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. 2023 May 22;28(10):4238. doi: 10.3390/molecules28104238

Exploring Echinops polyceras Boiss. from Jordan: Essential Oil Composition, COX, Protein Denaturation Inhibitory Power and Antimicrobial Activity of the Alcoholic Extract

Hazem S Hasan 1, Ashok K Shakya 2,*, Hala I Al-Jaber 3,*, Hana E Abu-Sal 3, Lina M Barhoumi 3
Editor: Vincenzo De Feo
PMCID: PMC10223352  PMID: 37241978

Abstract

In this article, we present the first detailed analysis of the hydro-distilled essential oil (HDEO) of the inflorescence heads of Echinops polyceras Boiss. (Asteraceae) from the flora of Jordan, offering observations at different growth (pre-flowering, full-flowering and post-flowering) stages. Additionally, we investigated the methanolic extract obtained from the aerial parts of the plant material at the full flowering stage in order to determine its inhibitory activity in terms of COX and protein denaturation and evaluate its antimicrobial effects against S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria. Performing GC/MS analysis of HDEO, obtained from the fresh inflorescence heads at the different growth stages, resulted in the identification of 192 constituents. The main class of compounds detected in these three stages comprised aliphatic hydrocarbons and their derivatives, which amounted to 50.04% (pre-flower), 40.28% (full-flower) and 41.34% (post-flower) of the total composition. The oils also contained appreciable amounts of oxygenated terpenoids, primarily sesquiterpenoids and diterpenoids. The pre-flowering stage was dominated by (2E)-hexenal (8.03%) in addition to the oxygenated diterpene (6E,10E)-pseudo phytol (7.54%). The full-flowering stage primarily contained (6E,10E)-pseudo phytol (7.84%), β-bisabolene (7.53%, SH) and the diterpene hydrocarbon dolabradiene (5.50%). The major constituents detected in the HDEO obtained at the post-flowering stage included the oxygenated sesquiterpenoid intermedeol (5.53%), the sesquiterpene hydrocarbon (E)-caryophyllene (5.01%) and (6E,10E)-pseudo phytol (4.47%). The methanolic extract obtained from air-dried aerial parts of E. polyceras displayed more COX-2 inhibition than COX-1 inhibition at a concentration level of 200 µg/mL. The extract exhibited a capacity to inhibit protein denaturation that was comparable with respect to the activity of diclofenac sodium and displayed moderate levels of antimicrobial activity against both bacterial species. The current results demonstrate the need to perform further detailed phytochemical investigations to isolate and characterize active constituents.

Keywords: Echinops polyceras, essential oil, GC/MS, aliphatic hydrocarbons and their derivatives, COX-1, COX-2, antimicrobial activity, protein denaturation

1. Introduction

The Asteraceae family, commonly known as Compositae, is among the largest families in the plant kingdom, with almost 1620 genera and 23,600 species distributed in almost all habitats worldwide, except underwater. Plants belonging to this family are commonly described as herbs, shrubs and trees. Echinops, one of the genera belonging to this family, is known to comprise approximately 120–130 distinct species [1,2,3]. Echinops plants, described as perennials, annuals and biennials, are known to grow wild in Eastern and Southern Europe, including the Mediterranean region, as well as in North Africa, the Afrotropical realm and the continent of Asia [1,4].

Several species of the Echinops genus have been long used in traditional medicine for the treatment of many ailments, primarily those illnesses related to inflammation, pain and fever [5,6]. Members of this genus have been extensively utilized in Ethiopia to treat of various ailments such as migraine, heart pain, diarrhea, hemorrhoid and intestinal worms [7]. In Ethiopian herbal medicine, chewing the roots of Echinops kebericho is prescribed to alleviate stomachache in humans, while roots decoction is employed to cure intestinal diseases in cattle [8]. Additionally, the flower heads and roots of many Echinops species find use in Arabian, Cameroonian, Chinese and Indian folk medicine to treat renal disorders and kidney stones [9], reduce asthma attacks [10], stimulate milk secretion [11], and alleviate sexual disability [12]. This wide range of bioactivities has been basically attributed to the wide spectrum of secondary metabolites, including the common members of this genus, i.e., terpenoids, sterols, flavonoids, alkaloids and thiophenes [1,13].

There are three Echinops species reported to grow wild in the flora of Jordan. These include Echinops polyceras Boiss., Echinops spinosissimus Turra. and Echinops glaberrimus DC. [14]. E. polyceras Boiss (Synonym: E. blancheanus Boiss., E. spinosus auct. non L.) is the most common species reported to grow wild in the flora of Jordan. This plant is commonly known as globe thistle. This species can best be described as a perennial, spiny, hairy plant that is 60–100 cm long. The leaves are long, dissected with spiny segments; their flower heads are spherical, spiky, 4–5 cm in diameter and characterized by their pale blue color. In the Arab region, primarily in Jordan, this plant is known as “chouk el Jemel” and is known to grow wild in waste places and hills of Irbid, Jarash, Al-Salt, Amman, Madaba, Al-Karak, and Al-Tafila. Flowering occurs during the period extending from July to October [13]. The plant is also reported to grow wild in the Mediterranean neighboring countries including Iraq, Lebanon, Syria, Jordan, Palestine and Saudi Arabia, as well as those located along the coast of the North African coast of the Mediterranean Sea.

E. polyceras has been applied in the folk medicine of many cultures in order to treat a wide variety of ailments. In the Mediterranean region, a decoction prepared from the roots is used to treat renal diseases and kidney stones [9]. In Saudi Arabia, the plant is used for the treatment of gastric pain, indigestion and spasmolytic problems [15]. In Algeria, the plant has been described as finding use in the treatment of dysmenorrhea and prostatism [16]. Previous phytochemical screening studies performed on this species revealed its richness in different classes of secondary metabolites, including flavonoids, sterols, terpenoids and quinolone alkaloids [1,17]. Despite the importance of this species, a thorough literature survey revealed that the plant has not previously been investigated in Jordan, neither for its phytochemical constituents (volatile and nonvolatile secondary metabolites) nor its bioactivity potentials. Accordingly, the current study aims to identify the chemical profile of the hydro-distilled essential oil (HDEO), obtained from the inflorescence heads of Jordanian E. polyceras, at different growth stages. In addition, we report on the determination of COX-1 and COX-2, in addition to the protein denaturation inhibitory activities and the antimicrobial potential of the methanolic extract (EPM) obtained from the aerial portions of E. polyceras.

2. Results

2.1. GC/MS Analysis

The results for the GC/MS analysis of the essential oils obtained from E. polyceras inflorescence heads at the different growth stages are shown in Table 1. Figure 1 indicates the different classes of volatile principles detected at the different growth stages in the analyzed essential oils. GC/MS chromatograms are available in Supplementary Figures S1–S3.

Table 1.

GC/MS analysis of the HDEO obtained from inflorescence heads of fresh E. polyceras at different growth stages.

No RI exp RI Theor Compound Class % Composition
PF FF Post-F
1 846 841 3-methyl-Pentanol AH&D 0.09 3.00 -
2 850 850 (2E)-Hexenal AH&D 8.03 - -
3 851 853 (3E)-Hexenol AH&D - - 0.23
4 861 862 (2E)-Hexenol AH&D 0.96 - 0.15
5 865 870 n-Hexanol AH&D 4.06 - 0.86
6 865 867 (2Z)-Hexenol AH&D - 0.08 -
7 884 888 Ethyl pent-4-enoate AH&D - 0.27 -
8 886 889 4-Heptanol AH&D - 0.15 -
9 888 892 2-Heptanone AH&D 0.13 - -
10 898 895 (4Z)-Heptenal AH&D - 0.13 -
11 899 900 n-Nonane AH&D 0.14 - -
12 904 902 Heptanal AH&D 0.43 0.10 0.03
13 916 916 (2E,4E)-Hexadienol AH&D 0.12 2.66 0.01
14 921 923 2-methyl-4-Heptanone AH&D - 0.59 -
15 932 944 5-methyl-3-Heptanone AH&D - 0.34 -
16 948 943 3-methyl-Cyclohexanol AH&D - 2.30 -
17 954 952 3-methyl-Cyclohexanone AH&D - 0.24 -
18 957 954 (2E)-Heptenal AH&D 0.12 - 0.03
19 963 960 Benzaldehyde AH&D - - 0.03
20 968 966 n-Heptanol AH&D - - 0.03
21 973 973 Hexanoic acid AH&D - - 0.05
22 978 979 1-Octen-3-ol AH&D - - 0.10
23 984 986 (3E)-Octen-2-ol AH&D - - 0.05
24 990 988 2-Pentyl furan Ar 6.32 - 0.15
25 999 1002 δ-2-Carene MH 0.75 - 0.04
26 1004 998 n-Octanal AH&D 0.14 - 0.10
27 1012 1016 (2E,4E)-Heptadienol AH&D - - 0.09
28 1027 1021 3-methyl-1,2-Cyclopentanedione AH&D - - 0.03
29 1034 1031 Eucalyptol OM - - 0.08
30 1038 1035 (3E)-Octen-2-one AH&D - - 0.09
31 1045 1042 Benzene acetaldehyde Ar - - 0.05
32 1055 1054 Prenyl isobutyrate AH&D 0.63 - 0.11
33 1059 1054 (2E)-Octen-1-al AH&D 0.27 - 0.26
34 1070 1068 n-Octanol AH&D - - 0.92
35 1082 1084 (2Z)-Hexenal diethyl acetal AH&D - - 0.05
36 1087 1086 trans-Linalool oxide OM - - 0.07
37 1090 1090 2-Nonanone AH&D - - 0.04
38 1094 1090 Isobutyl tiglate AH&D - - 0.41
39 1095 1098 2-Nonanol AH&D 0.11 - -
40 1100 1096 Linalool OM 1.44 0.45 3.13
41 1105 1100 n-Nonanal AH&D 3.47 0.27 2.17
42 1111 1108 cis-Rose oxide OM - - 0.20
43 1116 1116 (2E,4E)-Octadienol AH&D - - 0.16
44 1124 1119 trans-p-Mentha-2,8-dien-1-ol OM - - 0.08
45 1127 1133 1-Terpineol OM - - 0.10
46 1139 1140 Nopinone OM - - 0.11
47 1144 1144 trans- p-Menth-2-en-1-ol OM - - 0.10
48 1147 1146 trans-Verbenol OM - - 0.09
49 1151 1154 Camphor OM - - 0.23
50 1155 1154 (2E,6Z)-Nonadienal AH&D 0.79 0.13 1.48
51 1156 1153 (3E,6Z)-Nonadienol AH&D - - 0.12
52 1161 1157 (2E)-Nonen-1-al AH&D 1.85 0.17 2.63
53 1167 1166 (2Z)-Nonenol AH&D - - 0.38
54 1172 1169 n-Nonanol AH&D 0.12 - 0.36
55 1176 1169 Borneol OM - - 0.11
56 1184 1177 Terpinen-4-ol OM - - 0.38
57 1189 1182 p-Cymen-8-ol Ar - - 0.49
58 1191 1192 2-Decanone AH&D - - 0.04
59 1199 1196 γ-Terpineol OM 0.45 0.23 1.09
60 1202 1169 Safranal OM - - 0.06
61 1207 1201 n-Decanal AH&D 0.70 0.70 0.87
62 1211 1208 trans-Piperitol OM - - 0.14
63 1215 1205 Verbenone OM 0.13 - 0.35
64 1218 1229 Nerol OM 1.03 - 1.51
65 1223 1219 β-Cyclocitral OM 0.24 - 0.33
66 1225 1225 Citronellol OM - - 0.36
67 1230 1229 (Z)-Ocimenone OM - - 0.30
68 1240 1232 exo-Fenchyl acetate OM - - 0.06
69 1250 1252 Geraniol OM - - 0.63
70 1259 1263 cis-Carvone oxide OM - - 0.07
71 1264 1263 (2E)-Decenal AH&D 0.33 - 0.81
72 1270 1269 n-Decanol AH&D - - 0.30
73 1281 1285 Isobornyl acetate OM 0.22 - 0.08
74 1284 1285 iso-Isopulegyl acetate OM - - 0.32
75 1287 1285 Bornyl acetate OM - - 0.14
76 1291 1290 Thymol Ar - - 0.15
77 1297 1293 (2E,4Z)-Decadienal AH&D 0.16 - 0.28
78 1300 1300 n-Tridecane AH&D 0.27 0.29 0.55
79 1309 1306 Undecanal AH&D 0.32 0.19 0.27
80 1313 1309 p-vinyl-Guaiacol Ar - - 0.22
81 1322 1321 (2E,4E)-Decadienol AH&D 0.78 0.59 1.67
82 1331 1332 Hexyl tiglate AH&D - - 0.08
83 1337 1338 δ-Elemene SH 0.11 - 0.16
84 1352 1359 Eugenol Ar - - 0.12
85 1363 1361 γ-Nonalactone AH&D - - 0.17
86 1367 1360 (2E)-Undecenal AH&D - 0.17 0.33
87 1375 1370 n-Undecanol AH&D - - 0.11
88 1382 1376 α-Copaene SH 0.23 0.31 0.27
89 1390 1390 β-Elemene SH 0.94 1.07 1.30
90 1399 1399 9-Decenyl acetate AH&D - - 0.22
91 1402 1400 Tetradecane AH&D - 0.23 0.10
92 1410 1408 Dodecanal AH&D 0.22 0.24 0.03
93 1414 1412 dihydro-α-Ionone OM 0.62 0.55 1.86
94 1429 1419 (E)-Caryophyllene SH 3.10 2.45 5.01
95 1434 1440 trans-Nerone OM - - 0.04
96 1437 1433 β-Gurjunene SH 0.23 0.11 0.07
97 1439 1441 Aromadendrene SH - - 0.05
98 1449 1453 Geranyl acetone OM 0.18 0.13 0.32
99 1461 1466 (2E)-Dodecenal AH&D 0.54 0.62 0.51
100 1463 1454 α-Humulene SH 0.12 0.28 0.42
101 1469 1470 n-Dodecanol AH&D - - 0.12
102 1479 1479 6-nonyl-5,6-dihydro-2H-Pyran-2-one AH&D 0.76 0.30 2.71
103 1482 1481 methyl-γ-Ionone OS 0.83 0.68 1.18
104 1486 1488 (E)-β-Ionone OS 0.10 - 0.44
105 1488 1492 δ-Selinene SH - 0.50 -
106 1493 1496 Valencene SH 0.43 0.33 0.20
107 1497 1490 β-Selinene SH 0.15 0.27 -
108 1497 1492 δ-Selinene SH - - 0.30
109 1500 1500 n-Pentadecane AH&D - 0.35 0.18
110 1503 1498 α-Selinene SH 0.10 0.31 0.40
111 1507 1497 Methyl p-tert-butylphenyl acetate Ar - 0.12 -
112 1511 1505 β-Bisabolene - 7.53 0.52
113 1513 1510 Tridecanal AH&D 0.39 - -
114 1516 1512 α-Alaskene SH 0.37 0.17 0.62
115 1522 1515 Cubebol OS 0.29 - 0.55
116 1528 1523 δ-Cadinene SH - 0.76 0.12
117 1543 1546 Selina-3,7(11)-diene SH - - 0.09
118 1561 1566 Dodecanoic acid AH&D 0.49 0.35 0.89
119 1577 1566 (3Z)-Hexenyl benzoate Ar 1.83 1.11 1.61
120 1584 1580 n-Hexyl benzoate Ar 1.32 0.84 1.33
121 1582 1590 Caryophyllene oxide OS 1.53 2.22 3.27
122 1600 1600 n-Hexadecane AH&D - 0.44 0.11
123 1604 1604 Khusimone OS - - 0.11
124 1615 1612 Tetradecanal AH&D 0.32 0.22 0.30
125 1621 1619 2,(7Z)-Bisaboladien-4-ol OS - - 0.42
126 1627 1632 γ-Eudesmol OS - - 0.05
127 1633 1637 Caryophylla-4(12),8(13)-dien-5β-ol OS - - 0.39
128 1647 1650 β-Eudesmol OS 0.40 0.24 1.50
129 1656 1653 α-Eudesmol OS - - 0.31
130 1657 1659 Selin-11-en-4-α-ol OS - 0.17 -
131 1665 1660 neo-Intermedeol OS 0.10 0.29 0.40
132 1669 1663 7-epi-α-Eudesmol OS 0.23 0.30 0.40
133 1673 1672 n-Tetradecanol OS 0.36 1.11 0.47
134 1677 1666 Intermedeol OS 4.19 4.14 5.53
135 1681 1676 Cadalene Ar - 1.07 -
136 1688 1692 4-Cuprenen-1-ol OS 0.15 0.18 0.36
137 1691 1689 Shyobunol OS - - 0.18
138 1702 1700 n-Heptadecane AH&D 0.23 0.56 0.54
139 1717 1703 (2E)-Tridecenol acetate AH&D 1.59 0.86 1.83
140 1721 1718 Methyl eudesmate Ar - 0.52 0.12
141 1725 1723 Methyl tetradecanoate AH&D - 1.71 0.51
142 1733 1729 iso-Longifolol OS 0.41 1.95 0.90
143 1746 1746 γ-Costol OS - 0.18 0.49
144 1760 1774 n-Pentadecanol AH&D 0.94 0.91 0.60
145 1766 1767 12-hydroxy-(Z)-Sesquicineole OS - - 0.08
146 1795 1796 Ethyl tetradecanoate AH&D - - 0.10
147 1802 1800 n-Octadecane AH&D - 0.47 0.12
148 1806 1808 Eudesm-11-en-4-α, 6-α-diol OS - 0.70 0.04
149 1819 1817 (2E,6E)-Farnesoic acid OS 0.29 0.61 0.16
150 1831 1829 Isopropyl tetradecanoate AH&D 0.56 - 0.52
151 1843 1845 Isoamyl dodecanoate AH&D 2.50 1.86 1.83
152 1901 1900 n-Nonadecane AH&D 0.26 0.24 0.34
153 1921 1930 Ambrettolide AH&D 0.21 - 0.18
154 1926 1921 Hexadecanoic acid, methyl ester AH&D 1.26 1.44 1.99
155 1964 1960 Hexadecanoic acid AH&D 4.61 0.53 2.04
156 1984 1974 Dolabradiene Ar 2.62 5.50 1.48
157 1996 2000 n-Eicosane AH&D 0.20 0.18 0.19
158 2001 2003 Hexadecyl acetate AH&D 0.24 0.57 0.18
159 2012 1989 Manoyl oxide OD 0.40 0.90 0.20
160 2017 2017 Phyllocladene SH - - 0.02
161 2024 2024 Isopropyl hexadecanoate AH&D 0.31 0.37 0.27
162 2033 2010 13-epi-Manool oxide OD - - 0.06
163 2060 2060 13-epi-Manool OD - 0.31 0.05
164 2070 2043? (6E,10E)-Pseudo phytol OD 7.54 7.84 4.47
165 2087 2077 n-Octadecanol AH&D - - 0.07
166 2094 2085 Methyl linoleate AH&D 0.45 0.59 0.64
167 2101 2100 n-Heneicosane AH&D 0.82 0.98 0.84
168 2111 2116 Laurenan-2-one OD 0.26 0.40 0.31
169 2127 2128 Methyl stearate AH&D - 0.35 0.26
170 2134 2133 Linoleic acid AH&D 1.09 1.37 0.60
171 2139 2142 Oleic acid AH&D 0.29 0.14 0.13
172 2143 2149 Abienol OD 0.15 - 0.14
173 2165 2173 Linoleic acid ethyl ester AH&D - - 0.08
174 2180 2189 1-Docosene AH&D - - 0.28
175 2181 2196 Ethyl octadecanoate AH&D 0.38 - -
176 2194 2198 Ugandensidial OD 0.85 0.81 0.43
177 2195 2200 n-Docosane AH&D 0.77 0.67 0.39
178 2209 2209 Octadecanol acetate AH&D 0.10 0.18 0.05
179 2232 2223 Sclareol OD 0.12 - 0.11
180 2283 2269 Sandaracopimarinol OD 0.99 0.70 0.93
181 2300 2297 3-α-hydroxy-Manool OD 0.32 - 0.08
182 2302 2300 n-Tricosane AH&D 1.38 1.66 1.41
183 2335 2338 3-α-14,15-dihydro-Manool oxide OD 3.43 2.60 1.53
184 2406 2400 n-Tetracosane AH&D 0.29 0.46 0.31
185 2502 2500 n-Pentacosane AH&D 1.18 1.33 0.78
186 2535 2531 Tricosanal AH&D 0.79 3.78 0.65
187 2597 2600 Hexacosane AH&D 0.12 1.08 0.14
188 2702 2700 Heptacosane AH&D 2.53 1.56 1.60
189 2803 2800 Octacosane AH&D 0.19 0.41 0.13
190 2815 2790 Squalene AH&D - 0.27 -
191 2903 2900 Nonacosane AH&D - 0.62 -
192 3101 3101 Untriacontane AH&D - - 0.09
Monoterpene hydrocarbons (MH) 0.75 - 0.04
Oxygenated monoterpenes (OM) 4.31 1.36 12.35
Sesquiterpene hydrocarbons (SH) 5.78 14.08 9.53
Oxygenated sesquiterpenes (OS) 8.88 12.76 17.22
Diterpene hydrocarbons (DH) 2.62 5.50 1.48
Oxygenated diterpenes (OD) 14.05 13.56 8.34
Aromatics (Ar) 9.48 3.66 4.24
Aliphatic hydrocarbons & their derivatives (AH&D) 50.04 40.28 41.34
Total Identified 95.91 91.20 94.50
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Figure 1.

Figure 1

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Different classes of compounds detected in the HDEOs of E. polyceras from Jordan collected at different growth stages viz. Pre-F: pre-flowering, Full-F: full-flowering and Post-F: post-flowering.

2.2. Bioactivity Results

2.2.1. COX-1, COX-2 and Protein Denaturation Inhibitory Activity

The methanolic extract obtained from the air-dried aerial parts of E. polyceras (EPM) was investigated for its capacity to inhibitory effects against COX-1, COX-2 and protein denaturation. Results are shown in Table 2.

Table 2.

COX-1 and COX-2 and protein denaturation inhibitory effects of EPM extract.

% Inhibition (Mean ± SD)
COX-1 COX-2 Protein Denaturation
EPM: (200 µg/mL) 64.98 ± 2.55 96.37 ± 0.85 -
EPM: (400 µg/mL) - 98.97 ± 0.45 -
SC-560 (5 ng/mL) 50.17 ± 1.25 - -
Celecoxib * - 88.63 ± 2.20 -
EPM (250 µg/mL) - - 64.34 ± 2.83
Diclofenac sodium (250 µg/mL) - - 68.75 ± 1.05
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* (5× dilution, as instructed), n = 3.

2.2.2. Antimicrobial Activity

The EPM extract was assayed for its antimicrobial activity against S. aureus and E. coli. Results are shown in Table 3 and Figure 2.

Table 3.

Antimicrobial activity of EPM extract against S. aureus and E. coli.

Sample Name Inhibition Zone (mm)
S. aureus E. coli
EPM extract (5000 µg/mL) 15 12
EPM extract (2500 µg/mL) 12 10
EPM extract (1250 µg/mL) 10 None
Moxifloxacin (40 µg/mL) 26 13
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n = two measurements; diameter of well = 6 mm.

Figure 2.

Figure 2

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Antibacterial activity of EPM extract: well 1:5000 µg/mL; well 2:2500 µg/mL; well 3:1250 µg/mL, and moxifloxacin (well 4:40 µg/mL) against S. aureus (left) and E. coli. (right). Control (5% DMSO) is marked “C”.

3. Discussion

GC/MS analysis of the HDEO obtained from the inflorescence parts of E. polyceras collected at the different growth stages resulted in the identification of a total of 192 compounds (Table 1). Figure 2 reveals the different classes of compounds detected at the different growth stages. The results of this analysis revealed simple nonterpenic aliphatic hydrocarbons and their derivatives (AH&D) to be the main class that dominated the composition in all growth stages.

During the pre-F stage, HDEO contained good amounts of oxygenated diterpenes (OD, 14.05%) in addition to AH&D (50.04%). The primary individual constituents detected in this stage included (2E)-hexenal (8.03%), (6E,10E)-pseudo phytol (7.54%), hexadecanoic acid (4.61%), n-hexanol (4.06%), n-nonanal (3.47%), 3-α-14,15-dihydro-manool oxide (3.43%) and heptacosane (2.53%). Additionally, aromatic compounds were detected in appreciable amounts (9.48%) and were represented mainly by 2-pentyl furan (6.32%).

During the full-flowering and post-flowering stages (full-F, post-F, respectively), AH&D had the highest contribution to both essential oils composition but was detected at slightly lower concentration levels as compared to what occurred at the pre-F stage (40.28%, 41.38%, respectively). The major chemical constituents detected in the EO at the full-F stage were (6E,10E)-pseudo phytol (7.84%), β-bisabolene (7.53%), dolabradiene (5.50%), intermedeol (4.14%) and tricosanal (3.78%). The principal compounds detected during the post-F stage were intermedeol (5.53%), (E)-caryophyllene (5.01%), (6E,10E)-pseudo phytol (4.47%), caryophyllene oxide, (3.27%) linalool (3.13%) and hexadecanoic acid (2.04%).

Via a thorough investigation of the literature, it was revealed that the essential oil composition of the flowering heads of E. polyceras had never previously been investigated before. Previous studies concentrated on evaluating the constituents of the essential oils extracted from the roots of most Echinops species, including E. polyceras [18]. Recently, the study of Belabbes et al. [18] identified 5-(but-1-yn-3-enyl)-2,2’bithiophene and α-terthienyle (54.4 and 26.3%, respectively) as the primary constituents of the essential oil extracted from the roots of E. spinousus from Algeria. These two compounds were also detected in the essential oil obtained from the roots of E. spinousus of Tunisian origin (21.334%, 18.024%., respectively) [19]. These studies confirm that the essential oils extracted from different plant organs can have quite different compositions. Other factors can also affect the essential oil composition, including harvesting time, extraction method, soil type and many other environmental factors.

The chemical compositions of the essential oils of other Echinops species were investigated [20,21,22,23,24,25]. In these studies, the essential oils were extracted from a variety of different organs including the roots and aerial organs (stems, leaves, flowers, tubers). Table 4 summarizes the results of these studies and compares them to our current investigation [20,21,22,23,24,25]. These data shown in Table 4 reveal great qualitative and quantitative differences between the constituents of the EOs among the different Echinops species. It was noticed that some chemical constituents were common to all species, including 1,8-cineole, E-caryophyllene, caryophellene oxide and hexadecanoic acid.

Table 4.

Comparative studies on the chemical composition of the HDEO obtained from the roots, different aerial parts (stems, leaves, flowers, tubers) of several Echinops species from different origins around the world and the current study.

Constituents Current Study E. Spinousus [19] E. Ilicifolius [20] E. grijsii [21] E. latifolius [22] E. kebericho [23] E. kebericho [8] E. ritro [24] E. graecus [25] E. ellenbeckii [25]
PF FF Pst F R L F R R AP Tu R Infl Infl L S F R
(2E)-Hexenal 8.03 - - - - - - - - - - 21.4 - - - - -
𝛽-Pinene - - - - - - - 3.92 15.56 3.62 - t - - - - -
1,8-Cineole 1.44 0.45 3.13 0.321 - 0.1 - 5.56 19.63 - tr 16.3 - - - - -
β-Phellandrene - - - 0.413 - - - - 0.15 10.84 - - - - - -
(Z)-𝛽-Ocimene - - - - 0.1 - - 5.01 18.44 0.83 rt t - - - - -
Linalool - - 0.08 0.549 16.4 58.6 0.9 1.54 2.74 - - 9.8 5.6 - - - -
Geraniol - - 0.63 - 8.3 17.4 - - - - - t - - - - -
p-Cymene - - - - - - 0.9 - - 0.14 0.1 12.1 t - - - -
Camphor - - 0.23 0.862 0.1 - 0.6 - 1.50 0.16 tr 2.6 6.5 - - - tr
Intermedeol 4.19 4.14 5.53 - - - - - - - - - - - - - -
(E)-Caryophyllene 3.10 2.45 5.01 2.736 - - - 3.84 3.58 - 1.7 - - - - 2.30 0.23
β-Bisabolene - 7.53 0.52 - - - - 0.46 - - 0.1 - - - - 4.40 -
Caryophyllene oxide 1.53 2.22 3.27 5.217 - - - 3.53 0.82 0.40 9.7 - - 4.93 2.26 - 1.01
γ-Cadinene - - - 27.224 - 2.32 1.52 - - - - - - - - - -
(6E,10E)-Pseudo phytol 7.54 7.84 4.47 - - - - - - - - - - - - - -
Dolabradiene 2.62 5.50 1.48 - - - - - - - - - - - - - -
n-Dodecane - - - - 0.2 10.9 14.5 - - - - - t 0.52 0.11 0.90 0.06
Nerol 1.03 - 1.51 - 2.6 5.4 - - - - - t - - - - -
n-Hexadecanoic acid 4.61 0.53 2.04 - 36.2 3.3 7.8 - - - - 2.6 - - - - 7.10
Dehydrocostus lactone - - - - - - - - - 41.83 - - - - - - -
Germacrene B - - - - - - - - - 5.38 - - - -
Tridecane 0.27 0.29 0.55 - - - 0.2 - - - tr - - 0.57 0.11 1.50 0.10
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AP: aerial parts, L: leaves; S: stem; PF: pre-flower; FF: full flower; F: flower; Infl: Inflorescence; Tu: tubers; Pst-F: post-flower; AP: Aerial Part; R: root.

In the current study, the methanolic extract (EPM) obtained from the air-dried aerial parts of E. polyceras was investigated for its COX-1, COX-2 and protein denaturation inhibitory effects in addition to its antimicrobial potential against the Gram-positive and Gram-negative bacteria. The preliminary screening results indicated that the extract displayed more COX-2 inhibition than COX-1 inhibition. The percentage inhibition of COX-2 was 96.4% and 99.0% at 200 µg/mL and 400 µg/mL EPM concentration levels, respectively. The standard drug used, celecoxib, produced an 88.6% inhibition under the same experimental conditions. It has been widely reported that the COX-1 enzyme is responsible for the synthesis of the constitutional prostaglandins that are responsible for maintaining the integrity of the stomach lining and kidney functions. The inhibition of COX-1 produced certain side effects such as bleeding and ulceration. In this study, it was observed that EPM extracted at a concentration level of 200 μg/mL inhibited only 65.0% of COX-1 enzyme, while the standard reference compound SC-560 (5 ng/mL) inhibited 50.2% COX-1. These findings suggest that fractionation of the extract is required to explore the selective COX-2 activity of the isolated compounds.

Moreover, the EPM extract displayed moderate antibacterial activity against Gram-positive S. aureus. The extract showed weak antibacterial activity against the Gram-negative E. coli (Figure 1), with lower levels of activity than those observed for the extract obtained from the E. polyceras from Saudi Arabia [26].

4. Materials and Methods

4.1. Plant Material

The plant material was collected from the area surrounding Al-Balqa Applied University, (32′02′11″76″ N; 35′43′43″82″ E), Al-Salt governorate, Jordan, during the summer season of the year 2021. The inflorescence heads of the plant material were collected at the pre-flowering (pre-F), full-flowering (full-F) and post-flowering (post-F) stages. The taxonomic identity of the plant was confirmed by Prof. Dr. Hala I. Al-Jaber, Department of Chemistry, Faculty of Science, Al-Balqa Applied University, Al-Salt, Jordan. A voucher specimen (No: Ast/Ep/2021) was deposited at the herbarium of the Faculty of Science (Natural Products Laboratory Herbarium), Al-Balqa Applied University, Al-Salt, Jordan.

4.2. Hydro-Distillation and Extraction of Essential Oils

Essential oils (EOs) were extracted from fresh inflorescence heads of E. polyceras at different growth stages and according to the procedure described in the literature [27,28]. Briefly, a 300 g sample of fresh inflorescence heads collected at each flowering stage was coarsely powdered and then subjected to hydro-distillation for 3 h in a Clevenger-type apparatus. The obtained essential oil (HDEO) from each growth stage was extracted (twice) with GC-grade n-hexane, dried using anhydrous MgSO4, and then stored in amber glass vials at 4 °C until analysis was performed.

4.3. GC-MS Analysis

GC/MS analysis was done according to the procedure previously described in the literature [29,30]. The analysis was performed on a Shimadzu QP2020 GC-MS equipped with GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) with a split–splitless injector, utilizing a DB5-MS fused silica column (5% phenyl, 95% polydimethylsiloxane, 30 m × 0.25 mm, 25 µm film thickness). Briefly, a linear temperature program was used to separate the different components. The oven temperature was set to 50 °C for 1 min, after which temperature programming was applied at 7 °C/min. The heating rate started from 50 °C (initial temperature) to 280 °C (final temperature); this was then held at 280 °C for 10 min, and the total run time was 44 min. The injector temperature was 260 °C with a split ratio of 20:1; an injection volume of 1 µL; a carrier gas: helium (flow rate 1.50 mL/min); and a flow control mode: pressure, 88.3 kPa. MS source temperature/detector temperature: 240 °C; interface temperature: 250 °C; ionization energy (EI): 70 eV; scan range 35–500 amu; scan speed 1666. The solvent cut was 3 min, while these data were acquired in 4.5 min. These data were collected using Windows-based Lab-Solution GC-MS version 4.45SP1 Software. The mass spectra of isolated components were compared to those reported in ADAMS-2007 and NIST 2017 mass spectrometry libraries. To confirm the identified compound, a comparison was performed between reported values and relative retention indices (RRI) with reference to n-alkanes (C8–C30) in addition to these data published in the literature [31].

4.4. Preparation of the Alcoholic Extract

The alcoholic extract was prepared according to the procedure described in the literature with slight modifications [27,32]. Air-dried aerial parts of E. polyceras (20 g) were ground down to fine powder and then soaked in methanol (400 mL) at room temperature (3 × 24 h). The solvent was then evaporated under reduced pressure at 55 °C. The obtained methanol extract (EPM, 2.2479 g; yield: 11.24%) was then used for bioactivity screening.

4.5. COX-1 and COX-2 Inhibitory Activity

To evaluate the COX activity and to judge the NSAID activity of the methanolic extract (EPM), a COX-1 inhibitory screening assay kit containing SC-560 as standard (Fluorometric, ab204698, Abcam, Tokyo, Japan) and COX-2 inhibitor screening kit (Fluorometric, ab283401, Abcam, Tokyo, Japan) containing celecoxib as standard were used as per the instruction provided with the kit without any modification [33,34,35]. Two different concentrations of the EPM extract were prepared for initial screening in the supplied buffer. The buffer solution was used as a control.

4.6. Inhibition of Protein Denaturation

The procedure described in [35,36], was used to estimate the inhibition of protein denaturation with slight modifications. Different solutions were prepared for the assay, including the test solution (EPM extract), test control, product control and standard solution. All solutions were prepared using a buffer with pH 6.3. These prepared samples were kept at 37 °C for 20 min; then, they were incubated at 50 °C for another 20 min. The absorbance was measured at 416 nm using a synergy HTC multimode reader (Viotek, Winooski, VT, USA). Absorbance measurements were performed after all samples had cooled down. The percent inhibition of protein denaturation was calculated using the given formula:

PercentInhibition=100−( Abs of test solution−Abs of Product controlAbs of Test control×100)

4.7. Antimicrobial Activity

Gram-positive (S. aureus ATCC 6538) and Gram-negative (E. coli ATCC 8739) bacteria were used to assess the antibacterial activity of the EPM extract and moxifloxacin (standard drug) according to the procedure described in the literature [34]. Nutrient agar was added to a sterile Petri disc (9 cm, diameter). The plates were inoculated with bacterial culture using sterilized cotton swaps. Subsequently, 6 mm wells were created with sterilized 6 mm surgical punches. The wells were filled with 60 µL of extract solution (5000, 2500 or 1250 µg/mL) and moxifloxacin standard solution (40 µg/mL). A 5% DMSO solution was used as a control. The plates were incubated at 37 °C for 24 h. Duplicate runs of each experiment were produced.

4.8. Statistical Analysis

The results obtained in the present study are expressed as mean ± standard deviation (SD). For the statistical analysis of the experimental data, Graph-Pad Prism 5 (Graph-Pad Software, San Diego, CA, USA) was used.

5. Conclusions

This is the first study to report on the chemical composition of hydro-distilled essential oil obtained from the inflorescence heads of Jordanian E. polyceras at different growth stages. When comparing our current results with those reported in the literature, it can be concluded that the essential oil obtained from flowering heads of E. polyceras had a quite different composition than that obtained from the roots. The EO was rich in chemical constituents such as (6E,10E)-Pseudo phytol, dolabradiene, isoamyl dodecanoate, intermedeol, β-bisabolene, linalool, (E)-caryophyllene, caryophyllene oxide, nerol, β-elemene, hexaxecanoic acid and iso-longifolol. The methanolic extract obtained from the aerial parts of E. polyceras showed significant COX-1, COX-2 and protein denaturation inhibitory activities, but moderate antimicrobial activity, against S. aureus and E. coli. The current results of this study encourage researchers to undertake further detailed phytochemical investigations of the alcoholic extract in order to isolate and characterize its active constituents.

Acknowledgments

We thank the Deanship, Faculty of Science, Al-Balqa University, Al-Salt, Jordan and Faculty of Pharmacy, Al-Ahliyya Amman University, Amman, Jordan for providing the necessary facilities.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28104238/s1. Figure S1: GC-MS analysis of GC-MS analysis of essential oil during pre-flowering stage of E. polyceras inflorescence; Figure S2: GC-MS analysis of essential oil during full-flowering stage of E. polyceras inflorescence; Figure S3: GC-MS analysis of essential oil during full-flowering stage of E. polyceras inflorescence.

Click here for additional data file. (162.4KB, zip)

Author Contributions

H.I.A.-J. and A.K.S. conceived and designed the experiments; H.S.H., H.I.A.-J. and A.K.S. performed the experiments; H.I.A.-J., A.K.S., H.S.H., H.E.A.-S. and L.M.B. analyzed the data; H.I.A.-J. and A.K.S. wrote the paper; H.I.A.-J. and A.K.S. edited the final manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be provided upon request.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research received no external funding.

Footnotes

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Associated Data

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Supplementary Materials

Click here for additional data file. (162.4KB, zip)

Data Availability Statement

Data will be provided upon request.

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