CN103405586B - Lemna minor total flavonoid extract and preparation method thereof - Google Patents
Lemna minor total flavonoid extract and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of Lemna minor total flavonoid extract, it is prepared from according to following step: 1) get Herba Spirodelae grass herb powder, add the ethanol of 8 ~ 15 times 20 ~ 90%, heating and refluxing extraction 1 ~ 3 time, each 0.5 ~ 2 hour, merge extractive liquid; 2) extracting solution is evaporated to without alcohol taste, pass through macroporous adsorbent resin, first be eluted to effluent with the alcoholic solution that water or concentration are no more than 20% transparent, then be alcoholic solution eluting 4 ~ 8 times of column volumes of 40 ~ 90% by concentration, collect the ethanol elution of 40 ~ 90%, concentrated, dry, containing mass percent in described Lemna minor total flavonoid extract is the total flavones of 50 ~ 99%.Extract of the present invention has antiallergic action, detumescence, antipruritic effect.
Description
Technical field
The invention belongs to pharmaceutical technology sectors, be specifically related to a kind of Lemna minor total flavonoid extract and preparation method thereof.
Background technology
Herba Spirodelae grass is plant and of its applications duckweed [spirodela polyrhiza (L.) Schleid.] or Herba Spirodelae (Lemnaminor L.), also cries (" Shengji Zonglu ") such as Herba Spirodelae, duckweed grass, fountain moss, cascade, nine sub-duckweeds, Tian Ping.Be widely distributed in size lake, each provinces and regions, China north and south, especially all produce in the south four seasons, agriculturally with its water purification, feeding fish and poultry, thus it has wide material sources, the feature that output is large.Herba Spirodelae grass has very high medical value, from ancient times to the present has a large amount of research and apply, its acrid in the mouth, cold in nature, energy relieving the exterior syndrome by diaphoresis, rash is antipruritic, inducing diuresis to remove edema, heat-clearing and toxic substances removing, can be used for the diseases such as wind heat exterior syndrome, measles without adequate eruption, urticaria pruritus, the edema difficulty in urination, skin ulcer tinea erysipelas and scald.
Summary of the invention
The object of this invention is to provide a kind of Lemna minor total flavonoid extract and preparation method thereof.
Lemna minor total flavonoid extract, it is prepared from according to following step:
1) get Herba Spirodelae grass herb powder, adding 8 ~ 15 times of concentration is by weight the alcoholic solution of 20 ~ 90%, heating and refluxing extraction 1 ~ 3 time, each 0.5 ~ 2 hour, merge extractive liquid;
2) extracting solution is evaporated to without alcohol taste, by macroporous adsorbent resin, is first eluted to effluent with the alcoholic solution that water or concentration are no more than 20% transparent, then be alcoholic solution eluting 4 ~ 8 times of column volumes of 40 ~ 90% by concentration, collect the ethanol elution of 40 ~ 90%, concentrated, dry
Containing percentage by weight in described Lemna minor total flavonoid extract is the total flavones of 50 ~ 99%.
The preparation method of Lemna minor total flavonoid extract, is characterized in that comprising the following steps:
1) get Herba Spirodelae grass herb powder, adding 8 ~ 15 times of concentration is by weight the alcoholic solution of 20 ~ 90%, heating and refluxing extraction 1 ~ 3 time, each 0.5 ~ 2 hour, merge extractive liquid;
2) extracting solution is evaporated to without alcohol taste, by macroporous adsorbent resin, is first eluted to effluent with the alcoholic solution that water or concentration are no more than 20% transparent, then be alcoholic solution eluting 4 ~ 8 times of column volumes of 40 ~ 90% by concentration, collect the ethanol elution of 40 ~ 90%, concentrated, dry.
Preferably, the preparation method of described Lemna minor total flavonoid extract comprises the following steps:
1) get Herba Spirodelae grass herb powder, adding 12 times of concentration is by weight the alcoholic solution of 70%, heating and refluxing extraction 2 times, each 1 hour, merge extractive liquid;
2) being evaporated to by extracting solution without alcohol taste, by macroporous adsorbent resin, first washing with water to effluent transparent, is then alcoholic solution eluting 5 times of column volumes of 70% by concentration, collects the ethanol elution of 70%, concentrated, dry.
Preferred further, described macroporous adsorbent resin is D101 type macroporous adsorbent resin.
The weight concentration that said in the present invention " concentration " all refers to, as " alcoholic solution of 70% concentration " refers in every 100g solution containing ethanol 70g.
Extract of the present invention has antiallergic action, detumescence, antipruritic effect.Acute inflammation for the swelling of mice caused by dimethylbenzene xylene ear has obvious inhibitory action, Cavia porcellus pruritus is caused to histamine hydrochloride and has good inhibitory action, DNFB can be suppressed to cause the ear swelling of the subacute contact eczema of mice, can also significantly lower IL-4 level, thus suppress Th
2function, illustrate that the present invention can suppress delayed allergy.
Accompanying drawing explanation
Fig. 1 is the influence curve figure of different concentration ethanol to extraction effect;
Fig. 2 is that different concentration ethanol is to flavone compound elute effect curve chart;
Fig. 3 is the impact of eluting agent on general flavone content.
Detailed description of the invention
Embodiment 1
The preparation method of Lemna minor total flavonoid extract, is prepared from according to following steps:
1) get Herba Spirodelae grass herb powder, adding 12 times of concentration is by weight the alcoholic solution of 70%, heating and refluxing extraction 2 times, each 1 hour, merge extractive liquid;
2) being evaporated to by extracting solution without alcohol taste, by macroporous adsorbent resin, first washing with water to effluent transparent, is then alcoholic solution eluting 5 times of column volumes of 70% by concentration, collects the ethanol elution of 70%, concentrated, dry.
The content assaying method of total flavones: adopt ultraviolet spectrophotometry, take rutin as standard substance, absorbance is measured at 505nm wavelength place, with rutin concentration and absorbance drawing standard working curve, testing sample is used 80% methanol extraction, then absorbance is measured, by the content of total flavones in regression equation calculation testing sample.
After testing, the total flavones of 71.2% is contained in said extracted thing.
Embodiment 2 technical study
1) concentration of alcohol impact that total flavones is extracted: take 6 parts, Herba Spirodelae grass herb powder, every part of 20g, respectively with 10%, 30%, 50%, 70%, the alcohol reflux of 80%, 95%, by the liquid ratio of 12 times amount, extract twice, each 1 hour, merge extracted twice liquid, concentrate to obtain extractum, preparation sample solution also measures its absorbance, is gone out the content of total flavones in test sample by regression equation calculation.
Result shows, and different concentration ethanol on the impact of extraction effect as shown in Figure 1.Experimental result shows, the extraction effect of 20 ~ 90% ethanol is better, and the extraction effect of 70% ethanol is best, backward along with the increasing of concentration of alcohol, the flavones content extracted reduces fast, is likely the increase along with concentration of alcohol, the amount of the stripping of some oil-soluble impurities increases, and interference factor increases.
2) selection of resin types
The pretreatment of resin: get macroporous adsorbent resin D101, D4020 and AB-8 of newly purchasing respectively appropriate, adds appropriate purified water and soaks 24h, 2mol/L soak with hydrochloric acid 12h, then be washed till neutrality by purified water in beaker.2mol/L sodium hydroxide solution soaks 12h, and purified water is washed till neutrality.Finally use soaked in absolute ethyl alcohol 24h, upper prop, purified water is cleaned, until effluent and water mixing are without white opacity, then is washed till without alcohol taste, for subsequent use.
Staticadsorption experiment: accurately take each 1g of wet resin D101, D4020 and AB-8 handled well, be placed in 3 100mL conical flasks, accurately add sample liquid 50mL, fully vibrate in water bath chader 20h, filters, and surveys flavones content in filtrate, calculates adsorbance.The general flavone content of the rear filtrate of the general flavone content-absorption of sample liquid before resin absorption amount=absorption.As shown in Table 1, in 3 kinds of model resins, the adsorbance of D101 macroporous adsorbent resin is maximum.
Table 13 kind of resin is to flavone compound static adsorbance in Herba Hyperici perforati
| Resin model | Filter liquor concentration (Mg/ml) | Static adsorbance (Mg/g) |
| D101 | 3.682 | 85.165 |
| D4020 | 4.251 | 72.514 |
| AB-8 | 3.985 | 76.533 |
Static desorption experiment: get each 1g of different model resin after fully adsorbing Herba Spirodelae grass total flavones, put 12 100mL conical flasks respectively, numbering, respectively add 20%, 40%, 60%, 80% ethanol 50mL, fully vibrate in water bath chader, survey stripping liquid general flavone content, calculate desorption efficiency.Adsorbance × 100% of the content/resin of total flavones in filtrate after resin desorption rate (%)=desorbing.In table 2.As shown in Table 2, the desorption efficiency of 20%, 40%, 60%, 80% alcoholic solution is all that D101 macroporous adsorbent resin is higher, and maximum with 40% ethanol elution desorption efficiency in D101 macroporous adsorbent resin.
Table 23 kind of resin is to Herba Hyperici perforati flavone compound desorption efficiency
3) different concentration ethanol is to the investigation of flavone compound elute effect: by sample liquid by being equipped with the chromatographic column of D101 macroporous adsorbent resin, after absorption, use 20% respectively, 40%, 60%, 70%, 80%, 90% ethanol elution, the every 100mL of eluent collects once, to the eluent numbering of collecting, (1 ~ 3 is 20% ethanol elution, 4 ~ 6 is 40% ethanol elution, 7 ~ 9 is 60% ethanol elution, 10 ~ 12 is 70% ethanol elution, 13 ~ 15 is 80% ethanol elution, 16 ~ 18 is 90% ethanol elution), calculate general flavone content in eluent respectively, draw elution curve.The results are shown in Figure 2.When different concentration ethanol is to the resin elution adsorbed after flavone compound, flavone compound mainly concentrates on 70% ethanol elution, accounts for 76.6% of all resins absorption flavone compound, therefore the best concentration of alcohol of eluent is 70%.
4) eluting agent is investigated: by sample liquid by being equipped with the chromatographic column of D101 macroporous adsorbent resin, after absorption, use 70% ethanol elution, the every 50mL of eluent collects once, to the eluent numbering of collecting, calculate flavonoid content in eluent respectively, draw elution curve.The results are shown in Figure 3.Adopt concentration to be 70% ethanol elution, when eluant is 5 times of column volumes, eluting flavones content is maximum, and in eluant, flavones content sharply reduces afterwards.Therefore determine that eluant dosage is 5 times of column volumes.
Embodiment 3 drug efficacy study
Eczema is clinical common allergic inflammation dermatoses, various with erythra, violent pruritus, recurrent exerbation, easily develops into chronic to be feature, to be one of clinical common, multiple dermatosis, to account for 15% ~ 30% of Dermatology Outpatient Department amount.Pathogenesis, a kind of delayed allergy that eczema is mainly caused by motivating factor inside and outside complexity.Invention has been the pharmacodynamic study of the aspects such as antiinflammatory, antipruritic, antiallergic action, inquire into the pharmacological action that it prevents and treats eczema.
1, itching-relieving action
Get Cavia porcellus 50, latter 24 hours of the right back foot depilation of animal, with instep scratch after sand paper, with a small amount of oozing of blood for degree, then animal is divided into 5 groups at random, respectively at its abrasion even spread extract 0.5g/kg, 1g/kg, 2g/kg, PIYANPING 2g/kg or substrate 2g/kg.0.01%, 0.02%, 0.03% is dripped successively at its abrasion after 30min ... 1% hydrochloric acid histamine 0.05ml/ only, each interval time is 3min, record each animal to occur licking Histamine concentrations when foot reacts, this concentration is itch-threshold, and result t checks (see table 3).
Table 3 causes Cavia porcellus foot pruritus to hydrochloric acid histamine and to itch the impact (X ± s) of threshold
* P<0.05 is compared with matched group.
As seen from Table 3, extract of the present invention can significantly improve the itch-threshold of hydrochloric acid histamine to Cavia porcellus, and in a certain amount of effect relationship.
2, xylol causes the impact of mice ear
Get mice 30, be divided into 5 groups at random: blank group, positive controls and high, medium and low dose drug group, often organize 6.Each group of mouse right ear is coated with and gives caused by dimethylbenzene xylene inflammation, every 0.05mL.After 30min, basic, normal, high dose drug group respectively auris dextra even spread extract 0.5,1.0,2.0g/kg, positive controls gives QUMIXIN emulsifiable paste 2.0g/kg, and blank group gives bare substrate 2.0g/kg.After coating 30min, each administration group washes away medicinal liquid with purified water, clean for cotton balls, put to death mice, cut left and right ear respectively, left ear compares, ears cut with position homalographic by 8mm card punch, then the weighed quality of difference on BS2202S type electronic balance, with the difference of left and right auricle quality for swelling degree, calculates each group of swelling.
The results are shown in Table 4.Compare with blank group, low-dose drugs group no significant difference, and height, middle dose drug group difference have statistical significance (P<0.01 or P<0.05).Point out xylol of the present invention to cause mice auricle swelling acute inflammation and have obvious inhibitory action.
Auricle edema situation caused by table 45 group mice dimethylbenzene
3, on the impact of the mice delayed hypersensitivity of DNFB induction
Get mice 30, by each group of mouse web portion depilation after grouping, area 3cm × 3cm, depilation district's administration 1 time, in every Mus dorsal sc injection 7%2,4-dinitrochlorobenzene acetone soln 0.02mL after 24h, 3 days repeat sensitization 1 time afterwards.Respectively organize morning and evening every day each coating 1 time subsequently, continuous 8 days.9th day, evenly be applied to mouse right ear with 1%2,4-dinitrochlorobenzene acetone soln 0.03mL, after 24h, adopt eyeball excise method to get blood each group of 6 mice, get serum after whole blood is centrifugal, adopt euzymelinked immunosorbent assay (ELISA) (ELISA) to measure the content of IL-4 in serum.Put to death mice, take off left and right auricle with card punch (diameter 8mm), electronic balance claims quality, with the difference of two ear quality for swelling, calculates each group of swelling.
The results are shown in Table 5.Compare with blank group, high, medium and low dose drug group can improve mouse right ear swelling situation, difference has statistical significance (P<0.05), extract of the present invention brings out mouse allelgic contact dermatitis to DNFB certain treatment and inhibitory action; IL-4 assay result shows, high, the middle dosage of the present invention can reduce IL4 level in serum, and with positive controls comparing difference without significance.
A situation arises for the delayed allergy of table 55 group mice
This research confirms that Lemna minor total flavonoid extract has antiallergic action, detumescence, antipruritic effect.Acute inflammation for the swelling of mice caused by dimethylbenzene xylene ear has obvious inhibitory action, is thus applicable to eczematous dermatitis.Cavia porcellus pruritus is caused to histamine hydrochloride and has good inhibitory action, infer that its itching-relieving efficacies may be relevant with the release reducing over sensitivity mediator.
The pathogenesis of eczema is complicated, thinks a kind of delayed hypersensitivity that the T cell caused due to inside and outside motivating factor mediates at present more.This result of study shows, and Lemna minor total flavonoid extract causes on the subacute contact eczema model of mice at DNFB, has similar inhibitory action, can not only suppress ear swelling with QUMIXIN emulsifiable paste, can also significantly lower IL-4 level, thus suppresses Th
2function, illustrate that the present invention can suppress delayed allergy.
Claims (1)
1. treat a preparation method for the Lemna minor total flavonoid extract of eczema, it is characterized in that comprising the following steps:
1) get Herba Spirodelae grass herb powder, adding 12 times of concentration is by weight the alcoholic solution of 70%, heating and refluxing extraction 2 times, each 1 hour, merge extractive liquid;
2) being evaporated to by extracting solution without alcohol taste, by D101 type macroporous adsorbent resin, first washing with water to effluent transparent, is then alcoholic solution eluting 5 times of column volumes of 70% by concentration, collects the ethanol elution of 70%, concentrated, dry.
Priority Applications (1)
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| CN104127845A (en) * | 2014-07-25 | 2014-11-05 | 河南科技大学第一附属医院 | Preparation method for external medicine used for treating verruca plana |
| CN108379546A (en) * | 2018-05-29 | 2018-08-10 | 广州聚澜健康产业研究院有限公司 | A kind of privates washing lotion and preparation method thereof |
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| CN101011545A (en) * | 2007-02-09 | 2007-08-08 | 华中科技大学 | Method for extracting effective sites group of smilax China root |
| CN101810812A (en) * | 2010-03-11 | 2010-08-25 | 闫明 | Use of rhizoma galangae effect part for preparing medicament for treating leucoderma |
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| CN101011545A (en) * | 2007-02-09 | 2007-08-08 | 华中科技大学 | Method for extracting effective sites group of smilax China root |
| CN101810812A (en) * | 2010-03-11 | 2010-08-25 | 闫明 | Use of rhizoma galangae effect part for preparing medicament for treating leucoderma |
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