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Original article
13 (
7
); 6256-6266
doi:
10.1016/j.arabjc.2020.05.043

Antioxidant activity of crude extracts and essential oils from flower buds and leaves of Cistus creticus and Cistus salviifolius

, , , , , , , ,
a Department of Chemistry, Faculty of Science, Yarmouk University, Irbid, Jordan
b Chemistry Department, College of Science, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
c Faculty of Engineering Technology, Al-Balqa Applied University, Amman, Jordan
d Department of Biological Sciences, Faculty of Science, Yarmouk University, Irbid, Jordan

⁎Corresponding author. mahmoud.qudah@yu.edu.jo (Mahmoud A. Al-Qudah)

Received: , Accepted: ,
© The Author(s) 2020
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Volatile oils from flowers and leaves of C. creticus L. and C. salviifolius L. were extracted by two extraction methods; namely, hydrodistillation and solid-phase micro-extraction (SPME). The chemical composition of essential oils was analyzed by GC and GC–MS. The volatile extracted from leaves and flowers of C. criticus using SPME was dominated by monoterpenes and sesquiterpenes hydrocarbon with α-pinene, camphene and α-cubebene as major components. In hydrodistillation, the oil extracted from leaves was dominated by oxygenated diterpenes and diterpenes hydrocarbon with manoyl oxide and sclarene as major components, whereas, the oil extracted from flowers was dominated by oxygenated diterpenes and diterpenes hydrocarbon with manoyl oxide and abietatriene as major components. The volatile from flowers and leaves of C. salviifolius obtained by SPME were dominated by monoterpenes and sesquiterpenes with δ-3-carene, α-pinene, β-pinene, and E-caryophyllene as major constituents. On the other hand, the oils from flowers and leaves of C. salviifolius obtained by hydrodistillation were dominated by oxygenated diterpenes, diterpenes hydrocarbon and esters with dehydro abietol, abietol, manoyl oxide and methyl octadecenoate as major components. In the leaves, the major components of the oil were manoyl oxide, E-ethyl cinnamate, and Z-ethyl cinnamate. These oils showed weak antioxidant activity when compared to the positive controls α-tocopherol, ascorbic acid, and EDTA, while the crude extracts aq. MeOH, butanol, and water showed good antioxidant activity. Discriminating between the studied plants based on the extraction method was also possible upon applying Principle component analysis (PCA) to the obtained GC–MS data.

Keywords

Antioxidant activity
Cistus creticus L.
Cistus salviifolius L.
PCA
Essential oils
1

1 Introduction

Rock-rose plants and shrubs consist of 175 species of eight herbaceous genera. Genera Cistus creticus L. and Cistus salviifolius L. of the species (Cistaceae) are grown in the Mediterranean region, Europe, North America temperate areas and a limited number of them is found in South America and the northern area of Jordan (Comandini et al., 2006; Al-Eisawi, 1982; Guimarães et al., 2009). Cistus species are commonly used in many Mediterranean countries in traditional folk medicine as antidiarrheic and for the treatment of various skin diseases (Madaus et al., 1938; Barrajón-Catalán et al., 2010). The use of the plant in this way has multiple functions as an antiinflammatory (Demetzos et al., 2001), antiulcerogenic, wound healing, antimicrobial (Demetzos et al., 1999), antifungal (Bayoub et al., 2010), antiviral, antitumor (Dimas et al., 2000), cytotoxic (Ben Jemia et al., 2013) and antinociceptive (Barrajón-Catalán et al., 2010). Phytochemical studies on different Cistus species revealed the presence of several flavonoid compounds (Pascual et al., 1977; Danne et al., 1994; Kreimyeret al., 1998; Vogt and Gulz, 1986; Petereit et al., 1991; Vogt et al., 1987; Santagati et al., 2008), labdane diterpenes (Demetzos et al., 1990; Anastasaki et al., 1999; Chinou et al., 1994; Demetzos et al., 1994) and polyphenolic glycosides (Demetzos et al., 1989). The studies on essential oil composition of Cistus species revealed the presence of oxygenated monoterpenes, sesquiterpenes, aromatics, oxygenated sesquiterpenes and traces of carbonyl compounds (Demetzos et al., 1997; Demetzos et al., 1995; Costa et al., 2009; Mastino et al., 2017; Maggi et al., 2016). Leaves of all Cistus species are covered with glands secreting resin and essential oil consisting mainly of terpenoids (Mastino et al., 2017).

In Jordanian traditional medicine Cistus species are used in the treatment of multiple ailments such as anti-inflammatory, gout, ulcers, gastrointestinal disorders, diabetes, as well as reduction of blood glucose (Farley and McNeilly, 2000; Al-Khalil, 1995; Yesilada et al., 1999). Previous studies on essential oil composition in Cistus salviifolius, and Cistus creticus showed that they are rich in oxygenated diterpenes such as manoyl oxide, labd-13-en-8-yl acetate, and 13-epi-manoyl oxide, as well as in oxygenated sesquiterpenes such as viridiflorol, caryophyllene oxide, vitispirane, and bulnesol and hydrocarbon sesquiterpenes such as α-cadinene and δ-cadinene (Demetzos et al., 1997; Demetzos et al., 1995; Maggi et al., 2016).

As part of our continuous effort in investigating the chemical composition of essential oil and antioxidant activity of medicinal plants (Al-Qudah, 2013, 2016; Al-Qudah et al., 2014, 2018). The objective of the present work is to investigate the chemical composition of essential oils extracted from fresh flower buds and leaves of C. creticus and C. salviifolius using hydro-distillation and solid-phase micro-extraction (SPME) followed by GC and GC–MS analysis. Also, we have examined the antioxidant activities of the oils and crude extract fractions prepared from these plants.

2

2 Materials and methods

2.1

2.1 Chemical reagents

Chemical reagents used in this study were: helium (high purity 99%), n-alkanes (C7-C30) GC grade AR., 5% diphenyl, 95% dimethyl polysiloxane (DP-5) grade AR, 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, a-tocopherol, methanol, potassium persulfate, ferrous chloride, ferrozine, Sodium sulfate, EDTA (Ethylenediaminetetraacetic acid) and internal reference compounds. All chemicals used in this investigation were purchased from Sigma-Aldrich (Buchs, Switzerland).

2.2

2.2 Sampling and materials

The present study was carried out in April 2016. Samples of fresh flower buds and leaves of Cistus creticus and C. salviifolius were collected at maturity stage from different sites in Ajloun and Jerash areas in the northern part of the Hashemite Kingdom of Jordan. A voucher specimen of each has been deposited in the herbarium of the Department of Biological Sciences-Yarmouk University, Irbid, Jordan, C. creticus L. (YU/1/CC/1001) and C. salviifolius L. (YU/1/CC/1002).

2.3

2.3 Preparation of essential oils

The essential oils from fresh leave and flower buds of C. creticus and C. salviifolius were isolated as described (Al-Qudah, 2013; Al-Qudah et al., 2014, 2018). Fresh leaves and flower buds of C. creticus and C. salviifolius (200 g), were chopped into small parts and hydrodistillation for 4 h using a Clevenger-type apparatus. Subsequently, oils were dried over anhydrous sodium sulfate and immediately stored in GC-grade n-hexane at 4 °C until the analysis by gas chromatography/mass spectrometry (GC/MS) is carried out.

2.4

2.4 Solid phase micro-extraction (SPME) of volatile oils

The SPME experiments were performed using the fiber assemblies (PDMS/DVB; df 65 μm, length 1 cm) for manual sampling (Supelco, USA). About 0.1 g of freshly leave and flower buds of C. creticus and C. salviifolius were put into 5.0 mL amber glass vials, tightly capped with PTFE-coated septa, and SPME extraction was performed for 2.0 min at RT. Desorption of the analytes was carried out at 240 °C for 60 s. Each sample was repeated twice.

2.5

2.5 GC and GC–MS analysis

For chemical identification, a small portion of 1 µL extracted oils were diluted to 10.0 μL with GC grade n-hexane, then analyzed by GC–MS (Model Varian Chrompack CP-3800 GC/MS, Saturn, Netherlands) system, equipped with a DB-5 GC capillary column (5% diphenyl, 95% dimethyl polysiloxane, 30 m × 0.25 mm i.d., 0.25 μm film thicknesses). For mass spectroscopy detection, an electron ionization mode of 70 eV energy was used with a specific mass range. The flame ionization detector (FID) and injector temperature in the MS source were set at 180 °C. The temperature column was also programmed from 60 °C for 1 min (isothermal) to 246 °C at a constant rate of 3 °C/min, with the lower and upper temperatures being held for 3 min. The carrier gas was helium and was set at a flow rate of 0.9 mL/min. Quantitative analysis was performed using the Hewlett-Packard HP-8590 gas chromatography (Hewlett-Packard Co., Palo Alto, CA, USA) equipped with a split-splitless injector (split ratio 1:50) and a flame ionization detector (FID) was used. The device was connected to a 5% diphenyl, 95% dimethyl polysiloxane (optima-5) fused silica capillary column (30 m × 0.25 mm, 0.25 μm film thickness) (Varian Capillary Column) and under the same conditions described for the GC/MS analysis part.

2.6

2.6 Identification of the chemical constituents

A hydrocarbon mixture of n-alkanes (C7–C30) was analyzed separately under similar chromatographic conditions using the same DP-5 column. The identification of separated volatile components was achieved by matching their recorded mass spectra with the built-in library spectra (NIST, Gaithersburg, MD, USA, and Wiley Co., Hoboken, NJ, USA) and by comparing their calculated Kovats retention index (KI) relative to (C7–C30) n-alkanes values measured with the column of identical polarity. Further identification of major components of the extracts was confirmed by injecting authentic standard reference compounds on the same chromatography column and comparing their retention times with those of their counterparts from the oil samples.

2.7

2.7 Preparation of crude fractions

The fresh plants material from the flowers and leaves of C. salviifolius and C. creticus were air-dried in the shade for 1 month as previously described (Al-Qudah et al., 2018). Afterward, they were ground to fine powders and defatted with petroleum ether in Soxhlet extractor. After this, the plant residue was extracted in the same apparatus in methanol. The obtained alcoholic gummy residue was then partitioned between CHCl3 and H2O (1:1). The dried chloroform residue was then subjected to partitioning between 10% aqueous methanol (aq.MeOH) and hexane. The polar organic compounds were extracted from water by n-butanol. The different fractions obtained were assayed for their total phenol contents (TPC), total flavonoid contents (TFC) and in vitro and antioxidant activities.

2.8

2.8 Determination of total flavonoid (TFC)and phenol contents (TPC)

The total flavonoids contents of the crudes (Aq. MeOH, Butanol and water extracts) from the flowers and leaves of C. salviifolius and C. creticus were determined by the Folin-Ciocalteu method (Al-Qudah et al., 2018). 1.0 mL aliquot from the stock solution (1 mg/mL) of each extract, diluted in 4.0 mL distilled water, were introduced into a 10.0 mL volumetric flask, to which 0.30 mL of sodium nitrite solution (5% NaNO2, w/v) were added. The resulting mixture was allowed to stand for 5 min and then, 0.30 mL of aluminum chloride solution (10% AlCl3, w/v) was added. The resulting solution was incubated for another 6 min after which, 2.0 mL of 1.0 M NaOH solution was added and the final volume was adjusted to 10.0 mL with distilled water. After 15 min, the absorbance was measured at the wavelength, λ, of 510 nm. Methanol was used as blank. The total flavonoids content is expressed, in mg/g, as mass of quercetin with respect to the mass of the dry extract.

The phenols' contents of the crudes from the flowers and leaves of C. salviifolius and C. creticus was determined by aluminum chloride assay (Al-Qudah et al., 2018) 0.5 mL aliquot from the stock solution (1 mg/mL) of each extract was treated with 2.5 mL of Folin–Ciocalteu reagent (2 N) (diluted ten folds) and 2 mL of Na2CO3 (75 g/L). The mixture was allowed to stand at room temperature for 15 min and the absorbance was then recorded at the wavelength, λ, of 765 nm. Methanol was used as a blank solution. The total phenol content in the different extracts of both plants was expressed as mg gallic/g dry extract.

2.9

2.9 Determination of antioxidant activity

Also, the antioxidant activity of the crude and essential oils was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) radical scavenging, hydrogen peroxide scavenging and ferrous ion chelating activity assay (Al-Qudah, 2013, 2016; Al-Qudah et al., 2014, 2018).

2.9.1

2.9.1 DPPH free radical scavenging activity

Briefly, to 1.0 mL of 0.1 mM DPPH• solution (dissolved in MeOH), 2.0 mL of various concentrations (0.005–0.5) mg/mL of each methanolic extract solutions were added. The solutions were allowed to stand at room temperature in dark for 30 min and then the absorbance of the solutions was measured at the wavelength, λ, of 517 nm against blank samples using UV–VIS spectrophotometer.

2.9.2

2.9.2 ABTS radical scavenging assay

The ABTS•+ cation radical solution was prepared by reaction of similar quantities of 7 mM of ABTS and 2.4 mM of potassium persulfate (K2S2O8) solution and allowed to react for 16 h at R.T in the dark. Before use; this solution was diluted with methanol to get an absorbance of 0.75 ± 0.02 at 734 nm. The reaction mixture comprised 3.0 mL of ABTS•+ solution and 1 mL of the extracts at various concentration (0.005–0.50) mg/mL. The absorbance of the mixture was measured at the wavelength, λ, of 734 nm by using a UV–Vis spectrophotometer. The blank was run in each assay and all measurements were done after at least 5 min.

2.9.3

2.9.3 Hydroxyl radical assay

1 mL of different concentrations of the extract solution in methanol (0.005–0.50) mg/mL were added to a 0.5 mL FeSO4 solution (6 mM). Then, 0.5 mL of 6 mM H2O2 was added to the mixture. Subsequently, after shaking and incubation of the reaction mixture for 10 min at room temperature, a 1 mL of 6 mM salicylic acid was added and further incubated for 30 min at room temperature. The absorbance was at the wavelength, λ, of 510 nm.

2.9.4

2.9.4 Ferrous ion chelating (FIC) effect

3.0 mL of methanol solution containing the different concentrations of crudes and essential oils (0.005–0.50) mg/mL was added to a 0.25 mL of 2 mM ferrous chloride (FeCl2) reagent. Subsequently, a 0.2 mL of 5 mM ferrozine solution was added to the mixture and allowed to stand at r.t. for 10 min after vigorous shaking. Then, the reduction in the absorbance of the visible radiation of red color was measured spectrophotometrically at 562 nm. The scavenging activity of the tested extracts and essential oils was compared to those of the positive control's ascorbic acid, α- tocopherol, and EDTA under similar conditions.

The percentage of scavenging activity of the tested extracts and essential oils was calculated using the equation: Scavenging activity (%) = (Ac − As/Ac) × 100; where Ac is the absorbance of the control and As is the absorbance in the presence of either extracts or control substance.

Non-linear regression analysis of GraphPad Prism 6 (GraphPad Software, San Diego, California, USA) was applied for the determination of IC50 in all of the antioxidant assays from the sigmoidal curve which was obtained by plotting the percentages of scavenging relative to the control versus logarithmic concentration of test compound. Each concentration was tested three times in 3 independent experiments.

2.10

2.10 Data analysis

MAT LAB 7.0.4 (Math Works, MA, USA) with PLS Toolbox 4.0 (Eigenvector Research, Inc, WA, and the USA) software were used for data processing and PCA analysis.

3

3 Results and discussion

3.1

3.1 Essential oil composition of flowers and leaves from Cistus salviifolius

The essential oils yield of leaves and flowers from C. salviifolius was 0.05% w/w and 0.03% w/w, respectively. Analysis of extracted oils from leaves and flowers of genus salviifolius was achieved by GC and GC–MS. Also, the 48 and 17 constituents which represent 99.9% and 99.8% of the oils obtained by hydrodistillation were identified, respectively (Table 1).

Table 1 Chemical composition of essential oils from flowers and leaves of C. salviifolius L. and C. creticus L. obtained by SPME and hydrodistillation.
No. KI (reported) KI (Exp.) Compound % Area
C. salviifolius L.		 % Area
C. creticus L.		 Method of identification
SPME Hydrodistillation SPME Hydrodistillation
Flowers Leaves Flowers Leaves Flowers Leaves Flowers Leaves
1 821 826a 2E-Octene 1.1 – – – 0.1 – 0.1 – MSb, RI,
2 830 835 2E-Hexenal 3.4 0.4 – 0.9 – – 2.8 1.3 MS, RI
3 856 863 E-Salvene – 2.3 – – – – 0.1 – MS, RI
4 888 895 santene 0.7 3.9 – – – – 0.1 – MS, RI
5 901 903 Ethyl pentanoate 1.0 – – – – – – – MS, RI
6 926 926 tricyclene 0.3 0.2 – – 0.8 0.7 – – MS, RI
7 939 934 α-pinene 12.8 11.9 – 0.3 25.9 19.4 – – MS, RI, RCc
8 952 951 α-fenchene 3.1 1.4 – – 0.8 – – MS, RI
9 954 952 camphene – – – 12.0 9.3 – – MS, RI
10 975 973 sabinene 0.9 0.8 – – 0.1 – – – MS, RI
11 979 984 β-pinene 7.0 5.2 – – 8.5 6.3 – – MS, RI, RC
12 990 990 myrcene – – – 3.0 7.5 – – MS, RI
13 1004 1007 p-mentha-1(7),8-diene – – – 0.8 0.8 – – MS, RI
14 1017 1023 α-terpinene 0.3 0.4 – – 0.7 0.6 – – MS, RI
15 1024 1029 p-cymene 4.8 5.7 – – 0.6 0.9 – – MS, RI
16 1031 1034 δ-3-carene 15.7 15.5 – – 6.4 5.9 – – MS, RI, RC
17 1050 1046 E-β-ocimene 0.7 0.5 – – 0.1 0.3 – – MS, RI
18 1059 1061 γ-terpinene 1.7 0.6 – – 1.6 1.2 – – MS, RI
19 1070 1064 cis-sabinene hydrate 0.6 0.4 – – – – MS, RI
20 1088 1087 terpinolene 0.2 0.3 – – 0.7 0.7 – – MS, RI
21 1091 1091 p-cymenene 0.7 – – – – – – – MS, RI
22 1096 1101 linalool 0.7 0.8 – – – – – – MS, RI
23 1100 1106 n-Undecane – 0.7 – – – – 0.6 MS, RI,
24 1100 1107 n-Nonanal 0.3 0.5 0.4 – – – – – MS, RI
25 1102 1108 cis-thujone 0.3 – – – – – – – MS, RI
26 1146 1147 camphor 0.2 – – 3.0 – – – – MS, RI
27 1177 1180 terpinen-4-ol – – – – 0.1 0.4 0.8 MS, RI
28 1188 1194 α-terpineol – – – – – 0.1 2.2 MS, RI
29 1200 1200 n-Dodecane 0.5 0.4 – – – – – – MS, RI
30 1201 1206 n-Decanal 0.4 0.7 – – – – – – MS, RI
31 1225 1232 citronellol – 2.0 – 0.4 – 0.1 – – MS, RI
32 1248 1249 Benzeneacetic acid, ethyl ester – – – 0.4 – – – – MS, RI
33 1258 1258 carvenone – – – 11.2 – – – – MS, RI, RC
34 1265 1264 cis-chrysanthenyl acetate – – – 0.2 – – – – MS, RI
35 1285 1288 bornyl acetate – – – 0.7 – – – – MS, RI
36 1299 1298 Z-Methyl cinnamate – – – 0.3 – – – – MS, RI
37 1300 1305 n-tridecane 0.2 0.5 – – – – – – MS, RI
38 1346 1346 Benzyl butanoate – – – 0.5 – – – – MS, RI
39 1351 1353 α-cubebene 1.4 1.0 – – 10.4 12.0 0.8 4.2 MS, RI, RC
40 1369 1356 eugenol – – – – – – 10.2 – MS, RI
41 1370 1360 silphiperfol-5,7(14)-diene – – – – 0.3 0.2 MS, RI
42 1371 1367 cyclosativene 0.4 1.3 – 0.3 – – – – MS, RI
43 1376 1374 α-copaene 1.1 3.3 – – – – – – MS, RI
44 1381 1375 daucene – – – 5.6 4.0 0.3 – MS, RI
45 1377 1383 Z-Ethyl cinnamate – – – 11.3 0.7 0.5 – – MS, RI, RC
46 1378 1386 E-Methyl cinnamate – – – 1.3 – – – – MS, RI
47 1388 1388 β-cubebene 0.3 0.3 – – 1.2 1.6 0.6 2.8 MS, RI
48 1398 1398 methyl eugenol – – – – – – 0.5 – MS, RI
49 1400 1400 n-Tetradecane 0.2 0.4 – – – – – – MS, RI
50 1409 1404 α-gurjunene – 0.4 – – 0.3 0.3 – – MS, RI
51 1419 1417 E-caryophyllene 2.9 10.4 0.58 1.6 4.4 8.4 0.6 2.7 MS, RI, RC
52 1420 1427 β-copaene – – – – 0.2 0.1 MS, RI
53 1450 1446 cis-muurola-3,5-diene 0.2 0.5 – – 0.6 0.6 0.4 1.4 MS, RI
54 1454 1453 α-humulene – 1.0 – 0.4 0.3 0.5 – – MS, RI
55 1460 1457 Allo-aromadendrene 0.6 1.4 – 0.4 0.1 – – – MS, RI
56 1467 1462 E-Ethyl cinnamate – – 0.66 17.5 – – – – MS, RI
57 1473 1467 drima-7,9(11)-diene – – – – – 0.3 0.8 3.1 MS, RI
58 1477 1469 trans-cadina-1(6),4-diene 0.4 0.6 – – 1.7 1.5 0.8 2.5 MS, RI
59 1480 1472 γ-muurolene 0.3 0.8 – – 0.3 0.3 – – MS, RI
60 1485 1478 germacrene D 0.4 – – 0.1 0.1 0.1 – – MS, RI
61 1484 1488 γ-amorphene – 0.4 – – 0.1 – 0.5 1.5 MS, RI
62 1492 1489 selinene 0.4 – – 0.5 1.7 2.2 – – MS, RI
63 1493 1489 trans-muurola-4(14),5 diene – – – – 0.5 0.6 0.8 2.8 MS, RI
64 1500 1495 n-Pentadecane 0.3 0.5 – – 0.7 0.9 – – MS, RI
65 1500 1498 bicyclogermacrene – – – 2.39 – – – – MS, RI
66 1500 1500 α-muurolene 0.4 1.5 – – 0.4 0.3 – – MS, RI
67 1505 1503 (E,E)-α-farnesene 4.8 1.2 0.8 – 0.1 – – – MS, RI,
68 1511 1508 δ-amorphene – – – – 0.2 0.1 – – MS, RI
69 1513 1510 γ-cadinene 0.4 1.8 – – 0.1 – 3.8 10.8 MS, RI
70 1523 1515 δ-cadinene 1.8 4.5 – – 4.2 3.8 0.9 2.1 MS, RI
71 1529 1520 zonarene 0.3 0.4 – – 1.5 1.3 – – MS, RI
72 1534 1529 trans-cadina-1(2),4-diene 0.4 0.4 – – 1.2 1.5 0.5 1.5 MS, RI
73 1532 1534 Z-nerolidol – – – – 0.1 0.2 0.4 – MS, RI
74 1538 1536 α-cadinene – 0.4 – – – – – – MS, RI
75 1566 1569 3Z-hexenyl benzoate 0.3 1.1 – 0.5 – – – – MS, RI
76 1571 1572 dendrolasin 0.8 0.5 – – – – – – MS, RI
77 1583 1582 caryophyllene oxide – 0.3 – 0.9 0.2 0.5 0.4 – MS, RI
78 1587 1593 davanone – – – 4.77 – – – – MS, RI
79 1619 1605 1,10-di-epi-cubenol 0.4 0.7 – – 0.2 – 3.9 5.3 MS, RI
80 1625 1613 citronellyl pentanoate 0.2 – – – – – – – MS, RI
81 1640 1634 caryophylla-4(14),8(15)-diene-5,α-ol – – – – – 0.2 – 0.4 MS, RI
82 1642 1640 α-muurolol (Torreyol) – – – – 0.2 – 1.9 3.1 MS, RI
83 1650 1652 7-epi-α-eudesmol – 0.4 – – 0.1 0.3 – – MS, RI
84 1668 1663 E-citronellyl tiglate 0.4 – – – – – – – MS, RI
85 1672 1677 n-Tetradecanol 2.5 – – – – – – – MS, RI
86 1845 1855 Z-ternine 0.3 – 2.0 0.4 – – – – MS, RI
87 1878 1879 cubitene – – – – 0.1 – 1.2 2.5 MS, RI
88 1894 1896 catalponone – – – – – – 2.7 – MS, RI
89 1900 1900 n-Nonadecane – – 0.4 – – – – – MS, RI,
90 1905 1907 isopimara-9(11),15-diene – – – 0.3 – – – – MS, RI
91 1922 1916 totarene – – – 0.3 – – 0.2 – MS, RI
92 1921 1922 methyl hexadecanoate – – 0.6 – MS, RI
93 1934 1929 isohibaene – – – 0.3 – – – – MS, RI
94 1938 1953 cembrene – – – 1.5 – – – – MS, RI
95 1948 1957 3E-cembrene A – – 0.5 0.1 – – 0.2 – MS, RI
96 1960 1959 nootkatin – – – – 0.2 – 0.2 – MS, RI
97 1974 1979 dolabradiene – – – – – – 0.5 – MS, RI
98 1974 1987 sclarene – – – 0.5 – – 7.2 13.3 MS, RI, RC
99 1978 1991 bifloratriene – – – 0.4 – – – – MS, RI
100 1988 1996 1-Eicosene – – – – – – 0.3 – MS, RI
101 2003 2007 manoyl oxide 4.5 0.5 4.1 13.2 0.9 2.7 19.5 27.0 MS, RI, RC
102 2010 2017 epi-13-manoyl oxide 0.4 – – – – – – – MS, RI
103 2056 2053 abietatriene 0.4 – 4.4 12.3 – – 12.0 – MS, RI
104 2057 2060 manool – – – – – 0.1 1.1 2.1 MS, RI
105 2077 2082 octadecanol – – – – – – 1.2 4.0 MS, RI
106 2087 2088 abietadiene – 1.0 – 0.9 – – 3.3 – MS, RI
107 2100 2098 n-heneicosane – – – – – – 1.2 – MS, RI
108 2116 2110 laurenan-2-one – – – 0.7 – – – – MS, RI
109 2125 2118 methyl octadecanoate – – 9.2 – – – – – MS, RI
110 2133 2125 nezukol – – 0.3 0.9 – – – – MS, RI
111 2141 2136 osthole 1.1 – – 0.6 – MS, RI,
112 2184 2175 incensole acetate – – 2.6 – – – 0.3 – MS, RI
113 2184 2180 sandaracopimarinal – – 0.5 0.5 – – – – MS, RI
114 2200 2198 n-docosane – – – – – 0.1 – MS, RI
115 2203 2204 α-santonine – – – – – 1.2 – MS, RI
116 2210 2212 phyllocladanol – – – 0.6 – – – – MS, RI
117 2237 2235 7-α-hydroxy-manool – – – 0.9 – – – MS, RI
118 2241 2242 Z-isoeugenyl phenylacetate – – – – – 1.2 – MS, RI
119 2275 2270 dehydroAbietal – – 0.4 0.4 – – – MS, RI
120 2310 2305 isopimarol – – – 0.6 – – 0.4 – MS, RI
121 2313 2310 abietal – – – 0.3 – – – – MS, RI
122 2314 2317 trans-totarol – – – 0.7 – – – – MS, RI
123 2332 2328 trans-ferruginol – – – 1.4 – – – – MS, RI
124 2360 2360 3-α-acetoxy-manool – – – 1.0 – – 5.9 5.6 MS, RI
125 2368 2371 dehydro abietol – – 59.0 0.9 – – – – MS, RI
126 2401 2408 abietol – – 12.7 0.5 – – – – MS, RI
127 2422 2420 labd-13E-8,15-diol – – 1.4 – – – – – MS, RI
Total 85.1% 92.1% 99.9% 99.8% 98.9% 98.0% 96.7% 100%
Monoterpenes Hydrocarbon 131 (48.8)2 13 (48.9) 0 1 (0.3) 13 (61.2) 13 (53.5) 2 (0.2) 0
Oxygenated monoterpenes 4 (1.8) 3 (3.2) 0 5 (15.5) 1 (0.1) 3 (0.6) 2 (3.0) 0
Sesquiterpenes Hydrocarbon 17 (16.5) 19 (31.6) 2 (1.4) 7 (5.7) 23 (34.2) 20 (38.6) 12 (11.1) 11 (35.2)
Oxygenated Sesquiterpenes 4 (1.9) 4 (1.8) 0 2 (5.6) 6 (0.9) 4 (1.0) 6 (8.1) 3 (8.7)
Diterpenes Hydrocarbon (1) 0.4 1 (1.0) 2 (4.9) 9 (16.6) 1 (0.1) 0 7 (24.7) 2 (16.0)
Oxygenated Diterpenes 4 (4.9) 1 (0.5) 7 (81.0) 15 (23.0) 1 (0.9) 2 (2.8) 6 (27.9) 3 (34.7)
Esters 2 (1.5) 1 (1.1) 2 (9.8) 7 (31.8) 1 (0.7) 1 (0.5) 2 (1.8)
Others 10 (9.3) 8 (4.0) 3 (2.8) 2 (1.3) 2 (0.8) 1 (0.9) 10 (19.9) 2 (5.3)
a KI (Exp.) refers to the Kovats retention index experimentally calculated using C7 – C30 n-alkanes on HP-5MS capillary column.
b MS, identification by mass spectrum (NIST and our local generated libraries were used for all MS comparisons).
c RC, the identity of the major components was confirmed by injecting authentic reference compounds on the same chromatography column.
1 Number of compounds.
2 % area of compounds.

The total high percentage of principal oxygenated diterpenes oil in flowers (81.0%), associated with dehydro abietol (59.0%) and abietol (12.7%), was detected. Esters were found in a percentage of (9.9%) associated with methyl octadecanoate (9.2%) and E-ethyl cinnamate (0.7%). The analysis of the components of hydrodistilled oil from flowers contained two diterpene hydrocarbons (4.9%) and two sesquiterpenes (1.4%) as seen in Table 1. The essential oil of the leaves has E-ethyl cinnamate (17.5%), manoyl oxide (13.2%), abietatriene (12.3%), Z-ethyl cinnamate (11.3%) and carvenone (11.2%), which seem to be the major components of the essentail oil from leaves of C. salviifolius.

The volatiles from the flowers and leaves of C. salviifolius were extracted and collected by the SPME method for analysis using the GC–MS technique, as mentioned in the literature (Saleh et al., 2017). The analysis of volatiles from were identified 53 components from flowers and 50 components from leaves, with a percentage of 85.1% up to 92.1% of the total composition, respectively. The results of the analysis are shown in Table 1. Table 1 shows flower oil consisting of 13 monoterpene hydrocarbons (48.8%), associated with δ-3-carene (15.7%), α-pinene (12.8%) and β-pinene (7.0%) as major constituents. The identification reveals 17 sesquiterpene hydrocarbons (16.5%), 10 other compounds (9.3%), two oxygenate diterpenes (4.9%), four oxygenated monoterpenes (1.8%), four oxygenated sesquiterpenes (1.9%), two ester compounds (1.5%) and one diterpene hydrocarbon (0.4) from flower oil of C. salviifolius, which indicates that there is a high similarity in active essential oil components with C. creticus L. A different set of extracted oils was detected from leaves, which gave different components including 13 monoterpene hydrocarbons (48.9%), associated with δ-3-carene (15.5%) and α-pinene (11.9%) along with 19 sesquiterpene hydrocarbons (31.6%), associated with E-caryophyllene (10.4%), three oxygenated monoterpenes (3.2%), 8 other compounds (4.0%) and four oxygenated sesquiterpenes (1.8%) all of which may be considered as major compounds.

3.2

3.2 Essential oil composition of flowers and leaves from C. creticus

The distilled essential oils from leaves and flower buds are characterized to range from yellowish to yellow color and the yields were 0.02% w/w and 0.01% w/w, respectively. In terms of chemical structure, volatiles and essential oil components are classified into (8) classes associated with the calculated Kovats indices and mass spectra compared to those stored in the GC–MS built in libraries. Table 1 represents the GC and GC–MS analysis of the C. creticus oils from leaves and flower buds, where the results led to the identification of 21 and 47 constituents (100%) and (96.7%), respectively.

Experimentally, flower oil contents are manoyl oxide and 3-α-acetoxy-manool with a percentage of (19.5%) and (5.9%), respectively, whereas oxygenated diterpenes (27.9%) represent the major content of the total oil weight. Diterpenes (24.7%), abietatriene (12.0%) and sclarene (7.2%) were found to be the major components of the oils. The percentage composition of flower oil constituents obtained by the hydrodistillation method are summarizes in Table 1. It contains eight categories of volatiles with six oxygenated sesquiterpenes (8.1%), twelve sesquiterpene hydrocarbons (11.1%), ten aliphatic compounds (19.9%), two oxygenated monoterpenes (3.0%) and two monoterpene hydrocarbons (0.2%).

In comparison, the essential oil of the leaves has manoyl oxide (27.0%), sclarene (13.3%), γ-cadinene (10.8%), and 3-α-acetoxy-manool (5.6%) as major components. Organic active components of volatile oils extracted from flowers and leaves of C. creticus L. were obtained by Solid Phase Micro-Extraction (SPME) method and GC–MS technique using the procedure mentioned in the literature (Saleh et al., 2017). Table 1 shows the results of volatile principle active components of C. creticus L., which was analyzed by using GC–MS techniques. The results led to the identification of forty-eight flower and forty-four leave constituents, amounting up to 98.9% and 98.0% of the total composition, respectively. There have been 13 monoterpene hydrocarbons (61.2%) in flower oil, associated with α-pinene (25.9%) and camphene (12.0%) as major constituents. Furthermore, the experimental analysis indicated twenty-three sesquiterpene hydrocarbons (34.2%), associated with α-cubebene (10.4%). Sex oxygenated sesquiterpenes (0.9%), one oxygenated diterpene (0.9%), one diterpene (0.1%), and two aliphatic compound (0.8%) are the main active compounds. Oil extracted from leaves contains 13 monoterpene hydrocarbons (53.5%), associated with α-pinene (19.4%), camphene (9.3%), 20 sesquiterpene hydrocarbons (38.6%), α-cubebene (12.0%) E-caryophyllene (8.4%), in addition to two oxygenated diterpenes (2.8%), four oxygenated sesquiterpenes (1.0%) and three oxygenated monoterpenes (0.6%) as major contents as seen in Table 1.

In comparison to the previously published data on the oil composition of C. criticus and C. salviifolius (Mastino et al., 2017; Morales-Soto et al., 2015), our results show significant differences in the concentrations of the reported main components. The volatile components of the aerial parts of C. salviifolius from Spain were reported camphor (43.86%), E-caryophyllene (19.26%), eucalyptol (19.14%) and (β-bourbonene) (13.27%) as mainly compounds (Morales-Soto et al., 2015). But the oil from the aerial parts of C. salviifolius from Sardinia showed a high quantity of manoyl oxide (Mastino et al., 2017). The chemical composition from the aerial parts of C. creticus from Torre Beregna showed a high quantity of linolenic (39.2%), hexadecanoic (14.7%) and linoleic (12.9%) (Maggi et al., 2016). Whereas from the essential oils of the leaves of C. creticus subsp. from Greece were rich in sesquiterpenes such as δ-cadinene (5.6%), a-cadinene (6.5%), bulnesol (6.3%), and viridiflorol (5.4%) (Demetzos et al., 1997). These changes in the essential oil compositions might arise from several factors such as geological, geographical, seasonal, and climatic (Perry et al., 1999).

3.3

3.3 Screening of the total phenolic and flavonoid contents and evaluation of antioxidant activity

Table 2 summarizes the total phenols in mg/g gallic acid for aq. MeOH, butanol and aqueous extracts, which varied among the different extracts of plant ranging from 111 to 183.8 mg/g gallic acid in flowers of C. salviifolius and from 196 to 283 mg gallic/g dry extract in flowers of C. creticus, as well as from 126 to 393 mg gallic/g dry extract in leaves of C. salviifolius. The screening of aq. MeOH fraction from flowers and leaves of C. salviifolius showed the highest phenolic and flavonoid compound concentrations, compared with aqueous methanol and water extracts. Whereas, the butanol extract from flowers and leaves of C. creticus. contained the highest phenolic and flavonoid concentrations concerning the two different extracts (mg quercetin/g of a plant).

Table 2 Total flavonoids and total phenolic content of crude extract fractions from flowers and leaves of C. salviifolius L. and C. creticus L.
Crude C. salviifolius L. C. creticus L.
Flowers Leaves Flowers Leaves
Total flavonoids
(mg quercetin/g of extract)		 Total phenol
mg gallic/g dry extract		 Total flavonoids
(mg quercetin/g of extract)		 Total phenol
mg gallic/g dry extract		 Total flavonoids
(mg quercetin/g of extract)		 Total phenol
mg gallic/g dry extract		 Total flavonoids
(mg quercetin/g of extract)		 Total phenol
mg gallic/g dry extract
aq. MeOH 393 ± 8 183.8 ± 0.1 313 ± 4 199 ± 2 288 ± 9 221 ± 5 261 ± 8 172 ± 3
BuOH 173 ± 6 149.5 ± 0.3 284 ± 5 187.0 ± 0.2 369 ± 9 283 ± 4 273 ± 8 182 ± 5
Water 125 ± 1 110.9 ± 0.4 223 ± 5 161.0 ± 0.6 217 ± 9 196 ± 2 208 ± 1 112.4 ± 0.6

Table 3 represents the evaluation results of antioxidant activity of essential oils and crude extracts from flowers and leaves of C. salviifolius and C. creticus using different methods described in (Al-Qudah, 2013, 2016; Al-Qudah et al., 2014, 2018). Weak scavenging effects are shown in all the different antioxidant assays. Generally, the oils from leaves of C. salviifolius and C. creticus exhibited a higher scavenging effect than the ones obtained from the flower parts. The antioxidant activities for the different extract fractions from flowers and leaves of C. salviifolius and C. creticus showed strong scavenging effects in all the different antioxidant assays. The aq. MeOH crude from flowers and leaves of C. salviifolius exhibited a higher scavenging effect than the other crudes. Butanol extracted from flowers and leaves of C. creticus showed a higher scavenging effect than the other crudes.

Table 3 IC50 ((mg/mL))values of essential oils and crude extracts from flowers and leaves of C. salviifolius L., C. criticus L. and standards by using DPPH, ABTS, hydroxyl, Ferrous Ion Chelating (FIC) methods.
C. salviifolius L. Parts Crude DPPH ABTS Hydroxyl FIC
Flowers Essentail Oil 0.15 ± 0.06 0.09 ± 0.02 0.36 ± 0.02 0.37 ± 0.02
aq.MeOH 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 0.04 ± 0.00
BuOH (1.6 ± 0.06) * 10−2 (1.5 ± 0.06) * 10−2 0.12 ± 0.04 0.07 ± 0.00
Water (1.5 ± 0.06) * 10−2 (1.9 ± 0.06) * 10−2 0.05 ± 0.00 0.04 ± 0.00
Leaves Essentail Oil 0.16 ± 0.01 0.13 ± 0.01 0.28 ± 0.05 0.29 ± 0.03
aq.MeOH (1.8 ± 0.06) * 10−2 0.01 ± 0.00 0.01 ± 0.00 0.11 ± 0.02
BuOH (2.4 ± 0.06) * 10−2 0.01 ± 0.00 0.11 ± 0.00 0.12 ± 0.00
Water (2.5 ± 0.06) * 10−2 0.01 ± 0.00 0.09 ± 0.01 0.14 ± 0.00
C. criticus L. Flowers Essentail Oil 0.36 ± 0.06 0.31 ± 0.01 0.61 ± 0.10 0.58 ± 0.07
aq.MeOH (1.5 ± 0.06) * 10−2 (1.2 ± 0.06) * 10−2 0.14 ± 0.01 0.21 ± 0.01
BuOH (2.3 ± 0.16) * 10−3 (6.0 ± 0.24) * 10−3 0.10 ± 0.00 0.10 ± 0.00
Water (6.3 ± 0.01) * 10−3 0.01 ± 0.00 0.15 ± 0.00 0.19 ± 0.00
Leaves Essentail Oil 0.33 ± 0.01 0.31 ± 0.11 0.57 ± 0.02 0.55 ± 0.07
aq.MeOH (2.4 ± 0.06) * 10−2 0.02 ± 0.00 0.06 ± 0.00 0.16 ± 0.01
BuOH (1.7 ± 0.06) * 10−2 (1.4 ± 0.06) * 10−2 0.03 ± 0.00 0.11 ± 0.00
Water (1.7 ± 0.06) * 10−2 (1.6 ± 0.06) * 10−2 0.04 ± 0.00 0.19 ± 0.00
Ascorbic acid (1.8 ± 0.06) * 10−3 (1.9 ± 0.06) * 10−3 (2.6 ± 0.03) * 10−3 (1.9 ± 0.02) * 10−3
α-tocopherol (2.3 ± 0.04) * 10−3 (1.8 ± 0.01) * 10−3 (2.8 ± 0.05) * 10−3 (2.9 ± 0.02) * 10−3
EDTA (1.3 ± 0.02) * 10−3 (2.2 ± 0.01) * 10−3

Principle component analysis (PCA), is a multivariate data analysis method that can be used for finding differences or similarities among a given dataset. In PCA, the score plot or model contains information about samples (objects), while the PCA loading plot involves variable information (Obeidat et al., 2014).

PCA has been applied to the data obtained from the mass spectrometry (Table 1) to investigate chemical composition differences between the two studied plants, or among the different organs within the plant using different extraction procedures. The first few PCs were studied; the best result was obtained upon having a two-dimensional PCA model that accounts for more than 45% of the total variation in the data set using the first and the fifth PCs. Fig. 1 represents the resulted score PCA model, as it appears in this figure the leaves and flowers from each plant were almost overlapped when extracted with the SPME method. In predicting the PCA score model, the greater the distance between the points in the model the more different they are (Al-Qudah et al., 2018). Therefore, it can be concluded that flowers and leaves of the same plant produced almost similar chemical compounds when extracted with the SPME method. On the other hand, the large distance in the PCA model between the points that represent SPME extracts of C. salviifolius. (leaves and flowers) and that of C. creticus. (leaves and flowers), reflects the variance in chemical composition between the two plants when extracted via the SPME method. Discriminating among plants based on chemical composition extracted via the hydrodistillation method was not possible using the score PCA model (Fig. 1) indicating that almost similar compounds were extracted from the leaves and flowers in the two studied plants when extracted with the hydro-distillation method.

The PCA score plot for chemical composition of the extracts obtained by hydrodistillation HD and solid phase micro-extraction (SPME), from flowers and leaves of C. creticus and C. salviIfolius.
Fig. 1
The PCA score plot for chemical composition of the extracts obtained by hydrodistillation HD and solid phase micro-extraction (SPME), from flowers and leaves of C. creticus and C. salviIfolius.

Further investigations of the PCA loading model (Fig. 2) showed that the responsible compound for distinguishing between C. saliifolius. and C. creticus. on the first PC was α-Pinene, while the compound δ-3-Carene had the greatest impact on the variation in the fifth PC, and for a less extent come to the compounds Camphene and α-Cubebene, respectively.

The PCA score plot for chemical composition of the extracts obtained by hydrodistillation HD and solid phase micro-extraction (SPME), from flowers and leaves of C. creticus and C. salviIfolius.
Fig. 2
The PCA score plot for chemical composition of the extracts obtained by hydrodistillation HD and solid phase micro-extraction (SPME), from flowers and leaves of C. creticus and C. salviIfolius.

4

4 Conclusions

The extraction methods used, such as solid-phase micro-extraction method, showed that monoterpenes and sesquiterpenes are the most volatile constituents. However, hydrodistillation method indicated that diterpenes and oxygenated diterpenes are the principal classes represented in the volatile constituents of the essential oils. There are differences in chemical composition between the two organs, leaves, and flower buds in both plants. These oils showed weak antioxidant activity when compared to the positive controls, α-tocopherol, ascorbic acid, and EDTA, while crude extracts (aq. MeOH, butanol, water) showed good antioxidant activity, which could advocate their use as a source of natural antioxidants. PCA score plot showed that compounds extracted from the leaves and flowers of each plant were different upon extracted with SPME, while almost no significant difference between the two plants (C. salviifolius and C. creticus) was detected when extracted using hydrodistillation method.

Acknowledgments

This work was generously supported by the Deanship of Scientific Research and Graduate Studies at Yarmouk University, Irbid, Jordan.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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