Preparation method of momordica grosvenori saponin phospholipid compound

Abstract

The invention relates to a preparation method of a mogroside phospholipid complex. The preparation method comprises the following steps: (1) mixing a mogroside extract and lecithin according to a certain proportion to obtain a first turbid solution; (2) adding tetrahydrofuran into the first turbid solution obtained in the step (1) to obtain a second turbid solution, and performing ultrasonic treatment on the second turbid solution to uniformly disperse the second turbid solution to obtain a first reaction solution; (3) stirring the first reaction solution obtained in the step (2) until the first reaction solution is clear and transparent to obtain a second reaction solution; (4) carrying out reduced pressure evaporation on the second reaction liquid obtained in the step (3) to remove tetrahydrofuran so as to obtain an oily film; and (5) adding a proper amount of chloroform into the oily film obtained in the step (4) to obtain a third turbid solution, filtering the obtained third turbid solution through a microporous filter membrane to obtain a filtrate, carrying out rotary evaporation to obtain the filtrate, and removing the chloroform to obtain the momordica grosvenori saponin phospholipid compound. Compared with the MGE, the fat solubility of the MGE-PC is improved by 4.34 times; and the absorption rate of MGE-PC is obviously improved by 2.7 times.

Description

Technical Field

[0001] The invention relates to the field of food processing, and in particular to a method for preparing a mogroside phospholipid complex. Background Art

[0002] Siraitia grosvenorii is a traditional medicinal and edible plant unique to southern my country. It has the effect of expectoration and cough relief in traditional medicine. Modern pharmacological studies have shown that mogrosides are its main active ingredients, which have multiple biological activities such as anti-diabetes, hypoglycemic, hypolipidemic, anti-inflammatory, and anti-cancer. Among them, mogroside V (MGV) is the main component of mogrosides, accounting for about one-fifth of the total amount of mogrosides. Its sweetness can reach 300 times that of sucrose, but its calories are only 1 / 50 of sucrose. It is a natural low-calorie sweetener approved by the US FDA as a "generally recognized as safe (GRAS)" substance, and many countries have approved it as a food sweetener. As a food sweetener, mogrosides are very sweet and the amount added to food is very low; in addition, the high water solubility of mogrosides leads to poor cell membrane permeability and low oral bioavailability. The above reasons make the concentration of mogrosides in the body very low, making it difficult to exert its biological activity. Therefore, improving the oral bioavailability of mogrosides will help to better exert its potential as a functional food sweetener.

[0003] At present, the methods for increasing the bioavailability of highly water-soluble compounds include nanoparticles, liposomes, tablets, and appropriate chemical modifications. Among them, the commonly used method is to combine with phosphatidylcholines (PC) to form a complex. Phospholipids are an important nutrient in food, with amphiphilic properties, and can combine with highly water-soluble compounds such as glycosides to form a complex. In the prior art, there is no preparation process for synthesizing mogroside phospholipid complex (MGE-PC) using mogroside extract (MGE) as raw material. Summary of the invention

[0004] The first technical problem to be solved by the present invention is to provide a method for preparing a mogroside phospholipid complex in view of the current status of the prior art. The mogroside phospholipid complex prepared by the method has high gastrointestinal absorption and bioavailability.

[0005] The second technical problem to be solved by the present invention is to provide a mogroside phospholipid complex prepared by the above preparation method in view of the current status of the prior art.

[0006] The third technical problem to be solved by the present invention is to provide an application of the above-mentioned momordica saponin phospholipid complex in view of the current status of the prior art.

[0007] The technical solution adopted by the present invention to solve the above-mentioned first technical problem is: a method for preparing a mogroside phospholipid complex, comprising the following steps:

[0008] (1) mixing the mogroside extract and lecithin in a certain ratio to obtain a first turbid liquid;

[0009] (2) adding tetrahydrofuran to the first turbid liquid obtained in step (1) to obtain a second turbid liquid, and subjecting the second turbid liquid to ultrasonic treatment so that the second turbid liquid is evenly dispersed to obtain a first reaction liquid;

[0010] (3) stirring the first reaction solution obtained in step (2) until it becomes clear and transparent to obtain a second reaction solution;

[0011] (4) evaporating the second reaction solution obtained in step (3) under reduced pressure to remove tetrahydrofuran, thereby obtaining an oily film;

[0012] (5) Adding an appropriate amount of chloroform to the oily film obtained in step (4) to obtain a third turbid liquid, filtering the obtained third turbid liquid through a microporous filter membrane to obtain a filtrate, and rotary evaporating the obtained filtrate to remove chloroform to obtain a mogroside phospholipid complex.

[0013] Preferably, in step (1), the total saponin content of the mogroside saponin extract is greater than 80%, wherein the MGV content is greater than 50%; and the phosphatidylcholine content of the lecithin is greater than 70%.

[0014] Preferably, in step (1), the mass ratio of the added mogroside extract to the lecithin is 1:1 to 4.

[0015] Preferably, in step (2), the concentration of the mogroside extract in the second turbid liquid is 1-10 mg / mL, and the ultrasonic conditions are as follows: frequency of 50 kHz, power of 200-750 W, and time of 15-30 min.

[0016] Preferably, in step (3), the first reaction solution obtained in step (2) is stirred at 25-55° C. and 100-500 rpm for 0.5-4 h until it becomes clear and transparent.

[0017] Preferably, in step (4), the temperature of the reduced pressure evaporation is 30 to 45°C.

[0018] Preferably, in step (5), the pore size of the microporous filter membrane is 0.45 μm; and the temperature of the rotary evaporation is 30 to 45°C.

[0019] Preferably, the following steps are also included

[0020] (6) freeze-drying the mogroside phospholipid complex obtained in step (5) for 8 to 24 hours to obtain a light yellow mogroside phospholipid complex powder.

[0021] The technical solution adopted by the present invention to solve the above second technical problem is: a mogroside phospholipid complex prepared by the above preparation method.

[0022] The technical solution adopted by the present invention to solve the third technical problem is: an application of the above-mentioned momordica saponin phospholipid complex in food.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] (1) The preparation process is simple and efficient. The use of ultrasonic technology promotes the uniform distribution of the mixture during the reaction, effectively increasing the yield of the product to more than 95%;

[0025] (2) The prepared MGE-PC is an amorphous powder with good stability under heating and neutral conditions and good processing adaptability;

[0026] (3) Compared with MGE, the lipid solubility of MGE-PC increased by 4.34 times;

[0027] (4) Caco-2 cell transport experiments revealed that MGE-PC has a higher absorption rate, which is significantly increased by 2.7 times compared to MGⅤ. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 UV images of different samples in Example 4 of the present invention, wherein A is MGE; B is PC; C is a physical mixture of MGE and PC; D is MGE-PC;

[0029] Figure 2 FT-IR images of different samples in Example 4 of the present invention, wherein A is MGE; B is PC; C is a physical mixture of MGE and PC; D is MGE-PC;

[0030] Figure 3 : are XRD patterns of different samples in Example 4 of the present invention, wherein A is MGE; B is PC; C is a physical mixture of MGE and PC; D is MGE-PC;

[0031] Figure 4 The scanning electron microscope images (×10 3 ), where A is MGE; B is PC; C is a physical mixture of MGE and PC; D is MGE-PC;

[0032] Figure 5 The DSC graphs of different samples in Example 4 of the present invention, wherein A is MGE; B is PC; C is a physical mixture of MGE and PC; D is MGE-PC;

[0033] Figure 6 is the interaction diagram between MGⅤ and PC in Example 4 of the present invention;

[0034] Figure 7 The solubility analysis diagram of MGE, MGE-PC and MGE / PC physical mixture in Example 5 of the present invention is shown in FIG. Figure 7 A uses water as solvent. Figure 7 B uses n-octanol as solvent;

[0035] Figure 8 The results of the states of MGE-PC in different solutions in Example 5 of the present invention are shown in FIG. 1 , wherein A is the particle size; B is the PDI; and C is the Zeta potential;

[0036] Fig. 9 is the cumulative transport amount and P of MGⅤ-PC in the bidirectional transport experiment in Caco-2 cell monolayer in Example 6 of the present invention app Result diagram; A is the AP→BL transport volume; B is the BL→AP transport volume; C is P app (AP→BL); D is P app (BL→AP);

[0037] Fig.10 The figure is a result diagram of the effect of different reaction conditions on the MGE-PC recombination rate in Example 7 of the present invention; wherein A is the solvent; B is the mass ratio; C is the MGE concentration; D is the temperature; and E is the time;

[0038] Fig.11 3D graph and contour lines of the response surface analysis in Example 7 of the present invention; wherein A represents the interaction between MGE concentration and time; B represents the interaction between mass ratio and time; and C represents the interaction between mass ratio and MGE concentration. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below with reference to the accompanying drawings.

[0040] Example 1

[0041] The preparation method of MGE-PC in this embodiment comprises the following steps:

[0042] (1) Weigh 100 mg of MGE (total saponins ≥ 80%) and 200 mg of PC, and mix them to obtain a first turbid solution;

[0043] (2) adding 20 mL of tetrahydrofuran to the first turbid liquid obtained in step (1) to obtain a second turbid liquid, and subjecting the second turbid liquid to ultrasonic treatment to uniformly disperse the second turbid liquid to obtain a first reaction liquid; wherein the ultrasonic frequency is 50 kHz, the power is 500 W, and the time is 20 min;

[0044] (3) The first reaction solution obtained in step (2) was magnetically stirred at 40° C. and 500 rpm for 3 h until it became clear and transparent, thereby obtaining a second reaction solution;

[0045] (4) subjecting the second reaction solution obtained in step (3) to reduced pressure evaporation at 45° C. to remove tetrahydrofuran, thereby obtaining an oily film;

[0046] (5) 20 mL of chloroform is added to the oily film obtained in step (4) to obtain a third turbid liquid, and the obtained third turbid liquid is filtered through a 0.45 μm organic filter membrane to obtain a filtrate, and the obtained filtrate is subjected to rotary evaporation at 45° C. to remove chloroform, thereby obtaining a mogroside phospholipid complex, and the obtained mogroside phospholipid complex (MGE-PC) has a complexation rate of 95.09%; wherein, the mogroside extract that is not complexed with lecithin is insoluble in chloroform, so the organic filter membrane filtration can remove the uncomplexed mogroside extract;

[0047] (6) The MGE-PC obtained in step (5) was freeze-dried for 8 h, and a light yellow MGE-PC powder was obtained by scraping.

[0048] Example 2

[0049] The preparation method of MGE-PC in this embodiment comprises the following steps:

[0050] (1) Weigh 100 mg of MGE (total saponins ≥ 80%) and 100 mg of PC, and mix them to obtain a first turbid solution;

[0051] (2) adding 10 mL of tetrahydrofuran to the first turbid liquid obtained in step (1) to obtain a second turbid liquid, and subjecting the second turbid liquid to ultrasonication to uniformly disperse the second turbid liquid to obtain a first reaction liquid; wherein the ultrasonication frequency is 50 kHz, the power is 750 W, and the time is 30 min;

[0052] (3) stirring the first reaction solution obtained in step (2) under the conditions of 55° C. and 400 rpm for 4 h until it becomes clear and transparent, thereby obtaining a second reaction solution;

[0053] (4) subjecting the second reaction solution obtained in step (3) to reduced pressure evaporation at 30° C. to remove tetrahydrofuran, thereby obtaining an oily film;

[0054] (5) 20 mL of chloroform is added to the oily film obtained in step (4) to obtain a third turbid liquid, and the obtained third turbid liquid is filtered through a 0.45 μm organic filter membrane to obtain a filtrate, and the obtained filtrate is subjected to rotary evaporation at 30° C. to remove chloroform, thereby obtaining a mogroside phospholipid complex, and the obtained mogroside phospholipid complex (MGE-PC) has a complexation rate of 76.04%; wherein, the mogroside extract that is not complexed with lecithin is insoluble in chloroform, so the organic filter membrane filtration can remove the uncomplexed mogroside extract;

[0055] (6) The MGE-PC obtained in step (5) was freeze-dried for 8 h, and a light yellow MGE-PC powder was obtained by scraping.

[0056] Example 3

[0057] The preparation method of MGE-PC in this embodiment comprises the following steps:

[0058] (1) Weigh 100 mg of MGE (total saponins ≥ 80%) and 400 mg of PC, and mix them to obtain a first turbid solution;

[0059] (2) adding 100 mL of tetrahydrofuran to the first turbid liquid obtained in step (1) to obtain a second turbid liquid, and subjecting the second turbid liquid to ultrasonic treatment to uniformly disperse the second turbid liquid to obtain a first reaction liquid; wherein the ultrasonic frequency is 50 kHz, the power is 200 W, and the time is 15 min;

[0060] (3) magnetically stirring the first reaction solution obtained in step (2) at 25° C. and 100 rpm for 0.5 h until it becomes clear and transparent, thereby obtaining a second reaction solution;

[0061] (4) subjecting the second reaction solution obtained in step (3) to reduced pressure evaporation at 40° C. to remove tetrahydrofuran, thereby obtaining an oily film;

[0062] (5) 20 mL of chloroform is added to the oily film obtained in step (4) to obtain a third turbid liquid, and the obtained third turbid liquid is filtered through a 0.45 μm organic filter membrane to obtain a filtrate, and the obtained filtrate is subjected to rotary evaporation at 40° C. to remove chloroform to obtain a mogroside phospholipid complex, and the obtained mogroside phospholipid complex (MGE-PC) has a complexation rate of 85.49%; wherein, the mogroside extract that is not complexed with lecithin is insoluble in chloroform, so the organic filter membrane filtration can remove the uncomplexed mogroside extract;

[0063] (6) The MGE-PC obtained in step (5) was freeze-dried for 8 h, and a light yellow MGE-PC powder was obtained by scraping.

[0064] Example 4

[0065] The structural analysis of the prepared MGE-PC includes the following steps:

[0066] (1) UV, IR, XRD, SEM and DSC were used to analyze the structural characteristics and formation mechanism of the prepared mogroside phospholipid complex. Figure 1 , 2 , 3, 4 and 5, it was found that MGE and PC formed an amorphous complex MGE-PC, which was significantly different from the MGE / PC physical mixture;

[0067] (2) Molecular docking results of MGⅤ and PC are as follows Figure 6 As shown, the hydroxyl group of the glucose group of the MGⅤ molecule and the polar group of acetylcholine of the phospholipid molecule (-P=O, -OH, -NH2, etc.) form a complex through non-covalent bonds such as hydrogen bonds, electrostatic interactions, and van der Waals forces, but no new covalent bonds are formed, suggesting that MGⅤ and PC form a phospholipid complex through non-covalent bonds.

[0068] Example 5

[0069] The prepared MGE-PC characteristics analysis includes the following steps:

[0070] (1) The characteristics of the prepared MGE-PC were analyzed by HPLC and solubility determination, indicating that the drug loading of MGE-PC was 15.657% and that of MGV-PC was 27.54%. The solubility of MGE-PC in n-octanol solution was 0.28 mg / mL. Compared with the solubility of MGE in n-octanol solution, the lipid solubility of MGE-PC was increased by 4.34 times. Figure 7 ;

[0071] Among them, the method for determining the drug loading of MGE-PC is as follows:

[0072] Sample treatment: Accurately weigh 15 mg of MGE-PC / MGⅤ-PC, demulsify with acetonitrile, filter the sample solution through a 0.22 μm microporous filter membrane, and make up the volume to 1 mg / mL with liquid acetonitrile for MGⅤ content determination.

[0073] HPLC elution conditions: chromatographic column Kromasil 100C18 (250 mm × 4.6 mm, 5.0 μm); mobile phase composed of acetonitrile (B) and pure water (D); elution flow rate of 0.8 mL / min; injection volume of 10 μL; detection wavelength: 203 nm; column temperature 35°C. 0 min-5 min, 20% B; 5 min-15 min, 20%-25% B; 15 min-20 min, 25%-30% B; 20 min-25 min, 30%.

[0074] MGⅤ standard curve: The above chromatographic conditions were used for determination and fitting was performed using peak area and concentration. The concentration ranged from 46 to 736 μg / mL and had a good linear relationship. The standard curve was: y=2.4328x-15.009, R2=0.999.

[0075] The MGV content in the MGV-PC complex is the drug loading, which is calculated according to the following formula:

[0076]

[0077] Wherein: X is the content of MGⅤ (μg / mL); A is the peak area of ​​MGⅤ; C0 is the concentration of the standard sample (μg / mL), V is the fixed volume (mL), A1 is the peak area of ​​the standard solution, and m is the weighed mass (g).

[0078] (2) Figure 8 As shown in the figure, the average particle size of MGE-PC in pure water was 421.3nm, while that of PC was 283.4nm, which indicated that the MGE-PC phospholipid complex formed a larger vesicle structure in water, giving it colloidal opalescence and Tyndall phenomenon. The PDI of MGE-PC in water environment was 0.48, and the particle size distribution was relatively uniform. The Zeta data showed that MGE-PC and PC were -43.51 and -37.83mv, respectively, indicating that the particles were negatively charged under neutral conditions, which helped the particles to repel each other and thus maintain stability in the solution.

[0079] (3) Figure 8 As shown in the figure, the states of the complex in different in vivo environments (blood environment, small intestine environment, stomach environment, gastric acid environment) were investigated at the same time, and it was found that MGE-PC showed good stability and dispersibility in a neutral pH environment. As the pH value decreased to the simulated environment of blood and small intestine, the particle size gradually increased, the absolute value of Zeta potential decreased, and the stability began to decrease. Further reducing the pH to the simulated environment of the stomach, the particle size increased to 1291.37nm (millimeter level), and the Zeta potential turned positive, indicating that the pH value has a significant effect on the stability of the MGE-PC complex.

[0080] Example 6 Determination of the intestinal absorption rate of the prepared MGE-PC in Caco-2 cells

[0081] Mogroside is the main active substance in MGE. It is usually a compound containing 30 carbon atoms composed of 6 isoprene units connected head to tail, with the general formula (C5H8)6. The hydroxyl group on the core structure is often connected to 1 to 6 sugar groups by glycosidic bonds to form triterpene glycosides (also known as triterpene saponins). The diversity of this type of compound comes from the differences in the aglycone structure and the number, position and type of sugar groups. However, the absorption and transport characteristics of tetracyclic triterpene glycosides are similar, and they all have the characteristics of low intestinal cell membrane permeability and bioavailability. Mogroside V is the main component of mogroside, with a content of more than 50%. Therefore, in the cell transport experiment, in order to facilitate the determination, MGV-PC after phospholipidation of MGV was selected for the experiment. The specific steps include:

[0082] (1) Caco-2 cell culture

[0083] Caco-2 cells (10-20 generations) frozen in liquid nitrogen were taken out and quickly thawed in a 37°C constant temperature water bath, and then quickly mixed with 5 mL of complete culture medium (20% fetal bovine serum, 1% non-essential amino acid DMEM culture medium), centrifuged at 1000 r / min for 5 min, the supernatant was discarded, 1 mL of complete culture medium was added to the cell pellet, and the cell suspension was gently pipetted to form a cell suspension, which was inoculated into a T25 culture flask and cultured in a carbon dioxide incubator (37°C, 5% CO2). The culture medium was replaced every other day. When the cell confluence was about 80%, the cells were passaged at a ratio of 1:3.

[0084] (2) Caco-2 cell monolayer model and its integrity verification

[0085] When the confluence of Caco-2 cells in step (1) was about 80%, the cells were digested with 0.25% trypsin for 5-10 min, 2 mL of complete culture medium was added to terminate the digestion and gently pipetted, centrifuged at low speed for 5 min, the culture medium was discarded, 1 mL of complete culture medium was added and gently pipetted into a cell suspension, the cells were counted and adjusted to an appropriate density, and 0.5 mL was taken at 2.0×10 5 100 cells / well were inoculated on the AP side of a Transwell 12-well plate chamber, and 1.5 mL of complete culture medium (with 1% penicillin-streptomycin) was added to the BL side. After 21 days of culture, Caco-2 cells were fully differentiated and the transmembrane resistance value exceeded 500Ω / cm. 2 The AKP activity on the AP / BL side was greater than 5, and the apparent permeability coefficient of fluorescein sodium was between 0.97 and 3.71×10 -6 Between cm / s.

[0086] (3) Determination of MGⅤ-PC absorption rate

[0087] The integrity of the Caco-2 cell monolayer was verified well, and the culture medium on the AP and BL sides was gently aspirated. The chamber was washed twice with HBSS buffer, and 0.5 mL and 1.5 mL of HBSS buffer were added to the AP and BL sides, respectively. The chamber was placed in an incubator for 30 minutes of equilibrium, and then an absorption experiment was performed. When MGⅤ-PC was absorbed from the apical side (AP) to the basal side (BL) in the Caco-2 cell monolayer, its cumulative absorption increased significantly compared with the original MGⅤ, reaching 22.47 μg in 2 hours, and the intestinal absorption rate was 1.32%; while the intestinal absorption rate of MGⅤ was 0.48%, and MGⅤ-PC was 2.7 times that of MGⅤ, indicating that the formation of phospholipid complexes by MGⅤ can significantly improve its absorption rate in the intestine, see Fig. 9 and Table 1.

[0088] Table 1 MGV-PC in Caco-2 cell monolayers app , PDR and absorption-excretion ratio (n=3)

[0089]

[0090] Example 7 Optimization of the preparation process of MGE-PC composite

[0091] (1) Single factor experiment

[0092] Selection of reaction solvent: fixed reaction temperature 55℃, reaction time 3h, mass ratio of MGE to PC 1:1, saponin concentration 1mg / mL, and investigated the effects of 4 different solvents (methanol, anhydrous ethanol, tetrahydrofuran, ethyl acetate) on the complexation rate of mogroside saponin phospholipids;

[0093] Selection of the mass ratio of MGE to PC: Using tetrahydrofuran as the reaction solvent, the reaction temperature was fixed at 55°C, the reaction time was 3h, and the MGE concentration was 1mg / mL. The effects of different mass ratios (1:1, 1:1.5, 1:2, 1:3 and 1:4) on the recombination rate were investigated.

[0094] Selection of reactant MGE concentration: Tetrahydrofuran was used as the reaction solvent, the reaction temperature was set at 55°C, the reaction time was 3h, the mass ratio of MGE to PC was 1:1.5, and the effect of different concentrations of mogroside extract (1, 3, 5, 7 and 10 mg / mL) on the recombination rate was investigated;

[0095] Selection of reaction temperature: Tetrahydrofuran was used as the reaction solvent, the reaction time was set to 3 h, the mass ratio of MGE to PC was 1:1.5, the saponin concentration was 5 mg / mL, and the effect of different temperatures (25, 35, 45 and 55 °C) on the recombination rate was investigated;

[0096] Selection of reaction time: Tetrahydrofuran was used as the reaction solvent, the reaction temperature was set at 45°C, the mass ratio of MGE to PC was 1:1.5, and the saponin concentration was 5 mg / mL. The effects of different reaction times (0.5, 1, 2, 3 and 4 h) on the recombination rate were investigated.

[0097] The experimental results are as follows Fig.10 As shown, Fig.10 A shows that different solvents have a significant effect on the MGE-PC recombination rate. The recombination rates of phospholipid complexes prepared by methanol, ethanol, tetrahydrofuran, and ethyl acetate are 47.24%±5.60%, 46.19%±3.64%, 60.19%±3.26%, and 12.21%±0.58%, respectively. The recombination rate of tetrahydrofuran is significantly higher than that of other solvents, reaching 60.19%. Therefore, tetrahydrofuran is selected as the reaction solvent.

[0098] Secondly, the mass ratio of MGE to PC has a significant effect on the recombination rate of MGE-PC. With the increase of PC content, the mass ratio and recombination rate show a positive correlation trend. However, when the mass ratio exceeds 1:1.5, the recombination rate begins to decrease with the increase of PC concentration and gradually stabilizes; and when the mass ratio exceeds 1:2, the generated complex is very viscous and difficult to dry. When the mass ratio is 1:1.5, the recombination rate of MGE-PC is the highest, which is 91.11%±3.56% ( Fig.10 B).

[0099] In addition, MGE concentration is also an important factor affecting the recombination rate; Fig.10 C shows that in the concentration range of 1 to 5 mg / mL, as the MGE concentration increases, the recombination rate shows a slow growth trend, but when the MGE concentration is higher than 5 mg / mL, the recombination rate decreases rapidly. This may be because when the MGE concentration is too high, it cannot be fully dissolved, thus affecting the formation of the complex.

[0100] Depend on Fig.10 D shows that temperature also significantly affects the formation of phospholipid complexes. When the temperature is not higher than 45°C, the recombination rate shows a rapid upward trend as the temperature increases, and decreases slightly at 55°C. It is the highest at 45°C, so 45°C is selected as the reaction temperature.

[0101] For the reaction time, within the range of 0.5 to 2 hours, the recombination rate increases rapidly with time, increases slowly within 2 to 3 hours, and decreases within 3 to 4 hours ( Fig.10 E), indicating that the composite reaction is close to completion around 3 h, and prolonged heating may have a destructive effect on the formed complex, so the reaction time is preferably 3 h.

[0102] (2) Central composite design (CCD)-response surface methodology (RSM) optimization experiment

[0103] According to the results of the single-factor test, three factors, reaction time (A), sample concentration (B) and the mass ratio of MGE to PC (C), which have a greater impact on the recombination rate, were selected for star-point design. Each factor was designed at three levels. The levels of each factor are shown in Table 2.

[0104] Table 2 Star point design factor level table

[0105]

[0106] The CCD test design and results are shown in Table 3. Taking the MGE-PC recombination rate (Y) as an indicator, the Design-Expert 10.0 software was used for multiple linear regression and binomial fitting, and the multiple quadratic regression equation model Y (%) = 91.94 + 4.76A-2.84B + 9.11C + 2.36AB-1.99AC + 0.26BC-2.75A was obtained. 2 -4.50B 2 -9.33C 2 The variance analysis is shown in Table 4. It can be seen from the table that the model fit is extremely significant (P < 0.01), the lack of fit value is not significant (P = 0.1835 > 0.05), R 2 =0.9566, effective coefficient (R 2 Adj ) is 0.9175, indicating that the model equation is significant and well-fitted. From the mean square sum, we can see that A, B, and C all have significant effects, among which the effects of A and C are extremely significant (P < 0.01), and the order of influence is C (mass ratio) > A (complexation time) > B (MGE concentration).

[0107] Table 3 Results of optimization experiment of central composite design-response surface methodology

[0108]

[0109] Table 4 Analysis of variance

[0110]

[0111]

[0112] Note: * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01)

[0113] The interaction between reaction time, MGE concentration and mass ratio is shown in Fig.11The shape of the contour line reflects whether the interaction between the factors is significant. The ellipse indicates a significant interaction, and the circle indicates an insignificant interaction. The steepness of the three-dimensional stereogram can reflect the influence of each factor on the response value. Fig.11 B and 11C present an elliptical shape, indicating that the interaction between mass ratio and time, and between mass ratio and MGE concentration is significant, but the interaction between MGE concentration and time is not significant ( Fig.11 A). The response surface analysis shows that under the interaction of appropriate mass ratio and MGE concentration and mass ratio and time, the recombination rate has a maximum value, that is, MGE concentration is 3.8-6.2 mg / mL, time is 2-2.6 h, and mass ratio is 1:1.5-1.8.

[0114] (3) Verification test

[0115] According to the above quadratic regression equation and response surface analysis, the optimal composite reaction conditions were determined to be MGE concentration 4.68 mg / mL, mass ratio 1:1.73, and reaction time 2.34 h. In order to verify the reliability of the model and take into account the convenience of actual operation, the optimal preparation conditions were corrected to reaction time 2.5 h, mass ratio 1:1.7, MGE concentration 5.0 mg / mL, reaction temperature 45 ° C, and three parallel tests were performed. The prepared composite rate was 95.09 ± 1.12%, which was close to the predicted value (95.58%), indicating that the model is reliable.

[0116] In the present invention, the free saponin is insoluble in chloroform, while the complex of saponin and PC is soluble in chloroform. A certain amount of MGE (m1) and PC are mixed in a reaction solvent and ultrasonicated for 15 minutes. After stirring in a water bath at a certain temperature until clear and transparent, the complex liquid is vacuum concentrated to remove the reaction solvent, and an appropriate amount of chloroform is added to the obtained solid powder to fully dissolve PC and the phospholipid complex. After filtering, the filtrate and precipitate (m2) are collected. After the filtrate is vacuum concentrated and freeze-dried, the mogroside saponin phospholipid complex is obtained. The complex rate is calculated according to the following formula:

[0117]

Claims

1. A method for preparing a mogroside phospholipid complex, characterized in that The following steps are involved: (1) mixing the mogroside extract and lecithin in a certain ratio to obtain a first turbid liquid; (2) adding tetrahydrofuran to the first turbid liquid obtained in step (1) to obtain a second turbid liquid, and subjecting the second turbid liquid to ultrasonic treatment so that the second turbid liquid is evenly dispersed to obtain a first reaction liquid; (3) stirring the first reaction solution obtained in step (2) until it becomes clear and transparent to obtain a second reaction solution; (4) evaporating the second reaction solution obtained in step (3) under reduced pressure to remove tetrahydrofuran, thereby obtaining an oily film; (5) Adding an appropriate amount of chloroform to the oily film obtained in step (4) to obtain a third turbid liquid, filtering the obtained third turbid liquid through a microporous filter membrane to obtain a filtrate, and rotary evaporating the obtained filtrate to remove chloroform to obtain a mogroside phospholipid complex.

2. The preparation method according to claim 1, characterized in that: In step (1), the total saponin content of the mogroside saponin extract is greater than 80%, wherein the MGV content is greater than 50%; and the phosphatidylcholine content of the lecithin is greater than 70%.

3. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of the added mogroside extract to the lecithin is 1:1 to 4.

4. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the mogroside extract in the second turbid liquid is 1-10 mg / mL, and the ultrasonic conditions are as follows: frequency is 50 kHz, power is 200-750 W, and time is 15-30 min.

5. The preparation method according to claim 1, characterized in that: In step (3), the first reaction solution obtained in step (2) is stirred at 25-55° C. and 100-500 rpm for 0.5-4 h until it becomes clear and transparent.

6. The preparation method according to claim 1, characterized in that: In step (4), the temperature of the reduced pressure evaporation is 30 to 45°C.

7. The preparation method according to claim 1, characterized in that: In step (5), the pore size of the microporous filter membrane is 0.45 μm; and the temperature of the rotary evaporation is 30 to 45°C.

8. The preparation method according to claim 1, characterized in that: The following steps are also included (6) freeze-drying the mogroside phospholipid complex obtained in step (5) for 8 to 24 hours to obtain a light yellow mogroside phospholipid complex powder.

9. A mogroside phospholipid complex prepared by the preparation method according to claims 1 to 8.

10. Use of the mogroside phospholipid complex as claimed in claim 9 in food.

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