CN102247401A - Low molecular weight glycosylated chondroitin sulfate and its purpose in preparation of anti-HIV-1 medicament - Google Patents

Abstract

The invention discloses a low-molecular-weight glycosylated chondroitin sulfate, the weight-average molecular weight of which is 3000-15000 Da, and the monosaccharide composition is acetylgalactosamine (D-GalNAc), glucuronic acid (D-GlcUA), and fucose (L-Fuc), or its sulfate ester (-OSO ₃ ^- ), the molar ratio of D-GalNAc:D-GlcUA:L-Fuc:-OSO ₃ ^- is about 1:(1±0.3):(1± 0.3): (3.5 ± 0.5). The low-molecular-weight glycosylated chondroitin sulfate has potent anti-HIV-1 virus activity, is a gp120 invasion inhibitor, and can be used for preventing and/or treating AIDS. The present invention also provides a method for preparing the low-molecular-weight glycosylated chondroitin sulfate and its pharmaceutical composition preparation. The peroxide method is used to depolymerize the glycosylated chondroitin sulfate to obtain a low-molecular-weight product, which is then separated by gel or The ultrafiltration method removes low-molecular and/or high-molecular impurity components in the product. The low molecular weight glycosylated chondroitin sulfate and the pharmaceutical composition thereof can be prepared into injections, freeze-dried powder injections or suppositories.

Description

Low molecular weight glycosylated chondroitin sulfate and its use in the preparation of anti-HIV-1 medicine

technical field

The invention relates to a low molecular weight Glycosylated Chondroitin sulfate (LGC) and its preparation method, a pharmaceutical composition containing LGC and its use in the preparation of AIDS prevention and/or treatment drugs.

Background technique

Acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (human immunodeficiency virus, HIV-1) infection is a major disease that seriously endangers human life and health. According to a report released by UNAIDS on November 23, 2010, it is estimated that 33.3 million people worldwide are infected with HIV, and 25 million people have died since AIDS was first identified in June 1981. In China, as of the end of 2009, an estimated 740,000 adults and children were infected with HIV, and 48,000 were newly infected in 2009. Of these patients, an estimated 105,000 were AIDS cases and 26,000 died of AIDS-related causes in 2009. In addition to the treatment of complications, anti-infection treatment, and immunomodulatory treatment, the most effective treatment method for AIDS clinical treatment is highly effective antiviral treatment. Data show that since the advent of combined antiretroviral therapy (HAART, a combination of at least three nucleoside/non-nucleoside antiretroviral drugs and protease inhibitors) in 1996 to 2006, AIDS patients have been diagnosed from the date of diagnosis. The average survival time of patients has increased from 0.26 years to 13.3 years. Although great progress has been made in the treatment of AIDS, there are still defects such as difficult to cure, non-response to treatment, emergence of drug resistance, and significant side effects. At present, HAART treatment is ineffective for about 30% of AIDS patients due to reasons such as treatment non-response or drug resistance.

In order to enable patients who are ineffective in HAART treatment to receive effective drug treatment, new anti-HIV-1 drugs are urgently needed clinically, and the new anti-HIV-1 drugs should have different pharmacological targets from antiretroviral drugs and protease inhibitors .

Glycosylated chondroitin sulfate (Glycosylated chondroitin sulfate, GCs) is a glycosaminoglycan analogue with fucose side chain substitution, its polysaccharide backbone is similar to chondroitin sulfate, and is composed of hexuronic acid and hexosamine The disaccharide structural units are connected sequentially, and the main chain and side chain sugar hydroxyl groups can have sulfated groups. Natural GCs are mainly derived from the body wall or viscera of echinoderms, which have the main chain structural unit of [→4)D-GlcUA(β1→3)D-GalNAc(1→], and the side chain fucose sulfate is (α1 →3) Glycosidic linkages to D-GlcUA (J Biol Chem, 1988, 263(34): 18176-83 and J Biol Chem, 1991, 266(21): 13530-6). Natural GCs have significant anticoagulant activity , which is characterized by potent inhibition of endogenous factor X enzyme (internal factor tenase, f.Xase) activity, and significant HCII-dependent antithrombin (f.IIa) activity. As disclosed in Chinese Patent Publication No. CN101735336A Provided is a method for preparing oligomeric fucosylated glycosaminoglycans, which are prepared by depolymerizing fucosylated glycosaminoglycans in an aqueous medium using a fourth-period transition metal ion-catalyzed peroxide depolymerization method. The preparation method The reaction conditions are mild, the reproducibility and stability are good, the cleavage selectivity is high, and the quality of the product is uniform and controllable. The obtained oligo-fucosylated glycosaminoglycans have GalNAc as the reducing end of the glycan molecule is not less than 80% , the weight average molecular weight is about 6,000Da-20,000Da, and the PDI is 1.0-2.0.

It can be seen that it is of great significance to study low molecular weight glycosylated chondroitin sulfate (LGC) and its preparation method, pharmaceutical composition containing LGC and its use in the preparation of drugs for preventing and/or treating AIDS.

Contents of the invention

The object of the present invention is first to provide a kind of low molecular weight glycosylated chondroitin sulfate (LGC) and pharmaceutically acceptable salt thereof in the purposes in anti-type 1 human immunodeficiency virus (HIV-1) medicine preparation, wherein said LGC It is a mixture of homologous glycosaminoglycan derivatives having a structure of formula (I),

In formula (I):

D-GlcUA-β1-, is β-D-glucuronic acid-1-yl;

D-GalNAc-β1-, is β-D-N-acetylgalactosamine-1-yl;

L-Fuc-α1-, is α-L-fucose-1-yl;

R ₁ is -H or D-GalNAc-β1-;

R ₃ , R ₅ , R ₅ , R ₆ , R ₇ are independently -H or -SO ₃ ^- ;

R ₂ can be -OH, -4-OD-GlcUA, or a group shown in formula (II) or (III):

Wherein, R' is an L-Fuc-α1-substituent group, and there is a sulfate group with the same structure as formula (1) on L-Fuc-α1-;

R" is -4-[L-Fuc(α1-3)]D-GlcUA-β1-group, and there is a sulfate group with the same structure as formula (1) on L-Fuc-α1-,

R ₃ and R ₄ are as defined above.

In terms of molar ratio, the ratio range of the three monosaccharide residues of D-GlcUA, D-GalNAc and L-Fuc contained in the LGC to the contained -OSO ₃ ^- group is 1: (1±0.3): (1±0.3):(1±0.3) 0.3): (3.5±0.5);

n is an integer with an average value of about 3 to 17;

The weight average molecular weight of the LGC ranges from 3000 to 15000 Da.

The molecular weight of the LGC of the present invention can be detected by high performance gel chromatography (HPLPC). Considering the intensity of anti-HIV-1 activity and avoiding hematological influence, the molecular weight range of the low molecular weight glycosylated chondroitin sulfate selected by the present invention is about 3,000～15,000Da (i.e. the homologous compound shown in formula (I) The average value of n of the compound is about 3 to 17), and the preferred molecular weight range is about 5,000 to 8,000 Da (the average value of n of the homologue shown in formula (I) is about 5 to 9).

The polydispersity index (PDI, weight average/number average molecular weight ratio, Mw/Mn) of the LGC of the present invention is generally between 1.0 and 1.8; the preferred LGC has a PDI between 1.1 and 1.5.

The LGC of the present invention may be a pharmaceutically acceptable salt of an alkali metal, alkaline earth metal, etc., and similarly, the LGC may also be an ester formed with a basic organic group. The preferred pharmaceutically acceptable salt of LGC according to the invention is the sodium, potassium or calcium salt of LGC.

The LGC in the present invention is the depolymerization product of GCs extracted from the body wall of the Echinodermata Holothurian class animal body wall and the product in which the end of the GCs depolymerization product is reductively tyrosinated.

The research of the present invention has found that the LGC has potent anti-HIV-1 activity, and its _IC50 value for inhibiting HIV-1 experimental strains and clinical isolates from infecting human lymphocytes can reach about 10-100 nmol/L. The LGC had no significant anticoagulant activity at the concentration.

Further studies of the present invention have found that the LGC is an HIV-1 invasion inhibitor, which is consistent with the demand for anti-HIV-1 drugs with new clinical targets; the present invention proves that the pharmacological properties of the LGC are The biological target is HIV-1 outer membrane glycoprotein gp120; the interaction detection using soluble CD4 (sCD4) and CD4i monoclonal antibody 14b proves that the remarkable feature of the LGC is that it binds to the conserved region CD4i of the glycoprotein gp120. CD4 is the CD4 ⁺ cell membrane surface receptor that mediates HIV-1 adhesion, and CD4i is the gp120-CD4 binding-induced exposure site, and the gp120 exposed at this site can further bind to the CD4 ⁺ cell membrane surface co-receptor CCR5 or CXCR4, and cause HIV -1 glycoprotein gp41 enters the cell membrane, and gp41 rearranges and folds so that the viral outer membrane fuses with the CD4 ⁺ cell membrane, thereby finally realizing the HIV-1 invasion step.

Apparently blocking CD4i blocks HIV-1 from invading CD4 ⁺ cells. Due to the highly conserved nature of CD4i, HIV-1 entry inhibitors against CD4i are beneficial to avoid drug resistance. At present, HIV-1 vaccines and HIV-1 entry inhibitors targeting CD4i have become key directions in the development of AIDS therapeutic drugs, so the LGC of the present invention has important application value as anti-drug-resistant HIV-1 entry inhibitors.

When _R2 can be -OH or -4-OD-GlcUA group, the LGC of the present invention only has ultraviolet absorption in the near-infrared region, which is not conducive to the establishment of an analysis and detection method based on UV detection technology. For the convenience of description below, this specification refers to this LGC homologue as LGC-1. Apparently, LGC-1 has -D-GlcUA or -D-GalNAc at the reducing glycosyl end.

When R ₂ is a group represented by formula (II) or (III), the LGC of the present invention has an ultraviolet absorption at about 280 nm, and this specification refers to such LGC homologues as LGC-2. Apparently, LGC-2 is the product of reductive amination of the reducing sugar terminal of the corresponding LGC-1. When a certain amount of LGC-2 is contained in the LGC of the formula (I) structure of the present invention, due to the ultraviolet absorption at 280nm, it is easy to establish a spectrophotometric detection method, and these methods are useful for establishing reliable and accurate quality control, pharmacokinetic Kinetic analysis techniques are of great significance. Generally speaking, when LGC-2 accounts for more than 30% of all LGCs, detection methods suitable for quality control and pharmacokinetic analysis can be easily established.

The LGC- ₁ whose R of the present invention is -OH or -4-OD-GlcUA is actually the depolymerization product of GCs extracted from the body wall of Echinodermata and Holothuria; the _R is formula (II) or ( The LGC-2 of the group III) is actually the product obtained by terminal reductive amination of the GCs depolymerization product.

The research results of the present invention show that the anti-HIV-1 activity of GCs depolymerization product (LGC-1) is close to and slightly stronger than that of the corresponding GCs depolymerization products. For this reason, the GCs depolymerization products and some or all of the terminal reductive amination products can be used to prepare anti-HIV-1 drugs, and thus are included in the scope of the present invention.

A further object of the present invention is to provide a low molecular weight glycosylated chondroitin sulfate (LGC) and a pharmaceutically acceptable salt thereof, said LGC being a homologous glycosaminoglycan derivative mixture of the formula (I) structure defined above , wherein, in molar percentage, LGC-2 whose R ₂ is a group of formula (II) or (III) accounts for 30% to 100% of all compounds of formula (I).

A further object of the present invention is also to provide a low molecular weight glycosylated chondroitin sulfate (LGC) and a pharmaceutically acceptable salt thereof, said LGC being a homologous glycosaminoglycan derivative of the formula (I) structure defined above Mixture, wherein, R ₂ is the LGC-2 of formula (II) or (III) group, namely R ₂ is not -OH or -4-OD-GlcUA.

A further object of the present invention is to provide a preparation method of the low molecular weight glycosylated chondroitin sulfate (LGC). The preparation method of the LGC is to extract GCs from the body wall of sea cucumbers, and then use metal ion-catalyzed peroxide depolymerization to obtain GCs depolymerization products (LGC-1), and the obtained LGC-1 can be selected for further processing. Reductive amination reaction to obtain all or part of the terminal tyrosylated LGC product (LGC-2 or containing LGC-2).

The preparation method may include but not limited to the following steps:

Step 1: Extract acidic mucopolysaccharides, ie, glycosylated chondroitin sulfate (GCs), from the body wall of Echinodermata and sea cucumbers.

Extraction of GCs from the body wall of sea cucumbers can be carried out with reference to known methods in the art, generally including but not limited to the following steps: degreasing, alkali and/or enzyme treatment to obtain extract, lower alcohol and/or ketone precipitation to obtain crude sugar extract After reconstitution, the obtained crude sugar extract can be selected for protein removal treatment (isoelectric point method, protein denaturant precipitation method, etc.), decolorization treatment (oxidation method, etc.), and removal of small molecule impurities (ultrafiltration dialysis, gel filtration, etc.) , purification (gel chromatography and/or ion chromatography), etc., the resulting purified product can also be selected to obtain specific types of metal ion salts (such as sodium salts, potassium salts, calcium salts, etc.) Dry and/or freeze-dry to obtain the GCs final product.

Step 2: Peroxidative depolymerization of GCs obtained in step (1) to obtain its depolymerization product LGC-1.

Due to the particularity of the chemical structure of GCs, commonly used glycosaminoglycan depolymerization methods such as enzymatic depolymerization, nitrous acid depolymerization, and acid-base depolymerization are difficult to apply to GCs depolymerization. At present, the depolymerization method of GCs is mainly peroxide depolymerization method. For avoiding the impact of the depolymerization process on the characteristic structure of GCs, the preferred LGC-1 preparation method of the present invention is the peroxide depolymerization method catalyzed by metal ions, which except reducing the degree of polymerization of the structural unit (in the formula (I) The value of n is reduced), basically does not affect the monosaccharide composition of the depolymerization product, the characteristics of repeating structural units, does not affect the characteristic chemical functional groups such as side chain fucose substituents and sulfate groups, that is, does not affect the general structure of formula (I) .

Specifically, the preferred GCs depolymerization method of the present invention is that the GCs obtained in step 1 is made into an aqueous solution with a mass fraction of 0.05 to 10%, and the metal ion catalyst (metal ion Salt: GCs), add about 0.5-5% peroxide (peroxide: GCs) in mass fraction, and react at about 20-60°C. When the molecular weight of the depolymerization product (in terms of Mw and/or Mn) reaches the desired molecular weight range, add a catalyst chelating agent to terminate the reaction or directly add lower alcohols/ketones to precipitate the product. After the precipitate is collected and redissolved, it is purified by gel chromatography, ion exchange chromatography or ultrafiltration dialysis, and the obtained product can optionally be obtained by ion exchange to obtain the corresponding alkali metal ion salt. The GCs depolymerization product LGC-1 was thus obtained.

Generally speaking, in the LGC-1 prepared by the method of the present invention, according to the calculation of the product NMR detection spectrum, the compound whose _R2 is -OH can account for more than 85% of the total amount of LGC-1 in terms of molar percentage.

In the depolymerization method described in step 2, the preferred peroxide of the present invention is hydrogen peroxide; the preferred metal ion catalyst is divalent copper ion (salt).

In the preparation method of LGC-1 described in the present invention, the depolymerization speed of the reactant GCs is stable, the molecular weight distribution of the product is narrow, the obtained product is easy to separate and purify, and high-purity LGC products can be produced in large quantities.

Step 3: LGC-1, the depolymerization product of GCs obtained in step 2, can optionally be subjected to terminal reductive amination treatment to obtain LGC-2 containing R ₂ as a group of formula (II) or (III).

In the present invention, the reductive amination step of the LGC-1 terminal is: dissolving the GCs depolymerization product in a buffer solution with a pH of 7-9, adding stoichiometric tyramide and sodium cyanoborohydride in sequence, and reacting at room temperature or by heating. After the reaction is completed, the reaction product is separated and purified, and converted into the desired alkali metal ion salt. The figure below takes the terminal-D-GalNAc as an example to illustrate the reductive amination process:

The separation and purification of the reaction product of the step (3) means that in the purified product, in molar ratio, R ₂ is the formula (I) compound of formula (II) or (III) group usually accounts for all formula (I) compounds More than 30%.

Since the LGC of the present invention has potent anti-HIV-1 activity, a further object of the present invention is to provide a pharmaceutical composition containing LGC, which contains an effective dose of the LGC of the present invention or a pharmaceutically acceptable salt thereof, and Pharmaceutically acceptable excipients.

In the present invention, the pharmaceutical composition containing LGC may be a preparation for systemic administration or a preparation for local administration.

In the preparation for systemic administration, the pharmaceutical composition of LGC may be a preparation suitable for intravenous administration, subcutaneous and/or intramuscular injection, or a preparation for respiratory tract administration. Among these preparations, the preferred dosage forms of the pharmaceutical composition are aqueous solution for injection, freeze-dried powder for injection, and oral or nasal spray suitable for respiratory administration. In the pharmaceutical composition for systemic administration, according to the anti-HIV-1 activity of LGC of the present invention and its bioavailability for injection, the content of LGC in a single dose preparation may be about 5-150 mg.

In topical preparations, suppository, gel, ointment, liniment and the like can be selected as the dosage form of the pharmaceutical composition. The content of the active ingredient LGC in these pharmaceutical preparations can be determined by those skilled in the art according to the effective drug concentration during topical administration.

Since the anti-HIV-1 mechanism target of the LGC of the present invention is different from existing clinical drugs, it can be used in combination with existing clinical drugs, including sequential administration with existing anti-HIV-1 drugs, and can also be combined with these The drug is administered at the same time, or forms a pharmaceutical composition preparation together with existing anti-HIV-1 drugs.

The low-molecular-weight glycosylated chondroitin sulfate (LGC) of the invention has strong anti-HIV-1 activity, and can be used as an anti-HIV-1 drug for treating and preventing AIDS.

Summary of the positive effects of the present invention: the present invention first finds that the LGC with strong anti-HIV-1 activity can inhibit HIV-1 experimental strains, clinical isolates and drug-resistant strains from infecting human lymphocytes with _IC50 values up to about 10-100nmol/L, and at this effective concentration, there is basically no anticoagulant activity in LGC. Further studies have found that the LGC is an HIV-1 invasion inhibitor, which is consistent with the clinical demand for new target anti-HIV-1 drugs. In addition, the present invention also found that, as an HIV-1 invasion inhibitor, the LGC is characterized by binding to the conserved region CD4i of the HIV-1 outer membrane glycoprotein gp120. Due to the high conservation of CD4i, HIV-1 vaccines and HIV -1 invasion inhibitors have become the key direction in the development of AIDS treatment drugs, therefore, the LGC of the present invention has important application value for the development of drug-resistant HIV-1 invasion inhibitors.

Description of drawings

Figure 1 is the HPGPC spectrum of GCs derived from Prunus ginseng and its low molecular weight product LGC.

Fig. 2 is the detection spectrum of LGC-1A ¹ H NMR.

Fig. 3 is the detection spectrum of LGC-1A ¹³ C NMR.

Figure 4 is the detection spectrum of LGC-1A DEPT135°.

Figure 5 is the detection spectrum of LGC-1A ¹ H- ¹ H COZY.

Figure 6 is the HSQC detection spectrum of LGC-1A ¹ H- ¹³ C.

Fig. 7 is a graph showing the inhibitory effect of LGC on HIV-1-induced syncytium formation, showing the inhibitory effect of LGC on HIV-1-induced syncytium formation.

Fig. 8 is a graph showing the toxic effect of LGC on C8166, showing the toxic effect of LGC on C8166.

Fig. 9 is a graph showing the interaction data of LGC-gp120 detected by surface plasmon resonance (SPR), showing the interaction of LGC-gp120 detected by surface plasmon resonance (SPR).

Detailed ways

The following examples are detailed descriptions of the content of the present invention, which are not intended to limit the scope of the present invention.

[Example 1] Preparation of low molecular weight glycosylated chondroitin sulfate (LGC)

1.1 Materials:

Prunus ginseng (Thelenota ananas Jaeger), commercially available, eviscerated and dried body wall;

Reagents used for H ₂ O ₂ , CH ₃ COONa·3H ₂ O, NaCl, NaOH, Cu(CH ₃ COO) ₂ ·H ₂ O, etc.: all commercially available analytical reagents.

1.2 Method:

(1) Preparation of glycosylated chondroitin sulfate (GCs): take the dried body wall of Echinodermata ginseng, and prepare GCs according to the literature method (J Biol Chem, 1991, 266(21): 13530-6), with a yield of 0.75% , purity 98% (HPGPC, area normalization method), weight average molecular weight (Mw), 65,820.

(2) Preparation of low-molecular-weight glycosylated chondroitin sulfate (LGC): Take 5.0 g of GCs obtained in step (1) and add them to a round-bottomed flask, add 180 ml of distilled water to dissolve them, keep warm in a water bath at 35 ° C, and add 1% chlorine while stirring at a constant speed copper chloride solution (5ml), and 10ml of 15% H ₂ O ₂ was added dropwise within 60min, and the pH range was controlled to be 7.2-7.8 with 1N NaOH solution during the reaction. After continuously stirring for 1 hour, 400 mg of ethylenediaminetetraacetic acid disodium salt was added to the reaction solution to terminate the reaction, then cooled with ice water, 540 ml of ethanol with a concentration of 95% (v/v) was added, and centrifuged to obtain a precipitate.

Wash the obtained precipitate twice with 100ml 80% ethanol, add 150ml water to dissolve it, exchange it with 001×7 type cationic resin into sodium salt, then dialyze with a dialysis membrane with a molecular weight cut-off of 3500Da for 6 hours, concentrate the retained product, and freeze-dry , to obtain 4.02 g of depolymerized sample LGC-1A, with a yield of 80.4%.

(3) The physical and chemical parameters, monosaccharide composition and structure analysis of GCs derived from plum blossom ginseng and its depolymerization product LGC-1A were detected by spectrum: high performance gel chromatography (HPGPC) was used to detect molecular weight and distribution; conductometric method was used to detect -OSO ₃ ^- / -COO ^- molar ratio; Optical rotation is measured according to the method of Appendix VI E of Part Two of the Chinese Pharmacopoeia (2005 edition); Intrinsic viscosity is measured with Ubbelohde viscometer.

Elson-Morgon method to detect acetylgalactosamine (D-GalNAc) content, carbazole method to detect glucuronic acid (D-GlcUA) content (Zhang Weijie, Glycocomplex Biochemical Research Technology (Second Edition), Zhejiang: Zhejiang University Press , 1999, 19-20); ¹ H NMR methyl peak integral area to calculate the D-GalNAc/L-Fuc molar ratio. Swiss Bruker company AVANCE AV 400 superconducting nuclear magnetic resonance spectrometer (400MHz) detects NMR spectrogram (detection condition, solvent D ₂ O, 99.9Atom%D (Norell company); Internal standard, trimethylsilyl-propionic acid (TSP-d4); temperature 45°C).

The HPGPC detection spectrum of GCs and its depolymerization product LGC-1A is shown in Attachment 1, and the detection results of physical and chemical parameters and monosaccharide composition are shown in Table 1; the detection spectrum of ¹ H/ ¹³ C NMR and its correlation spectrum is shown in Attachment 2～ 6. See Table 2 for the signal assignment of ¹ H/ ¹³ CNMR spectrum data.

The test results in Table 1 show that compared with GCs, the molecular weight and intrinsic viscosity of LGC-1A are significantly reduced, while the monosaccharide composition remains stable (about 1:1:1:3.5).

Table 1. Physicochemical parameters and monosaccharide composition detection results of GCs and LGC-1A derived from plum blossom ginseng

The signal assignment of ^{the 1} H/ ¹³ C NMR spectrum data shown in Table 2 shows that the characteristic chemical structures of GCs and LGC-1A are basically the same. The following takes LGC-1A as an example to briefly describe its signal attribution and structure analysis.

In ¹ H NMR, there are three sets of strong signal peaks at 5.2-5.7ppm, which are hydrogen signals of different types of sulfated α-fucose end groups, and the signals at about 5.6 and 5.3ppm correspond to Fuc2S4S and Fuc4S end groups respectively hydrogen. The 5.35ppm peak with obvious shoulders is the superposition of Fuc3S and Fuc4S terminal hydrogen signals; the signal at about 5.18ppm is the α-terminal hydrogen signal of the free end of the main chain. The backbone β-terminal hydrogen signal appears at about 4.4-4.6 ppm. Fuc methyl proton signals appear at about 1.0-1.3 ppm; GalNAc acetyl protons appear at about 1.9-2.0 ppm. The sugar ring hydrogen at the sulfate ester substituent appears at about 4.6-5.0 ppm, while the signal at about 3.6-4.6 ppm is the superposition of the sugar ring hydrogen at the non-sulfate substituent.

It can be seen from ^{the 1} H- ¹ H COZY spectrum that the H2 and H3 signal peak positions of GlcUA are both > 3.6ppm, which is about 0.3-0.4ppm shifted to the downfield relative to the corresponding signal (3.35-3.55ppm) of the chondroitin sulfate compound GlcUA, It indicates that there is a side chain substitution at this position, and it is a sugar ring substitution rather than a sulfated substitution. ^{The 1} H- ¹ HTOCSY spectrum clearly shows the coupling correlation between GlcUA further proton signals; the spectrum also clearly shows the coupling between Fuc terminal hydrogen and H3. ^{The 1} H- ¹ H NOESY spectrum shows the coupling between each Fuc end group hydrogen and GlcUA H3, GlcUA end group hydrogen and GalNAc H3, and GalNAc end group hydrogen and GlcUA H4, respectively, indicating the connection between monosaccharides Relationship: GlcUA and GalNAc are connected to each other by β(1→3) and β(1→4) glycosidic bonds to form the main chain, and Fuc is connected to GlcUA by α(1→3) glycosidic bonds.

In the ¹³ C-NMR spectrum, the C1 peaks of GlcUA and GalNAc appear at about 102-106 ppm, indicating the β-configuration of their bridgehead hydrogens; the C1 signal of Fuc at a relatively high field (about 101-99 ppm) and its bridgehead hydrogen α- configuration matches. The carbonyl carbon (C6) in the GlcUA carboxyl group and the carbonyl carbon (C7) in the GalNAc acetamido group appear in almost the same position (~177ppm); the Fuc methyl carbon signal appears at about 18~19ppm; the methyl group in the GalNAc acetamido group The carbon signal appears around 25ppm. The C2 signal of GalNAc4S6S appeared at about 54ppm, indicating its characteristics of deoxygenation and acetamido substitution; the weaker GalNAc4S C2 signal appeared at 55ppm.

The DEPT 90° spectrum shows a primary carbon nature at -177 ppm carbonyl quaternary carbons and methyl carbons at about 18 and 25 ppm. In the DEPT135° spectrum, it can be seen that the secondary carbon (C6, CH ₂ ) signal of GalNAc4S6S appears at 70.1ppm, and the secondary carbon signal of GalNAc4S without the 6-sulfate group appears at 64.0ppm. According to the calculation of the two C6 signal intensities, GalNAc4S only accounts for about 5% of the total GalNAc, which is close to the proportion calculated according to the C2 signal intensity.

The coupling between C1 of Fuc and GlcUAH3, the coupling between GlcUAC1 and GalNAcH-3; the coupling between GalNAc C1 and GlcUAH4 can be seen in the HMBC spectrum; Fuc, GlcUA, GalNAc bridgehead hydrogen and GlcUAC3, The coupling between GalNAc C3 and GlcUAC3 further confirms the connection relationship between the monosaccharides of THG.

Based on THG-1 hydrogen spectrum, carbon spectrum and correlation spectrum, it can be seen that among the three constituent monosaccharides of this product, GlcUA and GalNAc are connected to each other through β(1→3) and β(1→4) glycosidic bonds to form the glycan backbone , thus forming the main chain disaccharide structural unit. Obviously, the main chain structure of THG is similar to chondroitin sulfate, and its hexosamine is mainly GalNAc4S6S, and only about 5% of the hexosamine has no 6-sulfate group substitution (GalNAc4S). According to the H2 and H3 chemical shifts of GlcUA combined with ¹ H- ¹ H NOESY and ¹ H- ¹³ C HMC, it can be judged that Fuc is linked to GlcUA by α(1→3) glycosidic bonds. Almost all GlcUAs have side chain fucosyl substitutions, while the ratio of GlcUA and Fuc monosaccharides in THG is close to 1:1, so each disaccharide structural unit of THG main chain has a side chain fucosyl substituent.

Table 2. ¹ H/ ¹³ CNMR detection data of GCs and LGC-1A derived from plum blossom ginseng (δ[ppm])

[Example 2] Preparation of series molecular weight LGC-1

2.1 Materials:

The GCs derived from Prunella ginseng were prepared with the method described in Example 1.

Reagents used for H ₂ O ₂ , CH ₃ COONa·3H ₂ O, NaCl, NaOH, Cu(CH ₃ COO) ₂ ·H ₂ O, etc.: all commercially available analytical reagents.

2.2 Method:

(1) Serial molecular weight LGC-1 sample preparation: 5 g of GCs derived from plum flower ginseng, each of which was depolymerized by the method described in step (2) of Example 1, but the time points for terminating the reaction were 90, 120, 360, and 420 minutes respectively . The LGC-1 products thus prepared are respectively numbered: LGC-1B, LGC-1C, LGC-1D and LGC-1E. The yields of the depolymerization reactions are all greater than 70%.

(2) Detection of LGC-1 products: HPGPC detection of molecular weight and distribution; conductometric detection -OSO ₃ ^- /-COO ^- molar ratio; optical rotation was determined according to the method of Appendix VI E of Part Two of the Chinese Pharmacopoeia (2005 edition); intrinsic viscosity Determination by Ubbelohde viscometer.

2.3 Results:

2.2 The detection results of the products LGC-1B-E obtained in the step (2) are shown in Table 3 below. The test results show that the yield of the product LGC-1 obtained by depolymerization of GCs derived from plum flower ginseng by hydrogen peroxide depolymerization method is relatively high, and the molecular weight distribution is narrow. Lower and lower.

Table 3. GCs derived from Prunella ginseng and their depolymerization products LGC

Detection of physical and chemical data such as molecular weight and its distribution, optical rotation and intrinsic viscosity

[Example 3] Terminal reductive amination of LGC-1A

3.1 Materials:

LGC-1A: Example 1 to prepare depolymerized GCs products.

Tyramine hydrochloride and sodium cyanoborohydride: both are commercially available reagents of analytical grade.

3.2 Method:

(1) Terminal reductive amination of LGC-1A: 1000 mg of GCs depolymerization product LGC-1A was dissolved in 35 ml of 0.2 mM phosphate buffer (PBS, pH 8.0), and excess 800 mg of tyramine and 300 mg of cyanoboron were added during stirring Sodium hydride, react in a constant temperature water bath at 35°C for about 100 hours. After the reaction, add 105ml of 95% ethanol, centrifuge to obtain a precipitate, wash the obtained precipitate twice with 30ml of 95% ethanol, redissolve the obtained precipitate with 35ml of 0.1% NaCl, centrifuge to remove insoluble matter, and put the supernatant on Sephadex ^™ G- 100 columns, 0.1% NaCl elution, UV detector detection and collection of eluted fractions with λ280nm light absorption, and the obtained fractions were freeze-dried to obtain 652 mg of LGC-2A.

(2) Physicochemical and spectral detection: molecular weight and distribution detected by HPGPC; molar ratio of -OSO ₃ ^- /-COO ^- detected by conductometric method; content of acetylgalactosamine (D-GalNAc) detected by Elson-Morgon method, glucose detected by carbazole method Alkyd acid (D-GlcUA) content, ¹ HNMR methyl peak integral area to calculate D-GalNAc/L-Fuc molar ratio (same as Example 1). AVANCE AV 400 superconducting nuclear magnetic resonance spectrometer detects NMR spectrum (solvent D ₂ O, internal standard TSP-d4, temperature 45° C.).

3.3 Results

Based on the amount of LGC-1A, the yield of LGC-2A is about 65%;

The detection results of product components showed that D-GalNAc:D-GlcUA:L-Fuc:-OSO ₃ ^- was about 1.00:1.13:1.00:3.80, Mw was about 14380, and PDI was about 1.42, which is the same as the degree of polymerization of the LGC-1A structural unit The theoretical calculation results of about 15 are basically consistent;

¹ H MR (D ₂ O, δ[ppm]): 7.25 (2', 6'H); 6.91 (3', 5'H); 5.65, 5.36, 5.28 (L-Fucα1H); 3.38 (8'H ); 2.82 (7'H); 2.02 (D-GalNAc, CH ₃ ); 1.30-1.32 (L-Fuc, CH ₃ ). The integral ratio of L-Fuc methyl hydrogen to benzene ring hydrogen is about 10.5, indicating that the reducing ends of the obtained product are all reductively tyrosinated.

[Example 4] Terminal reductive amination of LGC-1B～1E

4.1 Materials

LGC-1: LGC-1B, LGC-1C, LGC-1D and LGC-1E prepared in Example 2.

Tyramine hydrochloride and sodium cyanoborohydride: both are commercially available reagents of analytical grade.

4.2 Method

Dissolve 500mg each of LGC-1A, 1C, 1D and 1E in 20ml of 0.2mM phosphate buffer (PBS, pH 8.0), add excess 400mg of tyramide and 150mg of sodium cyanoborohydride respectively during stirring, and place in a constant temperature water bath at 35°C React in about 60h. After the reaction is complete, centrifuge to take the supernatant, add 80ml of 95% ethanol, and centrifuge to obtain a precipitate. After washing twice with 15ml of 95% ethanol, the obtained precipitate is redissolved with 20ml of distilled water, centrifuged to remove insoluble matter, and the supernatant is frozen. Dry, 468, 452, 410 and 422 mg each of LGC-2B, 2C, 2D and 2E were obtained, respectively.

4.3 Results

Based on LGC-1B～1E, the yield of LGC-2B～2E products is about 84%～93%;

Calculated based on the number average molecular weight of LGC-1B～1D and the integral ratio of L-Fuc methyl hydrogen and benzene ring hydrogen detected by AVANCE AV 400 superconducting NMR spectrometer in ^{the 1} H NMR spectrum, LGC-2B, 2C, 2D and 2E The proportions of reducing ends reductively tyrosinated were about 52%, 43%, 37% and 32%, respectively.

[Example 5] Anti-HIV-1 activity of low molecular weight glycosylated chondroitin sulfate LGC-1A

5.1 Materials and reagents

(1) Test sample LGC-1A (Mw 13820Da) prepared in Example 1 was dissolved in water for injection, prepared into a storage solution with a concentration of 25 mg/ml, and stored at 4°C.

(2) Reagents MTT (3-(4,5)-dimethylthiahiazo-2-y1)-2,5-diphenyltetrazolium bromide, SDS (Sodium Dodecyl Sulfate), DMSO (Dimethyl sulfoxide) and other reagents are commercially available and analytically pure. Standard rabbit plasma, rabbit platelet-poor plasma, Guangzhou Ruite Biotechnology Co., Ltd.; APTT detection kit (ellagic acid), product of Shanghai Sun Biotechnology Co., Ltd., batch number 090327.

(3) Cells and viruses C8166, HIV-1 _{IIIB/2802/2840/9495} and HIV-1-2 _CBL-20/ROD were donated by the British Medical Research council, AIDS Reagent Project. Prepare HIV-1 _IIIB according to conventional methods, titrate and calculate the TCID ₅₀ of the virus. After aliquoting the virus stock solution, store it at -70°C. Cells and viruses were frozen and recovered according to conventional methods.

5.2 Method

(1) Cytotoxicity detection of LGC-1A on C8166: 100 μl of C8166 cell suspension (4×10 ⁵ /ml) was mixed with 100 μl of serial concentrations of LGC-1A, cultured at 37°C in 5% CO ₂ for three days, and detected by MTT method Cytotoxicity. 570/630nm ELx800ELISA Reader was used to measure the OD value, and the CC ₅₀ (50% Cytotoxic concentration, that is, the drug concentration causing half of the cell death) was calculated.

(2) Detection of the inhibitory activity of LGC-1A on HIV-1-induced C8166 lesions: Mix 50 μl of C8166 cell suspension (8×10 ⁵ /ml) with 100 μl of different concentrations of LGC-1A, add 50 μl of HIV-1 to dilute the supernatant , the MOI was 0.0091, and blank control wells were set at the same time, cultured at 37° C. in 5% CO ₂ for three days, and the syncytia were counted under an inverted microscope at a magnification of 100 and five fields of view. Calculate the EC ₅₀ (50% Effective Concentration, that is, the concentration of LGC-1A that inhibits half of the syncytia formation) of LGC inhibiting HIV-1-induced C8166 lesions. Combined with the CC ₅₀ value obtained in (1) above, the selectivity index (CC ₅₀ /EC ₅₀ value, Therapeutic Index, TI) was calculated.

(3) Detection of the inhibitory activity of LGC-1A on HIV-2-induced C8166 lesions: The inhibitory activity of LGC-1A on HIV-2-induced C8166 lesions was detected by the same method as described in (2) above.

(4) Effect of LGC-1A on blood coagulation function: Activated partial thromboplastin time (APTT) method was used to detect the effect of LGC-1A on blood coagulation function. Take an appropriate amount of LGC-1A and prepare a sample solution with a concentration of 25 μg/ml in physiological saline. Take 20 μl of the prepared sample solution to be tested, add 180 μl of standard human plasma, mix immediately, and follow the instructions attached to the APTT detection kit (ellagic acid), in a BICO-dual-channel hemagglutination analyzer (Minivolt Company, Italy) The effects of LGC on blood coagulation function under effective anti-HIV-1 drug concentration were detected above.

(5) SPR method to detect the interaction between LGC-1A and gp120: The interaction between LGC and gp120 is detected by the surface plasmon resonance technology SPR (BIAcore 3000 biosensor system), and the CM-5 chip is activated to fix gp120 on the CM-5 On the chip surface, samples with different concentrations were diluted with buffer HBS-EP. Put the diluted sample on the sample rack, set the flow rate of the sample to 10 μl/min, and select the inject method to inject the sample. After the end, the kinetic constants were analyzed by the BIAcore 3000 software analysis system.

5.3 Results

(1) Antiviral activity of LGC-1A: The results in accompanying drawings 7 and 8 and Table 4 show that LGC-1A is a new structurally active ingredient that potently inhibits HIV-1, and its anti-HIV-1 _IC50 is as low as about 9.4 nM (0.13 μ g/ml); No significant cytotoxicity (referring to accompanying drawing 8 of the present invention) according to preparation highest concentration medicine (25mg/ml), its therapeutic index can reach more than 150,000; To HIV-1-2 Inhibitory activity is weak.

Table 4 LFC-1A anti-HIV activity detection results

(2) Effect of LGC-1A on blood coagulation function: the results in Table 5 show that no significant APTT prolonging activity was found for LGC-1A under the effective anti-HIV-1 virus dose (0.1-8 μg/ml).

Table 5 LFC-1A anticoagulant activity detection results

LFC-1A (μg/ml) 0 0.1 1 4 8 APTT(s) 23.2 23.2 23.6 24.9 25.1

(3) The target of LGC-1A anti-HIV-1 activity: SPR test results show that LGC-1A can bind gp120 with high affinity, but has no significant binding to CD4; after soluble CD4 (sCD4) binds to gp120, LGC-1A The binding affinity to CD4i was further enhanced, indicating that LGC-1A could bind to CD4i, a conserved region of pg120 exposed by CD4 induction. Since CD4i is highly conserved, gp120 inhibitors that bind to this region can effectively avoid drug resistance caused by viral DNA mutations (see Figure 9).

[Example 6] Detection of Anti-HIV-1 Activity of Series Molecular Weight LGC-1 and LGC-2

6.1 Materials:

LGC-1A, 1B, 1C, 1D, 1E and LGC-2A, 2B, 2C, 2D, 2E series molecular weight LGC samples, products obtained in Examples 1-4; other reagents are the same as 5.1 (3) of Example 5.

6.2 Method:

Detect the inhibitory activity and cytotoxicity of the LGC series samples on HIV-1IIIB experimental strain-induced C8166 cell pathology, and the detection method is the same as 5.2 of Example 5.

6.3 Results:

The experimental results are shown in Table 6. The experimental results show that under the experimental conditions, LGC-1 and LGC-2 series samples have less toxicity to C8166 cells, and their CC ₅₀ are all greater than about 25 mg/ml; in vitro anti-HIV-1 activity is strong, and their IC ₅₀ The values are between about 8.3nM (LGC-2A) and 30.8nM (LGC-1E); the therapeutic index is higher, all greater than 15000.

In addition, the experimental results also show that (1) comparing the anti-HIV-1 activity of LGC-1 series and LGC-2 series samples, it can be seen that the anti-HIV-1 activity increases with the average molecular weight; (2) LGC-1 series Compared with the anti-HIV-1 activity between LGC-2 series samples, it can be seen that the activity of LGC-2 samples is slightly stronger than that of the corresponding LGC-1 samples.

Since the effect of LGC samples on the blood coagulation system is weakened with the decrease of molecular weight, the description of the present invention indicates that the preferred LGC molecular weight (Mw) is about 5000-8000.

Table 6. In vitro anti-HIV-1 activity of a series of molecular weight LGCs

[Example 7] Preparation of LGC from different species and detection of its anti-HIV-1 activity

7.1 Materials

Dry body wall of sea cucumber (Stichopus japonicus) and jade foot sea cucumber (Holothuria leucospilota), commercially available.

7.2 Preparation of LGC from sea cucumber and yuzu sea cucumber

LGC derived from Apostichopus japonicus and sea cucumber yuzu were prepared in the same way as in Example 1 (1) and (2), and were referred to as LGC- _st and LGC- _hl for short, respectively.

7.3 Physicochemical and anti-HIV-1 activity detection

The results of physical and chemical testing show that the molecular weights of LGC- _st and LGC- _hl are 8650Da and 8595Da respectively; ¹ H, ¹³ C NMR and their correlation spectrum analysis show that LGC- _st and LGC- _hl have similar properties to LGC-1A described in Example 1. The basic structural characteristics of the glycosylated chondroitin sulfate compounds, but the position and number of sulfate groups on the monosaccharide residues are different. The D-GalNAc in the main chain of LGC- _st and LGC-1A is mainly D -GalNAc4S6S, and there is a certain amount of D-GalNAc4S in the structure of LGC- _hl ; different from LGC- _st and LGC- _hl , the side chain fucose substituent types besides L-Fuc2S4S and L-Fuc4S, LGC-1A also contains L-Fuc3S side chain, while lacking L-Fuc3S4S side chain.

The results of anti-HIV-1 activity detection showed that the activity intensity of LGC- _st was similar to that of LGC-1C with similar molecular weight, and slightly stronger than that of LGC- _hl with similar molecular weight.

Table 7 Effect of different chemical structural features of LGC on anti-HIV-1 activity

+: there is a side chain group of the type; -: there is no or less side chain group of the type

[Example 8] Freeze-dried product of low molecular weight glycosylated chondroitin sulfate

8.1 Materials

The low molecular weight glycosylated chondroitin sulfate (LGC-hl) obtained from Yuzu sea cucumber obtained in Example 7 has a weight average molecular weight of 9500 Da.

8.2 Prescription

The name of the raw material Dosage LGC- _hl 50g Water for Injection 500ml Co-made 1000 pieces

8.3 Preparation process

Weigh the prescribed amount of yuzu sea cucumber low molecular weight glycosylated chondroitin sulfate and add water for injection to the full amount, stir to dissolve completely. Add 0.6% medicinal activated carbon, stir for 20 minutes; use a Buchner funnel and a 3.0 μm microporous filter membrane to decarbonize and filter. Measure the content of intermediates. Filter with a 0.22μm microporous membrane after passing the test; fill in controlled vials, 0.5ml per bottle, monitor the filling volume during the filling process, half-tighten the stopper, put it in a freeze-drying box, and proceed according to the set freeze-drying curve Freeze-drying, corking, out of the box, capping, and visual inspection to obtain the finished product.

Freeze-drying process: Put the sample into the box, lower the temperature of the partition to -40°C, and keep it for 3 hours; drop the cold trap to -50°C, and start vacuuming to 300μbar. Start sublimation: heat up to -30°C at a constant rate for 1h, keep for 2h; heat up to -20°C for 2h, keep for 8h, and keep a vacuum at 200-300μbar; then dry: heat up to -5°C for 2h, keep for 2h, and keep a vacuum at 150-200μbar ; Raise the temperature to 10°C for 0.5h, keep it for 2h, and keep the vacuum at 80-100μbar; raise the temperature to 40°C for 0.5h, keep it for 4h, and pump it to the minimum.

[Example 9] Vaginal suppository of low molecular weight glycosylated chondroitin sulfate

9.1 Materials

The low molecular weight glycosylated chondroitin sulfate (LGC-2A obtained in Example 3) derived from plum blossom ginseng has a weight average molecular weight of 14380 Da. Reagents such as ethylparaben and glycerinated gelatin are commercially available and of pharmaceutical grade.

9.2 Prescription

The name of the raw material Dosage LGC-2A 100g Water for Injection 400ml Ethylparaben 2.0g Glycerin gelatin added to 4000g Co-made 1000 pieces

9.3 Preparation method

Take low molecular weight glycosylated chondroitin sulfate and add water for injection to dissolve, add ethylparaben and stir to dissolve, then add an appropriate amount of glycerin and stir well, slowly add to the gelatin glycerin matrix, stir well, keep warm at 55°C, fill the mold, each piece weighs 4g.

[Example 10] Gel of low molecular weight glycosylated chondroitin sulfate

10.1 Materials

The low molecular weight glycosylated chondroitin sulfate derived from sea cucumber (LGC-2C obtained in Example 4) has a weight average molecular weight of 9250Da. Reagents such as cross-linked sodium polyacrylate, polyethylene glycol 400, benzalkonium bromide and glycerin are commercially available and of pharmaceutical grade.

10.2 Prescription

Raw material name Dosage LGC-2C 10.0g Cross-linked sodium polyacrylate (SDB-L-400) 10.0g Polyethylene glycol 400 (PEG-400) 80.0g Benzalkonium Bromide 10.0ml Glycerin 100.0g Add distilled water to 1000g

10.3 Preparation method

Take low molecular weight glycosylated chondroitin sulfate and add distilled water to dissolve, weigh PEG-400 and glycerin in a beaker and heat until completely dissolved, add low molecular weight glycosylated chondroitin sulfate solution and mix well, add 700ml water to SDB-L-400 After grinding in a mortar, mix the matrix with PEG-400, glycerin, and low molecular weight glycosylated chondroitin sulfate, add water to 1000g, and fill it.

Claims (15)

1. low-molecular-weight glycosyl chondroitin sulfate and pharmaceutically acceptable salt thereof the purposes in anti-1 type HIV (human immunodeficiency virus) medication preparation, it is characterized in that: described LGC is the mixture with homology glycosaminoglycans derivant of formula (I) structure,

In the formula (I):

D-GlcUA-β 1-is β-D-glucuronic acid-1-base;

D-GalNAc-β 1-is β-D-N-acetylamino galactosamine-1-base;

L-Fuc-α 1-is α-L-fucose-1-base;

R ₁For-H or D-GalNAc-β 1-;

R ₃, R ₄, R ₅, R ₆, R ₇Be independently of each other-H or-SO ₃ ^-

R ₂For-OH ,-4-O-D-GlcUA or formula (II) or (III) shown in group:

Wherein, R ' is a L-Fuc-α 1-substituted radical, and has the sulfate group of cotype (1) structure on the L-Fuc-α 1-; R " be-4-[L-Fuc (α 1-3)] D-GlcUA-β 1-group, there are the sulfate group of cotype (1) structure, R on its L-Fuc-α 1- ₃, R ₄With above-mentioned definition;

With molar ratio computing, the contained D-GlcUA of described LGC, D-GalNAc, three kinds of monosaccharide residues of L-Fuc and contained-OSO ₃ ^-The proportion of base is 1: (1 ± 0.3): (1 ± 0.3): (3.5 ± 0.5);

N is that average is about 3～17 integer;

The weight average molecular weight range of described LGC is 3000～15000Da.

2. LGC and pharmaceutically acceptable salt thereof the purposes in the anti-HIV-1 medication preparation according to claim 1 is characterized in that described LGC is the mixture with homology glycosaminoglycans derivant of formula (I) structure, wherein contains the R of qualification ratio ₂Be formula (II) or (III) formula of group (I) chemical compound.

3. as LGC and the purposes of pharmaceutically acceptable salt in the anti-HIV-1 medication preparation thereof as described in the claim 2, it is characterized in that described qualification ratio is meant, in molar percentage, R ₂For formula (II) or (III) formula of group (I) chemical compound account for 30%～100% of whole formulas (I) chemical compound.

4. as LGC and the purposes of pharmaceutically acceptable salt in the anti-HIV-1 medication preparation thereof as described in the claim 1 to 3, it is characterized in that described LGC is that the depolymerization product of the glycosylation chondroitin sulfate that extracts of Echinodermata Holothuroidea animal body wall and/or described depolymerization product reducing end are by the amidized product of all or part of reduction cheese.

5. low-molecular-weight glycosyl chondroitin sulfate and pharmaceutically acceptable salt thereof, described LGC are the mixture of homology glycosaminoglycans derivant of formula (I) structure of claim 1 definition, and, in molar percentage, R ₂For formula (II) or (III) formula of group (I) chemical compound account for 30%～100% of whole formulas (I) chemical compound.

6. LGC as claimed in claim 5 and pharmaceutically acceptable salt thereof is characterized in that, in the mixture of the homology glycosaminoglycans derivant of described formula (I) structure, and R ₂Be formula (II) or (III) group.

7. as described LGC of claim 5 to 6 and pharmaceutically acceptable salt thereof, it is characterized in that described LGC weight average molecular weight (Mw) scope is 5000～8000Da.

8. as claim 5 to 7 described low-molecular-weight glycosyl chondroitin sulfate and pharmaceutically acceptable salt thereof, it is characterized in that described LGC is that the depolymerization product of the glycosylation chondroitin sulfate that extracts of Echinodermata Holothuroidea animal body wall and/or described depolymerization product reducing end are by the amidized product of all or part of reduction cheese.

9. as the preparation method of described LGC of claim 1 to 8 and pharmaceutically acceptable salt thereof, comprise the steps:

(1) from Echinodermata Holothuroidea animal body wall, extracts acquisition acid mucopolysaccharide, i.e. prototype glycosylation chondroitin sulfate GCs;

(2) peroxidating depolymerization step (1) gained GCs, the GCs depolymerization product of collection and purification desired molecule weight range further is converted into required alkali metal ion salt with the gained depolymerization product, obtains the described LGC of claim 1 thus;

(3) the optional reductive amination that carries out terminal saccharide of the described GCs depolymerization product of step (2), its method is, described GCs depolymerization product is reacted with stoichiometric tyramine in the presence of sodium cyanoborohydride, obtains the described reducing end of claim 4～5 by the amidized product of all or part of reduction cheese.

10. as LGC preparation method as described in the claim 9, it is characterized in that: the described peroxidating depolymerization method of described step (2) is: it is 0.05%～10% aqueous solution that the GCs in Holothuroidea animal body wall source is made into mass fraction, in obtained aqueous solution, add peroxide GCs to mass fraction 0.05%～5%, add transition metal ions salt GCs as catalyst to mass fraction 0.01%～1%, under 20～60 ℃, react to GCs by depolymerization to the desired molecule weight range; Add ethanol or acetone and make the depolymerization product precipitation in reactant liquor, after precipitation was dissolved again, ultrafilter membrane was dialysed and/or is crossed the gel filtration chromatography method and remove little minute impurity, and ion exchange further is converted into required alkali metal ion salt with products therefrom.

11. as LGC preparation method as described in the claim 9, it is characterized in that: the method for the terminal reductive amination of the described GCs depolymerization product of described step (3) is: step (2) gained GCs depolymerization product is dissolved in the buffer of pH 7～9, add stoichiometric tyramine and sodium cyanoborohydride successively, room temperature or reacting by heating.After reaction finishes, the separation and purification product, and be translated into required alkali metal ion salt.

12. as LGC preparation method as described in the claim 11, it is characterized in that: the separation and purification of the product of described step (3) is meant in the purified product, with molar ratio computing, and R ₂For formula (II) or (III) formula of group (I) chemical compound account for 30%～100% of whole formulas (I) chemical compound.

13. the pharmaceutical composition of an anti-HIV-1, described pharmaceutical composition contain the described LGC of claim 1～7 or its pharmaceutically acceptable salt of effective anti-HIV-1 dosage, and pharmaceutical excipient.

14., it is characterized in that the dosage form of described pharmaceutical composition is aqueous solution for injection, freeze-dried powder injection or topical with suppository, ointment or gel as pharmaceutical composition as described in the claim 13.

15. as pharmaceutical composition as described in the claim 10 to 11 as aids prevention and/or medicine.

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