CN106490136B - Method for extracting natural antibacterial substance from folium Mussaendae, extract and application thereof - Google Patents
Method for extracting natural antibacterial substance from folium Mussaendae, extract and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a method for extracting natural antibacterial substances from folium sennae, the natural antibacterial substances and application of the natural antibacterial substances in disease prevention and fresh keeping of picked vegetables. The method comprises the following steps: drying and crushing the kenaf leaves; carrying out ultrasonic extraction on the crushed kenaf leaves by using ethanol; centrifugally separating the ethanol extract, collecting supernatant, and rotationally evaporating to obtain a first extract; preparing the first extract into a suspension liquid medicine, and then passing through a macroporous adsorption resin adsorption column; eluting the adsorption column with first eluent until the liquid flow is colorless, collecting the eluent, and rotary evaporating to obtain a second extract; dissolving the second extract, and passing through an octadecyl reverse phase silica gel column; eluting with a second eluent until the liquid flow is colorless, collecting the eluent, and performing rotary evaporation to obtain a third extract; dissolving the third extract in an organic solvent, and passing through a normal phase silica gel column; and eluting the normal phase silica gel column by using a third eluent until the flow liquid is colorless, collecting the eluent and drying to obtain natural antibacterial substance powder.
Description
Technical Field
The invention belongs to the field of extraction of natural antibacterial substances, and particularly relates to a method for extracting natural antibacterial substances from folium sennae, a natural antibacterial substance extract obtained by the method, and application of the natural antibacterial substance extract in disease prevention and fresh keeping of picked fruits and vegetables.
Background
The Muscovy (Agave sisalana Perrine) is a plant of Agavaceae (Agavaceae) genus Agave, and is a perennial hard fiber crop unique to subtropics. The common flax has the characteristics of temperature preference and drought tolerance, is suitable for cultivation in tropical and subtropical regions, is mainly produced in countries such as Mexico, Brazil and tanzania, and is commercially cultivated in provinces such as Hainan, Guangdong, Guangxi, Yunnan and Fujian in the tropical and subtropical regions of China.
The leaves of the Musca domestica are sword-shaped, hard and long, the length of the leaves is generally 100-140 cm, the width is 13-15 cm, and the leaves are grayish green to bluish green. The senna leaf contains rich fiber, the fiber cell is in a long structure, the cell cavity is large and long, the wall thickness is thick, the senna leaf has the characteristics of long fiber, white color, tough texture, high elasticity, strong tension, friction resistance, acid and alkali resistance, corrosion resistance, difficult slippage and the like, and is widely applied to industries such as fishery, navigation, industrial and mining, transportation, oil field and the like, and is used for weaving sisal carpet, sisal cloth, filters, artware and the like.
Because the content of pectin, saponin and the like in the senna leaves is high, the research of extracting saponin, pectin and the like by using the senna has been carried out at present, but the research has not been reported in the aspects of extracting natural antibacterial substances and controlling and preserving diseases of fruits and vegetables after picking.
Disclosure of Invention
The invention aims to provide a method for extracting natural antibacterial substances from kenaf leaves, which can obtain the natural antibacterial substances with high activity by combining ultrasonic extraction with multi-step separation and purification steps of macroporous resin adsorption, reverse-phase silica gel column, normal-phase silica gel column and the like and corresponding gradient elution.
According to one aspect of the invention, the method for extracting the natural bacteriostatic substance from the senna leaves comprises the following steps: drying folium Murrayae at 55 + -2 deg.C, and pulverizing; according to the ratio of the mass g of the Muscovy leaves to the volume ml of the extraction solvent of 1: 10-15, adding chromatographic grade 95% ethanol into the crushed Mucuna anax leaves, and leaching in an ultrasonic water bath at 35-45 ℃ for 2-5 hours to obtain an ethanol extracting solution; centrifuging the ethanol extract, and collecting supernatant; repeating the leaching and centrifugal separation steps for at least 2 times in sequence, combining the supernatants, and rotatably evaporating at 50 +/-2 ℃ to dryness to obtain a first extract; preparing the first extract into a suspension liquid medicine with the liquid material concentration of 200mg/ml by using water, and then passing the suspension liquid medicine through an adsorption column of macroporous adsorption resin; eluting the adsorption column by using a first eluent until the flow liquid is colorless, repeating the elution step for 3 times, sequentially using water, 45 v/v% ethanol water solution and 60 v/v% ethanol water solution as the first eluent, collecting the eluent of the 60 v/v% ethanol water solution, and rotatably evaporating to dryness at the temperature of 50 +/-2 ℃ to obtain a second extract; dissolving the second extract by using methanol and then passing through an octadecyl reverse phase silica gel column; eluting the octadecyl reverse phase silica gel column by using a second eluent until the flow liquid is colorless, repeating the elution step for 2 times, sequentially using 50 v/v% methanol aqueous solution and 90 v/v% methanol aqueous solution for the second eluent, collecting the eluent of the 90 v/v% methanol aqueous solution, and performing rotary evaporation to obtain a third extract; dissolving the third extract in methanol, and passing through a normal phase silica gel column; and eluting the normal phase silica gel column by using a third eluent until the flow liquid is colorless, repeating the elution step for 2 times, sequentially using chloroform-methanol solutions with volume ratios of 25:1 and 15:1 for the third eluent, collecting the eluent of the chloroform-methanol solution with the volume ratio of 15:1, and carrying out vacuum freeze drying to obtain natural antibacterial substance powder.
Preferably, the natural bacteriostatic substance comprises a triterpenoid saponin substance.
According to one embodiment of the invention, the macroporous adsorbent resin is a D101 type macroporous resin.
According to another embodiment of the invention, the flow rate of the first eluent is 5 ± 0.5 mL/min.
According to another embodiment of the invention, the vacuum freeze drying time is 12-18 h, the temperature is-45 +/-2 ℃, and the vacuum degree is 12 +/-1 Pa.
According to another aspect of the present invention, there is provided a natural bacteriostatic substance extract obtained by the above method.
According to another aspect of the invention, the application of the natural bacteriostatic substance extract in disease prevention and fresh keeping of the picked vegetables is provided.
Preferably, the natural bacteriostatic substance extract is used in combination with tween 80 in an amount of 0.05% based on the mass of the natural bacteriostatic substance extract.
According to the extraction method, ultrasonic extraction, multi-step separation and purification steps and corresponding gradient elution steps are combined, and the natural antibacterial substances in the kenaf leaves are highly selective, so that high extraction rate and high purity are realized. The obtained extract has high content of active ingredients of natural antibacterial substances and strong activity.
The natural antibacterial substance extracted by the method can be used for keeping fruits and vegetables fresh and preventing diseases after picking.
According to the invention, the method for extracting the natural antibacterial substances from the flax leaves is provided, and green and healthy materials are provided for disease control of picked fruits and vegetables. The method has simple operation and high practicability, and the raw material can also be waste hemp residue generated in the production process of the hemp, thereby reducing the production cost. The extract has good application value in the process of fruit and vegetable postharvest preservation.
Drawings
FIG. 1 is a high performance liquid chromatogram of a natural bacteriostatic substance obtained according to examples 1 and 2 of the present invention;
FIG. 2 is a nuclear magnetic carbon spectrum of a natural bacteriostatic substance obtained according to examples 1 and 2 of the present invention;
FIG. 3 is nuclear magnetic hydrogen spectra of natural bacteriostatic substances obtained according to examples 1 and 2 of the present invention;
fig. 4 is a high performance liquid chromatogram of the extract according to comparative example 1.
Fig. 5 is a high performance liquid chromatogram of the extract according to comparative example 2.
Detailed Description
According to one embodiment of the invention, the method for extracting the natural bacteriostatic substance from the senna leaves comprises the following steps: drying folium Murrayae at 55 + -2 deg.C, and pulverizing; according to the ratio of the mass g of the Mucuna anax leaves to the volume ml of the extraction solvent of 1: 10-15, adding chromatographic grade 95% ethanol into the crushed Mucuna anax leaves, and leaching in an ultrasonic water bath at 35-45 ℃ for 2-5 hours to obtain an ethanol extracting solution; centrifuging the ethanol extract, and collecting the supernatant; repeating the leaching and centrifugal separation steps for at least 2 times in sequence, combining the supernatants, and rotatably evaporating at 50 +/-2 ℃ to dryness to obtain a first extract; preparing the first extract into a suspension liquid medicine with the liquid concentration of 200mg/ml by using water, and then passing the suspension liquid medicine through an adsorption column of macroporous adsorption resin; eluting the adsorption column with a first eluent until the liquid flow is colorless, repeating the elution step for 3 times, sequentially using water, 45 v/v% ethanol water solution and 60 v/v% ethanol water solution as the first eluent, collecting the eluent of the 60 v/v% ethanol water solution, and rotationally evaporating at 50 +/-2 ℃ to obtain a second extract; dissolving the second extract with methanol, and passing through octadecyl reverse phase silica gel column; eluting the octadecyl reverse phase silica gel column by using a second eluent until the flow liquid is colorless, repeating the elution step for 2 times, sequentially using 50 v/v% methanol water solution and 90 v/v% methanol water solution for the second eluent, collecting the eluent of the 90 v/v% methanol water solution, and performing rotary evaporation to obtain a third extract; dissolving the third extract in methanol, and passing through a normal phase silica gel column; and eluting the normal phase silica gel column by using a third eluent until the flow liquid is colorless, repeating the elution step for 2 times, sequentially using chloroform-methanol solutions with volume ratios of 25:1 and 15:1 for the third eluent, collecting the eluent of the chloroform-methanol solution with the volume ratio of 15:1, and carrying out vacuum freeze drying to obtain natural antibacterial substance powder.
Preferably, the natural bacteriostatic substance may comprise a triterpenoid saponin substance, preferably a triterpenoid saponin substance.
In the above macroporous resin adsorption and elution steps, the above macroporous adsorption resin may be a D101 type macroporous resin, and the macroporous adsorption resin may be packed in a column using a conventional method in the art. Preferably, the D101 macroporous resin can be filled into a column by a wet method, and the liquid level is kept. The suspension solution preferably passes through an adsorption column at a flow rate of 5. + -. 0.5 mL/min. Specifically, after the suspension liquid medicine is completely adsorbed on the adsorption column, the column body is washed by water until the flowing liquid is colorless. Eluting with 45 v/v% ethanol water solution until the eluate is colorless; and finally, eluting with 60 v/v% ethanol water solution, for example, eluting by 8-10 times of column volume (BV).
Further, the flow rate of the first eluent can be 5. + -. 0.5 mL/min. The first eluent is sequentially water, 45 v/v% ethanol water solution and 60 v/v% ethanol water solution, so that the polarity of the eluent is gradually enhanced, the elution capacity is gradually enhanced, other components which are not required in the extract are sufficiently removed in the first two elution processes, and the purity and the extraction efficiency of the target antibacterial substance (such as the triterpenoid saponin compound) are improved in the 60 v/v% ethanol water solution elution process.
And in the step that the methanol solution of the second extract passes through an octadecyl reversed phase silica gel (ODS) column and is eluted by using a second eluent until the liquid flow is colorless, 50 v/v% methanol water solution is used for eluting to remove impurities contained in the second extract, and then 90 v/v% methanol water solution is used for eluting, so that the elution efficiency and the yield of the antibacterial substance can be improved.
Specifically, on the ODS column, the more polar and less hydrophobic components in the second extract are less likely to be bound to the non-polar immobilization, and thus are eluted first. When the concentration of methanol aqueous solution is below 50 v/v%, the eluted components are mainly impurities, and when the methanol aqueous solution is eluted by 90 v/v%, the extract with outstanding antibacterial property can be obtained.
Preferably, the second extract methanol solution is passed through ODS column (preferably 50 μm particle size), wet loaded, the loading amount is 0.5 times the column volume, and after 0.45 μm membrane filtration, injected into ODS-C18 reverse phase silica gel column with column volume of 150 ml. And then, performing gradient elution by using a water-methanol system, namely eluting with 50 v/v% methanol aqueous solution until the mixture is colorless, then eluting with 90 v/v% methanol aqueous solution for 8-10 times of column volume, collecting eluent of the 90% methanol aqueous solution, and performing rotary evaporation to obtain a third extract.
Similarly, in the step of dissolving the third extract in methanol and then eluting the third extract with a third elution solution through a normal-phase silica gel column, the third elution solution is firstly a 25:1(v/v) chloroform methanol solution to remove impurities contained in the third extract, and then a 15:1(v/v) chloroform methanol solution is used to improve the elution efficiency and yield of the antibacterial substance.
The present inventors found that when the above chloroform-methanol solution has a volume ratio of 25:1 or more, for example, 40:1 or 30:1, the target product eluted is very little and the product is not pure; when the volume ratio of the chloroform-methanol solution is 15:1 or less, the target product is eluted little and the product is not pure.
Preferably, in the step of dissolving the third extract in methanol and passing through the normal phase silica gel column, for example, 300-400 mesh chromatography silica gel with the mass ratio of 1:1.2 to the third extract is added and uniformly stirred, and the mixture is dried by spinning under reduced pressure. Weighing 500-800 meshes of silica gel with 8-10 times of sample mass in advance, filling the silica gel into a column by adopting a chloroform-methanol solution wet method with a volume ratio of 50:1, filling the mixed sample silica gel into a chromatographic column, eluting with a chloroform methanol solution (25:1, v/v) until the silica gel is colorless, eluting with a chloroform methanol solution (15:1v/v), and collecting the eluent of the chloroform methanol solution (15:1, v/v).
The vacuum freeze-drying step described above can be carried out using conventional methods and conditions. For example, the vacuum freeze drying time is 12-18 h, the temperature is-45 +/-2 ℃, and the vacuum degree is 12 +/-1 Pa.
According to a preferred embodiment of the present invention, there is provided a method for extracting natural bacteriostatic substances from kenaf leaves, comprising the steps of:
(1) crushing: cutting cleaned folium Mussaendae Caudatae into pieces of 2 × 2cm, oven drying at 55 + -2 deg.C in a constant temperature oven, and pulverizing into powder with a pulverizer;
(2) ultrasonic extraction: extracting with chromatographic grade 95% ethanol as an extraction solvent in an ultrasonic water bath at 35-45 ℃ for 2-5 h, wherein the using amount ratio of the folium sennae powder to the chromatographic grade ethanol is 10-15 ml of chromatographic grade ethanol used for every 1g of powder, so as to obtain an ethanol extract;
(3) centrifugal separation: centrifuging the extractive solution at 6000r/min for 20min, separating supernatant, and discarding precipitate;
(4) repeating the above steps of extracting and centrifuging for at least 2 times, mixing the obtained supernatants, and rotary evaporating at 50 + -2 deg.C to obtain a first extract of crude extract;
(5) and (3) separation by an adsorption column: preparing the first extract into a suspension liquid medicine with the liquid concentration of 200mg/mL by using water, filling the macroporous adsorption resin (D101) into a column by adopting a wet method, keeping the liquid level, passing the suspension liquid medicine through the adsorption column, and passing the suspension liquid medicine through the adsorption column at the flow rate of 5 +/-0.5 mL/min; after complete adsorption, the column body is washed by water again until the flowing liquid is colorless; eluting with 45 v/v% ethanol water at the flow rate of 5 +/-0.5 mL/min, eluting with 60 v/v% ethanol water at the flow rate of 5 +/-0.5 mL/min for 8-10 BV after the flow liquid is colorless, collecting the eluent of the 60 v/v% ethanol water, and rotationally evaporating the solvent at the temperature of 50 +/-2 ℃ to obtain a second extract;
(6) reverse phase silica gel column separation: dissolving the second extract obtained in the step (5) with methanol, and passing through an ODS column (particle size of 50 μm); wet loading, wherein the loading amount is 0.5 times of the column volume, filtering with a 0.45-micron filter membrane, injecting into an ODS-C18 reverse phase silica gel column with the column volume of 150ml, performing gradient elution with a water-methanol system, eluting with 50 v/v% methanol aqueous solution until the column volume is colorless, then eluting with 90 v/v% methanol aqueous solution for 8-10 BV, collecting the eluent of the 90 v/v% methanol aqueous solution, and performing rotary evaporation to obtain a third extract;
(7) normal phase silica gel column separation: dissolving the third extract obtained in the step (6) by using methanol, passing through a normal-phase silica gel column, adding 300-400-mesh chromatographic silica gel with the mass ratio of the third extract to the normal-phase silica gel column, uniformly stirring, and performing reduced pressure spin drying; weighing 500-800 meshes of silica gel with the mass 8-10 times of that of a sample in advance, filling the silica gel into a column by adopting a chloroform-methanol mixed solvent wet method with the volume ratio of 50:1, filling the well-mixed sample silica gel into a chromatographic column, eluting the silica gel with chloroform methanol (25:1, v/v) until the silica gel is colorless, eluting the silica gel with chloroform methanol (15:1, v/v) for 8-10 BV, and collecting the eluent of the chloroform methanol (15:1, v/v);
(8) vacuum freeze drying: and (4) cooling the eluent collected in the step (7), and then carrying out freeze drying under vacuum to obtain natural antibacterial substance powder, wherein the time of vacuum freeze drying is 12-18 h, the temperature of freeze drying is-45 +/-2 ℃, and the vacuum degree is 12 +/-1 Pa.
The natural bacteriostatic substance obtained by the method can be used for keeping freshness of vegetables and fruits after picking and preventing and treating diseases. Preferably, the natural bacteriostatic substance is combined with tween 80, and the using amount of the tween 80 is 0.05% of the mass of the natural bacteriostatic substance.
Example 1
(1) Placing 10g of dry and crushed Mucuna anax leaf powder at 55 +/-2 ℃ and 100ml of chromatographic grade 95% ethanol in a beaker, sealing, uniformly mixing by using a vortex instrument, and then carrying out ultrasonic water bath extraction, wherein the ultrasonic water bath temperature is controlled at 38 +/-2 ℃ and the ultrasonic time is 4 hours, so as to obtain an ethanol extract;
(2) cooling the ethanol extract, centrifuging at 6000rpm for 20min, and separating to obtain supernatant;
(3) repeating the leaching and centrifugal separation steps twice in sequence, and combining the supernatants;
(4) rotatably evaporating the combined supernatant at 48 ℃ to obtain 1.96g of a first extract;
(5) dissolving the first extract with water to obtain suspension solution with concentration of 200mg/ml, passing through macroporous resin column (D101), and washing the column with water until the flow is colorless after the sample is completely adsorbed. Eluting with 45 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min until the eluate is colorless, eluting with 60 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min for 8BV, collecting the eluate of 60 v/v% ethanol water solution, and rotary evaporating to obtain second extract;
(6) dissolving the second extract obtained in the step (5) by using methanol, passing the second extract through a reversed-phase ODS column, eluting the second extract by using a 50 v/v% methanol aqueous solution until the second extract is colorless, eluting the second extract by using a 90 v/v% methanol aqueous solution for 9BV, collecting the eluent of the 90 v/v% methanol aqueous solution, and performing rotary evaporation to obtain a third extract;
(7) dissolving the third extract obtained in the step (6) with methanol, passing through a normal phase silica gel column, eluting with chloroform methanol solution (25:1, v/v) until colorless, eluting with chloroform methanol solution (15:1, v/v) for 8BV, and collecting the eluate of chloroform methanol solution (15:1, v/v);
(8) vacuum freeze drying: and (3) cooling the eluent collected in the step (7), and then carrying out freeze-drying under vacuum for 16h at the temperature of minus 45 +/-2 ℃ and the vacuum degree of 12 +/-1 Pa to obtain 0.40g of powdery natural antibacterial active substance with the yield of 4.10%.
Example 2
(1) Placing 10g of dry and crushed Mucuna anax leaves at 55 +/-2 ℃ and 140ml of chromatographic grade 95% ethanol in a beaker, sealing, uniformly mixing by using a vortex instrument, and then carrying out ultrasonic water bath extraction, wherein the ultrasonic water bath temperature is controlled at 42 +/-2 ℃, and the ultrasonic time is 3 hours, so as to obtain an ethanol extract;
(2) cooling the ethanol extract, centrifuging at 6000rpm for 20min, and separating to obtain supernatant;
(3) repeating the leaching and centrifugal separation steps twice in sequence, and combining the supernatants;
(4) rotatably evaporating the combined supernatant at 50 +/-2 ℃ to obtain 2.06g of a first extract;
(5) dissolving the first extract with water to obtain suspension liquid of 200mg/ml, passing through macroporous resin column (D101), and washing the column with water again until the liquid is colorless after the sample is completely adsorbed. Eluting with 45 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min until the eluate is colorless, eluting with 60 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min for 9BV, collecting eluate of 60 v/v% ethanol water solution, and rotary evaporating to obtain second extract;
(6) dissolving the second extract obtained in the step (5) by using methanol, passing the second extract through a reversed-phase ODS column, eluting the second extract by using a 50 v/v% methanol aqueous solution until the second extract is colorless, eluting the second extract by using a 90 v/v% methanol aqueous solution for 10BV, collecting the eluent of the 90 v/v% methanol aqueous solution, and performing rotary evaporation to obtain a third extract;
(7) dissolving the third extract obtained in the step (6) with methanol, passing through a normal phase silica gel column again, eluting with chloroform methanol (25:1, v/v) until colorless, eluting with chloroform methanol solution (15:1, v/v) for 9BV, and collecting the eluate of chloroform methanol solution (15: 1);
(8) vacuum freeze drying: and (4) cooling the eluent collected in the step (7), and then carrying out freeze drying under vacuum, wherein the time of vacuum freeze drying is 16h, the temperature of freeze drying is-45 +/-2 ℃, and the vacuum degree is 12 +/-1 Pa. 0.46g of powdery natural antibacterial active substance is obtained, and the yield is 4.6%.
Comparative example 1
(1) Placing 10g of dried and crushed Mucuna anax leaves and 140ml of chromatographic grade 95% ethanol in a beaker, sealing, uniformly mixing by using a vortex instrument, and then carrying out ultrasonic water bath extraction, wherein the ultrasonic water bath temperature is controlled at 42 +/-2 ℃, and the ultrasonic time is 3 hours, so as to obtain an ethanol extract;
(2) cooling the ethanol extract, centrifuging at 6000rpm for 20min, and separating to obtain supernatant;
(3) repeating the leaching and centrifugal separation steps twice in sequence, and combining the supernatants;
(4) rotatably evaporating the combined supernatant at 50 +/-2 ℃ to obtain 2.06g of a first extract;
(5) dissolving the first extract with water to obtain suspension liquid of 200mg/ml, passing through macroporous resin column (D101), and washing the column with water again until the liquid is colorless after the sample is completely adsorbed. Eluting with 50 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min until the eluate is colorless, eluting with 60 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min for 9BV, collecting eluate of 60 v/v% ethanol water solution, and rotary evaporating to obtain second extract;
(6) dissolving the second extract obtained in the step (5) by using methanol, passing through a reversed-phase ODS column, eluting by using a 65 v/v% methanol aqueous solution until the second extract is colorless, eluting by using a 90 v/v% methanol aqueous solution for 10BV, collecting the eluent of the 90 v/v% methanol aqueous solution, and performing rotary evaporation to obtain a third extract;
(7) dissolving the third extract obtained in the step (6) with methanol, passing through a normal phase silica gel column again, eluting with chloroform methanol solution (20:1, v/v) until colorless, eluting with chloroform methanol solution (15:1, v/v) for 9BV, and collecting the eluate of chloroform methanol solution (15:1, v/v);
(8) vacuum freeze drying: and (4) cooling the eluent collected in the step (7), and then carrying out freeze-drying under vacuum, wherein the time of vacuum freeze-drying is 16h, the temperature of freeze-drying is-45 +/-2 ℃, and the vacuum degree is 12 +/-1 Pa. The obtained powdery extract 0.31g, yield 3.1%.
Comparative example 2
(1) Placing 10g of dried and crushed Mucuna anax leaves and 140ml of chromatographic grade 95% ethanol in a beaker, sealing, uniformly mixing by using a vortex instrument, then carrying out ultrasonic water bath extraction, controlling the temperature of the ultrasonic water bath to be 42 +/-2 ℃ and the ultrasonic time to be 3 hours to obtain an ethanol extracting solution;
(2) cooling the extractive solution, centrifuging at 6000rpm for 20min to obtain supernatant;
(3) repeating the leaching and centrifugal separation steps one time in sequence, and combining the supernatant;
(4) rotatably evaporating the combined supernatant at 50 +/-2 ℃ to obtain 2.06g of a first extract;
(5) dissolving the first extract with water to obtain suspension liquid of 200mg/ml, passing through macroporous resin column (D101), and washing the column with water again until the liquid is colorless after the sample is completely adsorbed. Eluting with 35 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min until the eluate is colorless, eluting with 60 v/v% ethanol water solution at flow rate of 5 + -0.5 mL/min for 9BV, collecting eluate of 60 v/v% ethanol water solution, and rotary evaporating to obtain second extract;
(6) dissolving the second extract obtained in the step (5) by using methanol, passing through a reversed-phase ODS column, eluting by using 40 v/v% methanol aqueous solution until the second extract is colorless, eluting by using 90 v/v% methanol aqueous solution for 10BV, collecting the eluent of the 90 v/v% methanol aqueous solution, and performing rotary evaporation to obtain a third extract;
(7) dissolving the third extract obtained in the step (6) with methanol, passing through a normal phase silica gel column again, eluting with chloroform-methanol solution (35:1, v/v) until colorless, eluting with chloroform-methanol solution (15:1, v/v) for 9BV, and collecting the eluate of chloroform-methanol solution (15:1, v/v);
(8) vacuum freeze drying: and (4) cooling the eluent collected in the step (7), and then carrying out freeze-drying under vacuum, wherein the time of vacuum freeze-drying is 16h, the temperature of freeze-drying is-45 +/-2 ℃, and the vacuum degree is 12 +/-1 Pa. The obtained powdery extract 0.59g, yield 5.9%.
Characterization of Natural bacteriostatic substances
The natural bacteriostatic substance powder prepared in example 2 was subjected to composition analysis using high performance liquid chromatography. The test conditions for high performance liquid chromatography (Agilent 1200) were as follows:
a chromatographic column: HIQ Sil C18V reversed phase column (250 mm. times.4.6 mm I.D.,5 μm)
Mobile phase: acetonitrile (A) -aqueous solution (B), 50/50(v/v)
Isocratic elution conditions: 50% (A)
Flow rate: 1mL/min
Column temperature: 30 deg.C
A detector: evaporative light scattering detector
Sample introduction amount: 10 μ L.
The results of the above tests are shown in fig. 1.
Meanwhile, the results of nuclear magnetic hydrogen spectroscopy and nuclear magnetic carbon spectroscopy measurements of the natural bacteriostatic substance powder prepared in example 2 are shown in fig. 2 and 3, respectively.
As is apparent from the spectra of fig. 1 to 3, the natural bacteriostatic substance obtained according to example 2 is mainly a triterpenoid saponin substance.
Also, the powdered extracts obtained in comparative examples 1 and 2 were subjected to compositional analysis using high performance liquid chromatography, respectively. The test conditions for high performance liquid chromatography (Agilent 1200) were as follows:
a chromatographic column: HIQ Sil C18V reversed phase column (250 mm. times.4.6 mm I.D.,5 μm)
Mobile phase: acetonitrile (A) -aqueous solution (B), 80/20(v/v)
Isocratic elution conditions: 80% (A)
Flow rate: 1mL/min
Column temperature: 30 deg.C
A detector: evaporative light scattering detector
Sample introduction amount: 10 μ L.
The results of the above test are shown in fig. 4 and 5. As is apparent from the spectra in FIGS. 4 and 5, the powdery extracts obtained according to comparative examples 1 and 2 contained a large amount of impurities, and the characteristic peaks of triterpenoid saponins were also not apparent. It can be seen that the extraction conditions of comparative example 1 were not effective for extracting triterpenoid saponins.
As can be seen from the analysis of the extracts of examples 1 and 2 and comparative examples 1 and 2, the extraction method of the invention adopts a specific gradient elution mode for the adsorption and separation of macroporous resin and the separation of a negative phase silica gel column and a positive phase silica gel column, thereby having specific extraction selectivity for the triterpenoid saponin bacteriostatic active substances in the senna leaves. While comparative examples 1 and 2 employed different elution schemes despite the same types of macroporous resin adsorption column, negative phase silica gel column and positive phase silica gel column, the triterpene saponin-based substances could not be efficiently extracted.
Verification of bacteriostatic ability and fresh-keeping effect
The powdery natural bacteriostatic substances obtained in the above examples 1 and 2 were mixed to obtain samples, and the bacteriostatic ability and the fresh-keeping effect of the mixture after picking vegetables and fruits were measured.
Test of bacteriostatic ability
The test strains were: banana colletotrichum, capsicum colletotrichum and cucumber fusarium wilt.
Preparing a natural bacteriostatic substance solution: the mixture of natural bacteriostatic substances extracted according to the above examples 1 and 2 was made up into a solution with a concentration of 8mg/L using 0.1% sterile Tween 80.
Determination of the bacteriostatic effect: sterilizing potato glucose agar (PDA) culture medium at high temperature, cooling to 60 deg.C, adding the above natural antibacterial substance solution, and making into 8mg/L or 4mg/L culture medium containing antibacterial substance. The medium was dispensed on a clean bench into glass plates (15 to 20ml per plate), and the plates were cooled and solidified. The test pathogen cake (diameter 0.6cm) cultured for 5 days was inoculated to the center of the petri dish by using a punch. Blank control is set. The culture dishes are put into an incubator at 28 ℃ for constant-temperature culture, and the bacteriostatic effect is measured by a cross method when the culture dishes are cultured on the 5 th day.
The test results show that when the use concentration of the natural bacteriostatic substance is 8mg/L, the inhibition rate of the natural bacteriostatic substance on three tested pathogenic bacteria reaches 100 percent, namely the pathogenic bacteria do not grow; when the using concentration of the compound is 4mg/L, the inhibition rate of the compound on banana anthracnose is 86.42%, the inhibition rate on pepper anthracnose is 82.14%, and the inhibition rate on cucumber fusarium oxysporum is 72.18%.
Test of preservation effect of picked vegetables and fruits
Respectively spraying 10kg of the prepared 8mg/L natural antibacterial substance solution 50ml onto the surface of banana, pepper and cucumber to be stored, ventilating, naturally drying, and drying 10kg of the natural antibacterial substance solution4cfu/ml of banana colletotrichum, pepper colletotrichum and cucumber fusarium wilt germ spore suspension 50ml are respectively sprayed on the surfaces of bananas, peppers and cucumbers, dried and stored at room temperature. The control is made by replacing the bacteriostatic substance solution with sterile water. On day 5 of storage, incidence was measured.
The results show that: the morbidity of the banana treated group is 15.0 percent, and the morbidity of the control group is 100 percent; the morbidity of the pepper treatment group is 11.69 percent, and the control group is 96.14 percent; the incidence rate of the cucumber treatment group is 20.46%, and the incidence rate of the control group is 86.47%.
Therefore, the sample has strong antibacterial effect and wide antibacterial spectrum.
In conclusion, the natural antibacterial substance prepared by the extraction method can be used for disease prevention and fresh keeping of picked fruits and vegetables, and is safe and harmless to human bodies.
Claims (7)
1. A method for extracting natural antibacterial substances from Musca domestica Linn leaves is characterized by comprising the following steps:
drying folium Murrayae at 55 + -2 deg.C, and pulverizing;
adding chromatographic grade 95% ethanol into the crushed folium cannabis according to the ratio of the mass g of the folium cannabis to the volume ml of the extraction solvent of 1: 10-15, and leaching in an ultrasonic water bath at 35-45 ℃ for 2-5 hours to obtain an ethanol extract;
centrifuging the ethanol extract, and collecting supernatant;
repeating the leaching and centrifugal separation steps for at least 2 times in sequence, combining the supernatants, and rotatably evaporating at 50 +/-2 ℃ to dryness to obtain a first extract;
preparing the first extract into a suspension liquid medicine with the liquid material concentration of 200mg/ml by using water, and then passing the suspension liquid medicine through an adsorption column of macroporous adsorption resin;
eluting the adsorption column by using a first eluent until the flow liquid is colorless, repeating the elution step for 3 times, sequentially using water, 45 v/v% ethanol water solution and 60 v/v% ethanol water solution as the first eluent, collecting the eluent of the 60 v/v% ethanol water solution, and rotatably evaporating to dryness at the temperature of 50 +/-2 ℃ to obtain a second extract;
dissolving the second extract by using methanol and then passing through an octadecyl reverse phase silica gel column;
eluting the octadecyl reverse phase silica gel column by using a second eluent until the flow liquid is colorless, repeating the elution step for 2 times, sequentially using 50 v/v% methanol aqueous solution and 90 v/v% methanol aqueous solution for the second eluent, collecting the eluent of the 90 v/v% methanol aqueous solution, and performing rotary evaporation to obtain a third extract;
dissolving the third extract by using methanol, and then passing through a normal phase silica gel column; and
eluting the normal phase silica gel column by using a third eluent until the flow liquid is colorless, repeating the elution step for 2 times, sequentially using chloroform-methanol solutions with volume ratios of 25:1 and 15:1 for the third eluent, collecting the eluent of the chloroform-methanol solution with the volume ratio of 15:1, and carrying out vacuum freeze drying to obtain natural antibacterial substance powder;
the natural bacteriostatic substance comprises a triterpenoid saponin substance.
2. The method for extracting natural bacteriostatic substances from the leaves of the senna as claimed in claim 1, wherein the macroporous adsorbent resin is D101 type macroporous resin.
3. The method for extracting natural bacteriostatic substances from senna leaves as claimed in claim 1, wherein the flow rate of the first eluent is 5 ± 0.5 mL/min.
4. The method for extracting natural bacteriostatic substances from the leaves of the senna as claimed in claim 1, wherein the time of vacuum freeze-drying is 12-18 h, the temperature is-45 ± 2 ℃, and the vacuum degree is 12 ± 1 Pa.
5. A natural bacteriostatic substance extract obtained by the method according to any one of claims 1 to 4.
6. The use of a natural bacteriostatic substance extract according to claim 5 in disease prevention and freshness preservation of harvested vegetables.
7. Use according to claim 6, wherein the natural bacteriostatic substance extract is used in combination with Tween 80 in an amount of 0.05% based on the mass of the natural bacteriostatic substance extract.
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| CN102119717A (en) * | 2010-12-27 | 2011-07-13 | 中国热带农业科学院南亚热带作物研究所 | Method for preventing and curing anthracnose after picking mango |
| CN105506051A (en) * | 2015-12-23 | 2016-04-20 | 北京颐方生物科技有限公司 | Method for extracting tigogenin and/or hecogenin with composite bacteria method |
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| CN102119717A (en) * | 2010-12-27 | 2011-07-13 | 中国热带农业科学院南亚热带作物研究所 | Method for preventing and curing anthracnose after picking mango |
| CN105506051A (en) * | 2015-12-23 | 2016-04-20 | 北京颐方生物科技有限公司 | Method for extracting tigogenin and/or hecogenin with composite bacteria method |
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| 7 种龙舌兰属植物提取物对两种炭疽病病菌抑菌活性初探;覃旭等;《中国植保导刊》;20160625;第36卷(第6期);第9-12页 * |
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