Exploring the phytochemical and biological activity of Cardaria draba: Insights into Volatile and Nonvolatile Compounds (2024)

Total phenolics (TPC) and total flavonoids (TFC) contents

The phytochemical evaluation of various extracts (Methanol, CHCl₃, and H₂O) from the leaves, flowers, stems, and roots of C. draba is summarized in Table1. Among these, the aqueous extract of the flower exhibited the highest TPC at 76.12 mg GAE/g, followed by the leaf extract at 58.25 mg GAE/g, the root extract at 39.12 mg GAE/g, and the stem extract at 35.28 mg GAE/g. Conversely, the aqueous extract of the leaves demonstrated a superior TFC of 112.36 mg QE/g DE, surpassing that of the flower (83.18 mg QE/g), stem (59.16 mg QE/g), and root (36.48 mg QE/g) extracts. The highest concentrations of total phenols and flavonoids were associated with the flower, leaf, and stem of C. draba, respectively. In research conducted by Eruygur et al., the aqueous extract of C. draba flowers revealed a maximum TFC of 64.32 GAE/mg, while the ethanol extract of the leaves recorded the highest flavonoid content at 141.47 QE/mg, exceeding the values found in the current study ²². The TPC of the ethanol, water, and DCM extracts from the leaves and stems of C. draba were also evaluated, with the ethanolic extract of the leaves yielding the highest TPC at 57.79 GAE/mg ²⁵. Additionally, the antioxidant capacities of acetone, methanol, and aqueous extracts of C. draba were assessed, revealing that the acetone extract had the highest TPC at 31.67 mg GAE/g, followed by the methanolic extract at 18.41 mg GAE/g and the aqueous extract at 12.52 mg GAE/g ⁴.

Antioxidant activity

The antioxidant properties of extracts from the leaves, flowers, stems, and roots of C. draba, obtained using methanol, chloroform, and water, were assessed through DPPH radical scavenging assays, with results reported as IC₅₀ values (see Table1). The antioxidant action of extracts from C. draba was weaker than that of Trolox. The presence of flavonoids and phenolic compound in extracts due to low solubility in the DPPH radical reduction assay elucidated the low antioxidant action for extract. Among the various extracts, the methanol extracts from the leaves and flowers demonstrated the highest scavenging activity, yielding IC₅₀ values of 4.24 ± 1.65 and 5.32 ± 0.54 mg/mL, respectively. In contrast, the stem and root extracts exhibited higher IC₅₀ values of 6.75 ± 1.54 and 3.78 ± 1.34 mg/mL, respectively. The pronounced free radical scavenging capacity of the water and methanol extracts is likely linked to their elevated TPC and TFC, which possess hydrogen-donating capabilities. In research conducted by Sharifi rad et al., the antioxidant activity of the ethanol extract from seeds (0.01 mg/mL) surpassed that of the ethanol extract from leaves (0.03 mg/mL), although both exhibited significantly lower antioxidant potential compared to BHA and ascorbic acid ²⁶. Additionally, another investigation evaluated the antioxidant properties of three extracts (acetone, methanol, and water) from C. draba using various assays, including DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and beta-carotene/linoleic acid inhibition. The findings indicated that the methanolic extract displayed the highest antioxidant activity in CUPRAC, FRAP, and phosphomolybdenum assays, while the aqueous extract was most effective in DPPH and ABTS assays ⁴. The antioxidant action of water extract from L. draba were determined and measured value was IC₅₀ = 168/21 µL/mL ²⁷. The antioxidant activity of methanol, ethanol, and water extracts from C. draba flowers, leaves, stems, and roots were examined. The methanol and aqueous extract of flowers showed highest DPPH radical scavenging activity with IC₅₀ = 1.57 ± 0.71 and 1.27 ± 1.69 mg/mL respectively which was similar to our findings ²².

Antibacterial activity

Table2 illustrates the antimicrobial properties of methanol, chloroform, and water extracts derived from the leaves, flowers, stems, and roots of C. draba. The findings indicate that these extracts exhibited significantly greater antibacterial efficacy against S. aureus and E. faecalis compared to E. coli, which can be attributed to the differing structural compositions of the bacterial cell walls. The outer membrane of E. coli, characterized by its lipid and polysaccharide components, acts as a barrier to the penetration of antimicrobial agents ²⁸. Notably, the methanol extracts from the leaves and flowers demonstrated markedly stronger antibacterial effects, with MIC of 6.25 and 12.5 µg/mL, respectively; however, the antibacterial activity of C. draba extracts was somewhat inferior to that of tetracycline. Tetracycline is known to diffuse passively through porin channels in the bacterial membrane and binds reversibly to the 30S ribosomal subunit, thereby obstructing the attachment of tRNA to the mRNA-ribosome complex and disrupting protein synthesis ²⁹. However, crude extracts with the MIC values of less than 1 mg/mL are categorized as exhibiting substantial antibacterial activity, whereas those with MIC values exceeding 1 mg/mL are deemed non inhibitory ³⁰. The results indicate that the majority of C. draba extracts displayed considerable inhibition. Sharifi-Rad reported that both ethanolic and aqueous extracts from the leaves and seeds of C. draba were bactericidal against four bacterial strains, with MIC values ranging from 3.1 to 134 µg/mL, with the ethanolic leaf extract showing superior activity ²⁶. In a separate investigation, the MIC value for the leaf extract of L. draba against Aspergillus niger and Pseudomonas aeruginosa was found to be 128 mg/mL, while against Bacillus subtilis and S. aureus, it was 256 mg/mL ²⁷. Additionally, research conducted by Radonic et al. revealed that the essential oil extracted from C. draba exhibited a broad spectrum of growth inhibitory activity against both Gram-positive and Gram-negative bacteria, with MIC values of 4 and 128 mg/mL, respectively ³¹.

GC-MS analysis of C.draba

This research employed gas chromatography-mass spectrometry (GC-MS) to analyse the essential oils derived from the leaves, flowers, stems, and roots of the C. draba plant, which accounted for 95.7%, 97.6%, 89.1%, and 88.4% of the total oil content, respectively. A total of 24 distinct compounds were identified from the C. draba extract through GC-MS analysis. As detailed in Table3, the leaf extract revealed the presence of 20 chemical compounds, with 3-butenyl isothiocyanate (26.4%) and 6, 10, 14-Trimethyl-2-pentadecanone (16.4%) being the most abundant constituents. Furthermore, the analysis of the flower extract identified 16 chemical compounds, where 6, 10, 14-Trimethyl-2-pentadecanone (28.6%) and (E)-phytol (21.8%) were found to be the predominant components. Additionally, in the extracts from the roots and stems of C. draba, the compound with the highest concentration was identified as 4-methylsulfinylbutyl isothiocyanate. The research conducted by Radonic et al. identified that the predominant compounds in the essential oil extracted from the aerial parts of C. draba are 4-methyl sulfanyl butyl isothiocyanate, comprising 28.0%, and 5-methyl sulfanyl pentanenitrile, which accounts for 13.8%. Additionally, another investigation focused on the chemical constituents of the leaves, roots, and fruits of C. draba revealed that the leaves predominantly contain 3-butenyl isothiocyanate at 80.5% and 4-methyl sulfinyl butyl isothiocyanate at 5.6%. In the fruit, 4-methyl sulfinyl butyl isothiocyanate was found to be the most abundant at 72.1%, while the root samples exhibited a composition of 4-methyl sulfinyl butyl isothiocyanate at 30.0%, hexadecanoic acid at 24.1%, and isobutyl isothiocyanate at 14.3% ²⁰. Furthermore, an analysis of the chemical compounds in the aerial parts and leaves of C. draba sourced from France indicated that the most significant constituents were 6,10,14-trimethyl-2-pentadecanone (20.61% in aerial organs and 11.08% in leaves) and (E)-phytol (11.38% in aerial organs and 67.39% in leaves)²⁵. The aroma volatiles of roots, stems, leaves, flowers, and fruits of Cardaria draba collected from Tunisia investigated that the principal components were hexadecanoic acid (34.6%), 6-methyl-5-hepten-2-one (18.3%), decanal (15.0%), 6,10,14-trimethyl-2-pentadecanone (13.2%), and n-pentacosane (13%) ³².

^aRT, retention time.

^bKI_exp, Experimental Kovats retention index.

^CKI_Lit, Kovats retention indices on CP-Sil 8CB capillary column

Metabolomics analysis of methanol extracts of C. draba flower, leaf, stem and root

The application of liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) was conducted for the first time on methanol extracts derived from the flowers, leaves, stems, and roots of C. draba, aiming to identify the phytochemicals potentially responsible for its antioxidant and antibacterial properties. Notably, the highest concentrations of phenolic and flavonoid compounds were detected in the methanolic and aqueous extracts, which can be attributed to the superior solubility of these compounds in methanol and water compared to other solvents evaluated ³³. In terms of antibacterial efficacy, the interaction between methanol and water molecules is stronger than that among water molecules alone which making methanol extracts a suitable choice for LC-ESI-MS analysis ³⁴. The characterization of different parts led to identifying 62 compounds, including 7 hydroxy benzoic acid derivatives, 10 hydroxycinnamic acid derivatives, 15 flavonol glucosides, 12 flavone glycosides, 4 flavanone glycoside, 1 flavanonol glycoside, 2 anthocyanin glycosides, 3 flavan-3-ol glycosides, 2 tannins and 6 other phenol derivatives (Table4) and (Supplementary materials: Table S1-S2 and Fig. S1-S4). Metabolites profile in plants is affected by factors such as species, type of organ, agronomic, genomic, climatic, harvest conditions, processing, etc., ³⁵ A total of 53, 47, 48, and 54 compounds were tentatively identified in the flower, leaf, stem, and root of this species, respectively. Proposed structures were characterized by correlating mass adducts such as [M-H]-, [M-2H]-, [2M]-, [2M-H]-, [M-2H + K]-, and [M-2H + Na]- with previously published mass spectrometry data and molecular weights. The total ion chromatograms (TIC) and examples of extracted ion chromatograms (XIC) along with their corresponding mass adducts for the flower, leaf, stem, and root of C. draba are illustrated in Figs.1A–D. The biological activity of this species was in accordance with amount of dominant constituents as relative ion intensity value (Eⁿ) including, astilbin (5.0 E⁶), myrtillin (3.9 E⁶), rutin (1.9 E⁶), hesperidin (1.8 E⁶), caffeoyl glucose (1.7 E⁶), vicenin 2 (1.6E⁶), kaempferol-3-O-rutinoside (1.5 E⁶), vitexin 4'-O-glucoside (1.5 E⁶), narirutin (1.4 E⁶), kaempferol-3-O- arabinoside (1.3 E⁶), vincetoxicoside B (1.3 E⁶), caftaric acid (1.2 E⁶), salvianolic acid D (1.2 E⁶), and quercetin-3-O-rhamnopyranosyl(1->2)- galactopyranosyl]-7-O- rhamnopyranoside (1.0 E⁶) in flower; astilbin (8.1E⁵), petunidin 3-galactoside (3.0 E⁵), vincetoxicoside B (2.3 E⁵), myrtillin (2.7 E⁵), ellagic acid 2-rhamnoside (2.1 E⁵), isorhamnetin (2.1 E⁵) and sinapine (2.0 E⁵) in leaf; caffeoyl glucose (9.6 E⁵), kaempferol-3-O- arabinoside (2.1 E⁶), myrtillin (2.1 E⁶), hyperoside (2.1 E⁶) and caftaric acid (1.6 E⁶) in stem; and myrtillin (4.3 E⁶), kaempferol-3-O- arabinoside (2.0 E⁶), 3,7-Di-O-methylquercetin (1.5 E⁶), 3'-O-methylepicatechin-7-O-glucuronide (7.0 E⁵) and vincetoxicoside B (4.6 E⁵) in root. Studies have demonstrated that these metabolites have biological activities ^36–40. As shown in Table4, all the detected phytochemicals were described for the first time from the root and stem of C. draba and other Cardaria spp., while compounds of 14, 20, 22, 23, 26, 28, 31, 32, 33, 36, 37, 40, 41, 51, 52, 54 and 61 were found for the first time in brassicaceae family. Also, metabolites of 1, 2, 5, 6, 8–13, 15, 16, 18, 24, 25, 27, 29, 30, 42, 43, 45–50 and 56–59 were reported from previous studies of C. draba. All the references of detected components in each species were listed in Table4 to compare with each other.

Principal Component Analysis (PCA) and Heat Map

Multivariate analyses are effective methods in herbal medicine for distinguishing chemical profiles of different morphological parts ^19,41. This research employed Principal Component Analysis (PCA) and heat mapping as multivariate analytical techniques, utilizing ion relative abundance and area percentage data processed through MZmine and GraphPad Prism software. The objective was to compare the metabolite profiles derived from the LC-MS dataset of methanol extracts with those from the GC-MS dataset of essential oils, specifically focusing on samples from flowers, leaves, stems, and roots (Fig.2–4).

The peak intensities of compounds, designated as variables 1–62 in the LC-MS dataset, alongside the area percentages of compounds, classified as variables 1–23 in the GC-MS dataset, were utilized in multivariate analyses to identify marker compounds across four distinct samples. Principal Component Analysis (PCA) accounted for 49.87% of the total variation in the LC-MS dataset (PC1) and 32.12% (PC2), while for the GC-MS dataset, it explained 65.04% (PC1) and 23.36% (PC2). The loading plot for hydro-methanol extracts (Fig.2A) indicated that PC1 was predominantly associated with the concentrations of stem and root components, whereas the variables with the highest loadings in PC2 were linked to the concentrations of flower and leaf components. In the case of essential oils (Fig.3A), the metabolite features of flower and root were more pronounced in PC1 compared to PC2, while those of leaf and stem were more significant in PC2 than in PC1. The score plots are illustrated in Fig.2B and Fig.3B, demonstrating a clear separation of the samples into four distinct categories, thereby confirming the efficacy of this method in differentiating the four parts of C. draba. The biplot concurrently illustrated the relationships between samples (score plot) and variables (loading plot) (Fig.2C and Fig.3C), highlighting the similarities and differences in the metabolite profiles of the four samples. In the LC-MS dataset, metabolites characterized in flower, leaf, stem, and root included variables 1–4, 6–7, 10–12, 19–25, 31–33, 38, 43–48, 51, 53, 59, and 61; those found in leaf, stem, and root were 27, 28, 30, 37, and 56; while 8, 14–16, 34–36, and 42 were identified in flower, stem, and root. Additionally, variables 13, 39–41, and 54 were present in flower, leaf, and root; variable 5 was found in flower, leaf, and stem; 49 and 50 were identified in flower and leaf; 9, 26, and 62 were present in flower and stem; 52 was found in flower and root; 29, 57, 58 and 60 in stem and root; and 55 in stem and root. The variables numbered 17 and 18 were exclusively detected in the flower samples. In the context of the GC-MS dataset, variables 1, 2, 5, 8, 11–13, and 20 were observed across flower, leaf, stem, and root samples; variables 7, 10, 14, 17, 21, and 22 were found in flower, leaf, and stem; variables 3 and 15 were present in flower, leaf, and root; variables 6, 9, 16, and 19 were identified in leaf, stem, and root; variable 4 was detected in both flower and leaf; while variables 18 and 23 were found in stem and root.

Additionally, a heat map, serving as a form of multivariate analysis, was utilized to compare and highlight significant variables based on relative intensity (Fig.4A) and area percentage (Fig.4B). The color gradient in the heat map illustrates the distribution of metabolites, ranging from low values (indicated by white) to high values (indicated by blue), thereby categorizing the variables from minor to major within the samples.