Citrus hystrix DC. (CH) is found in many countries in Southeast Asia. This plant has been reported for anti-microbial, anti-cancer and anti-inflammatory bioactivities. However, the anti-inflammatory and anti-inflammasome properties of the leaves remain poorly understood. This study aimed to investigate the effect of CH leaves on NLRP3 and NF-κB signaling pathways. CH leaves were sequentially extracted using hexane, ethyl acetate and 95% ethanol to give three crude extracts. An active compound, lupeol was fractionated from the ethanolic extract using chromatographic techniques, and its structure was identified and confirmed by spectroscopic methods. Anti-inflammatory activities were observed on both lipopolysaccharide-stimulated and NLRP3 adenosine triphosphate-induced macrophages. The release of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) was analyzed by Enzyme-Linked Immunosorbent Assay (ELISA). Real-time qRT-polymerase chain reaction (PCR) was used to measure inflammatory-associated gene expression. NF-κB protein expressions were investigated using the immunoblotting technique. The active fraction of ethanolic CH leaves and lupeol significantly reduced the release of pro-inflammatory cytokines and suppressed the expression of both inflammasome genes and NF-κB proteins. The ethanolic extract of CH leaves and lupeol showed potent anti-inflammatory activities by targeting NF-κB and NLRP3 signaling pathways.

Oral hygiene and control of microbial plaque biofilm formation are effective methods for preventing gingivitis. Mouthwashes containing leaf extracts of the medicinal plants Citrus hystrix DC. (KL), Moringa oleifera Lam. (MO) and Azadirachta indica A. Juss. (NE) were assessed for oral healthcare and gingivitis adjunctive treatment. Three types of mouthwash were developed; KL, a combination of KL and MO (KL + MO), and a combination of KL, and NE (KL + NE). The mouthwashes were tested in vivo on 47 subjects with gingivitis who were allocated into five groups as (i) placebo, (ii) KL, (iii) KL + MO, (iv) KL + NE, and (v) 0.12% chlorhexidine gluconate (CHX). Participants were instructed to rinse with herbal mouthwash twice daily for two weeks. Gingival index (GI), plaque index (PI), and oral microbial colonies were measured at baseline and 15 days. Results showed that GI and PI of groups (ii)–(iv) significantly decreased over the placebo group, while accumulative reduction percentages of both Staphylococcus spp. and Candida spp. were found in groups (iii) and (iv). Findings indicated that the herbal mouthwashes reduced GI and PI, and showed potential as oral healthcare products.

Liver fibrosis, characterized by excessive extracellular matrix deposition, is driven by activated hepatic stellate cells (HSCs). Due to the limited availability of anti-fibrotic drugs, the research on therapeutic agents continues. Here we have investigated Moringa oleifera Lam. (MO), known for its various bioactive properties, for anti-fibrotic effects. This study has focused on 1-phenyl-2-pentanol (1-PHE), a compound derived from MO leaves, and its effects on LX-2 human hepatic stellate cell activation. TGF-β1-stimulated LX-2 cells were treated with MO extract or 1-PHE, and the changes in liver fibrosis markers were assessed at both gene and protein levels. Proteomic analysis and molecular docking were employed to identify potential protein targets and signaling pathways affected by 1-PHE. Treatment with 1-PHE downregulated fibrosis markers, including collagen type I alpha 1 chain (COL1A1), collagen type IV alpha 1 chain (COL4A1), mothers against decapentaplegic homologs 2 and 3 (SMAD2/3), and matrix metalloproteinase-2 (MMP2), and reduced the secretion of matrix metalloproteinase-9 (MMP-9). Proteomic analysis data showed that 1-PHE modulates the Wnt/β-catenin pathway, providing a possible mechanism for its effects. Our results suggest that 1-PHE inhibits the TGF-β1 and Wnt/β-catenin signaling pathways and HSC activation, indicating its potential as an anti-liver-fibrosis agent.

Liver fibrosis, a consequence of chronic liver damage or inflammation, is characterized by the excessive buildup of extracellular matrix components. This progressive condition significantly raises the risk of severe liver diseases like cirrhosis and hepatocellular carcinoma. The lack of approved therapeutics underscores the urgent need for novel anti-fibrotic drugs. Hepatic stellate cells (HSCs), key players in fibrogenesis, are promising targets for drug discovery. This study investigated the anti-fibrotic potential of Citrus hystrix DC. (KL) and its bioactive compound, β-citronellol (β-CIT), in a human HSC cell line (LX-2). Cells exposed to TGF-β1 to induce fibrogenesis were co-treated with crude KL extract and β-CIT. Gene expression was analyzed by real-time qRT-PCR to assess fibrosis-associated genes (ACTA2, COL1A1, TIMP1, SMAD2). The release of matrix metalloproteinase 9 (MMP-9) was measured by ELISA. Proteomic analysis and molecular docking identified potential signaling proteins and modeled protein–ligand interactions. The results showed that both crude KL extract and β-CIT suppressed HSC activation genes and MMP-9 levels. The MAPK signaling pathway emerged as a potential target of β-CIT. This study demonstrates the ability of KL extract and β-CIT to inhibit HSC activation during TGF-β1-induced fibrogenesis, suggesting a promising role of β-CIT in anti-hepatic fibrosis therapies.

Moringa oleifera (MO) is a prominent plant in traditional medicine, widely recognized for its phytochemicals with anti-inflammatory properties. Liver fibrosis characterized by chronic inflammation and excessive extracellular matrix deposition may benefit from the therapeutic properties of MO. This report focuses on the potential of oleamide (OLA), a bioactive compound identified from MO, in mitigating liver fibrosis. The anti-fibrotic effects of OLA were evaluated by assessing the production of pro-inflammatory cytokines, gelatinase activity and the expression of genes and proteins associated with the TGF-β/SMAD2/3 pathway. The LX-2 human hepatic stellate cell line, in conjunction with TGF-β1, was employed to model fibrotic conditions. OLA treatment significantly reduced the production of pro-fibrotic effectors in the activated LX-2 cells. Molecular docking analysis demonstrated a high binding affinity of OLA to key proteins in the TGF-β/SMAD2/3 pathway, while qRT-PCR and Western blotting revealed that OLA suppressed the expression of COL1A1, COL4A1, SMAD2, SMAD3, SMAD4, MMP2, MMP9, ACTA2 and TIMP1. These findings indicate that OLA effectively attenuates the pro-inflammatory responses induced by TGF-β1 and inhibits the activation of LX-2 cells. Collectively, OLA holds significant potential as a therapeutic agent for the prevention and treatment of liver fibrosis via the modulation of the TGF-β/SMAD2/3 signaling pathway.

Liver fibrosis can progress to irreversible cirrhosis if the underlying causes remain, and this can in turn develop into hepatocellular carcinoma (HCC). Despite these adverse outcomes, liver fibrosis can be reversed. Consequently, research has focused on substances that target liver fibrosis to prevent or reduce its progression. This study deals with the potential anti-fibrotic action of 3-hydroxy-β-ionone (3-HBI), a bioactive compound found in many plants. To assess the putative effects of 3-HBI, pro-inflammatory cytokine production and the expression of genes and proteins associated with the TGF-β/SMAD2/3 pathway were monitored following exposure to 3-HBI. Initially, cells of the human hepatic stellate cell line LX-2 were treated with TGF-β1 to simulate fibrogenesis. Following the exposure of activated LX-2 cells to 3-HBI, the production of pro-fibrotic substances was significantly reduced. Molecular docking studies revealed that 3-HBI exhibited a high binding affinity for key proteins in the TGF-β/SMAD2/3 pathway. Analyses using qRT-PCR and Western blotting revealed that 3-HBI suppressed the expression of TIMP1, MMP2, MMP9, COL1A1, COL4A1, SMAD2, SMAD3, SMAD4, MMP2, and ACTA2. Together, these findings demonstrate that 3-HBI inhibited the activation of LX-2 cells and significantly reduced the proinflammatory responses triggered by TGF-β1. Accordingly, we confirmed the noteworthy potential of 3-HBI as a therapeutic agent to prevent and treat liver fibrosis, effected by its modulation of the TGF-β/SMAD2/3 signaling pathway.