CN101224215B - Uses of ursolic acid saponin and oleanolic acid saponin in preparing medicine for increasing white blood cell and/or blood platelet - Google Patents

Abstract

The invention provides an application of ursolic acid saponin and oleanolic acid saponin with a general formula I in the preparation of medicines for increasing leucocyte and/or blood platelet. The invention also provides a medicine combination with the chemical compound. The invention adopts the low-cost ursolic acid and oleanolic acid which is easily available from natural products and introduces monosaccharide and oligosaccharyl through structure modification. Pharmacology experiments prove that the invention can notably increase the leucocyte and/or the blood platelet.

Description

Ursolic acid saponin, the application of oleanolic acid saponin in preparation leukocyte increasing and/or platelet medicine

Technical field

The invention belongs to technical field of pharmaceuticals.Specifically, relate to ursolic acid saponin, the application of oleanolic acid saponin in preparation leukocyte increasing and/or platelet medicine.

Background technology

Leukocyte has phagocytic activity, can eliminate external pathogen, and the health of defendance health is so people are described as the bodyguard of health with it.Leukocyte in the normal human blood circulation is 4000～10000/ cubic millimeters.The most important thing is to have the neutrophilic granulocyte of defense function in the leukocyte, account for the 50-70% of sum.Generally speaking, the most common topmost granulocytopenia that is meant of leukopenia.When total white blood cells was lower than 4000/ cubic millimeter, this moment, human body was lower for the resistance, caused bacterial infection extremely easily, life-threatening when serious.Platelet is one of visible component in the blood, and hematoblastic number change is very big in the human blood, and normal value is 15～300,000 a/microlitre.Platelet has the function of hemostatic function, phagocytic virus, antibacterial and other particulate matters, and energy nutrition and support capillary endothelial cell, and the fragility of blood capillary is reduced.If platelet counts significantly reduces or function has obstacle, will cause bleeding tendency.Leukocyte and/or thrombocytopenia are clinical common diseases, comprise constitutional, concurrency and Secondary cases.Except that geneogenous leukocyte and/or thrombocytopenia, the cause of disease that causes is many-sided.Disease, medicine, lonizing radiation, infection, toxin, chemical substance, operative treatment, environmental factors etc. all might cause leukocyte and/or hematoblastic minimizing.The disease that causes leukocyte and/or thrombocytopenia comprises disease of hematopoietic system, disease of immune system, infectious disease, systemic lupus erythematosus (sle), anaphylactic shock, thrombocytopenic purpura etc.Myeloproliferative disorder, aplastic anemia disease all may occur together leukocyte and/or thrombocytopenia.And bone marrow transplantation, Liver and kidney are transplanted and are also directly caused leukocyte and/or hematoblastic minimizing.For cancer patient, chemicotherapy regular meeting causes the inhibition of bone marrow hematogenesis especially, shows peripheral leukocytes and platelet obviously reduces.The medicine of all kinds of leukocyte of treatment and/or thrombocytopenia is also few at present.Gene engineering product granulocyte colony-stimulating factor GCSF, granulocyte be huge, and to bite the effect of colony stimulating factor GMCSF leukocyte increasing more remarkable, the Chinese medicine DIYU SHENGBAI PIAN has also obtained extensive use, but Western medicine has the medicine of significant curative effect then relatively more rare, so clinically, leukocyte and/or thrombocytopenia curative effect are clear and definite to treating, taking convenience, be easy to quality control, the synthetic Western medicine that is easy to get also has demand widely.

Pentacyclic triterpenoid is a kind of in the triterpenoid compound of natural distributed, is found to have physiologically active widely.Ursolic acid (English name ursolic acid) and oleanolic acid (oleanolic acid) are exactly wherein most representative chemical compound, extensively exist in plant kingdom, can obtain in a large number.The medicine that oleanolic acid is used to hepatopathy has had history for many years.Both are except having beneficial effect to liver, and other various active also has been in the news; People such as Lin are at article Effects ofoleanolic acid and ursolicacid on inhibiting tumor growth and enhancing the recovery of hematopoieticsystem postirradiation in mice (Cancer letter 7-13,111,1997) studied ursolic acid in, oleanolic acid is to suppressing the effect of growth of tumour cell, and find that the murine interleukin that the radiotherapy model is caused reduces symptom, ursolic acid, oleanolic acid is 25,50, during 100mg/kg dosage group intraperitoneal administration, effect with leukocyte increasing, and high dose administration group is apparent in view, and wherein the ursolic acid effect is better than oleanolic acid.Japan Patent JP7048260 also discloses ursolic acid and has had rising erythrocyte, hematoblastic effect.Chinese patent CN03135776 discloses a class to be separated from the Chinese medicine Radix Sanguisorbae and obtains, mainly (have another name called pomolic acid by 19-hydroxyl ursolic acid, English name pomolic acid) has leukocyte increasing and/or hematoblastic remarkable effect for the triterpene saponin of aglycon, owing to have this saponins that series structure is similar, polarity is close in the Chinese medicine Radix Sanguisorbae, and content is limited, separation and purification goes out the effective monomer hyoscine in batches certain difficulty, add that this distribution in natural product of pomolic acid or its glycosides is few, therefore the angle from separation and Extraction is not easy to obtain in a large number; And from synthetic angle, from the ursolic acid raw material of easy acquisition, the 19-hydroxyl is not easy to transform easily to obtain, thereby there is certain limitation in its exploitation use.

Up to now, do not see ursolic acid saponin, oleanolic acid saponin as yet at the application report for preparing leukocyte increasing and/or platelet medicine.The invention land productivity is used in the natural product and extensively distributes and ursolic acid, oleanolic acid cheap and easy to get is raw material, introduced hydrophilic group by structural modification, be monosaccharide or oligosaccharyl, find that resulting saponin is improving the water miscible while of parent nucleus, the pharmacological evaluation proof has remarkable leukocyte increasing and/or biologically active pdgf effect.Compare with separating the pomolic acid saponin that obtains in the Chinese medicine Radix Sanguisorbae, not only pharmacologically active is stronger, also has synthetic be easy to get, be fit to commercial production, advantage such as with low cost.

Summary of the invention

Technical scheme of the present invention has provided ursolic acid saponin, oleanolic acid saponin or its pharmaceutically acceptable salt or the purposes of ester in preparation leukocyte increasing and/or platelet medicine of general formula (I), and general formula (I) is:

Wherein, R1 is hydrogen, glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, xylosyl, ribosyl, lysol glycosyl, mannose group, allose base, altrose base, gulose base, fructosyl, Pyrusussuriensis glycosyl, quinovose base, husband's glycosyl, psicose base, 2-aminoglucose base, galacturonic acid acidic group, glucal acidic group, or by these monosaccharide 2-5 the oligosaccharyl of forming;

R2 is a hydrogen, glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, xylosyl, ribosyl, lysol glycosyl, mannose group, allose base, altrose base, gulose base, fructosyl, Pyrusussuriensis glycosyl, quinovose base, husband's glycosyl, psicose base, 2-aminoglucose base, galacturonic acid acidic group, glucal acidic group, or by these monosaccharide 2-5 the oligosaccharyl of forming;

And R1, R2 are not hydrogen simultaneously.

R3 is CH ₃, R4 is ursolic acid saponin during for H, sees general formula (II):

Further, among the general formula I I, when monosaccharide that G1, G2 representative is identical or oligosaccharide, be called two glycosylation ursolic acid saponins.G1, G2 are hydrogen or monosaccharide or oligosaccharide, and G1 is called non-pair of glycosylation ursolic acid saponin when being different from G2.

Wherein, described glycosyl is: glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, xylosyl, glucal acidic group, or by these monosaccharide 2-5 the oligosaccharyl of forming.

Further, two glycosylation ursolic acid saponins are selected among the general formula I I:

3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(α-L-pyrans rhamnose) ursolic acid-28-O-(α-L-pyrans rhamnose) ester,

3-O-(β-D-Glucopyranose .) ursolic acid-28-O-(β-D-Glucopyranose .) ester.

Further, non-pair of glycosylation ursolic acid saponin is selected among the general formula I I:

3-O-(β-D-galactopyranose) ursolic acid,

3-O-(α-L-pyrans rhamnose) ursolic acid,

3-O-(α-L-arabopyranose) ursolic acid,

3-O-(β-D-Glucopyranose .) ursolic acid,

3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-D-Glucopyranose .) ester,

3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-Xian Weisu disaccharide) ester,

Ursolic acid-28-O-(α-L-arabopyranose) ester.

Further, among the general formula I I ursolic acid saponin preferably from:

3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(α-L-arabopyranose) ursolic acid and sodium salt thereof,

3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-D-Glucopyranose .) ester,

3-O-(β-D-Glucopyranose .) ursolic acid-28-O-(β-D-Glucopyranose .) ester.

Further, chemical compound is further certainly preferred among the general formula I I:

3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester.

The present invention also provides the purposes of above-mentioned general formula I I ursolic acid saponin in preparation leukocyte increasing and platelet medicine.

The purposes of the described ursolic acid saponin of general formula (I), oleanolic acid saponin, wherein, R3 is that H, R4 are CH ₃The time, be oleanolic acid saponin, see general formula III:

Wherein, G3, G4 represent hydrogen, glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, xylosyl, glucal acidic group, or by these monosaccharide 2-5 the oligosaccharyl of forming, and G3, G4 are not hydrogen simultaneously.

Further, chemical compound is selected from the general formula (III):

3-O-(beta d glucopyranosiduronic acid) oleanolic acid,

3-O-(β-D-xylopyranose) oleanolic acid,

3-O-(α-L-galactopyranose) oleanolic acid,

3-O-(α-L-arabopyranose) oleanolic acid and sodium salt thereof,

3-O-(β-D-Glucopyranose .) oleanolic acid and sodium salt thereof,

3-O-(α-L-arabopyranose) oleanolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(β-D-Glucopyranose .) oleanolic acid-28-O-(β-D-Glucopyranose .) ester,

Oleanolic acid-28-O-(β-gentiobiose) ester,

Oleanolic acid-28-O-(β-D-Glucopyranose .) ester.

Further, chemical compound is selected from the general formula III:

3-O-(α-L-arabopyranose) oleanolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(β-D-Glucopyranose .) oleanolic acid-28-O-(β-D-Glucopyranose .) ester.

The present invention also provides the purposes of above-mentioned general formula III oleanolic acid saponin in preparation increased platelets counts medicine.

Wherein, described leukocyte increasing and/or platelet medicinal application are in the control of mammal constitutional, concurrency or Secondary cases leukocyte and/or thrombocytopenia.

Also can be separately or merge and be used in the control infectious disease with leukocyte increasing and/or platelet medicine.

Wherein, described leukocyte and/or thrombocytopenia are to be caused by disease, medicine, lonizing radiation, infection, toxin, chemical substance, operative treatment, environmental factors.

Wherein, the disease that causes leukocyte and/or thrombocytopenia comprises disease of hematopoietic system, disease of immune system, infectious disease, systemic lupus erythematosus (sle), anaphylactic shock, thrombocytopenic purpura and cancer patient chemicotherapy.

The invention provides a kind of pharmaceutical composition with rising human leukocyte and/or platelet effect, it is by any one of general formula (I), (II), (III) described chemical compound, or hydrate, solvate, ester and the prodrug of its pharmaceutically acceptable salt, salt be active component, adds the medicament that acceptable accessories or preparing carriers become.

Further, described medicament is tablet, capsule, pill, injection, slow releasing preparation, controlled release preparation and various particulate delivery system.

Universal law according to internal metabolism, and the technical staff in pharmaceutical chemistry field general knowledge to understand, chemical compound among general formula I-III can connect by ester bond derive, and have acid among general formula I-III, the chemical compound of alkali root also can be prepared to pharmaceutically can received salt.These are hydrate, solvate or the ester of acceptable salt, salt pharmaceutically, can be metabolized in vivo particularly that chemical compound is also covered by this patent when being used for the application for preparing leukocyte increasing and/or platelet medicine among above-mentioned general formula I-III.And the Chinese medicine, plant amedica and the effective site thereof that contain any chemical compound among general formula I-III are also covered by this patent when being used for the application for preparing leukocyte increasing and/or platelet medicine.Further, comprise pharmaceutical composition as the arbitrary chemical compound among general formula I-III and pharmaceutically acceptable salt, hydrate, solvate, ester and the prodrug of salt, the Chinese medicine that contains any chemical compound among general formula I-III, plant amedica and effective site thereof and pharmaceutically acceptable carrier composition, this Pharmaceutical composition can be tablet, capsule, pill, injection, slow releasing preparation, controlled release preparation and various particulate delivery system, is also covered by this patent when being used for the application for preparing leukocyte increasing and/or platelet medicine.

The specific embodiment

Further specify this patent with embodiment below, but this and do not mean that limitation of the present invention.Synthesis material ursolic acid, oleanolic acid that the present invention adopts are the commercial goods.

The preparation of embodiment 1:3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester:

Ursolic acid 1g (2.2mmol) is at the dry CH of 20ml ₂Cl ₂In the solution, N ₂Under the atmosphere, with arabinose glycosylation donor 2,3; 4-three-O-benzoyl-β-L-arabopyranose base tribromo-acetyl imines ester 3.4g (5.7mmol; 2.6eq) (this chemical compound synthesizes method with reference to J.Org.Chem.1999, and 64,7265-7266); after Powdered 4 dust molecular sieve 1.5g mix; in-10 ℃ drip trifluoromethanesulfonic acid trimethyl silicane alcohol ester TMSOTf (0.03ml, 0.15eq), reaction is warmed up to room temperature gradually; stirring is spent the night, and adds a small amount of Et ₃N (0.3ml) cancellation reaction, filter, after filtrate concentrates, silica gel column chromatography, petrol ether/ethyl acetate system (4/1-2/1) eluting, obtain two glycosylation intermediate 1.47g (1.1mmol), with its be dissolved in ethanol/methylene (2/1,30ml) and add 378mgNaOMe (7mmol, 6.4eq), room temperature reaction 4 hours, acidic resins DowexH ⁺After the neutralization, filtering and concentrating, silica gel column chromatography (chloroform/methanol 10/1-6/1) obtains white powder 0.61g, and total recovery is 39%.

${\left[\mathrm{\α}\right]}_D^{20}=+29.0(C0.72,\mathrm{MeOH})$

IR(KBr)cm ^-1：3330，2958，1724，1068，926，852

¹HNMR(600MHz，CD ₃OD)：δppm 5.40(d，J＝5.8Hz，1H，H-1″)，5.28(t，1H，H-12)，4.30(d，J＝6.8Hz，1H，H-1′)，3.81-3.90(m，4H)，3.67-3.71(m，2H)，3.51-3.60(m，4H)，3.15(dd，J＝11.8，4.4Hz，1H，H-3)，2.29(d，J＝11.6Hz，1H，H-18)，1.13，1.06，0.98，0.86，0.84(s each，3H each，Me×5)，0.97(3H，d，J＝4.0Hz)，0.91(3H，d，J＝6.4Hz)，0.79(d，J＝11.6Hz，1H，H-5)

¹³CNMR(150MHz，CD ₃OD)：δppm 176.5(C-28)，137.9(C-13)，125.9(C-12)，105.7(C-1′)，94.4(C-1″)，89.3(C-3)，72.9，72.2，71.4，69.9，68.1，67.0，64.9(d)，55.6，52.7，48.2，48.0，41.9，39.6，39.0，38.8(d)，38.6，36.5，36.2，32.9，30.3，27.9，27.2，25.7，23.7，23.0，22.6，20.1，17.9，16.5，16.2，15.6，14.7

ESI-MS(m/z)：743.3[M+Na] ⁺

The preparation of embodiment 2:3-O-(α-L-pyrans rhamnose) ursolic acid-28-O-(α-L-pyrans rhamnose) ester:

The method of similar embodiment 1,2,3,4-three-benzoyl-β-L-rhamnopyranosyl tribromo-acetyl imines ester is the glycosylation donor, can obtain 3-O-(α-L-pyrans rhamnose) ursolic acid-28-O-(α-L-pyrans rhamnose) ester.

¹HNMR(600MHz，d ₅-pyridine)：δppm 6.75(1H，s)，5.46(1H，m)，5.32(1H，s)，4.56-4,57(2H，m)，4.52(1H，dd，J＝9.12，3.2Hz)，4.47(1H，dd，J＝8.9，3.2Hz)，4.38(1H，t，J＝9.3Hz)，4.29-4.34(3H，m)，3.17(H，dd，J＝11.8，4.44Hz)，2.44(1H，d，J＝11.3Hz)，1.81(1H，m)，1.70(3H，d，J＝6Hz)，1.67(3H，d，J＝6Hz)，1.17(3H，s)，1.08(1H，m)，0.93(3H，s)，0.92(6H，s)，0.90(3H，d，J＝6.4Hz)，0.88(3H，d，J＝6.4Hz)，0.87(3H，s)，0.70(1H，d，J＝11.7Hz)

¹³CNMR(150MHz，CD ₃OD)：δppm 175.6(C-28)，138.1(C-13)，126.1(C-12)，103.0(C-1′)，93.6(C-1″)，89.0(C-3)，72.7，72.0，71.2，71.1(d)，71.0，70.0，68.5，55.3，53.1，48.5，48.2，41.9，40.0，39.5，39.0，38.6，38.4，36.7，36.5，33.0，30.2，27.6，27.4，25.2，23.8，23.0，22.7，20.0，18.0，16.8，16.4，16.1，15.6，14.6

ESI-MS(m/z)：771[M+Na] ⁺

The preparation of embodiment 3:3-O-(β-D-Glucopyranose .) ursolic acid-28-O-(β-D-Glucopyranose .) ester:

The method of similar embodiment 1, glycosylation donor are 2,3,4, and 6-four-O-benzoyl-alpha-D-glucose base tribromo-acetyl imines ester can obtain 3-O-(β-D-Glucopyranose .) ursolic acid-28-O-(β-D-Glucopyranose .) ester.

IR(KBr)cm ^-1：3417，2925，1727，1672，1456，1377，1226，1075，1027，896，831

¹HNMR(600MHz，CD ₃OD)：δppm5.33(d，J＝8.3Hz，1H，H-1″)，5.24(t，1H，H-12)，4.31(d，J＝7.8Hz，1H，H-1′)，3.77-3.84(m，2H)，3.65-3.68(m，2H)，3.28--3.39(m，7H)，3.16-3.18(m，2H)，2.22(d，J＝11.2Hz，1H，H-18)，1.12，1.05，0.96，0.84，0.83(s each，3Heach，Me×5)，0.88(d，J＝6.4Hz，3H)，0.78(d，J＝11.6Hz，1H，H-5)

¹³CNMR(150MHz，CD ₃OD)：δppm 176.5(C-28)，137.7(C-13)，125.9(C-12)，105.3(C-1′)，94.3(C-1″)，89.4(C-3)，77.2，76.9(d)，76.3，74.3，72.5，70.3，69.8，61.4，61.1，55.7，52.8，48.7，48.0，41.9，39.6，39.0，38.9，38.8，38.6，36.4，36.1，32.9，30.3，27.9，27.2，25.7，23.9，23.0，22.6，20.1，17.9，16.5，16.2，15.7，14.8

ESI-MS(m/z)：803[M+Na] ⁺

Embodiment 4: the preparation of ursolic acid-28-O-(α-L-arabopyranose) ester:

(195mg, 0.43mmol) with 2,3,4-three-O-benzoyl-(316mg 1.4eq) is mixed in CH to L-arabopyranose bromine glycosides with ursolic acid ₂Cl ₂/ water (1/1,10ml) in the system, add K ₂CO ₃(151mg, 2.5eq) and Bu ₄(56mg, 0.4eq), reflux adds 20ml CH to reacting completely to NBr ₂Cl ₂, tell water, organic facies water and salt water washing, drying is spin-dried for, and residue is directly used in the next step.Above-mentioned intermediate is dissolved in CH ₂Cl ₂In/MeOH (5ml, the 1/1) system, with Feldalat NM (130mg, 2.5mmol) room temperature reaction spends the night, and adds after shrend goes out, with ethyl acetate extraction repeatedly, organic facies merges after washing, after the drying, is spin-dried for silicagel column, CHCl ₃/ MeOH system eluting obtains the 200mg white powder.Yield 79%. ${\left[\mathrm{\α}\right]}_D^{20}=+43.0(C0.97,\mathrm{MeOH})$

¹HNMR(600MHz，CD ₃OD)：δppm 5.38(1H，d，J＝5.8Hz)，5.26(1H，t，J＝3.6Hz)，3.85-3.88(2H，m)，3.64-3.69(2H，m)，3.54-3.56(1H，m)，3.14(1H，dd，J＝11.6，4.5Hz)，2.26(1H，d，J＝11.0Hz)，1.11(3H，s)，0.97(3H，s)，0.96(3H，d，J＝5.9Hz)，0.95(3H，s)，0.88(3H，d，J＝6.5Hz)，0.82(3H，s)，0.77(3H，s)，0.74(1H，d，J＝11.8Hz)

¹³CNMR(150MHz，CD ₃OD)：δppm176.5，137.9，125.8，94.4，78.3，72.2，69.9，67.0，64.9，55.4，52.7，48.4，41.9，39.6，39.0，38.8，38.7，38.4，36.7，36.2，33.0，30.3，29.2，27.9，27.4，26.5，23.7，23.0，22.6，20.1，18.1，16.5，16.2，15.0，14.7

ESI-MS(m/z)：611.0[M+Na] ⁺

The preparation of embodiment 5:3-O-(α-L-arabopyranose) ursolic acid and sodium salt thereof:

Ursolic acid (1g, 2.2mmol), K ₂CO ₃(0.6g), benzyl chloride 0.3ml, be mixed among the 10mlDMF, 100 ℃ of reacting by heating are complete up to raw material reaction, cooled and filtered, filter cake washs with DMF, after merging organic facies, with it to going in the 100ml water, filter to obtain white crude product, silica gel column chromatography, petrol ether/ethyl acetate system eluting obtains Folium Vaccinii vitis-idaeae acid benzyl ester 1.09g (2.0mmol).With the above-mentioned dry intermediate of crossing, glycosyl donor 2,3, (2.8mmol, 1.4eq), mealy molecular sieve 1g is mixed in the dry CH of 15ml to 4-three-O-benzoyl-β-L-arabopyranose base tribromo-acetyl imines ester 1.7g ₂Cl ₂In, N ₂Atmosphere, (78ul, 0.43mmol), TLC follows the tracks of benzyl ester raw material, after reaction is finished, adds Et to drip trifluoromethanesulfonic acid trimethyl silicane alcohol ester TMSOTf in-10 ℃ ₃The N cancellation is filtered, and filtrate concentrates the back silica gel column chromatography, and petrol ether/ethyl acetate system eluting obtains glycosylation intermediate 3-O-(2,3,4-three-O-benzoyl-α-L-arabopyranose)-Folium Vaccinii vitis-idaeae acid benzyl ester 1.65g (1.67mmol).It is dissolved in 15ml methanol/CH ₂Cl ₂(1/2) in, under the 0.1g5%Pd/C catalysis, normal pressure hydrogenation spends the night, filtering and concentrating, and silica gel column chromatography, petrol ether/ethyl acetate system eluting can get 3-O-(2,3,4-three-O-benzoyl-α-L-arabopyranose) ursolic acid 1.26g (1.4mmol).It is dissolved in 25ml MeOH/CH ₂Cl ₂(2/1), and adding NaOMe (35mg, 0.65mmol), room temperature reaction spends the night, with dilute acetic acid solution neutralization reaction liquid, after concentrating, silica gel column chromatography, chloroform/methanol system eluting obtains 3-O-(α-L-arabopyranose) ursolic acid 0.737g (1.25mmol), four-step reaction total recovery 57%.

¹HNMR(600MHz，CD ₃OD)：δppm 5.22(1H，t，J＝3.6Hz，H-12)，4.27(1H，d，J＝6.7Hz，H-1′)，3.82(1H，dd，J＝12.2，3.4Hz)，3.79-3.80(m，1H)，3.49-3.58(3H，m)，3.14(1H，dd，J＝11.4，4.14Hz，H-3)，2.19(1H，d，J＝11.0Hz，H-18)，1.11(3H，s)，1.04(3H，s)，0.96(3H，s)，0.84(3H，s)，0.84(3H，s)，0.87(3H，d，J＝6.5Hz)，0.78(1H，d，J＝11.2Hz，H-5)

¹³CNMR(150MHz，CD ₃OD)：δppm175.0，139.6，126.9，107.1，90.7，74.3，72.8，69.5，66.4，57.0，54.4，48.0，42.2，40.8，40.4，40.2，39.9，38.1，37.8，34.3，31.8，29.2，28.6，27.0，25.3，24.4，24.1，21.6，19.3，17.8，17.7，17.0，16.1

ESI-MS(m/z)：611.0[M+Na] ⁺

30mg3-O-(α-L-arabopyranose) ursolic acid is dissolved in methanol/chloroform (4ml, 3/1), adds the 5mg Feldalat NM, stirring at room 30min, the white solid that filtration is separated out, methanol wash, drying obtain 3-O-(α-L-arabopyranose) ursolic acid sodium salt.

The preparation of embodiment 6:3-O-(β-D-Glucopyranose .) ursolic acid and sodium salt thereof:

With the ursolic acid is initiation material, and with 2,3,4,6-four-O-benzoyl-α-D-glucopyranosyl tribromo-acetyl imines ester is the glycosylation donor, and four-step reaction can obtain 3-O-(β-D-Glucopyranose .) ursolic acid.

¹HNMR(600MHz，CD ₃OD)：δppm5.22(t，1H，H-12)，4.32(d，J＝7.8Hz，1H，H-1′)，3.83(dd，J＝11.8，2.2Hz，1H，H-6′-1)，3.67(dd，J＝12，5.2Hz，1H，H-6′-2)，3.17-3.35(m，5H)，2.20(d，J＝11.4，1H，H-18)，1.11(3H，s)，1.06(3H，s)，0.97(6H，s Me×2)，0.90(3H，d，J＝5.8Hz)，0.85(6H，s)，0.78(d，J＝12.4Hz，1H，H-5)

¹³CNMR(150MHz，CD ₃OD)：δppm 178.9(C-28)，137.0(C-13)，126.2(C-12)，103.8(C-1′)，88.0(C-3)，75.4，74.8，72.8，68.8，59.9，54.2，51.5，46.7，45.0，40.4，37.9，37.5，37.2，37.0，35.2，34.9，31.4，28.9，27.8，26.3，25.7，24.2，22.5，21.5，21.2，18.7，16.4，14.9，14.8，14.1，13.1

ESI-MS(m/z)：641.5[M+Na] ⁺

Its sodium salt preparation method is with embodiment 5.

The preparation of embodiment 7:3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-D-Glucopyranose .) ester:

Ursolic acid is a raw material; the similar method that obtains embodiment 4; obtain intermediate ursolic acid-28-O-(β-D-Glucopyranose .) ester; again with 2; 3; 4-three-O-benzoyl-β-L-arabopyranose base tribromo-acetyl imines ester is a secondary glycosylation donor, similar glycosylation condition, and obtain 3-O (α-L-arabopyranose) ursolic acid-28-O-(β-D-Glucopyranose .) ester with the NaOMe deprotection.

¹HNMR(600MHz，CD ₃OD)：δppm 5.36(d，J＝8.2Hz，1H，H-1″)，5.26(t，1H，H-12)，4.29(d，J＝6.7Hz，1H，H-1′)，3.80-3.86(m，3H)，3.69(dd，J＝12，4.6Hz，1H，H-6′-1)，3.57-3.58(m，1H，H-6′-2)，3.32-3.43(m，4H)，3.15(dd，J＝11.6，2.4Hz，1H)，2.24(d，J＝11.2Hz，1H，H-18)，1.13(3H，s)，1.06(3H，s)，0.99(6H，s)，0.91(d，J＝6.3Hz，3H)，0.86(3H，s)，0.85(3H，s)，0.79(d，J＝11.7Hz，1H，H-5)

¹³CNMR(150MHz，CD ₃OD)：δppm 176.5(C-28)，137.7(C-13)，125.9(C-12)，105.7(C-1′)，94.3(C-1″)，89.3(C-3)，77.2，76.9，72.9，72.5，71.4，69.8，68.1，64.9，61.1，55.6，52.8，48.2，41.9，39.6，39.0，38.9，38.8，38.6，36.5，36.1，32.9，30.3，29.3，27.9，27.2，25.7，23.9，23.0，22.6，20.1，17.9，16.5，16.2，15.6，14.7

ESI-MS(m/z)：773.4[M+Na] ⁺。

Obtain the highest active chemical compound 3-O-(α-L-arabopyranose)-19-hydroxyl-ursolic acid-28-O-(β-D-Glucopyranose .) ester and compare with separating from Radix Sanguisorbae, the chemical compound in the present embodiment is the most approaching, only lacks 19 hydroxyls.But the synthetic method of the chemical compound in the present embodiment can as above be described, and is raw material with the ursolic acid, the easy acquisition of easy industrialized method.And this chemical compound in the Radix Sanguisorbae, because 19-hydroxyl-ursolic acid raw material is not easy to obtain, and be not easy from ursolic acid is synthetic, so the synthetic of this chemical compound acquires a certain degree of difficulty, adding directly to separate in a large number from natural product is also had certain limitation, so limited its application in medicine to a certain extent.One of creativeness of the present invention just provides more facile series compound, can be synthetic from ursolic acid cheap and easy to get, oleanolic acid, and activity also is enhanced simultaneously, therefore can obtain widely applying in medicine.

The preparation of embodiment 8:3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-Xian Weisu disaccharide) ester:

Similar approach can get.

¹HNMR(600MHz，CD3OD)：δppm 5.36(1H，d，J＝8.3Hz)，5.24(1H，t)，4.43(1H，d，J＝7.9Hz)，4.28(1H，d，J＝6.7Hz)，3.21-3.89(16H，m)，3.13(1H，dd，J＝11.4，4.1Hz)，2.22(1H，d，J＝11.4Hz)，1.11(3H，s)，1.04(3H，s)，0.96(6H，s)，0.89(3H，d，J＝6.4Hz)，0.84(3H，s)，0.83(3H，d.J＝6.9Hz)，0.78(1H，d，J＝11.2Hz)

ESI-MS(m/z)：912[M] ⁺

The preparation of embodiment 9:3-O-(α-L-pyrans rhamnose) ursolic acid:

With the method for similar embodiment 5,2,3,4-three-O-benzoyl-β-L-rhamnopyranosyl tribromo-acetyl imines ester is the glycosylation donor, deprotection base after the glycosylation can obtain 3-O-(α-L-pyrans rhamnose) ursolic acid.

¹HNMR(600MHz，d ₅-pyridine)：δppm 5.48(1H，m)，5.32(1H，s)，4.55(1H，t，J＝1.6Hz)，4.46(1H，dd，J＝10.4，3.4Hz)，4.26-4.34(2H，m)，3.17(1H，dd，J＝11.8，4.4Hz)，2.62(1H，d，J＝11.4Hz)，2.31(1H，dt，J＝13.4，4.4Hz)，2.12(1H，dt，J＝4.0，12.6Hz)，1.87-2.15(6H，m)，1.66(3H，d，J＝5.8Hz)，1.56(1H，m)，1.24(s，3H)，1.02(3H，s)，1.00(3H，d，J＝6.4Hz)，0.96(3H，d，J＝6.4Hz)，0.93(3H，s)，0.83(3H，s)，0.79(3H，s)，0.72(1H，d，J＝11.6Hz)

¹³CNMR(150MHz，d ₅-pyridine)：δppm180.2，138.2，125.5，103.0，89.0，72.7，71.2，71.1，68.5，55.3，53.0，48.2，48.0，47.2，41.9，39.4，39.0，38.6，38.4，36.7，36.5，32.9，30.4，27.8，27.4，25.2，23.9，23.0，22.7，20.2，18.0，16.4(d)，16.2，15.6，14.6

ESI-MS(m/z)：1203[2M-1] ⁺，601[M-1] ⁺

The preparation of embodiment 10:3-O-(β-D-galactopyranose) ursolic acid:

The method of similar embodiment 5,2,3,4,6-four-O-benzoyl-α-D-galactopyranose base tribromo-acetyl imines ester is the glycosylation donor, deprotection base after the glycosylation can obtain 3-O-(β-D-galactopyranose) ursolic acid.

¹HNMR(600MHz，d5-pyridine)：δppm 5.48(1H，t，J＝3.2Hz)，4.93(1H，d，J＝6.6Hz)，4.57(1H，d，J＝3.2Hz)，4.43-4.48(3H，m)，4.16(1H，dd，J＝9.6，3.4Hz)，4.11(1H，t，J＝6.2Hz)，3.41(1H，dd，J＝11.8，4.4Hz)，2.62(1H，d，J＝11.6Hz)，2.20-2.32(2H，m)，2.12(1H，m)，1.57(1H，t，J＝8.0Hz)，1.31(3H，s)，1.26(3H，s)，1.01(3H，s)，1.00(3H，d，J＝5.8Hz)，0.96(3H，d，J＝5.2Hz)，0.96(3H，s)，0.85(3H，s)，0.80(1H，d，J＝11.8Hz)

ESI-MS(m/z)：641[M+Na] ⁺

The preparation of embodiment 11:3-O-(α-L-arabopyranose) oleanolic acid-28-O-(α-L-arabopyranose) ester:

The method of similar embodiment 1,2,3,4-three-O-benzoyl-β-L-arabopyranose base tribromo-acetyl imines ester is the glycosylation donor, can get with the Feldalat NM deprotection behind the glycosylation.

${\left[\mathrm{\α}\right]}_D^{20}=+35.5(C0.71,\mathrm{MeOH})$

¹HNMR(600MHz，CD ₃OD)：δppm 5.41(1H，d，J＝5.7Hz，H-1”)，5.27(1H，t，H-12)，4.27(1H，d，J＝6.7Hz)，3.79-3.90(4H，m)，3.66-3.70(2H，m)，3.55-3.58(2H，m)，3.49-3.52(2H，m)，3.13(1H，dd，H-3)，2.00-2.06(1H，m，H-18)，1.15，1.04，0.95，0.94，0.90，0.83，0.79(each 3H，s，Me×7)

ESI-MS(m/z)：743.5[M+Na] ⁺

The preparation of embodiment 12:3-O-(β-D-Glucopyranose .) oleanolic acid-28-O-(β-D-Glucopyranose .) ester:

2,3,4,6-four-O-benzoyl-α-D-glucopyranosyl tribromo-acetyl imines ester is the glycosylation donor, and similar embodiment 1 can get.

¹HNMR(600MHz，CD ₃OD)：δppm5.37(d，J＝8.1Hz，1H，H-1″)，5.25(m，1H，H-12)，4.31(d，J＝7.86Hz，1H，H-1′)，3.17-3.84(13H，m)，2.85(1H，m)，1.16，1.05，0.95，0.93，0.89，0.84，0.80(s each，3H each，Me×7)

ESI-MS(m/z)：803[M+Na] ⁺

The preparation of embodiment 13:3-O-(β-D-Glucopyranose .) oleanolic acid:

Similar embodiment 5.

¹H NMR(600MHz，CD ₃OD)：δppm 5.26(1H，t，J＝3.6Hz)，4.34(1H，d，J＝7.9Hz)，3.86(1H，dd，J＝11.8，2.3Hz)，3.68(1H，dd，J＝11.8，5.3Hz)，3.19-3.37(5H，m)，2.88(1H，m)，1.18，1.08，0.98，0.97，0.92，0.88，0.86(each 3H，s)，0.81(1H，d，J＝11.6Hz)

¹³C NMR(150MHz，CD ₃OD)：δppm 181.5(C-28)，144.1(C13)，122.0(C12)，105.3(C’1)，89.4(C3)，76.9-70.3(C’4，C’5，C’3，C’2)，61.4(C6)，55.7(C5)，48.2(C9)，47.2(C19)，46.5(C17)，46.1(C18)，41.5(C14)，39.2(C8)，38.8(C1)，38.4(C4)，36.5(C10)，33.7(C21)，32.7(C29)，32.6(C7)，32.2(C22)，30.4(C20)，27.5(C23)，27.2(C15)，25.6，25.0(C27，C25)，23.1(C-30)，22.8(C11)，22.7(C16)，18.0(C6)，16.4(C26)，15.6(C24)，14.5(C25)

Its sodium salt preparation method is the same.

The preparation of embodiment 14:3-O-(β-D-galactopyranose) oleanolic acid:

Similar embodiment 5.

¹H NMR(600MHz，d ₆-DMSO)：δppm 12.00(brs，1H)，5.16(s，1H，H12)，4.71(d，J＝4.7Hz，1H)，4.60(d，J＝5.5Hz，1H)，4.50(t，J＝5.8Hz，1H)，4.29(d，J＝4.4Hz，1H)，4.10(d，J＝7.3Hz，1H)，3.6(t，J＝3.7Hz，1H，H4)，3.54-3.50(m，1H)，3.39-3.42(m，1H)，3.23-3.29(m，3H)，3.02(dd，J＝4.7，11.4Hz，1H，H3)，2.80(dd，J＝3.3，13.6Hz，1H，H18)，1.89-1.93(m，1H)，1.80-1.77(m，3H)，1.09(s，3H)，0.98(S，3H)，0.87(S，9H)，0.75(s，3H)，0.71(s，3H)

¹³CNMR(150MHz，d6-DMSO)：δppm 178.6(C28)，143.8(C13)，121.5(C12)，106.0(C’1)，87.8(C3)，74.8-68.0(C’4，C’5，C’2，C’3)，60.3(C’6)，54.9(C5)，47.0(C9)，45.7(C19)，45.2(C17)，41.2(C18)，40.8(C14)，38.8(C8)，38.7(C1)，38.1(C4)，36.3(C10)，33.3(C21)，32.8(C29)，32.3(C7)，32.1(C22)，30.4(C20)，27.6(C23)，27.1(C15)，25.6(C27)，25.5(C25)，23.3(C30)，22.9(C11)，22.6(C16)，17.7(C6)，16.8(C26)，16.4(C24)，15.1(C25)

The preparation of embodiment 15:3-O-(β-D-xylopyranose) oleanolic acid:

With 2,3,4-three-O-benzoyl-α-D-xylopyranosyl tribromo-acetyl imines ester is the glycosylation donor, and similar embodiment 5 obtains 3-O-(β-D-xylopyranose) oleanolic acid.

¹HNMR(600MHz，d ₆-DMSO)：δppm 12.00(brs，1H，COOH)，5.16(brs，1H，H12)，4.89-4.91(m，3H)，4.11(d，J＝7.8Hz，1H，H 1)，3.63(dd，J＝5.0，11.0Hz，1H)，3.21-3.26(m，1H)，2.92-3.07(m，4H)，2.73(dd，J＝3.7，13.7Hz，1H)，1.89-1.93(m，1H)，1.79(brd，J＝8.7Hz，2H)，1.09(s，3H)，0.97(s，3H)，0.87(s，6H)，0.85(s，3H)，0.75(s，3H)，0.71(s，3H)

¹³CNMR(150MHz，d ₆-DMSO)：δppm 178.6(C28)，143.8(C13)，121.5(C12)，106.2(C’1)，87.6(C3)，76.7(C’3)，73.7(C’2)，69.6(C’4)，65.5(C’5)，54.9(C5)，46.9(C9)，45.7(C19)，45.4(C17)，41.3(C18)，40.8(C14)，38.8(C8)，38.7(C1)，37.9(C4)，36.3(C10)，33.3(C21)，32.8(C29)，32.3(C7)，32.1(C22)，30.3(C20)，27.4(C23)，27.2(C15)，25.7(C27)，25.5(C2)，23.4(C30)，22.9(C11)，22.6(C16)，17.7(C6)，16.8(C26)，16.4(C24)，15.0(C25)

The preparation of embodiment 16:3-O-(α-L-arabopyranose) oleanolic acid:

Similar embodiment 5.

¹HNMR(600MHz，d ₆-DMSO)：δppm 12.01(s，1H，COOH)，5.15(t，J＝3.6Hz，1H，H12)，4.81(d，J＝4.7Hz，1H)，4.52(d，J＝5.9Hz，1H)，4.47(d，J＝4.4Hz，1H)，4.11(d，J＝6.2Hz，1H，H1)，3.64(dd，J＝3.3，12.5Hz，1H)，3.58(brs，1H)，3.33-3.34(m，3H)，3.00(dd，J＝4.4，11.4Hz，1H，H3)，2.74(dd，J＝5.4，13.6Hz，1H)，1.88-1.93(m，1H)，1.79(dd，J＝3.3，8.4Hz，2H，H11)，1.09(s，3H，H-27)，0.96(s，3H，H23)，0.87(s，9H)，0.75(s，3H)，0.71(s，3H)

¹³CNMR(150MHz，d ₆-DMSO)：δppm 178.6(C28)，143.8(C13)，121.5(C12)，105.8(C1)，87.6(C3)，72.7，70.9，67.6，65.1，54.9，46.9，45.7，45.4，41.3，40.8，38.8，38.7，38.0，36.3，33.3，32.8，32.3，32.0，30.4，27.6，27.2，25.7，25.5，23.4，22.9，22.6，17.7，16.8，16.4，15.1

Its sodium salt preparation method is the same.Chemical compound spectrum data and document among the embodiment 13,14,15,16 (Chinese Marine University's journal, 2005,35 (4): 635-640) unanimity.

Embodiment 17: the preparation of oleanolic acid-28-O-(β-D-Glucopyranose .) ester:

With 2,3,4,6-four-O-benzoyl-α-D-Glucopyranose. bromine glycosides, oleanolic acid are raw material, and after the phase transfer method glucosidesization of similar embodiment 4, the Feldalat NM deprotection can get.

m.p.180-183℃，

IR(KBr)cm ^-1：3435，2933，2862，1695，1461，1386，1181，1030，996，762

¹HNMR(600MHz，d ₅-pyridine)：δppm 6.19(1H，d，J＝8Hz，H’-1)，5.35(1H，brs，H12)，1.18，1.17，1.13，1.06，0.91，0.90，0.86(21H，s，7Me)

¹³CNMR：δppm176.9，144.7，123.4，92.3，79.8，79.4，78.6，74.6，71.6，62.7，56.3，48.7，47.5，46.7，42.3，40.4，40.0，39.5，37.9，34.5，33.6，33.0，31.3，30.5，29.3，28.8，28.6，26.6，24.3，23.9，24.3，24.2，23.9，18.0，17.0，16.1

ESI-MS(m/z)：641[M+Na] ⁺

Embodiment 18: the preparation of oleanolic acid-28-O-(β-gentiobiose) ester:

With full acetylated gentiobiose bromine glycosides, oleanolic acid is a raw material, and after the phase transfer method glucosidesization of similar embodiment 4, the Feldalat NM deprotection can get.m.p.248-250℃

¹H NMR(600MHz，CD ₃OD)：δppm 5.41(1H，d，J＝7.8Hz，H’1)，5.32(1H，d，H12)，4.41(1H，d，J＝8.7Hz，H”-1)，4.18(1H，d，J＝11.6Hz)，3.91(1H，d，J＝13.6Hz)，3.83(1H，dd，J＝6，5.5Hz)，3.73(1H，dd，J＝5.5，5.5Hz)，1.22，1.03，1.01，1.00，0.97，0.87，0.84(21H，s，7×Me)

The preparation of embodiment 19:3-O-(beta d glucopyranosiduronic acid) oleanolic acid:

The reaction condition of similar embodiment 5 can get after the glycosylation and behind the deprotection base.

¹HNMR(600MHz，d ₅-pyridine)：δppm 5.65(1H，brs，H12)，4.82(1H，d，J＝7Hz，H’-1)，1.33，1.32，1.29，1.04，0.99，0.98，0.79(21H，s，7×Me)

ESI-MS(m/z)：655[M+Na] ⁺

Below further prove beneficial effect of the present invention by pharmacodynamics test.

Chemical compound among the embodiment has been made myelomonocyte propagation increment rate experiment in vitro, and part of compounds among the embodiment carried out testing in mice leukocyte increasing and/or the hematoblastic body, contrasted the drug effect of a monomer saponin 3-O-(α-L-arabopyranose)-19-hydroxyl-ursolic acid-28-O-(β-D-Glucopyranose .) ester separated from the Chinese medicine Radix Sanguisorbae and ursolic acid, oleanolic acid, its result has proved beneficial effect of the present invention.

The effect of the monocytic promotion propagation of the mouse bone marrow cells of 1 pair of In vitro culture of test example

Get Kunming mouse 15-20 only (available from Henan Zhengzhou University Experimental Animal Center), the male and female dual-purpose takes off cervical vertebra and puts to death, separate the both sides femur, cut off femoral head, wash bone marrow repeatedly with serum-free RPMI1640 (Gibco company), collection contains the flushing liquor of medullary cell, blow and beat repeatedly so that cell disperses with suction pipe, leave standstill and moments later draw supernatant, the centrifugal 3min of 100g, collecting cell precipitation, it is resuspended and be settled to 5mL to add culture medium, this cell suspension is added to the centrifuge tube upper strata of 5mL mouse lymphocyte separating medium, the centrifugal 30min of 400g, the milky myelomonocyte of interface (BMMNC) layer in the middle of gathering is with serum-free RPMI1640 culture medium washing three times, get the myelomonocyte suspension, the counting back is inoculated in 96 orifice plates by 400,000/hole, adds the positive drug rhG-CSF (reorganization granulocyte colony-stimulating factor, Shandong Qilu Pharmaceutical Co., Ltd.) or the testing sample of variable concentrations, set up the blank group of not dosing simultaneously, put 37 ℃, 5% CO ₂Cultivated 5 days in the incubator, measure the cell proliferation situation, after the SRB dyeing, measure the OD value at 490nm place with conventional SRB (Sigma) method.Cell proliferation increment rate (cell proliferation increment rate=experimental group-blank group/blank group * 100%) after the calculating drug treating, experimental result sees Table one.

Table one sample is numbered and the mouse bone marrow cells mononuclear cell of In vitro culture is bred increment rate (%)

Sample number into spectrum	Chemical compound	Propagation increment rate %, 1 μ g/ml
			Blank		0
G-1	3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester	31.3
			G-2	3-O-(α-L-pyrans rhamnose) ursolic acid-28-O-(α-L-pyrans rhamnose) ester	19.9
G-3	3-O-(β-D-Glucopyranose .) ursolic acid-28-O-(β-D-Glucopyranose .) ester	25.2
			G-4	3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-Xian Weisu disaccharide) ester	18.0
G-5	Ursolic acid-28-O-(α-L-arabopyranose) ester	20.1
			G-6	3-O-(α-L-arabopyranose) ursolic acid	29.9
G-7	3-O-(β-D-Glucopyranose .) ursolic acid	23.7
			G-8	3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-D-Glucopyranose .) ester	29.5
G-9	3-O-(α-L-pyrans rhamnose) ursolic acid	23.4

Sample number into spectrum	Chemical compound	Propagation increment rate %, 1 μ g/ml
			G-10	3-O-(β-D-galactopyranose) ursolic acid	24.2
G-11	3-O-(α-L-arabopyranose) oleanolic acid-28-O-(α-L-arabopyranose) ester	26.8
			G-12	3-O-(β-D-Glucopyranose .) oleanolic acid-28-O-(β-D-Glucopyranose .) ester	25.4
G-13	3-O-(β-D-Glucopyranose .) oleanolic acid	21.6
			G-14	3-O-(β-D-galactopyranose) oleanolic acid	19.9
G-15	3-O-(β-D-xylopyranose) oleanolic acid	20.6
			G-16	3-O-(α-L-arabopyranose) oleanolic acid	21.6
G-17	Oleanolic acid-28-O-(β-D-Glucopyranose .) ester	20.6
			G-18	Oleanolic acid-28-O-(β-gentiobiose) ester	19.0
G-19	3-O-(beta d glucopyranosiduronic acid) oleanolic acid	20.9
			G-20	Oleanolic acid	4.5
G-21	Ursolic acid	7.9
			G-22	3-O-(α-L-arabopyranose)-19-hydroxyl-ursolic acid-28-O-(β-D-Glucopyranose .) ester	15.2
Positive	G-CSF	33.2 (5pg/ml)

Result and discussion

As shown in Table 1, the propagation increment rate of sample G-1～G-19 of the present invention is higher more than 10% than the propagation increment rate of ursolic acid, oleanolic acid, significant difference.Separating the G-22 that obtains in the Radix Sanguisorbae is the strongest active chemical compound in the disclosed chemical compound of Chinese patent CN03135776, and sample G-8 and G-22 structure are extremely similar, and G-8 has only lacked 19 hydroxyls.Can point out from the result of last table, the present invention is that the saponin of aglycon is that the saponin of aglycon not only has the synthetic advantage that is easy to get with respect to pomolic acid with ursolic acid, oleanolic acid, and activity has also obtained enhancing simultaneously.

From above-mentioned data also as can be seen, for ursolic acid saponin G-1, G-3, G-6, G-8, oleanolic acid saponin G-11, G-12 propagation increment rate numerical value is higher relatively, thus further preferred above-mentioned several chemical compounds, and adopt the method for experiment in the body to study.

Further, the chemical compound that relates among the present invention has been carried out testing in the body, adopted cyclophosphamide to cause murine interleukin and reduce the disease animal model.Experimental technique is summarized as follows: get healthy Kunming mouse, ♀

Half and half, 18 every group, the screening body weight is that the 18-22g mice is by the body weight random packet.Each experimental group gives different pharmaceutical, and negative control group, model control group give the normal saline with volume, positive control drug subcutaneous injection granulocyte colony-stimulating factor 40ug/kg (volumetric injection: 0.1ml/10g).Except that negative control group, other administration group administration pneumoretroperitoneum injection in three days cyclophosphamide (CY) 100mg/kg, once a day, caused murine interleukin to reduce in continuous three days, in the last intraperitoneal injection of cyclophosphamide one day after, three days and/or five days eye socket rear vein beards get one (20ul) and survey its hemogram (be modeling one day after, three days and/or five days), detect and understand leukocyte and platelet situation.(annotate; The all uninterrupted administration of each administration group during this time).Five days (after being modeling five days) puts to death mice behind the last intraperitoneal injection of cyclophosphamide, and leukocyte (WBC), platelet (PLT) in the hemogram index are counted.

Test example 2 ursolic acid saponins cause the influence that murine interleukin reduces disease animal model leukocyte (WBC) to cyclophosphamide

The main research of this experiment ursolic acid saponin G-1, G-6, G-8 leukogenic effect have compared come into the open the drug effect of compound G-22 of aglycon ursolic acid, oleanolic acid and patent.Result such as following table two:

Table two ursolic acid saponin causes murine interleukin to CY and reduces the leukocytic influence of disease animal model

Annotate: and between the model control group group relatively, * P＜0.05, * * P＜0.01, △ is for having good trend,

And compare ★ P＜0.05, ★ ★ P＜0.01 between the G-22 group.

As can be seen from the above table, ursolic acid saponin G-1 provided by the invention, G-6, G-8 have significant difference with respect to the G-22 leukocyte increasing that discloses under 0.8mg/kg dosage, after modeling the 3rd day especially.And aglycon G-20, G-21 no obvious function of increasing leukocyte under this dosage.

Test example 3 ursolic acid saponins, oleanolic acid saponin cause murine interleukin to cyclophosphamide and reduce disease animal model leukocyte and hematoblastic influence

This experiment purpose is, research is with respect to the patent compound G-22 of having come into the open, and the platelet-increasing role of ursolic acid saponin, oleanolic acid saponin G-1, G-3, G-11, G-12 is also observed oleanolic acid saponin G-11, G-12 simultaneously and whether had leukogenic effect.Experimental technique is the same, result such as following table:

Table three ursolic acid saponin, oleanolic acid saponin cause murine interleukin to CY and reduce the leukocytic influence of disease animal model

Annotate: and compare * P＜0.05, * * P＜0.01 between the model control group group;

Table four ursolic acid, oleanolic acid saponin cause murine interleukin to CY and reduce the hematoblastic influence of disease animal model

Annotate: and between the model control group group relatively, * P＜0.05, * * P＜0.01, △ is for having good trend,

And compare ★ P＜0.05, ★ ★＜0.01 between the G-22 group.

From testing 3 table three as a result, table four as can be seen, ursolic acid saponin G-1 leukocyte increasing and platelet effect are obvious.The significant difference that ursolic acid saponin G-1, G-3 had increased platelets counts with respect to G-22 at the 5th day.Function of increasing leukocyte is obvious one day after in modeling for oleanolic acid saponin G-11, G-12, has certain leukogenic effect.The first, five days platelet-increasing roles are obvious, and also there were significant differences with respect to G-22 in the 5th day.Positive drug G-CSF does not have the obvious effect of increased platelets counts.

Test example 4 is for illustrating the present invention further, with ⁶⁰G-1 and G-22 cause the leukopenia murine interleukin to radiotherapy effect has been compared in the modeling of Co-gamma-rays.Concrete operations and result thereof are as follows:

Dosage regimen sees the following form

Table five drug administration scheme

Experimental technique and result

Get Kunming mouse, male and female half and half are divided into 6 groups at random, 17～18 every group.The tail vein is got the normal hemogram of hematometry (leukocyte, erythrocyte, platelet and hemoglobin), adjusts according to body weight and hemogram result.Press medicine shown in the table five and dosage ig administration, normal control group and model group give the isometric(al) normal saline, and every day 1 time, successive administration is measured the pre-irradiation hemogram after 7 days.All the other are respectively organized mice to carry out accumulated dose are 3.0Gy's except that the normal control group ⁶⁰The total irradiation of Co-gamma-rays once, the preparation murine interleukin reduces the disease model, continues administration 14 days, G-CSF organizes in irradiation continuous subcutaneous injection administration on the same day 5 days, and gets the hematometry hemogram in 3,7 and 14 days respectively in the irradiation back.Concrete outcome sees the following form:

Table six is subjected to the reagent thing right ⁶⁰The Co-gamma-rays causes the influence (10 of leukopenia mice WBC ⁹/ L)

(compare with normal group, △ △ is P＜0.01, and △ is P＜0.05; Compare with model group, * * is P＜0.01, and * is P＜0.05.)

By table six as seen: the peripheral blood WBC that the model group mice is shone back 3 days, 7 days and 14 days all obviously reduces, and is remarkable with the normal group comparing difference; After G-1 group is irritated stomach, obvious leukocyte increasing minimizing disease murine interleukin number, back 7 days of irradiation with compared that there were significant differences in 14 days with model group, G-22 does not have obvious effect.

Test example 5 hemolytics relatively

Testing sample is configured to 40,80,120,160 with solvent DMSO/ normal saline (1: 4) dissolving, and 200ug/ml is tried Concentraton gradient.From rabbit heart extracting blood 25ml, put into the conical flask jolting 10 minutes gently that contains bead, remove Fibrinogen, make into defibrinated blood.Blood is moved into graduated centrifuge tube, add the about 10 times of amounts of normal saline, shake up, centrifugal 5 minutes of 2000rpm removes supernatant, and sedimentary erythrocyte reuse normal saline washs as stated above, totally 4 times, be water white transparency to supernatant till.The gained erythrocyte is made into 10% suspension with normal saline, is for experiment.In 2.5ml given the test agent, solvent (blank) or distilled water (positive control), add 10% red cell suspension, 250 μ l, behind the mixing, put immediately in 37 ℃ of calorstats and carry out incubation; Take out after 1 hour, centrifugal 5 minutes of 3000rpm gets supernatant in spectrophotometer 540nm place's colorimetric determination OD value.Haemolysis degree (hemolysis rate)=(sample absorbance-blank absorbance) * 100% ÷ (positive control absorbance-blank absorbance); Date processing adopts the SPSS13.0 statistical software, is independent variable x with the drug level, and the haemolysis degree draws equation of linear regression for dependent variable y carries out linear regression; Going out to cause the haemolysis degree according to Equation for Calculating again is 50% drug level, i.e. HD ₅₀With P＜0.05 for significance,statistical is arranged.

Table seven: G series of samples regression equation and HD ₅₀Information slip

As can be seen from the above table, G-1 has maximum HD ₅₀, show in several samples, to cause haemolysis least easily, so obtain further preferred.

Prove by above-mentioned pharmacological evaluation, The compounds of this invention has remarkable leukocyte increasing and/or biologically active pdgf effect, compares with separating the pomolic acid saponin that obtains in the Chinese medicine Radix Sanguisorbae, and pharmacologically active is all stronger, and there were significant differences, especially two glycosylation ursolic acid saponin G1 drug effect the bests.Two as can be known glycosylation ursolic acid saponin G1 have maximum HD50 value The compounds of this invention from the hemolytic experiment, and safety is good; Comprehensive drug and safety testing, two glycosylation ursolic acid saponin G1 are optimum selection in the The compounds of this invention.

Preparation of drug combination of the present invention:

Tablet:

Composition: quantity (mg/ sheet)

G-1 5

Starch 50

Microcrystalline Cellulose 40

Magnesium stearate 2

Sodium carboxymethyl cellulose 5

According to above proportioning, with G-1 and starch, microcrystalline Cellulose, sodium carboxymethyl cellulose mixing, use water-wet, the system granule, drying, granulate adds magnesium stearate, and tabletting can get behind the mixing.

Injection:

Take by weighing 50 milligrams of G-12, use an amount of anhydrous alcohol solution, add 1 gram polyoxyethylene castor oil acid esters, it is dissolved fully, add glucose injection and be diluted to 100ml, add 0.05% injection active carbon, 80 degree insulations 15 minutes, incipient fusion glass and filtering with microporous membrane, filtrate is distributed into the 2ml injection.The per unit preparation contains 1mgG-12.

Capsule:

Get 10gG-6, microcrystalline Cellulose 400g, behind the abundant mixing, 2000 of canned No. 1 capsules.The per unit preparation contains 5mgG-6.

Claims (12)

1. the ursolic acid saponin of general formula (I), oleanolic acid saponin or its pharmaceutically acceptable salt purposes in preparation leukocyte increasing and/or platelet medicine is characterized in that general formula (I) is:

Wherein, R1 is hydrogen, glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, xylosyl, glucal acidic group;

R2 is a hydrogen, glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, glucal acidic group, or when R3 be CH ₃, when R4 is H, R2 is a cellobiosyl; R3 is that H, R4 are CH ₃The time, R2 is the gentiobiose base;

And R1, R2 are not hydrogen simultaneously;

R3 is CH ₃, R4 is ursolic acid saponin during for H; R3 is that H, R4 are CH ₃The time be oleanolic acid saponin.

2. the purposes of ursolic acid saponin according to claim 1, oleanolic acid saponin is characterized in that: R3 is CH ₃, R4 is H, is ursolic acid saponin, sees general formula I I:

Wherein, G1, G2 are H, monosaccharide or oligosaccharide, and G1, G2 are not H simultaneously; The glycosyl of described monosaccharide or oligosaccharide is: glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, xylosyl, glucal acidic group, or cellobiosyl.

3. the purposes of ursolic acid saponin according to claim 2, oleanolic acid saponin is characterized in that: G1, G2 are identical monosaccharide or oligosaccharide, and described chemical compound is:

3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(α-L-pyrans rhamnose) ursolic acid-28-O-(α-L-pyrans rhamnose) ester,

3-O-(β-D-Glucopyranose .) ursolic acid-28-O-(β-D-Glucopyranose .) ester.

4. the purposes of ursolic acid saponin according to claim 2, oleanolic acid saponin is characterized in that, G1, G2 are group inequality, and chemical compound is selected among the general formula I I:

3-O-(β-D-galactopyranose) ursolic acid,

3-O-(α-L-pyrans rhamnose) ursolic acid,

3-O-(α-L-arabopyranose) ursolic acid,

3-O-(β-D-Glucopyranose .) ursolic acid and sodium salt thereof,

3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-D-Glucopyranose .) ester,

3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-Xian Weisu disaccharide) ester,

Ursolic acid-28-O-(α-L-arabopyranose) ester.

5. the purposes of ursolic acid saponin according to claim 4, oleanolic acid saponin is characterized in that, chemical compound is among the general formula I I:

3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(α-L-arabopyranose) ursolic acid and sodium salt thereof,

3-O-(α-L-arabopyranose) ursolic acid-28-O-(β-D-Glucopyranose .) ester,

3-O-(β-D-Glucopyranose .) ursolic acid-28-O-(β-D-Glucopyranose .) ester.

6. the purposes of ursolic acid saponin according to claim 5, oleanolic acid saponin is characterized in that, chemical compound is among the general formula I I:

3-O-(α-L-arabopyranose) ursolic acid-28-O-(α-L-arabopyranose) ester.

7. the purposes of ursolic acid saponin according to claim 1, oleanolic acid saponin is characterized in that: R3 is that H, R4 are CH ₃The time, be oleanolic acid saponin, see general formula III:

Wherein G3, G4 are: hydrogen, glucosyl group, Arabic glycosyl, rhamanopyranosyl, galactosyl, xylosyl, glucal acidic group, or gentiobiose base, and G3, G4 are not hydrogen simultaneously.

8. the purposes of ursolic acid saponin according to claim 7, oleanolic acid saponin is characterized in that, chemical compound is in the general formula III:

3-O-(beta d glucopyranosiduronic acid) oleanolic acid,

3-O-(β-D-xylopyranose) oleanolic acid,

3-O-(α-L-galactopyranose) oleanolic acid,

3-O-(α-L-arabopyranose) oleanolic acid and sodium salt thereof,

3-O-(β-D-Glucopyranose .) oleanolic acid and sodium salt thereof,

3-O-(α-L-arabopyranose) oleanolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(β-D-Glucopyranose .) oleanolic acid-28-O-(β-D-Glucopyranose .) ester,

Oleanolic acid-28-O-(β-gentiobiose) ester,

Oleanolic acid-28-O-(β-D-Glucopyranose .) ester.

9. the purposes of ursolic acid saponin according to claim 8, oleanolic acid saponin is characterized in that, oleanolic acid saponin is:

3-O-(α-L-arabopyranose) oleanolic acid-28-O-(α-L-arabopyranose) ester,

3-O-(β-D-Glucopyranose .) oleanolic acid-28-O-(β-D-Glucopyranose .) ester.

10. according to any described purposes of claim 1-9, it is characterized in that: described medicine is by any one of described chemical compound, or its pharmaceutically acceptable salt is active component, adds the medicament that acceptable accessories or preparing carriers become.

11. purposes according to claim 10 is characterized in that, described medicament is tablet, capsule, pill, injection.

12. purposes according to claim 10 is characterized in that, described medicament is slow releasing preparation, controlled release preparation or particulate delivery system.