Cytotoxic Effects of Some Nepeta Species against Breast Cancer Cell Lines and Their Associated Phytochemical Properties

Nepeta is one of the largest genera of the Lamiaceae family. Nepeta species are commonly employed in traditional medicine for a variety of ailments, as well as food additives. In addition, they also come to the fore with their rich phytochemical content. In the present study, the quantitative phytochemical content of methanolic extracts and infusions prepared from the aerial parts of 14 Nepeta taxa collected from Turkey and their cytotoxic effects on two breast cancer cell lines, MCF-7 and MDA-MB-231, were investigated by using the MTT (3-(4,5-dimethylthiazol-2-yl))-2,5-diphenyltetrazolium-bromide) test. According to HPLC-PDA analysis, N. racemosa methanolic extract had the highest ursolic acid content with 165.9 mg/g extract. Total sterol, total iridoid, and total triterpenoid content were determined to be greatest in the methanolic extracts of N. meyeri, N. trichocalyx and N. phyllochlamys. The MTT experiment demonstrated that certain Nepeta species suppressed the growth of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. Statistical analysis revealed a strong correlation between the cytotoxic effects of the extracts and their triterpene content. In conclusion, the data obtained from this study are important in terms of forming a basis for advanced anticancer activity studies on breast cancer with Nepeta sp.


Introduction
Nepeta L. is one of the largest genera of the Lamiaceae family, which is represented by approximately 300 species worldwide and is generally distributed in Central and Southern Europe, the Near East, Central and South Asia, and some parts of Africa [1]. It is represented in Turkey by 44 taxa, 20 of which are endemic [2]. Species belonging to the genus have different uses such as traditional medicine, agrochemical, and food additives [3]. It has been reported that the fresh or dried flowering top leaves of Nepeta cataria are used as flavoring agents in dishes, especially sauces, soups, and cheeses [3,4]. Because of their antispasmodic, expectorant, diuretic, antiseptic, emenagogue, antitussive, antiasthmatic, anticonvulsant, sedative, diaphoretic, analgesic, antipyretic, antifungal, antiviral, anti-inflammatory, antihemorrhoidal, antirheumatic activities Nepeta species are commonly used to treat a variety of ailments in the regions where they grow [5][6][7][8]. Numerous phytochemical studies with the genus have shown that the main constituents of Nepeta species are nepetalactones, iridoids, and their glycosides as well as diterpenes, triterpenes, and flavonoids. Moreover, most Nepeta species are rich in essential oils [9][10][11]. In parallel with their rich chemical content, including medicinally important compounds, and traditional uses, plants belonging to the genus have been shown to have many important biological activities. In respect to several in vitro and in vivo studies, different Nepeta species have been reported to possess antitumoral [12], anticancer [13], antiangiogenic [14], cytotoxic [15], antibacterial [16], antiparasitic [17], antioxidant [18], anti-Alzheimer [19], allelopathic [20], immunomodulator [21], antiaggregant [22], insecticide [23], spasmolytic, bronchodilator [24], anxiolytic [25] and antidiabetic [26] activities.
Breast cancer is the second most common type of cancer in the world after lung cancer and is a common malignancy in women. Breast cancers can be classified according to stage, pathology, grade, and expression of estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor (Her2/neu) [27]. Two types of breast cancer cells that have attracted attention by researchers for their differences are MDA-MB-231 and MCF-7. Both MCF-7 and MDA-MB-231 cells, despite being invasive ductal/breast carcinoma cells, represent a conspicuous example as they have phenotypic/genotypic differences: while MCF-7 is hormone-dependent (negative for HER-2 receptor, positive for estrogen and progesterone receptor), MDA-MB-231 is triple negative [28].
The traditional use of Nepeta betonicifolia against cancers in the form of a decoction or powder was documented in an earlier ethnobotanical study carried out in Turkey [29]. In addition, in a study conducted with Nepeta binaloudensis, it was reported that the extracts prepared from its aerial part decreased the cell viability on breast cancer cell lines (MCF-7 and MBA-MD-231) in a dose-and time-dependent manner [30]. The presence of diverse classes of terpenes in different species of the genus Nepeta and some data related to the cytotoxicity of some of these compounds such as nepetalactone (iridoid), ursolic acid (triterpene), and nepetolide (diterpene) directed us to examine total sterol, total iroid, and total triterpenoid content of these species [31][32][33]. In addition, ursolic acid content was quantitatively analyzed by HPLC-PDA. Finally, Spearman's correlation analysis was performed in order to correlate the observed biological effects with the phytochemical content of the species. The biological activities and contents of the majority of the species we investigated are not found in the literature and were evaluated in depth in this study for the first time. The scientific information acquired from this study is likely to contribute to the current database on the biochemical characteristics of Nepeta species.
There are various phytochemical studies in the literature regarding some of the species we included in our study.
Similarly, an investigation about analysis of N. humilis extracts with RP-HPLC-DAD showed that the major component in all parts of the plant, especially in flowers, is rosmarinic acid, while flowers contain phenolic compounds such as chlorogenic acid, luteolin and apigenin [38].
Various monoterpenes have been also detected in N. lepthenta and N. phyllochlamys species with some studies, but a detailed phytochemical investigation has not been found in the literature, except essential oil analyses of them. Similarly, no research has been found on the phytochemical properties of N. aristata and N. crinita species, except for their fatty acid content [35,43,44].
To our knowledge, neither Nepeta stricta var. stricta nor Nepeta stricta var. curvidens have been evaluated for their chemical composition or biological activity. The records in the literature about these species include a limited number of articles focused on their anatomical and morphological features.
Within the scope of the present study, total sterol, total iroid, and total triterpenoid assays of the above-mentioned species were performed and their ursolic acid content was also analyzed by HPLC-PDA. According to the phytochemical analyses, the total sterol content of N. meyeri methanolic extract was found to be the highest in the quantitative analysis, with 403.09 ± 15.24 mg cholesterol equivalents/g extract. Iridoid presence was detected in both methanolic extracts and infusions of N. trichocalyx and N. crinita species. The methanolic extract of N. stricta var. curvidens, on the other hand, contained iridoid, whereas the infusion did not. In contrast, iridoid appeared to be present in the infusion of N. racemosa, but not in the methanolic extract. Among these extracts, the total amount of iridoid was found to be highest in the N. trichocalyx methanolic extract with 85.60 ± 4.45 mg aucubin equivalents/g extract. The methanolic extract of N. stricta var. curvidens had the highest total amount of triterpenoids, with 928.07 ± 22.26 mg of oleanolic acid equivalents/g extract. Infusions of N. transcaucasica were found to be devoid of triterpenoids (Table 1). In addition, the amounts of pentacyclic triterpene ursolic acid in the extracts were investigated by HPLC-DAD analysis ( Table 2). As a result of the analysis, while ursolic acid could not be detected in the infusions, it was shown that N. racemosa methanolic extract had the highest ursolic acid content with 165.9 ± 0.6 mg/g extract ( Figure 1).  The presence of ursolic acid has been reported previously in many Nepeta species such as N. binaludensis, N. aragonensis, N. cataria, N. clarkei, N. crassifolia, N. eriostachya, N.  faassenii, N. grandiflora, N. heliotropifolia, N. juncea, N. leucophylla, N. mussinii, N. nuda ssp.  albiflora, N. obtusicrena, N. pannonica, N. prattii, and N. teydea. However the findings in the present study are the first examination (to our knowledge) on ursolic acid content of the species included in the study.  The presence of ursolic acid has been reported previously in many Nepeta species such as N. binaludensis, N. aragonensis, N. cataria, N. clarkei, N. crassifolia, N. eriostachya, N. faassenii, N. grandiflora, N. heliotropifolia, N. juncea, N. leucophylla, N. mussinii, N. nuda ssp. albiflora, N. obtusicrena, N. pannonica, N. prattii, and N. teydea. However the findings in the present study are the first examination (to our knowledge) on ursolic acid content of the species included in the study.

Cytotoxic Effect of Extracts
The MTT assay indicated that some Nepeta species inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. In MCF-7 cells, among the methanol extracts, N. argolica, N. stricta var. curvidens, N.leptentha, N. phyllochlamys, N. racemosa, and N. transcaucasica showed stronger cytotoxic activity than other extracts. All methanolic extracts were found to have significant antiproliferative activity compared to the control at the highest concentration (p < 0.001). N. racemosa and N. stricta var. curvidens showed activity with a significance of p < 0.01 at a concentration of 62.5 µg/mL. IC 50 values of the active species were calculated as follows: N. argolica 186.66 ± 13.32 µg/mL, N. stricta var. curvidens 213.14 ± 17.65 µg/mL, N. leptentha 190.99 ± 13.41 µg/mL, N. phyllochlamys 79.91 ± 9.47 µg/mL, N. racemosa 61.38 ± 1.38 µg/mL, and N. transcaucasica 220.66 ± 9.03 µg/mL. It was determined that infusion extracts did not have as much activity as methanolic extracts in the same cell line. N. racemosa was the most potent infusion extract inhibiting viability by approximately 34% at 62.5 µg/mL concentration (p < 0.05). While cisplatin used as a positive control affected the viability with a significance of p < 0.001 in the range of 31.25-250 µg/mL, ursolic acid showed an antiproliferative effect with a significance of p < 0.01 even at 3.9 µg/mL. The methanolic extract of N. racemosa, the extract with the highest ursolic acid content, showed the highest cytotoxic effect in the MCF-7 cell line ( Table 2).
N. argolica, N. phyllochlamys, N. racemosa, and N. transcaucasica showed more pronounced antiproliferative activity in the MDA-MB-231 cell line. IC 50 values were 166.06 ± 7.9 µg/mL, 90.80 ± 1.67 µg/mL, 94.26 ± 2.85 µg/mL, and 178.07 ± 9.81 µg/mL, respectively (Table 3). It was observed that methanolic extracts of N. phyllochlamys and N. racemosa significantly decreased the viability in this cell line at a concentration of 125 µg/mL (p < 0.01). It was ascertained that infusion extracts in the same cell line did not have a significant viability-reducing effect. Infusion extracts showed antiproliferative effect at a concentration of 250 µg/mL with a significance of p < 0.05 were N. cadmea, N. crinata, N. italica, and N. phyllochlamys. In the MDA-MB-231 cell line, cisplatin was able to significantly inhibit viability at all concentrations studied (p < 0.001), while ursolic acid did not affect viability at concentrations of 0.975 µg/mL and 1.95 µg/mL compared to the control. MCF-7 is an estrogen receptor positive (ER+) cancer cell line and is a good model for identifying the molecular events of some ER+ human breast cancers. The MDA-MB-231 (estrogen receptor-negative; ER−) cell line is more aggressive and does not respond to antiestrogens. Chemopreventive compounds must have stronger toxicity in order to exhibit an antiproliferative effect in this cell line [46,47]. According to the study results, the IC 50 values of the extracts except for N. argolica and N. transcaucasica, were found to be lower in the MCF-7 cell line. Ursolic acid likewise exhibited lower activity in the MDA-MB-231 cell line. Saponins and terpenoids in the composition of the extracts are thought to be responsible for the cytotoxic activity, and studies have shown that these compounds are cytotoxic [48][49][50].
As a consequence of the investigation into the literature, it was determined that the cytotoxic activities of the essential oils of Nepeta species were studied in different cancer cell lines [51][52][53][54]. Few studies have been conducted to assess the cytotoxic activities of Nepeta species extracts. It has been reported that Nepeta deflersiana chloroform extract does not affect viability in the MCF-7 cell line in the range of 10-250 µg/mL [13]. In the study by Orfali et al., it was stated that N. deflersiana ethyl acetate and n-butanol extracts were not cytotoxic against SK-OV-3, SK-MEL, KB, and BT-549 cancer cell lines [55]. In another study with the aerial parts of Nepeta teucriifolia, extracts were prepared with solvents of different polarities and their antiproliferative activities against C6 and HeLa cell lines were evaluated. Three new iriodite teucriifolian A-C were isolated from the active extract and their antiproliferative effects on HeLa and C6 cells were also evaluated. Among these compounds, only teucriifolian B compound at a dose of 250 µg/mL has been reported to have moderate antiproliferative activity against HeLa cells [40]. The toxicity profiles of ethanol, water, and n-hexane extracts of Nepeta binaloudensis in MCF-7, MBA-MD-231 cancer cells, and healthy MCF-10A cells were investigated. According to the findings, the most effective extract was n-hexane extract, with an IC 50 value of 60.89 µg/mL for MCF-7, 92.18 µg/mL for MBA-MD-231, and >300 µg/mL for MCF-10A evaluated for 48 h. It was concluded that N. binaloudensis extract was more effective in MCF-7 cells, and this result was consistent with our results [30]. Data are expressed as mean ± standard error (n = 3). Significant differences are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. MEOH: Methanolic extract. INF: Infusion.

Correlations between the Phytochemical Content and Cytotoxic Activities of Extracts
The relationship between the phytochemical content of Nepeta extracts and their effects on cell viability in breast cancer cell lines was evaluated with the nonparametric Spearman's correlation test and the results are shown in Figures 2 and 4. According to the statistical evaluation, there is no significant correlation between the total iridoid, total sterol and ursolic acid amounts of the extracts, and their cytotoxic effects. Spearman's ranking analysis revealed a strong correlation between the triterpenoid amounts of the extracts and their cytotoxic effects in both breast cancer cell lines MCF-7 (r = −0.6687; p = 0.0001) and MDA-MB-231(r = −0.8073; p < 0.0001).
Triterpenes are important natural compounds with anti-cancer and chemopreventive effects. They not only inhibit the survival of neoplastic cell lines, but also induce apoptosis in cancer cells, without affecting normal cells [56].
The most common triterpene identified in the genus Nepeta is ursolic acid. In addition, nepeticin, oleanolic acid, lupeol, betulin, nepehinol and triterpene nepetalic acids are among the important triterpenic compounds defined in this genus ( Figure 5) [36]. No correlation was found between the cytotoxic effects of the extracts and their amounts of ursolic acid, but the methanolic extract of N. racemosa, which has the highest ursolic acid content among all samples, showed the highest cytotoxic effect in MCF-7 cells. N. phyllochlamys extract, which has the highest triterpene content, strongly inhibited cell proliferation in both cell lines. This shows that although ursolic acid contributes to the cytotoxic effect, it cannot be held responsible for the effect alone, and other compounds, especially other triterpenic structures in the extracts, also contribute to the effect. Triterpenes are important natural compounds with anti-cancer and chemopreventive effects. They not only inhibit the survival of neoplastic cell lines, but also induce apoptosis in cancer cells, without affecting normal cells [56].
The most common triterpene identified in the genus Nepeta is ursolic acid. In addition, nepeticin, oleanolic acid, lupeol, betulin, nepehinol and triterpene nepetalic acids are among the important triterpenic compounds defined in this genus (Figure 4) [36]. No correlation was found between the cytotoxic effects of the extracts and their amounts of ursolic acid, but the methanolic extract of N. racemosa, which has the highest ursolic acid content among all samples, showed the highest cytotoxic effect in MCF-7 cells. N. phyllochlamys extract, which has the highest triterpene content, strongly inhibited cell proliferation in both cell lines. This shows that although ursolic acid contributes to the cytotoxic effect, it cannot be held responsible for the effect alone, and other compounds, especially other triterpenic structures in the extracts, also contribute to the effect. filtered while hot. The extracts concentrated in the evaporator were dried with a lyophilizer. The prepared extracts were stored in the refrigerator at +4 • C. In this report, plant names were abbreviated as NAT (Nepeta argolica subsp. tumeniana), NTX (Nepeta trichocalyx), NCD (Nepeta cadmea), NCR (Nepeta crinata), NHM (Nepeta humilis), NIT (Nepeta italica), NLP (Nepeta leptentha), NMY (Nepeta meyeri), NPH (Nepeta phyllochlamys), NRC (Nepeta racemosa), NSC (Nepeta stricta var. curvidens), NSS (Nepeta stricta var. stricta), NTC (Nepeta teucriifolia), and NTR (Nepeta transcaucasica).

Total Triterpenoid Assay
The total triterpenoid content was investigated using the method by Zhang et al., (2010). According to this method, vanillin-glacial acetic acid (5%, w/v, 0.5 mL) and 1 mL perchloric acid were added to the sample solution (0.5 or 2 mg/mL; 500 µL) and mixed in a test tube. It was left to cool in an ice bath for 15 min and then 5 mL of glacial acetic acid was added and mixed. After standing for 6 min, absorbances at 538 nm were measured. The total triterpenoid content of the extracts was calculated with a calibration curve obtained with oleanolic acid solution and the results were given as equivalents (OAE, mg/g extract) to oleanolic acid [57].

Total Iridoid Assay
The total iridoid content was determined by the Trim-Hill test. According to this assay, 400 µL of the extract was treated with 4 mL of Trim-Hill reagent for 5 min at 100 • C. At the end of the reaction, absorbances were measured at 609 nm. Aucubin was used as standard. Total iridoid content of the extracts was calculated as mg aucubin equivalent/g extracts [58].

Total Sterol Assay
The total sterol content was determined by Liebermann-Burchard assay. The extracts were stirred with chloroform and filtered. The Liebermann-Burchard reagent was prepared by adding 5 mL of concentrated H 2 SO 4 to 50 mL of acetic anhydrous incubated in an ice bath for 30 min [59,60]. The quantitative assay was carried out according to the method reported by Johnson et al. (2020). Briefly, after mixing the sample solutions with Liebermann-Burchard reagent for 1 min, they were kept at room temperature (26 • C) for 13 min. Absorbances were measured at 650 nm and cholesterol was used as a standard. The total sterols content of the extracts was calculated as mg cholesterol equivalent/g extracts [61].

Quantitation of Ursolic Acid with HPLC Analyses
The PDA detector combined with an Agilent 1100 liquid chromatography system was used to perform a qualitative and quantitative ursolic acid content analysis of the extracts. In the analysis, Thermo C18 analytical column (250 × 4.6 mm, 5 µm particle diameter) as the stationary phase, acetonitrile: methanol (80: 20) system was preferred as the mobile phase. The mobile phase passed through the system isocritically at a flow rate of 1 mL/min and the total analysis time for one sample was set for 15 min. The detection wavelength was 210 nm. Solutions were prepared at concentrations ranging from 7.8-2000 µg/mL to obtain a calibration curve for ursolic acid (Sigma Chemical Company, St. Louis, MO, USA) purchased as standard. The regression equation was obtained as y = 8.9087x + 143.24 and the calibration plot showed good linearity with a correlation coefficient of 0.9998. Limits of detection (LOD) and limit of quantification (LOQ) of the ursolic acid standard were found 0.045 and 0.136 mg/mL respectively. Extracts were prepared at a concentration of 10 mg/mL, filtered, and then injected into the system. Each analysis was repeated three times [62].

Cell Culture
To investigate the cytotoxic effects of the extracts on breast cancer cells, two different human breast adenocarcinoma cell lines, MCF-7, and MDA-MB-231 were used. For both cell lines, DMEM containing 10% FBS (fetal bovine serum), and 1% penicillin-streptomycin were used as the medium. Cells cultured in the flask were passaged at the end of the two-day incubation period.

MTT Assay
The cytotoxic effect of the extracts on MCF-7, and MDA-MB-231 cells was determined by the colorimetric MTT method. After the cells in the flask were counted, they were distributed into 96-well microplates and then incubated in a CO 2 incubator at +37 • C. At the end of 24 h, the supernatant of the cells was discarded and 100 µL of the sample was added to the microplate wells at concentrations varying between 3.906-1000 µg/mL for extract and 0.975-250 µg/mL for ursolic acid and cisplatin (as a positive control), and then exposed for 48 h. Untreated cells were used as controls. At the end of the time, the wells were emptied. The MTT solution prepared at a concentration of 5 mg/mL was diluted 10 times with a culture medium and 100 µL was added to each well. After 4 h of incubation, the wells were emptied and 100 µL of dimethyl sulfoxide (DMSO) was added and left for 10 min [63]. Finally, absorbance was read using ELISA (Biotek Synergy HT) at 570 nm wavelength. Equation (1) was used to compute the percentage of viability relative to untreated controls. % Viability = [(Abs sample × 100)/Abs control ] (1)

Statistical Analysis
The Levene test was performed for variance homogeneity. One-way analysis of variance was used among multiple groups. The Dunnett test was applied for multiple comparison tests at p < 0.05 level. Quantitative phytochemical analysis experiments were repeated three times and results are expressed as mean ± standard deviation. The non-parametric Spearman's correlation test was used to assess the correlations between antiproliferative effect and total triterpene, total sterol, total iridoid, and ursolic acid contents of the extracts.

Conclusions
In summary, the phytochemical content richness and inter-species secondary metabolite variability of Nepeta species were comparatively demonstrated in this study. The statistically significant decreases in proliferation of breast cancer cells, especially in the MCF-7 cell line, caused by the Nepeta species are striking. Despite a crude herbal extract being generally regarded as having a cytotoxic effect when the IC50 < 20 µg/mL after an incubation period of 48 to 72 h by the US NCI plant screening program, the results obtained from this study are expected to shed light on cancer research that can be carried out with these plants [61]. Considering the statistical analysis, there is a strong correlation between the cytotoxic effects of these plants and their triterpenoid content. However, bioactivity-guided fractionation studies are needed to fully elucidate the compound or compounds responsible for the effect. The cytotoxic effects of plants should be further supported by detailed in vitro biochemical and in vivo studies.