Protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis

OBJECTIVE To explore the protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis.METHODS INS-1cells were cultured in media containing 3n M STZ and different doses of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja.The proliferation of cells was examined by MTT assay,ROS content were detected by fluorescence enzyme label.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),hydrogen peroxidase(CAT),malondialdehyde(MDA)were also measured by colorimetry.The activities of caspase-3,9 were also observed by Caspase colorimetric assay kit in each group.The expressions of Bcl-2 m RNA and Bax m RNA were detected by Real time PCR,protein expression of LC3-Ⅱand PARP were detected by Western blotting.RESULTS Compared with the control group,total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja could promote the proliferation of INS-1 cells no matter with STZ or not when its concentration lower than 25μg·m L-1;but when its concentration higher than 100μg·m L-(1use individually)or 50μg·m L-1(combined use),total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja might significantly inhibite the growth of the cells whether STZ existed or not.Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja(6.25,12.5,25μg·m L-1)might inhibit INS-1cell apoptosis,decrease intra-cellular ROS contents,improve the s upernatant liquid SOD,GSH-PX,CAT activities,decrease MDA level,promote INS-1 cell secreting insulin,decrease the protein expressions of autophagy protein LC3Ⅱand apoptosis regulating protein cleaved PARP protein,up-regulate the anti-apoptotic protein Bcl-2 m RNA expression,down-regulate the pro-apoptotic protein Bax m RNA expression and Bcl-2and Bax,reduce the activity of caspase-9 and caspase-3(P<0.05 or P<0.01).CONCLUSION Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja has good protective effect on STZ-stimulated INS-1cells.It can inhibit STZ injured INS-1 cells to overproduce ROS production,enhance endogenous antioxidant enzymes(GSH-Px,SOD,CAT activities),reduce the expression of autophagy protein LC3Ⅱand apoptosisregulating protein cleaved PARP,up-reglute the antiapoptosis protein Bcl-2 expression,down-regulate the pro-apoptotic protein Bax expression,decrease the caspase-9 and caspase-3 activities,and improve the INS-1cell survival rate,and then play a protective effect on damaged INS-1 cells.

THE MECHANISM OF ANTITUMOR ACTIVITY OF TOTAL GLUCOSIDES EXTRACTED FROM CYNANCHUM AURICULA-TUM ROYLE (CA)

Since the chemical structure of total glucosides from Cynanchum Auriculatum Royle (CA) is similar to that of the steroline of Marsdenia Condurago Reich, a compound which exhibits antitumor activity, research into the antitumor activity of CA was carried out. Its mechanism of action was studied in vivo with C 57BL/6 mice bearing Lewis lung carcinoma and in vitro, with two mouse tumor cell lines: S180 and EAC. CA inhibited to a certain extent the growth of subcutaneously inoculated Lewis lung carcinoma and its pulmonary metastasis, and augmented the antitumor effect of cyclophosphamide. It showed a killing effect on the EAC and S180 tumor cells of mice in vitro as well. It blocked the tumor cells of solid Lewis lung carcinoma from entering into the S stage from G1 and inhibited DNA synthesis of S180 and EAC tumor cells of mice in vitro. It also markedly increased the number of mononuclear Mφ of tumor bearing mice, stimulated the macrophagic activity of their intraperitoneal Mφ, raised the percentage of ANAE(+) lymphocytes in peripheral blood and enhanced the ABC reaction and antibody formation in tumor bearing mice.

The Optimization of Ultrasonic Extraction Technology of Total Flavonoids in Leaves of Mallotus apelta by Response Surface Analysis Methodology

[Objectives] Response surface analysis methodology was used to optimize the extraction technology of total flavonoids from Mallotus apelta leaves. [Methods] The total flavonoids were extracted using ultrasonic assisted ethanol extraction,and extraction rate of total flavonoids was taken as evaluation index. On the basis of single-factor experiment,Box-Behnken response surface methodology was employed for selecting the optimum extraction process. [Results]The optimum extraction conditions of total flavonoids were as follows: 75% of ethanol concentration,1∶ 25 of ratio of material to liquid,31 min of ultrasonic time. Under these conditions,the extraction rate of total flavonoids was 1. 115%.[Conclusions] The extraction process obtained by response surface methodology was stable,reasonable,accurate and reliable. It was a feasible method to extract the total flavonoids from M. apelta leaves.

Extraction Process and Content Determination of Total Flavonoids in Ginkgo( Ginkgo biloba L. ) Leaves

[Objectives]This study was conducted to study the extraction process and the content determination of flavonoids in ginkgo( Ginkgo biloba L.) leaves.[Methods]Ethanol extraction and methanol extraction of total flavonoids in ginkgo leaves were studied,and the optimal extraction conditions for flavonoids were determined by orthogonal test; and with quercetin as reference substance,total flavonoid content in ginkgo leaves was determined by UV spectrophotometry.[Results]The optimal extraction process was 4 h of Soxhlet extraction with methanol; and the total flavonoid contents had a good linear relation in the range of 0. 006 5-0. 039 mg/ml( R^2= 0. 999 9),the average content was stabilized at 1. 135%,and the average recovery of the method was 102. 0%. [Conclusions]This study selected the optimal extraction process for total flavonoids in ginkgo leaves. The test method is simple with high accuracy and precision,and is suitable for the extraction and determination of total flavonoids in ginkgo leaves.

Extraction and separation of total flavonoids from Flos Puerariae and its antioxidant activity

Objective To study the optimum extraction and separation process of total flavonoids in the flowers of Flos Puerariae and its antioxidative activity.Methods The total flavonoids were extracted with assistance of ultrasonic wave and the content was determined at 265 nm wavelength by Spectrophotometric method.Orthogonal experiment L9(34)was carried out to investigate the effects of concentration of solvent,the ratio of material to liquid,time length of extraction,and frequency of extraction on extraction results of total flavonoids from Flos Puerariae.The extracted solution was purified by petroleum ether and ethanol sequently.Under these conditions,the total flavonoids was eluted gradiently with mixed mobile phase of methanol-chloroform solution in the silica gel column system,and then determined by UV scanning and HPLC.Fenton reaction was used to produce and detect hydroxyl radicals(·OH),and pyrogallol system was used to produce and detect the superoxide radical anion(O2-·).Results The optimum conditions were as follows;using 40%(V/V)methanol as extractor with the ratio of material to liquid at 1∶30,and extracting for 2.5 h a time for 3 times.The extraction yield of total flavonoids was 13.6%.Six isoflavone compounds were isolated from the flowers of Flos Puerariae by the method of silica gel column chromatography.Antioxidative test results showed good performance of flavonoid scavenging capacity in both hydroxyl radical system and superoxide radical system and its IC50 was 7.65 μg·mL-1 and 0.18 mg·mL-1,respectively.Conclusions This study provided scientific basis for further development and utilization of Flos Puerariae and make preparation for later pharmacological research.

Optimization of Extraction Process of Clerodendrum philippinum Schauer var. simplex Mlodenke Total Flavonoids(CPTF) by Central Composite Design-Response Surface Methodology

[Objectives] The research aimed to optimize extraction process of Clerodendrum philippinum Schauer var. simplex Mlodenke total flavonoids( CPTF),and provide reference for its development and utilization. [Methods] Based on single-factor test,ethanol concentration,extraction temperature and extraction time were taken as independent variables,and total flavonoids yield was taken as dependent variable. The test was conducted according to central composite design principle. Multivariate linear regression and binomial equation fitting of the result were conducted,and extraction process of CPTF was optimized by using response surface methodology. [Results]The optimal extraction process of CPTF was as below: ethanol concentration 54. 76%,extraction temperature 83. 92℃,extraction time 102. 64 min,solid-liquid ratio 1:20,extraction for twice. [Conclusions] The extraction process of CPTF by central composite design-response surface methodology was simple and feasible,with reliable prediction result,which was suitable for industrial production.

The Optimization of Ultrasonic Extraction Process of Total Flavonoids from Rhodiola Crenulata by Response Surface Method

[Objectives]To study the optimal extraction process of total flavonoids in Rhodiola rosea L.,and further facilitate the development and utilization of Rhodiola rosea L. [Methods]With Rhodiola crenulata as raw material,ethanol as extractant,ultrasonic extraction as the extraction method,the single factor method was first used for preliminary investigation of effect of ethanol volume fraction,solid-liquid ratio,extraction temperature and extraction time on the flavonoids extraction rate,and then the Box-Behnken response surface method was used to optimize the extraction process of total flavonoids in R. crenulata. [Results] The optimal extraction conditions: ethanol concentration of 72%;solid-liquid ratio of 1∶ 43; extraction temperature of 66℃; extraction time of 50 min. Under these conditions,the extraction rate of total flavonoids from R. crenulata was 2. 591%. [Conclusions] The results showed that the method of response surface was reasonable and feasible for the optimization of ultrasonic extraction of total flavonoids from R. crenulata.

Study on Ultrasonic Extraction of Total Flavonoids from Angelica keiskei Koidzumi

[Objective] This study aimed to optimize the ultrasonic extraction process of total flavonoids from Angelica keiskei Koidzumi. [Method] Using the yield of total flavonoids as an indicator, the ultrasonic extraction process of total flavonoids from A. keiskei was optimized by orthogonal experimental design. [ Result ] Among four factors in orthogonal experimental design, extraction time exhibited the most significant effect on the extraction result; ethanol concentration exhib- ited a significant effect on the extraction result; ultrasonic power and solid-to-liquid ratio had no significant effect. The optimal conditions for ultrasonic extraction of total flavonoids from A. keiskei were extraction time of 25 min, ethanol concentration of 80%, ultrasonic power of 60 W, solid-to-liquid ratio of 1：20 （g/ml）, under which the yield of total flavonoids reached 1.56%. [ Conclusion] This study provides the basis for further development and utilization of total flavonoids from A. keiskei.

Study on Extraction Technology of Total Flavonoids from Vitex rotundifolia

[ Objective ] This study aimed to establish the optimal extraction technology of total flavonoids from Vitex rotundifolia and provide a theoretical basis for the utilization and development of V. rotundifolia. [ Method ] With the extraction temperature, extraction time, solid-liquid ratio and ethanol concentration as various factors, the content of total flavonoids as an indicator, the extraction technology of total flavonoids from V. rotundifolia was optimized by orthogonal experiment. [ Result] The optimal combination of extraction conditions was extraction temperature 70℃, extraction time 1 h, solid-liquid ratio 1： 12, ethanol concentration 70%. [Condusion] The optimized extraction technology was simple and efficient for extracting total flavonoids from V. rotundifolia.

In vitro total phenolics,flavonoids contents and antioxidant activity of essential oil,various organic extracts from the leaves of tropical medicinal plant Tetrastigma from Sabah

Objective:To detect the in vitro total phenolics,flavonoids contents and antioxidant activity of essential oil,various organic extracts from the leaves of tropical medicinal plant Tetrastigma from Sabah.Methods:The dry powder leaves of Tetrastigma were extracted with different organic solvent such as hexane.ethyl acetate,chloroform,butanol and aqueous methanol.The total phenolic and total flavonoids contents of the essential oil and various organic extracts such as hexane,ethyl acetate,chloroform,butanol and aqueous ethanol were determined by Folin-Ciocalteu method and the assayed antioxidant activity was determined in vitro models such as antioxidant capacity by radical scavenging activity usingα,α-diphenyl-β-picrylhydrazyl (DPPH) method.Results:The total phenolic contents of the essential oil and different extracts as gallic acid equivalents were found to be highest in methanol extract(386.22 mg/g) followed by ethyl acetate(190.89 mg/g).chloroform(175.89 mg/g).hexane(173.44 mg/g).and Imtanol extract (131.72 mg/g) and the phenolic contents not detected in essential oil.The antioxidant capacity of the essential oil and different extracts as ascorbic acid standard was in the order of methanol extract】ethyl acetate extract】chloroform】butanol】hexane extract also the antioxidant activity was not detected in essential oil.Conclusions:The findings show that the extent of antioxidant activity of the essential oil and all extracts are in accordance with the amount of phenolics present in that extract.Leaves of Tetrastigma being rich in phenolics may provide a good source of antioxidant.

Optimization on Ultrasonic Extraction Method of Total Flavonoids for Liriodendron chinense Sarg.×L.tulipifera L. by Response Surface Methodology (RSM)

With the leaves,bark and roots of Liriodendron chinense Sarg.× L.tulipifera L.as experiment materials,the ultrasonic-assisted extraction conditions of total flavonoids were optimized by response surface Box-Behnken test design.Ultrasonic time and liquid-to-solid ratio had significant effects on the extraction amount of flavonoids from the leaves of L.chinense Sarg.× L.tulipifera L.;ultrasonic temperature and liquid-to-solid ratio had an interaction on the extraction amount of flavonoids;and the optimum ultrasonic time,temperature and liquid-to-solid ratio for the extraction of flavonoids from leaves of L.chinense Sarg.× L.tulipifera L.were 19.82 min,28.60 ℃ and 9.48 ml/g,respectively.Ultrasonic time,temperature and liquid-to-solid ratio had significant effects on the extraction amount of flavonoids from L.chinense Sarg.× L.tulipifera L.;there was a significant interaction between any two of the factors,which had a significant effect on the extraction of flavonoids;and the optimum extraction parameters for flavonoids in the bark were the ultrasonic time of 33.66 min,the ultrasonic temperature at 32.85 ℃ and the liquid-to-solid ratio of 11.39 ml/g.Ultrasonic time and liquid-to-solid ratio had significant effects on the extraction of flavonoids from roots;there was a significant interaction between ultrasonic time and liquid-to-solid ratio and between ultrasonic temperature and liquid-to-solid ratio;and the optimum extraction parameters for flavonoids from the roots of L.chinense Sarg.× L.tulipifera L.were the ultrasonic time of 32.38 min,the ultrasonic temperature at 25.0 ℃ and the liquid-to-solid ratio of 8.00 ml/g.The results of the three models were stable.The models are feasible and have good application value.

Optimization of Extraction Process of Total Flavonoids from Akebia trifoliata (Thunb.) Koidz. by Central Composite Design-Response Surface Methodology

[Objectives]To optimize extraction process of total flavonoids from Akebia trifoliata( Thunb.) Koidz.,so as to provide references for development and use of Akebia trifoliata( Thunb.) Koidz. [Methods]The extraction rate of total flavonoids of Akebia trifoliata( Thunb.)Koidz. was taken as observation indicator. On the basis of single factor experiment,central composite design( CCD) was used to evaluate the effects of the extraction temperature,extraction time,and ethanol concentration on the extraction process. Multiple linear regression and binomial fitting were used,and response surface methodology( RSM) was used to select the optimum extraction process. [Results] The optimum extraction process conditions for total flavonoids of Akebia trifoliata( Thunb.) Koidz. was extraction temperature: 83. 92 ℃; extraction time:96. 47 min; ethanol concentration: 63. 92%; extraction times: two times; solid to liquid ratio: 1 ∶ 20; extraction rate of total flavonoids:4. 55%. [Conclusions] The central composite design-response surface methodology( CCD-RSM) is simple,convenient,and feasible for extraction of total flavonoids from Akebia trifoliata( Thunb.) Koidz.,and the prediction results are reliable.

A Comparative Study on Extraction Methods of Total RNA from Leaves of Acer truncatum ‘Luhong No.1’

Leaves of Acer truncatum ＇ Luhong No. 1 ＇ contain large amounts of polysaccharides and polyphenols, which seriously affect extraction yield and quality of total RNA. In order to explore the appropriate total RNA extraction method, total RNA was extracted from leaves of A. truncatum ＇ Luhong No. 1 ＇ with three methods, including kit method, Trizol method and modified CTAB method. The results showed that ODE6o/OD2so and OD^o/OD2ao ratios of total RNA extracted from leaves of A. truncatum ＇ Luhong No. 1 ＇ with kit method were higher than 1.8, with a general yield and certain level of DNA contamination ; OD^o/OD~ and OD^o/OD^o ratios of total RNA extracted with Trizol method were about 1.5, with the lowest yield; OD260/OD280 and OD260/OD230 ratios of total RNA extracted with modified CTAB method were about 2.0, with the highest yield and distinct eleetrophoresis patterns. The results demonstrated that total RNA extracted with modified CTAB method exhibited high yield and purity, which could meet the demands of subsequent molecular biology research. Thus, modified CTAB method is the appro- oriate method for extracting total RNA from leaves of A. truncatum ＇ Luhong No. 1 ＇

Laparoscopic total mesorectal excision with natural orifice specimen extraction

AIM:To introduce transvaginal or transanal specimen extraction in laparoscopic total mesorectal excision surgery to avoid an abdominal incision. METHODS:Between January 2009 and December 2011,21 patients with rectal cancer underwent laparoscopic radical resection and the specimen was retrieved by two different ways:transvaginal or transanal rectal removal.Transvaginal specimen extraction approach was strictly limited to elderly post-menopausal women who need hysterectomy.Patients aged between 30 and 80 years,with a body mass index of less than 30 kg/m2, underwent elective surgery.The surgical technique and the outcomes related to the specimen extraction,such as duration of surgery,length of hospital stay,and the complications were retrospectively reviewed. RESULTS:Laparoscopic resection using a natural orifice removal approach was successful in all of the 21 patients.Median operating time was 185 min(range,122-260 min)and the estimated blood loss was 48 mL. The mean length of hospital stay was 7.5 d(range,2-11 d).One patient developed postoperative ileus and had an extended hospital stay.The patient complained of minimal pain.There were no postoperative complications or surgery-associated death.The mean size of the lesion was 2.8 cm(range,1.8-6.0 cm),and the mean number of lymph nodes harvested was 18.7(range, 8-27).At a mean follow-up of 20.6 mo(range,10-37 mo),there were no functional disorders associated with the transvaginal and transanal specimen extraction. CONCLUSION:Transvaginal or transanal extraction in L-TME is a safe and effective procedure.Natural orifice specimen extraction can avoid the abdominal wall incision and its potential complications.

Extraction Technology of Total Flavonoids from Pueraria lobata (Willd.) Ohwi and Content Determination of Pueraria lobata (Willd.) Ohwi at Different Altitudes

[Objectives]To optimize the extraction technology of total flavonoids from Pueraria lobata( Willd.) Ohwi. [Methods]The ultraviolet-visible spectrophotometry was used to determine the content of total flavonoids in Pueraria lobata( Willd.) Ohwi. With the puerarin as index,the reflux extraction and single factor test were employed to investigate the effects of temperature,time,ethanol concentration and solid-liquid ratio on the content of total flavonoids in Pueraria lobata( Willd.) Ohwi,respectively. Under the optimal extraction technology,the content of total flavonoids in Pueraria lobata( Willd.) Ohwi at different altitudes was determined.[Results] The optimum extraction process was as follows: 70%ethanol; solid-liquid ratio of 1∶ 30; 1 h reflux extraction. Under these conditions,the extraction rate of flavonoids in Pueraria lobata( Willd.) Ohwi was 11. 48%,the total flavonoids content of different kudzu parts was in the order of roots > stems > leaves,and the total flavonoids content of the sample at about an altitude of 1000 m was significantly higher than at the altitudes of 1400 m and 1700 m.[Conclusions]It was suggested that the Pueraria lobata( Willd.) Ohwi should not be cultivated as medicinal plant in too high mountains,and the stems and leaves of Pueraria lobata( Willd.) Ohwi could be used as raw materials for extracting total flavonoids.

Optimization of Extraction Process and Content Determination of Total Flavonoids from Sanguisorbae Radix and Carbonized Sanguisorba Root by Orthogonal Experimental Design

[Objectives] To optimize the extraction process of total flavonoids from Sanguisorbae Radix and carbonized Sanguisorba root,compare quality of different batches of Sanguisorbae Radix,study the effects of processing on the content of flavonoids,and provide scientific basis for reasonable utilization of Sanguisorbae Radix. [Methods] Test samples were prepared by heating,refluxing,and extraction,the extraction process was optimized by orthogonal experiment design,color was developed by NaNO_2-Al( NO_3)3-NaOH,and total flavonoids were measured by UV method at the wavelength of 510 nm. [Results] The linear relationship of rutin was excellent in the concentration range of 0. 1248 mg/mL-0. 5712 mg/mL,R^2= 0. 9997; the average recovery was 99. 67% and the RSD was 0. 70%. The optimum extraction conditions were as follows: the volume fraction of ethanol was 50%,the extraction temperature was 90℃,the extraction time was 90 min,and the solid-to-liquid ratio was 1∶ 20( g/mL). [Conclusions] After optimization of the extraction process,the extraction rate of total flavonoids in samples of Sanguisorbae Radix was significantly increased; there was certain difference in the content of total flavonoids between different batches of Sanguisorbae Radix and processed products; the total flavonoids significantly declines in carbonized sanguisorba root,and the influence of processing on its curative effect was to be further studied.

Extraction of Antioxidant Total Phenol from Lees of Xiaoqu Spirits

By the Soxhlet extraction, ethanol is used as extraction solvent of lees to determinate the total phenol content of Xiaoqu spirits lees. Folin-phenol method is handled to extract and to determine the reducing ability of extraction. The total phenolic content in the most suitable condition is determinated by the Folin-phenol method and the optimum extraction technological condition of total phenol is determinated by the experiment. The experimental results show that the optimization conditions of the determination methods of total phenolic content are determined by 3.5 mL for the Folin phenol reagent, 1:1 for the 10% Na2CO3 ratio and 30 min for the chromogenic reaction at room temperature. The optimum extraction technological condition of total phenol from lees is as following: solid-liquid ratio 1:12, ethanol concentration 100%, extraction temperature 80℃, extraction time 10 h.

Optimization of Extraction Process of Total Flavonoids and Chlorogenic Acid from Flos Lonicerae by Orthogonal Experiment

[Objectives]To explore the process of simultaneous extracting total flavonoids and chlorogenic acid from Flos Lonicerae by pressure assisted extraction.[Methods]Based on the single factor experiment,orthogonal experiments were carried out to investigate the effects of solvent concentration,solid-to-liquid ratio,extraction pressure,extraction time and extraction temperature on the extraction rate of total flavonoids and chlorogenic acid.[Results]The results showed that the extraction pressure had a significant effect on the extraction of two kinds of chemical constituents from F.Lonicerae.The optimum process conditions were as follows:solvent concentration was 60%,solid-liquid ratio was 1∶20(g/mL),extraction pressure was 2 MPa,extraction time was 30 min,extraction temperature was 70℃.The process conditions are stable,and the extraction efficiency is higher than that of ultrasonic assisted extraction method.Under the test conditions,the extraction rates of total flavonoids and chlorogenic acids were respectively 15.66%and 3.89%.[Conclusions]This study provides a new theoretical basis for the development and utilization of F.Lonicerae.

Optimization of Ultrasonic Extraction Process of Total Flavonoids from Mulberry Leaves by Orthogonal Experiment

In this study, by using mulberry leaves as raw materials, ultrasonic extraction process of total flavonoids from mulberry leaves was optimized by orthogonal experiment. Results showed that the maximum yield of total flavonoids （6.1% ） was obtained from mulberry leaves with addition of 45 times the volume of water by ultrasonic extraction at 55 ℃ for three times, 55 min each. According to the verification test, the optimized technical process was reasonable and feasible with high stability.

Development of an Efficient Method for Extracting Total DNA of Intestinal Microflora

[ Objective] The aim was to develop a fast and effective DNA extraction method of intestinal microflora, a modified method of chloroform extraction, and to provide the basis for quantitative and qualitative detection. [ Method] Through the improvement of conventional DNA extraction method, a rapid and efficient DNA extraction method was developed. Compared with the real-time PCR result of control sample and the result of QIAamp DNA Stool Mini kit, the developed method was verified. [ Result] The DNA yield of the developed method was 100 times as much as that of QIAamp DNA Stool Mini kit. And the real-time PCR result showed that the efficiency of DNA extraction of the developed method was higher than that of the QIAamp DNA Stool Mini kit. [ Conclusion] This modified method is inexpensive, efficient and rapid, and it is suitable for large quantities of feces samples.