CN101415332A - Extractions and methods comprising elder species - Google Patents

Extractions and methods comprising elder species Download PDF

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CN101415332A
CN101415332A CNA2007800094717A CN200780009471A CN101415332A CN 101415332 A CN101415332 A CN 101415332A CN A2007800094717 A CNA2007800094717 A CN A2007800094717A CN 200780009471 A CN200780009471 A CN 200780009471A CN 101415332 A CN101415332 A CN 101415332A
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rutin
virus
fraction
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R·T·高
G·W·塞佩尔特
D·李
R·S·阿尔伯特
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HerbalScience Singapore Pte Ltd
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Abstract

The present invention relates to extracts of elder species plant material prepared by supercritical CO2 extractions methods, methods of treating viruses in a subject and methods of inhibiting viral infections in cells thereof.

Description

The extract and the method that comprise the Sambucus species
Related application
The application requires the U.S. Provisional Patent Application the 60/783rd of submission on March 17th, 2006, No. 453,60/846 of submission on September 22nd, 2006, No. 412, with the U.S. Provisional Patent Application of submitting on December 7th, 2006 the 60/873rd, No. 473 priority, the application incorporates it into this paper in full by reference.
Invention field
The present invention relates to extract and its method derived from Sambucus species (elder sambucus species), and by the extract of this class methods extraction and the using method of this class extract, described extract has Essential Oil Chemistry composition, phenolic acid chemical composition, anthocyanidin (anthocyanidin) or the proanthocyanidin chemical composition that significantly improves, perhaps phytohemagglutin phytolectin-polysaccharide chemistry composition.
Background of invention
Sambucus, golden elder (Sambuca nigra L.), former Europe, North America and the West Asia be born in is a kind of wild bush.Sambucus is further formed by surpassing 20 kinds of elder species (Sambuca species), and its major part has similar chemical composition.Most scientific research is carried out around golden elder.It is a deciduous tree, can grow to 10 meters, and the early sucking white flowers is also tied black-and-blue fruit (elder berry).Its flower, leaf and fruit all include the chemical composition of important medical value, comprise essential oil compound, phenolic acid particularly flavonoids and anthocyanidin, phytohemagglutin phytolectin albumen composition, and polysaccharide compound.
The medicinal history of Sambucus species can be traced back to the 15th century BCE, is included in the works of Hippocrates, Dioscorides and Pliny.In the middle of America aboriginal and European herb doctor, the tradition of elder is used with a long history.Traditional medicine is used and the modern study activity all concentrates on the flower extract.
Pluck flower in spring, and be lower than under 40 ℃ the sunlight dry so that the loss of aroma minimum.Pluck fruit in the time of full ripe in autumn.Most flower and fruit are from the Russian Federation, Poland, Hungary, Bulgaria and Portugal's import on the market.Fruit also is used for to wine, winter pick-me-up, jam, food and flavouring seasoning and colouring.Flower and fruit have had very long history as medicinal reagent.
The chemical composition of golden elder flower and fruit comprises biologically active phenolic acid (flavonoids and anthocyanidin), protein, polysaccharide and vitamin (C, P, B1, B2 and B6).Though about the information of Sambucus species flower and chemical composition really is incomplete, table 1 has been enumerated known chemical composition.Stand in commercial and biological angle, think that traditionally flavonoids and anthocyanidin are more important than other compositions.
Table 1: the chemical composition of golden elder flower and fruit
Figure A200780009471D00081
Figure A200780009471D00091
The medicinal character of Sambucus species causes it to have the existence of the chemical composition of pharmacological activity.As the general rule of chemical content, fruit beautiful in colour contains high-caliber anthocyanidin (as pigment), and flavonol glycosides and aglycone (Espin JC or the like, J Agric FoodChem 48:1588-1592,2000; Kahkonen MP or the like, J Agric Food Chem49:4076-4082,2001).
Anthocyanidin be flavylium ion mutter (pyrylium) salt glycosylation-polyhydroxy and-Polymethoxylated derivative (Brouillard KaHSH, Chemical Structure of Anthocyanins.Academic Press, New York, 1982).Elder berry is one of rich in natural resources of these pigments, has the content of 0.2-1%, and this is higher than the content (Bronnum-HansenK or the like, J Food Technology 20:703-711,1985) in the grape far away.Elder berry comprises several different anthocyanins, wherein anthocyan-3-elder bioside (compound 1) and anthocyan-3-glucosides (compound 2) quantitatively are most important, what account for anthocyanidin content surpasses 85%, and anthocyan-3-elder bioside-5-glucosides (compound 3) and anthocyan-3,5 diglucosides (compound 4) only are trace (Bronnum-Hansen K or the like, J Chromatography 262:393-396,1983; Drdak M ﹠amp; Daucik P., Acta Aliment 19:3-7,1990).Anthocyanidin has shown very wide biologically active.One of foremost attribute is the antioxidant activity, especially the antioxidant activity of anthocyan derivative (Drdak M ﹠amp; Daucik P., Acta Aliment 19:3-7,1990; Tsuda T or the like, J Agric Food Chem 42:2407-2410,1994).
Figure A200780009471D00101
By the ability of growing in the body of testing different types of blueberry compound of phenolic acid inhibition colon cancer cell, find that anthocyanidin is powerful phenols (Kamei H or the like, Cancer Invest13:590-594,1995).Particularly, anthocyan is still very effective aspect the cell growth inhibiting when being low to moderate the concentration of 2 μ g/ml, and this concentration only is 1/10 of required powerful anticancer agent genistein concentration.Found that the anthocyanin in the blueberry has active anticancer (J FoodSci 65:352-356 such as Smith MAL, 2000).
Rutin and isoquercitrin are flavonol glycosides (PiettaP ﹠amp main in the Sambucus species plant corpus; Bruno A., J Chromatography 593:165-170,1992).These compounds can serve as powerful free radical scavenger (Shahidi F ﹠amp; Wannasundra PK., Crit Rev FoodSci Nutr 32:67-103,1992; Rice-Evans CA or the like, Free Radical Biol ﹠amp; Med20:933-956,1996), suppress plurality of enzymes (Formica JV ﹠amp; Regelson W., Food andChem Toxic 33:1061-1080,1995), and have antihaemorrhagics activity (Dawidowicz AJ or the like, J Liquid Chromotog ﹠amp by tightening up blood vessel; Related Technologies26:2381-2397,2003).In the research of the accelerated solvent extract of the flower, fruit and the leaf that use golden elder, the discovery rutin is main flavonoids.Flower has flavonoids and the isoquercitrin that concentration is the high-load of 2-3% and 0.1%.Elder berry and leaf have the rutin of similar content, concentration about 0.2%.The result is as shown in table 2.
Figure A200780009471D00102
Rutin: R=rutinoside isoquercitrin: R=glucosides
Rutin and isoquercitrin recovery rate that table 2. obtains from the different parts of golden elder with 80% methyl alcohol.
Rutin (%) Isoquercitrin (%)
Flower 2-2.88 0.114
Leaf 0.14-0.2 0.003-0.005
Really 0.16-0.19 0.02-0.03
Because its volatile ingredient, elder plantation object has pleasant overpowering odor.Identified multiple alkane in williams elder leaf, wherein the content of heptacosane, nonacosane and hentriacontane is most important.The flores sambuci essential oil has the fatty acid (66%) and the normal alkane (7.2%) of high-load.79 kinds of compounds (Toulemonde B or the like, J Agric Food Chem 31:365-370,1983) from the steaming thing of flores sambuci essential oil, have been identified.The main component of essential oil is anti--3,7-dimethyl-1,3, and 7-sarohornene (octatrien)-3-alcohol (13%), palmitic acid (11.3%), linalool (3.7%), suitable-hexenol (2.5%) and suitable-and anti--rose ethers (being respectively 3.4% and 1.7%).
To comprise concentration be 0.5% anthocyanidin (EspinJC or the like, J Agric Food Chem 48:1588-1592,2000) to main elder berry extract on the market.Main anthocyanidin is anthocyan-3-monoglycosides (97%) and anthocyan-3,5-bioside (3%).The feature of concentrate also is to exist caffeic acid derivative (0.011%) and rutin (0.055).
Triterpene and flavonoids are considered to main chemical compositions (Blumenthal M or the like that the biologically active of elder species is made contributions for a long time, Herbal Medicine:ExpandedCommission E Monographs, Integrative Medicine Communications, Newton, MA, 2000, pp.103-105).As if yet four kinds of main anthocyanidin play an important role in the anti-influenza activity of Sambucus species.These anthocyanidin are incorporated into (Youdin KA or the like, Free Radic Biol Med 29:51-60,2000) in the after birth and cell sap that is exposed to the endothelial cell of Sambucus species extract after 4 hours.As if the human and animal's endothelial cell that is rich in Sambucus species anthocyanidin be endowed the protective effect of opposing oxidative stress.In addition, the extract of Sambucus species fruit has demonstrated and has had the ability (Roy S or the like, Free Radical Res 36:1023-1031,2002) that absorbs oxygen radical.The hemagglutinin of Sambucus species and ribosome-deactivation protein has also been showed antiviral activity (Vanderbussche F or the like, Eur J Biochem 27:1508-1515,2004; De BenitoFM or the like, FEBS Lett 428:75-79,1998; Fujimura Y or the like, Virchows Arch444:36-42,2003).Standardized extract (the Sambucol of golden elder fruit
Figure A200780009471D0012081647QIETU
.RazeiBar, Jerusalem) (adult's dosage 4 grams), comprise 38% the black elder berry extract that combines with Echinacea angustifolica (rhizome) extract, Echinacea purpura (stem, Ye Hehua) extract, vitamin C (100mg) and zinc (10 milligrams) with anthocyanidin, this standard extract shows following character: suppress hemagglutination (Zakay-RonesZ or the like, J Alternative ﹠amp by human influenza virus's generation; Complementary Medicine 1:361-369,1995); Suppress in the human body and external virus replication (Zakay-Rones Z or the like, J Alternative ﹠amp; Complementary Medicine 1:361-369,1995); Improve the generation (Barak V or the like, Isr Med Assoc J 4 (suppl 11): 919-922,2002) of interior inflammatory of human body and anti-inflammatory cytokines; Reduce hemagglutination and inhibition A type and Type B human influenza virus at external duplicating (Zakay-RonesZ or the like, J Alternative ﹠amp; Complementary Medicine1:361-369,1995); Reduce the external infection (Zakay-Rones Z or the like, JInternational Med Res 32:132-140,2004) of HIV; The replication in vitro (Zakay-Rones Z or the like, JInternational Med Res 32:132-140,2004) that suppresses the HSV-1 bacterial strain; Reduce the colitis (Bobek P or the like, Biologia Bratislavia 56:643-648,2002) in the rat model; Alleviate the flu-like symptom (Gray AM or the like, J Nutr 30:15-20,2000) of chimpanzee; And clinical trial certificate has alleviated human A type and Type B flu-like symptom (Zakay-Rones Z or the like, JInternational Med Res 32:132-140,2004) at random.Other other discoveries of extracting composition that come from golden elder comprise: increase lysosomal enzymes, reduce the generation of lipoxygenase effect (lipoxygenation) product, with external activity (the Bobek P or the like that reduces myeloperoxidase, Biologia Bratislavia 56:643-648,2002); Prevent external response to oxidative stress (stress) (Brouillard KaHSH., Chemical Structure ofAnthocyanins., Academic Press, New York, 1982); Promote para-insulin and insulin releasing activity (Gray AM or the like, J Nutr 30:15-20,2000) in external oxygen radical absorbing capacity (Bronnum-Hansen K or the like, J Chromatography 262:393-396,1983) and the body.
Therapeutic value for brief overview golden elder chemical composition, Science Explorations and clinical research have proved all cpds, chemical group of Sambucus species or the following therapeutic action of extracting composition, comprising: antiviral, anti-flu, influenza emit, anti-HIV, anti-HSV (triterpene, anthocyanidin, phytohemagglutin phytolectin albumen, polysaccharide, crude extract); Antioxidant and removing oxygen radical (flavonoids, anthocyanidin, crude extract); Antiphlogistic activity (crude extract); Anti-diabetic activity (polysaccharide, water-soluble extractive); Regulate the movable and mitigation diarrhoea (extract) of intestines; Excited and the insomnia (extract) with minimizing.In addition, it has been generally acknowledged that it is safe that golden elder flores sambuci or elder berry extract composition, and do not have known contraindication.
Summary of the invention
On the one hand, the present invention relates to comprise and have among Figure 36-70 the Sambucus species extract of directly analyzing the fraction of (DART) mass spectrum chromatogram shown in arbitrary figure in real time.In further embodiment, this fraction has the DART mass spectrum chromatogram shown in arbitrary figure among Figure 46-50.In further embodiment, this fraction has the DART mass spectrum chromatogram of Figure 48.
On the one hand, the present invention relates to comprise the IC that the H1N1 influenza virus is recorded 50Sambucus species extract for the fraction of 150-1500 μ g/mL.In further embodiment, this fraction has the IC of 150-750 μ g/mL 50In further embodiment, this fraction has the IC of 150-300 μ g/mL 50In further embodiment, this fraction has the IC of at least 195 μ g/mL 50
In another embodiment, the present invention relates to Sambucus species extract of the present invention, wherein said fraction comprises anthocyanin; Flavonoids; C16 or C18 saturated or unsaturated fatty acid, alcohol or ester; And/or polysaccharide.In further embodiment, described anthocyanin is selected from anthocyan-3-glucosides and anthocyan-3-elder bioside.In further embodiment, the content of described anthocyanin surpasses 10,20,30,40 or 50 weight %.In further embodiment, described flavonoids is a rutin.In further embodiment, described C16 or C18 saturated or unsaturated fatty acid, alcohol or ester are selected from hexadecanol, hexadecylic acid, methyl palmitate, ethyl palmitate, hexadecylic acid butyl ester, stearic acid, Ethyl Stearate, butyl octadecanoate, 9-vaccenic acid-1-alcohol, 9,12-octadecadienoic acid, and their combination.In further embodiment, described C16 or C18 saturated or unsaturated fatty acid, alcohol or ester are 2,4,6,8 or 10 weight %.In further embodiment, described polysaccharide is selected from dextran, glucose, arabinose, galactose, rhamnose, wood sugar, uronic acid, and their combination.In further embodiment, the content of described polysaccharide is 10,15,20,25,30,35 or 40 weight %.
In another embodiment, the present invention relates to elderberry extract of the present invention, wherein said fraction comprises anthocyanin; C16 or C18 saturated or unsaturated fatty acid, alcohol or ester; And polysaccharide.In further embodiment, described anthocyanin is selected from anthocyan-3-glucosides and anthocyan-3-elder bioside.In further embodiment, the content of described anthocyanin surpasses 10,20,30,40 or 50 weight %.In further embodiment, described C16 or C18 saturated or unsaturated fatty acid, alcohol or ester are selected from hexadecanol, hexadecylic acid, methyl palmitate, ethyl palmitate, hexadecylic acid butyl ester, stearic acid, Ethyl Stearate, butyl octadecanoate, 9-vaccenic acid-1-alcohol, 9, the acid of 12-octadecadienoic acid, and their combination.In further embodiment, described C16 or C18 saturated or unsaturated fatty acid, alcohol or ester are 2,4,6,8 or 10 weight %.In further embodiment, described polysaccharide is selected from dextran, glucose, arabinose, galactose, rhamnose, wood sugar, uronic acid, and their combination.In further embodiment, the content of described polysaccharide is 10,15,20,25,30,35 or 40 weight %.
On the other hand, the present invention relates to comprise the food or the medicament of Sambucus species extract of the present invention.
On the other hand, the present invention relates to treat the method for patient's virus infections, comprise Sambucus species extract of the present invention patient's effective dosage of the above-mentioned treatment of needs.In further embodiment, this virus infections is caused by enveloped virus (envelope virus).In further embodiment, this enveloped virus is flavivirus (flavie virus).In another embodiment, described virus infections is caused by nonenveloped virus.In further embodiment, described virus infections is caused by influenza virus, A type and Type B human influenza virus, avian influenza virus, H1N1, H5N1, human immunodeficiency virus (HIV), SARs, herpes simplex virus (HSV), flavivirus, dengue fever, yellow fever, West Nile Virus (West Nile) and encephalitis viruses.In another embodiment, described virus infections is caused by C (Norwalk) virus, hepatitis A, poliomyelitis, andoviruses or rhinovirus.In further embodiment, described patient is primate, Bovidae, birds, sheep section, equine, Suidae, rodent, cat family or canid.In further embodiment, described patient is human.
In another embodiment, the present invention relates to suppress the method for the virus infections of cell, this method comprises makes described cell contact with Sambucus species extract of the present invention.In further embodiment, this virus infections is an infection of coated virus.In further embodiment, this infection of coated virus is a flaviviridae infections.In another embodiment, this virus infections is that nonenveloped virus infects.In another embodiment, this virus infections is that influenza virus, A type and Type B human influenza virus, avian influenza virus, H1N1, H5N1, human immunodeficiency virus (HIV), SARs, herpes simplex virus (HSV), flavivirus, dengue fever, yellow fever, West Nile Virus and encephalitis viruses infect.In yet another embodiment, this virus infections is norwalk virus, hepatitis A, poliomyelitis, andoviruses or rhinovirus infection.
On the other hand, the present invention relates to prepare the method for Sambucus species extract with at least a predetermined characteristic, this method comprises: extract Sambucus species plant corpus in proper order by following steps, to produce essential oil fraction, polyphenol fraction and polysaccharide fractions: a) extract Sambucus species plant corpus, to produce the essential oil fraction and first residue by supercritical carbon dioxide extracting; B) by extracting first residue that obtains in Sambucus species plant corpus or the step a) to about 70 ℃ water or water-alcohol (hydro-alcoholic) with about 40 ℃, to produce the polyphenol fraction and second residue; And c) come extraction step b at about 70 ℃ to about 90 ℃ of extractions by water) second residue that obtains, to produce polysaccharide fractions.In another embodiment, described extracting method can be applied to any species that are rich in anthocyanidin and/or proanthocyanidin, for example currant fruit, red currant fruit, gooseberry, wineberry (bilberry), blackberry, blueberry, blueberry, cherry, european cranberry (cranberry), haw berry, the sweet certain kind of berries of sieve, raspberry, serviceberry (chokeberry), apple, pomegranate, Wen Bai and plum.
In another embodiment, obtaining the essential oil fraction comprises: the digester of 1) the Sambucus species plant corpus that grinds being packed into; 2) under super critical condition, add carbonic acid gas; 3) Sambucus species plant corpus is combined a period of time with carbonic acid gas; And 4) in collector, collect the essential oil fraction.
In yet another embodiment, method of the present invention also comprises by change the compound ratio (compound ratios) of Essential Oil Chemistry composition with the described essential oil extract of supercritical carbon dioxide fractionated (fractional separation) system's fractionation.
In yet another embodiment, obtain the polyphenol fraction by following steps: 1) residue that the Sambucus species plant corpus that grinds or step a) are obtained contacts one enough period of extraction polyphenol chemical composition with about 40 ℃ to about 70 ℃ water or water-alcohol solution; 2) make the water-alcohol solution of the polyphenol chemical composition of extracting of step a) acquisition pass through the affinity adsorbent resin column, be adsorbed in interior polyphenol acid comprising anthocyanidin; With 3) from the affinity adsorbent resin with one or more polyphenol chemical composition fraction wash-outs of purifying.
In yet another embodiment, the method that obtains polysaccharide fractions comprises: 1) second residue that step b) is obtained contacts one enough period of extraction polysaccharide with about 70 ℃ to about 90 ℃ water; With 2) with ethanol precipitation polysaccharide is precipitated out from the aqueous solution.
On the other hand, the present invention relates to the Sambucus species extract that makes by any means of the present invention.
On the other hand, the present invention relates to Sambucus species extract, its cinnamic acid and weight that comprises the 2-metoxyphenol of methyl cinnamic acid that 1,2,3,-thrihydroxy-benzene (pyrogallol), weight are the 15-25% of 1,2,3,-thrihydroxy-benzene, cinnamamide that weight is the 1-4% of 1,2,3,-thrihydroxy-benzene, 5-10% that weight is 1,2,3,-thrihydroxy-benzene, benzaldehyde that weight is the 1-2% of 1,2,3,-thrihydroxy-benzene, 5-10% that weight is 1,2,3,-thrihydroxy-benzene is the cinnamyl acetate of the 5-15% of 1,2,3,-thrihydroxy-benzene.
On the other hand, the present invention relates to Sambucus species extract, its shikimic acid and weight that comprises forulic acid that rutin, weight are the 20-30% of rutin, cinnamic acid that weight is the 25-35% of rutin, 15-25% that weight is rutin is the phenyllactic acid of the 55-65% of rutin.
On the other hand, the present invention relates to Sambucus species extract, it comprises the anthocyan of texifolin (taxifolin) that rutin, weight are the 1-10% of rutin, forulic acid that weight is the 1-5% of rutin, cinnamic acid that weight is the 1-5% of rutin, shikimic acid that weight is the 0.5-5% of rutin, phenyllactic acid that weight is the 1-5% of rutin, 5-15% that weight is rutin and the petunidin (petunidin) that weight is the 15-25% of rutin.
On the other hand, the present invention relates to Sambucus species extract, the forulic acid and the weight that comprise rutin, weight and be the anthocyan of the 30-40% of rutin, petunidin that weight is the 75-85% of rutin, vanillic acid that weight is the 5-10% of rutin, 1-5% that weight is rutin are the cinnamic acid of the 1-10% of rutin.
On the other hand, the present invention relates to Sambucus species extract, its comprise to coumaric acid/phenylpyruvic acid, weight for to the rutin of the 65-75% of coumaric acid/phenylpyruvic acid, weight for to the vanillic acid of the 65-75% of coumaric acid/phenylpyruvic acid, weight for to the forulic acid of the 35-45% of coumaric acid/phenylpyruvic acid, weight for the cinnamic acid of the 65-75% of coumaric acid/phenylpyruvic acid, weight are the shikimic acid to the 45-55% of coumaric acid/phenylpyruvic acid.
On the other hand, the present invention relates to Sambucus species extract, its vanillic acid and weight that comprises aurantiamarin that rutin, weight are the 20-30% of rutin, 70-80% that weight is rutin is the cinnamic acid of the 40-50% of rutin.
On the other hand, the present invention relates to Sambucus species extract, its vanillic acid and weight that comprises rutin that petunidin, weight are the 85-95% of petunidin, 55-65% that weight is petunidin is the cinnamic acid of the 30-40% of petunidin.
On the other hand, the present invention relates to Sambucus species extract, it comprises the petunidin of anthocyan that rutin, weight are the 5-15% of rutin, texifolin that weight is the 1-10% of rutin, caffeic acid that weight is the 5-15% of rutin, forulic acid that weight is the 1-10% of rutin, shikimic acid that weight is the 1-10% of rutin, 25-35% that weight is rutin and eriodictyol or the young fustic of the 1-5% that weight is rutin.
On the other hand, the present invention relates to Sambucus species extract, the naringenin and the weight that comprise rutin, weight and be the eriodictyol of the anthocyan of the 10-20% of rutin, 1-5% that weight is rutin or young fustic, weight and be the 10-20% of rutin are the texifolin of the 1-10% of rutin.
By following description, accompanying drawing and claim, these embodiments of the present invention, other embodiments and feature thereof and characteristics are conspicuous.
Brief description of drawings
Fig. 1 has shown the exemplary schematic diagram of Sambucus species extracting method of the present invention.
Fig. 2 has shown the exemplary schematic diagram of Sambucus species extracting method of the present invention.
Fig. 3 has shown the exemplary schematic diagram of Sambucus species extracting method of the present invention.
Fig. 4 has shown the illustrative diagram of Sambucus species extracting method of the present invention.
Fig. 5 has shown that the virus of end user A type H1N1 virus enters analytical system (viral entryassay system).Only using virus (upper left; 10-4 influenza A), do not use virus (lower-left; PBS), use and concentration 1:1,000 is (upper right; 1:1000Ab) and 1:500 (bottom right; Hatch mdck cell under the condition of the virus that resisiting influenza virus antibody 1:500Ab) mixes.Each experiment is done three times.Each reddish brown spot is represented a virus infections incident.In antibody control, detect the minimizing of virus inhibition or color spot quantity.
Fig. 6 has shown the embodiment that uses the inhibition analysis of elder berry B anthocyanin fraction ADS5 desorb F4 and human influenza A type H1N1 virus.Before hatching, with the serial dilutions (undiluted) and the viral preincubate of elder berry B anthocyanin fraction ADS5 desorb F4 fraction to the 1:32 dilution with mdck cell.Each test is done three times.Spot is corresponding to a virus infections incident.The minimizing of amount of speckle represents that virus suppresses.
Fig. 7 has shown the inhibition analysis that uses elder berry B anthocyanin fraction ADS5 desorb F4 and human influenza A type H1N1 virus.Before hatching, with the serial dilutions (undiluted) and the viral preincubate of elder berry B anthocyanin fraction ADS5 desorb F4 fraction to the 1:32 dilution with mdck cell.Each test is done three times.The brownish red spot is corresponding to a virus infections incident.The minimizing of color spot quantity represents that virus suppresses.
Fig. 8 has shown the inhibition analysis that uses elder berry B anthocyanin fraction ADS5 desorb F4 and human influenza A type H5N1 virus.Before hatching, with the serial dilutions (undiluted) and the viral preincubate of elder berry B anthocyanin fraction ADS5 desorb F4 fraction to the 1:32 dilution with mdck cell.Each test is done three times.The brownish red spot is corresponding to a virus infections incident.The minimizing of color spot quantity represents that virus suppresses.
Fig. 9 has shown the inhibition analysis of chimeric HIV-1 SG3 (genome) C hypotype (coating).+, positive infection contrast; F4 is elder berry extract fraction F4; And T is the titration of used virus in analyzing.
Figure 10 has shown MTT viability (viability) analysis of elder berry B anthocyanin fraction ADS5 desorb F2 fraction in the 293T cell.
Figure 11 has shown the MTT viability analysis of elder berry B anthocyanin fraction ADS5 desorb F2 fraction in mdck cell.
Figure 12 has shown the MTT viability analysis of elder berry B anthocyanin fraction ADS5 desorb F4 fraction in the 293T cell after 24 hours.
Figure 13 has shown the MTT viability analysis of elder berry B anthocyanin fraction ADS5 desorb F4 fraction in the 293T cell after 44 hours.
Figure 14 has shown that the contagiosity of elder berry B anthocyanin fraction ADS5 desorb F2 fraction suppresses dose response curve and 50% inhibition concentration.
Figure 15 has shown that the contagiosity of elder berry B anthocyanin fraction ADS5 desorb F3 fraction suppresses dose response curve and 50% inhibition concentration.
Figure 16 has shown that the contagiosity of elder berry B anthocyanin fraction ADS5 desorb F4 fraction suppresses dose response curve and 50% inhibition concentration.
Figure 17 has shown that the contagiosity of flores sambuci XAD 7HP desorb F2 fraction suppresses dose response curve and 50% inhibition concentration.
Figure 18 has shown that the contagiosity of flores sambuci XAD 7HP desorb F3 fraction suppresses dose response curve and 50% inhibition concentration.
The contagiosity that the nothing of using H1N1 virus to obtain that shown Figure 19 cushions elder berry B anthocyanin fraction ADS5 desorb F3 fraction suppresses dose response curve and 50% inhibition concentration.
The contagiosity that the nothing of using H1N1 virus to obtain that shown Figure 20 cushions elder berry B anthocyanin fraction ADS5 desorb F3 fraction suppresses dose response curve and 50% inhibition concentration.
The contagiosity that the nothing of using H1N1 virus to obtain that shown Figure 21 cushions elder berry B anthocyanin fraction ADS5 desorb F2 fraction suppresses dose response curve and 50% inhibition concentration.
The contagiosity that the nothing of using H1N1 virus to obtain that shown Figure 22 cushions elder berry B anthocyanin fraction ADS5 desorb F4 fraction suppresses dose response curve and 50% inhibition concentration.
Figure 23 has shown that the contagiosity of the elder berry B anthocyanin fraction ADS5 desorb F4 fraction of the buffering that use H1N1 virus obtains suppresses dose response curve and 50% inhibition concentration.
The contagiosity that the nothing of using H1N1 virus to obtain that shown Figure 24 cushions elder berry B anthocyanin fraction ADS5 desorb F4 fraction suppresses dose response curve and 50% inhibition concentration.
Figure 25 has shown that the contagiosity of the elder berry B anthocyanin fraction ADS5 desorb F4 fraction of the buffering that the use H5N1 virus obtains suppresses dose response curve and 50% inhibition concentration.
Figure 26 has shown that the contagiosity of the elder berry B anthocyanin fraction ADS5 desorb F4 fraction of the buffering that the use H5N1 virus obtains suppresses dose response curve and 50% inhibition concentration.
Figure 27 has shown that (combined) contagiosity of the combination of institute's Test extraction thing suppresses dose response curve.
Figure 28 has shown that the contagiosity of the buffering flores sambuci ADS5 desorb F2 fraction that use H1N1 virus obtains suppresses dose response curve and 50% inhibition concentration.
Figure 29 has shown the IC that flores sambuci F2 fraction is calculated 50H1N1.
Figure 30 has shown the IC of elder berry F4 fraction and flores sambuci F2 fraction 50The contrast of H1N1.
Figure 31 has shown the IC of elder berry F4 fraction and flores sambuci F2 fraction 90The contrast of H1N1.
Figure 32 has shown that the contagiosity of using the elder berry B anthocyanin fraction ADS5 desorb F2 that 2 type dengue fever viruss obtain suppresses dose response curve and 50% inhibition concentration.
Figure 33 has shown that the contagiosity of the elder berry B anthocyanin fraction ADS5 desorb F4 fraction that use HIV virus obtains suppresses dose response curve.This curve shown in demonstrate 100% inhibition under the concentration.
Figure 34 has shown that the contagiosity of the elder berry B anthocyanin fraction ADS5 desorb F4 fraction that use HIV virus obtains suppresses dose response curve.This curve shown in demonstrate 100% inhibition under the concentration.
Figure 35 has shown that the contagiosity of the elder berry B anthocyanin fraction ADS5 desorb F4 fraction that use HIV virus obtains suppresses dose response curve and 50% inhibition concentration.
Figure 36 has shown the AccuTOF-DART mass spectrum (positive ion mode) of elder berry polysaccharide.
Figure 37 has shown the AccuTOF-DART mass spectrum (negative ion mode) of elder berry polysaccharide.
Figure 38 has shown the AccuTOF-DART mass spectrum (positive ion mode) of flores sambuci polysaccharide.
Figure 39 has shown the AccuTOF-DART mass spectrum (negative ion mode) of flores sambuci polysaccharide.
Figure 40 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the full elder berry charging with described possibility structure.Detect methyl cinnamic acid (163.0688) (abundance (abund.)=19.47), cinnamamide (148.0826) (abundance=2.63), 2-metoxyphenol (125.0599) (abundance=7.34), 3-methoxyl group-1-tyrosine (212.0985) (abundance=17.42), benzaldehyde (107.0422) (abundance=1.10), cinnamic acid (133.0568) (abundance=6.56), cinnamyl acetate (177.0956) (abundance=8.51), and 1,2,3,-thrihydroxy-benzene (127.0344) (abundance=93.67).Also detect unacknowledged Compound C 6H 8O 4+ H +(145.0469) and C 6H 6O 3+ H +(127.0344).
Figure 41 has shown the AccuTOF-DART mass spectrum (negative ion mode) of the full elder berry charging with described possibility structure.Detect cinnamic acid (147.0385) (abundance=5.57), cinnamic acid (131.04) (abundance=5.57), 1,2,3,-thrihydroxy-benzene (125.024) (abundance=3.54), quercetin (301.0253) (abundance=0.73), ursolic acid (455.3518) (abundance=10.99), and shikimic acid (173.0454) (abundance=7.18).
Figure 42 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the full elder berry charging extract that use 80%EtOH solution obtains.Detect unacknowledged Compound C 6H 10O 5+ H +(163.0601) (abundance=17.19) and C 14H 15NO+H +(214.1266) (abundance=24.06).
Figure 43 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the F2 column chromatography fraction that use ADS5 desorb filler obtains.Detect rutin or delphinin (303.0541) (abundance=59.28), forulic acid (195.0755) (abundance=13.54), cinnamic acid (149.0572) (abundance=19.55), shikimic acid (175.0699) (abundance=11.72) and phenyllactic acid (167.0793) (abundance=36.17).Also detect unacknowledged Compound C 6H 6O 3+ H +(127.0348) (abundance=100) and C 7H 6O 4+ H +(155.0335) (abundance=59.18).
Figure 44 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the F3 column chromatography fraction that use ADS5 desorb filler obtains.Detect rutin or delphinin (303.0521) (abundance=100), texifolin (305.0693) (abundance=4.25), forulic acid (195.075) (abundance=1.34), cinnamic acid (149.0552) (abundance=3.32), shikimic acid (175.0696) (abundance=0.96), phenyllactic acid (167.0701) (abundance=3.97), anthocyan (287.0622) (abundance=8.36) and petunidin (317.0707) (abundance=21.71).Also detect unacknowledged Compound C 10H 12O 3+ H +(181.0854) (abundance=9.71) and C 13H 14N 2O 2+ H +(231.1163) (abundance=5.85).
Figure 45 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the F4 column chromatography fraction that use ADS5 desorb filler obtains.Detect rutin or delphinin (303.0534) (abundance=100), forulic acid (195.0744) (abundance=3.32), cinnamic acid (149.057) (abundance=6.36), anthocyan (287.0608) (abundance=36.44), petunidin (317.0691) (abundance=78.75) and vanillic acid (169.0524) (abundance=7.75).Also detect unacknowledged Compound C 29H 18O 7+ H +(479.1218) (abundance=22.62) and C 12H 14O 4+ H +(223.0994) (abundance=21.56).
Figure 46 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the F2 column chromatography fraction of the elder berry B anthocyanin that use ADS5 desorb filler obtains.This fraction is used to use the antiviral analysis of H1N1 to obtain IC 50=333 μ g/mL.Detect rutin or delphinin (303.0566) (abundance=18.33), forulic acid (195.0724) (abundance=10.32), to coumaric acid/phenylpyruvic acid (165.0639) (abundance=25.54), cinnamic acid (149.0573) (17.86), shikimic acid (175.0633) (abundance=12.62) and vanillic acid (169.0575) (abundance=18.01).Also detect unacknowledged Compound C 13H 11O+H +(183.0818) (abundance=43.33) and C 14H 17NO 3+ H +(248.1271) (abundance=60.28).
Figure 47 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the F3 column chromatography fraction of the elder berry B anthocyanin that use ADS5 desorb filler obtains.This fraction is used to use the antiviral analysis of H1N1 to obtain IC 50=294 μ g/mL.Detect rutin or delphinin (303.0553) (abundance=41.74), aurantiamarin (hesperin) (287.0936) (abundance=10.41), cinnamic acid (149.0584) (abundance=17.85) and vanillic acid (169.0571) (abundance=31.09).Also detect unacknowledged Compound C 8H 8O+H +(121.0586) (abundance=29.36) and C 14H 20N 2O 3+ H +Or C 15H 20O 4+ H +(265.1469) (abundance=26.23).
Figure 48 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the F4 column chromatography fraction of the elder berry B anthocyanin that use ADS5 desorb filler obtains.This fraction is used to use the antiviral analysis of H1N1 to obtain IC 50=195 μ g/mL.Detect rutin or delphinin (303.0557) (abundance=20.27), cinnamic acid (149.0593) (abundance=7.94), petunidin (317.071) (abundance=22.09) and vanillic acid (169.0538) (abundance=12.82).Also detect unacknowledged Compound C 6H 10O 5+ H +(163.076) (abundance=63.28) and C 17H 18O+H +(239.1531) (abundance=26.32).
Figure 49 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the flores sambuci F2 column chromatography fraction that use XAD7HP desorb filler obtains.This fraction is used to use the antiviral analysis of H1N1 to obtain IC 50=1592 μ g/mL.Detect anthocyan (287.0588) (abundance=10.92), rutin or delphinin (303.0531) (abundance=100), texifolin (305.0651) (abundance=4.69), caffeic acid/4-hydroxyphenyl lactic acid (181.0589) (abundance=9.45), forulic acid (195.0741) (abundance=3.33), shikimic acid (175.0645) (abundance=3.11), petunidin (317.0689) (abundance=29.48) and eriodictyol or young fustic (288.0709) (abundance=2.36).Also detect unacknowledged Compound C 10H 13NO 2+ H +(180.1024) (abundance=15.98) and C 8H 6N 2O+H +Or C 9H 6O 2+ H +(147.0545) (abundance=73.50).
Figure 50 has shown the AccuTOF-DART mass spectrum (positive ion mode) of the F3 column chromatography fraction of the flores sambuci that use XAD7HP desorb filler obtains.This fraction is used to use the antiviral analysis of H1N1 to obtain IC 50=582 μ g/mL.Detect anthocyan (287.0574) (abundance=17.16), rutin or delphinin (303.0518) (abundance=100), texifolin (305.0658) (abundance=5.54), naringenin/butein/phloretin (273.0797) (abundance=16.06) and eriodictyol or young fustic (289.0795) (abundance=3.14).Also detect unacknowledged Compound C 10H 16O+H +(153.1268) (abundance=30.96) and C 23H 14O 4+ H +(355.1048) (abundance=30.03).
Figure 51 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #185.Detect cinnamic acid (149.0616) (abundance=3.82), shikimic acid (175.0613) (abundance=14.71), and phenyllactic acid (167.074) (abundance=5.35).Also detect unacknowledged Compound C 30H 46O 2+ H +(439.3625) (abundance=16.49) and C 39H 68O 5+ H +(617.5151) (abundance=4.09).
Figure 52 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #319.Detect coumaric acid/phenylpyruvic acid (165.0604) (abundance=3.96), cinnamic acid (149.0579) (abundance=0.48), 3,5-dimethoxy-4 '-hydroxycinnamic acid (225.0816) (abundance=10.59), shikimic acid (175.0699) (abundance=5.37), and phenyllactic acid (167.0773) (abundance=2.71).Also detect unacknowledged Compound C 6H 8O 4+ H +(145.0507) (abundance=100) and C 12H 12O 6+ H +(253.0708) (abundance=35.27).
Figure 53 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #322.Detect delphinin (304.0576) (abundance=8.75), rutin (303.057) (abundance=49.28), eriodictyol/young fustic (289.0752) (abundance=13.50), texifolin (305.0638) (abundance=3.41), forulic acid (195.0745) (abundance=7.15), to coumaric acid/phenylpyruvic acid (165.0613) (abundance=16.91), cinnamic acid (149.0695) (abundance=3.20), shikimic acid (175.067) (abundance=8.34) and phenyllactic acid (167.0722) (abundance=8.84).Also detect unacknowledged Compound C 6H 6O 3+ H +(127.0413) (abundance=100) and C 11H 15O 5+ H +(227.0876) (abundance=29.26).
Figure 54 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #324.Detect unacknowledged Compound C 37H 66O 4+ H +(575.51) (abundance=5.42) and C 59H 88O 5+ H +(877.67) (abundance=15.46).
Figure 55 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #325.Detect shikimic acid (175.0658) (abundance=6.05).Also detect unacknowledged Compound C 16H 14O 4+ H +(271.0941) (abundance=22.24) and C 16H 16O 5+ H +(289.0983) (abundance=15.76).
Figure 56 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #326.Detect cinnamic acid (149.0681) (abundance=2.67).Also detect unacknowledged Compound C 22H 42O 4+ H +(371.3196) (abundance=46.60) and C 18H 30O 2+ H +(279.2346) (abundance=20.28).
Figure 57 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #327.Detect unacknowledged Compound C 8H 8O+H +(121.0663) (abundance=66.34) and C 8H 8O 2+ H +(137.065) (abundance=20.16).
Figure 58 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #328.Detect forulic acid (195.0737) (abundance=4.04), to coumaric acid/phenylpyruvic acid (165.0604) (abundance=3.67), cinnamic acid (149.0691) (abundance=3.49), 3,5-dimethoxy-4 '-hydroxycinnamic acid (225.0817) (abundance=5.18), shikimic acid (175.0616) (abundance=4.88), and phenyllactic acid (167.0786) (abundance=2.63).Also detect unacknowledged Compound C 6H 10O 5+ H +(163.0602) (abundance=10.84) and C 12H 14O 7+ H +(271.0829) (abundance=21.7).
Figure 59 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #329.Detect cinnamic acid (149.0621) (abundance=1.43) and shikimic acid (175.0633) (abundance=3.23).Also detect unacknowledged Compound C 21H 36O 3+ H +(337.2763) (abundance=13.38) and C 39H 66O 4+ H +(599.507) (abundance=5.53).
Figure 60 has shown the AccuTOF-DART mass spectrum (positive ion mode) of #330.Detect forulic acid (195.0747) (abundance=2.76), to coumaric acid/phenylpyruvic acid (165.0608) (abundance=2.42), cinnamic acid (149.0616) (abundance=0.79), 3,5-dimethoxy-4 '-hydroxycinnamic acid (225.0824) (abundance=2.98), shikimic acid (175.0604) (abundance=2.55), and phenyllactic acid (167.078) (abundance=1.95).Also detect unacknowledged Compound C 14H 14O 4+ H +(247.0895) (abundance=4.28) and C 30H 46O 2+ H +(439.3619) (abundance=5.98).
Figure 61 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #185.Detect aurantiamarin (285.0841) (abundance=0.44) and phloridzin (255.0711) (abundance=0.71).Also detect unacknowledged Compound C 4H 6O 5-H +(133.0134) (abundance=100) and C 10H 8O 4-H +(191.0325) (abundance=25.34).
Figure 62 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #319.Detect cinnamic acid (147.0358) (abundance=0.67).Also detect unacknowledged Compound C 4H 6O 5-H +(133.0135) (abundance=86.11) and C 10H 8O 4-H +(191.0195) (abundance=100).
Figure 63 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #322.Detect anthocyan (286.0502) (abundance=5.30), delphinin (302.0388) (abundance=18.51), pelargonidin (270.0512) (abundance=0.34), myricetin (317.0315) (abundance=13.27), rutin (301.0324) (abundance=100), silymarin (silybin)/genistein (269.0399) (abundance=0.42), 3-OH flavones (237.0587) (abundance=0.89), eriodictyol/young fustic (287.0592) (abundance=7.09), catechin/epicatechin (epitcatechin) (289.0784) (abundance=5.29), texifolin (303.0468) (abundance=5.31), phloridzin (255.0614) (abundance=0.81), vanillic acid (167.0416) (abundance=4.07), to coumaric acid/phenylpyruvic acid (163.0307) (abundance=12.95), 3,5-dimethoxy-4 '-hydroxycinnamic acid (223.054) (abundance=0.80), gallic acid (169.0166) (abundance=1.73) and shikimic acid (173.0475) (abundance=1.11).Also detect unacknowledged Compound C 10H 8O 4-H +(191.0532) (abundance=31.51) and C 22H 22O 13-H +(493.0955) (abundance=4.42).
Figure 64 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #324.Detect eriodictyol/young fustic (287.0655) (abundance=0.99), catechin/epicatechin (289.0726) (abundance=0.92), ursolic acid (455.3465) (abundance=0.87), vanillic acid (167.0388) (abundance=1.89), forulic acid (193.0478) (abundance=7.35), to coumaric acid/phenylpyruvic acid (163.0404) (abundance=5.66), cinnamic acid (147.0373) (abundance=5.97), and shikimic acid (173.0373) (abundance=10.00).Also detect unacknowledged Compound C 16H 14O 4-H +(269.0878) (abundance=21.98) and C 23H 18O 3-H +(341.1193) (abundance=12.27).
Figure 65 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #325.Detect unacknowledged Compound C 4H 6O 5-H +(133.0118) (abundance=100) and C 10H 8O 4-H +(191.0183) (abundance=81.19).
Figure 66 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #326.Detect rutin (301.0441) (abundance=31.62), 3-OH flavones (237.062) (abundance=0.74), catechin/epicatechin (289.079) (abundance=2.70), phloridzin (255.0687) abundance=2.24), ursolic acid (455.3556) (abundance=7.43), caffeic acid/4-hydroxyphenyl lactic acid acid (179.0398) (abundance=12.26), forulic acid (193.051) (abundance=7.63), to coumaric acid/phenylpyruvic acid (163.0405) (abundance=8.75), cinnamic acid (147.0414) (abundance=3.24) and shikimic acid (173.0452) (abundance=23.59).Also detect unacknowledged Compound C 5H 6O 4-H +(129.0178) (abundance=100) and C 16H 16O 8-H +(335.0807) (abundance=25.82).
Figure 67 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #327.Detect 3-OH flavones (237.0524) (abundance=0.26), aurantiamarin (285.0822) (abundance=0.63), catechin/epicatechin (289.0732) (abundance=0.11), phloridzin (255.0706) (abundance=0.82), 3,5-dimethoxy-4 '-hydroxycinnamic acid (223.0543) (abundance=0.09), and chorismic acid (chorismicacid) (225.0489) (abundance=0.10).Also detect unacknowledged Compound C 4H 6O 5-H +(133.0117) (abundance=100) and C 20H 20O 7-H +(371.1175) (abundance=2.39).
Figure 68 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #328.Detect rutin (301.0446) (abundance=0.62), phloridzin (255.0744) (abundance=0.05) and to coumaric acid/phenylpyruvic acid (163.0386) (abundance=0.36).Also detect unacknowledged Compound C 5H 8O 5-H +(147.0293) (abundance=7.50) and C 6H 6O 6-H +(173.0099) (abundance=7.84).
Figure 69 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #329.Detect unacknowledged Compound C 6H 10O 5-H +(161.04) (abundance=2.97) and C 8H 12O 7-H +(219.05) (abundance=3.64).
Figure 70 has shown the AccuTOF-DART mass spectrum (negative ion mode) of #330.Detect unacknowledged Compound C 5H 4O 3-H +(111.01) (abundance=12.32) and C 6H 12O 6-H +(179.05) (abundance=1.20).
Detailed description of the invention
Definition
This paper uses article " a " and " an " to refer to a kind of or surpasses the grammatical object of a kind of (namely at least a) this article. For example: " an element (an element) " meaning is a kind of element or surpasses a kind of element.
Term " anthocyanidin " is well known in the art, refers to comprise the compound of yellow detailed salt (flavylium) cation derivative.
Term " anthocyanin " is well known in the art, refers to have the anthocyanidin of glycosyl group. It is mainly the 3-glucosides in the anthocyanidin. Described anthocyanidin is subdivided into sugar-free anthocyanidin aglycone and anthocyanin glucosides.
Term " virocapsid (capsid) " is well known in the art, refers to around and protect the protein shell of viral nucleic acid (DNA or RNA).
Term " comprise " and " comprising " with its implication that included, open, namely also can comprise other key elements.
Term " by ... form " be used for key element is limited to except the common impurity that interrelates with it clearly those key elements of appointment.
Term " substantially by ... form " be used for key element is limited to those key elements of clearly appointment, and can not affect in itself those key elements of the fundamental sum novel feature of described material or step.
Term " anthocyanidin " or " flavones-3-alcohol " are well known in the art, refer to be divided into the natural organic-compound of flavonoids and anthocyanin. It is a kind of pigment that all can find in many haws (redberries), these haws include but not limited to blueberry, blackberry, blueberry, blueberry, cherry, european cranberry fruit, elder berry, hawthorn, the sweet certain kind of berries of sieve, the Brazilian certain kind of berries (acai berry), and raspberry. It also can find in other fruit such as apple and plum.
Term used herein " effective dose " has guided the essential amount of required biological response. The effective dose that it will be understood by those skilled in the art that composition or bioactive agent can be along with the factors vary such as required biology terminal point, bioactive agent to be conveyed, the composition that forms the matrix of capsule, destination organization etc.
" elder " used herein refers to come from the Sambucus species plant of Sambucus species plant. Term " elder " also can with Sambucus species, elder species and elder berry Alternate, and the meaning refers to these plants, replisome, mutation and mutant etc.
Term used herein " elder composition " meaning is the compound of finding in the Sambucus species, and comprise all these compounds of above-mentioned identification and other compounds of in the Sambucus species, finding, include but not limited to Chemical Composition of The Essential Oil, Polyphenol Acids and polysaccharide.
Term used herein " enveloped virus " refers to comprise the virus of bimolecular lipid membrane, and this bimolecular film comprises the VGP class that derives from host cell membrane. In enveloped virus, found that in coating mediation is to the adhesion of host cell and the virus protein that penetrates. The example of enveloped virus comprises people and avian influenza virus, HIV, SARs, HPV, herpes simplex virus (HSV), dengue fever virus, and such as yellow fever, West Nile Virus, and the flavivirus of encephalitis viruses.
Term used herein " essential oil fraction " comprises fat-soluble, the water-insoluble compound that derives from or come from elder and relative species, includes but not limited to be divided into the compound of linolelaidic acid (Linoelaidic acid).
Term used herein " essential oil segmentation fraction (sub-fraction) " comprises fat-soluble, the water-insoluble compound that derive from or come from elder and relative species, include but not limited to be divided into the compound of linolelaidic acid, it has the particular compound of finding that improves or reduce concentration in Sambucus species essential oil.
Term used herein " charging (feedstock) " is often referred to unprocessed plant, comprise independent whole plant, or the combination of one or more parts of plant comprise leaf, root (including but not limited to main root, root of the tail and adventitious fiber root (fiber roots)), stem, skin, leaf, really, seed and flower, that wherein said plant or part can comprise is unprocessed, dry, that steam, heating or through the material of Physical Processing, being conducive to processing, its also can comprise intact, that mince, stripping and slicing, that pulverize, grind or through processing with the size that affects plant and the material of physical integrity. Sometimes, term " charging " can be used to expression and extracts product, and this product will be as the feed source of other leaching process.
" flavivirus " is the hypotype (subset) of enveloped virus. They are to be typically found in the animal, and infect human virus by obtaining the bimolecular lipid membrane coating. The example of flavivirus comprises yellow fever, dengue fever, West Nile Virus and encephalitis viruses.
Term used herein " fraction " refers to comprise the extraction composition of one group of specific compound, and described compound is take character certain physics, chemistry or physics or chemical property as feature.
Term used herein " comprises " that the meaning is " including but not limited to ". " comprise " and " including but not limited to " is used interchangeably.
Term used herein " nonenveloped virus " refers to the virus without bimolecular lipid membrane. In nonenveloped virus, virocapsid has mediated the adhesion of host cell and has penetrated. The example of nonenveloped virus comprises norwalk virus, hepatitis A, poliomyelitis and rhinovirus.
Term used herein " one or more compounds " refers at least a compound, such as but not limited to, the polysaccharide molecule of linolelaidic acid (the fat-soluble Chemical Composition of The Essential Oils of Sambucus species) or Cyanidin-3-glycoside (the water-soluble polyphenol of Sambucus species) or Sambucus species, or surpass a kind of compound, for example: linolelaidic acid and Cyanidin-3-glycoside. As known to those skilled in the art, term " compound " does not refer to single kind molecule, and refers to one or more multiple or a large amount of compounds. As known to those skilled in the art, term " compound " refers to have the specific chemical composition of unique chemistry and physical property, and wherein " compound " refers to one or more chemical compositions.
" patient ", " patient " or " host " with the goal approach treatment can be primate (such as the people), Bovidae, sheep section, equine, Suidae, rodent, cat family or canid.
Term " the acceptable salt of pharmacy " is well known in the art, refers to relatively nontoxic, the inorganic and organic acid adduct of compound, comprises for example being included in the present composition those.
Term used herein " polyphenol fraction " comprises the polyphenolic substance of the water-soluble and ethanol solubility that derives from or come from elder and relevant species, includes but not limited to the compound such as rutin and anthocyan-3-glucosides.
Term used herein " polysaccharide fractions " comprises water-soluble-ethanol insoluble plant hemagglutinin matter and the compound of polysaccharide that derives from or come from elder and relevant species.
Other chemical compositions of elder also can appear in these extract fractions.
Term used herein " anthocyanin (proanthocyanins) " refers to dimer, trimer and the tetramer of anthocyanin.
Term used herein " proportioning (profile) " refers to the ratio of quality percentage by weight of compound in extract fraction or the segmentation fraction, or final elder is extracted in three kinds of elder fraction chemical compositions in the composition ratio of every kind quality percentage by weight.
Term used herein " purifying " fraction or extract refer to comprise the fraction or the extract of the one group of specific compound that is concentrated into the 10% quality weight that is higher than described fraction or extract chemical composition, and described compound is a feature with character some physical-chemical property or physics or chemistry.In other words, the fraction of purifying or extract comprise be lower than 80% be not the chemical composition compound of feature with character some the required physical-chemical property that limits described fraction or extract or physics or chemistry.
It is known in the field that term " is worked in coordination with ", refers to that two or more compositions one work, thereby total effect is greater than each composition effect sum.
Term " treatment " is known in the field, refers to treat and improve at least a symptom of any morbid state or illness.
Term " virus " is known in the field, refers to the acellular organism that self lacks metabolic mechanism and breeds by the metabolic mechanism that uses host cell.Virus comprises nucleic acid (DNA or RNA) molecule, and can be enveloped virus or nonenveloped virus.
Composition
The present invention includes and derive from composition one or more Sambucus species, that separate and essential oil (or essential oil segmentation fraction), polyphenol acid and polysaccharide fractions purifying.The combination of these ratios (proportioning) that independently the fraction composition can be specific so that useful complex composition to be provided, and is provided at still undiscovered, reliable or reproducible extraction product in the current known extraction product.For example, essential oil fraction or segmentation fraction from species can be divided combination with essential oil fraction or the subfractionation from same species or different plant species, or with from the combination of the polyphenol of same species or different plant species acid fraction, and this combination can with or do not combine with polysaccharide fractions from same Sambucus species or different Sambucus species.
Useful one or more biological actions required, the Sambucus species composition of extraction according to given product can comprise in the concentrated extract fraction any, two kinds or all three kinds.Usually, the general preferred new compositions that first high-purity Sambucus species that composition that all three kinds of Sambucus species extract fractions is included in the useful chemical composition of all three kinds of main biology of finding in the natural frond as this class representative extract product that comprises.Embodiment of the present invention comprise method, and wherein Yu Ding feature is included in the concentration of the Sambucus species Essential Oil Chemistry composition, polyphenol-anthocyanidin and the polysaccharide that respectively extract preset selection raising in the fraction.
Particularly, at interior known combination thing, the present composition has the anthocyanin content of raising with respect to the composition that is included in the occurring in nature discovery.Anthocyanin is powerful antioxidant, the high activity chemicals close day by day with getting in touch of numerous health advantages, comprises that the protection health avoids heart disease and cancer.Except its antioxidant character, according to reports, anthocyanin also can be used for treating diabetes, and the insulin generation is increased to 50%.The present composition can comprise the anthocyanin of the unique active component of conduct that improves content, and perhaps said composition can comprise other active components relevant with elder.The example of other active components is included in C16 or C18 fatty acid, alcohol or the ester of finding in the essential oil fraction, or the polysaccharide of finding in polysaccharide fractions.
Can concentrate and proportioning anthocyanin and flavonoids by polymer absorbant (PA) technology.The scope of spendable polymer absorbant is very extensive in this class is used, for example: Amberlite XAD4, XAD7HP (Rohm-Hass), Dialon HP20, HP21, SP825 (Mitsubishi), ADS 5, ADS 17 (Naikai university).The operating principle that PA handles is based on " suction of phase patibhaga-nimitta " (absorbate is to keep adhering on the adsorbent or being dissolved into depending on relative intensity in the eluent).This paper provides the embodiment that uses XAD 7HP and ADS5.
The result is as shown in the table:
Table 3 extracts the weight % of back anthocyanin composition
XAD7HP polymer absorbant ADS5 polymer absorbant
Charging F2 F3 F4 F5 F6 F2 F3 F4
Total anthocyanin 0.06 2.43 2.99 2.92 1.29 0.80 2.2 0.03 0.003
CY-3，5-GLU 0.02 1.04 0.83 0.56 0.06 0.07 0.8 0
CY-3-SAM 0.01 0.37 0.48 0.45 0.22 0.14 0.33 0
CY-3-GLU 0.04 1.01 1.67 1.91 1.01 0.59 1.06 0.03 0.003
Rutin 0.27 0.23 2.60 5.74 16.28 17.01 0.41 29.12 11.2
Total polyphenols acid 1.55 27.81 31.02 40.49 31.87 36.87 41.8 34.3 20.7
Table 4 anthocyanin proportioning
XAD7HP polymer absorbant ADS5 polymer absorbant
Charging F2 F3 F4 F5 F6 F2 F3 F4
Total anthocyanin 88.1 100.0 100.0 100.0 100.0 100.0 100 100 100
CY-3，5-GLU 25.8 43.0 27.9 19.2 4.8 9.1 36.6 11.5
CY-3-SAM 9.0 15.3 16.1 15.5 16.8 16.9 15 11.1
CY-3-GLU 53.2 41.8 56.0 65.3 78.3 73.9 48.4 77.4 100
Table 5 rutin is to the ratio of total anthocyanin
XAD7HP polymer absorbant ADS5 polymer absorbant
Charging F2 F3 F4 F5 F6 F2 F3 F4
Rutin is to total pattern 3.8 0.10 0.87 1.96 12.5 21.3 0.20 885 3267
The ratio 81 of plain glycosides
Table 6 proportioning %
XAD7HP polymer absorbant ADS5 polymer absorbant
Feed F2 F3 F4 F5 F6 F2 F3 F4
Total anthocyanin 4.6 8.7 9.6 7.2 4.1 2.2 5.3 0.1 0.02
CY-3，5-GLU 1.2 3.8 2.7 1.4 0.2 0.2 1.9 0
CY-3-SAM 0.4 1.3 1.5 1.1 0.7 0.4 0.8 0
CY-3-GLU 2.5 3.6 5.4 4.7 3.2 1.6 2.5 0.1 0.02
Rutin 17.6 0.8 8.4 14.2 51.1 46.2 1 84.9 54.1
The percentage by weight of compound tells us in processing procedure, and compound is purified the degree of (concentrating): anthocyan-3,5-glucosides are purified to 56.2 times (F2 XAD7HP PA) in the charging; Anthocyan-3-elder bioside is purified to 74 times (F3, XAD7HP PA) in the charging; Anthocyan-3-glucosides is purified to 50 times (F4, XAD7HP PA) in the charging; Total anthocyanin is purified to 46-47 times (F2 and F3 XAD 7HP PA) in the charging; Rutin be purified to 107 times (F3, ADS5 PA) in the charging and total phenolic acid be purified in the charging 13-17 doubly.
Anthocyanin proportioning data show, can adjust the proportioning of anthocyanin in processing procedure: the proportioning of anthocyan-3-glucosides is adjusted to 42%-100%; The proportioning of anthocyan-3-elder bioside can tune to 9%-17%; Anthocyan-3, the proportioning of 5-glucosides can tune to 4.8%-43%.
Rutin and anthocyanin are medical compounds important in the Sambucus species.In processing procedure, rutin is whole to the adjustable ratio of total anthocyanin to be 0.10-3267.
In processing procedure, also can adjust the anthocyanin in the total polyphenols acid and the proportioning of rutin concentration: the proportioning of anthocyan-3-glucosides can tune to 0.02-5.4%; The proportioning of anthocyan-3-elder bioside can tune to 0-1.5%; Anthocyan-3, the proportioning of 5-glucosides can tune to 0-3.8%; The proportioning of total anthocyanin can tune to 0.02-9.6%; The proportioning of rutin can tune to 0.8-84.9%.
In one embodiment, the present composition comprises the anthocyanin that improves content and pharmaceutical carrier as described below.In another embodiment, the present composition comprises another kind of elder composition, as from the C16 of essential oil fraction with C18 is saturated and unsaturated fatty acid, pure and mild ester.
Being compared as follows shown in the table between the data in literature (Toulemonde 1983) of dry flores sambuci volatile ingredient and the present research:
The comparison of table 7 data in literature and experimental data
The data in literature experimental data
Fatty acid essential oil isopentane ethanol T4P1 T4P3 T4P5 T6P3 T6P5 T8P3 T8P5
The extract concentrate
Hendecanoic acid 3 0.06 0.19 0.35 2.78 0 0.87 1.6
Dodecylic acid 2 0.07 0.31 0.3 1.02 0.87 0.74 0.92
Myristic acid 2.1 0.4 1 0.17 0.15 00 0.12 0.29 0.5
Pentadecanoic acid 0.8 0.2 1.2 0.13 0.29 0 0.92 0.42 00
Palmitic acid 37.8 16.6 19.4 20.57 15.04 11.91 11.79 22.36 22.64 21.39
Octadecanoid acid 0.4 0.7 0.7 6.36 5.95 4.63 3.53 5.58 7.51 4.56
Oleic acid 5.7 7.9 18 5.91 8.82 6.08 8.54 9.14 12.71 8.01
Linoleic acid 9 17.5 19 31.99 5.24 11.33 5.65 2.87 11.93 4.89
Linolenic acid 9.1 24 16 2.73 2.12 00 1.88 2.3 1.31
Linolelaidic acid 22.11 8.5 1.33 1.52 2.38 1.19
Total fatty acids 69.9 67.3 75.3 71.41 17.81 17.54 9.77 8.8 22.62 10.08
The present composition can comprise anthocyanin and the polysaccharide that improves content.In the water crude extract, the protein yield in the flores sambuci is 0.09%, and the protein yield in the elder berry is 0.59%.Ethanol by 80% can make 95% protein precipitation in the crude extract.Therefore, 80% sediment is a glycocalix.The mean molecule quantity of these compounds is～2000KDa.In one embodiment, described composition comprises phytohemagglutin phytolectin-polysaccharide fractions composition, the purity based on colourity (colormetric) analytical method of said composition is 100-170mg/g dextran equivalent, is higher than 4-50% quality weight based on the phytohemagglutin phytolectin purity of protein of the Bradford analysis of protein of the present invention instruction.
The present composition can comprise the saturated or unsaturated fatty acid of the anthocyanin, C16 or the C18 that improve content, pure and mild polysaccharide.
Extract with respect to natural Sambucus species
The present composition also can define by the relative concentration with the composition of finding in natural Sambucus species.For example, the concentration of essential oil is 0.001 to 10000 times of natural Sambucus species concentration, and/or the concentration of required polyphenol acid is 0.001 to 40 times composition of natural Sambucus species concentration, and/or the concentration of water-soluble-ethanol insoluble polysaccharide is 0.001 to 40 times composition of natural Sambucus species concentration, and/or the phytohemagglutin phytolectin protein concentration is 0.001 to 100 times a composition of natural Sambucus species concentration.The present composition comprises that essential oil concentration is 0.01 to 10000 times composition of natural Sambucus species concentration, and/or required polyphenol acid concentration is 0.01 to 40 times a composition of natural Sambucus species concentration, and/or polysaccharide concentration is 0.01 to 40 times composition of natural Sambucus species concentration, and/or the phytohemagglutin phytolectin protein concentration is 0.01 to 100 times a composition of natural Sambucus species plant corpus concentration.In addition, the present composition comprises the segmentation fraction of Essential Oil Chemistry composition, this Essential Oil Chemistry composition has at least a or multiple compound that is present in the natural frond essential oil, and the content of these compounds is higher or lower than the content in natural elder plant corpus Essential Oil Chemistry composition.For example, described compound, the concentration of linolelaidic acid in essential oil segmentation fraction can 2% quality weight of total Essential Oil Chemistry composition be increased to the 22% quality weight of segmenting fraction from account for natural elder plant corpus, and concentration has increased by 10 times.With it relatively, the concentration of linolelaidic acid in essential oil segmentation fraction can be from account for natural elder plant corpus about 2% quality weight of total Essential Oil Chemistry composition, be reduced to the 0.01% quality weight that is lower than the segmentation fraction, concentration has reduced by 100 times.The present composition comprises following composition, and the concentration in the natural elder Essential Oil Chemistry of the concentration ratio composition of the specific compound in said composition in the novel essential oil segmentation of this class fraction improves about 1.1 to about 10 times, or reduces about 0.1 to about 100 times.
Extract purity
In the process of carrying out aforementioned extracting method, find at essential oil SCCO 2Can extract the dried fruit of the original Sambucus species that surpass 80% quality weight yield or the Essential Oil Chemistry composition in the flower charging in the extract fraction, it has the Essential Oil Chemistry composition (step 1A) that surpasses 95% purity.Use the method for instructing among step 1A and the 1B, because the segmentation fraction of Essential Oil Chemistry composition becomes the high-purity essential oil segmentation fraction with new chemical composition proportioning, thereby the output of described essential oil may reduce.In addition, the SCCO of the present invention's instruction 2Extract and the feasible ratio (proportioning) that can change each compound that constitutes Essential Oil Chemistry composition fraction of fractional method, thereby be the concrete unique essential oil subfractionation distribution ratio of goals of medicine structure.For example, the concentration that can improve the ether oil chemical composition reduces the concentration of fatty acid cpds simultaneously, and vice versa.
The method of using step 2 of the present invention to instruct, the water alcohol leaching fraction that quality weight yield with 35.6% obtains from the original Sambucus species charging of total phenolic acid with 4.3% concentration, the yield of the phenolic acid chemical composition in the natural elder berry charging is about 60% quality weight.In addition, this water-alcohol solvent extract also comprises valuable anthocyanidin chemical composition.
The method (affinity adsorbent extraction method or on-line chromatograph method) of using step 3 of the present invention to instruct can obtain 40% dry mass that purity surpasses the extract fraction, and wherein anthocyanidin surpasses the polyphenol fraction of 2.5% quality weight.May from the charging of water alcohol leaching extract, extract about 60% of polyphenol acid.This is equivalent to the polyphenol acidifying composition in the natural Sambucus species plant corpus of 40% yield.Also may produce and contain high concentration phenolic acid (〉 30% quality weight), with anthocyanidin (〉 the 2.9% quality weight of relative high concentration) or the relative phenolic acid segmentation fraction of the purifying of the anthocyanidin (＜0.05% quality weight) of low concentration.
The method (water extraction and ethanol precipitation) of using step 4 of the present invention to instruct, can in phytohemagglutin phytolectin-polysaccharide fractions, extract and purifying above water-soluble-ethanol insoluble plant hemagglutinin matter and the polysaccharide chemistry composition in the original dry Sambucus species feed material of 90% quality weight yield.Use 80% ethanol to precipitate phytohemagglutin phytolectin and polysaccharide, can from the water extraction extract, collect the phytohemagglutin phytolectin-polysaccharide fractions of purifying.The yield of phytohemagglutin phytolectin-polysaccharide fractions is the about 3.45% quality weight based on the charging of natural elder plant corpus.Based on the colorimetric analysis method of using dextran as the reference standard, can obtain purity of polysaccharide is 100-170mg/gm dextran equivalent.Based on Bradford protein analysis method, can obtain phytohemagglutin phytolectin purity is 16% quality weight of fraction.Obtainable evidence shows that compound remaining in the fraction is polysaccharide (about 83% quality weight).Use 60% precipitation with alcohol, can make phytohemagglutin phytolectin albumen reduce to about 5%, perhaps by after extracting at 60% precipitation with alcohol and polysaccharide, residual solution is carried out stage by stage 80% precipitation with alcohol, phytohemagglutin phytolectin albumen is further brought up to account for about 50% quality weight of segmentation fraction.
At last, the method of the present invention instruction makes described Sambucus species Essential Oil Chemistry composition fraction, novel polyphenol fraction or segmentation fraction, can reach high required chemical composition in the essential oil fraction with the purity (concentration) of novel plant hemagglutinin-polysaccharide fractions to 99% quality weight, high phenolic acid in the phenolic acid fraction to 41% quality weight, high in the polyphenol fraction to 3% anthocyanidin, high phytohemagglutin phytolectin in phytohemagglutin phytolectin-polysaccharide fractions, and high polysaccharide in phytohemagglutin phytolectin-polysaccharide fractions to 90% quality weight to 50% quality weight.Employed concrete extraction environment, extraction rate, solvent and extractive technique depend on the original chemical composition proportion of raw material and finally extract the required purity level of product.Those skilled in the art only need adopt normal experiment can determine the concrete grammar that the present invention instructed at an easy rate, and the experiment of this class is generally used for method of adjustment, to adapt to the sample deviation of raw material attribute that one-tenth to be processed has the output material of particular community.For example, in the Sambucus species plant corpus of big measuring, use the method that well known to a person skilled in the art of the present invention's instruction, can determine the initial concentration of Essential Oil Chemistry composition, polyphenol acid, anthocyanidin, phytohemagglutin phytolectin and polysaccharide.Those skilled in the art can determine, for arriving required concentration and/or stoicheiometry in the final Sambucus species composition product, from the initial concentration of Essential Oil Chemistry composition to the Essential Oil Chemistry composition scheduled volume of the final extraction product that for example adopts extracting method described herein or the variable quantity of distribution (proportioning).
The segmentation fraction
Another embodiment of the present invention is the composition that comprises the novel segmentation fraction of described Essential Oil Chemistry composition, wherein raises or reduction such as, but not limited to the concentration in each comfortable this novel extraction composition product of particular chemical group of alcohol, aldehyde, ester or fatty acid.
Another embodiment of the present invention is the composition that comprises the novel segmentation fraction of described purifying polyphenol chemical composition, wherein raises or reduction such as, but not limited to concentration in each comfortable novel extraction composition of particular chemical group of anthocyanidin.
Another embodiment of the present invention is the composition that comprises the novel segmentation fraction of described purifying phytohemagglutin phytolectin-polysaccharide chemistry composition, wherein raises or reduction such as, but not limited to the concentration in each comfortable novel extraction composition of particular chemical group of phytohemagglutin phytolectin.
Extracting method
The inventive method provides the novel elder composition of treatment and prevention human diseases.For example, with comparing of in Sambucus natural frond or generally well-known extraction product, finding, the essential oil fraction composition concentration (weight %) that the novel Sambucus species composition of treatment influenza can have the polyphenol fraction composition concentration (weight %) of raising, the polysaccharide composition concentration (weight %) that improves and reduce.With comparing of in natural Sambucus species plant corpus or generally well-known extraction product, finding, the novel Sambucus species composition that is used for antioxidant, anti-angiogenic damage and ischemic cerebrovascular disease can have the essential oil of raising and the polysaccharide fractions composition (weight %) of polyphenol fraction composition (weight %) and reduction.With comparing of in natural Sambucus species plant corpus or generally well-known extraction product, finding, another example that is used for the treatment of the novel Sambucus species composition of diabetes comprises the polysaccharide composition of the polyphenol fraction composition concentration of raising, reduction and the essential oil fraction composition that reduces.
Other embodiment comprises the composition that the proportioning (ratio distribution) of Sambucus species chemical composition changes with respect to the proportioning of finding in natural frond or existing Sambucus species extraction product.For example, with respect to polyphenol acid and/or polysaccharide concentration, described essential oil fraction increases or reduces.Similarly, with respect to other extract component fractions, described polyphenol acid or polysaccharide can increase or reduce, and make to have the novel components stoicheiometry composition that is used for the particular organisms effect.By in conjunction with in essential oil, polyphenol and/or the polysaccharide one or more separation and the fraction of purifying, can make new compositions.
The following method of being instructed can be used separately, or is used in combination with the disclosed method or the method that well known to a person skilled in the art.
The raw material that is used to extract is the plant corpus from one or more Sambucus species.This plant corpus can be any position of described plant, yet fruit and flower are most preferred raw material.
The present invention's expection, described Sambucus species plant corpus can make described material become any particular form and any form that helps extracting through step before extracting.Step included but not limited to before this class was extracted, and mince, shred, tear up, mill, grind, cut, or the step of the described material of tearing, and before extraction before the step, described raw material be drying or fresh plant corpus.Step comprises and described Sambucus species plant corpus is milled and/or grinds to form fine powder before preferred the extraction.Before extraction after the step, with described raw material or material is dry or to wherein adding moisture.In case described Sambucus species plant corpus becomes the form that is used to extract, extracting method is desired for the present invention.
The supercritical fluid extraction of elder
Extracting method of the present invention comprises method disclosed herein.Usually, method of the present invention comprises following method as a part, uses carbonic acid gas as solvent (SCCO in this method 2) use supercritical liq extraction (SFE) that elder species plant corpus is extracted, carry out one or more solvent extraction step subsequently, such as but not limited to water, water alcohol and affinity polymer absorbing agent leaching process.The additive method of the present invention expection comprises and uses other organic solvents, refrigeration chemicals, compressible gas, ultrasonic, fluid under pressure extraction, high speed adverse current chromatogram, molecularly imprinted polymer, and other known extraction methods are extracted synthetism wood plant body.This class technology is known by the art technology people.On the one hand, can prepare composition of the present invention by the method that comprises step shown in Fig. 1-4.
The present invention includes and use SCCO 2Technology concentrates (purifying) and the proportioning method from essential oil He other fat-soluble compounds of elder plant corpus.The fat-soluble chemical composition that the present invention includes elder is classified into, for example, and highly purified essential oil fraction (high Essential Oil Chemistry constituent concentration).In addition, the present invention includes SCCO 2Method, wherein each chemical composition in the extract fraction can have different chemical composition ratio or proportioning.For example, SCCO 2Feasible some essential oil compounds of the preferred extraction of other essential oil compounds relatively of chemical composition in the fractionated essential oil fraction, thus the essential oil thing extraction segmentation fraction that some compound concentration is higher than other compound concentrations can be produced.Instruct as the present invention, use SCCO 2The Essential Oil Chemistry composition of described Sambucus species extracted do not use poisonous organic solvent, and the fractionation of extract is provided simultaneously.Carbonic acid gas is the composition in natural and biologic safety and the many F﹠B.
Fig. 1-4 has shown the schematic diagram of the extracting method of elder biologically active chemical composition.This extracting method normally but is not limited to 5 steps.At embodiment the analytical method of using in the extracting method has been described partly.
Step 1: the supercritical fluid carbon dioxide extraction of elder essential oil
Because the hydrophobicity of essential oil, this extracting method can use non-polar solution, includes but not limited to SCCO 2, hexane, benzinum and ethyl acetate.Because some composition tool volatility of essential oil, steaming also can be used as extracting method.
Fig. 1 has described use SCCO 2Extract the Essential Oil Chemistry composition of Sambucus species rhizome.Charging 10 is dry elder berry of milling or flower (about 140 orders).Extracting solvent 210 is pure carbon dioxide.Ethanol can be used as cosolvent.Described charging is packed in the SFE extraction vessel 20.After purging the test of (purge) and seepage, described method comprises the CO that makes liquefaction 2Flow to CO from hold-up vessel by cooler 2Pump.With this CO 2Be compressed to required pressure, and flow through the charging in the extraction vessel, wherein pressure and temperature maintains desired level.The pressure limit of extracting is clung to 800 from about 60 and is clung to, and temperature range is from about 35 ℃ to about 90 ℃.SCCO teaching herein 2Extract preferably under the temperature of the pressure of at least 100 crust and at least 35 ℃ and carry out, more preferably cling to 500 pressure that cling to and about 40 ℃ and under about 80 ℃ temperature, carry out about 60.The extraction time that single phase extracts is from about 30 minutes to about 2.5 hours, by about 1 hour.Solvent to the ratio of charging usually to each SCCO 2Be extracted as about 60 to 1.Recycle described CO 2Collect extracted, Essential Oil Chemistry composition 30 purifying and proportioning with collector or separator then, in the lucifuge vial, keep, and be stored in 4 ℃ of unglazed refrigerators.Elder charging 10 materials can use one-step method (Fig. 1) to extract, and wherein the elder essential oil fraction 30 of gained extraction and purifying is collected in single collector SFE or SCCO 2In the system 20, perhaps extract (Fig. 1, step 1B) with multistep processes, wherein the elder essential oil of purifying that is extracted and proportioning segmentation fraction 50,60,70,80 is collected in respectively in the single collector SFE system 20 successively.Selectively, in classification SFE system, described SCCO 2The elder feed material of extracting is isolated in a plurality of collection containers (separator), thereby in each collector, the Essential Oil Chemistry that the purifying essential oil of every kind of collection segmentation fraction has different relative percentages becomes to be grouped into (proportioning).Collect, preserve described residue (residue) 40, and be used for further handling to obtain the purifying fraction of acid of Sambucus species polyphenol and polysaccharide.An embodiment of the present invention is included in 60 and clings under the pressure of 500 crust and 35 ℃ to 90 ℃ the temperature and use multistep SCCO 2Extraction method is extracted Sambucus species feed material, and collects the elder material that is extracted after per step.Second embodiment of the present invention is included in 60 and clings under the pressure of 500 crust and 35 ℃ to 90 ℃ the temperature and use classification SCCO 2Extraction method is extracted described Sambucus species feed material, and with under predetermined condition (pressure, temperature and density) and the predetermined space (time), collects the elder material that is extracted in different collection containers.From each multistep extractor or be collected in the elder purifying essential oil subfractionation that the gained in the different acquisition container (fractionating system) extracts and divide composition to reclaim separately and to use, perhaps make up to form one or more elder essential oil compositions, said composition comprises the predetermined Essential Oil Chemistry constituent concentration that is higher or lower than the concentration of finding in natural frond or conventional elder extraction product.Usually, use the maximum SCCO of single step 2The essential oil fraction total recovery that extraction method really obtains from the Sambucus species is the Essential Oil Chemistry composition of about 9 weight % (〉 95%), Essential Oil Chemistry composition purity is higher than 95% quality weight of described extract.In contrast, use the maximum SCCO of single step 2The essential oil fraction total recovery that extraction method obtains from Sambucus species flower is the Essential Oil Chemistry composition of about 1.5 weight % (〉 95%), Essential Oil Chemistry composition purity is higher than 95% quality weight of described extract.These digital proofs described elder berry compare the essential oil compounds that comprises about 6 times of concentration with flores sambuci.As embodiments of the invention, use elder berry as natural Sambucus species feed material.In embodiment 1, can find the example of this extracting method.
In this experiment embodiment, use elder berry as charging, set described extraction conditions, wherein temperature range is 40-80 ℃, pressure limit is the 80-500 crust.Described CO 2Flow rate is 10gm/min.Experimental result is shown in table 8 and 9.
Table 8. temperature, pressure and time are to using the SCCO of elder berry as charging 2The effect of essential oil recovery rate
Figure A200780009471D00391
The table 9. elder berry SCCO that (pressure that temperature-T and Israel and Palestine are represented) extracts under different SFE conditions 2Essential oil extracts the GC-MS chemical composition of fraction
Figure A200780009471D00401
Figure A200780009471D00411
Figure A200780009471D00421
Table 10. is by the elder berry essential oil compounds of GC-MS identification
Figure A200780009471D00432
Figure A200780009471D00441
Figure A200780009471D00451
Figure A200780009471D00461
These result verification pressure to extracting dynamic (dynamical) effect.
Higher extraction pressure causes system to arrive balance with the short time, consumes more a spot of CO 2Owing to density increases along with pressure raises, total recovery rate increases along with the rising of extracting pressure.What is interesting is that as the 100-300 crust, temperature is low more under lower pressure, because the higher yield of density is high equally more.Under higher pressure, as the 300-500 crust, temperature is much smaller to the effect of extract yield.Though under the pressure that surpass 300 crust, reach higher extract yield and bigger extraction efficiency, but under the temperature of the pressure that are lower than 300 crust and about 40-60 ℃, the purity of Essential Oil Chemistry composition can reach 95%.
In the trial stretch of research, clearly illustrated between temperature and density to have competitive effect.This respect has sufficient definition and record in the literature, and wherein because the solvability of overcritical and near critical fluids improves, under stationary temperature, the pressure increase causes yield to increase.The increase of temperature impels the increase that helps the compound steam that extracts to press.In addition, along with temperature is brought up to high value, the increase of diffusion coefficient and the reduction of solvent viscosity also help from the draft porous matrix extracts compound.On the other hand, under constant system pressure, the increase of temperature causes the reduction of solvent density.
According to the mass spectrum of every kind of compound, use the GC-MS analytical method can from the elder berry essential oil, separate and identify 67 kinds of compounds (table 9 and 10).Described compound changes in 23 carbon compounds (C23) scope at 7 carbon compounds (C7), comprising: retention time is 9 kinds of aldehydes (C7-C15) of 7-50 minute, is mainly unsaturated C7 and C10 aldehyde (the compound #1,2 in the table 5,6 and 8); 111 kinds of alcohols (C13-C20); 12 kinds of ester classes (C13-C22); 7 kinds of fatty acid (C14-C22); With other aromatic series and aliphatic compounds.Based on known biologically active, most important compound is saturated and unsaturated fatty acid, alcohol and the ester thereof of C16 and C18.For example, hexadecanol (#30), hexadecylic acid (#34), methyl palmitate (#32), ethyl palmitate (#35) and hexadecylic acid butyl ester (#52) all belong to the C16 compound.Saturated octadecanoid acid and ester thereof (octadecanoid acid ethyl ester (#53) and butyl octadecanoate), monounsaturated fatty acids 9-vaccenic acid-1-alcohol isomer (#38,39), polyunsaturated fatty acid 9,12-octadecadienoic acid isomer (#46,48) belongs to the C18 compound.The common first names of C16 and C18 fatty acid is palmitic acid and stearic acid
In table 9, the compound that highlights is the compound of the higher concentration found in the essential oil fraction.The ratio that should be noted that described compound is with different SCCO 2Extraction conditions and changing.For example, under the low pressure such as 100 crust, C16 and C18 fatty acid are in higher concentration, yet total recovery rate is lower.In contrast, it is higher to extract the concentration of finding C16 and C18 fatty acid ester under the temperature at height.
Be that the squalene of described extraction has about 23% high concentration in the essential oil fractions of 40 ℃ and 300 crust, and has about 8% low concentration in 40 ℃ and 500 fractions of clinging to enjoyably.Squalene is studied as the supplemental treatment of some human cancer.In animal model, proved that it can suppress lung cancer effectively.In animal model, shown that also it has the chemoprophylaxis effect to colon cancer.In animal model, show that replenishing squalene can improve immunologic function and cholesterol reducing level.
In a word, the concentration of some Sambucus species Essential Oil Chemistry composition can change along with the difference of used SFE condition.By using as step 1B, the SCCO of multistep in proper order shown in Figure 1 2Fractional method or many collectors fractionating system, the SFE extraction property that this class is different can be used to further improve or reduce some compound concentrations in the purifying essential oil segmentation fraction.
Step 2 is extracted the water alcohol leaching of crude phenols acid fraction and is followed the example of
On the one hand, the present invention comprises the extract and the concentrate of biologically active phenolic acid chemical composition, has kept simultaneously to be used for extracting separately and purifying (phytohemagglutin phytolectin of the residue of step 4) and polysaccharide.Fig. 2 illustrates the generality of this step and describes.The extracting method of step 2 is a solvent leaching method.The charging of this extraction is the Essential Oil Chemistry composition SCCO of dried plant corpus 10 of milling of Sambucus species or step 1 2The residue 40 that extracts.Extracting solvent 220 is hydrous ethanol.Extract the hydrous ethanol that solvent can be 10-95%, 80% hydrous ethanol is preferred.In the method, pack described elder feed material and described extraction solvent into heating and the extraction vessel 100,150 that stirs in.Can be heated to 100 ℃, about 90 ℃, about 80 ℃ about 70 ℃ or about 60-90 ℃.The about 1-10 of this extraction hour, about 1-5 hour, about 2 hours.Fluid-the extract of gained is filtered 110 and centrifugal 120.Collect filtrate (supernatant) 310,320,330 as product, behind the described solvent of evaporation, measurement volumes and dry weight solids content.Keep and store described extraction residual materials 160 and be used for further processing (referring to step 4).If necessary or desired, repeat this extraction for several times.Can repeat 1 time or repeatedly, 2 times or more times, 3 times or more times, or the like.For example, Fig. 2 has shown three-step approach, and wherein second step was used identical method and condition with the 3rd step.The example of having showed this extraction step among the embodiment 2.The result as shown in figure 11.
Table 11 elder berry lixiviate crude phenols acid yield and purity
Figure A200780009471D00481
Total crude phenols acid extractants yield is about 35% quality weight of original natural elder berry charging, total phenolic acids extraction yield be 1.6% and phenolic acid purity be 4.3% quality weight of described fraction.Anthocyanidin extract yield in the described crude phenols acid fraction is 0.06% a quality weight of original elder berry charging, and purity (concentration) is 0.18 quality weight of described fraction.Main phenolic acid is a rutin, and main anthocyanidin is anthocyan-3-glucosides.These data all with document in consistent.This crude phenols acid composition can be used as end product, or the charging that conduct is further handled is with the required phenolic acid chemical composition (step 3) of purifying.
Step 3. affinity adsorbent extraction method
As teaching herein, can contact with the affine polymer absorbant resin of solid by water-alcohol extraction the elder charging, so that active phenolic acid contained in the described water-alcohol extraction is adsorbed onto on the affinity adsorbent, obtain the elder of purifying and the phenolic acid fraction extract of relevant species.Chemical composition by the described combination of method wash-out teaching herein subsequently.Before the described phenolic acid fraction of wash-out chemical composition, available any mode easily, the affinity adsorbent that will have the required chemical composition of absorption separates with the extract residue, preferably, realize separating with described with the process of adsorbent contact by making aqueous extract flow through extraction column or sorbent material bed.
Various affinity adsorbents all can be used to the phenolic acid chemical composition of purifying Sambucus species, such as but not limited to: " Amberlite XAD-2 " (Rohm ﹠amp; Hass), " Duolite S-30 " (Diamond Alkai Co.), " SP207 " (Mitsubishi Chemical), ADS-5 (Nankai University, China Tianjin), ADS-17 (Nankai University, China Tianjin), Dialon HP 20 (Mitsubishi, Japanese) and Amberlite XAD7 HP (Rohm ﹠amp; Hass).Because the high-affinity to the phenolic acid chemical composition of elder and relevant species preferably uses AmaberliteXAD7 HP.
Though can use various eluents to reclaim described phenolic acid chemical composition from described adsorbent, on the one hand, this eluent comprises low-molecular-weight alcohol, includes but not limited to methyl alcohol, ethanol or propyl alcohol.Second aspect, described eluent is included in the low mass molecule alcohol in the aqueous mixture.On the other hand, described eluent comprises low-molecular-weight alcohol, second organic solvent, and water.
Preferably, one or more prepurification processes have been experienced in the charging of described Sambucus species, such as but not limited to, describe in the step 1 and 2, in aqueous phenol acidifying composition that will comprise extract and process before described affinity adsorbent material contacts.
Use as the affinity adsorbent that the present invention instructed, obtain high-purity Sambucus species phenolic acid chemical composition, it does not obviously contain other chemical compositions that are present in usually in natural frond or the commercially available extraction product.For example, the method that the present invention instructed can obtain the phenolic acid extract of purifying, and it comprises total phenolic acid chemical composition that surpasses 40% dry mass weight and the total anthocyanidin that surpasses 2% dry mass weight.
Fig. 3 diagram has shown that use polymer affinity adsorbent resin bead is extracted and the generality of purifying phenolic acid is described from Sambucus species leaf.The charging of this extracting method can be the flooding 310+ that comprises from step 2/-ethanol water of the phenolic acid of 320+/-330.Before the post 410,420 of packing into and afterwards, with the absorbent resin pearl (every mg polymeric adsorbent 5mg phenolic acid) of 4-5BV ethanol 230 and the suitable weight of 4-5BV distilled water 240 washings.The aqueous solution 310+320 that will comprise phenolic acid subsequently with 3-5 bed volume (BV)/hour flow rate be loaded on the post 430.In case this post is filled, with distilled water 250 flow rate washing 450 these posts, to remove any impurity in the adsorbed phenolic acid with 2-3BV/ hour.Collect the thing residue 440 and the debris 460 that flow out, measure its mass content, phenolic content, and abandon.As elute soln 260,,, realize the wash-out of absorption phenolic acid 470 with 40 or 80% ethanol/water in degree such as grade (isocratic) mode with 3-4BV/ hour flow rate, and the elution curve of record wash-out extract (multiple extract) 480.Can collect elution volume 480 in per approximately 25 minutes, and use HPLC to analyze these samples, and measure its solids content and purity.In embodiment 3, can find the example of this extracting method.The result is shown in table 12 and 13.
The mass balance and the HPLC analysis result of table 12. different fractions of wash-out from XAD 7HP post
Figure A200780009471D00491
Figure A200780009471D00501
The mass balance of table 13. different fractions of wash-out from the ADS5 post and HPLC analysis result
Figure A200780009471D00511
As teaching herein, described affinity adsorbent XAD7HP and ADS5 can be further purified the flavonoids (flavanoid) and the anthocyanidin phenolic acid of (concentrating) Sambucus species plant corpus.The purity of total phenolic acid, total anthocyanidin and rutin surpasses 40%, 2.8%, 29% quality weight of wash-out segmentation fraction separately.This explanation has improved above 10 times with specific concentration mutually that find in the Sambucus natural frond or known, or has improved above 5 times with the specific concentration of finding in existing elder extraction product mutually.In eluent, reclaimed the phenolic acid chemical composition that surpasses 60% quality weight yield in institute's filling solution.Based on original elder charging, total phenolic acid yield is about 4.2% quality weight of original feed material.In fact, in effluent or wash solution, almost detect less than rutin or anthocyanidin.What is interesting is that ADS5 has very unique advantage, promptly rutin is separated with anthocyanidin by using Different concentrations of alcohol solution to segment in the fraction in difference.For example, described ADS5 40% ethanol elution fraction (F2) has concentrated anthocyanidin and has surpassed 10 times, and the segmentation fraction (F3+F4) of combination has concentrated rutin and surpasses 25 times, and anthocyanidin concentration is few or do not have.Therefore, the affinity adsorbent method of step 3 can produce the novel purifying phenolic acid subfractionation branch with new chemical composition proportioning.
Step 4. phytohemagglutin phytolectin-polysaccharide fractions extraction method
Phytohemagglutin phytolectin-polyoses extract the fraction that will connect thing bone wood species chemical composition in the scientific and technical literature is defined as " the insoluble extract fraction of water-soluble, ethanol ".Fig. 4 illustrates the generality of using aqueous solvent leaching and ethanol precipitation to extract polysaccharide fractions from Sambucus species extract and describes.Charging 160 is the solid residue from the water alcohol extraction of step 2.With this charging of lixiviate of two steps.Described solvent is a distilled water 270.Use the method, with described Sambucus species residue 160 with extract solvent 270 and pack in the extraction vessel 500,520, and heating and stirring.Can be heated to 100 ℃, to about 80 ℃ or to about 70-90 ℃.The about 1-5 of this extraction hour, about 2-4 hour or about 2 hours.Described two steps are extracted solution 600+610 combination, and with dope filtration 540, centrifugal 550 and evaporate 560 to remove moisture, the chemical composition concentration in solution 620 improves about 8 times.Use the recombinate solution of initial volume of absolute ethyl alcohol 280 then, make the ultimate density of ethanol arrive 60-80%.Observe a large amount of sediment 570.With solution centrifugal 580, pour out 590, and abandon supernatant residue 730.Precipitated product 640 is the phytohemagglutin phytolectin-polysaccharide fractions of purifying, can use with molecular weight 5, and 000-410,000 dextran is analyzed its polysaccharide as the chromatmetry of reference reference material, and uses Bradford analysis of protein method to analyze its protein.The purity of the polysaccharide fractions that is extracted is about 100-170mg/g dextran standardization equivalent, and total recovery is the 2.4-3.5% quality weight of original natural elder plant corpus charging.The purity of the phytohemagglutin phytolectin protein that is extracted is about 16% quality weight of phytohemagglutin phytolectin-polysaccharide fractions, and total recovery is 0.56% a quality weight of original natural elder plant corpus charging.Embodiment 4 has provided the example of this method.The result is shown in table 14 and 15.In addition, use AccuTOF-DART mass spectrum (referring to the embodiment part) to come further proportioning to constitute the molecular weight of the compound of described purified polysaccharide fraction.
The polysaccharide analysis of table 14. elder berry phytohemagglutin phytolectin-polysaccharide fractions
The protein analysis of table 15. elder berry phytohemagglutin phytolectin-polysaccharide fractions
Sample Lipidated protein (%) Protein yield (%)
The water crude extract of elder berry 5.63 0.59
60% sediment of elder berry 4.81 0.12
80% sediment of elder berry 16.17 0.56
The total elder phytohemagglutin phytolectin-polysaccharide yield of 60% precipitation with alcohol and 80% precipitation with alcohol is respectively 2.43% and 3.45% quality weight of original natural elder berry feed material.Based on the test of many times and the scientific and technical literature of Sambucus species plant corpus and other plant medicinal material, the water-soluble-ethanol insoluble polysaccharide in 3.5% yield of phytohemagglutin phytolectin-polysaccharide fractions and the raw material Sambucus species plant corpus is very close with the concentration of phytohemagglutin phytolectin protein.
The purity of described polysaccharide is 100-170mg/gm dextran equivalent.Though as if the dextran equivalent of polysaccharide fractions slightly lower with comparing of finding, also do not know the molecular weight of polysaccharide in the Sambucus species plant corpus in coming from the purified polysaccharide fraction of other plant medicinal material.Therefore, the purity of the polysaccharide chemistry composition in the Sambucus species purified polysaccharide fraction may be than using estimated much higher of dextran equivalent colorimetric analysis.
The purity of the phytohemagglutin phytolectin protein of the elder phytohemagglutin phytolectin-polysaccharide fractions of 60% precipitation with alcohol and 80% precipitation with alcohol is respectively 4.8% and 16.2% quality weight of this fraction.Total phytohemagglutin phytolectin protein yield of 80% precipitation with alcohol is 0.56% quality weight of original natural elder charging and about 95% quality weight of thick flooding extract.Total phytohemagglutin phytolectin yield of 60% precipitation with alcohol only is about 20% quality weight of thick flooding extract.60% precipitation with alcohol obtains higher polysaccharide chemistry composition purity and lower phytohemagglutin phytolectin lipidated protein.Therefore, use described two step precipitation with alcohols, can realize following result: use that 60% ethanol obtains having high polysaccharide concentration, the segmentation fraction of low phytohemagglutin phytolectin protein concentration proportioning (～0/1), use 80% ethanol to carry out the second step precipitation subsequently, to produce the segmentation fraction of low polysaccharide/high phytohemagglutin phytolectin protein concentration proportioning (～2/1).
Many methods of removing alcohol from solution are known in the field.Keep this alcohol to recycle if desired, can come from described solution, to remove this alcohol by normal pressure or decompression distillation after the extraction.This alcohol is recycling.In addition, also have and manyly well known in the artly from solution (perhaps the aqueous solution or remove the solution of alcohol), remove the method for anhydrating.These class methods include, but are not limited to: the aqueous solution are spray dried on the suitable carrier, and such as but not limited to magnesium carbonate or maltodextrin, perhaps selectively can be dry with described liquid dried by freeze-drying or reflection window (refractive window).
Food and medicament
As food form of the present invention, it can be mixed with any optional form, for example, and particulate, particulate form, paste attitude, gel state, solid-state, or liquid.In these forms, can comprise the generally well-known various types of materials of those skilled in the art that allow to add in the food arbitrarily, for example: adhesive, disintegrant, thickener, dispersant, sorbefacient, flavor enhancement, buffer, surfactant, dissolution aids, preservative, emulsifier, isotonic agent, stabilizing agent or pH controlling agent or the like again.The amount that joins the elder berry extract in the food is not particularly limited, and for example, as adult's intake of the about 60kg of body weight, it can be every day about 10mg to 5g, and preferred 50mg is to 2g.
When it used as health food, functional food etc., it preferably comprised the active ingredient of the present invention of the amount that can fully show predetermined action of the present invention especially.
Medicament of the present invention can at random be prepared into solid chemicals according to generally well-known method, for example such as tablet, particulate, powder, capsule or the like, or such as the liquid preparation of injection etc.In these medicaments, can prepare any normally used material, for example adhesive, disintegrant, thickener, dispersant, sorbefacient, flavor enhancement, buffer, surfactant, dissolution aids, preservative, emulsifier, isotonic agent, stabilizing agent or pH controlling agent again.
The dosage of active ingredient in the described medicament (elder berry extract) can change according to kind, medicine type, patient's age, body weight or symptom to be used or the like, for example, when oral administration, the per day for adults of the about 60kg of body weight is administered once or several times, dosage is about every day 10mg to 5g, and preferably about 50mg is to 2g.Described active ingredient can be one or more compositions of described elderberry extract.
Induction system
Help carrying the mode to patient's administration of the present composition to comprise the generally well-known administering mode of those skilled in the art, for example powder, spraying, ointment, paste, emulsifiable paste, washing lotion, gel, solution, patch and inhalant.
In one embodiment, this administering mode is an inhalant, and it can comprise such as the time-delay release of little fat body (liposomal) preparation or controlled release inhalant form.This class induction system can be used for treating SARS, bird flu of patient or the like.In this embodiment, preparation of the present invention can be used for being fit to any dosage-dispensing device of intranasal administration.For nasal preparation, should consider when this device makes up guarantee optimum metering accuracy and with the compatibility of its composed component, described element is container, valve and actuator for example, and can be based on the mechanical pump system, for example dosing sprayer, Diskus, soft mist inhalator, or the mechanical pump system of sprayer.Because big dosage, preferred embodiment comprise jet nebulizer (for example, PARI LC Star, AKITA), soft mist inhalator (for example, PARI e-Flow) and based on Diskus (for example, the PH ﹠amp of capsule; T Turbospin).The propellant that is fit to can be selected from the gas such as fluorocarbon, hydrocarbon, nitrogen and nitrous oxide or its mixture.
Described suction conveying plant can be sprayer known in those skilled in the art or metered dose inhaler (MDI), or any other suitable suction conveying plant.This device can comprise and be used to carry the preparation of single dose, and maybe this device can comprise and be used to carry the present composition of multiple dose.
The suction conveying plant of sparger type can comprise the present composition of solution (being generally the aqueous solution) or form of suspension.When producing the vaporific spraying of composition for inhalation, can be by ultrasonic, compressed air, other gases, electronic or mechanical system drives this aerosol type conveying plant.Ultrasonic spray apparatus applies the waveform of rapid vibration usually on the preparation liquid film by electrochemical vibration surface.At given amplitude, it is unstable that this waveform becomes, thereby it decomposes this liquid film and produces the droplet preparation.The sprayer unit of air or other gas-powered carries out work for the basis as follows, and high pressure draught produces partial drop of pressure, and this pressure drop is drawn liquid formulation in the air-flow via capillarity.Decompose this thin liquid stream by shearing force then.Described sprayer can be designed to portable and portable, and can be equipped with supporting electronic component.This sprayer unit can comprise and has nozzle two predetermined pore sizes and that deposit (coincident) exit passageway, and liquid preparation can be accelerated during by these passages.This causes the atomizing of the bump and the preparation of two bursts of logistics.Described sprayer can use mechanical actuator to force the jet rose of liquid preparation by predetermined pore sizes, the preparation aerosol that is used to suck with generation.In the design of single dose sprayer, can adopt the blister package (blister packs) that comprises single-dose preparations.
In the present invention, adopting size that sprayer can guarantee particle is best for described particle in the location in the lung film for example.
Metered dose inhaler (MDI) can be used as the suction conveying plant of the present composition.This device is pressurizeed (pMDI), and its basic structure comprises metering valve, actuator and container.Use propellant to come delivery formulations from this device.Described composition can be made of the particle of the preliminary dimension in one or more propellant liquid that are suspended in pressurization, or described composition can be in the solution or suspension of one or more liquid propellant that pressurize.Used propellant mainly is atmosphere close friend's hydrogen fluorine carbon (HFC), for example 134a and 227.Only ought just use conventional Chlorofluorocarbons, as CFC-11,12 and 114 in case of necessity.The device of intake system can be carried single dose via for example blister package, or it can be designed to multiple dose.The pressurised metered inhalator of intake system can contain the fat preparation by what respiration drive was carried exact dose.For guaranteeing the accuracy of dosage, the conveying of said preparation can be programmed by microprocessor, with certain some realization in imbibition cycle.This MDI can be portable or portable.
In another embodiment, described induction system can be the transdermal delivery system, for example hydrogel, emulsifiable paste, washing lotion, ointment, or patch.When the time-delay that needs several weeks even several months is carried, can use patch especially.
In another embodiment, can use the parenteral approach.The outer approach of stomach and intestine comprises the injection to the health various piece.The outer route of stomach and intestine comprises intravenous (iv), promptly by directly administration in vascular system of vein; Intra-arterial (ia) is promptly by directly administration in vascular system of artery; In the peritonaeum (ip), promptly to intraperitoneal administration; Subcutaneous (sc) is promptly at subcutaneous administration; In the muscle (im), i.e. administration in muscle; And intracutaneous (id), i.e. administration between cortex.When the part of institute's drug-delivery preparation can partially or completely be degraded in intestines and stomach, the outer route of stomach and intestine was more more preferred than oral route sometimes.Similarly, when needing to respond fast in emergency treatment, parenteral is usually also than more preferably oral.
The method of treatment influenza
Quantized the inhibition activity of elder berry fraction to A type H1N1 influenza virus.The serial dilutions of each fraction is hatched with the virus of known quantity, and be transported to cell culture individual layer (referring to Fig. 5).Draw dose response curve, and measured 50% inhibition concentration (IC of each fraction anti-A type people H1N1 virus 50).IC 50Value is referring to Fig. 6-11 and following table 16.Determined that also elder berry B anthocyanin fraction ADS5 desorb F2 has suppressed dengue fever virus and A type H1N1 human influenza virus (referring to Figure 12).Experimental program is referring to embodiment 9.
Table 16. uses A type H1N1 human influenza virus's inhibition analysis result general introduction
Elder berry fraction IC 50(μ g/mL)
Elder berry B anthocyanin fraction ADS5 desorb F2 333
Elder berry B anthocyanin fraction ADS5 desorb F3 521
Elder berry B anthocyanin fraction ADS5 desorb F4 195
Flores sambuci XAD 7HP desorb F2 1,592
Flores sambuci XAD 7HP desorb F3 582
The method of treatment HIV
Quantitatively the inhibition activity of elder berry fraction to HIV-1 virus.Known extract dilution is hatched with chimeric HIV-1 SG3 (genome) C hypotype (coating) virus of known quantity.Referring to Fig. 9.Draw dose response curve, and determined 50% inhibition concentration (IC of extrapolation 50).Referring to Figure 32-34 and following table 17.Experimental program is referring to embodiment 10.
Table 17. uses the inhibition analysis result general introduction of HIV-1 virus
Cytotoxic dosage IC is observed in experiment 50(μ g/mL)
1 8,182μg/mL 500
2 6,550μg/mL 153
Embodiment
Material
Botanical: from Blessed Herbs, the wild golden elder (elder) that Inc.Elder (Cincinnati) buys really (Product #:724, Lot#:L10379w, Hungary) and the colored (Product#:725 of golden elder (elder), Lot#:L01258W, Poland).
Organic solvent: acetone (67-64-1), 〉=99.5%, ACS reagent (179124); HPLC acetonitrile (75-05-8), gradient level 〉=99.9% (GC) (000687); Hexane (110-54-3), 95+%, spectrophotometric level (248878); Ethyl acetate (141-78-6), 99.5+%, ACS level (319902); Ethanol, the isopropyl alcohol with 4.8% (02853) sex change; Ethanol (64-17-5), anhydrous (02883); Methyl alcohol (67-56-1), 99.93%, ACS HPLC level, (4391993); And water (7732-18-5), HPLC level, (95304).All solvents are all bought from Sigma-Aldrich.
Bronsted lowry acids and bases bronsted lowry: buy formic acid (64-18-6), 50% solution (09676) from Fisher company; Acetate (64-19-7), 99.7+%, ACS reagent (320099); Hydrochloric acid (7647-01-0), volumetric(al) standards (volumetric standard) the 1.0N aqueous solution (318949); Folin-Ciocalteu phenol reagent (2N) (47641); Phenol (108-95-2) (p3653); Sulfuric acid (7664-93-9), ACS reagent, 95-97% (44719); And sodium carbonate (s263-1, Lot#:037406).
Chemistry reference standard thing: buy seralbumin (9048-46-8) from Chromadex, bovine serum albumin(BSA) fraction V powder cell culture test (A9418); Rutin (CAS#153-18-4); And anthocyan-3-glucosides chloride (CAS#7084-24-4).Buy the dextran reference material of identifying according to DIN [5000 (00269), 50,000 (00891) and 410,000 (00895)] from Fluka company.The structure of HPLC chemistry reference standard thing is as follows.
Rutin anthocyan-3-glucosides chloride
Figure A200780009471D00581
The polymer affinity adsorbent: Amberlite XAD 7HP (Rohm ﹠amp; Haas, France), with the macrolattice aliphatic acrylic crosslinking polymer that the translucent globule form of white is used, granularity is that 560-710nm, surface area are 380m 2/ g's.ADS-5 (Nankai University, China), the polystyrene that ester group is modified, granularity is that 300-1200nm, surface area are 500-600m 2/ g.
Method
High performance liquid chromatography (HPLC) method
Chromatographic system: the Shimadzu high performance liquid chromatography LC-10AVP system that is equipped with the LC10ADVP pump of band SPD-M 10AVP photodiode array detector.
In anti-phase Jupiter C18 post (250 * 4.6mm I.D., 5,300 ) (Phenomenex, Part#:00G-4053-E0, series number: 2217520-3, lot number: measured alcohol extract product of the present invention 5243-17).Volume injected is 10 μ l, and the flow rate of flowing phase is 1ml/ minute.Column temperature is 25 ℃.This flowing phase by A (5% arboxylic acid, v/v) and B (methyl alcohol) constitute.Gradient is set as follows: initial 2 minutes, B maintained 5%, 2-10 minute, solvent B is increased to 24% from 5% linearity, and 10-15 minute, B maintained 24%, 15-30 minute, B was increased to 35% from 24% linearity, 30-35 minute, B maintains 35%, and 35-50 minute, B was increased to 45% from 35% linearity, form maintenance 5 minutes at this, 55-56 minute then, B was reduced to 5% from 45% linearity, 65-68 minute, B maintained 5%.The detection wavelength of flavonoids is 350nm, and the detection wavelength of anthocyanidin is 520nm.
Be dissolved in the ethanol with 5mg/ml by the n-compound of will weigh, prepare the methyl alcohol stock solution of two reference standard things.Then the normative reference thing solution that mixes is diluted step by step, produce a series of ultimate densities and be respectively 1.0,0.5,0.25,0.1 and the solution of 0.05mg/ml.All stock solutions and working solution all used in 7 days ,+4 ℃ of storages, and were warmed up to room temperature before use.This solution is used for discerning and quantizes compound in described elder berry and the flores sambuci.About respectively 13.27 and 20.20 minutes of the retention time of the rutin of the anthocyan of 520nm-3-glucosides (CY3glu) and 350nm.Find that linear fit (linear fit) scope is 0.01-20 μ g.Regression equation and correlation coefficient are as follows: anthocyanidin-3-glucosides: area/100=20888 ** C (μ g)+502.21, R 2=0.9994 (N=5); And rutin: area/100=11573 * C (μ g)+584.57, R 2=0.9996 (N=5).HPLC result is shown in table 18.Based on peak area, calculate the content of the reference standard thing in each sample by corresponding calibration curve (calibration curves) by interpolation method.
Table 18. concentration in methyl alcohol is the HPLC analysis result of the elder reference standard thing of 0.1mg/ml
ID Retention time (minute) Area (mAumin) Highly (mAu) Width (min) Time started (minute) Dwell time (minute) Number of theoretical plate *
Anthocyan-3-glucosides 13.312 1391742 104526 1.37 12.46 13.82 1510
Rutin 20.181 768924 21934 3.69 19.32 23.01 479
*By N=16 * (tR/w) 2Theory of computation plate number.t RBe retention time, w is the width at peak,
https://www.mn-net.com/web％5CMN-WEB
HPLCKatalog.nsf/WebE/GRUNDLAGEN
Gas chromatography-mass spectrum (GC-MS) method
Using Shimadzu GCMS-QP2010 system to carry out GC-MS analyzes.This system comprise high resolution gas chromatography instrument, directly coupling (coupled) the GC/MS interface, have electron collision (EI) ion gun of independent temperature control and quadrupole mass filter.This system is subjected to GCMSsolution Ver.2 software control, carries out the data post analysis that obtains and turn round.At Agilent J﹠amp; WDB-5 fused silica capillary column (30m * 0.25mm i.d., 0.25 μ m film (5% phenyl, 95% dimethyl silicone polymer) thickness) (catalogue: 1225032, series number US5285774H) on, use following temperature program(me) to separate.Initial temperature is 60 ℃, keeps 2 minutes, is elevated to 120 ℃ with 4 ℃/minute speed then, keeps 15 minutes, is elevated to 200 ℃ with 4 ℃/minute speed then, keeps 15 minutes, is elevated to 240 ℃ with 4 ℃/minute speed then, keeps 15 minutes again.Total run time is approximately 92 minutes.The sample implantation temperature is 250 ℃.In 1 minute, do not inject 1 μ l sample by automatic injector not shunt (splitless) mode.Vector gas is a helium, by the pressure control flow rate of 60KPa.Under this pressure, described flow rate is 1.03ml/ minute, and linear velocity is 37.1cm/ minute, and total flow rate is 35ml/ minute.The MS ion source temperature is 230 ℃, and the GC/MS interface temperature is 250 ℃.The MS detector scans between 50-500m/z, and sweep speed is 1000AMU/ second, and ionization voltage is 70eV.It is 3.5 minutes that solvent is held back (cutoff) temperature.
By Folin-Ciocalteu method (Markar, H.P.S., Bluemmel, M., Borowy, N, K. And Becker, K., 1993, J.Sci.Food Agric.61:161-165) total phenolic acid concentration
Instrument: Shimadzu UV-Vis spectrophotometer (the UV 1700:S/N:A1102421982LP that has the UV probe)
Reference standard thing: deposit gallic acid/aqueous solution of preparation concentration 1mg/ml.Gallic acid solution in vitro packing into an amount of complements to 0.5ml with distilled water with volume, adds the Folin Ciocalteu reagent of 0.25ml, adds the 20wt% sodium carbonate liquor of 1.25ml then.Abundant this test tube of vibration is 40 minutes in ultra sonic bath, and the absorbance at record 725nm place.Described reference standard thing data are shown in table 19.
The calibration curve data of the gallic acid reference standard thing that uses in the table 19.Folin-Ciocalteu method
Figure A200780009471D00611
*: according to the gallic acid solution amount of absorbance information
Unknown sample: the suitable aliquot (aliquots) that will comprise the extract of tannic acid is put into test tube, supplies volume to 0.5ml with distilled water, adds 0.25ml Folin Ciocalteu reagent, adds the 1.25ml sodium carbonate liquor then.After 40 minutes, rotate this test tube and write down the absorbance of 725nm.Calculate total phenols content of representing with gallic acid equivalant from above-mentioned calibration curve.
Protein content by Bradford reagent method mensuration
Instrument: Shimadzu UV-Vis spectrophotometer (the UV 1700:S/N:A1102421982LP that has the UV detector)
Standard calibration curve: preparation is dissolved in the protein reference material of the debita spissitudo in the identical buffer as unknown sample.In the present invention, deionized water is replaceable is buffer.By the BSA protein reference material solution of serial dilution 2mg/ml, making the BSA reference material is 0.1-1.4mg/ml.Then, with the Bradford reagent mix of BSA reference material and the 3ml of 0.1ml.The mixture eddy current is mixed (vortex), and at room temperature hatched sample 5-45 minute.The absorbance at record 595nm place.The absorbance of sample must be before 60 minutes time restriction, and record in 10 minutes each other.The result is shown in table 20.
The standard calibration data of table 20.Bradford protein analysis
The test tube sequence number BSA normal concentration (mg/ml) BSA solution (2mg/ml) (μ l) Distilled water (μ l) Bradford reagent (ml) BSA content (μ g) The absorbance of 595nm
0 0 0 100 3 0 0.415
1 0.1 5 95 3 10 0.497
2 0.3 15 85 3 30 0.672
3 0.5 25 75 3 50 0.818
4 1.0 50 50 3 100 1.169
The analysis of unknown sample: the suitable aliquot that will contain the protein sample is put into test tube; Supply volume to 0.1ml with distilled water.The Bradford reagent that adds 3ml then.In 5-45 minute, this test tube that vibrates, and the absorbance of record 595nm.From above-mentioned calibration curve, computational chart is shown the protein content of BSA reference material equivalent.
The polysaccharide analysis of use chromatmetry (Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956, Analytical Chemistry 28 (3): 350-356).
Spectrophotometer system: in this research, used Shimadzu UV-1700 ultraviolet ray-visible spectrophotometer (190-1100nm, 1mm resolution).
Colorimetric (colorimetric) method has been used for the polysaccharide analysis.Make deposit dextran (Mw=5000,50,000 and 410, the 000) solution of 0.1mg/ml.Get 0.08,0.16,0.24,0.32, the stock solution of 0.40ml, and supply volume to 0.4ml with distilled water.The phenol solution and the 1ml concentrated sulfuric acid that add 0.2ml5% then.Before carrying out UV scanning, this mixture was left standstill 10 minutes.Find maximum absorbance at 488nm.Then with wavelength set at 488nm, and measure the absorbance of each sample.The result is shown in table 21.Every kind of following dextran solution is obtained standard calibration curve: dextran 5000, absorbance=0.01919+0.027782C (μ g), R 2=0.97 (N=5); Dextran 50,000, absorbance=0.0075714+0.032196C (μ g), R 2=0.96 (N=5); With dextran 410,000, absorbance=0.03481+0.036293C (μ g), R 2=0.98 (N=5).
The colorimetric analysis of table 21. dextran reference standard thing
Test tube Dextran solution (ml) Distilled water (ml) 5% phenol (ml) Sulfuric acid (ml) Abs(Mw＝5K) Abs(Mw＝50K) Abs(Mw＝410K)
Blank 0 0.40 0.2 1 0 0 0
1 0.08 0.32 0.2 1 0.238 0.301 0.335
2 0.16 0.24 0.2 1 0.462 0.504 0.678
3 0.24 0.16 0.2 1 0.744 0.752 0.854
4 0.32 0.08 0.2 1 0.907 1.045 1.247
5 0.40 0.00 0.2 1 1.098 1.307 1.450
Direct in real time (DART) mass spectrum of analyzing that polysaccharide is analyzed
All DART chromatograms particularly all use instrument as described below and method to move from the fraction F1-F6 of XAD7HP filler with from the DART chromatogram of the fraction F1-F4 of ADS5 filler.
Instrument: JOEL AccuTOF DART LC flight time (time of flight) mass spectrograph (Joel USA, Inc., Peabody, Massachusetts, USA).This flight time (TOF) mass spectrometry art does not require any sample preparation, and produces the quality that accuracy reaches 0.00001 mass unit.
Method: being used for the instrument of Collection and analysis polysaccharide fractions is provided with as follows: for positive ion mode, the voltage of DART pin is 3000V, and heating element heater is at 250 ℃, and electrode 1 is at 100V, and electrode 2 is at 250V, and the helium flow rate is 7.45 liters/minute (L/min).For mass spectrograph, hole 1 is 10V, and lens ring (ring lens) is 5V, and hole 2 is 3V.In order to obtain the initial resolution capability of about 60m/z, still have enough resolution in high-quality scope more simultaneously, crest voltage is arranged to 600V.Microchannel plate wave detector (MCP) voltage is arranged to 2450V.Before the every morning sample introduction, (Sigma-Aldrich Co., St.Louis USA) calibrates to use the caffeine solution reference material of 0.5M.Calibration tolerance remains on≤5mmu.
Tweezers with sterilization are introduced sample in the DART helium plasma, are exposed to the surface area maximum of helium plasma ray to guarantee sample.For sample is introduced ray, adopt oscillating motion.This motion allows sample to expose repeatedly, and prevent the pyrolytic of sample with about 0.5 second/wheel (swipe) when swing.Repeat this motion up in wave detector, observing appreciable total ion current (TIC) signal, remove sample then, to carry out the standardization of baseline/background.
For negative ion mode, described DART and AccuTOF MS are transformed into negative ion mode.Described pin voltage is that 3000V, 250 ℃ of heating element heaters, electrode 1 is 250V for 100V, electrode 2, and the helium flow rate is 7.45L/min.For mass spectrograph, hole 1 is-20V, lens ring are-13V, and hole 2 is-5V.Crest voltage is 200V.MCP voltage is set to 2450V.With introducing sample with the identical mode of positive ion mode.All data analyses use the MassCenterMain Suite software that provides simultaneously with instrument to carry out.
Embodiment 1
The embodiment of step 1A: the single step SFE maximum extracted and the purifying of elder berry
Design pressure and temperature be respectively 690 crust at the most and 200 ℃ SFT 250 (Supercritical Fluid Technologies, Inc., Newark, Delaware carries out all SFE extractions on USA).This device allows under super critical condition neatly with dynamic or static schema is simple to operate and extraction effectively.This device mainly is made of three modules; Baking oven, pump and controller and collection module.This baking oven has the extraction vessel of a pre-plume and a 100ml.This pump module has the air operated pump that constant flow rate is 300ml/min.This collection module is the 40ml vial with lid and partition sealing, the recovery that is used to extract product.This equipment is equipped with little metering valve and flowmeter.In extraction vessel pressure and temperature monitoring and being controlled at ± 3 crust and ± 1 ℃.
In typical embodiment, the golden elder fruit (elder berry) that 5 grams are milled in each experiment or the powder of flower (flores sambuci) are packed in the 100ml extraction vessel, and the size that described powder is surveyed with screen cloth (140 order) sieve surpasses 105 μ m.Mineral wool is placed on the two ends of post, to avoid carrying secretly of any possible solid material.Before loading filling container, described baking oven is preheating to the temperature of requirement.After this container is connected to baking oven, by using CO 2(～850psig) pressurizeed to system and tested the seepage of described extraction system, and purges.Use pneumatic liquid pump with this system lock and be forced into required extraction pressure.Allow this system balancing～3 minute then.Sampling bottle (40ml) is weighed and be connected to sample tap.Cross CO by velocity flow with～5SLPM (10g/ minute) 2(by metering valve control) begins to extract.Yield is defined as the part by weight of total extract to the raw material charging.Yield is defined as the oil phase that the extracted percentage by weight for the raw material of packing at first in extractor.Take complete classification (fullfactorial) extraction scheme, change in 40-80 ℃ temperature and 100-500 crust.Analyze for gaschromatographic mass spectrometry (GC-MS), in the carrene that the extract that obtains under every kind of condition is dissolved in, concentration is 400ppm.
Embodiment 2
The embodiment of step 2: water alcohol lixiviate
The representative instance of two step solvent extractions of Sambucus species phenolic acid chemical composition is as described below: raw material is the elder berry SFE residue that the 17.6gm of the essential oil SCCO2 extraction (60 ℃, 300 crust, 90 minutes) from step 1 mills.Solvent is 25% ethanol water of 300ml.In the method, feed material and 80% ethanol water are respectively charged in the extraction vessel of 500ml, and mix 4 hours in 60 ℃ hot bath.The Fisherbrand P4 filter paper that uses particle to keep to be of a size of 4-8 μ m filters and extracts solution, and at 2000rpm centrifugal 20 minutes, and the graininess residue is used for further extraction.Collection and merging filtrate (supernatant) carry out yield calculating, HPLC analyzes and produce F1-F4 and F1-F6 fraction (referring to the following examples 3).Use said method to extract 2 hours (stage 2) of residue in stage 1.
Embodiment 3
The embodiment of step 3: the affinity adsorbent of phenolic acid fraction extracts (F1-F4 and F1-F6 fraction Preparation)
In typical experiment, working solution is the transparent water alcoholic solution of the Sambucus species ethanol water leaching extract of step 2.The affinity adsorbent polymer resin is XAD7HP or ADS5.After reaching before being filled into the post that ID is 25mm, long 500mm, ethanol with 95% (4-5BV) and distilled water (4-5BV) wash the ADS5 affinity adsorbent of 15gm or the XAD7HP affinity adsorbent of 20gm in advance.The solution that loads is thick 80% ethanol leaching phenolic acid solution, and wherein chemical composition reclaims by rotation vacuum distillation and ethanol and concentrates.Load for XAD7HP, last loading solution concentration is 29.03mg/ml, loads for ADS5, and its concentration is 34.90mg/ml.Under the 0.3BV/hr flow rate, 50ml is loaded solution be carried on the XAD7HP post, and 60ml loading solution is carried on the ADS5 post.About 50-60 of load time minute.Distilled water with 2BV cleans the post that loads with the flow rate of 0.2BV/hr, and scavenging period is 13 minutes.40% and 80% ethanol water wash-out with 40ml should load post successively, for the flow rate wash-out of XAD7HP with 2ml/ minute, for the flow rate wash-out of ADS5 with 1.5ml/ minute.In elution process, from the XAD7HP post, collect 6 kinds of eluent fraction (F1-F6:F1～20mL respectively, F2～20mL, F3～18mL, F4～10mL, F5～17mL and F6～27mL), from the ADS5 post, collect 4 kinds of eluent fraction (F1-F4:F1～20mL, F2～20mL, F3～17mL and F4～17mL).For the XAD7HP post, use 40% ethanol elution to go out F1-F3, use 80% ethanol to collect F4-F6.For the ADS5 post, use 40% ethanol elution to go out F1-F2, use 80% ethanol elution to go out F3-F4.Remove remaining chemicals on this post with 95% ethanol of 4-5BV with the 3.6BV/hr flow rate then, wash with the 3.8BV/hr flow rate with 4-5BV distilled water subsequently.Total processing time was less than 2 hours.During entire process, use FPU252
Figure A200780009471D00661
Speed change (3-50ml/ minute) peristaltic pump control flow rate.By DART mass balance and each elutriated fraction of HPLC Collection and analysis.
Embodiment 4
The embodiment of step 5: polysaccharide fractions extracts
Water-soluble, the insoluble purifying phytohemagglutin phytolectin of the ethanol-solvent extraction of polysaccharide fractions chemical composition and the exemplary embodiments of precipitation of Sambucus species are as described below: divide two sections, will extract 2 hours at 80 ℃ from the 15gm solid residue of the water alcohol lixiviate (step 2) in stage 2 distilled water with 300ml.Merge twice extraction solution, and filter this slurries with Fisherbrand P4 filter paper (aperture 4-8 μ m), and with 2, centrifugal 20 minutes of 000rpm.Compound concentrations is 3.8mg/ml in the solution.Get this solution of 300ml, add 456ml or 1200ml absolute ethyl alcohol subsequently, so that final concentration of alcohol is complemented to 60% or 80%.When precipitation took place, with this solution left standstill 1 hour.Extract solution with 3 with this, centrifugal 20 minutes of 000rpm, and pour out and abandon supernatant.Collect this sediment, and with baking oven 50 ℃ of dryings 12 hours.The polysaccharide fractions of the drying of weighing also is dissolved in the water, uses with dextran and analyzes purity of polysaccharide as the chromatmetry of reference standard thing, and use Bradford protein analysis method to analyze the phytohemagglutin phytolectin purity of protein.Use the AccuTOF-DART mass spectrum to come further proportioning to constitute the molecular weight of the compound of purified polysaccharide fraction.The result of elder berry as Figure 36,37 and table 22 shown in.The result of flores sambuci as Figure 38,39 and table 22 shown in.
The polysaccharide DART of table 20. elder berry and flores sambuci analyzes
The elder berry flores sambuci
Cation anion cation anion
(m+H)/z relative intensity (m-H)/z relative intensity (m+H)/z relative intensity (m-H)/z relative intensity
59.1 309.9 89.0 622.5 61.0 490.0 89.0 368.5
73.1 332.1 121.0 556.6 65.1 96.1 94.0 142.7
74.1 204.9 143.1 98.4 70.1 116.6 111.0 52.0
89.1 157.2 165.0 711.5 74.1 148.3 112.0 104.2
101.1 556.5 179.1 105.2 78.1 116.5 113.0 410.9
111.1 356.6 637.1 46.5 84.1 107.2 133.0 122.2
113.1 127.2 825.2 68.5 90.1 401.5 171.0 128.2
114.1 207.3 98.1 262.3 191.1 112.2
115.1 107.7 110.1 70.1
119.1 136.2 146.1 142.9
121.1 153.4 228.2 68.9
124.1 404.1 269.2 278.1
125.1 93.7 271.3 517.4
135.1 187.0 272.3 121.2
136.1 84.4 273.3 676.9
138.1 143.6 283.2 850.1
141.1 89.1 284.2 164.7
143.1 241.9 285.2 269.3
144.1 67.8 286.2 167.0
145.1 737.2 287.3 356.4
151.1 162.5 288.3 4144.0
152.1 196.1 289.3 2578.7
153.1 649.2 290.3 521.0
155.1 174.0 291.3 112.9
157.1 178.8 295.2 90.8
163.1 413.8 300.3 112.7
167.1 90.3 301.2 472.6
169.1 120.4 302.2 200.1
171.1 123.5 303.2 719.0
The elder berry flores sambuci
Cation anion cation anion
(m+H)/z relative intensity (m-H)/z relative intensity (m+H)/z relative intensity (m-H)/z relative intensity
173.1 159.9 305.3 1332.0
174.1 102.4 306.3 361.8
179.1 191.2 307.3 6262.5
180.2 912.9 308.3 1781.9
181.1 195.4 309.3 95.0
185.1 102.0 316.3 1114.4
186.1 123.7 317.3 189.6
195.1 528.5 319.2 627.1
198.1 85.0 320.3 247.6
199.2 143.6 321.2 1612.0
211.1 130.5 322.3 521.6
217.2 428.7 323.3 1510.2
219.2 131.2 324.3 358.6
223.1 264.7 335.2 140.7
279.2 229.8 337.3 805.3
287.2 365.1 338.3 429.6
288.3 848.4 339.3 1079.5
289.3 93.5 340.3 546.7
304.2 703.1 344.3 192.4
305.2 77.7 347.3 1100.0
316.3 200.2 348.3 235.6
371.1 534.9 349.3 4638.4
372.1 130.1 350.3 1002.4
373.1 107.3 351.3 113.1
388.1 164.0 353.3 306.8
391.3 405.3 354.3 238.3
409.4 451.1 355.3 417.2
356.3 584.2
357.3 134.8
The elder berry flores sambuci
Cation anion cation anion
(m+H)/z relative intensity (m-H)/z relative intensity (m+H)/z relative intensity (m-H)/z relative intensity
363.3 628.0
364.3 127.8
365.3 725.6
366.3 243.5
367.3 108.1
368.3 141.9
370.3 378.9
372.3 686.7
379.3 278.8
380.3 70.5
381.3 252.7
382.3 330.0
386.3 141.3
388.3 198.4
391.3 167.3
396.3 188.3
397.3 138.7
398.3 501.2
412.3 133.1
414.3 235.2
425.4 85.7
430.3 89.8
438.3 70.7
Embodiment 5
Mix following ingredients by prescription:
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Golden elder berry extract 150.0mg
Essential oil fraction (10mg, 6.6% dry weight)
Polyphenol fraction (120mg, 80% dry weight)
Polysaccharide (40mg, 26.6% dry weight)
Stevioside (qualities of stevia extract) 12.5mg
Carboxymethyl cellulose 35.5mg
Lactose 77.0mg
----------------------------------------------------------------
------
Total amount 275.0mg
The novel extract of Sambucus species comprises essential oil fraction, phenolic acid-essential oil fraction and the polysaccharide fractions of % quality weight greater than the numerical value of finding in natural rhizome material or conventional extraction product.Said preparation can be made into any oral dosage form, and for required physiology and psychology effect (reducing excited and insomnia) and medical function (viral disease, for example common cold, influenza, herpes simplex, herpes zoster and HIV, the prevention and the treatment of diabetes, cardiovascular and cranial vascular disease, antiatherosclerosis, antioxidant and radicals scavenging, anti-inflammatory, Antiarthritic, wind resistance diseases caused by dampness and gastrointestinal disease) administration every day or reach 15 administrations every day as required.
Embodiment 6
Mix following ingredients by prescription:
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------
Golden elder berry extract 150.0mg
Essential oil fraction (6mg, 4% dry weight)
Polyphenol fraction (30mg, 20% dry weight)
Polysaccharide (114.0mg, 76% dry weight)
Vitamin C 15.0mg
Sucralose
35.0mg
Green Gram Seed 10:1 50.0mg
Mocha spices (Mocha Flavor) 40.0mg
Chocolate flavoring (Chocolate Flavor) 10.0mg
------------------------------------------------------------
------
Total amount 300.0mg
The novel extraction composition of elder Ligusticum wallichii (chuangxiong) comprises essential oil, phenolic acid-essential oil and the polysaccharide chemistry composition fraction of % quality weight greater than the numerical value of finding in natural frond or conventional extraction product.Said preparation can be made into any oral dosage form, and according to demand every day of required physiology, psychology and medical function safely administration up to 15 times (referring to the foregoing description 5).
Embodiment 7
The MTT that determines cell quantity to be used analyzes
Purpose: this is the control experiment that is used for determining the cell quantity that uses in the later MTT/ cytotoxicity experiment.Each used cell-line only need be done once.
The JD that gives birth to the antiviral activity of body active substance estimates
First day
Cell (using MDCKs to be write as this scheme) from fusion (confluent) T-75 flask
1. absorption medium, and in flask, add 2mL trypsase, hatched 5 minutes at 37 ℃.
2. firmly impact the sidewall of flask, and trypsase is transferred in the 50cc conical pipe.The growth medium (DMEM+P/S+Glutamax+FBS) that also in this pipe, adds 0.5mL.
3. in flask, add 2ml trypsase again, hatched 3-5 minute at 37 ℃.
4. firmly impact the flask sidewall, and trypsase is transferred in the 50cc pipe of step 2.In flask, add 10mL growth medium, rinsing drag 2 times.This 10mL medium is put into identical 50cc pipe.Use the microexamination flask, look at whether removed cell.
5. 4 ℃, 1000rpm rotation 5 minutes.Absorb supernatant.
6. shift out particle, and this particle is resuspended in the growth medium of 5mL.
7. 4 ℃, 1000rpm rotation 5 minutes.Absorb supernatant.
8. shift out particle, and this particle is resuspended in the growth medium of 1mL.
9. in microcentrifugal tube, by 500 μ l cells being joined in the 500 μ l growth mediums to come with the 1:2 diluting cells.If from the very high plate of cell density, may be with the 1:4 diluting cells in growth medium.
10. on hemocytometer, verify the diluting cells of 10 μ l.With the cell counting of having write down 3 big lattice, get the mean of these three groups of numerals.Obtain cell counting like this: mean x 10 4Cell/mL.Need be from about 5 x 10 6Cell/mL begins.If too many cell is arranged, after another dilution, carry out cell counting again.
11. using altogether, 11 microcentrifugal tubes reach 2 times of dilutions.Be an example below:
Pipe # Cell/mL Add medium Add cell
1 1.34 x 10 6 ---------- ----------
2 6.7 x 10 5400 μ l, 400 μ l are from managing 1
3 3.35 x 10 5400 μ l, 400 μ l are from managing 2
4 1.68 x 10 5400 μ l, 400 μ l are from managing 3
5 8.4 x 10 4400 μ l, 400 μ l are from managing 4
6 4.2 x 10 4400 μ l, 400 μ l are from managing 5
7 2.1 x 10 4400 μ l, 400 μ l are from managing 6
8 1.05 x 10 4400 μ l, 400 μ l are from managing 7
9 5.25 x 10 3400 μ l, 400 μ l are from managing 8
10 2.63 x 10 3400 μ l, 400 μ l are from managing 9
The 11 contrast 400 μ l of medium only-------------
12. this analysis is carried out three times, so add 100 μ l from each pipe in the hole A-C of 96-orifice plate, the numbering of each row is corresponding to the pipe of its current contained sample on the plate.
13. with plate at 37 ℃ of overnight incubation w/CO 2, perhaps incubated cell recovers or adheres to the required time (12-18 hour usually) again.
Second day
Approximately the morning 9:00, the cell of verifying on the plate at microscopically adheres to confirm them, they are (confluent) that merges at row 1 at least, and when you swept entire plate, you saw that the cell in every hole is less.Medium in the 2-3 row that begin most should be for orange; Other should be pink.
2. every hole adds 10 μ l MTT reagent (it is stored in 4 ℃), changes between every hole most advanced and sophisticated (tips), and takes care not to pollute the storing solution of MTT reagent.Plate was hatched 2 hours at 37 ℃.
3. precipitate in the purple point-like that on microscopically verification plate, occurs, the cell.If do not see this, continued to hatch 24 hours at the most.
In a single day 4. see and add 100 μ l cleaning agents (being stored in room temperature) to every hole by sediment.After this do not shake this plate.Cover described plate with aluminium foil, plate is placed in room temperature spent the night.
The 3rd day
1. use Tecan to read the plate device, measure the absorbance of each hole at 560nm, reference wavelength is 620nm.If use the program of any being called " MTT " among the XFluor4, need do like this.Need guarantee that filter slide plate (filter slide) C is in this Tecan.
2. determine mean value by three readings, and deduct the mean value that on average obtains by the blank that only contains medium (row 11).Mark absorbance on the Y-axis and on X-axis, marking every milliliter cell number.
Select to produce the cell number of 0.75-1.25 absorbance, in subsequent analysis, to use.Selected cell number should drop on the straight line portion of curve.
Embodiment 8
MTT analyzes
Purpose: determine whether the extract pair cell has cytotoxic effect
The antiviral activity JD assessment of bioactivator
First day
1. use super quick balance by the window among the WH265, measure the extract of 0.01g, and be dissolved among the aseptic PBS of 100 μ l.Make it accurately may allow people's madness, therefore make it approaching as far as possible, and on notebook recording quality (mass), and the label details of extracting property management.This is " a undiluted extract ", and concentration is about 0.1g/mL.As berry extract be not dissolve fully soluble, then in microcentrifuge with 13krpm rotation precipitation 30 seconds, supernatant is transferred in the aseptic microcentrifugal tube as using the same day, and particle is used for possible follow-up use-20 ℃ of preservations.
Cell (using MDCK to be write as this scheme) from a fusion T-75 flask:
1. absorption medium, and in flask, add 2mL trypsase, hatched 5 minutes at 37 ℃.
2. firmly impact the sidewall of flask, and trypsase is transferred in the 50cc conical pipe.The growth medium (DMEM+P/S+Glutamax+FBS) that also in this pipe, adds 0.5mL.
3. in flask, add 2ml trypsase again, hatched 3-5 minute at 37 ℃.
4. firmly impact the flask sidewall, and trypsase is transferred in the 50cc pipe of step 2.In flask, add 10mL growth medium, rinsing drag 2 times.This 10mL medium is put into identical 50cc pipe.Use the microexamination flask, look at whether removed cell.
5. 4 ℃, 1000rpm rotation 5 minutes.Absorb supernatant.
6. shift out particle, and this particle is resuspended in the growth medium of 5mL.
7. 4 ℃, 1000rpm rotation 5 minutes.Absorb supernatant.
8. shift out particle, and this particle is resuspended in the growth medium of 1mL.
9. in microcentrifugal tube, by 500 μ l cells being joined in the 500 μ l growth mediums to come with the 1:2 diluting cells.If from the very high plate of cell density, may be with the 1:4 diluting cells in growth medium.
10. on hemocytometer, verify the diluting cells of 10 μ l.With the cell counting of having write down 3 big lattice, get the mean of these three groups of numerals.Obtain cell counting like this: mean x 10 4Cell/mL.During beginning, need about 1-1.6 x 10 5Mdck cell/mL, or 1.3-2.1 x 10 5293T cell/mL; This can realize in the following manner:
To MDCK:
A. 1:4 dilution
B. carry out cell counting.Usually obtain about 360 cells/big lattice.
C. 1:4 is diluted with 1:3.Subsequently with 1:10 dilution (400 μ l cells are in the 3.6mL medium).
D. carry out cell counting.Need 10-16 cell/big lattice.
For 293T:
A. 1:8 dilution.
B. carry out cell counting.Usually obtain about 300 cells/big lattice.
C. 1:8 is diluted with 1:2.Subsequently with 1:10 dilution (400 μ l cells are in the 3.6mL medium).
D. carry out cell counting.Need 13-21 cell/big lattice.
11. use as follows 9 microcentrifugal tubes altogether reaches 2 times of dilutions of extract:
Pipe # The extract dilution rate Add PBS Add extract
1 is undiluted--------------------------
2 1:2,50 μ l, 50 μ l are from managing 1
3 1:4,50 μ l, 50 μ l are from managing 2
4 1:8,50 μ l, 50 μ l are from managing 3
5 1:16,50 μ l, 50 μ l are from managing 4
6 1:32,50 μ l, 50 μ l are from managing 5
7 1:64,50 μ l, 50 μ l are from managing 6
8 1:128,50 μ l, 50 μ l are from managing 7
9 1:256,50 μ l, 50 μ l are from managing 8
10 1:512,50 μ l, 50 μ l are from managing 9
In the 96-orifice plate, row
The only contrast of PBS/ solvent of 11=(cell is arranged but do not have extract)
The only contrast of medium of 12=(blank-acellular, no extract)
Therefore 12. this analysis is carried out three times, to the capable cell that adds the suitable dilution that the firm eddy current of 100 μ l (vortexed) handle of the A-C of the row 1-11 of aseptic 96-orifice plate, has filled 3 row backs and the cell in managing is carried out eddy current has handled.
13. to the capable medium that adds 100 μ l of the A-C of row 12.
14. the capable extract dilution that adds 6 μ l of A-C of 1-10 row on plate then.(note: the sequence number of each row should corresponding above-mentioned pipe # on the plate)
15. to the capable 6 μ l solvents that add of the A-C of row 11.
16. access panel, and impact gently guaranteeing that extract is arranged in the liquid in every hole, rather than on the sidewall in hole.
17. with plate at 37 ℃ of overnight incubation w/CO 224 hours.
18. from initial microcentrifugal tube, get the 10mL growth medium that 500 μ l cells (just eddy current is handled) are put into the T-75 flask, cut apart (split) to carry out 1:2, be placed on 37 ℃ up to cutting apart once more.
19. utilize during this period of time, calculate μ g/mL extract definite in every row according to the volume of measuring and in every row, add.
Second day
1. absorb the liquid in every hole.Use the multichannel pipette, wash every hole once with the aseptic PBS of 200 μ l.Add 100 μ l aseptic culture mediums to every hole.
2. at the test under microscope cell, guaranteeing them still there, and they show purple because of the extract of internalization.
3. from bottle, shift out 400 μ l MTT reagent (its BSL3 that is kept at 4 ℃ is indoor) in microcentrifugal tube.Use conventional pipette to add 10 μ l MTT reagent, change the tip between every hole, and carefully do not pollute MTT reagent storing solution to every hole.Plate was hatched 2 hours at 37 ℃.
4. use the multichannel pipette, every hole adds 100 μ l cleaning agents (at room temperature storage).After this do not shake described plate.Cover described plate with aluminium foil, and with plate 37 ℃ of placements up to 3:00 in afternoon, this moment should on Tecan, read plate.
Read plate:
1. use Tecan to read the plate device, measure the absorbance of each hole at 560nm.Use the program that is called " MTT " among the XFluor4.Guarantee that filter slide plate (filter slide) C is in this Tecan.
Determine mean value by three readings, and deduct the mean value that on average obtains by the blank that only contains medium (row 12).Mark absorbance on the Y-axis and on X-axis, marking μ g/mL extract.
Embodiment 9
The inhibition analysis that the elder berry extract infects influenza virus A
The 1st day
1. measure extract by the window in the WH265 with super quick balance.From 40mg/ml at least.This should be the aseptic PBS of the per 125 μ l of 5mg (or 0.005g).
2. eddy current is handled with dissolving.If do not enter in the solution, add the PBS of same amount.Repeat again if desired.If after attempting three times like this, it can not enter in the solution fully, then in microcentrifuge with 10-13,000rpm rotation 30 seconds.Take out supernatant and use as an alternative.But, with insoluble part mark and be stored in-20 ℃.
3. repeating step 1 ﹠amp; 2, and merge the extract of the dissolving measure, to prepare the extract solution of 250 μ l.
4. two aseptic microcentrifugal tubes are labeled as " Ab 1:1000 " and " Ab 1:500 ".In " Ab 1:1000 " pipe, add the aseptic PBS of 999 μ l and the anti influenza A one anti-(primary antibody) of 1 μ l.Eddy current is handled.The anti influenza A one that adds 998 μ l PBS and 2 μ l in " Ab 1:500 " pipe is anti-.Eddy current is handled.
5. virus dilution:
A. 4 microcentrifugal tubes are labeled as " UV ", " 1 ", " 2 " and " 3 ".In " UV " pipe, add 990 μ l PBS, and add 900 μ l PBS to other pipes.
B. in " UV " pipe, adding the virus of 10 μ l on ice.Eddy current is handled.Change most advanced and sophisticated.From this pipe, take out in 100 μ l and adding " 1 " pipe.Eddy current is handled.Continue, take out 100 μ l and add in next pipe from every pipe, eddy current is handled, and changes most advanced and sophisticated between each dilution.
6. dilution extract:
A. 5 microcentrifugal tubes are designated as " 1:2 ", " 1:4 ", " 1:8 ", " 1:16 " and " 1:32 ".The PBS that in every pipe, adds 125 μ l.
B. eddy current is handled extract solution.The extract solution that in " 1:2 " pipe, adds 125 μ l.Eddy current is handled and changes most advanced and sophisticated.From " 1:2 ", add 125 μ l in " 1:4 ".Eddy current is handled and changes most advanced and sophisticated.From " 1:4 ", add 125 μ l in " 1:8 ".Remaining pipe is repeated, and eddy current is handled and changes most advanced and sophisticated between each dilution.
7. set up and analyze:
A. 7 microcentrifugal tubes are designated as " undiluted ", " 1:2 ", " 1:4 ", " 1:8 ", " 1:16 ", " 1:32 " and " PBS ".
B. add the PBS of 600 μ l in all pipes except that " PBS " pipe, " PBS " pipe adds the PBS of 1000 μ l.
C. (just eddy current is handled to add " 3 " viral dilution liquid of 100 μ l in all 6 pipes of non-" PBS " pipe! ).
D. " 1:2 " extract solution being carried out eddy current handles.The extract solution that in new " 1:2 " pipe, adds 100 μ l " 1:2 ".Eddy current is handled.
E. " 1:4 " arrived the pipe repeating step d of " 1:10 ", in the new pipe that contains PBS and virus that they mark separately, add the extract dilution.
F. (just eddy current is handled to add the undiluted extract solution of 100 μ l in " undiluted " pipe that contains PBS and virus! ).Eddy current is handled.
G. set up another pipe of the PBS of-3 viruses contain 100 μ l and 700 μ l, and be labeled as " 4 virus ".Eddy current is handled.
H. from " Ab 1:1000 " and " Ab 1:500 " pipe, discard 300 μ l immediately, and add in each pipe in " Ab1:1000 " and " Ab 1:500 " 100 μ l " 3 " viral dilution liquid (eddy current processing just! ).Eddy current mixes.
I. setting timer is 1 hour.
J. hatch the last stage at this, close the lamp in the fume hood (hood).
K. the described plate of mark, wherein " undiluted extract ", " 1:2 ", " 1:4 ", " 1:8 ", " 1:16 ", " 1:32 ", " 1:1000 " and " 1:500 " each mark three row are corresponding to antibody control, " only-4 viruses+PBS " and " only PBS ".
L. enter and hatch about 50 minutes of last stage, use PBS washed cell three times, the hole emptying is used for subsequent step.
M. after the time of hatching the last stage has crossed, every pipe is carried out eddy current handle, add 200 μ l in the hole of mark from each pipe respectively to each immediately.
N. on Belly Dancer incubated at room 30 minutes, half-twist after 15 minutes, and also prepare agar over lay (overlay) this moment.
8. when infecting about 15 minutes, agar over lay is set:
A. in water-bath, add the DMEM bottle, make its intensification.
B. with 5%SeaPlaque stock solution microwave treatment 1.5-2 minute.
C. following composition is mixed in preserving the aseptic vial of 100mL at least.
Agar over lay
To the 16-orifice plate: To the 56-orifice plate:
DMEM is warming up to 50 ℃ of 11.56mL 57.8mL
Antibiotic-antimycotic 150 μ l, 750 μ l
7.5％
Figure A200780009471D0078090918QIETU
0.576μl 2.88mL
Glutamax 150μl 750μl
Trypsase (1mg/ml) *14.4 μ l 72 μ l
5％ Sea Plaque
Figure A200780009471D0078090940QIETU
2.55mL 12.75mL
15mL cumulative volume 75mL cumulative volume
Figure A200780009471D0078091138QIETU
For preparation BSA, 0.75g BSA is added among the 10mL CaMg-PBS, and in fume hood inner filtration-sterilization.Be divided into the sample of 1.5-mL and-20 ℃ of storages.
*Trypsase is made 8.5g/L NaCl-H 2O solution, filter sterilization in fume hood is divided into the sample of 1mL, and-20 ℃ of storages.
Figure A200780009471D0079091202QIETU
To 100mL H 2Add the 5g agarose in O and the autoclave.Room temperature storage.
D. remove inoculum, and every hole substitutes with the 2ml agar over lay.Plate is faced up about 20 minutes of 4 ℃ of placements.
E. infect back (in step m, in cell, adding the virus back), from refrigerator, take out plate, and face up and in 37 ℃ couveuse, placed 27 hours
The 2nd day
Infect after 27 hours, every hole adds the Formafresh of 0.5-1mL.Plate is spent the night 4 ℃ of placements.
The 3rd day
1. absorb Formafresh.
2. remove the agar plug with spatula.
3. add 0.5mL70%EtOH, and incubated at room at least 20 minutes.Simultaneously, in the 50cc conical pipe of upper strata (upstairs) anti-the supplying of one among the Blotto is 1:1000, eddy current is handled to mix each composition.
15.5mL PBS
0.775g dry milk
15.5μl Tween 20
15.5 μ l anti influenza A antibody (remaining on 4 ℃)
4. absorb EtOH.Clean once with PBS.
5. be arranged in the anti-of Blotto to what every hole added that the firm eddy current of 500 μ l handles.Shaking the upper strata at 4 ℃ on BellyDancer spends the night.
The 4th day
1. upper strata resists two to be mixed to 1:500 in Blotto.(thereby supply Blotto as previously mentioned, only add 62 μ l two anti-(it is freezing in glycerine, and five equilibrium also is stored in-20 ℃) alternative and resist.)
2. plate is put into lower floor, and absorbs one anti-.
3. with the PBS washing once.
4. every hole adds 500 μ l and is arranged in the two anti-of Blotto, and on Belly Dancers in incubated at room 5 hours.
5. absorbing two resists.With the PBS washing once.
6. every hole adds 6 Dakko substrates (substrate) (being kept at 4 ℃ of lower floors in the indoor P3)
7. be placed on immediately on the Belly Dancer, and incubated at room 10-15 minute, or up to seeing focus.
8. absorption substrate, and with the PBS washing once.Be stored among the PBS.
9. on light box, take pictures and count focus.
Embodiment 10
The HIV that analyzes elder berry extract activity suppresses scheme
The production of pseudotype (pseudotyped) HIV-1
By with the 293T cell at the pSG3 that contains 6 μ g EenvCotransfection can produce pseudotype HIV-1 virus in the T75 Tissue Culture Flask of (plasmid that comprises the genomic scarce coating copy of HIV-1 bacterial strain SG3), 2 μ g coatings clone ZM53M.PB12 (coding is from the coating of the HIV-1 bacterial strain C hypotype of Zambia).(Qiagen, Valencia CA) come transfectional cell to use the Effectene transfection reagent.After 18 hours, substitute the culture and the medium that contain the Effectene transfection reagent.Collected supernatant in 48 hours after the transfection, clarify by low-speed centrifugal, five equilibrium, and freezing at-18 ℃.By infecting the GHOST cell, be seeded on the 96-orifice plate, 37 ℃ kept 2 hours and 10 times of titer of measuring viral storing solution of serial dilution.After hatching 2 hours, contain viral medium with the Eagle medium of the fresh Dulbecco modification that contains 10% hyclone is alternative, and hatched 48 hours at 37 ℃.(Amersham Bioscience, Piscataway NJ) scans plate, and carries out the focus counting by ImageQuant software with Typhoon phosphorus angiographic instrument (phosphorimager).
Elder berry extract preparation (F4 fraction) and infection inhibition analysis
Be resuspended in by freeze-drying elder berry extract among the PBS (pH7.2) of 1mL, and by NaOH the pH value be adjusted to 7.0 it is entered in the solution fully, thereby make elder berry extract (F4) with 40 μ L0.625M with 40mg.In order to analyze the antiviral activity of F4, with 5 x 10 to HIV-1 4In GHOST cell inoculation each hole in 96 hole tissue culturing plates.Second day, exist or do not have 6.55,3.28,1.64,0.82, under the condition of the F4/mL of 0.41 and 0.20 μ g, in every hole, add～1, the pseudotype virus of 000p.f.u..37 ℃ hatch 2 hours after, remove the medium that contains virus, and add the Eagle medium that 200 μ l contain the Dulbecco modification of 10% hyclone, and continued to hatch 48 hours at 37 ℃ to every hole.Subsequently, by ImageQuant software (AmershamBioscience) plate is scanned, and carry out the focus counting with Typhoon phosphorus angiographic instrument.
HIV-1 subtype C inhibition analysis
The inhibition analysis of chimeric HIV-1SG3 (genome) subtype C (coating).This specific envelope protein is cloned ZM135M.PB12 from coating, and GeneBank registration number AY423984 comes from Zambia, and circulation way is female to male, and E.Hunter and doctor C.Derdeyn provide.Bright white point (referring to Fig. 9) on the micro emulsion white background is a focus.Background is caused by the slight fluorescence of host cell, and can not further weaken.+, the positive infection contrasts; F4, elder is crossed extract fraction F4; T, the titration of used virus in the analysis.
Introducing is as reference
All United States Patent (USP)s and U.S. Patent Application Publication that this paper quotes all are incorporated herein by reference
Equivalent
Those skilled in the art will recognize that, or use the means that are no more than routine test can find many equivalents of specific embodiments of the present invention described herein.This class equivalent should be encompassed in the scope of following claim.

Claims (52)

1. comprise and have arbitrary Sambucus species extract of directly analyzing the fraction of (DART) mass spectrum chromatogram in real time shown in the drawings among Figure 36-70.
2. Sambucus species extract according to claim 1, wherein said fraction have arbitrary DART mass spectrum chromatogram shown in the drawings among Figure 46-50.
3. Sambucus species extract according to claim 1, wherein said fraction have DART mass spectrum chromatogram shown in Figure 48.
4. comprise IC 50Be the Sambucus species extract of the fraction of 150-1500 μ g/mL, wherein said IC 50In H1N1 influenza inhibition analysis, record.
5. Sambucus species extract according to claim 4, the IC that wherein said fraction records in H1N1 influenza inhibition analysis 50Be 150-750 μ g/mL.
6. Sambucus species extract according to claim 4, wherein said fraction has the IC of 150-300 μ g/mL 50
7. Sambucus species extract according to claim 4, wherein said fraction has the IC at least about 195 μ g/mL 50
8. according to claim 1 or 4 described Sambucus species extracts, wherein said fraction comprises anthocyanin; Flavonoids; Saturated or unsaturated fatty acid, alcohol or the ester of C16 or C18; And/or polysaccharide.
9. Sambucus species extract according to claim 8, wherein said anthocyanin are selected from anthocyan-3 glucosides and anthocyan-3-elder bioside.
10. Sambucus species extract according to claim 8, the content of wherein said anthocyanin is higher than 10 weight %.
11. Sambucus species extract according to claim 8, wherein said flavonoids is a rutin.
12. Sambucus species extract according to claim 8, saturated or unsaturated fatty acid, alcohol or the ester of wherein said C16 or C18 is selected from hexadecanol, hexadecylic acid, methyl palmitate, ethyl palmitate, hexadecylic acid butyl ester, stearic acid, Ethyl Stearate, butyl octadecanoate, 9-vaccenic acid-1-alcohol, 9,12-octadecadienoic acid, and combination.
13. Sambucus species extract according to claim 8, wherein said C16 or C18 content saturated or unsaturated fatty acid, alcohol or ester is at least about 2 weight %.
14. Sambucus species extract according to claim 8, wherein said polysaccharide is selected from dextran, glucose, arabinose, galactose, rhamnose, wood sugar, uronic acid, and combination.
15. Sambucus species extract according to claim 8, wherein polyoses content is at least about 10 weight %.
16. the described Sambucus species of claim 8 extract, it comprises anthocyanin; C16 or C18 saturated or unsaturated fatty acid, alcohol or ester; And polysaccharide.
17. Sambucus species extract according to claim 16, wherein said anthocyanin are selected from anthocyan-3-glucosides and anthocyan-3-elder bioside.
18. Sambucus species extract according to claim 16, the content of wherein said anthocyanin is higher than 10 weight %.
19. Sambucus species extract according to claim 16, wherein saturated or unsaturated fatty acid, alcohol or ester of C16 or C18 is selected from hexadecanol, hexadecylic acid, methyl palmitate, ethyl palmitate, hexadecylic acid butyl ester, stearic acid, Ethyl Stearate, butyl octadecanoate, 9-vaccenic acid-1-alcohol, 9,12-octadecadienoic acid, and combination.
20. Sambucus species extract according to claim 16, wherein C16 or C18 content saturated or unsaturated fatty acid, alcohol or ester is at least about 2 weight %.
21. Sambucus species extract according to claim 16, wherein said polysaccharide is selected from dextran, glucose, arabinose, galactose, rhamnose, wood sugar, uronic acid, and combination.
22. Sambucus species extract according to claim 16, wherein the content of polysaccharide is at least about 10 weight %.
23. comprise the food or the medicament of claim 1 or 4 described Sambucus species extracts.
24. the patient's that treatment is infected by the virus method comprises claim 1 or 4 described Sambucus species extracts to patient's effective dosage of the described treatment of needs.
25. method according to claim 24, wherein said virus are enveloped virus.
26. method according to claim 25, wherein said enveloped virus are flavivirus.
27. method according to claim 24, wherein said virus are nonenveloped virus.
28. method according to claim 24, wherein said virus are selected from influenza virus, A type and Type B human influenza virus, avian influenza virus, H1N1, H5N1, human immunodeficiency virus (HIV), SARs, herpes simplex virus (HSV), flavivirus, dengue fever, yellow fever, West Nile Virus and encephalitis viruses.
29. method according to claim 24, wherein said virus is selected from norwalk virus, hepatitis A, poliomyelitis, andoviruses and rhinovirus.
30. method according to claim 24, wherein said patient is primate, birds, Bovidae, sheep section, equine, Suidae, rodent, cat family or canid.
31. method according to claim 24, wherein said patient is human.
32. suppress the method for the virus infections of cell, comprise described cell is contacted with claim 1 or 4 described Sambucus species extracts.
33. method according to claim 32, wherein said virus are enveloped virus.
34. method according to claim 33, wherein said enveloped virus are flavivirus.
35. method according to claim 32, wherein said virus are nonenveloped virus.
36. method according to claim 32, wherein said virus are selected from influenza virus, A type and Type B human influenza virus, avian influenza virus, H1N1, H5N1, human immunodeficiency virus (HIV), SARs, herpes simplex virus (HSV), flavivirus, dengue fever, yellow fever, West Nile Virus and encephalitis viruses.
37. method according to claim 32, wherein said virus is selected from norwalk virus, hepatitis A, poliomyelitis, andoviruses and rhinovirus.
38. preparation has the method for the Sambucus species extract of at least a predetermined characteristic, comprising: extract Sambucus species plant corpus sequentially by following steps, to produce essential oil fraction, polyphenol fraction and polysaccharide fractions:
A) extract Sambucus species plant corpus by supercritical carbon dioxide extracting, to produce the described essential oil fraction and first residue;
B) by with about 40 ℃ to about 70 ℃ water, or the water alcohol extracting fetches extraction Sambucus species plant corpus or derives from first residue of step a), to produce the described polyphenol fraction and second residue; And
C) by extracting second residue that derives from step b) to about 90 ℃ water with about 70 ℃, to produce described polysaccharide fractions.
39. according to the described method of claim 38, wherein step a) comprises:
1) the Sambucus species plant corpus of milling is packed in the extraction vessel;
2) under super critical condition, add carbonic acid gas;
3) make described Sambucus species plant corpus contact a period of time with carbonic acid gas; And
4) in collection container, collect the essential oil fraction.
40. according to the described method of claim 39, also comprise, change the compound ratio of described Essential Oil Chemistry composition by making the classification of described essential oil extract with supercritical carbon dioxide fractionated system.
41. according to the described method of claim 38, wherein step b) comprises:
1) make Sambucus species plant corpus of milling or the residue that derives from step a) and about 40 ℃ to about 70 ℃ water, or contact the sufficiently long time, to extract the polyphenol chemical composition with water-alcohol solution;
2) make water or water-alcohol solution from the polyphenol chemical composition of extracting of step a) pass through the affinity adsorbent resin column, be adsorbed at interior phenolic acid comprising anthocyanidin; And
3) the polyphenol chemical composition fraction of the one or more purifying of wash-out from described affinity adsorbent resin.
42. according to the described method of claim 38, wherein the extracting method of polysaccharide fractions comprises:
1) makes and to contact the sufficiently long time with about 70 ℃ to about 90 ℃ water, with the extraction polysaccharide from second residue of step b); And
2) with ethanol precipitation polysaccharide is precipitated out from the aqueous solution.
43. Sambucus species extract by the described method preparation of arbitrary claim among the claim 38-42.
44. Sambucus species extract, the cinnamic acid and the weight that comprise 1,2,3,-thrihydroxy-benzene, weight and be the 2-metoxyphenol of the methyl cinnamic acid of the 15-25% of 1,2,3,-thrihydroxy-benzene, cinnamamide that weight is the 1-4% of 1,2,3,-thrihydroxy-benzene, 5-10% that weight is 1,2,3,-thrihydroxy-benzene, benzaldehyde that weight is the 1-2% of 1,2,3,-thrihydroxy-benzene, 5-10% that weight is 1,2,3,-thrihydroxy-benzene are the cinnamyl acetate of the 5-15% of 1,2,3,-thrihydroxy-benzene.
45. Sambucus species extract, comprising rutin, weight and be the forulic acid of the 20-30% of rutin, the cinnamic acid that weight is the 25-35% of rutin, shikimic acid, the weight that weight is the 15-25% of rutin is the phenyllactic acid of the 55-65% of rutin.
46. Sambucus species extract, the anthocyan and the weight that comprise rutin, weight and be the texifolin of the 1-10% of rutin, forulic acid that weight is the 1-5% of rutin, cinnamic acid that weight is the 1-5% of rutin, shikimic acid that weight is the 0.5-5% of rutin, phenyllactic acid that weight is the 1-5% of rutin, 5-15% that weight is rutin are the petunidin of the 15-25% of rutin.
47. Sambucus species extract, the forulic acid and the weight that comprise rutin, weight and be the anthocyan of the 30-40% of rutin, petunidin that weight is the 75-85% of rutin, vanillic acid that weight is the 5-10% of rutin, 1-5% that weight is rutin are the cinnamic acid of the 1-10% of rutin.
48. Sambucus species extract, comprise to coumaric acid/phenylpyruvic acid, weight for to the rutin of the 65-75% of coumaric acid/phenylpyruvic acid, weight for to the vanillic acid of the 65-75% of coumaric acid/phenylpyruvic acid, weight for to the forulic acid of the 35-45% of coumaric acid/phenylpyruvic acid, weight for the cinnamic acid of the 65-75% of coumaric acid/phenylpyruvic acid and weight are the shikimic acid to the 45-55% of coumaric acid/phenylpyruvic acid.
49. Sambucus species extract comprises rutin, weight and is the aurantiamarin of the 20-30% of rutin, vanillic acid and the weight of 70-80% that weight is rutin is the cinnamic acid of the 40-50% of rutin.
50. Sambucus species extract comprises petunidin, weight and is the rutin of the 85-95% of petunidin, vanillic acid and the weight of 55-65% that weight is petunidin is the cinnamic acid of the 30-40% of petunidin.
51. Sambucus species extract comprises rutin, weight and is the petunidin of the anthocyan of the 5-15% of rutin, texifolin that weight is the 1-10% of rutin, caffeic acid that weight is the 5-15% of rutin, forulic acid that weight is the 1-10% of rutin, shikimic acid that weight is the 1-10% of rutin, 25-35% that weight is rutin and eriodictyol or the young fustic of the 1-5% that weight is rutin.
52. Sambucus species extract, the naringenin and the weight that comprise rutin, weight and be the eriodictyol of the anthocyan of the 10-20% of rutin, 1-5% that weight is rutin or young fustic, weight and be the 10-20% of rutin are the texifolin of the 1-10% of rutin.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102807544A (en) * 2012-08-07 2012-12-05 宁波杰顺生物科技有限公司 Method for extracting anthocyanin from elderberry fruits
CN101773490B (en) * 2010-01-25 2013-02-20 香港理工大学深圳研究院 Active part of Sambucus williamsii Hance for reducing risk of bone-related diseases of menopausal women and application thereof
CN103097334A (en) * 2010-06-16 2013-05-08 马来西亚棕榈油协会 Compositions comprising shikimic acid obtained from oil palm based materials and method of producing thereof
CN112730152A (en) * 2021-01-12 2021-04-30 中国石油大学(华东) Experimental device and method for testing miscible viscosity of carbon dioxide and crude oil in rock core
CN115487170A (en) * 2022-11-04 2022-12-20 高州市人民医院 Application of pyrogallol in preparation of medicine for preventing or treating influenza virus infection

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101773490B (en) * 2010-01-25 2013-02-20 香港理工大学深圳研究院 Active part of Sambucus williamsii Hance for reducing risk of bone-related diseases of menopausal women and application thereof
CN103097334A (en) * 2010-06-16 2013-05-08 马来西亚棕榈油协会 Compositions comprising shikimic acid obtained from oil palm based materials and method of producing thereof
CN102807544A (en) * 2012-08-07 2012-12-05 宁波杰顺生物科技有限公司 Method for extracting anthocyanin from elderberry fruits
CN102807544B (en) * 2012-08-07 2015-03-11 宁波杰顺生物科技有限公司 Method for extracting anthocyanin from elderberry fruits
CN112730152A (en) * 2021-01-12 2021-04-30 中国石油大学(华东) Experimental device and method for testing miscible viscosity of carbon dioxide and crude oil in rock core
CN115487170A (en) * 2022-11-04 2022-12-20 高州市人民医院 Application of pyrogallol in preparation of medicine for preventing or treating influenza virus infection

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