CN113827629A - Application of folium Artemisiae Argyi in treating and preventing stomach diseases caused by helicobacter pylori - Google Patents
Application of folium Artemisiae Argyi in treating and preventing stomach diseases caused by helicobacter pylori Download PDFInfo
- Publication number
- CN113827629A CN113827629A CN202110630742.2A CN202110630742A CN113827629A CN 113827629 A CN113827629 A CN 113827629A CN 202110630742 A CN202110630742 A CN 202110630742A CN 113827629 A CN113827629 A CN 113827629A
- Authority
- CN
- China
- Prior art keywords
- folium artemisiae
- artemisiae argyi
- helicobacter pylori
- extract
- diseases caused
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/282—Artemisia, e.g. wormwood or sagebrush
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to application of folium artemisiae argyi in preparing a medicine for treating and preventing stomach diseases caused by helicobacter pylori. Application of folium Artemisiae Argyi in preparing medicine for treating and preventing stomach diseases caused by helicobacter pylori is provided. Tests show that the folium artemisiae argyi extract, the total flavonoids and the flavonoid compounds have a good prevention and treatment effect on gastric ulcer caused by helicobacter pylori, and the 3 compounds have remarkable inhibitory activity of toxic factors in an Hp induced AGS process, and can be used for preparing medicines or lead compounds for treating gastric diseases caused by the helicobacter pylori.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of folium artemisiae argyi in preparing a medicine for treating and preventing stomach diseases caused by helicobacter pylori.
Background
The factors causing gastric mucosa damage and gastric ulcer mainly have several aspects: alcohol, helicobacter pylori infection, drugs, stress, diseases such as cirrhosis, etc., other diseases such as gastroesophageal reflux, chemical factors, age, etc. Among them, infection with alcohol, helicobacter pylori and drugs are the most important factors.
The mechanism of ethanol-induced damage to the gastric mucosa includes direct damage and indirect damage. Direct damage includes physical and chemical damage, i.e. as an organic solvent, ethanol has dehydration function, can coagulate tissue protein, can corrode gastric mucosa after entering stomach, destroy surface mucus layer and mucus cells, and further destroy physiological environment required by normal metabolism of gastric mucosa, resulting in damage of gastric mucosa cell membrane. The indirect injury is biological injury, and ethanol can generate a series of effects on gastric mucosa and the physiological metabolic environment thereof, including changing the balance of gastric mucosal barriers, affecting gastric acid secretion, stimulating infiltration and release of inflammatory cells and inflammatory factors, changing the microcirculation state of the gastric mucosa, affecting the release of gastrointestinal hormones such as Nitric Oxide (NO) and prostaglandin (PGE2) and the like, so that a series of gastric mucosal damages are caused, proinflammatory cytokines are released, oxidative stress is induced, apoptosis is mediated, and finally biological injury of the gastric mucosa is caused.
Helicobacter pylori (Hp) infection is one of the highest incidence chronic bacterial infections in humans currently prevalent worldwide. Hp mainly parasitizes mucous membranes near the pylorus and antrum of the stomach and is closely related to the occurrence of various diseases of the stomach. The mechanism of gastric disease resulting therefrom is very complex and is a process in which multifactorial, multistage, polygenic variations are involved. It has been found that the pathogenic factors of helicobacter pylori can be classified into colonization factors (flagella and Urease) and virulence factors (serine protease (HtrA), vacuolar cytotoxin a (vaca), cytotoxin-related protein gene a (caga)) according to their properties. The fixed planting factor has strong adhesive capacity, and after Hp enters the stomach, the special spiral structure formed by the flagella and the thallus thereof provides convenient conditions for the flagella and the thallus to shuttle and fix the gastric mucus layer on the gastric mucosa. After the Hp reaches the epithelial surface, it is firmly attached to the epithelial cells by adhesins and, by means of its rich Urease (Urease), hydrolyzes urea to produce ammonia, forming a "ammonia cloud" protective layer around the cells to resist the killing action of gastric acid. Later, Hp secretes a highly evolved conserved heat shock-induced serine protease (HtrA). HtrpA secreted by Hp is actively secreted into extracellular environment, and reacts with E-cadherin (E-cadherin) which is one of cell adhesion molecule members, so that intercellular connection is opened, and a plurality of toxic pathogenic factors such as cytovacuole toxin A (VacA), cytotoxin-related protein A (CagA), gastric epithelium after-contact induced expression factor (IceA), blood group antigen binding adhesin (BabA), coded proinflammatory outer membrane protein (OiA), duodenal bulb ulcer initiation factor (DupA) and the like are secreted into cells, and finally peptic ulcer diseases such as gastric mucosa injury and the like are caused, and further, diseases such as acute gastritis, chronic gastritis and gastric cancer and the like are caused along with the development process of the diseases. Thus, one of the key features of helicobacter pylori survival under acidic conditions is the production of a large amount of Urease (Urease), the key protein for releasing virulence factors of which is serine protease (HtrA). VacA and CagA play important roles in the induction of gastric ulcer by H.pylori as toxic factors secreted in large quantities by H.pylori. The former promotes vacuolation of cells and induces apoptosis through mitochondrial pathways; the latter is involved in the production of various inflammatory factors and is involved in various carcinogenic mechanisms. Therefore, inhibition of the colonization of Hp and the release and invasion of cells by dual inhibition of both Urease and HtrA, and thus VacA or CagA, is an important strategy for the treatment of Hp-induced gastric ulcers.
Folium artemisiae argyi is derived from Artemisia argyi (Artemisia argyi Levl. et Vant.) of the Compositae family, has the effects of warming channels and stopping bleeding, dispelling cold and relieving pain, warming stomach and eliminating dampness, and enters liver, spleen and kidney channels. Is mainly used for treating hematemesis, epistaxis, metrorrhagia, menorrhagia, fetal leakage, hypogastrium psychroalgia, irregular menstruation and infertility due to cold womb; it can be used for treating skin pruritus. Vinegar moxa charcoal can warm meridians and stop bleeding, and is indicated for bleeding due to deficiency-cold. Modern pharmacological studies prove that the folium artemisiae argyi has broad-spectrum antibacterial, antiviral, hemostatic and blood coagulation effects. Although the folium artemisiae argyi has great medicinal value, so far, few basic material researches on the aspect of inhibiting the helicobacter pylori are carried out at home and abroad, and the clinical application of the folium artemisiae argyi is greatly limited.
Disclosure of Invention
The invention aims to provide an artemisia leaf active extract, an artemisia leaf total flavone, a flavonoid monomer compound in artemisia leaf and application thereof in preparing a medicine for treating stomach diseases caused by helicobacter pylori based on the current situation and the foundation of the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
application of folium Artemisiae Argyi in treating and preventing stomach diseases caused by helicobacter pylori, and application of folium Artemisiae Argyi in preparing medicine for treating and preventing stomach diseases caused by helicobacter pylori.
The folium Artemisiae Argyi is Artemisia argyi Levl. et Vant.
The folium Artemisiae Argyi is folium Artemisiae Argyi extract, folium Artemisiae Argyi total flavone purified from the extract or chemically synthesized folium Artemisiae Argyi total flavone.
The folium artemisiae argyi total flavonoids after the purification of the extract are one or more of flavonoids. Flavones (including flavone (alcohol), flavanone (alcohol), isoflavone, flavan, etc.).
The folium Artemisiae Argyi total flavone after the extract purification is one or more of 5, 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone and 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin).
The folium artemisiae argyi extract, the application of folium artemisiae argyi total flavonoids after the extract is purified, the extracted and purified or chemically synthesized 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), the extracted and purified or chemically synthesized 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone, the extracted and purified or chemically synthesized 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin) and other folium artemisiae argyi flavonoid components in preparing medicines for treating and preventing gastritis, gastric cancer and gastric ulcer caused by helicobacter pylori.
The folium artemisiae argyi extract, namely folium artemisiae argyi total flavonoids after the extract is purified, 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin) which is extracted, purified or chemically synthesized, 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone which is extracted, purified or chemically synthesized, 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin) which is extracted, purified or chemically synthesized and other folium artemisiae argyi flavonoid components are mixed with acceptable excipients in pharmacy, functional food, health care products or food to prepare the medicine for treating and preventing gastritis, gastric cancer and gastric ulcer caused by helicobacter pylori.
The folium Artemisiae Argyi extract is obtained by extracting dried leaf, stem, or whole aerial part of Artemisia argyi Levl. et Vant. of Compositae with alcohol.
The folium Artemisiae Argyi extract is prepared by extracting dried folium Artemisiae Argyi (Artemisia argyi Levl. et Vant.) with alcohol, concentrating the extractive solution, recovering solvent, and drying to obtain folium Artemisiae Argyi extract.
The alcohol is 60-95% alcohol water solution, preferably 75-95% alcohol water solution, and the extraction method comprises soaking, percolating, heating reflux, continuous heating reflux (Sabouraud extraction method), and ultrasonic extraction.
The alcohol is an alcohol solvent such as methanol or ethanol; the extraction times are 2-4 times; the ratio of the solvent dosage to the medicinal material mass is 1:4-1:12, wherein the ratio of the solvent dosage to the medicinal material mass in each immersion or percolation extraction is 1:4-1: 30; the soaking extraction time is 4-20 hr each time, and the heating reflux, or continuous heating reflux (Sabouraud extraction method), or ultrasonic extraction method is 0.5-4 hr each time.
The concentration mode is normal pressure distillation or reduced pressure distillation, and the drying mode is normal temperature heating drying, reduced pressure drying, freeze drying or spray drying, etc.
The folium artemisiae argyi total flavonoids obtained after the purification of the folium artemisiae argyi extract are obtained by further separating and purifying the folium artemisiae argyi total extract through four methods, namely a macroporous resin method, a polyamide column chromatography method, a silica gel column chromatography method or an alkali-acid method.
The macroporous adsorption resin method comprises the steps of sequentially eluting with water, 40-60% ethanol-water and 70-95% ethanol-water in volume ratio, and enriching 70-95% ethanol-water eluent. The macroporous resin is nonpolar resin, weak polar resin or medium polar resin, the nonpolar resin is selected from XAD-4, Diaion HP-20, D101, D102, D401, D1, D2, D3, D4, HPD-100 or X-5, the weak polar resin is selected from D-201, HPD-300 or AB-8, and the medium polar resin is selected from XAD-6, XAD-7 or XAD-8.
The polyamide column chromatography is sequentially eluted by 40 percent, 60 percent and 80 percent of ethanol-water according to the volume ratio, and 60 to 80 percent of ethanol-water eluate is enriched. The polyamide resin is 30-60 meshes.
The silica gel column chromatographic separation adopts a solvent gradient elution method comprising the following steps: gradient eluting with dichloromethane (or chloroform) -methanol system at volume ratio of 50:1-10: 1; or gradient eluting with petroleum ether (or n-hexane) -acetone system at volume ratio of 12:1-3: 1.
The alkali-acid method comprises the steps of dissolving folium artemisiae argyi total extract in dichloromethane-methanol solution with the volume ratio of 20:1, adding 5% of sodium carbonate solution with the same volume, extracting for 3-4 times, taking an alkali water layer, acidifying with hydrochloric acid to the pH value of 4-5, extracting for 3-4 times with ethyl acetate, concentrating under reduced pressure to recover extract, dissolving concentrate with methanol, mixing polyamide with a sample, and eluting with dichloromethane-methanol with the volume ratio of 20:1 to obtain folium artemisiae argyi total flavone.
The known flavonoid monomer compound is prepared by the following method:
performing gradient elution with solvent, performing gradient elution with dichloromethanol (chloroform) -methanol system, and performing Sephadex LH-20 gel column chromatography, ODS reverse phase silica gel column chromatography and high performance liquid chromatography to obtain compounds (I), (II) and (III); the structure of the compound (I) is 5, 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), the structure of the compound (II) is 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone, and the structure of the compound (III) is 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin) which are identified by a spectrum method.
(I)5, 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin)
(II)5,7,3',4' -tetrahydroxy-6, 5' -dimethoxyflavone
(III)5,7,4 '-Tetrahydroxy-6, 5' -Dimethoxyflavone (Brown cyanidin)
The dichloromethanol (chloroform) -methanol gradient: 100:0-100:100.
The Sephadex LH-20 gel column chromatography is carried out by using methanol or acetone as an eluting solvent.
The ODS reversed phase silica gel column chromatography adopts 50:50-100:0 methanol-water as eluent.
The high performance liquid chromatography is SHIMADZU LC-6AD (Shimadzu Japan); the column is YMC-Pack ODS-A (YMC, Japan); the eluent is methanol-water.
The invention has the advantages that:
the invention provides pharmaceutical uses of the compounds; the substances are folium artemisiae argyi total extracts, folium artemisiae argyi total flavonoids and flavonoid monomeric compounds in folium artemisiae argyi obtained by further purifying the folium artemisiae argyi total extracts, or chemically synthesized folium artemisiae argyi total flavonoids and flavonoid monomeric compounds in folium artemisiae argyi, and the obtained substances are used for measuring the Minimum Inhibitory Concentration (MIC) of helicobacter pylori and evaluating the treatment activity of rat gastric ulcer caused by the helicobacter pylori. And performing in-vitro anti-gastric mucosal injury activity evaluation by establishing an in-vitro helicobacter pylori co-incubated AGS gastric cancer cell model. And (3) testing the inhibitory activity of the toxic factor in the AGS induction process of the monomeric compound helicobacter pylori in the folium artemisiae argyi by taking the release amounts of VacA and CagA as indexes. The results show that the folium artemisiae argyi total extract, the folium artemisiae argyi total flavonoids and the flavonoid monomeric compounds in the folium artemisiae argyi have obvious treatment effect on rat gastric ulcer caused by helicobacter pylori, and the compounds in the formula (I), the formula (II) and the formula (III) have obvious inhibitory activity on toxic factors in the process of inducing AGS by helicobacter pylori, and can be used for preparing medicines or lead compounds for treating gastric diseases caused by the helicobacter pylori.
The folium artemisiae argyi extract, the folium artemisiae argyi total flavone and the flavonoid monomeric compound which have obvious treatment effect on the gastric ulcer of a rat caused by helicobacter pylori and have the inhibition activity on the toxic factor in the AGS induction process of the helicobacter pylori can be used for preparing the medicine or the lead compound for the gastric ulcer caused by the helicobacter pylori.
Drawings
FIG. 1 is a graph showing the effects of mugwort leaf extract, mugwort leaf total flavonoids and compound (I) of the present invention on the contents of TNF- α (FIG. 1A), IL-1 β (FIG. 1B) and IL-10 (FIG. 1C) in rat stomach tissue homogenate in example 3; p <0.05, P <0.01, P < 0.001.
FIG. 2 is a graph showing the effect of the compounds (I) (FIG. 2A), (II) (FIG. 2B) and (III) (FIG. 2C) of the present invention on the rate of Hp-induced suppression of AGS cells in example 4; wherein P <0.05, P <0.01, P < 0.001.
FIG. 3 is a graph showing the effect of the compounds (I) of the present invention (FIG. 3A), (II) (FIG. 3B) and (III) (FIG. 3C) on the release of the virulence factors VacA and CagA in example 5; wherein P <0.05, P <0.01, P < 0.001.
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
The invention extracts folium artemisiae argyi extract, folium artemisiae argyi total flavone and natural flavonoid monomer compounds separated from folium artemisiae argyi, including but not limited to 5, 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone or 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin) and the like; tests show that the folium artemisiae argyi extract, the total flavonoids and the compound (I) have better prevention and treatment effects on gastric ulcer caused by helicobacter pylori, and the 3 compounds have remarkable inhibitory activity of toxic factors in the process of inducing AGS by Hp and can be used for preparing medicines or lead compounds for treating gastric diseases caused by the helicobacter pylori.
Example 1 preparation of Total extract of Artemisia princeps Pampanini
Taking dried folium Artemisiae Argyi (Artemisia argyi Levl. et Vant.), drying, soaking 24kg folium Artemisiae Argyi in 95% ethanol at room temperature for 2 times, each time for 20 hr, recovering solvent under reduced pressure, and concentrating to obtain folium Artemisiae Argyi total extract (1458 g).
Wherein the folium Artemisiae Argyi material comprises dried leaf, stem, or whole aerial part of Artemisia argyi Levl. et Vant.
The mass ratio of the folium artemisiae argyi to the ethanol is 1: 10.
example 2 preparation of folium Artemisiae Argyi Total Flavonoids
Taking part of the folium artemisiae argyi total extract in the example 1, adsorbing resin color through HPD100 type macropore, eluting with water with 60% ethanol-water and 95% ethanol-water in volume ratio, concentrating and drying the eluent with 95% ethanol/water to obtain the folium artemisiae argyi total flavone.
Example 3 separation of monomeric flavonoids from mugwort leaves
Part of the total mugwort leaf extract of example 1 was dispersed in water, extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in this order, and concentrated under reduced pressure. Subjecting the ethyl acetate layer to silica gel column chromatography repeatedly, performing gradient elution with dichloromethane-methanol system (100:0 → 100:5 → 100:10 → 100:20 → 100:33 → 100:100) with each gradient dosage of 5L, collecting fractions, combining dichloromethane-methanol (100:5) and dichloromethane-methanol (100:10) fractions, subjecting to silica gel column chromatography, performing gradient elution with petroleum ether-acetone system (9:1 → 7:1 → 3:1 → 1:1), with each gradient dosage of 2L, and collecting fractions; wherein the petroleum ether-acetone (9:1) flow part is subjected to Sephadex LH-20 gel column chromatography, eluted by methanol, the flow rate is 0.8-1.5ml/min, every 5ml is one flow part, similar flow parts are combined according to TLC color development to obtain 3 components, the component 2 is subjected to semi-preparative HPLC purification to obtain the compound (I)5, 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), and the eluent is methanol-water (55: 100); purifying the component 3 by semi-preparative HPLC to obtain compound (II)5,7,3',4' -tetrahydroxy-6, 5' -dimethoxyflavone, and eluting with methanol-water (60: 100); the petroleum ether-acetone (7:1) fractions were purified by semi-preparative HPLC to give compound (III)5,7,4 '-tetrahydroxy-6, 5' -dimethoxyflavone (jaceosidin) as eluent methanol-water (58: 100). Column chromatography silica gel (200-300 mesh, Qingdao ocean chemical plant); sephadex LH-20 (GE Healthcare, Sweden); the high performance liquid chromatography is SHIMADZU LC-6AD (Shimadzu Japan); the column was YMC-Pack ODS-A (YMC, Japan).
The physicochemical properties and spectral data of the compounds of formula (I) are as follows: yellow powder (methanol) with dark spots at 254nm, ferric chloride-potassium ferricyanide reaction positive, and nuclear magnetic resonance hydrogen spectrum data are shown in Table 1.
The physicochemical properties and spectral data of the compounds of formula (II) are as follows: yellow powder (methanol) has dark spots at 254nm, has fluorescence at 365nm, is yellow in volume fraction of 10% sulfuric acid-ethanol, and is positive in ferric trichloride, and shows the existence of phenolic hydroxyl groups. The nmr data are shown in table 1.
The physicochemical properties of the compound of formula (III) are as follows: yellow powder (methanol). The ferric chloride-potassium ferricyanide reaction is positive, which indicates that phenolic hydroxyl exists, and the phenolic hydroxyl may be a flavonoid compound. The nmr data are shown in table 1.
TABLE 1 NMR spectra (600MHz, DMSO-d) of Compounds of formula (I), formula (II) and formula (III)6)
Experimental example 4 measurement of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration of the folium artemisiae argyi extract, the folium artemisiae argyi total flavonoids and the flavonoid monomeric compounds on the helicobacter pylori is determined by adopting a 96-well plate microdilution method.
The helicobacter pylori solution was diluted with Brucella broth medium at 100. mu.L/well to a final concentration of 1X 10/well6And (4) CFU. Each test compound was diluted with 100. mu.L of Brucella broth containing 5% fetal bovine serum and added to a 96-well plate, and co-cultured at 37 ℃ for 24 hours in a microaerophilic environment. The minimum inhibitory concentration was determined at 625nm using a multifunctional microplate reader.
As shown in Table 2, the compounds (I) (II) (III) showed good anti-helicobacter pylori activity, and the MICs were 16, 37 and 29. mu.g/ml, respectively.
TABLE 2 MIC values of Artemisia princeps Pampanini extract, Total Flavonoids and Compounds (I), (II) and (III) against helicobacter pylori
Experimental example 5 inhibition of in vivo gastric ulcer with mugwort leaf extract
Randomly selecting 10 rats as normal control groups, and modeling the rest rats: fasting for 12h, then gavage with 2ml 5% NaNO3After 15min, 1.5ml of helicobacter pylori is administered by intragastric administration, and after 30min, the food is taken. Infection was performed 2 times per week for a total of 3 weeks. 7 days after the last infection, tail blood of the rat is taken for serological examination, anti-helicobacter pylori related antibodies are examined, and the molding is successful if the antibody examination is positive.
100 rats successfully molded are selected and randomly divided into 10 groups, namely a model control group, a folium artemisiae argyi extract high-dose group, a folium artemisiae argyi extract medium-dose group and a folium artemisiae argyi extract low-dose group, a folium artemisiae argyi total flavone high-dose group, a folium artemisiae argyi extract medium-dose group and a eupatilin (I) high-dose group, a eupatilin (I) medium-dose group and a eupatilin (I) low-dose group, distilled water is administrated to the model control group in a gastric irrigation mode at 1.0ml/100g, folium artemisiae argyi extract 3.0g/kg (high-dose group), folium artemisiae argyi extract 1.0g/kg (medium-dose group) and eupatilin (I) low-dose group are administrated at 3.0g/kg, folium artemisiae argyi extract 1.0g/kg, eupatilin 0g/kg, eupatilin 100mg/kg and 50mg/kg are prepared into liquid medicines with corresponding concentrations, and the medicine is administrated in a gastric irrigation mode at 1 time per day for continuous administration for 15 days. After the last administration1h, killing the rat by breaking neck, opening abdominal cavity, ligating cardia and pylorus, taking out stomach, cutting along greater curvature of stomach, washing residues in stomach with cold 0.9% sodium chloride solution, measuring transverse diameter and vertical diameter of ulcer, and multiplying the two (mm)2) As an ulcer index. Ulcer index scoring criteria:
1 minute: the area of the ulcer is 1-12 mm2；
And 2, dividing: the area of the ulcer is 13-25 mm2；
And 3, dividing: the ulcer area is 26-37 mm2；
And 4, dividing: the area of the ulcer is 38-50 mm2；
And 5, dividing: the area of the ulcer is more than or equal to 50mm2Or a perforated ulcer.
For the area less than or equal to 1mm 210 points of the ulcer correspond to 1mm of ulcer area2And (6) counting. Percent ulcer inhibition ═ (ulcer index of model control group-ulcer index of administration group)/ulcer index of model control group × 100%.
Treatment of the homogenate of gastric tissue: accurately weighing the tissue weight, adding 9 times of physiological saline according to the weight volume ratio to prepare 10% tissue homogenate, centrifuging at 2500r/min for 10min, taking supernatant, subpackaging by an EP tube, and storing at-20 ℃ for later use to be tested.
TNF-alpha, IL-1 beta and IL-10 content determination: the rat TNF-alpha, IL-1 beta and IL-10 are detected by an enzyme linked immunosorbent assay (Elisa), and the operation is carried out according to the requirements of a kit instruction.
The experimental results are shown in fig. 1 and table 3, and compared with the model control group, the high and medium dose groups of the folium artemisiae argyi extract, the folium artemisiae argyi total flavone and the compound (I) have obvious prevention and treatment effects on rat gastric ulcer caused by helicobacter pylori and are in a dose-dependent relationship.
TABLE 3 Effect of mugwort leaf extract, Total Flavonoids and Compound (I) on gastric ulcer in rats caused by helicobacter pylori
Note: p <0.05, P <0.01, compared to model control group
Example 6 inhibition of Hp-induced AGS cells by monomeric flavonoid Compounds from Artemisia princeps Pampanini in vitro
AGS gastric cancer cells cultured in logarithmic growth phase are taken, and the cell density is adjusted to 3 x 10 by using fresh RPMI1640 medium containing 5% fetal bovine serum4cells/mL, seeded in 96-well plates at 100. mu.L/well at 37 ℃ in 5% CO2Culturing in the incubator. After the cells are cultured for 24 hours adherent, the cells are changed into fresh culture solution, and meanwhile, different dosage treatment is carried out. Clarithromycin (CLA), Omeprazole (OME) and the monomeric compound of Artemisia princeps obtained in the above examples were 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M and 12.5. mu.M. After 1h, the ratio of AGS/Hp quantity is 1: the Hp bacterial suspension is added according to the proportion of 50, and the mixture is co-cultured for 24 hours. CCK-8 solution was then added to the cell broth and incubated at 10. mu.L/well for 2h at 37 ℃. The absorbance OD was measured at 450 nm. The cellular inhibition rate calculation formula is as follows:
cell inhibition%
TABLE 3 inhibition of Hp-induced AGS cells in vitro by Compounds of formula (I), formula (II) and formula (III)
The experimental results are shown in fig. 2 and table 3, and the results show that the IC50 value of (II) is lower than that of the positive drug data of the same group, which indicates that the positive drug data has better performance in inhibiting Hp-induced AGS cell proliferation. And the IC50 values of (I) and (III) are only slightly higher than the positive drug data, indicating that they also play a good role in inhibiting Hp-induced AGS cell proliferation. Omeprazole, a proton pump inhibitor, is used as a clinical medicine for gastric ulcer, and has no obvious inhibition effect on AGS cell proliferation induced by Hp.
Example 7 Effect of monomeric Compounds in Artemisia princeps Pampanini on the Release amounts of the virulence factors VacA and CagA
AGS cells were plated in 96-well plates at a density of 4X 10 cells per ml4Culturing the individual cells for 24h, and diluting with RPMI1640 culture solution to different concentrations(different concentrations of 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M) monomeric compound of Artemisia princeps Pampanini and omeprazole were co-cultured for 1h, then Hp (AGS/Hp ratio 1:50) was added for co-incubation, and after 24h, the supernatant was collected. The content of VacA in the samples was determined by ELISA kit (see fig. 3), and CagA was performed as above. The specific operation method comprises the following steps:
(1) reagents, samples and standards were prepared.
(2) The prepared sample and standard were added and reacted at 37 ℃ for 30 min.
(3) Washing the plate for 5 times, adding an enzyme-labeled reagent, and reacting for 30min at 37 ℃.
(4) Washing the plate for 5 times, adding A, B color developing solution, and developing at 37 deg.C for 10 min.
(5) Adding the stop solution.
(6) Detecting and reading OD value with a microplate reader under 450nm within 15 min.
(7) And (4) drawing a standard curve by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate, and calculating the corresponding concentration according to the OD value of the sample.
As shown in FIG. 3, the compounds of formula (I) and formula (III) have superior inhibitory effects to the positive drug during the release of VacA, which are about 1.6 times and 1.5 times, respectively, that of the positive drug. The compounds of formula (I), formula (II) and formula (III) have better inhibition effect than positive drug in the CagA release process, and the action effect is about 1.4 times of the positive drug. Omeprazole has no obvious influence on the release of toxic factors VacA and CagA.
Experimental results show that the folium artemisiae argyi extract, the folium artemisiae argyi total flavonoids and the compound (I) have a remarkable prevention and treatment effect on gastric ulcer caused by helicobacter pylori. The compounds (I), (II) and (III) have obvious effect of inhibiting toxic factors in the process of inducing AGS by helicobacter pylori, and can be used for preparing medicaments or lead compounds for treating gastric ulcer caused by helicobacter pylori.
Claims (8)
1. The application of the folium artemisiae argyi in treating and preventing stomach diseases caused by helicobacter pylori is characterized in that: application of folium Artemisiae Argyi in preparing medicine for treating and preventing stomach diseases caused by helicobacter pylori is provided.
2. Use of mugwort leaves according to claim 1 for the treatment and prevention of gastric diseases caused by helicobacter pylori, characterized in that: the folium Artemisiae Argyi is Compositae plant Artemisia argyi Levl. et Vant.
3. Use of mugwort leaves according to claim 2 for the treatment and prevention of gastric diseases caused by helicobacter pylori, characterized in that: the folium Artemisiae Argyi is folium Artemisiae Argyi extract, folium Artemisiae Argyi total flavone purified from the extract or chemically synthesized folium Artemisiae Argyi total flavone.
4. Use of mugwort leaves according to claim 3 for the treatment and prevention of gastric diseases caused by helicobacter pylori, characterized in that: the folium artemisiae argyi total flavonoids after the purification of the extract are one or more of flavonoids.
5. Use of mugwort leaves according to claim 4 for the treatment and prevention of gastric diseases caused by helicobacter pylori, characterized in that: the folium Artemisiae Argyi total flavone after the extract purification is one or more of 5, 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone and 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin).
6. Use of mugwort leaves according to any one of claims 1 to 6 for the treatment and prevention of gastric diseases caused by helicobacter pylori, characterized in that: the folium artemisiae argyi extract, the application of flavonoids such as total flavonoids in folium artemisiae argyi after the extract is purified, extracted and purified or chemically synthesized 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), extracted and purified or chemically synthesized 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone, extracted and purified or chemically synthesized 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin) and the like in preparing medicines for treating and preventing gastritis, gastric cancer and gastric ulcer caused by helicobacter pylori.
7. The use of mugwort leaves according to claim 6 for the treatment and prevention of gastric diseases caused by helicobacter pylori, characterized in that: the folium artemisiae argyi extract, the total flavonoids of the folium artemisiae argyi after the extract is purified, the extracted and purified or chemically synthesized 7-dihydroxy-6, 3',4' -trimethoxy flavone (eupatilin), the extracted and purified or chemically synthesized 5,7,3',4' -tetrahydroxy-6, 5' -dimethoxy flavone, the extracted and purified or chemically synthesized 5,7,4' -tetrahydroxy-6, 5' -dimethoxy flavone (jaceosidin) and other flavonoid compounds are mixed with acceptable excipients in pharmacy, functional food, health care product or food to prepare the medicine for treating and preventing gastritis, gastric cancer and gastric ulcer caused by helicobacter pylori.
8. Use of mugwort leaves according to claim 7 for the treatment and prevention of gastric diseases caused by helicobacter pylori, characterized in that: the folium Artemisiae Argyi extract is obtained by extracting dried leaf, stem, or whole aerial part of Artemisia argyi Levl. et Vant. of Compositae with alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110630742.2A CN113827629A (en) | 2021-06-07 | 2021-06-07 | Application of folium Artemisiae Argyi in treating and preventing stomach diseases caused by helicobacter pylori |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110630742.2A CN113827629A (en) | 2021-06-07 | 2021-06-07 | Application of folium Artemisiae Argyi in treating and preventing stomach diseases caused by helicobacter pylori |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113827629A true CN113827629A (en) | 2021-12-24 |
Family
ID=78962633
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110630742.2A Pending CN113827629A (en) | 2021-06-07 | 2021-06-07 | Application of folium Artemisiae Argyi in treating and preventing stomach diseases caused by helicobacter pylori |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113827629A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114949081A (en) * | 2022-05-31 | 2022-08-30 | 海南娜古芳沉香科技有限公司 | Preparation method and application of anti-helicobacter pylori agilawood extract |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101396435A (en) * | 2008-10-27 | 2009-04-01 | 刘智谋 | Traditional Chinese medicine for treating gastrosis and preparation method and use thereof |
| CN105770673A (en) * | 2016-04-12 | 2016-07-20 | 广州三得医疗科技有限公司 | Application of Chinese herba preparation containing folium artemisiae argyi in preparing drug for treating chronic gastritis |
| CN106668110A (en) * | 2015-11-06 | 2017-05-17 | 沈阳药科大学 | Artemisia argyi extract, eupatilin, jaceosidin and preparation method and application thereof |
| CN107854507A (en) * | 2017-11-06 | 2018-03-30 | 山东省中医药研究院 | A kind of method that flavones ingredient is extracted from folium artemisiae argyi |
| CN112370476A (en) * | 2020-12-24 | 2021-02-19 | 南开大学 | Method for purifying flavonoid compounds in folium artemisiae argyi |
-
2021
- 2021-06-07 CN CN202110630742.2A patent/CN113827629A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101396435A (en) * | 2008-10-27 | 2009-04-01 | 刘智谋 | Traditional Chinese medicine for treating gastrosis and preparation method and use thereof |
| CN106668110A (en) * | 2015-11-06 | 2017-05-17 | 沈阳药科大学 | Artemisia argyi extract, eupatilin, jaceosidin and preparation method and application thereof |
| CN105770673A (en) * | 2016-04-12 | 2016-07-20 | 广州三得医疗科技有限公司 | Application of Chinese herba preparation containing folium artemisiae argyi in preparing drug for treating chronic gastritis |
| CN107854507A (en) * | 2017-11-06 | 2018-03-30 | 山东省中医药研究院 | A kind of method that flavones ingredient is extracted from folium artemisiae argyi |
| CN112370476A (en) * | 2020-12-24 | 2021-02-19 | 南开大学 | Method for purifying flavonoid compounds in folium artemisiae argyi |
Non-Patent Citations (5)
| Title |
|---|
| 张芷嫣等: "云艾散对大鼠胃溃疡愈合的影响", 《四川中医》 * |
| 戴小军等: "野艾组分对幽门螺杆菌的体外抑菌作用", 《世界华人消化杂志》 * |
| 杨楠等: "黄酮类化合物抗肿瘤活性及机制研究进展", 《中国中药杂志》 * |
| 耿文慧等: "抗疫植物艾的本草考证及艾叶化学成分与抗菌抗病毒最新研究", 《亚热带植物科学》 * |
| 胡厚才等: "蒿属植物在胃肠道疾病防治中的药理作用及其机制研究进展", 《现代药物与临床》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114949081A (en) * | 2022-05-31 | 2022-08-30 | 海南娜古芳沉香科技有限公司 | Preparation method and application of anti-helicobacter pylori agilawood extract |
| CN114949081B (en) * | 2022-05-31 | 2024-01-26 | 海南娜古芳沉香科技有限公司 | Preparation method and application of agilawood extract for resisting helicobacter pylori |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Deng et al. | Anneslea fragrans Wall. ameliorates ulcerative colitis via inhibiting NF-κB and MAPK activation and mediating intestinal barrier integrity | |
| Yesilada et al. | Anti-ulcerogenic activity and isolation of the active principles from Sambucus ebulus L. leaves | |
| CN113150048B (en) | Cyclocarya paliurus extract and application thereof in resisting rheumatoid arthritis | |
| CN101434592B (en) | Novel flavonoid extracted from Maackia amurensis | |
| CN109674802A (en) | Steroid compound purposes in preparing anti-inflammatory drugs | |
| CN113827629A (en) | Application of folium Artemisiae Argyi in treating and preventing stomach diseases caused by helicobacter pylori | |
| CN109320578B (en) | Triterpenoid saponin compound and extraction method thereof | |
| CN103191143B (en) | New application of cardiac glycoside compound | |
| CN104945460A (en) | Preparation method and applications of roxburic acid | |
| CN112876469B (en) | Lupine derivative with effects of relieving cough and reducing phlegm and preparation method thereof | |
| CN111349134B (en) | Preparation method of dammarane type triterpene compound in walnut green husk | |
| CN108148105B (en) | Dimeric iridoid compound and preparation method and application thereof | |
| CN105732736B (en) | A kind of preparation method of phenylpropanoids | |
| CN105884841B (en) | A kind of preparation method of phenylpropanoids | |
| CN106046071B (en) | A kind of preparation method of phenylpropanoids | |
| CN112898358B (en) | New compound NBY-4 extracted from folium Arctii and having antiinflammatory activity, and its preparation method and application | |
| CN109517023B (en) | Separation and purification method of chemical components of Sanjin preparation | |
| CN114773304B (en) | Linderane type sesquiterpene compound separated from herba Lespedezae Cuneatae extract and its application in preparing medicine for treating liver cancer | |
| CN113999245B (en) | Natural compound with anti-pancreatic cancer activity and separation method and application thereof | |
| CN112778255B (en) | Centipeda minima lactone L and extraction method and application thereof | |
| CN108623645A (en) | A kind of flavone compound and the preparation method and application thereof | |
| CN113072492B (en) | Isoquinoline alkaloid compound, preparation method and application | |
| CN114478700B (en) | Preparation method of nettle type cyclic peptide in cockscomb seed and application of nettle type cyclic peptide in anti-tumor drugs | |
| CN115433152B (en) | Compound separated from golden silk plum fruit, preparation method and application | |
| CN111419864B (en) | Application of currant anthocyanin in preparation of medicines for inducing apoptosis of colon cancer cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |