Erzhiwan Ameliorates Restraint Stress- and Monobenzone-Induced Depigmentation in Mice by Inhibiting Macrophage Migration Inhibitory Factor and 8-Hydroxy-2-Deoxyguanosine.

Purpose: Vitiligo is an autoimmune disease that results in the loss of epidermal melanocytes. The treatments for patients with vitiligo remain lacking. Erzhiwan (EZW), a traditional Chinese Medicine composed of Ligustri Lucidi Fructus and Ecliptae Herba, was used to ameliorate depigmentation since ancient China. This study aims to investigate the effect of EZW on vitiligo-related depigmentation. Methods: A vitiligo-related depigmentation mouse model was induced by monobenzone and restraint stress. The experimental depigmentation mice were treated with EZW. Histological observation of skin was conducted. Cutaneous oxidative damage and inflammation were determined. A network pharmacology analysis was carried out. Results: EZW reduced depigmentation score (p<0.01), cutaneous inflammatory infiltration (p<0.01), and CD8α-positive expression (p<0.01), and increased cutaneous melanin content in experimental depigmentation mice. EZW reduced stress reaction in experimental depigmentation mice (p<0.01). EZW inhibited 8-hydroxy-2-deoxyguanosine (8-OHdG)-related DNA oxidative damage in the skin (p<0.05, p<0.01). In addition, EZW reduced cutaneous macrophage migration inhibitory factor (MIF)-CD74-NF-κB signaling (p<0.01). The network pharmacology analysis demonstrated that EZW regulated necroptosis, apoptosis, and FoxO signaling pathways in vitiligo. An in vitro experiment showed that the main ingredient of EZW, specnuezhenide, protected against monobenzone and MIF-induced cell death in HaCaT cells (p<0.01). Conclusion: EZW ameliorates restraint stress- and monobenzone-induced depigmentation via the inhibition of MIF and 8-OHdG signaling. The findings provide a data basis of an utilization of EZW in vitiligo.

Rapid screening of active ingredients and action mechanisms of Ecliptae Herba for treating Alzheimer's disease by UPLC-Q-TOF/MS and "component-target-pathway" network.

Alzheimer's disease (AD) is a common neurodegenerative disease. The drugs widely used in clinic are mainly single-target drugs for symptomatic treatment, which can only alleviate symptoms to a certain extent. Ecliptae Herba (EH) is considered a potential therapeutic drug for AD due to its neuroprotective effects. Although EH has a clear anti-AD effect, the material basis and mechanism remain unclear. Therefore, we adopted an efficient analytical technique, namely ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), combined with "component-target-pathway" network to explore the active components and potential mechanisms of EH in treating AD. Due to the high sensitivity of UPLC-Q-TOF/MS, a total of 50 components were identified in EH. Among them, 20 and 12 compounds were found in plasma and brain samples, respectively. The network pharmacology analysis revealed that apigenin, luteolin, ecliptasaponin A, chlorogenic acid, wedelolactone, and quercetin were the active components, which could affect the serotonergic synapse, calcium and cAMP signaling pathways by regulating related targets such as EGFR, PRKCA, BRAF and ERBB2. This study clarified that EH can exert anti-AD effect through multi-component, multi-target and multi-pathway characteristics. Furthermore, it offers a good foundation for further in-depth research on the anti-AD effects of EH, and provides a valuable approach for the rapid screening of active components and potential mechanisms of other medicinal plants, potentially bringing changes to the discovery and development of novel therapeutics for neurodegenerative disorders.

Chinese Ecliptae herba (Eclipta prostrata (L.) L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Ecliptae herba (Eclipta prostrata (L.) L.) is an ethnomedicinal herb, which is used mainly to nourish kidney and thus strengthen bones according to traditional Chinese medicine theory. Pharmacological studies have supported the ethnomedicine use, showing that Ecliptae herba extract has an anti-osteoporotic effect in vivo and promoted osteoblast proliferation and activity in vitro. However, the molecular mechanism of Ecliptae herba on osteoblast differentiation from bone marrow mesenchymal stem cells (BMSC), the progenitors of osteoblasts, is still unclear. AIM OF THE STUDY: N6-methyladenosine (m6A) mRNA epigenetic modification may play a key role in promoting osteoblastic differentiation, and thus treating osteoporosis. This study sought to assess the mechanism through which Eclipate herba and its component wedelolactone influence m6A modification during the process of osteoblastogenesis from BMSC. MATERIAL AND METHODS: The alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were applied to determine osteoblastogenesis from BMSC. Western blot and quantitative real-time PCR were performed. RNA sequencing analysis was used to determine the characteristics of m6A methylation. Stable knocking down of METTL3 using lentiviral-based shRNA was performed. RESULTS: Upon 9 d treatment of BMSC with ethyl acetate extract of Ecliptae herba (MHL), ALP activity and ossification level increased in comparison with osteogenic medium (OS)-treated control. The expression of methyltransferase METTL3 and METTL14 was significantly increased, but WTAP expression had no change in response to MHL treatment. Knocking down of METTL3 resulted in a decrease in MHL-induced ALP activity, ossification level as well as mRNA expression of Osterix and Osteocalcin, two bone formation-related markers. The level of m6A increased when BMSC was treated with MHL for 9 d. RNA sequencing analysis indicated that MHL treatment altered mRNA m6A modification of genes associated with osteoblastogenesis. By kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, HIF-1α, PI3K/Akt, and Hippo signaling pathways were enriched and associated with m6A modification. The expression of m6A-modified genes including HIF-1α, VEGF-A, and RASSF1, was upregulated by MHL, but the upregulation was reversed after METTL3 knockdown. Additionally, the enhanced expression of METTL3 was also observed after treatment with wedelolactone, a component from MHL. CONCLUSIONS: These results suggested a previously uncharacterized mechanism of MHL and wedelolactone on osteoblastogenesis, by which METTL3-mediated m6A methylation is involved and thus contributes to the enhancement of osteoblastogenesis.

Combination of Ligustri Lucidi Fructus with Ecliptae Herba and their phytoestrogen or phytoandrogen like active pharmaceutical ingredients alleviate oestrogen/testosterone-induced benign prostatic hyperplasia through regulating steroid 5-α-reductase.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a urinary system disease with high prevalence among the middle and elder men. In BPH, proliferation of prostate cells and the imbanlance between androgen and estrogen are both important inducers. Previous studies have demonstrated that compounds from Ligustri Lucidi Fructus (LLF) and Ecliptae Herba (EH) are of phytoestrogenic or phytoandrogenic activities. The combination of LLF with EH at the ratio of 1:1 on crude drugs quantity is called Erzhi formula (EZF), which is used for in vivo research of our study. PURPOSE: This study aimed to investigate potential mechanisms of EZF and its active pharmaceutical ingredients on BPH in vitro and in vivo. METHODS: Therapeutic effects of EZF was evaluated in E2/testosterone (1:100) induced BPH rats model. The pathological changes of prostate, concentrations of testosterone, DHT, E2, PSA in rats' plasma and prostate were detected. The expressions of PCNA, AR, ERα, ERß, SRD5A1, SRD5A2 were measured in BPH rat prostates and E2-stimulated human benign prostatic epithelial cells (BPH-1). RESULTS: EZF treatment significantly attenuated rat prostate enlargement, alleviated BPH pathological features, and decreased the expression of PCNA. The up-regulation of AR, ERα, SRD5A1/2 expressions, and down-regulation of ERß expression at prostate of rat BPH model were significantly blocked by EZF administration. The expression levels of testosterone, DHT, E2, PSA were strongly inhibited by EZF treatment. At the cellular level, ligustrosidic acid and echinocystic acid inhibited E2-induced BPH-1 cell proliferation and PCNA expressions, which were consistent with the results in vivo. And these two ingredients also down-regulated the expressions of AR, ERα, SRD5A1/2 and up-regulated the expression of ERß in BPH-1 cells. CONCLUSION: EZF, ligustrosidic acid from LLF and echinocystic acid from EH showed inhibitive effects on BPH via down-regulating prostatic AR, ERα, SRD5A1/2 expressions and up-regulating ERß expression.

Enhanced identification of the in vivo metabolites of Ecliptae Herba in rat plasma by integrating untargeted data-dependent MS2 and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan.

Detection and identification of the in vivo metabolites of traditional Chinese medicine by untargeted profiling strategies are often confronted with severe interference from complex endogenous substances. Here we developed an integral approach, by combining untargeted data-dependent MS2 (dd-MS2) of Q-Orbitrap mass spectrometry and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan (pMRM-IDA-EPI) of triple quadrupole-linear ion trap (QTRAP) mass spectrometry, aiming to detect and identify more extensive metabolites in bio-samples. Ecliptae Herba (EH) is a widely consumed medicinal herb with the effects of nourishing liver/kidney, but its metabolites in vivo have not been fully elucidated. Firstly, after UHPLC separation on an HSS T3 column, chemical fingerprinting of 70% ethanolic extract of EH was performed by untargeted dd-MS2 in negative ion mode. We could characterize 41 compounds from EH, and 24 were detectable in the plasma of rats (prototypes) after oral administration of EH extract (1 g/kg). Secondly, using echinocystic acid (triterpene), wedelolactone (coumarin), and apigenin (flavonoid) as the different parent templates, an MRM list containing 150 predicted ion-pairs was established to enhance MS2 scan by pMRM-IDA-EPI, which enabled the primary identification of up to 200 metabolites. The biotransformations mainly involve oxidation, hydrogenation, methylation, glucuronidation, sulfonation etc. Thirdly, the rat plasma samples obtained after oral administration of three pure compounds (echinocystic acid, wedelolactone and apigenin) were analyzed to verify the reliability of metabolites identification, and 11, 4, and 10 metabolites were found individually. This is the first comprehensive research on the metabolism of EH in vivo.

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.

Effects of aqueous extracts of Ecliptae herba, Polygoni multiflori radix praeparata and Rehmanniae radix praeparata on melanogenesis and the migration of human melanocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygoni multiflori radix praeparata (PMRP), Ecliptae herba (EH) and Rehmanniae radix praeparata (RRP) are the most frequently-used herbs by Traditional Chinese Medicine practitioners for the treatment of vitiligo. Their abilities to stimulate melanogenesis, melanocyte migration and MITF (microphthalmia associated transcription factor) protein expression were evaluated in this study. MATERIALS AND METHODS: The effects of aqueous extracts of PMRP, EH and RRP on human melanocytes in vitro were examined by MTT assay, tyrosinase activity, melanin synthesis, migration assay and Western blot. RESULTS: Treatment with EH (at 100µg/ml and 400µg/ml) significantly increased intracellular tyrosinase activity in accordance with the elevation of melanin content at the same concentrations. Treatment with RRP (at 100µg/ml and 400µg/ml) promoted melanin production but had no stimulatory effect on tyrosinase activity. Treatment with PMRP and EH (at 100µg/ml) promoted the migration of human melanocytes in a type IV collagen-coated transwell migration assay. Western blot analysis showed MITF protein expression was elevated by PMRP, EH and RRP (at 100µg/ml). CONCLUSION: An aqueous extract of EH has a synergistic effect on melanocytes by up-regulating tyrosinase activity, enhancing melanin synthesis and promoting melanocyte migration as well as elevating MITF protein expression. RRP exhibits a significant stimulating effect on melanogenesis and MITF protein expression. These results suggest that EH and RRP contain substances with direct enhancing effects on melanogenesis and migration, possibly via their effects on MITF protein expression.

Studies on the quality standard of Ecliptae herba / 国际中医中药杂志

Objective To set up and improve the quality standards of Ecliptae herba through determination of effective components, moisture, total ashes, acid insoluble ashes, extracts and heavy metals in this herb.Methods Thin Layer Chromatogram (TLC) and HPLC were used for qualitative and quantitative identification of Ecliptae Herba. Routine examinations were determined based on the procedures recorded in the Appendix of Ch.P (2010) for foreign matter, moisture, ashes, extracts determination and heavy metal test respectively.Results The content of wedelolactone in MHLS-005 was lower than the standards of Chinese Pharmacopoeia, water insoluble ash in NHLC-1 and water content in MHLS-004 and MHLS-006 exceeded standards of Chinese Pharmacopoeia.Conclusion The established method was simple, accurate and can be used as the quality standard for the quality control of Ecliptae Herba.

Inhibitory effect of Ecliptae herba extract and its component wedelolactone on pre-osteoclastic proliferation and differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ecliptae herba, also known as "Mo-Han-Lian", has long been used in China to nourish Kidney and thereafter strengthen bones. Accumulating evidence indicates that extracts of Ecliptae herba have antiosteoporotic effect. However, the effective compounds and cellular mode of action are still unclear. To investigate the effect of ethyl acetate extract of Ecliptae herba (EAE) and its component wedelolactone on proliferation and differentiation of preosteoclastic RAW264.7 cells as well as proliferation of bone marrow stromal cells (BMSC). MATERIALS AND METHODS: RAW264.7 and BMSC were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Tartrate-resistant acid phosphatase (TRAP) activity of RAW264.7 was measured by using p-nitrophenyl sodium phosphate (pNPP) assay after the cells were treated with 30ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) plus various concentrations of EAE, wedelolactone or alendronate. The formation of multinucleated TRAP-positive RAW264.7 cells was observed by using a TRAP-staining kit. RESULTS: Treatment of RAW264.7 cells with EAE at high doses (20µg/ml and 40µg/ml) or wedelolactone at 10µg/ml resulted in a decrease in proliferation of RAW264.7 cells. Low doses of EAE (5, 10µg/ml) and wedelolactone (2.5µg/ml) inhibited RANKL-induced TRAP activity by 20.3%, 37.9%, and 48.3%. The inhibitory effect of wedelolactone is more potent than that of alendronate, an anti-resorptive drug. Morphological changes revealed that 5µg/ml EAE and 2.5µg/ml wedelolactone reduced the number of multinucleated osteoclast-like cells. At the high doses, EAE (20µg/ml) and wedelolactone (10µg/ml) inhibited the growth of BMSC. CONCLUSIONS: EAE and its component wedelolactone inhibited osteoclast RAW264.7 proliferation and differentiation at the low doses, but at the high doses, showed cytotoxic effect on BMSC. These results indicated that EAE and wedelolatone might be potential alternative therapy for osteoporosis.