Immunostimulating effect of polysaccharides isolated from Ma-Nuo-Xi decoction in cyclophosphamide-immunosuppressed mice.

Ma-Nuo-Xi Decoction (MNXD) is well-known in Tibetan medicine to be in line with the theory of treatment determination based on syndrome differentiation. However, the components responsible for its immunomodulating effect are unknown. In this study, three polysaccharide components-MNXD-P, MNXD-BD-P, and MNXD-AD-P-were isolated from MNXD and its basic and auxiliary prescription decoctions, of which MNXD-BD-P is composed of ß-(1,4)-d-glucan and RG-I pectin, MNXD-AD-P contains mainly α-(1,4)-d-glucan and some amount of arabinogalactan and/or arabinorhamnogalactan, and MNXD-P contains components of both MNXD-BD-P and MNXD-AD-P. And treatment with these polysaccharides could significantly improve the host's specific and non-specific immunity, including cellular and humoral immunities, as well as promote recovery from myelosuppression in cyclophosphamide (CTX)-immunosuppressed mice. To our knowledge, this is the first report on chemical and immunoactivity study on polysaccharides from traditional Tibetan medicine compounds, which may provide a new idea for development of carbohydrate drugs from them.

Ma-Nuo-Xi decoction has an immunostimulating effect in cyclophosphamide-immunosuppressed mice.

BACKGROUND: Ma-Nuo-Xi decoction (MNXD), as well as its hundreds of derivative preparations, has been used in Tibetan medicine since the 14th century. MNXD is in accordance with the theory of treatment determination based on syndrome differentiation. This study aimed to compare the effect of the auxiliary MNXD prescription (MNXD-AD) with that of the basic MNXD prescription (MNXD-BD) on the immunostimulating activity of MNXD. METHODS: The immunopotentiation of MNXD, MNXD-BD, and MNXD-AD was evaluated using a cyclophosphamide (CTX)-immunosuppressed mouse model. Their influences on non-specific and specific immunity were evaluated using immune organ indexes, peripheral white blood cell (WBC) count, red blood cell (RBC) count, platelet count, phagocytosis, macrophage-secreted nitric oxide (NO) and cytokines, natural killer (NK) cytotoxic activity, lymphocyte proliferation, serum cytokines, splenic T-lymphocyte subpopulations, and quantitative hemolysis of sheep red blood cell (QHS SRBC) assays. RESULTS: MNXD, MNXD-BD, and MNXD-AD increased the spleen and thymus indexes, as well as the peripheral WBC, RBC, and platelet counts. They also promoted phagocytosis, NO and cytokine secretion from macrophages, NK cytotoxic activity, and lymphocyte proliferation, and also raised the CD4+ /CD8+ T-cell ratio, serum cytokine concentrations, and haemolysin formation in CTX-treated immunosuppressed mice. Compared with MNXD-BD and MNXD-AD, MNXD was superior in restoring the phagocytic index, concanavalin A (ConA)-induced T-lymphocyte proliferation, NO secretion from macrophages, and haemolysin formation, as well as the levels of interleukin 1 beta (IL-1ß), and serum interleukin-2 (IL-2) and interferon gamma (INF-γ). CONCLUSIONS: MNXD, MNXD-BD, and MNXD-AD have excellent immunostimulating and myelosuppression-restoring activities on CTX-immunosuppressed mice. Among them, MNXD-AD might be an immunomodulator, which may happen to be in line with the clinical experience of Tibetan medicine physicians of using it to promote the efficacy of MNXD-BD.

Content Determination of 6 Kinds of Triterpene Acid in Tibetan Medicine Rubus biflorus by Pre-column Derivatization HPLC/ FLD-APCI/MS / 中国药房

OBJECTIVE： To establish the method for content determination of 6 kinds of triterpene acids such as haw acid， corosolic acid， betula acid， betulonic acid， oleanolic acid and ursolic acid in Tibetan medicine Rubus biflorus. METHODS： Pre-column devrivatization HPLC-FLD-APCI/MS method was adopted. 2-（7H-dibenzo[a，g]carbazol-7-yl） ethyl-4-methylbenzene- sulfonate was used as the pre-column derivatization reagent．Hypersil C18 column was used with the mobile phase consisted of 5％ acetonitrile water solution-acetonitrile （gradient elution） at the flow rate of 1.0 mL/min. The excitation wavelength of fluorescence was 300 nm and the emission wavelength was 395 nm. The column temperature was 35 ℃， and sample size was 10 μL. Under atmospheric-pressure chemical ionization （APCI） source in positive-ion mode， pressure was 60 psi， the drying gas flow rate was 9 L/min， the dry gas temperature was 350 ℃， the gasification temperature was 450 ℃， and the capillary voltage was 3 500 V. RESULTS： The linear range of haw acid，corosolic acid，betula acid，betulonic acid，oleanolic acid and ursolic acid were 0.025-6.4 μg/mL（r≥0.999 6）. The quantitative limits were 5.11， 4.78， 4.42， 4.22， 4.29， 4.51 ng/mL； and detection limits were 1.42， 1.27， 1.30， 1.28， 1.16， 1.22 ng/mL， respectively. RSD of precision test was less than 5％， stability and repeatability tests were all less than 2％（no betulonic acid detected）. The recovery rates were 97.90％-100.55％（RSD＝1.00％，n＝6）， 97.95％-102.95％（RSD＝1.74％，n＝6）， 96.00％-101.20％（RSD＝2.00％，n＝6）， 93.25％-104.20％（RSD＝4.25％，n＝6）， 92.20％-103.30％（RSD＝3.58％，n＝6）， 97.80％-103.50％（RSD＝2.03％，n＝6）， respectively. CONCLUSIONS： The method is accurate， reliable and exclusive， and can be used for simultaneous determination of 6 kinds of triterpene acids in Tibetan medicine R. biflorus.

Study on Quality Standard of Tibetan Medicine Rubus biflorus / 中国药房

OBJECTIVE:To establish the quality standard of Tibetan medicine Rubus biflorus.METHODS:The qualitative identification was conducted from characters characteristics,microscopic characteristics,TLC.The contents of moisture,ash and extract were determined.HPLC method was adopted for content determination of rutin and hyperoside.The determination was performed on Diamonsil C18 column with mobile phase consisted of acetonitrile-0.1％ phosphoric acid solution(18∶82,V/V) at the flow rate of 1.0 mL/min.The detection wavelength was set at 360 nm,column temperature was 30 ℃,and sample size was 10 μL.RESULTS:The surface of the medicinal material was grayish red to grey red brown,with longitudinal wrinkle grooves,and the peel was easy to peel off.The pith of the medicinal material was large,loose,sponge-like.The powder of the medicine was light yellow;wood fiber was bundle or scattered individually;bast fiber was thick.TLC spot of hyperoside was clear and well-separated.The contents of moisture,total ash,acid-insoluble ash,water-soluble extract and ethanol extract were 6.71％-7.55％,1.43％-1.67％,0.41％-0.48％,17.70％-19.31％ and 13.76％-16.99％.The linear range was 3.13-50 μg/mL for rutin (r=0.999 2)and hyperoside (r=0.999 5).The limits of quantitation were 0.5,1.8 μg/mL,and the limits of detection were 0.2,0.9 μg/mL.RSDsof precision,stability and reproducibility tests were all lower than 2.0 ％ (n =6).The recoveries were 92.8 ％-98.8 ％ (RSD =2.25 ％,n=9),94.0％-98.6％ (RSD=l.71％,n=9).CONCLUSIONS:The moisture content,total ash and acid insoluble ash content of medicinal materials is not more than 10.0％,3.0％ and 0.6％,respectively.The total amount of water extract,alcohol extract,rutin and hyperoside is not less than 15.0％,12.0％,1.0 mg/g,respectively.Established standard can be used for quality control of Tibetan medicine R.biflorus.

Clinical characteristics and meteorological factors of patients with chronic heart failure in Qinghai plateau area / 公共卫生与预防医学

Objective To analyze the clinical characteristics and meteorological factors of patients with chronic heart failure (CHF) in Qinghai Plateau area, and to provide a theoretical basis for the diagnosis and treatment of patients with chronic heart failure. Methods A total of 356 patients with CHF admitted to our hospital in Qinghai Plateau area from 2019 to 2020 were selected, and the number of cases, age, gender, comorbidity, NYHA cardiac function classification of CHF patients in Qinghai Plateau area from 2019 to 2020 were counted. To compare NYHA cardiac function classification and underlying diseases in CHF patients at different ages and altitudes in Qinghai Plateau area. The daily average temperature (℃), daily temperature and daily range (℃), daily average wind speed (m/s), daily average air pressure (kPa) and daily average relative humidity (%) in Qinghai Plateau from 2019 to 2020 were collected. According to the general clinical data of patients, the changes of the monthly incidence of CHF patients were analyzed. Pearson correlation analysis was used to compare the correlation between the monthly incidence of CHF patients and meteorological factors, and to explore the influence of meteorological factors on the incidence of CHD in Qinghai Plateau. Results Among the 356 CHF patients, there were 181 males and 175 females, with an average age of (72.47 ±6.49) years old. The NYHA classification of cardiac function was grade II in 103 cases, grade ⅲ in 198 cases, and grade ⅳ in 55 cases. NYHA cardiac function classification increased with age, and there was significant difference in NYHA cardiac function classification among CHF patients of different ages (P<0.05). Among 356 CHF patients, 91 cases (25.56%) were complicated with hypertension, 63 cases (17.70%) were complicated with coronary heart disease, 57 cases (16.01%) were complicated with diabetes, and 40 cases (11.24%) were complicated with hypergenic heart disease. 25 cases (7.02%) were complicated with heart valve disease. Hypertension, coronary heart disease, diabetes mellitus, hypergenic heart disease and valvular heart disease increased with age, and there were statistically significant differences between CHF patients with different ages and underlying diseases (P<0.05). CHF patients in Qinghai Plateau region were mainly concentrated in January-March and October-December. Taking the number of CHD admissions as the dependent variable and each meteorological factor as the independent variable, the correlation between each factor was compared. The results showed that the number of CHF cases was positively correlated with the daily temperature range and daily mean air pressure, and negatively correlated with the average daily temperature (P<0.05). Daily temperature range (OR=2.61) and daily mean air pressure (OR=1.89) were independent risk factors for CHF in Qinghai Plateau area (P<0.05). Conclusion CHF is more prevalent in elderly people in high altitude areas in Qinghai Plateau. For months with high temperature changes, wind speed, we should pay attention to weather changes and take preventive measures in advance.

Study on Quality Standard of Tibetan Medicine Myricaria germanica / 中国药房

OBJECTIVE:To establish the quality standard of Tibetan medicine Myricaria germanica. METHODS:M. germani-ca was identified in respects of properties,microscopic characteristics and TLC. The contents of moisture,ash and extract were de-termined. HPLC method was adopted for content determination of gallic acid. The determination was performed on Diamonsil C18 column with mobile phase consisted of acetonitrile-0.1% phosphoric acid solution(5:95,V/V)at the flow rate of 1.0 mL/min. The detection wavlengths were set at 270 nm. The column temperature was 30 ℃ and the sample size was 10 μL. RESULTS:M. ger-manica was cylindrical in shape,and there were many narrow strip alternate leaflets,which were brittle,easily broken,weak in smell,mild in flavor. Cork cell surface and lower epidermal cell surface showed polygon;most of xylon were bunched;there were many clusters of calcium oxalate. TLC spots were clear and well-separated. The contents of moisture,total ash,acid-insoluble ash, water-soluble extract and ethanol were 6.83%-8.12%,4.01%-5.01%,1.06%-1.98%,17.91%-22.65%,11.29%-15.51%. The linear range of gallic acid were 3.13-50 μg/mL(r=0.9997);RSDs of precision,stability and reproducibility tests were lower than 1.0%;the recoveries were 94.0%-100.8%(RSD=2.27%,n=9). CONCLUSIONS:Established standard can be used for quality evalua-tion of Tibetan medicine M. germanica.

Effects of Tibetan medicineManu-Xitang granules on the expression of prostaglandin E2 and TNF-? on adjuvant arthritis rats / 国际中医中药杂志

Objective To study the effect of Tibetan MedicineManu-Xitanggranules on the expression of prostaglandin E2 and TNF-a on adjuvant arthritis (AA) rats.Methods A total of 60 rats were randomly divided into the control group, the model group, the Aspirin group, the low-, medium- and high- dose Manu-Xitang granules groups (each group with 10). Complete Freund's adjuvant method was used for the adjuvant arthritis model except the control group. All the groups started treatment at 8th day, and the treatment last 20 days. The low-, middle- high-dosage groups were treated with 2.0, 1.0, 0.5g/(kg body weight) Manu-Xitang granules, while the the Aspirin group with ASP (0.27 g/kg), control and medol groups with the equal Volume saline. The body weight, the swelling of primary side of arthritis index were observed. The levels of PGE2 and TNF-α were measured by ELISA.Results At 6th, 12th, 18th day, compared with the model group, the body weights in Aspirin group, the low-, middle-, high-dosage groups significantly increased (P<0.05 or P<0.01); at the 6th day, the foot swelling (0.40% ± 0.18%, 0.50% ± 0.25%, 0.55% ± 0.35%vs. 0.85% ± 0.15%) in the Aspirin group, middle-, and high-dosage groups significantly decreased (P<0.05 or P<0.01). And the PGE2 (0.66 ± 0.31vs. 0.39 ± 0.11) and TNF-a (0.72 ± 0.24vs. 0.50 ± 0.15) in the high- dose group were significantly lower than the model group (P<0.05).Conclusions The Tibetan medicineManu-Xitanggranules could treat AA through the changes of PGE2 and TNF-a.

Study on safety of Tibetan medicine zuotai and preliminary study on clinical safety of its compound dangzuo / 中国中药杂志

Zuotai (gTso thal) is a typical representative of Tibetan medicines containing heavy metals, but there is still lack of modem safety evaluation data so far. In this study, acute toxicity test, sub-acute toxicity test, one-time administration mercury distribution experiment, long-term mercury accumulative toxicity experiment and preliminary study on clinical safety of Compound Dangzuo were conducted in the hope of obtain the medicinal safety data of Zuotai. In the acute toxicity test, half of KM mice given the lethal dose of Zuotai were not died or poisoned, and LD50 was not found. The maximum tolerated dose of Zuotai was 80 g x kg(-1). In the subacute toxicity test, Zuotai could reduce ALT, AST, Crea levels in serums under low dose (13.34 mg x kg(-1) x d(-1)) and medium dose (53.36 mg x kg(-1) x d(-1)), with significant difference under low dose, and increase the levels of ALT, AST, MDA, Crea in serums under high dose (2 000 mg x kg(-1) x d(-1)); besides, the levels of BUN and GSH in serums reduced with the increase in dose of Zuotai, indicating a significant dose-effect relationship. In the one-time administration distribution experiment, the content of mercury in rat kidney, liver and lung increased after the one-time administration with Zuotai, with a significant dose-dependent relationship in kidney. In the long-term mercury accumulative toxicity experiment, KM mice were administered with equivalent doses of Zuotai for 4.5 months and then stopped drug administration for 1.5 months. Since the 2.5th month, they showed significant mercury accumulation in kidney, which gradually reduced after drug withdrawal, without significant change in mercury content in liver, spleen and brain and ALT, AST, TBIL, BUN and Crea in serum. At the 4.5th month after drug administration, KM mice showed slight structural changes in kidney, liver and spleen tissues, and gradually recovered to normal after drug withdrawal. Besides, no significant difference in weight gain was found between the Zuotai group and the control group. According to the findings of the clinical safety study of Dangzuo, after subjects administered Dangzuo under clinical dose for one month, their serum biochemical indicators, blood routine indicators and urine routine indicators showed no significant adverse change. This study proved that traditional Tibetan medicine Zuotai was slightly toxic, with a better safety in clinical combined administration and no adverse effects on bodies under the clinical dose and clinical medication cycle. However, long-term high-dose administration of Zuotai may have a certain effect on kidney.