Establishment of Specific Chromatogram and Thin-layer Identification Method for Kaixinsan / 中国实验方剂学杂志

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） identification method of Kaixinsan（KXS） samples， in order to clarify the key quality attributes and provide reference for the quality evaluation of KXS. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of KXS was developed with YMC Hydrosphere C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-15 min， 2%-20%A； 15-25 min， 20%-25%A； 25-30 min， 25%-30%A； 30-45 min， 30%-31%A； 45-50 min， 31%-44%A； 50-65 min， 44%-45%A； 65-73 min， 45%-75%A； 73-95 min， 75%-100%A； 95-105 min， 100%A； 105-105.1 min， 100%-2%A； 105.1-120 min， 2%A）， the detection wavelength was 320 nm. Ultra high performance liquid chromatography-linear ion trap-electrostatic field orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） was used to identify the chemical components of KXS with electrospray ionization（ESI）， negative ion mode and scanning range of m/z 50-2 000. TLC identification methods for Poria and Ginseng Radix et Rhizoma in KXS were established. ResultThere were 11 common peaks in the specific chromatogram of KXS， attributed to Polygalae Radix， Poria and Acori Tatarinowii Rhizoma. Taking peak 9（α-asarone） as the reference peak， the relative standard deviations of the retention times of 15 batches of KXS samples were<0.2%. A total of 34 compounds were identified by UHPLC-LTQ-Orbitrap MS， including terpenoids， phenylpropanoids， oligosaccharides and ketones. The established TLC had good separation and was rapid， reliable， simple， feasible， suitable for the identification of Poria and Ginseng Radix et Rhizoma in KXS. ConclusionThe specific chromatogram and TLC of KXS are stable and reproducible. The material basis of KXS is basically clarified by MS， which can provide a reference for the development and quality control of KXS.

Effect of Kaixinsan on Keap-1/Nrf2/MnSOD Signaling Pathway in Rats with Alzheimer's Disease Induced by Aβ1-42 / 中国实验方剂学杂志

Objective:To explore the possible mechanism of Kaixinsan in improving cognitive impairment in Alzheimer's disease （AD） model rats based on the epichlorohydrin associated protein-1 （Keap-1）/nuclear factor E2 related factor （Nrf2）/manganese superoxide dismutase （MnSOD） signaling pathway. Method:The AD model was established by injecting Amyloid <italic>β</italic>_1-42 （A<italic>β</italic>_1-42， 5 μL） into the lateral ventricle. After modeling， the experimental rats were randomly divided into model group， donepezil group， and Kaixinsan low dose， medium dose and high dose groups. Another normal control group was also established. The donepezil group received donepezil tablets （1.8 g·kg^-1·d^-1）， Kaixinsan low dose， medium dose and high dose groups received corresponding doses of Kaixinsan （10， 20， 40 g·kg^-1·d^-1， respectively）， and the normal control group and model group were given with equal volume of pure water. Morris water maze was used to test the learning and memory ability of rats. The pathological morphology of hippocampal CA3 area was observed by Nissl staining. The expression levels of myeloperoxidase （MPO）， inducible nitric oxide synthase （iNOS） and superoxide dismutase （SOD） in serum were detected by colorimetry， and the protein expression levels of Keap-1， Nrf2 and MnSOD in hippocampus were detected by immunohistochemistry （IHC） and Western bolt. Result:Compared with the normal control group， the escape latency， total swimming distance and first arrival time of the plateau in the model group increased （<italic>P</italic><0.01）， while the times of crossing the plateau and the time in target quadrant decreased （<italic>P</italic><0.01）. Compared with the model group， the rats in donepezil group and Kaixinsan groups showed less latency， lower total swimming distance and first arrival time on the platform （<italic>P</italic><0.05， <italic>P</italic><0.01）， while the times of crossing the platform and time in target quadrant increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the normal control group， the expression levels of MPO and iNOS in serum of the model group increased （<italic>P</italic><0.01）， while the expression levels of SOD decreased （<italic>P</italic><0.01）. Compared with model group， the expression of MPO and iNOS in serum of donepezil group and Kaixinsan groups decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）， while the expression of SOD increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. In the normal control group， the neurons in the hippocampal CA3 of the rats were arranged neatly， without obvious Nissl body shrinkage. The neurons in the CA3 of the hippocampus of the model group were not arranged neatly， with obvious neuron loss and pyknosis of Nissl body. The neurons in the CA3 of the hippocampus of the rats in the donepezil group and Kaixinsan groups were arranged neatly， with increased number of neurons and decreased Nissl body shrinkage. Compared with the normal control group， the integrated optical density （<italic>IA</italic>） and protein level of Keap-1 in the hippocampus of the model group decreased（<italic>P</italic><0.01）， while the <italic>IA</italic> and protein level of Nrf2 and MnSOD increased （<italic>P</italic><0.01）. Compared with model group， <italic>IA</italic> and protein levels of Keap-1 and MnSOD in hippocampus of rats in donepezil group and Kaixinsan groups increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， while <italic>IA</italic> and protein levels of Nrf2 decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:Kaixinsan could alleviate memory impairment in AD rats， and its mechanism may be related to its regulation of Keap-1/Nrf2/MnSOD signaling pathway.

Textual Research on Ancient Literature of Classical Prescription Kaixinsan and Its Associated Prescription / 中国实验方剂学杂志

The classical prescription Kaixinsan，which is recorded in an ancient medical book named Beiji Qianjin Yaofang，is one of the famous prescriptions used by ancient physicians to treat amnesia. Research on classical prescriptions has attracted more and more attention from scientific research institutions and related enterprises. Based on ancient books，textual research on origins and development of prescriptions，combing the evolution of prescriptions，preparations，oral ways，taboos and others are the important contents of the study on classical prescriptions. The research results show that the creation of Kaixinsan in Beiji Qianjin Yaofang can be traced back to Kaixinsan recorded in Jiyanfang and Dingzhiwan recorded in Gujinluyanfang. Later generations of physicians created many associated prescriptions in the process of applying Kaixinsan，and the efficacy of these prescriptions was constantly expanded with the development of the times. In the Tang and Song dynasties，Kaixinsan and its associated prescriptions were mainly used to treat amnesia，sorrow，fear，and other diseases. In the Jin and Yuan dynasties，these prescriptions were also used to treat convulsions and yawning. In the Ming dynasty， they were mainly for the treatment of hyperopia， myopia， sprematorrhea，and constipation. In the Qing dynasty，these herbs could be used to treat auricular deafness， aging and sweating. The dosage of Ginseng Radix et Rhizoma and Poria should be increased in the treatment of farsightedness，spermatorrhea and blurred urine，and in the treatment of nearsightedness，the dosage of Polygalae Radix and Haliotidis Concha should be increased. The main pathogenesis of the disease that Kaixinsan and its associated prescriptions treated could be summarized as the deficiency of heart and spleen，imbalance between heart-Yang and kidney-Yin，and the internal resistance of phlegm stagnation. By summarizing the contents of the preparation of tradition Chinese medicine products for Kaixinsan and its associated prescriptions，it is suggested that the dosage form of Kaixinsan can be pills，with the specification size confroming to the most record of ancient generations of physicians，as big as Firmiana platanifolia's fruit.The volume of a single pill is about 0.25 mL and the weight is about 0.3 g. The initial dosage is fifteen pills，which can be modified according to the severity of the illness，with no more than forty pills for each time，three times a day. Also，some excitant food like the sour food，sweet food and mutton should be avoided during the medication. The above research results can provide literature basis for the development of compound tradition Chinese Medicine preparation of Kaixinsan.

Relation Textual Research Between Effect Variation and Dosage of Famous Classical Formula Kaixinsan / 中国实验方剂学杂志

Kaixinsan recorded in an ancient medical book named Beiji Qianjin Yaofang is one of the famous classical formula，which is one of the common prescriptions used by ancient physicians to treat amnesia. In the process of using this prescription，later generations of physicians derived many kinds of associated prescriptions. The effect and indications of these prescription have been inherited and expanded from those in the Beiji Qianjin Yaofang because of the changes in dosages. Therefore，it is necessary to verify the effect and indications of the formulas and the rules of dosage changes. The research results showed that its basic effects included to nourish the mind，induce resuscitation，strengthen the spleen and calm the mind，and keep balance between heart-Yang and kidney-Yin. The main indications included amnesia，sorrow and sadness，fright and fear，and so on，which may differ slightly in different dynasties. In Song，Jin and Yuan dynasties，it also demonstrated the effect of warming the heart and Yang，clearing away heat and relieving wind besides the basic effects， with basically the same indications （slightly different from those in previous dynasties）. In Ming dynasty，it demonstrated the effect of nourishing Yin，clearing away heat and nourishing blood besides the basic effects，and the indications expanded to farsightedness，nearsightedness，spermatorrhea and blurred urine. In the Qing dynasty，its effect also included to nourish the heart and kidney on the basis of the Ming dynasty，and the indications were basically the same with those in the previous dynasties. The compatibility ratio of ancient physicians in the application of this prescription and its associated prescriptions showed some remarkable features，for example，Ginseng Radix et Rhizoma and Poria should be increased and their ratio was≈1∶1 in the treatment of amnesia，sorrow，sadness，fright，fear，farsightedness，spermatorrhea and blurred urine，with ratio of Polygalae Radix to Acori Tatarinowii Rhizoma≈1∶1； the dosage of Polygalae Radix and Acori Tatarinowii Rhizoma should be increased in the treatment of nearsightedness，and their ratio was≈1∶1. The compatibility ratio of Polygalae Radix-Ginseng Radix et Rhizoma-Poria-Acori Tatarinowii Rhizoma=2∶3∶3∶2 was the most frequent，which basically included the indications of this prescription and its associated prescriptions. According to statistics，the average dosages that ancient physicians used were significantly higher than those in the modern times，Polygalae Radix 57 g，Ginseng Radix et Rhizoma 62 g，Poria 70 g，and Acori Tatarinowii Rhizoma 54 g，respectively in ancient times，while Polygalae Radix 11 g，Ginseng Radix et Rhizoma 15 g，Poria 17 g，and Acori Tatarinowii Rhizoma 9.5 g，respectively in modern times. The above textual research results can provide some reference for preparation of tradition Chinese medicine products of Kaixinsan.

Mechanism of Kaixinsan on Learning and Memory Ability and Synaptic Function of APP/PS1 Mice / 中国实验方剂学杂志

Objective:To explore the underlying protective mechanism of Kaixinsan on learning， memory， and synaptic function in APP/PS1 mice. Method:Sixty APP/PS1 mice were randomly divided into a model group， a donepezil （2 mg·kg^-1·d^-1） group， and low- （0.7 g·kg^-1·d^-1）， medium- （1.4 g·kg^-1·d^-1）， and high-dose （2.8 g·kg^-1·d^-1） Kaixinsan groups， and the wild-type mice of the same age in the same litter were assigned to the normal group， with 12 mice in each group. After continuous intragastric administration for two months， the Morris water maze experiment was performed. The ultrastructure of hippocampal neurons was observed by transmission electron microscopy. The colorimetric assay was used to detect serum content of acetylcholine （ACh）， choline acetyltransferase （ChAT）， acetylcholinesterase （AChE）， and levels of hippocampal reactive oxygen species （ROS）， malondialdehyde （MDA）， superoxide dismutase （SOD）， and glutathione peroxidase （GSH-Px）. Real-time fluorescence-based quantitative polymerase chain reaction （Real- time PCR） was used to detect the mRNA expression of hippocampal brain-derived neurotrophic factor （BDNF）， beta-nerve growth factor （NGFB）， discs large homolog (DLG)2， DLG4， and synaptophysin （SYP）. Result:Compared with the normal group， the model group showed prolonged escape latency， reduced number of crossing platforms， shortened stay in the target quadrant （<italic>P</italic><0.01）， decreased number of mitochondria with different shapes and irregular arrangement， some swollen and deformed mitochondria with broken mitochondrial cristae， endolysis， and cytoplasm vacuole， and more cell debris. Additionally， the model group also displayed reduced serum levels of ACh and ChAT， increased AChE （<italic>P</italic><0.01）， elevated hippocampal ROS and MDA （<italic>P</italic><0.05，<italic>P</italic><0.01）， declining SOD and GSH-Px （<italic>P</italic><0.01）， and diminished hippocampal BDNF， NGFB， DLG2， DLG4， and SYP mRNA levels （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the model group， the donepezil group， and the medium- and high-dose Kaixinsan groups showed shortened escape latency， increased number of crossing platforms， prolonged stay in the target quadrant （<italic>P</italic><0.05，<italic>P</italic><0.01）， improved mitochondrial damage with a regular shape （mainly oval shape）， relieved mitochondrial swelling and deformation， and clear mitochondrial cristae. Furthermore， the donepezil group， and the medium- and high-dose Kaixinsan groups also exhibited increased serum ACh and ChAT levels （<italic>P</italic><0.05，<italic>P</italic><0.01）， blunted AChE activity （<italic>P</italic><0.05）， reduced hippocampal ROS level （<italic>P</italic><0.05，<italic>P</italic><0.01）， declining MDA level （<italic>P</italic><0.05）， potentiated SOD and GSH-Px activities， and up-regulated hippocampal BDNF， NGFB， DLG2， DLG4， and SYP mRNA levels （<italic>P</italic><0.05，<italic>P</italic><0.01）. In the low-dose Kaixinsan group， the stay time in the target quadrant was prolonged and the expression of hippocampal SYP mRNA was elevated significantly （<italic>P</italic><0.05）. There was no statistical difference in swimming speed between the groups. Conclusion:Kaixinsan can improve the learning and memory ability of APP/PS1 mice by increasing the expression of synaptic plasticity-related proteins， reducing the ultrastructural damage to hippocampal neurons， resisting oxidative stress， and regulating cholinergic neurotransmitters， thereby exerting neuroprotective effects.

Research progress on pharmacodynamic material basis and pharmacological action mechanism of Kai-Xin-San / 中草药

Kai-Xin-San (KXS) is a well-known formula that was first recorded in an ancient Chinese book "Important Prescriptions Worth a Thousand Gold for Emergency" by Si-miao Sun from the Tang Dynasty. KXS is composed of Panax ginseng, Poria cocos, Polygala tenuifolia, and Acorus tatarinowii at a ratio of 3:3:2:2. The material basis of the pharmacological action of KXS is mainly related to ginsenosides, polygala saponins, polygala oligosaccharide esters, and polygalactone. However, the active components of P. tenuifolia and A. tatarinowii are less studied, and the research scope is limited. The pharmacological researches of KXS are mainly focused on antidepressant effect, antisenile dementia, improving learning and memory ability, antifatigue, and sedation. The mechanisms involved include nervous system, immune system and endocrine system. The material basis, pharmacological effects and known mechanisms of KXS are systematically described in this paper in order to provide some ideas for the clinical application of KXS in the future.

Material Basis and Mechanism of Kaixinsan for Treatment of Alzheimer's Disease Based on Integrated Pharmacological Platform of Chinese Medicine / 中国实验方剂学杂志

Objective: To explore the interrelation of "composition-target-disease" of Kaixinsan on treatment of Alzheimer's disease. Method: Through the integrated pharmacological platform of Chinese medicine V1.0,the active ingredients and potential targets of four Chinese herbs in Kaixinsan were collected,disease targets of Alzheimer's disease were searched,and enriched by the gene ontology database and the Kyoto encyclopedia of genes and genomes at hubs. Result: Among the 250 compounds of Kaixinsan,2 877 targets were associated with Alzheimer's disease.The key targets,such as mitochondrial trifunctional enzyme subunit alpha(HADHA),hydroxyacyl coenzyme A dehydrogenase(HADH),sterol-4-alpha-carboxylate 3-dehydrogenase(NSDHL) and others,played their pharmacological effects mainly through regulating purine and nucleotide metabolism,Huntington's disease,Alzheimer's disease,neurodegenerative diseases,oxidative phosphorylation,and endocrine and metabolic diseases in molecular reactions,such as cytoplasm,mitochondria,adenosine triphosphate binding,and mitochondrial matrix. Conclusion: The platform can predict the key targets and related pathways of Kaixinsan for treatment of Alzheimer's disease,which lays the foundation for further revealing material basis and mechanism of this formula,and plays an important role in digging and developing this classic and famous formula.

Study on Material Basis and Action Mechanism of Chinese Formula Kai-Xin-San on Anti-depression via Regulation of Neurotrophic Factors / 世界科学技术-中医药现代化

Objective: To observe the material basis and mechanism of action of Kai-Xin-San (KXS) in regulating antidepression of neurotrophic factors. Methods: KXS eluted by ethanol on macroporous resin was prepared. The antidepressive effect of different components was compared by tailing suspension test and forced swimming test of mice. The levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in hippocampus were determined by ELISA. The rat astrocyte glioma C6 cell line and the rat adrenal pheochromocytoma PC12 cell line were used to evaluate the effects of different ethanol elution sites on the expression of NGF and BDNF and the differentiation of PC12 cells.Results: All of the ethanol elution components from KXS exerted anti-depressive effects by shorting the immobile time of tailing suspension and forced swimming of mice and 70％ ethanol elution components exerted best efficacy. This site also could increase expressions of NGF and BDNF on C6 glioma cell line. The 10％ ethanol elution site had the strongest ability to promote PC12 cell differentiation. Ginsenosides were the main effectuve ingredients for promoting the expression of neurotrophic factors. Conclusion: Regulation of neurotrophic factors might be the prominent action mechanism of KXS exerting anti-depressive effects.

Antidepressant effects of Kai-Xin-San in fluoxetine-resistant depression rats

This study aimed to investigate the antidepressant effect and the mechanism of action of Kai-Xin-San (KXS) in fluoxetine-resistant depressive (FRD) rats. Two hundred male Wistar rats weighing 200±10 g were exposed to chronic and unpredictable mild stresses (CUMS) for 4 weeks and given fluoxetine treatment simultaneously. The rats that did not show significant improvement in behavioral indexes were chosen as the FRD model rats. These rats were randomly divided into four groups: FRD model control; oral fluoxetine and aspirin; oral KXS at a dose of 338 mg·kg-1·day-1; and oral KXS at a dose of 676 mg·kg-1·day-1. Rats continued to be exposed to CUMS and underwent treatment once a day for 3 weeks, then cytokine (COX-2, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, TGF-β, and TNF-α) levels in the hippocampus and serum, and organ coefficients were measured. Both doses of KXS improved the crossing and rearing frequencies, sucrose-preference index, and body weight in FRD rats. KXS at a dose of 338 mg·kg-1·day-1reduced COX-2, IL-2, IL-6, TNF-α levels, increased IL-10 level in the hippocampus, and reduced IL-2 and TNF-α levels in serum. KXS at a dose of 676 mg·kg-1·day-1reduced TNF-α level in the hippocampus, reduced IL-2 and TNF-α levels in serum, and increased IFN-γ and IL-10 levels in the hippocampus and serum. There were no significant differences in organ-coefficients of the spleen among and between groups. The results suggested that oral administration of KXS in FRD rats was effective in improving behavior disorders by influencing various inflammatory pathways.

Effect of Kaixinsan on monoamine oxidase activity / 中国中药杂志

To observe the effect of antidepressant medicine prescription, Kaixinsan (KXS) on monoamine oxidase (MAO) activity, and explore the mechanism of KXS in elevating the levels of monoamine neurotransmitter from the perspective of metabolism, in vitro enzyme reaction system and C6 neuroglial cells, the effect of KXS at different concentrations on MAO-A and MAO-B activity was observed. In animal studies, the effect of KXS at different concentrations on MAO-A and MAO-B activities of brain mitochondrialin normal rats and solitary chronic unpredictable moderate stress (CMS) model rats after intragastric administration for 1, 2, 3 weeks. Results showed that 10 g•L⁻¹ KXS could significantly reduce the activity of MAO-A and MAO-B in enzyme reaction system; and in C6 cells, KXS within 0.625-10 g•L⁻¹ concentration range had no significant effect on the activity of MAO-A, but had obvious inhibitory effect on the activity of MAO-B in a dose dependent manner. KXS had no significant effect on the activity of MAO-A and MAO-B in brains of normal rats after action for 1, 2, 3 weeks. After 2 and 3 weeks treatment with 338 mg•kg⁻¹ dose KXS, MAO-A activity in the brain of CMS rats was decreased as compared with the model group (P<0.05), while KXS had no significant effect on MAO-B activity after 1, 2, 3 weeks of treatment. The results indicated that KXS had certain effect on in vitro MAO-A and MAO-B activity, had no effect on brain MAO-A and MAO-B activity in vivo in normal rats, and had certain inhibitory effect on MAO-A activity in brains of CMS rats.

Effect of Kaixin-San on depression behavior and hippocampal brain-derived neurotrophic factor in mice / 第二军医大学学报

Objective To investigate the effect of Kaixin-San (KXS) on behavior, brain monoamine transmitters, and hippocampal brain-derived neurotrophic factor (BDNF) in mice receiving tail suspension test (TST), so as to explore the correlation of the antidepressant effect of KXS and hippocampal BDNF level. Methods The effect of KXS on the immobility time of mice in the TST was observed. The levels of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) in the brain were determined by high-performance liquid chromatography (HPLC). Western blotting analysis was used to examine the expressions of cAMP-response element-binding protein (CREB), p-CREB, and BDNF in the hippocampus. BDNF mRNA expression was also examined by RT-PCR. Pearson's correlation analysis was used to evaluate relationship between anti-depression effect of Kaixin-San and hippocampal BDNF level. Results KXS reduced the immobility time of mice in the TST (P<0.05); it also increased the brain levels of monoamine transmitters (DA and 5-HT) and hippocampal levels of CREB, p-CREB and BDNF compared with the control mice(P<0.05). Moreover, the anti-depression effect of KXS was correlated with BDNF level (protein:r=-0.694, P<0.01; mRNA:r=-0.547, P<0.01) in the hippocampus. Conclusion The anti-depression effect of KXS is positively correlated with the hippocampal BDNF level, indicating KXS may exert anti-depression effect via increasing hippocampal BDNF.