Sage Journals: Discover world-class research

Abstract

This work investigated the safety of extracts obtained from plants growing in Colombia, which have previously shown UV-filter/antigenotoxic properties. The compounds in plant extracts obtained by the supercritical fluid (CO₂) extraction method were identified using gas chromatography coupled to mass spectrometry (GC/MS) analysis. Cytotoxicity measured as cytotoxic concentration 50% (CC₅₀) and genotoxicity of the plant extracts and some compounds were studied in human fibroblasts using the trypan blue exclusion assay and the Comet assay, respectively. The extracts from Pipper eriopodon and Salvia aratocensis species and the compound trans-β-caryophyllene were clearly cytotoxic to human fibroblasts. Conversely, Achyrocline satureioides, Chromolaena pellia, and Lippia origanoides extracts were relatively less cytotoxic with CC₅₀ values of 173, 184, and 89 μg/mL, respectively. The C. pellia and L. origanoides extracts produced some degree of DNA breaks at cytotoxic concentrations. The cytotoxicity of the studied compounds was as follows, with lower CC₅₀ values representing the most cytotoxic compounds: resveratrol (91 μM) > pinocembrin (144 μM) > quercetin (222 μM) > titanium dioxide (704 μM). Quercetin was unique among the compounds assayed in being genotoxic to human fibroblasts. Our work indicates that phytochemicals can be cytotoxic and genotoxic, demonstrating the need to establish safe concentrations of these extracts for their potential use in cosmetics.

Keywords

Cytotoxicity genotoxicity trypan blue stain Comet assay human fibroblasts phytochemicals

Introduction

Phytochemicals are widely used as raw materials in the cosmetic industry.¹ The extensive use of plants as medicine has shown that they are not as safe as frequently claimed in the past.^2,3 They can produce various toxic effects, including genotoxic damage (e.g., DNA adducts, DNA strand breaks, DNA-protein crosslinks, and chromosomal breakage).⁴ Therefore, it is essential to study the adverse effects of plants before their use as a source of cosmetic ingredients.

Different guides or procedures using animal-free safety testing of cosmetic ingredients (e.g., colorants, preservatives, and UV filters) have been implemented in the European Union since the ban on animal use to assay cosmetic ingredients.^5-9 There are different in vitro genotoxicity assays that provide information on relevant endpoints such as mutagenicity, clastogenicity, and aneugenicity.¹⁰ According to our knowledge, no standard safety assay exists for testing phytochemicals used as cosmetic ingredients. However, it has been shown that the most commonly used genotoxicity assays for testing phytochemicals are those recommended by regulatory agencies for chemicals.¹¹ Among them, the Comet assay^12,13 is a method widely used for genotoxicity evaluation of medicinal plants¹¹ and pharmaceuticals with inclusion into the ICH S2R1 guidance.¹⁴ The use of a high-throughput platform (96-well microplate) has notably increased the overall capacity, reproducibility, and robustness of the Comet assay.^15-18

We recently showed that different plant extracts and major compounds have UV-filter/antigenotoxic properties¹⁹; therefore, they could potentially be used as cosmetic sunscreen ingredients. We hypothesize that these plant extracts and their constituents are neither cytotoxic nor genotoxic in human fibroblasts. To test this hypothesis, we evaluated the cytotoxicity and genotoxicity of these plants and constituents in human fibroblasts using the trypan blue exclusion and Comet assays, respectively. Here, we showed that some of the plant extracts under study were cytotoxic and genotoxic in human fibroblasts. We also provided evidence for the need for genotoxicity testing of plant-derived ingredients and for establishing safe phytochemical concentrations for use in cosmetics.

Materials and Methods

Plant Materials and Extracts

We studied extracts obtained from five plant species growing in Colombia, which have previously shown UV-filter/antigenotoxic properties.¹⁹ The plants and their Colombian National Herbarium voucher numbers (in parenthesis) were as follows: Achyrocline satureioides (COL579420), Chromolaena pellia (COL559437), Lippia origanoides (COL560259), Piper eriopodon (COL578364 and COL578974), and Salvia aratocensis (COL560246). The plant species were collected in different areas of Colombia as follows: Los Santos – Santander (COL559437, COL560246, and COL560259); Zapatoca – Santander (COL579420 and COL578364); and Tame – Arauca (COL578974). Plant species collection was supported by collection permit and granted by the “Ministerio de Ambiente y Desarrollo Sostenible” of Colombia (Contract N^o. 270).

For each specimen, fresh leaves and flowers were used for supercritical fluid (CO₂) extraction.²⁰ Extract stock solutions were prepared at 30 mg/mL, dissolved in methanol (1 mL), vortexed (3 min), exposed to ultrasound (10 min, 40°C), and centrifuged at 5000 r/min (10 min). Then, the supernatant (1 mL) was filtered and stored until use at 80°C. Before being used, the extracted stock solutions were defrosted and placed under refrigeration (5-8°C) for 24 h.

Gas Chromatography Coupled to Mass Spectrometry (GC/MS) Analysis

For the determination of major compounds in plant extracts, gas chromatography (GC) analyses were performed using a 6890 System Plus gas chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a mass selective detector MSD 5975 (Electron ionization, EI, 70 eV, AT, Palo Alto, CA, USA) and an automatic injector 7863 (Agilent Technologies, Palo Alto, CA, USA) that was operated in split mode (1:30), and its temperature was 250°C. A fused-silica capillary column DB-5MS (J&W Scientific, Folsom, CA, USA) of 60 m × .25 mm, i.d. x .25 μm, d_f, with stationary phase of 5%-phenyl poly (methysiloxane) was used for metabolite secondary separation. Chromatographic conditions were as follows: the initial column head pressure was 16.43 psi, and the operating mode was constant flow (1.0 mL/min) using helium as a carrier gas (99.995%, Messer, Bogotá, Colombia). The GC oven temperature was programmed from 45°C (5 min) to 150°C (2 min) at 4^oC/min and then was increased to 250°C (5 min) at 5^oC/min and finally to 300°C (5 min) at 10^oC/min. The mass range used for mass spectra was m/z 45-450 with an acquisition speed of 3.58 scans/s using the MSD Chem Station G1701DA software (AT, Palo Alto, CA, USA).

The identification of major compounds from plant extracts was based on chromatographic retention lineal indices (LRI) and interpretation and comparison of mass spectra with databases. The LRI index was calculated using the retention times of a linear hydrocarbon standard (C₈ to C₄₀) mixture as indicated by the following equation: $LRI = 100 n + 100 \frac{t_{Rx} - t_{Rn}}{t_{RN} - t_{Rn}}$ , where n is the number of carbon atoms in the n-paraffin that elutes before the compound of interest (its retention time is t_Rx) and t_Rn and t_RN are retention times of the n-paraffins with the numbers of carbon atoms n and N, respectively, which elute immediately before and after the analyte of interest. Mass spectra obtained experimentally were compared with those reported in the Adams 2007, NIST 2017, and Wiley 2008 databases.

Chemicals, Buffer, Enzymes, and Culture Media

Five plant compounds (trans-β-caryophyllene, squalene, pinocembrin, quercetin, and resveratrol), the filter titanium dioxide, the mutagens 4-nitroquinoline 1-oxide (4NQO) and bleomycin (BLM), trypan blue solution (.4%), lyophilized proteinase K, and high-resolution agarose, were obtained from Sigma-Aldrich Co. Inc. (Milwaukee, WI, USA). Quercetin and resveratrol have been chosen for comparative purposes as plant compounds that are frequently used in cosmetic industry for their antioxidant properties. YOYO solution was purchased from Thermo Scientific (MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), ethylenediaminetetraacetic acid (EDTA)-trypsin solution, and antibiotic mixture (penicillin/streptomycin) were purchased from Gibco (Grand Island, New York, USA). The remaining reagents were obtained from J.T. Baker (Phillipsburg, New Jersey, USA) or Merck (Kenilworth, New Jersey, USA). Other reagents and solvents were obtained from the commercial houses J.T. Baker (Phillipsburg, NJ, USA) or Merck (Kenilworth, NJ, USA). Stock compound solutions were prepared in ethanol or DMSO, depending on their solubility. From these solutions, working solutions for the tests were prepared. The standard mutagen stock solutions (4NQO and BLM) were prepared in acetone. Titanium dioxide stock solution was prepared in methanol.

Extract and Compound Cytotoxicity in Human Fibroblast (MRC5) Cells

Plant extracts and compounds were evaluated using the trypan blue exclusion assay.²¹ Lung embryonic fibroblast (MRC5) cells,²² graciously provided by Dr. Carlos Frederico Martins Menck from Universidade de Sao Paulo (Sao Paulo, Brazil), were grown in DMEM/F-12 medium (5 mL) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% GIBCO penicillin-streptomycin at 37°C under CO₂ (5%) conditions in a Midi 40 incubator (Thermo Scientific, Marietta, OH, USA). Every 3 days, cells were grown in fresh medium to reach a confluence of 80%. Cell cultures were mixed with each extract, compound, or mutagen at different concentrations and kept at 37°C (24 h) under CO₂ (5%) atmosphere conditions. After 24 h, trypsin-EDTA-treated cells were centrifuged (2000 r/min, 6 min), dissolved in PBS buffer (100 μL), and mixed (10 μL) with the same volume of trypan blue (.4%) to assess cell viability. Live and dead cells were counted using a Neubauer chamber and an Eclipse E200 optical microscope (Nikon Instruments Inc., NY, USA). At least three independent experiments were carried out for each treatment. The results were expressed as percentages of cell viability (% CV) as follows: % CV = (living cells)/(total cells) x 100. The cytotoxic concentrations 50% (CC₅₀) and 30% (CC₃₀) were calculated using the graphic method.²³ Extracts were considered cytotoxic at values ≥ CC₅₀ and noncytotoxic at values ≤ CC₃₀.

Extract and Compound Genotoxicities in Human Fibroblast (MRC5) Cells

The genotoxicity of plant extracts and major compounds was evaluated in MRC-5 cells using the Comet assay.¹² For this purpose, a high-throughput Trevigen CometChip^® platform (Gaithersburg, Maryland, USA) was used as indicated by Sykora et al,¹⁸ with minor modifications. First, a CometChip^® slide, cleaned previously with ethanol, was covered with an agarose solution prepared in PBS at 1.3% and tempered at 45°C; then, the agarose was solidified for 24 h at 4°C. Trypsin-treated cells (3 mL) were collected using centrifugation (2000 r/min, 6 min), washed two times using NaCl solution (.75%), centrifuged and suspended in fresh NaCl solution (3 mL), and then quantified using a Neubauer counting chamber. A cell suspension (3 mL) prepared at 4.4 × 10⁴ cells/mL was mixed with an equal volume of low-melting-point agarose prepared in PBS at 1%, and the mix was poured on a CometChip^® slide. The agarose was solidified for 15 min at 4°C. Finally, the CometChip^® chamber was assembled by hermetically sealing to prevent mixing between the wells. For cell treatments, 100 μL of extract, compound, or standard mutagen prepared as indicated above, were loaded into wells of the CometChip^® chamber. A sample of DMEM (100 μL) was considered a negative control. The chamber with treatments was incubated for 30 min at 37°C under CO₂ (5%) atmosphere conditions. The solutions of each treatment were removed from the wells, and 30 μL of Bioultra Proteinase K (.19 mg/mL) were loaded in each well for cell enzymatic lysis (1 h, 37°C).

After lysis, the CometChip^® slide was removed from the CometChip^® chamber and submerged for 15 min at 4°C in a comet electrophoresis tank (Cleaver Scientific Ltd, Rugby, Warwickshire,UK), which contained alkaline buffer (.3 N NaOH, 1 mM EDTA, and pH 13). Electrophoresis was carried out for 30 min at 300 mA and 25 V. The CometChip^® slide was submerged for 15 min in a tray containing neutralizing solution (.4 M TRIS and pH 7.5), then washed with distilled water, and dried at 37°C in a Midi 40 incubator. Finally, cell nuclei contained in each microgel were stained with 7 μL of YOYO solution (1 mM prepared in 5% DMSO) and were immediately scored using a Zeiss Axio Observer 7 fluorescence microscope (GmbH, Oberkochen, Germany), as shown in Figure 1.

Figure 1.

From left (non-damaged nuclei) to right (highly damaged nuclei), the figure shows increases in the number of DNA breaks. The intensity of the comet’s tail with respect to the head reflects the number of DNA breaks. Comets were classified into five categories based on this intensity of DNA damage, as was proposed by Collins et al.²⁴.

DNA damage was expressed in arbitrary units based on the classification of Comets into five categories (0-4), as proposed by Collins et al.²⁴ A genetic damage index (GDI) was calculated for each treatment as follows: GDI = (N₀x0 + N₁x1 + N₂x2 + N₃x3 + N₄x4)/n, where N is the number of nuclei scored in each category and n is the number of scored cells per slide.²⁵ Two hundred cells per slide and two slides per treatment were analyzed. The results from at least three independent experiments were averaged to obtain the GDI values for each treatment.

Statistical Analysis

The survival (%) and GDI values and their corresponding standard errors were calculated. In all cases, the data passed the Kolmogorov–Smirnov and F-maximum tests for normality and variance homogeneity, respectively; therefore, parametric tests were used in subsequent data analyses. When a significant F-value was obtained in one-way analysis of variance (ANOVA), the groups were subsequently compared with Tukey’s test. A Pearson correlation was performed to measure the relationship between GDI values and extract concentrations. For all statistical analyses, a value of P < .05 indicated significance. The R program²⁶ was used for all analyses.

Results

During cytotoxicity assays, we observed three well-differentiated responses of the fibroblasts to extract treatments: cells with intact membranes that were not stained, cells with disturbed membranes that were stained but kept their shape, and stained cell fragments that resulted from cell destruction (data not shown). None of the cells survived treatments with extracts from S. aratocensis and P. eriopodon species at the concentrations studied. Using graphical interpolation, the concentrations of the other plant extracts that produced 50% (CC₅₀) and 30% (CC₃₀) of cell cytotoxicity in human fibroblasts were calculated. The cytotoxicity (CC₅₀) of these extracts was as follows: L. origanoides (89 μg/mL; most cytotoxic) > A. satureioides (173 μg/mL) > C. pellia (184 μg/mL). These extracts were innocuous to fibroblast cells at concentrations less than or equal to (≤) the CC₃₀ values as follows (Figure 2): L. origanoides (74 μg/mL), A. satureioides (140 μg/mL), and C. pellia (149 μg/mL).

Figure 2.

Viability of untreated (controls) MRC5 human fibroblasts and viability of cells treated with different non-cytotoxic extract concentrations. The cell viability (%) mean values and their corresponding standard errors (SEM), calculated from at least three independent experiments (n = 3), are presented.

A characterization of chemical compositions of these extracts determined by GC/MS analysis is presented in Table 1. The major compounds (relative amount ≥10%) identified in the extracts were as follows: trans-β-caryophyllene (25.0%) and caryophyllene oxide (13.4%) in A. satureioides; α-amyrin (22.0%) and squalene (18.5%) in C. pellia; and pinocembrin (54.9%) and trans-β-caryophyllene (10.9%) in L. origanoides.

Table 1.

Major Compounds in Plant Extracts Obtained by Supercritical Fluid (CO₂) Extraction From: A: Achyrocline satureioides COL579420, B: Chromolaena pellia COL 559437, and C: Lippia origanoides COL 560259.

Compounds^a	LRI (DB-5MS)		Relative Amount (%)
Compounds^a	Exp.	Lit.	A	B	C
α- Pinene	0937	0932 [1]	7.4	—	—
p-Cymene	1029	1020 [1]	—	—	6.2
1,8-Cineol	1037	1026 [1]	—	—	3.5
Thymol	1290	1289 [1]	—	—	4.6
Carvacrol	1300	1298 [1]	—	—	6.3
trans-β-Caryophyllene	1433-35	1417 [1]	25.0	8.1	10.9
α-Humulene	1471	1452 [1]	—	—	4.6
γ-Muurolene	1487	1478 [1]	8.9	—	—
γ-Cadinene	1522-27	1513 [1]	7.5	—	—
Caryophyllene oxide	1597-99	1582 [1]	13.4	5.3	—
C₁₅H₂₆O (sesquiterpenol)^b	1859	—	—	11.6
Phytol	2118	2122 [2]	—	4.8	—
α-Linolenic acid	2144	2139 [1]	—	4.7	—
Ethyl α-linolenate	2170	2173 [2]	—	2.1	—
Pinocembrin	2034	2513 [2]	—	—	54.9
Squalene	2837	2832 [2]	—	18.5	—
α-Amyrin	3450	3376 [2]	—	22.0	—

LRI, Linear retention indices. Exp., Experimental, Lit., Literature. (−), Not detected in the extract.

[1] Adams P. 2017. Identification of essential oil components by gas chromatography/mass spectrometry. Allured Publishing Corporation, (4th ed.), Carol Stream, Illinois, USA.

[2] NIST Mass Spectrometry Data Center (https://webbook.nist.gov/chemistry).

^aOnly the components whose relative amount was higher than 2% in at least one specimen were considered.

^bMass spectrum: M⁺ 220 (15%), (M-CH₃)⁺ 205 (10%), (M-H₂O)⁺ 202 (45%), (M-CH₃-H₂O)⁺ 189 (19%), (M-H₂O-C₃H₇)⁺ 159 (100%), 93 (55%), and (C₃H₇)⁺ 43 (65%).

Some of these compounds (trans-β-caryophyllene, squalene, and pinocembrin), which have shown protective properties against UVB radiation,²⁷ were assayed for cytotoxicity. Quercetin, resveratrol, and filter titanium dioxide were also included for comparison. Trans-β-caryophyllene was cytotoxic at concentrations between 918 and 14680 μM (data not shown). We excluded this compound from further studies because lower concentrations (<7631 μM) are not relevant for photoprotection. The cell viability percentage values obtained for the remaining compounds are presented in Table 2. Except for squalene, all compounds showed some cytotoxicity at the concentrations studied. Based on the CC₅₀ values, the compound cytotoxicity was as follows: resveratrol (91 μM) > pinocembrin (144 μM) > quercetin (222 μM) > titanium dioxide (704 μM). As expected, these compounds were less toxic to human fibroblasts than the reference mutagen used (e.g., 4NQO, CC₅₀ = 2.5 μM). The compounds were safe to fibroblasts at concentrations ≤ CC₃₀ as follows: resveratrol (70 μM), pinocembrin (102 μM), quercetin (141 μM), titanium dioxide (455 μM), and squalene (820 μM). Thus, only the compound squalene could be used at concentrations higher than titanium dioxide.

Table 2.

Cell Viability of Human Fibroblast Treated With Different Concentrations of the Compounds are presented.

Pinocembrin		Quercitin		Resveratrol		Squalene		Titanium Dioxide		4NQO^a
Conc. (μM)	S ± SEM	Conc. (μM)	S ± SEM	Conc (μM)	S ± SEM	Conc. (μM)	S ± SEM	Conc. (μM)	S ± SEM	Conc. (μM)	S ± SEM
0.0	93 ± 3	0.0	96 ± 1	0.0	91 ± 2	0.0	90 ± 2	0.0	81 ± 8	0.0	87 ± 3
24.4	90 ± 1	20.7	92 ± 1	27.4	89 ± 3	58.6	90 ± 2	78.3	88 ± 3	0.3	84 ± 2
48.8	86 ± 2	41.4	92 ± 1	54.8	85 ± 4	117.1	90 ± 1	156.5	86 ± 2	0.6	76 ± 1
97.6	72 ±10	82.7	87 ± 1	109.5	32 ± 1	234.2	88 ± 1	313.0	81 ± 3	1.2	68 ± 1
195.1	25 ± 7	165.4	63 ± 4	219.0	2 ± 2	468.4	86 ± 1	626.0	57 ± 10	2.4	54 ± 3
390.2	0 ± 0	330.9	26 ± 5	438.1	1 ± 1	936.9	65 ± 10	1252.1	0 ± 0	4.7	2 ± 1

The survival (S) mean values and their corresponding standard errors (SEM) calculated from at least three independent experiments (n = 3) are presented.

^aThe standard mutagen 4-nitro-quinoline-1-oxide (4NQO) was used as a positive control (PC).

Genotoxicity dose–response relations were studied for A. satureioides, C. pellia, and L. origanoides extracts (Table 3). According to genotoxicity criteria (Figure 1), the L. origanoides and C. pellia extracts produced some degree of DNA damage from 375.0 μg/mL, while the A. satureioides extract was non-genotoxic at the concentration assayed. Evaluation of the equivalent solvent concentrations (dilutions) in extracts indicated that the solvent (methanol) was not genotoxic in human fibroblasts in the concentration range tested. Therefore, except for A. satureioides, the extracts showed low-to-moderate genotoxicity at the concentration range studied. Such genotoxicity showed a clear dose–response relationship (extract concentration–DNA damage). Compared with known mutagens (Figure 3), these data also suggest a genotoxic mode of action depending on cytotoxicity of the extracts.

Table 3.

Genotoxicity of the Flower Extracts in MRC5 Human Fibroblasts Cells.

Conc. (μg/mL)	Genetic Damage Index (GDI ± SEM)^a
Conc. (μg/mL)	A. satureioides	C. pellia	L. origanoides	SEC^b
0.0	.19 ± .07	.29 ± .07	.16 ± .11	.19 ± .05
46.9	.40 ± .07	.79 ± .29	.41 ± .08	.80 ± .28
93.7	.41 ± .01	.98 ± .28	.64 ± .14	.84 ± .28
187.5	.49 ± .02	.93 ± .02	.90 ± .12	.83 ± .28
375.0	.70 ± .08	1.36 ± .30	1.50 ± .14	.85 ± .30
750.0	.87 ± .20	1.80 ± .35	2.72 ± .48	.91 ± .28
PC	3.91 ± .03	3.90 ± .03	3.92 ± .04	3.90 ± .08
R =	.95 (P < .05)	.93 (P < .05)	.99 (P < .05)	.53 (P < .28)

The genetic damage index (GDI) mean values and their corresponding standard errors (SEM) calculated from at least three independent experiments (n=3) are given. The Pearson correlation coefficient (R) showing the relationship between GDI values and extract concentrations is also presented.

The standard mutagen 4-nitro-quinoline-1-oxide (.89 μg/mL) was used as a positive control (PC).

^aThe DNA damage criteria were as follows: (i) GDI values between 0 and 1 (no DNA damage), (ii) GDI values between 1 and 2 (little DNA damage), (iii) GDI values between 2 and 3 (moderate DNA damage), and (iv) GDI values between 3 and 4 (severe DNA damage). In addition, a clear dose-response relationship (concentration-DNA damage) must exist.

^bGDI values for solvent (methanol) equivalent concentrations (between 39 and 618 mM) in the extracts.

Figure 3.

Cytotoxicity versus genotoxicity curves in human fibroblasts (MRC5) of the standard mutagens 4-nitro-quinoline-N-oxide (above) and bleomycin (below). The mean values of cell viability (%), genetic damage index (GDI), and their corresponding standard errors (SEM), calculated from at least three independent experiments (n = 3), are given.

Genotoxicity dose–response relations were studied for plant compounds and a standard filter (titanium dioxide) (Table 4). According to the established criteria, the genotoxicity of the compounds studied was as follows: (i) moderate (quercetin), (ii) low (resveratrol), and (iii) non-genotoxic (pinocembrin, squalene, and titanium dioxide). Genotoxicity at non-cytotoxic concentrations was only evidenced for quercetin. In general, the compounds pinocembrin, squalene, and titanium dioxide were innocuous for human fibroblasts.

Table 4.

Genotoxicity of the Polyphenol Compounds in MRC5 Human Fibroblasts Cells.

Pinocembrin		Quercitin		Resveratrol		Squalene		Titanium Dioxide
Conc. (μM)	GDI ± SEM^a	Conc. (μM)	GDI ± SEM	Conc (μM)	GDI ± SEM	Conc. (μM)	GDI ± SEM	Conc. (μM)	GDI ± SEM
0.0	.12 ± .03	0.0	.09 ± .01	0.0	.23 ± .10	0.0	.12 ± .05	0.0	.11 ± .03
9.8	.52 ± .23	14.1	.45 ± .13	6.2	.64 ± .07	92.8	.33 ± .11	50.9	.28 ± .13
19.5	.51 ± .24	28.1	.64 ± .06	12.5	.68 ± .12	187.5	.32 ± .04	101.7	.26 ± .16
39.0	.52 ± .25	56.2	.87 ± .18	25.0	.77 ± .05	374.9	.33 ± .10	203.5	.25 ± .20
78.0	.51 ± .27	112.5	1.27 ± .15	49.9	.86 ± .03	749.9	.48 ± .07	406.9	.31 ± .22
156.1	.54 ± .23	225.0	2.15 ± .21	99.9	1.19 ± .04	1499.8	.52 ± .13	813.9	.28 ± .20
PC	3.54 ± .18	PC	3.85 ± .07	PC	3.96 ± .03	PC	3.87 ± .08	PC	3.63 ± .11
R = .46 (P = .40)		R = .99 (P < .05)		R = .89 (P < .05)		R = .84 (P < .05)		R = .5 (P = .3)

The genetic damage index (GDI) mean values and their corresponding standard errors (SEM) calculated from at least three independent experiments (n = 3) are given. The Pearson correlation coefficient (R) showing the relationship between GDI values and extract concentrations is also presented.

The standard mutagen 4-nitro-quinoline-1-oxide (.89 μg/mL) was used as a positive control (PC).

Discussion

This work focused on the safety of plants with previously demonstrated cosmetic use potential.¹⁹ Among the five plant species studied, two (P. eriopodon and S. aratocensis) were cytotoxic to human fibroblasts at all concentrations under study (between 125 and 750 mg/mL). This means that the extracts produce non-cell viability value at relevant antigenotoxic/photoprotective concentrations (Table 5) as a consequence of their membrane disruption ability. These findings provide new evidence on the cytotoxicity of the P. eriopodon and S. aratocensis plant species. At the relevant concentrations, the A. satureioides, C. pellia, and L. origanoides extracts produced relatively less cytotoxicity to human fibroblasts. In this way, our work provides new data on safety of these three plant extracts in human skin cells useful in photoprotection.

Table 5.

Relevant Information Available in the Literature on Activities of the Studied Plants.

Plant Species	Relevant Extract Concentrations [Sources]	Relevant Plant Properties [Sources]
Piper eriopodon	CGI = 125 μg/mL¹⁹CIP = 250 μg/mL¹⁹CC₃₀ = ND¹⁹CC₅₀ = ND [this work]CGD = ND [this work]	In vitro photoprotection/antigenotoxicity against UVB radiation¹⁹In vitro cytotoxicity in human fibroblast cells [¹⁹, this work]In vitro genotoxicity in human fibroblast cells [this work]In vitro cytotoxicity in human breast and cervical cancer cell lines [28]
Salvia aratocensis	CGI = 125 μg/mL¹⁹CIP = 250 μg/mL¹⁹CC₃₀ = ND¹⁹CC₅₀ = ND [this work]CGD = ND [this work]	In vitro photoprotection/antigenotoxicity against UVB radiation¹⁹In vitro cytotoxicity in human fibroblast cells [¹⁹, this work]In vitro genotoxicity in human fibroblast cells [this work]In vitro antibacterial activity against mycobacteria²⁹
Chromolaena pellia	CGI = 125 μg/mL¹⁹CIP = 125 μg/mL¹⁹CC₃₀ = 145-149 μg/mL [¹⁹, this work]CC₅₀ = 184 μg/mL [this work]CGD = 375 μg/mL [this work]	In vitro photoprotection/antigenotoxicity against UVB radiation¹⁹In vitro cytotoxicity in human fibroblast cells [¹⁹, this work]In vitro genotoxicity in human fibroblast cells [this work]
Lippia origanoides	CGI = 62 μg/mL¹⁹CIP = 62 μg/mL¹⁹CC₃₀ = 74 μg/mL [¹⁹, this work]CC₅₀ = 89 μg/mL [this work]CGD = 375 μg/mL [this work]	In vitro photoprotection/antigenotoxicity against UVB radiation¹⁹In vitro cytotoxicity in human fibroblast cells [¹⁹, this work]In vitro genotoxicity in human fibroblast cells [this work]In vitro cytotoxicity in Trypanosoma cruzi cells³⁴Acute toxicity activity against insects^35-37Acute toxicity activity against Artemia franciscana³⁸
Achyrocline satureioides	CGI = 125 μg/mL¹⁹CIP = 125 μg/mL¹⁹CC₃₀ = 135-140 μg/mL [¹⁹, this work]CC₅₀ = 173 μg/mL [this work]CGD = NG [this work]	In vitro photoprotection/antigenotoxicity against UVB radiation¹⁹In vitro cytotoxicity in human fibroblast cells [¹⁹, this work]In vitro genotoxicity in human fibroblast cells [this work]In vitro immunomodulatory activity in human leukocytes⁴⁰Neuroprotective activity against cerebral ischemic in rats⁴¹In vitro anticarcinogenic activity in human glioma cells⁴²In vitro anticarcinogenic activity in human breast cancer cell⁴³

CGI: Minimal concentration that produces significant (P ≤ .05) UVB-induced genotoxicity inhibition.

CIP: Minimal concentration that produces some degree of in vitro photoprotection (SPF_{in vitro} ≥ 6.0) against UVB radiation.

CC₃₀ and CC₅₀: Cytotoxic concentrations at which 70% and 50% of human fibroblast cells survive, respectively.

ND: Not determined because the extract resulted totally toxic at concentration ≥125 μg/mL.

CGD: Minimal concentration that produces relevant genetic damage index (GDI >1) values in human fibroblast cells.

NG: Not genotoxic in human fibroblast cells at concentration ≤750 μg/mL.

There are some reports on cytotoxicity of these plant species, or closely related, on human non-skin cells. For example, the essential oils obtained from P. eriopodon and Salvia officinalis, a closely related species to S. aratocensis, showed high cytotoxicity in human breast and cervical cancer cell lines.²⁸ Although there are no reports in humans, the S. aratocensis essential oil has demonstrated cytotoxicity to mycobacteria.²⁹ The essential oil from C. odorata, a related species to C. pellia, resulted toxic in human breast and cervical cancer and embryonic kidney cell lines,³⁰ whereas the flavonoids and steroids from C. hirsute and C. squalida species show antimicrobial activity.³¹ The synonymous genus Eupatorium has extensively demonstrated antimicrobial properties.³² Finally, L. origanoides essential oil bioactivity has been widely studied,³³ including its toxic effects in different models such as protozoa,³⁴ insects,^35–37 and crustaceans.³⁸

Conversely, we do not know positive cytotoxicity or toxicity reports of the A. satureioides species. This plant is used in Brazilian folk medicine as a digestive, eupeptic, emmenagogue, antispasmodic, anti-inflammatory, expectorant, and antidiarrheal agent.³⁹ The immunomodulatory and neuroprotective properties of this plant have been confirmed in pharmacological studies.^40,41 This plant also shows anticancer activity^42,43 and is non-cytotoxic and non-irritating to human skin,⁴⁴ supporting its use in topical administration. All these findings support the potential use of A. satureioides in cosmetic photoprotection.

On the other hand, the C. pellia and L. origanoides extracts also produced low-to-moderate DNA damage at cytotoxic concentrations. This genotoxic pattern contrasted with the results observed with known mutagens (Figure 3) that were genotoxic at cell viabilities ≥70%. This indicated that genotoxicity in C. pellia and L. origanoides extracts was related to cytotoxicity.⁴⁵ Previous studies^46-48 have recommended testing concentrations presenting viabilities ≥70-75% to avoid false genotoxicity results with the Comet assay. In this sense, trypan blue exclusion and comet assays, which measure cytotoxicity (membrane disruption) and genotoxicity (DNA breaks), respectively, were complementary for this purpose. As in previous studies,^15-18 our work demonstrated the usefulness of the high-throughput (96-well microplate) Comet platform, in this case, for safety testing of phytochemicals. Here, we replaced alkaline per enzymatic (proteinase K) lysis of the cells since step alkaline lysis produced high levels of DNA breaks in untreated fibroblast cells (data not shown), as previously observed by García-Forero et al.⁴⁹ Proteinase K also digests proteins associated with nuclei,^50,51 an action that facilitates DNA unwinding similar to alkaline treatments. Our data support the idea that enzymatic lysis and alkaline incubation prior to electrophoresis are sufficient to unwind DNA, as previously shown in human buccal⁵² and keratinocytes⁵³ cells.

We determined chemical composition of the studied plant extracts using gas chromatography coupled to mass spectrometry (GC/MS) analysis (Table 1). In accordance with previous studies,¹⁹ we have confirmed their chemical composition. Some of the major compounds present in these plants were evaluated for their cytotoxicity and genotoxicity. Two relevant findings resulted from this analysis. First, trans-β-caryophyllene was the only compound that was cytotoxic over the entire range of concentrations evaluated. That is, the compound destroyed the membranes of human fibroblasts at all concentrations as was previously demonstrated^54-56 These studies have explained cytotoxic activity of plant terpenoid compounds by their membrane disrupter capability that increase its permeability. Like previous studies,^57,58 pinocembrin and resveratrol also showed some degree of cytotoxicity depending on concentration. Second, pinocembrin, resveratrol, squalene, and titanium dioxide were non-genotoxic and basically innocuous to human fibroblasts. Overall, the genotoxicity was related to cytotoxicity, except for quercetin, whose genotoxicity has been well documented.^59,60 Our findings suggest that cytotoxic/genotoxic activities of the plant extracts could be related to a synergistic effect between its major compounds, as previously proposed,⁶¹ but this hypothesis requires confirmation in further studies. In addition, the results emphasize the necessity of establishing safe plant extract and compound concentrations for their potential use in cosmetics.

Conclusion

We showed that the P. eriopodon and S. aratocensis extracts were clearly cytotoxic in human fibroblast cells. The A. satureioides, C. pellia, and L. origanoides extracts showed different degrees of cytotoxicity (CC₅₀) as follows: L. origanoides (89 μg/mL) > A. satureioides (173 μg/mL) > C. pellia (184 μg/mL). L. origanoides and C. pellia extracts showed genotoxicity in human fibroblasts related to their cytotoxicity. A. satureioides, C. pellia, and L. origanoides extracts were neither cytotoxic nor genotoxic at relevant concentrations for photoprotection. Pinocembrin, squalene, and titanium dioxide were basically innocuous to human fibroblasts. Our findings unequivocally indicated the necessity of establishing safe plant extract concentrations for their potential use in cosmetics. That is, the plant extracts should be used at noncytotoxic (≤CC₃₀ values) and nongenotoxic (GDI values ≤1) concentrations. Our data were based on fibroblast cell assays; therefore, at least a second evaluation in different skin cell models (e.g., keratinocytes and melanocytes) is recommended before its use for skin photoprotection.

Footnotes

Acknowledgements

The authors thank funding from the Ministry of Science, Technology, and Innovation, the Ministry of Education, the Ministry of Industry, Commerce, and Tourism, and ICETEX, Programme Ecosistema Científico-Colombia Científica, from the Francisco José de Caldas Fund, Grant RC-FP44842-212-2018. The Ministry of Environment and Sustainable Development of Colombia supported the Universidad Industrial de Santander through access permits to genetic resources and derivatives for bioprospecting (contract no. 270). The authors thank Carlos Frederico Martins Menck from Universidade de Sao Paulo (Sao Paulo, Brazil) for supplying the human fibroblast (MRC5) cell line and thank Dr. Robert Tulio González from Universidad del Pacífico (Buenaventura, Colombia) for supplying the Achyrocline satureioides photo.

Author Contributions

Flórez González, S. J. contributed to acquisition, analysis, and interpretation; Stashenko, E. E. contributed to analysis and interpretation; Ocazionez, R. E. contributed to analysis and interpretation; Vinardell, M. P. contributed to analysis and interpretation; Fuentes, J. L. contributed to conception and design, contributed to analysis and interpretation, and drafted the manuscript. All authors critically revised the manuscript, gave final approval, and agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Ministry of Science, Technology, and Innovation, the Ministry of Education, the Ministry of Industry, Commerce, and Tourism, and ICETEX, Programme Ecosistema Científico-Colombia Científica, from the Francisco José de Caldas Fund, Grant RC-FP44842-212-2018.

Live Subject Statement

The project RC-FP44842-212-2018 was approved by the Scientific Research Ethical Committee (Record No. 15 – 2017 and File No. 4110) from Universidad Industrial de Santander. The experiments and chemical management were performed according to the national law (Resolution No. 008430- 1993) from the Ministry of Health of Colombia and the Institutional Manual of Integrated Management and Processes (PGIR–PGGA.05).

ORCID iD

Jorge Luis Fuentes

References

Mileva

Ilieva

Jovtchev

, et al.

Rose flowers–a delicate perfume or a natural healer?

Biomolecules. 2021;11:127.

Capasso

Izzo

Pinto

Bifulco

Vitobello

Mascolo

. Phytotherapy and quality of herbal medicines. Fitoterapia. 2020;71:S58-S65.

Fennell

Elgorashi

Lindsey

, et al. Assessing African medicinal plants for efficacy and safety: pharmacological screening and toxicology. J Ethnopharmacol. 2004;94:205-217.

Prinsloo

Nogemane

Street

. The use of plants containing genotoxic carcinogens as foods and medicine. Food Chem Toxicol. 2018;116:27-39.

Pauwels

Rogiers

. Human health safety evaluation of cosmetics in the EU: a legally imposed challenge to science. Toxicol Appl Pharmacol. 2010;243:260-274.

Pfuhler

Fautz

Reisinger

, et al. The Cosmetics Europe strategy for animal-free genotoxicity testing: project status up-date. Toxicol Vitro. 2014;28:18-23.

Vinardell

. The use of non-animal alternatives in the safety evaluations of cosmetics ingredients by the Scientific Committee on Consumer Safety (SCCS). Regul Toxicol Pharmacol. 2015;71:198-204.

Vinardell

Mitjans

. Alternative methods to animal testing for the safety evaluation of cosmetic ingredients: an overview. Cosmetics. 2017;4:30.

Kim

Kwack

Lee

Kacew

Lee

. Current opinion on risk assessment of cosmetics. J Toxicol Environ Health B. 2021;24:137-161.

10.

Pfuhler

Kirst

Aardema

, et al. A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. A COLIPA analysis. Regul Toxicol Pharmacol. 2010;57:315-324.

11.

Sponchiado

de Mello-Sampayo

Adam

, et al. Quantitative genotoxicity assays for analysis of medicinal plants: a systematic review. J Ethnopharmacol. 2016;178:289-296.

12.

Singh

McCoy

Tice

Schneider

. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175:184-191.

13.

Collins

. The Comet assay for DNA damage and repair. Principles, applications and limitations. Mol Biotechnol. 2004;26:249-261.

14.

Frötschl

. Experiences with the in vivo and in vitro comet assay in regulatory testing. Mutagenesis. 2015;30:51-57.

15.

Gutzkow

Langleite

Meier

Graupner

Collins

Brunborg

. High-throughput comet assay using 96 minigels. Mutagenesis. 2013;28:333-340.

16.

Chow

Fessler

Weingeist

Wood

Engelward

. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification. Mutagenesis. 2015;30:11-19.

17.

Albert

Robaire

Reintsch

Chan

. HT-COMET: a novel automated approach for high throughput assessment of human sperm chromatin quality. Hum Reprod. 2016;31:938-946.

18.

Sykora

Witt

Revanna

, et al. Next generation high throughput DNA damage detection platform for genotoxic compound screening. Sci Rep. 2018;8:2771.

19.

Fuentes

Villamizar-Mantilla

Flores-González

Núñez

Stashenko

. Plants growing in Colombia as sources of active ingredients for sunscreens. Int J Radiat Biol. 2021;97:1705-1715.

20.

Stashenko

Martínez

Cala

Durán

Caballero

. Chromatographic and mass spectrometric characterization of essential oils and extracts from Lippia (Verbenaceae) aromatic plants. J Sep Sci. 2013;36:192-202.

21.

Strober

. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2015;111:A3B1-A3B3.

22.

Jacobs

Jones

Baille

. Characteristics of a human diploid cell designated MRC- smelly emma 5. Nature. 1970;227:168-170.

23.

Ara

Nur

Amran

Wahid

MII

Ahmed

. In vitro antimicrobial and cytotoxic activities of leaves and flowers extracts from Lippia alba. Pak J Biol Sci. 2009;12:87-90.

24.

Collins

Dušinská

Franklin

, et al. Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ Mol Mutagen. 1997;30:139-146.

25.

Pitarque

Creus

Marcos

Hughes

Anderson

. Examination of various biomarkers measuring genotoxic endpoints from Barcelona airport personnel. Mutat Res. 1999;440:195-204.

26.

R Core Team. R: A Language and Environment for Statistical Computing. Vienna (Austria): R Foundation for Statistical Computing. 2013; Available online: https://www.R-project.org (accessed on 31 03 2022).

27.

Fuentes

García-Forero

Quintero-Ruiz

, et al. The SOS Chromotest applied for screening plant antigenotoxic agents against ultraviolet radiation. Photochem Photobiol Sci 2017;16(9):1424-1434.

28.

Velandia

Quintero

Stashenko

Ocazionez

. Actividad antiproliferativa de aceites esenciales de plantas cultivadas en Colombia. Acta Biol Colomb. 2018;23:189-198.

29.

Bueno

Escobar

Martínez

Leal

Stashenko

. Composition of three essential oils, and their mammalian cell toxicity and antimycobacterial activity against drug resistant-tuberculosis and nontuberculous mycobacteria strains. Nat Prod Commun. 2011;6:1743-1748.

30.

Velandia

Flechas

Ocazionez

. Proposal to select essential oils from Colombian plants for research based on its cytoxicity. VITAE. 2016;23:18-29.

31.

Taleb-Contini

Salvador

Watanabe

Yoko-Ito

Rodrigues de Oliveira

. Antimicrobial activity of flavonoids and steroids isolated from two Chromolaena species. Braz J Pharm Sci. 2003;39:403-408.

32.

Nogueira-Sobrinho

Fontenelle

ROS

Maia de Morais

de Souza

. The genus Eupatorium L. (Asteraceae): a review of their antimicrobial activity. J Med Plant Res. 2017;11:43-57.

33.

Oliveira

Leitão

Fernandes

Leitão

. Ethnopharmacological studies of Lippia origanoides. Rev Bras Farmacogn. 2014;24:206-214.

34.

Raposo-Borges

de Albuquerque Aires

Higino

TMM

, et al. Trypanocidal and cytotoxic activities of essential oils from medicinal plants of Northeast of Brazil. Exp Parasitol. 2012;132:123-128.

35.

Nerio

Olivero-Verbel

Stashenko

. Repellent activity of essential oils from seven aromatic plants grown in Colombia against Sitophilus zeamais Motschulsky (Coleoptera). J Stored Prod Res. 2009;45:212-214.

36.

Sivira

Sanabria

Valera

Vásquez

. Toxicity of ethanolic extracts from Lippia origanoides and Gliricidia sepium to Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae). Neotrop Entomol. 2011;40:375-379.

37.

Caballero-Gallardo

Olivero-Verbel

Stashenko

. Repellency and toxicity of essential oils from Cymbopogon martinii, Cymbopogon flexuosus and Lippia origanoides cultivated in Colombia against Tribolium castaneum. J Stored Prod Res. 2012;50:62-65.

38.

Olivero-Verbel

Güette-Fernandez

Stashenko

. Acute toxicity against Artemia franciscana of essential oils isolated from plants of the genus Lippia and Piper collected in Colombia. BLACPMA. 2009;8:419-427.

39.

Gonçalves

Ferreira

Ming

. Achrocline satureiodes Lam. (DC.). In: Medicinal and Aromatic Plants of South America. Brazil.

40.

Cosentino

Bombelli

Crema

, et al. Immunomodulatory properties of Achyrocline satureioides (Lam.) D.C. infusion: a study on human leukocytes. J Ethnopharmacol. 2008;116:501-507.

41.

Rivera-Megret

Tejera-Correa

Abin-Carriquiri

dos Santos

Martínez-Busi

Dajas-Méndez

. Achyrocline satureioides reduces brain damage in permanent focal ischemia in rats. Rev Cuba Plantas Med. 2013;18:445-460.

42.

Oliveira de Souza

Bianchi

Figueiró

, et al. Anticancer activity of flavonoids isolated from Achyrocline satureioides in gliomas cell lines. Toxicol Vitro. 2018;51:23-33.

43.

Bianchi

Mascia

Pegues

, et al. Achyrocline satureioides compounds, achyrobichalcone and 3-O-methylquercetin, induce mitochondrial dysfunction and apoptosis in human breast cancer cell lines. IUBMB Life. 2020;72:2133-2145.

44.

Balestrin

Kreutz

Silveira-Fachel

, et al. Achyrocline satureioides (Lam.) DC (Asteraceae) extract-loaded nanoemulsions as a promising topical wound healing delivery system: in vitro assessments in human keratinocytes (HaCaT) and HET-CAM irritant potential. Pharmaceutics. 2021;13:1241.

45.

Vock

Lutz

Hormes

Hoffmann

Vamvakas

. Discrimination between genotoxicity and cytotoxicity in the induction of DNA double-strand breaks in cells treated with etoposide, melphalan, cisplatin, potassium cyanide, triton x-100, and Γ-irradiation. Mutat Res. 1998;413:83-94.

46.

Anderson

McGregor

. Comet assay responses as indicators of carcinogenic exposure. Mutagenesis. 1998;13:539-555.

47.

Henderson

Wolfreys

Fedyk

Bourner

Windebank

. The ability of the comet assay to discriminate between genotoxins and cytotoxins. Mutagenesis. 1998;13:89-94.

48.

Frieauff

Hartmann

Suter

. Automatic analysis of slides processed in the Comet assay. Mutagenesis. 2001;16:133-137.

49.

García-Forero

Villamizar-Mantilla

Núñez de Villavicencio-Martínez

Ocazionez

Stashenko

Fuentes

. Photoprotective and antigenotoxic effects of the flavonoids apigenin, naringenin and pinocembrin. Photochem Photobiol 2019;95:1010-1018.

50.

Merk

Reiser

Speit

. Analysis of chromate induced DNA-protein crosslinks with the comet assay. Mutat Res. 2000;471:71-80.

51.

Singh

. Microgels for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. Mutat Res. 2000;455:111-127.

52.

Rojas

Valverde

Sordo

Ostrosky-Wegman

. DNA damage in exfoliated buccal cells of smokers assessed by the single cell gel electrophoresis assay. Mutat Res. 1996;370:115-120.

53.

Decome

De Méo

Geard

Doucet

Duménil

Botta

. Evaluation of photolyase (Photosome) repair activity in human keratinocytes after a single dose of ultraviolet B irradiation using the comet assay. J Photochem Photobiol B. 2005;79:101-108.

54.

Ultee

Kets

EPW

Smid

. Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Appl Environ Microbiol. 1999;65:4606-4610.

55.

Lambert

RJW

Skandamis

Coote

Nychas

GJE

. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J Appl Microbiol. 2001;91:453-462.

56.

Leal

Pino

Stashenko

Martínez

Escobar

. Antiprotozoal activity of essential oils derived from Piper spp. grown in Colombia. J Essent Oil Res. 2013;25:512-519.

57.

Chen

Rasul

Zhao

, et al. Antiproliferative and apoptotic effects of pinocembrin in human prostate cancer cells. Bangladesh J Pharmacol. 2013;8:255-262.

58.

Aggarwal

Bhardwaj

Aggarwal

Seeram

Shishodia

Takada

. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 2004;4:2783-2840.

59.

Gaspar

Rodrigues

Laires

, et al. On the mechanisms of genotoxicity and metabolism of quercetin. Mutagenesis. 1994;9:445-449.

60.

Engen

Maeda

Wozniak

, et al. Induction of cytotoxic and genotoxic responses by natural and novel quercetin glycosides. Mutat Res. 2015;784–785:15-22.

61.

Quintero-Ruiz

Cordoba-Campo

Stashenko

Fuentes

. Antigenotoxicity effect against ultraviolet radiation-induced DNA damage of the essential oils from Lippia species. Photochem Photobiol. 2017;93:1063-1072.

In vitro Safety Assessment of Extracts and Compounds From Plants as Sunscreen Ingredients

Abstract

Keywords

Introduction

Materials and Methods

Plant Materials and Extracts

Gas Chromatography Coupled to Mass Spectrometry (GC/MS) Analysis

Chemicals, Buffer, Enzymes, and Culture Media

Extract and Compound Cytotoxicity in Human Fibroblast (MRC5) Cells

Extract and Compound Genotoxicities in Human Fibroblast (MRC5) Cells

Statistical Analysis

Results

Discussion

Conclusion

Footnotes

Acknowledgements

Author Contributions

Declaration of Conflicting Interests

Funding

Live Subject Statement

ORCID iD

References