CN110483657B - Chinese lobelia homogeneous polysaccharide and preparation method and application thereof - Google Patents
Chinese lobelia homogeneous polysaccharide and preparation method and application thereof Download PDFInfo
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- CN110483657B CN110483657B CN201910863145.7A CN201910863145A CN110483657B CN 110483657 B CN110483657 B CN 110483657B CN 201910863145 A CN201910863145 A CN 201910863145A CN 110483657 B CN110483657 B CN 110483657B
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- polysaccharide
- lobelia
- chinese lobelia
- water
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- 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
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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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Abstract
The invention provides a Chinese lobelia homogeneous polysaccharide and a preparation method and application thereof. The preparation method of the lobelia chinensis homogeneous polysaccharide comprises the following steps: selecting Chinese lobelia herb, performing water extraction and alcohol precipitation on the Chinese lobelia herb, and performing freeze drying to obtain a Chinese lobelia polysaccharide crude product; dissolving the crude lobelia chinensis polysaccharide product with water, loading the dissolved crude lobelia chinensis polysaccharide product onto a DEAE-Fast-Flow anion exchange column, eluting with distilled water, then eluting with NaCl solutions with different concentrations, and simultaneously detecting by adopting a phenol concentrated sulfuric acid method to obtain the polysaccharide at the water-eluted part of the lobelia chinensis; loading the water-eluted part of herba Lobeliae chinensis polysaccharide to Sephadex chromatographic column, eluting with NaCl solution, collecting effective part according to peak position, dialyzing with dialysis bag, and freeze drying the dialyzed solution to obtain herba Lobeliae chinensis homogeneous polysaccharide. The Chinese lobelia homogeneous polysaccharide can obviously reduce the content of total cholesterol and triglyceride in serum and liver of obese mice, and is used for preparing medicaments for preventing or/and treating obesity and hyperglycemia.
Description
Technical Field
The invention relates to the field of Chinese lobelia homogeneous polysaccharide, and particularly relates to Chinese lobelia homogeneous polysaccharide and a preparation method and application thereof.
Background
In recent years, more and more obese patients are available, obese people are more prone to cause hypertension, hyperlipidemia, hyperglycemia and other 'three-high' symptoms, and the plant polysaccharide has biological activities of regulating blood sugar, reducing blood fat and the like, and can be used for improving and treating the 'three-high' symptoms. Chinese herba Lobeliae chinensis is a plant of Campanulaceae, has effects of clearing heat and detoxicating, inducing diuresis and relieving swelling, and has been researched more by modern science for small molecule alkaloids contained therein, especially plant polysaccharide has become a research hotspot for searching for therapeutic drugs for obesity.
Lobelia chinensis Lour (Lobelia chinensis Lour) is a perennial herb of the genus Lobelia of the family Campanulaceae, also known as JIEJISUO, FINGMEICAO and GUANRENCAO. Produced in China east China, the middle, lower and south provinces of Yangtze river. The Lobelia chinensis whole plant can be used for medicine, and has effects of clearing heat and detoxicating, inducing diuresis and relieving swelling, and stopping bleeding. As early as in Ben Cao gang mu of Li Shizhen in Ming Dynasty, there are records of snake poisonous snake bite, juice pounding and juice drinking, and residue smearing. Herba Lobeliae chinensis is special in flavor, sweet and pungent in flavor, and is prepared into decoction pieces by traditional Chinese medicine, decocted with water for oral administration, and the residue is mixed with human milk to obtain paste for applying on affected part for treating carbuncle, furuncle, poisonous insect, poisonous snake bite, herpes zoster, etc.
With the application of modern technology, people separate various active ingredients in Chinese lobelia, mainly comprising alkaloids, flavonoids, saponins, amino acids, polysaccharides and some small molecules which can be used as industrial raw materials, such as p-aminobenzoic acid, fumaric acid and succinic acid. At present, researches on the active ingredients of Chinese lobelia are mainly focused on alkaloids and flavonoids. The research on the Chinese lobelia polysaccharide is not deep, most of the research on the Chinese lobelia polysaccharide is stopped at the extraction and purification of the polysaccharide and the optimization of the process, and the research on the biological activity of the Chinese lobelia polysaccharide is rare. For example, CN201110134569.3 discloses a lobelia polysaccharide extracted from lobelia and its application, and CN102204946B discloses an application of the lobelia polysaccharide extracted from lobelia in preparing medicine for enhancing immune function. However, the structure of the lobelia uniform polysaccharide is not reported in the prior art (1); (2) there is no report on the research of the application of the Chinese lobelia homopolysaccharide in the treatment of obesity; (3) no research report of the application of the Chinese lobelia homogeneous polysaccharide in preventing and treating hyperglycemia is reported.
Disclosure of Invention
The present invention is intended to solve at least the problems of obesity and hyperglycemia existing in the prior art. The object of the present invention is to provide a compound which is effective for the prevention and treatment of obesity and hyperglycemia.
In a first aspect, the invention provides a lobelia homogeneous polysaccharide.
A Chinese medicinal preparation, BANBIANLIAN homogeneous polysaccharide, is prepared from herba Lobeliae chinensis by extracting with water, precipitating with ethanol, separating and purifying by column chromatography, and is linear straight-chain polysaccharide composed of terminal group alpha-D-glucose and D-fructose residue connected by beta-2, 1 glycosidic bond, and has relative molecular weight of 1-20 kDa. Preferably 2 to 4kDa, most preferably 2.7 kD.
In a second aspect, the invention provides a preparation method of Chinese lobelia homogeneous polysaccharide.
The preparation method of the Chinese lobelia homogeneous polysaccharide comprises the following specific steps:
a) taking the Chinese lobelia herb, extracting with water, precipitating with ethanol, and freeze-drying to obtain a Chinese lobelia polysaccharide crude product;
b) dissolving the crude Chinese lobelia polysaccharide obtained in the step a) with water, loading the dissolved crude Chinese lobelia polysaccharide to an anion exchange column, eluting with distilled water, then eluting with NaCl solution, and detecting by a phenol-sulfuric acid method to obtain polysaccharide at the water-eluted part of the Chinese lobelia;
c) loading polysaccharide at the water-eluted part of the Chinese lobelia obtained in the step b) onto a sephadex chromatographic column, and eluting with NaCl solution; collecting effective parts according to the peak position, dialyzing by using a dialysis bag with the molecular weight cutoff of 1000-14000Da, and freeze-drying the dialyzed inner liquid to obtain the lobelia uniform polysaccharide.
In the step a), degreasing the dried Chinese lobelia herb with ethanol, drying the residue, and then performing water extraction and alcohol precipitation.
The step a) comprises the following operations: firstly, carrying out reflux degreasing on the whole plant of the Chinese lobelia herb with ethanol, drying degreased residues, then adding water for heating and extracting for 2-5 times, combining extracting solutions, carrying out reduced pressure concentration and centrifugation, collecting supernate, adding 2-4 times of ethanol for carrying out alcohol precipitation, wherein the mass percent of the ethanol is 90-100%, and carrying out vacuum freeze drying on the collected precipitate to obtain a Chinese lobelia polysaccharide crude product.
The mass percentage of the ethanol used for degreasing treatment is 75-100%.
The material-liquid ratio (g/mL) during reflux degreasing is 1: 5-1: 20, and the reflux degreasing is carried out for 1-20 hours.
The feed-liquid ratio (g/mL) of the degreased dry residue to the water added each time is 1: 8-1: 30, the heating extraction temperature is 60-100 ℃, and the extraction time is 1-5 hours each time. Preferably, the method comprises the following steps: the heating extraction temperature is 85-100 ℃, and the extraction time is 1-3 hours each time.
And during alcohol precipitation, adding 90-100% by mass of 2-4 times of ethanol into the supernatant while stirring until the final mass fraction of the ethanol is 75-85%, and standing for 8-36 hours.
The step b) comprises the following operations: dissolving the prepared crude lobelia chinensis polysaccharide product with water, loading the dissolved crude lobelia chinensis polysaccharide product onto an anion exchange column, eluting with water, tracing an elution curve by a phenol-sulfuric acid method, collecting a sugar-containing solution, concentrating under reduced pressure, dialyzing a concentrate, and freeze-drying to obtain the polysaccharide at the water-eluted part of the lobelia chinensis.
The anion exchange column is DEAE-Fast Flow.
The step c) comprises the following operations: dissolving the prepared water-eluted part polysaccharide of the Chinese lobelia by using 0.1-0.4 mol/L NaCl solution, centrifuging, taking supernate to sample on a sephadex chromatographic column, eluting by using 0.1-0.4 mol/L NaCl solution, detecting by using a differential refraction detector, and collecting an effective part according to the peak position; concentrating, dialyzing with dialysis bag with molecular weight cutoff of 1000-.
In a third aspect, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises the Chinese lobelia homogeneous polysaccharide. According to the embodiment of the invention, the lobelia chinensis homopolysaccharide can obviously reduce the content of total cholesterol and triglyceride in serum and liver of an obese mouse, and has no toxic or side effect, and the pharmaceutical composition can effectively prevent and treat obesity; according to the embodiment of the invention, the lobelia chinensis homopolysaccharide can obviously reduce fasting blood glucose of mice, has very good glucose tolerance and blood glucose regulation effects, and the pharmaceutical composition can effectively prevent and treat hyperglycemia.
In a fourth aspect, the present invention provides the use of a lobelia homopolysaccharide in the manufacture of a medicament for the prevention and/or treatment of obesity, for the prevention and/or treatment of hyperglycemia.
In a fifth aspect, the present invention provides a food composition. The food composition comprises a lobelia uniform polysaccharide. According to the embodiment of the invention, the lobelia chinensis homopolysaccharide can obviously reduce the content of total cholesterol and triglyceride in serum and liver of an obese mouse, and has no toxic or side effect, and the food composition can effectively prevent and/or improve obesity; according to the embodiment of the invention, the lobelia chinensis homopolysaccharide can obviously reduce fasting blood glucose of mice, has very good glucose tolerance and blood glucose regulating effect, and the food composition can effectively prevent hyperglycemia and improve hyperglycemia symptoms.
In a sixth aspect, the present invention provides use of the homogeneous polysaccharide of Chinese lobelia for the preparation of a food for preventing and/or ameliorating obesity, and for preventing and/or ameliorating hyperglycemia symptoms.
The food composition or pharmaceutical composition of the present invention may be in various dosage forms as long as the active ingredient can be efficiently delivered into the body. Such as may be selected from: tablet, capsule, powder, granule, syrup, solution, suspension, injection, tincture, oral liquid, aerosol, buccal agent, granule, pill, powder, or sustained release preparation such as nanometer preparation.
Compared with the prior art, the invention has the following advantages:
(1) the Chinese lobelia homogeneous polysaccharide can obviously reduce total cholesterol and triglyceride in serum and liver of obese mice, has no toxic or side effect, can be expected to be used as a main or only active ingredient for preparing health-care food or pharmaceutical preparation for preventing or/and treating obesity, and has remarkable progress and unexpected effect compared with the prior art.
(2) The Chinese lobelia homogeneous polysaccharide provided by the invention can obviously reduce fasting blood glucose of mice, has very good glucose tolerance and blood glucose regulation effects, can be expected to be used as a main or only active ingredient for preparing health-care food or pharmaceutical preparation for preventing or/and treating hyperglycemia, and has significant progress and unexpected effect compared with the prior art.
(3) The preparation method is simple, can obtain high-purity lobelia chinensis homogeneous polysaccharide, and is suitable for large-scale production.
Drawings
FIG. 1 is a high performance liquid gel chromatogram of a Chinese lobelia homogeneous polysaccharide obtained in the present invention;
FIG. 2 is an infrared spectrum of the homogeneous polysaccharide of Chinese lobelia obtained in the present invention;
FIG. 3.1 is a hydrogen spectrum and a carbon spectrum of the homogeneous lobelia polysaccharide obtained in the present invention;
FIG. 3.2 is the HSQC spectrum of the Chinese lobelia homopolysaccharide obtained in the present invention;
FIG. 3.3 is a HHCOSY spectrum of the Lobelia chinensis homopolysaccharide obtained in the present invention;
FIG. 4.1 is a graph showing the effect of the homopolysaccharide of Chinese lobelia on TG (A) in the serum of an obese mouse obtained in the present invention;
FIG. 4.2 is a graph showing the effect of the homopolysaccharide of Chinese lobelia on TC (B) in the serum of obese mice;
FIG. 5.1 is a graph showing the effect of the homopolysaccharide of Chinese lobelia obtained in the present invention on TG (A) in the liver of an obese mouse;
FIG. 5.2 is a graph showing the effect of the homopolysaccharide of Chinese lobelia obtained in the present invention on TC (B) in the liver of an obese mouse;
FIG. 6 is a graph showing the relationship between the homopolysaccharide of Chinese lobelia obtained in the present invention and the effect of liver weight of mice;
FIG. 7 is a graph showing the relationship between the homopolysaccharide of Chinese lobelia obtained in the present invention and the effect of fat granules in the liver lobe of an obese mouse;
FIG. 8 is a graph showing the effect of the homopolysaccharide of Chinese Lobelia on TG (A) and TC (B) in the serum of an obese mouse obtained in the present invention;
FIG. 9 is a graph showing the results of measurement of the homogeneous polysaccharides of Lobelia chinensis obtained in the present invention on fasting plasma glucose at 5 to 8 weeks in obese mice.
Detailed Description
To facilitate an understanding of the present invention, the following description is made in conjunction with the accompanying drawings and examples.
The invention discloses a preparation method of Chinese lobelia homogeneous polysaccharide, which is simple, can obtain high-purity Chinese lobelia homogeneous polysaccharide and is suitable for large-scale production.
The first embodiment is as follows: preparation method of Chinese lobelia homogeneous polysaccharide
(1) Weighing 1kg of dry Lobelia chinensis whole plant, cutting into segments, adding 10 times of absolute ethanol with the mass percent of 95-100% for reflux degreasing (to remove low-polarity components), carrying out reflux degreasing for 2 hours at 80 ℃, filtering to obtain ethanol-insoluble residues, drying the residues in a 50 ℃ oven, adding the residues into a traditional Chinese medicine extraction tank, then adding 10 times of water, boiling and extracting for two times, each time for 3 hours, filtering, combining the two extracting solutions, concentrating, adding 95-100% by mass of ethanol while stirring until the final mass percent of the ethanol is diluted to 75%, standing, carrying out alcohol precipitation for 24 hours, filtering, collecting precipitates and volatilizing residual ethanol; centrifuging the precipitate, adding appropriate amount of distilled water for redissolving after centrifuging, and freeze-drying at-80 deg.C to obtain herba Lobeliae chinensis polysaccharide crude product 126g, with yield about 12.6% of the whole plant;
(2) taking 20g of the prepared crude lobelia chinensis polysaccharide, dissolving the crude lobelia chinensis polysaccharide in 200mL of distilled water, centrifuging (12000rpm for 10min), absorbing supernatant, loading the supernatant on a DEAE-Fast Flow column (10 multiplied by 40cm), eluting by using distilled water and NaCl solution with various concentrations, detecting by using a sulfuric acid-phenol method, detecting the sugar content of each tube at 480nm on an enzyme labeling instrument, drawing an elution curve, collecting sugar-containing solution, concentrating under reduced pressure, dialyzing the concentrate by using a dialysis bag with the molecular weight cutoff of 3500Da, and then freeze-drying to prepare the polysaccharide at the eluting part of the lobelia chinensis;
(3) dissolving 120mg of prepared water-eluted part polysaccharide of the Chinese lobelia by using 0.1, 0.2 or 0.4mol/L NaCl solution, centrifuging, taking supernate to sample on a sephadex chromatographic column, and eluting by using 0.1-0.4 mol/L NaCl solution; detecting by using a differential refraction detector, and collecting effective parts according to the peak positions; concentrating under reduced pressure, dialyzing the concentrate with dialysis bag with molecular weight cutoff of 3500Da, and freeze drying to obtain herba Lobeliae chinensis homogeneous polysaccharide (LCPW for short).
Identification and analysis of prepared Chinese lobelia homogeneous polysaccharide
(1) Purity and molecular weight determination
Using gel chromatography (GPC): high performance liquid chromatography (Shimadzu LC-10A); the chromatographic column is KS805-804-802 series gel column (8.0X 300 mm); the detector is a differential detector; the mobile phase is 0.05mol/L sodium chloride aqueous solution; column temperature: 40 ℃; flow rate: 0.6 mL/min; sample introduction volume: 20 μ L.
Taking dextran (Mw1152, 5200, 11600, 23800, 148000, 273000, 410000) with known molecular weight as standard, preparing 5mg of each standard solution with mobile phase 0.05mol/L sodium chloride (NaCl) aqueous solution to obtain 5mg/mL solution; centrifuging at 10000rpm for 10 min; taking the supernatant, and injecting 20 mu L of sample each time; the retention time is plotted as a log of molecular weight.
Taking 5mg of a sample to be detected, and preparing a 5mg/mL solution by using a sodium chloride aqueous solution with 0.05mol/L of a mobile phase; centrifuging at 10000rpm for 10 min; and taking the supernatant, injecting 20 mu L of sample, and carrying out high performance liquid gel chromatography test.
The average molecular weight of the sample can be determined from the standard curve on the basis of the measured retention time of the sample.
The standard curve drawing process and the molecular weight calculation are automatically completed by GPC software. FIG. 1 is a high performance liquid gel chromatography (HPGPC) chart of the resulting Lobelia chinensis homopolysaccharide (LCPW), with a single symmetrical peak indicating that it is a homopolysaccharide. The average molecular weight of the lobelia uniform polysaccharide (LCPW) is measured by GPC software to be 2.7 kDa.
(2) Structural identification
1) Sugar composition analysis:
analysis of sugar composition by hydrolysis: taking 1mg of polysaccharide sample, dissolving in 3ml of 2M TFA solution in an ampoule bottle, sealing, hydrolyzing at 121 ℃ for 2h, taking out, decompressing, evaporating to dryness, adding methanol, evaporating to dryness for three times to completely remove TFA; after hydrolysis, the residue was dissolved in deionized water and passed through a 0.22 μm filter.
The chromatographic conditions were as follows:
agilent high performance liquid chromatography system (Agilent 1100);
a chromatographic column: NH 2P-504E chromatographic column (250nm multiplied by 4.6nm)
The column temperature is 30 ℃; the mobile phase is a mixed solution of deionized water and acetonitrile (21: 79; V/V), the flow rate is 0.6mL/min, and the sample injection amount is 20 mu L; the detector used an ELSD evaporative light scattering detector.
And (3) detection results: the results for the lobelia polysaccharide showed two peaks, which were presumed to consist of fructose as well as glucose by comparison with standard monosaccharides, including fructose and literature reference.
2) Infrared spectrum analysis: a sample of 1mg was dried overnight under vacuum in a desiccator containing phosphorus pentoxide, tableted with KBr and subjected to infrared spectroscopic analysis.
FIG. 2 shows the obtained Lobelia chinensis homogeneous polysaccharide (LCPW) infrared spectrum at 3367cm-1The absorption peak has a characteristic absorption peak with large absorption intensity and wide peak value, and is apparently caused by the stretching vibration of hydroxyl groups of sugar molecules. At 2932cm-1A narrow peak with weaker absorption intensity is positioned, and belongs to a characteristic absorption peak caused by C-H stretching vibration on a sugar molecule. The two absorption peaks belong to the characteristic absorption peak of polysaccharide compound, and are at 1639cm-1The absorption peak is attributed to the vibration of the polysaccharide bound with H-O-H in water. 1470cm-1～1200cm-1The less sharp signal peaks are caused by the variable angle C-H oscillations. 1200cm-1～1000cm-1The presence of a larger signal peak within the range indicates the presence of stretching vibrations caused by C-O-H or C-O-C. At 936cm-1The absorption peak is caused by symmetric stretching vibration of furan ring, 874cm-1The absorption peak at (A) is caused by the transverse vibration of-CH 2, and 819cm-1The absorption peak is caused by the C-H angular vibration of the furan ring, and the three characteristic peaks indicate that the LCPW sample has furanose residues.
3) C spectrum analysis: 30mg of the sample was placed in a desiccator containing phosphorus pentoxide and dried overnight under vacuum, and 400. mu.LD was added2Dissolving O, and measuring at room temperature13C-NMR spectrum.
FIG. 3.1 shows the hydrogen spectrum and carbon spectrum of LCPW obtained from Lobelia chinensis homogeneous polysaccharide, and in FIG. 3.1, LCPW1The H-NMR spectrum shows that the peak has a weak signal peak at delta 5.44ppm, and the methylation analysis result of the LCPW shows that the delta 5.44ppm is supposed to belong to anomeric hydrogen of alpha-D-Glcp-1 → the peak is determined to be the peak. And stronger signal peaks exist in the ranges of delta 4.24, 4.08 and delta 3.98-3.68 ppm, and are based on the principle that1H-NMR is difficult to assign and we will analyze and assign later on with reference to its fig. 3.2 and 3.3 maps.
Of LCPW13C-NMR As shown in B of FIG. 3.1, there are two signal peaks in the anomeric carbon signal region, δ 104.5ppm and δ 93.76ppm, respectively, and their peak area integral ratio is about 14:1, so that the signal peak at δ 104.5ppm belongs to the major difference of LCPWThe head carbon signal. From the results of methylation analysis of LCPW, the signal peak at δ 104.5ppm should belong to fructose residues, and the signal peak at δ 93.76ppm should belong to glucose residues. In addition, the off-head signal of LCPW undergoes a significant downward shift, indicating that LCPW contains a β -type glycosidic linkage.
DEPT-135 of LCPW As shown by C in FIG. 3.1, the methylene signal peak at δ 62.13ppm should belong to C1 of fructose residue and the methylene signal peak at δ 63.34ppm should belong to C6 of fructose residue.
HSQC according to LCPW (as shown in FIG. 3.2) and1H-1two-dimensional spectrum of H COSY (shown in FIG. 3.3), which is obtained by analyzing the main residues1H NMR and13chemical shifts of C NMR were assigned respectively as shown in Table 1.
Its anomeric carbon regions 1104.5 and 93.77ppm correspond to → 1) -Fruf- (2 → C-2 and Glcp- (1 → C-1 signal → 1) -Fruf- (2 → C-1, C-2, C-3, C-4, C-5 and C-6 signals correspond to 62.13, 104.5, 78.5, 75.5, 82.3 and 63.34ppm, respectively.
In combination with the above analysis: the Chinese lobelia polysaccharide prepared in the first embodiment of the invention is linear straight-chain polysaccharide, consists of terminal group alpha-D-glucose and D-fructose residues connected by beta-2, 1 glycosidic bonds, and is homogeneous polysaccharide.
TABLE 1
In-vivo pharmacodynamic experiment of Chinese lobelia homogeneous polysaccharide in obese mice
(1) Establishing a mouse obesity model fed with high-fat feed:
animals: male ICR mice of 20 mice at 6-8 weeks old;
reagent: a TG detection reagent and a TC detection reagent;
grouping: one week after acclimation feeding, 4 groups of 5 mice were randomized. The first group was fed with normal diet (fat 10%, protein 20%, carbohydrate 70%), the remaining three groups were fed with high fat diet (fat 60%, protein 20%, carbohydrate 20%), no restriction to food intake and water intake, temperature 25-30 ℃, circadian rhythm, each 12h, mouse cage was cleaned once a week, wood chip bedding was replaced once, average daily diet intake was recorded, and body weight was measured once a week. After feeding for four weeks, randomly selecting a group of mice fed with high-fat feed, and intragastrically irrigating the mice with a Chinese lobelia polysaccharide solution, wherein the dosage is 300 mg/(kg. d), and the mice are marked as BC groups; a group of mice fed with high-fat feed is selected, and the mice are gazed with glimepiride tablet solution as a positive control, wherein the dose is 5 mg/(kg. d).
(2) Determination of serum TG and TC content in experimental mice
Collecting blood in a 1.5mL centrifuge tube, placing in a refrigerator at 4 ℃, standing, and detecting the content of triglyceride and total cholesterol in the blood serum of each group of animals by using TG and TC detection kits after blood cells precipitate serum.
As a result: the results of the detection of TG and TC in the sera of the animals in each group are shown in FIGS. 4.1 and 4.2, and compared with the FC group, the TG and TC contents of the BC group and the PC group are both significantly reduced, and the NC group is used as a normal control group, and the TG and TC contents are both in normal level.
(3) Liver TG and TC content determination
After the treatment of each group of mice is finished, 0.5g of liver of 2mL of EP intraductal mouse is taken, 4.5mL of absolute ethyl alcohol is added, mechanical homogenization is carried out under the ice-water bath condition, centrifugation is carried out for 10min at 2500rpm, and the content of triglyceride and total cholesterol in the liver of each group of animals is detected by TG and TC detection kit after supernatant fluid is taken.
As a result: the results of the detection of TG and TC in the livers of the animals in each group are shown in fig. 5.1 and 5.2, the TG and TC contents in the FC group are relatively dispersed, which may be related to the self-regulation of mice, the TG and TC contents in the BC group and the PC group are significantly reduced compared to the FC group, and the TG and TC contents in the NC group as a normal control group are both at normal levels and have relatively low dispersion.
In conclusion, LCPW has significant biological activity on TG and TC contents in the serum of an obese mouse, which shows that LCPW has the function of reducing blood fat; the effect of LCPW on reducing TG and TC contents in the liver of an obese mouse shows that LCPW can improve visceral obesity of an obese patient.
(4) Liver weight and morphological characteristics of liver tissue
After the liver of the dissected experimental mouse is taken out, the dissected experimental mouse is put into normal saline, blood in the liver is washed away, then the normal saline is absorbed by filter paper, and the weight of the liver is weighed. The liver large leaves fixed in 10% formaldehyde solution are taken out to prepare paraffin embedded sections, observed by a microscope after HE staining, and photographed.
As a result: as shown in FIG. 6, the liver weight of the FC group mice is significantly higher than that of the other three groups, and the liver weights of the BC group mice and the PC group mice are closer and slightly higher than that of the NC group mice.
After the mice are dissected, the livers of the mice are taken out for visual observation, and the livers of the mice in the NC group are reddish brown, have thinner edges and are soft; the liver of the FC group mouse is yellow-white, the edge is thick, the texture is greasy, and the whole body is fat; the livers of the BC and PC two groups of mice were dark red, slightly thick at the edges, and shiny on the surface.
Representative photomicrographs of mouse liver lobes as shown in fig. 7, HE stained sections of liver lobes showed no inflammation, necrosis and apoptosis of the liver in each group of mice. Wherein, the liver section of the NC group mouse in the A shows that the liver architecture of the mouse is complete and the liver cells are normal; the FC group mouse liver section in B shows that the mouse liver generates visible diffuse cytoplasm vacuolization and steatosis, which indicates that the liver generates degenerative change and a large number of lipid drops exist in liver cells; BC group mouse liver sections in C showed mouse livers exhibiting moderate steatosis and the presence of lipid droplets within hepatocytes; the liver sections of the PC group mice in D show that the liver of the mice has relatively less steatosis, and a small amount of lipid drops exist in the liver cells. The results of the above experiments further prove that LCPW has the biological activity of losing weight, reducing fat and improving visceral adiposity of obese patients.
In summary, it can be seen that: the novel inulin type Chinese lobelia homogeneous polysaccharide separated and purified from Chinese medicine Chinese lobelia can obviously reduce the content of total cholesterol and triglyceride in serum and liver of obese mice, has no toxic or side effect, and is expected to be used as a main or only active ingredient for preparing health-care food or pharmaceutical preparation for preventing and/or treating obesity.
The medicinal effect experiment of the Chinese lobelia homogeneous polysaccharide in reducing blood sugar of mice
(1) Experimental materials: white mouse
(2) Mouse raising and modeling
One week after 20 mice were acclimatized, they were randomly divided into 4 groups of 5 mice each. The first group was fed with normal diet (fat 10%, protein 20%, carbohydrate 70%), the remaining three groups were fed with high fat diet (fat 60%, protein 20%, carbohydrate 20%), no restriction to food intake and water intake, temperature 25-30 ℃, circadian rhythm, each 12h, mouse cage was cleaned once a week, wood chip bedding was replaced once, average daily diet intake was recorded, and body weight was measured once a week. After feeding for four weeks, randomly selecting a group of mice fed with high-fat feed, and intragastrically irrigating the mice with a Chinese lobelia polysaccharide solution, wherein the dosage is 300 mg/(kg. d), and the mice are marked as BC groups; selecting a group of mice fed with high-fat feed, and intragastrically administering the glimepiride tablet solution as a positive control, wherein the dosage is 5 mg/(kg. d), and the result is recorded as a PC group; the other two groups were separately gazed with physiological saline of the same volume, and the group fed with normal diet was recorded as NC group as normal control, and the group fed with high-fat diet was recorded as FC group as obesity group. The mice were fed by gastric gavage for 5 weeks, Fasting Blood Glucose (FBG) was measured once a week for the first 4 weeks, Oral Glucose Tolerance Test (OGTT) was performed for the last week, gavage feeding was continued for one week after the end of the OGTT test, and then the experimental mice were sacrificed and Fasting treatment was performed one night before the mice were sacrificed.
(3) Determination of fasting plasma glucose in mice
The test mice were subjected to fasting treatment one night before blood collection, and a drop of peripheral venous blood was collected the next morning and measured with a glucometer.
As a result: after the mice are fed with different feeds for the previous 4 weeks, the grouped administration is started at the 5 th week, the fasting blood sugar of the mice is respectively measured at the 5 th, 6 th, 7 th and 8 th weeks, the measurement result is shown as A-D in fig. 8, the 5 th week is the first week of administration, probably because the mice eat high fat feed for a short time, the fasting blood sugar content of the two groups of administration group BC and PC is obviously lower than that of the normal control group NC, the normal fasting blood sugar level of the mice is 3.89-6.11 mol/L, and the fasting blood sugar of the 5 th week of the two groups FC and NC is in the interval, which indicates that an obesity model is not established yet; with the extension of the feeding time, the mouse obesity model is gradually formed, as shown in B and C in fig. 8, the fasting plasma glucose of the FC group mouse is gradually increased, the fasting plasma glucose content is greater than 7mol/L at 7 weeks and exceeds the threshold value, the fasting plasma glucose of the NC group is always maintained at a normal level of 4-5 mol/L, the fasting plasma glucose content of the BC and PC group exceeds the NC group, but is lower than the FC group and within the normal level due to administration; at 8 weeks, the fasting blood sugar content of the FC group mice is close to 8mol/L, the blood sugar content of other three groups is obviously different from the fasting blood sugar content of the FC group mice, and the fasting blood sugar content of the other three groups is in a normal level fasting blood sugar range, which shows that the obesity model of the mice is successfully established, and the blood sugar content of the BC group is less than 6.11mol/L, which shows that the LCPW has a regulating effect on the blood sugar of the obese mice and can play a role in reducing the fasting blood sugar of the obese mice.
(4) Oral glucose tolerance test
The experimental mice were fasted one night before the OGTT was made, the mice were gazed with a glucose solution the following morning at a dose of 2g/kg, and then the tail vein blood of each group of mice was collected immediately, and the blood glucose levels were measured with a glucometer for 0, 30, 60, 90, 120 min.
As a result: the results of oral glucose tolerance experiments on mice are shown in fig. 9, after 30min of intragastric administration of glucose to mice, the blood glucose content of the mice has a peak value, the peak value of the blood glucose content of the FC group is the highest, the peak value of the blood glucose content of the NC group is the lowest, after 60min, the blood glucose content of the FC group is still maintained at a higher level, the blood glucose content of the other three groups begins to decrease, LCPW exhibits a good blood glucose increase inhibition effect, the effect is comparable to that of the hypoglycemic agent glimepiride, and the inhibition effect lasts for 120min, so that the blood glucose level is restored to the initial measurement value. In the OGTT experiment, BC group mice gazed with LCPW showed good glucose tolerance, indicating that LCPW has physiological effect of regulating blood sugar to maintain the normal level.
In summary, it can be seen that: the novel inulin type Chinese lobelia homogeneous polysaccharide separated and purified from Chinese medicine Chinese lobelia can obviously reduce fasting blood sugar of mice, has very good glucose tolerance and blood sugar regulation effect, and is expected to be used as a main or only active ingredient for preparing health-care food or pharmaceutical preparation for preventing and/or treating hyperglycemia.
The formulation of the health food or pharmaceutical preparation prepared by using the Chinese lobelia polysaccharide as an active ingredient in the invention can be various, and any formulation can be used as long as the active ingredient can effectively reach the body. Such as may be selected from: tablet, capsule, powder, granule, syrup, solution, suspension, injection, tincture, oral liquid, aerosol, buccal agent, granule, pill, powder, or sustained release preparation such as nanometer preparation.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
It should be noted that the structures, ratios, sizes, and the like shown in the drawings attached to the present specification are only used for matching the disclosure of the present specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions of the present invention, so that the present invention has no technical essence, and any structural modification, ratio relationship change, or size adjustment should still fall within the range covered by the technical contents disclosed by the present invention without affecting the efficacy and the achievable purpose of the present invention.
Claims (6)
1. The application of the Chinese lobelia homogeneous polysaccharide in the preparation of medicines and/or foods is characterized in that: the Chinese lobelia herb is obtained by water extraction, alcohol precipitation and column chromatography separation and purification, is linear straight-chain polysaccharide, consists of terminal group alpha-D-glucose and D-fructose residue connected by beta-2, 1 glycosidic bond, and has the relative molecular weight of 1-20 kDa; the medicine is used for preventing and/or treating obesity, and the food is used for preventing and/or improving obesity.
2. The application of the Chinese lobelia homopolysaccharide in the preparation of medicines and/or foods as claimed in claim 1, wherein the preparation method of the Chinese lobelia homopolysaccharide comprises the following steps:
a) taking the Chinese lobelia herb, extracting with water, precipitating with ethanol, and freeze-drying to obtain a Chinese lobelia polysaccharide crude product;
b) dissolving the crude Chinese lobelia polysaccharide obtained in the step a) with water, loading the dissolved crude Chinese lobelia polysaccharide to an anion exchange column, eluting with distilled water, then eluting with NaCl solution, and detecting by a phenol-sulfuric acid method to obtain polysaccharide at the water-eluted part of the Chinese lobelia;
c) loading polysaccharide at the water-eluted part of the Chinese lobelia obtained in the step b) onto a sephadex chromatographic column, and eluting with NaCl solution; collecting effective parts according to the peak position, dialyzing by using a dialysis bag with the molecular weight cutoff of 1000-14000Da, and freeze-drying the dialyzed inner liquid to obtain the lobelia uniform polysaccharide.
3. The use of the barbadosnut homopolysaccharide according to claim 2 for the preparation of a medicament and/or food, wherein: in the step a), degreasing the dried Chinese lobelia herb with ethanol, drying the residue, and then performing water extraction and alcohol precipitation.
4. The use of the barbadosnut homopolysaccharide according to claim 2 for the preparation of a medicament and/or food, wherein: the step a) comprises the following operations: firstly, carrying out reflux degreasing on the whole plant of the Chinese lobelia herb with ethanol, drying degreased residues, then adding water for heating and extracting for 2-5 times, combining extracting solutions, carrying out reduced pressure concentration and centrifugation, collecting supernate, adding 2-4 times of ethanol for carrying out alcohol precipitation, wherein the mass percent of the ethanol is 90-100%, and carrying out vacuum freeze drying on the collected precipitate to obtain a Chinese lobelia polysaccharide crude product.
5. The use of the barbadosnut homopolysaccharide according to claim 2 for the preparation of a medicament and/or food, wherein: the step b) comprises the following operations: dissolving the prepared crude lobelia chinensis polysaccharide product with water, loading the dissolved crude lobelia chinensis polysaccharide product onto an anion exchange column, eluting with water, tracing an elution curve by a phenol-sulfuric acid method, collecting a sugar-containing solution, concentrating under reduced pressure, dialyzing a concentrate, and freeze-drying to obtain the polysaccharide at the water-eluted part of the lobelia chinensis.
6. The use of the barbadosnut homopolysaccharide according to claim 2 for the preparation of a medicament and/or food, wherein: the step c) comprises the following operations: dissolving the prepared water-eluted part polysaccharide of the Chinese lobelia by using 0.1-0.4 mol/L NaCl solution, centrifuging, taking supernate to sample on a sephadex chromatographic column, eluting by using 0.1-0.4 mol/L NaCl solution, detecting by using a differential refraction detector, and collecting an effective part according to the peak position; concentrating, dialyzing with dialysis bag with molecular weight cutoff of 1000-.
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