WO2017190422A1 - Method for preparing novel mogrol derivative from total mogroside - Google Patents

Method for preparing novel mogrol derivative from total mogroside Download PDF

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WO2017190422A1
WO2017190422A1 PCT/CN2016/089211 CN2016089211W WO2017190422A1 WO 2017190422 A1 WO2017190422 A1 WO 2017190422A1 CN 2016089211 W CN2016089211 W CN 2016089211W WO 2017190422 A1 WO2017190422 A1 WO 2017190422A1
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optionally
composition
alcohol
han guo
volume ratio
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PCT/CN2016/089211
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French (fr)
Chinese (zh)
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余佩华
邓跃敏
钟牧源
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深圳以诺生物制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)

Definitions

  • the invention relates to the field of medicine, in particular to a method for synthesizing a mogroside derivative.
  • ginsenoside is the main ginseng (three-seven) Active ingredients, more than 40 monomer components have been isolated and identified from ginseng (37) (eg Ra, Rb1) Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, Rf, etc.), after being metabolized by the human body, will produce some minimal amounts of new metabolic oligo monomers Rg3, Rh2, C-K, aPPD and aPPT.
  • Mangosteen is a precious medicinal material belonging to the perennial vine of Cucurbitaceae. It is cool and sweet.
  • the non-sugar sweet saponin component of Luo Han Guo extract also known as Luo Han Guo total saponin, has been isolated and identified in the total saponin of Siraitia grosveni according to the number of saponin-linked glycosyl groups and the position of the saponin.
  • mogroside II, III, IV, V, VI mogroside II, III, IV, V, VI
  • mogroside V-MogrosideV 1A
  • Mogroside IV-Mogroside IV 1B
  • Mogrosides are two glucoside side chains consisting of four or fewer glucose units linked by a ⁇ -glycosidic bond to C-3 and C-24 of the sapogenin, Lo Hanol Mogrol (2) (as shown in Figure 2).
  • the linkage between the side chain glucoses is ⁇ -1,6 and ⁇ -l,2 glycosidic bonds.
  • mogroside can increase the utilization of glucose and fat and increase the sensitivity of insulin, but the active ingredients and mechanism of action of mogroside in hypoglycemic effect are not clear.
  • Chinese Academy of Sciences Shanghai The research on the hypoglycemic effect of Luo Han Guo was studied by the research group Hu Lihong and Shen Xu. Firstly, the highest content of mogroside V (1A) in Luo Han Guo was tested in HepG2 cells, and it was found to be inactive to AMPK.
  • mogrostat Mogrol (2) and its derivative 3 ⁇ -hydroxy-25-dehydroxy-24-oxomogrol (3) can activate AMPK (Chen XB, Et al; Bioorganic & Medicinal Chemistry 2011, 19: 5776).
  • the technical problem to be solved by the present invention is to provide a synthesis method comprising a composition of a mogroside derivative monomer (3) and a mogroside derivative monomer (4).
  • the present invention further provides a mogroside A method for producing an alcohol derivative monomer (3) and a mogroside derivative monomer (4).
  • a first aspect of the invention provides a composition comprising at least the structure
  • a method for synthesizing the above composition comprising: dissolving a Luo Han Guo extract containing Luo Han Guo total saponin in an aqueous solution of an acidic alcohol for heating reaction.
  • the aqueous solution of the acidic alcohol is prepared by adjusting the aqueous solution of the alcohol to an acidic state with an acidic substance.
  • the alcohol is at least one of methanol, ethanol, n-propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerin, and polyethylene glycol.
  • the volume ratio of alcohol to water in the aqueous solution of the alcohol is from 1:10 to 10:1.
  • the volume ratio of alcohol to water in the aqueous solution of the alcohol is from 1:3 to 3:1.
  • the volume ratio of alcohol to water in the aqueous solution of the alcohol is 1:1.
  • the acidic substance is an inorganic acid or an organic acid.
  • the inorganic acid is at least one of hydrochloric acid, sulfuric acid, and phosphoric acid.
  • the organic acid is at least one of formic acid, acetic acid, haloacetic acid, oxalic acid, malonic acid, and citric acid.
  • the acidity is represented by a pH of from 2.5 to 3.5.
  • the acidity is represented by a pH of 2.8 to 3.0.
  • the Luo Han Guo total saponin-containing Luo Han Guo extract is acidic.
  • the mass ratio of the aqueous solution of the alcohol is from 1:10 to 1:3.
  • the mass-to-volume ratio of the Luo Han Guo extract of the Luo Han Guo total saponin to the aqueous solution of the acidic alcohol is 1:5.
  • the heating reaction is carried out under the conditions of heating at 100 to 140 ° C for 1 to 3 hours.
  • the heating reaction is carried out under the conditions of heating at 120 ° C for 2 hours.
  • the reaction liquid obtained by the heating reaction is neutralized with an alkaline substance, and the alcohol is evaporated by heating to obtain a concentrate containing the composition.
  • neutralization with an alkaline material is neutralized to a pH of 6.5 to 7.5.
  • neutralization with a basic substance is neutralized to a pH of 7.0.
  • the basic substance is an inorganic base and/or a salt.
  • the inorganic base and/or salt is at least one of NaOH, KOH, and Na 2 CO 3 .
  • the method for synthesizing the composition further comprises the step of obtaining a brown extract after washing the concentrate containing the composition.
  • the step of washing the concentrate containing the composition to obtain a brown extract comprises: adding distilled water to the concentrate containing the composition, cooling and standing, discarding the upper aqueous layer to obtain a brown color containing the composition. extract.
  • the volume ratio of the concentrate to distilled water is from 1:5 to 1:3.
  • the method of synthesizing the composition further comprises extracting and concentrating the brown extract containing the composition to obtain a light brown solid containing the composition.
  • the extraction solvent used in the extraction is ethyl acetate.
  • the solid to liquid ratio of each extraction is from 1:6 to 1:3.
  • the number of extractions is 2 to 5 times, and each extract is combined.
  • the number of extractions is three.
  • the volume of the extraction solvent used in the first extraction is twice the volume of the extraction solvent used in the second extraction or the third extraction.
  • the concentration is: concentration at 100-120 ° C using a rotary evaporator, and the extraction solvent is completely evaporated to dryness.
  • a third aspect of the present invention provides a structure having the following structure
  • the preparation method of the mogroside derivative monomer (3) comprises dissolving the Luo Han Guo extract containing the Luo Han Guo total saponin in an aqueous solution of an acidic alcohol, heating, and then separating and purifying.
  • the step of separating and purifying the light brown solid containing the composition of claim 7 is included.
  • the separation is chromatographic separation.
  • the chromatographic separation is performed by silica gel column chromatography.
  • the silica gel column chromatography separation uses an acetonitrile and water
  • the volume ratio of acetonitrile to water is 95:5 to 20:1;
  • the light brown solid containing the composition is further concentrated after separation
  • the concentration is followed by chromatographic purification
  • the chromatographic purification is purified by silica gel column chromatography
  • the eluate used in the silica gel column chromatography purification is chloroform and ethanol.
  • the volume ratio of chloroform to ethanol is from 16:1 to 8:1.
  • the volume ratio of chloroform to ethanol is 12:1.
  • a fourth aspect of the present invention provides a structure having the following structure
  • the preparation method of the mogroside derivative monomer (4) comprises dissolving the Luo Han Guo extract containing the Luo Han Guo total saponin in an acidic alcohol/water solution for heating reaction, and then separating and purifying.
  • the step of separating and purifying the above-described light brown solid containing the composition is included.
  • the separation is chromatographic separation.
  • the chromatographic separation is performed by silica gel column chromatography.
  • the silica gel column chromatography separation uses an acetonitrile and water.
  • the volume ratio of acetonitrile to water is from 95:5 to 20:1.
  • the light brown solid containing the composition is further concentrated after separation.
  • the residue is purified by chromatography.
  • the chromatographic purification is purified by silica gel column chromatography.
  • the silica gel column chromatography purification is carried out using petroleum ether and ethyl acetate.
  • the volume ratio of petroleum ether to ethyl acetate is from 3:2 to 4:1.
  • the volume ratio of petroleum ether to ethyl acetate is 3:1.
  • rohansol derivative monomer (3) as used herein means 3 ⁇ -hydroxy-25-dehydroxy-24-oxomogrol; the term is used herein.
  • New romanol derivative monomer (4) means 3 ⁇ -hydroxy-22,24-diene-24,25-dehydroxy-rohansol (3 ⁇ -hydroxy-22, 24-diene-24, 25-dehydroxy -mogrol)(4).
  • the present invention provides a method for synthesizing a composition comprising a monomer component of the mogroside derivative (3) and a mogroside derivative monomer (4), as compared with the conventional method for synthesizing the mogroside derivative monomer (3),
  • the synthesis method is simple, low in cost, and can synthesize a new substance, mogroside derivative monomer (4), and on the basis of the above, the invention also provides preparation of mogroside derivative monomer (3) and rohanol derivative.
  • the monomer monomer (4) method which can prepare the mogroside derivative monomer (3) and the mogroside derivative monomer (4) by low-cost, high-efficiency and large-scale separation by a simple column chromatography separation technique (4) ), laying the foundation for the further development of a new class of related drugs.
  • Fig. 1 is a schematic view showing the molecular structure of mogroside V(1A) and VI(1B).
  • Figure 2 is a schematic diagram showing the molecular structure of mogroside Mogrol (2).
  • Fig. 3 is a schematic view showing the synthesis of a composition of the mogroside derivative monomer (3) and the mogroside derivative monomer (4).
  • Luo Han Guo total saponin forms an intermediate of mogroside (2) by acid degradation under acidic conditions
  • Luohan Fructol (2) is a relatively unstable intermediate compound which is not directly isolated in the present invention. It can be dehydrated by hydroxyl groups on the side chain to form derivatives (3) and (4) under acidic conditions (Fig. 3)), unlike most other high-resorpene arsenic tetracyclic triterpenoids extracted and separated from most Cucurbitaceae plants, the mogroside derivative monomer (3) and the mogroside derivative monomer (4) are both low.
  • Oxidized cucurbitane tetracyclic triterpene compound characterized by -OH attached to C-3 and C-11 instead of O, the degree of oxidation is greatly reduced, especially the side chain of compound (4) contains a total
  • the yoke double bond has a lower degree of oxidation than the compound (3) and is more resistant to oxidation.
  • the present invention first provides a composition comprising at least the following structure
  • the present invention also provides a method for synthesizing the above composition, comprising: dissolving a Luo Han Guo extract containing Luo Han Guo total saponin in an aqueous solution of an acidic alcohol to obtain a heating reaction.
  • the alcohol is at least one of methanol, ethanol, n-propanol, isopropanol, butanol; ethylene glycol, propylene glycol, glycerin, and polyethylene glycol.
  • the aqueous solution of the acidic alcohol is prepared by adjusting the aqueous solution of the alcohol to an acidic state with an acidic substance.
  • the volume ratio of the alcohol to the water in the aqueous solution of the alcohol is 1:10 to 10:1; preferably 1:3 to 3:1; further preferably 1:1.
  • the acidic substance is an inorganic acid or an organic acid; further preferably, the inorganic acid is at least one of hydrochloric acid, sulfuric acid and phosphoric acid; the organic acid is formic acid, acetic acid, haloacetic acid, oxalic acid At least one of malonic acid and citric acid.
  • the acidity is represented by a pH of 2.5 to 3.5; further preferably, the acidity is represented by a pH of 2.8 to 3.0. Too strong acidity will result in too many side reactions, and the acidity is too weak to carry out the reaction incompletely, so the pH must be controlled within the above range.
  • the quality of the aqueous extract of the Luo Han Guo total saponin and the acidic alcohol is determined.
  • the volume ratio is 1:10 to 1:3; further preferably, the mass-to-volume ratio of the Luo Han Guo extract of the Luo Han Guo total saponin to the aqueous solution of the acidic alcohol is 1:5.
  • the heating reaction is carried out under the conditions of heating at 100 to 140 ° C for 1 to 3 hours; further preferably, the heating reaction is carried out by heating at 120 ° C for 2 hours. Heating reaction If the temperature is low, the reaction is slow and incomplete.
  • the heating reaction further comprises neutralizing the acidic reaction solution obtained by the heating reaction with an alkaline substance, and heating to evaporate the alcohol to obtain a concentrate containing the composition; the neutralization is for further facilitating the subsequent separation and purification.
  • neutralization with a basic substance is neutralization to a pH of 6.5 to 7.5; further preferably, neutralization with a basic substance is neutralization to pH to 7.0.
  • the basic substance is an inorganic base and/or a salt; further preferably, the inorganic base and/or salt is at least one of NaOH, KOH and Na 2 CO 3 .
  • the method further comprises washing the concentrated liquid containing the composition.
  • the step of washing the concentrate containing the composition to obtain a brown extract is: adding distilled water to the concentrate containing the composition, cooling and standing, and discarding the upper aqueous layer to obtain a brown extract containing the composition.
  • the volume ratio of the concentrated liquid to distilled water is 1:5 to 1:3.
  • the method further comprises extracting and concentrating the coffee extract containing the composition to obtain a light brown solid containing the composition.
  • the extraction solvent used is ethyl acetate.
  • the solid-liquid ratio of each extraction is 1:6 to 1:3.
  • the number of extractions is 2 to 5, and each extract is combined; further preferably, the number of extractions is 3, and the volume of the extraction solvent used for the first extraction is the second extraction or the first extraction.
  • the extraction solvent used twice was twice the volume of the extraction solvent.
  • the concentration is concentrated by a rotary evaporator at 100 to 120 ° C, and the extracted solvent is completely evaporated to dryness.
  • the invention also provides a structure having the following structure
  • the preparation method of the mogroside derivative monomer (3) comprises dissolving the Luo Han Guo extract containing Luo Han Guo total saponin in an acidic alcohol/water solution for heating reaction, and then separating and purifying.
  • the step of separating and purifying the above-described light brown solid containing the composition is included.
  • the separation is chromatographic separation; further preferably, the separation is silica gel column chromatography; further preferably, the silica gel column chromatography separation is performed using acetonitrile and water; further preferred The volume ratio of the acetonitrile to the water is from 95:5 to 20:1; further preferably, the light brown solid containing the composition is further concentrated after separation; further preferably, after concentration, purification by chromatography;
  • the chromatographic purification is purified by silica gel column chromatography; further preferably, the silica gel column chromatography purification is performed using chloroform and ethanol; further preferably, the volume ratio of the chloroform to ethanol is 16 : 1 to 8:1; further preferably, the volume ratio of the chloroform to ethanol is 12:1.
  • the invention also provides a structure having the following structure
  • the preparation method of the mogroside derivative monomer (4) comprises dissolving the Luo Han Guo extract containing the Luo Han Guo total saponin in an acidic alcohol/water solution for heating reaction, and then separating and purifying.
  • the step of separating and purifying the above-described light brown solid containing the composition is included.
  • the separation is chromatographic separation
  • the separation is performed by silica gel column chromatography; further preferably, the eluent used in the silica gel column chromatography is acetonitrile and water; further preferably, the volume ratio of the acetonitrile to water is 95: 5-20:1; further preferably, the light brown solid containing the composition is further concentrated after separation; further preferably, after concentration, purification by chromatography;
  • the chromatographic purification is purified by silica gel column chromatography; and the silica gel column chromatography is used for purification.
  • the eluent is petroleum ether and ethyl acetate;
  • the volume ratio of the petroleum ether to ethyl acetate is from 3:2 to 4:1; further preferably, the volume ratio of the petroleum ether to ethyl acetate is 3:1.
  • Example 1 Preparation of mogroside derivative monomers (3) and (4) from 98% of the total saponins of Siraitia grosvenorum
  • Luo Han Guo extract 98% of Luo Han Guo total saponin, containing 55% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • the hydrochloric acid was adjusted to a pH of 3.0), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature at 25 ° C, the reaction solution was neutralized with a 1 M NaOH lye to a pH of 7. Evaporate the ethanol as much as possible at 90-95 ° C. Add the remaining concentrate to 200 mL of distilled water, stir well, cool, and let stand.
  • Example 2 Preparation of mogroside derivative monomers (3) and (4) from 95% of the total saponins of Siraitia grosvenor
  • Luo Han Guo extract 95% of Luo Han Guo total saponin, containing 45% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • methanol solution methanol and water volume ratio of 3:2
  • the sulfuric acid was adjusted to a pH of 2.8), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature, the reaction solution was neutralized with a 1 M KOH lye to a pH of 7 at 90- Methanol was distilled off as much as possible at 95 ° C. The remaining concentrate was added to 200 mL of distilled water, stirred thoroughly, cooled, and allowed to stand.
  • the upper aqueous layer was separated and discarded to obtain a brown extract; 100 mL, 50 mL, 50 mL of ethyl acetate were used respectively.
  • the coffee extract was extracted 3 times, and after 3 times of extracting, the mixture was concentrated at 100-120 ° C with a rotary evaporator, and the ethyl acetate was completely evaporated to obtain a monomer containing the mogroside derivative (3) and rohanol.
  • the light brown solid of the monomer (4) was 15.5 g; the light brown solid was loaded onto a silica gel column with methanol, eluted with a gradient of acetonitrile:water (19:1), and the different effluent was collected in order, and concentrated to give A. (6.0g), B (5.2g) two components; first collected A (6 The .0g) component was separated by a silica gel column (chloroform: ethanol/60:5 elution) to obtain a mannose alcohol derivative monomer (3) 3.9 g (HPLC>99%); HPLC, 13 C-NMR, ESESI detection The result was identical to the compound (3) in Example 1.
  • Luo Han Guo extract 90% of Luo Han Guo total saponin, containing 40% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • n-propanol the ratio of n-propanol to water was 2:3.
  • the remaining concentrate was added to 200 mL of distilled water, stirred thoroughly, cooled, and allowed to stand. The upper aqueous layer was separated and discarded to obtain a brown extract; 100 mL, 50 mL, respectively. Extracting the brown extract with 50 mL of ethyl acetate 3 times, combining the extracts three times, concentrating at 100-120 ° C with a rotary evaporator, and evaporating the ethyl acetate completely to obtain a monomer containing rohanol derivative (3) And light brown solid of 14.1 g of the mogroside derivative monomer (4); the light brown solid was loaded onto a silica gel column with methanol/ethanol, and eluted with a gradient of acetonitrile:water (20:1).
  • Example 4 Preparation of mogroside derivative monomers (3) and (4) from an extract of 80% of total saponins of Siraitia grosvenorum
  • Luo Han Guo extract 80% of Luo Han Guo total saponin, containing 20% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • ethanol the volume ratio of ethanol to water is 1:1
  • the oxalic acid was adjusted to a pH of 2.6), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature, the reaction solution was neutralized to a pH of 7 with 1 M Na 2 CO 3 . Evaporate the ethanol as much as possible at 90-95 ° C. Add the remaining concentrate to 200 mL of distilled water, stir well, cool, and let stand.
  • Example 5 Monocrotool derivative monomer (4) inhibiting cell proliferation experiment
  • Test drug the mogroside derivative monomer prepared in Example 1 (4);
  • Control drug paclitaxel (SELLECK; Cat. #S1150); ginsenoside monomer Rg3 (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number B21059).
  • Paclitaxel was added at a concentration ranging from a high concentration of 1 ⁇ M to a low concentration of 0.0014 ⁇ M. After test/control drug solution for 72 hours, use (Promega; Cat. #G7573) Luminescent cell viability assay to determine the percentage inhibition of proliferation of each cell of each cell in each cell line, and to plot the dose-effect relationship, and finally calculate the IC according to the curve in the figure 50 and the highest percent inhibition (E max ), as shown in Tables 1 and 2.
  • Table 1 Experimental results of inhibition of cell proliferation activity by the mogroside derivative monomer (4) and the control drug Rg3.
  • Table 2 Experimental results of the effectiveness of the mogroside derivative monomer (4) and the control drugs paclitaxel and Rg3 inhibiting cell proliferation.
  • the independent-sample T-test was used to determine whether the difference between the mean of the drug group and the control group was statistically significant (P ⁇ 0.05 or P ⁇ 0.01).
  • the average IC50 of the mogroside derivative monomer (4) of the present invention in 24 cell lines was statistically significant when compared with the average IC50 of the control drug ginsenoside monomer Rg3 in 24 cell lines (P ⁇ 0.05). At the same time P ⁇ 0.01).
  • the average maximum percent inhibition of the mogroside derivative monomer (4) of the present invention in 24 cell lines is compared with the average highest percent inhibition of the control drug paclitaxel and the control drug ginsenoside monomer Rg3 in 24 cell lines. The difference was statistically significant (P ⁇ 0.05 and P ⁇ 0.01).
  • the cellulose-inhibiting activity of the mogroside derivative monomer (4) of the present invention in all 24 cell lines is higher than that of the similar control drug ginsenoside monomer Rg3 (the composition of the mogroside derivative monomer (4)) 50 is less than the IC 50 of the ginsenoside monomer Rg3.
  • the highest inhibition rate (effectiveness) of the romanol derivative monomer (4) of the present invention on all 24 tested cell lines is very close to 100%; and the reference drug paclitaxel and ginsenoside monomer Rg3 are measured 24 In the cell line, only about 100% inhibition rate was observed for the two cell lines. This indicates that the mogroin derivative monomer (4) is significantly more effective than the control drug, and its high anticancer effectiveness is applicable to a plurality of cancer types.

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Abstract

A synthesis method for a composition comprising a mogrol derivative monomer (3) and a mogrol derivative monomer (4), compared with an existing method for synthesizing the mogrol derivative monomer (3), the present synthesis method is simple and inexpensive and also allows the synthesis of a novel substance, the mogrol derivative monomer (4). Also provided on such basis is a method for preparing the mogrol derivative monomer (3) and the mogrol derivative monomer (4). The method, by means of a simple column chromatography separation technique, allows inexpensive, highly efficient, and large-scale separate preparation of the mogrol derivative monomer (3) and the mogrol derivative monomer (4), thus laying a foundation for further development of related novel medicaments.

Description

一种从罗汉果总皂苷中制备罗汉果醇新型衍生物的方法Method for preparing novel derivative of mogroside from saponin of Luo Han Guo 技术领域Technical field
本发明涉及医药领域，具体涉及罗汉果醇衍生物的合成方法。The invention relates to the field of medicine, in particular to a method for synthesizing a mogroside derivative.
背景技术Background technique
近年来，世界各国对癌症和其它疾病(包括心脑血管疾病、糖尿病、精神忧郁、老年性痴呆症等)的看法正在发生重大的转变，越来越多的医生和研究人员，正在寻找可预防或延缓这些疾病发生的天然药物。目前，天然、高效、广谱、低毒的用于预防及治疗癌症和其它疾病的临床研究化合物药品，很多为天然药物或来自天然原料的半合成产品，是当前生物医药领域的研发热点。In recent years, the world's views on cancer and other diseases (including cardiovascular and cerebrovascular diseases, diabetes, mental depression, Alzheimer's disease, etc.) are undergoing major changes. More and more doctors and researchers are looking for preventable. Or delay the natural medicines that occur in these diseases. At present, natural, high-efficiency, broad-spectrum, low-toxic clinical research compound drugs for preventing and treating cancer and other diseases, many of which are natural drugs or semi-synthetic products derived from natural raw materials, are current research and development hotspots in the field of biomedicine.
最新的生物医学研究表明，许多来自天然植物的单体活性成分药物或多组分活性成分药物经人体胃肠道代谢或经肝脏代谢后所产生的各种代谢衍生物，被吸收入血后，才成为人体内真正起药效作用的活性成分，且具有比原形植物活性成分更显著疗效的特性。比如：大鼠口服放射性标记的地奥心血康胶囊24h后，大鼠粪便中出现了2-3个新的衍生物，同时也存在少量未改变的原型药物，进一步的研究表明，这些新出现的衍生物是甾体皂甙糖基选择性水解的一系列去糖基的次生甾体皂苷，说明地奥心血康在肠内菌代谢后真正被吸收入血的是代谢衍生物；人参皂苷是人参(三七)的主要活性成分，目前从人参(三七)中已经分离和鉴定了40多种单体成分(如：Ra、Rb1、 Rb2、Rb3、Rc、Rd、Re、Rg1、Rg2、Rf...等)，它们经过人体代谢后会产生一些极微量的新型代谢寡单体Rg3、Rh2、C-K、aPPD和aPPT等。大量的研究表明，未经人体代谢的原形人参皂苷对癌细胞没有抑制作用，而这些新产生的极微量的新型代谢寡单体(Rg3、Rh2、Rh1、C-K、aPPT和aPPD)以及转化人参皂苷Rk1和Rk2等对癌细胞却有杀伤作用，其中尤其以原人参二醇aPPD对癌细胞的抑制作用最为显著。The latest biomedical research shows that many monomeric active ingredient drugs or multi-component active ingredient drugs derived from natural plants are absorbed into the blood after being metabolized by the human gastrointestinal tract or metabolized by the liver. It becomes an active ingredient that is truly effective in the human body and has a more remarkable therapeutic effect than the active ingredient of the original plant. For example, after oral administration of radiolabeled Diao Xinxuekang Capsules for 24 hours, 2-3 new derivatives appeared in rat feces, and a small amount of unaltered prototype drugs were also present. Further studies showed that these newly emerging derivatives were A series of deglycosyl secondary steroidal saponins selectively hydrolyzed by steroidal saponin, indicating that the scorpion saponin is actually absorbed into the blood after metabolism by intestinal bacteria is a metabolic derivative; ginsenoside is the main ginseng (three-seven) Active ingredients, more than 40 monomer components have been isolated and identified from ginseng (37) (eg Ra, Rb1) Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, Rf, etc.), after being metabolized by the human body, will produce some minimal amounts of new metabolic oligo monomers Rg3, Rh2, C-K, aPPD and aPPT. Numerous studies have shown that the original ginsenosides that have not been metabolized by humans have no inhibitory effect on cancer cells, and these newly produced very small amounts of novel metabolic oligo monomers (Rg3, Rh2, Rh1, CK, aPPT and aPPD) and transformed ginsenosides Rk1 and Rk2 have a killing effect on cancer cells, especially the original ginseng diol aPPD has the most significant inhibitory effect on cancer cells.
由于上述原因，天然抗肿瘤药物的研发取得了越来越多的关注。无论是抑制或杀伤肿瘤细胞、调整机体免疫功能、改善症状与特征、减轻放化疗毒副作用，还是肿瘤的病后调理，天然抗癌药物具有重要作用。由此，天然植物新疗法将成为继手术、放疗、化疗、生物疗法的第五大治疗方法。Due to the above reasons, the development of natural anti-tumor drugs has attracted more and more attention. Whether it is to inhibit or kill tumor cells, adjust the body's immune function, improve symptoms and characteristics, reduce the side effects of radiotherapy and chemotherapy, or post-operative conditioning of tumors, natural anticancer drugs play an important role. Therefore, the new natural plant therapy will become the fifth major treatment after surgery, radiotherapy, chemotherapy and biological therapy.
罗汉果是一种名贵药材，属于葫芦科多年生藤本植物，性凉味甘。罗汉果提取物中含非糖甜味的皂苷成分，又称罗汉果总皂苷，根据其中皂苷元连接糖基的数目以及连接位置的不同已经在罗汉果总皂苷中分离鉴定出罗汉果二糖苷、三糖苷、四糖苷、五糖苷、六糖苷等多种皂苷单体，分别命名为罗汉果甜苷II、III、IV、V、VI(MogrosideII、III、IV、V、VI)等，其中罗汉果甜苷V–MogrosideV(1A)(如图1所示)的甜度是蔗糖的256-344倍，罗汉果甜苷IV–MogrosideIV(1B)(如图1所示)的甜度为蔗糖的126倍，其热量为零，具有清热润肺镇咳、润肠通便之功效，对肥胖、便秘、糖尿病等具有防治作用。Mangosteen is a precious medicinal material belonging to the perennial vine of Cucurbitaceae. It is cool and sweet. The non-sugar sweet saponin component of Luo Han Guo extract, also known as Luo Han Guo total saponin, has been isolated and identified in the total saponin of Siraitia grosveni according to the number of saponin-linked glycosyl groups and the position of the saponin. Various saponin monomers such as glycoside, pentaglucoside and hexaglucoside are named as mogroside II, III, IV, V, VI (Mogroside II, III, IV, V, VI), among them, mogroside V-MogrosideV ( 1A) (as shown in Figure 1) has a sweetness of 256-344 times that of sucrose, and Mogroside IV-Mogroside IV (1B) (shown in Figure 1) has a sweetness of 126 times that of sucrose, and its calorie is zero. It has the effects of clearing away heat, moistening lungs, relieving cough, and relaxing laxatives. It has preventive effects on obesity, constipation and diabetes.
罗汉果皂苷Mogrosides是两条由四个以下葡萄糖单位组成的葡萄糖苷侧链以β-糖苷键与皂苷元罗汉果醇Mogrol(2)(如图2所示)的C-3、C-24相连，在侧链葡萄糖之间的连接键为β-1,6和β-l,2糖苷键。Mogrosides are two glucoside side chains consisting of four or fewer glucose units linked by a β-glycosidic bond to C-3 and C-24 of the sapogenin, Lo Hanol Mogrol (2) (as shown in Figure 2). The linkage between the side chain glucoses is β-1,6 and β-l,2 glycosidic bonds.
许多研究表明，罗汉果皂苷能提高葡萄糖和脂肪的利用，增加胰岛素的敏感性，但罗汉果降血糖作用的有效成分和作用机制并不明确。中科院上海 药物研究所胡立宏课题组和沈旭课题组对罗汉果的降血糖作用进行了研究，首先在HepG2细胞中，对罗汉果中含量最高的罗汉果甜苷V(1A)进行测试，发现它对AMPK没有活性；而罗汉果醇Mogrol(2)及其衍生物3α-羟基-25-脱羟基-24-酮-罗汉果醇(3α-hydroxy-25-dehydroxy-24-oxomogrol)(3)却能够激活AMPK(Chen X.B.，et al；Bioorganic&Medicinal Chemistry 2011,19:5776)。虽然文献报道了罗汉果醇衍生物单体(3)(Chen X.B.，et al；Bioorganic&Medicinal Chemistry 2011,19:5776)的制备，但产率低、分离纯化困难，难以工业化生产，另外该方法无法同时制备罗汉果醇衍生物单体(4)；目前也没有任何专利和文献公开报道通过罗汉果提取物同时合成罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的方法。Many studies have shown that mogroside can increase the utilization of glucose and fat and increase the sensitivity of insulin, but the active ingredients and mechanism of action of mogroside in hypoglycemic effect are not clear. Chinese Academy of Sciences Shanghai The research on the hypoglycemic effect of Luo Han Guo was studied by the research group Hu Lihong and Shen Xu. Firstly, the highest content of mogroside V (1A) in Luo Han Guo was tested in HepG2 cells, and it was found to be inactive to AMPK. However, mogrostat Mogrol (2) and its derivative 3α-hydroxy-25-dehydroxy-24-oxomogrol (3) can activate AMPK (Chen XB, Et al; Bioorganic & Medicinal Chemistry 2011, 19: 5776). Although the preparation of the mogroside derivative monomer (3) (Chen XB, et al; Bioorganic & Medicinal Chemistry 2011, 19: 5776) has been reported in the literature, the yield is low, separation and purification are difficult, and industrial production is difficult, and the method cannot be simultaneously prepared. The mogroside derivative monomer (4); there is currently no patent or literature publicly reported on the simultaneous synthesis of the mogroside derivative monomer (3) and the mogroside derivative monomer (4) by the mogroside extract.
发明内容Summary of the invention
本发明要解决的技术问题是提供了包含有罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的组合物的合成方法，在此基础上，本发明还分别提供了罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的制备方法。The technical problem to be solved by the present invention is to provide a synthesis method comprising a composition of a mogroside derivative monomer (3) and a mogroside derivative monomer (4). On the basis of the present invention, the present invention further provides a mogroside A method for producing an alcohol derivative monomer (3) and a mogroside derivative monomer (4).
本发明第一方面提供了一种组合物，至少包括如下结构A first aspect of the invention provides a composition comprising at least the structure
Figure PCTCN2016089211-appb-000001
的罗汉果醇衍生物单体(3) 和如下结构
Figure PCTCN2016089211-appb-000001
The mogroside derivative monomer (3) and the following structure
Figure PCTCN2016089211-appb-000002
的罗汉果醇衍生物单体(4)。
Figure PCTCN2016089211-appb-000002
The mogroside derivative monomer (4).
本发明第二方面提供了上述组合物的合成方法，包括：将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇的水溶液中进行加热反应即得。According to a second aspect of the present invention, there is provided a method for synthesizing the above composition, comprising: dissolving a Luo Han Guo extract containing Luo Han Guo total saponin in an aqueous solution of an acidic alcohol for heating reaction.
在一优选例中，所述呈酸性的醇的水溶液的制备方法为：用酸性物质将醇的水溶液调节为酸性。In a preferred embodiment, the aqueous solution of the acidic alcohol is prepared by adjusting the aqueous solution of the alcohol to an acidic state with an acidic substance.
在一优选例中，所述醇为甲醇、乙醇、正丙醇、异丙醇、丁醇、乙二醇、丙二醇、丙三醇和聚乙二醇中的至少一种。In a preferred embodiment, the alcohol is at least one of methanol, ethanol, n-propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerin, and polyethylene glycol.
在一优选例中，所述醇的水溶液中醇与水的体积比为1：10～10：1。In a preferred embodiment, the volume ratio of alcohol to water in the aqueous solution of the alcohol is from 1:10 to 10:1.
在一优选例中，所述醇的水溶液中醇与水的体积比为1：3～3：1。In a preferred embodiment, the volume ratio of alcohol to water in the aqueous solution of the alcohol is from 1:3 to 3:1.
在一优选例中，所述醇的水溶液中醇与水的体积比为1：1。In a preferred embodiment, the volume ratio of alcohol to water in the aqueous solution of the alcohol is 1:1.
在一优选例中，所述酸性物质为无机酸或有机酸。In a preferred embodiment, the acidic substance is an inorganic acid or an organic acid.
在一优选例中，所述无机酸为盐酸、硫酸和磷酸中的至少一种。In a preferred embodiment, the inorganic acid is at least one of hydrochloric acid, sulfuric acid, and phosphoric acid.
在一优选例中，所述有机酸为甲酸、乙酸、卤代乙酸、乙二酸、丙二酸和柠檬酸中的至少一种。In a preferred embodiment, the organic acid is at least one of formic acid, acetic acid, haloacetic acid, oxalic acid, malonic acid, and citric acid.
在一优选例中，所述呈酸性体现为PH值为2.5～3.5。In a preferred embodiment, the acidity is represented by a pH of from 2.5 to 3.5.
在一优选例中，所述呈酸性体现为PH值为2.8～3.0。In a preferred embodiment, the acidity is represented by a pH of 2.8 to 3.0.
在一优选例中，当所述含罗汉果总皂苷的罗汉果提取物中罗汉果总皂苷的重量百分比为70％以上时，所述含罗汉果总皂苷的罗汉果提取物与呈酸性 的醇的水溶液的质量体积比为1：10～1：3。In a preferred embodiment, when the weight percentage of the total saponin of Siraitia grosvenorii in the Luo Han Guo extract of Luo Han Guo total saponin is 70% or more, the Luo Han Guo total saponin-containing Luo Han Guo extract is acidic. The mass ratio of the aqueous solution of the alcohol is from 1:10 to 1:3.
在一优选例中，所述含罗汉果总皂苷的罗汉果提取物与呈酸性的醇的水溶液的质量体积比为1：5。In a preferred embodiment, the mass-to-volume ratio of the Luo Han Guo extract of the Luo Han Guo total saponin to the aqueous solution of the acidic alcohol is 1:5.
在一优选例中，所述加热反应的条件是：在100～140℃下加热1～3小时。In a preferred embodiment, the heating reaction is carried out under the conditions of heating at 100 to 140 ° C for 1 to 3 hours.
在一优选例中，所述加热反应的条件是：在120℃下加热2小时。In a preferred embodiment, the heating reaction is carried out under the conditions of heating at 120 ° C for 2 hours.
在一优选例中，所述加热反应后还包括对加热反应得到的反应液用碱性物质进行中和，加热将醇蒸发，得到含组合物的浓缩液。In a preferred embodiment, after the heating reaction, the reaction liquid obtained by the heating reaction is neutralized with an alkaline substance, and the alcohol is evaporated by heating to obtain a concentrate containing the composition.
在一优选例中，用碱性物质进行中和是中和至PH到6.5～7.5。In a preferred embodiment, neutralization with an alkaline material is neutralized to a pH of 6.5 to 7.5.
在一优选例中，用碱性物质进行中和是中和至PH到7.0。In a preferred embodiment, neutralization with a basic substance is neutralized to a pH of 7.0.
在一优选例中，所述碱性物质为无机碱和/或盐。In a preferred embodiment, the basic substance is an inorganic base and/or a salt.
在一优选例中，所述无机碱和/或盐为NaOH、KOH和Na2CO3中的至少一种。In a preferred embodiment, the inorganic base and/or salt is at least one of NaOH, KOH, and Na 2 CO 3 .
在一优选例中，上述组合物的合成方法，还包括对含组合物的浓缩液洗涤后得到咖啡色浸膏的步骤。In a preferred embodiment, the method for synthesizing the composition further comprises the step of obtaining a brown extract after washing the concentrate containing the composition.
在一优选例中，所述对含组合物的浓缩液洗涤后得到咖啡色浸膏的步骤：在含组合物的浓缩液中加入蒸馏水冷却静置，弃去上面水层，得到含组合物的咖啡色浸膏。In a preferred embodiment, the step of washing the concentrate containing the composition to obtain a brown extract comprises: adding distilled water to the concentrate containing the composition, cooling and standing, discarding the upper aqueous layer to obtain a brown color containing the composition. extract.
在一优选例中，所述浓缩液与蒸馏水的体积比为1:5～1:3。In a preferred embodiment, the volume ratio of the concentrate to distilled water is from 1:5 to 1:3.
在一优选例中，上述的组合物的合成方法，还包括对含组合物的咖啡色浸膏进行萃取和浓缩，得到含组合物的浅咖啡色固体。In a preferred embodiment, the method of synthesizing the composition further comprises extracting and concentrating the brown extract containing the composition to obtain a light brown solid containing the composition.
在一优选例中，所述萃取所采用的萃取溶剂为乙酸乙酯。In a preferred embodiment, the extraction solvent used in the extraction is ethyl acetate.
在一优选例中，每次萃取的固液比1:6～1:3。In a preferred embodiment, the solid to liquid ratio of each extraction is from 1:6 to 1:3.
在一优选例中，所述萃取的次数为2～5次，将每次萃取液合并。 In a preferred embodiment, the number of extractions is 2 to 5 times, and each extract is combined.
在一优选例中，所述萃取的次数为3次。In a preferred embodiment, the number of extractions is three.
在一优选例中，第一次萃取所用的萃取溶剂的体积是第二次萃取或第三次萃取所用的萃取溶剂的体积的2倍。In a preferred embodiment, the volume of the extraction solvent used in the first extraction is twice the volume of the extraction solvent used in the second extraction or the third extraction.
在一优选例中，所述浓缩是：100-120℃下用旋转蒸发仪浓缩，把萃取溶剂完全蒸干。In a preferred embodiment, the concentration is: concentration at 100-120 ° C using a rotary evaporator, and the extraction solvent is completely evaporated to dryness.
本发明第三方面提供了一种具有如下结构A third aspect of the present invention provides a structure having the following structure
Figure PCTCN2016089211-appb-000003
的罗汉果醇衍生物单体(3)的制备方法，包括将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇的水溶液中进行加热反应，然后分离纯化即得。
Figure PCTCN2016089211-appb-000003
The preparation method of the mogroside derivative monomer (3) comprises dissolving the Luo Han Guo extract containing the Luo Han Guo total saponin in an aqueous solution of an acidic alcohol, heating, and then separating and purifying.
在一优选例中，包括对权利要求7的含组合物的浅咖啡色固体进行分离和纯化的步骤。In a preferred embodiment, the step of separating and purifying the light brown solid containing the composition of claim 7 is included.
在一优选例中，所述分离为层析分离。In a preferred embodiment, the separation is chromatographic separation.
在一优选例中，所述层析分离为硅胶柱层析分离。In a preferred embodiment, the chromatographic separation is performed by silica gel column chromatography.
在一优选例中，所述硅胶柱层析分离采用的洗脱液为乙腈和水；In a preferred embodiment, the silica gel column chromatography separation uses an acetonitrile and water;
在一优选例中，所述乙腈和水的体积比为95:5～20:1；In a preferred embodiment, the volume ratio of acetonitrile to water is 95:5 to 20:1;
在一优选例中，对含组合物的浅咖啡色固体进行分离后还要进一步浓缩；In a preferred embodiment, the light brown solid containing the composition is further concentrated after separation;
在一优选例中，浓缩后进行层析纯化；In a preferred embodiment, the concentration is followed by chromatographic purification;
在一优选例中，所述层析纯化为硅胶柱层析纯化； In a preferred embodiment, the chromatographic purification is purified by silica gel column chromatography;
在一优选例中，所述硅胶柱层析纯化采用的洗脱液为氯仿和乙醇。In a preferred embodiment, the eluate used in the silica gel column chromatography purification is chloroform and ethanol.
在一优选例中，所述氯仿和乙醇的体积比为16:1～8:1。In a preferred embodiment, the volume ratio of chloroform to ethanol is from 16:1 to 8:1.
在一优选例中，所述氯仿和乙醇的体积比为12:1。In a preferred embodiment, the volume ratio of chloroform to ethanol is 12:1.
本发明第四方面提供了一种具有如下结构A fourth aspect of the present invention provides a structure having the following structure
Figure PCTCN2016089211-appb-000004
Figure PCTCN2016089211-appb-000004
的罗汉果醇衍生物单体(4)的制备方法，包括将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇/水溶液中进行加热反应，然后分离纯化即得。The preparation method of the mogroside derivative monomer (4) comprises dissolving the Luo Han Guo extract containing the Luo Han Guo total saponin in an acidic alcohol/water solution for heating reaction, and then separating and purifying.
在一优选例中，包括对上述含组合物的浅咖啡色固体进行分离和纯化的步骤。In a preferred embodiment, the step of separating and purifying the above-described light brown solid containing the composition is included.
在一优选例中，所述分离为层析分离。In a preferred embodiment, the separation is chromatographic separation.
在一优选例中，所述层析分离为硅胶柱层析分离。In a preferred embodiment, the chromatographic separation is performed by silica gel column chromatography.
在一优选例中，所述硅胶柱层析分离采用的洗脱液为乙腈和水。In a preferred embodiment, the silica gel column chromatography separation uses an acetonitrile and water.
在一优选例中，所述乙腈和水的体积比为95:5～20:1。In a preferred embodiment, the volume ratio of acetonitrile to water is from 95:5 to 20:1.
在一优选例中，对含组合物的浅咖啡色固体进行分离后还要进一步浓缩。In a preferred embodiment, the light brown solid containing the composition is further concentrated after separation.
在一优选例中，浓缩后进行层析纯化。In a preferred embodiment, the residue is purified by chromatography.
在一优选例中，所述层析纯化为硅胶柱层析纯化。In a preferred embodiment, the chromatographic purification is purified by silica gel column chromatography.
在一优选例中，所述硅胶柱层析纯化采用的洗脱液为石油醚和乙酸乙酯。 In a preferred embodiment, the silica gel column chromatography purification is carried out using petroleum ether and ethyl acetate.
在一优选例中，所述石油醚和乙酸乙酯的体积比为3:2～4:1。In a preferred embodiment, the volume ratio of petroleum ether to ethyl acetate is from 3:2 to 4:1.
在一优选例中，所述石油醚和乙酸乙酯的体积比为3:1。In a preferred embodiment, the volume ratio of petroleum ether to ethyl acetate is 3:1.
本发明使用的术语具有以下定义，除非另有描述：Terms used in the present invention have the following definitions unless otherwise stated:
本文所用的术语“罗汉果醇衍生物单体(3)”意指3α-羟基-25-脱羟基-24-酮-罗汉果醇(3α-hydroxy-25-Dehydroxy-24-oxomogrol)；本文所用的术语“新型罗汉果醇衍生物单体(4)”意指3α-羟基-22、24-二烯-24、25-脱羟基-罗汉果醇(3α-hydroxy-22、24-diene-24、25-dehydroxy-mogrol)(4)。The term "rohansol derivative monomer (3)" as used herein means 3α-hydroxy-25-dehydroxy-24-oxomogrol; the term is used herein. "New romanol derivative monomer (4)" means 3α-hydroxy-22,24-diene-24,25-dehydroxy-rohansol (3α-hydroxy-22, 24-diene-24, 25-dehydroxy -mogrol)(4).
本发明提供了包含有罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的组合物的合成方法，相比现有的合成罗汉果醇衍生物单体(3)的方法，此合成方法简单、成本较低，而且还能合成新物质罗汉果醇衍生物单体(4)，同时在此基础上，本发明还提供了制备罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的方法，此方法通过简单的柱层析分离技术，可以低成本、高效率和大规模地分离制备罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)，为进一步开发相关的一类新药打下基础。The present invention provides a method for synthesizing a composition comprising a monomer component of the mogroside derivative (3) and a mogroside derivative monomer (4), as compared with the conventional method for synthesizing the mogroside derivative monomer (3), The synthesis method is simple, low in cost, and can synthesize a new substance, mogroside derivative monomer (4), and on the basis of the above, the invention also provides preparation of mogroside derivative monomer (3) and rohanol derivative. The monomer monomer (4) method, which can prepare the mogroside derivative monomer (3) and the mogroside derivative monomer (4) by low-cost, high-efficiency and large-scale separation by a simple column chromatography separation technique (4) ), laying the foundation for the further development of a new class of related drugs.
附图说明DRAWINGS
图1为罗汉果甜苷V(1A)、VI(1B)的分子结构示意图。Fig. 1 is a schematic view showing the molecular structure of mogroside V(1A) and VI(1B).
图2为罗汉果醇Mogrol(2)的分子结构示意图。Figure 2 is a schematic diagram showing the molecular structure of mogroside Mogrol (2).
图3为罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)形成的组合物的合成方法示意图。Fig. 3 is a schematic view showing the synthesis of a composition of the mogroside derivative monomer (3) and the mogroside derivative monomer (4).
具体实施方式detailed description
罗汉果总皂苷在酸性条件下通过糖降解形成中间体罗汉果醇(2)，罗汉 果醇(2)是一个相对不稳定的中间体化合物，在本发明中不直接进行分离，它在酸性条件下可以通过侧链上的羟基脱水形成衍生物(3)和(4)(如图3所示)，和其它多数葫芦科植物中直接提取分离的高氧化葫芦烷类四环三萜化合物不同，罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)均是低氧化的葫芦烷类四环三萜化合物,其特点是C-3和C-11上连接的是-OH,而不是＝O,氧化程度大大减低，特别是化合物(4)的侧链含有一个共轭双键，氧化程度比化合物(3)更低，抗氧化性更强。Luo Han Guo total saponin forms an intermediate of mogroside (2) by acid degradation under acidic conditions, Luohan Fructol (2) is a relatively unstable intermediate compound which is not directly isolated in the present invention. It can be dehydrated by hydroxyl groups on the side chain to form derivatives (3) and (4) under acidic conditions (Fig. 3)), unlike most other high-resorpene arsenic tetracyclic triterpenoids extracted and separated from most Cucurbitaceae plants, the mogroside derivative monomer (3) and the mogroside derivative monomer (4) are both low. Oxidized cucurbitane tetracyclic triterpene compound characterized by -OH attached to C-3 and C-11 instead of =O, the degree of oxidation is greatly reduced, especially the side chain of compound (4) contains a total The yoke double bond has a lower degree of oxidation than the compound (3) and is more resistant to oxidation.
具体来说，本发明首先提供了一种组合物，至少包括如下结构In particular, the present invention first provides a composition comprising at least the following structure
Figure PCTCN2016089211-appb-000005
的罗汉果醇衍生物单体(3)和如下结构
Figure PCTCN2016089211-appb-000005
The mogroside derivative monomer (3) and the following structure
Figure PCTCN2016089211-appb-000006
的罗汉果醇衍生物单体(4)。
Figure PCTCN2016089211-appb-000006
The mogroside derivative monomer (4).
本发明还提供了上述组合物的合成方法，包括：将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇的水溶液中进行加热反应即得。The present invention also provides a method for synthesizing the above composition, comprising: dissolving a Luo Han Guo extract containing Luo Han Guo total saponin in an aqueous solution of an acidic alcohol to obtain a heating reaction.
所述的醇为甲醇、乙醇、正丙醇、异丙醇、丁醇；乙二醇、丙二醇、丙三醇；聚乙二醇中的至少一种。 The alcohol is at least one of methanol, ethanol, n-propanol, isopropanol, butanol; ethylene glycol, propylene glycol, glycerin, and polyethylene glycol.
所述呈酸性的醇的水溶液的制备方法为：用酸性物质将醇的水溶液调节为酸性，优选的，所述醇的水溶液中醇与水的体积比为1：10～10：1；优选为1：3～3：1；，进一步优选为1：1。The aqueous solution of the acidic alcohol is prepared by adjusting the aqueous solution of the alcohol to an acidic state with an acidic substance. Preferably, the volume ratio of the alcohol to the water in the aqueous solution of the alcohol is 1:10 to 10:1; preferably 1:3 to 3:1; further preferably 1:1.
优选的，所述酸性物质为无机酸或有机酸；进一步优选的，所述无机酸为盐酸、硫酸和磷酸中的至少一种；所述有机酸为甲酸、乙酸、卤代乙酸、乙二酸、丙二酸和柠檬酸中的至少一种。Preferably, the acidic substance is an inorganic acid or an organic acid; further preferably, the inorganic acid is at least one of hydrochloric acid, sulfuric acid and phosphoric acid; the organic acid is formic acid, acetic acid, haloacetic acid, oxalic acid At least one of malonic acid and citric acid.
优选的，所述呈酸性体现为PH值为2.5～3.5；进一步优选的，所述呈酸性体现为PH值为2.8～3.0。酸性太强会导致副反应太多，酸性太弱反应进行的不彻底，因此其PH必须控制在上述范围内。Preferably, the acidity is represented by a pH of 2.5 to 3.5; further preferably, the acidity is represented by a pH of 2.8 to 3.0. Too strong acidity will result in too many side reactions, and the acidity is too weak to carry out the reaction incompletely, so the pH must be controlled within the above range.
优选的，所述含罗汉果总皂苷的罗汉果提取物中罗汉果总皂苷的重量百分比是70％以上时，在此基础上，所述含罗汉果总皂苷的罗汉果提取物与呈酸性的醇的水溶液的质量体积比为1：10～1：3；进一步优选的，所述含罗汉果总皂苷的罗汉果提取物与呈酸性的醇的水溶液的质量体积比为1：5。Preferably, when the weight percentage of the total saponin of the Luo Han Guo extract of the Luo Han Guo total saponin is more than 70%, the quality of the aqueous extract of the Luo Han Guo total saponin and the acidic alcohol is determined. The volume ratio is 1:10 to 1:3; further preferably, the mass-to-volume ratio of the Luo Han Guo extract of the Luo Han Guo total saponin to the aqueous solution of the acidic alcohol is 1:5.
优选的，所述加热反应的条件是：在100～140℃下加热1～3小时；进一步优选的，所述加热反应的条件是：在120℃下加热2小时。加热反应如果温度较低，反应慢且进行的不完全。Preferably, the heating reaction is carried out under the conditions of heating at 100 to 140 ° C for 1 to 3 hours; further preferably, the heating reaction is carried out by heating at 120 ° C for 2 hours. Heating reaction If the temperature is low, the reaction is slow and incomplete.
所述加热反应后还包括对加热反应得到的酸性反应液用碱性物质进行中和，加热将醇蒸发，得到含组合物的浓缩液；中和是为了更有利于后面的分离和纯化。The heating reaction further comprises neutralizing the acidic reaction solution obtained by the heating reaction with an alkaline substance, and heating to evaporate the alcohol to obtain a concentrate containing the composition; the neutralization is for further facilitating the subsequent separation and purification.
优选的，用碱性物质进行中和是中和至PH到6.5～7.5；进一步优选的，用碱性物质进行中和是中和至PH到7.0。Preferably, neutralization with a basic substance is neutralization to a pH of 6.5 to 7.5; further preferably, neutralization with a basic substance is neutralization to pH to 7.0.
优选的，所述碱性物质为无机碱和/或盐；进一步优选的，所述无机碱和/或盐为NaOH、KOH和Na2CO3中的至少一种。Preferably, the basic substance is an inorganic base and/or a salt; further preferably, the inorganic base and/or salt is at least one of NaOH, KOH and Na 2 CO 3 .
优选的，所述得到含组合物的浓缩液后，还包括对含组合物的浓缩液洗 涤后得到咖啡色浸膏的步骤。Preferably, after the obtaining the concentrated solution containing the composition, the method further comprises washing the concentrated liquid containing the composition. The step of obtaining a brown extract after washing.
所述对含组合物的浓缩液洗涤后得到咖啡色浸膏的步骤为：在含组合物的浓缩液中加入蒸馏水冷却静置，弃去上面水层，得到含组合物的咖啡色浸膏。The step of washing the concentrate containing the composition to obtain a brown extract is: adding distilled water to the concentrate containing the composition, cooling and standing, and discarding the upper aqueous layer to obtain a brown extract containing the composition.
优选的，所述浓缩液与蒸馏水的体积比为1:5～1:3。Preferably, the volume ratio of the concentrated liquid to distilled water is 1:5 to 1:3.
所述得到含组合物的咖啡色浸膏后，还包括对含组合物的咖啡色浸膏进行萃取和浓缩，得到含组合物的浅咖啡色固体。After obtaining the brown extract containing the composition, the method further comprises extracting and concentrating the coffee extract containing the composition to obtain a light brown solid containing the composition.
优选的，所述萃取是采用的萃取溶剂为乙酸乙酯。Preferably, the extraction solvent used is ethyl acetate.
优选的，每次萃取的固液比1:6～1:3。Preferably, the solid-liquid ratio of each extraction is 1:6 to 1:3.
优选的，所述萃取的次数为2～5，将每次萃取液合并；进一步优选的，所述萃取的次数为3次，第一次萃取所用的萃取溶剂的体积是第二次萃取或第三次萃取所用的萃取溶剂的体积的2倍。Preferably, the number of extractions is 2 to 5, and each extract is combined; further preferably, the number of extractions is 3, and the volume of the extraction solvent used for the first extraction is the second extraction or the first extraction. The extraction solvent used twice was twice the volume of the extraction solvent.
优选的，所述浓缩是在100～120℃下用旋转蒸发仪浓缩，把萃取的溶剂完全蒸干。Preferably, the concentration is concentrated by a rotary evaporator at 100 to 120 ° C, and the extracted solvent is completely evaporated to dryness.
本发明还提供了一种具有如下结构The invention also provides a structure having the following structure
Figure PCTCN2016089211-appb-000007
的罗汉果醇衍生物单体(3)的制备方法，包括将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇/水溶液中进行加热反应，然后分离纯化即得。
Figure PCTCN2016089211-appb-000007
The preparation method of the mogroside derivative monomer (3) comprises dissolving the Luo Han Guo extract containing Luo Han Guo total saponin in an acidic alcohol/water solution for heating reaction, and then separating and purifying.
优选的，包括对上述含组合物的浅咖啡色固体进行分离和纯化的步骤。 Preferably, the step of separating and purifying the above-described light brown solid containing the composition is included.
进一步优选的，所述分离为层析分离；进一步优选的，所述分离为硅胶柱层析分离；进一步优选的，所述硅胶柱层析分离采用的洗脱液为乙腈和水；进一步优选的，所述乙腈和水的体积比为95:5～20:1；进一步优选的，对含组合物的浅咖啡色固体进行分离后还要进一步浓缩；进一步优选的，浓缩后进行层析纯化；Further preferably, the separation is chromatographic separation; further preferably, the separation is silica gel column chromatography; further preferably, the silica gel column chromatography separation is performed using acetonitrile and water; further preferred The volume ratio of the acetonitrile to the water is from 95:5 to 20:1; further preferably, the light brown solid containing the composition is further concentrated after separation; further preferably, after concentration, purification by chromatography;
优选的，所述层析纯化为硅胶柱层析纯化；进一步优选的，所述硅胶柱层析纯化采用的洗脱液为氯仿和乙醇；进一步优选的，所述氯仿和乙醇的体积比为16:1～8:1；进一步优选的，所述氯仿和乙醇的体积比为12:1。Preferably, the chromatographic purification is purified by silica gel column chromatography; further preferably, the silica gel column chromatography purification is performed using chloroform and ethanol; further preferably, the volume ratio of the chloroform to ethanol is 16 : 1 to 8:1; further preferably, the volume ratio of the chloroform to ethanol is 12:1.
本发明还提供了一种具有如下结构The invention also provides a structure having the following structure
Figure PCTCN2016089211-appb-000008
Figure PCTCN2016089211-appb-000008
的罗汉果醇衍生物单体(4)的制备方法，包括将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇/水溶液中进行加热反应，然后分离纯化即得。The preparation method of the mogroside derivative monomer (4) comprises dissolving the Luo Han Guo extract containing the Luo Han Guo total saponin in an acidic alcohol/water solution for heating reaction, and then separating and purifying.
优选的，包括对上述含组合物的浅咖啡色固体进行分离和纯化的步骤。Preferably, the step of separating and purifying the above-described light brown solid containing the composition is included.
进一步优选的，所述分离为层析分离；Further preferably, the separation is chromatographic separation;
进一步优选的，所述分离为硅胶柱层析分离；进一步优选的，所述硅胶柱层析分离采用的洗脱液为乙腈和水；进一步优选的，所述乙腈和水的体积比为95:5～20:1；进一步优选的，对含组合物的浅咖啡色固体进行分离后还要进一步浓缩；进一步优选的，浓缩后进行层析纯化；Further preferably, the separation is performed by silica gel column chromatography; further preferably, the eluent used in the silica gel column chromatography is acetonitrile and water; further preferably, the volume ratio of the acetonitrile to water is 95: 5-20:1; further preferably, the light brown solid containing the composition is further concentrated after separation; further preferably, after concentration, purification by chromatography;
优选的，所述层析纯化为硅胶柱层析纯化；，所述硅胶柱层析纯化采用 的洗脱液为石油醚和乙酸乙酯；Preferably, the chromatographic purification is purified by silica gel column chromatography; and the silica gel column chromatography is used for purification. The eluent is petroleum ether and ethyl acetate;
进一步优选的，所述石油醚和乙酸乙酯的体积比为3:2～4:1；进一步优选的，所述石油醚和乙酸乙酯的体积比为3:1。Further preferably, the volume ratio of the petroleum ether to ethyl acetate is from 3:2 to 4:1; further preferably, the volume ratio of the petroleum ether to ethyl acetate is 3:1.
除非特殊说明，本发明所用术语具有本发明所属领域中的一般含义。Unless otherwise stated, the terms used in the present invention have their ordinary meanings in the art to which the invention pertains.
下面参考具体实施例，对本发明进行说明，需要说明的是，这些实施例仅仅是说明性的，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The invention is described below with reference to the specific embodiments, which are intended to be illustrative, and are not to be construed as limiting. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the art or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
实施例1：从罗汉果总皂苷98％的提取物制备罗汉果醇衍生物单体(3)和(4)Example 1: Preparation of mogroside derivative monomers (3) and (4) from 98% of the total saponins of Siraitia grosvenorum
将罗汉果提取物50g(罗汉果总皂苷98％，含55％罗汉果甙V，购买于桂林莱茵生物科技股份有限公司)加入250mL的乙醇的水溶液(乙醇和水的体积比为1:1)(预先用盐酸调节至PH值为3.0)，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温25℃后，反应液用1M的NaOH碱液中和至PH值为7，在90-95℃下将乙醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静置，将上面的水层分离弃去，得到咖啡色浸膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干，即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体18.2g；用乙醇将浅咖啡色固体载入硅胶柱，用乙腈：水(体积比20:1)梯度洗脱，按先后顺序收集不同的流出液，浓缩后得到A(6.8g)、B(5.6g)两组分；先收集得到的A(6.8g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)4.1g(HPLC>99％)。ESIMS m/z 503(458+45)[M+HCOO-]-(calcd for C31H51O5)；13C-NMR(600MHz，MeOD)δppm:29.1(C-1),31.7(C-2),79.4(C-3),42.8(C-4),144.1(C-5),120.7(C-6),25.2(C-7),44.8(C-8),41.1(C-9),35.4(C-10),77.7(C-11),30.6(C-12),48.3(C-13),49.6(C-14),34.9(C-15),27.5(C-16),50.7(C-17),17.2(C-18),26.6(C-19),41.1(C-20),19.0(C-21),37.8(C-22),40.3(C-23),218.2(C-24),35.4(C-25),18.8(C-26),18.8(C-27),26.4(C-28),19.9(C-29),19.3(C-30)；与文献报道的3a-羟基-25-脱羟基-24-羰基-罗汉果醇(3a-hydroxy-25-Dehydroxy-24-oxomogrol)碳谱数据比较，二者基本一致(Chen X.B.，et al；Bioorganic&Medicinal Chemistry 2011,19:5776)。50 parts of Luo Han Guo extract (98% of Luo Han Guo total saponin, containing 55% Luo Han Guo 甙V, purchased from Guilin Rhein Biotechnology Co., Ltd.) was added to 250 mL of ethanol (the volume ratio of ethanol to water was 1:1) (pre-use) The hydrochloric acid was adjusted to a pH of 3.0), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature at 25 ° C, the reaction solution was neutralized with a 1 M NaOH lye to a pH of 7. Evaporate the ethanol as much as possible at 90-95 ° C. Add the remaining concentrate to 200 mL of distilled water, stir well, cool, and let stand. Discard the upper aqueous layer to obtain a brown extract; use 100 mL, 50 mL, 50 mL of acetic acid, respectively. The ethyl ester was extracted three times with a brown extract, and after three times of extracting, the mixture was concentrated at 100-120 ° C with a rotary evaporator, and the ethyl acetate was completely evaporated to dryness to obtain a monomer containing the mogroside derivative (3) and mangosteen. 18.2 g of a light brown solid of the alcohol derivative monomer (4); a light brown solid was loaded onto a silica gel column with ethanol, and eluted with a gradient of acetonitrile:water (20:1 by volume), and the different effluents were collected in the order of After concentration, two components of A (6.8g) and B (5.6g) are obtained; Obtained A (6.8g) component separated by silica gel column (chloroform: ethanol / 60: 5) to give the alcohol derivative monomer Mangosteen (3) 4.1g (HPLC> 99%). ESIMS m/z 503 (458+45) [M+HCOO - ] - (calcd for C 31 H 51 O 5 ); 13 C-NMR (600 MHz, MeOD) δ ppm: 29.1 (C-1), 31.7 (C- 2), 79.4 (C-3), 42.8 (C-4), 144.1 (C-5), 120.7 (C-6), 25.2 (C-7), 44.8 (C-8), 41.1 (C-9) ), 35.4 (C-10), 77.7 (C-11), 30.6 (C-12), 48.3 (C-13), 49.6 (C-14), 34.9 (C-15), 27.5 (C-16) , 50.7 (C-17), 17.2 (C-18), 26.6 (C-19), 41.1 (C-20), 19.0 (C-21), 37.8 (C-22), 40.3 (C-23), 218.2 (C-24), 35.4 (C-25), 18.8 (C-26), 18.8 (C-27), 26.4 (C-28), 19.9 (C-29), 19.3 (C-30); The carbon spectrum data of 3a-hydroxy-25-dehydroxy-24-oxomogrol reported in the literature are basically the same (Chen XB, et al; Bioorganic & Medicinal Chemistry 2011) , 19:5776).
后收集得到的B(5.6g)组分再用硅胶柱分离(石油醚：乙酸乙酯/5:2洗脱)，得到罗汉果醇衍生物单体(4)。3.2g(HPLC>99％)。ESIMS m/z 441[M+H]+(calcd for C30H49O2)；13C-NMR(600MHz，MeOD)δppm:23.5(C-1),29.5(C-2),87.8(C-3),44.2(C-4),144.1(C-5),121.8(C-6),25.5(C-7),40.9(C-8),40.8(C-9),51.3(C-10),71.9(C-11),41.5(C-12),48.2(C-13),52.2(C-14),35.3(C-15),24.9(C-16),54.3(C-17),15.2(C-18),18.6(C-19),40.2(C-20),20.3(C-21),137.4(C-22),128.8(C-23),125.4(C-24),143.3(C-25),19.7(C-26),25.7(C-27),21.7(C-28),21.7(C-29),18.6(C-30)。The obtained B (5.6 g) fraction was separated by a silica gel column (petroleum ether: ethyl acetate/5:2) to obtain a mogroside derivative monomer (4). 3.2 g (HPLC > 99%). ESIMS m/z 441 [M+H] + (calcd for C 30 H 49 O 2 ); 13 C-NMR (600 MHz, MeOD) δ ppm: 23.5 (C-1), 29.5 (C-2), 87.8 (C) -3), 44.2 (C-4), 144.1 (C-5), 121.8 (C-6), 25.5 (C-7), 40.9 (C-8), 40.8 (C-9), 51.3 (C- 10), 71.9 (C-11), 41.5 (C-12), 48.2 (C-13), 52.2 (C-14), 35.3 (C-15), 24.9 (C-16), 54.3 (C-17) ), 15.2 (C-18), 18.6 (C-19), 40.2 (C-20), 20.3 (C-21), 137.4 (C-22), 128.8 (C-23), 125.4 (C-24) , 143.3 (C-25), 19.7 (C-26), 25.7 (C-27), 21.7 (C-28), 21.7 (C-29), 18.6 (C-30).
实施例2：从罗汉果总皂苷95％的提取物制备罗汉果醇衍生物单体(3)和(4)Example 2: Preparation of mogroside derivative monomers (3) and (4) from 95% of the total saponins of Siraitia grosvenor
将罗汉果提取物50g(罗汉果总皂苷95％，含45％罗汉果甙V，从桂林莱茵生物科技股份有限公司购买)加入250mL的甲醇的水溶液(甲醇和水的体 积比为3:2)(预先用硫酸调节至PH值为2.8)，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温后，反应液用1M的KOH碱液中和至PH值为7，在90-95℃下将甲醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静置，将上面的水层分离弃去，得到咖啡色浸膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干，即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体15.5g；用甲醇将浅咖啡色固体载入硅胶柱，用乙腈：水(19:1)梯度洗脱，按先后顺序收集不同的流出液，浓缩后得到A(6.0g)、B(5.2g)两组分；先收集得到的A(6.0g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)3.9g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的化合物(3)一致。后收集得到的B(5.2g)组分再用硅胶柱分离(石油醚：乙酸乙酯/3:1洗脱)，得到罗汉果醇衍生物单体(4)。3.0g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的罗汉果醇衍生物单体(4)一致。50 parts of Luo Han Guo extract (95% of Luo Han Guo total saponin, containing 45% Luo Han Guo 甙V, purchased from Guilin Rhein Biotechnology Co., Ltd.) was added to 250 mL of methanol solution (methanol and water volume ratio of 3:2) (pre-use The sulfuric acid was adjusted to a pH of 2.8), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature, the reaction solution was neutralized with a 1 M KOH lye to a pH of 7 at 90- Methanol was distilled off as much as possible at 95 ° C. The remaining concentrate was added to 200 mL of distilled water, stirred thoroughly, cooled, and allowed to stand. The upper aqueous layer was separated and discarded to obtain a brown extract; 100 mL, 50 mL, 50 mL of ethyl acetate were used respectively. The coffee extract was extracted 3 times, and after 3 times of extracting, the mixture was concentrated at 100-120 ° C with a rotary evaporator, and the ethyl acetate was completely evaporated to obtain a monomer containing the mogroside derivative (3) and rohanol. The light brown solid of the monomer (4) was 15.5 g; the light brown solid was loaded onto a silica gel column with methanol, eluted with a gradient of acetonitrile:water (19:1), and the different effluent was collected in order, and concentrated to give A. (6.0g), B (5.2g) two components; first collected A (6 The .0g) component was separated by a silica gel column (chloroform: ethanol/60:5 elution) to obtain a mannose alcohol derivative monomer (3) 3.9 g (HPLC>99%); HPLC, 13 C-NMR, ESESI detection The result was identical to the compound (3) in Example 1. The obtained B (5.2 g) fraction was separated by a silica gel column (petroleum ether: ethyl acetate / 3:1 elution) to give the mogroside derivative monomer (4). 3.0 g (HPLC>99%); HPLC, 13 C-NMR, and ESIMS results were consistent with the mogroside derivative monomer (4) in Example 1.
实施例3：从罗汉果总皂苷90％的提取物制备罗汉果醇衍生物单体(3)和(4)Example 3: Preparation of mogroside derivative monomers (3) and (4) from 90% extract of Luo Han Guo total saponin
将罗汉果提取物50g(罗汉果总皂苷90％，含40％罗汉果甙V，从桂林莱茵生物科技股份有限公司购买)加入250mL的正丙醇的水溶液(正丙醇和水的体积比为2:3)(预先用甲酸调节至PH值为3.2，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温后，反应液用1M的NaOH碱液中和至PH值为7，在90-95℃下将正丙醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静置，将上面的水层分离弃去，得到咖啡色浸 膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干，即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体14.2g；用甲醇/乙醇将浅咖啡色固体载入硅胶柱，用乙腈：水(20:1)梯度洗脱，按先后顺序收集不同的流出液，浓缩后得到A(5.4g)、B(4.3g)两组分；先收集得到的A(5.4g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)3.5g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的化合物(3)一致。后收集得到的B(4.3g)组分再用硅胶柱分离(石油醚：乙酸乙酯/6:4洗脱)，得到罗汉果醇衍生物单体(4)2.8g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的罗汉果醇衍生物单体(4)一致。50 kg of Luo Han Guo extract (90% of Luo Han Guo total saponin, containing 40% Luo Han Guo 甙V, purchased from Guilin Rhein Biotechnology Co., Ltd.) was added to 250 mL of an aqueous solution of n-propanol (the ratio of n-propanol to water was 2:3). (Previously adjusted to a pH of 3.2 with formic acid, stirred and dissolved in a reaction vessel, then heated to 120 ° C for 2 hours, cooled to room temperature, and then neutralized with a 1 M NaOH lye to a pH of 7. The n-propanol was distilled off as much as possible at 90-95 ° C. The remaining concentrate was added to 200 mL of distilled water, stirred thoroughly, cooled, and allowed to stand. The upper aqueous layer was separated and discarded to obtain a brown extract; 100 mL, 50 mL, respectively. Extracting the brown extract with 50 mL of ethyl acetate 3 times, combining the extracts three times, concentrating at 100-120 ° C with a rotary evaporator, and evaporating the ethyl acetate completely to obtain a monomer containing rohanol derivative (3) And light brown solid of 14.1 g of the mogroside derivative monomer (4); the light brown solid was loaded onto a silica gel column with methanol/ethanol, and eluted with a gradient of acetonitrile:water (20:1). Liquid, concentrated to obtain A (5.4g), B (4.3g) two components; The resulting set of A (5.4g) component separated by silica gel column (chloroform: ethanol / 60: 5) to give the alcohol derivative monomer Mangosteen (3) 3.5g (HPLC> 99 %); HPLC, 13 C The results of NMR and ESIMS were the same as those of the compound (3) in Example 1. The obtained B (4.3 g) fractions were separated by a silica gel column ( petroleum ether: ethyl acetate / 6:4 elution). The mogroside derivative monomer (4) was 2.8 g (HPLC>99%); the results of HPLC, 13 C-NMR and ESIMS were consistent with the mogroside derivative monomer (4) in Example 1.
实施例4：从罗汉果总皂苷80％的提取物制备罗汉果醇衍生物单体(3)和(4)Example 4: Preparation of mogroside derivative monomers (3) and (4) from an extract of 80% of total saponins of Siraitia grosvenorum
将罗汉果提取物50g(罗汉果总皂苷80％，含20％罗汉果甙V，从桂林莱茵生物科技股份有限公司购买)加入250mL的乙醇的水溶液(乙醇和水的体积比为1:1)(预先用乙二酸调节至PH值为2.6)，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温后，反应液用1M的Na2CO3碱液中和至PH值为7，在90-95℃下将乙醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静置，将上面的水层分离弃去，得到咖啡色浸膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干，即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体8.8g；用甲醇/乙醇将浅咖啡色固体载入硅胶柱，用乙腈：水(95:5)梯度 洗脱，按先后顺序收集不同的流出液，浓缩后得到A(3.8g)、B(3.0g)两组分；先收集得到的A(3.8g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)2.1g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的化合物(3)一致。后收集得到的B(3.0g)组分再用硅胶柱分离(石油醚：乙酸乙酯/4:1洗脱)，得到罗汉果醇衍生物单体(4)1.8g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的罗汉果醇衍生物单体(4)一致。Add 50g of Luo Han Guo extract (80% of Luo Han Guo total saponin, containing 20% Luo Han Guo 甙V, purchased from Guilin Rhein Biotechnology Co., Ltd.) to 250mL of ethanol (the volume ratio of ethanol to water is 1:1) (pre-use The oxalic acid was adjusted to a pH of 2.6), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature, the reaction solution was neutralized to a pH of 7 with 1 M Na 2 CO 3 . Evaporate the ethanol as much as possible at 90-95 ° C. Add the remaining concentrate to 200 mL of distilled water, stir well, cool, and let stand. Discard the upper aqueous layer to obtain a brown extract; use 100 mL, 50 mL, respectively. Extracting the brown extract with 50 mL of ethyl acetate 3 times, combining the extracts three times, concentrating at 100-120 ° C with a rotary evaporator, and evaporating the ethyl acetate completely to obtain a monomer containing rohanol derivative (3) And light brown solid 8.8 g of the mogroside derivative monomer (4); light brown solids were loaded onto a silica gel column with methanol/ethanol, and eluted with a gradient of acetonitrile:water (95:5). Liquid, concentrated to obtain A (3.8g), B (3.0g) two components; Obtained A (3.8g) component separated by silica gel column (chloroform: ethanol / 60: 5) to give the alcohol derivative monomer Mangosteen (3) 2.1g (HPLC> 99 %); HPLC, 13 C- The results of NMR and ESIMS were consistent with the compound (3) in Example 1. The collected B (3.0 g) fraction was separated by a silica gel column (petroleum ether: ethyl acetate / 4:1 elution) to obtain 1.8 g of the mogroside derivative monomer (4) (HPLC>99%); The results of HPLC, 13 C-NMR and ESIMS were consistent with the mogroside derivative monomer (4) in Example 1.
实施例5：罗汉果醇衍生物单体(4)抑制细胞增殖实验Example 5: Monocrotool derivative monomer (4) inhibiting cell proliferation experiment
实验样品：Experimental sample:
测试药物：实施例1制备得到的罗汉果醇衍生物单体(4)；Test drug: the mogroside derivative monomer prepared in Example 1 (4);
对照药物：紫杉醇(SELLECK；Cat.#S1150)；人参皂苷单体Rg3(自上海源叶生物科技有限公司购买，商品货号为B21059)。Control drug: paclitaxel (SELLECK; Cat. #S1150); ginsenoside monomer Rg3 (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number B21059).
实验步骤：Experimental steps:
以24种细胞系(包括23种肿瘤细胞系和1种人脐静脉内皮细胞系)为实验用细胞系，取对数生长期细胞(3x104/mL至2.5x105/mL)，以每孔100μL接种在96孔板内，每个细胞系用一个96孔板；然后，除对照药物紫杉醇以外，以高浓度150μM至低浓度2μM取7个对数递减浓度(每个浓度设两个复孔)，分别加入测试药物溶液和对照药物溶液500nL(测试药物溶液或对照药物溶液配制：分别采用测试药物或对照药物溶于0.5％的DMSO溶液)。紫杉醇的加入浓度为由高浓度1μM至低浓度0.0014μM的7个三倍递减浓度。经测试/对照药物溶液作用72小时后，用
Figure PCTCN2016089211-appb-000009
(Promega；Cat.#G7573)发光细胞活力检测法求出各细胞系中的每种药物的每个浓度对该系细胞的增殖抑制百分率，并绘制量效关系图，最后根据图中曲线测算IC50和最高抑制 百分率(Emax)，如表1和表2所示。24 cell lines (including 23 tumor cell lines and 1 human umbilical vein endothelial cell line) were used as experimental cell lines, and logarithmic growth phase cells (3x10 4 /mL to 2.5x10 5 /mL) were taken per well. 100 μL was seeded in a 96-well plate, and each cell line was plated with a 96-well plate; then, in addition to the control drug paclitaxel, 7 logarithmic decreasing concentrations were taken at a high concentration of 150 μM to a low concentration of 2 μM (two replicate wells per concentration) ), 500 nL of the test drug solution and the control drug solution were respectively added (test drug solution or control drug solution preparation: respectively, the test drug or the control drug was dissolved in a 0.5% DMSO solution). Paclitaxel was added at a concentration ranging from a high concentration of 1 μM to a low concentration of 0.0014 μM. After test/control drug solution for 72 hours, use
Figure PCTCN2016089211-appb-000009
(Promega; Cat. #G7573) Luminescent cell viability assay to determine the percentage inhibition of proliferation of each cell of each cell in each cell line, and to plot the dose-effect relationship, and finally calculate the IC according to the curve in the figure 50 and the highest percent inhibition (E max ), as shown in Tables 1 and 2.
表1：罗汉果醇衍生物单体(4)以及对照药Rg3抑制细胞增殖活性实验检测结果。Table 1: Experimental results of inhibition of cell proliferation activity by the mogroside derivative monomer (4) and the control drug Rg3.
Figure PCTCN2016089211-appb-000010
Figure PCTCN2016089211-appb-000010
Figure PCTCN2016089211-appb-000011
Figure PCTCN2016089211-appb-000011
表2：罗汉果醇衍生物单体(4)以及对照药紫杉醇、Rg3抑制细胞增殖有效性实验检测结果。Table 2: Experimental results of the effectiveness of the mogroside derivative monomer (4) and the control drugs paclitaxel and Rg3 inhibiting cell proliferation.
Figure PCTCN2016089211-appb-000012
Figure PCTCN2016089211-appb-000012
Figure PCTCN2016089211-appb-000013
Figure PCTCN2016089211-appb-000013
使用SPSS Statistics软件(提供者：IBM corporation,软件版本:XL fit 21)提供统计学分析。Statistical analysis was provided using SPSS Statistics software (provided by: IBM corporation, software version: XL fit 21).
用假设方差不相等的独立样本检验(independent-sample T-test)来检测药物组与对照组平均值的差异是否呈现统计显著(P<0.05或P<0.01)。The independent-sample T-test was used to determine whether the difference between the mean of the drug group and the control group was statistically significant (P<0.05 or P<0.01).
分析结果：Analysis results:
1.本发明罗汉果醇衍生物单体(4)在24个细胞系中的平均IC50与对照药物人参皂苷单体Rg3在24个细胞系中的平均IC50比较时，差异呈现统计显著(P<0.05同时P<0.01)。1. The average IC50 of the mogroside derivative monomer (4) of the present invention in 24 cell lines was statistically significant when compared with the average IC50 of the control drug ginsenoside monomer Rg3 in 24 cell lines (P<0.05). At the same time P <0.01).
2.本发明罗汉果醇衍生物单体(4)在24个细胞系中的平均最高抑制百分率与对照药物紫杉醇以及对照药物人参皂苷单体Rg3在24个细胞系中的平均最高抑制百分率比较时，差异呈现统计显著(P<0.05同时P<0.01)。2. The average maximum percent inhibition of the mogroside derivative monomer (4) of the present invention in 24 cell lines is compared with the average highest percent inhibition of the control drug paclitaxel and the control drug ginsenoside monomer Rg3 in 24 cell lines. The difference was statistically significant (P<0.05 and P<0.01).
结论：in conclusion:
1.本发明罗汉果醇衍生物单体(4)在所有24种所测细胞系中的抑制细胞增殖活性均高于同类对照药物人参皂苷单体Rg3(罗汉果醇衍生物单体(4)的IC50小于人参皂苷单体Rg3的IC50)。1. The cellulose-inhibiting activity of the mogroside derivative monomer (4) of the present invention in all 24 cell lines is higher than that of the similar control drug ginsenoside monomer Rg3 (the composition of the mogroside derivative monomer (4)) 50 is less than the IC 50 of the ginsenoside monomer Rg3.
2.在所有被测细胞系中，本发明罗汉果醇衍生物单体(4)在人脐血静脉内皮细胞系HUVEC中具有最高抑制细胞增殖活性(IC50＝10.72μM)，表现出其强大的抗肿瘤血管生成潜力；罗汉果醇衍生物单 体(3)在结直肠腺癌细胞系HCT-8中抑制细胞增殖活性排在其次(IC50＝14.83μM)。2. In all the cell lines tested, the moultanol derivative monomer (4) of the present invention has the highest inhibitory cell proliferation activity (IC 50 = 10.72 μM) in the human umbilical vein endothelial cell line HUVEC, showing its strong Anti-tumor angiogenesis potential; the mogroside derivative monomer (3) inhibited cell proliferation activity in the colorectal cancer cell line HCT-8 (IC 50 = 14.83 μM).
3.本发明罗汉果醇衍生物单体(4)对所有24种被测细胞系的最高抑制率(有效性)都非常接近100％；而对照药紫杉醇和人参皂苷单体Rg3在已测的24种细胞系中，均只对2种细胞系有近100％的抑制率。这表明罗汉果醇衍生物单体(4)的有效性明显高于对照药物，且其高抗癌有效性适用于多类癌种。 3. The highest inhibition rate (effectiveness) of the romanol derivative monomer (4) of the present invention on all 24 tested cell lines is very close to 100%; and the reference drug paclitaxel and ginsenoside monomer Rg3 are measured 24 In the cell line, only about 100% inhibition rate was observed for the two cell lines. This indicates that the mogroin derivative monomer (4) is significantly more effective than the control drug, and its high anticancer effectiveness is applicable to a plurality of cancer types.

Claims (9)

  1. 一种组合物，至少包括如下结构A composition comprising at least the following structure
    Figure PCTCN2016089211-appb-100001
    的罗汉果醇衍生物单体(3)和如下结构
    Figure PCTCN2016089211-appb-100001
    The mogroside derivative monomer (3) and the following structure
    Figure PCTCN2016089211-appb-100002
    的罗汉果醇衍生物单体(4)。
    Figure PCTCN2016089211-appb-100002
    The mogroside derivative monomer (4).
  2. 根据权利要求1的组合物的合成方法，其特征在于，包括：将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇的水溶液中进行加热反应即得。The method for synthesizing a composition according to claim 1, which comprises: dissolving a Luo Han Guo extract containing the total saponin of Luo Han Guo in an aqueous solution of an acidic alcohol for heating reaction.
  3. 根据权利要求2的组合物的合成方法，其特征在于，所述呈酸性的醇的水溶液的制备方法为：用酸性物质将醇的水溶液调节为酸性；The method for synthesizing a composition according to claim 2, wherein the aqueous solution of the acidic alcohol is prepared by adjusting an aqueous solution of the alcohol to an acidic state with an acidic substance;
    任选的，所述醇为甲醇、乙醇、正丙醇、异丙醇、丁醇、乙二醇、丙二醇、丙三醇和聚乙二醇中的至少一种。Optionally, the alcohol is at least one of methanol, ethanol, n-propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, and polyethylene glycol.
    任选的，所述醇的水溶液中醇与水的体积比为1：10～10：1；Optionally, the volume ratio of alcohol to water in the aqueous solution of the alcohol is 1:10 to 10:1;
    任选的，所述醇的水溶液中醇与水的体积比为1：3～3：1； Optionally, the volume ratio of alcohol to water in the aqueous solution of the alcohol is 1:3 to 3:1;
    任选的，所述醇的水溶液中醇与水的体积比为1：1；Optionally, the volume ratio of alcohol to water in the aqueous solution of the alcohol is 1:1;
    任选的，所述酸性物质为无机酸或有机酸；Optionally, the acidic substance is an inorganic acid or an organic acid;
    任选的，所述无机酸为盐酸、硫酸和磷酸中的至少一种；Optionally, the inorganic acid is at least one of hydrochloric acid, sulfuric acid, and phosphoric acid;
    任选的，所述有机酸为甲酸、乙酸、卤代乙酸、乙二酸、丙二酸和柠檬酸中的至少一种；Optionally, the organic acid is at least one of formic acid, acetic acid, haloacetic acid, oxalic acid, malonic acid, and citric acid;
    任选的，所述呈酸性体现为PH值为2.5～3.5；Optionally, the acidity is represented by a pH of 2.5 to 3.5;
    任选的，所述呈酸性体现为PH值为2.8～3.0；Optionally, the acidity is represented by a pH of 2.8 to 3.0;
    任选的，当所述含罗汉果总皂苷的罗汉果提取物中罗汉果总皂苷的重量百分比为70％以上时，所述含罗汉果总皂苷的罗汉果提取物与呈酸性的醇的水溶液的质量体积比为1：10～1：3；Optionally, when the weight percentage of the total saponin of the Lo Han Guo fruit extract of the Luo Han Guo total saponin is 70% or more, the mass to volume ratio of the Luo Han Guo total saponin-containing Luo Han Guo extract and the acidic alcohol aqueous solution is 1:10~1:3;
    任选的，所述含罗汉果总皂苷的罗汉果提取物与呈酸性的醇的水溶液的质量体积比为1：5。Optionally, the mass-to-volume ratio of the Luo Han Guo extract of the Luo Han Guo total saponin to the aqueous solution of the acidic alcohol is 1:5.
  4. 根据权利要求2的组合物的合成方法，其特征在于，所述加热反应的条件是：在100～140℃下加热1～3小时；The method for synthesizing the composition according to claim 2, wherein the heating reaction is carried out under conditions of heating at 100 to 140 ° C for 1 to 3 hours;
    任选的，所述加热反应的条件是：在120℃下加热2小时。Optionally, the heating reaction is carried out under the conditions of heating at 120 ° C for 2 hours.
  5. 根据权利要求2的组合物的合成方法，其特征在于，所述加热反应后还包括对加热反应得到的反应液用碱性物质进行中和，加热将醇蒸发，得到含组合物的浓缩液；The method for synthesizing a composition according to claim 2, wherein the heating reaction further comprises neutralizing the reaction liquid obtained by the heating reaction with an alkaline substance, and heating to evaporate the alcohol to obtain a concentrated liquid containing the composition;
    任选的，用碱性物质进行中和是中和至PH到6.5～7.5；Optionally, neutralization with an alkaline material is neutralization to a pH of 6.5 to 7.5;
    任选的，用碱性物质进行中和是中和至PH到7.0。Optionally, neutralization with an alkaline material is neutralized to a pH of 7.0.
    任选的，所述碱性物质为无机碱和/或盐；Optionally, the basic substance is an inorganic base and/or a salt;
    任选的，所述无机碱和/或盐为NaOH、KOH和Na2CO3中的至少一种；Optionally, the inorganic base and / or salt is at least one of NaOH, KOH, and Na 2 CO 3 ;
  6. 根据权利要求5的组合物的合成方法，其特征在于，还包括对含组合物的浓缩液洗涤后得到咖啡色浸膏的步骤， A method of synthesizing a composition according to claim 5, further comprising the step of obtaining a brown extract after washing the concentrate containing the composition,
    任选的，所述对含组合物的浓缩液洗涤后得到咖啡色浸膏的步骤：在含组合物的浓缩液中加入蒸馏水冷却静置，弃去上面水层，得到含组合物的咖啡色浸膏；Optionally, the step of washing the concentrate containing the composition to obtain a brown extract: adding distilled water to the concentrated solution containing the composition, cooling and standing, discarding the upper aqueous layer to obtain a brown extract containing the composition. ;
    任选的，所述浓缩液与蒸馏水的体积比为1:5～1:3。Optionally, the volume ratio of the concentrate to distilled water is 1:5 to 1:3.
  7. 根据权利要求6的组合物的合成方法，其特征在于，还包括对含组合物的咖啡色浸膏进行萃取和浓缩，得到含组合物的浅咖啡色固体；The method of synthesizing a composition according to claim 6, further comprising extracting and concentrating the brown extract containing the composition to obtain a light brown solid containing the composition;
    任选的，所述萃取所采用的萃取溶剂为乙酸乙酯；Optionally, the extraction solvent used in the extraction is ethyl acetate;
    任选的，每次萃取的固液比1:6～1:3；Optionally, the solid-liquid ratio of each extraction is 1:6 to 1:3;
    任选的，所述萃取的次数为2～5次，将每次萃取液合并；Optionally, the number of extractions is 2 to 5 times, and each extract is combined;
    任选的，所述萃取的次数为3次；Optionally, the number of extractions is 3 times;
    任选的，第一次萃取所用的萃取溶剂的体积是第二次萃取或第三次萃取所用的萃取溶剂的体积的2倍；Optionally, the volume of the extraction solvent used in the first extraction is twice the volume of the extraction solvent used in the second extraction or the third extraction;
    任选的，所述浓缩是：100-120℃下用旋转蒸发仪浓缩，把萃取溶剂完全蒸干。Optionally, the concentration is: concentration at 100-120 ° C using a rotary evaporator, and the extraction solvent is completely evaporated to dryness.
  8. 一种具有如下结构One having the following structure
    Figure PCTCN2016089211-appb-100003
    的罗汉果醇衍生物单体(3)的制备方法，其特征在于，包括将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇的水溶液中进行加热反应，然后分离纯化即得；
    Figure PCTCN2016089211-appb-100003
    The method for preparing the mogroside derivative monomer (3), comprising: dissolving the Luo Han Guo extract containing the total saponin of Luo Han Guo in an aqueous solution of an acidic alcohol, heating, and then separating and purifying;
    任选的，包括对权利要求7的含组合物的浅咖啡色固体进行分离和纯化 的步骤；Optionally, comprising isolating and purifying the light brown solid containing the composition of claim 7 A step of;
    任选的，所述分离为层析分离；Optionally, the separation is chromatographic separation;
    任选的，所述层析分离为硅胶柱层析分离；Optionally, the chromatographic separation is performed by silica gel column chromatography;
    任选的，所述硅胶柱层析分离采用的洗脱液为乙腈和水；Optionally, the eluent used in the silica gel column chromatography is acetonitrile and water;
    任选的，所述乙腈和水的体积比为95:5～20:1；Optionally, the volume ratio of the acetonitrile to water is 95:5 to 20:1;
    任选的，对含组合物的浅咖啡色固体进行分离后还要进一步浓缩；Optionally, the light brown solid containing the composition is further concentrated after separation;
    任选的，浓缩后进行层析纯化；Optionally, after concentration, chromatographic purification;
    任选的，所述层析纯化为硅胶柱层析纯化；Optionally, the chromatographic purification is purified by silica gel column chromatography;
    任选的，所述硅胶柱层析纯化采用的洗脱液为氯仿和乙醇；Optionally, the eluent used in the silica gel column chromatography purification is chloroform and ethanol;
    任选的，所述氯仿和乙醇的体积比为16:1～8:1；Optionally, the volume ratio of the chloroform to ethanol is from 16:1 to 8:1;
    任选的，所述氯仿和乙醇的体积比为12:1。Optionally, the volume ratio of chloroform to ethanol is 12:1.
  9. 一种具有如下结构One having the following structure
    Figure PCTCN2016089211-appb-100004
    Figure PCTCN2016089211-appb-100004
    的罗汉果醇衍生物单体(4)的制备方法，其特征在于，包括将含罗汉果总皂苷的罗汉果提取物溶于呈酸性的醇的水溶液中进行加热反应，然后分离纯化即得；The method for preparing a mogroside derivative monomer (4), comprising: dissolving a Luo Han Guo extract containing Luo Han Guo total saponin in an aqueous solution of an acidic alcohol for heating reaction, and then separating and purifying;
    任选的，包括对权利要求7的含组合物的浅咖啡色固体进行分离和纯化的步骤；Optionally, comprising the step of isolating and purifying the light brown solid containing the composition of claim 7;
    任选的，所述分离为层析分离； Optionally, the separation is chromatographic separation;
    任选的，所述层析分离为硅胶柱层析分离；Optionally, the chromatographic separation is performed by silica gel column chromatography;
    任选的，所述硅胶柱层析分离采用的洗脱液为乙腈和水；Optionally, the eluent used in the silica gel column chromatography is acetonitrile and water;
    任选的，所述乙腈和水的体积比为95:5～20:1；Optionally, the volume ratio of the acetonitrile to water is 95:5 to 20:1;
    任选的，对含组合物的浅咖啡色固体进行分离后还要进一步浓缩；Optionally, the light brown solid containing the composition is further concentrated after separation;
    任选的，浓缩后进行层析纯化；Optionally, after concentration, chromatographic purification;
    任选的，所述层析纯化为硅胶柱层析纯化；Optionally, the chromatographic purification is purified by silica gel column chromatography;
    任选的，所述硅胶柱层析纯化采用的洗脱液为石油醚和乙酸乙酯；Optionally, the eluent used in the silica gel column chromatography purification is petroleum ether and ethyl acetate;
    任选的，所述石油醚和乙酸乙酯的体积比为3:2～4:1；Optionally, the volume ratio of the petroleum ether to ethyl acetate is from 3:2 to 4:1;
    任选的，所述石油醚和乙酸乙酯的体积比为3:1。 Optionally, the volume ratio of petroleum ether to ethyl acetate is 3:1.
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