Isolation And Analysis Of Chemical Constituents In Chinese Toon

Toona sinensis leaves, which is bitter in flavor and warm in property, have efficacies in clearing heat and toxic materials, diminishing inflammatory and eliminating dampness, etc. It is also widely used in treating enteritis, dysentery and other diseases in clinic. Moreover, Toona sinensis leaves have an edible history over two thousand years, which is preferred deeply by both Chinese and foreigners.Modern medicine indicates that Toona sinensis leaves extract contains several biological activities, such as antioxidant, antitumor and hypolipidemic. Several domestic and foreign scholars have studied chemical compositions in Toona sinensis leaves and found they contain many types of compounds, mainly including flavonoids and gallic acids. Therefore, Toona sinensis leaves as edible Chinese medicine deserve further study and have broad prospects. The extraction, isolation, identification and content determination of flavonoids in Toona sinensis leaves have been studied. In addition, proteins in these leaves have been analyzed in this paper. The main research contents and conclusions are as follows:(1) Extraction, isolation and identification of flavonoids in Toona sinensis leavesUtilizing silica gel column chromatography and Pre-HPLC to study the chemical compositions which were extracted with ethyl acetate in Toona sinensis leaves and 11 compounds were obtained. Structures of these compounds were identified by MS and NMR, the compounds were 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(Ⅰ), Gallic acid(Ⅱ), Ethyl gallate(Ⅲ), Rutin(Ⅳ), Myricitrin(Ⅴ),quercetin-3-O-β-D-galactoranoside(Ⅵ), quercetin-3-O-β-D-glucopyranose(Ⅶ),quercetin-3-O-α-L-arabinopyranoside(Ⅷ), astragalin(Ⅸ),quercetin-3-O-α-L-rhamopyranoside(Ⅹ), kaempferol-3-O-α-L-rhamopyranoside(Ⅺ),respectively.(2) Content determination of flavonoids in Toona Sinensis leavesFirstly, a flavonoid glycoside(quercetin-3-O-α-L-rhamopyranoside), which is the main compound in Toona sinensis leaves, was assayed. Chromatography condition is as follows: XBridge Shield RP18 column(4.6 mm × 150 mm, 5 μm); mobile phase:0.1% aqueous formic acid(A)- acetonitrile(B) and A is 20%, B is 80%; isocratic program: 0 ~ 30 min; flow rate: 1.0 m L/min; detection wavelength: 254 nm; column temperature: 40℃; Injection volume: 20 μL; flow rate: 1.0 m L·min-1. Under this condition, quercetin-3-O-α-L-rhamopyranoside exhibits a good linear relationship between 5.260 and 105.2 ug·m L-1(r2=0.9991), the range of precision(n=6) is 0.90%~ 1.95%. In addition, this method has a good repeatability(RSD<2%, n=6) and sample solution is stable within 48 hrs. Moreover, average recovery of this method is from 95% to 110%(n=3) and RSD is less than 5%. Contents of quercetin-3-O-α-L-rhamopyranoside in 6 areas are 0.271%(Henan Xiping), 0.826%(Shanxi Yuncheng), 0.222%(Sichun Jianyang), 0.459%(Henan Jiaozuo), 0.808%(Shandong Ximou) and 0.293%(Jiangsu Zhenjiang), respectively. As a result, there are some differences in the content of this flavonoid glycoside in Toona sinensis leaves from these areas, and Toona sinensis leaves of Shanxi Yuncheng contains the most fquercetin-3-O-α-L-rhamopyranoside while Sichuan Jianyang contains the least.Afterwards, contents of 8 flavonoid glycosides, which are separated from Toona sinensis leaves, are determined. These compounds are Rutin(Ⅳ), Myricitrin(Ⅴ),quercetin-3-O-β-D-galactoranoside(Ⅵ), quercetin-3-O-β-D-glucopyranose(Ⅶ),quercetin-3-O-α-L-arabinopyranoside(Ⅷ), astragalin(Ⅸ),quercetin-3-O-α-L-rhamopyranoside(Ⅹ) and kaempferol-3-O-α-L-rhamopyranoside(Ⅺ), respectively. Orthogonal design is used to optimize extraction condition and influence factors such as mobile phase composition, column temperature and flow rate are also optimized. Eventually, a simple, reliable and efficient method is established with advantages such as good separation and accuracy. Extraction condition is solid-liquid ratio as 1:40; 60% methanol; ultrasonic extraction: 30 min;Waters RP18(2.1 mm×150 mm, 1.7 μm) column; mobile phase: 0.05% TFA H2O(A)-0.05% TFA ACN(B); gradient elution program is that from 0 to 10 min, the ratio of A decreases from 88% to 70% and from 10 min to 17 min, maintaining the ratio of A; flow rate is 0.350 m L/min; detection wavelength is 350 nm; column temperature was 30℃and injection volume is 5 μL. Under this condition, compounds Ⅳ, Ⅴ, Ⅵ,Ⅶ, Ⅷ, Ⅸ, Ⅹ and Ⅺ exhibit good linear relationships in the range of 0.906~58.0,0.0813~5.20, 0.0813~5.20, 1.05~67.40, 0.113~7.20, 0.263~16.8, 2.47~158 and1.45~92.6 μg, respectively. In addition, the range of precision(n=6) is 0.10%~3.28%and r2 of this method is 1.0000, 1.0000, 0.9999, 1.0000, 0.9992, 0.9992, 1.0000 and1.0000, respectively. Furthermore, this method has a good repeatability(RSD<5%,n=6) and sample solution is stable within 48 hrs. Besides, average recovery of this method is from 95% to 110%(n=3) and RSD is less than 5%. Contents of 8 flavonoid glycosides in Toona sinensis leaves from 10 areas(Henan Xiping, A; Sichuan Mianyang, B; Shandong Yantai, C; Shanxi Yuncheng, D; Henan Zhoukou, E;Shandong Ximou, F; Jiangsu Zhenjiang, G; Jiangsu Danyang, H; Henan Wenxian, J and Shangdong Yuncheng, K) are determined. As a result, the content of compoundⅣ, Ⅴ, Ⅵ, Ⅶ, Ⅷ, Ⅸ, Ⅹand Ⅺ is 0.023%~0.16%, 0.003%~0.16%, 0.018%~0.111%,0.071%~0.358%, 0.007%~0.067%, 0.011%~0.104%, 0.415%~1.270% and0.59%~0.795%, respectively. On the other hand, compounds contained in Toona sinensis leaves during different months are also determined(2013.03~2013.11). It shows that the content of compound Ⅳ, Ⅴ, Ⅵ, Ⅶ, Ⅷ, Ⅸ, Ⅹ and Ⅺ is0.047%~0.131%, 0.002%~0.016%, 0.015%~0.060%, 0.081%~0.305%,0.007%~0.337%, 0.024%~0.932%, 0.069%~0.747% and 0.146%~0.746%,respectively. Consequently, the content of 8 flavonoids glycoside in the leaves of Toona sinensis from different areas is different. Toona sinensis leaves from Shandong Yantai contain 5 flavonoids glycosides and flavonoid(Rutin, Myricitrin,quercetin-3-O-β-D-galactoranoside, quercetin-3-O-β-D-glucopyranose,kaempferol-3-O-α-L-rhamopyranoside) which are the least compounds while Shandong Yantai contains the most flavonoid. In April, Toona sinensis leaves have the highest percentage of 3 flavonoid glycosides(Rutin,quercetin-3-O-β-D-galactoranoside, quercetin-3-O-α-L-arabinopyranoside) and in June, they have 2 flavonoid glycosides(astragalin,quercetin-3-O-α-L-rhamopyranoside) at a maximum. However, Toona sinensis leaves in July have 6 flavonoids(Rutin, Myricitrin, quercetin-3-O-β-D-galactoranoside,quercetin-3-O-β-D-glucopyranose, quercetin-3-O-α-L-arabinopyranoside) glycosides and in August there exists 2 flavonoid glycosides(astragalin,quercetin-3-O-α-L-rhamopyranoside) which are at its lowest content.(3) Preliminary analysis of the protein in Toona sinensis leavesUsing SDS-PAGE to analyze Toona sinensis leaves harvested during different months. We found that the molecular weight of the protein in Toona sinensis leaves is almost less than 35 k D, and in different months, proteins in Toona sinensis leaves are different. Afterwards, 6 bands were cut, digested and determined by MALDI-TOF-MS. The result illustrates that the m/z of peptides generated from proteins in band 1 mainly concentrate in the range of 2200~2450 Da; band 2 in2100~4980 Da; band 3 in 2400~4200 Da; band 4 in 2100~2550 Da and band 5,6mainly in 2100~4980 Da. Searching the Mascot database according to the result obtained by using MALDI-TOF-MS and six major protein bands are likely to be Hypothetical protein, Phosphodiesterase, Dihydroxy-acid dehydratase, Hypothetical protein, uncharacterized protein LOC106503571 and ABC transporter substrate-binding protein.