CN108586626B - Preparation method and application of tetrastigma hemsleyanum Diels et Gilg oligosaccharide - Google Patents
Preparation method and application of tetrastigma hemsleyanum Diels et Gilg oligosaccharide Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicines, and discloses a preparation method of tetrastigma hemsleyanum michaux oligosaccharide and an application preparation method thereof, wherein the preparation method comprises the following steps: 1) picking whole plant of radix tetrastigme, washing, drying in the sun, drying to constant weight, pulverizing, and sieving; weighing coarse powder of radix tetrastigme, adding into an ethanol solution, and performing ultrasonic extraction; carrying out suction filtration, and discarding filtrate; 2) adding the filter residue into double distilled water for reflux extraction, filtering the extracting solution after each extraction, combining the filtrates, concentrating under reduced pressure, and removing protein by Sevag method; 3) adding ethanol solution into the deproteinized extracting solution, standing overnight, centrifuging, and removing precipitate; adding ethanol solution into the supernatant, precipitating again, standing overnight, centrifuging, and drying the precipitate under reduced pressure to obtain radix Apioris Fortunei oligosaccharide. The tetrastigma hemsleyanum michaux oligosaccharide prepared by the method is safe and nontoxic, has the effects of resisting tumors, improving intestinal flora, protecting gastrointestinal tracts and the like, and expands the medicinal application of the tetrastigma hemsleyanum hemsle.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to a preparation method and application of tetrastigma hemsleyanum Diels et Gilg oligosaccharide.
Background
Radix tetrastigme (Tetrastigma hemsleyanum Diels el Gilg) is a unique rare and rare plant of the vitidae cliff vine, and underground root tuber or whole grass can be used as a medicine. The radix tetrastigme is used as a common Chinese herbal medicine in folk, has the reputations of 'Yaowang' and 'anticancer magic grass' and has the effects of clearing away heat and toxic materials, promoting blood circulation, dispelling wind and the like.
Modern pharmacological research also proves that the radix tetrastigme has various pharmacological effects of relieving fever, resisting inflammation, resisting tumor, resisting virus, protecting liver, regulating immunity and the like, and has good development and utilization prospects. The variety of chemical components in the radix tetrastigme is various, researches on the pharmaceutical effective substances of the radix tetrastigme mainly focus on flavonoids, phenolic acids, fatty acid compounds and the like in the radix tetrastigme, the active ingredients with the anti-tumor effect and the like in the radix tetrastigme are generally considered to be the compounds at present, and for example, Chinese patent with application number of CN201210252136.2 discloses a preparation method of radix tetrastigme tea with high contents of flavone, polysaccharide and kaempferol in the radix tetrastigme. The radix tetrastigme tea product prepared by the method is natural green, the soup color is bright green, the leaf bottom is light green, caffeine is not contained, and the radix tetrastigme tea product contains abundant radix tetrastigme flavone, polysaccharide, kaempferol and other components, can effectively inhibit tumor cell proliferation and promote tumor cell apoptosis, has a good anti-tumor effect, is free of toxic and side effects, is easy to absorb, and is beneficial to health.
However, no other component, especially oligosaccharide, has been reported. Oligosaccharides, also known as oligosaccharides, are widely found in nature and have the advantages of low calorie, stability, safety, no toxicity, no residue and the like. Some researches on the regulation effect of oligosaccharides in certain plants on gastrointestinal tracts have been reported, such as banana oligosaccharide, asparagus oligosaccharide, soybean oligosaccharide and the like, but only a few plant oligosaccharides have similar effects, and whether the tetrastigma hemsleyanum michaux oligosaccharide has the same effect is not reported.
With the improvement of living standard and the influence of factors such as the change of dietary structure, the incidence of diseases such as tumor and gastrointestinal dysfunction is gradually increased, and patients need medication. Most of the medicines have dependence and broad spectrum, so that a plurality of adverse reactions such as headache, gastrointestinal hemorrhage, cognitive dysfunction and the like are easy to generate. Therefore, it is necessary to develop and research a safe drug with a definite drug effect.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method and application of tetrastigma hemsleyanum nakai oligosaccharide. The tetrastigma hemsleyanum michaux oligosaccharide prepared by the method is a natural substance, is safe and nontoxic, has the effects of resisting tumors, improving intestinal flora, protecting gastrointestinal tracts and the like, and expands the medicinal application of the tetrastigma hemsleyanum hem.
The specific technical scheme of the invention is as follows: a preparation method of radix tetrastigme oligosaccharide comprises the following steps:
1) picking whole plant of radix tetrastigme, washing, drying in the sun, drying to constant weight, pulverizing, and sieving; weighing coarse powder of radix tetrastigme, adding the coarse powder into an ethanol solution according to the material-liquid ratio of 1g:15-25mL, and performing ultrasonic extraction for at least 2 times; and (5) carrying out suction filtration, and discarding filtrate.
2) Taking filter residue, adding into double distilled water according to the material-liquid ratio of 1g:10-20mL, refluxing and extracting for at least 2 times, filtering the extracting solution after each extraction, combining the filtrates, concentrating under reduced pressure, and removing protein by Sevag method for at least 3 times.
3) Adding ethanol solution into the deproteinized extracting solution, standing overnight, centrifuging, and removing precipitate; adding ethanol solution into the supernatant, precipitating again, standing overnight, centrifuging, and drying the precipitate under reduced pressure to obtain radix Apioris Fortunei oligosaccharide.
Conventionally, methods for extracting flavonoids, phenolic acids, fatty acids, and the like from tetrastigma hemsleyanum Diels et Gilg have been disclosed, and generally, ethanol extraction, water extraction, and the like are also used. However, since the research on oligosaccharide in radix tetrastigme in the prior art is almost absent, no document discloses a method for extracting oligosaccharide in radix tetrastigme. The invention adopts an alcohol extraction method and a water extraction method in sequence, although the extraction solvent is similar to the prior art, the invention discovers that different extraction targets, different parameters such as material-liquid ratio, solvent concentration, time and the like can directly cause different solubilities of active substances in the radix tetrastigme in the solvent, so that the substances obtained by final extraction are completely different. Since there is no precedent for extracting oligosaccharide from radix tetrastigme, those skilled in the art do not know how to design an extraction process to extract oligosaccharide from radix tetrastigme.
Preferably, in the step 1), the drying temperature is 50-60 ℃.
Preferably, in step 1), the powder is ground and then sieved by a 40-mesh sieve.
Preferably, the ethanol solution has a concentration of 90 to 98wt% in step 1) and step 3).
Preferably, in the step 2), the extraction time of each reflux is 1-2 h.
Preferably, in step 3), the first addition of the ethanol solution is performed such that the volume of ethanol is 75%.
Preferably, after the step 3), the method further comprises a step 4): the molecular weight of radix tetrastigme oligosaccharide is determined by high performance liquid chromatography.
Further preferably, the specific process of step 4) is as follows: preparing tetrastigma hemsleyanum nakai oligosaccharide into a test solution of 1mg/mL, and setting chromatographic conditions: the chromatographic column is Waters Ultrahydrogel 120 with specification of 7.8 × 300mm, the column temperature is 35 deg.C, the drift tube temperature is 100 deg.C, the mobile phase is 300mmol/L ammonium acetate, the flow rate is 0.8mL/min, and the sample injection amount is 10 μ L.
The research of the group of the invention discovers that the tetrastigma hemsleyanum michaux oligosaccharide has the anti-tumor effect, so the tetrastigma hemsleyanum hemsle.
The research of the invention group discovers that the tetrastigma hemsleyanum p.f. oligomeric confectionary is not digested and absorbed by small intestine due to lack of metabolic enzyme system of human body, can directly enter large intestine to be utilized by microbial flora widely existing in the large intestine, and produces organic acid through fermentation, so that the pH value in the intestinal tract is reduced, the putrefaction is inhibited, the generation of toxic and harmful substances is reduced, the growth and activity of beneficial bacteria such as bifidobacterium and the like are promoted, the intestinal flora is further regulated, and the gastrointestinal tract is protected. Therefore, the tetrastigma hemsleyanum oligosaccharide also has the effect of improving the gastrointestinal tract function, and therefore can be used as a medicine for improving the gastrointestinal tract function.
In the prior art, although the radix tetrastigme has been disclosed to have the functions of resisting tumor and improving gastrointestinal tract, people have not fully mastered active ingredients playing key roles, and according to a general view, the substances playing the effects in the radix tetrastigme are generally considered to be concentrated in flavonoids, phenolic acids, fatty acid compounds and the like (the contents of the substances are high) in the radix tetrastigme, but no report is found at present on the research of other components, particularly oligosaccharides.
According to the invention, through establishing a 4T1 breast cancer mouse model, an in vitro intestinal canal microorganism experiment, a mouse constipation model prepared from loperamide hydrochloride and the like, the radix tetrastigme oligosaccharide is found to have various pharmacological actions of resisting tumors, promoting the proliferation of beneficial bacteria in human intestinal canals, protecting intestinal canal microecology, relaxing bowel, enhancing immunity and the like, so that the radix tetrastigme oligosaccharide can be used as an active ingredient and pharmaceutically acceptable auxiliary materials to be prepared into various dosage forms such as oral agents, capsules, tablets and the like, and has a certain application value in the prevention and treatment of diseases such as tumors, autoimmunity, intestinal diseases and the like.
Compared with the prior art, the invention has the beneficial effects that:
1. the method can effectively extract the tetrastigma hemsleyanum Diels et Gilg oligosaccharide, and the average extraction rate of the tetrastigma hemsleyanum Diels et Gilg oligosaccharide is 1.586%.
2. Pharmacological experiments show that the tetrastigma hemsleyanum michaux oligosaccharide prepared by the invention has the physiological functions of resisting tumors, promoting the proliferation of beneficial bacteria in human intestinal tracts, protecting intestinal microecology, relaxing bowel, enhancing immunity and the like, and has wide application prospects in the medical industry.
Drawings
FIG. 1 is a calibration curve of the molecular weight of tetrastigma hemsleyanum oligosaccharide prepared in example 1;
FIG. 2 is the HPLC analysis chart of the 95% alcohol precipitation site of the tetrastigma hemsleyanum Diels et Gilg oligosaccharide separated by water extraction and alcohol precipitation in example 1;
FIG. 3 is a photograph of breast cancer tumor entities in groups of mice administered tetrastigma hemsleyanum Diels et Gilg oligosaccharide;
FIG. 4 is a 24h growth curve for Lactobacillus casei;
FIG. 5 is a graph showing the effect of different concentrations of tetrastigma hemsleyanum oligosaccharide on the growth of lactobacillus casei in vitro.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A preparation method of radix tetrastigme oligosaccharide comprises the following steps:
1) picking whole plant of radix tetrastigme, cleaning, sun drying, oven drying at 50-60 deg.C to constant weight, pulverizing, and sieving with 40 mesh sieve; weighing coarse powder of radix tetrastigme, adding the coarse powder into 90-98wt% ethanol solution according to the material-liquid ratio of 1g:15-25mL, and performing ultrasonic extraction for at least 2 times; and (5) carrying out suction filtration, and discarding filtrate.
2) Taking filter residue, adding into double distilled water according to the material-liquid ratio of 1g:10-20mL, reflux extracting for at least 2 times, each time extracting for 102h, filtering the extracting solution after each extraction, combining the filtrates, concentrating under reduced pressure, and deproteinizing by Sevag method for at least 3 times.
3) Adding 90-98wt% ethanol solution into the deproteinized extractive solution to make ethanol volume up to 75%; standing overnight, centrifuging, and removing precipitate; adding 90-98wt% ethanol solution into the supernatant, precipitating again, standing overnight, centrifuging, and drying the precipitate under reduced pressure to obtain radix Apioris Fortunei oligosaccharide.
4) Determining the molecular weight of tetrastigma hemsleyanum hemsl oligosaccharide by using a high performance liquid chromatography: preparing tetrastigma hemsleyanum nakai oligosaccharide into a test solution of 1mg/mL, and setting chromatographic conditions: the chromatographic column is Waters Ultrahydrogel 120 with specification of 7.8 × 300mm, the column temperature is 35 deg.C, the drift tube temperature is 100 deg.C, the mobile phase is 300mmol/L ammonium acetate, the flow rate is 0.8mL/min, and the sample injection amount is 10 μ L.
Example 1
The preparation method of the tetrastigma hemsleyanum Diels et Gilg oligosaccharide comprises the following steps:
(1) picking the whole plant of radix tetrastigme, cleaning, drying in the sun, drying in an oven at 55 ℃ to constant weight, pulverizing, sieving with a 40-mesh sieve, weighing coarse powder of radix tetrastigme, adding 95% ethanol according to a material-liquid ratio of 1g: 20mL, and extracting in an ultrasonic device for 2 times; and (5) carrying out suction filtration, and discarding filtrate.
(2) Taking filter residue, adding double distilled water according to the material-liquid ratio of 1g:15 mL, reflux-extracting for 2 times, each time for 1.5h, filtering with eight layers of gauze, combining the filtrates, concentrating under reduced pressure to appropriate concentration, and removing protein by Sevag method for 3 times;
(3) adding 95% ethanol into the deproteinized extractive solution until the ethanol content (volume ratio) reaches 75%, standing overnight, centrifuging, and removing precipitate; adding 95% ethanol into the supernatant, precipitating again, standing overnight, centrifuging, and drying the precipitate under reduced pressure to obtain radix Apioris Fortunei oligosaccharide.
(4) Determining radix Apioris Fortunei oligosaccharide molecular weight of radix Apioris Fortunei oligosaccharide extract by high performance liquid chromatography.
Preparing a test solution: accurately weighing radix tetrastigme oligosaccharide, preparing into test solution with concentration of 1mg/mL, and filtering with 0.22 μm microporous membrane.
Determining the molecular weight of tetrastigma hemsleyanum Diels et Gilg oligosaccharide: taking the retention time of the standard solution as an abscissa and the logarithm value of the molecular weight as an ordinate to perform linear regression to obtain a standard curve of-0.6182X +9.9008, R20.9927 as shown. And carrying out water extraction and fractional alcohol precipitation until the ethanol concentration is 95%, thus obtaining an HPLC (high performance liquid chromatography) picture of the tetrastigma hemsleyanum Diels et Gilg oligosaccharide. Substituting into standard curve to obtain radix Apioris Fortunei 95% ethanol precipitation fraction composed of oligosaccharide with molecular weight of 115.54 and 580.35 as main components (see figure 2).
The drug effect experiment of the tetrastigma hemsleyanum Diels et Gilg oligosaccharide anti-tumor effect:
the radix tetrastigme oligosaccharide is dried according to the preparation method, and then dissolved by a certain amount of distilled water to obtain the low, medium and high concentration gradient solutions of the radix tetrastigme oligosaccharide.
Successfully modeled 4T1 breast cancer model mice were randomly divided into a model group, a cyclophosphamide (50mg/kg) group, a tetrastigma hemsleyanum oligosaccharide low (10mg/kg), a medium (50mg/kg) and a high dose (150mg/kg) group. The blank group and the model group are perfused with normal saline every day, the cyclophosphamide group is intraperitoneally injected once every two days, and the radix tetrastigme oligosaccharide low, medium and high dose groups are perfused with radix tetrastigme oligosaccharide solution every day for 28 days. After the mice in each group were fasted for 12 hours after the last administration, the mice were sacrificed by removing the neck, tumor masses were dissected out, and the tumor volume and the tumor inhibition rate were calculated (see table 1 and fig. 3).
Tumor volume (major diameter. times. minor diameter)2)/2
Tumor inhibition rate ═ [ (model group average tumor mass-administration group average tumor mass)/model group average tumor mass ] × 100%
TABLE 1 Tetrastigma hemsleyanum Diels et Gilg oligosaccharide to tumor growth transplantation in 4T1 breast cancer miceInfluence of the action
| Tumor weight | Tumor volume | Tumor inhibition Rate (%) | Lung metastasis range | |
| Model set | 1.097±0.200 | 920.22±172.75 | —— | 2.30±0.21 |
| Cyclophosphamide derivatives of formula (I) | 0.298±0.077** | 214.98±80.96** | 72.85 | 0.20±0.13** |
| Oligosaccharide low dose group | 0.722±0.267** | 595.07±196.72** | 34.21 | 0.50±0.22** |
| Oligosaccharide medium dose group | 0.843±0.328* | 602.60±179.75** | 23.12 | 0.50±0.17** |
| High dose group of oligosaccharides | 0.943±0.243 | 765.36±213.43 | 14.06 | 0.30±0.15** |
Note: in comparison with the set of models,*P＜0.05，**P＜0.01
the results show that the mouse state of each administration group is better than that of the model group, and the tumor has no ulceration phenomenon or light ulceration degree. Compared with a model group mouse, the tetrastigma hemsleyanum michaux oligosaccharide has low and medium dosage, can obviously reduce the tumor volume and weight of the mouse, wherein the tumor inhibition rate of a low-dosage group is highest and reaches 34.21%, and the tetrastigma hemsleyanum michaux oligosaccharide with different concentrations.
The radix tetrastigme oligosaccharide is used as a natural drug effect component, and can obviously inhibit the growth of the tumor of a 4T1 breast cancer mouse according to the result of an anti-tumor drug effect experiment. The pharmacological action of the tetrastigma hemsleyanum dielset et f.f. oligosaccharide on resisting tumor is continuously and deeply explored, and theoretical basis is provided for developing novel antitumor drugs.
Experiment on influence of tetrastigma hemsleyanum michaux oligosaccharide on in-vitro intestinal microorganisms:
radix tetrastigme oligosaccharide (all the radix tetrastigme oligosaccharides used in the experiment are prepared according to the preparation method).
The activated lactobacillus casei was inoculated in MRS liquid medium at an inoculum size of 1%, cultured in a constant temperature incubator at 37 ℃, and after 0, 1, 2, 3, 6, 12, and 24 hours of culture, the OD value was measured at a wavelength of 600nm, and the viable count was measured by a plate count method, and a growth curve was plotted (see fig. 4).
Adding radix tetrastigme oligosaccharide into MRS liquid culture medium instead of glucose as carbon source, making the mass concentration of radix tetrastigme oligosaccharide respectively be 0g/L, 1g/L, 5g/L, 10g/L and 20g/L, inoculating lactobacillus casei in the culture medium with 1% inoculation amount, culturing in a 37 ℃ constant temperature incubator, respectively culturing for 0, 1, 2, 3, 6, 12 and 24h, measuring bacterial liquid OD value at 600nm, measuring viable count by plate counting method, and drawing growth curve (see figure 5).
Selecting several colonies of Escherichia coli and Staphylococcus aureus incubated for 16-24 hr respectively, placing in physiological saline tube, and correcting to 0.5 McLeod standard (1.5 × 10)8CFU/mL); sterilizing filter paper wafer under high pressure, and soaking in 0.5%, 1.0%, 2.0% radix Apioris Fortunei oligosaccharide solution and sterile water for 3 hr; immersing a sterile cotton swab into the prepared bacterial suspension, uniformly smearing the surface of an M-H agar plate for 3 times in a continuous marking mode, and standing for 5 min; the antibacterial paper sheets were attached to the surface of M-H agar with sterile forceps, 3 sheets were attached to each plate, and the sterile water group was used as a blank control. The plates were allowed to stand at room temperature for 15min, the plates were then inverted and incubated in a 37 ℃ incubator for 16-18h, and the zone diameters were measured with a vernier caliper or ruler and recorded (see Table 2).
TABLE 2 diameter of oligosaccharide bacteriostatic circle (mm)
According to the influence result of the tetrastigma hemsleyanum michaux oligosaccharide on in-vitro intestinal microorganisms, the tetrastigma hemsleyanum oligosaccharide with a certain concentration has a good proliferation promoting effect on lactobacillus casei and escherichia coli in vitro, and the experimental result is exact and reliable. In recent years, with the increasing promotion of modernization of Chinese traditional medicine, the screening of active ingredients from natural Chinese traditional medicine becomes a research hotspot. The tetrastigma hemsleyanum Diels et Gilg oligosaccharide is used as a natural drug effect component and has better research and development prospects.
Effect of tetrastigma hemsleyanum oligosaccharide on gastrointestinal function of mice:
radix tetrastigme oligosaccharide (all the radix tetrastigme oligosaccharides used in the experiment are prepared according to the preparation method).
After the experimental mice are adaptively fed for one week, the experimental mice are randomly divided into four groups, namely a constipation model group, a normal group and a tetrastigma hemsleyanum oligosaccharide low, medium and high dose group, and the experimental mice are fasted without water supply for 16 hours. The four groups except the normal group were administered with 20mg/kg loperamide hydrochloride, and 30min later, each of the four groups was administered with 0.8mL of a carbon ink suspension containing the corresponding test substance. The time required for each mouse to firstly discharge the black feces and the number of the black feces discharged in 6 hours are observed and recorded, the wet weight of the black feces is collected and weighed, and the dry weight is weighed after drying at 60 ℃ (see table 3).
TABLE 3 Effect of Tetrastigma hemsleyanum Diels et Gilg oligosaccharides on defecation function of Constipated mice: (
n＝10)
| Group of | First time (min) | 6h number of black feces (granule) | Water content of excrement (%) |
| Normal group | 113.75±25.59 | 10.75±7.89 | 38.65±19.28 |
| Model set | 179.75±25.84## | 6.50±3.51# | 34.98±17.95 |
| Radix tetrastigme oligosaccharide low-dose group | 121.25±30.11** | 11.44±3.16* | 49.00±9.83* |
| Tetrastigma hemsleyanum Diels et Gilg oligosaccharide medium dose group | 145.62±24.17* | 9.94±2.70 | 54.38±9.03** |
| Tetrastigma hemsleyanum hemsl oligosaccharide high-dose group | 127.50±27.52** | 15.38±2.67** | 53.82±11.73* |
Note: in comparison with the set of models,*P＜0.05，**p is less than 0.01; in comparison with the normal group,#P＜0.05，##P＜0.01
the results show that compared with the normal control group, the first time black stool time of the model group mice is obviously delayed, and the number of black stool particles in 6h is obviously reduced. Compared with the model group, the first excrement blackening time of mice in each dose of tetrastigma hemsleyanum Diels et Gilg oligosaccharide group is shortened, the number of excrement granules is increased within 6h, the water content of the excrement is increased, and the excrement is soft. The results show that the radix tetrastigme oligosaccharide at each dose can improve temporary constipation caused by loperamide hydrochloride.
After the experiment is finished, the mice of each group are normally raised for one week, and the gavage administration is continued. After the 6 th day, the patient is fasted for 24h and then given another time at 7 th day. After 30min of administration, each group of mice was given 0.8 mL/mouse of the semi-solid nutritional paste. After another 25min, the mouse is killed by removing the neck, the abdominal cavity is cut open to separate mesentery, the cardia of the mouse is cut to the pylorus and the stomach, the gastric contents are washed off and wiped off, the net weight is weighed, and the gastric residual rate is calculated; and (3) cutting an intestinal canal from the cardia to the ileocaecal region, wherein the length of the intestinal canal is the total length of the small intestine, the length of the intestinal canal from the pylorus to the front edge of the carbon end is the advancing length of the carbon end, and the advancing rate of the small intestine is calculated.
The gastric residual rate was [ (total weight of stomach-net weight)/total weight of stomach ] × 100%
The small intestine propulsion rate is (total length of ink propulsion intestinal tract/small intestine) × 100%
Table 4 influence of tetrastigma hemsleyanum michaux oligosaccharides on the intestinal carbon ink propelling function of experimental mice: (
n＝10)
Note: in comparison with the normal group,*P＜0.05，**P＜0.01
compared with the normal control group mice, the intestinal propulsion rates of the mice in the low, medium and high dose groups of tetrastigme oligosaccharide are remarkably increased (P is less than 0.05) compared with the normal group, and are respectively increased by 18.31%, 14.6% and 19.00%, which shows that the peristalsis of the small intestine of the mice can be remarkably promoted by the tetrastigme oligosaccharide in each dose. Wherein the effect of improving the intestinal propulsion capacity of mice by low and high doses is better than that of the medium dose.
TABLE 5 Tetrastigma hemsleyanum Diels et Gilg oligosaccharideEffect on gastric emptying function in Experimental mice: (
n＝10)
| Group of | Stomach full weight (g) | Net weight of stomach (g) | Gastric residual ratio (%) |
| Normal group | 0.54±0.10 | 0.21±0.03 | 51.08±12.72 |
| Radix tetrastigme oligosaccharide low-dose group | 0.48±0.07 | 0.21±0.03 | 34.63±8.61** |
| Tetrastigma hemsleyanum Diels et Gilg oligosaccharide medium dose group | 0.53±0.06 | 0.22±0.02 | 41.14±6.91* |
| Tetrastigma hemsleyanum hemsl oligosaccharide high-dose group | 0.50±0.07 | 0.23±0.02 | 34.46±8.00** |
Note: in comparison with the normal group,*P＜0.05，**P＜0.01
compared with the normal control group mice, the gastric residual rate of the low, medium and high dose tetrastigma oligose group mice is remarkably reduced (P is less than 0.05), and is respectively reduced by 16.45%, 9.94% and 16.62%, which shows that the gastric residual rate of the mice can be effectively reduced by the tetrastigma oligose of each dose, the gastric emptying capability of the mice is improved, and the effect of improving the gastric emptying function of the mice by the low and high doses is better than that of the medium dose.
The experimental result of the influence of the tetrastigma hemsleyanum L oligosaccharide on the gastrointestinal tract function of a mouse shows that the tetrastigma hemsleyanum L oligosaccharide can obviously shorten the first defecation time of a normal mouse and a constipation model mouse caused by loperamide hydrochloride, increase the number of black stool particles and the moisture content of the black stool, and can obviously promote the small intestine peristalsis and the gastric emptying of the normal mouse, which indicates that the tetrastigma hemsleyanum L oligosaccharide has the effect of relaxing bowel to a certain. The method provides a research basis for the application of the traditional Chinese medicine radix tetrastigme in the aspect of regulating gastrointestinal tract functions, improves the additional value for the fine processing and comprehensive utilization of the radix tetrastigme, and has great significance.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (7)
1. A preparation method of tetrastigma hemsleyanum Diels et Gilg oligosaccharide is characterized by comprising the following steps:
1) picking whole plant of radix tetrastigme, washing, drying in the sun, drying to constant weight, pulverizing, and sieving; weighing coarse powder of radix tetrastigme, adding the coarse powder into an ethanol solution according to the material-liquid ratio of 1g:15-25mL, and performing ultrasonic extraction for at least 2 times; carrying out suction filtration, and discarding filtrate;
2) taking filter residue, adding the filter residue into double distilled water according to the material-liquid ratio of 1g:10-20mL, and carrying out reflux extraction for at least 2 times, wherein the reflux extraction time is 1-2h each time; filtering the extractive solution after each extraction, mixing filtrates, concentrating under reduced pressure, and deproteinizing by Sevag method for at least 3 times;
3) adding ethanol solution into the deproteinized extractive solution to make ethanol volume reach 75%; standing overnight, centrifuging, and removing precipitate; adding ethanol solution into the supernatant, precipitating again, standing overnight, centrifuging, and drying the precipitate under reduced pressure to obtain radix Apioris Fortunei oligosaccharide;
in the step 1) and the step 3), the concentration of the ethanol solution is 90-98 wt%.
2. The method for preparing tetrastigma hemsleyanum Diels et Gilg oligosaccharide of claim 1, wherein in the step 1), the drying temperature is 50-60 ℃.
3. The method for preparing tetrastigma hemsleyanum Diels et Gilg oligosaccharide according to claim 1 or 2, wherein in the step 1), the tetrastigma hemsleyanum Diels et Gilg oligosaccharide is sieved by a 40-mesh sieve after being pulverized.
4. The method for preparing tetrastigma hemsleyanum michaux oligosaccharide according to claim 1, further comprising step 4) after step 3): the molecular weight of radix tetrastigme oligosaccharide is determined by high performance liquid chromatography.
5. The method for preparing tetrastigma hemsleyanum michaux oligosaccharide according to claim 4, wherein the specific process of the step 4) is as follows: preparing tetrastigma hemsleyanum nakai oligosaccharide into a test solution of 1mg/mL, and setting chromatographic conditions: the chromatographic column is Waters Ultrahydrogel 120 with specification of 7.8 × 300mm, the column temperature is 35 deg.C, the drift tube temperature is 100 deg.C, the mobile phase is 300mmol/L ammonium acetate, the flow rate is 0.8mL/min, and the sample injection amount is 10 μ L.
6. The use of tetrastigma hemsleyanum michaux oligosaccharide prepared by the preparation method of claim 1 in preparing antitumor drugs.
7. The use of tetrastigma hemsleyanum Diels et Gilg oligosaccharide prepared by the preparation method of claim 1 in preparing a medicament for relaxing bowel.
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| CN104031169A (en) * | 2014-07-02 | 2014-09-10 | 河南中医学院 | Preparation method of bitter almond polysaccharide and bitter almond oligosaccharide |
| CN105504076A (en) * | 2015-12-01 | 2016-04-20 | 浙江中医药大学 | Tetrastigma hemsleyanum Diels et Gilg root tuber polysaccharide with anti-pyretic and anti-inflammatory functions and application of Tetrastigma hemsleyanum Diels et Gilg root tuber polysaccharide |
| CN105816430A (en) * | 2016-03-16 | 2016-08-03 | 浙江中医药大学 | Preparation method of radix tetrastigme polysaccharide particle with antitumor function |
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| CN105504076A (en) * | 2015-12-01 | 2016-04-20 | 浙江中医药大学 | Tetrastigma hemsleyanum Diels et Gilg root tuber polysaccharide with anti-pyretic and anti-inflammatory functions and application of Tetrastigma hemsleyanum Diels et Gilg root tuber polysaccharide |
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