CN107050301B - Bamboo leaf orchid extract and preparation method and application thereof - Google Patents
Bamboo leaf orchid extract and preparation method and application thereof Download PDFInfo
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- CN107050301B CN107050301B CN201710208324.8A CN201710208324A CN107050301B CN 107050301 B CN107050301 B CN 107050301B CN 201710208324 A CN201710208324 A CN 201710208324A CN 107050301 B CN107050301 B CN 107050301B
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- 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)
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- 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
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- 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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- A61K2236/30—Extraction of the material
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- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
The invention discloses a arundina graminifolia extract as well as a preparation method and application thereof, belonging to the field of research of natural medicines or natural products. The method comprises the following steps: drying and pulverizing herba Arundinis Purpureae, soaking in ethanol solvent, extracting under reflux, filtering, mixing extractive solutions, and concentrating under reduced pressure to obtain herba Arundinis Purpureae ethanol extract concentrated solution; mixing the concentrated solution of the ethanol extract of arundina graminifolia with silica gel, evaporating to dryness, sequentially eluting with petroleum ether under reflux, and eluting with ethyl acetate under reflux; finally, acetone or methanol is used for hot reflux elution, acetone or methanol eluent is collected, and the acetone or methanol eluent is decompressed, concentrated and dried to obtain the arundina graminifolia extract; or, eluting with acetone under reflux, eluting with methanol under reflux, collecting methanol eluate, concentrating under reduced pressure, and drying to obtain herba Arundinis Purpureae extract. The method has the advantages of simple operation, strong continuity, short time consumption, high efficiency and solvent saving. The extract can inhibit the proliferation of salmonella, and can be used for preparing products for preventing and treating salmonella infection.
Description
Technical Field
the invention belongs to the field of research on natural medicines or natural products, and particularly relates to a arundina graminifolia extract as well as a preparation method and application thereof.
Background
the Arundina graminifolia (Arundina graminifolia), also known as tulip orchid, is a perennial herb of the genus phyllostachys of the family orchidaceae, growing in a grass slope, beside a stream valley, under a bush or in a forest. The people of the Dai nationality are called "agricultural hi" and "Baiyijie". "nongshanhi" means that the food is well eaten and "Baizhi" means that the toxin is removed, and it can be used for curing food poisoning, snake bite, sore and carbuncle and pyogenic infections, and is a special good medicine for removing toxic substance for Dai medicine.
Salmonella belongs to the enterobacteriaceae family, and is a common food-borne pathogenic bacterium. In bacterial food poisoning species of various countries in the world, food poisoning caused by salmonella is often best shown in leaderboard. Food poisoning in inland areas of China also takes salmonella as the first place. In addition to infecting humans, salmonella can infect many animals including mammals, birds, reptiles, fish, amphibians, and insects. Infection of humans and animals can be manifested as clinically symptomatic, fatal diseases that can exacerbate illness or mortality, or reduce reproductive productivity in animals.
disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a preparation method of a arundina graminifolia extract.
Another object of the present invention is to provide an arundina graminifolia extract prepared by the above preparation method. The extract has effect in inhibiting Salmonella.
the invention also aims to provide application of the arundina graminifolia extract.
The purpose of the invention is realized by the following technical scheme:
a preparation method of arundina graminifolia extract comprises the following steps:
(1) Drying and crushing the arundina graminifolia, adding an ethanol solvent with the volume concentration of 10-100% according to the dosage of 1-100 mL per gram of raw material medicine, soaking for 0-24 h, performing hot reflux extraction for 1-4 times, each time for 0.5-6 h, filtering, combining extracting solutions, and performing reduced pressure concentration to obtain an arundina graminifolia ethanol extract concentrated solution;
(2) Uniformly stirring and evaporating the concentrated solution of the arundina graminifolia ethanol extract obtained in the step (1) according to the mass ratio of the dry paste to the silica gel of 1: 0.1-1: 100, sequentially eluting by using petroleum ether under thermal reflux for 0.5-48 h, then eluting by using ethyl acetate under thermal reflux for 0.5-48 h, and removing impurities;
(3) finally, acetone or methanol is used for hot reflux elution for 0.5-30 h, acetone or methanol eluent is collected, and the acetone or methanol eluent is subjected to reduced pressure concentration and drying to obtain the arundina graminifolia extract;
Or, performing hot reflux elution for 2-20 h by using acetone, performing hot reflux elution for 10-20 h by using methanol, collecting methanol eluent, performing reduced pressure concentration, and drying to obtain the arundina graminifolia extract.
Preferably, the crushing in the step (1) is to be crushed into small sections with the length of 2-3 cm.
Preferably, the dosage of the ethanol solvent in the step (1) is 6-12 mL/g of arundina graminifolia.
preferably, the volume concentration of the ethanol solvent in the step (1) is 70-95%.
Preferably, the soaking time in the step (1) is 1-12 h; more preferably 2 to 12 hours.
Preferably, the extraction in the step (1) is performed for 2-3 times, and each time lasts for 2 hours; more preferably, the number of extractions is 3, each for 2 hours.
Preferably, the silica gel in the step (2) is 100-300 mesh silica gel; more preferably 100-200 mesh silica gel;
Preferably, the mass ratio of the concentrated solution dry paste to the silica gel in the step (2) is 1: 1-1: 5; more preferably 1:1 to 1: 3.
Preferably, the time of the petroleum ether hot reflux elution in the step (2) is 2-16 h; more preferably 2-10 h;
Preferably, the time of the hot reflux elution with ethyl acetate in the step (2) is 2-16 h; more preferably 10 to 16 hours.
Preferably, the time of hot reflux elution by acetone or methanol in the step (3) is 2-20 h; more preferably 15 to 20 hours.
preferably, the acetone is firstly used for hot reflux elution for 10-15 h in the step (3), and then the methanol is used for hot reflux elution for 20 h.
a herba Arundinis Purpureae extract is prepared by the above preparation method.
the arundina graminifolia extract can inhibit the proliferation of salmonella, and can be used for preparing products for preventing and treating salmonella infection.
Compared with the prior art, the invention has the following advantages and effects:
(1) the preparation method comprises the steps of extracting with ethanol with a certain concentration, adsorbing and dispersing the extracted concentrated solution with crude silica gel, loading the concentrated solution into a constant-pressure separating funnel, eluting and removing impurities by using petroleum ether and ethyl acetate in combination with a solid-liquid extraction and continuous reflux elution technology, finally performing reflux elution by using acetone or methanol, and collecting the eluate to obtain the arundina graminifolia extract which has an antibacterial effect on salmonella.
(2) The preparation method disclosed by the invention is simple to operate, strong in continuity, short in time consumption, high in efficiency and capable of saving the solvent. The extract can be used for preparing products for preventing and treating salmonella infection.
Drawings
FIG. 1 shows the bacteriostatic effect of Arundina graminifolia extract A, Arundina graminifolia extract B and ampicillin sodium on Salmonella.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
example 1 preparation of extract of arundina graminifolia
Soaking 100g of dried and pulverized arundina graminifolia in 1000mL of ethanol solvent with volume fraction of 70% for 2h, heating and refluxing for extraction for 3 times, each time for 2h, combining the extracts, and concentrating under reduced pressure. Adsorbing and uniformly mixing the obtained concentrated solution and 100-200-mesh silica gel according to a mass ratio of 1:1, evaporating to dryness, loading into a constant-pressure separating funnel, performing thermal reflux elution by using petroleum ether for 10 hours, then performing thermal reflux elution by using ethyl acetate for 16 hours, then performing thermal reflux elution by using acetone for 10 hours, finally performing thermal reflux elution by using methanol for 20 hours, collecting methanol eluent, and performing reduced pressure concentration and drying to obtain a arundina graminifolia extract, wherein the reference is as follows: herba Arundinis Purpureae extract A.
Example 2 preparation of extract of arundina graminifolia
Soaking 100g of dried and pulverized arundina graminifolia in 1100mL of ethanol solvent with volume fraction of 70% for 4h, heating and refluxing for extraction for 3 times, 2h each time, combining the extracts, and concentrating under reduced pressure. Adsorbing and uniformly mixing the obtained concentrated solution and 100-200-mesh silica gel according to a mass ratio of 1:1, evaporating to dryness, loading into a constant-pressure separating funnel, performing thermal reflux elution by using petroleum ether for 10 hours, then performing thermal reflux elution by using ethyl acetate for 16 hours, then performing thermal reflux elution by using acetone for 20 hours, collecting acetone eluent, performing reduced pressure concentration and drying to obtain a arundina graminifolia extract, wherein the content is recorded as: and (4) bamboo leaf orchid extract B.
Example 3 preparation of extract of arundina graminifolia
Taking 100g of dried and crushed arundina graminifolia, soaking the 100g of arundinacea graminifolia in 600mL of ethanol solvent with the volume fraction of 80% for 6h, heating and refluxing for 3 times, extracting for 2h each time, combining extracting solutions, concentrating under reduced pressure, adsorbing and uniformly mixing the obtained concentrated solution and 100-200-mesh silica gel according to the mass ratio of 1:2, evaporating to dryness, putting the mixture into a constant-pressure separating funnel, performing thermal reflux elution by using petroleum ether for 5h, then performing thermal reflux elution by using ethyl acetate for 14h, then performing thermal reflux elution by using methanol for 15h, collecting methanol eluent, and performing reduced pressure concentration and drying to obtain.
example 4 preparation of extract of arundina graminifolia
Taking 100g of dried and crushed arundina graminifolia, soaking the arundinacea graminifolia in 1200mL of ethanol solvent with volume fraction of 95% for 12h, heating and refluxing for extraction for 3 times, each time for 2h, combining the extracting solutions, concentrating under reduced pressure, adsorbing and uniformly mixing the obtained extracting solution and 200-300-mesh silica gel according to the mass ratio of 1:3, evaporating to dryness, putting the mixture into a constant-pressure separating funnel, performing thermal reflux elution by using petroleum ether for 2h, then performing thermal reflux elution by using ethyl acetate for 10h, then performing thermal reflux elution by using acetone for 15h, finally performing thermal reflux elution by using methanol for 20h, collecting methanol eluent, concentrating under reduced pressure and drying to obtain.
Example 5 Experimental study of Arundina graminifolia extract for in vitro inhibition of Salmonella
salmonella s.cholereas ATCC13312 was purchased from guangzhou institute of microorganisms; preparing a beef extract peptone culture medium according to pharmacopoeia; ampicillin sodium was supplied by Shanghai MYM Biotechnology, Inc.; DMSO was obtained from Fuyu Fine chemical Co., Ltd, Tianjin.
And selecting a single colony from the well-stored solid culture medium, inoculating the single colony in a beef extract peptone liquid culture medium, culturing at the constant temperature of 37 ℃ for 16-18 h at the rotating speed of 100rpm, and adjusting the concentration of the bacterial liquid to 10 6 CFU/mL.
And (3) processing of a sample: the arundina graminifolia extract A obtained in example 1 and the arundina graminifolia extract B obtained in example 2 were dissolved in DMSO in an appropriate amount to give a concentration of 10 mg/mL.
The size of the zone of inhibition is determined by an Oxford cup method, 100 mu L of 10 6 CFU/mL bacterial suspension is added into a sterilized and solidified beef extract peptone solid culture medium and is uniformly coated, Oxford cups with the diameter of 6mm are placed on a flat plate, 100 mu L of 10mg/mL test solution is added into each Oxford cup, the size of the zone of inhibition is measured by a ruler after the Oxford cups are cultured at the constant temperature of 37 ℃ for 24h, three Oxford cups are placed on each flat plate as parallel, DMSO is used as a negative control, ampicillin sodium is used as a positive control, and the results are shown in Table 1 and figure 1.
TABLE 1 Aphyllophora Indusiata extract for Salmonella zone diameter (n ═ 3)
| Diameter/mm of bacteriostatic circle | |
| Bamboo leaf orchid extract A (10mg/mL) | 16 |
| Bamboo leaf orchid extract B (10mg/mL) | 13 |
| Ampicillin sodium (10 ug/mL) | 14 |
| DMSO | 0 |
And (3) measuring the minimum bacteriostatic concentration and the minimum bactericidal concentration of the arundina graminifolia extract on the salmonella.
The minimum inhibitory concentration is measured by a two-fold dilution method, namely, 100 mu L of beef extract peptone liquid culture medium is added into a 96-well plate one by one, then 100 mu L of test solution (10mg/mL) is added into a 1 st well, after the test solution is uniformly blown, 100 mu L of beef extract peptone liquid culture medium is sucked and added into a 2 nd well, after the 2 nd well is uniformly mixed, 100 mu L of beef extract liquid is added into a 3 rd well, and the process is repeated until a 6 th well, no liquid medicine is added into a 7 th well and an 8 th well, 100 mu L of prepared 10 5 CFU/mL bacterial suspension is added into the 1 st to 7 th wells, no liquid medicine is added into the 8 th well, the 96-well plate is placed into a constant temperature shaker for 24h culture, the culture solution is clarified after the culture, namely, the minimum inhibitory concentration of no obvious bacterial growth is the minimum inhibitory concentration, 100 mu L of culture solution which is still clarified after 24h of suction culture is coated on a solid plate, the culture solution is placed into the constant temperature shaker for 24h culture at 37 ℃, the minimum bactericidal concentration of the sterile colony growth is recorded.
TABLE 2 minimal inhibitory and minimal bactericidal concentrations of Arundina graminifolia extract against Salmonella
| Minimum inhibitory concentration (μ g/mL) | Minimum bactericidal concentration (μ g/mL) | |
| Arundina graminifolia extract A | 2500 | >2500 |
| Arundina graminifolia extract B | 2500 | >2500 |
| DMSO | Is free of | Is free of |
As can be seen from tables 1 and 2 and fig. 1, the experimental results of the zone of inhibition and the results of the determination of the minimum inhibitory concentration and the minimum bactericidal concentration are consistent, which indicates that the arundina graminifolia extract provided by the invention can inhibit the proliferation of salmonella.
the above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. The application of the arundina graminifolia extract in preparing products for preventing and treating salmonella infection is characterized in that: the preparation method of the arundina graminifolia extract comprises the following steps:
(1) Drying and crushing the arundina graminifolia, adding an ethanol solvent with the volume concentration of 10-100% according to the dosage of 1-100 mL per gram of raw material medicine, soaking for 0-24 h, performing hot reflux extraction for 1-4 times, each time for 0.5-6 h, filtering, combining extracting solutions, and performing reduced pressure concentration to obtain an arundina graminifolia ethanol extract concentrated solution;
(2) Uniformly stirring and evaporating the concentrated solution of the arundina graminifolia ethanol extract obtained in the step (1) according to the mass ratio of the dry paste to the silica gel of 1: 0.1-1: 100, sequentially eluting by using petroleum ether under thermal reflux for 0.5-48 h, and then eluting by using ethyl acetate under thermal reflux for 0.5-48 h;
(3) finally, acetone or methanol is used for hot reflux elution for 0.5-30 h, acetone or methanol eluent is collected, and the acetone or methanol eluent is subjected to reduced pressure concentration and drying to obtain the arundina graminifolia extract;
Or, performing hot reflux elution for 2-20 h by using acetone, performing hot reflux elution for 10-20 h by using methanol, collecting methanol eluent, performing reduced pressure concentration, and drying to obtain the arundina graminifolia extract.
2. Use according to claim 1, characterized in that:
The crushing in the step (1) is to be crushed into small sections with the length of 2-3 cm.
3. Use according to claim 1, characterized in that:
The dosage of the ethanol solvent in the step (1) is 6-12 mL/g of arundina graminifolia.
4. Use according to claim 1, characterized in that:
The volume concentration of the ethanol solvent in the step (1) is 70-95%.
5. Use according to claim 1, characterized in that:
The soaking time in the step (1) is 1-12 h;
the extraction in the step (1) is carried out for 2-3 times, and each time lasts for 2 hours.
6. Use according to claim 1, characterized in that:
the silica gel in the step (2) is 100-300 meshes of silica gel;
The mass ratio of the concentrated solution dry paste to the silica gel in the step (2) is 1: 1-1: 5.
7. use according to claim 1, characterized in that:
The time for thermal reflux elution with petroleum ether in the step (2) is 2-16 h;
And (3) performing hot reflux elution by using ethyl acetate for 2-16 h in the step (2).
8. use according to claim 1, characterized in that:
The time for hot reflux elution by acetone or methanol in the step (3) is 2-20 h;
And (4) eluting by acetone hot reflux for 10-15 h, and then eluting by methanol hot reflux for 20h in the step (3).
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