CN116854833B - Tartary buckwheat leaf polysaccharide extract and preparation method for selectively extracting active polysaccharide of tartary buckwheat leaves rich in RG-I - Google Patents
Tartary buckwheat leaf polysaccharide extract and preparation method for selectively extracting active polysaccharide of tartary buckwheat leaves rich in RG-I Download PDFInfo
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- CN116854833B CN116854833B CN202310830050.1A CN202310830050A CN116854833B CN 116854833 B CN116854833 B CN 116854833B CN 202310830050 A CN202310830050 A CN 202310830050A CN 116854833 B CN116854833 B CN 116854833B
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Polymers & Plastics (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Materials Engineering (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Sustainable Development (AREA)
- Medicines Containing Plant Substances (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention provides a polysaccharide extract extracted from tartary buckwheat leaves, and also provides a preparation method and application of the tartary buckwheat leaf polysaccharide which is selectively extracted and rich in RG-I. The invention adopts the eutectic solvent auxiliary pressurizing extraction method to extract polysaccharide from the tartary buckwheat leaves, optimizes the optimal extraction process parameters of the eutectic solvent auxiliary pressurizing extraction of the tartary buckwheat leaf polysaccharide through single-factor multi-level experiment combined with Box-Behnken center combination design-response surface method, realizes the efficient extraction of RG-I pectin polysaccharide in the tartary buckwheat leaves, and has the advantages of high extraction efficiency, low extraction cost, short extraction time, stability and reliability, higher antioxidant activity and immune activity of the extracted polysaccharide, and the like.
Description
Technical Field
The invention relates to a polysaccharide extract extracted from tartary buckwheat leaves and an extraction method thereof, in particular to a preparation method for selectively extracting active polysaccharides of tartary buckwheat leaves rich in RG-I, and a tartary buckwheat leaf polysaccharide extract with remarkable antioxidant activity and immunoregulation effect, belonging to the field of extraction of plant polysaccharides.
Background
Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) is annual herb plant of Fagopyrum of Polygonaceae, and is used as both medicine and food. The stems are upright and branched; the leaf width is triangular, the lower part of the leaf is provided with a long leaf stalk, the upper part She Jiao of the leaf is provided with a short stalk, and the base part of the leaf stalk is yellow brown; inflorescence is in a general shape, corolla and calyx are white or light red, oval and shorter in colume; the thin fruit is oval, the upper edge is sharp, and the lower edge is round and blunt. Flowering period is 6-9 months, and fruit period is 8-10 months. The tartary buckwheat has the characteristics of short growth period, cold resistance, barren resistance, low requirements on soil nutrition conditions, extremely strong stress resistance and adaptability and the like, so that the tartary buckwheat can be widely planted in the world. The current research is mainly focused on the tartary buckwheat seeds which are rich in a large amount of bioactive compounds such as flavonoids, polysaccharides, proteins and the like, and have the functions of resisting oxidation, reducing blood fat, resisting diabetes, resisting cancer, resisting inflammation, protecting liver and the like. Tartary buckwheat leaves are main byproducts in the tartary buckwheat industry, and also contain a large amount of bioactive compounds (such as flavonoid compounds and polysaccharides), and have the functions of resisting oxidation, reducing blood fat and the like. However, tartary buckwheat leaves are often used as functional tea leaves, or for cooking, such as vegetable soups, hot and cold sauces, and the like, for which there is an underutilization.
Pectin is a hydrocolloid polysaccharide, an important component of plant cell walls, and has a complex structure, and mainly consists of homogalacturonic acid (HG), rhamnogalacturonate I (RGI) and rhamnogalacturonate II (RGII), which may be affected by an extraction method. Pectin is generally extracted primarily from various fruits, vegetables and industrial byproducts. Pectin, which has the functional properties of antioxidant, hypoglycemic and hypolipidemic, antiallergic, cholesterol-reducing, colon cancer-preventing, radioprotective, intestinal beneficial growth promoting, is often added as a component to a myriad of food, pharmaceutical industries and other applications, for example in the development of edible films, plasticizers, paper substitutes, emulsifiers, and the like. The arabinose side chain and the galactan side chain in the RG-I pectin side chain can target and regulate probiotics such as Bacteroides thetaiotaomicron (Bacteroides polymorpha), can form gel in a sugar-free or low-sugar system, has a very outstanding thickening and stabilizing effect, can be used as a novel functional food additive with new prebiotic potential, and compared with the research on physiological activity, the prior art for extracting RG-I pectin is very deficient. The traditional pectin polysaccharide extraction generally adopts hot water extraction, and raw materials are extracted by water at a certain temperature and time, so that the polysaccharide separation is realized. The traditional hot water extraction method has the advantages of simple required equipment and operation steps, long extraction time, high energy consumption and low extraction rate. Therefore, improvements in the extraction process are often needed using some auxiliary methods. The eutectic solvent (DES) is generally composed of one hydrogen bond acceptor and one hydrogen bond donor. The DES is used as a green solvent and has the advantages of low economic cost, safety, biodegradability, high solubility and the like. DES can be used as extraction solvent for various bioactive components, such as flavonoids, phenolic acids, alkaloids, etc. Meanwhile, DES has also been used to extract various polysaccharides from plants, which has been shown to have higher extraction rates than conventional extraction solvents.
The pressurization is taken as a non-thermal processing technology, has obvious advantages, has the characteristics of environmental protection, green, cleanness and the like, and can better maintain the primary color, fragrance and natural nutrition of substances. The pressurization treatment at normal temperature can improve the extraction rate of the plant material because the pressurization can cause cell deformation, cell membrane damage and even cell rupture, while improving the rate of cell mass transfer, the permeability of the solvent in the cells and the diffusion of the secondary metabolite.
At present, related literature reports on tartary buckwheat leaf polysaccharide are not relevant, so that the method for preparing tartary buckwheat leaf polysaccharide with high efficiency and green color is provided, and the primary problem to be solved in the industrialization development of tartary buckwheat leaf polysaccharide is solved.
Disclosure of Invention
The invention provides a preparation method for extracting polysaccharide from tartary buckwheat leaves and selectively extracting active polysaccharide of tartary buckwheat leaves rich in RG-I.
The invention provides a tartary buckwheat leaf polysaccharide extract, which is tartary buckwheat leaf polysaccharide extracted and separated by taking tartary buckwheat leaves as raw materials; its molecular weight is 0.907×10 5 Da-1.663×10 5 Da; polydispersity of 1.881-2.890; mainly contains the following monosaccharides: galA, gal, ara, rha, xyl, glc, glcA and Man;
the molar ratio of the monosaccharides is as follows: (1.71-3.35):1.56-1.83 (0.97-1.0) 1 (0.17-0.24) (0.24-0.28) (0.22-0.32) 0.31-0.37);
the tartary buckwheat leaf polysaccharide is acidic polysaccharide; the DE value is 21.65% + -0.16% -42.13% + -0.10%;
the tartary buckwheat leaf polysaccharide contains polygalacturonic acid (HG) and rhamnogalacturonate I (RG-I), wherein galactose or arabinose is a side chain of RG-I.
Further preferably, the molecular weight thereof is: 0.907X 10 5 Da-0.973×10 5 Da; the monosaccharide: the molar ratio of GalA, gal, ara, rha, xyl, glc, glcA to Man is:
(1.71-1.93)︰(1.56-1.83)︰(0.97-1.0)︰1︰(0.17-0.20)︰(0.24-0.28)︰(0.22-0.29)︰(0.31-0.33)。
the invention also provides a preparation method for selectively extracting the Tartary buckwheat leaf polysaccharide rich in RG-I, which comprises the following steps:
a. pulverizing Fagopyrum tataricum leaf into powder, and sieving;
b. removing fat-soluble compounds in the tartary buckwheat leaf powder;
c. performing pressure extraction by taking a DES eutectic solvent as an extraction solvent;
d. centrifuging the extract to obtain supernatant to obtain Fagopyrum tataricum leaf polysaccharide extract.
Wherein, the method for removing the fat-soluble compound in the step b comprises the following steps: adding 70-95% ethanol into Tartary buckwheat leaf powder, and performing ultrasonic treatment for 1-2 times at ultrasonic power of 400-600W for 30-60 min.
The adding amount of the extraction solvent in the step c is calculated by the using amount of the tartary buckwheat leaf powder, wherein the eutectic solvent DES consists of choline chloride (ChCl) and Ethylene Glycol (EG) and ultrapure water, and the proportion of the choline chloride to the ethylene glycol is (1-3) based on the molar ratio: (2-5), preferably, the ratio of choline chloride to ethylene glycol is 1:3; the water content of DES is 10-70%, v/v; preferably 40-55%, v/v; most preferably 49-50%, v/v. Wherein, the ratio of the extraction solvent to the tartary buckwheat leaf powder in the step c is 20-60 mL/g; preferably 40-50, mL/g; most preferably 40-41, mL/g.
Wherein the pressure extraction time in the step c is 7-19 min; preferably 7-13 min; most preferably 9-10min.
Wherein the pressure extraction pressure in the step c is 50-130 MPa; preferably 110-130,MPa; and most preferably from 110 to 120,MPa.
Wherein, after the extract obtained in the step d is centrifuged, the supernatant is used for removing starch, and amylase and saccharifying enzyme are added into the supernatant.
And d, centrifuging the extract in the step, removing starch, adding 95% ethanol for precipitating, centrifuging, adding 76% ethanol for washing precipitate, re-dissolving at 60 ℃, centrifuging, dialyzing, and freeze-drying to obtain the tartary buckwheat leaf polysaccharide with the molecular weight cut-off of 3.5 kDa.
The invention also provides the application of the tartary buckwheat leaf polysaccharide extract with remarkable antioxidant activity and immunoregulation effect in preparing medicines or functional foods with high antioxidant activity and immunoregulation effect.
The invention adopts the eutectic solvent auxiliary pressurizing extraction method to extract polysaccharide from the tartary buckwheat leaves, optimizes the optimal extraction process parameters of the eutectic solvent auxiliary pressurizing extraction of the tartary buckwheat leaf polysaccharide through single-factor multi-level experiment combined with Box-Behnken center combination design-response surface method, realizes the efficient extraction of RG-I pectin polysaccharide in the tartary buckwheat leaves, and has the advantages of high extraction efficiency, low extraction cost, short extraction time, stability and reliability, higher antioxidant activity and immune activity of the extracted polysaccharide, and the like.
Drawings
FIG. 1 is a flow chart of a method for preparing Tartary buckwheat leaf polysaccharide by eutectic solvent assisted pressure extraction
FIG. 2 shows the effect of extraction time (A), DES moisture content (B), feed to liquid ratio (C) and extraction pressure (D) on the extraction yield of Fagopyrum tataricum leaf polysaccharide (note: different letters indicate that the difference between samples is significant (p <
0.05))
FIG. 3 is a three-dimensional plot of the response surface of each factor to the extraction rate of Fagopyrum tataricum leaf polysaccharide.
FIG. 4 is a chart showing the size exclusion chromatograms of Tartary buckwheat leaf polysaccharide prepared by the eutectic solvent assisted pressure extraction method and the conventional water extraction method
FIG. 5 is a high performance liquid chromatogram of monosaccharides comprising Fagopyrum tataricum leaf polysaccharide prepared by eutectic solvent assisted pressure extraction and conventional water extraction
FIG. 6 is a FT-IR spectrum of Fagopyrum tataricum leaf polysaccharide prepared by eutectic solvent assisted pressure extraction and conventional water extraction
FIG. 7 shows the Tartary buckwheat leaf polysaccharide prepared by the eutectic solvent assisted pressure extraction method and the conventional water extraction method 1 HNMR spectrogram
FIG. 8 shows the ability of the Tartary buckwheat leaf polysaccharide prepared by the eutectic solvent assisted pressure extraction method and the conventional water extraction method to remove ABTS free radicals
FIG. 9 shows DPPH radical scavenging ability of Tartary buckwheat leaf polysaccharide prepared by eutectic solvent assisted pressure extraction and conventional water extraction
FIG. 10 shows the effects of eutectic solvent assisted pressure extraction and conventional water extraction on RAW 264.7 macrophage cytotoxicity (A), NO production (B), IL-6 production (C) and TNF-alpha production (D) of Tartary buckwheat leaf polysaccharide
Detailed Description
EXAMPLE 1 Co-pressurizing extraction of Tartary buckwheat leaf polysaccharide (TBP-PD) with the eutectic solvent of the present invention
(1) Freeze-drying the tartary buckwheat leaves in a SCIENTZ-12N/C freeze dryer, pulverizing with a pulverizing machine, sieving with a 60-mesh sieve, packaging the tartary buckwheat leaves powder into a sealed bag, and storing in a drying dish for standby.
(2) Accurately weighing 2.0g of tartary buckwheat leaf powder, adding 20mL of 80% ethanol (1:10, W/v), and performing ultrasonic treatment for 30min under 480W power by using an ultrasonic cleaner. Centrifuging at 5000g for 10min, removing the supernatant, adding 20mL 80% ethanol, ultrasonic cleaning with ultrasonic cleaner at 480W for 30min, centrifuging at 4000g for 10min, and removing the supernatant to obtain precipitate.
(3) Preparing a eutectic solvent: first, choline chloride (ChCl) was mixed with Ethylene Glycol (EG) at 1:3, then continuously stirring for 60min at 80 ℃ until a clear homogeneous solution is obtained, adding ultrapure water to ensure that the water content of the DES is 50%, and uniformly mixing to obtain the DES eutectic solvent.
(4) Mixing the precipitate obtained in the step (2) with DES solution in a ratio of 1:41, and extracting with ATS high pressure homogenizer (AH-NANO, suzhou, china) under 110MPa for 10min. After centrifugation (4000 Xg, 15 min), the supernatant was collected. The precipitate was extracted once more according to the above procedure, and finally, the two extracts were mixed and the supernatant was rotary evaporated to one third of the original volume at 60 ℃.
(5) The above mixed supernatant was treated with high temperature alpha-amylase (5.0U/mL) and saccharifying enzyme (5.0U/mL) to remove starch, inactivating enzyme in a water bath at 95℃for 30min, removing denatured enzyme by centrifugation (4000 Xg, 15 min), and collecting supernatant.
(6) The supernatant from step (5) was ethanol precipitated with 4 volumes of 95% (v/v) ethanol and then at 4℃overnight. The precipitate was centrifuged (4000 Xg, 15 min) and washed with ethanol (76%, v/v). Then, the precipitate was redissolved in ultrapure water.
(7) Dialyzing the redissolved Tartary buckwheat leaf polysaccharide extract with distilled water (with molecular weight cut-off of 3.5 kDa) until the conductivity is unchanged (below 3). Finally, the tartary buckwheat leaf polysaccharide (TBP-PD) is obtained through vacuum freeze drying (-80 ℃ for 48 hours). Immediately after drying, the samples were collected and weighed with an analytical balance. The extraction rate calculation formula is as follows:
extraction ratio = a/B x 100%
Wherein A is the weight of tartary buckwheat leaf polysaccharide; b is the weight (2 g) of the tartary buckwheat leaf powder.
The polysaccharide extraction yield of this example was 3.94% and the polysaccharide extraction yield was 78.8mg. FIG. 1 is a flow chart of a method for eutectic solvent assisted pressure extraction of Fagopyrum tataricum leaf polysaccharides.
Example 2 pressure-assisted eutectic solvent extraction of Tartary buckwheat leaf polysaccharide according to the invention
The difference from example 1 is that: the water content of DES in the step (3) is 30%, the liquid-solid ratio of high-pressure extraction in the step (4) is 30 (mL/g), the extraction time is 10min, and the extraction pressure is 120MPa; the remainder was the same as in example 1.
The polysaccharide extraction yield of this example was 2.56% and the polysaccharide extraction yield was 51.2mg.
Example 3 pressure-assisted eutectic solvent extraction of Tartary buckwheat leaf polysaccharide according to the invention
The difference from example 1 is that: in the step (3), the water content of DES is 55%, the liquid-solid ratio of high-pressure extraction in the step (4) is 40 (mL/g), the extraction time is 13min, and the extraction pressure is 90MPa; the remainder was the same as in example 1.
The polysaccharide extraction yield of this example was 2.83% and the polysaccharide extraction yield was 56.6mg.
Example 4 pressure-assisted eutectic solvent extraction of Tartary buckwheat leaf polysaccharide according to the invention
The difference from example 1 is that: the water content of DES in the step (3) is 40%, the liquid-solid ratio of high-pressure extraction in the step (4) is 40 (mL/g), the extraction time is 7min, and the extraction pressure is 110MPa; the remainder was the same as in example 1.
The polysaccharide extraction yield of this example was 3.16% and the polysaccharide extraction yield was 63.2mg.
Example 5 pressure-assisted eutectic solvent extraction of Tartary buckwheat leaf polysaccharide according to the invention
The difference from example 1 is that: the water content of DES in the step (3) is 40%, the liquid-solid ratio of high-pressure extraction in the step (4) is 30 (mL/g), the extraction time is 7min, and the extraction pressure is 120MPa; the remainder was the same as in example 1.
The polysaccharide extraction yield of this example was 2.65% and the polysaccharide extraction yield was 53.0mg.
EXAMPLE 6 pressure-assisted eutectic solvent extraction of Tartary buckwheat leaf polysaccharide according to the invention
The difference from example 1 is that: the water content of DES in the step (3) is 30%, the liquid-solid ratio of high-pressure extraction in the step (4) is 50 (mL/g), the extraction time is 10min, and the extraction pressure is 120MPa; the remainder was the same as in example 1.
The polysaccharide extraction yield of this example was 3.41% and the polysaccharide extraction yield was 68.2mg.
EXAMPLE 7 pressure-assisted eutectic solvent extraction of Tartary buckwheat leaf polysaccharide according to the invention
The difference from example 1 is that: the water content of DES in the step (3) is 40%, the liquid-solid ratio of high-pressure extraction in the step (4) is 50 (mL/g), the extraction time is 10min, and the extraction pressure is 90MPa; the remainder was the same as in example 1.
The polysaccharide extraction yield of this example was 2.57% and the polysaccharide extraction yield was 51.4mg.
Comparative example traditional Water extraction of Tartary buckwheat leaf polysaccharide (TBP-W)
The difference from example 1 is that: the eutectic solvent was replaced with ultrapure water. Mixing the precipitate obtained in the step (2) with ultrapure water at a ratio of 1:30, mixing the materials and the liquid, heating and extracting the tartary buckwheat leaf polysaccharide in a water bath by using a constant-temperature water bath kettle, setting the extraction time to be 2 hours, and setting the extraction temperature to be 95 ℃; extracting twice as well; the remainder was the same as in example 1.
The polysaccharide extraction yield of this comparative example was 3.21% and the polysaccharide extraction yield was 64.2mg.
EXAMPLE 8 characterization of Tartary buckwheat leaf polysaccharide of the invention
The water-extracted tartary buckwheat leaf polysaccharide (TBP-W) and the tartary buckwheat leaf polysaccharide (TBP-PD) of the invention are respectively researched on molecular weight and molecular weight distribution, monosaccharide composition and FT-IR spectrum thereof to analyze chemical structures thereof. FIG. 4 shows size exclusion chromatograms of TBP-W and TBP-PD, and shows that the molecular weight of TBP-W (1.663X 10 5 Da) is much higher than the molecular weight of TBP-PD (0.907X 10) 5 Da). Meanwhile, the polydispersity of TBP-W and TBP-PD were 2.890 and 1.881, respectively, indicating that the molecular weight of TBP-PD may be degraded during the extraction process.
As shown in FIG. 5, the monosaccharide composition types of TBP-W and TBP-PD are the same, galA, gal, ara, rha, xyl, glc, glcA and Man, respectively. However, the molar ratio of monosaccharides in TBP-W is very different from that in TBP-PD, and the results indicate that the molar ratios of GalA, gal, ara, rha, xyl, glc, glcA and Man in TBP-W and TBP-PD, respectively, are; 3.35:1.83:1.00:1.00:0.24:0.28:0.32:0.37 and 1.71:1.56:0.97:1.00:0.17:0.24:0.22:0.31. From the results, it can be obtained that the RG-I structure ratio of the pressurizing assisted eutectic solvent extracted polysaccharide is significantly higher than that of the conventional water extraction.
FIG. 6 is FT-IR spectra of TBP-W and TBP-PD, from which it can be seen that TBP-W and TBP-PD exhibit similar FT-IR spectra, indicating that TBP-W and TBP-PD possess similar functional groups. Specifically 3434.3cm -1 And 2929.0cm -1 The strong absorption band at this point is due to the stretching vibration of the O-H and C-H groups; 1743.0cm -1 And 1630.0cm -1 Respectively due to esterified carboxylic acidsC=o vibration of the radical and free carboxyl groups, indicating that both TBP-W and TBP-PD are acidic polysaccharides. Furthermore, 1438.3cm -1 Further confirming the presence of uronic acid. Furthermore, at 1237.2cm -1 And 1022.0cm -1 The absorption bands of (2) are C-O-C tensile vibration and the presence of pyranose, respectively. According to 1743.0cm -1 And 1630.0cm -1 The Degree of Esterification (DE) value is calculated for the absorption band of (C). In fact, the DE values of TBP-W and TBP-PD were 42.13% + -0.10% and 21.65% + -0.16%, respectively, indicating that the eutectic solvent assisted pressure extraction had a significant effect on the DE values of Tartary buckwheat leaf polysaccharides.
In addition, as shown in FIG. 7, TBP-W and TBP-PD are present in 1 H NMR spectra showed similar characteristic signals, indicating that their main structures are similar. More specifically, the signal of 4.97pm is H-1 of 1,4- α -D-GalAMep, while the signal of 4.46ppm is related to H-1 of 1,3,6- β -D-Galp. A signal of 4.65ppm indicates the presence of H-1 of 1, 4-. Beta. -D-Galp. A signal of 4.52ppm indicates the presence of H-1 of 1, 3-. Beta. -D-Galp. In addition, a signal of 3.81ppm confirms GalA-OCH 3 Is present. A signal of 5.04ppm indicates the presence of H-1 of 1,4- α -D-GalAp. Signals of 2.07ppm and 2.00ppm indicate the presence of O-acetyl groups. The signals at 5.25 and 1.25ppm are H-1 and H-6 of 1,2, 4-alpha-L-Rhap, respectively. Furthermore, 5.10ppm of the signal is ascribed to H-1 of 1, 5-alpha-L-Araf, while 5.16ppm of the signal is related to H-1 of T-alpha-L-Araf. 5.45ppm indicates the presence of H-1 of 1, 3-alpha-L-Araf. Overall, these results indicate that large amounts of polygalacturonic acid (HG) and rhamnogalacturonate I (RG-I) may be present in TBP-W and TBP-PD, with galactose or arabinose as side chains of RG-I.
Test example 1 optimization test of the extraction Process conditions of Tartary buckwheat leaf polysaccharide of the invention
1. Single factor multiple level experiment
According to the pressurized auxiliary eutectic solvent extraction method for preparing the tartary buckwheat leaf polysaccharide of the embodiment 1,4 factors influencing the yield of the tartary buckwheat leaf polysaccharide are screened, namely: extraction time, water content in DES, feed-to-liquid ratio and extraction pressure. In addition, the extraction temperature was set to a fixed setting of 4.0 ℃ and a one-factor, multi-level experiment was performed.
1.1 Single factor experiment of extraction time
For optimization of extraction time, the water content, the liquid-solid ratio and the extraction pressure of DES are respectively set to be 30% and 30mL/g and 120MPa, and the extraction rate of the tartary buckwheat leaf polysaccharide is calculated when the extraction time is respectively 7, 10, 13, 16 and 19min.
As shown in FIG. 2A, the effect of the extraction time on the extraction rate of the tartary buckwheat leaf polysaccharide is about 2.50% when the extraction time is 7min, and about 2.78% when the extraction time is 10min, and the extraction rate of the tartary buckwheat leaf polysaccharide is obviously reduced along with the increase of the extraction time, so that the optimal extraction time is 10min.
1.2 single factor experiment of DES moisture content
For optimization of the DES water content, the extraction time, the liquid-solid ratio and the extraction pressure are respectively set to be 10min, 30mL/g and 120MPa, and the extraction rate of the tartary buckwheat leaf polysaccharide is calculated when the DES water content is respectively 10%, 25%, 40%, 55% and 70%.
As shown in FIG. 2B, the effect of DES water content on the extraction rate of Fagopyrum tataricum leaf polysaccharide can be obtained, wherein the extraction rate is increased along with the increase of water content within the range of 10% -55%, and the extraction rate is suddenly reduced after 55% of water content, so that the optimal DES water content is 55%.
1.3 Single factor experiment with liquid-solid ratio
For optimization of the liquid-solid ratio, the extraction time, the water content of DES and the extraction pressure are respectively set to be 10min, 30% and 120MPa, and the extraction rate of the tartary buckwheat leaf polysaccharide is calculated when the liquid-solid ratio is respectively 20, 30, 40, 50, 60 and mL/g.
As shown in FIG. 2C, the effect of the liquid-solid ratio on the extraction rate of the tartary buckwheat leaf polysaccharide can be obtained, the extraction rate of TBP-PD is obviously improved within the range of 20-40 mL/g, and the extraction rate of TBP-PD is obviously reduced within the range of 40-60 mL/g, so that the optimal liquid-solid ratio is 40mL/g.
1.4 Single factor experiment of extraction pressure
For optimization of extraction pressure, the extraction time, the water content and the liquid-solid ratio of DES are respectively set to be 10min, 30% and 30mL/g, and the extraction rate of the tartary buckwheat leaf polysaccharide is calculated when the extraction pressure is respectively 50, 70, 90, 110 and 130MPa.
As shown in FIG. 2D, the effect of the extraction pressure on the polysaccharide extraction rate of the tartary buckwheat leaves can be obtained, when the pressure is between 50 and 110MPa, the pressure has positive effect on the polysaccharide extraction rate, the extraction rate reaches the maximum value at 110MPa, and then gradually decreases along with the increase of the pressure, so that the optimal pressure is 110MPa.
And further evaluating the influence of the extraction parameters on the extraction rate of the tartary buckwheat leaf polysaccharide by adopting a four-factor three-level Box-Behnken design according to the result of the single-factor multi-level experiment. The argument includes the extraction time (X 1 7, 10, 13 min), DES moisture content (X 2 40, 55, 70%) and liquid-solid ratio (X) 3 30, 40, 50 mL/g) and extraction pressure (X 4 、90、110、130MPa)。
2. Response surface analysis test
The BBD matrix and experimental results are shown in Table 1, and a second-order polynomial equation is generated to express the predictive mathematical model through multiple regression analysis, as follows:
Y＝3.91+0.0992X 1 -0.5417X 2 +0.1867X 3 +0.1542X 4 +0.0475X 1 X 2 -0.0525X 1 X 3 -
0.0775X 1 X 4 -0.0675X 2 X 3 -0.1150X 2 X 4 +0.0850X 3 X 4 -0.04344X 1 2 -0.8282X 2 2 -0.7407X 3 2 -0.63442X 4 2
wherein Y is extraction rate, X 1 、X 2 、X 3 And X 4 The extraction time (min), the water content (%) in DES, the liquid-solid ratio (mL/g), and the extraction pressure (MPa), respectively.
The effect of the variables on TBP-PD extraction was assessed by analysis of variance (ANOVA) and the validity of the fitted model. As shown in Table 2, the predictive mathematical model of the eutectic solvent assisted pressure extraction method has a higher F value (534.40) and a lower P value (P<0.0001 The regression model was shown to be very significant. F value (0.9082) and P value (0.5922) (P)>0.05 Indicating failure to fitThe term is not significant. The result shows that the model equation can better predict the yield of TBP-PD. In addition, a lower coefficient of variation (CV, 1.44%) and a higher sufficient accuracy (adeq. Precision, 76.2316) also indicate that the model has good repeatability and reliability. Determining coefficient (R) 2 ) And the adjusted decision coefficient (R 2 adj ) 0.9981 and 0.9963, respectively, also indicate that the model has sufficient accuracy. Furthermore, the linear coefficient (X 1 、X 2 、X 3 And X 4 ) Interaction coefficient (X) 1 X 2 、X 1 X 3 、X 1 X 4 、X 2 X 3 、X 2 X 4 And X 3 X 4 ) And quadratic term coefficient (X 1 2 、X 2 2 、X 3 2 And X 4 2 ) Are all very remarkable (P<0.05). The results show that all selected extraction parameters can significantly affect the TBP-PD extraction yield.
A three-dimensional response surface diagram of the predictive model is shown in fig. 3. By analysis of the regression model equation, the predicted optimal extraction conditions are: the extraction time was 9.46min, the water content in DES was 49.20% (v/v), the liquid-solid ratio was 40.99mL/g, and the extraction pressure was 117.05MPa. In view of the operability of the actual process, the verification experiment was conducted under conditions of an extraction time of 10.0min, a DES water content of 50.0% (v/v), a liquid-solid ratio of 41.0mL/g, and an extraction pressure of 110.0 MPa. Three validation runs were performed under these conditions with an actual yield of 3.94% + -0.10% for TBP-PD, which is very consistent with the predicted yield (3.98%). The process for extracting the tartary buckwheat leaf polysaccharide by using the pressurizing auxiliary eutectic solvent after the optimization of the Box-Behnken center combination design-response surface method is illustrated to be feasible and can be popularized to industrial application. Furthermore, the yield of TBP-PD was higher than that of TBP-W in the present study (3.21% + -0.06%). Meanwhile, the extraction time of TBP-PD is 10.0min, which is obviously shorter than the extraction time of TBP-W (120 min), and the optimized pressurized auxiliary eutectic solvent extraction is more effective than hot water extraction.
TABLE 1Box-Behnken design experimental design results
TABLE 2 analysis of variance fitted to second order polynomial model
R 2 ＝0.9981，R 2 adj 0.9963, coefficient of Variation (CV) =1.44%, adeq.precision= 76.2316. And (3) injection: * p<0.05, ** p<0.01
test example 2 antioxidant Activity of Tartary buckwheat leaf polysaccharide of the present invention
And the determination of the in vitro antioxidant capacity of TBP-W and TBP-PD comprises the free radical scavenging capacity of ABTS and DPPH. The ABTS radical cation solution was dark reacted for more than 16 hours at room temperature from 7mM ABTS solution and 2.45mM potassium persulfate aqueous solution. The ABTS radical cation solution was diluted with phosphate buffer (0.05 m, ph 6.6) and the absorbance at 734nm was 0.750±0.02. Then, 200. Mu.LABSS free radical cationic working solution and 20. Mu.L of samples of different concentrations were shake protected from light for 6min in 96-well microwell plates and absorbance values were determined at 734 nm. ABTS radical scavenging activity was calculated using vitamin C (Vc) as positive control as follows:
200. Mu.L of 0.35mM DPPH solution was mixed with 25. Mu.L of samples of different concentrations in 96-well microwell plates, reacted at 37℃in the absence of light for 30min and absorbance values were measured at 517 nm. DPPH radical scavenging activity was calculated using 2, 6-di-tert-butyl-4-methylphenol (BHT) as a positive control as follows:
as shown in fig. 8 and 9, the results show that both the conventional water-extracted tartary buckwheat leaf polysaccharide (TBP-W) and the tartary buckwheat leaf polysaccharide (TBP-PD) of the present invention exhibit significant antioxidant activity as compared to the positive controls (VC and BHT). IC for determining the free radical scavenging ability of TBP-W and TBP-PD 50 Values 4.467 + -0.064 and 1.300+ -0.044 mg/mL (ABTS), respectively; 5.945.+ -. 0.233 and 1.564.+ -. 0.013mg/mL (DPPH). The result shows that compared with the traditional water-extracted tartary buckwheat leaf polysaccharide, the tartary buckwheat leaf polysaccharide has better antioxidant activity.
Test example 3 immunomodulatory effects of Tartary buckwheat leaf polysaccharide of the invention
The in vitro immunostimulatory activity of TBP-W and TBP-PD was determined using the RAW 264.7 macrophage model. RAW 264.7 macrophages were cultured in an RPMI-1640 medium anaerobic incubator at 37 ℃. Cells were treated with TBP-W and TBP-PD at concentrations of 50, 100 and 200. Mu.g/mL, respectively, and cultured for 24h. Medium and lipopolysaccharide (LPS, 1.0. Mu.g/mL) served as blank and positive controls, respectively. The effect of TBP-W and TBP-PD on RAW 264.7 macrophage viability was determined using MTT colorimetric method. In addition, the generation of Nitric Oxide (NO) by macrophages was detected using Griess reagent. Secretion of leukopenia-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) by RAW 264.7 macrophages after incubation of TBP-W and TBP-PD was detected by ELISA kit.
As shown in FIG. 10, both TBP-W and TBP-PD have no toxic effect on RAW 264.7 macrophages. TBP-W and TBP-PD significantly promote the production of NO, IL-6 and TNF- α, respectively, in a dose-dependent manner. However, compared with the traditional water-extracted tartary buckwheat leaf polysaccharide, the tartary buckwheat leaf polysaccharide also has stronger immunoregulation effect.
Claims (20)
1. The polysaccharide extract extracted from the tartary buckwheat leaves is characterized in that: the tartary buckwheat leaf polysaccharide is obtained by extracting and separating tartary buckwheat leaves as raw materials;
the molecular weight is as follows: 0.907 X 10 5 Da-0.973× 10 5 Da; polydispersity 1.881; mainly contains the following monosaccharides: galA, gal, ara, rha, xyl, glc, glcA and Man; the monosaccharide: galA (GalA),The molar ratio of Gal, ara, rha, xyl, glc, glcA to Man is: (1.71-1.93):1.56-1.83 (0.97-1.0) 1 (0.17-0.20) (0.24-0.28) (0.22-0.29) 0.31-0.33);
the tartary buckwheat leaf polysaccharide is acidic polysaccharide; the DE value is 21.65% + -0.16%;
the tartary buckwheat polysaccharide contains polygalacturonic acid (HG) and rhamnogalacturonate I (RG-I), wherein galactose or arabinose is a side chain of RG-I;
the preparation method of the tartary buckwheat polysaccharide comprises the following steps:
a. pulverizing Fagopyrum tataricum leaf into powder, and sieving;
b. removing fat-soluble compounds in the tartary buckwheat leaf powder;
c. performing pressure extraction by taking a DES eutectic solvent as an extraction solvent;
d. centrifuging the extract to obtain supernatant to obtain Fagopyrum tataricum leaf polysaccharide extract.
2. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 1, which is characterized in that: it comprises the following steps:
a. pulverizing Fagopyrum tataricum leaf into powder, and sieving;
b. removing fat-soluble compounds in the tartary buckwheat leaf powder;
c. performing pressure extraction by taking a DES eutectic solvent as an extraction solvent;
d. centrifuging the extract to obtain supernatant to obtain Fagopyrum tataricum leaf polysaccharide extract.
3. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 2, which is characterized in that: the method for removing the fat-soluble compounds in the step b is as follows: adding 70-95% ethanol into Tartary buckwheat leaf powder, and performing ultrasonic treatment for 1-2 times at ultrasonic power of 400-600W for 30-60 min.
4. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 2, which is characterized in that: the adding amount of the extraction solvent in the step c is calculated by the using amount of the tartary buckwheat leaf powder, wherein the eutectic solvent DES consists of choline chloride ChCl, ethylene glycol EG and ultrapure water, and the proportion of the choline chloride to the ethylene glycol is (1-3) in terms of molar ratio: (2-5).
5. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 4, which is characterized in that: the proportion of the choline chloride to the ethylene glycol is 1:3; the moisture content of DES is 10-70% v/v.
6. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 5, which is characterized in that: the moisture content of the DES is 40-55% v/v.
7. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 6, which is characterized in that: the DES water content is 49-50% v/v.
8. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 2, which is characterized in that: and c, the ratio of the extraction solvent to the tartary buckwheat leaf powder in the step is 20-60mL/g.
9. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 8, which is characterized in that: and c, the ratio of the extraction solvent to the tartary buckwheat leaf powder in the step is 40-50mL/g.
10. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 9, which is characterized in that: and c, the ratio of the extraction solvent to the tartary buckwheat leaf powder in the step is 40-41mL/g.
11. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 2, which is characterized in that: and c, the pressure extraction time in the step is 7-19min.
12. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 11, which is characterized in that: and c, the pressure extraction time in the step is 7-13min.
13. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 12, which is characterized in that: and c, the pressure extraction time in the step is 9-10min.
14. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 2, which is characterized in that: the pressure of the pressurized extraction in the step c is 50-130MPa.
15. The method for preparing tartary buckwheat leaf polysaccharide according to claim 14, wherein: the pressure of the pressurized extraction in the step c is 110-130MPa.
16. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 15, wherein: the pressure of the pressurized extraction in the step c is 110-120MPa.
17. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 2, which is characterized in that: d, centrifuging the extract in the step, and removing starch from the supernatant by adding amylase and saccharifying enzyme into the supernatant.
18. The method for preparing the tartary buckwheat leaf polysaccharide according to claim 2, which is characterized in that: d, adding 95% ethanol into the extract after the starch is removed by centrifugation, adding 76% ethanol for washing the precipitate after centrifugation, re-dissolving at 60 ℃, dialyzing after centrifugation, and obtaining the tartary buckwheat leaf polysaccharide after freeze drying, wherein the molecular weight cut-off is 3.5 kDa.
19. Use of the tartary buckwheat leaf polysaccharide of claim 1 in the preparation of a medicament having high antioxidant activity or immunomodulating effect.
20. Use of the tartary buckwheat leaf polysaccharide of claim 1 in the preparation of a food product.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103919051A (en) * | 2014-05-07 | 2014-07-16 | 山东阿如拉药物研究开发有限公司 | Fructus lycii oatmeal with hypoglycemic effect and preparation method thereof |
| CN107410517A (en) * | 2017-05-16 | 2017-12-01 | 昆明理工大学 | A kind of preparation method of bitter buckwheat polysaccharide inner ester bean curd |
| CN108112973A (en) * | 2017-12-25 | 2018-06-05 | 大连深蓝肽科技研发有限公司 | A kind of auxiliary lower hyperlipidemia, hypertension, hyperglycemia composition and preparation method thereof |
| CN110240626A (en) * | 2019-06-25 | 2019-09-17 | 中藜农业科技(江苏)有限公司 | A kind of quinoa polysaccharide polypeptide production method |
| CN111995697A (en) * | 2020-10-10 | 2020-11-27 | 成都农业科技中心 | Extraction and purification method of Lithocarpus litseifolius polysaccharide |
| CN112262890A (en) * | 2020-10-25 | 2021-01-26 | 丁宏石 | Bovine colostrum yoghourt and preparation method thereof |
| CN113061195A (en) * | 2021-04-01 | 2021-07-02 | 天津市食品安全检测技术研究院 | Compound polysaccharide with blood fat reducing effect and preparation method and application thereof |
| CN113214413A (en) * | 2021-06-02 | 2021-08-06 | 成都农业科技中心 | Sweet tea polysaccharide extract with high antioxidant activity and extraction method thereof |
| CN113729124A (en) * | 2021-11-03 | 2021-12-03 | 茂名加大饲料有限公司 | Compound plant extract, preparation method and application thereof in feed |
-
2023
- 2023-07-07 CN CN202310830050.1A patent/CN116854833B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103919051A (en) * | 2014-05-07 | 2014-07-16 | 山东阿如拉药物研究开发有限公司 | Fructus lycii oatmeal with hypoglycemic effect and preparation method thereof |
| CN107410517A (en) * | 2017-05-16 | 2017-12-01 | 昆明理工大学 | A kind of preparation method of bitter buckwheat polysaccharide inner ester bean curd |
| CN108112973A (en) * | 2017-12-25 | 2018-06-05 | 大连深蓝肽科技研发有限公司 | A kind of auxiliary lower hyperlipidemia, hypertension, hyperglycemia composition and preparation method thereof |
| CN110240626A (en) * | 2019-06-25 | 2019-09-17 | 中藜农业科技(江苏)有限公司 | A kind of quinoa polysaccharide polypeptide production method |
| CN111995697A (en) * | 2020-10-10 | 2020-11-27 | 成都农业科技中心 | Extraction and purification method of Lithocarpus litseifolius polysaccharide |
| CN112262890A (en) * | 2020-10-25 | 2021-01-26 | 丁宏石 | Bovine colostrum yoghourt and preparation method thereof |
| CN113061195A (en) * | 2021-04-01 | 2021-07-02 | 天津市食品安全检测技术研究院 | Compound polysaccharide with blood fat reducing effect and preparation method and application thereof |
| CN113214413A (en) * | 2021-06-02 | 2021-08-06 | 成都农业科技中心 | Sweet tea polysaccharide extract with high antioxidant activity and extraction method thereof |
| CN113729124A (en) * | 2021-11-03 | 2021-12-03 | 茂名加大饲料有限公司 | Compound plant extract, preparation method and application thereof in feed |
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