CN1313098C - New use of achyranthes polysaccharide sulphate for anti Aids and immune regulation - Google Patents

Abstract

The present invention relates to a new purpose of achyranthes polysaccharide sulphate. The pharmacodynamic experiment of the achyranthes polysaccharide sulphate proves that the compound has good functions of resisting AIDS viruses and regulating immunity, and can be prepared into medicines or disinfecting agents for treating or preventing AIDS virus infection.

Description

The anti-AIDS of achyranthes polysaccharide sulphate and immunoregulation novel use

Technical field:

The present invention relates to the application of achyranthes polysaccharide sulphate in AIDS virus resisting disease and immunomodulating.

Background technology:

Acquired immune deficiency syndrome (AIDS) is since 1981 find, rapid spread becomes human serious disaster in the world.Though 20 kinds of the medicines of anti-HIV are arranged at present in the world, comprising 11 kinds of reverse transcriptase inhibitors, 8 kinds of albumen enzyme inhibitors and a kind of fusion inhibitor, use efficient anti-reverse transcription conjoint therapy, can successfully reduce plasma viral load to the acquired immune deficiency syndrome (AIDS) patient, the rising cd4 cell is rebuild immunologic function, prolongs life, but still can not heal the sick, control popular.Because virus produces drug resistance and toxic and side effects, some medicines lost efficacy gradually to aids patient.Increase immune drug such as interleukin-22 and interferon etc. in the therapeutic alliance, can heighten the effect of a treatment, but toxicity also increases, influenced effect.Some Chinese medicine has immunoregulation effect, can improve the aids patient immunologic function in clinic trial, improves symptom.But Chinese medicine mostly is compound recipe or single medicinal material preparation, composition complexity, not approval listing as yet so far.

Since the eighties in 20th century, HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) was found, many scholars noticed that sulfated polysaccharide has film virus to comprise that herpes simplex virus (HSV) and HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) (HIV) etc. have the broad-spectrum disease resistance toxic action to multiple.About the sulfated polysaccharide class material of many animal and plant and microorganism and chemosynthesis has anti-AIDS, antitumor and immunoregulation effect, existing patent and reported in literature.Heparin (heparin) as animal origin, the sulphation dextran of chemosynthesis (Sulfated dextran), the sulfated polysaccharide carrageenin (Carrageenans) in Sargassum source, the Sulfation Strobilus Pini polysaccharide of the lentinan of originated from fungus and plant origin etc. all have the effect that suppresses HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) for tens of kinds.German Bayer in 1987 and Hoechst have developed xylan sulfuric ester (Hoe/Bay946), and Japan has developed Curdlan suldate CRDS, and the U.S. has developed immunoregulation medicaments such as DS2, in clinical trial treatment HIV sufferers, have obtained certain curative effect.The AIDS virus resisting Sargassum polysaccharides 911 of Chinese Qingdao Haiyang academy development in 2004 suppresses HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) in cell culture, also effective to simian acquired immunodeficiency syndrome viral infection monkey, ratifies to enter the clinical I phase and tests in 2002.But, do not see achyranthes polysaccharide sulphate AIDS virus resisting, immunoregulatory up to now about report and patent.

Inokopolyose is an isolating micromolecular polysaccharide (molecular weight 1476KDa) from Chinese crude drug Radix Achyranthis Bidentatae (Achyranthes bidentata Blum), and toxicity is little, antitumor, enhance immunity is arranged and rise the leukocyte effect, but do not have antivirus action.Chinese Academy of Sciences's Shanghai organic chemistry priority was applied for a patent in 1988,1993, and authorized the patent No. respectively at 1992,1999: 88105524.7 and 93112588.X.The achyranthes polysaccharide sulphate that the present invention relates to (S-AbPS), be professor Tian Gengyuan of Shanghai Organic Chemistry Institute, Chinese Academy of Sciences etc. to making after Inokopolyose (Abps) sulfonation, its structural formula is shown in (I):

Wherein: R=SO ₃Na, R '=R or wherein: R=SO ₃Na, MW=3414KDa, sulfur content is approximately

Be 21.26%.

N=1 → 9, sulfur content are 4-21%.The Fructoan sulfate that its Main Ingredients and Appearance is made up of 9 sugar, structural formula is shown in (II).

Because of its good water solubility, stable, have effects such as anti-herpes simplex virus 1 type, Coxsackie virus and hepatitis B virus, apply for a patent 2002 mandates, the patent No.: 99113444.3 in 1999.Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences discovered in 1999, achyranthes polysaccharide sulphate (S-AbPS) has the AIDS virus resisting effect, the HIV-1 replicative enzyme there is the wide spectrum inhibitory action: not only suppress the HIV-1 reverse transcriptase, also suppress intergrase and protease; In cultivating, suppresses human lymphocyte MT-4 cytopathy (mtt assay); (PBMC) suppresses the P24 antigen of HIV in the human peripheral blood mononuclear cell cultivates; Little to mouse toxicity, oral back serum has to raise and suppresses the effect of hiv reverse transcriptase, the lumbar injection lymphocyte that can raise; Can improve therapeutic effect to diseases such as opportunistic infection such as HIV (human immunodeficiency virus) (Human Immunodeficiency Virus), herpesvirus, Coxsackie virus or hepatitis B virus and tumors, be expected to develop into the newtype drug of tool extensive use.

The object of the present invention is to provide the new purposes of achyranthes polysaccharide sulphate in viruses such as anti-AIDS, raising immunologic function or disinfectant.

Summary of the invention:

The present invention has carried out serial experimentation to achyranthes polysaccharide sulphate (S-AbPS) pharmacologically active and mechanism of action.Result of study shows: S-AbPS can multipath suppresses AIDS viral infection and duplicates, and HIV (human immunodeficiency virus)-resistant activity is stable, and Mus serum has the effect that suppresses hiv reverse transcriptase behind the oral and lumbar injection, can regulate immune function of mice behind the lumbar injection.

Advantage of the present invention and good effect are, said achyranthes polysaccharide sulphate (S-AbPS) has the activity that many target spots multipath suppresses AIDS viral infection and duplicates, if be prepared into medicine, with the conjoint therapy of existing treatment acquired immune deficiency syndrome (AIDS) the wonderful of different Qu Tonggong arranged, and toxicity is low, antiviral activity is stable, also has immunoregulatory activity simultaneously, both can be used for treating AIDS viral infection, also can be used for the viral infection that prevents AIDS, also can prevent and treat the disinfectant of AIDS viral infection in order to preparation.

Specific embodiments:

Following for embodiment, just illustrative for the purpose of the present invention, and nonrestrictive.

Embodiment 1:S-AbPS is to the inhibition activity of HIV-1 intergrase (HIV-1IN), reverse transcriptase (HIV-1RT) and protease (HIV-1PR)

1.ELISA detection method is measured the activity that medicine suppresses HIV-1IN

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, measurement result sees Table 1.

2. ³The H mark detection method is measured medicine and is suppressed the HIV-1RT activity

With demarcate genetic engineering HIV-1 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 ₅₀Activity, measurement result sees Table 1.

3.ELISA measuring medicine, detection method suppresses the HIV-1PR 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 1.

4. result

S-AbPS suppresses HIV-1IN, HIV-1RT and the HIV-1PR activity the results are shown in Table 1.By table 1 as seen, S-AbPS three key enzymes that HIV is duplicated are that intergrase, reverse transcriptase and protease all have certain inhibitory action.

Table 1:S-AbPS suppresses HIV-1IN, RT and PR activity

Mechanism of action	IC 50(μM)
		Suppress the active HIV-1PR of inhibition of the active HIV-1RT of inhibition of HIV-1IN activity	0.067±0.015 5.156±2.230 0.188±0.088

Embodiment 2:S-AbPS suppresses the active mechanism of action of HIV-1 intergrase (HIV-1IN)

1.ELISA detection method is measured the gross activity that medicine suppresses HIV-1IN

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, measurement result sees Table 2.

2. suppressing HIV-1IN 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, and measurement result sees Table 2.

3. suppressing the HIV-1IN 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, and measurement result sees Table 2.

4. suppress HIV-1IN 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, and measurement result sees Table 2.

5. result

The mechanism of action that S-AbPS suppresses HIV-1IN sees Table 2.As shown in Table 2, S-AbPS has inhibitory action to three functions of HIV-1IN, and is active in suppressing the HIV-1IN chain transfer activity to the active main inhibition of HIV-1IN, promptly suppresses the process of HIV gene integration in host cell chromosome.

Table 2:S-AbPS suppresses the mechanism of action of HIV-1IN

Mechanism of action	Drug level (μ M)	Suppression ratio (%)
			It is active to suppress the active HIV-1IN 3 ' of inhibition of HIV-1IN (always)-cleavage activity inhibition HIV-1IN chain transfer activity inhibition HIV-1IN assembling	0.12 0.20 0.12 0.12	45.3±0.7 82.8±1.3 81.1±2.1 61.5±1.6

Embodiment 3: the activity of the anti-HIV-1 IN of the S-AbPS that different time is measured

Experimental technique the results are shown in Table 3 with embodiment 1.The activity of the anti-HIV-1 IN of the same crowd of S-AbPS that measures in different time does not as seen from Table 3 have significant change, and is activity stabilized.

Table 3: the activity of the anti-HIV-1 IN of the S-AbPS that different time is measured

Minute (year-moon-Ri)	Activity (the IC of anti-HIV-1 IN 50μM)
		2002-04-03	0.204
2002-07-14	0.038
		2002-12-31	0.118
2003-05-23	0.219
		2003-08-25	0.211
2003-12-25	0.090
		2004-03-27	0.139
2004-09-07	0.132
		2005-01-20	0.098
2005-03-03	0.158

The effect of embodiment 4:S-AbPS anti-HIV-1 in cell culture

1.MTT staining is measured the toxicity of S-AbPS to the MT-4 cell culture

In 96 porocyte culture plates, the S-AbPS that adds 2 times of dilutions simultaneously respectively is the medicinal liquid of totally 8 concentration with the MT-4 cell inoculation, 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.Supernatant 100 μ l are abandoned in suction in the 7th day 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 ₅₇₀The nm value is calculated the poisonous concentration (TC of medicine half ₅₀), the results are shown in Table 4.

2.MTT staining is measured S-AbPS inhibitory action to HIV-1IIIB in the MT-4 cell culture

The MT-4 cell inoculation in 96 porocyte culture plates, is added HIV-1 zoo virus strain IIIB culture fluid, add variable concentrations S-AbPS 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 was inhaled in the 7th day and to be abandoned supernatant 100 μ l, every hole adds 10 μ l 5mg/mlMTT dyeing, 37 ℃, 5%CO ₂Continue in the saturated humidity incubator to cultivate 4 hours, every hole adds 100 μ l 50%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 medicine medium effective concentration (EC ₅₀), and calculate therapeutic index (SI), the results are shown in Table 4.

3. the separation and the cultivation of peripheral blood lymphocytes (PBMC)

Get healthy people's fresh venous, with FiColl separating medium separation of human peripheral blood lymphocytes (PBMC), with the inoculum preparation 1.5 * 10 that contains PHA ⁵Cell/ml suspension inoculation was cultivated 72 hours in 37 ℃ of 5%CO2 incubators in culture bottle.Scrape collecting cell with cell, be made into 10 ⁶/ ml.

4. ³The H-thymus pyrimidine method of mixing is measured the toxicity of S-AbPS to the PBMC cell culture

The PBMC cell of 0.1ml and the S-AbPS of variable concentrations are cultivated in 96 orifice plates.After 4 days, use ³The H-thymus pyrimidine is added each hole, continues to cultivate results and detection CPM value after 4-6 hour, calculates TC ₅₀The results are shown in Table 4.

5.P24 the antigen measuring method is measured S-AbPS suppresses HIV-1AZT persister (HIV-1018c) in the PBMC cell culture activity

With AZT multidrug resistant disease strain (HIV-1018c) 100TCID ₅₀Viral liquid inductance transfect cell, adsorbed 2 hours.With serum-free 1640 culture medium flush awaies viral adsorption not, the cell of infective virus is made into 1 * 10 with culture fluid ⁶/ ml.Plant 96 well culture plates, 100 μ l/ holes.The S-AbPS medicinal liquid 100 μ l that add variable concentrations simultaneously respectively.If cell contrast and virus infected cell matched group group.37 ℃ of 5%CO2 cultivate the 4th day (96 hours), and the sucking-off supernatant is pressed test kit institute and measured HIV-1P24 antigen to method.Calculate medicine medium effective concentration (EC ₅₀), and calculate therapeutic index (SI), the results are shown in Table 4.

6. result

S-AbPS in the MT-4 cell culture toxicity and the action effect of anti-HIV-1 IIIB and the toxicity in the PBMC cell culture and the activity that suppresses HIV-1 018c see Table 4.

The table toxicity of 4:S-AbPS in the MT-4 cell culture and the effect of anti-HIV-1 IIIB

Cell	TC 50(μM)	EC 50(μM)	SI
				MT-4 PBMC	201.7 43.8	0.37 3.85	545 11

The medicine test card is bright in the cell culture, and S-AbPS has the effect of obvious suppression HIV-1IIIB at the MT-4 cell culture, and the effect of obvious suppression HIV-1 018c is arranged in the PBMC cell culture.

Mus serum active after the embodiment 5:S-AbPS administration is measured

Behind mouse stomach and the lumbar injection serum to the inhibitory action of HIV-1RT

Male mice in kunming 18 ~ 20 grams (5 every group), oral administration S-AbPS 1.25mmol/kg 1 time, after 1 hour, or S-AbPS 25 μ mol/kg are through lumbar injection after 30 minutes, eye socket is got blood, serum is through 56 ℃ of deactivations in 30 minutes and dilute the activity of measuring its inhibition HIV-1RT after 60 times, the results are shown in Table 5.

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

Administering mode	Get the blood time	Medicine	To HIV-1RT suppression ratio (%)
				Lumbar injection	30 minutes	Normal saline contrast S-AbPS 25 μ mol/kg	22.3±2.3 26.9±5.9
Irritate stomach	1 hour	Distilled water contrast S-AbPS 1.25mmol/kg	19.2±5.1 24.9±4.6

2. serum suppresses the HIV-1RT activity after the rat administration

Male SD rat 190 ~ 210 grams (3 every group), oral administration S-AbPS 1.25mmol/kg 1 time, after 1 hour, or S-AbPS 25 μ mol/kg are through lumbar injection after 30 minutes, eye socket is got blood, serum is through 56 ℃ of deactivations in 30 minutes and dilute the activity of measuring its inhibition HIV-1RT after 60 times, the results are shown in Table 6.

Serum suppresses the HIV activity behind table 6:SD rat oral gavage and the intraperitoneal injection of drugs

Dosage	Administering mode	To HIV-1RT suppression ratio (%)
					15 minutes	30 minutes	1 hour
		S-AbPS1.25mmol/kg	Irritate stomach					20.15±9.93	24.99±6.00
The distilled water contrast					15.91±5.55	19.77±15.36
		S-AbPS25μmol/kg		Lumbar injection			21.86±8.30	8.79±5.71
The normal saline contrast	4.57±4.52		6.26±3.82

The anti-HI-1RTV activity test of Mus serum shows after the above-mentioned administration, behind S-AbPS oral administration or the lumbar injection, all can detect the activity that it suppresses HIV-1 in large and small Mus serum.

Embodiment 6:S-AbPS is to the influence of mouse immune cell

1. experimental technique

Select 18-20 gram BALB/C mice for use, experiment divides 5 groups, 5 every group.Intraperitoneal injection, administration volume are 0.2ml/20g.5 groups is respectively physiology saline control group, cyclophosphamide-a control group, cyclophosphamide+thymosin positive drug control group, cyclophosphamide+S-AbPS medicine group and S-AbPS medicine matched group.Cyclophosphamide disposable celiac injection in the 1st day 80mg/kg, the 7th day 40mg/kg strengthen once; Thymosin 500 μ g/kg are subcutaneous injection, and except that cyclophosphamide, other is every day 1 time, successive administration 12 days, and wherein preceding 7 days dosage of S-AbPS is 4mg/kg, changes 8mg/kg in back 5 days into.Get blood, the EDTA-K3 anticoagulant in the 12nd day mouse orbit.Earlier the anticoagulation sample is diluted 5 times with normal saline before the labelling.Prepare two groups of fluidic cells mensuration and manage, first group of every pipe adds CD3, CD4, each 3 μ l of CD8 fluorescent antibody, and second group of every pipe adds respectively 3 μ l of CD3, CD19 fluorescent antibody, adds the anticoagulation sample after 100 μ l dilute then, lucifuge labelling 20 minutes.Add 0.5ml Lysing Solution and make erythrocytolysis, centrifugal 5 minutes of 1500rpm, PBS washes twice, uses the 0.4mlPBS suspension cell at last, with cells were tested by flow cytometry CD3 (T cell), CD4, the relative populations of CD8 and CD19 (B cell).Carry out the multifactor analysis of variance with SPSS10.0 software.

2. experimental result

S-AbPS the results are shown in Table 7 to the influence of mouse immune cell.By table as seen, compare, do not change for each cell percentage ratio of S-AbPS mice with each cell percentage ratio of normal saline control group mice; Significantly reduce for cyclophosphamide mouse B cell (CD19) percentage ratio.Compare with each cell percentage ratio of cyclophosphamide group, cyclophosphamide+thymosin mice CD3 and cd4 cell percentage significantly raise, and B cell percentage ratio significantly reduces; Cyclophosphamide+S-AbPS mice CD3 and cd4 cell percentage significantly raise.This shows: S-AbPS is to the not influence of normal mouse lymphocyte subpopulation percentage ratio, but can increase peripheral blood T (CD3) the cell percentage ratio of immunocompromised (reduction of the B cell) mice that cyclophosphamide causes, cd4 cell percentage ratio significantly increases in the T cell, cd8 cell percentage ratio is constant, do not have reverse effect but cyclophosphamide is reduced B cell percentage ratio, its result is identical with the effect of positive drug thymosin.

Table 7:S-AbPS is to the influence of mice peripheral blood T, B cell percentage ratio

Medicine	Cell percentage ratio (%)
					CD3	CD4	CD8	CD19
	Normal saline cyclophosphamide cyclophosphamide+thymosin cyclophosphamide+S-AbPS S-AbPS	56.5±5.24 64.6±15.55 72.9±9.38 82.6±3.25 65.1±5.80	41±2.90 45.6±10.19 53.7±7.45 62.8±0.92 46.6±4.53	15.2±2.72 19±5.37 19.2±2.23 19.9±4.17 18.5±1.27					33.2±4.68 14.3±11.21 11.4±0.20 11.8±2.83 27.4±5.66

Claims (3)

1, the application of chemical compound shown in the structural formula (1) in the preparation treatment or the virus infective medicament that prevents AIDS.

Wherein: R=SO ₃Na

2. the application of chemical compound shown in the structural formula (1) in the preparation treatment or the viral infection disinfectant that prevents AIDS.

Wherein: R=SO ₃Na

3. the opportunistic infection due to the preparation treatment or the virus that prevents AIDS of chemical compound shown in the structural formula (1)

Application in the medicine.

Wherein: R=SO ₃Na