CN1911258A - Extractive of parasitic loranthus, prepn. method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine, and provides a mulberry root extract which has a therapeutic effect on human and animal cell proliferation diseases and a preparation method of the extract.

Description

Mulberry extract and its preparation method and application

Technical field:

The invention belongs to the technical field of medicine, and in particular relates to an extract of mulberry plants, which has therapeutic effects on cell proliferation diseases of animals and humans. In addition, the present invention also relates to the preparation method of these extracts.

Background technique:

Sangjisheng is a commonly used traditional Chinese medicine. It has the functions of nourishing liver and kidney, dispelling rheumatism, lowering blood pressure, calming blood and nourishing fetus. There is no report of its anti-tumor effect. Several species of plants in the same family of Morushiaceae, such as Taxillus chinensis, Scurrula parasitica L., Taxillus sutchuenensis Danser, etc., are often used as Chinese medicinal materials.

Malignant tumors are diseases that seriously endanger human health and life. Epidemiological surveys show that there are about 1.6 million new cancer patients in my country every year, and about 1.3 million people die of cancer every year. These numbers have continued to rise in recent years due to an aging population and environmental degradation. Finding natural anti-tumor active substances from plants is an important way to study anti-tumor drugs. Herbal drugs such as paclitaxel, camptothecin, and vincristine are commonly used clinical anti-tumor drugs with good effects.

Invention content:

The object of the present invention is to provide an extract of mulberry parasites whose host is apocynaceae.

Another object of the present invention is to provide methods for the preparation of these extracts from Morus parasites whose host is plants of the family Apocynaceae.

Another object of the present invention is to provide the application of the extract in the treatment of human and animal cell proliferation diseases.

One aspect of the present invention relates to an extract of Mori parasites whose host is an Apocynaceae plant for the treatment of cell proliferative diseases in animals and humans.

Yet another aspect of the present invention relates to the host of the mulberry extract of Apocynaceae as a sensitizer or detoxifier for the treatment of cell proliferation diseases in animals and humans when used in combination with other antineoplastic drugs.

Another aspect of the present invention relates to a pharmaceutical composition for the treatment of animal and human cell proliferative diseases, comprising a combination of the Morus alba extract and other drugs, drug carriers or excipients for the treatment of animal and human cell proliferative diseases. Role in human cell proliferative disorders. Generally, the pharmaceutical composition of the present invention contains 0.1-99.9% by weight of the Mori parasitic extract, and the pharmaceutical composition can be prepared according to methods known in the art. When used for this purpose, the Morus japonica extract can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if necessary, into a suitable form or dosage form for human use. The route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal cavity, oral mucosa, skin, peritoneal or rectal, etc. Dosage forms such as tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, lyophilized powder injections etc., which can be common preparations, sustained-release preparations, controlled-release preparations and various microparticle drug delivery systems. Various carriers known in the art can be widely used in order to prepare unit dosage forms into the above-mentioned various dosage forms. In addition, colorants, preservatives, fragrances, correctives, sweeteners or other materials can also be added to the pharmaceutical preparations, if necessary.

The present invention also relates to a preparation method of the Morus sativa extract for treating animal and human cell proliferative diseases.

The mulberry parasite referred to in the present invention refers to any plant derived from Loranthaceae (Loranthaceae) Taxillus Van Tiehg., Scurrula Linn. or Loranthus Jacq, It is preferably any plant of the genus Pymeparasitic, most preferably the safflower mulberry parasite (Scurrula parasitica L.) of the genus Pymeparasitic, and the most fundamental feature they have in common is that their host is an Apocynaceae plant, preferably The most popular are oleander (Nerium indicum) and yellow phlox (Thevetia peruviana). For scientific definitions and categories of Loranthaceae, Taxillus Van Tiehg., Scurrula Linn. or Loranthus Jacq, and Apocynaceae, see Chinese Dictionary of Higher Plants (Revised Edition) (Edited by Hou Kuanzhao, Beijing: Science Press, 1984, 12) and "Flora of China" Volume 24 (Beijing: Science Press, 1986). The parts of medicinal materials used are generally leaves, branches, stems, flowers and fruits.

The host is the preparation method of the extract of mulberry parasites on Apocynaceae plants:

(1) The picking host is the mulberry medicinal material on the Apocynaceae plant, which is washed, dried or dried, then pulverized, heated with a polar or non-polar solvent or extracted at room temperature, and the preferred extraction solvent is 60-80% ethanol Or other alcoholic solvents of the same concentration, the preferred extraction temperature is in the range of 60-80°C; 1-10 times of extraction, the preferred number of extraction times is 3-5 times, and the extraction time of each time is generally 1-4 hours for heating and extraction Or longer, normal temperature extraction time is generally 1 day or longer each time. The medicinal material can also be extracted with an organic solvent (such as petroleum ether, benzene, toluene or ethane, etc.), filtered, the filtrate is discarded and the filter residue is retained to remove fat-soluble substances such as pigments, and the filter residue is extracted by the above method.

(2) Adjust the filtrate to contain about 50-70% alcohol, and put it in a 4°C refrigerator overnight or longer to precipitate the pigment and oil; filter to remove the pigment and oil; and then extract with petroleum ether to further remove the pigment and oil. The solvent was recovered from the filtrate and concentrated to obtain the extract.

(3) The extract is mixed into a paste with polyamide powder or silica gel powder, dried to remove moisture, ground into a fine powder, purified through a polyamide column (the preferred polyamide particle size is 100-200 mesh), water, 10%- The 90% gradient alcohol solution is eluted, the eluate is collected, the solvent is recovered and concentrated to obtain an extract, and the extract is further dried to remove water to obtain the desired extract.

(4) When concentrating in step (2), also can only be concentrated into thicker aqueous solution, and the concentrated solution at this moment also can directly pass polyamide column purification.

After component analysis, the 30-90% alcohol eluate mainly contains flavonoids (more than 50% flavonoids), cardiac glycosides and alkaloids and other chemical components; water and 10% alcohol eluate contain polysaccharides (containing sugar 56% or more), glycoproteins and peptides and other chemical components.

The composition feature of the extract obtained in the present invention is that it contains cardiac glycosides, while the Morus sativa extract that parasitizes on other hosts (such as mulberry, longan, osmanthus, etc.) does not contain cardiac glycosides. This type of cardiac glycoside composition is the same as the cardiac glycoside composition contained in the host Apocynaceae plant, because it was detected and analyzed by the 1100 type high performance liquid chromatography-ion trap mass spectrometer (LC-MSD Trap) of Agilent Company, from The cardiac glycosides extracted from the leaves of Safflower mulberry are different from those extracted from the leaves of its host oleander.

The mulberry extract prepared by the above method parasitized on the Apocynaceae plant was found to be a broad-spectrum high-efficiency and low-toxic anti-tumor component through in vivo and in vitro pharmacodynamic experiments. In vitro, it has strong cytotoxic activity against various tumor cells (such as leukemia, lymphoma, liver cancer, lung cancer, cervical cancer, nasopharyngeal carcinoma, etc.), and its IC ₅₀ is generally 0.1-1 μg/ml; /ml concentration also has no cytotoxic activity. It inhibits the growth of various tumors in vivo, and has a strong sensitization and attenuation effect on other clinically used antitumor drugs (such as doxorubicin).

Description of drawings:

Figure 1 The inhibitory rate of different concentrations of Safflower mulberry extracts on NB4 cell lines.

Figure 2 The inhibitory rate of different concentrations of Safflower mulberry extracts on Jurtet cell lines.

Fig. 3 Inhibition rate of U266 cell line by different concentrations of Safflower mulberry extracts.

Fig. 4 The inhibitory rate of different concentrations of Safflower mulberry extracts on HL-60 cell line.

Fig. 5 Inhibitory rate of different concentrations of safflower mulberry extracts on K562 cell line.

Fig. 6 The inhibitory rate of different concentrations of safflower mulberry extracts on NCI-H446 cell line.

Fig. 7 Inhibition rate of different concentrations of safflower mulberry parasite extracts on chronic myeloid primary cells.

Fig. 8 Inhibition rate of different concentrations of Safflower mulberry extracts on Hela cell line.

Fig. 9 Inhibition rate of different concentrations of safflower mulberry extracts on CNE cell lines.

Fig. 10 Inhibition rate of different concentrations of Safflower mulberry extracts on 293T cell line.

Fig. 11 Changes in light absorption value (OD value) of 293T cell line treated with different concentrations of Safflower mulberry extract.

Fig. 12 Changes in light absorption value (OD value) of NCI-446 cell line treated with different concentrations of Safflower mulberry extract.

Figure 13 The DNA ladder pattern of HL-60 cell line treated with different concentrations of safflower mulberry extract for 24 hours

In Figure 13: 1-negative control; 2-1 μg/mL group; 3-0.5 μg/mL group; 4-0.25 μg/mL group

Detailed ways:

The following examples and biological activity experiments are used to further illustrate the present invention.

Embodiment 1: adopt heating reflux extraction method to prepare the safflower mulberry parasitism (Scurrula parasitica L.) branches and leaves extract that parasitizes on oleander (Nerium indicum)

(1) Pick the leaves and twigs of safflower mulberry parasitic on oleander, wash, cool (or dry) and dry, crush them, and pass through a 60-mesh sieve. Take 1000 grams of coarse powder and add 15 times of 80% ethanol for three reflux extractions, each time for 4 hours, and the extraction temperature is 65°C. Add water until the alcohol content is about 70%, put it in the refrigerator (4°C) overnight, filter to remove the precipitate, and then extract the filtrate with petroleum ether to further remove pigment and grease. Take the ethanol solution to recover ethanol under reduced pressure, and concentrate to extract.

(2) The polyamide powder for the extract is mixed in a ratio of about 1:1, dried at 60°C, ground into powder, passed through a polyamide column (100-200 mesh), and mixed with distilled water, 10% ethanol, 30% ethanol , 50% ethanol, 70% ethanol and 90% ethanol for step-by-step gradient elution, and when the eluent of each gradient is eluted to basically no color, change to the next gradient. Concentrate under reduced pressure respectively. Distilled water and 10% ethanol eluate are viscous large colloidal substances, mainly containing polysaccharides (about 56%) through component detection, and also containing glycoproteins, polypeptides, amino acids and other substances; while 30%, 50%, 70% and The 90% ethanol eluate mainly contains flavonoids (about 54%), and also contains cardiac glycosides, alkaloids and other substances. The above-mentioned eluates are mixed in different proportions or used alone, and all are the Morus parasite extracts referred to in the present invention.

Embodiment two: the safflower mulberry (Scurrula parasitica L.) branches and leaves extract parasitic on oleander (Nerium indicum) is prepared by heating reflux extraction method

(1) Pick the leaves and twigs of safflower parasitic on oleander, wash, cool (or dry) and dry, then pulverize, and pass through a 60-mesh sieve. Take 1000 grams of coarse powder and add 15 times of 80% ethanol to reflux three times, each time for 4 hours, and the extraction temperature is 65°C. Add water until the alcohol content is about 70%, put it in the refrigerator (4°C) overnight, filter to remove the precipitate, and then extract the filtrate with petroleum ether to further remove pigment and grease. Take the ethanol solution and recover the ethanol under reduced pressure. During this process, as the concentration of ethanol in the concentrated solution gradually decreases, there will be continuous precipitation, and the precipitation will be separated and taken out; continue to concentrate to extract.

(2) The polyamide powder for the extract is mixed in a ratio of about 1:1, dried at 60°C, ground into powder, passed through a polyamide column (100-200 mesh), and mixed with distilled water, 10% ethanol, 30% ethanol , 50% ethanol, 70% ethanol and 90% ethanol step by step gradient elution, each eluent is eluted to the next gradient when there is almost no color. Concentrate under reduced pressure respectively. Distilled water and 10% ethanol eluate are viscous large colloidal substances, which mainly contain polysaccharides (about 56%) after testing, and also contain glycoproteins, polypeptides, amino acids and other substances; while 30%, 50%, 70% and 90% The ethanol eluate mainly contains flavonoids (about 54%), and also contains cardiac glycosides, alkaloids and other substances. The above-mentioned eluates are mixed in different proportions or used alone, and all are the Morus parasite extracts referred to in the present invention.

Embodiment three: adopt the percolation extraction method to prepare the safflower mulberry (Scurrulaparasitica L.) branches and leaves extract parasitic on oleander (Nerium indicum)

(1) Pick the leaves and twigs of safflower parasitic on oleander, wash, cool (or dry) and dry, then pulverize, and pass through a 60-mesh sieve. Take 3000 grams of coarse powder, put it in the percolation cylinder, add 80% ethanol to soak for 10 days, release the percolation liquid at a rate of about 1mL per minute, add 80% ethanol at any time to keep the medicinal materials completely soaked in the ethanol solution, until the percolation The extraction was considered complete when the filtrate was substantially colorless. After recovering part of the ethanol in the extract under reduced pressure, the alcohol content is adjusted to about 70%. Other steps are with embodiment 2.

Example 4: Pharmacodynamic study of safflower parasitic extract combined with doxorubicin against transplanted tumor of human acute leukemia

1. Experimental materials:

Tumor strain: Highly tumorigenic human promyelocytic leukemia cell line HL-60 was obtained from Institute of Hematology, Union Hospital of Fujian Province. Nude mice: BALB/c, nu/nu, age 6-8w, body weight 18-22g, purchased from The Shanghai Experimental Animal Center of the Chinese Academy of Sciences was raised in a constant temperature (25-270C), constant humidity (45%-50%) SPF air laminar flow cabinet, and the males and females were reared separately. Free intake of food and water, drinking water, feed, bedding and other items in contact with nude mice were all autoclaved. Adriamycin (ADR), product of Shanghai Hualian Pharmaceutical Co., Ltd., batch number 04-04-30.

2. Experimental method:

(1) Take HL-60 cells in the logarithmic growth phase to make a cell suspension, adjust the cell concentration to 1×107ml-1, and inoculate 0.2ml subcutaneously on the dorsal side of the right hind limb of each nude mouse. On the 5th day after inoculation, when the tumor grows to about the size of mung bean, the drug is given for treatment. Nude mice were weighed, tumor diameters were measured, and randomly divided into 5 groups, with 5 animals in each group. Observe and record the diet and activity status of the nude mice and the growth of the transplanted tumor every day, measure the body weight of the nude mice with a precision electronic balance every 2 days, and conduct a preliminary evaluation of drug toxicity, that is, final body weight (FBW)/initial body weight (IBW) ) ≥ 0.8 means no toxic reaction, <0.8 means toxic reaction. Measure the tumor volume every week, measure the long diameter (a) and short diameter (b) of the tumor with a vernier caliper, calculate the tumor volume according to the formula V=ab2/2, and draw the tumor volume growth curve. The nude mice were killed by dislocation of the neck on the second day after the treatment, the tumors were taken and weighed, and the tumor inhibition rate was calculated. Tumor inhibition rate (IR)=(1-average tumor weight of experimental group/average tumor weight of control group)×100%. The formula for calculating the Drug Interaction Index (CDI) is: CDI=AB/(A×B). In this experiment, AB is the ratio of the tumor weight of the two-combination group to the control group, and A or B is the ratio of each drug alone. The ratio of tumor weight to that of the control group. When CDI<1, the two drugs have synergistic effect, and when CDI<0.75, the synergistic effect is very significant.

Grouping: ① normal saline group: intraperitoneal injection of normal saline containing 1% DMSO; ② ADR group: 1.5 mg/ml doxorubicin; ③ extract intraperitoneal group: intraperitoneal injection of 1 mg/ml safflower mulberry extract; ④ extract tumor Weekly group: injection of 1 mg/ml safflower mulberry extract around the tumor ⑤ extract+ADR group: intraperitoneal injection of 1 mg/ml safflower mulberry extract, tail vein injection of 1.5 mg/ml doxorubicin. The injection dose was 0.1ml/10g body weight. Doxorubicin was injected into the caudal vein at one time, and safflower mulberry extract and normal saline were injected intraperitoneally once a day for a total of 14 days.

(2) Pathological examination: At the end of the experiment, the animals were dissected, and visceral lesions were observed with the naked eye; the heart, liver, kidney, and spleen were taken out, rinsed with normal saline, and fixed in 10% formaldehyde solution for 24 hours for pathological examination.

3. Experimental results:

(1) After drug treatment, the FBW/IBW value of each group was >0.8, showing no toxic reaction. (Table 1)

Table 1 drug toxicity evaluation table Drug Toxicity Evaluation saline group ADR group extract celiac group extract peritumoral group Extract + ADR group Initial Body Weight (IBW) 22.3±1.7 22.6±1.2 23.1±1.5 19.7±1.1 20.2±0.6 Final Body Weight (FBW) 30.1±4.8 25.7±2.1 28.9±2.4 24.8±3.2 20.8±0.7 FBW/IBW 1.35 1.13 1.24 1.25 1.02

(2) Changes in volume of subcutaneous transplanted tumors in HL-60 nude mice (Table 2):

Table 2 Changes in the volume of subcutaneous transplanted tumors in HL-60 nude mice (unit: cm ³ ) group 0 days 7 days 14 days control group 0.025±0.026 1.267±0.776 8.939±2.942 ADR group 0.028±0.025 0.197±0.111 3.527±1.822 extract celiac group 0.024±0.033 0.889±0.517 7.041±3.964 extract peritumoral group 0.035±0.017 0.237±0.144 5.085±3.094 Extract + ADM group 0.037±0.018 0.026±0.016 0.767±0.539

Note: 0 day means the time of starting administration

It can be seen that all the extract treatment groups significantly inhibited tumor growth on the seventh day, but the intraperitoneal injection group had a poor inhibitory effect on the late tumor growth; and the peritumoral injection also had a certain inhibitory effect on the late tumor growth. The extract combined with doxorubicin can significantly inhibit tumor growth.

(3) Weight changes of subcutaneous transplanted tumors in HL-60 nude mice (Table 3):

Table 3 Changes in the weight of subcutaneous transplanted tumors in HL-60 nude mice group Number of animals (only) Change in body weight after removal of tumor tumor weight Tumor inhibition rate P CDI beginning/end (g) (x±s, g) (%) negative control group 5/5 +1.394 7.52±1.36 ADR group 5/5 +0.532 2.57±1.38 65.8 0.001<0.01 ^* extract celiac group 5/5 +0.360 5.48±2.46 27.1 0.184>0.05 ^* extract peritumoral group 5/5 +1.510 3.55±2.29 52.8 0.019＜0.05 ^* Extract + ADR group 5/4 +0.145 0.46±0.23 93.9 0.002＜0.01 ^* 0.026＜0.05 ^** 0.009＜0.01 ^*** 0.24

Note: ^* The experimental group is compared with the control group (normal saline group), ^** The experimental group is compared with the doxorubicin single use group, ^*** The experimental group is compared with the intraperitoneal injection group.

The antitumor rate of 10 mg/kg intraperitoneal injection of Safflower mulberry extract was 27.1% (compared with the control group, P>0.05), and there was no significant difference with the control group. However, this dose of peritumoral injection has a significant tumor-inhibiting effect, and the tumor-inhibiting rate is 52.8% (compared with the control group, P<0.05). This dose combined with doxorubicin can significantly enhance the tumor inhibitory effect of doxorubicin. In the experiment, the tumor inhibition rate of the ADR15mg/kg single drug group was 65.8% (compared with the control group, P<0.05), and the combined use of the two drugs The inhibition rate was 93.8%, compared with the normal saline group and intraperitoneal injection group, P<0.01, the difference reached a very significant level; compared with the doxorubicin group, there was also a significant difference, P<0.05, the two drugs had a significant synergistic effect CDI=0.24 <0.75.

(4) There was no obvious change in the viscera of each treatment group; the pathological section showed that there were lesions in the heart tissue of the doxorubicin treatment group alone, the cardiomyocytes were enlarged, and the cells were arranged in disorder, while the cardiomyocytes in the group treated with Sangjisheng were normal.

Example 5: In vivo anti-mouse sarcoma S180 research of safflower parasitic extract

1. Experimental materials:

The mouse sarcoma S180 ascites type was obtained from the College of Pharmacy, Fujian Medical University. Kunming mice, weighing 18-22 g, were 6-8 weeks old, male and female, and were purchased from the Experimental Animal Center of Fujian Medical University. Adriamycin (ADR), product of Shanghai Hualian Pharmaceutical Co., Ltd., batch number 04-04-30.

2. Experimental method:

2.1 Drug preparation: 30% alcohol eluate powder, 50% alcohol eluate powder, 70% alcohol eluate powder each 100mg are mixed in a ratio of 1:1:1, diluted with normal saline to 10.0mg/ml stock liquid. Weigh 5 g of the distilled water eluate powder of Safflower mulberry and dissolve it in 200 ml of normal saline to form a 25.0 mg/ml distilled water eluate group.

Negative control group (A): normal saline;

Positive control group (B): 1.5mg/ml doxorubicin, one-time intraperitoneal injection;

Ethanol eluate group (C): dilute the stock solution to 1.0 mg/ml with physiological saline;

Ethanol eluate + distilled water eluate group (D): Dilute the stock solution with distilled water eluate to contain (1.0 mg ethanol eluate + 25.0 mg distilled water eluate) per ml;

Distilled water eluate group (E): 25.0 mg/ml distilled water eluate.

2.2 Experimental method: The ascites of ascites type sarcoma SI80 that had been passaged for 7 days was taken, diluted with sterile normal saline at a ratio of 1:3 to form a tumor cell suspension, and inoculated into the peritoneal cavity of mice, 0.2ml per mouse. On the next day after inoculation, they were randomly divided into 5 groups of A, B, C, D, E, and 10 rats in each group. They were administered by intraperitoneal injection at a volume of 0.1 ml per 10 g body weight, once a day, for 14 consecutive days. The survival days of the mice were counted (if more than 28 days were counted as 28 days), the average survival time of each group was calculated to obtain the life extension rate.

2.3 Experimental results: See Table 4.

As can be seen from the results, the life extension rates of the positive control group, the ethanol eluate group, the distilled water eluate group, the ethanol eluate+distilled water eluate group, and the S180 mice were 25.5%, 24.0%, respectively. 23.0%h and 43%. The distilled water eluate group has the effect of improving the life extension of mice. The mixed use of distilled water eluate and ethanol eluate has a very significant effect on the life extension of mice, and it has stronger anti-mouse S180 in vivo than the two alone. Sarcoma effect.

Table 4 The effect of safflower mulberry extract on the life extension rate of tumor-bearing mice group number of animals Survival time (d) Life extension rate (%) ABCDE 1010101010 16.5±1.2 0.7±4.7 ^* 20.5±3.6 ^* 23.6±4.0 ^** 20.3±4.6 ^* -25.524.043.023.0

Note: Compared with the control group, ^* p<0.05, ^** p<0.01

Example 6: Effect of Safflower Morus parasitic Extract on the Colony Formation Rate of NB4 and Jurkat Cells

Tumor lines are composed of tumor cell populations with different proportions, proliferation and differentiation abilities. Only a small fraction of them has the ability to self-renew, the so-called stem cells, about 1% of the entire cell population. Stem cells are the target cells of radiation therapy and anticancer drug therapy, and are closely related to the cure, recurrence or metastasis of tumors. It is generally believed that only stem cells have the ability to differentiate and form colonies. Therefore, the colony assay can be used as one of the methods for detecting the susceptibility of anticancer drugs in vitro.

1. Experimental materials: methyl cellulose was purchased from sigma, and others were the same as in Example 6.

2. Experimental method:

(1) Cell suspension is prepared, and the culture medium is RPMI1640 culture medium containing 20% fetal bovine serum. Count and dilute to 0.45ml containing about 300 cells.

(2) Inoculate cells: Add 0.45ml of 1.6% methylcellulose (prepared with 1640) to each well of a 24-well plate to drive away air bubbles; then add the cell suspensions of NB4 and Jurkat prepared in step 1, 0.45ml ml, about 300 cells.

(3) Dosing: Add 0.1ml of drug to each well of the suspended cells, with 6 drug concentrations, set up two replicate wells, and set up a parallel control group without drug.

(4) Culture in an incubator at 37° C., 5% CO 2 , and saturated humidity for 7 days, and count the number of CFU cells (≥30 as a colony). The colony inhibition rate was calculated according to the formula, namely: colony inhibition rate=(1-T/C)×100%.

3. Experimental results:

Table 5 Safflower mulberry extracts inhibit the formation of tumor cell colonies group Number of colonies in drug-dosed group (units) The number of colonies in the control group (units) colony formation inhibition rate NB4 cells 5μg/ml2.5μg/ml1μg/m0.5μg/ml0.25μg/ml0.1μg/ml 0011926 484745752987 100% 100% 97.78% 98.67% 68.97% 70.12% Jurkqat cells 5 μg/ml 2.5 μg/ml 1 μg/m 0.5 μg/ml 0.25 μg/ml 0.1 μg/m 0112423 323845564238 100% 97.37% 97.78% 96.43% 91.00% 39.47%

Compared with the MTT experiment, the same concentration of drugs has a higher inhibition rate on colony formation, indicating that the extract of Safflower mulberry is more sensitive to the effect of proliferating cells in tumor cells.

Example 7: Study on Inhibition of Various Human Tumor Cells in Vitro by Safflower Sangpara Extract

1. Experimental materials: human tumor cell lines: NB4 (human acute promyelocytic leukemia cell line M3), Jurket (human T-lymphoblastic leukemia), U266 (human myeloma cell line), HL-60 (human acute promyelocytic leukemia cell line), HL-60 (human acute promyelocytic leukemia cell line) Myeloid leukemia cell line M2), K562 (human chronic myeloid leukemia cell line), NCI-H446 (human lung cancer cell line), Hela (human cervical cancer cell line), CNE (human nasopharyngeal carcinoma cell line), blood from Fujian Province Disease Research Institute; primary cultured chronic myeloid leukemia cells were obtained from the bone marrow of chronic myeloid patients; RPMI1640 culture medium (GIBCO), fetal bovine serum (Hangzhou Sijiqing), MTT (sigma), safflower mulberry extract.

2. Experimental method: MTT method, take the cultured cells in the logarithmic growth phase and adjust the density to 1×104～2×104 living cells per 180 μl, inoculate them in 96-well culture plates (3 replicate wells for each group, repeat 3 times, take the average value), the suspension cells can be added after inoculation, and the adherent cells can be cultured for one day, after the cells adhere to the wall, the drug is added, and 20 μl of drug is added to each well, and a no-drug control group and a blank control group are set. , and then placed in a CO2 incubator at 37°C, 5% CO2, and saturated humidity for 24h, 48h, 72h, 96h, and 120h, respectively, and then added 20μl MTT, cultured for 4h, removed the supernatant, added 150μlDMSO, and placed in a microplate reader (StatFax-2100) was shaken for 3-4 minutes, and the OD value was measured with dual wavelengths of 492nm and 630nm, and the inhibition rate was calculated. Inhibition rate=(1-OD value of experimental group/OD value of untreated group)×100%.

3. Experimental results: As shown in Figures 1 to 9, the extract of Safflower mulberry has strong anti-proliferation and killing effects on human tumor cell lines and primary cultured chronic myelogenous leukemia cells, and has a strong anti-proliferation and killing effect on various tumors. The cell lines all have good effects, and it is a broad-spectrum antitumor drug. Microscopic observation revealed that most of the cells had increased in size, and a large number of vacuoles could be seen in the cytoplasm. Some cells shrink in size, the cells show budding changes, the nuclei are condensed, and the cell morphology is intact. Using DNA ladder detection, it was found that the killing effect of the safflower mulberry extract on tumor cells was to induce apoptosis ( FIG. 13 ).

Example 8: Study on the effect of the extract of Safflower mulberry parasite on human normal cells in vitro

1. Experimental materials: normal human embryonic kidney fibroblast cell line 293T, from the School of Life Sciences, Fujian Normal University; other colleagues

Example 6.

2. Experimental method: with embodiment 6.

3. Experimental results: the safflower mulberry extract also has inhibitory effect on the proliferation of normal human embryonic kidney fibroblast cell line 293T in vitro (Figure 10), but it has no killing effect, and the cell activity is normal, but it cannot adhere to the wall. Even after treatment with a drug concentration as high as 50 μg/ml, the light absorption value remained basically unchanged (Figure 11), it can be seen that the cells basically did not die or proliferate, while tumor cells such as lung cancer cell line NCI-446 were treated with 2.5 μg/ml When the drug concentration is treated, the light absorption value also decreases (Figure 12), indicating that there is a killing effect. The above results show that the extract of Safflower mulberry has little effect on normal cells and has low cytotoxicity.

Claims (14)

1. A mulberry extract, characterized in that it contains cardiac glycosides, and the host of the mulberry plant is Apocynaceae. 2. The mulberry extract according to claim 1, wherein said mulberry refers to a plant of the family Morus. 3. The mulberry parasite extract according to claim 1, wherein said Apocynaceae plant refers to Nerium indicum and Nerium indicum (Thevetia peruviana). 4. The mulberry parasite extract according to claim 1, characterized in that the chemical composition of the extract contains flavonoids. 5. The mulberry parasite extract according to claim 1, characterized in that the chemical composition of the extract contains polysaccharide compounds. 6. the mulberry parasitism extract as claimed in claim 2, is characterized in that wherein said mulberry parasitica plant refers to safflower mulberry parasitica (Scurrula parasitica L.), mulberry parasitism (Taxillus chinensis) and refers to Sichuan parasitism (Taxillus sutchuenensis Danser) . 7. prepare the method for the mulberry parasite extract described in claim 1, it is characterized in that the method comprises the following steps: (1) The medicinal materials are washed and dried, crushed, extracted with solvent for 1 to 10 times, and the filtrate is filtered; the medicinal materials can be extracted with an organic solvent first, filtered, and the filtrate is discarded to retain the filter residue to remove fat-soluble substances such as pigments. method extraction; (2) Adjust the filtrate to about 50-70% alcohol, put it in the refrigerator for more than one hour to precipitate the pigment and oil, and filter to remove the pigment and oil; then extract with petroleum ether to further remove the pigment and oil; take the filtrate to recover the solvent ,concentrate; (3) The concentrated solution is purified through a polyamide column, eluted with water and 10% to 90% gradient alcohol solution, the eluate is collected, the solvent is recovered and concentrated, and the concentrated solution is further dried to remove water to obtain the mulberry extract. 8. The method for preparing the Mori parasitic extract according to claim 7, wherein said solvent refers to 1% to 99% alcohol solvent. 9. The method for preparing the Mori parasitic extract according to claim 8, wherein said alcoholic solvent refers to 1-99% ethanol. 10. The method for preparing the Morus parasite extract according to claim 7, wherein said solvent refers to water. 11. The method for preparing the Mori parasitic extract according to claim 7, wherein said drying method refers to freeze-drying, spray-drying, and evaporation-drying. 12. The use of the Mori parasitic extract according to claim 1, characterized in that it has a therapeutic effect on cell proliferative diseases of humans and animals. 13. the purposes of the mulberry parasite extract as claimed in claim 12, it is characterized in that wherein said therapeutic effect refers to the therapeutic effect when using this medicine alone, when being used as the antitumor effect of sensitizer sensitization other medicines Effect, therapeutic effect as a composition of an active ingredient with other drugs, drug carriers or excipients. 14. The use of the mulberry root extract according to claim 12, characterized in that the therapeutic effect refers to reducing the toxicity of other anti-tumor drugs when used in combination with other anti-tumor drugs.

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