CN112823666A - Nano-liposome simultaneously containing xanthophyll and cordyceps militaris alcohol extract and preparation method thereof - Google Patents
Nano-liposome simultaneously containing xanthophyll and cordyceps militaris alcohol extract and preparation method thereof Download PDFInfo
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- CN112823666A CN112823666A CN201911145526.8A CN201911145526A CN112823666A CN 112823666 A CN112823666 A CN 112823666A CN 201911145526 A CN201911145526 A CN 201911145526A CN 112823666 A CN112823666 A CN 112823666A
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- alcohol extract
- cordyceps militaris
- lutein
- militaris alcohol
- liposome
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Abstract
A nano liposome simultaneously containing lutein and alcohol extract of cordyceps militaris relates to the field of food nutrition and health. In the nanoliposome, the mass ratio of the cordyceps militaris alcohol extract is 4-8%, and the mass ratio of lutein to the cordyceps militaris alcohol extract is 1: 3-1: 5. The invention adopts an ethanol injection method and a micro-jet technology to realize that two active substances with different hydrophobicity, namely lutein and cordyceps militaris alcohol extract, are co-loaded in the nano liposome, the particle size of the prepared liposome is less than 200nm, the lutein loading rate is higher than 90%, the cordyceps militaris alcohol extract loading rate is higher than 85%, the stability is good, the preparation process is safe and simple, the cost is low, and the industrial production is facilitated.
Description
Technical Field
The invention relates to the field of food nutrition and health, in particular to a nanoliposome containing lutein and cordyceps militaris alcohol extract and a preparation method thereof.
Technical Field
Studies have shown that dietary intake of lutein in certain amounts is associated with a reduced risk of AMD, cancer and cardiovascular disease; the alcohol extract of Cordyceps militaris can inhibit proliferation of various human tumor cell lines, wherein cordycepin is regarded as main antitumor component, and can cooperate with other anticancer components to more effectively induce apoptosis of cancer cells.
In order to improve the in vivo stability, solubility and bioavailability of the two natural active substances and better develop the pharmacological effects of the two natural active substances, in recent years, a novel nano-drug delivery system becomes a research hotspot and difficulty of pharmaceutics and food industry. Lipid-based self-assembling colloid delivery systems are favored by manufacturers for safety of lipid molecules, enhanced transcellular transport, and improved paracellular drug transport. Liposomes have a structure similar to biological membranes, which can improve the in vivo solubility of active substances, reduce toxicity caused by active substances, and provide sustained and targeted delivery; capable of delivering both lipophilic and hydrophilic compounds. At present, 29 nanoliposome medicines are approved to be on the market globally, and the wide application prospect is shown. The literature reports that the lutein nano-liposome with the particle size of 230nm prepared by an ethanol injection method can improve the dispersibility and the bioavailability of lutein (the preparation process optimization and the oxidation stability of the lutein nano-liposome [ J ] food science, 2017, 38 (18): 259 265). The lutein and lycopene mixture co-supported on liposomes play a synergistic antioxidant role (Carotenoid mixtures protect multilamellar lipids: synthetic effects of lycopen and lutein [ J ]. FEBS Letters, 1998, 427 (2): 305-. In intellectual property, chinese patent (publication No. CN105726482A, 2016, 7, 6) discloses a lutein nanoliposome and its preparation method, which is prepared from lutein crystal powder, egg yolk lecithin and cholesterol by high pressure homogenization, ultrasonic dispersion hydration and microfiltration. Chinese patent (publication No. CN106798725A, 6.6.2017) discloses a preparation method of cordycepin nanoliposome, the liposome prepared by combining a pH gradient method and a reverse evaporation method well protects the in vivo activity of cordycepin, and obviously inhibits hepatoma cells, and the liposome obtained by the method has high encapsulation rate (more than or equal to 90%), uniform particle size and good stability.
In the prior art and the technical scheme, two different hydrophobic drugs of lutein and cordyceps militaris alcohol extract are not considered to be co-loaded in the liposome, so that the problems of single active substance embedding, relatively poor pharmacological effect and the like exist. The development of functional foods with various health benefits necessitates the design of carriers that simultaneously entrap multiple active substances.
Disclosure of Invention
The invention aims to provide a nanoliposome simultaneously containing lutein and cordyceps militaris alcohol extract and a preparation method thereof, so as to realize the co-loading of different hydrophobic active substances in the nanoliposome and improve the loading capacity and stability of the lutein and cordyceps militaris alcohol extract.
The purpose of the invention is realized by the following modes:
a preparation method of a nano liposome simultaneously containing lutein and cordyceps militaris alcohol extract comprises the following preparation steps:
1) weighing soybean phospholipid, cholesterol and tween 80 according to the mass ratio of 4: 1, and fully dissolving in absolute ethanol to obtain a soybean phospholipid-cholesterol-tween 80 mixed solution;
2) slowly adding 5-10mL of lutein-ethanol solution into 15-25mL of the soybean lecithin-cholesterol-tween 80 mixed solution obtained in the step 2) under stirring at a magnetic force of 400 revolutions per minute, and homogenizing and emulsifying for 2-4min at 9000 revolutions per minute to obtain lutein suspension;
3) decompressing and concentrating the lutein suspension obtained in the step 2) at 35 ℃, and performing vacuum drying in a dark place to form a light yellow transparent film;
4) adding 20-30mL of phosphate buffer solution containing the cordyceps militaris alcohol extract into the dry film formed in the step 3), and performing reduced pressure rotary hydration at 35 ℃ at 40 rpm to obtain crude liposome suspension containing lutein and cordyceps militaris alcohol extract;
5) and (3) circulating the crude liposome suspension obtained in the step 4) for 2 times through 9000-12000psi high-pressure microjet to obtain a nano liposome solution containing lutein and cordyceps militaris alcohol extract.
The mass ratio of the cordyceps militaris alcohol extract in the nano liposome is 4% -8%, and the mass ratio of the lutein to the cordyceps militaris alcohol extract is 1: 3-1: 5.
The cordyceps militaris alcohol extract is prepared by carrying out ultrasonic-assisted extraction on 50-75% by volume of ethanol solution, and the alcohol extract contains cordycepin, adenosine and cordycepic acid.
Advantageous effects
The ethanol injection method and the micro-jet technology are combined to realize that two substances with different hydrophobicity, namely lutein and cordyceps militaris alcohol extract, are co-loaded in the nano-liposome according to a certain mass ratio and an optimal degree, the particle size of the prepared liposome is less than 200nm, the lutein loading rate is higher than 90%, the cordyceps militaris alcohol extract loading rate is higher than 85%, and the stability is good. Also, the bilayer embedded liposomes (figure 1) may be more potent in synergistically exerting antitumor activity and exert a variety of beneficial health effects in vivo than liposomes containing a single active substance.
Drawings
FIG. 1 is a schematic diagram of the structure of a nanoliposome containing lutein and alcohol extract of Cordyceps militaris
FIG. 2 shows the distribution diagram of the particle size of the nanoliposome containing lutein and alcohol extract of Cordyceps militaris
FIG. 3 photograph of lutein-Cordyceps militaris alcohol extract nanoliposome solution
Detailed Description
The invention provides a nano liposome containing lutein and cordyceps militaris alcohol extract and a preparation method thereof, and the invention is further explained by specific examples.
Example 1
1) Weighing soybean phospholipid, cholesterol and tween 80 according to the mass ratio of 4: 1, and fully dissolving in absolute ethanol to obtain a soybean phospholipid-cholesterol-tween 80 mixed solution;
2) slowly adding 5mL of lutein-ethanol solution into 20mL of the soybean lecithin-cholesterol-tween 80 mixed solution obtained in the step 2) under stirring at a magnetic force of 400 r/min, and homogenizing and emulsifying for 2min at 9000 r/min to obtain lutein suspension;
3) decompressing and concentrating the lutein suspension obtained in the step 2) at 35 ℃, and performing vacuum drying in a dark place to form a light yellow transparent film;
4) adding 20mL of phosphate buffer solution containing the cordyceps militaris alcohol extract into the dry film formed in the step 3), and performing reduced pressure rotary hydration at 35 ℃ at 40 rpm to obtain crude liposome suspension containing lutein and cordyceps militaris alcohol extract;
5) circulating the crude liposome suspension obtained in the step 4) for 2 times by 10000psi high-pressure micro-jet to obtain nano liposome solution containing lutein and Cordyceps militaris alcohol extract (figure 3).
The particle size of the liposome is measured by using a PSS nano laser particle sizer, and the measuring method comprises the following steps: diluting the liposome solution sample to 100 times with deionized water, adding 4mL in polystyrene tank, balancing for 2min, testing at 25 deg.C, and measuring with water property as measuring parameters, wherein the solution viscosity and refractive index are 0.933CP and 1.333 respectively. The particle size distribution diagram is shown in figure 2.
Taking 500 mu L of the freshly prepared lutein-cordyceps militaris extract nanoliposome, placing the 500 mu L of the liposome in a 2mL centrifuge tube, centrifuging at 10000 rpm for 30min, taking 250 mu L of supernatant after centrifuging, placing the supernatant in a 10mL centrifuge tube, adding 2mL of DCM (dichloromethane: methanol is 2: 1, V: V) solvent, repeatedly extracting for 3 times by using 1mL of n-hexane after vortex oscillation for 5min, mixing extract liquor, filtering by using a 0.45 mu m organic filter head, measuring a light absorption value at the maximum absorption wavelength of 460nm, and calculating the content of lutein, namely the amount of free lutein; and adding methanol into 500 mu L of liposome to a constant volume of 5mL, performing 100W ultrasonic treatment for 30min to break emulsion, putting 250 mu L of broken emulsion into a 10mL centrifuge tube, repeating the above lutein extraction method, and calculating to obtain the lutein content, namely the total lutein content in 250 mu L of liquid. The calculation formula of the lutein loading rate (%) is as follows:
taking cordycepin as an embedding rate index of the alcohol extract of cordyceps militaris, taking 500 mu L of lutein-alcohol extract nanoliposome of cordyceps militaris which is just prepared in a 2mL centrifuge tube, centrifuging for 30min at 10000 r/min, taking supernatant after centrifugation, filtering by a 0.45 mu m filter membrane, filtering by a 0.22 mu m filter membrane to remove solid impurities, and analyzing by a high performance liquid chromatography-ultraviolet detector to obtain the detection wavelength of 260 nm. The content of free cordycepin is obtained by calculation. In addition, 500. mu.L of liposome is added with 0.1 percent Triton X-100 to be constant volume to 5mL, and 100W is subjected to ultrasonic treatment for 30min to demulsify. Repeating the above filtration process, analyzing by high performance liquid chromatography-ultraviolet detector, and calculating to obtain total cordycepin content in liposome of 500 μ L. The calculation formula of the cordyceps militaris alcohol extract loading rate (%) is as follows:
the average particle size of the lutein-cordyceps militaris alcohol extract composite nano-liposome measured by the method is 131 +/-72 nm, the loading rate of lutein is 98.2%, and the loading rate of cordyceps militaris alcohol extract is 89.1%.
Example 2
1) Weighing soybean phospholipid, cholesterol and tween 80 according to the mass ratio of 4: 1, and fully dissolving in absolute ethanol to obtain a soybean phospholipid-cholesterol-tween 80 mixed solution;
2) slowly adding 10mL of lutein-ethanol solution into 20mL of the soybean lecithin-cholesterol-tween 80 mixed solution obtained in the step 2) under stirring at a magnetic force of 400 r/min, and homogenizing and emulsifying for 3min at 9000 r/min to obtain lutein suspension;
3) decompressing and concentrating the lutein suspension obtained in the step 2) at 35 ℃, and performing vacuum drying in a dark place to form a light yellow transparent film;
4) adding 30mL of phosphate buffer solution containing the cordyceps militaris alcohol extract into the dry film formed in the step 3), and performing reduced pressure rotary hydration at 35 ℃ at 40 rpm to obtain crude liposome suspension containing lutein and cordyceps militaris alcohol extract;
5) circulating the crude liposome suspension obtained in the step 4) for 1 time by 12000psi high-pressure microjet to obtain a nano liposome solution containing lutein and cordyceps militaris alcohol extract, wherein the particle size of the liposome is 163 +/-69 nm, the loading rate of the lutein is 93.7%, and the loading rate of the cordyceps militaris alcohol extract is 88.4%.
Example 3
1) Weighing soybean phospholipid, cholesterol and tween 80 according to the mass ratio of 4: 1, and fully dissolving in absolute ethanol to obtain a soybean phospholipid-cholesterol-tween 80 mixed solution;
2) slowly adding 8mL of lutein-ethanol solution into 15mL of the soybean lecithin-cholesterol-tween 80 mixed solution obtained in the step 2) under the magnetic stirring of 400 r/min, and homogenizing and emulsifying for 4min at 9000 r/min to obtain lutein suspension;
3) decompressing and concentrating the lutein suspension obtained in the step 2) at 35 ℃, and performing vacuum drying in a dark place to form a light yellow transparent film;
4) adding 25mL of phosphate buffer solution containing the cordyceps militaris alcohol extract into the dry film formed in the step 3), and performing reduced pressure rotary hydration at 35 ℃ at 40 rpm to obtain crude liposome suspension containing lutein and cordyceps militaris alcohol extract;
5) circulating the crude liposome suspension obtained in the step 4) for 3 times by 9000psi high-pressure microjet to obtain a nano liposome solution containing lutein and cordyceps militaris alcohol extract, wherein the particle size of the liposome is 203 +/-89 nm, the loading rate of the lutein is 92.5%, and the loading rate of the cordyceps militaris alcohol extract is 87.3%.
Claims (3)
1. A nanoliposome containing lutein and cordyceps militaris alcohol extract simultaneously is characterized in that the mass ratio of the cordyceps militaris alcohol extract in the nanoliposome is 4% -8%, and the mass ratio of the lutein to the cordyceps militaris alcohol extract is 1: 3-1: 5.
2. The nanoliposome containing lutein and cordyceps militaris alcohol extract according to claim 1, wherein the cordyceps militaris alcohol extract is prepared by performing ultrasonic assisted extraction with 50-75% by volume of ethanol solution, and the alcohol extract contains cordycepin, adenosine and cordycepic acid.
3. The preparation method of the nanoliposome containing lutein and cordyceps militaris alcohol extract simultaneously according to claim 1 is characterized by comprising the following preparation steps:
1) weighing soybean phospholipid, cholesterol and tween 80 according to the mass ratio of 4: 1, and fully dissolving in absolute ethanol to obtain a soybean phospholipid-cholesterol-tween 80 mixed solution;
2) slowly adding 5-10mL of lutein-ethanol solution into 15-25mL of the soybean lecithin-cholesterol-tween 80 mixed solution obtained in the step 2) under stirring at a magnetic force of 400 revolutions per minute, and homogenizing and emulsifying for 2-4min at 9000 revolutions per minute to obtain lutein suspension;
3) decompressing and concentrating the lutein suspension obtained in the step 2) at 35 ℃, and performing vacuum drying in a dark place to form a light yellow transparent film;
4) adding 20-30mL of phosphate buffer solution containing the cordyceps militaris alcohol extract into the dry film formed in the step 3), and performing reduced pressure rotary hydration at 35 ℃ at 40 rpm to obtain crude liposome suspension containing lutein and cordyceps militaris alcohol extract;
5) and (3) circulating the crude liposome suspension obtained in the step 4) for 2 times through 9000-12000psi high-pressure microjet to obtain a nano liposome solution containing lutein and cordyceps militaris alcohol extract.
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| CN201911145526.8A CN112823666A (en) | 2019-11-21 | 2019-11-21 | Nano-liposome simultaneously containing xanthophyll and cordyceps militaris alcohol extract and preparation method thereof |
| LU101863A LU101863B1 (en) | 2019-11-21 | 2020-05-25 | Nano-liposome simultaneously containing lutein and cordyceps militaris alcohol extract, and preparation method therefor |
| PCT/CN2020/092080 WO2021098174A1 (en) | 2019-11-21 | 2020-05-25 | Nanoliposome containing lutein and cordyceps militaris alcohol extract, and method for preparation thereof |
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