Anti-Inflammatory Activity of Labdane and Norlabdane Diterpenoids from Leonurus sibiricus Related to Modulation of MAPKs Signaling Pathway

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Plant material
• Chemicals and reagents
• Extraction and isolation
• Cell culture
• Cell cytotoxicity assay
• Griess assay
• Quantitative real-time polymerase chain reaction
• Western blotting
• Statistical Analysis
• Contributorsʼ Statement
• References

Abstract

Leonurus sibiricus, a widely cultivated herbaceous plant in Asian countries, exhibits diverse medicinal applications. Recent studies emphasize its pharmacological properties and efficacy in promoting bone health. In addition to the known compounds and their pharmacological activities, in this study, we isolated and elucidated two new labdane-type diterpenoids, (3R,5R,6S,10S)-3,6-dihydroxy-15-ethoxy-7-oxolabden-8(9),13(14)-dien-15,16-olide (1) and (4R,5R,10S)-18-hydroxy-14,15-bisnorlabda-8-en-7,13-dione (2), a new natural phenolic compound, and a known compound from L. sibiricus using advanced spectroscopic techniques, including circular dichroism spectroscopy, high-resolution mass spectrometry, and 1- and 2-dimensional NMR. Among these, compound 1 demonstrated potent inhibition of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression levels, followed by compound 2. Whereas compounds 3 and 4 did not exhibit effectiveness in RAW264.7 macrophages. Moreover, compound 1 suppressed pro-inflammatory markers induced by lipopolysaccharide (LPS) stimulation. Compound 1 also suppressed iNOS and cyclooxygenase-2 (COX-2) protein levels and downregulated pro-inflammatory cytokines. Additionally, compound 1 showed inhibition of the phosphorylation of p38, JNK, and ERK, key mediators of the MAPK signaling pathway. These findings indicate that a natural-derived product, compound 1, might be a potential candidate as an anti-inflammation mediator.

Introduction

Inflammation serves as a natural mechanism employed by the immune system to protect the body from harmful agents [1], [2], [3]. Dysregulation of this process can give rise to various diseases, including rheumatoid arthritis, chronic inflammatory bowel disorders, neurodegenerative conditions, and septic shock syndrome [4], [5]. Inflammation is initiated by a range of physical, chemical, or biological agents and results from a combination of genetic predisposition and numerous paracrine and autocrine responses [6]. While the specifics of inflammatory reactions may vary across diseases, they commonly involve a spectrum of genes and endogenous mediators, such as growth factors, inflammatory cytokines like interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and toxic molecules like nitric oxide (NO) [7], [8].

Numerous studies have emphasized the crucial role of various signaling pathways, particularly the mitogen-activated protein kinases (MAPKs), in regulating inflammatory responses [9], [10]. MAPKs, a family of serine/threonine protein kinases, coordinate fundamental biological processes and cellular reactions to external stress signals [11]. Specifically, the regulation of several cellular processes, including proliferation, differentiation, apoptosis, cell survival, motility, metabolism, stress response, and inflammation, is facilitated by the MAPK pathway, which is essential in conveying signals from cell surface receptors to DNA [12]. In addition, MAPKs serve as regulators in the creation of pro-inflammatory cytokinase and downstream signaling events such as COX-2, inducible nitric oxide synthase (iNOS), and a nitric oxide (NO) synthesis enzyme [13]. The elevated activity of MAPKs and their involvement in modulating the synthesis of inflammatory mediators at both transcriptional and translational levels make them promising targets for anti-inflammatory therapeutics [14], [15].

Leonurus sibiricus L. (Lamiaceae) is a widely distributed herbaceous plant cultivated in crop fields across Asian countries and regions, including Siberia, China, Korea, Japan, and Vietnam [16], [17]. L. sibiricus is an aromatic herb, typically characterized by its deflexed lower lip, and it can be an annual, biennial, or perennial plant [18], [19]. This plant typically sprouts from seeds and has an average height ranging from 40 to 120 cm. L. sibiricus has been used extensively in traditional herbal medicine and culinary practices [19]. A recent study indicates that the methanolic extract of L. sibiricus aerial parts exhibits promising inhibitory effects against human HCC cell lines, Huh-7 and HSC-T6 [20]. Furthermore, it upregulates the expression of proapoptotic genes such as p53, Bax, and caspase 9, while downregulating the activation of ERK and Akt. Notably, caspase 3 activity and ROS generation increase significantly in a dose-dependent manner compared to the control. Additionally, ethanolic extract from L. sibiricus demonstrates significant potential in promoting osteoblast differentiation and suppressing osteoclast differentiation in vitro [21]. It effectively inhibits LPS-induced bone loss in in vivo models. Previous phytochemical studies have identified labdane-type diterpenoids, flavonoids, iridoids, and phenolic compounds as some of the major secondary metabolites isolated from L. sibiricus, such as sibiricusins K – U, leonosides A and B, leucosceptoside A, rutin, hyperoside, isoquercitrine, chlorogenic acid, caffeic acid, acetylharpagide, lavandulifolioside, and verbascoside [22], [23]. A previous study indicates that L. sibiricus root extract exhibited potential anti-inflammatory properties, with a NO-scavenging activity (IC₅₀) of 48.0 µg/mL [24]. Additionally, leonurine hydrochloride, isolated from L. sibiricus, inhibits the expression of proinflammatory factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-2, IL-6, and toll-like receptor 4 (TLR4), in 120 one-day-old male Ross broiler chicks [25].

In our ongoing effort to discover bioactive compounds from medicinal plants, this study resulted in the isolation of two new labdane-type diterpenoids (1 and 2), a new natural phenolic compound (3), and a known compound (4) from the aerial parts of L. sibiricus. Their chemical structures were elucidated using spectroscopic techniques, including NMR, HR-MS, and electronic circular dichroism (CD) spectra. Additionally, the anti-inflammatory activity of all isolated compounds (1–4) was evaluated. Among them, new compound 1 strongly inhibited NO production in macrophage cells via in vitro assays. Furthermore, we investigated the inhibitory effects of compound 1 on the LPS-induced expression of protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, as well as its suppression of the phosphorylation of p38, JNK, and ERK, three members of MAPKs.

Results and Discussion

In this study, we conducted a phytochemical investigation of L. sibiricus aerial parts. This effort led to the isolation and structural elucidation of two new labdane-type diterpenoids (1 and 2), a new natural phenolic compound (3), and a known compound, (R)-methyl 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoate (4) [26]. This identification was accomplished through analyses of their NMR spectra, as well as comparison with values found in the literature ([Fig. 1]).

Compound 1 was isolated as a yellow oil. Its molecular formula was determined to be C₂₃H₃₂O₆ by HR-ESI-MS analysis, with a sodium adduct molecular ion peak at m/z 415.2097 [M + Na]⁺ (calcd. for C₂₃H₃₂O₆Na, 415.2077). The ¹H-NMR spectrum of compound 1 showed the signals of four tertiary methyl groups [δ _H 1.86 (3H, s, H-17), 1.11 (3H, s, H-18), 1.32 (3H, s, H-19), and 1.37 (3H, s, H-20)], two olefin methine protons [δ _H 6.86 (1H, br s, H-14) and 5.84 (1H, br s, H-15)], two aliphatic methine protons [δ _H 3.46 (1H, br s, H-3) and 4.22 (1H, d, J = 3.8 Hz, H-6)], and an ethoxy group [δ _H 3.96 (1H, m, H-1′a), 3.77 (1H, m, H-1′b), 1.29 (3H, t, J = 7.0 Hz, H-2′)] ([Table 1]). The ¹³C-NMR and DEPT spectra of compound 1 exhibited the presence of 22 carbon signals, including five methyl groups (δ _C 27.2, 24.4, 22.3, 11.8, and an ethoxy group at δ _C 15.2), five methylene groups (δ _C 30.0, 27.7, 25.3, 24.9, and an ethoxy group at δ _C 66.5), five methine groups (including two oxymethine groups at δ _C 76.4 and 71.4), and seven quaternary carbons (including two carbonyl groups at δ _C 199.5 and 171.2). These spectroscopic characteristics indicate that compound 1 belongs to a labdane-type diterpenoid, which is commonly found in the genus Leonurus [27], [28]. Detailed analysis of NMR chemical shifts revealed that compound 1 was similar to those of 15,16-epoxy-3α,6β-dihydroxylabda-8(9),13(16),14-trien-7-one [29], except that compound 1 possessed a 3-substituted 5-ethoxyfuran-2(5H)-one moiety at C-12 rather than the furan ring of 15,16-epoxy-3α,6β-dihydroxylabda-8(9),13(16),14-trien-7-one. This was supported by the HMBC correlations of δ _H 6.86 (H-14) with δ _C 24.9 (C-12) and δ _C 137.7 (C-13), and δ _H 2.52 (H-12) with C-13, as well as the ¹H-¹H COSY correlations between H-14 and H-15 ([Fig. 2]). The position of a carbonyl group at C-16 was determined by the HMBC correlations of H-12 with δ _C 171.2 (C-16), whereas the linkage of an ethoxy group with C-15 was determined by the HMBC correlations of H-1′ with δ _C 101.8 (C-15). The relative configuration of compound 1 was identified by the NOESY spectrum. The NOESY correlations of H-3 with H-19, H-19 with H-20, H-5 with H-6, and H-6 with H-18, along with the small coupling constant of J _5,6 = 3.8 Hz, indicated the configurations of H-5, H-18, and 3-OH as α-orientations, and H-19, H-20, and 6-OH as β-orientations ([Fig. 3]). In addition, compound 1 exhibits the same structural backbone with 15,16-epoxy-3α,6β-dihydroxylabda-8(9),13(16),14-trien-7-one and leojapone A [29], suggesting that the relative configuration of compound 1 was (3R*,5R*,6S*,10S*). To determine the absolute configuration of compound 1, the experimental CD spectrum of compound 1 was obtained (Fig. S9, Supporting Information), and the calculated CD spectra were conducted using Gaussian 16 with time-dependent density functional theory (TDDFT) at the IEFPCM-MeOH/B3LYP/6 – 31 G(d,p) level, following our reported procedure [30]. As shown in [Fig. 4], the absolute configuration of compound 1 was elucidated as 3R,5R,6S,10S. The configuration of C-15 was not determined because there was no observed NOE correlations between the 3-substituted 5-ethoxyfuran-2 (5H)-one unit and the decalin unit. Thus, the structure of compound 1 was deduced as (3R,5R,6S,10S)-3,6-dihydroxy-15-ethoxy-7-oxolabden-8(9),13(14)-dien-15,16-olide.

Compound 2 was isolated as a yellow oil. Its molecular formula was determined to be C₁₈H₂₈O₃ by HR-ESI-MS analysis, with a protonated molecule [M + H]⁺ at m/z 293.2124 (calcd. for C₁₈H₂₉O₃, 293.2117). The ¹H-NMR spectrum of compound 2 showed the signals of four tertiary methyl groups [δ _H 2.17 (3H, s, H-16), 1.70 (3H, s, H-17), 0.84 (3H, s, H-18), and 1.11 (3H, s, H-20)] and a hydroxymethyl group [δ _H 3.41 (1H, d, J = 10.9 Hz, H-19a), 3.09 (1H, d, J = 10.9 Hz, H-19b)] ([Table 1]). The ¹³C-NMR and DEPT spectra of compound 2 exhibited the presence of 18 carbon signals, including four methyl groups (δ _C 29.9, 18.5, 17.5, and 11.5), seven methylene groups (δ _C 42.4, 35.5, 35.0, 34.7, 22.9, 18.0, and a hydroxymethyl group at δ _C 70.6), a methine group (δ _C 43.4), and six quaternary carbons (including two carbonyl groups at δ _C 207.1 and 199.8). These NMR spectroscopic characteristics revealed that compound 2 was highly similar to those reported of 14,15-bisnorlabda-8-en-7,13-dione, a 14,15-bisnor labdane diterpene previously isolated from Nicotiana tabacum and Leonurus japonicus [31], [32]. The main difference is that compound 2 exhibited an additional hydroxy group compared to that of 14,15-bisnorlabda-8-en-7,13-dione. The position of an additional hydroxy group at C-19 was determined by the HMBC correlations of H-19 with δ _C 34.7 (C-3), 37.6 (C-4), 43.4 (C-5), and 17.5 (C-18) ([Fig. 2]). The relative configuration of compound 2 was determined by the NOESY correlations of δ _H 2.40 (H-6a) with H-19, H-19 with H-20, and δ _H 2.36 (H-6b) with H-5 ([Fig. 3]), indicating that H-5 is in the α-orientation and H-19 and H-20 are in the β-orientations. The absolute configuration of 2 was determined as 4R,5R,10S by comparison of the experimental and calculated CD spectra ([Fig. 4]). Thus, compound 2 was elucidated to be (4R,5R,10S)-18-hydroxy-14,15-bisnorlabda-8-en-7,13-dione.

Compound 3 was isolated as a light yellow amorphous powder. Its molecular formula was determined to be C₁₁H₁₄O₅ by HR-ESI-MS analysis, with a deprotonated molecule [M – H]⁻ at m/z 225.0745 (calcd. for C₁₁H₁₃O₅, 225.0763). The ¹H- and ¹³C-NMR spectra of compound 3 showed signals of an ABX pattern [δ _H 6.84 (1H, d, J = 8.0 Hz, H-5′), 6.74 (1H, d, J = 1.9 Hz, H-2′), 6.68 (1H, dd, J = 8.0, 1.9 Hz, H-6′)], a methylene group [δ _H 3.06 (1H, dd, J = 14.1, 4.3 Hz, H-3a), 2.90 (1H, dd, J = 14.1, 6.6 Hz, H-3b)], a hydroxymethylene group [δ _H 4.43 (1H, dd, J = 10.6, 6.2 Hz, H-2)], two methoxy groups [δ _H 3.87 (3H, s, 3′-OCH₃), 3.78 (3H, s, 1-OCH₃)], and a carbonyl group [δ _C 174.7 (C-1)]. The NMR spectroscopy data of compound 3 were similar to those of oresbiusin A, except for the presence of an additional methoxy group [33]. The position of a methoxy group with C-1 was determined through HMBC correlations of the proton at δ _H 3.78 (1-OCH₃) with the carbonyl group C-1. Additionally, the linkage of an additional methoxy group at C-3′ was determined based on HMBC correlations of δ _H 3.87 (3′-OCH₃) with the carbon at δ _C 146.5 (C-3′) ([Fig. 2]). The optical rotation of compound 3 was determined as [α]_D ²⁰ − 11.6 (c 0.1, MeOH), which is similar to that of (−)-(R)-methyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate ([α]_D ²¹ − 7.8 (c 0.6, MeOH) [33]. This suggests that the configuration at C-2 of compound 3 is R. To elucidate the absolute configuration at C-2, the CD spectra of two possible isomers, 3-(2R) and 3-(2S), were calculated using Gaussian 16. The experimental CD spectrum of compound 3 displayed a positive Cotton effect at the 230 – 232 nm region, similar to the calculated CD of the 3-(2R) configuration ([Fig. 4]). On the contrary, the 3-(2S) configuration exhibited the opposite calculated CD spectrum. Based on the above evidence, the structure of compound 3 was established as (R)-methyl 2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoate, a compound previously reported as a product of synthetic reactions (Patent No. CN105168196). In this study, compound 3 was isolated from a natural source for the first time.

To investigate the potential anti-inflammatory properties of isolated compounds 1–4, their effects on NO production were examined in RAW264.7 cells. These cells were treated with 40 µM of compounds 1–4 for 2 h, followed by stimulation with LPS for an additional 24 h. As shown in [Table 2], compounds 1 and 2 displayed strong inhibition of NO production, with IC₅₀ values of 7.20 ± 1.85 and 36.65 ± 1.35 µM, respectively, whereas compounds 3 and 4 did not exhibit inhibitory effects on NO production. Notably, compound 1 exhibited a more potent suppression of NO production compared to quercetin (10.86 ± 1.43 µM), which served as a positive control. In addition, NO, which is generated by intracellular NOS and synthesized by iNOS, has the ability to interact with other free radicals to form cytotoxic molecules leading to inflammatory responses. To ascertain whether the suppression of NO synthesis by compound 1–4 could be linked to the decrease in iNOS expression, the mRNA expression levels of this enzyme were assessed in LPS-induced RAW264.7 cells. Compound 1 is the most effective in suppressing the mRNA expression level of iNOS, followed by compound 2, while compounds 3 and 4 do not show a decrease in iNOS mRNA levels (Fig. S27, Supporting Information). Consequently, based on these findings, compound 1 was selected for further anti-inflammatory evaluations.

To confirm whether treatment with compound 1 is cytotoxic to RAW264.7 macrophage cells, cell viability was assessed using the methyl thiazol tetrazolium (MTT) assay at four concentrations: 5, 10, 20, and 40 µM. [Fig. 5 a] shows that compound 1 is not associated with cell toxicity at the indicated concentrations. To ensure the absence of toxicity for further investigation, cells were treated with compound 1 at different doses prior to incubation with LPS (1 µg/mL). Even at the highest concentration tested (40 µM), cell viability remained unchanged ([Fig. 5 b]). These data indicate that the viability of RAW264.7 macrophage cells is unaffected by treatment with compound 1 at concentrations up to 40 µM.

COX-2 and iNOS are recognized as crucial enzymes involved in regulating the progression of inflammation [34]. To assess the anti-inflammatory properties of compound 1, its effect on iNOS and COX-2 protein levels was investigated. The protein levels of iNOS and COX-2 were significantly elevated in the LPS-treated group; however, they were gradually suppressed with increasing concentrations of compound 1 in a concentration-dependent manner ([Fig. 6 A]). Furthermore, the upregulation of pro-inflammatory cytokines induced by LPS, including IL-6, TNF-α, and IL-1β, was significantly inhibited by pretreatment with compound 1, as determined by quantitative real-time polymerase chain reaction (PCR) analysis ([Fig. 6 b]).

MAPKs, including three representative members, p38, JNK, and ERK, constitute signaling cascades activated by various extracellular stimuli, eventually leading to inflammation [35], [36]. Therefore, we investigated whether pretreatment with compound 1 is associated with the phosphorylation of p38, ERK, and JNK in LPS-induced RAW264.7 cells. As shown in [Fig. 7], the phosphorylation of ERK, p38, and JNK, key components of the MAPK signaling pathway, was activated by LPS treatment, contributing to an inflammatory response. However, in cells pretreated with compound 1 at the indicated concentrations, the levels of p-ERK, p-p38, and p-JNK were significantly inhibited in a concentration-dependent manner. Specifically, at the concentration of 5 µM, compound 1 slightly decreases the phosphorylation of p38, ERK, and JNK in LPS-induced RAW264.7 cells, followed by the treatment with compound 1 at the concentration of 10 µM. Notably, at the concentrations of 20 µM and 40 µM, the phosphorylation of p38, ERK, and JNK was strongly inhibited compared to the LPS-stimulated cells group. These findings demonstrate that compound 1 attenuates inflammation in LPS-stimulated RAW264.7 cells by suppressing the phosphorylation of the MAPKs signaling pathway.

Many mediators are produced throughout the inflammatory processes. Two of these mediators–iNOS, an enzyme that synthesizes nitric oxide, and COX-2, an essential enzyme for prostaglandin synthesis–have been linked to a range of inflammatory responses. NO induced iNOS and COX-2 to produce their inflammatory effects [13]. Our data indicated that compound 1 reduced the NO production, and both COX-2 and iNOS protein expression levels were elevated in the LPS-stimulated RAW264.7 murine macrophage cells; however, it was suppressed in the treatment with compound 1 in a concentration-dependent manner. Subsequent NO activation can trigger the transcription of downstream inflammatory genes, including pro-inflammatory cytokines like IL-1β, TNF-α, and IL-6, as well as the activation of enzyme mediators like COX-2 and iNOS, which are regulated via signaling intermediation of the MAPK pathways [13]. In this study, the mRNA level of IL-1β, TNF-α, and IL-6 was decreased in the treatment with compound 1 at the indicated concentration, and the protein expression level of the phosphorylation of three main types of MAPK, including p38, ERK, and JNK was significantly inhibited compared with LPS stimulation alone.

In conclusion, two new labdane-type diterpenoids (1 and 2), a new natural compound (3), along with a known compound (4), were isolated from the aerial parts of L. sibiricus. Modern spectroscopic techniques, including NMR, HR-MS, and ECD spectra, were conducted to structurally elucidate their chemical structures. Among them, the new compound (3R,5R,6S,10S)-3,6-dihydroxy-15-ethoxy-7-oxolabden-8(9),13(14)-dien-15,16-olide (1) strongly inhibited LPS-induced NO production in macrophage cells via in vitro assays. Compound 1 also inhibited the LPS-induced expression of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α. Furthermore, the treatment of compound 1 suppressed the phosphorylation of p38, JNK, and ERK, three members of MAPKs. Taken together, the new compound 1 isolated from L. sibiricus shows potential anti-inflammatory effects in LPS-treated RAW264.7 cells, and it could be promising as a natural anti-inflammatory therapy.

Materials and Methods

General experimental procedures

Optical rotation was measured using a P-2000 digital polarimeter (JASCO, Tokyo, Japan) equipped with a tungsten-halogen lamp (WI) at a 589 nm wavelength, a photomultiplier tube (1P28 – 01) detector, and a CG2-100 cylindrical glass cell (2.5 Ø × 100 mm). CD spectra were recorded in the 190 – 400 nm range using a JASCO J-1500 CD spectrophotometer, equipped with a PM-539 detector, 0.025 nm wavelength resolution, 0.00 001 mdeg CD resolution, and a scanning speed of 50 nm/min, with measurements taken at room temperature (20 – 25 °C) using a 0.1 cm path length quartz cuvette. HR-ESI-MS was conducted using a 6530 Accurate-Mass Q-TOF LC/MS system (Agilent, Santa Clara, CA, USA). The 1D and 2D NMR spectra were measured with an Avance III HD 500 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) at 294 K, using tetramethylsilane as an internal standard, with J in Hz. MPLC was conducted on an Isolera One flash chromatography system (Biotage, Uppsala, Sweden). Column chromatography (CC) was performed using silica gel (Kieselgel 60 Å, 70 – 230 mesh) and RP-18 gel (LiChroprep RP-18, 40 – 63 µm) (Merck, Darmstadt, Germany). TLC was conducted on glass plates pre-coated with silica gel 60 F₂₅₄ and RP-18 F_254S (Merck, Darmstadt, Germany), with spots visualized under UV light and then detected by spraying with an aqueous 10% sulfuric acid reagent followed by heating at 120 °C for 1 – 2 min.

Plant material

The aerial parts of L. sibiricus were collected in Thai Binh province, Vietnam, in July 2019. The identification of the plant was performed by one of the authors, N. V. P., using both morphological characteristics, such as leaf shape, flower structure, and stem features, and comparison with authenticated herbarium specimens and the relevant botanical literature [18]. A voucher specimen of L. sibiricus (LS-2019-001) was maintained at the Institute of Marine Biochemistry, VAST, for future reference.

Chemicals and reagents

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbeccoʼs modified Eagleʼs medium (DMEM), fetal bovine serum (FBS), streptomycin, and penicillin were obtained from Life Technologies (Carlsbad, CA, USA).

Extraction and isolation

The air-dried aerial parts of L. sibiricus (3.0 kg) were extracted with MeOH (20 L × 3) under reflux for 5 h at room temperature (20 – 25℃). The solvent was evaporated under vacuum to obtain a MeOH residue (290 g). This was suspended in distilled water and then partitioned with n-hexane and EtOAc to yield the n-hexane extract (42 g), EtOAc extract (60 g), and water layer.

The EtOAc extract (60 g) was separated by silica gel MPLC using a mixture of n-hexane–acetone (gradient 100 : 1 – 1 : 1, v/v) as the mobile phase to obtain nine fractions, E1 – E9. Fraction E7 (6.1 g) was subjected to silica gel CC using an eluent of CH₂Cl₂–MeOH (gradient 20 : 1 – 1 : 1, v/v) to obtain four fractions, E7.1–E7.4. Compound 1 (4.2 mg) was purified from fraction E7.3 (166.7 mg) by RP-18 CC using acetone–H₂O (1 : 2, v/v) as the eluent. Fraction E6 (3.2 g) was separated on silica gel CC using a mixture solvent of CH₂Cl₂–MeOH (gradient 25 : 1 – 1 : 1, v/v) as the eluent, to give five fractions, E6.1–E6.5. From fraction E6.3 (101.4 mg), compound 2 (6.1 mg) was isolated by RP-18 CC eluting with acetone–H₂O (1 : 2.5, v/v). Fraction E4 (2.1 g) was separated by silica gel CC, using a mixture of CH₂Cl₂–MeOH (gradient 25 : 1 – 5 : 1, v/v) to obtain six fractions, E4.1–E4.6. From fraction E4.2 (83.5 mg), compounds 3 (3.1 mg) and 4 (2.5 mg) were isolated by silica gel CC using a mixture of n-hexane–acetone (4 : 1, v/v) as the mobile phase. All chemicals used for the extraction and isolation procedures were obtained from Daihan Scientific (Wonju, Gangwon, Korea) at the highest grade commercially available (min. 94%).

(3R,5R,6S,10S)-3,6-dihydroxy-15-ethoxy-7-oxolabden-8(9),13(14)-dien-15,16-olide (1): yellow oil; [α] _d − 58.6 (c 0.1, MeOH); CD (c 0.01 mM, MeOH) Δε _359 nm + 0.36, Δε _284 nm + 0.40, Δε _250 nm − 0.60, Δε _214 nm + 2.37; ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data, see [Table 1]; HR-ESI-MS m/z 415.2097 [M + Na]⁺ (calcd. for C₂₃H₃₂O₆Na, 415.2077).

(4R,5R,10S)-18-Hydroxy-14,15-bisnorlabda-8-en-7,13-dione (2): yellow oil; [α] _d + 14.6 (c 0.1, MeOH); CD (c 0.01 mM, MeOH) Δε _336 nm + 1.56, Δε _293 nm − 0.52, Δε _260 nm − 0.44, Δε _214 nm + 6.92; ¹H (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃) data, see [Table 1]; HR-ESI-MS m/z 293.2124 [M + H]⁺ (calcd. for C₁₈H₂₉O₃, 293.2117).

(R)-Methyl 2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoate (3): Light yellow amorphous powder; [α] _d − 11.6 (c 0.1, MeOH); CD (c 0.01 mM, MeOH) Δε _283 nm − 1.87, Δε _231 nm + 15.73; ¹H-NMR (CDCl₃, 500 MHz): δ _H 6.84 (1H, d, J = 8.0 Hz, H-5′), 6.74 (1H, d, J = 1.9 Hz, H-2′), 6.68 (1H, dd, J = 8.0, 1.9 Hz, H-6′), 5.51 (1H, br s, 4′-OH), 4.43 (1H, dd, J = 10.6, 6.2 Hz, H-2), 3.87 (3H, s, 3′-OCH₃), 3.78 (3H, s, 1-OCH₃), 3.06 (1H, dd, J = 14.1, 4.3 Hz, H-3a), 2.90 (1H, dd, J = 14.1, 6.6 Hz, H-3b), 2.69 (1H, d, J = 6.1 Hz, 2-OH); ¹³C-NMR (125 MHz, CDCl₃) δ _C 174.7 (C-1), 146.5 (C-3′), 144.8 (C-4′), 128.2 (C-1′), 122.3 (C-6′), 114.5 (C-5′), 112.2 (C-2′), 71.6 (C-2), 56.0 (3′-OCH₃), 52.6 (1-OCH₃), 40.3 (C-3); HR-ESI-MS m/z 225.0745 [M – H]⁻ (calcd. for C₁₁H₁₃O₅, 225.0763).

Cell culture

RAW 264.7 macrophage cells (ATCC TIB-71, RRID: CVCL_0493, Manassas, VA, USA) were cultured in DMEM medium containing 10% FBS, penicillin G (100 units/mL), streptomycin (100 µg/mL). Cells were nurtured at 37 °C, 5% CO₂, and 95% humidity. Cells were treated with various concentrations (5, 10, 20, and 40 µM/mL) of compound 1 and/or LPS (1 µg/mL).

Cell cytotoxicity assay

RAW264.7 murine macrophage cells (5 × 10³ cells/well) were seeded in a 96-well plate and nurtured for 24 h in the incubator before being treated with compound 1 (5, 10, 20, and 40 µM) and/or LPS (1 µg/mL) for an additional 24 h. After the media was discarded from the cells, MTT (5 mg/mL) was added to each well for 4 h before the supernatant was discarded with dimethyl sulfoxide (DMSO) to dissolve formazan crystals [37]. Cells were exposed at the 540 nm absorbance using a BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA) to detect cell viability based on the absorbance of treated cells relative to DMSO-only exposed cells.

Griess assay

RAW264.7 cells were seeded at a density of 5 × 10³ cells/well in 96-well plates. Cells were pre-treated at the concentration 40 µM of compounds 1–4 for 2 h, then stimulated with 1 µg/mL LPS for an additional 24 h at 37 °C with 5% CO₂. The culture supernatant (100 µL) was mixed with Griess reagent (100 µL) at an equal volume in a 96-well plate and incubated for 15 min at room temperature. The absorbance was measured at 540 nm using a BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA).

Quantitative real-time polymerase chain reaction

For total RNA isolation, harvested cells were lysed with TRIZOL reagent (Welgene Biosciences; Daegu, Korea). Then, the total RNA was isolated, and cDNA was synthesized with 500 ng of RNA using an RT PreMix kit (Enzynomics, Daejeon, Korea). Reverse transcription of the total RNA was performed using RT PreMix kit (Enzynomics, Korea). The total reaction volume was 10 µL containing 1 µL of cDNA/control and gene-specific primers. The gene expression was calculated using the equation as follows: gene expression = 2^−ΔΔCT, where ΔΔCT = (CT target−CT gapdh). The details of primer sequences are described in Table S1 (Supporting Information). The results are presented as the mean ± SD of three independent replicates.

Western blotting

Briefly, RAW264.7 cells were harvested for lysis in RIPA buffer with 1× protease inhibitor cocktail on ice for 30 min. After centrifugation at 16 000 g for 20 min at 4 °C, lysates were loaded for separation on SDS–PAGE gels. After transferring proteins to PVDF membranes, the membranes were blocked for 1 h using 5% skim milk in 0.1% TBS-T. Then, membranes were incubated with the indicated primary antibodies overnight. iNOS (cat. #2982, 1 : 1000), COX-2 (cat. #4842, 1 : 1000), phospho-p38 (cat. #9211, 1 : 1000), p38 (cat. #9212, 1 : 1000), phospho-p44/42 MAPK (Erk1/2) (cat. #9101, 1 : 1000), p44/42 MAPK (Erk1/2) (cat. #9102, 1 : 1000), phospho-SAPK/JNK (cat. #9251, 1 : 1000), SAPK/JNK (cat. #9252, 1 : 1000), and β-actin (cat. #4967, 1 : 1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). After washing three times with TBS-T, the membrane was rid of any extra primary antibodies before being treated for 2 h with secondary antibodies (against mouse or rabbit). Bands were identified using ECL Western blot detection reagents and an ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA) following three washes with 0.1% TBS-T.

Statistical Analysis

SPSS Statistics 27.0 (IBM, Armonk, NY, USA) was used for data analysis. The data gathered from three independent experiments were used to compute the mean values ± SD. One-way ANOVA was used to determine significance (p value) followed by Tukeyʼs post hoc test.

Contributorsʼ Statement

Data collection: N. M. Trang, L. B. Vinh, N. V. Phong; design of the study: N. V. Phong, S. Y. Yang; statistical analysis: N. M. Trang; analysis and interpretation of the data: N. M. Trang; drafting the manuscript: N. M. Trang; critical revision of the manuscript: N. V. Phong, S. Y. Yang.

Conflict of Interest

The authors declare that they have no conflict of interest.

• References

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  CrossrefPubMedSearch in Google Scholar
• 23 Pitschmann A, Zehl M, Heiss E, Purevsuren S, Urban E, Dirsch VM, Glasl S. Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae). Phytochem Anal 2016; 27: 23-31
  CrossrefPubMedSearch in Google Scholar
• 24 Merecz-Sadowska A, Sitarek P, Kowalczyk T, Palusiak M, Hoelm M, Zajdel K, Zajdel R. In vitro evaluation and in silico calculations of the antioxidant and anti-inflammatory properties of secondary metabolites from Leonurus sibiricus L. root extracts. Molecules 2023; 28: 6550
  CrossrefPubMedSearch in Google Scholar
• 25 Yang L, Liu G, Zhu X, Luo Y, Shang Y, Gu XL. The anti-inflammatory and antioxidant effects of leonurine hydrochloride after lipopolysaccharide challenge in broiler chicks. Poult Sci 2019; 98: 1648-1657
  CrossrefPubMedSearch in Google Scholar
• 26 Kuo HT, Peng CF, Huang HY, Lin CH, Chen IS, Tsai IL. Chemical constituents and antitubercular activity of Formosan Pisonia umbellifera . Planta Med 2011; 77: 736-741
  Thieme ConnectPubMedSearch in Google Scholar
• 27 Zhang RH, Liu ZK, Yang DS, Zhang XJ, Sun HD, Xiao WL. Phytochemistry and pharmacology of the genus Leonurus: The herb to benefit the mothers and more. Phytochemistry 2018; 147: 167-183
  CrossrefPubMedSearch in Google Scholar
• 28 Wang C, Tian J, Liu C, He Y, Li J, Zhang Q, Xiao T, Xie C, Yang C. Labdane and ent-halimane diterpenoids with STAT3-inhibitory activity from Leonurus sibiricus . Phytochemistry 2023; 214: 113802
  CrossrefPubMedSearch in Google Scholar
• 29 Zhang XJ, Zhong WM, Liu RX, Wang YM, Luo T, Zou Y, Qin HY, Li XL, Zhang R, Xiao WL. Structurally diverse labdane diterpenoids from Leonurus japonicus and their anti-inflammatory properties in LPS-induced RAW264.7 cells. J Nat Prod 2020; 83: 2545-2558
  CrossrefPubMedSearch in Google Scholar
• 30 Huong PTM, Phong NV, Huong NT, Trang DT, Thao DT, Cuong NX, Nam NH, Van Thanh N. Aplydactylonins A–C, three new sesquiterpenes from the Vietnamese sea hare Aplysia dactylomela and their cytotoxicity. J Nat Med 2022; 76: 210-219
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 32 Nguyen LTT, Vo HKT, Dang SV, Le TH, Ha LD, Nguyen LTT, Nguyen LHD. Labdane and norlabdane diterpenoids from the aerial parts of Leonurus japonicus . Phytochem Lett 2017; 22: 174-178
  CrossrefPubMedSearch in Google Scholar
• 33 Hwu JR, Varadaraju TG, Abd-Elazem IS, Huang RCC. First total syntheses of oresbiusins A and B, their antipodes, and racemates: Configuration revision and anti-HIV activity. Eur J Org Chem 2012; 2012: 4684-4688
  CrossrefPubMedSearch in Google Scholar
• 34 Wang HMD, Fu L, Cheng CC, Gao R, Lin MY, Su HL, Belinda NE, Nguyen TH, Lin WH, Lee PC, Hsieh LP. Inhibition of LPS-induced oxidative damages and potential anti-inflammatory effects of Phyllanthus emblica extract via down-regulating NF-κB, COX-2, and iNOS in RAW 264.7 cells. Antioxidants 2019; 8: 270
  CrossrefPubMedSearch in Google Scholar
• 35 Trang NM, Kim EN, Pham TH, Jeong GS. Citropten ameliorates osteoclastogenesis related to MAPK and PLCγ/Ca²⁺ signaling pathways through the regulation of amyloid beta. J Agric Food Chem 2023; 71: 10037-10049
  CrossrefPubMedSearch in Google Scholar
• 36 Trang NM, Kim EN, Lee HS, Jeong GS. Effect on osteoclast differentiation and ER stress downregulation by amygdalin and RANKL binding interaction. Biomolecules 2022; 12: 256
  CrossrefPubMedSearch in Google Scholar
• 37 Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Med 2002; 68: 445-448
  Thieme ConnectPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 16 May 2024

Accepted after revision: 12 October 2024

Accepted Manuscript online:
12 October 2024

Article published online:
04 November 2024

© 2024. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

• References

• 1 Megha KB, Joseph X, Akhil V, Mohanan PV. Cascade of immune mechanism and consequences of inflammatory disorders. Phytomedicine 2021; 91: 153712
  CrossrefPubMedSearch in Google Scholar
• 2 Viet Phong N, Thi Nguyet Anh D, Yeong Chae H, Young Yang S, Jeong Kwon M, Sun Min B, Ah Kim J. Anti-inflammatory activity and cytotoxicity against ovarian cancer cell lines by amide alkaloids and piperic esters isolated from Piper longum fruits: In vitro assessments and molecular docking simulation. Bioorg Chem 2022; 128: 106072
  CrossrefPubMedSearch in Google Scholar
• 3 Ba Vinh L, Jang HJ, Viet Phong N, Dan G, Won Cho K, Ho Kim Y, Young Yang S. Bioactive triterpene glycosides from the fruit of Stauntonia hexaphylla and insights into the molecular mechanism of its inflammatory effects. Bioorg Med Chem Lett 2019; 29: 2085-2089
  CrossrefPubMedSearch in Google Scholar
• 4 Wang S, Lei B, Zhang E, Gong P, Gu J, He L, Han L, Yuan Z. Targeted therapy for inflammatory diseases with mesenchymal stem cells and their derived exosomes: From basic to clinics. Int J Nanomedicine 2022; 17: 1757-1781
  CrossrefPubMedSearch in Google Scholar
• 5 Zhang Y, Zhao W, Ruan J, Wichai N, Li Z, Han L, Zhang Y, Wang T. Anti-inflammatory canthin-6-one alkaloids from the roots of Thailand Eurycoma longifolia Jack. J Nat Med 2020; 74: 804-810
  CrossrefPubMedSearch in Google Scholar
• 6 Zhang H, Wang M, Xu Y. Understanding the mechanisms underlying obesity in remodeling the breast tumor immune microenvironment: from the perspective of inflammation. Cancer Biol Med 2023; 20: 268-286
  CrossrefPubMedSearch in Google Scholar
• 7 Seo S, Lee KG, Shin JS, Chung EK, Lee JY, Kim HJ, Lee KT. 6′-O-Caffeoyldihydrosyringin isolated from Aster glehni suppresses lipopolysaccharide-induced iNOS, COX-2, TNF-α, IL-1β and IL-6 expression via NF-κB and AP-1 inactivation in RAW 264.7 macrophages. Bioorg Med Chem Lett 2016; 26: 4592-4598
  CrossrefPubMedSearch in Google Scholar
• 8 Serhan CN. Treating inflammation and infection in the 21st century: New hints from decoding resolution mediators and mechanisms. FASEB J 2017; 31: 1273-1288
  CrossrefPubMedSearch in Google Scholar
• 9 Vendrame S, Klimis-Zacas D. Anti-inflammatory effect of anthocyanins via modulation of nuclear factor-κB and mitogen-activated protein kinase signaling cascades. Nutr Rev 2015; 73: 348-358
  CrossrefPubMedSearch in Google Scholar
• 10 Park J, Min JS, Kim B, Chae UB, Yun JW, Choi MS, Kong IK, Chang KT, Lee DS. Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways. Neurosci Lett 2015; 584: 191-196
  CrossrefPubMedSearch in Google Scholar
• 11 Qiao Y, Yan W, He J, Liu X, Zhang Q, Wang X. Identification, evolution and expression analyses of mapk gene family in Japanese flounder (Paralichthys olivaceus) provide insight into its divergent functions on biotic and abiotic stresses response. Aquat Toxicol 2021; 241: 106005
  CrossrefPubMedSearch in Google Scholar
• 12 Bahar ME, Kim HJ, Kim DR. Targeting the RAS/RAF/MAPK pathway for cancer therapy: From mechanism to clinical studies. Signal Transduct Target Ther 2023; 8: 455
  CrossrefPubMedSearch in Google Scholar
• 13 Kim HK. Role of ERK/MAPK signalling pathway in anti-inflammatory effects of Ecklonia cava in activated human mast cell line-1 cells. Asian Pac J Trop Med 2014; 7: 703-708
  CrossrefPubMedSearch in Google Scholar
• 14 Zhao H, Wu L, Yan G, Chen Y, Zhou M, Wu Y, Li Y. Inflammation and tumor progression: Signaling pathways and targeted intervention. Signal Transduct Target Ther 2021; 6: 263
  CrossrefPubMedSearch in Google Scholar
• 15 Behl T, Upadhyay T, Singh S, Chigurupati S, Alsubayiel AM, Mani V, Vargas-De-La-Cruz C, Uivarosan D, Bustea C, Sava C, Stoicescu M, Radu AF, Bungau SG. Polyphenols targeting MAPK mediated oxidative stress and inflammation in rheumatoid arthritis. Molecules 2021; 26: 6570
  CrossrefPubMedSearch in Google Scholar
• 16 Sitarek P, Skała E, Toma M, Wielanek M, Szemraj J, Nieborowska-Skorska M, Kolasa M, Skorski T, Wysokińska H, Śliwiński T. A preliminary study of apoptosis induction in glioma cells via alteration of the Bax/Bcl-2-p 53 axis by transformed and non-transformed root extracts of Leonurus sibiricus L. Tumor Biol 2016; 37: 8753-8764
  CrossrefPubMedSearch in Google Scholar
• 17 Narukawa Y, Niimura A, Noguchi H, Tamura H, Kiuchi F. New diterpenoids with estrogen sulfotransferase inhibitory activity from Leonurus sibiricus L. J Nat Med 2014; 68: 125-131
  CrossrefPubMedSearch in Google Scholar
• 18 Pitschmann A, Waschulin C, Sykora C, Purevsuren S, Glasl S. Microscopic and phytochemical comparison of the three Leonurus species L. cardiaca, L. japonicus, and L. sibiricus . Planta Med 2017; 83: 1233-1241
  Thieme ConnectPubMedSearch in Google Scholar
• 19 Sayed MA, Alam MA, Islam MS, Ali MT, Ullah ME, Shibly AZ, Ali MA, Hasan-Olive MM. Leonurus sibiricus L. (honeyweed): A review of its phytochemistry and pharmacology. Asian Pac J Trop Biomed 2016; 6: 1076-1080
  CrossrefPubMedSearch in Google Scholar
• 20 Krishnan V, Subramaniam S, Chia-Chuan C, Venkatachalam B, Thomas Cheeran A, Chi-Ying FH. Anticancer activity of Leonurus sibiricus L.: Possible involvement of intrinsic apoptotic pathway. Nutr Cancer 2022; 74: 225-236
  CrossrefPubMedSearch in Google Scholar
• 21 Kim JH, Kim M, Jung HS, Sohn Y. Leonurus sibiricus L. ethanol extract promotes osteoblast differentiation and inhibits osteoclast formation. Int J Mol Med 2019; 44: 913-926
  PubMedSearch in Google Scholar
• 22 Li J, Niu L, Huang H, Li Q, Xie C, Yang C. Anti-inflammatory labdane diterpenoids from the aerial parts of Leonurus sibiricus. Phytochemistry 2024; 217: 113927
  CrossrefPubMedSearch in Google Scholar
• 23 Pitschmann A, Zehl M, Heiss E, Purevsuren S, Urban E, Dirsch VM, Glasl S. Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae). Phytochem Anal 2016; 27: 23-31
  CrossrefPubMedSearch in Google Scholar
• 24 Merecz-Sadowska A, Sitarek P, Kowalczyk T, Palusiak M, Hoelm M, Zajdel K, Zajdel R. In vitro evaluation and in silico calculations of the antioxidant and anti-inflammatory properties of secondary metabolites from Leonurus sibiricus L. root extracts. Molecules 2023; 28: 6550
  CrossrefPubMedSearch in Google Scholar
• 25 Yang L, Liu G, Zhu X, Luo Y, Shang Y, Gu XL. The anti-inflammatory and antioxidant effects of leonurine hydrochloride after lipopolysaccharide challenge in broiler chicks. Poult Sci 2019; 98: 1648-1657
  CrossrefPubMedSearch in Google Scholar
• 26 Kuo HT, Peng CF, Huang HY, Lin CH, Chen IS, Tsai IL. Chemical constituents and antitubercular activity of Formosan Pisonia umbellifera . Planta Med 2011; 77: 736-741
  Thieme ConnectPubMedSearch in Google Scholar
• 27 Zhang RH, Liu ZK, Yang DS, Zhang XJ, Sun HD, Xiao WL. Phytochemistry and pharmacology of the genus Leonurus: The herb to benefit the mothers and more. Phytochemistry 2018; 147: 167-183
  CrossrefPubMedSearch in Google Scholar
• 28 Wang C, Tian J, Liu C, He Y, Li J, Zhang Q, Xiao T, Xie C, Yang C. Labdane and ent-halimane diterpenoids with STAT3-inhibitory activity from Leonurus sibiricus . Phytochemistry 2023; 214: 113802
  CrossrefPubMedSearch in Google Scholar
• 29 Zhang XJ, Zhong WM, Liu RX, Wang YM, Luo T, Zou Y, Qin HY, Li XL, Zhang R, Xiao WL. Structurally diverse labdane diterpenoids from Leonurus japonicus and their anti-inflammatory properties in LPS-induced RAW264.7 cells. J Nat Prod 2020; 83: 2545-2558
  CrossrefPubMedSearch in Google Scholar
• 30 Huong PTM, Phong NV, Huong NT, Trang DT, Thao DT, Cuong NX, Nam NH, Van Thanh N. Aplydactylonins A–C, three new sesquiterpenes from the Vietnamese sea hare Aplysia dactylomela and their cytotoxicity. J Nat Med 2022; 76: 210-219
  CrossrefPubMedSearch in Google Scholar
• 31 Wahlberg I, Eklund AM, Nordfors K, Vogt C, Enzell CR, Berg JE. Tobacco Chemistry. 69. Five new labdanic compounds. Acta Chem Scand 1988; 42: 708-716
  CrossrefPubMedSearch in Google Scholar
• 32 Nguyen LTT, Vo HKT, Dang SV, Le TH, Ha LD, Nguyen LTT, Nguyen LHD. Labdane and norlabdane diterpenoids from the aerial parts of Leonurus japonicus . Phytochem Lett 2017; 22: 174-178
  CrossrefPubMedSearch in Google Scholar
• 33 Hwu JR, Varadaraju TG, Abd-Elazem IS, Huang RCC. First total syntheses of oresbiusins A and B, their antipodes, and racemates: Configuration revision and anti-HIV activity. Eur J Org Chem 2012; 2012: 4684-4688
  CrossrefPubMedSearch in Google Scholar
• 34 Wang HMD, Fu L, Cheng CC, Gao R, Lin MY, Su HL, Belinda NE, Nguyen TH, Lin WH, Lee PC, Hsieh LP. Inhibition of LPS-induced oxidative damages and potential anti-inflammatory effects of Phyllanthus emblica extract via down-regulating NF-κB, COX-2, and iNOS in RAW 264.7 cells. Antioxidants 2019; 8: 270
  CrossrefPubMedSearch in Google Scholar
• 35 Trang NM, Kim EN, Pham TH, Jeong GS. Citropten ameliorates osteoclastogenesis related to MAPK and PLCγ/Ca²⁺ signaling pathways through the regulation of amyloid beta. J Agric Food Chem 2023; 71: 10037-10049
  CrossrefPubMedSearch in Google Scholar
• 36 Trang NM, Kim EN, Lee HS, Jeong GS. Effect on osteoclast differentiation and ER stress downregulation by amygdalin and RANKL binding interaction. Biomolecules 2022; 12: 256
  CrossrefPubMedSearch in Google Scholar
• 37 Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Med 2002; 68: 445-448
  Thieme ConnectPubMedSearch in Google Scholar

• Supplementary Material