Bithiophene and coumestan derivatives from Eclipta prostrata (L.) L. and their hepatoprotective activity.

One new bithiophene derivative, 5-(but-3-en-1-yn-1-yl)-5'-(methoxymethyl)-2,2'-bithiophene (1), along with twelve known compounds, senecioester (2), tiglinsaureester (3), 5-acetoxymethyl-2'-(but-3-en-1-yn-1-yl)-2,5'-bithiophene (4), 5-(4-isovaleroyloxybut-1-ynyl)-2,2'-bithiophene (5), 5-hydroxymethyl-(2,5':2',5'')-terthienyl tiglate (6), 5-hydroxymethyl-(2,5':2',5'')-terthienyl agelate (7), 5- hydroxymethyl-2,5':2',5''-terthiophene dimethylacrylate (8), 5-methoxymethyl-2,2':5',2''-terthiophene (9), α-terthiophene (10), 1,3,8,9-tetrahydroxycoumestan 3-sulfate (11), demethylwedelolactone (12), and wedelolactone (13) were isolated from the methanol extract of aerial parts of Eclipta prostrata (L.) L. All isolated compounds were evaluated for the protective ability on the HepG2 cells. At the concentration of 100 µM, compounds 11-13 showed the highest hepatoprotective effects, with HepG2 cell viability ranging from 38.68% to 48.54%. Bithiophenes showed higher hepatoprotective cell viability than terthiophenes.

Assessment of hemostatic ability of biomaterial based on chitosan andEclipta prostrataL. extract.

The biomaterials based on chitosan andEclipta prostrataL. extract have been prepared by microemulsion method and solution method (with and without sodium tripolyphosphate (STPP) as a cross-linking agent). The main component inEclipta prostrataL. extract is flavonoid groups. The structure of the chitosan/extract biomaterials was studied by infrared spectroscopy. The chitosan/extract biomaterial using STPP cross-linker appeared an absorption band at 1152 cm-1attributed to the vibrations of C-O-P bonds, which proved that chitosan has crosslinked with STPP. The morphology of the biomaterials was investigated by the dynamic light scattering technique and field emission scanning electron microscopy. The obtained results showed that the particle size of the chitosan/extract biomaterials prepared by microemulsion method and solution method with STPP ranged from 68.06 nm to 1484 nm, with an average particle size of 304.9-1019 nm. The microemulsion method produced biomaterials with much smaller average particle size than the solution method using cross-linkers. The hemostatic ability of the biomaterials was better than that of the control sample based on the time of blood clotting formation and glomerular aggregation ability. The sample with the ratio ofE. prostrataL. extract: chitosan of 1:30 had the lowest hemostasis time (6 min 46 s) and its glomerular aggregation rate after 5 min was 13.05%. This indicated that the biomaterials based on chitosan andE. prostrataL. extract are promising for application in biomedicine as hemostatic materials.

Antioxidant and Antiproliferative Activities of Eclipta prostrata (L.) L. Extract and Isolated Compounds.

The primary objective of this study was to elucidate the chemical composition, antioxidant properties, and antiproliferative activities of Eclipta prostrata extracts. Two flavonoids, 3'-O-methylorobol and apigenin 7-sulfate, were isolated from the ethyl acetate (EtOAc) extract of E. prostrata. The total phenolic and flavonoid contents of the E. prostrata extracts, as well as their overall antioxidant activities as measured using the 2,2-diphenyl-1-picrylhydrazyl and reducing power assays, were investigated. The E. prostrata EtOAc extract exhibited significantly greater antioxidant activities in both assays and higher phenol and flavonoid contents than the other extracts. The potential antiproliferative properties of the E. prostrata extracts and isolated compounds were investigated in vitro against the AGS, A549, and HT-29 cancer cell lines and the normal human HEK-293 cell line using the MTT assay. Annexin V-FITC/PI staining analysis and quantitative real-time PCR were used to assess AGS cell apoptosis. At a concentration of 100 µg/mL, the EtOAc extract of E. prostrata reduced AGS cell viability and proliferation by inducing apoptosis through the alteration of gene expression in the apoptotic cascade. These results highlight E. prostrata as a promising source of anticancer compounds.

Assessment of the Chemical Profile and Potential Medical Effects of a Flavonoid-Rich Extract of Eclipta prostrata L. Collected in the Central Highlands of Vietnam.

Eclipta prostrata L. (EPL), a medicinal plant, is widely utilized in the central highlands of Vietnam. This study aims to assess the chemical profile and potential medical effects of an EPL extract rich in flavonoids. A total of 36 secondary metabolites were identified from the EPL extract through GC-MS and UHPLC-UV analysis. Among them, 15 volatile compounds and several phenolic and flavonoid chemicals, including salicylic acid, epicatechin gallate, isovitexin, and apigetrin, were reported in EPL extract for the first time. This herbal extract demonstrated moderate inhibition against α-amylase and α-glucosidase, and high anti-oxidant and anti-acetylcholinesterase activities (IC50 = 76.8 ± 0.8 µg/mL). These promising attributes can be likely attributed to the high levels of major compounds, including wedelolactone (1), chlorogenic acid (3), epicatechin gallate (6), salicylic acid (8), isovitexin (9), apigetrin (11), and myricetin (12). These findings align with the traditional use of EPL for enhancing memory and cognitive function, as well as its potential benefits in diabetes management. The results of the molecular docking study reveal that the major identified compounds (1, 6, 9, and 11) showed a more effective acetylcholinesterase inhibitory effect than berberine chloride, with good binding energy (DS values, -12.3 to -14.3 kcal/mol) and acceptable values of RMSD (1.02-1.67 Å). Additionally, almost all the identified major compounds exhibited good ADMET properties within the required limits.

Discovery of the radio-protecting effect of Ecliptae Herba, its constituents and targeting p53-mediated apoptosis in vitro and in vivo.

Radiation protection drugs are often accompanied by toxicity, even amifostine, which has been the dominant radio-protecting drug for nearly 30 years. Furthermore, there is no therapeutic drug for radiation-induced intestinal injury (RIII). This paper intends to find a safe and effective radio-protecting ingredient from natural sources. The radio-protecting effect of Ecliptae Herba (EHE) was discovered preliminarily by antioxidant experiments and the mouse survival rate after 137Cs irradiation. EHE components and blood substances in vivo were identified through UPLC‒Q-TOF. The correlation network of "natural components in EHE-constituents migrating to blood-targets-pathways" was established to predict the active components and pathways. The binding force between potential active components and targets was studied by molecular docking, and the mechanism was further analyzed by Western blotting, cellular thermal shift assay (CETSA), and ChIP. Additionally, the expression levels of Lgr5, Axin2, Ki67, lysozyme, caspase-3, caspase-8,8-OHdG, and p53 in the small intestine of mice were detected. It was found for the first time that EHE is active in radiation protection and that luteolin is the material basis of this protection. Luteolin is a promising candidate for RⅢ. Luteolin can inhibit the p53 signaling pathway and regulate the BAX/BCL2 ratio in the process of apoptosis. Luteolin could also regulate the expression of multitarget proteins related to the same cell cycle.

Discovery of the radio-protecting effect of Ecliptae Herba, its constituents and targeting p53-mediated apoptosis in vitro and in vivo

Radiation protection drugs are often accompanied by toxicity, even amifostine, which has been the dominant radio-protecting drug for nearly 30 years. Furthermore, there is no therapeutic drug for radiation-induced intestinal injury (RIII). This paper intends to find a safe and effective radio-protecting ingredient from natural sources. The radio-protecting effect of Ecliptae Herba (EHE) was discovered preliminarily by antioxidant experiments and the mouse survival rate after 137Cs irradiation. EHE components and blood substances in vivo were identified through UPLC‒Q-TOF. The correlation network of "natural components in EHE-constituents migrating to blood-targets-pathways" was established to predict the active components and pathways. The binding force between potential active components and targets was studied by molecular docking, and the mechanism was further analyzed by Western blotting, cellular thermal shift assay (CETSA), and ChIP. Additionally, the expression levels of Lgr5, Axin2, Ki67, lysozyme, caspase-3, caspase-8,8-OHdG, and p53 in the small intestine of mice were detected. It was found for the first time that EHE is active in radiation protection and that luteolin is the material basis of this protection. Luteolin is a promising candidate for RⅢ. Luteolin can inhibit the p53 signaling pathway and regulate the BAX/BCL2 ratio in the process of apoptosis. Luteolin could also regulate the expression of multitarget proteins related to the same cell cycle.

Anti-inflammatory effects of Eclipta prostrata Linné on house dust mite-induced atopic dermatitis in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Atopic dermatitis (AD) is a kind of inflammation on the skin following with swollen, itchy, dryness and cracked skin. Though the exact cause of AD is unknown, there are evidence that people with AD have a compromised skin barrier along with inflammation. Eclipta prostrata Linné is a traditional herbal medicinal plant, has been used for the diabetes, obesity, jaundice, and inflammation. We supposed E. prostrata L. has an anti-inflammatory effect on the skin. AIM OF THE STUDY: We aimed to assess the effect of E. prostrata L. EtOH extract (EP) and elucidate the associated molecular mechanisms. MATERIALS AND METHODS: The effect of EP and the molecular mechanisms were eluciated in house dust mite (HDM)-induced AD mice model and TNF-α/IFN-γ-stimulated HaCaT keratinocytes by histological analysis, enzyme-linked immunosorbent assay, quantitative real time polymerase chain reaction, and Western blot. RESULTS: The results revealed that EP improved the progression of AD symptoms, decreasing epidermis/dermis thickness, infiltrated immune cells, and restored the skin barrier dysfunction and imbalanced immune response. EP suppressed the expressions of T helper (Th)1, Th2, Th17 cytokines, phosphorylation of extracellular signal-regulated kinase/signal transducer and activator of transcription 1 in skin of HDM-induced AD mice as well as inhibition the translocation of nuclear factor-κB in HaCaT keratinocytes. CONCLUSIONS: Collectively, EP improved the allergic inflammation of the skin through recovery the skin barrier, and regulation the immune balance. These results suggest EP may have therapeutic potential as an anti-atopic agent.

Jasmonates promote enhanced production of bioactive caffeoylquinic acid derivative in Eclipta prostrata (L.) L. hairy roots.

Eclipta prostrata (L.) L. is widely used in traditional medicine for treatment of hepatitis, poisoning from snake bites and viral infections. Pharmacological studies confirmed its antioxidant, anti-inflammatory and anticancer activities. The efficacy of E. prostrata (L.) L. extracts has been correlated to phenylpropanoids such as flavonoids, coumestans and caffeoylquinic acid derivatives. In this work, the production of wedelolactone, demethylwedelolactone and 3,5-di-O-caffeoylquinic acid (3,5-diCQA) in hairy root cultures of E. prostrata (L.) L. C19 clone was increased after addition of eliciting agents jasmonic acid (JA) or methyl jasmonate (MeJA) at multiple concentrations. Cultures elicited with 100 µM of JA saw a 5.2 fold increase in wedelolactone (from 0.72 to 3.72 mg/g d.w.), a 1.6 fold increase in demethylwedelolactone (from 5.54 to 9.04 mg/g d.w.) and a 2.47 fold increase in 3,5-diCQA (from 18.08 to 44.71 mg/g d.w.). Obtained data validate the potential of E. prostrata (L.) L. hairy root cultures as a production system of wedelolactone, demethylwedelolactone and especially 3,5-diCQA, which has recently been reported to possess activity against coronavirus disease (Covid-19) by in silico computational studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s11240-021-02201-4.

A network pharmacology-based approach to explore the active ingredients and molecular mechanism of Lei-gong-gen formula granule on a spontaneously hypertensive rat model.

BACKGROUND: Lei-gong-gen formula granule (LFG) is a folk prescription derived from Zhuang nationality, the largest ethnic minority among 56 nationalities in China. It consists of three herbs, namely Eclipta prostrata (L.) L., Smilax glabra Roxb, and Centella asiatica (L.) Urb. It has been widely used as health protection tea for hundreds of years to prevent hypertension in Guangxi Zhuang Autonomous Region. The purpose of this study is to validate the antihypertensive effect of LFG on the spontaneously hypertensive rat (SHR) model, and to further identify the effective components and anti-hypertension mechanism of LFG. METHODS: The effects of LFG on blood pressure, body weight, and heart rate were investigated in vivo using the SHR model. The levels of NO, ANG II, and ET-1 in the serum were measured, and pathological changes in the heart were examined by H&E staining. The main active components of LFG, their corresponding targets, and hypertension associated pathways were discerned through network pharmacology analysis based on the Traditional Chinese Medicine Systems Pharmacology (TCMSP), Traditional Chinese Medicine Integrated Database (TCMID), and the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM). Then the predicted results were further verified by molecular biology experiments such as RT-qPCR and western blot. Additionally, the potential active compounds were predicted by molecular docking technology, and the chemical constituents of LFG were analyzed and identified by UPLC-QTOF/MS technology. Finally, an in vitro assay was performed to investigate the protective effects of potential active compounds against hydrogen peroxide (H2O2) induced oxidative damage in human umbilical vein endothelial cells (HUVEC). RESULTS: LFG could effectively reduce blood pressure and increase serum NO content in SHR model. Histological results showed that LFG could ameliorate pathological changes such as cardiac hypertrophy and interstitial inflammation. From network pharmacology analysis, 53 candidate active compounds of LFG were collected, which linked to 765 potential targets, and 828 hypertension associated targets were retrieved, from which 12 overlapped targets both related to candidate active compounds from LFG and hypertension were screened and used as the potential targets of LFG on antihypertensive effect. The molecular biology experiments of the 12 overlapped targets showed that LFG could upregulate the mRNA and protein expressions of NOS3 and proto-oncogene tyrosine-protein kinase SRC (SRC) in the thoracic aorta. Pathway enrichment analysis showed that the PI3K-AKT signaling pathway was closely related to the expression of NOS3 and SRC. Moreover, western blot results showed that LFG significantly increased the protein expression levels of PI3K and phosphorylated AKT in SHR model, suggesting that LFG may active the PI3K-AKT signaling pathway to decrease hypertension. Molecular docking study further supported that p-hydroxybenzoic acid, cedar acid, shikimic acid, salicylic acid, nicotinic acid, linalool, and histidine can be well binding with NOS3, SRC, PI3K, and AKT. UPLC-QTOF/MS analysis confirmed that p-hydroxybenzoic acid, shikimic acid, salicylic acid, and nicotinic acid existed in LFG. Pre-treatment of HUVEC with nicotinic acid could alleviate the effect on cell viability induced by H2O2 and increase the NO level in cell supernatants. CONCLUSIONS: LFG can reduce the blood pressure in SHR model, which might be attributed to increasing the NO level in serum for promoting vasodilation via upregulating SRC expression level and activating the PI3K-AKT-NOS3 signaling pathway. Nicotinic acid might be the potential compound for LFG antihypertensive effect.

Combined Extract of Leonurus japonicus Houtt, Eclipta prostrata L., and Pueraria lobata Ohwi Improved Hot Flashes and Depression in an Ovariectomized Rat Model of Menopause.

Menopause leads to ovarian hormone loss, which causes symptoms such as weight gain, hot flashes, and depression. Exploring nutraceuticals is important for treating menopausal symptoms that extensively impact women's quality of life. We hypothesized that a combination of Leonurus japonicus Houtt, Eclipta prostrata L., and Pueraria lobata Ohwi (LEPE) would alleviate menopausal symptoms in an ovariectomized menopausal rat model. Bilateral ovariectomy was performed and animals were assigned to five groups: (1) Sham, (2) Vehicle, (-) Control, (3) LEPE (100 mg/kg bw), (4) LEPE (200 mg/kg bw), and (5) Estradiol (3 µg/kg bw). LEPE was orally administered daily for 12 weeks. LEPE supplementation did not affect growth performance (body weight and feed intake) or body composition (lean mass and fat in tissue). LEPE did not cause deviations in aspartate aminotransferase, alanine aminotransferase, estradiol, and follicle-stimulating hormone levels, indicating no hepatotoxicity or endocrine disturbance. LEPE decreased type I collagen (CTX-1) but did not affect bone mineral density or osteocalcin. LEPE decreased tail temperature and increased rectal temperature, improving menopause-related vasomotor symptoms. Furthermore, LEPE ameliorated depression-related behavior, including in forced swimming and tail suspension tests. Thus, LEPE may improve menopausal symptoms by enhancing vasomotor symptoms and depression in an ovariectomized rat menopause model.

PTP1B Inhibitory and Anti-inflammatory Properties of Constituents from Eclipta prostrata L.

The white-flowered leaves of Eclipta prostrata L. together with leaves of Scoparia dulcis and Cynodon dactylon are mixedly boiled in water and given to diabetic patients resulting in the significant improvement in the management of diabetes. However, the active constituents from this plant for antidiabetic and anti-obesity properties are remaining unclear. Thus, this study was to discover anti-diabetes and anti-obesity activities through protein tyrosine phosphatases (PTP)1B inhibitory effects. We found that the fatty acids (23, 24) showed potent PTP1B inhibition with IC50 values of 2.14 and 3.21 µM, respectively. Triterpenoid-glycosides (12-15) also exhibited strong to moderate PTP1B inhibitory effects, with IC50 values ranging from 10.88 to 53.35 µM. Additionally, active compounds were investigated for their PTP1B inhibitory mechanism and docking analysis. On the other hand, the anti-inflammatory activity from our study revealed that compounds (1-4, 7, 8, 10) displayed the significant inhibition nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Especially, compound 9 showed the potent inhibitory effects in LPS-induced NO production on RAW264.7 cell. Therefore, further Western blot analysis was performed to identify the inhibitory expression including heme oxygenase-1 (HO-1) and inhibitor of kappaB (IκB) phosphorylation.

The combination of HPLC and biological analysis to determine the quality markers and its structural composition of Eclipta prostrata L.

INTRODUCTION: The diversity and complexity of components are important reasons for the unstable efficacy and safety of Chinese materia medica (CMM) in quality control. The good and stable quality control may be related to the quality markers with structural composition of multi-components. OBJECTIVE: In the present study, we take Eclipta prostrata L. as a representative example. The 11 samples were collected from the different areas in China, and the discrepancy in bioactivity and chemical composition of these samples were compared. DOX (doxorubicin hydrochloride)-induced ICR mice were established for in vivo nephrotic syndrome experiments. The biochemical indicators including 24-h urine protein, triglyceride (TG), etc. were measured and the pathological change of kidney tissue was observed. MPC-5 cells damage model was induced to compare the difference of these samples in bio-activity. High-performance liquid chromatography (HPLC) method for 11 EEP (extract of Eclipta prostrata L.) samples were performed to analyse the content of the quality markers. RESULTS: In vivo experiments, EEP could mitigate the content or activity of urine protein, TG, etc. compared with the positive group (TG content was 2.53 ± 0.39 mmol/L, urinary protein quantification on 35th day was 16.79 ± 2.32 mg). In vitro experiments, CCE (coumestans component of Ecliptae) and EEP had equivalent effects on biochemical indicators such as cell viability, etc. According to the HPLC analysis, the content of wedelolactone was 45.88% and demethylwedelolactone was 23.74% in CCE. CONCLUSION: The CCE with a ratio of 2:1 can be considered as a quality marker of Eclipta prostrata L.. This research may provide a perspective for quality control of CMM.

Lei-gong-gen formula granule attenuates hyperlipidemia in rats via cGMP-PKG signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Lei-gong-gen formula granule (LFG) is a folk prescription derived from Zhuang nationality, the largest ethnic minority among the 56 nationalities in China. It is composed of three herbs, namely Centella asiatica (L.) Urb., Eclipta prostrata (L.) L., Smilax glabra Roxb. It has been widely used as health protection tea for many years to prevent cardiovascular and cerebrovascular diseases such as hyperlipidemia and hypertension. AIM OF THE STUDY: This study validated the lipid-lowering effect of LFG in a hyperlipidemia rat model. Then we employed network pharmacology and molecular biological approach to identify the active ingredients of LFG, corresponding targets, and its anti-hyperlipidemia mechanisms. MATERIALS AND METHODS: Hyperlipidemia rat model was established by feeding male Sprague-Dawley rats with high-fat diet for two weeks. LFG (two doses of 10 and 20 g/kg) was administered orally to hyperlipidemia rat model for 4 weeks, twice per day. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) were monitored in rats pre and post-treatment. Hematoxylin-eosin staining was applied to observe the pathology and lipid accumulation of liver. We then performed network pharmacology analysis to predict the ingredients, their associated targets, and hyperlipidemia associated targets. Pathway analysis with significant genes was carried out using KEGG pathway. These genes and proteins intersectioned between compound targets and hyperlipidemia targets were further verified with samples from hyperlipidemia rats treated with LFG using Real-time RT-PCR and Western Blot. RESULTS: LFG attenuated hyperlipidemia in rat model, and this was characterized with decreased serum levels of TC, LDL-C, liver wet weight, and liver index. LFG alleviated the hepatic steatosis in hyperlipidemia rats. Network pharmacology analysis identified 53 bioactive ingredients from LFG formula (three herbs), which link to 765 potential targets. 53 hyperlipidemia associated genes were retrieved from public databases. There were 10 common genes between ingredients-targets and hyperlipidemia associated genes, which linked to 20 bioactive ingredients. Among these 10 genes, 3 of them were validated to be involved in LFG's anti-hyperlipidemia effect using Real-time RT-PCR, namely ADRB2 encoding beta-2 adrenergic receptor, NOS3 encoding nitric oxide synthase 3, LDLR encoding low-density lipoprotein receptor. The cGMP-PKG signaling pathway was enriched for hyperlipidemia after pharmacology network analysis with ADRB2, NOS3, and LDLR. Interestingly, expression of cGMP-dependent protein kinase (PKG) was downregulated in hyperlipidemia rat after LFG treatment. Molecular docking study further supported that ferulic acid, histidine, p-hydroxybenzoic acid, and linalool were potential active ingredients for LFG's anti-hyperlipidemia effect. LC-MS/MS analysis confirmed that ferulic acid and p-hydroxybenzoic acid were active ingredients of LFG. CONCLUSION: LFG exhibited the lipid-lowering effect, which might be attributed to downregulating ADRB2 and NOS3, and upregulating LDLR through the cGMP-PKG signaling pathway in hyperlipidemia rat. Ferulic acid and p-hydroxybenzoic acid might be the underlying active ingredients which affect the potential targets for their anti-hyperlipidemia effect.

Ultra-high-pressure-assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high-speed counter-current chromatography.

Ultra-high-pressure extraction combined with high-speed counter-current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra-high-pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid-liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra-high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (3:7:5:5, v/v) was used for high-speed counter-current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one-step separation. This research demonstrated that ultra-high-pressure extraction combined with high-speed counter-current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.

A new stigmasterol glucoside from aerial parts of Ecliptae Herba / 中草药

Objective: To investigate the bioactive chemical constituents of the aerial parts of Eclipta prostrata. Methods: The compounds were isolated and purified by macroporous absorption resin, silica gel, Sephadex LH-20 and semi-preparative HPLC chromatography. Their structures were determined by MS and NMR data. The α-glucosidase inhibitory activities of compounds 1 and 2 were tested by in vitro screening assay. Results: A total of eight compounds were isolated from the ethyl acetate partition of the ethanol extract of E. prostrata. They were identified as 7β-hydroxystigmasterol 3-O-β-D-glucopyranoside (1), 7α-hydroxystigmasterol 3-O-β-D-glucopyranoside (2), 7α-hydroxysitosterol 3-O-β-D-glucopyranoside (3), 3β,23-dihydroxy-30-norolean-12,20 (29)-dien-28- oic acid (4), camellenodiol (5), echinocystic acid-3-O-(6-O-acetyl)-β-D-glucopyranoside (6), eclalbasaponin I (7) and eclalbasaponin IV (8). Compound 2 exhibited strong inhibition against α-glucosidase with an IC50 value of (11.7 ± 4.2) μmol/L. Conclusion: Compound 1 is a new compound named eclalbasaponin XIV and compounds 3-5 are reported from this herb for the first time. Steroidal glycosides could be the anti-hyperglycaemic components in E. prostrata by inhibiting α-glucosidase.

Regulation of Eclipta prostrata L. components on cigarette smoking-induced autophagy of bronchial epithelial cells via keap1-Nrf2 pathway.

Cigarette smoking extract (CSE)-induced autophagic injury has been regarded as an important contributor to the pathogenesis of lung cancer. We previously found that Eclipta prostrata L. component (CCE) reduced CSE-induced bronchial epithelial cells damage. However, the mechanism remains unknown. Human normal bronchial epithelial cells (NHBE) were exposed to CSE to establish stress model. Nrf2-siRNA and Keap1-siRNA transfection were performed. mRFP-GFP-LC3 dual fluorescence and transmission electron microscopy were used to observe the autophagic characteristics. CCE prevented CSE-induced Nrf2 transfer into cytoplasm and up-regulated Keap1 level of NHBE cells. Furthermore, CCE significantly increased p-p16, p-p21 and p-p53 phosphorylation levels in Nrf2-siRNA- or Keap1-siRNA-transfected cells. As demonstrated by transmission electron microscopy and mRFP-GFP-LC3 dual fluorescence assays, CCE mitigated autophagic injury, and also down-regulated autophagy-related Beclin-1, LC3II/LC3I ratio, Atg5 and ATF4 levels. Our findings showed the attenuation of CCE on CSE-induced NHBE cells injury was associated with Nrf-2-mediated oxidative signaling pathway.

Autophagy Flux Contributes to Regulation of Components of Eclipta prostrata L. on Cigarette Smoking-Induced Injury of Bronchial Epithelial Cells.

Excessive autophagy plays a crucial role in cigarette smoking extract (CSE)-induced inflammation response and oxidative damage of respiratory epithelial cells. The components from Eclipta prostrata L. (CCE) have been shown to be beneficial for CSE-induced epithelial cells injury. However, whether its protection on CSE-stress injury is related to its regulation on autophagy remains still unclear. In this study, CCE, containing mainly wedelolactone of 45.88% and demethylwedelolactone of 23.74%, could improve significantly 10%CSE-induced cell viability of normal human bronchial epithelial (NHBE) cells using CCK-8 kit. We revealed that CCE could remarkably increase autophagic factors Beclin-1, Atg5, ATF4 proteins expression levels and the transformation of LC3-I to LC3-II. Additionally, CCE up-regulated significantly p-p16 and p-p21 phosphorylation levels whereas down-regulated p-p53 in NHBE cells. The changes of typical autolysosom and representative autophagosome in the presence of CCE or/and autophagy inhibitor chloroquine (CQ) were also observed by transmission electron microscopy. These data demonstrated that CCE reduced CSE-induced autophagy flux activation in NHBE cells. The blockade of CCE on autophagy flux contributes to its protection against CSE-induced NHBE cells damage, and CCE is promising to be combination therapeutic molecules to excessive autophagic damage in respiratory diseases.

Validated UPLC-MS/MS method for quantification of seven compounds in rat plasma and tissues: Application to pharmacokinetic and tissue distribution studies in rats after oral administration of extract of Eclipta prostrata L.

A rapid, sensitive and specific ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed to investigate the pharmacokinetics and tissue distribution of Eclipta prostrata extract. Rats were orally administrated the 70% ethanol extract of E. prostrata, and their plasma as well as various organs were collected. The concentrations of seven main compounds, ecliptasaponin IV, ecliptasaponin A, apigenin, 3'-hydroxybiochanin A, luteolin, luteolin-7-O-glucoside and wedelolactone, were quantified by UPLC-MS/MS through multiple reactions monitoring method. The precisions (RSD) of the analytes were all <15.00%. The extraction recoveries ranged from 74.65 to 107.45% with RSD ≤ 15.36%. The matrix effects ranged from 78.00 to 118.06% with RSD ≤ 15.04%. To conclude, the present pharmacokinetic and tissue distribution studies provided useful information for the clinical usage of Eclipta prostrata L.

The ethanolic extract of the Eclipta prostrata L. ameliorates the cognitive impairment in mice induced by scopolamine.

ETHNOPHARMACOLOGICAL RELEVANCE: Eclipta prostrata L. (Asteraceae) has been prescribed for whole body nourishment and nervine tonic in Asia. However, the effects of E. prostrata in learning and memory have not been fully explored. AIM OF THE STUDY: To scientifically elucidate the effects of E. prostrata on cognitive functions, we examined whether E. prostrata could ameliorate a cholinergic blockade-induced memory impairment, and we also investigated the effects of E. prostrata on the synaptic plasticity in the hippocampus. MATERIALS AND METHODS: Memory impairment was induced by scopolamine, a cholinergic muscarinic receptor antagonist. The anti-amnesic effects of the ethanolic extract of Eclipta prostrata L. (EEEP) were measured in mice by the passive avoidance, Y-maze and Morris water maze tasks. To test the effects of EEEP on synaptic plasticity, we measured long-term potentiation (LTP) in the hippocampus. We also studied several signaling molecules related to learning and memory, such as phosphorylated protein kinase B (Akt) or phosphorylated glycogen synthase kinase-3ß (GSK-3ß). RESULTS: In the passive avoidance task, EEEP (50 or 100mg/kg, p.o.) significantly ameliorated the shortened step-through latency induced by scopolamine. EEEP (100mg/kg, p.o.) also showed significant increase in alternation behavior during the Y-maze task. In the Morris water maze task, scopolamine-induced a decrease in both the swimming time within the target zone and the number of crossings where the platform had been placed were significantly reversed by EEEP (50 or 100mg/kg, p.o.). Moreover, EEEP (100µg/ml) significantly enhanced hippocampal LTP without affecting basal synaptic transmission. The administration of EEEP (100mg/kg) increased the phosphorylation levels of Akt and GSK-3ß in the hippocampal region. CONCLUSION: These results suggest that EEEP has memory-ameliorating activity against scopolamine-induced cognitive impairment and facilitates LTP in the hippocampus. This could be, at least in part, mediated by the activation of the Akt-GSK-3ß signaling pathway.

Simultaneous extraction, identification and quantification of phenolic compounds in Eclipta prostrata using microwave-assisted extraction combined with HPLC-DAD-ESI-MS/MS.

A simple and rapid method was developed using microwave-assisted extraction (MAE) combined with HPLC-DAD-ESI-MS/MS for the simultaneous extraction, identification, and quantification of phenolic compounds in Eclipta prostrata, a common herb and vegetable in China. The optimized parameters of MAE were: employing 50% ethanol as solvent, microwave power 400 W, temperature 70 °C, ratio of liquid/solid 30 mL/g and extraction time 2 min. Compared to conventional extraction methods, the optimized MAE can avoid the degradation of the phenolic compounds and simultaneously obtained the highest yields of all components faster with less consumption of solvent and energy. Six phenolic acids, six flavonoid glycosides and one coumarin were firstly identified. The phenolic compounds were quantified by HPLC-DAD with good linearity, precision, and accuracy. The extract obtained by MAE showed significant antioxidant activity. The proposed method provides a valuable and green analytical methodology for the investigation of phenolic components in natural plants.