CN1679795A - Anti-tumor extract of salicymus root and its preparation method and use - Google Patents

Abstract

An anticancer extract of thinleaf adina root for preparing the medicines in the form of injection, etc to treat tumors is prepared from the root of thinleaf adina through extracting in water or organic solvent, flocculating deposition, regulating acidity, purifying, recovering solvent, extracting, concentrating and drying.

Description

Anti-tumor extract of salicymus root and its preparation method and use

Technical field

The present invention relates to the field of medicine, in particular to an anti-tumor extract of salicybean root and its preparation method and application.

Background technique

The root of Myrica rubella is the root of Adina rubella (Sieb. et Zucc.) Hance, a deciduous shrub of the Rubiaceae family. Yu Rencun et al. reported in "Common Chinese Herbal Medicine for Tumors", Oncology of Traditional Chinese Medicine, 1997, page 72, that salicylic radix root alcoholic extract was administered orally or intraperitoneally at 5g/Kg daily for 6 days, and it had inhibitory effect on mouse leukemia L615 , and the inhibition rate was 21.4%; it had inhibitory effects on mouse cervical cancer cells, mouse sarcoma AK, and rat sarcoma; this suggested that the alcoholic extract of Salicymus root had a broad-spectrum anti-tumor effect. The inflorescences of Myrica salicifolia contain catechins, ursolic acid, oleanolic acid, β-sitosterol, salicyrin A, and alkaloids, etc., but there are few reports on the effective components of Myrica rubra root . He Zhisheng and others reported in "Research on the Chemical Constituents of Myrica rubra", Chinese Herbal Medicine, 1996, 26(6): 285-288, that they isolated several new Quinovic acid glycoside compounds from the root of Myrica rubra, but did not carry out Antitumor activity studies. Quinovic acid glycosides are also found in other anti-tumor plants. It was mentioned in the literature Raffauf RF, etal.Lloydia, 1978, 41 (5): 432-434 that Quinovic acid glycosides were found in cinchona bark. The compound has anti-tumor activity, and later Raffauf RF also found this kind of compound in other plant medicinal materials with anti-tumor activity, which indicates that Quinovic acid glycoside compound may also be the main anti-tumor active substance of salicymus root.

At present, the research on the root of salicylum only stays in the superficial understanding of its medicinal materials, and there are no reports on the anti-tumor effective parts, component separation methods and preparation techniques of the root of salicylate.

Contents of Invention

The purpose of the present invention is to overcome the shortcomings in the prior art, and to provide a method for preparing the anti-tumor extract of salicylum root which can effectively purify the effective part of the traditional Chinese medicine salicylaceae root and is convenient for industrial production.

Another object of the present invention is to provide an anti-tumor extract of salicyrdium root prepared by the above method.

Another object of the present invention is to provide a use of the anti-tumor extract of the above-mentioned salicylic bayberry root.

The object of the present invention is achieved through the following technical scheme: the preparation method of the anti-tumor extract of the salicymus root comprises the following steps:

(1) extracting the root medicinal material of Myrica rubra with water or an organic solvent at a temperature of 70-100° C. to obtain an extract;

(2) Adding a flocculant to the leaching solution to precipitate and remove impurities to obtain an extract;

(3) Adjust the pH value of the extract to 2-5, let stand, and obtain the supernatant;

(4) passing the supernatant through a macroporous resin, eluting with a solvent, and collecting the eluate;

(5) After recovering the solvent from the eluent, extract it with ethyl acetate to obtain an extract;

(6) Concentrate and dry the extract to obtain the anti-tumor extract of the salicylate root.

In the step (1), the organic solvent is 30-70% (V/V) ethanol; the medicinal material of salicylum root can be block or pulverized; the extraction temperature is preferably 70-80°C .

In step (1), the number of extractions is 1 to 3 times; each extraction time is 1 to 2 hours; the weight ratio of the root of the salicymus root extracted for the first time to water or organic solvent is 1: 8～ 10: The weight ratio of the salicylate root extracted for the second and third times to water or organic solvent is 1:4-5.

In step (2), the leachate can be concentrated first, and then flocculated and precipitated; the flocculating agent can be chitin, ZTC flocculating agent or gelatin, and the dosage is 0.1 to 1% of the weight of the root medicinal material of salicymus; Filter or centrifuge the precipitate.

In step (3), an acidic reagent is used to adjust the pH value of the extract, preferably hydrochloric acid or acetic acid.

In step (4), the macroporous resin can be various nonpolar or weakly polar macroporous resins; the solvent is an aqueous solvent, preferably ethanol water or acetone water.

In step (5), the amount of ethyl acetate used is 1 to 3 times the volume of the eluent.

In step (6), the drying may be vacuum drying, spray drying or freeze drying.

The above-mentioned method is used to prepare the anti-tumor extract of the salicylic root; the active ingredient (polyphenols) content of the prepared anti-tumor extract can reach 80-90%.

The anti-tumor extract of salicymus root is used as an active ingredient in combination with an acceptable drug carrier to make oral or injection single and compound preparations that can be used to treat various tumors, such as tablets made with starch and other auxiliary materials, microcrystalline Capsules made of cellulose as auxiliary materials, injections made of mannitol as a scaffold, etc.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention adopts high temperature, multiple times, and long-time leaching to the raw medicinal material, and the extraction effect is good, and the transfer rate of the anti-tumor active ingredients in the raw medicinal material is high;

(2) The process is simple, and the precipitation step adopts the flocculation precipitation method, which can avoid the loss caused by incomplete precipitation and the increased process steps of ethanol recovery in the alcohol precipitation method;

(3) product purity is high, is convenient to suitability for industrialized production, adopts acid adjustment method to remove acid-insoluble matter on the one hand, improves the adsorption effect of macroporous resin; The ethyl ester extraction method further improves the purity of the effective parts, and improves the content and precision of the active ingredients.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

Example 1

Add 10L of water to 1Kg of salicylum root medicinal material, keep it at 100°C for 120 minutes, and then filter to obtain the first leachate; then add 5L of water to the filter residue, keep it at 100°C for 90 minutes Minutes, then filter to obtain the second leachate; combine the two leachates.

Concentrate the leachate under reduced pressure until the thermal specific gravity is 1.05, add 1g of chitin, centrifuge to remove the precipitate after stirring, and obtain the extract; adjust the pH value of the extract to 2 with 10% hydrochloric acid, then let it stand, and centrifuge to remove the precipitate, and obtain the above supernatant; the supernatant was passed through the macroporous resin D101, then eluted with 70% ethanol water, and then the eluate was decompressed to recover ethanol, and then extracted 3 times with 1 times the volume of ethyl acetate; finally the extract Concentrate under normal pressure and dry in vacuum to prepare 30 g of the anti-tumor extract of the salicylate root. Its polyphenol content is determined to be 85%.

Example 2

Grind 1Kg of salicylate root medicinal material into coarse powder, add 8L of water, keep at 80°C for 60 minutes, then filter to obtain the first leachate; then add 4L of water to the filter residue, keep at 80°C Under leaching for 60 minutes, and then filtered to obtain the second leaching solution; the two leaching solutions were combined.

Add 10g of gelatin to the leachate, stir and centrifuge to remove the precipitate to obtain the extract; use 20% acetic acid to adjust the pH value of the extract to 5, then let it stand and centrifuge to remove the precipitate to obtain the supernatant; pass the supernatant through the large hole Resin HPD300, then eluted with 70% acetone water, then the eluent was decompressed to recover acetone, and then extracted once with 3 times the volume of ethyl acetate; finally, the extract was concentrated under reduced pressure and freeze-dried to obtain 35g Antineoplastic extract of salicylate root. Its polyphenol content is determined to be 87%.

Example 3

Grind 1Kg of salicylate root medicinal material into coarse powder, add 9L of water, keep at 90°C for 90 minutes, then filter to obtain the first leachate; then add 5L of water to the filter residue, keep at 90°C Extract at low temperature for 90 minutes, then filter to obtain the second leach solution; then add 5 L of water to the filter residue, keep it at 90°C for 90 minutes, then filter to obtain the third leach solution; combine the three leach solutions.

Concentrate the leachate under normal pressure to a thermal specific gravity of 1.03, add 3g of ZTC flocculant, stir and centrifuge to remove the precipitate to obtain the extract; use 5% hydrochloric acid to adjust the pH value of the extract to 3, then let stand, centrifuge to remove the precipitate, and obtain the above supernatant; the supernatant was passed through the macroporous resin HPD600, then eluted with 50% acetone water, and then the eluate was decompressed to recover acetone, and then extracted 3 times with 2 times the volume of ethyl acetate; finally the extract Atmospheric pressure concentration, spray drying, prepared 36g of salicylate root anti-tumor extract. Its polyphenol content is determined to be 83%.

Example 4

The root medical material of salicymus root of 1Kg is pulverized into coarse powder, adds 70% ethanol of 10L, keeps leaching 90 minutes at 70 ℃ of temperature, then filters and obtains the leachate for the first time; Leach at a temperature of 70°C for 60 minutes, and then filter to obtain a second leach solution; combine the two leach solutions.

Concentrate the leachate under reduced pressure to a thermal specific gravity of 1.05, add 3g of chitin, stir and centrifuge to remove the precipitate to obtain the extract; use 5% hydrochloric acid to adjust the pH value of the extract to 2, then stand still and centrifuge to remove the precipitate, and obtain the supernatant liquid; the supernatant was passed through the macroporous resin HPD300, then eluted with 50% ethanol water, and then the eluent was decompressed to reclaim ethanol, and then extracted twice with ethyl acetate of 3 times the volume; finally the extract was reduced Concentrate under pressure and freeze-dry to obtain 38g of the anti-tumor extract of the salicylate root. Its polyphenol content is determined to be 93%.

Example 5

The root medical material of salicymus root of 1Kg is pulverized into coarse powder, adds 50% ethanol of 8L, keeps leaching at 80 ℃ of temperature for 60 minutes, then filters to obtain the leachate for the first time; then adds 50% ethanol of 4L in filter residue, keeps Extract at 80°C for 60 minutes, then filter to obtain the second leachate; then add 4L of 50% ethanol to the filter residue, keep it at 80°C for 60 minutes, then filter to obtain the third leachate; combine three times Leachate.

Concentrate the leachate under normal pressure to a thermal specific gravity of 1.03, add 8g of gelatin, centrifuge to remove the precipitate after stirring, and obtain the extract; use 20% acetic acid to adjust the pH value of the extract to 4, then stand still, and centrifuge to remove the precipitate to obtain the supernatant ; The supernatant was passed through the macroporous resin HPD600, then eluted with 70% acetone water, and then the eluent was decompressed to reclaim acetone, and then extracted twice with 2 times the volume of ethyl acetate; finally the extract was extracted under normal pressure Concentrate and spray-dry to prepare 36 g of the anti-tumor extract of the salicylate root. Its polyphenol content is determined to be 96%.

Example 6

Add 9L of 30% ethanol to 1Kg of salicylate root medicinal material, keep it at 80°C for 120 minutes, then filter to obtain the first leachate; then add 5L of 30% ethanol to the filter residue, keep at 80°C Under leaching for 90 minutes, and then filtered to obtain the second leaching solution; the two leaching solutions were combined.

Add 5g of ZTC flocculant to the leaching solution, centrifuge to remove precipitation after stirring, and obtain the extract; use 10% hydrochloric acid to adjust the pH value of the extract to 3, then let stand, centrifuge to remove precipitation, and obtain the supernatant; Pass through the macroporous resin D101, then elute with 70% ethanol water, then reduce the eluent to recover ethanol, then extract with 1 times the volume of ethyl acetate for 3 times; finally, concentrate the extract under normal pressure and dry it in vacuum. 32 g of the anti-tumor extract of salicylaceae root were prepared. Its polyphenol content is determined to be 90%.

Example 7

Cellular experiment of anti-tumor extract of salicymus root:

Method: Put tumor cells (2×10 ⁶ /ml) into a sterile 96-well culture plate, add 50 μl of tumor cell suspension, add 50 μl of 1640 culture solution containing 15% calf serum, add Example 1 ～3 Prepare 10 μl of the aqueous solution of the anti-tumor extract of salicymus root, add 3 wells in each group, add an equal amount of normal saline to the wells, shake and mix well, then put them in a 5% _CO2 incubator (37°C) for 24 hours, add Tetrazolium salt (MTT) phosphate buffer, 10 μl per hole (each ml containing 5 mg MTT) continued to incubate for 4 hours, then add 100 ml of dimethyl sulfone to terminate the reaction, and measure the optical density at wavelengths of 570 nm and 630 nm with an enzyme gel immunoassay instrument OD value, calculation result.

Calculation formula:

Results: In the tumor cell culture experiment of gastric cancer RF cell line and rectal cancer LS174T cell line, it was found that the anti-tumor rate of the anti-tumor extract of salicymus root was significantly higher than that of the water extract of its medicinal materials (without precipitation, acid adjustment). , macroporous resin adsorption and extraction water extract concentrate), and the dosage is increasing. The results are shown in Table 1.

Attached table 1 Inhibition experiment of effective fractions of salicymus root on gastric cancer and colorectal cancer cells

deal with Concentration (mg/ml) Inhibitory rate of gastric cancer % ( x±SD) Inhibition rate of colon cancer %( x±SD) The aqueous solution of the water extract of the salicyrdium root medical material Example 1 The aqueous solution of Example 2 The aqueous solution of Example 3 5125 23.5±4.543.7±6.272.7±4.996.3±8.4 25.3±5.745.3±5.176.8±6.495.9±9.3

Example 8

Animal Experiments on Inhibiting Tumor by the Effective Fractions of Salicylica Root Compound

Methods: Take the S180 tumor mass of the mouse, dilute it after homogenization and adjust the cell number (1×10 ⁷ /ml), inject 0.2ml into the right axillary subcutaneous of the mouse, and randomly divide into groups for administration after 24 hours. Control group intragastric administration; CTX (cyclophosphamide) group intraperitoneal administration; the water extract (water extraction concentrate that has not been through precipitation, acid adjustment, macroporous resin adsorption and extraction) and embodiment 3 6. All the anti-tumor extracts prepared in 6 were intragastrically administered. On the 11th day, the mice were weighed and sacrificed to take tumor pieces, the tumor weight and volume were measured, and the tumor inhibition rate was calculated.

Result: the antitumor rate of S180 by the water extract of the root of the salicymus root medicinal material is 38.3%, and the antitumor rate of the antitumor extract of the root of the salicymus root prepared by the present invention is 66.4%, which is significantly different from that of the control group, while The difference with the CTX group was not significant, suggesting that it has a good anti-tumor effect. The results are shown in Table 2.

Attached Table 2 Animal Experiments on the Inhibition of S180 Sarcoma by Effective Fractions of Salicymus Root

group Dose (g/Kg) Tumor weight (g) ( x±SD) Tumor volume (cm ³ ) ( x±SD) Tumor inhibition rate (%) Control group CTX group water bayberry root medical material embodiment 3 embodiment 6 25201020 3.42±0.461.12±0.262.11±0.261.45±0.411.15±0.29 2.85±0.730.81±0.372.01±0.281.49±0.520.89±0.23 67.238.351.266.4

Claims (10)

1, a kind of preparation method of salicylate root anti-tumor extract, is characterized in that comprising the steps: (1) extracting the root medicinal material of Myrica rubra with water or an organic solvent at a temperature of 70-100° C. to obtain an extract; (2) Adding a flocculant to the leaching solution to precipitate and remove impurities to obtain an extract; (3) Adjust the pH value of the extract to 2-5, let stand, and obtain the supernatant; (4) passing the supernatant through a macroporous resin, eluting with a solvent, and collecting the eluate; (5) After recovering the solvent from the eluent, extract it with ethyl acetate to obtain an extract; (6) Concentrate and dry the extract to obtain the anti-tumor extract of the salicylate root. 2. The preparation method of the anti-tumor extract of salicymus root according to claim 1, characterized in that in step (1), the organic solvent is 30-70% ethanol. 3. The preparation method of the anti-tumor extract of salicymus root according to claim 1, characterized in that in step (1), the extraction temperature is 70-80°C. 4. The preparation method of the anti-tumor extract of salicymus root according to claim 1, characterized in that: in step (1), the extraction times are 1 to 3 times; each extraction time is 1 to 2 hours; the weight ratio of the water bayberry root and water or organic solvent extracted for the first time is 1: 8～10; the weight ratio of the water bayberry root and water or organic solvent extracted for the second and third time is 1: 4~5. 5. The preparation method of the anti-tumor extract of salicylum root according to claim 1, characterized in that in step (2), the amount of the flocculant is 0.1-1% of the weight of the salicylic root medicinal material. 6. The preparation method of the anti-tumor extract of salicymus root according to claim 1, characterized in that: in step (3), hydrochloric acid or acetic acid is used to adjust the pH value of the extract. 7. The preparation method of the anti-tumor extract of salicymus root according to claim 1, characterized in that: in step (4), the solvent is ethanol water or acetone water. 8. The method for preparing the anti-tumor extract of salicymus root according to claim 1, characterized in that in step (5), the amount of ethyl acetate used is 1 to 3 times the volume of the eluent. 9. An anti-tumor extract of salicylaceae root prepared according to any one of claims 1-8. 10. The application of the anti-tumor extract of salicylum root according to claim 9, characterized in that: the anti-tumor extract of salicymus root is used as an active ingredient in combination with an acceptable drug carrier to make a medicine for treating various tumors. Oral or injection single and compound preparations.