WO2012034519A1

WO2012034519A1 - Tannin extract of sanguisorba officinalis l. and preparation method and use thereof

Info

Publication number: WO2012034519A1
Application number: PCT/CN2011/079633
Authority: WO
Inventors: 杨明; 苏柘僮; 杨胜; 王达宾; 邹文铨
Original assignee: 成都科尔医药技术有限公司
Priority date: 2010-09-14
Filing date: 2011-09-14
Publication date: 2012-03-22
Also published as: MY173437A; GB2497899B; CN103079560A; CN103784434A; CN103948710A; SG10201505752QA; SG188529A1; GB201306569D0; GB2497899A

Abstract

A tannin extract of Sanguisorba officinalis L. contains tannin in a range of 35%-100% (w/w), wherein the content of total saponin is less than 10% (w/w), and the content of sanguisorbin I is less than 5% (w/w). The extract can protect bone marrow hematopoietic stem cells, relieve the inhibition of mouse bone marrow DNA caused by drugs for radiation therapy and chemotherapy, as well as increase numbers of leucocytes.

Description

Earthworm extract, preparation method and use thereof

The invention relates to a mantle extract, a preparation method thereof and use thereof, and belongs to the field of medicine. Background technique

Myelosuppression refers to a decrease in the activity of blood cell precursors in the bone marrow. Both red blood cells and white blood cells in the blood are derived from hematopoietic stem cells in the bone marrow. The blood cells in the blood have a short life span and often need to be replenished. In order to achieve timely replenishment, stem cells that are precursors of blood cells must divide rapidly. Chemotherapy and radiation therapy, as well as many other anti-tumor treatments, are directed at rapidly dividing cells, which often lead to inhibition of normal bone marrow cells, causing hematopoietic dysfunction in patients with bone marrow, resulting in lowering of red blood cells, white blood cells, and platelets in patients, leading to anemia. , bleeding, decreased immune mechanisms and other phenomena.

Leukopenia, a common blood disease, means that white blood cells in the peripheral blood continue to be less than 4 X 10 ⁹ /L. White blood cells are patrols that the body defends against bacterial invasion. A decrease in the number of white blood cells weakens the body's antibacterial ability and is susceptible to infection. In particular, the use of immunosuppressive agents such as radiotherapy, chemotherapy, infection, systemic lupus erythematosus, and the use of the anti-tuberculosis drug rifampicin in infectious diseases can reduce the peripheral blood leukocytes, thereby weakening the body's antibacterial ability and leading to immunity. Adverse reactions such as decreased force. At present, leukopenia is mainly divided into two types: unexplained and secondary. The former is more common, while the latter is caused by bone marrow damage caused by radiotherapy, chemotherapy, infection, and immune factors.

Both leukopenia and myelosuppression have a significant phenomenon of leukopenia. However, there are still some differences in the administration of drugs. At present, some drugs with whitening effect do not have the effect of bone marrow protection, such as rhG-CSF. Strong whitening effect, but its damage to bone marrow cells can not be ignored. Therefore, it is necessary to find drugs that can increase the concentration of white blood cells and effectively protect bone marrow cells.

The mantle is the dry root of Sanguisorba officinalis L. or Sanguisorbad officinalis L.var. longifolia (Bert.) Yu et Li. It has the effect of cooling blood to stop bleeding and detoxification. The mantle medicinal material contains a large amount of enamel components (about 20%), and in addition, it also contains compounds such as saponins and flavonoids. Most of the current studies on mantle are carried out around mantle saponins. It is generally believed that the effective component of mantle-elevating leukocyte action and bone marrow protection is saponin, and the material basis research on the effect of mantle on the mantle in the country is that mantle Saponin is the main active site for promoting the proliferation of mouse bone marrow cells in vitro. Gentiana saponin can also increase the number of leukocytes, red blood cells and platelets in mice with myelosuppression, while mantle and mantle flavonoids can not promote the proliferation of bone marrow cells. At high concentrations, it can produce myelosuppressive effects (high-parity, effective site screening for hematopoiesis, Chinese natural medicine, Vol. 4, No. 2, 2006). At present, studies on the efficacy of mantle are rare, and there are reports in foreign countries that mantle has anticancer effects [Bastow kF.Bort LD.Fukushrma Y et al.Inhibition of DNA topoisomerases by sanguim H-6, a cytotoxic dimeric ella-gitan-ton from sanguisorba officinalis. Planta Med, 1993; 59(3): 240-245.]. There is also a partial report on the extraction of mantle. For example, Gao Xiaoping, the extract of the mantle is extracted with 70% aqueous acetone, and the content of tannin in the extract is 43.86% (China Natural Medicine, 2006, Vol. 4) Because the method is extracted by acetone, without separation, the extract contains saponin in addition to enamel, and the saponin content is about 15% detected; Wang Manli et al. also use alumina column. Separation of mantle and mantle saponins, and finally separation of the two (Journal of Guizhou Institute of Technology, 1993, Volume 22, Issue 2).

There have been no reports of the application of mantle in bone marrow protection and treatment of leukopenia.

Summary of the invention

It is an object of the present invention to provide a enamel extract; another object of the present invention is to provide a novel use of the enamel extract, a compound contained in the enamel.

The present invention provides a mantle extract having a content of tannin of 35%-100% w/w; the total saponin content of the extract is <10% w/w and The content of saponin I is <5% w/w.

Further preferably, the extract has a enamel content of 50% to 100% by weight. Further, the weight percentage of the tannin is 55% to 100%.

Further preferably, in the extract, the total saponin content of the extract in the extract is <7.2% w/w, and the content of the saponin I is < 4.9% w/w. 0001%w/w。 The content of the total saponin content of the extract is 0. 001% w / w, the content of the saponin I is < 0.0001% w / w.

Further, the mantle contains mantle, catechin, proanthocyanidin B2, which accounts for 0. 05%-3. 0% w/w, catechin. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 %% 0%w/w, catechin 2. 0%-4. 0% w/w, proanthocyanidin B2 0. 05%-0. 4% w/w.

Further, in the extract, the weight percentage of the tannin is 62% to 100%.

The extract is derived from the dried root of the Sangui sorba officinal is L. or the long-leaved mantle Sanguisorba officinal is L. var. longifol ia (Bert.) Yu et Li.

Among them, the extract is prepared by the following method:

(1) Extraction: The ground medicinal material is pulverized into a coarse powder, and extracted with water, ethanol, aqueous ethanol, acetone or water-containing acetone, filtered, and concentrated to obtain a concentrated liquid;

(2) Purification: After taking the concentrate and treating it with the conventional separation method of tannin, it is obtained. Extract.

Wherein the conventional separation method of the enamel comprises chromatography, protein precipitation, solvent method, or a combination of the above methods.

Further, the chromatography is adsorption chromatography, and a gel or macroporous adsorption resin is preferably used. Further, the protein precipitation method is preferably carried out by gelatin.

Further, the solvent method refers to degreasing the aqueous solution containing the concentrated liquid, and then extracting with ethyl acetate to obtain an extract of the earthworm;

Alternatively, the concentrate may be dissolved in ethanol and ethyl acetate, and diethyl ether or petroleum ether may be precipitated to precipitate the enamel extract.

Further, the extract is prepared by the following method:

(1) Extraction: The medicinal material is pulverized into a coarse powder, extracted with water, ethanol, 10%-90% aqueous ethanol, acetone or 50-90% aqueous acetone, filtered, and concentrated to obtain a concentrated liquid;

(2) Purification: Take the concentrate, use macroporous adsorption resin for adsorption and impurity removal, first elute with water to colorless, then elute with 10% ethanol, and finally elute with 60% ethanol to collect 60% ethanol eluent. , dry, that is, the extract of the earthworm; or,

The concentrate was taken, and after degreasing, it was extracted with ethyl acetate to obtain an extract of the earthworm.

Preferably, the aqueous ethanol has a concentration of 70%; and the aqueous acetone has a concentration of 70%.

Wherein, the macroporous adsorption resin is a non-polar resin or a low-polar resin, preferably a DA-201, D-101, LSA-20, HP-10 or AB-8 type macroporous adsorption resin.

The invention also provides a preparation method of the above-mentioned mantle extract, which comprises the following steps:

(2) Purification: The concentrate is taken, and after treatment with the conventional separation method of tannin, the extract of the earthworm is obtained.

Further, the extract is prepared by the following method:

(1) Extraction: The medicinal material is pulverized into a coarse powder, extracted with water, ethanol, 10% _90% aqueous ethanol, acetone or 50-90% aqueous acetone, filtered, and concentrated to obtain a concentrated liquid;

The concentrate was taken, and after degreasing, it was extracted with ethyl acetate to obtain an extract of the earthworm. Preferably, the concentration of the aqueous ethanol is 70%; the concentration of the aqueous acetone is 70%.

Wherein, the macroporous adsorption resin is a non-polar resin or a low-polar resin, preferably DA-201.

D-101, LSA-20, HP-10 or AB-8 macroporous resin.

The present invention also provides the use of the above extract of the enamel extract for the preparation of a medicament having bone marrow protection.

Wherein the drug is a drug for preventing or/and treating myelosuppression.

Further, the medicament is a medicament for preventing or/and treating normal bone marrow cell suppression caused by radiotherapy or chemotherapy.

Further, the drug is a drug for preventing or/and treating anemia, leukopenia or thrombocytopenia caused by radiotherapy or chemotherapy, and preferably a drug for preventing or/and treating leukopenia caused by radiotherapy or chemotherapy.

The present invention also provides the use of the above-described enamel extract for the preparation of a medicament for treating leukopenia.

The present invention also provides the use of quercetin, proanthocyanidin B2 for the preparation of a medicament having bone marrow protection.

Wherein the drug is a drug for preventing or/and treating myelosuppression.

The present invention also provides the use of quercetin, proanthocyanidin B2 for the preparation of a medicament for treating leukopenia.

The present invention also provides a pharmaceutical composition for preventing or treating myelosuppression, leukopenia, which comprises an effective amount of scutellarin, proanthocyanidin B2 or the above-mentioned mantle extract as an active ingredient, and is added pharmaceutically The excipients or auxiliary ingredients received are prepared into pharmaceutically acceptable preparations.

Wherein the preparation is an oral preparation.

The present invention also provides the use of a tumor chemotherapeutic drug and the above-described mantle extract for the preparation of a combined anti-tumor drug.

Wherein, the tumor chemotherapy drug is a sputum agent antitumor drug.

Further, the tumor chemotherapy drug is cyclophosphamide.

The present invention also provides an antitumor pharmaceutical composition which is prepared by an effective amount of a tumor chemotherapeutic drug and the above-mentioned mantle extract as an active ingredient together with a pharmaceutically acceptable adjuvant.

Wherein, the tumor chemotherapy drug is a sputum agent antitumor drug.

Further, the tumor chemotherapy drug is cyclophosphamide.

The active ingredient in the enamel extract of the present invention is mantle, which has the function of protecting bone marrow hematopoietic stem cells, and has significant protective effect on chemical DNA and radiation-induced inhibition of mouse bone marrow DNA, and is clinically relieved by radiotherapy and chemotherapy. The resulting inhibition of normal bone marrow cells provides a new medication option. In addition, the mantle can also raise white blood cells and has a clear whitening effect. When the enamel extract of the present invention is combined with a chemotherapeutic drug, it not only enhances the curative effect on the tumor, but also protects the bone marrow cells and the whole blood cells, and reduces or avoids the damage of the chemotherapeutic drugs on the bone marrow cells and the whole blood cells. Therefore, before, after, or during the treatment of cancer by radiotherapy or chemotherapy, a combination of radiotherapy and chemotherapy with the extract of the enamel extract of the present invention can be used to reduce or avoid radiotherapy and chemotherapy for the patient. Damage caused by bone marrow cells and white blood cells.

detailed description

1. Determination of tannin content in the present invention, according to the Chinese Pharmacopoeia 2010 edition of an appendix X B enamel content determination:

This experiment should be protected from light.

Preparation of reference solution: Precisely weigh 50mg of gallic acid reference substance, put it into 100ml brown volumetric flask, add water to dissolve and dilute to the mark, accurately measure 5ml, set it in 50ml brown volumetric flask, dilute with water to the mark, shake well, ie得含含酸酸0. 05mg).

Preparation of the standard curve: Precisely weigh the reference solution 0. 5ml, 1. 0ml, 2. 0ml, 3. 0ml, 4. 0ml, 5. 0ml, respectively, in a 25ml brown volumetric flask, each added phosphorus molybdenum tungstic acid test 1 ml of liquid, then add 11.50ml, l lml, 10ml, 9ml, 8ml, 7ml, respectively, dilute to the mark with 29% sodium carbonate solution, shake, place for 30 minutes with the corresponding reagent blank, according to UV-visible spectrophotometry (Appendix VA), absorbance is measured at a wavelength of 760 nm, and the standard curve is plotted with absorbance as the ordinate and concentration as the abscissa.

Preparation of the test solution: Take the extract powder about 0. lg, accurately weighed, placed in a 250ml brown volumetric flask, add 150ml of water, leave it overnight, sonicate for 10 minutes, let cool, dilute with water to the mark, shake, static Set (to precipitate solid matter), filter, discard 50ml of the initial filtrate, accurately measure 20ml, place it in a 100ml brown volumetric flask, dilute with water to the mark, shake well, that is.

Assay

Total phenol: Precisely measure 2ml of the test solution, place it in a 25ml brown volumetric flask, according to the method under the preparation of the standard curve, from the "addition of phosphomolybdate solution 1ml", add water 10ml, measure the absorbance according to law, from The standard curve reads the amount (mg) of gallic acid in the test solution, which is calculated and obtained.

Polyphenols that are not adsorbed: Precisely measure 25ml of the test solution, add it to a 100ml stoppered conical flask containing 0. 6g of casein, and put it in a 30°C water bath for 1 hour. Shake, take out, let cool, shake, filter, discard the first filtrate, accurately measure the 2ml of the filtrate, place it in a 25ml brown volumetric flask, according to the method under the preparation of the standard curve, from the "addition of phosphotungstic acid test Liquid lml "from, add water 10ml, absorb the absorbance according to law, read the amount of gallic acid (mg) in the test solution from the standard curve, calculate, that is.

Calculate the content of tannin by the following formula: Tannin content = total phenol amount - the amount of polyphenol that is not adsorbed.

2. Determination of the content of scutellarin, catechin and proanthocyanidin B2 can be found in the literature: Composition analysis and functional study of mantle polyphenols, Liu Haiying, Shaanxi Normal University, 2009. The specific measurement methods are: 2. Instrument and test materials

2. 1. 1 instrument Agi lent 1200 High Performance Liquid Chromatograph (Agi lent 1200 Diode Array Detector; Quaternary Pump Online Degassing System; Agi lent 1200 Chromatography Workstation) (Agilent Technologies, Inc., USA);

Mettler AE240 One hundred thousandth electronic analytical balance (Mettler, Germany);

FA1104 million electronic analytical balance (Shanghai Tianping Instrument Factory);

KQ3200 Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

2. 1. 2 test drugs

Proanthocyanidin B2 (purity: HPLC 99%);

Catechins (purity: HPLC^99%);

Glutin (purity: HPLC 99%);

Methanol is chromatographically pure, water is re-distilled water (home-made), and other reagents are of analytical grade.

2. 1. 3 earthworm extract

The mantle extract prepared by the method of the examples was taken.

2. 2. Preparation of test solution

Take about lg of extract of enamel, accurately weighed into a lOOmL volumetric flask, add 90mL of water, ultrasonic treatment (power 250W, frequency 20KHz) for 30 min, take out, let cool, add water to volume, filter, and get.

2. 3. Study of chromatographic conditions

2. 3. 1 Selection of detection wavelength

In order to ensure the maximum information quantification of the map, the whole composition of the mantle component is reflected as much as possible, and the components are roughly separated by a binary gradient elution of methanol: phosphate buffer salt, and the column temperature is 30 °C. Collect the chromatographic components at 195 ηπ using a diode array detector (DAD)! The information of all the chromatographic spectra in the ~400 nm spectral region was compared. The absorption of each peak in the chromatogram at each wavelength was compared. The absorption at each peak was larger at 265 nm, and the baseline performance was stable. Therefore, 265 nm was finally determined as the detection wavelength.

2. 3. 2 chromatographic conditions

Several mobile phases were tested during the test to determine the optimum mobile phase, see Table 1.

Mobile phase and elution procedures

2. 3. 3 durability inspection

The same batch of enamel extracts were taken and tested using three different brands and manufacturers' columns. The chromatographic peak resolution and the number of chromatographic peaks were used as evaluation indexes to examine the durability of the instrument.

Under the selected chromatographic conditions, the obtained chromatogram and peak separation effect of the Global Column is superior to the other two columns. Therefore, considering the comprehensive consideration, choose the Global Column.

Through the above tests, the chromatographic conditions were determined as follows: Global chromatography column (4.6 mm X 250 mm, 5 m) _; DAD detector; methanol_0. 05% phosphate buffer salt gradient elution procedure: 0 min→80 min, methanol 5%→45%, 0. 05 % phosphate buffer salt 95% → 55%; 80 min → 90 min, methanol 45% → 55 %, 0. 05% phosphate buffer salt 55 % → 45%; 90 min → 91 min, methanol 55 % → 5%, 0 05% phosphate buffer salt 45% → 95%; 91min → 100min, methanol 5% → 5%, 0. 05% phosphate buffer salt 95% → 95%; detection wavelength 265nm; column temperature 30 ° C; O ml/min; Operating time: 100 min.

2. 3.4. Specificity research

Prepare the test solution according to the method of "2", and check according to the chromatographic method under "3". Record the peak and its retention time and peak area within 90 min. The aqueous solution and the mixed reference solution ΙΟ μ Ι were separately extracted and injected into a high performance liquid chromatograph. The 90 min chromatogram was recorded, and the spectrum of each control substance was recorded. The results showed that there was no interference under the conditions.

2. 3. 5 Determination of content

3. Determination of total saponins and saponins I in mantle extracts. The determination methods are as follows:

1) Method for determination of total saponin content in mantle

Preparation of the reference solution Take 5mg of saponin I in a 10ml volumetric flask, add about 9ml of absolute ethanol, solubilize in the air, let cool, add anhydrous ethanol to the mark, that is, (0.5mg per lml).

Preparation of the standard curve Precision draw reference solution 0, 200, 300, 400, 500, 600, 800, 1000 μ ΐ separately placed in a 10 ml stoppered test tube, precision added to the newly prepared 8% vanillin ethanol solution (take vanilla 8g of aldehyde, make up to 100ml with absolute ethanol) lml, 70% sulfuric acid 10ml (take 70ml sulfuric acid, slowly inject into 30ml water, that is), shake well, close the plug, put 60 ° C water bath for 15min, put into ice water After cooling for 2 min, shake well, take the colorimetric solution without reference to the blank, and measure the absorbance at 530 nm by spectrophotometry (Appendix VIB). The absorbance (A) is the ordinate and the concentration (C) is On the abscissa, draw a standard curve.

Prepare the test solution for about 5g of extract of earthworm extract, grind finely, accurately weigh it, place it in a 250ml volumetric flask, add about 200ml of absolute ethanol, ultrasonically extract for 30min, filter, and collect the filtrate in a 250ml volumetric flask. Make up to 250 ml with absolute ethanol.

Determination Precision ligation of the test solution lml, placed in a 10ml test tube with a plug, according to the method under the standard curve to determine the absorbance according to law, substituted into the regression equation calculation, that is.

2) Study on the determination method of the content of saponin I

Chromatographic conditions Column, Agilent-Zorbax (4. 6mm X 250mm, 5 m); mobile phase, acetonitrile: water (32: 68); flow rate, lml/min; column temperature 40 ° C; evaporative light scattering detector.

Preparation of reference solution Take 25mg of saponin I in a 25ml volumetric flask, add about 20ml of absolute ethanol, dissolve in the ultrasonic solution, let cool, add the absolute volume of absolute ethanol, that is, get 1mg per lml.

Preparation of the standard curve accurately draw the above reference solution 1, 2, 3, 4, 5ml, respectively, placed in a 5ml volumetric flask, add absolute ethanol to the mark, accurately absorb each solution 10 μ ΐ, according to the above The chromatographic conditions were determined by taking the natural logarithm (Y) of the peak area as the ordinate and the natural logarithm (X) of the injection amount as the abscissa to draw a standard curve.

Prepare the test solution for about 5g of extract of earthworm extract, grind finely, accurately weigh it, place it in a 100ml volumetric flask, add about 90ml of absolute ethanol, ultrasonically extract for 30min, let cool, and dilute to volume with absolute ethanol. Shake well and filter through 0.45μπι microporous filter.

Determination Precision extraction of the test solution ΙΟμ Ι, according to the above chromatographic conditions, determination, substituted into the regression equation calculation, that is. Example 1 Preparation of the enamel extract of the present invention

The medicinal materials were pulverized into coarse powder, and 70% acetone was extracted twice by flash, 2 min each time, the amount of solvent was 10 times for the first time, and the second time was 8 times. The filtrate was combined, concentrated under reduced pressure, and diethyl ether and concentrate 1 : 1 volume ratio extraction degreasing to ether layer colorless, then extracting total phenol 6 times with ethyl acetate and mother liquor 1:1 volume ratio, combined with ethyl acetate, concentrated at 45 ° C under reduced pressure to an appropriate amount, 45 ° C Dry under reduced pressure.

The results of the content determination were: extract containing 62% enamel, 1.32% gerberin, 2.61% catechin, proanthocyanidin B20.08%, saponin: 6.2%, saponin 1: 3.7%, all weight Percentage. Example 2 Preparation of the enamel extract of the present invention

The medicinal materials were pulverized into coarse powder, and 70% ethanol was refluxed for 3 times, each time for 2 hours, the amount of solvent was 10 times for the first time, the second time was 8 times, the third time was 6 times, the filtrate was combined, and concentrated under reduced pressure. , diethyl ether and concentrate 1:1 volume ratio degreasing to ether layer colorless, and then extracting total phenol 6 times with ethyl acetate and mother liquor 1:1 volume ratio, combined with ethyl acetate, concentrated at 45 ° C under reduced pressure To the right amount, dry at 45 ° C under reduced pressure.

The results of the content determination were as follows: extract containing 65% enamel, 1.41% gerberin, 2.65% catechin, proanthocyanidin B 20.09%, saponin: 5.4%, saponin 1: 3.1%, all weight Percentage. Example 3 Preparation of the enamel extract of the present invention

The medicinal materials were pulverized into coarse powder, and 70% ethanol was refluxed for 3 times, each time for 2 hours, the amount of solvent was 10 times for the first time, the second time was 8 times, the third time was 6 times, the filtrate was combined, and concentrated under reduced pressure. , diethyl ether and concentrate 1:1 volume ratio degreasing to ether layer colorless, the concentrate is separated by macroporous resin, first eluted with water to colorless, then eluted with 2 column volumes of 10% ethanol, and finally 2 The column volume was eluted with 60% ethanol, and the 60% ethanol eluate was collected, concentrated under reduced pressure at 55 ° C to an appropriate amount, and spray-dried.

The results of the content determination were: extract containing 73% enamel, mantle pigment 1.52%, catechin 2.88%, proanthocyanidin B 20.11%, saponin: 3.2%, saponin 1: 1.7%, all weight Percentage. Example 4 Preparation of the enamel extract of the present invention

The medicinal materials were pulverized into coarse powder, and 70% ethanol was refluxed for 3 times, each time for 2 hours, the amount of solvent was 10 times for the first time, the second time was 8 times, the third time was 6 times, the filtrate was combined, and concentrated under reduced pressure. The precipitate was removed, and the supernatant was separated by macroporous resin, first eluted with water until colorless, and then washed with 2 column volumes of 10% ethanol. The mixture was finally eluted with 2 column volumes of 60% ethanol, and a 60% ethanol eluate was collected, concentrated to a suitable amount by a membrane, and then spray-dried to obtain.

The results of the determination were as follows: extract containing 79% enamel, citrate 1. 72%, catechin 3.24%, proanthocyanidin B2 0. 13%, saponin: 2. 4%, saponin 1 : 1. 3%, all in weight percent. Example 5 Preparation of the enamel extract of the present invention

The ground medicinal herbs are pulverized into coarse powder, decoctioned for 3 times, each time for 2 hours, water is added for the first time 10 times, the second time is 8 times, the third time is 6 times, the filtrate is combined, concentrated under reduced pressure, concentrated to Appropriate amount, remove the precipitate, the liquid is separated by macroporous resin, first eluted with water to colorless, then eluted with 2 column volumes of 10% ethanol, and finally eluted with 2 column volumes of 60% ethanol to collect 60%. The ethanol eluate was concentrated to a suitable amount under reduced pressure at 55 ° C, and then spray dried to obtain.

The results of the determination were as follows: extract containing 61% enamel, citrate 1.22%, catechin 2.43%, proanthocyanidin B2 0. 07%, saponin: 6. 9%, saponin 1 : 4. 3%, all in weight percent. Example 6 Preparation of the enamel extract of the present invention

The ground medicinal herbs are pulverized into coarse powder, decoctioned for 3 times, each time for 2 hours, water is added for the first time 10 times, the second time is 8 times, the third time is 6 times, the filtrate is combined, concentrated under reduced pressure, concentrated to Appropriate amount, remove the precipitate, the liquid is separated by macroporous resin, first eluted with water to colorless, then eluted with 2 column volumes of 10% ethanol, and finally eluted with 2 column volumes of 60% ethanol to collect 60%. The ethanol eluate is concentrated to a suitable amount through a membrane, and then spray-dried to obtain.

The results of the determination were: extract containing 67% enamel, citrate 1. 34%, catechin 2. 59%, proanthocyanidin B2 0. 08%, saponin: 5. 5%, saponin 1 : 3. 4%, all in weight percent. Example 7 Preparation of the enamel extract of the present invention

The medicinal materials were pulverized into coarse powder, and 70% ethanol was extracted twice by flash, 2 min each time, the amount of solvent was 10 times for the first time, and the second time was 8 times. The filtrate was combined, and the membrane was concentrated to an appropriate amount. The pore resin was separated, first eluted with water until colorless, then eluted with 2 column volumes of 10% ethanol, and finally eluted with 2 column volumes of 60% ethanol. The 60% ethanol eluate was collected and concentrated through the membrane. Appropriate amount, then spray drying, that is.

The results of the determination were as follows: extract containing 78% enamel, gentiana 1.69%, catechin 3. 35%, proanthocyanidin B2 0. 13%, saponin: 3. 1%, saponin 1 : 1. 4%, all in weight percent. Example 8 Preparation of the enamel extract of the present invention

Take the medicinal material coarse powder, dilute with 12 times the amount of water at 60 ° C for 2 times, each time for 3 hours, filter, combine the filtrate, concentrate to 0. 5g crude drug / ml, placed in a centrifuge, centrifuged at 4000 / rpm for 10min Take the supernatant and slowly apply to the D-101 type macroporous adsorption resin at a flow rate of 2BV/h. The aspect ratio is 1:5. After static adsorption for 30 minutes, wash with 2BV/h with 2BV/h, and then use 1BV for 10%. The ethanol was washed at 2 BV/h, washed with 2 BV 60% ethanol at 1 BV/h, and the eluate was collected, concentrated, and dried.

The content determination results were as follows: The extract contained 55% enamel. The extraction rate of the enamel of the invention is as high as 13% On. Saponin: 7. 2%, saponin I: 4.9%.

Among them, 10 batches of the mantle prepared in this example were selected, and the contents of the genistein, catechin, proanthocyanidin B2 were determined, and the results were as follows:

Table 2 Determination of extracts from ten batches of mantle extract

The average content of scutellarin, catechin and proanthocyanidin B2 were: 1.61%, 3.02%, 0.12%, all in weight percent.

Example 9 Preparation of enamel extract of the present invention

The ground medicinal material is pulverized into a coarse powder, and decoctioned twice a time, each time 1. 5h, adding water for the first time 10 times, the second 8 times, combining the filtrate, concentrating under reduced pressure, concentrating to an appropriate amount, removing the precipitate, The drug solution was separated by macroporous resin, eluted with water to colorlessness, eluted with 2 column volumes of 10% ethanol, and finally eluted with 3 column volumes of 60% ethanol to collect 60% ethanol eluate. Concentrate to a suitable amount under reduced pressure, then spray-dry, add water to dissolve, place for lh, filter the precipitate, the filtrate is placed in an evaporating dish, slowly sprayed into the gelatin solution, placed for 1 h, collected and precipitated, freeze-dried for 12 h, taken out, ground into subdivided, 5%。 The lyophilic content of the enamel is 98.5%. The enamel content is 98.5%.

1. The content of scutellarin, catechin and proanthocyanidin B2 is: scorpion 2.29%, catechin 4. 85%, proanthocyanidin B2 0. 38%

2, determination of tannin content

According to the Chinese Pharmacopoeia 2010 edition of an appendix X B enamel content determination:

This experiment should be protected from light.

Preparation of reference solution: Accurately weigh 48. 30mg of gallic acid reference substance, place it in a 100ml brown volumetric flask, dissolve in water and dilute to the mark, accurately measure 5ml, place in a 50ml brown volumetric flask, dilute with water to the mark, shake up , that is, (0,0483mg per gallon). Preparation of standard curve: Accurately measure the reference solution 0.5ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, respectively, in a 25ml brown volumetric flask, add 1ml of phosphomolybdate solution, add water separately 11.50ml, llml, 10ml, 9ml, 8ml, 7ml, dilute to the mark with 29% sodium carbonate solution, shake well, place for 30 minutes with the corresponding reagent as blank, according to UV-Vis spectrophotometry (Appendix VA), at 760nm The absorbance is measured at the wavelength, and the absorbance is plotted on the ordinate and the concentration is plotted on the abscissa. A standard curve is drawn.

Concentration (yg/mi) absorbance (A) regression equation

0.966 0.154

1.932 0.276

3.864 0.511 Y =0.118 X + 0.046 {if = 0.999)

5.796 0.731

7.728 0.961

9.66 1.184 Preparation of test solution: Take three extracts of 0.101, 0.105, 0.997g, accurately weighed, place in a 250ml brown volumetric flask, add 150ml of water, leave it overnight, sonicate for 10 minutes, let cool, dilute with water until Scale, shake, stand still (precipitate solid matter), filter, discard 50ml of the initial filtrate, accurately measure 20ml, place it in a 100ml brown volumetric flask, dilute with water to the mark, shake well, that is.

Assay

Total phenol: Precisely measure 2ml of the test solution, place it in a 25ml brown volumetric flask, according to the method under the preparation of the standard curve, add 10ml of water from the "addition of phosphomolybdate test solution lml", and measure the absorbance according to law. The standard curve reads the amount (mg) of gallic acid in the test solution, which is calculated and obtained.

Polyphenols that are not adsorbed: Precisely measure 25ml of the test solution, add it to a 100ml stoppered conical flask containing 0.6g of casein, and put it in a 30°C water bath for 1 hour. Shake, take out, let cool, shake well, filter, discard the primary filtrate, accurately measure 2ml of the filtrate, place it in a 25ml brown volumetric flask, according to the method under the preparation of the standard curve, from "adding phosphotungstic acid test solution From lml", add 10ml of water, measure the absorbance according to law, read the amount (mg) of gallic acid in the test solution from the standard curve, and calculate.

Sample Sample size (g) Total phenol (%) Polyphenols that are not adsorbed (%) Tannins contain i 1 (%) Average

1 0.101 98.9 0.6 98. 3

2 0.105 98.7 0.3 98. 4 98.5

3 0.997 99.3 0.5 98. 8

3. Determination of total saponins and saponins I in mantle extract:

Three batches of enamel extract were determined, and the peak of saponin I was not detected by HPLC analysis. 0001%。 The content of the total saponin content of the mantle is 0. 001, it can be inferred that the total saponin content of the mantle < 0. 001% and the content of saponin I <0. 0001%. Therefore, when the content of tannin is 98% or more, the contents of total saponins and saponins I of the mantle are almost zero.

According to the results of the determination of quercetin, catechin and proanthocyanidin B2 in the examples 1-9, the content of the quercetin, catechin and proanthocyanidin B2 in the enamel extract of the present invention is relatively stable, which accounts for The 5% of the extract is 0. 05% - 3. 0% w / w, catechin 1. 0% - 5. 0% w / w, proanthocyanidin B2 0. 05%_1. 5% w/w. The phytocyanin B2 is 0. 0-2. 0% w/w, proanthocyanidin B2, respectively, the content of the quercetin, the catechin, and the proanthocyanidin B2 is 1. 0-2. 0% w/w, catechin 2. 0-4. 0% w/w, proanthocyanidin B2

Example 10 Optimization of enamel purification by macroporous resin

1 inspection of resin types

1. 1 Test method After the macroporous resin is dried, weigh 15g separately, put it into a 100ml stoppered conical flask, soak it in 50ml of 2% hydrochloric acid for 3h, then rinse it with water until the pH is neutral, then add 2 separately. The %Na0H solution was soaked in 50 ml for 3 h, then rinsed with water until the pH was neutral, and activated by adding 60 ml of 95% alcohol, respectively. Separately extract the mantle extract (0.33 g/ml) and place 50 ml in an activated conical flask filled with macroporous resin to completely immerse the macroporous resin in the macroporous resin for 24 hours. Then determine the volume of the remaining liquid in the conical flask, determine the adsorption amount of the macroporous resin; remove the macroporous resin in the conical flask, soak the ultrasonic with 95% ethanol for 1 h, and then determine the content of tannin in the eluate, and then according to The elution rate of the liquid enamel before and after adsorption was determined.

Maximum adsorption amount = (pre-adsorption enamel content - enamel content after adsorption) / macroporous resin quality

Elution rate = enamel content in the eluent / (the enamel content of the drug solution before adsorption - the enamel content after adsorption) According to DA-201, D-101, LSA-20, HP-10 and AB-8 The maximum adsorption amount and elution rate of the macroporous resin determine the type of the resin.

1. 2 Test results The test data is shown in Table 3.

Table 3 Screening test of resin types

Resin type Maximum adsorption capacity (g / g) Elution rate (%)

DA-201 0. 106 72. 41

D-101 0. 120 76. 52

LSA-20 0. 097 62. 76

HP-10 0. 102 68. 45

AB-8 0. 104 64. 33 Through the above experiments, it can be confirmed that the macroporous resin has a good adsorption and elution ability to the mantle. Therefore, a macroporous resin can be selected for the purification of the tannin. In the present invention, D-101 is selected as the preferred resin. .

2 D-101 macroporous resin leakage curve

The pretreated D-101 macroporous resin was packed in a column of 20 g, the column volume was 30. 4 cm ³ , and the aspect ratio was 1:4. The cellar extract 60 ml (0.33 g of raw medicinal material/ml) was dynamically loaded. The flow rate was controlled to be 0.77 ml/min, and it was made constant. The effluent was collected from the bottom of the column, and 5 ml was collected per bottle. A total of 11 bottles were collected, and the content of enamel in each effluent was measured. Further, the leakage curve of the macroporous resin was determined. The leakage curve of the macroporous resin is plotted with the volume as the abscissa and the tannin content as the ordinate.

Accurately remove 1ml from each bottle, dilute to 100ml with water, accurately measure 2ml of test solution, place 25ml brown volumetric flask, add 1ml of phosphomolybdate solution, add 10ml of water, then use 29% sodium carbonate solution The volume is allowed to stand, and the absorption value is measured after standing for 30 minutes, and the amount (mg) of gallic acid in the test solution is calculated from the standard curve, that is, it is obtained.

Table 4 D-101 macroporous resin leakage curve data

In summary, the drug solution began to leak after 30ml. The optimal loading volume is 30ml, the concentration of the chemical solution is 0.33g/ml, and the amount of macroporous resin is 20g. Therefore, the ratio of the best medicinal material to the macroporous resin is 1:2.

3 inspection of the elution curve

0. The diameter of the column is 30. 4cm ³ , the diameter-to-height ratio is 0. 3cm ³ , the diameter of the column is 30. 4cm ³ , the diameter-to-height ratio is 1 : 4, adsorption lh, make it saturated, elute with 1 column volume of water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol, control the flow rate constant, collect and elute The liquid was measured for its tannin content, and the effect of different eluent concentrations on the adsorption of macroporous resin was compared.

Take 0.5 ml of eluent, dilute to 100 ml, accurately measure 2 ml of the test solution, place 25 ml of brown volumetric flask, add 1 ml of phosphomolybdate solution, add 10 ml of water, and then dilute with 29% sodium carbonate solution. The scale was measured after 30 minutes of standing, and the amount (mg) of gallic acid in the test solution was read from the standard curve. The test data is shown in Table 5. Table 5 Effect of different elution solvents on the elution curve

Considering the elution curve and the actual production cost, the tannin was eluted with 60% ethanol.

4 uniform design optimization process parameters

Taking the tannin content, transfer rate and total evaluation value (0D value) as the evaluation index, U ₅ (5 ⁴ ) uniform design table was used to investigate the ratio of diameter to height ratio, concentration of chemical solution, flow rate and 60% alcohol consumption. The effect of quality purification. The uniform design table arrangement factors and levels are shown in Table 6. The test data is shown in Table 7:

Table 7 Uniform design test data sheet

After analysis by spssl7.0 software, the main output results were selected, R2=0. 85, and the analysis showed that F=16. 192, P=0. 026, stepwise regression was statistically significant.

a. Predictor variables: (constant), x3.

b. Dependent variable: y

Equation coefficient, x ₃ partial regression coefficient t statistic = -4.11, unilateral P = 0.026, should be retained, ie The regression equation is y=1. 092— 0. 172. Since the regression equation x ₃ is a negative sign and does not contain _Xl , x ₂ , x ₄ terms, χ ₃ should take the minimum value of the test range. According to the actual, _Xl =l: 5, _3⁄4 =2, χ ₄ =0. 5 ο The approximate estimation of the most advantageous is: xw, SP, the optimum process parameters are: aspect ratio 1: 5, the concentration of the chemical solution is 0.5. , flow rate lBV / h,

The amount of 60% alcohol is 2 times.

a. Dependent variable: y

5 Macroporous Resin Resin Purification Mantle Process Verification Test

According to the above optimal process, the predicted value of the 0D value is 0.92, and the result is verified.

Table 8 Process Verification (HPLC)

The results show that the purification process results are more stable.

6 summary

According to the above experimental results, the purification conditions of the proposed mantle are: taking the mantle extract, concentrating to 0. 5g crude drug / ml, placed in a centrifuge, centrifuged at 4000/rpm for 10 minutes, the supernatant was taken, and the flow rate was 2 BV/h. Slowly apply to D-101 macroporous adsorption resin, diameter to height ratio 1: 5, static adsorption for 30min, wash with 2BV/h with 2BV water, then wash with 2BV10% ethanol at 2 BV/h, then use 2 BV60 The % ethanol was washed at 1 BV/h, and the eluate was collected, concentrated, and dried.

In the application process, other macroporous adsorption resins can also be selected to purify the mantle. The yield of the mantle extracting method reported by the prior art is low, all below 10%; the extraction rate of the method of the present invention is high, reaching 13% or more. The beneficial effects of the present invention are demonstrated by specific pharmacodynamic tests below.

Test Example 1 The protective effect of the enamel bone defect of the present invention

1. Earthworm extract

5%。 The high purity of the enamel prepared in Example 9 having a tannin content of 98.5%.

2. Study on the effect of enamel on chemical inhibition caused by osteophytes

2. 1 Experimental materials KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Japan Photoelectric Industry Co., Ltd.; Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; high purity enamel (more than 98% enamel content); cyclophosphamide for injection Recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75 g); normal saline.

2. 2 Experimental methods

2. 2. 1 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. Sixty mice were completely randomized into 6 groups according to gender (see Table 9 for specific grouping and administration, and ig administration for 3 days before modeling). On the 4th day, except for the blank group, ip was given the same volume of normal saline. The other groups were given ip cyclophosphamide 100 mg/kg-d on the day of the mice, except for the western medicine positive group, and the ig administration was continued for 6 days from the day of modeling. RhG_CSF was injected subcutaneously on the 7th to 9th day.

On the 7th day after model establishment, blood was collected from the eyelids. The peripheral blood routine parameters including white blood cell WBC, red blood cell RBC, hemoglobin HGB, and platelet PLT were measured by automatic blood cell counter. The spleen was weighed and the spleen coefficient was calculated. The mice were sacrificed and the left femur was removed. The soft tissue was removed. All the bone marrow was flushed into the test tube with 0. 005 mol/L CaC12 10 mL, placed in a refrigerator at 30 °C for 30 min, centrifuged at 2500 r/min for 15 min, and the supernatant was discarded. The precipitate was added with 0.2 mol/L HC104 5 mL, mixed well, heated at 90 ° C for 15 min, centrifuged at 3500 r / min for 10 min after cooling, and the supernatant was taken, and the 0D value was measured at a wavelength of 260 nm using a microplate reader.

The experimental data were expressed as mean±S.D., using SPSS software, statistical test using group t test and single factor F test, test level α =0. 05.

2. 2. 3 results analysis

The effects of mantle on leukocyte, erythrocyte, thrombocytopenia and spleen coefficient in peripheral blood of mice induced by cyclophosphamide are shown in Table 10. The intraperitoneal injection of cyclophosphamide in mice significantly reduced the number of peripheral blood leukocytes, red blood cells, hemoglobin and platelets, and the spleen coefficient and bone marrow DNA content were significantly lower than those in the blank group. Compared with the model group, the high, medium and low dose groups of the mantle and rhG-CSF can significantly increase the number of white blood cells (P<0.05) and increase the spleen coefficient. The high dose of mantle can significantly increase the number of peripheral red blood cells and platelets, and the content of hemoglobin in rhythm and rhG-CSF. The effect was not significant; the high dose group of mantle could significantly increase the DNA content of bone marrow (P<0.05), while rhG-CSF would damage the bone marrow DNA content.

Table 10 Study on the effect of enamel on chemical substances caused by myelosuppression

(mean + S. D., n=10 )

△: Compared with the model group P<0.05

It can be seen from the above experiments that rhG-CSF has a strong whitening effect, which can significantly increase the number of white blood cells over the normal group (P < 0.05), but rhG-CSF does not protect the bone marrow, and accelerates the differentiation of bone marrow cells by stimulation. Further aggravating the load of hematopoietic stem cells, which is one of the causes of side effects such as bone pain. rhG-CSF is one of the main cytokines regulating granulocyte hematopoiesis in bone marrow. It has significant therapeutic effects on leukocyte, erythrocyte and thrombocytopenia caused by radiotherapy and chemotherapy. Its whitening mechanism is selective for granulocyte hematopoietic progenitor cells. It directly promotes its proliferation and differentiation, and can increase the function of the terminally differentiated cells of the granulosa, so that the white blood cells rise rapidly.

The enamel of the present invention has significant protective effect on the bone marrow inhibition of the chemical substance cyclophosphamide, including the peripheral blood leukocytes, red blood cells, hemoglobin and platelet count, and the decrease of bone marrow DNA content (P<0.05). , and presents a certain dose-effect relationship: high > medium > low. The purity of the mantle produced by the present invention is as high as 98% or more. The test proves that the mantle has obvious therapeutic effects on the inhibition of bone marrow caused by chemicals and leukocyte, red blood cell and thrombocytopenia.

3. Study on the effect of earthworm enamel on osteophyll inhibition caused by radiation

3. 1 experimental materials

KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Japan Photoelectric Industry Co., Ltd.; Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; mantle (more than 98% tannin content); cobalt 60-ray external camera; Reorganization Human granulocyte colony-stimulating factor (rhG-CSF, 75 g); normal saline.

3. 2 experimental methods

3. 2. 1 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. 60 mice were completely randomized into 6 groups according to gender (specific grouping and administration were the same as "the study of the effect of enamel on chemical substances caused by bone marrow suppression"), and prevention of ig administration before modeling 3d. 5cy, the dose rate is 82. 6cGy / min, the dose is 82. 6cGy / min, the dose is 82. 6cGy / min , the irradiation distance is 80cm. Except for the western medicine positive group, ig administration was continued for 6 days from the day of modeling. RhG_CSF was injected subcutaneously on days 7~9.

3. 2. 2 Evaluation indicators and data processing

In this experiment, the number of white blood cells WBC was used as an observation index.

The experimental data are expressed as mean±S.D., using SPSS software, statistical test using group t test and single factor F test, test level α =0. 05.

3. 3 results analysis

The effect of mantle on the bone marrow suppression induced by cobalt ray in mice is shown in Table 11.

Table 11 Effect of mantle on bone marrow suppression induced by cobalt ray (mean ± S. D., n=10 )

Δ : P<0.05, compared with the model group; in addition, the mantle also has a rising trend on red blood cells, platelets, etc. (P<0.05) o

From the results, it can be seen that rhG-CSF has a strong whitening effect, which can significantly increase the number of white blood cells over the normal group (P<0.05), but with the study on the effect of enamel on chemical substances caused by bone marrow suppression. "Same, the bone marrow DNA content of the rhG-CSF group was decreased.

The enamel had significant protective effects on the DNA content of bone marrow and leukopenia in mice induced by chemical substances (P<0.05), and showed a dose-effect relationship: high>medium>low. The purity of the enamel of the present invention is higher than 98%, and the test proves that the enamel inhibits the bone marrow caused by radiation and white fine Cell, erythrocyte and thrombocytopenia have significant therapeutic effects.

Test Example 2 Study on the efficacy of the enamel and its components of the present invention

It has been reported in the literature that the mantle contains mantle, catechin, proanthocyanidin B2, gallocatechin, catechin gallate, epicatechin, epigallocatechin gallate (mantle) Component Analysis and Functional Study of Phenol, Liu Haiying, Shaanxi Normal University, 2009) Therefore, the following experiments were designed to reveal the main active substances in the tannin of the present invention and to compare the activity with a single compound.

1. Study on the effect of mantle and its components on bone marrow suppression caused by chemical substances

1. 1 Experimental materials

KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Japan Photoelectric Industry Co., Ltd.; Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; cyclophosphamide for injection; recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75 g ; physiological saline; high-purity enamel (prepared in Example 9, enamel content 98% or more); quercetin (HPLC > 99%); catechin (HPLC > 99%); proanthocyanidin B2 (HPLC 〉99%); gallocatechin (HPLC>99%); catechin gallate (HPLC>99%); epicatechin (HPLC >99%); epigallocatechin gallate (HPLC) 〉99%), the above monomer compounds were purchased from Chengdu Purifa Technology Co., Ltd.

1. 2 Experimental methods

1. 2. 1 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. 110 mice were completely randomized into 11 groups according to gender (see Table 12 for specific grouping and administration), and ig administration was prevented for 3 days before modeling. On the 4th day, except for the blank group ip to give an equal volume of normal saline, the other groups were given the weight of the mouse ip cyclophosphamide 100 mg/kg - d for 3 days, except for the western medicine positive group, and continued from the day of modeling. Medicine 6d. 7th~

The experimental data is expressed as mean±S. D., using SPSS

And single factor F test, test level α = 0.05.

1.2.3 Analysis of results

Table 13 Effect of enamel and its components on chemical substances caused by myelosuppressive drugs

(mean + S. D., n=10)

Δ : Ρ < 0.05, compared with the model group; A: P < 0.05, the mantle is compared with catechin; in addition, the mantle also has an increasing trend on red blood cells, platelets, etc. (P < 0.05).

The above experimental results show that the components of catechin, quercetin, gallocatechin, catechin gallate and other components contained in the mantle have certain inhibition of mouse bone marrow induced by cyclophosphamide. The therapeutic effect, in which quercetin, catechin and proanthocyanidin B2 had significant effects (P<0.05), and the geranin was the most potent, the catechin was the second, and the proanthocyanidin B2 was slightly weak. In addition, at the same dose, the effect of the mantle is stronger than that of the elements such as quercetin and catechin, revealing a synergistic effect between the components.

2. Study on the effect of mantle and its components on radiation-induced myelosuppression

2.1 Experimental materials

KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Japan Photoelectric Industry Co., Ltd.; Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; cobalt 60-ray external camera; recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75) g); physiological saline; high-purity enamel (obtained in Example 9, having a tannin content of 98% or more); Hirudin (HPLC >99%); catechin (HPLC >99%); proanthocyanidin B2 (HPLC >99%); gallic catechin (HPLC >99%); catechin gallate (HPLC > 99) %); epicatechin (HPLC)

>99%); Epigallocatechin gallate (HPLC > 99%).

2. 2 experimental methods

2. 2. 1 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. 110 mice were completely randomized into 11 groups according to gender (specific grouping and administration were the same as "Study on the effect of sputum and its components on chemical substances caused by bone marrow suppression"), before modeling Prevention of ig administration for 3d. 5cy, the dose rate is 82. 6cGy / min, the dose is 82. 6cGy / min, the dose is 82. 6cGy / min , the irradiation distance is 80cm. Except for the western medicine positive group, ig administration was continued for 6 days from the day of modeling. RhG_CSF was injected subcutaneously on days 7~9.

2. 2. 2 Evaluation indicators and data processing

Same as test case 1.

2. 3 results analysis

Table 14 Study on the effect of enamel and its components on radiation-induced myelosuppression

(mean Shi S. D., n=10 )

Δ : Ρ<0. 05 , compared with the model group; A : P<0.05, the comparison of the mantle and catechin; The extract of the earthworm also has an increasing trend on red blood cells, platelets, etc. (P < 0.05).

Experiments have shown that catechins, scutellarin, gallocatechin, catechin gallate and other components contained in the mantle have certain therapeutic effects on radiation-induced mouse bone marrow suppression. The scutellarin, catechin and proanthocyanidin B2 had significant effects (P<0.05), and the geranin had the strongest effect, the proanthocyanidin B2 was the second, and the catechin was slightly weaker. In addition, at the same dose, the effect of the mantle is stronger than that of the elements such as stilbenin, catechin, and proanthocyanidin B2, and there is a synergistic effect between the components.

In summary, the main active components of the sputum caused by chemical damage and radiation damage to myelosuppression and leukocyte, erythrocyte and thrombocytopenia are scutellarin, proanthocyanidin B2, catechin, and the treatment of two models of scorpion The effects are strong, while catechin and proanthocyanidin B2 have different emphasis, catechin has a better protective effect on chemical damage, and proanthocyanidin B2 focuses on the protection of physical damage. It has also been shown by the above studies that there is a synergistic effect between the components of the mantle.

The above experiments fully demonstrate that the mantle can counteract the myelosuppression caused by chemical damage and radiation damage, and the resulting white blood cells, red blood cells, and thrombocytopenia, which can be predicted to be anti-tumor drugs due to its protective effect on bone marrow. When used in combination, it has a very optimistic clinical application prospect.

Since the prior art all considers that the saponin is an active substance for raising white blood cells, the inventors have carefully conducted a series of experiments to further study the material basis of the mantle whitening effect.

Test Example 3 Material basis study on the whitening effect of the enamel of the present invention

1. Experimental materials

KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Nippon Optoelectronics Co., Ltd.); Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; high purity enamel (more than 98% enamel content, prepared in Example 9); Cyclophosphamide for injection; recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75 g); physiological saline.

2, the experimental method

2.1 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. Seventy mice were completely randomized into 7 groups according to gender (see Table 15 for specific grouping and administration), and ig was given for 3 days before modeling. On the 4th day, except for the blank group ip to give an equal volume of normal saline, the other groups were given the same day weight ip cyclophosphamide 100 mg/kg - d for 3 consecutive days, except for the western medicine positive group, and continued from the day of modeling. Medicine 6d. 7th~

The experimental data were expressed as mean±S.D., using SPSS software, statistical test using group t test and single factor F test, the test level α = 0.05.

3, the result analysis

According to the results of WBC experiment in Table 16, the results of WBC experiment showed that there was a significant difference between the blank group and the model group (/0.05), indicating that the animal model of leukopenia caused by cyclophosphamide was successful. 2 There was a significant difference between the high and low dose groups, the positive group and the model group (<0.05), indicating that the mantle had a good therapeutic effect on cyclophosphamide-induced leukopenia in mice; There was no significant difference between the drug-administered group and the model group (>0.05), and the other drug-administered groups had no dose-effect relationship on the efficacy of cyclophosphamide-induced leukopenia in mice. : High dose > low dose (^Ο. 05).

Table 16 White blood cell count WBC test results (mean Shi S.D., n=10)

△: Compared with the model group P<0.05

☆: Compared with the enamel group? <0. 05

Test Example 4 Material basis study on the whitening effect of the extract of the enamel extract of the present invention

1. Experimental materials

KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Nippon Optoelectronics Co., Ltd.); Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; mantle extract (manufactured in Example 8, extract containing enamel 55 % , , , 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 2 2 2 7 2 2 2 2 2 2 2 2 2 7 2 2 2 2 2 7 %, saponin I was 4.9%); cyclophosphamide for injection; recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75 g); physiological saline.

2, the experimental method

2. 1 grouping and administration

The experimental animals were kept in the Laboratory of Pharmacological Experimental Animals of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally given. Seventy mice were completely randomized into 7 groups according to gender (see Table 17 for specific grouping and administration), and ig administration was prevented for 3 days before modeling. 4th day except blank Group ip was given an equal volume of normal saline, and the other groups were given ip cyclophosphamide 100 mg/kg -d on the day of the mice, except for the western drug positive group for 3 consecutive days. The ig administration was continued for 6 days from the day of modeling. Subcutaneous injection of rhG_CS on days 7 to 9

3, the result analysis

According to the results of WBC experiment in Table 18, there was a significant difference between the blank group and the model group (/ 0.05), indicating that the animal model of leukopenia caused by cyclophosphamide was successful. 2 There were significant differences between the high and low dose groups, the positive group and the model group (< 0.05), indicating that the mantle had a good therapeutic effect on cyclophosphamide-induced leukopenia in mice; There was no significant difference between the group and the model group (>0.05), and the other drug-administered groups had no dose-effect relationship on the efficacy of cyclophosphamide-induced leukopenia in mice. Low dose (^Ο.05).

Table 18 White blood cell count WBC test results (mean±S.D.,

△: Compared with the model group Ρ<0.05

☆: Compared with the enamel group? <0.05

4 Conclusion

The tests of Test Example 3 and Test Example 4 showed that the enamel extract of the present invention has a significant therapeutic effect on cyclophosphamide-induced leukopenia (<0.05), and at the same time, gelatin removal is used. After the enamel component in the mantle extract, there is no therapeutic effect, further demonstrating that the enamel is an effective component for treating leukopenia.

Test Example 5 Study on the whitening of earthworm saponins and the protection of osteophytes

1 experimental material

KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Experimental Animal Research Institute of Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Japan Photoelectric Industry Co., Ltd.; Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; mantle saponin extract (98.18%); cyclophosphamide for injection; recombinant human granulocyte Colony stimulating factor (rhG-CSF, 75 g); saline.

Preparation of mantle saponin: The medicinal materials of the mantle are pulverized into coarse powder, and the mixture is extracted with 70% ethanol for 3 times, 1.5 h each time, concentrated at 70 ° C under reduced pressure to an appropriate amount (no ethanol flavor), and transferred to a separatory funnel. First, degrease twice with water-saturated diethyl ether. The mother liquor is used to recover the mixed ether under reduced pressure at 70 ° C, then dissolved in water, and extracted twice with water-saturated n-butanol. The extract is concentrated under reduced pressure at 80 ° C. Ethanol dissolves the solids, transfers to the beaker, adds water to the appropriate amount, adjusts the pH to 10-14, places, centrifuges, removes the precipitate, vacuums at 70 ° C for 2 h, transfers to a stoppered flask, and ultrasonically extracts with absolute ethanol. After filtration, the filtrate was recovered under reduced pressure at 70 ° C until the solid matter was precipitated, transferred to an evaporating dish, and the ethanol was evaporated in a water bath at 80 ° C, dried under vacuum at 70 ° C for 12 h, taken out, and ground. The content of saponin was 98.18%.

2 experimental methods

2.1 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. Sixty mice were completely randomized into 6 groups according to gender (see Table 19 for specific grouping and administration), and ig administration was prevented for 3 days before modeling. On the 4th day, except for the blank group, ip was given an equal volume of normal saline, and the other groups were given ip cyclophosphamide 100 mg/kg-d for 3 days. Except for the western medicine positive group, ig administration was continued for 6 days from the day of modeling. RhG-CSF was injected subcutaneously on days 7 to 9.

2.2 Evaluation indicators and data processing

On the 7th day after model establishment, blood was collected from the eyelids. The peripheral blood routine parameters including white blood cell WBC, red blood cell RBC, hemoglobin HGB, and platelet PLT were measured by automatic blood cell counter. The spleen was weighed and the spleen coefficient was calculated. The mice were sacrificed and the left femur was removed. In addition to the soft tissue, all the bone marrow was flushed into the test tube with 0.005 mol/L CaC12 10 mL, placed in a refrigerator at 30 °C for 30 min, centrifuged at 2500 r/min for 15 min, and the supernatant was discarded to precipitate. Add 0.2mol/L HC104 5mL and mix well, heat at 90 °C After 15 min, the cells were centrifuged at 3500 r/min for 10 min, and the supernatant was taken, and the 0D value was measured at a wavelength of 260 nm using a microplate reader.

The experimental data are expressed as mean±S.D., using SPSS software, statistical test using group t test and single factor F test, test level α = 0.05

2.3 Analysis of results

The effects of saponin extract on leukocyte, erythrocyte, platelet reduction and spleen coefficient in peripheral blood of mice induced by cyclophosphamide are shown in Table 20. Compared with the model group, the high, medium and low doses of the extract of the saponin had no significant increase in the number of white blood cells (Ρ>0.05), and had no significant therapeutic effect on the bone marrow suppression induced by cyclophosphamide (Ρ>0.05). .

Table 20 Study on the efficacy of saponin extract (mean± S.D. n=10)

△: compared with the model group P>0.05

It is generally believed that "the active ingredient of leukocyte-elevating leukocyte action and bone marrow protection is mantle saponin, which can increase the number of white blood cells, red blood cells and platelets in mice with myelosuppression, while mantle and mantle flavonoids cannot Promotes the proliferation of bone marrow cells, but at the high concentration, it produces myelosuppressive effect." In the production of mantle extract, of course, the target of mantle saponin is obtained. However, the inventors unexpectedly discovered that in the mantle by the above experiment. The active ingredient that raises white blood cells is not saponin, but scorpion.

Test Example 6 Effects of mantle and mantle saponins on tumors

1 experimental material

C57 mice (early half, 18 22g, purchased from the Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, purchased from Nippon Optoelectronics Co., Ltd.); electronic analytical balance (BP211D, Purchased from the German company Sartorius); cyclophosphamide for injection; mantle extract (prepared in Example 9, tannin content 98% or more); physiological saline.

Lewis lung cancer cell suspension: 7 days after inoculation of the tumor cells, the mice were sacrificed by cervical dislocation, ascites was aseptically aspirated, centrifuged, the supernatant was removed, and the underlying tumor cells were diluted with physiological saline to 250 mg/ml. Tumor cell fluid.

Preparation of saponin: The medicinal material is pulverized into a coarse powder, and 70% ethanol is refluxed for 3 times, each time 1. 5h, concentrated at 70 ° C under reduced pressure to an appropriate amount (no ethanol flavor), transferred to a separatory funnel First, degrease twice with water-saturated ether. The mother liquor is used to recover the mixed ether under reduced pressure at 70 ° C. Then, it is dissolved in water and then extracted twice with water-saturated n-butanol. The extract is concentrated under reduced pressure at 80 ° C. Ethanol dissolve the solids, transfer to a beaker, add water, adjust the pH to 10-14, place, centrifuge, remove the precipitate, dry at 70 ° 2 vacuum for 2 h, transfer to a stoppered flask, and extract with absolute ethanol. After filtration, the filtrate was recovered under reduced pressure at 70 ° C until the solid matter was precipitated, transferred to an evaporating dish, and the ethanol was evaporated in a water bath at 80 ° C, dried under vacuum at 70 ° C for 12 h, taken out, and ground. The content of saponin is 98. 18%.

2 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. Sixty C57 mice were randomly divided into model group, cyclophosphamide group, mantle group, mantle + cyclophosphamide group, saponin group, saponin + cyclophosphamide group. Group (see Table 21 for specific grouping and administration). Each group of animals was inoculated with 0.2 ml of Lewis tumor strain suspension by subcutaneous subcutaneous injection, and molded one time. Except for administration of cyclophosphamide by ip., each group of animals was intragastrically administered with 0.2 ml of drug solution per 10 g of body weight, and the model group was given corresponding physiological saline for 14 days.

Table 21 Animal grouping and dose

Inch

0 0

o

3 evaluation indicators and data processing

Animals of each group were sacrificed on the 15th day, the tumors of the animals were harvested, weighed, and the tumor inhibition rate was recorded. The experimental data was expressed as meani S D., using SPSS software, and the statistical test was performed by a group t test and a single factor F test, and the test level α = 0.05.

Tumor inhibition rate (%) = [model group average (tumor weight/body weight) - experimental group average (tumor weight/body weight)] / model group average (tumor weight/body weight) Χ 100%

4 results analysis

Table 22 Earthworm and mantle saponin effects on tumors Groups Dose (mg/kg) Tumor weight/body weight inhibition rate (%) Model group 0. 0976

Cyclophosphamide group 20 mg/kg · d 0. 0415 57. 48 ⁴ mantle extract group 10 mg/kg · d 0. 0758 22. 34 mantle extract + cyclophosphamide group lOmg/kg · d +20mg/kg · d 0. 0294 69. 88 ^A

The saponin group lOmg/kg · d 0. 1063 - 8. 91 saponin + cyclophosphamide group lOmg / kg · d +20mg / kg · d 0. 0568 △: Compared with the model group ^O. 05

Although the literature reports that mantle saponin has an anti-tumor effect, it can be seen from the above experimental results that the saponin group has no inhibitory effect on tumors, but has side effects of stimulating tumor growth, and, in addition, saponin combined with cyclophosphamide After use, the tumor inhibition rate is lower than that of cyclophosphamide, which indicates that saponin antagonizes the therapeutic effect of chemotherapeutic drugs on tumors; while using sputum, it not only enhances the efficacy of tumors, but also Protect bone marrow cells and whole blood cells, reduce or avoid damage to bone marrow cells and whole blood cells by chemotherapy drugs. Therefore, it is necessary to control the content of the saponin in the enamel extract of the present invention to prevent the side effects of saponin production. According to the results of the examples and the test examples, the weight percentage of total saponins of the mantle extract in the mantle extract is <10% w/w, the weight percentage of saponin I is <5% w/w, preferably, extraction. 0001%w/w。 The content of the total saponin content of the mantle <0. 001% w / w, the content of the saponin I <0. 0001% w / w.

Test Example 7 The efficacy test of the enamel extract of the present invention on bone marrow protection

Experimental material

KM mice (early half, 18~22g, clean grade, certificate number: SCXK (chuan) 2004-15, purchased from Institute of Laboratory Animals, Sichuan Academy of Medical Sciences); automatic blood cell counter (MEK-6318, Purchased from Japan Photoelectric Industry Co., Ltd.; Electronic analytical balance (BP211D, purchased from Sartorius, Germany); microplate reader; cobalt 60-ray external camera; recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75) g); physiological saline; extract of the extract obtained in Examples 1-8 (wherein the weight percentage of tannin is between 55% and 79%, and the weight percentage of saponin is <10% w/w).

2. Experimental method

2. 1 grouping and administration

The experimental animals were housed in the Pharmacological Laboratory Animal Observation Room of Chengdu University of Traditional Chinese Medicine. The environment was adapted to the environment for 3 days before the start of the experiment, and the diet and water were normally administered. 110 mice were completely randomized into 11 groups according to gender (see Table 23 for specific grouping and administration), and ig administration was prevented for 3 days before modeling. 5cy, the dose rate is 82. 6cGy / min, the dose is 82. 6cGy / min, the dose is 82. 6cGy / min, the dose is 82. 6cGy / min, the dose is 82. 6cGy / min, the dose is 82. 6cGy / min, The irradiation distance is 80 cm. Except for the western medicine positive group, ig administration was continued for 6 days from the day of modeling. RhG_CSF was injected subcutaneously on days 7~9.

Table 23 grouping and administration

Group name given liquid dose n blank group equal volume distilled water 10 model group equal volume distilled water 10 positive group rhG-CSF (30 μg/kg 'd) 10 Example 1 lOmg/kg · d 10 Example 2 lOmg/kg · d 10 Example 3 lOmg/kg · d 10 Example 4 lOmg/kg · d 10 Example 5 lOmg/kg · d 10 Example 6 lOmg/kg · d 10 Example 7 lOmg/kg · d 10 Example 8 lOmg/kg · d 10 2.2 Evaluation indicators and data processing

On the 7th day after model establishment, blood was collected from the eyelids. The peripheral blood routine parameters including white blood cell WBC, red blood cell RBC, hemoglobin HGB, and platelet PLT were measured by automatic blood cell counter. The spleen was weighed and the spleen coefficient was calculated. The mice were sacrificed and the left femur was removed. In addition to the soft tissue, all the bone marrow was flushed into the test tube with 0.005 mol/L CaC12 10 mL, placed in a refrigerator at 30 °C for 30 min, centrifuged at 2500 r/min for 15 min, and the supernatant was discarded to precipitate. Add 0.2mol/L HC104 5mL and mix well, heat at 90 °C for 15min, centrifuge at 3500r/min for 10min after cooling, take the supernatant, and measure the 0D value at 260nm wavelength with the microplate reader.

2.3 Analysis of results

The effect of mantle on the bone marrow inhibition induced by cobalt ray in mice is shown in Table 24.

Table 24 Comparison of the efficacy of the mantle of different preparation methods (meaniS.D., n=10) Group dose WBC RBC HGB PLT Spleen coefficient Bone marrow DNA

(mg/kg) (109/L) (1012/L) (g/D (109/L) (mg/lOg) Content (μg) Blank group 6.55 ± 5.21 ± 0.37 157.43 + 521.27 ± 35.19 + 129.48

0.32 13.76 43.45 3.98 ±7.65 model group 2.62 + 3.89±0.24 121.39 士 276.89 + 21.26 士 69.92 +

0.22 10.11 34.33 2.56 5.58 Positive group 30μg/kg'd 7.43 + 3.76±0.46 133.58 423.25 + 34.21士 52.29 +

0.27 Δ 11.88 34.56 2.29 Δ 5.69 Example 1 10 4.44 士 4.02±0.25 137.76 士 433.23 + 28.27士 99.67 +

0.33 Δ 14.54 38.87 3.32 Δ 6.59 Δ Example 2 10 4.72 士 4.12±0.23 132.84 + 398.52 士 29.32 + 96.47 +

0.26 Δ 8.65 41.65 2.79 Δ 6.55 Δ Example 3 10 5.45 + 4.18±0.43 136.48 + 402.64 士 31.21士 109.76

0.31 Δ 15.07 38.57 3.09 Δ ±8.53

Δ

Example 4 10 5.63 + 3.98±0.26 131.55 399.07 士 32.45 + 115.54

0.38 Δ 7.69 35.11 3.14Δ ±8.12

Δ

Example 5 10 4.25 + 3.87±0.34 138.27 士 421.09 + 28.38士 97.56士

0.37 Δ 7.55 30.98 3.45 Δ 6.27 Δ Example 6 10 4.64 士 4.07±0.28 130.29 387.43 + 29.57 + 99.89 +

0.29 Δ 9.42 29.75 2.67 Δ 5.29 Δ Example 7 10 5.75 + 4.22±0.18 135.69 + 409.28 + 32.87 + 121.55

0.43 Δ 12.38 21.98 3.34 Δ + 9.60

Δ

Example 8 10 4.25 + 3.54 ± 0.32 127.34 394.76 士 28.65 + 102.37

0.28 Δ 13.09 24.55 3.07 Δ ±8.44

Δ △: Compared with the model group ^O. 05

It can be seen from the results that the enamel extract of the present invention has a significant protective effect on the DNA content of the bone marrow and the decrease of leukocytes in the mouse caused by the chemical substance (P<0.05), and the higher the content of the enamel, the ground The higher the content of components such as alizarin, catechin and proanthocyanidin B2, the higher the drug efficacy. Test Example 8 Side effects of extract of mantle extract

Acute toxicity test

KM mice were randomly divided into a blank group and a drug-administered group, 20 animals in each group. The blank group was intragastrically administered with normal saline, and the drug-administered group was intragastrically administered to the animals at the maximum concentration and maximum volume that the animal can tolerate. It was converted into extract 2g /ml and observed continuously for two weeks. As a result, there were no obvious toxic side effects. The maximum tolerated dose of the mouse extract to the mantle extract (prepared in Example 9) was calculated to be 80 g/kg.

2. Different routes of administration test

KM mice were randomly divided into intramuscular injection group, intragastric group, intraperitoneal injection group, subcutaneous injection, intravenous injection group, 10 animals in each group, and the same dose of mantle extract was given except blank group (Example 9 Preparation) 10 mg/kg, for 14 consecutive days, the animals were sacrificed, and the morphological changes of the animal tissues were observed and photographed.

As a result, other routes of administration of enamel have strong side effects, which are manifested as:

Intraperitoneal injection: tissue, muscle adhesion lesions

Intramuscular injection: muscle has many blood stasis

Subcutaneous injection: subcutaneous tissue congestion

Intravenous injection: mice die immediately

However, there was no obvious adverse reaction during gavage, indicating that the enamel was suitable for intragastric administration.

It can be proved by the above pharmacodynamic experiments that the enamel extract of the present invention contains enamel in a weight percentage of 35%-100% w/w; the total saponin content in the mantle is <10% w/w. The content of saponin I is <5% w/w, and the active ingredient is enamel, and the higher the enamel content, the higher the medicinal effect, and the high purity enamel is At the same dose, it is superior to the compounds contained in it, such as quercetin and catechin. It has the function of protecting bone marrow hematopoietic stem cells. It has a significant protective effect on chemical and radiation-induced inhibition of mouse bone marrow DNA and can raise white blood cells. When used in combination with chemotherapeutic drugs, it not only enhances the efficacy of tumors, but also protects bone marrow cells and whole blood cells, reduces or avoids the damage of bone marrow cells and whole blood cells by chemotherapy drugs, and has a simple preparation process and high yield for clinical treatment. And to prevent the inhibition of normal bone marrow cells caused by radiotherapy and chemotherapy to provide a new medication option.

Industrial applicability

The enamel of the present invention has the function of protecting bone marrow hematopoietic stem cells, and has a significant protective effect against chemical and radiation-induced inhibition of mouse bone marrow DNA. At the same time, the enamel can also raise white blood cells and has a clear whitening effect. This product provides a new drug selection for clinical remission of normal bone marrow cells caused by radiotherapy and chemotherapy, and has excellent clinical application and industrialization prospects.

Claims

Claim

1. A mantle extract, characterized in that: the extract contains tannin in a weight percentage of 35% to 100% w/w; and the mantle total saponin has a weight percentage of < 10% w /w, saponin I weight percentage <5% w / w.

The extract according to claim 1, wherein the extract has a weight percentage of enamel of 50% to 100%.

The extract according to claim 2, wherein the extract has a bismuth content of 55% to 100% by weight.

4. The extract according to claim 1 or 2, wherein: in the extract, the total saponin content of the extract is < 7. 2% w / w, the content of saponin I < 4. 9 %w/w.

The content of the total saponin of the extract is < 0.001% w/w, and the content of the saponin I is < 0.001% w/w.

The extract according to claim 1 or 2, wherein: the mantle contains mantle, catechin, proanthocyanidin B2, and the weight percentage of the extract is scorpion 0 05%-3. 0%w/w, catechin 1. 0%-5. 0%w/w, proanthocyanidin B2 0. 05%- 1. 5% w/w.

The extract of claim 6, wherein the mantle contains mantle, catechin, proanthocyanidin B2, and the weight percentage of the extract is scorpion 1. 0. %-2. 0% w/w, catechin 2. 0%-4. 0% w/w, proanthocyanidin B2 0. 05%_0. 4% w/w.

The extract according to any one of claims 1 to 7, wherein the extract is derived from the genus Sanguisorba officinalis L. or the Sanguisorba officinalis L. var. longifolia (Bert. ) The dry root of Yu et Li.

The extract according to any one of claims 1 to 7, wherein the extract is prepared by the following method:

(2) Purification: The concentrate of the step (1) is taken, and then treated with the conventional separation method of the tannin to obtain the extract of the earthworm.

The extract according to claim 9, wherein the conventional separation method of the enamel comprises chromatography, protein precipitation, a solvent method, or a combination of the above methods.

The extract according to claim 10, wherein the chromatography is carried out by using a gel or a macroporous adsorption resin.

The extract according to claim 10, wherein the protein precipitation method is carried out by gelatin.

The extract according to claim 10, wherein the solvent method is obtained by degreasing an aqueous solution containing a concentrated solution, and then extracting with ethyl acetate to obtain an extract of the earthworm; Alternatively, the concentrate may be dissolved in ethanol and ethyl acetate, and diethyl ether or petroleum ether may be added to precipitate the extract of the extract.

The extract according to any one of claims 9-13, wherein the extract is prepared by the following method:

The extract according to claim 14, wherein the macroporous adsorption resin is a nonpolar resin or a low polarity resin.

The extract according to claim 15, wherein the macroporous adsorption resin is a DA-201, D-101, LSA-20, HP-10 or AB-8 macroporous adsorption resin.

17. The method of preparing a mantle extract according to claim 1, wherein: the method comprises the following steps:

The preparation method according to claim 17, wherein the conventional separation method of the enamel comprises chromatography, protein precipitation, a solvent method, or a combination of the above methods.

The preparation method according to claim 18, wherein the chromatography is carried out by using a gel or a macroporous adsorption resin.

The process according to claim 18, wherein the protein precipitation method is carried out by gelatin.

The preparation method according to claim 18, wherein: the solvent method is: degreasing the aqueous solution containing the concentrated liquid, and then extracting with ethyl acetate to obtain an extract of the earthworm; or The concentrate was dissolved in ethanol and ethyl acetate, and diethyl ether or petroleum ether was added to precipitate the extract of the extract.

The preparation method according to any one of claims 17 to 21, wherein the extract is prepared by the following method:

(2) Purification: Take the concentrate, use macroporous adsorption resin for adsorption and impurity removal, first elute with water to Colorless, eluted with 10% ethanol, finally eluted with 60% ethanol, collected 60% ethanol eluate, dried, to obtain the extract of the earthworm; or,

The preparation method according to claim 22, wherein the macroporous adsorption resin is a non-polar resin or a low-polar resin, preferably DA-201, D-10K LSA-20, HP-10 or AB_8.

24. Use of the extract of the genus of any of claims 1-16 for the preparation of a medicament having bone marrow protection.

The use according to claim 24, wherein the drug is a drug for preventing or/and treating myelosuppression.

Use according to claim 24 or 25, characterized in that the medicament is a medicament for preventing or/and treating normal bone marrow cell suppression caused by radiation or chemotherapy.

The use according to claim 26, wherein the drug is an agent for preventing or/and treating anemia, leukocyte or thrombocytopenia caused by radiotherapy or chemotherapy, preferably for preventing or/and treating radiotherapy or chemotherapy. A drug that causes leukopenia.

28. Use of the enamel extract of any of claims 1-16 for the manufacture of a medicament for the treatment of leukopenia.

29. Use of scutellarin and proanthocyanidin B2 for the preparation of a medicament having bone marrow protection.

30. Use according to claim 29, characterized in that the medicament is a medicament for preventing or/and treating myelosuppression.

The use according to claim 29 or 30, wherein the drug is a drug for preventing or/and treating normal bone marrow cell suppression caused by radiotherapy or chemotherapy.

The use according to claim 31, characterized in that the drug is a drug for preventing or/and treating anemia, leukocyte or thrombocytopenia caused by radiotherapy or chemotherapy, preferably for preventing or/and treating radiotherapy or chemotherapy. A drug that causes leukopenia.

33. Use of scutellarin and proanthocyanidin B2 for the preparation of a medicament for treating leukopenia.

A pharmaceutical composition for preventing or treating myelosuppression, leukopenia, which is active by an effective amount of scorpion, proanthocyanidin B2 or the extract of the genus of any one of claims 1-16. Ingredients, pharmaceutically acceptable excipients or auxiliary ingredients are added to prepare a pharmaceutically acceptable preparation.

The pharmaceutical composition according to claim 34, wherein the preparation is an oral preparation.

Use of a tumor chemotherapeutic drug and the enamel extract according to any one of claims 1 to 16 for the preparation of a combination drug for antitumor.

37. The use according to claim 36, wherein: said tumor chemotherapy drug is Anti-tumor drugs.

38. Use according to claim 37, characterized in that the tumor chemotherapeutic agent is cyclophosphamide.

The use according to claim 36, wherein the antitumor combination is a combination drug for treating lung cancer.

40. An antitumor pharmaceutical composition, which comprises: an effective amount of a tumor chemotherapeutic drug and the enamel extract according to any one of claims 1-16 as an active ingredient, plus pharmaceutically acceptable A preparation prepared from the accepted excipients.

The pharmaceutical composition according to claim 40, wherein the tumor chemotherapeutic drug is a sputum agent antitumor drug.

The pharmaceutical composition according to claim 41, wherein the tumor chemotherapeutic drug is cyclophosphamide.