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12changes will happen in the body, which all need to be further studied. Finally, how does the interaction occur between traditional Chinese medicine and western medicine? What is the mutual effect? After drug enters the body, what kind of change will happen upon two drugs, and what is the influence between them? In the process of disease treatment, by what mechanism does the synergy or antagonistic action happen between them? On these issues I'm afraid all medical research practice can only give limited answer in the future. Although we need very long time to clear these problems, but I believe people will make it clear one day in the future with the deepening of medical studies. If these problems really research clearly, I think traditional Chinese pharmaceutical and western pharmaceutical may appear in one prescription in future clinical treatment, and the former probably won't appear in a traditional drug forms, but in modern drug forms in the prescription. At that time we will truly achieve "a dialectical medicine". [4] References:
[1]. Ni Cheng. Clinical research prospects about rational use of combining traditional Chinese and western pharmaceutical [J]. Chinese Journal of Information On Traditional Chinese Medicine, 1999, (12): 22-23.
[2] Xu Xian-dian. the preliminary report on treating beef tapeworm trough combining Betel nut and quinacrine [J], National Medical Journal of China, 1954, 12 (9) : 715-716.
[3] Yang Yun-song. Several problems need to be considered in combining Traditional Chinese and western pharmaceutical [J]. Harbin Medical Journal, 2012, (4) : 299-300.
[4] Yang Yun-song, discussion and analysis about the guiding ideology's success or failure for combining traditional Chinese and western medicine, [J]. Information On Traditional Chinese Medicine, 2009, 26 (6) : 136-139.
Author introduction: YangYun-Song (1979 - ), male, medical doctor. Main research areas: scientific research design about combination of traditional Chinese and western medicine; theclinical path to control medical miscellaneous disease bycombining traditional Chinese and western medicine.
Correspondence address: No.24#,HepingRoad, Xiang Fang District, Harbin City, Hei Long-jiang Prov.China. Teaching and research section of Medical History, BasicMedicalSchool, Heilong-jiangUniversity of Traditional Chinese Medicine. Zip code: 150040. Telephone: 13624505169
Preparation of Cnidium monnieri Emulsion and Investigation of itsin vitro Antitrichomonas Vaginalis
YANG Jing, QIU Jin-shuang, JIANG Ji-ming, WANG Ya-xian, WANG Rui
HeilongjiangUniversity Of Chinese Medicine,Harbin 150040,China
Abstracts Objective:To prepare o/w type emulsion with total coumarin from Cnidium monnieri, and investigate its in vitro anti-trichomonas vaginalis activity.Methods:Total coumarin from C. monnieri was extracted by ultrasonic method with emulsification time,times and pressure,adding order of emulsifier as factors,single factor test was used to optimize preparation technology of C. monnieri emulsion,and its pharmacodynamic trial of in vitro anti-trichomonas vaginalis was investigated. Result:Preparation technology of C. monnieri emulsion was ascertained as follows: emulsification 3 times at 150 MPa,15 min each time,adding order of emulsifier with oil phase added to aqueous phase;and this emulsion had good inhibition on trichomonas vaginalis.Conclusion:This preparation technology was stable and feasible,minimum drug
concentration of this emulsion had good inhibition effect to trichomonas vaginalis was 0.15-0.62 gL-1.
Key words Cnidium monnieri; Emulsion; Anti-Trichomonas vaginalis
Trichomonas vaginitis is one of the common diseases of gynaecology,happens in women of 20~40 years old [1] .Cnidium monnieri has warming the kidney,removing the wind, eliminating dampness and insecticidal efficacy, and treats impotence,vaginal itching,etc.Studies have found that its chemical composition is complicated, mainly including volatile oil and total coumarin from C. monnieri (TCFC) [2]. Total coumarin from C. monnieri was extracted by ultrasonic method and prepared after the o/w type emulsion in this paper.Our purpose is to provide experiment basis for the development of C. monnieri through investigating its in vitro anti-trichomonas vaginalis activity.
1 Equipment and Materials
1.1 Equipment Waters-2695 HPLC;KQ-250B-type Ultrasonic Cleaner(Kun Shan Ultrasonic Instruments Co., Ltd.);APV-type High Pressure Homogenizer;TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory).
1.2 material (purchased from Harbin Pharmaceutical Group Shiyitang Chinese Herbal Slices Co., Ltd., and the results of its tests were in conformity with the standards of Chinese Pharmacopoeia(2010 Edition).);reference substance (the National Institute for the Control of Pharmaceutical and Biological Products,lot number: 201004); Methanol (DIKMA); Activated carbon(China National Chemical Reagent Co., Ltd.); Trichomonad culture (Heilongjiang University of Chinese Medicine Experiment Center); Metronidazole (Hongda Pharmaceutical Co., Ltd.); Methylene blue (Tianjin Kemiou Chemical Reagent Co., Ltd.).
2 Methods and Results
2.1 Study on preparation of Cnidium monnieri extract[3-5]
50g of Cnidium monnieri, grinding, adding 95% ethanol 100ml, power 360w conditions under ultrasonic reflux extraction 1.5h, residue extraction was repeated twice. The combined extracts were concentrated under reduced pressure for recovering ethanol, 1.25% active carbon was added in the sample solution, filtration, 70°C vacuum drying to constant weight, that is the Total coumarins of Fructus Cnidii
2.2 single factor investigated to determine the emulsion preparation process
2.2.1 establishment of the method
Emulsion was added sequentially:under high-speed stirring, the water phase was added to the oil phase method and the oil phase was added to the water phase two processes separately prepared into colostrum,then transferred to the centrifuge, 4000r/min centrifugal 15min observed after colostrum stratification and floating oil.
The emulsification time study:high agitation, start the time after its preparation in colostrum, stirring 5 min respectively, 10 min, 15 min.Were transferred to the centrifuge to 4000r/min, centrifugal 15min, observed the colostrum stratification and floating oil to examine different emulsification time colostrum stability.
Emulsification pressure study: in the 1000,1500,2000 bar pressure level, prepared colostrum, go to the centrifuge, 4000r/min, centrifuged for 15 min was observed after the colostrum stratification and the floating oil, study different pressure emulsion impact of colostrum stability.
Emulsified number of visits: emulsification process in the preparation of colostrum, set the number of homogeneous 1,3,5, each interval of 3 minutes. transferred to the centrifuge to 4000r/min, centrifugal 15min observed after the colostrum stratification and floating oil, to inspection of different emulsion frequency colostrum stability.
2.2.2 Experimental Results
The test results are shown in Table 1. The test shows that the sequence of adding slightly influence the stability of the emulsion.In order to guarantee the quality and stability,water phase was added to the oil phase. Emulsifying time within a certain range, the longer is conducive to the
stability of the colostrum, 10min and 15 min were not stratified, but floating oil situation, better for a long time, so choose the emulsifying time for 15min. Emulsification pressure analysis result shows that using 1500bar. The stability of the emulsion was positively correlated with the emulsion frequency,but considering more single emulsification time is shorter, is not conducive to the preparation of the comprehensive consideration given 3 times.
Table 1 single factor experiment method to determine the process parameters
Factor level hierarchical oil droplets
Order of adding Oil to water no stratification occasional floating
Water to oil no stratification No floating
Emulsification time 5min no stratification More floating
10min no stratification Little floating
15min no stratification No floating
Emulsification 1000bar Stratification occasional floating
pressure 1500bar no stratification No floating
2000bar no stratification No floating
Emulsification 1 time no stratification Occasional floating
frequency 3 times no stratification No floating
5 times no stratification No floating
To sum up: take sodium saccharin was added to distilled water to dissolve. Take the tragacanth, arabic gum 10g each, made into mucilage. Put the extract 20g, glue slurry, syrup liquid and about 1000ml distilled water into the high-pressure homogenization machine according to the process
parameters are made from cnidium monnieri o/w emulsions. The process parameters are shown in Table 2.
Table 2 cnidium monnieri emulsion parameters
emulsification Emulsification Emulsification Order of adding
time(min) pressure (bar) frequency
parameters 15 1500 3 Oil to water
2.3 The quality of the emulsion
2.3.1 Cnidium monnieri element content in emulsion method
Chromatographic conditions: Column DiamonsilC18(5p,m,250mm*4.6mm);mobile phase: methanol-water(80:20);column temperature:30°C;flow rate: 1mL/min; measurement wavelength: 323nm. Figure 1.
_tj_
■ — i - ■ ■ i ■ ■ ■ i ■ ■ i ■ ■—r^-■—i - ■ - i -— 2.DO 4.1X1 &.00 8.0C 10.00 12.00 14.00 It 00
Figure 1 osthol standard HPLC
The preparation of standard curve:reference substance 1.0 mg, 25 mL volumetric flask, made with the capacity and the methanol concentration is 40.0p,g/mL standard stock solution. Precision, respectively, from the standard stock solution 0.1, 0.5, 1, 1.5, 2, 2.5 mL, were put in 5 mL volumetric flask, dilute methanol to scale, shake well, with 0.22^m microporous membrane filter. Precision from the filtrate 20p,L, in turn into the highly effective liquid phase color spectrometer, measuring the peak area. By peak area (A) as the ordinate, the concentration (C) as the abscissa, osthol linear regression equation: A=32999C-4632.7, R =0.9994, showed that osthol concentration in the range 0.8~20.0^gmL - 1 has good linear relationship.
Precision test: precision drawing of osthole test solution 20^L,6 replicate injections in the chromatographic conditions,RSD = 1.45% .Precision is good.
Recovery and stability test:Precision according to take a certain amount of reference substance, added to the known concentration of osthole in the solution, in the above chromatography conditions were determined. Experiment results show that the method is of high accuracy, in line with the requirements. Another 20p,L of test sample solution precision from osthole 20^L, at 0, 6, 12, 18, 24 h was measured.The results showed that the reference substance solution is stable within 24 h.
Emulsion samples determination: draw about 0.1mL emulsion into 50mL volumetric flask, with methanol after ultrasonic constant volume, then 0.22p,m microporous filter.Precision from the filtrate 20^L, into the High performance liquid chromatography, measuring the peak area and calculate the content.
2.3.2 Determination of the emulsion type
With glue dropper drain emulsion 1 ~ 2 drops, coated on glass slide, methylene blue solution and in the microscopic observation, if only foreign minister dyeing, for o/w type emulsion.
2.3.3 Quality Determination of stability
Emulsion into the centrifuge, 4000r/min, centrifugal 15min, Observe whether stratified, if stratified argues that its quality is not stable.
2.3.4 Emulsion quality determination results
According to the optimum process,Preparation of osthol emulsion. measured at the method for determination of 2.3, the result shows that: Osthol concentration was 12.36 ± 0.13gL-1. Emulsion of methylene blue staining for type identification, When foreign minister is blue, while the inner phase is a transparent circular bright spot, can be determined which is the o/w type emulsion. So 4000r/min centrifugal 15min no stratification and oil droplets phenomenon, quality stable and reliable.
2.4 Cnidium monnieri emulsion anti-Trichomonas vaginalis test in vitro
2.4.1 Create test method
The liquid preparation: take emulsion 10ml, measured and calculated concentration of the drug-containing and set aside. Take metronidazole 20mg diluted with water volume to 10 mL volumetric flask, get the content of 2 gL-1 liquid.
Preparation of specimen Cultured liquid: Put Trichomonad culture in the autoclave sterilization 30 min,then add sterile bovine serum according to culture broth: sterile bovine serum = 4:1, penicillin and streptomycin were added again.All dubbed the concentration of the drug-containing specimens 0.1gL-1 culture medium was used to inhibit the breeding of bacteria other than Trichomonas vaginalis [3].
Specimens culture: Take the vaginal secretions of patients,examine under the endoscopic to confirme the Trichomonas vaginalis, added to the specimen culture 48 h at 37°C, take the culture fluid with a white blood cell counter counting under the microscope.According to the count results, formulate it into Trichomonas atoms containing 105 / mL of solution.
Test Method: Take no osthol gum arabic and tragacanth solution as the blank solution. Take Trichomonas solution without drugs as the negative solution.0.1mL solution,drawn from the emulsion and metronidazole respectively,were added to the specimens liquid of 0.9 mL. The trichomoniasis liquid of 105 cells/mL 0.1ml were added to each solution, and incubated at 37°Cafter 24 hours.Remove the culture medium and observe results under the microscope.When culture medium had no Trichomonas,the minimum concentration of the drug is its inhibition of the concentration of Trichomonas.
2.4.2 minimum inhibitory concentration measurement results in vitro
According to the method of 2.4.1, the experiment results are shown in Table 3.
Experimental results show that the the osthol emulsion of 10 clinical isolated Trichomonas
vaginalis have a better inhibition. The lowest drug concentration of inhibiting the growth of Trichomonas is 0.15 ~ 0.62gL-1.
Table 3 minimum inhibitory concentration of Trichomonas measurement result in vitro (gL-1)
1 2 3 4 5 6 7 8 9 10
Metronidazole 0.05 0.025 0.1 0.1 0.05 0.0125 0.025 0.05 0.0125 0.05
Osthol 0.309 0.154 0.618 0.618 0.618 0.154 0.309 0.618 0.154 0.618
emulsion
Blank control + + + + + + + + + +
group
Negtive + + + + + + + + + +
control group
3 Discussion
The process of the Osthol fat-soluble extracts ,made of oil-in-water emulsion, is simple, stable and reliable. Not only it can effectively improve its compliance and appropriate to use in patients, but also by inhibition of trichomoniasis test in vitro, it can inhibit Trichomonas vaginalis. References
[1] Zhang Li-juan, Sun Bao-qing, Tan Gui-lian MTT method determination of osthole inclusion complex role in the killing of Trichomonas vaginalis [J]. .2011.33 Of proprietary Chinese medicines (12):2163-2165
[2] Gong Zhu-yun, Zhang Gao-Yong, Nie Yong-liang, et al. Supercritical CO2 extraction process Cnidium extract the material extraction rate of [J]. .2006.28 Of proprietary Chinese medicines (8) :1102-1106.
[3] Lin Xiu-xian, Li Jing, Zhang Shu-hua, et al. The Kochia scoparia supercritical CO2 extract antiTrichomonas vaginalis pharmacodynamic studies [J] Chinese herbal medicines .2005.28 (1) :44-45
[4] Wu Yan-fang, Wang Xin-sheng, Yin Yan-yan ultrasonic assisted extraction the the forsythia Total Flavonoids preferred [J]. Chinese Journal of Experimental Traditional Medical Formulae .2011.17 (14) :30-32
[5] Su Xiu-fang, Qin Jian-mei ultrasound-assisted extraction process of total flavonoids cochinchinenin roots [J] Chinese Journal of Experimental Traditional Medical Formulae .2011.17 (10) :97-100
Effects of SHENLIJIA Granule on Tie2 expression in heart failure rats screened
by protein chips
Jiang Deyou,Liu Dezhu,Pang Zuowei,Chang Jiayi(corresponding author)
JIN GUI Section of Heilongjiang University of Chinese Medicine,Harbin,150040,China
Abstracts:Objective:To assess the effects of SHENLIJIA Granule on Tie2 expression in heart failure rats by protein chips.Methods: Selected fifteen rats randomly into blank group from total of 70 Wistar rats.The others were induced by intraperitoneal injection of doxorubicin,and divided into model group and treatment group randomly after 6 weeks.After the model establishment, the rats have been fed for 28 days.Meanwhile,the rats in the treatment group were treated with gastric perfusion of Chinese herb SHENLIJIA Granule once a day for twenty days.And those in the blank and modle group were treated with gastric perfusion saline.Four weeks later,cut out specimens from the myocardium of left ventricle for HE staining and protein microarray analysis.Results:HE staining displayed that the blank group was normal;the myocardium of the model group was coarse,the interstitial substance was proliferative andthere were a few inflammatory cells infiltration;the treatment group had no obvious proliferation or inflammatory cells infiltration.The protein microarray analysis indicated that the Tie2 expressions in those three groups were different.Compared with blank group,the Tie2 expression of model group was decreased,while that of treatment group was increased.There was nodifference between treatment group and blank
