• Users Online: 265
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 6  |  Issue : 2  |  Page : 62-71

Protective effect of flavonoids from Foeniculum vulgare against ultraviolet-B-induced oxidative stress in human dermal fibroblasts


Department of Biological Sciences, Sunandan Divatia School of Science, NMIMS (Deemed-to-be) University, Vile Parle (West), Mumbai, Maharashtra, India

Date of Submission11-Sep-2019
Date of Decision10-Oct-2019
Date of Acceptance18-Oct-2019
Date of Web Publication22-Nov-2019

Correspondence Address:
Dr. Purvi Bhatt
Department of Biological Sciences, Sunandan Divatia School of Science, Vile Parle (W), Mumbai - 400 056, Maharashtra
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/BMRJ.BMRJ_22_19

Rights and Permissions
  Abstract 


Background: Traditionally, Foeniculum vulgare (fennel seeds) has been used for its antimicrobial, analgesic, antipyretic, antiflatulence, antispasmodic, and antiandrogenic activities. Materials and Methods: In the present study, the protective effect of flavonoids from fennel seeds was investigated against ultraviolet (UV)-B radiation-induced cell damage and oxidative stress in human dermal fibroblast (HDF) cells. Results: Flavonoid-enriched fraction (FEF) of fennel seeds showed high flavonoid content and antioxidant potential as well as the presence of a marker compound rutin. Pretreatment of HDF cells with the FEF (15–45 μg/ml) significantly protected against UV-B-induced cytotoxicity, endogenous enzymatic antioxidant depletion, oxidative DNA damage, intracellular reactive oxygen species generation, and apoptotic morphological changes. Conclusion: The current study proved for the first time that the FEF of fennel seeds reduced oxidative stress through the nuclear factor E2-related factor 2-antioxidant response elements pathway. Flavonoids from fennel seeds have a potential as UV-B protectants and can be explored against diseases, in which oxidative stress is closely implicated.

Keywords: Antioxidants, DNA damage, nuclear factor E2-related factor 2-antioxidant response elements pathway, oxidative stress


How to cite this article:
Patwardhan J, Bhatt P. Protective effect of flavonoids from Foeniculum vulgare against ultraviolet-B-induced oxidative stress in human dermal fibroblasts. Biomed Res J 2019;6:62-71

How to cite this URL:
Patwardhan J, Bhatt P. Protective effect of flavonoids from Foeniculum vulgare against ultraviolet-B-induced oxidative stress in human dermal fibroblasts. Biomed Res J [serial online] 2019 [cited 2019 Dec 16];6:62-71. Available from: http://www.brjnmims.org/text.asp?2019/6/2/62/271484




  Introduction Top


Ultraviolet (UV) radiation is a type of electromagnetic radiation and is divided into three types – UV-C (100–280 nm), UV-B (280–315 nm), and UV-A (315–400 nm). UV radiation has ionizing as well as mutagenic potentials causing various immune-mediated diseases or even cancer. Out of the total radiation reaching the earth, 95% is UV-A, 5% is UV-B, but UV-B is significantly erythematogenic and genotoxic than UV-A [1] causing direct DNA damage by formation of cyclobutane pyrimidine dimers, pyrimidine-pyrimidone photoproducts, mutations in genes; and indirect damage to various biomolecules by free radical production, which causes protein modifications, lipid peroxidation damaging the cells, and disruption of the oxido-redox status.[2],[3],[4]

Oxidative stress depletes endogenous enzymatic and nonenzymatic antioxidants; hence, exogenous antioxidants are essential to neutralize the free radicals. Synthetic antioxidants and sunscreens are used against UV-induced oxidative stress but are reported to have damaging effects on continual use involving the conversion of sunscreen components into free radicals and increase the risk of Vitamin D deficiency.[5] Free radicals escape the antioxidant defense, damaging collagen and elastin, causing photoaging. Fibroblasts present in the subcutaneous and submucosal tissues are necessary for tissue repair.[4]

Therefore, plant molecules are being used as effective antioxidants against oxidative stress. Due to the physiological processes, small doses of orally supplemented antioxidants reach the skin; however, topical antioxidants are now being developed which can overcome the limitations.[2]

Nuclear factor E2-related factor 2-antioxidant response elements (Nrf2-ARE) pathway is an important pathway for antioxidant defense. Nrf2 is a transcription factor bound to its inhibitor protein Keap1 in the cytosol undergoing constant ubiquitination. Oxidative stress makes the bond between Nrf2 and Kelch-like ECH-associated protein-1 (Keap1) unstable, increasing level of free Nrf2 facilitating its translocation in the nucleus and binding to ARE in the promoter regions of antioxidant enzyme, and Phase II detoxification enzyme genes such as heme oxygenase-1 (HO-1), glutathione S-transferase, and NAD(P)H: quinine oxidoreductase 1.[6],[7] In the current study, regulation of HO-1 gene was studied through the Nrf2-ARE pathway.

Foeniculum vulgare (fennel seeds) has been traditionally used as antimicrobial, analgesic, antipyretic, antispasmodic, cardiovascular, antiandrogenic, and antioxidant agent.[8],[9],[10] It contains 3% of essential oils with anethole as a prominent constituent (70%). Some compounds, such as quercetin, rutin, isoquercitrin, and rosaminaric acid, have been identified in fennel seeds.[9] UV-B protective effect of various plant molecules such as silymarin,[11] ursolic acid,[12] ferulic acid,[13] sesamol,[4] and epicatechin gallate [14] have been studied extensively. In the present study, UV-B protective potential of flavonoid-enriched fraction (FEF) from fennel seeds has been studied against UV-B-induced cytotoxicity, endogenous enzymatic antioxidant depletion, generation of reactive oxygen species (ROS), DNA damage, apoptotic morphological changes, and regulation of HO-1 through Nrf2-ARE pathway in human dermal fibroblast (HDF) cells.


  Materials and Methods Top


Chemicals

HDF was obtained from the Scientific Research Centre, VG Vaze College, Mumbai; Silica Gel 60 F254 precoated plates, goat antimouse HRP conjugated secondary antibody from Merck (NJ, USA); Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), Dulbecco's phosphate-buffered saline (DPBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), penicillin-streptomycin from Genetix Biotech (New Delhi, India); 2',7'-dichlorofluorescein diacetate (DCFH-DA) dye, monoclonal antibodies against Nrf2 and HO-1 from Abcam (MA, USA); Natural product reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), monoclonal antibody against β-actin, silymarin, TRI reagent from Sigma (St. Louis, MO, USA); MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)from Himedia Labs (Mumbai, India); ethidium bromide (EtBr) and acridine orange (AO) from SRL (Mumbai, India); cDNA synthesis kit from TAKARA (Shiga, Japan); Primers for Nrf2 and HO-1 from Eurofins (Luxembourg, Germany); SYBR green real-time polymerase chain reaction (PCR) master mix from Roche (BASEL, Switzerland); enhanced chemiluminescence (ECL) detection kit from Biorad (Berkeley, California); X-ray films from Kodak (NY, US); and protease inhibitor cocktail from Amresco (OH, USA). All other chemicals, reagents, and solvents were of analytical grade from S.D. Fine Chemicals (Mumbai, India) and Fischer Inorganic and Aeromatic Limited (Mumbai, India).

Extraction and enrichment of flavonoids

The fennel seeds subjected to cold extraction using n-hexane, chloroform, and alcohol successively as previously reported.[15] Preliminary phytochemical analysis of crude extracts detected flavonoids in the alcoholic extract, and hence, it was used for enrichment of flavonoids.

Alcoholic extract (10 g) was dissolved in 100 ml of distilled water and fractioned thrice using ethyl acetate.[16] Flavonoid-enriched ethyl acetate fraction (FEF) was pooled, concentrated, and stored in vacuum till further use.

Thin-layer chromatography, thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl, and high-performance thin-layer chromatography analysis

Flavonoids from the crude alcoholic extract and the separated fractions were detected by thin-layer chromatography (TLC) analysis using rutin as a standard. The samples were spotted on silica gel 60 F254 precoated plates and the solvent system used was as reported previously.[15] The plates were derivatized using 1% natural product reagent and observed under UV light (254 nm and 366 nm) as previously reported.[17] Another derivatizing reagent used was 0.1% DPPH and the plates were observed under visible light.[18] High-performance thin-layer chromatography (HPTLC) was performed for FEF after derivatization with natural product reagent. Plates were scanned at 420 nm using DESAGA HPTLC system and Rf values and spectra were recorded using ProQuant software (DESAGA, Germany).

Determination of flavonoid content

Flavonoid content of the crude extract and FEF were evaluated by a previously described method.[18],[19] Quercetin was used as the standard in the concentration range of 10–100 μg/ml.

Antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl, ferric reducing antioxidant power, and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) assay

The antioxidant potential of FEF was determined by DPPH, ABTS, and ferric reducing antioxidant power assays as previously described [20],[21],[22] and compared to crude alcoholic extract.[15]

UV-B protective potential of FEF was further studied in HDF.

Cell culture

HDF (ATCC No. PCS-201-012) were grown in DMEM containing 10% FBS, 100 units/ml of penicillin, 0.1 mg/ml of streptomycin, and 2.5 μg/ml of amphotericin at 37°C, 5% CO2.

Experimental groups

HDF was divided into seven different treatment groups as follows:

  • Group 1 – Untreated control
  • Group 2 – FEF treated (45 μg/ml FEF-treated fibroblasts)
  • Group 3 – UV-B irradiated
  • Group 4 – UV-B-irradiated cells treated with 5 μg/ml silymarin
  • Group 5 – UV-B-irradiated cells treated with 15 μg/ml FEF
  • Group 6 – UV-B-irradiated cells treated with 30 μg/ml FEF
  • Group 7 – UV-B-irradiated cells treated with 45 μg/ml FEF.


Treatment of cells

Cultured HDF cells were treated with various concentrations of FEF (Group 2, 5, 6, 7) and silymarin (Group 4) 24 h prior to UV-B irradiation. Preliminary studies were carried out using MTT assay to confirm that these concentrations had no toxic effect. After 24 h of FEF and silymarin treatment, cells were washed once with DPBS and covered with minimum volume of serum-free DMEM followed by UV-B irradiation.

Irradiation procedure

A UV-B tube (Sankyo Denki, Japan) served as a UV-B source with a wavelength range of 280–315 nm which peaked at 312 nm. Cells were irradiated at an intensity of 5 mW/cm 2 for 500 s with 2.5 J/cm 2 radiation reaching the cells, decreasing the viability to 50%. After irradiation, cells were incubated at room temperature for 30 min and then used for further studies.

MTT assay

Cultured cells (5 × 104) were seeded in each well of the 96 well plate followed by FEF (15, 30, 45 μg/ml) and silymarin (5 μg/ml) treatment and incubation for 24 h at 37°C and 5% CO2. After incubation, wells were washed once with DPBS and UV-B irradiated (500 s, i.e., 2.5J/cm 2) followed by MTT addition (5 mg/ml) and incubation at 37°C for 4 h.[23] The absorbance was read on an enzyme-linked immunosorbent assay reader at 570 nm.

Estimation of levels of endogenous antioxidants

Cell lysates from the treatment groups were prepared using a lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 50 mM HEPES, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 1 mM PMSF, and ×1 protease inhibitor cocktail. Protein quantification was done by Folin–Lowry's assay followed by estimating levels of SOD, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) by previously described method.[24],[25],[26],[27]

Comet assay

Cultured HDF (1 × 106) were seeded in 6 well plate and treated with FEF and silymarin followed by UV-B irradiation (500 s, i.e., 2.5 J/cm 2). Comet assay was performed as previously described.[4] 100 comets of each treatment group were visualized at ×400 magnification using a fluorescence microscope and analyzed using Casp software version 2.0 (Krzysztof Konca, CaspLab.com), percent head DNA was calculated and statistically analyzed.

Ethidium bromide/acridine orange staining

After FEF pretreatment and UV-B treatment, the cells were stained using 1:20 diluted mixture of EtBr (100 μg/ml) and AO (100 μg/ml). Cells were observed under × 400 magnification using a fluorescence microscope.

Measurement of intracellular reactive oxygen species by 2',7'-dichlorofluorescein diacetate

After FEF pretreatment and UV-B treatment, cells were washed and resuspended in DPBS. DCFH-DA dye (1 μM, 10 μl) was added to the cells and incubated for 45 min at 37°C. The cells were then analyzed by flow cytometry using 488 nm laser wavelength and 535 nm detection wavelength.

Ames test

Mutagenic potential of FEF was determined by Ames test using  Salmonella More Details typhimurium strain TA100. The strain identification tests (histidine requirement, Rfa mutation, UVrB mutation, and R-factor assay) were carried out as previously described [28] to ensure the properties of the strain have been retained. Ames test was performed as previously described.[29],[30]

Quantitative polymerase chain reaction analysis for nuclear factor E2-related factor 2 and heme oxygenase-1

Quantitative PCR (qPCR) analysis was performed for Nrf2 and HO-1 genes using 18S rRNA as internal control. Total RNA was extracted from treatment groups using TRI reagent method followed by cDNA synthesis using TAKARA PrimeScript first-strand cDNA synthesis kit. qPCR analysis was done using the SYBR Green real-time PCR Master Mix. The designed primer sequences and the standardized cycling conditions for the real-time analysis are given in [Table 1] and [Table 2].
Table 1: Primer sequences and product sizes of nuclear factor E2-related factor 2, heme oxygenase-1, and 18S rRNA

Click here to view
Table 2: Standardized cycling conditions for nuclear factor E2-related factor 2, heme oxygenase-1, and 18S rRNA

Click here to view


Western blot analysis for nuclear factor E2-related factor 2 and heme oxygenase-1

Cell lysates were prepared and quantified, as mentioned earlier. They were electrophoresed and transferred on the nitrocellulose membrane. For Nrf2, the membrane was blocked using 5% BSA followed by probing with 1:1000 diluted monoclonal Nrf2 antibody and 1:2000 diluted goat antimouse HRP conjugated secondary antibody. The expression of the protein was detected by ECL technique. Membrane was stripped using stripping buffer (10% SDS, 0.5 M Tris-Cl, β-mercaptoethanol) at 55°C for 30 min and washed thrice using phosphate-buffered saline with 0.1% tween20. The membrane was blocked using 5% BSA, reprobed using 1:500 diluted monoclonal HO-1 antibody followed by the above-given procedure. The blot was restripped and reprobed for internal control β-actin using 5% NFDM as blocking reagent and 1:1000 diluted monoclonal β-actin antibody followed by the above-given procedure.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). by one-way ANOVA followed by Dunnett's and Tukey's posttest, ***P < 0.001. Results are expressed as Mean ± SD.


  Results Top


Thin-layer chromatography, thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl, and high-performance thin-layer chromatography analysis

Qualitative separation of flavonoids in crude alcoholic extract and FEF was done by TLC using rutin as a standard. In [Figure 1]a and [Figure 1]b, lanes 1–4 demonstrate rutin, crude alcoholic extract, FEF, and water fraction, respectively. Presence of rutin was observed in crude alcoholic extract as well as the FEF. FEF indicated the presence of all the flavonoid bands observed in the crude alcoholic extract indicating a successful separation. Blue fluorescence seen in lane 4 may be due to the presence of impurities. Presence of broader and intense bands in FEF indicated enrichment of flavonoids. TLC-DPPH analysis is a qualitative test to detect antioxidant potential which illustrated an increased potential of FEF probably due to enrichment of flavonoids. No yellow coloration was observed in lane 4 ensuring that antioxidant potential was retained in FEF. [Figure 1]c and [Figure 1]d demonstrate the HPTLC profile and densitogram analysis of FEF. Five peaks were detected in the analysis with their area, area % and Rf values given in [Table 3]. Out of the 5 peaks, peak 3 was confirmed as rutin.
Figure 1: Thin-layer chromatography, thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl, and high-performance thin-layer chromatography analysis. (a) Comparative thin-layer chromatography profile of rutin, crude alcoholic extract, and fractions after derivatization with 1% natural product reagent under ultraviolet light at 366 nm, lane 1-rutin, lane 2-crude alcoholic extract, lane 3-flavonoid-enriched fraction, and lane 4-water fraction; (b) Comparative thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl profile of rutin, crude alcoholic extract, and fractions after derivatizing with 0.1% 2,2-diphenyl-1-picrylhydrazyl under visible light, lane 1-rutin, lane 2-crude alcoholic extract, lane 3-flavonoid-enriched fraction, lane 4-water fraction; (c) High-performance thin-layer chromatography profile of flavonoid-enriched fraction; (d) High-performance thin-layer chromatography spectra of flavonoid-enriched fraction at 420 nm

Click here to view
Table 3: Area, Area %, Rf values, and identified compounds detected in the HPTLC spectra of flavonoid-enriched fraction after densitometric analysis at 420 nm

Click here to view


Flavonoid content and antioxidant activity

The flavonoid content and antioxidant activity of FEF were estimated and compared with that of crude alcoholic extract [15], as illustrated in [Table 4]. The flavonoid content and antioxidant potential of FEF were higher than crude alcoholic extract confirming successful separation of flavonoids.
Table 4: Flavonoid content and effective concentration50 values of crude alcoholic extract and flavonoid-enriched fraction

Click here to view


Initial results indicate that the FEF possess a higher flavonoid content as well as better antioxidant potential. Hence, it was studied for its UV-B protective potential.

Cytoprotection by MTT assay

Results demonstrate that cell viability is greatly reduced in the UV-B irradiated cells [Figure 2]. Pretreatment of cells with 45 μg/ml FEF significantly maintained their viability to 72% as compared to 50% viability of UV-B control cells. This increase in cell viability was concentration dependent from 15 μg/ml to 45 μg/ml (***P < 0.001), indicating that FEF protected the cells from UV-B-induced damage and injury.
Figure 2: MTT assay for the cytoprotective ability of flavonoid-enriched fraction. Statistical analysis was performed by GraphPad Prism software version 2.0 using one-way ANOVA followed by Dunnett's and Tukey's posttest, **P < 0.01, ***P < 0.001

Click here to view


Estimation of levels of endogenous antioxidants

In the current study, results demonstrated significantly depleted levels of all the four antioxidant enzymes (SOD, CAT, GPx, and GR) due to UV-B-induced oxidative stress. FEF pretreatment significantly retained the levels of the endogenous enzymes, indicating a potential to protect against UV-B-induced oxidative stress through the endogenous enzymatic antioxidant system [Figure 3]. Statistical analysis demonstrated that the levels of enzymes increased from 15 μg/ml to 45 μg/ml (***P < 0.001).
Figure 3: Levels of endogenous enzymatic antioxidants. (a) SOD, (b) CAT, (c) glutathione peroxidase, (d) glutathione reductase. Statistical analysis was performed by GraphPad Prism software version 2.0 using one-way ANOVA followed by Dunnett's and Tukey's posttest, *P < 0.05, **P < 0.01, ***P < 0.001

Click here to view


Comet assay

Comet assay was performed to determine the protective ability of FEF against UV-B-induced DNA damage. Percent head DNA was greatly decreased in case of UV-B control group indicating UV-B-induced DNA damage and fragmentation. FEF pretreatment demonstrated a significant increase in the percent head DNA which was concentration dependent from 15 μg/ml to 45 μg/ml (***P < 0.001), indicating that DNA damage and fragmentation is significantly reduced due to the protective effect of FEF [Figure 4].
Figure 4: Comet assay for assessment of DNA damage. Cells observed under fluorescence microscope, ×400; (a) comparative graph showing percent head DNA in all treatment groups. Statistical analysis was performed by GraphPad Prism software version 2.0 using one-way ANOVA followed by Dunnett's and Tukey's posttest, *P < 0.05, ***P < 0.001

Click here to view


Ethidium bromide/acridine orange staining

UV-B-induced early apoptosis, loss of membrane integrity, loss of spindle shape, and orange fluorescent cells were observed in UV-B-irradiated group [Figure 5]. Presence of bright green and orange fluorescent spots in cells pretreated with 15 μg/ml of FEF indicated chromatin condensation and nuclear fragmentation, which disappeared after increasing concentration to 45 μg/ml. FEF is, therefore, able to protect the cells against apoptosis restoring cell membrane integrity in a concentration-dependent manner.
Figure 5: Ethidium bromide/acridine orange staining for detection of apoptotic morphological changes. Cells observed under fluorescence microscope, ×400

Click here to view


Measurement of intracellular reactive oxygen species

Results demonstrate a high percentage of ROS-positive cells in the UV-B control group possibly due to the UV-B-induced excessive generation of intracellular ROS. FEF pretreatment indicated a significant decrease in the percentage of ROS-positive cells (***P < 0.001) in a concentration-dependent manner from 15 μg/ml to 45 μg/ml [Figure 6]. The free radical scavenging ability of FEF was confirmed through the results.
Figure 6: Measurement of intracellular reactive oxygen species by 2',7'-dichlorofluorescein diacetate using flow cytometry. (a) comparative graph showing percentage reactive oxygen species-positive cells in treatment groups. Statistical analysis was performed by GraphPad Prism software version 2.0 using one-way ANOVA followed by Dunnett's and Tukey's posttest, *P < 0.05, **P < 0.01, ***P < 0.001

Click here to view


Ames test

S. typhimurium TA100 identity tests were carried out to confirm the genotype of the strain. It showed the presence of colonies in the medium containing histidine and absence in the medium without histidine, indicating that the strain is auxotrophic for the amino acid. Along with histidine dependence, the strain also showed the presence of an Rfa mutation which decreases the liposaccharide barriers and allows permeability of larger molecules, which was confirmed by observing a clear zone around a disc of crystal violet. Its compromised DNA repair mechanism was confirmed by the absence of colonies in the UV irradiated region of the plate. In the R-factor assay, the strain showed resistance to antibiotic ampicillin. These results were in accordance with the genotype of S. typhimurium TA100.

Ames test results [Table 5] showed that all tested concentrations of FEF of fennel seeds (10-100 μg/ml) had similar number of revertant colonies as compared to the negative control indicating that they are unable to induce a mutation in the histidine gene. Sodium azide, a known mutagen, showed a four-fold increase in the number of colonies of S. typhimurium TA100, indicating that it induces mutation in the histidine gene producing the amino acid.
Table 5: Mutagenic activity of flavonoid-enriched fraction by Ames test

Click here to view


Western blot and quantitative polymerase chain reaction analysis for nuclear factor E2-related factor 2 and heme oxygenase-1

UV-B induced 1.8- and 3.3-fold increase in expression of Nrf2 and HO-1 genes, respectively, with respect to the control [Figure 7] and [Figure 8]. Pretreatment of cells with 45 μg/ml FEF exhibited a 1.4- and 2.0-fold decreased expression of Nrf2 and HO-1 genes as compared to that in UV-B control group. Western blot and qPCR analyses indicated that the decreased expression in pretreated cells was concentration dependent from 15 μg/ml to 45 μg/ml (***P < 0.001) proving that FEF minimizes oxidative stress in the cell. FEF might be working through the Nrf2-ARE pathway to confer UV-B protection.
Figure 7: Quantitative polymerase chain reaction analysis for nuclear factor E2-related factor 2 and heme oxygenase-1 expression at mRNA level. (a) Fold change in nuclear factor E2-related factor 2 gene expression, (b) Fold change in heme oxygenase-1 gene expression, *P < 0.05, **P < 0.01, ***P < 0.001

Click here to view
Figure 8: Western blot analysis for nuclear factor E2-related factor 2 and heme oxygenase-1 expression at protein level. Densitometric analysis of nuclear factor E2-related factor 2 (a) heme oxygenase-1 (b) β-actin (c) where 1-control, 2-flavonoid-enriched fraction treated, 3-ultraviolet-B irradiated, 4-ultraviolet-B + silymarin (5 μg/ml), 5-ultraviolet-B ±15 μg/ml flavonoid-enriched fraction, 6-ultraviolet-B ± 30 μg/ml flavonoid-enriched fraction, and 7- ±45 μg/ml flavonoid-enriched fraction. Results were statistically analyzed by GraphPad Prism software version 2.0 using one-way ANOVA followed by Dunnett's and Tukey's posttest, *P < 0.05, **P < 0.01, ***P < 0.001

Click here to view



  Discussion Top


UV-B radiation reaches the Earth's surface affecting the epidermis and dermis of the skin by modulating immune responses, DNA damage, and generation of ROS, which lead to protein modification, lipid peroxidation, and mutations, causing ailments of various systems of the body. UV-B radiation is reported to induce death in fibroblasts, melanocytes, and keratinocytes along with disintegration of collagen and elastin.[2] Hence, protection of skin against UV-B radiation is essential. Sunscreens and synthetic antioxidants are used against UV-B-induced oxidative stress but exhibit limitations after continual use; hence, botanical antioxidants are now developed for UV-B protection. UV-B protective effect of plant polyphenols such as silymarin,[11] ursolic acid,[12] ferulic acid,[13] sesamol,[4] and epicatechin gallate [14] have been reported.

We have also reported the UV-B protective effect of flavonoids from Eugenia caryophylata[31] and Abelmoschus esculentus,[32] which have shown a comparative UV-B protection to that of FEF of fennel seeds. Flavonoids from E. caryophylata protected against UV-B-induced cell injury, DNA damage, and were nongenotoxic to HDF. They prevented excessive formation of intracellular ROS, hence maintained the levels of endogenous antioxidant enzymes. They reduced oxidative stress through the Nrf2-ARE pathway and increased the oxidative stress tolerance of the cell through priming effect.

Flavonoids are a type of polyphenols widely studied for their beneficial pharmacological activities; therefore, in the current study, flavonoids from fennel seeds were studied for their protective potential against UV-B-induced damage. Along with the presence of rutin, FEF also exhibited a significant flavonoid content and antioxidant potential than that in crude extract demonstrating free radical scavenging ability and reducing power. Antioxidant ability of flavonoids depends on the structure and substitution pattern of hydroxyl groups of flavonoids, i.e., 3′, 4′-orthodihydroxy configuration in B ring, 4-carbonyl group in C ring, and 3-OH or 5-OH groups are necessary.[33] Rutin exhibits a similar configuration in its structure along with other unidentified flavonoids and can be a possible reason for antioxidant potential.

Researchers have already reported that UV-B induces cytotoxicity and has been established that polyphenolic compounds reverse the UV-B-induced damage.[4],[12],[13] UV-B radiations initiate the generation of excessive intracellular ROS leading to oxidative stress and redox imbalance in the cell. FEF pretreatment demonstrated a statistically significant decrease in the level of ROS which reflects its free radical scavenging property as also seen in the antioxidant assays. FEF may also interact with the cellular chromophores and photosensitizers, limiting the formation of free radicals. Excessive ROS also depletes endogenous enzymatic antioxidants, as observed in the results. Endogenous antioxidants form the first line of defense against oxidative stress. SOD dismutates the superoxide anion into molecular oxygen and hydrogen peroxide. CAT and GPx convert this hydrogen peroxide into water and molecular oxygen. GPx and GR maintain a ratio of reduced glutathione and oxidized glutathione which is an important index of oxidative stress.[34] Decreased levels of SOD may be due to formation of superoxide anions, direct absorption of UV-B by heme depletes CAT levels. GPx and GR levels are reduced due to the various antioxidant recycling mechanisms.[35] FEF pretreatment retained the levels of endogenous enzymatic antioxidants significantly in a concentration-dependent manner.

DNA absorbs UV-B radiations directly and induce the formation of cyclobutanepyrimidine dimers, thymine glycols, 8-hydroxyguanine, which are highly mutagenic and are present in UV-induced cancer cells.[36] FEF pretreatment protected the cell against UV-B-induced DNA damage, probably due to a UV-B-masking effect preventing the interaction of UV-B with DNA. FEF did not show genotoxicity in the FEF-treated cells as well as does not show a mutagenic potential till 100 μg/ml concentration in S. typhimurium TA100 strain. Cell membrane integrity is also lost due to UV-B-induced peroxidation of lipid bilayer in the cell membrane due to increased free radicals in the cell. As already demonstrated, FEF has a potential to scavenge UV-B-induced free radicals minimizing lipid peroxidation, and hence, retaining the cell membrane integrity. It also decreased the apoptotic morphological changes in the cell in a concentration-dependent manner as observed in EtBr/AO staining.

Nrf2-ARE pathway is important in cellular defense. As observed in the results, UV-B-induced excessive generation of ROS leading to increase in oxidative stress which can be correlated to the increase in expression of Nrf2 and HO-1 in the UV-B control group. Whereas, FEF pretreatment leads to reduction in expression of Nrf2 and HO-1 which may be due to the decrease in oxidative stress in the cells.

In the current study, flavonoids from fennel seeds effectively reduced UV-B-induced cytotoxicity, oxidative DNA damage, intracellular ROS generation, apoptotic morphological changes, and overexpression of (Nrf2) and HO-1; and increased the endogenous antioxidant levels as compared to those in the UV-B control group. Flavonoids from fennel seeds could be considered as potential UV-B protectants and developed into natural topical sunscreen or photoprotectant.


  Conclusion Top


Our studies demonstrated for the first time that the flavonoids from fennel seeds could protect against UV-B-induced damage, probably through the Nrf2-ARE pathway. Flavonoids from fennel seeds can be further isolated, studied, and developed into herbal sunscreen or photoprotectant and can be used for treating skin ailments caused due to UV-B exposure.

Acknowledgments

The authors are grateful to Dr. Kshitij Satardekar for providing the HDF cell line. We are also thankful to Dr. Laddha, Institute of Chemical Technology, Mumbai, for gifting a sample of Rutin. Acknowledgments are also due to Dr. Ganesh Vishwanathan and Mrs. Madhura Joshi, IIT Bombay for helping with the flow cytometer facilities. The authors are thankful to Dr. Nancy Pandita, Dr. Krutika Desai, ad Dr. R. Joshi for their critical inputs during the study as well as Dr. Aparna Khanna, Dean, Sunandan Divatia School of Science, Mumbai, for providing necessary facilities for the experimental work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Maverakis E, Miyamura Y, Bowen MP, Correa G, Ono Y, Goodarzi H. Light, including ultraviolet. J Autoimmun 2010;34:J247-57.  Back to cited text no. 1
    
2.
Pinnell SR. Cutaneous photodamage, oxidative stress, and topical antioxidant protection. J Am Acad Dermatol 2003;48:1-9.  Back to cited text no. 2
    
3.
Svobodova A, Walterova D, Vostalova J. Ultraviolet light induced alteration to the skin. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2006;150:25-38.  Back to cited text no. 3
    
4.
Ramachandran S, Rajendra Prasad N, Karthikeyan S. Sesamol inhibits UVB-induced ROS generation and subsequent oxidative damage in cultured human skin dermal fibroblasts. Arch Dermatol Res 2010;302:733-44.  Back to cited text no. 4
    
5.
Rai R, Shanmuga SC, Srinivas C. Update on photoprotection. Indian J Dermatol 2012;57:335-42.  Back to cited text no. 5
[PUBMED]  [Full text]  
6.
Ma Q, He X. Molecular basis of electrophilic and oxidative defense: Promises and perils of nrf2. Pharmacol Rev 2012;64:1055-81.  Back to cited text no. 6
    
7.
Alrawaiq N, Abdullah A. Dietary phytochemicals activate the redox-sensitive transcription factor Nrf2. Int J Pharm Pharm Sci 2014;6:11-6.  Back to cited text no. 7
    
8.
Kaur GJ, Arora DS. Antibacterial and phytochemical screening of Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi. BMC Complement Altern Med 2009;9:30.  Back to cited text no. 8
    
9.
He W, Huang B. A review of chemistry and bioactivities of medicinal spice: Foeniculum vulgare. J Med Plants Res 2011;5:3595-600.  Back to cited text no. 9
    
10.
Devika V, Mohandass S, Aiswarya PR. Screening of methanolic extract of Foeniculum vulgare for hepatoprotective activity. Int J Pharm Pharm Sci 2013;5:56-9.  Back to cited text no. 10
    
11.
Svobodová A, Psotová J, Walterová D. Natural phenolics in the prevention of UV-induced skin damage. A review. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2003;147:137-45.  Back to cited text no. 11
    
12.
Ramachandran S, Prasad NR. Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity, lipid peroxidation and DNA damage in human lymphocytes. Chem Biol Interact 2008;176:99-107.  Back to cited text no. 12
    
13.
Prasad NR, Ramachandran S, Pugalendi KV, Menon VP. Ferulic acid inhibits UV-B–induced oxidative stress in human lymphocytes. Nutr Res 2007;27:559-64.  Back to cited text no. 13
    
14.
Huang CC, Wu WB, Fang JY, Chiang HS, Chen SK, Chen BH, et al. (-)-epicatechin-3-gallate, a green tea polyphenol is a potent agent against UVB-induced damage in haCaT keratinocytes. Molecules 2007;12:1845-58.  Back to cited text no. 14
    
15.
Patwardhan J, Pandita N, Bhatt P. Comparative study of antioxidant potential of two Indian medicinal plants Foeniculum vulgare and Eugenia caryophylata. Int J Pharm Sci Rev Res 2013;21:312-6.  Back to cited text no. 15
    
16.
Lee GS, Shim H, Lee KM, Kim SH, Yim D, Cheong JH, et al. The role of the ethylacetate fraction from hydnocarpi semen in acute inflammation in vitro model. Immune Netw 2012;12:291-5.  Back to cited text no. 16
    
17.
Wagner H, Bladt S. Plant Drug Analysis a Thin Layer Chromatography Atlas. Germany: Springer-Verlag; 1996.  Back to cited text no. 17
    
18.
Sethiya NK, Raja MK, Mishra SH. Antioxidant markers based TLC-DPPH differentiation on four commercialized botanical sources of Shankhpushpi (A Medhya rasayana): A preliminary assessment. J Adv Pharm Technol Res 2013;4:25-30.  Back to cited text no. 18
[PUBMED]  [Full text]  
19.
Chang CC, Yang MH, Wen HM, Chern JC. Estimation of total flavonoid content in propolis by two complementary colorimetric method. J Food Drug Anal 2002;10:178-82.  Back to cited text no. 19
    
20.
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 1999;26:1231-7.  Back to cited text no. 20
    
21.
Kedare SB, Singh RP. Genesis and development of DPPH method of antioxidant assay. J Food Sci Technol 2011;48:412-22.  Back to cited text no. 21
    
22.
Fakruddin M, Mannan KS, Mazumdar RM, Afroz H. Antibacterial, antifungal and antioxidant activities of the ethanol extract of the stem bark of Clausena heptaphylla. BMC Complement Altern Med 2012;12:232.  Back to cited text no. 22
    
23.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.  Back to cited text no. 23
    
24.
Chance B, Herbert D. The enzymesubstrate compounds of bacterial catalase and peroxides. Biochem J 1950;46:402-14.  Back to cited text no. 24
    
25.
Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469-74.  Back to cited text no. 25
    
26.
Awasthi YC, Beutler E. Purification and properties of human erythrocyte glutathione peroxidease. J Biol Chem. 1975; 250, 5144-5149.  Back to cited text no. 26
    
27.
Goldberg DM, Spooner RJ. Oxidoreductases acting on groups other than CHOH Glutathione reductase. In: Bergmeyer HU, Bergmeyer J, Grassl M, editors. Methods of Enzymatic Analysis. Weinheim: Verlag Chemie; 1983. p. 258-65.  Back to cited text no. 27
    
28.
Issazadeh K, Aliabadi MA, Darsanaki RK, Pahlaviani M. Antimutagenicity activity of olive leaf aqueous extract by Ames test. Adv Studies Biol 2012;4:397-405.  Back to cited text no. 28
    
29.
Magesh V, Raman D, Pudupalayam KT. Genotoxicity studies of dry extract of Boswellia serrata. Trop J Pharm Res 2008;7:1129-35.  Back to cited text no. 29
    
30.
Sundaram SG, Vijayalakshmi M, Nema RK. Antimutagenicity of ethanol extract of Derris brevipes. J Chem Pharm Res 2010;2:598-603.  Back to cited text no. 30
    
31.
Patwardhan J, Bhatt P. Ultraviolet-B protective effect of flavonoids from Eugenia caryophyllata on human dermal fibroblast cells. Pharmacogn Mag 2015;11:S397-406.  Back to cited text no. 31
    
32.
Patwardhan J, Bhatt P. Flavonoids derived from Abelmoschus esculentus attenuates UV-B induced cell damage in human dermal fibroblasts through nrf2-ARE pathway. Pharmacogn Mag 2016;12:S129-38.  Back to cited text no. 32
    
33.
Wojdyla A, Oszmianski J, Czemerys R. Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem 2007;105:940-9.  Back to cited text no. 33
    
34.
Lü JM, Lin PH, Yao Q, Chen C. Chemical and molecular mechanisms of antioxidants: Experimental approaches and model systems. J Cell Mol Med 2010;14:840-60.  Back to cited text no. 34
    
35.
Shindo Y, Witt E, Han D, Tzeng B, Aziz T, Nguyen L, et al. Recovery of antioxidants and reduction in lipid hydroperoxides in murine epidermis and dermis after acute ultraviolet radiation exposure. Photodermatol Photoimmunol Photomed 1994;10:183-91.  Back to cited text no. 35
    
36.
Ambothi K, Nagarajan RP. Ferulic acid prevents UV-B radiation induced oxidative damage in human dermal fibroblasts. Int J Nutr Pharmacol Neurol Dis 2014;4:203-13.  Back to cited text no. 36
  [Full text]  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed483    
    Printed11    
    Emailed0    
    PDF Downloaded21    
    Comments [Add]    

Recommend this journal