CN1846717A - Medicine for treating influenza and bird flu - Google Patents
Medicine for treating influenza and bird flu Download PDFInfo
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- CN1846717A CN1846717A CN 200610011341 CN200610011341A CN1846717A CN 1846717 A CN1846717 A CN 1846717A CN 200610011341 CN200610011341 CN 200610011341 CN 200610011341 A CN200610011341 A CN 200610011341A CN 1846717 A CN1846717 A CN 1846717A
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Abstract
The present invention provides medicine for treating influenza and bird flu with effective component extracted from basket kern. The present invention proposes the application of basket kern extract in preparing medicine for treating influenza and bird flu for the first time, and provides the high efficiency liquid chromatography-mass spectrography analysis process to constitute the fingerprint of basket fern rhizome extract. The process includes the HPLC-MS analysis condition of basket fern rhizome extract, chromatographic peak characteristic, compound structure information of the chromatographic components, etc. The fingerprint and technology of the present invention may be used in the variety identification of basket fern and the quality control of the basket fern rhizome extract.
Description
Technical field
The present invention relates to field of medicaments, specifically, relate to the medicine of a kind of influenza and bird flu virus and preparation thereof, detection method.
Background technology
Influenza is called for short influenza (Influenza), is that (Influenza Virus, the acute respiratory disease that a kind of people, animal that IFV) cause are suffered from altogether have characteristics such as strong, popular wide, the M ﹠ M height of infectiousness by influenza virus.Evidence suggests that annual flu outbreak phase in the winter whole world has people's infection morbidity of 10% approximately, the complication that annual nearly 500,000 people in the whole world die from various influenzas and cause because of influenza.(Avian influenza AI) is a kind of birds deadly infectious disease that is caused by A type influenza virus to bird flu, and is huge to aviculture harm, and threatening human life and health.Since in the Decembers, 2003, at first the highly pathogenic H5N1 subtype avian influenza that breaks out in the Asia has spread to a plurality of countries and regions except that Africa, causes tens million of avian to infect and dead, and the people was dead because of infecting bird flu virus surplus whole world priority had 70.This bird flu epidemic situation that have swepts the globe also is not effectively controlled so far.
At present both at home and abroad the control measure of poultry bird flu are mainly comprised slaughter, sterilization, bio-safety and vaccination.The effective measures that birds flu-preventing takes place are vaccinations, when the vaccine of inoculation is consistent with clinical popular strain hypotype, can obtain ideal protection effect; But the vaccine of inoculation and epidemic isolates hypotype are not simultaneously, just can not obtain immunoprotection.The bird flu virus blood serum subtype is numerous, will the producer reorganization during A type influenza virus mixed infection of different subtype, forms new bird flu virus, and this development and application to vaccine brings very big difficulty.The case that vaccine and epidemic isolates hypotype because of inoculation are unworthy of causing immuning failure often takes place in the production practices.Therefore only rely on generation that avian influenza vaccine is difficult to control bird flu fully with popular, medical treatment also is the generation of control bird flu, spread and the important measures of birds-to-human transmission.
Virus is the strict endotrophic noncellular microorganism of a class, and the cellular metabolism that relies on the host duplicates.The difficulty of antiviral treatment is that medicine is when blocking virus duplicates and can not influence the homergy of host cell.Therefore present most of viral infection still do not have effective medicine, and the research of anti-influenza virus medicament also never has big breakthrough.In recent years, people provide new approach to the deep exploitation for the influenza new drug of influenza virus surface antigen " neuraminidase (NA) " understanding.Two kinds of NA inhibitor successively went on the market in 1999: the Oseltamivir (Ao Simiwei, trade name Tamiflu) of the Zanamivir (zanamivir) of Glaxo Wellcome company and Roche company.Clinical trial proves that these two kinds of NA inhibitor can be blocked the reproduction process of influenza virus effectively, and prophylactic and early stage medication are evident in efficacy, be difficult for producing drug resistance, and all effective to all influenza virus sub-strains.But, only be used for the control of people's parainfluenza at present in minority developed country owing to these two kinds of medicine synthesis step complexity, cost an arm and a leg.
Chinese medicine is the exclusive medical system of China, extensive knowledge and profound scholarship.Over the past thousands of years, anti-cure cold and the struggle of influenza in, Chinese medicine has been brought into play important function.Rhizoma Osmundae is the dry rhizome of multiple pteridophyta, is stated from Shennong's Herbal as Chinese medicine beginning, bitter in the mouth, be slightly cold, slightly poisonous, have effects such as heat-clearing and toxic substances removing, anthelmintic, hemostasis.Be used for the treatment of anemopyretic cold, hepatitis, damp and hot skin eruption clinically, spit blood, have blood in stool and disease such as metrorrhagia.Pharmacological actions such as that modern medicine study proof Rhizoma Osmundae has is antibiotic, antiviral, leukemia, antitumor, the contraction of promotion uterine smooth muscle, anthelmintic.Although the medicinal history of Rhizoma Dryopteris Crassirhizomatis is long, relevant its research report of active ingredient, pharmacological action etc. is less.(number of patent applications: 03108749.3) with the Rhizoma Dryopteris Crassirhizomatis be monarch drug such as the Zhu Rong ancestral of LanZhou FoCi Pharmacy Co., Ltd, be equipped with the Chinese medicine and western medicine compound preparation " compound recipe Rhizoma Osmundae aspirin tablet " of developments such as Radix Isatidis, Flos Chrysanthemi Indici, Radix Sophorae Tonkinensis, aspirin, chlorphenamine maleate, be used for anti-cure cold and influenza has remarkable result, but this patent does not relate to the active ingredient of Rhizoma Dryopteris Crassirhizomatis resisiting influenza virus.
Summary of the invention
(1) technical problem that will solve
Purpose of the present invention aims to provide the medicine of a kind of influenza and bird flu virus, and its preparation and detection method are provided.
(2) technical scheme
The effective ingredient of influenza provided by the invention and bird flu virus and/or pharmaceutically the acceptable adjuvant form, described effective ingredient is a Rhizoma Osmundae extract, it prepares by the following method:
With Rhizoma Osmundae tuber powder essence, with the ethanol water reflux, extract, of envelope-bulk to weight ratio 4-10 50-90% doubly 2-4 time, merge extractive liquid,, concentrated, with water dissolution concentrated solution to aqueous solution weight be Rhizoma Osmundae medical material weight 1-1.5 doubly, extract 2-3 time respectively with ethyl acetate and n-butyl alcohol successively, volume and the Rhizoma Osmundae aqueous solution volume with extract is 1: 1 at every turn, merges butanol extraction liquid, the volatilization n-butyl alcohol, concentrate paste liquid, drying, promptly.
The drying of described paste liquid can be oven drying at low temperature or spray drying, and the Rhizoma Osmundae extract of dry back gained is rufous or brownish black.
The Rhizoma Osmundae extract of gained of the present invention has prevention and therapeutical effect to humans and animals influenza, bird flu.Can Rhizoma Osmundae extract of the present invention be prepared into any preparation that is suitable for clinical use, for example powder, tablet, capsule, oral liquid etc. according to conventional formulation technology.
The present invention also provides the detection method of the medicine of described influenza and bird flu virus, is with high performance liquid chromatography-esi-msn combined instrument Rhizoma Osmundae extract to be analyzed to obtain the finger printing of extract.Its chromatographiccondition is: chromatographic column: and the XDB-C18 post (150mm * 2.1mmi.d., 3.5um); Binary gradient elution, A are acetonitrile mutually, and B is water mutually, all contains 1% formic acid, and flow velocity is 0.5mL/min.The mass spectrum condition is: ESI ion source, positive and negative ion mode detection; The APCI ion source, positive ion mode detects; Automatic 3 grades of mass spectrums (selecting the strongest ion to carry out inducing cracking in the source automatically).
Preferred chromatographic condition: chromatographic column: the XDB-C18 post (150mm * 2.1mm i.d., 3.5um); Binary gradient elution, A are acetonitrile mutually, and B is water mutually, all contains 1% formic acid; The gradient program: the 20%A phase, keep 2min, increase A phase ratio gradually, A is 100% mutually during to 50min, keeps 10min; Flow velocity: 0.5mL/min; Sample size 20 μ L.
Preferred mass spectrum condition: ESI and APCI ion source, the positive and negative ion mode detection; Nebulizer N
2Pressure 40psi; Dry gas N
2Flow velocity 9L/min; Mass scanning scope 100～1000u; Automatic 3 grades of mass spectrums (selecting the strongest ion to carry out inducing cracking in the source automatically).
Analyze through the liquid phase chromatogram-mass spectrometry combination machine, main component in the Rhizoma Osmundae extract is the oligomer of phloroglucinol derivant, comprises root bark pyrone PB, flavaspidic acid AA, albaspidin AA, pannol AA, pannol AP, flavaspidic acid AB, Filixic Acids ABA, filixic acid ABA, Filixic Acids ABB, Filixic Acids PBP, dryocrassin ABAA Dryocrassin., dryocrassin ABPP, dryocrassin ABBP, dryocrassin PBBP etc.Wherein 9 kinds of chemical compounds such as root bark pyrone PB, flavaspidic acid AA, pannol AA, pannol AP, Filixic Acids ABA, Filixic Acids ABB, Filixic Acids PBP, dryocrassin ABPP, dryocrassin PBBP are the noval chemical compound of finding in Rhizoma Dryopteris Crassirhizomatis first.
In the total ion current chromatograph that APCI (+) and two kinds of detecting patterns of ESI (-) obtain, the component peaks area sum that contains poly phloroglucinol derivant accounts for 61.6% and 77.8% of whole component chromatographic peak areas respectively.Wherein molecular weight is that the pannol AA of 404Da or albaspidin AA, molecular weight are that the filixic acid ABA of 612 Da or Filixic Acids ABP, molecular weight are that the content of Filixic Acids ABB in Rhizoma Osmundae extract of 626Da is higher.
(3) beneficial effect
The present invention provides the application of Rhizoma Osmundae extract in the medicine of preparation influenza and bird flu virus first, and provides application high performance liquid chromatography-mass spectrum on line analytical processing and result to make up the method for Rhizoma Dryopteris Crassirhizomatis extract finger printing.This method comprises compound structure information that the mass spectrum of feature, the chromatographic component of HPLC-MS analysis condition, the chromatographic peak of Rhizoma Osmundae extract provides etc.Finger printing disclosed by the invention and technology thereof can be used for the kind discriminating of Rhizoma Dryopteris Crassirhizomatis and the quality monitoring of Rhizoma Osmundae extract.
Description of drawings
Fig. 1 is that Rhizoma Osmundae extract HPLC-APCI-MS (+) analyzes total ions chromatogram
Fig. 2 is that Rhizoma Osmundae extract HPLC-ESI-MS (+) analyzes total ions chromatogram
Fig. 3 is that Rhizoma Osmundae extract HPLC-ESI-MS (-) analyzes total ions chromatogram
Fig. 4 is that the quasi-molecular ion structure and the cleavage of mass spectrum approach thereof of poly phloroglucinol derivant inferred
The specific embodiment
Can further be expressly understood the present invention by specific embodiments of the invention given below, but following embodiment not a limitation of the invention.
The preparation of embodiment 1 Rhizoma Osmundae extract
Get the Rhizoma Dryopteris Crassirhizomatis tuber, powder is pure in whole mistake 40 mesh sieves, ethanol water reflux, extract, with 80% 2 times, Rhizoma Osmundae powder account for 15% (W/V) of ethanol water, merge extractive liquid,, distilling under reduced pressure concentrates, ethanol is reclaimed in volatilization, and the residue extract adds the suitable quantity of water dissolving, and the weight of Rhizoma Osmundae aqueous solution is 1.5 times of Rhizoma Osmundae, use ethyl acetate and n-butanol extraction successively, extract respectively 3 times, the volume of each used extract is suitable with the Rhizoma Osmundae aqueous solution, merges butanol extraction liquid, distilling under reduced pressure concentrates, volatilize the recovery n-butyl alcohol, get paste liquid, oven drying at low temperature, powder essence, get rufous or rice-pudding black powder, be Rhizoma Osmundae extract.
Experimental example 1 medicine of the present invention is to influenza virus (H in the Embryo Gallus domesticus
9N
2Strain) inhibition of proliferation effect
1. experiment material
Avian influenza strain: A/Chicken/Guangdong/3/2004 (H
9N
2).Agricultural University Of South China's veterinary college separation, purification, preservation and evaluation.(this virus is Lowly Pathogenic Avian Influenza Virus, except that Agricultural University Of South China, all there is preservation in units such as Harbin Veterinary Medicine Inst., China Academy of Agriculture, Chinese disease prevention and control centre institute of viruses, Wuhan Virology Institute,Chinan academy of Sciences virus preservation center.There is the production licence (No. the 35th, 2003 new veterinary drugs card words) of the production H9 subtype avian influenza vaccine that the national Ministry of Agriculture issues in applicant unit one belongs to.Its separate and authentication method referring to document: 1. Bi Yingzuo, Cao Yongchang, thiocarbonyl U.S. etc. the serosurvey of Guangdong bird flu [J], Chinese poultry infectious disease, 1998,20 (1): 32-34; 2. the Qin likes to build Shao Hongxia, Qian Kun etc. the development and the application [J] of anti-avian influenza virus H5 and H9 hypotype hemagglutinin monoclonal antibody specific, Chinese Preventive Veterinary Medicine newspaper, 2003,25 (3): 161-163.)
Embryo Gallus domesticus is used in test: 9 age in days SPF Embryo Gallus domesticus, veterinary Science ﹠ Technology Center of Agricultural University Of South China provides.
Medicine is used in test: Rhizoma Osmundae extract prepares voluntarily, and selecting medical material for use is Rhizoma Dryopteris Crassirhizomatis, and the Rhizoma Dryopteris Crassirhizomatis in source should all can use at this arbitrarily.
Erythrocyte use in HA test: be 0.5% chicken red blood cell, prepare voluntarily.
2. test method
Half lethal dose LD with Embryo Gallus domesticus test determination Rhizoma Osmundae extract
50, determine suitable drug dose; With the median infective dose EID of Embryo Gallus domesticus test determination bird flu virus to Embryo Gallus domesticus
50, determine suitable counteracting toxic substances concentration.Adopt earlier and carry out the Embryo Gallus domesticus test, determine the prevention and the therapeutic effect of medicine to two kinds of different modes of administration of administration behind counteracting toxic substances behind the medicine and the first counteracting toxic substances.
2.1 medicine is to the half lethal dose LD of Embryo Gallus domesticus
50Mensuration
Rhizoma Osmundae extract is earlier with a small amount of 1, and 2-propylene glycol (be no more than quantity of solvent 20%) dissolves, and adds normal saline and is made into 5.00% solution, doubling dilution is 2.50%, 1.25%, 0.62%, 0.32% totally 5 Concentraton gradient, 9 embryo/groups, the 0.2mL/ embryo was observed 72 hours.Embryo is not caused death, do not influence idiosome normal development person and be judged to safety.Press Reed-Muench Liang Shi method and calculate half poisoning (causing death) dosage.
2.2 the selection of drug dose
According to the LD of 2.1 Rhizoma Osmundae extracts that record to Embryo Gallus domesticus
50As a result, the highest safe drugs concentration of all surviving by Embryo Gallus domesticus is to suppress the initial concentration of influenza virus in the Embryo Gallus domesticus, and doubling dilution is established 5 concentration groups and experimentized.Establish normal saline matched group and Western medicine amantadine matched group simultaneously.
2.3 influenza virus (H
9N
2Strain) Embryo Gallus domesticus median infective dose (EID
50) mensuration
With virus stock solution used (H
9N
2Strain) presses 10 with sterile saline
-5, 10
-6, 10
-7, 10
-8, 10
-9, 10
-10, 10
-11, 10
-12Instar chicken embryo on the 9th is inoculated in totally 8 Concentraton gradient dilutions, 9 pieces of embryos of each dilution factor inoculation, 0.2mL/ embryo was hatched 96 hours for 37 ℃, embryo dead in 24 hours discards, measure that the HA of dead embryo tires after 24 hours, positive greater than 4 holes, press Reed-Muench Liang Shi method and calculate EID
50
2.4 medicine is to influenza virus (H in the Embryo Gallus domesticus
9N
2) inhibitory action measure
Every embryo counteracting toxic substances amount is 100 times of EID
50(0.2mL), allantoic cavity inoculation and administration, 12 embryo/groups, administration 0.2mL/ embryo.Prophylactic is counteracting toxic substances behind 1h after the administration, and medicine for treatment is the 1h administration behind counteracting toxic substances.Hatched 96 hours for 37 ℃, during every day according to embryo 2 times, dead embryo discards in 24 hours.The death condition of statistics embryo, and the HA of mensuration chick embryo allantoic liquid tires.
3. result
Record the LD of Rhizoma Osmundae extract by 2.1 to Embryo Gallus domesticus
50For the 10.0mg/ embryo (5.0%, 0.2mL), the highest safe drugs concentration be the 5.0mg/ embryo (2.5%, 0.2mL).The amantadine aqueous solution is to the LD of Embryo Gallus domesticus
50Be the 3.5mg/ embryo.Record influenza virus (H by 2.3
9N
2Strain) to the EID of Embryo Gallus domesticus
50Be 10
-9, therefore testing counteracting toxic substances concentration is 10
-7(100EID
50).The test of pesticide effectiveness is respectively organized chicken embryo death situation and various dose Rhizoma Osmundae extract to influenza virus (H
9N
2Strain) the inhibitory action measurement result of Embryo Gallus domesticus internal breeding sees Table 1-1.The result shows: 100EID
50The counteracting toxic substances amount cause the negligible amounts of chicken embryo death, have only amantadine high dose group mortality rate higher (3/12), 1-2 piece of other test group dead germ or not dead shows the H that this test is adopted
9N
2The virulence of strain a little less than.Meansigma methods is 11.3 but the HA that infects matched group tires, and shows that the reproductive capacity of this strain is stronger.No matter Rhizoma Osmundae extract 5.00mg, 2.50mg, 1.25mg three dosage groups are prophylactic or medicine for treatment, the HA of blastochyle tires and is 0; 0.625mg the prevention group HA of dosage is 0, treatment group HA is 2.3; And the HA of amantadine 1.25mg dosage prevention group and treatment group to tire be respectively 1.8 and 3.2, it is respectively 4.6 and 5.8 that the HA of 0.625mg dosage prevention group and treatment group tires, all higher than the HA of the Rhizoma Osmundae extract of same concentrations, shows that Rhizoma Osmundae extract is to H
9N
2Influenza virus has remarkable inhibitory action, and it suppresses effect and obviously is better than amantadine.
Table 1-1 various dose Rhizoma Osmundae extract is to influenza virus (H
9N
2Strain) the inhibitory action measurement result of Embryo Gallus domesticus internal breeding
| Medicine grouping and dosage (mg/ embryo) | Each organizes Embryo Gallus domesticus number (only) | Dead embryo number in the 24h | 24h-96h dead germ number | 96h embryo number alive | HA tire (X, log2) | |||||
| Prevention | Treatment | Prevention | Treatment | Prevention | Treatment | Prevention | Treatment | |||
| Rhizoma Osmundae extract | 5.00 | 12 | 0 | 1 | 1 | 1 | 11 | 10 | 0 ** | 0 ** |
| 2.50 | 12 | 0 | 0 | 0 | 1 | 12 | 11 | 0 ** | 0 ** | |
| 1.25 | 12 | 0 | 0 | 1 | 0 | 11 | 12 | 0 ** | 0 ** | |
| 0.625 | 12 | 0 | 0 | 0 | 0 | 12 | 12 | 0 ** | 2.3 ** | |
| 0.312 | 12 | 0 | 0 | 0 | 1 | 12 | 11 | 3.5 ** | 4.2 ** | |
| Amantadine | 1.25 | 12 | 1 | 2 | 0 | 1 | 11 | 9 | 1.8 ** | 3.2 ** |
| 0.625 | 12 | 0 | 0 | 0 | 0 | 12 | 12 | 4.6 * | 5.8 * | |
| Infect matched group | 12 | 0 | 2 | 10 | 11.3 | |||||
| The blank group | 12 | 0 | 0 | 12 | 0 ** | |||||
Annotate: compare with the infection matched group,
*P＜0.05,
*P＜0.01
Experimental example 2 medicines of the present invention are to the pharmacodynamics test of mice artificial challenge influenza virus
1. experiment material
Experimental animal: kunming mice is provided by Nanfang Medical Univ (Guangzhou) Experimental Animal Center.
Trial drug: the Rhizoma Osmundae extract powder, preparation voluntarily, every gram Rhizoma Osmundae extract is equivalent to the 2.6g crude drug.Selecting medical material for use is Rhizoma Dryopteris Crassirhizomatis.
A type influenza virus FM1 Mus lung adapted strain is from biotechnology research institute of the Chinese Academy of Medical Sciences.
2. test method and result
2.1 Rhizoma Osmundae extract is to the acute toxicity test of mice
20 of kunming mices, weight 19～21 gram, random packet is 4 groups, 5 every group, irritate and feed the method administration, according to pre-test result, select for use horn method with 2.15 times group apart from the design dosage, observed 7 days continuously behind the single administration, dead mouse and survival condition see Table 2-1.
Table 2-1 Rhizoma Osmundae extract is to mice LD
50Measurement result
| Group | Number of animals | Drug level (g/mL) | Dosage (g/kg) | Administration volume (mL) | The dead mouse number | The survival mice situation |
| 1 | 5 | 0.086 | 2.15 | 0.5 | 0 | |
| 2 | 5 | 0.186 | 4.64 | 0.5 | 0 | |
| 3 | 5 | 0.400 | 10.0 | 0.5 | 3 | The movable minimizing |
| 4 | 5 | 0.86 | 21.5 | 0.5 | 5 |
Annotate: dosage calculates by every mice body weight 20g.
Behind the animals administer, high dose group mice spirit is depressed, it is in heaps not eat, assemble, and occurs deadly after 20 hours, and all the other group mices spirit are good.Result of the test is tabled look-up and is drawn the LD of Rhizoma Osmundae extract to mice
50Be 9.26g/kg, the 95% credible 6.36-13.5g/kg that is limited to, show Rhizoma Osmundae extract belong to low toxicity, near the avirulence material.
2.2 Rhizoma Osmundae extract is to the influence of influenza virus infecting mouse lung weight
60 of kunming mices, body weight 13～15g, the male and female dual-purpose is divided into 6 groups at random by body weight, is respectively normal control group, virus control group, amantadine group, the high, medium and low dosage group of Rhizoma Osmundae extract, 10 every group.Infect and play administration the previous day, morning and afternoon every day, each administration was 1 time, successive administration 5 days.Normal control group and virus control group are all irritated and feed to be waited the appearance normal saline.After the administration second day, except that the normal control group, all the other respectively organized mice under the slight anesthesia of ether, with 15 times of LD
50Influenza virus Mus lung adapted strain FM1 collunarium infects, and the normal control group is used the normal saline collunarium under identical conditions.Respectively organize mice fasting 12 hours after the last administration, take off cervical vertebra after weighing and put to death, the anatomic observation pulmonary lesion takes out full lung with clear water washing 3 times, uses the filter paper suck dry moisture, and precision balance is weighed, and is calculated as follows lung exponential sum suppression ratio.The results are shown in Table 2-2.
Lung index=(mouse lung weight/mice body weight) * 100%
Lung index suppression ratio=(the average lung index of the average lung index-test group of matched group)/average lung index of matched group
Table 2-2 Rhizoma Osmundae extract is to the influence of influenza virus Lung Index of mice infected by Influenza virus
| Group | Dosage (g/kg) | Number of animals | Lung index (%) | Lung index suppression ratio (%) |
| Normal control | Deng the capacity normal saline | 10 | 0.86±0.10 ** | |
| Virus control | Deng the capacity normal saline | 8 | 1.43±0.26 | |
| Amantadine | 0.1 | 9 | 1.27±0.17 * | 11.18 |
| The Rhizoma Osmundae extract low dosage | 0.3 | 9 | 1.36±0.22 * | 4.90 |
| Dosage in the Rhizoma Osmundae extract | 0.9 | 10 | 1.15±0.18 **# | 19.58 |
| The Rhizoma Osmundae extract high dose | 2.7 | 10 | 1.03±0.16 **# | 27.97 |
Annotate: compare with the virus control group
*P＜0.01,
*Compare with the amantadine group P＜0.05
#P＜0.05.
The result shows: Rhizoma Osmundae extract 0.9g/kg and 2.7g/kg dosage group can obviously reduce A type influenza infection mouse lung exponential quantity, and lung index suppression ratio is respectively 19.58% and 27.97%.It reduces effect than amantadine group effective (P＜0.05).
2.3 protective effect to the death of influenza virus infecting mouse
Choose 110 of the Kunming mouses of 11-13g body weight, the male and female dual-purpose is divided into 6 groups at random by body weight, is respectively normal control group, virus control group, amantadine group, 10 of the high, medium and low dosage group of Rhizoma Osmundae extract, normal control groups, and all the other each groups are 20.Infect and play administration the previous day, morning and afternoon every day, each administration was 1 time, successive administration 5 days.Normal control group and virus control group are all irritated and feed to be waited the appearance normal saline.After the administration second day, except that the normal control group, all the other respectively organized mice under the slight anesthesia of ether, with 20 times of LD
50Influenza virus Mus lung adapted strain FM1 collunarium infects.Incidence behind the observation mouse infection, record infects the dead number of elements of mice in back 15 days, the results are shown in Table 2-3.The result shows; Rhizoma Osmundae extract 0.9g/kg and 2.7g/kg dosage group have significant protective effect to A type influenza infection dead mouse; dead protective rate is respectively 50% and 65% (P＜0.01), and the average survival natural law of mice is not brought up to 11.5 days and 12.9 days (P＜0.01) by 7.3 natural gift that infect matched group.
Table 2-3 Rhizoma Osmundae extract is to the protective effect of influenza virus infecting mouse death
| Group | Drug dose (g/kg) | Number of animals (only) | Death toll (only) | Mortality rate (%) | Protective rate (%) | Average survival natural law (d) |
| Normal control | Deng capacity | 10 | 0 | 0 | ＞15 | |
| Virus control | Deng capacity | 20 | 17 | 85 | 7.3 |
| Amantadine | 0.1 | 20 | 12 | 60 | 40 | 10.3 ** |
| The Rhizoma Osmundae extract low dosage | 0.3 | 20 | 15 | 75 | 25 | 8.6 * |
| Dosage in the Rhizoma Osmundae extract | 0.9 | 20 | 10 | 50 | 50 | 11.5 ** |
| The Rhizoma Osmundae extract high dose | 2.7 | 20 | 7 | 35 | 65 | 12.9 **# |
Annotate: compare with the virus control group
*P＜0.01,
*Compare with the amantadine group P＜0.05
#P＜0.05.
The fingerprint characteristic analysis of experimental example 3 Rhizoma Osmundae extract HPLC-ESI/APCI-MS analysis and collection of illustrative plates
1. experimental section
1.1 reagent and sample
Acetonitrile is the HPLC level, and water is the MiliQ ultra-pure water, and formic acid, petroleum ether (60-90 ℃ of boiling range), ethyl acetate, n-butyl alcohol and 95% ethanol are analytical pure.
1.2 sample treatment
The 50g Rhizoma Dryopteris Crassirhizomatis is ground into coarse powder, ethanol water with 500mL50% divides heating and refluxing extraction 3 times, merge extractive liquid,, vacuum rotary steam is used the 50mL water dissolution to paste, use 50mL ethyl acetate, n-butanol extraction each 3 times respectively, the nearly back drying under reduced pressure of doing of steaming is revolved in the butanol extraction liquid merging, add an amount of dissolve with methanol, carry out the liquid chromatography-mass spectrography analysis.
1.3 instrument and condition
U.S. Agilent company 1100 series liquid chromatograph-ion trap mass spectrometry coupling instrument and work station.Chromatographic column: the XDB-C18 post (150mm * 2.1mm i.d., 3.5um); Binary gradient elution, A are acetonitrile mutually, and B is water mutually, all contains 1% formic acid; The gradient program: the 20%A phase, keep 2min, increase A phase ratio gradually, A is 100% mutually during to 50min, keeps 10min; Flow velocity: 0.5mL/min; Sample size 20 μ L.
Mass spectrum condition: ESI ion source, positive and negative ion mode detection; The APCI ion source, positive ion mode detects; Nebulizer N
2Pressure 40psi; Dry gas N
2Flow velocity 9L/min; Mass scanning scope 100-1000u; Automatic 3 grades of mass spectrums (selecting the strongest ion to carry out inducing cracking in the source automatically).
2. result and analysis
2.1 HPLC-APCI-MS (+) analysis result and finger print information thereof
The total ion current chromatograph that the HPLC-APCI-MS of Rhizoma Osmundae extract analyzes is seen Fig. 1.After normalization, the relative area of each chromatograph component peaks sees Table 3-1 among Fig. 1.Table 3-1 gives secondary and three grades of mass spectrums of leading ion in ion that each chromatographic component one-level mass spectrum comprises and the one-level mass spectrum.The detected ion of APCI positive ion mode generally is [M+H]
+Ion also has a small amount of [M+NH
4]
+Therefore ion can infer the possible molecular weight that each chromatographic component according to the one-level mass spectrum.The one-level mass spectrum of part chromatographic component comprises a plurality of ions, and these ionic second order mses show and do not have primary and secondary relation between the mass spectral different ions of its one-level, illustrate that these chromatographic components contain chemical constituent more than 2 kinds or 2 kinds.Table 3-1 shows that HPLC-APCI-MS (+) can detect the material of 25 kinds of different molecular weights in the Rhizoma Osmundae extract.Wherein molecular weight is its chromatographic peak area maximum of composition of 404, is 17.9%, secondly is respectively several compositions of 612 (15.7%), 626,418 (8.3%), 265 (8.0%) for molecular weight.The peak area of each component depends on ionizing efficiency and the concentration thereof of this component in mass spectrograph, though can not infer the relative amount of this component according to the peak area of component, if but each component similar homologue that is molecular structure, their ionizing efficiency is suitable, then can infer the relative amount of component according to the total ion current chromatographic peak area.
Table 3-1 Rhizoma Osmundae extract HPLC-APCI-MS (+) analyzing total ion flow chromatographic peak component finger print information
| Sequence number | Retention time tR/min | Relative peak area (%) | MS 1Leading ion (m/z) | Main daughter ion | The relative molecular mass that component is possible | |
| MS 2(m/z) | MS 3(m/z) | |||||
| 1 | 0.8 | 8.0 | 266 *，248 | 248 | 230 | 265 |
| 2 | 1 1.1 | 4.6 | 355 | 289 *，337，319，259 | 205 | 354 |
| 3 | 12.8 | 2.7 | 453 *，333，373 | 283，301，315 | 452 | |
| 4 | 15.2 | 4.0 | 349 *，331 | 209，331 *，221 | 285 | 348 |
| 5 | 17.3 | 2.6 | 453 *，317，306，290 | 435 *，322 | 322，209 | 452 |
| 6 | 18.8 | 6.2 | 289 *，435，451 | 163，179，153，271 | 288 | |
| 7 | 20.0 | 1.1 | 517，373 *，535，355 | 355，337，277 | 517，373 | |
| 8 | 29.6 | 1.8 | 295 | 277 | 259，241 | 294 |
| 9 | 35.2 | 1.4 | 391 *，451 | 183 *，197，209 | 165，141 | 390 |
| 10 | 37.5 | 2.4 | 405 | 387，371 | 404 | |
| 11 | 38.8 | 7.6 | 419 | 211 | 193，165 | 418 |
| 12 | 39.6 | 1.5 | 405 *，295，209 | 209 *.191 | 191 | 404 |
| 13 | 41.9 | 17.9 | 405 | 197 *，209 | 179 | 404 |
| 14 | 42.9 | 1.9 | 413 | 412 | ||
| 15 | 44.7 | 1.9 | 419 | 197 *，209，223，401 | 179，155 | 418 |
| 16 | 47.3 | 4.3 | 463； 405； 613 | 443 *，167； 209，197，223 *； 403，417 | 415； 205； | 462； 404； 612 |
| 17 | 47.9 | 8.3 | 627； 419 | 417； 209 *，223，197 | 209，197； 191 | 626； 418 |
| 18 | 49.0 | 2.5 | 599 | 417，403，391 * | 209，195 | 598 |
| 19 | 50.0 | 15.7 | 613 *，405，417 | 417 *，405 | 209，197 | 612 |
| 20 | 55.2 | 3.5 | 697； 397 | 516 *，534，262； 243 *，257，287 | 262 161，187 | 696； 396 |
*. secondary and three grades of mass spectrometry precursor ions of expression respective components.
2.2 HPLC-ESI-MS (+) analysis result and finger print information thereof
The total ion current chromatograph that the HPLC-ESI-MS (+) of Rhizoma Osmundae extract analyzes is seen Fig. 2.After normalization, the relative area of each chromatograph component peaks sees Table 3-2 among Fig. 2.Table 3-2 gives secondary and three grades of mass spectrums of leading ion in ion that each chromatographic component one-level mass spectrum comprises and the one-level mass spectrum.The detected ion of ESI positive ion mode generally is [M+H]
+Ion, [M+Na] appears in part molecule simultaneously
+Ion, the m/z of the two differs 22.Infer the possible molecular weight that each chromatographic component according to the one-level mass spectrum.In table 3-2, there is the one-level mass spectrum of 18 chromatographic components to comprise m/z simultaneously and differs two kinds of ions of 22, be that this component ionization has formed [M+H] simultaneously
+, [M+Na]
+Ion.Such ion pair appears at the molecular weight that helps more accurate supposition component in the same chromatographic peak simultaneously.Among the table 3-2 in the one-level mass spectrum of retention time 21.2min chromatographic component m/z679,701 may be respectively component molecule ([M+H]
+: [2M+H] 340)
+[2M+Na]
+The peak.Table has the one-level mass spectrum of 31 chromatographic peaks to comprise a plurality of ions among the 3-2, and these ionic second order mses show and do not have the primary and secondary relation between the mass spectral different ions of its one-level, illustrate that these chromatographic components contain chemical constituent more than 2 kinds or 2 kinds.Though among the table 3-2 a plurality of chromatographic peak retention time differences are arranged, but the molecular mass of inferring according to their mass spectrum is identical, obviously they are not the identical in structure materials, but the different composition of structure, some chromatographic components with identical one-level, secondary and three grades of ionic different retention times of mass spectrum are likely isomers.Molecular weight is its chromatographic peak area maximum of composition of 452 among the table 3-2, is 12.8%, secondly is respectively several compositions of 339,348 (10.9%), 380,265,364 (8.8%), 474,226 (5.4%) for molecular weight.
Table 3-2 Rhizoma Osmundae extract HPLC-ESI-MS (+) analyzing total ion flow chromatographic peak component finger print information
| Sequence number | Retention time t R/min | Relative peak area (%) | MS 1Leading ion (m/z) | Main daughter ion | The relative molecular mass that component is possible | |
| MS 2(m/z) | MS 3(m/z) | |||||
| 1 | 1.0 | 8.8 | 381； 266； 365 | 200，219，236； 248； 203 *，236 | 230，182； 167，185 | 380； 265； 364 |
| 2 | 3.9 | 2.9 | 475 *，249，227 | 249 | 474，226# | |
| 3 | 4.5 | 5.4 | 475 *，249，227 | 249 | 474，226# | |
| 4 | 10.8 | ＜0.1 | 355 *，377，311 | 337，289，319 | 354# | |
| 5 | 12.6 | 3.1 | 453； 362； 340 | 301，163，247，315； 344； 322 | 326，298 | 452； 339# |
| 6 | 13.4 | 1.2 | 362 *，340 | 344，249 | 339 | |
| 7 | 13.7 | 10.9 | 362，340 *； 349 | 322； 209，221，331，313 | 209，226 | 339#； 348 |
| 8 | 14.9 | 2.5 | 349 *，371，387 | 209 *，221，331，313 | 191 | 348#，386 |
| 9 | 16.1 | 2.0 | 343，457，475 * | 457 *，362，313 | 439，295 | 342，474 |
| 10 | 17.3 | 12.8 | 475； 453 | 457，362 *； 435 | 249； 322，209 | 452# |
| 11 | 18.6 | 1.2 | 457 *，597 289； | 279； 163，153； | 456，596； 288 | |
| 12 | 19.3 | 1.9 | 588； 566 | 476； 548，435，322，209 | 565# | |
| 13 | 19.5 | 3.4 | 588，566 * | 548 *，435，322 | 435，322，209 | 565# |
| 14 | 21.2 | 2.8 | 340； 701 *，679 | 331； 685 | 322，209 | 339#§ |
| 15 | 21.6 | 2.7 | 441 *，457，221 | 263，201 | 220§ | |
| 16 | 22.4 | 0.5 | 397 *，407 | 387 *，322，209 | 322，209 | 396 |
| 17 | 22.8 | ＜0.1 | 441 *，267 | 263 | 440 | |
| 18 | 23.3 | 2.0 | 441，453 *，475 | 444，387 | 452#，440 | |
| 19 | 23.9 | 0.7 | 441 *，457 | 279，351 | 440 | |
| 20 | 25.4 | 0.9 | 279 *； 221 *，203，239 | 149，205； 203 | 147 | 278； 220 |
| 21 | 28.5 | ＜0.1 | 427； 497 *，369 | 269，409，327； 209 | 191，149 | 426； 496，368 |
| 22 | 29.5 | 2.8 | 353； 295 *，277 | 335； 277 | 317； 259，241 | 352； 294 |
| 23 | 30.8 | ＜0.1 | 497 *，352，311，211 | 289，301，479 | 496；210；310 | |
| 24 | 31.3 | 1.3 | 353； 311，557 * | 335，261； 349 *，539，405 | 331，313，285 | 352； 556，310 |
| 25 | 31.6 | ＜0.1 | 415 *，237 | 396 | 414，236 | |
| 26 | 32.2 | ＜0.1 | 409； 499； | 391，299； 347，291； | 408； 498 | |
| 27 | 32.8 | ＜0.1 | 409；351 * | 333； | 408，350 | |
| 28 | 33.3 | ＜0.1 | 579 *，619 | 561 | 337 | 578，618 |
| 29 | 33.9 | ＜0.1 | 555； 595 | 537； 415 | 177 | 554； 594 |
| 30 | 34.4 | ＜0.1 | 579； 574 *，539 | 353，561； 556，331 | 578； 573，538 | |
| 31 | 34.9 | ＜0.1 | 355 *，391 | 337，263； | 354，390 | |
| 32 | 35.2 | ＜0.1 | 531，509 | 508# | ||
| 33 | 35.5 | ＜0.1 | 478 | 460，337 | 477 | |
| 34 | 35.9 | ＜0.1 | 355，377 | 354# | ||
| 35 | 36.3 | 2.6 | 520 | 502 *，184 | 443，163 | 519 |
| 36 | 37.3 | ＜0.1 | 529，267 * | 211，155 | 528，266 | |
| 37 | 37.9 | 3.2 | 279； 496 | 243，261，209； 478 *，184 | 419 | 278； 495 |
| 38 | 38.5 | 2.0 | 419 | 211 | 193 | 418 |
| 39 | 39.4 | 1.3 | 405 *，295 | 209 | 191 | 404 |
| 40 | 40.3 | 1.3 | 301； 279 | 245，283； 149，205 | 300； 278 | |
| 41 | 40.7 | ＜0.1 | 205 *，405 | 149 | 404 | |
| 42 | 41.5 | 3.4 | 405 *，427； 419 | 197 *，209，179； 223 | 179 205 | 404#； 418 |
| 43 | 42.1 | 2.8 | 495 *，517 | 477 *，459，409 | 459，441 | 494# |
| 44 | 42.7 | 1.8 | 413； 453 | 331，275 *； 245 | 257 | 412； 452 |
| 45 | 44.3 | 0.8 | 419 *，377，355 | 197，211，401 | 418，354# | |
| 46 | 46.1 | 1.0 | 481，459 * | 441 * | 413 | 480，458 |
| 47 | 47.0 | 2.2 | 613 *，585 | 403，417，391 | 612，584 | |
| 48 | 47.3 | 2.1 | 463 *，485 | 167，443 | 462# | |
| 49 | 48.4 | ＜0.1 | 461，483 | 460# | ||
| 50 | 49.1 | 1.3 | 641 *，613，627 | 431 | 640，612，626 |
| 51 | 49.5 | 2.2 | 613 | 417 | 612 | |
| 52 | 52.5 | 2.2 | 413，391 *，429 | 149，279，260 | 412，390，428 |
Annotate:
*. secondary and three grades of mass spectrometry precursor ions of expression respective components; #. represents that this relative molecular mass value is that one-level mass spectrum according to respective components contains m/z and differs 22 ion pair and infer; §. represent that this relative molecular mass value is that one-level mass spectrum according to respective components contains the ion pair that m/z differs a times (dimer) and infers.
2.3 HPLC-ESI-MS (-) analysis result and finger print information thereof
The total ion current chromatograph that the HPLC-ESI-MS (-) of Rhizoma Osmundae extract analyzes is seen Fig. 3.After normalization, the relative area of each chromatograph component peaks sees Table 3-3 among Fig. 3.Table 3-3 gives secondary and three grades of mass spectrums of leading ion in ion that each chromatographic component one-level mass spectrum comprises and the one-level mass spectrum.The ESI negative ion mode is detected to be [M-H]
-Ion, the molecular weight that component is possible are the mass spectral m/z+1 of one-level.Though among the table 3-3 a plurality of chromatographic peak retention time differences are arranged, but the molecular mass of inferring according to their mass spectrum is identical, show that they are the different compositions of structure, the chromatographic component that wherein has identical one-level, secondary and three grades of ionic different retention times of mass spectrum is likely isomers.Among the table 3-3 in the one-level mass spectrum of retention time 14.8min chromatographic component m/z 695 are m/z 347 ionic two times, in the HPLC-ESI-MS of identical retention time (+) one-level mass spectrum, comprise m/z 349, therefore can infer that the relative molecular mass of this component is 348Da, m/z 695 is m/z 347 ionic dimers.Molecular weight is that the chromatographic peak area of several compositions of 612,626,404,418 is bigger among the table 3-3.
Table 3-3 Rhizoma Osmundae extract HPLC-ESI-MS (-) analyzing total ion flow chromatographic peak component finger print information
| Sequence number | Retention time t R/min | Relative peak area (%) | MS 1Leading ion (m/z) | Main daughter ion | The relative molecular mass that component is possible | |
| MS 2(m/z) | MS 3(m/z) | |||||
| 1 | 0.7 | 2.2 | 377 *，538 | 341 | 179 | 378，539 |
| 2 | 12.5 | 0.5 | 517； 453； 535 | 451，331； 331，361； 413 | 518； 454； 536 | |
| 3 | 14.8 | 1.0 | 695； 347 | 347； 328，259，303 | 348§ | |
| 4 | 16.0 | 642，662，865 | 643，663，866 | |||
| 5 | 17.1 | 519； 595 | 399，279，309，501； 459 | 520； 596 | ||
| 6 | 18.6 | 0.6 | 596 *，659 | 459 | 357，235 | 597，660 |
| 7 | 21.6 | 0.4 | 463； 581 *，480 | 417； 397 | 464； 582，481 | |
| 8 | 29.6 | 0.6 | 513 *，330 | 195 | 151 | 514，331 |
| 9 | 31.2 | 0.9 | 555 | 347 | 259，329 | 556 |
| 10 | 32.2 | 0.3 | 497 | 195，289 | 498 | |
| 11 | 32.6 | 0.5 | 691 | 495 *，483 | 207，343 | 692 |
| 12 | 33.9 | 0.9 | 571 *，639，433 | 255，391，315 | 572，640，434 | |
| 13 | 34.4 | 0.8 | 555 | 225，299 | 556 | |
| 14 | 34.9 | 1.5 | 389 *，581 | 195，204 | 390，582 | |
| 15 | 37.1 | 1.5 | 403 *，375 | 195 *，207 | 151，139 | 404，376 |
| 16 | 38.5 | 4.3 | 417 | 209 *，221，195 | 165，139 | 418 |
| 17 | 39.3 | 2.3 | 403 | 207 *，195 | 137，163 | 404 |
| 18 | 41.5 | 4.6 | 403 | 195 *，207 | 151 | 404 |
| 19 | 43.3 | 0.5 | 431； 641 | 221； 433 | 151，203 | 432； 642 |
| 20 | 44.3 | 2.3 | 417 | 209 *，221，195 | 165，189 | 418 |
| 21 | 44.8 | 583 | 375，387，207 | 584 | ||
| 22 | 45.5 | 2.7 | 597； 637 | 389 *，401； 430，441 | 221 | 598； 638 |
| 23 | 46.6 | 2.5 | 583 | 375，387，207 | 584 | |
| 24 | 46.9 | 5.6 | 611； 585 | 403 *，414； 375，387 | 207，221 | 612； 586 |
| 25 | 47.5 | 9.4 | 625 | 417 *，429 | 221，207 | 626 |
| 26 | 48.5 | 3.1 | 597 *，611 | 389 *，401，415 | 207 | 598；612 |
| 27 | 49.0 | 3.3 | 639 *，611 | 417 *，429 | 221，207 | 640；612 |
| 28 | 49.5 | 17.2 | 611 | 403 *，414 | 207 | 612 |
| 29 | 49.8 | 1.0 | 611，653 * | 417 | 221 | 612；654 |
| 30 | 50.5 | 1.4 | 611，625 * | 417，403 | 612；626 | |
| 31 | 51.4 | 8.0 | 625； 791 | 403 *，417，207； 583 | 207； 389，400，193 | 626； 792 |
| 32 | 52.2 | 3.2 | 639 | 403 | 207 | 640 |
| 33 | 52.7 | 3.1 | 833 | 625 | 417，429 | 834 |
| 34 | 53.6 | 2.4 | 619； 819 *，847 | 412 *，423； 611 *，403 | 367，208； 403，415 | 620； 820；848 |
| 35 | 54.2 | 2.2 | 819 | 611 *，403 | 403，415 | 820 |
| 36 | 56.7 | 6.0 | 611 *，403 | 403 | 195，207 | 612 |
| 37 | 57.8 | 1.9 | 637 | 429 *，207 | 207 | 638 |
| 38 | 58.5 | 1.4 | 637 | 429，207 | 638 |
Annotate: §. represent that this relative molecular mass value is that one-level mass spectrum according to respective components contains the ion pair that m/z differs a times (dimer) and infers.
2.4 total ion current chromatograph that different ionization modes obtain and component mass spectrum are relatively
Table 3-4 two or three ionization pattern is detected Rhizoma Osmundae extract component simultaneously
| Sequence number | Retention time tR/min | MS 1Leading ion | Component relative molecular mass Da | ||
| APCI(+) | ESI(+) | ESI(-) | |||
| 1 | 0.8(APCI)，1.0(ESI+) | 266 *，248 | 381，266 *，365 | 265 | |
| 2 | 11.1(APCI)，10.8(ESI+) | 355 * | 355 *，377，311 | 354 | |
| 3 | 12.8(APCI)，1 2.6(ESI+) | 453 *，333，373 | 453 *，362，340 | 452 | |
| 4 | 15.2(APCI)，14.9(ESI+)， 14.8(ESI-) | 349 *，331 | 349 *，371，387 | 695，347 * | 348 |
| 5 | 17.3 | 453 *，317，306 | 475，453 * | 453 | |
| 6 | 18.8(APCI)，18.6(ESI+) | 289 *，435，451 | 457，597，289 * | 288 | |
| 7 | 29.6(APCI)，29.5(ESI+) | 295 * | 353，295 *，277 | 294 | |
| 8 | 31.3(ESI+)，31.2(ESI-) | 353，311，557 * | 555 * | 556 | |
| 9 | 35.2(APCI)，34.9(ESI+)， 34.9(ESI-) | 391 *，451 | 355，391 * | 389 *，581 | 390 |
| 10 | 37.5(APCI)，37.1(ESI-) | 405 * | 403 *，375 | 404 | |
| 11 | 38.8(APCI)，38.5(ESI+)， 38.5(ESI-) | 419 * | 419 * | 417 * | 418 |
| 12 | 39.6(APCI)，39.4(ESI+)， 39.3(ESI-) | 405 *，295，209 | 405 *，295 | 403 * | 404 |
| 13 | 41.9(APCI)，41.5(ESI+)， 41.5(ESI-) | 405 * | 405 *，427，419 | 403 * | 404 |
| 14 | 42.9(APCI)，42.7(ESI+) | 413 * | 413 *，453 | 412 | |
| 15 | 44.7(APCI)，44.3(ESI+)， 44.3(ESI-) | 419 * | 419 *，377，355 | 417 * | 418 |
| 16 | 47.3(APCI)，47.0，47.3(ESI+)， 46.6，46.9(ESI-) | 463 *，405，613 * | 613 *，585 *； 463 *，485 | 583 *； 611 *，585 | 462，612， 584 |
| 17 | 47.9(APCI)，47.5(ESI-) | 627 *，419 | 625 * | 626 | |
| 18 | 49.0(APCI)，48.5(ESI-) | 599 * | 597 *，611 | 598 | |
| 19 | 50.0(APCI)，49.5(ESI+)， 49.0，49.5(ESI-) | 613 *，405，417 | 613 * | 639，611 * | 612 |
*. infer the ion of component relative molecular mass foundation.
Although the chromatographic separation condition of Rhizoma Osmundae extract is in full accord, there were significant differences for the total ion current chromatograph that different ionization patterns obtain and the mass spectrum of each chromatographic component.The chromatographic component that ESI cation detecting pattern obtains is maximum, totally 52; Secondly be ESI anion detecting pattern, detect 38 kinds of chromatographic components altogether; The chromatographic peak component that APCI cation detecting pattern obtains is minimum, only has 20.The chromatographic component retention time that compares three kinds of different detecting patterns, the detected component major part of ESI negative ion mode is the component that goes out the peak at retention time 30min later on as can be seen, and the detected component of ESI positive ion mode is distributed in the scope of retention time 0-55min, show that the bigger component of polarity is difficult for losing the proton belt negative charge in the ESI ionization source, the component of opposed polarity is more easily caught H in the ESI ionization source
+And Na
+Ion generates quasi-molecular ion.Most of component of Rhizoma Osmundae extract in the APCI ionization source can not with H
+In conjunction with the generation ion, so the component that APCI (+) mode detection arrives is less.More different ionization modes are at the one-level mass spectrum of the chromatographic component of identical retention time, three kinds of detected ions of ionization mode are not quite identical as can be seen, table 3-4 has compared three kinds of detecting patterns at the detected one-level mass spectrum of same retention time ion, and all can detected ion under two or three ionization pattern infers the relative molecular mass that the composition that this component contains according to identical retention time chromatographic component.Table 3-4 result show, having 9 components can be detected under APCI (+), ESI (+) and three kinds of ionization patterns of ESI (-) simultaneously, having 8 components can be detected under APCI (+) and two kinds of ionization patterns of ESI (+) simultaneously, and having 3 components can be detected under APCI (+) and two kinds of ionization patterns of ESI (-) simultaneously.APCI (+) and ESI (+) simultaneously detected component mainly preceding half section of chromatographic isolation (R.T＜32.0min), three kinds of patterns simultaneously detected component mainly at second half section of chromatographic isolation (R.T＞32.0min).The one-level mass spectrum of the chromatographic component that three kinds of ionization mode detection of more identical retention time arrive, the bigger component of the easy detected mass number of ESI (-) as can be seen, and APCI (+) and ESI (+) easily detect the less component of mass number.
2.5 the structure of poly phloroglucinol analog derivative is inferred
Existing studies show that, poly phloroglucinol analog derivative is widely distributed at the Aspidium plant species, is the biomarker of Aspidium plant.Wu Shoujin, height increase equality and discover that the Rhizoma Dryopteris Crassirhizomatis kind contains 9 kinds of phloroglucinol analog derivatives such as albaspidin AA, flavaspidic acid AB, Filixic Acids ABP, Filixic Acids ABA, filixic acid ABA, dryocrassin ABBA, dryocrassin ABAA Dryocrassin., dryocrassin ABBP, dryocrassin ABPA.This experiment utilizes the HPLC-ESI/APCI-MS coupling to analyze basket fern effective parts, has obtained the multi-stage ms figure of total ions chromatogram and each chromatographic component of effective site.Under the detecting pattern of ESI (+) and APCI (+), comprise the characteristic ion of phloroglucinol derivants such as m/z 191,193,197,209,211,223,391,403,417,431 in the mass spectrum of a plurality of chromatographic components; Under the detecting pattern of ESI (-), comprise the characteristic ion of phloroglucinol derivants such as m/z 195,207,209,211,389,401,403,417,429 in the mass spectrum of a plurality of chromatographic components.Forefathers to the Aspidium plant on the research report basis of phloroglucinol analog derivative chemical constitution, the present invention carries out structure according to the mass spectrum of basket fern effective parts chromatographic component to the component that contains above characteristic ion and derives, determined to contain in the Rhizoma Dryopteris Crassirhizomatis effective site 14 kinds of poly phloroglucinol analog derivatives, be respectively: root bark pyrone PB, flavaspidic acid AA, albaspidin AA, pannol AA, pannol AP, flavaspidic acid AB, Filixic Acids ABA, filixic acid ABA, Filixic Acids ABB, Filixic Acids PBP, dryocrassin ABAA Dryocrassin., dryocrassin ABPP, dryocrassin ABBP, dryocrassin PBBP etc.Wherein 9 kinds of chemical compounds such as root bark pyrone PB, flavaspidic acid AA, pannol AA, pannol AP, Filixic Acids ABA, Filixic Acids ABB, Filixic Acids PBP, dryocrassin ABPP, dryocrassin PBBP are the noval chemical compound of finding in Rhizoma Dryopteris Crassirhizomatis first.Their quasi-molecular ion structure and possible lytic pathway thereof are seen Fig. 4.
Three kinds of mass spectrum detecting patterns all detect Rhizoma Osmundae drug effect position and contain multiple poly phloroglucinol derivant.Wherein in the total ion current chromatograph that APCI (+) detecting pattern obtains, 12 chromatographic components contain the feature mass spectrum ion of phloroglucinol derivant, its chromatographic peak area sum accounts for 61.6% of whole component chromatographic peak areas, and wherein retention time is that the component peaks area of 41.9min accounts for 17.9%, [M+H]
+Be m/z 405, its chemical constituent is that molecular weight is 404 pannol AA or albaspidin AA; Retention time is that the component peaks area of 50.0min accounts for 15.7%, [M+H]
+Be m/z 613, its chemical constituent is that molecular weight is 612 filixic acid ABA or Filixic Acids ABP.In the total ion current chromatograph that ESI (-) detecting pattern obtains, 19 chromatographic components contain the feature mass spectrum ion of phloroglucinol derivant, its chromatographic peak area sum accounts for 77.8% of whole component chromatographic peak areas, and wherein retention time is that the component peaks area of 49.5min accounts for 17.2%, [M-H]
-Be m/z 611, its chemical constituent is that molecular weight is 612 filixic acid ABA or Filixic Acids ABP, and the result is consistent with APCI (+); Retention time is that the component peaks area of 47.5min accounts for 9.4%, [M-H]
-Be m/z 625, its chemical constituent is that molecular weight is 626 Filixic Acids ABB.In the total ion current chromatograph that three kinds of ionization patterns obtain, the chromatographic component that a plurality of retention times are different contains the ion of identical mass-to-charge ratio, in the total ions chromatogram that obtains in APCI (+) pattern, in retention time is the ion that the chromatographic component of 37.5min, 39.6min, 41.9min, 47.3min all contains m/z 405, and the chemical substance of their correspondences is that molecular weight is albaspidin AA, pannol AA and other isomers of 404Da.In addition, molecular mass is 418,612,626, the pairing poly phloroglucinol of 640Da derivant also has isomers more than 2 kinds or 2 kinds.
In the total ion current chromatograph that two kinds of detecting patterns of APCI (+) and ESI (-) obtain, the component peaks area that contains poly phloroglucinol derivant accounts for 61.6% and 77.8% of whole component chromatographic peak areas respectively, shows that poly phloroglucinol analog derivative is the main component of Rhizoma Osmundae resisiting influenza virus effective site.The chromatographic component that ESI (+) detecting pattern obtains more (52), but contain the ionic chromatographic component of phloroglucinol derivant feature mass spectrum then less (8), and its chromatographic peak area sum only accounts for 12.4% of whole component chromatographic peak areas, be starkly lower than APCI (+) (61.6%) and the detected poly phloroglucinol of ESI (-) (77.8%) derivant chromatographic component peak area, show that the ionizing efficiency of phloroglucinol derivant under APCI (+) and ESI (-) is significantly higher than the ionizing efficiency under ESI (+).
Claims (9)
1, a kind of effective composition and/or the pharmaceutically influenza of acceptable adjuvant and medicine of bird flu virus of containing, it is characterized in that: described effective ingredient is a Rhizoma Osmundae extract.
2, medicine as claimed in claim 1, Rhizoma Osmundae extract wherein prepares by the following method: with Rhizoma Osmundae tuber powder essence, with the ethanol water reflux, extract, of envelope-bulk to weight ratio 4-10 50-90% doubly 2-4 time, merge extractive liquid,, concentrate, with water dissolution concentrated solution to aqueous solution weight be Rhizoma Osmundae medical material weight 1-1.5 doubly, extract 2-3 time respectively with ethyl acetate and n-butyl alcohol successively, volume and Rhizoma Osmundae aqueous solution volume with extract is 1: 1 at every turn, merge butanol extraction liquid, the volatilization n-butyl alcohol, concentrate paste liquid, drying.
3, medicine as claimed in claim 2, the drying that it is characterized in that described paste liquid can be oven drying at low temperature or spray drying.
4,, it is characterized in that containing in the Rhizoma Osmundae extract oligomer of phloroglucinol derivant as the arbitrary described medicine of claim 1-3.
5, as the arbitrary described medicine of claim 1-3, it is characterized in that containing in the Rhizoma Osmundae extract root bark pyrone PB, flavaspidic acid AA, albaspidin AA, pannol AA, pannol AP, flavaspidic acid AB, Filixic Acids ABA, filixic acid ABA, Filixic Acids ABB, Filixic Acids PBP, dryocrassin ABAA Dryocrassin., dryocrassin ABPP, dryocrassin ABBP, dryocrassin PBBP.
6, medicine as claimed in claim 5 is characterized in that it is pharmaceutically acceptable dosage form of powder, tablet, capsule, oral liquid or other.
7, a kind of test right requires the method for the arbitrary described medicine of 1-5, be with high performance liquid chromatography-esi-msn combined instrument Rhizoma Osmundae extract to be analyzed to obtain the finger printing of extract, wherein chromatographiccondition is: chromatographic column: the XDB-C18 post; Binary gradient elution, A are acetonitrile mutually, and B is water mutually, all contains 1% formic acid, and flow velocity is 0.5mL/min; The mass spectrum condition is: ESI ion source, positive and negative ion mode detection; The APCI ion source, positive ion mode detects; Automatic 3 grades of mass spectrums.
8, detection method as claimed in claim 7, the gradient program that it adopts is: the 20%A phase, keep 2min, increase A phase ratio gradually, A is 100% mutually during to 50min, keeps 10min; Flow velocity: 0.5mL/min; Sample size 20 μ L.
9, detection method as claimed in claim 7 is characterized in that the mass spectrum condition is: ESI and APCI ion source, positive and negative ion mode detection; Nebulizer N
2Pressure 40psi; Dry gas N
2Flow velocity 9L/min; Mass scanning scope 100-1000u; Automatic 3 grades of mass spectrums.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698465A (en) * | 2013-12-20 | 2014-04-02 | 新疆医科大学 | Quality detecting method of cyrtomium rhizome medical material and thin-layer plate special for cyrtomium rhizome |
| CN104068277A (en) * | 2014-07-04 | 2014-10-01 | 芜湖欧标农业发展有限公司 | Application of adiantum soboliferum in preparation of chicken feed additive for resisting bird flu |
| CN107661322A (en) * | 2017-01-13 | 2018-02-06 | 北京中医药大学 | The new application of phloroglucinol derivatives compound in thick wood-fern rhizome |
| CN109908120A (en) * | 2019-04-22 | 2019-06-21 | 井冈山大学 | The narrow top shield-fern effective component of one kind, extracting method and its application |
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2006
- 2006-02-22 CN CNB2006100113414A patent/CN100556415C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698465A (en) * | 2013-12-20 | 2014-04-02 | 新疆医科大学 | Quality detecting method of cyrtomium rhizome medical material and thin-layer plate special for cyrtomium rhizome |
| CN104068277A (en) * | 2014-07-04 | 2014-10-01 | 芜湖欧标农业发展有限公司 | Application of adiantum soboliferum in preparation of chicken feed additive for resisting bird flu |
| CN107661322A (en) * | 2017-01-13 | 2018-02-06 | 北京中医药大学 | The new application of phloroglucinol derivatives compound in thick wood-fern rhizome |
| CN107661322B (en) * | 2017-01-13 | 2019-09-13 | 北京中医药大学 | The new application of phloroglucinol derivatives compound in thick wood-fern rhizome |
| CN109908120A (en) * | 2019-04-22 | 2019-06-21 | 井冈山大学 | The narrow top shield-fern effective component of one kind, extracting method and its application |
| CN109908120B (en) * | 2019-04-22 | 2022-04-19 | 井冈山大学 | Effective component of Dryopteris stenoptera, extraction method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN100556415C (en) | 2009-11-04 |
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