CN1269491C - Isatis root extract composition, its preparation method and application - Google Patents

Abstract

The present invention relates to a method for preparing isatis root extract compositions, which comprises the steps: (a) 30 to 95 weight percentage of ethanol water of which the weight is from 6 to 12 times than that of isatis root is used for reflowing extraction, and ethanol extracting solution is merged; (b) medical liquid can be obtained by filtering and recovering the ethanol; (c) the obtained medical liquid passes through macroporous resin adsorbing columns; (d) the macroporous resin adsorbing columns are washed by washing liquid to the state that effluent liquid is colorless; (e) 30 to 95 weight percentage of the ethanol water is used for elution, and eluent is collected; (f) the eluent can be obtained by being dried. The present invention also provides isatis root extract compositions prepared by the method and use thereof. The medicine is adopted from fever relieving medicine, anti-inflammatory medicine, anodyne medicine, medicine for promoting lymphocyte proliferation and antivirus infection medicine.

Description

Radix Isatidis extract composition and method of making the same and purposes

Technical field

The present invention relates to the extraction process field of effective ingredient in Chinese and composition, specifically, is the extraction and the preparation of the total alkaloids extract of Radix Isatidis.The invention still further relates to the compositions of Radix Isatidis extract.

Background technology

Radix Isatidis is a kind of conventional Chinese medicine material, is the root of Isatis indigotica Fort. (Isatis indigotica).It in state-owned extensive cultivation, have the effect (China book on Chinese herbal medicine, pp.2346,3709) of heat-clearing and toxic substances removing.Existing report shows that it has antiviral (Radix Isatidis chemical constitution study (I), Chinese herbal medicine, the 32nd the 12nd phase of volume of calendar year 2001, pp1057), antibacterium endotoxin (Herald of Medicine, volume 21, No.2, pp.74,2002), anticancer, antidepressant (Chinese herbal medicine, the 32nd the 7th phase of volume of calendar year 2001, effect pp.670-671).But these methods are based on injection (Chinese herbal medicine, the 32nd the 7th phase of volume of calendar year 2001 of crude extract, pp.670-671) or chloroform extract (CHINA JOURNAL OF CHINESE MATERIA MEDICA, in June, 2002, Vol.27, No.6, p.439-442), the report at the smart alkaloid position of extracting of not useful as yet macroporous resin.

At present the main production to Radix Isatidis is thick extraction, with the extractum amount of gained as the foundation of investigating.See for example Chinese Pharmacopoeia, 2000, pp.490 wherein mainly is that Radix Isatidis is decocted, and gets supernatant after the soak with ethanol, compacting obtains common isatis root tea and granule, does not have the separation to effective site in this traditional handicraft.Stipulate in the Pharmacopoeia of People's Republic of China of nineteen ninety-five, measure that 45% ethanol soluble extraction is no less than 25% and is qualified products with hot dipping.The product effective site content of Huo Deing is low like this, and quality control method is rough.Huang Qiao Shu etc., Planta Medica, 1981,42 (3), 308-310 has described and has a kind ofly extracted the method for pure Beilstein from the Chinese crude drug Radix Isatidis, and its method mainly is with hexane and dichloromethane extracting, carries out silica gel column chromatography and monitors with TLC.This method only is fit to laboratory to be extracted, and its purpose also is only to use for research, owing to reasons such as cost can not be used for large-scale production.

Summary of the invention

An object of the present invention is to provide a kind of preparation Radix Isatidis extract method for compositions, wherein by alcohol reflux, column chromatography, ethanol elution, the dry effective site that obtains.

Another object of the present invention provides the Radix Isatidis extract compositions that obtains with said method.Wherein the total alkali extractive content of Radix Isatidis is a 50-90% weight, and the content of Beilstein in total composition is 10-30% weight.

The present invention provides also that said composition is analgesic in preparation, the purposes aspect the medicine of antiinflammatory, analgesia, promotion lymphopoiesis, viral infection resisting.

In one aspect of the invention, provide a kind of preparation Radix Isatidis extract method for compositions, the method comprising the steps of:

(a) with the ethanol water reflux, extract, of the 30-95% weight of 6-12 times of Radix Isatidis weight, merge ethanol extract;

(b) filter, reclaim ethanol, obtain medicinal liquid;

(c) the gained medicinal liquid is crossed the macroporous resin adsorption post;

(d) using the cleaning mixture column scrubber, is colourless to effluent;

(e) with the ethanol water eluting of 30%-95% weight, collect eluent;

(f) eluent that obtains of drying.

In the preference of the present invention aspect this, the ethanol water in the described step (a) be medical material weight 8-12 doubly.In another preference of the present invention, the ethanol water concentration in the step (a) is preferably 50-80% weight, is more preferably 75% weight.

In another preference of the present invention, step (a) preferably repeats 1-4 time, and more preferably 1.5-3 hour, most preferably 2 hours.

In another preference of the present invention, each reflux, extract, 0.5-5 hour of step (a), preferred reflux, extract, 1-4 hour, more preferably 1.5-3 hour, most preferably 2 hours.

In another preference of the present invention, the consumption of macroporous resin is preferably the 1/3-4/3 of medical material weight, most preferably 2/3 times in the step (c).

In another preference of the present invention, the ethanol water concentration in the step (e) is preferably 65-80% weight, is more preferably 75% weight.

In another preference of the present invention, also comprise in the described step (e) with high performance liquid chromatography (HPLC) and under the 245nm wavelength, flow velocity 1.0ml/ minute, under 25 ℃, use CH ₃OH: H ₂O=25: 75 detect the content of Beilstein.

In another preference aspect this of the present invention, in described step (f), also comprise the step of filtering described eluent earlier.

In another preference aspect this of the present invention, the drying in the described step (f) is by water bath method, and oven dry is carried out then.Preferred temperature is 60-100 ℃, more preferably 70-90 ℃, and most preferably 80 ℃.

In another aspect of the present invention, a kind of Radix Isatidis extract compositions is provided, said composition is to use:

(a) with the ethanol water reflux, extract, of the 30-95% weight of 6-12 times of Radix Isatidis weight, merge ethanol extract;

(b) filter, reclaim ethanol, obtain medicinal liquid;

(c) the gained medicinal liquid is crossed the macroporous resin adsorption post;

(d) using the cleaning mixture column scrubber, is colourless to effluent;

(e) with the ethanol water eluting of 30%-95% weight, collect eluent;

(f) eluent that obtains of drying

Method preparation, wherein the total alkali extract concentrations is a 50%-90% weight, Beilstein concentration is 10%-30% weight, by total extract weight.

In another aspect of the present invention, the purposes of above-mentioned composition also is provided, said composition is used to prepare medicine, and described medicine is selected from antipyretic analgesics, antibiotic medicine, analgesic, the lymphopoietic medicine of promotion and viral infection resisting medicine.

Description of drawings

Fig. 1 is the Radix Isatidis extract compositions causes rabbit fever models to Typhoid Vi Polysaccharide Vaccine Detoxication (body temperature changing value-hour figure).

The specific embodiment

Radix Isatidis is conventional Chinese crude drug, it simply can be cut into decoction pieces and use.This medical material can be buied from each Radix Isatidis place of production, for example Haozhou, Anhui medical material market, sky, Suzhou Ling Zhongyaoyinpianchang etc.Maybe can use its other form, for example clean and cut into inch strips or be ground into the coarse granule form.

Radix Isatidis extracting method of the present invention adopts ethanol as extracting reagent, slightly to leach supernatant different but with the ethanol of routine, also different with other chloroform extraction method, and ethanol slightly leaches supernatant and this law at extraction ratio, total alkaloids and main effective ingredient also have very big-difference in the extract.

Recovery ethanol step in the step among the present invention (b) makes the medicinal liquid that obtains concentrate, and effectively separates required component on the macroporous resin thereby can be adsorbed on.Recovery is undertaken by conventional method, for example distills etc.

Column chromatography of the present invention uses the macroporous resin adsorption post of various routines, can adopt various pharmaceutical grade macroporous adsorbent resins, and resin commonly used is selected from D101, AB-8, LSA-10.Preferred resin is available from the blue dark LSA-10 in Xi'an.Before use can first watering balance.Last sample, washing, elution speed is unrestricted, but preferably between 30-300ml/ minute, most preferably is 60ml/min.

Cleaning mixture of the present invention can use polar solvent, and for example coupled columns such as deionized water washs.

Comprised the detection step among another embodiment of this aspect, this detection step is to use conventional HPLC (high performance liquid chroma-tography) in step (e) elution process, silicagel column etc. for example, Beilstein composition in the eluent is detected, the detection wavelength is 245nm, flow velocity is 1.0ml/ minute, under 25 ℃, and CH ₃OH: H ₂O=25: 75.As in this eluent, there not being the Beilstein peak to occur proving that then its eluting is complete.

Employed defecator can be various conventional filtration devices among the present invention (f).Filter paper vacuum suction filter for example, porous resin, molecular sieve, packed column etc.

The total alkali extractive content is 50%-9% in the present composition, is more than 20 times of conventional ethanol solvent extraction method, and extraction efficiency reaches more than 60%, effective component yield is more than 0.3%, simultaneously, because operating procedure is simple, do not have TLC, expensive step such as electrophoresis can be used for commercial production in enormous quantities.

The effective site that the present invention obtains mainly is total alkaloids extract, has various antiinflammatories, and is antibiotic, analgesic, antivirus action.The main position toxicity that the present invention obtains is low, can high dose uses and has no side effect.Its available various conventional methods are detectable concentration easily.These conventional methods comprise non-aqueous titration, HPLC method etc.

Patent medicine can be made in the main position that the present invention obtains, for example add sweeting agent, flavoring agent, coloring agent and antiseptic, tablet, lozenge, water or oil suspension, dispersible powders or the granule of preparation oral administration, Emulsion, hard or soft capsule or syrup or elixir.Tablet contains and the active component that is applicable to the nontoxic acceptable mixed with excipients of making tablet.These excipient can be inert diluents for example, as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; Granulating agent and disintegrating agent, for example alginic acid; Binding agent is as starch, gelatin or arabic gum; And lubricant, as magnesium stearate, stearic acid or Talcum.Tablet can not have sugar-coat or uses the known technology coating, postpones disintegration and absorption in gastrointestinal tract, thereby continuous action is provided in a long time.For example, can use the time-delay material, as glyceryl monostearate or distearin.The preparation that orally uses also can be made into hard gelatin capsule, wherein active component and inert solid diluent mix as calcium carbonate, calcium phosphate or Kaolin, or make Perle, wherein active component and water or oil medium, for example Oleum Arachidis hypogaeae semen, liquid paraffin or mixed with olive oil.

The invention effect

Use preparation method of the present invention, can obtain having the Radix Isatidis extract compositions of the total alkali extract and the Beilstein of high concentration, this method can detect its extraction efficiency with HPLC easily.That compositions of the present invention has is analgesic significantly, antiinflammatory, analgesia, antivirus action, is better than general Radix Isatidis decoction pieces etc.

Embodiment

Embodiment 1

The extraction at main position

1. material and instrument

Material: the Radix Isatidis 20kg that originates from Chinese Jiangsu.

Resin: LSA-10 is blue dark available from Xi'an

Instrument: multi-function extractor (500L), broad-mouthed receptacle for holding liquid river, Wujin, Changzhou pharmaceutical machine factory

2. extracting method:

Radix Isatidis is cut into decoction pieces, and 50% ethanol extraction pot reflux, extract, with 8 times of medical material amounts repeats each 0.5 hour 1 time; Merge ethanol extract;

Use the qualitative filter paper vacuum draw, ethanol is reclaimed in 75 ℃ of distillations;

To remain medicinal liquid is added on the LSA-10 resin absorption post;

With deionized water wash post bed (flow velocity 60ml/ branch); Detect to colourless until naked eyes;

With the ethanol water eluting (flow velocity 60ml/ branch) of 50% weight, to high performance liquid chroma-tography post (C18Kromasil post, Chinese Academy of Sciences's main road Chemical Physics institute) under the 245nm wavelength, flow velocity 1.0ml/ minute, under 25 ℃, use CH ₃OH: H ₂O=25: 75 detect the content of Beilstein, to its peak not occurring;

Vacuum draw filters;

90 ℃ of water bath methods, 60 ℃ of oven dry.

Each preparation repeats 3 times, averages.

Embodiment 2-4

With the method for embodiment 1, prepare Radix Isatidis extract as table 1 change condition, and with conventional its content of determination of acid-basetitration, every group is carried out three times, averages.(make standard curve with Beilstein standard substance concentration-peak area, go out its concentration (weight %)) according to the Beilstein calculated by peak area in the final sample

The selection of table 1 reaction condition

Embodiment	Medical material amount (multiple)	Concentration of alcohol (wt%)	Extraction time	Time (hour)	Concentration of alcohol	Bake out temperature	Beilstein content (weight %)	Extraction ratio (weight %)
									2	9	65	2	1	65	75	11.5	60
3	10	75	2	1.5	75	80	13	75
									4	12	50％	2	2	50％	60	12.5	65

From the result of three tests as seen, wherein the yield of effective site is all more than 60%, and Beilstein content is all more than 10% in wherein total solution.

Contain the Radix Isatidis extract powder that the total alkali extract is 65% weight (called after SY-1) with embodiment 4, through following evidence the drug action of effective site.

The refrigeration function of embodiment 5 rabbit fever models:

1 experiment material:

1.1 animal:

New Zealand's white big ear rabbit, male.Available from animal reproduction field, Green Dragon mountain, Jiangning, Nanjing, the quality certification number: SCXK (Soviet Union) 2002-00271.

1.2 equipment:

DT-ITB type electronic thermometer, Shanghai Medical Instrument and Meter Factory, amount system Shanghai word 0003591.

1.3 medicine and reagent:

SY-1

Aspirin (acetic acid salicylic acid enteric coatel tablets), space pharmaceutical factory is respected in Nanjing in vain, lot number: 20020701,

Typhoid Vi Polysaccharide Vaccine, 1ml/ props up, Shanghai Vaccine and Serum Institute, lot number: 20010601032.

2. experimental technique:

2.1 Typhoid Vaccine causes house and exempts from fever model: select about body weight 2kg, body temperature is at the white male rabbit of the big ear of 38.5-39.6 ℃ New Zealand, 21 ± 2 ℃ of following constant temperature adapt to 7 days, and adaptability is measured the rabbit rectum temperature with clinical thermometer for three days on end.The same day was measured rabbit body temperature twice in 1 hour in experiment at interval, chose the body temperature fluctuating margin and used for experiment with interior rabbit at 0.3 ℃.

The meansigma methods of getting twice body temperature is as basal body temperature, be divided into 5 groups by the basal body temperature stratified random, every group 5, irritate stomach for 2 times respectively at-2 and 0 hours branches and give medicine, twice accumulated dose is aspirin 100mg/kg, SY-1-low dosage (2.5mg/kg), dosage among the SY-1 (7.5mg/kg), SY-1 high dose (22.5mg/kg), matched group gives isopyknic water.In administration in 0 hour, every rabbit was respectively surveyed body temperature 1 time in 0.5,1,2,3,4 and 5 hour all from the quiet notes Typhoid Vi Polysaccharide Vaccine of ear edge 1.0ml/kg in Typhoid Vi Polysaccharide Vaccine injection back; The body temperature of being surveyed with different time and the difference of the basal body temperature before the administration be as the changing value of body temperature (δ T, ℃), with δ T (℃) be vertical coordinate, the time is abscissa, makes body temperature change curve (Fig. 1).

3. experimental result

Table 2 SV-1 causes the refrigeration function (n=5) of rabbit fever models to Typhoid Vi Polysaccharide Vaccine

Group	Basal body temperature (℃, X ± SD)	Different time body temperature changing value after the modeling (℃, X ± SD)
								0.5h	1h	2h	3h	4h	5h
		Model group	39.14±0.18	0.56±0.21	1.12±0.27	0.98±0.19	0.72±0.18							0.46±0.23	0.34±0.11
Aspirin	39.12±0.18							0.34±0.20	0.58±0.16 **	0.42±0.16 **	0.38±0.14 *	0.28±0.21	0.20±0.18
		The SY low dosage	39.11±0.19	0.55±0.20	1.07±0.08	0.95±0.17	0.61±0.17							0.45±0.22	0.27±0.19
Dosage among the SY	39.16±0.22							0.50±0.11	0.82±0.25	0.62±0.25 *	0.46±0.19	0.36±0.15	0.30±0.15
		The SY high dose	39.10±0.16	0.44±0.12	0.70±0.10 *	0.52±0.15 **	0.32±0.06 **							0.24±0.06	0.14±0.06

Carry out statistical procedures (t check),

^*P＜0.05, ^*P＜0.01 is compared with matched group.

As can be seen from Table 2, rabbit is after the intravenous injection Typhoid Vi Polysaccharide Vaccine, and 0.5 hour body temperature has begun to rise, peaked rapidly in 1 hour, continue to maintain higher level during by 2 hours, descend rapidly since 3 hours body temperature, body temperature is near foundation level during by 5 hours.Be subjected to reagent thing SY-1 that the pyrogenicity rabbit is had tangible refrigeration function, and show certain dose-dependence, the high dose group refrigeration function is particularly outstanding, and is suitable with the aspirin group.From whole process, the performance of aspirin refrigeration function is very fast, the suppression ratio of 1 hour body temperature rise is near 50% after the modeling, the SY-1 high dose group is 37%, but hold time from refrigeration function, the SY-1 high dose group is better than the aspirin group, and during by 5 hours, SY-1 high dose group body temperature is compared with model group and still had significant difference.

The influence of embodiment 6, SY-1 xylol induced mice auricle edema

1. experiment material

1.1 animal: 60 of male mice in kunming, body weight 22-26 gram is provided by Nanjing University of Traditional Chinese Medicine's Animal House

1.2 equipment: irritate the stomach syringe needle, micropipette rifle (20 microlitre), micrometer caliper.

1.3 medicine and reagent:

SY-1

Aspirin (diethylstilbestrol salicylic acid enteric coatel tablets), space pharmaceutical factory is respected in Nanjing in vain, lot number: 20020701,

Radix Isatidis granules, the Nanchang Ji is given birth to pharmaceutical factory, lot number: 011207

Dimethylbenzene, Chinese Hangzhou chemical reagent factory, lot number: 20001129.

2. experimental technique

Mice is divided into six groups at random by body weight: model control group, aspirin 50mg/kg group, Radix Isatidis granules 3g/kg, SY-1 5,15, the 45mg/kg group, water, aspirin, Radix Isatidis granules, SY-1 according to dosage irritate and emit 1 time respectively the same day in experiment, irritate stomach more once after 5 hours, the accumulated dose of twice administration of each medicine group is 100mg/kg, 6g/kg, 10,30 and 90mg/kg.

Last is irritated behind the stomach and only evenly to be coated with 100% dimethylbenzene, 20 μ l/ in the wide positive and negative two sides of mouse right ear in one hour, after 1 hour the mice cervical vertebra is dislocated till death, measure the thickness of left and right sides auricular concha with micrometer caliper, as the swelling degree, the swelling degree of matched group and administration group is carried out statistical procedures (t check) with its thickness difference.

3. experimental result

The influence of table 3 SY-1 xylol induced mice auricle edema

Group	Dosage (in the m)	Number of mice (only)	Auricle swelling degree (mm) (X ± SD)	Suppression ratio (%)
					Model group	-	9	0.119±0.031	-
The SY-1 low dose group	10	10	0.062±0.035 **	47.6
					Dosage group among the SY-1	30	10	0.05±0.032 **	57.7
The SY-1 high dose group	90	10	0.045±0.018 **	62.6
					The aspirin group	100	10	0.058±0.022 **	51.5
Plate basket root group	6000	10	0.067±0.026	43.9

^*P＜0.01 is compared with matched group.

As shown in table 3, compare with model group, SY-1 has presented the inhibitory action of dose dependent to ease auricle swelling, and three dosage groups all have extremely significant difference (P＜0.01), and its suppression ratio reaches 47.6,57.7 and 62.6% respectively.Aspirin group and Radix Isatidis group have also obviously reduced auricle swelling degree, and suppression ratio is respectively 51.5% and 43.9%.

Embodiment 7, SY-1 are to the influence of Ovum Gallus domesticus album induced mice pedal swelling

1. experiment material:

1.1 laboratory animal: 70 of male mouse of kunming, body weight 22-26 gram.Provide by Nanjing University of Traditional Chinese Medicine's Animal House.

1.2 equipment: irritate the stomach syringe needle, syringe (1.0ml), No. 4 injection needles, micrometer caliper.

1.3 medicine and reagent;

SY-1。

Aspirin (diethylstilbestrol salicylic acid enteric coatel tablets), space pharmaceutical factory is respected in Nanjing in vain, lot number: 20020701,

Radix Isatidis granules, the Nanchang Ji is given birth to pharmaceutical factory, lot number: 011207

Freshly-slaughtered poultry Ovum Gallus domesticus album (10%): get fresh Ovum Gallus domesticus album, make with 1: 10 by volume mix homogeneously of normal saline.

2. experimental technique

Mice is divided into six groups at random by body weight; Model control group, aspirin 50mg/kg group, Radix Isatidis granules 3g/kg, SY-1 5,15, the 45mg/kg group, the same day, water, aspirin, Radix Isatidis granules, SY-1 according to dosage irritated stomach 1 time respectively in testing, irritate stomach more once after 5 hours, the accumulated dose of twice administration of each medicine group is 100mg/kg, 6g/kg.10,30 and 90mg/kg.Last is irritated behind the stomach and respectively to be organized mice in one hour in right back sufficient plantar subcutaneous injection 10% freshly-slaughtered poultry Ovum Gallus domesticus album 20 microlitres/only cause inflammation.Injection back 30min measures the thickness that left and right metapedes is stepped respectively, as the swelling degree, the swelling degree of matched group and administration group is carried out statistical procedures (t check) with its thickness difference.

3 experimental results

Table 4 SY-1 is to the influence of Ovum Gallus domesticus album induced mice pedal swelling

Group	Dosage (mg/kg)	Number of mice (only)	Claw swelling degree (mm) (X ± SD)	Suppression ratio (%)
					Model group	-	10	0.285±0.103	-
The SY-1 low dose group	10	10	0.134±0.083 **	53.2
					Dosage group among the SY-1	30	10	0.124±0.065 **	56.4
The SY-1 high dose group	90	10	0.119±0.049 **	58.3
					The aspirin group	100	10	0.106±0.037 **	62.9
The Radix Isatidis group	6000	10	0.142±0.052 **	50.1

^*Compare with matched group P＜0.01.

As shown in table 3, compare with model control group, SY-1 has presented the inhibitory action of dose dependent to pedal swelling, and three dosage groups all have extremely significant difference (P＜0.01).Aspirin group and Radix Isatidis group have also reduced swelling degree of the paw significantly.But its effect is all not as SY-1 of the present invention, even used dosage has also substantially exceeded SY-1.

The influence of embodiment 8SY-1 Dichlorodiphenyl Acetate induced mice writhing response

1. experiment material

1.1 animal: 60 of kunming mices, male and female half and half, body weight 18-22 gram is provided by Nanjing University of Traditional Chinese Medicine's Animal House

1.2 equipment: irritate the stomach syringe needle, syringe (1.0ml); No. 4 injection needles.

1.3 medicine and reagent:

SY-1

Aspirin (diethylstilbestrol salicylic acid enteric coatel tablets), space pharmaceutical factory is respected in Nanjing in vain; Lot number: 20020701,

Radix Isatidis granules, the Nanchang Ji is given birth to pharmaceutical factory, lot number: 011207

Glacial acetic acid, Shanghai chemical reagent company limited, lot number: 0201101.

2 experimental techniques

Mice is divided into six groups at random by body weight: model control group, aspirin 50mg/kg group, Radix Isatidis granules 3/kg, SY-1 5,15, the 45mg/1cg group, the same day, water, aspirin, Radix Isatidis granules, SY-1 according to dosage irritated stomach 1 time respectively in testing, irritate stomach more once after S hour, the accumulated dose of twice administration of each medicine group is 100mg/kg, 6g/kg, 10,30 and 90mg/kg.Last is irritated the acetic acid of respectively organizing mouse peritoneal injection 0.6% behind the stomach in one hour, every 0.2M, behind the record injection algogen, in 15 minutes each Mus turn round the body number of times, calculate medicine analgesia percentage rate.With t check carrying out statistical analysis, comparable group differences.

Behind the acetic acid of lumbar injection 0.6%, can cause deep, large tracts of land and more persistent pain stimulation, it is as shown in table 5 to cause mice to produce " turn round body ' " reaction (abdominal part indent, trunk and back leg extension, hips up), compare with model control group, SY-1 has presented significant analgesic effect, three dosage groups all have significant difference, and it has reached 67.9,42.9 and 77.1% respectively to turning round body number of times suppression ratio in 15 minutes.The aspirin group has also reduced significantly turns round the body number in the 15min, the analgesia rate has reached 69.3% respectively, and the Radix Isatidis group is 32.3% to the suppression ratio of writhing response, but it compares there was no significant difference with model group.

The influence of table 5 SY-1 Dichlorodiphenyl Acetate induced mice writhing response

Group	Dosage (mg/kg)	Number of mice (only)	Turn round body number (X ± SD) in the 15min	Analgesia rate (%)
					Model group	-	9	42.4±18.512	-
The SY-1 low dose group	10	10	13.6±12.258 **	67.9
					Dosage group among the SY-1	30	10	24.2±18.96 *	42.9
The SY-1 high dose group	90	10	9.7±17.101 **	77.1
					The aspirin group	100	10	13±8.981 **	69.3
The Radix Isatidis group	6000	10	28.7±24.788	32.3

^*P＜0.01 * P＜0.05 is compared with matched group.

Embodiment 9SY-1 is to the influence of lymphopoiesis function

1 experiment material

1.1 animal: some of Kunming mouses, female, body weight 18-22 gram.

1.2 equipment: operating theater instruments one cover, centrifuge tube, 200 eye mesh screens, plunger, ice chest.

1.3 medicine and reagent:

SY-1

Aspirin (diethylstilbestrol salicylic acid enteric coatel tablets Nanjing respect space pharmaceutical factory in vain, lot number: 20020701,

RPMI-1640，GIBICO，Lot?NO.1120035

Three (methylol) amido methane (Tris), China Medicine (Group) Shanghai Chemical Reagent Co.,, lot number F20020730.

New-born calf serum (NBS).Hangzhou Sijiqing Biological Engineering Material Co., Ltd., lot number: 021008.

2. experimental technique

Get mice, aseptic extraction spleen.Extruding is dispersed into single splenocyte suspension in cold Hank ' s liquid, through washing, Tris-NH ₄After Cl removes erythrocyte, be suspended in RPMI 1640 culture medium that contain 10%NBS, make 5 * 10 ⁴The concentration of/ml is used for cultivating.In 96 ghost culture plates, every hole adds the splenocyte suspension of 100 microlitres, and (final concentration of cells is 2.5 * 10 ⁵/ ml).Add 100 microlitre culture medium (blank), 5 mcg/ml ConA or 100 mcg/ml LPS again, or contain the culture medium of variable concentrations medicine.Put 37 ℃, 5%CO ₂Incubator in cultivated 72 hours, stop preceding 4 hours, add 2mg/ml MTT 40 microlitres, after 4 hours, centrifugal (1000rpm 5min), inhales and abandons supernatant, and every hole adds 200 microlitre DMSO, vibrates 10 minutes.Read the OD value at microplate reader 540nm place.Each tests equal triplicate, and each experiment is used three multiple holes, is calculated as follows stimulation index.

Stimulation index (SI)=OD (dosing hole)/OD (blank)

3 experimental results

3.1SY-1 to normal lymphopoietic influence

Table 6 SY-1 is to normal lymphopoietic influence

Group	Concentration (g/ml)	SI
			N		1.0±0.12
F	10 -3	1.1±0.05
			SY-1	10 -8	1.14±0.14
10 -7	1.19±0.2
		10 -6		1.2±0.18
10 -5	1.3±0.16 *
		10 -4		1.7±0.03 *

^*P＜0.01 VS N group

As known from Table 6, SY-1 has the normal lymphopoietic trend of certain promotion.

3.2SY-1 lymphopoietic influence to the ConA stimulation

The lymphopoietic influence that table 7 SY-1 stimulates ConA

Group	Concentration (g/ml)	SI
			ConA	2.5×10 -6	5.6±0.5 #
F	10 -3	5.6±0.45
			SY-1	10 -8	6.3±0.56
10 -7	6.5±0.98
		10 -6		7.0±1.17 *
10 -5	6.6±0.19 *
		10 -4		6.9±0.77 *

^#P＜0.01 VS N group, ^*P＜0.05 VS ConA group

As shown in table 7, the SI of ConA group reaches 5.6 ± 0.5, and SY-1 can promote the lymphocytic propagation that ConA stimulates, 10 ^-6Significant difference is arranged during the above concentration of g/ml.

3.3SY-1 lymphopoietic influence to the LPS stimulation

The lymphopoietic influence that table 8 SY-1 stimulates LPS

Group	Concentration (g/ml)	SI
			LPS	5×10 -5	3.6±0.6 *
F	10 -3	3.6±0.8
			SY-1	10 -8	4.6±1.2
10 -7	4.2±0.9
		10 -6		4.6±1.2
10 -5	5.2±1.4 *
		10 -4		4.5±1.0

^#P＜0.01 VS N group, ^*P＜0.05 VS ConA group

As shown in table 8, the SI of LPS group reaches 3.6 ± 0.57, and SY-1 can promote the lymphocytic propagation that LPS stimulates, 10 ^-5There is significant difference at g/ml concentration place.

The experiment of the anti-mice viral infection of embodiment 10SY-1

One, the result of resisiting influenza virus

1. material and method:

1. medicine:

SY-1

Positive control: ribavirin injection (the accurate word XF19990719 of traditional Chinese medicines, lot number 20020511).Hubei Qianjiang Pharmaceutical Co., Ltd..

Is control drug: Radix Isatidis (defended the accurate word 1999 of medicine No. 129203, manufacturer?)

2. animal:

ICR mice: 14-16g, male and female half and half are provided by Jiangsu Province's Experimental Animal Center.

3. viral:

FMI Mus lung adapted strain A/FM1/1/47/ (H1N1): Nanjing Medical University's microbiology and immunology system preserve.

4. mice is to the death protection experiment of influenza infection

Experiment grouping for the first time and drug dose are as follows:

First group: SY-1 high dose group (90mg/kg)

Second group: dosage group (30mg/kg) among the SY-1

The 3rd group: SY-1 low dose group (10mg/kg)

The 4th group; Ribavirin group (100mg/kg)

The 5th group: Radix Isatidis group (3g/kg)

The 6th group: the virus control group

The 7th group: the normal control group

Every group of number of mice sees Table, and all under the slight anesthesia of ether, intranasal vaccination 0.03ml/ only is equivalent to the chick embryo allantoic liquid that 2-5LD50 contains influenza virus to mice.

Each group of medication: SY-1, Radix Isatidis group and ribavirin group are in viral infection clysmata administration in preceding 2 hours 1 time, and later 2 times/day, 0.2ml/ time, administration is 5 days altogether.Virus control group and normal control group give the equivalent distilled water by the same method and substitute.

Mouse invasion and death toll are observed and write down to continuous the observation 15 days day by day behind viral infection, calculates mortality rate, dead protective rate, average life day and prolong vital rates.

5. lung index and pneumonopathy become degree change behind the mouse infection influenza virus

Experiment divides 6 groups, and group technology, counteracting toxic substances and medication are all identical with death protection experiment.

Infected the back the 6th day, and put to death mice, full lung is taken out in the back of weighing, clean with normal saline, and blot, claim lung heavy with clean filter paper, calculate lung exponential sum lung index suppression ratio, observe pneumonopathy and become, no pathological changes is designated as (-),＜25% pathological changes is designated as (1+), the 25-50% pathological changes is designated as (2+), and the 50-75% pathological changes is designated as (3+), and＞75% pathological changes is designated as (4+), dead mouse lung pathological changes is designated as (5+), calculates pneumonopathy and becomes the rate that alleviates.

2.2 result

The dead protective effect of table 9 pair mice influenza infection

Group	Number of animals (only)	Death toll (only)	Mortality rate (%)	Dead protective rate (%)	Average life day (my god)	Prolong vital rates (%)
							1	10	7	70	22.2	8.80±4.59	37.50
2	10	5	50	44.4	10.40±4.93 *	62.50
							3	10	5	50	44.4	9.80±5.49	53.13
4	10	3	30 *	66.7	12.20±4.52 **	90.63
							5	10	6	60	33.3	9.90±4.53	54.69
6	10	9	90		6.40±3.20
							7	10	0	0 **		15.00±0.00 **

Annotate: compare with the 6th group, ^*Be P＜0.05, ^*P＜0.01

The influence that table 10 pair mice influenza infection lung index and pneumonopathy become

Group	Number of animals (only)	The lung index	Suppression ratio (%)	Pneumonopathy becomes	Alleviate rate (%)
						1	10	2.76±0.95	-3.76	3.90±1.73	0.00
2	10	2.45±0.76	7.89	3.60±1.43	7.69
						3	10	2.28±1.03	14.29	3.00±2.21	23.08
4	10	1.43±0.55 **	46.24	0.90±1.73 **	76.92
						5	10	2.26±1.02	15.04	3.20±1.99	17.95
6	10	2.66±0.85		3.90±1.20
						7	10	0.80±0.16 **		0±0.00 **

Annotate: compare with the 6th group, ^*Be P＜0.05, ^*＜0.01

3. conclusion

The death protection of the pneumonia that SY1 causes the mice influenza virus, pneumonopathy become and alleviate degree and reduce lung index aspect and do not have clear meaning, but dosage group significance aspect average life day prolongation wherein.

Two, the anti-Sendai virus test of SY1

1. material and method

Virus: Sendai virus Mus lung adapted strain, Nanjing Medical University's microorganism and immunology system preserve.

The animal grouping of positive control drug, test and dosage, experimental technique are the same.

2. result

Table 11 pair mice Sendai virus infects dead protective effect

Group	Number of animals (only)	Death toll (only)	Mortality rate (%)	Dead protective rate (%)	Average life day (my god)	Prolong vital rates (%)
							1	10	6	60	33.33	9.00±5.29	40.63
2	10	5	50	44.4	10.50±5.04 *	64.06
							3	10	9	90	0.00	5.90±3.28	7.81
4	10	1	10 **	88.89	14.00±3.16 **	118.73

5	10	5	50	44.44	10.50±4.93 *	70.31
							6	10	9	90		6.40±3.44
7	10	0	0 **		15.00±0.00 **

Annotate: compare with the 6th group, ^*Be P＜0.05, ^*P＜0.01

The influence that table 12 pair mice influenza infection lung index and pneumonopathy become

Group	Number of animals (only)	The lung index	Suppression ratio (%)	Pneumonopathy becomes	Alleviate rate (%)
						1	10	1.72±0.66 **	36.99	2.70±2.11 **	43.75
2	10	2.11±0.84	22.71	3.00±2.16 **	37.50
						3	10	2.35±0.65	13.92	4.30±1.57	10.42
4	10	1.63±0.75 **	40.92	2.20±2.30 **	54.17
						5	10	2.52±0.62	7.69	4.20±1.62	12.50
6	10	2.70±0.47		4.80±0.42
						7	10	0.81±0.09 **		0±0.00 **

Annotate: compare with the 6th group, ^*Be P＜0.05, ^*P＜0.01

3. conclusion

The average life of the pneumonia that SY1 causes little murine sendai virus day prolongs significance, and high dose and middle dosage are alleviating significance aspect pneumonopathy range degree and the reduction lung index.

As mentioned above, method of the present invention can be extracted Radix Isatidis extract efficiently, and technology is simple, the active constituent content height.In addition, resulting product all has remarkable effect on antiviral, antibiotic, antiinflammatory, clearing away heat and alleviating pain.

Product of as above having described Radix Isatidis extract preparation method disclosed in this invention and having made and uses thereof.But need to understand and wherein comprised variation understood by one of ordinary skill in the art and change.These variations and change are also contained in the scope of the present invention that claim limits.

Claims (7)

1. method for preparing Radix Isatidis extract is characterized in that the method comprising the steps of:

(a) with the ethanol water reflux, extract, of the 30-95% weight of 6-12 times of Radix Isatidis weight 1-4 time, each 0.5-5 hour, merge ethanol extract;

(b) filter, reclaim ethanol, obtain medicinal liquid;

(c) the gained medicinal liquid is crossed the macroporous resin adsorption post;

(d) using the cleaning mixture column scrubber, is colourless to effluent;

(e) with the ethanol water eluting of 50%-95% weight, collect eluent;

(f) filter the eluent that drying obtains.

2. the method for claim 1 is characterized in that, the ethanol water in the described step (a) is 8-12 a times of medical material weight.

3. the method for claim 1 is characterized in that, the ethanol water concentration in the described step (a) is 50-80% weight.

4. the method for claim 1 is characterized in that, the ethanol in the step (e) is the aqueous solution of 65-80% weight.

5. the method for claim 1 is characterized in that, also comprises in the described step (e) with high performance liquid chromatography (HPLC) flow velocity 1.0ml/ minute, under 25 ℃, using CH under the 245nm wavelength ₃OH: H ₂O=25: 75 detect the content of Beilstein.

6. Radix Isatidis extract is characterized in that: this extract is with the described method preparation of claim 1, and wherein the total alkali extract concentrations is a 50%-90% weight, and Beilstein concentration is 10%-30% weight.

7. the purposes of the described Radix Isatidis extract of claim 6 is characterized in that, is used to prepare medicine, and described medicine is selected from antipyretic analgesics, antibiotic medicine, analgesic, the lymphopoietic medicine of promotion and viral infection resisting medicine.

Images