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In vitro skin permeation and cellular protective effects of flavonoids isolated from Suaeda asparagoides extracts
Journal of Industrial and Engineering Chemistry 18 (2012) 680–683
Contents lists available at SciVerse ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
In vitro skin permeation and cellular protective effects of flavonoids isolated from Suaeda asparagoides extracts Soo Nam Park *, Sun Young Kim, Gyu Nam Lim, Na Rae Jo, Min Hye Lee Department of Fine Chemistry, College of Nature and Life Science, Seoul National University of Science and Technology, 232, Gongreung-ro, Nowon-gu, Seoul 139-743, Republic of Korea
A R T I C L E I N F O
Article history: Received 3 May 2011 Accepted 29 June 2011 Available online 20 November 2011 Keywords: Suaeda asparagoides Kaempferol Astragalin Cellular protective effect
A B S T R A C T
The objective of this study was to compare astragalin (kaempferol-3-O-glucoside) isolated from Suaeda asparagoides extract with kaempferol, an aglycone of astragalin showing the anti-oxidative activity, the cellular protective effect, and skin permeability. Kaempferol was superior to (+)-a-tocopherol and Lascorbic acid known as strong antioxidants in the antioxidative activity by DPPH method and chemiluminescence assay. Astragalin and kaempferol exhibited more prominent cellular protective effect than the lipid peroxidation chain blocker, (+)-a-tocopherol. In this in vitro permeation experiment of astragalin and kaempferol through the ICR mouse skin, kaempferol permeated deeper into the skin than astragalin. These results indicate that kaempferol and astragalin can function as antioxidants in biological systems, particularly skin exposed to solar radiation, and protect cellular membranes against reactive oxygen species. ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
1. Introduction The skin is sensitive to stress by various environment factors (UV, pollution, oxidants and so on). The most important factor of oxidative stress is exposure of skin to UVA or UVB, which produces reactive oxygen species (ROS). ROS includes singlet oxygen (1O2), superoxide anion radical (O2 ), hydroxyl radical (OH) and hydrogen peroxide (H2O2). These can be produced significantly in cells by a variety of processes including high energy irradiation, photosensitization, phagocytosis, and several enzymatic reactions [1]. Excessive production of ROS in skin may accelerate skin aging by inducing mutations, inflammation, and degradation of collagen or elastin, carcinogenesis, and protein denaturation [2–5]. ROS including 1O2 leads expression of matrix metalloproteinase and UVA-induced 1O2 can mediate synthesis of MMP-1 (collagenase) in human skin fibroblast [6–8]. Therefore it is obvious that reduction of ROS by antioxidant can prevent [9–11] and minimize photoaging [12,13]. Several lines of evidence support the view that 1O2, among the reactive oxygen species, has the greatest significance in the cells which have endogenous photosensitizable molecules, and frequent opportunities of being exposed to a variety of xenobiotics. Tyrrell and Pidoux proposed that 1O2 but not H2O2 or OH, plays an important role in the inactivation of cultured human cells by UV-A
[14]. Foote reviewed that protein damage in UV-exposed skin is the result of 1O2-mediated polymerization and the photocarcinogenesis has been attributed to the action of 1O2 [15]. Flavonoids widely used as therapeutic agents are known to act as strong scavengers of the reactive oxygen species, and react with peroxyradicals involving termination of radical chain reactions during the auto-oxidation of polyunsaturated fatty acids [16–18]. Kaempferol is a natural flavonol, a type of flavonoid, that has been isolated from tea [19], broccoli, grapefruit, brussels sprouts, apples, and other plants. It has been reported to have antioxidative activity, anti-mutation, and protective activity against cancer [20–22]. Anti-oxidative activities of extract/fractions of Suaeda asparagoides and the stability of the cream containing S. asparagoides extract were previously investigated [23,24]. A principal component of ethyl acetate fraction of S. asparagoides extract was identified as kaempferol-3-O-glucoside (astragalin) [25]. In this study, astragalin isolated from S. asparagoides extracts was compared with kaempferol, an aglycone of astragalin showing the anti-oxidative activity, the cellular protective effect, and the skin permeability. 2. Experimental 2.1. Plant material
* Corresponding author. E-mail address: [email protected] (S.N. Park).
The air-dried S. asparagoides used in the experiment was purchased from the Kyungdong local market (Seoul, South Korea).
1226-086X/$ – see front matter ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jiec.2011.11.126
S.N. Park et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 680–683
Fig. 1. Chemical structures of the kaempferol and kaemferol-3-O-glucoside (astragalin).
2.2. Extraction and isolation The air-dried S. asparagoides (500 g) was extracted with 50% ethanol (5 L). In order to remove the non-polar components, 50% ethanol extract was fractionated with n-hexane and ethyl acetate and then was dried. The ethyl acetate fraction was dissolved in 100% ethanol and was chromatographed using the elution solvent composed of ethyl acetate/acetic acid/formic acid/distilled water (10:1:1:1) in volume ratio. TLC chromatogram of the ethyl acetate fraction of S. asparagoides extract revealed 5 bands. The band of astragalin (Rf value, 0.86) was extracted with 50% ethanol, and then dried and dissolved in 100% ethanol. Astragalin was processed by the acid hydrolysis method to obtain kaempferol. Astragalin was added 5% H2SO4 solution, and then heated on a boiling water bath under a reflux condenser for 4 h. Refluxed solution was neutralized with 5% KOH in methanol and washed with distilled water and fractionated with ethyl acetate and then dried. Obtained kaempferol powder was dissolved in 100% ethanol for use in the experiment. The chemical structures of kaempferol and astragalin were shown in Fig. 1. 2.3. DPPH free radical scavenging assay The free radical scavenging activity was determined by the modified method [26] using 1,1-diphenyl-2-picrylhydrazyl (DPPH). 1.0 mL of ethanol sample solution was added to 1.0 mL of 0.2 mM DPPH methanol solution, and allowed to stand for 10 min at 25 8C. The amount of free radicals was measured by absorbance at 517 nm. The free radical scavenging activity (FSC50) was defined as the concentration of the sample required for 50% of the free radicals to be scavenged. 2.4. Luminol-dependent chemiluminescence assay The ROS scavenging activity was examined in Fe3+-EDTA/H2O2 system by the luminol-dependent chemiluminescence assay. The reaction mixture consisted of distilled water 1.78 mL, sample solution 50 mL, 2.5 mM EDTA 40 mL, 5 mM FeCl36H2O 10 mL and 35 mM luminol 80 mL. After incubation at 35 8C for 5 min, 150 mM H2O2 40 mL was added to reaction mixture and incubated at 35 8C for 25 min. The chemiluminescence was measured by a luminescence spectrometer LB9505. The ROS scavenging activity (OSC50) was defined as the concentration of the sample required for 50% of ROS to be scavenged. 2.5. Photohemolysis assay (for cellular membrane protective effect against ROS) 2.5.1. Preparation of erythrocytes The blood collected from a human was centrifuged at 3000 rpm for 5 min to separate erythrocytes and serum, then washed and diluted with 0.9% saline phosphate buffer, pH 7.4 to prepare the erythrocyte suspension (15.0 million cells/mL). The erythrocyte was used either immediately or within a few hours, while stored at 4 8C in a refrigerator.
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2.5.2. Measurement of photohemolysis The reaction mixture for photohemolysis determination consisted of a test group containing 3.5 mL of erythrocyte suspension, 50 mL of ethanol sample solution and a control group containing 3.5 mL of erythrocyte suspension, 50 mL of ethanol. After the mixture was pre-incubated for 30 min in the dark room, 0.5 mL of 12 mM rose-bengal as a photosensitizer was added per tube, and then sealed with parafilm. Then the tubes placed in 50 20 25 rectangular hexahedron box of which the inside had been painted black were irradiated for 15 min by a 20 W fluorescent lamp at a distance of 5 cm. The degree of hemolysis of the erythrocytes was measured by % transmittance at 700 nm at intervals of 15 min. Every step of the above experiment was carried out at room temperature. The activity of the sample in protecting the cells against active oxygen species was defined as half-time of hemolysis (t50), the time required for 50% of the erythrocytes to be hemolysed. 2.6. In vitro skin permeation study The in vitro skin permeation study for kaempferol and astragalin was carried out using Franz-type diffusion cells [27–29]. 2.6.1. Skin preparation Full-thickness skin was removed from the dorsal side of a freshly excised ICR albino mouse (8 weeks, female). Subcutaneous fat and bristles were carefully removed from the skin, which was then stored at 70 8C. 2.6.2. Permeation experiments 3.0 mM Astragalin and kaempferol were prepared in stock solution (1,3-butylene glycol:ethanol = 4:1), respectively. A specimen of the skin was sandwiched between the 2 halves of the Franz cell, with the stratum corneum side facing the donor compartment. The receptor compartment has a volume of 5 mL and was filled with phosphate buffered saline (PBS). It was kept at 37 8C by circulating water. 0.2 mL of the sample stock solution was applied in the donor compartment and then the donor compartment was covered with parafilm to prevent evaporation of the solvent. The receptor solution was continuously stirred by means of a spinning bar magnet. Receptor solution samples, 0.5 mL aliquots, were withdrawn through the sampling port of the receptor compartment at intervals of 4 h. The receptor compartment was refilled with PBS solution to keep the volume of receptor solution constant during the experiment. The experiments were run for 24 h. And at the end of the experiment (24 h), the 0.5 mL of each receptor solution samples withdrawn at intervals of 4 h were concentrated and dissolved in 100% ethanol and then analyzed for astragalin and kaempferol content (Fig. 4, Transdermal) using HPLC (Shimadzu, Japan) equipped with a UV detector and computer integrating apparatus. The column was a Shim-pack VP-ODS C18 (250 mm 4.6 mm). Transdermal flux results were expressed as mg cm 2 h 1. The skin surface was washed with 3 mL PBS solution on each side to remove the residual donor sample. The stratum corneum was removed by the stripping method using 3 strips of 3 M scotch tape (Korea 3M) [30]. The tape strips were dissolved in 100% ethanol at 70 8C and analyzed for flavonoids content (Fig. 4, Tape) using HPLC. The skin was cut into small pieces and dissolved in 100% ethanol at 70 8C and analyzed for flavonoids content (Fig. 4, Skin) using HPLC. 3. Results and discussion 3.1. Antioxidative activities of kaempferol and astragalin The free radical scavenging activities (FSC50) of astragalin (FSC50, 408.00 mM), kaempferol (FSC50, 6.45 mM), and references
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S.N. Park et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 680–683
Table 1 Free radical scavenging activities of astragalin, kaempferol, and reference compounds. Compound Flavonoid Astragalin (kaempferol-3-O-glucoside) Kaempferol Reference (+)-a-Tocopherol L-Ascorbic acid
Free radical scavenging activity (FSC50, mM) 408.00 6.45 13.40 8.40
Table 2 Reactive oxygen species (ROS) scavenging activities of astragalin, kaempferol, and reference compounds by luminol-dependent chemiluminescence assay. Compound Flavonoid Astragalin (kaempferol-3-O-glucoside) Kaempferol Reference (+)-a-Tocopherol L-Ascorbic acid
ROS scavenging activity (OSC50, mM) 8.55 0.38 9.00 5.20
were shown in Table 1. Particularly, the free radical scavenging activity of kaempferol was comparable to that of L-ascorbic acid (FSC50, 8.4 mM) known as a strong antioxidant. The ROS scavenging activities (OSC50) of astragalin and kaempferol were 8.55 mM and 0.38 mM, respectively. Kaempferol was superior to (+)-a-tocopherol (OSC50, 9.00 mM) and L-ascorbic acid (OSC50, 5.20 mM) in the ROS scavenging activity (Table 2).
Table 3 Cellular protective effect of astragalin, kaempferol, and reference compounds on the rose-bengal sensitized photohemolysis of human erythrocytes. Concentration (mM)
Flavonoid Astragalin (kaempferol-3-O-glucoside) Kaempferol Reference (+)-a-Tocopherol L-Ascorbic acid
t50 (Half-time of hemolysis) 1
5
25
50
46.2 46.9
55.2 66.6
78.2 301.0
90.3 435.0
– –
– –
40.0 –
58.0 32.0
control = 32 min) than a lipid peroxidation chain blocker, (+)-atocopherol (t50, 58.0 min). A flavonoid glycoside, astragalin (t50, 90.3 min) isolated from S. asparagoides extract also showed a good protective effect (Table 3). In the same concentrations (5, 25, and 50 mM), kaempferol exhibited better cellular protective effect than astragalin. These results indicate that kaempferol is more efficient than astragalin at approaching the cell membrane, inhibiting lipid peroxidation, and suppressing damage of cellular membrane induced by ROS. Astragalin is a glycoside and because of its glucose, a water shell is formed around it. Thus, astragalin has difficulty approaching the cell membrane. 3.3. Skin permeation study
The cellular membrane protective effects of astragalin, kaempferol, and references were investigated. The protective effect of kaempferol on the rose-bengal sensitized photohemolysis of human erythrocytes was shown in Fig. 2. Kaempferol and astragalin reduced 1O2-induced photohemolysis in a concentration dependent manner (1–50 mM). Particularly, kaempferol exhibited better cellular protective effects (t50, 435.0 min at 50 mM,
In vitro permeability study of astragalin and kaempferol through ICR mouse skin was carried out using Franz diffusion cells. The in vitro permeation profiles of astragalin and kaempferol for 24 h were shown in Fig. 3. The cumulative degrees of permeation increased linearly throughout the experimental period with the slope of kaempferol much steeper than astragalin. Kaempferol showed greater cumulative permeation at 24 h than astragalin. Fig. 4 showed the results of permeation after 24 h in Franz diffusion cell. The amount of kaempferol in the receptor chamber was higher than that of astragalin. 30% of astragalin remained into the skin (dermis plus epidermis without stratum corneum). These results indicate that the differences in permeation between astragalin and kaempferol were solely due to their
Fig. 2. Cellular protective effect of kaempferol on the rose-bengal sensitized photohemolysis of human erythrocytes. Concentration of kaempferol; X–X: control, ~–~: 1 mM, &–&: 5 mM, ^–^: 25 mM, *–*: 50 mM.
Fig. 3. In vitro skin permeation profiles of kaempferol and astragalin through ICR albino mouse skin.
3.2. Cellular membrane protective effects of astragalin and kaempferol
S.N. Park et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 680–683
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Acknowledgement This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A103017). References
Fig. 4. Proportions of permeated amount of kaempferol and astragalin through ICR albino mouse skin after 24 h incubation.
molecular structure. A flavonoid glycoside, astragalin, has a higher molecular weight and polarity than kaempferol. 4. Conclusions In this study, the anti-oxidative activity, the cellular protective effect, and the skin permeability of astragalin isolated from S. asparagoides extract and kaempferol, an aglycone of astragalin, were investigated. As a result, kaempferol was superior to (+)-a-tocopherol and L-ascorbic acid in the anti-oxidative activity. Astragalin and kaempferol exhibited better cellular protective effect than (+)-a-tocopherol. In this in vitro permeation experiment of astragalin and kaempferol, kaempferol permeated deeper into the skin than astragalin. These results indicate that kaempferol and astragalin can be applicable to new functional cosmetics for antioxidant and a nti-aging.
[1] C.S. Foote, Photosensitized Oxidation and Singlet Oxygen, 2nd ed., Academic press, New York, 1976. [2] S.N. Park, Ph.D. Thesis, Seoul National University, Seoul, Korea, 1989. [3] S.N. Park, J. Korean Cosmet. Sci. 23 (1997) 75. [4] S.N. Park, Korean J. Food Sci. Technol. 35 (2003) 510. [5] S.N. Park, J. Korean Ind. Eng. Chem. 14 (2003) 657. [6] G.F. Vile, R.M. Tyrrell, Free Radic. Biol. Med. 18 (1995) 721. [7] K. Scharffetter-Kochanek, M. Wlaschek, K. Briviba, H. Sies, FEBS Lett. 331 (1993) 304. [8] M. Wlaschek, K. Briviba, G.P. Stricklin, H. Sies, K.K. Scharffetter-Kochanek, J. Invest. Dermatol. 104 (1995) 194. [9] A. Oikarinen, M. Kallioinen, Photodermatology 6 (1989) 24. [10] L.H. Kligman, UVA-induced biochemical changes in hairless mouse skin collagen: a contrast to F U.V.B. effects, in: F. Urbach (Ed.), Biologic Responses to UVA Radiation, Valdenmar, Overland, 1992, pp. 209–215. [11] J.W. Choi, S.I. Kim, J.Y. Kim, H.J. Yang, K.H. Lee, S.N. Park, J. Korean Cosmet. Sci. 32 (2006) 181. [12] H.J. Yang, S.N. Park, J. Korean Cosmet. Sci. 33 (2007) 61. [13] J.Y. Kim, S.N. Park, J. Korean Cosmet. Sci. 34 (2008) 83. [14] R.M. Tyrrell, M. Pidoux, Photochem. Photobiol. 49 (1989) 407. [15] C.S. Foote, Photosensitized Oxidation and Singlet Oxygen: Consequences in Biological Systems in Free Radical in Biology, 2nd ed., Academic press, New York, 1976. [16] B. Havsteen, Plant Cell Physiol. 28 (1983) 1141. [17] S.R. Husain, J. Gillard, P. Cillard, Phytochemistry 26 (1987) 2489. [18] J. Torel, J. Cillard, P. Cillard, Phytochemistry 25 (1986) 383. [19] J.S. Park, H.S. Rho, D.H. Kim, I.S. Chang, J. Agric. Food Chem. 54 (2006) 2951. [20] R. Singh, B. Singh, S. Singh, N. Kumar, S. Kumar, S. Arora, Toxicol. In Vitro 22 (2008) 1965. [21] H. Luo, G.O. Rankin, Z. Li, L.D. Priest, Y.C. Chen, Food Chem. 218 (2011) 513. [22] I. Mylonis, A. Lakka, A. Tsakalof, G. Simos, Biochem. Biophys. Res. Commun. 398 (2010) 74. [23] S.M. Jeon, S.I. Kim, J.Y. Ahn, S.N. Park, J. Korean Cosmet. Sci. 33 (2007) 145. [24] S.M. Jeon, J.Y. Ahn, S.N. Park, J. Korean Cosmet. Sci. 33 (2007) 231. [25] H.J. Yang, S.N. Park, J. Korean Cosmet. Sci. 34 (2007) 157. [26] Y. Fujita, L. Uehara, Y. Morimoto, M. Nakashima, T. Hatano, T. Okuda, Yakugaku Zasshi 108 (1988) 129. [27] Y.H. Tsai, K.F. Lee, Y.B. Huang, C.T. Huang, P.C. Wu, Int. J. Pharm. 388 (2010) 257. [28] C.M. Heard, S. Johnson, G. Moss, C.P. Thomas, Int. J. Pharm. 317 (2006) 26. [29] R.J. Batchelder, R.J. Calder, C.P. Thomas, C.M. Heard, Int. J. Pharm. 283 (2004) 45. [30] W.G. Cho, J. Korean Oil Chem. Soc. 24 (2007) 410.
Contents lists available at SciVerse ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
In vitro skin permeation and cellular protective effects of flavonoids isolated from Suaeda asparagoides extracts Soo Nam Park *, Sun Young Kim, Gyu Nam Lim, Na Rae Jo, Min Hye Lee Department of Fine Chemistry, College of Nature and Life Science, Seoul National University of Science and Technology, 232, Gongreung-ro, Nowon-gu, Seoul 139-743, Republic of Korea
A R T I C L E I N F O
Article history: Received 3 May 2011 Accepted 29 June 2011 Available online 20 November 2011 Keywords: Suaeda asparagoides Kaempferol Astragalin Cellular protective effect
A B S T R A C T
The objective of this study was to compare astragalin (kaempferol-3-O-glucoside) isolated from Suaeda asparagoides extract with kaempferol, an aglycone of astragalin showing the anti-oxidative activity, the cellular protective effect, and skin permeability. Kaempferol was superior to (+)-a-tocopherol and Lascorbic acid known as strong antioxidants in the antioxidative activity by DPPH method and chemiluminescence assay. Astragalin and kaempferol exhibited more prominent cellular protective effect than the lipid peroxidation chain blocker, (+)-a-tocopherol. In this in vitro permeation experiment of astragalin and kaempferol through the ICR mouse skin, kaempferol permeated deeper into the skin than astragalin. These results indicate that kaempferol and astragalin can function as antioxidants in biological systems, particularly skin exposed to solar radiation, and protect cellular membranes against reactive oxygen species. ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
1. Introduction The skin is sensitive to stress by various environment factors (UV, pollution, oxidants and so on). The most important factor of oxidative stress is exposure of skin to UVA or UVB, which produces reactive oxygen species (ROS). ROS includes singlet oxygen (1O2), superoxide anion radical (O2 ), hydroxyl radical (OH) and hydrogen peroxide (H2O2). These can be produced significantly in cells by a variety of processes including high energy irradiation, photosensitization, phagocytosis, and several enzymatic reactions [1]. Excessive production of ROS in skin may accelerate skin aging by inducing mutations, inflammation, and degradation of collagen or elastin, carcinogenesis, and protein denaturation [2–5]. ROS including 1O2 leads expression of matrix metalloproteinase and UVA-induced 1O2 can mediate synthesis of MMP-1 (collagenase) in human skin fibroblast [6–8]. Therefore it is obvious that reduction of ROS by antioxidant can prevent [9–11] and minimize photoaging [12,13]. Several lines of evidence support the view that 1O2, among the reactive oxygen species, has the greatest significance in the cells which have endogenous photosensitizable molecules, and frequent opportunities of being exposed to a variety of xenobiotics. Tyrrell and Pidoux proposed that 1O2 but not H2O2 or OH, plays an important role in the inactivation of cultured human cells by UV-A
[14]. Foote reviewed that protein damage in UV-exposed skin is the result of 1O2-mediated polymerization and the photocarcinogenesis has been attributed to the action of 1O2 [15]. Flavonoids widely used as therapeutic agents are known to act as strong scavengers of the reactive oxygen species, and react with peroxyradicals involving termination of radical chain reactions during the auto-oxidation of polyunsaturated fatty acids [16–18]. Kaempferol is a natural flavonol, a type of flavonoid, that has been isolated from tea [19], broccoli, grapefruit, brussels sprouts, apples, and other plants. It has been reported to have antioxidative activity, anti-mutation, and protective activity against cancer [20–22]. Anti-oxidative activities of extract/fractions of Suaeda asparagoides and the stability of the cream containing S. asparagoides extract were previously investigated [23,24]. A principal component of ethyl acetate fraction of S. asparagoides extract was identified as kaempferol-3-O-glucoside (astragalin) [25]. In this study, astragalin isolated from S. asparagoides extracts was compared with kaempferol, an aglycone of astragalin showing the anti-oxidative activity, the cellular protective effect, and the skin permeability. 2. Experimental 2.1. Plant material
* Corresponding author. E-mail address: [email protected] (S.N. Park).
The air-dried S. asparagoides used in the experiment was purchased from the Kyungdong local market (Seoul, South Korea).
1226-086X/$ – see front matter ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jiec.2011.11.126
S.N. Park et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 680–683
Fig. 1. Chemical structures of the kaempferol and kaemferol-3-O-glucoside (astragalin).
2.2. Extraction and isolation The air-dried S. asparagoides (500 g) was extracted with 50% ethanol (5 L). In order to remove the non-polar components, 50% ethanol extract was fractionated with n-hexane and ethyl acetate and then was dried. The ethyl acetate fraction was dissolved in 100% ethanol and was chromatographed using the elution solvent composed of ethyl acetate/acetic acid/formic acid/distilled water (10:1:1:1) in volume ratio. TLC chromatogram of the ethyl acetate fraction of S. asparagoides extract revealed 5 bands. The band of astragalin (Rf value, 0.86) was extracted with 50% ethanol, and then dried and dissolved in 100% ethanol. Astragalin was processed by the acid hydrolysis method to obtain kaempferol. Astragalin was added 5% H2SO4 solution, and then heated on a boiling water bath under a reflux condenser for 4 h. Refluxed solution was neutralized with 5% KOH in methanol and washed with distilled water and fractionated with ethyl acetate and then dried. Obtained kaempferol powder was dissolved in 100% ethanol for use in the experiment. The chemical structures of kaempferol and astragalin were shown in Fig. 1. 2.3. DPPH free radical scavenging assay The free radical scavenging activity was determined by the modified method [26] using 1,1-diphenyl-2-picrylhydrazyl (DPPH). 1.0 mL of ethanol sample solution was added to 1.0 mL of 0.2 mM DPPH methanol solution, and allowed to stand for 10 min at 25 8C. The amount of free radicals was measured by absorbance at 517 nm. The free radical scavenging activity (FSC50) was defined as the concentration of the sample required for 50% of the free radicals to be scavenged. 2.4. Luminol-dependent chemiluminescence assay The ROS scavenging activity was examined in Fe3+-EDTA/H2O2 system by the luminol-dependent chemiluminescence assay. The reaction mixture consisted of distilled water 1.78 mL, sample solution 50 mL, 2.5 mM EDTA 40 mL, 5 mM FeCl36H2O 10 mL and 35 mM luminol 80 mL. After incubation at 35 8C for 5 min, 150 mM H2O2 40 mL was added to reaction mixture and incubated at 35 8C for 25 min. The chemiluminescence was measured by a luminescence spectrometer LB9505. The ROS scavenging activity (OSC50) was defined as the concentration of the sample required for 50% of ROS to be scavenged. 2.5. Photohemolysis assay (for cellular membrane protective effect against ROS) 2.5.1. Preparation of erythrocytes The blood collected from a human was centrifuged at 3000 rpm for 5 min to separate erythrocytes and serum, then washed and diluted with 0.9% saline phosphate buffer, pH 7.4 to prepare the erythrocyte suspension (15.0 million cells/mL). The erythrocyte was used either immediately or within a few hours, while stored at 4 8C in a refrigerator.
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2.5.2. Measurement of photohemolysis The reaction mixture for photohemolysis determination consisted of a test group containing 3.5 mL of erythrocyte suspension, 50 mL of ethanol sample solution and a control group containing 3.5 mL of erythrocyte suspension, 50 mL of ethanol. After the mixture was pre-incubated for 30 min in the dark room, 0.5 mL of 12 mM rose-bengal as a photosensitizer was added per tube, and then sealed with parafilm. Then the tubes placed in 50 20 25 rectangular hexahedron box of which the inside had been painted black were irradiated for 15 min by a 20 W fluorescent lamp at a distance of 5 cm. The degree of hemolysis of the erythrocytes was measured by % transmittance at 700 nm at intervals of 15 min. Every step of the above experiment was carried out at room temperature. The activity of the sample in protecting the cells against active oxygen species was defined as half-time of hemolysis (t50), the time required for 50% of the erythrocytes to be hemolysed. 2.6. In vitro skin permeation study The in vitro skin permeation study for kaempferol and astragalin was carried out using Franz-type diffusion cells [27–29]. 2.6.1. Skin preparation Full-thickness skin was removed from the dorsal side of a freshly excised ICR albino mouse (8 weeks, female). Subcutaneous fat and bristles were carefully removed from the skin, which was then stored at 70 8C. 2.6.2. Permeation experiments 3.0 mM Astragalin and kaempferol were prepared in stock solution (1,3-butylene glycol:ethanol = 4:1), respectively. A specimen of the skin was sandwiched between the 2 halves of the Franz cell, with the stratum corneum side facing the donor compartment. The receptor compartment has a volume of 5 mL and was filled with phosphate buffered saline (PBS). It was kept at 37 8C by circulating water. 0.2 mL of the sample stock solution was applied in the donor compartment and then the donor compartment was covered with parafilm to prevent evaporation of the solvent. The receptor solution was continuously stirred by means of a spinning bar magnet. Receptor solution samples, 0.5 mL aliquots, were withdrawn through the sampling port of the receptor compartment at intervals of 4 h. The receptor compartment was refilled with PBS solution to keep the volume of receptor solution constant during the experiment. The experiments were run for 24 h. And at the end of the experiment (24 h), the 0.5 mL of each receptor solution samples withdrawn at intervals of 4 h were concentrated and dissolved in 100% ethanol and then analyzed for astragalin and kaempferol content (Fig. 4, Transdermal) using HPLC (Shimadzu, Japan) equipped with a UV detector and computer integrating apparatus. The column was a Shim-pack VP-ODS C18 (250 mm 4.6 mm). Transdermal flux results were expressed as mg cm 2 h 1. The skin surface was washed with 3 mL PBS solution on each side to remove the residual donor sample. The stratum corneum was removed by the stripping method using 3 strips of 3 M scotch tape (Korea 3M) [30]. The tape strips were dissolved in 100% ethanol at 70 8C and analyzed for flavonoids content (Fig. 4, Tape) using HPLC. The skin was cut into small pieces and dissolved in 100% ethanol at 70 8C and analyzed for flavonoids content (Fig. 4, Skin) using HPLC. 3. Results and discussion 3.1. Antioxidative activities of kaempferol and astragalin The free radical scavenging activities (FSC50) of astragalin (FSC50, 408.00 mM), kaempferol (FSC50, 6.45 mM), and references
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S.N. Park et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 680–683
Table 1 Free radical scavenging activities of astragalin, kaempferol, and reference compounds. Compound Flavonoid Astragalin (kaempferol-3-O-glucoside) Kaempferol Reference (+)-a-Tocopherol L-Ascorbic acid
Free radical scavenging activity (FSC50, mM) 408.00 6.45 13.40 8.40
Table 2 Reactive oxygen species (ROS) scavenging activities of astragalin, kaempferol, and reference compounds by luminol-dependent chemiluminescence assay. Compound Flavonoid Astragalin (kaempferol-3-O-glucoside) Kaempferol Reference (+)-a-Tocopherol L-Ascorbic acid
ROS scavenging activity (OSC50, mM) 8.55 0.38 9.00 5.20
were shown in Table 1. Particularly, the free radical scavenging activity of kaempferol was comparable to that of L-ascorbic acid (FSC50, 8.4 mM) known as a strong antioxidant. The ROS scavenging activities (OSC50) of astragalin and kaempferol were 8.55 mM and 0.38 mM, respectively. Kaempferol was superior to (+)-a-tocopherol (OSC50, 9.00 mM) and L-ascorbic acid (OSC50, 5.20 mM) in the ROS scavenging activity (Table 2).
Table 3 Cellular protective effect of astragalin, kaempferol, and reference compounds on the rose-bengal sensitized photohemolysis of human erythrocytes. Concentration (mM)
Flavonoid Astragalin (kaempferol-3-O-glucoside) Kaempferol Reference (+)-a-Tocopherol L-Ascorbic acid
t50 (Half-time of hemolysis) 1
5
25
50
46.2 46.9
55.2 66.6
78.2 301.0
90.3 435.0
– –
– –
40.0 –
58.0 32.0
control = 32 min) than a lipid peroxidation chain blocker, (+)-atocopherol (t50, 58.0 min). A flavonoid glycoside, astragalin (t50, 90.3 min) isolated from S. asparagoides extract also showed a good protective effect (Table 3). In the same concentrations (5, 25, and 50 mM), kaempferol exhibited better cellular protective effect than astragalin. These results indicate that kaempferol is more efficient than astragalin at approaching the cell membrane, inhibiting lipid peroxidation, and suppressing damage of cellular membrane induced by ROS. Astragalin is a glycoside and because of its glucose, a water shell is formed around it. Thus, astragalin has difficulty approaching the cell membrane. 3.3. Skin permeation study
The cellular membrane protective effects of astragalin, kaempferol, and references were investigated. The protective effect of kaempferol on the rose-bengal sensitized photohemolysis of human erythrocytes was shown in Fig. 2. Kaempferol and astragalin reduced 1O2-induced photohemolysis in a concentration dependent manner (1–50 mM). Particularly, kaempferol exhibited better cellular protective effects (t50, 435.0 min at 50 mM,
In vitro permeability study of astragalin and kaempferol through ICR mouse skin was carried out using Franz diffusion cells. The in vitro permeation profiles of astragalin and kaempferol for 24 h were shown in Fig. 3. The cumulative degrees of permeation increased linearly throughout the experimental period with the slope of kaempferol much steeper than astragalin. Kaempferol showed greater cumulative permeation at 24 h than astragalin. Fig. 4 showed the results of permeation after 24 h in Franz diffusion cell. The amount of kaempferol in the receptor chamber was higher than that of astragalin. 30% of astragalin remained into the skin (dermis plus epidermis without stratum corneum). These results indicate that the differences in permeation between astragalin and kaempferol were solely due to their
Fig. 2. Cellular protective effect of kaempferol on the rose-bengal sensitized photohemolysis of human erythrocytes. Concentration of kaempferol; X–X: control, ~–~: 1 mM, &–&: 5 mM, ^–^: 25 mM, *–*: 50 mM.
Fig. 3. In vitro skin permeation profiles of kaempferol and astragalin through ICR albino mouse skin.
3.2. Cellular membrane protective effects of astragalin and kaempferol
S.N. Park et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 680–683
683
Acknowledgement This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A103017). References
Fig. 4. Proportions of permeated amount of kaempferol and astragalin through ICR albino mouse skin after 24 h incubation.
molecular structure. A flavonoid glycoside, astragalin, has a higher molecular weight and polarity than kaempferol. 4. Conclusions In this study, the anti-oxidative activity, the cellular protective effect, and the skin permeability of astragalin isolated from S. asparagoides extract and kaempferol, an aglycone of astragalin, were investigated. As a result, kaempferol was superior to (+)-a-tocopherol and L-ascorbic acid in the anti-oxidative activity. Astragalin and kaempferol exhibited better cellular protective effect than (+)-a-tocopherol. In this in vitro permeation experiment of astragalin and kaempferol, kaempferol permeated deeper into the skin than astragalin. These results indicate that kaempferol and astragalin can be applicable to new functional cosmetics for antioxidant and a nti-aging.
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