Anti-perimenopausal osteoporosis effects of Erzhi formula via regulation of bone resorption through osteoclast differentiation: A network pharmacology-integrated experimental study.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhi formula (EZF) consists of Ecliptae herba (EH) and Fructus Ligustri Lucidi (FLL) at a ratio 1:1, and constitutes a well-known formula in China that is commonly used for treating menopausal diseases. AIM OF THE STUDY: In this study, we explored the pharmacologic actions and potential molecular mechanisms underlying EZF's action in preventing and treating osteoporosis. MATERIALS AND METHODS: The active components and related targets of EZF's anti-osteoporotic effects were predicted by network pharmacology, and functional enrichment analysis was also performed. We then used an osteoporosis model of ovariectomized (OVX) mice to detect the effects of EZF on osteoporosis. RESULTS: The results from network pharmacology identified a total of 10 active ingredients from EH and 13 active ingredients from FLL that might affect 65 potential therapeutic targets. GO enrichment analysis revealed that EZF affected bone tissue primarily via hormone (particularly estradiol)-related pathways and bone resorption by osteoclast differentiation. KEGG analysis demonstrated that bone-related factors such as Runt-related transcription factor 2 (Runx2), Ca2, estrogen receptor1 (ESR1), androgen receptors (AR), and TNFα served as the primary targets during osteoclastic differentiation. In vivo experiments showed that the formula significantly improved the diminution in estrogen and the subsequent uterine atrophy induced by ovariectomy (P < 0.01 or 0.05), implying that the EZF exerted its actions via regulation of estradiol and the nourishing effects of the uterus in OVX mice. Dual-energy X-ray absorptiometry and micro-CT showed that EZF significantly inhibited bone loss and improved bone micro-architecture by statistically increasing the number of bone trabeculae and decreasing the separation of bone trabeculae in OVX mice (P < 0.01 or 0.05); EZF also inhibited bone loss and enhanced bone-fracture load. Furthermore, we confirmed that EZF reduced the calcium concentrations, augmented protein and mRNA levels for Runx2 in the bone marrow, and reduced PPARγ levels. RANKL-a key downstream regulatory protein of many targets that was referred to in our results of network pharmacology as being involved in the regulation of osteoclastogenesis-was significantly diminished by EZF; it also elevated OPG content. In addition, we used monocytes of bone-marrow origin to detect the effects of the potential components of EZF on osteoclast differentiation and found that wedelolactone, oleanolic acid, echinocystic acid, luteolin, and luteolin-7-o-glucoside significantly inhibited osteoclast differentiation from monocytes induced by 25 ng/mL MCSF and 50 ng/mL RANKL (P < 0.01 or 0.05). CONCLUSIONS: Our present study indicated that EZF significantly inhibited the bone loss induced by OVX in mice by its regulation of estradiol combined with the nourishing effect of the uterus, and that it also attenuated bone resorption by decreasing the RANKL/OPG ratio so as to inhibit osteoclast maturation.

Advances in the Study of the Potential Hepatotoxic Components and Mechanism of Polygonum multiflorum.

The roots of Polygonum multiflorum (PM) (He Shou Wu in Chinese) are one of the most commonly used tonic traditional Chinese medicines (TCMs) in China. PM is traditionally valued for its antiaging, liver- and kidney-tonifying, and hair-blackening effects. However, an increasing number of hepatotoxicity cases induced by PM attract the attention of scholars worldwide. Thus far, the potential liver injury compounds and the mechanism are still uncertain. The aim of this review is to provide comprehensive information on the potential hepatotoxic components and mechanism of PM based on the scientific literature. Moreover, perspectives for future investigations of hepatotoxic components are discussed. This study will build a new foundation for further study on the hepatotoxic components and mechanism of PM.

[Recent advances in treatment of viral pneumonia using Chinese patent medicine].

Viral pneumonia is caused by a spreading of lung infection caused by respiratory viruses. Some virus infections were found to be highly aggressive, leading to lung inflammation and severe damage in respiratory system with high fatality rate. Currently, there is no effective therapeutic drugs in the clinic. The common clinical symptoms of viral pneumonias include fever, rhinitis, runny nose, nonproductive cough, fatigue, myalgias and headaches after the immune system being tricked by driving cytokines and overactivated immune response induced by cytokine storms. Patients with severe symptoms could get persistent high fever, dysfunctional breathing, consciousness disorders and even respiratory failure, post-inflammatory pulmonary fibrosis, multi-organ damages, shock and so on. Most clinical treatments are used to inhibit virus replication, relieve symptoms, inhibit excessive inflammatory response, regulate immune balance and protect organs. Both applied and basic research demonstrate that Chinese patent medicine has certain anti-viral effects, effectively inhibiting viral pneumonia transiting from mild to severe, rapid relieving of patient symptoms because of their multi-component and multi-target integrated roles. This review has summarized the reports on the treatment of viral pneumonia. Based on the pathogenic characteristics of viral pneumonia, this paper summarizes the diverse roles of the marketed Chinese patent medicine, such as their effects in inhibiting the progress of viral replication and overactivated inflammatory response, regulating immune balance, attenuating pulmonary fibrosis and so forth. Our paper summarizes the advantages of Chinese patient medicine in the treatment of viral pneumonia, based on which improvements of clinical therapy are expected to be made soon.

[N-containing compounds from seeds of Paganum harmala].

To investigate the N-containing compounds in the seeds of Paganum harmala,fourteen compounds were finally isolated from the 95% Et OH extract of P. harmala seeds by using various chromatographic techniques including silica gel,MCI resin,and ODS column chromatography as well as the semi-preparative HPLC. Depending on spectroscopic techniques and comparison with the reported data in the literatures,the structures of all compounds were identified as N-[3-(2-amino-4-methoxyphenyl)-3-oxopropyl]acetamide(1),dehydroharmalacidine(2),harmalacidine(3),harmine N-oxide(4),harmine(5),tetrahydroharmine(6),demethylharmalacidine(7),harmol(8),tetrahydroharmol(9),harmindol ß-D-glucopyranoside(10),tryptophyl ß-D-glucopyranoside(11),pegamineß-D-glucopyranoside(12),vasicol(13) and vasicinone(14). Among them,1 was a new compound,and 2 and 10 were obtained as natural products for the first time.

Two new naphthalene glycosides from the seeds of Cassia obtusifolia.

A phytochemical study on the seeds of Cassia obtusifolia was carried out, which finally led to obtain two naphthalenes (1 and 2), two naphthopyrans (3 and 4) and twelve anthraquinones (5-16). The structures of all compounds were established mainly by NMR and MS experiments as well as the necessary chemical evidence. Among them, 1 and 2 (obtusinaphthalensides A and B) were identified to be new naphthalene glycosides.

Simultaneously targeted and untargeted multicomponent characterization of Erzhi Pill by offline two-dimensional liquid chromatography/quadrupole-Orbitrap mass spectrometry.

Large-scale targeted and untargeted metabolites characterization can be achieved by feat of different liquid chromatography/mass spectrometry (LC-MS) platforms by multiple MS experiments or using data-independent acquisition followed by precursor-product ions matching based on certain algorithms. The resulting insufficiency in efficiency and availability greatly restricts the applicability of these strategies in large-scale profiling and identification of various metabolites. A strategy simultaneously enabling both the targeted and untargeted metabolites characterization is established on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer, by integrating precursor ions list-triggered data-dependent MS2 acquisition (PIL/dd-MS2) of the targeted components and using the "If idle-pick others" (IIPO) function to induce untargeted metabolites fragmentation. A compounds-specific mass defect filter (MDF) algorithm is proposed as a method to generate the PIL. As a proof of concept, this strategy coupled with offline two-dimensional liquid chromatography (2D-LC) was applied to identify the multicomponents of a traditional Chinese medicine formula Erzhi Pill (EZP). A rigid MDF vehicle was elaborated by orthogonal screening of the integer mass and integer mass-dependent dynamic mass defects considering a variation of 20 ppm. The Full MS/dd-MS2 method enabling PIL and IIPO exhibited better performance than Full MS/dd-MS2 and Targeted SIM/dd-MS2 (selected ion monitoring) in respect of the sensitivity in identifying the targeted components and the ability to characterize more untargeted ones. As a consequence, 270 components were separated from EZP, and 146 thereof were selectively characterized. In conclusion, it is a practical, multifaced strategy facilitating the in-depth metabolites profiling and characterization of complex herbal and biological samples.

[Chemcial constituents of Eclipta prostrata].

A total of twenty-two compounds were isolated from the 95% EtOH extract of Eclipta prostrata by various purification steps, and their structures were established as ecliptalignin A (1)，ecliptasaponin Ⅰ (2), ecliptasaponin Ⅱ (3), echinocystic acid (4), 3-oxo-16α-hydroxy-olean-12-en-28-oic acid (5), acacetin-7-O-rutinoside (6), luteoloside (7), apigenin (8), luteolin (9), acacetin (10), skullcapflavone Ⅱ (11), kaempferol (12), kaempferide (13), quercetin (14), 4',7-dihydroxyl-3',6'-dimethoxylisoflavone-7-O-glucoside (15), ecliptal (16), 5-hydroxymethyl-(2,2',5',2″)-terthienyl tiglate (17), psoralen (18), isopsoralen (19), wedelolactone (20), crinumaquine (21), and 2,3,9,12-tetramethoxyprotoberberine (22) mainly based on the spectroscopic techniques, of which 1 was a new lignin analogue, and 5, 6, 10-13, 15, 18, 19, 21 and 22 were isolated form this plant for the first time.

C21 steroid derivatives from the Dai herbal medicine Dai-Bai-Jie, the dried roots of Marsdenia tenacissima, and their screening for anti-HIV activity.

Twenty-three new C21 steroidal glycosides, marstenacissides C1-C10 (1-10), D1-D7 (11-17) and E1-E6 (18-23), and four new C21 steroids, 11α,12ß-O-ditigloyl-tenacigenin C (24), 11α-O-benzoyl-12ß-O-tigloyl-tenacigenin C (25), 11α-O-tigloyl-12ß-O-benzoyl-tenacigenin C (26) and 11α-O-tigloyl-12ß-O-benzoyl-marsdenin (27), were isolated from the Dai herbal medicine Dai-Bai-Jie, derived from the roots of Marsdenia tenacissima. The chemical structures of all compounds were established by spectroscopic techniques, including high-resolution mass spectrometry and NMR spectroscopy, as well as by comparison with reported spectral data. The anti-HIV activities of these compounds were screened, and the compounds obtained displayed inhibitory effects against HIV-1 with inhibition rates of 36.4-81.3% at 30 µM.

Polyoxypregnane Glycosides from the Roots of Marsdenia tenacissima and Their Anti-HIV Activities.

A continuous phytochemical study on the roots of Marsdenia tenacissima led to the isolation and identification of 13 new polyoxypregnane glycosides named marstenacissides B10-B17 (1, 2, 4, 7, 8, 11, 12, and 14) and marstenacissides A8-A12 (3, 9, 10, 13, and 15) in addition to two known polyoxypregnane glycosides marsdenosides M and L (5 and 6). Their structures were established by spectroscopic techniques and by comparison with the reported data in the literature. Moreover, the anti-HIV activities of these isolates and the previous isolated marstenacissides A1-A7 and B1-B9 were assessed, some of which exhibited slight or negligible effects against HIV-1.

A randomized, double-blind, placebo-controlled trial of Chinese herbal medicine granules for the treatment of menopausal symptoms by stages.

OBJECTIVE: The study aims to evaluate the effectiveness and safety of Chinese herbal medicine granules Danzhi Qing'e formula (DZQE), Erzhi formula (EZ), and their combination (Combined formula) in the treatment of menopausal symptoms at different stages of menopause. METHODS: Women between the ages of 40 to 60 years, who met menopausal symptoms diagnostic criteria and experienced hot flushes at least 14 times/week in the last 4 weeks, were recruited to participate in a stratified randomized, double-blind, placebo-controlled clinical trial (n = 389). They received a treatment period of 8 weeks and were followed up for 4 weeks. Participants were categorized into two subgroups: 197 in the perimenopausal subgroup (menstrual disorder to 1 y after amenorrhea) and 192 in the early postmenopausal subgroup (1-5 y after amenorrhea). Participants were randomly assigned to placebo or one of the three herbal formula treatments. The primary outcome instrument was the Menopause-Specific Quality of Life (MENQOL) questionnaire. RESULTS: When analyzing the two subgroups together, DZQE markedly decreased the MENQOL total score at the end of 12th week with statistical significance (P = 0.02) and improved vasomotor symptoms after 8 weeks treatment and 4 weeks follow-up (P < 0.05). What is more, the combined formula also greatly improved the participants' vasomotor symptoms compared with placebo after the 4 weeks follow-up. No statistically meaningful difference was observed in any other outcomes among the groups. The results of subgroup analysis showed that DZQE and Combined formula were more effective than placebo in improving MENQOL total score for perimenopausal women at the end of week 12. For typical menopausal symptoms such as hot flushes and night sweats, DZQE displayed more favorable effects on early postmenopausal participants. Compared to placebo, the DZQE both showed statistically significant differences after 8 weeks treatment and 4 weeks follow-up. Although at the end of 12th week, DZQE also had better effects than placebo in the perimenopausal subgroup on vasomotor symptoms. Participants in the EZ group did not show a significant difference of any domains in MENQOL compared with participants in the placebo group. CONCLUSIONS: The DZQE formula improves the quality of life for menopausal women, especially for those with vasomotor symptoms during the whole menopausal period. The DZQE and EZ combination formula is effective only on perimenopausal symptoms.

Analysis of the Constituents in "Zhu She Yong Xue Shuan Tong" by Ultra High Performance Liquid Chromatography with Quadrupole Time-of-Flight Mass Spectrometry Combined with Preparative High Performance Liquid Chromatography.

"Zhu She Yong Xue Shuan Tong" lyophilized powder (ZSYXST), consists of a series of saponins extracted from Panax notoginseng, which has been widely used in China for the treatment of strokes. In this study, an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) combined with preparative high performance liquid chromatography (PHPLC) method was developed to rapidly identify both major and minor saponins in ZSYXST. Some high content components were removed through PHPLC in order to increase the sensitivity of the trace saponins. Then, specific characteristic fragment ions in both positive and negative mode were utilized to determine the types of aglycone, saccharide, as well as the saccharide chain linkages. As a result, 94 saponins, including 20 pairs of isomers and ten new compounds, which could represent higher than 98% components in ZSYXST, were identified or tentatively identified in commercial ZSYXST samples.

[Identification of Chinese Traditional Medicine Cistanches Herba from Different Places by HPLC-ESI-MS and FTIR Methods].

Five samples of Cistanches Herba from different places were analyzed by HPLC-ESI-MS and FTIR methods. The effective compositions in Cistanches Herba including cistanoside A, echinacoside, acteoside , isoacteoside, 2'-actylacteoside, cistanoside C and tubluoside B were determined by HPLC-MS. The common peak ratio and variant peak ratio were calculated by FTIR spectroscopy of the five samples and the dual index sequence of common peak ratio and variant peak ratio were established. The results showed that the evaluation results of the samples by the two methods were the same. The general fake plant Cynomorii Herba could be identified by FTIR. HPLC-ESI-MS, which has high sensitivity and rapid determination procedure, can be used to evaluate quality of Cistanches Herba by quantitative analysis of the primary compositions. FTIR is a non-destructive analysis method. without complicated extraction and separation procedures to the samples. The absorption strength and the absorption shape were the synergistic effect of the functional groups and the nestification of the components in Cistanches Herba. The provided method has some advantages such as rapid analysis process, good reproducibility, non-destructive, small quantity of sample, simple treatment, good specificity, low-cost and environment-friendly. The method meets the trend of complex analysis and whole analysis for the Chinese medicines. Combination of FTIR and HPLC-ESI-MS was a good method for identification and evaluation of quality of Chinese medicines.

New polyoxypregnane glycosides from the roots of Marsdenia tenacissima.

For the first time, a systematic phytochemical study was performed on the roots of Marsdenia tenacissima. Finally, sixteen new polyoxypregnane glycosides, marstenacissides A1-A7 (1-7) and marstenacissides B1-B9 (8-16), were isolated from M. tenacissima roots. The structures of these new compounds were established by various spectroscopic techniques, including 1D and 2D NMR spectroscopy and mass spectrometry.

Steroidal saponins from Tribulus terrestris.

Sixteen steroidal saponins, including seven previously unreported compounds, were isolated from Tribulus terrestris. The structures of the saponins were established using 1D and 2D NMR spectroscopy, mass spectrometry, and chemical methods. They were identified as: 26-O-ß-d-glucopyranosyl-(25R)-furost-4-en-2α,3ß,22α,26-tetrol-12-one (terrestrinin C), 26-O-ß-d-glucopyranosyl-(25R)-furost-4-en-22α,26-diol-3,12-dione (terrestrinin D), 26-O-ß-d-glucopyranosyl-(25S)-furost-4-en-22α,26-diol-3,6,12-trione (terrestrinin E), 26-O-ß-d-glucopyranosyl-(25R)-5α-furostan-3ß,22α,26-triol-12-one (terrestrinin F), 26-O-ß-d-glucopyranosyl-(25R)-furost-4-en-12ß,22α,26-triol-3-one (terrestrinin G), 26-O-ß-d-glucopyranosyl-(1→6)-ß-d-glucopyranosyl-(25R)-furost-4-en-22α,26-diol-3,12-dione (terrestrinin H), and 24-O-ß-d-glucopyranosyl-(25S)-5α-spirostan-3ß,24ß-diol-12-one-3-O-ß-d-glucopyranosyl-(1→4)-ß-d-galactopyranoside (terrestrinin I). The isolated compounds were evaluated for their platelet aggregation activities. Three of the known saponins exhibited strong effects on the induction of platelet aggregation.

Pharmacokinetics study of asperosaponin VI and its metabolites cauloside A, HN saponin F and hederagenin.

This experiment's with aim was to study the pharmacokinetics of asperosaponin VI and its three metabolites (cauloside A, HN saponin F and hederagenin) via a sensitive high performance liquid chromatography connected with electrospray ionization triple quadrupole mass spectrum (HPLC-ESI-MS/MS). Chromatographic separation was achieved on a reverse phase C18 column with a gradient mobile phase of CH3CN-water with 0.1 % HCOOH at a flow rate of 0.3 mL/min. Sample analysis was simultaneously performed with a multiple reaction monitoring mode using target determination ions at m/z 927.5 â†’ 603.4 for asperosaponin VI, m/z 811.1 â†’ 603.4 for cauloside A, m/z 649.4 â†’ 603.4 for HN saponin F, m/z 71.4 â†’ 393.3 for hederagenin and m/z 307.0 â†’ 161.1 for warfarin as the internal standard. The calibration curve was linear at the range of 0.25-500 ng/mL, and the lower limit of quantification was 0.25 ng/mL for each compound. While the precisely intra-assay and inter-assay variabilities were <9.5 and 7.8 %, respectively; accuracy was determined at the concentrations of 5, 25, 100 ng/mL for all the analytes with the relative standard deviation (%) no more than 15.0 %. Consequently, the validated method could be successfully and precisely applied to the pharmacokinetic study of asperosaponin VI and its metabolites. As a result, the pharmacokinetic parameters of cauloside A, HN saponin F and hederagenin such as T max were obtained at 9.33 ± 2.49, 7.33 ± 0.47 and 12.33 ± 2.36 h, respectively.

A new phenolic amide glycoside from Cimicifuga dahurica.

A new phenolic amide glycoside, cimicifugamide A (1) along with four known compounds, trans-feruloyl tyramine 4-O-beta-D-glucopyranoside (2), (+)-isolariciresinol 3-O-beta-D-glucopyranoside (3), cimidahurine (4), and 24-epi-7, 8-didehydrocimigenol-3-O-beta-D-xylopyranoside (5) were isolated from the rhizomes of Cimicifuga dahurica. Compound 3 was identified as a lignan and has been obtained from Cimicifuga genus for the first time. The structure of compound 1 was elucidated by IR, UV, HR-MS and NMR spectroscopic methods.

[Chemical constituents of Dipsacus asper].

To study the chemical constituents of Dipsacus asper, chromatographic methods such as D101 macroporous resin, silica gel, octadecylsilyl (ODS) column chromatographic techniques and preparative HPLC were used, and five compounds were isolated from 70% (v/v) ethanol extract of the plant. By using spectroscopic techniques including 1H NMR, 13C NMR, 1H-1H COSY, HSQC, HMBC and TOF-MS, the compounds were identified as 3beta-hydroxy-24-nor-urs-4 (23), 12-dien-28-oic acid (1), ursolic acid (2), oleanolic acid (3), 3-O-alpha-L-rhamnosyl(1 --> 3)-beta-D-glucopyranosyl (1 --> 3)-alpha-L-rhamnosyl (1 --> 2)-alpha-L-arabinopyranosyl hederagenin 28-O-beta-D-glucopyranosyl (1 --> 6)-beta-D-glucopyranosyl ester (4), 3-O-[beta-D-xylopyranosyl (1 --> 4)-beta-D-glucopyranosyl (1 --> 4)] [alpha-L-rhamnosyl(1 --> 3)]-beta-D-glucopyranosyl (1 --> 3)-alpha-L-rhamnosyl(1 --> 2)-alpha-L-arabinopyranosyl hederagenin (5), separately. Among them, 1 is a new compound, and 2 is isolated from this plant for the first time.

Bioactive protopanaxatriol type saponins isolated from the roots of Panax notoginseng (Burk.) F. H. Chen.

Seven new protopanaxatriol type saponins, 20S-sanchirhinosides A1 (1), A2 (2), A3 (3), A4 (4), A5 (5), and A6 (6), and sanchirhinoside B (7) were obtained as minor constituents from the root extract of Panax notoginseng (Burkill, F. H. Chen), which showed protection effects against antimycin A induced mitochondrial oxidative stress. Their structures were elucidated by chemical and spectroscopic methods (IR, HRESI-TOF-MS, 1D and 2D NMR). Among them, compounds 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury.

Rare syringyl acylated flavonol glycosides from the aerial parts of Leonurus japonicus Houtt.

Five new syringyl acylated flavonol glycosides, named leonurusoides A (1), B (2), C (3), D (4), and E (5), together with one known one 6 were obtained from the aerial parts of Leonurus japonicus. Their structures were elucidated by chemical and spectroscopic methods (UV, IR, HRESI-TOF-MS, 1D and 2D NMR). Compounds 1-6 showed triglyceride (TG) accumulation inhibitory effects in free fatty acid-induced HepG2 cells.

[Identification of Chinese traditional medicine Angelicae Pubescentis Radix from different places by fourier transform infrared spectrometry].

Common and variation peak rates were calculated as dual-index for drug samples based on the infrared spectrogram of Angelicae Pubescentis Radix samples from different places. The results indicated that the common peak rates of 4 samples from Sichuan province, 2 samples from Gansu province, and 2 samples from Hubei province were higher than 73% , 79. 5% and 76. 3%, respectively. Also, the variation peak rates of 2 samples from Gansu province were lower than 12. 9%. Therefore, there is similar germplasm resources and similar chemical composition in the samples originated from the same place. However, for the samples originated from different places, their germplasm resources and chemical composition maybe have been changed. Fourier transform infrared spectrometry could be used to evaluate the rule of the fingerprint and overview of Angelicae Pubescentis Radix originated from different places by its changes of germplasm resources and chemical composition.