<i>Nelumbo nucifera </i>callus extract having increased content of gallic acid, method for preparing same, and whitening cosmetic composition containing same

Abstract

The present invention relates to Nelumbo nucifera callus having an increased content of gallic acid or an extract thereof and to a method for preparing the same. The Nelumbo nucifera callus extract according to the present invention has an excellent whitening effect by containing a large amount of gallic acid, and thus can be advantageously used as a cosmetic composition.

Claims

1. A method for preparing a Nelumbo nucifera callus extract having an increased gallic acid content, the method comprising the steps of: (a) germinating a Nelumbo nucifera seed to obtain germinated Nelumbo nucifera tissue; (b) inducing a Nelumbo nucifera callus from the germinated Nelumbo nucifera tissue; (c) culturing the Nelumbo nucifera callus in a medium containing methyl jasmonic acid; and (d) extracting the Nelumbo nucifera callus culture having an increased gallic acid content.

2. The method of claim 1, wherein the medium contains the methyl jasmonic acid at a concentration of 100 to 400 M.

3. The method of claim 1, wherein the gallic acid content of the extract obtained by the method is increased at least 10-fold compared to the gallic acid content of the extract of a Nelumbo nucifera callus cultured in a medium containing no methyl jasmonic acid.

4. The method of claim 1, wherein the Nelumbo nucifera is Ara Hongnyon lotus.

5. The method of claim 1, wherein the methyl jasmonic acid is added to the medium at 4 to 6 weeks from the start of the culturing in step (c).

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the results of HPLC analysis performed to analyze the gallic acid content of a Nelumbo nucifera callus extract prepared in Comparative Example 1.

(2) FIG. 2 shows the results of HPLC analysis performed to analyze the gallic acid content of a Nelumbo nucifera callus extract prepared in Example 2-2.

(3) FIG. 3 shows the results of HPLC analysis performed to analyze the gallic acid contents of Nelumbo nucifera extracts prepared without callus induction (Comparative Examples 5 to 7).

(4) FIG. 4 shows the results of measuring a change in skin brightness (L value) after applying a cream containing 0.5% lyophilisate of a Nelumbo nucifera callus extract (obtained through treatment with 200 M of methyl jasmonic acid).

(5) FIG. 5 shows the results of measuring a change in skin melanin index after applying a cream containing 0.5% lyophilisate of a Nelumbo nucifera callus extract (obtained through treatment with 200 M of methyl jasmonic acid).

MODE FOR INVENTION

(6) Hereinafter, the present invention will be described in detail with reference to examples.

(7) However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Reference Example: Obtaining of Nelumbo nucifera Seeds

(8) In this Example, 30 Nelumbo nucifera seeds, harvested during 3 years (2010 to 2012) after cultivation of an about 700-year-old lotus seed (which dates back to Korea's Goryeo Dynasty) excavated from the Haman Seongsan mountain fortress in 2009, were obtained from the Haman Museum located at Gaya-Eup, Haman-gun, Gyeongsangnam-do, South Korea.

Example 1: Preparation of Nelumbo nucifera Callus

(9) Sterilized Nelumbo nucifera seeds were sowed on MS (Murashige-Skoog) basal solid medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) containing no hormone and were germinated under a long-day condition (18 hours) at 25 to 28 C.

(10) The cotyledon attached to the germinated seedling was cut about 3-5 cm with a sharp knife and placed on an MS basal solid medium (pH 5.8) obtained by adding 40.0 g/L sucrose, 5-10.0 g/L agar, 0.5 mg/L -naphthaleneacetic acid and 1.0 mg/L 6-benzylaminopurine to MS basal solid medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221). After about 2 weeks, a shoot together with a callus was produced from the hypocotyl and subcultured with a rooting medium while the callus was maintained.

(11) On the above-described medium prepared by adding plant growth regulating substances (40.0 g/L sucrose, 5-10.0 g/L agar, 0.5 mg/L -naphthaleneacetic acid and 1.0 mg/L 6-benzylaminopurine to MS basal solid medium) to MS basal solid medium, culture was performed at 25 C. for 4 weeks. At 5 weeks of the culture, methyl jasmonic acid was added to the medium at concentrations of 100 M, 200 M and 400 M, followed by culture.

Example 2: Preparation of Nelumbo nucifera Callus Extracts

(12) Each callus obtained as described above was harvested, washed several times with water, and dried in a vacuum freeze dryer (Ilshin, Cat no. LP-50) for 3 days. 1 kg of the dried Nelumbo nucifera callus was placed in a container, and 100 L of purified water was added thereto, followed by hot-water extraction at 90 to 120 for 48 hours. After the extraction, the solid was removed by filtration, thereby preparing Nelumbo nucifera callus extracts.

(13) The Nelumbo nucifera callus extracts obtained by culturing in the media containing 100 M, 200 M and 400 M of methyl jasmonic acid, respectively, were named Example 2-1, Example 2-2 and Example 2-3, respectively.

Comparative Examples 1 to 4: Preparation of Comparative Examples According to Elicitor Treatment

(14) Nelumbo nucifera callus extracts were prepared by Nelumbo nucifera callus preparation and hot-water extraction in the same manner as described in Examples 1 and 2 above, except that the elicitor methyl jasmonic acid was not added or salicylic acid (SA) in place of methyl jasmonic acid was added to the medium at concentrations of 100 M, 200 M and 400 M.

(15) The Nelumbo nucifera callus extract obtained by culturing in the medium containing no methyl jasmonic acid was named Comparative Example 1, and the Nelumbo nucifera callus extract obtained by culturing in the media containing 100 M, 200 M and 400 M of salicylic acid, respectively, were named Comparative Example 2, Comparative Example 3 and Comparative Example 4, respectively.

Comparative Examples 5 to 7: Preparation of Comparative Examples Using Various Nelumbo nucifera Parts without Callus Induction

(16) Nelumbo nucifera seeds and leaves were collected from Haman, Korea. The Nelumbo nucifera leaves were dried at 55 C. for 8 hours and ground in a mixer, and 750 ml of water as an extraction solvent was added to 50 g of the ground material, followed by extraction for 6 hours. The resulting extract was named Comparative Example 5.

(17) To obtain Comparative Examples 6 and 7, Nelumbo nucifera seeds were cut and separated into seed embryos and flesh, and water was added thereto, followed by extraction for 6 hours. Next, 1% filtration aid was added, followed by filtration and then drying at 80 C. The water extract of the Nelumbo nucifera seed embryo was named Comparative Example 6, and the water extract of the Nelumbo nucifera seed flesh was named Comparative Example 7.

Experimental Example 1: Analysis of Increase in Gallic Acid Content of Nelumbo nucifera Callus Extracts Prepared Using Different Kinds and Concentrations of Elicitor

(18) In order to examine whether the gallic acid content of the Nelumbo nucifera callus extracts increases depending on the kind and concentration of elicitor, the content of gallic content in each of the Nelumbo nucifera callus extracts prepared in Example 2 and Comparative Examples 1 to 4 was measured by HPLC.

(19) Specifically, 0.1 g of each of lyophilisates of the Nelumbo nucifera callus extracts prepared in Examples 2-1, 2-2 and 2-3 above was dissolved in purified water, thereby obtaining 10 ml of each sample solution. In addition, 10 mg of a gallic acid (C.sub.7H.sub.6O.sub.5: 170.12) standard was dissolved in purified water to a final volume of 100 ml, and then 0.5 ml of the solution was dissolved again in purified to a final volume of 100 ml and diluted to and , thereby obtaining standard solutions. 20 l of each of the above-described sample solutions and standard solutions was analyzed by HPLC.

(20) The HPLC analysis was performed using Agilent 1260 Infinity (Agilent, USA). The column used was CAPCELL PAK C18 UG120 (250 mm4.6 mm; 5 m). As a mobile phase, a mixture of 0.1% trifluoroacetic acid (TFA) and 0.1% acetonitrile (B) was used. The content of B was increased from 0% to 90% with a constant gradient over 35 minutes. The flow rate was 1.0 ml/min, and the detection wavelength of the UV detector was set at 270 nm. The gallic acid content of each of the Nelumbo nucifera callus extracts, measured by HPLC, is shown in FIGS. 1 and 2 and Table 1 below.

(21) TABLE-US-00001 TABLE 1 Methyl jasmonic acid Salicylic acid Nelumbo nucifera concentration concentration Gallic acid callus extract (M) (M) (g/mg) Wt % Example 2-1 100 7.36 0.74 Example 2-2 200 27.83 2.78 Example 2-3 400 14.18 1.42 Comparative 0.00 0.00 Example 1 Comparative 100 0.00 0.00 Example 2 Comparative 200 0.00 0.00 Example 3 Comparative 400 0.00 0.00 Example 4

(22) As a result, as shown in FIGS. 1 and 2, no gallic acid was detected in the Nelumbo nucifera callus extract prepared in Comparative Example 1 (FIG. 1), whereas gallic acid was detected in the Nelumbo nucifera callus extract prepared through treatment with methyl jasmonic acid in Example 2-2 (FIG. 2).

(23) In addition, as shown in Table 1 above, the results of HPLC indicated that the peak of gallic acid did not appear not only in the Nelumbo nucifera callus extract prepared in Comparative Example 1, but also in Comparative Examples 2 to 4 in which treatment with salicylic acid was performed, indicating that no gallic acid was also detected in the Nelumbo nucifera callus extracts obtained through treatment with salicylic acid.

(24) However, it was shown that the Nelumbo nucifera callus extracts prepared through treatment with methyl jasmonic acid in Examples 2-1 to 2-3 had increased gallic acid contents. In particular, it was shown that the Nelumbo nucifera callus extract prepared by culturing in the medium containing 200 M of methyl jasmonic acid (Example 2-2) had the highest gallic acid content.

Experimental Example 2: Analysis of Gallic Acid Content in Extract Prepared from Each Nelumbo nucifera Part without Callus Induction

(25) In order to measure the gallic acid contents of the extract samples of Example 2-2 and Comparative Examples 5 to 7, HPLC analysis was performed.

(26) The HPLC analysis was performed using Agilent 1260 Infinity (Agilent, USA). The column used was CAPCELL PAK C18 UG120 (250 mm4.6 mm; 5 m). As a mobile phase, a mixture of 0.1% trifluoroacetic acid (TFA) and 0.1% acetonitrile (B) was used. The content of B was increased from 0% to 90% with a constant gradient over 35 minutes. The flow rate was 1.0 ml/min, and the detection wavelength of the UV detector was set at 270 nm. The gallic acid content of each of the extracts of Comparative Examples 5 to 7, measured by HPLC, is shown in FIG. 3.

(27) TABLE-US-00002 TABLE 2 Gallic acid Test group (g/mg) Example 2-2 (Nelumbo nucifera callus extract prepared 27.83 through treatment with 200 M of methyl jasmonic acid) Comparative Example 5 (water extract of 0.00 Nelumbo nucifera leaf) Comparative Example 6 (water extract of 0.00 Nelumbo nucifera seed embryo) Comparative Example 7 (water extract of 0.00 Nelumbo nucifera seed flesh)

(28) As a result, it was shown that gallic acid was detected in the Nelumbo nucifera callus extract prepared in Example 2-2 (see FIG. 2), whereas no gallic acid was detected in the extracts of Comparative Examples 5 to 7, prepared without callus induction (see FIG. 3). The gallic acid content of Example 2-2 and Comparative Examples 5 to 7 are shown in Table 2 above.

Experimental Example 3: Examination of Whitening Effect of Nelumbo nucifera Callus Extract Having Increased Gallic Acid Content

(29) In order to evaluate the whitening effect of the Nelumbo nucifera callus extract having an increased gallic acid content, prepared through treatment with methyl jasmonic acid in Example 2 above, the effect of the extract on the inhibition of melanin production was evaluated using B16F1 melanocytes.

(30) Specifically, B16F1 melanocytes, cells of mouse origin, were obtained from the ATCC (American Type Culture Collection) and used in this Example. The B16F1 melanocytes were dispensed into a 6-well plate at a density of 210.sup.5 cells per well, attached to each well, and then dispensed at a concentration that caused no toxicity. Next, the cells were treated with each of 300 g/ml of a lyophilisate of the Nelumbo nucifera callus extract, prepared in Comparative Example 1, and 300 g/ml of a lyophilisate of the Nelumbo nucifera callus extract having an increased gallic acid content, prepared in Example 2, and were incubated in a CO.sub.2 incubator at 37 C. for 72 hours. As a control, 10 nM of -melanocyte-stimulating hormone (-MSH) was used and incubated in the same manner as the test substance. After 72 hours of the incubation, the cells were detached by trypsin-EDTA, counted, and then collected by centrifugation. The quantification of melanin in the cells was performed using a modification of the method of Lotan (Cancer Res., 40: 3345-3350, 1980). The cell pellets were washed once with PBS, and then 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added thereto, and the cells were lysed by vortexing for 5 minutes. To the cell filtrate obtained by centrifugation (at 3,000 rpm for 10 min), 1N NaOH (10% DMSO) was added to extract melanin. The extracted melanin was dissolved, measured for its absorbance at 490 nm by microplate reader, and then quantified, and the protein amount was measured.

(31) Melanin production (%) in the sample was evaluated by calculating the amount of melanin per unit protein and calculating the change relative to the control by use of the following Equation 1. The results are shown in Table 3.
Melanin production (%)=B/A*100Equation 1

(32) A: the amount of melanin in control well (control)/total protein;

(33) B: the amount of melanin in Nelumbo nucifera callus extract-containing well (Example)/total protein.

(34) TABLE-US-00003 TABLE 3 Methyl jasmonic acid Conc. aMSH level Melanin/protein (M) (g/ml) (mM) (% control) Control 10 100% Comparative 300 97% Example 1 Example 2-1 100 84% Example 2-2 200 80% Example 2-3 400 125%

(35) As a result, as shown in Table 3 above, the lyophilisate of the Nelumbo nucifera callus extract of Comparative Example 1 showed a melanin synthesis inhibitory effect of about 3%, which was insignificant. In comparison with this, among the lyophilisates of the Nelumbo nucifera callus extracts having an increased gallic acid content, the extracts prepared through treatment with 100 M or 200 M of methyl jasmonic acid as an elicitor in Examples 2-1 and 2-2 inhibited melanin synthesis by about 20%. However, the group treated with 400 M of methyl jasmonic acid as an elicitor showed increased melanin synthesis, and this result was believed to be because the number of the cells was decreased due to high cytotoxicity, and thus melanin synthesis seemed to be relatively increased.

Experimental Example 4: Test for Whitening Effect on Human Skin

(36) On 24 healthy 20-60-year old women, the whitening improvement effect of a cream containing 0.5% lyophilisate of the Nelumbo nucifera callus extract (prepared through treatment with 200 M methyl jasmonic acid) was evaluated. The face of each of the selected subjects was washed with a cleanser, and then the subjects were maintained in a waiting room under constant-temperature and constant-humidity conditions (temperature: 222 C., and humidity: 5010%) for 30 minutes so that the skin surface temperature and humidity would be adapted to the environment of the measurement space.

(37) Using a cream containing 0.5% lyophilisate of the Nelumbo nucifera callus extract and a placebo cream, a whitening effect test was performed. On first visit, the test cream and the control cream were randomly assigned to the left and right areas of the face, and the skin brightness was measured. Thereafter, the cream formulations were applied to the left and right facial areas in the morning and evening.

(38) After 4 and 8 weeks of use of each cream, the skin brightness (L-value) was quantitatively measured using CM-2600d (Minolta, Japan), and the skin melanin index was evaluated by measuring light, emitted at 568 nm (green), 660 nm (red) and 880 nm (infrared) from a probe and reflected from the skin, by use of a mexameter and a narrow-band reflectance spectrophotometer.

(39) The L-value is a brightness parameter, L in the L-value indicates brightness. The L-value is expressed as a number from 0 to 100.

(40) Instrumental evaluation was performed on visit, and the test areas were measured three times with a chromameter, and the measured values were averaged. The increase rate in the skin brightness measured using the chromameter was calculated using the following Equation 2:
Rate of increase (%) in L-value=((L-value after sample application)(L-value before sample application))/(L-value before sample application)*100Equation 2

(41) As a result, as can be seen in FIG. 4, the L-value (average) measured after 8 weeks of application of the cream containing 0.5% increased from 59.8 before application to 61.8 after 8 weeks of application (3.4% increase). The P-value of the value after 8 weeks of application was less than 0.05 as tested by paired samples t-test according to normality test, indicating that the value was increased to a statistically significant level.

(42) In addition, as can be seen in FIG. 5, the skin melanin index (average) decreased from 197.7 before application to 174.9 after 8 weeks of application (11.8% decrease). The P-value of the melanin index after 8 weeks of application was less than 0.05 as tested by paired samples t-test according to normality test, indicating that the value was increased to a statistically significant level.

Formulation Examples: Preparation of Formulations to Confirm Stability of Cosmetic Composition

(43) To confirm the stability of a cosmetic composition containing the Nelumbo nucifera callus extract having an increased gallic acid content, formulations (solubilized formulation, essence formulation and cream formulation) containing a lyophilisate of the Nelumbo nucifera callus extract of Example 2-2 above were prepared according to the following Formulation Examples 1 to 3.

Formulation Example 1: Preparation of Solubilized Formulation

(44) TABLE-US-00004 TABLE 4 Content Phase Component (%) Aqueous phase Purified water To 100 Glycerin 10-25 Dipropylene glycol Betaine D-Panthenol Sodium hyaluronate Thickener q.s. Chealating agent q.s. Solubilizing PEG-60 hydrogenated castor oil 0-2 phase PEG-40 hydrogenated castor oil 0-2 Polyol 0-10 Fragrance q.s. Ethanol 0-20 Addition I Lyophilisate of Nelumbo nucifera callus 0.5 extract prepared through treatment with 200 M methyl jasmonic acid in Example 2-2 Addition II Preservative q.s. Other additives q.s.

Preparation Method

(45) (1) The components of each of the aqueous phase and the solubilizing phase were uniformly mixed and dissolved.

(46) (2) The solubilizing phase was added to and mixed with the aqueous phase, followed by solubilization.

(47) (3) Addition I, a phase containing a lyophilisate of the Nelumbo nucifera callus extract prepared through treatment with 200 m methyl jasmonic acid in Example 2-2, was solubilized, added and mixed, after which addition II was added.

Formulation Example 2: Preparation of Essence Formulation

(48) TABLE-US-00005 TABLE 5 Content Phase Component (wt %) Aqueous phase Purified water To 100 Ceteareth-6 olivate 0.1-3 Glycerin 10-25 1,2-propanediol Sodium hyaluronate Thickener q.s. Chelating agent q.s. Oil phase PEG-100 stearate 0.1-1 Glyceryl stearate 0.1-1 Polysorbate 60 0.1-1 Cetyl alcohol 0.1-1 Behenyl alcohol 0.1-1 Squalane 5-20 Tocopheryl acetate 0.1-0.5 Addition phase I Lyophilisate of Nelumbo nucifera callus 0.5 extract prepared through treatment with 200 uM methyl jasmonic acid in Example 2-2 Addition phase II Fragrance q.s. Preservative q.s. Other additives q.s.

Preparation Method

(49) (1) The components of each of the aqueous phase and the oil phase were heated, mixed uniformly and mixed.

(50) (2) At 75 C., the oil phase was added to the aqueous phase, followed by emulsification.

(51) (3) Addition phase I, a phase containing a lyophilisate of the Nelumbo nucifera callus extract prepared through treatment with 200 uM methyl jasmonic acid in Example 2-2, was added and mixed at 50 C., after which addition phase II was added thereto.

Formulation Example 3: Preparation of Cream Formulation

(52) TABLE-US-00006 TABLE 6 Content Phase Component (wt %) Aqueous phase Purified water To 100 Glycerin 10-25 Betaine Sodium hyaluronate Thickener q.s. Chelating agent q.s. Oil phase PEG-100 stearate 0.1-2 Glyceryl stearate 0.1-2 Polysorbate 60 0.1-2 Stearic acid 0.1-2 Cetearyl alcohol 0.1-2 Capric/caprylic triglyceride 10-30 Tocopheryl acetate 0.1-0.5 Addition I Lyophilisate of Nelumbo nucifera callus 0.5 extract prepared through treatment with 200 uM methyl jasmonic acid in Example 2-2 Addition II Fragrance q.s. Preservative q.s. Other additives q.s.

Preparation Method

(53) (1) The components of each of the aqueous phase and the oil phase were heated, mixed uniformly and dissolved.

(54) (2) At 75 C., the oil phase was added to the aqueous phase, followed by emulsification.

(55) (3) Addition phase I, a phase containing a lyophilisate of the Nelumbo nucifera callus extract prepared through treatment with 200 uM methyl jasmonic acid in Example 2-2, was added and mixed at 50 C., after which addition phase II was added thereto.

Experimental Example 5: Evaluation of Stability of cosmetic composition containing Nelumbo nucifera Callus Extract Having Increased Gallic Acid Content

(56) 1) Evaluation of Raw Material Stability of Nelumbo nucifera Callus Extract Having Increased Gallic Acid Content

(57) To examine raw material stability, the solubilized

(58) formulation, essence formulation and cream formulation of Formulation Examples 1 to 3 were stored under conditions of 4 C., 30 C., 45 C. and sunlight for 12 weeks. Then, time-dependent changes in the gallic acid content of the lyophilisate of the Nelumbo nucifera callus extract having an increased gallic acid content was observed by HPLC analysis according to the same method as described in Experimental Example 1, and the results are shown in Table 7 below.

(59) TABLE-US-00007 TABLE 7 Gallic acid (% relative to initial content) Elapsed Solubilized formulation Essence formulation Cream formulation time 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight Initial 99.9 100 99 99.2 99 99.8 100 99.3 100 99.5 99.3 100 1 week 96.2 96.7 97.2 97 97.3 96.6 96 97.7 97 96.2 95.7 94.4 3 weeks 97 97.6 97.3 94.3 97 98.1 98.5 97.1 96.7 97.7 98.3 92.4 5 weeks 97.6 98.2 97.7 90.9 96.1 96.1 98.8 96.3 95.8 96.8 99.4 92 8 weeks 96.6 99 94.6 86.4 94.3 96 96.6 96.9 96.4 95.2 100.2 90.9 12 weeks 94.9 94.2 98.7 92.3 97 95.7 98.1 94.2 95.9 93.9 101.7 88.9 Result Stable Stable Stable Stable Stable Stable Stable Stable Stable Stable Stable stable

(60) As a result, as shown in Table 7 above, in all the solubilized formulation, the essence formulation and the cream formulation, the active ingredient gallic acid of the lyophilisate of the Nelumbo nucifera callus extract having an increased gallic acid content was highly stable under high-temperature and sunlight conditions.

(61) 2) Evaluation of Formulation Stability of Cosmetic Composition Containing Nelumbo nucifera Callus Extract Having Increased Gallic Acid Content

(62) To evaluate formulation stability, the solubilized formulation, essence formulation and cream formulation of Formulation Examples 1 to 3 were stored under conditions of 4 C., 30 C., 45 C. and sunlight for 12 weeks. Then, changes in the color, odor and formulation were observed by visual evaluation and sensory evaluation and the results are shown in Tables 8 to 10 below. In this regard, the changes in the color, odor and formulation were evaluated according to the following criteria.

Evaluation Criteria

(63) No change: ;

(64) Slight change: ;

(65) Significant change: X.

(66) TABLE-US-00008 TABLE 8 Solubilized formulation Elapsed Odor change Color change Formulation change time 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight 1 week 2 weeks 4 weeks 8 weeks 12 weeks

(67) TABLE-US-00009 TABLE 9 Essence formulation Elapsed Odor change Color change Formulation change time 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight 1 week 2 weeks 4 weeks 8 weeks 12 weeks

(68) TABLE-US-00010 TABLE 10 Cream formulation Elapsed Odor change Color change Formulation change time 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight 4 C. 30 C. 45 C. Sunlight 1 week 2 weeks 4 weeks 8 weeks 12 weeks

(69) As a result, as shown in Tables 8 to 10 above, the solubilized formulation, the essence formulation and the cream formulation were all highly stable under high-temperature and sunlight conditions.

Experimental Example 6: Evaluation of Skin Irritation of Cosmetic Composition Containing Nelumbo nucifera Callus Extract Having Increased Gallic Acid Content

(70) In order to evaluate the skin irritation of a cosmetic composition containing the Nelumbo nucifera Callus extract having an increased gallic acid content, the skin irritation of the cream formulation of Formulation Example 3 was evaluated.

(71) Specifically, a predetermined amount (20 l) of the cream formulation (prepared in Formulation Example 3) containing the lyophilisate of the Nelumbo nucifera callus extract having an increased gallic acid content was applied to a 520 cm area on the back of each of 30 adult persons for 24 hours, and the removed. After 1 and 24 hours, changes in the skin condition were visually observed, and the results are shown in Table 11 below. In this regard, the changes in the skin condition were evaluated according to the following criteria.

Criteria for Evaluation of Skin Condition

(72) 0: no change;

(73) 1: very slight change;

(74) 2: slight change;

(75) 3: slightly serious change;

(76) 4: serious change;

(77) 5: very serious change.

(78) TABLE-US-00011 TABLE 11 Subject (sex, Skin condition at various extract contents skin type) Elapsed time 3.2% 6.4% 16% 1 (male, 1 hour 0 0 0 neutral) 24 hours 0 0 0 2 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 3 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 4 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 5 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 6 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 7(male, 1 hour 0 0 0 neutral) 24 hours 0 0 0 8 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 9 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 10 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 11 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 12 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 13 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 14 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 15 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 1 16 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 17 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 18 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 19 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 20 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 21 (male, 1 hour 0 0 0 neutral) 24 hours 0 0 0 22 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 23 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 1 24 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 25 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 26 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 27 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 28 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 29 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 0 30 (female, 1 hour 0 0 0 neutral) 24 hours 0 0 1

(79) As a result, as shown in Table 11 above, the cream formulation containing the lyophilisate of the Nelumbo nucifera callus extract having an increased gallic acid content also had no skin irritation.