WO2020042665A1

WO2020042665A1 - Shark cartilage glycoprotein and anticancer use thereof

Info

Publication number: WO2020042665A1
Application number: PCT/CN2019/086298
Authority: WO
Inventors: 车路平
Original assignee: 车路平
Priority date: 2018-08-27
Filing date: 2019-05-09
Publication date: 2020-03-05
Also published as: CN108939043A

Abstract

Disclosed are a shark cartilage glycoprotein and an anticancer use thereof. The shark cartilage glycoprotein is a shark cartilage active ingredient obtained after shark cartilage has been pulverized, extracted with alkaline water, salted out, freed of free proteins and purified. Pharmacological experiments have shown that the shark cartilage glycoprotein has anti-angiogenic effects, and can inhibit cancer cells from proliferating, such as those of skin cancer, breast cancer, non-small-cell lung cancer, liver cancer, stomach cancer and ovarian cancer. In vivo experiments have also demonstrated tumor suppression effects, showing that the invention can be applied to cancer prevention and treatment.

Description

Shark cartilage glycoprotein and its anticancer use

Technical field

The invention belongs to the field of medicine, and particularly relates to a shark cartilage glycoprotein and its anticancer use.

Background technique

Cancer, also known as malignant tumor, refers to a class of diseases in which the uncontrolled proliferation of cells caused by the abnormal mechanism of control of cell proliferation and local invasion of surrounding normal tissues, or even metastasis to other parts of the body via the internal circulatory system or lymphatic system . Because cancer has no obvious clinical symptoms in its early stages, it is often at the advanced stage of cancer when it is discovered, which often leads to the best treatment window missed at the time of discovery, making its mortality rate high and becoming one of the top five terminal illnesses in the world. China is one of the regions with high incidence of cancer. In recent years, the incidence of cancer is increasing year by year. In 2014, there were about 3.8 million new cancers and 2.29 million deaths. As of now, the exact pathogenesis of cancer is still unclear. The factors related to the onset of cancer mainly include exogenous and endogenous factors. Among them, exogenous factors include lifestyle habits, environmental pollution, and iatrogenic factors. Source factors include genetic factors, immune factors, and endocrine factors. Although the overall incidence of cancer is not much different, there is a significant difference in the incidence of different cancers between men and women. Due to smoking and the increase in environmental pollution in recent years, lung cancer is the most prevalent cancer among men, and breast cancer is female. The highest incidence of cancer.

At present, the treatment of cancer mainly includes surgical treatment, radiotherapy, chemotherapy, etc. Among them, surgical treatment can theoretically completely remove cancer tissue, thereby achieving cure of cancer, but since cancer is mostly in advanced stage when cancer is discovered, cancer cells have invaded the surrounding normal. Tissue or spread to other organs of the body with the circulatory system, making it impossible to completely understand cancer cells through surgery, and surgery is an invasive treatment. During the surgery, the destruction of the circulatory system surrounding cancer tissue may promote cancer cells to a certain extent The spread of the disease, combined with the adverse effects of wound infections and the destruction of the human immune system caused by surgery, has led to the limited role of surgical treatment in cancer treatment. Radiation therapy is a method of using radiation to kill cancer cells. However, due to the lack of targeting, radiation therapy can cause large-scale damage to normal cells, affect the patient's digestive system, destroy the patient's immune system, and make the patient's ability to resist cancer greater. The decrease in amplitude, although widely used in the treatment of cancer in modern radiation therapy, is only an expedient measure and is not an ideal method for treating cancer. Chemotherapy, or chemotherapeutics, refers to taking drugs that kill cancer cells and is used to treat cancer. Chemotherapy drugs also have damage to normal cells, but through the continuous development of chemotherapeutic drugs, people are working hard to develop drugs with less effect on normal tissues through targeted administration to avoid the damage to normal tissue cells during chemotherapy. Damage, which can effectively treat cancer.

In the 1980s, shark cartilage was found to have an effect of inhibiting angiogenesis, so it was gradually applied to the prevention and treatment of cancer, but its overall therapeutic effect was still not ideal. In recent years, people have studied the anti-cancer effects of shark cartilage extract, shark chondroitin, and shark cartilage angiogenesis inhibitors, and have not achieved breakthrough progress. Even the view that shark cartilage does not have anti-cancer effect has appeared.

Based on a thorough investigation of the existing technology, the inventors of the present invention have isolated a shark cartilage glycoprotein from shark cartilage through a large number of experiments. Pharmacological experiments have shown that it has excellent inhibition of vascularization and The effect of promoting cancer cell apoptosis can be used for cancer prevention and treatment.

Summary of the Invention

The object of the present invention is to provide a use of shark cartilage glycoprotein in preparing an anticancer medicine.

Preferably, the shark cartilage glycoprotein is prepared by the following method:

(1) The shark cartilage is crushed and passed through a 10-30 mesh sieve to obtain shark cartilage particles;

(2) Shark cartilage granules are added with 3-8 times purified water, adjusted to pH 10-13 with NaOH, heated to 50-70 ° C, immersed for 3-10 hours and filtered, the filtrate is reserved, and the filter residue is repeatedly extracted 1-2 times The filtrates are combined;

(3) After the filtrate was subjected to salting out, free protein was removed by Sevage method, and then dialysis and DEAE-C32 anion interaction resin column chromatography were performed, followed by freeze-drying to obtain shark cartilage glycoprotein.

It is still another object of the present invention to provide an anticancer pharmaceutical composition comprising shark cartilage glycoprotein or shark cartilage.

Preferably, the anti-cancer pharmaceutical composition further comprises turtle plate, abalone shell, baking soda, clam shell, purslane, beef bone meal, Poria cocos, Shanci mushroom, sage grass, Houttuynia cordata, astragalus, Qumai, zero Lingxiang.

Preferably, the anti-cancer drug composition comprises 5-10 parts of shark cartilage, 2-5 parts of turtle plate, 1-3 parts of abalone shell, 1-3 parts of baking soda, 1-3 parts of clam shell, and purslane 3- 8 servings, 8-15 servings of beef bone powder, 2-5 servings of Poria cocos, 5-10 servings of Shan Ci mushrooms, 2-5 servings of Saguaris, 1-3 servings of Houttuynia cordata, 1-3 servings of Astragalus, 1 serving of Qumai Made from 3 servings and 1-3 tonka incense.

More preferably, the anticancer drug composition is composed of 80 g of shark cartilage, 30 g of tortoise shell, 20 g of abalone shell, 20 g of baking soda, 20 g of clam shell, 50 g of purslane, 100 g of beef bone powder, and 30 g of Poria 30 It is made from 80 grams, 80 grams of Shanci mushroom, 30 grams of misograss, 10 grams of houttuynia, 10 grams of astragalus, 10 grams of qumai, and 10 grams of tonka.

In the pharmaceutical composition of the present invention:

Shark cartilage: It has multiple functions such as anti-tumor, rheumatism, analgesia, and diarrhea. It is often used in the treatment of rheumatoid arthritis, headache, diarrhea and other symptoms.

Turtle plate: It is the plastron and carapace of the tortoise chinemys reevesii, mainly distributed in Zhejiang, Hubei, Hunan and other places. The turtle plate tastes salty, sweet, and has the effects of nourishing yin, Qianyang, kidney, and bones. It is often used in Bone steaming fever, hot flashes, night sweats, bleeding, nocturnal emission, seminal discharge, epiphysis, vomiting and other symptoms. Modern pharmacological studies have shown that turtle plate can improve the body's immune function and anti-tumor effects.

Abalone shell: also known as stone cassia, it is a precious medicinal material, which has the functions of heat-clearing and liver-relieving, nourishing yin and aphrodisiac, abalone and abalone shell contain rich protein, and the abalone shell itself has very high nutritional value. Abalone shell can be treated with medicine. Chitin, which is extracted from abalone shells, can be used for the treatment of gastric, duodenal ulcers, and hyperacidity.

Baking soda: Sodium bicarbonate, the chemical formula is NaHCO _{3. The} baking soda solution is alkaline. It can be sterilized and disinfected externally in medicine. Oral can be used to treat hyperacidity. Intravenous administration can also be used to correct acidosis. Modern pathological studies show that the tumor has an acidic environment. Taking baking soda can break the acidic environment of tumor tissues and inhibit tumor cell proliferation.

Clam shells: Clams are salty and cold, and they have the functions of nourishing yin, moisturizing, diuretic, swelling, soft and firm. "Ben Cao Jing Shu" records: "Clams are moisturizing and help tonic fluid, so they can moisten the internal organs, quench thirst, and appetite. Salty can soften blood, so the housewife's blood clots and old habits are also cold and hot." And ascites tumors have inhibitory and relieving effects. Clam shells are salty, cold, and can enter the lung, kidney, and liver meridians. They can clear heat, relieve phlegm, dampness, and softness. They are often used to treat stomach pain, cough, cough, edema, and urination. Bloated and scalded. The well-known herbal clam powder is that the clam shells are washed and then charcoal fired and roasted for the treatment of phlegm, fire and cough.

Purslane: It is the dry aerial part of purslane, purslane, sour, cold, returning to the liver, large intestine, and has the effects of clearing heat and dampness, detoxification and swelling, anti-inflammatory, thirst quenching, and diuretic. It is commonly used in Treatment of fever, blood poisoning, carbuncle, scabies, eczema, erysipelas, snake bite, blood in the stool, and leakage.

Bovine Bone Meal: It is the bone of the bovine bison ox or buffalo buffalo. It is sweet and warm. It has the functions of paralysis, truncation, and astringent sores. It is often used for arthritis, diarrhea, malaria, scabies, etc. Treatment. The bone marrow contained in the bone has been recorded since ancient times as "replenishing the essence, strengthening the bones, and prolonging life". The ancient book "Shou Shi Zhen Yuan" believes that the bone marrow "specially repairs damage, promotes blood circulation, strengthens the skin, rejuvenates the skin, and returns to the age of children", and is anti-aging Hematopoietic organs can enhance the body's hematopoietic function and improve the body's immunity. It contains all the nutritional essentials needed by the human body, and has lecithin that promotes children's brain development and brain health. It also has moisturizing skin, beauty and anti-aging bone. Collagen (multiple amino acids), mucopolysaccharides and multiple protein peptides. Beef bone also stores a wealth of nutrients. Its protein, iron, sodium, and energy are much higher than fresh meat. The protein content is 23% higher than milk powder, twice that of pork, and 61% higher than beef. It is egg. More than 1 time, the content of phosphorus and calcium is unmatched by other foods. Particularly rare is that the nutritional components of bovine bone are more easily absorbed by the body than plant proteins.

Poria cocos: It is a dry rhizome of Liliaceae, Phyllostachys pubescens, which is sweet, light and flat. It has the effects of detoxification and dehumidification, and the same joints. It is often used for the treatment of limb constriction caused by syphilis and mercury poisoning.

Shanci mushroom: It is a dried pseudobulb of the orchid family Rhododendron Orchid, Orchid Orchid, or Yunnan Orchid Orchid. It is sweet, slightly spicy, cool, and belongs to the liver and spleen. It has the effect of clearing heat and detoxifying, eliminating spleen and dissolving stagnation, and is often used for the treatment of bloated and swollen venom, rickets, and snake bite.

Scaly grass: The dried whole grass of Scalyaceae white flower Scaly sauce, tastes bitter, bitter, cool, and returns to the liver, stomach, and large intestine meridian. It has the functions of clearing heat and detoxifying, expectorant and purulent, and is often used in intestines and lungs. Treatment of dysentery, dysentery, swollen scabies.

Houttuynia cordata: It is the dry aerial part of amaranthaceae plant Amaranth. It has the functions of clearing heat and detoxifying, reducing swelling and purging sore, diuretic and dehumidifying, clearing heat and stopping dysentery, strengthening stomach and digesting food, and can be used to treat lung pus ulcer, lung heat cough and asthma, fever, hot shower, edema, beriberi, urinary infection Excessive, swollen, sore, and so on. Modern pharmacological studies have shown that Houttuynia cordata also has antibacterial, antiviral, body immunity, and diuretic effects. Clinical practice has proven that Houttuynia cordata has good effects on upper respiratory tract infections, bronchitis, pneumonia, chronic bronchitis, chronic cervicitis, and pertussis, and it also has certain effects on acute conjunctivitis and urinary tract infections.

Astragalus membranaceus: Mongolian astragalus root is a legume, sweet and warm, and belongs to the lungs, spleen, liver and kidney. It has the effects of replenishing qi and solid surface, detoxifying and purging, diuretic, and muscle growth. It is often used for the treatment of qi deficiency and fatigue, chronic diarrhea and prolapse, spontaneous sweating, edema, uterine prolapse, diabetes, and long-term non-healing of wounds.

Qumai: It is the dry aerial part of the Dianthus plant Qumai, which has bitter taste, coldness, homesickness, and small intestine meridian. It has the effects of diuresis, phlegm and blood circulation, and is often used in the treatment of hot leaching, blood leaching, stone leaching, dysuria, leaching astringent pain, and amenorrhea.

Tonka incense: also known as smoky grass, bell scented, is a primrose plant, the herb of the genus vanillin, with roots, sweet, warm, and full of Taiyin, Yangming Jing, has the effect of removing wind cold, clearing turbid , Commonly used to treat typhoid fever, cold headache, fullness of the chest and abdomen, adverse, nocturnal emission, nasal congestion, toothache and other symptoms.

The anti-cancer pharmaceutical composition of the present invention is an oral pharmaceutical composition or a topical pharmaceutical composition.

Preferably, the oral pharmaceutical composition is a tablet, capsule, granule, pill, powder, decoction; the topical pharmaceutical composition is an external application cream.

Another object of the present invention is to provide a method for preparing an oral anticancer pharmaceutical composition, which includes the following steps:

After the shark cartilage glycoprotein is added with a pharmaceutically acceptable excipient, an oral anticancer pharmaceutical composition is prepared according to a conventional method in the art. ,

Or follow these steps:

(1) Crush the shark cartilage, tortoise shell, abalone shell, baking soda, clam shell, purslane, beef bone meal, Poria cocos, Shanci mushroom, sauerkraut, Houttuynia cordata, Astragalus membranaceus, Qumai, Tonka fragrant into each Mix after 20 mesh particles;

(2) Add the mixed particles obtained in step (1) to 5 times of purified water, adjust the pH to 12 with NaOH, heat to 60 ° C, and soak for 5 hours and filter. The filtrate is reserved. The filter residue is repeatedly extracted twice. The filtrates are combined and reduced. Pressure concentrated to extracts with a density of 1.02-1.05 at 25 ° C;

(3) The extract in step (2) is prepared with a pharmaceutically acceptable auxiliary material according to a conventional method in the art to obtain an oral anticancer pharmaceutical composition.

Yet another object of the present invention is to provide a method for preparing an external cancer drug composition external plaster, which includes the following steps:

(1) Crush shark cartilage, tortoise shell, abalone shell, baking soda, clam shell, purslane, beef bone meal, earthy Poria, Shanci mushroom, sauerkraut, Houttuynia cordata, Astragalus, Qumai, Linglingxiang to 150 -200 mesh particles;

(2) Soak the water of Houttuynia cordatum, Tonka fragrant, Shanci mushroom, and rotten grass with sea salt for 48 hours. When using, use this water to shark cartilage, turtle plate, abalone shell, baking soda, clam shell, horse teeth Loquat, beef bone powder, Poria cocos, Astragalus and Qumai granules are prepared into ointments to obtain an external plaster.

Another object of the present invention is to provide a use of a composition comprising the shark cartilage glycoprotein or shark cartilage in the preparation of an anticancer drug. The shark cartilage glycoprotein is prepared by the following method:

Beneficial effects of the present invention

Shark cartilage glycoprotein in shark cartilage is isolated by the present invention. In vitro experiments show that shark cartilage glycoprotein can inhibit the proliferation of human umbilical vein endothelial cells, and can also inhibit skin cancer cells, breast cancer cells, liver cancer cells, lung cancer cells, and gastric cancer. Cell proliferation.

On the basis of finding that shark cartilage glycoprotein can effectively resist angiogenesis and induce apoptosis of cancer cells, the present invention also proposes an anticancer drug composition. The anticancer drug composition passes shark cartilage glycoprotein or shark cartilage and Used in other ingredients, it has achieved better anti-angiogenesis and induction of apoptosis of cancer cells than shark cartilage glycoprotein, especially the addition of tonka makes the pharmaceutical composition of the present invention unexpected. Anti-cancer effect.

detailed description

The invention is described in more detail below to facilitate an understanding of the invention.

Example 1: A shark cartilage glycoprotein

The shark cartilage glycoprotein is prepared according to the following method:

(1) Shark cartilage 200g, crushed and passed through a 20 mesh sieve to obtain shark cartilage particles;

(2) Shark cartilage particles are added with 5 times purified water, adjusted to pH 12 with NaOH, heated to 60 ° C, and immersed for 5 hours, then filtered, and the filtrate is reserved. The filter residue is repeatedly extracted twice, and the filtrates are combined;

(3) After salting out the filtrate, use Sevage method to remove free protein, and then dialysis and DEAE-C32 anion interaction resin column chromatography, freeze-drying to obtain 5.6 g of shark cartilage glycoprotein.

Example 2: An anticancer tablet

30 g of shark cartilage glycoprotein, 230 g of microcrystalline cellulose, 25 g of hydroxypropyl cellulose, 15 g of sodium carboxymethyl starch, and appropriate amount of magnesium stearate were prepared according to the following method:

Shark cartilage glycoprotein was smashed through a 60-mesh sieve, after adding microcrystalline cellulose, hydroxypropylcellulose was used for softening, and after 14-mesh sieve was used for granulation. After drying, it was sieved into a whole particle with carboxymethyl starch sodium and hard After mixing the magnesium stearate, it was compressed to obtain 1000 anticancer tablets.

Example 3: An anti-cancer external application cream

3 grams of shark cartilage glycoprotein, 30 grams of turtle plate, 20 grams of abalone shell, 20 grams of baking soda, 20 grams of clam shells, 50 grams of purslane, 100 grams of beef bone meal, 30 grams of Poria cocklebur, 80 grams of mountain mushroom 30 grams of grass, 10 grams of houttuynia cordata, 10 grams of astragalus, 10 grams of qumai, 10 grams of tonka, prepared according to the following method:

(1) Crush the turtle plate, abalone shell, baking soda, clam shell, purslane, beef bone meal, earth Poria, Shanci mushroom, mischievous grass, houttuynia cordata, astragalus, qumai, tonka incense into 180 mesh particles;

(2) Use Houttuynia cordata, Tonka fragrant, Shanci mushroom, and Saponaria granules to soak water in sea salt for 48 hours. During use, use this water to combine shark cartilage glycoprotein with turtle plate, abalone shell, baking soda, clam shell, Portulaca oleracea, bovine bone powder, Poria cocos, Astragalus and Qumai granules are prepared into ointments to obtain an external plaster.

Example 4: An anti-cancer external plaster

Shark cartilage 80g, turtle plate 30g, abalone shell 20g, baking soda 20g, clam shell 20g, purslane 50g, beef bone powder 100g, earthy poria 30g, mountain citron 80g G, Houttuynia cordata 10 g, Astragalus 10 g, Qumai 10 g, Tonka incense 10 g, prepared according to the following method:

(1) Crush shark cartilage, tortoise shell, abalone shell, baking soda, clam shell, purslane, beef bone meal, earthy Poria, Shanci mushroom, sauerkraut, Houttuynia cordata, Astragalus, Qumai, Tonka fragrant into 180 Mesh particle

Example 5: An anti-cancer capsule

(1) Crush the shark cartilage, turtle plate, abalone shell, baking soda, clam shell, purslane, beef bone meal, earth Poria, Shanci mushroom, sauerkraut, Houttuynia cordata, astragalus, qumai, tonka, respectively. Mix after 20 mesh particles;

(3) Add the microcrystalline cellulose to the extract in step (2) to make soft materials, granulate through a 14-mesh sieve, and dry the whole granules through a 12-mesh sieve. Add an appropriate amount of magnesium stearate and fill the gelatin capsule shell. 100 caps of anti-cancer capsules were prepared.

Comparative Example 1: An anticancer drug composition

Shark cartilage 80g, turtle plate 30g, abalone shell 20g, baking soda 20g, clam shell 20g, purslane 50g, beef bone powder 100g, earthy poria 30g, mountain citron 80g G, Houttuynia cordata 10 g, Astragalus 10 g, Qumai 10 g were prepared according to the procedure of Example 4 to obtain an external application cream.

Comparative Example 2: An anticancer drug composition

Shark cartilage 80g, turtle plate 30g, abalone shell 20g, baking soda 20g, clam shell 20g, purslane 50g, beef bone powder 100g, earthy poria 30g, mountain citron 80g Grams, 10 grams of Houttuynia cordata, 10 grams of Astragalus membranaceus, and 10 grams of Qumai were prepared according to the steps of Example 5 to obtain an extract.

Effect Example 1 Anti-angiogenic effect of the pharmaceutical composition of the present invention

1.1 Experimental drugs

Example 1 The shark cartilage glycoprotein was prepared as a suspension of 200 mg / L in sterilized PBS, the extract obtained in step (2) of Example 5, and the extract of Comparative Example 2 was suspended in 5 g of crude drug / mL using sterilized PBS. liquid.

1.2 Experimental methods

Fresh human umbilical cord 15cm. Wash the umbilical cord's appearance with sterilized PBS and rinse the umbilical cord veins to no residual blood. Ligation one end of the umbilical cord. Inject 2.5 g / L trypsin into the open end of the umbilical cord and digest for 20 min. Shake the umbilical cord every 3 minutes during the period. Once, after digestion is completed, the digestive solution is poured into a 50 mL sterile centrifuge tube, and the umbilical vein is rinsed twice with sterile PBS. The rinse solution is also injected into the sterile centrifuge tube, centrifuged, the supernatant is discarded, and 30 mL of sterile PBS is added and pipetted. After homogenization, centrifuge again, discard the supernatant, add DMEM medium containing 10% newborn bovine serum, add it to a 24-well plate, and place it in a CO ₂ incubator at 37 ° C and 5% humidity for 24 hours. Replace with 10% newborn cattle The serum was further cultured in DMEM medium for 24 hours to obtain primary isolated human umbilical vein endothelial cells.

Primary isolated human umbilical vein endothelial cells were digested with 0.05% trypsin, and then seeded into 96-well plates at a density of 2 × 10 ⁴ cells / mL, 200 μl / well, in a total of 4 groups, specifically blank group, shark cartilage sugar Protein group, Example 5 group, Comparative Example 2 group, each group is provided with 3 parallel wells, placed in a CO ₂ incubator at 37 ° C. and 5% humidity for 24 hours, and added with a test drug 50 μl / well, specifically: a blank group PBS was added, the shark cartilage glycoprotein group was added with a 200 mg / L suspension of shark cartilage glycoprotein, the suspension of the extract obtained in Example 5 was added with 5 g of crude drug / mL in the group of Example 5, and the control group 2 was added with 5 g of crude drug. / mL of a suspension of the extract obtained in Comparative Example 2. After 24 hours of incubation, the supernatant was discarded, and 5 mg / ml MTT reagent 20 μL and 200 μL DMSO were added. After 3 hours, the absorbance value at 570 nm was measured using a microplate reader, and the inhibition rate of human umbilical vein endothelial cells in each group was calculated. The specific results are shown in Table 1. ,among them:

Proliferation inhibition rate = (absorbance value of control group-absorbance value of experimental group) / absorbance value of control group × 100%.

1.3 Experimental results

Table 1 Inhibitory effect of the pharmaceutical composition of the present invention on human umbilical vein endothelial cells

The experimental results in Table 1 show that the extract extract of the pharmaceutical composition of the comparative group 2 without tonka also showed a certain effect of inhibiting the proliferation of human umbilical vein endothelial cells, while shark cartilage glycoprotein has a significant human umbilicus Inhibition effect of venous endothelial cells. The pharmaceutical composition of the group 5 of Example 5 of the present invention shows a more excellent effect of inhibiting the proliferation of human umbilical vein endothelial cells. Its inhibition rate is about 1.7 times that of shark cartilage glycoprotein alone. The inhibitory rate of the control group 2 containing tonka was 2.7 times. The experimental results also suggested that the addition of tonka significantly enhanced the anti-angiogenic effect of the shark cartilage-containing pharmaceutical composition.

Effect Example 2: Growth inhibition effect of the pharmaceutical composition of the present invention on tumor cells

2.1 Experimental drugs

2.2 Experimental methods

Cell lines: HS-4 (human skin cancer cells), MCF-7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), HepG2 (human liver cancer cells), MGC80-3 (human gastric cancer cells)

Take the above cell lines in the logarithmic growth phase and inoculate them into 96-well plates at a density of 2 × 10 ⁴ cells / mL at 200 μl / well in a total of 4 groups, specifically the blank group, the shark cartilage glycoprotein group, the Example 5 group, Comparative example 2 groups, each group is provided with 3 parallel wells, placed in a CO ₂ incubator at 37 ° C. and 5% humidity for 24 hours, and the test drug is added at 50 μl / well, specifically: the blank group is added with PBS and the shark cartilage glycoprotein group A suspension of 200 mg / L of shark cartilage glycoprotein was added, the suspension of the extract obtained in Example 5 was added with 5 g of crude drug / mL in the group of Example 5, and the extract obtained in Comparative Example 2 was added with 5 g of crude drug / mL in the Comparative Example 2 group. Suspension of cream. After 24 hours of incubation, the supernatant was discarded, 20 μL and 200 μL DMSO of 5 mg / ml MTT reagent was added, and the absorbance at 570 nm was measured using a microplate reader after 3 hours to calculate the tumor cell proliferation inhibition rate of each group. The specific results are shown in Table 2, where:

2.3 Experimental results

Table 2 Inhibitory effect of the pharmaceutical composition of the present invention on tumor cell proliferation

Table 2 shows the results of the shark cartilage glycoprotein and the extract of the pharmaceutical composition of Example 5 of the present invention against human skin cancer cells, human breast cancer cells, human lung adenocarcinoma cells, human liver cancer cells, and human gastric cancer cells. All showed obvious proliferation inhibitory effects, especially against human skin cancer cells, human breast cancer cells, and human lung adenocarcinoma cell proliferation inhibition effects, indicating that the pharmaceutical composition of the present invention has excellent tumor cell proliferation inhibition effects, and Comparative group 2 of the same composition, but without tonka, has inhibitory effects on the proliferation of each tumor cell compared to the group of Example 5. The experimental results also prove that the addition of tonka can The combination of shark cartilage-containing drugs significantly inhibited tumor cell proliferation.

Effect Example 3: Therapeutic effect of the external application cream of the present invention on tumor-bearing mice

3.1 Experimental drugs:

Example 4: External application cream.

3.2 Experimental methods

Male Balb / c mice 20 mice weighing 20 ± 2g, adaptive feeding for one day, mice forelimb armpit alcohol disinfectant to 5 × 10 ⁴ cells were seeded at a concentration of mouse melanoma B16 0.2mL, successful inoculation 10 days after inoculation selection Ten mice with approximately the same tumor volume were randomly divided into two groups, specifically the model group and the group of Example 4, with five in each group, in which the model group was coated with the tumor site with physiological saline, and the group of Example 4 would be implemented The external dressing cream prepared in Example 4 was applied to the tumor site once a day. After the application was completed, the coated site was covered with gauze to prevent the coating material caused by the mouse from licking, and the previous application was removed before each application. The drug residue was continuously administered for 15 days, and the number of surviving mice in each group was recorded 2 hours after the last administration. The mice were sacrificed by neck dissection, the tumor tissue was stripped and weighed, and the tumor suppression rate was calculated. The specific experiment is shown in Table 3, where:

Tumor inhibition rate = (tumor weight in model group-tumor weight in experimental group) / tumor weight in model group × 100%.

3.3 Experimental results

Data analysis was performed using the multivariate analysis of variance module of statistical software SPSS. P <0.05 indicated that the difference was statistically significant.

The experimental results in Table 3 show that two of the five mice in the model group have died at the end of the experiment, while all the five mice in the group of Example 4 survived. The tumor growth of the tumor-bearing mice was also significantly inhibited and the tumor was inhibited. The rate exceeded 50%, showing excellent antitumor effect in vivo.

Table 3 Therapeutic effect of the external application cream of the present invention on tumor-bearing mice

^** , P <0.01 compared with the model group.

The preferred embodiments of the present invention have been described above, but they are not intended to limit the present invention. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

Application of shark cartilage glycoprotein in preparing anticancer medicine.
The use according to claim 1, characterized in that the shark cartilage glycoprotein is prepared by the following method:

(1) Shark cartilage particles after shark cartilage crushed through a 10-30 mesh sieve;

(2) Shark cartilage granules are added with 3-8 times purified water, adjusted to pH 10-13 with NaOH, heated to 50-70 ° C, immersed for 3-10 hours and filtered, the filtrate is reserved, and the filter residue is repeatedly extracted 1-2 times The filtrates are combined;

(3) After the filtrate is subjected to salting-out, the Sevage method is used to remove free proteins, and then the filtrate is obtained after dialysis and DEAE-C32 anion interaction resin column chromatography.
An anticancer pharmaceutical composition, characterized in that it comprises shark cartilage glycoprotein or shark cartilage.
The anticancer pharmaceutical composition according to claim 3, further comprising a turtle plate, abalone shell, baking soda, clam shell, portulaca oleracea, beef bone meal, Poria cocos, Shanci mushroom, sage grass, Houttuynia cordata , Astragalus, Qumai, Lingling.
The anticancer pharmaceutical composition according to claim 4, characterized in that the pharmaceutical composition comprises 5-10 parts of shark cartilage, 2-5 parts of turtle plate, 1-3 parts of abalone shell, 1-3 parts of baking soda, 1-3 shells of clam shells, 3-8 shells of purslane, 8-15 shells of beef bone powder, 2-5 shells of Poria cocos, 5-10 shells of Shanci mushrooms, 2-5 shells of misograss, 1-Houttuynia cordata Made from 3 servings, 1-3 servings of astragalus, 1-3 servings of qumai, and 1-3 servings of tonka.
The anticancer pharmaceutical composition according to claim 5, characterized in that the pharmaceutical composition comprises 80 grams of shark bone, 30 grams of turtle plate, 20 grams of abalone shell, 20 grams of baking soda, 20 grams of clam shell, and purslane It is made of 50 grams, 100 grams of beef bone powder, 30 grams of Poria cocos, 80 grams of Shan Ci mushroom, 30 grams of spoiled grass, 10 grams of Houttuynia cordata, 10 grams of astragalus, 10 grams of qumai, and 10 grams of tonka.
The anticancer pharmaceutical composition according to any one of claims 4 to 6, characterized in that the pharmaceutical composition is an oral pharmaceutical composition or a topical pharmaceutical composition.
The anticancer pharmaceutical composition according to claim 7, characterized in that: the oral pharmaceutical composition is a tablet, capsule, granule, pill, powder, decoction; and the topically applied pharmaceutical composition is an external plaster .
The anticancer pharmaceutical composition according to claim 8, characterized in that the external application cream is prepared by the following method:

(1) Crush shark cartilage, tortoise shell, abalone shell, baking soda, clam shell, purslane, beef bone meal, earthy Poria, Shanci mushroom, sauerkraut, Houttuynia cordata, Astragalus, Qumai, Linglingxiang to 150 -200 mesh particles;

(2) Soak the water of Houttuynia cordatum, Tonka fragrant, Shanci mushroom, and rotten grass with sea salt for 48 hours. When using, use this water to shark cartilage, turtle plate, abalone shell, baking soda, clam shell, horse teeth Loquat, beef bone powder, Poria cocos, Astragalus and Qumai granules are prepared into ointments to obtain an external plaster.
Use of the anticancer pharmaceutical composition according to any one of claims 4-9 in the preparation of an anticancer pharmaceutical composition.