GC/MS and LC-MS/MS phytochemical evaluation of the essential oil and selected secondary metabolites of Ajuga orientalis from Jordan and its antioxidant activity

2.1 General

Gas chromatography-Mass spectrometry (GC–MS) analysis was performed using Agilent 6890 series II – 5973 mass spectrometers interfaced with HP chemstation. UV–vis spectra were recorded on Shimadzu UV-1800 UV/Visible Scanning Spectrophotometer. Detection of the selected phenolic acids, flavonoids and nonphenolic acids and compounds was done utilizing a Bruker Daltonik (Bremen, Germany) Impact II ESI-Q-TOF System equipped with Bruker Dalotonik Elute UHPLC system (Bremen, Germany). n-Hexane (GC-grade), the n-alkanes (C₈-C₂₀) standard mixture, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, purity > 99 %), 2,2-diphenyl-2-picrylhydrazyl ((DPPH, purity > 99 %), ascorbic acid (purity > 98 %), methanol, potassium persulfate, sodium carbonate, Folin and Ciocalteu's Phenol reagent, sodium nitrite, aluminum chloride, and sodium hydroxide were all products of Sigma-Aldrich.

2.2 Plant material

Fresh aerial parts of A. orientalis were collected at full flowering stage in April/2018 from Ajloun city, north of Jordan (N 32.363932; E 35.775043). The plant identity was confirmed by Prof. Dr. Jamil Laham, Yarmouk University, Irbid, Jordan. A voucher specimen (AO/L/2018) was deposited in Prof. Mahmoud A. Al-Qudah Laboratory, Department of Chemistry, Faculty of Science, Yarmouk University, Irbid, Jordan.

2.3 Hydro-distillation of essential oil and extracts preparation

Fresh aerial parts of A. orientalis (200 g) were minced, suspended in 250 mL distilled water and the mixture was subjected to hydro-distillation in a Clevenger type apparatus for 4 h. The obtained yellow oil was dissolved in n-hexane (GC-grade), dried over anhydrous sodium sulfate, and then stored in amber glass vial at 4–6 °C until analysis.

Extraction and fractionation of the aerial parts of A. orientalis was performed according to the procedure listed in the literature (Al-Jaber et al., 2012). Each of the aqueous methanol (AO-A) and the butanol (AO-B) fractions were then assayed for their TPC, TFC, antioxidant activity (by the DPPH and ABTS assay methods) and then were subjected to LC-ESI-MS/MS analysis for the detection of selected phenolic acids, flavonoids and nonphenolic compounds.

2.4 Determination of essential oil constituents and their % concentration

The chemical constituents of A. orientalis hydro-distilled essential oil (AO-HDEO) and their relative percentage composition were determined according to the procedure listed in the literature in the literature using the same instruments and under identical chromatographic conditions (Abu-Orabi et al., 2020).

Identification of chemical constituents was achieved by comparing their calculated Kovats retention index (KI) values relative to (C₈–C₂₀) n-alkanes literature values measured with columns of identical polarity, or by matching their recorded mass spectra with the built-in mass spectral libraries (NIST, Gaithhersburg, MD, USA, and Wiley Co., Hoboken, NJ, USA) in addition to mass spectrum matching to the available authentic standards.

2.5 Total phenols and total flavonoids contents

The total phenols and total flavonoids contents of the AO-A and AO-B extracts were determined by Folin-Ciocalteu method and aluminum chloride assay, respectively as previously described (Al-Humaidi., 2017).

2.6 Antioxidant activity

The antioxidant activity of the AO-HDEO and each AO-A and AO-B fractions was determined by the DPPH and ABTS methods according to the procedure listed in the literature (Al-Qudah 2016; Al-Qudah et al., 2015; Al-Qudah et al., 2014; Teismann and Ferger., 2000). The ability of the AO-HDEO/fractions to scavenge radicals was calculated using the following equation: $$Scavenging\left(\%\right)=[(A_c-A_s)/A_c]\times 100$$

Where A_c is the absorbance of the blank and A_s is the absorbance in the presence of essential oil/extract.

2.7 LC-MS analysis of secondary metabolites

Analysis of selected secondary metabolites was performed on Bruker Daltonik (Bremen, Germany) Impact II ESI-Q-TOF System equipped with Bruker Daltonik Elute UHPLC system (Bremen, Germany) in both positive (M + H) and negative (M−H) electrospray ionization modes. Chromatographic separation was performed on a C-18 reversed phase column (100 × 2.1 mm, 2.0 μm) from Bruker Daltonik (Germany) at 40 °C, with an autosampler temperature of 8 °C. The elution gradient consisted of mobile phase A: water with 0.05 % formic acid and mobile phase B: acetonitrile. The gradient elution program was: linear gradient 5–80 % B (0 – 27 min); 95 % B (27 – 29 min); 5 % B (29.1–35.0 min). The flow rate of the solvent was 0.51 mL/min and the injection volume of the sample was 3.0 μL. Mass spectrum was operating at the following conditions: the capillary voltage was 2500 V, the nebulizer gas was 2.0 bar, dry gas (N₂) gas flow was 8.0 L/min and the dry temperature was 200 °C. The mass accuracy was < 1 ppm; the mass resolution was 50,000 FSR (Full Sensitivity Resolution) and the TOF repetition rate was up to 20 kHz.

A stock solution containing standard compounds (0.5 mg/mL) was prepared in HPLC-grade. Plant samples were dissolved with 2.0 mL DMSO, the volume was completed to 50 mL by acetonitrile, then each sample was centrifuged at 4000 rpm for 2 min and 3.0 µL was injected. The composition of the samples was identified based on the identification of m/z ratio with reference to the retention time of the used standards.

3.1 Essential oil

Hydro-distillation of the fresh aerial parts of A. orentalis afforded a yellow oil (yield 0.05 %, w/w). GC–MS analysis of the obtained HD-AOEO (Fig. 1) resulted in the identification of a total of 92 compounds amounting to 90.49 % of the total oil content (Table 1). The HD-AOEO was dominated by different classes of terpenoids, aliphatic hydrocarbons and their derivatives (Table 1), mainly oxygenated sesquiterpenoids (27.29 %). Individual main components included tiglic acid (18.90 %), ageratochromene (8.09 %), α-thujene (6.20 %), and 5-cedranone (5.82 %). Moreover, the obtained HDEO was assayed for its antioxidant activity using the DPPH and ABTS methods, results (Table 2) indicated a relatively high activity as compared to the employed positive controls (DPPH: (6.92 ± 0.22) × 10^-3 mg/mL; ABTS: 6.44 ± 0.18) × 10^-3 mg/mL).

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Fig. 1

GC–MS, peaks were numbered as reported in Table 1.

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The chemical composition of the essential oils of A. orientals from Turkey (Küçükbay et al., 2013) and from Iran (Sajjadi and Ghannadi, 2004); was quite different when compared to current results. The essential oil obtained from Turkish A. orientalis was dominated by phytol (36.7 %) while Iranian A. oreintalis EO was dominated by germacrene (24.2 %). Fig. 2 shows the main variations among the different classes of constituents detected in the essential oils A. orientalis from Jordan (current study), Iran, and Turkey. This variation in composition could be attributed to the different climatic conditions, different soil properties in addition to other factors like time of collection and different extraction procedures (Mercy and David Udo., 2018).

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Fig. 2

A classification of the constituents of the A. orientalis L. and their % composition from Jordan, Iran, and Turkey. Hemiterpenoids oxygenated (HO), monoterpene hydrocarbons (MH), monoterpenes oxygenated (OM), sesquiterpene hydrocarbons (SH), sesquiterpenes oxygenated (SO), diterpene oxygenated (DO).

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3.2 TPC, TFC, antioxidant activity

In the current study, each of the aqueous methanol (AO-A) and butanol (AO-B) fractions were investigated for their TPC, TFC and antioxidant activity using two assay methods and according to the procedures listed in the literature ((Al-Qudah, 2016; Al-Qudah et al., 2014, 2015; Govindan et al., 2016; Sanchez-Moreno, 2002). As could be deduced from the results shown in Table 2, AO-B fraction had the highest TPC and TFC (217.63 ± 2.65 mg gallic acid/g extract; 944.41 ± 4.77 mg quercetin/g extract, respectively). This extract had also the highest antioxidant activity as measured by the DPPH ((4.00 ± 0.20) × 10^-2 mg/mL) and ABTS ((3.00 ± 0.20) × 10^-2 mg/mL) assay methods.

3.3 LC-MS/MS profiling of selected secondary metabolites

In the current investigation, AO-M and AO-B fractions were screened for the presence of a selected set of secondary metabolites by LC-ESI-MS/MS using both, the positive and negative ionization modes. The list of the 33 different phenolic and nonphenolic compounds detected in both extracts are shown in Table 3, chromatograms are shown in Fig. 3. Both extracts were found to contain acteoside as a major phenolic acid derivative. It was noticed that each of 8-Prenylnaringenin, 3-gal(1–2)gluA soyasapogenol B, hederagenin, myristic acid and (Z)-3-hydroxyoctadec-7-enoic acid were detected in the AO-A fraction only. The phenolic and flavonoids profiles detected in the extracts of A. orientalis from Jordan in our current study were completely different from those reported for the plant from Turkish origin (Göger et al., 2015; Zengin et al., 2018).

Table 3 Compounds identified in the AO-M and AO-B extracts from A. orientalis from Jordan.

No.	Rt	Name	Structure	Molecular Formula	m/z meas.	Mwt	Compounds*
							AO-M	AO-B
1	0.99	Succinic acid		C4H6O4	117.0193	118.0266	+	+
2	1.8	2,5-Dihydroxybenzoic acid		C7H6O4	153.0192	154.0265	+	+
3	2.81	Caffeic Acid		C9H8O4	179.0343	180.0415	+	+
4	3.16	Vanillic acid		C8H8O4	167.0351	168.0423	+	+
5	4.4	p-Coumaric acid		C9H8O3	163.0399	164.0471	+	+
6	4.44	Ethyl gallate		C9H10O5	197.0457	198.053	+	+
7	4.82	3,5-Dimethoxy-4-hydroxyacetophenone		C10H12O4	195.0644	196.0716	+	+
8	5.45	Vitexin		C21H20O10	431.0976	432.1049	–	+
9	5.51	Eriodictyol-7-neohesperidoside		C27H32O15	595.167	596.1743	+	+
10	5.89	Salicylic acid		C7H6O3	137.0243	138.0315	+	+
11	5.89	Luteolin 7-O-glucoside (Cynaroside)		C21H20O11	447.0931	448.1004	+	+
12	5.95	Acteoside = Verbascoside		C29H36O15	623.1981	624.2056	+	+
13	6.05	3-O-Neohesperidoside Kaempferol		C27H30O15	593.1511	594.1583	+	+
14	6.17	Rutin		C27H30O16	609.145	610.1523	+	+
15	6.77	Kaempferol-3-O-glucoside		C21H20O11	447.0931	448.1004	+	+
16	6.78	3,6,2′,4′-Tetrahydroxyflavone		C15H10O6	285.0399	286.0472	+	+
17	7.00	Diosmin		C28H32O15	607.166	608.1733	+	+
18	7.09	7-Glu-Chrysoeriol		C22H22O11	461.1085	462.1157	+	+
19	7.23	Kaempferol-7-O-glucoside		C21H20O11	447.0928	448.1	+	+
20	8.55	Luteolin		C15H10O6	285.0403	286.0475	+	+
21	10.24	Hispidulin		C16H12O6	299.0557	300.063	+	+
22	13.73	Caffeic acid phenethyl ester		C17H16O4	283.1011	284.1084	+	+
23	14.65	8-Prenylnaringenin		C20H20O5	339.1235	340.1308	+	–
24	16.46	3-Gal(1–2)GluA Soyasapogenol B		C42H68O14	795.4529	796.4602	+	–
25	21.63	Hederagenin		C30H48O4	471.3472	472.3545	+	–
26	22.58	Glc-octadecatrienoyl-sn-glycerol		C27H46O9	513.3099	514.3172	+	+
27	26.4	(Z)-3-Hydroxyoctadec-7-enoic acid		C18H34O3	297.2435	298.2508	+	+
28	26.7	Myristic acid		C14H28O2	227.2012	228.2085	+	–
29	28.78	Pentadecanoic acid		C15H30O2	241.2176	242.2249	+	+

* (+): Detected; (-): not detected; samples were verified against authentic samples isolated in our labs or purchased from Sigma-Aldrich.

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Fig. 3

LC-MS/MS chromatograms of AO-B and AO-M fractions of A. orientalis from Jordan.

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