KR20220086467A - Nanocomposites for enhancing stability and bioavailability of curcumin using polysaccharide and preparation method thereof - Google Patents

Abstract

The present invention relates to a nanocomposite using the polysaccharide carrageenan to improve the stability and bioavailability of curcumin. A high encapsulation rate of 90% or more was confirmed, and as it was confirmed that the stability and cell membrane permeability of curcumin encapsulated in the nanocomposite increased, the nanocomposite using carrageenan could improve the water solubility of curcumin and improve the bioavailability. , it can be provided as a pharmaceutical, cosmetic, and health food containing curcumin as an active ingredient.

Description

Nanocomposites for enhancing stability and bioavailability of curcumin using polysaccharide and preparation method thereof

The present invention relates to a nanocomposite with improved stability and bioavailability of curcumin using carrageenan, a polysaccharide, and a method for preparing the same.

Curcumin, a compound present in the spice turmeric, has been shown to have pharmacological effects such as antioxidant, anti-inflammatory, anti-proliferative and anti-angiogenic activities in many studies. As such, curcumin represents a target for fighting diseases such as cancer, heart disease, diabetes, Crohn's disease and various neurological diseases. For this reason, considerable research is being done on curcumin.

An important benefit of curcumin is its widespread acceptance due to being a natural compound that has been used for centuries as a spice in foods such as curry. A further advantage is that even at high doses there are few or no side effects. It is also relatively inexpensive to supply and can be stored well at room temperature.

Despite these advantages, a top priority still to be addressed is the well-known problem of curcumin, low bioavailability in animals. This is believed to be due to a combination of factors including poor solubility and thus poor absorption, clearance from the system and/or rapid metabolism.

In the past, this poor solubility has been addressed by adding a carrier that helps to increase the solubility of curcumin, at least in in vitro studies, such as DMSO or Tween 80. However, there are problems that such carriers, such as DMSO, induce an unpleasant taste, add to manufacturing costs and processes, and undermine the benefits of curcumin being a natural product (which consumers want).

Republic of Korea Patent Publication No. 10-2008-0112224 (published on December 24, 2008)

An object of the present invention is to provide a method for preparing a curcumin nanocomposite using a polysaccharide, carrageenan, in order to improve the stability and bioavailability of curcumin exhibiting various physiological activities.

The present invention comprises the steps of dissolving curcumin in an organic solvent to prepare an organic solvent phase (first step); preparing an aqueous phase by dissolving carrageenan in water (second step); mixing and homogenizing the organic solvent phase of the first step and the aqueous phase of the second step (third step); sonicating the homogenized mixture (fourth step); and evaporating the organic solvent from the sonicated mixture (step 5).

In addition, the present invention provides a curcumin nanocomposite according to the above preparation method.

According to the present invention, in the nanocomposite using the sulfated polysaccharide carrageenan, the encapsulation rate of curcumin increased in a concentration-dependent manner of carrageenan, and a high encapsulation rate of 90% or more was confirmed. As the rate improvement was confirmed and the stability and cell membrane permeability of curcumin encapsulated in the nanocomposite were confirmed to increase, the nanocomposite using carrageenan can improve the water solubility of curcumin and improve the bioavailability, so curcumin is effective It can be provided as pharmaceuticals, cosmetics, and health foods as ingredients.

1 is a schematic diagram illustrating the prepared curcumin nanocomposite and improved intestinal permeability.
2 is an image result confirming the prepared curcumin nanocomposite.
3 is a result confirming the stability of curcumin in the prepared curcumin nanocomposite.
4 is a result of confirming the solvent affinity according to the ratio of the prepared curcumin nanocomposite and the solvent.
5 is a result of confirming the solvent affinity according to the ratio of the prepared curcumin nanocomposite and the solvent in the condition containing curcumin.
6 is a result confirming the enhanced antioxidant effect of the prepared curcumin nanocomposite.
7 is a result of confirming the enhanced antioxidant effect of the prepared curcumin nanocomposite through a cell experiment.
8 is a result of confirming the anti-inflammatory effect of the prepared curcumin nanocomposite through a cell experiment.
9 is a result confirming the enhanced interaction between sulfated polysaccharides and small intestine cells among various polysaccharides constituting the curcumin nanocomposite.
10 is a result confirming the bioavailability (Pharmacokinetics) of the prepared curcumin nanocomposite.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for preparing a nanocomposite using carrageenan, a sulfated polysaccharide, in order to increase the water dispersibility and stability of curcumin. And it can contribute to increasing the utilization as food.

The present invention comprises the steps of dissolving curcumin in an organic solvent to prepare an organic solvent phase (first step); preparing an aqueous phase by dissolving the sulfated polysaccharide in water (second step); mixing and homogenizing the organic solvent phase of the first step and the aqueous phase of the second step (third step); sonicating the homogenized mixture (fourth step); and evaporating the organic solvent from the sonicated mixture (the fifth step) may provide a method for preparing a curcumin nanocomposite.

The organic solvent phase may be selected from the group consisting of dichloromethane, ethanol, and chloroform.

The sulfated polysaccharide may be selected from the group consisting of carrageenan, fucoidan, chondroitin sulfate, and dextran sulfate.

The sulfated polysaccharide may have a viscosity of 0.6 to 0.7 wt%.

The sulfated polysaccharide may have a molecular weight of 100 KDa or more or 100 KDa to 800 KDa.

The organic solvent phase may contain 0.01 to 10 parts by weight of curcumin based on 100 parts by weight of the organic solvent.

The aqueous phase may contain 0.1 to 0.8 parts by weight of carrageenan based on 100 parts by weight of water.

In addition, the present invention may provide a curcumin nanocomposite according to the above preparation method.

The "carrageenan" of the present invention is a polysaccharide component obtained by extraction from various red algae belonging to the Dolomaceae family or the Gossiaceae family. It exhibits high viscosity under alkaline conditions and is a hydrophilic colloidal component having the property of easily combining with various cations to form a gel. .

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

< Example 1> curcumin Nanocomposite Manufacturing

Curcumin (Cur) was dissolved in dichloromethane at 5 mg/mL, iota-Carrageenan (ι-CRN (H); High molecular weight (100K ~ 800K), sigma-aldrich, kappa-Carrageenan ( κ-CRN; High molecular weight (100K ~ 800K), sigma-aldrich, iota-Carrageenan (ι-CRN (L); Low molecular weight (1K), JBK-LAB) and ramda-Carrageenan (λ-CRN; molecular weight unknown , JBK-LAB) were dissolved in distilled water at concentrations of 0.4% (CRN 100 mg/25 mL) and 0.66% (CRN 100 mg/15 mL), respectively.

After injecting 2 mL of 5 mg/mL curcumin into the carrageenan solution using a syringe, it was homogenized for 3 minutes at 8,000 rpm through a homogenizer.

Thereafter, ultrasonication was performed for 3 minutes, dichloromethane was evaporated at 600 rpm for 4 hours, and then unsealed curcumin was removed using a centrifuge (3,000 rpm, 3 minutes) to obtain the final nanocomposite as shown in FIG. got it

The size of the nanocomposite as shown in Table 1 was confirmed by measuring the size at 0.1 mg/mL based on Cur using dynamic light scattering (DLS).

Nunber mean (d.nm) Cur@ι-CRN (H) 65.97 ± 19.82 Cur@κ-CRN 217.39 ± 22.54 Cur@ι-CRN (L) 11.25 ± 0.42 Cur@λ-CRN 77.62 ± 10.22

< Example 2> Using ethanol curcumin Nanocomposite Manufacturing

Curcumin (Cur) was dissolved in ethanol (Ethanol) at 2.5 mg/mL, and 100 mg of carrageenan was sufficiently dissolved in 15 mL of distilled water at 600 rpm with a stirrer for 2 hours. After injecting 2.5 mg/mL of curcumin 4 mL into the carrageenan solution using a syringe, it was homogenized for 3 minutes at 8,000 rpm through a homogenizer.

Then, ultrasonication was performed for 6 minutes, and the ethanol was evaporated for 10 minutes under reduced pressure using a round rotary evaporator to completely evaporate.

After evaporating the ethanol, the unencapsulated curcumin was removed using a centrifuge (3,000 rpm, 3 minutes) to obtain a final nanocomposite.

< comparative example 1> curcumin Twin 80 ( Tween80 ) Nanocomposite Manufacturing

Curcumin (Cur) was dissolved in dichloromethane at 5 mg/mL, and Tween80 was dissolved in distilled water at a concentration of 0.66% (Tween80 100 mg/15 mL). After injecting 2 mL of 5 mg/mL curcumin into the Tween80 solution using a syringe, it was homogenized for 3 minutes at 8,000 rpm through a homogenizer.

Thereafter, ultrasonication was performed for 3 minutes, dichloromethane was evaporated at 600 rpm for 4 hours, and then unencapsulated curcumin was removed using a centrifuge (3,000 rpm, 3 minutes) to obtain a final nanocomposite.

< comparative example 2> curcumin Hydroxypropyl Cellulose (HPC) Nanocomposite Preparation

Curcumin (Cur) was dissolved in dichloromethane at 5 mg/mL, and HPC (molecular weight 80K, Sigma-aldrich) was dissolved in distilled water at a concentration of 0.66% (Tween80 100 mg/15 mL). After injecting 2 mL of 5 mg/mL curcumin into the HPC solution using a syringe, it was homogenized for 3 minutes at 8,000 rpm through a homogenizer.

Thereafter, ultrasonication was performed for 3 minutes, dichloromethane was evaporated at 600 rpm for 4 hours, and then unencapsulated curcumin was removed using a centrifuge (3,000 rpm, 3 minutes) to obtain a final nanocomposite.

< Example 3> carrageenan Viscosity check

ι-CRN (H), κ-CRN, ι-CRN (L) and λ-CRN were dissolved in distilled water at a concentration of 0.4% (CRN 100 mg/25 mL) and 0.66% (CRN 100 mg/15 mL), respectively, and , the viscosity (mPa·sec) of carrageenan was measured while changing the rpm at 25 °C, and the spindle of the viscosity meter was used No. 21.

As a result, as shown in Table 2, it was confirmed that the higher the concentration of carrageenan, the higher the viscosity.

(mPa sec) RPM
Conc. (%) 10 20 50 80 100 120 200 ι-CRN
(H) 0.4% 145.00 110.00 72.00 56.30 45.50 42.50 33.50 0.66% 880.00 557.80 297.00 222.00 188.50 167.50 142.00 κ-CRN 0.4% 50.00 32.50 32.00 25.90 27.00 25.40 23.00 0.66% 70.00 47.50 44.00 43.80 38.00 36.20 33.80 ι-CRN
(L) 0.4% 20.00 12.50 15.00 11.90 5.50 5.84 8.75 0.66% 45.00 32.50 29.00 27.50 25.50 24.60 22.80 λ-CRN 0.4% 50.00 42.50 35.00 31.30 30.00 28.80 25.80 0.66% 135.00 107.56 69.00 61.90 59.00 57.10 49.00

< Example 4> inclusion rate Confirm

To check the curcumin encapsulation rate of actual carrageenan, the prepared nanocomposite was diluted 1/10 with ethanol to dissolve curcumin inside the nanoparticles, and curcumin (Cur) was dissolved in ethanol to confirm the calibration curve, 425 It was quantified by measuring the absorbance at nm.

As a result, in dichloromethane (Dichloromethane) In the case of dissolving and encapsulating curcumin, as shown in Table 3, the encapsulation of curcumin in 0.4% carrageenan was not effective due to the low viscosity, whereas in the case of 0.66% carrageenan, ι - CRN (H) and κ - CRN were more than 90% λ- CRN showed a high encapsulation efficiency of 88.30% .

From the above results, it was confirmed that carrageenan is suitable for the production of curcumin nanocomposites, and carrageenan with a high molecular weight (100 KDa to 800 KDa) shows higher encapsulation efficiency than carrageenan with a small molecular weight (1 KDa) .

Cur:X ( curcumin 10 mg ) 15 mL (0.66 %, w/v) 25 mL (0.40 %, w/v) Loading efficiency (%) Loading content (%) Loading efficiency (%) Loading content (%) Cur@ι-CRN (H) 97.81 ± 2.10 8.89 ± 0.19 11.49 ± 0.15 1.04 ± 0.01 Cur@κ-CRN 95.79 ± 4.36 8.71 ± 0.40 14.26 ± 0.36 1.30 ± 0.03 Cur@ι-CRN (L) 41.76 ± 0.31 3.80 ± 0.03 9.85 ± 0.23 0.89 ± 0.02 Cur@λ-CRN 88.30 ± 1.55 8.03 ± 0.14 37.49 ± 3.16 3.41 ± 0.29

< Example 5> Check stability

Curcumin (Cur) was dissolved in dichloromethane at 5 mg / mL, and ι-CRN (H), κ-CRN, ι-CRN (L) and λ-CRN were each 0.4% (CRN 100 mg) /25 mL), dissolved in distilled water to a concentration of 0.66% (CRN 100 mg/15 mL).

2 mL of 5 mg/mL curcumin was injected into the carrageenan solution using a syringe, and then homogenized at 8,000 rpm for 3 minutes using a homogenizer.

Thereafter, ultrasonication was performed for 3 minutes, dichloromethane was evaporated at 600 rpm for 4 hours, and then unencapsulated curcumin was removed using a centrifuge (3,000 rpm, 3 minutes) to obtain a final nanocomposite.

The final nanocomposite was stored in a refrigerator (4 ℃) and the stability of the nanocomposite was tested over time (1, 3, 6, 10 days), and the measurement was repeated 7 times to quantify the amount of curcumin remaining. did

As a result, as shown in FIGS. 2 and 4, Cur@ι- CRN (H) and Cur@κ- CRN decreased by about 7% on the 10th day with time, and 90% and 88% of curcumin were maintained , respectively. It was confirmed that Cur@ι- CRN (L) decreased by about 11% and curcumin was maintained at 30%, and Cur@λ-CRN decreased by 49.48% and curcumin was maintained at 38%.

0 day 10 days difference Cur@ι-CRN (H) 97.81 90.67 7.14 Cur@κ-CRN 95.79 88.80 6.99 Cur@ι-CRN (L) 41.77 30.44 11.33 Cur@λ-CRN 88.30 38.81 49.49

< Example 6> carrageenan according to molecular weight with organic solvents Affinity check

In order to confirm the affinity between carrageenan and the organic solvent, each experiment was carried out using curcumin (100KDa ~ 800KDa) having a large molecular weight. The total volume of DW and EtOH was maintained at 5 mL, and the volume ratio was 10:1, 8:2, 6:4, 4:6, 2:8, 1:10. In a total volume of 5 mL, 33.33 mg/6.33 mL of carrageenan and 3.33 mg/6.33 mL of curcumin were mixed.

After mixing, the solvent affinity according to the ratio of the solvent was confirmed. 4 and 5 show the results of the experimental group not containing curcumin and the experimental group containing curcumin, respectively, and carrageenan was dispersed in water and precipitated in an instant when it met ethanol.

In addition, the transmittance was measured using a UV-vis spectrometer to confirm the state change of the emulsion. In carrageenan with a large molecular weight, as the specific gravity of ethanol increased, the decrease in light transmittance by precipitation was more steep. When the ratio of DW:EtOH is 4:6, it can be seen from FIG. 4 that carrageenan is strongly precipitated and subsides while the transmittance temporarily increases.

800nm DW: EtOH I-CAR(H) 10:0 8:2 6:4 4:6 2:8 0:10 transmission (%) 100.2 84.2 77.2 91.6 63.0 38.3 (%) 100 84.0 77.0 91.4 62.9 38.2

< Example 7> Check the antioxidant effect

The antioxidant effect in the aqueous phase according to the solubility improvement of curcumin (Cur) was confirmed. The antioxidant ability of curcumin or curcumin nanocomposite itself was confirmed by the DPPH (2,2-diphenyl-1-picryl hydrazyl) test method. Each sample was dissolved in distilled water based on Cur 80 μg/mL, and diluted in DPPH solution based on Cur 2 μg/mL. Since DPPH shows UV absorbance at 515 nm, the change in absorbance was measured once every 30 seconds for 30 minutes and converted into a relative value. In addition, the solvent, which was purple due to the DPPH offset with time, changed to yellow, and the color change after 30 minutes was measured with a photograph. Ascorbic acid, a well-known antioxidant, was used as a positive control.

As the solubility of curcumin was improved, DCF-DA (2,7-dichlorodihydrofluucine diacetate) test method was performed to confirm the enhanced antioxidant effect through cell activity. First, HaCa-T cells were aliquoted at 1 × 10 ⁴ cells/well in a 96-well cell culture plate, and after 1 day, the generation of active oxygen in the cells was artificially generated through tBHP (tetrabutyl hydrogen peroxide, positive control). induced. In order to evaluate the antioxidant ability of curcumin or curcumin nanocomposite in the intracellular environment induced by free radicals, each sample was treated at 5 μg/mL of Cur.

As a result, as shown in FIG. 7 , DCF-DA fluorescence intensity dyed with active oxygen was decreased due to the antioxidant effect of curcumin, and the antioxidant of the nanocomposite (Cur@CRN) using double sulfated polysaccharide with high molecular weight was most effective. could check

< Example 8> Check the anti-inflammatory effect

In order to confirm the anti-inflammatory effect of curcumin solubility improvement, the expression levels of major factors of the NFκB/IκBα signaling system were compared through Western blot. In a normal state, NFκB forms a complex with IκBα and is inactivated, but in an inflammatory state, phosphorylated IκBα (pIκBα) increases by IκBα kinase. An increase in pIκBα and degradation of pIκBα dissociate the complex, and a single NFκB binds in the nucleus and transcribes inflammatory factors. Therefore, in the positive control (Lipopolysaccharide, LPS), the expression levels of NFκB and pIκBα increase, and the expression levels of IκBα decrease. However, the expression levels of NFκB and pIκBα, which were increased according to the anti-inflammatory effect of carrageenan or carrageenan nanocomposite, decrease again, and the expression level of IκBα increases again.

To confirm this, HaCa-T cells were aliquoted at 5 × 10 ⁵ cells/well in a 6-well cell culture plate, and after 1 day, each sample was dissolved in a medium based on Cur 5 μg/mL. After incubating each sample for 4 hours, LPS was incubated at a concentration of 10 μg/mL for 6 hours to induce immune activity. After 12 hours, the cells were extracted and the expression levels of NFκB, IκBα and pIκBα were confirmed through Western blot.

As a result, as shown in FIG. 8 , it was confirmed that the anti-inflammatory effect was enhanced in the experimental group using the curcumin nanocomposite compared to when curcumin was used alone, and in particular, sulfated It was confirmed that the anti-inflammatory effect was the highest in the polysaccharide (Cur@CRN) group.

< Example 9> Check cell membrane permeability

12 MDCK (Madin-Darby Canine Kidney) cells were seeded in a trans well apical plate at 1 × 10 ⁵ cells/well, and after 4 days, transepithelial electrical resistance (TEER) was measured and 250 Ω cm ² It was confirmed that

Each sample was treated on an apical (A) or basolateral (B) plate for 2 hours based on Cur 150 μM, and after collecting the apical (A) and basolateral (B) samples, LC-MS/MS analysis was used to By quantifying the amount of curcumin, the apparent permeability (Paap) of curcumin was measured, and the Efflux ratio was confirmed accordingly.

As a result, as shown in Table 6, the Paap values were 44.54 times in Cur@ι-CRN (H), 48.3 times in Cur@κ-CRN, 16.21 times in Cur@ι-CRN (L), and 16.21 times in Cur@λ-, compared to curcumin. It increased 8.33 times in CRN.

From the above results, as significant results were confirmed in Cur@ι- CRN (H) and Cur@κ- CRN, which are high molecular weight, it can have a positive effect on in vivo bioavailability.

Compounds P _aap (Х10 ^-6 cm/sec) Efflux ratio
(B to A/A to B) Cur (A to B) 0.0185 ± 0.0021 0.0809 Cur (B to A) 0.0014 ± 0.0004 Cur@ι- CRN (H) (A to B) 0.8228 ± 0.0620 0.0005 Cur@ι- CRN (H) (B to A) 0.0048 ± 0.0015 Cur@κ- CRN (A to B) 0.8922 ± 0.3829 0.0031 Cur@κ- CRN (B to A) 0.0028 ± 0.0008 Cur@ι- CRN (L) (A to B) 0.2994 ± 0.1354 0.0817 Cur@ι- CRN (L) (B to A) 0.0244 ± 0.0134 Cur@λ- CRN (A to B) 0.1538 ± 0.1016 0.0779 Cur@λ- CRN (B to A) 0.0120 ± 0.0118

< Example 10> sulfation Interaction of polysaccharides with gut-expressed solutes

To investigate the cause of the excellent cell membrane permeability of sulfated polysaccharide (Cur@CRN), the interaction between the solute carrier transporter expressed in the intestine and sulfated polysaccharide was confirmed. A confocal microscope was used to visually confirm the interaction between polysaccharides and cells, and a fluorescent substance (FITC) was conjugated to polysaccharides for use under a confocal microscope. FITC-conjugated polysaccharide was treated based on FITC 5 μg/mL, and the degree of absorption for 2 hours was compared. Caco-2 cells are cells expressing solute carriers (Solute carrier 26A2, SLC26A2) as positive controls, and MDCK and L929 cells are cells that do not express solute carriers as negative controls.

Referring to FIG. 9 , the solute carrier expressed only in Caco-2 cells could be identified through antibody staining, and it was confirmed that the fluorescence of FITC conjugated to sulfated polysaccharide (CRN) exhibited the highest intensity.

From the above results, it was confirmed that the interaction between the solute carrier (SLC26A2) that specifically recognizes sulfur and the sulfated polysaccharide could be the cause of enhancing the intestinal permeability of the curcumin nanocomposite.

< Example 11> Check bioavailability

Rats acclimatized for one week were maintained in a fasting state for 12 hours, and then Cur and Cur@ι-CRN were orally administered (based on Cur 500 mg/kg). Blood from rats is collected every 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, 360 and 480 minutes, plasma is separated at 13,000 rpm for 5 minutes, and Cur present in plasma was quantified using LC-MS/MS analysis.

As a result, as shown in FIG. 10 , it was confirmed that Cur@ι- CRN (H) increased the AUC value by about 2.5 times compared to Cur. In addition, as it was confirmed that the T _max value was delayed by 7.49 times compared to Cur, it was confirmed that absorption proceeded slowly in the intestine for a long time.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

preparing an organic solvent phase by dissolving curcumin in an organic solvent (first step);
preparing an aqueous phase by dissolving the sulfated polysaccharide in water (second step);
mixing and homogenizing the organic solvent phase of the first step and the aqueous phase of the second step (third step);
sonicating the homogenized mixture (step 4); and
A method for producing a curcumin nanocomposite prepared by evaporating an organic solvent from the sonicated mixture (step 5). The method according to claim 1, wherein the organic solvent is selected from the group consisting of dichloromethane, ethanol and chloroform. The method according to claim 1, wherein the sulfated polysaccharide is selected from the group consisting of carrageenan, fucoidan, chondroitin sulfate and dextran sulfate. The method according to claim 1, wherein the sulfated polysaccharide has a viscosity of 0.6 to 0.7 wt%. The method according to claim 1, wherein the sulfated polysaccharide has a molecular weight of 100 KDa or more. The method according to claim 1, wherein the organic solvent phase contains 0.01 to 10 parts by weight of curcumin based on 100 parts by weight of the organic solvent. The method according to claim 1, wherein the aqueous phase contains 0.1 to 0.8 parts by weight of carrageenan based on 100 parts by weight of water. The curcumin nanocomposite according to any one of claims 1 to 7.

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