CN101628075A

CN101628075A - Method for preparing lilium total saponin extract by using macroporous resin and application thereof

Info

Publication number: CN101628075A
Application number: CN200910139706A
Authority: CN
Inventors: 李卫民; 朱贺年; 贾金良
Original assignee: HOMALOMENA MEDICINE RESEARCH DEVELOPMENT Co Ltd BAOTOU CITY
Current assignee: HOMALOMENA MEDICINE RESEARCH DEVELOPMENT Co Ltd BAOTOU CITY
Priority date: 2009-06-22
Filing date: 2009-06-22
Publication date: 2010-01-20

Abstract

The invention belongs to the field of pharmaceutical research, in particular to a method for preparing lilium total saponin extract brownie by using macroporous resin and the application thereof, aiming at extracting and purifying total lilium saponin from lilium by using macroporous resin and the application of the obtained lilium extract. The obtained lilium extract containing lilium saponins is proved by research to have the function of improving the symptoms of chronic stress depression rats, and can be used for treating human depression. The method for preparing the lilium saponins is convenient, and the prepared lilium saponin has the purity of more than 50 percent; and the invention has low cost and is easily implemented.

Description

Method of a kind of preparing lilium total saponin extract by using macroporous resin and uses thereof

Technical field:

The invention belongs to the pharmacy research field, related to method of a kind of preparing lilium total saponin extract by using macroporous resin and uses thereof specifically.

Background technology:

Bulbus Lilii is liliaceous plant tiger lily Lilium lancifolium Thunb., the dry meat scale leaf of Bulbus Lilii Lilium brownii var. or Lilium tenuifolium Lilium pumilum DC., has nourishing YIN and moistening the lung, the function of clearing away heart-fire for tranquillization, contained Bulbus Lilii Saponin is one of its main component, but the purity that the traditional method separation obtains is far below 50%, resulting material is except that containing the Bulbus Lilii Saponin, most compositions all are other materials, because composition is various, the physicochemical property of adding many compositions is close, does not use any special measures to be difficult to Bulbus Lilii Saponin and other composition are separated.

Summary of the invention:

The objective of the invention is to be to remedy the deficiencies in the prior art, the purity of Bulbus Lilii saponin is extracted in raising from Bulbus Lilii, specifically from Bulbus Lilii, extract the method for purification Bulbus Lilii saponin with macroporous resin, and the purposes of resulting Bulbus Lilii extract, the resulting Bulbus Lilii extract that contains the Bulbus Lilii saponin, finding after deliberation has the effect that improves chronic stress depression model symptom of rats, can be used for the treatment of human depression, to overcome above-mentioned deficiency.

The objective of the invention is to be achieved through the following technical solutions:

A kind of method of preparing lilium total saponin extract by using macroporous resin, its characteristics are:

(1) Bulbus Lilii is prepared into the crude extract that contains Bulbus Lilii total saponins;

(2) with the Bulbus Lilii crude extract by being filled with the chromatographic column of macroporous resin, use the eluant eluting, collect the eluting part, reclaim eluant, collect the gained Bulbus Lilii extract, promptly;

Described macroporous resin is a polystyrene resin type macroporous resin; The model of described macroporous resin is one or more the mixture among D101, SA40, HP20, SP825, XAD16, XDA1, XDA5, LX18, AB8, the LSA7; Used eluant is a polar solvent; Used eluant is water and 5～20BV ethanol; The crude extract that contains described Bulbus Lilii total saponins is prepared from by following method: get the Bulbus Lilii medical material,, concentrate or reclaim ethanol with 65%～95% ethanol 70～90 ℃ of extractions of 6～10 times of amounts, concentrated solution or continue dry cream powder promptly; With the Bulbus Lilii crude extract by being filled with the chromatographic column of macroporous resin, the load sample weight ratio is with resin: crude drug is counted: 0.1: 1～2: 1, last sample concentration is with crude drug: the solution weight volume ratio counts 0.01: 1～and 1: 1, flow velocity is 1～10ml/min, with distilled water and ethanol elution, collect the ethanol elution part, reclaim ethanol, collect the gained Bulbus Lilii extract, promptly; Lilium total saponin extract is used to prepare the medicine for the treatment of depression.

Beneficial effect:

The objective of the invention is to remedy the deficiencies in the prior art, the purity of Bulbus Lilii saponin is extracted in raising from Bulbus Lilii, it is convenient to use the inventive method to prepare Bulbus Lilii Saponin method, purity can reach more than 50%, with low cost, and easy to implement, the resulting Bulbus Lilii extract that contains the Bulbus Lilii saponin, finding after deliberation has the effect that improves chronic stress depression model symptom of rats, can be used for the treatment of human depression.

Description of drawings:

Fig. 1 is that Bulbus Lilii total saponins of the present invention sees through curve chart;

Fig. 2 is a macroporous resin adsorption Bulbus Lilii total saponins gradient elution curve of the present invention;

Fig. 3 is the influence curve figure of Bulbus Lilii extract of the present invention to the rat model body weight change;

Fig. 4 is the influence curve figure that Bulbus Lilii extract of the present invention changes rat model consumption appetite:

The specific embodiment:

One, Bulbus Lilii is prepared into the crude extract that contains Bulbus Lilii total saponins

Precision takes by weighing Bulbus Lilii medical material 10.0g, adds 70 ℃ of reflux, extract, of 10 times of amount 80% ethanol respectively 3 times, each 3h, merging filtrate is settled to 500ml, and merging filtrate is settled to 500ml with coordinative solvent, the accurate 10ml evaporate to dryness of drawing, water 10ml dissolving, n-butyl alcohol 15ml extraction is reclaimed n-butyl alcohol to doing, dissolve with methanol 10ml, the accurate 1ml evaporate to dryness of drawing, vanillin-ice acetic acid method is measured absorbance in 540nm wavelength place.The in addition accurate 25ml that draws puts in the evaporating dish that is dried to constant weight by the drying means operation of (Chinese Pharmacopoeia version I in 2005 portion, appendix XA) extractum, calculates total saponins leaching rate and total saponins purity.The results are shown in Table 1.

Table 1 extraction process result of the test (n=6)

Two, purification by macroporous resin technology

1, macroporous adsorbent resin pretreatment

Cleaning equipment and pipeline before the dress post, in case nuisance is to the pollution of resin, the water of net plant side by side.In resin, add the ethanol that is equivalent to 0.4～0.5 times of loaded resin volume earlier, then resin is dropped in the post, make its liquid level be higher than resin bed 0.3m, soak 24h.Use 2BV ethanol, by resin bed, and soak 4～5h with the flow velocity of 2BV/h.Use ethanol, by resin bed, be washed till effluent and add water and be not white in color till the muddiness, and clean ethanol by same flow velocity with water with the flow velocity of 2BV/h.Resin is in service continuously needn't to carry out pretreatment again, should consider pretreatment again when mistake out of service is long-time.Want abundant desorbing before stopping, washing quiet, and with solution soaking, in order to avoid the germ contamination resin greater than 10% NaCl.

2, the preparation of sample solution

Take by weighing the Bulbus Lilii medical material, extract by above-mentioned preferred ethanol extraction technology, filter, merge extractive liquid, reclaims ethanol to there not being the alcohol flavor.Add 10 times of water gaging suspendibles, high speed centrifugation, decompress filter.Filtrate is preserved in addition, and is standby.(concentration is counted 0.1g/ml with the crude drug amount)

Embodiment 1: one, with Bulbus Lilii be prepared into contain described in the crude extract of pharmaceutically active ingredient

Table 1 extraction process result of the test (n=6)

Two, purification by macroporous resin technology

1 macroporous adsorbent resin pretreatment

The preparation of 2 sample solutions

The test of 3 purifying process

Precision takes by weighing the AB-8 type resin of handling well (being equivalent to dried resin 2.0g), the accurate sample liquid 40ml (upper prop of 0.10g crude drug/ml) of drawing, flow speed control is at 2ml/min, after the absorption fully, use 10BV distilled water and 20BV80% ethanol elution successively, collect 80% ethanol elution part, measure total saponin content behind the standardize solution in accordance with the law.A certain amount of the putting in the evaporating dish that is dried to constant weight of accurate in addition absorption by the drying means operation of (Chinese Pharmacopoeia version I in 2005 portion, appendix X A) extractum, measured the weight of extractum, calculates Bulbus Lilii extract purity, the results are shown in Table 1.

Table 1 purifying process result of the test (n=6)

Embodiment 1:

Adopt the different tests condition to test by table 2 tested number, each precision takes by weighing Bulbus Lilii medical material 10.0g.

Table 2 test method

Merging filtrate is settled to 500ml with coordinative solvent, the accurate 10ml evaporate to dryness of drawing, water 10ml dissolving, n-butyl alcohol 15ml extraction is reclaimed n-butyl alcohol to doing, with methanol 10ml dissolving, the accurate 1ml evaporate to dryness of drawing, vanillin-ice acetic acid method is measured absorbance in 540nm wavelength place.Accurate in addition absorption 25ml puts in the evaporating dish that is dried to constant weight, by the drying means operation of (Chinese Pharmacopoeia version I in 2005 portion, appendix XA) extractum, calculates total saponins leaching rate and yield of extract.Following table as a result.

Table 3 experimental result

Embodiment 2:

Precision takes by weighing Bulbus Lilii medical material 10.0g, adds 70 ℃ of reflux, extract, of 10 times of amount 65%, 80% and 95% ethanol respectively 3 times, each 3h, merging filtrate is settled to 500ml with coordinative solvent, the accurate 10ml evaporate to dryness of drawing, water 10ml dissolving, n-butyl alcohol 15ml extraction, reclaim n-butyl alcohol to doing, with methanol 10ml dissolving, the accurate 1ml evaporate to dryness of drawing, vanillin-ice acetic acid method is measured absorbance in 540nm wavelength place.Accurate in addition absorption 25ml puts in the evaporating dish that is dried to constant weight, by " total saponins leaching rate and total saponins purity are calculated in the drying means operation of Chinese pharmacopoeia (the appendix XA of version I portion in 2005) extractum.The results are shown in Table 4.

Table 4 different ethanol concentration lixiviate total saponins (n=6)

Embodiment 3:

Precision takes by weighing D101, SA40, HP20, SP825, XAD16, XDA1, XDA5, LX18, AB8, each 2.0g of LSA7 resin that has handled well, carries out following test respectively:

Above resin is put respectively in the 100ml conical flask, the accurate sample liquid 20ml that adds, shaking table jolting 24h makes the absorption that reaches capacity, and filters, and the filtrate precision is settled to proper volume, measures total saponin content.Resin after filtering is put in the 100ml conical flask in addition, the accurate 95% ethanol 50ml that adds, shaking table jolting 24h filters, the filtrate standardize solution, the content of total saponins is measured in the dilution back.Be calculated as follows static saturated adsorption rate of each resin and static eluting rate.The results are shown in Table 5.

The static saturated adsorption rate of table 510 kind of macroporous resin and eluting rate result

Annotate: adsorption capacity=(sample liquid total saponins amount-not adsorbance)/dried resin heavy (mg/g dried resin)

Saturated adsorption rate (%)=(sample liquid total saponins amount-not adsorbance)/sample liquid total saponins amount x100%

Eluting rate (%)=desorption amount/(sample liquid total saponins amount-not adsorbance) x100%

Embodiment 4:

Take by weighing macroporous resin 5g, wet method dress post, last sample, concentration is 0.1g crude drug/ml, connects with 20ml scale test tube and spills liquid, every 10ml adorns 1 test tube, connects 20 pipes, gets the content that 10ml measures total saponins, the results are shown in Figure 1 Bulbus Lilii total saponins and sees through curve chart.

Embodiment 5:

Precision takes by weighing the AB-8 type resin of handling well (being equivalent to the about 10.0g of dried resin), the accurate sample liquid 60ml upper prop of drawing, flow speed control is at 1ml/min, after the absorption fully, use distilled water 200ml, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, each 100ml gradient elution of 95% ethanol successively.The distilled water eluent is collected 50ml2 part earlier, 25ml4 part, and 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol are collected 25ml4 part respectively.Measure Bulbus Lilii total saponins concentration in accordance with the law, be numbered abscissa (X) with volumetric flask, total saponins concentration is that vertical coordinate (Y) is drawn elution curve, sees Fig. 2 macroporous resin adsorption Bulbus Lilii total saponins gradient elution curve.

Embodiment 6:

Precision takes by weighing the AB-8 type resin of handling well (being equivalent to the about 5.0g of dried resin), the accurate sample liquid 20ml upper prop of drawing, flow speed control is at 1ml/min, after the absorption fully, be eluted to no reducing sugar reaction with distilled water successively, use 100ml80%, 95% ethanol elution respectively.Measure Bulbus Lilii total saponins concentration in accordance with the law, the results are shown in Table 6.

Table 680% ethanol and 95% ethanol elution result

Embodiment 7:

Take by weighing 3 parts of the AB-8 type resins of handling well (being equivalent to dried resin 10.0g), wet method dress post, the accurate sample liquid 60ml that draws is with 1ml/min flow velocity upper prop.After the absorption fully, collected post liquid, respectively with 100ml, 150ml, 200ml distilled water eluting, use respectively then 50% ethanol 200ml (100ml, 50ml, 50ml) eluting successively, flow speed control is pressed 100ml at 2ml/min, 50ml, 50ml divide 3 parts of collections.Total saponin content is measured in the dilution of each several part eluent in accordance with the law behind the standardize solution, calculate the ratio of each several part total saponins.Accurate in addition absorption 80% ethanol 100ml eluting part 50ml puts in the evaporating dish that is dried to constant weight, by the drying means operation of (Chinese Pharmacopoeia version I in 2005 portion, appendix XA) extractum, measures the weight of extractum, calculates total saponins purity.The results are shown in Table 7.

Table 7 eluting solvent different volumes result of the test

Embodiment 8:

Take by weighing 6 parts of the AB-8 type macroporous resins of handling well (being equivalent to dried resin 2.0g), wet method dress post is prepared 0.32g/ml, 0.16g/ml, 0.08g/ml3 plant the sample liquid of concentration, each 2 parts of (all containing total saponins 67.59mg) upper prop, after the absorption, the resin column that sample liquid concentration is identical carries out eluting by 2 kinds of flow velocitys (1ml/min, 3ml/min) fully, after being eluted to no reducing sugar reaction with distilled water, use the 20BV80% ethanol elution, collect 80% ethanol elution part, measure total saponin content in accordance with the law.A certain amount of the putting in the evaporating dish that is dried to constant weight of accurate in addition absorption by the drying means operation of (Chinese Pharmacopoeia version I in 2005 portion, appendix XA) extractum, measured the weight of extractum, calculates Bulbus Lilii extract yield and purity, the results are shown in Table 8.

Different sample liquid concentration of table 8 and elution flow rate result of the test

Annotate: Bulbus Lilii extract yield (%)=eluent total saponins/sample liquid total saponins x100%

Embodiment 9:

Bulbus Lilii extract is to the influence of chronic stress depression model rat

Test a Bulbus Lilii extract to the ethological influence of rat model

1 animal

The SD rat: male, body weight 220-270g.

2 methods

2.1 model preparation

Matched group is by 7/cage, and normal diet drinking-water is not given any stimulation.Other five groups, 1 raising of every cage, with reference to the method for Willner, the gentleness stimulation with chronic Unpredictability cooperates lonely supporting, and 7 kinds of stress factors are used in 21d by random method: 1. put upside down (24h) round the clock; 2. fasting (24h); 3. prohibit water and accept 21 days long-term stress stimulations, comprising: 3 fasting in 24 hours, prohibited water in 3 times 24 hours, 3 illuminations all night, 34 ℃ of cold water swimming 5 minutes, 3 45 ℃ of baking oven baking the affected part after applying some drugs 5 minutes, 3 times 1 minute folder tail, high speed level vibration in 3 times 30 minutes.Give a kind of stimulation every day at random.

Every kind stress operational approach:

(1) fasting: 24 hours fractures; (2) prohibit water: cut off the water supply in 24 hours; (3) illumination: animal put into during 7:00 morning and to put upside down case round the clock, not turning on light makes animal be in dark state; To put upside down the daylight lamp of case during to late 19:00 round the clock and open, and make animal be in the illumination state, take out until early 7 o'clock next day; (4) cold water swimming: animal is put into the bucket (depth of water 15cm) that fills 4 ℃ of cold water, and the back toe of rat just can touch drum head, after 5 minutes the animal taking-up is put back in the cage; (5) baking the affected part after applying some drugs: oven temperature transfer to 45 ℃ constant, animal is put into baking oven, after 5 minutes animal is taken out; (6) folder tail: rat is put into fixedly cage, expose afterbody, it is lasting after 1 minute to clamp apart from tail point 1cm place (firmly not excessive, rat is sent wail get final product) with mosquito forceps, rat is pulled out put back in the cage; (7) vibration: rat is put into agitator, and the vibration of high speed level was taken out after 30 minutes.

2.2 medication

In the time of modeling, rat gastric infusion every day.Normal group, model group are irritated the stomach distilled water, and the fluoxetine group is irritated stomach fluoxetine 2mg/kg, are equivalent to 10 times of 60kg body weight adult daily dose; The heavy dose of group of Bulbus Lilii extract is irritated stomach 48mg/kg, be equivalent to 20 times of 60kg body weight adult daily dose, the dosage group is irritated stomach 24mg/kg in the Bulbus Lilii extract, be equivalent to 10 times of 60kg body weight adult daily dose, the Bulbus Lilii extract small dose group is irritated stomach 12mg/kg, is equivalent to 5 times of 60kg body weight adult daily dose.

2.2 weight gain value

The 0th week, the 1st week, the 2nd week, the 3rd week take by weighing the body weight of respectively organizing rat respectively, are that index is added up with the rat body weight increasing value, i.e. weight gain value=each rat the 3rd all day body weight-Di 0 all body weight.

2.3 consumption appetite increasing value

The 0th week, the 1st week, the 2nd week, the 3rd week, respectively group was respectively at the jejunitas back of rat survey consumption on the 2nd appetite, and concrete grammar: every rat gives quantitative Mus grain, the weight of title residue Mus grain behind the 24h, consumption appetite=administered dose-surplus.

2.3 sucrose solution consumption experiment

2d measures rat sucrose diseases caused by retention of fluid consumption after the 0th week, the 1st week, the 2nd week, the 3rd week prohibiting water respectively at rat, measure the rat clear water amount of drinking the 3rd week simultaneously, concrete grammar is: allow each treated animal drink two bottles of different water arbitrarily: wherein one bottle is tap water for the tap water that contains 1% sucrose, one bottle.Test the amount of drinking (by claiming a drinking-water bottle weight) of 4:00PM to the second day 8:00 tap water and sucrose water, with sucrose partially degree of having a liking for (sucrose preference) (sucrose is degree of having a liking for (%)=sucrose diseases caused by retention of fluid consumption/(sucrose diseases caused by retention of fluid consumption+tap water amount of drinking) partially) as criterion, respectively organize the sucrose variation of degree of having a liking for (%) partially.

2.4 spacious case test (Open field)

22d, rat opens the case experiment.Used spacious case (self-control) is that high 40cm, length and width are 80cm, and the column body of interior sky, perisporium are black, and ground is divided into 25 that area equates with black line.Single rat is placed bright and clean spacious case central authorities, passing through ground square number as the horizontal anomalous movement score with animal, is vertical activity score with upright number of times, observes rat in the center lattice time of staying, and fecal grains simultaneously, every animal is only once measured, each 3 minutes.

3 results

3.1 the variation of weight gain value

Table 9-1 Bulbus Lilii extract is to the influence of rat model body weight change

^▲Compare with normal group: P＜0.05, ^{▲ ▲}Compare with normal group: P＜0.01;

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

Annotate: the g of body weight unit

From table 9-1 as can be seen, the model group rat body weight increasess slowly, and compares with normal group, and significant difference (P＜0.01) is arranged; It is slow that the Bulbus Lilii extract group can be improved the body weight gain of model group, and weight gain value is compared with model group apparently higher than model group, and utmost point significant difference (P＜0.01) is arranged; The positive drug fluoxetine also can increase the body weight gain of rat model, but not as Bulbus Lilii extract obvious, may be relevant with its side effect of gastrointestinal reaction.

Fig. 3 as can be seen, the normal rats body weight constantly increases, the 1st week of model group rat body weight, the 2nd week slowly increase, the 3rd all body weight descended to some extent than the 2nd week, illustrate the depression model rat normally rat body weight alleviate; Bulbus Lilii extract group rat body weight also constantly increases, but increasing degree is not as normal group; In addition, the 1st week of positive drug fluoxetine group rat body weight increases not obvious, and the 2nd week, the 3rd all body weight increase to some extent, prompting, and Bulbus Lilii extract can obviously improve the body weight gain of depression model rat, and is certain dose dependent.

3.2 the variation of consumption appetite increasing value

The influence that table 9-2 Bulbus Lilii extract changes rat model consumption appetite

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

Annotate: the consumption g of appetite unit

From table 9-2, Fig. 4 Bulbus Lilii extract rat model is consumed the influence curve figure that appetite changes; As can be seen, the big wastage of grain, etc. caused by rats appetite of model group increasess slowly, and compares with normal group, consumption appetite increasing value has significant difference, P＜0.01, and Bulbus Lilii extract can improve rat model consumption appetite and increases slowly, consumption appetite increasing value is compared with model group, utmost point significant difference is arranged, P＜0.01; The positive drug fluoxetine also can improve rat model consumption appetite and increases slowly, but effect is remarkable not as good as Bulbus Lilii extract.

In conjunction with the influence of Bulbus Lilii extract to the rat model body weight change, can draw, Bulbus Lilii extract can increase the consumption appetite of depression model rat, thereby increases the body weight of rat model; The positive drug fluoxetine increases consumption appetite not as good as Bulbus Lilii extract, thereby makes the also too late Bulbus Lilii extract of weight increase of depression model rat.

3.3 sucrose solution consumption experiment

The influence that table 9-3 Bulbus Lilii extract changes rat model sucrose solution consumption

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

Annotate: consume the sucrose solution unit/ml

Table 9-4 Bulbus Lilii extract is to the influence of degree of having a liking for partially of rat model sucrose solution

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

Annotate: consume the sucrose solution unit/ml

From table 9-3 as can be seen, since model group rat sucrose solution consumption reduced in the 2nd week, sucrose solution consumes increasing value and compares with normal group, utmost point significant difference is arranged, Bulbus Lilii extract has the effect that improves this symptom, but because the increasing value standard deviation is bigger, except that the Bulbus Lilii extract low dose, there was no significant difference as a result.

From table 9-4 sucrose solution partially degree of having a liking for as can be seen, the model group rat than the normal rats sucrose solution partially degree of having a liking for reduce, prompting, the model group rat has not had the pleasant sensation when drinking sucrose solution, and depressive symptom is arranged, Bulbus Lilii extract can obviously improve this symptom, and is certain dose-effect relationship.

3.4 spacious case experiment

Table 9-5 Bulbus Lilii extract is to the influence of the spacious case experiment of rat model

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

From table 9-5 as can be seen, in the spacious case experiment, the rat model horizontal movement, move both vertically, defecation grain number average reduces, the center lattice time of staying obviously prolongs, and compares with normal group, and utmost point significant difference is arranged, the Bulbus Lilii extract heavy dose can obviously increase rat model horizontal movement, move both vertically, defecation grain number, and can shorten the center lattice time of staying, and compare with model group, significant difference is arranged.

Test of the influence of two Bulbus Lilii extracts to rat model COR, ACTH

1 material

1.1 reagent

Chloral hydrate: Tianjin Kermel Chemical Reagent Co., Ltd., lot number: 20080118.

COR radioimmunological kit: Beijing North biotechnology research institute, lot number: 080520.

ACTH radioimmunological kit: Beijing North biotechnology research institute, lot number: 080520.

1.2 instrument

The SN-695B type is put and exempted from the γ measuring instrument: Shanghai day is encircled instrument one factory.

2 methods

2.1 the mensuration of change of serum C OR

Behind the rat anesthesia, the ventral aorta blood sampling, 2ml puts into the 4ml centrifuge tube that does not add anticoagulant, and the centrifugal 10min of 3000rpm 4c gets supernatant and moves into the 1.5ml centrifuge tube, and the content that the method for exempting from is measured rat blood serum COR is put in-20 ℃ of preservations.

2.2 plasma ACTH detects

Behind the rat anesthesia, the ventral aorta blood sampling, 3ml puts into and adds 10%EDTA-Na ²⁺30ul, in the 4ml centrifuge tube of aprotinin 30ul, the centrifugal 10min of 3000rpm 4c gets supernatant and moves in the 1.5ml centrifuge tube, and the content that the method for exempting from is measured rat plasma ACTH is put in-20 ℃ of preservations.

3 results

3.1 Bulbus Lilii extract is to the influence of rat model change of serum C OR

Table 9-6 Bulbus Lilii extract is to the influence of rat model COR

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

The result shows that model group rat blood serum COR level obviously increases, and compares with normal group, and significant difference is arranged, P＜0.01, and Bulbus Lilii extract can reduce the change of serum C OR level of rat model, compares with model group, and significant difference is arranged, P＜0.01; The positive drug fluoxetine also can reduce rat model change of serum C OR level.

3.2 Bulbus Lilii extract is to the influence of rat model plasma ACTH

Table 9-7 Bulbus Lilii extract is to the influence of rat model ACTH

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

The result shows that model group rat plasma ACTH level obviously increases, and compares with normal group, and significant difference is arranged, P＜0.01, and Bulbus Lilii extract can reduce the plasma ACTH level of rat model, compares with model group, and significant difference is arranged, P＜0.01; The positive drug fluoxetine also can reduce rat model plasma ACTH level.

Test the influence that three Bulbus Lilii extracts are expressed rat model CRF mRNA, GR mRNA, MR mRNA

1 material

1.1 reagent

TRNzol-A+ total RNA extraction reagent: TIANGEN BIOTECH COMPANY LIMITED, lot number: G5411.

DEPC:AMRESCO, lot number: 071210.

EB substituted dyes: Beijing Puli's lema gene technology company limited, lot number: D1210.

DNA Marker:TIAN GEN BIOTECH COMPANY LIMITED, lot number: G6207.

R Nasin:TIAN GEN BIOTECH COMPANY LIMITED, lot number: H6312.

5-First strand Buffer:TIAN GEN BIOTECH COMPANY LIMITED, lot number: H6121.

DTT:TIAN GEN BIOTECH COMPANYLIMITED, lot number: H6118.

Rnase-free dd H2O:TIAN GEN BIOTECH COMPANY LIMITED, lot number: G6206.

DNTP Mixture:TIAN GEN BIOTECH COMPANY LIMITED, lot number: G5723.

Transcript M MLV:TIAN GEN BIOTECH COMPANY LIMITED, lot number: H6227.

Oligo (dT): TIAN GEN BIOTECH COMPANY LIMITED, lot number: G5418.

2 Taq PCR Master Mix:TIAN GEN BIOTECH COMPANY LIMITED, lot number: H6331.

Tris-base:HOPE company, lot number: 080417.

Disodiumedetate: Tianjin Da Mao chemical reagent factory: lot number: 20080321.

1.2 instrument

01J2003-05 type vertical pressure steam sterilization tube: Shanghai Boxun Industrial Co., Ltd..

Thermostatic drying chamber: Shanghai Fuma Experiment Equipment Co., Ltd..

FM ventilated chamber: Pan American research laboratory, male city, Guangzhou equipment company limited.

BCD-215KAN DL type refrigerator: Qingdao HaiEr Co., Ltd.

LX-100 palm centrifuge: its woods Bel instrument Manufacturing Co., Ltd of Haimen City, Jiangsu.

High speed refrigerated centrifuge 1-15k:sigma company.

Christ RVC2-18 type blower fan: the sharp Science and Technology Ltd. of new east station of Guangzhou.

BS2002s electronic balance: Beijing Sai Duolisi instrument system company limited.

VORTEX-5 QILINBIER vortice: its woods Bel instrument Manufacturing Co., Ltd of Haimen City, Jiangsu.

AL1296 DNA Engine (PCR instrument): BIO-RAD company.

Gene Quant pro (nucleic acid determination instrument): Amersham bioscience.

Magnetic agitation instrument KT/C:IKA company.

Dry type thermostat K300: Hangzhou Lanyan Science and Technology Co., Ltd.

EPS301 electrophresis apparatus: Amersham bioscience.

Image capture system VTLBER LOURMAT0614503:Amersham bioscience.

Microwave oven PT021 TP-6:Galanz.

1.3 experiment utensil:

Liquid-transfering gun: 1ml, 200ul, 10ul; Suction nozzle: 1ml, 200ul, 20ul; The suction nozzle platform: place the 1ml suction nozzle one, place 200ul and 20ul suction first; EP manages 1.5ml, 100ul; Volumetric flask: 1000ml; Saline bottle: 100ml; 5ml centrifuge tube (joining 75% ethanol uses)

1.4 primer is synthetic: TaKaRa Biotechnology (DaLian) Co.Ltd.

2 methods

2.1 the processing and the preparation of experiment utensil

2.1.1 plastic: (comprising suction nozzle, EP pipe etc.) is soaked in the 1 ‰ DEPC water (the lancet head need be squeezed into DEPC water with suction pipe in case of necessity) 37 ℃ one by one with plastic and spends the night, deliver to high pressure then, back oven dry in 80 ℃ of baking boxes (or placing 37 ℃ of oven dry about 8 hours) is put into the suction nozzle platform with the rifle head before the test.

2.1.2 glass: (mainly being the glass grinding device) bubble acid is earlier spent the night, and after rinsing well, about 8 hours, 37 ℃ of oven dry are with covering tinfoil parcel, 240 ℃ of roasting 4h at 1 ‰ DEPC bubbly waters.

2.1.3 metallic article: (tweezers etc.) first wash clean, with covering tinfoil parcel, 240 ℃ of roasting 4h.(not needing to steep DEPC water)

2.2 the preparation of reagent and preparation

2.2.1DEPC water: the preparation of the DEPC water of bubble experiment utensil: add 1mlDEPC in the 1000ml distilled water, be placed on leave standstill in the 1000ml volumetric flask after 4 hours standby.The preparation of joining 75% alcoholic acid DEPC water: dress 40ml distilled water in the 100ml saline bottle, add 40ulDEPC, 37 ℃ are spent the night, deliver to high pressure.

2.2.275% ethanol (will when extracting, now join): with dehydrated alcohol and the preparation of DEPC water (DEPC water: dehydrated alcohol=1: 3), be put in then-20 ℃ standby.

2.3 total RNA extracts

2.3.1 rat cerebral tissue's (survey CRFmRNA and take off thalamus, survey GRMRmRNA and go Hippocampus) adds (100mg tissue/1ml Trizol) behind the Trizol, fully blows and beats so that the body thickness with the rifle head, in vortex mixer spiral 30s mixing; Placed 5 minutes, and made its abundant cracking for-20 ℃.

Add chloroform (pre-cooling) 2.3.2 press 200ul chloroform/mlTrizol, in vortex mixer spiral 30s mixing; Placed 15 minutes for-20 ℃.

2.3.34 the centrifugal 15min of ℃ 12000g.

2.3.4 draw the upper strata water, to the centrifuge tube of another pre-cooling.

2.3.5 add isopyknic isopropyl alcohol (pre-cooling) mixing, room temperature is placed 30min.

2.3.64 the centrifugal 10min of ℃ 12000g abandons supernatant, RNA is sunken to the pipe end.

2.3.7 add 75% ethanol 1ml (pre-cooling), gentle concussion centrifuge tube, precipitation suspends.

2.3.84 the centrifugal 5min of ℃ 12000g abandons supernatant as far as possible.

2.3.9 room temperature is dried or is dried up with blower fan.

2.3.10 with 30ul RNase-free H2O dissolving RNA sample.

2.3.11 the nucleic acid determination instrument is measured RNA concentration, the integrity of RNA degeneration electrophoresis detection RNA.

2.4 the mensuration of nucleic acid concentration

2.4.1 draw 5ul RNA sample, add depc water to the 100ul mixing, change in the quartz cuvette.

2.4.2 earlier with 100ulDEPC water suppressed zero.

2.4.3 sample determination, the sample of purity between 1.5-1.9 of choosing RNA carries out reversion rate, i.e. λ 260nm/ λ 280nm=1.5-1.9.

2.4.4RNA degeneration electrophoresis 5S band mays be seen indistinctly, 28S and 18S band are bright, prove the integrity that extracts total RNA, and do not see that genomic DNA pollutes.Can be used for the RT-PCR experiment.

2.5RNA reverse transcription

2.5.1 in the PCR pipe of the nuclease free of ice bath, add following reactant mixture:

The total RNA of 1-5ug;

2uloligo(dT)；

2uldNTP；

Mend Nasefree ddH2O and be settled to 13.5ul.

2.5.2 simple centrifugal rapidly at cooled on ice 2min, adds following each component behind 70 ℃ of heating 5min:

4ul5-First?strand?Bufier；

1ul0.1MDTT；

0.5ulRNasin；

1ulTRANScript?M-MLV。

2.5.342 a ℃ temperature is bathed 45min.

2.5.495 ℃ heating 5min cessation reaction.

2.5.5 with Nasefree ddH2O reaction system is diluted to 50ul, gets 2-5ul and carry out pcr amplification reaction.

2.6 design of primers, synthetic

2.6.1 primer sequence: provide the gene nucleic acid sequence according to U.S. gene biological storehouse, with PRIMER 5.0 software design primers, choose the no hairpin structure of G+C content about 50, do not have two sections specific and conserved sequence of homologous sequence each other, the design primer is as follows:

Beta-actin-FP：5-GAG?ACC?TTC?AAC?ACC?CCA?GCC-3

Beta-actin-RP:5-AAT GTC ACG CAC GAT TTC CC-3 amplified fragments is 263bp.

CRF-FP：5-CAG?AAC?AAC?AGT?GCG?GGC?TCA-3

CRF-RP:5-GGAAAAAGT TAG CCG CAG CCT-3 amplified fragments is 365bp.

GR-FP：5-AGAAAC?TTA?CAC?CTG?GAT?GAC-3

GR-RP:5-TGG AGT TCC CTT CCC TTT T-3 amplified fragments is 373bp.

MR-FP：5-TTC?TTC?CCG?CTT?CCA?TCC?G-3

MR-RP:5-GGG TCT CCA CGC CAT TCT G-3 amplified fragments is 362bp.

2.6.2 primer dilution:

At first with dd H2O primer is diluted to the preservation liquid of 100uM, redilution is to experimental concentration 10uM before using, and concrete grammar is: the use amount V of dd H2O (μ l)=nmol counts * 10

2.7PCR amplified reaction

2.7.1 in the PCR pipe of the nuclease free of ice bath, add following reactant mixture:

Forward primer: 1ul

Downstream primer: 1ul

2*Taq enzyme reaction system: 12.5ul

DNA product: 2-5ul

Nase?free?dd?H2O：5.5-8.5ul

The volume that finally makes reaction system is 25ul.

2.7.2 increase on the PCR instrument, amplification condition is as follows:

2.8 electrophoretic analysis:

2.8.1 the preparation of electrophoretic buffer (50*TAE)

2.8.2Loading the preparation of Buffer

It is an amount of to take by weighing agarose gel, is mixed with 1.5% solution with TAE.

2.8.4,, repeat 2-3 time to the gel boiling with microwave oven heating agarose gel solution.

2.8.5 take out, room temperature is put cold, adds an amount of EB replacement liquid, shakes up.

2.8.9 water plate, put coldly, solidify to gel.

2.8.10 point sample carries out electrophoresis in electrophoresis tank, condition: 120v, 100mA, electrophoresis 40min.

2.8.11 with gel images analyser images acquired.

2.8.12 graphical analysis: ImageMasterTMTotalLab Software software analysis gray value, between organizing with the gray level ratio of genes of interest and B-actin relatively.

3 results

3.1 the extraction of total RNA

The extraction result shows that nucleic acid purity 260/280 major part is all between 1.5-2.0.

RNA degeneration electrophoresis result: the 5s band is clear, and 18s, 28s band are all high-visible.

3.2 respectively organize the gray level ratio result

3.2.1 the influence that Bulbus Lilii extract is expressed CRFmRNA

The influence that table 9-8 Bulbus Lilii extract is expressed rat model CRFmRNA

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

The result shows that model group rat CRFmRNA expresses and increases, and compares with normal group, and utmost point significant difference is arranged, and Bulbus Lilii extract can reduce the expression of CRFmRNA, compares with model group, and significant difference is arranged, the expression that fluoxetine also can less CRFmRNA.

3.2.2 the influence that Bulbus Lilii extract is expressed GR, MRmRNA

The influence that table 9-9 Bulbus Lilii extract is expressed rat model GR, MRmRNA

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

The result shows that model group rat GR, MRmRNA express reduction, compare with normal group, and utmost point significant difference is arranged, and Bulbus Lilii extract can increase the expression of GR, MRmRNA, with model group significant difference is arranged relatively, and fluoxetine also can increase the expression of GR, MRmRNA.

Test of the influence of four Bulbus Lilii extracts to the rat model monoamine neurotransmitter

1 material

1.1 reagent

5-HT:sigma company, lot number: 046k7038.

5-HIAA:sigma company, lot number: 046k7059.

Dopamine hydrochloride: the brilliant pure reagent company limited in Shanghai, lot number: 200803051.

L-norepinephrine: the brilliant pure reagent company limited in Shanghai, lot number: 200805172.

1-sodium heptanesulfonate: the brilliant pure reagent company limited in Shanghai, lot number: 2008050125.

3,4-dihydroxy Bian amine: the brilliant pure reagent company limited in Shanghai, lot number: 200805078.

Disodiumedetate: Tianjin Da Mao chemical reagent factory, lot number: 20080321.

Citric acid: Tianjin Da Mao chemical reagent factory, lot number: 20080411.

Sodium hydrogen phosphate: Tianjin Da Mao chemical reagent factory, lot number: 20080411.

1.2 instrument

Esa C4-926 electrochemical detector: ESA Bioscience Lnc.

2 methods

2.1 the preparation of solution

2.1.1 the preparation of standard solution: precision takes by weighing NE, DA, 5-HT, the 5-HIAA standard substance are an amount of, dissolve with 0.1mol/l HC1, be made into the storing solution that concentration is 1g/L respectively, face with preceding with 0.1mol/L HC1 diluted concentration be 0.019,0.038,0.065,0.13,0.65, the series standard liquid of 1.25ul/mL.

2.1.2 the preparation of albumen precipitation liquid: get perchloric acid solution and 0.01mol/L EDTA solution is an amount of, dilute with water is the albumen precipitation liquid that contains perchloric acid 0.1mol/L and EDTA 0.1mmol/L.

2.1.3 the preparation of interior mark working solution: it is an amount of that precision takes by weighing DHBA, is made into the interior mark working solution that concentration is 3.9mg/L with the dissolving of albumen precipitation liquid.

2.2 the processing of sample

Get rat cerebral cortex, add the interior mark working solution of 1000ul albumen precipitation liquid and 50ul, homogenate under the ice bath, homogenate is in 15000r/min low-temperature centrifugation 30min, and supernatant is got supernatant in 15000r/min low-temperature centrifugation 25min once more, 0.22um membrane filtration, sample detection.

2.3 the chromatographic condition chromatographic column be Kromasil C18 reversed-phase column (150mm * 4.6mm, 5um); Mobile phase is 75mmol/L Na ₂HPO4: 150mmoL/L citric acid: 0.1mol/LEDTA: 150mmol/L B7: methanol: water: 80: 40: 2: 7: 32: 89, before using through 0.45u m filtering with microporous membrane 2 times, and ultrasonic degas 20min; Flow velocity is 1.0ml/min; Column temperature: room temperature.Electrochemical Detection is the glass working electrode, the Ag/AgCl reference electrode, and running voltage is+0.25V.Sample size: 15ul.

2.4 the drafting of standard curve

The interior mark work that each concentration standard liquid 1ml of NE, DA, 5-HT, 5-HIAA is added 50ul respectively also, 0.22um membrane filtration, sample introduction 15ul, ratio with the peak area of each standard substance and interior mark peak area is a vertical coordinate, the concentration of each standard substance is abscissa, carry out linear regression, the result is as table 9-10.

Regression equation, correlation coefficient, the range of linearity of table 9-10 monoamine neurotransmitter

2.5 precision experiment

With DA is example, continuous sample introduction 5 times, and with the ratio calculation RSD of DA peak area and interior mark peak area, the result, RSD is 2.16%,＜3%, illustrates that instrument precision is good.

2.6 extraction recovery experiment

Take by weighing an amount of cerebral cortex, mark in adding adds the quantitative standards product, presses sample treatment and handles, as molecule, take by weighing an amount of cerebral cortex, mark in adding is pressed sample treatment and is handled, the supernatant that obtains adds the standard substance of equivalent, as denominator, calculate extraction recovery, the results are shown in Table 9-11.

The experiment of table 9-11 extraction recovery

2.7 repeated experiment

Get the cerebral cortex of same rat, be divided into 3 parts, press sample treatment and handle, carry out repeated experiment, the result shows, RSd DA=2.56%, NE=1.63%, 5-HIAA=2.28%, 5-HT=3.27% all＜5.0%, illustrates that this method repeatability is good.

2.8 stability experiment

Get the sample of having handled well, respectively 0,12h, 24h measure, and the results are shown in Table 9-12.

Table 9-12 neurotransmitter stability experiment (unit: ng/ml/g)

From table 9-12 as can be seen, neurotransmitter is made behind the sample unstable, is preferably in 12h and has surveyed.

2.9 sample determination

Table 9-13 respectively organizes neurotransmitter measurement result (unit: ng/ml/g)

^*Compare with model group: P＜0.05, ^*Compare with model group: P＜0.01.

The result shows, model group rat cerebral cortex DA, NE, 5-HT reduce, prompting patients with depression monoamine neurotransmitter insufficiency of function, 5-HIAA increases, prompting 5-HT metabolism becomes 5-HIAA, and Bulbus Lilii extract can improve the content of DA, NE, 5-HT, compares with model group, significant difference is arranged, and the positive drug fluoxetine also can improve the content of DA, NE, 5-HT.

3.2.1 maze experiment

3.2.2 outstanding tail experiment

3.2.3 Open-filed performance testing

3.2.4 sucrose water consumption experiment

3.2.5 detect following index:

3.2.5.1 influence: the mensuration of monoamine transmitters total amount in the brain to monoamine neurotransmitter in the brain

High performance liquid chromatography-electrochemical method (HPLC-ECD) is measured mediator and the interior 5-HT of metabolite brain thereof in rat brain prefrontal cortex and the Hippocampus, 5-HIAA, the content of NE and MAO.

3.2.5.2 influence to hpa axis

3.2.5.2.1 the mensuration of corticotropin releasing hormone (CRF) mRNA

(1) takes out rapidly full brain after the rat sacrificed by decapitation,, hypothalamus and the cortex of every group of rat mixed, extract cortex and the total RNA of hypothalamus, carry out the reverse transcriptase polymerase connection and react (RT-PCR) with the Trizol test kit at selective separating and hypothalamus rapidly on ice.

(2) extraction of total RNA

(3) RNA purity and integrity detection

(4) design of primers, synthetic (cDNA PCR)

(5) RNA reverse transcription

3.2.5.2.2 the mensuration (putting the method for exempting from) of blood plasma thyroliberin ACTH

3.2.5.2.3 the mensuration of blood plasma cortisol (putting the method for exempting from)

3.2.5.2.4 the mensuration of glucocorticoid receptor (GR) (GR) and mineralcorticoid receptor (RT-PCR method)

Above content is to further describing that the present invention did in conjunction with concrete embodiment, can not assert that concrete enforcement of the present invention is confined to these explanations, for the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, its embodiment can be flexible and changeable, can derive from many series methods.Just make some simple deduction or replace, all should be considered as belonging to the scope of patent protection that the present invention is determined by claims of being submitted to.

Claims

1, a kind of method with preparing lilium total saponin extract by using macroporous resin is characterized in that:

(2) with the Bulbus Lilii crude extract by being filled with the chromatographic column of macroporous resin, use the eluant eluting, collect the eluting part, reclaim eluant, collect the gained Bulbus Lilii extract, promptly.

2, according to claims 1 described a kind of method with preparing lilium total saponin extract by using macroporous resin, it is characterized in that: described macroporous resin is a polystyrene resin type macroporous resin.

3, according to the described a kind of method with preparing lilium total saponin extract by using macroporous resin of claims 1, it is characterized in that: the model of described macroporous resin is one or more the mixture among D101, SA40, HP20, SP825, XAD16, XDA1, XDA5, LX18, AB8, the LSA7;

4, according to claims 1 described a kind of method with preparing lilium total saponin extract by using macroporous resin, it is characterized in that: used eluant is a polar solvent.

5 methods according to claims 1 described a kind of preparing lilium total saponin extract by using macroporous resin is characterized in that: used eluant is water and 5～20BV ethanol.

6, according to the method for claims 1 described a kind of preparing lilium total saponin extract by using macroporous resin, it is characterized in that: the crude extract that contains described Bulbus Lilii total saponins is prepared from by following method: get the Bulbus Lilii medical material, with 70～90 ℃ of extractions of 6～10 times of amount 65%～95% ethanol, concentrate or reclaim ethanol, concentrated solution or continue dry cream powder promptly.

7, according to the method for claims 1 described a kind of preparing lilium total saponin extract by using macroporous resin, it is characterized in that: with the Bulbus Lilii crude extract by being filled with the chromatographic column of macroporous resin, the load sample weight ratio is counted with the portions of resin crude drug: 0.1: 1～2: 1, last sample concentration is with crude drug: the solution weight volume ratio counts 0.01: 1～and 1: 1, flow velocity is 1～10ml/min, with distilled water and ethanol elution, collect the ethanol elution part, reclaim ethanol, collect the gained Bulbus Lilii extract, promptly.

8, a kind of purposes of preparing lilium total saponin extract by using macroporous resin is characterized in that: lilium total saponin extract is used to prepare the medicine for the treatment of depression.