COMPOSITION FOR IMPROVING SKIN COMPRISING STEAMED CITRUS PEEL EXTRACT AS ACTIVE INGREDIENT

Abstract

The present invention relates to cosmetic, food, and quasi-drug compositions having antioxidant, anti-inflammatory, and wrinkle or elasticity improvement effects, comprising citrus peel extracts as an active ingredient, which increase phenolic compounds, increase the generation of flavonoid aglycones, and reduce furanocoumarin, a harmful substance, a method for preparing the same, and citrus peel extracts prepared by the method. The present invention has excellent antioxidant, anti-inflammatory, and wrinkle or elasticity improvement effects, and thus can be used as a cosmetic composition, a food composition, and a quasi-drug composition that are safe for the skin and have excellent skin condition improvement effects.

Claims

1. A method for preparing a citrus peel extract, comprising: steaming citrus peel; and extracting the steamed citrus peel.

2. The method of claim 1, wherein the steaming is performed in an airtight container at 103° C. to 150° C. for an hour to 15 hours.

3. The method of claim 1, wherein the peel is the peel of satsuma mandarin (Citrus unshiu), yuzu (Citrus junos), orange (Citrus sinensis), bitter orange (Citrus aurantium), grapefruit (Citrus paradisi), lemon (Citrus limonum) or lime (Citrus aurantifolia).

4. The method of claim 1, wherein the steaming is characterized by converting flavonoid glycones to low-molecular flavonoid aglycones or reducing the generation of furanocoumarin.

5. The method of claim 1, wherein the steamed citrus peel extract has a higher content of flavonoid aglycones as compared to when it is not steamed.

6. The method of claim 5, wherein the flavonoid glycone is narirutin, naringin, or hesperidin, and the flavonoid aglycone is naringenin or hesperetin.

7. The method of claim 4, wherein the furanocoumarin is bergapten or bergamottin.

8. The method of claim 1, wherein the steamed citrus peel extract has a higher total phenolic content as compared to when it is not steamed.

9. The method of claim 1, wherein the method further comprises drying the steamed citrus peels.

10. The method of claim 1, wherein the steamed citrus peel extract has an antioxidant use.

11. The method of claim 1, wherein the steamed citrus peel extract has an anti-inflammatory use.

12. The method of claim 1, wherein the steamed citrus peel extract is used for improving elasticity or wrinkles.

13. A citrus peel extract prepared by the method of claim 1.

14. A composition comprising the citrus peel extract of claim 13 as an active ingredient.

15. The composition of claim 14, wherein the composition is a cosmetic, food or quasi-drug composition.

16. An antioxidant method, comprising applying the citrus peel extract of claim 13 or a composition comprising the citrus peel extract as an active ingredient to a subject.

17. A method for preventing or improving inflammation, comprising applying the citrus peel extract of claim 13 or a composition comprising the citrus peel extract as an active ingredient to a subject.

18. A method for improving elasticity or wrinkles, comprising applying the citrus peel extract of claim 13 or a composition comprising the citrus peel extract as an active ingredient.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

[0110] Hereinafter, the present invention will be described in more detail by way of Examples. However, these Examples are given for illustrative purposes only, and the scope of the invention is not intended to be limited by these Examples.

Example 1. Preparation of Steamed Citrus Peel Extracts

Example 1-1. Preparation of Citrus Peels and Steaming Thereof

[0111] The contaminants on the surface of satsuma mandarin (Citrus unshiu), yuzu (Citrus junos), orange (Citrus sinensis), bitter orange (Citrus aurantium), grapefruit (Citrus paradisi), lemon (Citrus limonum) and lime (Citrus aurantifolia) distributed in Korea were carefully washed, and the peels were separated from the inner pulp. After completely drying the separated peels at 40° C., they were cut into a uniform size of about 0.5 cm.

[0112] In addition, the cut citrus peels were placed in a thick airtight stainless-steel container under the condition where they could be moistened with a small amount of purified water, and the container was sealed so that outside air could not pass through. Thereafter, steaming was carried out at 120° C. under various steaming time conditions (untreated, 30 minutes, 2 hours, 4 hours, 8 hours and 12 hours). Then, the peels were sufficiently dried at 40° C. until completely dried.

Example 1-2. Preparation of Steamed Citrus Peel Extracts for Evaluation of Components

[0113] 10 g of the steamed citrus peels were powdered, mixed with 100 mL of high-performance liquid chromatography (HPLC) grade methanol, and sonicated for 1 hour. The extraction process was repeated three times to obtain a total of 300 mL of extraction solvent. 300 mL of the thus-obtained extraction solvent was concentrated and re-adjusted to 100 mL, and filtered through a PTFE filter (0.45 μm) to prepare citrus peel extracts.

[0114] In the case of evaluating the flavonoid content or furanocoumarin content described below, the steamed citrus peel extract solution, which was concentrated by the method above, was used, and in the case of evaluating the total content of phenolic compounds, the concentration of the concentrated steamed citrus peel extract solution was diluted by half with methanol and used.

Example 1-3. Preparation of Steamed Citrus Peel Extracts for Efficacy Evaluation

[0115] 10 g of the steamed citrus peels were powdered, mixed with 100 mL of ethanol, and sonicated for 1 hour. The extraction process was repeated three times to obtain a total of 300 mL of extraction solvent. 300 mL of the thus-obtained extraction solvent was concentrated and re-adjusted to 100 mL, and filtered through a PTFE filter (0.45 μm) to prepare citrus peel extracts.

[0116] In the case of confirming the antioxidant effect described below, the concentration of the concentrated steamed citrus peel extract solution was diluted by half with ethanol and used.

[0117] In the case of confirming anti-inflammatory effect and collagen synthesis-promoting effect, ethanol was completely removed from the steamed citrus peel extract solution using a rotary evaporator, and the residue was redissolved in DMSO so that the extract solution had a final concentration of 50,000 ppm and used.

Example 2. Change in Components According to Steaming Treatment Time and Heat Treatment Conditions and Comparison of Contents

Example 2-1. Analysis of Peel Components According to Citrus Species

[0118] In order to analyze the peel components according to the citrus species, the contents of flavonoid glycones, flavonoid aglycones, and furanocoumarins of non-steamed satsuma mandarin, yuzu, orange, bitter orange, grapefruit, lemon or lime were compared (Table 1).

[0119] The content (mg) of narirutin, naringin and hesperidin, which are three flavonoid glycones; naringenin and hesperetin, which are two flavonoid aglycones; and bergapten and bergamottin, which are two furanocoumarins, contained in 100 g of dried citrus peels was evaluated using high-performance liquid chromatography (HPLC) and diode array detector (DAD) manufactured by Shimadzu.

[0120] Specifically, the components in the extracts were analyzed by a gradient elution method using ZORBAX Eclipse Plus reversed-phase column (4.6×150 mm, 3.5 μm particle size) manufactured by Agilent as the stationary phase, and a combination of water added with a small amount of formic acid and acetonitrile as the mobile phase. The sample input amount was 10 μL, the flow rate was 1 mL/min, and the detection wavelengths were 283 and 310 nm. Standards for qualitative and quantitative analyses were purchased from Sigma-Aldrich, and a calibration curve was prepared using absorbance values according to five or more concentrations to calculate the content in the extract (average value, n=3).

TABLE-US-00001 TABLE 1 Contents Satsuma Bitter (mg/100 g) mandarin Yuzu Orange orange Grapefruit Lemon Lime Narirutin 1064.2 473.4  134.2 6.4 282.7 18.5 55.9 Naringin 14.6 525.5 N.D. 1975.0   3131.4 13.4 3.6 Hesperidin 2149.6 536.7 1629.9 N.D. N.D. 640.0 486.2 Total 3228.4 1535.6 1764.1 1981.4   3414.1 671.9 545.7 Naringenin N.D. N.D. N.D. N.D. N.D. N.D. N.D. Hesperetin N.D. N.D. N.D. N.D. N.D. N.D. N.D. Total N.D. N.D. N.D. N.D. N.D. N.D. N.D. Bergapten N.D. N.D. N.D. 6.4 N.D. 0.3 19.6 Bergamottin N.D. N.D. N.D. N.D. 2.5 1.6 8.1 Total N.D. N.D. N.D. 6.4 2.5 1.9 27.7

[0121] As shown in Table 1, there were differences in the content or composition of flavonoid glycones for each citrus species, and flavonoid aglycones were not detected in the peels that were not steamed (Not Detected, N.D.). Additionally, bergapten or bergamottin, a furanocoumarin, was detected in 4 of the 7 types of peels (bitter orange, grapefruit, lemon, lime).

[0122] In Examples 2-2 to 2-8 below, changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins (mg contained in 100 g of citrus) according to the steaming treatment time conditions for each citrus species were confirmed.

Example 2-2. Change in Components According to Steaming Treatment Time of Satsuma Mandarin Peels

[0123] The changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins were compared under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours) of satsuma mandarin, a citrus species (Table 2).

[0124] As a result, in the case of flavonoid glycones (narirutin, hesperidin), they were decomposed and reduced by heat in the initial stage with increasing steaming time (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours). However, after that (steamed for more than 4 hours, steamed for 8 hours), it was confirmed that the content of flavonoid glycones, which had been reduced, was increased and partially recovered as the extraction efficiency was increased due to the physicochemical change of the peels by the steaming treatment. After 8 hours of steaming, it showed a tendency to decrease again. However, naringin tended to increase because the increase in the extraction efficiency was superior due to the physicochemical changes than the decomposition by heat.

[0125] In the case of flavonoid aglycones, they started to appear from 2 hours of steaming, and at 8 hours of steaming, the content thereof was increased by about 1.6 to 2 times or more compared to the initial stage, confirming that the flavonoid glycones were converted to low-molecular aglycones. After 8 hours of steaming, it showed a tendency to decrease again.

[0126] The components of furanocoumarins were not detected under untreated and steam-treated conditions.

TABLE-US-00002 TABLE 2 Steamed Steamed Steamed Steamed Steamed Contents for for for for for (mg/100 g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Narirutin 1064.2 998.3 669.2 622.7 655.9 415.1 Naringin 14.6 17.1 43.2 55.5 98.3 70.8 Hesperidin 2149.6 1893.4 879.4 700.5 1092.2 693.1 Total 3228.4 2908.8 1591.8 1378.7 1846.4 1179.0 Naringenin N.D. N.D. 10.1 15.0 22.9 13.1 Hesperetin N.D. N.D. 8.5 16.4 28.5 13.8 Total N.D. N.D. 18.6 31.4 51.4 26.9 Bergapten N.D. N.D. N.D. N.D. N.D. N.D. Bergamottin N.D. N.D. N.D. N.D. N.D. N.D. Total N.D. N.D. N.D. N.D. N.D. N.D.

Example 2-3. Change in Components According to Steaming Treatment Time of Yuzu Peels

[0127] The changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins were compared under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours) of yuzu, a citrus species (Table 3).

[0128] As a result, in the case of flavonoid glycones, it was confirmed that the content of flavonoid glycones decreased due to decomposition by heat as the steaming time increased.

[0129] In the case of flavonoid aglycones, they started to appear from 2 hours of steaming, and at 8 hours of steaming, the content thereof was increased by about 1.6 to 2 times or more compared to the initial stage, confirming that the flavonoid glycones were converted to low-molecular aglycones. After 8 hours of steaming, it showed a tendency to decrease again.

[0130] The components of furanocoumarins were not detected under untreated and steam-treated conditions.

TABLE-US-00003 TABLE 3 Steamed Steamed Steamed Steamed Steamed Contents for for for for for (mg/100 g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Narirutin 473.4 451.9 397.6 363.8 247.3 249.5 Naringin 525.5 518.7 478.9 490.2 410.8 404.8 Hesperidin 536.7 511.0 453.6 398.0 282.4 283.5 Total 1535.6 1481.6 1330.1 1252.0 940.5 937.8 Naringenin N.D. N.D. 15.9 25.6 26.5 21.9 Hesperetin N.D. N.D. 14.0 23.6 30.1 25.4 Total N.D. N.D. 29.9 49.2 56.6 47.3 Bergapten N.D. N.D. N.D. N.D. N.D. N.D. Bergamottin N.D. N.D. N.D. N.D. N.D. N.D. Total N.D. N.D. N.D. N.D. N.D. N.D.

Example 2-4. Change in Components According to Steaming Treatment Time of Orange Peels

[0131] The changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins were compared under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours) of orange, a citrus species (Table 4).

[0132] As a result, in the case of flavonoid glycones (narirutin, hesperidin), it was confirmed that the content of hesperidin was more than 12 times higher compared to that of narirutin. Additionally, the two substances were decomposed and reduced by heat in the initial stage with increasing steaming time (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours). However, after that (steamed for more than 4 hours, steamed for 8 hours), it was confirmed that the content of flavonoid glycones, which had been reduced, was increased and partially recovered as the extraction efficiency was increased due to the physicochemical change of the peels by the steaming treatment.

[0133] In the case of flavonoid aglycones, the amount of hesperidin was high in the orange peels, but the total amount of narirutin and naringin was small, and thus, only hesperetin was detected as a result of steaming. Hesperetin started to appear from 2 hours of steaming, and at 8 hours of steaming, the content thereof was increased by about 3 times or more compared to the initial stage, confirming that the flavonoid glycones were converted to low-molecular aglycones. After 8 hours of steaming, it showed a tendency to decrease again.

[0134] The components of furanocoumarins were not detected under untreated and steam-treated conditions.

TABLE-US-00004 TABLE 4 Steamed Steamed Steamed Steamed Steamed Contents for for for for for (mg/100 g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Narirutin  134.2  127.7  57.3 49.3 20.6 66.9 Naringin N.D. N.D. N.D. N.D. N.D. N.D. Hesperidin 1629.9 1496.5 407.8 329.1  527.0  623.2  Total 1764.1 1624.2 465.1 378.4  547.6  690.1  Naringenin N.D. N.D. N.D. N.D. N.D. N.D. Hesperetin N.D. N.D.  5.5 10.4 17.5 11.0 Total N.D. N.D.  5.5 10.4 17.5 11.0 Bergapten N.D. N.D. N.D. N.D. N.D. N.D. Bergamottin N.D. N.D. N.D. N.D. N.D. N.D. Total N.D. N.D. N.D. N.D. N.D. N.D.

Example 2-5. Change in Components According to Steaming Treatment Time of Bitter Orange Peels

[0135] The changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins were compared under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours) of bitter orange, a citrus species (Table 5).

[0136] As a result, in the case of flavonoid glycones (narirutin, naringin), it was confirmed that the content of naringin was significantly higher compared to that of narirutin. Additionally, the two substances were decomposed and reduced by heat in the initial stage with increasing steaming time (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours). However, after that (steamed for more than 4 hours, steamed for 8 hours), it was confirmed that the content of flavonoid glycones, which had been reduced, was increased and partially recovered as the extraction efficiency was increased due to the physicochemical change of the peels by the steaming treatment. After 8 hours of steaming, naringin showed a tendency to decrease again, and narirutin was not detected.

[0137] In the case of flavonoid aglycones, they started to appear from 2 hours of steaming, and at 8 hours of steaming, the content thereof was increased by about 4 times or more compared to the initial stage, confirming that the flavonoid glycones were converted to low-molecular aglycones. After 8 hours of steaming, it showed a tendency to decrease again.

[0138] In the case of furanocoumarin (bergapten), from 2 hours of steaming, the content thereof was reduced to less than half compared to the initial amount, confirming that harmful substances were reduced according to the steaming treatment. At 8 hours of steaming, it was confirmed that the content of bergapten was partially recovered, but after 8 hours of steaming, it showed a tendency to decrease again, confirming that it had a similar pattern to the change in the flavonoid glycones.

TABLE-US-00005 TABLE 5 Steamed Steamed Steamed Steamed Steamed Contents for for for for for (mg/100 g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Narirutin 6.4 6.0 1.4 0.9 1.4 N.D. Naringin 1975.0   1853.2   625.1  518.0 936.7 561.8  Hesperidin N.D. N.D. N.D. N.D. N.D. N.D. Total 1981.4   1859.2   626.5  518.9 938.1 561.8  Naringenin N.D. N.D. 9.6 14.5 40.8 18.9 Hesperetin N.D. N.D. 9.7 14.8 41.0 21.1 Total N.D. N.D. 19.3  29.3 81.8 40.0 Bergapten 6.4 5.9 2.1 1.6 2.3  1.6 Bergamottin N.D. N.D. N.D. N.D. N.D. N.D. Total 6.4 5.9 2.1 1.6 2.3  1.6

Example 2-6. Change in Components According to Steaming Treatment Time of Grapefruit Peels

[0139] The changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins were compared under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours) of grapefruit, a citrus species (Table 6).

[0140] As a result, in the case of flavonoid glycones, narirutin and naringin were detected, while hesperidin was not detected. Additionally, narirutin and naringin were decomposed and reduced by heat in the initial stage with increasing steaming time (untreated, steamed for 30 minutes, steamed for 2 hours). However, after that (steamed for more than 2 hours), it was confirmed that the content of flavonoid glycones, which had been reduced, was increased and partially recovered as the extraction efficiency was increased due to the physicochemical change of the peels by the steaming treatment. After 4 hours of steaming, it showed a tendency to decrease again.

[0141] In the case of flavonoid aglycones, since hesperidin was not detected, hesperetin was also not detected, and only naringenin was detected. Naringenin started to appear from 2 hours of steaming, and at 8 hours of steaming, the content thereof was increased by about 2.5 times or more compared to the initial stage, confirming that the flavonoid glycones were converted to low-molecular aglycones. After 8 hours of steaming, it showed a tendency to decrease again.

[0142] In the case of furanocoumarin (bergamottin), it was detected only when not treated, steamed for 30 minutes, and steamed for 2 hours, and was not detected after 4 hours of steaming, confirming that the harmful substances were reduced according to the steaming treatment.

TABLE-US-00006 TABLE 6 Steamed Steamed Steamed Steamed Steamed Contents for for for for for (mg/100 g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Narirutin 282.7 269.5 211.4 237.5  203.4  152.3  Naringin 3131.4 2767.1 2443.8 2822.8  2679.9  2164.8  Hesperidin N.D. N.D. N.D. N.D. N.D. N.D. Total 3414.1 3036.6 2655.2 3060.3  2883.3  2317.1  Naringenin N.D. N.D. 27.7 51.7 72.6 51.3 Hesperetin N.D. N.D. N.D. N.D. N.D. N.D. Total N.D. N.D. 27.7 51.7 72.6 51.3 Bergapten N.D. N.D. N.D. N.D. N.D. N.D. Bergamottin 2.5 2.1 0.8 N.D. N.D. N.D. Total 2.5 2.1 0.8 N.D. N.D. N.D.

Example 2-7. Change in Components According to Steaming Treatment Time of Lemon Peels

[0143] The changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins were compared under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours) of lemon, a citrus species (Table 7).

[0144] As a result, in the case of flavonoid glycones, it was confirmed that the content of hesperidin was significantly higher compared to that of narirutin and naringin. Additionally, the flavonoid glycones were decomposed and reduced by heat in the initial stage with increasing steaming time (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours). However, after that (steamed for more than 4 hours, steamed for 8 hours), it was confirmed that the content of flavonoid glycones, which had been reduced, was increased and partially recovered as the extraction efficiency was increased due to the physicochemical change of the peels by the steaming treatment. After 8 hours of steaming, naringin showed a tendency to decrease again, and narirutin was not detected.

[0145] In the case of flavonoid aglycones, since the content of narirutin and naringin in the Lime peel was significantly lower compared to that of hesperidin, only hesperetin was detected as a result of steaming. Hesperetin started to appear from 2 hours of steaming, and at 8 hours of steaming, the content thereof was increased by about 6.5 times or more compared to the initial stage, confirming that the flavonoid glycones were converted to low-molecular aglycones. After 8 hours of steaming, it showed a tendency to decrease again.

[0146] In the case of furanocoumarin, it was detected only when not treated and steamed for 30 minutes, and was not detected after 2 hours of steaming, confirming that the harmful substances were reduced according to the steaming treatment.

TABLE-US-00007 TABLE 7 Steamed Steamed Steamed Steamed Steamed Contents for for for for for (mg/100 g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Narirutin 18.5 15.2 4.9 6.0 7.8 6.5 Naringin 13.4 12.9 4.1 6.3 5.5 2.8 Hesperidin 640.0 580.8 178.8  201.1  263.9 243.7  Total 671.9 608.9 187.8  213.4  277.2 253.0  Naringenin N.D. N.D. N.D. N.D. N.D. N.D. Hesperetin N.D. N.D. 1.9 6.3 12.7 6.9 Total N.D. N.D. 1.9 6.3 12.7 6.9 Bergapten 0.3 0.3 N.D. N.D. N.D. N.D. Bergamottin 1.6 1.4 N.D. N.D. N.D. N.D. Total 1.9 1.7 N.D. N.D. N.D. N.D.

Example 2-8. Change in Components According to Steaming Treatment Time of Lime Peels

[0147] The changes in the components of flavonoid glycones, flavonoid aglycones, and furanocoumarins were compared under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours) of lime, a citrus species (Table 8).

[0148] As a result, in the case of flavonoid glycones, they were decomposed and reduced by heat in the initial stage with increasing steaming time (untreated, steamed for 30 minutes, steamed for 2 hours). However, after that (steamed for more than 2 hours), it was confirmed that the content of flavonoid glycones, which had been reduced, was increased and partially recovered as the extraction efficiency was increased due to the physicochemical change of the peels by the steaming treatment.

[0149] In the case of flavonoid aglycones, they started to appear from 2 hours of steaming, and at 8 hours of steaming, the content thereof was increased by about 2.2 to 3.5 times or more compared to the initial stage, confirming that the flavonoid glycones were converted to low-molecular aglycones. After 8 hours of steaming, it showed a tendency to decrease again.

[0150] In the case of furanocoumarin, the content of bergapten was significantly reduced from 2 hours of steaming, but was partially recovered due to the increase in the extraction efficiency after 4 hours of steaming. Additionally, the content of bergamottin was also significantly reduced to less than half within 2 hours of steaming, and bergamottin was not detected after 4 hours of steaming unlike bergapten, confirming that the harmful substances were reduced according to the steaming treatment.

TABLE-US-00008 TABLE 8 Steamed Steamed Steamed Steamed Steamed Contents for for for for for (mg/100 g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Narirutin 55.9 51.5 26.7 31.1 30.2 37.2 Naringin 3.6 4.0 6.7 15.5 26.1 21.7 Hesperidin 486.2 440.6 216.6 229.3 283.1 403.6 Total 545.7 496.1 250.0 275.9 339.4 462.5 Naringenin N.D. N.D. 1.4 3.3 4.9 3.2 Hesperetin N.D. N.D. 7.0 20.6 37.8 25.8 Total N.D. N.D. 8.4 23.9 42.7 29.0 Bergapten 19.6 18.4 12.9 17.2 18.2 15.3 Bergamottin 8.1 7.7 1.6 N.D. N.D. N.D. Total 27.7 26.1 14.5 17.2 18.2 15.3

[0151] In Examples 2-2 to 2-8, the contents of flavonoid glycones and flavonoid aglycones were changed according to the steaming treatment time. In particular, the content of flavonoid glycones decreased and flavonoid aglycones increased, until 8 hours of steaming treatment, confirming that the flavonoid glycones were converted to low-molecular aglycones. In addition, in the case of furanocoumarin, a harmful substance, it was confirmed that the content thereof decreased when steamed for 2 hours or more.

Example 2-9. Changes in Properties and Components According to Heat Treatment Conditions

[0152] In order to confirm whether the changes in the components shown in Examples 2-2 to 2-8 are characteristics that appear only during steaming (105-130° C.) under specific conditions of the present invention, the changes in properties and components were compared during roasting at high temperatures or steaming at low temperatures of 100° C. or less.

[0153] Typically, the components of hesperidin and hesperetin were compared (mg contained in 100 g of satsuma mandarin) under various heat treatment conditions (untreated, roasted at 120° C. for 8 hours, roasted at 200° C. for 5 minutes, steamed at 80° C. for 48 hours, steamed at 100° C. for 24 hours, and steamed at 120° C. for 8 hours) of satsuma mandarin, a citrus species, and the changes in appearance were confirmed (average value, n=3, Table 9).

TABLE-US-00009 TABLE 9 Roasted at Roasted at Steamed at Steamed at Steamed at Content 120° C. for 200° C. for 80° C. for 100° C. for 120° C. for (mg/100 g) Untreated 8 hours 5 minutes 48 hours 24 hours 8 hours Hesperidin 2149.6 2625.1 2237.3 566.9 873.8 1092.2 Hesperetin N.D. 4.5 N.D. 1.2 1.9 28.5 Color Orange Dark brown Black Dark brown Dark brown - Dark brown - Black Black Odor Unique Combination Burning Combination Odor of Odor of odor of unique odor of unique steaming steaming odor and odor and nutty flavor odor of steaming

[0154] As shown in Table 9, when roasting was performed at 120 t for 8 hours instead of steaming, it was confirmed that the amount of hesperidin converted to hesperetin was not significant. When roasted for 5 minutes at a higher temperature of 200° C., there was no significant change in the components, and it was confirmed that hesperidin could not be converted to hesperetin because the outer surface of the raw material was easily burned. That is, it was confirmed that the steaming process plays an important role in changing the contents of hesperidin and hesperetin.

[0155] Meanwhile, when steamed at 80° C. for 48 hours and at 100° C. for 24 hours, hesperidin, a flavonoid glycone, was greatly reduced as compared when steamed for 8 hours at 120° C., and the amount of hesperetin, a flavonoid aglycone, was also significantly reduced. Based on the results, it can be implied that since the pressure was not high during steaming at 100° C. or lower in an airtight container, not only hesperidin was not converted to hesperetin, but also, the flavonoid glycones were not converted to low-molecular aglycones even when steamed for a longer period of time at low temperatures and thus subjected to more heat treatment. Additionally, it was confirmed that the aglycones were significantly generated only when steamed at a high temperature of 105° C. to 130° C. in an airtight container.

Example 3. Antioxidant Effect According to Change in Phenolic Compound Content and Free Radical Scavenging According to Steaming Treatment

Example 3-1. Change in Phenolic Compound Content According to Steaming Treatment Time Conditions of Citrus Peels

[0156] The evaluation of total phenolic contents (TPC) was conducted using the Folin-Ciocalteu colorimetric method under various steaming time conditions (untreated, steamed for 30 minutes, steamed for 2 hours, steamed for 4 hours, steamed for 8 hours, and steamed for 12 hours).

[0157] Specifically, the reaction was initiated by mixing 2 mL of distilled water, 250 μL of citrus peel extract, and 250 μL of Folin-Ciocalteu's phenol reagent (2 N), and after 5 minutes, 500 μL of 7.5% sodium carbonate solution was added thereto, and a blue discoloration was observed. After reacting for 90 minutes, the absorbance was measured at 760 nm.

[0158] The absorbance was evaluated at concentrations of 25, 50, 100, 200, 400, and 800 ppm of gallic acid to prepare a calibration curve in the same manner. The total phenolic contents calculated by substituting the absorbance of the extract for the calibration curve were expressed as mg gallic acid equivalents/g of dried peel (average value, n=3).

[0159] The total phenolic contents of satsuma mandarin, yuzu, orange, grapefruit, and lime were compared (Table 10).

TABLE-US-00010 TABLE 10 TPC Steamed Steamed Steamed Steamed Steamed (mg gallic acid for for for for for equivalents/g) Untreated 30 minutes 2 hours 4 hours 8 hours 12 hours Satsuma 23.9 23.7 23.6 21.8 31.8 15.8 mandarin Yuzu 15.8 16.2 17.4 20.3 25.4 28.2 Orange 19.3 18.8 10.1 11.7 20.2 22.4 Grapefruit 17.7 15.9 11.0 15.5 22.0 19.1 Lime 10.0 9.3 4.9 9.0 18.0 24.0

[0160] As shown in Table 10, for the phenolic acid present in the citrus peel extracts and the flavonoid glycones, which are the main ingredient, as phenolic compounds, and the changes in the total phenolic contents showed a similar tendency to the changes in the flavonoid glycones. That is, it decreased at the beginning of steaming, and after that, the reduced amount partially recovered.

[0161] Unlike the flavonoid glycones, the TPC was mostly increased at 8 hours of steaming compared to the initial stage, thereby confirming that the steaming process increased the total phenolic contents.

[0162] Hereinafter, it was attempted to confirm whether the increase in the total phenolic contents of the citrus peel extracts contributed to the increase in antioxidant effect.

Example 3-2. Antioxidant Effect According to Free Radical Scavenging

[0163] In order to confirm the antioxidant effect of citrus peel extracts, the extracts were untreated, steamed for 30 minutes or steamed for 8 hours, and thereafter, the free radical scavenging ability was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. As a relatively stable free radical, DPPH, which exhibits the maximum absorption at 517 nm when present in a radical state and loses its absorption ability when the radical is scavenged, was dissolved in methanol at a concentration of 0.12 mM and used. As a positive control, Trolox, a water-soluble analog of vitamin E, was used.

[0164] 100 μL of Trolox solutions (0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 mg/mL) for each concentration was seeded into a 24-well plate, and DPPH solution was added at 1,900 μL. The plate was left for 1 hours by blocking light at room temperature, and the absorbance was measured at 517 nm using an ELISA reader to prepare calibration curves according to the concentration using the absorbance values at 7 concentrations. Similarly, 100 μL of the citrus peel extract and 1,900 μL of DPPH solution were reacted. By substituting the absorbance of the extract for the calibration curve, the degree of antioxidant effect of 1 g of the citrus peel was expressed as Trolox μmol equivalents/g. The experiment was performed three times and the average value was calculated.

TABLE-US-00011 TABLE 11 Antioxidant Efficacy Steamed for Steamed for (μmol Trolox equivalents/g) Untreated 30 minutes 8 hours Satsuma mandarin 10.8 11.0 18.6 Yuzu 11.1 11.9 21.3 Orange 7.0 6.5 16.3 Bitter orange 10.5 8.6 10.6 Grapefruit 4.9 4.4 15.1 Lemon 5.0 4.1 11.4 Lime 3.7 3.5 14.1

[0165] As a result, it was confirmed that the antioxidant efficacy greatly increased when steamed for 8 hours in all citrus species. In particular, the orange, grapefruit, lemon, and lime had low antioxidant efficacy when untreated and steamed for 30 minutes, but when steamed for 8 hours, the efficacy increased by about 2 to 4 times or more, confirming that the antioxidant effect was excellent.

Example 4. Anti-Inflammatory Effect by Inhibition of NO Production

Example 4-1. Anti-Inflammatory Effect by Inhibition of NO Production

[0166] In order to confirm the anti-inflammatory effect of the citrus peel extracts, NO production inhibition rate was measured after the extracts were untreated, steamed for 30 minutes, or steamed for 8 hours. In order to culture Raw264.7 cells, a mixture of DMEM (Dulbecco's Modified Eagle's Medium) and FBS (Fetal Bovine Serum) was used as a basic medium, and the Raw264.7 cells were seeded into a 24-well plate at a concentration of 1 to 2×10.sup.5 cells/mL and cultured for 24 hours. The medium was removed and the cells were starvated in serum-free medium for 12 hours, and thereafter, the DMSO lysate of each extract was treated so that the concentration was 200 ppm, and after 30 minutes, lipopolysaccharide was added at a concentration of 500 ng/ml and cultured for 18 hours. L-NMMA, a positive control, was treated at 20 ppm. After completion of the culture, the supernatant was taken and transferred to a 96-well plate, reacted at room temperature by adding the GRIESS reagent, and the absorbance at 540 nm was measured using an ELISA reader. The amount of NO inhibited compared to the control group was calculated as NO production inhibition rate (%), and the average value was calculated after the experiment was performed three times.

TABLE-US-00012 TABLE 12 NO Production Steamed for Steamed for Inhibition Rate (%) Untreated 30 minutes 8 hours L-NMMA 55.1% (Positive control) Satsuma mandarin 12.2% 13.1% 18.2% Yuzu 14.9% 15.1% 17.5% Orange 10.3% 10.9% 12.4% Bitter orange 8.2% 7.9% 11.1% Grapefruit 6.7% 6.3% 8.3% Lemon 4.4% 4.5% 6.3% Lime −2.1% −1.5% 2.9%

[0167] As a result, as shown in Table 12, when the extracts were steamed for 8 hours, it was confirmed that the anti-inflammatory effect was superior compared to when they were untreated and steamed for 30 minutes.

Example 4-2. Anti-Inflammatory Effect According to Components and Concentration of Citrus Peel

[0168] In order to identify the cause of the change in anti-inflammatory efficacy among the steamed citrus peel extract components, the NO production inhibition rates of hesperidin, a flavonoid glycone, and hesperetin, a a flavonoid aglycone, were compared (Table 13)

TABLE-US-00013 TABLE 13 NO Production Inhibition Rate (%) Hesperidin Hesperetin L-NMMA 51.3% (Positive control) 0.1 ppm −5.5% −3.0% 1 ppm 0.6% 2.6% 10 ppm 1.8% 8.1% 20 ppm 2.4% 35.6%

[0169] As a result, as shown in Table 13, when hesperidin and hesperetin were treated at higher concentrations, the NO production inhibition rate was further increased, and in particular, at a certain concentration or higher (20 ppm), the NO production inhibition rate of hesperetin was significantly increased. Accordingly, it was confirmed that the flavonoid aglycones generated from the steamed citrus peel extracts could be the cause of the increased anti-inflammatory efficacy.

Example 5. Collagen Synthesis-Promoting Effect

Example 5-1. Collagen Synthesis-Promoting Effect

[0170] In order to confirm the collagen synthesis promoting effect of the citrus peel extracts, the amount of additional collagen production was measured after the extracts were untreated, steamed for 30 minutes, or steamed for 8 hours. In order to culture human dermal fibroblasts, a mixture of DMEM and FBS was used as a basic medium, and the skin fibroblasts were seeded into a 48-well plate at a concentration of 2 to 5×10.sup.4 cells/mL and cultured for 24 hours. The medium was removed, and the DMSO lysate of each extract was treated in serum-free medium so that the concentration was 100 ppm, and cultured for 24 hours. A positive control that promotes collagen synthesis was treated with TGF-β to a concentration of 5 ppb. The cell culture solution was taken and the synthesized collagen was measured using the Human procollagen 1α1 duoset ELISA Kit (R&D Systems) and ELISA reader. A calibration curve was prepared by measuring the isolated and purified collagen at concentrations of 0.25, 0.5, 1, 2, 4, and 8 ng/mL, and based on the results, the amount of collagen (Type I collagen) produced in the DMSO lysate of the citrus peel extracts was calculated. The amount of collagen produced compared to the control group (DMSO) was calculated as collagen production rate (%), and the average value was calculated after the experiment was performed three times (Table 14).

TABLE-US-00014 TABLE 14 Additional Collagen Steamed for Steamed for Production Rate (%) Untreated 30 minutes 8 hours TGF-β 25.2% (Positive control) Satsuma mandarin 19.5% 18.7% 27.9% Yuzu 13.7% 14.2% 18.1% Orange 5.1% 5.0% 6.6% Bitter orange 12.8% 13.6% 17.4% Grapefruit 7.3% 6.5% 9.3% Lemon −1.0% −1.5% 2.6% Lime 0.5% 2.1% 5.2%

[0171] As a result, as shown in Table 14, it was confirmed that the amount of collagen produced when steamed for 8 hours was superior compared to when untreated and steamed for 30 minutes.

Example 5-2. Collagen Synthesis-Promoting Effect According to Components and Concentration of Citrus Peel

[0172] In order to identify the cause of the change in collagen synthesis promoting efficacy among the steamed citrus peel extract components, the amount of additional collagen production of hesperidin, a flavonoid glycone, and hesperetin, a flavonoid aglycone, was compared (Table 15).

TABLE-US-00015 TABLE 15 Additional Collagen Production Rate (%) Hesperidin Hesperetin TGF-β 23.7% (Positive control) 0.1 ppm −0.2% 17.0% 1 ppm 0.2% 16.8%

[0173] As a result, as shown in Table 15, unlike hesperidin, the collage production was significantly increased in hesperetin at lower concentrations (0.1 ppm, 1 ppm). Accordingly, it was confirmed that the flavonoid aglycones produced in the steamed citrus peel extracts may be the cause of the increase in collagen synthesis promoting efficacy.

[0174] From the foregoing, a skilled person in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.