CN115572332B - Tobacco leaf active polysaccharide and extraction method and application thereof - Google Patents
Tobacco leaf active polysaccharide and extraction method and application thereof Download PDFInfo
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- CN115572332B CN115572332B CN202211054531.XA CN202211054531A CN115572332B CN 115572332 B CN115572332 B CN 115572332B CN 202211054531 A CN202211054531 A CN 202211054531A CN 115572332 B CN115572332 B CN 115572332B
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- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 129
- 150000004676 glycans Chemical class 0.000 title claims abstract description 117
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- 238000000605 extraction Methods 0.000 title claims abstract description 58
- 244000061176 Nicotiana tabacum Species 0.000 title 1
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- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
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- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
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Classifications
-
- 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
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- 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
-
- 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)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Polymers & Plastics (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Sustainable Development (AREA)
- Mycology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Hospice & Palliative Care (AREA)
- Psychiatry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a tobacco leaf active polysaccharide and an extraction method and application thereof. The method of the invention obviously improves the extraction rate of the waste and inferior tobacco leaf active polysaccharide, the extraction rate is as high as 6.42 percent, and the obtained tobacco leaf active polysaccharide has high purity and the function of improving memory damage, and can be applied to foods, medicines and health care products.
Description
Technical Field
The invention belongs to the field of natural product extraction, and particularly relates to a separation and extraction method of waste and inferior tobacco leaf active polysaccharide.
Background
The plant polysaccharide is a natural macromolecular polymer and is formed by connecting aldose or ketose through glycosidic bond. Polysaccharides can be categorized into four groups according to source: plant sources (pectin, cellulose, starch, etc.), microbial sources (dextran, cellulose, etc.), animal sources (chitosan, etc.), algae sources (agar, alginate, etc.). The plant polysaccharide has wide biological activity, and has anti-inflammatory, anti-tumor and immunoregulatory activities besides the common antioxidant activity. In particular, plant polysaccharide is taken as a special form of dietary fiber, and can produce relatively broad-spectrum improvement effect on various intestinal, metabolic and neurological diseases, such as intestinal stress syndrome, diabetes, obesity, asthma and the like after long-term intake. Another reason why researchers continue to rise in enthusiasm for the study of different plant polysaccharides is that these plant polysaccharides are mostly composed of a plurality of sugar units. They have strong intermolecular forces, higher molecular weights and stable structures, and the molecular structure of plant polysaccharides contains a large number of functional groups which can be changed or modified to produce high quality polysaccharides. In addition, plant polysaccharides have unique advantages in the food and pharmaceutical fields because of their significantly superior availability, biocompatibility, biodegradability, low toxicity, swelling and water solubility to other classes of polysaccharides.
Tobacco polysaccharides are important components in tobacco. Qualitative and quantitative analysis of polysaccharide species and content in tobacco has been a research hotspot over the last few years. It has been shown that 30.25% of tobacco polysaccharides are from tobacco leaves, whereas 22.58% of tobacco polysaccharides are reducing sugars, in addition to raffinose, glucose, sucrose, maltose, xylose and fructose. Although the development of research is slow due to the very complex chemical structure of the polysaccharide with biological activity, the biological activity of tobacco polysaccharide has not been intensively studied, but many potential anti-pathological functions and physiologically relevant activities thereof have been discovered. In addition to the antioxidant activity, antitumor and anti-inflammatory properties of tobacco polysaccharides, the broad prebiotic effect of tobacco polysaccharides also contributes to improving intestinal health.
At present, waste and inferior tobacco leaves in China are not effectively utilized, and most of the tobacco leaves are abandoned, so that great waste of resources and serious environmental pollution are caused. Therefore, the functional components extracted from tobacco waste have great potential value and deserve further research. CN201410070873.X discloses that two tobacco polysaccharide components obtained by ultrasonic extraction can be used as an antioxidant, but the extraction efficiency of the method is not high, and the extraction rate of the tobacco polysaccharide is only 2.1%; CN201510148793.6 discloses 400-600W ultrasonic extraction and purification by column chromatography to obtain refined polysaccharide, however, the method has higher energy consumption, more steps and extraction rate of only 3.22+/-0.4 percent; CN201710076652.7 discloses that cellulase and pectase are adopted as complex enzymes for enzymolysis, then ultrasonic extraction and alkali treatment are adopted to obtain crude polysaccharide, but the method has higher cost of extracting by using a plurality of enzymes, and the obtained crude polysaccharide has more impurities and can only be used in food preservation, and meanwhile, the method uses a large amount of alkali NaOH for extraction, so that the activity of the tobacco polysaccharide is greatly destroyed although the extraction rate is high. The tobacco polysaccharide with new structural characteristics is extracted from the waste and inferior tobacco leaves, which is favorable for further returning to the key active ingredients and improving the application of the active substances in the fields of food, nutrition and the like.
Disclosure of Invention
In order to improve the utilization rate of the waste and inferior tobacco leaves, the invention obtains a new high-activity tobacco polysaccharide with health regulation activity by optimizing the extraction method of the tobacco active polysaccharide in the waste and inferior tobacco leaves, and provides a new scheme for the utilization of the waste and inferior tobacco leaves.
The invention adopts the following technical scheme for realizing the purpose:
the extraction method of the tobacco leaf active polysaccharide comprises the following steps:
s1, degreasing tobacco leaves to obtain degreased tobacco leaf powder;
s2, mixing the defatted tobacco leaf powder with distilled water and protease enzyme solution, regulating pH, and then performing enzymolysis to obtain enzymolysis solution; inactivating protease in the enzymolysis liquid, and then extracting under ultrasonic conditions to obtain ultrasonic extracting liquid;
s3, centrifuging the ultrasonic extracting solution, taking supernatant, precipitating with alcohol, and centrifugally collecting alcohol precipitate;
s4, redissolving the alcohol precipitate, deproteinizing by using an organic solvent, then carrying out secondary alcohol precipitation, and centrifugally collecting the secondary alcohol precipitate;
s5, re-dissolving the secondary alcohol precipitate, and dialyzing to obtain crude tobacco leaf polysaccharide;
s6, purifying the crude tobacco leaf polysaccharide by adopting DEAE cellulose column chromatography to obtain the tobacco leaf active polysaccharide.
Further, the method of step S1 is: drying the waste and inferior tobacco leaves in a baking oven at 60 ℃, removing tobacco stems, grinding and sieving with a 80-mesh sieve to obtain tobacco leaf powder; adding petroleum ether into tobacco leaf powder, soaking for 12h, pouring petroleum ether, adding petroleum ether again, and pouring petroleum ether after soaking for 12h, wherein the mass-volume ratio of the added petroleum ether to the tobacco leaf powder is 5 mL/1 g; and then carrying out suction filtration by using a Buchner funnel, repeating the operation until the filter paper is free of oil stains, thoroughly removing petroleum ether, and finally drying in a 60 ℃ oven to obtain defatted tobacco powder, and placing the defatted tobacco powder in a dryer for standby.
Further, the method of step S2 is: mixing the defatted tobacco leaf powder, protease enzyme liquid and distilled water according to the mass volume ratio of 1g to 1mL to 60mL of protease enzyme liquid and distilled water with the mass concentration of 1.5%, adding carbonic acid and sodium bicarbonate to adjust the pH value to 5.5-6.0, and then carrying out enzymolysis for 80min at 55 ℃ to obtain an enzymolysis liquid; boiling the enzymolysis liquid in boiling water for 10min to inactivate protease, extracting under ultrasonic condition for 20min at ultrasonic power of 200W and ultrasonic temperature of 45deg.C to obtain ultrasonic extractive solution.
Further, the method of step S3 is: centrifuging the ultrasonic extracting solution at 12000r/min for 15min at 4 ℃, taking supernatant, precipitating with 4 times of absolute ethyl alcohol at 4 ℃ for 48h, centrifuging at 12000r/min at 4 ℃ for 15min, and collecting precipitate to obtain an alcohol precipitate.
Further, the method of step S4 is: adding ultrapure water into the alcohol precipitate according to the mass ratio of the alcohol precipitate to the ultrapure water of 1:10 to redissolve the alcohol precipitate, adding the Sevage reagent according to the volume ratio of the ultrapure water to the Sevage reagent of 3:1, carrying out shaking extraction for 20min, standing for separation, removing an organic phase, and repeating the operations of adding the Sevage reagent, carrying out shaking extraction for 20min, standing for separation and removing the organic phase in the obtained water phase until no white precipitate exists at the boundary of the liquid level; separating the water phase, precipitating with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min at 4deg.C for 15min, and collecting the precipitate to obtain secondary alcohol precipitate.
Further, the method of step S5 is: re-dissolving the secondary alcohol precipitate in the step S4 with ultrapure water, dialyzing in distilled water for 24h by using a dialysis bag with molecular retention of 8000-14000Da, repeating dialysis for 3 times, collecting the dialyzed product, rotationally evaporating redundant solution, and freeze-drying at-80 ℃ for 48h to obtain crude tobacco leaf polysaccharide.
Further, the method of step S6 is: dissolving crude polysaccharide of tobacco leaves in distilled water to prepare 30mg/mL crude polysaccharide solution of tobacco leaves, centrifuging for 10min at 4 ℃ at 4500r/min, filtering supernatant with a 0.45 mu m filter head, loading to a DEAE-52 cellulose column for chromatography, eluting with 0.1mol/L sodium chloride aqueous solution, eluting with 5 times of column volume, and at a flow rate of 0.8mL/min, collecting eluate eluting components, mixing the eluate components, and concentrating under reduced pressure to 1/10 of the volume; and (3) placing the concentrated eluent into a dialysis bag with the molecular weight of 8000-14000, dialyzing in distilled water for 36-48 h, changing water every 4h, and freeze-drying the dialyzate at-80 ℃ for 48h to obtain the tobacco leaf active polysaccharide.
The present invention provides leaf active polysaccharide obtained by the extraction method, the configuration of the anomeric carbon in the structure is mainly beta type, and the molecular weight is 6.85 multiplied by 10 4 Contains hydroxyl, C-H bond, c=o bond, c= C, C-O bond. The leaf active polysaccharide has the function of improving memory injury, and can be applied to foods, medicines and health care products.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the tobacco leaf active polysaccharide is prepared by the ultrasonic-assisted enzymolysis method, and the related extraction conditions are optimized, so that the extraction rate of the waste and inferior tobacco leaf active polysaccharide is remarkably improved to 6.42%, the extraction rate is remarkably higher than that of the existing extraction method, the purity of the obtained tobacco leaf active polysaccharide is high, technical support is provided for green development and utilization of waste and inferior tobacco leaf resources, the process conditions can be extended to the application of surplus tobacco leaves, and a new way is provided for comprehensively utilizing the surplus tobacco leaves. The tobacco leaf active polysaccharide extracted by the invention is a novel polysaccharide with high purity, strong activity and molecular weight of 6.85 multiplied by 10 4 Has the function of improving memory injury, and has the potential of being used for foods, health products and medicines.
Drawings
FIG. 1 shows a glucose standard curve, and the extraction yield of tobacco polysaccharide can be calculated by the phenol-sulfuric acid method.
Figure 2 is a one-factor analysis of ultrasound conditions.
Fig. 3 is a combination factor analysis of ultrasonic conditions, according to the requirements of orthogonal experiments, the conditions used were A1B1C1D1, A2B2C2D2, A3B3C3D3, A2B1C2D3, A2B2C3D1, A2B3C1D2, A3B1C3D2, A3B2C1D3, A3B3C2D1, respectively, the combination corresponding to table 1.
FIG. 4 is a one-factor analysis of enzymatic hydrolysis conditions.
Fig. 5 is a combination factor analysis of enzymolysis conditions, and according to the requirements of orthogonal experiments, the conditions used are A1B1C1D1, A2B2C2D2, A3B3C3D3, A2B1C2D3, A2B2C3D1, A2B3C1D2, A3B1C3D2, A3B2C1D3, A3B3C2D1, and A3B3C2D1, respectively, the combination corresponding to table 2.
FIG. 6 is a condition optimization analysis of ultrasound-assisted enzymolysis.
FIG. 7 shows the chromatographic peak of crude polysaccharide from tobacco in DEAE-52 cellulose column, and the elution effect of 0.1mol/L sodium chloride aqueous solution is optimal as shown by drawing elution curves by phenol-sulfuric acid method after gradient elution with 0, 0.1, 0.2 and 0.3mol/L sodium chloride aqueous solution in the elution process.
FIG. 8 is an ultraviolet scan spectrum of tobacco active polysaccharide in the 190-400 nm range.
FIG. 9 is a spectrum of tobacco active polysaccharide detected by HPLC.
FIG. 10 is an infrared spectrum of tobacco active polysaccharide.
FIG. 11 is a statistical plot of the regulatory function of tobacco active polysaccharide on animal spatial memory.
Detailed Description
The invention is further described below in connection with specific embodiments.
Example 1 establishment of tobacco active polysaccharide extraction method
1. Degreasing the tobacco leaves to obtain degreased tobacco leaf powder:
drying the waste and inferior tobacco leaves in a baking oven at 60 ℃, removing tobacco stems, grinding and sieving with a 80-mesh sieve to obtain tobacco leaf powder; adding petroleum ether into tobacco leaf powder, soaking for 12h, pouring petroleum ether, adding petroleum ether again, and pouring petroleum ether after soaking for 12h, wherein the mass-volume ratio of the added petroleum ether to the tobacco leaf powder is 5 mL/1 g; and then carrying out suction filtration by using a Buchner funnel, repeating the operation until the filter paper is free of oil stains, thoroughly removing petroleum ether, and finally drying in a 60 ℃ oven to obtain defatted tobacco powder, and placing the defatted tobacco powder in a dryer for standby.
2. Ultrasonic method for extracting tobacco leaf polysaccharide
1g of defatted tobacco leaf powder is accurately weighed, placed in a beaker, distilled water is added, and extracted under ultrasonic conditions (feed liquid ratio, ultrasonic time, ultrasonic power and ultrasonic temperature are shown in table 1) to obtain ultrasonic extract.
Centrifuging the ultrasonic extraction solution at 12000r/min for 15min at 4deg.C, collecting supernatant, precipitating with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min for 15min at 4deg.C, and collecting precipitate to obtain alcohol precipitate.
Adding ultrapure water into the alcohol precipitate according to the mass ratio of the alcohol precipitate to the ultrapure water of 1:10 to re-dissolve the alcohol precipitate, adding a Sevage reagent (chloroform: n-butanol=4:1) according to the volume ratio of the ultrapure water to the Sevage reagent of 3:1, carrying out oscillation extraction for 20min, standing for separation, removing an organic phase, and repeating the operations of adding the Sevage reagent, carrying out oscillation extraction for 20min, standing for separation, and removing the organic phase in the obtained water phase until no white precipitate exists at the liquid level junction; separating the water phase, precipitating with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min at 4deg.C for 15min, and collecting the precipitate to obtain secondary alcohol precipitate.
Re-dissolving the secondary alcohol precipitate with ultrapure water, dialyzing in distilled water for 24h by using a dialysis bag with molecular retention of 8000-14000Da, repeating dialysis for 3 times, collecting dialyzed product, rotary evaporating redundant solution, and freeze-drying at-80deg.C for 48h to obtain crude tobacco leaf polysaccharide.
In order to complete the optimization of various process conditions, an accurate extraction rate calculation means is required to be established. Thus, a standard curve for glucose was first obtained using the phenol-sulfuric acid method, as shown in FIG. 1. And then respectively carrying out single-factor experiments on a plurality of levels of a plurality of factors of the feed liquid ratio, the ultrasonic time, the ultrasonic power and the ultrasonic temperature, and determining the optimal ultrasonic treatment condition. As shown in FIG. 2, the extraction of tobacco polysaccharide at 50deg.C under ultrasonic power of 200W for 20min at a feed-liquid ratio of 1:50 can obtain good extraction rate of 1.82%, and the higher or lower feed-liquid ratio can reduce the extraction rate. The single-factor experimental result aiming at ultrasonic time shows that the ultrasonic time of 20min is the optimal treatment condition, and the extraction rate of 1.67% can be obtained; the single-factor experimental result aiming at ultrasonic power shows that the ultrasonic power of 175W is the optimal condition, and the crude polysaccharide yield of 1.81% can be obtained; in terms of ultrasonic temperature, the treatment at 50 ℃ can obtain the highest crude polysaccharide yield, which reaches 1.75%.
According to the single-factor experimental result, the optimal four-factor three-level (table 1) is selected, an L9 (34) orthogonal table is selected for carrying out an ultrasonic extraction orthogonal test, and the analysis result is compared to determine the optimal ultrasonic condition combination.
TABLE 1 ultrasonic extraction orthogonal test factor level
According to the setting rule of the orthogonal experiment, the group conditions adopted respectively are as follows: A1B1C1D1, A2B2C2D2, A3B3C3D3, A2B1C2D3, A2B2C3D1, A2B3C1D2, A3B1C3D2, A3B2C1D3, A3B3C2D1. As can be seen from fig. 3, the extraction yield of polysaccharide is significantly affected by the combination of conditions. Relatively, the polysaccharide content obtained by the ultrasonic treatment for 20min is higher in the whole body, wherein the feed liquid ratio is 1:60. After orthogonal analysis, the extreme differences of the four conditions A-D are 0.25, 1.14, 0.80 and 0.51 respectively, so that the primary and secondary orders of factors are B > C > D > A, and the theoretical optimal condition is A3B3C3D2. A new polysaccharide extraction experiment was performed under this condition, and it was found that the optimum extraction rate obtained by the ultrasonic method was 3.15% (see FIG. 6)
3. Enzymatic hydrolysis method for extracting tobacco leaf polysaccharide
1g of defatted tobacco leaf powder is accurately weighed and placed in a beaker, 60mL of distilled water and 1mL of protease enzyme solution are added, and the mixture is extracted under the enzymolysis condition (the enzyme concentration, the enzymolysis time, the enzymolysis temperature and the enzymolysis pH are shown in Table 1) to obtain an enzymolysis solution.
Boiling the enzymolysis solution in boiling water for 10min to inactivate protease, rapidly cooling to room temperature, centrifuging at 12000r/min at 4deg.C for 15min, and collecting supernatant.
Precipitating the supernatant with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min at 4deg.C for 15min, and collecting precipitate to obtain alcohol precipitate.
Adding ultrapure water into the alcohol precipitate according to the mass ratio of the alcohol precipitate to the ultrapure water of 1:10 to redissolve the alcohol precipitate, adding a Sevage reagent (chloroform: n-butanol=4:1) according to the volume ratio of the ultrapure water to the Sevage reagent of 3:1, carrying out vibration extraction for 20min, standing for separation, removing an organic phase, and repeating the operations of adding the Sevage reagent, carrying out vibration extraction for 20min, standing for separation, and removing the organic phase in the obtained water phase until no white precipitate exists at the liquid level junction; separating the water phase, precipitating with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min at 4deg.C for 15min, and collecting the precipitate to obtain secondary alcohol precipitate.
Re-dissolving the secondary alcohol precipitate with ultrapure water, dialyzing in distilled water for 24h by using a dialysis bag with molecular retention of 8000-14000Da, repeating dialysis for 3 times, collecting dialyzed product, rotary evaporating redundant solution, and freeze-drying at-80deg.C for 48h to obtain crude tobacco leaf polysaccharide.
And (3) respectively carrying out single-factor experiments on a plurality of levels of a plurality of factors of enzyme concentration, enzymolysis time, enzymolysis temperature and enzymolysis pH, and determining the better enzymolysis treatment condition. As shown in FIG. 4, the extraction rate of the tobacco polysaccharide can be better and can reach 4.26% under the treatment condition that the enzyme concentration is 1.5% and the enzymolysis pH is 6.0 for 80min, and the extraction rate is reduced due to the higher or lower enzymolysis pH. The single-factor experimental result aiming at the enzymolysis time shows that the ultrasonic time of 80min is the optimal treatment condition, and the extraction rate of 4.17 percent can be obtained; the single factor experiment result aiming at the enzymolysis concentration shows that the enzymolysis concentration of 1.5 percent is the optimal condition, and the crude polysaccharide yield of 4.29 percent can be obtained; in the aspect of enzymolysis temperature, the treatment at 55 ℃ can obtain the highest crude polysaccharide yield which reaches 4.37 percent.
According to the single-factor experimental result, the optimal four-factor three-level (table 2) is selected, the L9 (34) orthogonal table is selected for enzymolysis extraction orthogonal test, and the analysis result is compared to determine the optimal enzymolysis condition combination.
TABLE 2 level of orthogonal test factors for enzymatic extraction
According to the setting rule of the orthogonal experiment, the group conditions adopted respectively are as follows: A1B1C1D1, A2B2C2D2, A3B3C3D3, A2B1C2D3, A2B2C3D1, A2B3C1D2, A3B1C3D2, A3B2C1D3, A3B3C2D1. As can be seen from fig. 5, the extraction yield of polysaccharide is significantly affected by the combination of the respective conditions. Relatively, the enzymolysis concentration is 1.5%, and the polysaccharide content obtained by the enzymolysis for 80-100 min is higher overall. After orthogonal analysis, the extreme differences of the four conditions A-D are 1.33, 1.59, 2.48 and 0.46 respectively, so that the primary and secondary orders of factors are C > B > A > D, and the theoretical optimal condition is A3B2C2D1. A new polysaccharide extraction experiment was performed under this condition, and the optimal extraction rate finally obtained by the enzymolysis method was found to be 4.75% (FIG. 6).
4. Ultrasonic-assisted enzymolysis method for extracting crude polysaccharide from tobacco leaves
Accurately weighing 1g of defatted tobacco leaf powder, placing the defatted tobacco leaf powder in a beaker, adding 60mL of distilled water and 1mL of protease enzyme solution with the mass concentration of 1.5%, adding carbonic acid and sodium bicarbonate to adjust the pH to 5.5, and then carrying out enzymolysis for 80min at 55 ℃ to obtain an enzymolysis solution. Boiling the enzymolysis solution in boiling water for 10min to inactivate protease, and ultrasonically extracting at ultrasonic power of 200w and ultrasonic temperature of 55deg.C for 20min to obtain ultrasonic extractive solution.
Centrifuging the ultrasonic extraction solution at 12000r/min for 15min at 4deg.C, collecting supernatant, precipitating with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min for 15min at 4deg.C, and collecting precipitate to obtain alcohol precipitate.
Adding ultrapure water into the alcohol precipitate according to the mass ratio of the alcohol precipitate to the ultrapure water of 1:10 to re-dissolve the alcohol precipitate, adding a Sevage reagent (chloroform: n-butanol=4:1) according to the volume ratio of the ultrapure water to the Sevage reagent of 3:1, carrying out oscillation extraction for 20min, standing for separation, removing an organic phase, and repeating the operations of adding the Sevage reagent, carrying out oscillation extraction for 20min, standing for separation, and removing the organic phase in the obtained water phase until no white precipitate exists at the liquid level junction; separating the water phase, precipitating with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min at 4deg.C for 15min, and collecting the precipitate to obtain secondary alcohol precipitate.
Re-dissolving the secondary alcohol precipitate with ultrapure water, dialyzing in distilled water for 24h by using a dialysis bag with molecular retention of 8000-14000Da, repeating dialysis for 3 times, collecting dialyzed product, rotary evaporating redundant solution, and freeze-drying at-80deg.C for 48h to obtain crude tobacco leaf polysaccharide.
One advantage of ultrasound-assisted enzymatic extraction is that the ultrasound treatment can increase the enzymatic efficiency, and consequently increase the yield of polysaccharide products. The different sequence of ultrasonic and enzymolysis treatments can lead to significant differences in extraction efficiency to a great extent. For this purpose, the present study further examined and optimized the order of setting up of ultrasound and enzymolysis. The experimental results are shown in FIG. 6. Compared with the single ultrasonic or enzymolysis treatment group, the ultrasonic auxiliary enzymolysis obviously improves the extraction efficiency of tobacco polysaccharide. And comparing the sequence, the ultrasonic treatment and the enzymolysis can improve the extraction rate of the polysaccharide to 5.11 percent, and the ultrasonic treatment and the enzymolysis can improve the extraction rate of the polysaccharide to 6.42 percent. This extraction rate is obtained on the basis of not using a structural, active destructive agent such as concentrated alkali, etc., so that a high extraction rate is an important innovation point of the present invention. Therefore, the highest extraction rate of tobacco leaf polysaccharide can be obtained by means of enzymolysis and then ultrasonic treatment, and the method is a preferred method for extracting the waste and inferior tobacco leaf active polysaccharide.
EXAMPLE 2 purification of tobacco active polysaccharide extracted by ultrasound-assisted enzymolysis
The crude polysaccharide of tobacco leaves extracted by the ultrasonic-assisted enzymolysis method in example 1 was purified and separated by using anion exchange resin DEAE-52 cellulose: dissolving crude polysaccharide of tobacco leaves in distilled water to prepare 30mg/mL crude polysaccharide solution of tobacco leaves, centrifuging for 10min at 4 ℃ at 4500r/min, filtering supernatant with a 0.45 mu m filter head, loading to a DEAE-52 cellulose column for chromatography, eluting with 0.1mol/L sodium chloride aqueous solution, eluting with 5 times of column volume, and at a flow rate of 0.8mL/min, collecting eluate eluting components, mixing the eluate components, and concentrating under reduced pressure to 1/10 of the volume; and (3) placing the concentrated eluent into a dialysis bag with molecular weight of 8000-14000, dialyzing in distilled water for 48h, changing water every 4h, and freeze-drying the dialyzate at-80 ℃ for 48h to obtain the tobacco leaf active polysaccharide.
Example 3 purity detection, molecular weight detection and Structure determination of tobacco active polysaccharide
(1) Purity detection of tobacco active polysaccharide
The tobacco leaf active polysaccharide obtained in the example 2 is prepared into 1mg/mL tobacco leaf active polysaccharide aqueous solution, ultraviolet scanning is carried out within the range of 190-400 nm, whether the sample to be detected has absorption peaks at 260nm and 280nm or not is observed, and as shown in the figure 8, the tobacco leaf active polysaccharide aqueous solution obviously has no ultraviolet absorption peaks at 260nm and 280nm, which indicates that the extracted and purified tobacco active polysaccharide does not contain nucleic acid and protein.
(2) Determination of molecular weight of tobacco active polysaccharide
The analysis uses chromatographic column to select two columns of UltraHydrogel 2000 and UltraHydrogel 500 connected in series, and the detector uses differential refraction detector. Dextran standard curves were drawn in advance after preparation of samples from a series of dextran of known molecular weight and analysis of the samples. Then preparing 1mg/mL tobacco leaf active polysaccharide aqueous solution by using the tobacco leaf active polysaccharide obtained in the example 2, centrifuging for 15min at 12000r/min, and taking the supernatant to pass through a micro-pore filtration membrane with the thickness of 0.22 mu m to prepare the sample liquid to be tested. And loading the obtained sample liquid on a high performance liquid chromatograph, and calculating according to the obtained peak time and a dextran molecular weight standard curve formula to obtain the molecular weight of the purified tobacco polysaccharide. Referring to FIG. 9, the isolated and purified tobacco active polysaccharide of the present invention exhibits a single symmetrical peak, indicating that the isolated tobacco active polysaccharide is a uniform component, depending on retention time and different known fractionsDextran standard curve Log (Mw) = -0.1472x+10.27 of molecular weight (X is the off-peak time, mw average molecular weight), calculated to have a molecular weight of 6.85X 10 4 。
(3) Infrared spectroscopic analysis of tobacco leaf active polysaccharide
1mg of tobacco active polysaccharide sample was taken according to 1:100 mass ratio, adding dry KBr powder, fully mixing, grinding uniformly, placing a pressurizing sheet into a Nicolet67 infrared spectrometer, and measuring the mass ratio at (600-4000) cm -1 Scanning is performed in a range. Referring to fig. 10, primary analyses of hydroxyl groups, carboxyl groups, and other main chemical groups of tobacco active polysaccharides were performed, and reference was made to the literature: at 3346cm -1 The strong signal at this point represents the hydroxyl (O-H) stretching vibration absorption peak of the polysaccharide; at 2922cm -1 The weaker shoulder belongs to the stretching vibration of the C-H bond; at 1644cm -1 The signal peak at is the stretching vibration of the c=o bond; at 1377cm -1 The signal at the position represents the stretching vibration of the C=C key, 1039cm -1 The signal at the point is caused by bending vibration of the C-O bond; at 899cm -1 The nearby absorption peaks indicate that the configuration of the anomeric carbon of the tobacco polysaccharide of the present invention is predominantly beta-type.
EXAMPLE 4 improving action of tobacco active polysaccharide on memory impairment due to sleep deficiency
Establishment and feeding of experimental animal models: after SD rats grow to 8 weeks, the rats are divided into different groups, and the number of animals in each group is more than 6, and the animals are male and female halves. The method for establishing the sleep deprivation animal model comprises the following steps: an improved multi-platform water environment method is adopted to build a sleep deprivation model, and a sleep deprivation box is made. The accommodating water tank with the diameter of 120 multiplied by 70 multiplied by 50cm is ordered for transformation, 10 small columns of organic glass with the diameter of 3cm and the height of 8cm are stuck on the bottom of the accommodating water tank, round pieces with the diameter of 6.5cm are stuck on the small columns, a small platform capable of enabling a rat to stand is formed, and the distance between each small platform is 15cm. During molding, water at 28-30 ℃ is injected into the water tank, and the horizontal plane is approximately 1cm away from the lower part of the rat platform. The water tank is provided with a cover, and enough food and water are placed on the water tank, so that rats can freely enter water and eat through the platform. The room for molding was kept at a proper temperature and humidity, and rats were placed in a water tank for 1 hour per day on a suitable platform before the final SD molding. Normal rats were placed in an anhydrous large platform sleep deprivation chamber with a circadian cycle of 12h light rhythms. The sleep deprivation group was placed in a box filled with water on a small platform, the deprivation time was from 8:00 a.m., and water was changed each time a day. Sleep was continuously deprived for 5d, and fluorescent lamps were used to continuously illuminate for 5d to exclude the effect of circadian rhythms on experimental results. Whereas the tobacco polysaccharide feeding group (200 mg/kg/d) was performed on the same day as sleep deprivation for the rats of the sleep deprivation group, and also for 5 days, using the other plant active polysaccharide purslane polysaccharide existing in the laboratory as a control (200 mg/kg/d). The feeding mode is to feed 3mL of each rat in a fixed time every day by adopting a separate container, and the feeding process of each rat can be completed in 1 minute generally due to the preference of the rats for beverages.
The behavioural experiments of spatial memory were completed using the Moris water maze. In Moris experiments, water with the depth of 400mm is firstly injected into a pool, the water temperature is kept consistent with the temperature of an animal house, and an edible caramel pigment is added to make the pool water black so as to make a platform below the water surface invisible to naked eyes; the circular pool area is divided into four equal-sized sector quadrants by software, and the position of the circled platform is the platform quadrant. The training process is as follows: the rats were placed on the platform for 30 seconds and then removed to place the rats facing the pool wall, and water entry immediately began to record the movement trace. Automatically ending the recording of the first motion trail if the platform is found within 90 seconds, and allowing the first motion trail to stand at the platform for 30 seconds; if the platform is not found, it is guided to find the platform and allowed to stand on the platform for 30 seconds. After one repetition the training experiment for the next animal was started. Each animal was trained four times a day for five consecutive days, and experiments were performed at the same time every day.
The testing process comprises the following steps: on the 6 th day, the rat is put into water from the middle position of the third quadrant towards the pool wall after the platform is taken out, and the data of the swimming track, the number of times of passing through the position of the platform, the swimming speed and the like of the rat within 90 seconds are automatically recorded by using software. The recorded motion trail and data are imported into a computer and analyzed by utilizing Anymaze software.
From the experimental results, as can be seen from fig. 11, in the index of the number of times of crossing the target platform, the ingestion of the tobacco active polysaccharide obtained by the present invention significantly improves the memory defect (P < 0.001) of animals, showing effective health adjustment. The tobacco active polysaccharide of the present invention has a superior memory modulating effect (p < 0.05) as compared to the control plant polysaccharide.
The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.
Claims (5)
1. The extraction method of the tobacco leaf active polysaccharide is characterized by comprising the following steps:
s1, degreasing tobacco leaves to obtain degreased tobacco leaf powder;
s2, mixing the defatted tobacco leaf powder, protease liquid and distilled water according to the mass volume ratio of 1g to 1mL to 60mL of the protease liquid with the mass concentration of 1.5%, adding carbonic acid and sodium bicarbonate to adjust the pH value to 5.5-6.0, and then carrying out enzymolysis for 80min at 55 ℃ to obtain an enzymolysis liquid; boiling the enzymolysis liquid in boiling water for 10min to inactivate protease, extracting under ultrasonic condition for 20min at ultrasonic power of 200W and ultrasonic temperature of 45deg.C to obtain ultrasonic extractive solution;
s3, centrifuging the ultrasonic extracting solution at the temperature of 4 ℃ for 15min at 12000r/min, taking supernatant, performing alcohol precipitation for 48h at the temperature of 4 ℃ by using 4 times of absolute ethyl alcohol, centrifuging at the temperature of 4 ℃ for 15min at 12000r/min, and collecting precipitate to obtain an alcohol precipitate;
s4, adding ultrapure water into the alcohol precipitate according to the mass ratio of the alcohol precipitate to the ultrapure water of 1:10 to re-dissolve the alcohol precipitate, adding the Sevage reagent according to the volume ratio of the ultrapure water to the Sevage reagent of 3:1, carrying out oscillation extraction for 20min, standing for separation, removing an organic phase, and repeating the operations of adding the Sevage reagent, carrying out oscillation extraction for 20min, standing for separation and removing the organic phase in the obtained water phase until no white precipitate exists at the boundary of the liquid level; separating the water phase, precipitating with 4 times volume of absolute ethanol at 4deg.C for 48 hr, centrifuging at 12000r/min at 4deg.C for 15min, and collecting precipitate to obtain secondary alcohol precipitate;
s5, re-dissolving the secondary alcohol precipitate in the step S4 by using ultrapure water, dialyzing for 24 hours in distilled water by using a dialysis bag with the molecular retention of 8000-14000Da, repeatedly dialyzing for 3 times, collecting a dialyzed product, rotationally evaporating redundant solution, and freeze-drying at-80 ℃ for 48 hours to obtain crude tobacco leaf polysaccharide;
s6, dissolving crude tobacco polysaccharide in distilled water to prepare 30mg/mL crude tobacco polysaccharide solution, centrifuging for 10min at 4 ℃ at 4500r/min, filtering supernatant by using a 0.45 mu m filter head, loading to a sample anion exchange column DEAE-52 cellulose column for chromatography, eluting by using 0.1mol/L sodium chloride aqueous solution, eluting by 5 times of column volume, connecting eluent eluting components at a flow rate of 0.8mL/min, combining the eluting components, and concentrating to 1/10 of volume under reduced pressure; and (3) placing the concentrated eluent into a dialysis bag with the molecular weight of 8000-14000, dialyzing in distilled water for 36-48 h, changing water every 4h, and freeze-drying the dialyzate at-80 ℃ for 48h to obtain the tobacco leaf active polysaccharide.
2. The extraction method according to claim 1, wherein the method of step S1 is: drying the waste and inferior tobacco leaves in a baking oven at 60 ℃, removing tobacco stems, grinding and sieving with a 80-mesh sieve to obtain tobacco leaf powder; adding petroleum ether into tobacco leaf powder, soaking for 12h, pouring petroleum ether, adding petroleum ether again, and pouring petroleum ether after soaking for 12h, wherein the mass-volume ratio of the added petroleum ether to the tobacco leaf powder is 5 mL/1 g; and then carrying out suction filtration by using a Buchner funnel, repeating the operation until the filter paper is free of oil stains, thoroughly removing petroleum ether, and finally drying in a 60 ℃ oven to obtain defatted tobacco powder, and placing the defatted tobacco powder in a dryer for standby.
3. A tobacco leaf active polysaccharide obtainable by the extraction process of any one of claims 1 to 2.
4. A tobacco leaf active polysaccharide according to claim 3, wherein: the molecular weight of the tobacco leaf active polysaccharide is 6.85 multiplied by 10 4 Contains hydroxyl, C-H bond, C=O bond, C= C, C-O bond, and the configuration of the anomeric carbon is mainly beta type.
5. Use of the tobacco leaf active polysaccharide according to claim 4 for preparing a medicament having an effect of improving memory impairment.
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| AU2002336535A1 (en) * | 2001-09-14 | 2003-04-01 | Momenta Pharmaceuticals, Inc. | Methods of making glycolmolecules with enhanced activities and uses thereof |
| CN103804507A (en) * | 2014-02-28 | 2014-05-21 | 河南中烟工业有限责任公司 | Maryland tobacco polysaccharide, extracting and purifying method and application thereof as antioxidant |
| CN106749744A (en) * | 2017-02-13 | 2017-05-31 | 重庆中烟工业有限责任公司 | A kind of tobacco leaf polyoses extract and its extracting method and application |
| CN110679997A (en) * | 2019-10-31 | 2020-01-14 | 湖北中烟工业有限责任公司 | Tobacco shred containing tobacco leaf polysaccharide and derived polysaccharide with high moisturizing property |
| CN110679996A (en) * | 2019-10-31 | 2020-01-14 | 湖北中烟工业有限责任公司 | Tobacco leaf containing selenium-enriched polysaccharide |
| CN110950972A (en) * | 2019-12-13 | 2020-04-03 | 贵州大学 | Polysaccharide extracted by ultrasonic-assisted enzyme method and application thereof |
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| AU2002336535A1 (en) * | 2001-09-14 | 2003-04-01 | Momenta Pharmaceuticals, Inc. | Methods of making glycolmolecules with enhanced activities and uses thereof |
| CN103804507A (en) * | 2014-02-28 | 2014-05-21 | 河南中烟工业有限责任公司 | Maryland tobacco polysaccharide, extracting and purifying method and application thereof as antioxidant |
| CN106749744A (en) * | 2017-02-13 | 2017-05-31 | 重庆中烟工业有限责任公司 | A kind of tobacco leaf polyoses extract and its extracting method and application |
| CN110679997A (en) * | 2019-10-31 | 2020-01-14 | 湖北中烟工业有限责任公司 | Tobacco shred containing tobacco leaf polysaccharide and derived polysaccharide with high moisturizing property |
| CN110679996A (en) * | 2019-10-31 | 2020-01-14 | 湖北中烟工业有限责任公司 | Tobacco leaf containing selenium-enriched polysaccharide |
| CN110950972A (en) * | 2019-12-13 | 2020-04-03 | 贵州大学 | Polysaccharide extracted by ultrasonic-assisted enzyme method and application thereof |
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