CN1286465C - Radix salviae miltiorrhizae oligosaccharide and its application for resisting acquired immunodeficiency syndrome, adjusting immunity and increasing leukocyte - Google Patents

Abstract

The present invention relates to radix oligosaccharide mixture and a method for preparing the effective part of the radix oligosaccharide mixture. The radix oligosaccharide mixture respectively has the molecular weight of 504, 666 and 904, and contains three kinds of glycosyls of mannose, galactoside and glucose. The relative molar ratio of the mannose to the galactoside to the glucose is 1 to 8 to 7. Proved by pharmacodynamic tests, the oligosaccharide mixture and the effective part thereof have good functions of resisting acquired immune deficiency syndrome, regulating immunity and increasing leukocytes after separation and purification.

Description

A kind of Radix Salviae Miltiorrhizae oligosaccharide and the purposes in anti-AIDS, adjusting immunity and leukocyte increasing thereof

Technical field:

The present invention relates to a kind of Radix Salviae Miltiorrhizae oligosaccharide mixture and and effective site and preparation method thereof and and the purposes in anti-AIDS, immunomodulating and leukocyte increasing.

Background technology:

Acquired immune deficiency syndrome (AIDS) is with the speed that is difficult to contain rapid spread in the world, the stern challenge that faces for the 21 century mankind.Though the medicine of 20 kinds of anti-HIV and the conjoint therapy of efficient anti-reverse transcription are arranged so far, successfully reduced the HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) plasma viral load, the rising cd4 cell, rebuild immunologic function, postpone morbidity, prolong life and reduce dead, but because of producing drug resistance and toxic and side effects, still can not significantly improve body's immunological function, can not heal the sick, control popular.Current trends are to increasing immune drug such as interleukin-22 and interferon etc. in therapeutic alliance, but because of toxicity occurring, and limit its application.Radix Salviae Miltiorrhizae for a long time, Radix Salviae Miltiorrhizae is in clinical cardiovascular and cerebrovascular disease and the hepatitis of being used for the treatment of.The present invention finds that Radix Salviae Miltiorrhizae oligosaccharide mixture and effective site thereof have AIDS virus resisting,, medicine effect such as immunomodulating and leukocyte increasing, HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) and opportunistic infection, hepatitis B, myocarditis and diseases such as other hematopathys and tumor to immunocompromised or leukocyte attenuating can improve therapeutic effect, are expected to develop into the newtype drug that extensive use is arranged.

Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences's Chen Hong coral equals the effect that discovery Radix Salviae Miltiorrhizae (Salviamiltiorrhiza) water extract has AIDS virus resisting and hepatitis B virus in 1989.HIV (human immunodeficiency virus) had inhibitory action in pair cell was cultivated, oral murine leukemia and the simian acquired immunodeficiency syndrome viral infection of suppressing of mice and monkey, in 2155 cell culture, suppress hepatitis B virus, and prove that danshensu can suppress HIV (human immunodeficiency virus) (Human Immunodeficiency Virus), obtained the Chinese patent (patent No.: ZL95105902.5) in 2000.This Chen Hong coral and the cooperation of U.S. Johns Hopkins university find that the Radix Salviae Miltiorrhizae AIDS virus resisting suppresses the composition alkannic acid and the alkannic acid B of hiv integrase, in application international monopoly (WO 02/26726) in 2002; This institute found that SHY-2 suppresses HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) and intergrase thereof, applied for a patent (application number: 200310117349.5) in December, 2003 in 2002 again; So far in Radix Salviae Miltiorrhizae, separated and obtained multiple monomeric compound, wherein relate to water-soluble substances with as the phenolic acid chemical compound, as danshensu, caffeic acid, salvianolic acid A～H and alkannic acid and rosmarinic acid etc. and polymer and derivant, a lot of to the patent of its preparation technology, resisting cardiovascular disease and hepatitis.To Radix Salviae Miltiorrhizae Injection, compound capsule etc. in the mice body and the immunoregulation effect of clinical patient reported in literature is also arranged.In addition, much have the report and the patent of anti-AIDS, antitumor and immunoregulation effect about the synthesis of oligose compounds in addition, wherein to have set forth be that the oligosaccharide that extracts in AIDS virus resisting Sargassum polysaccharides 911 has the effect of the HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) of inhibition to the patent of Qingdao ocean research institute in 2004.But do not see that so far Radix Salviae Miltiorrhizae oligosaccharide and effective site thereof suppress the relevant report and the patent of AIDS virus resisting, immunomodulating and leukocyte increasing.

The present invention has prepared active component of red sage root A and B, isolation identification effective ingredient Radix Salviae Miltiorrhizae oligosaccharide mixture, its molecular mass number is respectively 504,666 and 944, its mixture contains mannose, three kinds of glycosyls of galactose and glucose, its relative mol ratio is a mannose: galactose: glucose is 1: 8: 7.The present invention shows its pharmacological activity test: Radix Salviae Miltiorrhizae oligosaccharide mixture and effective site thereof can suppress HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) intergrase and reverse transcriptase activity and virus replication, behind the oral and lumbar injection of mice, serum has the hiv reverse transcriptase of inhibition and HIV virus replication, regulates immunocyte and function of increasing leukocyte.One of purpose of the present invention provides anti-AIDS, the Radix Salviae Miltiorrhizae oligosaccharide of immunomodulating and leukocyte increasing and effective site thereof and and preparation technology: two of purpose of the present invention provide Radix Salviae Miltiorrhizae oligosaccharide and effective site thereof in viruses such as preparation anti-AIDS, improve the purposes in immunity and the leucocyte increasing agent.

Summary of the invention:

The said Radix Salviae Miltiorrhizae oligosaccharide mixture of the present invention, its molecular mass number is respectively 504,666 and 944, and its mixture contains mannose, three kinds of glycosyls of galactose and glucose, its relative mol ratio is a mannose: galactose: glucose is 1: 8: 7

The preparation method of said Radix Salviae Miltiorrhizae oligosaccharide of the present invention and effective site thereof mainly may further comprise the steps:

A. crude extract is extracted in water logging;

B. crude extract obtains total effective parts A through the macroporous adsorbent resin column chromatography separation and purification;

C. the further isolation and purification of effective site obtains effective site B;

D. be further purified the back and obtain effective ingredient Radix Salviae Miltiorrhizae oligosaccharide mixture.

Step 1: crude extract is extracted in water logging

Leaching process is that red sage root is pulverized, and adopts salt-free water retting method, and percolation extracts, and natural sedimentation, centrifugal or filter method are tentatively removed solid impurity, obtain crude extract.Behind the crude extract concentrating under reduced pressure, drying is obtained crude extract.

Step 2: the macroporous adsorbent resin column chromatography purifies and separates is obtained effective site A

Adopt filler preparative hplc post, filler is X-5, SP825, macroporous adsorbent resins such as HP20.

1) chromatographic column activation: adopt water, diluted acid water, diluted acid buffer thorough washing, swelling and balance;

2) crude extract is added capital, after salt-free water washing, inhale, collect stripping liquid with the lower alcohol hydrolysis.Stripping liquid concentrating under reduced pressure, freezing or spray drying obtain effective site A.

Step 3: effective site A isolation and purification obtains effective site B

Get effective site A, behind salt-free water dissolution, the reuse ethanol precipitation, precipitation part is with going up gel resin behind the salt-free water dissolution such as sephadex LH-20 carries out column chromatography, salt-free water elution, portioning is collected, and TLC checks, the same section merging.Eluent concentrating under reduced pressure, freezing or spray drying obtain effective site B.

Step 4: effective site B isolation and purification obtains the Radix Salviae Miltiorrhizae oligosaccharide mixture

The back upward sephadex G-50 gel resin of effective site B dissolving etc. carries out column chromatography, and salt-free water elution, portioning are collected, FeCl ₃With anthrone test check, same section merges.Eluent concentrating under reduced pressure, freezing or spray drying obtain the Radix Salviae Miltiorrhizae oligosaccharide mixture.

Concrete preparation method may further comprise the steps:

Extract raw material Radix Salviae Miltiorrhizae powder is extracted with 10～80 ℃ water retting or diafiltration, natural sedimentation, solid impurity is tentatively removed in centrifugal or filtration, obtains crude extract; Crude extract is used salt-free water washing through macroporous adsorbent resin column chromatography, inhales with the lower alcohol hydrolysis, collects stripping liquid; The stripping liquid concentrating under reduced pressure, freezing or spray drying obtains effective site A; Behind salt-free water dissolution effective site A, the reuse ethanol precipitation, the precipitation part is carried out column chromatography with going up sephadex LH-20 gel resin behind the salt-free water dissolution, salt-free water elution, portioning is collected, the TLC monitoring, same section merges, the eluent concentrating under reduced pressure, freezing or spray drying obtains effective site B; Carry out column chromatography with going up sephadex G-50 gel resin behind the salt-free water dissolution effective site B, salt-free water elution, portioning is collected, the TLC monitoring, same section merges, the eluent concentrating under reduced pressure, freezing or spray drying obtains the Radix Salviae Miltiorrhizae oligosaccharide mixture.

The discriminating of effective site and Radix Salviae Miltiorrhizae oligosaccharide mixture:

Effective site A and B be pale brown toner not, FeCl ₃Test is positive, and the anthrone test is positive, and Molisch test is weak positive reaction, and the negative reaction of Fehlings reagent detection reaction, the negative reaction of 1,2,3-indantrione monohydrate chromogenic reaction.

The Radix Salviae Miltiorrhizae oligosaccharide mixture also be pale brown toner not, FeCl ₃Test is positive (blue brown), and anthrone is tested be positive (palm fibre is blue).

Measure through MALDI-TOF MS, the Radix Salviae Miltiorrhizae oligosaccharide mixture is a mixture, contains molecular weight and is respectively 504,666 and 944 material (Fig. 1).Infrared spectrum analysis contains sugared hydroxyl composition (Fig. 2).Sample carries out gas chromatographic analysis and shows that the Radix Salviae Miltiorrhizae oligosaccharide mixture contains mannose through the chemistry dirt that spreads out, three kinds of glycosyls of galactose and glucose, and its relative mol ratio is a mannose: galactose: glucose is 1: 8: 7 (Fig. 3).

Radix Salviae Miltiorrhizae oligosaccharide mixture and effective site A thereof and B resisting HIV (HIV) activity:

1. to the inhibition activity and the mechanism of action thereof of HIV-1 intergrase

1.1 suppress the gross activity of HIV-1 intergrase

Donor substrate bag by bread board, is added after washing plate: reaction buffer, variable concentrations medicinal liquid and demarcate the genetic engineering hiv integrase of concentration, 37 ℃ of reactions 1 hour.Add the target substrate, mix, 37 ℃ were reacted 1 hour, and washed plate.Add BSA (bovine serum albumin), room temperature 30 minutes is washed plate.Add the Avidin of alkali phosphatase enzyme mark, room temperature reaction 1 hour is washed plate.Add chromogenic substrate colour developing 30 minutes, add the reaction of 0.1N NaOH color development stopping.Microplate reader 405nm measures the OD value.Establish enzyme contrast and blank simultaneously, calculate IC ₅₀

1.2 suppressing HIV-1 intergrase 3 ' cleavage activity measures

With bag by the microwell plate of donor substrate 1 wash plate 2 times with PBS, reuse 1 * reaction buffer is washed once.Add 2 * reaction buffer, different diluted concentration medicinal liquid and genetic engineering hiv integrase respectively, mixing, 37 ℃ were reacted 1 hour.PBS washes plate, unconjugated enzyme of flush away and medicine.Add 1 * reaction buffer and target substrate, mixing, 37 ℃ of water-baths continue reaction 1 hour.Subsequent step is the same.

1.3 suppressing HIV-1 intergrase chain transfer activity measures

With bag by the microwell plate of donor substrate 2 wash plate 2 times with PBS, reuse 1 * reaction buffer is washed once.Add 2 * reaction buffer, water and genetic engineering hiv integrase respectively, mixing, 37 ℃ of water-baths 1 hour.Wash plate, the unconjugated enzyme of flush away.Add 2 * reaction buffer, water and variable concentrations medicinal liquid, add the target substrate, mixing, 37 ℃ of water-baths continue reaction 1 hour.Subsequent step is the same.

1.4 suppress HIV-1 intergrase assembling determination of activity

With bag by the microwell plate of donor substrate 2 wash plate 2 times with PBS, reuse 1 * reaction buffer is washed once, adds the medicinal liquid and the genetic engineering hiv integrase of 2 * reaction buffer, variable concentrations respectively, mixing, 37 ℃ of water-baths 1 hour.Wash plate, unconjugated enzyme of flush away and medicine add 1 * reaction buffer and target substrate, mixing, and 37 ℃ of water-baths continue reaction 1 hour.Subsequent step is the same.

1.5 suppressing the exercising result of HIV-1IN, Radix Salviae Miltiorrhizae oligosaccharide mixture and effective site A, B see Table 1

Table 1. Radix Salviae Miltiorrhizae oligosaccharide mixture and effective site A, B suppress the effect of HIV-1IN

Mechanism of action	IC 50(μg/ml)
				Effective site A	Effective site B	The Radix Salviae Miltiorrhizae oligosaccharide mixture
	Suppressing HIV-1IN gross activity inhibition HIV-1IN 3 ＇-cleavage activity inhibition HIV-1IN chain transfer activity suppresses HIV-1IN and assembles active	2.86±1.99 - - -	1.18±0.04 1.59±0.28 ＜0.48 1.79±0				0.89±0.13 - - -

Annotate :-for not doing this

2. effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture suppress HIV-1RT and PR activity

2.1 ³The H mark detection method is measured extract and is suppressed the hiv reverse transcriptase activity

With demarcate genetic engineering hiv reverse transcriptase (p66/51), the variable concentrations of concentration medicinal liquid, reaction buffer and ³After H-dTTP adds, mix, 37 ℃ were reacted 0 ℃ of cessation reaction 30 minutes.Reactant liquor drips on filter paper, and cold trichloroacetic acid is washed 3 times, dries after the ethanol dehydration.Liquid scintillation instrument is measured the cpm value, establishes enzyme contrast, blank and medicine contrast simultaneously, calculates IC ₅₀Determination of activity the results are shown in Table 2 (RT).

2.2ELISA measuring extract, detection method suppresses the hiv protease activity

Microwell plate adds reaction buffer and substrate, adds an amount of genetic engineering hiv protease and variable concentrations medicinal liquid, mixing, and 37 ℃ were reacted 60 minutes.Add the DMSO cessation reaction.Add sodium iodoacetate, mixing, 37 ℃ were reacted 45 minutes.Add Dig-NHS, mixing, 37 ℃ were reacted 50 minutes.Add glycine and interrupt reaction.Add in 96 orifice plates of streptavidin labelling, add reaction buffer simultaneously, mixing, room temperature reaction 60 minutes is attached to substrate on the ELISA Plate, washes plate.Add blocker room temperature blocking-up 50 minutes.Add the anti digoxin antibody Fab fragment of alkali phosphatase enzyme mark, room temperature 1 hour is washed plate.Add substrate pNPP colour developing, microplate reader 405nm surveys the OD value.Establish enzyme contrast, blank and positive drug contrast simultaneously, calculate IC ₅₀Determination of activity the results are shown in Table 2 (PR).

2.3. result

Effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture suppress HIV-1RT and the PR activity the results are shown in Table 2.

Table 2: effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture suppress HIV-1RT and PR activity

Medicine	IC 50(μg/ml)
			RT	PR
	Effective site A effective site B Radix Salviae Miltiorrhizae oligosaccharide mixture	26.94±4.92 13.58±0.69 11.12±2.50			＞200 ＞200 ＞200

Above-mentioned experiment in vitro shows that effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture have the activity of anti-hiv integrase, and its mechanism of action mainly is to suppress viral integrase in the process of host cell chromosome.Effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture suppress the hiv reverse transcriptase activity simultaneously in addition.

The 3 couples of HIV-1 in cell culture to the effect of HIV-1

3.1MTT staining measure extract in the MT-4 cell culture toxicity and to the inhibitory action of HIV-1IIIB

The MT-4 cell inoculation in 96 porocyte culture plates, is added extraction medicine or 2 times of dilutions of positive drug medicinal liquid of totally 8 concentration respectively simultaneously, and each dilution factor repeats 3 holes, establishes the cell matched group.Put 37 ℃, 5%CO ₂With cultivate in the saturated humidity incubator, every day the observation of cell pathological changes.The 4th day (96 hours) back is inhaled and is abandoned supernatant 100 μ l after the dosing, and every hole adds 10 μ l5mg/mlMTT dyeing, 37 ℃, 5%CO ₂Continue in the saturated humidity incubator to cultivate 4 hours, every hole adds 100 μ l50%DMF-17%Triton X-100 destaining solution, and 37 ℃ are spent the night, and measuring wavelength on enzyme connection instrument is the OD of 570nm _570nmValue is calculated the poisonous concentration (TC of medicine half ₅₀).

The MT-4 cell inoculation in 96 porocyte culture plates, is added HIV-1 zoo virus strain IIIB culture fluid, add variable concentrations extract or the positive control drug AZT and the NVP medicinal liquid of the following dilution of maximal non-toxic concentration simultaneously, at 37 ℃, 5%CO ₂With cultivate under the saturated humidity, the observation of cell pathological changes, in the 7th day with mtt assay dyeing, measure OD _570nm, calculate medicine medium effective concentration (EC ₅₀), and calculate therapeutic index (SI).

3.2 result: effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture in the MT-4 cell culture toxicity and the effect of anti-HIV-1 III see Table 3.

Table 3: effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture toxicity in the MT-4 cell culture and the effect of anti-HIV-1 IIIB

Medicine	TC 50(μg/ml)	EC 50(μg/ml)	SI
				Effective site A effective site B Radix Salviae Miltiorrhizae oligosaccharide mixture	5.8±1.36 10.6±1.81 11.1±3.83	0.76 0.78 1.06	7.6 13.6 10.5

The medicine test card is bright in the cell culture, and effective site A, B and Radix Salviae Miltiorrhizae oligosaccharide mixture all have the effect of obvious suppression HIV-1 at the MT-4 cell culture.

4 effective site A and B are at the intravital acute toxicity of mice

Kunming kind female mice (body weight 20 ± 1.0g), difference oral administration (administration volume 1.0ml/20g), tail vein injection (administration volume 0.4ml/20g) and lumbar injection (administration volume 0.4ml/20g), administration 1 time was observed 10 days, and the record dead mouse is counted situation.The results are shown in Table 4.

Table 4: extract is to the acute toxicity of Kunming kind female mice

Route of administration	LD 50(g/kg)
			Effective site A	Effective site B
	Irritate stomach tail vein injection lumbar injection	＞20 2.02 4.04			- - ＞0.12

The mice serum determination of activity of 5 effective site A and Radix Salviae Miltiorrhizae oligosaccharide mixture

5.1 serum is to the inhibitory action of HIV-1-RT behind mouse stomach and the lumbar injection

Kunming mouse (5 every group) oral administration effective site A 5.0g/kg 1 time after 1 hour, after eye socket is got blood, measures the activity that mice serum suppresses HIV-1RT after diluting 60 times after 56 ℃ of deactivations in 30 minutes.The results are shown in Table 5.After 30 minutes, mice pastille serum is measured the activity that its blood medicine suppresses HIV-1IIIB with the MTT staining in the MT-4 cell culture after different extension rate dilutions, the results are shown in Table 5 through lumbar injection for effective site A and Radix Salviae Miltiorrhizae oligosaccharide mixture.

Table 5: serum suppresses the HIV activity behind mouse stomach and the intraperitoneal injection of drugs

Medicine	Administering mode	Get the blood time	Suppression ratio (%) to HIV-1	To HIV-1RT suppression ratio (%)
					Active component A 500mg/kg red sage root oligosaccharide mixture 100mg/kg active component A 5.0g/kg	The lumbar injection lumbar injection is irritated stomach	30 minutes 30 minutes 1 hour	47.3±28.9 23.1±20.6 -	- - 12.3±5.1

5.2 behind mouse stomach and the lumbar injection serum in cell culture to the inhibitory action of HIV-1-:

Kunming mouse after different approaches is given effective site A 1 hour the time blood medicine suppress HIV-1RT active and in the PBMC cell effect of anti-HIV-1 see Table 6.

Table 6: Kunming mouse administration effective site A serum HIV (human immunodeficiency virus)-resistant activity after 1 hour

Administering mode and dosage	Inhibition percentage rate to HIV-RT	Anti-HIV-1 activity in the PBMC cell
			Normal control is irritated stomach 1g/kg and is irritated stomach 10g/kg lumbar injection 400mg/kg	56.5 50.0 64.2 77.8	1.8 49.4 68 58.2

The test of mice serum HIV (human immunodeficiency virus)-resistant activity shows after the above-mentioned administration, behind effective site and Radix Salviae Miltiorrhizae oligosaccharide mixture oral administration or the lumbar injection, can detect the activity that suppresses HIV in mice serum.

6 brief summaries

Above-mentioned all anti-HIV result of the tests show, the activity of the existing anti-HIV-1 IN of extract also has the activity of anti-HIV-1 RT, and the effect that suppresses HIV-1 is also arranged in cell culture; Extract toxicity is low, after the administration, can determine the activity that it suppresses HIV-1 in the mice serum.

Radix Salviae Miltiorrhizae oligosaccharide mixture and effective site A thereof influence mouse immune cell ground

1 method

Select for use 18-20 gram BALB/C mice to divide 12 groups, 6 every group.6 groups is normal mouse, immunosuppressed mice intraperitoneal injection of cyclophosphamide 100mg/kg 1 time.Effective site A and Radix Salviae Miltiorrhizae oligosaccharide mixture: each 2 dosage group: Radix Salviae Miltiorrhizae oligosaccharide mixture: 50mg/kg and 150mg/kg; Effective site A.:100mg/kg and 300mg/kg.Positive drug lentinus edodes polysaccharide injecta 2mg/kg; The negative control normal saline.Medication is a lumbar injection: the grouping administration is as follows:

Normal mouse: 1. normal saline contrast, 2. lentinan 2mg/kg; 3. effective site A:100mg/kg; 4. effective site A:300mg/kg; 5. Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg; 6. Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg;

Immunosuppressed mice: 7. cyclophosphamide 100mg/kg; 8. cyclophosphamide 100mg/kg+ effective site A100mg/kg; 9. cyclophosphamide 100mg/kg+ effective site A 300mg/kg lumbar injection; 10. Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg lumbar injection; 11. cyclophosphamide 100mg/kg+ Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg; 12. cyclophosphamide 100mg/kg+ Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg.

Each organizes the mouse peritoneal drug administration by injection, 7 days every days 1 time.Pluck eyeball in administration after 7 days and get peripheral blood, make blood cell suspension with the EDTA-K3 anticoagulant.Mensuration routine blood test and flow cytometer carry out fluorescent antibody marker determination mouse lymphocyte and make lymphocyte subgroup analysis and NK raji cell assay Raji.Adopt SPSS10.0 software to carry out Kruskal-Wallis H check relatively of many groups and the Mann-Whitney U check of comparing between any two.

2. result

Each is organized mice lymphocyte subpopulation data and is summarized in table 7 (wherein CD3 is total T lymphocyte, and CD4 is complementary Th lymphocyte, and CD8 is for killing and wounding/inhibition Tc/s lymphocyte, and CD19 is a bone-marrow-derived lymphocyte).

Table 7: Radix Salviae Miltiorrhizae oligosaccharide and effective site A are to the influence of mice lymphocyte subpopulation

Group		CD3(T) (×10 9/L)	CD4(Th) (×10 9/L)	CD8(Tc/s) (×10 9/L)	CD19(B) (×10 9/L)	CD4/CD8
							Normal saline		2.58±0.68	1.98±0.52	0.58±0.17	1.00±0.29	3.43
Lentinan 2mg/kg		3.95±0.85 *↑	3.05±0.63 *↑	0.87±0.27	2.35±1.29 *↑	3.51
							Effective site A 100mg/kg		3.88±0.34 *↑	3.01±0.28 *↑	0.86±0.07 *↑	1.21±0.34	3.52
Effective site A 300mg/kg		5.46±1.08 *↑	4.24±0.90 **↑	1.18±0.18 **↑	2.55±1.20 **↑	3.61
							Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg		3.80±0.96 *↑	3.02±0.85 *↑	0.77±0.13	0.82±0.21	3.93
Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg		6.53±0.79 *↑	4.97±0.62 **↑	1.52±0.20 *↑	2.90±1.12 **↑	3.26
							Endoxan 100 mg/ kg	Contrast	3.25±0.56	2.57±0.32	0.78±0.14 *↑	0.28±0.04 **↓	3.29
Lentinan 2mg/kg	3.38±0.81	2.60±0.58	0.82±0.19	0.28±0.09 **↓	3.18
						Effective site A 100mg/kg		5.06±1.29 *↑+	3.95±1.05 *↑+	1.10±0.24 *↑+	0.05±0.02 **↓++	3.58

	Effective site A 300mg/kg	10.9±8.45* ↑++	8.37±6.28 **↑++	2.46±2.17 **↑++	0.34±0.39 *↓	3.40
							Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg	10.5±2.73* ↑+	8.28±2.18 *↑+	2.22±0.57 *↑+	0.37±0.21 *↓	3.72
	Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg	9.1±2.09* ↑++	6.87±1.49 **↑++	2.17±0.60 **↑++	0.51±0.14* ↓++	6.16

*: compare P＜0.01, *: compare P＜0.05 with the normal saline group with the normal saline group

++: compare P＜0.01 with the cyclophosphamide group ,+: compare P＜0.05 with the cyclophosphamide group

2.1 influence to the normal mouse lymphocyte subpopulation

By table 7 as seen, administration once a day is after 7 days:

(1) the lentinan administration is 7 days, and mice peripheral blood T, Th, B cell absolute quantity significantly raise.

(2) T6 100mg/kg significantly raises the absolute quantity of T, Th, Tc/s cell and the ratio of T, Th; T6 300mg/kg ratio is constant, and the absolute quantity of T, Th, Tc/s, B all significantly raises.

(3) T6AC 50mg/kg significantly raises the absolute quantity of T, Th, Tc/s cell and the ratio of T, Th, and the B cell proportion descends, and absolute value is constant; T6AC 150mg/kg ratio is constant, and the absolute quantity of T, Th, Tc/s, B all significantly raises.

2.2 influence to cyclophosphamide immunosuppressed mice lymphocyte subpopulation

(1) the disposable administration of cyclophosphamide raises T, Th, Tc/s ratio, and the B cell proportion descends; The Tc/s absolute quantity raises, and B cell absolute quantity descends.

(2) cyclophosphamide+lentinan group result is with basic identical to the cyclophosphamide group separately.

(3) each administration group T6 of cyclophosphamide+T6 and T6AC and T6AC raise T, Th, Tc/s ratio, and the B cell proportion descends, and wherein the result of T, Th and B cell and cyclophosphamide group all have marked difference; T, Th, Tc/s absolute quantity raise and with the cyclophosphamide group marked difference are arranged, and B cell absolute quantity descends, and wherein T6 100mg/kg group is lower than the cyclophosphamide group, and T6AC 150mg/kg group is higher than the cyclophosphamide group.

(4) the CD4/CD8 ratio of each group is constant.

Radix Salviae Miltiorrhizae oligosaccharide mixture and effective site A thereof are to the influence of normal and cyclophosphamide mice blood leukocytes

1 method

The mice medication is with two.Each mice blood specimen carries out routine blood test to be measured, and calculates leukocyte, lymphocyte, and mononuclear cell and neutrophilic granulocyte and erythrocyte and hematochrome are observed the influence of medicine to normal mouse and cyclophosphamide immunosuppressed mice peripheral blood leucocyte

2 the results are shown in Table 8 and 9.

2.1 influence to the normal mouse peripheral blood leucocyte

Table 8. medicine is to the influence of normal mouse peripheral blood leucocyte

Grouping	Leukocyte (* 10 9/L)	Lymphocyte (* 10 9/L)	Mononuclear cell (* 10 9/L)	Neutrophilic granulocyte (* 10 9/L)	Erythrocyte (* 10 12/L)	Platelet (* 10 9/L)
							Normal saline	4.75±1.04	3.96± 0.91	0.02± 0.02	0.77±0.16	9.82±0.28	505±41.45
Lentinan 2mg/kg	7.98±1.95 *↑	6.58± 2.14*↑	0.02± 0.02	1.37±0.86	9.78±0.25	431.17± 34.27*↓
							Effective site A 100mg/kg	6.51±0.50 *↑	5.39± 0.36*↑	0.14± 0.07*↑	0.99±0.18 *↑	9.73±0.26	599.67± 76.96*↑
Effective site A 300mg/kg	11.59±3.57 **↑	8.40± 2.19** ↑	0.47± 0.27**↑	2.71±1.56 **↑	9.53±0.31	530.33± 100.98
							Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg	6.52±1.24	4.87± 1.09	0.11± 0.10	1.14±0.82	10.09±0.13	507.4± 71.21
Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg	13.89±2.67 **↑	9.96± 1.73**↑	0.58± 0.20**↑	3.34±0.97 *↑	10.45±0.28 *↑	457.20± 72.94

*: compare P＜0.01, *: compare P＜0.05 with the normal saline group with the normal saline group

By table 8 as seen, the normal mouse intraperitoneal injection of drugs is after 7 days every days 1 time:

(1) lentinan 2mg/kg can make murine interleukin and total lymphocyte count significantly raise, and platelet significantly descends.

(2) mouse peritoneal injection effective site A 100, and behind the 300mg/kg, not only leukocyte and total lymphocyte count, mononuclear cell and neutrophilic granulocyte sum also significantly raise, and the ratio that mononuclear cell increases is very surprising.Show that effective site A has the facilitation of highly significant to leukocyte, and the effect of 300mg/kg is greater than 100mg/kg.Facilitation to mononuclear cell and neutrophilic granulocyte is that lentinan is not available.Effective site A 100mg/kg group platelet raises.

(3) with the effective site category-A seemingly, behind the mouse peritoneal injection Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg, total white blood cells, total lymphocyte count, mononuclear cell sum, neutrophilic granulocyte sum all significantly raise, and effect is better than effective site A 300mg/kg, and this group erythrocyte also significantly raises.The result of Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg also has rising, but does not have statistical significance owing to standard deviation is excessive.

2.2 the influence to caused by cyclophosphamide immunocompromised mice peripheral blood leucocyte the results are shown in Table 9.

Table 9. medicine is to the influence of caused by cyclophosphamide immunocompromised mice peripheral blood leucocyte

Group and processing		Leukocyte (* 10 9/L)	Lymphocyte (* 10 9/L)	Mononuclear cell (* 10 9/L)	Neutrophilic granulocyte (* 10 9/L)	Erythrocyte (* 10 12/L)	Platelet (* 10 9/L)
								Normal saline		4.75±1.04	3.96±0.91	0.02±0.02	0.77±0.16	9.82±0.28	505±41.4 5
Cyclophosphamide 100mg/kg		4.16±0.45	3.88±0.48	0.02±0.02	0.26±0.05 **↓	8.84±0.35 **↓	535.67± 48.54
								Endoxan 100 mg/ kg	Lentinan 2mg/kg	4.19±0.83	3.92±0.84	0.04±0.04	0.22±0.14 **↓	9.32±0.42 *↓+	502.67± 46.13
Effective site A 100mg/kg	11.51±2.83 **↑++	5.34±1.32	0.94±0.89 **↑++	4.11±1.21 **↑++	9.91±0.34 ++	419.33± 42.80 *↓++
							Effective site A 300mg/kg		26.1±6.34 **↑++	11.6±9.16 **↑++	6.24±4.64 **↑++	8.32±4.98	9.36±1.13	190.17± 51.77 **↓++
Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg	15.3±4.56 **↑++	11.32±3.10 *↑+	4.03±3.95 **↑++	0.27±0.35	9.49±0.57	415.25± 77.71 ↓
							Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg		29.65±7.15 **↑++	9.91±2.26 **↑++	7.33±4.66 **↑++	12.20±1.97 **↑++	10.1±0.17 ++	214.6± 58.58 **↓++

*: compare P＜0.01, *: compare P＜0.05 with the normal saline group with the normal saline group

++: compare P＜0.01 with the cyclophosphamide group ,+: compare P＜0.05 with the cyclophosphamide group

(1) cyclophosphamide immunosuppressed mice: neutrophilic granulocyte and erythrocyte obviously descend.

(2) neutrophilic granulocyte and the erythrocyte of cyclophosphamide+lentinan 2mg/kg group also obviously descend, so the immunocompromised mice not effect of lentinan to having injected cyclophosphamide.

(3) total white blood cells of cyclophosphamide mouse peritoneal injection effective site A 300mg/kg, total lymphocyte count, mononuclear cell sum, neutrophilic granulocyte sum significantly raise.The neutrophilic granulocyte sum of the total lymphocyte count of effective site A 100mg/kg and effective site A300mg/kg also raises, but does not all have statistical significance.Erythrocyte does not descend, and with the normal group indifference, shows that effective site A has recovered cyclophosphamide to erythrocytic inhibitory action.Effective site A two dosage group platelet all descend.

(3) total white blood cells of (4) cyclophosphamide mouse peritoneal injection Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg, total lymphocyte count, mononuclear cell sum, neutrophilic granulocyte sum significantly raise.The total white blood cells of Radix Salviae Miltiorrhizae oligosaccharide mixture 50mg/kg, total lymphocyte count, mononuclear cell sum significantly raise, and the neutrophilic granulocyte sum descends but do not have statistical significance.Erythrocyte does not descend yet, and with the normal group indifference, shows that the Radix Salviae Miltiorrhizae oligosaccharide mixture has recovered cyclophosphamide to erythrocytic inhibitory action.Radix Salviae Miltiorrhizae oligosaccharide mixture 150mg/kg group platelet descends.

In a word, effective site A and Radix Salviae Miltiorrhizae oligosaccharide mixture all have obvious facilitation to the peripheral blood leucocyte of normal and cyclophosphamide immunocompromised mice.

Advantage of the present invention

The Radix Salviae Miltiorrhizae active component is a lot, but Radix Salviae Miltiorrhizae oligosaccharide effective constituents does not still have report so far.Radix Salviae Miltiorrhizae oligosaccharide mixture of the present invention has three kinds of pharmacologically actives: in the activity of external existing anti-HIV-1 IN the activity of anti-HIV-1 RT is arranged also, the effect of drug combination is arranged in cell culture; Mouse peritoneal injection and filling stomach are after 30 minutes or 1 hour, and serum is measured its inhibition HIV RT activity and measure the activity that it suppresses HIV in cell culture external.Injection has remarkable rising CD4, CD8 and NK cell to mouse peritoneal for this oligosaccharide mixture and effective site thereof, keeps the immunoregulation effect of B cell.In addition and rising lymphocyte, mononuclear cell and the granulocytic effect in increasing leukocyte of highly significant arranged.This can play a significant role in the treatment acquired immune deficiency syndrome (AIDS), also may be used for the treatment of other immune disease simultaneously, does not still have similar medicine at present.

Description of drawings:

The mass spectrogram of Fig. 1 Radix Salviae Miltiorrhizae oligosaccharide mixture

The infrared spectrum of Fig. 2 Radix Salviae Miltiorrhizae oligosaccharide mixture

The gas chromatogram of Fig. 3 Radix Salviae Miltiorrhizae oligosaccharide mixture, wherein 1 is internal standard substance, and 2 for glycosides reveals sugar, and 3 is glucose, and 4 is galactose

Claims (2)

1, a kind of Radix Salviae Miltiorrhizae oligosaccharide class mixture, it is characterized in that containing in the described mixture molecular weight and be respectively 504,666 and 944 material, described mixture contains mannose, galactose and three kinds of glycosyls of glucose, its relative mol ratio mannose: galactose: glucose is 1: 8: 7, and concrete preparation method may further comprise the steps:

Extract raw material Radix Salviae Miltiorrhizae powder is extracted with 10～80 ℃ water retting or diafiltration, natural sedimentation, solid impurity is tentatively removed in centrifugal or filtration, obtains crude extract; Crude extract is used salt-free water washing through macroporous adsorbent resin column chromatography, inhales with the lower alcohol hydrolysis, collects stripping liquid; The stripping liquid concentrating under reduced pressure, freezing or spray drying obtains effective site A; Behind salt-free water dissolution effective site A, the reuse ethanol precipitation, the precipitation part is carried out column chromatography with going up sephadex LH-20 gel resin behind the salt-free water dissolution, salt-free water elution, portioning is collected, the TLC monitoring, same section merges, the eluent concentrating under reduced pressure, freezing or spray drying obtains effective site B; Carry out column chromatography with going up sephadex G-50 gel resin behind the salt-free water dissolution effective site B, salt-free water elution, portioning is collected, the TLC monitoring, same section merges, the eluent concentrating under reduced pressure, freezing or spray drying obtains the Radix Salviae Miltiorrhizae oligosaccharide mixture.

2, the application of Radix Salviae Miltiorrhizae oligosaccharide class mixture in preparation AIDS virus resisting, adjusting immunity or leucocyte increasing agent according to claim 1.

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