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ISSN: 2157-2518
Journal of Carcinogenesis & Mutagenesis
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Calendula officinalis Extracts Protect against H2O2 Induced Chromosome Damage on HacaT Human Skin Cells

Abdullah M Alnuqaydan1, Claire E Lenehan2, Rachel R Hughes2 and Barbara J Sanderson3*
1Medical Biotechnology, School of Medicine, Faculty of Medicine, Nursing and Health Sciences, Flinders University, Australia
2School of Chemical and Physical Sciences, Faculty of Science and Engineering, Flinders University, Australia
3Department of Medical Biotechnology, School of Medicine, Flinders University, Australia
Corresponding Author : Sanderson BJ
GPO Box 2100, Adelaide, SA 5001, Australia
Tel: +61087221 8556
Fax: +61(08) 7221 8555
E-mail: [email protected]
Received: October 16, 2015 Accepted: November 19, 2015 Published: November 21, 2015
Citation: Alnuqaydan AM, Lenehan CE, Hughes RR, Sanderson BJ (2015) Calendula officinalis Extracts Protect against H2O2 Induced Chromosome Damage on HacaT Human Skin Cells. J Carcinog Mutagene 6:245. doi:10.4172/2157-2518.1000245
Copyright: © 2015 Alnuqaydan AM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: Calendula officinalis extracts contain antioxidant compounds. The extracts tested in the current study protect against reactive oxygen species (ROS) induced cytotoxic activity towards human cells. Objective: To determine the ability of Calendula officinalis extracts to protect against H2O2 induced chromosome damage on HaCaT human skin cells. Methodology: The chromosome damage protection by four Calendula officinalis extracts was investigated in vitro using a dose-response (0.125, 0.5 and 1.0% (v/v)) on HaCaT human skin cells. Two water/ethanol based proprietary calendula extracts A and B and two proprietary aqueous calendula extracts C and D from Biodynamically grown plants were examined. Extracts were characterised using Folin-Ciocalteau and DPPH assays. Protection of extracts against chromosome damage was measured via micronuclei induction using the cytokinesis-blocked micronucleus assay. No genotoxicity was induced by the extracts themselves. Cells were also exposed to the Calendula extracts for 47 h before being exposed to an oxidative stress (300-μM hydrogen peroxide for 1 h). Results: The oxidative stress induced micronuclei (MNi) at a frequency significantly higher (P<0.05; range=20-25 MNi/1000 binucleated cells, n=3) than the background frequency (media alone control; MNi range=7-13 MNi/1000 binucleated cells, n=3). When the cells were pre-treated with extracts before the oxidative stress, the level of protection against chromosomal damage by all Calendula extracts was significant (P<0.05). The frequency of MNi in the presence of extracts was reduced by an average increment of 20 MNi/1000 BN cells to 2-9 MNi/1000 BN cells at all doses tested. Conclusion: The Calendula extracts protected against the chromosomal damage induced by oxidative stress in vitro. Such form of genetic damage has been linked with carcinogenesis.

Calendula officinalis; Genotoxic protection; Chromosomal damage; Oxidative stress; CBMN (cytokinesis-block micronucleus) assay; Micronuclei (MNi)
Alteration of genetic material can lead to cancer and can include chromosome breakage and loss, dicentric chromosomes and DNA damage, including mutations and gene amplification. The number of chromosome alteration is correlated within tumour process and malignant potential of the cancer [1]. DNA damage occurs from cellular sources (endogenous) and environmental agents (exogenous) [1]. Exogenous agents such as chemicals or radiation can interact with genetic material and induce genetic damage (genotoxicity), chronic toxicity and/or cell death (cytotoxicity) [2]. Exogenous agents can promote the formation of free radicals. Ultraviolet light (UV light) can lead to the formation of single oxygen (O) and other reactive oxygen species in the skin. Other atmospheric pollutants, such as ozone and nitrogen peroxide, lead to radical formation and may cause respiratory disease. Cigarette smoke also contains free radicals [3,4]. Oxidative stress arises from an imbalance between free radicals including reactive oxygen species (ROS), and antioxidant defences. ROS can damage cellular components including lipids, proteins and nucleic acids [5]. Oxidative damage to proteins or nucleic acids often increases the variety of damage products due to modifications in amino acids and nucleotides [6]. There are many conditions where oxidative damage is involved. Example includes the aging process, cancer, atherosclerosis, inflammatory conditions, viral infections, trauma, effects of cigarette smoke and interaction with environmental pollution [7-10]. An antioxidant is a substance which can inhibit or delay oxidative damage occurring and can therefore potentially protect against the damaging effects of ROS. Many studies have examined the chemical nature of fruits, vegetables, herbs and grains for their natural antioxidant activity [5,11] The majority of antioxidants isolated from higher plants are polyphenols with the main biological activity identified as antiinflammatory, anti-carcinogenic, anti-bacterial and anti-viral [11-13]. The activity of different extracts of Calendula officinalis flower includes anti-tumoral, anti-inflammatory, anti-viral activity and anti-HIV properties [10,14-16]. Calendula officinalis extracts contain antioxidant compounds and these include phenolic, Tocopherols and Carotenes [17]. Individual components of relevance to this study are quercetin, vitamin E, beta-Carotene and amino acids [18]. The extracts tested in the current study have been shown to protect human cells in vitro against ROS induced cytotoxic activity [17]. We tested four extracts of Calendula officinalis for protection against chromosome damage caused by free radicals using an in vitro human skin cell model.
Materials and Methods
Materials used were RPMI 1640 media and foetal bovine serum (FBS) were purchased from Gibco® Cell Culture. Cytochalasin B (Cyt- B) solution was purchased from Sigma™. Sodium dodecyl sulphate (SDS, approximately 99%), Phosphate buffered saline (PBS) was purchased from Sigma™. Hydrogen peroxide (H2O2) and Quercetin were purchased from Sigma™. Folin-ciocalteu reagent was purchased from Sigma™. Diff-Quik stains were purchased from Lab Aids (Australia). Media prepared for growth and treatment was RPMI medium supplemented with 10% FBS.
It is shown the results of density determination; total dry weight after freeze drying and Gallic acid aqueous (GAE) results performed by Folin-Ciocalteu assay.
Plant extracts
Plants extract preparation and characterization was as previous published [18]. Briefly, dried Calendula officinalis flowers were supplied by an industry partner (Jurlique International, Mount Barker, South Australia) from Biodynamically grown plants. Four different extracts were prepared independently. Extracts A and B were prepared as water/ethanol based proprietary calendula extracts and extracts C and D proprietary aqueous calendula extracts. When required, extracts were diluted in media to prepare the treatment solution. Total phenolic content; dry weight and density determination were used to characterise the plant extracts. The determination of density for all extracts was carried out at 20°C from the average of 10 independent measurements. Table 1 is presented data as mean ± SD, with errors calculated using the propagation of uncertainty equation (density=mass/volume). The percentage of total dry weights of all extracts (A,B,C or D) were determined after extracts samples were freeze dried at -56°C at 3.4 Pa for 24 h. The total dry solids were weighed once all volatiles were removed from samples.
Cell line
A human non-cancer keratinocytes cell line (HaCaT) was as a gift from the department of Haematology and Genetic Pathology-Flinders Medical Centre, school of medicine at Flinders University, Adelaide, South Australia. The cell line was maintained in RPMI-1640 media (RPMI-1640 powder reconstituted medium supplemented with Lglutamine 2 mM, penicillin 100U/ml, streptomycin 100 μg/ml and 10% FBS). Cells were seeded in tissue culture flasks at 5×105 cells/ml and incubated in a fully humidified atmosphere at 37°C with 5% CO2. The cells were subcultured twice a week when they reached 60-80% confluence or greater.
Cell treatment with Calendula extracts
Cells were treated in vitro for extract exposure as part of the genetic protection assay. Six wells of a 6-wells plate were seeded with 3 × 105 cells/well in a 2 ml volume and incubated for 19 h to allow the adherence of cells at 37°C in 5% CO2. The media were aspirated and replaced with 2 ml of the treatment solution per well. The cells were treated for 48 h prior to CBMN assay analysis at doses derived from the MTT assay results for extracts A, B, C and D extracts (data not shown). Treatment solutions included A,B,C or D at 0, 0.125, 0.5 and 1 (v/v). The positive control compound used was Quercetin (16 μg/ml final concentration) with known antioxidant capacity. Media were used as a negative control.
Induction of oxidative stress by hydrogen peroxide (H2O2) to determine chromosome protection activity
Following incubation with or without extracts, oxidative stress was induced by H2O2 as previously published [18].
Folin-Ciocalteu assay
To prepare the calendula extracts, they were diluted 50-fold in 10 ml water. Gallic acid aqueous standards were prepared by adding 5.8, 12.8, 16.4, 22.8 and 27.8 mg in 250 ml of water. Then the Folin–Ciocalteu assay was carried out as previously described by [18,19]. For the assay, the mixtures were incubated 2 h at room temperature. Using a UV-Vis spectrophotometer the absorbance of each solution at 765 nm and 750 nm was measured. A calibration curve of gallic acid concentration versus absorbance was constructed from the Gallic acid aqueous standards. The gallic acid equivalents of the extracts was then determined in triplicate.
DPPH assay
The DPPH● assay was performed to determine antioxidant activities of the extracts via radical scavenging ability using stable DPPH● radical. A methanolic stock solution of Quercetin (1.6 mg/ml) was the positive control with dilutions in methanol (5, 4, 3, 2 and 1% (v/v)) used to determine the linear range of the DPPH● assay. Calendula extracts were prepared and analysed as previously reported [18,20]. The decrease in absorbance at 517 nm was determined with a Thermo Scientific Evolution Array UV-Visible spectrophotometer after 15 minutes for all samples.
Cytokinesis-block micronucleus (CBMN)
The CBMN assay was carried out as previously described [21] with the minor modifications was reported [22]. Briefly, after treatment Cyt-B was added at 4.5 μg/ml in media and the cultures were incubated at 37°C for 23 h. Cells were harvested by trypsinization and collected onto slides by centrifuging at 47 Xg (@6000 rpm) for 5 minutes using a cytospin. Slides were air-dried, fixed for 10 min in DiffQuick Fixative, and then stained with stain 1 (red DiffQuick Stain) and then Stain 2 (blue DiffQuick Stain). Slides were scored at a magnification of 250X or 40 X. Criteria for scoring micronuclei MNi, nucleoplasmic bridge (NPB) or nuclear buds (NBUDs) were as described [23]. The chromosomal damage induced by treatment, total number of MNI in BN cells were counted in total of 1000 BN.
Statistical analysis
Triplicate assays were performed for all experiments, and data is presented as the mean ± S.E.M. One way ANOVA (SPSS software, version 19.0) followed by Tukey’s HSD post was used. T-test for independent samples was also used. Differences were considered statistically significant when the value of P<0.05.
Characteristics of Calendula extracts
The physico-chemical properties of Calendula officinalis extract A, B, C and D were characterised (Table 1). The Folin-Ciocalteu assay was used to estimate polyphenol composition and is expressed using per dry weight determinations and gallic acid equivalents (Table 1). DPPH● assay characterization is also included (Table 3).
Genotoxicity of Calendula plant extracts on Human Skin cells HaCaT
The in vitro genotoxicity and protective effects of Calendula plant extracts A, B, C and D on HaCaT Human Skin cells was performed after incubating cells with extracts for 48h. No genotoxicity was induced by the extracts themselves shown in (Figures 1-4). At no dose of any extract alone is the frequency of micronuclei greater than that of the zero dose control.
Protection induced by extracts against H2O2 induced oxidative stress on human skin cells
Oxidative stress induced micronuclei frequency by 300-μM hydrogen peroxide (H2O2) with no pre-treatment was significantly higher than the zero dose (untreated control) (P<0.05) in all experiments. Calendula extracts A,B,C and D induced protection against H2O2 induced micronuclei, as shown in (Figures 1-4). This was, at all doses, and statistically significant (P<0.05) for Human skin cells after treatment with each of the extracts for 48 h followed by 1h treatment with 300-μM H2O2. This is shown for all extracts in (Figs. 1- 4) by the solid lines (ROS challenge series of experiments) where there is a significant difference (P<0.05) between cells in the absence of extract but with H2O2 (0 dose of extract) and all other doses of extracts in the presence of H2O2. The positive control of a known antioxidant, Quercetin significant protected against H2O2 induced micronuclei (Table 2, P<0.05). The protection level of all extracts was similar to the protection level of Quercetin against H2O2 induced oxidative stress genetic damage.
A previous study demonstrated protection by Calendula extracts against H2O2 induced cell killing [18]. Further work was required to cover the characterization of additional aspects of the calendula extracts protective action. The current study investigated the protection against chromosome damage by four calendula flower extracts on human skin cells (HaCaT) in vitro. Based on the cytotoxicity data for extracts A, B, C and D (data not shown) non-toxic doses were chosen for use in the CBMN assay. Firstly, the study showed the safety of calendula extracts as has been shown for medicinal plants [16,18,24,25]. No extracts were genotoxic by themselves at any dose tested on HaCaT skin cells. Total phenolics in extracts were determined by the Folin-Ciocalteu assay. The values for extracts A and B were 28 mg GAE/g dry weight and 26 mg GAE/g dry weights, and were 10.83 mg GAE/g dry weight for C and 11.73 mg GAE/g dry weights for D (Table 1). The levels of polyphenols obtained are equal to or greater than that obtained by others for extracts of calendula flower and other medicinal plants [19,26]. Consistently with this, the DPPH assay demonstrated that all extracts and the positive control quercetin, exhibited free radical inhibition at 2.5% dose ranging from 29-55% (Table 3). The genotoxicity protection of HaCaT skin cells in vitro against chromosome damage caused by ROS was explored by pre-treating cells with Calendula extracts for 48h then challenging with H2O2. Calendula flower extracts were negative in the Ames Salmonella/microsome mutagenicity assay and the extracts contained carotenes and flavonoids thus indicating high antioxidant activities [24]. Laser calendula extracts has shown significant inhibition of tumor growth of tumor cell proliferation when tested on human and murine tumor cell lines, therefore calendula flower extracts could be used in treating cancers [16]. Findings in the current study were consistent with previously observed antioxidant activity of calendula flower extracts, and the four extracts protected against chromosome damage from oxidative stress induced by H2O2 (known to induce free radicals including ROS). Genetic damage from oxidative stress induced by 300 μM H2O2 was significantly different from the background level of MNi in the negative control (un-treated control). The number of MNi/1000 BN in the un-treated control populations ranged from 7-13 MNi/1000 BN, n=3. Hydrogen peroxide, however, induced up to 24 MNi/1000 BN (range= 20-24 MNi), n=3. The four extracts offered protection against genetic damage induced by ROS with significant decreases in the number of MNi/1000 BN when HaCaT cells treated with extracts were challenged for 1h with H2O2 post-treatment. These results are similar to Quercetin the positive control for protection, which reduced genetic damage to only 8.7 MNi/ 1000 BN, as shown in Table 2. The bioactivity level of extracts appears high enough that there is no significant difference between all the treatment doses for each extract. Extracts A, B, C and D offered protection against MNi induction for all doses tested (Figures 1-4) related to DPPH and Folin-Ciocalteu results. The extracts were also characterized for Folin-Ciocalteu and consistently D extract was also lower than extracts A and B for this test as well. C extract varied in its performance in these two assays indicating it has a different composition of bioactives to the other extracts. This needs further investigation as the protection offered by the extracts did not fully reflect the chemical characterization results.
Increases in total number of MNi per 1000 BN cells indicates changes at the chromosome level, reflecting damage that is induced by H2O2 due to oxidative stress from free radicals. Free radical induced damage to DNA and chromosomes by a cellular redox imbalance from oxidative stress can result in cancer [27,28]. Micronuclei arise from either whole chromosome losses or acentric chromosome fragments. To investigate the specific type of chromosome damage responsible for the increases of MNi there is a need to use molecular techniques in combination with the CBMN assay e.g. probe for presence of centromeres in MNi using fluorescence probes [21]. Nucleoplasmic bridge (NPB) and Nucleoplasmic buds (NBUDs) were not observed after were cells exposed to hydrogen peroxide (H2O2). NPB can be due to dicentric chromosome and NBUDs can arise from gene amplification. The development of cancer is a result of the long term accumulation of mutations in human genes [29]. Also, some cancers are diagnosed with a genetic instability and of these, most are observed with instability at the chromosome level that can be chromosome breakage or whole chromosome losses [29]. Calendula extracts examined in this study offered protection against chromosomal damage induced by oxidative stress. Preventing induction of chromosomal damage using calendula extracts could reduce the risk of cancer. Interestingly, although not being used for protection, there is Calendula officinalis versus Trolamine in phase III randomized trial of Calendula officianalis for breast cancer [30].
In conclusion, H2O2 damage can lead to oxidative stress and cause human cancer via chromosomal breakage or loss and DNA damage [21]. Calendula flower extracts protect HaCaT skin cells against an oxidative stress induced (by H2O2) chromosome damage by a decreased frequency of MNi. Further work on calendula officinalis extracts should include studying protection against UV light induced chromosome and mutation damage on human skin cells in vitro [31]. The extracts tested in this paper have potential applications as protectants due to antioxidants properties, including bio actives that act against oxidative stress induced damage to human cells.
Conflict of Interest Statement
The authors declare no conflict of interest
We thank the Ministry of Education, Saudi Arabia, for its partial support of this project.

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