CN104758340A - Caffeoylquinic acid extract in Lonicera japonica Thunb leaf, preparation method and application thereof - Google Patents
Caffeoylquinic acid extract in Lonicera japonica Thunb leaf, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method for a caffeoylquinic acid extract in Lonicera japonica Thunb leaf. The method includes the steps of: (1) taking the Lonicera japonica Thunb leaf, using a 8-10 times amount of ethanol solution to conduct reflux extraction, performing filtration and combining the filtrate, carrying out vacuum concentration to a relative density of 1.0-1.1 so as to obtain an originally extracted liquid; (2) adding the originally extracted liquid into distilled water of the same volume to prepare a suspension liquid, extracting the suspension liquid with n-butanol, and performing vacuum concentration on the n-butanol phase to a relative density of 1.2-1.3, thus obtaining an n-butanol extract; (3) employing a polyamide filler to conduct enrichment and separation on the n-butanol extract, and adopting ethanol and water as the elution solvent, with the volume ratio of ethanol to water being 10:90-20:80; and (4) combining the eluted solutions in step (3), and performing concentration to obtain an extract, i.e. the caffeoylquinic acid extract. In the extract prepared by the method provided by the invention, the weight percent content of caffeoylquinic acid in the extract is 65-76%. The caffeoylquinic acid extract provided by the invention has strong anti-Newcastle disease virus activity, and has potential development and application value.
Description
Technical field
The present invention relates to coffee mesitoyl quinine acid extract and its production and use in a kind of Folium Lonicerae, belong to natural product chemistry technical field.
Background technology
Radix Ophiopogonis (Lonicera japonica Thunb.) is Caprifoliaceae (Caprifoliaceae) Lonicera (Lonicera) perennial evergreen winding liana, be China's Chinese medicine, there is the multiple medicinal efficacy such as heat-clearing and toxic substances removing, anti-inflammation.Radix Ophiopogonis, medicinal part experienced by the transition course of stem and leaf-stem and leaf flower-flower in history.At present, Radix Ophiopogonis Different plant parts make a distinction in " pharmacopeia ", Flos Lonicerae (Jinyinhua, Lonicera japonica flos), this product is the flower that the dry flower of caprifoliaceae plant Radix Ophiopogonis Lonicera japonicaThunb. or band are just opened.Early summer the flowers are in blossom put before gather, dry ".Caulis Lonicerae (Rendongteng, Lonicerajaponica caulis), this product is the dry stem branch of caprifoliaceae plant Radix Ophiopogonis Lonicera japonica Thunb..Autumn, season in winter two tap, and dry ".Flos Lonicerae is because of its limits throughput, and often supply falls short of demand.And Folium Lonicerae and flower homophyletic, in order to ensure the normal growth of alabastrum, Radix Ophiopogonis needs repeatedly trimming and finishing, leaf in a large number can be obtained every year, and these Folium Lonicerae are Radix Ophiopogonis traditional discarded part, be regarded as dis-medicinal part always and utilized for a long time, wasting phenomenon is very serious.
Newcastle (Newcastle disease, ND) be by Avian pneumo-encephalitis virus (Newcastle disease virus, the one of the main birds such as infringement chicken and turkey etc. NDV) caused is acute, high degree in contact sexually transmitted disease, is classified as the maximum category-A infectious disease of animal injury by World Organization for Animal Health.Although take intensive preventive measure domestic to ND, lack effectively preventing medicine, its sickness rate remains high always, is still one of Infectious Diseases affecting poultry husbandry sound development at present.
At present, home and abroad about the medicinal report of Radix Ophiopogonis and extract thereof mainly in AIDS preventing and controlling, anticancer, antiinflammatory and pain relieving etc., there are no about coffee mesitoyl quinine acid extract in Folium Lonicerae for preventing and treating the report of Avian pneumo-encephalitis virus.In view of the generation of current Avian pneumo-encephalitis virus disease and epidemic rate have exceeded the development speed of vaccine, and the antiviral drugs of forbidding the mankind being used of Ministry of Agriculture's dispatch in 2012, as amantadine, rimantadine, acyclovir, ribavirin etc. are applied in livestock medicine, to ensure livestock products safety; Therefore, natural plant extracts is developed to the medicine being suitable for Avian pneumo-encephalitis virus control imperative.
Summary of the invention
For the deficiency that above-mentioned prior art exists, the object of this invention is to provide coffee mesitoyl quinine acid extract and preparation method thereof in a kind of Folium Lonicerae.
Another object of the present invention is to provide this caffeic acid quininic acid extract and is preparing the application in anti-new castle disease virus medicine.
For achieving the above object, the present invention adopts following technical proposals:
Coffee mesitoyl quinine acid extract in a kind of Folium Lonicerae, in described extract, the weight percentage of caffeoylquinic acids is 65-76%.
The preparation method of coffee mesitoyl quinine acid extract in Folium Lonicerae of the present invention, step is as follows:
(1) get Folium Lonicerae, by the alcoholic solution reflux, extract, of 8-10 times amount (g/ml), filter, merging filtrate, being evaporated to relative density is 1.0-1.1 (50 DEG C of surveys), obtains just extract;
(2) by first extract prepared by step (1), add isopyknic distilled water and make suspension, extracted by suspension n-butyl alcohol, it is 1.2-1.3 (50 DEG C of surveys) that n-butyl alcohol is evaporated to relative density mutually, obtains n-butyl alcohol extract;
(3) n-butyl alcohol extract step (2) prepared adopts polyamide filler to carry out concentration and separation, and eluting solvent is alcohol-water, and the volume ratio of ethanol, water is 10:90 ~ 20:80;
(4) solution after combining step (3) eluting, concentrated, obtain extractum, be coffee mesitoyl quinine acid extract.
After testing, in extract prepared by the method, the weight percentage of coffee mesitoyl quinine acid compounds is 65-76%, described coffee mesitoyl quinine acid compounds mainly comprises chlorogenic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acids, 4-dicaffeoylquinic acid and neochlorogenic acid.
Present invention also offers the application of coffee mesitoyl quinine acid extract in the medicine preparing anti-new castle disease virus in this Folium Lonicerae.
The present invention also provides a kind of pharmaceutical preparation of anti-new castle disease virus, this pharmaceutical preparation with containing coffee mesitoyl quinine acid extract in the Folium Lonicerae of pharmacy effective dose for effective ingredient.
This pharmaceutical preparation can mix with pharmaceutic adjuvant further, makes capsule, tablet, granule, powder or injection in conventional manner; Form of medication is oral administration or drug administration by injection.
Described pharmaceutic adjuvant is one or more in mannitol, starch, stevioside, Sorbitol, cellulose derivative, magnesium stearate, Polyethylene Glycol or solvent for injection;
Described cellulose derivative is crystalline cellulose, hydroxypropyl cellulose or sodium carboxymethyl cellulose;
Described solvent for injection is water, ethanol or glycerol.
Beneficial effect of the present invention:
(1) coffee mesitoyl quinine acid method for preparing extractive energy consumption of the present invention low, pollute little, technique simple, be applicable to carrying out suitability for industrialized production.
(2), in the coffee mesitoyl quinine acid extract that preparation method of the present invention obtains, the weight percentage of caffeoylquinic acids is 65-76%, can directly be used as medicine, without the need to being further purified.
(3) test proves that coffee mesitoyl quinine acid extract of the present invention has the activity of stronger anti-new castle disease virus, has potential development and application values.
Detailed description of the invention
The present invention is further illustrated in conjunction with the embodiments, should be noted that following explanation is only to explain the present invention, not limiting its content.
Embodiment 1: the preparation of coffee mesitoyl quinine acid extract in Folium Lonicerae
(1) get dried Folium Lonicerae 100 grams, by 800mL alcoholic solution reflux, extract, filter, merging filtrate, being evaporated to relative density is 1.1 (50 DEG C of surveys), obtains just extract;
(2) by first extract prepared by step (1), add isopyknic distilled water and make suspension, extracted by suspension n-butyl alcohol, it is 1.3 (50 DEG C of surveys) that n-butyl alcohol is evaporated to relative density mutually, obtains n-butyl alcohol extract;
(3) n-butyl alcohol extract step (2) prepared adopts polyamide filler to carry out concentration and separation, and eluting solvent is alcohol-water, and the volume ratio of ethanol, water is 10:90 ~ 20:80.
(4) solution after combining step (3) eluting, being evaporated to relative density is 1.3 (50 DEG C of surveys), obtains extractum, is coffee mesitoyl quinine acid extract.
The feature of this extract: extract is faint yellow, UV-VIS spectrophotometry is adopted to detect extract, reference substance is chlorogenic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 4-dicaffeoylquinic acid and neochlorogenic acid, determined wavelength 325nm.In extract, the weight percentage of coffee mesitoyl quinine acid compounds is 72.5% mainly comprise chlorogenic acid, 3,4-O-dicaffeoylquinic acids, 3,5-O-dicaffeoylquinic acids, 4,5-O-dicaffeoylquinic acids, 4-dicaffeoylquinic acid and neochlorogenic acid.
Embodiment 2: the preparation of the pharmaceutical preparation of anti-new castle disease virus
Medicament composing prescription: coffee mesitoyl quinine acid extract 80 parts in Folium Lonicerae, astragalus polysaccharides 10 parts, mannitol 20 parts, Polyethylene Glycol 15 parts.
In Folium Lonicerae, the preparation method of coffee mesitoyl quinine acid extract is with embodiment 1.
Said medicine prescription is prepared into granule according to a conventional method.
Embodiment 3: coffee mesitoyl quinine acid extract anti-new castle disease virus Embryo Gallus domesticus test in Folium Lonicerae
One, to the toxicity test of Embryo Gallus domesticus
(1) trial drug: coffee mesitoyl quinine acid extract prepared by the embodiment of the present invention 1, is made into 200,100,50 and 25mg/mL with PBS;
(2) test method: adopted by the medicine of preparation chorioallantoic cavity inocalation method to inoculate 11 age in days SPF Embryo Gallus domesticus in Biohazard Safety Equipment, inoculum concentration 0.2mL/ embryo, each concentration connects embryo 5 pieces of Embryo Gallus domesticus, paraffin sealing after inoculation, be put in 37 DEG C of incubators and hatch, Fan Pei 2 times, observed Embryo Gallus domesticus growing state and recorded chicken embryo death situation every day, discard the dead germ in 24h, Continuous Observation 5d.The Embryo Gallus domesticus of dying after 24h is put into 4 DEG C of refrigerators, the centrifugal 15min of sterile collection allantoic fluid 3000r/min next day, gets each chick embryo allantoic liquid and carries out hemagglutination test.After 5d, all Embryo Gallus domesticus are put into 4 DEG C of refrigerator overnight, next day carries out hemagglutination test.
The method of hemagglutination test is:
1. get 96 hole V-type blood-coagulation-boards, every hole adds the PBS that 25 μ L newly prepare;
2. draw allantoic fluid 25 μ L in the 1st hole, fully inhale 25 μ L in the 2nd hole, by that analogy doubling dilution from the 1st hole, to the 10th hole after mixing; 11st, 12 holes add the contrast of negative allantoic fluid and positive-virus contrast respectively;
3. every hole adds 25 μ L chicken red blood cells suspension (1%);
4., after concussion shakes up, put into 37 DEG C of incubators and hatch 15min, obvious for one-tenth button-type sinks at the bottom of hole by control wells erythrocyte.
5. result judges: tilted by plate, observes erythrocyte with or without the trickling in teardrop shaped.The most highly diluted multiple of virus of complete blood clotting (not trickling) represents a HAU (HAU).
(3) result of the test
In the Folium Lonicerae of variable concentrations, the toxicity test of coffee mesitoyl quinine acid extract to Embryo Gallus domesticus the results are shown in Table 1.
The chicken embryo death quantity (5 pieces/group) of the coffee mesitoyl quinine acid extract inoculated into chick embryo of table 1 variable concentrations
| Concentration of component (mg/mL) | Chicken embryo death quantity |
| 200 | 1 |
| 100 | 0 |
| 50 | 0 |
| 25 | 0 |
As can be seen from Table 1, in the Folium Lonicerae of 100mg/mL and following concentration, coffee mesitoyl quinine acid extract is safe to Embryo Gallus domesticus, and illustrate that the toxicity of coffee mesitoyl quinine acid extract in Folium Lonicerae is lower, safety is higher.
Two, anti-new castle disease virus Embryo Gallus domesticus test
(1) trial drug: coffee mesitoyl quinine acid extract prepared by the embodiment of the present invention 1, is made into 10,2,0.4,0.08 and 0.016mg/mL respectively with PBS.
(2) test method: respectively the medicine 1mL of different parts, variable concentrations and equivalent newcastle disease virus strain (100EID50/0.1mL) are mixed, establish the virus control of saline control and inoculation 0.1mL/ embryo simultaneously, under room temperature, 1h is made in sense, and inoculation method is identical with embryonic toxicity test with Embryo Gallus domesticus observational technique.
(3) result of the test:
In the Folium Lonicerae of variable concentrations, coffee mesitoyl quinine acid extract anti-new castle disease virus test Embryo Gallus domesticus death condition and coagulation result of the test are in table 2.
Coffee mesitoyl quinine acid extract anti-new castle disease virus Embryo Gallus domesticus result of the test (5 pieces/group) of table 2 variable concentrations
Result display 10,2,0.4,0.08 and 0.016mg/mL with Avian pneumo-encephalitis virus after, within 5 days, Embryo Gallus domesticus embryo rate alive is respectively 100%, 100%, 0,0 and 0, HA average titer is respectively 0,0,7.5,7.3 and 7.6, illustrate that coffee mesitoyl quinine acid extract is killed completely or suppresses viral least concentration to be 2mg/mL, 0.4mg/mL concentration also can extend the Embryo Gallus domesticus time-to-live, and coffee mesitoyl quinine acid extract has reasonable anti-new castle disease virus effect in Embryo Gallus domesticus.
Embodiment 4: coffee mesitoyl quinine acid extract anti-new castle disease virus test cell line in Folium Lonicerae
(1) trial drug: coffee mesitoyl quinine acid extract prepared by the embodiment of the present invention 1, is made into 752,376,188,94,47 and 24 μ g/mL respectively with PBS.
(2) virus preparation: select Avian pneumo-encephalitis virus (NDVJN strain exists-30 DEG C after new propagation): Avian pneumo-encephalitis virus being measured cell median infective dose (TCID50), is 10
8tCID50/0.1mL.Be diluted to 10TCID50/0.1mL with PBS, then dilute 5 times, become 2TCID50/0.1mL, same method dilutes 10 times again, becomes 0.2TCID50/0.1mL.Mix with medicine equal-volume after feeling and doing, access in 96 orifice plates, its final concentration is 1TCID50/0.1mL or 0.1TCID50/0.1mL.
(3) preparation of CEF cell: prepare according to a conventional method.
(4) antiviral activity Cellular Assay Experimental Method: adopt CPE Inhibition test to evaluate.Be inoculated in by CEF cell in 96 well culture plates, 37 DEG C of cultivations, make it to cover with monolayer.Mixed with the Avian pneumo-encephalitis virus equal-volume of 1TCID50/0.1mL respectively by the test medicine of doubling dilution, 60min is made in 37 DEG C of lucifuge senses, and each dilution factor inoculates 4 holes, 200 μ L/ holes, virus control 100 μ L/ hole.Continue in 37 DEG C of incubators to cultivate 120h, observed and recorded cytopathy (CPE), after inoculating cell freeze thawing 2 times, it is coagulation to carry out HA test determination, HA assay method and Embryo Gallus domesticus hemagglutination experimental technique similar.
(5) result of the test:
In the Folium Lonicerae of variable concentrations, the anti-new castle disease virus test cell line of coffee mesitoyl quinine acid extract the results are shown in Table 3.
The coffee mesitoyl quinine acid extract anti-new castle disease virus test cell line result of table 3 variable concentrations
| Concentration of component (μ g/mL) | CPE number | HA tires |
| 752 | 1/4 | 0、0、4、0 |
| 376 | 3/4 | 4、4、5、0 |
| 188 | 2/4 | 5、5、0、0 |
| 94 | 3/4 | 6、5、0、6 |
| 47 | 4/4 | 6、6、6、6 |
| 24 | 4/4 | 6、6、6、6 |
| Virus control | 4/4 | 7、7、7、7 |
After the coffee mesitoyl quinine acid extract antiviral of 188 more than μ g/mL as can be seen from Table 3, cell HA tires and all drops to less than 6, has anti-virus ability.
Embodiment 5: the effect experimental for the treatment of newcastle disease virus
1. materials and methods
1.1 laboratory animals: 5 week age SPF chicken, purchased from Jinan Si Pafasi poultry company limited.
1.2 medicines: coffee mesitoyl quinine acid extract in Folium Lonicerae prepared by embodiment 1; The pharmaceutical preparation of anti-new castle disease virus prepared by embodiment 2; Fuzheng Jiedu powder (production of Nuo Weikang bio tech ltd, Funan).
1.3 experimental techniques:
Chicken is divided into test group I, test group II, matched group, virus control group and blank group, often organize 20, the chicken of test group I, test group II, matched group, virus control group carries out counteracting toxic substances process, strain is selected from Avian pneumo-encephalitis virus (NDVJN strain,-30 DEG C are had after new propagation), purchased from China Veterinary Drugs Supervisory Inst.; Blank group does not process.
Counteracting toxic substances is after 12 hours, coffee mesitoyl quinine acid extract in the Folium Lonicerae of test group I administration embodiment 1 preparation, and administration concentration is 40mg/kg, and route of administration is oral, and administration number of times is 2 times/day, continuous use 5 days;
The pharmaceutical preparation of the anti-new castle disease virus of test group II administration embodiment 2 preparation, administration concentration is 40mg/kg, and route of administration is oral, and administration number of times is 2 times/day, continuous use 5 days;
Matched group administration Fuzheng Jiedu powder, by specification administration, continuous use 5 days;
Virus control group and the not administration of blank group.
1.4 evaluation indexes:
Survival rate and weight gain index: duration of test, observe the spirit of chicken, appetite and active situation every day, on-test and at the end of weigh, calculate rate of body weight gain and survival rate.
2. experimental result:
2.1 impacts on chicken weight gain
Chicken weight gain situation after duration of test different disposal is in table 4.
Chicken weight gain situation after table 4 different disposal
| Grouping | Original body mass (gram) | Whole opisthosoma heavy (gram) | Rate of body weight gain (%) |
| Test group I | 342.5 | 416.8 | 21.7 |
| Test group II | 354.2 | 425.3 | 20.1 |
| Matched group | 348.6 | 395.2 | 13.4 |
| Virus control group | 351.3 | 385.5 | 9.7 |
| Blank group | 358.4 | 453.8 | 26.6 |
2.2 impacts on adult livability
Chicken survival rate after duration of test different disposal is in table 5.
Chicken survival rate after table 5 different disposal
| Grouping | Amount of survival (only) | Survival rate (%) |
| Test group I | 18 | 90 |
| Test group II | 19 | 95 |
| Matched group | 15 | 75 |
| Virus control group | 9 | 45 |
| Blank group | 20 | 100 |
As can be seen from table 4 and table 5, the pharmaceutical preparation of anti-new castle disease virus prepared by coffee mesitoyl quinine acid extract and embodiment 2 in Folium Lonicerae prepared by the embodiment of the present invention 1, compared with drugs compared, the survival rate after chicken infection Avian pneumo-encephalitis virus can be significantly improved, and the growth Be very effective to chicken body weight.
Claims (9)
1. the preparation method of coffee mesitoyl quinine acid extract in Folium Lonicerae, it is characterized in that, step is as follows:
(1) get Folium Lonicerae, by the alcoholic solution reflux, extract, of 8-10 times amount, filter, merging filtrate, being evaporated to relative density is 1.0-1.1, obtains just extract;
(2) by first extract prepared by step (1), add isopyknic distilled water and make suspension, extracted by suspension n-butyl alcohol, it is 1.2-1.3 that n-butyl alcohol is evaporated to relative density mutually, obtains n-butyl alcohol extract;
(3) n-butyl alcohol extract step (2) prepared adopts polyamide filler to carry out concentration and separation, and eluting solvent is alcohol-water, and the volume ratio of ethanol, water is 10:90 ~ 20:80;
(4) solution after combining step (3) eluting, concentrated, obtain extractum, be coffee mesitoyl quinine acid extract.
2. coffee mesitoyl quinine acid extract in the Folium Lonicerae that obtains of preparation method according to claim 1, it is characterized in that, in described extract, the weight percentage of caffeoylquinic acids is 65-76%.
3. coffee mesitoyl quinine acid extract in Folium Lonicerae as claimed in claim 2, it is characterized in that, described coffee mesitoyl quinine acid compounds comprises chlorogenic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acids, 4-dicaffeoylquinic acid and neochlorogenic acid.
4. the application of coffee mesitoyl quinine acid extract in the medicine preparing anti-new castle disease virus in Folium Lonicerae according to claim 2.
5. a pharmaceutical preparation for anti-new castle disease virus, is characterized in that, this pharmaceutical preparation with containing coffee mesitoyl quinine acid extract in the Folium Lonicerae according to claim 2 of pharmacy effective dose for effective ingredient.
6. pharmaceutical preparation as claimed in claim 5, it is characterized in that, this pharmaceutical preparation mixes with pharmaceutic adjuvant, makes capsule, tablet, granule, powder or injection in conventional manner.
7. pharmaceutical preparation as claimed in claim 6, it is characterized in that, described pharmaceutic adjuvant is one or more in mannitol, starch, stevioside, Sorbitol, cellulose derivative, magnesium stearate, Polyethylene Glycol or solvent for injection.
8. pharmaceutical preparation as claimed in claim 7, it is characterized in that, described cellulose derivative is crystalline cellulose, hydroxypropyl cellulose or sodium carboxymethyl cellulose.
9. pharmaceutical preparation as claimed in claim 7, it is characterized in that, described solvent for injection is water, ethanol or glycerol.
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| CN108836990A (en) * | 2018-09-05 | 2018-11-20 | 河南大学 | A kind of honeysuckle-leaf anti-inflammatory active ingredient and its preparation method and application |
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| CN1839815A (en) * | 2006-01-20 | 2006-10-04 | 深圳市生物谷科技有限公司 | Pharmaceutical composition containing caffeoylquinic acids |
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| CN1839815A (en) * | 2006-01-20 | 2006-10-04 | 深圳市生物谷科技有限公司 | Pharmaceutical composition containing caffeoylquinic acids |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108836990A (en) * | 2018-09-05 | 2018-11-20 | 河南大学 | A kind of honeysuckle-leaf anti-inflammatory active ingredient and its preparation method and application |
| CN108836990B (en) * | 2018-09-05 | 2021-06-11 | 河南大学 | Anti-inflammatory active component of honeysuckle leaves and preparation method and application thereof |
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Application publication date: 20150708 Assignee: JINAN HEBAO CHINESE MEDICINAL HERBS Co.,Ltd. Assignor: SHANDONG ANALYSIS AND TEST CENTER Contract record no.: X2020980009421 Denomination of invention: The invention relates to caffeoylquinic acid extract from Lonicera japonica Leaves, a preparation method and application thereof Granted publication date: 20181019 License type: Exclusive License Record date: 20201215 |