Parameter optimization method of cistanche ethanol glycoside compound extraction process

Abstract

The invention relates to the technical field of cistanche deserticola extraction, in particular to a parameter optimization method of a cistanche deserticola ethanol glycoside compound extraction process, a first-order model obtained by adopting a screening test design method is as follows: YPhGs = 95.8991-12.6459 X1-2.8928 X2 + 3.7767 X3 + 9.1595 X4 + 6.7017 X5, YPhGs represents the content of phenylethanoid glycoside compounds, X1 represents the ratio of an ethanol water solution to desert cistanche deserticola, X2 represents the ratio of ethanol water solution to desert cistanche deserticola, X3 represents the ratio of ethanol water solution to desert cistanche deserticola, X3 represents the ratio of ethanol water solution to desert cistanche deserticola, and X4 represents the ratio of ethanol water solution to desert cistanche X2 represents the mixed treatment temperature of the ethanol aqueous solution and the desert cistanche, X3 represents the mixed treatment time of the ethanol aqueous solution and the desert cistanche, X4 represents the ultrasonic oscillation power of the desert cistanche in the ethanol aqueous solution, and X5 represents the concentration of the ethanol aqueous solution. According to the method, the relationship between the content of phenylethanoid glycoside and various parameters can be accurately predicted by adopting a screening test design method, and a better parameter value range is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of Cistanche deserticola extraction, and particularly relates to a method for optimizing parameters of an extraction process of Cistanche deserticola ethanol glycoside compounds. Background Art

[0002] Cistanche deserticola is a perennial parasitic herb of the genus Cistanches in the family Orobanchaceae, and is mostly distributed in dry areas such as warm deserts and wastelands in the Northern Hemisphere. Cistanche deserticola and its active ingredients have functions such as antibacterial, anti-aging, anti-fatigue, antioxidant, liver protection, immune enhancement, endocrine regulation, anti-tumor, memory improvement, laxative, reproductive function improvement, osteoporosis and Alzheimer's disease prevention. The active ingredients of Cistanche deserticola mainly include phenyl ethanol glycosides, iridoids and their glycosides, lignans and their glycosides, oligosaccharide esters, polyols and polysaccharides, etc. Among them, phenyl ethanol glycoside compounds usually contain rich phenolic hydroxyl groups, and these groups can act as free radical scavengers, thereby reducing oxidative stress and protecting cells from damage. In addition, phenyl ethanol glycoside compounds may reduce the inflammatory response by inhibiting the production of inflammatory mediators such as prostaglandins and cytokines. Therefore, phenyl ethanol glycoside compounds have potential therapeutic effects on inflammatory diseases such as arthritis and intestinal inflammation; at the same time, some phenyl ethanol glycoside compounds may provide protection to the cardiovascular system through mechanisms such as reducing blood pressure, improving vascular endothelial function, and reducing atherosclerosis; phenyl ethanol glycoside compounds may protect neurons through antioxidant and anti-inflammatory mechanisms and reduce the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. At present, water extraction method is mostly used to extract phenyl ethanol glycoside substances from Cistanche deserticola, but the content of the extracted phenyl ethanol glycoside compounds is relatively low.

[0003] The prior art discloses a method for extracting phenyl ethanol glycoside substances from Cistanche tubulosa, which combines the solvent extraction method and the high-shear homogenization and emulsification technology, and improves the yield of phenyl ethanol glycoside compounds in Cistanche deserticola. However, only the yield at high temperature is considered in the prior art, and the antioxidant activity of phenyl ethanol glycoside compounds in Cistanche deserticola is related to the key structures in the molecule, such as 3,4-dihydroxy phenethyl alcohol group and caffeoyl group. These structures are sensitive to temperature changes. When using the high-shear homogenization and emulsification technology, due to high temperature, the above-mentioned phenyl ethanol glycoside compounds are inactivated, the extraction rate of phenyl ethanol glycoside compounds is low, and the influence of other parameters on the extraction of phenyl ethanol glycoside substances is not considered. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect that the parameter optimization of the extraction process of phenyl ethanol glycoside compounds of Cistanche deserticola in the prior art is inaccurate, so as to provide a method for optimizing parameters of an extraction process of Cistanche deserticola ethanol glycoside compounds.

[0005] Therefore, another technical problem to be solved by the present invention is to overcome the defect of low extraction rate of phenyl ethanol glycosides in Cistanche deserticola in the prior art, so as to provide a method for optimizing the parameters of the extraction process of phenyl ethanol glycosides in Cistanche deserticola.

[0006] For this purpose, the present invention provides a method for optimizing the parameters of the extraction process of phenyl ethanol glycosides in Cistanche deserticola. The first-order model obtained by using the screening test design method is Y PhGs = 95.8991 - 12.6459X 1 - 2.8928X 2 + 3.7767X 3 + 9.1595X 4 + 6.7017X 5 , where Y PhGs represents the content of phenyl ethanol glycosides, and X 1 represents the ratio of the ethanol aqueous solution to Cistanche deserticola Y.C.Ma, in units of mL:g. X 2 represents the temperature of the mixture of the ethanol aqueous solution and Cistanche deserticola, X 3 represents the time of the mixture of the ethanol aqueous solution and Cistanche deserticola, X 4 represents the power of ultrasonic oscillation of Cistanche deserticola in the ethanol aqueous solution, X 5 represents the concentration of the ethanol aqueous solution.

[0007] In some embodiments, the response surface method is used to determine the second-order model as Y PhGs = 122.4750 + 1.2995X 1 + 5.1491X 4 + 5.4530X 5 + 2.7358X 1 X 4 + 3.1125X 1 X 5 - 3.0989X 4 X 5 - 7.3914X 2 X 1 - 15.1039X 2 X 4 - 11.7645X 2 X 5 , where X 1 X 4 represents the interaction term between the ratio of the ethanol solution to Cistanche deserticola and the power of ultrasonic oscillation, and X 1 X 5 represents the interaction term between the ratio of the ethanol solution to Cistanche deserticola and the concentration of the ethanol aqueous solution, in units of mL:g. X 4 X5 Represents the interaction term of the power of ultrasonic oscillation and the concentration of the ethanol aqueous solution, X 2 X 1 Represents the quadratic term of the proportion of the ethanol aqueous solution concentration and Cistanche deserticola, X 2 X 4 Represents the quadratic term of the power of ultrasonic oscillation, X 2 X 5 Represents the quadratic term of the concentration of the ethanol aqueous solution.

[0008] In some embodiments, the method for extracting phenylpropanoid glycosides from Cistanche deserticola includes the following steps: mixing Cistanche deserticola (Cistanche deserticola Y.C.Ma) and an ethanol aqueous solution under ultrasonic oscillation conditions, where the power of the ultrasonic oscillation is 50 - 500 W, the ratio of the ethanol aqueous solution to Cistanche deserticola is (5 - 80):1, in units of mL:g, and the concentration of the ethanol aqueous solution is 10 - 100 Vol%.

[0009] Preferably, the power of the ultrasonic oscillation is 100 - 300 W. More preferably, the power of the ultrasonic oscillation is 215 - 216 W.

[0010] Preferably, the ratio of the ethanol aqueous solution to Cistanche deserticola is (5 - 20):1, in units of mL:g. More preferably, the ratio of the ethanol aqueous solution to Cistanche deserticola is (13 - 14):1, in units of mL:g.

[0011] Preferably, the concentration of the ethanol aqueous solution is 30 - 70 Vol%. More preferably, the concentration of the ethanol aqueous solution is 54 - 55 Vol%.

[0012] Preferably, the temperature of the mixing treatment is 25 - 95 °C. More preferably, the temperature of the mixing treatment is 40 - 70 °C.

[0013] Preferably, the time of the mixing treatment is 5 - 120 min. More preferably, the time of the mixing treatment is 30 - 90 min.

[0014] In some embodiments, the temperature of the mixing treatment is 55 °C.

[0015] In some embodiments, the time of the mixing treatment is 60 min.

[0016] In some embodiments, the extraction process of cistanche phenyl ethanol glycosides further includes solid-liquid separation of the mixed solution after the mixing treatment, mixing the obtained filter residue with an ethanol solution under ultrasonic oscillation conditions, repeating at least 3 times, and combining the filtrates.

[0017] In some of these embodiments, the solid-liquid separation is centrifugation, the rotation speed of the centrifugation is 3000-5000 rpm, and the time of the centrifugation is 20-40 min.

[0018] In some of these embodiments, the extraction method further includes a step of freeze-drying the combined filtrate, and the temperature of the freeze-drying is -85 to -75 °C.

[0019] The technical solution of the present invention has the following advantages:

[0020] 1. A method for optimizing the parameters of an extraction process of cistanche phenylethanoid glycosides. By using a screening experimental design method, the first-order model is obtained as Y PhGs = 95.8991 - 12.6459X 1 - 2.8928X 2 + 3.7767X 3 + 9.1595X 4 + 6.7017X 5 , where Y PhGs represents the content of cistanche phenylethanoid glycosides, X 1 represents the ratio of the ethanol aqueous solution to Cistanche deserticola Y.C.Ma, in the unit of mL:g, X 2 represents the temperature of the mixture of the ethanol aqueous solution and Cistanche deserticola, X 3 represents the time of the mixture of the ethanol aqueous solution and Cistanche deserticola, X 4 represents the power of ultrasonic oscillation of Cistanche deserticola in the ethanol aqueous solution, X 5 represents the concentration of ethanol in the ethanol aqueous solution. The present invention uses a screening experimental design method to evaluate the effects of the ratio of the ethanol solution to Cistanche deserticola, the power of ultrasonic oscillation, the concentration of ethanol in the ethanol solution, the time of mixing treatment, and the temperature of mixing treatment on the extraction of cistanche phenylethanoid glycosides from Cistanche deserticola, accurately determines that the ratio of the ethanol solution to Cistanche deserticola, the power of ultrasonic oscillation, and the concentration of ethanol in the ethanol solution are parameters that significantly affect the content of cistanche phenylethanoid glycosides, and can accurately predict the relationship between the content of cistanche phenylethanoid glycosides and various parameters, and obtain a better parameter value range.

[0021] 2. The extraction method of cistanche phenylethanoid glycosides from Cistanche deserticola provided by the present invention uses the response surface method to determine the second-order model as Y PhGs = 122.4750 + 1.2995X 1 + 5.1491X 4 + 5.4530X 5 + 2.7358X 1 X 4 + 3.1125X 1 X 5-3.0989X 4 X 5 -7.3914X 2 X 1 -15.1039X 2 X 4 -11.7645X 2 X 5 , wherein, X 1 X 4 represents the interaction term of the ratio of the ethanol solution and Cistanche deserticola and the power of ultrasonic oscillation, X 1 X 5 represents the interaction term of the ratio of the ethanol solution and Cistanche deserticola and the concentration of the ethanol aqueous solution, X 4 X 5 represents the interaction term of the power of ultrasonic oscillation and the concentration of the ethanol aqueous solution, X 2 X 1 represents the quadratic term of the ratio of the ethanol solution and Cistanche deserticola, with the unit of mL:g, X 2 X 4 represents the quadratic term of the power of ultrasonic oscillation, X 2 X 5 represents the quadratic term of the concentration of the ethanol aqueous solution. The present invention uses the response surface method to determine the optimization parameters of the ratio of the ethanol solution and Cistanche deserticola, the power of ultrasonic oscillation, and the concentration of ethanol in the ethanol solution. Through the response surface method, considering the influence of each factor and the interaction of pairwise factors on the yield of phenylpropanoid glycosides, more accurate parameters can be optimized to make the extraction method of phenylpropanoid glycosides in Cistanche deserticola efficient, stable, suitable for popularization, without repeatedly repeating the corresponding experiments, and shortening the time for parameter optimization.

[0022] 3. An extraction method of phenylpropanoid glycosides from Cistanche deserticola provided by the present invention comprises the following steps: mixing Cistanche deserticola (Cistanche deserticola Y.C.Ma) and an ethanol solution under the condition of ultrasonic oscillation, wherein the power of the ultrasonic oscillation is 50 - 500 W, the ratio of the ethanol solution and Cistanche deserticola is (5 - 80):1, with the unit of mL:g, and the concentration of ethanol in the ethanol solution is 10 - 100 Vol%. The present invention combines an ethanol solution and ultrasonic oscillation, which can reduce the loss of heat-sensitive components and improve the yield of phenylpropanoid glycosides in Cistanche deserticola.

[0023] 4. The extraction method of phenylpropanoid glycosides from Cistanche deserticola provided by the present invention, wherein the power of ultrasonic oscillation is 215 - 216W, the concentration of ethanol in the ethanol solution is 54 - 55Vol%, and the ratio of the ethanol solution to Cistanche deserticola is (13 - 14):1, with the unit of mL:g. By defining the parameters of the significant influencing factors of the extraction method, the content of phenylpropanoid glycosides extracted from Cistanche deserticola can be significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 shows the content of phenyl ethanol glycosides in Cistanche deserticola obtained in Examples 1 - 6 of Experimental Example 1 of the present invention. Figure 1 In [the figure], A represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 1. Figure 1 In [the figure], B represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 2. Figure 1 In [the figure], C represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 3. Figure 1 In [the figure], D represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 4. Figure 1 In [the figure], E represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 5. Figure 1 In [the figure], F represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 6.

[0026] Figure 2 shows the content of phenyl ethanol glycosides in Cistanche deserticola obtained in Examples 1, 7 - 11 of Experimental Example 1 of the present invention. Figure 2 In [the figure], A represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 1. Figure 2 In [the figure], G represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 7. Figure 2 In [the figure], H represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 8. Figure 2 In [the figure], I represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 9. Figure 2 In [the figure], J represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 10. Figure 2 In [the figure], K represents the extract of phenyl ethanol glycosides from Cistanche deserticola obtained in Example 11.

[0027] Figure 3 is the content of phenylethanoid glycosides in Cistanche deserticola obtained from Examples 1, 12 - 16 in Experimental Example 1 of the present invention, Figure 3 where A in Figure 3 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 1, Figure 3 L in Figure 3 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 12, Figure 3 M in Figure 3 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 13,

[0028] Figure 4 is the content of phenylethanoid glycosides in Cistanche deserticola obtained from Examples 1, 17 - 21 in Experimental Example 1 of the present invention, Figure 4 where A in Figure 4 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 1, Figure 4 Q in Figure 4 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 17, Figure 4 R in Figure 4 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 18,

[0029] Figure 5 is the content of phenylethanoid glycosides in Cistanche deserticola obtained from Examples 1, 22 - 26 in Experimental Example 1 of the present invention, Figure 5 where A in Figure 5 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 1, Figure 5 V in Figure 5 represents the extract of phenylethanoid glycosides from Cistanche deserticola obtained in Example 22, Figure 5 W in Figure 5In this, Z represents the phenyl ethanol glycoside compound extract of Cistanche deserticola obtained in Example 26;

[0030] Figure 6 is the Pareto chart drawn in Experimental Example 2 of the present invention;

[0031] Figure 7 is the three-dimensional diagram of the content of phenyl ethanol glycoside compounds and the optimized parameters drawn in Experimental Example 2 of the present invention. Among them, Figure 7 in this, a represents the ultrasonic power, the response surface diagram of the liquid-solid ratio (the volume ratio of the ethanol aqueous solution to the mass of Cistanche deserticola, unit mL:g) and phenyl ethanol glycoside compounds, b represents the contour diagram of the ultrasonic power and the liquid-solid ratio (the volume ratio of the ethanol aqueous solution to the mass of Cistanche deserticola, unit mL:g), c represents the response surface diagram of the ethanol aqueous solution concentration, the liquid-solid ratio (the volume ratio of the ethanol aqueous solution to the mass of Cistanche deserticola, unit mL:g) and phenyl ethanol glycoside compounds, d represents the contour diagram of the ethanol aqueous solution concentration and the liquid-solid ratio (the volume ratio of the ethanol aqueous solution to the mass of Cistanche deserticola, unit mL:g), e represents the response surface diagram of the ethanol aqueous solution concentration, the ultrasonic power and phenyl ethanol glycoside compounds, f represents the contour diagram of the ethanol aqueous solution concentration and the ultrasonic power;

[0032] Figure 8 is the volcano plot of differential metabolites drawn in Experimental Example 3 of the present invention. Detailed implementation manners

[0033] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal implementation manners, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0034] For those steps or conditions not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specified for the manufacturers, they are all conventional reagent products that can be obtained through commercial purchase.

[0035] Example 1

[0036] This example provides a method for extracting phenylpropanol glycoside compounds from Cistanche deserticola. The specific steps and parameters are as follows:

[0037] (1) Mix a 50Vol% ethanol aqueous solution and desert cistanche in a ratio of 20:1 (in mL:g) under ultrasonic oscillation. The power of ultrasonic oscillation is 200W, the temperature during treatment is 55°C, and the treatment time is 60 min. After mixing, centrifuge at 4000 rpm for 30 min to obtain filter residue and filtrate, and retain the filtrate.

[0038] (2) Re-extract the filter residue 3 times according to the method in step (1), and combine the filtrates to obtain an extract of phenyl ethanol glycosides from desert cistanche.

[0039] Freeze-dry the obtained extract of phenyl ethanol glycosides from desert cistanche at -80°C to obtain a freeze-dried product of phenyl ethanol glycoside extract.

[0040] Example 2

[0041] This example provides a method for extracting phenylpropanol glycosides from desert cistanche. The specific steps and parameters are the same as those in Example 1, except that the ratio of ethanol solution to desert cistanche in step (1) is 5:1.

[0042] Example 3

[0043] This example provides a method for extracting phenylpropanol glycosides from desert cistanche. The specific steps and parameters are the same as those in Example 1, except that the ratio of ethanol solution to desert cistanche in step (1) is 10:1 (in mL:g).

[0044] Example 4

[0045] This example provides a method for extracting phenylpropanol glycosides from desert cistanche. The specific steps and parameters are the same as those in Example 1, except that the ratio of ethanol solution to desert cistanche in step (1) is 40:1 (in mL:g).

[0046] Example 5

[0047] This example provides a method for extracting phenylpropanol glycosides from desert cistanche. The specific steps and parameters are the same as those in Example 1, except that the ratio of ethanol solution to desert cistanche in step (1) is 60:1 (in mL:g).

[0048] Example 6

[0049] This example provides a method for extracting phenylpropanol glycosides from desert cistanche. The specific steps and parameters are the same as those in Example 1, except that the ratio of ethanol solution to desert cistanche in step (1) is 80:1 (in mL:g).

[0050] Example 7

[0051] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the temperature during the treatment in step (1) is 25 °C.

[0052] Embodiment 8

[0053] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the temperature during the treatment in step (1) is 40 °C.

[0054] Embodiment 9

[0055] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the temperature during the treatment in step (1) is 70 °C.

[0056] Embodiment 10

[0057] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the temperature during the treatment in step (1) is 85 °C.

[0058] Embodiment 11

[0059] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the temperature during the treatment in step (1) is 95 °C.

[0060] Embodiment 12

[0061] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the time during the treatment in step (1) is 5 min.

[0062] Embodiment 13

[0063] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the time during the treatment in step (1) is 10 min.

[0064] Embodiment 14

[0065] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1. The difference lies in that the time during the treatment in step (1) is 30 min.

[0066] Embodiment 15

[0067] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the time for treatment in step (1) is 90 min.

[0068] Embodiment 16

[0069] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the time for treatment in step (1) is 120 min.

[0070] Embodiment 17

[0071] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the power of ultrasonic oscillation in step (1) is 50 W.

[0072] Embodiment 18

[0073] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the power of ultrasonic oscillation in step (1) is 100 W.

[0074] Embodiment 19

[0075] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the power of ultrasonic oscillation in step (1) is 300 W.

[0076] Embodiment 20

[0077] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the power of ultrasonic oscillation in step (1) is 400 W.

[0078] Embodiment 21

[0079] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the power of ultrasonic oscillation in step (1) is 500 W.

[0080] Embodiment 22

[0081] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the concentration of the ethanol aqueous solution in step (1) is 10 Vol%.

[0082] Embodiment 23

[0083] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the concentration of the ethanol aqueous solution in step (1) is 30 Vol%.

[0084] Embodiment 24

[0085] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the concentration of the ethanol aqueous solution in step (1) is 70 Vol%.

[0086] Embodiment 25

[0087] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that the concentration of the ethanol aqueous solution in step (1) is 90 Vol%.

[0088] Embodiment 26

[0089] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are the same as those in Embodiment 1, except that an equal volume of absolute ethanol is used to replace the ethanol aqueous solution in step (1).

[0090] Embodiment 27

[0091] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are as follows:

[0092] (1) According to the ratio of ethanol solution to Cistanche deserticola of 20:1, in units of mL:g, mix 50 Vol% ethanol aqueous solution and Cistanche deserticola under ultrasonic oscillation conditions, where the power of ultrasonic oscillation is 200 W, the temperature during treatment is 55 °C, and the treatment time is 60 min. After mixing treatment, centrifuge at 3000 rpm for 40 min to obtain filter residue and filtrate, and retain the filtrate;

[0093] (2) Redo the extraction of the filter residue 3 times according to the method in step (1), and combine the filtrates to obtain an extract of phenyl ethanol glycosides from Cistanche deserticola.

[0094] The obtained extract of phenyl ethanol glycosides from Cistanche deserticola is freeze-dried at -85 °C to obtain a freeze-dried product of phenyl ethanol glycoside extract.

[0095] Embodiment 28

[0096] This embodiment provides a method for extracting phenylpropanoid glycosides from Cistanche deserticola, and the specific steps and parameters are as follows:

[0097] (1) Mix a 50Vol% ethanol aqueous solution and desert cistanche in a ratio of 20:1 (in mL:g) under ultrasonic oscillation. The power of ultrasonic oscillation is 200W, the temperature during treatment is 55°C, and the treatment time is 60min. After mixing, centrifuge at 5000rpm for 20min to obtain filter residue and filtrate, and retain the filtrate.

[0098] (2) Re-extract the filter residue 3 times according to the method in step (1), and combine the filtrates to obtain an extract of phenyl ethanol glycosides from desert cistanche.

[0099] Freeze-dry the obtained extract of phenyl ethanol glycosides from desert cistanche at -75°C to obtain a freeze-dried product of phenyl ethanol glycoside extract.

[0100] Experimental Example 1

[0101] Use ultraviolet spectrophotometry to determine the content of phenyl ethanol glycosides (PhGs) in the phenyl ethanol glycoside extract. The phenyl ethanol glycoside extract is the freeze-dried product of the phenyl ethanol glycoside extract prepared in Examples 1-26. The specific determination method is as follows:

[0102] Take echinacoside as the standard compound of PhGs, absorb the echinacoside standard solution, and prepare a series of reference substance solutions with concentrations of 0.004, 0.008, 0.012, 0.016, and 0.02mg / mL with 50% methanol. Measure the absorbance at a wavelength of 330nm respectively, and draw the standard curve of phenyl ethanol glycosides.

[0103] Dilute the freeze-dried products of the phenyl ethanol glycoside extracts obtained in Examples 1-26 with a 50vol% methanol aqueous solution to a solution with a concentration of 0.1mg / mL, and measure the absorbance of the solution at a wavelength of 330nm by ultraviolet-visible spectrophotometry to calculate the content of PhGs in each example.

[0104] The results are shown in Figures 1 - 5 It can be seen that the content of phenyl ethanol glycosides in the phenyl ethanol glycoside extract obtained in the examples of the present invention is 70-130mg / g.

[0105] Experimental Example 2

[0106] Use the Plackett-Burman design (PBD) to evaluate the effects of five variables, namely the ratio of ethanol aqueous solution to desert cistanche, the concentration of ethanol aqueous solution, the power of ultrasonic oscillation, the temperature during treatment, and the treatment time, on the content of PhGs in the phenyl ethanol glycoside extract. The content of PhGs in the phenyl ethanol glycoside extract is used to determine significant factors.

[0107] Based on the test results of Examples 1-26 in Experimental Example 1, the Box-Behnken design (BBD) was used to evaluate the important factors obtained from the previous analysis and the content of PhGs in the phenylethanoid glycoside compound extract. The influence levels of these important influencing factors were further examined at three levels (-1, 0, +1), where the factor level range was defined by the single-factor and PBD results.

[0108] Continue to refer to Figures 1 - 5 It can be seen that the effects of the five variables, namely the ratio of ethanol solution to Cistanche deserticola, the concentration of ethanol aqueous solution, the power of ultrasonic oscillation, the temperature during treatment, and the treatment time, on the content of PhGs in the phenylethanoid glycoside compound extract show a trend of first increasing and then decreasing. Therefore, based on the content of PhGs in the phenylethanoid glycoside compound extract as the leading result, the ratio of ethanol solution to Cistanche deserticola (5-20):1 (unit: mL:g), the treatment temperature of 40-70 °C, the treatment time of 30-90 min, the power of ultrasonic oscillation of 100-300 W, and the concentration of ethanol aqueous solution of 30-70 Vol% were determined as the factor ranges to be evaluated.

[0109] According to the PBD method, the experimental conditions of the 5 factors used in the single-factor experiment were screened. According to the steps of Example 1 and the parameter settings in Table 1, the phenylethanoid glycoside compound extract was obtained from Cistanche deserticola. After freeze-drying the obtained phenylethanoid glycoside compounds, the content of PhGs in the phenylethanoid glycoside compound extract was determined by the test method of Experimental Example 1, and the results are shown in Table 1.

[0110] Table 1 PBD experimental design and results

[0111]

[0112]

[0113] According to the results in Table 1, it can be seen that the ratio of ethanol solution to Cistanche deserticola (X 1 ) and the treatment temperature (X 2 ) have a negative impact on the content of PhGs, while the treatment time (X 3 ), the power of ultrasonic oscillation (X 4 ), and the concentration of ethanol aqueous solution (X 5 ) all have a positive impact on the 3 factors. The results of variance analysis (Table 2) show that X 1 , X 3 , X 4 and X 5 have statistical significance on the content of PhGs (p<0.05), among which X 1 , X 4 and X 5The influence was extremely significant (p<0.01). The first-order model of the PhGs content obtained from PBD is as follows:

[0114] Y PhGs = 95.8991 - 12.6459X 1 - 2.8928X 2 + 3.7767X 3 + 9.1595X 4 + 6.7017X 5 .

[0115] Table 2 ANOVA and regression analysis of PBD data

[0116]

[0117] By analyzing and plotting a Pareto chart using Design Expert software, see Figure 6 , where the goodness of fit of the PhGs content is 0.96, indicating that the Pareto chart analysis is reliable. Taking the Bonferroni limit (4.4047) and t value limit (2.4469) as reference standards, factors with t values higher than the Bonferroni limit value are extremely significant, factors with t values between the Bonferroni limit value and the t value limit are considered significant, and factors with t values lower than the t value limit are not significant. The results show that the influencing factors X 1 , X 4 and X 5 are extremely significant, and X 3 is significant.

[0118] According to Table 1 and Figure 6 Overall, the influencing factors of the ratio of aqueous ethanol solution to Cistanche deserticola, the power of ultrasonic oscillation, and the concentration of aqueous ethanol solution are more important for the content of phenylpropanoid glycosides in the phenylpropanoid glycoside compound extract. According to the results of the single-factor experiment for the treatment time and treatment temperature, the treatment time was determined to be 60 min and the treatment temperature was determined to be 55 °C as the optimized parameters.

[0119] Using response surface (BBD) to further optimize the ratio of ethanol solution to Cistanche deserticola, the power of ultrasonic oscillation, and the concentration of aqueous ethanol solution. According to the steps of Example 1, with the parameter settings in Table 3 (treatment time is 60 min, treatment temperature is 55 °C), extract phenylpropanoid glycosides from Cistanche deserticola. After freeze-drying the obtained extract, use the test method in Experimental Example 1 to determine the content of phenylpropanoid glycosides in the phenylpropanoid glycoside compound extract, and the results are shown in Table 3.

[0120] Table 3 Response surface (BBD) experimental design and PhGs content

[0121]

[0122] Analysis of variance (Table 4) results showed that the linear terms (X 1 , X 4 and X 5 ), interaction terms (X 1 X 4 , X 1 X 5 and X 4 X 5 ) and quadratic terms (X 2 X 1 , X 2 X 4 and X 2 X 5 ) had a significant (p < 0.05) effect on the PhGs content. The p-value of this model < 0.0001, indicating that it significantly and accurately predicted the PhGs content. The lack-of-fit F-value of this model (0.0598) > 0.05, indicating that the experimental results fitted well with the second-order mathematical model of BBD. Since there was no significant difference between the lack-of-fit term and the pure error, this mathematical model could be used for the statistical analysis of experimental results. The second-order mathematical model of the PhGs content (Y PhGs ) established by BBD was as follows:

[0123] Y PhGs = 122.4750 + 1.2995X 1 + 5.1491X 4 + 5.4530X 5 + 2.7358X 1 X 4 +

[0124] 3.1125X 1 X 5 - 3.0989X 4 X 5 - 7.3914X 2 X 1 - 15.1039X 2 X 4 - 11.7645X 2 X 5

[0125] The coefficient of variation can indicate the correctness of the model, and the smaller the coefficient of variation value, the higher the precision. As shown in Table 4, for the BBD model of the present invention, the coefficient of variation for predicting the PhGs content was 1.30, indicating that this model accurately and effectively captured the experimental data. The higher the value of the coefficient of determination (R 2 ), the higher the credibility of the model. R 2Close to 1 (0.9944), indicating that the BBD model is reasonable. Predicting R 2 (0.9180) is in good agreement with the adjusted R 2 (0.9873), indicating that the model can predict the actual situation well.

[0126] Table 4 Analysis of variance of the response surface quadratic model

[0127] Factor Sum of squares Degree of freedom Mean square F value P value Model 2368.50 9 263.17 139.31 <0.0001 <![CDATA[X 1 > 13.51 1 13.51 7.15 <![CDATA[0.0318 c > <![CDATA[X 4 > 201.50 1 201.50 106.67 <0.0001 <![CDATA[X 5 > 225.99 1 225.99 119.63 <0.0001 <![CDATA[X 1 X 4 > 31.60 1 31.60 16.73 0.0046 <![CDATA[X 1 X 5 > 40.90 1 40.90 21.65 0.0023 <![CDATA[X 4 X 5 > 38.41 1 38.41 20.33 0.0028 <![CDATA[X 2 X 1 > 171.71 1 171.71 90.90 <0.0001 <![CDATA[X 2 X 4 > 960.54 1 960.54 508.48 <0.0001 <![CDATA[X 2 X 5 > 582.75 1 582.75 308.49 <0.0001 Residual 13.22 7 1.89 - - Lack of fit 10.78 3 3.59 5.89 0.0598 Pure error 2.44 4 0.61 - - Standard deviation 1.37 - - - - Mean 105.69 - - - - Coefficient of variation % 1.30 - - - - <![CDATA[R 2 > 0.9944 - - - - <![CDATA[Adjusted R 2 > 0.9873 - - - - <![CDATA[Predict R 2 > 0.9180 - - - -

[0128] Using Design Expert software to draw a three-dimensional graph of the response surface optimization parameters for extracting the content of PhGs, and obtaining Figure 7 , the steeper the three-dimensional surface and the more elliptical the contour lines, the more significant the interaction between the two. According to Figure 7 , it can be seen that the interactions between the three factors all have a significant impact on the content of PhGs, which is consistent with the results of the variance analysis (Table 3). Among Figure 7 , the content of PhGs in each group shows a trend of first rising to a peak and then decreasing. This is because as the ethanol content increases, the polarity of the solvent increases and approaches that of PhGs, thus increasing the content of PhGs. However, when the ethanol concentration in the extraction solvent is too high, many other alcohol-soluble compounds may be dissolved, causing the extraction solvent to become saturated and unable to dissolve more flavonoid compounds; as the ratio of the ethanol solution to Cistanche deserticola increases, the contact area between the powder and the extraction solvent increases, which is beneficial to the dissolution of PhGs. But when the liquid-solid ratio is too high, the content of PhGs in the unit volume of the extraction solvent is relatively low, and the dissolved PhGs are prone to decomposition; increasing the temperature can accelerate the migration speed of molecules, thus helping the dissolution and diffusion of PhGs. But when the temperature is too high, it will accelerate the volatilization of ethanol in the extraction solvent, and high temperature will also damage the molecular structure of flavonoid compounds, resulting in a decrease in the yield of PhGs.

[0129] In summary, in this experimental example, through the response surface method, it is predicted that when the ratio of the ethanol solution to Cistanche deserticola is 13 - 14:1, the power of ultrasonic oscillation is 215 - 216 W, and the concentration of the ethanol aqueous solution is 54 - 55 Vol%, the content of phenyl ethanol glycosides extracted from Cistanche deserticola according to the steps provided by the present invention is 122 - 124 mg / g.

[0130] Experimental Example 3

[0131] Extract phenyl ethanol glycoside compounds from Cistanche deserticola in the manner of the following test example, and analyze the components in the phenyl ethanol glycoside extract.

[0132] Test Example 1: According to the ratio of ethanol solution to Cistanche deserticola of 14:1, in units of mL:g, mix a 54Vol% ethanol aqueous solution and Cistanche deserticola under ultrasonic oscillation conditions. Among them, the power of ultrasonic oscillation is 215W, the temperature during treatment is 55°C, and the treatment time is 60min. After mixing and treatment, centrifuge at 4000rpm for 30min to obtain filter residue and filtrate, and retain the filtrate; repeat the extraction of the filter residue 3 times according to the above extraction method, combine the filtrates to obtain an extract of phenylethanoid glycosides from Cistanche deserticola, and perform freeze-drying treatment at -80°C to obtain a freeze-dried product.

[0133] Test Example 2: According to the ratio of ethanol solution to Cistanche deserticola of 14:1, in units of mL:g, mix a 54Vol% ethanol aqueous solution and Cistanche deserticola at 55°C for 60min. After mixing and treatment, centrifuge at 4000rpm for 30min to obtain filter residue and filtrate, and retain the filtrate; repeat the extraction of the filter residue 3 times according to the above extraction method, combine the filtrates to obtain an extract of phenylethanoid glycosides from Cistanche deserticola, and perform freeze-drying treatment at -80°C to obtain a freeze-dried product.

[0134] Obtain the content of the extracted phenylethanoid glycosides according to the method of Experimental Example 1. Among them, the content of phenylethanoid glycosides in Test Example 1 is 122.51±0.72mg / g, and the content of phenylethanoid glycosides in Test Example 2 is 108.67±1.65mg / g.

[0135] Accurately weigh 50mg of the freeze-dried products of the above two groups of test examples respectively, add 1200μL of 70% methanol aqueous internal standard extraction solution pre-cooled at -20°C, filter the samples with a microporous membrane (0.22μm), and store them in a sample injection bottle for the analysis of the components of phenylethanoid glycosides by UPLC-MS / MS.

[0136] The chromatographic conditions are as follows:

[0137] Use an Agilent SB-C18 1.8μm (2.1mm×100mm) chromatographic column.

[0138] Mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile.

[0139] The elution gradient is as follows: 0.00min→9min, mobile phase B 5vol%→95vol%,

[0140] 9min→10min, mobile phase B 95vol%,

[0141] 10min→11min, mobile phase B 95vol%→5vol%,

[0142] 11min→14min: mobile phase B 5vol%, for equilibration.

[0143] Sample loading volume: 2 μL;

[0144] Flow rate: 0.35 mL / min;

[0145] Column temperature: 40 °C.

[0146] The results are shown in Table 5.

[0147] Table 5 Identification of PhGs in the extract and the corresponding peak area percentages

[0148]

[0149]

[0150]

[0151] * in the table indicates the existence of isomers.

[0152] According to Table 5, in the mixture of phenylethanoid glycosides extracted in Test Example 1, cistanoside F is the most abundant component in the mixture, with a peak area of 48407566.53, which is the highest among all PhGs, indicating that it occupies a considerable proportion in the mixture; followed by echinacoside, with a peak area of 38919334.17, which is also an important component in the mixture; in addition, the peak area percentage of acteoside is 21590104.82, which also occupies a significant proportion in the mixture. The high contents of these three compounds indicate that they are the main active components or marker components of the mixture. Moreover, the peak areas of these three components are all higher than those of the three components in the phenylethanoid glycoside mixture extracted by solvent extraction in Test Example 2, indicating that ultrasonic-assisted extraction helps to increase the content of PhGs in the ethanol extract of Cistanche deserticola.

[0153] Although the peak areas of other components such as magnolioside D, cistanoside A, tubuloside C, etc. are not among the top three, they cannot be ignored. Their peak areas are 15199898.94, 7191533.235 and 4827300.333 respectively. These components may contribute to the overall characteristics and functions of the mixture, and their contents in the ethanol extract of Cistanche deserticola by ultrasonic-assisted extraction are higher than those in the solvent extraction group of Test Example 2.

[0154] Based on the variable importance in projection (VIP) obtained from the OPLS-DA model (biological replicates ≥ 3), metabolites with differences between different varieties or tissues can be preliminarily screened out. The results are shown in Figure 8, Through this difference analysis, it can be seen that compared with the ethanol extract obtained by solvent extraction, the contents of 42 phenolic substances in the ethanol extract obtained by ultrasonic-assisted extraction show an upward trend but there is no significant difference; three components increase significantly, and the three components are angoroside B, magnolioside A and tubuloside A respectively.

[0155] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. A parameter optimization method for the extraction process of phenylethanoid glycosides from Cistanche deserticola, characterized in that: The first-order model obtained by the screening test design method is: PhGs =95.8991-12.6459X1-2.8928X2+3.7767X3+9.1595X4+6.7017X5, Among them, Y PhGs Indicates the content of phenylethanoid glycosides. X1 represents the ratio of ethanol aqueous solution to Cistanche deserticola YCMa, in mL:g. X2 represents the temperature of the mixed treatment of ethanol aqueous solution and Cistanche deserticola, X3 represents the time of mixing ethanol aqueous solution and desert Cistanche deserticola. X4 represents the power of ultrasonic oscillation of Cistanche deserticola in ethanol aqueous solution, X5 represents the concentration of the ethanol aqueous solution.

2. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 1, characterized in that: The response surface method was used to determine the second-order model: Y PhGs =122.4750+1.2995X1+5.1491X4+5.4530X5+2.7358X1X4+3.1125X1X5-3.0989X4X5-7.3914X2X1-15.1039X2X4-11.7645X2X5, Among them, X1X4 represents the interaction term between the ratio of ethanol solution and Cistanche deserticola and the power of ultrasonic oscillation, X1X5 represents the interaction term between the ratio of ethanol solution to Cistanche deserticola and the concentration of ethanol solution, with the unit of mL:g. X4X5 represents the interaction term between the power of ultrasonic oscillation and the concentration of ethanol aqueous solution, X2X1 represents the quadratic term of the ratio of the ethanol aqueous solution concentration to Cistanche deserticola, X2X4 represents the quadratic term of the power of ultrasonic oscillation, X2X5 represents the quadratic term of the concentration of the ethanol aqueous solution.

3. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to any one of claims 1 to 2, characterized in that: The extraction process of phenylethanoid glycosides from Cistanche deserticola comprises the following steps: Cistanche deserticola YCMa and ethanol aqueous solution were mixed under ultrasonic oscillation conditions. The power of the ultrasonic oscillation is 50-500W, The ratio of the ethanol aqueous solution to the deserticola is (5-80):1, in units of mL:g. The concentration of the ethanol aqueous solution is 10-100 Vol%.

4. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 3, characterized in that: The power of the ultrasonic oscillation is 100-300W; and / or, The ratio of the ethanol aqueous solution to the deserticola is (5-20):1, in units of mL:g; and / or, The concentration of the ethanol aqueous solution is 30-70 Vol%.

5. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 4, characterized in that: The power of the ultrasonic oscillation is 215-216W; and / or, The concentration of the ethanol aqueous solution is 54-55 Vol%; and / or, The ratio of the ethanol aqueous solution to the deserticola is (13-14):1, with the unit being mL:g.

6. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 5, characterized in that: The mixing temperature is 25-95°C; and / or, The mixing time is 5-120 min.

7. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 6, characterized in that: The mixing temperature is 40-70°C; and / or, The mixing time is 30-90 minutes.

8. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 7, characterized in that: The mixing temperature is 55°C; and / or, The mixing time is 60 minutes.

9. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 3, characterized in that: The extraction process of phenylethanol glycoside compounds from Cistanche deserticola also includes solid-liquid separation of the mixed liquid after the mixing treatment, mixing the obtained filter residue with the ethanol solution under ultrasonic oscillation conditions, repeating at least 3 times, and combining the filtrate.

10. The method for optimizing the parameters of the extraction process of phenylethanoid glycosides from Cistanche deserticola according to claim 9, characterized in that: The solid-liquid separation step includes centrifugation, the centrifugal speed is 3000-5000 rpm, and the centrifugal time is 20-40 min; and / or, The extraction process of phenylethanoid glycoside compounds from Cistanche deserticola also includes a step of freeze-drying the combined filtrate, and the freeze-drying temperature is -85 to -75°C.

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