Systematic DOE Approach for Dry Granulation Study at Early Clinical Stage of Novel Drugs: An Industrial Case.

In order to introduce a cost-effective strategy method for commercial scale dry granulation at the early clinical stage of drug product development, we developed dry granulation process using formulation without API, fitted and optimized the process parameters adopted Design of Experiment (DOE). Then, the process parameters were confirmed using one formulation containing active pharmaceutical ingredient (API). The results showed that the roller pressure had significant effect on particle ratio (retained up to #60 mesh screen), bulk density and tapped density. The roller gap had significant influence on particle ratio and specific energy. The particle ratio was significantly affected by the mill speed (second level). The tabletability of the powder decreased after dry granulation. The effect of magnesium stearate on the tabletability was significant. In the process validation study, the properties of the prepared granules met the requirements for each response studied in the DOE. The prepared tablets showed higher tensile strength, good content uniformity of filled capsules, and the dissolution profiles of which were consistent with that of clinical products. This drug product process development and research strategies could be used as a preliminary experiment for the dry granulation process in the early clinical stage.

Wine- and stir-frying processing of Cuscutae Semen enhance its ability to alleviate oxidative stress and apoptosis via the Keap 1-Nrf2/HO-1 and PI3K/AKT pathways in H2O2-challenged KGN human granulosa cell line.

BACKGROUND: Cuscutae Semen (CS) has been prescribed in traditional Chinese medicine (TCM) for millennia as an aging inhibitor, an anti-inflammatory agent, a pain reliever, and an aphrodisiac. Its three main forms include crude Cuscutae Semen (CCS), wine-processed CS (WCS), and stir-frying-processed CS (SFCS). Premature ovarian insufficiency (POI) is a globally occurring medical condition. The present work sought a highly efficacious multi-target therapeutic approach against POI with minimal side effects. Finally, it analyzed the relative differences among CCS, WCS and SFCS in terms of their therapeutic efficacy and modes of action against H2O2-challenged KGN human granulosa cell line. METHODS: In this study, ultrahigh-performance liquid chromatography (UPLC)-Q-ExactiveTM Orbitrap-mass spectrometry (MS), oxidative stress indices, reactive oxygen species (ROS), Mitochondrial membrane potential (MMP), real-time PCR, Western blotting, and molecular docking were used to investigate the protective effect of CCS, WCS and SFCS on KGN cells oxidative stress and apoptosis mechanisms. RESULTS: The results confirmed that pretreatment with CCS, WCS and SFCS reduced H2O2-induced oxidative damage, accompanied by declining ROS levels and malondialdehyde (MDA) accumulation in the KGN cells. CCS, WCS and SFCS upregulated the expression of antioxidative levels (GSH, GSH/GSSG ratio, SOD, T-AOC),mitochondrial membrane potential (MMP) and the relative mRNA(Nrf2, Keap1, NQO-1, HO-1, SOD-1, CAT). They inhibited apoptosis by upregulating Bcl-2, downregulating Bax, cleaved caspase-9, and cleaved caspase-3, and lowering the Bax/Bcl-2 ratio. They also exerted antioxidant efficacy by partially activating the PI3K/Akt and Keap1-Nrf2/HO-1 signaling pathways. CONCLUSIONS: The results of the present work demonstrated the inhibitory efficacy of CCS, WCS and SFCS against H2O2-induced oxidative stress and apoptosis in KGN cells and showed that the associated mechanisms included Keap1-Nrf2/HO-1 activation, P-PI3K upregulation, and P-Akt-mediated PI3K-Akt pathway induction.

Structural and Compositional Changes in Two Marine Shell Traditional Chinese Medicines: A Comparative Analysis Pre- and Post-Calcination.

BACKGROUND: Arcae concha and Meretricis concha cyclinae concha are two marine shellfish herbs with similar composition and efficacy, which are usually calcined and used clinically. OBJECTIVE: This study investigated variations in the inorganic and organic components of Arcae concha and Meretricis concha cyclinae concha from different production regions, both Arcae concha and Meretricis concha cyclinae concha. The aim was to enhance the understanding of these two types of marine shell traditional Chinese medicine (msTCM) and provide a foundation for their future development and application. METHOD: Spectroscopic techniques, including infrared spectroscopy, X-ray spectroscopy, and X-ray fluorescence spectroscopy, were used to analyze the calcium carbonate (CaCO3) crystal and trace elements. Thermogravimetric analysis was used to investigate the decomposition process during heating. The proteins were quantified using the BCA protein assay kit. Principal component analysis (PCA) was used to classify inorganic elements in the two marine shellfish traditional Chinese medicines. RESULTS: No significant differences were found among the various production regions. The crystal structure of CaCO3 in the raw products was aragonite, but it transformed into calcite after calcination. The contents of Ca, Na, Sr, and other inorganic elements were highest. The protein content was significantly reduced after calcination. Therefore, these factors cannot accurately reflect the internal quality of TCM, rendering qualitative identification challenging. CaCO3 dissolution in the decoction of Arcae concha and Meretricis concha cyclinae concha increased after calcination, aligning with the clinical application of calcined shell TCM. PCA revealed the inorganic elements in them, indicating that the variation in trace element composition among different drugs leads to differences in their therapeutic focus, which should be considered during usage. CONCLUSIONS: This study clarifies the composition and structure changes of corrugated and clam shell before and after calcining, and it lays the foundation for the comprehensive utilization of marine traditional Chinese medicine. HIGHLIGHTS: These technical representations reveal the differences between raw materials and processed products, which will provide support for the quality control of other shellfish TCM.

The modern scientific mystery of traditional Chinese medicine processing--take some common traditional Chinese medicine as examples.

The processing of traditional Chinese medicine (TCM) is a unique traditional pharmaceutical technology in China, which is the most important feature that distinguishes Chinese medicine from natural medicine and plant medicine. Since the record in Huangdi Neijing (Inner Canon of the Yellow Emperor), till now, the processing of TCM has experienced more than 2000 years of inheritance, innovation, and development, which is a combination of TCM theory and clinical practice, and plays an extremely important position in the field of TCM. In recent years, as a clinical prescription of TCM, Chinese herbal pieces have played a significant role in the prevention and control of the COVID-19 and exhibited their unique value, and therefore they have become the highlight of China's clinical treatment protocol and provided Chinese experience and wisdom for the international community in the prevention and control of the COVID-19 epidemic. This paper outlines the research progress in the processing of representative TCM in recent years, reviews the mechanism of the related effects of TCM materials after processing, such as changing the drug efficacy and reducing the toxicity, puts forward the integration and application of a variety of new technologies and methods, so as to reveal the modern scientific mystery of the processing technology of TCM.

Study on the preparation and enzyme inhibitory activity of polyphenols from Sargassum pallidum.

This study aimed to obtain a high yield and purity of Sargassum pallidum polyphenol extracts (SPPE) and study its enzyme activity. Fresh Sargassum pallidum seaweed was selected for optimization of ultrasound-assisted extraction (UAE) conditions and purification conditions using macroporous resin and Sephadex LH20 to obtain SPPE. The SPPE was characterized using UPLC-QTOF-MS/MS and α-amylase, α-glucosidase, tyrosinase, and AchE inhibitory activity were determined. The maximum extraction rate of SPPE was 7.56 mg GAE/g and the polyphenol purity reached 70.5% after macroporous resin and Sephadex LH-20 purification. A total of 50 compounds were identified by UPLC-QTOF-MS/MS. The IC50 values of SPPE were 334.9 µg/mL, 6.290 µg /mL, 0.834 mg /mL and 0.6538 mg /mL for α-amylase, α-glucosidase, tyrosinase and AchE, respectively. Molecular docking technology further revealed the effects of SPPE on the above enzymes. This study provided information on the potential hypoglycemic, whitening and anti-Alzheimer's disease biological activities of SPPE, which had guiding significance for the purification and development of other seaweed polyphenols.

Berberine inhibits Candida albicans growth by disrupting mitochondrial function through the reduction of iron absorption.

AIMS: This study aimed to investigate whether berberine (BBR) can inhibit the iron reduction mechanism of Candida albicans, lowering the iron uptake of the yeast and perhaps having antimicrobial effects. METHODS AND RESULTS: We determined that BBR may cause extensive transcriptional remodeling in C. albicans and that iron permease Ftr1 played a crucial role in this process through eukaryotic transcriptome sequencing. Mechanistic research showed that BBR might selectively inhibit the iron reduction pathway to lower the uptake of exogenous iron ions, inhibiting C. albicans from growing and metabolizing. Subsequent research revealed that BBR caused significant mitochondrial dysfunction, which triggered the process of mitochondrial autophagy. Moreover, we discovered that C. albicans redox homeostasis, susceptibility to antifungal drugs, and hyphal growth are all impacted by the suppression of this mechanism by BBR. CONCLUSIONS: The iron reduction mechanism in C. albicans is disrupted by BBR, which disrupts mitochondrial function and inhibits fungal growth. These findings highlight the potential promise of BBR in antifungal applications.

Liensinine pretreatment reduces inflammation, oxidative stress, apoptosis, and autophagy to alleviate sepsis acute kidney injury.

Liensinine is mainly derived from alkaloids extracted and isolated from lotus seeds (Nelumbo nucifera Gaertn). It possesses anti-inflammatory, and antioxidant, according to contemporary pharmacological investigations. However, the effects and therapeutic mechanisms of liensinine on acute kidney injury (AKI) models of sepsis are unclear. To gain insight into these mechanisms, we established a sepsis kidney injury model by LPS injection of mice treated with liensinine, and stimulation of HK-2 with LPS in vitro and treated with liensinine and inhibitors of p38 MAPK, JNK MAPK. We first found that liensinine significantly reduced kidney injury in sepsis mice, while suppressing excessive inflammatory responses, restoring renal oxidative stress-related biomarkers, reducing increased apoptosis in TUNEL-positive cells and excessive autophagy, and that this process was accompanied by an increase in JNK/ p38-ATF 2 axis. In vitro experiments further demonstrated that lensinine reduced the expression of KIM-1, NGAL, inhibited pro- and anti-inflammatory secretion disorders, regulated the activation of the JNK/p38-ATF 2 axis, and reduced the accumulation of ROS, as well as the reduction of apoptotic cells detected by flow cytometry, and that this process played the same role as that of p38 MAPK, JNK MAPK inhibitors. We speculate that liensinine and p38 MAPK, JNK MAPK inhibitors may act on the same targets and could be involved in the mechanism of alleviating sepsis kidney injury in part through modulation of the JNK/p38-ATF 2 axis. Our study demonstrates that lensinine is a potential drug and thus provides a potential avenue for the treatment of AKI.

Dynamic changes in marker components during the stir-frying of Pharbitidis Semen, and network analysis of its potential effects on nephritis.

Introduction: Pharbitidis Semen (PS) has been widely used in traditional Chinese medicine to treat several diseases such as nephritis. PS is usually stir-fried to enhance its therapeutic efficacy before use in clinical practice. However, the changes in phenolic acids during stir-frying and the mechanisms of their therapeutic effects on nephritis are still unclear. Methods: Here, we studied the processing-induced chemical changes and elucidated the mechanism of PS in the treatment of nephritis. We determined the levels of the 7 phenolic acids in raw PS (RPS) and stir-fried PS (SPS) using high-performance liquid chromatography, analyzed the dynamic compositional changes during stir-frying, and used network analysis and molecular docking to predict and verify compound targets and pathways corresponding to nephritis. Results: The dynamic changes in the 7 phenolic acids in PS during stir-frying are suggestive of a transesterification reaction. Pathway analysis revealed that the targets of nephritis were mainly enriched in the AGE-RAGE, hypoxia-inducible factor-1, interleukin-17, and tumor necrosis factor signaling pathways among others. Molecular docking results showed that the 7 phenolic acids had good binding ability with the key nephritic targets. Discussion: The potential pharmaceutical basis, targets, and mechanisms of PS in treating nephritis were explored. Our findings provide a scientific basis for the clinical use of PS in treating nephritis.

In vitro and in vivo evaluation of a water-in-oil microemulsion of platycodin D.

Platycodin D (PD) is the active metabolite of Platycodon grandiflorum. The main purpose of this study was to develop and evaluate a water-in-oil (W/O) microemulsion formulation of PD (PD-ME). The PD-ME was successfully prepared by the water titration method at K m = 2, to draw the pseudoternary phase diagrams. Physical characterization including the particle size, pH, refractive index, average viscosity, and polydispersity index (PDI) was performed. The in vivo characteristics were evaluated by intestinal permeability and pharmacokinetic studies. The optimized microemulsion formulation consisted of 100 mg/ml PD aqueous solution, soybean phospholipids, ethanol, and oleic acid (27:39:19:15, w/w). The average viscosity, pH, droplet size, PDI, and zeta potential of the PD-ME were 78.65 ± 0.13 cPa•s, 5.70 ± 0.05, 30.46 ± 0.20 nm, 0.33 ± 0.00, and -3.13 mV, respectively. The drug concentration of the PD-ME was 26.3 ± 0.6 mg/ml. The PD-ME showed significantly higher apparent permeability coefficients than PD (p < .01). The pharmacokinetic studies showed that the PD-ME had significantly higher values of T 1/2 (2.26-fold), AUC0-24h (area under the curve; 1.65-fold), and MRT0-24h (1.58-fold) than PD (p < .01). It can be seen that W/O ME presents a strategy with great promise for enhancing the intestinal permeability and better oral absorption of drugs with high polarity and poor permeability.

Mechanism of enhanced oral absorption of akebia saponin D by a self-nanoemulsifying drug delivery system loaded with phospholipid complex.

Akebia saponin D (ASD) exhibits a variety of pharmacological activities, such as anti-osteoporosis, neuroprotection, hepatoprotection, but has poor oral bioavailability. A self-nanoemulsifying drug delivery system loaded with akebia saponin D - phospholipid complex (APC-SNEDDS) (composition: Peceol: Cremophor® EL: Transcutol HP: ASD: phospholipid; ratio: 10:45:45:51:12.3, w:w:w:w:w) was first developed to improve the oral absorption of saponins and it was found to significantly enhance ASD's oral bioavailability by 4.3 - fold (p < .01). This study was conducted to elucidate the mechanism of enhanced oral absorption of ASD by the drug delivery system of APC-SNEDDS. The aggregation morphology and particle size of ASD and APC-SNEDDS prepared in aqueous solutions were determined by transmission electron microscope and particle size analyzer, respectively. Stability of ASD and APC-SNEDDS in gastrointestinal luminal contents and mucosa homogenates were also explored. The differences of in situ intestinal permeability of ASD and APC-SNEDDS were compared. APC-SNEDDS reduced the aggregation size from 389 ± 7 nm (ASD) to 148 ± 3 nm (APC-SNEDDS). APC-SNEDDS increased the remaining drug in large intestine luminal contents from 47 ± 1% (ASD) to 83 ± 1% (APC-SNEDDS) during 4 h incubation. APC-SNEDDS provided an 11-fold increase in Ka value and an 11-fold increase in Peff value compared to ASD. In summary, APC-SNEDDS improved ASD's oral bioavailability mainly by increasing membrane permeability, destroying self-micelles and inhibiting the intestinal metabolism.

Akebia saponin D reverses corticosterone hypersecretion in an Alzheimer's disease rat model.

BACKGROUND: Glucocorticoid hormones are implicated in the pathogenesis of Alzheimer's disease (AD) and other diseases including diabetes, hyperlipidemia, and osteoporosis. Akebia saponin D (ASD) possesses numerous pharmacological activities, including as an anti-AD, anti-hyperlipidemia, anti-diabetes, and anti-osteoporosis agent. The anti-AD effect of ASD is possibly through its regulation of glucocorticoid levels. PURPOSE: The present study was undertaken to investigate the neuroprotective effects of ASD on Aß25-35-induced cognitive deficits and to elucidate its underlying mechanism of action. METHODS: The AD rat model was established by an intracerebroventricular injection of Aß25-35 into the lateral ventricles. Spatial learning and anxiety state were assessed by Morris water maze task and elevated plus-maze assay, respectively. The degree of hypertrophy of adrenal gland was analyzed using the viscera coefficient. Corticosterone and ACTH concentrations in the plasm were measured using biochemical assay kits. The activity of 11ß-hydroxysteroid dehydrogenase type-1 (11ß-HSD1) in liver and groin fat pad was assessed by measuring cortisol production. RESULTS: Compared with the control group, AD rats displayed significant spatial learning and reference memory impairments, serious anxiety disorders, obvious hypertrophy of adrenal gland, elevated corticosterone and ACTH levels in the plasma, and increased 11ß-HSD1 activity in liver and groin fat pad. ASD could significantly ameliorate the memory deficits and anxiety symptoms, markedly reduce the viscera coefficient of adrenal gland, observably decrease corticosterone and ACTH concentrations, and showed no effect on the activity of 11ß-HSD1. CONCLUSIONS: These results indicate that ASD might exert a significant neuroprotective effect on cognitive impairment, driven in part by reducing systemic corticosterone level by down-regulation of the hypothalamic-pituitary-adrenal (HPA) axis.

Protodioscin ameliorates fructose-induced renal injury via inhibition of the mitogen activated protein kinase pathway.

BACKGROUND: High dietary fructose can cause metabolic syndrome and renal injury. PURPOSE: The effects of protodioscin on metabolic syndrome and renal injury were investigated in mice receiving high-dose fructose. METHODS: Mice received 30% (w/v) fructose in water and standard chow for 6 weeks to induce metabolic syndrome and were divided into four groups to receive carboxymethylcellulose sodium, allopurinol (5 mg/kg) and protodioscin (5 and 10 mg/kg) continuously for 6 weeks, respectively. The glucose intolerance, serum uric acid (UA), blood urea nitrogen (BUN), creatinine (Cr), total cholesterol (TC), triglyceride (TG), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined. RESULTS: Protodioscin significantly improved glucose intolerance and reduced the levels of serum UA, BUN, Cr, TC and TG. Histological examinations showed that protodioscin ameliorated glomerular and tubular pathological changes. Protodioscin significantly reduced renal concentrations of IL-1ß, IL-6 and TNF-α by inhibiting the activation of nuclear factor-κB, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. In addition, the effect of protodioscin on the mitogen activated protein kinases (MAPK) pathway was examined. CONCLUSION: Taken together, protodioscin is a potential drug candidate for high dietary fructose-induced metabolic syndrome and renal injury.

Preparation and evaluation of a self-nanoemulsifying drug delivery system loaded with Akebia saponin D-phospholipid complex.

BACKGROUND: Akebia saponin D (ASD) exerts various pharmacological activities but with poor oral bioavailability. In this study, a self-nanoemulsifying drug delivery system (SNEDDS) based on the drug-phospholipid complex technique was developed to improve the oral absorption of ASD. METHODS: ASD-phospholipid complex (APC) was prepared using a solvent-evaporation method and characterized by infrared spectroscopy, differential scanning calorimetry, morphology observation, and solubility test. Oil and cosurfactant were selected according to their ability to dissolve APC, while surfactant was chosen based on its emulsification efficiency in SNEDDS. Pseudoternary phase diagrams were constructed to determine the optimized APC-SNEDDS formulation, which was characterized by droplet size determination, zeta potential determination, and morphology observation. Robustness to dilution and thermodynamic stability of optimized formulation were also evaluated. Subsequently, pharmacokinetic parameters and oral bioavailability of ASD, APC, and APC-SNEDDS were investigated in rats. RESULTS: The liposolubility significantly increased 11.4-fold after formation of APC, which was verified by the solubility test in n-octanol. Peceol (Glyceryl monooleate [type 40]), Cremophor® EL (Polyoxyl 35 castor oil), and Transcutol HP (Diethylene glycol monoethyl ether) were selected as oil, surfactant, and cosurfactant, respectively. The optimal formulation was composed of Glyceryl monooleate (type 40), Polyoxyl 35 castor oil, Diethylene glycol monoethyl ether, and APC (1:4.5:4.5:1.74, w/w/w/w), which showed a particle size of 148.0±2.7 nm and a zeta potential of -13.7±0.92 mV after dilution with distilled water at a ratio of 1:100 (w/w) and good colloidal stability. Pharmacokinetic studies showed that APC-SNEDDS exhibited a significantly greater Cmax1 (733.4±203.8 ng/mL) than ASD (437.2±174.2 ng/mL), and a greater Cmax2 (985.8±366.6 ng/mL) than ASD (180.5±75.1 ng/mL) and APC (549.7±113.5 ng/mL). Compared with ASD, Tmax1 and Tmax2 were both remarkably shortened by APC-SNEDDS. The oral bioavailability in rats was enhanced significantly to 183.8% and 431.8% by APC and APC-SNEDDS, respectively. CONCLUSION: These results indicated that APC-SNEDDS was a promising drug delivery system to enhance the oral bioavailability of ASD.

Neuroprotective effects of polygalacic acid on scopolamine-induced memory deficits in mice.

BACKGROUND: Polygala tenuifolia Willd is a Traditional Chinese Medicine used for the treatment of learning and memory deficits. Triterpenoid saponins, the main bioactive compounds of Polygala tenuifolia Willd, are easily hydrolyzed to polygalacic acid (PA). PURPOSE: The present study was undertaken to investigate the neuroprotective effects of PA on scopolamine-induced cognitive dysfunction and to elucidate its underlying mechanisms of action. METHODS: PA (3, 6, and 12 mg/kg) was administered orally to mice for fourteen days, and scopolamine (1 mg/kg) was injected intraperitoneally for fourteen days to induce memory impairment. Memory-related behaviors were evaluated using the Morris water maze. Cholinergic and neuroinflammatory activities were measured in brain tissue. Superoxide dismutase activities, malondialdehyde and reduced glutathione contents were also measured in the brains. RESULTS: Treatment with scopolamine significantly increased the escape latency time, decreased the number of crossings, and shortened the time spent in the target quadrant, while PA reversed these scopolamine-induced effects. PA significantly improved cholinergic system reactivity, as indicated by decreased acetylcholinesterase (AChE) activity, increased choline acetyltransferase (ChAT) activity, and elevated levels of acetylcholine (ACh) in the hippocampus and frontal cortex. PA also significantly ameliorated neuroinflammation and oxidative stress in mice. CONCLUSION: These results suggest that PA might exert a significant neuroprotective effect on cognitive impairment, driven in part by the modulation of cholinergic activity and neuroinflammation.

Enhancement of absorption and bioavailability of echinacoside by verapamil or clove oil.

PURPOSE: This present study investigated the absorption kinetics of echinacoside (ECH) in situ and in vitro and its oral bioavailability in rats. Additional aim was to find an agent(s) to promote ECH absorption and oral bioavailability among two efflux proteins and three absorption promoters. METHODS: ECH absorption behaviors were investigated by everted gut sac model in vitro and single-pass intestinal perfusion model in situ. Pharmacokinetics study was performed to investigate the influences of verapamil and clove oil on ECH bioavailability in vivo. All samples were measured at different time intervals by high performance liquid chromatography. RESULTS: The results showed that the effective permeability coefficient (P eff) and apparent permeability coefficient of ECH were 0.83×10(-6)-3.23×10(-6) cm/s and 2.99×10(-6)-9.86×10(-6) cm/s, respectively. The P eff among duodenum, jejunum, and ileum were not statistically different, but they were higher than colon (P<0.01), which demonstrated that intestinal ECH absorption was poor and site dependent. Additionally, verapamil and clove oil significantly increased the jejunal P eff of ECH both in situ and in vitro. Moreover, the bioavailability of ECH in combination with verapamil and clove oil were increased by 1.37-fold (P<0.05) and 2.36-fold (P<0.001), respectively, when compared to ECH group. Overall, verapamil and clove oil facilitated ECH absorption and oral bioavailability. CONCLUSION: The absorption and bioavailability of ECH were enhanced by verapamil and clove oil, respectively, both in vitro and in vivo. Consequently, the combination of verapamil and clove oil with ECH will be a promising and effective approach to promote intestinal absorption and oral bioavailability of ECH.

Efficacy and safety of echinacoside in a rat osteopenia model.

This study aimed to investigate the efficacy and safety of echinacoside (ECH) using an osteopenia rat model. Forty-eight 6-month-old female Sprague-Dawley rats were randomly divided into one sham-operated group (SHAM) and five OVX (ovariectomized) subgroups: SHAM with vehicle 0.5% carboxymethylcellulose sodium (0.5% CMC-Na) and OVX with vehicle (OVX), OVX with 17 ß -estradiol (E2), and OVX with ECH of graded doses (ECH-L, ECH-M, and ECH-H). The effects of ECH and E2 on serum biochemical parameters, bone mineral density (BMD), bone biomechanical properties, bone microarchitecture, and immunohistochemistry were examined, and safety assessments were also evaluated. The results showed that ECH treatments improved total femur BMD, bone microarchitecture, and biomechanical properties and decreased serum marker levels in comparison to OVX group. Moreover, ECH administration significantly increased osteoprotegerin (OPG) level, and decreased receptor activator of nuclear factor- κ B ligand (RANKL) level in serum, as well as in proximal femur. Importantly, ECH treatment ameliorated the lipid parameters without the overall incidences of adverse events of uterus and mammary gland compared to OVX and SHAM groups. This study demonstrated that administration of ECH for 12 weeks can effectively and safely prevent OVX-induced osteoporosis in rats via increasing the OPG/RANKL ratio.