Application of beautiful millettia root polysaccharide in preparation of synergist of tumor treatment drug

Abstract

The invention discloses a novel application of beautiful millettia root polysaccharide as a synergist of an anti-tumor drug, and particularly relates to a combination of the beautiful millettia root polysaccharide and the anti-tumor drug paclitaxel, so that the anti-tumor effect of the paclitaxel can be improved. The invention further discloses a preparation method of the beautiful millettia root polysaccharide, and a detection method of the antitumor effect of the beautiful millettia root polysaccharide. The preparation method comprises the following steps: boiling a beautiful millettia root medicinal material, filtering, concentrating and centrifuging to obtain a first supernatant and a first precipitate; adding a proper amount of water into the first precipitate for dissolving, adjusting the pH to 2-4 by using hydrochloric acid, and centrifuging again to obtain a second supernatant; combining the first supernatant and the second supernatant, adding NaOH to adjust the pH value to 6-7, performing vacuum concentration, adding anhydrous 90% ethanol for precipitation, and performing suction filtration to obtain a second precipitate; washing the second precipitate with a solvent to obtain crude extract beautiful millettia root polysaccharide; finally, step-by-step elution is conducted through an ion exchange column, and purified beautiful millettia root polysaccharide is obtained after vacuum drying.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a new use of a polysaccharide derived from a plant of the genus Millettia in Fabaceae as an antitumor drug synergist, and also relates to a preparation method of polysaccharide of Millettia speciosa Champ. and a detection method for its antitumor activity. Background Art

[0002] China is extremely rich in natural resources and has unique advantages. The number of plant varieties identified so far ranks third in the world, second only to Brazil and Indonesia. Coupled with the precious experience of traditional Chinese medicine and folk medicine left by our ancestors for thousands of years, the success rate of developing new drugs from them is relatively high, and the cost can be significantly reduced. Developing and utilizing natural drugs is a unique advantage of China. Based on the original medicinal functions of traditional Chinese medicine, guided by a multi-index activity screening system, modern research means are used to extract, separate, identify the structure and study the pharmacological effects of the active ingredients of natural drugs. Then, appropriate pharmacological models are selected to conduct further experiments, evaluate the activity and explore the mechanism of action, and find the action targets. Combining the drug structure and the mechanism of action organically to clarify its activity is conducive to discovering clinically useful prototype drugs and laying a foundation for new drug research and development.

[0003] Millettia speciosa Champ. is the dried root of Millettia speciosa Champ. in the genus Millettia of Fabaceae Papilionoideae. It is mild in nature, sweet in taste, and belongs to the lung, spleen, and kidney meridians. Millettia speciosa Champ. has a long history of medicinal use. As early as in "Herbal Medicine Properties", it was recorded that it "strengthens tendons and activates collaterals, tonifies deficiency and moistens the lungs. Treats low back pain, rheumatic arthralgia, chronic hepatitis, and pulmonary tuberculosis." "Luchuan Materia Medica" also recorded that it "clears the lungs and relieves cough, cools and detoxifies. Treats hemoptysis, dysentery, fever and thirst in febrile diseases, and dizziness." Clinically, it has been proven to have therapeutic effects on a variety of chronic diseases, mainly used to treat low back pain, kidney deficiency leukorrhea, rheumatoid arthritis, lumbar muscle strain, chronic hepatitis, and weakness after illness. As early as in the 1970s, Millettia speciosa Champ. was used as a raw material for the production of Chinese patent medicines such as Zhuangyao Jianshen Pills and Qiangli Jianshen Capsules. The main chemical component of Millettia speciosa Champ. is polysaccharide of Millettia speciosa Champ. Modern pharmacological studies have found that it has functions of improving immune function, anti-fatigue, antioxidant, anti-inflammatory, expectorant, antitussive, anti-asthmatic, and liver protection. Only the activity against cervical cancer has been reported for its antitumor effect (Zheng Yuansheng. Extraction and Pharmacological Activity Research of Polysaccharide of Millettia speciosa Champ. [D]. Jinan University, 2009).

[0004] Millettia speciosa Champ. is a well-known medicinal and edible plant in Guangdong and Guangxi regions. In order to make full use of this genuine medicinal material resource, and since common tumor drugs and treatment methods such as radiotherapy and chemotherapy have good effects but large side effects, the inventor intends to further explore more antitumor biological activity uses of Millettia speciosa Champ.

[0005] Chinese Patent No. 201911238835.X discloses a polysaccharide from Millettia speciosa Champ. and its preparation method and application in antibacterial aspects. The method includes the following steps: (1) cleaning, slicing, drying, pulverizing and sieving the root tubers of Millettia speciosa Champ., and then performing hot water reflux extraction to obtain a crude extract of polysaccharide from Millettia speciosa Champ.; (2) removing pigments from the crude extract of polysaccharide from Millettia speciosa Champ. with macroporous resin, removing residual proteins with Sevag reagent, and then performing alcohol precipitation to obtain crude polysaccharide from Millettia speciosa Champ.; (3) dissolving the crude polysaccharide from Millettia speciosa Champ. in water to prepare a crude polysaccharide solution of Millettia speciosa Champ., then adding it to a DEAE-52 cellulose anion exchange column, and eluting successively with distilled water and 0.2 mol / L NaCl solution, and collecting the eluates respectively to obtain polysaccharide MSCP1 and MSCP2 of Millettia speciosa Champ. The prepared polysaccharide from Millettia speciosa Champ. has a certain inhibitory effect on Gram-negative bacteria, especially Escherichia coli and Salmonella. Therefore, it can only be used for developing antibacterial materials or antibacterial agents. Summary of the Invention

[0006] On the one hand, the present invention provides an application of a polysaccharide from Millettia speciosa Champ. in preparing a synergist for anti-tumor drugs.

[0007] Further, the synergist for anti-tumor drugs refers to the combination of the above-mentioned polysaccharide from Millettia speciosa Champ. and the anti-tumor drug paclitaxel, which can enhance the anti-tumor effect of paclitaxel.

[0008] Further, the above-mentioned drug includes polysaccharide from Millettia speciosa Champ. and pharmaceutically acceptable acids, bases, salts, hydrates or esters and other excipients.

[0009] Further, the above-mentioned tumors are ovarian cancer, lung cancer and breast cancer.

[0010] On the other hand, the present invention provides a method for preparing the above-mentioned polysaccharide from Millettia speciosa Champ., including the following steps:

[0011] a. After drying and pulverizing the Millettia speciosa Champ. medicinal materials, boiling, filtering and concentrating, adding 0.3 mol / L hydrochloric acid to adjust the pH to 2-4, and centrifuging to obtain a first supernatant and a first precipitate;

[0012] b. Dissolving the first precipitate obtained by centrifugation in step a in an appropriate amount of water, and then using 0.3 mol / L hydrochloric acid to adjust the pH to 2-4, and centrifuging again to obtain a second supernatant;

[0013] c. Combining the first supernatant and the second supernatant, and adding NaOH to adjust the pH to 6-7;

[0014] d. After concentrating under reduced pressure, adding anhydrous -90% ethanol for precipitation, and filtering to obtain a second precipitate;

[0015] e. Washing the second precipitate with acetone, ether and anhydrous ethanol to obtain the crudely extracted polysaccharide from Millettia speciosa Champ.;

[0016] f. Dissolve the crude polysaccharide of Millettia speciosa Champ. obtained in step e in water, perform stepwise elution through an ion exchange column, and obtain purified polysaccharide of Millettia speciosa Champ. after vacuum drying. Preferably, the ion exchange column is a DEAE Sephadex A-25 ion exchange column; elute with a 1.5 mol / L NaCl solution, and use the purified polysaccharide of Millettia speciosa Champ. obtained after elution for subsequent experiments.

[0017] Preferably, the pH in steps a and b is adjusted to 3.

[0018] Furthermore, the concentration of NaOH in step c is 0.05 mol / L - 0.3 mol / L. Preferably 0.1 mol / L.

[0019] Another aspect of the present invention provides a method for detecting the anti-tumor activity of the above-mentioned polysaccharide of Millettia speciosa Champ., including:

[0020] a. Inoculate tumor cells into fresh medium, culture until the logarithmic growth phase, and prepare a cell suspension, then inoculate it into a multi-well plate;

[0021] b. After the cells adhere to the wall, replace the medium with a medium containing polysaccharide of Millettia speciosa Champ. at different concentrations, and continue to culture for 24 hours, 48 hours or 72 hours;

[0022] c. Replace the medium in step b with a medium containing CCK-8, continue to culture and then detect the absorbance value;

[0023] d. Calculate the influence of the polysaccharide of Millettia speciosa Champ. on the proliferation activity of tumor cells at different time periods, and determine the IC50 value.

[0024] Furthermore, the final concentration of the above-mentioned CCK-8 is 8% - 12%, and detect the absorbance value at a detection wavelength of 450 nm. Preferably, the final concentration of CCK-8 is 10%.

[0025] Furthermore, the above-mentioned tumor cells are ovarian cancer cells Caov-3, lung cancer cells A549 and breast cancer cells MCF7.

[0026] The present invention investigated the pharmacodynamics of the synergistic anti-tumor effect of the polysaccharide of Millettia speciosa Champ. on paclitaxel through Examples 2 - 4. The results showed that when paclitaxel and the polysaccharide of Millettia speciosa Champ. were used in combination, the effect of inhibiting tumor cell proliferation was significantly enhanced and greater than that when paclitaxel was used alone. Paclitaxel is a first-line drug for various solid tumors. The polysaccharide of Millettia speciosa Champ. can enhance the anti-tumor effect of paclitaxel, especially the synergistic effect on the anti-tumor effect of breast cancer with a very high incidence, and is expected to become an adjuvant drug for the prevention and treatment of malignant tumors.

[0027] The following will further illustrate the concept, specific steps and technical effects generated by the present invention to fully understand the purpose, features and effects of the present invention. Detailed implementation mode

[0028] All kinds of reagents used in the present invention can be directly purchased commercially, unless otherwise specified.

[0029] The polysaccharide of Millettia speciosa in the present invention is a polysaccharide extracted and preliminarily purified from the dried roots of Millettia speciosa Champ., a plant of the genus Millettia in the subfamily Papilionoideae of the Leguminosae family. Millettia speciosa can be purchased from the Guangdong and Guangxi regions without special requirements, and regular commercially available products can be used.

[0030] Example 1 Extraction and purification of polysaccharide of Millettia speciosa

[0031] Extraction: Take 100 g of Millettia speciosa and crush it into coarse powder, add 3000 mL of pure water and boil for 4 hours; filter with double-layer 200-mesh silk cloth to obtain a medicinal liquid containing the water extract of Millettia speciosa; concentrate the medicinal liquid to 1000 mL; after cooling to room temperature, adjust the pH to 3 with 0.3 mol / L hydrochloric acid, keep it warm in a water bath at 60 °C for 6 hours, stir slowly continuously during this period, centrifuge the above solution at 8000 rpm / min for 15 min, and take the supernatant; add pure water to the remaining precipitate to 700 mL, add 0.3 mol / L hydrochloric acid again to adjust the pH to 3, keep it warm in a water bath at 60 °C for 6 hours, stir slowly continuously during this period, centrifuge the above solution at 8000 rpm / min for 15 min, and take the supernatant again; combine the two supernatants, add 0.3 mol / L NaOH to adjust the pH to 6-7; add anhydrous ethanol - 90% ethanol to make the final ethanol concentration 70%, let it stand overnight, filter by suction to obtain the second precipitate; wash the precipitate with acetone, ether, and anhydrous ethanol respectively, and freeze-dry to obtain the crude polysaccharide of Millettia speciosa in powder form.

[0032] Purification: Dissolve the powdered crude polysaccharide extracted by the above method in water, take 3 ml of the sample and pass it through a DEAE Sephadex A-25 ion exchange column, elute with distilled water and 1.5 mol / L NaCl solution at a flow rate of 0.5 ml / min, collect 100 tubes with 5 ml per tube, and determine the polysaccharide concentration of each tube by the anthrone-sulfuric acid method. After concentrating the eluate with a higher sugar content under reduced pressure, dialyze to remove salts and vacuum dry to obtain the polysaccharide. Add the polysaccharide to pure water to prepare a 1 mg / mL mother liquor, filter and sterilize, dispense, and store at 4 °C for later use, and conduct anti-tumor activity detection with this.

[0033] Example 2 Synergistic effect of polysaccharide of Millettia speciosa on paclitaxel in inhibiting ovarian cancer cell Caov-3

[0034] After resuscitating ovarian cancer cell Caov-3, seed it at 5*10 6Inoculated at a density of / L into fresh 1640 medium (containing 89% RPMI 1640 medium, 10% fetal bovine serum FBS, 1% penicillin-streptomycin at 1 U / mL), and cultured in an incubator at 37°C, saturated humidity, and 5% CO2. The medium was changed and the cells were passaged once every 2 days. Logarithmic growth phase ovarian cancer cells Caov-3 were digested with 0.25% trypsin, counted, diluted with RPMI-1640 culture medium containing 10% fetal bovine serum to prepare a cell suspension of 1×104 / mL, and inoculated into 3 96-well culture plates, 200 μL per well, so that the cell density per well was 2×103, with 4 parallel replicate wells in each group, and a blank group without cells was set. After culturing for 24 h, when the cells adhered to the wall, 200 μL of medium containing different concentrations of paclitaxel (0, 2, 5, 8, 10 μg / mL) was added to each well for medium change, and the 0 μg / mL paclitaxel group was the control group. After continuous culture for 48 h respectively, 200 μL of medium containing 10% CCK-8 was added for medium change, and the culture was continued for 2.5 h. The absorbance value A of each well at a wavelength of 450 nm was detected using an automatic microplate reader, and the effect of paclitaxel on the proliferation viability of ovarian cancer Caov-3 cells after 48 h was calculated to determine the IC50 concentration.

[0035] At the same time, polysaccharide of Millettia speciosa Champ. (40, 50, 60, 80, 100 μg / mL) was added according to the above method, and the effect of polysaccharide of Millettia speciosa Champ. on the proliferation viability of ovarian cancer cells Caov-3 after 48 h was calculated to determine the IC50 concentration after 48 h.

[0036] According to the above method, the IC50 concentration of paclitaxel on ovarian cancer Caov-3 cells was taken, and the IC50 concentration of polysaccharide of Millettia speciosa Champ. on ovarian cancer Caov-3 cells and two groups of concentrations of its adjacent higher and lower levels were used for combined drug use to measure the IC50 value after another 48 h. It was compared with the paclitaxel group. The results are shown in Table 1:

[0037] Table 1 IC50 of paclitaxel on ovarian cancer Caov-3 before and after adding polysaccharide of Millettia speciosa Champ.

[0038] Paclitaxel Millettia speciosa polysaccharide Combined use of paclitaxel and Millettia speciosa polysaccharide IC50 7.47±1.47 85.26±2.45 6.85±2.58

[0039] It can be seen that when paclitaxel and polysaccharide of Millettia speciosa Champ. are used in combination, the inhibitory effect on ovarian cancer Caov-3 cells is significantly enhanced and greater than the inhibitory effect of paclitaxel alone on the proliferation of ovarian cancer Caov-3 cells.

[0040] Example 3 Synergistic effect of polysaccharide of Millettia speciosa Champ. on the inhibition of lung cancer cells A549 by paclitaxel

[0041] The culture method of lung cancer cell A549, the detection method of the proliferation activity of paclitaxel on lung cancer cell A549, the detection method of the proliferation activity of polysaccharide from Millettia speciosa Champ. on lung cancer cell A549, and the detection method of the proliferation activity of the combined use of paclitaxel and polysaccharide from Millettia speciosa Champ. on lung cancer cell A549 are the same as those in Example 2. The test results are as follows:

[0042] Table 2 IC50 of paclitaxel on lung cancer A549 before and after adding polysaccharide from Millettia speciosa Champ.

[0043] Paclitaxel Millettia speciosa polysaccharide Combined use of paclitaxel and Millettia speciosa polysaccharide IC50 3.56±0.24 84.55±2.12 2.99±2.35

[0044] It can be seen that when paclitaxel and polysaccharide from Millettia speciosa Champ. are used in combination, the inhibitory effect on lung cancer cell A549 is significantly enhanced and greater than the inhibitory effect of paclitaxel alone on the proliferation of lung cancer cell A549.

[0045] Example 4 Synergistic effect of polysaccharide from Millettia speciosa Champ. on the inhibition of breast cancer cell MCF7 by paclitaxel

[0046] The culture method of ovarian cancer cell MCF7, the detection method of the proliferation activity of paclitaxel on ovarian cancer cell MCF7, the detection method of the proliferation activity of polysaccharide from Millettia speciosa Champ. on ovarian cancer cell MCF7, and the detection method of the proliferation activity of the combined use of paclitaxel and polysaccharide from Millettia speciosa Champ. on ovarian cancer cell MCF7 are the same as those in Example 2. The test results are as follows:

[0047] Table 3 IC50 of paclitaxel on breast cancer cell MCF7 before and after adding polysaccharide from Millettia speciosa Champ.

[0048] Paclitaxel Millettia speciosa polysaccharide Combined use of paclitaxel and Millettia speciosa polysaccharide IC50 0.42±1.98 82.13±2.12 0.28±1.53

[0049] It can be seen that when paclitaxel and polysaccharide from Millettia speciosa Champ. are used in combination, the inhibitory effect on breast cancer cell MCF7 is significantly enhanced and greater than the inhibitory effect of paclitaxel alone on the proliferation of breast cancer cell MCF7. The above results show that when paclitaxel and polysaccharide from Millettia speciosa Champ. are used in combination, the tumor inhibitory effect on ovarian cancer cell Caov-3, lung cancer cell A549 and breast cancer cell MCF7 is better than that of paclitaxel alone. This indicates that polysaccharide from Millettia speciosa Champ. increases the inhibitory effect of paclitaxel on tumor cell proliferation and plays a role as a synergist for paclitaxel.

Claims

1. Use of polysaccharide from Millettia speciosa Champ. in preparing a synergist for anti-tumor drugs.

2. The use according to claim 1, characterized in that The synergist for the anti-tumor drug refers to the combination of millettia speciosa champ polysaccharide and the anti-tumor drug paclitaxel, which enhances the anti-tumor effect of paclitaxel.

3. The use according to claim 1 or 2, characterized in that The drug includes the millettia speciosa champ polysaccharide and pharmaceutically acceptable acids, bases, salts, hydrates or esters, and other excipients.

4. The use according to claim 1 or 2, characterized in that The tumors are ovarian cancer, lung cancer and breast cancer.

5. The use of the polysaccharide from Millettia speciosa Champ. according to claim 1, wherein the preparation method of the polysaccharide from Millettia speciosa Champ. is as follows: a. After drying, pulverizing the Millettia speciosa Champ. medicinal materials, boiling in water, filtering and concentrating, adding 0.3 mol / L hydrochloric acid to adjust the pH to 2 - 4, and centrifuging to obtain a first supernatant and a first precipitate; b. The first precipitate obtained by centrifugation in step a is dissolved in an appropriate amount of water, and the pH is adjusted to 2-4 with 0.3 mol / L hydrochloric acid, and then centrifuged again to obtain a second supernatant; c. The first supernatant and the second supernatant are combined, and NaOH is added to adjust the pH to 6-7; d. After concentration under reduced pressure, 90% anhydrous ethanol is added for precipitation, and the second precipitate is obtained by suction filtration; e. The second precipitate is washed with acetone, ether and anhydrous ethanol to obtain the crude extracted millettia speciosa champ polysaccharide; f. The crude millettia speciosa champ polysaccharide in step e is dissolved in water, fractionally eluted through an ion exchange column, and purified millettia speciosa champ polysaccharide is obtained after vacuum drying.

6. The use according to claim 1, characterized in that The method for detecting the anti-tumor activity of millettia speciosa champ polysaccharide is as follows: a. Tumor cells are inoculated into fresh medium, cultured until the logarithmic growth phase, and made into a cell suspension, which is inoculated into a multi-well plate; b. After the cells adhere to the wall, the medium is replaced with a medium containing different concentrations of millettia speciosa champ polysaccharide, and the culture is continued for 24 hours, 48 hours or 72 hours; c. The medium in step b is replaced with a medium containing CCK-8, and the absorbance value is detected after continuous culture; d. Calculate the influence of the millettia speciosa champ polysaccharide on the proliferation activity of tumor cells at different time periods, and determine the IC50 value.