Protective effects and mechanism of icariin against vascular function in diabetic mice / 中国药科大学学报

@#To explore the effects and molecular mechanism of icariin on the vascular function of mice with type 1 diabetes induced by alloxan, type 1 diabetic mice model was established by intraperitoneal injection with 200 mg/kg alloxan.After oral administration with icariin (60, 120 mg/kg) daily for 2 weeks, blood glucose, body weight, food intake and water intake were detected.To evaluate the impact of icariin on the function of isolated vascular ring contraction and relaxation, thoracic aortas of mice were removed and the Ach-induced vascular ring relaxation, Phe-induced vascular ring contraction, SNP-induced vascular ring relaxation and KCl-induced vascular ring contraction response were detected.To further confirm the mechanism of icariin to improve vascular function, human umbilical vein endothelial cells (HUVECs) were induced by high glucose (HG) in vitro.Western blot was used to detect the effect of icariin on eNOS, p-eNOS, p38 MAPK and p-p38 MAPK expressions in HG-induced human umbilical vein endothelial cells (HUVECs).The results indicated that icariin significantly ameliorated the weight loss and dampened the increase in water intake of the diabetic mice.Meanwhile, icariin had a certain ameliorative effect on blood glucose and food intake without significant difference.The results of isolated thoracic aortas vascular rings contraction and vasodilation function indicated that icariin significantly improved Phe-induced vascular contraction and Ach‐induced vascular relaxation.Meanwhile, icariin had a certain ameliorative effect on KCl-induced vascular contraction response without significant difference.However, no significant change was observed on endothelium‐independent vascular rings relaxation response induced by SNP after treatment with icariin.Results of Western blot showed that icariin inhibited the expression of p-p38 MAPK and induced expression of p-eNOS in the high glucose-induced HUVECs cell model.Therefore, icariin may attenuate alloxan-induced type 1 diabetic mice vascular diastolic function by inhibiting expression of p-p38 MAPK and inducing expression of p-eNOS.

Mechanism of Icariin in Ameliorating Neuronal and Dendritic Damage in Alzheimer's Disease by Regulating RhoA/ROCK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of icariin on the recombinant Ras homolog family member A （RhoA）/Rho-associated coiled-coil forming protein kinase （ROCK） signaling pathway in rats with Alzheimer's disease （AD）， and to explore the mechanism of icariin in ameliorating the neuronal and dendritic damage. MethodThe β-amyloid 1-42 （Aβ1-42， 2.5 g·L-1） was used to induce AD in rats via lateral ventricle injection， and the rats were divided into a model group， a low-dose icariin group （0.03 g·kg-1）， a middle-dose icariin group （0.06 g·kg-1）， a high-dose icariin group （0.09 g·kg-1）， and a control group. The control group and the model group were given an equal volume of normal saline at a dose of 10 mL·kg-1. The cognitive function of rats was assessed by the Morris water maze. The pathological morphology of the rat hippocampal CA1 area was observed by Nissl staining. Dendritic spine density and dendritic length in the CA1 region of the hippocampus were observed by Golgi-Cox staining. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tumor necrosis factor-α （TNF-α）， interleukin （IL）-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus. Western blot assay was used to detect the protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus. ResultAs compared with the control group， the escape latency of the rats in the model group was increased （P<0.01）， while the number of crossing the platform and the dwelling time in the target quadrant were decreased （P<0.01）. As compared with the model group， the escape latency of the rats in the middle and high-dose icariin groups was decreased （P<0.05， P<0.01）， while the number of crossing the platform and the dwelling time in the target quadrant were increased （P<0.05， P<0.01）. As compared with the control group， the number of neurons， dendritic spine density， and dendritic length in the hippocampal CA1 area of the rats in the model group were decreased （P<0.01）. As compared with the model group， the number of neurons， dendritic spine density， and dendritic length in the hippocampus of the rats in the middle and high-dose icariin groups were increased （P<0.05， P<0.01）. As compared with the control group， the mRNA and protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus of the rats in the model group were increased （P<0.01）. As compared with the model group， the mRNA and protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus of the rats in the middle and high-dose icariin groups were decreased （P<0.05， P<0.01）. ConclusionIcariin improves cognitive function and neuronal and dendritic damage in AD by inhibiting the RhoA/ROCK signaling pathway.

Icariin Inhibits Proliferation of CLC5 Hepatoma Cells via Akt/GSK3β/CDK Pathway / 中国实验方剂学杂志

ObjectiveTo study the effect of icariin on the proliferative capacity of hepatocellular carcinoma cell line CLC5 and the underlying mechanism. MethodThe targets of icariin were screened out by network pharmacology， and the target network and protein-protein interaction （PPI） network were constructed to predict the possible targets and pathways of icariin. CCK-8 assay was employed to explore the effects of different concentrations （0， 6.25， 12.5， 25， 50 μmol·L-1） of icariin on the viability of CLC5 cells. Further， CLC5 cells were treated with 0， 25， 50 μmol·L-1 icariin， and the effect of icariin on CLC5 cell proliferation was examined by Edu-488 assay and clone formation assay （CFA）. Western blot was employed to measure the expression levels of proteins in the protein kinase B （Akt）/glycogen synthase kinase 3β （GSK3β）/cell cycle-dependent kinase （CDK） pathway in the CLC5 cells exposed to different concentrations of icariin. ResultNetwork pharmacological analysis revealed that icariin may inhibit the hepatocellular carcinoma via cell cycle arrest and inhibition of tumor cell proliferation. Compared with the blank group， icariin decreased the viability of CLC5 cells in a time- and concentration-dependent manner （P<0.01） and reduced the positive rate of Edu-488 and the colonies in CFA （P<0.05， P<0.01）. Moreover， icariin down-regulated the protein levels of p-Akt， p-GSK3β， CDK4， and CyclinD1 （P<0.05， P<0.01）. ConclusionIcariin may block cell cycle to suppress the proliferation of CLC5 cells via inhibiting the Akt/GSK3β/CDK pathway.

Icariin improves renal interstitial fibrosis in a rat model of chronic renal failure by regulating mitochondrial dynamics / 中国中药杂志

This study aims to explore the effect of icariin(ICA) on mitochondrial dynamics in a rat model of chronic renal failure(CRF) and to investigate the molecular mechanism of ICA against renal interstitial fibrosis(RIF). CRF was induced in male Sprague-Dawley(SD) rats with 5/6(ablation and infarction, A/I) surgery(right kidney ablation and 2/3 infarction of the left kidney). Four weeks after surgery, the model rats were randomized into the following groups: 5/6(A/I) group, 5/6(A/I)+low-dose ICA group, and 5/6(A/I)+high-dose ICA group. Another 12 rats that received sham operation were randomly classified into 2 groups: sham group and sham+ICAH group. Eight weeks after treatment, the expression of collagen-Ⅰ(Col-Ⅰ), collagen-Ⅲ(Col-Ⅲ), mitochondrial dynamics-related proteins(p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2), and mitochondrial function-related proteins(TFAM, ATP6) in the remnant kidney tissues was detected by Western blot. The expression of α-smooth muscle actin(α-SMA) was examined by immunohistochemical(IHC) staining. The NRK-52 E cells, a rat proximal renal tubular epithelial cell line, were cultured in vitro and treated with ICA of different concentration. Cell viability was detected by CCK-8 assay. In NRK-52 E cells stimulated with 20 ng·mL~(-1) TGF-β1 for 24 h, the effect of ICA on fibronectin(Fn), connective tissue growth factor(CTGF), p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 was detected by Western blot, and the ATP content and the mitochondrial morphology were determined. The 20 ng·mL~(-1) TGF-β1-stimulated NRK-52 E cells were treated with or without 5 μmol·L~(-1) ICA+10 μmol·L~(-1) mitochondrial fusion promoter M1(MFP-M1) for 24 h and the expression of fibrosis markers Fn and CTGF was detected by Western blot. Western blot result showed that the levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were increased and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were decreased in 5/6(A/I) group compared with those in the sham group. The levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were significantly lower and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were significantly higher in ICA groups than that in 5/6(A/I) group. IHC staining demonstrated that for the expression of α-SMA in the renal interstitium was higher in the 5/6(A/I) group than in the sham group and that the expression in the ICA groups was significantly lower than that in the 5/6(A/I) group. Furthermore, the improvement in the fibrosis, mitochondrial dynamics, and mitochondrial function were particularly prominent in rats receiving the high dose of ICA. The in vitro experiment revealed that ICA dose-dependently inhibited the increase of Fn, CTGF, and p-Drp1 S616, increased p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6, elevated ATP content, and improved mitochondrial morphology of NRK-52 E cells stimulated by TGF-β1. ICA combined with MFP-M1 further down-regulated the expression of Fn and CTGF in NRK-52 E cells stimulated by TGF-β1 compared with ICA alone. In conclusion, ICA attenuated RIF of CRF by improving mitochondrial dynamics.

Simultaneous Determination of the Contents of 6 Components in Fuzheng Guben Granules by QAMS / 中国药房

OBJECTIVE：To establish the method for content determination of 6 components in Fuzheng guben granules ，such as 2，3，5，4′-tetrahydroxystilbene glucoside ，baicalin，icariin，scutellarin，baicalein and wogonin. METHODS ：HPLC method was adopted. The determination was performed on Dikma Diamonsil C 18 column with mobile phase consisted of acetonitrile- 0.1％ phosphoric acid aqueous solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 275 nm （0-8 min），320 nm（8-9 min）and 275 nm（9-33 min）. The column temperature was set at 25 ℃，and sample size was 10 μL. With baicalin as reference material ，the relative corr ection factors （fk/s） of other five components were calculated by multi-point correction method and slope correction method ；the retention time difference method was used to locate the chromatographic peaks ； the calculation values obtained by above 2 QAMS were compared with measured values of external standard method. RESULTS ： The linear range of 2，3，5，4′-tetrahydroxystilbene glucoside ，baicalin，icariin，scutellarin，baicalein and wogonin were 0.053-2.12， 0.163-6.52，0.059-2.36，0.021 6-0.864，0.03-1.2，0.021-0.84 μg（r＞0.999），respectively. RSDs of precision ，stability（12 h）and reproducibility tests were all lower than 3％. Average recoveries were 98.72％-99.82％（RSDs were 0.89％-1.24％，n＝9）. Using baicalin as reference material ，fk/s of multi-point correction method for 2，3，5，4′-tetrahydroxystilbene glucoside ，icariin，scutellarin， baicalein and wogonin were 1.172，0.528，1.479，1.820 and 2.534，respectively；fk/s of slope correction method were 1.234， 0.550，1.559，1.939，2.664. RSDs of 6 components in 10 batches of Fuzheng guben granules by 3 methods were 0.29％-2.77％ （n＝10），respectively. Pearson correlation coefficient was not lower than 0.999 9（P＜0.001）in measured values between QAMS and external standard method. CONCLUSIONS ：QAMS method is established successfully for simultaneous determination of 6 components in Fuzheng guben granules.

Molecular mechanism of icariin effects on articular chondrocytes, subchondral bone and synovium in the treatment of osteoarthritis / 中国组织工程研究

BACKGROUND: Icariin is the main effective component of Epimedium, which functions to tonify the kidney, and strengthen tendons and bones. In recent years, a large number of studies have found that icariin plays a significant role in the treatment of osteoarthritis. OBJECTIVE: To review the research progress in the molecular mechanism of icariin in the treatment of osteoarthritis. METHODS: The first author used “Icariin, Osteoarthritis, Cartilage, Subchondral bone, Synovial membrane, synovium, Inflammation" as search words in English and Chinese to search PubMed, CNKI, WanFang, and VIP databases. According to the inclusion criteria and exclusion criteria, 42 articles were included for final analysis. RESULTS AND CONCLUSION: Icariin can promote the cartilage differentiation of bone marrow mesenchymal stem cells and enhance the proliferation of cartilage cells and osteoblasts, to inhibit the degradation of cartilage extracellular matrix, reduce the activity of osteoclasts and alleviate synovial inflammation caused by inflammatory factors. It is an effective treatment for osteoporosis. However, the optimal effective dose and concentration safety of icariin still need a large number of experimental studies. Currently, most of the experiments are still in animal and tissue cell experiments. Numerous clinical studies are needed to continue to explore its specific mechanism in order to provide evidence-based medical evidence for icariin in the treatment of osteoarthritis.

Effects and mechanism of epimedium polysaccharide on solubility of icariin and baohuoside Ⅰ / 中国中药杂志

This study evaluated the effects of epimedium polysaccharide(EPS) on the solubility of icariin and baohuoside Ⅰ so as to preliminary explore its solubilization function and the underlying mechanism. The solubility of these two insoluble flavonoids in water and polysaccharide solutions was compared by high performance liquid chromatography, and the mechanism was investigated by diffe-rential scanning calorimetry(DSC) and critical micelle concentration determination. The results indicated that their solubilization in crude EPS solutions was concentration-dependent. The solubility of icariin and baohuoside Ⅰ in 20 mg·mL~(-1) EPS-1-1 was 9.05 times and 5.76 times that in water, respectively; while their solubility in 20 mg·mL~(-1) EPS-2-1 was 10.55 and 8.39 times that in water, respectively. The change of the DSC thermograms suggested the formation of new complexes from icariin and baohuoside Ⅰ with polysaccharides. The critical micelle concentrations proved the micellar properties of both EPS-1-1 and EPS-2-1. In short, EPS can significantly increase the solubility of icariin and baohuoside Ⅰ, the mechanism of which may be related to the formation of micellar complexes between EPS and insoluble flavonoids.

Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells / 中国中药杂志

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.

Study on Improvement Effects of Icariin on Cognitive Function in Schizophrenia Model Rats and Its Mechanism / 中国药房

OBJECTIVE：To study the improvement effects of ica riin（ICA）on cognitive function in schizophrenia model rats and its mechanism. METHODS ：SD rats were divided into blank control group ，model group ，ICA low-dose ，medium-dose and high-dose groups （15，30，60 mg/kg）. Except for blank control group ，other groups were given N-methyl-D-aspartate receptor antagonist MK- 801（0.2 mg/kg）intraperitoneally to induce schizophrenia rats models ，once a day ，for consecutive 14 days. After modeling，ICA groups were intragastrically administered with the corresponding drugs ，while blank control group and model group were intragastrically administered with the same volume of water ，once a day ，for consecutive 7 days. The behavioral com changes of rats were detected by Morris water maze test ，open field test ， forced swimming test and Y maze test pathological changes of hippocampus were observed by Nissl staining；the levels of cholinergic indexes [acetylcholine （Ach），choline acetyltransferase （ChAT） and acetylcholinesterase （AchE）] in cerebral tissues were detected by ELISA. The expression of BDNF ，ERK and CREB mRNA in cerebral tissue were detected by RT-PCR ；expression or phosphorylation level of BDNF ，ERK，CREB protein ，apoptosis related proteins （Bcl-2，Bax and Caspase- 3）were detected by Western blot. RESULTS ：Compared with blank control group ，escape latency ，distance at T 1-T3， cumulative immobility time and the expression of Caspase- 3 protein in cerebral tissues were significantly increased in model group （P＜0.05）；the times of crossing platform ，alternation rate ，the number of Nissl staining positive neurons in hippocampus tissues ， the levels of Ach and ChAT in cerebral tissues ，Bcl-2/Bax ratio ，mRNA and protein expression of BDNF ，mRNA expression of ERK and CREB ，the phosphorylation of ERK 1/2 and CREB were significantly decreased （P＜0.05）.Compared with model group ， escape latency ，distance at T 1-T3，cumulative immobility time ，the number of Nissl staining positive neurons ，AchE level in cerebral tissues and relative expression of Caspase- 3 protein were significantly decreased in ICA high-dose group （P＜0.05）；the times of crossing platform ，alternation rate ，levels of Ach and ChAT in cerebral tissues ，Bcl-2/Bax ratio ，mRNA and protein expression of BDNF ，mRNA expression of ERK and CREB ，the phosphorylation of ERK 1/2 and CREB were increased significantly（P＜0.05）. Above indexes in ICA low-dose and medium-dose groups were partially improved significantly than model group（P＜0.05）. CONCLUSIONS ：ICA can improve cognitive function in schizophrenia model rats.Its mechanism may be related to regulating cholinergic system ，inhibiting neuronal apoptosis ，and promoting the expression of BDNF/ERK/CREB signaling pathway.

Protection of icariin on impairment of rat bone mesenchymal stem cells induced by 5-fluorouracil in vitro / 中国药理学通报

Aim To study the protective effect of icariin on the impairment of rat bone mesenchymal stem cells (BMSCs) induced by 5-fluorouracil (5-FU) and the underlying mechanism. Methods The proliferation of BMSCs was evaluated by MTT assay, the morphological change of apoptotic cells was tested by using the chromatin dye Hoechst 33258, and the apoptotic rate was analyzed by flow cytometry. The proteins expression of cleaved caspase-3 was detected with Western blotting, the activity of caspase-3 was detected by Activity Assay Kit; iNOS and TNOS were detected by nitric oxide synthase (NOS) assay kit. Results MTT detection showed that 5-FU could reduce the survival rate of BMSCs, 10, 20, 40 μmol · L-1ICA could promote the proliferation of BMSCs, and blocked the inhibition of BMSCs cells growth induced by 5-FU. When BMSCs cells were treated by both ICA (10, 20 p, mol · L-1) and 5-FU (2.5×l0-2g· L-1) for 48 hours, compared with model group, the early apoptotic rates of ICA test groups were significantly reduced (P < 0. 01), the expression of cleaved caspase-3 and the activity of caspase-3 were significantly reduced (P < 0. 01), and the activity of iNOS and TNOS was inhibited in a certain degree (P < 0. 05). Conclusions ICA could protect BMSCs against 5-FU-induced damage. The protective role of ICA is probably related to reducing the activity of iNOS and TNOS, inhibiting the activation of caspase-3, and inhibiting the occurrence of apoptosis.

Icariin promotes differentiation of MC3T3-E1 pre-osteoblasts through Hedgehog signaling pathway / 中国药理学通报

Aim To investigate the effect of icariin (ICA) on the proliferation and differentiation of MC3T3-E1 pre-osteoblast cell line. Methods CCK-8 assay was used to detect the proliferation activity of MC3T3-E1 cells. Alizarin red staining (AR-S) assay was used to detect osteoblast differentiation and the formation of calcium nodules. Western blot assay was used to examine the protein expression of osteocalcin, ALP and shh. ALP assay was used to detect its activity. Results The effect of CCK-8 assay showed that ICA (5 ∼ 40 μmol · L-1) significantly promoted MC3T3-E1 cell proliferation compared with control group. The activity and protein expression of ALP and the quantitative analysis of mineralization deposition significantly increased in ICA group compared with control group. The ALP activity, protein expression level of shh and the quantification of alizarin red staining showed a significant decrease in ICA + CYC group compared with ICA group. Conclusions ICA promotes osteoblast proliferation and differentiation through the Hedgehog signaling pathway.

Effect of icaritin on ferroptosis of bone marrow mesenchymal stem cells and their differentiation into cardiomyocytes / 中国组织工程研究

BACKGROUND: Iron accumulation or iron overload is closely related to the proliferation and apoptosis of bone marrow mesenchymal stem cells, which may play an important role in the pathological process of cardiovascular diseases. OBJECTIVE: To investigate the regulatory effect of icariin on ferroptosis of bone marrow mesenchymal stem cells and its differentiation into cardiomyocytes. METHODS: Taking bone marrow mesenchymal stem cells as the research object, the blank group was cultured for 14 days. The model group was treated with 0.4 μmol/L Erastin for 14 days to establish ferroptosis model. The treatment group was treated with 80 nmol/L icariin and 0.4 μmol/L Erastin for 14 days. CCK-8 method was used to detect the proliferation of cells in each group, and the levels of malondialdehyde and superoxide dismutase were detected by related kits. Western blot assay was used to detect the expression of TFR1, FPN1, Bcl-2 and Bax protein. RT-PCR was used to detect the mRNA expression of GATA4 and cTnT. RESULTS AND CONCLUSION: (1) Compared with the model group, the cell proliferation ability of the treatment group was significantly improved (P < 0.05). (2) Compared with the model group, the level of malondialdehyde in the lysate of bone marrow mesenchymal stem cells in the treatment group was significantly lower (P < 0.05), while the level of superoxide dismutase was significantly increased (P < 0.05). (3) Compared with the model group, TFR1 protein expression was decreased (P < 0.05), FPN1 protein expression was increased (P < 0.05), and Bcl-2/Bax protein expression ratio was increased significantly (P < 0.05) in the treatment group. (4) Compared with the model group, the expression of GATA4 and cTnT mRNA was significantly increased in the treatment group (P < 0.05). (5) The results showed that icariin could inhibit the ferroptosis of bone marrow mesenchymal stem cells and improve the ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells.

Molecular mechanism of icariin in the treatment of bone nonunion / 中国组织工程研究

BACKGROUND: Nonunion is a common clinical complication in orthopedics, which seriously impacts the physical and mental health and quality of life of patients. In recent years, a large number of studies have found that icariin plays a significant role in promoting fracture healing and treating bone defects. Bone nonunion and fracture healing coexist, and the research on the mechanism of fracture healing actually focuses on the treatment of bone nonunion. OBJECTIVE: To review the research progress in the molecular mechanism of icariin in the treatment of bone nonunion. METHODS: The first author used “icariin, bone nonunion, bone marrow mesenchymal stem cells, periosteal cell, osteoblasts, osteoclast” as key words in English and Chinese to search PubMed, CNKI, WanFang and VIP databases. A total of 542 articles were retrieved and screened manually according to the selection criteria and exclusion criteria. Finally, 44 articles were included for result analysis. RESULTS AND CONCLUSION: Icariin can effectively promote fracture healing and treat bone nonunion by promoting the proliferation and differentiation of bone marrow mesenchymal stem cells and periosteal cells, promoting the proliferation and maturation of osteoblasts and inhibiting the osteoclast effect of osteoclasts. However, most of the experiments are still in the basic experimental research, and there is still a need for a large number of clinical studies as well as studies on related proteins and genes, to provide a new idea for the clinical use of Chinese herbs in the treatment of bone nonunion.

Osteogenesis of bone marrow mesenchymal stem cells on hydroxyapatite/icariin/poly(Lactic-co-glycolic acid) scaffolds / 中国组织工程研究

BACKGROUND: Bone tissue engineering has provided a novel ideal for treating bone defects in clinic. This study is the first to combine traditional Chinese medicine with the nanostructures of tissue-engineered scaffolds in order to explore and construct a new bone tissue substitute material for the treatment of bone defects. OBJECTIVE: To investigate the osteogenic activity of icariin (ICA)/hydroxyapatite (HA)/poly(lactic-co-glycolic acid) (PLGA) composite scaffolds. METHODS: A HA/PLGA composite scaffold was prepared by physical blending of HA and PLGA, and was then soaked in ICA solution of different concentrations to obtain the HA/ICA/PLGA scaffold. Rabbit bone marrow mesenchymal stem cells were used to evaluate the cell adhesion, proliferation, osteogenesis and cytotoxicity of the composite scaffold. The cell adhesion, proliferation and cytotoxicity were detected by MTT method. The activities of alkaline phosphatase and osteocalcin were detected by ELISA. The expression levels of osteogenic genes and proteins were detected by fluorescence quantitative PCR and western blot assay, respectively. RESULTS AND CONCLUSION: Adding appropriate amount of HA into PLGA could improve the mechanical strength of the scaffold, and 10% HA had the best effect with tensile strength of (1.67±0.37) MPa, and compression modulus of (4.17±1.62) MPa, and nanostructure would be formed on the surface of the scaffold. The nanostructure could promote the adhesion of bone marrow mesenchymal stem cells on the surface of the scaffold. ICA did not affect the proliferation of bone marrow mesenchymal stem cells on the composite scaffold. However, the HA/PLGA composite scaffold soaked in 1.00 µmol/L ICA aqueous solution had the optimal osteogenic differentiation function, and the expression levels of alkaline phosphatase, osteocalcin, osteogenic related genes and proteins (Runx-2 and COL I) were increased. The ICA/HA/PLGA scaffold had no cytotoxicity. These results suggest that HA (10%)/ICA (1.00 µmol/L)/PLGA scaffold has good mechanical properties, osteogenesis and biocompatibility, which has the potential to be a favorable scaffold for bone tissue engineering.

Quality evaluation of different parts of multiple botanical origins of Epimedii Folium / 中草药

Objective: To compare the flavonoids contained in different parts of different botanical origins of Epimedii Folium, and provide a basis for the quality evaluation of Epimedii Folium and the reasonable selection of medicinal parts. Methods: The aerial parts of 13 batches of Epimedii Folium were collected and divided into three parts: leaf, petiole and stem. The HPLC fingerprint and content of five flavonoids, including epimedin A, epimedin B, epimedin C, icariin and baohuoside I, were analyzed. Then the analysis of variance and the similarity evaluation software of traditional Chinese medicine chromatographic fingerprint were used. Combined with cluster analysis (HCA), the content differences of flavonoids in leaf, petiole and stem of Epimedii Folium were evaluated. Results: The fingerprints showed that the chemical constituents in Epimedii Folium leaves were richer than stems and petioles, and the chemical constituents in petioles and stems were basically the same. The content of five components in leaves was significantly higher than that of petiole and stem, even up to 10 times. Cluster analysis also showed that the leaves were obviously distinct from the petiole and stem. Conclusion: The quality differences of Epimedii Folium leaves, petioles and stems were clarified, and this study can provide the scientific evidence for the selection of medicinal parts and quality control of Epimedii Folium.

Analysis changes of Epimedii Folium's flavonoids before and after processing based on UPLC-Q/TOF-MS / 中草药

Objective: Based on UPLC-Q/TOF-MS technology, the fingerprint of Epimediumkoreanum before and after processing was established to analyze the whole composition and find out the iconic chemical composition to clarify the change rule of flavonoids. Methods: The data of E.koreanum raw and processed products were collected in positive ion mode using UPLC-Q/TOF-MS technology, and orthogonal partial least least squares-discriminant analysis (OPLS-DA) method was used to explore the differences in chemical composition of E.koreanum before and after processing in nine different origins and batches. Results: Nine iconic chemical constituents from E.koreanum raw and processed products were found and identified, including 8-ethylene-kaempferol, icaritin, icariin I, icartin-3-O-glucoside, isoamyl alcohol sagittatoside B,1,3-prenyl epimedin C, 1,3-prenyl-sagittatoside B-7-O-glucuronic acid, 3-O-((4-acetoxy) rhamnose-2-O-(m-2acetoxy) glucose) icariin and its isomers. Conclusion: The structures of E.koreanum's flavonoids changed after the processing, the secondary glycosides of flavonoids increased, the polyglycosides decreased, and the flavonoids were generally converted to low glycoside components, which further clarified the change rule of E.koreanum's flavonoids after processing.

Purification process of epimedin and icariin from Epimedii Folium by macroporous resin / 中草药

Objective: To investigate the best technological conditions for the purification of epimedin and icariin from Epimedii Folium by macroporous resin, and preliminarily characterize the purification fraction of the best technological conditions. Methods: Five kinds of macroporous resins were screened by static adsorption experiment with the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin as indexes. The best purification conditions were optimized by the concentration of upper column solution, the maximum sample volume, the upper column flow rate, the volume of water washing, the concentration of removing impurity ethanol and elution ethanol, the volume of removing impurity ethanol and elution ethanol, the column diameter-height ratio through dynamic adsorption experiment. Finally, UPLC-Q-TOF/MS, HPLC and ultraviolet spectrophotometry were used to characterize the purification fraction of the best technological conditions. Results: The best macroporous resin was AB-8, column diameter-height ratio was 1:7, 6 BV of upper column solution (crude drug 0.5 g/mL) was used for dynamic adsorption at a flow rate of 6 BV/h, 5 BV of water and 5 BV of 20% ethanol were used for impurity removal, and 6 BV of 50% ethanol was used for elution. The flow rate of impurity removal and elution was 6 BV/h. After purification, the total flavonoids content was 63.29%, the total content of epimedin A1, A, B, C and icariin was 40.48%, the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin was 1.63%, 2.52%, 16.36%, 5.51% and 14.46%, respectively. Conclusion: The purification process of epimedin and icariin from Epimedii Folium by AB-8 macroporous resin is stable, reasonable and feasible. The chemical characterization indicated that the purification fraction was mainly flavonoids, mainly consisting of epimedin and icariin. The optimized purification process can be used for the purification and enrichment of such ingredients.

HPLC characteristic chromatograms and quantitative analysis of Kunxian Capsules / 中草药

Objective: In order to provide a scientific basis for the quality control of Kunxian Capsules (KC), HPLC characteristics chromatogram combined with multi-components determination was established. Methods: The analysis was performed on Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm), using acetonitrile and 0.1% phosphoric acid solution as the mobile phase at a flow rate of 0.8 mL/min for gradient elution, the column temperature was 33 ℃, and the detection wavelength was 270 nm. The fingerprints of 15 batches of KC were established and evaluated by the similarity evaluation system of TCM (2012A version), hierarchical cluster analysis and discriminant analysis of partial least squares. Furthermore, the content of hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ were determined. Results: The HPLC fingerprint with 21 common peaks of KC was established, and the similarities of samples were over 0.9. The linearity relationships separated with hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ were good, and the contents of the above-mentioned components in 15 batches of preparations were 2.817-7.527, 7.287-9.103, 8.730-18.675, 33.377-70.371, 35.297-50.291 and 4.059-9.079 mg/g, respectively. Conclusion: The combination methods of HPLC characteristic chromatograms and simultaneous determinations of multiple components are rapid, simple and reproducible, which can provide methodological reference for the quality control of KC.

Comparative study on icariin and Bu-Shen-Gu-Chi-Wan for alveolar reconstruction in chronic periodontitis in rats / 口腔疾病防治

Objective@#To observe the effects of icariin (ICA) and Bu-Shen-Gu-Chi-Wan on the alveolar bone absorption of chronic periodontitis in rats, and to explore the effect and possible mechanism of ICA in the treatment of chronic periodontitis. @*Methods@#After the establishment of the periodontitis model, the rats were divided into the periodontitis group (group P), ICA high dose group (group H), ICA low dose group (group L) and Bu-Shen-Gu-Chi-Wan treatment group (group B). Each group received treatment for one month and two months, separately, and the serum osteocalcin (OCN) level was measured. Three-dimensional reconstruction was performed after micro computed tomography (micro CT) scanning to measure the bone parameters of specific points, and the distance between the enamel cementum boundary and alveolar crest (CEJ-ABC) was recorded as alveolar bone resorption value. Tissue sections were generated to evaluate the effect of ICA on alveolar bone repair and reconstruction in rats with experimental periodontitis. @*Results@# Compared with the periodontitis group (group P), OCN levels in the serum in treatment groups (groups H, L and B) were decreased significantly (P < 0.05); the values of bone volume/tissue volume (BV/TV) and trabecular number (Tb.N) in treatment groups (groups H, L and B) were significantly higher than that in group P (P < 0.05). Compared with group P, trabecular thickness (Tb.Th) values of groups H and B significantly increased, and trabecular separation (Tb.Sp) of groups H and B significantly decreased (P < 0.05). The changing trend of parameters in group L was the same as that in group H but only after two months of administration. The difference between Tb.Sp values in groups L and P was statistically significant (P < 0.05). Compared with group P, the CEJ-ABC distance significantly reduced in group L (1 month and 2 months after administration), group H (1 month and 2 months after administration), and group B (only 2 months after administration) (P < 0.05).@*Conclusion@#ICA and Bu-Shen-Gu-Chi-Wan improve the alveolar bone resorption in an experimental model of chronic periodontitis in rats, and the mechanism may be related to the regulation of osteocalcin serum levels.

Effects of light quality on growth and icariin flavonoid content of Epimedium pseudowushanense under different light intensity / 中国中药杂志

In this study, the growth index including plant height, compound leaf area, specific leaf area, leaf water content, number of branches, and leaf biomass per plant and the icariin flavonoids such as epimedin A, epimedin B, epimedin C and icariin of Epimedium pseudowushanense were determined on 30 d and 60 d under light intensity(18.2±2.5) μmol·m~(-2)·s~(-1)(L1) and(90.9 ±2.5) μmol·m~(-2)·s~(-1)(L2), and white light as control, red light, blue light and yellow light were used as three light quality treatments, to study the effect of light quality on the growth and flavonoids accumulation of E. pseudowushanense. The E. pseudowushanense was sui-table for growth under L1 light intensity, the blue light treatment significantly reduced the leaf area, but had little effect on the stem height, the red light treatment and the yellow light treatment had no obvious effect on the stem height and leaf area, but the yellow light treatment significantly increased the germination of new branches, and had a sustained promoting effect, and the biomass was significantly higher than the white light treatment at 60 d. The content of icariin flavonoids in red light, blue light and yellow light treatment was higher than that in white light treatment at 30 d and 60 d under L1 light intensity, while yellow light treatment promoted the synthesis of icariin flavonoids to the largest extent, which was 1.8 and 1.9 times of white light treatment(30 d and 60 d).Under L2 light intensity, the effect of strong light on promoting stem germination became the main factor, while the yellow light treatment showed no significant effect on promoting stem germination, and the red light treatment exhibited a significant effect on reducing leaf area. Icariin flavonoids under red light, blue light and yellow light treatment were all lower than that under white light treatment, that is, the effect of white light treatment on the synthesis of icariin flavonoids is better than red light, blue light and yellow light treatment. When the time of strong light treatment was longer, the degradation range of icariin flavonoids in other light treatment appeared, while red light treatment promotes the synthesis of icariin flavonoids. Therefore, the influence of light quality on E. pseudowushanense is quite different under different light intensity, no matter from growth index or flavonoid content index. The results support that the biomass and icariin flavonoid content can be increased by providing appropriate red and yellow light.