阅读说明：本技术 一种复方褐藻糖胶组合物 (Compound fucoidan composition ) 是由 朱荣 于 2021-07-30 设计创作，主要内容包括：本申请公开了一种复方褐藻糖胶组合物,所述复方褐藻糖胶包括以下组分：褐藻糖胶和法地榄仁果提取物。本发明在直接对癌细胞进行抑制和消灭的同时可激发其他免疫细胞的活性,进一步对抗癌细胞,通过两种途径共同抑制和杀灭癌细胞,提高抗癌作用和效果,疗效较佳。(The application discloses a compound fucoidan composition, which comprises the following components: fucoidan and Terminalia catappa fruit extract. The invention can directly inhibit and eliminate the cancer cells and simultaneously stimulate the activity of other immunocytes to further resist the cancer cells, inhibit and kill the cancer cells by two ways, improve the anticancer effect and have better curative effect.)

1. The compound fucoidin composition is characterized by comprising the following components: fucoidan and Terminalia catappa fruit extract.

2. The compound fucoidan composition of claim 1, wherein the weight ratio of the fucoidan to the terminalia faba fruit extract is 2: 1.

3. The compound fucoidan composition according to claim 1 or 2, wherein the fucoidan and the terminalia faba fruit extract are formulated into a tablet, a capsule or a granule.

4. The compound fucoidan composition of claim 3, wherein the fucoidan and the Terminalia catappa fruit extract are formulated into a capsule.

5. The method for preparing the compound fucoidan composition according to claim 4, wherein the capsule preparation method comprises the following steps:

s1: extracting a crude product of fucoidin from the algae powder by a complex enzyme enzymolysis method;

s2: further precipitating, deproteinizing, decolorizing and removing small molecular substances from the crude product of fucoidan to obtain relatively pure fucoidan for later use;

s3: the method comprises the steps of taking Terminalia catappa fruit as a raw material, cleaning, removing kernels, drying by hot air, cooling, crushing fresh fruits, removing dregs by pressure filtration, and sterilizing at high temperature to obtain Terminalia catappa fruit extract for later use;

s4: preparing capsule body, taking gelatin, glycerol and purified water as capsule body raw materials, injecting the glycerol and the purified water into a sol tank, stirring and heating to 50-70 ℃, adding the gelatin, continuously stirring for 20-30min, performing vacuum defoaming to obtain the capsule body, and keeping the temperature at 50-60 ℃ for later use;

s5: and (4) taking the extracts in the steps S2 and S3 as contents, and carrying out pelleting, shaping, pill washing, air drying, pill refining and subpackaging on the extracts and the capsule body prepared in the step S4 to prepare the soft capsule containing the fucoidin and the terminalia faba fruit extract.

6. The method for preparing a compound fucoidan composition according to claim 5, wherein in step S1, the extracted enzymes obtained by the complex enzymatic hydrolysis are cellulase, pectinase, papain and xylanase, respectively, and the dosage ratio is 1:2:2: 2.

7. The method for preparing the compound fucoidan composition according to claim 5, wherein the step S2 is performed by ethanol precipitation with a concentration of 70% -80%.

8. The method of claim 5, wherein the step S5, the forming machine is turned on, the capsule shell thickness is adjusted, the gelatin box temperature is set at 60-70 ℃, the loading capacity of the mold is adjusted, the gelatin solution is spread on a rotating gelatin wheel to automatically form a gelatin skin, then the gelatin skin is loaded between a pair of rolling molds on the machine head, the paraffin oil is used to lubricate the gelatin skin, the feeding system injects the weighed fucoidan and the extracted solution of Terminalia faba fruit through the injector, the rolling molds spin-press the capsule to form a capsule with a certain loading capacity and shape.

9. The method of claim 5, wherein the fucoidan and the Terminalia catappa fruit extract are separately encapsulated in soft capsules in step S5.

Technical Field

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to a compound fucoidan composition.

Background

Breast cancer is one of the most common malignant tumors that endanger women's health. The incidence of breast cancer has been found to be increasing over the past 50 years, accounting for about 31% of women's tumors. Although the mortality rate of breast cancer is greatly reduced by the comprehensive treatment methods (operation, chemotherapy, radiotherapy and endocrine treatment) widely used at present, about more than one third of breast cancer patients have the characteristics of easy relapse and easy distant metastasis after operation, are not sensitive to endocrine treatment, have poor treatment effect and prognosis and obvious chemotherapy toxic and side effects, and a satisfactory comprehensive treatment scheme is still lacked so far. The natural compound has small toxic and side effects, has the characteristic of target killing of tumor cells, and is a hotspot of the current antitumor research. Therefore, the search for natural medicines for resisting breast cancer metastasis is of great significance.

Research reports that the incidence rate of breast cancer only accounts for 1/9 in western countries in japan and korea where kelp is consumed in large quantities, particularly, the number of kelp consumed in okinawa prefecture in japan is the top of japan, but the incidence rate of cancer in okinawa prefecture is the lowest in japan. The kelp contains a polysaccharide substance consisting of repeated arrangement of monosaccharides such as uronic acid, mannose and fucose, and can induce cancer cell suicide and further play a role in resisting cancer, wherein the polysaccharide substance is fucoidan.

In recent years, researches show that fucoidan has obvious inhibition effect on breast cancer cells, lung cancer cells, human bladder cancer cells, melanoma cells and the like, can only directly act on cancer cells, has little influence on activity excitation of other immune cells, and has insignificant anti-cancer effect.

Disclosure of Invention

The invention aims to provide a compound fucoidan composition aiming at the defects of the prior art, which can directly inhibit and eliminate cancer cells and simultaneously can stimulate the activity of other immunocytes to further resist the cancer cells, inhibit and kill the cancer cells together through two ways, improve the anticancer effect and have better curative effect.

In order to achieve the purpose, the invention adopts the following technical scheme:

a compound fucoidan composition comprises the following components: fucoidan and Terminalia catappa fruit extract.

As a further improvement of the invention, the weight ratio of the fucoidan to the kernel fruit extract of Terminalia faba is 2: 1.

As a further improvement of the invention, the fucoidan and the terminalia faba fruit extract are prepared into tablets, capsules or granules.

As a further improvement of the invention, the fucoidan and the Terminalia catappa fruit extract are prepared into capsules.

As a further improvement of the invention, the preparation method of the capsule comprises the following steps:

s1: extracting a crude product of fucoidin from the algae powder by a complex enzyme enzymolysis method;

s2: further precipitating, deproteinizing, decolorizing and removing small molecular substances from the crude product of fucoidan to obtain relatively pure fucoidan for later use;

s3: the method comprises the steps of taking Terminalia catappa fruit as a raw material, cleaning, removing kernels, drying by hot air, cooling, crushing fresh fruits, removing dregs by pressure filtration, and sterilizing at high temperature to obtain Terminalia catappa fruit extract for later use;

s4: preparing capsule body, taking gelatin, glycerol and purified water as capsule body raw materials, injecting the glycerol and the purified water into a sol tank, stirring and heating to 50-70 ℃, adding the gelatin, continuously stirring for 20-30min, performing vacuum defoaming to obtain the capsule body, and keeping the temperature at 50-60 ℃ for later use;

s5: and (4) taking the extracts in the steps S2 and S3 as contents, and carrying out pelleting, shaping, pill washing, air drying, pill refining and subpackaging on the extracts and the capsule body prepared in the step S4 to prepare the soft capsule containing the fucoidin and the terminalia faba fruit extract.

As a further improvement of the present invention, in step S1, the extracted enzymes of the complex enzymatic hydrolysis method are cellulase, pectinase, papain and xylanase, respectively, and the dosage ratio thereof is 1:2:2: 2.

As a further improvement of the present invention, in step S2, ethanol is used for precipitation by fractionation, and the concentration of ethanol is 70% to 80%.

As a further improvement of the present invention, in the step S5, the forming machine is started, the thickness of the capsule shell is adjusted, the temperature of the gelatin box is set to 60-70 ℃, the loading amount of the mold is adjusted, the gelatin solution is spread on a rotating gelatin wheel to automatically form a gelatin skin, then the gelatin skin is loaded between a pair of rolling molds on the machine head, the paraffin oil is used to lubricate the gelatin skin, the weighed fucoidan and the extracted solution of the terminalia faberi fruit are injected through the injection body by the feeding system, and at the same time, the rolling molds are spun to complete the capsule encapsulation, and the capsule with a certain loading amount and shape is pressed.

As a further improvement of the present invention, in step S5, the fucoidan and the terminalia faba fruit extract are separately sealed in soft capsules.

Compared with the prior art, the invention has the beneficial effects that:

the invention directly inhibits and kills cancer cells by fucoidin, simultaneously stimulates the activity of other immunocytes by the terminalia faba fruit to further resist the cancer cells, inhibits and kills the cancer cells by two ways, can obviously control vascular proliferation, reduces the probability of cancer cell diffusion and metastasis, induces the self-destruction of the cancer cells, inhibits the proliferation and metastasis of the cancer cells, relieves the side effect of radiotherapy and chemotherapy, strengthens the inhibition of inflammatory reaction of the whole body, increases the immunity of the whole body and has better curative effect.

Detailed Description

For better understanding of the present application, the following will clearly and completely describe the technical solutions in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Examples

The invention provides a compound fucoidin composition, which comprises the following components: the weight ratio of the fucoidan to the terminalia faba fruit extract is 2:1, namely when the fucoidan is 75%, the terminalia faba fruit extract can be 25% or less, other pharmaceutical excipients can be added into the remaining volume, the fucoidan and the terminalia faba fruit extract are prepared into soft capsules, and the fucoidan and the terminalia faba fruit extract are respectively and independently sealed in the soft capsules to prevent the occurrence of chemical reactions among related substances, which causes the change of components and influences the later curative effect, and the preparation method comprises the following steps:

a) extracting a crude product of fucoidin from the algae powder by a complex enzyme enzymolysis method; adding cellulase, pectinase, papain and xylanase into algae powder for enzymolysis, wherein the dosage ratio is 1:2:2:2, the pH is 5.0, the temperature is 30 ℃, the extraction time is 3h, and then heating and inactivating enzymes and filtering.

b) Further precipitating, deproteinizing, decolorizing and removing small molecular substances from the crude product of fucoidan to obtain relatively pure fucoidan for later use; in the precipitation stage, ethanol is adopted for fractional precipitation, fucoidan is separated and purified by utilizing the properties of different solubilities of alginic acid, kelp starch and fucoidan in ethanol with different concentrations, the optimal ethanol concentration is 70-80%, alginic acid is easy to separate out when the ethanol concentration is 20-30%, kelp starch is easy to dissolve when the ethanol concentration is 60%, and fucoidan can separate out when the ethanol concentration is 70-80%; in the deproteinization stage, deproteinization is carried out by a hydrochloric acid method, a trichloroacetic acid method or a Savage method, preferably the Savage method, and the conditions of the method are mild, so that the damage to the polysaccharide structure is small; in the decolorizing stage, mainly alcohol washing method, active carbon adsorption method, strong oxidant oxidation decolorizing method and chromatographic column adsorption decolorizing method are adopted, preferably chromatographic column adsorption decolorizing method, i.e. adsorption decolorizing by ion exchange resin or macroporous resin, etc., DEAE-cellulose ion exchanger is mainly adopted, the main steps are that 10mm x 300mm chromatographic column is adopted, the sample loading amount is about 50mg of fucoidan each time, the fucoidan is dissolved in about 2mL of equilibrium solution, the flow rate of eluent is 15mL/h, 10min is collected into one tube, 0.1mL of each tube is taken, and the detection is carried out by sulfuric acid benzene-phenol method.

c) The method comprises the steps of taking Terminalia catappa fruit as a raw material, cleaning, removing kernels, drying by hot air, cooling, crushing fresh fruits, removing dregs by pressure filtration, and sterilizing at high temperature to obtain Terminalia catappa fruit extract for later use;

d) preparing a capsule body, taking gelatin, glycerol and purified water as capsule body raw materials, injecting the glycerol and the purified water into a sol tank, stirring and heating to 60 ℃, adding the gelatin, continuously stirring for 25min, then carrying out vacuum defoaming to obtain the capsule body, and keeping the temperature at 55 ℃ for later use;

e) taking the extracts in the step b) and the step c) as contents, and performing pelleting, sizing, pill washing, air drying, pill refining and subpackaging with the capsule prepared in the step d) to prepare the soft capsule containing the fucoidan and the terminalia faberi fruit extract, wherein in the pelleting step, a forming machine is started, the thickness of a capsule shell is adjusted, the temperature of a gelatin box is set to be 65 ℃, the filling amount of a mould is adjusted, the gelatin solution is spread on a rotating gelatin wheel to automatically prepare a gelatin skin, then the gelatin skin is filled between a pair of rolling dies on a machine head and lubricated by paraffin oil, the weighed fucoidan and the terminalia faberi fruit extract are injected through a spray body by a feeding system, the rolling dies are spun to complete capsule sealing, and the capsule with a certain filling amount and shape is compacted; in the shaping step, the pressed capsule is put into a roller drying device for drying by blowing, so that the capsule is hardened and shaped, the ambient temperature is 25 ℃, the humidity is 30%, and the drying time is 20 h.

Test examples

1. Laboratory animal

The experimental animals used in the study were 28 clean-grade female SD rats, each weighing 100g, purchased from national animal center, exposed to natural light, raised in cages, 7 animals per cage, given appropriate temperature and humidity control tubes, and provided with adequate feed and drinking water.

2. Medicine and food additive

1) Adopting the fucoidan extracted by the method and the terminalia fruit extract extracted by the method, 2) preparing MTT (thiazole blue) solution: weighing 250mg MTT, placing into a small beaker, adding 50 ml PBS (pH7.4), stirring and mixing for 30min, filtering with 0.22 μm microporous filter for sterilization, subpackaging under aseptic condition, and storing at 4 deg.C.

3. Cell culture

Breast cancer cells 4T1 were purchased from national cell banks, and 4T1 cells were cultured in DMEM high-glucose medium (pH: 7.2-7.5) containing 15% fetal bovine serum, which contained 0.2% NaHCO₃100. mu.g/ml penicillin, 100. mu.g/ml streptomycin in RPMI1640 complete medium in 5% CO₂And culturing in a 37 ℃ incubator saturated with water vapor, replacing the culture solution once a day, carrying out passage for 1 time for 2 days, and observing the integrity and growth speed of the cell morphology by an inverted microscope regularly during the culture period.

3. Establishment of animal model

(1) Taking 4T1 cells growing in logarithmic phase, digesting with 0.25% pancreatin and 0.02% EDTA for 1min, stopping digestion of culture solution, centrifuging at low speed of 1000/min for 2-5min at 800-.

(2) The transplantation method adopts subcutaneous injection, a 1m syringe No. 6 needle is used for inoculating cells on the left forelimb armpit backrest part of a rat, and 28 rats are inoculated in total, wherein each inoculation part is 1m 1.

(3) The general life of the mice was observed daily, and the tumor growth rate of each group of mice was observed after one week.

(4) Tumor body diameter =3mm is taken as the tumor forming positive, all the 28 experimental mice form tumors, and the tumor forming positive mice are randomly divided into 4 groups of 7 mice each.

(5) When the transplanted tumor grows to be visible by naked eyes 3 weeks after tumor inoculation, namely the tumor body diameter reaches 2-3mm, the intervention experiment is started, the medicine is administered in groups by adopting an intraperitoneal injection mode, and the experimental animals are divided into the following four groups

a) The low dose group is 5mg/kg. bw fucoidan, which is prepared by normal saline, 0.2 ml/piece;

b) high dose group, 10mg/kg. bw fucoidan, prepared with normal saline, 0.2 ml/piece;

c) negative control group is physiological saline 0.2 ml/mouse;

d) experimental groups: mixing fucoidan and Terminalia catappa fruit extractive solution with normal saline 0.2 ml/piece; the administration was performed every two days for 20 days.

(6) The growth of transplanted tumors of rats in each group is observed, such as tumor formation rate, incubation period, tumor weight, volume, presence or absence of metastasis, and the like. The maximum diameter a and the maximum transverse diameter b of the tumor were measured every 3 days by a vernier caliper according to V =1/2 × a × b²The tumor volume V is calculated and the growth curve of the transplanted tumor is plotted according to the change of the tumor volume.

(7) 48 hours after the last administration, the mice are killed by pulling the neck, tumor bodies are obtained by separation, the tumor bodies are weighed, and partial tumor tissues are added with liquid nitrogen for homogenate and protein extraction; fixing part of tumor tissue in 10% neutral formalin for 24 hr, gradient dehydrating with normal alcohol, clearing with xylene, embedding in paraffin, making 4 μm thick continuous section, and staining paraffin section.

4. Results

4.1 Effect of fucoidan and Terminalia catappa fruit extract on tumor growth of laboratory mice

When the transplanted tumor growth of experimental mice reaches 2-3mm 3 weeks after tumor inoculation, there is no significant difference between tumor volumes of groups of mice after random grouping (P > 0.05). With the progress of the fucoidan intervention experiment, the activity of the control group mice is gradually reduced, the hair color becomes dark, and the reaction is not sensitive, while the reaction of the experimental group rats of the fucoidan and the terminalia faberi fruit extracting solution is sensitive, and the food intake and the activity are not obviously different from those before the treatment. The tumor volumes of the treatment groups are gradually increased, wherein the tumor volume of the control group is increased most rapidly, the tumor volume of the fucoidan treatment group is increased gradually and slowly, the high-dose fucoidan (10mg/kg. bw) group is slower than that of the fucoidan treatment group, the fucoidan and the Terminalia faba fruit extract are slowest, and the tumor volumes of the low-dose fucoidan group, the high-dose fucoidan group and the experiment group after the intervention experiment are respectively 1.44+0.26cm³、1.10±0.21cm³And 0.8. + -. 0.16cm³Comparison with control group (2.10 + -0.48 cm)³) The comparison difference has statistical significance (P is less than 0.05). In addition, the average weights of the tumors in each group after the experiment are finished are remarkably different (P < 0.05).

4.2 Effect of fucoidan and Terminalia catappa fruit extract on apoptosis of transplanted tumor tissue cells

The tumor tissues of rats in each group are subjected to paraffin section, TUNEL kit staining is used for detecting apoptotic cells, the number of apoptotic cells in the tumor tissues of an experimental group is obviously more than that of a control group and other groups, the difference has statistical significance (P is less than 0.01), and the number of apoptotic cells in a high-dose alginate (10mg/kg.bw) treatment group is also obviously different from that of a low-dose alginate (5mg/kg.bw) treatment group (P is less than 0.01).

4.3 expression of Bc-2, Bax proteins in transplanted tumor tissue

And extracting proteins of transplanted tumor tissues of the mice of each group, and carrying out Western blot detection. The results show that compared with the control group, the Bcl-2 protein level in the tumor tissues of each fucoidan-treated group is obviously reduced (P is less than 0.05), the Bax expression of the experimental group is increased (P is less than 0.05), the Bcl-2/Bax ratio in the tumor tissues of each fucoidan-treated group is obviously reduced, and the Bcl-2/Bax ratio of the experimental group is obviously lower than that of the low-dose fucoidan-treated group (P is less than 0.05).

4.4 Effect of fucoidan and Terminalia catappa fruit extract on growth of breast cancer in rats

The average tumor weight of an experimental group under the combined action of the fucoidin and the terminalia faba fruit extracting solution is lower than that of a model group, the tumor weight inhibition rate of the experimental group reaches 54.8%, the tumor weight inhibition rate of a high-dose fucoidin group reaches 44.7%, and the tumor weight inhibition rate of a low-dose fucoidin group is 28.2%.

4.5 Effect of fucoidan and Terminalia catappa fruit extract on peripheral blood NK cells

NK cells play a key role in mediating cell immunity and killing tumor cells, so a flow cytometer is adopted to detect the activity of peripheral blood NK cells so as to determine the influence of the fucoidan and the terminalia faba fruit extracting solution on the activation of the NK cells, the activities of the peripheral blood TCR alpha beta and CD161a positive NK cells of the interference group of the fucoidan and the terminalia faba fruit extracting solution are obviously higher than those of other three groups, and the activity of the peripheral blood NK cells can be activated by the terminalia faba fruit extracting solution.

4.6 Effect of Terminalia catappa fruit extract on T lymphocyte subpopulations

To investigate the effect of Terminalia catappa fruit extract on T cell mediated immune response, the number of peripheral blood T lymphocyte subsets, including CD4+, CD8+ and Foxp3+ Treg cells, was examined. The results show that compared with the model group, the proportion of CD4+ and CD8+ T lymphocytes in the peripheral blood of the pre-treated group of the fucoidan and the terminalia fabiana fruit extract is increased compared with the proportion of the CD4+ and the CD8+ T lymphocytes in the single-use fucoidan, and the proportion of Foxp3+ Treg cells with immunosuppressive activity is reduced, so that the terminalia fabiana fruit extract can enhance the antitumor immune response in a rat body and reduce immunosuppression.

5. Conclusion

Combining the above research results, in this embodiment, the combined action of fucoidan and the extract of terminalia faberi can regulate the immunity of rats, activate the activity of immune cells, enhance anti-tumor immunity, and alleviate the immune suppression of tumors.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to limit the scope of the present invention, and those skilled in the art can make various changes and modifications to the embodiments without departing from the spirit and scope of the present invention.