Gynostemma pentaphyllum glucosidase and application of gynostemma pentaphyllum glucosidase in hydrolysis

Abstract

The invention discloses gynostemma pentaphyllum glucosidase which is prepared by a preparation method comprising the following steps: (1) adding ammonium sulfate into a gynostemma pentaphyllum crude enzyme solution, uniformly stirring and mixing to obtain a first mixed solution with the ammonium sulfate saturation of A1, standing and centrifuging to obtain a first supernatant and a first precipitate; and (2) adding ammonium sulfate into the first supernatant, uniformly stirring and mixing to obtain a second mixed solution with the ammonium sulfate saturation degree of A2, standing and centrifuging to obtain a second supernatant and a second precipitate which is gynostemma pentaphyllum glucosidase. According to the method, the extraction condition and the enzymolysis reaction condition of the gynostemma pentaphyllum glucosidase are optimized, the characteristics of the enzymatic reaction are defined, and the rare saponin is produced by fully utilizing enzymolysis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a gypenoside glucosidase and application of hydrolysis thereof. BACKGROUND

[0002] Dammarane-type triterpenoid saponins are widely distributed in Gynostemma pentaphyllum, Panax ginseng, Panax notoginseng, Panax quinquefolium and other plants, and are the main active ingredients. Dammarane-type saponins have diverse structures, including the structure of aglycone, the connection position of sugar, the type of sugar, the connection mode of sugar and the variation of side chain, which all constitute the diversity of dammarane-type triterpenoid saponins.

[0003] A large number of previous studies have shown that the saponins in plants of the Araliaceae family, such as Panax ginseng, Panax notoginseng and Panax quinquefolium, will undergo hydrolysis, C-20 configuration conversion and other chemical reactions after being subjected to heat treatment such as high-temperature continuous steaming.

[0004] Numerous studies have shown that, compared with the prototype saponins, rare saponins generally have higher biological activity and better application prospects. The application of gypenoside glucosidase and hydrolysis thereof claimed in the present application has not been found in the prior art. SUMMARY

[0005] Therefore, the present application provides a gypenoside glucosidase, which is obtained by a preparation method comprising the following steps:

[0006] (1) adding ammonium sulfate to a gypenoside crude enzyme solution, stirring and mixing to obtain a first mixed solution with an ammonium sulfate saturation degree of A1, standing, centrifugation to obtain a first supernatant and a first precipitate; and

[0007] (2) adding ammonium sulfate to the first supernatant, stirring and mixing to obtain a second mixed solution with an ammonium sulfate saturation degree of A2, standing, centrifugation to obtain a second supernatant and a second precipitate, wherein the second precipitate is the gypenoside glucosidase.

[0008] Further, the mixing is performed under water bath.

[0009] Further, the standing is performed under water bath.

[0010] Further, the water bath is an ice water bath.

[0011] Further, the ice water bath is at 0℃.

[0012] Further, the ammonium sulfate saturation degree A1 in the first mixed solution is 50% to 70%.

[0013] Further, the ammonium sulfate saturation degree A1 in the first mixed solution is about 60%.

[0014] Further, the saturation A2 of ammonium sulfate in the second mixed solution is 60% to 80%.

[0015] Further, the saturation A2 of ammonium sulfate in the second mixed solution is about 70%.

[0016] Further, the saturation A2 is greater than the saturation A1.

[0017] Further, the saturation A2 minus the saturation A1 is about 10%.

[0018] Further, the standing time is 0.5 to 1.5 h, for example, about 1 h.

[0019] Further, the centrifugation speed is 10,000 to 15,000 r / min, for example, about 13,000 r / min.

[0020] Further, the centrifugation temperature is 0 to 10℃, for example, about 4℃.

[0021] Further, the centrifugation time is 10 to 20 min, for example, about 15 min.

[0022] Further, the preparation method of the G. pectinatum crude enzyme solution comprises the following steps:

[0023] (a) weighing a proper amount of G. pectinatum leaves into a container, adding a proper amount of excipient and a first solvent, grinding, and centrifuging for the first time to obtain a first supernatant; and

[0024] (b) collecting the first supernatant and centrifuging for the second time to collect a second supernatant, thereby obtaining the G. pectinatum crude enzyme solution.

[0025] Further, the G. pectinatum leaves are fresh G. pectinatum leaves.

[0026] Further, the container is a mortar.

[0027] Further, the mortar is a pre-cooled mortar.

[0028] Further, the excipient is cross-linked povidone.

[0029] Further, the first solvent is a buffer saline solution.

[0030] Further, the buffer salt is citric acid-sodium citrate, disodium hydrogen phosphate-potassium dihydrogen phosphate, or sodium hydroxide-potassium dihydrogen phosphate.

[0031] Further, the buffer salt is citric acid-sodium citrate.

[0032] Further, the concentration of the citric acid-sodium citrate buffer saline solution is 0.01 to 1 mol / L, for example, about 0.1 mol / L.

[0033] Further, the pH of the citric acid-sodium citrate buffer saline solution is 5-7, for example, about 6.

[0034] Further, the ratio of the volume / mass (mL / g) of the buffer saline solution to the Gynostemma leaves is 7-20, for example, about 8.

[0035] Further, the ratio of the mass of the cross-linked povidone to the Gynostemma leaves is 1-2, for example, about 1.5.

[0036] Further, the grinding is performed on ice.

[0037] Further, the speed of the first centrifugation is 8000-12000 r / min, for example, about 10000 r / min.

[0038] Further, the temperature of the first centrifugation is 0-10℃, for example, about 4℃.

[0039] Further, the time of the first centrifugation is 2-10 min, for example, about 5 min.

[0040] Further, the speed of the second centrifugation is 12000-16000 r / min, for example, about 14000 r / min.

[0041] Further, the temperature of the second centrifugation is 0-10℃, for example, about 4℃.

[0042] Further, the time of the second centrifugation is 5-15 min, for example, about 10 min.

[0043] According to another aspect of the present application, there is provided a use of the above-mentioned Gynostemma glucosidase in hydrolyzing 1,2-linked diglucosyl.

[0044] Further, the hydrolysis comprises the following steps: weighing an appropriate amount of a substrate, adding the first solvent and the Gynostemma crude enzyme solution, mixing, water bath reaction for a period of time, and obtaining a rare saponin product.

[0045] Further, the substrate is a Gynostemma saponin extract.

[0046] Further, the substrate concentration is 1-3 mg / mL, for example, about 2 mg / mL.

[0047] Further, the first solvent is a citric acid-sodium citrate buffer saline solution.

[0048] Further, the ratio of the volume of the citric acid-sodium citrate buffer solution to the crude enzyme solution is 8-10, for example, about 9.

[0049] Further, the temperature of the water bath reaction is 40-60℃, for example, about 50℃.

[0050] Further, the time of the water bath reaction is 3-5h, for example, about 4h.

[0051] Further, the action of the gypenoside enzyme is to hydrolyze the glucose group at C-3.

[0052] Further, the action substrate of the gypenoside enzyme solution is the original gypenoside of the protopanaxadiol type.

[0053] Further, the gypenoside enzyme solution does not hydrolyze the gypenoside with acetyl or malonyl modification.

[0054] Further, the action site of the gypenoside enzyme solution is the glycosidic bond at C-3 of the gypenoside of the protopanaxadiol type.

[0055] Further, the glycosidic bond is C3-(1,2) diglucosidic bond.

[0056] Further, the application includes the conversion of rare saponins, deglucosylation of glycosides and / or structural identification of glycosides.

[0057] Further, the rare saponin is a C-3 deglucosylated rare saponin.

[0058] Further, the rare saponin includes gypenoside LXXIV, gypenoside XIII, gypenoside TN-1 and / or ginsenoside CK.

[0059] The beneficial effects of the present application are:

[0060] (1) Innovative rare saponin conversion method: the efficacy of dammarane type rare saponin is better than that of the original saponin. In the past, physical methods such as high-temperature cooking were usually used to obtain rare saponins by deglucosylation. The present application finds that the hydrolysis characteristics of gypenoside enzyme can be used to convert rare saponins. Enzymatic conversion of rare saponins has low energy consumption, high efficiency, high product conversion rate and few impurities.

[0061] (2) Innovative hydrolysis site: the hydrolysis and deglucosylation site of gypenoside enzyme is different from that of the past high-temperature cooking deglucosylation. The gypenoside enzyme of the present application hydrolyzes the glucose group at C-3, while the high-temperature cooking deglucosylation removes the sugar group at C-20.

[0062] (3) Hydrolysis of glucose substrate species: the extracted Gypenoside glucosidase of the application has high activity compared with enzymes on the market, and can specifically hydrolyze 1,2-linked diglucosyl; the starch hydrolyzing enzyme on the market mainly hydrolyzes 1,6-linked glucosyl, and the glucanase mainly hydrolyzes 1,3-linked glucosyl. The enzyme can be applied to rare saponin conversion and other glycoside-containing deglycosylation conversion, and can be used for structural identification of various glycosides such as saponins and flavones with 1,2-linked glucosyl, and for determining the connection mode of glycosides, thus having wide commercial application value.

[0063] (4) Rare saponin source innovation: the rare saponin of Gypenoside is similar in structure to the rare saponin of ginseng, and has better potential application value. Therefore, attention is paid to the Gypenoside glucosidase, the saponin enzymatic conversion rule is studied, and new drug sources rich in rare saponin components of ginseng are found from plants outside the Araliaceae family, which not only has important academic significance, but also has great economic value.

[0064] (5) How to obtain rare saponin is currently a hot research topic, and currently, physical method, chemical method and biological conversion method are usually used to produce rare saponin. The physical method usually uses high-temperature cooking to remove sugar, which has the disadvantages of high energy consumption and low yield, and most of the hydrolysis is C-20 glycosyl; the chemical method has the disadvantages of violent reaction, easy change of aglycone structure, no selectivity of glycosyl hydrolysis, low yield of product, and high impurities; the enzyme hydrolysis of the application is green, environmentally friendly, has high hydrolysis specificity and high efficiency, has wide plant sources, and has many advantages such as various substrates. BRIEF DESCRIPTION OF DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art according to these drawings, without exceeding the scope of the application.

[0066] Figure 1 The schematic diagram of the PVPP dosage investigation results in the extraction process.

[0067] Figure 2 The schematic diagram of the buffer salt type investigation results in the extraction process.

[0068] Figure 3 The schematic diagram of the buffer salt pH value investigation results in the extraction process.

[0069] Figure 4 The schematic diagram of the effect of buffer volume on enzyme activity in the extraction process.

[0070] Figure 5The results of the optimum temperature for enzyme reaction are shown in the following figure.

[0071] Figure 6 The mass spectrum of different reaction proportions is shown in the following figure.

[0072] Figure 7 The results of the peak area of saponin conversion of different reaction proportions are shown in the following figure.

[0073] Figure 8 The mass spectrum of the blank control group is shown in the following figure.

[0074] Figure 9 The peak area chart of different reaction times is shown in the following figure.

[0075] Figure 10 The BPI chart of the in-vitro simulation reaction at different times is shown in the following figure. Wherein, A: before reaction; B: 30 minutes after reaction; C: 4 hours after reaction.

[0076] Figure 11 The compound structure diagram of the action site of the enzymatic conversion of gypenoside is shown in the following figure.

[0077] Figure 12 The BPI chart of various enzyme reaction groups is shown in the following figure. Wherein, A: 0h reaction of the crude enzyme group; a: 4h reaction of the crude enzyme group; B: 0h reaction of the snail enzyme group; b: 4h reaction of the snail enzyme group; C: gypenoside substrate; D-H: 4h reaction of the β-amylase (aspergillus source) group, the β-amylase (bacillus source) group, the β-glucanase group, the β-glucosidase (aspergillus source) group, and the β-glucosidase (almond source) group.

[0078] Figure 13 The BPI chart before and after the enzymatic reaction is shown in the following figure. Wherein, A: comparison chart of the crude enzyme and notoginsenoside before and after 4h reaction; B: comparison chart of the crude enzyme and ginsenoside before and after 4h reaction.

[0079] Figure 14 The conversion structure diagram of notoginsenoside and ginsenoside components is shown in the following figure.

[0080] Figure 15 The protein gel electrophoresis chart is shown in the following figure. Wherein, M: protein marker (Marker); S: pure enzyme protein sample. DETAILED DESCRIPTION

[0081] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0082] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application, or that are given in the specification. Although in principle any method, condition, material or substance similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods, conditions, materials or substances are described herein.

[0083] The present application is intended to embrace all options, variations and equivalents that can be encompassed by the present invention as defined by the claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The application is not to be limited in scope by the methods and materials described.

[0084] As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0085] In the present application, the term "comprising" is synonymous with "including", "containing", or "comprehending". As used herein, the terms "comprise", "comprising", "include", "including", "contain", "containing", "comprehend" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0086] As described in the background section, the existing saponins are subjected to heat treatment such as continuous steaming at high temperature, which causes various chemical reactions such as hydrolysis and C-20 configuration conversion of ginsenosides. In order to solve the above problems, the present application provides a gypenoside glucosidase obtained by a preparation method comprising the steps of:

[0087] (1) adding ammonium sulfate to a gypenoside crude enzyme solution, stirring and mixing to obtain a first mixed solution having a saturation degree of ammonium sulfate of A1, standing, and centrifuging to obtain a first supernatant and a first precipitate; and

[0088] (2) adding ammonium sulfate to the first supernatant, stirring and mixing to obtain a second mixed solution having a saturation degree of ammonium sulfate of A2, standing, and centrifuging to obtain a second supernatant and a second precipitate, wherein the second precipitate is the gypenoside glucosidase.

[0089] In a preferred embodiment, the stirring and mixing is performed under a water bath.

[0090] In a preferred embodiment, the standing is performed under a water bath.

[0091] In a preferred embodiment, the water bath is an ice water bath.

[0092] In a preferred embodiment, the ice water bath is 0°C.

[0093] In a preferred embodiment, the saturation of ammonium sulfate in the first mixed solution A1 is about 60%.

[0094] In the present application, where a saturation, time, speed, temperature, concentration, ratio, frequency, or other value or parameter is expressed in a range, a preferred range, or a range having an upper preferred value and a lower preferred value, it is understood that all ranges formed by any pair of an upper range limit or preferred value and a lower range limit or preferred value are specifically disclosed, whether the range is explicitly disclosed or not. For example, where a range of "50% to 70%" is disclosed, the described range should be interpreted to include ranges of "50% to 70%", "50% to 65%", "50% to 60%", "50% to 55%", "55% to 70%", "55% to 65%", "55% to 60%", "60% to 70%", "60% to 65%", "65% to 70%", etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include the end values and all integers and fractions within that range.

[0095] In a preferred embodiment, the saturation of ammonium sulfate in the first mixed solution A1 is about 60%.

[0096] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 60%" includes ±5% of 60%, or from 57% to 63%.

[0097] In a preferred embodiment, the saturation of ammonium sulfate in the second mixed solution A2 is about 70%.

[0098] In a preferred embodiment, the saturation of ammonium sulfate in the second mixed solution A2 is about 70%.

[0099] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 70%" includes ±5% of 70%, or from 66.5% to 73.5%.

[0100] In a preferred embodiment, the saturation A2 is greater than the saturation A1.

[0101] In a preferred embodiment, the saturation A2 minus the saturation A1 is about 10%.

[0102] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 10%" includes ±5% of 10%, or from 9.5% to 10.5%.

[0103] In a preferred embodiment, the time of standing is 0.5-1.5 h, such as about 1 h.

[0104] In the present application, "about" means a value within ±5% of a specified value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0105] In a preferred embodiment, the centrifugation is at a speed of 10000-15000 r / min, such as about 13000 r / min.

[0106] In the present application, "about" means a value within ±5% of a specified value. For example, "about 13000" includes ±5% of 13000, or from 12350 to 13650.

[0107] In a preferred embodiment, the centrifugation is at a temperature of 0-10 °C, such as about 4 °C.

[0108] In the present application, "about" means a value within ±5% of a specified value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0109] In a preferred embodiment, the centrifugation is for a time of 10-20 min, such as about 15 min.

[0110] In the present application, "about" means a value within ±5% of a specified value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0111] In a preferred embodiment, the method of preparing the crude gynostemma enzyme solution comprises the following steps:

[0112] (a) weighing an appropriate amount of gynostemma leaves into a container, adding an appropriate amount of an excipient and a first solvent, grinding, and centrifuging for the first time to obtain a first supernatant; and

[0113] (b) collecting the first supernatant and centrifuging for the second time, collecting a second supernatant, to obtain the crude gynostemma enzyme solution.

[0114] In a preferred embodiment, the gynostemma leaves are fresh gynostemma leaves.

[0115] In a preferred embodiment, the container is a mortar.

[0116] In a preferred embodiment, the mortar is a pre-cooled mortar.

[0117] In a preferred embodiment, the excipient is cross-linked polyvinylpyrrolidone.

[0118] In a preferred embodiment, the first solvent is a buffered saline solution.

[0119] In a preferred embodiment, the buffer salt is citric acid-sodium citrate, disodium hydrogen phosphate-potassium dihydrogen phosphate, or sodium hydroxide-potassium dihydrogen phosphate.

[0120] In a preferred embodiment, the buffer salt is citric acid-sodium citrate.

[0121] In a preferred embodiment, the concentration of the aqueous citric acid-sodium citrate buffer salt solution is 0.01-1 mol / L, such as about 0.1 mol / L.

[0122] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0123] In a preferred embodiment, the pH of the aqueous citric acid-sodium citrate buffer salt solution is 5-7, such as about 6.

[0124] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0125] In a preferred embodiment, the ratio of the volume of the aqueous buffer salt solution to the mass of the G. peltatum leaves (mL / g) is 7-20, such as about 8.

[0126] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4.

[0127] In a preferred embodiment, the ratio of the mass of the cross-linked povidone to the mass of the G. peltatum leaves is 1-2, such as about 1.5.

[0128] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575.

[0129] In a preferred embodiment, the grinding is performed on ice.

[0130] In a preferred embodiment, the speed of the first centrifugation is 8000-12000 r / min, such as about 10000 r / min.

[0131] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 10000" includes ±5% of 10000, or from 9500 to 10500.

[0132] In a preferred embodiment, the temperature of the first centrifugation is 0-10°C, such as about 4°C.

[0133] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0134] In a preferred embodiment, the time of the first centrifugation is 2-10 min, such as about 5 min.

[0135] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0136] In a preferred embodiment, the speed of the second centrifugation is 12000-16000 r / min, such as about 14000 r / min.

[0137] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 14000" includes ±5% of 14000, or from 13300 to 14700.

[0138] In a preferred embodiment, the temperature of the second centrifugation is 0-10°C, such as about 4°C.

[0139] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0140] In a preferred embodiment, the time of the second centrifugation is 5-15 min, such as about 10 min.

[0141] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0142] According to another aspect of the present application, there is provided a use of the above-mentioned gypenoside glucosidase in hydrolyzing 1,2-linked diglucosyl.

[0143] In a preferred embodiment, the hydrolyzing comprises the following steps: weighing a proper amount of a substrate, adding the first solvent and the gypenoside crude enzyme solution, mixing, water-bath reacting for a period of time, and obtaining a rare saponin product.

[0144] In a preferred embodiment, the substrate is a gypenoside extract.

[0145] In a preferred embodiment, the substrate concentration is 1-3 mg / mL, such as about 2 mg / mL.

[0146] In the present invention, "about" means a value within ±5% of a specified value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0147] In a preferred embodiment, the first solvent is a citric acid-sodium citrate buffer aqueous solution.

[0148] In a preferred embodiment, the ratio of the citric acid-sodium citrate buffer solution to the crude enzyme solution is 8-10, for example about 9.

[0149] In the present invention, "about" means a value within ±5% of a specified value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0150] In a preferred embodiment, the temperature of the water bath reaction is 40-60°C, for example about 50°C.

[0151] In the present invention, "about" means a value within ±5% of a specified value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0152] In a preferred embodiment, the time of the water bath reaction is 3-5h, for example about 4h.

[0153] In the present invention, "about" means a value within ±5% of a specified value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0154] In a preferred embodiment, the action of the gypenoside glucosidase is to hydrolyze the C-3 glucose group.

[0155] In a preferred embodiment, the action substrate of the gypenoside glucosidase solution is a protopanaxadiol-type gypenoside.

[0156] In a preferred embodiment, the gypenoside glucosidase solution does not hydrolyze gypenosides with acetyl or malonyl modification.

[0157] In a preferred embodiment, the action site of the gypenoside glucosidase solution is the C-3 glycosidic bond of a protopanaxadiol-type gypenoside.

[0158] In a preferred embodiment, the glycosidic bond is a C3-(1,2) diglucosidic bond.

[0159] In a preferred embodiment, the application includes conversion to generate rare saponins, deglycosylation of glycosides, and / or structural identification of glycosides.

[0160] In a preferred embodiment, the rare saponin is a C-3 desglycosylated rare saponin.

[0161] In a preferred embodiment, the rare saponin comprises Gypenoside LXXIV, Gypenoside XIII, Gypenoside TN-1 and / or Ginsenoside CK.

[0162] In a preferred embodiment, the Gypenoside glucosidase comprises a protein having an amino acid sequence as set forth in SEQ ID NO: 1, a protein having an amino acid sequence as set forth in SEQ ID NO: 2, a protein having an amino acid sequence as set forth in SEQ ID NO: 3, a protein having an amino acid sequence as set forth in SEQ ID NO: 4, a protein having an amino acid sequence as set forth in SEQ ID NO: 5, a protein having an amino acid sequence as set forth in SEQ ID NO: 6, a protein having an amino acid sequence as set forth in SEQ ID NO: 7, a protein having an amino acid sequence as set forth in SEQ ID NO: 8, a protein having an amino acid sequence as set forth in SEQ ID NO: 9, a protein having an amino acid sequence as set forth in SEQ ID NO: 10 and a protein having an amino acid sequence as set forth in SEQ ID NO: 11.

[0163] The application is further described in conjunction with the following examples. It should be understood that these examples are merely for illustrative purposes and do not limit the scope of the application. The experimental methods in the following examples, unless otherwise specified, were generally carried out according to conventional conditions or as recommended by the manufacturer.

[0164] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are for illustrative purposes only and are not intended to limit the scope of the application.

[0165] Any of the features mentioned in relation to the above described aspects of the application, or features mentioned in relation to the examples, can be provided in any combination. All features disclosed in the specification and all features of the claims are expressly incorporated by reference. Each feature disclosed in the specification and all features of the claims can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, the features disclosed in the specification and the features of the claims are not exhaustive.

[0166] Examples

[0167] Experimental Instruments

[0168] TGL-20M benchtop high-speed refrigerated centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), Multiskan FC microplate reader (Thermo Fisher Scientific), LC3000 preparative HPLC (Beijing Innovative Technology Co., Ltd.), ODS packing (50 μm, YMC Corporation, Japan), electrophoresis instrument (Beijing Liyi Instrument), Xevo G2-S QTof mass spectrometer (Waters Corporation, USA), ACQUITY UPLC liquid chromatograph (Waters Corporation, USA), Diamonsil C18 chromatographic column (5 μm, 250 mm x 4.6 mm), DFY-5L / 20 low-temperature constant-temperature reaction bath (Gongyi Yuhua Instrument Co., Ltd.), BSA423S-CW electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry (Thermo Fisher Scientific), protein electrophoresis instrument (BIORAD).

[0169] Experimental reagents

[0170] Fresh leaves of Gynostemma pentaphyllum were collected from Yingfu Village, Zhangzhou City, Fujian Province. Cross-linked povidone (BASF), citric acid (National Pharmaceutical Group Chemical Reagent Co., Ltd.), sodium citrate (National Pharmaceutical Group Chemical Reagent Co., Ltd.), sodium carbonate (National Pharmaceutical Group Chemical Reagent Co., Ltd.), disodium hydrogen phosphate (National Pharmaceutical Group Chemical Reagent Co., Ltd.), potassium dihydrogen phosphate (National Pharmaceutical Group Chemical Reagent Co., Ltd.), sodium hydroxide (National Pharmaceutical Group Chemical Reagent Co., Ltd.), p-nitroaminophenol-β-glucoside (Shanghai Yuanye Biological Technology Co., Ltd.), p-nitroaminophenol (Shanghai Yuanye Biological Technology Co., Ltd.), purified water (Guangzhou Watsons Food and Beverage Co., Ltd.), β-glucosidase (Shanghai Yuanye Biological Technology Co., Ltd.), glucanase (Shanghai Yuanye Biological Technology Co., Ltd.), β-amylase (Shanghai Yuanye Biological Technology Co., Ltd.), snailase (Shanghai Yuanye Biological Technology Co., Ltd.), total ginsenoside extract (National Pharmaceutical Group Chemical Reagent Co., Ltd.), total notoginsenoside extract (National Pharmaceutical Group Chemical Reagent Co., Ltd.), ammonium sulfate (MERCK), BCA kit (Bi Yun Tian), SDS-PAGE buffer (Bi Yun Tian), Blue IV Protein Marker (Beijing Quanshi Gold Biological), ACN (MERCK), FA (J&K), NH4HCO3 (Aldrich), IAM (Aldrich), PNGaseF (Sigma), Trypsin enzyme (Promega), DTT (Thermo Fisher Scientific).

[0171] 1. Optimization of glucoside hydrolysis enzyme extraction method

[0172] 1.1, Extraction method

[0173] Take 1 g of fresh Gynostemma pentaphyllum leaves, add a certain amount of cross-linked povidone (PVPP), add 15 mL of pre-cooled buffer, grind into paste on ice, pre-cool the low-temperature centrifuge, centrifuge at 10000 r / min at 4°C for 5 min, collect the supernatant; centrifuge at 14000 r / min at 4°C for 10 min, take the supernatant to obtain the crude enzyme solution, and store at -25°C.

[0174] Take 0.1 mL of fresh leaf crude enzyme extract, take 0.4 mL of 10 mmol / L pNPG as substrate, dilute with 4 mL of buffer salt solution, react for 1 h, take 0.45 mL of reaction solution, add 2.5 mL of 1 mol / L Na2CO3 solution to stop the reaction and develop color, then detect the absorbance value at 400 nm wavelength, and measure the absorbance by colorimetry to compare the enzyme activity. The higher the absorbance, the higher the enzyme activity.

[0175] 1.2, Extraction process optimization

[0176] 1.2.1, PVPP addition amount

[0177] Take 5 portions of 1 g of fresh Gynostemma pentaphyllum leaves, add different proportions of cross-linked povidone (PVPP) to the pre-cooled mortar, control the variable, and add 15 mL of pre-cooled 0.1 mol / L citric acid-sodium citrate buffer (pH = 6.0) in parallel, grind into paste on ice, pre-cool the low-temperature centrifuge, centrifuge at 10000 r / min at 4°C for 5 min, collect the supernatant; centrifuge at 14000 r / min at 4°C for 10 min, take the supernatant to obtain the crude enzyme solution.

[0178] Detect the enzyme activity respectively, the operation is the same as 1.1, and the results are shown in Figure 1 When the PVPP dosage is 1:1.5 of the fresh leaf amount, the enzyme activity reaches a high level, and the enzyme activity does not increase significantly with the increase of PVPP dosage. In order to save cost and avoid waste, and at the same time the extracted enzyme has high enzyme activity, the mass ratio of fresh Gynostemma pentaphyllum leaves to PVPP is selected as 1:1.5 as the best extraction ratio.

[0179] 1.2.2, Buffer salt solution type

[0180] Investigate the effects of 0.1 mol / L citric acid-sodium citrate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and sodium hydroxide-potassium dihydrogen phosphate buffer salt solution types on the enzyme activity of the extracted glycoside hydrolase.

[0181] Take 3 parts of 1 g fresh Gynostemma leaves, add 1.5 g of equal amount of PVPP, respectively, use the above three kinds of buffer solution pH 6.0 15 mL grinding, from the fresh leaves of Gynostemma extract enzyme, operation with 1.1. Repeat three times.

[0182] The results are shown in the following table. The absorbance of the reaction with pNPG was detected, and the results are shown in the following table. Figure 2 The citric acid-sodium citrate buffer solution was selected to extract glycoside hydrolase, and the absorbance after reaction was the highest, and the enzyme activity was the best.

[0183] 1.2.3, pH value of buffer salt solution

[0184] Take 3 parts of 1 g fresh Gynostemma leaves, add 1.5 g of equal amount of PVPP, respectively, use 0.1 mol / L pH 5.0; 6.0; 6.5 citric acid-sodium citrate buffer solution 15 mL grinding, from the fresh leaves of Gynostemma extract enzyme. Operation with 1.1. Repeat three times.

[0185] The results are shown in the following table. The absorbance of the reaction with pNPG was detected, and the results are shown in the following table. Figure 3 When the pH value of the citric acid-sodium citrate buffer salt solution is 6.0, the activity of the extracted glycoside hydrolase is the best.

[0186] 1.2.4, volume of buffer salt solution

[0187] Take 4 parts of 1 g fresh Gynostemma leaves, add 1.5 g of equal amount of PVPP, respectively, add 8 mL, 9 mL, 10 mL, 15 mL of 0.1 mol / L, pH=6.0 citric acid-sodium citrate buffer solution grinding, from the fresh leaves of Gynostemma extract enzyme. Operation with 1.1. Repeat three times.

[0188] The results are shown in the following table. The absorbance of the reaction with pNPG was detected, and the results are shown in the following table. Figure 4 Under the premise of ensuring that the sample is ground into a flow state, as little buffer salt solution as possible is added, and the crude enzyme solution obtained by extraction has a higher enzyme concentration, so it shows higher enzyme activity.

[0189] Finally, the fresh leaves of Gynostemma and PVPP were ground into a paste with a mass ratio of 1:1.5, and pH=6.0 citric acid-sodium citrate buffer solution was added. The low-temperature centrifuge was precooled, and the supernatant was collected after centrifugation at 4°C 10000 r / min for 5 min. The supernatant was collected after centrifugation at 4°C 14000 r / min for 10 min, and the crude enzyme solution was obtained. Store at -25°C.

[0190] The following materials are all Gynostemma pentaphyllum glycoside hydrolase crude enzyme solution obtained under the best extraction process conditions, which is simply referred to as "crude enzyme".

[0191] 2, enzyme property investigation and optimization of enzyme reaction system

[0192] 2.1, Determination of the optimum temperature of enzyme reaction

[0193] Take 0.1 mL of crude enzyme solution in parallel, take 0.4 mL of 10 mmol / L pNPG as substrate, add 4 mL of 0.1 mol / L citric acid-sodium citrate buffer solution with pH value of 6.0, and react in water bath at 30, 40, 50, 60°C respectively for 1 h. Take 0.45 mL of reaction solution respectively, add 2.5 mL of 1 mol / L Na2CO3 solution to stop the reaction and develop color, and then measure the absorbance value at 400 nm wavelength. The experiment is repeated three times in parallel, and the results are shown in Figure 5 The optimum reaction temperature of the enzyme extracted from fresh Gynostemma pentaphyllum leaves is 50°C.

[0194] 2.2, Investigation of optimum substrate concentration and enzyme dosage ratio

[0195] Take a certain amount of gypenoside extract as substrate, dissolve completely in 9 mL of 0.1 mol / L citric acid-sodium citrate buffer solution with pH value of 6.0 in a test tube with a stopper, and then add 1 mL of crude enzyme solution. Set three groups of parallel experiments:

[0196] Reaction group (three groups): add 10 mg, 20 mg, 30 mg of saponin + 1 mL of crude enzyme solution + 9 mL of buffer salt solution respectively;

[0197] Enzyme blank group: 1 mL of crude enzyme solution + buffer salt solution to 9 mL;

[0198] Substrate blank group: 20 mg of saponin + buffer salt solution to 10 mL.

[0199] According to the results of "determination of the optimum temperature of enzyme reaction" in item 2.1, set the reaction temperature to 50°C water bath heating. The specific design is shown in Table 1.

[0200] Table 1 Design of enzyme reaction groups

[0201]

[0202]

[0203] Note: - No addition in the reaction system.

[0204] After 0.5 h of reaction, take 0.5 mL of reaction solution respectively, add 1 mL of methanol to terminate the reaction, and then perform mass spectrometry analysis respectively. The mass spectrum is shown in Figure 6 . Calculate the peak area increase value of gypenoside enzymatic product under different reaction ratios at the same time. The peak area increase value of the product is calculated as Figure 7As shown, when the reaction system ratio is 20 mg saponin substrate + 1 mL crude enzyme solution + 9 mL buffer salt solution, the target hydrolysis product generation is the largest, and the saponin conversion efficiency is the highest, and the reaction system is determined as the optimal reaction system.

[0205] The liquid chromatography-mass spectrometry analysis conditions are as follows:

[0206] Chromatographic conditions: chromatographic column, ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 μm); mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid water; gradient elution: 0-3 min 30%-35% A, 3-8 min 35%-45% A, 8-12 min 45%-60% A, 12-15 min 60%-80% A; flow rate is 0.4 mL·min -1 ; injection volume 2 μL; column temperature: 35°C, automatic sampler temperature 20°C, PDA detector scan range is 200-400 nm.

[0207] Mass spectrometry conditions: ESI ion source, negative ion mode scanning, capillary voltage is 2 kV, cone hole voltage is 40 V, ion source temperature is 120°C, desolvation gas volume flow rate is 600 L·h -1 , desolvation gas temperature is 400°C, cone hole gas volume flow rate is 50 L·h -1 , parent ion collision energy is 6 eV, fragment ion collision energy is 20-50 eV, mass scan range is 100-2000 Da, scan time is 0.2 s.

[0208] The blank control mass spectrometry analysis is as shown in Figure 8 , the enzyme blank group and the substrate blank group do not produce component conversion during the reaction process, and do not affect the saponin component conversion in the reaction system.

[0209] 2.3, optimization of the optimal reaction length

[0210] Under the optimal substrate concentration and enzyme ratio, incubate at the optimum reaction temperature, that is, the reaction system of 20 mg saponin substrate + 1 mL crude enzyme solution + 9 mL buffer salt solution, take samples at 0 h, 2 h, 4 h, and 12 h, the sampling volume is 0.5 mL, add 1 mL methanol to terminate the reaction and dilute, pass through 0.22 μm filter membrane for mass spectrometry analysis. The peak area values of gypenoside substrate and enzymatic conversion products at different reaction time points are as shown in Figure 9 . The results are as follows, after 4 hours of reaction, the hydrolysis conversion tends to be stable.

[0211] The optimal reaction conditions are finally determined as follows: saponin substrate concentration in the reaction system is 2 mg / mL, buffer salt solution: crude enzyme solution is 9:1, reaction temperature is 50°C, and reaction length is 4 h.

[0212] 3. Exploration of the Enzymatic Conversion Mechanism of Gynostemma pentaphyllum Saponins

[0213] 3.1 Substrates for the enzymatic transformation of Gynostemma pentaphyllum saponins

[0214] Under the optimal reaction conditions determined in the previous step, 20 mg of Gynostemma pentaphyllum saponin extract was used as a substrate and added to a stoppered test tube with 0.1 mol·L⁻¹. -1 The enzyme solution was completely dissolved in 9 mL of a citrate-sodium citrate buffer solution (pH=6), then 1 mL of enzyme solution was added, mixed well, and heated in a 50°C water bath. At 0 h, 0.5 h, and 4 h of reaction time, 0.5 mL of the reaction solution was taken, and 1 mL of methanol was added to terminate the reaction and dilute the solution. The terminated reaction solution was filtered through a 0.22 μm filter membrane to prepare the test solution for the reaction group. UPLC-Q-TOF-MS was used for analysis. n The technology is used to analyze the composition of compounds in a sample solution.

[0215] The baseline ion chromatograms (BPI) of the reaction solution before reaction, after 30 minutes of reaction, and after 4 hours of reaction are shown below. Figure 10 As shown in Table 2, peak marking and mass spectrometry identification of the main compounds were performed.

[0216] Table 2 Mass spectrometry analysis and identification of Gynostemma pentaphyllum saponin transformation substrates and products

[0217]

[0218]

[0219]

[0220] The compounds were identified based on information such as the form of molecular ions, functional group fragmentation patterns, and characteristic fragments of each component in the mass spectrometry results. A total of 15 saponin components in 4 groups were identified, serving as reaction substrates, intermediates, and final products, respectively.

[0221] Peaks 1, 3, 4, and 7 of the protopanaxadiol-type neutral saponin compounds successively lost two glucose molecules, yielding intermediate product peaks 9, 10, and 11, and final product peaks 12, 13, 14, and 15. Peaks 2, 5, 6, and 8 of the compounds modified with acetyl or malonyl groups did not undergo transformation.

[0222] This indicates that the substrate of gypenosinolate hydrolase is protopanaxadiol-type gypenosinoside, and it does not hydrolyze gypenosinoside modified with acetyl or malonyl groups.

[0223] 3.2. Enzymatic transformation sites of Gynostemma pentaphyllum saponins

[0224] With the guidance of LC-MS peak, the gypenoside extract was taken and ODS Flash column chromatography was used to elute with 60%, 70%, 90% methanol gradient, and the 70% methanol elution part was taken and semi-preparative liquid chromatography was used to elute with 35% acetonitrile water isocratic elution to obtain the peak 1 compound; semi-preparative liquid chromatography was used to elute with 38% acetonitrile isocratic elution to obtain the peak 3 compound.

[0225] Another part of the original gypenoside crude extract in the above step "2.1" was taken and reacted with the crude enzyme solution, and then concentrated to recover to obtain a 1-2 sugar gypenoside crude extract, which was subjected to ODS Flash column chromatography to elute with 70%, 80%, 90% methanol gradient, and the 80% methanol elution part was taken and subjected to semi-preparative liquid chromatography to elute with 35%-45% acetonitrile water gradient to obtain the peak 12 compound and the peak 13 compound. The four compounds obtained by separation and purification were identified, and the chemical structures were as shown in Figure 11 .

[0226] Determination of hydrolysis site: peaks 1 and 3 are original gypenoside substrates, and peaks 12 and 13 are rare gypenoside products. The peak 1 compound and the peak 12 compound are substrates and products of each other, and the difference between the two is two molecules of glucose at the C-3 position; the peak 3 compound and the peak 13 compound are also substrates and products of each other, and the difference between the two is also two molecules of glucose at the C-3 position. We have determined that the hydrolysis of gypenoside hydrolytic enzyme is mainly on the C-3 position of the original gypenoside glycoside, and the acetyl or malonyl substituted glucose group on the gypenoside and the C-20 position glucose group are not easy to be hydrolyzed. Therefore, it is determined that the hydrolysis site of gypenoside hydrolytic enzyme is C-3; the hydrolysis glycosyl type is determined: the structural characteristics of the substrate peaks 1 and 13 are that the C-3 position is connected with 1,2 connected glucose, so it is determined that the gypenoside glycosyl hydrolytic enzyme mainly hydrolyzes 1,2 connected diglucosyl.

[0227] 4. Application of gypenoside hydrolytic enzyme

[0228] 4.1. Comparison of enzymolysis efficiency of gypenoside hydrolytic enzyme and other commercial enzymes

[0229] Select β-glucosidase (aspergillus source), β-glucosidase (almond source), glucanase, snail enzyme, β-amylase (aspergillus source), β-amylase (bacillus source) and gypenoside hydrolytic enzyme crude enzyme, and take 10U units of each enzyme, respectively, and react with 0.5mL of 2mg / mL substrate solution, and the solvent is selected as 0.1mol·L -1, citric acid-sodium citrate buffer solution, pH = 6. After mixing, place in a 50°C water bath and heat for 4h. Take 0.5mL of the sample solution at 0h and 4h respectively, add 1mL of methanol to terminate the reaction and dilute, take the terminated reaction liquid through a 0.22μm filter membrane, and prepare the test solution sample for liquid chromatography-mass spectrometry analysis. Investigate the hydrolysis of various commercial enzymes on gypenoside, and the mass spectrum results are shown in Figure 12 .

[0230] The hydrolysis characteristics of various commercial enzymes and crude enzymes in the known experiment are shown in Table 3.

[0231] Table 3 Hydrolysis characteristics of various glycoside hydrolytic enzymes

[0232]

[0233]

[0234] Within 4 hours of reaction, 10U of gypenoside hydrolytic enzyme can completely hydrolyze and convert gypenoside into a large amount of rare saponins, and 10U of snailase can only hydrolyze and remove one molecule of glucose from gypenoside, and the ability to continue to convert to rare saponins is weaker than gypenoside hydrolytic enzyme. However, 10U of commercial enzymes including β-glucosidase (Aspergillus source), glucanase, and β-amylase (Aspergillus source) do not show obvious hydrolysis and conversion to gypenoside. The main reason is that the current market glucose glycosidase mainly hydrolyzes 1,3, 1,4 or 1,6 glucose glycosidic bond, and there are few enzymes that can hydrolyze glycosides containing 1,2 glucose glycosidic bond. Unlike existing commercial enzymes, the gypenoside hydrolytic enzyme extracted from gynostemma pentaphyllum mainly hydrolyzes glycosides containing 1,2 glucose glycosidic bond. The structural characteristics of gypenoside are that the connection mode of two molecules of glucose at C-3 site is 1,2 connection. Therefore, compared with other commonly used commercial enzymes on the market, gypenoside hydrolytic enzyme can significantly improve the hydrolysis and conversion efficiency of gypenoside, and has specificity and high efficiency in removing glucose from C-3 1,2 connection of protopanax diol type gypenoside.

[0235] 4.2, Application of gypenoside glucosidase in hydrolysis of other dammarane type saponin medicinal materials

[0236] The main components of ginseng and panax notoginseng are ginsenosides and notoginsenosides, which are dammarane type tetracyclic triterpenoid saponin compounds. Their structural types are similar to gypenosides, and they have the same parent nucleus structure, but the positions, types and numbers of the sugars connected to the parent nucleus are different.

[0237] Take 2 mg of total ginsenoside extract and total notoginsenoside extract, respectively, add 0.1 mL of crude enzyme solution and 0.9 mL of citric acid-sodium citrate buffer. Mix immediately, then take 0.5 mL of the mixed solution, add 1 mL of methanol to terminate the reaction, and use as the sample test solution for 0 h of reaction.

[0238] Put the remaining mixed solution in a 50℃ water bath for 4 h, then add 1 mL of methanol to terminate the reaction, and use as the sample test solution for 4 h of enzymatic reaction.

[0239] Pass through a 0.22 μm filter, and use UPLC-Q-TOF-MS n technology to analyze the transformation of ginsenoside and notoginsenoside compounds before and after the enzymatic reaction, and mark the peaks of the main compound peaks. The obtained BPI diagram is shown in Figure 13 .

[0240] It is found that the main component peaks 1, 2, and 3 are completely hydrolyzed after 4 hours of crude enzyme hydrolysis reaction, and a large amount of compound peaks 4, 5, and 6 are generated, indicating that the gypenoside hydrolytic enzyme also has high efficiency in the hydrolysis and transformation of notoginsenoside and ginsenoside.

[0241] According to the comparison of the base peak ion mass of the known reference substance with Figure 14 each compound in the table, each main compound peak is identified, and the structure diagram of the compound is drawn according to the characteristics of the gypenoside hydrolytic enzyme in hydrolyzing the glucose connected to C-3 of the original ginsenoside diol type. The identification results are shown in Table 4, and the structure diagram of the component transformation is shown in Figure 14 .

[0242] Peak 1, m / z 1153 [M+HCOO] - is ginsenoside Rb1; peak 2, m / z

[0243] 1123 [M+HCOO] - is ginsenoside Rb3; peak 3, m / z 991 [M+HCOO] - is ginsenoside Rd; peak 4, m / z 829 [M+HCOO] - is gypenoside LXXIV; peak 5, m / z 799 [M+HCOO] - is gypenoside XIII; peak 6, m / z 667 [M+HCOO] - is ginsenoside CK.

[0244] The main component peaks 1, 2, and 3 as substrates are completely hydrolyzed and transformed into rare saponin peaks 4, 5, and 6 after 4 hours of crude enzyme hydrolysis reaction, indicating that the gypenoside hydrolytic enzyme has a wide range of hydrolysis substrates and high hydrolysis efficiency. It can efficiently transform a large amount of C-3 desugar rare saponin using gypenoside hydrolytic enzyme.

[0245] Table 4 Mass spectrometric analysis and identification of the transformation of notoginsenosides and ginsenoside components

[0246]

[0247] 5. Purification and identification of the enzyme

[0248] 5.1. Purification of the enzyme

[0249] 5.1.1. Ammonium sulfate fractionation

[0250] By using the salting-out characteristics of proteins, different types of proteins have different salting-out saturations. With the change of the ammonium sulfate saturation, the proteins in the crude enzyme solution are well separated by fractionation. According to the "Ammonium Sulfate Saturation Calculation Table (0°C)", eight levels of precipitated proteins with ammonium sulfate saturation intervals of 0-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, and 80-90% are prepared, and the enzyme specific activities of the proteins precipitated in each ammonium sulfate saturation interval are compared.

[0251] Method 1:

[0252] 1. The crude enzyme solution is extracted according to the optimal extraction method determined in Section 1. That is, 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone are added to 30 mL of a 0.1 M, pH 6.0 citric acid-sodium citrate buffer solution, and ground into a paste in an ice bath. A low-temperature centrifuge is pre-cooled, and centrifuged at 10,000 r / min for 5 min at 4°C. The supernatant is collected. Centrifugation is performed at 14,000 r / min for 10 min at 4°C, and the supernatant is collected to obtain the crude enzyme solution, which is stored at -25°C.

[0253] 2. Take 20 mL of the crude enzyme solution and place it in a 0°C ice water bath. Add 2.12 g of ammonium sulfate while stirring until the ammonium sulfate is completely dissolved, and the ammonium sulfate saturation of the solution reaches 20%. After 1 h of standing in the 0°C ice water bath, centrifuge at 13,000 r / min for 15 min at 4°C in a low-temperature centrifuge. The precipitate obtained is the protein precipitated at an ammonium sulfate saturation interval of 0-20%.

[0254] 3. Dissolve the precipitated protein with 1 mL of a 0.1 mol / L pH 6 citric acid-sodium citrate buffer solution. Apply colorimetry and BCA method to detect and calculate the protease specific activity of the protein precipitated at an ammonium sulfate saturation interval of 0-20%, which is 0.003 x 10 6 .

[0255] Method 2:

[0256] 1. The crude enzyme solution is extracted according to the optimal extraction method determined in Section 1. That is, take 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone, add 30 mL of 0.1 M, pH = 6.0 citric acid-sodium citrate buffer solution to ice bath and grind into paste, pre-cool the low-temperature centrifuge, centrifuge at 10,000 r / min at 4℃ for 5 min, collect the supernatant; centrifuge at 14,000 r / min at 4℃ for 10 min, take the supernatant to obtain the crude enzyme solution, and store at -25℃.

[0257] 2. Take 20 mL of the crude enzyme solution and place it in a 0℃ ice water bath, add 2.12 g of ammonium sulfate, stir while adding ammonium sulfate until the ammonium sulfate is completely dissolved, and the ammonium sulfate saturation of the solution reaches 20%. After standing in a 0℃ ice water bath for 1 h, centrifuge at 13,000 r / min at 4℃ for 15 min in a low-temperature centrifuge. Discard the precipitate, take 20 mL of the supernatant, add 1.1 g of ammonium sulfate, and the ammonium sulfate saturation of the solution reaches 30%. After standing in a 0℃ ice water bath for 1 h, centrifuge at 13,000 r / min at 4℃ for 15 min in a low-temperature centrifuge. The precipitate obtained is the protein precipitated at an ammonium sulfate saturation interval of 20-30%.

[0258] 3. Dissolve the precipitated protein with 1 mL of 0.1 mol / L pH 6 citric acid-sodium citrate buffer solution. Apply colorimetric method and BCA method to detect and calculate the specific activity of the protease precipitated at an ammonium sulfate saturation interval of 20-30% to be 0.001 x 10 6 .

[0259] Method Three:

[0260] 1. The crude enzyme solution is extracted according to the optimal extraction method determined in Section 1. That is, take 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone, add 30 mL of 0.1 M, pH = 6.0 citric acid-sodium citrate buffer solution to ice bath and grind into paste, pre-cool the low-temperature centrifuge, centrifuge at 10,000 r / min at 4℃ for 5 min, collect the supernatant; centrifuge at 14,000 r / min at 4℃ for 10 min, take the supernatant to obtain the crude enzyme solution, and store at -25℃.

[0261] 2. Take 20 mL of the crude enzyme solution and place it in a 0℃ ice water bath, add 3.28 g of ammonium sulfate, stir while adding ammonium sulfate until the ammonium sulfate is completely dissolved, and the ammonium sulfate saturation of the solution reaches 30%. After standing in a 0℃ ice water bath for 1 h, centrifuge at 13,000 r / min at 4℃ for 15 min in a low-temperature centrifuge. Discard the precipitate, take 20 mL of the supernatant, add 1.12 g of ammonium sulfate, and the ammonium sulfate saturation of the solution reaches 40%. After standing in a 0℃ ice water bath for 1 h, centrifuge at 13,000 r / min at 4℃ for 15 min in a low-temperature centrifuge. The precipitate obtained is the protein precipitated at an ammonium sulfate saturation interval of 30-40%.

[0262] 3. Use 1 mL of 0.1 mol / L pH 6 citric acid-sodium citrate buffer solution to dissolve the precipitated protein. Apply colorimetry and BCA method to detect and calculate the specific activity of the proteinase precipitated in the ammonium sulfate saturation interval of 30-40% as 0.006 x 10 6 .

[0263] Method four:

[0264] 1. Extract the crude enzyme solution according to the optimal extraction method determined in Section 1. That is, take 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone, add 30 mL of 0.1 M, pH = 6.0 citric acid-sodium citrate buffer solution to ice bath and grind into paste, pre-cool the low-temperature centrifuge, centrifuge at 10000 r / min for 5 min at 4°C, collect the supernatant; centrifuge at 14000 r / min for 10 min at 4°C, take the supernatant to obtain the crude enzyme solution, and store at -25°C.

[0265] 2. Take 20 mL of the crude enzyme solution, place it in a 0°C ice water bath, add 4.52 g of ammonium sulfate, and stir while adding ammonium sulfate until the ammonium sulfate is completely dissolved, and the ammonium sulfate saturation of the solution reaches 40%. After standing in a 0°C ice water bath for 1 h, centrifuge at 13000 r / min at 4°C in a low-temperature centrifuge for 15 min. Discard the precipitate, take 20 mL of the supernatant, add 1.16 g of ammonium sulfate, and the ammonium sulfate saturation of the solution reaches 50%. After standing in a 0°C ice water bath for 1 h, centrifuge at 13000 r / min at 4°C in a low-temperature centrifuge for 15 min. The precipitate obtained is the protein precipitated in the ammonium sulfate saturation interval of 40-50%.

[0266] 3. Use 1 mL of 0.1 mol / L pH 6 citric acid-sodium citrate buffer solution to dissolve the precipitated protein. Apply colorimetry and BCA method to detect and calculate the specific activity of the proteinase precipitated in the ammonium sulfate saturation interval of 40-50% as 0.047 x 10 6 .

[0267] Method five:

[0268] 1. Extract the crude enzyme solution according to the optimal extraction method determined in Section 1. That is, take 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone, add 30 mL of 0.1 M, pH = 6.0 citric acid-sodium citrate buffer solution to ice bath and grind into paste, pre-cool the low-temperature centrifuge, centrifuge at 10000 r / min for 5 min at 4°C, collect the supernatant; centrifuge at 14000 r / min for 10 min at 4°C, take the supernatant to obtain the crude enzyme solution, and store at -25°C.

[0269] 2. Take 20 mL of crude enzyme solution, place it in a 0°C ice water bath, add 5.82 g of ammonium sulfate, and stir while adding ammonium sulfate until the ammonium sulfate is completely dissolved and the solution reaches 50% ammonium sulfate saturation. After standing in the 0°C ice water bath for 1 h, centrifuge at 13000 r / min for 15 min at 4°C in a low-temperature centrifuge. Discard the precipitate, take 20 mL of supernatant, add 1.2 g of ammonium sulfate, and the solution reaches 60% ammonium sulfate saturation. After standing in the 0°C ice water bath for 1 h, centrifuge at 13000 r / min for 15 min at 4°C in a low-temperature centrifuge. The precipitate obtained is the protein precipitated at an ammonium sulfate saturation interval of 50-60%.

[0270] 3. Dissolve the precipitated protein with 1 mL of 0.1 mol / L pH 6 citric acid-sodium citrate buffer solution. Use colorimetry and BCA method to detect and calculate the specific activity of the protease precipitated at an ammonium sulfate saturation interval of 50-60% to be 0.115 x 10 6 .

[0271] Method Six:

[0272] 1. Extract the crude enzyme solution according to the optimal extraction method determined in Section 1. That is, take 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone, add 30 mL of 0.1 M, pH 6.0 citric acid-sodium citrate buffer solution, grind into a paste in an ice bath, pre-cool the low-temperature centrifuge, centrifuge at 10000 r / min for 5 min at 4°C, collect the supernatant; centrifuge at 14000 r / min for 10 min at 4°C, take the supernatant to obtain the crude enzyme solution, and store at -25°C.

[0273] 2. Take 20 mL of crude enzyme solution, place it in a 0°C ice water bath, add 7.22 g of ammonium sulfate, and stir while adding ammonium sulfate until the ammonium sulfate is completely dissolved and the solution reaches 60% ammonium sulfate saturation. After standing in the 0°C ice water bath for 1 h, centrifuge at 13000 r / min for 15 min at 4°C in a low-temperature centrifuge. Discard the precipitate, take 20 mL of supernatant, add 1.24 g of ammonium sulfate, and the solution reaches 70% ammonium sulfate saturation. After standing in the 0°C ice water bath for 1 h, centrifuge at 13000 r / min for 15 min at 4°C in a low-temperature centrifuge. The precipitate obtained is the protein precipitated at an ammonium sulfate saturation interval of 60-70%.

[0274] 3. Dissolve the precipitated protein with 1 mL of 0.1 mol / L pH 6 citric acid-sodium citrate buffer solution. Use colorimetry and BCA method to detect and calculate the specific activity of the protease precipitated at an ammonium sulfate saturation interval of 60-70% to be 0.244 x 10 6 .

[0275] Method Seven:

[0276] 1. The crude enzyme solution was extracted according to the optimal extraction method determined in Section 1. That is, 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone were added to 30 mL of a citric acid-sodium citrate buffer solution with a concentration of 0.1 M and pH = 6.0, and were ground into a paste in an ice bath. The low-temperature centrifuge was pre-cooled, and the mixture was centrifuged at 10,000 r / min at 4°C for 5 min. The supernatant was collected. The mixture was centrifuged at 14,000 r / min at 4°C for 10 min, and the supernatant was collected to obtain the crude enzyme solution, which was stored at -25°C.

[0277] 2. 20 mL of the crude enzyme solution was placed in an ice water bath at 0°C, and 8.72 g of ammonium sulfate was added while stirring until the ammonium sulfate was completely dissolved, and the ammonium sulfate saturation of the solution reached 70%. After standing in the ice water bath at 0°C for 1 h, the mixture was centrifuged at 13,000 r / min at 4°C for 15 min. The precipitate was discarded, and 20 mL of the supernatant was taken and 1.3 g of ammonium sulfate was added to reach an ammonium sulfate saturation of 80% in the solution. After standing in the ice water bath at 0°C for 1 h, the mixture was centrifuged at 13,000 r / min at 4°C for 15 min. The precipitate obtained was the protein precipitated at an ammonium sulfate saturation of 70-80%.

[0278] 3. The precipitated protein was dissolved in 1 mL of a citric acid-sodium citrate buffer solution with a concentration of 0.1 mol / L and pH 6. The protease specific activity of the protein precipitated at an ammonium sulfate saturation of 70-80% was calculated to be 0.103 × 10 6 .

[0279] Method Eight:

[0280] 1. The crude enzyme solution was extracted according to the optimal extraction method determined in Section 1. That is, 3 g of fresh Gynostemma leaves and 4.5 g of cross-linked povidone were added to 30 mL of a citric acid-sodium citrate buffer solution with a concentration of 0.1 M and pH = 6.0, and were ground into a paste in an ice bath. The low-temperature centrifuge was pre-cooled, and the mixture was centrifuged at 10,000 r / min at 4°C for 5 min. The supernatant was collected. The mixture was centrifuged at 14,000 r / min at 4°C for 10 min, and the supernatant was collected to obtain the crude enzyme solution, which was stored at -25°C.

[0281] 2. 20 mL of the crude enzyme solution was placed in an ice water bath at 0°C, and 10.32 g of ammonium sulfate was added while stirring until the ammonium sulfate was completely dissolved, and the ammonium sulfate saturation of the solution reached 80%. After standing in the ice water bath at 0°C for 1 h, the mixture was centrifuged at 13,000 r / min at 4°C for 15 min. The precipitate was discarded, and 20 mL of the supernatant was taken and 1.34 g of ammonium sulfate was added to reach an ammonium sulfate saturation of 90% in the solution. After standing in the ice water bath at 0°C for 1 h, the mixture was centrifuged at 13,000 r / min at 4°C for 15 min. The precipitate obtained was the protein precipitated at an ammonium sulfate saturation of 80-90%.

[0282] 3. Dissolve the precipitated protein with 1 mL of 0.1 mol / L citric acid-sodium citrate buffer solution at pH 6. Apply colorimetric method and BCA method to detect and calculate the specific activity of the precipitated protease at the ammonium sulfate saturation interval of 80-90% as 0.022 x 10 6 .

[0283] The specific activity of the protein obtained by each of the above eight preparation methods is shown in Table 5. Among them, the protein precipitated at the ammonium sulfate saturation interval of 60-70% in preparation method six has the highest enzyme specific activity.

[0284] Table 5 Enzyme specific activity of the protein obtained by each preparation method

[0285]

[0286] The purity and molecular weight of the preliminarily purified gypenoside hydrolase obtained by preparation method six were determined by SDS-PAGE gel electrophoresis, and the electropherogram is shown in Figure 15 The main band of the enzyme protein sample is the purified gypenoside hydrolase protein. By comparing with the protein molecular weight standard band, it is determined that the molecular weight of the gypenoside hydrolase protein is about 60 kDa.

[0287] 5.1.2, Colorimetric method for measuring enzyme activity

[0288] Take 20 μL of the protein solution after each level of precipitation and resolubilization, add 80 μL of pNPG (10 mmol / mL), and then add 900 μL of buffer salt solution. After mixing, take 0.5 mL of the mixed solution and add 5 mL of Na2CO3 (1 mol / L) solution as a blank control before reaction. The remaining 0.5 mL of the mixed solution is placed in a 50°C water bath for reaction for 30 min, then 5 mL of Na2CO3 (1 mol / L) solution is added to terminate the reaction and develop color. The absorbance value is measured at 400 nm wavelength and recorded.

[0289] The standard curve of low concentration of p-nitrophenol is y = 1.7114x + 0.0049, R 2 = 0.9994

[0290] The standard curve of high concentration of p-nitrophenol is y = 1.6386x + 0.012, R 2 = 0.9998.

[0291] The amount of p-nitrophenol generated after enzyme hydrolysis reaction is calculated according to the standard curve of p-nitrophenol, and the enzyme activity and enzyme activity concentration are calculated according to the following formula.

[0292] Enzymatic activity (U) definition: 1 enzyme unit is defined as the amount of enzyme that catalyzes the production of 1 μmol / L p-nitrophenol per minute.

[0293]

[0294] c (μmol / L): The concentration of p-nitrophenol produced after enzymatic reaction

[0295] t (min): Reaction time

[0296] Enzymatic activity concentration (U / V) definition: The amount of enzyme units contained in 1 L of enzyme solution.

[0297]

[0298] U: Enzymatic activity

[0299] V (L): The volume of enzyme solution contained in the color developing system

[0300] Total enzymatic activity (U x V) definition: The amount of enzyme contained in the total volume of enzyme solution.

[0301] U: Enzymatic activity

[0302] V (L): Total volume of enzyme solution

[0303] 5.1.3, Protein concentration determination by BCA method

[0304] The protein concentration of the protein solution after resolubilization of the precipitate at each stage was determined using a BCA kit.

[0305] 5.1.4, Calculation of specific enzymatic activity and purification fold

[0306] Specific enzymatic activity (U / mg): The amount of enzyme activity per mg of protein. That is, the ratio of enzymatic activity concentration to protein concentration.

[0307] Purification fold: The specific enzymatic activity of the pure enzyme is calculated as a multiple of the specific enzymatic activity of the crude enzyme.

[0308] 5.1.5, SDS-PAGE gel electrophoresis

[0309] The purified protein was subjected to SDS-PAGE gel electrophoresis. The precast gel and SDS-PAGE electrophoresis solution were placed in an electrophoresis apparatus, the purified protein sample solution was mixed with the loading buffer at a volume ratio of 1:5, and the metal bath was heated at 100°C for 3 min to obtain the loading solution. 30 μL of the loading solution was loaded together with 10 μL of protein Marker for electrophoresis. The electrophoresis conditions were as follows: constant voltage 120 V, 120 min.

[0310] 5.2, Enzyme identification

[0311] The purified gypenoside hydrolytic enzyme collected in the previous step was subjected to amino acid sequencing. Mass spectrometry was used to analyze the protein sample, and the characteristic peptide fragments were compared and annotated with the self-built protein library of gynostemma.

[0312] The operation steps of the pretreatment experiment of the purified gypenoside hydrolytic enzyme sample are as follows:

[0313] An appropriate amount of purified gypenoside hydrolytic enzyme sample obtained by the preparation method six was placed in a 3kDa ultrafiltration centrifuge tube and ultrafiltrated six times, with deionized water as the displacement solvent and the protein concentration adjusted to about 1.00mg / mL.

[0314] 100μL of the above sample was taken, 200mM NH4HCO3 was added to adjust the pH to 7-8. 100mM DTT was added, and the reduction reaction was carried out at 56℃ for 1h. Then 250mM IAM was added, and the alkylation reaction was carried out at room temperature in the dark for 1h. PNGaseF was continuously added to cut the sugar, and the reaction was carried out at 37℃ for 8h. Trypsin was added for enzymatic digestion, and the reaction was carried out at 37℃ for 8h. Finally, 1% FA was added to neutralize the solution, and the enzymatic digestion reaction was terminated. The enzymatic hydrolysate was collected by LC-MS / MS, and the sample was collected twice.

[0315] Chromatographic conditions: chromatographic column, ACQUITY UPLC BEH C18 (2.1mm×100mm, 1.7μm); mobile phase A: 90% ACN + 10% H2O + 0.1% FA, mobile phase B: 98% H2O + 2% ACN + 0.1% FA; gradient elution: 0-10min 3%-3% A, 10-60min 3%-50% A, 60-70min 50%-90% A, 70-80min 90%-90% A, 80-85min 90%-3% A, 85-90min 3%-3% A; column temperature: 40℃; detection wavelength: 214nm.

[0316] Mass spectrometry conditions: HESI ion source, ion source voltage 3.8kV, scanning mode: Full MS / dd MS 2 ; capillary temperature: 320℃; collision energy: 27eV; nebulizing gas N2 (L / min): 35arb; auxiliary gas N2 (L / min): 10arb.

[0317] The data of the gypenoside self-built protein library is from the genomic sequencing of fresh leaves of G. peltatum, and the CDS sequence is extracted and translated into amino acid sequence, and then the protein annotation is performed. The proteins and amino acid sequences annotated as glycoside hydrolase are selected, and the mass spectrum information of the purified gypenoside hydrolase in the laboratory is compared. The more the number of matched unique peptides is, the higher the matching degree with the protein is, and the more accurate the protein amino acid sequence obtained by matching is. Finally, 11 proteins are obtained. The specific amino acid sequences are shown in Table 6.

[0318] Table 6 Amino acid sequences of gypenoside hydrolases

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325] The above has carried out the detailed introduction to the embodiment of the present application, the principle and the implementation mode of the present application have been described in this paper by applying specific examples, the above embodiment is only used for helping understanding the method of the present application and its core idea. Meanwhile, the change or deformation of the person skilled in the art according to the idea of the present application, based on the specific implementation mode and the application range of the present application, all belong to the scope of the present application. In summary, the content of the specification should not be understood as the limitation of the present application.

Claims

1. A gypenoside glucosidase, characterized in that, The gypenoside enzyme is obtained by a preparation method comprising the following steps: (1) adding ammonium sulfate into the gynostemma pentaphyllum crude enzyme solution, stirring and mixing to obtain a first mixed solution with ammonium sulfate saturation degree A1, standing, centrifuging to obtain a first supernatant and a first precipitate; and (2) adding ammonium sulfate into the first supernatant, stirring and mixing to obtain a second mixed solution with ammonium sulfate saturation degree A2, standing, centrifuging to obtain a second supernatant and a second precipitate, wherein the second precipitate is the gypenoside enzyme.

2. The Gypenoside glucosidase according to claim 1, characterized in that, The mixing is preferably performed under water bath; The standing is preferably performed under water bath; The water bath is preferably an ice water bath; The ice water bath is preferably 0℃.

3. The Gypenoside glucosidase according to claim 1, characterized in that, The ammonium sulfate saturation degree A1 in the first mixed solution is 50% to 70%; The ammonium sulfate saturation degree A1 in the first mixed solution is preferably about 60%; The ammonium sulfate saturation degree A2 in the second mixed solution is preferably 60% to 80%; The ammonium sulfate saturation degree A2 in the second mixed solution is preferably about 70%; The saturation degree A2 is preferably greater than the saturation degree A1; The saturation degree A2 minus the saturation degree A1 is preferably about 10%; The standing time is preferably 0.5 to 1.5 hours, for example about 1 hour; The centrifuging speed is preferably 10000 to 15000 r / min, for example about 13000 r / min; The centrifuging temperature is preferably 0 to 10℃, for example about 4℃; The centrifuging time is preferably 10 to 20 minutes, for example about 15 minutes.

4. The Gypenoside glucosidase according to claim 1, characterized in that, The preparation method of the gynostemma pentaphyllum crude enzyme solution comprises the following steps: (a) weighing a proper amount of gynostemma pentaphyllum leaves into a container, adding a proper amount of auxiliary materials and a first solvent, grinding, first centrifuging to obtain a first supernatant; and (b) collecting the first supernatant and performing second centrifuging to collect a second supernatant, thereby obtaining the gynostemma pentaphyllum crude enzyme solution.

5. The method of claim 4, wherein, The gynostemma pentaphyllum leaves are fresh gynostemma pentaphyllum leaves; The container is preferably a mortar; The mortar is preferably a pre-cooled mortar; The auxiliary materials are preferably cross-linked povidone; The first solvent is preferably a buffer salt aqueous solution; The buffer salt is preferably citric acid-sodium citrate, disodium hydrogen phosphate-potassium dihydrogen phosphate, or sodium hydroxide-potassium dihydrogen phosphate; The buffer salt is more preferably citric acid-sodium citrate; The concentration of the citric acid-sodium citrate buffer salt aqueous solution is preferably 0.01 to 1 mol / L, for example about 0.1 mol / L; The pH of the citric acid-sodium citrate buffer salt aqueous solution is preferably 5 to 7, for example about 6; The volume / mass (mL / g) ratio of the buffer salt aqueous solution to the gynostemma pentaphyllum leaves is preferably 7 to 20, for example about 8; The mass ratio of the cross-linked povidone to the gynostemma pentaphyllum leaves is preferably 1 to 2, for example about 1.5; The grinding is more preferably performed on ice; The first centrifuging speed is more preferably 8000 to 12000 r / min, for example about 10000 r / min; More preferably, the temperature of the first centrifugation is 0-10℃, for example about 4℃; More preferably, the time of the first centrifugation is 2-10min, for example about 5min; More preferably, the speed of the second centrifugation is 12000-16000r / min, for example about 14000r / min; More preferably, the temperature of the second centrifugation is 0-10℃, for example about 4℃; More preferably, the time of the second centrifugation is 5-15min, for example about 10min.

6. Use of the gypenoside glucosidase of any one of claims 1 to 5 in hydrolyzing 1,2 linked diglucosyl.

7. Use according to claim 6, characterized in that, The hydrolysis comprises the following steps: weighing an appropriate amount of substrate, adding the first solvent and the gypenoside crude enzyme solution, mixing, water bath reaction for a period of time, and obtaining the rare saponin product.

8. Use according to claim 7, characterized in that, The substrate is gypenoside extract; Preferably, the substrate concentration is 1-3mg / mL, for example about 2mg / mL; Preferably, the first solvent is a citric acid-sodium citrate buffer saline solution; Preferably, the volume ratio of the citric acid-sodium citrate buffer solution to the crude enzyme solution is 8-10, for example about 9; Preferably, the temperature of the water bath reaction is 40-60℃, for example about 50℃; Preferably, the time of the water bath reaction is 3-5h, for example about 4h.

9. Use according to claim 6, characterized in that, The gypenoside glucosidase hydrolyzes the C-3 position glucosyl; Preferably, the substrate of the gypenoside glucosidase solution is the protopanaxadiol type gypenoside; Preferably, the gypenoside glucosidase solution does not hydrolyze the gypenoside with acetyl or malonyl modification; Preferably, the action site of the gypenoside glucosidase solution is the C-3 position glucosidic bond of the protopanaxadiol type gypenoside; Preferably, the glucosidic bond is C3-(1,2) diglucosidic bond.

10. Use according to claim 6, characterized in that, The application comprises the conversion to generate rare saponins, the deglucosylation of glycosides and / or the structural identification of glycosides; Preferably, the rare saponin is a C-3 position deglucosylated rare saponin; Preferably, the rare saponin comprises gypenoside LXXIV, gypenoside XIII, gypenoside TN-1 and / or ginsenoside CK.

Images