Primary Research on UVB-irradiation Protective Effect of Total Flavonoids from Large-leaved Kuding Tea (Ilex latifolia Thunb.)

Liyan LI Tao HUANG Sen PANG Xiaoning LIU

Abstract Total flavonoids extracted from large-leaved Kuding tea (Ilex latifolia Thunb.) were investigated to develop anti-UV sun-screening agents. The results showed that total flavonoids of I. latifolia Thunb. (TFILT) showed maximum absorb peak at 325 nm between UVB and UVA by wavelength scanning spectrum. TFILT could inhibit the decrease of cell viability induced by UVB irradiation by CCK-8 method, and 0.25 mg/ml of TFILT showed the most significant protective activity on cells. The dermal thickness and collagen fiber density as skin structural characteristic were analyzed by Histological staining. The results showed that TFILT protected mice skin by significantly attenuating the dermal thickness and inhibiting collagen fiber degradation. The mechanism in vivo needs to be further confirmed in future.

Key words Large-leaved Kuding tea; Ilex latifolia Thunb.; Total flavonoids; Anti-UV

Received: June 9, 2022  Accepted: August 16, 2022

Supported by Henan Provincial Department of Education (21B350001); Zhengzhou Science and Technology Department (ZZSZX202109; ZZSZX202108).

Liyan LI (1974-), female, P. R. China, associate professional, devoted to research about anti-cancer and anti-UV irradiation of natural product.

*Corresponding author. E-mail: liyanli0921@163.com.

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 earths surface comprises approximately 90%-99% UVA and 1%-10% of UVB［1-2］. UVB is more genotoxic and about 1 000 times more capable of causing sunburn than UVA, so most research about anti-UV was focused on UVB. Recently, attention has been focused on natural products or extracts from plants due to some disadvantages of chemical absorbers and physical shielding reagents such as TiO2 and ZnO.

Kuding tea is a particularly bitter-tasting tea used widely in China throughout history. The large-leaved Kuding tea latin named Ilex latifolia Thunb was certified to be the original Kuding Tea species［3］. Large-leaved Kuding tea showed many abilities such as antioxidant, lipid metabolism, hepatoprotective and anti-atheosclerotic activity, ameliorate lipid accumulation［4-11］, but the anti-UV ability has not been reported.

Therefore, ultraviolet absorption characteristic, protective activity on cells and mice skin of total flavonoids of I. latifolia Thunb (TFILT) was investigated primarily in the present research, in order to develop a kind of sun-screening agent derived from plant source.

Materials and Methods

Chemicals and reagents

Leaves of large-leaved Kuding tea (I. latifolia Thunb) was purchased from Yihongtang Pharmaceutical Co. Ltd of Bozhou, Anhui. Modified Eagles medium (MEM), penicillin-streptomycin solution, and non-animal L-glutamine and trypsin-EDTA solution was purchased from Life Technologies Company. Fetal bovine serium (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. HaCaT cells were purchased from Cell Resource Center of Chinese Union Medical College. CCK-8 kit was purchased from Beijing Sorlabio Biological Co., Ltd. All other reagents were analytical pure.

Methods

Extract of TFILT

Leaves of I. latifolia Thunb was dried and crushed into 200 meshes. The powder was soaked with petroleum ether in room temperature for 48 h to remove fats from plant. The powder was dried and soaked with 60% ethanol at a ratio of 1 g∶40 ml (w/v) for 2 h and then ultrasonic-assisted extracted at 30 ℃ for 0.5 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 freeze-dried.

Full UV wavelength scanning

TFILT was resolved with methanol to 1 mg/ml. Then, 5 μl of the solution was loaded on ultra-micro spectrophotometer (Nanodrop 2000, Thermo Scientific, USA) to scan from 190-480 nm.

Cell culture

HaCaT cells line and Raw 264.7 cell lines were purchased from Procell company (Wuhan, China). HaCaT cells were cultured in MEM supplemented with 10% FBS, 4 mM non-animal L-glutamine, 1% penicillin-streptomycin solution, in a humidified incubator aerated with 5% CO2, at 37 ℃. Raw 264.7 cells were cultured in the condition mentioned above except DMEM instead of MEM. The culture medium was changed twice a week.

Cytotoxicity of TFILT

The cells were exposed for 48 h to TFILT (0.25-4.00 mg/ml) in complete medium, and CCK-8 method was used to determine the cytotoxicity of TFILT. Results are shown as percentage of viable cells (% cell viability) compared to negative control cells (ctr) set on 100%.

Cell viability after UVB irradiation

HaCaT cells were seeded in 96-well plates at a density of 1×105 cells/ml. The cells were exposed to TFILT (0.25-2.00 mg/ml) for 1 h after proliferating to 80% confluence. The culture medium was removed. Cells were rinsed once with phosphate-buffered saline (PBS), covered with a thin PBS layer, and irradiated with UVB in doses of 30, 60, 90 and120 mJ/cm2, respectively. The plates were kept on ice in case of cells overheating. Control cells were treated in the same way but were not exposed to UVB rays. The cells were incubated for another 24 h in MEM. Cell viability was estimated by CCK-8 method. 10% CCK-8 solution was used to incubate cells for 1 h and OD450 was measured (Multimode Plate Reader, Envision, PerkinElmer, USA). Viable cells were calculated as a percentage of the negative control cells set at 100%.

Determination of NO production

Raw 264.7 cells were seeded in a 96 well plate with density of 2 ×105 cells/ml for overnight. The cells were then treated with LPS (1 μg/ml), TFILT (1.0, 0.5, 0.25, 0.125 mg/ml) containing LPS (the final concentration was 1 μg/ml), DXM (20 μg/ml) as positive control containing LPS (the final concentration was 1 μg/ml) for 24 h. The cells without any drugs were as control. Then the supernatant was collected by centrifuging at 1 000 rpm at 4 ℃ for 10 min for Griess assay.

Griess assay［12］ was used to evaluate the level of nitrite in culture media which was a major stable product of NO. The protocol has been reported in 2021［13］.

Animal management and UV irradiation

Five-week-old female C576B/L mice (weight, 21.5-27.5 g) were purchased from Experiment Animal center of Zhengzhou University. The trial was approved by the Institutional Animal Care and Use Committee of Huanghe Science and Technology University (approval no. 2022-009, 20, April, 2022). Six mice per group, 30 mice were divided into five groups randomly. They were raised in a stationary condition (24 ℃, 50% humidity) in 12 h light /12 h dark cycles. The skin on the dorsal of mice was exposed by depilation cream and then irradiated by different doses of UV light to determine one MED. The erythema formation was estimated after 24 h. The sites of hair removal were external applied with TFILT (0.25, 0.5 and 1 mg/cm2, respectively) for 0.5 h and then radiated by 100 mJ/cm2 of UVB, the process applied again 24 h later. The mice skin tissues were obtained after sacrificed mice using CO2 gas.

Histological staining

The dermal thickness and collagen fiber density as skin structural characteristic could be analyzed by Histological staining. The skin samples for each experiment group were fixed in 10% formalin at 4 ℃ for 2 d and then washed, dehydrated, permeated and embedded using paraffin. Hematoxylin & eosin (H & E) staining was performed to determine the dermal thickness. Massons trichrome and toluidine blue staining (MT staining) was performed to compare the collagen fiber density among different experiment groups, P<0.01 as extremely significant difference.

Statistical analysis

Values were expressed as mean±SEM. The analysis of variance (AVONA) and the Tukey test were used to assess biological activity data, with *P<0.05 established as statistically significant, **P<0.01 established as statistically extremely significant.

Results and Discussion

Full UV wavelength scanning

The ultraviolet scanning Spectrum of TFILT is shown in Fig.1. The spectrum showed that TFILT had a strong absorb peak between 290 to 355 which were UVB and UVA waveband range, which provides an evidence for TFILT about its anti-UVB and UVA ability.

Cytotoxicity of TFILT

HaCaT cells were exposed to different concentrations of the TFILT and cytotoxicity was assessed after exposure 48 h later. The result is shown in Fig. 2a. The cell viability was 100%, 116.25%, 126.58%, 114.26%, 32.24% and 25.82% following with the increased concentration, respectively. Less than 1 mg/ml of TFILT showed its pro-proliferative activity, and more than 2 mg/ml of TFILT inhibited the cell proliferation and show cytotoxicity. For Raw 264.7 cells, TFILT showed similar tendency, and showed significant proliferative activity at 0.25 mg/ml and the viability decreased following with the increased concentration.

Cell viability after UVB irradiation

UVB significantly reduced HaCaT viability from 90.22% with UVB dose of 30 mJ/cm2 to 36.72% at dose of 120 mJ/cm2 and showed in a dose-dependent manner which we had reported. Comparing with UVB-vacuum group, pretreatment of TFILT significantly decreased UVB-induced cytotoxicity and showed a negative correlation with concentration in range of 0.25 to 1.0 mg/ml (P<0.05). 0.25 mg/ml of TFILT showed the best cell viability of 87.10% at 120 mJ/cm2, and cell viability of 1.0 mg/ml of TFILT at 120 mJ/cm2 was 77.06%. For 2.0 mg/ml of TFILT, although it showed cell cytotoxicity to a certain extent, the cell viability was about 81.36% at 120 mJ/cm2. It was possible that 2.0 mg/ml of TFILT preferentially absorbed UVB to protective cells from UVB irradiation damage.

Effects of TFILT on NO production in LPS-Stimulated RAW 264.7 cells

The measurement of NO production was used to reflect indirectly anti-inflammatory ability of TFILT in vitro. After the treatment with TFILT and LPS stimulation, NO concentration in the cultured medium was determined here. For the control group, the basal concentration of NO in Raw 264.7 macrophages was 0.03 μM, but this value was significantly increased to 0.285 μM after the treatment with 1 μg/ml of LPS. TFILT treatment could decrease the NO level to 0.13 and 0.23 at concentration of 1.0 and 0.5 mg/ml, respectively, which were more significant than DXM group. It confirmed that TFILT had anti-inflammatory activity.

Liyan LI et al. Primary Research on UVB-irradiation Protective Effect of Total Flavonoids from Large-leaved Kuding Tea (Ilex latifolia Thunb.)

Effects of TFILT on skin histology

In order to confirm the results of TFILT in vitro, C576B/L mice was used to build UVB-irradiation skin model. Dermal thickness of the dorsal skin was measured via HE staining (Fig.5 A and B) and collagen fiber structures/density were assayed by MT staining (Fig. 6). Comparing with the control group, the dermal thickness of UVB group (Fig. 5A-b) increased about 36.7 nm. The pretreatment of TFILT on skin decreased significantly the dermal thickness (Fig.5A-c, d, e), and for 1 mg/cm2 of TFILT group (Fig.5A-e), the dermal thickness was similar to that of the normal group (Fig. 5A-a). For MT staining groups, comparing with the normal group (Fig.6-a), the density of collagen fiber in UVB vehicle group significantly decreased (Fig.6-b).  The pretreatment of TFILT on UVB-radiated mice skin could inhibit the decreasing density of collagen fiber in concentration-dependent relationship. The results suggested that TFILT significantly inhibited UVB-induced changes in dermal thickness and collagen fiber degradation.

Many researches on plants extracts or their single constituents have been reported for their UV-protective potentials due to their abilities to reduce the skin damage of UV ray and prevent or treat UV mediated dermatological diseases［14-18］. The large-leaved Kuding tea (I. latifolia Thunb.) was reported to have antioxidant, lipid metabolism, hepatoprotective and anti-cancer activity［4-7,19］. However, the photo-protective effects of I. latifolia Thunb. on UVB-irradiated keratinocytes are not reported ever. Therefore, we investigated primarily the photoprotecive ability of TFILT from three ways including its UV absorb property, experiments in vitro and in vivo in present research. UV scanning results identified that TFILT had UVB and UVA absorb property, which is important for screening anti-UV potential plant extract or natural products quickly. Then, in vitro experiment identified that TFILT had photoprotective and anti-inflammatory abilities on cell model, which were further identified in vivo by mice skin UV-irradiation model. TFILT could protect skin from UVB damage by decreasing the dormal thickness and inhibiting collagen fiber degradation. Wrinkle formation was also significantly reduced in mice skin compared with UVB group (data not shown). It is similar with the report by Choi SL et al.［20］ who identified some processed products from agricultural produce such as fermented rice bran (FRB), soybean cake (FSB) and sesame seed cake (FSC) inhibited collagen degradation and mast cell infiltration were reduced in hairless mouse skin.

Our experiment in vivo and in vitro preliminarily identified the anti-UVB irradiation activity of TFILT, the further mechanism needs to be confirmed in future.

Conclusion

Our results showed that total flavonoids of I. latifolia Thunb. (TFILT) has UVB and UVA absorb properties by wavelength scanning spectrum. Pretreatment with TFILT attenuated UVB-induced cytotoxicity significantly. The anti-inflammatory activity of TFILT was confirmed by NO production measurement. TFILT also protected mice skin by significantly attenuating the dermal thickness and inhibiting collagen fiber degradation in vivo. The results suggest that TFILT has great potential as a sunscreen additive and topical anti-inflammatory drug.

References

［1］ VOSTALOVA J, ZDA ILOV A, SVOBODOV A. Prunella vulgaris extract and rosmarinic acid prevent UVB-induced DNA damage and oxidative stress in HaCaT keratinocytes［J］. Arch Dermatol Res, 2010(302): 171-181.

［2］ YAMADA Y, YASUI H, SAKURAI H. Suppressive effect of caffeic acid and its derivatives on the generation of UVA-induced reactive oxygen species in the skin of hairless mice and pharmacokinetic analysis on organ distribution of caffeic acid in ddY mice［J］. Phtotchem. Photobiol, 2006(82): 1668-1676.

［3］ HE ZD, LAU KM, BUT PPH, et al. Antioxidative glycosides from the leaves of Ligustrum robustum［J］. J Nat Prod, 2003(66): 851-854.

［4］ NISHIMURA K, FUKUDA T, MIYASE T, et al. Activity-guided isolation of triterpenoid acyl CoA cholesteryl acyl transferase (ACAT) inhibitors from Ilex kudincha［J］. J. Nat. Prod.,1999(62): 1061-1064.

［5］ NISHIMURA K, MIYASE T, NOGUCHI H. 1999b. Triterpenoid saponins from Ilex kudincha［J］. J. Nat. Prod. (62): 1128-1133.

［6］ WOO AY, JIANG JM, CHAU CF, et al. Inotropic and chronotropic actions of Ilex latifolia inhibition of adenosine-5-triphosphatases as a possible mechanism［J］. Life Sci. 2001(68): 1259-1270.

［7］ Li L, XU LJ, MA GZ, DONG YM. The large-leaved Kudingcha (Ilex latifolia Thunb. and Ilex kudingcha C.J. Tseng): A traditional Chinese tea with plentiful secondary metabolites and potential biological activities［J］. J. Nat. Med., 2013(67): 425-437.

［8］ ZHANG TT, HU ZT, JIANG JG, et al. Polyphenols from Ilex latifolia Thunb. (a Chinese bitter tea) exert anti-atherosclerotic activity through suppressing NF-κB activation and phosphorylation of ERK1/2 in macrophages［J］. Med. Chem. Comm. 2018(9): 254-263.

［9］ WU H, CHEN YL, YU Y, et al. Ilex latifolia Thunb. protects mice from HFD-induced body weight gain［J］. Sci. Rep. 2017(7): 14660.

［10］ FENG RB, FAN CL, LIU Q, et al. Crude triterpenoid saponins from Ilex latifolia (Da Ye Dong Qing) ameliorate lipid accumulation by inhibiting SREBP expression via activation of AMPK in a non-alcoholic fatty liver disease model［J］. Chin. Med., 2015(10): 23-35.

［11］ FAN S, ZHANG Y, HU N. Extract of Kuding tea prevents high-fat diet-induced metabolic disorders in C57BL/6 mice via liver X receptor (LXR) β antagonism［J］. PLoS ONE, 2012(7): e51007.

［12］ SITTISART P, CHITSOMBOON P, KAMINSKI NE. Pseuderanthemum palatiferum leaf extract inhibits the proinflammatory cytokines, TNF-α and IL-6 expression in LPS-activated macrophages［J］. Food Chem Toxicol, 2016(97): 11-22.

［13］ LI LY, HUANG T, LAN C. Anti-inflammatory activity and mechanism of total flavonoids from the phloem of Paulownia elongate S.Y. Hu in LPS-stimulated RAW 264.7 macrophages［J］. Agricultural biotechnology, 2021, 10(3): 117-120, 124.

［14］ CHEN F, TANG Y, SUN YJ. 6-shogaol, an active constituents of ginger prevents UVB radiation mediated inflammation and oxidative stress through modulating NrF2 signaling in human epidermal keratinocytes (HaCaT cells)［J］. J. Photochem. Photobiol. B, 2019(197): 1115-1118.

［15］ KIM JK, KIM Y, NA KM, et al. Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo［J］. Free Radic. Res., 2007(41): 603-614.

［16］ HASOVA M, CRHAK T, SAFRANKOVA B, et al. Hyaluronan minimizes effects of UV irradiation on human keratinocytes［J］. Arch. Dermatol. Res., 2011(303): 277-284.

［17］ LIN P, HWANG E, NGO HTT, et al. Sambucus nigra L. ameliorates UVB-induced photoaging and inflammatory response in human skin keratinocytes［J］. Cytotechnology, 2019(71): 1003-1017.

［18］ AZAHARA RL, JAVIER BR, HELENA O. Fucoxanthin and rosmarinic acid combination has anti-inflammatory effects through regulation of NLRP3 inflammasome in UVB-exposed HaCaT keratinocytes［J］. Mar. Drugs, 2019(17): 451-464.

［19］  KIM JY, JEONG HY, LEE HK, et al. Protective effect of Ilex latifolia, a major component of "kudingcha", against transient focal ischemia-induced neuronal damage in rats［J］. J. Ethnopharmacol., 2011(133): 558-564.

［20］ CHOI SI, JUNG TD, CHO BY, et al. Anti-photoaging effect of fermented agricultural by-products on ultraviolet B-irradiated hairless mouse skin［J］. International Journal of Molecular Medicine, 2019(44): 559-568.