CN111205376B - Preparation method and application of wart litchi and snail polysaccharide - Google Patents
Preparation method and application of wart litchi and snail polysaccharide Download PDFInfo
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- CN111205376B CN111205376B CN202010016291.9A CN202010016291A CN111205376B CN 111205376 B CN111205376 B CN 111205376B CN 202010016291 A CN202010016291 A CN 202010016291A CN 111205376 B CN111205376 B CN 111205376B
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
Abstract
The invention discloses a preparation method and application of wart snail polysaccharide, which comprises the steps of taking fresh wart snail (Thais clavigera Kuster) as a starting material, boiling with water, shelling and taking meat, carrying out vacuum freeze drying treatment, extracting with alkaline water, precipitating with ethanol, carrying out protease enzymolysis, decolorizing with macroporous resin, and obtaining a refined polysaccharide component which is obtained after ultrafiltration and has protein and pigment removed and molecular weight larger than 10 kD. The wart lychee snail polysaccharide prepared by the invention has obvious anti-hepatitis B activity, can obviously reduce the content of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in serum, and has important effect on protecting liver.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of wart litchi snail polysaccharide.
Background
Hepatitis B Virus (HBV) is a typical hepadnavirus, and chronic infection of Hepatitis B virus develops into liver fibrosis, cirrhosis, liver dysfunction, and even liver cancer. Hepatitis b is a chronic necrotic disease caused by Hepatitis B Virus (HBV) infection, has become a major public health problem worldwide, and the malignant tumor of the liver caused by hepatitis b is the fifth place of the malignant tumor. Inhibiting or killing hepatitis B virus has very important function for treating hepatitis B. The current clinical drugs for treating chronic hepatitis B virus are divided into two types: one is an immunomodulator, which has low response rate and great side effect; the other is nucleotide analogues which are prone to accumulate drug resistance and have long treatment courses and require life-long treatment, so that the development of new safe and effective anti-HBV drugs is urgent.
The research proves that the polysaccharide has excellent effects of resisting oxidation, removing free radicals and the like, has the characteristics of high efficiency, low toxicity and high bioavailability, shows unique superiority in preventing and treating diseases caused by the free radicals, such as tumors, inflammations and the like, and has an important effect in improving the immunity of the organism. At present, researches show that polysaccharide extracts such as polyporus polysaccharide, spirulina polysaccharide, seaweed sulfated polysaccharide and the like have better anti-HBV activity.
Wart lychee snail is also called bitter snail and peppery snail, is an edible mollusk very popular in southern Fujian area, and people have habit of eating the conch to achieve the purpose of protecting liver, and also have cases of successfully turning hepatitis B into negative by eating the conch. Modern medical research shows that the conch has better anti-inflammatory effect, but the research results in the aspects of anti-hepatitis B virus and liver protection are less, and no relevant report exists so far.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of wart litchi snail polysaccharide.
The invention also aims to provide application of the wart lychee snail polysaccharide.
The technical scheme of the invention is as follows:
a preparation method of wart lychee snail polysaccharide comprises the following steps:
(1) degreasing the molluscum contagiosum powder by using normal hexane to obtain degreased molluscum contagiosum powder;
(2) mixing the defatted molluscum powder with 0.8-1.2wt% sodium hydroxide solution, leaching at 70-80 deg.C for 2-3 times, each time for 1-2 hr, and performing solid-liquid separation to obtain molluscum aqueous extract;
(3) adjusting the pH of the water extract of the wart litchi chinensis snail to be neutral, and then placing the water extract of the wart litchi chinensis snail in a rotary evaporator for reduced pressure concentration to obtain a concentrated water extract of the wart litchi chinensis snail;
(4) mixing the concentrated wart litchi chinensis snail water extract with absolute ethyl alcohol, standing at room temperature until precipitates appear, and centrifuging and filtering to obtain a first precipitate;
(5) freeze-drying the first precipitate, re-dissolving in distilled water, centrifuging, and filtering to remove water insoluble substances to obtain a first filtrate;
(6) mixing the first filtrate with absolute ethyl alcohol, standing at room temperature until a precipitate appears, and centrifuging and filtering to obtain a second precipitate;
(7) freeze-drying the second precipitate to obtain first wart litchi chinensis crude polysaccharide;
(8) dissolving the first wart litchi chinensis crude polysaccharide in distilled water, and adding trypsin or papain for enzymolysis to obtain an enzymolysis solution;
(9) adding Sevage reagent into the enzymolysis liquid, fully stirring and uniformly mixing, centrifuging to remove insoluble substances, collecting supernatant, and then replacing the enzymolysis liquid with the supernatant to repeat the step until no obvious precipitate exists, thereby obtaining supernatant;
(10) concentrating the supernatant, and freeze drying to obtain second wart litchi chinensis crude polysaccharide;
(11) dissolving the second wart litchi chinensis snail crude polysaccharide in distilled water, adsorbing and decoloring by macroporous resin, centrifuging by a 10kD ultrafiltration tube, collecting liquid on the upper layer of the 10kD ultrafiltration tube, and concentrating, freezing and drying the liquid to obtain the wart litchi chinensis snail polysaccharide.
In a preferred embodiment of the present invention, the preparation method of the wart lychee snail powder comprises the following steps: cleaning fresh wart-containing litchi chinensis with uniform size, removing silt, boiling in clear water for 3-5min, rapidly cooling with cold water, picking out edible meat part, and discarding the rest parts; freezing the edible meat in refrigerator for at least 24h, quickly taking out, vacuum freeze drying for 24-48h until completely drying, pulverizing in Chinese medicinal pulverizer, and sieving with 75-85 mesh sieve.
In a preferred embodiment of the invention, the feed-liquid ratio of the defatted wart lychee snail powder to the sodium hydroxide solution in the step (1) is 1: 8-12.
In a preferred embodiment of the present invention, the conditions of the enzymatic hydrolysis in the step (7) are: the enzyme is used in an amount of 3-5% for 1.8-2.2h, at 58-61 deg.C and pH of 7.4-7.6.
The other technical scheme of the invention is as follows:
the application of the wart litchi chinensis snail polysaccharide prepared by the preparation method in preparing the medicine for preventing and treating the viral hepatitis B.
In a preferred embodiment of the invention, the dosage form of the medicament for preventing and treating the viral hepatitis B is an injection preparation or an oral preparation.
The invention adopts another technical scheme as follows:
the application of the wart litchi chinensis snail polysaccharide prepared by the preparation method in preparing a health-care product for protecting the liver.
The invention has the beneficial effects that: the invention takes fresh wart lychee snail (Thais clavigera Kuster) as a starting material, and the fresh wart lychee snail is boiled in water, shelled to take meat, is subjected to vacuum freeze drying treatment, then is extracted by alkaline water, is precipitated by ethanol, is subjected to protease enzymolysis, is decolorized by macroporous resin, and is subjected to ultrafiltration to obtain a refined polysaccharide component with protein and pigment removed and molecular weight more than 10 kD.
Drawings
FIG. 1 shows the effect of wart litchi chinensis helical polysaccharide on the appearance of liver pathological changes of HBV transgenic mice in example 1 of the present invention.
FIG. 2 shows the effect of wart litchi chinensis helical polysaccharide on liver weight of HBV transgenic mice in example 1 of the present invention.
FIG. 3 is the H.E staining of liver tissue after the effect of wart lychee snail polysaccharide on HBV transgenic mice in example 1 of the invention.
FIG. 4 shows the effect of wart lychee snail polysaccharide on liver function indexes AST and ALT of HBV transgenic mice in example 1 of the invention.
FIG. 5 shows the effect of Conus pumila Linnaeus polysaccharide on the serum markers HBsAg, HBeAGg and HBVDNA of HBV transgenic mice in example 1 of the present invention.
FIG. 6 shows the effect of Conus chinensis polysaccharide on the liver tissue indexes HBsAg, HBeAGg, HBVDNA and HBVRNA of HBV transgenic mice in example 1 of the present invention.
FIG. 7 is an HPLC chromatogram of a wart litchi spiro polysaccharide sample and a monosaccharide standard after PMP derivatization in example 1 of the invention. Wherein, (a) is an HPLC mixed spectrum of a monosaccharide standard substance after PMP derivatization; (b) is an HPLC chromatogram of a polysaccharide sample after PMP derivatization; 1-mannose; 2-glucosamine; 3-glucuronic acid; 4-galacturonic acid; 5-glucose; 6-galactose; 7-rhamnose; 8-arabinose.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1
A preparation method of wart lychee snail polysaccharide comprises the following steps:
(1) degreasing the wart lychee snail powder by using normal hexane to obtain degreased wart lychee snail powder, which specifically comprises the following steps: adding n-hexane into 300g of the wart oncomelania littoralis powder according to a proper proportion, extracting for 1-2min each time, repeating for 3 times, filtering, removing a clear bright yellow solution, taking a precipitate, namely the degreased wart oncomelania littoralis powder, and airing for later use;
the preparation method of the wart lychee snail powder comprises the following steps: cleaning fresh wart-containing litchi chinensis with uniform size, removing silt, boiling in clear water for 3-5min, rapidly cooling with cold water, picking out edible meat part, and discarding the rest parts; freezing the edible meat in refrigerator for at least 24h, quickly taking out, putting into vacuum freeze drying oven for 24-48h until completely drying, pulverizing in Chinese medicinal pulverizer, and sieving with 80 mesh sieve;
(2) mixing the defatted molluscacide powder with 0.1 wt% sodium hydroxide solution at a ratio of 1: 10, leaching at 70 deg.C for 2-3 times, each time for 50min, and performing solid-liquid separation to obtain molluscacide liquid extract;
(3) cooling the wart litchi chinensis snail water extract, adjusting the pH value to be neutral by using 0.1mol/L HCl, and then placing the cooled wart litchi chinensis snail water extract in a rotary evaporator for reduced pressure concentration to obtain a concentrated wart litchi chinensis snail water extract;
(4) slowly mixing the concentrated wart litchi chinensis snail water extract with absolute ethyl alcohol, standing at room temperature until precipitates appear, and centrifuging and filtering to obtain 73.0g of first precipitates;
(5) freeze-drying the first precipitate, re-dissolving in distilled water, centrifuging, and filtering to remove water insoluble substances to obtain a first filtrate;
(6) mixing the first filtrate with absolute ethyl alcohol, standing at room temperature until a precipitate appears, and centrifuging and filtering to obtain a second precipitate;
(7) freeze-drying the second precipitate to obtain 118.15g of first wart litchi chinensis crude polysaccharide;
(8) dissolving the first wart litchi chinensis crude polysaccharide in distilled water, and adding papain for enzymolysis to obtain an enzymolysis solution; the enzymolysis conditions are as follows: the enzyme dosage is 4%, the time is 2h, the temperature is 60 ℃, the pH value is 7.5, then the temperature is increased to 90 ℃ to act for 15min to inactivate the enzyme, the temperature is cooled to room temperature, and the pH value is adjusted to be neutral by 0.1mol/L HCl;
(9) adding Sevage reagent (CHCl) into the above enzymolysis solution3Mixing n-butanol at volume ratio of 5: 1), stirring, centrifuging to remove insoluble substances, collecting supernatant, and repeating the above steps to obtain supernatant;
(10) concentrating the supernatant, and freeze drying to obtain second wart litchi chinensis crude polysaccharide;
(11) dissolving the crude second wart litchi chinensis polysaccharide in distilled water, adsorbing and decoloring by macroporous resin (passing through a D101 macroporous adsorption resin packed column with r being 2.5cm and h being 35cm twice, eluting by distilled water, identifying the polysaccharide in the eluent by a sulfuric acid-phenol method, stopping eluting until the polysaccharide is not detected in the eluent, concentrating the eluent, passing through a clean D101 macroporous adsorption resin packed column with r being 2.5cm and h being 35cm again, decoloring for the second time, concentrating the eluent to obtain crude polysaccharide after pigment removal, and decoloring again by using active carbon if the color is dark), then removing small molecular impurities from the decolored crude polysaccharide by using ultrafiltration tubes of 3kD and 10kD respectively, and the polysaccharide is subjected to primary segmentation and centrifugation, the upper layer solution is freeze-dried and weighed to obtain 18.77g of total polysaccharide, and the polysaccharide yield is calculated to be 6.25%. Wherein 13.90g of purified polysaccharide having a molecular weight of 10kD or more, 3.73g of purified polysaccharide having a molecular weight of 3kD to 10kD, and 1.14g of purified polysaccharide having a molecular weight of less than 3kD are obtained. Redissolving the refined polysaccharide with the molecular weight of more than 10kD with distilled water, removing insoluble substances, taking supernatant, and freeze-drying to obtain 9.7g of the wart litchi snail polysaccharide with the molecular weight of more than 10 kD.
In vivo pharmacodynamics of HBV transgenic mouse model
Experimental design and methods: the experimental animals were SPF-level HBV transgenic mice (C57BL/6-HBV, beijing wituda biotechnology limited) 25, randomly divided into 5 groups of 5 animals each, each group consisting of Tenofovir Alafenamide (TAF) group (positive control group), low dose group, medium dose group, high dose group and vehicle group (negative control), and raised in the barrier environment of SPF-level secondary biosafety laboratory. The positive control group is administered with Tenofovir Alafenamide (TAF) at a dose of 10mg/kg, the high, middle and low groups of the experimental group are sequentially administered with doses of 50mg/kg, 100mg/kg and 150mg/kg, the negative control group is used as a solvent, and the administration route is intraperitoneal injection. The administration was carried out at the same time every day for 28 days.
Preparation of serum and liver tissue samples: after 28 days of intraperitoneal injection administration of experimental mice, all experimental mice are fasted and fed without water for 12 hours, beard is cut off, 0.5-1 mL of blood is taken out from eyeballs, standing is carried out at 4 ℃, the cervical vertebrae of the mice are dislocated and killed, the complete liver is dissected and taken out, the mice are rinsed by precooled physiological saline, water on the surface of the liver is sucked by absorbent paper, the mice are weighed, the left leaves of the liver are taken and placed in 10% neutral formalin for fixation, and the left leaves of the liver are used for preparing pathological sections in the later period. Taking the tissue at the same position of the right lobe of the liver tissue, cleaning, mixing with the homogenate, fully crushing, centrifuging, and taking the supernatant.
The experimental results are as follows:
1. appearance, weight and pathological section H.E. staining of experimental animal liver
Mouse liver appearance observation: FIG. 1 is an appearance diagram of mouse liver, the liver color of each group of mice is ruddy, the negative control group, i.e. the model group, has a few needle point-shaped necrotic foci and a few micro nodules on the surface, is not smooth, and has obvious blood vessel congestion on the edge. The liver of the positive control group and the liver of the treatment group have luster and have no necrotic spots or bleeding. The liver weights of HBV transgenic mice after 28 days of administration are shown in Table 1.
TABLE 1
Note:*the analysis of the significant difference from the solvent group is that p is less than 0.05;**analysis of significant difference from the solvent group, p is less than 0.01
The results in table 1 show that, compared with the negative control in the vehicle group, the weight average of the liver in the positive control TAF group and the liver in the treatment group are reduced, and the significant difference exists, wherein the liver weight reduction effect in the wart litchi spiroglycan high-dose group is the most significant (p is less than 0.01). The HBV transgenic mouse can generate complete HBV virus particles, has strong killing effect on liver cells, and causes liver re-compensation hyperplasia and inflammatory edema, thereby increasing the liver weight. The reduction of the liver weight of the model animal can intuitively reflect the reduction of liver inflammatory edema or hyperplasia, and the liver weight experiment result shows that the wart lychee snail polysaccharide has a certain therapeutic effect on the HBV transgenic mouse at the organ level (figure 2).
2. H.E. staining of liver tissue of experimental group HBV transgenic mouse
The negative control group has the defects of liver cell swelling, more deeply-stained cell nucleus, disordered liver lobule structure, almost invisible liver cord and no false lobule. The liver tissues of the experimental group and the positive control TAF group are recovered to a certain degree, the swelling degree of liver cells is greatly reduced, partial liver chorda structure can be seen, partial liver sinus can be seen, but the whole liver state is not reached to the normal liver state. Wherein, the effect of the high-dose group and the medium-dose group is better than that of the positive control group, the swelling degree of the liver cells is lighter, and the liver cords are more obvious and ordered. The section H.E. staining result shows that the wart litchi chinensis spiroglycan has obvious improvement effect on the liver of the HBV transgenic mouse and is superior to the positive control TAF under the experimental dose. (see FIG. 3)
3. Influence of experimental group on biochemical indexes of liver function (AST and ALT) of HBV transgenic mice
Glutamate-oxaloacetate transaminase (AST) and glutamate-pyruvate transaminase (ALT) are known as human body substance metabolism catalysts, are rich in the cytoplasm of liver cells and are also important indexes for measuring whether the liver is normal or not, and when HBV virus particles are released from the liver cells to cause cell rupture and damage, the AST and ALT contents in serum can rise, so that clinical diagnosis is carried out. The liver function (AST, ALT) index test results are shown in Table 2.
TABLE 2
Note:*the analysis of the significant difference from the solvent group is that p is less than 0.05;**analysis of significant difference from the solvent group, p is less than 0.01
Fig. 4 shows the change of the content of AST and ALT in the serum of the mouse, and the results show that the content of AST and ALT in the experimental group is obviously reduced compared with the negative control of the vehicle group, and especially the effect of the high-dose group is better than that of the positive control TAF group under the experimental dose. The results show that the wart litchi polysaccharide has obvious effects of reducing transaminase and improving liver function damage caused by HBV, has obvious liver protection effect, and is consistent with the results expressed at an organ level and a cell level in the fifth embodiment.
4. Effect of the experimental group on serum markers (HBsAg, HBeAg and HBV DNA) of HBV transgenic mice
Hepatitis B Virus (HBV) antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) are important indexes for clinically judging HBV infection. Hepatitis B surface antigen (HBsAg) is the coat protein of HBV, is antigenic in itself and not infectious, but its appearance is often accompanied by the presence of HBV and is therefore a hallmark of infection with HBV. HBeAg is the core internal component of the virus and is considered to be a marker of viral replication in viral serological assays, indicating the presence of progressive damage and high infectivity of hepatocytes. HBV DNA is HBV deoxyribonucleic acid and is also a virus core part, and quantitative HBV DNA examination is the most direct index for detecting HBV infection in order to judge the gold index of virus replication. The current infection situation of a patient can be judged and the treatment effect of the medicine can be accurately evaluated by quantitatively detecting the content of HBV in the blood of a hepatitis B patient clinically. The above three indexes are currently the most commonly used important indexes for evaluating anti-HBV activity. The effect of the experimental group on serum indicators of HBV transgenic mice is shown in Table 3.
TABLE 3
Note:*the analysis of the significant difference from the solvent group is that p is less than 0.05;**analysis of significant difference from the solvent group, p is less than 0.01
The results show that the experimental group has a remarkable inhibitory effect compared with the vehicle group, and each polysaccharide group shows better dose dependence. Wherein the positive control group has the same inhibitory effect on HBsAg and HBeAg as the low dose group and the medium dose group, but weaker than the high dose group; the inhibitory effect of HBV DNA is better than that of wart litchi and snail polysaccharide group. (see FIG. 5)
5. Effect of the experimental groups on HBV transgenic mouse liver markers (HBsAg, HBeAg, HBV DNA and RNA)
HBV is a hepatotropic virus that can proliferate in large amounts in the liver, the content of HBV virions in liver tissue can be reflected by the level of HBsAg, HBeAg and HBV DNA in liver tissue, while the level of HBV RNA in liver cells can reflect the replication of HBV. The effect of the experimental group on the liver index of HBV transgenic mice is shown in Table 4.
TABLE 4
Note:*the analysis of the significant difference from the solvent group is that p is less than 0.05;**analysis of significant difference from the solvent group, p is less than 0.01
The results in table 4 show that the levels of HBsAg, HBeAg, and HBV DNA and HBV RNA in the liver tissue were significantly reduced compared to the vehicle group, and the polysaccharide groups showed better dose dependence. Except HBV DNA, other indexes show that the medium-dose group and the high-dose group of the polysaccharide are superior to the positive control TAF group, which shows that the polysaccharide plays a certain role in inhibiting the synthesis of HBV RNA, and compared with the role mechanism of inhibiting DNA replication by TAF, the polysaccharide has a new role mechanism. (see FIG. 6)
Wart litchi and snail polysaccharide monosaccharide composition analysis
Acid hydrolysis of wart litchi spiropolysaccharide: accurately weighing the wart litchi chinensis snail polysaccharide (10 +/-0.1 mg) with the molecular weight of more than 10kD, placing the wart litchi chinensis polysaccharide into a 10mL volumetric flask, adding distilled water to a constant volume, taking 1mL polysaccharide solution into a 5mL round-bottom flask, adding 2mL of 2mol/mL trifluoroacetic acid solution, heating and refluxing in an oil bath kettle at 105 ℃ for 12 hours, taking out the solution, evaporating the solvent, dropwise adding methanol into the flask, evaporating and repeating for three times. And transferring the solid into a 5mL volumetric flask, and adding distilled water to constant volume to obtain the polysaccharide hydrolysate.
Derivatization and purification of polysaccharide: accurately transferring 100 μ L of the polysaccharide hydrolysate into a 2.5mL centrifuge tube, adding 100 μ L of 0.5mol/mL 1-phenyl-3 methyl-5-pyrazolone (PMP) methanol solution and 100 μ L of 0.3mol/L sodium hydroxide solution, vortex mixing, reacting in 70 deg.C water bath for 2h, taking out, cooling to room temperature, adding 100 μ L of 0.3mol/L hydrochloric acid solution, vortex mixing, adding 1mL chloroform, fully oscillating, centrifuging with an ultracentrifuge, standing for layering, discarding the lower CCl3Layer, using CCl according to the above method3Extracting for three times, filtering the upper water phase with 0.45 μm filter membrane, and performing high performance liquid chromatography.
Derivatization of monosaccharide standard working solutions: respectively and accurately transferring monosaccharide [ D-mannose (Man), L-rhamnose (Rha), D-glucose (Glc), DL-arabinose (Arab), galacturonic acid, D-galactose (Gal), D-glucuronic acid (Glc-UA) and glucosamine with the concentration of 10mg/mL]Adding 100 mu L of standard working solution into a 2.5mL centrifuge tube, adding 100 mu L of 0.5 mol/L1-phenyl-3 methyl-5-pyrazolone methanol solution and 100 mu L of 0.3mol/L sodium hydroxide solution, mixing by vortex, reacting in 70 ℃ water bath for 2h, taking out, cooling to room temperature, adding 100 mu L of 0.3mol/L hydrochloric acid solution, mixing by vortex, adding 1mL CCl3Fully oscillating, centrifuging for 2min at 12000r/min by an ultracentrifuge, standing for layering, and discarding the lower CCl3Layer, using CCl according to the above method3Fully extracting for 3 times, taking a water layer, and preparing the monosaccharide series standard solution derivatives. The derivatives of the monosaccharide series standard solution are filtered through a 0.45-micron filter membrane and subjected to high performance liquid chromatography. Peaks derived from standard solutions of the monosaccharide seriesThe area is the ordinate, and the concentration of the corresponding monosaccharide is the abscissa to draw a standard curve.
Chromatographic conditions
A chromatographic column: c18 reverse phase chromatography column (4.6 mm. times.150 mm, 5 μm).
Column temperature: at 40 ℃.
Flow rate: 0.9 mL/min.
Detection wavelength: 250 nm.
Sample introduction amount: 20 μ L.
Mobile phase: a: phosphate buffer salt-acetonitrile solution A [ (KH)2PO4NaOH, pH 6.9: acetonitrile (85: 15)]
B: phosphate buffer salt-acetonitrile solution B [ (KH)2PO4NaOH, pH 6.9: acetonitrile (60: 40)]
TABLE 5 procedure for mobile phase gradient elution
HPLC (high performance liquid chromatography) chromatogram HPLC results of the wart litchi snail polysaccharide and monosaccharide after PMP derivatization are shown in a figure 7, and monosaccharide component analysis results are shown in a table 6:
TABLE 6
As shown in fig. 7, the HPLC chromatogram after derivatization of the wart litchi snail polysaccharide was compared with the standard, and the results showed that the wart litchi snail polysaccharide mainly contains monosaccharides: mannose, glucosamine, glucuronic acid, glucose, galactose and arabinose. The simplest molar ratio of the above monosaccharides is about 9: 16: 4: 512: 9: 4.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (9)
1. The application of the wart litchi snail polysaccharide in preparing the medicine for preventing and treating the viral hepatitis B is characterized in that: the preparation method of the wart litchi snail polysaccharide comprises the following steps:
(1) degreasing the molluscum contagiosum powder by using normal hexane to obtain degreased molluscum contagiosum powder;
(2) mixing the defatted molluscum powder with 0.8-1.2wt% sodium hydroxide solution, leaching at 70-80 deg.C for 2-3 times, each time for 1-2 hr, and performing solid-liquid separation to obtain molluscum aqueous extract;
(3) adjusting the pH of the water extract of the wart litchi chinensis snail to be neutral, and then placing the water extract of the wart litchi chinensis snail in a rotary evaporator for reduced pressure concentration to obtain a concentrated water extract of the wart litchi chinensis snail;
(4) mixing the concentrated wart litchi chinensis snail water extract with absolute ethyl alcohol, standing at room temperature until precipitates appear, and centrifuging and filtering to obtain a first precipitate;
(5) freeze-drying the first precipitate, re-dissolving in distilled water, centrifuging, and filtering to remove water insoluble substances to obtain a first filtrate;
(6) mixing the first filtrate with absolute ethyl alcohol, standing at room temperature until a precipitate appears, and centrifuging and filtering to obtain a second precipitate;
(7) freeze-drying the second precipitate to obtain first wart litchi chinensis crude polysaccharide;
(8) dissolving the first wart litchi chinensis crude polysaccharide in distilled water, and adding trypsin or papain for enzymolysis to obtain an enzymolysis solution;
(9) adding Sevage reagent into the enzymolysis liquid, fully stirring and uniformly mixing, centrifuging to remove insoluble substances, collecting supernatant, and then replacing the enzymolysis liquid with the supernatant to repeat the step until no obvious precipitate exists, thereby obtaining supernatant;
(10) concentrating the supernatant, and freeze drying to obtain second wart litchi chinensis crude polysaccharide;
(11) dissolving the second wart litchi chinensis snail crude polysaccharide in distilled water, adsorbing and decoloring by macroporous resin, centrifuging by a 10kD ultrafiltration tube, collecting liquid on the upper layer of the 10kD ultrafiltration tube, and concentrating, freezing and drying the liquid to obtain the wart litchi chinensis snail polysaccharide.
2. The use of claim 1, wherein: the preparation method of the wart lychee snail powder comprises the following steps: cleaning fresh wart-containing litchi chinensis with uniform size, removing silt, boiling in clear water for 3-5min, rapidly cooling with cold water, picking out edible meat part, and discarding the rest parts; freezing the edible meat in refrigerator for at least 24h, quickly taking out, vacuum freeze drying for 24-48h until completely drying, pulverizing in Chinese medicinal pulverizer, and sieving with 75-85 mesh sieve.
3. The use of claim 1, wherein: the material-liquid ratio of the defatted wart lychee snail powder and the sodium hydroxide solution in the step (2) is 1: 8-12.
4. The use of claim 1, wherein: the enzymolysis conditions in the step (8) are as follows: the enzyme dosage is 3-5%, the time is 1.8-2.2h, the temperature is 58-61 deg.C, and pH = 7.4-7.6.
5. The use according to any one of claims 1 to 4, wherein: the dosage form of the medicament for preventing and treating the viral hepatitis B is an injection preparation or an oral preparation.
6. The application of the wart lychee snail polysaccharide in preparing the health care product for protecting the liver is characterized in that: the preparation method of the wart litchi snail polysaccharide comprises the following steps:
(1) degreasing the molluscum contagiosum powder by using normal hexane to obtain degreased molluscum contagiosum powder;
(2) mixing the defatted molluscum powder with 0.8-1.2wt% sodium hydroxide solution, leaching at 70-80 deg.C for 2-3 times, each time for 1-2 hr, and performing solid-liquid separation to obtain molluscum aqueous extract;
(3) adjusting the pH of the water extract of the wart litchi chinensis snail to be neutral, and then placing the water extract of the wart litchi chinensis snail in a rotary evaporator for reduced pressure concentration to obtain a concentrated water extract of the wart litchi chinensis snail;
(4) mixing the concentrated wart litchi chinensis snail water extract with absolute ethyl alcohol, standing at room temperature until precipitates appear, and centrifuging and filtering to obtain a first precipitate;
(5) freeze-drying the first precipitate, re-dissolving in distilled water, centrifuging, and filtering to remove water insoluble substances to obtain a first filtrate;
(6) mixing the first filtrate with absolute ethyl alcohol, standing at room temperature until a precipitate appears, and centrifuging and filtering to obtain a second precipitate;
(7) freeze-drying the second precipitate to obtain first wart litchi chinensis crude polysaccharide;
(8) dissolving the first wart litchi chinensis crude polysaccharide in distilled water, and adding trypsin or papain for enzymolysis to obtain an enzymolysis solution;
(9) adding Sevage reagent into the enzymolysis liquid, fully stirring and uniformly mixing, centrifuging to remove insoluble substances, collecting supernatant, and then replacing the enzymolysis liquid with the supernatant to repeat the step until no obvious precipitate exists, thereby obtaining supernatant;
(10) concentrating the supernatant, and freeze drying to obtain second wart litchi chinensis crude polysaccharide;
(11) dissolving the second wart litchi chinensis snail crude polysaccharide in distilled water, adsorbing and decoloring by macroporous resin, centrifuging by a 10kD ultrafiltration tube, collecting liquid on the upper layer of the 10kD ultrafiltration tube, and concentrating, freezing and drying the liquid to obtain the wart litchi chinensis snail polysaccharide.
7. The use of claim 6, wherein: the preparation method of the wart lychee snail powder comprises the following steps: cleaning fresh wart-containing litchi chinensis with uniform size, removing silt, boiling in clear water for 3-5min, rapidly cooling with cold water, picking out edible meat part, and discarding the rest parts; freezing the edible meat in refrigerator for at least 24h, quickly taking out, vacuum freeze drying for 24-48h until completely drying, pulverizing in Chinese medicinal pulverizer, and sieving with 75-85 mesh sieve.
8. The use of claim 6, wherein: the material-liquid ratio of the defatted wart lychee snail powder and the sodium hydroxide solution in the step (2) is 1: 8-12.
9. The use of claim 6, wherein: the enzymolysis conditions in the step (8) are as follows: the enzyme dosage is 3-5%, the time is 1.8-2.2h, the temperature is 58-61 deg.C, and pH = 7.4-7.6.
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| CN1749282A (en) * | 2005-10-11 | 2006-03-22 | 大连轻工业学院 | Belly bottom snail polysaccharide and its preparing method |
| CN107955080A (en) * | 2017-11-20 | 2018-04-24 | 淮阴工学院 | It is a kind of that there is the anti-oxidant and method of the white shellfish acidic polysaccharose of liver protection effect from Bai Beizhong extractions |
| CN109402206A (en) * | 2018-11-22 | 2019-03-01 | 浙江海洋大学 | A kind of preparation method of Thais clavgeria Antihypertensive Peptides |
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| CN107955080A (en) * | 2017-11-20 | 2018-04-24 | 淮阴工学院 | It is a kind of that there is the anti-oxidant and method of the white shellfish acidic polysaccharose of liver protection effect from Bai Beizhong extractions |
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