Preparation Technique And Determination Of Flavonoids In The Leaves Of Toona Sinensis Roemer

Toona sinensis leaves have the functions of clearing heat,removing inflammation and detoxification,clearing damp,which are bitter in flavor and warm in property,which are commonly used for the treatment of colitis and diarrhea in clinic.Moreover,Toona sinensis sprouts have an edible history over a long time,which is pretty popular in China.Modern researches indicates that Toona sinensis leaves extract has pharmacological activities,such as antioxidant,anti-inflammatory,relieving hyperglycemia,regulating lipid metabolism,and inducing apoptosis of cancer cells,etc.This paper studied the extraction,isolation,transformation and identification of flavonoids in Toona sinensis leaves,which is one of the main active ingredients in Toona sinensis leaves.Work completed and conclusions made are as follows:1.Integrated extraction and purification of total flavonoids from Toona sinensis leavesPressurized liquid extraction was performed to extract flavonoids in Toona sinensis leaves using dilute ethanol as extraction solution,and single factor experiments were carried out to optimize the extraction conditions with total flavonoids yield as the index,including extraction temperature,liquid/solid ratio as well as amount of TSL powder.Then,the extraction liquid was directly loaded onto a glass column packed with certain amount of macroporous resin for subsequent adsorption.After the completeness of the integrated extraction-adsorption process,the column was washed and then flushed successively,with total flavonoids recovery and purity as the indexes to optimize the integrated extraction-purification conditions,including amount of macroporous resin/amount of powder,flow rate of extraction solvent,elution volume and flow rate of elution solvent.Eight flavonol glycosides were identified in purified extract using UPLC-ESI-MS,includingrutin,myricitrin,quercetin-3-O-?-D-galactoside,quercetin-3-O-?-D-glucoside,quercetin-3-O-?-L-arabinoside,astragalin,quercetin-3-O-?-L-rhamnoside and kaempferol-3-O-?-L-rhamnoside.Consequently,the optimal extraction conditions were performed at 90?by 10%EtOH constantly pumped at 1.0 mL/min,and liquid/solid ratio was 30:1 mL/g.For the purification of flavonoids,solid/resin ratio was set at 1:6 g/g,and 2.0 BV of 70%EtOH was used to flush the macroporous resin column at 1 BV/h.Comparing the integrated procedure with the conventional method,some advantages were observed,the recovery of total flavonoids in the product was increased from 61.6%to 71.1%,and the purity of total flavonoids was increased from 54.9%to 66.6%.Moreover,through this way,products of high quality were obtained in an environmentally friendly process with lower consumption of solvents and time,which decreased 48.1%and 45.3%.2.Simultaneous determination of four flavonoid glycosides in Toona sinensis leaves by UPLC methodAn UPLC method was established,namely,chromatographic column:Waters Xbridge Shield RP18?2.1 mm×150 mm,1.7?m?,mobile phases:0.05%TFA H₂O?A?-0.05%TFA ACN?B?;gradient elution program is that from 0 to 8 min,the ratio of A maintains 80%,from 8to 10 min,the ratio of A decreases from 80%to 70%and from 10 to 15 min,maintaining the ratio of A;the flow rate is 0.30 mL/min;column temperature is 40?;the detection wavelength is 350 nm and injection volume is 5?L.The linearity,precision,repeatability,stability and accuracywereinvestigated.Resultsshowedthatthelinearityofrutin,quercetin-3-O-?-D-glucoside,quercetin-3-O-?-L-rhamnosideand kaempferol-3-O-?-L-rhamnoside was good within the range of 0.469¹20?R²=0.9994?,0.547¹40?R²=0.9994?,1.25³20?R²=0.9994?and 0.781²00?R²=0.9994?.The method was precise?RSD<3.5%?,repeatable?RSD<3%?and accurate due to the recoveries were with the range of100%¹05%?n=6?.The sample was stable within 48 h?RSD<2%?.Contents of the four flavonoid glycosides in Toona sinensis leaves from 9 areas?Shanxi Jinzhong,1;Shandong Jinan,2;Jiangsu Suzhou,3;Jiangsu Wuxi,4;Hebei Baoding,5;Anhui Wuhu,6;Henan Nanyang,7;Jiangsu Zhenjiang,8;Anhui Fuyang,9?were determined.As a result,the content of the four flavonoids in the TSL were various.Among them,the total amount of the four flavonoids in TSL from Anhui Fuyang were the highest.The total content of the four flavonoid glycosides in TSL from Anhui Wuhu,Henan Nanyang and Jiangsu Zhenjiang were similar.While the content of the four flavonoid glycosides from Shanxi Jinzhong were the lowest relatively.The content of rutin and quercetin-3-O-?-D-glucoside in TSL from Anhui Wuhu were the highest.The content of quercetin-3-O-?-L-rhamnoside in TSL from Anhui Fuyang was the highest,followed by that from Hebei Baoding and Jiangsu Zhenjiang.The content of kaempferol-3-O-?-L-rhamnoside in TSL from Henan Nanyang was the highest,followed by that from Anhui Fuyang.Then,SPSS software was used to analyze the content of four flavonoid glycosides in TSL from the nine areas by one-way ANOVA.It turned out that at the significance level of 0.05,there was a significant difference?P<0.001?in the content of four flavonoid glycosides in TSL from nine areas.In order to determine whether there was significant difference between the each two origins,the content of four flavonoid glycosides in TSL from each two origins were compared by least significant difference method.It was found that the P values of the four flavonoid glycosides in the nine areas were all less than 0.05,indicating that there was significant difference between each two origins.Then,correlation analysis of four flavonoid glycosides in TSL was conducted.And it showed that at the significance level of 0.01,there was a highly significant positive correlation between the contents of rutin and quercetin-3-O-?-D-glucoside in TSL from the nine areas.At the significance level of 0.05,there was a significant positive correlation between the contents of quercetin-3-O-?-L-rhamnoside and kaempferol-3-O-?-L-rhamnoside in the samples of TSL from the nine areas.Finally,the Euclidean distance squared between groups was used as the interval of the measurement standard to systematically cluster the contents of four flavonoid glycosides in the TSL from the nine areas.And it turned out that samples from the nine origins can be divided into three categories.The first category is TSL from Anhui Fuyang,due to its obviously highest total content of the four flavonoid glycosides;the second category is TSL from Henan Nanyang,due to its relatively higher content of the four flavonoid glycosides;the third category is TSL from Shanxi Jinzhong,Shandong Jinan,Jiangsu Suzhou,Jiangsu Wuxi,Hebei Baoding,Anhui Wuhu and Jiangsu Zhenjiang,the quality of them were relatively poor.3.Simultaneous determination of quercetin and kaempferol in Toona sinensis leaves by biphasic acid hydrolysis-HPLCA HPLC-UV method was established,chromatographic column:Waters Xbridge Shield R18?4.6 mm×150 mm,3.5?m?,mobile phases:0.1%Formic Acid H₂O?A?-ACN?B?;gradient elution program is that from 0 to 5 min,the ratio of A decreases from 75%to 70%,from 5 to 15min,the ratio of A decreases from 70%to 60%and from 15 to 25 min,the ratio of A decreases from 60%to 15%;the flow rate is 1.0 mL/min;column temperature is 35?;the detection wavelength is 254 nm and injection volume is 10?L.The linearity,precision,repeatability,stability and accuracy were investigated.Results showed that the linearity of quercetin and kaempferol was good within the range of 0.313⁴0.0?g/mL?R²=0.9999?.The method was precise?RSD<2.5%?,repeatable?RSD<4%?and accurate due to the recoveries were with the range of 95%¹05%?n=9?.The sample was stable within 24 h?RSD<2%?.A biphasic acid hydrolysis system was established and applied to obtain flavonoid aglycone from flavonoid glycosides in TSL and single factor experiments were carried out to optimize the extraction conditions with the yield of quercetin and kaempferol as indexes,including extraction time,liquid/solid ratio as well as ethanol concentration.The optimal extraction conditions were as follows,namely,extraction time was 2 h,solid/liquid ratio was 1:30 g/mL and the ethanol concentration was 60%.Then,single factor experiments were carried out to optimize the hydrolysis conditions with the yield of quercetin and kaempferol as indexes,including hydrolysis time,hydrolysis temparature,hydrochloric acid concentration,powder amount as well as ethyl acetate volume.The optimal hydrolysis conditions were as follows,namely,hydrolysis time was 1 h,hydrolysis temparature was 80?,hydrochloric acid concentration was 6%,powder amount was 100 mg as well as ethyl acetate volume was 10 mL.Afterwards,hydrolysis conditions were optimized through orthogonal design,where three factors including hydrolysis time,hydrolysis temparature and hydrochloric acid concentration were investigated.As a result,the best conditions,namely,time was 1.5 h,temperature was 80?,hydrochloric acid concentration was 4%.Compared to the conventional method,the yield of quercetin and kaempferol increased around 40.7%and 52.7%separately while the hydrochloric acid concentration used was much less using the new method.Contents of quercetin and kaempferol in TSL from 9 areas?Shanxi Jinzhong,1;Shandong Jinan,2;Jiangsu Suzhou,3;Jiangsu Wuxi,4;Hebei Baoding,5;Anhui Wuhu,6;Henan Nanyang,7;Jiangsu Zhenjiang,8;Anhui Fuyang,9?were determined.It turned out that content of quercetin was much more than the content of kaempferol in TSL from the nine areas,and the total content of quercetin and kaempferol in TSL from Anhui Fuyang was significantly higher than that from other areas.Moreover,the total content of quercetin and kaempferol in TSL from Jiangsu Zhenjiang,Shanxi Jinzhong,Shandong Jinan,Jiangsu Suzhou,Jiangsu Wuxi,Hebei Baoding,Anhui Wuhu and Henan Nanyang showed the similar,among them,the total content of quercetin and kaempferol in TSL from Shanxi Jinzhong was the lowest.