CN117882848A - Application of saponins compound, preparation method of saponins compound, sweetener and sweetness enhancer - Google Patents

Application of saponins compound, preparation method of saponins compound, sweetener and sweetness enhancer Download PDF

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CN117882848A
CN117882848A CN202311642228.6A CN202311642228A CN117882848A CN 117882848 A CN117882848 A CN 117882848A CN 202311642228 A CN202311642228 A CN 202311642228A CN 117882848 A CN117882848 A CN 117882848A
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compound
methanol
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杜芝芝
张红霞
陈文允
李志坚
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Kunming Institute of Botany of CAS
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Kunming Institute of Botany of CAS
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Abstract

The invention relates to application of a saponin compound, a preparation method thereof, a sweetener and a sweetness enhancer, and relates to the technical field of foods and medicines. Wherein the saponin compound is used as or for preparing sweetener or sweetness enhancer. The saponin compounds include:

Description

Application of saponins compound, preparation method of saponins compound, sweetener and sweetness enhancer
Technical Field
The invention relates to the technical field of foods and medicines, in particular to application of a saponin compound, a preparation method of the saponin compound, a sweetener and a sweetness enhancer.
Background
Taste refers to: a unique sensation is caused on the mouth and tongue when eating foods and various compounds. Taste sensation affects the overall comfort of the mouth and is a fundamental attribute in assessing food products. Taste is one of the basic sensations of humans and is critical to the overall well-being and quality of life of an individual.
Five basic tastes are perceived by humans, including sweetness, umami, bitterness, salty, and sourness, where sweetness is important, and humans are inherently (including animals) preferred for sweetness, while avoiding ingestion of typical bitter foods. Materials capable of producing sweetness are known as sweeteners, including sugar sweeteners (e.g., sucrose, glucose, maltose, fructose, etc.) and non-sugar sweeteners, and are classified by source as synthetic sweeteners (e.g., saccharin, aspartame, acesulfame k, sucralose, etc.) and natural sweeteners.
Sucrose is the longest and most widely used sugar sweetener in daily life. With the rapid increase of the technological development and production level, people can more easily obtain sugar sweeteners such as sucrose, however, excessive intake of sugar sweeteners causes more and more people to suffer from diseases such as diabetes, cardiovascular diseases, obesity, hyperlipidemia, caries and the like. Thus, there has been an effort to find sugar substitutes that are high sweetness, low calorie, non-nutritive.
Non-sugar high-power sweeteners such as acesulfame-K, aspartame, neotame, saccharin, sucralose, alitame, cyclamate and the like are present in people's daily lives by means of artificial synthesis. They have the advantages of high sweetness, small volume, low cost, etc., and occupy a larger market share. However, these synthetic sweeteners also have many disadvantages such as impure sweetness, having various degrees of bitter and astringent taste, metallic aftertaste or off-flavor, being very different from sucrose flavor, and also having a lot of problems in safety. Many countries, particularly developed countries, have been continually outsourced regulatory measures for chemically synthesized sweeteners. The national regulations that the use of non-nutritive high-intensity sweeteners such as saccharin, cyclamate, etc. must not exceed the use ranges and amounts specified by GB2760 (food additive use sanitation standard) and strictly prohibit the use of related chemical synthetic sweeteners in infant foods.
In summary, there is a need for natural compounds with sweetness from natural plants for use as sweeteners or sweetness enhancers to meet the requirements of safety, high sweetness, low calorie.
Disclosure of Invention
In view of the above, the present invention provides an application of a saponin compound, a preparation method thereof, a sweetener and a sweetness enhancer, and mainly aims to provide a natural saponin compound which can be used as or prepared into a sweetener or sweetness enhancer.
In order to achieve the above purpose, the present invention mainly provides the following technical solutions:
in one aspect, embodiments of the present invention provide an application of a saponin compound in the use as or in the preparation of a sweetener or sweetness enhancer, wherein the saponin compound is any one of the following compounds:
preferably, the saponin compounds are separated from gynostemma pentaphylla; preferably, the gynostemma pentaphylla is gynostemma pentaphylla produced by Fujian; preferably, the separation site is an aerial part of gynostemma pentaphylla.
Preferably, the preparation method comprises the following steps:
step 1): carrying out alcohol extraction treatment on gynostemma pentaphylla to be extracted to obtain an alcohol extract; then, extracting the alcohol extract to obtain a target part;
step 2): after dissolving the target part, performing column chromatography separation and segmentation, and performing gradient elution to obtain a plurality of fractions; two streams are divided into a stream 2 and a stream 3;
step 3): separating the fraction 2 to obtain a compound 2, a compound 5, a compound 6, a compound 4 and a compound 7; and separating the fraction 3 to obtain a compound 8, a compound 1 and a compound 3.
Preferably, in the step 1): the gynostemma pentaphylla to be extracted is dried gynostemma pentaphylla powder.
Preferably, in the step 1):
carrying out cold leaching extraction treatment on the gynostemma pentaphylla to be extracted by adopting ethanol to obtain an extracting solution; concentrating the extractive solution under reduced pressure to obtain alcohol extract; and/or
Extracting the alcohol extract by petroleum ether to obtain petroleum ether extract and a water phase part; wherein the aqueous phase portion is a target portion after drying.
Preferably, in said step 2):
dissolving the target part with water, separating and segmenting by using a D101 macroporous resin column chromatography, then performing gradient elution by using an ethanol-water solvent system, and combining and separating according to TLC detection results after recovering the solvent to obtain a fraction 1, a fraction 2, a fraction 3, a fraction 4, a fraction 5, a fraction 6, a fraction 7 and a fraction 8. Preferably, the fraction 2 is a fraction collected after elution with an ethanol-water solvent system having an ethanol volume fraction of 30-50%; the fraction 3 is collected after elution by an ethanol-water solvent system with the ethanol volume fraction of 50-70%.
Preferably, in the step 3):
dissolving the fraction 2, performing column chromatography separation, then performing gradient elution by using a methanol-water solvent system, recovering the solvent, and combining the fractions according to the TCL detection result to obtain a plurality of fractions; one of the streams is split into stream 2E; preferably, the fraction 2E is a fraction collected after elution with a methanol-water solvent system having a methanol volume fraction of 60-80%;
gradient elution is carried out on the fraction 2E by adopting a dichloromethane-methanol system, and fractions are combined according to a TCL detection result to obtain a plurality of fractions; two of the streams are divided into a stream 2E8 and a stream 2E6; preferably, the elution ratio corresponding to the fraction 2E8 is: the volume ratio of the dichloromethane to the methanol is 6:2-6:5; the elution ratio corresponding to fraction 2E6 is: the volume ratio of the dichloromethane to the methanol is 4:1-3:1;
after dissolving the fraction 2E8, segmenting by using a medium-pressure liquid phase system, performing gradient elution by using a methanol-water system, and combining the fractions according to a TCL detection result to obtain a plurality of fractions; one of the streams is split into stream 2E8B; purifying the fraction 2E8B to obtain a compound 2; preferably, the fraction 2E8B is a fraction collected after elution by a methanol-water system with a methanol volume fraction of 50-60%;
after dissolving the fraction 2E6, segmenting by using a medium-pressure liquid phase system, performing gradient elution by using a methanol-water system, and combining the fractions according to TLC detection results to obtain a plurality of fractions; two streams are divided into a stream 2E6E and a stream 2E6G; wherein, after separating the fraction 2E6E, compound 5 and compound 6 are obtained; after separation of the fraction 2E6G, compound 4 and compound 7 are obtained. Preferably, the fraction 2E6E is a fraction collected after elution with a methanol-water system having a methanol volume fraction of 50-60%; preferably, the fraction 2E6G is a fraction collected after elution with a methanol-water system having a methanol volume fraction of 60-65%.
Preferably, in said step 3):
performing gradient elution on the fraction 3 by using a dichloromethane-methanol system, and combining the fractions according to a TCL detection result to obtain a plurality of fractions; wherein one stream is split into stream 3G; preferably, the elution ratio corresponding to the fraction 3G is: the volume ratio of the dichloromethane to the methanol is 4:1-3-1;
after dissolving the fraction 3G, segmenting the fraction by using a medium-pressure liquid phase system, performing gradient elution by using a methanol-water system, and combining the fractions according to a TCL detection result to obtain a plurality of fractions; three of these streams are divided into: fraction 3G1, fraction 3G3; preferably, the fraction 3G1 is a fraction collected after elution by a methanol-water system with a methanol volume fraction of 50-55%; preferably, the fraction 3G3 is a fraction collected after elution by a methanol-water system with a methanol volume fraction of 60-65%;
wherein, after the fraction 3G1 is purified and separated, a compound 8 is obtained; and purifying and separating the fraction 3G3 to obtain a compound 1 and a compound 3.
In still another aspect, an embodiment of the present invention provides a sweetener, where the sweetener includes the above-mentioned saponin compound.
In yet another aspect, the present invention provides a sweetness enhancer, which is characterized in that the sweetness enhancer comprises the above-mentioned saponins.
Compared with the prior art, the application of the saponin compounds, the preparation method of the saponin compounds, the sweetener and the sweetness enhancer have at least the following beneficial effects:
in one aspect, the present invention first discovers 8 new natural saponins compounds that can be used as sweeteners or sweetness enhancers (wherein, compound 1 and compound 4 are new natural compounds, which are new compounds extracted for the first time in the present application), and the 8 natural saponins compounds have a sweet taste or a slightly astringent or licorice taste, and although individual compounds have a certain astringent or licorice taste or other off-flavors, they have a higher sweet taste with a sweetness 20-166 times that of sucrose, and can be used in the fields of foods and pharmaceuticals as sweeteners/sweetness enhancers.
On the other hand, the invention extracts 8 new natural saponins which can be used as sweeteners or sweetness enhancers from gynostemma pentaphylla produced in Fujian for the first time, and the report of the 8 natural saponins which can be used as sweeteners or sweetness enhancers is not related to the prior art.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Detailed Description
In order to further describe the technical means and effects adopted for achieving the preset aim of the present invention, the following detailed description will refer to the specific implementation, structure, characteristics and effects according to the present application in combination with the preferred embodiments. In the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Furthermore, the particular features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.
Gynostemma pentaphylla (Gynostemma pentaphyllum (thunder.) Makino) is a herb climbing plant of Gynostemma of Cucurbitaceae, and is also called herba Gynostemmatis, root of herba Gynostemmatis, etc. Researches show that the main components in the gynostemma pentaphylla are 2 alpha-hydroxy ginseng diol and ginseng diol triterpenoid saponins, and the saponins which are separated and reported from the gynostemma pentaphylla are more than 300. A dammarane type triterpene saponin compound, gypenoside XX, identified by Japanese scholars Takemoto in 1983 was isolated from Gynostemma Pentaphyllum extract and was confirmed to have sweetness in later studies reports, but not clear its sweetness (A.D. Kinghorn; N.—C.Kim; D.S. H.L.Kim, terpenoid Glycoside Sweeteners. In Naturally Occurring Glycosides: chemistry, distribution and Biological Properties; R.Ikan, ed.; john Wiley & Sons: chichester, UK,1999; pp 399-429.). Subsequent related studies reported that 12 dammarane-type triterpenoids in gynostemma pentaphylla could be used as sweeteners or sweetness enhancers, but there was no clear sweetness and sweetness threshold data. In the earlier study: the inventors found when comparing the sweetness of gynostemma pentaphylla in different places of origin: the sweet taste of the gynostemma pentaphylla produced in Yunnan is obviously superior to that of gynostemma pentaphylla produced in other places, and 15 new natural products which have sweet taste with different degrees and sweetness which is 10-100 times that of sucrose are found after systematic research. In a further subsequent study of the invention, the inventors found that gynostemma pentaphylla produced in Fujian province has a sweet and bitter taste, but has a higher sweet taste, and has a sweetness higher than that of yunnan sweet gynostemma pentaphylla, and found that 8 gynostemma pentaphylla saponins with sweetness, which is 20-166 times of that of sucrose, have a sweetness much higher than that of yunnan sweet gynostemma pentaphylla saponins, through systematic study, wherein compounds 1 and 4 are novel natural products.
Example 1
The embodiment is mainly used for extracting natural sweet compounds from gynostemma pentaphylla produced in Fujian province.
1. Instrument and materials
Specific rotation: autopol VI, serial #91058; infrared spectroscopy: thermo NICOLET iS10; ultraviolet spectrum: UV-2700 series; ESI and HRESI: the Shimadzu UPLC-IT-TOF-type liquid phase-ion trap time-of-flight chromatograph-mass spectrometer; nuclear magnetic data: bruker AVANCE III 500MH and AV-600, TMS as internal standard, chemical shift delta unit is ppm, coupling constant J unit is HZ.
Rotary evaporator (eyella and switzerland B chi company, japan); medium pressure liquid chromatograph (Buchi, switzerland); semi-preparative high performance liquid chromatograph (Jiangsu Hanbang technology Co., ltd.); the column (YMC-Pack ODS-AQ,10 mm. Times.250 mm; YMC-Pack CN,10 mm. Times.250 mm).
Normal phase silica gel (80-100 mesh, 200-300 mesh, qingdao ocean chemical plant); normal phase silica gel plate (Qingdao ocean chemical plant); reverse phase silica gel (YMC Co.); reverse phase silica gel plate (Merck company, germany); gel Sephadex LH-20 (GE healthcare, sweden); macroporous adsorbent resin D101 (Tianjin Xing Nanchun high molecular technology Co., ltd.); macroporous adsorbent resin HP-20 (Tianjin Xing Nanchun high molecular technology Co., ltd.); color-developing agent: sulfuric acid-vanillin solution.
The gynostemma pentaphylla plant material required for the experiment in this example was collected in the city of Zhangzhou, fujian province in 2021.
2. The preparation steps of the natural sweet compound are as follows:
and (3) drying and crushing 4 kg of the aerial parts of the gynostemma pentaphylla, carrying out cold leaching extraction for three times by using ethanol with the volume fraction of 95%, each time for 48 hours, and concentrating the combined extracting solution under reduced pressure by a rotary evaporator to recover ethanol to obtain an ethanol extract. To the resulting ethanol extract, 2L of distilled water was added to suspend, followed by extraction with an equal volume of petroleum ether for 6 times to obtain petroleum ether extract (24.61 g, denoted as GPPP) and aqueous phase fraction (450 g, denoted as GPMP).
GPMP (450 g) was dissolved in water, partitioned by D101 macroporous resin column chromatography, and gradient eluted using ethanol-water solvent system (0%, 30%,50%,70%,90% M/W). The solvents were recovered by rotary evaporator and combined to give 8 fractions after TLC detection: fraction 1 (fr.1, 21 g), fraction 2 (fr.2, 81 g), fraction 3 (fr.3, 122 g), fraction 4 (fr.4, 10 g), fraction 5 (fr.5, 9 g), fraction 6 (fr.6, 6 g), fraction 7 (fr.7, 2 g), fraction 8 (fr.8, 3 g). What should be stated here is: after elution with each elution ratio, it is necessary to detect with TCL, possibly dividing it into multiple fractions depending on the composition; and finally, combining the same or similar parts in the parts obtained after elution and detection of all elution ratios to obtain the 8 parts.
Fraction 2 (81 g) was dissolved in water and separated by Diaion HP-20 column chromatography, and eluted with a gradient of methanol-water solvent system (0%, 30%,50%,70%,90% M/W). The solvents were recovered by rotary evaporator and combined to give 8 fractions after TLC detection: fraction 2A (2G), fraction 2B (11G), fraction 2C (6G), fraction 2D (1.6G), fraction 2E (20.6G), fraction 2F (9.5G), fraction 2G (8G), fraction 2H (6G).
Mixing the flow 2E (20 g) with 80-100 mesh silica gel, and a 200-300 mesh silica gel column with dichloromethane: methanol (8:2, 7:3, 6:4) gradient elution. TLC detection and combining gave 12 fractions: fraction 2E1 (800 mg), fraction 2E2 (560 mg), fraction 2E3 (735 mg), fraction 2E4 (340 mg), fraction 2E5 (690 mg), fraction 2E6 (2 g), fraction 2E7 (3 g), fraction 2E8 (5 g), fraction 2E9 (986 mg), fraction 2E10 (500 mg), fraction 2E11 (2 g), fraction 2E12 (350 mg).
Fraction 2E8 (5 g), dissolved in methanol, was segmented with a medium pressure liquid phase system and eluted with a methanol-water (50%, 60%,70%, 100%) gradient, and finally combined to give 8 fractions: fraction 2E8A (800 mg), fraction 2E8B (2G), fraction 2E8C (800 mg), fraction 2E8D (21 mg), fraction 2E8E (46 mg), 2E8F (86 mg), fraction 2E8G (514 mg), fraction 2E8H (86 mg). Wherein fraction 2E8B (2 g) was dissolved in methanol, and Compound 2 (178 mg) was isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ,58% methanol-water). Wherein fraction 2E8G (514 mg) was dissolved in methanol, and Compound 9 (256 mg) was isolated by semi-preparative high performance liquid chromatography (YMC-Pack C18, 78% methanol-water).
Fraction 2E6 (2 g), dissolved in methanol, was segmented with a medium pressure liquid phase system, gradient eluted with methanol-water (50%, 60%, 100%) and finally combined to give 8 fractions: fraction 2E6A (32 mg), fraction 2E6B (85 mg), fraction 2E6C (800 mg), fraction 2E6D (18 mg), fraction 2E6E (890 mg), fraction 2E6F (500 mg), fraction 2E6G (96 mg), fraction 2E6H (150 mg). Wherein fraction 2E6E (89 mg) was dissolved in methanol, and compounds 5 (38 mg) and 6 (45 mg) were separated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ,63% methanol-water). Wherein fraction 2E6G (96 mg) was dissolved in methanol, and compounds 4 (46 mg) and 7 (22 mg) were separated by semi-preparative high performance liquid chromatography (YMC-Pack C18, 68% methanol-water).
Sample 3 (122 g) is mixed with 80-100 mesh silica gel, a 200-300 mesh silica gel column is prepared by using methylene dichloride: methanol (15:1, 9:1,8:2,7:3, methanol) gradient elution. TLC detection and combining gave 8 fractions: fraction 3A (2G), fraction 3B (1G), fraction 3C (2.9G), fraction 3D (5.2G), fraction 3E (7.2G), fraction 3F (36.4G), fraction 3G (12.3G), fraction 3H (37.9G).
Fraction 3G (12G) was dissolved in methanol, segmented with a medium pressure liquid phase system, gradient eluted with methanol-water (50%, 60%,65%,70%, 100%) and finally combined to obtain 11 fractions: fraction 3G1 (940 mg), fraction 3G2 (673 mg), fraction 3G3 (711 mg), fraction 3G4 (830 mg), fraction 3G5 (193 mg), fraction 3G6 (2.9G), fraction 3G7 (870 mg), fraction 3G8 (600 mg), fraction 3G9 (2.37G), fraction 3G10 (580 mg), fraction 3G11 (820 mg).
Fraction 3G1 (331 mg) was dissolved in methanol, and Compound 8 (40 mg) was isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ,63% methanol-water). Fraction 3G2 (673 mg) was dissolved in methanol, and Compound 12 (48 mg) was isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ,70% methanol-water).
Fraction 3G3 (711 mg) was dissolved in methanol, and Compound 1 (107 mg) and Compound 3 (144 mg) were separated by semi-preparative high performance liquid chromatography (YMC-Pack SB-C18, 58% methanol-water).
3G7 (870 mg) is mixed with 80-100 mesh silica gel, 200-300 mesh silica gel column is mixed with dichloromethane: methanol (8:2, 7:3, methanol) gradient elution. TLC detection and combining gave 7 fractions: fraction 3G7A (62 mg), fraction 3G7B (129 mg), fraction 3G7C (41 mg), fraction 3G7D (45 mg), fraction 3G7E (31 mg), fraction 3G7F (38 mg), fraction 3G7G (303 mg). Wherein fraction 3G7B (129 mg) was dissolved in methanol, and Compound 11 (80 mg) was isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ,70% methanol-water).
3G9 (2.37G) is mixed with 80-100 mesh silica gel, 200-300 mesh silica gel column is mixed with dichloromethane: methanol (8:2, 7:3, methanol) gradient elution. TLC detection and combining gave 2 fractions: fraction 3G9A (1.5G) and fraction 3G9B (700 mg), wherein 3G9A is compound 10.
The structural formula of the above compounds 1-12 is as follows:
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wherein, the nuclear magnetic data of the novel natural compound 1 and compound 4 are as follows:
compound 1, white powder,；UV(MeOH)λmax(nm)(logε)：203.00(3.07)；IR(KBr)：3432，2943，2879，1640，1453，1421，1385，1261，1165，1078，1039，895，845，630，577，425cm -1 ；ESI-MS m/z 1133[M+Na] + ；HRESIMS m/z 1109.5742(calcd for C 53 H 90 O 24 ,1108.5749).
compound 4, white powder,；UV(MeOH)λmax(nm)(logε)：203.00(2.94)；IR(KBr)：3433，2927，2877，1639，1455，1419，1343，1265，1162，1077，1042，895，659，615cm -1 ；ESI-MS m/z 1256[M-H] - ；HRESIMS m/z 947.5222(calcd for C 47 H 80 O 19 ,947.5221).
table 1 shows the nuclear magnetic data of compound 1 and compound 4:
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example 2
The present example is directed to taste sensory evaluation of monomeric compounds 1-12 isolated from gynostemma pentaphylla in example 1.
Compound solution preparation: accurately weighing a certain amount of compound, adding water to dissolve the compound, diluting the compound to prepare a mother solution with the concentration of 1mg/mL, diluting the mother solution again to prepare a series of tasting product solutions with concentration gradients, wherein the concentration of the tasting product solutions is sequentially 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.2mg/mL, 0.125mg/mL, 0.1mg/mL, 0.04mg/mL and the like.
Preparing a sucrose solution: a certain amount of sucrose is accurately weighed and prepared into 10mg/mL and 20mg/mL solution by water, and is used for comparison with a sample to determine the relative sweetness of the compound and the sucrose.
Sensory panel members tasted the compound and sucrose solution to determine the compound concentration C at a sweetness level equivalent to sucrose Compounds of formula (I) ，C Compounds of formula (I) And C Sucrose The multiple of the two is the relative sucrose sweetness of the compound.
The panelists tasted each sample until a solution without sweetness was tasted. Concentration C of the sample solution n With penultimate sample concentration C n-1 The arithmetic mean of (2) is the sweetness threshold of the compound.
Table 2 shows the results of taste sensory evaluation of the compounds
Note that: * Sweetness: the ratio of the sweetness sample such as sucrose to the concentration of sucrose is the relative sweetness of sucrose.
As can be seen from table 2:
(1) The compound 1-8 has sweet taste mainly, is bitter, has higher sweet taste, has sweetness which is 20-166 times of that of sucrose, and can be used as sweetener/sweetness enhancer in the fields of food and medicine.
(2) Although the compounds 9-12 are also extracted from gynostemma pentaphylla, their structures are very similar to those of the compounds 1-8 of the present invention, the differences are mainly that the individual groups are different; the following is explained: the change of the structural groups of the compound has a great influence on the sweetness of the compound, and even changes from sweet to bitter.
In fact, the present inventors have conducted extensive studies on gynostemma pentaphylla, and in the early stage technique, the inventors extracted various sweet and bitter compounds from gynostemma pentaphylla; the inventors have found that there is little commonality to sweet compounds because individual groups or amounts of these compounds change and easily become bitter. Therefore, the sweet compounds extracted from gynostemma pentaphylla cannot be summarized as a skeleton. It is also not possible to directly determine whether a compound is sweet or not from its structure.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention in any way, but any simple modification, equivalent variation and modification made to the above embodiments according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims (10)

1. Use of a saponin compound as or in the preparation of a sweetener or sweetness enhancer, wherein the saponin compound is any one of the following compounds:
2. the method for preparing a saponin compound according to claim 1, wherein the saponin compound is isolated from gynostemma pentaphylla; preferably, the gynostemma pentaphylla is gynostemma pentaphylla produced by Fujian; preferably, the separation site is an aerial part of gynostemma pentaphylla.
3. The method for preparing a saponin compound according to claim 2, wherein the method comprises the steps of:
step 1): carrying out alcohol extraction treatment on gynostemma pentaphylla to be extracted to obtain an alcohol extract; then, extracting the alcohol extract to obtain a target part;
step 2): after dissolving the target part, performing column chromatography separation and segmentation, and performing gradient elution to obtain a plurality of fractions; two streams are divided into a stream 2 and a stream 3;
step 3): separating the fraction 2 to obtain a compound 2, a compound 5, a compound 6, a compound 4 and a compound 7; and separating the fraction 3 to obtain a compound 8, a compound 1 and a compound 3.
4. A process for the preparation of saponins according to claim 3, characterized in that in step 1): the gynostemma pentaphylla to be extracted is dried gynostemma pentaphylla powder.
5. A process for the preparation of saponins according to claim 3, characterized in that in step 1):
carrying out cold leaching extraction treatment on the gynostemma pentaphylla to be extracted by adopting ethanol to obtain an extracting solution; concentrating the extractive solution under reduced pressure to obtain alcohol extract; and/or
Extracting the alcohol extract by petroleum ether to obtain petroleum ether extract and a water phase part; wherein the aqueous phase portion is the target portion after drying.
6. A process for the preparation of saponins according to claim 3, characterized in that in step 2) the following steps are carried out:
dissolving the target part with water, separating and segmenting by using a D101 macroporous resin column chromatography, then performing gradient elution by using an ethanol-water solvent system, and combining and separating according to TLC detection results after recovering the solvent to obtain a fraction 1, a fraction 2, a fraction 3, a fraction 4, a fraction 5, a fraction 6, a fraction 7 and a fraction 8; preferably, the fraction 2 is a fraction collected after elution with an ethanol-water solvent system having an ethanol volume fraction of 30-50%; the fraction 3 is collected after elution by an ethanol-water solvent system with the ethanol volume fraction of 50-70%.
7. The method for producing a saponin compound according to claim 3, wherein in the step 3):
dissolving the fraction 2, performing column chromatography separation, then performing gradient elution by using a methanol-water solvent system, recovering the solvent, and combining the fractions according to the TCL detection result to obtain a plurality of fractions; one of the streams is split into stream 2E; preferably, the fraction 2E is a fraction collected after elution with a methanol-water solvent system having a methanol volume fraction of 60-80%;
gradient elution is carried out on the fraction 2E by adopting a dichloromethane-methanol system, and fractions are combined according to a TCL detection result to obtain a plurality of fractions; two of the streams are divided into a stream 2E8 and a stream 2E6; preferably, the elution ratio corresponding to the fraction 2E8 is: the volume ratio of the dichloromethane to the methanol is 6:2-6:5; the elution ratio corresponding to fraction 2E6 is: the volume ratio of the dichloromethane to the methanol is 4:1-3:1;
after dissolving the fraction 2E8, segmenting by using a medium-pressure liquid phase system, performing gradient elution by using a methanol-water system, and combining the fractions according to a TCL detection result to obtain a plurality of fractions; one of the streams is split into stream 2E8B; purifying the fraction 2E8B to obtain a compound 2; preferably, the fraction 2E8B is a fraction collected after elution by a methanol-water system with a methanol volume fraction of 50-60%;
after dissolving the fraction 2E6, segmenting by using a medium-pressure liquid phase system, performing gradient elution by using a methanol-water system, and combining the fractions according to TLC detection results to obtain a plurality of fractions; two streams are divided into a stream 2E6E and a stream 2E6G; wherein, after separating the fraction 2E6E, compound 5 and compound 6 are obtained; separating the fraction 2E6G to obtain a compound 4 and a compound 7; preferably, the fraction 2E6E is a fraction collected after elution with a methanol-water system having a methanol volume fraction of 50-60%; preferably, the fraction 2E6G is a fraction collected after elution with a methanol-water system having a methanol volume fraction of 60-65%.
8. A process for the preparation of saponins according to claim 3, characterized in that in said step 3):
performing gradient elution on the fraction 3 by using a dichloromethane-methanol system, and combining the fractions according to a TCL detection result to obtain a plurality of fractions; wherein one stream is split into stream 3G; preferably, the elution ratio corresponding to the fraction 3G is: the volume ratio of the dichloromethane to the methanol is 4:1-3:1;
after dissolving the fraction 3G, segmenting the fraction by using a medium-pressure liquid phase system, performing gradient elution by using a methanol-water system, and combining the fractions according to a TCL detection result to obtain a plurality of fractions; three of these streams are divided into: fraction 3G1, fraction 3G3; preferably, the fraction 3G1 is a fraction collected after elution by a methanol-water system with a methanol volume fraction of 50-55%; preferably, the fraction 3G3 is a fraction collected after elution by a methanol-water system with a methanol volume fraction of 60-65%;
wherein, after the fraction 3G1 is purified and separated, a compound 8 is obtained; and purifying and separating the fraction 3G3 to obtain a compound 1 and a compound 3.
9. A sweetener comprising the saponin compound of claim 1.
10. A sweetness enhancer comprising the saponin compound of claim 1.
CN202311642228.6A 2023-12-04 2023-12-04 Application of saponins compound, preparation method of saponins compound, sweetener and sweetness enhancer Pending CN117882848A (en)

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