Nymphaea hybrid polysaccharide extract and preparation method and application thereof
1. The preparation method of the nymphaea hybrid polysaccharide extract is characterized by comprising the following steps:
A. pulverizing nymphaea hybrid, adding water for extraction, and concentrating the extract to obtain extract;
B. adding ethanol solution into the extract, stirring, filtering, and freeze drying the precipitate to obtain polysaccharide crude extract I;
C. dissolving polysaccharide crude extract I in water, deproteinizing by Sevag method, washing out precipitate, filtering, and drying precipitate to obtain polysaccharide crude extract II;
D. removing monosaccharide and oligosaccharide: dissolving polysaccharide crude extract II in water, dialyzing with 8000-14000D dialysis membrane with cut-off molecular weight, adding ethanol solution into dialyzed polysaccharide crude extract liquid, stirring until precipitate is sucked out, filtering, and drying precipitate to obtain polysaccharide crude extract III;
E. purifying by using a gel column: purifying the polysaccharide crude extract III with gel chromatography column, tracking the fraction with sulfuric acid-phenol method, collecting main peak, mixing, adding ethanol solution, stirring, filtering, precipitating, and freeze drying to obtain the polysaccharide extract of herba Eichhorniae.
2. The method of claim 1, wherein the nymphaea hybrid is a dried or fresh flower.
3. The method of claim 1, wherein the pulverizing is to 20 mesh.
4. The preparation method of claim 1, wherein the water extraction is carried out by soaking the pulverized nymphaea hybrid with 5-10 times of water, mechanically stirring, extracting for 1-12 hours each time for 3-5 times in water bath, filtering, and combining the filtrates.
5. The method of claim 1, wherein the Sevag deproteinization is carried out by dissolving crude polysaccharide extract I in water, adding chloroform at a volume ratio of 5: 1: and mixing the n-butanol with a solvent, stirring, layering, recovering the supernatant, adding an ethanol solution into the supernatant, stirring until a precipitate is separated out, filtering, and drying the precipitate to obtain the polysaccharide crude extract II.
6. The method of claim 1, wherein the dialysis is dialysis for 2 days.
7. A nymphaea hybrid polysaccharide extract prepared according to the preparation method of claim 1, 2, 3, 4, 5 or 6.
8. The use of the nymphaea hybrid polysaccharide extract according to claim 7 for preparing cosmetics, beverages or health foods with antibacterial, whitening, moisturizing and antioxidant effects.
9. An antibacterial, whitening, moisturizing and antioxidant cosmetic, beverage or health food, characterized by containing the nymphaea hybrid polysaccharide extract according to claim 7 as an active ingredient.
Background art:
the nymphaea hybrid is large-scale nymphaea hybrid, and belongs to perennial aquatic perennial root herbaceous plants of Nymphaeaceae. The perfume lotus has light fragrance and long flowering phase, and is the highest grade variety of urban garden waterscape, courtyard radiography scenery and potted ornamental precious aquatic flowers at home and abroad. In the last 70 th century, the American globeflower was introduced into Taiwan of China, and on the basis of the Taiwan, nine major color systems such as gold, yellow, purple, blue, red, tea, green, red, white and the like are developed through cultivation, so that the Taiwan is also named as 'Jiupin globeflower'. The water lily is suitable for tropical to temperate climates, so that the water lily can be widely planted in places such as Guangdong, Hunan, Zhejiang, Jiangsu, Hainan and the like. The flowers of the nymphaea hybrid have very remarkable ornamental property and are high-grade aquatic ornamental aquatic plant species in China.
The flower of the lotus tea can be directly used for making fresh perfume lotus tea, can be used for raw eating, tea making, wine soaking, stewing and boiling food and the like, and can be used as a new resource plant with homology of medicine and food. In addition, the nymphaea hybrid can be prepared into a nine-grade nymphaea hybrid tea by sterilizing and baking at the high temperature of 120 ℃, and has the summer heat relieving and health care effects. The nymphaea hybrid flowers contain plant placentin and natural pollen, have good tranquilizing and calming effects, and also have the effects of softening skin and removing dead skin. In addition, the nymphaea hybrid also has the effects of beautifying, enhancing immunity and promoting metabolism. Currently, the perfume lotus has formally become the etiquette flower of the olympic games.
In the extraction and purification process of the nymphaea hybrid polysaccharide, the existing technology relies on macroporous resin adsorption, and the prepared polysaccharide has obvious color, so that the color is easy to change after long-term storage, and the subsequent development of related products, particularly whitening products, is disturbed. At present, the extraction of the nymphaea hybrid polysaccharide generally adopts a high-temperature and ultrasonic-assisted extraction method, however, the polysaccharide structure is easy to break and denature under the action of high temperature or ultrasonic waves.
Meanwhile, the extraction of the nymphaea hybrid polysaccharide is mainly concentrated on a crude extract or a high molecular weight polysaccharide, and the content of the polysaccharide is not high or the property of the polysaccharide cannot meet the requirement of subsequent product development.
The invention content is as follows:
based on the defects of the current extraction method of the nymphaea hybrid polysaccharide, the invention aims to provide the nymphaea hybrid polysaccharide extract and the preparation method thereof, so that the nymphaea hybrid polysaccharide with high purity, stable property, white color and medium molecular weight (the molecular weight is mainly 80-360KD) is efficiently extracted, and the nymphaea hybrid polysaccharide is more conveniently absorbed through the body surface of the skin on the basis of keeping the effects of moisture preservation, whitening, antibiosis, antioxidation and the like.
The preparation method of the nymphaea hybrid polysaccharide extract comprises the following steps:
A. pulverizing nymphaea hybrid, adding water for extraction, and concentrating the extract to obtain extract;
B. adding ethanol solution into the extract, stirring, filtering, and freeze drying the precipitate to obtain polysaccharide crude extract I;
C. dissolving polysaccharide crude extract I in water, deproteinizing by Sevag method, washing out precipitate, filtering, and drying precipitate to obtain polysaccharide crude extract II;
D. removing monosaccharide and oligosaccharide: dissolving polysaccharide crude extract II in water, dialyzing with 8000-14000D dialysis membrane with cut-off molecular weight, adding ethanol solution into dialyzed polysaccharide crude extract liquid, stirring until precipitate is sucked out, filtering, and drying precipitate to obtain polysaccharide crude extract III;
E. purifying by using a gel column: purifying the polysaccharide crude extract III with gel chromatography column, tracking the fraction with sulfuric acid-phenol method, collecting main peak, mixing, adding ethanol solution, stirring, filtering, precipitating, and freeze drying to obtain the polysaccharide extract of herba Eichhorniae.
The nymphaea hybrid is dry or fresh flowers.
The pulverization is to 20 meshes so as to ensure that the polysaccharide can be quickly extracted.
The water extraction is to soak the smashed nymphaea hybrid with 5-10 times of water, mechanically stir, extract for 3-5 times in water bath for 1-12 hours each time, then filter and combine the filtrate. Extracting at room temperature to 50 deg.C to ensure stable property of polysaccharide; in addition, the amount of water is 5-10 times of the amount of perfume.
The Sevag method deproteinization is to take polysaccharide crude extract I to be dissolved in water, and chloroform with the volume ratio of 5: 1 is added: and mixing the n-butanol with a solvent, stirring, layering, recovering the supernatant, adding an ethanol solution into the supernatant, stirring until a precipitate is separated out, filtering, and drying the precipitate to obtain the polysaccharide crude extract II.
The dialysis is dialysis for 2 days.
The method has the advantages that the extraction at room temperature or low temperature ensures that the original structure of the nymphaea hybrid polysaccharide is not degraded, and the prepared polysaccharide is pure white in color, high in purity (94% -99%), stable in property and easy to dissolve in water. The prepared nymphaea hybrid polysaccharide has controllable quality, high active ingredient content (94% -99%), and pharmacological functions of resisting bacteria, whitening skin, keeping moisture, resisting oxidation, etc. In addition, the preparation process is convenient to operate, and the product can be widely applied to cosmetics, beverages and health-care foods.
The specific implementation mode is as follows:
the following will further illustrate the essence and effect of the present invention with reference to the embodiment, which is only used to illustrate the process and result of the present invention, but not to limit the present invention.
Example 1 preparation of polysaccharide extract of nymphaea hybrid
Taking 100g of freeze-dried nymphaea hybrid, crushing the 100g of nymphaea hybrid by using a crusher, and filtering the crushed powder by using a 20-mesh iron sieve to obtain nymphaea hybrid micropowder. Adding 10 times volume (1L) of water into the nymphaea hybrid micropowder, and leaching in a water bath with mechanical stirring (400 r/min) at room temperature for 4-5 h. Primarily filtering the leaching solution through gauze, and collecting filtrate. Extracting the residue with the same method for 2 times, and mixing the filtrates for 3 times. The filtrate was concentrated under reduced pressure by means of a rotary evaporator to 1/10(100mL) in the original volume, and a 95 vol% ethanol solution (130mL) was added to the concentrated solution and stirred to effect precipitation, and when the concentration of the added ethanol reached 60% (by volume), a large amount of flocculent precipitates appeared. The precipitate was filtered through gauze and dried under vacuum to give crude polysaccharide extract I (27 g).
Crude polysaccharide extract I was dissolved in water (100mL) and purified by adding chloroform in a 5: 1 volume ratio: and (3) mixing a n-butanol mixed solvent (100mL), fully stirring, demixing, recovering a supernatant, adding an ethanol aqueous solution (130mL) with the volume fraction of 95% into the supernatant, stirring until a precipitate is separated out, filtering, and drying the precipitate to obtain the polysaccharide crude extract II (16 g).
Dissolving the crude polysaccharide extract II in water (100mL), dialyzing with dialysis membrane with molecular weight cut-off of 8-14KD for 2 days, adding 95% ethanol water solution (130mL) with volume fraction into the dialyzed crude polysaccharide extract liquid, stirring, sucking out the precipitate, filtering, and drying the precipitate to obtain crude polysaccharide extract III (11 g);
purifying the polysaccharide crude extract III with gel chromatography column, tracking the fraction with sulfuric acid-phenol method, collecting main peak, and mixing. Mixing the eluates, concentrating under pressure at room temperature with a rotary evaporator, concentrating to 50mL, adding 95% ethanol water (80mL) at one time, stirring, and filtering. Freeze-drying the filter cake for 12h to obtain the nymphaea hybrid polysaccharide extract (7 g). The lotus polysaccharide is white solid powder, the purity is 94% -99%, and the molecular weight is mainly 80-360 KD. The lotus polysaccharide can be rapidly dissolved in water at room temperature (10min), and can be placed in a sealed manner at room temperature for 30 days without color and physicochemical property changes.
Example 2: evaluation of antibacterial Activity of Compound
In this example, the Minimum Inhibitory Concentration (MIC) of the antibacterial activity of a sample is determined by the Resazurin color development method. The experimental strains are gram-positive bacteria or gram-negative bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus Aureus (SA), Bacillus cereus (BC, Bacillus cereus), B.subtilis (BS, Bacillus subtilis), B.thuringiensis (BT, Bacillus thuringiensis) or Escherichia coli (EC, Escherichia coli).
The assay will use a 96-well plate dilution titer technique, with the Minimum Inhibitory Concentration (MIC) of the various substances being determined simultaneously. Firstly, 7.5mL of indicator solution (100 mu g/mL of resazurin aqueous solution) and 5mL of bacteria solution to be tested (10)8CFU/mL) and 100 μ L of the mixed bacteria solution was added to each of all the test wells of columns 1 to 8. Then 100. mu.L of the aqueous solution (32mg/mL) of the sample to be tested was sequentially added to each well of the first row, after uniform mixing, 100. mu.L of the solution was taken out and transferred to the corresponding well of the second row, and the solution was diluted to the 8 th row in the same manner. Finally, the well plate with the added sample is placed into a constant temperature incubator and cultured for 10-12h at 37 ℃. The bacteria liquid turns red into no bacteriostatic activity, blue is bacteriostatic activity, and the lowest dilution concentration of the bacteria liquid maintaining blue is considered as the lowest bacteriostatic concentration of the compound to be detected. Each sample was made in 3 replicates.
The results show that: the nymphaea hybrid polysaccharide extract has obvious antibacterial activity on gram-positive bacteria, and almost has obvious inhibition effects on Methicillin-resistant Staphylococcus aureus (MRSA), Methicillin-resistant Staphylococcus Aureus (SA), Bacillus cereus (BC, Bacillus cereus), B.subtilis (BS, Bacillus subtilis) and B.thuringiensis (BT, Bacillus thuringiensis) when the concentration is more than 2 mg/mL. The results show that the nymphaea hybrid polysaccharide extract has good application prospect in the aspects of antibiosis and antiphlogosis. The specific experimental results are shown in table 1 below.
TABLE 1 in vitro antibacterial Activity (MIC) of the nymphaea hybrid polysaccharides
Example 3: determination of lipid peroxidation inhibition Activity
0.50mL of lecithin solution was weighed and added to 1.0mL of PBS buffer with pH 7.0. 1.0mL of sample solutions containing different concentrations was mixed uniformly with 1.0mL of an aqueous solution containing 2.5mmol/L of EDTA-Fe (II), which was then added to the aforementioned PBS mixed solution of lecithin. After mixing, the mixture is placed in a water bath at 37 ℃ for reaction for 45 min. Then, 2.0mL of 28% (w/v) trichloroacetic acid and 1.0mL of 1% (w/v) thiobarbituric acid were added successively. After mixing uniformly, the mixed solution is placed in a boiling water bath at 100 ℃ for heating for 10min, and after cooling, the absorbance is measured at 532 nm. The absorbance A0 was measured by zeroing with PBS buffer and replacing the sample with PBS buffer for blank tubes. 3 replicates of each sample were taken and averaged for A1. The lipid peroxidation inhibition was calculated as follows: inhibition ratio (%) [ (a0-a1)/a0] × 100 in the formula: a0 is the absorbance without sample; a1 is the absorbance of the sample.
The results show that: the nymphaea hybrid polysaccharide extract has obvious inhibitory activity on lipid peroxidation. The results show that the nymphaea hybrid polysaccharide extract has certain potential in the aspect of being used as an antioxidant. The specific experimental results are shown in table 2 below.
TABLE 2 in vitro anti-lipid peroxidation Activity of Nelumbo Nucifera polysaccharide
Example 4: activity measurement of trollius chinensis polysaccharide for inhibiting tyrosinase in cells
Adjusting the number of melanoma cells in mice to 5 × 104One cell/mL, 100. mu.L of cell fluid was added to each well of a 96-well plate, 100. mu.L of the culture solution was supplemented, and after plating for 12 hours, the supernatant was discarded. Thereafter, 200. mu.L of culture medium containing samples of different mass concentrations (mass concentration gradient of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2mg/mL) was added to the system at 37 ℃ with a volume fraction of 5% CO2Culturing under saturated wet condition, setting 6 multiple wells per mass concentration, culturing for 48h with cells cultured without adding sample as blank control, and changing liquid every day. Tyrosinase activity was measured by the dopa oxidation method. Discarding supernatant in the well plate, rinsing with PBS buffer solution, adding 1% Triton-X10040 μ L into each well, rapidly placing into-80 deg.C refrigerator, thawing at 37 deg.C after 30min, disrupting cells, and adding 1% L-DOAnd (3) performing correction on 10 mu L/hole of PA solution by using L-DOPA as a background, performing reaction at 37 ℃ for 30min by using cells cultured without adding a whitening agent as a control, and measuring the A450 value at 450nm by using an enzyme-labeling instrument. The inhibition rate of the test solution (including the positive control) on the tyrosinase is calculated by using the following formula with the corresponding negative control as a reference during the determination. Inhibition (%) - (a-B)/a × 100% where a is the absorbance of the standard control and B is the absorbance of the positive control. Each experiment was done in 3 replicates. The high inhibition rate indicates that the inhibition intensity of the compound on tyrosinase activity is high.
The results show that: the nymphaea hybrid polysaccharide extract has obvious inhibitory activity on tyrosinase, particularly shows good anti-tyrosinase activity when the concentration is more than 1.6 mg/mL, and the specific experimental results are shown in the following table 3. Tyrosinase is an oxidase and is the rate-limiting enzyme in the regulation of melanin production. It is present in plant and animal tissues and catalyzes the production of melanin and other pigments from the oxidation of tyrosine. Tyrosinase inhibitors are generally excellent whitening agents, and can inhibit the production of skin melanin. The results show that the nymphaea hybrid polysaccharide extract can be widely applied as a whitening agent and used for developing whitening products.
TABLE 3 in vitro antityrosinase Activity of nymphaea hybrid polysaccharides
Example 5: preparation method of nymphaea hybrid polysaccharide essence with whitening, antibacterial, moisturizing and antioxidant functions
Dissolving 200mg of the trollius chinensis bunge polysaccharide extract in 100mL of ultrapure water, stirring for 30min, and sequentially adding phenoxyethanol (0.5mL), butanediol (0.2mL), catechin (1.0g) and orange flower essence (0.2 mL). And fully and uniformly stirring to obtain the nymphaea hybrid polysaccharide essence similar to gel.
Example 6: preparation method of nymphaea hybrid polysaccharide beverage
Dissolving 200mg of the trollius chinensis bunge polysaccharide extract in 100mL of ultrapure water, stirring for 30min, and sequentially adding polylysine (0.5g), sodium dehydroacetate (0.5g), sucrose (2.0g), stevioside (0.2g) and orange juice (10 mL). After fully and uniformly stirring, obtaining the nymphaea hybrid polysaccharide functional beverage with the taste similar to orange juice.
Example 7: preparation of nymphaea hybrid polysaccharide buccal tablet
Taking 1.0g of the trollius chinensis bunge polysaccharide extract, 4.0g of xylitol, 50mg of stevioside, 50mg of orange juice powder and 50mg of magnesium stearate, stirring uniformly, and crushing into micro powder with the granularity of more than 300 meshes by a crusher. After adding 3mL of ultrapure water and stirring the mixture uniformly, the mixture was granulated (about 25 granules). Freeze-drying the prepared granules, and tabletting by a tabletting machine to obtain the functional trollius chinensis polysaccharide buccal tablet.
