CN108530550A - A kind of oil tea loranthus parasiticus polysaccharides and its extracting method inhibiting blood clotting - Google Patents
A kind of oil tea loranthus parasiticus polysaccharides and its extracting method inhibiting blood clotting Download PDFInfo
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Abstract
The present invention relates to a kind of oil tea loranthus parasiticus polysaccharides and its extracting method for having and inhibiting blood clotting function, the oil tea loranthus parasiticus polysaccharides are water-soluble polysaccharide, acid-soluble polysaccharide, the alkali-soluble polysaccharides extracted from Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf, they can extend thrombin time (TT), prothrombin time（PT）With active partial thromboplastin time (APTT), has the function of preferable external inhibition blood clotting, extracting method is simple and practical, has development and application values.
Description
Technical field
The present invention relates to a kind of oil tea loranthus parasiticus polysaccharides and its extracting method for having and inhibiting blood clotting function.
Background technology
Blood clotting or blood coagulation refer to the process of that blood is changed into immobilising gel state by liquid condition, are raw
The important link of rationality hemostasis.Blood clotting is a complicated process, is amplified step by step using cascade mechanism inside clotting mechanism
Coagulation signal, and reinforce coagulation process by multiple positive feedback links.At the same time, unexpected under normal circumstances in order to prevent to be formed
The thrombus that thrombus or bleeding part are formed is uncontrolled, and body also has anticoagulant mechanism and fibrinolytic mechanism to anticlotting mechanism.
Anticoagulant is a kind of drug for inhibiting blood clotting by influencing the different links of coagulation process, anticoagulant
It is widely used in the prevention and treatment of thrombotic disease, pre- anti-stroke or other thrombotic diseases.In traditional anticoagulant
Most common oral anticoagulation warfarin, Clinical practice for many years, but it is there are many limitations, delays, resists as worked
Solidifying therapeutic window is narrow, it is mutual easily to need frequently to exist between monitoring and regulating dosage and a variety of drugs during by food effect, medication
The side effects such as effect and massive haemorrhage.Therefore for a long time, people always search for it is a kind of safely, effectively with convenient anticoagulation
Object.Ideal anticoagulation should have high effective and safe coefficient, predictable dose response (without monitoring), rapid-action, drug
Between interact the features such as few.It is always the hot spot paid close attention to that safely and effectively anticoagulating active substance is found from natural plants.
Ramulus Taxilli, it is parasitic on original name mulberry, it is also known as wide parasitic.Ramulus Taxilli is parasitize Moraceae, Theaceae, pulse family etc. a kind of half
Parasitic plant, for same Ramulus Taxilli, its host can reach tens kinds or more.Ramulus Taxilli main product in China Fujian,
The ground such as Guangxi, Guangdong, the most area in China all have presence.The Ramulus Taxilli beginning is recorded in《Sheng Nong's herbal classic》, quilt《Legendary god of farming's sheet
Grass warp》It is classified as top grade, effect has that " main pain in the back, children's back of the body is strong, carbuncle swells, tocolysis, fills skin, heavily fortified point hair tooth, long man." as normal
To see in the clinical application of Chinese medicine, Ramulus Taxilli has the primary efficacies such as tocolysis, cough-relieving, strengthening the bones and muscles, and modern pharmacology research is shown,
Ramulus Taxilli has for hypertension, apoplexy, inflammation, hyperlipidemia, miscarriage and all therapeutic effect to arrhythmia cordis etc..
Polysaccharide is a kind of natural macromolecular compound with extensive bioactivity, is distributed widely in the plants such as nature height
In object, algae, microorganism (bacterium and fungi) and animal body.Polysaccharose substance is the another class weight except isolating protein and nucleic acid
The large biological molecule wanted is the material base that many autonomic drugs play drug effect.Plant polyose has complicated, various biology
Activity and function such as immunoloregulation function, anti-infective, radioresistance, anticoagulation, hypoglycemic, reducing blood lipid, promote nucleic acid and protein
Biosynthesis and the effects that anti-aging.As licorice polysaccharide has apparent antiviral and antitumor action；Blackfungus polyhexose,
Gingko exocarp polysaccharide and aloe polysaccharide can antitumor and enhancing immune function of human body；There is Bupleurum chinense polysaccharide radioresistance, enhancing to exempt from
The biological actions such as epidemic disease function；Ophiopogonpolysaccharide has hypoglycemic and immunological enhancement；Boston ivy polysaccharide has antiviral and anti-
Aging acts on；The plant polyoses such as Tea Polysaccharides, ganoderma lucidum polysaccharide, garlic polysaccharide and Blackfungus polyhexose have blood coagulation resisting function etc..Mesh
Preceding existing part natural polysaecharides compound shows good efficacy for clinic.Polysaccharide has become natural drug and health products
Pith in research and development.
Invention content
The technical problem to be solved by the present invention is to：It is a kind of have inhibit blood clotting function oil tea loranthus parasiticus polysaccharides and its
Extracting method
The technical scheme is that：
1. a kind of oil tea loranthus parasiticus polysaccharides inhibiting blood clotting, it is characterised in that：
The oil tea loranthus parasiticus polysaccharides are extracted from Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf
Water-soluble polysaccharide, acid-soluble polysaccharide, alkali-soluble polysaccharide, their main component be rhamnose, arabinose, xylose, mannose,
Glucose, six kinds of monosaccharide of galactolipin and protein, acid-soluble polysaccharide is also containing 18.52% uronic acid, this in water-soluble polysaccharide
The quality molar ratio of six kinds of monosaccharide is 5.09:21.06:6.49:6.65:10.70:9.19, this six kinds of monosaccharide in acid-soluble polysaccharide
Quality molar ratio be 7.15:1.82:15.36:5.42:61.08:35.35, the quality of this six kinds of monosaccharide is rubbed in alkali-soluble polysaccharide
You are than being 0.47:0.66:6.81:0.34:0.77:0.73, water-soluble polysaccharide is the proteoglycan containing α configuration pyranoid rings, acid
Soluble polysaccharide and alkali-soluble polysaccharide are the pyranoid ring proteoglycan of beta comfiguration, and water-soluble polysaccharide weight average molecular weight is 3.61E+04,
Acid-soluble polysaccharide weight average molecular weight is 4.64E+04, alkali-soluble polysaccharide weight average molecular weight is 1.21E+04；
The oil tea loranthus parasiticus polysaccharides have the function of inhibiting blood clotting, specially water-soluble polysaccharide that can extend activation portion
Divide prothrombin time (APTT), there is significant difference；Acid-soluble polysaccharide can extend active partial thromboplastin time
(APTT), extend to dose-dependant thrombin time (TT) and prothrombin time (PT), there is significant difference；Alkali solubility is more
Sugar can extend thrombin time (TT), extend to dose-dependant active partial thromboplastin time (APTT) and prothrombin time
(PT), there is significant difference.
2. the extracting method of the oil tea loranthus parasiticus polysaccharides according to 1 is：
(1) after crushing Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf, with the matter containing ethyl alcohol of 5 times of volumes
The ethanol-water solution of amount percentage 80-85% impregnates 110-130min at 40-60 DEG C, filters, abandons filtrate, 10 times of bodies of filter residue a
Long-pending deionized water leaches 2-3 times at 90-100 DEG C, and each 110-130min, solution filters after being cooled to room temperature, filtered
Filter residue b continues extraction for (2) in next step, and filtrate adds in 50-60 DEG C of vacuum distillation concentration and is equivalent to concentrate triploid
Long-pending absolute ethyl alcohol is stood overnight in 4 DEG C of immersions, is centrifuged, is obtained sediment a, weigh, and is replaced with acetone, ether, ethyl alcohol
Washing precipitate a three times, adds deionized water dissolving sediment a, add be equivalent to sediment a mass monoploids product contain trichlorine
Trichloroacetic acid-aqueous solution of quality of acetic acid percentage 2-3% impregnates in 4 DEG C and stands 90-150min, centrifuge, stands, take
Supernatant is placed in distilled water the 20-30h that dialyses, and a dialyzate is changed every 8h, and solution after dialysis is freeze-dried to get to institute
The water-soluble oil tea loranthus parasiticus polysaccharides stated；
(2) the 0.3mol/L hydrochloric acid solutions of 10 times of volumes, 80- are added in the filter residue b in (1) after deionized water is extracted
90 DEG C of stirring 110-130min, cooling, suction filtration repeats 2-3 times, and filtered filter residue c continues extraction, filter for (3) in next step
Liquid is 7 with 20% sodium hydroxide tune pH value, and in 50-60 DEG C of vacuum distillation concentration, the nothing for being equivalent to 3 times of volumes of concentrate is added
Water-ethanol precipitates, and centrifuges, obtains sediment b, weigh, and alternately washing 3 times of sediment b acetone, ether, ethyl alcohol add
Ion water dissolution adds three chloroethenes of the 2-3% of mass percent containing trichloroacetic acid for the monoploid product for being equivalent to precipitation b mass
Acid-aqueous solution impregnates in 4 DEG C and stands 90-150min, centrifuge, stands, supernatant is taken to be placed in distilled water the 20- that dialyses
30h changes a dialyzate every 8h, and solution after dialysis is freeze-dried to get to the acid-soluble oil tea loranthus parasiticus polysaccharides；
(3) the 0.3mol/L NaOH aqueous solutions of 10 times of volumes are added in the filter residue c in (2) after hydrochloric acid extraction, in
50-60 DEG C in N2Flow down immersion and stand 110-130min, it is cooling, filter, repeat 2-3 time, centrifuge, standing, supernatant with
Glacial acetic acid tune pH value is 5-6, in 50-60 DEG C of vacuum distillation concentration, the absolute ethyl alcohol for being equivalent to 3 times of volumes of concentrate is added, sinks
It forms sediment, centrifuges, obtain sediment c, alternately washing 3 times of sediment c acetone, ether, ethyl alcohol add deionized water dissolving, add
Trichloroacetic acid-the aqueous solution for entering to be equivalent to the 2-3% of mass percent containing trichloroacetic acid of one times of precipitation c mass, in 4 DEG C of standings
90-150min is centrifuged, and is stood, is taken supernatant to be placed in distilled water the 20-30h that dialyses, change a dialyzate every 8h, thoroughly
Solution after analysis is freeze-dried to get to the alkali solubility oil tea loranthus parasiticus polysaccharides；
It is described be under N2 streams avoid the oxidation of oil tea loranthus parasiticus polysaccharides under alkaline condition, and (2) aqueous extraction,
(3) acidic extraction all need not be in the N2 gas shieldeds under N2 streams
3. the structure feature of the oil tea loranthus parasiticus polysaccharides according to 1 is detected according to following (1)-(4) method：
(1) detection of total sugar content, glucuronic acid content, protein content
The total sugar content that oil tea loranthus parasiticus polysaccharides are measured using phend-sulphuric acid, using an xenol colorimetric method for determining
Its glucuronic acid content measures its protein content using BCA protein quantification methods.
(2) detection of monosaccharide composition
It is formed using the monosaccharide of gas Chromatographic Determination oil tea loranthus parasiticus polysaccharides.
(3) infrared spectrum detects
Using the molecular structure and chemical composition of infrared spectrum analysis oil tea loranthus parasiticus polysaccharides.
(4) detection of average molecular weight and distribution
Molecular weight and its distribution of oil tea loranthus parasiticus polysaccharides are measured by High Performance Gel Permeation Chromatography.
4. the oil tea loranthus parasiticus polysaccharides according to 1 have the experimental method for inhibiting blood clotting function
Pass through thrombin time (TT), the measurement of prothrombin time (PT) and active partial thromboplastin time (APTT)
To investigate the anticoagulant active of oil tea loranthus parasiticus polysaccharides.
The effect of the present invention：
From Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf extract water-soluble polysaccharide, acid-soluble polysaccharide,
Alkali-soluble polysaccharide can extend TT, PT and APTT, wherein most with the alkali-soluble polysaccharide effect of oil tea Ramulus Taxilli dried leaf extraction
By force, the very strong external effect for inhibiting blood clotting is shown.Compared to for example most common oral anticoagulation of traditional anticoagulant
Warfarin, oil tea loranthus parasiticus polysaccharides then more safety and conveniently, has the characteristics that higher effective/safety coefficient；Compared to facing
The most common anticoagulation heparin of bed does not absorb since heparin is oral, needs injecting drug use, and oil tea loranthus parasiticus polysaccharides not only take orally
It absorbs well, and good mouthfeel, is suitable for prolonged application；Compared to the tea polysaccharide that can only extend APTT, oil tea loranthus parasiticus polysaccharides were both
APTT can be extended, can also extend TT and PT, show that oil tea loranthus parasiticus polysaccharides can be by influencing endogenous and exogenous solidifying
Blood approach and play blood coagulation resisting function, also can play blood coagulation resisting function by the way that reinforcing fiber protein dissolution is active.Oil tea mulberry is posted
Raw polysaccharide as anticoagulant have the characteristics that safely, effectively, cheap and easy to use, tool Development volue, application prospect is fine.
Specific implementation mode
With reference to embodiment, the present invention is described further.
Embodiment
1. more from polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf extraction water-soluble polysaccharide, acid-soluble polysaccharide and alkali solubility
Sugar method be：
(1) after crushing polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf, with the mass percent containing ethyl alcohol of 5 times of volumes
The ethanol-water solution of 80-85% impregnates 110-130min at 40-60 DEG C, filters, abandons filtrate, 10 times of volumes of filter residue a go from
Sub- water leaches 2-3 times at 90-100 DEG C, and each 110-130min, solution filters after being cooled to room temperature, under filtered filter residue b is supplied
One step (2) continues extraction and uses, and filtrate adds in 50-60 DEG C of vacuum distillation concentration and is equivalent to the anhydrous of concentrate three times volume
Ethyl alcohol is stood overnight in 4 DEG C of immersions, is centrifuged, is obtained sediment a, weigh, and with acetone, ether, ethyl alcohol, alternately washing precipitates
Object a three times, adds deionized water dissolving sediment a, adds the quality containing trichloroacetic acid for being equivalent to sediment a mass monoploids product
Trichloroacetic acid-aqueous solution of percentage 2-3% impregnates in 4 DEG C and stands 90-150min, centrifuge, stands, supernatant is taken to set
Dialyse 20-30h in distilled water, and a dialyzate is changed every 8h, and solution after dialysis is freeze-dried to get to described water-soluble
Property oil tea loranthus parasiticus polysaccharides；
(2) the 0.3mol/L hydrochloric acid solutions of 10 times of volumes, 80- are added in the filter residue b in (1) after deionized water is extracted
90 DEG C of stirring 110-130min, cooling, suction filtration repeats 2-3 times, and filtered filter residue c continues extraction, filter for (3) in next step
Liquid is 7 with 20% sodium hydroxide tune pH value, and in 50-60 DEG C of vacuum distillation concentration, the nothing for being equivalent to 3 times of volumes of concentrate is added
Water-ethanol precipitates, and centrifuges, obtains sediment b, weigh, and alternately washing 3 times of sediment b acetone, ether, ethyl alcohol add
Ion water dissolution adds three chloroethenes of the 2-3% of mass percent containing trichloroacetic acid for the monoploid product for being equivalent to precipitation b mass
Acid-aqueous solution impregnates in 4 DEG C and stands 90-150min, centrifuge, stands, supernatant is taken to be placed in distilled water the 20- that dialyses
30h changes a dialyzate every 8h, and solution after dialysis is freeze-dried to get to the acid-soluble oil tea loranthus parasiticus polysaccharides；
(3) the 0.3mol/L NaOH aqueous solutions of 10 times of volumes are added in the filter residue c in (2) after hydrochloric acid extraction, in
50-60 DEG C is impregnated under N2 streams and stands 110-130min, cooling, is filtered, is repeated 2-3 time, centrifuges, standing, supernatant with
Glacial acetic acid tune pH value is 5-6, in 50-60 DEG C of vacuum distillation concentration, the absolute ethyl alcohol for being equivalent to 3 times of volumes of concentrate is added, sinks
It forms sediment, centrifuges, obtain sediment c, alternately washing 3 times of sediment c acetone, ether, ethyl alcohol add deionized water dissolving, add
Trichloroacetic acid-the aqueous solution for entering to be equivalent to the 2-3% of mass percent containing trichloroacetic acid of one times of precipitation c mass, in 4 DEG C of standings
90-150min is centrifuged, and is stood, is taken supernatant to be placed in distilled water the 20-30h that dialyses, change a dialyzate every 8h, thoroughly
Solution after analysis is freeze-dried to get to the alkali solubility oil tea loranthus parasiticus polysaccharides；
2. the water-soluble polysaccharide extracted from polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf, acid-soluble polysaccharide, alkali solubility
Polysaccharide, structure feature are measured according to following (1)-(4) method：
(1) total sugar content, glucuronic acid content, protein content
Water-soluble polysaccharide, the acid of the extraction of polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf are measured using phend-sulphuric acid
The total sugar content of soluble polysaccharide, alkali-soluble polysaccharide, using its glucuronic acid content of xenol colorimetric method for determining, using BCA eggs
White determination of quantitative analysis protein content, the results are shown in Table 1.
Total reducing sugar, uronic acid, the protein content for the polysaccharide that table 1 is extracted from oil tea Ramulus Taxilli dried leaf
(2) monosaccharide forms
Water solubility using the extraction of gas Chromatographic Determination Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf is more
Sugared, acid-soluble polysaccharide, alkali-soluble polysaccharide monosaccharide composition, as a result show extracted water-soluble polysaccharide, acid-soluble polysaccharide, alkali solubility
Polysaccharide is mainly made of 6 kinds of rhamnose, arabinose, xylose, mannose, glucose and galactolipin monosaccharide, wherein water-soluble more
The molar ratio of sugared 6 kinds of monosaccharide is 5.09：21.06：6.49：6.65：10.70：9.19, the molar ratio of 6 kinds of monosaccharide of acid-soluble polysaccharide
It is 7.15：1.82：15.36：5.42：61.08：35.35, the molar ratio of 6 kinds of monosaccharide of alkali-soluble polysaccharide is 0.47：0.66：6.81：
0.34：0.77：0.73.
(3) infrared spectrum
Larger absorption peak has 3336.12cm from the water-soluble polysaccharide that oil tea Ramulus Taxilli dried leaf is extracted-1、
2921.40cm-1、2853.68cm-1、1596.99cm-1、1394.65cm-1、1249.16cm-1、1056.02cm-1、852.84cm-1、650.50cm-1, as shown in attached drawing Fig. 1.3336.12cm-1It is the O-H and protein due to carbohydrate nearby strong absworption peak occur
Caused by N-H stretching vibrations, there are intramolecular hydrogen bonds；In 3000-2800cm-1Between there are small absorption peak, be that carbohydrate C-H stretches
Absorption peak caused by contracting vibration (- CH2-)；1596.99cm-1Neighbouring absorption peak belongs to amino deformation vibration peak；1394.65cm-1、1249.16cm-1Neighbouring absorption peak is generated by C-C-H or H-C-O, 1056.02cm-1Nearby shake for two kinds of C-O are flexible
Absorption peak caused by dynamic；In 852.84cm-1The characteristic absorption peak at place shows that the polysaccharide has α-glycosidic bond；650.50cm-1Place
There is absorption peak, shows that there are acylamino-s in the polysaccharide structures, can speculate that oil tea Ramulus Taxilli dried leaf carries by infrared spectrum analysis
The water-soluble polysaccharide taken is the proteoglycan containing α configuration pyranoid rings.
Larger absorption peak has 3337.02cm from the acid-soluble polysaccharide that oil tea Ramulus Taxilli dried leaf is extracted-1、2921.36cm-1、1752.38cm-1、1613.55cm-1、1406.14cm-1、1326.44cm-1、1227.88cm-1、1148.18cm-1、
1089.05cm-1、1019.62cm-1、891.07cm-1、623.68cm-1, as shown in attached drawing Fig. 2.3337.02cm-1There is strong inhale
It is caused by the O-H of carbohydrate and protein N-H stretching vibrations to receive peak, and there are intramolecular hydrogen bonds；2921.36cm-1Place occurs
Small absorption peak is absorption peak caused by carbohydrate C-H stretching vibrations (- CH2-)；1752.38cm-1、1613.55cm-1It is amino
Strong absworption peak caused by the change angular oscillation of the C=O and N-H of acid, it was demonstrated that have the presence of protein；1406.14cm-1、
1326.44cm-1、1227.88cm-1This several groups of peaks are the O-H inner faces bending vibration of carbohydrate ,-CH2Wave and torsional vibration；
1148.18cm-1、1089.05cm-1、1019.62cm-1Three neighbouring strong absworption peaks illustrate that sweetness agent classification extraction oxalates is molten
Pectic polysaccharides are pyranose；1089.05cm-1It is nearby the characteristic absorption peak of uronic acid；891.07cm-1Neighbouring absorption peak
Show that the polysaccharide is to show that the component contains β-D- glucopyra saccharide rings caused by the change angular oscillation of β-D- pyranoses C-H, point
Son is with β-glucosides key connection；623.68cm-1There is absorption peak in place, shows that there are acylamino-s in the polysaccharide structures, by infrared spectrum
Figure may infer that the acid-soluble polysaccharide extracted from oil tea Ramulus Taxilli dried leaf is β-D- pyranoid ring proteoglycans.
Larger absorption peak has 3311.56cm from the alkali-soluble polysaccharide that oil tea Ramulus Taxilli dried leaf is extracted-1、
2923.08cm-1、1544.73cm-1、1375.73cm-1、1036.99cm-1、902.38cm-1、639.77cm-1, such as attached drawing Fig. 3 institutes
Show.In 3311.56cm-1The absorption peak of appearance is in carbohydrate molecule or the stretching vibration of intermolecular O-H keys, 2923.08cm-1Place
There is a weak absorbing peak, is the stretching vibration of carbohydrate asymmetry C-H, 1544.73cm-1The absorption peak at place is that amino deformation is shaken
Dynamic absorption peak, 1375.73cm-1The absorption peak at place belongs to the angle vibration absorption peak of C-H；1036.99cm-1The absorption peak at place is
C-O-C groups vibrate caused or hexatomic ring C-H in-plane bending vibrations, 902.38cm-1The characteristic absorption peak at place is β-D- pyranoses
Caused by the change angular oscillation of C-H, show that the component contains β-D- glucopyra saccharide rings, molecule with β-glucosides key connection,
639.77cm-1There is absorption peak in place, shows there are acylamino- in structure, the alkali that thus deducibility oil tea Ramulus Taxilli dried leaf is extracted
Soluble polysaccharide is a kind of pyranoid ring proteoglycan containing beta comfiguration.
(4) average molecular weight and distribution
It is measured from Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf and is carried by High Performance Gel Permeation Chromatography
The molecular weight of the water-soluble polysaccharide, acid-soluble polysaccharide, alkali-soluble polysaccharide that take, as a result as shown in attached drawing Fig. 4-6 and table 2.
The polysaccharide molecular weight and distribution that table 2 is extracted from oil tea Ramulus Taxilli dried leaf
Note：Mw is weight average molecular weight, and Mn is number-average molecular weight, and Mz is matter average molecular weight
3. the water-soluble polysaccharide, acid-soluble more extracted from Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf
Sugar, alkali-soluble polysaccharide have the function of inhibiting blood clotting
(1) platelet poor plasma is prepared
20 week old male rabbits, anesthesia, venous blood sampling are placed in sodium citrate anticoagulation test tube, anti-coagulants sodium citrate
Ratio with blood is 1:9, it mixes well, 2800-3200rpm centrifuges 8-12min, and separated plasma is dispensed to centrifuge tube, system
Platelet poor plasma (PPP) is obtained, -20 DEG C of preservations are spare.
(2) inhibit blood clotting
1) sample preparation：Water-soluble polysaccharide, acid-soluble polysaccharide, the alkali-soluble polysaccharide that oil tea Ramulus Taxilli dried leaf is extracted are taken,
Be dissolved in physiological saline respectively, be configured to 0.5,2.0, the water-soluble polysaccharide of 8.0g/L, acid-soluble polysaccharide, each solution of alkali-soluble polysaccharide
For sample to be tested, carries out extracorporeal blood solidification inhibitory activity and measure, sample to be tested is with platelet poor plasma (PPP) with 1：4 body
Product is than mixing, this is test plasma, and the heparin of 0.03g/L concentration is as positive control, and normal saline is as solvent control.
2) measurement of thrombin time (Thrombin time, TT)：The test plasma 0.2mL for taking 37 DEG C of pre-temperature 3min, adds
Enter the TT reagent 0.2mL of 37 DEG C of pre-temperatures, mixing starts manual time-keeping, constantly tilts test tube and slowly tends to stop to liquid flowing
When only, timing is terminated, the time required to record, replication 3 times or more.
3) measurement of prothrombin time (Prothrombin time, PT)：Take the test plasma of 37 DEG C of pre-temperature 3min
0.1mL, is added the PT reagent 0.2mL of 37 DEG C of pre-temperatures, and mixing starts manual time-keeping.It is slow to liquid flowing constantly to tilt test tube
It is slow when tending to stop, terminating timing, the time required to record, replication 3 times or more.
4) survey of active partial thromboplastin time (Aetived partial thromboplastin time, APTT)
It is fixed：The test plasma 0.1mL of 37 DEG C of pre-temperature 3min is taken, APTT reagents 0.1mL, the 37 DEG C of incubation 5min of 37 DEG C of pre-temperatures are added, then
The 0.025mol/L CaCl of 37 DEG C of pre-temperatures are added2Solution 0.1mL, mixing start manual time-keeping, constantly tilt test tube to liquid
When body flowing slowly tends to stop, timing is terminated, the time required to record, replication 3 times or more.
5) statistical procedures：Data are indicated with mean ± standard deviation, and variance analysis is carried out with SPSS8.0 statistics softwares,
Comparison among groups are examined using Dunett ' s, P<0.05 has statistical significance.
6) pass through thrombin time (TT), the survey of prothrombin time (PT), active partial thromboplastin time (APTT)
It is fixed the result shows that：The water-soluble polysaccharide of oil tea Ramulus Taxilli dried leaf extraction can extend APTT at higher dosage (8.0g/L), and
With significant difference (P≤0.05), certain external effect for inhibiting blood clotting is shown, the results are shown in Table 3；Oil tea
The acid-soluble polysaccharide of Ramulus Taxilli dried leaf extraction (8.0g/L) in higher dosage can extend APTT, the dosage used in experiment
Range (0.5-8.0g/L) can extend PT to dose-dependant, and all have significant difference (P≤0.05), in 2.0,8.0g/L two
A dosage can extend TT, and dose-effect relationship is apparent, and all have significant difference (P≤0.05), show stronger external suppression
The effect of blood clotting processed, the results are shown in Table 4；The alkali-soluble polysaccharide of oil tea Ramulus Taxilli dried leaf extraction, in 2.0,8.0g/L
Two dosage can be obviously prolonged APTT, in 8.0g/L dosage groups close to the level of heparin, all have significant difference (P≤
0.05), the dosage range used in experiment (0.5-8.0g/L) can extend PT, and dose-effect relationship is apparent, in 8.0g/L dosage
Group all has significant difference (P≤0.05) close to the level of heparin, can extend TT at higher dosage (8.0g/L), have
Significant difference (P≤0.05) shows the very strong external effect for inhibiting blood clotting, and the results are shown in Table 5.
The water-soluble polysaccharide of 3 various concentration oil tea Ramulus Taxilli dried leaf of table extraction inhibits blood coagulation activity (* and physiology salt in vitro
Water compares P≤0.05)
Inhibit blood coagulation activity (* and physiology salt outside the acid-soluble polysaccharide body of 4 various concentration oil tea Ramulus Taxilli dried leaf of table extraction
Water compares P≤0.05)
The alkali-soluble polysaccharide of 5 various concentration oil tea Ramulus Taxilli dried leaf of table extraction inhibits blood coagulation activity (* and physiology salt in vitro
Water compares P≤0.05)
Description of the drawings
Fig. 1 is the water-soluble polysaccharide infrared spectrogram of oil tea Ramulus Taxilli dried leaf extraction.
Fig. 2 is the acid-soluble polysaccharide infrared spectrogram of oil tea Ramulus Taxilli dried leaf extraction.
Fig. 3 is the alkali-soluble polysaccharide infrared spectrogram of oil tea Ramulus Taxilli dried leaf extraction.
Fig. 4 is the water-soluble polysaccharide High Performance Gel Permeation chromatogram of oil tea Ramulus Taxilli dried leaf extraction, and M is water-soluble polysaccharide
Molecular weight, W represent water-soluble polysaccharide sample quality, it is seen that dW/d (logM) is mainly distributed on logM=3 to logM=3.7 ranges
It is interior.
Fig. 5 is the acid-soluble polysaccharide High Performance Gel Permeation chromatogram of oil tea Ramulus Taxilli dried leaf extraction, and M is acid-soluble polysaccharide
Molecular weight, W represent acid-soluble polysaccharide sample quality, it is seen that dW/d (logM) is mainly distributed on logM=3 to logM=5.5 ranges
It is interior.
Fig. 6 is the alkali-soluble polysaccharide High Performance Gel Permeation chromatogram of oil tea Ramulus Taxilli dried leaf extraction, and M is alkali-soluble polysaccharide
Molecular weight, W represent alkali-soluble polysaccharide sample quality, it is seen that dW/d (logM) is mainly distributed on logM=3 to logM=4.3 ranges
It is interior.
Claims (2)
1. a kind of oil tea loranthus parasiticus polysaccharides inhibiting blood clotting, it is characterised in that：
The oil tea loranthus parasiticus polysaccharides be extracted from Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf it is water-soluble
Property polysaccharide, acid-soluble polysaccharide, alkali-soluble polysaccharide, their main component is rhamnose, arabinose, xylose, mannose, grape
Sugar, six kinds of monosaccharide of galactolipin and protein, acid-soluble polysaccharide is also containing 18.52% uronic acid, this six kinds of lists in water-soluble polysaccharide
The quality molar ratio of sugar is 5.09:21.06:6.49:6.65:10.70:9.19, the quality of this six kinds of monosaccharide in acid-soluble polysaccharide
Molar ratio is 7.15:1.82:15.36:5.42:61.08:35.35, the quality molar ratio of this six kinds of monosaccharide is in alkali-soluble polysaccharide
0.47:0.66:6.81:0.34:0.77:0.73, water-soluble polysaccharide is the proteoglycan containing α configuration pyranoid rings, acid-soluble more
Sugar and alkali-soluble polysaccharide are the pyranoid ring proteoglycan of beta comfiguration, and water-soluble polysaccharide weight average molecular weight is 3.61E+04, acid-soluble
Polysaccharide weight average molecular weight is 4.64E+04, alkali-soluble polysaccharide weight average molecular weight is 1.21E+04；
The oil tea loranthus parasiticus polysaccharides have the function of inhibiting blood clotting, specially water-soluble polysaccharide that it is solidifying to extend activated partial
Blood zymogen time (APTT) has significant difference；Acid-soluble polysaccharide can extend active partial thromboplastin time (APTT), agent
Amount relies on ground and extends thrombin time (TT) and prothrombin time (PT), has significant difference；Alkali-soluble polysaccharide can extend
Thrombin time (TT) extends to dose-dependant active partial thromboplastin time (APTT) and prothrombin time (PT), tool
There is significant difference.
2. the extracting method of oil tea loranthus parasiticus polysaccharides according to claim 1 is：
（1）After Loranthaceae polypetalous parasitism platymiscium oil tea Ramulus Taxilli dried leaf is crushed, with the quality containing ethyl alcohol hundred of 5 times of volumes
The ethanol-water solution of score 80-85% impregnates 110-130 min at 40-60 DEG C, filters, abandons filtrate, 10 times of volumes of filter residue a
Deionized water leaches 2-3 times at 90-100 DEG C, and each 110-130 min, solution filters after being cooled to room temperature, filtered filter residue
B is in next step（2）Continue extraction to use, filtrate adds in 50-60 DEG C of vacuum distillation concentration and is equivalent to concentrate three times volume
Absolute ethyl alcohol is stood overnight in 4 DEG C of immersions, is centrifuged, is obtained sediment a, weigh, and is alternately washed with acetone, ether, ethyl alcohol
Sediment a three times, adds deionized water dissolving sediment a, add be equivalent to sediment a mass monoploids product contain trichloroacetic acid
Trichloroacetic acid-aqueous solution of mass percent 2-3% impregnates in 4 DEG C and stands 90-150 min, centrifuge, stands, take supernatant
Liquid is placed in distilled water the 20-30h that dialyses, and a dialyzate is changed every 8h, and solution after dialysis is freeze-dried to get to described
Water-soluble oil tea loranthus parasiticus polysaccharides；
（2）To（1）The 0.3mol/L hydrochloric acid solutions of 10 times of volumes of addition in the middle filter residue b after deionized water is extracted, 80-90 DEG C
110-130 min are stirred, it is cooling, it filters, repeats 2-3 times, filtered filter residue c is in next step（3）Continue extraction to use, filtrate is used
20% sodium hydroxide tune pH value is 7, and in 50-60 DEG C of vacuum distillation concentration, the anhydrous second for being equivalent to 3 times of volumes of concentrate is added
Alcohol precipitates, and centrifuges, obtains sediment b, weigh, and alternately washing 3 times of sediment b acetone, ether, ethyl alcohol add deionization
Water dissolution adds trichloroacetic acid-water of the 2-3% of mass percent containing trichloroacetic acid for the monoploid product for being equivalent to precipitation b mass
Solution impregnates in 4 DEG C and stands 90-150 min, centrifuge, stands, take supernatant to be placed in distilled water the 20-30h that dialyses, often
A dialyzate is changed every 8h, solution after dialysis is freeze-dried to get to the acid-soluble oil tea loranthus parasiticus polysaccharides；
（3）To（2）0.3 mol/L NaOH aqueous solutions of 10 times of volumes are added in the middle filter residue c after hydrochloric acid extraction, in 50-60
DEG C in N2It flows down immersion and stands 110-130 min, it is cooling, it filters, repeats 2-3 times, centrifuge, stand, supernatant is with ice vinegar
Acid tune pH value is 5-6, in 50-60 DEG C of vacuum distillation concentration, the absolute ethyl alcohol for being equivalent to 3 times of volumes of concentrate is added, precipitates, from
The heart detaches, and obtains sediment c, and alternately washing 3 times of sediment c acetone, ether, ethyl alcohol add deionized water dissolving, are added suitable
In trichloroacetic acid-aqueous solution of the 2-3% of mass percent containing trichloroacetic acid of one times of c mass of precipitation, 90-150 is stood in 4 DEG C
Min is centrifuged, and is stood, is taken supernatant to be placed in distilled water the 20-30h that dialyses, a dialyzate is changed every 8h, after dialysis
Solution freeze-drying is to get to the alkali solubility oil tea loranthus parasiticus polysaccharides；
It is described in N2Flow down be avoid the oxidation of oil tea loranthus parasiticus polysaccharides under alkaline condition, and（2）Aqueous extraction,（3）It is acid
Extraction all need not be in N2The N flowed down2Gas shielded.
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