Cimicifugoside H-2 as an Inhibitor of IKK1/Alpha: A Molecular Docking and Dynamic Simulation Study.

One of the most challenging issues scientists face is finding a suitable non-invasive treatment for cancer, as it is widespread around the world. The efficacy of phytochemicals that target oncogenic pathways appears to be quite promising and has gained attention over the past few years. We investigated the effect of docking phytochemicals isolated from the rhizomes of the Cimicifuga foetida plant on different domains of the IκB kinase alpha (IKK1/alpha) protein. The Cimicifugoside H-2 phytochemical registered a high docking score on the activation loop of IKK1/alpha amongst the other phytochemicals compared to the positive control. The interaction of the protein with Cimicifugoside H-2 was mostly stabilized by hydrogen bonds and hydrophobic interactions. A dynamic simulation was then performed with the Cimicifugoside H-2 phytochemical on the activation loop of IKK1/alpha, revealing that Cimicifugoside H-2 is a possible inhibitor of this protein. The pharmacokinetic properties of the drug were also examined to assess the safety of administering the drug. Therefore, in this in silico study, we discovered that the Cimicifugoside H-2 phytochemical inhibits the actively mutated conformation of IKK1/alpha, potentially suppressing the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway.

Transfer of preclinical study data on the influence of cimicifuga racemosaon functional changes in the hippocampus during menopause.

Menopausal transition in women involves complex neurobiochemical changes linked to ovarian dysfunction, resulting in symptoms like vasomotor symptoms (VMS), sleep disturbances, anxiety, and cognitive impairments. Hormone replacement therapy is the first-line treatment. However, many women are reluctant to use HRT or have contraindications toward HRT and seek for alternatives. Non-hormonal therapies with extracts of Cimicifuga racemosa rhizomes like the isopropanolic extract (iCR, black cohosh) offer a promising alternative. A preclinical pilot study exploring iCR's effects on gene expression in the hippocampus and hypothalamus of ovarectomized (OVX) rats mimicking menopausal conditions identified important signaling pathways and CNS-based contributions to the multitargeted modes of action of iCR. Especially in the hippocampus, iCR compensated effects of OVX on gene expression profiles. These changes are reflected by the genes AVPR1A, GAL, CALCA, HCRT, PNOC, ESR1, ESR2 and TAC3 contributing to the formation of hot flushes or thermoregulation as well as to secondary effects such as blood pressure, metabolism, hormonal regulation, homeostasis, mood regulation, neuroendocrine modulation, regulation of sleep and arousal, and in learning, memory and cognition. To understand the mechanisms in the brain of estrogen-depressed animals (OVX) and subsequent iCR treatment we combined the results of the pilot study with those of up-to-date literature and tried to transfer the current knowledge to humans during menopausal transition and adaptation. Focus was laid on changes in the hippocampal function, that is disturbed by hormonal fluctuations, but can also be brought back into balance by iCR.

In-depth characterization of cycloartane triterpenoids and discovery of species-specific markers from three Cimicifuga species guided by a strategy that integrates in-source fragment elimination, diagnostic ion recognition, and feature-based molecular networking.

Characterization studies of the plant metabolome are crucial for revealing plant physiology, developing functional foods, and controlling quality. Mass spectrometry-based metabolite profiling allows unprecedented qualitative coverage of complex biological extract composition. However, the electrospray ionization used in metabolite profiling generates multiple artifactual signals for a single analyte, which makes it challenging to filter out redundant signals and organize the signals corresponding to abundant constituents. This study proposed a strategy integrating in-source fragments elimination, diagnostic ions recognition, and feature-based molecular networking (ISFE-DIR-FBMN) to simultaneously characterize cycloartane triterpenoids (CTs) from three medicinal Cimicifuga species. The results showed that 63.1 % of the measured ions were redundant. A total of 184 CTs were annotated, with 27.1 % being reported for the first time. It presents a promising approach to assess the composition of natural extracts, thus facilitating new ingredient registrations or natural-extracts-based drug discovery campaigns. Besides, chemometrics analysis of the three Cimicifuga species identified 32 species-specific markers, highlighting significant differences among them. The valuable information can enhance the sustainable utilization and further development of Cimicifuga resources. The codes involved in ISFE-DIR-FBMN are freely available on GitHub (https://github.com/LHJ-Group/ISFE-DIR-FBMN.git).

Hydroxy(phenyl)pyruvic acid reductase in Actaea racemosa L.: a putative enzyme in cimicifugic and fukinolic acid biosynthesis.

MAIN CONCLUSION: Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as ß-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration. The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or ß-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (Km 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with ß-hydroxypyruvic acid (Km 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (Km 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of ß-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

Variation in Botanical Reference Materials: Similarity of Actaea Racemosa Analyzed by Flow Injection Mass Spectrometry.

BACKGROUND: Botanical reference materials (BRMs) generally account for the species, cultivar, and year and location of harvest that result in variability in the chemical composition that may lead to statistically significant differences using chemometric methods. OBJECTIVE: To compare the chemical composition of five species of Actaea root BRMs, four herbal sources of A. racemosa root BRMs, and A. racemosa BRMS, and commercial roots and supplements using chemometric methods and selected pre-processing approaches. METHOD: Samples were analyzed by flow injection mass spectrometry (FIMS), principal component analysis (PCA), and factorial multivariate analysis of variance (mANOVA). RESULTS: Statistically significant (P = 0.05) compositional differences were found between three genera (Actaea, Panax, and Ginkgo), five species of Actaea (A. racemosa, A. cimicifuga, A. dahurica, A. pachypoda, and A. rubra) root BRMs, four herbal sources of A. racemosa root BRMs, and A. racemosa BRMS and commercial roots and supplements. The variability of 6% of the BRM variables was found to be quantitatively conserved and reduced the compositional differences between the four sources of root BRMs. Compositional overlap of A. racemosa and other Actaea BRMs was influenced by variation in technical repeats, pre-processing methods, selection of variables, and selection of confidence limits. Sensitivity ranged from 94 to 97% and specificity ranged from 21 to 89% for the pre-processing protocols tested. CONCLUSIONS: Environmental, genetic, and chemometric factors can influence discrimination between species and authentic botanical reference materials. HIGHLIGHTS: Frequency distribution plots derived from soft independent modeling of class analogy provide excellent means for understanding the impact of experimental factors.

NMR-tracking for 15,16-seco-cycloartane triterpenes from Cimicifuga acerina.

Twelve undescribed 15,16-seco-cycloartane triterpenoids, 15,16-seco-cimiterpenes C-N, as well as five previously reported analogs were isolated by NMR-tracking methods from the rhizomes of Cimicifuga acerina (Sieb. et Zucc.) Tanaka. Among them, 15,16-seco-cimiterpenes C-N were the first 15,16-seco-cycloartane triterpenoids featuring acetal or hemiacetal structures at C-15. The chemical structures of 15,16-seco-cimiterpenes C-N were determined based on comprehensive spectroscopic analysis, chemical method, and comparison with the previous literature data. After that, all these compounds were evaluated for their lipid-lowering effects on 3T3-L1 adipocytes.15,16-seco-cimiterpene D was found to exhibit a comparable reducing lipid effect at the concentration of 50 µM, with an inhibition rate at 35.96%.

A Preliminary Study on the Effects of Black Cohosh Preparations on Bone Metabolism of Rat Models With GnRH-a-Induced Peri-Menopausal Symptoms.

Background: Endometriosis (EMS) is a relapsing and estrogen-dependent disease. For endometriosis such as deep endometriosis and ovarian endometrioid cysts, surgery is the most effective treatment. Long-term follow-up showed that the recurrence rate of endometriosis after surgical treatment was high, so postoperative drugs were needed to reduce recurrence, and Gonadotropin-releasing hormone agonists (GnRH-a) were the most commonly used drug for postoperative management.GnRH-a may reduce the post-treatment endometriosis relapses by lowering the hormone levels in the body. However, the use of GnRH-a can give rise to perimenopausal symptoms, especially osteoporosis, bone loss, and bone pain, for which reason GnRH-a use is often limited. The add-back therapy is often used to alleviate the untoward effects caused by GnRH-a. However, long-term use of hormone drugs may lead to EMS recurrence, thrombosis, and breast cancer. Therefore, a safer and more effective drug is urgently needed to alleviate the untoward effects caused by GnRH-a. In recent years, scholars at home and abroad have found that isopropanolic Cimicifuga racemosa extract (ICR), as a plant extract, can better relieve the symptoms of perimenopausal women. At the same time, some studies have initially confirmed that black cohosh preparations can relieve the perimenopausal symptoms caused by GnRH-a treatment in EMS patients. Objective: To investigate the effect of black cohosh preparations on the bone metabolism of rat models with GnRH-a-induced perimenopausal symptoms. Methods: The rat models of perimenopausal symptoms were established by GnRH-a injection. and normal saline (NS injection) was used as the control. According to the modeling method and drug intervention, the rats were randomly divided into four groups: GnRH-a injection + saline intervention group (GnRH-a + NS), saline injection control + saline intervention group (NS + NS), GnRH-a injection + estradiol intervention group (GnRH-a + E2), and GnRH-a injection + black cohosh preparation intervention group (GnRH-a + ICR). The rat models were identified with the vaginal smear method, and then the corresponding drug intervention was administrated for 28 days. After the intervention, the rats were sacrificed. The rats' bone mineral density (BMD) of the distal femur was detected by a dual-energy X-ray bone density scanner. Rat tibia bone tissues were decalcified and made into slices. The pathological and morphological changes of rat tibial bones in each group were observed through HE staining. Histomorphometry parameters of rat tibial bones in each group, such as trabecular bone volume (TBV), trabecular thickness (TbTh), trabecular number (TbN), and trabecular spacing (TbSp), were detected and analyzed by using an automatic image analysis system. Results: (1) The BMD level of the distal femur in the GnRH-a + NS group was significantly lower than the NS + NS, GnRH-a + E2, and GnRH-a + ICR groups (P<0.01), the BMD levels in GnRH-a + E2 and GnRH-a + ICR groups were slightly lower than the NS + NS group, but there was no significant difference among the three groups (P>0.05). (2) The pathological changes of the tibia bones under the microscope in different groups were as follows: The tibia bone trabecular structure was normal in the NS + NS group, without trabecular thinning or fracture, and the arch structure was normal. In the GnRH-a + NS group, some trabecular structures tapered, the arch structure disappeared, but no obvious bone fracture was observed in the trabecula. In the GnRH-a + E2 and GnRH-a + ICR groups, the trabecular structures were normal, without trabecular bone thinning or fracture, and the arch structures were normal. (3) The TBV level of the GnRH-a + INS group was significantly lower than that of the NS + NS, GnRH-a + E2 and GnRH-a + ICR groups (P<0.01, P<0.05, P<0.01), while there was no significant difference among NS + NS, GnRH-a + E2 and GnRH-a + ICR groups (P>0.05). (4) The TbTh levels in the four groups had no significant difference (P>0.05). Compared with the NS + NS group, the TbTh levels in the GnRH-a + NS, GnRH-a + E2, and GnRH-a + ICR groups showed a descending tendency, while the TbTh levels in the GnRH-a + E2 and GnRH-a + ICR groups were slightly higher than that of the GnRH-a + NS group. However, such differences were not significant statistically (P>0.05). (5) Compared with the NS + NS group, the TbN levels in the GnRH-a + NS, GnRH-a + E2, and GnRH-a + ICR groups decreased remarkably (P<0.05). Compared with the GnRH-a + NS group, the TbN levels in the GnRH-a + E2 and GnRH-a + ICR groups showed a mild descending tendency, but such differences were not significant statistically (P>0.05). (6) The TbSp level of the GnRH-a + NS group was significantly higher than that of the NS + NS, GnRH-a + E2, and GnRH-a + ICR groups (P<0.01), while there was no significant difference among NS + NS, GnRH-a + E2 and GnRH-a + ICR groups (P>0.05). Conclusion: The GnRH-a injection could achieve the desired effect. GnRH-a injection may lead to the loss of bone mass in rats. Black cohosh preparations, like estrogen, may have a protective effect on bone mass loss caused by GnRH-a injection.

Fibrous Roots of Cimicifuga Are at Risk of Hepatotoxicity.

The cause of liver damage by using black cohosh preparation has been concerned but remains unclear. After a preliminary investigation, the black cohosh medicinal materials sold in the market were adulterated with Asian cohosh (Cimicifuga) without removing the fibrous roots. The safety of Cimicifuga rhizome and fibrous roots is unknown and has not been reported. Therefore, in this paper, the rhizome and fibrous roots of Cimicifuga dahurica (Turcz.) Maxim (C. dahurica) were completely separated, extracted with 70% ethanol, and freeze-dried to obtain crude rhizome extract (RC) and fibrous roots extract (FRC). UHPLC-Q-TOF-MS was used to identify 39 compounds in the rhizome and fibrous roots of Cimicifuga, mainly saponins and phenolic acids. In the L-02 cytotoxicity experiment, the IC50 of fibrous roots (1.26 mg/mL) was slightly lower than that of rhizomes (1.417 mg/mL). In the 90-day sub-chronic toxicity study, the FRC group significantly increased the level of white blood cells, ALP, ALT, AST, BILI and CHOL (p < 0.05); large area of granular degeneration and balloon degeneration occurred in liver tissue; and the expression of p-NF-kB in the nucleus increased in a dose-dependent manner. Overall, Fibrous roots of Cimicifuga are at risk of hepatotoxicity and should be strictly controlled and removed during the processing.

Two new chemical constituents from the rhizomes of Actaea dahurica.

A new phenylpropanoid allopyranoside (1) and a new indolinone alkaloid (2) were isolated from the rhizomes of Actaea dahurica (syn. Cimicifuga dahurica). The structures of those two compounds were deduced as cimicifugaside F (1) and 3E,11E-(3-methyl-2-butenylidene acid)-2-indolinone-1-O-ß-d-glucopyranoside (2) by detailed analysis of their MS, 1D and 2D NMR data and comparison with literatures. Additionally, the isolates were evaluated for their inhibitory effects on the production of NO by LPS-stimulated RAW 264.7 macrophages.

Gene expression analyses on multi-target mode of action of black cohosh in menopausal complaints - a pilot study in rodents.

PURPOSE: This study aimed at assessing gene expression profiles in hippocampus and hypothalamus of ovariectomized (OVX) rats with or without treatment with an isopropanolic extract of Cimicifuga racemosa rhizomes (iCR) in comparison to intact rats. METHODS: Exploration of hippocampal (Hi) and hypothalamic (Hy) tissue from Sprague Dawley rats: without OVX (NHi = NHy = 4), tissues 3 months after OVX (NHi = 4, NHy = 3), or tissues of rats after their treatment with iCR for 3 months after OVX (NHi = NHy = 2). Gene expression profiles in these tissues were investigated by RNA-microarray-analysis and subsequent verification by qPCR. RESULTS: 4812 genes were differentially regulated when comparing the three groups in hippocampus and hypothalamus. iCR compensated the effects of OVX in 518 genes. This compensatory effect was most prominent in hippocampal signalling pathways, thereof genes (GAL, CALCA, HCRT, AVPR1A, PNOC, etc.) involved in thermoregulation, regulation of sleep and arousal, blood pressure regulation, metabolism, nociception, hormonal regulation, homeostasis, learning and cognition, mood regulation, neuroendocrine modulation, etc.. In the hypothalamus, iCR compensated OVX-effects at TAC3 and OPRM1 but not at KISS1. These genes are involved in the pathophysiology of hot flashes. CONCLUSIONS: Our pilot study findings support a multifaceted mode of action of iCR in menopausal complaints on a tissue-specific brain gene expression level.

Working with the natural complexity: Selection and characterization of black cohosh root extract for use in toxicology testing.

Black cohosh (Actaea racemosa L.) is a botanical supplement marketed to women of all ages. Due to paucity of data to assess the safe use, the National Toxicology Program (NTP) is evaluating the toxicity of black cohosh. The use of an authentic, quality material is imperative to generate robust data. Because botanical materials are complex mixtures with variable composition, the selection of a material is challenging. We describe selection and phytochemical characterization of an unformulated black cohosh root extract (i.e., an extract that serves as source material for a formulated product) to be used in the NTP assessments. A material was selected using a combination of non-targeted and targeted chemical analyses, including confirmation of authenticity, absence of contaminants and adulterants, and similarity to a popular black cohosh product used by consumers. Thirty-nine constituents covering three major classes, triterpene glycosides, phenolic acids, and alkaloids were identified. Among constituents quantified, triterpene glycosides made up approximately 4.7% (w/w) with total constituents quantified making up 5.8% (w/w) of the extract. Non-targeted chemical analysis followed by chemometric analysis of various materials sold as black cohosh, and reference materials for black cohosh and other Actaea species further confirmed the suitability of the selected extract for use.

Systematically identifying the anti-inflammatory constituents of Cimicifuga dahurica by UPLC-Q/TOF-MS combined with network pharmacology analysis.

Cimicifuga dahurica (Turcz.) Maxim, which is also regarded as the main origin of "Shengma" in the Chinese Pharmacopoeia, has been used as a cooling and detoxification agent for thousands of years. Our previous phytochemical investigations of C. dahurica extracts (CDEs) led to the isolation of a series of 9,19-cycloalkane triterpenoids and phenolic acids showing a potential anti-inflammatory activity. However, the chemical profiling of CDEs and the material basis of its anti-inflammatory effect in vivo has not been clarified. In the present study, the CDE chemical profile and prototype components in rat plasma were identified via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. As a result, a total of 106 components were identified or tentatively characterized in CDEs, including 54 triterpenoids, 35 phenolic acids, eight amides and nine other type constituents (39 compounds were confirmed with the reference standards). In addition, 20 prototype components (15 triterpenoids and five phenolic acids) were identified in rat plasma, which potentially related to the anti-inflammatory effects of CDEs. Moreover, the anti-inflammatory activities of the main prototype components were further evaluated by their inhibitory effects on the production of NO, as well as the expressions of iNOS and COX-2 in lipopolysaccharide-stimulated RAW264.7 cells, which indicated that 9,19-cycloalkane triterpenoids may play an anti-inflammatory role by down-regulating the expression of iNOS.

Cimicifuga dahurica extract inhibits the proliferation, migration and invasion of breast cancer cells MDA-MB-231 and MCF-7 in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Cimicifuga dahurica (Turcz.) Maxim (C. dahurica) has a long history of treating breast cancer. From the Qing Dynasty to the Tang Dynasty and even earlier, C. dahurica has been documented in the treatment of breast carbuncle (Breast cancer is classified as breast carbuncle in Chinese medicine). In traditional prescriptions such as "Sheng Ge Decoction", "Sheng Ma Powder" and "Breast Carbuncle Pill", as the main medicine, C. dahurica plays an important role. At present, the systematic studies on the in vitro and in vivo effects of Cimicifuga against breast cancer are rare, especially the C. dahurica. AIM OF THE STUDY: In this article, we evaluated the in vitro activity and in vivo effects of CREE (extract of the root of C. dahurica) against breast cancer, and discussed the possible mechanism of CREE in promoting breast cancer cell apoptosis. MATERIALS AND METHODS: The main component in the CREE was analyzed by HPLC. The effects of CREE on the proliferation, migration and invasion of human breast cancer cells were evaluated through SRB, colony assay, LDH release, wound healing and transwell assay. The pro-apoptotic effect of CREE was investigated in Hochest33342 and Annexin V-FITC/PI assay. To verify the results of CREE in vivo effects, we applied nude mice subcutaneous xenograft experiments. The possible mechanism of CREE treating breast cancer was investigated through mitochondrial membrane potential and western blot experiments. RESULTS: CREE contains cycloartane triterpene saponins. CREE can significantly inhibit the proliferation, migration and invasion of human breast cancer MCF-7 and MDA-MB-231 cells in vitro and it can effectively inhibit the growth of MDA-MB-231 cell subcutaneous tumors in vivo. Besides, we also found that CREE up-regulated the expression levels of Bax, caspase-9/3 and cytochrome C, and down-regulated the expression of Bcl-2. Therefore, regulation of the mitochondrial pathway may be one of the mechanisms by which CREE promotes breast cancer cell apoptosis. CONCLUSIONS: CREE exhibits sufficient anti-breast cancer activity in vivo and in vitro, this study provides persuasive evidence for the further research and development of C. dahurica.

GnRH-a-Induced Perimenopausal Rat Modeling and Black Cohosh Preparations' Effect on Rat's Reproductive Endocrine.

Background: Endometriosis (EMS) is an estrogen-dependent disease, which easily recurs after operation. Gonadotropin-releasing hormone agonist (GnRH-a), an estrogen-inhibiting drug, can effectively inhibit the secretion of gonadotropin by pituitary gland, so as to significantly decrease the ovarian hormone level and facilitate the atrophy of ectopic endometrium, playing a positive role in preventing postoperative recurrence. The application of GnRH-a can lead to the secondary low estrogen symptoms, namely the perimenopausal symptoms, and is a main reason for patients to give up further treatment. The add-back therapy based on sex hormones can well address the perimenopausal symptoms, but long-term use of hormones may cause the recurrence of EMS, as well as liver function damage, venous embolism, breast cancer and other risks, which has long been a heated topic in the industry. Therefore, it is necessary to find effective and safe anti-additive drugs soon. Studies at home and abroad show that, as a plant extract, isopropanolic extract of cimicifuga racemosa (ICR) can well relieve the perimenopausal symptoms caused by natural menopause. Some studies have preliminarily confirmed that black cohosh preparations can antagonize perimenopausal symptoms of EMS patients treated with GnRH-a after operation. Objective: To establish a rat model of perimenopausal symptoms induced by GnRH-a injection, for the purposes of laying a foundation for further research and preliminarily exploring the effect of black cohosh preparations on reproductive endocrine of the rat model. Method: The rat model of perimenopausal symptoms was established by GnRH-a injection, and normal saline (NS injection) was used as the control. The rats were randomly divided into four groups according to different modeling methods and drug intervention schemes. GnRH-a injection + normal saline intervention group (GnRH-a + NS), normal saline injection control + normal saline intervention group (NS + NS), GnRH-a injection + estradiol intervention group (GnRH-a + E2), and GnRH-a injection + black cohosh preparations intervention group (GnRH-a + ICR). After modelling was assessed to be successful with the vaginal smear method, the corresponding drugs were given for intervention for 28d. In the process of rat modeling and drug intervention, the skin temperature and anus temperature of the rat tails were measured every other day, the body weights of the rats were measured every other day, and the dosage was adjusted according to the body weight. After the intervention was over, the serum sex hormone level, the uterine weight, the uterine index, and the endometrial histomorphology changes, as well as the ovarian weight, the ovarian index, and the morphological changes of ovarian tissues of each group were measured. Results: (1) The vaginal cell smears of the control group (NS + NS) showed estrous cycle changes, while other model rats had no estrous cycle of vaginal cells. (2) The body weight gains of the GnRH-a + NS, GnRH-a + E2 and GnRH-a + ICR groups were significantly higher than that of the NS + NS control group. The intervention with E2 and ICR could delay the weight gain trend of rats induced by GnRH-A. (3) After GnRH-a injection, the temperature of the tail and anus of rats showed an overall upward trend, and the intervention with E2 and ICR could effectively improve such temperature change. (4) The E2, FSH, and LH levels in the GnRH-a + NS, GnRH-a + E2, and GnRH-a + ICR groups were significantly lower than those in the NS + NS group (P < 0.01). The E2 level was significantly higher and the LH level was significantly lower in the GnRH-a + E2 group than those in the GnRH-a + NS and GnRH-a + ICR groups (P < 0.05). Compared with those of the GnRH-a + NS and GnRH-a + ICR groups, the FSH level of the GnRH-a + E2 group showed a slight downward trend, but the difference was not statistically significant (P > 0.05). There was no significant difference in the levels of sex hormones between the GnRH-a + NS group and GnRH-a + ICR group (P > 0.05). (5) Compared with those of the NS + NS group, the uterine weight and uterine index of the GnRH-a + NS, GnRH-a + E2 and GnRH-a + ICR groups significantly decreased (P < 0.01). In a comparison between the groups, the uterine weight and uterine index in the GnRH-a + NS and GnRH-a + ICR groups were significantly lower than those in the GnRH-a + E2 group (P < 0.01). There was a statistical difference in the uterine weight and uterine index between the GnRH-a + NS group and GnRH-a + ICR group (P > 0.05). (6) Compared with those of the NS + NS group, the ovarian weight and ovarian index of the GnRH-a + NS, GnRH-a + E2 and GnRH-a + ICR groups significantly decreased (P < 0.01). There was no statistical difference in the ovarian weight and ovarian index among the GnRH-a + E2, GnRH-a + NS and GnRH-a + ICR groups (P > 0.05). (7) Compared with those in the NS + NS group, the number of primordial follicles increased significantly, while the number of growing follicles and mature follicles decreased significantly in the GnRH-a + NS, GnRH-a + E2, and GnRH-a + ICR groups (P < 0.01), but there was a statistical difference in the total number of follicles among the four groups (P > 0.05). Conclusions: The GnRH-a injection could achieve the desired effect. The animal model successfully achieved a significant decrease in the E2, FSH, and LH levels in rats, and could cause the rats to have rising body surface temperature similar to hot flashes in the perimenopausal period. The intervention with E2 and ICR could effectively relieve such "perimenopausal symptoms", and ICR had no obvious effect on the serum sex hormone level in rats.

Chemical profile of Cimicifuga heracleifolia Kom. And immunomodulatory effect of its representative bioavailable component, cimigenoside on Poly(I:C)-induced airway inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried rhizome of Cimicifuga heracleifolia Kom. (C. heracleifolia) is a popular traditional Chinese medicine, which has been extensively used in Asian countries for its anti-inflammatory, antipyretic and analgesic activities. However, further utilization and application of C. heracleifolia have been hampered due to a lack of full understanding of its active ingredients. AIM OF STUDY: The present study aims for clarification of the systematical chemical profile of C. heracleifolia and the immunomodulatory effect of its main bioavailable component. MATERIALS AND METHODS: Comprehensive chemical profile of C. heracleifolia was systematically analyzed by ultra-performance liquid chromatography hyphenated with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). Xenobiotics after oral administration of C. heracleifolia extracts were investigated to hunt for bioavailable components. The immunomodulatory activity evaluation of cimigenoside was achieved on poly(I:C)-induced airway inflammation mouse and BEAS-2B cell models from aspects of neutrophil infiltration, lung inflammation by using microscope analysis, quantification of production and expression of inflammatory cytokine and chemokines by using ELISA and quantitative PCR. RESULTS: By UPLC-Q-TOF/MS analysis, 110 compounds (including 81 triterpenoids, 21 cinnamic acid derivatives, and 8 other structure types) were identified or tentatively characterized in ethanolic extract of C. heracleifolia. Based on the data of chemical profile, xenobiotics of C. heracleifolia were subsequently analyzed, and triterpene glycosides were detected as the major bioavailable ingredients. Oral administration of cimigenoside, a representative triterpene glycoside, could prevent neutrophils infiltration in the lung due to suppression of the production of CXCL2 and CXCL10, and the expression of P-selectin, VCAM1 in poly(I:C)-induced airway inflammation model mice. Moreover, cimigenoside also inhibited the productions of inflammatory cytokines and chemokines from human airway epithelial cell line (BEAS-2B cells) induced by poly(I:C). CONCLUSION: Triterpene glycosides were the main components of C. heracleifolia extract, and cimigenoside was considered as the effective component with immunomodulatory effect on the pulmonary immune system by oral administration.

Actaea racemosa L. extract inhibits steroid sulfation in human breast cancer cells: Effects on androgen formation.

BACKGROUND: Actaea racemosa L., also known as black cohosh, is a popular herb commonly used for the treatment of menopausal symptoms. Because of its purported estrogenic activity, black cohosh root extract (BCE) may trigger breast cancer growth. STUDY DESIGN/METHODS: The potential effects of standardized BCE and its main constituent actein on cellular growth rates and steroid hormone metabolism were investigated in estrogen receptor alpha positive (ERα+) MCF-7 and -negative (ERα-) MDA-MB-231 human breast cancer cells. Cell numbers were determined following incubation of both cell lines with the steroid hormone precursors dehydroepiandrosterone (DHEA) and estrone (E1) for 48 h, in the presence and absence of BCE or actein. Using a validated liquid chromatography-high resolution mass spectrometry assay, cell culture supernatants were simultaneously analyzed for the ten main steroids of the estrogen pathway. RESULTS: Inhibition of MCF-7 and MDA-MB-231 cell growth (up to 36.9%) was observed following treatment with BCE (1-25 µg/ml) or actein (1-50 µM). Incubation of MCF-7, but not of MDA-MB-231 cells, with DHEA and BCE caused a 20.9% reduction in DHEA-3-O-sulfate (DHEA-S) formation, leading to a concomitant increase in the androgens 4-androstene-3,17-dione (AD) and testosterone (T). Actein was shown to exert an even stronger inhibitory effect on DHEA-S formation in MCF-7 cells (up to 89.6%) and consequently resulted in 12- to 15-fold higher androgen levels compared with BCE. The formation of 17ß-estradiol (E2) and its glucuronidated and sulfated metabolites was not affected by BCE or actein after incubation with the estrogen precursor estrone (E1) in either cell line. CONCLUSIONS: The results of the present study demonstrated that actein and BCE do not promote breast cancer cell growth or influence estrogen levels. However, androgen formation was strongly stimulated by BCE and actein, which may contribute to their ameliorating effects on menopausal symptoms in women. Future studies monitoring the levels of AD and T upon BCE supplementation of patients are warranted to verify an association between BCE and endogenous androgen metabolism.

Differentiation of Actaea species by NMR metabolomics analysis.

The 1H NMR spectra of crude extracts from a total of 33 Actaea samples were acquired and analyzed for their species- and plant part-specific metabolomic characteristics by identifying fingerprint resonances via visual observation as well as a chemometric approach using principal component analysis (PCA). The main study subjects were the roots/rhizomes and aerial parts of three American species, Actaea racemosa (AR), Actaea podocarpa (AP) and Actaea cordifolia (AC). AP exhibited an already visually distinct chemical profile from those of the other two species. The species-characteristic resonances were identified as analytical chemotaxonomic markers. AR and AC exhibited visually similar 1H NMR spectral profiles that required statistical analysis for differentiation. Several characteristic peaks and peak patterns were identified for each group of samples. Together with the three American Actaea species, the characteristics of the 1H NMR spectra of Asian species are also discussed. A statistical analysis method using PCA was employed to provide the metabolomic profile for visually minor but analytically significant chemotaxonomic differences. PCA scores allowed differentiation between the three American Actaea species, as well as the ability to differentiate between the various plant parts (aboveground vs. roots/rhizomes).

Cycloartane triterpene glycosides from rhizomes of Cimicifuga foetida L. with lipid-lowering activity on 3T3-L1 adipocytes.

Six previously undescribed cycloartane triterpenes glycosides, cimimanols A-F (1-6), together with thirteen known analogues (7-19) were isolated from the rhizomes of Cimicifuga foetida. Among them, cimimanol A (1) was the first example of cycloartane triterpene glycoside featuring a unique cyclic carbonate, and cimimanol B (2) was a rare trinortriterpene glycoside. The chemical structures and absolute configurations of new compounds were determined on the basis of comprehensive spectroscopic analysis, chemical method, and X-ray crystal diffraction, as well as quantum chemistry calculations. Finally, all these compounds were evaluated for their lipid-lowering effect on 3T3-L1 adipocytes. Compounds 1-3, 6-10, 12-16, 18-19 could significantly reduce the fat accumulation in 3T3-L1 adipocytes, especially compounds 8, 9, 14, and 15 exhibited strong lipid-lowering effect at the concentration of 10 µM, with inhibition rates ranging from 8.35% to 12.07%.

Comparative in vivo pharmacokinetics study of affeic acid, isoferulic acid and ferulic acid in crude and three different prepared Cimicifuga foetida L.

Our study investigated the differences in pharmacokinetics of three major components of crude Cimicifuga foetida L. and its fried product and honey- and liquor-prepared products. A rapid and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry approach was established for determing caffeic acid, isoferulic acid and ferulic acid in rat plasma. The approach has good linearity, precision, accuracy, recovery and stability. Phenolic acid was rapidly absorbed. The times to peak concentration were shorter in the processed group than those for the crude product, with their values of <30 min. The peak concentration values of caffeic acid and isoferulic acid were higher in the crude group than in the processed groups (p < 0.05). Area under the curve values of the three phenolics in the crude group were significantly higher than those of the processed groups (p < 0.05).

Structure-Guided Identification of Black Cohosh (Actaea racemosa) Triterpenoids with In Vitro Activity against Multiple Myeloma.

Black cohosh is a well-established medicinal plant and preparations of its rootstock are used for the treatment of mild climacteric complaints. The compounds considered responsible for the therapeutic effect are triterpene glycosides, characterized by a cycloartane scaffold and a pentose moiety. Because some of these triterpenoids were found to exhibit relevant cytotoxic effects against human breast cancer cells, we decided to investigate their activity on multiple myeloma cell lines NCI-H929, OPM-2, and U266. In a systematic approach, we initially tested three known cytotoxic compounds of three different triterpenoid types, revealing the cimigenol-type triterpenoid as the most active constituent. In a second round, seven naturally occurring cimigenol derivatives were compared with respect to their sugar moiety and their substitution pattern at position C-25, leading to 25-O-methylcimigenol-3-O-α-L-arabinopyranoside as the most potent candidate. Interestingly, not only the methyl group at position C-25 increased the cytotoxic effect but also the arabinose moiety at position C-3 had an impact on the activity. The variety of cimigenol derivatives, moreover, allowed a detailed discussion of their structure-activity relationships, not only for their effect on multiple myeloma cells but also with regard to previous studies on the cytotoxicity of black cohosh triterpenoids.