CN113024627B - Dammarane type triterpenoid saponin compound and preparation method and application thereof - Google Patents
Dammarane type triterpenoid saponin compound and preparation method and application thereof Download PDFInfo
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- CN113024627B CN113024627B CN202110180000.4A CN202110180000A CN113024627B CN 113024627 B CN113024627 B CN 113024627B CN 202110180000 A CN202110180000 A CN 202110180000A CN 113024627 B CN113024627 B CN 113024627B
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- dammarane
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- methanol
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- -1 triterpenoid saponin compound Chemical class 0.000 title claims abstract description 19
- OORMXZNMRWBSTK-LGFJJATJSA-N dammarane Chemical compound C1CCC(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@H](C)CCCC(C)C)[C@H]4CC[C@@H]3[C@]21C OORMXZNMRWBSTK-LGFJJATJSA-N 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000003765 sweetening agent Substances 0.000 claims abstract description 23
- 235000003599 food sweetener Nutrition 0.000 claims abstract description 22
- 239000003623 enhancer Substances 0.000 claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 135
- 150000001875 compounds Chemical class 0.000 claims description 79
- 238000004809 thin layer chromatography Methods 0.000 claims description 34
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 33
- 238000000926 separation method Methods 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 238000010828 elution Methods 0.000 claims description 27
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000741 silica gel Substances 0.000 claims description 24
- 229910002027 silica gel Inorganic materials 0.000 claims description 24
- 235000002956 Gynostemma pentaphyllum Nutrition 0.000 claims description 22
- 241001065361 Gynostemma Species 0.000 claims description 21
- 229930182493 triterpene saponin Natural products 0.000 claims description 21
- 239000002904 solvent Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 14
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 13
- 239000000287 crude extract Substances 0.000 claims description 10
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 claims description 8
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 claims description 8
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- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 claims description 7
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
- C07J17/005—Glycosides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
- A23L27/11—Natural spices, flavouring agents or condiments; Extracts thereof obtained by solvent extraction
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention provides a dammarane type triterpenoid saponin compound and a preparation method and application thereof; wherein the structural general formula of the dammarane type triterpenoid saponin compound is shown as a formula (I) or a formula (II):
a novel dammarane type triterpenoid saponin compound is provided in stems and leaves, and the application of the novel dammarane type triterpenoid saponin compound as one or more of sweeteners or sweetness enhancers is found for the first time.
Description
Technical Field
The invention relates to the field of natural medicinal chemistry, in particular to a dammarane type triterpenoid saponin compound and a preparation method and application thereof.
Background
Among the five fundamental tastes perceived by humans, including sweet, fibrous, bitter, salty, and sour, sweet taste dominates. The substance capable of sensing sweetness is called sweetener, is an indispensable substance in daily life, and can be divided into sugar sweeteners (such as sucrose, glucose, fructose, maltose, xylose and the like) and non-sugar sweeteners.
With the improvement of the living standard of people, nowadays, more and more people suffer from many diseases, such as diabetes, cardiovascular diseases, obesity, hyperlipidemia, decayed teeth and the like, which are related to the excessive intake of sucrose more or less. Thus, there has been a constant search for high-sweetness, low-calorie, non-nutritive sugar substitutes. Non-sugar high sweeteners such as acesulfame-K, aspartame, neotame, saccharin, sucralose, alitame, cyclamate and the like are found in people's daily lives by artificial synthesis methods. They have the advantages of high sweetness, small volume, low cost and the like, and occupy larger market share. However, these synthetic sweeteners also have many disadvantages, such as an unsatisfactory sweetness, various degrees of bitterness, metallic aftertaste or off-taste, a great difference from the sucrose flavor, and safety problems, and many countries, especially developed countries, have been under continuous control of chemical synthetic sweeteners. According to the national regulation, the use range and the use amount of non-nutritive high-potency sweeteners such as saccharin, sodium cyclamate and the like do not exceed the use range and the use amount specified in GB2760 (the hygienic standard of food additives), and related chemical synthetic sweeteners are strictly forbidden to be used in infant food.
Gynostemma pentaphylla (Gynostemma pentaphylum (Thunb.) Makino) is a grass climbing plant of Gynostemma of Cucurbitaceae, also called Hemsleya paniculata, ubiquitously rooted roots and the like, widely distributed in southern Shaanxi and southern Yangtze provinces in China, also distributed in Korea, Japan and some southeast Asia countries, is a common medicinal and edible plant in China, and can be divided into two varieties of sweet taste and bitter taste according to taste; it is called sweet tea in Japan as a natural sweet tea. Researches show that the main components of the gynostemma pentaphylla are 2 alpha-hydroxy panaxadiol and panaxadiol triterpenoid saponin compounds, more than 200 saponin compounds are separated and reported from the gynostemma pentaphylla at present, but the reports on sweet components are less. A dammarane-type triterpene saponin compound gypenoside XX isolated and identified by Japanese scholars in 1983 was confirmed to have sweet taste in later research reports but its sweetness was not clarified (A.D.Kinghorn; N. -C.Kim; D.S.H.L.Kim, Terphenoid Glycoside Sweeteners. in Naturally Ocurring Glycosides: Chemistry, Distribution and Biologica1 Properties; R.Ikan, Ed.; John Wiley & Sons: Chichester, UK, 1999; pp. 429). An international patent publication (EP3501296a1) in 2017 reported that 12 dammarane-type triterpene saponin compounds in gynostemma pentaphylla can be used as a sweetener or a sweetness enhancer, but there is no clear sweetness and sweetness threshold data.
The applicant compares the sweetness of gynostemma pentaphylla from different producing areas to find that the sweetness of gynostemma pentaphylla from Yunnan is obviously superior to that of gynostemma pentaphylla from other producing areas, and finds 15 new natural products through system research, wherein the natural products have different degrees of sweetness, and the sweetness is 10-100 times of sucrose.
Disclosure of Invention
In view of the above, the invention provides dammarane type triterpenoid saponin compounds and a preparation method and application thereof, and mainly aims to: a series of novel dammarane type triterpenoid saponin compounds are innovated for the first time, and the application of the series of novel dammarane type triterpenoid saponin compounds in serving as or preparing a sweetening agent or a sweetness enhancer is provided.
In order to achieve the purpose, the invention mainly provides the following technical scheme:
on one hand, the dammarane type triterpenoid saponin compound has a structural general formula shown as a formula (I) or a formula (II):
in said formula (I): r1Is H or beta-D-xylosyl- (1 → 2) -beta-D-glucosyl; r2Is any one of OH, H and O; r3Is H, OH, OMe, OOH, or a sugar chain formed with any one or more of beta-D-xylosyl, beta-D-glucosyl, alpha-L-rhamnosyl, beta-D-glucuronyl; wherein the dotted line in formula (I) is a double bond or a single bond;
in said formula (II): r1Is H or beta-D-glucosyl; r2Is H or acetyl; r3Is any one of H, beta-D-glucosyl, beta-D-xylosyl, alpha-L-rhamnosyl, beta-D-xylosyl- (1 → 3) -alpha-L-rhamnosyl, alpha-L-rhamnosyl- (1 → 2) -beta-D-xylosyl or beta-D-glucosyl- (1 → 3) -alpha-L-rhamnosyl; r4Is any one of OH, H and O; wherein the dotted line in formula (II) is a double bond or a single bond.
On the other hand, the dammarane-type triterpene saponin compound is any one of the following compounds:
wherein compounds 1, 5, 6, 16 are based on formula (I); compounds 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15 are based on formula (II).
On the other hand, in the preparation method of the dammarane-type triterpene saponin compound, the dammarane-type triterpene saponin compound is separated from gynostemma pentaphylla; preferably, the gynostemma pentaphylla is produced in Yunnan (compared with gynostemma pentaphylla produced in other producing places, the sweetness of the gynostemma pentaphylla produced in Yunnan is obviously higher); the separation part is the overground part of the gynostemma pentaphylla;
the preparation method comprises the following steps:
1) pulverizing stem and leaf of herba Gynostemmatis, and extracting with ethanol to obtain crude extract of herba Gynostemmatis; performing column separation on the gynostemma pentaphylla crude extract, performing spot-plate combination on flow components, and determining a target separation section;
2) dissolving the target separation section, performing column chromatography separation and gradient elution, recovering the solvent, and combining the fractions to obtain fraction A, fraction B, fraction C, fraction D, fraction E and fraction F (by sensory evaluation, fraction C, D, E is sweet, and fraction A, B, F is bitter, so that fraction C, D, E is selected for subsequent operation);
3) after the separation treatment is carried out on the flow part C, a compound 4, a compound 9, a compound 3, a compound 7, a compound 6, a compound 14, a compound 1, a compound 2 and a compound 8 can be obtained;
4) after the fraction D is subjected to separation treatment, a compound 5, a compound 10, a compound 11, a compound 12 and a compound 13 can be obtained;
5) after separation treatment of the fraction E, compounds 15 and 16 can be obtained.
On the other hand, in the above method for preparing dammarane-type triterpene saponin compounds, in the step 1):
the alcohol extraction treatment steps are as follows:
reflux-extracting pulverized aerial parts of herba Gynostemmatis with 65-100% methanol-water solution or 65-100% ethanol-water solution at 30-70 deg.C to obtain herba Gynostemmatis crude extract;
preferably, the column separation is normal phase silica gel column, and the silica gel used in the chromatographic column is 200-300 mesh silica gel; wherein, when the normal phase silica gel column is used for separation, a petroleum ether-acetone gradient elution solvent system is adopted; wherein, petroleum ether: the volume ratio of acetone is respectively 10:1, 7:1, 3:1 and 1:1, 0:1, the acetone is counted according to per 200g crude extract, each gradient is 20L, and finally 10L methanol is used for elution;
preferably, the point-plate combined flow treatment comprises the following steps: and (3) carrying out thin-layer chromatography (TLC) spot plate treatment on the fractions after column separation, and combining to obtain 8 fractions which are respectively: fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr 8; then, since only Fr8 had a sweet taste as evaluated by taste activity, Fr8 was identified as a target isolated fragment.
In another aspect, in the above method for preparing dammarane-type triterpene saponin compounds, in the step 2):
dissolving the target separation section in water according to the mass-volume ratio of (1-4) to 1(g/ml), and then performing column chromatography separation by using macroporous adsorption resin; preferably, the macroporous adsorption resin is Diaion HP-20 with the specification of 7.7cm multiplied by 65.5 cm;
preferably, in the solvent system of gradient elution, the ratio of methanol: the water accounts for 0 percent, 10-30 percent, 30-50 percent, 50-70 percent and 70-100 percent respectively according to volume percentage, and each solution has 8L gradient;
preferably, the solvent is recovered by a rotary evaporation method, and the fractions A, B, C, D, E and F are obtained by combining after TLC detection.
On the other hand, in the above method for preparing dammarane-type triterpene saponin compounds, in the step 3):
dissolving the fraction C in water, segmenting by using a medium-pressure liquid phase system, performing gradient elution, and finally combining to obtain 8 fractions C1, C2, C3, C4, C5, C6, C7 and C8; preferably, the mass volume ratio of the flow part C to the water is 2: 3 g/ml; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 4.0 multiplied by 25cm, and the flow rate of the upper column is 20 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: the water accounts for 0 percent, 30 percent, 50 percent, 70 percent and 100 percent by volume percent respectively;
preferably, the fraction C2 is segmented by the medium-pressure liquid phase system, gradient elution is carried out, and TLC detection is combined, so that 7 fractions C2a, C2b, C2C, C2d, C2e, C2f and C2g are obtained; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 2.0 x 45cm, and the flow rate of the upper column is10 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: the water accounts for 30 percent, 40 percent, 50 percent, 60 percent and 100 percent by volume percent respectively; wherein the fraction C2d is separated into compound 4 and compound 9 by semi-preparative high performance liquid chromatography; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 58% methanol-water (volume percent); separating the flow part C2e into a compound 3 and a compound 7 by semi-preparative high performance liquid chromatography; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 62% methanol-water (volume percent);
preferably, the fractions C4 are segmented by a medium-pressure liquid phase system, subjected to gradient elution and combined by TLC detection to obtain 6 fractions C4a, C4b, C4C, C4d, C4e and C4 f; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 2.0 x 45cm, and the flow rate of the upper column is10 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: 50%, 52%, 54%, 56%, 58%, 100% (volume percent) of water, respectively; wherein the fraction C4e is separated into compound 6 and compound 14 by semi-preparative high performance liquid chromatography; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 70% methanol-water (volume percent);
preferably, the fractions C5 are segmented by a medium-pressure liquid phase system, subjected to gradient elution and combined by TLC detection to obtain 5 fractions C5a, C5b, C5C, C5d and C5 e; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 2.0 x 45cm, and the flow rate of the upper column is10 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: 50 percent, 58 percent, 54 percent, 60 percent and 100 percent of water (volume percentage) respectively; wherein the fraction C5d is separated by semi-preparative high performance liquid chromatography to obtain compound 1; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack CN, 53% methanol-water (volume percent);
preferably, the fraction C8 is fractionated by a medium-pressure liquid phase system, and is subjected to gradient elution and TLC detection to obtain 4 fractions: c8a, C8b, C8C, C8 d; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 2.0 x 45cm, and the flow rate of the upper column is10 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: water is 45%, 47%, 49%, 51%, 70%, 100% (volume percentage) respectively; wherein the fraction C8C is separated by semi-preparative high performance liquid chromatography to give Compound 2; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 68% methanol-water (volume percent); wherein the fraction C8d is separated by semi-preparative high performance liquid chromatography to give Compound 8; preferably, the semi-preparative HPLC is YMC-Pack ODS-AQ, 70% methanol-water (volume percent).
On the other hand, in the above method for preparing dammarane-type triterpene saponin compounds, in the step 4):
dissolving the fraction D in water, performing segmentation and gradient elution by using a medium-pressure liquid phase system, performing dot-plate detection after concentration, and combining to obtain 9 fractions D1, D2, D3, D4, D5, D6, D7, D8 and D9; preferably, the mass volume ratio of the flow part D to the water is 2: 1 g/ml; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 4.0 multiplied by 25cm, and the flow rate of the upper column is 20 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: water is 0%, 30%, 50%, 60%, 70%, 100% (volume percentage) respectively; preferably, the elution fractions obtained after gradient elution are subjected to rotary evaporation concentration, and then are combined to obtain 9 fractions after thin-layer chromatography (TLC) detection;
preferably, the fractions D7 are subjected to TLC detection after silica gel column separation and combined to obtain 9 fractions D7a, D7b, D7c, D7D, D7e, D7f, D7g, D7h and D7 i;
wherein said fraction D7b is subjected to semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 60% methanol-water) to isolate Compound 5; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 60% methanol-water (volume percent);
wherein said fraction D7e is separated by semi-preparative high performance liquid chromatography to give compound 10; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 60% methanol-water (volume percent);
wherein, 3 fractions are obtained after the fraction D7g passes through a negative pressure column: d7g1, D7g2, D7g 3; wherein fraction D7g1 was separated by semi-preparative high performance liquid chromatography to give compound 11; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 70% methanol-water (volume percent); wherein fraction D7g3 was separated by semi-preparative high performance liquid chromatography to give compound 12; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-AQ, 78% methanol-water (volume percent);
preferably, the fraction D8 is fractionated by a medium-pressure liquid phase system, and is subjected to gradient elution and TLC detection to obtain 2 fractions: d8a, D8 b; preferably, the medium-pressure liquid phase system is RP-18, Merck, 2.0 × 45cm, and the upper column flow is10 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: 65% and 70% of water (volume percentage); wherein the fraction D8b was partially crystallized to give compound 13; preferably, the crystallization condition is that methanol is dissolved, and the crystallization is carried out after 25 percent by volume to 70 percent by volume of methanol is slowly volatilized in a room temperature environment.
On the other hand, in the above method for preparing dammarane-type triterpene saponin compounds, in the step 5):
mixing the fraction E with silica gel, loading the column, washing the column, eluting until the end, detecting by TLC, and combining to obtain 7 fractions E1, E2, E3, E4, E5, E6 and E7; preferably, the sample mixing silica gel is 80-100 meshes, and the column filling silica gel is 200-300 meshes; preferably, the column washing adopts chloroform: methanol (volume ratio 8: 1, 8: 2); preferably, chloroform, methanol and water are adopted for elution (the volume ratio is 6-8: 2-4: 0.1-1); preferably, the detection condition of thin layer chromatography TLC is (7: 3: 0.5);
preferably, the fraction E2 is sampled by silica gel, loaded on a column, eluted, and detected and combined by a TLC point plate to obtain 9 fractions E2a, E2b, E2c, E2c, E2d, E2E, E2f, E2g and E2 i; preferably, the sample mixing silica gel is 80-100 meshes, and the column filling silica gel is 200-300 meshes; preferably, the elution is performed using chloroform: methanol: water (volume ratio 3: 1: 0.1);
further preferably, fractions E2d were fractionated in a medium pressure liquid phase system, gradient eluted, and combined by TLC to give 3 fractions E2d1, E2d2, and E2d 3; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 2.0 x 45cm, and the flow rate of the upper column is10 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: water is 70%, 75%, 78% and 100% (volume percentage) respectively; wherein said fraction E2d2 is separated by semi-preparative high performance liquid chromatography to give compound 15; preferably, the semi-preparative high performance liquid chromatography specification is YMC-Pack ODS-A, 50% acetonitrile-water (volume ratio);
more preferably, E2f is segmented by a medium-pressure liquid phase system, eluted by gradient, and detected by TLC to be combined to obtain 4 fractions E2f1, E2f2, E2f3 and E2f 4; preferably, the specification of the medium-pressure liquid phase system is RP-18, Merck, 2.0 x 45cm, and the flow rate of the upper column is10 ml/min; preferably, in the solvent system of gradient elution, the ratio of methanol: water is 60%, 65%, 70%, 72%, 75% and 100% (volume percentage) respectively; wherein, E2f3 is separated into compound 16 by semi-preparative high performance liquid chromatography (YMC-Pack ODS-A, 50% acetonitrile-water);
preferably, the semi-preparative HPLC is YMC-Pack ODS-A, 50% acetonitrile-water (by volume).
On the other hand, the dammarane type triterpenoid saponin compound with the structural general formula of the formula (I) and/or the formula (II) is applied to one or more of a sweetening agent or a sweetness enhancer;
preferably, the compound 1, the compound 2, the compound 3, the compound 4, the compound 5, the compound 6, the compound 7, the compound 8, the compound 9, the compound 10, the compound 11, the compound 12, the compound 13, the compound 14 and the compound 15 are used as or used for preparing one or more of a sweetener or a sweetness enhancer.
In still another aspect, a sweetener or sweetness enhancer comprises dammarane-type triterpenoid saponins compound with the structural general formula of formula (I) and/or formula (II);
preferably, the sweetener or sweetness enhancer comprises: one or more of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14 and compound 15.
Compared with the prior art, the dammarane type triterpenoid saponin compound and the preparation method and application thereof provided by the invention have at least the following beneficial effects:
on one hand, 15 new natural products are separated from the Yunnan gynostemma pentaphylla for the first time, and all the products have sweet taste with different degrees, and the sweetness is 10-100 times of that of cane sugar.
On the other hand, the 15 new sweet natural products are sweeteners derived from natural sources, and can be applied to foods, medicines and the like as sweeteners or sweetness enhancers singly or after being combined.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a flow chart of the preparation process of dammarane type triterpene saponin compounds.
Detailed Description
To further explain the technical means and effects of the present invention adopted to achieve the predetermined object, the following detailed description of the embodiments, structures, features and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The invention is further illustrated in detail below by means of specific examples:
example 1:
1. instruments and materials:
specific optical rotation: autopol VI, Serial # 91058; infrared spectrum: thermo NICOLET iS 10; ultraviolet spectrum: UV-2700 series; ESI and HRESI: shimadzu UPLC-IT-TOF liquid phase-ion trap time-of-flight chromatography-mass spectrometry instrument; nuclear magnetic data: BrukeraVANCE III500MH and AV-600, TMS as internal standards, chemical shifts δ in ppm and coupling constants J in HZ.
A rotary evaporator (company EYELA, Japan, and Switzerland Buchi); medium pressure liquid chromatograph (switzerland buchi company); semi-preparative high performance liquid chromatography (Jiangsu Hanbang science and technology Co., Ltd.); a 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 factory); reversed phase silica gel (YMC Co.); reversed phase silica gel plates (Merck, germany); gel Sephadex LH-20 (Swedish GE healthcare); macroporous adsorbent resin D101 (tianjin south china high molecular technology limited); macroporous adsorption resin HP-20 (Technology Co., Ltd., Nanyu south of Tianjin); color developing agent: sulfuric acid-vanillin solution.
2. Preparation of natural sweetener:
pulverizing stem and leaf of herba Gynostemmatis 15kg, reflux extracting with 95% methanol at 50 deg.C for 72 hr to obtain 900g crude extract of herba Gynostemmatis. A normal phase silica gel column is adopted, a solvent system of petroleum ether-acetone (10: 1, 7:1, 3:1, 1: 1), acetone and methanol is adopted for elution, and 900g of crude extract is subjected to primary separation. The 8 fractions were pooled by TLC spotting (Fr1-Fr 8). Fr1-Fr6 was insoluble in water, Fr7 was poorly soluble in water, and Fr8(702.95g) was soluble in water and had a sweet taste, and therefore Fr8 was selected as the target fraction.
Fr8(200g) was dissolved in 200mL of water, and subjected to column chromatography using Diaion HP-20(7.7 cm. times.65.5 cm) and gradient elution using a methanol-water solvent system (0%, 30%, 50%, 70%, 90% M/W). The solvent was recovered by rotary evaporator and combined after TLC detection to give 6 fractions: a (4.70g), B (7.50g), C (13.47g), D (33.70g), E (38.93g), F (0.56 g).
The C (10g) fractions were taken, dissolved in 15mL of water, fractionated using a medium pressure liquid phase system (RP-18, Merck, 4.0X 25cm), eluted with a methanol-water (0%, 30%, 50%, 70%, 100%) gradient and finally combined to give 8 fractions: c1(0.25g), C2(4.58g), C3(0.76g), C4(1.08g), C5(0.38g), C6(0.23g), C7(0.04g), C8(1.05 g).
Fractions of C2(4.58g) were fractionated again using a medium pressure liquid phase system (RP-18, Merck, 2.0X 45cm), eluted with a methanol-water (30%, 40%, 50%, 60%, 100%) gradient and combined by TLC to give 7 fractions: c2a (0.29g), C2b (0.20g), C2C (0.37g), C2d (1.44g), C2e (0.72g), C2f (1.851g), C2g (0.10 g).
C2d (1.44g) was partially separated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 58% methanol-water) to give compounds 4(239mg) and 9(357 mg).
The C2e (0.72g) fraction was subjected to semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 62% methanol-water) to isolate compounds 3(24mg) and 7(13 mg).
Fractions of C4(1.08g) were fractionated using a medium pressure liquid phase system (RP-18, Merck, 2.0X 45cm), eluted with a methanol-water (50%, 52%, 54%, 56%, 58%, 100%) gradient and combined by TLC to give 6 fractions: c4a (0.01g), C4b (0.03g), C4C (0.01g), C4d (0.488g), C4e (0.14g), C4f (0.15 g).
C4e (0.14g) was isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 70% methanol-water) to give compounds 6(39mg) and 14(11 mg).
Fractions of C5(0.38g) were fractionated using a medium pressure liquid phase system (RP-18, Merck, 2.0X 45cm), eluted with a methanol-water (50%, 58%, 54%, 60%, 100%) gradient and combined by TLC to give 5 fractions: c5a (0.04g), C5b (0.04g), C5C (0.06g), C5d (0.10g), C5e (0.07 g). A C5d (0.10g) fraction was isolated using semi-preparative high performance liquid chromatography (YMC-Pack CN, 53% methanol-water) to give compound 1(47 mg).
Fractions of C8(1.05g) were fractionated using a medium pressure liquid phase system (RP-18, Merck, 2.0X 45cm), eluted with a methanol-water (45%, 47%, 49%, 51%, 70%, 100%) gradient and combined by TLC to give 4 fractions: c8a (0.16g), C8b (0.17g), C8C (0.43g), C8d (0.20 g).
Compound 2(216mg) was isolated from the C8C (0.43g) fraction by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 68% methanol-water).
C8d (0.20g) fraction was subjected to semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 70% methanol-water) to isolate Compound 8(136 mg).
A portion of D (30g) was dissolved in 15mL of water, fractionated using a medium pressure liquid phase system (RP-18, Merck, 4.0X 25cm) and eluted with a methanol-water (0%, 30%, 50%, 60%, 70%, 100%) gradient. After concentration by a rotary evaporator, detection is carried out by a TLC spot plate, and 9 fractions are obtained by combination: d1(0.67g), D2(0.69g), D3(0.75g), D4(2.28g), D5(1.91g), D6(21.42g), D7(1.77g), D8(0.37g), D9(1.42 g).
Part of D7(1.70g) was separated on a silica gel column and checked by TLC to give 9 fractions: d7a (0.12g), D7b (0.04g), D7c (0.15g), D7D (0.06g), D7e (0.14g), D7f (0.16g), D7g (0.51g), D7h (0.32g), D7i (0.13 g).
Compound 5(6mg) was isolated from D7b (0.04g) fraction by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 60% methanol-water).
A D7e (0.14g) fraction was subjected to semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 60% methanol-water) to isolate compound 10(13 mg).
Fraction D7g (0.51g) was passed through a counter pressure column to give 3 fractions: d7g1(0.25g), D7g2(0.23g), D7g3(0.07 g).
D7g1(0.25g) was then subjected to semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 70% methanol-water) to isolate compound 11(195 mg).
D7g3(0.07g) was isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-AQ, 78% methanol-water) to give compound 12(12 mg).
Fractions of D8(0.37g) were fractionated using a medium pressure liquid phase system (RP-18, Merck, 2.0X 45cm), eluted with a methanol-water (65%, 70%) gradient and combined by TLC to give 2 fractions: d8a (0.04g), D8b (0.13 g).
D8b (0.13g) was partially crystallized to give compound 13(68 mg).
Mixing the E (38.93g) part with 80-100 mesh silica gel, loading the mixture into a 200-300 mesh silica gel column, washing the column with chloroform-methanol (8: 1, 8: 2), and finally eluting with chloroform-methanol-water (7: 3: 0.5) until the elution is finished. TLC detection and combining gave 7 fractions: e1(1.2g), E3(7.5g), E4(4.4g), E5(3.9g), E6(2.2g), E7(0.3 g).
E2(12g) was sampled with 24g silica gel, packed on a 200-mesh 300-mesh silica gel column and eluted with chloroform: methanol: water (3: 1: 0.1). TLC plates were checked and pooled to give 9 fractions: e2a (1.2g), E2b (1.5g), E2c (2.0g), E2d (2.1g), E2E (1.0g), E2f (1.3g), E2g (0.5g), E2h (1.0g), E2i (0.25 g).
E2d (2.1g) was fractionated using a medium pressure liquid phase system (RP-18, Merck, 2.0X 45cm), eluted with a methanol-water (70%, 75%, 78%, 100%) gradient and combined by TLC to give 3 fractions: e2d1(0.83), E2d2(1.03g), E2d3 (0.24).
E2d2(1.03g) 50mg was collected and isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-A, 50% acetonitrile-water) to give 15(14 mg).
E2f (1.3g) was fractionated using a medium pressure liquid phase system (RP-18, Merck, 2.0X 45cm), eluted with a methanol-water (60%, 65%, 70%, 72%, 75%, 100%) gradient and combined by TLC to give 4 fractions: e2f1(0.12g), E2f2(0.20g), E2f3(0.55g), E2f4 (0.36).
E2f3(0.55g) 50mg was isolated by semi-preparative high performance liquid chromatography (YMC-Pack ODS-A, 50% acetonitrile-water) to give compound 16(10 mg).
3. Physicochemical properties of the sweetener:
the compound 1, a white powder,
(c＝0.102，Methanol)；UV(MeOH，λmax，nm)：202.00(0.260)；IR(KBr)：3428，2973，2926，1631，1450，1380，1312，1260，1156，1082，1049，881，841，805，557，544，532，514，496，478，466，450，435，413cm-1；ESI-MS m/z 1117[M+Na]+；HRESI-MS 1093.5805[M-H]-(pred.for C53H90O23m/z 1093.5800[M-H]-)。
the compound 2, a white powder,
(c＝0.110，Pyridine)；UV(MeOH，λmax，nm)：195.50(0.319)；IR(KBr)：3417，2939，2882，1633，1450，1382，1314，1258，1200，1160，1044，897，840，807，583，531cm-1；ESI-MS m/z 1256[M-H]-；HRESI-MS 1255.6323[M-H]-(pred.for C59H100O28m/z 1255.6328[M-H]-)。
the amount of compound 3, a white powder,
(c＝0.106，Methanol)；UV(MeOH，λmax，nm)：197.00(0.409)；IR(KBr)：3418，2927，1667，1631，1453，1415，1382，1346，1314，1257，1159，1076，1045，892，838，809，593cm-1；ESI-MS m/z 1416[M-H]-；HRESI-MS1415.6706[M-H]-(pred.for C65H108O33m/z 1415.6700[M-H]-)。
the amount of compound 4, a white powder,
(c＝0.108，Methanol)；UV(MeOH，λmax，nm)：202.00(0.214)，268.00(0.012)；IR(KBr)：3406，2928，1635，1450，1416，1384，1314，1258，1199，1159，1076，1046，896，838，809，579cm-1；ESI-MS m/z 1418[M-H]-；HRESI-MS 708.3394[M-2H]2-(pred.for C65H110O33m/z 708.3392[M-2H]2-)。
the amount of compound 5, a white powder,
(c＝0.106，Methanol)；UV(MeOH，λmax，nm)：202.00(0.628)；IR(KBr)：3853，3727，3425，2926，2068，1632，1458，1384，1314，1261，1156，1075，1051，918，894，877，842，808，561，544，452，422cm-1；ESI-MSm/z 1108[M-H]-；HRESI-MS553.2940[M-2H]2-(pred.for C54H92O23 m/z 553.2942[M-2H]2-)。
the compound 6, a white powder,
(c＝0.108，Methanol)；UV(MeOH，λmax，nm)：196.50(0.323)；IR(KBr)：3418，2969，2939，1632，1458，1383，1314，1250，1154，1076，1048，921，894，840，809，574，560，533，468，419，403cm-1；ESI-MSm/z 1093[M-H]-；HRESI-MS 1093.5801[M-H]-(pred.for C53H90O23 m/z 1093.5801[M-H]-)。
the amount of compound 7, a white powder,
(c＝0.104，Methanol)UV(MeOH，λmax，nm)：195.50(0.234)；IR(KBr)：3419，2968，2927，2880，1636，1454，1416，1382，1313，1233，1164，1077，1044，923，891，865，579，467，413cm-1；HRESI-MS 1139.5859[M-H]-(pred.for C54H92O25 m/z 1139.5859[M-H]-)。
the amount of compound 8, a white powder,
(c＝0.107，Pyridine)；UV(MeOH，λmax，nm)：195.50(0.222)；IR(KBr)：3417，2970，2935，1633，1454，1381，1313，1235，1157，1075，1044，894，839，809，575，532，412cm-1；ESI-MS m/z 1256[M-H]-；HRESI-MS 1255.6326[M-H]-(pred.for C59H100O28m/z 1255.6328[M-H]-)。
the amount of compound 9, a white powder,
(c＝0.124，Methanol)；UV(MeOH，λmax，nm)：195.50(0.263)；IR(KBr)：3421，2969，2931，1636，1455，1419，1382，1315，1235，1158，1076，1044，893，865，837，809，622，578，542，467，413，403cm-1；ESI-MS m/z 1418[M-H]-；HRESI-MS 1417.6858[M-H]-(pred.for C65H110O33m/z 1417.6857[M-H]-)。
the amount of compound 10, a white powder,
(c＝0.108，Methanol)；UV(MeOH，λmax，nm)：202.50(0.404)；IR(KBr)：3440，2924，2856，1632，1418，1384，1317，1261，1164，1077，1038，890，563cm-1；ESI-MS m/z 1148[M+Na]+；HRESI-MS 1123.5902[M-H]-(pred.for C54H92O24 m/z 1123.5906[M-H]-)。
the amount of compound 11, a white powder,
(c＝0.110，Methanol)；UV(MeOH，λmax，nm)：202.50(0.495)；IR(KBr)：3441，2970，2925，1638，1453，1425，1384，1318，1262，1162，1075，1045，878，613，411cm-1；ESI-MS m/z 1427[M+Na]+；HRESI-MS 700.3419[M-2H]2-(pred.for C65H110O32m/z 700.3417[M-2H]2-)。
the amount of compound 12, a white powder,
(c＝0.107，Pyridine)；UV(MeOH，λmax，nm)：202.50(0.419)；IR(KBr)：3440，2970，2926，1634，1550，1453，1385，1317，1160，1076，1044，924，894，875，620，569，475，401cm-1；ESI-MS m/z 1386[M-H]-；HRESI-MS 692.8447[M-2H]2-(pred.for C65H110O31m/z 692.8443[M-2H]2-)。
the amount of compound 13, a white powder,
(c＝0.100，Pyridine)；UV(MeOH，λmax，nm)：202.50(0.546)；IR(KBr)：3424，2975，2924，2029，1639，1550，1453，1417，1384，1317，1265，1160，1076，1046，879，620，414cm-1；ESI-MS m/z 1240[M-H]-；HRESI-MS 1239.6373[M-H]-(pred.for C59H100O27m/z1239.6379[M-H]-)。
the amount of compound 14, a white powder,
(c＝0.108，Methanol)；UV(MeOH，λmax，nm)：195.50(0.261)；IR(KBr)：3425，2925，1632，1450，1383，1314，1254，1158，1076，1046，891，839，806，574，467，438cm-1；HRESI-MS 1431.7014[M-H]-(pred.for C66H112O33m/z 1431.7013[M-H]-)。
4. Compound nuclear magnetic data:
table 1 shows compounds 1 to 41H NMR and13c NMR data.
5. The structural formula of the compound:
6. sensory evaluation of taste of monomeric compound isolated from Gynostemma pentaphyllum:
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, and diluting the mother solution to prepare a series of concentration gradient sample solutions with the concentrations of 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.2mg/mL, 0.125mg/mL, 0.1mg/mL, 0.04mg/mL and the like in sequence.
Preparing a sucrose solution: accurately weighing a certain amount of sucrose, preparing into 10mg/mL and 20mg/mL solutions with water, and comparing with the sample to determine the relative sweetness of the compound and the sucrose.
The concentration of the compound C which gives a sweetness equal to that of sucrose was determined by tasting the compound and sucrose solution by a sensory panelCompound (I)，CCompound (I)And CSucroseThe multiple between the two is the relative sweetness of the compound to sucrose.
The sensory panelists tasted each sample until a non-sweet solution was tasted. Concentration C of the sample solutionnAnd the penultimate sample concentration Cn-1The arithmetic mean of (d) is the sweetness threshold of the compound.
Table 4 shows the sensory evaluation of taste of the compounds in gynostemma pentaphylla.
TABLE 4
| Compound (I) | Taste evaluation characteristics | Sweetness level* | Threshold (mg/mL) |
| 1 | Sweet and |
20 | 0.17 |
| 2 | Sweet, slightly astringent | 20 | 0.17 |
| 3 | Sweet and good taste | 100 | 0.03 |
| 4 | Sweet, slightly astringent | 20 | 0.17 |
| 5 | Sweet, bitter and |
30 | 0.07 |
| 6 | Sweet, slightly astringent | 10 | 0.1 |
| 7 | Sweet and |
20 | 0.07 |
| 8 | Sweet and |
10 | 0.3 |
| 9 | Sweet and |
10 | 0.3 |
| 10 | Sweet and slightly |
10 | 0.07 |
| 11 | Sweet, bitter and |
50 | 0.10 |
| 12 | Sweet, slightly astringent | 50 | 0.07 |
| 13 | Sweet and slightly |
30 | 0.1 |
| 14 | The sweet taste is obtained by the following steps of,slightly bitter aftertaste | 80 | 0.03 |
| 15 | Sweet, bitter and |
50 | 0.1 |
| 16 | Bitter taste | - | - |
Note: sweetness: the ratio of the sweetness sample such as sucrose to the sucrose concentration is the relative sweetness of sucrose.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.
Claims (10)
1. The application of dammarane-type triterpenoid saponin compounds in serving as or preparing sweeteners or sweetness enhancers, wherein the structural general formula of the dammarane-type triterpenoid saponin compounds is shown as a formula (I) or a formula (II):
in said formula (I): r1Is H or beta-D-xylosyl- (1 → 2) -beta-D-glucosyl; r2Is any one of OH, H and O; r3Is H, OH, OMe, OOH, or a sugar chain formed with any one or more of beta-D-xylosyl, beta-D-glucosyl, alpha-L-rhamnosyl, beta-D-glucuronyl; wherein,the dotted line in formula (I) is a double bond or a single bond;
in said formula (II): r1Is H or beta-D-glucosyl; r2Is H or acetyl; r3Is any one of H, beta-D-glucosyl, beta-D-xylosyl, alpha-L-rhamnosyl, beta-D-xylosyl- (1 → 3) -alpha-L-rhamnosyl, alpha-L-rhamnosyl- (1 → 2) -beta-D-xylosyl or beta-D-glucosyl- (1 → 3) -alpha-L-rhamnosyl; r4Is any one of OH, H and O; wherein the dotted line in formula (II) is a double bond or a single bond;
wherein the dammarane type triterpenoid saponin compound is any one of the following compounds:
2. the method for preparing dammarane-type triterpene saponin compounds according to claim 1, wherein the dammarane-type triterpene saponin compounds are obtained by separating from gynostemma pentaphylla;
the preparation method comprises the following steps:
1) pulverizing stem and leaf of herba Gynostemmatis, and extracting with ethanol to obtain crude extract of herba Gynostemmatis; performing column separation on the gynostemma pentaphylla crude extract, performing spot-plate combination on flow components, and determining a target separation section;
2) dissolving the target separation section, performing column chromatography separation and gradient elution, recovering the solvent, and combining the fractions to obtain fraction A, fraction B, fraction C, fraction D, fraction E and fraction F;
3) after the separation treatment is carried out on the flow part C, a compound 4, a compound 9, a compound 3, a compound 7, a compound 6, a compound 14, a compound 1, a compound 2 and a compound 8 can be obtained;
4) after the fraction D is subjected to separation treatment, a compound 5, a compound 10, a compound 11, a compound 12 and a compound 13 can be obtained;
5) after the fraction E is subjected to separation treatment, a compound 15 and a compound 16 can be obtained;
wherein the gynostemma pentaphylla is produced in Yunnan province; the separation part is the overground part of gynostemma pentaphylla.
3. The method for preparing dammarane-type triterpene saponin compounds according to claim 2, wherein in the step 1):
extracting ground herba Gynostemmatis aerial part with 65-100% methanol-water solution or 65-100% ethanol-water solution at 30-70 deg.C under reflux to obtain herba Gynostemmatis crude extract.
4. The method for preparing dammarane-type triterpene saponin compounds according to claim 3,
the column separation adopts a normal phase silica gel column, and the silica gel is 200-300 meshes; wherein, when the normal phase silica gel column is used for separation, a petroleum ether-acetone gradient elution solvent system is adopted; in the gradient system, petroleum ether: acetone was added in a volume ratio of 10:1, 7:1, 3:1 and 1:1, acetone was added in a gradient of 20L per 200g of crude extract, and finally 10L of methanol was used for elution.
5. The method for preparing dammarane-type triterpene saponin compounds according to claim 3,
the point-plate combined flow treatment comprises the following steps: and (3) carrying out thin-layer chromatography (TLC) spot plate treatment on the fractions after column separation, and combining to obtain 8 fractions which are respectively: fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr 8; the fraction Fr8 having a sweet taste was selected as a target fraction.
6. The method for preparing dammarane-type triterpene saponin compounds according to claim 3, wherein in the step 2):
dissolving the target separation section in water according to the mass-volume ratio (1-4):1g/ml, and then performing column chromatography separation by using macroporous adsorption resin.
7. The method for preparing the dammarane-type triterpene saponin compound according to claim 6, wherein the macroporous adsorption resin is Diaion HP-20 with a specification of 7.7cm x 65.5 cm.
8. The method for preparing dammarane-type triterpene saponin compounds according to claim 6, wherein in a solvent system of gradient elution, methanol: the water is 0 percent, 10-30 percent, 30-50 percent, 50-70 percent and 70-100 percent by volume percentage respectively, and each solution has 8L of gradient.
9. The method for preparing dammarane-type triterpene saponin compounds according to claim 6,
after removing the organic solvent by rotary evaporation, detecting by thin layer chromatography TLC, and combining to obtain stream A, stream B, stream C, stream D, stream E and stream F.
10. A sweetener or sweetness enhancer comprising the dammarane-type triterpene saponin compound of claim 1;
wherein the sweetener or sweetness enhancer comprises: one or more of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14 and compound 15.
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