CN116919842A - Stable and safe phycocyanin nanoemulsion preparation and preparation method thereof - Google Patents
Stable and safe phycocyanin nanoemulsion preparation and preparation method thereof Download PDFInfo
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- PRXRUNOAOLTIEF-ADSICKODSA-N Sorbitan trioleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCC\C=C/CCCCCCCC)[C@H]1OC[C@H](O)[C@H]1OC(=O)CCCCCCC\C=C/CCCCCCCC PRXRUNOAOLTIEF-ADSICKODSA-N 0.000 description 4
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- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 2
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicinal Preparation (AREA)
Abstract
The application relates to the technical field of nano materials, in particular to a stable and safe phycocyanin nanoemulsion preparation and a preparation method thereof. The phycocyanin nanoemulsion preparation comprises a surfactant, a cosurfactant, an oil phase and a water phase, wherein the mass ratio of the surfactant to the cosurfactant is 1-5:1, the mass ratio of the surfactant to the oil phase is 1-9:1, the phycocyanin nanoemulsion preparation also comprises an phycocyanin aqueous solution, the concentration of the phycocyanin aqueous solution is 0.5% -8%, and the mass ratio of the surfactant to the oil phase to the water phase is 25-55:33:8-30. The prepared stable and safe nanoemulsion preparation improves the stability and biological activity of the nanoemulsion.
Description
Technical Field
The application relates to the technical field of nano materials, in particular to a stable and safe phycocyanin nanoemulsion preparation and a preparation method thereof.
Background
Nanoemulsions are discovered from 1943, develop rapidly after the period of 1943, are widely used as drug carriers in the medicine field after the period of 80 years, and research in the field in China just begins, and application research of nanoemulsions is more rapid after the period of 90 years.
Nanoemulsion refers to an emulsion with a particle size in the range of 20-200 nanometers, and has the following advantages: the thermodynamics and dynamics are stable, and flocculation, aggregation, layering and other phenomena do not occur; no toxicity and no irritation; the administration route is diversified, such as oral administration, transdermal administration, gastrointestinal administration, topical administration, etc.; can entrap hydrophobic or hydrophilic drugs; the particle size is small, the surface area is large, so that the absorption can be promoted, the variability is reduced, and the bioavailability of the medicine is improved; has no harm to human or animal, and is suitable for treating human or animal diseases; the medicine is entrapped so that the medicine is not easy to hydrolyze or oxidize and can mask bad taste; can enhance transdermal absorption of the drug.
The current preparation method of nanoemulsion comprises a desolventizing chemical crosslinking method, a self-assembly method, a high-pressure homogenizing emulsification method and the like. In addition, some novel techniques such as reverse micelle method, high gravity field method, etc. are also used for the preparation of nanoemulsion. In addition, a phase transition temperature method (PIT method) was studied, which was proposed by Shinoda et al in 1969, and mainly uses nonionic surfactant molecules whose spontaneous curvature is zero and surface tension is reduced to very low at the phase transition temperature to promote emulsification. The acyclovir W/O/W bicontinuous nanoemulsion prepared by a PIT method has the advantages of uniform and stable particle size distribution of about 100nm and good transdermal performance, and the nanoemulsion is used as an excellent carrier to greatly improve the skin permeability of acyclovir.
The nanoemulsion has wide application prospects in the fields of foods, medicines and the like. In foods, nanoemulsions can be used as emulsifiers, stabilizers, nutritional additives, and the like; in the medicine, the nanoemulsion can be used for targeted administration, slow release, controlled release and the like.
With the continuous deep understanding of the characteristics and functions of phycocyanin, the application prospect of the phycocyanin is wider and wider. Phycocyanin belongs to polypeptide substances, marine bioactive peptides have wide prospects in the fields of antioxidant health-care foods, medicines, cosmetics and the like at present, and with the arrival of aging of population, antioxidant functional foods and the like are more and more favored by people, but the polypeptide substances have instability, and stability becomes a bottleneck for limiting the application of the polypeptide substances, so the solution of stability problems is promoted to the application range and scale of the phycocyanin.
Disclosure of Invention
The present disclosure provides a stable and safe phycocyanin nanoemulsion formulation and a preparation method thereof, and the stable and safe nanoemulsion formulation is prepared, so that the stability and the bioactivity of the nanoemulsion are improved.
In a first aspect, the present disclosure provides a stable and safe phycocyanin nanoemulsion formulation, the phycocyanin nanoemulsion formulation includes a surfactant, a cosurfactant, an oil phase and a water phase, the mass ratio of the surfactant to the cosurfactant is 1-5:1, the mass ratio of the surfactant to the oil phase is 1-9:1, the phycocyanin nanoemulsion formulation further includes an aqueous phycocyanin solution, the concentration of the aqueous phycocyanin solution is 0.5% -8%, and the formula mass ratio of the surfactant, the oil phase and the water phase is 25-55:33:8-30.
Phycocyanin is a dark blue powder isolated from spirulina. Mainly in blue algae, red algae and hidden algae. The main component of the medicine is porphyrin pigment protein, has pharmacological actions of antioxidation, anti-inflammatory, anticancer, neuroprotection, immunoregulation and the like, and is often applied to the field of medical care. Phycocyanin can be used as a natural pigment in cosmetics industry as colorant, food additive, and anti-inflammatory agent, antioxidant, anticancer agent, immunomodulator and fluorescent detection probe. Phycocyanin is a natural protein extracted from blue algae, and has various biological activities such as anti-inflammatory, antioxidant and immunosuppression.
According to the characteristics that phycocyanin is easy to dissolve in water, easy to degrade and easy to decompose in visible light, the prescription of the phycocyanin nano-preparation is researched, the optimal prescription is discussed, and the phycocyanin nano-preparation is prepared into a stable and safe nano-emulsion preparation, so that the stability and the bioactivity of the phycocyanin nano-preparation are improved.
Preferably, the compound surfactant is Tween80-Span80, wherein hlb=4-7.
Preferably, the cosurfactant is one of ethanol, propylene glycol and glycerol.
In a second aspect, the present disclosure provides a method of preparing a stable and safe phycocyanin nanoemulsion formulation, comprising the steps of:
(1) At room temperature, adding the cosurfactant and the oil phase into the surfactant, fully mixing the three phases, and uniformly stirring by using a magnetic stirrer to prepare a mixed system;
(2) Dropwise adding the water phase into the mixed system, wherein when dropwise adding is started, the system presents a clear oil phase with smaller viscosity due to less oil phase and water phase, the system becomes turbid as the water phase is continuously added, and the system gradually becomes clear from turbid after continuous dropwise adding, so that the W/O type nanoemulsion is formed;
(3) Gradually and slowly adding the phycocyanin aqueous solution into the W/O type nanoemulsion, uniformly stirring by using a magnetic stirrer, and gradually dropwise adding distilled water until clear and transparent nanoemulsion is formed.
The nanoemulsion is prepared by adopting a phase inversion emulsification method. At room temperature, adding a proper cosurfactant and an oil phase into a proper surfactant, fully mixing three phases, uniformly stirring by a magnetic stirrer, then dropwise adding the water phase into a mixed system, and when the dropwise adding is started, the system presents a clear oil phase with smaller viscosity due to less oil phase and water phase, and the system becomes cloudy as the water phase is continuously added, and continuously dropwise adding, the system gradually becomes clear from cloudy, thus the W/O nanoemulsion is formed. The nanoemulsion prepared by visual inspection should be transparent or semitransparent, should have thermodynamic stability, and cannot be layered by hot press sterilization or centrifugation. The water carrying capacity is high, the water carrying capacity is generally represented by a phase diagram, and the larger the range of the formable nanoemulsion region is, the easier the nanoemulsion is formed, and the nanoemulsion prepared under the condition has more stable properties.
The used reagents are nontoxic reagents, and the prepared phycocyanin nanoemulsion has high safety. After the nanoemulsion is prepared, enzymolysis of the medicine in vivo is reduced, so that the protection effect on the medicine can be formed and the absorption of the gastrointestinal tract on the medicine can be improved.
Preferably, in the step (1), the oil phase is one of liquid paraffin, soybean oil, olive oil, and isopropyl myristate (IPM).
Preferably, in the step (2), the phycocyanin aqueous solution is prepared by adding pure water.
Preferably, the particle size of the prepared phycocyanin nanoemulsion is 180nm-220nm.
In a third aspect, the present disclosure provides the use of an phycocyanin nanoemulsion formulation in health foods, pharmaceuticals, cosmetics for antioxidant, anti-inflammatory, anticancer, neuroprotection and immunomodulation.
The stability is good, and the physical stability of the phycocyanin nanoemulsion is good. Belongs to a thermodynamic stability system, and can not be delaminated after hot press sterilization or centrifugation. No toxic and side effects and safety. The reagent has no toxic or side effect. Can improve bioavailability of the medicine, reduce enzymolysis of the medicine in vivo, protect the medicine and improve absorption of gastrointestinal tract to the medicine. Prolonging half-life of the medicine in vivo.
In summary, the application has the following beneficial effects:
1. since phycocyanin is a dark blue powder isolated from spirulina in the present application. Mainly in blue algae, red algae and hidden algae. The main component of the medicine is porphyrin pigment protein, has pharmacological actions of antioxidation, anti-inflammatory, anticancer, neuroprotection, immunoregulation and the like, and is often applied to the field of medical care. Phycocyanin can be used as a natural pigment in cosmetics industry as colorant, food additive, and anti-inflammatory agent, antioxidant, anticancer agent, immunomodulator and fluorescent detection probe. Phycocyanin is a natural protein extracted from blue algae, and has various biological activities such as anti-inflammatory, antioxidant, immunosuppression and the like;
2. the reagent used in the application is nontoxic, the prepared phycocyanin nanoemulsion has high safety, and the enzymolysis of the medicine in the body is reduced after the phycocyanin nanoemulsion is prepared, so that the protection effect on the medicine can be formed and the absorption of the gastrointestinal tract on the medicine can be improved;
3. the phycocyanin nanoemulsion has good stability, belongs to a thermodynamic stability system, can not be layered after hot press sterilization or centrifugation, has no toxic or side effect, is safe, has no toxic or side effect, can improve the bioavailability of the medicament, reduces the enzymolysis of the medicament in vivo, can form the protection effect on the medicament, improves the absorption of the gastrointestinal tract on the medicament, and prolongs the half life of the medicament in vivo.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the disclosure.
Drawings
1. Fig. 1 is a pseudo ternary phase diagram for a blank nanoemulsion single surfactant selection: fig. 1 is a pseudo ternary phase diagram for a blank nanoemulsion single surfactant selection: a: tween80 (hlb=15), B: span80 (hlb=4.3), C: span85 (hlb=1.8);
2. fig. 2 is a pseudo ternary phase diagram for a blank nanoemulsion formulated surfactant selection: tween80-Span80 (a: hlb=8, b: hlb=7, c: hlb=6), tween80-Span85 (D: hlb=7, e: hlb=6, f: hlb=5);
3. FIG. 3 is a blank nanoemulsion surfactant selected pseudo-ternary phase diagram milk domain area;
4. FIG. 4 is a pseudo ternary phase diagram for a surfactant blank selection;
5. FIG. 5 is a schematic illustration of the area of a pseudo ternary phase diagram emulsion region for a blank nanoemulsion co-surfactant (A: ethanol, B: glycerol, C: propylene glycol);
6. fig. 6 is a pseudo ternary phase diagram for selection of k m values for a blank nanoemulsion: a:4:1, B:3:1, C:2:1, D:1:1;
7. FIG. 7 is a graph of dummy ternary phase diagram milk area for selection of Km values for blank nanoemulsions;
8. fig. 8 is a blank nanoemulsion phase selection pseudo ternary phase diagram: a: liquid paraffin, B: soybean oil, C: olive oil, D: isopropyl myristate (IPM);
9. FIG. 9 is a blank nanoemulsion phase selection pseudo ternary phase diagram emulsion area;
10. fig. 10 is a blank nanoemulsion aqueous phase selection pseudo ternary phase diagram: a:0%, B:0.5%, C:1%, D:2%, E:3%, F:4%, G:5%, H:6%, I:7%, J:8%;
11. FIG. 11 is a blank nanoemulsion phase selection pseudo ternary phase diagram milk area;
12. FIG. 12 is a graph of particle size and PDI of nanoemulsions formulated in different proportions in the influence of mixed surfactant on the physicochemical properties of nanoemulsions;
13. fig. 13 is a graph of particle size and PDI of nanoemulsion formulations of different ratios in the influence of phycocyanin aqueous solution on the physicochemical properties of nanoemulsion.
Detailed Description
The application is further described in detail below with reference to the following examples, which are specifically described: the following examples, in which no specific conditions are noted, are conducted under conventional conditions or conditions recommended by the manufacturer, and the raw materials used in the following examples are commercially available from ordinary sources except for the specific descriptions.
The preparation method of the nanoemulsion comprises the following steps:
the embodiment adopts a phase inversion emulsification method to prepare the nanoemulsion. At room temperature, adding a proper cosurfactant and an oil phase into a proper surfactant, fully mixing three phases, uniformly stirring by a magnetic stirrer, then dropwise adding the water phase into a mixed system, and when the dropwise adding is started, the system presents a clear oil phase with smaller viscosity due to less oil phase and water phase, and the system becomes cloudy as the water phase is continuously added, and continuously dropwise adding, the system gradually becomes clear from cloudy, thus the W/O nanoemulsion is formed.
Evaluation criteria for blank nanoemulsion:
the nanoemulsion prepared by visual inspection should be transparent or semitransparent, should have thermodynamic stability, and cannot be layered by hot press sterilization or centrifugation. The higher the water carrying capacity (Liu Kai, etc., 2016), which is generally represented by a phase diagram, it is generally believed that the larger the range of nanoemulsion regions that can be formed, the more likely nanoemulsion is formed and that nanoemulsions produced under these conditions are more stable in character (Pan Haimin, etc., 2006).
Establishing a pseudo ternary phase diagram:
establishing a pseudo ternary phase diagram is the most basic method for evaluating nanoemulsion systems. In the screening of the blank nanoemulsion formulation, the oil phase was thoroughly mixed with the surfactant and cosurfactant, followed by thorough stirring at room temperature using a magnetic stirrer. And respectively dripping pure water into each mixed system, observing the change of the clarity of the system, forming nanoemulsion, and recording the volume fractions of each phase. When the mass fraction of the three phases of oil, water and mixed surfactant reaches a critical point when a pseudo-ternary phase diagram is drawn, the range of the formed emulsion region can be used for determining the basic formula composition of the nanoemulsion.
Examples
Example 1
Screening of surfactant and mixed surfactant:
screening surfactants by taking an HLB value as an index, respectively selecting Tween80 (HLB=15), span80 (HLB=4.3) and Span85 (HLB=1.5) as single surfactants for screening, and then respectively screening the compound surfactants with the HLB value of 4-7. When the surfactant is screened, absolute ethyl alcohol is fixed as cosurfactant, km value is set to be 3:1, and soybean oil is used as oil phase. Mixing surfactant and oil phase according to the ratio of agent to oil (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1) for 1h, magnetically stirring, dropwise adding pure water by a water dripping method, immediately oscillating after each dripping to fully and uniformly mix the mixture, repeating the above operation until the solution becomes turbid from clarification, recording the adding amount of the pure water at the critical point, and drawing a pseudo ternary phase diagram.
Although nanoemulsions can be formed using a single surfactant, there are few nanoemulsion regions formed. Studies have shown that more stable nanoemulsions can be formed using mixed surfactants. Based on single surfactant screening, the influence of the single surfactant and the mixed surfactant on the formation of nanoemulsion is compared by compounding Tween80 and Span80 and Tween80 and Span85 according to an HLB value compounding principle.
Effect of surfactant and mixed surfactant on nanoemulsion formation:
first, the effect of three single surfactants on nanoemulsion formation was compared with the HLB value as a reference. As a result, three single surfactants can form W/O type nanoemulsions, but different surfactants are selected, so that the formed nanoemulsion areas are different. As shown in fig. 1, three different surfactants form nanoemulsion domains of: tween80> Span85> Span80, it can be seen from the phase diagram that the selection of Tween80 can form a large number of regions of nanoemulsion, while the selection of two other surfactants can form a small number of regions.
As shown in FIG. 2, tween80-Span80 was selected and the HLB value was formulated to be 7 to maximize the milk area. As is evident from fig. 3, the milk area formed by using the single surfactant Tween80 is the largest, and the milk area formed by using the mixed surfactant Tween80-Span80 (hlb=7) is larger. Thus, the final selection was made of Tween80 or the mixed surfactant Tween80-Span80 (hlb=7).
Example 2
Screening of cosurfactant:
ethanol, propylene glycol and glycerol are selected as cosurfactants. The surfactant and the cosurfactant are uniformly mixed according to the proportion of 1:1, 2:1, 3:1, 4:1 and 5:1, and the influence of the cosurfactant on the formation of nanoemulsion is observed. Fully mixing a cosurfactant, a surfactant and oil phase (soybean oil) at room temperature, fully stirring by a magnetic stirrer, then gradually dripping pure water into a mixed system, continuously stirring, observing whether the mixed system becomes turbid, and when the turbid system becomes clear, the cosurfactant is suitable for preparing nanoemulsion, after nanoemulsion is formed, recording the volume fractions of each phase, and determining the optimal cosurfactant.
Effect of cosurfactants on nanoemulsion formation:
as shown in fig. 4 and 5, three cosurfactants can promote the formation of nanoemulsion, and the area of the milk zone formed is as follows: ethanol > propylene glycol > glycerol. This is probably because ethanol contains a large amount of hydroxyl groups, and propylene glycol and glycerol contain a large amount of water, which affects the formation of nanoemulsion. It follows that ethanol is a better cosurfactant.
Example 3
Screening of Km values:
km value indicates the ratio of surfactant to cosurfactant, which is also important in the process of preparing nanoemulsions. The surfactant and cosurfactant are mixed with the set oil phase (soybean oil) according to the ratio of 2:1, 3:1 and 4:1. At room temperature, uniformly stirring by using a magnetic stirrer, gradually dropwise adding pure water into the mixed system, observing whether the system becomes turbid, judging that the Km value is suitable for preparing nanoemulsion when the turbid system is recovered to be clear after stirring, recording the volume fractions of each phase after nanoemulsion is formed, and determining the optimal Km value.
Screening results of Km values:
km is the ratio of surfactant to cosurfactant. Related studies have shown that Km values also have a certain influence on nanoemulsion formation. In the experiment, a pseudo ternary phase diagram is successfully drawn by adopting a mixed surfactant of Tween80-Span80 (HLB=7) and ethanol as an auxiliary surfactant and screening different Km ratios. On the basis, the influence of the emulsifier on the performance of the emulsion is studied by changing the dosage of the emulsifier. As shown in fig. 6 and 7, as the Km ratio increases, the milk area increases. However, the milk area reaches a maximum value when Km is 3:1, and decreases as Km continues to increase to 4:1. From this, it can be seen that the Km value has a certain influence on the preparation of nanoemulsion, and is optimal when the Km value is set to 3:1. The reason for this is believed by the pen to be that a certain amount of surfactant will improve the fluidity and elasticity of the system and thus promote the formation of nanoemulsion, but a high content of surfactant will cause the fluidity of nanoemulsion to exceed the desired value and thus affect the stability of nanoemulsion and even the formation of nanoemulsion.
Example 4
Screening of oil phase:
liquid paraffin, soybean oil, olive oil, isopropyl myristate (IPM) are selected as oil phase. These oil phases are mixed uniformly with the selected surfactant and cosurfactant at the selected Km value. At room temperature, uniformly stirring by using a magnetic stirrer, gradually dropwise adding pure water into a mixed system, gradually turning the system into turbidity, continuously stirring, observing whether the turbid system becomes clear or not, and when the turbid system becomes clear, recording volume fractions of each phase after the nanoemulsion is formed, and determining the optimal oil phase.
Screening results of oil phase:
the oil phase is an integral part of the nanoemulsion. Tween80-Span80 (HLB=7) was chosen as the co-surfactant, ethanol as co-surfactant, and Km value was 3:1 to compare the effect of different oil phases on nanoemulsion preparation, as shown in FIG. 8, and a corresponding pseudo-ternary phase diagram was plotted. The oil phase selected as shown in the figures all contribute to nanoemulsion formation, but with differences in impact. As can be intuitively seen from fig. 9, the area of the milk area formed by using IPM as the oil phase is the largest, and the area of the milk area formed by using olive oil as the oil phase is the smallest. From the related studies, it is known that the formation of nanoemulsion is affected by the fatty chain of the oil phase. Olive oil belongs to long-chain oil phase, has large volume, so the contact area with the surfactant is large, and the interfacial film is not easy to form. IPM has a suitable fatty chain length and can be fully contacted with a surfactant, so that the emulsion area for forming nanoemulsion is maximum, and IPM is safe and nontoxic, so that IPM is selected as an oil phase.
Example 5
Screening of aqueous phase:
and fully mixing the screened surfactant and cosurfactant with the oil phase at room temperature according to a proper Km value, fully stirring by using a magnetic stirrer to achieve a uniform mixing effect, and gradually dripping pure water and phycocyanin aqueous solutions with the concentrations of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% into a mixed system respectively. The system becomes turbid gradually, stirring is continued, whether the turbid system becomes clear gradually is observed, when the turbid system becomes clear, the concentration of the phycocyanin aqueous solution is suitable for preparing the nanoemulsion, after the nanoemulsion is formed, the volume fractions of all phases are recorded, and the concentration of the phycocyanin aqueous solution which can be borne by the nanoemulsion is determined.
Screening results of aqueous phase:
as shown in fig. 10, the experiment uses mixed surfactant Tween80-Span80 (hlb=7), ethanol as cosurfactant, km value is 3:1, IPM is used as oil phase, and the cosurfactant is mixed with phycocyanin aqueous solutions with different concentrations to form nanoemulsion, the influence of the phycocyanin aqueous solutions with different concentrations on the preparation of nanoemulsion and the maximum concentration which can be contained in the nanoemulsion are compared, and a pseudo ternary phase diagram is drawn. As can be seen from fig. 10 and 11, the milk area is the highest when the concentration of the phycocyanin aqueous solution is 5%, and thus the optimal concentration. The concentration continues to increase and the milk area decreases dramatically. Therefore, the maximum concentration of phycocyanin which can be contained in the nanoemulsion is about 5%.
Example 6
Preparation of phycocyanin nanoemulsion:
on the basis of preparing the hollow white nano preparation, proper surfactant, cosurfactant and oil phase are proportionally used, an phycocyanin aqueous solution is gradually and slowly added, and distilled water is gradually and dropwise added after the phycocyanin aqueous solution is uniformly stirred by a magnetic stirrer until clear and transparent nano emulsion is formed.
Optimization of phycocyanin nanoemulsion formulation:
from the determined base formula, a preliminary milk area range may be derived. In order to obtain a larger milk area, an optimal prescription is obtained, the prescription is to be optimized, and the influence of the mixed surfactant content and the water phase content on the nanoemulsion is examined. And fixing the ratio of the water phase content, and screening surfactants with different ratios, or fixing the ratio of the surfactant content, and screening the water phase content with different ratios. And the PDI and the particle size distribution are used as indexes respectively.
Nanoemulsion particle size distribution and PDI determination:
the Mean Droplet Diameter (MDD) and polydispersity index (PDI) of the nanoemulsion were measured using a Zetasizer rnano-zs 2000. The detection angle was set to 173 °, the temperature was set to 25 ℃, and all samples were allowed to equilibrate in the test cell for 2min prior to measurement.
Influence of mixed surfactants on nanoemulsion physicochemical properties:
the fixed aqueous phase (W) ratio in this example was 20%, the mixed surfactant (Smix) ratios in nanoemulsion formulations were screened for 25, 30, 35, 38, 40, 41, 43, 45, 46, 47, 49, 50, 55 (%), and particle size and PDI index were measured. As shown in fig. 12, when the mixed surfactant accounts for 45% of the nanoemulsion formulation, the particle size of the obtained nanoemulsion is minimum, and the PDI at this time is minimum, the system is most stable, and the change of the particle size and the PDI with time is not obvious. In addition, the particle size of the nanoemulsion prepared by all the formulas is larger than 200nm, and the particle size is overlarge, and the main reasons include: (1) in the process of preparing the nanoemulsion, if the operation is incorrect, such as too slow stirring speed, too short stirring time, too high or too low temperature and the like of the emulsion, the particle size of the nanoemulsion is too large; (2) raw material problems: if the quality of the emulsifier or surfactant in the raw material is poor, or if the addition is excessive or insufficient, the nanoemulsion particle size may be excessively large. The preparation of nanoemulsion is a relatively complex process, and the particle size and stability of nanoemulsion need to comprehensively consider various factors, and the preparation process is carefully controlled and monitored.
Influence of phycocyanin aqueous solution on nanoemulsion physicochemical properties:
fixing the mixed surfactant (Smix) 45%, screening the ratio of the aqueous phase (W) in the nanoemulsion formulation (1) (10, 15, 20; (2) 8, 12, 16, 20, 25, 30; (3) 20, 21, 22, 23, 24, 25 (%), and particle size and PDI index were measured. As shown in fig. 13, when the phycocyanin content is 22% in the nanoemulsion formulation, the PDI of the obtained nanoemulsion is minimum, and the particle size of the nanoemulsion is 220nm, which indicates that the prepared nanoemulsion has better dispersibility, but the particle size is larger and not smaller than 200nm. When the phycocyanin content is 25% in the nanoemulsion formulation, the nanoemulsion particle size is smaller than 200nm, but the PDI is higher, which indicates that the nanoemulsion droplet dispersibility is general.
A compound surfactant Smix:45%, aqueous phase W:22%, oil phase O:33%, total amount of about 10g, magnetic stirring for 1h, constant temperature water bath temperature 28,32,36 ℃ (thermometer, + -1 ℃), dropwise adding water phase for 3 s/drop. Particle size measurement parameters solvent water.
TABLE 1
Combining table 1, a basic formula for preparing nanoemulsion is obtained by using a pseudo ternary phase diagram, a compound surfactant Tween80-Span80 (HLB=7) is used as a surfactant, absolute ethyl alcohol is used as a cosurfactant, and Km value is set to be 3:1, isopropyl myristate (IPM) is used as an oil phase, and an aqueous phycocyanin solution with a concentration of 5% is used as an aqueous phase.
The optimal proportion obtained by optimizing the phycocyanin nanoemulsion formula on the basis of blank nanoemulsion preparation is as follows: a compound surfactant Smix: oil phase O: aqueous phase w=45: 33:22.
while the present disclosure has been described with respect to exemplary embodiments thereof, it should be understood that the scope of the present disclosure is not limited thereto, but rather, any changes or substitutions that would occur to one skilled in the art within the scope of the present disclosure should be included in the scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.
Claims (8)
1. The stable and safe phycocyanin nanoemulsion preparation is characterized by comprising a surfactant, a cosurfactant, an oil phase and a water phase, wherein the mass ratio of the surfactant to the cosurfactant is 1-5:1, the mass ratio of the surfactant to the oil phase is 1-9:1, the phycocyanin nanoemulsion preparation also comprises an phycocyanin aqueous solution, the concentration of the phycocyanin aqueous solution is 0.5% -8%, and the formula mass ratio of the surfactant to the oil phase to the water phase is 25-55:33:8-30.
2. The stable and safe phycocyanin nanoemulsion formulation according to claim 1, characterized in that the formulated surfactant is Tween80-Span80, wherein HLB = 4-7.
3. The stable and safe phycocyanin nanoemulsion formulation according to claim 1, characterized in that said cosurfactant is one of ethanol, propylene glycol and glycerol.
4. A method of preparing the stable and safe phycocyanin nanoemulsion formulation of claims 1-3, characterized in that the preparation method comprises the following steps:
(1) At room temperature, adding the cosurfactant and the oil phase into the surfactant, fully mixing the three phases, and uniformly stirring by using a magnetic stirrer to prepare a mixed system;
(2) Dropwise adding the water phase into the mixed system, wherein when dropwise adding is started, the system presents a clear oil phase with smaller viscosity due to less oil phase and water phase, the system becomes turbid as the water phase is continuously added, and the system gradually becomes clear from turbid after continuous dropwise adding, so that the W/O type nanoemulsion is formed;
(3) Gradually and slowly adding the phycocyanin aqueous solution into the W/O type nanoemulsion, uniformly stirring by using a magnetic stirrer, and gradually dropwise adding distilled water until clear and transparent nanoemulsion is formed.
5. The method of claim 4, wherein in step (1), the oil phase is one of liquid paraffin, soybean oil, olive oil, and isopropyl myristate (IPM).
6. The method of claim 4, wherein in step (2), the aqueous phycocyanin solution is prepared by adding pure water.
7. The method for preparing a stable and safe phycocyanin nanoemulsion formulation according to claim 4, wherein the particle size of the prepared phycocyanin nanoemulsion is 180nm-220nm.
8. The application of the phycocyanin nanoemulsion preparation prepared according to claims 1-7, characterized in that the phycocyanin nanoemulsion preparation is applied to health-care foods, medicines and cosmetics with oxidation resistance, inflammation resistance, cancer resistance, neuroprotection and immunoregulation.
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