A biosynthetic method for mogrosanol

Abstract

The present invention relates to a biosynthesis method of mogrosanol; mogrosanol is a kind of Siraitia the grosvenors ) is a cucurbitane-type tetracyclic triterpenoid compound, which is aglycone of mogroside compounds and has multiple pharmacological activities such as anti-cancer, anti-pulmonary fibrosis, and neuroprotection. However, the content of mogroside alcohol in Momordica grosvenori is very small, and large-scale preparation is difficult and costly. Therefore, the present invention discloses a new biocatalytic process for preparing mogroside alcohol by hydrolyzing crude extract of mogroside using snail enzyme as a catalyst, which provides a simple and quick method for solving the problem of the source of mogroside alcohol.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product biosynthesis, and specifically relates to the preparation of aglycone mogroside alcohol by hydrolyzing a crude extract of mogroside catalyzed by snail enzyme. The biosynthesis efficiency of mogroside alcohol is further improved by constructing a biphasic enzymatic reaction system, the product purification procedure is simplified, and a material source is provided for the development of drugs based on mogroside alcohol. Background Art

[0002] Momordica alcohol is a cucurbitane-type tetracyclic triterpenoid compound derived from Siraitia grosvenorii, and is the aglycone of momordica glycosides. Studies have shown that momordica alcohol has multiple biological activities such as anti-cancer, anti-cancer, anti-viral, anti-pulmonary fibrosis, and neuroprotection (Liu C, et al. Future Med. Chem., 2018, 10: 845-850). In addition, studies on the activity of momordica glycosides have shown that most momordica glycosides are excreted in the blood in the form of momordica alcohol after oral administration (Xu F, et al. J. Pharm. Biomed. Anal., 2015, 115: 418-430). It can be seen that momordica alcohol is a drug lead compound with great development value and potential. Analysis of the biosynthetic pathway of mogrosides showed that mogroside alcohol is the precursor of mogrosides with different numbers of biosynthetic sugar units and diverse glycosidic bonds (Itkin M, et al. Proc. Natl. Acad. Sci. USA, 2016, 113: E7619-E7628).

[0003] However, the content of mogrosanol in Momordica grosvenori is very small, and it is difficult to prepare it on a large scale with high cost. In addition to direct extraction from Momordica grosvenori, acid / base hydrolysis of mogrosan glycosides is an important means to prepare mogrosanol. However, the conditions of acid / base hydrolysis reaction are relatively severe, which may destroy the skeleton structure of mogrosanol, cause product loss or produce unexpected impurities (Chen X, et al. Bioorg. Med. Chem., 2011, 19: 5776-5781). In contrast, enzyme-catalyzed reaction is more gentle and has strong position selectivity, which is expected to overcome the shortcomings of acid / base hydrolysis and become a new method for preparing mogrosanol by hydrolysis of mogrosan glycosides. However, due to the substrate-specific selectivity of biological enzymes, there is currently no report on the method of preparing mogrosanol by completely hydrolyzing mogrosan glycosides by enzyme method. Summary of the invention

[0004] The purpose of the present invention is to establish a new method for biosynthesis of mogroside alcohol by using glycosidase with mild reaction conditions, obtain a large amount of high-quality mogroside alcohol, and solve the problem of its source.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A biosynthesis method for mogrosanol is characterized in that mogrosanol is obtained by catalyzing the hydrolysis of a crude extract of mogrosanol using glycosidase, wherein the glycosidase used is pectinase or snailase.

[0007] The biosynthesis method of mogrosanol is characterized in that a biphasic enzymatic reaction system is constructed by using an organic reagent and a reaction buffer to hydrolyze the mogrosanol extract to prepare mogrosanol; the organic reagent is selected from methyl tert-butyl ether or toluene.

[0008] The biosynthesis method of mogrosanol is characterized in that the buffer is selected from a buffer with a pH of 4.0-7.0, and the buffer is selected from sodium citrate-citric acid buffer, disodium hydrogen phosphate-citric acid buffer and sodium acetate-acetic acid buffer.

[0009] The biosynthesis method of the mogrosanol is characterized in that snail enzyme is used in the two-phase enzymatic reaction system, the dosage of snail enzyme is 5 mg / mL-15 mg / mL; the concentration of the mogrosanol extract is 10-20 mg / mL; and the volume concentration of toluene is 10%-70% V / V.

[0010] The biosynthesis method of mogrosanol is characterized in that the reaction pH of the two-phase enzymatic reaction system is 4.0-7.0; the reaction temperature is 35° C.-60° C.; and the reaction time is 0-24 hours.

[0011] The biosynthesis method of mogrosanol is characterized in that the steps include:

[0012] Snail enzyme and monk fruit extract are added to a buffer solution, and toluene is added and mixed to obtain a two-phase reaction system; after the reaction is completed, the two phases are separated after standing for 2 hours, and the toluene layer is collected; the aqueous layer is extracted with ethyl acetate; toluene and ethyl acetate are removed, and the crude extract of monk fruit alcohol is combined; the crude extract is dissolved in a 20% V / V ethanol solution and then separated and purified using a polyamide resin column, and eluted with an ethanol-water solution in a gradient manner, and the eluent containing high-purity monk fruit alcohol is combined and collected, and dried to obtain monk fruit alcohol.

[0013]

[0014] Beneficial Effects

[0015] Cellulase, pectinase, snail enzyme, β-glucosidase are common glycosidases that hydrolyze glycosidic bonds (Li W, et al. Molecules, 2011, 16: 10093-10103), wherein snail enzyme is a mixed enzyme composed of pectinase, cellulase, amylase and other hydrolases. Glycosidase is an important enzyme for hydrolyzing glycosidic bonds to prepare corresponding hydrolyzates, but each glycosidase has a relatively serious substrate selectivity. After testing the hydrolysis effects of cellulase, pectinase, snail enzyme, and β-glucosidase on the crude extract of mogroside, the present invention compared that not all glycosidases can enzymolyze the monk fruit extract to obtain a single product. The present invention first found that only snail enzyme and pectinase can hydrolyze the monk fruit extract to obtain monk fruit alcohol.

[0016] In the aqueous phase reaction system, the yield of preparing mogrosanol by hydrolyzing 3 mg / mL monk fruit extract catalyzed by snail enzyme is less than 75%, which cannot meet the needs of large-scale preparation from the perspective of substrate concentration and efficiency. The two-phase reaction system composed of organic solvent and reaction buffer is one of the means to improve substrate concentration and catalytic efficiency, but since the presence of organic solvent often leads to enzyme inactivation, it is generally difficult to construct a two-phase reaction system in the enzymatic reaction. In view of the difference in water solubility between mogrosanol and mogroside, the present invention screened a variety of organic solvents, and through system optimization, the substrate concentration of the reaction was increased to 15 mg / mL, and the yield of mogrosanol reached 93.1%, which basically met the large-scale preparation. In addition, the high solubility of mogrosanol in organic solvents effectively simplifies the extraction and purification steps of the product.

[0017] Therefore, the present invention has the following advantages:

[0018] 1) In the aqueous phase reaction system, the efficiency of synthesizing mogrosanol catalyzed by snail enzyme is low and cannot meet the needs of large-scale preparation. The biphasic enzymatic reaction system constructed by the present invention has a higher efficiency in synthesizing mogrosanol, and the high solubility of mogrosanol in the organic phase further simplifies the separation and purification process.

[0019] 2) Compared with the acid / base hydrolysis reaction, the method for preparing mogrosanol by the snail enzyme method established in the present invention has milder reaction conditions, avoids the structural destruction of mogrosanol, and is conducive to obtaining high-quality products.

[0020] 3) The present invention adopts polyamide resin to purify and prepare mogrosanol, which has the advantages of large sample loading capacity and reusability, and is conducive to low-cost large-scale preparation of mogrosanol.

[0021] In summary, the biphasic preparation system of mogroside alcohol established by the present invention using snail enzyme as a biocatalyst is of great significance to the in-depth development of mogroside alcohol and mogroside glycosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The HPLC spectrum of the synthesis of mogroside alcohol from monk fruit extract (containing 50% mogroside V) catalyzed by snail enzyme, wherein i is the HPLC spectrum of the reference substance of mogroside alcohol, and ii is the HPLC spectrum of the system after the reaction. DETAILED DESCRIPTION

[0023] The specific steps of the present invention are described below by way of examples, but the scope of the present invention is not limited by these examples.

[0024] Example 1 Screening of glycosidase

[0025] The ability of several common glycosidases, including cellulase, pectinase, snailase, and β-glucosidase, to hydrolyze the crude extract of mogroside to synthesize mogroside alcohol was investigated. 2mL reaction system contained 10mg of crude extract of mogroside (containing 50% mogroside V), 10mg of glycosidase, and 50mM disodium hydrogen phosphate-citric acid buffer (pH=4.5, added to 2mL of reaction system). After shaking the reaction at 45℃ for 24h, the reaction solution was boiled for 5min, cooled to room temperature, an equal volume of methanol was added, and after centrifugation at 12000rpm for 15min, the supernatant was detected by HPLC.

[0026] HPLC detection conditions are as follows:

[0027] Chromatographic column: Inertsil ODS-3V HPLC Column, 5μm, 150×4.6mm

[0028] Mobile phase: A: ultrapure water (containing 0.1% formic acid) B: acetonitrile (containing 0.1% formic acid)

[0029] Analysis time: 28min Injection volume: 10μL Column temperature: 30℃ Detection wavelength: 205nm

[0030] Flow rate: 1 ml / min Gradient elution: 10% B-90% B

[0031] Under the above HPLC analysis conditions, the retention time of mogrosanol was 13.4 min ( Figure 1 The production rate of mogrosanol = A m / A t ×100%,A m is the peak area of ​​mogrosanol, A t is the total area of ​​mogroside and mogrool. The results show that β-glucosidase and cellulase have no ability to hydrolyze mogroside to synthesize mogrool. The production rate of mogrool synthesized by snail enzyme is 72.1%, which is significantly higher than 32.0% of pectinase. Taking the production rate of mogrool as a reference indicator, the present invention preferably uses snail enzyme as a biocatalyst to synthesize mogrool.

[0032] Table 1 Screening results of glycosidase

[0033] Glycosidase type Monk Fruit Extract Concentration Yield of mogrosanol (%) cellulose enzyme 5mg / mL 0 Snail enzyme 5mg / mL 72.1％ Pectinase 5mg / mL 32％ β-Glucosidase 5mg / mL 0

[0034] Example 2 Investigation of Momordica grosvenori Extract

[0035] In a 2mL reaction system, different types of monk fruit extracts were 10mg, snail enzyme was 10mg and 50mM sodium dihydrogen phosphate-citric acid buffer (pH = 4.5), and the reaction was shaken at 45°C for 24 hours. The results showed that the extract containing 25% mogroside V, the extract containing 50% mogroside V and mogroside V could all be hydrolyzed by snail enzyme to produce mogroside alcohol. In view of the high cost of mogroside V and the high impurities in the extract containing 25% mogroside V, the substrate containing 50% mogroside V was used in subsequent investigations.

[0036] Example 3 Investigation of Buffer for Synthesis of Mogroside Alcohol by Snail Enzyme

[0037] In order to improve the efficiency of snail enzyme hydrolysis of crude extract of mogroside to synthesize mogroside alcohol, the present invention investigates the influencing factors such as buffer type, pH, reaction temperature, substrate concentration, etc. In a 2mL reaction system, 10mg of momordica grosvenori extract, 10mg of snail enzyme and 50mM buffer were used, and the reaction was shaken at 45℃ for 24 hours.

[0038] The results showed that the mogrosanol production rates of sodium citrate-citric acid buffer, disodium hydrogen phosphate-citric acid buffer and sodium acetate-acetic acid buffer were 68.5%, 72.1% and 67.8%, respectively. Disodium hydrogen phosphate-citric acid buffer was preferred for subsequent experiments.

[0039] Table 2 Screening results of aqueous reaction buffer

[0040] Buffer Type Monk Fruit Extract Concentration Yield of mogrosanol (%) Sodium citrate-citric acid buffer 5mg / mL 68.5％ Sodium hydrogen phosphate-citrate buffer 5mg / mL 72.1％ Sodium acetate-acetic acid buffer 5mg / mL 67.8％

[0041] Example 4 pH Investigation of the Synthesis of Momordica grosvenori Alcohol by Snail Enzyme

[0042] In the 2mL reaction system, the extract of Monk Fruit was 10mg, the snail enzyme was 10mg and the 50mM sodium dihydrogen phosphate-citric acid buffer (pH = 4.0-7.0), and the reaction was shaken at 45°C for 24 hours. The results showed that the production rates of Monk Fruit alcohol at pH = 4.0, pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5 and pH = 7.0 were 50.2%, 72.1%, 60.5%, 55.8%, 52.4%, 41.6% and 27.5%, respectively, and the subsequent experiments were preferably carried out with pH = 4.5 and 50mM sodium dihydrogen phosphate-citric acid buffer.

[0043] Table 3 Screening results of aqueous phase reaction pH

[0044] pH Monk Fruit Extract Concentration Yield of mogrosanol (%) 4.0 5mg / mL 50.2％ 4.5 5mg / mL 72.1％ 5.0 5mg / mL 60.5％ 5.5 5mg / mL 55.8％ 6.0 5mg / mL 52.4％ 6.5 5mg / mL 41.6％ 7.0 5mg / mL 27.5％

[0045] Example 5 Investigation of the reaction temperature of snail enzyme synthesis of mogrosanol

[0046] In a 2mL reaction system, 10mg of monk fruit extract, 10mg of snail enzyme and 50mM sodium dihydrogen phosphate-citrate buffer (pH=4.5) were shaken at 25℃-55℃ for 24 hours. The results showed that the production rates of monk fruit alcohol at 25℃, 30℃, 35℃, 40℃, 45℃, 50℃ and 55℃ were 32.6%, 49.6%, 60.5%, 64.3%, 69.3%, 72.6% and 67.6%, respectively, and 45℃ was preferred for subsequent experiments.

[0047] From the above records, snail enzyme is not like conventional enzymes, which have the strongest enzyme activity at 37°C. As the temperature rises, the enzyme activity is strongest at 50°C, and then decreases as the temperature rises.

[0048] Table 4 Water phase reaction temperature investigation

[0049]

[0050]

[0051] Example 6 Investigation of substrate concentration for the synthesis of mogrosanol by snail enzyme

[0052] In a 2mL reaction system, the extract of Monk Fruit was 2-10mg, the snail enzyme was 10mg and 50mM sodium dihydrogen phosphate-citric acid buffer (pH=4.5), and the reaction was shaken at 50°C for 24 hours. The results showed that the production rates of Monk Fruit alcohol at 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL and 5mg / mL of Monk Fruit extract were 69.3%, 72.5%, 74.7%, 72.8% and 70.3%, respectively, indicating that the Monk Fruit extract was not completely hydrolyzed at a concentration of 1-5mg / mL.

[0053] Table 5 Screening results of aqueous reaction substrate concentration

[0054] Monk Fruit Extract Concentration Yield of mogrosanol (%) 1mg / mL 69.3％ 2mg / mL 72.5％ 3mg / mL 74.7％ 4mg / mL 72.8％ 5mg / mL 70.3％

[0055] Example 7 Organic solvent screening for biphasic reaction system

[0056] Example 6 shows that a single solvent cannot completely enzymolyze the monk fruit extract, so the present invention will investigate the tolerance of snail enzyme to organic solvents, construct a two-phase enzymatic reaction system, improve the synthesis efficiency of monk fruit alcohol, and meet the needs of large-scale preparation. In order to construct a two-phase enzymatic reaction system and improve the yield of monk fruit alcohol, the present invention investigates the effect of ethyl acetate, n-butanol, toluene, n-hexane, petroleum ether and methyl tert-butyl ether (MTBE) on the efficiency of snail enzyme in preparing mogroside. In a 2mL reaction system, the monk fruit extract is 10mg, the snail enzyme is 10mg, the organic solvent is 0.6mL, 50mM sodium dihydrogen phosphate-citric acid buffer (pH=4.5), and the reaction is shaken at 45°C for 24 hours. The results show that when ethyl acetate, n-butanol, toluene, n-hexane, petroleum ether and methyl tert-butyl ether are organic phases, the production rates of monk fruit alcohol are 32.4%, 22.8%, 96.8%, 45.6%, 35.4% and 82.4%, respectively. According to the formation rate of mogrosanol, the two-phase mixed solvent formed by ethyl acetate, n-butanol, n-hexane and petroleum ether has an inhibitory effect on snail enzyme, and toluene and MTBE are obviously better than the single water phase reaction system, so the present invention will conduct subsequent dosage investigation.

[0057] Table 6 Results of snail enzyme tolerance to organic solvents

[0058]

[0059]

[0060] Example 8 Investigation of the amount of organic solvent used in a two-phase reaction system

[0061] In the 2mL reaction system, the extract of Momordica grosvenori is 10mg, the snail enzyme is 10mg, the organic solvent is 0.2-1.4mL, 50mM sodium dihydrogen phosphate-citric acid buffer (pH=4.5), and the reaction is shaken at 45°C for 24 hours. The results show that when the amount of toluene is 10%, 20%, 30%, 40%, 50%, 60% and 70%, the formation rate of Momordica grosvenori alcohol is 80.1%, 83.5%, 96.8%, 91.3%, 90.6%, 87.3% and 86.3% respectively. When the amount of MTBE is 10%, 20%, 30%, 40%, 50%, 60% and 70%, the formation rate of Momordica grosvenori alcohol is 85.4%, 89.6%, 82.4%, 80.6%, 78.9%, 76.5% and 72.1% respectively. According to the production rate of mogrosanol, the present invention selects 30% toluene and 50mM disodium hydrogen phosphate-citric acid buffer (pH=4.5) to construct a two-phase reaction system. The above shows that the two-phase reaction system containing 10-70% organic solvent is better than the control, among which toluene has the best effect. As the organic concentration increases, the catalytic efficiency increases. Toluene reaches 30% and is the highest (96.8%), while MTBE is 20% and is the highest (89.6%). The catalytic efficiency gradually decreases as the concentration of the organic solvent increases, but it is still higher than the control.

[0062] Table 7 Results of investigation on the amount of organic solvents

[0063]

[0064]

[0065] Example 9 Reaction Temperature Optimization of Dual-Phase Reaction System

[0066] In the 2mL reaction system, the extract of Momordica grosvenori was 15mg, the snail enzyme was 10mg, the toluene was 0.6mL, the 50mM sodium dihydrogen phosphate-citric acid buffer (pH=4.5), and the reaction was shaken at 35℃-60℃ for 24 hours. The results showed that the production rates of Momordica grosvenori alcohol at 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃ were 90.0%, 91.5%, 92.6%, 94.5%, 92.9%, and 75.5%, respectively. Therefore, 50℃ was selected as the optimal reaction temperature for the two-phase reaction system. As the temperature increased to 50℃, the catalytic efficiency was the highest, and then the catalytic efficiency decreased as the temperature increased.

[0067] Table 8 Screening results of biphasic reaction temperature

[0068] temperature Monk Fruit Extract Concentration Yield of mogrosanol (%) 35℃ 7.5 mg / mL 90.0％ 40℃ 7.5 mg / mL 91.5％ 45℃ 7.5 mg / mL 92.6％ 50℃ 7.5 mg / mL 94.5％ 55℃ 7.5 mg / mL 92.9％ 60℃ 7.5 mg / mL 75.5％ Water phase reaction system (control) 5mg / mL 72.6％

[0069] Example 10 Optimization of snail enzyme concentration

[0070] In a 2mL reaction system, the extract of Momordica grosvenori is 10-40mg, the snail enzyme is 10-30mg, the toluene is 0.6mL, the buffer of 50mM sodium hydrogen phosphate-citric acid (pH=4.5), and the reaction is shaken at 50°C for 24 hours. The results are shown in Table 1. When the concentration of snail enzyme is 11mg / mL, 15mg / mL of Momordica grosvenori extract can be catalyzed to be converted into Momordica grosvenori alcohol, and the generation rate reaches 94.5%. Therefore, the present invention finally selected 15mg / mL Momordica grosvenori extract and 11mg / mL snail enzyme for amplification reaction.

[0071] Table 9 Investigation on the concentration of monk fruit extract and the dosage of snail enzyme

[0072]

[0073]

[0074] Example 11 Preparation of Momordica alcohol

[0075] In a 0.5L two-phase reaction system, the extract of monk fruit (containing 50% mogroside V) is 7.5g, the snail enzyme is 5.5g, the toluene is 0.15L, the 50mM sodium dihydrogen phosphate-citric acid buffer (pH=4.5), and the reaction temperature is 50°C. Samples are taken every hour, and the formation of monk fruit alcohol is detected by HPLC. The formation rates of monk fruit alcohol after 4, 8, 12, 16, 20 and 24 hours of reaction are 45.6%, 80.3%, 90.4%, 96.7%, 97.8% and 98.0%, respectively. Taking the formation rate of monk fruit alcohol>95% as an indicator, the preferred reaction time is greater than or equal to 16 hours. The above experimental results show that the preparation of monk fruit alcohol by a large-volume two-phase system is practical.

[0076] Table 10 Biphasic reaction time investigation

[0077]

[0078] Example 12 Purification and Preparation of Momordica grosvenori Alcohol

[0079] After the reaction in Example 11 is completed, the reaction solution is allowed to stand until stratification, the toluene phase is collected, and the crude extract 1 of Momordica grosvenori alcohol is obtained. The aqueous phase is extracted twice with an equal volume of ethyl acetate, and the ethyl acetate phases are combined and dried under reduced pressure to obtain a crude extract 2 of Momordica grosvenori alcohol. Crude extracts 1 and 2 are combined and dissolved in a 20% ethanol solution (V / V) to obtain a crude extract solution. The crude extract solution is loaded onto a polyamide resin chromatography column (column diameter ratio = 10:1, column volume of 40 mL), and then washed with 20% ethanol solution (V / V) for 2 column volumes, 30% ethanol solution (V / V) for 4 column volumes, and 35% ethanol solution (V / V) for 4 column volumes. Collect and combine Momordica grosvenori alcohol solutions with a purity of >95%, and after drying, 1.24 g of pure Momordica grosvenori alcohol is obtained.

Claims

1. A biosynthetic method of mogrosanol, characterized in that: In a two-phase enzymatic reaction system, glycosidase is used to catalyze the hydrolysis of a crude extract of Momordica grosvenori to obtain Momordica alcohol; the glycosidase used is snail enzyme; the two-phase enzymatic reaction system is constructed by an organic reagent and a reaction buffer; the organic reagent is selected from methyl tert-butyl ether or toluene; the amount of snail enzyme is 5 mg / mL-15 mg / mL; the concentration of Momordica grosvenori extract is 10-20 mg / mL; the volume concentration of toluene is 10%-70% V / V; the reaction pH of the two-phase enzymatic reaction system is 4.0-7.0; the reaction temperature is 35°C-60°C; and the reaction time is 8-24 hours.

2. The biosynthesis method of mogrosanol according to claim 1, characterized in that: The buffer is selected from a buffer having a pH of 4.0-7.0, and the buffer is selected from a sodium citrate-citric acid buffer, a disodium hydrogen phosphate-citric acid buffer and a sodium acetate-acetic acid buffer.

3. The biosynthesis method of mogrosanol according to claim 2, characterized in that the steps include: Snail enzyme and monk fruit extract were added to the buffer solution, toluene was added, and the mixture was evenly mixed to obtain a two-phase reaction system. After the reaction was completed, the two phases were separated after standing for 2 hours, and the toluene layer was collected. The aqueous layer was extracted with ethyl acetate. Toluene and ethyl acetate were removed, and the crude extract of monk fruit alcohol was combined. The crude extract was dissolved in 20% V / V ethanol solution and then separated and purified using a polyamide resin column, and the ethanol-water solution was gradient eluted. The eluates containing high-purity monk fruit alcohol were combined and collected, and dried to obtain monk fruit alcohol.

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