Cl₂, and aqueous extracts. The CH₂Cl₂ extract (ca. 47.6 g) was fractionated by a C₁₈ gel flash column (15 × 25 cm) eluting with a 20%, 40%, 60%, 80%, 90%, and 100% MeOH/H₂O, successively, to yield six fractions (Fr. I‒VI). Fr. IV was separated by chromatography on a flash column (silica gel, 60–230 mesh, 15 × 20 cm) eluting with CH₂Cl₂ and acetone (from 5% to 100% acetone) to obtain eight subfractions (Fr. IVA ∼ H). Fr. IVF was separated by preparative HPLC on a Cosmosil 5C₁₈ AR-II column (250 × 20 mm, flow rate: 10.0 ml/min) with 60% acetonitrile (ACN) in H₂O to afford seven subfractions (Fr. IVF-1∼7). Fr. IVF-4 was further purified by HPLC with Cosmosil 5C₁₈ AR-II column (flow rate: 10.0 ml/min), eluting with 45% ACN to afford 2 (2.1 mg, R_t: 33.7 min). Using the same HPLC and RP column, eluting with 50% ACN, compound 5 (2.6 mg, R_t: 21.3 min) was purified. Fr. IVE was fractionated by HPLC (Cosmosil 5C₁₈ AR-II column, 250 × 20 mm, flow rate: 10.0 ml/min) with 55% ACN to afford eight subfractions (Fr. IVE-1∼8). Fr. IVE-3 was further purified again with 45% ACN (flow rate: 10.0 ml/min) to yield 3 (1.9 mg, R_t: 18.3 min). Fr. IVD was subjected to preparative HPLC with 45% ACN (flow rate: 10.0 ml/min) to yield six subfractions (Fr. IVD-1∼6). Fr. IVD-4 was further repeatedly purified with 40% ACN (flow rate: 10.0 ml/min) to afford compounds 4 (2.3 mg, R_t: 17.9 min) and 7 (4.8 mg, R_t: 26.2 min). Fr. IVC was subjected to HPLC with 45% ACN (flow rate: 10.0 ml/min) to divide into eight subfractions (Fr. IVC-1∼5)_. Fr. IVC-2 was separated by a Cosmosil 5C₁₈ AR-II column (flow rate: 10.0 ml/min) to afford 1 (1.6 mg, R_t: 31.2 min). Fr. IVC-4 was also purified by HPLC using the same RP column with 40% ACN (flow rate: 10.0 ml/min) to afford 6 (2.5 mg, R_t: 49.9 min).

2.4 Spectroscopic data

2.4.1 Mesonaic acid D (1)

White amorphous powder; [α] +12.9 (c 0.5, MeOH); IR (KBr) ν_max 3402, 2927, 1694, 1437, 1316, 1017 cm⁻¹; ¹H- (600 MHz) and ¹³C- (150 MHz) NMR spectroscopic data (methanol-d₄) are shown in Tables 1, 2, respectively; HRESIMS m/z 495.3097 [M + Na]⁺ (calcd. for C₂₉H₄₄O₅Na, 495.3081).

TABLE 1

TABLE 1. ¹H-NMR spectroscopic data of 1‒6 in methanol-d₄.

TABLE 2

TABLE 2. ¹³C-NMR spectroscopic data of 1‒6 in methanol-d₄.

2.4.2 Mesonaic acid E (2)

White amorphous powder; [α] +19.7 (c 0.5, MeOH); IR (KBr) ν_max 3399, 2930, 1744, 1390, 1245, 1012 cm⁻¹; ¹H- (600 MHz) and ¹³C- (150 MHz) NMR spectroscopic data (methanol-d₄) are shown in Tables 1, 2, respectively; HRESIMS m/z 469.2965 [M ‒ H]^‒ (calcd. for C₂₉H₄₁O₅, 469.2949).

2.4.3 Mesonaic acid F (3)

White amorphous powder; [α] +25.1 (c 0.5, MeOH); IR (KBr) ν_max 3369, 2936, 1712, 1630, 1385, 1170, 1017 cm⁻¹; ¹H- (600 MHz) and ¹³C- (150 MHz) NMR spectroscopic data (methanol-d₄) are shown in Tables 1, 2, respectively; HRESIMS m/z 485.2919 [M ‒ H]^‒ (calcd. for C₂₉H₄₁O₆, 485.2898).

2.4.4 Mesonaic acid G (4)

White amorphous powder; [α] +23.4 (c 0.5, MeOH); IR (KBr) ν_max 3402, 2927, 1687, 1235, 1022 cm⁻¹; ¹H- (600 MHz) and ¹³C- (150 MHz) NMR spectroscopic data (methanol-d₄) are shown in Tables 1, 2, respectively; HRESIMS m/z 517.3176 [M ‒ H]^‒ (calcd. for C₃₀H₄₅O₇, 517.3160).

2.4.5 Mesonaic acid H (5)

White amorphous powder; [α] +27.3 (c 0.5, MeOH); IR (KBr) ν_max 3414, 2952, 1707, 1462, 1163, 1027 cm⁻¹; ¹H- (500 MHz) and ¹³C- (125 MHz) NMR spectroscopic data (methanol-d₄) are shown in Tables 1, 2, respectively; HRESIMS m/z 533.3124 [M ‒ H]^‒ (calcd. for C₃₀H₄₅O₈, 533.3109).

2.4.6 2α,3α,19α-Trihydroxy-24-norursa-4 (23),12-dien-28-oic acid (6)

White amorphous powder; [α] +21.8 (c 0.5, MeOH); IR (KBr) ν_max 3470, 2947, 1697, 1459, 1247, 1039 cm⁻¹; ¹H- (600 MHz) and ¹³C- (150 MHz) NMR spectroscopic data (methanol-d₄) are shown in Tables 1, 2, respectively; HRESIMS m/z 495.3072 [M + Na]⁺ (calcd. for C₂₉H₄₄O₅Na, 495.3081).

2.4.7 Mesoeudesmol B (7)

Colorless oil; [α] +9.1 (c 0.5, MeOH); IR (KBr) ν_max 3389, 2932, 1716, 1455, 1388, 1279, 1116, 1025 cm⁻¹; UV λ_max (MeOH) (log ε) 273 (2.98), 229 (3.82) nm; ¹H- (600 MHz) and ¹³C- (150 MHz) NMR spectroscopic data (methanol-d₄) are shown in Table 3; HRESIMS m/z 399.2148 [M + Na]⁺ (calcd. for C₂₂H₃₂O₅Na, 399.2142).

TABLE 3

TABLE 3. ¹H- and ¹³C-NMR spectroscopic data of 7 in methanol-d₄.

2.5 Dimolybdenum tetraacetate [Mo₂(OAc)₄]-modified circular dichroism analysis

The determination of the absolute configuration of cyclic and acyclic vic-diols was achieved by employing a transition metal chelate reagent, dimolybdenum tetraacetate [Mo₂(OAc)₄]. Compound 7 was directly dissolved in a solution of Mo₂(OAc)₄ complex in DMSO in a molar ratio of Mo₂(OAc)₄/compound of about 1:0.3–1:0.7, and the mixture was subsequently measured for the induced CD spectra without the preparation and isolation of the complexes.

2.6 Cell culture and viability assay

The RAW264.7 mouse macrophages were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in DMEM supplemented with 10% heat-inactivated FBS in a 5% CO₂ humidified incubator at 37°C. For viability assay, RAW264.7 cells were pretreated with various concentrations of the isolated compounds (0, 5, 10, and 20 μM) 1 h prior to LPS (1 μg/ml) stimulation. After 24 h treatment, cell viability was determined by Cell Counting Kit-8 (Dojindo, Rockville, MD, United States) according to the manufacturer’s instructions.

2.7 NO releasing inhibition assay

Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) was used to determine NO production. RAW264.7 cells were pretreated in the same manner described in cell viability assay. 100 µL of supernatant of each pretreated cell solution was transferred to a new microtiter plate, and each supernatant was mixed with 100 µL of Griess reagent. The microtiter plate was left at room temperature for 10 min for color development, and each solution was measured by a microplate reader at UV 540 nm. All experiments were performed in triplicate.

2.8 Western blot analysis

RAW264.7 cells were pretreated with compound 7 (0, 5, 10, or 20 μM) or quercetin (25 μM, Sigma-Aldrich) for 1 h and then stimulated with LPS for an additional 24 h. The cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer and protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Munich, Germany). The protein lysates were separated by SDS-PAGE and then transferred to a PVDF membrane. The blot was blocked with TBS containing 5% nonfat milk for 1 h at room temperature and then incubated with primary antibodies to iNOS, COX-2 or β-actin at 4°C overnight. The blots were washed three times with 0.1% TBST (0.1% Tween 20 in TBS) and then incubated with a peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing, the protein bands were detected using an ECL reagent and X-ray film.

2.9 Determination of IL-6 and TNF-α levels

The measurement of the IL-6 and TNF-α levels was performed using commercial ELISA kits (RAB0308-1KT and RAB0477-1KT (Sigma Chemical Co., St. Louis, MO, United States), respectively) according to the manufacturer’s instructions. Briefly, samples and standards were added to antibody-coated 96 wells and were incubated for 2.5 h at room temperature. After incubation, wells were washed with wash buffer four times, and then detection antibody was added and incubated for another 1 h at room temperature. After washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added and incubated in the dark for 30 min at room temperature followed by adding stop solution and reading absorbance at 450 nm immediately.

2.10 Statistical analysis

Statistical analyses were performed using SPSS (SPSS, Chicago, IL, United States). Data are expressed as the mean ± standard deviations. Statistical significance was determined using one-way ANOVA analysis followed by Tukey’s test. p-values < 0.05 were considered statistically significant.

3 Results and discussion

The methanolic extract of M. procumbens was dried and resuspended in H₂O; this aqueous solution was extracted by n-hexane and CH₂Cl₂ to give rise to two organic layers. The CH₂Cl₂ portion was subjected to chromatography by a flash column and preparative RP-HPLC to afford five new and one known triterpene acids (1–6), together with one brand new sesquiterpene (7) (Figure 1). All pure components (1–7) were then evaluated for their anti-inflammatory activities using an in vitro LPS-stimulated murine macrophage model.

FIGURE 1

FIGURE 1. Chemical structures of compounds 1‒7 isolated from M. procumbens.

3.1 Structural elucidation of the isolated compounds

Compound 1 ([α] +12.9, c = 0.5, MeOH), a colorless amorphous powder, has a molecular formula of C₂₉H₄₄O₅ with 8 degrees of unsaturation (DOU) deduced from the sodiated HRESIMS pseudo-ion at m/z 495.3097 [M + Na]⁺ (calcd. for C₂₉H₄₄O₅Na, 495.3081). In ¹H-NMR spectrum (Table 1), four tertiary methyl signals (δ_H 0.76, 0.86, 0.92, and 1.21 s), one olefinic methylene signal (δ_H 4.67 brs and 5.00 brs), and one olefinic methine signal (δ_H 5.28 t, J = 3.6 Hz) were observed. Based on ¹³C-NMR and DEPTs spectra (Table 2), the 29 carbons can be categorized into 4 methyls, 11 methylenes (one oxymethylene at δ_C 73.0 and one olefinic methylene at δ_C 109.4), 6 methines (two oxymethines at δ_C 68.7 and 75.6 as well as one olefinic methine at δ_C 122.4), and 8 quaternary carbons (two sp² quaternary carbons at δ_C 144.1 and 151.0 and one carboxylic acid group at δ_C 180.3). The above two olefins and one carboxyl group accounted for three DOU, and the remaining five constrained 1 to a pentacyclic structure. The assignments of ¹H- and ¹³C-NMR spectroscopic data were completed by a combination of HSQC, ¹H–¹H COSY, and HMBC experiments. By comparison of carbon signals of 1 with that of oleanolic acid along with the analysis of the COSY and HMBC correlations of 1 altogether determined the carbon skeleton of 1, an oleanane-type nortriterpenoid (Kashiwada et al., 1998). In Figure 2, the COSY correlations of H-2 (δ_H 3.65 ddd, J = 3.6, 4.8, 12.0 Hz)/H-3 (δ_H 4.12 d, J = 3.6 Hz) confirmed that the attachments of these two hydroxyls are at C-2 and C-3, whereas the HMBC correlations from H₂-30 (δ_H 3.18 brs) to C-19 (δ_C 39.9), C-20 (δ_C 35.4), and C-21 (δ_C 27.9) revealed a primary alcohol at C-30. Furthermore, an exo-4 (23)-double bound was verified according to the HMBC correlations of olefinic methylene H₂-23 to C-3 (δ_C 75.6), C-4 (δ_C 151.0), and C-5 (δ_C 44.4); the tri-substituted Δ¹² double bond was confirmed by the COSY correlations of H-9 (δ_H 1.82 m)/H₂-11 (δ_H 1.94 and 2.02 m)/H-12 (δ_H 5.28 t, J = 3.6 Hz) and by the HMBC correlations from H-18 (δ_H 2.88 dd, J = 4.8 and 14.4 Hz) to C-12 (δ_C 122.4), C-13 (δ_C 144.1), and C-14 (δ_C 41.8). The spectroscopic evidence altogether indicated that 1 is a new 24-noroleanane-type triterpene acid.

FIGURE 2

FIGURE 2. ¹H–¹H COSY and key HMBC correlations of triterpene acids 1‒5.

The pentacyclic moiety of oleanane-type triterpenoids is rigid, showing the configurations with those of regular oleanane-type triterpenoids based on NOESY experiment, which provides clear assignments. The NOESY correlations of H-5/H-9, H-9/CH₃-27, CH₃-27/H_α-19, and H_α-19/H₂-30 indicated α-orientations, whereas those of CH₃-25, CH₃-26, H-18, and CH₃-29 presented β-directions with regard to H-18/CH₃-25, CH₃-25/CH₃-26, and H-18/CH₃-29 (Figure 3). Meanwhile, the NOESY correlations of H-2/H-3 and H-2/CH₃-25 and the small proton constant of H-3 (³J_H-2,H-3 = 3.6 Hz) agreed with the α-oriented OH-2 and OH-3 within a cis-relationship. As a result, the chemical structure of mesonaic acid D (1) was established as 2α,3α,30-trihydroxy-24-norolean-4 (23),12-dien-28-oic acid.

FIGURE 3

FIGURE 3. Main NOESY correlations of triterpene acids 1‒5.

Mesonaic acid E (2) was white amorphous powders with [α] +19.7 (c 0.5, MeOH). The molecular formula C₂₉H₄₂O₅ on par with 9 DOU was deduced based on a quasimolecular ion at m/z 469.2965 [M ‒ H]^‒ (calcd. for C₂₉H₄₁O₅, 469.2949) in the HRESIMS experiment. The characteristic ¹H- and ¹³C-NMR signals (Tables 1, 2), four tertiary methyls (δ_H 0.66 s/δ_C 13.5; δ_H 0.94 s/δ_C 32.0; δ_H 0.98 s/δ_C 22.7; δ_H 1.17 s/δ_C 20.0), two oxymethines C-2 (δ_H 3.68 ddd, J = 3.6, 4.8, 11.4 Hz/δ_C 68.8) and C-3 (δ_H 4.11 d, J = 3.6 Hz/δ_C 75.4), one exocyclic methylene involving C-4 (δ_C 150.5) and C-23 (δ_H 4.85 brs and 4.99 brs/δ_C 109.0), and one COOH-28 (δ_C 180.3) suggested that compound 2 is a derivative of 24-nor-oleanane triterpene acid possessing the same 2,3-diol ring A as 1. Deduction of DOU from the 4 (23)-double bond, COOH-28, and the pentacyclic ring moiety, there are two DOU unassigned. After a detailed comparison of ¹H-, ¹³C-NMR, and DEPTs spectra, we found that a tri-substituted Δ¹² double bond and the primary alcohol of C-30 disappeared, while two oxymethines (δ_H 4.00 dd, J = 2.4 and 5.4 Hz/δ_C 73.0 and δ_H 5.02 d, J = 5.4 Hz/δ_C 78.0), one aliphatic methylene (δ_H 0.74 d, J = 7.2 Hz/δ_C 15.7), and one aliphatic quaternary carbon (δ_C 33.8) emerged. By a combination of the COSY correlations of H-9/H₂-11/H-12 (indicating that olefinic methine C-12 converts to an oxymethine) and the HMBC correlations from another oxymethine H-15 to C-12, one DOU was contributed to an oxygen-bridge between C-12 and C-15. The remaining DOU is resulted from a cyclopropane unit assembled by one aliphatic methylene C-27 and two aliphatic quaternary carbons C-13 and C-14, which were confirmed by the HMBC correlations (Figure 2) of H-18 (δ_H 1.88 dd, J = 4.2 and 12.6 Hz)/C-12, C-13, C-17 (δ_C 45.2), C-27, and COOH-28 and H₃-26 (δ_H 1.17 s)/C-7 (δ_C 37.0), C-8 (δ_C 39.5), C-9 (δ_C 43.2), and C-14 (δ_C 36.1).

In terms of stereochemistry, the chiral centers at ring junctions of 2 are carried on as those at compound 1; The α-side of the cyclopropane unit (C-13, C-14, and C-27) which formed via a si-attack by α-oriented CH₃-27 were supported by the NOESY correlations (H-9/H₂-27, H₂-27/H_α-19, and H_α-19/CH₃-30) (Figure 3). The 2α,3α-diol is also confirmed by the NOESY correlations of H-2/H-3 and H-2/CH₃-25 as well as the small proton constant of H-3 (J = 3.6 Hz). The configurations of the oxygen bridge heads, C-12 and C-15, were determined to be at the β-side based on the NOESY correlations of H-12/H-18 and H-15/CH₃-26. Thereby, compound 2 was clearly elucidated as 2α,3α-dihydroxy-12 (15)-epoxy-13α,27-cycloolean-4 (23)-en-28-oic acid.

Mesonaic acid F (3) was obtained as a white powder with specific rotation [α] +25.1 (c 0.5, MeOH). Its IR spectrum showed the presence of a hydroxyl group (3369 cm⁻¹), a carboxyl group (1712 cm⁻¹), a conjugated carbonyl group (1630 cm⁻¹), and an olefinic group (1385 cm⁻¹). The molecular formula of 3 was determined to be C₂₉H₄₂O₆ (9 DOU) by the given HRESIMS ion at m/z 485.2919 [M ‒ H]^‒ (calcd. 485.2898 for C₂₉H₄₁O₆). The 29 carbons (Table 2) can be separated into 6 methyls (δ_C 11.9, 12.5, 13.0, 16.3, 22.5, and 25.1), 7 methylenes (δ_C 20.5, 23.4, 27.6, 28.4, 32.0, 43.4, and 51.8), 6 methines (δ_C 41.8, 43.4, 48.5, 54.1, 73.1, and 128.0), and 10 quaternary carbons (δ_C 39.1, 40.9, 41.6, 47.2, 75.2, 131.6, 138.2, 143.9, 180.1, and 194.2) by ¹³C-NMR and DEPTs spectra of 3. The protons of five methyls [δ_H 0.88, 0.93, 1.36 s, 1.16 d, (J = 6.6 Hz), and 1.84 d (J = 1.8 Hz)], one oxymethine (δ_H 3.90 dd, J = 3.0 and 6.0 Hz), and one olefinic methine (δ_H 5.33 t, J = 3.6 Hz) can be clearly observed in ¹H-NMR spectrum (Table 1). Combining the ¹H-, ¹³C-NMR, and HSQC experiments, compound 3 was figured out to be a pentacyclic triterpene acid containing one conjugated carbonyl group, one tri-substituted double bond, and one carboxylic acid, reconciling these functional groups and pentacyclic fused ring system with the 9 DOU. The COSY correlations of H-9/H₂-11/H-12 and the HMBC correlations from a junction proton H-18 (δ_H 2.69 brs) to C-12, C-13, C-14, C-17 and C-28 together made a tri-substituted double bond at the Δ¹² position clear; the ¹H–¹H COSY correlations of CH₃-30/H-20/H-21/H₂-22 alongside the HMBC correlations of CH₃-29/C-18, C-19, C-20 illustrated that 3 is a derivative of ursane-type triterpenoids. The most difficult part is that the α-hydroxy-β-methyl substitution in the α,β-unsaturated conjugated carbonyl part in ring A is deduced by the HMBC correlations of CH₃-23 to C-3, C-4, C-5 and CH₃-25 to C-1, C-9, C-10, revealing the ursane triterpenoid without the C-24 carbon in 3 (Figure 2). The chiral configurations of 3 were built up by the NOESY correlations (Figure 3) and comparison with a pomolic acid (Kashiwada et al., 1998). As a result, 3 is identified as 3,19α,21α-trihydroxy-2-oxo-24-norurs-3,12-dien-28-oic acid.

Compound 4, [α] +23.4 (c 0.5, MeOH), was purified as white amorphous powders. The molecular formula C₃₀H₄₆O₇ (8 DOU) was determined by the HRESIMS molecular ion at m/z 517.3176 [M-H]^‒ (calcd. for C₃₀H₄₅O₇, 517.3160). The ¹H-, ¹³C-NMR (Tables 1, 2), and HSQC spectra of 4 as a whole revealed the presence of six methyls (δ_H 0.79 s/δ_C 13.6; δ_H 0.92 d, J = 6.6 Hz/δ_C 15.2; δ_H 1.03 s/δ_C 15.9; δ_H 1.18 s/δ_C 25.6; δ_H 1.19 s/δ_C 16.2; and δ_H 1.38 s/δ_C 23.6), one tri-substituted olefinic double bond (δ_H 5.28 t, J = 3.6 Hz/δ_C 127.8 and δ_C 138.8), and two carboxyl groups (δ_C 178.5 and 180.8), the characteristics of urs-12-ene or olean-12-ene derivatives. In addition, two secondary hydroxyl groups at the C-2 and C-3 positions were amended for 4 because of the (C)−CH₂−CHOH−CHOH−(C) structural moiety (δ_H 1.34 and 1.53 m; 3.89 ddd, J = 3.0, 4.8, and 12.0 Hz; δ_H 3.73 brd, J = 3.0 Hz) established by COSY spectrum. The HMBC spectrum of 4 confirmed the above deductions. Furthermore, the two carboxyl groups were assigned at C-24 (δ_C 180.8) and C-28 (δ_C 178.5) according to the HMBC correlations from H-18 (δ_H 2.49 brs) toC-17 (δ_C 47.6) and carboxyl carbon C-28 (δ_C 180.8), from CH₃-23 to two oxymethine carbons [C-3 (δ_C 75.4) and C-4 (δ_C 51.8)], carboxyl carbon C-24 (δ_C 178.5), respectively. The HMBC correlation from CH₃-29 to oxygen-bearing quaternary carbon C-19 (δ_C 72.1) put forward that the tertiary hydroxyl group is at C-19 (Figure 2). The configuration of 4 was established by the NOESY correlations (Figure 3) and the coupling constant interpretations for the relevant protons. The 2α,3α-configurations of the two secondary hydroxyl groups of 4 were ascertained by the NOESY correlations of H-2/H-3, H-2/CH₃-25, H-3/CH₃-23, CH₃-23/CH₃-25, and the small proton coupling constant of H-3 (J = 3.6 Hz) agreed with their directions. These elucidations leaded compound 4 to 2α,3α,19α-trihydroxyurs-12-ene-24,28-dioic acid, a triterpene acid monomer named mesonaic acid G, the first of its kind isolated from M. procumbens (it was reported to be a unit in the dimeric triterpene glucoside sanguidioside A) (Liu et al., 2004).

Mesonaic acid H (5) was obtained as white powders ([α] +27.3 (c 0.5, MeOH) with a molecular formula C₃₀H₄₆O₈ (8 DOU) determined by the HRESIMS ion at m/z 533.3124 [M ‒ H]^‒ (calcd. for C₃₀H₄₅O₈, 533.3109). Compound 5 was thought to be a urs-12-ene derivative where one methyl doublet (δ_H 0.98 d, J = 6.5 Hz), seven methyls, one tri-substituted olefinic double bond (δ_H 5.31 t, J = 4.0 Hz/δ_C 128.4 and δ_C 138.0), and three carboxyl groups (δ_C 174.2, 179.0 and 182.4) were deduced by ¹H- and ¹³C-NMR spectra (Tables 1, 2). Except for one secondary hydroxyl group and two carboxyl groups, the overall similarity concerning the chemical shifts of remaining carbons made the chemical skeleton of ursane-type triterpene acid with seven unmodified methyls. Based on HMBC correlations (Figure 2), the secondary OH group was assigned at C-22 by the resonances of H-22 (δ_H 3.71 dd, J = 4.5 and 12.0 Hz)/C-17 (δ_C 53.5) and carboxyl C-28 (δ_C 179.0). The HMBC correlations of both CH₃-23 (δ_H 1.29 s) and CH₃-24 (δ_H 1.28 s) to carboxyl C-3 (δ_C 182.4), C-4 (δ_C 46.1), and C-5 (δ_C 48.4) pointed out that there are two carboxyl groups one at C-3 and the other at C-2. It means that the C‒C bond between C-2 and C-3 is broken likely by an oxidative cleavage, and compound 5 has a seco-ring A. The stereochemistry of 5 is identical to regular ursane triterpene acids in whichα-orientation of OH-22 is supported by the NOESY correlations as shown in Figure 3. Added together, compound 5 was determined to be 19α,22α-dihydroxy-2,3-secours-12-ene-28-oic acid.

Compound 6, [α] +21.8 (c 0.5, MeOH), was isolated as white amorphous powders and had a molecular formula of C₂₉H₄₄O₅ determined by sodiated quasimolecular ion at m/z 495.3072 [M + Na]⁺ (calcd. for C₂₉H₄₄O₅Na, 495.3081) in the HRESIMS experiment. The IR, ¹H, and ¹³C-NMR spectroscopic data are very similar to compound 4. Detailed analysis of their data, compound 6 possesses an exocyclic double bond (δ_H 4.68 brs, 5.01 brs; δ_C 151.1, 109.3), which replaced a carboxyl group and a singlet methyl in 4. The complete chemical structure and NMR assignment (Tables 1, 2) were further established and confirmed by 1D and 2D NMR to be identified as 2α,3α,19α-trihydroxy-24-norursa-4 (23),12-dien-28-oic acid (Jang et al., 2005).

Compound 7 was collected as an oil sample ([α] +9.1, c 0.5 in MeOH) with a molecular formula C₂₂H₃₂O₅ (on par with 7 DOU) deduced from the HRESIMS ion at m/z 399.2148 [M + Na]⁺ (calcd. for C₂₂H₃₂O₅Na, 399.2142). According to ¹³C-NMR and DEPT spectra (Table 3), the 22 carbons can be divided into three methyls, six methylenes (one oxymethylene at δ_C 67.7), eight methines [including one oxymethine at δ_C 69.3 and five sp² methines at δ_C 128.1 (2), 129.0 (2), and 132.7], and five quaternary carbons (two oxygenated ones at δ_C 72.0 and 74.0, one sp2 quaternary carbon at δ_C 130.4, and one ester carbonyl at δ_C 166.0). In the ¹H-NMR spectrum (Table 3), three methyl singlets (δ_H 1.02 s, 1.12 s, and 1.19 brs), one oxygenated methylene [δ_H 3.46 s (2H)] and one oxygenated methine [δ_H 3.46 s (2H) and 5.18 ddd, J = 4.2, 7.8, and 12.0 Hz] together with the multiple peaks of mono-substituted benzene protons (δ_H 7.45–7.98) distributed spread from upfield to downfield. Combining the information, compound 7 was reasoned to have a double-ring moiety in addition to a benzene and an ester carbonyl portion. A comprehensive analysis of ¹H–¹H COSY and HMBC spectra, an eudesmane-type sesquiterpene (Evans et al., 1982) was put forward by incorporating two COSY fragments (H₂-1/H-2/H₂-3 and H-5/H₂-6/H-7/H₂-8/H₂-9) and the correlations of HMBC at CH₃-14/C-1, C-5, C-9, and C-10 and CH₃-15/C-3, C-4, and C-5. Moreover, the attachments of an O-benzoyl (OBz) group and a propane-1,2-diol group were confirmed to be at C-2 and C-7 by the HMBC correlations: H-2, H-2′, and H-3′ to ester carbonyl C-1′; H₂-12 and H₃-13 to C-7 and C-11 (Figure 4A). Concerning the stereochemistry of 7, it was concluded to be a regular eudesmane-type sesquiterpene with anti-relationships of α-oriented H-5 and β-oriented CH₃-14. Furthermore, both the cross peaks of NOESY correlations for H-2/H_β-3, H-2/CH₃-14, and H-2/CH₃-15 suggested the β-orientation of H-2 and CH₃-14 but α-orientation of H-7 given H_α-3/H-5, and H-5/H-7 (Figure 4B). However, the conformation of C-11 was not unambiguous because it cannot be differentiated directly by the NOESY correlations of H-7/CH₃-13, H_α-8/CH₃-13, H_β-8/CH₃-14, and H_β-8/H₂-12. For this reason, the dimolybdenum tetra-acetate [Mo₂(OAc)₄]-modified CD analysis was applied to resolve the steric assignment of the acyclic propane-1,2-diol group, a prim, sec-glycol (Frelek et al., 1997). As in many vic-glycols with a rigid conformation, one can follow the “helicity rule” to interpret the CD curves, whereby a positive (negative) torsional angle in the (HO)‒C‒C‒(OH) moiety should lead to a positive (negative) Cotton effect of 300 nm. In Figure 4C, a negative CD band at 300 nm is obvious and accompanied by a second Cotton effect of the same sign at around 400 nm corresponding to a negative torsional angle in the (HO)‒C‒C‒(OH) moiety. A Newman projection for the propane-1,2-diol moiety in chelation with Mo₂(OAc)₄ demonstrated a counterclockwise (a negative torsional angle) relationship from OH-12 to OH-11. Finally, compound 7 was clearly identified as 2-O-benzoyl-proximadiol, named mesoeudesmol B.

FIGURE 4

FIGURE 4. 2D correlations and CD spectrum of 7. (A) Key HMBC and ¹H–¹H COSY correlations. (B) Main NOESY correlations. (C) CD spectrum of 7 with Mo₂(OAc)₄ in DMSO with the inherent CDs subtracted.

3.2 Effects of compounds 1‒7 on RAW264.7 macrophage cell viability and NO production

M. procumbens extracts have been shown with pharmacological potentialities for many inflammation-associated disorders (Huang et al., 2012; Huang et al., 2021). Given the isolated compounds 1‒7 from M. procumbens, we examined these isolates to see whether they similarly exhibit anti-inflammatory activities by using the LPS-induced RAW 264.7 macrophage cell model. As shown in Table 4, mesonaic acids D (1) and E (2), 2α,3α,19α-trihydroxy-24-norursa-4 (23),12-dien-28-oic acid (6), and mesoeudesmol B (7) show lower EC₅₀ values than the positive control, quercetin (Li et al., 2016), suggesting that these four compounds possess better anti-inflammatory activity than those previously reported. Among them, mesoeudesmol B (7), a proximadiol derivative, displayed the highest inhibition activity on NO production with an EC₅₀ value of 12.88 ± 0.23 μM than that of quercetin (an EC₅₀ value of 24.12 ± 0.21 μM). Furthermore, all these seven compounds (at a concentration of 30 μΜ) showed an approximate 100% survival rate in cell viability assay, suggesting that they are, in general, safe with no major cytotoxicity (to RAW264.7 cells).

TABLE 4

TABLE 4. Anti-NO production activity of compounds 1‒7.

3.3 Effects of compound 7 on the protein expression of iNOS and COX-2

To better understand the anti-inflammatory mechanism of compound 7, we examined two key inflammation-mediated proteins in LPS-induced RAW264.7 cells, iNOS and COX-2 at the protein level (Murakami and Ohigashi, 2007). In terms of the expression level of iNOS and COX-2 shown in Figure 5A, the LPS-treated cells display a higher level as opposed to the non-treated cells, which show a low expression level. In addition, the protein level of the LPS-induced iNOS and COX-2 is negatively proportional to the addition of mesoeudesmol B (7) (5–20 μM) into the LPS-treated cells in a dose-dependent manner. Notably, 10 μM of 7 exhibits the lowest iNOS/COX-2 than 25 μM of quercetin does; moreover, the protein level of iNOS and COX-2 in the LPS-treated cells with the addition of 7 (20 μM) is nothing more than that in the non-treated cells, confirming that sesquiterpene 7 possesses stronger inhibitory activity than quercetin.

FIGURE 5

FIGURE 5. Effects of 7 on the expression of the iNOS and COX-2 proteins and cytokine secretion. RAW 264.7 cells were pretreated with the compounds for 1 h followed by stimulation with LPS (1 μg/ml) for an additional 24 h. (A) The expression of iNOS and COX-2 was determined using western blot analysis. The relative levels of iNOS and COX-2 were quantified by normalization to the β-actin levels. (B) The levels of TNF-α and IL-6 were measured using ELISA kits. Cells were treated with 7 at a concentration of 5–20 μM. Q: quercetin (25 μM). The data shown here represent the mean values of three independent experiments. ***p < 0.001 compared with the group treated with LPS.

3.4 Effects of compound 7 on the secretion of cytokines TNF-α and IL-6

Given that TNF-α and IL-6 are major pro-inflammatory cytokines, they were further measured by ELISA in the LPS-stimulated murine macrophages. As shown in Figure 5B, it shows a dose-dependent suppression of the LPS-induced secretion of cytokines TNF-α and IL-6 with the addition of compound 7, which shows a similar trend to that of the LPS-induced expression of iNOS and COX-2. Likewise, 10 μM of 7 exhibits a lower level of TNF-α and IL-6 than 25 μM of quercetin does; the level of TNF-α and IL-6 in the LPS-treated cells with the addition of 7 (20 μM) is close to the basal one, once again confirming that 7 is a stronger inflammatory inhibitor than quercetin.

4 Conclusion

Several lines of evidence have underscored that the phenolics (i.e. kaempferol and caffeic acid), triterpenoids (i.e. oleanolic acid and ursolic acid), and polysaccharides in M. procumbens Hemesley are functional ingredients with strong antioxidant and anti-inflammatory properties, thus making Hsian-tsao a superb heat-clearing (Qingre) and detoxifying (Jiedu) herb. Having gone through rigorous and comprehensive interrogations against the literature and chemical databases, the five triterpene acids (1–5) and one sesquiterpene (7) discovered herein are new chemical entities; mesoeudesmol B (7) featuring a proximadiol core structure, in particular, is the first of its kind isolated from the extract of M. procumbens Hemesley. Of them, three triterpene acids (1, 2, and 6) showed strong anti-inflammatory activities with the EC₅₀ values of 20.3, 21.1, and 20.2 μM comparable to quercetin (EC₅₀ values of 24.1 μM) and 15 triterpene acids isolated from our previous study, all presenting strong and promising anti-inflammatory potentials (Huang et al., 2021).

Of them, mesoeudesmol B (7), a 2-OBz proximadiol, outperformed others with an EC₅₀ value of 12.9 μM 2-fold higher than quercetin in terms of anti-inflammation. Proximol^® (cryptomeridiol, a eudesmane sesquiterpenoid) is a well-known Egyptian folk medicine effective as a renal antispasmodic and diuretic agent extracted from the desert weed Cymbopogon proximus Stapf (Gramineae) but also from other species, such as Dysphania graveolens (Gui et al., 2020) and Chenopodium vulvariap (Locksley et al., 1982). This active sesquiterpene is best known for its high antidiabetic activity, whereas the C. proximus extracts are more versatile exhibiting multiple bioactivities, including antihypertensive activity and relaxation of the smooth muscle fibers (El-Nezhawy et al., 2014).

We believe that mesoeudesmol B (7) is the dominant agent in M. procumbens. The anti-inflammation mechanism of 7 is that it suppresses inflammation-mediated proteins (iNOS and COX-2) and pro-inflammatory cytokines (TNF-α and IL-6), thereby underscoring Hsian-tsao (the grass jelly herb) a superb medicinal herb worth of further studies for advanced pharmacologic applications and biosynthetic diversifications.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Author contributions

H-TH: performing the experiments of the isolation and bioassay and writing the article. I-WL: elucidating the chemical structures and writing the article. G-YL and Y-CS: analyzing and interpreted the bioassay data and writing the article. T-LL: advising on the experiment and revising the article. Y-CL and H-CH: analyzed and interpreted the NMR data. C-CL: conceiving and designing the experiments and editing the article. Y-HK and K-TL: funding acquisition. All authors listed approved it for publication.

Funding

This research was financially supported by the Ministry of Science and Technology (MOST 107-2320-B-077-003-MY3) and China Medical University (CMU109-MF-02), Republic of China.

Acknowledgments

The authors sincerely thank all members of the C-CL, Y-HK, and K-TL Laboratories who supported and helped this research.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2022.1003356/full#supplementary-material

References

Azab, A., Nassar, A., and Azab, A. N. (2016). Anti-inflammatory activity of natural products. Molecules 21 (10), 1321. doi:10.3390/molecules21101321

PubMed Abstract | CrossRef Full Text | Google Scholar

El-Nezhawy, A. O., Maghrabic, I. A., Mohamed, K. M., and Omar, H. A. (2014). Cymbopogon proximus extract decreases LNAME-induced hypertension in rats. Int. J. Pharm. Sci. Rev. Res. 27 (1), 66–69.

Google Scholar

Evans, F. E., Miller, D. W., Cairns, T., Baddeley, G. V., and Wenkert, E. (1982). Structure analysis of proximadiol (cryptomeridiol) by ¹³C NMR spectroscopy. Phytochemistry 21 (4), 937–938. doi:10.1016/0031-9422(82)80097-6

CrossRef Full Text | Google Scholar

Fan, S. L., Lin, J. A., Chen, S. Y., Lin, J. H., Lin, H. T., Chen, Y. Y., et al. (2021). Effects of hsian-tsao (Mesona procumbens hemsl.) extracts and its polysaccharides on the promotion of wound healing under diabetes-like conditions. Food Funct. 12 (1), 119–132. doi:10.1039/D0FO02180F

PubMed Abstract | CrossRef Full Text | Google Scholar

Feng, T., Ye, R., Zhuang, H., Fang, Z., and Chen, H. (2012). Thermal behavior and gelling interactions of Mesona blumes gum and rice starch mixture. Carbohydr. Polym. 90 (1), 667–674. doi:10.1016/j.carbpol.2012.05.094

PubMed Abstract | CrossRef Full Text | Google Scholar

Frelek, J., Ikekawa, N., Takatsuto, S., and Snatzke, G. (1997). Application of [Mo₂(OAc)₄] for determination of absolute configuration of brassinosteroid vic-diols by circular dichroism. Chirality 9 (5-6), 578–582. doi:10.1002/(SICI)1520-636X(1997)9:5/6<578::AID-CHIR27>3.0.CO;2-K

CrossRef Full Text | Google Scholar

Gui, Y. H., Liu, L., Wu, W., Zhang, Y., Jia, Z. L., Shi, Y. P., et al. (2020). Discovery of nitrogenous sesquiterpene quinone derivatives from sponge Dysidea septosa with anti-inflammatory activity in vivo zebrafish model. Bioorg. Chem. 94, 103435. doi:10.1016/j.bioorg.2019.103435

PubMed Abstract | CrossRef Full Text | Google Scholar

Huang, G. J., Liao, J. C., Chiu, C. S., Huang, S. S., Lin, T. H., and Deng, J. S. (2012). Anti‐inflammatory activities of aqueous extract of Mesona procumbens in experimental mice. J. Sci. Food Agric. 92 (6), 1186–1193. doi:10.1002/jsfa.4682

PubMed Abstract | CrossRef Full Text | Google Scholar

Huang, H. T ., Liaw, C. C., Chiou, C. T., Kuo, Y. H., and Lee, K. T. (2021). Triterpene acids from Mesona procumbens exert anti-inflammatory effects on LPS-stimulated murine macrophages by regulating the MAPK signaling pathway. J. Agric. Food Chem. 69 (22), 6271–6280. doi:10.1021/acs.jafc.1c01810

PubMed Abstract | CrossRef Full Text | Google Scholar

Hung, C. Y., and Yen, G. C. (2002). Antioxidant activity of phenolic compounds isolated from Mesona procumbens hemsl. J. Agric. Food Chem. 50 (10), 2993–2997. doi:10.1021/jf011454y

PubMed Abstract | CrossRef Full Text | Google Scholar

Jang, D. S., Kim, J. M., Kim, J. H., and Kim, J. S. (2005). 24-nor-Ursane type triterpenoids from the stems of Rumex japonicas. Chem. Pharm. Bull. 53 (12), 1594–1596. doi:10.1248/cpb.53.1594

PubMed Abstract | CrossRef Full Text | Google Scholar

Kashiwada, Y., Wang, H. K., Nagao, T., Kitanaka, S., Yasuda, I., Fujioka, T., et al. (1998). Anti-AIDS agents. 30. Anti-HIV activity of oleanolic acid, pomolic acid, and structurally related triterpenoids. J. Nat. Prod. 61 (9), 1090–1095. doi:10.1021/np9800710

PubMed Abstract | CrossRef Full Text | Google Scholar

Lai, L. S., and Liao, C. L. (2002). Dynamic rheology of structural development in starch/decolourised hsian-tsao leaf gum composite systems. J. Sci. Food Agric. 82 (10), 1200–1207. doi:10.1002/jsfa.1171

CrossRef Full Text | Google Scholar

Lai, L. S., Liu, Y. L., and Lin, P. H. (2003). Rheological/textural properties of starch and crude hsian-tsao leaf gum mixed systems. J. Sci. Food Agric. 83 (10), 1051–1058. doi:10.1002/jsfa.1506

CrossRef Full Text | Google Scholar

Li, Y., Yao, J., Han, C., Yang, J., Chaudhry, M. T., Wang, S., et al. (2016). Quercetin, inflammation and immunity. Nutrients 8 (3), 167. doi:10.3390/nu8030167

PubMed Abstract | CrossRef Full Text | Google Scholar

Lin, K. H., Shih, M. C., Wang, P., Yu, Y. P., and Lu, C. P. (2018). Effect of different ethanolic concentrations on antioxidant properties and cytoprotective activities of Platostoma palustre blume. Nat. Prod. Res. 32 (24), 2959–2963. doi:10.1080/14786419.2017.1392952

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, X., Shi, B. F., and Yu, B. (2004). Four new dimeric triterpene glucosides from Sanguisorba officinalis. Tetrahedron 60 (50), 11647–11654. doi:10.1016/j.tet.2004.09.045

CrossRef Full Text | Google Scholar

Locksley, H. D., Fayez, M. B. E., Radwan, A. S., Chari, V. M., Cordell, G. A., and Wagner, H. (1982). Constituents of local plants. XXV, constitution of the antispasmodic principle of Cymbopogon proximus. Planta Med. 45 (5), 20–22. doi:10.1055/s-2007-971233

PubMed Abstract | CrossRef Full Text | Google Scholar

Murakami, A., and Ohigashi, H. (2007). Targeting NOX, INOS and COX-2 in inflammatory cells: Chemoprevention using food phytochemicals. Int. J. Cancer 121 (11), 2357–2363. doi:10.1002/ijc.23161

PubMed Abstract | CrossRef Full Text | Google Scholar

Shyu, M. H., Kao, T. C., and Yen, G. C. (2008). Hsian-tsao (Mesona procumbens heml.) prevents against rat liver fibrosis induced by CCl₄ via inhibition of hepatic stellate cells activation. Food Chem. Toxicol. 46 (12), 3707–3713. doi:10.1016/j.fct.2008.09.051

PubMed Abstract | CrossRef Full Text | Google Scholar

Singh, N., Baby, D., Rajguru, J. P., Patil, P. B., Thakkannavar, S. S., and Pujari, V. B. (2019). Inflammation and cancer. Ann. Afr. Med. 18 (3), 121–126. doi:10.4103/aam.aam_56_18

PubMed Abstract | CrossRef Full Text | Google Scholar

Yang, M., Xu, Z. P., Xu, C. J., Meng, J., Ding, G. Q., Zhang, X. M., et al. (2008). Renal protective activity of hsian-tsao extracts in diabetic rats. Biomed. Environ. Sci. 21 (3), 222–227. doi:10.1016/S0895-3988(08)60033-1

PubMed Abstract | CrossRef Full Text | Google Scholar

Yeh, C. T., Huang, W. H., and Yen, G. C. (2009). Antihypertensive effects of hsian-tsao and its active compound in spontaneously hypertensive rats. J. Nutr. Biochem. 20 (11), 866–875. doi:10.1016/j.jnutbio.2008.07.015

PubMed Abstract | CrossRef Full Text | Google Scholar

Yeh, Y. H., Liang, C. Y., Chen, M. L., Tsai, F. M., Lin, Y. Y., Lee, M. C., et al. (2019). Apoptotic effects of hsian-tsao (Mesona procumbens Hemsley) on hepatic stellate cells mediated by reactive oxygen species and ERK, JNK, and caspase-3 pathways. Food Sci. Nutr. 7 (5), 1891–1898. doi:10.1002/fsn3.1046

PubMed Abstract | CrossRef Full Text | Google Scholar

Yen, G. C., Duh, P. D., and Hung, Y. L. (2001). Contributions of major components to the antimutagenic effect of hsian-tsao (Mesona procumbens hemsl.). J. Agric. Food Chem. 49 (10), 5000–5004. doi:10.1021/jf0103929

PubMed Abstract | CrossRef Full Text | Google Scholar

Yen, G. C., and Hung, C. Y. (2000). Effects of alkaline and heat treatment on antioxidative activity and total phenolics of extracts from hsian-tsao (Mesona procumbens hemsl.). Food Res. Int. 33 (6), 487–492. doi:10.1016/S0963-9969(00)00073-9

CrossRef Full Text | Google Scholar

Yen, G. C., Hung, Y. L., and Hsieh, C. L. (2000). Protective effect of extracts of Mesona procumbens hemsl. On DNA damage in human lymphocytes exposed to hydrogen peroxide and UV irradiation. Food Chem. Toxicol. 38 (9), 747–754. doi:10.1016/S0278-6915(00)00069-7

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhuang, H., Feng, T., Xie, Z., Toure, A., Xu, X., Jin, Z., et al. (2010). Effect of Mesona blumes gum on physicochemical and sensory characteristics of rice extrudates. Int. J. Food Sci. Technol. 45 (11), 2415–2424. doi:10.1111/j.1365-2621.2010.02426.x

CrossRef Full Text | Google Scholar