Selection and evaluation of quality markers (Q-markers) of vladimiriae radix extract for cholestatic liver injury based on spectrum-effect relationship, pharmacokinetics, and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: As a representative local medicinal herb produced in China, Vladimiriae Radix (VR) has been proven to exert hepatoprotective and choleretic effects, with particular therapeutic efficacy in cholestatic liver injury (CLI), as demonstrated by the VR extract (VRE). However, the quality markers (Q-markers) of VRE for the treatment of CLI remain unclear. AIM OF THE STUDY: A new strategy based on the core element of "efficacy" was proposed, using a combination of spectrum-effect relationship, pharmacokinetics, and molecular docking methods to select and confirm Q-markers of VRE. MATERIAL AND METHODS: First, the HPLC fingerprinting of 10 batches of VRE was studied, and the in vivo pharmacological index of anti-CLI in rats was determined. The spectrum-effect relationship was utilized as a screening method to identify the Q-markers of VRE. Secondly, Q-markers were used as VRE pharmacokinetic markers to measure their concentrations in normal and CLI rat plasma, and to analyze their disposition. Finally, molecular docking was utilized to predict the potential interaction between the identified Q-markers and crucial targets of CLI. RESULTS: The fingerprints of 10 batches of VRE was established. The in vivo pharmacological evaluation of rats showed that VRE had a significant therapeutic effect on CLI. The spectrum-effect correlation analysis showed that costunolide (COS) and dehydrocostus lactone (DEH) were the Q-markers of VRE anti-CLI. The pharmacokinetic results showed that AUC(0-t), Cmax, CLZ/F, and VZ/F of COS and DEH in CLI rats had significant differences (P < 0.01). They were effectively absorbed into the blood plasma of CLI rats, ensuring ideal bioavailability, and confirming their role as Q-markers. Molecular docking results showed that COS, DEH had good affinity with key targets (FXR, CAR, PXR, MAPK, TGR5, NRF2) for CLI treatment (Binding energy < -4.52 kcal mol-1), further verifying the correctness of Q-marker selection. CONCLUSIONS: In this study, through the combination of experimental and theoretical approaches from the aspects of pharmacodynamic expression, in vivo process rules, and interaction force prediction, the therapeutic effect of VRE and Q-markers (COS、DEH) were elucidated. Furthermore, a new idea based on the principle of "efficacy" was successfully proposed for screening and evaluating Q-markers.

Exploration of costunolide derivatives as potential anti-inflammatory agents for topical treatment of atopic dermatitis by inhibiting MAPK/NF-κB pathways.

Atopic dermatitis (AD) is a common inflammatory disease and it is very difficult to treat. In the present work, a series of costunolide derivatives have been prepared, and in vitro and in vivo anti-inflammatory activities have evaluated. The results showed that most derivatives displayed good inhibition of NO generation with low cytotoxicity, and 7d could inhibit the phosphorylation of P38, P65 NF-κB and IκB-α in LPS-induced RAW264.7 model. The in vivo researches showed that 7d could improve skin injury symptoms, decrease Th2-type cytokine levels, inhibit HIS levels, alleviate scratching and repaire the damaged skin barrier through the inhibition of phosphorylation of MAPK and NF-κB signaling pathways on MC903-induced AD model. Therefore, costunolide derivatives may be new potent anti-AD agents for further study.

Three sesquiterpene lactones suppress lung adenocarcinoma by blocking TMEM16A-mediated Ca2+-activated Cl- channels.

Transmembrane protein TMEM16A, which encodes calcium-activated chloride channel has been implicated in tumorigenesis. Overexpression of TMEM16A is associated with poor prognosis and low overall survival in multiple cancers including lung adenocarcinoma, making it a promising biomarker and therapeutic target. In this study, three structure-related sesquiterpene lactones (mecheliolide, costunolide and dehydrocostus lactone) were extracted from the traditional Chinese medicine Aucklandiae Radix and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on the proliferation and migration of lung adenocarcinoma cells were examined. Whole-cell patch clamp experiments showed that these sesquiterpene lactones potently inhibited recombinant TMEM16A currents in a concentration-dependent manner. The half-maximal concentration (IC50) values for three tested sesquiterpene lactones were 29.9 ± 1.1 µM, 19.7 ± 0.4 µM, and 24.5 ± 2.1 µM, while the maximal effect (Emax) values were 100.0% ± 2.8%, 85.8% ± 0.9%, and 88.3% ± 4.6%, respectively. These sesquiterpene lactones also significantly inhibited the endogenous TMEM16A currents and proliferation, and migration of LA795 lung cancer cells. These results demonstrate that mecheliolide, costunolide and dehydrocostus lactone are novel TMEM16A inhibitors and potential candidates for lung adenocarcinoma therapy.

Costunolide alleviated DDC induced ductular reaction and inflammatory response in murine model of cholestatic liver disease.

Purpose: Cholestatic liver diseases are groups of hepatobiliary diseases without curative drug-based therapy options. Regulation of bile acid (BA) metabolism, hepatoperiductal fibrosis, and inflammatory response indicated present novel methods for the treatment of cholestatic liver disease. Costunolide (COS) from herb Saussurea lappa exerts a pharmacological effect of regulation of BA metabolism, liver fbrosis and inflammatory response. The present study aimed to clarify the pharmacodynamic effects of COS against the murine model of cholestatic liver disease. Methods: We established a murine model of cholestatic liver disease through chronic feeding of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 28 days. Two independent in vivo experiments were designed to reveal the pharmacological effect of COS against cholestatic liver disease. In the first experiment, two dosages of COS (10 and 30 mg/kg) were intraperitoneally injected into model mice daily for 14 days. In the second experiment, high dosage of COS (30 mg/kg) was intraperitoneally injected into control and model mice daily for 28 days. Results: In the evaluation of the hepatoprotective effect of COS, COS showed dosage-dependent improvement of cholestatic liver disease, including ductular reaction, hepatoperiductal fibrosis, and inflammatory response. The mechanism of COS-mediated hepatoprotective effects mainly relies on the regulation of BA metabolism, and the inflammatory response. DDC diet feed induced hepatic BA metabolism, transport and circulation dysfunction. COS treatment not only regulated the BA metabolism and transport gene, but also reprogrammed hepatic primary and secondary BA concentrations. DDC induced hepatic infiltrated monocytes derived macrophages and lymphocytes were inhibited, while Kupffer cells were preserved by COS treatment. The liver elevating inflammatory cytokines of DDC diet feed were alleviated by COS. Moreover, high dosage of 30 mg/kg COS treatment for 28 days resulted in no significant serological changes and no obvious hepatic histopathological changes when compared with control mice. Conclusion: COS protected against DDC diet feeding-induced cholestatic liver disease since COS regulated BA metabolism, ductular reaction, hepatoperiductal fibrosis and inflammatory response. COS is suggested as a potential natural product for the treatment of cholestatic liver disease.

HPLC-PDA Method for Quantification of Bioactive Compounds in Crude Extract and Fractions of Aucklandia costus Falc. and Cytotoxicity Studies against Cancer Cells.

Aucklandia costus Falc. (Synonym: Saussurea costus (Falc.) Lipsch.) is a perennial herb of the family Asteraceae. The dried rhizome is an essential herb in the traditional systems of medicine in India, China and Tibet. The important pharmacological activities reported for Aucklandia costus are anticancer, hepatoprotective, antiulcer, antimicrobial, antiparasitic, antioxidant, anti-inflammatory and anti-fatigue activities. The objective of this study was the isolation and quantification of four marker compounds in the crude extract and different fractions of A. costus and the evaluation of the anticancer activity of the crude extract and its different fractions. The four marker compounds isolated from A. costus include dehydrocostus lactone, costunolide, syringin and 5-hydroxymethyl-2-furaldehyde. These four compounds were used as standard compounds for quantification. The chromatographic data showed good resolution and excellent linearity (r2 ˃ 0.993). The validation parameters, such as inter- and intraday precision (RSD < 1.96%) and analyte recovery (97.52-110.20%; RSD < 2.00%),revealed the high sensitivity and reliability of the developed HPLC method. The compounds dehydrocostus lactone and costunolide were concentrated in the hexane fraction (222.08 and 65.07 µg/mg, respectively) and chloroform fraction (99.02 and 30.21 µg/mg, respectively), while the n-butanol fraction is a rich source of syringin (37.91 µg/mg) and 5-hydroxymethyl-2-furaldehyde (7.94 µg/mg). Further, the SRB assay was performed for the evaluation of anticancer activity using lung, colon, breast and prostate cancer cell lines. The hexane and chloroform fractions show excellent IC50 values of 3.37 ± 0.14 and 7.527 ± 0.18 µg/mL, respectively, against the prostate cancer cell line (PC-3).

Costunolide covalently targets and inhibits CaMKII phosphorylation to reduce ischemia-associated brain damage.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) is a leading cause of disability and mortality worldwide. Restoring cerebral blood flow (CBF) through vasodilatation is particularly important in the treatment of CCH. Costunolide (Cos) is a natural sesquiterpenoid compound with vasodilatory effect, but its mechanism is unclear. PURPOSE: This study aimed to investigate the vasodilatory mechanism of Cos and provide a new therapeutic regimen for treating CCH. METHODS: The therapeutic effect of Cos on CCH was assessed in a rat model of permanent common carotid artery occlusion. The direct target protein for improving CBF was identified by drug affinity responsive target stability combined with quantitative differential proteomics analysis. The molecular mechanism of Cos acting on its target protein was analyzed by multidisciplinary approaches. The signalling involved was assessed using site-directed pharmacological intervention. RESULTS: Cos has a significant therapeutic effect on ischemic brain injury by restoring CBF. Multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) was identified as a direct target of the natural small molecule Cos with a therapeutic effect on CCH. Mechanistic studies revealed that the α,ß-unsaturated-γ-lactone ring of Cos covalently binds to the Cys116 residue of CaMKII. It then inhibits the phosphorylation of CaMKII and reduces the calcium concentration in vascular smooth muscle cells, thus playing a role in vasodilation and increasing CBF. Notably, this covalent binding between Cos and CaMKII can exert a long-term vasodilator activity. CONCLUSION: We reported for the first time that Cos reduced ischemia-associated brain damage by covalently binding to the Cys116 residue of CaMKII, inhibiting CaMKII phosphorylation, and exerting long-term vasodilatory activity. This study not only found a new covalent inhibitor against the phosphorylation of CaMKII but also suggested that pharmacologically targeting CaMKII is a promising therapeutic strategy for CCH.

Costunolide covalently targets NACHT domain of NLRP3 to inhibit inflammasome activation and alleviate NLRP3-driven inflammatory diseases.

The NLRP3 inflammasome's core and most specific protein, NLRP3, has a variety of functions in inflammation-driven diseases. Costunolide (COS) is the major active ingredient of the traditional Chinese medicinal herb Saussurea lappa and has anti-inflammatory activity, but the principal mechanism and molecular target of COS remain unclear. Here, we show that COS covalently binds to cysteine 598 in NACHT domain of NLRP3, altering the ATPase activity and assembly of NLRP3 inflammasome. We declare COS's great anti-inflammasome efficacy in macrophages and disease models of gouty arthritis and ulcerative colitis via inhibiting NLRP3 inflammasome activation. We also reveal that the α-methylene-γ-butyrolactone motif in sesquiterpene lactone is the certain active group in inhibiting NLRP3 activation. Taken together, NLRP3 is identified as a direct target of COS for its anti-inflammasome activity. COS, especially the α-methylene-γ-butyrolactone motif in COS structure, might be used to design and produce novel NLRP3 inhibitors as a lead compound.

Inhibition of TAK1/TAB2 complex formation by costunolide attenuates obesity cardiomyopathy via the NF-κB signaling pathway.

BACKGROUND: Chronic and persistent obesity can lead to various complications, including obesity cardiomyopathy. Inhibition of the inflammatory response is an effective measure for the intervention of obesity cardiomyopathy. Numerous studies indicate that costunolide (Cos) can reduce inflammation. However, the role of Cos in obesity cardiomyopathy and its molecular targets remains unknown. HYPOTHESIS/PURPOSE: We aimed to clarify potential cardioprotective effects and mechanism of Cos against obesity cardiomyopathy. METHODS: The model of obesity cardiomyopathy was established by feeding mice with a high-fat diet for 24 weeks. Cos at 10 and 20 mg/kg or vehicle (1% CMCNa solution) was administered once every two days via oral gavage from the 17th to 24th week. Body weight, heart weight/tibia length, cardiac function, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers, inflammatory factors were assessed. The targets of Cos were predicted through molecular docking. Pull-down assay and biolayer interferometry were used to confirm the target of Cos. RESULTS: Cos effectively reduces obesity-induced cardiomyocyte inflammation, cardiac hypertrophy and fibrosis, thereby improving cardiac function. We confirmed that Cos can interact with TAK1 and inhibit downstream NF-κB pathway activation by blocking the formation of the TAK1/TAB2 complex, thus inhibiting inflammatory cytokine release in cardiomyocytes. CONCLUSION: Our results demonstrated that Cos significantly improved myocardial remodeling and cardiac dysfunction against obesity cardiomyopathy by reducing myocardial inflammation. Therefore, Cos may serve as a promising therapeutic agent in obesity cardiomyopathy.

Costunolide covalently targets NACHT domain of NLRP3 to inhibit inflammasome activation and alleviate NLRP3-driven inflammatory diseases

The NLRP3 inflammasome's core and most specific protein, NLRP3, has a variety of functions in inflammation-driven diseases. Costunolide (COS) is the major active ingredient of the traditional Chinese medicinal herb Saussurea lappa and has anti-inflammatory activity, but the principal mechanism and molecular target of COS remain unclear. Here, we show that COS covalently binds to cysteine 598 in NACHT domain of NLRP3, altering the ATPase activity and assembly of NLRP3 inflammasome. We declare COS's great anti-inflammasome efficacy in macrophages and disease models of gouty arthritis and ulcerative colitis via inhibiting NLRP3 inflammasome activation. We also reveal that the α-methylene-γ-butyrolactone motif in sesquiterpene lactone is the certain active group in inhibiting NLRP3 activation. Taken together, NLRP3 is identified as a direct target of COS for its anti-inflammasome activity. COS, especially the α-methylene-γ-butyrolactone motif in COS structure, might be used to design and produce novel NLRP3 inhibitors as a lead compound.

Epithelial Cell Adhesion Molecule (EpCAM) Expression Can Be Modulated via NFκB.

The epithelial cell adhesion molecule (EpCAM) is considered an essential proliferation signature in cancer. In the current research study, qPCR induced expression of EpCAM was noted in acute lymphoblastic leukemia (ALL) cases. Costunolide, a sesquiterpene lactone found in crepe ginger and lettuce, is a medicinal herb with anticancer properties. Expression of EpCAM and its downstream target genes (Myc and TERT) wasdownregulated upon treatment with costunolide in Jurkat cells. A significant change in the telomere length of Jurkat cells was not noted at 72 h of costunolide treatment. An in silico study revealed hydrophobic interactions between EpCAM extracellular domain and Myc bHLH with costunolide. Reduced expression of NFκB, a transcription factor of EpCAM, Myc, and TERT in costunolide-treated Jurkat cells, suggested that costunolide inhibits gene expression by targeting NFκB and its downstream targets. Overall, the study proposes that costunolide could be a promising therapeutic biomolecule for leukemia.

Mushroom-brush transitional conformation of mucus-inert PEG coating improves co-delivery of oral liposome for intestinal metaplasia therapy.

The blocking of gastric mucosal intestinal metaplasia (IM) has been considered to be the pivotal method to control the occurrence of gastric cancer. However, there is still a lack of effective therapeutic agent. Here, we developed mucus-penetrating liposome system by covering surface with polyethylene glycol (PEG) chains (hydrophilic and electroneutral mucus-inert material) to co-delivery candidate drugs combination. Then studied the impact on the transmucus performance of different conformations, which were constructed by controlling the density of PEG chains on the surface. The results showed that the particle size of 5%PEG-Lip was less than 120 nm, the polydispersity index was less than 0.3, and the surface potential tended to be neutral. The D value (long chain spacing) of 5% PEG-Lip was 3.25 nm, which was close to the RF value (diameter of spherical PEG long chain group without external force interference) of 3.44 nm, and the L value (extended length) was slightly larger than 3.44 nm. In this case, PEG showed mushroom-brush transitional conformation on the surface of liposomes. This conformation was not only promoted stable delivery, but also shielded the capture of mucus more favorably, leading to a more unrestricted transportation in mucus. The further in vivo experimental results demonstrated the rapid distribution of liposomes, which gradually appeared both in the superficial and deep glandular of mucosa and gland cells at 1 h and absorbed into the cell cytoplasm at 6 h. The 5% PEG-Lip with the mushroom-brush transitional configuration recalled abnormal organ index and improved inflammation and intestinal metaplasia. The modified PEG conformation assay presented here was more suitable for liposomes. This PEG-modified liposome system has potential of mucus-penetrating and provides a strategy for local treatment of gastric mucosal intestinal metaplasia.

Natural essential oils efficacious in internal organs fibrosis treatment: Mechanisms of action and application perspectives.

Internal organs fibrosis (IOF) is the leading cause of morbidity and mortality in most chronic inflammatory diseases, which is responsible for 45% of deaths due to disease. However, there is a paucity of drugs used to treat IOF, making it urgent to find medicine with good efficacy, low toxic side effects and good prognosis. Essential oils (EOs) extracted from natural herbs with a wide range of pharmacological components, multiple therapeutic targets, low toxicity, and broad sources have unique advantages and great potential in the treatment of IOF. In this review, we summarized EOs and their monomeric components with anti-IOF, and found that they work mainly through inhibiting TGF-ß-related signaling pathways, modulating inflammatory cytokines, suppressing NF-κB, and anti-oxidative stress. The prognostic improvement of natural EOs on IOF was further discussed, as well as the quality and safety issues in the current development of natural EOs. This review hopes to provide scientific basis and new ideas for the development and application of natural medicine EOs in anti-IOF.

Dynamics of germination stimulants dehydrocostus lactone and costunolide in the root exudates and extracts of sunflower.

Orobanche cumana Wallr. (Orobanche cernua Loefl.) causes severe yield losses of confectionary sunflower in China. While germination of O. cumana is stimulated by sesquiterpene lactones (STLs) from host sunflower (Helianthus annuus L.). Dehydrocostus lactone and costunolide isolated from sunflower root exudates are known as STLs to specifically induce O. cumana germination. Two major confectionary sunflower cultivars, SH363 (highly susceptible to O. cumana) and TH33 (resistant to O. cumana), were planted in China. However, STLs in these two sunflower cultivars has remained unknown. To identify STLs from root and exudates of sunflower for better understanding the role of stimulants in parasitic interaction of sunflower and O. cumana, we tested dehydrocostus lactone (DCL) and costunolide (CL) in root and root exudates of susceptible and resistant sunflower cultivars. The stimulant activity of sunflower root exudate and root extract to germination of O. cumana were also determined. Dehydrocostus lactone and costunolide were identified through ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). Both DCL and CL were found in root extracts and root exudates in the whole tested time point from two sunflower cultivars. The concentration of dehydrocostus lactone was higher than that of costunolide at the same tested growth stage of each sunflower cultivar. It was observed that higher quantity of dehydrocostus lactone in susceptible cultivar than resistant cultivar of root and root exudates at later tested developmental stages. However, the amount of CL was no significant difference between SH363 and TH33 at all tested stages. The release amount of DCL from susceptible cultivar is 3.7 folds that of resistant cultivar at 28 DAT. These findings suggested that DCL was the one of the major signal compound in these two sunflower cultivars, and lower dehydrocostus lactone might contribute to the resistance of sunflower TH33 to O. cumana.

Costunolide protects against alcohol-induced liver injury by regulating gut microbiota, oxidative stress and attenuating inflammation in vivo and in vitro.

Costunolide (cos) derived from the roots of Dolomiaea souliei (Franch.), which belongs to the Dolomiaea genus in the family Compositae, exert the anti-inebriation effect mainly by inhibiting the absorption of alcohol in the gastrointestinal tract. However, the protective effect of cos against alcohol-induced liver injury (ALI) remains obscure. The present study was aimed to evaluate the hepatoprotective effects of cos (silymarin was used as positive control) against ALI and its potential mechanisms. MTT was used to examine the effect of cos on the cell viability of L-02 cells. Plasma was separated from blood that used to test the levels of TNF-α, IL-6 and IL-12, and LPS while serum separated from blood which used to detect the level of ALT and AST. Liver tissues were obtained for histopathological examination and western blot analysis. Fresh mice feces samples were collected for the detection of bacterial composition. Cos exhibited protective effect against alcoholic-induced liver injury by regulating gut microbiota capacities (higher relative abundance of Firmicutes and Actinobacteria while lower in Bacteroidetes and Proteobacteria), adjusting oxidative stress (reduced the activities of MDA and ROS while promoted SOD, GSH and GSH-PX in L-02 cells) and attenuating inflammation (decreased the levels of ALT, AST, LPS, IL-6, IL-12 and TNF-α) via LPS-TLR4-NF-κB p65 signaling pathway, which might be an active therapeutic agent for treatment of ALI.

Effect of explant type (leaf, stem) and 2,4-D concentration on callus induction: influence of elicitor type (biotic, abiotic), elicitor concentration and elicitation time on biomass growth rate and costunolide biosynthesis in gazania (Gazania rigens) cell suspension cultures.

Gazania rigens (L.) Gaertn. (Asteraceae) is a medicinal plant with high ornamental potential and use in landscaping. The therapeutic potential of sesquiterpene lactones (SLs) as plant natural products for pharmaceutical development has gained extensive interest with costunolide (chemical name: 6E,10E,11aR-6,10-dimethyl-3-methylidene-3a,4,5,8,9,11a-hexahydrocyclodeca[b]furan-2-one) used as a popular herbal remedy due to its anti-cancer, antioxidant, anti-inflammatory, anti-microbial, anti-allergic, and anti-diabetic activities, among others. In the present study, two explant types (leaf, stem) and four 2,4-dichlorophenoxy acetic acid (2,4-D) concentrations (0, 0.5, 1 and 2 mg/L) were tested for callusing potential. The results showed that stem explants treated with 1.5 mg/L 2,4-D exhibited higher callus induction percentage (90%) followed by leaf explants (80%) with 1 mg/L 2,4-D, after a 4-week period. Cell suspension cultures were established from friable callus obtained from stem explants following a sigmoid pattern of growth curve with a maximum fresh weight at 20 days of subculture and a minimum one at 5 days of subculture. In the following stage, the effects of elicitation of cell suspension cultures with either yeast extract (YE) or methyl jasmonate (MeJA), each applied in five concentrations (0, 100, 150, 200 and 250 mg/L) on cell growth (fresh and dry biomass) and costunolide accumulation were tested. After 20 days of culture, YE or MeJA suppressed cell growth as compared to the non-elicited cells, while costunolide accumulation was better enhanced under the effect of 150 mg/L MeJA followed by 200 mg/L YE, respectively. In the subsequent experiment conducted, the optimal concentration of the two elicitors (200 mg/L YE, 150 mg/L MeJA) was selected to investigate further elicitation time (0, 5, 10, 15 and 20 days). The results revealed that YE biotic elicitation stimulated cell growth and costunolide production, being maximum on day 20 for fresh biomass, on day 5 for dry biomass and on day 15 for the bioactive compound. Accordingly, cell growth parameters were maximized under the effect of abiotic elicitation with MeJA for 15 days, while highest costunolide content was achieved after 10 days. Overall, MeJA served as a better elicitor type than YE for biomass and costunolide production. Irrespective of elicitor type, elicitor concentration and elicitation time, maximal response was obtained with 150 mg/L MeJA for 10 days regarding costunolide accumulation (18.47 ppm) and 15 days for cell growth (fresh weight: 954 mg and dry weight: 76.3 mg). The application of elicitors can lead the large quantity of costunolide to encounter extensive range demand through marketable production without endangering of G. rigens.

Phenotype-Based HPLC-Q-TOF-MS/MS Coupled With Zebrafish Behavior Trajectory Analysis System for the Identification of the Antidepressant Components in Methanol Extract of Anshen Buxin Six Pills.

Phenotype screening has become an important tool for the discovery of active components in traditional Chinese medicine. Anshen Buxin Six Pills (ASBX) are a traditional Mongolian medicine used for the treatment of neurosis in clinical settings. However, its antidepressant components have not been explicitly identified and studied. Here, the antidepressant effect of ASBX was evaluated in adult zebrafish. High performance liquid chromatography-mass spectrometry (HPLC-Q-TOF-MS/MS) was combined with zebrafish behavior trajectory analysis to screen and identify the antidepressant-active extract fraction and active components of ASBX. Finally, the antidepressant effect of the active ingredients were verified by the behavior, pathology, biochemical indices and protein level of adult fish. The novel tank driving test (NTDT) showed that ASBX can effectively improve the depressive effect of reserpine on zebrafish. Petroleum ether and dichloromethane extracts of ASBX were screened as antidepressant active extracts. Costunolide (COS) and dehydrocostus lactone (DHE) were screened as the active components of ASBX. COS had been shown to significantly improve the depressive behavior, nerve injury and neurotransmitter levels (5-hydroxytryptamine (5-HT) and norepinephrine (NE)) of zebrafish by inhibiting the high expression of serotonin transporter and norepinephrine transporter induced by reserpine suggesting the antidepressant effect of COS may be related to its effect on 5-HT and NE pathways. This study provided a phenotype based screening method for antidepressant components of traditional Chinese medicines, so as to realize the separation, identification and activity screening of components at the same time.

Costunolide Loaded in pH-Responsive Mesoporous Silica Nanoparticles for Increased Stability and an Enhanced Anti-Fibrotic Effect.

Liver fibrosis remains a significant public health problem. However, few drugs have yet been validated. Costunolide (COS), as a monomeric component of the traditional Chinese medicinal herb Saussurea Lappa, has shown excellent anti-fibrotic efficacy. However, COS displays very poor aqueous solubility and poor stability in gastric juice, which greatly limits its application via an oral administration. To increase the stability, improve the dissolution rate and enhance the anti-liver fibrosis of COS, pH-responsive mesoporous silica nanoparticles (MSNs) were selected as a drug carrier. Methacrylic acid copolymer (MAC) as a pH-sensitive material was used to coat the surface of MSNs. The drug release behavior and anti-liver fibrosis effects of MSNs-COS-MAC were evaluated. The results showed that MSNs-COS-MAC prevented a release in the gastric fluid and enhanced the dissolution rate of COS in the intestinal juice. At half the dose of COS, MSNs-COS-MAC still effectively ameliorated parenchymal necrosis, bile duct proliferation and excessive collagen. MSNs-COS-MAC significantly repressed hepatic fibrogenesis by decreasing the expression of hepatic fibrogenic markers in LX-2 cells and liver tissue. These results suggest that MSNs-COS-MAC shows great promise for anti-liver fibrosis treatment.

Thymoquinone and Costunolide Induce Apoptosis of Both Proliferative and Doxorubicin-Induced-Senescent Colon and Breast Cancer Cells.

Doxorubicin (Dox) induces senescence in numerous cancer cell types, but these senescent cancer cells relapse again if they are not eliminated. On this principle, we investigated the apoptotic effect of thymoquinone (TQ), the active ingredient of Nigella sativa seeds and costunolide (COS), the active ingredient of Costus speciosus, on the senescent colon (Sen-HCT116) and senescent breast (Sen-MCF7) cancer cell lines in reference to their corresponding proliferative cells to rapidly eliminate the senescent cancer cells. The senescence markers of Sen-HCT116 and Sen-MCF7 were determined by a significant decrease in bromodeoxyuridine (BrdU) incorporation and significant increases in SA-ß-gal, p53, and p21 levels. Then proliferative, Sen-HCT116, and Sen-MCF7 cells were subjected to either TQ (50 µM) or COS (30 µM), the Bcl2-associated X protein (Bax), B-cell lymphoma 2 (Bcl2), caspase 3 mRNA expression and its activity were established. Results revealed that TQ significantly increased the Bax/Bcl2 ratio in HCT116 + Dox5 + TQ, MCF7 + TQ, and MCF7 + Dox5 + TQ compared with their corresponding controls. COS significantly increased the Bax/Bcl2 ratio in HCT116 + Dox5 + TQ and MCF7 + Dox5 + TQ compared with their related controls. Also, TQ and COS were significantly increased caspase 3 activity and cell proliferation of Sen-HCT116 and Sen-MCF7. The data revealed a higher sensitivity of senescent cells to TQ or COS than their corresponding proliferative cells.

Dolomiaea souliei ethyl acetate extract protected against α-naphthylisothiocyanate-induced acute intrahepatic cholestasis through regulation of farnesoid x receptor-mediated bile acid metabolism.

BACKGROUND: Cholestasis is characterized by accumulation of bile components in liver and systemic circulation. Restoration of bile acid homeostasis via activating farnesoid x receptor (FXR) is a promising strategy for the treatment of cholestasis. FXR-SHP (small heterodimer partner) axis plays an important role in maintaining bile acid homeostasis. PURPOSE: To investigate the anti-cholestasis effect of Dolomiaea souliei (Franch.) C.Shih (D. souliei) and clarify its underlying mechanism against α-naphthylisothiocyanate (ANIT) induced acute intrahepatic cholestasis. METHODS: ANIT-induced Sprague-Dawley rats were employed to investigate the anti-cholestasis effect of D. souliei ethyl acetate extract (DSE). Ursodeoxycholic acid (UDCA) was used as positive control. Bile flow and blood biochemical parameters were measured. Liver histopathological examination was conducted via hematoxylin-eosin staining. Western blot analysis was carried out to evaluate the protein levels related to bile acids metabolism and inflammation. The interactions between FXR and costunolide or dehydrocostus lactone, were conducted by molecular docking experiments. The effect of costunolide and dehydrocostus lactone on aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels and FXR expression were also evaluated using guggulsterone-induced L02 cells. RESULTS: DSE could promote bile excretions and protect against ANIT-induced liver damage in cholestasis rats. Protein levels of FXR, SHP, Na+/taurocholate cotransporter (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2) were increased and the expressions of cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) were decreased by DSE. Meanwhile, the anti-inflammatory factors, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) were also significantly increased, and the pro-inflammatory factor, interleukin-10 (IL-10), was significantly decreased in rats of DSE groups. Molecular docking revealed that costunolide and dehydrocostus lactone could be well docked into the FXR protein molecule, and hydrophobic interactions played the main function. Costunolide could reverse the increased AST and ALT levels and increase the FXR expression in guggulsterone-induced L02 cells. CONCLUSION: DSE had an anti-cholestasis effect by activating FXR-SHP axis, inhibiting synthesis of bile acid, and increasing bile secretion, together with inflammatory response and improving liver injury. Costunolide may be the main active component. This study provided a potential therapeutic mechanism for D. souliei as an anti-cholestasis medicine in the treatment of cholestasis liver diseases.

Targeting AKT with costunolide suppresses the growth of colorectal cancer cells and induces apoptosis in vitro and in vivo.

BACKGROUND: Colorectal cancer (CRC) is a clinically challenging malignant tumor worldwide. As a natural product and sesquiterpene lactone, Costunolide (CTD) has been reported to possess anticancer activities. However, the regulation mechanism and precise target of this substance remain undiscovered in CRC. In this study, we found that CTD inhibited CRC cell proliferation in vitro and in vivo by targeting AKT. METHODS: Effects of CTD on colon cancer cell growth in vitro were evaluated in cell proliferation assays, migration and invasion, propidium iodide, and annexin V-staining analyses. Targets of CTD were identified utilizing phosphoprotein-specific antibody array; Costunolide-sepharose conjugated bead pull-down analysis and knockdown techniques. We investigated the underlying mechanisms of CTD by ubiquitination, immunofluorescence staining, and western blot assays. Cell-derived tumour xenografts (CDX) in nude mice and immunohistochemistry were used to assess anti-tumour effects of CTD in vivo. RESULTS: CTD suppressed the proliferation, anchorage-independent colony growth and epithelial-mesenchymal transformation (EMT) of CRC cells including HCT-15, HCT-116 and DLD1. Besides, the CTD also triggered cell apoptosis and cell cycle arrest at the G2/M phase. The CTD activates and induces p53 stability by inhibiting MDM2 ubiquitination via the suppression of AKT's phosphorylation in vitro. The CTD suppresses cell growth in a p53-independent fashion manner; p53 activation may contribute to the anticancer activity of CTD via target AKT. Finally, the CTD decreased the volume of CDX tumors without of the body weight loss and reduced the expression of AKT-MDM2-p53 signaling pathway in xenograft tumors. CONCLUSIONS: Our project has uncovered the mechanism underlying the biological activity of CTD in colon cancer and confirmed the AKT is a directly target of CTD. All of which These results revealed that CTD might be a new AKT inhibitor in colon cancer treatment, and CTD is worthy of further exploration in preclinical and clinical trials.