Isolation, Characterization, and Breast Cancer Cytotoxic Activity of Gyrophoric Acid from the Lichen Umbilicaria muhlenbergii

: Lichens produce a large variety of secondary metabolites with diverse bioactivities, chemical structures, and physicochemical properties. For this reason, there is a growing interest in the use of lichen-derived bioactive molecules for drug discovery and development. Here, we report on the isolation, identiﬁcation, and cytotoxic evaluation of gyrophoric acid (GA) from the lichen Umbilicaria muhlenbergii , a largely unexplored and scantly described lichen species. A simple puriﬁcation protocol was developed for the fractionation of lichen crude extracts with silica gel column chromatography using solvents with changing polarity. GA was identiﬁed in one of the fractions with Fourier trans-form infrared spectroscopy (FTIR), ion trap mass spectrometry (MS), and nuclear magnetic resonance spectroscopy ( 1 H-NMR and 13 C-NMR). The FTIR spectra demonstrated the presence of aromatic and ester functional groups C=C, C-H, and C=O bonds, with the most remarkable signals recorded at 1400 cm − 1 for the aromatic region, at 1400 cm − 1 for the CH 3 groups, and at 1650 cm − 1 for the carbonyl groups in GA. The MS spectra showed a molecular ion [M-1] − at ( m / z ) 467 with a molecular weight of 468.4 and the molecular formula C 24 H 20 O 10. that correspond to GA. The 1 H-NMR and 13 C-NMR spectra veriﬁed the chemical shifts that are typical for GA. GA reduced the cell viability of breast cancer cells from the MCF-7 cell line by 98%, which is indicative of the strong cytotoxic properties of GA and its signiﬁcant potential to serve as a potent anticancer drug.


Introduction
Cancer is a complex disease with a strong relationship between genetic and environmental factors [1]. One of the hallmarks of cancer is that cancer cells can penetrate adjacent parts of the body and lead to invasion and metastasis [2]. Although cancer research has advanced considerably over the years, cancer mortality remains high [3]. A report showed that the cancer rate has been escalating since 1990, with the most common types being lung, colorectal, and stomach cancers [4]. Between 2012 and 2020, new cancer cases increased by 37% to reach 19.3 million [5,6]. In the U.S. alone, 1.8 million new cancer cases occurred, representing 9% of the global cancer occurrence in 2020 [7]. Among cancer types, breast cancer remains one of the most common lethal types of cancer for women [8], resulting in the deaths of 1 out of 10 women diagnosed with breast cancer [9]. In 2020, there were 2.3 million women diagnosed with breast cancer and 685,000 deaths globally [10]. The

Cell Culture and Maintenance
The human breast cancer cell line MCF-7 (purchased from American Type Culture Collection, ATCC, Manassas, VA, USA) was grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The cell line was maintained in a humidified atmosphere containing 5% CO 2 at 37 • C.

Cell Viability Assay and IC 50
The MTT method [41] was used to measure cell viability. The MCF-7 cells were seeded in a 96-well plate (Corning@Costar, 96-well plate) at a concentration of 1 × 10 4 cells/well and incubated in an atmosphere of 5% CO 2 and 37 • C for 24 h to obtain good adherence. The initial medium was then replaced with fresh medium supplemented with 300-500 µg/mL of lichen extract. The MTT substrate, prepared in a physiologically balanced solution, was added to the cell in culture at a final concentration of 5 mg/mL and incubated at 37 • C for 4 h. Thereafter, 50 µL of DMSO was added to each well to dissolve the formazan crystals [13]. The absorbance of each well was measured with a BioTek Microplate Reader Spectrophotometer (San Francisco, CA, USA) at 490 nm (using a reference wavelength of 690 nm). The cells in the medium alone and those in hydrogen peroxide (H 2 O 2 ) served as a negative and positive control, respectively [42]. The percentage cell viability was calculated as per Equation (1): where At is the absorbance of treated cells, Ac is the absorbance of background controls, and ADMSO is the absorbance of matched DMSO concentration controls. The halfmaximal inhibitory concentration (IC 50 ) was determined from the cell viability assay using 300-500 µg/mL of lichen extract [43].

Extraction and Lichenochemical Analysis
The lichen samples were air-dried and ground using a mortar and pestle. A 50 g dried lichen powder sample was soaked in 500 mL acetone (HPLC grade) and placed in an airtight screw cap bottle on an Innova 44 orbital shaker (New Brunswick Scientific, Edison, NJ, USA) at 150 rpm and at room temperature for 24 h. The crude acetone extract was then dried by vacuum evaporation using a rotary evaporator (Buchi, New Castle, DE, USA). Thereafter, the extract powder was subjected to preliminary lichenochemical screening and analysis using different reagents as described in [35] to identify the presence of phytochemicals (alkaloids, hydrocarbon, terpenoids, proteins, tannins, flavonoids, saponins, phenols, and glycosides).

Statistical Analysis
All experiments were carried out in triplicate, and each result was the average of three independent experiments. Statistical analysis was performed with Microsoft Excel 2010, and the data are presented as the mean standard deviation (±SD) of triplicates. Oneway Analysis of Variance (ANOVA) with p-values were calculated from https://goodcalculators.com/one-way-anova-calculator/ (accessed on 6 June 2022).

Lichen Identification and Anti-Proliferation Screening
A lichen specimen was collected from the area of Tamblyn Lake in Northwestern Ontario, and following organoleptic macroscopic examination, it was identified as Umbilicaria muhlenbergii. Acetone extraction of the lichen yielded 14.3% (w/w) dried crude acetone extract. The cytotoxic activity of acetone extracts from U. muhlenbergii against MCF-7 breast cancer cell line has been described in our previous study [41].

Lichenochemical Analysis
Lichenochemical screening studies on the crude extract of U. muhlenbergii revealed the presence of alkaloids and phenols as major components, with tannins, saponins, and

Statistical Analysis
All experiments were carried out in triplicate, and each result was the average of three independent experiments. Statistical analysis was performed with Microsoft Excel 2010, and the data are presented as the mean standard deviation (±SD) of triplicates. One-way Analysis of Variance (ANOVA) with p-values were calculated from https://goodcalculators. com/one-way-anova-calculator/ (accessed on 6 June 2022).

Lichen Identification and Anti-Proliferation Screening
A lichen specimen was collected from the area of Tamblyn Lake in Northwestern Ontario, and following organoleptic macroscopic examination, it was identified as Umbilicaria muhlenbergii. Acetone extraction of the lichen yielded 14.3% (w/w) dried crude acetone extract. The cytotoxic activity of acetone extracts from U. muhlenbergii against MCF-7 breast cancer cell line has been described in our previous study [41].

Lichenochemical Analysis
Lichenochemical screening studies on the crude extract of U. muhlenbergii revealed the presence of alkaloids and phenols as major components, with tannins, saponins, and hydrocarbons present in smaller quantities, whereas flavonoids, terpenoids, proteins, and glycosides were not detected (data not shown). A qualitative analysis of two Sri Lankan extracts from the lichens Parmotrema tinctorum and Parmotrema rampoddense revealed the presence of saponins, flavonoids, and polyphenols [44]. Anthracene glycosides were present in P. tinctorum but not in P. rampoddense, whereas proteins, alkaloids, tannins, reducing sugars, cyanogenic glycosides, and cardenolide glycosides were absent from both lichen extracts [44]. The presence of phenols and alkaloids with strong bioactivities is also in agreement with the published literature [17,45,46]. It has been reported that most of the lichen phenolics isolated from Parmelia caperata, Cladonia convoluta, C. rangiformis, Platisma glauca, and Ramalina cuspidata exhibited strong antioxidant activities and anticancer activities [47]. Lichen-derived compounds have been extensively discussed in previous reviews [48,49], and they provide in-depth information on their bio-therapeutic potential.

Purification and Characterization of GA
A detailed schematic diagram of purification of GA is shown in Figure 1. The bioactive compound(s) responsible for the anticancer activity of U. muhlenbergii was purified with successive solvent elution using nonpolar (hexane and ethyl ether) and polar (methanol, ethyl acetate) solvents as well as their combinations. It appeared that hexane (nonpolar solvent) and methanol (polar solvent) did not produce any bioactive fractions; however, the solvent elution with ethyl acetate alone, and in combination with methanol, yielded chemical containing fractions (F1-F4), as shown in Table 1. In many cases, the purification of secondary metabolites from lichens is tedious and complex. The product yield is affected by the type of solvent used; the presence of other compounds with similar structures and colored substances (pigments); possible interactions; and/or the formation of hydrogen bonding between the targeted molecule, the solvent, and other molecules. All this may create a masking effect due to the overlapping of compounds of different solubility and polarity with the molecule of interest, gyrophoric acid in our case, thereby altering the molecule's polarity and reducing its purification yield [50]. A maximum eluent yield of 0.14% (w/w of lichen biomass) was obtained in fraction F1 using ethyl acetate as the solvent. Fractions F2-F4 contained extraction yields that decreased as the polarity of the mixed solvent increased. Fractions F1-F4 were examined for their cytotoxic effect on MCF-7 breast cancer cells. Fractions F1 and F2 showed a maximum of 98% and 96% cytotoxicity, respectively ( Figure 2). These results were comparable with the positive control of hydrogen peroxide (H 2 O 2 ). In comparison, fractions F3 and F4 had a much lower antiproliferative activity, thereby reducing the MCF-7 cell viability by only 26% and 21%, respectively. Thus, fraction F1 was selected for further purification and identification studies as it contained the highest eluent yield (Table 1) and highest anti-proliferative activity (Figure 2). These studies included spectroscopic MS, UV, FTIR, and NMR data for F1 [19,[51][52][53][54][55][56][57].
The UV absorbance of F1 ( Figure S1) was measured in the wavelength range of 240-340 nm. The UV absorbance peaks for GA were detected at 210, 240, 310, and 340 nm, with high similarity for the depside groups [48] and in good agreement with previous findings [58][59][60].
The IR spectrum revealed functional groups and strength binding [13,61]. From the IR spectra at 600-4000 cm −1 (Figure S2), it is evident that the peaks at approximately 3300, 3200, and 3665 cm −1 reflect the stretching vibration of hydroxyl (OH) groups. The bands at 670-900 cm −1 result from the presence of aromatic groups and C-H bending in the compound. The UV absorbance of F1 ( Figure S1) was measured in the wavelength range of 240-340 nm. The UV absorbance peaks for GA were detected at 210, 240, 310, and 340 nm, with high similarity for the depside groups [48] and in good agreement with previous findings [58][59][60].
The IR spectrum revealed functional groups and strength binding [13,61]. From the IR spectra at 600-4000 cm −1 (Figure S2), it is evident that the peaks at approximately 3300, 3200, and 3665 cm −1 reflect the stretching vibration of hydroxyl (OH) groups. The bands at 670-900 cm −1 result from the presence of aromatic groups and C-H bending in the compound.
The electron ionization mass spectroscopy (Figure 3) was recorded in the negative ionization mode [M-1] − (m/z) at 467.0, with an error of 1.4 ppm, corresponding to GA with an exact molecular weight of 468.4 and molecular formula of C24H20O10 (Table 2). Its fragmentation produced an ion at (m/z) 316.9 due to ester cleavage, which may indicate lecanoric acid in trace amounts with an error of 1.1 ppm, as presented in Table 2 [55,62,63]. Similar fragmentation patterns for GA were also observed during the rapid identification of lichen compounds based on the structure-fragmentation relationship using ESI-MS/MS analysis [52]. Other studies also demonstrated that GA is present in high concentrations and is the prevalent depside in the Umbilicaria genus [19,53,[64][65][66][67]. The chemical formula C24H20O10 for GA was verified from MS data in several reports [52,68,69].  (Table 2). Its fragmentation produced an ion at (m/z) 316.9 due to ester cleavage, which may indicate lecanoric acid in trace amounts with an error of 1.1 ppm, as presented in Table 2 [55,62,63]. Similar fragmentation patterns for GA were also observed during the rapid identification of lichen compounds based on the structure-fragmentation relationship using ESI-MS/MS analysis [52]. Other studies also demonstrated that GA is present in high concentrations and is the prevalent depside in the Umbilicaria genus [19,53,[64][65][66][67]. The chemical formula C 24 H 20 O 10 for GA was verified from MS data in several reports [52,68,69].    The 1 H-NMR and 13 C-NMR spectra verified the chemical shifts that are typical for GA [52,61,69,70]  Evidence for the localization of the methyl and hydroxyl groups on the aromatic rings of GA was obtained from heteronuclear multiple bond correlation (HMBC) experiments ( Figure S5). The HMBC spectra showed HMBC correlations between the protons of the methyl groups/hydroxyl groups and the corresponding carbon atoms. In addition, the heteronuclear single quantum coherence (HSQC) spectrum provided data of direct determination of carbon and attached protons (hydrogen), which belong to the same spin system ( Figure S6). Based on these observations, the molecular structure of GA can be presented as shown in Figure 4. The GA purity was further confirmed by HPLC (data not shown). GA, isolated from U. hirsuta, was likewise identified by 1 H-NMR and 13 C-NMR, and validated with HPLC [19].

Cytotoxicity of GA
Lichen secondary metabolites are known to have diverse biological activities, including antibacterial, antitumor, antiproliferative, and cytotoxic effects [18,48]. Anticancer activities have been reported for several lichen-derived compounds, such as stictic acid [31], physodic acid [13], and parietin, to name a few. As evident from Figure 5 of this study, GA isolated from U. muhlenbergii crude extracts decreased the cell viability of MCF-7 cells. At 300 µg/mL GA, the cell proliferation was inhibited by approximately 45%, and thereafter, with a further increase in the GA dose up to 500 µg/mL, it reached a plateau. Keeping in mind the SD of the data presented in Figure 5, no significant difference in the cell viability was observed in the 300-500 µg/mL GA concentration range. This might be due to the fact that the MCF-7 cancer cells were exposed to GA for only 4 h. The low exposure time did not allow for the development of a clear dose-dependent correlation between cell viability and GA concentration using the MTT assay. Similar observations were reported for the impact of the GA dose on the inhibition of cell proliferation for HDF cells after their incubation with GA for 24 h [19]. Increasing the incubation time to 72 h produced a well-pronounced dose-dependent effect of GA on cell proliferation. The IC50 value of 478

Cytotoxicity of GA
Lichen secondary metabolites are known to have diverse biological activities, including antibacterial, antitumor, antiproliferative, and cytotoxic effects [18,48]. Anticancer activities have been reported for several lichen-derived compounds, such as stictic acid [31], physodic acid [13], and parietin, to name a few. As evident from Figure 5 of this study, GA isolated from U. muhlenbergii crude extracts decreased the cell viability of MCF-7 cells. At 300 µg/mL GA, the cell proliferation was inhibited by approximately 45%, and thereafter, with a further increase in the GA dose up to 500 µg/mL, it reached a plateau. Keeping in mind the SD of the data presented in Figure 5, no significant difference in the cell viability was observed in the 300-500 µg/mL GA concentration range. This might be due to the fact that the MCF-7 cancer cells were exposed to GA for only 4 h. The low exposure time did not allow for the development of a clear dose-dependent correlation between cell viability and GA concentration using the MTT assay. Similar observations were reported for the impact of the GA dose on the inhibition of cell proliferation for HDF cells after their incubation with GA for 24 h [19]. Increasing the incubation time to 72 h produced a well-pronounced dose-dependent effect of GA on cell proliferation. The IC 50 value of 478 µM of GA from U. muhlenbergii, calculated from the cell viability data on MCF-7 cells, was comparatively close to the 384 µM reported for GA from U. hirsuta on the same cell line [19]. In comparison, a standard GA showed >200 µM on the HT-29 human colon adenocarcinoma cell line [70]. Previous work has identified GA as a major bioactive compound present in 31 of the 33 studied Umbilicaria species, including U. muhlenbergii [66,71]. The present data are consistent with our previous findings that suggested the inhibition of cellular proliferation and induction of apoptosis of U. muhlenbergii crude extracts as possible causes for MCF-7 cell death [41], and they provide further evidence that GA can act as a potent anticancer agent.
GA isolated from U. muhlenbergii crude extracts decreased the cell viability of MCF-7 cells. At 300 µg/mL GA, the cell proliferation was inhibited by approximately 45%, and thereafter, with a further increase in the GA dose up to 500 µg/mL, it reached a plateau. Keeping in mind the SD of the data presented in Figure 5, no significant difference in the cell viability was observed in the 300-500 µg/mL GA concentration range. This might be due to the fact that the MCF-7 cancer cells were exposed to GA for only 4 h. The low exposure time did not allow for the development of a clear dose-dependent correlation between cell viability and GA concentration using the MTT assay. Similar observations were reported for the impact of the GA dose on the inhibition of cell proliferation for HDF cells after their incubation with GA for 24 h [19]. Increasing the incubation time to 72 h produced a well-pronounced dose-dependent effect of GA on cell proliferation. The IC50 value of 478 µM of GA from U. muhlenbergii, calculated from the cell viability data on MCF-7 cells, was comparatively close to the 384 µM reported for GA from U. hirsuta on the same cell line [19]. In comparison, a standard GA showed >200 µM on the HT-29 human colon adenocarcinoma cell line [70]. Previous work has identified GA as a major bioactive compound present in 31 of the 33 studied Umbilicaria species, including U. muhlenbergii [66,71]. The present data are consistent with our previous findings that suggested the inhibition of cellular proliferation and induction of apoptosis of U. muhlenbergii crude extracts as possible causes for MCF-7 cell death [41], and they provide further evidence that GA can act as a potent anticancer agent.

Conclusions
This study demonstrates that the lichen U. muhlenbergii is capable of producing GA as a predominant secondary metabolite with strong anticancer activity against MCF-7 breast cancer cells. A simplified extraction and purification protocol to obtain pure GA from U. muhlenbergii crude extract was developed. The molecular structure of GA was validated through spectroscopic analyses that included UV, FTIR, MS, NMR, HSQC, and HMBC. GA, 4-({4-[(2,4-dihydroxy-6-methylphenoxy)carbonyl]-2-hydroxy-6-methylbenzoyl}oxy)-2hydroxy-6-methylbenzoic acid, is a depside molecule that, in addition to the three aromatic rings, contains functional hydroxyl and carboxyl groups. While the polyaromatic structure of GA facilitates free radical scavenging activity, the functional side-groups impart selective reactivity of GA toward biochemical molecules, such as enzymes and cell-bound proteins, thereby promoting a dynamic interaction with different cell types. Due to the unique properties and functionalities that the molecular structure of GA presents, its potential therapeutic applications are expanding beyond the promise it holds as a cytotoxic and antitumor agent to encompass new medicinal uses that take advantage of the antioxidant, antimicrobial, and anti-inflammatory utilities of GA. Further assessment of the cytotoxic effects of GA in clinical studies is needed to establish the potential for anticancer drug development and application.