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Abstract To promote the recovery of saponins from Tribulus terrestris L.， the water dissolution and ethanol precipitation method was adopted to extract saponins from the powder prepared from T. terrestris fruit. The optimal process was to extract with 9 fold of the extracting water at the ethanol concentration of 85 for 3 times， each time 3.5 h. The effects of the factors ranked as the extracting water quantity > treatment time > treatment times > ethanol concentration. Under these optimal conditions， the recovery of saponins was 2.81%. After these crude saponins were purified by D101 macroporous adsorptive resin， the purity was found to be promoted through the UV scanning method， and the recovery of the refined saponins was up to 1.53%. Both of the recovery values were higher than that of the existing process.
Key words Tribulus terrestris L.; Saponins; Orthogonal experiment; Recovery; Macroporous adsorptive resin
Saponins are a kind of secondary metabolites widely present in plants. They are natural active substances formed by plants during the long term evolution to protect against pathogenic microorganism damage and herbivore damage[1]. Saponins are glycocalyx composed of terpenes or steroidal sapogenins and saccharides， with anti inflammatory[1]， anti cancer[2]， blood viscosity reducing[3-4]， antioxidant[5]， antidiabetic[6] and other effects， and thus are widely used in the pharmaceutical industry and food industry. Saponins also have pesticidal and germicidal effects， and can also be used as natural bio pesticides and germicides[7-8]. Due to its strong polarity， saponins are natural detergents and surfactants. In recent years， saponins have also been used as bio agricultural pesticides and environmental remediation agents[9-10].
Tribulus terrestris L. is an annual herb belonging to Tribulus in Polygonaceae. It is native to the Mediterranean region and is now distributed throughout the warm tropical regions of the world. It is a traditional Chinese herbal medicine that can be used in health foods. It has the effects of calming the liver， invigorating blood circulation and improving eyesight[11]. Steroidal saponins are composed of six rings （27 carbon atoms） and saccharides， and the active ingredients of T. terrestris are mainly steroidal saponins[12]. Currently， there are 16 kinds of steroidal saponins which have been found in the whole class of T. terrestris， among which eleven are closed loop spirostanol saponins， and five are furan saponins in which the F ring is open[13]. According to the characteristic of saponins that they can be dissolved in water and diluted alcohol， the saponin components in T. terrestris fruit are often extracted by the water dissolution and alcohol precipitation method and ethanol refluxing extraction method in industrial production. Due to the strong polarity of saponins， the efficiency of ethanol refluxing extraction method is not high; although the water dissolution and alcohol precipitation method has certain advantages， the current industrially used water dissolution and alcohol precipitation method only optimizes the amount of extracting water， extraction times and extraction time， and the recovery is not ideal. The recovery of the crude saponins extracted by the above two methods is all below 2%. Through previous experiments， it is found that ethanol concentration has a certain effect on the extraction efficiency of saponins from T. terrestris by the water dissolution and ethanol precipitation method. Therefore， in this study， taking ethanol concentration as one of the investigation factors， the orthogonal experiment method was used to optimize the method of extracting saponins from T. terrestris fruit by the water dissolution and ethanol precipitation method， and the extraction product was partially purified， so as to provide reference for the optimization of the extraction parameters of saponins in industrial production.
Materials and Methods
Main materials
Raw materials
T. terrestris fruit： Liping Boyuan Ecological Agriculture Technology Co.， Ltd.
Reagents and instruments
Cobalt chloride： analytically pure， Jiangsu Yongfeng Chemical Reagent Factory; p dimethylaminobenzaldehyde： analytically pure， Tianjin Damao Chemical Reagent Factory; macroporous resin： D101 type， Tianjin Haiguang Chemical Co.， Ltd.; other reagents： domestic， analytically pure.
Alcohol meter： 40°-70°， 70°-100°， Hebei Wuqiang Huapeng Instrument Factory; ultraviolet scanning spectrophotometer： UVmini 1240， Shimadzu， Japan; electronic analytical balance： AUY120， Shimadzu， Japan; electric thermostat drying oven： 101， Beijing Zhongxing Weiye Instrument Co.， Ltd.; electric thermostatic water bath： HH 8 Type， Changzhou Nuoji Instrument Co.， Ltd.; refrigerated centrifuger： L530 type， Hunan Xiangyi Laboratory Instrument Development Co.，Ltd.; rotary evaporator： RE 52C type， Shanghai Biaohe Instrument Co.， Ltd.
Experimental methods
Preparation of T. terrestris saponins
T. terrestris saponins were prepared from T. terrestris fruit through drying， pulverization to 40 meshes， water extraction， centrifugation， concentration by rotary evaporation， ethanol precipitation at 30 ℃， centrifugation， concentration by rotary evaporation and purification of the macroporous resin portion.
Determination of T. terrestris saponin content
The content of T. terrestris saponins was determined by Yin[4]. The to be detected sample （2 ml） was added into a 25 ml volumetric flask， and diluted with methanol to constant volume， followed by shaking well to mix the solution and standing for 10 min. The supernatant （2 ml） was taken and added into a 10 ml test tube， and added with 5 ml of the p dimethylaminobenzaldehyde solution and 3 ml of methanol. The obtained solution was shaken well， sealed， and heated in a 60 ℃ water bath for 2 h. Then， the solution was rapidly cooled to room temperature， and with methanol as a substitute for the same treatment as a blank group which was used for zero setting， the absorbance was determined at 515 nm. Furthermore， distilled water was also used for zero setting， and the absorbance of the cobalt chloride solution was measured at 515 nm. For the preparation of p dimethylaminobenzaldehyde test solution： 1 g of p dimethylaminobenzaldehyde was dissolved in 9.0 ml of ethanol， added with 2.3 ml of concentrated hydrochloric acid and mixed well. For the preparation of the cobalt chloride solution： 0.6 g of cobalt chloride （CoCl 2·6H 2O） was dissolved in a 25 ml volumetric flask， added with distilled water to constant volume， and shaken well. Percentage content of T. terrestris saponins was calculated according to following equation：
Y=A S×5W R×0.009 3A R×W S×100%（1）
Wherein Y is the content of T. terrestris saponins， %; A S is the absorbance of the sample; A R is the absorbance of cobalt chloride; W R is the mass of cobalt chloride， g; and W S is the volume of the sample， ml.
The recovery was calculated according to following equation：
R=WW t×100%（2）
Wherein W is the mass of T. terrestris saponins， g; and W t is the mass of the sample， g.
Purification of the macroporous resin portion
T. terrestris fruit （200 g） powder was extracted by the water extraction and alcohol precipitation method. The saponin extract was adsorbed on the column at a flow rate of 2 ml/min. The effluent was collected and adsorbed once again on the column. The adsorption column was activated D101 type macroporous resin （80 cm×3 cm） which was eluted with 80% ethanol at a flow rate of 1.5 ml/min， and the eluate was collected according to 6 ml per tube. Finally， the T. terrestris content in the liquid of each tube was determined.
Detection of T. terrestris saponins by ultraviolet scanning
Referring the method of Guo[14]， 1 ml of the saponin solution was taken and scanned at a wavelength of 200-300 nm with ethanol used as a blank， to detect whether there was an absorption peak.
Single factor experiment
The extracting water quantity： 40 g of T. terrestris powder was added to each of five conical flasks. With the extraction times， extraction time and ethanol concentration fixed as 1 time， 2 h and 75， respectively， the extracting water quantity was set to be 3， 5， 7， 9 and 11 fold of the mass of the T. terrestris powder， respectively. The saponins were extracted under above conditions， and the saponin contents were finally determined.
Extraction times： 40 g of T. terrestris powder was added to each of five conical flasks. With the extracting water quantity， extraction time and ethanol concentration fixed as nine fold of the mass of the T. terrestris powder， 2 h and 75， respectively， the saponins in the powder was extracted for 1， 2， 3， 4， and 5 times， respectively. The saponin contents were finally determined.
Extraction time： 40 g of T. terrestris powder was added to each of five conical flasks. With the amount of the extracting water， extraction times and ethanol concentration fixed as 7 fold of the mass of the T. terrestris powder， 1 time and 75， respectively， the saponins in the powder were extracted for 1.5， 2.0， 2.5， 3.0 and 3.5 h， respectively. The saponin contents were finally determined. Ethanol concentration： 40 g of T. terrestris powder was added to each of five conical flasks. With the amount of the extracting water， extraction times and extraction time fixed as 7 fold of the mass of the T. terrestris powder， 1 time and 2 h， respectively， the saponins in the powder were extracted at the ethanol concentrations of 55， 65， 75， 85 and 95， respectively. The saponin contents were finally determined.
Orthogonal test
Through single factor experiments， the extracting water quantity， extraction times， extraction time and ethanol concentration were selected as the investigation factors. With saponin content as the investigation index， and a 4 factor 3 level orthogonal experiment was carried out to optimize the process conditions， and the optimal conditions were then verified.
Data processing
Data were expressed as mean ± SD （n=3）， and SPSS 19.0 was used for comparison of significant differences （P<0.05） （LSD method）， with significant differences in lowercase letters.
Analysis on Results of Single Factor Experiment
Effect of extracting water quantity on the recovery of T. terrestris saponins
As can be seen from Fig. 1， when the extracting water quantity was 9 fold of the mass of the powder， the recovery of saponins was the largest， being 2.67%， and the recovery was 2.2 times of that when the extracting water quantity was 3 fold. When the extracting water quantity was too low， saponins cannot be sufficiently dissolved， but when the extracting water quantity was too large， the extraction efficiency was also lowered. Therefore， the extracting water was selected to be 9 fold.
Effect of extraction times on recovery of T. terrestris saponins
As can be seen from Fig. 2， when the extraction was performed for 2 times， the recovery of saponins was the highest， being 2.13% ， and when extracting for 1 time， the recovery was only 1.25% . When the number of extraction times was greater than 2， the recovery did not increase significantly. Therefore， the number of extraction times was selected as 2.
Effect of extraction time on recovery of T. terrestris saponins
As can be seen from Fig. 3， with the extraction time increasing， the recovery of saponins increased. When the extraction time was 3 h， the recovery of saponins was the highest， reaching 1.57% . However， when the extraction time was further prolonged， the recovery decreased， so the extraction time was selected as 3 h. Effect of ethanol concentration on recovery of T. terrestris saponins
It can be seen from Fig. 4 that the recovery of saponins gradually increased with the increase of ethanol concentration， and the recovery was the highest when the ethanol concentration was 85， which was 1.77%. Steroidal saponins have a longer carbohydrate chain， and although they are strongly polar， they also have greater solubility in alcohols. Higher ethanol concentrations should be used for ethanol extraction. Therefore， the ethanol concentration was selected as 85.
Results of the orthogonal experiment
The orthogonal test was designed according to the results of the single factor experiments. The levels of the factors are shown in Table 1.
The 4 factor 3 level orthogonal experiment was performed according to Table 1， in which the extracting water quantity， extraction time， ethanol concentration and extraction times were used as factors. The experimental results are shown in Table 2.
The intuitive analysis of Table 2 showed that the optimal conditions for the extraction of saponins were A 2B 3C 2D 3， and the factors affecting the extraction effects of saponins ranked as the extracting water quantity > extraction time > extractions times > ethanol concentration.
Taking the C factor with the smallest range as the error term[15]， the results in Table 2 was subjected to variance analysis and F test. It was found that there was a significant difference in the extracting water quantity between any two of the levels. The extraction time， ethanol concentration and extraction times had no significant effects （Table 3）.
Three times of verification experiments were carried out under the optimized process conditions， and the recovery of saponins was （2.81±0.16）%， which was higher than that of reported methods in previous studies[4，16].
Purification of the macroporous resin portion
D101 type macroporous resin has stable physical properties and is a low cost method for preparing water soluble Chinese herbal medicines， which can be scaled up for large scale production[17]. The experiment was carried out with a constant concentration of 80% ethanol （Fig. 5）， and the peak was obtained at the elution volume of 66 ml. The recovery of the saponin product was 1.53% （Table 4）， which was significantly better than the existing process[4].
Ultraviolet scanning was carried out on the crude saponins obtained by the water dissolution and ethanol precipitation method and the refined saponins purified by D101 type macroporous resin， and it was found that there were absorption peaks at 265-275 nm， which accords with the ultraviolet absorption characteristics of saponins. After resin chromatography， the extract was not absorbed at 300 nm， indicating that the purity of the saponin extract was higher[14].
Conclusions
Based on the single factor experiments， the optimal conditions of the water dissolution and ethanol precipitation method were obtained by orthogonal experiment and variance analysis. Under the test conditions， the recovery of saponins was 2.81%. After the crude product was purified by D101 macroporous resin， the recovery of saponins was 1.53%， which was higher than that of the existing process. The experimental process parameters have strong reference value for the improvement of the existing industrial saponin preparation.
References
[1] CHEOK CY， SALMAN HAK， SULAIMAN R. Extraction and quantification of saponins： A review[J]. Food Research International， 2014， 59： 16-40.
[2] KIM TDK， THANH HN， THUY DN， et al. Anticancer effects of saponin and saponin–phospholipid complex of Panax notoginseng grown in Vietnam[J]. Asian Pacific Journal of Tropical Biomedicine， 2016， 6（9）： 795-800.
[3] SINGH B， SINGH JP， SINGH N， et al. Saponins in pulses and their health promoting activities： A review[J]. Food Chemistry， 2017， 233： 540-549.
[4] YIN X， LIU W， YAN WM. Study on water extraction of saponins from Tribulus terrestris L.[J]. Shanghai Medical & Pharmaceutical Journal， 2004， 25（5）： 231-232. （in Chinese）
[5] SINGH D， CHAUDHURI PK. Structural characteristics， bioavailability and cardioprotective potential of saponins[J]. Integrative Medicine Research， 2018， 7（1）： 33-43.
[6] YANG J， ZHU LL， YANG Z， et al. Separation of furostanol saponins by supercritical fluid chromatography[J]. Journal of Pharmaceutical and Biomedical Analysis， 2017， 145： 71-78.
[7] JIANG XG， CAO YI， Jrgensen LG， et al. Where does the toxicity come from in saponin extract[J]. Chemosphere， 2018， 204： 243-250.
[8] JIANG XG， HANSEN HCB， STROBEL BW， et al. What is the aquatic toxicity of saponin rich plant extracts used as biopesticides[J]. Environmental Pollution， 2018， 236： 416-424.
[9] WANG MX， WU BO， SHAH SN， et al. Saponins as natural adjuvant for antisense morpholino oligonucleotides delivery in vitro and in mdx mice[J]. Molecular Therapy Nucleic Acid， 2018， 11： 192-202.
[10] LIU ZF， LI ZG， ZHONG H， et al. Recent advances in the environmental applications of biosurfactant saponins： A review[J]. Journal of Environmental Chemical Engineering， 2017， 5（6）： 6030-6038.
[11] WANG FX， WANG ZF， KANG KP， et al. Comparative analysis on chemical constituents in different parts of Tritulus terrestris from different areas[J]. Chinese Traditional and Herbal Drugs， 2016， 47（6）： 897-904. （in Chinese） [12] XU YJ， XU TH， YANG JY， et al. Two new furostanol saponins from Tribulus terrestris L.[J]. Chinese Chemical Letters， 2010， 21（5）： 580-583.
[13] KANG LI PING， WU KE LEI， YU HE SHUI， et al. Steroidal saponins from Tribulus terrestris[J]. Phytochemistry， 2014， 107： 182-189.
[14] GUO Z. Applications of UV spectrometry in the structural identification of natural product[J]. Chinese Journal of Spectroscopy Laboratory， 2006， 23（3）： 594-597. （in Chinese）
[15] ZHANG XF， CHENG G， HE Q， et al. Separation of procyanidin from grape seed by aqueous two phase system[J]. Food & Machinery， 2017， 3（33）： 168-173. （in Chinese）
[16] SONG YX， MIN YT. Study on extract and partial purification of Tribulus terrestris[J]. Journal of Zhongzhou University， 2014， 31（2）： 117-120. （in Chinese）
[17] WUN QW， WU S， FANG Y. Process for purifying flavonoids from cigar formula by D101 macroporous resin[J]. Chinese Traditional Patent Medicine， 2014， 36（10）： 2209-2211. （in Chinese）
Key words Tribulus terrestris L.; Saponins; Orthogonal experiment; Recovery; Macroporous adsorptive resin
Saponins are a kind of secondary metabolites widely present in plants. They are natural active substances formed by plants during the long term evolution to protect against pathogenic microorganism damage and herbivore damage[1]. Saponins are glycocalyx composed of terpenes or steroidal sapogenins and saccharides， with anti inflammatory[1]， anti cancer[2]， blood viscosity reducing[3-4]， antioxidant[5]， antidiabetic[6] and other effects， and thus are widely used in the pharmaceutical industry and food industry. Saponins also have pesticidal and germicidal effects， and can also be used as natural bio pesticides and germicides[7-8]. Due to its strong polarity， saponins are natural detergents and surfactants. In recent years， saponins have also been used as bio agricultural pesticides and environmental remediation agents[9-10].
Tribulus terrestris L. is an annual herb belonging to Tribulus in Polygonaceae. It is native to the Mediterranean region and is now distributed throughout the warm tropical regions of the world. It is a traditional Chinese herbal medicine that can be used in health foods. It has the effects of calming the liver， invigorating blood circulation and improving eyesight[11]. Steroidal saponins are composed of six rings （27 carbon atoms） and saccharides， and the active ingredients of T. terrestris are mainly steroidal saponins[12]. Currently， there are 16 kinds of steroidal saponins which have been found in the whole class of T. terrestris， among which eleven are closed loop spirostanol saponins， and five are furan saponins in which the F ring is open[13]. According to the characteristic of saponins that they can be dissolved in water and diluted alcohol， the saponin components in T. terrestris fruit are often extracted by the water dissolution and alcohol precipitation method and ethanol refluxing extraction method in industrial production. Due to the strong polarity of saponins， the efficiency of ethanol refluxing extraction method is not high; although the water dissolution and alcohol precipitation method has certain advantages， the current industrially used water dissolution and alcohol precipitation method only optimizes the amount of extracting water， extraction times and extraction time， and the recovery is not ideal. The recovery of the crude saponins extracted by the above two methods is all below 2%. Through previous experiments， it is found that ethanol concentration has a certain effect on the extraction efficiency of saponins from T. terrestris by the water dissolution and ethanol precipitation method. Therefore， in this study， taking ethanol concentration as one of the investigation factors， the orthogonal experiment method was used to optimize the method of extracting saponins from T. terrestris fruit by the water dissolution and ethanol precipitation method， and the extraction product was partially purified， so as to provide reference for the optimization of the extraction parameters of saponins in industrial production.
Materials and Methods
Main materials
Raw materials
T. terrestris fruit： Liping Boyuan Ecological Agriculture Technology Co.， Ltd.
Reagents and instruments
Cobalt chloride： analytically pure， Jiangsu Yongfeng Chemical Reagent Factory; p dimethylaminobenzaldehyde： analytically pure， Tianjin Damao Chemical Reagent Factory; macroporous resin： D101 type， Tianjin Haiguang Chemical Co.， Ltd.; other reagents： domestic， analytically pure.
Alcohol meter： 40°-70°， 70°-100°， Hebei Wuqiang Huapeng Instrument Factory; ultraviolet scanning spectrophotometer： UVmini 1240， Shimadzu， Japan; electronic analytical balance： AUY120， Shimadzu， Japan; electric thermostat drying oven： 101， Beijing Zhongxing Weiye Instrument Co.， Ltd.; electric thermostatic water bath： HH 8 Type， Changzhou Nuoji Instrument Co.， Ltd.; refrigerated centrifuger： L530 type， Hunan Xiangyi Laboratory Instrument Development Co.，Ltd.; rotary evaporator： RE 52C type， Shanghai Biaohe Instrument Co.， Ltd.
Experimental methods
Preparation of T. terrestris saponins
T. terrestris saponins were prepared from T. terrestris fruit through drying， pulverization to 40 meshes， water extraction， centrifugation， concentration by rotary evaporation， ethanol precipitation at 30 ℃， centrifugation， concentration by rotary evaporation and purification of the macroporous resin portion.
Determination of T. terrestris saponin content
The content of T. terrestris saponins was determined by Yin[4]. The to be detected sample （2 ml） was added into a 25 ml volumetric flask， and diluted with methanol to constant volume， followed by shaking well to mix the solution and standing for 10 min. The supernatant （2 ml） was taken and added into a 10 ml test tube， and added with 5 ml of the p dimethylaminobenzaldehyde solution and 3 ml of methanol. The obtained solution was shaken well， sealed， and heated in a 60 ℃ water bath for 2 h. Then， the solution was rapidly cooled to room temperature， and with methanol as a substitute for the same treatment as a blank group which was used for zero setting， the absorbance was determined at 515 nm. Furthermore， distilled water was also used for zero setting， and the absorbance of the cobalt chloride solution was measured at 515 nm. For the preparation of p dimethylaminobenzaldehyde test solution： 1 g of p dimethylaminobenzaldehyde was dissolved in 9.0 ml of ethanol， added with 2.3 ml of concentrated hydrochloric acid and mixed well. For the preparation of the cobalt chloride solution： 0.6 g of cobalt chloride （CoCl 2·6H 2O） was dissolved in a 25 ml volumetric flask， added with distilled water to constant volume， and shaken well. Percentage content of T. terrestris saponins was calculated according to following equation：
Y=A S×5W R×0.009 3A R×W S×100%（1）
Wherein Y is the content of T. terrestris saponins， %; A S is the absorbance of the sample; A R is the absorbance of cobalt chloride; W R is the mass of cobalt chloride， g; and W S is the volume of the sample， ml.
The recovery was calculated according to following equation：
R=WW t×100%（2）
Wherein W is the mass of T. terrestris saponins， g; and W t is the mass of the sample， g.
Purification of the macroporous resin portion
T. terrestris fruit （200 g） powder was extracted by the water extraction and alcohol precipitation method. The saponin extract was adsorbed on the column at a flow rate of 2 ml/min. The effluent was collected and adsorbed once again on the column. The adsorption column was activated D101 type macroporous resin （80 cm×3 cm） which was eluted with 80% ethanol at a flow rate of 1.5 ml/min， and the eluate was collected according to 6 ml per tube. Finally， the T. terrestris content in the liquid of each tube was determined.
Detection of T. terrestris saponins by ultraviolet scanning
Referring the method of Guo[14]， 1 ml of the saponin solution was taken and scanned at a wavelength of 200-300 nm with ethanol used as a blank， to detect whether there was an absorption peak.
Single factor experiment
The extracting water quantity： 40 g of T. terrestris powder was added to each of five conical flasks. With the extraction times， extraction time and ethanol concentration fixed as 1 time， 2 h and 75， respectively， the extracting water quantity was set to be 3， 5， 7， 9 and 11 fold of the mass of the T. terrestris powder， respectively. The saponins were extracted under above conditions， and the saponin contents were finally determined.
Extraction times： 40 g of T. terrestris powder was added to each of five conical flasks. With the extracting water quantity， extraction time and ethanol concentration fixed as nine fold of the mass of the T. terrestris powder， 2 h and 75， respectively， the saponins in the powder was extracted for 1， 2， 3， 4， and 5 times， respectively. The saponin contents were finally determined.
Extraction time： 40 g of T. terrestris powder was added to each of five conical flasks. With the amount of the extracting water， extraction times and ethanol concentration fixed as 7 fold of the mass of the T. terrestris powder， 1 time and 75， respectively， the saponins in the powder were extracted for 1.5， 2.0， 2.5， 3.0 and 3.5 h， respectively. The saponin contents were finally determined. Ethanol concentration： 40 g of T. terrestris powder was added to each of five conical flasks. With the amount of the extracting water， extraction times and extraction time fixed as 7 fold of the mass of the T. terrestris powder， 1 time and 2 h， respectively， the saponins in the powder were extracted at the ethanol concentrations of 55， 65， 75， 85 and 95， respectively. The saponin contents were finally determined.
Orthogonal test
Through single factor experiments， the extracting water quantity， extraction times， extraction time and ethanol concentration were selected as the investigation factors. With saponin content as the investigation index， and a 4 factor 3 level orthogonal experiment was carried out to optimize the process conditions， and the optimal conditions were then verified.
Data processing
Data were expressed as mean ± SD （n=3）， and SPSS 19.0 was used for comparison of significant differences （P<0.05） （LSD method）， with significant differences in lowercase letters.
Analysis on Results of Single Factor Experiment
Effect of extracting water quantity on the recovery of T. terrestris saponins
As can be seen from Fig. 1， when the extracting water quantity was 9 fold of the mass of the powder， the recovery of saponins was the largest， being 2.67%， and the recovery was 2.2 times of that when the extracting water quantity was 3 fold. When the extracting water quantity was too low， saponins cannot be sufficiently dissolved， but when the extracting water quantity was too large， the extraction efficiency was also lowered. Therefore， the extracting water was selected to be 9 fold.
Effect of extraction times on recovery of T. terrestris saponins
As can be seen from Fig. 2， when the extraction was performed for 2 times， the recovery of saponins was the highest， being 2.13% ， and when extracting for 1 time， the recovery was only 1.25% . When the number of extraction times was greater than 2， the recovery did not increase significantly. Therefore， the number of extraction times was selected as 2.
Effect of extraction time on recovery of T. terrestris saponins
As can be seen from Fig. 3， with the extraction time increasing， the recovery of saponins increased. When the extraction time was 3 h， the recovery of saponins was the highest， reaching 1.57% . However， when the extraction time was further prolonged， the recovery decreased， so the extraction time was selected as 3 h. Effect of ethanol concentration on recovery of T. terrestris saponins
It can be seen from Fig. 4 that the recovery of saponins gradually increased with the increase of ethanol concentration， and the recovery was the highest when the ethanol concentration was 85， which was 1.77%. Steroidal saponins have a longer carbohydrate chain， and although they are strongly polar， they also have greater solubility in alcohols. Higher ethanol concentrations should be used for ethanol extraction. Therefore， the ethanol concentration was selected as 85.
Results of the orthogonal experiment
The orthogonal test was designed according to the results of the single factor experiments. The levels of the factors are shown in Table 1.
The 4 factor 3 level orthogonal experiment was performed according to Table 1， in which the extracting water quantity， extraction time， ethanol concentration and extraction times were used as factors. The experimental results are shown in Table 2.
The intuitive analysis of Table 2 showed that the optimal conditions for the extraction of saponins were A 2B 3C 2D 3， and the factors affecting the extraction effects of saponins ranked as the extracting water quantity > extraction time > extractions times > ethanol concentration.
Taking the C factor with the smallest range as the error term[15]， the results in Table 2 was subjected to variance analysis and F test. It was found that there was a significant difference in the extracting water quantity between any two of the levels. The extraction time， ethanol concentration and extraction times had no significant effects （Table 3）.
Three times of verification experiments were carried out under the optimized process conditions， and the recovery of saponins was （2.81±0.16）%， which was higher than that of reported methods in previous studies[4，16].
Purification of the macroporous resin portion
D101 type macroporous resin has stable physical properties and is a low cost method for preparing water soluble Chinese herbal medicines， which can be scaled up for large scale production[17]. The experiment was carried out with a constant concentration of 80% ethanol （Fig. 5）， and the peak was obtained at the elution volume of 66 ml. The recovery of the saponin product was 1.53% （Table 4）， which was significantly better than the existing process[4].
Ultraviolet scanning was carried out on the crude saponins obtained by the water dissolution and ethanol precipitation method and the refined saponins purified by D101 type macroporous resin， and it was found that there were absorption peaks at 265-275 nm， which accords with the ultraviolet absorption characteristics of saponins. After resin chromatography， the extract was not absorbed at 300 nm， indicating that the purity of the saponin extract was higher[14].
Conclusions
Based on the single factor experiments， the optimal conditions of the water dissolution and ethanol precipitation method were obtained by orthogonal experiment and variance analysis. Under the test conditions， the recovery of saponins was 2.81%. After the crude product was purified by D101 macroporous resin， the recovery of saponins was 1.53%， which was higher than that of the existing process. The experimental process parameters have strong reference value for the improvement of the existing industrial saponin preparation.
References
[1] CHEOK CY， SALMAN HAK， SULAIMAN R. Extraction and quantification of saponins： A review[J]. Food Research International， 2014， 59： 16-40.
[2] KIM TDK， THANH HN， THUY DN， et al. Anticancer effects of saponin and saponin–phospholipid complex of Panax notoginseng grown in Vietnam[J]. Asian Pacific Journal of Tropical Biomedicine， 2016， 6（9）： 795-800.
[3] SINGH B， SINGH JP， SINGH N， et al. Saponins in pulses and their health promoting activities： A review[J]. Food Chemistry， 2017， 233： 540-549.
[4] YIN X， LIU W， YAN WM. Study on water extraction of saponins from Tribulus terrestris L.[J]. Shanghai Medical & Pharmaceutical Journal， 2004， 25（5）： 231-232. （in Chinese）
[5] SINGH D， CHAUDHURI PK. Structural characteristics， bioavailability and cardioprotective potential of saponins[J]. Integrative Medicine Research， 2018， 7（1）： 33-43.
[6] YANG J， ZHU LL， YANG Z， et al. Separation of furostanol saponins by supercritical fluid chromatography[J]. Journal of Pharmaceutical and Biomedical Analysis， 2017， 145： 71-78.
[7] JIANG XG， CAO YI， Jrgensen LG， et al. Where does the toxicity come from in saponin extract[J]. Chemosphere， 2018， 204： 243-250.
[8] JIANG XG， HANSEN HCB， STROBEL BW， et al. What is the aquatic toxicity of saponin rich plant extracts used as biopesticides[J]. Environmental Pollution， 2018， 236： 416-424.
[9] WANG MX， WU BO， SHAH SN， et al. Saponins as natural adjuvant for antisense morpholino oligonucleotides delivery in vitro and in mdx mice[J]. Molecular Therapy Nucleic Acid， 2018， 11： 192-202.
[10] LIU ZF， LI ZG， ZHONG H， et al. Recent advances in the environmental applications of biosurfactant saponins： A review[J]. Journal of Environmental Chemical Engineering， 2017， 5（6）： 6030-6038.
[11] WANG FX， WANG ZF， KANG KP， et al. Comparative analysis on chemical constituents in different parts of Tritulus terrestris from different areas[J]. Chinese Traditional and Herbal Drugs， 2016， 47（6）： 897-904. （in Chinese） [12] XU YJ， XU TH， YANG JY， et al. Two new furostanol saponins from Tribulus terrestris L.[J]. Chinese Chemical Letters， 2010， 21（5）： 580-583.
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