Pinoresinol diglucoside mitigates dexamethasone-induced osteoporosis and chondrodysplasia in zebrafish.

BACKGROUND: The global increase in the aging population has led to a higher incidence of osteoporosis among the elderly. OBJECTIVE: This study aimed to evaluate the protective properties of pinoresinol diglucoside (PDG), an active constituent of Eucommia ulmoides, against dexamethasone-induced osteoporosis and chondrodysplasia. METHODS: A zebrafish model of osteoporosis was established by exposing larval zebrafish to dexamethasone. The impact of PDG on bone mineralization was assessed through alizarin red and calcein staining. Alkaline phosphatase activity was quantified to evaluate osteoblast function. The influence of PDG on chondrogenesis was estimated using alcian blue staining. Fluorescence imaging and motor behavior analysis were employed to assess the protective effect of PDG on the structure and function of dexamethasone-induced skeletal teratogenesis. qPCR determined the expression of osteogenesis and Wnt signaling-related genes. Molecular docking was used to assess the potential interactions between PDG and Wnt receptors. RESULTS: PDG significantly increased bone mineralization and corrected spinal curvature and cartilage malformations in the zebrafish model. Furthermore, PDG enhanced swimming abilities compared to the model group. PDG mitigated dexamethasone-induced skeletal abnormalities in zebrafish by upregulating Wnt signaling, showing potential interaction with Wnt receptors FZD2 and FZD5. CONCLUSION: PDG mitigates dexamethasone-induced osteoporosis and chondrodysplasia by promoting bone formation and activating Wnt signaling.

Pinoresinol diglucoside ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

This study is aimed to investigate the effect of pinoresinol diglucoside (PDG) in ameliorating myocardial ischemia-reperfusion injury (MIRI). Hypoxia/reperfusion (H/R)-induced H9c2 cardiomyocytes were used to establish an in-vitro ischemia-reperfusion injury model of cardiomyocytes. Cells were treated with 1 µmol/L of PDG. Reactive oxygen species (ROS) level was detected by a 2',7'-dichlorofluorescein-diacetate assay. The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was examined by enzyme-linked immunosorbent assay. The viability and apoptosis of H9c2 cells were probed by MTT assay and flow cytometry. Besides this, Western blot and quantitative real-time PCR were used to detect microRNA-142-3p (miR-142-3p) and hypoxia-inducible factor 1 subunit alpha inhibitor (HIF1AN) expression levels. The binding sequence between miR-142-3p and HIF1AN 3'-untranslated region was validated by a dual-luciferase reporter gene assay. PDG treatment significantly reduced the level of ROS, LDH, and CK-MB, promoted viability, and inhibited the apoptosis of H9c2 cells. PDG treatment promoted miR-142-3p expression and inhibited HIF1AN expression in H9c2 cells. MiR-142-3p overexpression enhanced the effects of PDG on ROS, LDH, CK-MB levels, cell viability, and apoptosis in H9c2 cardiomyocytes, while overexpression of HIF1AN reversed the above effects. PDG ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

Effects of rosin diglucoside on bone metabolism in young rats / 中国药理学通报

Aim To research the effect of PDG on bone metabolism in young rats. Methods The experimental rats were randomly divided into contro group, PDG-25 group and PDG-50 group. PDG-25 group and PDG-50 group were given PDG at the dose of 25 mg·kg

Pinoresinol diglucoside alleviates ischemia/reperfusion-induced brain injury by modulating neuroinflammation and oxidative stress.

Brain ischemia/reperfusion (I/R) injury is a common pathological process after ischemic stroke. Pinoresinol diglucoside (PDG) has antioxidation and anti-inflammation activities. However, whether PDG ameliorates brain I/R injury is still unclear. In this study, middle cerebral artery occlusion (MCAO) model was established with male C57BL/6 mice, and the mice were treated with 5 and 10 mg/kg PDG via intravenous injection, respectively. The neurological deficit, infarct volume, and brain water content were then evaluated. HE staining and Nissl staining were used to analyze neuron injury. Besides, enzyme-linked immunosorbent assay and colorimetry assay were used to examine the level of inflammatory markers and oxidative stress markers, and Western blot was used to detect the expressions of p-p65, Nrf2, and HO-1. It was revealed that PDG could significantly alleviate the MCAO-induced neurological dysfunction of the mice and reduce the infarct volume, brain water content, and neuron injury. PDG treatment decreased the levels of TNF-α, IL-1ß, IL-6, NO, ROS, and MDA, and significantly increased the activities of SOD, GSH, and GSH-Px in the brain tissue of the mice. Additionally, PDG could repress the activation of p65 and promote Nrf2 and HO-1 expressions. In conclusion, PDG exerts anti-inflammatory and antioxidation effects via regulating the NF-κB pathway and Nrf2/HO-1 pathway, thereby reducing the I/R-induced brain injury of mice.

Comparison of Pinoresinol and its Diglucoside on their ADME Properties and Vasorelaxant Effects on Phenylephrine-Induced Model.

Pinoresinol (PINL) and pinoresinol diglucoside (PDG), two natural lignans found in Eucommia ulmoides Oliv. (Duzhong), have several pharmacological activities. However, there is no report available on their absorption, distribution, metabolism, and elimination (ADME) properties. Given the possible wide spectrum of their application in therapeutic areas, this area should be investigated. This work studied the in vitro ADME properties of PDG and PINL, including their kinetic solubility, permeability across monolayer cells (PAMPA), protein binding, and metabolic stabilities in liver microsomes. The in vivo pharmacokinetic study and in vitro vasorelaxant effects on isolated phenylephrine-induced aortic rings of PINL and PDG were also investigated. It was found that both of their kinetic solubility in PBS (pH 7.4) was greater than 100 µM, indicating that they are both soluble compounds. The permeability investigations (P eff ) by PAMPA indicated that PINL had higher permeability than PDG (p < 0.05). Both components represented moderate plasma protein binding activities (average binding rate in human plasma: PINL 89.03%, PDG 45.21%) and low metabolic rate (t 1/2 in human liver microsome: PINL 1509.5 min, PDG 1004.8 min). Furthermore, the results of pharmacokinetic studies indicated that PINL might be eliminated less quickly than PDG from the rat plasma, and its cumulative urinary excretion was much lower than that of PDG. The phenylephrine-induced aortic rings demonstrated concentration-dependent vasorelaxation in PDG, PINL, or their combination group. The vasorelaxant effects of PINL were more obvious than those of PDG, whereas the vasorelaxant effect of the combinations was significantly better than that of the single component (p < 0.05). The similarity or difference between PINL and its diglucoside in these pharmaceutical aspects may offer valuable insights into the further exploration of lignans and might contribute to relevant studies involving natural products with similar molecular structure and their glucosides.

Pinoresinol diglucoside (PDG) attenuates cardiac hypertrophy via AKT/mTOR/NF-κB signaling in pressure overload-induced rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pinoresinol diglucoside (PDG), the active compound extracted from Eucommia ulmoides, Styrax sp. and Forsythia suspensa, plays the roles in regulating hypertension, inflammation and oxidative stress. AIMS: Considering that hypertension and inflammation has been proved to contribute to cardiac remodeling, we tested the effects of PDG on cardiac hypertrophy (CM). METHODS: Male Sprague Dawley (SD) rats were used to construct hypertrophic rats by partial abdominal aortic constriction (AAC)-surgery. PDG solution (2 mg/ml) was used to treat AAC-induced rats by intraperitoneal injection at low dose (L-PDG, 2.5 mg/kg per day), medium dose (M-PDG, 5 mg/kg per day), and high dose (H-PDG, 7.5 mg/kg per day) for 3 weeks post AAC-surgery. CM was evaluated by the ratio of left ventricular weight to body weight ratio (LVW/BW), left ventricular wall thickness by H&E staining, and collagen content deposit by Masson's staining. Further, isoproterenol (ISO) and phenylephrine (PE) were used to produce cellular models of CM in neonatal rat ventricular cardiomyocytes (NRVMs). PDG pre-treated NRVMs 2 h at low dose (L-PDG, 2.5 µg/ml), medium dose (M-PDG, 5 µg/ml), and high dose (H-PDG, 7.5 µg/ml) for 24 h with or without PE- and ISO-stimulation. CM was evaluated by the expressions of hypertrophic biomarkers. Next, the hypertrophic biomarkers and pro-inflammatory cytokines were measured using quantitative real-time PCR (qRT-PCR), the expressions of protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/transcription factor nuclear factor-kappa B (NF-kB) signaling pathway were determined by Western blotting. RESULTS: PDG treatment prevented cardiac histomorphology damages, decreased upregulations of hypertrophic biomarkers, and prevented fibrosis and inflammation after pressure overload resulting from AAC-surgery. Consistently, PDG remarkably inhibited the changes of cardiomyocyte hypertrophic biomarkers and inflammatory responses in cellular models of CM. Interestingly, PDG administration inhibited the activation of AKT/mTOR/NF-kB signaling pathway both in vivo and in vitro. CONCLUSIONS: PDG prevents AAC-induced CM in vivo, PE- and ISO-induced CM in vitro. The AKT/mTOR/NF-kB signaling pathway could be the potential therapeutic target involved in the protection of PDG. These findings provide novel evidence that PDG might be a promising therapeutic strategy for CM.

Aqueous extract from You-Gui-Yin ameliorates cognitive impairment of chronic renal failure mice through targeting hippocampal CaMKIIα/CREB/BDNF and EPO/EPOR pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: You-Gui-Yin (YGY) is a traditional Chinese recipe used for reinforcing kidney essence which is recorded in Jingyue Quanshu written by Zhang Jingyue in Ming dynasty. According to traditional Chinese medicine theory, kidney essence is associated with brain and without sufficient kidney essence, cognitive impairment may occur. AIM OF THE STUDY: In this study, we aimed to investigate the effect of YGY extract on cognitive impairment of chronic renal failure (CRF) mice and explore the mechanisms involved. MATERIALS AND METHODS: Aqueous extract of YGY was prepared from crude drugs and was quality controlled by high-performance liquid chromatography (HPLC). CRF was induced by 0.2% adenine in mice and CRF mice were intragastrically administered with 1.5 g kg-1, 3.0 g kg-1, and 6.0 g kg-1 of YGY extract. Mice were identified with CRF by determining several biochemical and physiological indexes, including creatinine clearance rate, serum creatinine, serum urea nitrogen, serum Ca, serum P, serum Mg, body weight and body temperature. Morris water maze and novel object recognition tests were conducted for evaluation of cognitive function. In addition, changes of CaMKIIα/CREB/BDNF and EPO/EPOR pathways in hippocampus were examined by detecting the protein expressions of CaMKIIα, p-CaMKIIα (Thr286), CREB1, p-CREB1 (Ser133), BDNF, EPO, EPOR, p-EPOR (Tyr485), STAT5, and AKT1 using western blotting assays. Also, the primary EPO-producing cells in brain (i.e. astrocytes) and EPO expression regulator HIF-2α were checked by fluorescence microscopy and western blotting assay, respectively. RESULTS: Nine components in YGY extract were figured out and monitored with their contents by HPLC for the quality control of YGY extract. Biochemical and physiological measurements validated the success of induction of CRF in mice, and YGY extract significantly retarded the CRF progression and ameliorated the CRF-induced cognitive impairment. The behavioral tests showed that compared with normal control mice, CRF mice had impaired cognitive function. However, treatment of YGY extract significantly ameliorated the cognitive impairment of CRF mice. Additionally, decreased expressions of hippocampal CaMKIIα, p-CaMKIIα (Thr286), CREB1, p-CREB1 (Ser133), and BDNF were observed in the hippocampus of CRF mice, but YGY extract significantly restored these protein expressions. Moreover, hippocampal EPO, EPOR, p-EPOR (Tyr485), STAT5, AKT1, and HIF-2α, as well as the number of astrocytes in CA1 zone of hippocampus were also decreased in CRF mice, while YGY extract prominently promoted the expressions of these proteins and increased the number of astrocytes. CONCLUSIONS: All the data in this study suggested that YGY extract ameliorated the cognitive impairment of CRF mice, and this amelioration was related to up-regulating the CaMKIIα/CREB/BDNF and EPO/EPOR pathways.

Quality Standards of Salt Eucommia Dispensing Granules / 医药导报

Objective To establish the quality standards for salt eucommia dispensing granules. Methods The extractives were obtained by alcohol extraction method. HPLC was applied for the determination of pinoresinol diglucoside in dispensing granules. HPLC fingerprints were established by the contrast of Agilent 1260 HPLC, Waters HPLC and various chromatogram column. Results Pinoresinol diglucoside showed a good linear relationship ( Y = 2. 9594X + 3. 2825,R2 =0.9999) at 102.8-2056.0 mg?L-1 with average recovery of 99.85％ (RSD = 0.31％,n = 9). Conclusion The method is easy-operated and accurate,which has a good specificity for the quality control of common salt eucommia dispensing granules.

Bioconversion of Pinoresinol Diglucoside from Glucose Using Resting and Freeze-Dried Phomopsis sp. XP-8 Cells.

Phomopsis sp. XP-8 (an endophytic fungus) was previously found to produce pinoresinol diglucoside (PDG), a major antihypertensive compound of Tu-Chung (the bark of Eucommia ulmoides Oliv.), which is widely used in Chinese traditional medicines. In the present study, two bioconversion systems were developed for the production of PDG in Tris-HCl buffer containing glucose and Phomopsis sp. XP-8 cells (both resting and freeze-dried). When other factors remained unchanged, the bioconversion time, glucose concentration, cell ages, cell dosage, pH, temperature, and stirring speed influenced PDG production in a similar and decreasing manner after an initial increase with increasing levels for each factor. Considering the simultaneous change of various factors, the optimal conditions for PDG production were established as 70 g/l cells (8-day-old), 14 g/l glucose, 28°C, pH 7.5, and 180 rpm for systems employing resting cells, and 3.87 g/l cells, 14.67 g/l glucose, 28°C, pH 7.5, and 180 rpm for systems employing freeze-dried cells. The systems employing freeze-dried cells showed lower peak PDG production (110.28 µg/l), but at a much shorter time (12.65 h) compared with resting cells (23.62 mg/l, 91.5 h). The specific PDG production levels were 1.92 and 24 µg per gram cells per gram glucose for freeze-dried cells and resting cells, respectively. Both systems indicated a new and potentially efficient way to produce PDG independent of microbial cell growth.

Regulative effect of pinoresinol diglucoside on gene of ESF-1 cells collagen secretion / 国际中医中药杂志

Objective To study the pinoresinol diglucoside (PDG) on gene regulation role of ESF-1 cells in collagen secretion, to reveal PDG repair mechanisms on scalded skin.Methods The cells cultured in vitro were divided into the control group, the estradiol group and the three different PDG doses groups. The concentration of the high, medium and low dose groups were 100, 10, 1μmol/L, and that of estradiol group were 10-3μmol/L. The activity of proliferation was detected by MTT. Then collagen type I (Col I), collagen typeⅢ (ColⅢ), tissue inhibitors of metalloproteinase 1 (TIMP-1), tissue inhibitors of metalloproteinase 2 (TIMP-2) and matrix metalloproteinase 1 (MMP-1) expression levels of mRNA after administration of cells were detected by RT-PCR.Results Compared with the control group, the proliferation of ESF-1 cells (0.559 ± 0.027, 0.552 ± 0.034vs. 0.489 ± 0.027,P<0.05) in the estradiol and medium-dose PDG was significantly higher. The expression level of mRNA of ColⅠ(0.958 ± 0.021, 0.929 ± 0.031, 0.916 ± 0.015vs. 0.844 ± 0.022), ColⅢ (0.783 ± 0.038, 0.918 ± 0.021, 0.855 ± 0.017vs. 0.678 ± 0.024), TIMP-1 (0.939 ± 0.025, 0.889 ± 0.036, 0.853 ±  0.015 vs. 0.780 ± 0.023), TIMP-2 (0.507 ± 0.024, 0.655 ± 0.037, 0.572 ± 0.025vs. 0.405 ± 0.062) in the estradiol, low-, medium-dose PDG groups were significantly higher than those in the control group (P<0.05 or P<0.01). Besides, the MMP-1 (0.343 ± 0.038, 0.407 ± 0.046, 0.435 ± 0.037vs.0.519 ± 0.041) mRNA expression level in the middle and low dose PDG groups significantly decrease (P<0.05 orP<0.01). Conclusions The PDG could enhance the activity of ESF-1 cell proliferation, increase the expression of related collagen and tissue inhibitor of metalloproteinases and inhibit that of matrix metalloproteinases to repair scalded skin.

Comparison of pinoresinol diglucoside production by Phomopsis sp. XP-8 in different media and the characterisation and product profiles of the cultivation in mung bean.

BACKGROUND: Phomopsis sp. XP-8 is an endophytic fungus with the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application in biosynthesis of PDG independent of plants. In order to enhance the production of PDG, 18 different natural materials were tested in solid-state cultivation of Phomopsis sp. XP-8. RESULTS: Most of the tested natural materials promoted the production of PDG. A supplement derived from mung beans produced the highest PDG yield and better fungal growth than the other materials. Also, pinoresinol monoglucoside, pinoresinol and other substrates (phenylalanine, p-coumaric acid, cinnamic acid, caffeic acid, and ferulic acid) were obtained after fermentation on mung beans. Furthermore, PDG production was much higher when mung beans were incorporated into solid state agar versus a liquid medium. The highest pinoresinol diglucoside production (72.1 mg kg(-1) in fresh culture) was obtained in 9 days using a solid state culture of Phomopsis sp. XP-8 on a mung bean grain medium containing 100 g kg(-1) glucose. Mung bean water-soluble polysaccharide was identified as a major promoter of PDG production by Phomopsis sp. XP-8. CONCLUSION: Mung bean, especially its water-soluble polysaccharide fraction, was an efficient natural material to promote PDG production by Phomopsis sp. XP-8. © 2015 Society of Chemical Industry.

Comparison on contents of four active constituents between slab and branch barks of Eucommia ulmoides / 中草药

Objective: To determine the contents of aucubin, chlorogenic acid, geniposide, and pinoresinol diglucoside in the slab and branch barks of Eucommia ulmoides. Methods: The separation was performed on a Cosmosil C18 (250 mm ×4.6 mm, 5 μm) column with the gradient elution acetonitrile -0.1% phosphoric acid; The flow rate was 0.8 mL/min; The detection wavelength was 208 nm; The column temperature was at 25 ℃. Results: The average content of aucubin: slab bark > branch bark; The average content of chlorogenic acid: branch bark > slab bark; The average content of geniposide: branch bark > slab bark; The average content of pinoresinol diglucosid: slab bark > branch bark. In different origins, the average contents of the above four constituents are more certain differences. Conclusion: The method is simple, rapid, accurate, and there are significant differences in the contents of aucubin, chlorogenic acid, geniposide, and pinoresinol diglucosid from the different parts of E. ulmoides, which would provide the better technique support for the quality control of E. ulmoides.

Determination of plasma protein binding rate of five components in Eucommia ulmoides extract / 中国药理学通报

Aim To determine the plasma protein binding rate of five components of Eucommia ulmoides extract. Methods The equilibrium dialysis method was used to study the plasma protein binding rate. The plasma samples were extracted by protein precipitation with methanol. With the use of puerarin as the internal standard, UPLC-MS/MS was carried out to determine the concentration of the five compounds in and out of the dialysis membrane. Results The average plasma protein binding rates of five compounds on the area of the concentration which was determinate were as fol-lows, respectively: geniposidic acid was ( 25. 77 ± 2. 68 )%, protocatechuic acid was ( 57. 54 ± 3. 79)%, chlorogenic acid was (53. 91 ± 3. 00)%, pinoresinol diglucoside was (24. 15 ± 4. 92)%, and pinoresinol singleglucoside was (49. 78 ± 3. 61)%. Conclusions The results show that the binding percentage of geniposidic acid and pinoresinol diglucoside is relatively low, but the binding rate of the others with rat plasma protein is moderate.

Study on HPLC Fingerprints of Eucommia Ulmoides / 中国药师

To establish the HPLC fingerprints of Eucommia ulmoides at different ages of different varieties with pi-noresinol diglucoside as the reference material to provide the basis for the HPLC fingerprints in the enterprise quality standards of Eu-commia ulmoides from the GAP base of Chenzhou Dacheng Chinese Herbal Medicine Co. Ltd. . Methods: A column of ZORBAX E-clipse XDB-C18 (250 mm × 4. 6 mm, 5μm) was used. The mobile phase consisted of methanol-0. 1% phosphoric acid with gradient e-lusion. The detection wavelength was 277 nm, the column temperature was 25℃,the flow rate was 1. 0 ml·min-1 , and the injection volume was 5 μl. Results:The Eucommia ulmoides HPLC fingerprints including 14 common peaks were established with pinoresinol diglucoside as the reference material. The similarity of the HPLC fingerprints of 10 samples was over 0. 939. Conclusion:The method is accurate and reliable. The HPLC fingerprints can be used as the enterprise quality standards of Eucommia ulmoides for Chenzhou Dacheng Chinese Herbal Medicine Co. Ltd. .

Simultaneous Determination of Pinoresinol Diglucoside and Chlorogenic Acid in Traditional Decoction and Its Solid Decoction of Sam Soup by HPLC / 中国中医药信息杂志

Objection To estabish a HPLC methold for determining the contents of pinoresinol diglucoside and chlorogenic acid in Sam Soup and its solid decoction. Methods Chromatographic column was Ultimate XB-C18 reversed-phase chromatography column (250 mm×4.6 mm, 5 μm). Mobile phase was acetonitrile-0.4% glacial acetic acid with gradient elutim. The flow rate was 1.0 mL/min, detection wavelength was 277 nm, and column temperature was 40 ℃. Results The pinoresinol diglucoside and showed good linear relationship in the range of 5.0-50.0 μg, r =0.999 9 (n =6), and chlorogenic acid showed linear relationship in the range of 5.4-54.0 μg, r=0.999 6 (n=6). The average recoveries of pinoresinol diglucoside and chlorogenic acid were 98.75% (RSD=1.39%) and 101.11% (RSD=2.69%), respectively. Conclusion The established method was accurate and reliable. The contents of pinoresinol diglucoside and chlorogenic acid in solid decoction are higher than traditional decoction of Sam Soup.

Simultaneous determination of five index components in Eucommiae Cortex by HPCE / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis (HPCE) method for simultaneous determination of aucubin, geniposide, pinoresinol diglucoside, harpagide, and chlorogenic acid in Eucommiae Cortex. METHODS: 60 mmol·L-1 sodium borate-20 mmol·L-1 sodium dihydrogen phosphate-10% methanol was used as buffer solution (pH 10.0), un-coated fused silica capillary (64.5 cm×75 μm id, 56 cm of effective length) was used, separation voltage was 20 kV, detection wavelength was 210 nm, column temperature was maintained at 25°C, and sample was injected at 5 kPa×6 s. RESULTS: Five index components showed good linearity (r>0.9973) in the range of the tested concentration. The average recoveries of the method were between 96.63%-103.73%. CONCLUSION: The method is simple, accurate and reproducible, and can be used for quality control of Eucommiae Cortex. Copyright 2012 by the Chinese Pharmaceutical Association.

Study on the Quality of Eucommia Ulmodies Processing in Planting by Different Methods / 中国中医药信息杂志

Objective To study on the quality of Eucommia ulmodies processing in planting by different methods.Methods The quantity percent of coarsed cortex,water and ethanol extract were compred,the contents of Pinoresinol diglucoside(PDG)were determined by HPLC,the residues of pesticides and the content of heavy metal element were carried out to investigate the quality.Results The quantity percent of coarsed cortex was 12.7%(n=3).Ethanol extract had no remarkable difference whether scrape the coarsed cortex or not.Water extract was higher 11.26%(P