Supercritical CO2 extraction process of Ganoderma lucidum spore oil with entrainer
Supercritical CO2 extraction of Ganoderma lucidum spore oil with entrainer was studied. Methods: The effects of the extraction pressure, the extraction temperature and the content of entrainer were studied with central composite experimental design, a surface response model was established. Results: Based on this model, the optimal conditions for SFE process are as followings: extraction pressure at 32MPa, extraction temperature at 57 ℃ , 95% alcohol as enrtainer (1:1w/v). Be compared with supercritical CO2 extraction, the optimum conditions of SFE not only can increase the content of active ingredients（triterpenoids, sterols） of Ganoderma lucidum spore oil, but guarantee its yield and quality. Conclusion Entrainer enhancing technology can significantly improve the Ganoderma lucidum spore oil separation efficiency of supercritical CO2 extraction.
Wang Wen-xuan,WU Yan,CHEN Fei-fei, GE Fa-huan
1.School of pharmaceutical sciences，Sun Yat-Sen University，Guangzhou 510006 ,China
2.College of Life science,Sun Yat-sen University,Guangzhou 510275,China
3. Nansha Research Institute of Sun Yat-Sen University, Guangzhou 510458,China
Key Words: Ganoderma lucidum spore oil, supercritical CO2 extraction, entrainer, triterpenoids
Ganoderma lucidum is one of the traditional Chinese herbal medicines in China. It has a long history of medicinal use in China and is called the top grade by Shennong Materia Medica . Ganoderma lucidium spore is a spore of Ganoderma lucidum from the cap of the Ganoderma lucidum. It is a propagule of Ganoderma lucidum and has all the genetic material of Ganoderma lucidum. The main pharmacological effects include anti-tumor, enhanced immune regulation, anti-virus, hypoglycemia, and hypolipidemic. Ganoderma lucidum spore oil is mainly used as a lipid active substance extracted from Ganoderma lucidum spores by supercritical CO2 extraction technology, and has similar pharmacological activities with Ganoderma lucidum spores. Among them, triterpenoids and sterols are the main components of pharmacological action. Since CO2 is a non-polar molecule, pure supercritical CO2 fluid has good solubility for small molecular weight and lipophilic components. For Ganoderma lucidum spores, triterpenoids and sterols have slightly larger molecular weight and certain polar components. It can be fully extracted and introduced into the entrainer under pure supercritical CO2 conditions, which is beneficial to increase the content of triterpenoids and sterols with strong pharmacological activity in Ganoderma lucidum spore oil, and has important application significance for enhancing the activity of Ganoderma lucidum spore oil.
In this study, the extraction rates of the main active constituents of Ganoderma lucidum spore oil, triterpenoids and ergosterol, were used as indicators. The response surface method was used to optimize the design of the main factors affecting the supercritical extraction process.
1 Instruments and materials
1L supercritical CO2 extraction equipment, designed by itself. The equipment allows a maximum working pressure of 50 MPa and uses two stages of separation).
UV-2550 UV-Vis Spectrophotometer (Shimadzu Corporation, Japan).
U-3000 High Performance Liquid Chromatograph (Dai'an, USA), mobile phase methanol is chromatographically pure.
Agilent 6890GC/5973MS Gas Chromatograph (Agilent, USA)
CO2 gas (purity of 99.9%, Guangzhou Gas Plant).
95% ethanol, absolute ethanol, ethyl acetate, vanillin, glacial acetic acid, perchloric acid, the above reagents are of analytical grade.
Ursolic acid reference product (provided by China National Institute for the Control of Pharmaceutical and Biological Products); ergosterol reference substance (available from sigma, USA).
Ganoderma lucidum spore powder was identified as a dried spore of the polyporaceae fungus Ganoderma lucium (Leyss. ex Fr.) Karst, which was prepared by breaking the wall and granulating.
2 methods and results
2.1 response surface method process optimization design
According to the response surface analysis method, the three factors of extraction pressure, extraction temperature and entrainer dosage were used as independent variables. Each factor took 3 levels, encoded by -1, 0, +1, and the total extraction rate was three. And the extraction rate of ergosterol was the response value. The effects of the above three factors on the extraction process were investigated. The test factors and codes are as follows.
A: Extraction pressure (MPa) 25 30 35
B: Extraction temperature (°C) 50 55 60
C: amount of entrainer (W/V) 1:0.5 1:1 1:1.5
2.2 Supercritical CO2 extraction of Ganoderma lucidum spore oil process
A certain amount of pre-broken granulated Ganoderma lucidum spores were weighed into a 1 L extraction vessel, and the extraction kettle, the separation vessel I and the separation vessel II were separately heated, and the refrigerator was started to be cooled. When the temperature of the extraction kettle, the separation kettle and the cold storage tank reaches the experimental requirements, the CO2 is condensed in the cold storage tank and then driven into the extraction kettle and the two separation kettles by the high pressure pump, and the valve is adjusted to bring the pressure to the set value. Close the CO2 cylinder and start the cycle extraction. After the pressure temperature is stable, open the entrainer metering pump, pump the required entrainer for extraction, and start timing. Each time interval, the separation kettle I and the separation kettle II were discharged and weighed.
2.3 Determination of total triterpenoids in Ganoderma lucidum spore oil
2.3.1 Preparation of reference substance and drawing of standard curve
Approximately 11.76 mg of ursolic acid standard was accurately weighed, placed in a 100 ml volumetric flask, dissolved in ethyl acetate and diluted to the mark, and shaken to prepare a reference solution of 0.1176 mg/ml/. Aspirate liquids of 0.00 ml, 0.10 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml, respectively, and evaporate to dryness on a water bath at 100 ° C, then add 0.40 ml of 5% vanillin-glacial acetic acid solution and 1.00 ml of perchloric acid was heated in a 60 ° C water bath for 45 min, then transferred to an ice water bath, and 5.00 ml of glacial acetic acid was added, shaken and allowed to stand at room temperature. After 15 min, the absorbance of the control solution was measured at 525 nm using an ultraviolet-visible spectrophotometer. The standard curve was drawn by concentration (C) and absorbance (A), respectively. The regression equation was obtained: A=6.5174C+0.0226, r=0.9991, and the total triaxes had a good linear relationship in the range of 11.76~235.2μg.
2.3.2 Preparation and determination of sample solution
Ganoderma lucidum spore oil extracted by the process was 0.08 g, accurately weighed, placed in a 100 ml volumetric flask, dissolved in ethyl acetate and diluted to the mark, and shaken to prepare a test solution. Accurately draw 0.5 ml of the test solution, evaporate dry on a 100 ° C water bath, then add 0.40 ml of 5% vanillin-glacial acetic acid solution and 1.00 ml of perchloric acid, heat in a 60 ° C water bath for 45 min, and then transfer to an ice water bath. Add 5.00 ml of glacial acetic acid, shake well and leave at room temperature. After 15 min, the absorbance was measured at 525 nm using an ultraviolet-visible spectrophotometer, and the total triterpenoid content was calculated.
2.4 Determination of ergosterol in Ganoderma lucidum spore oil
2.4.1 Chromatographic conditions
The column was Kromasil 100-5C18, (250 mm × 4.6 mm, 5 μm), the column temperature was 30 ° C; methanol was used as the mobile phase, the flow rate was 0.8 mL / min; the detection wavelength was 282 nm; the injection volume was 10 μl. The results show that ergosterol in the test solution can be well separated from other components, and no interference peaks exist.
2.4.2 Preparation of reference solution
Accurately weigh 9.93mg of ergosterol reference substance, put it in a 10ml volumetric flask, dissolve it in methanol and dilute to the mark, and dilute to volume. Shake well to prepare a reference stock solution containing ergosterol 0.993mg per ml of methanol. Solution; accurately draw 1 ml of the reference stock solution, place it in a 10 mL volumetric flask, add methanol to the mark, shake well, and prepare a reference solution containing 0.0993 mg of ergosterol per ml of methanol.
2.4.3 Preparation of test solution
Take 1.0g of Ganoderma lucidum spore oil extracted by the process, accurately weighed, placed in a 25mL volumetric flask, dissolved in a methanol solution containing 50% ethyl acetate and diluted to the mark, shake well, through a microporous membrane (0. 45μm) Filtered, that is.
2.4.4 Investigation of linear relationship
Take the ergosterol reference solution at a concentration of 0.0993 mg/ml, and inject 2 μl, 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, respectively, with the peak area (y) as the ordinate and the injection amount (x) as the abscissa. Linear regression obtained linear regression equation: y = 19.427x + 0.1424, r = 1.0000. The results showed that ergosterol showed a good linear relationship with the peak area in the 0.199~2.483μg injection range.
2.4.5 Precision Experiment
Under the above chromatographic conditions, accurately draw 10 μl of the same reference solution and measure continuously.
The peak area was recorded and the RSD value of the ergosterol peak area was 0.03%, indicating that the instrument precision was good.
2.4.6 Stability test
Ganoderma lucidum spore oil was accurately extracted for test solution, and analyzed at 0h, 2h, 4h, 6h, 8h, 10h, 12h after preparation, and the peak area was recorded. The RSD of ergosterol peak area was 0.38%, indicating that the test sample was obtained. Ergosterol in the solution was stable in 12 h.
2.4.7 Repeatability test Take 6 parts of the same batch of Ganoderma lucidum spore oil, accurately weighed, prepare the sample according to the preparation method of the test solution, and determine the RSD value of the ergosterol peak area is 0.89%. The results show that the method has good repeatability.
2.4.8 Sample recovery test
Take the same batch of 6 kinds of extracts of Ganoderma lucidum spore oil, about 0.5g each, accurately weighed, respectively, add 1.2ml of ergosterol reference solution with a concentration of 0.993mg/ml, and add 50% ethyl acetate in methanol to the mark. Shake well, determine, calculate the recovery rate of the sample, the average recovery of ergosterol was 101.2, and the RSD value was 0.72%, indicating that the determination method was accurate.