Efficient mogroside biotransformation utilization method and application

Abstract

The invention discloses a method for efficient conversion and utilization of mogroside. According to the method, mogroside V is converted through fermentation of candida parapsilosis L7, and siamenoside I is directionally synthesized. According to the invention, Candida parapsiloides L7 is utilized for fermentation conversion, and beta-1, 6-glucosidic bonds in glucose branched chains connected with C3-site hydroxyl of a mogroside V structure parent nucleus can be directionally hydrolyzed with high regioselectivity (the beta-1, 6-glucosidic bonds on glucose branched chains connected with C24-site hydroxyl are hardly hydrolyzed), so that one molecule of glucose is removed, and siamenoside I is obtained. The siamenoside I provided by the invention can be efficiently prepared, and the maximum molar yield of the siamenoside I can reach 87.40%. The siamenoside I has more obvious selective hydrolysis characteristic, higher space time yield of the siamenoside I, no cytotoxicity of the siamenoside I and better anti-inflammatory and antioxidant activity, provides a new drug choice for diseases such as inflammation and the like, and provides technical support for development of high-valued products of mogroside.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and relates to a method for efficient conversion and utilization of mogrosides, in particular to a method for preparing Siamenoside I and its application in the preparation of anti-inflammatory and antioxidant drugs. Background Art

[0002] Momordica grosvenori Swingle is a traditional Chinese medicine belonging to the category of "medicinal and edible herbs", with the effects of clearing heat and detoxifying, antibacterial and anti-inflammatory, and lowering blood sugar. It is rich in triterpenoid sweet compounds, and mogroside V is one of the main components. As a natural sugar substitute, mogroside has the characteristics of high sweetness (100-300 times that of sucrose) and does not affect blood sugar metabolism, so it is widely used in the fields of food and health products [1]. In addition, studies have shown that mogroside series compounds have various pharmacological activities such as anti-inflammatory, anti-diabetic, and anti-pulmonary fibrosis [2-4], and have important application values in the medical field.

[0003] Due to its high sweetness characteristics, mogroside V is highly favored in the food industry, and its derivative Siamenoside I not only has a higher sweetness, but also is superior in taste and flavor [5]. At present, the conversion of mogroside V mainly relies on chemical or enzymatic catalysis technologies, but these methods are often accompanied by environmental pollution, high costs, and limitations in conversion efficiency; in addition, existing bioconversion technologies perform poorly in terms of substrate selectivity, and it is easy to produce more by-products during the conversion of mogroside V, which is not conducive to the separation and preparation of subsequent single products. Therefore, developing a highly efficient and green microbial catalytic conversion method to achieve the directional bioconversion of mogroside V to Siamenoside I has important industrial application values.

[0004] References

[0005] [1].Partap, M., et al., Fruit derived callus and cell suspensionculture as promising alternative sources for mogrosides production inSiraitia grosvenorii (Swingle) C. Jeffrey: a zero-caloric natural sweetener.Journal of Food Composition and Analysis, 2022. 108: p. 104450.

[0006] [2]. Yanan, S., et al., Inhibition of Mogroside IIIE on isoproterenol-induced myocardial fibrosis through the TLR4 / MyD88 / NF-kappaB signaling pathway. Iran J Basic Med Sci, 2023. 26(1): p. 114-120.

[0007] [3]. Xue, W., et al., Mogroside IIIE Alleviates High Glucose-Induced Inflammation, Oxidative Stress and Apoptosis of Podocytes by the Activation of AMPK / SIRT1 Signaling Pathway. Diabetes Metab Syndr Obes, 2020. 13: p. 3821-3830.

[0008] [4]. Tao, L., et al., Mogroside IIIE Attenuates LPS-Induced Acute Lung Injury in Mice Partly Through Regulation of the TLR4 / MAPK / NF-kappaB Axis via AMPK Activation. Phytother Res, 2017. 31(7): p. 1097-1106.

[0009] [5]. Su, Y., et al., Enzymatic hydrolyzation of mogrosides in Luo Han Guo extract by NKA-adsorbed snailase improves its sensory profile. Food Chem, 2022. 390: p. 133205. Summary of the Invention

[0010] The object of the present invention is to provide a method for efficient biotransformation and utilization of mogrosides in view of the problem that it is difficult to obtain a large amount of mogroside I by only physical adsorption, purification and other means directly from Siraitia grosvenorii fruits, and the economic efficiency is low. The technical problem to be solved by the present invention is a method of pressure stress type microbial culture and screening by adding Siraitia grosvenorii and mogroside V step by step, obtaining Candida orthopsilosis L7 with both Siraitia grosvenorii resistance and the function of directionally and efficiently synthesizing mogroside I, and using Candida orthopsilosis L7 for fermentation conversion, highly regioselectively and directionally hydrolyzing the β-1,6-glycosidic bond in the glucose branch chain connected to the hydroxyl group at the C3 position of the mogroside V structural nucleus (almost not hydrolyzing the β-1,6-glycosidic bond in the glucose branch chain connected to the hydroxyl group at the C24 position), and then removing one molecule of glucose to finally achieve the efficient synthesis of mogroside I, and the molar yield of mogroside I can reach up to 87.40% at most. Mogroside I has no cytotoxicity and has good anti-inflammatory and antioxidant activities.

[0011] To solve the above problems, the present invention provides the following solution: A method for efficient transformation and utilization of mogrosides, characterized in that the specific biotransformation and preparation method comprises the following steps:

[0012]

[0013] In the MG fermentation medium, Candida orthopsilosis L7 can selectively remove the β-1,6-glycosidic bond in the glucose branch chain connected to the hydroxyl group at the C3 position of the substrate mogroside V, so as to efficiently and directionally synthesize mogroside I. The said Candida orthopsilosis, deposit name: Candida orthopsilosis L7, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, China, deposit date: April 9, 2025, deposit number: CCTCC NO: M 2025731.

[0014] Preferably, the MG fermentation medium uses maltose as the carbon source.

[0015] Preferably, the concentration of Siraitia grosvenorii extract in the MG fermentation medium is 12 g / L.

[0016] The specific biotransformation method is: Pick a single colony of Candida orthopsilosis L7 and inoculate it into a potato liquid (PD) medium, culture it at 30 °C in a shaker at 150 - 200 rpm / min for 1 - 2 days, centrifuge at 4000 rpm for 10 min to collect the wet cells, inoculate them into the MG fermentation medium containing 4 - 20 g / L Siraitia grosvenorii extract (mogroside V content ≥ 50%) at 1 - 3% (w / v), and ferment in a shaking flask at 150 - 200 r / min at 30 °C for 1 - 3 days. The molar yield of mogroside I can reach up to 87.40% at most.

[0017] The formulation of the MG fermentation medium is as follows: 1.0 - 8.0 g / L maltose, 1.0 - 8.0 g / L (NH4)2SO4 (w / v), 0.5 - 4.5 g / L KH2PO4, 0.1 - 1.0 g / L NaCl, 0.1 - 1.0 g / L MgSO4·7H2O, 0.01 - 0.10 g / L ZnSO4·7H2O. Sterilize it with high-pressure steam at 121°C for 20 min and cool it to room temperature for standby.

[0018] This invention studies the anti-inflammatory effect of semonoside I on LPS-induced macrophages RAW264.7 to verify the application of semonoside I in the preparation of anti-inflammatory drugs. The method is to adopt a lipopolysaccharide (LPS)-induced inflammation model of mouse macrophages (RAW264.7) to investigate the anti-inflammatory activity of semonoside I. It is shown by qPCR that semonoside I has a significant inhibitory effect on inflammation. Semonoside I has a significant and non-toxic anti-inflammatory effect on LPS-induced mouse macrophages RAW264.7.

[0019] This invention studies the antioxidant ability of semonoside I by the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging method to verify the application of semonoside I in the preparation of antioxidant drugs. The method is to mix semonoside I and DPPH in a solution, detect the change in the absorbance of the sample, and investigate the antioxidant activity of semonoside I. Semonoside I can effectively scavenge DPPH free radicals and has good antioxidant activity.

[0020] Beneficial effects

[0021] 1. To meet the increasing demand for mogrosides in recent years in the fields of the food industry, clinical medicine, etc., this invention adopts a pressure stress-type microbial culture method of stepwise adding Momordica grosvenori and mogroside V, screens the microorganism Candida parapsilosis L7 with both Momordica grosvenori resistance and the function of directionally and efficiently synthesizing semonoside I from soil samples in the origin area of Momordica grosvenori, and uses a commercially available and inexpensive Momordica grosvenori extract (mogroside V content ≥ 50%, Chengdu Lemeitian Medical Technology Co., Ltd., batch number: DST230220-025) as the raw material, and realizes the high-efficient, non-toxic and large-batch preparation of semonoside I through screening and obtaining microbial fermentation and directional transformation.

[0022] 2. This invention provides a method for the directional synthesis of semonoside I by fermenting and transforming mogroside V with Candida parapsilosis L7.

[0023] 3. Semonoside I has no cytotoxicity and has good anti-inflammatory and antioxidant activities, providing a new drug option for diseases such as inflammation. The results of the DPPH free radical scavenging experiment of semonoside I show that semonoside I has good antioxidant activity. Figure 6 It is the antioxidant effect of semonoside I.

[0024] 4. The method for preparing semonoside I by biotransformation provided by the present invention. The inoculum amount of Candida parapsilosis is 1 - 3% (w / v), the concentration of Siraitia grosvenorii extract (the mogroside V content is ≥50%) is 4 - 20 g / L, and it is subjected to shaking flask fermentation at 150 - 200 rpm / min for 1 - 3 days at 30°C. The highest yield of semonoside I can reach 87.40%.

[0025] 5. The present invention provides a strain of Candida parapsilosis that has resistance to Siraitia grosvenorii and can efficiently and directionally transform mogroside V into semonoside I. The above strain can ferment and directionally hydrolyze the β-1,6-glycosidic bond in the glucose branch chain connected to the C3 hydroxyl group of the mogroside V structural nucleus (hardly hydrolyzing the β-1,6-glycosidic bond in the glucose branch chain connected to the C24 hydroxyl group), and then remove 1 molecule of glucose, ultimately achieving the efficient synthesis of semonoside I. It is isolated from soil samples from the origin of Siraitia grosvenorii, enriched and cultured in LB liquid medium and potato liquid (PD) medium supplemented with Siraitia grosvenorii (50 - 250 g / L) for 1 - 2 days, diluted and spread on LB solid medium and potato solid (PDA) medium for 1 - 2 days, and the significantly growing colonies are selected and transferred to a screening medium with Siraitia grosvenorii extract (the mogroside V content is ≥50%) as the sole carbon source, continuously fermented for 1 - 2 days, sampled every 12 h, and the fermentation products are detected by UPLC. The strains with the ability to directionally transform mogroside V into semonoside I are selected as candidate strains, identified as Candida parapsilosis by 18srDNA, named Candida parapsilosis L7, and the strain is preserved.

[0026] 6. The present invention obtains 4 strains with the ability to biotransform mogroside V, which are identified as Aspergillus terreus, Aspergillus niger, Candida utilis, and Candida parapsilosis respectively. Among them, the main hydrolysis products of 3 strains (Aspergillus terreus, Aspergillus niger, Candida utilis) are mogroside IIIE, and only 1 strain (Candida parapsilosis L7) has the ability to selectively remove the β-1,6-glycosidic bond in the glucose branch chain connected to the C3 hydroxyl group of mogroside V to form semonoside I. In addition, there is 1 strain of Candida albicans, and it is detected that it does not have the ability to biotransform mogroside V.

[0027] 7. The results of the 3-day fermentation and transformation of mogroside V by Candida parapsilosis L7 of the present invention show that the molar content of semonoside I is above 82%. The process for the efficient and directional synthesis of semonoside I by Candida parapsilosis L7 fermenting and transforming mogroside V has more obvious characteristics of selective hydrolysis, higher space-time yield of semonoside I, and is innovative.

[0028] 8. Detection was performed by UPLC. After UPLC detection, when fermented for 3 days in the presence of glucose, maltose, and sucrose respectively, the molar yields of mogroside V converted to neomogroside I by this strain were 83.97%, 86.32%, and 79.09% respectively. Adding maltose was the optimal choice.

[0029] After UPLC detection, when the substrate concentration was 12 g / L, the highest molar yield of mogroside V converted to neomogroside I by the strain could reach 87.40% (Table 1), and both the biomass and the substrate utilization efficiency were at relatively optimal levels. Brief Description of the Drawings

[0030] Figure 1 Front view of the PDA medium plane of Candida parapsilosis L7

[0031] Figure 2 UPLC detection chart of microbial fermentation conversion of mogroside V (2 days) a: Mogroside series standards (V: mogroside V; SI: neomogroside I; IVA: mogroside IVA; IV: mogroside IV; IIIE: mogroside IIIE); b: Momordica grosvenori extract (mogroside V content ≥ 50%); c - g: Aspergillus terreus, Aspergillus niger, Candida utilis, Candida albicans, Candida parapsilosis L7 respectively

[0032] Figure 3 UPLC detection chart of Candida parapsilosis L7 fermentation conversion of mogroside V (3 days) a: Mogroside series standards (V: mogroside V; SI: neomogroside I; IVA: mogroside IVA; IV: mogroside IV; IIIE: mogroside IIIE); b and c: Samples of Candida parapsilosis L7 fermentation conversion of Momordica grosvenori extract (mogroside V content ≥ 50%) at 0 day and 3 days respectively

[0033] Figure 4 Cytotoxic effect of neomogroside I on macrophage RAW264.7

[0034] Figure 5 Anti - inflammatory effect of neomogroside I on LPS - induced macrophage RAW264.7

[0035] Figure 6 Antioxidant effect of neomogroside I (1, 2, 3, 4, 5 respectively represent the final concentration of neomogroside I being 1, 2, 3, 4, 5 μg / mL)

[0036] The present invention provides a Candida orthopsilosis, with a preservation name of Candida orthopsilosis L7, a preservation unit of China Center for Type Culture Collection, a preservation address of Wuhan University, China, a preservation date of April 9, 2025, and a preservation number of CCTCC NO: M 2025731. DETAILED DESCRIPTION

[0037] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0038] Example 1: Screening and identification of strains for directed synthesis of simenoside I

[0039] 5 g of soil samples from each place of origin of Momordica grosvenori were added to 3 100 mL LB liquid medium containing Momordica grosvenori (200 g / L) and 3 100 mL PD liquid medium containing Momordica grosvenori (200 g / L), respectively, and enriched and cultured at 30℃, 150 r / min, for 2 days, and diluted and spread on LB solid medium and PDA solid medium, respectively, and cultured in a constant temperature incubator at 30℃ for 2 days. The colonies with significant growth were selected and inoculated into blank LB solid medium and PDA solid medium by the plate streak method, and cultured for 2 days; the above steps were repeated until single colonies of different strains were obtained. Single colonies with different growth characteristics were selected and inoculated into LB liquid medium and PD liquid medium, respectively, and cultured at 30℃ for 3 days. The growth trends of different strains were comprehensively evaluated, and 17 strains were screened and stored in ultra-low temperature after being numbered.

[0040] PD liquid culture medium: add 1000 mL of deionized water to 200 g of fresh potatoes and boil for 30 min. Add 20 g of glucose to the liquid obtained by filtering through 8 layers of gauze and add deionized water to 1000 mL (18 g / L of agar needs to be added to PDA solid culture medium). Sterilize with high pressure steam at 121°C for 20 min and cool to room temperature for use.

[0041] LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl (18 g / L agar needs to be added to LB solid culture medium), sterilize with high-pressure steam at 121°C for 20 min, and cool to room temperature for use.

[0042] MG selective medium: 5.0 g / L (NH4)2SO4 (w / v), 2.0 g / L KH2PO4, 0.5 g / L NaCl, 0.4 g / L MgSO4·7H2O, 0.04 g / L ZnSO4·7H2O, autoclaved at 121 °C for 20 min, cooled to room temperature for standby.

[0043] The preserved strain was inoculated into PD liquid medium at a ratio of 1:1000 (v / v) from the cryotube, cultured at 30 °C in a shaker at 150 r / min for 1 day, and the wet cells were collected by centrifugation or filtration. Then, they were added to the MG selective medium at a concentration of 2% (w / v). At the same time, mogroside extract (mogroside V content ≥ 50%) was added to a concentration of 5 g / L, and fermentation was carried out at 30 °C in a shaker at 150 r / min for 2 days. Using mogroside extract as the sole carbon source, strains with the ability to transform mogroside V were screened. The fermentation time was calculated from the time when mogroside extract was added to the selective medium. At fermentation times of 0, 12, 24, 36, and 48 h, the supernatant of the fermentation broth was taken, centrifuged at 12000 r / min for 5 min, the supernatant was taken, an equal volume of chromatographically pure methanol was added, and centrifugation was carried out again under the same conditions for 5 min. The supernatant was filtered through a 0.22 mm filter membrane and detected by UPLC. After the reaction, 4 strains with the ability to biotransform mogroside V were obtained by UPLC detection. After identification, they were Aspergillus terreus, Aspergillus niger, Candida utilis, and Candida parapsilosis, respectively. Among them, the main hydrolysis products of 3 strains (Aspergillus terreus, Aspergillus niger, Candida utilis) were mogroside IIIE, and only 1 strain (Candida parapsilosis) had the ability to selectively remove the β-1,6-glycosidic bond in the glucose branch chain connected to the C3 hydroxyl group of mogroside V to form neomogroside I. In addition, there was 1 strain of Candida albicans, which was detected to have no ability to biotransform mogroside V (as Figure 2 shown in Fig. f).

[0044] One strain with the ability to selectively remove the β-1,6-glycosidic bond in the glucose branch chain connected to the C3 hydroxyl group of mogroside V to form neomogroside I obtained by the above screening was cultured on PDA solid medium at 30 °C for 3 days. The colony was milky white, shiny, relatively flat, and had a neat edge, showing a yeast-like shape. Through 18S rDNA homology identification and phylogenetic analysis, this strain was identified as Candida parapsilosis and named Candida parapsilosis L7. Deposit name: Candida parapsilosis L7, Deposit unit: China Center for Type Culture Collection, Deposit address: Wuhan University, China, Deposit date: April 9, 2025, Deposit number: CCTCC NO: M 2025731.

[0045] Example 2: Effect of fermentation carbon source on the directed synthesis of neomogroside I by Candida parapsilosis L7

[0046] Take the stored strain Candida parapsilosis L7 in the strain bank, dip the bacterial liquid and inoculate it into a PDA solid medium plate by the streaking method, culture and activate it in a constant temperature incubator at 30 °C for 3 days, pick the activated single colony, transfer it into a PD liquid medium, culture it at 30 °C in a shaker at 150 r / min for 1 day, centrifuge to collect the wet bacterial cells, and inoculate them into an MG selective medium containing 5 g / L of different carbon sources (glucose, maltose, sucrose) at a concentration of 2% (w / v). At the same time, add mogroside extract (using a commercially available and inexpensive mogroside extract (mogroside V content ≥ 50%, Chengdu Lemeitian Medical Technology Co., Ltd., batch number: DST230220 - 025) as the raw material) to a concentration of 5 g / L, and ferment at 30 °C in a shaker at 150 r / min for 3 days. The fermentation time starts from the addition time of the mogroside extract in the medium. Take the supernatant of the fermentation broth at fermentation times of 0, 12, 24, 36, 48, 60, and 72 h respectively, centrifuge at 12000 r / min for 5 min, take the supernatant, add an equal volume of chromatographically pure methanol, centrifuge again under the same conditions for 5 min, filter the supernatant with a 0.22 mm filter membrane, and detect it by UPLC. After UPLC detection, under the conditions of the presence of glucose, maltose, and sucrose respectively, after 3 days of fermentation, the molar yields of the conversion of mogroside V to neomogroside I by this strain are 83.97%, 86.32%, and 79.09% respectively.

[0047] Example 3: Effect of Substrate Concentration on the Directed Synthesis of Neomogroside I by Candida parapsilosis L7

[0048] Substrate concentration is an important factor affecting the rate of enzyme-catalyzed reactions. Within a certain range, an increase in substrate concentration can increase the rate of enzyme-catalyzed reactions. Obtain wet bacterial cells according to the same method as in Example 2, and inoculate them into an MG fermentation medium with a mogroside extract (mogroside V content ≥ 50%) concentration of 4, 8, 12, 16, and 20 g / L at a concentration of 2% (w / v), culture at 30 °C in a shaker at 150 r / min, and continuously ferment for 3 days. Starting from the time of adding the mogroside extract, take the supernatant of the fermentation broth at fermentation times of 0, 12, 24, 36, 48, 60, and 72 h respectively, centrifuge at 12000 r / min for 5 min, take the supernatant, add an equal volume of chromatographically pure methanol, centrifuge again under the same conditions for 5 min, filter the supernatant with a 0.22 mm filter membrane, and detect it by UPLC. After UPLC detection, when the substrate concentration is 12 g / L, the highest molar yield of the conversion of mogroside V to neomogroside I by the strain can reach 87.40% (Table 1), and both the biomass and the substrate utilization efficiency are at relatively optimal levels.

[0049] MG fermentation medium: 5 g / L maltose, 5.0 g / L (NH4)2SO4 (w / v), 2.0 g / L KH2PO4, 0.5 g / L NaCl, 0.4 g / L MgSO4·7H2O, 0.04 g / L ZnSO4·7H2O, autoclaved at 121 °C for 20 min, cooled to room temperature for standby.

[0050] Table 1 Fermentation conversion of mogroside V by Candida pseudotrichopsilosis L7 (3 days)

[0051]

[0052] Example 4: Preparation of separation by column chromatography of neomogroside I

[0053] After Candida pseudotrichopsilosis L7 was fermented and converted the Momordica grosvenori extract (mogroside V content ≥ 50%) in the MG fermentation medium for 3 days, methanol was added at a volume ratio of 1:1 to terminate the reaction. The supernatant was collected by centrifugation at 4000 rpm for 10 min, and vacuum concentrated under reduced pressure using a rotary evaporator at a heating temperature of 40 °C. After rotary evaporation to 1 / 10 of the volume, it was separated by macroporous resin AB-8. An appropriate amount of resin was soaked in methanol for 24 h, then packed into a column by the wet method, rinsed with 2 column volumes (BV) of methanol to remove resin fragments and debris, and then rinsed with distilled water containing 0.1% glacial acetic acid until there was no alcohol smell; the sample was loaded onto the column at 20 mg / g wet resin, incubated overnight, and then eluted with a mobile phase of methanol plus deionized water at a rate of 2 BV / h. After washing away impurities with 5 BV of 40% methanol, the crude separation solution of neomogroside I was eluted with 5 BV of 60% - 70% methanol; the crude separation solution was vacuum concentrated under reduced pressure using a rotary evaporator at a heating temperature of 40 °C. After rotary evaporation to 1 / 10 of the volume, it was finely separated by MCI small pore resin. The method of packing the MCI small pore resin into the column was the same as that of the macroporous resin. The sample loading amount was 20 mg / L wet resin, and it was eluted with 50% methanol. Neomogroside I with a purity of more than 90% was obtained by UPLC detection. The elution solutions were combined, vacuum concentrated under reduced pressure using a rotary evaporator at a heating temperature of 40 °C, evaporated to dryness, then 5 mL of deionized water was added to dissolve it. The sample dissolved in water was frozen at -80 °C overnight and then freeze-dried in a freeze dryer for 3 days.

[0054] Example 5: Application of neomogroside I in anti-inflammatory

[0055] (1) Cell viability experiment

[0056] RAW264.7 cells were placed in a 10 cm culture dish with DMEM medium containing 10% FBS and 1% double antibody, and cultured in a cell incubator at 37 °C and 5% CO2. The RAW264.7 cells were seeded at 1×10 4Inoculate cells at a density into a 96-well plate. After culturing for 24 h until they adhere to the wall, add Symendoside I (1, 10, 50 μM), and culture in a 37 °C, 5% CO₂ cell culture incubator for 24 h. Then add 10 μL of CCK8 to each well of the 96-well plate containing the cultured cells, place it in a 37 °C, 5% CO₂ cell culture incubator for 2 h, and detect the absorbance value at a wavelength of 450 nm using a full-wavelength microplate reader.

[0057] (2) Seeding the plate and mRNA extraction

[0058] Select RAW264.7 cells in good growth state, gently blow the cells off with cell culture medium, collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 3 min, carefully aspirate the supernatant, resuspend the cells, perform cell counting using a hemocytometer, and inoculate 1 × 10 5 cells into each well of a 6-well plate. After culturing in a carbon dioxide cell culture incubator for 24 h, set up 5 drug administration groups, namely a control group (serum-free medium), a model group (serum-free medium containing 5 μg / mL LPS), and drug administration groups (serum-free medium containing 5 μg / mL LPS and curcumin glycoside complex at three concentrations).

[0059] Pre-protect the cells with the drug for 6 h, then add 5 μg / mL LPS and stimulate for 3 h. Discard the cell culture medium, add 300 μL of RNAiso Plus reagent, and repeatedly pipette to fully lyse the cells. Transfer the cell lysate to an RNase-free 1.5 mL tube;

[0060] Then add chloroform (1 / 5 volume of RNAiso Plus) to the cell lysate, tightly cap the EP tube, and invert the centrifuge tube up and down to fully mix until the solution becomes milky white, and let it stand for 15 min;

[0061] Centrifuge at 12000 rpm at 4 °C for 15 min. Carefully take out the EP tube from the low-temperature high-speed centrifuge. At this time, the homogenate is divided into three layers, namely a colorless supernatant (containing RNA), a middle white protein layer (mostly DNA), and a colored lower organic phase. Carefully aspirate the supernatant and transfer it to another new EP tube, taking care not to aspirate the white middle layer;

[0062] Precipitate the RNA. Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube to mix well, let it stand at room temperature for 10 min, centrifuge at 12000 rpm at 4 °C for 15 min, observe the appearance of RNA precipitate at the bottom of the tube, carefully discard the supernatant, and do not touch the precipitate;

[0063] Wash the RNA. Add 500 μL of 75% ethanol, gently invert the tube up and down to wash the RNA pellet, centrifuge at 9000 rpm at 4 °C for 5 min, discard the supernatant without touching the pellet, and repeat the RNA washing 2 times;

[0064] Dissolve the RNA. Open the lid of the EP tube to dry the pellet. Observe that the pellet changes from white to transparent, and add an appropriate amount of DEPC-treated water to dissolve the pellet;

[0065] RNA quantification and purity analysis. Take 2 μL of the dissolved RNA solution, use a multifunctional microplate reader to detect the concentration, and measure the absorbance values at wavelengths of 260 nm and 280 nm respectively. An OD260 / OD280 ratio between 1.8 - 2.1 indicates high RNA purity, OD260 / OD280 < 1.8 indicates possible residual protein impurities, and OD260 / OD280 > 2.1 indicates possible RNA degradation.

[0066] (3)Reverse transcription

[0067] The reverse transcription reagent for mRNA, HiScript® II Q RT SuperMix for qPCR (+ gDNA wiper), was purchased from Nanjing Novoprotein Scientific Co., Ltd., product number R223, and is suitable for two-step qRT-PCR detection. The 4× gDNA wiper Mix in the product can completely remove the residual genomic DNA contamination in the RNA template, and the program is 42 °C for 2 min; the 5× HiScript qRT SuperMix Ⅱ contains all the components required for the reverse transcription reaction. Adding the template RNA and water can initiate the reaction quickly, and at the same time terminate the action of the gDNA wiper to ensure the integrity of the cDNA. Reverse transcribe the RNA into cDNA according to the kit instructions, and the reverse transcription system is 20 μL. The specific reverse transcription reaction system and reaction program are shown in Table 2.

[0068] Table 2 Reverse transcription reaction system and reaction program

[0069]

[0070] Note: The 4 × gDNA wiper Mix and 5 × HiScript qRT SuperMix Ⅱ contain a high concentration of glycerol. Before use, briefly centrifuge to collect it at the bottom of the tube, and gently pipette and mix well before accurately pipetting.

[0071] (4)Real-time fluorescence q-PCR

[0072] The Q-PCR reagent AceQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novoprotein Scientific Inc., with the product number Q511. It is a dedicated reagent for qPCR using the SYBR Green I chimeric fluorescence method. Prepare the q-PCR reaction system according to the kit instructions. The total volume of the system is 20 μL, and the specific volumes of each reactant are shown in Table 3 as follows.

[0073] Table 3 q-PCR reaction system

[0074]

[0075] Prepare the above reagents with each reactant according to the kit instructions. After thoroughly mixing them by inverting the tube up and down, centrifuge the reagents on the tube wall to the bottom of the tube, and place it in the StepOne Real Time PCR instrument. Perform the q-PCR reaction using the program shown in Table 4. For data analysis, normalize by subtracting the Ct value of β-actin in the same group from the Ct value of each gene, and perform the conversion using the 2 -△△ Ct method, and then calculate the corresponding expression levels of each gene.

[0076] Table 4 q-PCR reaction conditions

[0077]

[0078] The primer sequences of each gene are shown in Table 5.

[0079] Table 5 q-PCR primer sequences

[0080]

[0081] The results showed that the obtained Saimenggan I prepared in Example x could dose-dependently inhibit the expression of TNF-α, IL-1β, and IL-6 in macrophages, showing a good anti-inflammatory effect.

[0082] Statistical method: The calculated data are expressed as [data representation]. Use SPSS statistical software for statistical analysis. One-way ANOVA is used for comparison among multiple groups, and the T-test is used for comparison between two groups. The test level is α = 0.05.

[0083] The results of the effect of Saimenggan I on the cell viability of macrophage RAW264.7 showed that Saimenggan I had no cytotoxicity. Figure 4 This is the effect of Saimenggan I on the cell viability of macrophage RAW264.7.

[0084] The anti-inflammatory effect of syenoside I on LPS-induced macrophages RAW264.7 was significant compared with the blank group, demonstrating the successful establishment of the cell inflammation model; compared with the model group, syenoside I had a significant anti-inflammatory effect. Figure 5 It is the anti-inflammatory effect of syenoside I on LPS-induced macrophages RAW264.7.

[0085] Example 6: Application of syenoside I in anti-inflammatory

[0086] The antioxidant activity of syenoside I was evaluated by the DPPH method. DPPH was dissolved in methanol aqueous solution (50%, v / v) to prepare a 0.1 mM DPPH solution. At the same time, solid syenoside I was mixed with 4 mL of DPPH solution to obtain a mixed system with a final concentration of syenoside I of 1, 2, 3, 4, 5 μg / mL. The reaction was carried out in the dark at room temperature for 60 minutes. The absorbance of the test sample was measured at a wavelength of 517 nm, and the antioxidant activity calculation formula was as follows:

[0087]

[0088] A0 represents the absorption value of the blank group (DPPH + methanol aqueous solution), and A1 represents the absorption value of the experimental group (DPPH + methanol aqueous solution + syenoside I).

[0089] Statistical method: Each experiment was repeated 5 times, and data were reported as mean ± standard deviation using SPSS 27.0 software. Statistical analysis was performed using GraphPad Prism 9 software, and one-way ANOVA was used.

[0090] The results of the DPPH free radical scavenging experiment of syenoside I showed that syenoside I had good antioxidant activity. Figure 6 It is the antioxidant effect of syenoside I.

Claims

1. A method for efficient biotransformation and utilization of mogrosides, characterized in that : The specific method for the efficient biotransformation of mogroside to synephrine I is as follows: Candida orthopsilosis L7 in the MG fermentation medium can selectively remove the β-1,6-glycosidic bond in the glucose branch chain linked to the C3 hydroxyl group of the substrate mogroside V, thus efficiently and directionally synthesizing synephrine I; The Candida orthopsilosis, deposit name: Candida orthopsilosis L7, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, China, deposit date: April 9, 2025, deposit number: CCTCC NO: M 2025731.

2. The method for efficient biotransformation and utilization of mogrosides according to claim 1, wherein: The MG fermentation medium uses maltose as the carbon source.

3. The method for highly efficient biotransformation and utilization of mogrosides according to claim 1, wherein: The concentration of momordica grosvenori extract in the MG fermentation medium is 12 g / L.

4. The method for highly efficient biotransformation and utilization of mogrosides according to claim 1, characterized in that: Specifically, it includes the following steps: Pick a single colony of Candida orthopsilosis L7 and inoculate it into the potato liquid PD medium. Culture it in a shaker at 150-200 rpm / min at 30°C for 1-2 days. Centrifuge at 4000 rpm for 10 min to collect the wet cells. Inoculate them at 1-3% (w / v) into the MG fermentation medium containing 4-20 g / L momordica grosvenori extract and the mass content of mogroside V ≥ 50%. Ferment in a shaking flask at 150-200 r / min at 30°C for 1-3 days. The highest molar yield of synephrine I can reach 87.40%.

5. The method for highly efficient biotransformation and utilization of mogrosides according to claim 1, characterized in that: The formula of the MG fermentation medium is: 1.0-8.0 g / L maltose, 1.0-8.0 g / L (NH4)2SO4 (w / v), 0.5-4.5 g / L KH2PO4, 0.1-1.0 g / L NaCl, 0.1-1.0 g / L MgSO4·7H2O, 0.01-0.10 g / L ZnSO4·7H2O. Sterilize at 121°C with high-pressure steam for 20 min and cool to room temperature for standby.

6. Use of the symenglycoside I prepared by the method according to claim 1, characterized in that: The application of the synephrine I in the preparation of antioxidant drugs.

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