Hippophae rhamnoides fruit water-soluble micromolecule and component extraction process thereof

Abstract

The invention discloses a sea buckthorn fruit water-soluble micromolecule and a component extraction process thereof, and relates to the technical field of natural plant chemistry. The method comprises the following steps: S1, pretreating the sea buckthorn fruits, namely crushing the sea buckthorn fruits, accurately weighing the crushed sea buckthorn fruits, adding ultrapure water according to a ratio of 1g: 10mL, carrying out hot water extraction and centrifugation, and filtering impurities by using a filter membrane; s2, extracting sea buckthorn fruit water-soluble small molecules, adding high-concentration absolute ethyl alcohol into centrifugally filtered supernate, sealing and standing overnight at 4 DEG C, centrifuging an alcohol precipitation sample, filtering, performing vacuum evaporation by using a rotary evaporator to remove ethyl alcohol and water, weighing, re-supplementing ultrapure water, heating in a water bath for dissolving, and filtering to obtain the sea buckthorn fruit water-soluble small molecules. Finally, filtering to obtain a sea buckthorn fruit water-soluble micromolecule extracting solution, and refrigerating and storing at 4 DEG C; according to the method, the water-soluble micromolecular chemical components of the sea buckthorn fruits are extracted by using a water extraction and high-concentration ethanol precipitation technology for the first time; the functional analysis shows that the water-soluble small molecule extract has antibacterial biological activity.

Description

Technical Field

[0001] The invention relates to the technical field of natural plant chemistry, in particular to a process for extracting water-soluble small molecules of seabuckthorn fruit and components thereof. Background Art

[0002] The Tibetan medicine seabuckthorn fruit is the mature fruit of the plant seabuckthorn (Hippophae rhamnoides L.) of the Elaeagnaceae family, and is widely distributed in northwest my country and the plateau regions. Seabuckthorn fruit is rich in nutrients and pharmacological effects, and was included in the "Yuewang Yaozhen" and "Four Medical Classics" as early as the 8th century AD. It has the effects of resolving phlegm and benefiting the lungs, promoting blood circulation and removing blood stasis. Seabuckthorn fruit contains a large amount of vitamin C, carotenoids, flavonoids, polyphenols, and organic acids. Modern research shows that seabuckthorn fruit has multiple biological activities such as regulating blood sugar, improving lipid metabolism, protecting the liver, promoting immune function, preventing alcoholic liver disease, anti-oxidation, and regulating intestinal flora. Currently, research on seabuckthorn fruit mainly focuses on the analysis of fat-soluble components and preliminary extracts, especially the study of its oil, polysaccharide and essential oil extracts. The extraction of water-soluble small molecules and analysis of active ingredients in seabuckthorn fruit have not been studied in depth, especially the specific chemical small molecule components with antibacterial activity have not been identified. In response to the above problems, the inventors proposed a process for extracting water-soluble small molecules from seabuckthorn fruit and their components to solve the above problems. Summary of the Invention

[0003] In order to solve the above problems, the present invention aims to provide a process for extracting water-soluble small molecules from seabuckthorn fruit and their components.

[0004] To solve the above technical problems, the present invention adopts the following technical solution: a process for extracting water-soluble small molecules from seabuckthorn fruit, comprising the following steps: S1. Seabuckthorn fruit pretreatment The sea buckthorn fruit was crushed and accurately weighed, and ultrapure water was added at a ratio of 1 g:10 mL. The mixture was heated and extracted with water, centrifuged, and impurities were filtered out using a filter membrane. S2. Extraction of water-soluble small molecules from seabuckthorn fruit High-concentration anhydrous ethanol was added to the supernatant after centrifugal filtration, and the mixture was sealed and allowed to stand overnight at 4°C. The alcohol-precipitated sample was centrifuged and filtered, and vacuum evaporated using a rotary evaporator to remove ethanol and water. After weighing, ultrapure water was added again, and the mixture was heated in a water bath to dissolve. Finally, the water-soluble small molecule extract of sea buckthorn fruit was obtained by filtration and stored in a refrigerator at 4°C.

[0005] Preferably, the analysis step of the seabuckthorn fruit water-soluble small molecule extract comprises: A1. Antibacterial function analysis The water-soluble small molecule extract was diluted twice with LB liquid medium and then used. The LB solid plate was streaked to activate the strain. A single colony was picked and transferred to 0.9% saline. The concentration of the bacterial solution was adjusted to 0.5 McFarland's concentration (1.5×10 8 CFU / mL), diluted 100 times, and added to each diluted concentration solution in a 96-well plate. Each group was repeated 3 times, and LB medium was used as a blank control. The cells were cultured at 37℃ for 18 to 24 hours, and the bacterial growth was observed by comparing the OD before and after culture. 600 The antibacterial effect was evaluated by the change of the value; A2. Identification and analysis of water-soluble small molecule components in seabuckthorn fruit Chromatographic conditions: A Waters ACQUITY™ Premier HSS T3 column (2.1 mm × 100 mm, 1.8 μm particle size) was used; mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of methanol. Separation was performed in gradient elution mode. Positive ion mode: 0-5 min, 0% B; 5-20 min, 0% to 20% B; 20-30 min, 20% to 40% B; 30-35 min, 40% to 100% B; 35-40 min, 100% to 0% B; Negative ion mode: 0-5 min, 0% B; 5-8 min, 0%-10% B; 8-15 min, 10%-20% B; 15-25 min, 20%-100% B; 25-30 min, 100-0% B; flow rate 0.3 mL / min, column temperature 30°C, injection 3 μL; all concentrations are volume percentages; Mass spectrometry conditions: An ESI electrospray ionization source was used, with nitrogen used as drying, nebulizing, and collision gas. Data were collected in both positive and negative ion modes. The acquisition conditions for both modes were a source temperature of 350°C, a capillary temperature of 320°C, a spray voltage of 3.8 kV, a sheath gas flow rate of 30 L / h, an auxiliary gas flow rate of 15 L / h, a capillary voltage of 35 V, a tube lens voltage of 110 V, a primary resolution of 70,000, a secondary resolution of 17,500, and stepped NCEs of 20, 40, and 60. In both MS and MS / MS modes, the mass range was scanned from m / z 50 to 1500. Mass spectral data were recorded. Data-dependent scanning was used for the secondary mass spectrometry. The precise molecular weights of ions of water-soluble substances in seabuckthorn fruit recorded in multiple literatures were selected for collision-induced dissociation fragmentation scanning. The data collected by mass spectrometry were analyzed to identify the water-soluble small molecule components of sea buckthorn fruit.

[0006] Preferably, in S1, water extraction is used for extraction, the ratio of sea buckthorn fruit to water is 1 g:10 mL, and the extraction time is 2 h.

[0007] Preferably, in S2, high concentration ethanol precipitation is used to remove macromolecular polysaccharides and proteins, thereby extracting water-soluble small molecules of sea buckthorn fruit.

[0008] Preferably, in S2, the centrifugation parameters used are 12000 g and the centrifugation time is 60 min, so as to separate and precipitate macromolecular polysaccharides and proteins with higher efficiency.

[0009] Preferably, in A1, it was found that the water-soluble small molecule extract of sea buckthorn fruit has significant antibacterial properties.

[0010] Preferably, in A2, a Waters ACQUITY™ Premier HSS T3 column is used to achieve better separation effect.

[0011] Preferably, in A2, the analysis and identification method is: First, the chemical component data of seabuckthorn fruit was obtained and the compound structures of the chemical components were input into the Compound Discover software to establish a primary database for comparison. At the same time, a high-resolution mass spectrometry database of traditional Chinese medicine components was imported to establish a local secondary database for secondary fragment comparison. Data analysis and component identification were carried out by combining the online search of the primary database ChemSpider and the secondary database MzCloud. Then, a comprehensive judgment was made based on the accurate mass, mass spectrometry fragments and characteristic fragment ions of the compounds in the HMDB and PMhub databases to screen out the final identification results.

[0012] The present invention also provides a seabuckthorn fruit water-soluble small molecule, including an extract obtained by the above-mentioned seabuckthorn fruit water-soluble small molecule component extraction process.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. In the present invention, for the first time, the water-soluble small molecule chemical components of seabuckthorn fruit are extracted by using water extraction and high-concentration ethanol precipitation technology; 2. In the present invention, functional analysis revealed that its water-soluble small molecule extract has antibacterial biological activity; 3. In the present invention, by optimizing the extraction conditions of active ingredients, chromatographic conditions and mass spectrometry conditions, a method for efficiently separating multiple active ingredients was established in positive and negative ion modes. The UPLC-Q-Exactive-Orbitrap-MS / MS technology was used to systematically and accurately identify the water-soluble small molecule chemical components in seabuckthorn fruit. In combination with multiple databases (ChemSpider, MzCloud, PMhub, OTCML, HMDB), the compound discovery 3.0 data analysis and processing software combined with fragment ion information for comparison improved the accuracy of the identification results. The positive ion scanning mode and negative ion scanning mode detected and identified 38 and 46 chemical components, respectively, of which 16 chemical components were repeated. After deducting the repeated parts, 68 chemical components were finally detected and identified from the sea buckthorn fruit extract, including 13 flavonoids, 12 organic acids, 11 amino acids and their derivatives, 10 phenols, 4 tannins, 3 sugars, 2 alkaloids and 13 other compounds. This provides data support for further research on the pharmacokinetics and pharmacodynamic material basis of sea buckthorn fruit, and lays a theoretical foundation for its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is a chart showing the experimental results of the water-soluble small molecule extract of seabuckthorn fruit inhibiting Staphylococcus aureus.

[0016] Figure 2 This is a chart showing the experimental results of the inhibition of Escherichia coli by the water-soluble small molecule extract of seabuckthorn fruit of the present invention.

[0017] Figure 3 This is a chart showing the experimental results of the water-soluble small molecule extract of seabuckthorn fruit of the present invention inhibiting Pseudomonas aeruginosa.

[0018] Figure 4 This is the total ion current of the mass spectrometer collected in the positive ion mode of the present invention.

[0019] Figure 5 This is the total ion current of the mass spectrometer collected in the negative ion mode of the present invention. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Example: Figure 1-5 As shown, the present invention provides a process for extracting water-soluble small molecules from seabuckthorn fruit, comprising the following steps: S1. Seabuckthorn fruit pretreatment The sea buckthorn fruit was crushed in a grinder, weighed, and added with ultrapure water at a ratio of 1 g:10 mL. The mixture was sealed and heated at 180°C until the surface boiled. After boiling, the temperature was adjusted to 110°C to 120°C and cooked for 2 hours. During the heating period, the water extract was centrifuged at 12,000 rpm for 60 minutes and filtered through a 0.45 μm filter membrane. S2. Extraction of water-soluble small molecules from seabuckthorn fruit Slowly add anhydrous ethanol (1:8, v / v) to the aqueous extract while stirring until the final ethanol concentration is 87.5%. Seal and let stand overnight at 4°C. Centrifuge the alcohol-precipitated sample at 12000g for 60min. After the supernatant is filtered through a 0.22μm membrane, ethanol and water are removed by vacuum evaporation at 75°C in a rotary evaporator. Add ultrapure water according to the original volume of the aqueous extract, stir and dissolve in a water bath, filter through a 0.22μm membrane to obtain the water-soluble small molecule extract of sea buckthorn fruit, and refrigerate at 4°C.

[0022] The analysis of the water-soluble small molecule extract of seabuckthorn fruit consists of the following steps: A1. Antibacterial function analysis The water-soluble small molecule extract was diluted twice in a 96-well plate with LB liquid medium (50%, 25%, 12.5%, 6.25%, 3.125%, 1.563%, 0.781%) for later use; After streaking activation of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa on LB solid plates, single colonies were picked and transferred to 0.9% saline solution. The concentration of the bacterial solution was adjusted to 0.5 McFarland's density (1.5 × 10 8 CFU / mL), diluted 100-fold, and added 10 μL of the diluted bacterial solution to each diluted concentration solution (volume 200 μL) in a 96-well plate. Each group was repeated 3 times, and LB medium was used as a blank control. The culture was carried out at 37°C for 18-24 hours, and the bacterial growth was observed. The OD before and after culture was compared using a microplate reader. 600 The value changes, the antibacterial results are shown in Figure 1 、 Figure 2 、 Figure 3 ; A2. Identification and analysis of water-soluble small molecule components in seabuckthorn fruit The water-soluble small molecule extract was filtered through a 0.22 μm filter membrane and then loaded; Chromatographic conditions: A Waters ACQUITY™ Premier HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used; mobile phase A was 0.1% formic acid in water, and mobile phase B was methanol. Gradient elution was performed. Positive ion mode: 0-5 min, 0% B; 5-20 min, 0% to 20% B; 20-30 min, 20% to 40% B; 30-35 min, 40% to 100% B; 35-40 min, 100% to 0% B; Negative ion mode: 0-5 min, 0% B; 5-8 min, 0%-10% B; 8-15 min, 10%-20% B; 15-25 min, 20%-100% B; 25-30 min, 100-0% B; flow rate 0.3 mL / min, column temperature 30°C, injection 3 μL; all concentrations are volume percentages; Mass spectrometry conditions: An ESI electrospray ionization source was used, with nitrogen used as drying, nebulizing, and collision gas. Data were collected in both positive and negative ion modes. The acquisition conditions for both modes were: ion source temperature of 350°C, capillary temperature of 320°C, spray voltage of 3.8 kV, sheath gas flow rate of 30 L / h, auxiliary gas flow rate of 15 L / h, capillary voltage of 35 V, tube lens voltage of 110 V, primary resolution of 70,000, secondary resolution of 17,500, and stepped NCE of 20, 40, and 60. Spectra were recorded in the mass range of m / z 50 to 1500 in MS and MS / MS modes. Data-dependent scanning was used for secondary mass spectrometry. The precise molecular masses of multiple water-soluble small molecules in seabuckthorn fruit were imported from the literature. Collision-induced dissociation (CID) fragmentation scanning was performed. The mass spectra of the total ion current changing with time were collected in both positive and negative ion scanning modes. Figure 4 、 Figure 5 ; The collected mass spectrometry data were analyzed using Compound Discover 3.0. Literature was searched for components of seabuckthorn fruit. Chemical formulas were entered into PMhub (https: / / pmhub.org.cn / # / ) to search for compound structures and establish a local primary structure library. The Traditional Chinese Medicine Component High-Resolution Mass Spectrum Database (OTCML) was imported to establish a local secondary library. Simultaneously, online searches were performed using the primary data resource library ChemSpider and the secondary data resource library MzCloud for analysis, comparison, and identification of components. Details of the analysis and identification results are shown in Tables 1 and 2 below. NO Compound δ / ppm Calc. MW m / z RT[min] ReferenceIon type 1 Arginine 0.94 174.11184 175.11912 0.84 [M + H]+1 amino acids 2 Aspartic acid 1.18 133.03767 134.04489 0.86 [M + H]+1 amino acids 3 Gamma-aminobutyric acid 4.84 103.06383 104.07111 0.87 [M + H]+1 Amino acid derivatives 4 Homoserine 2.47 119.05854 120.06581 0.88 [M + H]+1 amino acids 5 Glycerolphosphocholine -0.23 257.10277 258.11008 0.90 [M + H]+1 other 6 Adenine 0.49 135.05456 136.06184 1.05 [M + H]+1 other 7 4-Guandinobutyric acid 0.03 145.08513 146.09241 1.05 [M + H]+1 organic acids 8 Methionine 0.89 149.05118 150.05846 1.30 [M + H]+1 amino acids 9 4-Amino-3-hydroxybenzoic acid 0.43 153.04266 154.04994 1.31 [M + H]+1 organic acids 10 Isoleucine 0.76 131.09473 132.10201 2.70 [M + H]+1 amino acids 11 Tyrosine 0.23 181.07394 182.08121 2.71 [M + H]+1 amino acids 12 Phenylalanine 0.05 165.07899 166.08626 8.47 [M + H]+1 amino acids 13 Guanosine -0.40 283.09156 284.09883 9.04 [M + H]+1 other 14 Quinic acid -0.24 192.06334 193.07062 11.10 [M + H]+1 organic acids 15 8-Hydroxyquinoline 0.37 145.05282 146.06009 12.72 [M + H]+1 other 16 Rosin hydrocarbon -0.03 202.11061 203.11788 13.28 [M + H]+1 other 17 Tryptamine 0.29 160.10009 161.10737 15.00 [M + H]+1 alkaloids 18 2-Naphthylamine 0.42 143.07356 144.08084 15.05 [M + H]+1 other 19 Tryptophan -0.03 204.08987 205.09715 15.10 [M + H]+1 amino acids 20 1,8-Diazacyclotetradecane-2,9-dione -0.35 226.16805 227.17533 18.07 [M + H]+1 other 21 catechins -0.39 290.07892 291.0862 19.94 [M + H]+1 Phenols 22 Epigallocatechin -0.6 306.07377 307.08105 20.04 [M + H]+1 Tannins 23 Dihydroquercetin -0.28 304.05822 305.06549 22.38 [M + H]+1 flavonoids 24 Halol 0.53 198.07942 199.08670 23.18 [M + H]+1 alkaloids 25 Sinapic acid 0.23 224.06853 225.07576 23.35 [M + H]+1 Phenolic acid 26 Dibenzylamine 0.31 197.12051 198.12779 25.56 [M + H]+1 other 27 Kaempferol-3-O-rutinoside -0.39 594.15824 595.16557 31.99 [M+H]+1 flavonoids 28 Narcissusin -0.46 624.16875 625.17609 32.11 [M + H]+1 flavonoids 29 Isoquercetin 0.18 464.09556 465.10286 32.11 [M + H]+1 flavonoids 30 Rutin -0.19 610.15327 611.16066 32.16 [M + H]+1 flavonoids 31 Typhaneroglucosides -0.21 770.22678 771.23415 32.34 [M + H]+1 flavonoids 32 Abscisic acid 0.40 264.13626 265.14354 32.51 [M + H]+1 other 33 Astragaloside 0.60 448.10083 449.10807 32.92 [M + H]+1 flavonoids 34 Isorhamnetin-3-O-glucoside -0.24 478.11101 479.11838 33.00 [M + H]+1 flavonoids 35 Isorhamnetin -1.39 316.05786 317.06514 33.20 [M + H]+1 flavonoids 36 Isorhamnetin-3-O-neohesperidin -0.62 624.16865 625.17611 33.06 [M + H]+1 flavonoids 37 Xanthoside -0.06 448.10053 449.10781 33.42 [M + H]+1 flavonoids 38 Dioctyl phthalate -0.51 390.27681 391.27605 36.37 [M +H]+1 other Table 1: Identification results in positive ion scanning mode NO Compound δ / ppm Calc. MW m / z RT [min] Reference Ion type 1 Asparagine 0.27 132.05353 131.04625 0.84 [MH]-1 amino acids 2 Aspartic acid -0.09 133.0375 132.03022 0.86 [MH]-1 amino acids 3 D-galacturonic acid -0.64 194.04253 193.03525 0.91 [MH]-1 carbohydrate 4 Mannose -0.42 180.06331 179.05604 0.92 [MH]-1 carbohydrate 5 gluconic acid -0.53 196.0582 195.05092 0.93 [MH]-1 organic acids 6 L-Threonate -0.87 136.03705 135.02978 0.97 [MH]-1 organic acids 7 5-Dehydroquinic acid -0.34 190.04767 189.04041 0.97 [MH]-1 organic acids 8 Quinic acid -1.35 192.06313 191.05585 1.02 [MH]-1 organic acids 9 Malic acid -1.57 134.02131 133.01404 1.10 [MH]-1 organic acids 10 Malonic acid -0.75 104.01088 103.0036 1.21 [MH]-1 organic acids 11 Citric acid 1.33 192.02726 191.01998 1.69 [MH]-1 organic acids 12 Acetyl-DL-glutamate 0.45 189.06381 188.05653 2.10 [MH]-1 Amino acid derivatives 13 Ethyl-β-D-glucopyranoside -0.26 222.0739 221.06662 2.33 [MH]-1 carbohydrate 14 Tyrosine -0.57 181.07379 180.06651 2.36 [MH]-1 amino acids 15 gallic acid 0.02 170.02153 169.01425 5.18 [MH]-1 Phenols 16 Guanosine 0.29 283.09175 282.08447 8.35 [MH]-1 other 17 Methylsuccinic acid -0.63 132.04218 131.0349 9.22 [MH]-1 organic acids 18 Gentisic acid -0.43 154.02654 153.01927 10.38 [MH]-1 Phenolic acid 19 Catechol -1 110.03667 109.02939 10.38 [MH]-1 Phenols 20 Epigallocatechin gallate 1.14 306.0743 305.06702 10.99 [MH]-1 Tannins 21 Pantothenic acid 0.4 219.11076 218.10348 11.21 [MH]-1 other 22 Tryptophan 0.51 204.08998 203.0827 12.22 [MH]-1 amino acids 23 Adipic acid 0.08 146.05792 145.05064 12.35 [MH]-1 organic acids 24 Proanthocyanidin B1 1.06 578.14304 577.13576 14.21 [MH]-1 Phenols 25 N-Acetyl-L-Tyrosine 0.67 223.08461 222.07733 14.30 [MH]-1 Amino acid derivatives 26 Epigallocatechin -0.06 306.07393 305.06666 15.89 [MH]-1 Tannins 27 catechins 0.61 290.07921 289.07194 15.90 [MH]-1 Phenols 28 Korilagin 1.26 634.08141 633.07414 18.45 [MH]-1 Tannins 29 Epicatechol 0.93 290.07931 289.07203 18.48 [MH]-1 Phenols 30 Ethyl gallate 0.18 198.05286 197.04558 19.01 [MH]-1 Phenols 31 Dihydroquercetin 0.63 304.0585 303.05122 19.69 [MH]-1 flavonoids 32 Myricetin-3-O-galactoside 0.72 480.09074 479.08346 20.00 [MH]-1 flavonoids 33 Suberic acid -0.81 174.08907 173.08179 20.00 [MH]-1 organic acids 34 Sinapic acid 0.46 224.06858 223.0613 20.16 [MH]-1 Phenolic acid 35 Momordica grosvenori 1.05 740.21716 739.20988 20.17 [MH]-1 flavonoids 36 Isorhamnetin-3-O-neohesperidin 0.33 624.16924 623.16197 20.44 [MH]-1 flavonoids 37 Rutin -0.15 610.15329 609.14602 20.62 [MH]-1 flavonoids 38 Ethyl protocatechuate -0.8 182.05776 181.05049 20.64 [MH]-1 Phenolic acid 39 Isoquercetin 0.61 464.09576 463.08848 20.65 [MH]-1 flavonoids 40 Ellagic acid 0.41 302.00639 300.99911 21.08 [MH]-1 Tannins 41 Kaempferol-3-O-rutinoside 0.6 594.15883 593.15155 21.18 [MH]-1 flavonoids 42 Azelaic acid -1.25 188.10462 187.09735 21.26 [MH]-1 organic acids 43 Narcissusin -0.09 624.16898 623.1617 21.29 [MH]-1 flavonoids 44 Abscisic acid 0.63 264.13633 263.12905 21.56 [MH]-1 other 45 Xanthoside 0.86 448.10095 447.09367 21.67 [MH]-1 flavonoids 46 6-Gingerol 1.07 294.18342 293.17615 23.90 Phenols Table 2: Identification results of negative ion scanning mode Among them, the chromatographic instrument used was the Thermo Scientific Vanquish UHPLC system; Thermo Fisher Scientific Q Exactive quadrupole-Orbitrap mass spectrometer was used; Anhydrous ethanol, ultrapure water, LB liquid medium, chromatography-grade methanol, chromatography-grade formic acid, chromatography-grade acetonitrile, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were all experimental grade materials. In S1, the centrifugation parameters are 12000g for 60 min to separate and precipitate macromolecular polysaccharides and proteins with greater efficiency.

[0023] In S1, water extraction was used for extraction, the ratio of sea buckthorn fruit to water was 1 g:10 mL, and the extraction time was 2 h.

[0024] In S2, high concentration ethanol precipitation is used to remove large molecular weight polysaccharides and proteins, and to extract the target sea buckthorn fruit water-soluble small molecules.

[0025] In A1, the antibacterial properties of a water-soluble small molecule extract of sea buckthorn fruit were discovered.

[0026] In A2, a Waters ACQUITY™ Premier HSS T3 column was used to achieve better separation.

[0027] In A2, the analytical and identification methods are: First, we obtained data on the chemical components of seabuckthorn fruit by consulting literature. We then entered the chemical compound structures into the Compound Discover software to establish a primary database for comparison. We also imported the Traditional Chinese Medicine High-Resolution Mass Spectrum Database (OTCML) to establish a local secondary database for secondary fragment comparison. We then combined online searches of the primary data resource ChemSpider and the secondary data resource MzCloud to analyze and identify the components. Then, the accurate mass, mass spectrometry fragments and characteristic fragment ions of the compounds in the HMDB and PMhub databases were used to make judgments and determine the final screening results.

[0028] The present invention also provides a seabuckthorn fruit water-soluble small molecule, including an extract obtained by the above-mentioned seabuckthorn fruit water-soluble small molecule component extraction process.

[0029] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A process for extracting water-soluble small molecules from seabuckthorn fruit, characterized in that: The following steps are involved: S1. Sea buckthorn fruit pretreatment The sea buckthorn fruit was crushed and accurately weighed, ultrapure water was added at a ratio of 1 g:10 mL, heated water extraction was performed, centrifuged, and impurities were filtered out using a filter membrane; S2. Extraction of water-soluble small molecules from seabuckthorn fruit High-concentration anhydrous ethanol was added to the supernatant after centrifugal filtration, and the mixture was sealed and allowed to stand overnight at 4°C. The alcohol-precipitated sample was filtered after centrifugation, and vacuum evaporated using a rotary evaporator to remove ethanol and water. Ultrapure water was added again after weighing, and the mixture was heated in a water bath to dissolve. Finally, the water-soluble small molecule extract of sea buckthorn fruit was obtained by filtration and stored in a refrigerator at 4°C.

2. The process for extracting water-soluble small molecules from seabuckthorn fruit according to claim 1, characterized in that: The analysis steps of the seabuckthorn fruit water-soluble small molecule extract include: A1. Antibacterial function analysis The water-soluble small molecule extract was diluted twice with LB liquid medium for later use. The LB solid plate was streaked to activate the strain. A single colony was picked and transferred to 0.9% saline. The concentration of the bacterial solution was adjusted to 0.5 McFarland concentration (1.5×10 8 CFU / mL), diluted 100 times, and added to each diluted concentration of the drug solution in a 96-well plate. Each group was set up with 3 replicates, and LB medium was used as a blank control. The cells were cultured at 37°C for 18 to 24 hours, and the bacterial growth was observed. The OD before and after the culture was compared. 600 The antibacterial effect was evaluated by the change of the value. A2. Identification and analysis of water-soluble small molecule components of seabuckthorn fruit Chromatographic conditions: A Waters ACQUITY™ Premier HSS T3 column with a size of 2.1 mm × 100 mm and a particle size of 1.8 μm was used; mobile phase A was 0.1% formic acid in water, mobile phase B was methanol, and gradient elution mode was used for separation; Positive ion mode: 0-5 min, 0% B; 5-20 min, 0% to 20% B; 20-30 min, 20% to 40% B; 30-35 min, 40% to 100% B; 35-40 min, 100% to 0% B; Negative ion mode: 0-5 min, 0% B; 5-8 min, 0%-10% B; 8-15 min, 10%-20% B; 15-25 min, 20%-100% B; 25-30 min, 100-0% B; flow rate 0.3 mL / min, column temperature 30°C, injection 3 μL; the above concentrations are all volume percentages; Mass spectrometry conditions: ESI electrospray ion source was used, nitrogen was used as drying, nebulizing and collision gas, data were collected in positive and negative ion modes respectively, and the acquisition conditions for both modes were ion source temperature of 350°C, capillary temperature of 320°C, spray voltage of 3.8 kV, sheath gas flow rate of 30 L / h, auxiliary gas flow rate of 15 L / h, capillary voltage of 35 V, tube lens voltage of 110 V, primary resolution of 70000, secondary resolution of 17500, stepped NCE 20,40,60, in MS and MS / MS modes, the scanning mass range was m / z 50-1500, mass spectrometry data were recorded, and secondary mass spectrometry adopted data-dependent scanning, and the ion accurate molecular weight of water-soluble substances of seabuckthorn fruit recorded in multiple literatures was selected for collision-induced dissociation fragment scanning; The data collected by mass spectrometry were analyzed to identify the water-soluble small molecule components of sea buckthorn fruit.

3. The process for extracting water-soluble small molecules from seabuckthorn fruit according to claim 1, characterized in that: In S1, water extraction was used for extraction, the ratio of sea buckthorn fruit to water was 1 g:10 mL, and the extraction time was 2 h.

4. The process for extracting water-soluble small molecules from seabuckthorn fruit according to claim 1, characterized in that: In S2, high concentration ethanol precipitation is used to remove large molecular weight polysaccharides and proteins, thereby extracting water-soluble small molecules of sea buckthorn fruit.

5. The process for extracting water-soluble small molecules from seabuckthorn fruit as claimed in claim 2, characterized in that: In S2, the centrifugation parameters used were 12000 g and a centrifugation time of 60 min to separate and precipitate macromolecular polysaccharides and proteins with higher efficiency.

6. The process for extracting water-soluble small molecules from seabuckthorn fruit as claimed in claim 2, characterized in that: In A1, it was found that the water-soluble small molecule extract of sea buckthorn fruit had significant antibacterial properties.

7. The process for extracting water-soluble small molecules from seabuckthorn fruit as claimed in claim 2, characterized in that: In A2, a Waters ACQUITY™ Premier HSS T3 column was used to achieve better separation.

8. The process for extracting water-soluble small molecules from seabuckthorn fruit as claimed in claim 2, characterized in that: In A2, the analysis and identification method is: Firstly, the chemical composition data of seabuckthorn fruit were obtained, and the compound structures of the chemical components were input into the Compound Discover software to establish a primary database for comparison. At the same time, the high-resolution mass spectrometry database of traditional Chinese medicine components was imported, and a local secondary database was established for secondary fragment comparison. The online search of the primary database ChemSpider and the secondary database MzCloud was combined for data analysis and component identification. Then, a comprehensive judgment was made based on the accurate mass, mass spectrometry fragments and characteristic fragment ions of the compounds in the HMDB and PMhub databases to screen out the final identification results.

9. A water-soluble small molecule of seabuckthorn fruit, characterized in that: The extract comprises the extract obtained by the process for extracting the water-soluble small molecules of seabuckthorn fruit as described in any one of claims 1 to 8.

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