Summary of the invention
One of technical problem of solution required for the present invention provides a kind of long stalk Fructus Schisandrae Sphenantherae extract; Two of the technical problem of solution required for the present invention provides a kind of preparation method of growing the stalk Fructus Schisandrae Sphenantherae extract; Three of the technical problem of solution required for the present invention provides a kind of purposes of growing the stalk Fructus Schisandrae Sphenantherae extract; Four of the technical problem of solution required for the present invention provides a kind of compositions that contains long stalk Fructus Schisandrae Sphenantherae extract; To overcome the deficiencies in the prior art and defective.
Technical scheme of the present invention is as follows:
Long stalk Fructus Schisandrae Sphenantherae extract of the present invention, its preparation method is as follows:
Obstruct RADIX KADSURAE LONGIPEDUN LATAE 12-48 hour with the water logging bubble of 6~14 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight is long, decoct and extract 2-4 time, extracted 0.5-1.5 hour at every turn, merge each time water extract; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 60~80%, placed precipitate with ethanol 8~24 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Said long stalk Fructus Schisandrae Sphenantherae is its root, but after the clean dry, pulverizes and use.
Said water consumption is 6~14 times of medical material weight, and preferably 8~12 times, most preferred is 10 times.Amount of water is too much, the workload when increase filtrate concentrates; Very few, then extracts active ingredients is incomplete.It is to extract solvent that extracting method of the present invention makes water, and different with the acetone and other organic solvent extraction method with ethanol, the effective ingredient of resulting extract also has essential distinction on kind.
Said decoction is extracted as 2~4 times, extracts 0.5~1.5 hour at every turn, and collecting decoction preferably extracts 3 times, extracts collecting decoction 1 hour at every turn.Extraction time is too much, overlong time, and impurity such as more starch, polysaccharide are easy to get; Extraction time is very few, the time is too short, and then extracts active ingredients is incomplete.
Said filtration can use the filter method of the various routines of the field of Chinese medicines to carry out, as filter paper filtering, membrane filtration etc.
Said concentrating can use the method for concentration of the various routines of the field of Chinese medicines to carry out, as decompression rotary evaporation, ultrafiltration etc.
The said ethanol that adds makes concentration of alcohol to 60~80% to the condensed water extract, and preferred concentration of alcohol is 70%.Concentration of alcohol is too high, easily causes loss of effective components; Cross lowly, can not effectively remove impurity such as protein, polysaccharide.
The said precipitate with ethanol time is 8~24 hours.Time is too short, effectively precipitated impurities.
Said drying can use the drying means of the various routines of the field of Chinese medicines to carry out, as water bath method, drying under reduced pressure etc.
Long stalk Fructus Schisandrae Sphenantherae extract of the present invention can use separately, also can become pharmaceutical composition with pharmaceutically acceptable vehicle group.Extract adds various conventional adjuvant required when preparing different dosage form, as diluent, disintegrating agent, adhesive, lubricant, correctives, antiseptic etc., method of Chinese medicinal with routine can be prepared into any peroral dosage form commonly used, as pill, powder, tablet, capsule, oral liquid etc.
Beneficial effect of the present invention is:
Simple, lower, the favorable reproducibility of cost of the preparation technology of long stalk Fructus Schisandrae Sphenantherae extract.
That resulting long stalk Fructus Schisandrae Sphenantherae water solubility extract has is significantly antibiotic, antioxidation, antiulcer and diarrhea effect, and the oral acute toxicity of rat is lower, can become the active drug of clinical treatment gastroenteropathy.
With long stalk Fructus Schisandrae Sphenantherae extract of the present invention is principal agent, can prepare the modern Chinese medicine preparation of multiple long stalk Fructus Schisandrae Sphenantherae, makes things convenient for the patient to use.
The specific embodiment
Embodiment
Can further understand the present invention by the following specific embodiments.But they are not limitation of the invention.
Embodiment 1:
Water logging bubble with 6 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 2 times, extracts collecting decoction 1.5 hours at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 60%, placed 8 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 2:
Water logging bubble with 8 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 3 times, extracts collecting decoction 1 hour at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 70%, placed 12 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 3:
Water logging bubble with 10 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 4 times, extracts collecting decoction 0.5 hour at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 80%, placed 24 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 4:
Water logging bubble with 6 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 3 times, extracts collecting decoction 1.5 hours at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 60%, placed 10 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 5:
Water logging bubble with 8 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 2 times, extracts collecting decoction 1 hour at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 70%, placed 16 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 6:
Water logging bubble with 10 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 4 times, extracts collecting decoction 1 hour at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 80%, placed 20 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 7:
Water logging bubble with 6 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 4 times, extracts collecting decoction 1.5 hours at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 70%, placed 8 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 8:
Water logging bubble with 8 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 3 times, extracts collecting decoction 0.5 hour at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 80%, placed 12 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Embodiment 9:
Water logging bubble with 10 double-lengths stalk RADIX KADSURAE LONGIPEDUN LATAE weight spends the night, and extracts 2 times, extracts collecting decoction 0.5 hour at every turn; Filter the condensed water extract; Add ethanol to the condensed water extract, make concentration of alcohol to 60%, placed 24 hours; Filter, reclaim ethanol, drying must long stalk Fructus Schisandrae Sphenantherae extract.
Employed medicine is the long stalk Fructus Schisandrae Sphenantherae extract among the embodiment 2 among the embodiment 10~14.
Embodiment 10:
External antibacterial, the bactericidal effect of long stalk Fructus Schisandrae Sphenantherae extract
1 strain: staphylococcus aureus, bacillus subtilis, escherichia coli, clostridium perfringen, saccharomyces cerevisiae
2 culture medium: nutrient agar, nutrient broth medium, sabouraud culture medium, Sha Shi culture fluid all have the disease prevention and control center, Shanghai to provide.
3 experiment equipments: pressure-steam sterilization device (Shanghai Medical Nuclear Instrument Factory, model YXQ-SG4b-280), constant incubator (Taicang experimental facilities factory, model THZ-C), clean work station (Suzhou instrument plant, model SW-CJ-2FD), slide gauge etc.
4 experimental techniques:
1) inhibition zone is measured
Add the solid medium that 20ml has melted in the aseptic plate, fixing back adds 100 μ l and contains bacterium (10
6Individual/milliliter) normal saline solution, with spreading rod it is dispersed on the solid medium.Interior even the punching 4~6 of aseptic metal card punch of using diameter 6mm of every plate removed agar in the hole, and adding is subjected to reagent liquid, and antibacterial is organized and puts the interior cultivation of 37 ℃ of incubators 18~24 hours, and the fungus group is put the interior cultivation of 30 ℃ of incubators 48 hours, observed result.
Antibacterial action shows as the area that occurs no bacterial growth around the hole, i.e. inhibition zone is selected evenly and the inhibition zone of integral asepsis growth is measured, and writes down the diameter (comprising the aperture) of inhibition zone with slide gauge.The experiment triplicate.The positive contrast of azithromycin (6.4 μ g/ sheet or hole), negative control is a water.The results are shown in Table 1.
2) minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are measured
The two-fold dilution tests medicine with distilled water, gets each dilution experiment medicinal liquid 2.5ml and joins in the test tube that contains the double concentration nutrient broth of 2.5ml.Getting 50 μ l bacteria containing amounts is 10
6Individual/milliliter bacteria suspension is inoculated in respectively and contains medicinal liquid (experimental group) and not containing in the test tube of medicinal liquid (positive controls), and other gets 3 test tubes that contain nutrient broth, as negative control.The antibacterial group is put in 37 ℃ of incubators and was cultivated 18~24 hours, and the fungus group is put in 30 ℃ of incubators and cultivated observed result 48 hours.The positive control pipe has bacteria growing (muddiness), negative control pipe asepsis growth (transparent), and experimental group is MIC with the liquor strength of perusal asepsis growth.Get 100 μ l from the experiment tube of asepsis growth, add respectively in the nutrient agar panel, 37 ℃ of cultivations were cultivated 48 hours in 24 hours or 30 ℃, observed the bacterial growth situation, and the medicinal liquid Cmin that no bacterium colony forms is MBC, experiment repetition 4 times.The results are shown in Table 2.
5 experimental results
1) bacteriostasis of long stalk Fructus Schisandrae Sphenantherae extract the results are shown in Table 1.Medicinal liquid is all had the obvious suppression effect to trying bacterium when 50mg/ml as can be known, and wherein the inhibition effect to gram positive bacteria significantly is better than gram negative bacteria and saccharomyces cerevisiae.
The bacteriostasis of the long stalk of table 1 Fructus Schisandrae Sphenantherae extract
Annotate: " NA " represents no obvious fungistatic effect.
2) minimum inhibitory concentration and bacteriocidal concentration measurement result see Table 2.Can play antibacterial and bactericidal action to gram positive bacteria when liquor strength is low as can be known, and, then need higher liquor strength gram positive bacteria and saccharomyces cerevisiae.
The MIC and the MBC of the long stalk of table 2 Fructus Schisandrae Sphenantherae extract
Annotate: "-" expression asepsis growth; "+" expression has bacteria growing.
Embodiment 11:
Long stalk Fructus Schisandrae Sphenantherae extract Antioxidtive Activities in Vitro
1 suppresses the autooxidation of liver homogenate lipid
Get the SD rat, sacrificed by decapitation is got liver, cleans in 4 ℃ normal saline, is cut into fine grained chippings; Take by weighing the 10g tissue, add the 100ml normal saline, make liver homogenate (8000-10000rpm) with refiner.Centrifuge tube is numbered, getting fresh rat liver homogenate (100mg/ml) 1.5ml adds respectively in each centrifuge tube, experiment tube adds variable concentrations long stalk Fructus Schisandrae Sphenantherae extract solution or vitamin c solution 0.1ml respectively, control tube adds normal saline 0.1ml, every group of parallel 3 pipes, 37 ℃ of vibration incubation 2h take out the trichloroacetic acid cessation reaction that the back adds 1.5ml20%.Other establishes the blank pipe, adds the trichloroacetic acid of 1.5ml20% before the incubation, 37 ℃ of vibration incubation 2h.Leave standstill 10min behind the mixing, the centrifugal 10min of 3000rpm.Get supernatant solution 1.5ml, add the 1ml thiobarbituricacid, boiling water bath heating 10min.532nm wavelength place, cooling back measures trap.
The long stalk of table 3 Fructus Schisandrae Sphenantherae extract is to the autoxidizable inhibitory action of rat liver homogenate
Annotate: this group experiment is not carried out in "-" expression.
The results are shown in Table 3, show that long stalk Fructus Schisandrae Sphenantherae extract has the obvious suppression effect to rat liver homogenate, and this inhibitory action strengthens with the increase of liquor strength.Its median effective dose (IC
50) be 0.2mg/ml, be lower than the IC of reference substance dimension C
50(0.8mg/ml).
2 suppress the rat oxidative hemolysis of erythrocyte effect that hydrogen peroxide brings out
Erythrocytic preparation: the SD rat tail vein is got blood, anticoagulant heparin, and the centrifugal 5min of 2000rpm, normal saline washs 3 times, is made into the red cell suspension of 10% (v/v).Experiment is divided into matched group (do not add medicinal liquid, add the normal saline of same amount), medicine group (drug level be respectively 50.0 and 25.0mg/ml), every group of parallel tee pipe.Get red cell suspension 0.5ml, add normal saline or drug solution 0.15ml respectively, behind 37 ℃ of incubation 10min, 0.5 milliliter of the hydrogenperoxide steam generator (blank pipe does not add hydrogen peroxide) that adds 100mmol/L, continue incubation 2h, add the normal saline dilution of 3 times of amounts, the centrifugal 6min of 3000rpm, get supernatant, 541nm wavelength place measures trap.With matched group is haemolysis degree and the inhibition degree that 100% haemolysis calculates matched group and medicine group.
The inhibitory action of the rat oxidative hemolysis of erythrocyte that the long stalk of table 4 Fructus Schisandrae Sphenantherae extract brings out hydrogen peroxide
The results are shown in Table 4, show that long stalk Fructus Schisandrae Sphenantherae extract has the obvious suppression effect to the rat oxidative hemolysis of erythrocyte that hydrogen peroxide brings out, when drug level was 50.0mg/ml, its suppression ratio reached 82%.Along with the increase of drug level, its inhibitory action to oxidative hemolysis of erythrocyte strengthens.
The mensuration of 3 extract reducing powers
Get the drug solution 0.1ml of variable concentrations, add phosphate buffer solution 0.5ml, 1% the potassium ferricyanide solution 0.5ml of 0.2mol/L pH6.6, mixing, 50 ℃ of insulation 20min, add 10% trichloroacetic acid solution 0.5ml again, behind the vibration mixing, centrifugal 10min under the 4000rpm.Get supernatant solution 1ml, add the ferric chloride solution of 1ml distilled water and 0.2ml 0.1%, leave standstill 10min after, system becomes blueness by yellow, 700nm place mensuration trap.Positive control is the vitamin c solution of 0.2mg/ml.
The reducing power of the long stalk of table 5 Fructus Schisandrae Sphenantherae extract
The results are shown in Table 5, show that its reducing power improves along with the increase of long stalk Fructus Schisandrae Sphenantherae extract concentration.When drug level was 2.5mg/ml, its reducing power was higher than positive control.
Embodiment 12:
Long stalk Fructus Schisandrae Sphenantherae extract causes the therapeutical effect of diarrhea mice to Radix Et Rhizoma Rhei
Get 40 of mices, male and female half and half, body weight 20 ± 2g is provided by Fudan University's Experimental Animal Center.Behind the fasting 12h, be divided into 4 groups at random: normal control group, Radix Et Rhizoma Rhei group, low dosage administration group and high dose administration group.Normal control group and Radix Et Rhizoma Rhei group are irritated stomach and are given normal saline, and low dosage administration group and high dose administration group are irritated stomach respectively and grown stalk Fructus Schisandrae Sphenantherae extract 4.8g/kg and 6.0g/kg.Behind the administration 1h, except that the normal control group, all the other each group is all irritated stomach and is given the Radix Et Rhizoma Rhei infusion (12.5g crude drug/kg of 1g crude drug/ml).Single cage that only divides is observed, give Radix Et Rhizoma Rhei decocting liquid 2h after, the record defecation is counted, and writes down 6h altogether.
The long stalk of table 6 Fructus Schisandrae Sphenantherae extract causes the inhibitory action of diarrhea of mouse to Radix Et Rhizoma Rhei
The results are shown in Table 6, show when dosage is 6.0g/kg, long stalk Fructus Schisandrae Sphenantherae extract has tangible anti-diarrhea effect to diarrhoea due to the Radix Et Rhizoma Rhei.
Embodiment 13:
Long stalk Fructus Schisandrae Sphenantherae extract causes the therapeutical effect of diarrhea mice to Radix Et Rhizoma Rhei
1) hydrochloric acid-ethanol is caused the influence of rat acute gastric mucosa injury
Get the SD rat, male and female half and half are provided by Fudan University's Experimental Animal Center.Be divided into 5 groups at random: normal rat group, high dose administration group, middle dosed administration group, low dosage administration group, losec group.Before the experiment, rat fasting 24h can freely drink water.The normal control group is irritated stomach and is given normal saline, and low dosage administration group, middle dosed administration group and high dose administration group are irritated stomach respectively and grown stalk Fructus Schisandrae Sphenantherae extract 0.7g/kg, 1.4g/kg and 2.0g/kg, and the losec group is irritated stomach and given magnesium omeprazole 10mg/kg.Behind the administration 1h, every rat oral gavage gives hydrochloric acid-alcoholic solution 1.5ml (hydrochloric 1.7 milliliters of per 100 milliliters of hydrochloric acid-alcoholic solution, 60 milliliters of dehydrated alcohol, distilled water preparation).Put to death rat behind the 1h, the ligation cardiac end injects 1% formalin 5ml from the pylorus end, ligation pylorus end, fixedly 10min takes out stomach, cut off coat of the stomach along greater gastric curvature, as ulcer index (diameter surpasses 1 millimeter person's length doubles), calculate ulcer and suppress percentage rate with gastric mucosa injury length summation.
The long stalk of table 7 Fructus Schisandrae Sphenantherae extract causes the inhibitory action of gastric mucosa injury to acidic alcohol
The results are shown in Table 7, when showing high, medium and low dosage, long stalk Fructus Schisandrae Sphenantherae extract has the obvious suppression effect to gastric mucosa injury due to the ethanol solution hydrochloride.When dosage was 2.0g/kg, it was close with the marketed drugs losec to suppress effect.
2) to the influence (method is restrained in water logging) of stress in rats gastric ulcer
Get the SD rat, male and female half and half are provided by Fudan University's Experimental Animal Center.Be divided into 5 groups at random: normal rat group, high dose administration group, middle dosed administration group, low dosage administration group, losec group.Before the experiment, rat fasting 24h can freely drink water.The normal control group is irritated stomach and is given normal saline, and low dosage administration group, middle dosed administration group and high dose administration group are irritated stomach respectively and grown stalk Fructus Schisandrae Sphenantherae extract 0.7g/kg, 1.4g/kg and 2.0g/kg, and the losec group is irritated stomach and given magnesium omeprazole 10mg/kg.Behind the administration 1h, rat is fixed in the little cage of wire netting system, is dipped in the water of (20 ± 1) ℃, liquid level remains on rat ensiform process of sternum level.Behind the 18h, take out rat, put to death the ligation cardiac end, inject 1% formalin 5ml from the pylorus end, ligation pylorus end, fixedly 10min takes out stomach, cut off coat of the stomach along greater gastric curvature, magnifier is measured ulcer length summation down, as ulcer index, calculates ulcer and suppresses percentage rate.
The long stalk of table 8 Fructus Schisandrae Sphenantherae extract is arrested cold water and is soaked the inhibitory action that causes gastric ulcer
The results are shown in Table 8, when showing high and low dosage, long stalk Fructus Schisandrae Sphenantherae extract is to cold water straining lasering type tool significant inhibitory effect.
Embodiment 14:
The acute toxicity of long stalk Fructus Schisandrae Sphenantherae extract
In the preliminary experiment, irritate stomach and award long stalk Fructus Schisandrae Sphenantherae extract solution, mice does not all occur dead, can't measure median lethal dose(LD 50), so measure its maximum tolerated dose.
Get kunming mice, 20 ± 2g is provided by Fudan University's Experimental Animal Center.Be divided into three groups, 15 every group, gastric infusion, every day 2 times, 8h observed 14 days continuously at interval, record dead mouse number.
The oral acute toxicity of table 9 long stalk Fructus Schisandrae Sphenantherae extract mice
Do not have after the mice administration lose weight, untoward reaction such as loss of appetite, observe and do not see dead mouse in 14 days, postmortem does not see that major organs is unusual.The result shows the oral maximum tolerated dose of long stalk Fructus Schisandrae Sphenantherae extract mice greater than 30g/kg, and toxicity is lower.
By embodiment 10~14 as can be known:
1) long stalk Fructus Schisandrae Sphenantherae extract of the present invention has obvious suppression and killing action to common microorganism, wherein the inhibitory action to gram positive bacteria is comparatively obvious, and it can give birth to inhibitory action to staphylococcus aureus and producing bacillus subtilis when 60 and 120 μ g.
2) long stalk Fructus Schisandrae Sphenantherae extract of the present invention has tangible antioxidant activity.Externally can significantly suppress the erythrocyte hemolysis that liver homogenate autoxidation degree, hydrogen peroxide cause, and have certain reducing power.
3) long stalk Fructus Schisandrae Sphenantherae extract of the present invention can alleviate Radix Et Rhizoma Rhei to a certain extent and causes the diarrhea of mouse symptom, and tool resists insufficiency of the spleen, diarrhea effect.
4) long stalk Fructus Schisandrae Sphenantherae extract antiulcer effect of the present invention is remarkable, can effectively suppress hydrochloric acid-alcoholic solution and cold water and arrest and soak the gastric mucosa injury that causes and the generation of ulcer.
5) rat acute toxicity test shows that long stalk Fructus Schisandrae Sphenantherae extract oral toxicity of the present invention is less, and maximum tolerated dose surpasses 30g/kg, tool excellent development prospect.
Embodiment 15
Long stalk Fructus Schisandrae Sphenantherae extract capsule
To grow the stalk Fructus Schisandrae Sphenantherae extract and pulverize, and sieve, and add 5% micropowder silica gel, mixing adds 60% ethanol system soft material, granulates, and drying is crossed 20~40 mesh sieves, packs snap fit capsule into promptly.
Embodiment 16
Long stalk Fructus Schisandrae Sphenantherae extract granule
To grow the stalk Fructus Schisandrae Sphenantherae extract and pulverize, and sieve, and add Icing Sugar and 1 times of amount dextrin of 4 times of amounts, mixing adds 60% ethanol system soft material, granulate, and drying, granulate, promptly.
Embodiment 17
Long stalk Fructus Schisandrae Sphenantherae extract tablet
To grow the stalk Fructus Schisandrae Sphenantherae extract and pulverize, and sieve, and add 10% starch, mixing adds 60% ethanol system system soft material, granulate, and drying, granulate adds 1% stearic acid, mixing, tabletting, promptly.
Embodiment 16
Long stalk Fructus Schisandrae Sphenantherae extract syrup
To grow the stalk Fructus Schisandrae Sphenantherae extract and pulverize, sieve, add the simple syrup and an amount of antiseptic of 100 times of amounts, heating for dissolving, cooling, promptly.