Cyclocarya paliurus mogroside as well as extraction method and application thereof

Abstract

The invention relates to plant extraction, and discloses cyclocarya paliurus mogroside as well as an extraction method and application thereof. The cyclocarya paliurus mogroside is selected from at least one of a compound as shown in a formula I, a compound as shown in a formula II, a compound as shown in a formula III, a compound as shown in a formula IV and a compound as shown in a formula V. The cyclocarya paliurus mogroside has high sweetness and can be used as a sweetening agent. # imgabs0 #

Description

Technical Field

[0001] The invention relates to plant extraction, and in particular to cyclocarya paliurus glycoside and an extraction method and application thereof. Background Art

[0002] Cyclocarya paliurus (Batal.) Iljinskaja is a deciduous tree of the genus Cyclocarya in the family Juglandaceae. It is a very unique tree species that survived the Ice Age and is one of the rare plants unique to my country. It is also known as the money tree and sweet tea tree because of its coin-like fruits and sweet leaves. It is widely distributed in Hunan, Jiangxi, Zhejiang, Anhui, Guangdong and other areas south of the Yangtze River. Modern pharmacological studies have shown that Cyclocarya paliurus has hypoglycemic and hypolipidemic, anti-cancer, antioxidant, anti-inflammatory, hepatoprotective and antibacterial activities.

[0003] So far, more than 210 secondary metabolites have been extracted and isolated from Cyclocarya paliurus. Through phytochemical research, the main chemical components of Cyclocarya paliurus include dammarane saponins, flavonoids, and phenolic acids, among which dammarane triterpenes are the indicators and active ingredients of Cyclocarya paliurus. The sweet compounds of Cyclocarya paliurus reported so far include cyclocarioside A and cyclocarioside I, whose sweetness is about 200 and 250 times that of sucrose, respectively. This type of compound is a 3,4-closed-ring dammarane triterpenoid, which is a novel type of triterpenoid and glycoside formed by condensing the hydroxyl groups at the C-20 and C-24 positions into ethers on the basis of the dammarane triterpene skeleton, and replacing the hydroxyl groups at C-3, C-11 or C-12 with sugars. However, the sweet components of Cyclocarya paliurus have yet to be developed.

[0004] The compound represented by Formula IV of the present invention is similar in structure to the sweet compounds cyclocarioside A and cyclocarioside I. In addition, a single crystal structure of this sweetener is obtained for the first time. Electronic tongue measurement shows that the compounds represented by Formula I and Formula III in large quantities have sweet taste, and the compound represented by Formula IV is molecularly docked with the sweet taste receptor TIR2 / TIR3 to clarify its sweetness mechanism. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem in the prior art that the sweet components of Cyclocarya paliurus have yet to be developed, and to provide a Cyclocarya paliurus glycoside and an extraction method and application thereof. The Cyclocarya paliurus glycoside has a sweet taste and can be used as a sweetener.

[0006] In order to achieve the above object, the first aspect of the present invention provides a cyclocarya paliurus glycoside, wherein the cyclocarya paliurus glycoside is selected from:

[0007]

[0008]

[0009] At least one of .

[0010] The second aspect of the present invention provides a method for extracting the above-mentioned Cyclocarya paliurus glycoside, comprising the following steps:

[0011] (1) extracting the leaves of Cyclocarya paliurus and a mixed solution of ethanol and water to obtain an extract, and concentrating the extract to obtain a total extract;

[0012] (2) dispersing the total extract with water, and then extracting with petroleum ether, chloroform and n-butanol to obtain a petroleum ether layer, a chloroform layer, an n-butanol layer and an aqueous layer;

[0013] (3) The chloroform layer is separated to obtain the Cyclocarya paliurus extract.

[0014] A third aspect of the present invention provides a cyclocarya paliurus glycoside extracted by the above extraction method.

[0015] A fourth aspect of the present invention provides a use of the above-mentioned Cyclocarya paliurus glycoside in a sweetener.

[0016] Through the above technical solution, the present invention has the following effective effects:

[0017] The invention extracts five new compounds from Cyclocarya paliurus, which have high sweetness and can be used as sweeteners, and the compounds shown in formula I are single crystal structures. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the HR-ESI-MS spectrum of the compound represented by Formula I; Figure 2 is a compound of formula I 1 H-NMR spectrum; Figure 3 is a compound of formula I 13 C-NMR spectrum; Figure 4 is a compound of formula I 1 H- 1 H COSY spectrum; Figure 5 is the HSQC spectrum of the compound shown in Formula I; Figure 6 is the HMBC spectrum of the compound shown in Formula I; Figure 7 is the ROESY relationship spectrum of the compound represented by formula I;

[0019] Figure 8 is a single crystal diffraction pattern of the compound represented by formula I;

[0020] Fig. 9is the HR-ESI-MS spectrum of the compound represented by formula II; Fig.10 is a compound of formula II 1 H-NMR spectrum; Fig.11 is a compound of formula II 13 C-NMR spectrum; Fig.12 is a compound of formula II 1 H- 1 H COSY spectrum; Fig.13 is the HSQC spectrum of the compound represented by formula II; Fig.14 is the HMBC spectrum of the compound represented by formula II; Fig.15 is the ROESY relationship spectrum of the compound represented by formula II;

[0021] Fig.16 is the HR-ESI-MS spectrum of the compound represented by formula III; Fig.17 is a compound of formula III 1 H-NMR spectrum; Fig.18 is a compound of formula III 13 C-NMR spectrum; Fig.19 is a compound of formula III 1 H- 1 H COSY spectrum; Fig. 20 is the HSQC spectrum of the compound represented by formula III; Fig.21 is the HMBC spectrum of the compound represented by formula III; Fig. 22 is the ROESY relationship spectrum of the compound represented by formula III;

[0022] Fig.23 is the HR-ESI-MS spectrum of the compound represented by Formula IV; Fig.24 is a compound of formula IV 1 H-NMR spectrum; Fig.25 is a compound of formula IV 13 C-NMR spectrum; Fig.26 is a compound of formula IV 1 H- 1 H COSY spectrum; Fig. 27 is the HSQC spectrum of the compound represented by formula IV; Fig.28 is the HMBC spectrum of the compound represented by formula IV; Fig.29 is the ROESY relationship spectrum of the compound represented by formula IV;

[0023] Fig.30 is the HR-ESI-MS spectrum of the compound represented by Formula V; Fig.31 is a compound of formula V 1 H-NMR spectrum; Fig.32 is a compound of formula IV13 C-NMR spectrum; Fig.33 is a compound of formula V 1 H- 1 H COSY spectrum; Fig.34 is the HSQC spectrum of the compound represented by Formula V; Fig.35 is the HMBC spectrum of the compound represented by formula V; Fig.36 is the ROESY relationship spectrum of the compound represented by formula V;

[0024] Fig.37 It is a structural diagram of the homology model of T1R2 and T1R3; Fig.38 are the Ramachandran plots of the homology models of T1R2 (A) and T1R3 (B); Fig.39 DoGSiteScorer predicts the active pockets of T1R2 and T1R3; Fig.40 is a molecular docking result diagram of the compound represented by formula IV and sweet taste receptors T1R2 and T1R3;

[0025] Fig.41 is a microscopic image of the compound shown in Formula I; Fig.42 is a picture of the compound represented by formula I. DETAILED DESCRIPTION

[0026] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0027] As described above, the first aspect of the present invention provides a Cyclocarya paliurus extract, wherein the Cyclocarya paliurus glycoside is selected from at least one of the compounds shown in Formula I, the compounds shown in Formula II, the compounds shown in Formula III, the compounds shown in Formula IV and the compounds shown in Formula V.

[0028] Specifically, the cyclocarya paliurus glycoside can be a compound shown in formula I, a compound shown in formula II, a compound shown in formula III, a compound shown in formula IV, or a compound shown in formula V, or a mixture of two of the compounds shown in formula I, the compounds shown in formula II, the compounds shown in formula III, the compounds shown in formula IV, and the compounds shown in V, or a mixture of three of the compounds shown in formula I, the compounds shown in formula II, the compounds shown in formula III, the compounds shown in formula IV, and the compounds shown in V, or a mixture of four of the compounds shown in formula I, the compounds shown in formula II, the compounds shown in formula III, the compounds shown in formula IV, and the compounds shown in V, or a mixture of the compounds shown in formula I, the compounds shown in formula II, the compounds shown in formula III, the compounds shown in formula IV, and the compounds shown in V. Preferably, the cyclocarya paliurus glycoside is a compound shown in formula I, a compound shown in formula II, a compound shown in formula III, a compound shown in formula IV, or a compound shown in formula V.

[0029] The inventors discovered during the research process that the present invention extracted five new compounds from Cyclocarya paliurus, which have high sweetness and can be used as sweeteners.

[0030] Preferably, the compound represented by formula I is a crystalline crystal. The present invention first discovered that the compound represented by formula I is a crystalline crystal, and the compound has a high sweetness.

[0031] In a second aspect, the present invention provides a method for extracting cyclocarya paliurus glycosides, comprising the following steps:

[0032] (1) extracting the leaves of Cyclocarya paliurus and a mixed solution of ethanol and water to obtain an extract, and concentrating the extract to obtain a total extract;

[0033] (2) dispersing the total extract with water, and then extracting with petroleum ether, chloroform and n-butanol to obtain a petroleum ether layer, a chloroform layer, an n-butanol layer and an aqueous layer;

[0034] (3) separating the chloroform layer to obtain the cyclocarya paliurus glycoside;

[0035] The cyclocarya paliurus glycoside is selected from at least one of the compounds shown in formula I, the compounds shown in formula II, the compounds shown in formula III, the compounds shown in formula IV and the compounds shown in formula V.

[0036] According to the present invention, in step (1), the leaves of Cyclocarya paliurus can be purchased commercially or collected. They can be dried leaves of Cyclocarya paliurus or fresh leaves of Cyclocarya paliurus. Preferably, they are dried leaves of Cyclocarya paliurus. Before extraction, the leaves of Cyclocarya paliurus can be crushed to improve the extraction efficiency of the effective ingredients in the leaves of Cyclocarya paliurus. In step (2), the dispersion method can be a conventional dispersion method, such as stirring, ultrasound, vibration, etc., preferably ultrasound, and the specific ultrasonic dispersion conditions can be determined by those skilled in the art according to actual conditions. The amount of petroleum ether, chloroform and n-butanol added can be determined by those skilled in the art according to actual conditions. Preferably, relative to 1g of total extract, the amount of petroleum ether added is 1-7mL, the amount of chloroform added is 3-9mL, and the amount of n-butanol added is 2-8mL. In a specific embodiment of the present invention, relative to 1g of total extract, the amount of petroleum ether added is 4mL, the amount of chloroform added is 6mL, and the amount of n-butanol added is 5mL.

[0037] During the research process, the inventors found that five new compounds were extracted from Cyclocarya paliurus in the present invention, which have high sweetness and can be used as sweeteners.

[0038] According to the present invention, the extraction method can adopt any feasible extraction method, such as high pressure extraction method, ultrasonic extraction method, etc. Preferably, in step (1), the extraction is percolation extraction. This method can further improve the extraction efficiency of the effective ingredients. From the perspective of further improving the extraction efficiency of the effective ingredients, preferably, the percolation extraction conditions include: a temperature of 10-40°C, specifically 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or any value between the two values; a time of 3-10 days, specifically 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any value between the two values. Further preferably, the percolation extraction conditions include: a temperature of 20-30°C and a time of 6-8 days. As a specific embodiment of the present invention, the percolation extraction conditions include: a temperature of 25°C and a time of 7 days.

[0039] The mass ratio of the Cyclocarya paliurus leaves to the ethanol-water mixture can be determined by a person skilled in the art according to actual conditions. In order to further improve the extraction efficiency, preferably, in step (1), the mass ratio of the Cyclocarya paliurus leaves to the ethanol-water mixture on a dry weight basis is 1:10-17, specifically 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or any value between the above two values.

[0040] The ethanol content in the ethanol-water mixture can be determined by those skilled in the art according to actual conditions. In order to further improve the extraction effect, preferably, in step (1), the ethanol content in the ethanol-water mixture is 90-98% by volume.

[0041] Preferably, in step (1), the concentration conditions include: a temperature of 40-60° C., more preferably 45-55° C. and a concentration time of 0.5-2 days, preferably 0.8-1.5 days.

[0042] Preferably, in step (2), the mass ratio of the total extract to the water is 1:0.5-2. Under this mass ratio condition, the total extract has a better dispersion effect in water, which is convenient for the subsequent extraction of active ingredients and further improves the extraction efficiency.

[0043] Preferably, the method further comprises, before step (3), concentrating the chloroform layer into a chloroform layer extract, and then subjecting the chloroform layer extract to separation treatment to obtain the Cyclocarya paliurus extract. The specific concentration conditions can be determined by those skilled in the art according to actual conditions.

[0044] In order to further improve the separation effect, preferably, in step (3), the separation includes: S1, separating the chloroform layer through a silica gel column I, and gradient eluting with a dichloromethane / methanol system to obtain nineteen fractions of Fr. (A) to Fr. (S); S2, purifying the Fr. (P) fraction.

[0045] According to the present invention, in step S1, the step of gradient elution using a dichloromethane / methanol system is to perform gradient elution using a dichloromethane / anhydrous methanol system with a volume ratio of 200:1 to 0:1 (without adding dichloromethane).

[0046] Preferably, in step S2, the purification comprises: separating the Fr.(P) fraction by silica gel column II-1, and eluting with petroleum ether / ethyl acetate / methanol system gradient to obtain sixteen fractions of Fr.(P)1-Fr.(P)16; separating the Fr.(P)8 fraction by silica gel column II-2, and eluting with dichloromethane / methanol system gradient to obtain six fractions of Fr.(P)8.1-Fr.(P)8.6; separating the Fr.(P)8.4 fraction by ODS reverse phase column II-3, and eluting with methanol / water system gradient to obtain six fractions of Fr.(P)8.4.1-Fr.(P)8.4.6; The Fr.(P)8.4.4 fraction was separated into II-4 using a semi-preparative HPLC-evaporative light scattering detector, and isocratically eluted with acetonitrile / water to obtain the compound of formula III; the Fr.(P)9 fraction was separated into III-1 using a silica gel column, and gradient eluted with a dichloromethane / anhydrous methanol system to obtain five fractions from Fr.(P)9.1 to Fr.(P)9.5; the Fr.(P)9.4 fraction was separated into III-2 using a gel column, and eluted with methanol to obtain four fractions from Fr.(P)9.4.1 to Fr.(P)9.4.4; the Fr.(P)9.4.3 fraction was separated into III-3 using a silica gel column, and isocratically eluted with dichloromethane / anhydrous methanol to obtain the compound of formula III; The elution was performed by gradient elution with methyl chloride / anhydrous methanol system to obtain five fractions Fr.(P)9.4.3.1-Fr.(P)9.4.3.5; the Fr.(P)9.4.3.2 fraction was separated by ODS reverse phase column III-4, and gradient eluted with methanol / water system to obtain the compound of formula II; the Fr.(P)9.4.3.3 fraction was separated by natural crystallization to obtain the compound of formula I; the Fr.(P)9.3 fraction was separated by gel column III-5, and eluted with methanol to obtain four fractions Fr.(P)9.3.1-Fr.(P)9.3.4; the Fr.(P)9 ... The fraction III-6 is separated by silica gel column, and gradient eluted with dichloromethane / anhydrous methanol system to obtain four fractions Fr.(P)9.3.2.1-Fr.(P)9.3.2.4; the Fr.(P)9.3.2.2 fraction is separated by ODS reverse phase column III-7, and gradient eluted with methanol / water system, and then separated by semi-preparative high performance liquid phase-evaporative light scattering detector III-8, and isocratically eluted with acetonitrile / water, isocratically eluted to obtain the compound shown in formula IV; the Fr.(P)9.3.2.3 fraction is separated by ODS reverse phase column III-9, and gradient eluted with methanol / water system to obtain the compound shown in formula V. The above method can accurately extract the corresponding compound and improve the purity of the extracted compound.

[0047] According to the present invention, in a petroleum ether / ethyl acetate / methanol system, the volume ratio of petroleum ether, ethyl acetate and methanol is 1:1:0 to 0:0:1; in a dichloromethane / methanol system, the volume ratio of dichloromethane and methanol is 1:0 to 0:1; in a methanol / water system, the volume ratio of methanol and water is 3:2 to 1:0; in a dichloromethane / methanol system, the volume ratio of dichloromethane and methanol is 1:0 to 0:1; in acetonitrile / water, the volume ratio of acetonitrile and water is 0.75:0.25.

[0048] According to the present invention, silica gel column separation is a column chromatography silica gel separation method disclosed in the prior art. Gel column separation is also a column chromatography gel separation method disclosed in the prior art. ODS reverse phase column separation is a column chromatography ODS reverse phase separation method disclosed in the prior art. Semi-preparative high performance liquid phase-evaporative light scattering detector separation.

[0049] The third aspect of the present invention provides a cyclocarya paliurus glycoside extracted by the above extraction method. The cyclocarya paliurus glycoside has a sweet taste.

[0050] A fourth aspect of the present invention provides a use of the above-mentioned Cyclocarya paliurus glycoside in a sweetener.

[0051] The above-mentioned Cyclocarya paliurus glycoside has a relatively high sweetness. The present invention extracts five new compounds from Cyclocarya paliurus, which have a relatively high sweetness and can be used as a sweetener.

[0052] According to a particularly preferred embodiment of the present invention, a method for extracting a Cyclocarya paliurus extract is provided, comprising the following steps:

[0053] (1) extracting the leaves of Cyclocarya paliurus and a mixed solution of ethanol-water having an ethanol content of 90-98% by volume at a mass ratio of 1:10-17 to obtain an extract, and concentrating the extract at a temperature of 40-60° C. for 0.5-2 days to obtain a total extract;

[0054] (2) dispersing the total extract with water (the mass ratio of the total extract to water is 1:0.5-2), and extracting with petroleum ether, chloroform and n-butanol in sequence to obtain a petroleum ether layer, a chloroform layer, an n-butanol layer and a water layer;

[0055] (3) The chloroform layer was separated by silica gel column I, and gradient eluted with dichloromethane / anhydrous methanol system with a volume ratio of 200:1 to 0:1, and 5% concentrated vanillin sulfate as a color developer, and TLC thin layer analysis was performed to obtain 19 fractions of Fr. (A) to Fr. (S);

[0056] The Fr.(P) fraction was separated into II-1 by a silica gel column, and gradient eluted with a petroleum ether / ethyl acetate / methanol system with a volume ratio of 1:1:0 to 0:0:1 to obtain sixteen fractions of Fr.(P)1-Fr.(P)16; the Fr.(P)8 fraction was separated into II-2 by a silica gel column, and gradient eluted with a dichloromethane / anhydrous methanol system with a volume ratio of 1:0 to 0:1 to obtain six fractions of Fr.(P)8.1-Fr.(P)8.6; the The Fr.(P)8.4 fraction was separated from II-3 using an ODS reverse phase column, and gradient eluted using a methanol / water system with a volume ratio of 3:2 to 1:0 to obtain six fractions Fr.(P)8.4.1-Fr.(P)8.4.6; the Fr.(P)8.4.4 fraction was separated from II-4 using a semi-preparative high performance liquid chromatography-evaporative light scattering detector, and isocratically eluted using acetonitrile / water (the volume ratio of acetonitrile to water was 0.75:0.25) to obtain a compound of formula III;

[0057] The Fr.(P)9 fraction was separated into III-1 by a silica gel column and eluted with a dichloromethane / anhydrous methanol system with a volume ratio of 1:0 to 0:1 to obtain five fractions Fr.(P)9.1-Fr.(P)9.5; the Fr.(P)9.4 fraction was separated into III-2 by a gel column and eluted with methanol to obtain four fractions Fr.(P)9.4.1-Fr.(P)9.4.4; the Fr.(P) The 9.4.3 fraction was separated from III-3 by a silica gel column, and gradient eluted with a dichloromethane / anhydrous methanol system with a volume ratio of 1:0 to 0:1 to obtain five fractions Fr.(P)9.4.3.1-Fr.(P)9.4.3.5; the Fr.(P)9.4.3.2 fraction was separated from III-4 by an ODS reverse phase column, and gradient eluted with a methanol / water system with a volume ratio of 3:2 to 1:0 to obtain the compound of formula II;

[0058] The Fr.(P)9.4.3.3 fraction is separated by natural crystallization to obtain the compound of formula I;

[0059] The Fr.(P)9.3 fraction is separated from III-5 by a gel column and eluted with methanol to obtain four fractions of Fr.(P)9.3.1-Fr.(P)9.3.4; the Fr.(P)9.3.2 fraction is separated from III-6 by a silica gel column and gradient eluted with a dichloromethane / anhydrous methanol system with a volume ratio of 1:0 to 0:1 to obtain four fractions of Fr.(P)9.3.2.1-Fr.(P)9.3.2.4; the Fr.(P)9.3.2.2 fraction is separated from III-7 by an ODS reverse phase column and gradient eluted with a methanol / water system with a volume ratio of 3:2 to 1:0, and then separated from III-8 by a semi-preparative high performance liquid phase-evaporative light scattering detector, and isocratically eluted with acetonitrile / water (the volume ratio of acetonitrile to water is 0.75:0.25) to obtain the compound of formula IV;

[0060] The Fr.(P)9.3.2.3 fraction was separated into III-9 by an ODS reverse phase column, and gradient eluted with a methanol / water system with a volume ratio of 3:2 to 1:0 to obtain a compound of formula V.

[0061] The compounds extracted by the above extraction method, five new compounds extracted from Cyclocarya paliurus in the present invention, have high sweetness and can be used as sweeteners.

[0062] The present invention will be described in detail below through examples. In the following examples, the Cyclocarya paliurus leaf medicinal material was collected from Huaihua City, Hunan Province in June 2015, and was identified as the dried leaf part of Cyclocarya paliurus of the genus Cyclocarya of the Juglandaceae by Professor Wang Wei of the International Joint Laboratory of Traditional Chinese Medicine and Ethnic Medicine of Hunan University of Chinese Medicine. The medicinal material specimen (20150629) is currently stored in the International Joint Laboratory of Traditional Chinese Medicine and Ethnic Medicine of Hunan University of Chinese Medicine.

[0063] The three-purpose ultraviolet analyzer was purchased from Shanghai Baoshan Gucun Electro-Optical Instrument Factory, and the instrument model is ZF-1; the Fourier transform infrared spectrometer was purchased from Thermo Fisher Scientific, and the instrument model is Nicolet iS5; the ultraviolet visible spectrophotometer was purchased from Shimadzu Corporation of Japan, and the instrument model is UV-1900i; the polarimeter was purchased from Rudolph Corporation of the United States, and the instrument model is AUTOPOLⅢ; the semi-preparative high performance liquid chromatograph was purchased from Beijing Qingbohua Technology Co., Ltd., and the instrument model is SEP LC-52; the evaporative light scattering detector was purchased from Wuxi Senar Instrument Equipment Manufacturing Co., Ltd., and the instrument model is E800; the nuclear magnetic resonance instrument was purchased from Bruker AG of Switzerland, and the instrument model is BRUKER-600; the mass spectrometer was purchased from Thermo Fisher Scientific, and the instrument model is OrbitrapExploris 120; the semi-preparative chromatographic column model is Fisher Wharton (10 mm × 250 mm, 5 μm), taste analysis system-SA402B electronic tongue was purchased from Japan insent Company, the instrument model is SA402B.

[0064] Petroleum ether, ethyl acetate, dichloromethane and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; chloroform was purchased from Hunan Huihong Reagent Co., Ltd.; chromatographic acetonitrile was purchased from Shanghai Sigma-Aldrich Trading Co., Ltd.; deuterated methanol was purchased from Cambridge Isotope Laboratory, USA; thin layer chromatography silica gel plates were purchased from Yantai Jiangyou Silica Gel Development Co., Ltd., with a specification of HSGF254 (5×10 cm); column chromatography silica gel was purchased from Qingdao Ocean Chemical Co., Ltd., with a specification of 80-100 mesh / 300-400 mesh.

[0065] Example 1

[0066] (1) 20 kg of dried Cyclocarya paliurus leaves were crushed, and then mixed with 270 L of ethanol-water mixture with an ethanol content of 95% by volume to obtain an extract, and the extract was concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 50° C. to obtain 5 kg of a black total extract;

[0067] (2) After the total extract was dispersed with 5 L of water, it was extracted with 20 L of petroleum ether, 30 L of chloroform and 25 L of n-butanol in sequence to obtain 0.53 kg of petroleum ether layer, 2.57 kg of chloroform layer, 1.21 kg of n-butanol layer and 0.56 kg of water layer;

[0068] (3) the chloroform layer was concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 50° C. to obtain 0.98 kg of chloroform layer extract;

[0069] (4) The chloroform extract was mixed into a 1.3 kg silica gel column (80-100 mesh), subjected to silica gel column chromatography (4.5 kg 300-400 mesh silica gel loading), and gradient eluted with a dichloromethane / anhydrous methanol system with a volume ratio of 200:1 to 0:1 to obtain 19 fractions of Fr.(A)-Fr.(S);

[0070] 41.0 g of Fr.(P) fraction was subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate / methanol in a volume ratio of 1:1:0 to 0:0:1 to obtain sixteen fractions of Fr.(P)1-Fr.(P)16; 1.5 g of Fr.(P)8 fraction was subjected to silica gel column chromatography and eluted with a gradient of dichloromethane / anhydrous methanol in a volume ratio of 1:0 to 0:1 to obtain six fractions of Fr.(P)8.1-Fr.(P)8.6; 570 mg of Fr.(P)8.4 fraction was subjected to ODS reverse phase column chromatography and eluted with a gradient of methanol / water in a volume ratio of 3:2 to 1:0 to obtain six fractions of Fr.(P)8.4.1-Fr.(P)8.4.6; 86.5 mg of The fraction Fr.(P)8.4.4 was detected by semi-preparative HPLC-evaporative light scattering detector (3.0 mL / min, acetonitrile:water volume ratio of 0.75:0.25, t R =9.6min), isocratically eluted to obtain 25.0mg of the compound represented by formula III ((3α,11α,25)-trihydroxy-(20S,24R)-epoxydammar-3-O-α-L-arabinopyranosyl-11-O-β-D-pyranose glycoside);

[0071] 6.9 g of Fr.(P)9 fraction was subjected to silica gel column chromatography and gradient eluted with a dichloromethane / anhydrous methanol system in a volume ratio of 1:0 to 0:1 to obtain five fractions Fr.(P)9.1-Fr.(P)9.5; 2.5 g of Fr.(P)9.4 fraction was subjected to gel column chromatography and eluted with methanol to obtain four fractions Fr.(P)9.4.1-Fr.(P)9.4.4; 2.1 g of Fr.(P)9.4.3 fraction was subjected to silica gel column chromatography and gradient eluted with a dichloromethane / anhydrous methanol system in a volume ratio of 1:0 to 0:1 to obtain five fractions Fr.(P)9.4.3.1-Fr.(P)9.4.3.5; 540 mg The fraction Fr.(P)9.4.3.2 was subjected to ODS reverse phase column chromatography and gradient eluted with a methanol / water system with a volume ratio of 3:2 to 1:0 to obtain 10 mg of the compound represented by formula II ((3α,11α,25)-trihydroxy-(20S,24R)-epoxydammar-3-O-α-L-(5′-O-acetyl)-arabinofuranosyl-11-O-β-D-pyranose);

[0072] 120 mg of the Fr.(P)9.4.3.3 fraction was separated after natural crystallization to obtain 30 mg of the compound represented by formula I ((3α,11α,25)-trihydroxy-(20S,24R)-epoxydammar-3-O-α-L-furanosyl-11-O-β-D-pyranose);

[0073] 2.9 g of Fr.(P)9.3 fraction was subjected to gel column chromatography and eluted with methanol to obtain four fractions Fr.(P)9.3.1-Fr.(P)9.3.4; 2.05 g of Fr.(P)9.3.2 fraction was subjected to silica gel column chromatography and gradient eluted with a dichloromethane / anhydrous methanol system with a volume ratio of 1:0 to 0:1 to obtain four fractions Fr.(P)9.3.2.1-Fr.(P)9.3.2.4; 774 mg of Fr.(P)9.3.2.2 fraction was subjected to ODS reverse phase column chromatography and gradient eluted with a methanol / water system with a volume ratio of 3:2 to 1:0, and then continuously eluted with a semi-preparative high performance liquid chromatography-evaporative light scattering detector (3.0 mL / min, acetonitrile / water system with a volume ratio of 0.75:0.25, t R =20.3min), and eluted isocratically to obtain the compound represented by formula IV ((3α,11α,25)-trihydroxy-(20S,24R)-epoxydam-3-O-α-L-(4′-O-acetyl)-arabinopyranosyl-11-O-β-D-pyranose);

[0074] 500 mg of Fr.(P)9.3.2.3 fraction was subjected to ODS reverse phase column chromatography and gradient eluted with a methanol / water system with a volume ratio of 3:2 to 1:0 to obtain 10 mg of the compound represented by formula V ((3α,11α,25)-trihydroxy-(20S,24R)-epoxydammar-3-O-α-L-(4′-O-acetyl)-pyranose-11-O-β-D-pyranose).

[0075] Test Example 1

[0076] Characterization of the compound shown in Formula I:

[0077] The compound is a white powder. The TLC color reaction of 5% vanillin-concentrated sulfuric acid is yellow-green. The UV spectrum of this compound has no obvious ultraviolet absorption in the range of 200-400nm, indicating that this compound has no chromophore; the IR spectrum shows that the -1 and 2962cm -1 There are absorptions at the positions respectively, indicating that the compound contains hydroxyl, methyl and methylene; HR-ESI-MS spectrum ( Figure 1 ) shows its quasi-molecular ion peak m / z 753.4797[MH] -(calcd.for C 41 H 69 O 12 - ,753.4795), it can be inferred that the molecular structure of the compound is C 41 H 70 O 12 , the unsaturation is 7.

[0078] like Figure 2 As shown, the compound 1 The H-NMR spectrum (600 MHz, CD3OD) showed eight angular methyl singlet proton signals as [δ H 1.23 (3H, s, H-27), 1.16 (3H, s, H-21), 1.15 (3H, s, H-26), 1.09 (3H, s, H-19), 1.00 (3H, s, H-18), 0.97 (3H, s, H-30), 0.95 (3H, s, H-28) and 0.89 (3H, s, H-29)]. In addition, there are a series of oxygen-containing methyl groups [δ H 4.93 (d, J = 1.4 Hz, H-1′), 4.34 (d, J = 7.8 Hz, H-1″), 4.00 (dd, J = 3.7, 1.4 Hz, H-2′), 3.95 (overlap, H-4′), 3.83 (dd, J = 6.8, 3.7 Hz, H-3′), 3.27 (t-like, J = 9.1 Hz, H-3″), 3.24 (overlap, H-5″), 3.09 (dd, J = 9.1, 7.8 Hz, H-2″) and 2.97 (t-like, J = 9.1 Hz, H-4″)]; one oxymethylene δ H 3.75 (dd, J = 11.9, 3.1 Hz, H-5′a) and 3.63 (dd, J = 11.9, 5.4 Hz, H-5′b), a methyl δ H 1.26 (d, J = 6.1 Hz, H-6″), indicating the presence of pentose and hexose. The compound was hydrolyzed with acid, and the sugar part was further derivatized and analyzed by HPLC and compared with standard sugar derivatives. It was determined that the compound contained L-arabinose and D-chinchonose.

[0079] like Figure 3 As shown, the compound 13 The C-NMR spectrum (150 MHz, CD3OD) showed 41 carbon signals, which, combined with the DEPT-135° spectrum, suggested that 9 of them were methyl carbon signals [δ C30.1 (C-28), 26.8 (C-27), 25.1 (C-21), 24.6 (C-26), 23.0 (C-29), 18.1 (C-6″), 17.3 (C-18), 17.1 (C-30) and 17.1 (C-19)], and the 12 oxygen-linked methine carbon signals are [δ C 106.2 (C-1′), 100.7 (C-1″), 85.0 (C-4′), 84.9 (C-24), 84.2 (C-2′), 80.5 (C-3), 79.2 (C-3′), 78.0 (C-3″), 77.2 (C-4″), 76.7 (C-11), 75.6 (C-2″), 73.0 (C-5″)], in addition to one oxymethylene carbon signal δ C 63.0 (C-5′), 9 methylene carbon signals [δ C 37.1 (C-7), 36.1 (C-1), 34.6 (C-22), 32.3 (C-15), 27.5 (C-16), 27.0 (C-23), 21.5 (C-2), 19.0 (C-6)] and two oxygen-linked quaternary carbons [δ C 88.0(C-20) and 72.9(C-25)].

[0080] according to 1 H and 13 C-NMR, 1 H- 1 H COSY( Figure 4 )、HSQC( Figure 5 ) and HMBC spectra ( Figure 6 ), the carbon and hydrogen signals of the compound were fully assigned (see Table 1). 1 H- 1 In the H COSY spectrum, six groups of spin coupling systems can be observed, namely:

[0081] In the HMBC spectrum, δ H 1.09(H-19) and δ C 54.8(C-9) / 51.5(C-5) / 36.1(C-1), δ H 1.00(H-18) and δ C 54.8(C-9) / 51.1(C-14) / 37.1(C-7), δ H 0.97(H-30) and δ C 42.4(C-8) / 41.8(C-13) / 32.3(C-15), δ H 1.16(H-21) and δ C50.1(C-17) / 34.6(C-22),δ H 1.23(H-27) and δ C 84.9(C-24) / 24.6(C-26),δ H 1.15(H-26) and δ C 84.9(C-24), δ H 3.79(H-24) and δ C 50.1(C-17),δ H 0.95(H-28) and δ C 51.5(C-5) / 80.5(C-3) / 23.0(C-29) and δ H 3.29(H-3) and δ C 51.5 (C-5) have long-range carbon-hydrogen correlation, indicating that the parent core is a triterpene with a dammarane skeleton. Among other related HMBC signals, δ H 4.34(H-1″) and δ C 76.7(C-11) / δ C 73.0 (C-5″) has a long-range correlation, which indicates the presence of a chinonose moiety on the hydroxyl group at position 11, δ H 4.93(H-1′) and δ C 80.5(C-3) / δ C There is a long-range correlation at 85.0 (C-4′), indicating that the arabinose moiety is in the furan configuration and is attached to the 3-position of the parent nucleus.

[0082] In summary, the above data suggest that the compound is a new 3,4-closed-ring dammarane-type triterpene, and its planar structure is as follows:

[0083]

[0084] The ROESY spectrum of this compound ( Figure 7 ) can be observed in δ H 4.06(H-11) and δ H 1.69(H-13) / 1.09(H3-19) / δ H 1.00 (H-18) has NOE correlation, indicating that H-11, H-13, H3-19, and H3-18 are facing the same side of the fragment, and it is determined to be β type. H 1.74 (H-9) and δ H There was NOE correlation at 1.90(H-17) / 0.97(H3-30), suggesting that H-9, H-17, and H3-30 were located on the same side, confirming that they were α-type.

[0085] Furthermore, we obtained a single crystal of compound I and performed single crystal X-ray diffraction analysis of compound I using a copper target κα-ray diffraction method. Although the crystal data [Flack parameter = 0.51 (15), Figure 8 ] is slightly larger, so only its relative configuration can be determined. However, based on a series of reported molecular skeletons of this type of compounds and the optical rotation, NMR and acid hydrolysis data of the compounds, it can still be determined that the absolute configurations of the chiral centers of compound I are 3R, 8R, 9R, 10S, 11R, 13R, 14R, 17S, 21S, and 24R.

[0086] Crystal data of the compound shown in formula I: Molecular formula is C 41 H 70 O 12 (M = 754.97 g / mol): orthorhombic symmetry, space group P21212 (no.18), Z=4, T=169.99(10)K, μ(Cu Kα)=0.687mm -1 , Dcalc=1.168g / cm 3 , 24895 reflection measurements (4.898≤2Θ≤147.266), 8476 unique reflection values ​​(R int =0.0447,R sigma =0.0386) were used for all calculations, and the final R1 was 0.0645 (I>2σ(I)), wR2 was 0.1738 (all data), and the Flack parameter was 0.51 (15). The microscopic image of the compound represented by formula I is shown in Fig.41 As shown, the photos Fig.42 shown.

[0087] By comparing with the literature and analyzing the spectra of the above compounds, the compound was finally named (3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-3-α-L-arabinofuranosyl-11-O-β-D-quinovopyranoside. Based on the above data, the compound is a 3,4-ring closed dammarane triterpene, a new compound named qingqianliutianoisde A.

[0088] The structural formula of the compound is:

[0089] Test Example 2

[0090] Characterization of the compound shown in formula II:

[0091] The compound shown in formula II is a white amorphous powder. The TLC color reaction of 5% vanillin-concentrated sulfuric acid is yellow-green. The UV spectrum of this compound has no obvious ultraviolet absorption in the range of 200-400nm, indicating that this compound has no chromophore; the IR spectrum shows that the hydroxyl group (3433cm -1 ) and carbonyl (1699cm -1 ) characteristic absorption; HR-ESI-MS spectrum ( Fig. 9 ) shows its quasi-molecular ion peak m / z 795.4904[MH] - (calcd.for C 43 H 71 O 13 - ,795.4900), it can be inferred that the molecular structure of the compound is C 43 H 72 O 13 , the unsaturation is 8.

[0092] like Fig.10 As shown, the compound 1 H-NMR spectrum (600 MHz, CD3OD) shows an additional methyl group compared to compound I. H 2.06(3H,s,5′-OCO CH3 ), the other 8 angular methyl singlet proton signals are [1.21 (3H, s, H-27), 1.16 (3H, s, H-21), 1.15 (3H, s, H-26), 1.09 (3H, s, H-19), 1.00 (3H, s, H-18), 0.97 (3H, s, H-30), 0.93 (3H, s, H-28) and 0.89 (3H, s, H-29)]. In addition, there are a series of connected oxygen methines [δ H 4.01 (dd, J = 3.8, 1.5 Hz, H-2′), 4.05 (overlap, H-4′), 3.79 (overlap, H-3′), 3.27 (t-like, J = 9.1 Hz, H-3″), 3.24 (overlap, H-5″), 3.09 (dd, J = 9.1, 7.8 Hz, H-2″) and 2.97 (t-like, J = 9.1 Hz, H-4″)], one oxymethylene δ H 4.30 (dd, J = 11.8, 3.4 Hz, H-5′a) and 4.14 (dd, J = 11.8, 6.4 Hz, H-5′b), a methyl δ H1.26 (d, J = 6.1 Hz, H-6″) and two sugar terminal proton signals δ H 4.91 (d, J = 1.5 Hz, H-1′) and 4.34 (d, J = 7.8 Hz, H-1″), indicating the presence of pentose and hexose. Similar to compound I, it can be determined that the compound contains L-arabinose and D-chinchonose.

[0093] like Fig.11 As shown, the compound 13 The C-NMR spectrum (150 MHz, CD3OD) showed 43 carbon signals, 2 more than compound I, including 1 ester carbonyl signal [δ C 172.6(5′-O CO CH3)] and 1 methyl 20.7 (5′-OCO CH3 ). Combined with the DEPT-135° spectrum, the remaining 8 methyl carbon signals are [δ C 30.0 (C-28), 26.8 (C-27), 25.1 (C-21), 24.6 (C-26), 23.0 (C-29), 17.3 (C-18), 17.1 (C-30) and 17.1 (C-19)], and the 12 oxygen-linked methine carbon signals are [δ C 106.4 (C-1′), 100.7 (C-1″), 84.9 (C-24), 84.2 (C-2′), 81.8 (C-4′), 80.9 (C-3), 79.8 (C-3′), 78.0 (C-3″), 77.2 (C-4″), 76.8 (C-11), 75.6 (C-2″), 72.9 (C-5″)], in addition to one oxymethylene carbon signal δ C 65.3 (C-5′), 9 methylene carbon signals [δ C 37.1 (C-7), 36.1 (C-1), 34.6 (C-22), 32.3 (C-15), 27.5 (C-16), 27.0 (C-23), 21.6 (C-2), 19.0 (C-6)] and two oxygen-linked quaternary carbons [δ C 88.0(C-20) and 73.0(C-25)].

[0094] according to 1 H and 13 C-NMR, 1 H- 1 H COSY( Fig.12 )、HSQC( Fig.13 ) and HMBC spectra ( Fig.14 ), the carbon and hydrogen signals of the compound were fully assigned (see Table 1). 1H-NMR and 13 C-NMR spectrum and 2D-NMR spectrum show that its basic skeleton is very similar to compound I, except for an additional set of acetyl signals. In the HMBC spectrum of compound II ( Fig.14 ) can be observed in H 4.30(H-5′a) / 4.14(H-5′b) / 2.06(5′-OCO CH3 ) and δ C 172.6(5′-O CO CH3) has obvious long-range correlation, which further indicates that the acetyl group is connected to the C-5′ position. The final planar structure of the compound is as follows, and the systematic name is (3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-5'-O-acetyl-3-α-L-arabinofuranosyl-11-O-β-D-quinovopyranoside ((3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-3-O-α-L-5′-O-acetyl-arabinofuranosyl-11-O-β-D-quinovopyranoside).

[0095]

[0096] The structural formula of the compound is:

[0097] Based on the above data, the compound is a 3,4-ring closed dammarane triterpene, a new compound named qingqianliutianoside B.

[0098] Table 1 Compounds I and II 1 H-NMR and 13 C-NMR data (CD3OD, J in Hz)

[0099]

[0100]

[0101] Test Example 3

[0102] Characterization of the compound shown in formula III:

[0103] The compound shown in formula III is a white amorphous powder. The TLC color reaction of 5% vanillin-concentrated sulfuric acid is yellow-green. The UV spectrum of this compound has no obvious ultraviolet absorption in the range of 200-400nm, indicating that this compound has no chromophore; the IR spectrum shows that the -1There is characteristic absorption at, indicating that the compound contains hydroxyl groups; HR-ESI-MS spectrum ( Fig.16 ) shows its quasi-molecular ion peak m / z 799.4841 [M+HCOO] - (calcd.for C 42 H 71 O 14 - ,799.4841), it can be inferred that the molecular structure of the compound is C 41 H 70 O 12 , the unsaturation is 7.

[0104] like Fig.17 As shown, the compound 1 The H-NMR spectrum (600 MHz, CD3OD) showed eight angular methyl singlet proton signals as [δ H 1.21 (3H, s, H-27), 1.16 (3H, s, H-21), 1.15 (3H, s, H-26), 1.10 (3H, s, H-19), 1.00 (3H, s, H-18), 0.99 (3H, s, H-30), 0.96 (3H, s, H-28) and 0.89 (3H, s, H-29)]. In addition, there are a series of oxygen-linked methyl groups [δ H 3.82 (overlap, H-4′), 3.59 (dd, J=8.4,6.2 Hz, H-2′), 3.56 (dd, J=8.4,3.3 Hz, H-3′), 3.27 (t-like, J=9.1 Hz, H-3″), 3.25 (overlap, H-5″), 3.09 (dd, J=9.1,7.8 Hz, H-2″) and 2.97 (t-like, J=9.1 Hz, H-4″), one methylene [δ H 3.85 (overlap, H-5′a) and 3.50 (overlap, H-5′b)], a methyl δ H 1.26 (d, J = 6.1 Hz, H-6″) and two sugar terminal proton signals δ H 4.28 (d, J = 6.2 Hz, H-1′) and 4.34 (d, J = 7.8 Hz, H-1″), indicating the presence of pentose and hexose. The compound was hydrolyzed with acid, and the sugar part was further derivatized and analyzed by HPLC and compared with standard sugar derivatives. It was determined that the compound contained L-arabinose and D-chinchonose.

[0105] like Fig.18 As shown, the compound 13The C-NMR spectrum (150 MHz, CD3OD) showed 41 carbon signals, which, combined with the DEPT-135° spectrum, indicated that there were 8 angular methyl carbon signals [δ C 30.0 (C-28), 26.8 (C-27), 25.2 (C-21), 24.6 (C-26), 23.0 (C-29), 17.3 (C-18), 17.1 (C-30) and 17.1 (C-19)], and the 12 oxygen-linked methine carbon signals are [δ C 101.5 (C-1′), 100.8 (C-1″), 84.9 (C-24), 82.4 (C-3), 78.0 (C-3″), 77.2 (C-4″), 76.9 (C-11), 75.6 (C-2″), 74.3 (C-3′), 72.9 (C-5″), 72.5 (C-2′), 69.3 (C-4′)], in addition to one oxymethylene carbon signal δ C 66.1 (C-5′), 9 methylene carbon signals [δ C 37.1 (C-7), 36.0 (C-1), 34.7 (C-22), 32.4 (C-15), 27.5 (C-16), 27.0 (C-23), 21.9 (C-2), 19.1 (C-6)] and two oxygen-linked quaternary carbons [δ C 88.0(C-20) and 73.0(C-25)].

[0106] according to 1 H and 13 C-NMR, 1 H- 1 H COSY( Fig.19 )、HSQC( Fig. 20 ) and HMBC spectra ( Fig.21 ), the carbon and hydrogen signals of the compound were fully assigned (see Table 2). 1 H- 1 In the H COSY spectrum, six groups of proton coupling systems can be observed, namely:

[0107] In the HMBC spectrum, δ H 1.10(H-19) and δ C 54.6(C-9) / 51.5(C-5) / 36.0(C-1), δ H 1.00(H-18) and δ C 54.6(C-9) / 51.2(C-14) / 37.1(C-7), δ H 0.99(H-30) and δ C42.4(C-8) / 41.8(C-13) / 32.4(C-15), δ H 1.16(H-21) and δ C 50.1(C-17) / 34.7(C-22),δ H 1.21(H-27) and δ C 84.9(C-24) / 24.6(C-26),δ H 1.15(H-26) and δ C 84.9(C-24), δ H 3.79(H-24) and δ C 50.1(C-17),δ H 0.96(H-28) and δ C 51.5(C-5) / 82.4(C-3) / 23.2(C-29) and δ H 3.32(H-3) and δ C 51.5 (C-5) have long-range carbon-hydrogen correlation, indicating that the parent core is a triterpene with a dammarane skeleton. Among other related HMBC signals, δ H 4.34(H-1″) and δ C 76.9(C-11) / δ C 72.9(C-5″),δ H 4.06 (H-11) has a long-range correlation, which indicates that the 11-hydroxyl group is connected to the chinonose group, δ H 4.28(H-1′) and δ C 82.4(C-3) / δ C There is a long-range correlation at 66.1 (C-5′), indicating that the arabinose moiety is in pyranose configuration and is attached to the 3-position of the parent nucleus.

[0108] In summary, the above data suggest that this compound is a new dammarane-type triterpene, and its planar structure is as follows:

[0109]

[0110] The ROESY spectrum of this compound ( Fig. 22 ) can be observed in δ H 4.06(H-11) and δ H 1.68(H-13) / 1.10(H3-19) / δ H 1.00 (H-18) has NOE correlation, indicating that H-11, H-13, H3-19, and H3-18 are facing the same side of the fragment, and it is determined to be β type. H 1.80(H-9) and δ HThere was NOE correlation at 1.90(H-17) / 0.99(H3-30), suggesting that H-9, H-17, and H3-30 were located on the same side, confirming that they were α-type.

[0111] Based on the above, the compound was systematically named (3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-3-α-L-arabinopyranosyl-11-O-β-D-quinovopyranoside ((3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-3-O-α-L-arabinopyranosyl-11-O-β-D-quinovopyranoside), and finally named qingqianliutianoside C.

[0112] The structural formula of the compound is:

[0113] Test Example 4

[0114] Characterization of the compound shown in formula IV:

[0115] The compound shown in formula IV is a white amorphous powder. The TLC color reaction of 5% vanillin-concentrated sulfuric acid is yellow-green. The UV spectrum of this compound has no obvious ultraviolet absorption in the range of 200-400nm, indicating that this compound has no chromophore; the IR spectrum shows that the -1 and 1699cm -1 There are absorptions at the positions respectively, indicating that the compound contains hydroxyl and carbonyl groups; HR-ESI-MS spectrum ( Fig.23 ) shows its quasi-molecular ion peak m / z 841.4946 [M+HCOO] - (calcd.forC 43 H 72 O 13 - ,841.4944), it can be inferred that the molecular structure of the compound is C 43 H 72 O 13 , the unsaturation is 8.

[0116] like Fig.24 As shown, the compound 1 H-NMR spectrum (600MHz, CD3OD) shows an additional methyl group compared to compound III. H 2.07(3H,s,4′-OCO CH3 ), and the other proton signals are very similar. The singlet proton signals of the 8 corner methyl groups are [δ H1.21 (3H, s, H-27), 1.16 (3H, s, H-21), 1.15 (3H, s, H-26), 1.09 (3H, s, H-19), 1.00 (3H, s, H-18), 0.99 (3H, s, H-30), 0.93 (3H, s, H-28) and 0.88 (3H, s, H-29)]. In addition, there are a series of oxygen-linked methyl groups [δ H 3.27 (dd, J = 9.5, 7.8 Hz, H-2′), 4.70 (td, J = 9.5, 5.4 Hz, H-4′), 3.55 (t-like, J = 9.5 Hz,, H-3′), 3.26 (t-like, J = 9.1 Hz,, H-3″), 3.25 (overlap, H-5″), 3.09 (dd, J = 9.1, 7.8 Hz, H-2″) and 2.96 (t-like, J = 9.1 Hz, H-4″), one methylene [δ H 3.93 (dd, J = 11.4, 5.4 Hz, H-5′a) and 3.20 (overlap, H-5′b)], one methyl group [δ H 1.25 (d, J = 6.1 Hz, H-6″)] and the two sugar terminal proton signals [δ H 4.26 (d, J = 7.8 Hz, H-1′), 4.34 (d, J = 7.8 Hz, H-1″)], indicating the presence of pentose and hexose. The compound was hydrolyzed with acid, and the sugar part was further derivatized and analyzed by HPLC and compared with standard sugar derivatives, which confirmed that the compound contained L-arabinose and D-chinchonose.

[0117] like Fig.25 As shown, the compound 13 The C-NMR spectrum (150 MHz, CD3OD) showed 43 carbon signals, 2 more than compound III, including 1 ester carbonyl signal [δ C 172.3(4′-O CO CH3)] and 1 methyl 20.8 (4′-OCO CH3 ). Combined with the DEPT-135° spectrum, the remaining 8 methyl carbon signals are [δ C 29.8 (C-28), 26.8 (C-27), 25.2 (C-21), 24.6 (C-26), 23.2 (C-29), 17.3 (C-18), 17.1 (C-19) and 17.0 (C-30)], and the 12 oxygen-linked methine carbon signals are [δ C102.1 (C-1′), 100.7 (C-1″), 84.9 (C-24), 82.5 (C-3), 78.0 (C-3″), 77.2 (C-4″), 76.9 (C-11), 75.6 (C-2″), 75.2 (C-3′), 75.1 (C-2′), 73.5 (C-4′), 72.9 (C-5″)], in addition to one oxymethylene carbon signal δ C 63.5 (C-5′), 9 methylene carbon signals [δ C 37.1 (C-7), 35.9 (C-1), 34.7 (C-22), 32.3 (C-15), 27.5 (C-16), 27.0 (C-23), 22.1 (C-2), 19.0 (C-6)] and two oxygen-linked quaternary carbons [δ C 88.0(C-20) and 73.0(C-25)].

[0118] according to 1 H and 13 C-NMR, 1 H- 1 H COSY( Fig.26 )、HSQC( Fig. 27 ) and HMBC spectra ( Fig.28 ), the carbon and hydrogen signals of the compound were fully assigned (see Table 2). 1 H-NMR and 13 C-NMR spectrum and 2D-NMR spectrum show that its basic skeleton is very similar to compound III, except for an additional set of acetyl signals. In the HMBC spectrum of compound IV ( Fig.28 ) can be observed in H 4.70(H-4′) / 2.07(4′-OCO CH3 ) and δ C 172.3(4′-O CO CH3) has obvious long-range correlation, which further indicates that the acetyl group is connected to the C-4′ position. The final planar structure of the compound is as follows, and the system is named (3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-4'-O-acetyl-3-α-L-arabinopyranosyl-11-O-β-D-quinovopyranoside ((3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-3-O-α-L-4′-O-acetyl-arabinopyranosyl-11-O-β-D-quinovopyranoside), and finally named qingqianliutianoside D.

[0119]

[0120] The structural formula of the compound is:

[0121] Table 2 Compounds III and IV 1 H-NMR and 13 C-NMR data (CD3OD, J in Hz)

[0122]

[0123]

[0124] Test Example 5

[0125] Characterization of the compound shown in Formula V:

[0126] The compound shown in formula V is a white amorphous powder. The TLC color reaction of 5% vanillin-concentrated sulfuric acid is yellow-green. The UV spectrum of this compound has no obvious ultraviolet absorption in the range of 200-400nm, indicating that this compound has no chromophore; the IR spectrum shows that the -1 and 1733cm -1 There is absorption at , indicating that the compound contains hydroxyl and carbonyl groups; HR-ESI-MS spectrum ( Fig.30 ) shows its quasi-molecular ion peak m / z 809.5060[MH] - (calcd.for C 44 H 73 O 13 - ,809.5060), it can be inferred that the molecular structure of the compound is C 44 H 73 O 11 , the unsaturation is 8.

[0127] like Fig.31 As shown, the compound 1 The H-NMR spectrum (600 MHz, CD3OD) showed eight methyl singlet proton signals as [δ H 1.21 (3H, s, H-27), 1.16 (3H, s, H-21), 1.15 (3H, s, H-26), 1.09 (3H, s, H-19), 1.00 (3H, s, H-18), 0.99 (3H, s, H-30), 0.93 (3H, s, H-28) and 0.88 (3H, s, H-29)]. In addition, there are a series of oxygen-linked methyl groups [δ H3.27 (dd, J = 9.5, 7.8 Hz, H-2′), 4.57 (t-like, J = 9.5 Hz, H-4′), 3.49 (t-like, J = 9.5 Hz, H-3′), 3.42 (dd, J = 9.5, 6.2 Hz, H-5′), 3.26 (t-like, J = 9.1 Hz, H-3″), 3.25 (overlap, H-5″), 3.08 (dd, J = 9.1, 7.8 Hz, H-2″) and 2.96 (t-like, J = 9.1 Hz, H-4″)], two methyl δ H 1.13 (d, J = 6.2 Hz, H-6′) and 1.26 (d, J = 6.1 Hz, H-6″) and two sugar terminal proton signals δ H 4.34 (d, J = 7.8 Hz, H-1″) and 4.28 (d, J = 7.8 Hz, H-1′), indicating the presence of two hexose sugars. The compound was hydrolyzed with acid, and the sugar part was further derivatized and analyzed by HPLC and compared with standard sugar derivatives, which confirmed that the compound contained two D-chinchonases.

[0128] like Fig.32 As shown, the compound 13 The C-NMR spectrum (150 MHz, CD3OD) showed 44 carbon signals, which, combined with the DEPT-135° spectrum, suggested that there was one ester carbonyl signal [δ C 172.2(4′-O CO CH3)], and the 9 corner methyl carbon signals are [δ C 29.9(C-28), 26.8(C-27), 25.1(C-21), 24.6(C-26), 23.2(C-29), 21.0(4′-OCO CH3 ), 17.1 (C-19), 17.1 (C-30) and 17.3 (C-18)], and the 13 oxygen-linked methine carbon signals are [δ C 101.4 (C-1′), 100.7 (C-1″), 84.9 (C-24), 82.5 (C-3), 78.0 (C-3″), 77.2 (C-4″), 76.9 (C-11), 75.6 (C-2″), 75.8 (C-3′), 72.9 (C-5″), 75.4 (C-2′), 77.6 (C-4′), 70.8 (C-5′)], in addition to 9 methylene carbon signals [δ C 37.1 (C-7), 36.0 (C-1), 34.6 (C-22), 32.4 (C-15), 27.6 (C-16), 27.0 (C-23), 22.2 (C-2), 19.0 (C-6)] and two oxygen-linked quaternary carbons [δC 88.1(C-20) and 73.0(C-25)].

[0129] according to 1 H and 13 C-NMR, 1 H- 1 H COSY( Fig.33 )、HSQC( Fig.34 ) and HMBC spectra ( Fig.35 ), the carbon and hydrogen signals of the compound were fully assigned (see Table 3). 1 H- 1 In the H COSY spectrum, six groups of proton coupling systems can be observed, namely:

[0130] In the HMBC spectrum, δ H 1.09(H-19) and δ C 54.4(C-9) / 51.4(C-5) / 36.0(C-1), δ H 1.00(H-18) and δ C 54.4(C-9) / 51.2(C-14) / 37.1(C-7), δ H 0.99(H-30) and δ C 42.4(C-8) / 41.8(C-13) / 32.4(C-15), δ H 1.16(H-21) and δ C 50.1(C-17) / 34.6(C-22),δ H 1.21(H-27) and δ C 84.9(C-24) / 24.6(C-26),δ H 1.15(H-26) and δ C 84.9(C-24), δ H 3.79(H-24) and δ C 50.1(C-17),δ H 0.93(H-28) and δ C 51.4(C-5) / 82.5(C-3) / 23.2(C-29) and δ H 3.32(H-3) and δ C 51.4 (C-5) have long-range carbon-hydrogen correlation, indicating that the parent core is a triterpene with a dammarane skeleton. Among other related HMBC signals, δ H 4.34(H-1″) and δ CThere is a long-range correlation at 76.9 (C-11) / 72.9 (C-5″), which indicates the presence of a chinonose moiety on the 11-hydroxyl group. H 4.28(H-1′) and δ C There is a long-range correlation at 82.5 (C-3) / 70.8 (C-5′), indicating that the chinonose moiety is attached to the 3-hydroxyl group.

[0131] In summary, the above data suggest that this compound is a dammarane-type triterpene. Compared with the known compound cypaliuruside M, its basic skeleton is very similar to compound V, except for an additional set of acetyl signals. Fig.35 ) can be observed in H 4.57(H-4′) / 2.09(4′-OCO CH3 ) and δ C 172.2(4′-O CO CH3) has obvious long-range correlation, which further indicates that the acetyl group is connected to the C-4' position. The planar structure is as follows:

[0132]

[0133] The ROESY spectrum of this compound ( Fig.36 ) can be observed in δ H 4.05(H-11) and δ H 1.68(H-13) / 1.09(H3-19) / δ H 1.00 (H-18) has NOE correlation, indicating that H-11, H-13, H3-19, and H3-18 are facing the same side of the fragment, and it is determined to be β type. H 1.89 (H-17) and δ H There was NOE correlation at 1.81(H-9) / 0.99(H3-30), suggesting that H-9, H-17, and H3-30 were located on the same side, confirming that they were α-type.

[0134] Based on the above, the compound was systematically named (3α,11α,25)-trihydroxy-(20S,24R)-epoxy-dammarane-4'-O-acetyl-3-α-L-quinovopyranosyl-11-O-β-D-quinovopyranoside ((3α,11α,25)-trihydroxy-(20S,24R)-epoxydammarane-3-O-α-L-4′-O-acetyl-pyranosyl-11-O-β-D-pyranosyl) and finally named qingqianliutianoside E.

[0135] The structural formula of the compound is:

[0136] Table 3 Compound V 1 H-NMR and 13 C-NMR data (CD3OD, J in Hz)

[0137]

[0138]

[0139] Test Example 6

[0140] Electronic tongue sweetness test:

[0141] (1) Preparation of positive electrode cleaning solution:

[0142] Accurately weigh 7.46 g of potassium chloride, stir and dissolve it with 500 ml of distilled water, then add 300 ml of anhydrous ethanol solution, and while stirring, add accurately weighed 0.56 g of potassium hydroxide. After dissolution is complete, transfer to a 1 L volumetric flask and make up to volume.

[0143] (2) Preparation of reference solution:

[0144] Accurately weigh 2.24 g potassium chloride and 0.045 g tartaric acid, dissolve them in 500 ml distilled water, transfer to a 1 L volumetric flask, and make up to volume.

[0145] (3) Preparation of test solution:

[0146] Referring to the requirements of GB / T 2760-2014 "Food Additives Usage Standard", the concentration gradient of the test sample was confirmed through preliminary experiments.

[0147] Weigh 3g, 10g, and 20g of sucrose and fully dissolve them in 100mL of 0.01mol / L KCl solution respectively;

[0148] Weigh 25 mg of each of the compounds represented by Formula I, Formula III and Cyclocarya paliurus glycoside I, and fully dissolve them in 50 mL of 0.01 mol / L KCl solution.

[0149] (4) Electronic tongue test method:

[0150] The electronic tongue sensor consists of a sweetness sensor (GL1), a ceramic reference electrode (Ag / AgCl) and a temperature sensor. The sweetness sensor GL1 and the reference electrode are used to detect the sweetness signal changes of the test solution. First, wash it in the positive cleaning solution for 90 seconds, then wash it in the reference solution for 3 times. The GL1 sweetness sensor is zeroed at the equilibrium position for 30 seconds. After reaching the equilibrium condition, the test is started and it is immersed in the reference solution and the sample solution to be tested in turn. After 30 seconds, the membrane potential values ​​Vr and Vs are measured respectively.

[0151] Each sample to be tested is tested 5 times in a cycle, the first and last cycles are removed, and the average data of the middle 3 times is taken as the test result. The liquid for each cleaning, balancing and testing is distributed in different sample cups.

[0152] (5) Measurement results:

[0153] Pour the test solution of each sample into a small plastic cup specially used for electronic tongue testing, 25 ml per cup, and test according to the method under (4). The difference between the measured membrane potential values ​​Vs and Vr is the sweetness value of the GL1 sweetness sensor. Collect data from three parallel measurements and calculate the average value. The larger the value, the more sensitive the GL1 sweetness sensor is and the sweeter the taste is.

[0154] The results show that by comparing with sucrose solutions with mass fractions of 3%, 10% and 20%, the compounds represented by Formula I and Formula III have a certain sweetness, and the sweetness is higher than that of Cyclocarya paliurus glycoside I. They have the potential to become a good substitute for sucrose and contribute to the development of high-sweetness and low-calorie natural sweeteners. Some of their sweetness values ​​are shown in Table 4.

[0155] Table 4 Sweetness value measured by electronic tongue

[0156] sample Sweetness value (mV) 3% sucrose solution 15.54±0.10 10% sucrose solution 17.22±0.18 20% sucrose solution 18.23±0.11 Compounds of formula I 17.45±0.46 Compound of formula III 17.59±0.23

[0157] Note: All values ​​are mean ± standard deviation (n = 3).

[0158] Test Example 7

[0159] Molecular docking and simulation testing:

[0160] The homology model of sweet taste receptor T1R2 / T1R3 was constructed using the I-TASSER online server (https: / / zhanggroup.org / I-TASSER / ). The amino acid sequences of T1R2 and T1R3 (T1R2: NP_689418.2; T1R3: NP_689414.2) were obtained from the National Center for Biotechnology Information (NCBI) platform (https: / / www.ncbi.nlm.nih.gov / ).

[0161] The C-score and TM-score values ​​of the model were calculated by the I-TASSER online server, and the ERRAT value and the percentage of amino acids in the allowed region of the Ramachandran plot were calculated by the SAVES v6.0 server, and then the best model was selected for subsequent molecular docking studies.

[0162] The binding pocket and active site of the best model were predicted using the DoGSiteScorer website (https: / / proteins.plus / ). Subsequently, the highly reliable homology models and energy-minimized ligands were imported into AutoDock Tools 1.5.6 software using ChemDraw 3D, converted into pdbqt format, and then molecular docking was performed using AutoDock Vina 1.1.2 software. The specific parameter settings were as follows: the initial search grid was set to (x, y, z): (106, 122, 114), and the default grid spacing was The search grid centers of T1R2 and T1R3 were defined as (x, y, z): (6.444, -13.444, 39.167), Exhaustiveness was set to 8, and the remaining parameters were default values. The docking process was completed using a Python script. In addition, PyMOL 2.2.0 software and LIGPLOT 2.2.8 software were used to analyze the binding sites on the receptors and the interaction forces between the receptors and the ligands.

[0163] The results showed that a homology model of the sweet taste receptor T1R2 / T1R3 containing the N-terminal Venus flytrap binding domain (VFTM) was established (see Fig.37 ). C-score, TM-Score and ERRAT are key indicators for evaluating the quality of T1R2 / T1R3 protein structure models. C-score values ​​are usually in the range of [-5,2]. The higher the value, the higher the confidence of the model. A TM-Score value greater than 0.5 indicates that the topological structure of the model is correct, and an ERRAT value greater than 80 indicates that the atomic distribution of the model is relatively reliable. According to the data in Table 5, these indicators all fall within a reasonable range, indicating that the established homology model has a high degree of confidence.

[0164] Table 5 Parameters for evaluating model reliability of the T1R2 / T1R3 homology model

[0165] Receptors ERRAT value C-score TM-score value T1R2 84.7375 0.06 0.72±0.11 T1R3 86.2816 -0.16 0.69±0.12

[0166] exist Fig.38 In the Ramachandran plot shown in Figure 2, 98.4% of the amino acid residues in the T1R2 receptor model are located in reasonable areas, and 1.6% are located in disallowed areas. At the same time, in the T1R3 receptor model, 1.8% were found to be located in disallowed areas, with a coverage rate of 98.2%. According to the 90% key evaluation principle, the structures of the T1R2 and T1R3 receptor models are reasonable.

[0167] like Fig.39As shown, the binding pockets of sweet taste receptors T1R2 and T1R3 are located in the VFTM part, in the middle cavity of the two larger leaves. The compounds shown in Formula IV were docked to the binding pockets in the VFTM domain of T1R2 / T1R3 by AutoDock Vina to determine the best binding posture. Fig.40 As shown, the three-dimensional (3D) and two-dimensional (2D) images of the docked conformation show important active sites and provide a basis for analyzing the interaction forces (hydrogen bonds and hydrophobic interactions). The calculated binding energy of the compound represented by Formula IV is shown in Table 6.

[0168] In addition, the docking results show (Table 6) that the amino acid active residues and hydrophobic amino acids of T1R2 / T1R3 interact with the compound shown in Formula IV. The binding sites for hydrogen bond formation mainly include Ser212, Ser105 and Thr239. Ser212 appears most frequently in the compound shown in Formula IV, followed by Thr239 and Ser105, which appear in the compounds shown in Formula I, III and V; while in the T1R3 receptor, Asn380, Thr305 and Val381 have higher binding frequencies. Most of the hydrophobic amino acids Arg256, Asp213, Leu243, Pro241, Thr242 and Tyr103 in T1R2 have higher binding frequencies, while Ala383, Asn68, Asn380, Asn386, Glu358, Leu308, Ser67 and Ser382 in T1R3 have higher binding frequencies. Therefore, hydrogen bonding and hydrophobic interaction are important factors in the binding between the compound represented by Formula IV and the sweet taste receptor T1R2 / T1R3.

[0169] Table 6 Molecular docking energy and binding sites of the compound represented by formula IV with T1R2 and T1R3

[0170]

[0171]

[0172] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A cyclocarya paliurus glycoside, characterized in that The cyclocarya paliurus glycoside is selected from: At least one of .

2. The Cyclocarya paliurus glycoside according to claim 1, characterized in that The compound shown in formula I is a single crystal structure.

3. The method for extracting cyclocarya paliurus glycosides as claimed in claim 1 or 2, characterized in that: The steps include: (1) extracting the leaves of Cyclocarya paliurus and a mixed solution of ethanol and water to obtain an extract, and concentrating the extract to obtain a total extract; (2) dispersing the total extract with water, and then extracting with petroleum ether, chloroform and n-butanol to obtain a petroleum ether layer, a chloroform layer, an n-butanol layer and an aqueous layer; (3) The chloroform layer is separated to obtain the Cyclocarya paliurus extract.

4. The extraction method according to claim 3, characterized in that In step (1), the extraction is percolation extraction; The ethanol content in the ethanol-water mixture is 90-98% by volume.

5. The extraction method according to claim 3, characterized in that In step (1), the concentration conditions include: a temperature of 40-60°C.

6. The extraction method according to claim 3, characterized in that In step (2), the mass ratio of the total extract to the water is 1:0.5-2.

7. The extraction method according to any one of claims 3 to 6, characterized in that In step (3), the separation comprises: S1, separating the chloroform layer through a silica gel column I, and eluting with a dichloromethane / methanol system gradient to obtain nineteen fractions of Fr.(A)-Fr.(S); S2. Purify the Fr.(P) fraction.

8. The extraction method according to claim 7, characterized in that In step S2, the purification includes: The Fr.(P) fraction was separated into II-1 by a silica gel column and gradient eluted with a petroleum ether / ethyl acetate / methanol system to obtain sixteen fractions of Fr.(P)1-Fr.(P)16; The Fr.(P)8 fraction is separated into II-2 by a silica gel column, and gradient eluted with a dichloromethane / methanol system to obtain six fractions of Fr.(P)8.1-Fr.(P)8.6; the Fr.(P)8.4 fraction is separated into II-3 by an ODS reverse phase column, and gradient eluted with a methanol / water system to obtain six fractions of Fr.(P)8.4.1-Fr.(P)8.4.6; the Fr.(P)8.4.4 fraction is separated into II-4 by a semi-preparative high performance liquid chromatography-evaporative light scattering detector, and isocratically eluted with acetonitrile / water to obtain the compound of formula III; The Fr.(P)9 fraction was separated into III-1 by a silica gel column and gradient eluted with a dichloromethane / anhydrous methanol system to obtain five fractions from Fr.(P)9.1 to Fr.(P)9.5; The Fr.(P)9.4 fraction is separated into III-2 by a gel column and eluted with methanol to obtain four fractions of Fr.(P)9.4.1-Fr.(P)9.4.4; the Fr.(P)9.4.3 fraction is separated into III-3 by a silica gel column and gradient eluted with a dichloromethane / anhydrous methanol system to obtain five fractions of Fr.(P)9.4.3.1-Fr.(P)9.4.3.5; the Fr.(P)9.4.3.2 fraction is separated into III-4 by an ODS reverse phase column and gradient eluted with a methanol / water system to obtain the compound of formula II; The Fr.(P)9.4.3.3 fraction is separated by natural crystallization to obtain the compound of formula I; The Fr.(P)9.3 fraction is separated into III-5 by a gel column and eluted with methanol to obtain four fractions of Fr.(P)9.3.1-Fr.(P)9.3.4; the Fr.(P)9.3.2 fraction is separated into III-6 by a silica gel column and gradient eluted with a dichloromethane / anhydrous methanol system to obtain four fractions of Fr.(P)9.3.2.1-Fr.(P)9.3.2.4; the Fr.(P)9.3.2.2 fraction is separated into III-7 by an ODS reverse phase column and gradient eluted with a methanol / water system, and then III-8 is separated by a semi-preparative high performance liquid phase-evaporative light scattering detector, and isocratically eluted with acetonitrile / water, and isocratically eluted to obtain the compound of formula IV; The Fr.(P)9.3.2.3 fraction was separated into III-9 by ODS reverse phase column and gradient eluted with methanol / water system to obtain the compound represented by formula V.

9. Cyclocarya paliurus glycoside extracted by the extraction method according to any one of claims 3 to 8.

10. Use of the Cyclocarya paliurus glycoside according to any one of claims 1, 2 and 9 in a sweetener.

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