CN1297568C - Echinacea polysaccharide, and its preparing method and use for preparing medicines for treating antivirus and antibacterial, especially anti SARS - Google Patents
Echinacea polysaccharide, and its preparing method and use for preparing medicines for treating antivirus and antibacterial, especially anti SARS Download PDFInfo
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Abstract
The present invention discloses pinecone chrysanthemum polysaccharide, a preparing method thereof, a medicinal preparation containing the pinecone chrysanthemum polysaccharide and an application of the medicinal preparation to the preparation of medicines for treating various inflammation, promoting immunity and resisting viruses, bacteria and SARS particularly. The pinecone chrysanthemum polysaccharide is extracted from pinecone chrysanthemum as raw material, the content of total sugar is from 75 to 95%, and the average molecular weight is between 30000 and 80000. Monosaccharide in the total sugar comprises 30 to 75% of glucose, 10 to 60% of fructose and 5 to 20% of arabinose. The preparing method comprises: pinecone chrysanthemum is boiled in water and precipitated by alcohol, and concentrated extract solution after the water boiling of the pinecone chrysanthemum is dissolved in a polar solvent to removing protein and other impurities; after the polar solvent is removed, alcohol is used for precipitation, and precipitates are collected; the precipitates are separated to obtain the pinecone chrysanthemum polysaccharide with the molecular weight of 3000 to 130000, and power of the pinecone chrysanthemum polysaccharide can be obtained through further treatment. The pinecone chrysanthemum polysaccharide does not contain other impurities and is directly prepared into medicines, so that the dosage is greatly reduced, the therapeutic effect is specific, and no toxic or side effect exists. The product quality is stable and is easy to control.
Description
Technical field
The present invention relates to a kind of Echinacea purpurea Moench polysaccharide, its preparation method, contain pharmaceutical preparation of Echinacea purpurea Moench polysaccharide and uses thereof, specifically, the present invention relates to Echinacea purpurea Moench polysaccharide, its preparation method that extracts by Echinacea purpurea Moench, the pharmaceutical preparation that contains the Echinacea purpurea Moench polysaccharide and preparing the various inflammation of treatment, promotion immunologic function, antiviral and antibiotic, purposes on the particularly anti-SARS medicine belongs to pharmaceutical field.
Background technology
Echinacea purpurea Moench is a composite family Compositae Echinacea Echinacea plant, is distributed in North America, and this platymiscium has 9 kinds approximately, and main medicinal kind is narrow leaf Echinacea purpurea Moench E.angustifolia, pale reddish brown Echinacea purpurea Moench E.purpurea and pale purple Echinacea purpurea Moench E.pallida.
Echinacea purpurea Moench is the plant amedica that external especially Europe is widely used for treating various inflammation and promotes immunologic function, its pharmacological action comprises immune enhancement, anti-inflammatory, antiviral and anti-microbial effect etc., therepic use is very extensive, and curative effect has obtained affirming of increasing clinician.
Preparation from the juice of Echinacea purpurea Moench is made shows through external and experimentation on animals, can increase the activity of granulocyte and scavenger cell.Contained alkylamide in the Echinacea purpurea Moench, alkenone, chicoric acid can strengthen granulocytic phagolysis.Polyose can strengthen the phagolysis of scavenger cell.1985, Stuppner found in experiment in vitro: juice (103～105mgmL of the cryodesiccated pale reddish brown Echinacea purpurea Moench of different concns
-1), all show tangible phagolysis enhanced activity, but when greater concn, show as immunosuppression and cytotoxicity.
Bibliographical information Echinacea purpurea Moench alcohol extract stimulates scavenger cell to produce a large amount of immunopotentiation factors, as tumour necrosis factor, and Interferon, rabbit, interleukin I, II, VI, X.
Unidasa is a microorganism secretion, can decompose hyaluronic acid, and tissue permeability is increased, and is easier to toxic product and organizes diffusion towards periphery.The coffic acid derivative can suppress Unidasa, keeps the structure and the integrity of reticular tissue and intercellular stroma thereof.J Jakupovic, R X Tan, F Bohlmann be at Pharmacol Res Commun, 1988,20 (Suppl, 5): reported in 87 that Echinacea purpurea Moench can suppress oedema, reduced inflammatory cell infiltration.Alkylamide can suppress cyclo-oxygenase, and synthesizing of blocking-up prostaglandin(PG) eases the pain, the effect of heating and inflammation.
Fresh pressure juice of Echinacea purpurea Moench over-ground part and root water extract show antiviral activity, and the virus that is suppressed in cell cultures has influenza virus, simplexvirus etc.Its mechanism of action is relevant with immunostimulation and antihyaluronidase.The direct anti-microbial activity of Echinacea purpurea Moench as mild as a dove.But Echinacea purpurea Moench is used for inside and outside infectation of bacteria effectively long history is arranged.Its clinical antibacterial effect, mechanism of action may be because its immunostimulatory activity.
In the prior art, it is directly medicinal or slightly carried and obtain the liposoluble part through pure and mild polar solvent by the Echinacea purpurea Moench raw material that the Echinacea purpurea Moench preparation is mainly crude drug in whole.In " CHINA JOURNAL OF CHINESE MATERIA MEDICA " 27 (5), main component is the mixture that unsaturated fatty acids acid amides, beta-unsaturated ketone, coffic acid derivative, sesquiterpenoids, alkaloids and other chemical ingredientss are formed in the Echinacea purpurea Moench extract, and C according to people such as Li Jiren
10-23Alkane, polyyne class, plant sterol, flavonoid and polyose.Wherein be mainly the unsaturated acyl amine, phenylethyl alcohol glycoside and polysaccharide, but the former two's content is less, and deterioration by oxidation very easily.
The medicinal compositions that contains Echinacea purpurea Moench is also disclosed in the prior art, as Chinese patent application 97195836 and WO/11778.
Up to the present, also do not have monomer of Echinacea purpurea Moench or chemical effective constituent directly to be used as the report of medicine with treatment.Extract polysaccharide and, there is no report especially at home and abroad with Echinacea purpurea Moench directly as medicinal.
Summary of the invention
The object of the present invention is to provide a kind of Echinacea purpurea Moench polysaccharide, is the main active polysaccharide composition that extracts from Echinacea purpurea Moench, directly is used as medicine with polysaccharide, has no side effect, and dose significantly reduces, and the curative effect of medicine is clear and definite, and is applied widely.
Another object of the present invention is to provide a kind of Echinacea purpurea Moench polyose medicinal composition, this pharmaceutical composition contains the extract of Echinacea purpurea Moench---Echinacea purpurea Moench polysaccharide, and safe, inexpensive, the drug composition effective of pharmaceutically acceptable other auxiliary materials, various pharmaceutical dosage forms.
A further object of the present invention is to provide the preparation method of above-mentioned Echinacea purpurea Moench polysaccharide, Echinacea purpurea Moench polyose medicinal composition, this method is simple, effectively, be suitable for suitability for industrialized production, constant product quality, be easy to control.
The present invention also has a purpose to be to provide Echinacea purpurea Moench polyoses extract, the Echinacea purpurea Moench polysaccharide medicine purposes on preparation treatment disease of immune system, anti-inflammatory, antiviral and antibacterials.
In addition, the present invention also provides the Echinacea purpurea Moench polysaccharide in the various inflammation of preparation treatment, promotion immunologic function, antiviral and antibiotic, the purposes on the particularly anti-SARS medicine.
To achieve these goals, the technical solution used in the present invention is: a kind of Echinacea purpurea Moench polysaccharide is that the Echinacea purpurea Moench raw material obtains through extraction, and its total sugar content is 75-95wt%, and molecular-weight average is between 3000-130000.
Preferred total sugar content is 75-85wt%.
The composition of monose and content are in its total reducing sugar: glucose 30-75wt%, fructose 10%-60wt%, pectinose 5%-20wt%.Preferred molecular-weight average is between the 3-8 ten thousand.
Echinacea purpurea Moench polysaccharide odorless of the present invention, no sweet taste, its solid be canescence to pale yellow powder, be soluble in hot water, be insoluble to ethanol.
Described Echinacea purpurea Moench polyose medicinal composition contains Echinacea purpurea Moench polysaccharide of the present invention and acceptable accessories.
In the Echinacea purpurea Moench polyose medicinal composition, the content of Echinacea purpurea Moench polysaccharide is 0.05-90wt%.
Described Echinacea purpurea Moench polyose medicinal composition also comprises at least a medicines such as antiviral, antibiotic or immunological reagent that are selected from.Include but not limited to: penicillin and derivative thereof, cephalosporin and derivative thereof, erythromycin and derivative thereof, antibacterial Chinese herbal medicines extract, ribavirin, vidarabine, acyclovir, valacyclovir, ganciclovir, trifluridine, Sorivudine, lamivudine, phosphine formic acid, Moroxydine, amantadine etc.
The preparation method of Echinacea purpurea Moench polysaccharide of the present invention is included as poach, alcohol precipitation and refining.Be specially:
Method A:
(1) with Echinacea purpurea Moench poach, concentrated, gets concentrated extracting solution, add the polar solvent precipitation, go precipitation and eliminate solvent, obtain the Echinacea purpurea Moench concentrated extracting solution of Deproteinization;
(2) the Echinacea purpurea Moench concentrated extracting solution that obtains to above-mentioned steps (1) adds alcohol, and precipitation is abandoned clear liquid, collects Echinacea purpurea Moench polysaccharide first lees;
(3) Echinacea purpurea Moench polysaccharide first lees is through the refining thick liquid of Echinacea purpurea Moench polysaccharide that obtains molecular weight 3000-130000.
Perhaps method B:
(1) with Echinacea purpurea Moench poach, concentrated, gets concentrated extracting solution, add the alcohol precipitation, the collecting precipitation thing;
(2) throw out that is dissolved in water adds polar solvent precipitation albumen wherein, filters and removes sedimentary albumen, and eliminate polar solvent, obtains the concentrated extracting solution behind the Deproteinization;
(3) concentrated extracting solution behind the Deproteinization is through the refining thick liquid of Echinacea purpurea Moench polysaccharide that obtains molecular-weight average 3000-130000.
The thick liquid of above-mentioned Echinacea purpurea Moench polysaccharide obtains the Echinacea purpurea Moench polysaccharide through further handling.
Among the method A, the poach of Echinacea purpurea Moench comprises Echinacea purpurea Moench decocted in distilled water, treated extracting solution supernatant liquor; The supernatant liquor of concentrated extracting solution obtains concentrated extracting solution.
The Echinacea purpurea Moench here is the Echinacea purpurea Moench herb, comprises narrow leaf Echinacea purpurea Moench E.angustifolia, pale reddish brown Echinacea purpurea Moench E.purpurea or pale purple Echinacea purpurea Moench E.pallida etc.
Described Echinacea purpurea Moench herb also can be treated Echinacea purpurea Moench herb, as its segment or make meal, is beneficial to extract more effectively activeconstituents.
This decoction is carried out under boiling, decoct to extract 1-4 time and fully extracts 1-5 hour at every turn to effective constituent.
Its supernatant liquid is got in being treated to united extraction liquid, filtering, leave standstill after the decoction.
In the decoction process, add the distilled water of 5-20 times of Echinacea purpurea Moench weight.
Wherein, the supernatant liquor of described simmer down to concentrated extracting solution is to the 1/2-1/5 of original volume; Concentrate and can adopt any method of the prior art, as heat concentrated, concentrating under reduced pressure, thin film evaporation concentrates.
Among the present invention, preferably adopt thin film evaporation to concentrate down at 50-70 ℃.
Then, in concentrated extracting solution, add the polar solvent of 1-5 times of volume of concentrated extracting solution, make protein and other insoluble contamination precipitations in polar solvent in the concentrated extracting solution.
Described polar solvent comprises for dissolving Echinacea purpurea Moench polysaccharide of the present invention and polar solvent that can protein precipitation: a kind of in chloroform, methylene dichloride, ether, sherwood oil, ethyl acetate, THF, the propyl carbinol or at least two kinds mixture wherein.
The preferred two kinds of mixed solvents that mix that are selected from chloroform, methylene dichloride and THF, the propyl carbinol that adopt, the volume ratio of two kinds of solvents is 2: 1-5: 1;
More preferably adopt the mixed solvent of chloroform and propyl carbinol, chloroform is 3-5 a times of propyl carbinol volume.
Above-mentioned precipitation is under agitation carried out, and fully post precipitation is by filtering or sedimentary albumen is removed in centrifugation, and the reclaim under reduced pressure polar solvent eliminates fully to the polarity solvent;
Adopt this method, can remove protein in the Echinacea purpurea Moench and the insolubles in other polar solvents, this sedimentary albumen can utilize by further extracting albumen processing again.
This precipitation is removed the process of albumen and other impurity, also can carry out on the isothermal vibration device.
In removing proteic concentrated extracting solution part, add alcohol, stir, filter or centrifugal collecting precipitate to be settled out Echinacea purpurea Moench polysaccharide precipitation thing.Also can solution mixture be left standstill 5-30 hour earlier, inclining supernatant liquid, again by filtering or centrifugal collecting precipitate.
Wherein the used alcohol of alcohol precipitation is methyl alcohol, ethanol or propyl alcohol; Generally in removing proteic concentrated extracting solution, add alcohol and make that the determining alcohol of mixed solution is 60-80wt%.The consumption of general alcohol is a 1-5 times of volume of concentrated solution, and certainly, those skilled in the art can be according to the consumption of undissolved concentrated extracting solution amount appropriate change alcohol partly.By alcohol precipitation, can further remove albumen and other impurity, purified product.
The ethanol sedimentation of preferred employing 75%-95% is removed albumen and other impurity in the proteic concentrated extracting solution among the present invention.
Wherein, in the treating process, earlier thick Echinacea purpurea Moench polysaccharide precipitation thing is dissolved in the Echinacea purpurea Moench amount 0.5-3 50-85 ℃ of distilled water doubly, while hot behind the filtering insolubles, method with chromatography, dialysis or ultrafiltration is separated, remove small molecular weight impurity, obtain the Echinacea purpurea Moench polysaccharide fraction of molecular-weight average, the Echinacea purpurea Moench polysaccharide fraction of preferred molecular-weight average 30,000-80,000 at 3000-130000.
The preferred method of dialysis that adopts is refining among the present invention, obtains the aqueous solution of Echinacea purpurea Moench polysaccharide.
The aqueous solution of above-mentioned Echinacea purpurea Moench polysaccharide obtains the Echinacea purpurea Moench polysaccharide through super-dry, and described dry employing method commonly known in the art is carried out, as heat drying, lyophilize, spraying drying etc.
Above-mentioned Echinacea purpurea Moench polysaccharide, Echinacea purpurea Moench polysaccharide solution or contain the pharmaceutical composition of this Echinacea purpurea Moench polysaccharide, can adopt the prior art known method to make to contain the pharmaceutical composition of Echinacea purpurea Moench polyoses extract or Echinacea purpurea Moench polysaccharide, and acceptable forms on any pharmaceutics, as pharmaceutical compositions such as granule, oral liquid, capsule, tablet, injection, powder pin etc.
Among the present invention, also comprise adding the alcohol precipitation in the Echinacea purpurea Moench polyoses extract solution that after refining, obtains again, abandon clear liquid, collect the Echinacea purpurea Moench polyoses extract; Described alcohol is methyl alcohol, ethanol or propyl alcohol, and the add-on of general alcohol is 0.6-1.5 a times of Echinacea purpurea Moench amount, preferred alcohol.If desired, can also obtain the powder of Echinacea purpurea Moench polyoses extract with the Echinacea purpurea Moench polysaccharide that is settled out through freezing, normal temperature or heat drying.
The Echinacea purpurea Moench polyoses extract odorless that adopts the inventive method to obtain, no sweet taste, for canescence to pale yellow powder, be soluble in hot water, be insoluble to ethanol, molecular-weight average is between 30,000-80,000, total sugar content is 75-95%, its monose consists of glucose 30-75%, fructose 10%-60%, pectinose 5%-20%.Can make said formulation on any pharmaceutics.
One of the present invention preferred embodiment may further comprise the steps:
1) get the Echinacea purpurea Moench herb, after treatment, the distilled water that adds 5-20 times of weight of Echinacea purpurea Moench medicinal material decocts, extracts 2-4 time after, abandon precipitation, the merging decoction liquor; Filter the decoction liquor that merges, get its supernatant liquid after leaving standstill, under 50-70 ℃, be concentrated into 1/3 of original volume with thin film evaporation;
2) add 4-5 chloroform-n-butanol mixed solvent doubly in concentrated extracting solution, mixing discards the albumen that is settled out, and reclaims solvent;
3) behind the removal albumen, add 1-5 and doubly measure alcohol, make that the mixed solution determining alcohol is 60-80wt%, after stirring, the precipitation polysaccharide leaves standstill hypsokinesis in 12-30 hour and goes out supernatant liquid, centrifugal collecting precipitation;
4) add 2-10 to precipitation and doubly measure distilled water, be heated to 60-90 ℃ and make resolution of precipitate, filtered while hot, filtrate is removed small molecular weight impurity through the dialysis of flowing, and obtains the Echinacea purpurea Moench polysaccharide fraction of molecular-weight average at 3000-130000.
Preferred preparation method of the present invention also comprises: stir down, partly to add alcohol be 80% to containing the alcohol amount to debris in film, leaves standstill and separate out Echinacea purpurea Moench polysaccharide precipitation thing.
Wherein said being extracted as 2-4 time, each extraction time is 1-3 hour.After the extraction, each extracting solution merges.When actually operating, common way is poach 3 times, and each time extraction time was respectively 2 hours, 2 hours, 1 hour.
Step 2) described extracting liquid filtering can use filter cloth to carry out, and the filter cloth specification is generally the 100-200 order.In the chloroform-propyl carbinol mixed solution that uses during the Deproteinization operation, the consumption of chloroform and propyl carbinol is 4: 1 a volume ratio.Deproteinization mixing under agitation or on constant temperature oscillator, preferably on constant temperature oscillator with 190r/min jolting 20min.
The alcohol that uses in the preparation process can be with containing the ethanol of alcohol amount as 75-95%, and described dialysis can be used tap water or deionized water.
Among the preparation method B of the present invention, can be with reference to the raw material among the preparation method A, consumption and treating processes, but concrete operating procedure process and order are slightly different.Here only introducing preferred preparation process is:
(1) extract: get the Echinacea purpurea Moench herb, the distilled water with 5-20 times of weight of Echinacea purpurea Moench medicinal material decocts after treatment, extracts 2-4 time after, abandon precipitation, the merging decoction liquor; Filter the decoction liquor after merging, get its supernatant liquid after leaving standstill, thin film evaporation is concentrated into the 1/2-1/5 of original volume under the heating; This technology is consistent with corresponding condition among the preparation method A.The temperature of thin film evaporation is 50-70 ℃.
(2) alcohol precipitation: stir the alcohol that in the Echinacea purpurea Moench concentrated extracting solution, adds 1-5 times down, the Echinacea purpurea Moench polysaccharide precipitation in the Echinacea purpurea Moench concentrated extracting solution is gone out, leave standstill, abandon supernatant liquor, centrifugal collection Echinacea purpurea Moench polysaccharide first lees;
(3) Deproteinization: in Echinacea purpurea Moench polysaccharide first lees, add 0.5-5 times of distilled water, and make its dissolving, under agitation to mixing solutions that wherein to add 1-5 times of volume ratio be chloroform-propyl carbinol of 4: 1, filter and remove albumen and other impurity that is settled out, organic solvent in the Echinacea purpurea Moench extracting solution behind the recovery removal albumen, to eliminating organic solvent, obtain removing proteic Echinacea purpurea Moench extracting solution.
(4) refining: refining with the mobile dialysis of deionized water, to remove inorganic salt and small-molecule substance, obtaining molecular-weight average is the thick liquid of Echinacea purpurea Moench polysaccharide of 3-8 ten thousand.
Among the above-mentioned preparation method B, the kind of the time of decoction, number of times, alcohol or polar solvent and consumption, and other processing parameters can be with reference to disclosed content among the preparation method A.
If desired, the thick liquid alcohol precipitation of this Echinacea purpurea Moench polysaccharide further can be made with extra care.
In one embodiment of the invention, adopt following preparation of compositions Echinacea purpurea Moench polyoses oral liquid:
Echinacea purpurea Moench polysaccharide: 10-40g
Distilled water: 500-1000ml
Honey: 70-120ml
Sanitas: 0.1-0.2g
Said components is mixed, filter, bottling, sterilization.
Equally, be raw material with the Echinacea purpurea Moench polyoses extract, can prepare the medicinal compositions and the various formulation of various Echinacea purpurea Moench polyoses extracts with reference to the method for prior art pharmaceutics.
Echinacea purpurea Moench polysaccharide suggestion consumption of the present invention is generally 0.01-0.05g/kg body weight every day.Can effectively treat flu, burn infection, streptococcal infection, infection of staphylococcus aureus, urinary tract infection, upper respiratory tract infection, trachitis, bleb, Whooping cough, leukopenia, rheumatoid arthritis, have a toothache and oral cavity gingival infection and snakebite etc., can also be used for the assisting therapy of chemotherapy.
The present invention adopts the effective constituent Echinacea purpurea Moench polysaccharide in the sedimentary method system extraction of the poach alcohol Echinacea purpurea Moench, in the leaching process, adopts albumen and other impurity in the former medicine of removal of solvents Echinacea purpurea Moench; The sedimentary process of alcohol can further purify and purify, and by separating, obtains the Echinacea purpurea Moench polysaccharide of purified certain molecular weight, and after testing, the total sugar content height does not contain other impurity, has avoided because the toxicity, side effect that albumen and impurity cause.Because product purity improves greatly, dose can significantly reduce relatively, and the curative effect of medicine is clear and definite more and be improved, and constant product quality, is easy to control.The toxicity test and the effect experiment result of the Echinacea purpurea Moench polysaccharide of employing the inventive method preparation are as follows:
1, toxicity is observed:
Get 64 of Kunming mouses, random packet, employing is taken and is not taken the Echinacea purpurea Moench polysaccharide and carries out toxicity contrast experiment research, the result shows, Echinacea purpurea Moench polysaccharide group is under the dosage up to 500mg/kg, without any side effects, by all no abnormal to the heart, liver, kidney, lung anatomic observation, and body weight all has increase.Experimental result sees Table 1.
Table 1: the toxicity test of Echinacea purpurea Moench polysaccharide
| The medicine group | Weight increase (g) | The heart | Liver | Kidney |
| Echinacea purpurea Moench polysaccharide (5mg/kg) Echinacea purpurea Moench polysaccharide (50mg/kg) Echinacea purpurea Moench polysaccharide (500mg/kg) normal control group | 4.2±2.4 5.4±3.2 5.6±2.7 4.2±2.6 | No abnormality seen no abnormality seen no abnormality seen no abnormality seen | No abnormality seen no abnormality seen no abnormality seen no abnormality seen | No abnormality seen no abnormality seen no abnormality seen no abnormality seen |
2, immunization experiment research:
Get the mouse spleen lymphocyte of fresh separated, adjustment concentration is 1 * 10y/ml, get 96 porocyte culture plates, every hole adds cell suspension 100uL/ hole, add stimulant ConA100ul/ hole and sample 100ul/ hole according to experimental design again, each sample is established 3 parallel holes, and is provided with control wells (only adding RPMT1640), and every hole cumulative volume is 200ul.Then culture plate is placed 5%CO
2Cultivated 48 hours in the incubator, take out culture plate, every hole is inhaled and is abandoned 100ul, each hole adds 5mg/mlMTT10ul, continues to cultivate 4 hours, takes out culture plate, each hole adds 10%SDS lysate 100ul, places in the incubator and spends the night, and surveys the OD value at 570nm place on the enzyme linked immunological instrument.Experimental result sees Table 2.
Table 2
| Medicine ug/ml | The OD value |
| Blank stimulant ConA 0.5 (Echinacea purpurea polysaccharide group) 2.5 (Echinacea purpurea polysaccharide group) 5.0 (Echinacea purpurea polysaccharide groups) 50 (Echinacea purpurea polysaccharide group) 100 (Echinacea purpurea polysaccharide group) | 0.06±0.04 0.18±0.07 0.28±0.05 *With the contrast of ConA group, each group has significant difference, 0.32 ± 0.07 * P＜0.01 0.35±0.07 * 0.43±0.04 * 0.41±0.06 * |
Above-mentioned experimental result shows that Echinacea purpurea Moench polysaccharide of the present invention has significant immunizing power under various dosage.
3. anti-inflammatory action research:
With body weight is that the male mice of 18-22g is divided into 5 groups at random, 10 every group, is respectively: the blank group, Sai Misong group and each dosage group of Echinacea purpurea Moench polysaccharide.Blank group is irritated the sodium carboxymethyl cellulose solution of stomach 0.5%, Sai Misong dosage be 1.5mg/kg.Be mixed with 0.15mg/ml respectively, 4.8mg/ml, 22mg/ml, the solution of 49mg/ml concentration is pressed the dose of 0.1ml/10g and is irritated stomach.Irritate stomach every day 1 time, continuous irrigation stomach 3 days, the last administration after 1 hour equal and quantitative smear dimethylbenzene in mouse right ear, put to death mouse after 1 hour, cut ears, the punch tool of 0.9cm has two ear antimeres to sweep away circular auricle, it is other to calculate its ear method of double differences.Experimental result sees Table 3.
Table 3
| Group | Dosage (mg/kg) | Number of animals | The ear method of double differences is (X ± S) not |
| The Sai Misong group polysaccharide group 1 polysaccharide group 2 polysaccharide groups 3 of blank group | 1.5 48 220 490 | 10 10 10 10 10 | 1.03±0.33 0.78±0.17 * 0.63±0.38 * 0.68±0.20 * 0.60±0.43 * |
Compare with the blank group,
*P＜0.05
Experimental result shows that each dosage group of polysaccharide has anti-inflammatory action.
3. anti-SARS effect research:
Experiment in vitro shows that the SARS virus that Echinacea purpurea Moench polysaccharide of the present invention is cultivated for chick embryo method is inhibited.
The coronavirus genome is RNA, and by respiratory tract, mucous membrane or contact, virus mediates the glycoprotein receptor that is adsorbed in sensitive cells film or cytolemma through acceptor, merges or melts in the film synthetic cell by film and swallow.The coronavirus geneome RNA enters the permissive cell kytoplasm, at first with himself geneome RNA as mRNA as early stage translation, required RNA polymerase is duplicated in generation, RNA polymerase is a template with viral genome+ssRNA more then, transcribe the generation strand RNA, with it is that template is transcribed the generation virus mRNA, forms progeny virus through translation and translation post-treatment packing then, and discharges in the mode of sprouting.The pathophysiological change of clinical symptom mainly is that the polarity cytocidal infection takes place target cell, and is further development of multiple organ failure.After Echinacea purpurea Moench polysaccharide of the present invention or its composition enter human body, be adsorbed in the glycoprotein receptor of sensitive cells film or cytolemma, suppress the synthetic of virosome nucleic acid, simultaneously, Echinacea purpurea Moench polysaccharide of the present invention has good bactericidal antiphlogistic effect and significant immunizing power, the complication and the superinfection that can prevent and effectively control SARS to cause, the treatment level that helps treating SARS reduces the acute death rate of severe acute respiratory syndrome simultaneously.
Embodiment
Embodiment 1
Get pale reddish brown Echinacea purpurea Moench herb 1kg, make meal, with 10kg distilled water reflux, extract 3 times, first, second time respectively extracted 2 hours, extracted for the third time 1 hour, merge No. three times extracting solution, filter extracting solution, get supernatant liquid after filtrate is left standstill with 200 order double-layer filter cloths, use thin film evaporation to be concentrated into 10kg down at 60 ℃, add the mixing solutions that the 30L volume ratio is chloroform-propyl carbinol of 4: 1, on constant temperature oscillator (TH2-82), with 190r/min jolting 20min, filter and remove albumen and the impurity that is settled out, obtain the Echinacea purpurea Moench concentrated extracting solution of Deproteinization; Organic solvent in the reclaim under reduced pressure Echinacea purpurea Moench concentrated extracting solution is to eliminating organic solvent; Stir that to add ethanol (95%) to mixed solution determining alcohol down in the Echinacea purpurea Moench concentrated extracting solution be 80%, the Echinacea purpurea Moench polysaccharide precipitation in the Echinacea purpurea Moench concentrated extracting solution gone out, leave standstill 24 hours after, abandon supernatant liquid, centrifugal collection Echinacea purpurea Moench polysaccharide first lees 345g.In Echinacea purpurea Moench polysaccharide first lees, add 1 liter of distilled water, be heated to 80 ℃ of dissolvings, use the filter paper filtered while hot to go out insolubles, filtrate is flowed to dialyse through deionized water and is made with extra care, to remove inorganic salt and small-molecule substance, obtaining molecular-weight average is the thick liquid of Echinacea purpurea Moench polysaccharide of 5-7 ten thousand.The thick liquid of this Echinacea purpurea Moench polysaccharide can further be prepared as various pharmaceutically acceptable compositions and various formulation.
If desired, the thick liquid of Echinacea purpurea Moench polysaccharide that obtains further alcohol precipitation is refining, method is: under agitation add 0.8 liter or an amount of ethanol (95%) again, making determining alcohol is 80%, leave standstill and separate out precipitation, abandon clear liquid, use freeze drier, get Echinacea purpurea Moench polysaccharide solid 60g in subzero 20 ℃ of lyophilizes.
Gained Echinacea purpurea Moench polysaccharide odorless, no sweet taste, color is yellowish, and molecular-weight average is 70,000, total sugar content 80%, wherein glucose is 61.1%, and fructose is 28.9%, and pectinose is 9.9%.
Embodiment 2
Get pale reddish brown Echinacea purpurea Moench herb 5kg, segment, extract with 25kg distilled water heated and boiled, extracting 5 effective constituents to the Echinacea purpurea Moench carries to the greatest extent substantially, filtering extracting solution goes residual, united extraction liquid, filter extracting solution with 100 order double-layer filter cloths, after leaving standstill, filtrate gets supernatant liquid, under 50 ℃, be evaporated to 25kg, obtain the Echinacea purpurea Moench concentrated extracting solution,, mixed 2 hours to wherein adding the 25L sherwood oil, albumen and other impurity that is settled out are removed in centrifugation, the reclaim under reduced pressure organic solvent, adding 75% ethanol to mixed solution determining alcohol under stirring in the Echinacea purpurea Moench concentrated extracting solution of Deproteinization is 60%, leaves standstill the Echinacea purpurea Moench polysaccharide precipitation is separated out, centrifugal collection Echinacea purpurea Moench polysaccharide first lees is abandoned clear liquid.In Echinacea purpurea Moench polysaccharide first lees, add 10L distilled water, be heated to 70 ℃ of dissolvings, leach insolubles while hot, filtrate is refining through ultrafiltration, further remove salt and small molecular weight impurity, obtain molecular weight for (greater than not all right, may be very big, be uncertain, can be adjusted into a scope according to embodiment) the thick liquid 1.5kg of heavy-gravity Echinacea purpurea Moench polysaccharide of 3-5 ten thousand, its total sugar content is 85%, glucose is 71.4%, fructose is 13.3%, and pectinose is 15.3%.
Embodiment 3
Get pale purple Echinacea purpurea Moench herb 1kg and pulverize or directly use herb, adding 20kg distilled water heated and boiled extracts, effective constituent to Echinacea purpurea Moench is carried to the greatest extent substantially, filter extracting solution and remove residue, after leaving standstill, filtrate gets supernatant liquid, be concentrated into 70 ℃ of following thin film evaporation and obtain the 4kg concentrated extracting solution, stir down, adding 20L methylene dichloride and ethyl acetate volume ratio are 3: 1 mixing solutions in concentrated extracting solution, fully be settled out to albumen and other impurity, left standstill 10 hours, and filtered precipitations such as removing albumen and impurity, reclaim under reduced pressure organic solvent, obtain the Echinacea purpurea Moench concentrated extracting solution of Deproteinization, adding 75% methyl alcohol mixed liquor determining alcohol under stirring in the Echinacea purpurea Moench concentrated extracting solution of Deproteinization is 70%, leaves standstill Echinacea purpurea Moench polysaccharide first lees is separated out centrifugal collection Echinacea purpurea Moench polysaccharide first lees after fully stirring.Add 0.5 liter of distilled water in Echinacea purpurea Moench polysaccharide first lees, be heated to 60 ℃ of dissolvings, leach insolubles while hot, filtrate is removed small molecular weight impurity through ultrafiltration, and obtaining molecular weight is the thick liquid of Echinacea purpurea Moench polysaccharide of 6-8 ten thousand.The ethanol that under agitation adds 1.5 liter 75% in the thick liquid of Echinacea purpurea Moench polysaccharide is again separated out precipitation after leaving standstill, and filtration collecting precipitation, Air drying get faint yellow odorless, tasteless Echinacea purpurea Moench polysaccharide powder 200g.Its total sugar content is 75%, and glucose is 36.3%, and fructose is 50.6%, and pectinose is 13.1%.
Embodiment 4
Get narrow leaf Echinacea purpurea Moench herb 1kg, reference example 1 described method is extracted 3 times, filtration, concentrated extracting solution, add the 40L ethyl acetate, under stirring albumen and other impurity fully are settled out, ultrafiltration gets the heavy-gravity Echinacea purpurea Moench polysaccharide 150g that molecular weight is 3000---30000, obtain odorless, tasteless, faint yellow Echinacea purpurea Moench polysaccharide powder through lyophilize, the total reducing sugar amount is 92%, and wherein glucose is 44.5%, fructose is 46.4%, and pectinose is 8.9%.
Embodiment 5
Get and mix Echinacea purpurea Moench herb 1kg, make meal,, extract 3 times with 15kg distilled water reflux, each extraction time was respectively 3,2,1 hours, merge No. three times extracting solution, cross the elimination residue, get supernatant liquid after filtrate is left standstill, filter extracting solution with 150 order double-layer filter cloths, get supernatant liquid after filtrate is left standstill, use thin film evaporation to be concentrated into 9kg down, obtain the concentrated extracting solution of Echinacea purpurea Moench at 65 ℃; Stir the ethanol that in the concentrated extracting solution of Echinacea purpurea Moench, adds 27L75% down, the Echinacea purpurea Moench polysaccharide precipitation in the Echinacea purpurea Moench concentrated extracting solution gone out, leave standstill 24 hours after, abandon supernatant liquid, centrifugal collection Echinacea purpurea Moench polysaccharide first lees; In Echinacea purpurea Moench polysaccharide first lees, add 2 liters of distilled water, be heated to 80 ℃ of dissolvings, and then under agitation to wherein adding the mixing solutions that the 8L volume ratio is chloroform-propyl carbinol of 4: 1, on constant temperature oscillator (TH2-82), speed jolting 30min with 150r/min, filter and remove albumen and other impurity that is settled out, obtain the Echinacea purpurea Moench extracting solution of Deproteinization; Organic solvent in the Echinacea purpurea Moench extracting solution of reclaim under reduced pressure Deproteinization is to eliminating organic solvent; Flow to dialyse with deionized water at last and make with extra care, to remove inorganic salt and small-molecule substance, obtaining molecular-weight average is the thick liquid of Echinacea purpurea Moench polysaccharide of 3.5-7.1 ten thousand.The thick liquid of this Echinacea purpurea Moench polysaccharide can further be prepared as various pharmaceutically acceptable compositions and various formulation.
If desired, the thick liquid of Echinacea purpurea Moench polysaccharide that obtains further alcohol precipitation is refining, and method is: under agitation add 0.8 liter or an amount of ethanol (95%) again, making determining alcohol is 80%, leave standstill and separate out precipitation, abandon clear liquid, the precipitation drying gets Echinacea purpurea Moench polysaccharide solid 100g.
Gained Echinacea purpurea Moench polysaccharide odorless, no sweet taste, color is yellowish, and molecular-weight average is 7.3 ten thousand, total sugar content 86%, wherein glucose is 57.1%, and fructose is 23.5%, and pectinose is 19.3%.
Embodiment 6
Get Echinacea purpurea Moench polysaccharide 1000g, pulverize, sieve, encapsulated, promptly get capsule.
Embodiment 7
Get Echinacea purpurea Moench polysaccharide 250g, add water 200ml, be heated to moltenly, become thick medicinal extract, add sucrose 750g, granulate, drying, packing promptly gets granule.
Embodiment 8
Get Echinacea purpurea Moench polysaccharide 1000g, add water for injection 1500ml, dissolving is filtered, and the filtrate can promptly gets oral liquid.
Embodiment 9
Get Echinacea purpurea Moench polysaccharide 1000g, pulverize, add the 10g Magnesium Stearate, mix, compressing tablet, the bag film-coat, drying promptly gets tablet.
Embodiment 10
Get Echinacea purpurea Moench polysaccharide 500g, add water for injection 1000ml, 70 ℃ of heating for dissolving add the 10g activated carbon, be incubateds 30 minutes, filtration, and can, 100 ℃ of flowing steam sterilizations 15 minutes promptly get injection.
Experimental example
Echinacea purpurea Moench polysaccharide determination method of the present invention is as follows:
1. the solution of the Echinacea purpurea Moench polysaccharide 1ml concentration 10% of embodiment of the invention 1-5 adds phenolsulfuric acid reagent and is orange, adds anthraquinone reagent and is blue-greenish colour, and the polysaccharide that is for preparing is described.
2. stability experiment: adopting the reserved sample observing method, is that 10%, 50%, 80% sample is preserved respectively in 3-5 ℃, 20-25 ℃, 33-37 ℃ thermostat container with concentration, and sampling is every other month observed, appearance luster no change in 1 year, sediment-free.
3. the measuring method of total reducing sugar amount:
Precision takes by weighing D-glucose 0.20g in the 250ml volumetric flask, and adding distil water is diluted to scale, is standard solution.Accurate standard solution 0.5,1,2,3,4, the 5ml of drawing, adding distil water is diluted to 25ml, gets the standard solution of different concns.The accurate standard solution 2ml that draws adds 5% phenol solution 1ml, slowly adds vitriol oil 5ml, shakes up, and put and be chilled to room temperature, be blank with the phenol solution of 1ml 5%, under 490nm, measure optical density, the drawing standard curve.Get the polysaccharide sample then, the method for preparing by reference liquid is mixed with sample liquid, with the method operation, measures optical density, calculates its total sugar content from typical curve.
Claims (10)
1. an Echinacea purpurea Moench polyoses extract is obtained by the extraction of Echinacea purpurea Moench raw material, it is characterized in that in the described polyoses extract, total sugar content is 75-95%, and molecular-weight average is 3000-130000, wherein consisting of of monose: glucose 30-75%, fructose 10%-60%, pectinose 5%-20%.
2. Echinacea purpurea Moench polyoses extract according to claim 1 is characterized in that, molecular-weight average is 3-8 ten thousand.
3. the pharmaceutical composition that contains claim 1 or 2 described Echinacea purpurea Moench polyoses extracts is characterized in that described Echinacea purpurea Moench polyoses extract is aided with acceptable accessories, and wherein the content of Echinacea purpurea Moench polyoses extract is 0.05-90%; Described composition also comprises at least a penicillin, cephalosporin, erythromycin, ribavirin, vidarabine, acyclovir, valacyclovir, ganciclovir, trifluridine, Sorivudine, lamivudine, phosphine formic acid, Moroxydine and the amantadine of being selected from.
4. the preparation method of the described Echinacea purpurea Moench polyoses extract of claim 1 is characterized in that:
Method A:
(1) with Echinacea purpurea Moench poach, concentrated, gets concentrated extracting solution, add the polar solvent precipitation, go precipitation and eliminate solvent, obtain the Echinacea purpurea Moench concentrated extracting solution of Deproteinization;
(2) the Echinacea purpurea Moench concentrated extracting solution to Deproteinization adds alcohol, and precipitation is abandoned clear liquid, collects Echinacea purpurea Moench polyoses extract first lees;
(3) Echinacea purpurea Moench polyoses extract first lees is dissolved in the Echinacea purpurea Moench amount 0.5-3 50-85 ℃ of distilled water doubly, while hot behind the filtering insolubles, method with chromatography, dialysis or ultrafiltration is separated, and removes small molecular weight impurity, and obtaining molecular weight is the thick liquid of Echinacea purpurea Moench polyoses extract of 3000-130000;
(4) the thick liquid drying of Echinacea purpurea Moench polyoses extract obtains the Echinacea purpurea Moench polyoses extract;
Perhaps method B:
(1) with Echinacea purpurea Moench poach, concentrated, gets concentrated extracting solution, add the alcohol precipitation, collection Echinacea purpurea Moench polyoses extract first lees;
(2) the Echinacea purpurea Moench polyoses extract first lees that is dissolved in water adds polar solvent precipitation albumen wherein, filters and removes sedimentary albumen, and eliminate polar solvent, obtains the Echinacea purpurea Moench polyoses extract concentrated extracting solution behind the Deproteinization;
(3) the Echinacea purpurea Moench polyoses extract concentrated extracting solution behind the Deproteinization separates through the method for chromatography, dialysis or ultrafiltration, removes small molecular weight impurity, and obtaining molecular-weight average is the thick liquid of Echinacea purpurea Moench polyoses extract of 3000-130000;
(4) the thick liquid drying of Echinacea purpurea Moench polyoses extract obtains the Echinacea purpurea Moench polyoses extract;
Described polar solvent be for dissolving described Echinacea purpurea Moench polyoses extract and polar solvent that can protein precipitation, is selected from a kind of in chloroform, methylene dichloride, ether, sherwood oil, ethyl acetate, tetrahydrofuran (THF), the propyl carbinol or at least two kinds mixture wherein;
Described alcohol is methyl alcohol, ethanol or propyl alcohol.
5. according to the preparation method of the described Echinacea purpurea Moench polyoses extract of claim 4, it is characterized in that:
Described Echinacea purpurea Moench is Echinacea purpurea Moench herb or treated Echinacea purpurea Moench herb;
The poach of described Echinacea purpurea Moench is that Echinacea purpurea Moench is decocted in distilled water; The distilled water that adds 5-20 times of Echinacea purpurea Moench weight; Decoction is carried out under boiling, decoct to extract 1-4 time and fully extracts 1-5 hour at every turn to effective constituent;
Simmer down among described method A (1) and the B (2) is the supernatant concentration of the extracting solution 1/2-1/5 to original volume, concentrates and adopts that heating concentrates, concentrating under reduced pressure or thin film evaporation concentrate;
Tap water or deionized water are used in described dialysis;
The consumption of described alcohol is a 1-5 times of volume of concentrated solution;
Precipitation among described method A (1) and the B (2) is under agitation carried out, or carries out on the isothermal vibration device;
Described drying is heat drying, lyophilize or spraying drying.
6. according to the preparation method of the described Echinacea purpurea Moench polyoses extract of claim 5, it is characterized in that:
Described polar solvent is by a kind of two kinds of solvent mixtures that mix in a kind of and tetrahydrofuran (THF) in chloroform, the methylene dichloride, the propyl carbinol, and the volume ratio of two kinds of solvents is 2: 1-5: 1;
Described ethanol is the ethanol of 75%-95%;
Described thin film evaporation is concentrated in 50-70 ℃ and carries out.
7. according to the preparation method of the arbitrary described Echinacea purpurea Moench polyoses extract of claim 4-6, it is characterized in that may further comprise the steps among the method A:
(1) get the Echinacea purpurea Moench herb, after treatment, the distilled water that adds 5-20 times of weight of Echinacea purpurea Moench medicinal material decocts, extracts 2-4 time after, abandon precipitation, the merging decoction liquor; Filter the decoction liquor that merges, get its supernatant liquid after leaving standstill, under 50-70 ℃, be concentrated into 1/3 of original volume with thin film evaporation;
(2) add the chloroform of 4-5 times of volume of concentrated extracting solution and the mixed solvent of propyl carbinol in concentrated extracting solution, chloroform is 3-5 a times of propyl carbinol volume, and mixing discards the albumen that is settled out, and reclaims solvent;
(3) behind the removal albumen, the alcohol of concentrated extracting solution 1-5 times volume makes that the mixed solution determining alcohol is 60-80wt% behind the adding Deproteinization, and after stirring, the precipitation polysaccharide leaves standstill hypsokinesis in 12-30 hour and goes out supernatant liquid, centrifugal collecting precipitation;
(4) add Echinacea purpurea Moench amount 2-10 distilled water doubly to precipitation, be heated to 60-90 ℃ and make resolution of precipitate, filtered while hot, filtrate is removed small molecular weight impurity through the dialysis of flowing, and obtaining molecular-weight average is the thick liquid of Echinacea purpurea Moench polyoses extract of 3000-130000;
(5) the spray-dried Echinacea purpurea Moench polyoses extract that obtains of the thick liquid of Echinacea purpurea Moench polyoses extract.
8. according to the preparation method of the arbitrary described Echinacea purpurea Moench polyoses extract of claim 4-6, it is characterized in that may further comprise the steps among the method B:
(1) get the Echinacea purpurea Moench herb, the distilled water with Echinacea purpurea Moench medicinal material 5-20 times of weight decocts after treatment, extracts 2-4 time after, abandon precipitation, the merging decoction liquor; Filter the decoction liquor after merging, get its supernatant liquid after leaving standstill, heating, under 50-70 ℃, thin film evaporation is concentrated into the 1/2-1/5 of original volume;
(2) stir the alcohol that in the Echinacea purpurea Moench concentrated extracting solution, adds 1-5 times of volume of Echinacea purpurea Moench concentrated extracting solution down, the Echinacea purpurea Moench polyoses extract in the Echinacea purpurea Moench concentrated extracting solution is settled out, leave standstill, abandon supernatant liquor, centrifugal collection Echinacea purpurea Moench polyoses extract first lees;
(3) distilled water of 0.5-5 times of weight of adding Echinacea purpurea Moench in Echinacea purpurea Moench polyoses extract first lees, and make its dissolving, the volume ratio that under agitation adds lysate 1-5 times volume is the mixing solutions of chloroform-propyl carbinol of 4: 1, filter and remove albumen and other impurity that is settled out, organic solvent in the Echinacea purpurea Moench extracting solution behind the recovery removal albumen, to eliminating organic solvent, obtain removing proteic Echinacea purpurea Moench extracting solution;
(4) separate with the mobile dialysis of deionized water, remove inorganic salt and small-molecule substance, obtaining molecular-weight average is the thick liquid of Echinacea purpurea Moench polyoses extract of 3-8 ten thousand;
(5) the spray-dried Echinacea purpurea Moench polyoses extract that obtains of the thick liquid of Echinacea purpurea Moench polyoses extract.
9. claim 1 or 2 described Echinacea purpurea Moench polyoses extracts promote application on immunologic function, antiviral, the antibacterials in preparation treatment.
10. the described pharmaceutical composition that contains the Echinacea purpurea Moench polyoses extract of claim 3 promotes purposes on immunologic function, antiviral, the antibacterials in the preparation treatment.
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| DE3217214A1 (en) * | 1982-05-07 | 1983-11-10 | Wagner, Hildebert, Prof. Dr., 8211 Breitbrunn | Polysaccharides obtained from plants of the Compositae family, process for their isolation, and pharmaceutical preparations containing them |
| DE3744345A1 (en) * | 1987-12-28 | 1989-07-06 | Lohmann Rudolf Lomapharm | Pharmaceutical composition for stimulating macrophage activity |
| US4857512A (en) * | 1985-11-27 | 1989-08-15 | Lomapharm, Rudolf Lohmann Gmbh Kg, Pharmazeutische Fabrik | Immunostimulating polysaccharides, method for using such, and pharmaceutical preparations containing them |
| US20020031524A1 (en) * | 2000-08-10 | 2002-03-14 | Kralovec Jaroslav A. | Chlorella preparations exhibiting immunomodulating properties |
| CN1376068A (en) * | 1999-09-30 | 2002-10-23 | 要素的研究与开发技术有限公司 | Echinacea supplement and method of manufacture |
| CN1424904A (en) * | 2000-03-06 | 2003-06-18 | 荷兰联合利华有限公司 | Echinacea extract as anti-irritant and anti-aging booster in cosmetic compositions |
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2003
- 2003-08-08 CN CNB031532314A patent/CN1297568C/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3217214A1 (en) * | 1982-05-07 | 1983-11-10 | Wagner, Hildebert, Prof. Dr., 8211 Breitbrunn | Polysaccharides obtained from plants of the Compositae family, process for their isolation, and pharmaceutical preparations containing them |
| US4857512A (en) * | 1985-11-27 | 1989-08-15 | Lomapharm, Rudolf Lohmann Gmbh Kg, Pharmazeutische Fabrik | Immunostimulating polysaccharides, method for using such, and pharmaceutical preparations containing them |
| DE3744345A1 (en) * | 1987-12-28 | 1989-07-06 | Lohmann Rudolf Lomapharm | Pharmaceutical composition for stimulating macrophage activity |
| CN1376068A (en) * | 1999-09-30 | 2002-10-23 | 要素的研究与开发技术有限公司 | Echinacea supplement and method of manufacture |
| CN1424904A (en) * | 2000-03-06 | 2003-06-18 | 荷兰联合利华有限公司 | Echinacea extract as anti-irritant and anti-aging booster in cosmetic compositions |
| US20020031524A1 (en) * | 2000-08-10 | 2002-03-14 | Kralovec Jaroslav A. | Chlorella preparations exhibiting immunomodulating properties |
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