CN100420459C - Lower alcohol-insoluble extract of hovenia dulcis thunb and a polysaccharide isolated therefrom - Google Patents
Lower alcohol-insoluble extract of hovenia dulcis thunb and a polysaccharide isolated therefrom Download PDFInfo
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Abstract
A pharmaceutical composition and health care food comprising a lower alcohol-insoluble extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom has a potent hepatoprotective and hangover-resolving activity.
Description
Invention field
The present invention relates to a kind ofly have the liver protecting and eliminate the active extract that is insoluble to the Semen Hoveniae (Fructus Hoveniae) (Hovenia dulcis Thunb) of lower alcohol of being still drank after a night, from isolating polysaccharide wherein with comprise its pharmaceutical composition or health food.
Background of invention
Hepatitis is tormenting increasing in the world people, and in default of effective medicine, often can develop into chronic hepatitis, liver cirrhosis, or cancer.When patient is subjected to multiple factor,, during the influencing of excessive consumption ethanol and/or Hepatoxic substance, may develop various types of hepatitis such as pressure.
The CCl that has that is considered to typical Hepatoxic substance
4, D-galactosamine, lipopolysaccharide (LPS), bromobenzene and aldehydes are as being considered to the acetaldehyde of alcohol metabolism approach intermediate.Therefore, seek and new can the liver protecting to avoid the Hepatoxic substance injury or after being subjected to this type of injury, can recover the trial of medicine of liver function a lot.
For example, isolating triterpene glucosides is considered to suppress the release and the alcoholic acid absorption (Yoshikawa, K.T. etc., (1995) Chem.Pharm.Bull.Tokyo, 43 (3), 532-534 page or leaf) of human histamine from the seed of Semen Hoveniae (Fructus Hoveniae) and fruit; The fruit of Semen Hoveniae (Fructus Hoveniae) is found and can suppresses the injury to liver (Hase K. etc., (1997) Chem.Pharm.Bull.Tokyo, 20 (4), the 381-385 page or leaf) that caused by carbon tetrachloride or D-galactosamine/lipopolysaccharide.Above-mentioned and other prior art document description the pharmacological activity of multiple extract of fruits and seeds of hair Semen Hoveniae (Fructus Hoveniae) (Japan cultivars) and Semen Hoveniae (Fructus Hoveniae) (Chinese variety); But, but not about the alcohol insoluble matter extract of Semen Hoveniae (Fructus Hoveniae) or the report of the application of therefrom isolating polysaccharide in the liver protecting and elimination are still drank after a night.
Summary of the invention
Therefore one object of the present invention just provides a kind of pharmaceutically active substance that extracts from Semen Hoveniae (Fructus Hoveniae), and this material shows the liver protecting and the elimination activity of being still drank after a night.
Another object of the present invention provides a kind of method of separating described material from Semen Hoveniae (Fructus Hoveniae).
Another object of the present invention provides a kind of pharmaceutical composition that suppresses ethanol dehydrogenase and lactic acid dehydrogenase, said composition comprise pharmaceutical carrier and above-mentioned from Semen Hoveniae (Fructus Hoveniae) isolating material.
Another purpose of the present invention provides a kind of liver related disease and can treat the pharmaceutical composition of being still drank after a night of being used to prevent or suppressing, and it comprises pharmaceutical carrier and isolating polysaccharide from Semen Hoveniae (Fructus Hoveniae).
A further object of the invention provides a kind of health food that comprises described material and/or derive from the polysaccharide of Semen Hoveniae (Fructus Hoveniae).
According to one aspect of the present invention, provide a kind of insoluble fraction of lower alcohol of handling hot water extract's acquisition of dried Semen Hoveniae (Fructus Hoveniae) with lower alcohol.
According to another aspect of the present invention, also provide a kind of from the insoluble fraction of described lower alcohol isolating polysaccharide with effective liver protecting activities.
The accompanying drawing summary
When in conjunction with the accompanying drawings, above-mentioned and other purpose of the present invention and feature will become from following description of the present invention obviously, and these accompanying drawings are represented respectively:
Fig. 1: the insoluble fraction of methanol of the carpopodium extract of Semen Hoveniae (Fructus Hoveniae) and the preparation flow sketch map of isolating polysaccharide therefrom;
Fig. 2: the DSC scanning of the insoluble fraction of methanol;
Fig. 3: the MALLS spectrum of the insoluble fraction of methanol;
Fig. 4: the IR spectrum of the insoluble fraction of methanol;
Fig. 5: the UV spectrum of the insoluble fraction of methanol;
Fig. 6: the GC spectrum of the insoluble fraction of methanol;
Fig. 7: methanol insoluble polysaccharide MALLS spectrum;
Fig. 8: the IR spectrum of the insoluble fraction of methanol;
Fig. 9: the NMR spectrum of the insoluble fraction of methanol;
Figure 10: the insoluble fraction of methanol is to the external albumen synthesizing activity of the liver section culture of tetrachloro-methane induction;
Figure 11: the insoluble fraction of methanol is to the external albumen synthesizing activity of the inductive liver section culture of galactosamine/LPS;
Figure 12: the insoluble fraction of methanol is to the external albumen synthesizing activity of the inductive liver section culture of bromobenzene;
Figure 13: the insoluble fraction of methanol discharges the inhibition activity to the LDH of the inductive liver section culture of bromobenzene;
Figure 14: the polysaccharide that shows among Fig. 7 is to the external albumen synthesizing activity of the inductive liver section culture of bromobenzene;
Figure 15: the polysaccharide that shows among the insoluble fraction of methanol and Fig. 7 reduces the activity of ethanol in blood concentration; With
Figure 16: the alcohol dehydrogenase activity of the polysaccharide that shows among the insoluble fraction of methanol and Fig. 7.
Detailed Description Of The Invention:
The hot water extract's of trifoliate orange Dulcis the fraction that is insoluble to lower alcohol can be by the preparation of two steps among the present invention. At first, the hot water extract of dried trifoliate orange Dulcis obtains with the high pressure extraction method, and then insoluble fraction obtains with the hot water extract that the lower alcohol processing so obtains.
In the first step, that the carpopodium section of trifoliate orange Dulcis is also dry in the cool. To the dry sliced middle adding suitable quantity of water of fruit stalk of Hovenia dulcis THunb, the mixture of gained is kept at temperature 110-150 ℃, and preferred 120-125 ℃, 1-3atm, the pressure of preferred 1.5atm lower 15 minutes-48 hours, preferred 30 minutes-12 hours period. Then mixture is cooled to room temperature and filters, filtrate according to conventional freeze drying process freeze-drying to obtain the hot water extract.
The hot water extract is at room temperature further dry, under the decompression as-evaporate under the 1.5atm, use lower alcohol, particular methanol, ethanol or butanols, most preferably 100% methyl alcohol extracts, and obtains the insoluble fraction of lower alcohol of wanting with the soluble fraction of removal lower alcohol wherein.
Above-mentioned trifoliate orange Dulcis is selected from Hovenia dulcis Thunb.var.Koreana NAKAI, Hovenia dulcis Thunb.var.tomentella Makino and Hovenia dulcis Thunb.
The insoluble fraction of the lower alcohol that so obtains has following characteristics: the peak value of gel permeation chromatography (GPC) is 1,330,000 dalton (Da) at mean molecule quantity, 142,800Da, 70,540Da and 102,400Da place; IR (KBr, nm) absorption band is at 1000-1300nm (ether, phenol, sulfoxide, the peak of vinyl); UV is absorbed in 200-300nm (peak of cyclic rings) and locates.
A kind of have high liver protecting and the polysaccharide of elimination activity of being still drank after a night can be separated by following steps from the insoluble fraction of lower alcohol.
The insoluble fraction of lower alcohol is dissolved in the distilled water, gained solution is filled ion exchange column, with the wash-out polysaccharide component wash-out progressively of the solution with the cumulative NaCl concentration of 0-5M, dialysis, concentrated and freeze-drying (seeing Fig. 1).
In the process of carrying out ion exchange, can use cation exchange resin or anion exchange resin.The example that can be used as the exchanger resin of this purpose has: strong-acid ion exchange resin such as AG50W-x8, Amberlite IR-120 and Dowex 50W-x8; Weak-acid ion exchange resin such as Amberlite IRC-50, Bio-Rex 70 and Duolite-436; Weak-base ion-exchange resin such as Amberlite IRA-67 and Dowex 3-x4A; Strong basic ion exchange resin such as AG 2x8, Amberlite IRA-400 and Dowex 2-x8; The cellulose cation exchange resin of modification such as CM-Cellulose and SE-Cellulose; A kind of cellulose anion exchange resin of modification such as DEAE cellulose; The cross-linking dextran resin of the resin of cationic crosslinked glucosan type such as G-25 and G-50 pearl type; With ion exchange resin such as the CepharoseCL by the pearl type of the modification of agarose preparation, Biogel A Cepharose resin, Fractogels and Toyopearl.。The exchanger resin of preferred ToyopearlDEAE type, the most preferably exchanger resin of Toyoprearl DEAE-650C type.
Can obtain multiple polysaccharide fractions according to above-mentioned separation method, with the highest liver protecting activities of a kind of polysaccharide demonstration of 0.2M NaCl eluant solution.The polysaccharide that obtains from the Semen Hoveniae (Fructus Hoveniae) of Korean native according to above-mentioned steps shows following characteristic: it is by mannose, glucose, and galactose, rhamnose (rhamanose) and arabinose are formed, and its ratio is 1: 2.51: 12.53: 187: 13.43; Absolute molecular weight is 114,500Da; IR (KBr, nm) spectrum 3550-3450Da (wide, OH), 1660-1600 Da (C=C) and 1290-1420 Da (=CH-OH) locate to show the peak;
1H-NMR (600MHz, D
2O) peak value is located at 4.4-4.8ppm (sugared peak).In addition, the polysaccharide that obtains in China domestic Semen Hoveniae (Fructus Hoveniae) shows following characteristic: it is by comprising galactose, rhamnose, and glucuronic acid, galacturonic acid, xylose, mannose and glucose are formed, and its ratio is 1: 0.84: 1.05: 0.87: 0.89: 2.05.
Various experiments clearly illustrate the insoluble fraction of lower alcohol and have high liver protecting and the elimination activity of being still drank after a night from isolating polysaccharide wherein, and to prevention and treatment hepatopathy be still drank after a night effectively.
Therefore, insoluble fraction of the lower alcohol among the present invention and polysaccharide can be used as prevention or treatment liver toxicity and hepatopathy such as hepatitis, hardened health food of fatty liver regulating liver-QI and medicament.
Therefore, the present invention also provides a kind of pharmaceutical composition that suppresses ethanol dehydrogenase and lactic acid dehydrogenase, and said composition comprises in the insoluble fraction of lower alcohol of the Semen Hoveniae (Fructus Hoveniae) that combines with pharmaceutical excipient, carrier or diluent or the fraction 3 from the insoluble fraction of methanol isolated polysaccharide as active component.
In addition, the invention provides a kind of health food that comprises said extracted thing and/or polysaccharide.
Equally, the pharmaceutical composition that the present invention also provides a kind of prevention and treatment hepatopathy and has been still drank after a night, said composition comprises the insoluble fraction of lower alcohol of the Semen Hoveniae (Fructus Hoveniae) that combines with pharmaceutical excipient, carrier or diluent and therefrom isolating polysaccharide as active component.In addition, the invention provides a kind of health food that comprises said extracted thing and/or polysaccharide.
Pharmaceutical preparation of the present invention can be according to any conventional method preparation.In the process of preparation preparation, active component is preferably mixed or dilution with carrier, perhaps is encapsulated in the carrier, and this carrier can be a capsule, the form of medicine bag or other container.When carrier was used as diluent, it can be solid, semisolid or the liquid substance of carrier, excipient or medium as active component.Therefore, said preparation can be tablet, pill, powder, medicine bag, elixir, suspensoid, Emulsion, solution, syrup, aerosol, soft capsule and hard capsule, aseptic parenteral solution, aseptic packaging powder etc.
Appropriate carriers, the example of excipient or diluent are lactose, glucose, sucrose, Sorbitol, mannitol, starch, Radix Acaciae senegalis, alginate, gelatin, calcium phosphate, calcium silicates, cellulose, methylcellulose, microcrystalline Cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, nipasol, Talcum, magnesium stearate and mineral oil.Said preparation can also contain filler, anti-agglutinant, lubricant, wetting agent, flavoring agent, emulsifying agent, antiseptic etc.By using any method well known in the art can prepare compositions of the present invention,, continue or the release of the active component that postpones so that provide later on fast it being applied to the patient.
Pharmaceutical composition of the present invention can be used by number of ways, comprise oral, percutaneous, subcutaneous, intravenous and intramuscular import.For the treatment of human patients, the typical daily dose of isolating above-mentioned fraction and polysaccharide is about 0.01-10g/kg body weight from Semen Hoveniae (Fructus Hoveniae), preferred 1-5g/kg body weight, and can single dose or divided dose use.But, be to be understood that the amount of the actual active component of using should comprise the symptom that will handle according to various correlative factors, the route of administration of selection, the age of individual patient, sex and body weight, and the seriousness of patient symptom decides; Therefore, above-mentioned dosage should not be used to limit the scope of the invention by any way.
Insoluble fraction of above-mentioned lower alcohol and therefrom isolating polysaccharide be introduced into and prevent various hepatopathys in the Foods or drinks and be still drank after a night.For preventing various hepatopathys or the amount that can join described fraction in the Foods or drinks and/or polysaccharide of being still drank after a night can be generally 0.1-15w/w% based on the food gross weight, preferred 1-10w/w% and based on 100ml beverage 1-30g, preferred 3-10g.
Health beverage compositions of the present invention can contain other component, for example, and as deodorizer in the conventional beverage and natural carbohydrate.This examples of carbohydrates is monosaccharide such as glucose and fructose; Disaccharide such as maltose and sucrose; Conventional sugar is as dextrin and cyclodextrin; And sugar alcohol such as xylitol, Sorbitol and erythritol.As deodorizer, a kind of natural deodorizer such as taumatin, levaudioside A and glycyrrhizin, perhaps synthetic deodorizer such as glucide and radix asparagi sweet extract (aspartam) may be used to.The consumption of above-mentioned natural carbohydrate is generally the about 1-20g based on the 100ml beverage, preferred 5-12g.
Other components that may be added in the Foods or drinks compositions of the present invention are various nutrients, vitamin, mineral; synthetic flavoring agent, coloring agent, pectic acid and salt thereof; alginic acid and salt thereof, organic acid, the gluey binding agent of protectiveness; the pH regulator agent, stabilizing agent, antiseptic; glycerol; alcohol, carburization agent (carbonizing agent), fruit juice and vegetable juice.
Following reference example, check embodiment and preparation embodiment are intended to further illustrate the present invention and do not limit its scope.
Reference example 1: measure absolute molecular weight by the Gel permeation chromatography
The measurement of molecular weight is to use is equipped with pump (spectroscopic system; the p2000 type), guard column (TSKPWH, Tosoh company); RI-detector (Shodex SE71 type); SEC (size exclusion chromatography) post (tsk gel 3000pw, 4000pw, 5000pw (7.8x300mm; Tosoh company) and MALLS (multi-angle laser disperses Dawn DSP-F; Wyatt technology company) the GPC equipment of detector, as developer solution, it comprises 0.15M NaNO with 0.02% Hydrazoic acid,sodium salt
3, be to carry out under the 0.5ml/min at flow velocity.
Reference example 2: reagent and material
The amount of LDH (lactic acid dehydrogenase) is to measure with 340-UV spectrometer (Sigma company, the U.S.).Be used for measuring and syntheticly have by the Hepatoxic substance infringement back liver active proteic amount that heals
3H-leucine (5.Ci/ plate) isotope and being used for is measured the amount of synthetic RNA
3H-uridnine isotope is all bought from Sigma company.The measurement that absorb to change is to use gas chromatogram head space (head-space) analytical equipment, is equipped with that HP 5890 gas chromatographys of FID (flame ionization detector) carry out, to measure alcohol dehydrogenase activity.
Embodiment 1: the preparation of the insoluble fraction of lower alcohol of Korean native Semen Hoveniae (Fructus Hoveniae)
The Korean native Semen Hoveniae (Fructus Hoveniae) stalk (1.5kg) of dry and section is carried out at 120 ℃ the extraction under the high pressure (1.5atm) 3 hours.The extraction solution of gained filters by Wattman paper.Filtrate (218.5g) lyophilizing with the Korean native Semen Hoveniae (Fructus Hoveniae), the powder of gained is through 3 circulation reflux, extract,, the pure methanol of each usefulness 31HPLC-level 1 hour, remaining powder is dried to obtain the insoluble fraction of methanol (dry weight 65.71g, the productive rate: 4.3%w/w) of Korean native Semen Hoveniae (Fructus Hoveniae).In addition, united extraction thing and lyophilizing are to obtain fraction (dry weight 152.29g, the productive rate: 10.27%w/w) that is dissolved in methanol.
Embodiment 2: the analysis of the insoluble fraction of Semen Hoveniae (Fructus Hoveniae) methanol
The property analysis of the insoluble fraction of methanol that obtains among the embodiment 1 is as follows.(1) mensuration of melting temperature and melting enthalpy (entalphy)
The mensuration of melting temperature and melting enthalpy is to measure by DSC (differential scanning calorimetry (DSC), the DSC6100 of Seiko Co., Ltd.).
The sample of the insoluble fraction of methanol of Korean native Semen Hoveniae (Fructus Hoveniae) is placed in the aluminum dish, and sealing is heated to 200 ℃ with the speed of 10 ℃/min from 20 ℃ then, obtains melting heat absorption curve and melting temperature respectively and therefrom records the degree of crystallinity of sample.
The DSC scanning of the insoluble fraction of methanol of Korean native shows that main peak starts from 164.9 ℃, reaches maximum melting temperature at 185.3 ℃.The insoluble fraction of methanol is made several carbohydrates (mp:60-100 ℃) and a small amount of protein (mp:60-100 ℃) (Fig. 2).
(2) analysis of sugar chain
For detecting in the above-mentioned main peak component whether contain sugar chain, according to Hakomori etc. (j.Biochem.Tolyo,
55, 205-209,1964) and Waeghe T.J. etc. (Carbohydrate Research,
123, 281-304,1983) and described method carries out methylation analysis.
The sample of 500.g is methylated, and methylated then product uses the C of ethanol absorption
188x10 barrel mast (Sep-Pak) is collected.The acid sugar of methylated product partly uses the LiB (C among the THF
2H
5)
3D (Super-Deupride, 1ml, Aldrich company) reduction, reduzate uses C
188x10 barrel mast (Sep-Pak) reclaims.Subsequently, the sample of handling carries out successively: 121 ℃ of following hydrolysis are 2 hours in 1.0M TFA; NaBD
4Reduction; Acetylation.The acetic acid sugar alcohol of the part methylization that obtains is analyzed by GLC and GC-EIMS, and peak area is measured with FID (flame ionization detector).
(3) by the GPC determining molecular weight
The result who measures as the GPC that carries out in the reference example 1 shows what the insoluble fraction of methanol was made up of 4 peak value components as shown in Fig. 3 and table 1.
The peak value component of the insoluble fraction of table 1. methanol
| | Peak | 1 | |
|
|
| Mean molecule quantity (Da) | 1,330,000 | 142,800 | 70,540 | 102,400 |
(4) IR spectrum (Fig. 4)
Sample is analyzed by IR spectrum (Vector 22 types, Bruker Analytische Messtechnik GMBH): resolution, 4.0; The source, sphere; Speed, 6,10KHz; Acquisition mode, dual wall (dual wall)/round condition)
IR (KBr, cm
-1): ether, phenol, sulfoxide and ethylene base peak (1000-1300nm; Main 1039nm), aromatic rings peak (665,939,1313,1663), hydroxyl peak (3435nm).
(5) UV spectrum (Fig. 5)
Sample is to analyze by UV-Vis spectroscopy (HP 8453 types, Hewlett Packard company), and the result shows absorption maximum at 200-300nm, and there is cyclic rings in prompting.
Embodiment 3: the separating of polysaccharide with the liver protecting and the elimination activity of being still drank after a night
Have liver protecting and the reactive compound of the elimination activity of being still drank after a night for separating in the insoluble fraction of methanol that from embodiment 1, obtains, the insoluble fraction of the methanol of 200mg is dissolved in distilled water, be filled into
In the DEAE-650C post (4.0x30cm), use 0,0.1,0.2,0.3 and 3M NaCI eluant solution successively.Fraction behind the eluting is used molecular weight 1000 and the dialysis of following penetrating dialyzer, concentrates, and lyophilizing is respectively 38mg, 64mg, 73mg, the purification fraction of 5mg and 4mg to obtain weight then.
Embodiment 4: separate the qualitative of fraction in the Korean native Semen Hoveniae (Fructus Hoveniae)
Among the embodiment 3 through 0,0.1,0.2,0.3 with 3M NaCl eluting after the fraction that separates that obtains be called as fraction 1-5 respectively.To each component, the content of total sugar and weight polyphenol fraction is measured by phenolsulfuric acid method (Dubois M. etc., Anal.Chem.28,350-356,1956), and thus obtained result is presented in the table 2.
Table 2: the content of total sugar and weight polyphenol fraction
(Varian CP-3800 type imposes a condition to carry out the GC analysis; Detector: FID, post: SP-2380 (30mx0.25mmx0.2 μ m), column temperature: 230 ℃, injector temperature: 250 ℃, detector temperature: 250 ℃, mobile phase: N
2Gas (1.0ml/min)) differentiate the saccharic composition in the above-mentioned fraction, measure the relative quantity of mannose, glucose, galactose, rhamnose, arabinose and xylose in each fraction.The result is presented in the table 3.
Table 3: the amount of saccharic composition (with respect to mannose)
| |
|
|
|
|
|
| |
1 | 1 | 1 | 1 | 1 |
| |
6 | 3.29 | 2.51 | 1.13 | 3.05 |
| Galactose | 3.1 | 2.04 | 12.53 | 7.81 | - |
| Rhamnose | - | 0.32 | 187 | 3.64 | - |
| Arabinose | 1.58 | 1.93 | 13.43 | 5.86 | 1.93 |
| Xylose | - | 0.26 | - | 0.75 | - |
The sign of fraction 3
The absolute molecular weight of the fraction of being measured by the method in the reference example 13 is 114,500Da (Fig. 7).IR analyzes (KBr) and shows peak (cm
-1) 3550-3450 (wide, OH), 1660-1600 (C=C) and 1290-1420 (=CH-OH) locate (Fig. 8).
1H-NMR (600MHz, D
2O) shown a peak (sugared peak) at the 4.4-4.8ppm place (Fig. 9).
Embodiment 5: China domestic Semen Hoveniae (Fructus Hoveniae) separates the sign of fraction
Step among the repetition embodiment 1 is to obtain the insoluble fraction of methanol (dry weight 61.5g, productive rate: 4.1%w/w) with the fraction (dry weight 133.5g, the productive rate: 8.9%w/w) that are dissolved in methanol from the carpopodium of China domestic Semen Hoveniae (Fructus Hoveniae).The insoluble fraction of methanol is carried out ion-exchange chromatography according to the method for embodiment 3, through 0,0.1,0.2,0.3 with 3M NaCl eluting after the fraction that separates that obtains be called as fraction 1 '-5 ' respectively.
Fraction 1 '-5 ' is characterized according to the method among the embodiment 4.Saccharic composition is by the GC Analysis and Identification, and the result is presented in the table 4.
The amount of table 4. saccharic composition (with respect to galactose)
| Fraction 1 ' | Fraction 2 ' | Fraction 3 ' | Fraction 4 ' | Fraction 5 ' | |
| Galactose | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| Rhamnose | 0.58 | 0.57 | 0.84 | 0.77 | - |
| Glucuronic acid | 0.87 | 0.78 | 1.05 | 1.47 | 0.87 |
| Galacturonic acid | 0.88 | 0.30 | 0.87 | 0.97 | 0.98 |
| Xylose | 0.55 | - | - | - | - |
| Mannose | 0.91 | - | 0.89 | - | 1.09 |
| Glucose | 1.50 | 0.41 | 2.05 | 4.00 | 1.88 |
SEC-RI peak and absolute molecular weight are according to the step measurements in the reference example 1.The result is presented at respectively in table 5 and 6.
Table 5 is by the peak component ratio of the insoluble fraction of methanol of SEC-RI detector
| Fraction 1 ' | Fraction 2 ' | |
Fraction 4 ' | Fraction 5 ' | |
| |
13％ | 35％ | 5％ | 2％ | 3 |
| Peak | |||||
| 2 | 64.6％ | 60％ | 89％ | 92％ | 89 |
| Peak | |||||
| 3 | 15％ | 1％ | 5％ | 4％ | 4 |
| Peak | |||||
| 4 | 7％ | 3％ | - | 2％ | 4％ |
Table 6 is measured the absolute molecular weight of the insoluble fraction of methanol by SEC-MALLS
Check embodiment 1: the liver protecting function of the insoluble fraction of methanol of Semen Hoveniae (Fructus Hoveniae)
(1)
To by the protection activity of the destructive liver of carbon tetrachloride
The liver that will take out from the Sprague-Dawley rat in five ages in week is cut into discoid sample with histotome (U.S. Brendel/Vitron company), and it is 200.m (weight in wet base: 18-22mg) that the diameter of each is about 0.8mm thickness.The sample that cuts is divided into four groups, and wherein two groups (group 3 and group 4) respectively with insoluble fraction of preparing among the embodiment 1 of methanol and the level divisional processing that is dissolved in methanol, every group concentration is 200.g/ml.Then, 4 samples that cut are at O
2/ CO
2Under=95%/5% the atmosphere in heat power organ-tissue slice culture instrument (Japanese Sankyo company) surface cultivate.After 1 hour, to group 3, group 4 with remain in two groups one (group 2) every group to add carbon tetrachloride to concentration be 4mM.Another group (group 1) substitutes carbon tetrachloride with distilled water and handles (contrast).All groups are preserved and were induced hepatic injury in 5 hours then.
Thereafter, the amount that the liver detoxicating functions that Semen Hoveniae (Fructus Hoveniae) extracts fraction is measured synthetic proteins by the method for describing according to Bonney etc. (" SomeCharacteristics and Function of Adult Rat Liver Pimary Culture; in GeneExpression and Carcinogenesis in Cultured Liver; (1975) Gerschenson; E and Thompson, E.B. (Eds), Academic Press; New York, 24-45 page or leaf) is assessed.Shown in Figure 10 a, prove that the fraction that the insoluble fraction of methanol is dissolved in methanol has much higher activity.
In addition, the result among Figure 10 b is by assigning to obtain with the insoluble fraction of methanol of preparation among the embodiment 5 or the level that is dissolved in methanol according to the method described above, and wherein the insoluble fraction of methanol shows more a lot than the fraction that is dissolved in methanol.
(2)
Recovery is by the activity of the liver function of D-galactosamine/LPS damage
The lipopolysaccharide (LPS) of having known D-galactosamine and antibacterial is used together and is caused hepatic injury, and this damage is similar to hepatic injury that human hepatitis causes on biochemistry and histology.The insoluble fraction of the methanol of Semen Hoveniae (Fructus Hoveniae) detects as follows to the Detoxication of the liver of this damage:
Substitute carbon tetrachloride with the D-galactosamine of 500.M and the LPS of 1.g/ml, the step among the duplicate test embodiment 1 is measured the activity of repairing liver injury.Shown in Figure 11 a, the insoluble fraction of methanol all has significant repairing activity with the fraction that is dissolved in methanol.In addition, the result among Figure 11 b shows that insoluble fraction of methanol that obtains among the embodiment 5 and the fraction that is dissolved in methanol also demonstrate significant repairing activity.
(3)
Repair the liver of bromobenzene damage
Except that the bromobenzene with 1mM replaces the carbon tetrachloride, the step evaluates of the detoxicating activity of Korea S or China domestic Semen Hoveniae (Fructus Hoveniae) extract by describing in (1).The fraction that result displayed points out the insoluble level of methanol proportion by subtraction to be dissolved in methanol among Figure 12 a (Korea S) and the 12b (China) is more effective aspect the impaired liver of reparation.
In addition, the amount of the LDH that discharges from culture medium (lactic acid dehydrogenase) is measured by using Sigma test kit 340-UV device, as a result, Figure 13 a (Korea S) and 13b (China), the fraction that insoluble grade of proportion by subtraction of demonstration methanol is dissolved in methanol is more effective aspect the release of inducing the LDH that is caused by bromobenzene.
Check embodiment 2: the liver protecting activities of isolating polysaccharide in the insoluble fraction of the methanol of Semen Hoveniae (Fructus Hoveniae).
The liver section that to win from the Sprague-Dawley rat in five ages in week with histotome (U.S. Brendel/Vitron company) is about 0.8mm to obtain diameter, and thickness is 200.m (weight in wet base: disk shaped samples 18-22mg).The sample that cuts is divided into 7 groups, handles with five polysaccharide fractions of preparation among the embodiment 4 respectively for wherein 5 groups, and the concentration of each is 200.g/ml (being respectively group 1-group 5).Then, the sample that cuts is at O
2/ CO
2Under=95%/5% the atmosphere, cultivate instrument (Japanese Sankyo company) surface, lining at the heat power organ-tissue and cultivated 1 hour.Then, each sample adding bromobenzene to the concentration among to group 1-5 and in remaining two groups (group 6) is 4mM.Last remaining one group (group 7) substitutes bromobenzene with distilled water and handles (contrast).
Thereafter, the amount of the synthetic proteins measured by the method for describing according to (on seeing) such as Bonney of the liver detoxification effect of each polysaccharide fractions of Korea S's local products Semen Hoveniae (Fructus Hoveniae) is assessed.The polysaccharide of result displayed proof fraction 3 has the highest activity among Figure 14.
Check embodiment 3: the elimination living-article of being still drank after a night of isolating polysaccharide in the insoluble fraction of Semen Hoveniae (Fructus Hoveniae)
In order to measure the elimination activity of being still drank after a night of the insoluble fraction of methanol of checking the Semen Hoveniae (Fructus Hoveniae) that obtains among the embodiment 1, the Sprague-Dawley rat in 15 three ages in week has been divided into 3 groups, and fasting still allowed to take in water in 24 hours.Then, the ethanol of 2ml 40% is with rustless steel probe (length: 10cm) by by oral administration to every rat.After 1 hour, with 2ml, insoluble fraction of the methanol of 500mg/ml or therefrom isolating polysaccharide (fraction 3) are applied to every rat of experimental group (group 2 and group 3), and the rat of contrast (group 1) is used 2ml water.After 4 hours, the ethanol content from the blood sample that every rat heart is taked is measured according to the method (In Methods of EnzymaticAnalysis, the 3rd edition, 598-6062,1984) that Bergmeyer describes.
Result (Figure 15) shows that the concentration of alcohol of the blood sample of taking is respectively 0.058%, 0.032% and 0.028% from group 1-group 3.Promptly compare with matched group, insoluble fraction of the methanol of Semen Hoveniae (Fructus Hoveniae) and therefrom isolating polysaccharide have reduced by 44.8% and 51.7% respectively with the concentration of alcohol of blood.This result has proved that the alcohol insoluble matter fraction of Semen Hoveniae (Fructus Hoveniae) and therefrom isolating polysaccharide have the ability of the concentration of alcohol in the remarkable reduction blood.
The detection of the alcohol dehydrogenase activity of impaired liver is as follows.
Rat is by CO
2Suffocate, therefrom take out its liver, use distilled water wash.Each liver sample is placed in the 0.1M kaliumphosphate buffer that contains 0.154M KCl (pH 7.4) of 10 times of volumes, and stirs evenly with the politef glass homogenizer.With the solution of homogenize under 4 ℃ 9, carried out under the 000xg centrifugal 30 minutes, the supernatant that obtains 110, carried out ultracentrifugation 1 hour under the 000xg under 4 ℃, to obtain supernatant kytoplasm fraction.The kytoplasm fraction of 2-3mg is added to comprises 55mM sodium pyrophosphate buffer solution (pH 7.4), in the reactant mixture of 20mM ethanol and 0.2mM NAD.The concentration of the NAD that uses in the reactant mixture changes between 0.025mM-2mM.Measure the absorbance of reactant mixture and change 3min, calculate the activity of ethanol dehydrogenase by the slope of absorption curve at the 340nm place.
As shown in figure 16, the insoluble fraction of methanol and from the fraction 3 of the insoluble fraction of methanol isolating polysaccharide show the alcohol dehydrogenase activity of increase, proved the elimination activity of being still drank after a night that they are superior thus.
Exemplify as following institute, insoluble fraction of lower alcohol of the present invention and therefrom isolating polysaccharide can be used for preparation effective powder, tablet, capsule, injection or fluid composition on the pharmacology according to any known conventional method.
[preparation embodiment 1]
The 2g dry extract that obtains among the embodiment 1 mixed obtaining powder with the 1g lactose, it is filled and is sealed in the airtight packing.
[preparation embodiment 2]
With the dry extract that obtains among the 100mg embodiment 1, the 100mg corn starch, 100mg lactose and the mixing of 2mg magnesium stearate and tabletting are to obtain tablet formulation.
[preparation embodiment 3]
With the dry extract that obtains among the 100mg embodiment 1, the 100mg corn starch, the 100mg lactose, the 2mg magnesium stearate is mixed in the gelatine capsule of packing into to obtain capsule preparations.
[preparation embodiment 4]
With the dry extract that obtains among the 100mg embodiment 5, distilled water and an amount of pH regulator agent dissolving to be obtaining ejection preparation, said preparation be loaded into that the peace of 2ml is cutd open in (ample) and according to the injection preparation method sterilization of routine to obtain ejection preparation.
Health food can typically prepare according to following method:
[preparation of health food]
With brown rice, Fructus Hordei Vulgaris, (the exsiccant dietary mixt of the adustion of Job ' s-tears) is pulverized and is sieved to obtain 60 orders or following cereal-granules for Oryza glutinosa and Semen Coicis.In addition, Semen sojae atricolor, the mixture of Semen Sesami Nigrum and Herba lamii barbati are steamed, dry, adustion, pulverize, and sieve to obtain 60 orders or following seed particle.
The insoluble fraction of the methanol of the exsiccant Semen Hoveniae (Fructus Hoveniae) that obtains among the embodiment 1 is pulverized and is sieved obtaining 60 orders and following granule, and itself and cereal-granules and seed grain are prepared corpuscular health food with following mixed.
Corn: brown rice 30w%, Semen Coicis 15w%, Fructus Hordei Vulgaris 20w%,
Seed: Herba lamii barbati 7w%, Semen sojae atricolor 8w%, Semen Sesami Nigrum 7w%,
The dried powder of Korea S's local products Semen Hoveniae (Fructus Hoveniae): 3w%,
Lentinus Edodes (Shiitake mushroom) 0.5w%, Radix Rehmanniae (rehmania root) 0.5w%
Although describe the present invention with reference to above-mentioned specific embodiments, be to be understood that those skilled in the art can carry out various improvement and variation to the present invention, in its scope of the present invention that also belongs to appended claim and limited.
Claims (14)
1. the insoluble fraction of Semen Hoveniae (Fructus Hoveniae) hot water extract's methanol.
2. polysaccharide, it separates from the insoluble fraction of the methanol of claim 1.
3. the polysaccharide of claim 2, it has following feature: by mannose, glucose, galactose, rhamnose and arabinose were with 1: 2.51: 12.53: 187: 13.43 ratio is formed; Absolute molecular weight: 114,500Da; IR (the OH broad peak of KBr, nm) peak: 3550-3450Da, the C=C peak of 1660-1600Da and 1290-1420Da=the CH-OH peak; With
1H-NMR (600MHz, D
2O) the sugared peak of peak: 4.4-4.8ppm.
4. the polysaccharide of claim 2, it has following feature: by galactose, rhamnose, glucuronic acid, galacturonic acid, xylose, mannose and glucose were with 1: 0.84: 1.05: 0.87: 0.89: 2.05 ratio is formed.
5. prepare the method for the insoluble fraction of methanol of claim 1, it comprises following steps: the hot water extract who obtains dry Semen Hoveniae (Fructus Hoveniae); Described hot water extract is carried out methanol extraction to be handled to obtain the insoluble fraction of methanol.
6. prepare the method for the polysaccharide of claim 2, it comprises following steps: the hot water extract who obtains dry Semen Hoveniae (Fructus Hoveniae); Handle described hot water extract to obtain the insoluble fraction of methanol with methanol; The insoluble fraction of methanol is carried out ion-exchange chromatography to obtain described polysaccharide.
7. the method for claim 6, wherein said ion-exchange chromatography is to use cation exchange resin or anion exchange resin to carry out.
8. the method for claim 7, wherein said ion-exchange chromatography is to use and is selected from CepharoseCL, Biogel A Cepharose resin, the ion exchange resin of the pearl type of the modification of Fractogels and Toyopearl resin carries out.
9. the method for claim 8, wherein said ion-exchange chromatography is to use the Toyopearl resin to carry out.
10. one kind is used to eliminate the compositions of being still drank after a night, and it comprises the insoluble fraction of methanol of claim 1 or the polysaccharide of claim 2, and pharmaceutical carrier.
11. a pharmaceutical composition that is used to prevent or treat hepatopathy, it comprises the insoluble fraction of methanol of claim 1 or the polysaccharide of claim 2, and pharmaceutical carrier.
12. the pharmaceutical composition of claim 11, wherein said hepatopathy are hepatitis or liver cirrhosis.
13. a health food, it comprises acceptable additive on the polysaccharide of the insoluble fraction of methanol of claim 1 or claim 2 and the sitology.
14. the health food of claim 13, wherein said food is type of beverage.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR2002/2772 | 2002-01-17 | ||
| KR10-2002-0002772A KR100403721B1 (en) | 2001-01-31 | 2002-01-17 | Lower alcohol insoluble extract and a polysaccharide therein isolated from hovenia dulcis var. koreana nakai having antihepatotoxic and anti-hangover activity and composition containing same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1615145A CN1615145A (en) | 2005-05-11 |
| CN100420459C true CN100420459C (en) | 2008-09-24 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB028271955A Expired - Lifetime CN100420459C (en) | 2002-01-17 | 2002-07-31 | Lower alcohol-insoluble extract of hovenia dulcis thunb and a polysaccharide isolated therefrom |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN100420459C (en) |
| AU (1) | AU2002367003A1 (en) |
| WO (1) | WO2003059369A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1718167A4 (en) | 2004-01-31 | 2010-07-14 | Kiyoung Kim | Composition comprising hovenia dulcis thunb. extract, lindera obtusiloba blume extract, or herbal mixture extract thereof |
| CN100396200C (en) * | 2005-11-24 | 2008-06-25 | 许滔 | Alcohol neutralize drink of Japanese raisin tree seed and its preparation method |
| KR100626167B1 (en) * | 2006-06-28 | 2006-09-20 | 조일환 | Anti-hangover composition |
| CN101336987B (en) * | 2008-08-12 | 2012-04-25 | 西北农林科技大学 | Preparation method of total flavone of Hovenia dulcisThunb |
| CN101423558B (en) * | 2008-12-17 | 2011-08-10 | 四川大学 | Eupatorium adenophorum spreng polysaccharide and preparation method and use |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3153256B2 (en) * | 1991-03-08 | 2001-04-03 | サントリー株式会社 | Sickness improver |
| KR20000051662A (en) * | 1999-01-25 | 2000-08-16 | 남종현 | A natural tea for helping recovery of liver function |
| KR19990073622A (en) * | 1999-07-27 | 1999-10-05 | 이군희 | Manufacture method of drink for white stone extract contain |
| KR100345798B1 (en) * | 1999-08-17 | 2002-07-24 | 현 용 이 | The food with intoxication dissolution function, using Hovenia dulcis THUNB, Alnus japonica, Pueraria thunbergiana BENTH |
| KR20010069022A (en) * | 2000-01-11 | 2001-07-23 | 문혜연 | Functional beverage containing water extract of Hovenia dulcis and process for preparation thereof |
-
2002
- 2002-07-31 CN CNB028271955A patent/CN100420459C/en not_active Expired - Lifetime
- 2002-07-31 AU AU2002367003A patent/AU2002367003A1/en not_active Abandoned
- 2002-07-31 WO PCT/KR2002/001449 patent/WO2003059369A1/en not_active Application Discontinuation
Non-Patent Citations (2)
| Title |
|---|
| 枳椇属植物研究进展 马林，宋万志.中草药，第31卷第11期. |
| 枳椇属植物研究进展 马林，宋万志.中草药，第31卷第11期. * |
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| Publication number | Publication date |
|---|---|
| WO2003059369A1 (en) | 2003-07-24 |
| AU2002367003A1 (en) | 2003-07-30 |
| CN1615145A (en) | 2005-05-11 |
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