Prunella vulgaris oligosaccharide, oligosaccharide composition and application

Abstract

The invention relates to the technical field of traditional Chinese medicine oligosaccharide, in particular to selfheal oligosaccharide, an oligosaccharide composition and application. The selfheal oligosaccharide provided by the invention is obtained through polysaccharide extraction, hydrolysis and preparative chromatographic separation, and through specific parameters such as hydrolysis conditions and chromatographic separation conditions, the obtained selfheal oligosaccharide has a good in-vitro anti-oxidation effect and an effect of regulating intestinal flora, and has a good application prospect and development value. The invention further provides the selfheal oligosaccharide with a specific structure, the antioxidant activity of the selfheal oligosaccharide also has a synergistic effect under the condition of combined use, and the selfheal oligosaccharide has a good application prospect in the fields of food, health care products, medical products and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine oligosaccharides, and particularly relates to a Prunella vulgaris oligosaccharide, an oligosaccharide composition and an application thereof. Background Art

[0002] Prunella vulgaris is one of the traditional Chinese medicines in China and is often used to treat symptoms such as liver-fire stagnation, red and swollen eyes, acute hepatitis and hypertension. So far, researchers have isolated about 200 compounds from Prunella vulgaris, most of which are triterpenoids, sterols and flavonoids. In addition, there are also coumarins, phenylpropanoids, polysaccharides and volatile oils. Compared with other components, there are few studies on the pharmacological effects of Prunella vulgaris polysaccharides at present. The proven pharmacological activities mainly include antioxidant, immunomodulatory, antiviral and antitumor effects.

[0003] Oligosaccharides are low-degree polymers formed by connecting 2-10 monosaccharide molecules through glycosidic bonds, and their structure is between monosaccharides and polysaccharides. Different oligosaccharides have different pharmacological activities due to differences in monosaccharide composition, monosaccharide arrangement order, glycosidic bond type, etc. According to biological functions, oligosaccharides can be divided into two categories: ordinary oligosaccharides and functional oligosaccharides. Some of these functional oligosaccharides have been proven by modern pharmacological research to have biological activities such as antitumor, anti-inflammatory, antibacterial and immune enhancement, showing good application prospects. However, there are few studies on Prunella vulgaris oligosaccharides at present. It is still unclear whether pharmacologically active oligosaccharides can be hydrolyzed from Prunella vulgaris polysaccharides, and further research and development are needed to obtain more oligosaccharide components with pharmacological activities and provide more data references for the pharmacological research of Prunella vulgaris. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a Prunella vulgaris oligosaccharide, an oligosaccharide composition and an application thereof. The Prunella vulgaris oligosaccharide provided by the present invention has good in vitro antioxidant effect and intestinal flora regulation effect, and has good application prospects and development value. In addition, when the Prunella vulgaris oligosaccharide with a specific structure provided by the present invention is used in combination, its antioxidant activity also has a synergistic effect.

[0005] To achieve the above invention purpose, the present invention adopts the following technical solutions: In the first aspect of the present invention, a Prunella vulgaris oligosaccharide is provided, and its preparation method includes the following steps: S1. Extract the spike of Prunella vulgaris with water. After solid-liquid separation, add ethanol to the obtained liquid phase until the alcohol concentration is 78%-82%, let it stand for precipitation, then remove the protein from the obtained precipitate, and retain the polysaccharide with a molecular weight greater than 3500 Da through dialysis; S2. Dissolve the polysaccharide obtained in S1 in an aqueous solution of 0.45 - 0.55 M trifluoroacetic acid (TFA), and then hydrolyze it at 80 ± 2 °C and 100 ± 2 °C for 2.5 - 3.5 h respectively. Recover the solvent, and dry the obtained samples respectively to obtain the mixed oligosaccharides ZTW-80 and ZTW-100; S3. Dissolve the mixed oligosaccharides ZTW-80 and ZTW-100 in water and perform preparative chromatography separation. The chromatographic conditions of the preparative chromatography are as follows: Chromatographic column: amino chromatographic column; Mobile phase A is an aqueous solution of 0.08% - 0.12% v / v formic acid, and mobile phase B is acetonitrile with 0.08% - 0.12% v / v formic acid; Negative ion mode; Elution gradient: 0–20.0 min: 85% phase B; 20.0–32.0 min: 85%–72% phase B; 32.0–42.0 min: 72% phase B; 42.0–57.0 min: 72%–69% phase B; 57.0–63.0 min: 69%–65% phase B; 63.0–73.0 min: 65%–62% phase B; 73.0–74.0 min: 62%–50.0% phase B; 74.0–114.0 min: 50% phase B; Flow rate is 2.4 - 2.6 mL / min; When performing the preparative chromatography separation on the mixed oligosaccharide ZTW-80, collect the eluate within 0 - 114.0 min; When performing the preparative chromatography separation on the mixed oligosaccharide ZTW-100, collect the eluate within 23.9 - 31.8 min; The obtained eluate contains the Prunella vulgaris oligosaccharide.

[0006] The Prunella vulgaris polysaccharide prepared by the above method has significant antioxidant effects, can regulate the intestinal flora, promote the growth of probiotics, and does not promote the growth of pathogenic bacteria, and has good application prospects and development value in the fields of food, health products, pharmaceutical products, etc.

[0007] Preferably, the water extraction method in S1 is ultrasonic extraction, the extraction temperature is 70 ± 5 °C, the ultrasonic frequency is 40 kHz, and the extraction time is 1 h.

[0008] Preferably, the solid-liquid separation method in S1 is centrifugation, the centrifugation speed is 4000 rpm, and the centrifugation time is 20 min.

[0009] Preferably, ethanol is added to the obtained liquid phase in S1 until the alcohol concentration reaches 80%.

[0010] Preferably, the concentration of the trifluoroacetic acid aqueous solution in S2 is 0.5 M.

[0011] Preferably, the hydrolysis time in S2 is 3 h.

[0012] Preferably, the chromatographic column in S3 is an Xbridge BEH Amide OBD Prep Column with a specification of 30×250 mm, 5 µm.

[0013] Preferably, the mobile phase A in S3 is an aqueous solution of 0.1% v / v formic acid, and the mobile phase B is acetonitrile of 0.1% v / v formic acid.

[0014] Preferably, the flow rate in S3 is 2.5 mL / min.

[0015] Preferably, when performing the preparative chromatography separation on the mixed oligosaccharide ZTW-80, the eluents at 0 - 20.0 min, 20.0 - 40.0 min, 40.0 - 60.0 min, or 60.0 - 80.0 min are collected. The prunella vulgaris oligosaccharides contained in the above eluents have excellent antioxidant activity, and the ability to scavenge DPPH free radicals at 0.01 mg / mL is almost equivalent to that of vitamin C.

[0016] Among them, the prunella vulgaris oligosaccharides contained in the eluent at 0 - 20.0 min have the strongest antioxidant activity. Its DPPH free radical scavenging activity at 0.01 mg / mL can reach 90.45% ± 0.98%, and its DPPH free radical scavenging activity at 0.1 mg / mL is 92.53% ± 0.57%. Moreover, the prunella vulgaris oligosaccharides contained in this eluent can significantly increase the expression level of Bacteroidetes at the phylum level of the flora, and can significantly increase the expression levels of Bifidobacterium and Akkermansia at the genus level of the flora.

[0017] The prunella vulgaris oligosaccharides contained in the eluent at 20.0 - 40.0 min also have high antioxidant activity. Its DPPH free radical scavenging activity at 0.05 mg / mL can reach 92.15% ± 0.42%, and its DPPH free radical scavenging activity at 0.1 mg / mL is 92.65% ± 0.40%. In addition, the prunella vulgaris oligosaccharides contained in this eluent can significantly increase the expression level of Lactobacillus at the genus level of the flora.

[0018] The prunella vulgaris oligosaccharides contained in the eluent at 40.0 - 60.0 min have a DPPH free radical scavenging activity of 90.95% ± 0.18% at 0.1 mg / mL, and can also significantly increase the expression level of Lactobacillus at the genus level of the flora.

[0019] The prunella vulgaris oligosaccharides contained in the eluent at 60.0 - 80.0 min have a DPPH free radical scavenging activity of 91.02% ± 0.83% at 0.1 mg / mL.

[0020] Preferably, when performing the preparative chromatography separation on the mixed oligosaccharide ZTW-80, collect the eluate at 40.0 - 60.0 min and perform the operation of S4: S4. Perform preparative chromatography separation on the eluate at 40.0 - 60.0 min. The chromatographic conditions of the preparative chromatography are as follows: Chromatographic column: amino chromatographic column; Mobile phase A is an aqueous solution of 0.08% - 0.12% v / v formic acid, and mobile phase B is acetonitrile with 0.08% - 0.12% v / v formic acid; Negative ion mode; Elution gradient: 0 - 40.0 min: 82% phase B; 40.0 - 50.0 min: 82% - 80% phase B; 50.0 - 60.0 min: 80% - 75% phase B; 60.0 - 80.0 min: 75% phase B; 80.0 - 90.0 min: 75% - 72% phase B; 90.0 - 95.0 min: 72% - 50% phase B; 95.0 - 105.0 min: 50% phase B; Flow rate is 2.4 - 2.6 mL / min; Collect the eluate at 39.0 - 41.0 min and 70.0 - 74.5 min, and the obtained eluate contains the Prunella vulgaris oligosaccharide.

[0021] Preferably, the chromatographic column in S4 is Xbridge BEH Amide OBD Prep Column, with a specification of 30×250 mm, 5 µm.

[0022] Preferably, in S4, the mobile phase A is an aqueous solution of 0.1% v / v formic acid, and the mobile phase B is acetonitrile with 0.1% v / v formic acid.

[0023] Preferably, the flow rate in S4 is 2.5 mL / min.

[0024] In the second aspect of the present invention, a Prunella vulgaris oligosaccharide is provided, and its structure is as shown in Formula I:

[0025] Formula I R1 is hydrogen or methyl; When R1 is hydrogen, R2 is hydrogen or methyl, where: when R2 is hydrogen, R3 is -Xyl (xylosyl), R4 is hydrogen, R5 is methyl, R6 is carboxyl, and R7 is hydrogen; when R2 is methyl, R3 is hydrogen, R4 is carboxyl, and R5, R6, R7 are hydrogen; When R1 is methyl, R2 and R3 are hydrogen, R4 is carboxyl, R5 and R6 are hydrogen, and R7 is -Xyl-Xyl-GlcA (glucuronyl).

[0026] The Prunella vulgaris oligosaccharide prepared by the above preparation method contains the Prunella vulgaris oligosaccharide shown in Formula I. This Prunella vulgaris oligosaccharide has good in vitro antioxidant effects, can effectively scavenge free radicals, and has good application prospects and development value in the fields of food, health products, pharmaceutical products, etc.

[0027] Preferably, when R1 is hydrogen and R2 is hydrogen, the structure of the Prunella vulgaris oligosaccharide is β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- β -D-GlcA p -4-OMe-(1→ or β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- α -D-GalA p -4-OMe-(1→. This Prunella vulgaris oligosaccharide has good in vitro antioxidant effects. Its DPPH free radical scavenging activity is 84.55% ± 4.02% at 0.1 mg / mL, and it also has the effect of regulating the intestinal flora, and can significantly increase the expression level of Bacteroidetes at the phylum level of the flora. When preparing this Prunella vulgaris oligosaccharide by the above preparation method, the mixed oligosaccharide ZTW-80 is subjected to preparative chromatography separation, and the eluate at 40.0 - 60.0 min is further subjected to preparative chromatography separation according to the method of S4 above, and the eluate at 39.0 - 41.0 min is collected, and the obtained eluate contains this Prunella vulgaris oligosaccharide.

[0028] Preferably, when R1 is hydrogen and R2 is methyl, the structure of the Prunella vulgaris oligosaccharide is α -D-GlcA p -3-OMe-(1→2)- β -D-Xyl p or α -D-GlcA p -3-OMe-(1→2)- α -D-Xyl p . This Prunella vulgaris oligosaccharide has in vitro antioxidant effects and can also significantly increase the expression level of Lactobacillus at the genus level of the flora. When preparing this Prunella vulgaris oligosaccharide by the above preparation method, when the mixed oligosaccharide ZTW-100 is subjected to preparative chromatography separation, the eluate at 23.9 - 31.8 min contains this Prunella vulgaris oligosaccharide.

[0029] Preferably, when R1 is methyl, the structure of the Prunella vulgaris oligosaccharide is α -D-GlcA p-2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- α -D-GlcA p- 3-OMe-(1→ or α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-GlcA p- 3-OMe-(1→. The Prunella vulgaris oligosaccharide has good in vitro antioxidant activity, and its DPPH free radical scavenging activity is 89.73% ± 1.94% at 0.1 mg / mL. When preparing the Prunella vulgaris oligosaccharide by the above preparation method, the mixed oligosaccharide ZTW-80 is subjected to preparative chromatography separation, and the eluate at 40.0 - 60.0 min is further subjected to preparative chromatography separation according to the method of S4 above, and the eluate at 70.0 - 74.5 min is collected, and the Prunella vulgaris oligosaccharide is contained in the obtained eluate.

[0030] The third aspect of the present invention provides an oligosaccharide composition comprising at least one of the above Prunella vulgaris oligosaccharides.

[0031] Preferably, the oligosaccharide composition is composed of the Prunella vulgaris oligosaccharide represented by the formula I.

[0032] More preferably, the oligosaccharide composition is composed of the Prunella vulgaris oligosaccharide with the structure of β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- β -D-GlcA p -4-OMe-(1→ or β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- α -D-GalA p -4-OMe-(1→, and the structure is α -D-GlcA p-3-OMe-(1→2)- β -D-Xyl p or α -D-GlcA p -3-OMe-(1→2)- α -D-Xyl p of Prunella vulgaris oligosaccharides and the structure is α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- α -D-GlcA p- 3-OMe-(1→or α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-GlcA p- 3-OMe-(1→of Prunella vulgaris oligosaccharides are composed in an equal mass ratio.

[0033] The fourth aspect of the present invention provides the application of the above Prunella vulgaris oligosaccharides or oligosaccharide compositions, including: In the preparation of antioxidant products; Application in the preparation of products for regulating intestinal flora.

[0034] The above Prunella vulgaris oligosaccharides have good antioxidant activity. In the case of combined use, their antioxidant activity also has a synergistic effect. Therefore, the above Prunella vulgaris oligosaccharides and oligosaccharide compositions can be used to prepare antioxidant products.

[0035] The above Prunella vulgaris oligosaccharides can significantly increase the abundance of probiotics and reduce the abundance of pathogenic bacteria. Therefore, they can be used to prepare products for regulating intestinal flora.

[0036] Preferably, the form of the product includes medicines, health foods, and foods.

[0037] The beneficial effects of the present invention are as follows: The present invention uses the spike of Prunella vulgaris as a raw material to prepare Prunella vulgaris polysaccharide with a molecular weight greater than 3500 Da, and then performs partial acid hydrolysis with TFA at 80 ± 2 °C and 100 ± 2 °C respectively to prepare oligosaccharide fragments, and then uses preparative chromatography for separation and purification to obtain purified oligosaccharides with different degrees of polymerization. The obtained Prunella vulgaris oligosaccharides have good antioxidant activity and also have a regulatory effect on the intestinal flora. The present invention provides new ideas for the preparation and application of Prunella vulgaris oligosaccharides and has reference significance for the development of Prunella vulgaris oligosaccharides. Brief Description of the Drawings

[0038] Figure 1 is the TIC diagram of dividing the ZTW-80 sample into 5 oligosaccharide samples in Example 1 of the present invention by HPLC-MS; Figure 2 is the TIC diagram of detecting the F3 segment sample by HPLC-MS in Example 1 of the present invention; Figure 3 is the TIC diagram of detecting the ZTW-100 sample by HPLC-MS in Example 1 of the present invention; Figure 4 is the UHPLC-UV (250 nm) spectrum of 10 mixed monosaccharide standards and the PMP derivative of DP2 in Example 1 of the present invention: (A) Monosaccharide composition of 10 mixed monosaccharide standards; (B) Monosaccharide composition of DP2 sample; Figure 5 is the UHPLC-UV (250 nm) spectrum of 10 mixed monosaccharide standards and the PMP derivative of DP3 in Example 1 of the present invention: (A) Monosaccharide composition of 10 mixed monosaccharide standards; (B) Monosaccharide composition of DP3 sample; Figure 6 is the UHPLC-UV (250 nm) spectrum of 10 mixed monosaccharide standards and the PMP derivative of DP5 in Example 1 of the present invention: (A) Monosaccharide composition of 10 mixed monosaccharide standards; (B) Monosaccharide composition of DP5 sample; Figure 7 is the base peak chromatogram of the DP2, DP3, and DP5 samples in Example 1 of the present invention under the negative ion HILIC / IM-QTOF-HDMS E mode; Figure 8 is the MS / MS diagram of the DP2, DP3, and DP5 samples in Example 1 of the present invention under the negative ion HILIC / IM-QTOF-HDMS E mode; Figure 9 is for DP2 in Example 1 of the present invention 1 1H NMR spectrum; Figure 10It is the 13 13C NMR spectrum of DP2 in Example 1 of the present invention; Figure 11 It is the 1 1H- 1 1H COSY spectrum; Figure 12 It is the 1 1H- 13 13C HSQC spectrum; Figure 13 It is the 1 1H- 13 13C HMBC spectrum; Figure 14 It is the DEPT135 spectrum of DP2 in Example 1 of the present invention; Figure 15 It is the 1 1H NMR spectrum of DP3 in Example 1 of the present invention; Figure 16 It is the 13 13C NMR spectrum; Figure 17 It is the 1 1H- 1 1H COSY spectrum; Figure 18 It is the 1 1H- 13 13C HSQC spectrum; Figure 19 It is the 1 1H- 13 13C HMBC spectrum; Figure 20 It is the DEPT135 spectrum of DP3 in Example 1 of the present invention; Figure 21 It is the 1 1H NMR spectrum of DP5 in Example 1 of the present invention; Figure 22 It is the 13 13C NMR spectrum; Figure 23 It is the 1 1H- 1 1H COSY spectrum; Figure 24 It is the 1 1H- 13 13C HSQC spectrum; Figure 25is DP5 in Example 1 of the present invention 1 H- 13 C HMBC diagram; Figure 26 is a diagram of DEPT135 of DP5 in Example 1 of the present invention; Figure 27 is the antioxidant activity of PVPs, ZTW-80, ZTW-100, F1–F5, and DP2, DP3, and DP5 in Example 5 of the present invention; Figure 28 is the in vitro antioxidant activity of DP2, DP3, DP5, and DP2+DP3+DP5 in Example 6 of the present invention; Figure 29 Schematic diagram of the effects of ZTW-80, ZTW-100, F1–F5, DP2, DP3, and DP5 on the richness of intestinal flora in vitro of mice at the phylum (A) and genus (B) levels in Example 7 of the present invention; compared with the 24 h blank: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0040] Oligosaccharides can be hydrolyzed from polysaccharides. Different oligosaccharides can be obtained under different hydrolysis conditions, and different oligosaccharides have different pharmacological activities due to differences in monosaccharide composition, monosaccharide arrangement order, glycosidic bond type, etc. Prunella vulgaris is one of the traditional Chinese medicines in my country. Currently, there are few studies on Prunella vulgaris oligosaccharides. It is not clear whether pharmacologically active oligosaccharides can be hydrolyzed from Prunella vulgaris polysaccharides. This field needs further research and development in order to obtain more pharmacologically active oligosaccharide components.

[0041] The embodiment of the present invention provides a self-peppermint oligosaccharide prepared by the following preparation method, wherein the preparation method comprises the following steps: S1. Extracting the ear of Prunella vulgaris with water, separating the solid from the liquid, adding ethanol to the obtained liquid phase until the alcohol concentration is 78% to 82%, allowing the obtained precipitate to stand for precipitation, removing protein from the obtained precipitate, and retaining polysaccharides with a molecular weight greater than 3500 Da by dialysis; S2. Dissolve the polysaccharide obtained in S1 in an aqueous solution of trifluoroacetic acid at 0.45 - 0.55 M, and then hydrolyze it at 80 ± 2 °C and 100 ± 2 °C for 2.5 - 3.5 h respectively. Recover the solvent, and dry the obtained samples respectively to obtain the mixed oligosaccharides ZTW-80 and ZTW-100; S3. Dissolve the mixed oligosaccharides ZTW-80 and ZTW-100 in water and perform preparative chromatography separation. The chromatographic conditions of the preparative chromatography are as follows: Chromatographic column: amino chromatographic column; Mobile phase A is an aqueous solution of formic acid at 0.08% - 0.12% v / v, and mobile phase B is acetonitrile with 0.08% - 0.12% v / v formic acid; Negative ion mode; Elution gradient: 0–20.0 min: 85% B phase; 20.0–32.0 min: 85%–72% B phase; 32.0–42.0 min: 72% B phase; 42.0–57.0 min: 72%–69% B phase; 57.0–63.0 min: 69%–65% B phase; 63.0–73.0 min: 65%–62% B phase; 73.0–74.0 min: 62%–50.0% B phase; 74.0–114.0 min: 50% B phase; Flow rate is 2.4 - 2.6 mL / min; When performing the preparative chromatography separation on the mixed oligosaccharide ZTW-80, collect the eluate within 0 - 114.0 min; When performing the preparative chromatography separation on the mixed oligosaccharide ZTW-100, collect the eluate within 23.9 - 31.8 min; The obtained eluate contains the Prunella vulgaris oligosaccharide.

[0042] The embodiment of the present invention also provides a Prunella vulgaris oligosaccharide with a specific structure, and its structure is shown in Formula I:

[0043] Formula I R1 is hydrogen or methyl; When R1 is hydrogen, R2 is hydrogen or methyl, where: when R2 is hydrogen, R3 is -Xyl (xylosyl), R4 is hydrogen, R5 is methyl, R6 is carboxyl, and R7 is hydrogen; when R2 is methyl, R3 is hydrogen, R4 is carboxyl, and R5, R6, and R7 are hydrogen; When R1 is methyl, R2 and R3 are hydrogen, R4 is carboxyl, R5 and R6 are hydrogen, and R7 is -Xyl-Xyl-GlcA. This Prunella vulgaris oligosaccharide has good in vitro antioxidant effects, can effectively scavenge free radicals, and also has the effect of regulating the intestinal flora, can significantly increase the abundance of probiotics and reduce the abundance of pathogenic bacteria.

[0044] The embodiment of the present invention also provides an oligosaccharide composition.

[0045] The embodiments of the present invention also provide the applications of the above-mentioned Prunella vulgaris oligosaccharides or oligosaccharide compositions.

[0046] The following specific examples are used for further illustration.

[0047] In the following examples, the Prunella vulgaris spikes used were purchased from Beijing Tongrentang Chinese Medicine Co., Ltd., with the origin in Anhui and the batch number 2024041602.

[0048] Example 1 The embodiments of the present invention provide Prunella vulgaris oligosaccharides and their preparation methods.

[0049] 1. Preparation of Prunella vulgaris polysaccharides 5 kg of Prunella vulgaris spike powder was added with 10 times the amount of water, and under the condition of a water bath at 70 ± 5°C, ultrasonic extraction was carried out at 40 kHz (400 W) for 1 h. After it was cooled to room temperature, centrifugation was carried out at 4000 rpm for 20 min, and the supernatant was collected. 4 times the amount of absolute ethanol was added, and it was allowed to stand for 48 h. The obtained precipitate was dried at 60°C for 30 min. The dried polysaccharide was completely dissolved in water, and Sevage reagent (chloroform∶n-butanol = 4∶1 ( v / v )) was added and vortexed thoroughly. Centrifugation was carried out at 4000 rpm for 10 min to remove proteins, and the supernatant was collected. A dialysis bag with a cut-off molecular weight of 3500 Da was selected for dialysis for 48 h, and the retentate was freeze-dried to obtain a Prunella vulgaris polysaccharide powder sample, named PVPs.

[0050] 2. Preparation of total extract of partially acid-hydrolyzed oligosaccharides Using the Prunella vulgaris polysaccharide powder sample obtained in step 1 as the substrate for oligosaccharide sample preparation: 20 g of the Prunella vulgaris polysaccharide powder sample was weighed in two portions, 1.2 mL of TFA (0.5 M) was added, and it was vortexed and dissolved thoroughly. The obtained solutions were heated and hydrolyzed at 80°C and 100°C in a water bath for 3 h respectively. A rotary evaporator was used to recover the solvent under reduced pressure at 60°C. During the evaporation process, absolute ethanol was added to accelerate the recovery rate. After the sample was recovered until there was no alcohol smell and no sour smell, it was freeze-dried to obtain hydrolyzed samples (respectively marked as: ZTW-80, ZTW-100).

[0051] 3. Preparation of oligosaccharide samples 3.1 Preparation of F1~F5, DP3, DP5 Dissolve the ZTW-80 sample in 100 mL of water to prepare a test solution of approximately 200 mg / mL, centrifuge at 14,000 rpm for 10 min and filter using a 0.22 μm microporous membrane. Use Agilent semi-preparative HPLC for the preparation of oligosaccharides. The chromatographic conditions are as follows: Chromatographic column: Xbridge BEH Amide OBD Prep Column (30×250 mm, 5 µm); Injection volume: 100 µL; Mobile phase: Phase A: 0.1% FA-H2O; Phase B: 0.1% FA-ACN; Negative ion mode; Flow rate: 2.5 mL / min. The elution gradient is as follows: 0–20.0 min: 85% (B); 20.0–32.0 min: 85%–72% (B); 32.0–42.0 min: 72% (B); 42.0–57.0 min: 72%–69% (B); 57.0–63.0 min: 69%–65% (B); 63.0–73.0 min: 65%–62% (B); 73.0–74.0 min: 62%–50.0% (B); 74.0–114.0 min: 50% (B). The TIC diagram of the ZTW-80 sample is as shown in Figure 1 shown. Collect the eluate at the following times: F1, 0–20 min; F2, 20–40 min; F3, 40–60 min; F4, 60–80 min; F5, 80–114 min. Use a rotary evaporator to recover the solvent from the collected eluate and lyophilize it to obtain Prunella vulgaris oligosaccharide samples F1~F5.

[0052] Dissolve the sample in section F3 in 100 mL of water to prepare a test solution of approximately 200 mg / mL, centrifuge at 14,000 rpm for 10 min and filter using a 0.22 μm microporous membrane. Use Agilent semi-preparative HPLC for the preparation of oligosaccharides DP3 and DP5. The chromatographic conditions are as follows: Chromatographic column: Xbridge BEH Amide OBD Prep Column (30×250 mm, 5 µm); Injection volume: 100 µL; Mobile phase: Phase A: 0.1% FA-H2O; Phase B: 0.1% FA-ACN; Negative ion mode; The elution gradient is as follows: 0–40.0 min: 82% (B); 40.0–50.0 min: 82%–80% (B); 50.0–60.0 min: 80%–75% (B); 60.0–80.0 min: 75% (B); 80.0–90.0 min: 75%–72% (B); 90.0–95.0 min: 72%–50% (B); 95.0–105.0 min: 50% (B). The TIC diagram of the sample in section F3 is as shown in Figure 2As shown, Prunella vulgaris oligosaccharides with different degrees of polymerization exist in the F3 segment. The eluates at 39.0 - 41.0 min and 70.0 - 74.5 min were collected, and the solvents were recovered and freeze-dried respectively to obtain Prunella vulgaris oligosaccharide samples DP3 and DP5.

[0053] 3.2 Preparation of DP2 The ZTW-100 sample was dissolved in 100 mL of water to prepare a test solution of about 200 mg / mL, centrifuged at 14,000 rpm for 10 min and filtered using a 0.22 μm microporous membrane. The oligosaccharide DP2 was prepared using Agilent semi-preparative HPLC. Chromatographic conditions: Column: Xbridge BEH Amide OBD Prep Column (30×250 mm, 5 µm); Injection volume: 100 µL; Mobile phase: Phase A: 0.1% FA-H2O; Phase B: 0.1% FA-ACN; Negative ion mode; Elution gradient as follows: 0–20.0 min: 85% (B); 20.0–32.0 min: 85–72% (B); 32.0–42.0 min: 72% (B); 42.0–57.0 min: 72–69% (B); 57.0–63.0 min: 69–65% (B); 63.0–73.0 min: 65–62% (B); 73.0–74.0 min: 62–50.0% (B); 74.0–114.0 min: 50% (B); Room temperature: 25°C; Flow rate: 2.5 mL / min. The TIC diagram is as Figure 3 shown. The eluate at 23.9–31.8 min was collected, the solvent was recovered and freeze-dried to obtain the Prunella vulgaris oligosaccharide sample DP2.

[0054] 4. Monosaccharide composition analysis 4.1 Liquid phase detection Data collection was carried out on an Agilent 1290 ultra-high performance liquid chromatograph equipped with a DAD detector; the detection wavelength was 250 nm, the chromatographic column was Waters XBridge® Shield RP18 (4.6×150 mm, 3.5 µm), the mobile phase was 10 mM AA-water (A) / ACN (B), the sample tray temperature was 10°C, the column temperature was 28°C, the flow rate was 1 mL / min, the injection volume was 3 µL, and the elution gradient: 0–45.0 min: 17% (B).

[0055] Analysis of the results of the monosaccharide composition: As Figure 4As shown in the figure, the DP2 sample of Prunella vulgaris oligosaccharide contains five monosaccharides: Man (25.71%), GlcA (11.23%), Glc (17.17%), Gal (28.13%) and Xyl (17.76%).

[0056] As Figure 5 shown, the DP3 sample of Prunella vulgaris oligosaccharide contains six monosaccharides: Rha (11.27%), GlcA (12.62%), GalA (14.53%), Gal (19.57%), Xyl (20.61%), Ara (21.41%).

[0057] As Figure 6 shown, the DP5 sample of Prunella vulgaris oligosaccharide contains five monosaccharides: Man (23.06%), GlcA (16.00%), Glc (5.63%), Gal (11.13%), Xyl (44.20%).

[0058] It is speculated from the above analysis results that the structures of DP2 (339.0926) sample, DP3 (471.1345) sample and DP5 (793.2273) sample are relatively complex and may contain other structural oligosaccharides. Since Prunella vulgaris polysaccharide is relatively complex, DP2, DP3 and DP5 may also contain free monosaccharides or other impurities. Therefore, it is necessary to comprehensively analyze the structural information by combining mass spectrometry and nuclear magnetic resonance.

[0059] 4.2 Mass Spectrometry Analysis Mass spectrometry analysis was carried out on a Waters ACQUITY UPLC I-Class / Vion IMS-QTOF system.

[0060] The specific chromatographic conditions are as follows: chromatographic column: BEH Amide (2.1×100 mm, 1.7 µm); mobile phase: 0.1% FA-water (A) / 0.1% FA-ACN (B); sample disk temperature: 4°C; column temperature: 30°C; flow rate: 0.3 mL / min; injection volume: 3 µL; elution gradient: 0–5.0 min: 90% (B); 5.0–7.0 min: 90%–85% (B); 7.0–10.0 min: 85%–80% (B); 10.0–18.0 min: 80%–76% (B); 18.0–28.0 min: 76%–70% (B); 28.0–38.0 min: 70%–60% (B); 38.0–41.0 min: 60% (B).

[0061] Mass spectrometry conditions: ion source: ESI source; acquisition mode: HDMS E (negative ion mode); capillary voltage: −1.5 kV; cone voltage: −60 V; MS1 Collision energy: 6 eV, MS 2 Collision dissociation energy: 20–40 eV; scanning range m / z : 150–2000, scanning time: 0.3 s; ion source temperature: 120 °C; desolvation gas ( N 2) flow rate: 800 L / h, temperature: 500 °C; cone gas ( N 2) flow rate: 50 L / h. External calibration solution: 200 ng / mL leucine enkephalin solution (LE, Leucine-Enkephalin, Sigma-Aldrich, St. Louis, MO, USA), flow rate: 10 μL / min, interval time: 1 min.

[0062] Experimental results: By collecting data on the samples, BPC graphs of DP2, DP3, and DP5 can be obtained ( Figure 7 ), and the structures of DP2, DP3, and DP5 oligosaccharides can be speculated based on the combined secondary fragments ( Figure 8 ). As shown in Figure A of Figure 8 , the MS 2 mass spectrum in the negative ion mode shows the fragmentation fragments of the parent ion 339.0926. The fragment 207.05 (C1 − ), and the mass number of the neutral loss on the sugar backbone of 90.03 Da ( 1,3 A2) are observed. It is speculated that the structure of DP2 is GlcA-Xyl. As shown in Figure B of Figure 8 , the MS 2 mass spectrum in the negative ion mode shows the fragmentation fragments of the parent ion 471.1372. The fragment 249.06 and the mass numbers of the neutral losses on the sugar backbone of 60.02 Da ( 2,4 A1), 78.03 Da ( 2,4 A1−H2O] − ) are observed. It is speculated that the structure of DP3 may be Xyl-Ara-GlcA or Xyl-Ara-GalA. As shown in Figure C of Figure 8 , the MS 2 mass spectrum in the negative ion mode shows the fragmentation fragments of the parent ion 793.2274. The fragments 249.06, 471.13 (C3 − ), 603.18 (C4 − ) and the mass numbers of the neutral losses on the sugar backbone of 60.02 Da ( 0,2 A5), 78.03 Da ( 0,2 A5−H2O] − ) are observed. It is speculated that the structure of DP5 may be GlcA-Xyl-Xyl-Xyl-GlcA.

[0063] 4.3 NMR Analysis Data Acquisition: Dissolve the DP2, DP3, and DP5 samples (about 35 mg) in D2O (99.9% atom % D, containing 0.05% TMSP, Tris(trimethylsilyl)phosphate, 3(3-methylsilyl)phosphate), centrifuge at 14,000 rpm for 10 min, and transfer to an NMR tube. Use a Bruker AvanceⅢ-600 nuclear magnetic resonance spectrometer to collect the one-dimensional spectra ( 1 H, 13 C, DEPT135) and two-dimensional correlation spectra ( 1 H- 1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC) of the samples. The results are processed and analyzed using MestReNova.

[0064] Data Analysis: The 1D / 2D NMR spectra and data of DP2 are shown in Figures 9 to 14 and Table 1. The 1D / 2D NMR spectra show that there are seven anomeric hydrogen signals in the range of 4.4–5.4 ppm at the low-field end. According to the monosaccharide composition of DP2, these seven anomeric carbon-hydrogen signals are: A1- α -D-GlcA ( δ 4.70 ppm; 96.86 ppm), B1- α -D-Xyl ( δ 5.07 ppm; 96.58 pmm), C1- α -D-GlcA ( δ 4.70 ppm; 96.86 ppm), D1- β -D-Xyl ( δ 4.63 ppm; 95.95 ppm), E1- α -D-Man ( δ 5.19 ppm; 94.07 ppm), F1- α -D-Gal ( δ 5.36 ppm; 97.74 ppm), G- α -D-Glc ( δ 4.58 ppm; 96.49 ppm). The E value shown on IM-QTOF-HDMS m / z is [M−H] −(Measured value 339.0926 / theoretical value 339.0928), the molecular formula of DP2 is C 12 H 20 O 11 , and according to the secondary fragment information, the structure is inferred to be GlcA-Xyl. In the hydrogen spectrum, 4.70, 5.07, and 4.63 are the anomeric hydrogen signals of two conformations of DP2, respectively, and 1 The integral ratio of H is approximately 0.70∶0.38∶0.32, which satisfies this inference. 1 H- 13 The H- δ C HMBC spectrum shows the correlation signals between C1 of residual sugar A / C and H2 of residual sugar B / D ( 1 H- 13 CHSQC spectrum shows the C / H correlation signal of -OCH3 ( δ 3.48 / 59.86 ppm), and in the 1 H- 13 C HMBC spectrum shows the correlation signal between -OCH3-H and A / C-C3 ( δ 3.47 / 72.74 ppm). To sum up, the structure of DP2 is inferred to be: α -D-GlcA p -3-OMe-(1→2)- β -D-Xyl p or α -D-GlcA p -3-OMe-(1→2)- α -D-Xyl p , and the two will dynamically interconvert in the solution state and show two forms in the NMR signals.

[0065] Table 1 Chemical shift assignment of DP2

[0066] The 1D / 2D NMR spectra and data of DP3 are shown in Figures 15 to 20 and Table 2. Similarly, the 1D / 2D NMR spectra show that there are six anomeric hydrogen signals in the range of 4.4–5.4 ppm at the low-field end. According to the monosaccharide composition of DP3, these six anomeric carbon-hydrogen signals are: A1- β -D-Xyl ( δ 4.46 ppm; 96.59 ppm), B1- β -D-Ara ( δ5.20 ppm; 97.60 ppm), C1- β -D-GlcA ( δ 4.47 ppm; 101.58 ppm), D1- β -D-Xyl ( δ 4.46 ppm; 96.59 ppm), E1- β -D-Ara ( δ 5.20 ppm; 97.60 ppm), F1- α -D-GalA ( δ 5.18 ppm; 97.60 ppm). Its E value shown on IM-QTOF-HDMS m / z is [M−H] − (measured value 471.1372 / theoretical value 471.1350), and the molecular formula of DP3 is determined to be C 17 H 28 O 15 . Based on the secondary fragment information, the structures are inferred to be Xyl-Ara-GlcA and Xyl-Ara-GalA. 1 H- 13 C HMBC spectrum shows the correlation signals between C1 of the residual sugar A / D and H4 of the residual sugar B / E ( δ 4.11 / 96.59 ppm), further confirming that the connection modes between the residual sugars A and B and D and E are 1→4 linkages. There is a correlation signal peak at δ 3.39 / 97.69 ppm between C1 of the residual sugar B and H2 of the residual sugar C, indicating the presence of →4)- β -D-Ara f- (1→2)- β -D-GlcA p -(1→ linkage. In addition, there is a correlation signal peak between C1 of the residual sugar E and H2 of the residual sugar F ( δ 3.41 / 97.60 ppm), indicating the presence of →4)- β -D-Ara f -(1→2)- α -D-GalA p -(1→ linkage. In 1 H- 13 C HSQC spectrum, there is a C / H correlation signal of -OCH3 ( δ 3.38 / 59.40 ppm). In 1 H- 13 C HMBC spectrum, it shows -OCH3-C and C-H4 ( δ3.36 / 59.40 ppm), -OCH3-C and F-H4 ( δ The correlation signal between 3.67 / 59.40 ppm) indicates the presence of →2)- β -D-GlcA p -4-OMe-(1→ and →2)- α -D-GalA p -4-OMe-(1→. In summary, the structure of DP3 is inferred to be: β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- β -D-GlcA p -4-OMe-(1→ or β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- α -D-GalA p -4-OMe-(1→.

[0067] Table 2 Chemical shift assignments of DP3

[0068] The 1D / 2D NMR spectra and data of DP5 are shown in Figures 21 to 26 and Table 3. Similarly, the 1D / 2D NMR spectra show that there are ten anomeric hydrogen signals in the range of 4.4–5.4 ppm at the low-field end. According to the monosaccharide composition of DP5, these ten anomeric carbon-hydrogen signals are: A1- α -D-GlcA ( δ 5.31 ppm; 97.91 ppm), B1- β -D-Xyl ( δ 4.63 ppm; 101.28pmm), C1- β -D-Xyl ( δ 4.64 ppm; 101.28 ppm), D1- β -D-Xyl ( δ 4.63 ppm; 101.14 ppm), E1- α -D-GlcA ( δ 5.34 ppm; 97.90 ppm), F1- α -D-GlcA ( δ 5.31 ppm; 97.91 ppm), G1- β -D-Xyl ( δ4.63 ppm; 101.28 ppm), H1- β -D-Xyl( δ 4.64 ppm; 101.28 ppm), I1- β -D-Xyl( δ 4.63 ppm; 101.14 ppm), J1- β -D-GlcA( δ 4.48 ppm; 101.69 ppm). The E value shown on IM-QTOF-HDMS m / z is [M−H] − (measured value 793.2274 / theoretical value 793.2250), and the molecular formula of DP5 is determined to be C 29 H 46 O 25 . Based on the secondary fragment information, the structure is inferred to be GlcA-Xyl-Xyl-Xyl-GlcA. 1 H- 13 C HMBC spectrum shows the correlation signals between H1 of the residual sugars A / F and C2 of the residual sugars B / G ( δ 5.32 / 76.84 ppm), further confirming that the linkage between the residual sugars A and B and F and G is 1→2 linkage. There is a correlation signal peak at δ 4.63 / 76.12 ppm between H1 of the residual sugars B / G and C4 of the residual sugars C / H, indicating the presence of →2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→ linkage. There is a correlation signal peak between H1 of the residual sugars C / H and C4 of the residual sugars D / I ( δ 4.64 / 76.23 ppm), indicating the presence of →4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→ linkage. The correlation signal between C1 of the residual sugars D / I and H4 of the residual sugars E / J ( δ 3.83, 3.80 / 101.14 ppm) further confirms that the linkage between the residual sugars D and E and I and J is 1→4 linkage. In 1 H- 13 C HSQC spectrum, there is a C / H correlation signal of -OCH3 ( δ 3.54 / 59.71 ppm). In 1 H- 13The HMBC spectrum shows the correlation signals between -OCH3-H and A / F-C2 ( δ 3.54 / 71.40 ppm), -OCH3-H and E-C3 ( δ 3.50 / 81.36 ppm), -OCH3-H and J-C3 ( δ 3.54 / 72.35 ppm). In summary, the structure of DP5 is deduced as: α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- α -D-GlcA p- 3-OMe-(1→ or α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-GlcA p- 3-OMe-(1→. This is because when the sample is in a solution state, the terminal OH shows α and β two configurations.

[0069] Table 3 Chemical shift assignments of DP5

[0070] Example 2 The embodiment of the present invention provides Prunella vulgaris oligosaccharide and its preparation method.

[0071] 1. Preparation of Prunella vulgaris polysaccharide 5 kg of Prunella vulgaris spike powder is added with 10 times the amount of water, and under the condition of a 70 ± 5 °C water bath, ultrasonic extraction is carried out at 40 kHz (400 W) for 1 h. After it is cooled to room temperature, it is centrifuged at 4000 rpm for 20 min, and the supernatant is collected. Anhydrous ethanol is added until the alcohol concentration is 78% v / v, and it is left standing for 48 h. The obtained precipitate is dried at 60 °C for 30 min. The dried polysaccharide is completely dissolved in water, and Sevage reagent (chloroform∶n-butanol = 4∶1 ( v / v))Perform sufficient vortex mixing, centrifuge at 4000 rpm for 10 min to remove proteins, collect the supernatant, select a dialysis bag with a molecular weight cut-off of 3500 Da and dialyze for 48 h, and lyophilize the retentate to obtain the Prunella vulgaris polysaccharide powder sample.

[0072] 2. Preparation of the total extract of partially acid-hydrolyzed oligosaccharides Using the Prunella vulgaris polysaccharide powder sample obtained in step 1 as the substrate, prepare the oligosaccharide sample: Weigh two portions of 20 g of the Prunella vulgaris polysaccharide powder sample respectively, add 1.2 mL of TFA (0.45 M), vortex and dissolve thoroughly, and heat and hydrolyze the resulting solutions in a water bath at 80 °C and 100 °C for 2.5 h respectively. Use a rotary evaporator to recover the solvent under reduced pressure at 60 °C, and accelerate the recovery rate by adding anhydrous ethanol during evaporation. After the sample recovers the solvent until there is no alcohol smell and no sour smell, lyophilize it to obtain the hydrolyzed samples (labeled as: ZTW-80 and ZTW-100 respectively).

[0073] 3. Preparation of the oligosaccharide sample 3.1 Preparation of F1~F5, DP3, and DP5 Dissolve the ZTW-80 sample in 100 mL of water to prepare a test solution of about 200 mg / mL, centrifuge at 14,000 rpm for 10 min and filter using a 0.22 μm microporous membrane. Use Agilent semi-preparative HPLC for the preparation of oligosaccharides. The chromatographic conditions are as follows: Chromatographic column: Xbridge BEH Amide OBD Prep Column (30×250 mm, 5 µm); Injection volume: 100 µL; Mobile phase: Phase A: 0.08% FA-H2O; Phase B: 0.12% FA-ACN; Negative ion mode; Flow rate: 2.4 mL / min. The elution gradient is as follows: 0–20.0 min: 85% (B); 20.0–32.0 min: 85%–72% (B); 32.0–42.0 min: 72% (B); 42.0–57.0 min: 72%–69% (B); 57.0–63.0 min: 69%–65% (B); 63.0–73.0 min: 65%–62% (B); 73.0–74.0 min: 62%–50.0% (B); 74.0–114.0 min: 50% (B). Collect the eluate at the following times: F1, 0–20 min; F2, 20–40 min; F3, 40–60 min; F4, 60–80 min; F5, 80–114 min. Use a rotary evaporator to recover the solvent from the collected eluate and lyophilize it to obtain the Prunella vulgaris oligosaccharide samples F1~F5. Prepare DP3 and DP5 from the F3 segment sample according to the method of Example 1.

[0074] 3.2 Preparation of DP2 Dissolve the ZTW-100 sample in 100 mL of water to prepare a test solution of approximately 200 mg / mL, centrifuge at 14,000 rpm for 10 min and filter using a 0.22 μm microporous membrane. Prepare the oligosaccharide DP2 using an Agilent semi-preparative HPLC. Chromatographic conditions: Column: Xbridge BEH Amide OBD Prep Column (30×250 mm, 5 µm); Injection volume: 100 µL; Mobile phase: Phase A: 0.08% FA-H2O; Phase B: 0.12% FA-ACN; Negative ion mode; Elution gradient as follows: 0–20.0 min: 85% (B); 20.0–32.0 min: 85–72% (B); 32.0–42.0 min: 72% (B); 42.0–57.0 min: 72–69% (B); 57.0–63.0 min: 69–65% (B); 63.0–73.0 min: 65–62% (B); 73.0–74.0 min: 62–50.0% (B); 74.0–114.0 min: 50% (B); Room temperature: 25°C; Flow rate: 2.4 mL / min. Collect the eluate from 23.9–31.8 min, recover the solvent, and lyophilize to obtain the Prunella vulgaris oligosaccharide sample DP2.

[0075] Example 3 The embodiments of the present invention provide Prunella vulgaris oligosaccharides and their preparation methods.

[0076] 1. Preparation of Prunella vulgaris polysaccharide Add 5 kg of Prunella vulgaris spike powder to 10 times the amount of water, under the condition of a 70 ± 5°C water bath, ultrasonically extract at 40 kHz (400 W) for 1 h. After cooling to room temperature, centrifuge at 4000 rpm for 20 min, collect the supernatant, add absolute ethanol until the alcohol concentration is 82% v / v, stand for 48 h, dry the obtained precipitate at 60°C for 30 min. The dried polysaccharide is completely dissolved in water, add Sevage reagent (chloroform∶n-butanol = 4∶1 ( v / v )) and vortex thoroughly, centrifuge at 4000 rpm for 10 min to remove proteins, collect the supernatant, select a dialysis bag with a molecular weight cut-off of 3500 Da and dialyze for 48 h, and lyophilize the retentate to obtain the Prunella vulgaris polysaccharide powder sample.

[0077] 2. Preparation of the total extract of partially acid-hydrolyzed oligosaccharides Using the Prunella vulgaris polysaccharide powder sample obtained in Step 1 as a substrate for oligosaccharide sample preparation: Weigh two portions of 20 g Prunella vulgaris polysaccharide powder sample respectively, add 1.2 mL TFA (0.55 M), vortex thoroughly to dissolve, and heat and hydrolyze the resulting solutions in a water bath at 80°C and 100°C for 3.5 h respectively. Use a rotary evaporator to recover the solvent under reduced pressure at 60°C, and add anhydrous ethanol during evaporation to accelerate the recovery rate. After the sample recovers the solvent until there is no alcohol smell and no sour smell, perform freeze-drying to obtain the hydrolyzed samples (labeled as: ZTW-80, ZTW-100).

[0078] 3. Preparation of oligosaccharide samples 3.1 Preparation of F1~F5, DP3, and DP5 Dissolve the ZTW-80 sample in 100 mL of water to prepare a test solution of approximately 200 mg / mL, centrifuge at 14,000 rpm for 10 min and filter using a 0.22 μm microporous membrane. Use Agilent semi-preparative HPLC for the preparation of oligosaccharides. The chromatographic conditions are as follows: Chromatographic column: Xbridge BEH Amide OBD Prep Column (30×250 mm, 5 µm); Injection volume: 100 µL; Mobile phase: Phase A: 0.12% FA-H2O; Phase B: 0.08% FA-ACN; Negative ion mode; Flow rate: 2.6 mL / min. The elution gradient is as follows: 0–20.0 min: 85% (B); 20.0–32.0 min: 85%–72% (B); 32.0–42.0 min: 72% (B); 42.0–57.0 min: 72%–69% (B); 57.0–63.0 min: 69%–65% (B); 63.0–73.0 min: 65%–62% (B); 73.0–74.0 min: 62%–50.0% (B); 74.0–114.0 min: 50% (B). Collect the eluate at the following times: F1, 0–20 min; F2, 20–40 min; F3, 40–60 min; F4, 60–80 min; F5, 80–114 min. Use a rotary evaporator to recover the solvent from the collected eluate and perform freeze-drying to obtain the Prunella vulgaris oligosaccharide samples F1~F5. Prepare DP3 and DP5 from the sample of the F3 segment according to the method of Example 1.

[0079] 3.2 Preparation of DP2 Dissolve the ZTW-100 sample in 100 mL of water to prepare a test solution of approximately 200 mg / mL, centrifuge at 14,000 rpm for 10 min and filter using a 0.22 μm microporous membrane. Prepare oligosaccharide DP2 using Agilent semi-preparative HPLC. Chromatographic conditions: Column: Xbridge BEH Amide OBD Prep Column (30×250 mm, 5 µm); Injection volume: 100 µL; Mobile phase: Phase A: 0.12% FA-H2O; Phase B: 0.08% FA-ACN; Negative ion mode; Elution gradient as follows: 0–20.0 min: 85% (B); 20.0–32.0 min: 85–72% (B); 32.0–42.0 min: 72% (B); 42.0–57.0 min: 72–69% (B); 57.0–63.0 min: 69–65% (B); 63.0–73.0 min: 65–62% (B); 73.0–74.0 min: 62–50.0% (B); 74.0–114.0 min: 50% (B); Room temperature: 25°C; Flow rate: 2.6 mL / min. Collect the eluate from 23.9–31.8 min, recover the solvent, freeze-dry to obtain the Prunella vulgaris oligosaccharide sample DP2.

[0080] Example 4 An embodiment of the present invention provides an oligosaccharide composition, which is composed of Prunella vulgaris oligosaccharides DP2, DP3, and DP5 prepared in Example 1 mixed in an equal mass ratio.

[0081] Example 5 An embodiment of the present invention investigated the antioxidant effect of the Prunella vulgaris oligosaccharide prepared in Example 1.

[0082] Experimental method: Take DPPH (3.9432 mg), dissolve it in absolute ethanol, and make up the volume to 100 mL in a volumetric flask, keep it away from light and store it at -4°C. Dissolve the Prunella vulgaris polysaccharide PVPs, hydrolyzed samples ZTW-80, ZTW-100, and Prunella vulgaris oligosaccharide samples F1~F4, DP2, DP3, and DP5 prepared in Example 1 in water to prepare sample solutions of Prunella vulgaris oligosaccharides DP2, DP3, and DP5 with concentrations of 0.005, 0.01, 0.02, 0.05, and 0.1 mg / mL respectively. Take the sample solutions of each concentration of Prunella vulgaris oligosaccharides DP2, DP3, and DP5 and mix them with 500 μL of DPPH solution. After reacting in the dark for 30 min, measure the absorbance value of the reaction solution at a wavelength of 517 nm. The control group is a mixed solution of different concentrations of the sample and absolute ethanol, and the blank group is a mixed solution of absolute ethanol and DPPH solution. And use vitamin C as a positive control. Calculate the DPPH radical scavenging rate according to the following formula:

[0083] In the formula, A S is the absorbance value of the experimental group; A C is the absorbance of the control group; A0 is the absorbance of the blank group.

[0084] The results are as Figure 27 shown. All samples exhibited a certain degree of hydroxyl radical scavenging ability. Except for DP2, the scavenging ability of other samples against DPPH radicals showed a good dose-dependence (0.005–0.1 mg / mL). For PVPs, ZTW-100, F1–F4, and DP2, DP3, DP5 samples, the scavenging activity > 40.0% at 0.005 mg / mL, showing good antioxidant activity, and the scavenging activity reached the maximum at 0.1 mg / mL. The antioxidant activities of ZTW-80, ZTW-100, F1–F4, DP3, and DP5 were comparable to that of VC at 0.1 mg / mL, and the scavenging activities were respectively: F1: 92.53% ± 0.57%, F2: 92.65% ± 0.40%, F3: 90.95% ± 0.18%, F4: 91.02% ± 0.83%, ZTW-80: 90.96% ± 0.97%, ZTW-100: 91.17% ± 0.30%, DP3: 84.55% ± 4.02%, DP5: 89.73% ± 1.94%.

[0085] Example 6 In this example of the present invention, the antioxidant effect of the Prunella vulgaris oligosaccharide composition was investigated.

[0086] Experimental method: Take DPPH (3.9432 mg) and dissolve it in absolute ethanol, and make up the volume to 100 mL in a volumetric flask, keep it away from light, and store it at −4°C. Dissolve the oligosaccharide composition of Example 2 in water to prepare oligosaccharide composition solutions of 0.005, 0.01, 0.02, 0.05, 0.1 mg / mL, and investigate their DPPH radical scavenging rates according to the experimental method of Example 5.

[0087] The results are as Figure 28 shown. The radical scavenging activity of the oligosaccharide composition was higher than that of the individual samples of DP2, DP3, and DP5, and showed a certain dose-dependence, with the highest at 0.1 mg / mL, which was 94.93% ± 0.42%, higher than that of VC, and the IC 50 was 0.001039 mg / mL. This indicates that there may be a synergistic effect in the in vitro antioxidant activities of Prunella vulgaris oligosaccharides DP2, DP3, and DP5, and the scavenging activity will be enhanced when used in combination.

[0088] Example 7 In the embodiments of the present invention, the effects of the Prunella vulgaris oligosaccharide samples F1-F5, DP2, DP3, and DP5 prepared in Example 1 on the richness of common phylum-level and genus-level bacteria in the intestinal flora were investigated.

[0089] Primer design and synthesis: Specific sequences were designed for common phylum-level and genus-level bacteria in the intestinal flora, and the results are shown in Table 4.

[0090] Table 4 Primer sequence table for common phylum-level and genus-level bacteria in the intestinal flora

[0091] In vitro co-culture of oligosaccharide samples and intestinal flora: An appropriate amount of anaerobic culture medium was prepared, autoclaved at 121 °C for 20 min, cooled, and stored in a refrigerator at 4 °C for later use. Subsequently, various acid hydrolysis sample suspensions with a concentration of 5-10 mg / mL were prepared using the sterilized anaerobic culture medium, vortexed and mixed evenly, and ultrasonicated for 30 min. They were prepared immediately before use. An appropriate amount of fresh mouse feces was placed in an anaerobic culture bag and then transferred to an anaerobic incubator, mixed evenly with sterile saline (feces: saline (m:v) = 1:5), centrifuged at 12000 rpm for 10 min, and the supernatant was taken to obtain the intestinal bacteria incubation solution. The intestinal bacteria incubation solution and the anaerobic culture medium containing the sample to be tested were mixed at a ratio of 1:9 respectively, and samples were taken after culturing under anaerobic conditions at 37 °C for 0 and 24 h. The blank group was the anaerobic culture medium without the sample to be tested and the intestinal bacteria incubation solution for culture.

[0092] Extraction of fecal genomic DNA from samples: The genomic DNA of the fermentation samples was extracted according to the fecal genomic DNA extraction kit, and then the concentration and purity of the DNA were measured with a ultra-micro spectrophotometer, marked on the outside of the centrifuge tube, and stored in a refrigerator at -20 °C.

[0093] Real-time fluorescence quantitative PCR: The qPCR reaction was carried out according to the instructions of the qPCR SuperMix(+Dyell) kit. The extracted genomic DNA of the sample was used as the template, and the template amount was uniformly set to 10 ng. The sample addition system is shown in Table 5. The qPCR program adopted a two-step method, as shown in Table 6, and the data was processed using the 2- △△Ct method.

[0094] Table 5 qPCR sample addition system

[0095] Table 6 qPCR program

[0096] Data processing and analysis: Data are expressed as mean ± standard deviation. One-way ANOVA test was performed using Graphpad Prism 10.0 (GraphPad Software, Inc., USA) for data analysis, and P < 0.05 was used as the standard for significant difference.

[0097] The results are as Figure 29 shown in Figure [Figure number not provided in the original] and Table 7. Compared with the blank group, there were certain differences in the regulatory effects of ZTW-80, ZIW-100, F1–F5, DP2, DP3, and DP5 on different flora after 24 h of administration.

[0098] Table 7 Flora expression levels

[0099] The results of the differences in flora at the phylum level showed ( Figure 29 Figure A in [Figure number not provided in the original]). After 24 h of administration, the richness of Bacteroidetes was significantly upregulated in F1 and DP3 (F1: P < 0.001, DP3: P < 0.05); the richness of Proteobacteria was significantly upregulated in ZTW-80 and ZIW-100 after 24 h of administration (ZTW-80: P < 0.01, ZTW-100: P < 0.0001); the richness of Actinobacteria was significantly downregulated in F2, F3, F4, F5, DP2, DP3, and DP5 after 24 h of administration (F2: P < 0.001, F3–F5, DP2, DP3, DP5: P < 0.0001). This indicates that the oligosaccharides obtained from Prunella vulgaris polysaccharide under different conditions have different effects on the intestinal flora, and some of the oligosaccharides may promote the proliferation of pathogenic bacteria. It is speculated that this may be related to the chemical structure and type of the polysaccharide.

[0100] The results of the differences in flora at the genus level showed ( Figure 29 Figure B in [Figure number not provided in the original]). After 24 h of administration, the abundances of Bifidobacterium and Akkermansia showed a significant upward trend in ZTW-80, ZIW-100, and F1 (P < 0.0001). Among them, Akkermansia has unique effects in metabolic diseases, cancer, and immunotherapy. This result indicates that F1 has the potential to be used in metabolic diseases, cancer, and immunotherapy by upregulating Akkermansia; after 24 h of administration, the richness of Lactobacillus showed a significant upward trend in F2, F5, and DP2 (P < 0.0001).

[0101] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Prunella vulgaris oligosaccharide, characterized in that, The preparation method comprises the following steps: S1. Extract the spikelets of Prunella vulgaris with water. After solid-liquid separation, add ethanol to the obtained liquid phase until the alcohol concentration is 78% - 82%. After standing for precipitation, remove the protein from the obtained precipitate, and retain the polysaccharide with a molecular weight greater than 3500 Da through dialysis; S2. Dissolve the polysaccharide obtained in S1 in 0.45 - 0.55 M trifluoroacetic acid aqueous solution, and then hydrolyze it at 80 ± 2 °C and 100 ± 2 °C for 2.5 - 3.5 h respectively. Recover the solvent, and dry the obtained samples respectively to obtain the mixed oligosaccharides ZTW-80 and ZTW-100; S3. Dissolve the mixed oligosaccharides ZTW-80 and ZTW-100 in water, and perform preparative chromatography separation. The chromatographic conditions of the preparative chromatography are as follows: chromatographic column: amino chromatographic column; mobile phase A is 0.08% - 0.12% v / v formic acid aqueous solution, and mobile phase B is 0.08% - 0.12% v / v formic acid acetonitrile; negative ion mode; elution gradient: 0–20.0 min: 85% B phase; 20.0–32.0 min: 85%–72% B phase; 32.0–42.0 min: 72% B phase; 42.0–57.0 min: 72%–69% B phase; 57.0–63.0 min: 69%–65% B phase; 63.0–73.0 min: 65%–62% B phase; 73.0–74.0 min: 62%–50.0% B phase; 74.0–114.0 min: 50% B phase; flow rate is 2.4 - 2.6 mL / min; when performing the preparative chromatography separation on the mixed oligosaccharide ZTW-80, collect the eluate within 0 - 114.0 min; when performing the preparative chromatography separation on the mixed oligosaccharide ZTW-100, collect the eluate within 23.9 - 31.8 min; the obtained eluate contains the Prunella vulgaris oligosaccharide.

2. The Prunella vulgaris oligosaccharide according to claim 1, wherein, The water extraction method in S1 is ultrasonic extraction, the extraction temperature is 70 ± 5 °C, the ultrasonic frequency is 40 kHz, and the extraction time is 1 h; and / or The solid-liquid separation method in S1 is centrifugation, the centrifugation speed is 4000 rpm, and the centrifugation time is 20 min; and / or In S1, add ethanol to the obtained liquid phase until the alcohol concentration is 80%; and / or The concentration of the trifluoroacetic acid aqueous solution in S2 is 0.5 M; and / or The hydrolysis time in S2 is 3 h; and / or The chromatographic column in S3 is Xbridge BEH Amide OBD Prep Column, with a specification of 30×250 mm, 5 µm; and / or In S3, the mobile phase A is 0.1% v / v formic acid aqueous solution, and the mobile phase B is 0.1% v / v formic acid acetonitrile; and / or In S3, the flow rate is 2.5 mL / min; and / or When performing the preparative chromatography separation on the mixed oligosaccharide ZTW-80, collect the eluate in the time ranges of 0 - 20.0 min, 20.0 - 40.0 min, 40.0 - 60.0 min, or 60.0 - 80.0 min.

3. The Prunella vulgaris oligosaccharide according to claim 1 or 2, characterized in that, When performing the preparative chromatography separation on the mixed oligosaccharide ZTW-80, collect the eluate in the time range of 40.0 - 60.0 min and perform the operation of S4: S4. Perform preparative chromatography on the eluate in the time range of 40.0 - 60.0 min. The chromatographic conditions for the preparative chromatography are as follows: Chromatographic column: amino chromatographic column; Mobile phase A is an aqueous solution of 0.08% - 0.12% v / v formic acid, and mobile phase B is acetonitrile with 0.08% - 0.12% v / v formic acid; Negative ion mode; Elution gradient: 0–40.0 min: 82% phase B; 40.0–50.0 min: 82%–80% phase B; 50.0–60.0 min: 80%–75% phase B; 60.0–80.0 min: 75% phase B; 80.0–90.0 min: 75%–72% phase B; 90.0–95.0 min: 72%–50% phase B; 95.0–105.0 min: 50% phase B; Flow rate is 2.4 - 2.6 mL / min; Collect the eluate in the time ranges of 39.0 - 41.0 min and 70.0 - 74.5 min. The obtained eluate contains the Prunella vulgaris oligosaccharide.

4. The prunella vulgaris oligosaccharide according to claim 3, wherein The chromatographic column in S4 is Xbridge BEHAmide OBD Prep Column, with a specification of 30×250 mm, 5 µm; and / or The mobile phase A in S4 is an aqueous solution of 0.1% v / v formic acid, and mobile phase B is acetonitrile with 0.1% v / v formic acid; and / or The flow rate in S4 is 2.5 mL / min.

5. A Prunella vulgaris oligosaccharide, characterized in that Its structure is shown in Formula I: Formula I R1 is hydrogen or methyl; When R1 is hydrogen, R2 is hydrogen or methyl, where: when R2 is hydrogen, R3 is -Xyl (xylosyl), R4 is hydrogen, R5 is methyl, R6 is carboxyl, and R7 is hydrogen; when R2 is methyl, R3 is hydrogen, R4 is carboxyl, and R5, R6, R7 are hydrogen; When R1 is methyl, R2 and R3 are hydrogen, R4 is carboxyl, R5 and R6 are hydrogen, and R7 is -Xyl-Xyl-GlcA.

6. The Prunella vulgaris oligosaccharide according to claim 5, characterized in that, When R1 is hydrogen and R2 is hydrogen, the structure of the Prunella vulgaris oligosaccharide is β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- β -D-GlcA p -4-OMe-(1→ or β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- α -D-GalA p -4-OMe-(1→; and / or When R1 is hydrogen and R2 is methyl, the structure of the Prunella vulgaris oligosaccharide is α -D-GlcA p -3-OMe-(1→2)- β -D-Xyl p or α -D-GlcA p -3-OMe-(1→2)- α -D-Xyl p ; and / or When R1 is methyl, the structure of the Prunella vulgaris oligosaccharide is α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- α -D-GlcA p- 3-OMe-(1→ and α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-GlcA p- 3-OMe-(1→.

7. An oligosaccharide composition, characterized in that Comprising at least one of the Prunella vulgaris oligosaccharides described in any one of claims 1 - 4 and / or at least one of the Prunella vulgaris oligosaccharides described in claim 5 or 6.

8. The oligosaccharide composition according to claim 7, characterized in that, Composed of the Prunella vulgaris oligosaccharide described in claim 5 or 6.

9. The oligosaccharide composition according to claim 7, characterized in that, The oligosaccharide composition consists of β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- β -D-GlcA p -4-OMe-(1→ or β -D-Xyl p -(1→4)- β -D-Ara f -(1→2)- α -D-GalA p -4-OMe-(1→ of Prunella vulgaris oligosaccharide, with the structure of α -D-GlcA p -3-OMe-(1→2)- β -D-Xyl p or α -D-GlcA p -3-OMe-(1→2)- α -D-Xyl p of Prunella vulgaris oligosaccharide and the structure of α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- α -D-GlcA p- 3-OMe-(1→ or α -D-GlcA p -2-OMe-(1→2)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-Xyl p -(1→4)- β -D-GlcA p- 3-OMe-(1→ of Prunella vulgaris oligosaccharide are composed in an equal mass ratio.

10. Use of the Prunella vulgaris oligosaccharide according to any one of claims 1 to 4, the Prunella vulgaris oligosaccharide according to claim 5 or 6, or the oligosaccharide composition according to any one of claims 7 to 9, characterized in that Including: In the preparation of antioxidant products; Use in the preparation of products for regulating the intestinal flora.

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