Supercritical Fluid Extraction Of Bioactive Compounds From Terminalia Chebula Retz And In Vitro Antioxidant Activity

Background and objectives:Terminalia chebula?T.chebula?is the dried immature fruit of plant Terminalia chebula Retz,which can clear away the heat-evil and expel superficial evils,used to treat Yin deficiency diphtheria cleinically.This fruit is widely distributed in Guangdong,Guangxi,Yunnan.The major phytochemicals reported from T.chebula are tannins,phenolic acids and triterpene compounds,and these compounds have many biological activities,such as antioxidant activities,anti-inflammatory,et al.Previous studies have reported the anti-inflammatory and anti-arthritic properties exhibited by chubulanin on a collageninduced arthritis?CIA?model in DBA/1 mice.Phenolic compounds are secondary metabolites of plants.Due to its antioxidant,antibacterial,anti-aging and other biological activities,it has become a hot research topic in field of medicine,food,daily chemical.Previous studies have indicated that T.chebula is rich in phenolic compounds,such as gallic acid and corilagin,chebulanin,chebulagic acid,ellagic acid,and et al.These compounds play an active effect in antibacterial,anticancer,anti-hyperglycemia and anti-oxidation.Modernization of traditional Chinese medicine is the focus of national planning in 12 th Five-Year.There are abundant natural Chinese medicines in China.How to extract bioactive compounds is one of the most important problems in the development of traditional Chinese medicine.Our previous study found that there are some problems with impregnation of bioactive compunds from T.chebula,such as long time-consuming and low yield.Therefore,it is most important to find an efficient and green extraction technology.Supercritical fluid extraction?SFE?is one of the most important technologies in the development of traditional Chinese medicine.It is widely used in the extraction of volatile,low polar compounds,especially suitable for the extraction of the components with unstable for temperature.There has studies showed that the supercritical fluid extraction technology and ethanol extraction and aqueous extraction of integrated use,can be used for the extraction of phenolics,anthocyanin,saponins and other high polar molecules compounds.The results indicate that the technique can effectively extract active ingredients from T.chebula.Our research aimed to integrate the supercritical CO2 extraction technology and acetone extraction and water extraction technology,which uses the sequential extraction method in fixed bed to obtain the maximum extraction yield and more concentrated extracts of phenolic compounds by the following solvents in order to increase the polarity: sc CO2 in the first step,acetone in the second step and water in the third step.Total phenolic compounds of T.chebula were determined by Folin-Ciocalteu reagent.High Performance Liquid Chromatography?HPLC?was established to determine the main phenolic compounds in the T.chebula extract.The antioxidant activity of different solvent extracts from T.chebula in vitro was studied.Provide the basis for the study of the development and application of traditional Chinese medicine from the T.chebula.Methods and Results:Part 1.Supercritical fluid extraction of bioactive compounds from Terminalia chebulaMethods1.Physical characteristics of T.chebulaThe geometric mean diameter distribution of the particles,moisture content,real density,apparent density,porosity of T.chebula fruits was detected.2.Sequential extraction in fixed bed processExtraction of bioactive compounds from the fruits of T.chebula by sequential extraction in fixed bed at 40°C and 300 bar,using supercritical carbon dioxide,acetone and water as solvents.The extraction time was 18 h,and the extract was collected every 2 0 min.The extract was concentrated and dried.3.Fixed bed?one-step?extraction processExtraction of bioactive compounds from the fruits of T.chebula in fixed bed at 40°C and 300 bar,using acetone/water as solvent.Extraction time was 12 h.The extract was collected every 30 min,and the acetone extract and water extract were obtained after concentration and drying.4.Conventional extraction processExtract of bioactive compounds from the fruits of T.chebula at 40°C and atmospheric pressure.The acetone extract and water extract were obtained after concentration and drying.Results1.Characterization of T.chebula fruitsThe particle size distribution of the dried and ground fruits which was distributed between 0 mm to 0.6 mm with few fine particles ranging between 0.6 mm to 1 mm: 34.913%?0-0.1 mm?,20.498%?0.1-0.2 mm?,16.09%?0.2-0.3 mm?,11.925%?0.3-0.4 mm?,5.299%?0.4-0.5 mm?,7.193%?0.5-0.6 mm?,1.939%?0.6-0.7 mm?,1.15%?0.7-0.8?,0.993%?0.8-1 mm?.Moisture content: 10.86±0.01%;Average particle size?Dav?: 212.47±0.03 ?m;Median diameter?D50?: 166.60±0.03 ?m;Real density: 1.54±0.01 g/cm3;Apparent density: 0.49±0.01 g/cm3;Bed porosity: 0.68±0.01.2.Results of sequential extraction in fixed bed processThe global yield of sequential extraction in fixed bed process: the yield of supercritical CO2 extraction was 1.25±0.04%,the yield of acetone extraction was 28.15±0.94%;the yield of aqueous extraction was about 33.44±0.56%;the total extraction yield was 62.84±1.54%.3.Results of one-step extraction in fixed bed processThe global yield of acetone extraction and water extraction in fixed bed process: The yield of acetone extraction was 41.28±0.84%;the yield of water extraction was about 55.60±1.41%.4.Results of conventional extraction processThe global yield of acetone extraction and water extraction in conventional process: the yield of acetone extraction was 37.46±1.56%;the yield of water extraction was about 45.52±1.52%.Part 2.Determination of total phenolics and major phenolic acids in Terminalia cheublaMethods1.Determination of total phenolic contentsStandard curve: the preparation of a series of concentrations of gallic acid standard solution,adding 0.5 m L of Folin-Ciocalteu reagent,then adding 2 m L 20% Na2CO3 solution.After mixing for 1 h at room temperature,the optical density of solution was measured at 760 nm by spectrophotometer.The mass density is the abscissa,the optical density is the ordinate,and the standard curve is drawn.Preparation and determination of sample solution: the extracts were prepared into 1 mg/m L of the extract liquor.The extract liquor was diluted to an appropriate multiple,and the optical density was measured according to the above method.The phenolic contents were calculated from the standard curve equation.Total phenolic contents were expressed as equivalents of gallic acid?mg/g?.2.Linear relationship investigationAccording to the chromatographic conditions,determine the chromatographic peak area of the standards of chebulagic acid,gallic acid and corilagin,chebulanin,chebulinic acid.The regression equation of standards and correlation coefficient were obtained by linear regression treatment.3.Precision experimentThe standard configuration of low concentration solution,medium concentration solution and high concentration solution,according to the provisions of the chromatographic conditions of every sampling continuously for 5 times a day,and continuous injection of 3 days for the study of its day precision.4.Stability experimentThe samples were divided into two parts,placed at-15? and-4?,respectively.Then placed on the first day of the sample and placed on the third day of the sample according to the chromatographic conditions were tested.The chromatographic peaks of chebulagic acid,chebulinic acid,gallic acid,chebulinin and corilagin were determined,respectively.5.HPLC for the major phenolic acids of extracts of three extraction methodsThe five phenolic acids extracted from T.chebula were loaded into chromatographed?Ultimate LP-C18?250 mm×4.6 mm,5 ?m??and successively eluted with methanol and 0.6% phosphoric acid at 1.2 m L/min on 40?.Data was collected at the wavelength of 278 nm.Results1.Results of total phenolic contentsGallic acid standard curve equation was Y=0.1089X+0.0056,correlation coefficient R2=0.9994.Gallic acid in the concentration range of 0-8 ?g/mL has a good linear relationship.Sequential extraction in fixed bed process: the total phenolic contents of acetone and water extracts were?656.73±1.90?mg/g and?459.24±2.89?mg/g,respectively.One-step in fixed bed process: the total phenol contents of acetone extracts and water extracts were?663.48±2.22?mg/g and?466.07±1.88?mg/g,respectively.Conventional extraction process: the total phenolic contents of acetone extracts and water extracts were?446.81±3.17?mg/g and?307.71±2.40?mg/g,respectively.When acetone was used as solvent,the total phenolic contents of acetone extracts were A>SA>CA?p<0.05?.When water was used as solvent,the total phenol contents of water extracts were W>SW>CW?p<0.05?.2.Results of linear relationship investigationGallic acid,corilagin,chebulinin,chebulagic acid and chebulinic acid were in a good linear relationship in the range of 0.75-10 ?g/m L,0.75-10 ?g/m L,1.2563-20.10 ?g/m L,2.5125-40.2 ?g/m L,0.75-10 ?g/m L.3.Results of precision experimentThe intraday precision and daytime precision of RSD were less than 5%,indicating that the precision of the method and instrument was good.4.Results of stability testAccording to the results,it was found that the standard was stable at-15?.5.The results of HPLC detection of total phenolic acids in three extraction processesAccording to the results,yields of low phenolic acids?g/100 g fruits?were also obtained in the sc CO2 extraction?1st step?,which is almost negligible.But high average yields of 17.61±0.37% and 10.64±0.43% were obtained in the acetone and aqueous extracts in the second and third steps,respectively.Acetone extracted most phenolic compounds?about 62.34%?,much higher than that of phenolic acids extracted from aqueous?about 37.66%?.The yields of major phenolic acids in one-step fixed bed process: the yield of acetone extraction was 23.26±0.87%;the yield of aqueous extraction was 16.08±0.29%.The yield of major phenolic acids in conventional process: the yield of acetone extraction was 14.57±0.51%;the yield of aqueous extraction was 12.19±0.14%Part 3.Antioxidant activities of different solvent extracts from Terminalia cheubla in vitroMethods1.Antioxidant activity by 2,2-diphenyl-1-picrylhydarzyl?DPPH?assayA DPPH solution with a concentration of 100 ?g/mL was prepared.Precision extraction of the extract of 25 mg,and the liquor was prepared by using ethanol as the mass concentration of 1 mg/mL.A sample volume of 5,10,25,50,125 and 250 ?g/mL was prepared in a 25 mL volumetric flask.Accurately remove the sample to be measured 3 mL of the solution and DPPH solution 3 m L,after mixing at room temperature for 30 min,the optical density of test solution were measured at 518 nm,recorded as Ai.Accurately remove the sample to be measured 3 mL of the solution and methanol solution 3 mL,after mixing at room temperature for 30 min,the optical density of test solution were measured at 518 nm,recorded as Aj.Accurate removal of DPPH solution 3 mL and methanol 3 mL,after mixing at room temperature for 30 min,the optical density of test solution were measured at 518 nm,recorded as A0.The experiment was carried out under dark conditions and ethanol was used as a blank control.SPSS18.0 statistical software was used to calculate the semi-inhibitory concentration(EC₅₀,?g/mL)of each extract under DPPH method.The clearance was calculated according to the formula.2.Determination of free radical scavenging activity of ABTSABTS+· solution were preparation,and the solution in the light of 16 h.The solution requirements the absorbance was 0.70±0.02 at the wavelength of 734 nm.Precision extraction of the acetone extract and aqueous extract and Vc of 40 mg,respectively.The liquor was prepared by using methanol as the mass concentration of 4 mg/mL.A sample volume of 5,10,20,40,80,160,320 and 640 ?g/mL was prepared.Accurately remove the sample to be measured 158 ?L of the solution and ABTS+· solution 3 m L,after mixing at room temperature for 6 min,the optical density of test solution were measured at 734 nm,recorded as B.Accurately remove the sample to be measured 158 ?L of the solution and ethanol solution 3 m L,after mixing at room temperature for 6 min,the optical density of test solution were measured at 734 nm,recorded as C.Accurate removal of ABTS+· solution 3 mL and ethanol 158 ?L,after mixing at room temperature for 6 min,the optical density of test solution were measured at 734 nm,recorded as A.The experiment was carried out under dark conditions and ethanol was used as a blank control.SPSS18.0 statistical software was used to calculate the semi-inhibitory concentration(EC₅₀,?g/mL)of each extract under ABTS method.The clearance was calculated according to the formula.3.Determination of antioxidant activity by reducing methodPrecision extraction of the acetone extract and aqueous extract and Vc of 40 mg,respectively.The liquor was prepared by using methanol as the mass concentration of 4 mg/m L.A sample volume of 0.005,0.01,0.02,0.04,0.08,0.16,0.32 and 0.64 mg/mL was prepared.The optical density of the solution was determined at 700 nm by ultraviolet spectrophotometry.The higher the value of the optical density is,the stronger the reduction ability of the compound is.Results1.Results of DPPH free radical scavenging activityThe EC₅₀ values of acetone extracts and aqueous extracts of sequential extraction in fixed bed process were 14.94 ?g/mL and 43.45 ?g/mL,respectively.The EC₅₀ values of acetone extracts and aqueous extracts of one-step extraction in fixed bed process were 8.73 ?g/mL and 24.53 ?g/mL,respectively.The EC₅₀ values of acetone extracts and aqueous extracts of conventional extraction process were 20.04 ?g/mL and 69.29 ?g/mL,respectively.When acetone was used as solvent,the antioxidant activity of each extract was as following results: A>SA>CA?p<0.05?.When water was used as solvent,the antioxidant activity of each extract was as follows: W>SW>CW?p<0.05?.The order of the results is consistent with the total phenolic content.It is suggested that the antioxidant activity of acetone and aqueous extracts of T.chebula may be related to the presence of phenolic compounds.2.Results of ABTS free radical scavenging activityThe EC₅₀ values of acetone extracts and aqueous extracts of one-step extraction in fixed bed process were 20.273 ?g/mL and 33.064 ?g/mL,respectively.The EC₅₀ values of Vc was 17.680 ?g/mL.According to the total phenolic contents and the value of EC₅₀ can calculate the EC₅₀ of acetone and aqueous extraction methods of T.chebula scavenging ABTS free radical values were 32.583 ?g/mL and 72.282 ?g/mL,respectively.Suggesting that using acetone as solvent of ABTS free radical scavenging effect is 2.22 times with water as solvent.3.Results of reducing capacityThe reduction ability of acetone and aqueous extracts of T.chebula showed correlation with the concentration.The higher the concentration,the reducing capability is strong.Acetone and aqueous extracts of T.chebula have reducing power,and reducing power of the acetone extract was higher than the same quality concentration of aqueous extract.When the concentration of 0.005⁰.04 mg/m L,the value of optical density of the extracts increased with the increase of concentration tends to be smooth;when the concentration of 0.04⁰.32 mg/mL,the extracts of the value of optical density increases rapidly;when the concentration of 0.64 mg/mL,acetone and aqueous extracts and Vc of the optical density values were 1.877,1.547 and 2.281,respectively.Conclusions:1.Sequential extraction in fixed bed process with higher efficiency and global yield.The total yield of the extract was 62.84±1.54%,the yield of supercritical CO2 extract was 1.25 ± 0.04%,the yield of acetone extracts was 28.15±0.94%,and the aqueous extraction yield was 33.44±0.56%.According to the results,the yield of T.chebula fruits was improved.2.Total phenolic contents were measured by Folin-Ciocalteu method,and the five phenolic acids of T.chebula were determined by HPLC method.The content of the total phenolic acids in the acetone extract is higher than others,suggesting that acetone can be used as an effective solvent for the extraction of phenolic acids.Sequential extraction in fixed bed process can extract more phenolic compounds than one-step in fixed bed and convention extraction process.3.Using DPPH and ABTS radical scavenging assay and reducing power to evaluate the vitro antioxidant activities of extracts from Terminalia chebula.It was confirmed that the acetone and aqueous extracts,especially acetone extracts,have better excellent scavenging ability of DPPH and ABTS free radical and vitro antioxidant capacity,suggesting that T.chebula fruits has the potential as a natural antioxidant development.Terminalia chebual has development potential as a natural antioxidant.