CN110538296B - Application of tsaoko amomum fruit extract - Google Patents
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- 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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/906—Zingiberaceae (Ginger family)
- A61K36/9064—Amomum, e.g. round cardamom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- 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
- A61P31/06—Antibacterial agents for tuberculosis
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- 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
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- 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
Abstract
The invention discloses application of a tsaoko amomum fruit extract. The invention provides application of tsaoko alcohol extract in preparation of drugs for resisting drug-resistant tuberculosis and application in preparation of drugs for inhibiting drug-resistant mycobacterium tuberculosis. The invention adopts a Soxhlet extraction method, uses ethanol with the concentration of 80-100% as a solvent for the tsaoko amomum fruit, and obtains the tsaoko amomum fruit ethanol extract by heating reflux extraction, filtration concentration and freeze drying. The test proves that the tsaoko alcohol extract has strong antituberculosis effect on mycobacterium tuberculosis, particularly multidrug-resistant mycobacterium tuberculosis, and in addition, the cytotoxicity evaluation result on the tsaoko alcohol extract proves that the tsaoko alcohol extract has no obvious cytotoxicity on human embryonic lung fibroblast MRC-5 when the concentration of the tsaoko alcohol extract is not more than 250 mu g/mL. Therefore, the invention has important significance for the development of anti-tubercle bacillus, in particular to the development of drugs for resisting drug-resistant tubercle bacillus.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of a tsaoko amomum fruit extract.
Background
Tuberculosis is a chronic infectious disease caused by infection of mycobacterium tuberculosis, is mainly a disease caused by respiratory tract transmission, and is one of the diseases which have the longest history and serious consequences and harm human life health. China is a large country with tuberculosis, and with the application of antibiotics, drug-resistant tuberculosis gradually becomes a difficult problem in tuberculosis prevention and control. At present, the tuberculosis prevention and control situation is still very severe. The worldwide tuberculosis report of 2018 provided by the world health organization shows that about 1000 million new tuberculosis patients are found in the world, and about 89 million people are found in China. It is estimated that there are 56 million drug-resistant tuberculosis occurring globally in 2017, of which about 7.3 occur in china, which is a major world of drug-resistant tuberculosis.
The western medicine chemotherapy technology can improve the cure rate of the pulmonary tuberculosis and reduce the death rate. For active tuberculosis and drug-sensitive tuberculosis, a 'governor-controlled short-range chemotherapy' strategy is generally adopted, namely four antibiotics (isoniazid, rifampin, ethambutol and pyrazinamide) are jointly treated for 6 months. Generally, most patients can be cured by conventional and normative drug therapy of the pulmonary tuberculosis, but more and more pulmonary tuberculosis patients are changed into drug-resistant pulmonary tuberculosis patients due to unreasonable drug combination, increase of immune damage hosts, interruption or self-reduction of drug caused by that part of patients cannot tolerate toxic and side effects of chemical drugs, and the like. The traditional Chinese medicine composition has limitations on the treatment scheme of multi-drug resistant tuberculosis, obvious side effects of the medicine, poor curative effect, long course of treatment and high price. Drug-resistant pulmonary tuberculosis is a difficult problem in controlling pulmonary tuberculosis, has a low cure rate, a success rate of less than 70 percent and a high death rate, is a difficult problem to be solved urgently before the medical interface, and is a social problem which is increasingly concerned. Therefore, the continuous research and development of new antituberculosis drugs has practical significance.
Traditional Chinese medicines and national medicines are treasure of Chinese culture and natural resources, and as early as two thousand years ago, the traditional Chinese medicines are recorded in ancient China for treating tuberculosis. At present, the current situation of treating tuberculosis in China is mainly chemical medicines and assisted by traditional Chinese medicines. The Chinese medicinal materials have the auxiliary effects of clearing away heat and toxic materials, softening and resolving hard mass, invigorating spleen, moistening lung, eliminating phlegm, and relieving cough. In recent years, the hot spot is focused at home and abroad to search for high-efficiency antitubercular drugs from rich Chinese herbal medicine resources. The traditional Chinese medicine has the characteristics of multiple components and multiple targets, can regulate the organism on the whole, promotes the recovery of the disease resistance of a patient, and relieves complications; the traditional Chinese medicine is not easy to cause drug resistance of pathogenic bacteria, can overcome the occurrence of drug-resistant tuberculosis to a certain extent, and has great potential in the aspect of treating drug-resistant tuberculosis. In recent years, the traditional Chinese medicine is further developed and tried to treat tuberculosis, and clinical reports show that the traditional Chinese medicine is combined with chemical medicines to treat pulmonary tuberculosis, so that the traditional Chinese medicine not only can promote the rate of converting sputum bacteria into negative, improve symptoms and treat lesion lesions, but also can help the healthy qi of a human body, improve the immunity of the organism and regulate the general state so as to achieve the purpose of treatment. In the aspect of tuberculosis treatment, the vast resource treasure of traditional Chinese medicines and national medicines is excavated and developed, and the traditional Chinese medicines have great potential for resisting tuberculosis, particularly drug-resistant tuberculosis.
The fructus Tsaoko is dry mature fruit of Amomum tsao-ko Crevost et Lemaire belonging to Zingiberaceae. Harvesting in autumn when the fruit is ripe, removing impurities, and drying in the sun or at low temperature. Warm in nature and pungent in flavor. Has the effects of eliminating dampness, warming the middle-jiao, checking malaria and eliminating phlegm. Can be used for treating cold-dampness retention, abdominal distention and pain, distention and fullness, emesis, malaria, fever, plague, fever, etc. Modern researches show that volatile oil components of tsaoko amomum fruit mainly comprise 1, 8-eucalyptol, 2-tetradecene-1-alcohol acetate, geranyl acetate, decanol acetate, geraniol and the like, and other main components comprise quercetin, curcumin and the like. Pharmacological effects are rarely reported. At present, reports about the application of the amomum tsao-ko extract in preparing a medicament for treating multi-drug resistant tuberculosis are not seen.
Disclosure of Invention
The invention aims to provide a new application of a tsaoko amomum fruit extract.
In a first aspect, the invention claims the application of the tsaoko alcohol extract in the preparation of drugs for resisting drug tuberculosis.
Wherein the drug-resistant tuberculosis is tuberculosis caused by drug-resistant mycobacterium tuberculosis infection.
Further, the tuberculosis is pulmonary tuberculosis.
In a second aspect, the invention claims the use of an alcoholic extract of tsaoko amomum fruit for the preparation of a product intended to inhibit drug-resistant mycobacterium tuberculosis.
In the first and second aspects, the drug-resistant mycobacterium tuberculosis may be a multi-drug resistant mycobacterium tuberculosis.
Further, the multi-drug resistant mycobacterium tuberculosis is resistant to isoniazid and rifampicin, at least predominantly two first-line antitubercular drugs, simultaneously. The multi-drug resistant mycobacterium tuberculosis can be resistant to ethambutol, rifabutin, levofloxacin, moxifloxacin, aminosalicylic acid and/or ethionamide besides isoniazid and rifampin.
In a particular embodiment of the invention, the multi-drug resistant mycobacterium tuberculosis is selected from the group consisting of: MDR-A07 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A08 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin), MDR-A09 (rifampicin + isoniazid + ethambutol + rifabutin), MDR-A11 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A12 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A15 (rifampicin + isoniazid + ethambutin + rifabutin + p-aminosalicylic acid). The drug resistance is shown in parentheses.
In the application of the two previous aspects, the tsaoko ethanol extract can be specifically an ethanol extract of tsaoko.
In the application of the two aspects, the tsaoko alcohol extract is prepared by mixing mature tsaoko fruits with 80% -100% (such as 80% -90% or 90% -100%) ethanol according to a mass-volume ratio of 1g (10-20) mL [ such as 1g: (10-15) mL or 1g: (15-20) mL, and heating by a Soxhlet extractor until boiling, refluxing and extracting for 2-4 h. Further, the method can also comprise the steps of filtering, retaining filtrate, concentrating and drying after heating to boiling reflux extraction for 2-4 hours.
Further, the tsaoko alcohol extract can be prepared according to a method comprising the following steps: mixing ripe tsaoko fruits with 90% ethanol according to the weight ratio of 10 g: heating to boiling and refluxing for 3h by using a Soxhlet extractor at a ratio of 150mL to obtain an extracting solution containing the tsaoko alcohol extract.
Still further, the method may further comprise the steps of: filtering the extractive solution, and concentrating the filtrate at 45 deg.C to obtain extract.
Still further, the method may further comprise the steps of: freezing the extract at-80 ℃, and then carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain the tsaoko alcohol extract.
In a third aspect, the invention claims a drug-resistant tuberculosis drug, the effective component of which is the tsaoko alcohol extract.
In a fourth aspect, the invention claims a drug-resistant tuberculosis drug, which consists of the alcohol extract of tsaoko amomum fruit and chemical drugs or traditional Chinese medicines or natural medicines.
In a fifth aspect, the invention claims a drug-resistant tuberculosis drug, which is prepared by adding pharmaceutically acceptable auxiliary materials into the alcohol extract of tsaoko amomum fruit to prepare pharmaceutical oral dosage forms such as tablets, granules or capsules.
In the third to fifth aspects, the tuberculosis is, for example, tuberculosis.
In the third to fifth aspects, the drug-resistant tuberculosis is tuberculosis caused by infection with drug-resistant mycobacterium tuberculosis.
Further, the drug-resistant mycobacterium tuberculosis may be a multi-drug resistant mycobacterium tuberculosis.
Further, the multi-drug resistant mycobacterium tuberculosis is a mycobacterium tuberculosis that is resistant to at least isoniazid and rifampicin. The multi-drug resistant mycobacterium tuberculosis can be resistant to ethambutol, rifabutin, levofloxacin, moxifloxacin, aminosalicylic acid and/or ethionamide besides isoniazid and rifampin.
In a particular embodiment of the invention, the multi-drug resistant mycobacterium tuberculosis is selected from the group consisting of: MDR-A07 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A08 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin), MDR-A09 (rifampicin + isoniazid + ethambutol + rifabutin), MDR-A11 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A12 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A15 (rifampicin + isoniazid + ethambutin + rifabutin + p-aminosalicylic acid). Drugs tolerated by the strains are in parentheses.
The invention adopts a Soxhlet extraction method, uses ethanol with the practical concentration of 90 percent of mature tsaoko fruit as a solvent, and obtains the tsaoko fruit ethanol extract by heating reflux extraction, filtration concentration and freeze drying. The Minimal Inhibitory Concentration (MIC) determination method proves that the tsaoko alcohol extract has strong antitubercular effect on mycobacterium tuberculosis, particularly multi-drug resistant mycobacterium tuberculosis, and the cytotoxicity evaluation result of the tsaoko alcohol extract proves that the tsaoko alcohol extract has no obvious cytotoxicity on human embryonic lung fibroblast MRC-5 when the Concentration of the tsaoko alcohol extract is not more than 250 mu g/mL. Therefore, the invention has important significance for the development of anti-tubercle bacillus, in particular to the development of drugs for resisting drug-resistant tubercle bacillus.
Drawings
FIG. 1 shows the fingerprint of the tsaoko alcohol extract under three conditions.
FIG. 2 is a graph showing the effect of alcohol extraction of Amomum tsao-ko on the growth of tubercle bacillus in vitro.
FIG. 3 is a graph showing the effect of alcohol extraction of Carpesium nasutum on the growth of tubercle bacillus in vitro.
FIG. 4 is a graph showing the effect of alcohol extract of tsaoko on MRC-5 cell proliferation.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of alcoholic extracts under different conditions
Taking 10g of mature fruit of tsaoko amomum fruits, adding 200mL of 80% ethanol (volume percentage content, the rest 20% is water), heating by a Soxhlet extractor until boiling and refluxing for 4h, filtering the extracting solution, concentrating the extracting solution at 45 ℃ to form an extract, freezing the extract at-80 ℃, and carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain a freeze-dried extract, namely the tsaoko amomum fruit alcohol extract S1.
Taking 10g of mature fruit of tsaoko amomum fruits, adding 150mL of 90% ethanol (volume percentage content, the rest 10% is water), heating by a Soxhlet extractor to boil and reflux for extraction for 3h, filtering the extracting solution, concentrating the extracting solution at 45 ℃ to form an extract, freezing the extract at-80 ℃, and carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain a freeze-dried extract, namely the tsaoko amomum fruit alcohol extract S2.
Taking 10g of mature fruit of tsaoko amomum fruits, adding 100mL of 100% ethanol, heating by a Soxhlet extractor until boiling and refluxing for 2h, filtering the extracting solution, concentrating the extracting solution at 45 ℃ to form an extract, freezing the extract at-80 ℃, and carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain a freeze-dried extract, namely the tsaoko amomum fruit ethanol extract S3.
HPLC analysis is carried out on the alcohol extract under different conditions, and the chromatographic conditions are as follows: a chromatographic column: waters Symmetry C18 column (4.6X 250mm,5 μm); mobile phase: gradient elution with 0.1% formic acid (A) -methanol (B) (elution procedure: 0-30min, 5% -95% B; 30-40min, 95% B, wherein% represents volume percentage); detection wavelength: 250 nm; the column temperature is 40 ℃; the flow rate is 1mL/min, the recording time is 40min, and the sample concentration is 5 mg/mL; the amount of the sample was 10. mu.L.
The control spectra were generated by the median method for the three alcohol extracts by the software for evaluation of similarity of chromatography fingerprints of chinese herbs (2012 edition), and the fingerprints of the alcohol extracts of tsaoko under the three conditions were obtained, as shown in fig. 1. Similarity calculation is carried out, and results show that the similarity of the 3 batches of extracts S1-S3 and a contrast map is 0.983, 0.973 and 0.978 in sequence. Therefore, the similarity of S1, S2 and S3 is greater than 0.97, which indicates that the extracts of 3 batches have higher uniformity and good similarity.
Example 2 preparation of extract
Taking 10g of mature fruit of tsaoko amomum fruits, adding 150mL of 90% ethanol (volume percentage content, the rest 10% is water), heating by a Soxhlet extractor until boiling and refluxing for 3h, filtering the extracting solution, concentrating the extracting solution at 45 ℃ to form an extract, freezing the extract at-80 ℃, and carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain a freeze-dried extract, namely the tsaoko amomum fruit alcohol extract.
Taking 10g of mature fruit of tsaoko amomum fruits, adding 150mL of deionized water, heating by a Soxhlet extractor until boiling and refluxing for extraction for 3h, filtering the extracting solution, concentrating the extracting solution at 45 ℃ to form an extract, freezing the extract at-80 ℃, and carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain a freeze-dried substance, namely the tsaoko amomum water extract.
Taking 10g of mature fruit of the Nanhuilus nasutus, adding 150mL of 90% ethanol (volume percentage content, the rest 10% is water), heating by a Soxhlet extractor until boiling and refluxing for 3h, filtering the extracting solution, concentrating at 45 ℃ to obtain an extract, freezing the extract at-80 ℃, and carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain a freeze-dried extract, namely the Nanhuilus nasutus alcoholic extract.
Example 3 anti-tubercle bacillus test of alcohol extract
1. Materials and methods
(1) Test drugs: the tsaoko alcohol extract prepared by the method of the present invention in example 2 was prepared into a mother liquor with a concentration of 50mg/mL using Dimethyl sulfoxide (DMSO). The tsaoko amomum fruit water extract prepared by the method of the embodiment 2 of the invention is prepared into mother liquor with the concentration of 50mg/mL by deionized water.
(2) Test of tuberculosis strains: standard strain H37Rv (no drug resistance); clinical strains 00419, 221090, 221114, 221136, 221173 and 222155 (drug-intolerant, provided by the tuberculosis prevention and control center of the Chinese disease prevention and control center); multidrug-resistant strain MDR-A07 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A08 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin), MDR-A09 (rifampicin + isoniazid + ethambutol + rifabutin), MDR-A11 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + moxifloxacin + p-aminosalicylic acid + ethionamide), MDR-A12 (rifampicin + isoniazid + ethambutol + rifabutin + levofloxacin + p-aminosalicylic acid + ethionamide), MDR-A15 (rifampicin + isoniazid + ethambutin + rifampicin + salicylic acid) (the drug-resistant strain, the drug-resistant strain is a drug-resistant strain, provided by the tuberculosis prevention and control center of the Chinese disease prevention and control center). All provided by the tuberculosis prevention and control center of the Chinese disease prevention and control center.
(3) Determination of minimum inhibitory concentration: fresh colonies growing on neutral Roche medium (Xenopt) for 2 weeks were scraped with an inoculating loop and placed in a turbidimetric tube, ultrasonically shaken for 30s to form a bacterial suspension, diluted by turbidimetric method to obtain a concentration of about 7.5X 105CFU/mL working solution; adding the gradient concentrations of 2000, 1000, 500, 250, 125 μ g/mL of the medicinal liquid into 100 μ L of liquid 7H9 culture medium per well in sequence by a multiple dilution method, and adding 100 μ L of bacterial liquid working solution (7.5 × 10)5CFU/mL) so that the final concentration of the drug solution is 1000, 500, 250, 125, 62.5 μ g/mL. Are simultaneously provided with pairsThe highest concentration DMSO control and water control should be used.
(4) The culture and observation were carried out at 37 ℃ based on the results obtained after 7 days of culture.
(5) Adding 70 μ L of color developing agent (prepared by mixing Alma blue and 5% Tween 80 at a ratio of 2: 5) into each well after the culture is finished, incubating for 24h, and reading the result; the color of the 96-well plate changed from blue to red (or purple) indicates bacterial growth, and the Minimum Inhibitory Concentration (MIC) is the lowest drug Concentration value at which blue color is not changed at all. If the MIC of the extract is less than or equal to 250 mu g/mL, the extract is considered to have the activity of inhibiting tubercle bacillus.
2. Results
Effect on bacterial growth in vitro: MIC assays were first performed for the tubercular standard strain H37Rv and clinical strain 221090, with the MIC concentrations of the tsaoko alcohol extract to both strains being 250. mu.g/mL (FIG. 2); no bacteriostatic action of the water extract of amomum tsao-ko on the two strains is detected. The enlarged tsaoko alcohol extract is detected for clinical strains, MICs of 5 clinical strains (00419, 221136, 221173, 221114 and 222155) are respectively 62.5, 125, 250 and 125 mu g/mL, which shows that the tsaoko alcohol extract has bacteriostasis to tuberculosis standard strains H37Rv and 6 clinical strains, and the inhibition rate to the 6 clinical strains is 100%.
Further, whether the tsaoko alcohol extract has an antibacterial effect on drug-resistant tubercle bacillus is analyzed, 6 drug-resistant strains are selected and respectively are multi-drug-resistant strains MDR-A07, MDR-A08, MDR-A09, MDR-A11, MDR-A12 and MDR-A15, an MIC detection result shows that the tsaoko extract also has a relatively obvious antibacterial effect on the drug-resistant bacteria, and MIC values of the 6 multi-drug-resistant strains are 125, 250, 125 and 62.5 mu g/mL respectively. Further indicates that the alcohol extract not only has the bacteriostasis to common tuberculosis clinical strains, but also has stronger bacteriostasis to drug-resistant tubercle bacillus, and the inhibition rate to 6 multi-drug resistant strains is 100 percent.
In order to detect whether the traditional Chinese medicine with the bacteriostatic action on the common tuberculosis strain generally has the bacteriostatic action on the drug-resistant tuberculosis strain, research on the bacteriostatic action of part of other traditional Chinese medicines on the tuberculosis strain is carried out, and the MICs of part of traditional Chinese medicines such as the alcohol extract of the Nanhuishu (prepared in example 2) on tuberculosis standard strains H37Rv and 6 clinical strains (221090, 00419, 221114, 221136, 221173 and 222155) are respectively 250, 62.5, 250 and 250 mu g/mL, and the inhibitory rate on the 6 clinical strains is 100%. However, when the drug-resistant tubercle bacillus (MDR-A07, MDR-A08, MDR-A09, MDR-A11, MDR-A12 and MDR-A15) is detected, the MIC values of the alcohol extract of the Carpesium nankinensis to 6 multi-drug resistant strains are respectively 500, 250 and 500 mu g/mL, which shows that the bacteriostasis of the alcohol extract of the Carpesium nankinensis to the drug-resistant tubercle bacillus is weaker than that to common clinical strains (figure 3), and the inhibition rate to the 6 drug-resistant strains is 16.7%. Further indicates the potential value of the tsaoko alcohol extract on resisting tubercle bacillus, in particular to drug-resistant tubercle bacillus.
Example 4 in vitro cell assay
In this example, the Cell Counting Kit CCK8(Cell Counting Kit-8) was used to examine the effect of the alcohol extract of tsaoko amomum fruit prepared in example 2 on the proliferation of human embryonic lung fibroblast MRC-5 cells.
Diluting the amomum tsao-ko alcohol extract mother liquor with the concentration of 50mg/mL prepared in the example 2 into different concentrations (1000, 500, 250, 125, 62.5 mu g/mL), treating MRC-5 cells in a growth log phase, setting DMSO control with corresponding concentration, measuring cell viability by using a CCK8 kit after 24 hours, and calculating the cell viability; the experiment was repeated three times. The results are shown in FIG. 4. As can be seen from the figure, the tsaoko amomum fruit extract has no obvious cytotoxicity to MRC-5 cells at a concentration of not more than 250. mu.g/mL.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (14)
1. The application of the tsaoko alcohol extract in preparing drugs for resisting drug tuberculosis;
the tsaoko alcohol extract is obtained by mixing mature tsaoko fruits with 80-100% ethanol according to the mass volume ratio of 1g (10-20) mL, and performing heating reflux extraction for 2-4 h.
2. The application of the tsaoko alcohol extract in preparing a product for inhibiting drug-resistant mycobacterium tuberculosis;
the tsaoko alcohol extract is obtained by mixing mature tsaoko fruits with 80-100% ethanol according to the mass volume ratio of 1g (10-20) mL, and performing heating reflux extraction for 2-4 h.
3. Use according to claim 1, characterized in that: the drug-resistant tuberculosis is tuberculosis caused by drug-resistant mycobacterium tuberculosis infection.
4. Use according to claim 2, characterized in that: the drug-resistant mycobacterium tuberculosis is multi-drug-resistant mycobacterium tuberculosis.
5. Use according to claim 4, characterized in that: the multi-drug resistant mycobacterium tuberculosis is at least mycobacterium tuberculosis resistant to isoniazid and rifampicin.
6. Use according to claim 1 or 2, characterized in that: the tsaoko alcohol extract is prepared by the method comprising the following steps: mixing mature fruit of Amomum tsao-ko with 90% ethanol according to a ratio of 10 g: mixing the components in a proportion of 150mL, and heating and refluxing for extraction for 3h to obtain an extracting solution containing the tsaoko alcohol extract.
7. Use according to claim 6, characterized in that: the method further comprises the steps of: filtering the extractive solution, and concentrating the filtrate at 45 deg.C to obtain extract.
8. Use according to claim 7, characterized in that: the method further comprises the steps of: freezing the extract at-80 ℃, and then carrying out vacuum freeze drying at-60 to-40 ℃ for 24h to obtain the tsaoko alcohol extract.
9. A drug-resistant tuberculosis drug, the active ingredient of which is the tsaoko alcohol extract of any one of claims 1 to 8.
10. The drug-resistant tuberculosis drug according to claim 9, wherein: the drug-resistant tuberculosis drug consists of the tsaoko alcohol extract and a chemical drug or a traditional Chinese medicine or a natural drug.
11. The drug-resistant tuberculosis drug according to claim 10, wherein: the drug-resistant tuberculosis drug is prepared by adding pharmaceutically-allowable auxiliary materials into the tsaoko alcohol extract and preparing into a pharmaceutically-allowable oral dosage form.
12. The drug-resistant tuberculosis drug according to any one of claims 9 to 11, wherein: the drug-resistant tuberculosis is tuberculosis caused by drug-resistant mycobacterium tuberculosis infection.
13. The drug-resistant tuberculosis drug according to claim 12, wherein: the drug-resistant mycobacterium tuberculosis is multi-drug-resistant mycobacterium tuberculosis.
14. The drug-resistant tuberculosis drug according to claim 13, wherein: the multi-drug resistant mycobacterium tuberculosis is at least mycobacterium tuberculosis resistant to isoniazid and rifampicin.
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| CN109200098A (en) * | 2018-10-22 | 2019-01-15 | 四川师范大学 | A kind of combination medicine inhibiting Multiple drug-resistan Tubercle bacillus |
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