HPLC–NMR-Based Chemical Proﬁling of Matricaria pubescens (Desf.) Schultz and Matricaria recutita and Their Protective Effects on UVA-Exposed Fibroblasts

: The present study aimed to investigate the chemical proﬁle and the protective activity on ﬁbroblasts of two Matricaria species: M. pubescens , which grows wild in Algeria, and M. recutita , which is cultivated in Greece. A comparative phytochemical investigation using High-Performance Liquid Chromatography, coupled with Photodiode Array Detection and Mass Spectrometry (HPLC– PDA–MS) combined with Nuclear Magnetic Resonance (NMR), was performed for the identiﬁcation of the main constituents in the ﬂowerheads of these medicinal plants. In M . pubescens more than 25 compounds were identiﬁed and/or isolated; among them are quercetagenin-3-O-glucopyranoside, reported for the ﬁrst time in Matricaria sp., and two polyamines previously reported in other Asteraceae species. In M. recutita , which is the ofﬁcially recognized species in Europe, 19 constituents were identiﬁed. To minimize time analysis, the structure elucidation was based on a multi-analytical approach directly on subfractions. Two representative polar extracts from each species were characterized chemically and further screened for their protective effects on 3T3 ﬁbroblasts. The cells were exposed to a mild toxic dose of UVA light (6 J/cm 2 ), in the presence of different concentrations of the extracts. Both M. recutita and M. pubescens extracts were effective. The methanolic extract was the best protective agent at lower concentrations (0.1 to 10 µ g/mL), and hydromethanolic was best at higher ones (100–200 µ g/mL). M. recutita exhibited the most enhanced cell viability in relation to those not exposed to UV control; it ranged from of 28 to 49% higher viability, depending on the dose, leading to the conclusion that the latter seems to exhibit potent cytoprotective activity and signiﬁcant regeneration activity. for ﬂavones, and at 350 nm for 3-O-substituted ﬂavonols. Results were adjusted using a molecular weight correction factor.


Introduction
Matricaria pubescens (Desf.) Schultz (Asteraceae), known as hairy chamomile, is endemic to North Africa and is used to treat rheumatic and muscular pains, coughs, allergies, ocular affections, dysmenorrhea, scorpion stings, and toothaches [1]. Ethnobotanical studies [1,2] have put in evidence the important role that this species holds in Algerian traditional medicine. Previous phytochemical work on M. pubescens [3] showed the presence of flavonoids, mainly apigenin and luteolin derivatives, and the same was shown for the well-known European species Matricaria recutita L. The latter is the officially recognized medicinal species in Europe. According to the European Medicines Agency (EMA), Sci 2022, 4, 14 2 of 16 chamomile preparations are used for mild gastrointestinal disorders, ulcers and inflammations of the mouth and throat, for irritated skin and mucosae, and the relief of common cold [4]. A variety of constituents are responsible for these activities, such as the essential oil components-bisabolol and chamazulene-as well as polar phenolic compounds such as apigenin-7-O-glucoside. Chamomile flowers have a broad spectrum of applications in the pharmaceutical and cosmetic industries, mainly due to their anti-inflammatory properties, which have been well known since antiquity. Chamomile products are often used to treat several skin conditions such as ultraviolet (UV)-induced erythema, pityriasis alba, peristomal lesions, contact dermatitis, eczema, atopic eczema, erythema induced by removal of adhesive tape, radiodermatitis, induced contact dermatitis, and wounds. In some cases, chamomile preparations have been shown to be superior to corticosteroids [5]. However, in many of these studies the specific species is not defined. For example, other members of the Asteraceae family, such as Chamaemelum spp. (known as Roman chamomile or Anthemis spp.), have similar chemistry and are often confused with Matricaria spp.; these might induce allergic cross-reactions with other Asteraceae members [6].
In the framework of a project aiming to study the chemical profile of Matricaria spp., we carried out chemical characterization of extracts of M. pubescens and M. recutita. The particular environmental conditions under which M. pubescens grows-a hot desert environment with mild winters and little rainfall-made this species an attractive target for studying its chemical and biological properties. In previous work Gherboudj et al. [3], reported the presence of apigenin and luteolin derivatives, showing chemical similarity to the European species M. recutita.
Fibroblasts, the main components of the dermis, have a crucial role in the wound healing process, and they also prevent photoaging by releasing tropocollagen and tropoelastin, precursors of the elastic skin fibers. Elevated reactive oxygen and nitrogen species (ROS and RNS), which occur in chronic wounds and after exposure to UV radiation and oxidative stress, lead to skin inflammation disrupting fibroblasts' normal functions [7]. When exposed to oxidative stress, the effectiveness of the skin's endogenic antioxidant system is decreased [8]. Plant extracts, rich in phenolic compounds, possess antioxidant properties and may inhibit ROS production, thus leading to decreased or non-inflammation and to the restoration of fibroblasts. In the present work, a detailed phytochemical profile of M. pubescens is reported for the first time. In parallel, M. recutita cultivated in Greece was chosen for comparison reasons. Comparative phytochemical and pharmacological investigations concerning their protective effect on fibroblast viability were designed. The identification of the constituents was based on chromatographic investigations and assisted by the application of two analytical platforms: the High-Performance Liquid Chromatography coupled with Photodiode Array Detection and Mass Spectrometry (HPLC-PDA-MS) and Nuclear Magnetic Resonance (NMR) techniques. Two polar extracts from each species, one methanolic and one hydromethanolic, were characterized qualitatively and quantitatively and their dried forms were tested for their activity on BALbC 3T3 mouse skin fibroblasts.

General Experimental Procedures
1 H, 13 C and 2D NMR experiments were recorded at 295 K in CD 3 OD on an Agilent DD2 500 (500.1 MHz for 1 H-NMR and 125.5 MHz 13 C-NMR) spectrometer (Palo Alto, CA, USA). Chemical shifts are given in parts per million (ppm) and were referenced to the solvent signals at 3.31 ppm and 49.5 ppm for 1 H and 13 C NMR, respectively. COSY, HSQC, HMBC and HSQC-TOCSY were performed using standard Varian microprograms. Column chromatography (CC) was performed on Sephadex LH-20 (Sigma-Aldrich, Darmstad, Germany) and Amberlite XAD7HP resin (Supelco, Bellefonte, PA, USA) with the solvent mixtures indicated in each case; TLC analyses were carried out using aluminum-coated silica gel plates 60 F 254 (Merck, Art. 5554, Darmstad, Germany). Detection was performed using UV light and Naturstoff reagent [9].

Isolation of the Compounds from M. pubescens
The dried and powdered aerial parts (347 g) of M. pubescens were extracted according to a protocol developed by Bohlmann and slightly modified [11]. According to this scheme, a 1:1:1 cyclohexane: diethylether: methanol (chex: Et 2 O: MeOH, 3 times, 3Lt in total) solvent mixture is used with the aim of extracting medium polarity constituents such as sesquiterpene lactones, which are often present in plants of the Asteraceae family. The extract was condensed to dryness (27.9 g) and then redissolved in 100 mL of the above system and partitioned with an equal volume of brine to obtain an organic layer (organic phase A, 15.2 g) and an aqueous phase. The aqueous phase was further extracted twice with ethylacetate 100% (EtOAc) and Butanol 100% (BuOH) and yielded two extracts: organic phase B (EtOAc, 5.10 g) and organic phase C (BuOH, 4.39 g). The plant material was further extracted three times with MeOH 100% and MeOH:H 2 O 75:25 and produced two extracts of 16.1 g and 20.8 g, respectively ( Figure S1, Supplementary Material).
M. recutita flowers (117.7 g) were treated in the same way and they produced the following extracts: organic phase A (9.0 g), organic phase B (EtOAc, 2.19 g), organic phase C (BuOH, 2.2 g), MeOH extract (7.17 g) and MeOH:H 2 O 50:50 extract (7.66 g). A scheme of the extraction procedure is available as supplementary material. From the above extracts, the MeOH and MeOH:H 2 O extracts of both plants were characterized qualitatively and quantitatively using HPLC-PDA-MS and tested for their protective activity in 3T3 fibroblasts.

Sample Preparation for HPLC Quantitative Analysis of Methanol and Hydromethanolic Extracts
Approximately 50 mg of each extract was diluted in a 100 mL volumetric flask with 70% methanol. The samples were filtered through Nylon filters (0.45 µm pore size) and immediately injected.

Chemicals and Standards
The solvents used for the isolation of the flavonoids were of reagent grade, whereas the solvents used for HPLC analysis were HPLC grade. All solvents were purchased from Sigma-Aldrich (Amersham, Sweden). Water was purified using a Milli-Q plus system from Millipore (Milford, MA, USA). Sephadex LH-20 was purchased from Sigma-Aldrich. Nylon filters (0.45 µm pore size) were from Agilent (Agilent Technologies, Palo Alto, CA, USA). Apigenin-7-O-glucoside (99% purity) was purchased form Extrasynthese (Genay, France). Rutin (95% purity) was purchased from Sigma-Aldrich, and apigenin (97% purity) and chlorogenic acid (98% purity) from Alfa Aesar (Kandel, Germany). A series of stock solutions were prepared and kept at −20 • C in 100% methanol. From these stock solutions, a series of fresh working solutions were prepared immediately prior to analysis.

HPLC-PDA-MS Analysis Instrumentation
Analysis was carried out using an HPLC-PDA-MS Thermo Finnigan system (LC Pump Plus, Autosampler, Surveyor PDA Plus Detector) interfaced with an ESI MSQ Plus (Thermo Finnigan) and equipped with Xcalibur software (2.1, Thermo Finnigan, MA, USA). The same column, timetable and flow rate were used during the HPLC-MS analyses. The mass spectrometer operated in both negative and positive ionization modes, scan spectra were from m/z 100 to 1000, gas temperature was at 350 • C, nitrogen flow rate at 10 L/min, and capillary voltage at 3000 V. The cone voltage was in the range 60-100 V. The column was an SB-Aq (Agilent) RP-C18 column (150 mm × 3 mm) with a particle size of 5 µm,

Activity of M. pubescens and M. recutita Extracts on Fibroblasts
Equipment and Reagents: The incubator was an InCO2 Memmert (Schwabach, Germany), and the abductor a Telstar PV100 (Terrassa, Spain). An Axiovert 25 ZEISS (Schwabach, Switzerland) inverted microscope and a Fluostar Galaxy BMG Microplate Photometer (Ortenberg, Germany) were used. The UVA source was an Astralux Type UVA MED, (UK), and the centrifugal was a Hettich Roto Silenta III (Tuttlingen, Germany). The Laboratory Oven was a Memmert (Schwabach, Germany) and the liquid nitrogen freezing cell container was a 34XT Taylor-Wharton (Cambridge Scientific, Merck KGaA, Darmstadt, Germany). The plate shaker was an MS2 Minishaker, Vortex-IKA, Staufen, Germany). The following reagents were used: DMEM 1X, FBS (Fetal Bovine Serum), PBS, Trypsin-EDTA and antibiotic-antimycotic solution were all purchased from Gibco (Thermo Fisher Scientific Inc., Palo Alto, CA, USA), whereas dimethyl sulfoxide, absolute ethanol and glacial acetic acid were of analytical grade and were purchased from Sigma (Merck KGaA, Darmstadt, Germany). Chlorpromazine hydrochloride was purchased from Thermo Fisher Scientific Inc. (Palo Alto, CA, USA). Neutral red solution was from Sigma (Merck KGaA, Darmstadt, Germany), and distilled water or purified water suitable for cell culture was from Millipore-Sigma (Merck KGaA, Darmstadt, Germany). BALB/c 3T3, Fibroblasts, were a gift from Biological Laboratory, Demokritos, Greece.
The possible protective role against cell-induced necrosis of methanolic and hydromethanolic extracts of the two chamomile species (M. pubescens and M. recutita) was tested in vitro. Both extracts were dried prior to use. More specifically, using BALbC 3T3 mouse skin fibroblasts (ATCC cell line) and UVA irradiation (6 J/cm 2 ), the effect of the two chamomile species was studied in a mild UVA-induced phototoxicity test. The selected UVA dose was slightly cytotoxic for the specific cell line, in order to reveal the possible cytoprotective efficacy of the extracts. The irradiance was adjusted to reach 6 J/cm 2 within a  The possible protective role against cell-induced necrosis of methanolic and hydromethanolic extracts of the two chamomile species (M. pubescens and M. recutita) was tested in vitro. Both extracts were dried prior to use. More specifically, using BALbC 3T3 mouse skin fibroblasts (ATCC cell line) and UVA irradiation (6 J/cm 2 ), the effect of the two chamomile species was studied in a mild UVA-induced phototoxicity test. The selected UVA dose was slightly cytotoxic for the specific cell line, in order to reveal the possible cytoprotective efficacy of the extracts. The irradiance was adjusted to reach 6 J/cm 2 within a time period of 60 min. The chamomile extracts were incorporated both in the culture

Results
In the present work, the chemical constitution and biological activity of M. pubescens from Algeria and M. recutita from Greece were assessed. Both plants were subjected to successive extractions, and the polar extracts, once characterized for their chemical content (Tables 1 and 2; Figures 1-4; Figures S1, S13-S15 and Tables S4-S6 of Supplementary Material), were tested for their activity on fibroblasts. M. pubescens is a sub-Saharan species used in Algerian traditional medicine, but its chemical content has not been sufficiently studied [3]. To fill this gap, the plant was further analyzed, and the results are herein reported. In contrast, M. recutita, which is widely distributed in Europe, is well-characterized and was chosen for comparison reasons. A cultivated population from Greece was used, which recently demonstrated rich polyphenolic content [12]. In the present study, polar methanol and hydromethanolic extracts were prepared and quantified in order to perform biological assays in a comparative manner with the Algerian species. To this end, a successive extraction scheme was applied for both plants.

Extraction, Isolation and Identification of the Constituents
The solvent system that was initially applied was chex:Et 2 O:MeOH 1:1:1. This system is appropriate for the removal of triterpenoids and sesquiterpene lactones, as well as medium-polarity compounds ( Figure S1, Supplementary Material) [11] and has been used extensively in the past for plants of the Asteraceae family containing sesquiterpene lactones [13]. The more polar fractions of this extract, which were obtained through liquidliquid extractions, served as a reservoir for the phytochemical isolations in order to create a small chemical and spectral library to use in further steps of this study ( Figure S1). Phytochemical isolations, although not initially considered, were mandatory; this is because several compounds were not commercially available as reference standards and the phytochemical profile of M. pubescens has been little explored. Fractionations of the ethyl acetate (organic phase B) and butanol phase (organic phase C) ( Figure S1, Supplementary Material) from M. pubescens produced eight compounds, which were identified by 1D and 2D NMR, namely: apigenin (21), luteolin (19), hispidulin (22), quercetin (18), quercetagetin (23), luteolin-4 -O-glucoside (13), p-coumaric acid (25) and the p-coumaroyl polyamine derivative 26 (Figure 1). In order to minimize the time of the analysis, a multi-analytical approach was applied, and selected subfractions were studied spectroscopically using a combination of NMR, HPLC-PDA-MS, reference standards where available. Subfractions ALG-CQ, ALG-CL and ALG-CP, obtained from the M. pubescens organic phase C, were studied in this way. Detailed information on the identification process is provided in the Supplementary Material (Figures S2-S12). Using this dereplication methodology, the following compounds were identified: (isoorientin) (3), 6-hydroxyluteolin-7-O-glucopyranoside (5), quercetin-7-O-glucoside (6), luteolin-7-O-glucopyranoside (10), luteolin-4 -O-glucoside (13), 6-hydroxykaempferol-3-hexoside (24). Compound 24 has been reported several times in plants of the Asteraceae family [14,15], while the presence of 6-hydroxyluteolin-7-Oglucopyranoside is considered characteristic of the European chamomile M. recutita [16]. Based on the in-house-created chemical library, the analysis of the polar extracts (methanolic, hydromethanolic) of M. pubescens and M. recutita was then performed (Tables 1 and 2,  Tables S4-S6 and Figures S13-S15, Supplementary Material). The discrimination between the isomers orientin (4) and isoorientin (3) was feasible by co-chromatography with the lab isolate. A series of caffeoylquinic acid derivatives such as chlorogenic acid (1), 3,5-dicaffeoyl Sci 2022, 4, 14 9 of 16 quinic acid (11) and 1,5-dicaffeoyl quinic acid (12) were confirmed by examining of the UV and MS data, and by using reference standards [17]. Indeed, is seems that compounds 11 and 12 co-elute under the present HPLC conditions. The presence of isovitexin (8) was suggested due to the lack of shoulder at 302 nm, which is observed in the isobaric vitexin. Instead, compound 7 was identified as a hydroxyluteolin-4 -O-glucoside derivative on the basis of its molecular weight and the hypsochromic shift of Band I at 337 nm, just like the similar luteolin-4 -O-glucoside (13), also isolated from this plant. However, the exact hydroxylation site could not be deduced. The peak at 30.72 min (17) had an absorption maximum typical of p-coumaroyl moiety and its fragmentation pattern was similar to that of polyamine 26. From its molecular weight, it is suggested that it is a tri-p-coumaroyl derivative of spermine/thermospermine. Unfortunately, the compound was isolated only in a small amount (1.5 mg), which did not permit further elucidation of its structure. Finally, for compound 20, UV, MS data and retention time suggested a caffeoyl-substituted polyamine derivative, but the structure needs further isolations and study.  A: aglycon; numbering is set as Mr-X, according to the retention time and in order to discriminate from the constituents of M. pubescens.
Concerning M. recutita, its identification ( Table 2) was based on reference standards, isolated compounds (where available) and data from the literature. Quercetagenin-3-Oglucoside (corresponding to peak number Mr-4 in Table 2 and Figure 3) was detected for the first time in M. recutita and was confirmed by co-elution of the isolated compound. Peaks  Mr-16, 17, 18 and 19 were identified as acetylated apigenin hexosides [21]. Constituents Mr-16 and Mr-19 had identical spectral data, suggesting that the isobaric constituents had different acetyl substitutions on the sugar moiety. Compound Mr-16 however, presented many differences. Its UV spectrum had an hypsochromic shift of Band I at 329 nm, indicating a 4 -substitution on ring B of the flavonoid. Likewise, its MS fragmentation pattern was different and the fragment at m/z = 473 [M-H] − was merely observable, while the fragment at m/z = 269 [A-H] − had the higher intensity. Therefore, the compound Mr-16 was tentatively identified as apigenin-4 O-acetylhexoside, reported here for the first time in Matricaria spp. Overall, 26 compounds were characterized (isolated and/or identified using HPLC-PDA-MS) in the extracts of M. pubescens, and 19 compounds were detected using HPLC-PDA-MS in hydromethanolic and methanolic extracts of M. recutita, obtained under the extraction scheme described above.

Quantitative Data
Quantitation of the major phenolic acids and flavonoids in the examined extracts (methanolic and hydromethanolic 50%) showed marked qualitative and quantitative differences (Table 3) recutita. This is of importance, since these two latter constituents have been linked to allergic reactions [22].

In Vitro Protective Activity on BALbC 3T3 Mouse Skin Fibroblasts
The administration of 6J/cm 2 of UVA induced a 10% mean fibroblast viability decrease. The mean positive control decrease in viability was 60% in the higher tested doses ( Figure 5). Both dried methanolic extracts provided significant cell protection at the lower concentrations, while in the higher ones, the viability in most cases slightly decreased ( Figure 5). Upon addition of M. recutita methanolic extract at doses of 0.1 to 10 µg/mL, a mean increase in viability is observed in relation to the control, ranging from of 28 to 49%, depending on the dose. In this case, apparently, in addition to the UVA protection there was an increase in the fibroblast mitosis rate. Correspondingly, M. pubescens methanolic extract showed an increase of 10% only with the lower dose of 0.1 µg/mL. Hydromethanolic extracts showed some protection at relatively higher doses; maximum viability was obtained by M. recutita at a dose of 100 µg/mL with a mean enhancement of 22%. Both extracts showed, at many concentrations, enhanced fibroblast cytoprotection. The phototoxicity protocol is apparently valid, as the addition of chlorpromazine showed an enhanced decrease in fibroblasts of 67% for the highest dose of 200 µg/mL. Qualitative viability appreciation under the microscope confirmed the quantitative measurements. concentrations, while in the higher ones, the viability in most cases slightly decreased ( Figure 5). Upon addition of M. recutita methanolic extract at doses of 0.1 to 10μg/mL, a mean increase in viability is observed in relation to the control, ranging from of 28 to 49%, depending on the dose. In this case, apparently, in addition to the UVA protection there was an increase in the fibroblast mitosis rate. Correspondingly, M. pubescens methanolic extract showed an increase of 10% only with the lower dose of 0.1 μg/mL. Hydromethanolic extracts showed some protection at relatively higher doses; maximum viability was obtained by M. recutita at a dose of 100μg/mL with a mean enhancement of 22%. Both extracts showed, at many concentrations, enhanced fibroblast cytoprotection. The phototoxicity protocol is apparently valid, as the addition of chlorpromazine showed an enhanced decrease in fibroblasts of 67% for the highest dose of 200 μg/mL. Qualitative viability appreciation under the microscope confirmed the quantitative measurements.

Discussion
In the present study, the phytochemical profile of M. pubescens was fully explored. In polar extracts, it consists mainly of flavonoid compounds, among which luteolin glycosides and luteolin prevail, and are followed by apigenin-7-O-glucoside and patuletin-3-O-glucoside. It should be noted that luteolin and its derivatives are present in negligible amounts in M. recutita, though the content of apigenin glycosides is almost twice that in M. pubescens. Quantitatively, the content of total phenolic acids is almost equal in both species.

Discussion
In the present study, the phytochemical profile of M. pubescens was fully explored. In polar extracts, it consists mainly of flavonoid compounds, among which luteolin glycosides and luteolin prevail, and are followed by apigenin-7-O-glucoside and patuletin-3-O-glucoside. It should be noted that luteolin and its derivatives are present in negligible amounts in M. recutita, though the content of apigenin glycosides is almost twice that in M. pubescens. Quantitatively, the content of total phenolic acids is almost equal in both species.
Phenolic compounds as functional ingredients are considered an important tool with many applications in skin-care products. The antioxidant properties with which these compounds are endowed play a crucial role in the restoration of fibroblasts. When the latter are exposed to phenolic compounds, a decrease in ROS production and an increase in collagen expression is observed, resulting in the acceleration of wound healing and protection against UV-induced photoaging [7]. Matricaria species are traditionally used for several skin ailments, such as ultraviolet (UV)-induced erythema, pityriasis alba, peristomal lesions, contact dermatitis, eczema, atopic eczema, radiodermatitis, induced contact dermatitis, and wounds. Recently, a dual-layered herbal biopolymeric patch based on chamomile extract (of which the chemical synthesis is not reported) increased collagen deposition and showed rapid re-epithelialization at a wound site as a potential wound dressing [23]. Chamomile hydroalcoholic extracts (ethanol: water 1:1, v/v) (no chemical analysis provided) have been found to improve wound healing by enhancing fibroblast proliferation and re-vascularization in diabetic skin injuries [24]. In another study (no chemical data provided) the wound-healing effects of chamomile have been demonstrated to be superior to those of corticosteroids [25]. All these effects are generally attributed to apigenin and its derivatives. Apigenin has notable anti-inflammatory activities such as inhibition of prostaglandin E2 (PG-E2), cyclooxygenase 2 (COX-2) and nitric oxide production (EMA). Furthermore, it has been found to interfere with leukocyte adhesion and adhesion-protein upregulation in human endothelial cells. It has also been shown to inhibit interleukin 1α (IL-1)-induced prostaglandin synthesis and tumor necrosis factor α (TNF-α), among others. Choi et al. [26] reported that apigenin restored the viability of normal human dermal fibroblasts exposed to UVA irradiation through suppression of the expression of the collagenase, matrix metalloproteinase (MMP)-1. Further in vivo tests with an apigenin-containing cream showed increased dermal density and elasticity, improved skin evenness, improved moisture content, and improved trans-epidermal water loss in the subjects who used it. A literature survey showed that other phenolic compounds, which are also present in chamomile extracts, act in a similar manner. Chlorogenic acid from Coffea arabica, administered in a dose-dependent manner, inhibited intracellular reactive oxygen species production in CCRF cells stimulated by UV radiation; suppressed the expression of the matrix metalloproteinases-1, 3, and 9; and increased synthesis of type-I procollagen [27]. Flavonol derivatives from Eriobotrya deflexa and especially hyperin reduced matrix metalloproteinase I and intracellular reactive oxygen species, and increased procollagen type-I and TIMP-1 in UVB-irradiated human fibroblasts (WS-1 cells) [28].
In view of the above data, the activity of our extracts on UVA-exposed fibroblasts might be explained. A comparison between the relation of the biological activity and the quantitative results corroborates the hypothesis that apigenin-7-O-glucoside accounts mostly for the UVA-protective activity of chamomile. M. pubescens methanol extracts, rich in luteolin-7-O-glucoside (up to 1.1% w/w), also showed UVA-protective potential. Luteolin-7-O-glucoside anti-inflammatory activity [29,30] could contribute significantly to the cytoprotective properties of M. pubescens methanol extracts. The slight regeneration properties of M. pubescens methanol extracts at low doses ( Figure 5) are in accordance with Ustuner et al. [31], who have shown that luteolin-7-O-glycoside was the major phenolic compound of Thymus sipyleus decoction and infusion; these were proven to be effective in the wound-healing process. Similarly, dicaffeoylquinic acid derivatives, which are also reported to have antioxidant, antiradical and hepatoprotective activities [32,33], are in accordance with the cytoprotective effect of M. pubescens methanolic extract.
M. recutita extracts contain a higher variety and content of flavonols (5.06 vs. 4.00% in M. pubescens), especially a considerable higher amount of apigenin-7-O-glucoside in methanolic extracts, and patuletin-3-O-glucoside in both the methanol and hydromethanolic extracts (1.91 and 1.89%, respectively); however, the phenolic acids content was almost equal in both extracts. This feature might explain the difference in the activity of the plant extracts on fibroblast protection in relation to M. pubescens extracts, as well in the difference obtained between M. recutita methanolic and hydromethanolic extract ( Figure 5). Additionally, anti-inflammatory activity of extracts rich in patuletin derivatives has been previously reported [34]. The anti-inflammatory effect of patuletin 3-O-β-D-glucopyranoside in vivo has been proven to be almost equal to that of dexamethasone [35], though previous studies show that it significantly inhibits histamine-induced hind-paw edema [36]. Apigenin possessing notable anti-inflammatory activity and collagenase and MMP-1 downregulation [26]-as well caffeic acid and patuletin glycosides, endowed with antioxidant and anti-inflammatory activity [37,38], which are contained in M. recutita methanolic extractcould explain its cytoprotective and regenerative activity ( Figure 5). The fact that the methanolic extract at concentrations >50 µg/mL showed relative cytotoxicity, up to a mean maximum of 27% for M. pubescens could be attributed to the phenolic antioxidants it contains, which often prevent pro-oxidant activity [39]. To sum up, the stronger protective potential against UV stress might be attributed to a combination of apigenin-7-O-glucoside with other flavonoids and phenolics present in the extracts. Further studies with the isolated constituents are needed in order to understand the contribution or the synergistic effect of each compound toward the protective outcome. Further studies are also needed, especially with M. recutita methanolic extract, in the field of UV-induced skin damage and wound healing.

Conclusions
In the present work, M. pubescens growing wild in Algeria was studied for the first time by a combination of HPLC-PDA-MS and chromatographic isolations, followed by NMR. For comparison reasons, extracts of Matricaria recutita, the officially recognized European species, were prepared under the same experimental conditions and analyzed using HPLC-PDA-MS. Overall, 26 compounds were characterized (isolated and/or identified using HPLC-PDA-MS) in the extracts of M. pubescens, and 19 compounds were detected using HPLC-PDA-MS in the hydromethanolic and methanolic extracts of M. recutita. Quantitation using HPLC-PDA-MS showed that M. pubescens extracts had a higher content of luteolin derivatives, while M. recutita extracts had notably higher concentrations of the anti-inflammatory apigenin-7-O-glucoside, as well as a higher variety of flavonols and caffeoylquinic acid derivatives. Two representative polar extracts from each species were screened for their protective effects on UVA-induced 3T3 fibroblast cytotoxicity. Both M. recutita and M. pubescens extracts were cytoprotective. The methanolic extracts had the best protective effect at the lower concentrations, while the hydromethanolic extracts had the best protective effect at the higher ones. M. recutita exhibited the higher cell viability, leading to the conclusion that the latter seems to exhibit potent cytoprotective activity and significant regeneration activity. The stronger protective potential against UV stress might be attributed to a combination of apigenin-7-O-glucoside with other flavonoids and phenolics present in the extracts. Further studies with the isolated constituents are underway in order to understand the contribution of each compound to the protective effect. Further studies are also needed, especially with M. recutita methanolic extract, in the field of UV-induced skin damage such as ageing, irritation, skin cancer and wound healing.