AU2021100407A4 - Mollusc heparin with a mild anticoagulant effect and preparation method and use thereof - Google Patents

Abstract

The present disclosure discloses a mollusc heparin with a mild anticoagulant effect and a preparation method and use thereof. The preparation method includes: raw materials treatment; autolysis; enzymolysis: adding NaOH to adjust a pH value to 7.8-8.2, adding trypsin, conducting enzymolysis for 3.5-6.5 h at 32-42°C, adding HCl to adjust the pH value to 6.8-7.2, adding papain, conducting enzymolysis for 4.5-5.5 h at 62-68°C to obtain an enzymatic hydrolysate; enzyme deactivation and centrifugation; concentration and ethanol precipitation; redissolving, deproteinization; desalination; and drying. The present disclosure takes molluscs such as Mactra antiquata, Panopea abrupta, Tapes dorsatus, Tegillarca granosa and Meretrix lusoria as sources for the first time to prepare the mollusc heparin with a mild anticoagulant effect; The heparin from marine molluscs has high safety and mild effects. The present disclosure not only broadens the source of heparin, but also provides a simple preparation method and stable product quality, and has potential use value in the preparation of heparin with low side effects or other medical products and foods with anticoagulant effects. 1/3 4.0 198nm 3. 5 3.0 Tapes 2.5 2 orsatus 2.0 Tegil rca rano a 1.5 niu ti er 1.0 nt ru 0.5Hepari lam 0.0 H 200 230 260 290 320 350 380 nm FIG.1I

Description

1/3

4.0 198nm

3. 5

3.0

Tapes 2.5 2orsatus

2.0 Tegil rca rano a 1.5 niu ti er 1.0 nt ru

0.5Hepari lam

0.0 H 200 230 260 290 320 350 380 nm

FIG.1I

I MOLLUSC HEPARIN WITH A MILD ANTICOAGULANT EFFECT AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

The present disclosure belongs to the technical field of biological medicine, more

specifically, relates to a mollusc heparin with a mild anticoagulant effect and a preparation

method and use thereof.

BACKGROUND

As a glycosaminoglycan that exists in animals, heparins have diverse biological activities

and are widely used in clinical practice, especially as an anticoagulant. Currently, there is no

product that can completely replace heparin. The heparin used in clinic is mostly derived from

porcine small intestine and bovine lung, which has so strong anticoagulant effect that is easy to

cause side effects such as hemorrhage effect and platelet reduction. In view of the problems of

strong anticoagulant activity and excessive side effects of porcine-derived and bovine-derived

heparins, it is necessary to develop a heparin with a mild anticoagulant effect. Marine-derived

heparin has the characteristics of good compatibility with human cells, small side effects, good

tissue structure and mild anticoagulant activity. Therefore, it is becoming increasingly important

to develop a marine-derived heparin with mild anticoagulant action that is not derived from

porcine small intestine and bovine lung.

Molluscs, belonging to the phylum Mollusca, are animals with trilaminar germs,

symmetrical sides and deuterocoel. As a large molluscs farming country in the world, the

production of marine molluscs aquaculture in China accounts for 80% of the total production of

marine aquaculture, which is an important part of the marine aquaculture industry. These marine

molluscs that are rich in active substances have biological activities of resisting aging, resisting

tumor, resisting virus, improving immunity, and reducing blood sugar, blood lipids, and blood

pressure. Due to high nutritional value and health benefits, marine molluscs have become a hot

spot in global research and development. Mactra antiquata, Tapes dorsatus, Panopea abrupta,

Tegillarca granosa and Meretrix lusoria are all molluscs. However, there has been no report on the extraction of heparin from Mactra antiquata, Tapes dorsatus, Panopea abrupta, Tegillarca granosa and Meretrix lusoria, and there are no reports on the anticoagulant activity of heparins in these five molluscs. Moreover, the existing heparin has the disadvantages of a long extraction production cycle, high energy consumption, and large loss of product activity.

Mactra antiquata, marine bivalves, commonly known as sea mussels in Fujian, Jiangsu and

Zhejiang, and Royal Mussel in Guangdong and Hong Kong, belong to the phylum Mollusca,

class Lamellibranchia, order Veneroida, and family Mactridae; it is crispy and tender with rich

nutrition, high-quality protein and amino acids, and has the effects of nourishing YIN and

tonifying deficiency, and clearing heat and cooling liver.

Tapes dorsatus is a marine molluscs belonging to the phylum Mollusca, class

Lamellibranchia, order Veneroida, and family Veneridae. The species occurs throughout the

Southwest Pacific, and generally inhabits sand to sand/silt or mud sediments in the intertidal zone,

the low-tide zone, the subtidal zone and the shallow sea (3-15 cm); it is common in the East

China Sea, the South China Sea and the coast of Taiwan Island in China, and is a common edible

coastal molluscs with delicious meat.

Panopea abrupta (biological name of Panopea abrupta), also known as king clam and

goddess clam, has two thin and brittle shells of the same size, with serrations, auxiliary shells,

and water pipes (also called tentacles) at the anterior end. It is rich in protein, and has the effects

of maintaining balance of potassium and sodium, eliminating edema, improving immunity,

lowering blood pressure, and buffering anemia.

Tegillarca granosa belongs to the phylum Mollusca, class Bivalvia, orde Taxodonta, family

Arcidae, and genus Anadara. Tegillarca granosa is red because of containing heme in blood, so it

is also called blood clams. Tegillarca granosa is rich in unique hemoglobin and vitamin B12,

which has the effects of nourishing blood, warming the middle and strengthening the stomach.

Meretrix lusoria is one of Chinese clam belonging to the class Bivalvia, order Veneroida,

and family Veneridae. The body is oblate, generally white, and the shell is bright. It is widely distributed in the coastal waters of Zhanjiang and Hainan, China. The meat is plump, fresh and sweet with high nutritional value. It has the effects of clearing heat and dampness, resolving phlegm, and dispelling nodules. It has a significant inhibitory effect on liver cancer, and obvious curative effects on asthma, chronic bronchitis, goiter, and lymphatic tuberculosis.

SUMMARY

The technical problem to be solved by the present disclosure is to overcome the

shortcomings and deficiencies of the prior art, and provide a simple and effective method for

preparing a mollusc heparin with a mild anticoagulant effect.

Another objective of the present disclosure is to provide a mollusc heparin obtained by the

aforementioned preparation method.

Another objective of the present disclosure is to provide use of the aforementioned mollusc

heparin in the preparation of products with a mild anticoagulant effect.

The objectives of the present disclosure are achieved by the following technical solutions.

A method for preparing a mollusc heparin includes the following steps:

Si. raw materials treatment: after rinsing molluscs and removing shells, conducting

homogenization by adding water to obtain a homogenate;

S2. autolysis: conducting autolysis on the homogenate for 3-7 h under a condition of

38-62°C water bath;

S3. enzymolysis: adding NaOH to adjust a pH value to 7.8-8.2, adding trypsin, conducting

enzymolysis for 3.5-6.5 h at 32-42°C, adding HCl to adjust the pH value to 6.8-7.2, adding

papain, and conducting enzymolysis for 4.5-5.5 h at 62-68°C to obtain an enzymatic hydrolysate;

S4. enzyme deactivation and centrifugation: putting the enzymatic hydrolysate in boiling

water for enzyme deactivation for 5-15 min, conducting centrifugation, and taking a supernatant;

S5. concentration and ethanol precipitation: concentrating the supernatant, adding ethanol,

standing for 20-28 h at 0.5-7.5°C, and conducting centrifugation to obtain a precipitate;

S6. redissolving and deproteinization: rinsing the precipitate with acetone and ethanol alternately for 2-3 times, dissolving the precipitate with water, and conducting centrifugation to remove insoluble substances; adding a n-butanol alcohol-chloroform mixed solution in the supernatant, fully shaking for 10-30 min, standing for 5-15 min, taking a supernatant, and repeating the operation for 3-4 times to obtain an extracted solution;

S7. desalination: putting the extracted solution into a dialysis bag for dialysis for 48-96 h to

obtain a dialyzate; and

S8. drying: freeze-drying the dialyzate to obtain the mollusc heparin.

The present disclosure takes molluscs such as Mactra antiquata, Tapes dorsatus, Panopea abrupta, Tegillarca granosa and Meretrix lusoria as sources for the first time to prepare the mollusc heparin with a mild anticoagulant effect; the heparins from marine molluscs have high safety and mild effects, and show a characteristic absorption peak at 185-220 nm wavelength in ultraviolet scanning; a strong absorption peak of O-S bond stretching vibration at 1240 cm-1

, characteristic absorption of heparin at 890 cm- and 940 cm-1 , and stretching vibration absorption bands of C-O-S system of sulfuric acid groups on hexosamine at 800-850 cm-1 in infrared spectrum scanning; and contain glucosamine, glucuronic acid and iduronic acid in the monosaccharide composition. Preferably, the molluscs are selected from the group consisting of Mactra antiquata, Tapes

dorsatus, Panopea abrupta, Tegillarca granosa and Meretrix lusoria.

Preferably, a material-to-liquid ratio in the homogenate in step S Iis 1:(1.5-4.5).

More preferably, a material-to-liquid ratio in the homogenate in step S Iis 1:3.

As a preferable scheme, the autolysis in step S2 includes the following steps: conducting

autolysis on the homogenate for 5 h in a water bath at 50°C.

As a preferable scheme, the enzymolysis in step S3 includes the following steps: adding

NaOH to adjust a pH value to 8, adding trypsin, performing enzymolysis for 5 h at 37°C, adding

HCl to adjust the pH value to 7.0, adding papain, and performing enzymolysis for 5 h at 65°C to

obtain an enzymatic hydrolysate.

Preferably, an amount of the trypsin added in step S3 is 0.3%-0.7%; and an amount of the

papain added in step S3 is 0.3%-0.7%.

Preferably, an amount of the trypsin added in step S3 is 0.5%; and an amount of the papain

added in step S3 is 0.5%.

Preferably, the centrifugation in step S4 is conducted at 6000-8000 rpm for 10-20 min.

More preferably, the centrifugation in step S4 is conducted at 8000 rpm for 20 min.

Preferably, the enzyme deactivation in step S4 lasts forlO min.

Preferably, an amount of the ethanol added in step S5 is 0.3-0.6 times the volume of the

concentrated solution.

Preferably, an amount of the ethanol added in step S5 is 0.4 times the volume of the

concentrated solution.

Preferably, after ethanol is added in step S5, the mixture is left standing for 24 h at 4°C.

Preferably, a volume ratio of n-butanol to chloroform in the n-butanol-chloroform mixed

solution in step S6 is 1:(4-5); and a volume ratio of the n-butanol-chloroform mixed solution to

the supernatant is 1:(4-5).

Preferably, a volume ratio of n-butanol to chloroform in the n-butanol-chloroform mixed

solution in step S6 is 1:4; and a volume ratio of the n-butanol-chloroform mixed solution to the

supernatant is 1:5.

Preferably, the dialysis is conducted for 72 h in step S7.

Preferably, in step Si, a degreasing treatment is further conducted; the degreasing treatment

includes: soaking the molluscs in acetone for 20-28 h at 2-6°C, conducting suction filtration at

room temperature to remove the acetone, and rinsing with water 3-5 times.

The present disclosure also provides a mollusc heparin prepared by the aforementioned

method; the heparin shows a characteristic absorption peak at 185-220 nm wavelength in

ultraviolet spectrum scanning; a strong absorption peak of O-S bond stretching vibration at 1240

cm 1, characteristic absorption of heparin at 890 cm and 940 cm1 , and stretching vibration

absorption bands of C--S system of sulfuric acid groups on hexosamine at 800-850 cm-' in infrared spectrum scanning; and contain glucosamine, glucuronic acid and iduronic acid in the monosaccharide composition.

Use of the mollusc heparin in the preparation of products with a mild anticoagulant effect is

also within the protection scope of the present disclosure.

The present disclosure has the following beneficial effects as compared with the prior art:

The present disclosure takes molluscs such as Mactra antiquata, Tapes dorsatus, Panopea

abrupta, Tegillarca granosa and Meretrix lusoria as sources for the first time to prepare the

mollusc heparin with a mild anticoagulant effect; the heparins from marine molluscs have high

safety and mild effects, and show a characteristic absorption peak at 185-220 nm wavelength in

ultraviolet scanning; a strong absorption peak of O-S bond stretching vibration at 1240 cm-1

, characteristic absorption of heparin at 890 cm- and 940 cm-1 , and stretching vibration absorption

bands of C-O-S system of sulfuric acid groups on hexosamine at 800-850 cm-1 in infrared

spectrum scanning; and contain glucosamine, glucuronic acid and iduronic acid in the

monosaccharide composition.

The present disclosure not only broadens the source of heparin, but also has a simple

preparation method and stable product quality. It has potential use value in the preparation of

heparin with low side effects or other medical products and foods with anticoagulant effects, and

has great significance for the prevention and treatment of thrombotic diseases.

General

Those skilled in the art will appreciate that the invention described herein is susceptible to

variations and modifications other than those specifically described. The invention includes all

such variation and modifications. The invention also includes all of the steps and features

referred to or indicated in the specification, individually or collectively and any and all

combinations or any two or more of the steps or features.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however be understood to be common general knowledge.

The present invention is not to be limited in scope by any of the specific embodiments

described herein. These embodiments are intended for the purpose of exemplification only.

Functionally equivalent products and methods are clearly within the scope of the invention as

described herein.

Throughout this specification, unless the context requires otherwise, the word "comprise" or

variations such as "comprises" or "comprising", will be understood to imply the inclusion of a

stated integer or group of integers but not the exclusion of any other integer or group of integers.

Other definitions for selected terms used herein may be found within the detailed description

of the invention and apply throughout. Unless otherwise defined, all technical terms used herein

have the same meaning as commonly understood to one of ordinary skill in the art to which the

invention belongs.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an ultraviolet spectrum of the mollusc heparin in the present disclosure.

FIG. 2 is an infrared spectrum of the mollusc heparin in the present disclosure.

FIG. 3 is a diagram of the monosaccharide composition of the mollusc heparin in the present

disclosure.

FIG. 4 is an analysis of the anticoagulant activity of the mollusc heparin in the present

disclosure.

DETAILED DESCRIPTION

The following clearly and completely describes the technical solutions in the examples of the present disclosure with reference to the examples of the present disclosure. Apparently, the described examples are merely a part rather than all of the examples of the present disclosure. All other examples obtained by a person of ordinary skill in the art based on the examples of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure. All other examples obtained by a person of ordinary skill in the art based on the examples of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.

Unless otherwise specified, the reagents, methods and apparatus employed in the present

disclosure are conventional in the art.

Example 1 Preparation of mollusc heparins

1. A method for preparing a mollusc heparin included the following steps:

S1. raw materials treatment: after rinsing the molluscs raw materials and removing shells,

the molluscs were soaked in acetone for 20 h at 2°C, suction filtration was conducted at room

temperature to remove the acetone, followed by rinsing with distilled water for 3-5 times,

homogenization was conducted by adding distilled water (a material-to-liquid ratio is 1:3) to

obtain a homogenate; where the molluscs were selected from any one of the group consisting of

Mactra antiquata, Tapes dorsatus, Panopea abrupta, Tegillarca granosa and Meretrix lusoria.

S2. autolysis: autolysis was conducted on the homogenate for 5 h under a condition of 50°C

water bath;

S3. enzymolysis: NaOH was added to adjust a pH value to 8.0, 0.5% trypsin was added,

enzymolysis was conducted for 5 h at 37°C, HCl was added to adjust the pH value to 7.0, 0.5%

papain was added, and enzymolysis was conducted for 5 h at 65°C to obtain an enzymatic

hydrolysate;

S4. enzyme deactivation and centrifugation: the enzymatic hydrolysate was put in boiling

water for enzyme deactivation for 10 min, and centrifugation was conducted for 20 min at 8000 rpm;

S5. concentration and ethanol precipitation: the supernatant was concentrated, ethanol with a

volume of 0.4 times of the supernatant was added, the mixture was left standing for 24 h at 4°C,

and centrifugation was conducted to obtain a precipitate;

S6. redissolving and deproteinization: the precipitate were rinsed with acetone and ethanol

alternately for 2-3 times and dissolved with water, and centrifugation was conducted to remove

insoluble substances; a n-butanol alcohol-chloroform mixed solution with a volume of 1/5 of the

supernatant was added (a volume ratio of n-butanol to chloroform of 1:4), followed by fully

shaking for 30 min, standing for 10 min, taking a supernatant, and repeating the operation for 4

times to obtain an extracted solution;

S7. desalination: the extracted solution was put into a dialysis bag for dialysis 72 h; and

S8. drying: the dialyzate was freeze-dried to obtain the mollusc heparin.

Example 2 Preparation of mollusc heparins

1. A method for preparing a mollusc heparin included the following steps:

Si. raw materials treatment: after rinsing the molluscs raw materials and removing shells,

homogenization was conducted by adding distilled water (a material-to-liquid ratio is 1:1.5) to

obtain a homogenate;

S2. autolysis: autolysis was conducted on the homogenate for 7 h under a condition of 38°C

water bath;

S3. enzymolysis: NaOH was added to adjust a pH value to 7.8, 0.3% trypsin was added,

enzymolysis was conducted for 6.5 h at 32°C, HCl was added to adjust the pH value to 6.8, 0.3%

papain was added, and enzymolysis was conducted for 5.5 h at 62°C to obtain an enzymatic

hydrolysate;

S4. enzyme deactivation and centrifugation: the enzymatic hydrolysate was put in boiling

water for enzyme deactivation for 5min, and centrifugation was conducted for 10 min at 8000 rpm;

S5. concentration and ethanol precipitation: the supernatant was concentrated, ethanol with a

volume of 0.3 times of the supernatant was added, the mixture was left standing for 20 h at 0.5°C,

and centrifugation was conducted to obtain a precipitate;

S6. redissolving and deproteinization: the precipitate were rinsed with acetone and ethanol

alternately for 3 times and dissolved with water, and centrifugation was conducted to remove

insoluble substances; a n-butanol alcohol-chloroform mixed solution with a volume of 1/4.5 of

the supernatant was added (a volume ratio of n-butanol to chloroform of 1:4), followed by fully

shaking for 20 min, standing for 10 min, taking a supernatant, and repeating the operation for 4

times to obtain an extracted solution;

S7. desalination: the extracted solution was put into a dialysis bag for dialysis 48 h; and

S8. drying: the dialyzate was freeze-dried to obtain the mollusc heparin.

Example 3 Preparation of mollusc heparins

1. A method for preparing a mollusc heparin included the following steps:

S1. raw materials treatment: after rinsing the molluscs raw materials and removing shells,

the molluscs were soaked in acetone for 28 h at 6°C, suction filtration was conducted at room

temperature to remove the acetone, followed by rinsing with distilled water for 3-5 times,

homogenization was conducted by adding distilled water (a material-to-liquid ratio is 1:4.5) to

obtain a homogenate;

S2. autolysis: autolysis was conducted on the homogenate for 3 h under a condition of 62°C

water bath;

S3. enzymolysis: NaOH was added to adjust a pH value to 8.2, 0.7% trypsin was added,

enzymolysis was conducted for 3.5 h at 42°C, HCl was added to adjust the pH value to 7.2, 0.7%

papain was added, and enzymolysis was conducted for 4.5 h at 68°C to obtain an enzymatic

hydrolysate;

S4. enzyme deactivation and centrifugation: the enzymatic hydrolysate was put in boiling water for enzyme deactivation for 15 min, and centrifugation was conducted for 20 min at 6000 rpm;

S5. concentration and ethanol precipitation: the supernatant was concentrated, ethanol with a

volume of 0.5 times of the supernatant was added, the mixture was left standing for 28 h at 7.5°C,

and centrifugation was conducted to obtain a precipitate;

S6. redissolving and deproteinization: the precipitate were rinsed with acetone and ethanol

alternately for 3 times and dissolved with water, and centrifugation was conducted to remove

insoluble substances; a n-butanol alcohol-chloroform mixed solution with a volume of 1/5 of the

supernatant was added (a volume ratio of n-butanol to chloroform of 1:4.5), followed by fully

shaking for 10 min, standing for 5 min, taking a supernatant, and repeating the operation for 4

times to obtain an extracted solution;

S7. desalination: the extracted solution was put into a dialysis bag for dialysis 96 h; and

S8. drying: the dialyzate was freeze-dried to obtain the mollusc heparin.

Table 1 Properties of mollusc heparins prepared in Examples 1-3 Yield (mg/kg) Heparin content (pg/mg) Example 1 Mactra antiquata 143.42 239.1 Tapes dorsatus 244.31 212.2 Panopea abrupta 206.21 163.4 Tegillarca granosa 288.24 234.8 Meretrix lusoria 373.39 214.8 Example 2 Mactra antiquata 112.53 217.2 Tapes dorsatus 186.42 200.9 Panopea abrupta 172.47 156.3 Tegillarca granosa 213.62 228.1 Meretrix lusoria 343.21 207.5 Example 3 Mactra antiquata 142.46 201.4 Tapes dorsatus 239.13 196.2 Panopea abrupta 199.81 143.1 Tegillarca granosa 276.33 216.3 Meretrix lusoria 351.31 197.2

Example 4 Identification of mollusc heparin structures

1. The mollusc heparin prepared in Examples 1-3 was mixed with the standard heparin to

form a 1 mg/L solution, and an ultraviolet scanning was conducted in a range of 190-400 nm.

In the ultraviolet spectrum (FIG. 1), the heparin standard product had a characteristic

absorption peak at 198 nm, crude heparins in Mactra antiquata, Panopea abrupta, Tegillarca

granosa, and Tapes dorsatus had an absorption peak at 198 nm, and heparin in Meretrix lusoria

had an absorption peak at 194 nm and 198 nm, according with the ultraviolet characteristic

absorption of heparin.

2. The mollusc heparin prepared in Examples 1-3 and heparin standards was mixed with

potassium bromide at a ratio of 1:100, and potassium bromide was used as a blank group, and an

infrared scanning was conducted in a range of 4000-400 cm-1 .

In the infrared spectrum (FIG. 2), mollusc heparins were mainly composed of hydroxyl,

amino, amide, carboxyl and sulfuric acid groups, showing a strong absorption peak of O-S bond

stretching vibration at 1240 cm- 1, showing the characteristic absorption of heparin at 890 cm-1

and 940 cm- 1, and stretching vibration absorption bands of C-0-S system of sulfuric acid groups

on hexosamine at 800-850 cm-1 , according with the infrared absorption and functional group

characteristics of heparin.

3. The monosaccharide composition of mollusc heparins prepared in Examples 1-3 was

analyzed by PMP-pre-column derivatization high performance liquid chromatography.

(1) Chromatographic conditions of high performance liquid chromatography:

chromatographic column: ZORBAX Eclipse XDB-C18 separation column (4.6x250nm, p[m); mobile phase: phosphate buffer (0.05 mol/L, pH 6.74)/acetonitrile (V:V=83:17); flowing rate (mL/min); column temperature: 30°C; detection wavelength: 245 nm, ultraviolet detector; injection volume: 10 pL.

(2) As shown in FIG. 3, the monosaccharide composition of heparins in Mactra antiquata,

Tegillarca granosa and Meretrix lusoria consists of 9 monosaccharides of Man, GlcN, GcA, IdoA,

GalA, Glc, Gal, Xyl, Ara and Fuc, and the monosaccharide composition of heparins in Panopea

abrupta and Tapes dorsatus consists of 6 monosaccharides of Man, GlcN, GIcA, IdoA, Glc, Gal

and Ara, all of which have IdoA, GlcA and GlcN, according with the main monosaccharide

composition of heparin.

Example 5 Anticoagulant activity of mollusc heparins

1. The titer of the standard heparin was determined by adopting an azure A colorimetric

method:

(1) 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of heparin sodium standard solution (2

U/mL) were pipetted into test tubes respectively, and distilled water was added to 2.0 mL;

(2) 2.0 mL of barbital sodium buffer solution (pH 8.6) and 0.5 mL of azure A staining

solution were added, followed by mixing uniformly and standing for 5 min, distilled water was

used as a blank group, and ultraviolet absorbance was measured at 505 nm;

(3) a standard curve was constructed by plotting the titer of heparin on the X-axis and the

absorbance on the Y-axis, and a regression equation was established;

the absorbance of the mollusc heparin prepared in Example 1 was measured in the same way

and the titre of the mollusc heparin was calculated according to the regression equation. The

regression equation was obtained as: Y=0.0676x+0.0005, R2=0.9979; y was absorbance, X was

heparin titer (U/mg).

Table 2 Titer of Mactra antiquata, Tapes dorsatus, Panopea abrupta, Tegillarca granosa and

Meretrix lusoria

Sources of Mactra Tapes Panopea Tegillarca Meretrix molluscs antiquata dorsatus abrupta granosa lusoria

Titer 67.11 39.29 27.23 29.61 44.61 (U/mg) 2. Influence of mollusc heparins prepared in Examples 1-3 on three indexes (APTT, PT and

TT) of blood plasma coagulation

(1) Determination of activation of partial thromboplastin time (APTT value)

0.1 mL of sheep plasma and 0.1 mL of APTT reagent were added into a test tube, followed

by mixing thoroughly, the test tube was placed in a water bath kettle at 37°C for warm bath for 3

min, followed by shaking gently; 0.1 mL of 0.025 mol/L calcium chloride solution was added,

followed by shaking up immediately and starting timing, and the test tube was placed in a water

bath kettle to shake continuously; the test tube was slowly tilted at about 30 s from time to time to

observe the flow state of the test tube, stop the timer when the liquid did not flow, and record the

time.

(2) Determination of prothrombin time (PT value)

PT reagent and sheep plasma were in a water bath kettle at 37°C for preheating for 3min, 0.1

mL of plasma was added into a preheated test tube, 0.2 mL of PT reagent was added, followed by

mixing thoroughly, and starting a stopwatch; after 8 s, the test tubes were removed to observe the

flow state of the plasma from time to time, and when the flow stopped, the number of seconds

was recorded, which was the PT value.

(3) Determination of thrombin time (TT value)

TT reagent and sheep plasma were in a water bath kettle at 37°C for preheating for 3min, 0.2

mL of plasma was added into a preheated test tube, 0.2 mL of TT reagent was added, followed by

mixing thoroughly, and starting a stopwatch; the test tubes were removed to observe the flow state of the plasma from time to time, and when the flow stopped, the number of seconds was recorded, which was the TT value.

The analysis of the anticoagulant effect was shown in FIG. 4, and tests showed that the

heparins extracted from Mactra antiquata, Tapes dorsatus, Panopea abrupta, Tegillarca granosa

and Meretrix lusoria in the present disclosure had mild anticoagulant activity, so the present

disclosure has good potential application value in preparing the heparins with low side effects or

other medical products with a mild anticoagulant effect.

The foregoing embodiments are preferred embodiments of the present disclosure. However,

the embodiments of the present disclosure are not limited by the foregoing embodiments. Any

other changes, modifications, replacements, combinations and simplifications made without

departing from the spirit and principle of the present disclosure should all be equivalent

replacement manners, and fall within the protection scope of the present disclosure.

Claims (5)

1. A method for preparing a mollusc heparin, comprising the following steps:

Si. raw material treatment: after rinsing molluscs and removing shells, conducting

homogenization by adding water to obtain a homogenate;

S2. autolysis: conducting autolysis on the homogenate for 3-7 h under a condition of

38-62°C water bath;

S3. enzymolysis: adding NaOH to adjust a pH value to 7.8-8.2, adding trypsin, conducting

enzymolysis for 3.5-6.5 h at 32-42°C, adding HCl to adjust the pH value to 6.8-7.2, adding

papain, and conducting enzymolysis for 4.5-5.5 h at 62-68°C to obtain an enzymatic hydrolysate;

S4. enzyme deactivation and centrifugation: putting the enzymatic hydrolysate in boiling

water for enzyme deactivation for 5-15 min, conducting centrifugation, and taking a supernatant;

S5. concentration and ethanol precipitation: concentrating the supernatant, adding ethanol,

standing at 0.5-7.5°C for 20-28 h, and conducting centrifugation to obtain a precipitate;

S6. redissolving and deproteinization: rinsing the precipitate with acetone and ethanol

alternately for 2-3 times, dissolving the precipitate with water, and conducting centrifugation to

remove insoluble substances; adding a n-butanol alcohol-chloroform mixed solution in the

supernatant, fully shaking for 10-30 min, standing for 5-15 min, taking a supernatant, and

repeating the operation for 3-4 times to obtain an extracted solution;

S7. desalination: putting the extracted solution into a dialysis bag for dialysis for 48-96 h to

obtain a dialyzate; and

S8. drying: freeze-drying the dialyzate to obtain the mollusc heparin.

2. The preparation method according to claim 1, wherein the molluscs are selected from the

group consisting of Mactra antiquata, Tapes dorsatus, Panopea abrupta, Tegillarca granosa and

Meretrix lusoria.

3. The preparation method according to claim 1, wherein a material-to-liquid ratio in the homogenate is 1:(1.5-4.5); wherein an amount of the trypsin added in step S3 is 0.3%-0.7%; and an amount of the papain added in step S3 is 0.3%-0.7%; wherein an amount of the ethanol added in step S5 is 0.3-0.6 times the volume of the concentrated solution; wherein a volume ratio of n-butanol to chloroform in the n-butanol-chloroform mixed solution in step S6 is 1:(4-5); and a volume ratio of the n-butanol-chloroform mixed solution to the supernatant is 1:(4-5); wherein the centrifugation in step S4 is conducted at 6000-8000 rpm for 10-20 min; wherein in step Sl, a degreasing treatment is further conducted, and the degreasing treatment comprises: soaking the molluscs in acetone for 20-28 h at 2-6°C, conducting suction filtration at room temperature to remove the acetone, and rinsing with water 3-5 times.

4. A mollusc heparin prepared by the preparation method according to any one of claims 1-3,

wherein the heparin shows a characteristic absorption peak at 185-220 nm wavelength in

ultraviolet spectrum scanning; a strong absorption peak of O-S bond stretching vibration at 1240

cm 1, characteristic absorption of heparin at 890 cm and 940 cm1 , and stretching vibration

absorption bands of C--S system of sulfuric acid groups on hexosamine at 800-850 cm-' in

infrared spectrum scanning; and comprises glucosamine, glucuronic acid and iduronic acid in the

monosaccharide composition.

5. Use of the mollusc heparin according to claim 4 in the preparation of products with a mild

anticoagulant effect.