Pharmacological Effects and Molecular Mechanism of Osthole

Osthole has various pharmacological effects such as anti-cancer,anti-inflammation,prevention and treatment of cardiovascular diseases and neuroprotection.This paper reviews the advances in the research of the pharmacological effects and molecular mechanisms of osthole,in order to provide new ideas for further research and clinical application of osthole.

Osthol和Icarin对甲减小鼠血清甲状腺激素的影响

目的：观察蛇床子素（Ｏｓｔｈｏｌ）和淫羊藿甙（Ｉｃａｒｉｎ）对甲减“肾阳虚”小鼠血清甲状腺激素的影响。方法：用丙基硫氧嘧啶（ＰＴＵ）制备甲减“肾阳虚”小鼠模型，用放射免疫法测定小鼠血清Ｔ３、ｒＴ３和Ｔ４的浓度。结果：模型鼠血清Ｔ３、ｒＴ３和Ｔ４浓度皆降低（Ｐ＜０．０１），死亡率升高（Ｐ＜０．０１），造模过程中同时分别给药Ｏｓｔｈｏｌ和Ｉｃａｒｉｎ能有效地阻止Ｔ３、ｒＴ３和Ｔ４浓度下降（Ｐ＜０．０１或Ｐ＜０．０５），死亡率也显著降低（Ｐ＜０．０１）。同时模型鼠分别经Ｏｓｔｈｏｌ和Ｉｃａｒｉｎ治疗后，Ｔ３、ｒＴ３和Ｔ４浓度得到明显提高（Ｐ＜０．０１或Ｐ＜０．０５）。结论：Ｏｓｔｈｏｌ和Ｉｃａｒｉｎ既能拮抗ＰＴＵ的甲状腺抑制作用，预防“肾阳虚”证的出现，又能促进ＰＴＵ所致甲减“肾阳虚”小鼠体内甲状腺激素水平的提高，具有补肾壮阳作用。

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri（L.） Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.

Osthol attenuates hepatic steatosis via decreased triglyceride synthesis not by insulin resistance

AIM: To evaluate the effects of osthol on intrahepatic fat synthesis, β-oxidation, inflammation, and insulin resistance by multifaceted analysis.METHODS: Sprague-Dawley rats(n = 30) were randomly divided into control, non-alcoholic fatty liver disease(NAFLD), and osthol groups. NAFLD and osthol groups were fed with a high-fat diet for 14 wk. After 8 wk of the high-fat diet, the osthol group also received osthol 20 mg/kg orally 5 times/wk. To assess the insulin resistance, oral glucose tolerance was performed at the end of 14 wk. Immunohistochemical(4-HNE, F4/80) and hematoxylin and eosin(HE) staining wereperformed on liver tissue extracts after animal sacrifice at 14 wk. SREBP1 c, FAS, SCD-1, PPAR-α, CROT, MCP-1, IRS-1, and IRS-2 mRNA expressions were assessed with reverse transcription-polymerase chain reaction.RESULTS: HE staining revealed that, compared with the NAFLD group, the osthol group showed significantly decreased intrahepatic fat content(39.4% vs 21.0%; P = 0.021). SREBP1 c expression in the NAFLD group increased compared to controls(P = 0.0001), while osthol treatment decreased SREBP1 c expression compared with the NAFLD group(P = 0.0059). In the osthol group, intrahepatic FAS and SCD-1, which act downstream of SREBP1 c, decreased significantly compared with the NAFLD group. Moreover, PPAR-α expression in the osthol group was also significantly higher than in the NAFLD group(P = 0.0147).CONCLUSION: Osthol treatment attenuated liver steatosis by decreasing de novo liver triglyceride synthesis and had nominal effects on insulin resistance and liver inflammation.

Osthole improves synaptic plasticity in the hippocampus and cognitive function of Alzheimer's disease rats via regulating glutamate

Osthole, an effective monomer in Chinese medicinal herbs, can cross the blood-brain barrier and protect against brain injury, with few toxic effects. In this study, a rat model of Alzheimer＇s disease was established after intracerebroventricular injection of β-amyloid peptide （25-35）. Subsequently the rats were intraperitoneally treated with osthole （12.5 or 25.0 mg/kg） for 14 successive days. Results showed that osthole treatment significantly improved cognitive impairment and protected hippocampal neurons of AIzheimer＇s disease rats. Also, osthole treatment alleviated suppressed long-term potentiation in the hippocampus of Alzheimer＇s disease rats. In these osthole-treated Alzheimer＇s disease rats, the level of glutamate decreased, but there was no significant change in y-amino-butyric acid. These experimental findings suggest that osthole can improve learning and memory impairment, and increase synaptic plasticity in Alzheimer＇s disease rats. These effects of osthole may be because of its regulation of central glutamate and y-amino-butyric acid levels.

Effect of osthole,aFructus Cnidii-derivednature coumarin,on osteogenesis and bone healing in mice

OBJECTIVE To investigate the effect of osthole,a natural coumarin isolated from traditional Chinese medicine Fructus Cnidii,on osteogenesis in vitro and bone fracture healing in vivo.METHODS Primary bone marrow mesenchymal stem cells(MSCs)were isolated from 6-week C57/B6 mice,and osteogenic differentiation was assessed by alkaline phosphate(ALP)activity and calcium nodule formation.Adult(12-week)C57mice were subjected to mid-shaft osteotomy on femur.The mice were oral administrated with osthole(5,20 or 50mg·kg-1)or vehicle solvent daily from post-operational week 1.Radiographic imaging,real time molecular imaging,micro computed tomography(μCT)and histology analysis were performed to evaluate the healing progress.RESULTS Results showed that osthole promoted osteogenesis of bone marrow MSCs by enhancing ALP activity and mineralization dose dependently in the range of 1-100μmol·L-1.Plain radiographs showed that administration of osthole at 20 and 50 mg·kg-1 significantly accelerated fracture healing by reducing the period of reparative phase.Further investigation withμCT and histology showed that osthole-treated group had high proportion of newly-formed woven bone and smaller cartilage island compare to control group at week 2;and treatment group had completed endochondral ossification and started remodeling phase at week 3.Molecular imaging of near-infrared(NIR)fluorescent labeled palmidronate depositing on newly formed bone suggested that osthole treatment(20 mg·kg-1)augmented callus mineralization process at both postoperative week 2 and week 3 by 80.72% and 25.95% respectively.CONCLUSION Osthole demonstrates significant osteopromotive effect in vitro and anabolic effect on bone formation in fracture repair,which makes it a potential agent for bone regeneration and against osteoporosis.

Osthole prevents cognitive impairment through modulating neuron cells in Aβ25-35-injected mice

OBJECTIVE To investigate the effects of osthole on learning and memory impairment of AD mice induced by injection of Aβ25-35 and the content of Ca2+, GLU and Aβ1-42 in the brain tissue and peripheral blood. METHODS Mice were randomly assigned to sham operation, Aβ25-35, Aβ25-35+Ost-L,Aβ25-35+Ost-M, and Aβ25-35+Ost-H group. Water maze test was performed to assessing spatial learning ability of mice. It is determined that the MDA level and the activity of SOD in the brain tissue of mice in each group by colorimetry. The GLU kit and Ca2 +kit were used to detect the GLU, Ca2 +in tissue and serum. ELISA was used to detect the expression of Aβ1-42 in the hippocampus and serum of mice. HE staining and silver staining were used to detect neuron apoptosis and pathological changes in brain slices and so on. RESULTS(1) Effects of osthole on learning and memory: With the increase of training day,the escape latencies continuously reduced in each experimental group, the escape latencies of the model group was longer on the 1 st, 2 nd, 3 rd, and 5 thdays than the normal group, the difference was statistically significant(day 3 and 4: P<0.05, day 5: P<0.01);compared with the model group, the escaping latency on the 5 thday of the OST low-medium high-dose group was significantly shortened, which was statistically significant(P<0.05).(2) Effects on oxidative stresspathway: the SOD activity of AD mice in the hippocampus model group was lower than that in the normal group, which was statistically significant(P<0.05);The SOD activity in the OST group was higher than that in the model group, which was statistically significant(P<0.05). The MDA content in the model group was significantly higher than that in the normal group(P<0.05). The MDA content in the OST high-dose group was lower than that in the model group, which was statistically significant(P<0.05).(3) Effects of GLU levels on neurotransmitters:the results of the detection of GLU in cortical area and GLU in serum of AD mice in OST dose groups showed that serum GLU levels in the model group were significantly lower than those in the sham group, which was statistically significant(P<0.05). GLU levels in the cortical area were also significantly higher than those in the sham group, which was statistically significant(P<0.05). Compared with the model group, GLU levels in the OST administration group were significantly down-regulated. Among the serum, the effect of medium dose group was obvious. Although there was a trend of down-regulation in the cortical administration group, there was no statistical significance.(4) Changes in Ca2+concentration in the brain. Detection of intracellular Ca2 +concentration in AD mice by OST doses showed that,compared with the sham group, the model group was significantly upregulated in cortical Ca2 +levels.There was no statistical difference in the administration group. Compared with the model group, the concentration of Ca2+in the OST-H group significantly decreased.(5) Effect on levels of Aβ1-42 in hippocampus and serum: model group had significantly higher Aβ1-42 levels in hippocampus than in sham operation group, which was statistically significant(P<0.05). Aβ1-42 in serum was also significantly upregulated compared to the sham group, which was statistically significant(P<0.05). Compared with the model group, the levels of Aβ1-42 in the OST administration group were significantly down-regulated,with the lower and middle doses in the hippocampus being more significant, while the serum was more pronounced at lower doses.(6) Silver staining to detect the tangles of hippocampal neurons: Neuron tangles in the hippocampal CA1 region showed a dark brown-yellow granule distribution in the nuclei of the model group(positive expression). Nerve cell body and dendrites, axons are black or black red,background light yellow. Compared with the model group, the administration group has improved significantly. CONCLUSION OST improves spatial learning and memory of dementia model mice injected with Aβ25-35 in both hippocampus. Experimental studies have shown that OST has different degrees of regulation on neuronal apoptosis, Ca2 +/GLU/oxidative stress and other pathways, and it plays a role in improving multiple AD pathological changes and delaying the pathogenesis of neurodegenerative diseases.

NP-3 Effects of Osthole Microemulsion by Nasal Administration on the Cholinergic Pathway in AD Mice

Objectives:Fructus Cnidii is the dry ripe fruit of Cnidium monnieri(L.)Cuss.,which belongs to the umbelliferous plant.It has long been used in clinic practice and has been found to have pharmacological activity in the central nervous system.Osthole is the main active component of Fructus Cnidii.However,it shows low bioavailability,fast distribution and elimination,and low concentration in the brain when given orally.In this study,we aimed to develop a new dosage form to increase the osthole concentration in the brain and enhance its pharmacological effects in the central nervous system through reducing the dosage while improving the stability and bioavailability.Thus,microemulsion containing osthole was prepared and the effects of osthole microemulsion were examined in the mouse model of Alzheimer’s disease(AD).Methods:On the basis of pseudo-ternary phase diagram,microemulsion was prepared by using polyoxyethlated Cremophor RH40 as emulsifiers,propylene glycol as assistant emulsifiers and ethyl acetate as the oil phase.The particle size and distribution of osthole microemulsion were detected by laser particle size analyzer and transmission election microscope.The content of osthole was determined by UV spectrophotometry.The effects of osthole microemulsion by nasal administration on the learning and memory abilities in scopolamine-treated mice were assessed by Morris water maze and novel object recognition tests.The superoxide dismutase(SOD)activity,glutathione(GSH)levels and malondialdehyde(MDA)contents in the serum were examined to evaluate the oxidant stress.Choline acetyltransferase(ChAT)and acetylcholinesterase(AChE)expression in the olfactory-basal forebrain pathway were detected by immunohistochemical analysis.We also investigated the acetylcholine(ACh)levels and the histological morphology in the brain.Results:The average particle size of 1μg·μL-1 osthole microemulsion was less than 15 nm.It was characterized as spheres under the transmission electron microscopy,and the osthole was completely encapsulated in the microemulsion core.Morris water maze and novel object recognition tests showed that osthole microemulsion improved spatial and object learning and memory in scopolamine-treated mice.Moreover,osthole microemulsion restored the abnormal activity of SOD and increased the levels of MDA and GSH in the serum.Brain immunohistochemistry staining showed that osthole microemulsion up-regulated ChAT expression,while down-regulated AChE in the olfactory-basal forebrain cholinergic pathway.Additionally,the ACh levels and pathological morphology in the brain were also reversed after nasal administration with osthole microemulsion.Conclusion:The 1μg·μL-1 osthole microemulsion is an ideal dosage form with a small particle size,uniform distribution and high permeation.Osthole microemulsion ameliorated memory impairment in scopolamine-teated mice,likely via the olfactory-basal forebrain cholinergic pathway and by reducing oxidative stress.The results implicate the development of intranasal brain targeting drugs as potential treatment of certain central nervous system diseases,including disorders affecting memory such as Alzheimer’s disease.

Fluorescence spectroscopy of osthole binding to human serum albumin

The interaction of human serum albumin （HSA） with osthole was investigated by fluorescence spectroscopy. Osthole can quench the fluorescence of HSA and the quenching mechanism is a static process. The binding site number n and apparent binding constant K were measured at different temperatures. The thermodynamic parameters △H^0, △G^0 and △S^0 were calculated at different temperatures. The results indicated that electrostatic forces played a major role in the interaction of osthole with HSA. Results of osthole synchronous fluorescence and UV absorption spectra showed that the microenvironment and conformation of HSA were changed.

Compatibility Screening of Plant Extracts Synergistic with Osthole

Plant pests have been a major problem in agricultural production. To establish sustainable methods for plant pest control, 20 of plant material extracts were selected which were synergistic with Osthole to improve the performance of the osthole existing in products. The preliminary screening results indicated that Zanthoxylum bungeanum Maxim.(Z. bungeanum Maxim) compounds exerted the highest synergism. A single dose(2 000 mg · L^(-1)) of Z. bungeanum Maxim. crude extracts was used against Plutella xyllostella(P. xyllostella), Lipaphis erysimi(L. erysimi), and Sitophilus zeamais(S. zeamais). For a further examination form Z. bungeanum Maxim., in which crude extracts were mixed with osthole at different ratios. The volume ratio of 7 : 3(Z. bungeanum Maxim. extract: osthole) exhibited a more pronounced synergistic effect, and all the values of the co-toxicity coefficients were above 120. Eight distinct compounds were derived from the ethanol crude extract from Z. bungeanum Maxim.Then, the toxicity of these components to pests, such as P. xyllostella, L. erysimi, S. zeamais, Rhizopertha dominica(R. dominica),and Tribolium castaneum Herbst(T. castaneum Herbst), was determined. The results indicated that Compound 4 and Compound 7 exerted lethal effects on pest investigation. Compound 4 had the most substantial insecticidal action, at a concentration of64 ug · mL^(-1), and a death ratio of 78.3% was achieved for P. xyllostella.

Osthole attenuates pulmonary arterial hypertension by modulation of phospholipid metabolism

OBJECTIVE Pulmonary arterial hypertension(PAH)is a malignant pulmonary vascular disease lacking efficacy therapeutics.Therefore,it urgently needs to develop safe and effective drugs for PAH treatment.Osthole derived from Cnidium monnieri(L.)Cusson(Shechuangzi)or Angelica pubescens Maxim(Duhuo)has the capacity to alleviate PAH by decreasing pulmonary arterial pressure and alleviating pulmonary vascular remodeling in rats,which is a candidate drug for the prevention of PAH,but the underlying modulatory mechanism is still unclear.Our study aims at investigating the metabolic modulatory mechanism of osthole against PAH employing functional metabolomics strategy.METHODS PAH model rats were successfully established with MCT,following osthole administration,then functional metabolomics based on untargeted metabolomics assay,targeted lipidomics analysis,qRT-PCR,Western blotting and ELISA were performed to investigate the modulatory mechanism of osthole against pulmonary arterial pressure and pulmonary vascular remodeling in PAH.RESULTS Untargeted metabolomics results found that sphingosine 1-phosphate(S1P)was the differential metabolites characterized PAH and reversed by osthole treatment.S1P is a crucial sphingolipid metabolite catalyzed by sphingosine kinases1(Sphk1)and functions as promoting PASMCs proliferation contributing to pulmonary vascular remodeling and pulmonary arterial pressure increase.We revealed that osthole reversed high level of S1P by modulating metabolic enzyme Sphk1 via inactivating microRNA-21-PI3K/Akt/mTOR signal pathway to decrease pulmonary arterial pressure in rats with PAH.Then,targeted phospholipid metabolomics results uncovered that decadienyl-L-carnitine(C10:2)was the differential metabolite characterized PAH and corrected by osthole treatment in rat with PAH.C10:2 is the intermediate metabolite of fatty acid oxidation(FAO),and C10:2 accumulation indicated mitochondrial dysfunction and FAO increase.CONCLUSION Osthole could block lipid metabolic reprogramming through functional modulating the expression of fatty acid translocase,fatty acid synthase,phospholipase A2,carnitine palmitoyltransferase 1A to inhibit C10:2,thus to improve mitochondrial dysfunction and inhibit utilizing lipid to biosynthesize necessary essence for pulmonary artery smooth muscle cells(PASMCs)proliferation.Moreover,we delineated that C10:2 and metabolic reprogramming enzymes were modulated by miRNA-22-3p which was involved in PASMCs proliferation and pulmonary vascular remodeling.Therefore,osthole inhibited miRNA-22-3p mediated lipid metabolic reprogramming to ameliorate pulmonary vascular remodeling.

Research progress on anticancer effects and molecular mechanisms of osthole

Cancer is a serious threat to human life and a big problem in clinical treatment.Some natural active substances extracted from Traditional Chinese Medicine can effectively inhibit the growth of cancer cells,which is a new direction for finding more effective anticancer drugs.Osthole is a natural coumarin compound extracted from Traditional Chinese Medicines such as Cnidium monnieri,Angelica pubescens and Peucedanum praeruptorum Dunn.It has significant inhibitory activity against a variety of cancers.This paper summarizes the anticancer effects and molecular mechanisms of osthole in the treatment of cancers in recent years in order to provide references for further research.

S1-5 Effects of Osthole on Learning and Memory and the Estrogen Pathway in Ovariectomized Rats

Objective:To investigate the effects of osthole,a natural coumarin first derived from the Cnidium plant,on learning and memory,physiological and pathological changes,and expression of estrogen receptor(ER)αandβin the brain of ovariectomized(OVX)rats of Alzheimer’s disease(AD)models.Methods:Female rats were randomly divided into six groups:①sham operation,and OVX plus:②saline,③Estradiol(0.1 mg·kg-1;positive control),④osthole at 12.5 mg·kg-1,⑤osthole at 25 mg·kg-1,and⑥osthole at 50 mg·kg-1;intragastric administration for 30 days.The Morris water-maze test was used to evaluate the learning and memory ability of rats,ELISA to measure the levels of estradiol in the serum,Western blotting to detect the expression of ERαand ERβin the hippocampus,and HE staining to determine the histopathological changes in the brain.Results:①Effects on learning and memory:compared to the OVX alone,osthole at 25 or 50 mg·kg-1 signifi cantly increased the number of entries and the duration in the target quadrant in the water-maze probe trial test(P<0.05).②Effects on the estrogen pathway in the brain:the level of estradiol in the serum and expression of ERβin the hippocampus in the OVX alone were signifi cantly lower,while the expression of ERαwas higher,relative to the sham operation control(P<0.01);osthole at 25 mg·kg-1 reversed the OVX-induced changes in expression of ERαand ERβ(P<0.01).③Effects on histopathological change in the brain:in comparison with the sham operation group,the OVX rats treated with saline displayed increases in the number of apoptotic cells in the hippocampus,which was reversed by osthole at 25 or 50 mg·kg-1(P<0.05),but not the lower dose of 12.5 mg·kg-1.Conclusion:Osthole produced enhancement of learning and memory in the ovariectomized dementia model,which was mediated,at least in part,by regulating neuronal apoptosis and the estrogen pathway.Therefore,osthole is potent in delaying the development of female neurodegenerative diseases,which provides a potential,new approach to treatment of female AD.

NP-5 Alleviating Effects of Osthol on Scopolamine-Induced Cholinergic Impairments in Mice

Objective:To investigate the effect of osthol on the central cholinergic nerve circuit in AD model mouses,,which were established by intraperitoneal injection of scopolamine.Methods:60 health female rats were randomly divided into six groups.The sham operation group and model group were given intragastric administration of normal saline;the positive control group was given Aricept(3 mg·kg-1)intragastric gavage;The high,middle and low doses of the osthole groups were treated by intragastric administration of 50 mg·kg-1,25 mg·kg-1,and 12.5 mg·kg-1 osthole,respectively.30 minutes before the water maze test,except for the sham operation group who was given intraperitoneal injection of normal saline,other groups were intraperitoneally injected with scopolamine hydrobromide(3 mg·kg-1)until the experiment was completed.The ability of spatial learening and memory in mice was evaluted by behavioral experiments and changes and changes in the central cholinergic function of mice were examined The ability of spatial learning and memory in mice was evaluated by behavioral experiments and changes of function of the central cholinergic cricuis of mice were detected by molecular biology and pathology.Results:1.Effects on cholinergic nerve pathways:Osthole can reduce escape latency and search distance in dementia mice,while the osthol can increase the level of ACH in the central cholinergic circuits of dementia mice.2 effects on oxidative stress pathway:The activity of SOD in the model was obvious lower than that in the normal group,while the SOD activity of each dose of osthole was higher than that in of the model group.The content of MDA in the model group was obvious higher than that in the normal group,while each dose of osthole was lower than that of the model group.The activity of GSH-Px in the model group was significantly lower than that in the normal group,while the activity of GSH-Px in the osthol group was higher than that in the model group.Conclusion:The scopolamine-induced mouse model of denentia can cause cognitivedysfunction of mice and reduce the content of acetylcholine and acetylcholinesterase(AchE)in the central cholinergic circuit in mice of denentia,Osthole has effect on improving cognitivein dysfunction,increasing content of ACH and improving the activity of acetylcholinesterase(AchE)in the central cholinergic nerve circuit in dementia mice.

Hypothesis of the osthol intervention in osteoarthritis treatment through COX-2 specific pathway

Osteoarthritis(OA)is a joint degenerative disease characterized by softening and peeling of articular cartilage,reactive hyperplasia of subchondral bone,and narrowing of joint space,which seriously affects the quality of daily life of patients.Drug therapy is one of the effective means to treat osteoarthritis,it mainly includes the use of nonsteroidal antiinflammatory drugs,hormones and painkillers.These methods have a certain effect on alleviating symptoms,but there are also some obvious adverse reactions,and are not easy to take for a long time.Osthol is a natural coumarin isolated from Pinaceae plants.It has pharmacological effects such as anti-inflammatory,antioxidant and neuroprotective.A large number of studies and experiments show that Osthol has a good effect in the treatment of osteoarthritis,and no obvious harmful effect has been found.Therefore,by analyzing and sorting out the relevant experiments and literatures published in recent years,this paper puts forward some scientific hypotheses on the possible specific ways of regulating COX-2 mRNA expression,one of the important roles of Osthol in the treatment of osteoarthritis,in order to clarify the possible potential mechanism and inspire subsequent related research.

NP-14 Effects of Osthole on the Improvement of Learning and Memory Impairment in A Mouse Model Injected with Aβ25-35

Objective:We aimed to investigate the effects of osthole on learning and memory impairment of AD mice induced by injection of Aβ25-35 and the content of Ca2+、GLU、Ab1-42 in the brain tissue and peripheral blood.Methods:Mice were randomly assigned to sham operation,Aβ25-35,Aβ25-35+Ost-L,Aβ25-35+Ost-M,and Aβ25-35+Ost-H group.Water maze test was performed to assessing spatial learning ability of mice.It is determined that the MDA level and the activity of SOD in the brain tissue of mice in each group by colorimetry.The GLU kit and Ca2+kit were used to detect the GLU,Ca2+in tissue and serum.Elisa was used to detect the expression of Aβ1-42 in the hippocampus and serum of mice.HE staining and silver staining were used to detect neuron apoptosis and pathological changes in brain slices.Results:①Effects of osthole on learning and memory:With the increase of training day,the escape latencies continuously reduced in each experimental group,the escape latencies of the model group was longer on the 1st,2nd,3rd,and 5th days than the normal group,the difference was statistically significant(day 3,4:P<0.05,day 5:P<0.01);compared with the model group,the escaping latency on the fifth day of the OST low-medium high-dose group was significantly shortened,which was statistically significant(P<0.05).②Effects on oxidative stresspathway:the SOD activity of AD mice in the hippocampus model group was lower than that in the normal group,which was statistically significant(P<0.05);The SOD activity in the OST group was higher than that in the model group,which was statistically significant(P<0.05).The MDA content in the model group was significantly higher than that in the normal group(P<0.05).The MDA content in the OST high-dose group was lower than that in the model group,which was statistically significant(P<0.05).③Effects of GLU levels on neurotransmitters:the results of the detection of GLU in cortical area and GLU in serum of AD mice in OST dose groups showed that serum GLU levels in the model group were significantly lower than those in the sham group,which was statistically significant(P<0.05).GLU levels in the cortical area were also significantly higher than those in the sham group,which was statistically significant(P<0.05).Compared with the model group,GLU levels in the OST administration group were significantly downregulated.Among the serum,the effect of medium dose group was obvious.Although there was a trend of down-regulation in the cortical administration group,there was no statistical significance.④Changes in Ca2+concentration in the brain;Detection of intracellular Ca ion concentration in AD mice by OST doses showed that,compared with the sham group,the model group was significantly upregulated in cortical Ca2+levels.There was no statistical difference in the administration group.Compared with the model group,the concentration of Ca2+in the OST-H group significantly decreased.⑤Effect on levels of Ab1-42 in hippocampus and serum:model group had significantly higher Ab1-42 levels in hippocampus than in sham operation group,which was statistically significant(P<0.05).Ab1-42 in serum was also significantly upregulated compared to the sham group,which was statistically significant(P<0.05).Compared with the model group,the levels of Aβ1-42 in the OST administration group were significantly down-regulated,with the lower and middle doses in the hippocampus being more significant,while the serum was more pronounced at lower doses.⑥Silver staining to detect the tangles of hippocampal neurons:Neuron tangles in the hippocampal CA1 region showed a dark brown-yellow granule distribution in the nuclei of the model group(positive expression).Nerve cell body and dendrites,axons are black or black red,background light yellow.Compared with the model group,the administration group has improved significantly.Conclusion:OST improves spatial learning and memory of dementia model mice injected with Ab25-35 in both hippocampus.Experimental studies have shown that OST has different degrees of regulation on neuronal apoptosis,Ca2+/GLU/oxidative stress and other pathways,and it plays a role in improving multiple AD pathological changes and delaying the pathogenesis of neurodegenerative diseases.

Experimental study of osthole on treatment of hyperlipidemic and alcoholic fatty liver in animals

瞄准：在脂肝上评估 osthole 的效果，并且调查可能的机制。方法：有 hyperlipidemic 脂肝的一个鹑模型和有含酒精的脂肝的老鼠模型被分别地喂多脂饮食和白酒建立。这些实验动物然后分别地为 6 wk 与 osthole 5-20 mg/kg 被对待。然后，在浆液的类脂化合物和肝的织物，和肝的重量的系数被测量。结果：胆固醇(TC ) ，甘油三酸酯(TG ) ，更低的密度脂蛋白胆固醇(LDL-C ) ，肝的重量的系数，和肝的织物 TC 和 TG 满足的有浆液的层次总计的 osthole 的术后疗法显著地被减少。在肝的超级氧化物歧化酶(草皮) 的活动被改进。在导致白酒的脂肝老鼠，在肝的 malondialdehyde (MDA ) 的水平被减少。在高导致脂肪的脂肝鹑，在肝的谷胱甘肽过氧化物酶(GSH-PX ) 显著地被改进。肝标本的组织学的评估证明 osthole 戏剧性地减少了类脂化合物累积。结论：这些结果建议 osthole 在酒精和高导致脂肪的脂肝上有治疗学的效果。机制可能与它的反氧化被联系。

In Vitro Skin Permeation of Osthol from Hydro-Alcohofic Gel Formulations

Aim To evaluate the in vitro percutaneous absorption behavior of osthol from a series of hydro-alcoholic gel formulations containing three penetration enhancers through excised human skin (stratum cormeum and epidermis,SCE). Methods Excised human skin was mounted in Franz-type diffusion cells. The samples withdrawn from the receptor cell were analyzed for osthol content by high-performance liquid chromatography (HPLC). Results The enhancers azone, menthol and chenopodium increased the osthol percutaneous steady-state fluxes 3.12, 2.00 and 1.25 times those of the enhancer-free formulations (controls), separately. Conclusions The main enhancement mechanism of the skin penetration enhancers azone, menthol and chenopodium is to destroy the barrier fimction of stratum corneum, reducing the resistance of drug transport through the skin and increasing the diffusion coefficients of osthol.

HPLC法同时测定驻春胶囊中4个成分的含量

目的:建立以高效液相色谱法(HPLC)同时测定驻春胶囊中异补骨脂素、补骨脂素、淫羊藿苷、蛇床子素含量的方法。方法:采用HPLC法,以异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素作为指标成分,采用Agilent ZORBAX SB-C_(18)色谱柱(4.6 mm×250 mm,5μm);流动相为甲醇-水,梯度洗脱,体积流量为1.0 m·min^(-1);柱温为30℃,检测波长为254 nm,进样量为5μL。结果:异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素质量浓度分别在0.0059~0.1770 mg/mL、0.0049~0.1470 mg/mL、0.0031~0.093 mg/mL和0.0048~0.1440 mg/mL范围内与峰面积呈良好的线性关系,其相关系数分别为0.9996、0.9995、0.9997和0.9995,平均加样回收率分别为101.45%、100.95%、100.46%和101.17%;RSD分别为2.19%、1.83%、1.71%和2.01%。结论:该分析方法适用于驻春胶囊中异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素的含量测定。