Method for Preparing Nanoparticle Composites Loaded with Extracts of Grapefruit

Abstract

The present disclosure describes a method for preparing a grapefruit extract-loaded nanoparticle complex that can effectively improve solubility and flavor by encapsulating a grapefruit extract containing low-solubility naringenin and bitter-tasting naringin in a complex form with cyclodextrin (CD) and a cationic biopolymer.

Description

Technology Field

[0001] The present disclosure relates to a method for preparing a grapefruit extract-loaded nanoparticle complex. More specifically, the present disclosure relates to a method for preparing a grapefruit extract-loaded nanoparticle complex capable of effectively improving solubility and flavor by encapsulating a grapefruit extract containing low-solubility naringenin and bitter-tasting naringin in a complex form using cyclodextrin (CD) and a cationic biopolymer. Background Technology

[0002] Grapefruit is gaining popularity as a fruit suitable for dieting because it is composed of over 90% water and contains about 30 kcal per 100g. In particular, grapefruit is rich in soluble dietary fiber and potassium, and consuming it promotes diuresis, which helps eliminate swelling and prevent constipation.

[0003] In addition, grapefruit extract is known to have excellent antioxidant and anti-inflammatory activities due to its high content of vitamin C, phenolic compounds, etc. In this regard, naringenin and naringin, represented by the following chemical formulas 1 and 2, are known as active ingredients in grapefruit extract.

[0004] [Chemical Formula 1]

[0005]

[0006] [Chemical Formula 2]

[0007]

[0008] Among these, naringenin is a colorless, flavorless flavanone (a type of flavonoid) that performs functions such as antioxidant activity, free radical scavenging, anti-inflammation, promotion of carbohydrate metabolism, and regulation of the immune system. Furthermore, it has been reported to reduce oxidative damage to DNA in the body and contribute to lowering serum and liver cholesterol levels, and is utilized in nutritional supplements. Meanwhile, naringin is a flavonoid component responsible for the bitter taste of grapefruit; it is known to exist in nature either in a free state or in plants as a glycoside combined with sugars such as glucose. Additionally, naringin is known as a non-toxic natural antimicrobial agent exhibiting antibacterial properties against various microorganisms, and due to its fat-degrading ability, it is effective for body fat reduction and is therefore used as a dietary supplement.

[0009] Despite the aforementioned advantages, there are limitations to applying grapefruit extract to food due to the low water solubility of naringenin and its bitter taste. Specifically, in the case of naringenin, its low water solubility makes it difficult to absorb into the body through simple ingestion; it has been reported that even in the most effective cases, only 15% of the ingested naringenin is absorbed in the intestines (Food Industry and Nutrition 19(2), 35-38, 2014). Furthermore, attempts have been made to add sugars and sweeteners to mitigate the problem of reduced palatability of grapefruit extract caused by the bitter taste derived from naringin. However, while these techniques can improve the palatability of grapefruit extract to some extent, they cannot be considered techniques that reduce the bitter taste itself.

[0010] Conventionally, attempts have been made to encapsulate naringenin or naringin via liposomes to address either the low water solubility or the bitterness of grapefruit extract; however, no method has been known to effectively improve both water solubility and bitterness simultaneously. Consequently, grapefruit extract is primarily applied in the pharmaceutical field, making it difficult to utilize widely in the food industry, where water solubility and flavor are critical.

[0011] Therefore, there is a need to overcome the limitations of the aforementioned conventional technology and expand the potential for applying grapefruit extract to food. The problem to be solved

[0012] In one embodiment of the present disclosure, a method is provided to expand the food applicability of grapefruit extract by improving the flavor degradation caused by low water solubility and bitterness resulting from active ingredients such as naringenin and naringin in the grapefruit extract. means of solving the problem

[0013] According to one embodiment of the present disclosure,

[0014] a) a step of forming a grapefruit extract-containing cyclodextrin solution by combining a grapefruit extract containing naringenin and naringin with a cyclodextrin; and

[0015] b) a step of adding a cationic biopolymer and a phosphate-based compound to the grapefruit extract-containing cyclodextrin solution to induce ionic gelation between the cationic biopolymer and the phosphate-based compound, which form an inclusion molecule with the cyclodextrin, thereby encapsulating the grapefruit extract in a cyclodextrin and biopolymer-based matrix;

[0016] A method for preparing a grapefruit extract-loaded nanoparticle complex comprising is provided.

[0017] According to an exemplary embodiment, the cationic biopolymer may be an amino group-containing biopolymer.

[0018] According to an exemplary embodiment, the amino group-containing biopolymer may be at least one selected from the group consisting of chitosan, chitooligosaccharide, gelatin, and polylysine.

[0019] According to an exemplary embodiment, the average particle size of the grapefruit extract-loaded nanoparticle complex can be controlled in the range of 150 to 800 nm.

[0020] According to an exemplary embodiment, the polydispersity index of the grapefruit extract-loaded nanoparticle complex can be controlled to 0.4 or less.

[0021] According to an exemplary embodiment, step a) above is,

[0022] a1) a step of adding cyclodextrin to an aqueous medium to form an aqueous cyclodextrin solution; and

[0023] a2) a step of adding grapefruit extract to the above-mentioned aqueous cyclodextrin solution to form a grapefruit extract-containing cyclodextrin solution;

[0024] It may include.

[0025] According to an exemplary embodiment, the concentration of cyclodextrin in the aqueous cyclodextrin solution is controlled within the range of 0.5 to 1.5 mg / mL, and

[0026] The concentration of grapefruit extract in the above grapefruit extract-containing cyclodextrin solution can be adjusted in the range of 0.1 to 1 mg / mL.

[0027] According to an exemplary embodiment, the amounts of cationic biopolymer and phosphate compound added in step b) can be controlled within the ranges of 0.5 to 3 mg / mL and 0.1 to 1.2 mg / mL, respectively.

[0028] According to an exemplary embodiment, in step b), the weight ratio of the cationic biopolymer to the phosphate compound can be controlled in the range of 1:0.4 to 0.8.

[0029] According to an exemplary embodiment, the weight ratio of cyclodextrin to cationic biopolymer in the matrix based on the cyclodextrin and cationic biopolymer can be controlled in the range of 1:0.2 to 1.5.

[0030] According to an exemplary embodiment, the content of grapefruit extract in the grapefruit extract-loaded nanoparticle complex can be determined in the range of 0.1 to 10 weight%.

[0031] According to an exemplary embodiment, the cyclodextrin may be at least one selected from the group consisting of α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and γ-cyclodextrin (γ-CD).

[0032] According to an exemplary embodiment, the molecular weight of the cationic biopolymer may be in the range of 30 to 250 kDa.

[0033] According to an exemplary embodiment, the phosphate-based compound may be at least one selected from the group consisting of tripolyphosphate and polyphosphoric acid.

[0034] According to an exemplary embodiment, step b) may further include a step of performing ultrasonic treatment.

[0035] According to an exemplary embodiment, during the ultrasonic treatment, the ultrasonic output can be adjusted to 200 to 700 W, the frequency to a range of 5 to 50 KHz, and the amplitude to 10 to 50%.

[0036] According to an exemplary embodiment, the ultrasonic irradiation time during the ultrasonic treatment can be adjusted in the range of 0.1 to 60 minutes.

[0037] According to an exemplary embodiment, the water solubility of naringenin in the grapefruit extract loaded into the nanoparticle complex may be at least 200 μg / mL. Effects of the invention

[0038] A method for preparing a grapefruit extract-loaded nanoparticle composite according to a specific embodiment of the present disclosure involves loading a grapefruit extract containing naringenin, which exhibits low water solubility, and naringin, a bitter-tasting component, into a nanomatrix based on cyclodextrin and an amino group-containing biopolymer. By treating the grapefruit extract in the order of cyclodextrin and the amino group-containing biopolymer during the manufacturing process, the water solubility of the grapefruit extract can be improved while effectively suppressing the bitter taste. As a result, the method offers a way to utilize grapefruit extract, which has relatively low applicability in the food industry, in various foods, thus demonstrating high potential for commercialization. Brief explanation of the drawing

[0039] FIG. 1 is a diagram schematically illustrating a series of processes for preparing a grapefruit extract-loaded nanoparticle complex according to an exemplary embodiment; FIG. 2 shows the physical properties of nanoparticle complexes containing naringenin, naringin, and grapefruit extract, respectively, according to the manufacturing sequence and the presence or absence of additional processing, and is a graph showing (a) the results of measuring the particle size and polydispersity index (PDI) of a naringenin-loaded nanoparticle complex, (b) the results of measuring the particle size and polydispersity index (PDI) of a naringin-loaded nanoparticle complex, and (c) the results of measuring the particle size and polydispersity index (PDI) of a grapefruit extract-loaded nanoparticle complex, respectively; Figure 3 is a graph showing the results of confirming the relative solubility by comparing the solubility of naringenin-loaded nanoparticle complexes and naringenin dispersed in ethanol (NRG-EtOH) according to the manufacturing order and the presence or absence of additional process treatment; Figure 4 is a graph showing the results of evaluating relative release characteristics by comparing the release characteristics of a grapefruit extract-containing nanoparticle composite and grapefruit extract (GE-EtOH) dispersed in ethanol under oral pH conditions according to the manufacturing sequence and the presence or absence of additional process treatment; FIG. 5 shows the results of measuring fluorescence images by treating HEK-293 cells with naringin and grapefruit extracts (NG-EtOH, GE-EtOH) dispersed in ethanol, respectively, and with naringin-loaded nanoparticle complexes and grapefruit extract-loaded nanoparticle complexes according to the preparation order and the presence or absence of additional processing, wherein (a) a fluorescence image of HEK-293 cells treated with a naringin-loaded nanoparticle complex, and (b) a fluorescence image of HEK-293 cells treated with a grapefruit extract-loaded nanoparticle complex; and Figure 6 shows the results of the fluorescence intensity of the fluorescence images according to Figure 5, (a) the fluorescence intensity of HEK-293 cells treated with a naringin-loaded nanoparticle complex, and (b) the fluorescence intensity of HEK-293 cells treated with a grapefruit extract-loaded nanoparticle complex. Specific details for implementing the invention

[0040] The present invention can be fully achieved by the following description. The following description should be understood as describing preferred embodiments of the present invention, but the present invention is not necessarily limited thereto. Furthermore, the attached drawings are for illustrative purposes only and do not limit the present invention; details regarding individual components can be appropriately understood in accordance with the specific intent of the relevant descriptions provided below.

[0041] Terms used in this specification may be defined as follows.

[0042] The term "extract" can be understood to include not only extracts obtained directly using an extraction solvent, but also purified extracts obtained by performing additional separation and purification treatments (e.g., fractionation).

[0043] "Biopolymer" can refer to natural polymers, specifically polymers derived from renewable sources such as plants.

[0044] "Nanoscale" may mean having morphological features of, for example, about 1,000 nm or less, specifically about 500 nm or less.

[0045] "Cationic polymer" refers to a polymer having a net positive charge, and "anionic polymer" may refer to a polymer having a net negative charge.

[0046] "Encapsulation" or "entrapment" may refer to the formation of a functional barrier on an active substance within a nanoparticle complex, specifically grapefruit extract.

[0047] "Particle size" can refer to the average particle diameter.

[0048] "Solubility" can be defined as the maximum amount of solute that can be dissolved in a solvent.

[0049] "Flavonoids" can be understood as polyphenol compounds that fall within the range of flavanones.

[0050] Terms such as "on" or "on the upper side" and "on the lower side" or "below" can be understood as describing the relative positional relationship between components or members, and the terms "located on the upper side" or "located on the lower side" can be understood as expressing the relative positional relationship not only in a state of contact with a specific object but also in a state of non-contact.

[0051] Where a numeric range is specified in this specification as a lower limit and / or an upper limit, it may be understood that any sub-combination within said numeric range is also disclosed. For example, where "1 to 5" is written, it may include 1, 2, 3, 4 and 5, as well as any sub-combination between them.

[0052] Where in this specification it is stated that any component or member is "connected" to another component or member, unless otherwise stated, this may be understood to include not only cases where it is directly connected to said other component or member, but also cases where it is connected through the interposition of said other component or member.

[0053] Similarly, the term "contact" can also be understood to include not only cases of direct contact, but also cases of contact involving other components or members.

[0054] When a component is said to "include," it means that, unless otherwise noted, it may include additional components.

[0055] Method for preparing a grapefruit extract-loaded nanoparticle complex

[0056] A series of processes for preparing a grapefruit extract-loaded nanoparticle complex according to an exemplary embodiment of the present disclosure is as illustrated in FIG. 1.

[0057] Referring to the drawing above, a step of preparing a grapefruit extract-containing cyclodextrin aqueous solution may be performed, wherein the cyclodextrin is first introduced into an aqueous medium (specifically, water) to prepare the cyclodextrin aqueous solution, and then the grapefruit extract may be combined.

[0058] According to an exemplary embodiment, cyclodextrin (CD) is a non-toxic sugar polymer stable in an aqueous medium, specifically a cyclic oligosaccharide comprising a plurality of glucose subunits, and can be broadly classified into alpha-cyclodextrin (α-CD), beta-cyclodextrin (β-CD), and gamma-cyclodextrin (γ-CD), each having 6, 7, and 8 glucose subunits, respectively. Cyclodextrin typically exhibits high crystallinity and does not absorb moisture.

[0059] In particular, cyclodextrin has a toroidal shape with a hydrophilic outer surface and a small number of inner cavities, and the hydrophobic cavities have the property of partially or completely trapping small organic molecules.

[0060] The cyclodextrin applicable in the present embodiment may be at least one selected from the group consisting of α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and γ-cyclodextrin (γ-CD). According to a specific embodiment, β-cyclodextrin (β-CD) represented by the following chemical formula 1 may be used as the cyclodextrin, and β-CD has a cavity size suitable for capturing various active ingredients than α-CD and γ-CD, and thus has the characteristic of having good ability to capture active ingredients.

[0061] [Chemical Formula 1]

[0062]

[0064] For example, β-cyclodextrin (CD) can generally be enzymatically produced from a starch source, and the enzyme used can be, for example, cyclodextrin glycosyltransferase with alpha-amylose. In particular, the cavity diameter of the β-CD is about 6 to 6.4 Å, the outer diameter is about 15.4 Å, and the height is about 7.8 Å.

[0065] According to an exemplary embodiment, in this embodiment, the cyclodextrin can be used without separate pretreatment or modification treatment (specifically chemical modification treatment), and by using a cyclodextrin that has not undergone pretreatment or modification treatment, applicability to food can be improved.

[0066] According to exemplary embodiments, the concentration of cyclodextrin in the aqueous solution of cyclodextrin can be adjusted in consideration of the fact that if the concentration is excessively high or low, aggregation may occur or the amount of grapefruit extract captured may decrease to a level below the desired level, for example, in the range of about 0.5 to 1.5 mg / mL, specifically about 0.75 to 1.25 mg / mL, more specifically about 0.9 to 1.1 mg / mL, but this can be understood as being for exemplary purposes.

[0067] As described above, when an aqueous cyclodextrin solution is provided, a grapefruit extract-containing aqueous cyclodextrin solution can be prepared by combining or mixing it with grapefruit extract.

[0068] In the present embodiment, the grapefruit extract typically contains naringenin and naringin and can be obtained according to extraction methods known in the art. Alternatively, a commercially available type of grapefruit extract may be used. The process of preparing the grapefruit extract can be carried out by finely grinding at least one part selected from, for example, grapefruit, such as seeds, pulp, peel, etc., and immersing it in at least one extraction solvent selected from, for example, water, alcohol (e.g., ethanol, etc.), glycerol, etc.

[0069] At this time, the extraction time can be adjusted in the range of, for example, about 0.1 to 5 days, specifically about 1 to 4 days, more specifically about 1.5 to 3 days, and if necessary, it can be performed under stirring conditions. Afterwards, the extract can be concentrated using a concentration means such as distillation to obtain a grapefruit extract stock solution, and furthermore, the grapefruit extract stock solution can be dried to obtain a grapefruit extract in powder form.

[0070] In the present embodiment, the grapefruit extract is not limited to a specific form and may be applied in liquid form or powder form. Alternatively, the grapefruit extract may be used in the form of an aqueous solution to facilitate smooth contact with cyclodextrin.

[0071] According to an exemplary embodiment, considering that it is difficult to exhibit physiological activity when the concentration of grapefruit extract in a grapefruit extract-containing cyclodextrin solution is excessively low, while the bitterness improvement effect may be reduced due to non-uniform particle characteristics when it is excessively high, the concentration may be adjusted in the range of, for example, about 0.1 to 1 mg / mL, specifically about 0.15 to 0.8 mg / mL, and more specifically about 0.2 to 0.6 mg / mL, but this can be understood as being for exemplary purposes.

[0072] According to an exemplary embodiment, the conditions for preparing a grapefruit extract-containing cyclodextrin solution are not particularly limited, but the mixing temperature may typically be about 20 to 40°C, specifically room temperature.

[0073] Meanwhile, referring again to Fig. 1, a cationic biopolymer is added to a grapefruit extract-containing cyclodextrin solution. Accordingly, the cyclodextrin and the cationic biopolymer form an inclusion. At this time, since the cyclodextrin does not carry an electric charge, it is difficult to effectively combine with the cationic biopolymer to form a nanocomposite matrix. Taking this into consideration, nanoparticles can be formed by inducing an ionic gelation reaction between cationic groups (e.g., amino groups) contained in the biopolymer and phosphate ions (anions) derived from the phosphate-based compound described later.

[0074] According to an exemplary embodiment, the biopolymer may be added in powder form, but it may be advantageous to add it in aqueous solution form for uniform mixing, and more specifically, it may be added dropwise.

[0075] In this regard, it is noteworthy that when grapefruit extract is combined in the order of CD-biopolymer, the water solubility of the grapefruit extract and the bitterness suppression effect can be improved to a significant level compared to when it is combined in the order of biopolymer-CD. Although the present disclosure is not bound by any specific theory, water solubility and the bitterness suppression effect can be effectively improved by adding a cationic biopolymer and a phosphate compound after combining grapefruit extract with cyclodextrin. Although the present disclosure is not bound by any specific theory, it can be explained that while the exterior of the cyclodextrin is hydrophilic, the internal cavity is hydrophobic, so when the grapefruit extract binds to the hydrophobic part within the cyclodextrin cavity and host-guest interactions are induced, the grapefruit extract is effectively captured, thereby enhancing its solubility and also suppressing contact with the external environment, thereby enhancing the bitterness improvement effect. Furthermore, through ionic bonding between cationic biopolymers and phosphate compounds, the water solubility of grapefruit extract can be further enhanced and the protective effect against the external environment can be further improved, thereby achieving a better bitterness suppression effect.

[0076] According to exemplary embodiments, the cationic biopolymer may be an amino group-containing biopolymer, and it may be particularly advantageous to use a naturally derived biopolymer that has not undergone chemical treatment.

[0077] According to an exemplary embodiment, the molecular weight (M) of the cationic biopolymer wThe molecular weight may be, for example, in the range of about 30 to 250 kDa, specifically about 50 to 190 kDa, and more specifically about 80 to 150 kDa. In this regard, the molecular weight of the cationic biopolymer is a factor that affects the solubility, stability, viscosity, etc. of the nanoparticle composite; if it is excessively large, it may cause a phenomenon where viscosity increases excessively or solubility decreases, whereas if it is excessively small, dispersion stability may decrease. Considering the above points, it may be advantageous to appropriately control it within the aforementioned molecular weight range.

[0078] According to exemplary embodiments, among cationic biopolymers, the amino group-containing biopolymer may be at least one selected from chitosan, chitooligosaccharide, gelatin, polylysine, etc., and more specifically, may be chitosan. The reason chitosan is advantageous as a cationic biopolymer is that the degree of protonation of the amine group is high.

[0079] In this regard, chitosan can be represented by the following chemical formula 2.

[0080] [Chemical Formula 2]

[0081]

[0082] In the above formula, n is the degree of polymerization, which is determined according to the molecular weight, and may be, for example, in the range of about 160 to 1,400, specifically about 270 to 1,060, more specifically about 440 to 840.

[0083] Chitosan is a polysaccharide composed of copolymers in which N-acetyl-d-glucosamine and D-glucosamine units are linked by β-(1,4)-glycosidic bonds. Due to the presence of amine groups, it is positively charged under neutral or acidic conditions and can form intermolecular complexes with other anionic polymers or polyanions.

[0084] In this regard, the form of chitosan is not particularly limited, but the chitosan chains within the molecular structure may include both crystalline and amorphous characteristics, and may also include not only chitosan but also derivatives thereof. Meanwhile, the viscosity of chitosan is related to the molecular weight, and as an example, it may be in the range of about 10 to 300 cps, specifically about 20 to 250 cps, more specifically about 20 to 200 cps.

[0085] According to an exemplary embodiment, chitosan is a natural polymer derived from chitin (poly-N-acetyl-D-glucosamine), and a major portion of the N-acetyl group can be removed by hydrolysis. In this case, the degree of deacetylation may be, for example, at least about 70%, specifically at least about 75%, more specifically in the range of about 75 to 95%.

[0086] According to another specific embodiment, chitosan can be used as a biopolymer, and typically, chitosan can be converted into chitosan by the action of a degrading enzyme (specifically, a hydrolytic enzyme). At this time, the degrading enzyme may be used in powder form, or alternatively, in the form of an aqueous solution or dispersion dissolved in water. Additionally, depending on the type of degrading enzyme, a small amount of acid or base may be added to promote the hydrolysis reaction by the action of the enzyme present inside the gel. As an example, chitosanase, chitinase, lysozyme, β-glucosidase, papain, etc., derived from fungi such as Aspargillus, Muco, and Penicillium genera, and bacteria such as Streptomyces and Bacillus genera, may be used as the degrading enzyme for chitosan, and the types listed above may be used individually or in combination. According to a specific embodiment, a chitosanase derived from the genus Bacillus may be used. In this regard, since the exo-type chitosanase may increase the production of monomers such as monosaccharides and glucosamine due to enzyme characteristics, it may be advantageous to use an endo-type chitosanase. According to an exemplary embodiment, the chitosan-degrading enzyme may be of a type having an enzyme activity in the range of, for example, about 10,000 to 50,000 unit / g, specifically about 20,000 to 45,000 unit / g, and more specifically about 25,000 to 40,000 unit / g. In addition, the optimal activation pH of the enzyme may be in the range of, for example, about 4 to 7, specifically about 4.5 to 6, and more specifically about 5 to 5.5. The characteristics of the enzyme described above should be understood as exemplary. As described above, chitooligosaccharides produced by enzymatic action can be obtained in powder form and used through freeze-drying, hot-air drying, spray drying, etc.

[0087] In the illustrated embodiment, the amount of cationic biopolymer (e.g., amino group-containing biopolymer) added to the grapefruit extract-containing cyclodextrin aqueous solution can be adjusted considering that aggregation may occur if the amount is excessively large, while the amount of grapefruit extract captured decreases if the amount is excessively small. For example, it may be in the range of about 0.5 to 3 mg / mL, specifically about 0.7 to 2.5 mg / mL, more specifically about 1 to 2 mg / mL, but this can be changed depending on the type of biopolymer, etc.

[0088] Referring again to Fig. 1, a phosphate-based compound is added as a cross-linker together with or after the addition of the biopolymer.

[0089] These phosphate compounds can be selected from those that exhibit anionic properties in an aqueous medium, and may be at least one selected from, for example, tripolyphosphate polyphosphate. According to a specific embodiment, it may be tripolyphosphate, specifically sodium tripolyphosphate.

[0090] According to the illustrated embodiments, the amount of a phosphate compound added to the grapefruit extract-containing cyclodextrin aqueous solution can be determined by taking into account particle characteristics, for example, in the range of about 0.1 to 1.2 mg / mL, specifically about 0.2 to 1 mg / mL, and more specifically about 0.3 to 0.8 mg / mL, but this should be understood as being for illustrative purposes. Additionally, according to certain embodiments, the phosphate compound may be added in powder form or in aqueous form, specifically in aqueous form, and more specifically in a dropwise manner.

[0091] In this way, a biopolymer and a phosphate-based compound are added to an aqueous solution of grapefruit extract-containing cyclodextrin and uniformly mixed to induce an ionic gelation reaction (specifically, an ionic gelation reaction between a cationic biopolymer and a phosphate compound). During this process, the cyclodextrin and the biopolymer form a nanocomposite as a matrix, which can be used to capture the grapefruit extract.

[0092] According to exemplary embodiments, the content ratio between the cationic biopolymer and the phosphate compound can be adjusted by considering the degree of ionic gelation, particle characteristics, etc., for example, in the range of 1: about 0.2 to 0.8 by weight, specifically 1: about 0.25 to 0.6, and more specifically 1: about 3 to 0.5. In this regard, if the ratio between the cationic biopolymer and the phosphate compound is excessively large or small, the smooth ionic gelation reaction may be hindered, leading to aggregation or almost no ionic gelation reaction; therefore, it may be advantageous to adjust the ratio appropriately within the aforementioned range in consideration of this. However, this should be understood as being for illustrative purposes only.

[0093] The aforementioned ionic gelation reaction conditions are not particularly limited, but the reaction temperature can be controlled, for example, in the range of about 20 to 40 °C, specifically about 22 to 35 °C, and the reaction time can be controlled, for example, in the range of about 1 to 20 minutes, specifically about 5 to 15 minutes. However, the above-described reaction conditions should be understood as exemplary.

[0094] Meanwhile, according to another exemplary embodiment, ultrasonic treatment may be optionally performed at or after the addition of biopolymers and phosphate-based compounds. As ultrasonic treatment is performed as in the present embodiment, the biopolymers and phosphate-based compounds capable of ionic bonding increase, and as a result, the capture ability for grapefruit extract is improved, thereby further enhancing water solubility and bitterness suppression effects.

[0095] In this regard, the ultrasonic irradiation may be of a type known in the art, for example, a vessel type (bath type) or a probe type (sonotrode type). In this regard, in the case of the vessel type, for example, an ultrasonic irradiation device may be fixedly positioned on one side of the vessel, for example, the lower side, and the ultrasonic waves irradiated therefrom may be directly transmitted to the liquid medium inside the vessel. On the other hand, in the case of the probe type, a relatively high output can be generated as the emitter surface comes into direct contact with the liquid medium.

[0096] For example, the following processing conditions can be set during ultrasound irradiation.

[0097] According to an exemplary embodiment, the ultrasonic output can be adjusted in the range of, for example, about 200 to 700 W, specifically about 300 to 600 W, more specifically about 400 to 500 W. In addition, the ultrasonic frequency can be set in the range of, for example, about 5 to 50 KHz, specifically about 10 to 40 KHz, more specifically about 15 to 25 KHz. Furthermore, the amplitude of the ultrasonic can be adjusted in the range of, for example, about 10 to 50%, specifically about 15 to 35%, more specifically about 20 to 25%.

[0098] According to exemplary embodiments, if the ultrasonic irradiation (treatment) time is excessively short, there are limitations in effectively reducing particle size, whereas if it is excessively long, the particle size may increase as nanoparticles are damaged and aggregated; therefore, the ultrasonic irradiation time can be adjusted in consideration of this. As an example, the ultrasonic irradiation time may be in the range of, for example, about 0.1 to 60 minutes, specifically about 0.5 to 20 minutes, more specifically about 1 to 2 minutes, but this can be understood as being for exemplary purposes.

[0099] As described above, after the process of encapsulating grapefruit extract in a cyclodextrin and biopolymer-based matrix through an ionic gelation reaction is performed, conventional post-processing processes (e.g., washing, solid-liquid separation (specifically centrifugation, filtration, etc.), drying, etc.) may be performed. Since such post-processing processes are known in the art, details are omitted.

[0100] Characteristics of grapefruit extract-loaded nanoparticle complex

[0101] As described above, the composite prepared according to the present embodiment has a nanoscale particle form in which grapefruit extract is encapsulated or coated in a matrix based on cyclodextrin and amino group-containing biopolymer.

[0102] According to an exemplary embodiment, the average particle size of the nanoparticle complex can be controlled in the range of, for example, about 150 to 800 nm, specifically about 250 to 680 nm, more specifically about 350 to 480 nm.

[0103] Particle size is one of the factors affecting the solubility and bitterness improvement of grapefruit extract, and it may be advantageous to appropriately control it within the aforementioned range. In addition, the nanoparticle composite prepared according to the present embodiment may exhibit a uniform size distribution, and the polydispersity index (PDI), which is an indicator thereof, may be, for example, about 0.4 or less, specifically about 0.3 or less, more specifically about 0.25 or less.

[0104] Meanwhile, the relative amounts of cyclodextrin and cationic biopolymer within the matrix of the nanoparticle complex can be determined by considering particle characteristics, as a tendency for aggregation to occur or a decrease in the amount of grapefruit extract captured may occur if the relative amount of cyclodextrin is excessively large or small. Taking this into consideration, according to an exemplary embodiment, the weight ratio of cyclodextrin to cationic biopolymer within the nanoparticle complex can be controlled in the range of, for example, 1:0.2 to 1.5, specifically about 0.3 to 1.4, and more specifically about 0.4 to 1.2, but this should be understood as being for illustrative purposes.

[0105] In addition, according to exemplary embodiments, the content of grapefruit extract in the grapefruit extract-loaded nanoparticle complex can be determined by considering that if it is too low, it is difficult to exhibit physiological activity, and if it is too high, the particle characteristics may not be uniform and the bitterness improvement effect may be reduced. For example, it can be determined in the range of about 0.1 to 10 weight%, specifically about 2 to 8 weight%, and more specifically about 4 to 6 weight%.

[0106] One of the main advantages of the nanoparticle complex prepared according to the present embodiment is that by encapsulating a grapefruit extract containing naringenin, which exhibits low water solubility, and naringin, a bitter-tasting component, as the main active ingredients into a nanoparticle complex, it is possible to simultaneously increase water solubility and improve bitterness (e.g., reduced release in an oral pH environment / reduced bitterness level).

[0107] As an example, when not captured by a nanoparticle complex, the water solubility of naringenin in grapefruit extract is at a level of about 40 to 50 μg / mL. In this regard, the ethanol solubility, which is relatively good for naringenin solubility, is at a level of about 180 to 200 μg / mL. On the other hand, the water solubility of naringenin captured by the nanoparticle complex prepared according to the present embodiment may be at least about 200 μg / mL, specifically about 250 to 270 μg / mL. It should be noted that this water solubility is at least about 5 times higher than when not captured.

[0108] In addition, regarding naringin, a bitter-tasting component in grapefruit extract, the relative release amount under oral pH conditions (approximately pH 6 to 7) can be, for example, about 25% or less, specifically about 20% or less, by being captured by the nanoparticle complex prepared according to the present embodiment compared to the uncaptured state, which indicates that the bitter taste is effectively suppressed by capture.

[0109] The present invention may be more clearly understood by the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the invention.

[0110] The materials used in this embodiment are as follows.

[0111] - Naringenin (NRG) was purchased from Sigma-Aldrich Co. (St Louis, MO, USA).

[0112] - Naringin (NG) was purchased from Sigma-Aldrich Co. (St Louis, MO, USA).

[0113] - Grapefruit extract (GE) was purchased from Huachengbio Inc. (Huacheng, China).

[0114] - β-cyclodextrin was purchased from Sigma-Aldrich Co. (St Louis, MO, USA).

[0115] - Chitosan was purchased from Sigma-Aldrich Co. (St Louis, MO, USA) (molecular weight: 50-190 kDa).

[0116] - Tripolyphosphate was purchased from Sigma-Aldrich Co. (St Louis, MO, USA).

[0117] - Ethanol and acetic acid were each purchased from Daejeonghwageum.

[0118] - HEK-293 cells were obtained from the Korean Cell Line Bank.

[0119] - Analytical grade was used for all chemical substances, and HPLC grade was used for all solvents.

[0120] Example 1

[0121] Preparation of chitosan nanoparticle complexes (NRG-CS, NG-CS, GE-CS) containing naringenin (NRG), naringin (NG), and grapefruit extract (GE), respectively

[0122] NRG-CS, NG-CS, and GE-CS (final concentration: 0.3 mg / mL) containing naringenin, naringin, and grapefruit extract, respectively, were prepared using the ionic gelation properties between chitosan (final concentration: 1.5 mg / mL) and tripolyphosphate (final concentration: 0.4 mg / mL).

[0123] Specifically, naringenin, naringin, and grapefruit extract were each dissolved in ethanol, the chitosan solution in a 1% (v / v) acetic acid solution, and the tripolyphosphate solution in distilled water. After mixing the chitosan solution with the solutions of naringenin, naringin, and grapefruit extract, the tripolyphosphate solution was added dropwise at a rate of 1.0 mL / min to prepare NRG-CS, NG-CS, and GE-CS, respectively.

[0124] Example 2

[0125] Preparation of chitosan nanoparticles and β-cyclodextrin complexes (NRG-CS-CD, NG-CS-CD, GE-CS-CD) containing naringenin (NRG), naringin (NG), and grapefruit extract (GE), respectively

[0126] NRG-CS-CD, NG-CS-CD, and GE-CS-CD (final concentration: 0.3 mg / mL) containing naringenin, naringin, and grapefruit extract, respectively, were prepared using the ionic gelation properties between chitosan (final concentration: 1.5 mg / mL) and tripolyphosphate (final concentration: 0.4 mg / mL) and the complex-forming ability of β-cyclodextrin (1.0 mg / mL).

[0127] Specifically, after mixing the chitosan solution with the solutions of naringenin, naringin, and grapefruit extract, a tripolyphosphate solution was added dropwise at a rate of 1.0 mL / min, and subsequently, a β-cyclodextrin solution was added dropwise at a rate of 1.0 mL / min to prepare NRG-CS-CD, NG-CS-CD, and GE-CS-CD.

[0128] Example 3

[0129] Preparation of chitosan and β-cyclodextrin nanoparticle complexes (NRG-CD-CS, NG-CD-CS, GE-CD-CS) containing naringenin (NRG), naringin (NG), and grapefruit extract (GE), respectively

[0130] NRG-CD-CS, NG-CD-CS, and GE-CD-CS (final concentration: 0.3 mg / mL) containing naringenin, naringin, and grapefruit extract, respectively, were prepared using the ionic gelation properties between chitosan (final concentration: 1.5 mg / mL) and tripolyphosphate (final concentration: 0.4 mg / mL) and the complex-forming ability of β-cyclodextrin (1.0 mg / mL).

[0131] Specifically, after mixing the β-cyclodextrin solution with the solutions of naringenin, naringin, and grapefruit extract, respectively, a chitosan solution was added dropwise at a rate of 1.0 mL / min, and subsequently, a tripolyphosphate solution was added dropwise at a rate of 1.0 mL / min to prepare NRG-CD-CS, NG-CD-CS, and GE-CD-CS.

[0132] Example 4

[0133] Preparation of chitosan nanoparticles and β-cyclodextrin complexes (ultrasonically treated) (NRG-CS-CD-S, NG-CS-CD-S, GE-CS-CD-S) containing naringenin (NRG), naringin (NG), and grapefruit extract (GE), respectively

[0134] NRG-CS-CD-S, NG-CS-CD-S, and GE-CS-CD-S (final concentration: 0.3 mg / mL) containing naringenin, naringin, and grapefruit extract, respectively, were prepared using the ionic gelation properties between chitosan (final concentration: 1.5 mg / mL) and tripolyphosphate (final concentration: 0.4 mg / mL) and the complex-forming ability of β-cyclodextrin (1.0 mg / mL).

[0135] Specifically, after mixing the chitosan solution with the solutions of naringenin, naringin, and grapefruit extract, respectively, a tripolyphosphate solution was added dropwise at a rate of 1.0 mL / min, and subsequently, a β-cyclodextrin solution was added dropwise at a rate of 1.0 mL / min to prepare NRG-CS-CD, NG-CS-CD, and GE-CS-CD. Then, NRG-CS-CD-S, NG-CS-CD-S, and GE-CS-CD-S were prepared by performing sonication (90 sec, frequency: 20 KHz, amplitude: 20%) using overhead sonication (CV18, Sonics & Materials Inc., Newtown, CT, USA).

[0136] Example 5

[0137] Preparation of chitosan and β-cyclodextrin nanoparticle complexes (ultrasonically treated) (NRG-CD-CS-S, NG-CD-CS-S, GE-CD-CS-S) containing naringenin (NRG), naringin (NG), and grapefruit extract (GE), respectively

[0138] NRG-CD-CS-S, NG-CD-CS-S, and GE-CD-CS-S (final concentration: 0.3 mg / mL) containing naringenin, naringin, and grapefruit extract, respectively, were prepared using the ionic gelation properties between chitosan (final concentration: 1.5 mg / mL) and tripolyphosphate (final concentration: 0.4 mg / mL) and the complex-forming ability of β-cyclodextrin (1.0 mg / mL).

[0139] Specifically, after mixing the β-cyclodextrin solution with the respective solutions of naringenin, naringin, and grapefruit extract, a chitosan solution was added dropwise at a rate of 1.0 mL / min, and subsequently, a tripolyphosphate solution was added dropwise at a rate of 1.0 mL / min to prepare NRG-CD-CS, NG-CD-CS, and GE-CD-CS. Then, NRG-CD-CS-S, NG-CD-CS-S, and GE-CD-CS-S were prepared by performing sonication (90 sec, frequency: 20 KHz, amplitude: 20%) using overhead sonication (CV18, Sonics & Materials Inc., Newtown, CT, USA).

[0140] Experimental Example

[0141] Experimental Example 1

[0142] Particle Characterization

[0143] Particle size and polydispersity index (PDI) were measured using a nanoparticle size analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK).

[0144] Experimental Example 2

[0145] Solubility measurement

[0146] In this experimental example, the solubility of naringenin was evaluated relatively by comparing the NRG-CS, NRG-CS-CD, NRG-CD-CS, NRG-CS-CD-S, and NRG-CD-CS-S prepared in Examples 1 to 5, respectively, with naringenin dissolved in ethanol (NRG-EtOH).

[0147] Each dispersion was filtered through a 0.45 μm syringe filter (Advantec, Japan) and then quantitatively analyzed using HPLC (Waters 486 Tunable Absorbance Detector, Waters Corporation, Milford, MA, USA). A reversed-phase C18 (4.6 × 150 mm, 5 μm) was used with the Waters HPLC, and the analysis was performed with an injection volume of 20 μL, a flow rate of 1.0 mL / min, and an ultraviolet absorption wavelength of 260 nm. As the mobile phase, (A) distilled water, 0.1% phosphate acid, and (B) acetonitrile were used, and a gradient method was used for analysis with A: 80%, B: 20% at 0 min; A: 30%, B: 70% at 10 min; A: 0%, B: 100% at 10.1 min; A: 0%, B: 100% at 15 min; A: 80%, B: 20% at 17 min; and A: 80%, B: 20% at 25 min.

[0148] Experimental Example 3

[0149] In vitro (of grapefruit extract) in vitro ) Evaluation of emission characteristics

[0150] In this experimental example, the release characteristics of grapefruit extract under oral pH conditions (pH 6.8) were relatively evaluated by comparing GE-CS, GE-CS-CD, GE-CD-CS, GE-CS-CD-S, and GE-CD-CS-S prepared in Examples 1 to 5, respectively, with grapefruit extract dissolved in ethanol (GE-EtOH).

[0151] Each dispersion was filtered through a 0.45 μm syringe filter (Advantec, Japan), and the naringin in the released grapefruit extract was quantitatively analyzed using HPLC (Waters 486 Tunable Absorbance Detector, Waters Corporation, Milford, MA, USA). A reversed-phase C18 (4.6 × 150 mm, 5 μm) was used with the Waters HPLC, and the analysis was performed with an injection volume of 20 μL, a flow rate of 1.0 mL / min, and a UV absorption wavelength of 260 nm. As the mobile phase, (A) distilled water, 0.1% phosphate acid, and (B) acetonitrile were used, and a gradient method was used for analysis with A: 80%, B: 20% at 0 min; A: 30%, B: 70% at 10 min; A: 0%, B: 100% at 10.1 min; A: 0%, B: 100% at 15 min; A: 80%, B: 20% at 17 min; and A: 80%, B: 20% at 25 min.

[0152] Experimental Example 4

[0153] Bitterness evaluation of naringin and grapefruit extract

[0154] In this experimental example, the NG-CS, GE-CS, NG-CS-CD, and GE-CS-CD prepared in Examples 1 to 5, respectively

[0155] The bitterness of naringin and grapefruit extract was evaluated by treating fluorescence-treated HEK-293 cells with NG-CD-CS, GE-CD-CS, NG-CS-CD-S, GE-CS-CD-S, NG-CD-CS-S, and GE-CD-CS-S, as well as naringin (NG-EtOH) and grapefruit extract (GE-EtOH) dissolved in ethanol, respectively, and then measuring the fluorescence intensity. Cultured HEK-293 cells were treated with fluo-3 / AM before the samples were administered. Subsequently, measurements were taken at 523 nm (λ) using a Deltavision Elite (Applied Precision, Issaquah, WA, USA). exe Measurements were taken at = 475nm. The results were analyzed using the program Soft worx 5.5.

[0156] In the cellular signaling pathway, G-proteins stimulated by external bitter substances stimulate phospholipase to break down phospho-inositol 2-phosphoric acid into diacyl glycerol and inositol triphosphate. At this time, Ca in the smooth endoplasmic reticulum by the broken-down inositol triphosphate 2+ It releases and Ca into the cytoplasm. 2+ ...is introduced. Using the aforementioned mechanism, intracytoplasmic Ca is introduced via fluo-3 / AM 2+ The degree of bitterness can be compared by staining and comparing the fluorescence intensity. In this case, a higher fluorescence intensity indicates a stronger bitter taste, whereas a lower fluorescence intensity indicates a weaker bitter taste.

[0157] - The evaluation of particle characteristics, solubility, and release characteristics was performed at least three times, and the experimental results were analyzed using SPSS 26.0 with one-way ANOVA followed by Duncan's analysis p Significance was determined at the < 0.05 level.

[0158] - The physical properties (particle size and polydispersity index of the nanoparticle complex) of nanoparticle complexes containing naringenin, naringin, and grapefruit extract, respectively, according to the manufacturing order and whether additional processing was performed, are shown in Figures 2a to 2c.

[0159] Referring to the above figure, when nanoparticle complexes were prepared according to the manufacturing order (CS, CS-CD, and CD-CS) and whether ultrasonic treatment was performed (CS-CD-S and CD-CS-S) after fixing the concentrations of naringenin, naringin, and grapefruit extract, respectively, and the concentrations of chitosan, tripolyphosphate, and β-cyclodextrin, it was confirmed that the particle size of the nanoparticle complexes (CS-CD and CD-CS) increased compared to the chitosan nanoparticles.

[0160] In addition, it was confirmed that the size of CS-CD combined with β-cyclodextrin after preparing chitosan nanoparticles increased compared to the particle size of CD-CS combined with tripolyphosphate after forming a chitosan-β-cyclodextrin complex.

[0161] Furthermore, it was confirmed that the additional ultrasonic treatment process significantly reduced the particle size and PDI of the nanoparticle composite, enabling the formation of a nanoparticle composite with small and uniform particle size.

[0162] Figure 3 shows the results of confirming the relative solubility by comparing the solubility of naringenin-loaded nanoparticle complexes and naringenin dispersed in ethanol (NRG-EtOH) according to the manufacturing order and whether additional processing was performed.

[0163] Referring to the figure above, the relative solubility was found to be significantly highest in NRG-CS-CD-S and NRG-CD-CS-S, which have the smallest particle sizes ( p < 0.05). Therefore, it was confirmed that the solubility of naringenin increased further as a result of performing additional processing (ultrasonic treatment) on the nanoparticle complex.

[0164] Figure 4 shows the results of evaluating the relative release characteristics by comparing the release characteristics of grapefruit extract-containing nanoparticle complexes (GE-CS, GE-CS-CD, GE-CD-CS, GE-CS-CD-S and GE-CD-CS-S) and grapefruit extract dispersed in ethanol (GE-EtOH) under oral pH conditions according to the manufacturing order and the presence or absence of additional process treatment.

[0165] According to the figure above, the relative release amount of naringin in grapefruit extract captured in GE-CS, GE-CS-CD, GE-CD-CS, GE-CS-CD-S, and GE-CD-CS-S, respectively, was significantly reduced compared to the release amount of GE-EtOH (100%). In particular, it was confirmed that GE-CD-CS-S was the most effective in inhibiting the release of naringin under oral pH conditions. This is attributed to the fact that GE-CD-CS, which is prepared by capturing grapefruit extract in a host-guest form through hydrophobic binding with β-cyclodextrin and then ionically binding with chitosan and tripolyphosphate, effectively protects the grapefruit extract compared to other nanoparticle complexes. Furthermore, in the case of GE-CD-CS-S, it is believed that the protective effect on the grapefruit extract was significantly increased as the amount of chitosan and tripolyphosphate capable of ionically binding increased through ultrasonic treatment.

[0166] Therefore, it can be seen that the CD-CS-S nanoparticle complex is most effective in reducing bitterness by inhibiting the release of grapefruit extract under oral pH conditions.

[0167] Figure 5 shows the results of measuring fluorescence images after treating HEK-293 cells with naringin and grapefruit extracts (NG-EtOH and GE-EtOH) dispersed in ethanol, respectively, and naringin-loaded nanoparticle complexes and grapefruit extract-loaded nanoparticle complexes (NG / GE-EtOH, NG / GE-CS, NG / GE-CS-CD, NG / GE-CD-CS, NG / GE-CS-CD-S and NG / GE-CD-CS-S) according to the preparation order and the presence or absence of additional processing.

[0168] Referring to the figure above, it was confirmed that the fluorescence intensity decreased as naringin was captured in the nanoparticle complex, and that the fluorescence intensity of the nanoparticle complex decreased even further, particularly when accompanied by ultrasonic treatment.

[0169] In addition, considering that NG-CD-CS-S, which was ultrasonically treated with NG-CD-CS, showed a more pronounced decrease in fluorescence intensity compared to NG-CS-CD-S, which was ultrasonically treated with NG-CS-CD, it was confirmed that NG-CD-CS-S was most effective in reducing the bitterness of grapefruit extract compared to NG-CS-CD-S.

[0170] - In Figure 6, the previously analyzed fluorescence image is quantified and displayed as fluorescence intensity.

[0171] Referring to the figure above, in the case of naringin, there was no significant difference in fluorescence intensity between NG-EtOH and NG-CS. However, in the case of NG-CS-CD, NG-CD-CS, NG-CS-CD-S, and NG-CD-CS-S, the fluorescence intensity values ​​decreased by approximately 11.18%, 28.39%, 29.53%, and 36.48%, respectively, compared to NG-EtOH. Thus, regarding the manufacturing order, NG-CD-CS was effective in reducing fluorescence intensity values ​​compared to NG-CS-CD, and the fluorescence intensity values ​​were further reduced due to the additional process treatment of ultrasonic treatment. This confirmed that the manufacturing order and the presence or absence of additional process treatment are important factors influencing the reduction of bitterness in naringin.

[0172] Meanwhile, in the case of grapefruit extract, compared to GE-EtOH, the fluorescence intensity values ​​increased in GE-CS, GE-CS-CD, and GE-CS-CD-S, whereas they decreased in GE-CD-CS and GE-CD-CS-S. In particular, the fluorescence intensity of GE-CD-CS-S decreased by 49.04% compared to GE-EtOH, confirming that the manufacturing order and the presence or absence of additional processing are important factors affecting the reduction of bitterness in grapefruit extract.

[0173] From the above results, it is determined that NG / GE-CD-CS, prepared by capturing naringin and grapefruit extracts in a host-guest form through hydrophobic bonding with β-cyclodextrin and then forming an ionic bond between chitosan and tripolyphosphate, provides an effective bitterness reduction effect by minimizing the surface area where naringin and grapefruit extracts react with cells compared to other nanoparticle complexes. Furthermore, in the case of NG / GE-CD-CS-S, it is determined that the ionic bonding capabilities of chitosan and tripolyphosphate are further increased through ultrasonic treatment, thereby effectively reducing the bitterness of naringin and grapefruit extracts.

[0174] Therefore, it was confirmed that to effectively reduce the bitterness of naringin and grapefruit extracts, it is suitable to use a CD-CS-S nanoparticle complex in which naringin and grapefruit extracts are each first mixed with cyclodextrin to encapsulate them in a host-guest form, followed by the addition of chitosan and tripolyphosphate to introduce ionic bonding, and additionally subjected to ultrasonic treatment.

[0175] Simple variations or modifications of the present invention can be easily utilized by those skilled in the art, and all such variations or modifications are considered to be included within the scope of the present invention.

Claims

Claim 1 a) a step of forming a grapefruit extract-containing cyclodextrin solution by combining a grapefruit extract containing naringenin and naringin with a cyclodextrin, thereby capturing the grapefruit extract within the cyclodextrin through host-guest interactions via hydrophobic bonding; and b) a step of adding a cationic biopolymer and a phosphate-based compound to the grapefruit extract-containing cyclodextrin solution to induce ionic gelation between the cationic biopolymer and the phosphate-based compound, which form an inclusion molecule with the cyclodextrin, thereby encapsulating the grapefruit extract in a matrix based on the cyclodextrin and the biopolymer; wherein the concentration of the grapefruit extract in the grapefruit extract-containing cyclodextrin solution is controlled in the range of 0.1 to 1 mg / mL, the weight ratio of the cationic biopolymer to the phosphate-based compound in step b) is controlled in the range of 1:0.4 to 0.8, the water solubility of naringenin in the grapefruit extract loaded in the nanoparticle complex is at least 200 μg / mL, and compared to the case where it is not loaded in the nanoparticle complex, the naringenin in the grapefruit extract loaded in the nanoparticle complex A method for preparing a grapefruit extract-loaded nanoparticle complex having a relative release amount in the oral cavity of 25% or less. Claim 2 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that the cationic biopolymer is an amino group-containing biopolymer. Claim 3 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 2, wherein the amino group-containing biopolymer is at least one selected from the group consisting of chitosan, chito-oligosaccharide, gelatin, and polylysine. Claim 4 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that the average particle size of the grapefruit extract-loaded nanoparticle complex is controlled within the range of 150 to 800 nm. Claim 5 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, wherein step a) comprises: a1) a step of adding cyclodextrin to an aqueous medium to form a cyclodextrin aqueous solution; and a2) a step of adding grapefruit extract to the cyclodextrin aqueous solution to form a grapefruit extract-containing cyclodextrin solution. Claim 6 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 5, characterized in that the concentration of cyclodextrin in the aqueous cyclodextrin solution is controlled within the range of 0.5 to 1.5 mg / mL. Claim 7 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that, in step b), the amounts of cationic biopolymer and phosphate-based compound added are controlled within the ranges of 0.5 to 3 mg / mL and 0.1 to 1.2 mg / mL, respectively. Claim 8 delete Claim 9 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that the weight ratio of cyclodextrin to cationic biopolymer in the matrix based on the cyclodextrin and cationic biopolymer is controlled in the range of 1:0.2 to 1.

5. Claim 10 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that the content of grapefruit extract in the grapefruit extract-loaded nanoparticle complex is determined in the range of 0.1 to 10 weight%. Claim 11 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, wherein the cyclodextrin is at least one selected from the group consisting of α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and γ-cyclodextrin (γ-CD). Claim 12 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that the molecular weight of the cationic biopolymer is in the range of 30 to 250 kDa. Claim 13 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that the phosphate-based compound is at least one selected from the group consisting of tripolyphosphate and polyphosphate. Claim 14 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, wherein step b) further comprises the step of performing ultrasonic treatment. Claim 15 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 14, characterized in that, during the ultrasonic treatment, the ultrasonic output is controlled to be 200 to 700 W, the frequency to be in the range of 5 to 50 KHz, and the amplitude to be controlled to be 10 to 50%. Claim 16 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 15, characterized in that the ultrasonic irradiation time during the ultrasonic treatment is controlled within the range of 0.1 to 60 minutes. Claim 17 delete Claim 18 A method for preparing a grapefruit extract-loaded nanoparticle complex according to claim 1, characterized in that, compared to the case where the grapefruit extract is not loaded into the nanoparticle complex, the water solubility of naringenin in the grapefruit extract loaded into the nanoparticle complex is at least 5 times.

Images