Study On Extraction,Purification,Identification And Vitro Activity Of Squalene And Phenols From Strobilanthes Tonkinensis Lindau

Strobilanthes tonkinensis Lindau is a genus of perennial plants, of the family Acanthaceae which originate in Jinghong of Yunnan province of China as well as Thailand and Vietnam. It is a natrural aromatic and medical plant which has been drinked as a tea beverage for a long time in the Dai ethnic of Yunnan province. S. tonkinensis is abundant with squalene and phenols with strong bioactivities and antioxidant activity. The cost of S. tonkinensis is considerably low since it grow fast and can be harvested all around the year which provide the enough raw material. However, the product development of S. tonkinensis is not adequate and more work is required to be done. The extraction, seperation process are optimizated in our study. The liposoluble substances, phenol substances and volatile substances extracted from S. tonkinensis have been identified by LC-MS. The bioactivities of inhibiting advanced glycation end products （AGEs） formation, glycosidase and a-amylase activities as well as activities of scavening free radical in vitro are evaluated for both squalene solution and phenols solution. The results are as follows:1. A high performance liquid chromatography （HPLC） method for determining squalene of S. tonkinensis has been studied. The chromatographic condition included a DiamonsilTM C18 column （200×4.6mm,5um particle size） and the mobile phase consisting of methanol/acetonitrile （60/40, v/v）, at a flow rate of 1.2 mL/min, the ultraviolet detector was set at 210nm and the column temperature was kept 40℃. The content of squalene extracted from 100 g S. tonkinensis using petroleum ether, n-hexane and acetone are 6.80±0.15 mg、7.10±0.26 mg and 3.32±0.05 mg, respectively. The squalene content of 100 g S. tonkinensis from Yunnan province is 12.39 mg and 7.17 mg from Hainan province.2. Response surface methodology （RSM） was employed to optimize the conditions of supercritical CO₂ extraction of squalene from S. tonkinensis. The effects of pressure, temperature and extraction time on the yield of non-saponification substances and squalene were investigated. It was predicted the optimum extraction conditions would be the extraction pressure of 25 MPa, temperature of 37.5℃ and extraction time of 90 min. Under such parameters, the squalene content of 100 g S. tonkinensis was predicated to be 21.13 mg which is 1.7 times of content of Soxhlet extraction.3. Equal volume of n-hexane and water are added into the extraction solution of S. tonkinensis obtained by supercritical fluid extraction. Both the n-hexane phase and water phase are collected separately. The n-hexane phase is used to obtain high purity squalene and the purity of squalene reached 95.31% treated with silica gel and 98.67% treated with semipreparative HPLC. The water phase is used to obtain phenols of S. tonkinensis extracted with ethyl acetate. The total phenol content is about 0.809±0.003 mg/mL.4. LC-APCI-MS/MS was performed to identify the liposoluble active compounds of 5. tonkinensis. Δ5-Avenasteryl-β-D-glucoside, a-tocoenol, Stigmasteryl-β-D-glucoside, stigmasterol-glucoside, Stigmasterol-dihydroxy, a-tocopherol, Δ5-Avenasterol, sitgmasterol, β-sitosterol and squalene were found in the hexane extract of S. tonkinensis. RP-HPLC was applied to determine the content of its main active compounds including 0.24%o squalene,0.11%。 α-tocopherol and 0.04%o stigmasterol. LC-ESI-MS/MS was performed to identify the flavonoids of S. tonkinensis. Luteolin diglycoside、 Forsythoside A、Frosythoside G、Frosythoside G+Na、Calceolarioside C and Galengin-7-O-glu acid were found in the ethyl acetate extract of S. tonkinensis. GC-MS was also applied to identify the composition of aroma constituent and 61 aroma compounds were identified.5. Squalene solution and phenol solution of S. tonkinensis were both evaluated to inhibit the formation of fluorescent AGEs in the BSA/glucose system. The inhibition rate of 0.04 mg/mL of phenols solution of S. tonkinensis increased from 83.1% to 90.5% when incubated from 1 to 7 week. While the inhibition rate of 0.04 mg/mL of squalene solution of S. tonkinensis was declined from 86.3% to 61.1%.6. For squalene solution of S. tonkinensis the IC50 value of inhibiting glycosidase and a-amylase are 5.2μg/mL and 0.078 mg/mL, respectively. For phenols solution of S. tonkinensis the IC50 value of inhibiting glycosidase is 9.8μg/mL and the most inhibition rate of inhibiting a-amylase is 37.9±3.2%. The results indicate the squalene solution and phenols solution of S. tonkinensis exhibited potential to prevent 2-type diabetes.7. The IC50 value of scavening DPPH free radical for squalene solution and phenols solution of S. tonkinensis are 0.026 mg/mL and 9.8μg/mL, respectively. The IC50 value of scavening OH free radical for squalene solution and phenols solution of S. tonkinensis are 0.77 mg/mL and 0.72 mg/mL, respectively.