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Research on Guidance of Prevention and Treatment of Vascular Diseases by Yingwei Theory of Vessel-collateral Theory

Yi-ling WU ; Zhen-hua JIA ; Li-ping CHANG ; Cong WEI

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):1-10. doi:10.13422/j.cnki.syfjx.20190199

The blood-vein theory is inherited and developed. Combined with the related theory features of Qi and blood of traditional Chinese medicine(TCM), the progress results in two national 973 plan projects undertaken are learned from, the vessel-collateral theory guiding the prevention and treatment of vascular lesions is systematically built, the academic development of TCM collateral disease discipline is pushed from "treatment of symptoms of collateral disease" to study on "vessel-collateral theory", a new stage in the history. The pathogenesis, basic pathogenesis, clinical syndrome and syndrome differentiation therapy of vessel-collateral lesions are systematically studied, Yingwei theory, the core content of vessel-collateral theory, is proposed, the syndrome differential diagnosis standard of "vessel-collateral-vascular system disease" is established, and the blood-vessel theory of TCM is improved. The development of Yingwei theory of vessel-collateral theory is deepened. The theory of Yingwei sympathia and Yingwei's "opening by collaterals, intersecting and generating" are proposed to guide the study on prevention and control of vascular lesions. With tertiary collaterals-microvessel as a breakthrough point of research, four types of microscopic pathological features of microvessels are proposed. It is confirmed that dredging collaterals and protecting tertiary collaterals-microvessel is the common key mechanism for the treatment of major diseases of heart, brain and kidney(sugar). It protects microvessels, improves blood flow perfusion, protects tissue cells, and improves the viscera function. Dredging collaterals and protecting microvascular endothelial cells is the core mechanism of treating tertiary collaterals-microvessel lesions. It is proposed that protection of microvascular endothelial cells is a new strategy to solve the problem of microvascular lesions. After more than ten years, four studies of evidence-based medicine have been completed to solve the clinical problem of acute myocardial infarction without reflow. New drugs are provided to the international medical problem of cardiac insufficiency with premature ventricular contraction. The gap of fast and slow treatment, integration and rhythm adjustment of sinus bradycardia with premature ventricular contraction is filled. The clinical treatment effect of chronic heart failure is significantly improved, a major breakthrough in the treatment of microvascular lesions by TCM is achieved, and it is fully recognized and highly evaluated by the medical profession at home and abroad. It promotes the specialized subject platform construction and society development under the guidance of Yingwei theory of vessel-collateral theory, as well as the clinical application of syndrome differentiation diagnosis and treatment method and drugs of vessel-collateral lesions, and has significant social and economic benefits.

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Coronary Heart Disease Phlegm-blood Stasis-stagnation-deficiency Innovation Theory and Clinical Application Based on Complex System

Jie WANG ; Guang CHEN

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):11-15. doi:10.13422/j.cnki.syfjx.20190750

There exits a trend of treatment for coronary heart disease(CHD) in terms of Chinese medicine(CM) in history,from warming Yang and dispelling cold,moving Qi and dissolving phlegm to invigorating blood and dissolving stasis,whose time-space development accords to the theory of complex system.Targeting the contemporary population of CHD,phlegm-blood stasis-stagnation-deficiency theory is proposed to direct clinical application to further improve effectiveness.From the perspective of theory,blood stasis-stagnation is the basic pathogenesis of CHD that runs through the whole period in the advance of CHD,while phlegm has been changing the feature of CM Zheng of CHD population with the development of the society,and nourishing the body according to the type of deficiency might delay the process of growing older,postpone the occur of disease and hinder the advance of disease,which genuinely differs from modern medicine to render the superiority of CM to work out the challenges of CHD.As for the clinical application,the treatment options could consider invigorating blood,dissolving stasis and removing stagnation as the basic method,such as CHD formula 2,Xuefu Zhuyutang,Zhizi Chitang and Danggui Niantongtang could be used in varied period of CHD.Moreover,boosting the body by herbal medicine could regulate the routine of body,then to prevent CHD and fear down the advance of CHD,for instance,Codonopsis Radix,Astragali Radix,Liuwei Dihuang pills,Zuoguiyin and Youguiyin.Furthermore,balmiest herbs could not only dissolve phlegm,unblock the meridian and relieve pain,but also calm the mind and eliminate the anxiety.For example,Aquilariae Lignum Resinatum,Santali Albi Lignum and Moschus could be used handily in the treatment of CHD.

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Establishment of DNA Fingerprints and Genetic Similarities Analysis of Dendrobium officinale

Wen-xia YU ; Zhou-xi LEI ; Yuan YUAN ; Yu-lin WU ; Qing-sheng SHUN ; Gang WEI ; Liu-ping HU ; Ju-ting HU ; Lu-qi HUANG

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):16-21. doi:10.13422/j.cnki.syfjx.20190161

This study adopted the 4 pairs of simple sequence repeats(SSR) primers selected by National Resource Center for Chinese Meteria Medica (CACMS) to detect 29 populations of Dendrobium officinale samples from 9 main places of production; 5,7,4 and 3 polymorphism bands were amplified from these 4 pairs of SSR primers. The DNA identities of different populations were constructed by SSR. The 29 D. officinale populations could be divided into 4 classes. The clustering result was related to the places of production. Samples from Yunnan,Guizhou,Sichuan provinces were classified into one category,while samples from Anhui and Guangxi provinces were classified into another category. Samples from Guangdong Danxia,Zhejiang Yongkang,Zhejiang Leqing and Taining belonged to a category. PopGene (version 1.32) software was applied to calculate the genetic similarity of the 29 D. officinale populations. The similarities was between 0.403 4 and 1.0.Based on the genetic similarity,the genetic consistency included three classes,A,B and C. Samples with a similar geographical location and landform environment have higher genetic similarities,which indicate the same genetic background. This paper provides reference information to study the identification, selection and breeding of good varieties.

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HPLC Characteristic Spectrum Optimization of Flavonoid Glycosides on Dendrobium officinale and Characteristics Analysis of Different Provenances

Zhi-yun LIANG ; Zhen-shan XIE ; Yue-chun HUANG ; Yuan YUAN ; Chu-juan ZHOU ; Ya-wen WANG ; Gang WEI

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):22-28. doi:10.13422/j.cnki.syfjx.20190112

Objective: To optimize the HPLC characteristic spectrum of flavonoid glycosides of Dendrobium officinale,and identify the common and specific components of different provenances. Method: Kromasil 100-5 C₁₈ column was adopted, with tetrahydrofuran-acetonitrile-methanol (10:22:5)-0.05% phosphoric acid as mobile phase (gradient elution). The detection wavelength was 340 nm,the column temperature was 30℃,and the flow rate was 1.0 mL ·min^-1. Result: 13 flavonoid characteristic peaks were marked in 27 batches of D. officinale,and 7 characteristic peaks of 6 flavonoid C-glycosides (vicenin Ⅱ,vicenin Ⅰ,schaftoside,isoschaftoside,violanthin and isoviolanthin) and one flavonoid O-glycosides (rutin) was identified. 7-11 characteristic peaks were detected in different batches of samples. Among them,vicenin Ⅱ was a relatively stable common peak in different source samples,and the characteristic peaks of rutin,schaftoside and isoschaftoside were quite different. According to the relative abundance of the characteristic peaks,the samples could be divided into three categories. Among them,the first category had 10 batches of samples,which mainly came from Danxia landforms of Guangdong,Jiangxi,Fujian and Zhejiang (Wuyi) Province (which called "Danxia landform species") and characterized by detection of obvious peak of rutin. The second category had 11 batches of samples,which mainly came from Yunnan and Guangxi Province (which included "Yunnan Guangnan species" and "Guangxi Tiepilan species") and characterized by detection of violanthin and isoviolanthin. And the third category had 6 batches of samples, which were mainly derived from Zhejiang Province (which called "native species from Zhejiang") and characterized by detection of different degrees of rutin peak, but it was difficult to detect the characteristic peaks of violanthin and isoviolanthin. HPLC characteristic chromatograms of D. officinale in bionics wild cultivation and greenhouse of "Danxia landform species" and "Guangxi Tiepilan species" were compared. The results showed that the characteristic peaks in D. officinale planted in greenhouse could be detected stably,which verified the reliability of the source in D. officinale. Conclusion: The analytical method has a better separation effect on flavonoids of D. officinale, with a good reproducibility. The commonness and specificities of flavonoid glycosides components of D. officinale from different categories have basically confirmed. This suggests that Vicenin Ⅱ is suitable to be a reference peak for characteristic chromatogram. Both the relative abundance of rutin and the detection or relative abundance of violanthin and isoviolanthin peaks could be used as a reference to judge the categories of D. officinale in "Danxia landform species" or "Tiepilan species from Yunnan, South Guangdong and Guangxi" or "native species from Zhejiang".

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Isolation and Identification of Main Flavonoid Glycosides of Dendrobium officinale from Danxia Species and Yunnan Guangnan Species

Xiao-feng ZHANG ; Chun-hua ZHOU ; Long-kai ZHANG ; Mei JIANG ; Zhen-shan XIE ; Yuan YUAN ; Yue-chun HUANG ; Ying-yi LUO ; Gang WEI

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):29-34. doi:10.13422/j.cnki.syfjx.20182417

Objective: To extract,isolate,purify and identify the structures of the flavonoid glycoside in Dendrobium officinale from two different origin places (Danxia species and Yunnan Guangnan species),and provide experimental reference for confirming the common flavonoid glycoside components in D. officinale. Method: ① 70% ethanol was applied to extract the total flavonoids in leaves of D. officinale from two different species. Organic solvents petroleum ether,acetic ether and water saturated n-butyl alcohol were used in turn to extract the crude extraction. Then AB-8 Macroporous resin,Sephadex LH-20 and ODS chromatographic column were applied to isolate and purify the water saturated n-butyl alcohol extraction fraction. The structures of flavonoid glycoside were identified by studying physicochemical property,applying modern spectroscopy method like HPLC,ESI-MSⁿ,¹H-NMR,¹³ C-NMR,etc. ② HPLC characteristic spectrum technique was used to analyse and compare the common flavonoid glycoside components in Dendrobium officinale from different origin places (Danxia species,Yunnan Guangnan species,Guangxi Tiepilan species and Zhejiang native species). Result: Five flavonoid glycoside compounds were isolated from the crude extractions of the leaves of D. officinale from two different species,and they were identified as rutin,vicenin Ⅱ,viceninⅠ,violanthin and isoviolanthin. The characteristic spectrum of vicenin Ⅱ and viceninⅠwere detected in stems of D. officinale from four different origin places (Danxia species,Yunnan Guangnan species,Guangxi Tiepilan species and Zhejiang native species),and vicenin Ⅱ had a better separation degree in the characteristic spectrum. However,the characteristic spectrum of violanthin and isoviolanthin were more obvious in Yunnan Guangnan species and Guangxi Tiepilan species,while rutin was obvious in the Danxia species. Conclusion: Vicenin Ⅱis the common flavonoid glycosides component in D. officinale from different origin places (Danxia species,Yunnan Guangnan species,Guangxi Tiepilan species and Zhejiang native species),and can be used as the internal reference material for the characteristic spectrum of D. officinale.

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Content Comparison of Mannose,Glucose and Narinhenin in Dendrobium officinale and Dendrobium huoshanense

Ya-wen WANG ; Zhi-yun LIANG ; Zhen-shan XIE ; Jia-hong YE ; Si-jian OU ; Chu-juan ZHOU ; Yuan YUAN ; Yue-chun HUANG ; Gang WEI

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):35-42. doi:10.13422/j.cnki.syfjx.20182418

Objective: To optimize the pre-column derivation high performance liquid chromatography (HPLC) content determination method of D-mannose and D-glucose as well as the content determination method of narinhenin in Dendrobium officinale and D. huoshanense, and compare the contents of D-mannose,D-glucose and narinhenin between D. officinale and D. huoshanense. Method: A pre-column derivation HPLC method modified by Chinese Pharmacopoeia(Ch.P) 2015 was used to simultaneously determine the contents of D-mannose and D-glucose,with acetonitrile-0.02 mol·L^-1 ammonium acetate solution as mobile phase for gradient elution. Kromasil 100-5 C₁₈ was performed with the wavelength set at 250 nm,and the flow rate was 1 mL·min^-1;column temperature was 30℃. HPLC content determination of narinhenin was performed on Kromasil 100-5 C₁₈ with the acetonitrile-methanol-0.4% phosphoric acid solution as mobile phase for gradient elution,and the wavelength was set at 290 nm; the flow rate was 0.8 mL·min^-1,and column temperature was 40℃. Result: D-mannose and D-glucose showed a good linear relationship within the range of 0.15-3.0 μg and 0.075-2.25 μg (r=0.999 9); and their average recoveries were 99.01% (RSD 2.1%) and 101.69% (RSD 2.0%) respectively. In addition, the other methodological researches such as repeatability and durability all met the requirements. The contents of D-mannose(C_m),D-glucose(C_g) and sum of them (C_m+C_g) were 12.75%-36.40%,2.93%-18.39% and 19.23%-54.58% in 43 batch of D. officinale. Almost all of the results except very few samples reached the D-mannose standard in Ch.P 2015, and the total content of D-mannose and D-glucose was also up to the total polysccharide standard in Ch.P. The correlation between content and origin was not significant. The contents of D-mannose(C_m),D-glucose(C_g) and sum of them (C_m+C_g) were 14.33%-29.47%,6.64%-15.20%,and 25.73%-44.37% in 12 batch of D. huoshanense. These contents and ratio of peak areas of D-mannose to D-glucose (A_m/A_g) were within the scope of D. officinale's; in addition, their average contents were basically the same with those in D. officinale (about 33%).Next,naringenin showed a good linear relationship within the range of 0.020 8-0.832 0 μg (r=0.999 9),and its average recovery was 101.96% (RSD 1.8%). The content of naringenin was 0.053 2-0.122 4 mg·g^-1 (average value of 0.081 0 mg·g^-1) in 11 batch of D. officinale, slightly higher than 0.040 3-0.090 0 mg ·g^-1 (average value of 0.068 3 mg ·g^-1) in 7 batch of D. huoshanense. All of these results of narinfenin did not reach the content lower limit in Ch.P. Conclusion: The method used to determinate the content of D-mannose and D-glucose is reproducible, and their sum content is possible to substitute the total polysccaride determination (with higher errors) in D. officinale; monosaccharide content determination can be used for quantitative quality control of D. huoshanense. However, it could not distinguish D. officinale and D. huoshanense by determining the contents of polysccharide,D-glucose,D-mannose and narinhenin, and shall be combined with other specificity methods for further identification.

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Antioxidation Activities in Vitro of Vicenin Ⅱ Isolated from Dendrobii Officinalis Caulis and Effect on HepG2 Cells

Ying-yi LUO ; Yun-rong LI ; Zhou-xi LEI ; Yuan YUAN ; Yue-chun HUANG ; Gang WEI

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):43-50. doi:10.13422/j.cnki.syfjx.20182210

Objective: To study the antioxidation activities in vitro of a comment flavonoid component named vicenin Ⅱ(Apigenin 6,8-di-C-glucoside) in Dendrobii Officinalis Caulis from different origin places and investigate its effects on apoptosis of HepG2 cells. Method: The antioxidation activities in vitro of vicenin Ⅱ (0.005-1 g·L^-1) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), salicylic acid and 2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid(ABTS) and copper ion reduction assays. Methye thiazolye telrazlium(MTT) assay was used to test the inhibitory effect of vicenin Ⅱ(12.5~100 μmol·L^-1) on proliferation of 6 tumour cells in vitro. In subsequent apoptosis experiment, the concentration of vicenin Ⅱ was 75 μmol·L^-1. The morphological changes of HepG2 cells were evaluated by Hoechst 33258 under fluorescence microscope; and the cell apoptosis rate was detected by flow cytometry with AnnexinV/PI apoptosis assay kit. The mRNA expressions of mitogen activated protein kinase (MAPK) pathway related apoptotic genes were detected by Real-time PCR assay. Result: The 1 g·L^-1 vicenin Ⅱ showed 48.82% and 22.01% for DPPH scavenging rate and Cu²⁺ reduction rate respectively(P<0.01). 0.5 g·L^-1 vicenin Ⅱ showed 86.88% for ABTS scavenging rate(P<0.01). vicenin Ⅱ could significantly inhibit the proliferation and increase the apoptosis rate on HepG2 cells; after treatment for 48 h with 75 μmol·L^-1 Vicenin Ⅱ, the cells survival rate was 45.69%(P<0.01) and apoptotic rate was 14.57%(P<0.01). Meanwhile, the mRNA expression levels of B-cell lymphoma-2 (Bcl-2) related X protein (Bax)/Bcl-2, Caspase-8, p38, extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and nuclear transcription factor (NF)-κB were increased(P<0.01). Conclusion: The general flavone glycosides component vicenin Ⅱ of Dendrobii Officinalis Caulis from different origins has a certain antioxidation effect and significant inhibitory effect on proliferation, and could induce apoptosis on HepG2 cells probably by regulating the expression of related genes in MAPK pathway and Bax/Bcl-2.

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Progress in Research of Kangxianling Decoction in Delaying Renal Fibrosis

Jing JI ; Li-qun HE

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):51-56. doi:10.13422/j.cnki.syfjx.20190121

Renal fibrosis is a common pathological change in the later stages of all kidney diseases. It is a multi-cytokine, multi-signal pathway, multi-factor driven chronic kidney disease. It includes renal interstitial fibrosis, tubular sclerosis and glomerular sclerosis, which eventually leads to chronic renal failure. From health through injury to loss of function, the disease process is closely related to the degree of deterioration of renal function and the prognosis of chronic kidney disease. There is no effective western medicine for the treatment of renal fibrosis. Professor HE Liqun from Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine has summarized the Kangxianling decoction through decades of long-term practical experience. It has the effects in strengthening the spleen and replenishing Qi, clearing away dampness and heat, promoting blood circulation and removing phlegm, and strengthening turbidity. It is composed of Salviae Miltiorrhizae Radix et Rhizoma 15 g, Persicae Semen 12 g, Angelicae Sinensis Radix 12 g, Achyranthis Bidentatae Radix 9 g, Rhei Radix et Rhizoma 15 g. Kangxianling can affect the synthesis and secretion of cytokines and inflammatory factors by expanding blood vessels, and can improve renal tubular fibrosis. It has a good multi-channel, multi-target and multi-directional protective effect on renal function. It also can delay the progress of chronic renal fibrosis by significantly alleviating such symptoms as fatigue and edema in patients with chronic renal fibrosis, and reducing serum creatinine, urea nitrogen and urine protein. In this paper, 5/6 nephrectomy, ischemia reperfusion injury, adriamycin-induced nephropathy, unilateral ureteral obstruction and other different modeling methods are listed. The mechanism of Kangxianling decoction in antagonizing renal fibrosis is discussed and summarized, which further provided new ideas and directions for future clinical and scientific research.

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Effect of Kangxianling Decoction on Renal Fibrosis and ACE-AngⅡ-AT1R Axis in 5/6 Nephrectomized Rats

Jing JI ; Li-qun HE

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):57-62. doi:10.13422/j.cnki.syfjx.20190122

Objective: To study the therapeutic effect of Kangxianling decoction on renal fibrosis induced by 5/6 nephrectomy, and angiotensin converting enzyme-angiotensin Ⅱ-angiotensin Ⅱ 1 receptor (ACE-AngⅡ-AT1R) axis. Method: Totally 50 SD rats were randomly divided into the following groups:control group (n=10), sham-operation group (n=10), 5/6 nephrectomized renal fibrosis model group (n=30). After two weeks, the rats in operation group were divided into the model group, Kangxianling group, and losartan potassium group, n=10 in each group. Rats in losartan potassium group were administered with losartan potassium by gastrogavage, and rats in Kangxianling group were administered with Kangxianling by gastrogavage. Equal volume of saline was administered to rats in the other groups. The rats were put to death after 16 weeks of consecutive medication, and serum creatinine(SCr), blood urea nitrogen(BUN), 24 h urine protein(24 h-Pro) were measured in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of kidney tissues, and the degree of renal fibrosis was observed by Masson staining. The expressions of ACE1 and AT1R were detected by immunohistochemistry. The protein expression levels of ACE1, AngⅡ and AT1R were determined by Western blot. Result: Compared with control and sham-operation groups, SCr, BUN and 24 h-Pro in model group were significantly increased (P<0.01). Compared with model group, SCr, BUN and 24 h-Pro levels in the Kangxianling group and the losartan potassium group were significantly lower, with statistically significant differences (P<0.05,P<0.01). HE and Masson staining showed that the degree of renal fibrosis was reduced in Kangxianling group and losartan potassium group compared with model group. Western blot showed that ACE1, AngⅡ and AT1R in Kangxianling group, and the losartan potassium were significantly lower than the model group, with statistically significant differences (P<0.01). Immunohistochemistry results showed that the levels of ACE1 and AT1R showed the same changes as Western blot. Conclusion: Kangxianling decoction can delay the progress of renal fibrosis in 5/6 nephrectomized rats, which is closely related to the inhibition of ACE-AngⅡ-AT1R axis activation.

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Effect of Kangxianling Decoction on Renal Function and Kidney ECM Expression in Rats with Renal Fibrosis

Jing JI ; Li-qun HE

Chinese Journal of Experimental Traditional Medical Formulae.2019;25(1):63-68. doi:10.13422/j.cnki.syfjx.20190123

Objective: To investigate the effect of Kangxianling decoction on renal function and expression of extracellular matrix(ECM) in renal tissue of rats with renal fibrosis induced by 5/6 nephrectomy. Method: Totally 50 SD rats were randomly divided into control group (n=7), sham-operation group (n=7), operation group (n=36). The 5/6 nephrectomized renal fibrosis model was established in operation group. After successful modeling, rats were randomly divided into model group, Kangxianling group and losartan potassium group. The losartan potassium group and the Kangxianling group were given losartan potassium (33.3 g·kg^-1) and Kangxianling decoction (21 g·kg^-1) every day. The control group, sham-operation group and model group were all treated with equal volume of normal saline. Rats were put to death after 24 weeks of consecutive medication, and renal function, 24 h urine volume and 24 h-Pro quantitation of each group were measured. The expressions of collagen Ⅰ(ColⅠ) and ColⅢ in renal tissues were determined by immunohistochemistry and Western blot. Result: Compared with the normal group and sham-operation group, serum creatinine(SCr), blood urea nitrogen(BUN), 24 h urine volume, 24 h-Pro quantitation, renal tissue ColⅠ and ColⅢ expression levels were significantly increased in the model group (P<0.01). Compared with the model group, the levels of SCr, BUN, 24 h-Pro quantitation, protein expression levels of renal tissue ColⅠ and ColⅢ were significantly decreased in the Kangxianling group and the losartan potassium group (P<0.05,P<0.01). Conclusion: Kangxianling decoction can down-regulate the expressions of ColⅠ and ColⅢ in kidney tissue, there by inhibiting the accumulation of ECM, and exerting the effect in delaying renal fibrosis and protecting renal function.

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