CN1686141A - Medicinal composition for treating hepatitis and diabetes - Google Patents
Medicinal composition for treating hepatitis and diabetes Download PDFInfo
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Abstract
A composite medicine for treating hepatitis and diabetes is prepared from twospot swertia herb through extracting its active components (oleanolic acid, etc).
Description
Background technology
The invention belongs to the plant amedica field, relate to a kind of preparation method of Herba Swertiae bimaculatae active component and the application in pharmacy thereof.
The Swertia plant has kind more than 70 in China, wherein more than 20 kind is used to treat liver and gall diseases or other inflammation (" Chinese medicine resource will is wanted " among the people, Science Press, 1974,957 pages), seldom by the plant variety of business development, only Herba Swertiae Mileensis is recorded (" 2000 editions one one the 155th page of Chinese pharmacopoeia) by national standard at present, other has Herba Swertiae bimaculatae, S.macrosperma, kinds such as surrounded swertia herb once were put into local medical material standard (Guizhou Province D/WS-208-88), the medical material or the patent medicine that derive from these Swertia plants are classes that has much characteristic in the existing hepatitis medicine, determined curative effect, side effect is slight, is subjected to patient's favor.There is report that this platymiscium has been carried out pharmacodynamic experiment research, confirms that further extract has hepatoprotective effect, as: new Chinese medicine and clinical pharmacology, 2003,14 (5): 304; New Chinese medicine and clinical pharmacology, 2002,13 (6): 376.
The Swertia plant has tangible chemotaxonomy feature, generally contain type composition [northwest Botany Gazettes such as oleanolic acid, ketone, flavone, iridoid glycoside, 2003,23 (12): 2235], there is data to think that oleanolic acid and iridoid glycoside are its active component, and developed oleanolic acid tablet, swertiamarin sheet, Herba Swertiae Mileensis total glycosides sheet (GANFUKANG sheet) and put on market, be used for the treatment of hepatitis or abdominal pain in children.In recent years, bibliographical information is arranged also, this genus plants extract and oleanolic acid are to artificial diabetes tool have certain effect [Chinese Medicine science and technology, 2003,10 (2): 96; Yunnan chemical, 2000,27 (4): 29; CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2002,27 (12): 884], but do not see medicine listing or clinical practice.Many chemical research documents clearly disclose, the chemical constituent of Swertia plant has multiformity, form very complicated, it should be noted that, which kind of effective ingredient plays a decisive role in the anti-hepatitis of Herba Swertiae bimaculatae and hypoglycemic activity actually, prior art does not have clear and definite instruction, does not provide the foundation of science for extracting active component effectively.
Summary of the invention
Main purpose of the present invention is exactly the effective substance that will further illustrate Herba Swertiae bimaculatae class plant, develops new Herba Swertiae bimaculatae class medicine, for this plant resources of industrialized developing is established technical foundation.
The inventor is by a large amount of creative research works, hepatic cholagogic active component with Herba Swertiae bimaculatae is locked as the effective site that mainly contains total ketone and oleanolic acid from numerous structure types of numerous and complicated first, at this object specialized designs two extraction process routes, and found the using value of said composition aspect treating diabetes first.Further screening active ingredients is found, and is the most key with protecting the liver with blood sugar reducing function of Radix Gentianae ketone phenol again in described total ketone, and and oleanolic acid between exist conspiracy relation.
One of technology path of the present invention is shown in accompanying drawing 1-a, concrete operations are: the dry herb of Swertia of getting the autumn and winter season collection, be ground into coarse powder, with lower alcohol or other polarity～middle polarity organic solvent (for example ethanol, methanol, acetone or ethyl acetate, preferred 60%～95% ethanol) extract 2～5 times, merge extractive liquid, concentrates, and gained extractum adds about 10 times of water gagings and stirs, the leaching insoluble matter, washing, drying, porphyrize, defat (for example adopting petroleum ether, gasoline to reflux), drain, wave most residual solvent, promptly.
Technology path of the present invention two shown in accompanying drawing 1-b, concrete operations are: get the dry herb of Swertia that autumn and winter season gathers, be ground into coarse powder, with aqueous alkali (for example 0.2%NaOH, 1%Na
2CO
3, 2%NaHCO
3) extract, merge extractive liquid,, adding dilute hydrochloric acid or dilute sulfuric acid make and are acidified to pH value about 3, and filter collection precipitate is washed to neutrality, drying, promptly.
The extract that above-mentioned two kinds of methods obtain has similar biological activity and physicochemical property, be the yellowish-brown powder, flavor is extremely bitter, be insoluble in water (dissolubility＜0.1%), be difficult for the moisture absorption, chemical property is stable, can make various oral formulations such as tablet, capsule, drop pill, granule easily by prior art.
This extract ingredient has phenolic hydroxyl group or carboxyl functional group, shows certain acidity.Advantageously, for improving its water solublity, can be by impelling its dissolving with the sodium hydroxide solution salify, solution gets its sodium salt through lyophilization or spray drying after filtering again, and its appearance character also is the yellowish-brown powder, good water solubility especially is fit to make injection liquid drugs injection or powder pin.
The inventive method has the different of essence with the decocting method of folk tradition.The latter uses ordinary water to decoct, and abstract mainly is a water soluble ingredient, and as iridoid glycosides, saccharide, inorganic salt, impurity is a lot, and extremely low ketone of water solublity and oleanolic acid are seldom; The inventive method serves as to extract refining object with ketone in the Herba Swertiae bimaculatae and oleanolic acid, adopt organic solvent or aqueous alkali to extract, fully object is extracted, further remove water soluble ingredient and oils impurity again, dosage is reduced significantly, improved the effectiveness of medication, safety and controllability, oral administration 30mg/kg on the gained active component animal model (is equivalent to 1～2g crude drug/kg) can produce obvious drug effect, and the effective dose of bibliographical information use Swertia punicea Hemsl. total extract administration is 7g crude drug/kg[new Chinese medicine and clinical pharmacology, 2002,13 (6): 376], be more than three times of the present composition.
The inventive method route is short, and is simple to operate, need not special installation, therefore is easy to industrialization.The more important thing is, according to the compositions of this method preparation be a kind of purity height, quality controllable, satisfy the plant amedica effective site that modern novel technique requires, controlled content of material reaches more than 50%, can be used as crude drug and is directly used in the various medicaments of preparation.
Pharmaceutical composition of the present invention is characterised in that, it is to be main active substances with ketone, oleanolic acid or the sodium salt of the two; The part by weight of ketone and oleanolic acid is 0.1～10: 1, preferred 1～4: 1. ketones component in the Swertia plant has multiple, and because of the difference of the place of production, collecting season, ecological environment, concrete number and content may have fluctuation and variation to a certain degree.Said ratio indication ketone is actual to comprise multiple ketones component, or claims total ketone.Total ketone in the active component of the present invention has significant chemical feature, and Radix Gentianae ketone phenol is its main active, and content is not less than 1% (weight), and for example 5%-80% is preferably 8%～16%; Also can further contain chimonin,, sweroside peaceful, methylswertianin, 1,3,5,8-tetrahydroxy ketone, 1-hydroxyl-2 when medicine, 3,5-trimethoxy ketone, 1-hydroxyl-2,3,5,7-tetramethoxy ketone, 1,7-dihydroxy-3,8-dimethoxy ketone, 1-hydroxyl-2,3,4,5-tetramethoxy ketone, 1-hydroxyl-3,7,8-trimethoxy ketone, 1,7-dihydroxy-3,4, analog such as 8-trimethoxy ketone, its content is 5%-85% (weight), is preferably 20-60%.The chemical constitution of active component of the present invention is seen accompanying drawing-2.
The inventor is a raw material with Swertia punicea Hemsl. (Swertia punicea Hemsl), Swertia franchetiana H.Smith (S.franchetianaH Smith), Guizhou Herba Swertiae bimaculatae (S.kauitchensis), under pilot plant conditions preparation method of the present invention has been carried out scale-up, test data sees Table-1.
Show-1. extract the pilot experiment result
| Batch | Medical material | Inventory | The compositions yield | Compositions oleanolic acid content (HPLC) | Compositions Radix Gentianae ketone phenol content (HPLC) | Total ketone content (UV is by Radix Gentianae ketone phenol) | Total ketone: oleanolic acid |
| ??1 | Swertia punicea Hemsl. | ??50kg | ??2.8％ | ??20.2％ | ??8.9％ | ??34.5％ | ??1.7∶1 |
| ??2 | Swertia punicea Hemsl. | ??50kg | ??2.9％ | ??19.5％ | ??9.5％ | ??39.2％ | ??2.0∶1 |
| ??3 | Swertia punicea Hemsl. | ??50kg | ??3.1％ | ??21.3％ | ??10.3％ | ??31.7％ | ??1.5∶1 |
| ??4 | Swertia punicea Hemsl. | ??50kg | ??3.0％ | ??18.2％ | ??9.0％ | ??38.6％ | ??2.1∶1 |
| ??5 | Swertia franchetiana H.Smith | ??50kg | ??2.1％ | ??18.3％ | ??3.8％ | ??45.9％ | ??2.5∶1 |
| ??6 | Swertia franchetiana H.Smith | ??50kg | ??2.5％ | ??16.8％ | ??3.4％ | ??52.7％ | ??3.1∶1 |
| ??7 | The Guizhou Herba Swertiae bimaculatae | ??50kg | ??1.8％ | ??8.2％ | ??2.5％ | ??42.5％ | ??5.2∶1 |
| ??8 | The Guizhou Herba Swertiae bimaculatae | ??50kg | ??1.9％ | ??10.4％ | ??2.2％ | ??40.5％ | ??3.9∶1 |
Preparation technology of the present invention proves through repetition test, the yield of pharmaceutical composition is stabilized in 1.5%～3.5%, and wherein Radix Gentianae ketone phenol content is all greater than 2%, and the ratio of total ketone and oleanolic acid is 0.1～10: in 1 scope, majority concentrates on 1～4: 1, and the two content sum is all greater than 50%.The Radix Gentianae ketone phenol content of Swertia punicea Hemsl. extract is higher, and therefore, Swertia punicea Hemsl. is a preferred medical material kind of extracting pharmaceutical composition of the present invention.
For confirming the advance of technology of the present invention, the inventor is solvent by prior art with water and 80% ethanol, to carrying out heating extraction with a collection of Swertia punicea Hemsl. medical material, has prepared crude extract respectively, and measured the wherein content of oleanolic acid, Radix Gentianae ketone phenol and total ketone, the results are shown in following table.
Table-2. the distinct methods extract compares
| Extracting method | The extract character | Extract yield | Oleanolic acid % | Radix Gentianae ketone phenol % | Total ketone % |
| Water extraction | The brown thick paste | ????14.3％ | ????0.3 | ????0.2 | ????4.5 |
| Alcohol extraction | The brown thick paste | ????12.5％ | ????4.6 | ????2.1 | ????8.3 |
| Technology of the present invention | The yellowish-brown powder | ????2.9％ | ????20.2 | ????9.8 | ????37.5 |
Above-mentioned experiment shows that in prior art gained Swertia punicea Hemsl. water extract and ethanol extract, oleanolic acid is respectively 4.8% and 12.9% with total ketone content sum, and controlled ratio is on the low side, contains a large amount of hygroscopicity impurity in the extract, is not suitable as crude drug; And technology of the present invention extract obtained in oleanolic acid and total ketone content sum up to 57.7%, extract is nonhygroscopic substantially, is suitable for preparation production.
Pharmaceutical composition chemical property of the present invention is stable, and the high temperature accelerated test shows that this product was placed 3 months under 50 ℃ of constant temperature and humidity conditions, and its appearance character and physicochemical property do not have any variation; Total ketone, oleanolic acid, Radix Gentianae ketone phenol equal size also do not have obvious decline (range of decrease＜1.0%).It should be noted that, the less stable of existing Herba Swertiae bimaculatae class medicine Herba Swertiae Mileensis total glycosides, under 50 ℃ of constant temperature and humidity conditions, placed 3 months, not only outward appearance moisture absorption variable color, more seriously, the content of main constituent swertiamarin reduces to 8% by 25%, and fall has surpassed 10% rotten limit up to 68%.By contrast, the stability advantage of the present composition is very outstanding.
In one embodiment of the invention, be provided for treating or preventing the pharmaceutical composition of hepatitis, diabetes or its complication, wherein contain said extracted thing (being effective site) and suitable pharmaceutical excipient, described effective site comprises that total ketone, oleanolic acid or the sodium salt of the two are active substance.Described total ketone contains Radix Gentianae ketone phenol, can also further contain chimonin,, sweroside peaceful when medicine, methylswertianin, 1,3,5,8-tetrahydroxy ketone and 1-hydroxyl-2,3,5-trimethoxy ketone, 1-hydroxyl-2,3,5,7-tetramethoxy ketone, 1,7-dihydroxy-3,8-dimethoxy ketone, 1-hydroxyl-2,3,4,5-tetramethoxy ketone, 1-hydroxyl-3,7,8-trimethoxy ketone, 1,7-dihydroxy-3,4, compositions such as 8-trimethoxy ketone.One of ordinary skill in the art will appreciate that and implement be, the source of described ketone, oleanolic acid or the sodium salt of the two is not limited to Swertia plants such as Swertia punicea Hemsl., Swertia franchetiana H.Smith, Guizhou Herba Swertiae bimaculatae, also can adopt the synthetics of high-purity extract, monomer or the above-mentioned substance of other plant origin of Gentianaceae.
Accompanying drawing 1 is two kinds of preparation method flow charts of pharmaceutical composition of the present invention
Accompanying drawing 2 is the molecular structure of pharmaceutical composition of the present invention
In order to reflect ins and outs of the present invention more fully, provide embodiment to be illustrated below.
The preparation of embodiment 1 Swertia punicea Hemsl. active component (method one)
Get Swertia punicea Hemsl. herb 25kg, pulverize, put in the rustless steel extraction pot, add 85% ethanol heat and carry 3 times, each ethanol consumption floods with liquid level and exceeds be degree about medical material 5～10cm, and refluxed 2 hours the first time, respectively refluxes 1 hour for the 2nd, 3 time; Merge alcoholic extract, concentrating under reduced pressure gets extractum 4.6kg, proportion 1.25; Add 40kg water in extractum, fully stir, visible a large amount of brown precipitations are separated out; Filter, water washing and precipitating is drained, vacuum drying, and porphyrize divides 3 backflow defats with 6 times of amount petroleum ether again, and the insoluble part of filter collection is drained, and waves most solvent, gets yellowish-brown powder 0.76kg, is the Swertia punicea Hemsl. active component, yield 3.04%.
Randomly, also can further add the about 1L of 5% sodium hydroxide solution and make dissolving, transfer pH7.5～8.0, filter, spray-dried its sodium salt 0.75kg that gets.
The preparation (method two) of embodiment 2 Swertia punicea Hemsl. active components
Get Swertia punicea Hemsl. herb 25kg, pulverize, put in the rustless steel extraction pot, add 0.2%NaOH, heating and refluxing extraction 3 times merges potass extraction liquid, adds 10%H
2SO
4To pH value 2～3, filter collection precipitation, washing, drying gets yellowish-brown powder 0.82kg, is the Swertia punicea Hemsl. active component, yield 3.28%.
Embodiment 3 assays
1. total ketone assay
Precision takes by weighing embodiment 1 gained Swertia punicea Hemsl. active component 30mg, puts in the 10ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up; The accurate 1ml solution of drawing is put in the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up; According to spectrophotography, measure trap at the wavelength place of 333nm, press the absorptance (E of Radix Gentianae ketone phenol
1cm 1%) be 330 calculating.Total ketone content is pressed Radix Gentianae ketone phenol (C in this sample
14H
10O
6) meter, measured result is 40.6%.
2. oleanolic acid assay
Precision takes by weighing embodiment 1 gained Swertia punicea Hemsl. active component 50mg and oleanolic acid reference substance 10mg, puts respectively in the 25ml measuring bottle, adds methanol and makes dissolving and be diluted to scale, shakes up, as need testing solution and reference substance solution.According to high performance liquid chromatography, above-mentioned two kinds of solution, the 10 μ l of accurate absorption inject chromatograph of liquid respectively, measure the peak area of oleanolic acid, calculate content by external standard method.Chromatographic condition: chromatographic column EclipesXDB C
18(4.6mm * 150mm, 5 μ m); Mobile phase CH
3CN-CH
3OH-H
2O-ammonium acetate (80: 10: 10: 0.5); Detect wavelength 207nm; Flow velocity 1ml/min; 40 ℃ of temperature.The actual measurement content of oleanolic acid is 20.18% in this test sample.
3. Radix Gentianae ketone phenol assay
Precision takes by weighing embodiment 1 gained Swertia punicea Hemsl. active component 50mg and Radix Gentianae ketone phenol reference substance 5mg, puts respectively in the 25ml measuring bottle, adds methanol and makes dissolving and be diluted to scale, shakes up, as need testing solution and reference substance solution.According to high performance liquid chromatography, above-mentioned two kinds of solution, the 10 μ l of accurate absorption inject chromatograph of liquid respectively, measure the peak area of Radix Gentianae ketone phenol, calculate content by external standard method.Chromatographic condition: chromatographic column PuritexC
18(4.6mm * 200mm, 5 μ m); Mobile phase CH
3CN-H
2O (8: 2); Detect wavelength 330nm; Flow velocity 1ml/min; 40 ℃ of temperature.The actual measurement content of Radix Gentianae ketone phenol is 10.50% in this test sample.
Embodiment 4 preparations
1. capsule is got embodiment 1 extract obtained 60g, adds starch 78g, magnesium stearate 2g, and mixing directly is filled to 1000 with Autocapsulefillingmachine, and polishing promptly.Every contains compositions 60mg.Become human oral every day 1～5 time, each 1～10.
2. tablet is got embodiment 2 extract obtained 60g, add starch 80g, dextrin 5g, mix homogeneously, add 10% starch slurry system soft material, granulate 60～70 ℃ of aeration-dryings with 14 order nylon screens, 16 mesh sieve granulate, add magnesium stearate 1.5g, carboxymethyl starch sodium 5g mixing, be pressed into 1000, coating promptly.Every contains compositions 60mg.Become human oral every day 1～5 time, each 1～10.
3. soft capsule is got PEG400 276g, put in the appropriate vessel, add PEG6000 6g, heating in water bath dissolves to PEG6000, stir evenly, add glycerol 18g, the content that add content and be 95% commercially available oleanolic acid 12g, derives from gentianaceae plant is that 90% Radix Gentianae ketone phenol 30g and the content that derives from Folium mangiferae are 95% chimonin 18g, grind to form even mastic with colloid mill, be transferred to the material storage barrel of encapsulating machine, be pressed into 1000 of soft capsules with the mould of 0.3ml, typing, the flush away surface lubricant, dry under 20 ~ 30% relative humiditys, 28 ℃ of conditions, promptly.Become human oral every day 1～5 time, each 1～10.
4. drop pill is got embodiment 1 extract obtained 10g, drop in the polyethylene glycol 6000 of 32g heating and melting, be stirred to dissolving, be transferred in the reservoir, airtight and insulation is regulated drop pill machine drop quantitative valve at 80～90 ℃, splash into from top to bottom in 10～15 ℃ the liquid Paraffin, make 1000 altogether, the drop pill that forms is drained and wipe liquid Paraffin, be drying to obtain.Every contains compositions 10mg.Become human oral every day 1～5 time, each 6～60.
Drug effect and toxicity test
1. the Swertia punicea Hemsl. active component is to the protective effect of rat chronic carbon tetrachloride hepatic injury
Get 140 of Wistar rats, body weight 170～200g, male and female half and half, be divided into seven groups at random, i.e. dosage group, Swertia punicea Hemsl. low dose group and Swertia punicea Hemsl. ethanol total extract group in blank group, carbon tetrachloride model group, oleanolic acid positive controls, Swertia punicea Hemsl. high dose group, the Swertia punicea Hemsl..Except that the blank group, all the other respectively organize subcutaneous injection of carbon tetrachloride 1.2ml/kg, and 2 times weekly, continuous 15 weeks.Get blood from the animal afterbody during the 6th week, measure sGPT and sGOT, and the anatomic part animal, degree of hepatic fibrosis observed.From the 7th week of injection carbon tetrachloride, by dosage group 60mg/kg, Swertia punicea Hemsl. low dose group 30mg/kg, Swertia punicea Hemsl. ethanol total extract group 300mg/kg gastric infusion (being mixed with suspension) in oleanolic acid 100mg/kg, Swertia punicea Hemsl. high dose group 120mg/kg, the Swertia punicea Hemsl. with soil temperature-80, once a day, continuous 60 days.Get blood after the last administration, measure sGPT, sGOT, and get liver and do histopathologic examination, the results are shown in Table-3.
Table-3. the Swertia punicea Hemsl. active component is to the influence of tetrachloro-methane induction chronic hepatic injury rat
| Group | Number of animals | ??????????????sGPT(u/L) | ??????????????sGOT(u/L) | ||
| Before the administration | After the administration | Before the administration | After the administration | ||
| The blank group | ????10 | ??16.82±10.33 | ??16.65±9.76 | ??59.45±6.98 | ??65.78±14.55 |
| The carbon tetrachloride group | ????10 | ??151.21±13.67 | ??195.28±27.33 | ??137.01±20.45 | ??171.46+38.38 |
| The oleanolic acid group | ????10 | ??152.34±16.56 | ??138.96±45.31 | ??142.44±32.76 | ??130.36±35.66 |
| The Swertia punicea Hemsl. high dose group | ????10 | ??160.14±26.32 | ??53.88±25.79 | ??139.68±24.25 | ??105.64±32.30 |
| Dosage group in the Swertia punicea Hemsl. | ????10 | ??159.22±36.77 | ??78.58±36.65 | ??132.69±28.42 | ??113.87±38.49 |
| The Swertia punicea Hemsl. low dose group | ????10 | ??153.24±35.23 | ??116.25±36.72 | ??129.57±31.?86 | ??123.65±30.12 |
| Swertia punicea Hemsl. total extract group | ????10 | ??155.24±32.83 | ??126.55±26.44 | ?135.50±21.02 | ??130.25±20.39 |
*. determination data is compared respectively with the carbon tetrachloride group with the blank group before the administration of each administration group, after the administration, significant differences is all arranged, p＜0.001.
Result of the test shows, the rat skin lower injection carbon tetrachloride can obviously damage liver function during to the 6th week, the transaminase is raise, visible hepatocellular degeneration necrosis of pathological tissue inspection and fibrosis.During off-test, carbon tetrachloride group sGPT and sGOT obviously raise, and liver cirrhosis forms, and the administration group was treated 60 days with Swertia punicea Hemsl. effective site, the transaminase that each dosage group is brought out carbon tetrachloride raises very significantly reduction effect is arranged, and obviously alleviates degree of hepatic fibrosis.The drug effect of Swertia punicea Hemsl. active component obviously is better than oleanolic acid group and Swertia punicea Hemsl. total extract group.
2. the choleretic effect of Swertia punicea Hemsl. active component
Get 30 of rabbit, be divided into 5 groups at random, 6 every group.1. be physiology saline control group; 2. be the positive group of anethol trithione sheet (6mg/kg); 3.～5. for organized by examination, give Swertia punicea Hemsl. active component (sodium salt) 120mg/kg, 60mg/kg, 30mg/kg respectively.The reference literature method experimentizes (herbal pharmacology research methodology, People's Health Publisher,, 448 pages in 1994), record bile flow index, and determination data sees Table-4.
Table 4. Swertia punicea Hemsl. active component is to the influence (n=6) of rabbit bile flow
| Group | Dosage mg/kg | Before the administration (ml) | Bile flow reduction rate (%) after the administration | |||
| ???0～0.5h | ??0.5～1h | ??1～1.5h | ??1.5～2h | |||
| Matched group | ????--- | ??4.16±1.89 | ??-27.82±15.60 | ??-34.02±12.58 | ??-43.08±14.37 | ??-50.00±11.35 |
| Anethol trithione | ????6 | ??4.07±1.93 | ??-15.72±8.86 1 | ??-15.88±10.95 1 | ??-29.04±8.99 1 | ??-43.46±17.38 1 |
| Active component | ????30 | ??3.98±1.60 | ??-17.60±11.53 1 | ??-19.74±8.57 1 | ??-27.08±6.64 2 | ??-37.37±15.26 1 |
| Active component | ????60 | ??3.25±1.53 | ??-15.27±8.17 2 | ??-19.04±9.53 2 | ??-20.29±9.50 2 | ??-31.09±12.27 2 |
| Active component | ????120 | ??3.47±1.46 | ??-21.36±10.56 | ??-23.11±3.91 2 | ??-26.78±14.72 2 | ??-32.97±9.52 2 |
T check between group: compare with matched group,
1P＜0.05,
2P＜0.01.
Table 4 shows that the matched group bile flow changes in time and descends; Comparing three dosage groups of Swertia punicea Hemsl. active component, 0.5～2h after administration with matched group all can make the bile flow reduction rate reduce.Show the effect that this product has increases bile flow.
The data acknowledgement of experiment 1 and 2, pharmaceutical composition of the present invention has the hepatic cholagogic activity, can be used for treating hepatitis, for example first, hepatitis B due to the virus.
3. the Swertia punicea Hemsl. active component is induced the hypoglycemic activity of diabetic mice to Streptozocin
Get NIH kind male mice, body weight 22～24g is divided into five groups at random: the 1st group is the normal control group, intraperitoneal injection of saline 0.15ml/10g; The 2nd group～the 5th group lumbar injection Streptozocin 150mg/kg after 48 hours, gets blood from mouse orbit, and according to the determination of glucose oxidase blood sugar concentration, mice all produces blood sugar increasing in various degree, shows the modeling success.Be made as model group with the 2nd group, mice is irritated stomach and give normal saline every day; 3rd, 4,5 groups of mices are irritated stomach respectively every day and give compositions 60mg/kg, 120mg/kg and glyburide 5mg/kg, and continuous 10 days, after administration, got blood from eye socket on the 11st day, measure blood sugar concentration.The results are shown in Table-5.
Table-5. the Swertia punicea Hemsl. active component is to the influence of blood glucose in diabetic mice
| Group | Number of animals | Blood sugar concentration (mg/dl) | |
| Before the administration | After the administration | ||
| The normal control group | ????10 | ????148.5±47.3 | ????120.2±45.2 |
| Model group | ????10 | ????260.2±40.2 1 | ????322.2±105.5 1 |
| Compositions 120mg/kg | ????10 | ????235.2±65.2 2 | ????180.2±85.2 3 |
| Compositions 60mg/kg | ????10 | ????240.2±49.2 1 | ????196.2±65.2 3 |
| Glyburide 5mg/kg | ????10 | ????249.2±75.2 1 | ????212.2±66.2 3 |
*Compare with the normal control group
1P＜0.001,
2P＜0.01; Compare with model group
3P＜0.01.
Data show all have after the horizontal administration of the mouse blood sugar of three administration groups in various degree and reduce, and the fall of two dosage groups of Swertia punicea Hemsl. is all greater than the glyburide group.
4. the Swertia punicea Hemsl. active component is to the inhibitory action of aldose reductase activity
Animal: get the Wistar rat, random packet is placed in 18～29 ℃ of temperature, relative humidity 50～70%, 40W fluorescent lamp lighting, the sound damping animal housing, and per 5 one cage group supports are freely ingested, drunk water, and is used for experiment after the week that conforms.
Erythrocytic In vitro culture: the socket of the eye venous plexus is got blood behind the big rathole, the EDTA anticoagulant, and 2500rpm * 5min is centrifugal for the gained whole blood, abandon blood plasma, usefulness normal saline Washed Red Blood Cells were 3 times in 1: 4 by volume, and each 2500rpm * 5min is centrifugal, abandon supernatant, stay packed red cells.Draw erythrocyte 0.5ml, add glucose solution (normal saline preparation) and the 1ml composition solution (being diluted to variable concentrations) of 0.5ml 20%, in 37 ℃ of 5%CO with normal saline
2Cultivated 3 hours in the incubator.Take out temperature and incubate liquid, centrifugal, abandon supernatant, with 4 ℃ of normal saline Washed Red Blood Cells 3 times, 2500rpm * 5min is centrifugal at every turn, abandons supernatant, stays packed red cells.Draw 10 μ l for measuring hemoglobin (Hb); Draw 200 μ l usefulness normal saline dilution in by volume 1: 3, slowly shake up, get 25% erythrocyte diluting fluid, for measuring sorbitol content.
The erythrocyte sorbitol content is measured: draw 25% erythrocyte diluting fluid 0.5ml, add 1.2ml 6% perchloric acid solution, jolting 1 minute, 4000rpm * 5min is centrifugal, draws supernatant 1ml and puts in the test tube, adds 50 μ l 0.025M periodic acid solution, mixing, kept in Dark Place 10 minutes, and added 50 μ, 1 0.25M sodium arsenite solution, left standstill behind the mixing 10 minutes, add 2ml 0.2% chromotropic acid-concentrated sulfuric acid solution, mixing, boiling water heating 30 minutes is put and is chilled to room temperature, in 570nm place colorimetric, read the optical density value of each pipe, calculate erythrocytic sorbitol content, calculate the suppression ratio that medicine generates sorbitol in the erythrocyte according to following formula:
Cc is the growing amount of sorbitol in the blank group erythrocyte in the formula; Cm is the growing amount of sorbitol in the model group erythrocyte; Cs is the growing amount of sorbitol in each administration group erythrocyte.Experimental result sees Table-6.
Table 2 data show, the present composition incubates to external temperature that the generation of sorbitol has remarkable inhibitory action in the erythrocyte, and is good dose relationship, and its suppression ratio equation is y=-0.2721x+1.3765, R
2=0.9356, IC
50Be 6.0 * 10
-4G/ml.
Table 6. compositions is incubated the influence that sorbitol generates in the erythrocyte to external temperature
| Group | Drug level (g/ml) | Sorbitol/hemoglobin (μ mol/g) | Suppression ratio (%) |
| Blank group | --- | ?11.11±0.31 ** | |
| Model group | --- | ?27.21±0.44 | |
| Compositions | 3×10 -2 | ?11.10±0.30 ** | 100 |
| Compositions | 1×10 -2 | ?11.93±1.14 ** | 95 |
| Compositions | 5×10 -3 | ?15.85±0.35 ** | 71 |
| Compositions | 1×10 -3 | ?19.33±0.05 ** | 49 |
| Compositions | 1×10 -4 | ?25.72±0.48 * | 9 |
| Compositions | 1×10 -5 | ?26.05±0.20 * | 7 |
Compare with model group:
*P＜0.05;
*P＜0.01.
The data acknowledgement of experiment 3 and 4, pharmaceutical composition of the present invention have blood sugar lowering and suppress aldose reductase activity, can be used for prevention and treatment diabetes and complication, for example type ii diabetes and cataract.
5. Swertia punicea Hemsl. active component acute toxicity test
Select the healthy mice of body weight for use, adopt the oral administration mode, determine the median lethal dose(LD 50) (LD of Swertia punicea Hemsl. active component at 18～22g
50) be 9.5g/kg, be clinical plan 158 times with dosage (60mg/kg).
6. Swertia punicea Hemsl. active component long term toxicity test
Get 180 of rats, body weight 70～90g, male and female half and half are divided into four groups at random: 50 of matched groups, 50 of heavy dose of groups, 40 of middle dosage groups, 40 of low dose group, every group of male and female half and half.The dosage of three dosage groups is respectively 50,30 and 10 times of clinical plan consumption, i.e. 3g/kg, 1.8g/kg, 0.6g/kg, medicine face with before adding soil temperature-80 suspendible, gastric infusion, once a day, continuous six months.Matched group gavages equivalent soil temperature-80.Experimental session observed and recorded every day animal general state, the weighing the weight of animals once weekly.When experiment finishes, each treated animal is carried out electrocardiogram, blood, biochemistry and pathological examination.When proceeding to three month, experiment puts to death each 10 of heavy dose of group and control animals; Each group is put to death 30 in the time of six month, keeps 10, and presumable toxic effect of medicine and recovery situation were observed in drug withdrawal in one month; All put to death in the time of seven month.The result shows, rats gavaged Swertia punicea Hemsl. active component is after six months, and there are no significant changes for the activity of three dosage treated animals, appetite, defecation and growth promoter; The hemogram of each administration treated animal, hepatic and renal function, electrocardiogram and main organs index and matched group relatively there are no significant difference (through t assay statistics, p＞0.05), and all in the normal physiological scope.Pathological examination shows that matched group and three each organs and tissues structures of administration treated animal there is no obvious pathologic and change.
Above result of the test shows, Herba Swertiae bimaculatae active component of the present invention have definite protect the liver, function of gallbladder promoting, blood sugar lowering, inhibition aldose reductase activity, and toxicity is little, safety is good, the high-efficiency low-toxicity advantage is outstanding, can be used as the medicine of hepatitis and diabetes and complication thereof.Compare respectively with existing medicine oleanolic acid, glyburide, the present composition still has certain advantage on drug effect.
Claims (9)
1. pharmaceutical composition contains part by weight and is 0.1～10: 1 total ketone and oleanolic acid active component.
2. the compositions of claim 1, wherein total ketone and oleanolic acid are sodium salt.
3. claim 1 and 2 compositions, described total ketone comprises Radix Gentianae ketone phenol.
4. the compositions of claim 3, described total ketone further comprise chimonin, when medicine is peaceful, sweroside, methylswertianin, 1,3,5,8-tetrahydroxy ketone, 1-hydroxyl-2,3,5-trimethoxy ketone, 1-hydroxyl-2,3,5,7-tetramethoxy ketone, 1,7-dihydroxy-3,8-dimethoxy ketone, 1-hydroxyl-2,3,4,5-tetramethoxy ketone, 1-hydroxyl-3,7,8-trimethoxy ketone, 1,7-dihydroxy-3,4,8-trimethoxy ketone.
5. the compositions of one of claim 1～4 is made by Swertia punicea Hemsl. or other kindred plant.
6. claim 5 preparation of drug combination method comprises: get the Swertia plant, pulverize, with lower alcohol or other polarity～middle polarity organic solvent extraction, concentrating under reduced pressure, gained alcohol extractum is dissolved in water, the leaching insoluble matter, and washing, drying, defat, promptly; Or get the Swertia plant, and pulverize, use potass extraction, merge extractive liquid,, acidify, filter collection precipitate, washing is drying to obtain.
7. the method for claim 6 further comprises products therefrom added making dissolving in the sodium hydroxide solution, filter, drying, its water solublity sodium salt.
8. the application of the pharmaceutical composition of one of claim 1-5 in the medicine of preparation treatment hepatitis.
9. the application of the pharmaceutical composition of one of claim 1-5 in the medicine of preparation treatment diabetes.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102106865B (en) * | 2009-12-29 | 2012-10-31 | 北京大学 | Application of bi-xanthone glycoside compound in oxidative damage resistance and apoptosis inhibition |
| CN107022569A (en) * | 2016-02-01 | 2017-08-08 | 华东师范大学 | A kind of gene loop induced expression regulator control system, microcapsules and its application |
| CN107022546A (en) * | 2016-02-01 | 2017-08-08 | 华东师范大学 | A kind of gene loop induced expression regulates and controls method |
| CN109908128A (en) * | 2018-11-07 | 2019-06-21 | 青海师范大学 | A kind of Bupleurum scorzonerifoliumWild Antibacterial Constituents formula and preparation method thereof |
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2005
- 2005-03-31 CN CN 200510059674 patent/CN1686141A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102106865B (en) * | 2009-12-29 | 2012-10-31 | 北京大学 | Application of bi-xanthone glycoside compound in oxidative damage resistance and apoptosis inhibition |
| CN107022569A (en) * | 2016-02-01 | 2017-08-08 | 华东师范大学 | A kind of gene loop induced expression regulator control system, microcapsules and its application |
| CN107022546A (en) * | 2016-02-01 | 2017-08-08 | 华东师范大学 | A kind of gene loop induced expression regulates and controls method |
| CN107022569B (en) * | 2016-02-01 | 2020-08-25 | 华东师范大学 | Gene loop induced expression regulation system, microcapsule and application thereof |
| CN109908128A (en) * | 2018-11-07 | 2019-06-21 | 青海师范大学 | A kind of Bupleurum scorzonerifoliumWild Antibacterial Constituents formula and preparation method thereof |
| CN109908128B (en) * | 2018-11-07 | 2021-07-23 | 青海师范大学 | Formula of antibiotic active ingredients of swertia mussotii and preparation method thereof |
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