Chemo-Diversity and Antiradical Potential of Twelve Matricaria chamomilla L. Populations from Iran: Proof of Ecological Effects

Matricaria chamomilla L. is a popular medicinal herb that is used for healing various diseases and is widely distributed worldwide in temperate climate zones, and even in the subtropical climate of Southern and Western Iran. This study was aimed at comparing the volatile oil constituents, along with antiradical potential and HPLC analysis of methanolic extracts from twelve plant samples growing in Iran. The present research was carried out for the first time on these populations. Among seventeen identified volatile chemicals evaluated by GC/MS and GC/FID, representing 92.73–97.71% of the total oils, α-bisabolone oxide A (45.64–65.41%) was the major constituent, except in case of “Sarableh” as a new chemotype, where (E)- and (Z)-γ-bisabolene (42.76 and 40.08%, respectively) were the predominant components. Oxygenated sesquiterpenes (53.31–74.52%) were the most abundant compounds in the samples excluding “Sarableh” with 91.3% sesquiterpene hydrocarbons. “Sarableh” also exerted the most potent antioxidant capacity with EC50 = 7.76 ± 0.3 µg/mL and 6.51 ± 0.63 mmol TE (Trolox® equivalents)/g. In addition, populations “Lali” and “Bagh Malek” contained the highest amounts of apigenin and luteolin with 1.19 ± 0.01 mg/g and 2.20 ± 0.0 mg/g of plant material, respectively. Our findings depict a clear correlation between phytochemical profiles and antiradical potential of M. chamomilla and geographical factors.


Introduction
Matricaria chamomilla L. (syn. Chamomilla recutita L. Rauschert, German chamomile), belonging to the Asteraceae family, is one of the well-known medicinal plant species which has been widely used for centuries. The herb is currently consumed around the world as herbal tea, with more than 1 million cups daily [1,2]. M. chamomilla is used in the treatment of many ailments and disorders; internally to facilitate digestion and as antispasmodic, externally to treat minor wounds. In folk medicine, its use spreads from the relief of various pains such as headaches and toothaches to the facilitation of menstruation [3]. Chamomile essential oil (EO) has been commonly applied in Iranian folk medicine as an anti-inflammatory, antispasmodic, anti-peptic ulcer, antibacterial and antifungal agent [4,5].
In the present study, EO compositions of twelve Iranian M. chamomilla populations collected from diverse regions were qualitatively and quantitatively assessed. Since the main radical scavengers are phenolics, flavonoids being the major group within phenolics [22], our study was also designed to determine apigenin and luteolin contents, as its main flavonoid aglycons [19,23], by utilizing analytical HPLC. Antiradical capacities of the extracts with DPPH and ORAC assays were also compared. In order to make comparison, the plants were harvested at the same flourishing period. To the best of our knowledge, this is the first report of these populations.

Essential Oil Contents
As shown in Table 1, the studied plant samples were characterized mostly with similar EO yields. Populations "B" (1.03 ± 0.003%) and "BM" (0.78 ± 0.017%) contained the highest and lowest amount of EO, respectively ( Table 1). As the plant sample "B" was cultivated in the university's garden and it was regularly irrigated, the highest EO content can be predicted.

Chemical Profiles of Volatile Oils
Seventeen compounds were detected in these twelve populations. The sesquiterpene α-bisabolone oxide A (45.64-65.41%) was the major EO constituent in the samples except "B" and "S", whereas its concentration was the highest in population "Mu" ( Table 1).
The cultivated sample "B" was rich in α-bisabolol oxide B (21.88%) and chamazulene (19.22%), while the percentages of these compounds were lower under the wild growth conditions. The blue colour of EOs in all samples except "S" represented their chamazulene contents, whereas, "S" due to lack of this compound showed a yellowish green colour. Accordingly, oxygenated sesquiterpenes (53.31-74.52%) were the predominant chemical group of EO constituents in all studied samples, excluding "S", which was rich in sesquiterpene hydrocarbons ( Figure 1).

Apigenin and Luteolin Quantification of Methanolic Extracts
Methanolic extracts of samples "L" and "A" contained the highest amounts of apigenin, with 1.19 ± 0.01 mg/g and 1.02 ± 0.01 mg/g, respectively. Luteolin was present in higher concentrations in "BM" (2.20 ± 0.0 mg/g) and "A" (1.01 ± 0.02 mg/g) extracts ( Figure 2).    PCA is a mathematical procedure that transforms several correlated variables into various uncorrelated variables called principal components (PC). PC1, PC2, and PC3 showed the highest variation of phytochemicals among the studied populations. PC1 explained 41.57% of total variation and had a positive correlation with α-bisabolol oxide A, (E)-β-farnesene, α-bisabolone oxide A and (E)-spiroether, and negative correlation with (Z)-γ-bisabolene and (E)-γ-bisabolene. The second PC (PC2), with 24.98% of variance, demonstrated positive correlation with chamazulene, α-bisabolol oxide B, α-bisabolone oxide A and the EO content. Furthermore, PC3 represented positive correlation in the case of apigenin and luteolin, which accounted for 12.02% of the total variance (Table 2). Table 2. Eigenvalues, variance and cumulative variance for three principal components.

Major Phytochemicals
Principal Components Regarding a significant contribution of phytochemical variation in PC1 and PC2, the scatter plot of PC1 and PC2 was used to specify phytochemical distance. The studied populations were classified in three groups, which confirmed the CA results.
In accordance with the CA, the populations "I", "DS", "Mo", "MS", "Mu", "G", "SS", "L", "A" and "BM" were classified into the same category, while, "B" and "S" were grouped into the individual subclasses. The first group possessed α-bisabolone oxide A and α-bisabolol oxide A as the major constituents as well as apigenin and luteolin (chemotype I). The second chemotype (II), was characterized by high amounts of chamazulene and α-bisabolol oxide B. The chemotype (III) was the richest in (Z) and (E)-γ-bisabolene.

Effect of Environmental Factors on Phytochemicals
In order to assess the effect of environmental factors on EO components, along with apigenin and luteolin contents, canonical correspondence analysis (CCA) was applied based on a matrix of three environmental factors including altitude, mean annual temperature (MAT), and mean annual precipitation (MAP) [24] and major EO compounds, along with apigenin and luteolin contents ( Figure 5).
According to the CCA, phytochemicals of populations in first group were significantly affected by ecological parameters (altitude, temperature and precipitation) while the main oil composition and flavonoids of "Sarableh" and "Boldgold" were changed by genetic factors. Therefore, the "Sarableh" population can be introduced as a new chemotype.

Antiradical Activity of the Extracts
In the evaluation of the antioxidant potential of the twelve selected populations, "S" showed the most significant antiradical capacity, with EC 50 = 7.76 ± 0.3 µg/mL and 6.51 ± 0.63 mmol TE/g measured by DPPH and ORAC assays, respectively. However, the extracts showed lower activity compared to ascorbic acid (EC 50 = 0.3 ± 0.02 µg/mL) in the DPPH and rutin (20.22 ± 0.63 mmol TE/g) and EGCG (11.97 ± 0.02 mmol TE/g) in the ORAC assay (Figures 6 and 7).

Discussion
The results of the present study confirm the variability of EO composition, flavonoid profile and antiradical activity, which are significantly affected by a diversity of ecological conditions. The abiotic factors (e.g., moisture, topography, temperature, and edaphic factors) highly impact the phytoconstituents variation, and/or biotic factors remarkably influenced the terpene biosynthesis pathways and chemotype profiles [25]. However, the role of other ecological effects such as climatic factors (e.g., humidity, wind, atmospheric gases, and light), physiographic factors (e.g., steepness and sunlight on vegetation and direction of slopes), edaphic factors (the soil attributes), and biotic factors (e.g., the existence of lianas, epiphytes, parasites etc.) may also be considerable in chemotype variations.
In accordance with our findings, oxygenated sesquiterpenes (53.31-74.52%) are the most dominant EO compounds of the selected Matricaria chamomilla L. samples, which corroborates previous reports. The wild populations were significantly different compared with the cultivated sample (Bodgold) in EO composition. α-Bisabolone oxide A was mostly the major EO constituent in the populations.
It is noteworthy, that the sample "Sarableh" (with the highest altitude and least minimum and maximum annual temperatures) as a new chemotype was the richest in two geometric isomers of γ-bisabolene (82.84%), whilst these sesquiterpenes were not markedly detected in the other plant populations. This population also demonstrated the most potent antiradical capacity compared with those samples.
According to the results, the abundance of luteolin was much higher than the apigenin content. The lack of luteolin and apigenin as the selected flavones was reported in populations "MS" and "Mo". These samples are most probably rich in other phenolic natural products.
As apigenin and luteolin were present in "Sarableh" in low concentrations, the high free radical scavenging capacity of this sample is obviously related to other polyphenolic compounds.
In a similar study, methanol extracts of M. chamomilla yielded from different samples indicated more potent antiradical activity than EOs analysed by the DPPH test, due to polyphenols present in extracts; although, they possessed a moderate effect in comparison with the standards [6].
The plants were identified by Dr. Mehrangiz Chehrazi affiliated with the same department, and a voucher specimen of each sample was deposited in the herbarium of the department. The voucher's codes and geographic coordinates including the latitude, longitude, altitude using the Global Positioning System (GPS), along with mean annual temperatures of the studied populations are given in Table 3. The meteorological data (MAP, MAT, MMaxAT, and MMinAT) were collected from September until May 2017 [24]. The flowers were shade dried and finely ground. Each powdered sample was individually well-mixed and subjected to analysis.

Extraction of Volatile Oils
To extract the EOs, 60 g of each powdered sample was individually subjected to Clevenger apparatus (hydro-distillation method) for 3 h. The obtained EOs were dried over anhydrous sodium sulphate and stored in refrigerator at 4 • C until analysis. Yields of extracted EOs were calculated by Equation (1): Diethyl ether was used to elute the whole amount of EOs from the apparatus, and the weighing process was performed after evaporating the solvent.

Gas Chromatographic Analysis (GC-FID)
In the case of GC-FID analysis, the EOs were analyzed by Shimadzu GC-17A (Kyoto, Japan) equipped with an FID detector and SGE™ BP5 capillary column (Trajan Scientific and Medical, Victoria, Australia) (30 m × 0.25 mm column with a 0.25 µm film thickness). The split mode in GC was a ratio of 1:100. Injector and FID detector temperatures were set at 280 and 300 • C, respectively. The oven temperature was kept at 60 • C for 1 min and then raised to 250 • C at 5.0 • C/min and held for 2 min, while the ambient oven temperature range was +4 to +450 • C. Helium gas was used at a flow rate of 1 mL/min as a carrier gas.

Gas Chromatography-Mass Spectrometric (GC-MS) Analysis
Analysis of the samples was carried out using an Agilent 7890B gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a 5977B mass spectrometry detector. The GC instrument was equipped with a split inlet, working in a split ratio of 100:1 mode with a 30 m × 0.25 mm HP-5MS capillary column with a 0.25 µm film thickness and 0.25 µm particle size (temperature range: −60 to +320/340 • C). The injection port temperature was 250 • C. The oven temperature was kept at 60 • C for 1 min and then programmed from 60 • C to 250 • C at 5 • C/min; then, the temperature was kept at 250 • C for 2 min. Helium (99.999%) was used as a carrier gas with a flow rate of 1 mL/min and inlet pressure 35.3 kPa. The mass spectrometer was operated in the electron impact mode at 70 eV, and the inert ion source (HES EI) temperature was set to 350 • C; the temperature of the quadrupole was set at 150 • C, while the MS interface was set to 250 • C. A scan rate of 0.6 s (cycle time: 0.2 s) was applied, covering a mass range from 35 to 600 amu.

Identification of Essential Oil Components
Most of the compounds were identified using two different analytical approaches: (a) comparison of Kovats indices of n-alkanes (C 8 -C 24 ) [42] and (b) comparison of mass spectra (using authentic chemicals and Wiley spectral library collection). Identification was considered tentative when based on mass spectral data alone. In GC-FID and GC-MS, data acquisition and analysis were performed using Chrom-card TM (Scientific Analytical Solutions, Zurich, Switzerland, version DS) and Xcalibur TM software (Thermo Fisher Scientific, Waltham, MA, USA, 4.0 Quick Start), respectively.

Preparation of Solvent Extracts
Five g of each sample was individually extracted with MeOH (3 × 75 mL) in an ultrasonic bath (VWR-USC300D) for 10 min, at 40 • C under power grade 9.
After removing the solvent under reduced pressure at 50 • C (Rotavapor R-114, Büchi), the concentrated extracts were subjected to evaluate the antiradical assays and HPLC analysis.

HPLC Analysis of Apigenin And Luteolin
Twenty µL of each extract (1 mg/mL) was separately injected into an analytical high-performance liquid chromatography system (HPLC) (Knauer, Berlin, Germany) by using an end capped Eurospher II 100-5 C18, Vertex Plus Column (Knauer, Berlin, Germany) (250 × 4.6 mm with precolumn) with particle size: 5 µm, pore size: 100 Å and temperature 30 • C; coupled to UV detector (Knauer GmbH-Smartline 2600, Berlin, Germany) at a wavelength range 190 to 500 nm (quantification at 330 nm), while MeOH/H 2 O was applied as the mobile phase with a gradient system, increasing MeOH from 30% to 70% within 40 min, with a flow rate of 1 mL/min, at ambient temperature. Analysis was performed using SAS software, version 9.2 (SAS Institute Inc., Cary, NC, USA).

DPPH Assay
Free radical scavenging activity of the plant extracts was assessed by DPPH assay [43]. The measurement was carried out on 96-well microtiter plates. In brief, Microdilution series of samples (1 mg/mL, dissolved in MeOH) were prepared starting with 150 µL. To gain 200 µL of sample, 50 µL of DPPH reagent (100 µM) was added to each sample. The microplate was stored at room temperature under dark conditions. The absorbance was measured after 30 min at 550 nm using the microplate reader. MeOH (HPLC grade) and ascorbic acid (0.01 mg/mL) were used as a blank control and standard, respectively. Antiradical activity was calculated using the following Equation (2): where A 0 is the absorbance of the control and A 1 is the absorbance of the standard sample. Anti-radical activity of the samples was expressed as EC 50 (concentration of the compounds that caused 50% inhibition). EC 50 (µg/mL) values were calculated using GraphPad Prism software version 6.05 (GraphPad Software, San Diego, CA, USA). Each sample was measured in triplicate.

ORAC Assay
Twelve extracts were subjected to ORAC assay [44] with slight modifications. Microtiter plates (96-well) were used for measurement of the samples. Briefly, 20 µL of extracts (0.01 mg/mL) was mixed with 60 µL of AAPH (peroxyl free radical generator) (12 mM) and 120 µL of fluorescein solution (70 mM). Then, the fluorescence was measured for 3 h at 1.5-min cycle intervals with the microplate reader. The standard Trolox ® was used. Activity of all samples was compared with rutin and EGCG as positive controls. Antioxidant capacities were reported as µmol TE (Trolox ® equivalents)/g of dry matter.

Statistical Analysis
All the experiments were done in triplicate, and the results are expressed as means ± SD. The data were assessed with one-way analysis of variance (ANOVA) using SAS software (version 9.2, SAS Institute Inc., Cary, NC, USA) and GraphPad Prism version 6.05. The means were compared using Duncan's comparisons test (p < 0.05).

Conclusions
According to our preliminary study, the chemo-diversity and antiradical potential of twelve studied Matricaria chamomilla populations were highly affected by a variety of ecological conditions. To acquire the best yield with a good profile of active principles, it is crucial to combine a good genotype with optimal environmental circumstances.
In accordance with our findings, oxygenated sesquiterpenes are the most dominant EO compounds of the selected Matricaria chamomilla samples. Almost all plant populations contained apigenin and luteolin. Since the plant sample "Sarableh" indicated the most potent capacity to scavenge free radicals and its apigenin and luteolin contents were insignificant, its activity undoubtedly refers to the presence of other polyphenolic compounds. Due to high amounts of apigenin and luteolin, populations "Lali" and "Bagh Malek" can be considered as a rich source of these compounds. Our results confirm the effects of growth conditions on the quantity and quality of aromatic phytochemicals. More investigations are required to study the amounts of other polyphenolic compounds in diverse populations and the correlations between secondary metabolite contents, bioactivities and ecological effects.