Abstract Houttuynia cordata flavonoid and Sophora japonica L. polysaccharide were investigated to develop antiUV sunscreening agent through UV wavelength scanning spectrum， antioxidant ability and cell viability after UVB irradiation. The results showed that the extracts had strong UVA and UVB absorption ability and hydroxyl free radical scavenging ability. The S. japonica L. polysaccharide showed strong antiUVB irradiation ability according to the cell viability experiment. It suggests that the H. cordata flavonoid and S. japonica L. polysaccharide could be combined as a candidate of antiUV sunscreening supplement and compensate for the shortcomings of chemical sunscreens against UVA ray.
　　Key words Houttuynia cordata; Flavonoid; Sophora japonica L.; Polysaccharide; AntiUV radiation
　　Life on the earth is impossible without solar light， while excessive exposure to ultraviolet radiation is a key risk factor associated with the initiation and development of various skin diseases. The ultraviolet wavelength is divided into three sections： UVA （320-400 nm）， UVB （290-320 nm）， and UVC （100-290 nm）. Solar UV radiation on the earthюs surface comprises approximately 90%-99% UVA and 1%-10% of UVB[1-2]. UVA causes darkening of the skin， pigmentation and skin aging， and even skin cancer， such as melanoma. UVA can also cause increasing of free radicals and reactive oxygen species in skin cells， indirectly causing damage to the skin. UVB mainly acts on the epidermis layer， causing immediate and serious skin damage， such as sunburn. Excessive radiation can cause skin immune response， leading to skin cancer， which is stronger than UVA.
　　Traditional sunscreening agents， such as UV absorber and UV shielding agent， may cause inflammation， allergies and other side effects. Therefore， the field of skin protection has shown a considerable interest in the use of botanicals recently， such as Lonicera caerulea and Vaccinium myrtillus fruit polyphenols[3]， Codium fragile extract[4]， exopolysaccharide from P. agglomerans[5]， Prunella vulgaris extract[1]， green tea polyphenols[6]， grape seed extract[7]and so on.
　　Sophora japonica L. is widely distributed in Korea， China and Japan. Its buds， dried flowers and fruits are a medicinal herb used as hemostatic agents in traditional korea medicine and flavonoids from the buds were reported as haemostatic constituent[8].
　　Main components of S. japonica flower buds include flavonoids， isoflavonoids， triterpenes， alkaloids and polysaccharides[9]. Some biological functions of compounds derived from S. japonica L. such as antiinflammatory， antioxidant， antitumor， antibacterial， antiviral， hemostatic effects have been reported[10-14]. 　　Houttuyniae cordata is a whole plant of the genus Amaranthus， which is one of the varieties officially identified by the Ministry of Health as a drug/food dualuse. It mainly contains flavonoids， volatile oils， organic acids and fatty acids， and has the pharmacological effects such as antiinflammatory， antitumor， antibacterial and antiviral effects and the effect of enhancing human immunity， while a few studies have been conducted on the antiionizing radiation effect[15-19].
　　In this research， H. cordata flavonoid and S. japonica L. polysaccharide were extracted and evaluated in protecting skin from UV radiation. It will be a basic research for the development of antiUV sunscreening supplement.
　　Materials and Methods
　　Chemicals and reagents
　　Modified Eagleюs Medium （MEM）， penicillinstreptomycin solution， nonanimal Lglutamine， MEM vitamin solution 100≠， nonessential amino acid solution 100≠， 3（45dimethylthiazol211）2，5diphenyl tetrazolium bromide （MTT）， dimethyl sulfoxide （DMSO） and trypsinEDTA solution 1 （trypsin） were obtained from Life Technologies Company. Propidium iodide （PI） kit and Annexin VFITC kit， ROS （reactive oxygen species） assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. Fetal bovine serium （FBS） was purchased from Gibco， USA.
　　Extract of H. cordata flovonoid
　　H. cordata whole grass was purchased from Pharmacy， dried and crushed into 200 meshes. The powder was soaked with 60% ethanol at a ratio of 1 g≥15 ml （w/v） for 2 h and ultrasonicassisted extracted at 70 for 1 h. The residue was removed by vacuum suction filter and extracted again according to the method mentioned above. The filtrates collected by the two extractions were combined and concentrated at 60 and then freezedried. The yield was 7.9%.
　　Extract of S. japonica L. flower buds polysaccharide
　　The flower buds of S. japonica L. purchased from Pharmacy was dried and crushed into 200 meshes. The powder was soaked with water at a ratio of 1 g≥20 ml for 2 h and mixed with a small amount of borax and CaO to regulate solution to pH 8.0-9.0. The mixed solution was heated to 100 for 10 min and the supernatant was removed. The residue was extracted again according to the method mentioned above. The combined supernatant was regulated to pH 4.0 and placed overnight to remove precipitation （Rutin）. The supernatant was regulated to pH 7.0 and concentrated to 1/10 of the original volume， and then added with precooled EtOH， followed by stirring and standing overnight at 4. The precipitation was collected by centrifuge at 4 000 rpm for 10 min and freezedried. The yield was 3.17%. 　　Full UV wavelength scanning
　　The H. cordata extract was resolved with methanol to 1 mg/ml and the S. japonica L. flower bud polysaccharide was resolved with water to 1 mg/ml. Then， 5 l of the solution was loaded on ultramicro spectrophotometer （Nanodrop 2000， Thermo Scientific， USA） to scan from 190 to 800 nm.
　　Assay of reactive oxygen species scavenging
　　Assay of hydroxyl radical scavenging
　　The hydroxyl radicals were generated in an H2O2FeSO4 and were assayed by the color change of salicylic acid[20]. In this experiment， the hydroxyl radicals were generated in 3.0 ml of reactive solution containing 25 mM FeSO4， 2 mM sodium salicylate， 6 mM H2O2 and samples to be tested at different amounts. The mixture was incubated at 37 for 1 h. The change in absorbance caused by the color change of salicylic acid was measured at 510 nm. The scavenging rate of hydroxyl radical production was calculated as follows： Scavenging rate （%） = （A-B）/A≠100， wherein A is the absorbance of the control group in the hydroxyl radical generation system and B is the absorbance of the test group.
　　Assay of superoxide radical scavenging
　　Superoxide radicals were generated in the system of pyrogallolюs autoxidation in an alkalescent condition[21]. In this experiment， the reaction was performed in 3.0 ml of TrisHCl buffer （50 mM， pH 8.2）， which contained 3 mM pyrogallol solution and the samples to be tested at different amounts. The change speed of absorbance （A， min） of the reactive solution was measured at 325 nm. The inhibition of superoxide radicals production was calculated as follows： Inhibition rate （%） = （A-B）/A≠100， wherein A is the change speed of absorbance of the control group in the superoxide radical generation system and B is the change speed of absorbance of the test group.
　　Cell culture
　　HaCaT keratinocytes， a spontaneously transformed human epithelial cell line， were purchased from Cell Resource Center of Peking Union Medical College， China. Cells were grown in DMEM supplemented with 10% heatinactivated FBS， 4 mM nonanimal Lglutamine， 1% penicillinstreptomycin solution， 1% MEM vitamin solution 100≠ and 1% nonessential amino acid solution 100≠， in a humidified incubator aerated with 5% CO2， at 37. Culture medium was changed twice a week.
　　Cell treatment with extract
　　HaCaT cells were exposed for 24 h to H. cordata flavonoid or S. japonica L. flower buds polysaccharide （0.5-4.0 mg/ml， 37） in MEM medium. Cytotoxicity was assessed by MTT later. 　　Then， to assess the protective effect of the extracts， HaCaT cells were pretreated （2 h， 37） with serial concentration of extract （0.25-2.00 mg/ml） in serumfree medium， irradiated， then incubated for another 24 h in culture medium.
　　UV irradiation system
　　The illuminator system consisted of one UVB lamp （peak 312 nm） and one UVA lamp （peak 365 nm） （ZF4， Shanghai Jinpeng company， China）. The irradiances of the UVA and UVB lamps were 156 and 230 w/cm2， respectively. The emitted intensity was measured using the UV 340B radiometer （Sanpometer Company， China）. Before irradiation， culture medium was removed. Cells were rinsed once with phosphatebuffered saline （PBS 1≠）， covered with a thin PBS layer， and irradiated. To prevent the PBS overheating during irradiation， plates were kept on ice. Control cells were treated the same way as the experimental ones but were not exposed to UV rays.
　　Cell viability
　　To assess the effect of H. cordata extract on cell viability and its protection against UVBinduced cytotoxicity， we used the MTT test， according to Mosmann[22]with some modifications. This colorimetric test measures the ability of mitochondrial dehydrogenase of viable cells to cleave the trtrazolium ring and yield purple formazan crystals which are insoluble in aqueous solutions. An increase or decrease in viable cells results in a concomitant change in the amount of formazan formed， indicating the degree of cytotoxicity caused by UV rays.
　　HaCaT cells were seeded in 96well plates and at 80% confluence were exposed to the extract （0.25-2.0 mg/ml） for 2 h， 37. Its effect on cell viability was studied 24 h after the end of the treatment.
　　In order to evaluate the protection effect of the extracts against UVBinduced cytotoxicity， we pretreated cells with each extract （0.25-2.0 mg/ml） for 2 h at 37 and then irradiated them with different doses of UVB （0.03-0.12 J/cm2）. Cell viability was assessed immediately and 24 h after irradiation. To evaluate cytotoxicity， we exposed the cells to the MTT solution （final concentration 0.5 mg/ml） and incubated them for 4 h at 37. The formazan crystals were then dissolved with DMSO and measured spectrophotometrically on a microplate reader at 490 nm （Multimode Plate Reader， Envision， PerkinElmer， USA）. Viable cells were calculated as a percentage of the negative control cells set at 100%.
　　Statistical analysis
　　Values were expressed as mean÷SEM. The analysis of variance （ANOVA） and the Tukey test were used to assess biological activity data， with P<0.05 established as statistically significant. 　　Results and Discussion
　　Full UV wavelength scanning
　　According to the method described in sunscreen cosmetics standard （QB/T24101998）， the UV absorbance of the sample in UVB region （280-320 nm） was determined using ultraviolet spectrophotometer as ultraviolet light source to evaluate the UVB protective ability of sample. 1 mg/ml of H. cordata flavonoid and flower buds of S. japonica L. crude polysaccharide showed high absorbance in UVB region （Fig. 1 and Fig. 2）. The mean absorbance of H. cordata flavonoid and flower buds of S. japonica L. polysaccharide in UVB region was 1.283÷0.401 and 2.517÷0.236， respectively. In additional to this， the UV absorbance of samples in UVA region from 320-400 nm was also determined （Fig. 1 a and b）. The results suggest that the two extracts have farranging and high absorbance in UVA and UVB， might be a candidate for UV sunscreening supplement to protect skin.
　　Assay of reactive oxygen species scavenging
　　Hydroxyl free radical ·OH） and superoxide anion radical （O-2） were evaluated the antioxidant ability of the extracts. Vc was a positive control group. Superoxide anion radical was initially generated from exposure to UVA. The generation of hydroxyl free radical was related to UVB irradiation. The scavenging ability of H. cordata flavonoid and S. japonica L. polysaccharide on hydroxyl free radical increased following with the increased concentration， and showed dosedependent relationship （Fig. 2a and Fig. 2b）， and S. japonica L. extract showed better ability than H. cordata extract to scavenge hydroxyl free radicals.
　　The scavenging rate of H. cordata extract and S. japonica L. extract on superoxide anion radical showed a dosedependent manner， but their scavenging ability was less than that of Vc （Fig. 3a and Fig. 3b）.
　　Cytotoxicity of extracts
　　The cell viability after drug administration was shown in Fig. 4a and Fig. 4b. For H. cordata flavonoid， the cell viability was 96.79%， 93.15%， 102.52% and 84.82% at a serial concentration. 4 mg/ml of H. cordata polysaccharide decreased the cell viability slightly， so 2 mg/ml was the highest concentration for the following experiments. For S. japonica L. polysaccharide， there was no significant cytotoxicity even at a concentration of 4 mg/ml. Therefore， 2 mg/ml was used as the maximal concentration for the further experiments.
　　HaCaT cells were exposed to different concentrations of the extract at 37， 5% CO2. Cytotoxicity was assessed immediately after the end of exposure and 24 h later. Results are shown as percentage of viable cells （% cell viability） compared to negative control cells （ctr） set on 100%. Each data point is the mean of three independently reproduced experiments （Oneway ANOVA test， Dunnettюs Multiple Comparison Test）. 　　HaCaT cells were pretreated with H. cordata extract （0.25-2.0 mg/ml） for 1 h and then irradiated by UVB （2.1-12.6 J/cm2）， respectively， and incubated in fresh cell culture medium at 37， 5% CO2 for another 24 h. Results are expressed as percentage of viable cells （%） compared to control cells without irradiation and H. cordata flavonoid on 100%. Each data point is the mean of three independently reproduced experiments. *P<0.05 vs. control without H. cordata extract （Oneway ANOVA test， Dunnettюs Multiple Comparison Test）.
　　Cell viability
　　UVB significantly reduced HaCaT viability and showed in a dosedependent manner. The cell viability was 54% with the UVB dose of 30 mJ/cm2 and decreased to 24.58% with UVB dose of 120 mJ/cm2. It suggests that UVB ray has a delayed effect on cell damage. However， pretreatment with S. japonica L. polysaccharide significantly decreased UVBinduced cytotoxicity and also showed a dosedependent relationship （Fig. 6）. For each UV irradiation group， the cell viability increased with the extract concentrations increasing （P<0.05）， while for H. cordata flavonoid （Fig. 5）， it had a similar protective effect on cells with S. japonica L. polysaccharide， but the activity was much less than that of the later one. At the doses of 30 and 60 mJ/cm2， the 1.0 and 2.0 mg/ml H. cordata flavonoid treatments showed good antiUVB ability， but for the high UVB dose， its antiUVB ability was slight.
　　HaCaT cells were exposed to different concentrations of extract （0.25-2.00 mg/ml） for 2 h at 37， 5% CO2 and then irradiated by UVB （30-120 mJ/cm2）， respectively. Cytotoxicity induced by UVB was assessed 24 h after the end. Results are expressed as percentage of viable cells （%） compared to control cells without irradiation and PS on 100%. Each data point is the mean of three independently reproduced experiments. *P<0.05 vs. the same treatment w/o extract （Oneway ANOVA test）.
　　Excessive UV irradiation can cause ROS accumulated in the body. ROS can attack， infiltrate and damage the structure of the skin cells， producing lipid peroxides in damaged parts of the cell membrane， causing a series of mutation processes. UVA rays account for up to 95% of the UV radiation that reaches the Earthюs surface. Exposure of the skin to UVA radiation increases the production of cellular reactive oxygen species （ROS）， indulging singlet oxygen （1O2）， superoxide anion （O-2）， while the generation of hydroxyl free radical is related to UVB irradiation[23]. Therefore， the scavenging ability of extracts to hydrogen free radical and superoxide anion radical was evaluated in this research. The results showed that the S. japonica L. polysaccharide had excellent clear activity on free radicals， especially on hydroxyl free radicals. This is consistent with the results of cell viability UVBinduced. The HaCaT cells pretreated by S. japonica L. polysaccharide had high viability after UVB radiation. It suggests that the high antiUVB ability of S. japonica L. polysaccharide might be obtained through scavenging hydrogen free radicals. 　　The scavenging activity of H. cordata extract and S. japonica L. extract on superoxide anion was less than that of Vc， while the extracts showed high absorbance in UVA region. It suggests that the extracts had directly strong UVA absorption ability， so they could protect skin cells by UVA absorption directly and scavenging superoxide anions induced by UVA radiation indirectly. It also compensate for the shortcomings of traditional chemical sunscreens against UVA. In brief， the H. cordata flavonoid and S. japonica L. polysaccharide could be good UV absorbers and combined together to protect skin cells from ultraviolet radiation damage.
　　Conclusions
　　H. cordata flavonoid and S. japonica L. polysaccharide were investigated in UV scanning spectrum， antioxidant ability and cell viability after UVB irradiation. The results showed that the extracts had strong UVA and UVB absorption ability， hydroxyl free radical scavenging ability. The S. japonica L. extract showed strong antiUVB irradiation ability according to the cell viability experiment. It suggests that the H. cordata extract and S. japonica L. extract could be combined as a candidate of antiUV sunscreening supplement and compensate for the shortcomings of traditional chemical sunscreens against UVA ray.
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