CN1281727A - Antiviral composite containing phycocyanin extract and extraction of phycocyanin - Google Patents

Abstract

The present invention provides an extract containing phycocyanin, antiviral composite and its method for extracting phycocyanin.

Description

Contain the extraction of the antiviral composition and the phycocyanin of phycocyanin extract

The present invention relates to the extraction and the application of the extract and the application thereof, particularly phycocyanin of algae bio body.

Phycocyanin extensively is present in the blue-green alge, and it was evolved tens years before chlorophyll occurs on earth.Because it not only contains magnesium but also contain ferrum, so phycocyanin matter is regarded as the pioneer of chlorophyll and haemachrome again, is the common Source of life of plant and animal.

Phycocyanin is gone back the multiple human body of enrichment needed mineral, particularly calcium except that containing abundant high quality protein matter, therefore exploitation and sharp phycocyanin are significant.

Extraction and application for phycocyanin rarely have bibliographical information at present.

One of purpose of the present invention provides a kind of antiviral agent that contains phycocyanin extract, wherein contains the blue egg extract of algae and/or the pharmaceutically acceptable carrier for the treatment of effective dose.

Another object of the present invention provides a kind of method of extracting phycocyanin, and the method comprises the following steps:

A. the blue-green alge powder is dissolved in the 5-20 times of water, low temperature kept 8-30 hour,

B. solid-liquid separation transfers to acidity with the pH value of solution, places,

C. collecting the gained solid is the phycocyanin crude product.

Fig. 1 is the proteic U.V. scanning spectra of algae;

Fig. 2 is the U.V. scanning spectra after the algae Protein Separation.The present invention describes in detail:

The invention provides a kind of antiviral composition that contains phycocyanin extract, comprise acceptable carrier on the phycocyanin extract for the treatment of effective dose and/or the materia medica in the described composition.

Described phycocyanin extract prepares with following steps:

A. the algae powder is dissolved in the 5-20 times of water, low temperature kept 8-30 hour,

B. Separation of Solid and Liquid transfers to acidity with the pH value of solution, places,

C. collecting the gained solid is the phycocyanin crude product. Wherein the algae powder among the step a is preferably spirulina powder, and wherein the consumption of water be preferably the algae powder 8-15 doubly, preferably 10 times, because phycocyanin easily at high temperature destroys, therefore whole leaching process must keep low temperature, usually is advisable for 10-15 ℃.

The pH value of the solution among the step b preferably is adjusted to 3.8-4.2.

Has the ability of duplicating and breeding that suppresses virus effectively by the antiviral composition that contains phycocyanin extract of the present invention.

Treatment effective dose described in the present invention is grasped easily by those of ordinary skills, usually according to patient's situation, and disease, age, situation such as body weight and deciding.Because phycocyanin extract is nontoxic, therefore, as required, pharmaceutical composition can directly be made by phycocyanin extract.When containing other medicines in the compositions and learn to go up acceptable carrier, they can be made by the pharmaceutical field conventional method.

Acceptable carrier comprises conventional those that use in this area on the described materia medica, and as solvent, various excipient are separated and collapsed agent etc.Described pharmaceutical composition can be made into solution, oral liquid, syrup, pill, tablet, electuary, powder, granule, dosage form such as suppository.

The invention will be further described below in conjunction with embodiment.

Embodiment 1

With the 2kg spirulina powder, add 20 premium on currency, soaked 15-20 hour down in 10-15 ℃.Get rid of filter and keep supernatant, filtering residue repeats to extract.Merging filtrate, with the HCl adjusting pH to 3.8 of filtrate with 0.1N, low temperature is placed and is spent the night.Remove supernatant with supercentrifugal process, be precipitated as the phycocyanin extract crude product, lyophilization.

1. amino acid analysis: accurately take by weighing the about 0.2mg product of embodiment 1 product. add 1ml 5.7N HCl, seal after bleeding, 110 ℃ of following hydrolysis 24 hours, the open pipe final vacuum is drained hydrochloric acid, add the 50ml distilled water, vacuum is drained again, and residue is diluted to 100ml or 00ml (concentration decision per sample) with distilled water. and the aminoacid of measuring each sample with Backman 121MB amino-acid analyzer is formed.Experimental result sees Table 1:

Table 1. analysis of amino acids tables of data (mg/g sample)

Aminoacid	Content	Aminoacid	Content
				????Asp	????181．98	????Met	????19．143
????Thr	????81．376	????Ile	????79．118
				????Ser	????80．634	????Leu	????125．43
????Glu	????176．68	????Tyr	????-
				????Pro	????35．343	????Phe	????48．639
????Gly	????143．88	????Lys	????59．721
				????Ala	????159．94	????His	????69．133
????Cys	????-	????Arg	????72．749
				????Val	????100．00

The amino acid analysis result shows and contains rich in amino acid in the sample, and wherein aspartic acid and tyrosine contents are high especially, and aspartic acid can quicken the synthetic of ATP in the muscle, CP and glycogen, helps S-acetyl-coenzyme-A again and enters tricarboxylic acid cycle.

2. results of elemental analyses:

Accurately take by weighing about 0.1 gram of embodiment 1 gained sample, after the heating of dropping concentrated nitric acid makes its hydrolysis, add the about 0.5ml of hydrogen peroxide, to 50ml, reuse plasma-speetrometer (JCAP 9000ST type) is carried out quantitative analysis with distilled water diluting.Test fluid concentration for this experiment usefulness is 2.42mg/ml, and constituent content is represented with ng/ml.Income analysis result is as follows:

Element	Content	Element	Content
				????Pb	????0．0621	????Mo	????0．0041
????Au	????0．0056	????Cr	????0．00271
				????Fe	????4．556	????Mn	????0．1821
????Mg	????13．62	????Co	????0．0113
				????Si	????6．920	????P	????50．18
????Al	????2．358	????Ba	????0．1946
				????Ti	????0．0755	????B	????0．3915
????Cu	????0．0972	????Sr	????0．1320
				????Ni	????0．0247	????In	????0．0639
????Zn	????0．4016	????La	????0．0698
				????Cd	????0．0613	????Ce	????0．0497
????Bi	????0．0454	????Y	????0．0055
				????Ga	????0．0747	????Sc	????0．0025
????Ag	????0．0152	????Be	????0．0041
				????Ca	????59．41	????As	????-
????V	????0．0149	????Nd	????0．2402

3. biochemical analysis:

(1) U.V. spectrum: algae albumen is divided into two main chromoproteins behind Sephadex G-100 column chromatography, i.e. the less blue albumen of xanthoprotein that molecular weight is bigger and molecular weight, as shown in Figure 1.(2 * 105cm), eluant adopts 0.05M phosphate buffer, PH8.0, flow velocity: 3.5ml/min wherein to use Sephadex G 100 posts.

As shown in Figure 2, in the composition of algae albumen after separating: Emax:670nm (xanthoprotein), Emax:615nm (phycocyanin), 650nm (allophycocyanin).

(2) chemical stability:

The phycocyanin extract lyophilized powder can be preserved and not fade in 2 years under exsiccant room temperature condition, never degenerates.

Quality detecting method:

1) determining the protein quantity:

[reagent] BSA is available from east instrument company of the Chinese Academy of Sciences, lot number 90090.

[instrument] Shimadzu UV-240 of company spectrophotometer.

[assay method] biuret method

(1) manufactures standard curve: measure 0,0.15,0.30 respectively, 0.60,0.90,1.20, the standard protein stock solution (1.2mg/ml) of 1.50ml, supply the 1.5ml/ pipe with distilled water, each adds 1.5ml6%NaOH and 0.15ml biuret reagent, and mixing at room temperature left standstill 20 minutes, in 310nm place colorimetric, the correlation curve of draw OD value and sample concentration.

(2) protein content in the working sample (phycocyanin): take by weighing sample 13.2mg, with distilled water dissolving and fixed molten to 50ml, measuring method is the same.

2) the ash content of coal is measured: the calcination method

[assay method] measures ash (comprising the inorganic salts material), adopts calcination, constant weight, weighing method to measure.

3) amino acid analysis: accurately take by weighing about 0.2mg sample, add 1ml5.7N HCl, seal after bleeding, 110 ℃ of following hydrolysis 24 hours.The open pipe final vacuum is drained hydrochloric acid, adds the 50ml distilled water, and vacuum is drained again.Residue is diluted to 100ml or 200ml (concentration decision per sample) with distilled water.Measure the aminoacid of each sample forms with Backman 121MB amino-acid analyzer.

4) aseptic experiment.

[testing index]

(1) sample is behind lactate fermentation and EMB culture medium culturing, and coliform count is no more than 3;

(2) sample is behind the steamed beef soup culture medium culturing, and total bacteria count is less than 100.

4. effect:

(1) quantity-T lymphocyte check that increases T-cell in the body is tested

Laboratory animal and medicine:

Laboratory animal: Kunming mouse, body weight 18～22 grams, the epidemic disease inspection is qualified.

Cyclophosphamide (injection): Shanghai No.12 Pharmaceutical Factory produces.

The dry powder that medicament: embodiment 1 makes is mixed with the medicinal liquid of 20 μ g/ml and 10 μ g/ml before experiment.

Experimental technique: mice is divided into four groups at random, every group 10, administration group intraperitoneal injection of cyclophosphamide 10mg/kg every every day, per os gastric infusion liquid again, every every day 0.2ml, promptly be equivalent to 0.2mg/kg (high dose group) and 0.1mg/kg (low dose group); Positive controls intraperitoneal injection of cyclophosphamide 10mg/kg every every day irritates stomach ordinary water 0.2ml at per os; The blank group is not injected, not administration, and a per os is irritated the stomach ordinary water 0.2ml//day.Behind the successive administration 10 days, drug withdrawal 3 days, after getting mouse orbit blood and doing the leukocyte push jack, hatch, dye, the T lymphocyte percentage.

Experimental result: should cause mice T-lymphocyte obviously to descend behind the injection cyclophosphamide, but rising appears in experimental result administration group on the contrary, especially its rising of high dose group result compares with matched group and presents significant difference (P＜0.01).Experimental result shows that medicinal liquid has the function of human body immunity improving power preferably.

It all has the good restraining effect to AIDS (HIV-1) virus, herpes simplex virus, influenza A virus, mumps virus, Measles virus etc. the clinic trial proof.

(2) to the influence of collagen content in the skin

The content of collagen protein is to reduce with advancing age and gradually in mammal and the human skin.In the aminoacid of collagen protein was formed, hydroxyproline accounted for 12～14%.And contain hydroxyproline in other tissue hardly.Content height and stable hydroxyproline composition are big characteristics of collagen protein.Therefore, can be used as an index measuring the skin aging degree by hydroxyproline content in the quantitative assay skin.Experiment material and instrument

1) laboratory animal: Kunming mouse, body weight 28-30g, male and female dual-purpose.

2) instrument: UV-visual scan instrument.

3) reagent: hydroxyproline (import packing);

Chloramine-T;

Right-dimethylaminobenzaldehyde;

Cross chloric acid;

Citrate buffer solution, pH 6

Test preparation: phycocyanin extract

Embodiment 1

Experimental technique

1) animal is handled:

With experiment mice, random packet, 10 every group.Remove the back hair with depilatory, make to expose about 1cm ²Skin.Medication spreads upon the depilation district, smears every day twice.Put to death animal after 28 days and draw materials, half gives over to histological observation; Dry after second half defat, do hydroxyproline determination.

2) sample treatment:

Get above-mentioned reagent sample of accurately weighing and put in the 10ml tool plug scale test tube, add 6NHCl2.0ml, mixing, close plug, put in the baking oven in 105 ℃ of acidolysis 18 hours, take out and put to room temperature, add 10N NaOH 2.0ml (making pH6), filter, the filtrate thin up is sample solution to 5.0ml.

3) determination step:

Standard curve: accurately take by weighing the hydroxyproline standard substance 50.0mg that is dried to constant weight, with 0.01N HCl dissolving and be diluted to 100ml (1ml is equivalent to 500 μ), this is a standard reserving solution.Get above-mentioned standard reserving solution 2.0ml, thin up is standard application liquid to 100ml (1ml is equivalent to 10 μ hydroxyprolines).

Get 10 μ g/ml titer 0,0.2,0.4,0.6,0.8 and 1.0ml, put respectively in the 10ml tool plug scale test tube, water is adjusted to 1.0ml with each pipe volume, respectively add citrate buffer solution 1.0ml and chlorine ammonia-T liquid 1.0ml again, mixing was placed 6 minutes, in each pipe, added chloric acid liquid 1.0ml again, mixing was placed 5 minutes, and each pipe adds P-DMAB liquid 1.0ml, mixing, 75 ℃ of water-baths were placed 10 minutes, were cooled to room temperature rapidly, and water adjustment cumulative volume is 5.0ml.Do blank with first pipe, measure trap in the 562nm wavelength, (μ g) is abscissa with hydroxyproline content, and trap is a vertical coordinate drawing standard curve.

Sample determination: accurately measure sample solution 200 μ l, to tool plug scale in vitro, add water to 1.0ml after, by the same operational approach of above-mentioned preparation standard curve, the trap of working sample solution, hydroxyproline content from the standard curve calculation sample again.Can obtain the percentage composition (is 13% in hydroxyproline content in the collagen protein) of hydroxyproline and collagen protein in experimental group and the control animals skin thus.

Experimental result: experimental result is shown in table 1 with the concentration of the hydroxyproline in every milligram of dry skin, and institute's column data is with every group of 10 meansigma methodss that animal is measured in the table.

Table 1 is respectively organized hydroxyproline content in the skin

	????Conc．／mg(±SD)	????conc．／mg(±SD)
			Group	For the first time	For the second time
The blank group	????0．0547±0．010
			Negative control group	????0．0739±0．015
Positive controls	????0．0759±0．017
			The Phc experimental group	????0．1000±0．023

Annotate: 1. the blank group is a normal group;

2. negative control group does not contain the medicine composition for only smearing the substrate of experimental group;

3. positive controls is for smearing the SOD sunscreen cream;

4.Phc experimental group is to smear test reagent and the prepared sample of substrate.

Experimental result shows that the hydroxyproline content of experimental group is higher than blank group and positive controls far away, adds up through biology to have very evident difference.

The histological observation result:

Get each fritter of skin of back of blank group, positive controls and three groups of experiment mices of experimental group, make the paraffin serial section, carry out microscopic examination after H.E. dyeing, the result shows:

(1) the experimental group cuticular layer is thinner than the cuticular layer of two matched groups, especially compares more obvious with the blank group.This explanation phycocyanin extract can promote the cutin degraded to make the attenuation of keratinization layer, and has certain function in delaying senility.

(2) volume of experimental group epidermal layer cells obviously increases, and Cytoplasm is abundant, and chromatin is slightly and many in the nuclear, and cell is educated well, and cell quantity also has increase, and based on stratum germinativum, these metabolism that cell all has been described are bred in order.

(3) the skin corium collagen fiber of experimental group are parallel and interlaced arrangement, and fibrocyte increases, and illustrate that collagenic supersession upgrades in order, and this has certain effect to strengthening skin resistance and elasticity.

Claims (10)

1. the antiviral composition that contains phycocyanin extract wherein contains acceptable carrier on the phycocyanin extract for the treatment of effective dose and/or the materia medica, and wherein phycocyanin extract makes with the following step:

A. the blue-green alge powder is dissolved in the 5-20 times of water, low temperature kept 8-30 hour,

B. solid-liquid separation transfers to acidity with the pH value of solution, places,

C. collecting the gained solid is the phycocyanin crude product.

2. according to the compositions of claim 1, wherein said algae powder is selected from Spirulina powder.

3. according to the compositions of claim 1, wherein among the step a consumption of water be the algae powder 8-15 doubly.

4. according to the compositions of claim 3, wherein the consumption of water is 10 times of algae powder among the step a.

5. according to the compositions of claim 1, wherein the pH value of solution is adjusted to 3.8-4.2 among the step b.

6. the preparation method of phycocyanin extract, comprising step:

A. the blue-green alge powder is dissolved in the 5-20 times of water, low temperature kept 8-30 hour,

B. solid-liquid separation transfers to acidity with the pH value of solution, places,

C. collecting the gained solid is the phycocyanin crude product.

7. according to the compositions of claim 6, wherein the algae powder is selected from Spirulina powder.

8. according to the compositions of claim 6, wherein among the step a consumption of water be the algae powder 8-15 doubly.

9. compositions according to Claim 8, wherein the consumption of water is 10 times of algae powder among the step a.

10. according to the compositions of claim 6, wherein the pH value of solution is adjusted to 3.8-4.2 among the step b.

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