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Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage
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Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage Keon Soo Lee, Soo Nam Park* Department of Fine Chemistry, Cosmetic R&D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 01811, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 July 2018 Received in revised form 17 October 2018 Accepted 10 November 2018 Available online xxx
The purpose of this study was to compare the anti-oxidative and cytoprotective effects of quercitrin and avicularin isolated from Lespedeza cuneata G. Don extract to those of quercetin, an aglycone of quercitrin, and avicularin. Quercetin had higher antioxidative activity and cell penetration ratio than its glycosides, resulting in greater cytoprotective effects against 1O2. The cytoprotective effects against cell damage seems to reflect 1O2 quenching rate, free radical and ROS scavenging activity, and cell permeability. Among them, cell permeability to block free radical initiation and chain reactions in cell membranes is considered to be the most important feature of cytoprotective activity. © 2018 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.
Keywords: Cytoprotective effect Quercetin Quercitrin Avicularin Antioxidant mechanism
Introduction With increased interest in skin aging, development of anti-aging materials and applications in cosmetic products are being continuously pursued. Intrinsically-aged skin appears dry, thin, and has fine wrinkles, and is caused by the passage of time. Alternatively, extrinsic aging is induced by chronic exposure to external environmental factors. Photoaging by UV exposure is an example of extrinsic aging. Exposed skin undergoes oxidative stress by radicals and reactive oxygen species (ROS). This oxidative stress causes oxidative damage of biomolecules such as lipids, proteins and DNA, which are components of skin cells, resulting in skin aging [1–5]. Reactive oxygen species include radical species such as superoxide anion radical (O2 ) and hydroxyl radical ( OH), non1 radical species such as singlet oxygen ( O2) and hydrogen peroxide (H2O2). There are peroxyl radical (ROO ) and alkoxyl radical (RO ) produced by the biological components reacting with the preceding ROS [6–8]. These ROS are also generated by photosensitization reactions with UV radiation. The photosensitization reaction generates various ROS as well as 1O2, which are highly reactive. Photosensitization reactions occur in the skin when exposed to UV light because there are compounds in the skin such as porphyrin and riboflavin that can induce this reaction. The
* Corresponding author. E-mail address: [email protected] (S.N. Park).
photosensitization reaction can be broadly divided into two types, Type I and Type II. Type I reactions produce radicals or radical ions, and the main product of Type II reaction is 1O2, and additionally various ROS are produced. Especially, 1O2 is a highly reactive and is not subject to enzymatic metabolism or scavenging in vivo, which can destroy cell membrane and produce tissue damage importantly [9–11]. The skin utilizes a complementary protection network of enzymatic antioxidants and non-enzymatic antioxidants to prevent damages [12–14]. When reactive oxygen species is continuously generated from the external environment via photosensitization reactions resulting from exposure to UV rays, the antioxidant in the body become depleted and the cells and tissues undergo damage, resulting in aging and adult diseases such as hypertension, atherosclerosis, and diabetes. Therefore, interest in the development of natural antioxidants for the supplementation of antioxidant defense networks that protect skin cells from UV and reactive oxygen species damage has increased. As these antioxidants not only possess hydrophilic and hydrophobic properties but also various antioxidant properties, they can exhibit synergistic antioxidant effects to protect skin cells from oxidative stress. Thus, the development of antioxidant materials and application studies for the cosmetics industry are required [15–19]. Prior to this study, the authors isolated and analyzed components from Lespedeza cuneata G. Don extract and reported that they have antioxidant and antiaging activites [20–22]. The major components of L. cuneata G. Don extracts are quercetin and its glycosides, quercitrin and avicularin. The chemical structure of
https://doi.org/10.1016/j.jiec.2018.11.018 1226-086X/© 2018 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.
Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
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Preparation of erythrocytes suspensions
quercetin, quercitrin, and avicularin was shown in Fig. 1. In previous studies, quercetin showed superior cytoprotective effects against cellular damage induced by photosensitization reactions, compared to its glycosides. However, there are no comparative studies on the cytoprotective effects of quercetin and its glycosides quercitrin and avicularin on cell damage induced by 1O2 using the photosensitization reaction. In this study, various types of antioxidative experiments were performed using three principal components, along with L-ascorbic acid and ()-α-tocopherol, which are well known as antioxidants, as a control group, to investigate the mechanism of cell protection in oxidative stress induced by ROS.
Erythrocytes used in this experiment were immediately added to heparinized test tubes after collection and used within 12 h. Blood was centrifuged at 3000 rpm for 5 min to separate erythrocytes and plasma. The separated erythrocytes were washed three times with 0.9% saline phosphate buffer (pH 7.4, 9.6 mM Na2HPO412H2O, 1.6 mM NaH2PO42H2O) prior to use. The optical density (OD) value of the erythrocyte suspension used in the experiment was 0.6 at 700 nm, and the number of erythrocytes was approximately 1.5 107 cells/mL.
Materials and methods
Measurement of photohemolysis inhibition
Materials
A total of 3.5 mL of erythrocyte suspension was placed in a Pyrex test tube and 50 mL of samples was added. After reaction in a black box for 30 min, 0.5 mL of the photosensitizer rose-bengal (15 mM) was added, and the mixture was irradiated for 15 min. Rose-bengal produces 1O2 in high yields [23]. After irradiation, the degree of destruction of erythrocytes was measured by transmittance (%) at 700 nm in 15 min intervals. At this wavelength, increased transmittance of the erythrocyte suspension is proportional to the degree of erythrocyte hemolysis. The degree of hemolysis was shown by plotting the transmittance versus time to determine t50 values (the time required for 50% hemolysis).
The whole part of L. cuneata G. Don. was purchased from the herbal medicine shop in the Kyungdong market located in Korea. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 1,3-diphenylisobenzofuran (DPBF), H2O2, luminol, rose-bengal were purchased from Sigma Chemical Co. (USA). FeCl36H2O was purchased from Junsei Chemical Co. (Japan). L-Ascorbic acid and ()-α-tocopherol were purchased from Sigma-Aldrich (Korea). Na2HPO412H2O, NaH2PO42H2O, NaCl, trizmabase, HCl, H2SO4, ethanol (EtOH), methanol (MeOH), ethyl acetate (EtOAc), acetic acid, and formic acid were extra pure grade in this study.
Measurement of singlet oxygen quenching rate constants Extraction, isolation and fractionation Dried L. cuneata G. Don (100 g) was finely cut and then extracted by immersion in 4 L of 70% ethanol for 1 week. The 70% ethanol extract was filtered under pressure and dried to obtain a powder. The 70% ethanol extract was treated 3 times with ethyl acetate, which separated out as a fraction that was then concentrated under use of a rotary evaporator to remove the organic solvent (the concentrated form the ethyl acetate fraction of L. cuneata). The ethyl acetate fraction was dissolved in ethanol and separated on TLC plates (Merck, USA) using ethyl acetate: acetic acid: formic acid: distilled water = 8: 1: 1: 1 (v/v) as mobile phase. In previous studies, the bands of avicularin (Rf 0.66) and quercitrin (Rf 0.54), which were previously determined to be the main components of the L. cuneata ethyl acetate fraction, were extracted with ethanol and then dried again [20]. To obtain quercetin, quercitrin was acid hydrolyzed. Quercitrin was added to a 5% H2SO4 solution and refluxed and cooled for 4 h. The reaction solution was neutralized with a 5% KOH–MeOH solution, washed with distilled water, and then partitioned with ethyl acetate and dried. The obtained quercetin was dissolved again in ethanol.
The singlet oxygen quenching rate constant is an indicator of how much 1O2, the product of the Type II reaction, is extinguished, and is an indicator of initiation of the radical chain reaction. This experiment was performed according to a previous study [17]. Rose-bengal was used as the photosensitizer, and the amount of 1 O2 produced was measured using 1,3-diphenylisobenzofuran (DPBF). DPBF chemically quenches 1O2 but does not physically quench 1O2. The reduction in absorbance following the reaction of DPBF with 1O2 allows indirect measurement of the amount of 1O2. Methanol was used as a solvent to prepare 24 mM rose-bengal, and DPBF was prepared at concentrations of 15, 30, 60, and 90 mM. A total of 1 mL of rose-bengal and 1 mL of DPBF were added to the test tube, along with 1 mL of various concentrations of the samples prepared in methanol, and light-irradiated for 1 min. Absorbance reduction was measured for four concentrations of DPBF before and after light irradiation. For samples, the same procedure was repeated for at least four concentrations, and the Kq value of the 1 O2 quenching rate constant was obtained using the Stern–Volmer equation. A blank was used to calibrate the effects of rose-bengal and sample without DPBF
Fig. 1. The chemical structure of quercetin (A), quercitrin (B) and avicularin (C).
Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
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Measurement of free radical scavenging activity 1,1-Diphenyl-2-picrylhydrazyl (DPPH), a relatively stable radical, was used to measure the free radical scavenging activity of the samples. For the experiment, 1 mL of ethanol was added to 1 mL of 0.2 mM DPPH dissolved in methanol, then 1 mL of the sample was added, mixed and allowed to stand at room temperature for 10 min. The absorbance was then measured at 517 nm with a UV– visible spectrophotometer Cary 50 (Varian, Australia). Controls consisted of sample-free solvent, resulting in maximal DPPH absorbance. In the experimental group, when DPPH is reduced by the sample, the absorbance at 517 nm is decreased. Blanks consisted of samples without DPPH. Free radical scavenging activity was expressed as the concentration of free radical scavenging activity (FSC50, mM) required to reduce the concentration of DPPH by 50%. DPPH radical scavenging activity (%) was obtained from each absorbance (A) by the following formula. Aexperiment Ablank 100 Radical scavening ð%Þ ¼ 1 Acontrol
Measurement of ROS scavenging activity In the Fe3+-EDTA/H2O2 system, Fenton reaction occurs through the reaction of Fe2+ and H2O2, generating various ROS (O2 , OH, and H2O2). Detection of the generated ROS can be confirmed by measuring chemiluminescence through the reaction of luminol with ROS. Various concentrations of the sample (50 mL) were mixed with 1.78 mL of distilled water in a tube for chemiluminescence measurement. A total of 40 mL of 2.5 mM EDTA and 10 mL of 5 mM FeCl36H2O were added, followed by 80 mL of 35 mM luminol, and mixed by shaking. The tube was placed in a chemiluminescent light-emitting device, LB9505 LT (Berthold, Germany) and reacted for 5 min. Then, 40 mL of 150 mM H2O2 was added to the tube, and chemiluminescence was measured for 25 min. In the control group, distilled water was added in place of the sample solution. Blanks were evaluated under the same conditions as the experimental group, except distilled water was added instead of H2O2 and FeCl3. ROS scavenging activity was expressed as the concentration of reactive oxygen species (OSC50, mM) required to reduce the chemiluminescence intensity (counts per minute, CPM) by 50%. The ROS scavenging activity (%) was determined by the following formula. ROS scavening ð%Þ ¼
cpmcontrol cpmexperiment 100 cpmcontrol cpmblank
Penetration rate of the erythrocyte membrane To investigate whether penetration of samples into erythrocyte membranes correlated with antioxidant activity, cell permeability experiment was evaluated. Experimental conditions were identical to those employed under conventional photohemolysis experiments. Samples were prepared at 25 mM, the highest concentration allowing efficient observation during photohemolysis measurements. Samples were incubated in a black box for 30 min. Then, 0.5 mL of 0.9% saline phosphate buffer was added in place of rose-bengal, which was used as a photosensitizer, and shaken to facilitate reaction. Unpenetrated samples were removed by centrifugation. After centrifugation at 3000 rpm for 5 min, the supernatant was removed and pellets washed three times with 4 mL of buffer. After the final supernatant was removed, 4 mL of ethanol was added, and sonicated for 10 min to destroy the erythrocytes. The ruptured red blood cells were centrifuged at 3000 rpm for 5 min to precipitate cell contents and then the
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supernatant was collected. The absorbance of the obtained supernatant was measured by using a UV–visible spectrophotometer and quantified. In the blank group, solvent was added instead of the sample; the value of the blank sample was used to normalize the experimental values and the effect of the erythrocytes. Results and discussion The cytoprotective effect of 1O2-induced erythrocyte destruction When skin is exposed to UV light, various types of reactive oxygen species are generated by photochemical and photobiologi cal reactions. Among these ROS, 1O2 and OH are highly reactive and play a leading role in photoaging by oxidation of biomolecules. Photosensitization reactions which generate 1O2 are particularly important in the photoaging process. Erythrocyte photohemolysis is a useful screening method substances that are cytoprotective against 1O2-induced cell damage [1,24]. Porphyrin or riboflavin present in human skin tissue can act as a photosensitizer when exposed to sunlight, leading to production of ROS such as 1O2. These ROS produce lipid radicals by dehydrogenating unsaturated fatty acids present in cell membranes, resulting in peroxyl lipid radicals that damage the cell membrane through a radical chain reaction process. In this experiment, rose-bengal was used as a photosensitizer. Rose-bengal is a compound with a high quantum yield that produces 1O2. The cytoprotective effect of natural products against cell membrane damage induced by 1O2 is regarded as a very useful method for the development of antioxidants for use in cosmetics. The cytoprotective effects of flavonoid components of L. cuneata G. Don were indicated by the time (t50) require to destroy 50% of the assayed erythrocytes (Table 1). In this experiment, the cytoprotective effect of quercetin, a flavonoid, and quercitrin and avicularin, its glycosides, was measured. Larger t50 values indicate greater cytoprotective effects. Erythrocyte suspensions were pretreated with 1, 5, 10 and 25 mM of quercetin (or its respective glycosides) for 30 min and irradiated with light, and the resulting t50 values were 35.7, 55.7, 88.2, and 252.4 min, respectively. Quercitrin t50 values were 31.3, 33, 33.7 and 35.6 min. Avicularin t50 values were 30.6, 31.3, 31.8 and 35.4 min. Each compound was cytoprotective in a concentrationdependent manner. In particular, quercetin exhibited a seven times greater protective effect than the glycosides at 25 mM (Fig. 2). For comparison, ()-α-tocopherol and L-ascorbic acid, known antioxidants, were used as positive control reference compounds. ()-α-Tocopherol showed a dose-dependent cytoprotective effect, but L-ascorbic acid showed no significant protective effect. In 1O2induced cell membrane damage, quercetin showed significant cytoprotective effects compared to quercitrin and avicularin. Quercetin easily penetrates into the red blood cell membrane composed of phospholipids, and efficiently transfers electrons to the radicals generated in the autoxidation reaction of unsaturated Table 1 Cellular protective effects of quercetin, quercitrin, avicularin, and two reference compounds on rose-bengal sensitized photohemolysis of erythrocytes. Compound
Quercetin Quercitrin Avicularin ()-α-Tocopherol L-Ascorbic acid
t50 (Half time of hemolysis)c 1 mM
5 mM
10 mM
25 mM
35.7 0.3b 31.3 0.8b 30.6 1.8b 38.6 3.5 –
55.7 2.4b 33.0 1.4b 31.3 0.6b 40.6 3.2 –
88.2 7.4b 33.7 0.8b 31.8 2.2b 43.2 2.2 –
252.4 3.2b,a 35.6 2.3b,a 35.4 1.5b,a 50.2 0.9 31.2 2.4
Values represent the mean SD of data from three independent experiments. a p < 0.05 compared with L-ascorbic acid. b p < 0.05 compared with ()-α-tocopherol. c Control, t50 = 30.9 1.9 min.
Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
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Fig. 2. Cellular protective effects of quercetin, quercitrin, avicularin and two reference compounds on rose-bengal sensitized photohemolysis of erythrocytes at 25 mM. (Control, t50 = 30.9 1.9 min). Data are presented as mean S.D. *p < 0.05 compared with L-ascorbic acid, #p < 0.05 compared with ()-α-tocopherol.
fatty acids, blocking the radical chain reaction and preventing the cell membrane damage. However, quercitrin and avicularin are less effective due to less efficient penetration into the cell membrane due to higher relative polarity compared to quercetin. These results are consistent with a previous report on cytoprotective effects of kaempferol and its glycoside nicotiflorin isolated from Annona muricata [15]. Singlet oxygen quenching rate constant In previous experiments, the protective effect against cell damage induced by 1O2 was significantly greater for quercetin than for its glycosides quercitrin and avicularin. Singlet oxygen is the main ROS produced in the rose-bengal photosensitization reaction (Type II reaction), which plays a crucial role in initiating the radical reaction in the cell membrane. Once radicals are formed, a chain reaction occurs in the cell membrane, which is an autoxidation reaction, followed by cell membrane damage and destruction. Therefore, the quenching effect of 1O2 by antioxidants is very important aspect of cytoprotective activity. Singlet oxygen quenching ability was examined by measuring the 1O2 quenching rate constant (kq). β-carotene, which is known as an excellent quenching agent of 1O2, was used as a control. The quenching rate constant of β-carotene measured in this experiment was 2.2 1010 M1S1, which is similar to the rate constant (2.6 1010 M1S1) cited in the literature [17]. The kq of L-ascorbic acid, a water-soluble antioxidant, and ()-α-tocopherol, a fat-soluble antioxidant, were 1.0 108 M1S1 and 1.8 108 M1S1, respectively. Quercetin, quercitrin, and avicularin kq were found to be 1.5 108 M1S1, 1.1 108 M1S1, and 1.0 108 M1S1, respectively (Table 2). Quercetin showed higher quenching ability than its glycosides, with 1.5 times higher quenching ability compared to L-ascorbic acid. However, ()-α-tocopherol and β-carotene exhibited greater quenching ability than quercetin. These results suggest that the 1 O2 quenching effect of quercetin is not the major mechanism of cytoprotective properties of quercetin.
Table 2 Singlet oxygen quenching rate constant of quercetin, quercitrin, avicularin and two reference compounds. Compound
Kq (M1S1)
Quercetin Quercitrin Avicularin ()-α-Tocopherol L-Ascorbic acid
1.5 108b,a 1.1 108b 1.0 108b 1.8 108 1.0 108
Values are presented as mean S.D (n = 3). a p < 0.05 compared with L-ascorbic acid. b p < 0.05 compared with ()-α-tocopherol.
Free radical scavenging activity Lipid radicals produced during oxidation of plasma membrane lipids induced by 1O2 in the type II reaction, and radicals or radical ions produced by the type I reaction in the skin contribute to autoxidation of cell membrane lipids. Therefore, removal of initially generated radicals is very important for the protection of cells from free radicals because it can block the initiation of radical-mediated cell membrane reactions. Measurement of the radical scavenging activity of the free radical DPPH can provide information on the ability of an antioxidant to block the radical chain reaction. The free radical scavenging activity (FSC50) of quercetin, quercitrin, and avicularin used in the experiments were 5.7 mM, 8.6 mM, and 18.0 mM, respectively. The FSC50 of ()-α-tocopherol and L-ascorbic acid, which were used as positive antioxidant controls, were 21.9 mM and 15.1 mM, respectively (Fig. 3). As a result, quercetin showed greater radical scavenging activity than quercitrin and avicularin, which are glycosides, and ()-α-tocopherol and L-ascorbic acid, which are fat soluble and water-soluble antioxidants, respectivley. As shown in Fig. 2, quercetin showed the greatest free radical scavenging activity and the greatest protective effect against 1O2 induced cell damage. This suggests that the free radical scavenging activity contributed
Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
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Fig. 3. Free radical scavenging activities of quercetin, quercitrin, avicularin and two reference compounds. Data are presented as mean S.D. *p < 0.05 compared with Lascorbic acid, #p < 0.05 compared with ()-α-tocopherol.
to the protective effect against ROS-induced cell damage. Quercitrin and avicularin showed radical scavenging activities similar to ()-α-tocopherol and L-ascorbic acid. The radical scavenging activities of quercetin and quercitrin were not significantly different. These results suggest that free radical scavenging activity does not seem to contribute significantly to the cytoprotective effects. This suggests that antioxidants should be present in the cell membrane to effectively block radical initiation and chain reaction in the cell membrane. In this experimental condition, glycosides of quercetin and L-ascorbic acid seem to have little cytoprotective effect because their penetration into cell membranes is minimal due to their structures. ROS Scavenging activity The Type II reaction of the photosensitization reaction produces various ROS such as O2 , OH, and H2O2 as well as 1O2. These ROS can damage cells and tissues. In particular, 1O2-induced cell damage results in the generation of lipid peroxidation radicals, alkoxyl radicals, lipids hydroperoxides, and other oxidation products due to autoxidation of unsaturated fatty acids in cell membranes. In addition, ROS such as O2 , H2O2, and OH are 2+ 3+ produced in the redox process of Fe /Fe in the red blood cells, and these ROS are also involved in cell damage. Therefore, the ROS scavenging activity of quercetin, quercitrin and avicularin (OSC50) showed the total antioxidative capacity against various types of ROS involved in cell damage. Quercetin, quercitrin and avicularin had very high ROS scavenging activity (0.78, 1.46 and 1.01 mM, respectively) (Fig. 4). The control group, L-ascorbic acid and ()-αtocopherol had an OSC50 value of 0.53 and 1.06 mM, respectively. Quercetin showed greater antioxidant capacity than its glycosides. Quercetin exhibited radical scavenging activity and general ROS scavenging activity. L-Ascorbic acid showed excellent scavenging ability for ROS but not for ROS-induced cell damage.
Penetration rate into the erythrocyte membrane Secondarily-generated free radicals and ROS in the course of O2-induced reactions can also contribute to cell damage. Therefore, antioxidants with the ability to scavenge free radical or ROS, and 1O2 quenching ability, can contribute to cell protection. Antioxidants must be present in the cell membrane to suppress cell membrane damage induced by 1O2. Therefore, it is important to investigate the extent to which antioxidants capable of scavenging radicals and ROS can penetrate cell membranes. The penetration rates of quercetin, quercitrin, and avicularin were 19.6, 8.3, and 3.9%, respectively. L-ascorbic acid, used as a positive antioxidant control, showed the lowest penetration rate (1.2%), and ()-αtocopherol showed a relatively high penetration rate (10.3%) (Fig. 5). Among measured samples, quercetin showed the greatest penetration rate (19.6%), which was 2.4 times greater than quercitrin and 5.0 times greater than avicularin. In addition, the penetration rate was 16 times greater than L-ascorbic acid. The 1O2 quenching ability, and free radical and ROS scavenging activities of quercetin were greater than the flavonoid glycosides, which contributed to the cytoprotective effect. However, cell membrane permeability was the largest contributor to cytoprotective effects. L-ascorbic acid, a positive antioxidant control, was found to have a great free radical and ROS scavenging ability, but cell membrane penetration was not efficient and showed little cytoprotective effects. This tendency was also observed for the quercetin glycosides. Their radical scavenging activity and ROS scavenging activity were large, but they were not effective cytoprotectants due to insufficient membrane permeability. In the case of ()-αtocopherol, radical scavenging ability was lower than the quercetin glycosides, but cell membrane penetration was more efficient, resulting in a greater cytoprotective effect. The above results suggest that the cytoprotective effect of ROS-induced cell damage induced by the photosensitization reaction is complex, and related to the radical and ROS scavenging ability, 1O2 quenching ability, 1
Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
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Fig. 4. ROS scavenging activities of quercetin, quercitrin, avicularin and two reference compounds. Data are presented as mean S.D. *p < 0.05 compared with L-ascorbic acid, # p < 0.05 compared with ()-α-tocopherol.
Fig. 5. Cell membrane penetration efficiency of quercetin, quercitrin, avicularin and two reference compounds. Data are presented as mean S.D. *p < 0.05 compared with Lascorbic acid, #p < 0.05 compared with ()-α-tocopherol.
and cell membrane permeability, with cell membrane permeability being particularly important. These results are consistent with the findings of Park et al. that kaempferol exerts greater cytoprotective effect than its glycosides because of greater cell membrane permeability [15].
Conclusion In this study, the cytoprotective effects against ROS-induced cell damage of quercitrin, avicularin and quercetin, which are major components of the L. cuneata G. Don extract, were evaluated, and
Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
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protective mechanisms investigated. We found that quercetin exerts greater cytoprotective effect than its glycosides quercitrin and avicularin against 1O2-induced cell damage. To further investigate this effect, we measured DPPH free radical scavenging ability, ROS scavenging ability, 1O2 quenching rate constant, and cell permeability. In DPPH free radical scavenging ability, ROS scavenging ability and 1O2 quenching rate constant, quercetin was more effective than quercitrin and avicularin. In addition, the penetration rate of quercetin cell membrane was 16 times greater than L-ascorbic acid, which had little effect on cell membrane protection. In conclusion, the cytoprotective effect against 1O2induced cytotoxicity was reflected by the 1O2 quenching ability, free radical and ROS scavenging activity, and cell permeability, with cell membrane permeability being the most significant contributor. Antioxidant ability and membrane penetration ability of antioxidants which can block the radical initiation reaction and the chain reaction in the cell membrane at the early stage of generation of UV-induced reactive oxygen species are the most important factors for suppressing photoaging. Acknowledgements This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Journal of Industrial and Engineering Chemistry xxx (2018) xxx–xxx
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Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage Keon Soo Lee, Soo Nam Park* Department of Fine Chemistry, Cosmetic R&D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 01811, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 July 2018 Received in revised form 17 October 2018 Accepted 10 November 2018 Available online xxx
The purpose of this study was to compare the anti-oxidative and cytoprotective effects of quercitrin and avicularin isolated from Lespedeza cuneata G. Don extract to those of quercetin, an aglycone of quercitrin, and avicularin. Quercetin had higher antioxidative activity and cell penetration ratio than its glycosides, resulting in greater cytoprotective effects against 1O2. The cytoprotective effects against cell damage seems to reflect 1O2 quenching rate, free radical and ROS scavenging activity, and cell permeability. Among them, cell permeability to block free radical initiation and chain reactions in cell membranes is considered to be the most important feature of cytoprotective activity. © 2018 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.
Keywords: Cytoprotective effect Quercetin Quercitrin Avicularin Antioxidant mechanism
Introduction With increased interest in skin aging, development of anti-aging materials and applications in cosmetic products are being continuously pursued. Intrinsically-aged skin appears dry, thin, and has fine wrinkles, and is caused by the passage of time. Alternatively, extrinsic aging is induced by chronic exposure to external environmental factors. Photoaging by UV exposure is an example of extrinsic aging. Exposed skin undergoes oxidative stress by radicals and reactive oxygen species (ROS). This oxidative stress causes oxidative damage of biomolecules such as lipids, proteins and DNA, which are components of skin cells, resulting in skin aging [1–5]. Reactive oxygen species include radical species such as superoxide anion radical (O2 ) and hydroxyl radical ( OH), non1 radical species such as singlet oxygen ( O2) and hydrogen peroxide (H2O2). There are peroxyl radical (ROO ) and alkoxyl radical (RO ) produced by the biological components reacting with the preceding ROS [6–8]. These ROS are also generated by photosensitization reactions with UV radiation. The photosensitization reaction generates various ROS as well as 1O2, which are highly reactive. Photosensitization reactions occur in the skin when exposed to UV light because there are compounds in the skin such as porphyrin and riboflavin that can induce this reaction. The
* Corresponding author. E-mail address: [email protected] (S.N. Park).
photosensitization reaction can be broadly divided into two types, Type I and Type II. Type I reactions produce radicals or radical ions, and the main product of Type II reaction is 1O2, and additionally various ROS are produced. Especially, 1O2 is a highly reactive and is not subject to enzymatic metabolism or scavenging in vivo, which can destroy cell membrane and produce tissue damage importantly [9–11]. The skin utilizes a complementary protection network of enzymatic antioxidants and non-enzymatic antioxidants to prevent damages [12–14]. When reactive oxygen species is continuously generated from the external environment via photosensitization reactions resulting from exposure to UV rays, the antioxidant in the body become depleted and the cells and tissues undergo damage, resulting in aging and adult diseases such as hypertension, atherosclerosis, and diabetes. Therefore, interest in the development of natural antioxidants for the supplementation of antioxidant defense networks that protect skin cells from UV and reactive oxygen species damage has increased. As these antioxidants not only possess hydrophilic and hydrophobic properties but also various antioxidant properties, they can exhibit synergistic antioxidant effects to protect skin cells from oxidative stress. Thus, the development of antioxidant materials and application studies for the cosmetics industry are required [15–19]. Prior to this study, the authors isolated and analyzed components from Lespedeza cuneata G. Don extract and reported that they have antioxidant and antiaging activites [20–22]. The major components of L. cuneata G. Don extracts are quercetin and its glycosides, quercitrin and avicularin. The chemical structure of
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Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
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Preparation of erythrocytes suspensions
quercetin, quercitrin, and avicularin was shown in Fig. 1. In previous studies, quercetin showed superior cytoprotective effects against cellular damage induced by photosensitization reactions, compared to its glycosides. However, there are no comparative studies on the cytoprotective effects of quercetin and its glycosides quercitrin and avicularin on cell damage induced by 1O2 using the photosensitization reaction. In this study, various types of antioxidative experiments were performed using three principal components, along with L-ascorbic acid and ()-α-tocopherol, which are well known as antioxidants, as a control group, to investigate the mechanism of cell protection in oxidative stress induced by ROS.
Erythrocytes used in this experiment were immediately added to heparinized test tubes after collection and used within 12 h. Blood was centrifuged at 3000 rpm for 5 min to separate erythrocytes and plasma. The separated erythrocytes were washed three times with 0.9% saline phosphate buffer (pH 7.4, 9.6 mM Na2HPO412H2O, 1.6 mM NaH2PO42H2O) prior to use. The optical density (OD) value of the erythrocyte suspension used in the experiment was 0.6 at 700 nm, and the number of erythrocytes was approximately 1.5 107 cells/mL.
Materials and methods
Measurement of photohemolysis inhibition
Materials
A total of 3.5 mL of erythrocyte suspension was placed in a Pyrex test tube and 50 mL of samples was added. After reaction in a black box for 30 min, 0.5 mL of the photosensitizer rose-bengal (15 mM) was added, and the mixture was irradiated for 15 min. Rose-bengal produces 1O2 in high yields [23]. After irradiation, the degree of destruction of erythrocytes was measured by transmittance (%) at 700 nm in 15 min intervals. At this wavelength, increased transmittance of the erythrocyte suspension is proportional to the degree of erythrocyte hemolysis. The degree of hemolysis was shown by plotting the transmittance versus time to determine t50 values (the time required for 50% hemolysis).
The whole part of L. cuneata G. Don. was purchased from the herbal medicine shop in the Kyungdong market located in Korea. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 1,3-diphenylisobenzofuran (DPBF), H2O2, luminol, rose-bengal were purchased from Sigma Chemical Co. (USA). FeCl36H2O was purchased from Junsei Chemical Co. (Japan). L-Ascorbic acid and ()-α-tocopherol were purchased from Sigma-Aldrich (Korea). Na2HPO412H2O, NaH2PO42H2O, NaCl, trizmabase, HCl, H2SO4, ethanol (EtOH), methanol (MeOH), ethyl acetate (EtOAc), acetic acid, and formic acid were extra pure grade in this study.
Measurement of singlet oxygen quenching rate constants Extraction, isolation and fractionation Dried L. cuneata G. Don (100 g) was finely cut and then extracted by immersion in 4 L of 70% ethanol for 1 week. The 70% ethanol extract was filtered under pressure and dried to obtain a powder. The 70% ethanol extract was treated 3 times with ethyl acetate, which separated out as a fraction that was then concentrated under use of a rotary evaporator to remove the organic solvent (the concentrated form the ethyl acetate fraction of L. cuneata). The ethyl acetate fraction was dissolved in ethanol and separated on TLC plates (Merck, USA) using ethyl acetate: acetic acid: formic acid: distilled water = 8: 1: 1: 1 (v/v) as mobile phase. In previous studies, the bands of avicularin (Rf 0.66) and quercitrin (Rf 0.54), which were previously determined to be the main components of the L. cuneata ethyl acetate fraction, were extracted with ethanol and then dried again [20]. To obtain quercetin, quercitrin was acid hydrolyzed. Quercitrin was added to a 5% H2SO4 solution and refluxed and cooled for 4 h. The reaction solution was neutralized with a 5% KOH–MeOH solution, washed with distilled water, and then partitioned with ethyl acetate and dried. The obtained quercetin was dissolved again in ethanol.
The singlet oxygen quenching rate constant is an indicator of how much 1O2, the product of the Type II reaction, is extinguished, and is an indicator of initiation of the radical chain reaction. This experiment was performed according to a previous study [17]. Rose-bengal was used as the photosensitizer, and the amount of 1 O2 produced was measured using 1,3-diphenylisobenzofuran (DPBF). DPBF chemically quenches 1O2 but does not physically quench 1O2. The reduction in absorbance following the reaction of DPBF with 1O2 allows indirect measurement of the amount of 1O2. Methanol was used as a solvent to prepare 24 mM rose-bengal, and DPBF was prepared at concentrations of 15, 30, 60, and 90 mM. A total of 1 mL of rose-bengal and 1 mL of DPBF were added to the test tube, along with 1 mL of various concentrations of the samples prepared in methanol, and light-irradiated for 1 min. Absorbance reduction was measured for four concentrations of DPBF before and after light irradiation. For samples, the same procedure was repeated for at least four concentrations, and the Kq value of the 1 O2 quenching rate constant was obtained using the Stern–Volmer equation. A blank was used to calibrate the effects of rose-bengal and sample without DPBF
Fig. 1. The chemical structure of quercetin (A), quercitrin (B) and avicularin (C).
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Measurement of free radical scavenging activity 1,1-Diphenyl-2-picrylhydrazyl (DPPH), a relatively stable radical, was used to measure the free radical scavenging activity of the samples. For the experiment, 1 mL of ethanol was added to 1 mL of 0.2 mM DPPH dissolved in methanol, then 1 mL of the sample was added, mixed and allowed to stand at room temperature for 10 min. The absorbance was then measured at 517 nm with a UV– visible spectrophotometer Cary 50 (Varian, Australia). Controls consisted of sample-free solvent, resulting in maximal DPPH absorbance. In the experimental group, when DPPH is reduced by the sample, the absorbance at 517 nm is decreased. Blanks consisted of samples without DPPH. Free radical scavenging activity was expressed as the concentration of free radical scavenging activity (FSC50, mM) required to reduce the concentration of DPPH by 50%. DPPH radical scavenging activity (%) was obtained from each absorbance (A) by the following formula. Aexperiment Ablank 100 Radical scavening ð%Þ ¼ 1 Acontrol
Measurement of ROS scavenging activity In the Fe3+-EDTA/H2O2 system, Fenton reaction occurs through the reaction of Fe2+ and H2O2, generating various ROS (O2 , OH, and H2O2). Detection of the generated ROS can be confirmed by measuring chemiluminescence through the reaction of luminol with ROS. Various concentrations of the sample (50 mL) were mixed with 1.78 mL of distilled water in a tube for chemiluminescence measurement. A total of 40 mL of 2.5 mM EDTA and 10 mL of 5 mM FeCl36H2O were added, followed by 80 mL of 35 mM luminol, and mixed by shaking. The tube was placed in a chemiluminescent light-emitting device, LB9505 LT (Berthold, Germany) and reacted for 5 min. Then, 40 mL of 150 mM H2O2 was added to the tube, and chemiluminescence was measured for 25 min. In the control group, distilled water was added in place of the sample solution. Blanks were evaluated under the same conditions as the experimental group, except distilled water was added instead of H2O2 and FeCl3. ROS scavenging activity was expressed as the concentration of reactive oxygen species (OSC50, mM) required to reduce the chemiluminescence intensity (counts per minute, CPM) by 50%. The ROS scavenging activity (%) was determined by the following formula. ROS scavening ð%Þ ¼
cpmcontrol cpmexperiment 100 cpmcontrol cpmblank
Penetration rate of the erythrocyte membrane To investigate whether penetration of samples into erythrocyte membranes correlated with antioxidant activity, cell permeability experiment was evaluated. Experimental conditions were identical to those employed under conventional photohemolysis experiments. Samples were prepared at 25 mM, the highest concentration allowing efficient observation during photohemolysis measurements. Samples were incubated in a black box for 30 min. Then, 0.5 mL of 0.9% saline phosphate buffer was added in place of rose-bengal, which was used as a photosensitizer, and shaken to facilitate reaction. Unpenetrated samples were removed by centrifugation. After centrifugation at 3000 rpm for 5 min, the supernatant was removed and pellets washed three times with 4 mL of buffer. After the final supernatant was removed, 4 mL of ethanol was added, and sonicated for 10 min to destroy the erythrocytes. The ruptured red blood cells were centrifuged at 3000 rpm for 5 min to precipitate cell contents and then the
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supernatant was collected. The absorbance of the obtained supernatant was measured by using a UV–visible spectrophotometer and quantified. In the blank group, solvent was added instead of the sample; the value of the blank sample was used to normalize the experimental values and the effect of the erythrocytes. Results and discussion The cytoprotective effect of 1O2-induced erythrocyte destruction When skin is exposed to UV light, various types of reactive oxygen species are generated by photochemical and photobiologi cal reactions. Among these ROS, 1O2 and OH are highly reactive and play a leading role in photoaging by oxidation of biomolecules. Photosensitization reactions which generate 1O2 are particularly important in the photoaging process. Erythrocyte photohemolysis is a useful screening method substances that are cytoprotective against 1O2-induced cell damage [1,24]. Porphyrin or riboflavin present in human skin tissue can act as a photosensitizer when exposed to sunlight, leading to production of ROS such as 1O2. These ROS produce lipid radicals by dehydrogenating unsaturated fatty acids present in cell membranes, resulting in peroxyl lipid radicals that damage the cell membrane through a radical chain reaction process. In this experiment, rose-bengal was used as a photosensitizer. Rose-bengal is a compound with a high quantum yield that produces 1O2. The cytoprotective effect of natural products against cell membrane damage induced by 1O2 is regarded as a very useful method for the development of antioxidants for use in cosmetics. The cytoprotective effects of flavonoid components of L. cuneata G. Don were indicated by the time (t50) require to destroy 50% of the assayed erythrocytes (Table 1). In this experiment, the cytoprotective effect of quercetin, a flavonoid, and quercitrin and avicularin, its glycosides, was measured. Larger t50 values indicate greater cytoprotective effects. Erythrocyte suspensions were pretreated with 1, 5, 10 and 25 mM of quercetin (or its respective glycosides) for 30 min and irradiated with light, and the resulting t50 values were 35.7, 55.7, 88.2, and 252.4 min, respectively. Quercitrin t50 values were 31.3, 33, 33.7 and 35.6 min. Avicularin t50 values were 30.6, 31.3, 31.8 and 35.4 min. Each compound was cytoprotective in a concentrationdependent manner. In particular, quercetin exhibited a seven times greater protective effect than the glycosides at 25 mM (Fig. 2). For comparison, ()-α-tocopherol and L-ascorbic acid, known antioxidants, were used as positive control reference compounds. ()-α-Tocopherol showed a dose-dependent cytoprotective effect, but L-ascorbic acid showed no significant protective effect. In 1O2induced cell membrane damage, quercetin showed significant cytoprotective effects compared to quercitrin and avicularin. Quercetin easily penetrates into the red blood cell membrane composed of phospholipids, and efficiently transfers electrons to the radicals generated in the autoxidation reaction of unsaturated Table 1 Cellular protective effects of quercetin, quercitrin, avicularin, and two reference compounds on rose-bengal sensitized photohemolysis of erythrocytes. Compound
Quercetin Quercitrin Avicularin ()-α-Tocopherol L-Ascorbic acid
t50 (Half time of hemolysis)c 1 mM
5 mM
10 mM
25 mM
35.7 0.3b 31.3 0.8b 30.6 1.8b 38.6 3.5 –
55.7 2.4b 33.0 1.4b 31.3 0.6b 40.6 3.2 –
88.2 7.4b 33.7 0.8b 31.8 2.2b 43.2 2.2 –
252.4 3.2b,a 35.6 2.3b,a 35.4 1.5b,a 50.2 0.9 31.2 2.4
Values represent the mean SD of data from three independent experiments. a p < 0.05 compared with L-ascorbic acid. b p < 0.05 compared with ()-α-tocopherol. c Control, t50 = 30.9 1.9 min.
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Fig. 2. Cellular protective effects of quercetin, quercitrin, avicularin and two reference compounds on rose-bengal sensitized photohemolysis of erythrocytes at 25 mM. (Control, t50 = 30.9 1.9 min). Data are presented as mean S.D. *p < 0.05 compared with L-ascorbic acid, #p < 0.05 compared with ()-α-tocopherol.
fatty acids, blocking the radical chain reaction and preventing the cell membrane damage. However, quercitrin and avicularin are less effective due to less efficient penetration into the cell membrane due to higher relative polarity compared to quercetin. These results are consistent with a previous report on cytoprotective effects of kaempferol and its glycoside nicotiflorin isolated from Annona muricata [15]. Singlet oxygen quenching rate constant In previous experiments, the protective effect against cell damage induced by 1O2 was significantly greater for quercetin than for its glycosides quercitrin and avicularin. Singlet oxygen is the main ROS produced in the rose-bengal photosensitization reaction (Type II reaction), which plays a crucial role in initiating the radical reaction in the cell membrane. Once radicals are formed, a chain reaction occurs in the cell membrane, which is an autoxidation reaction, followed by cell membrane damage and destruction. Therefore, the quenching effect of 1O2 by antioxidants is very important aspect of cytoprotective activity. Singlet oxygen quenching ability was examined by measuring the 1O2 quenching rate constant (kq). β-carotene, which is known as an excellent quenching agent of 1O2, was used as a control. The quenching rate constant of β-carotene measured in this experiment was 2.2 1010 M1S1, which is similar to the rate constant (2.6 1010 M1S1) cited in the literature [17]. The kq of L-ascorbic acid, a water-soluble antioxidant, and ()-α-tocopherol, a fat-soluble antioxidant, were 1.0 108 M1S1 and 1.8 108 M1S1, respectively. Quercetin, quercitrin, and avicularin kq were found to be 1.5 108 M1S1, 1.1 108 M1S1, and 1.0 108 M1S1, respectively (Table 2). Quercetin showed higher quenching ability than its glycosides, with 1.5 times higher quenching ability compared to L-ascorbic acid. However, ()-α-tocopherol and β-carotene exhibited greater quenching ability than quercetin. These results suggest that the 1 O2 quenching effect of quercetin is not the major mechanism of cytoprotective properties of quercetin.
Table 2 Singlet oxygen quenching rate constant of quercetin, quercitrin, avicularin and two reference compounds. Compound
Kq (M1S1)
Quercetin Quercitrin Avicularin ()-α-Tocopherol L-Ascorbic acid
1.5 108b,a 1.1 108b 1.0 108b 1.8 108 1.0 108
Values are presented as mean S.D (n = 3). a p < 0.05 compared with L-ascorbic acid. b p < 0.05 compared with ()-α-tocopherol.
Free radical scavenging activity Lipid radicals produced during oxidation of plasma membrane lipids induced by 1O2 in the type II reaction, and radicals or radical ions produced by the type I reaction in the skin contribute to autoxidation of cell membrane lipids. Therefore, removal of initially generated radicals is very important for the protection of cells from free radicals because it can block the initiation of radical-mediated cell membrane reactions. Measurement of the radical scavenging activity of the free radical DPPH can provide information on the ability of an antioxidant to block the radical chain reaction. The free radical scavenging activity (FSC50) of quercetin, quercitrin, and avicularin used in the experiments were 5.7 mM, 8.6 mM, and 18.0 mM, respectively. The FSC50 of ()-α-tocopherol and L-ascorbic acid, which were used as positive antioxidant controls, were 21.9 mM and 15.1 mM, respectively (Fig. 3). As a result, quercetin showed greater radical scavenging activity than quercitrin and avicularin, which are glycosides, and ()-α-tocopherol and L-ascorbic acid, which are fat soluble and water-soluble antioxidants, respectivley. As shown in Fig. 2, quercetin showed the greatest free radical scavenging activity and the greatest protective effect against 1O2 induced cell damage. This suggests that the free radical scavenging activity contributed
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Fig. 3. Free radical scavenging activities of quercetin, quercitrin, avicularin and two reference compounds. Data are presented as mean S.D. *p < 0.05 compared with Lascorbic acid, #p < 0.05 compared with ()-α-tocopherol.
to the protective effect against ROS-induced cell damage. Quercitrin and avicularin showed radical scavenging activities similar to ()-α-tocopherol and L-ascorbic acid. The radical scavenging activities of quercetin and quercitrin were not significantly different. These results suggest that free radical scavenging activity does not seem to contribute significantly to the cytoprotective effects. This suggests that antioxidants should be present in the cell membrane to effectively block radical initiation and chain reaction in the cell membrane. In this experimental condition, glycosides of quercetin and L-ascorbic acid seem to have little cytoprotective effect because their penetration into cell membranes is minimal due to their structures. ROS Scavenging activity The Type II reaction of the photosensitization reaction produces various ROS such as O2 , OH, and H2O2 as well as 1O2. These ROS can damage cells and tissues. In particular, 1O2-induced cell damage results in the generation of lipid peroxidation radicals, alkoxyl radicals, lipids hydroperoxides, and other oxidation products due to autoxidation of unsaturated fatty acids in cell membranes. In addition, ROS such as O2 , H2O2, and OH are 2+ 3+ produced in the redox process of Fe /Fe in the red blood cells, and these ROS are also involved in cell damage. Therefore, the ROS scavenging activity of quercetin, quercitrin and avicularin (OSC50) showed the total antioxidative capacity against various types of ROS involved in cell damage. Quercetin, quercitrin and avicularin had very high ROS scavenging activity (0.78, 1.46 and 1.01 mM, respectively) (Fig. 4). The control group, L-ascorbic acid and ()-αtocopherol had an OSC50 value of 0.53 and 1.06 mM, respectively. Quercetin showed greater antioxidant capacity than its glycosides. Quercetin exhibited radical scavenging activity and general ROS scavenging activity. L-Ascorbic acid showed excellent scavenging ability for ROS but not for ROS-induced cell damage.
Penetration rate into the erythrocyte membrane Secondarily-generated free radicals and ROS in the course of O2-induced reactions can also contribute to cell damage. Therefore, antioxidants with the ability to scavenge free radical or ROS, and 1O2 quenching ability, can contribute to cell protection. Antioxidants must be present in the cell membrane to suppress cell membrane damage induced by 1O2. Therefore, it is important to investigate the extent to which antioxidants capable of scavenging radicals and ROS can penetrate cell membranes. The penetration rates of quercetin, quercitrin, and avicularin were 19.6, 8.3, and 3.9%, respectively. L-ascorbic acid, used as a positive antioxidant control, showed the lowest penetration rate (1.2%), and ()-αtocopherol showed a relatively high penetration rate (10.3%) (Fig. 5). Among measured samples, quercetin showed the greatest penetration rate (19.6%), which was 2.4 times greater than quercitrin and 5.0 times greater than avicularin. In addition, the penetration rate was 16 times greater than L-ascorbic acid. The 1O2 quenching ability, and free radical and ROS scavenging activities of quercetin were greater than the flavonoid glycosides, which contributed to the cytoprotective effect. However, cell membrane permeability was the largest contributor to cytoprotective effects. L-ascorbic acid, a positive antioxidant control, was found to have a great free radical and ROS scavenging ability, but cell membrane penetration was not efficient and showed little cytoprotective effects. This tendency was also observed for the quercetin glycosides. Their radical scavenging activity and ROS scavenging activity were large, but they were not effective cytoprotectants due to insufficient membrane permeability. In the case of ()-αtocopherol, radical scavenging ability was lower than the quercetin glycosides, but cell membrane penetration was more efficient, resulting in a greater cytoprotective effect. The above results suggest that the cytoprotective effect of ROS-induced cell damage induced by the photosensitization reaction is complex, and related to the radical and ROS scavenging ability, 1O2 quenching ability, 1
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Fig. 4. ROS scavenging activities of quercetin, quercitrin, avicularin and two reference compounds. Data are presented as mean S.D. *p < 0.05 compared with L-ascorbic acid, # p < 0.05 compared with ()-α-tocopherol.
Fig. 5. Cell membrane penetration efficiency of quercetin, quercitrin, avicularin and two reference compounds. Data are presented as mean S.D. *p < 0.05 compared with Lascorbic acid, #p < 0.05 compared with ()-α-tocopherol.
and cell membrane permeability, with cell membrane permeability being particularly important. These results are consistent with the findings of Park et al. that kaempferol exerts greater cytoprotective effect than its glycosides because of greater cell membrane permeability [15].
Conclusion In this study, the cytoprotective effects against ROS-induced cell damage of quercitrin, avicularin and quercetin, which are major components of the L. cuneata G. Don extract, were evaluated, and
Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018
G Model JIEC 4255 No. of Pages 7
K.S. Lee, S.N. Park / Journal of Industrial and Engineering Chemistry xxx (2018) xxx–xxx
protective mechanisms investigated. We found that quercetin exerts greater cytoprotective effect than its glycosides quercitrin and avicularin against 1O2-induced cell damage. To further investigate this effect, we measured DPPH free radical scavenging ability, ROS scavenging ability, 1O2 quenching rate constant, and cell permeability. In DPPH free radical scavenging ability, ROS scavenging ability and 1O2 quenching rate constant, quercetin was more effective than quercitrin and avicularin. In addition, the penetration rate of quercetin cell membrane was 16 times greater than L-ascorbic acid, which had little effect on cell membrane protection. In conclusion, the cytoprotective effect against 1O2induced cytotoxicity was reflected by the 1O2 quenching ability, free radical and ROS scavenging activity, and cell permeability, with cell membrane permeability being the most significant contributor. Antioxidant ability and membrane penetration ability of antioxidants which can block the radical initiation reaction and the chain reaction in the cell membrane at the early stage of generation of UV-induced reactive oxygen species are the most important factors for suppressing photoaging. Acknowledgements This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Please cite this article in press as: K.S. Lee, S.N. Park, Cytoprotective effects and mechanisms of quercetin, quercitrin and avicularin isolated from Lespedeza cuneata G. Don against ROS-induced cellular damage, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.11.018