CN101560266B - Oil-tea camellia husk polysaccharide and application thereof - Google Patents

Abstract

The invention discloses an oil-tea camellia husk polysaccharide which has the following characteristics: a. physicochemical properties: the polysaccharide is brown powder with small odor and is easily-soluble in water and not soluble in organic solvents such as chloroform and ether, the polysaccharide is orange if reacting with phenol-sulfuric acid, shows a negative reaction with Fehling reagent and shows a negative reaction with iodine-potassium iodide; b. composition of the polysaccharide: the content of the polysaccharide is 30 percent to 50 percent; the distribution range of molecular weight is 7,000 to 200,000,0Da; the polysaccharide is prepared by the following six kinds of monosaccharide: glucose, fucose, arabinose, galactose, seminose and rhamnose. The oil-tea camellia husk polysaccharide is used as free radical scavenger or used for preparing medicaments with anti-tumor function for curing tumors.

Description

A kind of oil-tea camellia husk polysaccharide and uses thereof

Technical field

The present invention relates to a kind of extract from fruit shell of camellia oleifera abel and uses thereof; In particular, relate to a kind of oil-tea camellia husk polysaccharide and uses thereof.

Background technology

Oil tea (Camellia oleifera Abel) is Theaceae (Theaceae) Camellia (Camellia) xylophyta, is the distinctive oil plant seeds of China, mainly is distributed in the ground such as Jiangxi, Hunan, Zhejiang, Guangxi, Guangdong, Fujian, Guizhou of China.Oil tea can be divided into and spends oil tea, safflower oil tea and chrysanthemum oil tea in vain by the color difference of flower, plant the widest in spending oil tea in vain, wherein the widest with common oil tea distribution.Camellia oil contains abundant unsaturated fatty acids, has very high nutritive value, is described as the sweet oil in east.Present Chinese oil tea cultivated area increases just year by year, and the output of tea seed is also progressively increasing.

The oil tea Pu claims tea bag again, is the shell of oil tea fruit, accounts for 2/3 of oil tea fruit gross weight, is the waste that oil tea produces, and annual output has more than 170 ten thousand tons approximately.In the oil tea producing region, oil tea Pu or act as a fuel or be dropped utilization ratio extremely low [Chinese oil, 1996,21 (4): 39-42].The research of relevant oil tea Pu has bibliographical information to utilize the oil tea Pu to produce salt of wormwood and potassium pyrophosphate, and its further investigation utilization is not appeared in the newspapers.As seen to the development and utilization of oil tea Pu to revitalize camellia oleiferaindustry, increase oil tea added value, realize the comprehensive utilization of oil tea by product having profound significance.Up to now, the extraction and the research thereof of oil-tea camellia husk polysaccharide are not seen that report is arranged.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of oil-tea camellia husk polysaccharide (being extract from fruit shell of camellia oleifera abel), and this oil-tea camellia husk polysaccharide has anti-oxidant function and anti-tumor function.

In order to solve the problems of the technologies described above, the invention provides a kind of oil-tea camellia husk polysaccharide, this oil-tea camellia husk polysaccharide has following characteristic:

A. physico-chemical property: this polysaccharide is a brown ceramic powder, and gas is little, and is soluble in water, is insoluble to chloroform, ether organic solvent, is orange-yellow with the phenolsulfuric acid reaction, with the fehling reagent reaction that is negative, with the IKI reaction that is negative.

B. polysaccharide is formed: polysaccharide content (phenolsulfuric acid method mensuration) is at 30%～50% (with glucose meter); Range of molecular weight distributions is 7,000～200,000,0Da; Polysaccharide is made up of following 6 kinds of monose: glucose, Fucose, pectinose, semi-lactosi, seminose and rhamnosyl.

The present invention also provides the purposes of above-mentioned oil-tea camellia husk polysaccharide simultaneously: as free-radical scavengers.

Improvement as oil-tea camellia husk polysaccharide purposes of the present invention: be used for makeup or healthcare products, contain the oil-tea camellia husk polysaccharide of 10～20000 μ g in every mL or the every gram makeup, contain the oil-tea camellia husk polysaccharide of 10～500mg in every mL or the every gram healthcare products.

Further improvement as oil-tea camellia husk polysaccharide purposes of the present invention: contain the oil-tea camellia husk polysaccharide of 50～10000 μ g in every mL or the every gram makeup, contain the oil-tea camellia husk polysaccharide of 50～300mg in every mL or the every gram healthcare products.

The present invention also provides the application of above-mentioned oil-tea camellia husk polysaccharide in preparation treatment anti-tumor function medicine simultaneously.

As the application of oil-tea camellia husk polysaccharide of the present invention in preparation treatment anti-tumor function medicine, this tumour is lung cancer, liver cancer, mammary cancer, cervical cancer, prostate cancer or cancer of the stomach.

Oil-tea camellia husk polysaccharide of the present invention adopts following method to make, and carries out following steps successively:

1), oil tea Pu powder water is extracted 1 time at least, extracting solution is through centrifugal or filtration treatment, and the supernatant liquor of gained is carried out vacuum concentration, concentrated solution;

2), in above-mentioned concentrated solution, add alcohol in 0～20 ℃ of precipitation process 12～48 hours, centrifugal then, after the gained throw out cleaned with dehydrated alcohol, anhydrous diethyl ether and acetone successively, vacuum-drying promptly got pulverous oil-tea camellia husk polysaccharide.

Improvement as the preparation method of above-mentioned oil-tea camellia husk polysaccharide:

Step 1) is: the oil tea Pu is pulverized back water extraction 1～4 time; All extracting solution merged after centrifugal or filtration treatment, with the supernatant liquor vacuum concentration of gained to 1/5～1/7 of original volume.Extracting mode is: extraction, water bath reflux method, countercurrent extraction method, ultrasonic extraction, enzyme assisted extraction method or microwave radiation extraction method; Solid-liquid ratio 1: 3～1: 30g/mL, extraction time is 0.1～5 hour; Extract temperature and be generally 0～100 ℃.Further preferred version is as follows: extraction is: extract 20～100 ℃ of temperature, 0.1～5 hour extraction time; The microwave radiation extraction method is: microwave irradiation power is 100～6000W, and radiation extraction time is 20 seconds～1 hour; Ultrasonic extraction is ultrasonic power 50～5000W, and the ultrasonic extraction time is 12～180 minutes; Enzyme assisted extraction method is selected papoid or cellulase for use.Further preferred version is as follows: oil tea Pu powder is meant the powder that can cross 40～60 mesh sieves after the oil tea Pu is pulverized, and extracting solution 3000～5000r/min carries out centrifugal; Centrifugal gained supernatant liquor is in 50～70 ℃ of vacuum concentration.During extraction, 80～100 ℃ of preferred temperature; Time is 2～3 hours; The microwave radiation extraction method is: preferred radiation extraction time is 2min～0.5h.

Step 2) be: add alcohol in concentrated solution, making the final concentration of alcohol is 30%～90%; Alcohol is selected ethanol or methyl alcohol for use.Further preferred version is as follows: after 12～48 hours, 4000～6000r/min is centrifugal in 0～10 ℃ of precipitation for adding alcohol in the concentrated solution; Vacuum drying temperature is: 40～70 ℃, perhaps select vacuum freezedrying for use.

The oil-tea camellia husk polysaccharide (being extract from fruit shell of camellia oleifera abel) that the present invention obtains after oil tea Pu powder is extracted carries out the cell in vitro experiment first, and the result shows that oil-tea camellia husk polysaccharide concentration has the effect of anticancer increment preferably in 1-1000 μ g/mL scope; Be applicable to cancer cells pearls such as suppressing lung carcinoma cell, liver cancer cell, breast cancer cell, cervical cancer cell, stomach cancer cell, prostate cancer.Oil-tea camellia husk polysaccharide of the present invention is carried out in the body experiment show that orally give oil-tea camellia husk polysaccharide 10-500mg/kgd is to S ₁₈₀The tumour of solid tumor mouse has obvious suppression growth effect, and has a dose-dependence, this just shows that oil-tea camellia husk polysaccharide has the good antitumor effect, be applicable to the prophylactic effect of assisting therapy such as chemotherapy and radiations such as lung cancer, liver cancer and cancer, can be applicable to the application and development of antitumor drug and functional food.Generally, patient carries out oral or injection according to the dosage of 10-500mg/kgd.

Oil-tea camellia husk polysaccharide of the present invention has the activity of removing free radical preferably, and has dose-dependence in 1-20000 μ g/mL scope, is suitable in healthcare products and the makeup.

Major advantage of the present invention is:

1, sets up the extraction process of effective oil-tea camellia husk polysaccharide, determined anti-oxidant, the radioprotective and the antitumor action effect of extract from fruit shell of camellia oleifera abel (oil-tea camellia husk polysaccharide);

2, can guarantee to reach three effective, safe, stable basic demands of medicine;

3, make full use of the oil tea Pu that is dropped in the past, reduced the wasting of resources, helped environment protection;

4, the availability of camellia resource is provided, has improved economic benefit, helped increasing income of peasant.

Description of drawings

Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.

Fig. 1 is the liquid phase collection of illustrative plates of CPPS;

Fig. 2 is the liquid phase collection of illustrative plates of CPPS-1;

Fig. 3 is the liquid phase collection of illustrative plates of CPPS-2;

Fig. 4 is a standard polysaccharide molecular weight canonical plotting;

Fig. 5 mixes the monose gas chromatogram;

Fig. 6 is polysaccharide sample 1 gas chromatogram;

Fig. 7 is polysaccharide sample 2 gas chromatograms;

Among Fig. 5,6,7:

1-rhamnosyl (Rha) 2-Fucose (Fuc) 3-pectinose (Ara) 4-wood sugar (Xyl)

5-seminose (Man) 6-glucose (Glu) 7-semi-lactosi (Gal);

Fig. 8 is that oil-tea camellia husk polysaccharide is to DPPH measured by esr technique figure;

Fig. 9 is that oil-tea camellia husk polysaccharide is to ABTS measured by esr technique figure.

Embodiment

The invention will be further elaborated below by example, further sets forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Unless otherwise defined, the same meaning that employed all specialties and scientific words and those skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

The preparation method of embodiment 1, a kind of oil-tea camellia husk polysaccharide, carry out following steps successively:

1), accurately took by weighing the oil tea Pu powder 20g of 50 mesh sieves,, above-mentioned powder was put into the distilled water of 400mL, extracted under 80 ℃ of conditions of temperature after 2 hours according to the solid-liquid ratio of 1g/20mL; Extract altogether 2 times according to above-mentioned condition, extracting solution cools off after 4000r/min carries out centrifugal; Merge above-mentionedly extract for 2 times, centrifugal gained supernatant liquor, and be concentrated in vacuo to 1/6 of its original volume in 60 ℃, concentrated solution;

2), add ethanolic soln in above-mentioned concentrated solution, control alcoholic acid final volume concentration is 80%, i.e. ethanol/(concentrated solution+ethanolic soln)=80%; 4 ℃ of refrigerator overnight precipitation process (being that sedimentation time is 20 hours), 5000r/min is centrifugal then, and the gained throw out is used dehydrated alcohol, anhydrous diethyl ether and washing with acetone successively, and 60 ℃ of vacuum-dryings get pulverous oil-tea camellia husk polysaccharide 1.12g.

The preparation method of embodiment 2, a kind of oil-tea camellia husk polysaccharide, make the extracting mode among the embodiment 1 into: in the ultrasonic circulating extractor under 80 ℃ of conditions of temperature, 1000w assisted extraction 0.5 hour; Extract altogether 2 times, all the other are all with embodiment 1.

Get pulverous oil-tea camellia husk polysaccharide 1.48g.

The preparation method of embodiment 3, a kind of oil-tea camellia husk polysaccharide makes the extracting mode among the embodiment 1 into: under the microwave power 2000w condition, extract 30s; Extract altogether 2 times, all the other are all with embodiment 1.

Get pulverous oil-tea camellia husk polysaccharide 1.63g.

From the result of embodiment 1～embodiment 3, we can learn: adopt microwave and the ultrasonic raising that helps polysaccharide extract rate.

The preparation method of embodiment 4, a kind of oil-tea camellia husk polysaccharide, make the extracting mode among the embodiment 1 into: 100 ℃ of heating in water bath backflow 5h, to extract altogether 2 times, all the other are all with embodiment 1.

Get pulverous oil-tea camellia husk polysaccharide 1.10g.

Polysaccharide content in experiment 1, the mensuration oil-tea camellia husk polysaccharide (polysaccharide content also adopts this method in the following experiment of the present invention):

The making of typical curve: accurately take by weighing 105 ℃ of standard glucose 40mg that are dried to constant weight, with distilled water dissolving, constant volume is to 500mL, draws 0.25,0.50,0.75,1.0,1.25,1.50 respectively, 1.75mL, respectively mends to 2.0mL with distilled water.Add vitriol oil 5.0mL rapidly after adding 5% phenol 1.0mL mixing then, leave standstill 10min, place 20min in room temperature behind the mixing, measure 490nm place light absorption value on the spectrophotometer, press with distilled water and operate as blank equally.With glucose sugar concentration (C) is X-coordinate, and absorbancy (A) is an ordinate zou, the production standard curve.The typical curve regression equation is: A=0.0135C+0.0022, and r=0.9998, wherein: A is a light absorption value, and r is a relation conefficient, and C is glucose concn (μ g/mL).

Determination of polysaccharide: the oil-tea camellia husk polysaccharide that above-mentioned Different Extraction Method (embodiment 1～embodiment 4) is extracted with distilled water is made into 1.0mg/mL, gets the light absorption value of 0.2mL by above time-and-motion study 490nm, calculates polysaccharide content according to typical curve.

The quality of contained polysaccharide/oil-tea camellia husk polysaccharide quality * 100% in oil-tea camellia husk polysaccharide content (%)=oil-tea camellia husk polysaccharide.

The result shows:

Among the embodiment 1 in the oil-tea camellia husk polysaccharide polysaccharide content be 37.2%;

Among the embodiment 2 in the oil-tea camellia husk polysaccharide polysaccharide content be 36.4%;

Among the embodiment 3 in the oil-tea camellia husk polysaccharide polysaccharide content be 36.8%;

Among the embodiment 4 in the oil-tea camellia husk polysaccharide polysaccharide content be 46.7%;

Oil-tea camellia husk polysaccharide to the foregoing description 1 gained carries out different purification process, obtains following examples respectively accordingly.

Embodiment 5, the oil-tea camellia husk polysaccharide of embodiment 1 gained is carried out following purification process:

Post (3.0cm * 80cm) will be adorned after the pre-treatment of DA-201 macroporous resin, after the distilled water balance, the oil-tea camellia husk polysaccharide of embodiment 1 gained is made into certain concentration solution (10mg/mL with distilled water, 50mL) upper prop absorption, after 12 hours, use distilled water respectively, 20% ethanolic soln (volumetric concentration), 40% ethanolic soln, 70% ethanolic soln and dehydrated alcohol be the elutriant of 2 column volumes of wash-out successively, collect different components, obtain the powder of different components after the lyophilize, measure the polysaccharide content of different components respectively, protein content and polyphenol pigment content.

In the present invention, protein content determination and polyphenol are measured and can be adopted following method respectively:

1), protein content determination: Xylene Brilliant Cyanine G method

The making of typical curve: accurately take by weighing bovine serum albumin 10.00mg, be settled to 100mL, be 0.1mg/mL protein reference liquid, preserve in 4 ℃ of-5 ℃ of refrigerators with a small amount of dissolved in distilled water.Accurate absorption standard bovine serum albumin solution 0,0.1,0.2,0.3,0.4,0.5,0.5,0.7,0.8,0.9,1.0mL are in 10mL tool plug test tube respectively, each pipe adds water to 1mL, add Xylene Brilliant Cyanine G G-250 solution 5mL, mixing, place 10min, measure its absorbancy, production standard curve in the 595nm place.The typical curve regression equation is: A=0.0055C-0.0223, and r=0.9951, wherein: A is a light absorption value, and r is a relation conefficient, and C is the concentration (μ g/mL) of bovine serum albumin.

Sample determination: getting concentration is different sample solution 0.1mL, 0.5mL, the 1mL of 1.0mg/mL, in tool plug chromoscope, mends to 1.0mL with distilled water, adds Xylene Brilliant Cyanine G G-250 solution 5mL, and mixing is placed 10min, measures its absorbancy in the 595nm place.

2), polyphenol is measured: forint phenol colorimetry

The preparation of standardized solution: reference substance standardized solution preparation accurately takes by weighing gallic acid standard substance 25mg, with water dissolution and constant volume to 250mL, reference substance standardized solution that must 0.1mg/mL.Accurate standard model solution 0.1,0.2,0.3,0.4,0.5,0.6,0.7, the 0.8mL of drawing is in the 10mL volumetric flask, add 1mL FC developer again, add 2mL massfraction 15% sodium carbonate solution after shaking up again, be settled to 10mL, measure A behind the reaction 2h under the room temperature ₇₆₀Place's absorbancy, the drawing standard curve, A=0.0129C+0.0253, r=0.99813, wherein: A is a light absorption value, and r is a relation conefficient, and C is gallic acid content (μ g).

Sample determination: get and be diluted to certain density sample solution 0.3mL, in the 10mL volumetric flask, add 1mL FC developer again, add 2mL massfraction 15% sodium carbonate solution after shaking up again, be settled to 10mL, reaction was measured A after 2 hours under the room temperature ₇₆₀Place's light absorption value.

Experiment 2,

The oil-tea camellia husk polysaccharide of embodiment 1 gained, distilled water elution fraction and 20% ethanol elution component in the foregoing description 5 are made into 1.0mg/mL with distilled water, get and measure protein content in right amount respectively, polyphenol content and polysaccharide content, result such as following table 1:

Table 1 DA-201 macroporous resin purification polysaccharide sample

Found out by above table 1 result, behind the polysaccharide sample behind the DA-201 macroporous resin purification, compare with the primary oil-tea camellia husk polysaccharide that washing fraction polysaccharide content has improved 24.5%, polyphenol content has descended 63.2%, and protein content has descended 23.7%; 20% ethanol elution fraction polysaccharide content has improved 5.5%, and polyphenol content has descended 26.0%, and protein content has descended 60.8%.

Embodiment 6, the oil-tea camellia husk polysaccharide of embodiment 1 gained carried out deproteinated handle:

Oil-tea camellia husk polysaccharide with dissolved in distilled water (being made into 5mg/mL), is got polysaccharide soln; Be divided into 8 parts then, every part of 30mL, the 1st part is carried out 1 deproteinated processing, and the 2nd part is carried out twice deproteinated processing, and the rest may be inferred, and the 8th part is carried out 8 deproteinated and handles.Each chloroform-propyl carbinol (4: 1) solution of 1/3 that adds the polysaccharide soln volume, centrifugal 5min (5000r/min) gets supernatant liquor behind the jolting 30min, shakes up the back and surveys polysaccharide and Protein content.Supernatant liquor is added chloroform-propyl carbinol (4: the 1) solution that is equivalent to its volume 1/3 again, repeat said process until the number of times of setting that removes.

Protein percentage before albumen decreasing ratio=(protein percentage behind the preceding protein percentage-deproteinated of deproteinated)/deproteinated

Polysaccharide percentage composition before polysaccharide loss rate=(polysaccharide percentage composition behind the preceding polysaccharide percentage composition-deproteinated of deproteinated)/deproteinated

Table 2 Savag method Deproteinization effect

By last table 2 as can be seen, behind 7 Deproteinizations of Savag method, polyprotein matter clearance only is 16.85%, and removal effect is not good, and the polysaccharide loss amount reaches 35.90%.

Embodiment 7, the polysaccharide sample through DA-201 macroporous resin distilled water elution fraction among the embodiment 5 is carried out membrane separation purification, carries out following steps successively:

Sample ligand is made 5mg/mL, adopt the rolling ultrafiltration membrane system to separate, working pressure is 0.4～1.2Mpa, 30 ℃ of service temperatures; The film of choosing molecular weight cut-off is: 1,000, and 0Da, 1,000,00Da, 5,000,00Da or 1,000,000Da.Collect different components, vacuum freezedrying is calculated yield.

The membrane sepn polysaccharide sample in the different apertures of table 3

	?＜?1,000,0Da	1,000,0～ 1,000,00Da	1,000,00～ 5,000,00Da	5,000,00～ 1,000,000Da	?＞1,000,000Da
						Yield/%	10.2	17.4	12.3	32.4	23.5

As can be seen from Table 3, polysaccharide molecular weight is distributed more widely: wherein, molecular weight distribution is 5,000, and 00～1,000,000Da accounts for 32.4% of total component, is molecular weight＞1,000 secondly, and 000Da accounts for 23.5% of total component.

Embodiment 8, the polysaccharide sample through DA-201 macroporous resin distilled water elution fraction among the embodiment 5 is carried out column chromatography DEAE-cellulose purifying, carries out following steps successively:

With the DEAE-cellulose dress post (2.6cm * 70cm), use the distilled water balance that handles well.With the polysaccharide sample ligand make finite concentration (10mg/mL, 2mL) solution upper prop is with 0, the NaCl solution of 0.1mol/L, 0.3mol/L, 0.5mol/L, 1.0mol/L carries out gradient elution, the fraction collection elutriant, the 5mL/ pipe, 1mL/min, pipe is surveyed light absorption value with the phenolsulfuric acid method at the 490nm place.Draw elution curve, merge same peak, amalgamation liquid is through 7000Da dialysis tubing dialysis 72 hours (except the water elution part), and the yield of variant component is calculated in lyophilize.

The NaCl eluant solution gained polysaccharide fraction of table 4 different concns

Be mainly neutral sugar through the washing part, through the acidic polysaccharose that is mainly of NaCl eluant solution part.By last table 4 as can be seen, wherein the highest behind the NaCl of different concns eluant solution with 0.5mol/LNaCl eluant solution part yield, reach 20.5%; Secondly reach 19.3% for 0.3mol/LNaCl eluant solution component, with this component called after: CPPS-1.

Embodiment 9, will carry out column chromatography Sephadex G-100 column purification purifying through DEAE-cellulose through 0.3mol/LNaCl eluant solution fraction polysaccharide sample among the embodiment 8, carry out following steps successively:

With the Sephadex G-100 that handles well dress post (1.6cm * 110cm), after the balance with finite concentration (3mg/mL, 0.3mol/LNaCl eluant solution fraction polysaccharide sample upper prop 2mL), NaCl solution with 0.3mol/L carries out gradient elution, the fraction collection elutriant, 5mL/ pipe, 1mL/min, pipe is surveyed light absorption value with the phenolsulfuric acid method and is drawn elution curve at the 490nm place, the result obtains two elution peaks.After two elution peaks collection merging dryings, more respectively successively as stated above, the upper prop wash-out is drawn elution curve, obtains simple spike respectively, obtains two single polysaccharide components after the drying, and the components that content is many are carried out subsequent experimental, called after: CPPS-2.

Test 3, oil-tea camellia husk polysaccharide carried out physico-chemical property and activity research:

Oil-tea camellia husk polysaccharide and the sample after macroporous resin and column chromatography carry out purifying are carried out the mensuration of liquid chromatography, explore the distribution of the molecular weight of polysaccharide.

Experiment material: oil-tea camellia husk polysaccharide (CPPS): by embodiment 1 method gained; Purifying oil-tea camellia husk polysaccharide 1 (CPPS-1): by among the embodiment 8 through DEAE-cellulose through 0.3mol/LNaCl eluant solution fraction polysaccharide; Purifying oil-tea camellia husk polysaccharide (CPPS-2): by the component of embodiment 9 method gained; Dextran standard Dextran10500 (T-10), Dextran43200 (T-40), Dextran76900 (T-70), Dextran188000 (T-110): buy from Sigma company.

Laboratory apparatus: Waters 2695 high performance liquid chromatographs (U.S. Waters company);

Experimental technique: chromatographic column: Japanese TOSOH company's T SK-gel 3000 SWXL (7.8 * 300mm); Moving phase is bi-distilled water; Flow velocity is 0.7mL.min ^-1Differential refraction detects; 30 ℃ of column temperatures; Detector temperature: 35 ℃, sample size is 10 μ L.

The retention time and the corresponding molecular weight of table 5, different mark product

By Fig. 1～Fig. 4 and table 5 as can be seen, the RT distribution range of oil-tea camellia husk polysaccharide of the present invention is: after 6.35～16min and standard substance correspondence analysis calculated, the range of molecular weight distributions of this polysaccharide was: 7, and 000-2,000,000Da.

Test 4, utilize the monose of gas Chromatographic Determination oil-tea camellia husk polysaccharide to form:

Test reagent: rhamnosyl (Rha), pectinose (Ara), wood sugar (Xyl), seminose (Man), glucose (Glu), semi-lactosi (Gal), Fucose (Fuc), oxammonium hydrochloride, trifluoroacetic acid, pyridine, acetic anhydride etc. are homemade analytical pure.

Test apparatus: 6890N type gas chromatograph (Agilent, fid detector; The DB-1701 capillary column);

Micro-analytical balance: Sartrius, West Germany;

Test method: take by weighing the 20mg sample, add 2M trifluoroacetic acid 6mL, tube sealing reacts 6h down at 110 ℃, dries up with nitrogen under 70 ℃ then, and is stand-by.Accurately weighing monose standard substance rhamnosyls (Rha), pectinose (Ara), wood sugar (Xyl), seminose (Man), glucose (Glu), semi-lactosi (Gal), each 10mg of Fucose and above-mentionedly be concentrated into dried polysaccharide sample, add 10mg oxammonium hydrochloride and 0.5mL pyridine, put into 90 ℃ of water-bath reacting by heating 30min and vibration.Be chilled to room temperature after the taking-up, add the 0.5mL acetic anhydride, continue reaction 30min down at 90 ℃ and carry out acetoxylation, reaction product is directly carried out gas chromatographic analysis behind membrane filtration.Monosaccharide derivatives is respectively got 10 μ L mixing be mixing monose.

Chromatographic column: DB-1701 capillary column (30.0m * 0.32mm * 0.25 μ m)

Constant current mode: flow is 1.5mL/min.Temperature programming: from 180 ℃ to 220 ℃, keep 5min with 5 ℃/min.250 ℃ of injector temperatures, 280 ℃ of the temperature of fid detector, carrier gas is a nitrogen, sample size 1 μ L.

The gas chromatogram that mixes monose, polysaccharide sample 1 (CPPS), polysaccharide sample 2 (CPPS-1) is respectively as Fig. 5～shown in Figure 7.

Table 6 mixes monose sample retention time and proportion

Experiment 5,

Oil-tea camellia husk polysaccharide (by embodiment 1 gained) is carried out following experiment:

Oil-tea camellia husk polysaccharide is mixed with the solution of mass concentration 1.0mg/mL with distilled water, and be diluted to different concentration gradient (25.0,20.0,15.0,10.0 and 5.0 μ g/mL), the sample solution of respectively getting the above-mentioned concentration of 1.0mL is in test tube, adding the 3.0mL mass concentration again is the DPPH (2 of 0.04g/L, 2-phenylbenzene-1-picryl phenylhydrazine) solution, mix, behind the lucifuge reaction 30min being A at 517nm place survey light absorption value _i, according to of the clearance rate mapping of different concns sample solution to DPPH.With xitix (Vc) in contrast, face with preceding and prepare with distilled water.Make three parallel samples, each parallel survey three times.

The clearance rate of DPPH is calculated by following formula:

Clearance rate (%)=[1-(A _i-A _j)/A ₀] * 100%

Wherein: A ₀The light absorption value of DPPH solution when not adding sample; A _iLight absorption value for solution behind the adding sample; A _jLight absorption value for sample solution.

As seen from Figure 8, the concentration of oil-tea camellia husk polysaccharide is proportionate to the clearance rate and the concentration of DPPH free radical in 5～25 μ g/mL.The clearance rate of DPPH free radical reaches 50% when polysaccharide concentration is 12.29 μ g/mL, and clearance rate is up to 87.72% when polysaccharide concentration is 25 μ g/mL; When the concentration of positive control xitix (Vc) is 4.44 μ g/mL, the clearance rate of DPPH free radical is reached 50%.

Test 6, oil-tea camellia husk polysaccharide (by embodiment 1 gained) carried out following experiment:

With deionized water with ABTS[2,2 '-azine-two (3-ethyl benzothiazoles-6-sulfonic acid)] dissolving, making ABTS concentration is 7mmol/L, adds K ₂S ₂O ₈, make K ₂S ₂O ₈Concentration be 2.45mmol/L, at room temperature place the dark place to spend the night (12～16h) this solution afterwards.With the ABTS that generates ⁺Free-atom aqueous solution is with phosphoric acid buffer (PBS) dilution, and making its absorbancy under 30 ℃, 734nm wavelength is 0.70 ± 0.02, promptly obtains ABTS ⁺The free radical working fluid.Oil-tea camellia husk polysaccharide is mixed with the solution of mass concentration 1.0mg/mL with PBS, and is diluted to different concentration gradient (20.0,16.0,12.0,8.0 and 4.0 μ g/mL), in test tube, add the above-mentioned oil-tea camellia husk polysaccharide solution of 1.0mL, add the ABTS of 3.0mL again ⁺The free radical working fluid mixes, and 30 ℃ leave standstill 6min, read light absorption value under the 734nm wavelength.With xitix (Vc) in contrast, face with preceding and prepare with PBS.Make three parallel samples, each parallel survey three times.

The ABTS free radical scavenging activity is calculated by following formula:

Clearance rate (%)=[1-(A _i-A _j)/A ₀] * 100%

Wherein: A ₀Blank; A _iLight absorption value for solution behind the adding sample; A _jLight absorption value for sample solution.As seen from Figure 9, the result of this method mensuration is similar to DPPH method result.Clearance rate to the ABTS free radical when polysaccharide concentration is 11.60 μ g/mL reaches 50%, and clearance rate is up to 75.95% when polysaccharide concentration is 20 μ g/mL; When the concentration of positive control xitix (Vc) is 6.65 μ g/mL, the clearance rate of ABTS free radical is reached 50%.

Test 7, oil-tea camellia husk polysaccharide (by embodiment 1 gained) carried out following experiment:

Oil-tea camellia husk polysaccharide is allocated in the sun-proof matrix of makeup, according to the prescription and the following method preparing cosmetics of table 7.

Table 7 sun care preparations allocation sheet

The preparation method: A liquid and B liquid are heated to 72～82 ℃ respectively, and continuously stirring is all dissolved until various compositions, while stirring A liquid is added B liquid, continues to stir to be cooled to room temperature (15-30 ℃) until formed emulsion, obtains the sun-proof standard substance of 100g at last.

The makeup of above acquisition are passed through to measure its SPF (sun protection factor) (sun protect factor, SPF) performance of evaluation oil-tea camellia husk polysaccharide anti-ultraviolet radiation.

Utilize the SPF (sun protection factor) of adhesive tape method working sample.

1. the 3M adhesive tape is cut into 1.1cm * 4.5cm size, sticks on the quartz colorimetric utensil transparent side surface.

2. connect power supply, the preheating spectrophotometer, selected multi-wavelength is measured, and measuring parameter is set at T% and Abs setting ultraviolet wavelength at twice respectively and is respectively 290nm-400nm, and the wavelength interval is 5nm.

The quartz colorimetric utensil that 3. will post adhesive tape places sample light path and reference light paths, adjusts instrument zero.

4. weighing 9.9mg testing sample is evenly coated in sample on the quartz colorimetric utensil 3M adhesive tape.

5. the sample cuvette for preparing is put in the loft drier with 37 ℃ dry 15 minutes.

6. the testing sample cuvette is put with the sample light path in, get another quartz cell that posts adhesive tape and place reference light paths, measure the UVB district respectively and set wavelength ultraviolet absorbance value and transmissivity, get the arithmetic equal value of respectively measuring numerical value.

7. 5 parallel sample of sequentially determining, computation of mean values is in the arithmetical mean of calculating average and be the absorbance of this sample and transmissivity.

8. utilize the spf value of following formula calculation sample.

$\mathrm{SPF}=\frac{{\mathrm{\Σ}}_{290}^{400}E\left(\mathrm{\λ}\right)I\left(\mathrm{\λ}\right)}{{\mathrm{\Σ}}_{290}^{400}E\left(\mathrm{\λ}\right)I\left(\mathrm{\λ}\right)T\left(\mathrm{\λ}\right)}$

Wherein: E (λ): north latitude 40 degree, sun drift angle 20 degree, noon in summer the sunlight different wave length yield of radiation.

I (λ): be the erythemal effect coefficient of different wavelengths of light.

T (λ): be the transmissivity of different wave length sample.

The spf value of the blank group of result is 8.72 ± 2.19, and the spf value of sample sets is 17.13 ± 3.38 (SPF 〉=15 are for super sun-proof), so the tea husk polysaccharide has stronger anti-ultraviolet radiation performance.

Test 8, oil-tea camellia husk polysaccharide (by embodiment 1 gained) carried out following experiment:

Vitro inhibition cancer cells increment experiment: mtt assay.

With JEG-3 lung carcinoma cell H460, liver cancer cell HepG2 and the cervical cancer cell Hela cell recovery of buying, 5% CO ₂, 37 ℃ cultivate, go down to posterity, the inverted microscope counting is adjusted cell concn.The cancer cells (1 * 10 that the phase of taking the logarithm grows ⁵Individual/as mL) to be inoculated in 96 orifice plates, (marginal pore is filled with aseptic PBS or substratum) every hole inoculating cell 100 μ L, 5% CO ₂, 37 ℃ hatched 24 hours.Change the substratum in 96 orifice plates into fresh substratum (marginal pore need not change), add the polysaccharide sample solution 100 μ L (ultimate density is 0,31.25,62.5,125,250,500 μ g/mL) of different concns, 5% CO ₂, 37 ℃ hatch 48 hours after, every hole adds 20 μ l MTT solution (5mg/mL, i.e. 0.5%MTT), continues to cultivate 4h.Stop to cultivate, the careful suction removed the nutrient solution hole in, and every hole adds the 200uL dimethyl sulfoxide (DMSO), crystallisate is fully dissolved after, the light absorption value in each hole of measurement, microplate reader OD 570nm place, calculating inhibiting rate.Each concentration is done 5 multiple holes, three parallel laboratory tests.Do blank and positive controls simultaneously.

Inhibiting rate IR (%)=(1-OD _Sample/ OD _Blank) * 100%

Table 8, polysaccharide are to the growth-inhibiting effect of different carcinoma cell

As can be seen from Table 8, oil-tea camellia husk polysaccharide concentration is at 31.25～500 μ g/mL, increase along with concentration, growth-inhibiting effect to 3 kinds of cancer cells strengthens gradually, when polysaccharide concentration is 500 μ g/mL, growth inhibition ratio to the H460 cell reaches 51.38%, and the growth inhibition ratio of Hela cell is reached 63.42%, and the growth inhibition ratio of HepG2 cell is reached 74.54%.

Test 9, oil-tea camellia husk polysaccharide (by embodiment 1 gained) carried out following experiment:

It is (male to choose the ICR mouse, about body weight 20g, derive from Chinese Academy of Sciences's Shanghai animal center) 50, aseptic ascitic type S180 knurl strain mouse of getting the 7d that goes down to posterity, after the cervical vertebra dislocation is put to death under the aseptic condition, routine disinfection is got ascites, with stroke-physiological saline solution by being made into cell suspension, mixing at 1: 3, this cell suspension 0.2mL is inoculated in every mouse right fore oxter, behind the inoculated tumour 24h, weigh and mouse is divided into 5 groups at random, 10 every group.Be made as basic, normal, high 3 the dosage experiments groups of blank group, positive controls and oil-tea camellia husk polysaccharide sample (50mg/kg.d, 150mg/kg.d and 450mg/kg.d) respectively.The blank group gavages physiological saline by 0.2mL/10g every day, gavages 10d continuously; According to dosage 50mg/kg.d was at the 1st day and the 2nd day continuous intraperitoneal injection of cyclophosphamide (CTX) for positive controls, and injection volume is by 0.2mL/10g, totally 2 times; Polysaccharide sample basic, normal, high 3 dosage experiments group every days gavage the soup of different concns, every day 1 time, 10d continuously by 0.2mL/10g.Write down the mouse body weight change every day in the experimentation, adjusts dosage.Behind the last administration 24h, mouse is put to death in the cervical vertebra dislocation, dissects and wins the knurl piece, calculates tumour inhibiting rate (%), calculates as follows: specifically as shown in table 9:

Tumour inhibiting rate (the %)=average knurl of (the average knurl of the average knurl weight-sample sets of blank group is heavy)/blank group heavy * 100%

Table 9, the effect of extract from fruit shell of camellia oleifera abel anti-tumor in vivo

The result shows that oil-tea camellia husk polysaccharide can effectively suppress the growth effect of mouse interior tumor, and the high dose group of oil-tea camellia husk polysaccharide has reached 38.38% to the inhibiting rate of tumour, and has dose-dependence.

The acute toxicological experiment of experiment 10, oil-tea camellia husk polysaccharide: by the Zhejiang Academy of Medical Sciences Entrustment Inspection.

Experimental technique: with reference to " food safety toxicity assessment program and method " " GB 15193.1-2003 "

Judgment basis: with reference to " food safety toxicity assessment program and method " " GB 15193.1-2003 "

Test-results shows: this oil-tea camellia husk polysaccharide (oil tea extract) is through mouse mouth acute toxicity test: female, male mouse LD50 is the 14.70g/kg body weight, presses acute toxicity grading criteria and judges, oil-tea camellia husk polysaccharide is real belong to nontoxic.

At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.

Claims (5)

1. oil-tea camellia husk polysaccharide is characterized in that this oil-tea camellia husk polysaccharide has following characteristic:

A. physico-chemical property: this polysaccharide is a brown ceramic powder, and gas is little, and is soluble in water, is insoluble to chloroform, ether organic solvent, is orange-yellow with the phenolsulfuric acid reaction, with the fehling reagent reaction that is negative, with the IKI reaction that is negative;

B. polysaccharide is formed: the polysaccharide mass content is 30%～50%; Range of molecular weight distributions is 7,000～200,000,0Da; Polysaccharide is made up of following 6 kinds of monose: glucose, Fucose, pectinose, semi-lactosi, seminose and rhamnosyl.

2. the purposes of oil-tea camellia husk polysaccharide as claimed in claim 1: as free-radical scavengers.

3. the purposes of oil-tea camellia husk polysaccharide according to claim 2 is characterized in that: be used for makeup or healthcare products, contain the oil-tea camellia husk polysaccharide of 10～20000 μ g in every mL or the every gram makeup, contain the oil-tea camellia husk polysaccharide of 10～500mg in every mL or the every gram healthcare products.

4. the purposes of oil-tea camellia husk polysaccharide according to claim 3 is characterized in that: the oil-tea camellia husk polysaccharide that contains 50～10000 μ g in every ml or the every gram makeup; The oil-tea camellia husk polysaccharide that contains 50～300mg in every mL or the every gram healthcare products.

5. the application of oil-tea camellia husk polysaccharide as claimed in claim 1 in preparation treatment anti-tumor function medicine, it is characterized in that: described tumour is lung cancer, liver cancer or cervical cancer.

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