WO2012034519A1 - Extrait de tanin de sanguisorba officinalis l., procédé de préparation et son utilisation - Google Patents

Extrait de tanin de sanguisorba officinalis l., procédé de préparation et son utilisation Download PDF

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WO2012034519A1
WO2012034519A1 PCT/CN2011/079633 CN2011079633W WO2012034519A1 WO 2012034519 A1 WO2012034519 A1 WO 2012034519A1 CN 2011079633 W CN2011079633 W CN 2011079633W WO 2012034519 A1 WO2012034519 A1 WO 2012034519A1
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extract
ethanol
drug
mantle
preparation
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PCT/CN2011/079633
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English (en)
Chinese (zh)
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杨明
苏柘僮
杨胜
王达宾
邹文铨
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成都科尔医药技术有限公司
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Priority to GB1306569.3A priority Critical patent/GB2497899B/en
Priority to SG2013018692A priority patent/SG188529A1/en
Priority to CN2011800389315A priority patent/CN103079560A/zh
Publication of WO2012034519A1 publication Critical patent/WO2012034519A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/739—Sanguisorba (burnet)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals

Definitions

  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the extract has a enamel content of 50% to 100% by weight. Further, the weight percentage of the tannin is 55% to 100%.
  • 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.
  • 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 0 0 0 0 0 0 0 0
  • 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.
  • the extract is prepared by the following method:
  • the conventional separation method of the enamel comprises chromatography, protein precipitation, solvent method, or a combination of the above methods.
  • 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.
  • 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;
  • the concentrate may be dissolved in ethanol and ethyl acetate, and diethyl ether or petroleum ether may be precipitated to precipitate the enamel extract.
  • the extract is prepared by the following method:
  • the concentrate was taken, and after degreasing, it was extracted with ethyl acetate to obtain an extract of the earthworm.
  • the aqueous ethanol has a concentration of 70%; and the aqueous acetone has a concentration of 70%.
  • 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:
  • the conventional separation method of the enamel comprises chromatography, protein precipitation, solvent method, or a combination of the above methods.
  • 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.
  • 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;
  • the concentrate may be dissolved in ethanol and ethyl acetate, and diethyl ether or petroleum ether may be precipitated to precipitate the enamel extract.
  • the extract is prepared by the following method:
  • the concentrate was taken, and after degreasing, it was extracted with ethyl acetate to obtain an extract of the earthworm.
  • concentration of the aqueous ethanol is 70%; the concentration of the aqueous acetone is 70%.
  • the macroporous adsorption resin is a non-polar resin or a low-polar resin, preferably DA-201.
  • 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.
  • the drug is a drug for preventing or/and treating myelosuppression.
  • the medicament is a medicament for preventing or/and treating normal bone marrow cell suppression caused by radiotherapy or chemotherapy.
  • 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.
  • the drug is a drug for preventing or/and treating myelosuppression.
  • the medicament is a medicament for preventing or/and treating normal bone marrow cell suppression caused by radiotherapy or chemotherapy.
  • 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 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.
  • 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.
  • the tumor chemotherapy drug is a sputum agent antitumor drug.
  • 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.
  • the tumor chemotherapy drug is a sputum agent antitumor drug.
  • 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.
  • the mantle can also raise white blood cells and has a clear whitening effect.
  • 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.
  • 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.
  • 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 ⁇ 0. 05mg).
  • 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.
  • 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.
  • Tannin content total phenol amount - the amount of polyphenol that is not adsorbed.
  • Mettler AE240 One hundred thousandth electronic analytical balance (Mettler, Germany);
  • Proanthocyanidin B2 (purity: HPLC 99%);
  • Methanol is chromatographically pure, water is re-distilled water (home-made), and other reagents are of analytical grade.
  • the mantle extract prepared by the method of the examples was taken.
  • 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.
  • DAD diode array detector
  • 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.
  • 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.
  • 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.
  • 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 ⁇ ⁇ , 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.
  • 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 0.45 ⁇ microporous filter.
  • 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 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 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 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 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 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 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%.
  • the average content of scutellarin, catechin and proanthocyanidin B2 were: 1.61%, 3.02%, 0.12%, all in weight percent.
  • 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.
  • 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).
  • 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.
  • 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.
  • Tannin content total phenol amount - the amount of polyphenol that is not adsorbed.
  • 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
  • Elution rate enamel content in the eluent / (the enamel content of the drug solution before adsorption - the enamel content after adsorption)
  • 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.
  • D-101 is selected as the preferred resin.
  • 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.
  • the drug solution began to leak after 30ml.
  • the optimal loading volume is 30ml
  • the concentration of the chemical solution is 0.33g/ml
  • the amount of macroporous resin is 20g. Therefore, the ratio of the best medicinal material to the macroporous resin is 1:2.
  • 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.
  • the tannin was eluted with 60% ethanol.
  • 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.
  • the predicted value of the 0D value is 0.92, and the result is verified.
  • 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.
  • the % ethanol was washed at 1 BV/h, and the eluate was collected, concentrated, and dried.
  • Test Example 1 The protective effect of the enamel bone defect of the present invention
  • 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.
  • 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.
  • mice 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.
  • test level ⁇ 0. 05.
  • mice 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.
  • 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.
  • 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.
  • 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.
  • SCXK SCXK
  • 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.
  • 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.
  • RhG_CSF was injected subcutaneously on days 7 ⁇ 9.
  • test level ⁇ 0. 05.
  • ⁇ 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
  • 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.
  • 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.
  • 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.
  • 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
  • test level ⁇ 0.05.
  • ⁇ ⁇ 0.05 compared with the model group;
  • 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)
  • 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.
  • 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.
  • 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.
  • 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
  • catechin and proanthocyanidin B2 have different emphasis
  • catechin has a better protective effect on chemical damage
  • 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 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
  • 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.
  • SCXK chuan 2004-15
  • 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
  • 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 ⁇
  • test level ⁇ 0.05.
  • Test Example 4 Material basis study on the whitening effect of the extract of the enamel extract of the present invention
  • 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. 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. 1. 1. 1. 1. 1. 1. 1.
  • saponin I was 4.9%
  • cyclophosphamide for injection recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75 g); physiological saline.
  • 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
  • test level ⁇ 0.05.
  • Test Example 3 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • mice 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.
  • 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.
  • test level ⁇ 0.05
  • 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.
  • mantle saponin the target of mantle saponin is obtained.
  • the active ingredient that raises white blood cells is not saponin, but scorpion.
  • 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%.
  • mice 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.
  • mice of each group were sacrificed on the 15th day, the tumors of the animals were harvested, weighed, and the tumor inhibition rate was recorded.
  • Tumor inhibition rate (%) [model group average (tumor weight/body weight) - experimental group average (tumor weight/body weight)] / model group average (tumor weight/body weight) ⁇ 100%
  • 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
  • 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).
  • 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 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.
  • mice 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.
  • 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.
  • test level ⁇ 0.05.
  • Example 4 10 5.63 + 3.98 ⁇ 0.26 131.55 399.07 ⁇ 32.45 + 115.54
  • Example 8 10 4.25 + 3.54 ⁇ 0.32 127.34 394.76 ⁇ 28.65 + 102.37
  • 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
  • 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.
  • 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.
  • Intraperitoneal injection tissue, muscle adhesion lesions
  • Intramuscular injection muscle has many blood stasis
  • Subcutaneous injection subcutaneous tissue congestion
  • 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.
  • 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.

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Abstract

L'invention concerne un extrait de tanin de Sanguisorba officinalis L. comprenant du tanin dans une proportion de 35 à 100 % (p/p), la teneur en saponine totale étant inférieure à 10 % (p/p), et la teneur en sanguisorbine I étant inférieure à 5 % (p/p). Selon l'invention, l'extrait peut protéger les cellules souches hématopoïétiques de la moelle épinière, supprimer l'inhibition de l'ADN de la moelle épinière d'une souris due aux médicaments utilisés en radiothérapie et chimiothérapie, et il peut également augmenter le nombre de leucocytes.
PCT/CN2011/079633 2010-09-14 2011-09-14 Extrait de tanin de sanguisorba officinalis l., procédé de préparation et son utilisation WO2012034519A1 (fr)

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GB1306569.3A GB2497899B (en) 2010-09-14 2011-09-14 Sanguiin for use in the prevention or treatment of bone marrow inhibition
SG2013018692A SG188529A1 (en) 2010-09-14 2011-09-14 A sanguisorba tannin extract, and preparation method and use thereof
CN2011800389315A CN103079560A (zh) 2010-09-14 2011-09-14 一种地榆鞣质提取物及其制备方法和用途

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CN104435220A (zh) * 2013-09-22 2015-03-25 成都百草和济科技有限公司 治疗骨髓抑制的联合用药物
CN104435220B (zh) * 2013-09-22 2018-01-05 成都百草和济科技有限公司 治疗骨髓抑制的联合用药物
CN104623015A (zh) * 2013-11-08 2015-05-20 成都百草和济科技有限公司 地榆和茜草组合物的新用途
CN105147804A (zh) * 2015-08-28 2015-12-16 四川英路维特医药科技有限公司 一种地榆总皂苷提取物及其制备方法和用途
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CN112876523A (zh) * 2021-03-30 2021-06-01 江西海富生物工程有限公司 一种从罗汉果、甜茶或甜叶菊絮凝渣中回收鞣酸的方法
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