Pimenta Dioica and Pimenta Racemosa

Food & Function

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Pimenta dioica and Pimenta racemosa: GC-based metabolomics for the assessment of seasonal and Cite this: Food Funct., 2021, 12, 5247 organ variation in their volatile components, Received 7th February 2021, in silico and in vitro cytotoxic activity estimation† Accepted 25th April 2021 DOI: 10.1039/d1fo00408e Fadia S. Youssef, a Rola M. Labib,a Haidy A. Gad,a SafaaY. Eid,b a,c d rsc.li/food-function Mohamed L. Ashour and Hanaa H. Eid *

Volatile constituents isolated from the stems (S) and leaves (L) of compounds detected in the oils by determining their inhibitory Pimenta dioica (PD) and Pimenta racemosa (PR) during the four effect on human DNA topoisomerase II (TOP-2), human cyclin- seasons were analyzed using GLC/FID (Gas liquid chromatography dependent kinase 2 (CDK-2) and matrix metalloproteinase 13 – flame ionization detector) and GLC/MS (Gas liquid chromato- (MMP-13). o-Cymene followed by eugenol showed the highest graphy – mass spectrometry). Eighty-nine compounds were ident- fitting with all of the examined proteins approaching doxorubicin. ified in all samples, in which oxygenated monoterpene represented It can be concluded that GC coupled with chemometrics provide a by eugenol was the major constituent in PDS-S3 (autumn) (88.71%) strong tool for the discrimination of samples, while Pimenta could and PDS-S2 (summer) (88.41%). Discrimination between P. dioica afford a natural drug that could alleviate cancer. and P. racemosa leaves and stems in different seasons was achieved by applying chemometrics analysis comprising Principal Component Analysis (PCA) and Hierarchal Cluster Analysis (HCA). Introduction For P. dioica, they were partially segregated where leaves collected from spring and autumn were superimposed, and similarly for Nowadays, spices play an important role worldwide as natural P. dioica stems collected in summer and autumn. For P. racemosa coloring, flavoring agents, as well as antimicrobials, antioxi- leaves, the PCA score plot showed that all seasons were comple- dants in the food and beverage industries, in addition to its Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM. tely segregated from each other, with the winter and autumn utilization in cosmetics and perfumery. Moreover, spices samples being in very close distance to each other. P. racemosa showed an immense potential in the nutraceutical industry stems collected in autumn and spring exhibited significant vari- owing to their beneficial health and physiological effects. ation, as they were completely detached from each other. Spices are commonly obtained from different plant parts, such Moreover, summer and winter fell in a near distance to each other. as seeds, fruits, leaves, flowers and subterranean organs, An in vitro cell viability assay was done to evaluate the variation in which are rich in essential oils.1 the cytotoxicity of the isolated essential oils against breast The essential oils of many herbs and spices are used nowa- (MCF-7), hepatic (HepG-2), and cervical (HeLa-2) cancer cell lines days in aromatherapy, as they showed many reported biologi- using the MTT assay. The maximum cytotoxic effect was observed cal activities, especially the significant potential as anti-

by PDL against HeLa, HepG-2 and MCF-7 cells with IC50 values microbial, antifungal, antiviral, analgesic, anticarcinogenic, − equal to 122.1, 139.6, and 178.7 µg mL 1, respectively. An in silico antiparasitic, anti-inflammatory and antioxidant agents.2 study was done to assess the cytotoxic eﬀect of the major Genus Pimenta Lindley (family Myrtaceae), comprises 18 species of aromatic trees, native to Tropical America and to the Caribbean, that are widely distributed in Central America. The most commonly known species are Pimenta dioica and aDepartment of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, 11566 Cairo, Egypt Pimenta racemosa, where the former is called allspice or bDepartment of Biochemistry, Faculty of Medicine, Umm Al-Qura University, Makkah, Jamaica pepper, while the latter is named by Bay or Bay-rum- 1,3 Saudi Arabia tree. cDepartment of Pharmaceutical Sciences, Pharmacy Program, Batterjee Medical Traditionally, the berries of Pimenta were used in the treat- College, 21442 Jeddah, Saudi Arabia ment of digestive disorders, such as nausea, cramp, flatulence, dDepartment of Pharmacognosy, Faculty of Pharmacy, Cairo University, 11562 Cairo, and colic, as well as for the management of diarrhea, dyspep- Egypt. E-mail: [email protected]; Tel: (+2) 01223907981 4 †Electronic supplementary information (ESI) available. See DOI: 10.1039/ sia, and rheumatism. Moreover, the essential oils are utilized 4–6 d1fo00408e in food industry, perfumery and cosmetics. In addition, the

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essential oils of Pimenta leaves and fruits can improve the GLC/FID and GLC/MS analyses neurological signs as tension due to stress, nervous collapse, The GLC/FID and GLC/MS analyses were carried out on a 5 tightness, neuralgia and anxiety. Varian 3400 (Varian GmbH, Darmstadt, Germany) and The bioactivities and the chemical composition of the Hewlett-Packard gas chromatograph (GC 5890 II, Hewlett- ff essential oils of the Pimenta species obtained from di erent Packard GmbH, Bad Homburg, Germany), respectively 5,6 locations have been recently reviewed. Concerning the bio- equipped with OV-5 fused bonded column (30 m × 0.25 mm × logical activities, many were reported regarding the essential 0.25 μm) (Ohio Valley, Ohio, USA). The GLC conditions were ff oils and the di erent extracts of various Pimenta species, conducted as previously reported by El-Readi et al.13 For GLC/ including antioxidant, anti-inflammatory, antibacterial, anti- FID, helium was used as a carrier gas with a flow rate of 2 mL fungal, antiviral, anticancer, hypoglycemic, antitermite, anti- −1 – min ; the initial temperature used was 45 °C, followed by ff 1,5 7 − nociceptive and antivenum e ects as well. 2 min isothermal, 300 °C, 4 °C min 1 300 °C, then 20 min iso- However, relatively limited information could be found in thermal. The detector and injector temperatures were 300 and 8,9 the literature concerning the essential oils of leaves, 250 °C, respectively. The split ratio was 1 : 20. The chromato- 8,10 8 flowers and fruits of P. racemosa and P. dioica, which are grams were recorded and integrated using the Peak Simple cultivated in Egypt. Moreover, the lack of published data con- 2000 chromatography data system (SRI Instruments, cerning the essential oils of the stem of P. racemosa and California, USA). All data were calculated from three indepen- P. dioica encouraged us to carry out this work. Hence, the dent chromatographic runs, and the averages of the area target of this study was to evaluate the influence of the harvest under the peak were used to calculate the abundance of each time on the essential oils of the stems and leaves of component relative to the total area under the peaks. For GLC/ P. racemosa and P. dioica, as regards to the composition and MS, the capillary column was directly coupled to a quadrupole their discrimination using multivariate analyses. Additionally, mass spectrometer (SSQ 7000, Thermo-Finnigan, Bremen, in vitro cytotoxic activity estimation for the oils, followed by Germany). The injector temperature was 250 °C. The helium − in silico molecular modelling studies were performed for the carrier gas flow rate was 2 mL min 1. The following conditions major compounds identified in the oils on human DNA topoi- were employed for all mass spectra: filament emission current, somerase II (TOP-2), human cyclin-dependent kinase 2 100 mA; electron energy, 70 eV; ion source, 175 °C; diluted (CDK-2) and matrix metalloproteinase 13 (MMP-13), as well as samples (0.5% v/v) were injected with split mode (split ratio, aiming to outline their probable mode of action as cytotoxic 1 : 15). The Wiley Registry of Mass Spectral Data 8th edition, agents. Additionally, the present work aimed to establish the NIST Mass Spectral Library14,15 was used to identify the com- best economic conditions for the oil production for further position of the tested essential oils, and confirmed by litera- application in pharmaceutical industries. ture data.11 All reagents and controls used for GLC analysis were obtained from Sigma (Sigma Aldrich GmbH, Sternheim, Germany).

Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM. Materials and methods Chemometric analysis and data analysis Plant material Both Principal component analysis (PCA) and Hierarchal Leaves (L) and stems (S) of Pimenta dioica (L.) Merr. (PD) Cluster Analysis (HCA) were applied as chemometric methods and Pimenta racemosa Mills J.W. Moore (PR) (Family for multivariate analysis of GC analyses for the metabolic pro- Myrtaceae) were collected (2017) from Alzohreya botanical filing of data from diﬀerent Pimenta species. Unscrambler® X garden (Cairo, Egypt) in spring S1 (April), summer S2 (July), 10.4 from Computer Aided Modeling, AS, Norway (CAMO) was autumn S3 (October) and winter S4 (January). The plants were used to apply PCA and HCA analyses. These multivariate dis- kindly authenticated morphologically by Dr Therese Labib; crimination techniques are considered a simplistic approach consultant of plant taxonomy at the Ministry of Agriculture, for the classification and grouping of diﬀerent species based Egypt. Voucher specimens of the plant material were deposited on the complete metabolic profile of the essential oils. The at the herbarium of the Pharmacognosy Department, Faculty percentage of compounds and the correlations between the – of Pharmacy, Cairo University (PHE. #1217 1224), (PHE. percentage abundance of the identified compounds are the – #1217 1232). major data used for classification and clustering.16,17

Essential oil preparations Cell culture and cytotoxicity assay About 0.5 kg of fresh plant materials (leaves and stems) of Human cervical (HeLa), breast (MCF-7), and hepatocellular each PD and PR were hydro-distillated using a Clevenger-type carcinoma (HepG-2) cancer cell lines were used to evaluate the apparatus for 4 h. The obtained essential oils were dehydrated cytotoxic activity of the Pimenta species. Dulbecco’s modified using anhydrous sodium sulfate.11,12 The yield was calculated Eagle’s medium (DMEM) and RPMI 1640 media were used to in triplicate on fresh weight basis (v/w), and is listed in cultivate and maintain cell lines. Some additives, such as fetal Table 1. The oil samples were stored in sealed vials at −4°C calf serum, penicillin/streptomycin, and L-glutamine for further analysis and investigation. (BioChrom KG, Berlin, Germany), were used as supplements

5248 | Food Funct.,2021,12,5247–5259 This journal is © The Royal Society of Chemistry 2021 Published on 26 April 2021. Downloaded on 7/9/2021 2:15:43 PM. od&Function & Food hsjunli h oa oit fCeity2021 Chemistry of Society Royal The © is journal This Table 1 Chemical composition of essential oils obtained from the leaves and stems of P. diocia and P. racemosa at diﬀerent seasons

Kovat’s index PDL PDS PRL PRS

# Compounds Calc. Rep. S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4

I-Aliphatic hydrocarbons 1 n-Octane 800 800 0.14 0.14 0.1 0.10 0.15 0.14 0.13 0.09 0.12 0.05 0.10 0.15 0.14 0.11 0.11 0.12 2 n-Nonane 900 900 0.03 0.04 0.02 0.03 0.01 0.04 0.05 0.03 0.04 0.02 0.03 0.04 0.04 0.03 0.03 0.03 II-Monoterpene hydrocarbons 3 β-Thujene 918 920 ——— 0.04 ——0.02 0.09 0.08 0.09 0.02 0.02 0.12 0.03 0.06 4 α-Pinene 923 932 0.41 0.23 0.27 1.29 0.01 0.12 0.30 1.46 1.15 0.76 1.08 0.38 1.18 2.20 3.07 4.36 5 Camphene 939 946 0.01 — 0.06 0.01 2.22 — 0.01 0.03 0.01 0.01 0.01 0.01 0.03 0.03 0.08 0.11 6 Sabinene 968 969 ——————0.07 0.34 0.08 0.07 0.12 — 0.02 0.02 —— 7 β-Pinene 969 974 0.09 0.07 0.06 0.08 0.05 0.03 ——— ——0.09 0.24 0.19 0.84 1.01 8 β-Myrcene 987 988 0.30 0.16 0.21 20.70 0.56 0.06 0.15 4.45 18.81 12.89 2.02 0.21 9.82 25.17 10.86 17.78 9 α-Phellandrene 1000 1002 0.03 0.03 0.02 1.54 0.38 0.02 0.02 0.24 1.23 1.03 1.49 0.04 0.48 1.45 0.57 0.86 10 Δ-3-Carene 1009 1013 0.01 — 0.01 0.01 ————0.03 ——————— 11 Δ-4-Carene 1012 1014 ———0.03 0.03 0.01 0.0|1 0.01 0.02 0.02 0.02 0.05 0.01 0.04 0.01 0.02 12 α-Terpinene 1013 1014 0.02 0.03 0.02 0.15 ———0.03 ————0.05 0.14 0.08 0.11 13 o-Cymene 1023 1022 0.11 0.06 0.07 7.87 0.02 0.03 0.03 0.45 1.12 0.54 0.96 0.05 1.10 2.26 1.19 2.03 14 Limonene 1027 1024 5.32 7.19 5.04 7.89 0.05 2.52 2.56 5.24 8.08 51.20 8.34 — 6.33 12.77 8.80 13.32 15 (Z)-β-Ocimene 1022 1037 ———————0.01 — ——————— 16 (E)-β-Ocimene 1052 1050 0.01 — 0.01 2.59 3.76 — 0.01 0.58 1.93 1.74 2.71 — 1.22 0.16 1.60 2.57 17 γ-Terpinene 1056 10–59 0.02 0.04 0.02 0.20 0.02 0.01 0.02 0.05 0.13 0.13 0.19 0.07 0.07 0.16 0.10 0.13 18 Terpinolene 1086 1088 0.04 0.03 0.03 0.22 0.03 ——0.08 0.30 0.25 0.41 0.05 — 0.24 0.17 0.21 III-Sesquiterpene hydrocarbons 19 α-Copaene 1378 1374 0.07 0.05 0.04 0.14 ————0.14 24.62 0.13 0.04 0.28 0.25 0.06 0.09 20 β-Cubebene 1385 1377 0.75 0.30 0.15 0.09 — 0.13 0.10 0.01 0.14 0.04 0.09 0.12 0.12 0.13 0.30 0.07 21 β-Bourbonene 1388 1387 0.01 0.03 0.02 —————— ——————— 22 β-Elemene 1394 1389 — 0.04 0.02 —————— ——————— 23 α-Cedrene 1403 1411 0.10 0.02 — 0.02 ————— ——————— 24 β-Caryophyllene 1420 1419 5.83 7.95 5.32 0.31 — 4.68 4.54 4.66 0.30 0.07 0.27 6.60 0.55 0.56 0.01 0.25 25 β-Copaene 1433 1432 0.03 0.05 0.03 0.01 6.31 0.02 0.02 0.03 ————0.03 0.03 0.13 0.02 26 α-Guaiene 1436 1439 ————————— ——0.01 ———— 27 Longifolene 1444 1448 ————————0.01 ——0.01 ———— 28 α-Humulene 1450 1452 — 2.29 1.53 0.13 0.01 1.34 1.35 1.27 0.12 0.01 0.11 1.81 0.23 0.23 58.50 0.15 29 Alloaromadendrene 1457 1458 1.65 0.07 0.05 0.01 1.83 0.03 0.04 1.27 ———0.06 ———— 30 β-Gurjunene 1462 1478 ————————0.02 ——————— 31 γ-Muurolene 1464 1478 0.04 0.15 0.11 0.20 0.05 0.08 0.08 0.13 0.10 — 0.08 0.13 0.51 0.50 0.30 0.25 — —————— ——————— odFunct. Food 32 Viridiflorene 1484 1496 0.16 0.10 33 β-Eudesmene 1488 1490 ————0.12 0.01 0.01 —— ——————— 34 γ-Gurjunene 1499 1475 — 0.02 ————0.07 0.08 ————0.09 0.09 0.59 0.03 35 Elixene 1502 1511 ————————— ——0.12 ———— 36 α-Muurolene 1505 1500 0.17 0.11 0.08 0.05 0.02 0.06 0.06 0.10 0.05 0.02 0.03 0.08 0.14 0.12 0.03 0.10 ,2021, 37 γ-Selinene 1512 1522 ——————0.01 —— ——————— 38 γ-Cadinene 1517 1513 0.01 0.09 0.06 — 0.09 0.05 ——0.12 0.06 0.06 0.07 ———— β — 12 39 -Cadinene 1517 1518 0.06 0.54 0.36 0.34 0.07 0.26 0.43 0.34 0.03 0.23 0.37 0.81 0.69 0.19 0.3 Communication ,5247 40 α-Cubenene 1526 1514 0.39 0.01 0.01 ————0.01 0.14 0.04 0.09 0.20 ———— 41 α-Cadinene 1542 1527 —————0.01 0.01 —— ———0.04 0.08 0.11 0.01 α — — —— View ArticleOnline – 42 -Calacorene 1546 1545 0.02 0.06 0.03 0.01 0.03 0.03 0.06 0.02 0.06 0.06 0.15 0.03 29| 5259 IV-Diterpene hydrocarbons 43 α-Springene 1956 1969 ————————0.02 ——————— 5249 Published on 26 April 2021. Downloaded on 7/9/2021 2:15:43 PM. Communication 5250 Table 1 (Contd.)

| Kovat’s index PDL PDS PRL PRS odFunct. Food # Compounds Calc. Rep. S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4

V-Oxygenated monoterpenes 44 Eucalyptol 1029 1031 ————————— ——9.03 ———— ,2021, 45 cis Linalool oxide 1067 1067 —————0.01 0.01 —— ——————— 46 trans Linalool oxide 1067 1084 —————0.03 0.02 —— ———————

12 47 β-Linalool 1097 1095 0.10 0.10 0.08 1.29 0.02 0.18 0.12 0.62 1.36 0.88 1.76 0.15 1.38 1.46 1.39 1.66 ,5247 48 cis-p-Menth-2-en-1-ol 1119 1118 ——0.01 0.03 ————0.04 0.02 0.04 — 0.05 0.05 0.02 0.07 49 cis-β-Terpineol 1125 1140 ————————0.02 ———————

– 50 trans-p-Menth-2-en-1-ol 1137 1136 ———0.02 ———0.02 — 0.01 0.03 — 0.05 0.04 0.06 0.06 29Ti ora s©TeRylSceyo hmsr 2021 Chemistry of Society Royal The © is journal This 5259 51 trans-Sabinol 1138 1140 ————————0.03 0.01 0.02 — 0.10 0.14 0.71 0.14 52 β-Pinene oxide 1160 1154 ———0.02 ———0.02 — ——————— 53 L-Borneol 1163 1169 ————————— ———0.04 0.35 — 0.04 54 Terpinen-4-ol 1171 1174 0.26 0.21 0.18 0.42 0.03 0.14 0.17 0.37 0.50 0.30 0.62 0.28 0.73 0.72 0.06 1.00 55 p-Cymen-8-ol 1171 1179 ———————0.02 0.04 0.01 0.03 — 0.08 0.06 —— 56 L-α-Terpineol 1185 1186 0.36 0.42 0.31 0.14 0.13 0.27 0.27 0.31 0.24 0.09 0.21 0.39 0.36 0.34 0.04 0.48 57 cis-Piperitol 1198 1197 ———0.01 ————0.02 — 0.02 — 0.01 ——— 58 trans-Piperitol 1204 1207 ————————— ————0.02 0.05 — 59 β-Citral 1215 1238 ———0.03 ——0.01 0.02 0.04 0.19 0.04 — 0.05 0.04 0.02 — 60 trans-Carveol 1215 1215 ————————— ———0.02 0.01 0.03 0.02 61 cis-Geraniol 1230 1229 ————————0.03 0.01 0.02 ————— 62 Chavicol 1253 1250 — 0.03 0.06 7.97 0.19 0.19 0.20 1.73 7.07 3.51 9.96 0.06 6.28 3.76 0.04 2.50 63 Thymol 1290 1290 — 0.01 —————0.01 0.04 — 0.02 0.02 0.25 0.07 0.05 0.08 64 Chavicol acetate 1330 1350 — 0.01 — 0.20 ———0.04 0.18 — 0.32 — 0.13 0.14 5.86 0.08 65 Eugenol 1368 1359 82.22 77.57 84.11 44.49 81.18 87.55 87.88 72.71 54.13 0.28 66.55 78.11 64.75 43.26 0.04 47.37 66 Chavibetol 1377 1362 ———0.12 0.04 0.03 0.06 0.01 ———0.04 — 0.01 — VI-Oxygenated sesquiterpenes 67 4-epi-Cubebol 1484 1494 ————————0.02 — 0.01 ————— 68 Epiglobulol 1530 1564 ——— —0.05 0.06 0.04 — ——————— 69 Caryophyllenyl alcohol 1543 1572 0.05 0.02 0.03 ——0.03 0.03 0.03 ———0.01 ———— 70 Caryophyllene oxide 1583 1583 0.12 0.25 — 0.02 — 0.34 0.03 0.33 0.05 0.01 0.03 0.11 0.08 0.09 0.08 0.12 71 Viridiflorol 1593 1592 0.43 0.06 0.04 ——0.03 0.05 —— ——0.04 ———— 72 Ledol 1608 1602 ——————0.06 0.05 — ——————— 73 Humulene-1,2-epoxide 1613 1608 ————————0.01 ——0.02 ———— 74 Selina-6-en-4-ol 1621 1624 ———————0.02 — ——————— 75 Humulane-1,6-dien-3-ol 1623 1619 — 0.05 ——————— ——————— 76 Cubenol 1633 1645 0.05 0.07 0.05 0.01 — 0.04 0.04 0.05 0.03 0.01 0.02 0.04 0.09 0.07 0.28 0.19 77 τ-Cadinol 1647 1640 ——————0.01 — 0.12 0.03 0.09 0.32 ———— 78 τ-Muurolol 1648 1640 0.05 0.48 0.39 ——0.56 0.55 0.45 0.13 0.05 0.08 — 0.30 0.25 0.86 0.35 79 δ-Cadinol 1652 1646 ————————0.04 0.06 0.04 — 0.02 — 0.24 — 80 α-Cadinol 1663 1652 0.55 0.51 0.43 0.09 — 0.60 0.57 0.45 0.01 0.04 — 0.38 0.29 0.27 0.03 0.34 81 cis,α-Santalol 1685 1674 ————————— ———0.14 0.01 0.11 0.17 82 trans Farnesol 1743 1715 ————————0.09 ——————— 83 Drimenol 1750 1766 ————————— —0.01 0.03 ————

VI-Aliphatic alcohols, aldehydes and ketones Function & Food 84 Hexanal 806 801 — 0.01 0.01 —————— ——————— — ————— —— ————

85 2-Methyl-4-pentenal 840 832 0.02 0.01 0.01 0.01 View ArticleOnline 86 3-Hexen-1-ol 844 853 0.04 0.02 0.03 0.03 0.01 0.01 ——0.01 — 0.01 0.04 ———— 87 1-Octen-3-ol 977 981 ——0.01 0.73 ———0.15 0.71 0.50 1.02 — 0.39 0.43 0.43 0.30 88 Octanal 1002 998 ———0.01 ————— ——————— 89 Decanal 1201 1201 ————————0.03 0.03 0.03 — 0.03 0.03 0.40 0.03 View Article Online Food & Function Communication

for the media. Optimum growth conditions, e.g., 37 °C, 5%

CO2, and 95% humidity were controlled during the entire experimental time. Trypsin/EDTA solution was used to detach the cells at the logarithmic growth phase. The cells (2 × 104 cells per well) were seeded in 96-well plates (Greiner Labortechnik), and incubated for 24 h. The stock solution − (100 mg mL 1) of essential oils in DMSO was diluted with media in serial dilution fashion, and each sample was incu- − bated with cells for 24 h. The MTT solution (0.5 mg mL 1)was added and incubated for 4 h. The DMSO (100 µL) was used to dissolve the formazan crystals. The light absorbance was measured at 570 nm using a microplate reader. Experimental sets were performed in triplicate and repeated three times.13 Cell viability was determined by this equation:

——————— Viability % ¼ ðOD sample treated cells‐‐OD sample medium controlÞ %

0.02 ðOD untreated cells‐‐OD medium controlÞ

0.15 0.75 0.53 1.06 0.05 0.42 0.46 0.83 0.33 Molecular modelling study The in silico molecular docking experiment was performed for

— the major identified compounds in the essential oils in the active center of human DNA topoisomerase II (TOP-2) (PDB ID 4G0U, 2.70 Å), human cyclin-dependent kinase 2 (CDK-2) (PDB ID 1PXP, 2.30 Å) and matrix metalloproteinase 13 (MMP-13) (PDB ID 1XUD, 1.8 Å) by Discovery Studio 2.5 (Accelrys, Inc., San Diego, CA, USA), employing the C-docker protocol on the proteins downloaded from the Protein Data Bank (http://www. pdb.org). This was done in order to determine the potential mode of cytotoxic behavior of these compounds, which greatly influences the activity of the overall oil. Free binding energies (ΔG) were determined for the most stable docking poses, as – previously described.18 20 Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM.

ΔGbinding ¼ Ecomplex ðEprotein þ EligandÞ; 82.94 78.351.25 84.750.04 1.44 54.62 81.67 0.05 0.94 88.41 0.06 0.12 88.71 75.93 0.78 0 63.75 0.01 5.31 0.01 1.65 79.64 88.04 1.4 74.32 50.46 1.42 8.38 0.5 53.5 0.2 0.28 0.95 0.92 0.69 1.6 1.17 ————————

where; ΔGbinding: the ligand–protein interaction binding

energy, Ecomplex: the potential energy for the complex of the s index PDL PDS PRL PRS ’ protein bound with the ligand, Eprotein: The potential energy of the protein alone and Eligand: the potential energy for the Kovat Calc. Rep. S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 ligand alone.

Results and discussion Yield and sensory characters The distilled oils of P. dioica (PD) and P. racemosa (PR) were ; cal, calculated; rep., reported; L, leaf; S, stem; Spring, S1; Summer, S2; Autumn, S3; Winter, S4. 9.13 11.96 7.91 1.31 8.5 6.7 6.32 8.05 1.48 24.91 1.09 9.62 2.86 2.74 60.37 1.3 6.37 7.84 5.82 42.58 7.17 2.8 3.18 12.99 32.98 68.72 17.44 0.97 20.57 44.95 27.4 42.57 0.17 0.18 0.12 0.13 0.16 0.18 0.18 0.12 0.16 0.07 0.13 0.19obtained 0.18 0.14 0.14 0.15 as pale yellow to dark yellow oily liquids with strong spicy aromatic clove-like odor. The essential oil yield (v/w fresh weight basis) of the leaves and stems of both species varied

P. racemosa with respective seasons. The highest yield was obtained from the leaves collected in summer for PD (1.46%) and PR (1.71%), and decreased rapidly to yield the lowest percentage of the and PR, essential oils in autumn (0.58% and 0.82%, respectively), and (Contd.) a continuous increase in the yield was noted in winter (1.04% P. diocia

Diterpene hydrocarbons and 0.96%, respectively) until spring (1.39% and 1.37%, Other Oxygenated components Oxygenated monoterpenes - Sesquiterpeneshydrocarbons - - Oxygenated sesquiterpenes - Monoterpene hydrocarbons Aliphatic hydrocarbons - - - Total oxygenated compoundsTotal identified compoundsTotal number of identified compoundsColorSpecific gravityYield % (v/w fresh plant)PD, 42 84.23 48 79.84 99.90 85.75 99.80 55.52 49 99.60 81.68 99.54 90.07 44 97.51 90.11 99.75 77.5 99.79 29 65 98.66 1.39 99.62 39 1.46 99.74 6.04 1.029 0.58 99.64 1.019 43 80.98 99.82 1.041 1.04 89.04 99.27 1.002 Pale yellow 75.66 0.21 52 99.44 1.039 51.61 98.72 1.043 0.24 10.81 99.02 1.043 63 55 0.37 1.018 0.9471 0.16 0.871 46 0.957 1.37 0.971 53 0.941 1.71 0.934 0.82 0.869 45 0.918 0.96 53 0.12 0.28 53 0.43 51 0.11 49 Bright yellow V VI Total hydrocarbonsIV 15.67 19.98 13.85 44.02 15.83 9.68 9.68 21.16 34.64 93.7 18.66 10.78 23.61 47.83 87.91 44.02 III II I Table 1 #I Compounds respectively). However, during autumn, the yield of the essen-

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tial oils from the stems of PD and PR remained high (0.37% showed the highest amount of monoterpene hydrocarbons and 0.43%, respectively), while the lowest percentage was (68.72%), as well as sesquiterpene hydrocarbons (24.91%). obtained in the winter (0.16% and 0.11%, respectively). Thus, However, PRS oil displayed significant amounts of monoter- to obtain high essential oil yields, summer was the best pene hydrocarbons both in summer (44.95%) and winter harvest time for the leaves, while autumn is the best season (42.57%). Moreover, considerable amounts of oxygenated for stem collection. monoterpenes were presented in all seasons, except for autumn, which revealed a significant amount of sesquitrepene Essential oil composition hydrocarbons estimated at 60.37%. Notable amounts of oxyge- A total of 89 compounds had been identified in all samples nated monoterpenes were observed in all of the examined (Table 1). FID and MS data provide quantitative and qualitative samples, especially eugenol, with PDS-S3 and PDS-S2 display- information, respectively. Thus, upon identification of the ing the highest concentrations reaching about 87.88% and compounds from MS data, they were quantified via FID data, 87.55%, respectively. In contrast, the PRL-S2 and PRS-S3 which were used mainly for chemometrics analysis. samples revealed traces of eugenol. In addition, chavicol exists Meanwhile, compounds that were present in trace amounts in all leaves and stem samples, except for PDL-S1. were identified only from MS data. Identifications were based Monoterpene hydrocarbons were detected in smaller amounts on the retention time only in GC-FID, although GC–MS has in comparison to oxygenated monoterpenes with a significant the additional data provided by the unit mass resolution frag- concentration of β-myrecene estimated at 25, 21 and 19% for mentation patterns usually at 70 eV. This was further con- PRS-S2, PDL-S4 and PRL-S1, respectively. Meanwhile, limonene firmed by determination of Kovat’s index.21 There is a very accounting for 51% represents one of the major compounds in close correlation between the essential oils obtained from PRL-S2. Other minor compounds were also observed in the P. dioica and P. racemosa (leaves and stem) collected in examined samples, such as β-ocimene, camphene and different seasons, where all of the samples showed their rich- o-cymene. The sesquiterpene hydrocarbon, α-copaene, exists ness in the classes of secondary metabolites represented by only in PRL-S2 (25%); α-humulene (59%) in PRS-S3; and monoterpene hydrocarbons, sesquiterpene hydrocarbons and β-copaene (6%) in PDS-S1. However, β-caryophyllene was oxygenated monoterpenes. The major constituents prevailing present in the leaves and stems of PD in all seasons except for in the essential oils are illustrated in Fig. 1. Concerning the S4 and S1. essential oil distilled from PDL, oxygenated monoterpenes rep- Our results are in accordance with many reports that pre- resent the highest amounts in autumn (84.75%), followed by viously studied the chemical composition of different organs spring (82.94%), then summer (78.35%), and the lowest value of different Pimenta species growing in different regions. The in winter (54.62%). In winter, monoterpene hydrocarbons con- essential oil of the P. dioica leaf from Jamaica was rich in stitute the highest amounts (42.58%). However, sesquiterpene eugenol (76%), methyl eugenol (7%), β-caryophyllene (6%), hydrocarbons represent the prevailing compounds in summer and traces of α-humulene and α-selinene.22 However, Brazilian (11.96%). Regarding PDS oil, there is no major difference in leaves have lesser amounts of eugenol, reaching (60%), while Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM. the composition among the seasons, except in the amount of leaves from Cuba contain (54%) eugenol. On the other side, monterpene hydrocarbons, which exists in higher amounts in Portuguese leaves contain the highest amounts of eugenol, winter (12.99%). PRL revealed considerable amounts of oxyge- reaching 90%. Regarding the Egyptian Pimenta species con- nated monoterpenes, except in summer (S2) samples that ducted in the foregoing study, results were in accordance to

Fig. 1 The major constituents prevailing in the diﬀerent samples of P. dioica and P. racemosa essential oil.

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what was previously reported, but with certain variations in measurements of time-evolving chemical systems. This type of composition that are mainly attributed to the seasonal and analysis was used for solving the overlapping gas chromato- organ variations, together with the age of both plants and the graphy spectral data into pure chromatogram and mass – place of cultivation.8,9 spectra.27 29 There are many methods for classification, such Gas chromatographic analyses showed that the essential oil as multivariate curve resolution alternating least squares obtained from the leaves of P. racemosa is rich in eugenol (MCR-ALS) and independent components analysis (ICA). PCA (52%), β-myrecene (24%), α-terpineol acetate (27%), and HCA are the most commonly used tools to determine α-terpineol (20%), and chavicol (8%), with traces of limonene similarities and hidden patterns between samples, where the and 1.8-cineole.23 Oil composition varied according to the geo- relationship on the data and grouping is unclear. Large chemi- graphical origin, where oils from Benin showed eugenol cal data sets, as well as bioactive compounds and functional (54.5%) and chavicol (10.3%) with low anti-inflammatory and properties, are the primary target of these methodologies.30 powerful antioxidant activities. In general, the proportion of PCA and HCA were used to reveal the metabolic profile differ- β-myrcene and chavicol were used as key markers to differen- ences and the inter-relationships between the oils in different tiate between the leaf oils of P. racemosa and P. dioica.24 seasons of Pimenta species using the GC chromatographic It has been proven that the alteration in the metabolic pro- data. The PCA score plot explained 100% of the variance of the filing of the essential oils of different organs of a plant is very data for both P. dioica leaves and stems (Fig. 2a and c). common. Basically, this can be explained by virtue of the P. dioica leaves were discriminated into three main groups, chemical composition variation based upon the site of biosyn- where leaves collected in summer and winter were completely thesis, as well as the sites of storage of the secondary metab- segregated, each in a separate quadrant. However, those col- olites.25 The significant differences found in the presence or lected in autumn and spring were superimposed over each absence of certain components, and the relative variation in other in a far distance from the other seasons. The loading the percentage of their content of the essential oils of leaves plot (Fig. 2b) showed that the main discriminating markers and stems can be used as a chemical marker to determine the were β-myrecene and β-caryopyllene for the winter and authenticity of the supplied oils, as previously reported by Gad summer seasons, respectively. However, eugenol was the et al.26 The major constituents prevailing in different samples leading metabolite discriminating spring and autumn. It is of Pimenta oil are represented in Fig. 1. the cause of the differentiation and placement of the spring and autumn samples at the bottom right quadrant, as illus- Chemometrics analysis trated in Fig. 2a, while the concentration of this substance in Multivariate resolution analysis is a group of mathematical the winter sample is very different from those of the other tools that clarified the underlying profiles from a group of three seasons. Concerning P. dioica stems, the PCA score plot Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM.

Fig. 2 PCA score and loading plots of P. dioca (PD) leaves (a and b) and stems (c and d) in diﬀerent seasons. L: leaf; S: stem; S1: Spring; S2: Summer; S3: Autumn; S4: Winter.

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Fig. 3 PCA score and loading plots of P. racemosa (PR) leaves (a and b) and stems (c and d) in diﬀerent seasons. L: leaf; S: stem; S1: Spring; S2: Summer; S3: Autumn; S4: Winter.

revealed significant variation between winter and spring. By significant observation of the loading plot (Fig. 3b), it was Conversely, autumn and summer resembled each other. The obvious that limonene, α-copaene, eugenol and β-myrecene loading plot (Fig. 2d) displayed eugenol, β-caryopyllene, were the loading metabolites explaining the pattern of the PCA β-myrecene, limonene and β-copaene as the main discriminat- score plot. On the contrary, P. racemosa stems collected in ing markers. autumn and spring exhibited significant variation, as they Concerning P. racemosa leaves, the PCA score plot (Fig. 3a) were completely detached from each other. Nevertheless, showed that all seasons were completely segregated from each summer and winter fell in a near distance to each other other in a separate quadrant. However, those collected from (Fig. 3c). Eugenol and α-humulene were the main markers seg-

Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM. winter and autumn were in very close distance to each other. regating spring and autumn, respectively, from each other.

Fig. 4 HCA dendrogram of P. dioica (PD) leaves (a); stems (b) and P. racemosa (PR) leaves (c) and stems (d) in diﬀerent seasons. L: leaf; S: stem; S1: Spring; S2: Summer; S3: Autumn; S4: Winter.

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Additionally, HCA was useful as an unsupervised pattern tissues with subsequent modification in plant biosynthesis recognition method to confirm the results obtained by PCA. processes, resulting in quantitative variations in the essential The dendrograms obtained for both P. diocia and P. racemosa oil components.33 leaves and stems in diﬀerent seasons endorsed the results of PCA (Fig. 4). Cytotoxicity study It is noteworthy to highlight that this is the first time to The cytotoxic activity of P. dioca and P. racemosa essential oils describe the seasonal variations between the essential oil com- and standard doxorubicin was assessed against three diﬀerent positions obtained from P. diocia and P. racemosa leaves and cancer cells; HeLa cells, HepG-2 and MCF-7 (Fig. 5). stems. However, a study was previously conducted on the vola- Srisawat and coworkers34 declared that the cytotoxic activity

tile constituents obtained from the leaves of of crude extracts has been classified based on the IC50 values ff ≤ μ −1 P. pseudocaryophyllus collected from di erent seasons, where into highly active = IC50 20 gmL , moderately active = IC50 −1 −1 the results declared that the oil varied in its composition from of 21–200 μgmL , weakly active = IC50 of 201–500 μgmL , −1 month to month. The optimum yield of oil was observed in and inactive = IC50 > 501 μgmL . Therefore, the leaves and November with chavibetol (50.2–70.9%) constituting the main stems of PD and PR essential oils showed variable moderate 31 component, followed by methyl eugenol (15.4–20.7%). The cytotoxicity on most of the examined cancer cells. It is note- variations in the essential oil compositions could be attributed worthy to highlight that the PR essential oils of both leaves to extensive variations in the microclimate conditions as temp- and stems showed weak cytotoxic effect with respect to MCF-7, −1 erature, precipitation and atmospheric pressure, in addition to showing IC50 values equal to 256.56 and 262.8 μgmL , the effect of winds, soil, and relative humidity, as well as the respectively (Table 2). In this study, the cytotoxic effects of the duration of sun exposure that undoubtedly reflected on the tested essential oils are attributed to the synergistic effect of 32 enzyme activity and the pattern of metabolism. Additionally, the various oil components, such as eugenol, eucalyptol, lina- the long dry season could be the reason for the production of lool, α-terpineol, terpinen-4-ol, β-myrcene, and geraniol.35 higher amounts of essential oils in some plants that was These components target different cytotoxic pathways inside explained by the virtue of the slow uptake of oxygen inside the the cancer cells, including apoptosis, cell cycle arrest, cell cycle, membrane depolarization, inhibition of DNA synthesis, and modulation of ABC transporters.36 HeLa cells were the most sensitive cells, while MCF-7 showed more resistance against the sample’s treatment. MCF-7 showed global resistance of approximately 1.5 times more than the other cells. The essential oils of PDL showed the maximum cytotoxic

eﬀect on the HeLa, HepG-2, and MCF-7 cells with IC50 values − of 122.1, 139.6, and 178.7 (µg mL 1), respectively. However, the ﬀ

Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM. oils of PRS showed the minimum cytotoxic e ects among the tested oils, as compared to the corresponding P. dioca oil. The high expression of ABC-transporters in MCF-7, especially

BCRP, might explain the increase of the IC50 values of MCF-7 cells compared to the other tested cells.13,37 P. dioca and P. racemosa previously showed potent cytotoxicity when tested versus diﬀerent cell lines, such as the −1 macrophage, colon, hepatic, pulmonary, and intestinal cancer Fig. 5 IC50 (µg mL ) of the essential oils of leaves (L) and stems (S) of 10,38 P. dioica (PD) and P. racemosa (PR) on human cervical (HeLa), hepato- cell lines. This diﬀerence in the potency may be due to the cellular (HepG-2) and breast (MCF-7) carcinoma cell lines in comparison use of other cell lines in our study. In addition, the specificity to doxorubicin. of targeting compounds and the sensitivity of cells are variable

−1 Table 2 IC50 (µg mL ) of the essential oils of leaves and stems of P. diocia and P. racemosa on human cervical (HeLa), hepatocellular (HepG-2) and breast (MCF-7) carcinoma cell lines

−1 IC50 (µg ml )

P. dioica P. racemosa

Human cell lines Doxorubicin PDL PDS PRL PRS

HeLa 0.41 ± 0.03 122.07 ± 0.23 131.56 ± 0.29 155.19 ± 0.41 169.37 ± 0.05 HepG-2 0.32 ± 0.02 139.61 ± 0.04 144.81 ± 0.26 173.98 ± 0.02 177.11 ± 0.13 MCF-7 1.23 ± 0.12 178.73 ± 0.11 191.23 ± 0.07 256.56 ± 0.17 262.80 ± 0.28

−1 IC50 values are expressed as Mean ± S.D. of the concentration (µg mL ).

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Table 3 Free binding energies (ΔG) in kcal mol−1 for major compounds from compound to others, and from cell to cell. The chemical identified from the different samples of P. dioica and P. racemosa essen- variations in the essential oils’ constituents could explain the tial oil in the binding sites of DNA topoisomerase II (TOP-2), human difference in the IC50 values of tested samples. The different cyclin-dependent kinase 2 (CDK-2), and Matrix metalloproteinase 13 ff (MMP-13) using in silico molecular modelling regional sources and di erent environmental conditions of the cultivation, collection, and isolation might explain both Compound TOP-2 CDK-2 MMP-13 chemical and biological activities of the essential oils. (E)-β-Ocimene 23.41 15.52 12.27 α-Copaene 11.11 6.51 11.52 Molecular modelling study α-Humulene 54.60 55.43 49.00 α-Phellandrene 17.01 8.47 4.99 An in silico study was performed to assess the cytotoxic effect Alloaromadendrene 35.75 33.87 30.71 β-Caryophyllene 22.28 18.34 17.97 of the major compounds detected in the oils. This was done β-Linalool 10.95 7.86 3.01 via determining their inhibitory effect on human DNA topoi- β-Myrcene 16.24 9.95 6.64 Chavicol −7.08 −13.75 −14.19 somerase II (TOP-2), human cyclin-dependent kinase 2 Eucalyptol 14.11 11.17 10.81 (CDK-2) and matrix metalloproteinase 13 (MMP-13), aiming to Eugenol −10.58 −18.84 −18.78 Limonene 26.92 19.03 16.37 outline their probable mode of action as cytotoxic agents. It is o-Cymene −12.55 −19.93 −22.57 noteworthy to highlight that the tested enzymes are effectively Doxorubicin −16.28 −40.79 −16.30 involved in the growth, division and function of cells.39 Positive values indicate unfavorable interaction. o-Cymene, followed by eugenol and chavicol, showed the Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM.

Fig. 6 The 2D, 3D binding modes and active pockets of o-Cymene within the binding sites of (A) TOP-2; (B) CDK-2 and (C) MMP-13; Dotted light green line indicates a π-donor hydrogen bond; dotted purple line indicates the alkyl and π-alkyl bonds.

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highest fitting with all examined proteins approaching doxo- o-Cymene forms multiple bonds at the site of action of the rubicin. o-Cymene showed ΔG of −12.55, −19.93 and diﬀerent enzymes represented by one π-alkyl bond with − −22.57 kcal mol 1 with respect to TOP-2, CDK-2 and MMP-13, Arg503, and two alkyl bonds with Pro455 and Arg503 at the respectively. Meanwhile, eugenol displayed ΔG of −10.58, TOP-2 active center (Fig. 6A). However, it forms two π-alkyl − −18.85 and −18.78 kcal mol 1 with TOP-2, CDK-2 and bonds with Ile10 and Leu134, in addition to five alkyl bonds MMP-13, respectively. However, chavicol showed −7.08, −13.75 with Ala31, Val18, Leu134 and Ala144 at the binding site of − and −14.19 kcal mol 1 with respect to TOP-2, CDK-2 and CDK-2 (Fig. 6B). Meanwhile, it forms one alkyl bond with MMP-13, respectively. In this aspect, o-cymene and eugenol Leu218 and a π-donor hydrogen bond with Thr245 at the showed inhibitory potential approaching that of doxorubicin, MMP-13 active center (Fig. 6C). Furthermore, o-cymene forms the standard anticancer drug, concerning TOP-2, and CDK-2 a large number of van der Waals interactions with the amino where doxorubicin displayed free binding energies of −16.28 acid residues present at the active sites of the three enzymes. and −40.79, respectively. However, both compounds revealed Regarding eugenol, which is the major constituent in superior activity relative to doxorubicin regarding MMP-13, nearly all samples of Pimenta oil, it forms one conventional − where doxorubicin showed ΔG of −16.31 kcal mol 1 (Table 3). H-bond with Arg503, two π-alkyl bonds with Pro455 and Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM.

Fig. 7 The 2D, 3D binding modes and active pockets of eugenol within the binding sites of (A) TOP-2; (B) CDK-2 and (C) MMP-13; dotted heavy green line indicates conventional H-bonds; dotted light green line indicates C–H bond; dotted purple line indicates π-alkyl bonds.

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Arg503, in addition to one C–H bond with Glu522 at the TOP-2 ponents: Review, J. Essent. Oil Res., 2012, 24, 203, DOI: binding site (Fig. 7A). Concerning CDK-2 and MMP-13, 10.1080/10412905.2012.659528. eugenol forms two H-bonds with Leu83 and Glu81, and four 3 E. A. Weiss, Essential Oil Crops, CABI Publishing, USA, π-alkyl bonds with Ala31, Val18, Leu134 and Ile10 with the 2002. former (Fig. 7B). Meanwhile, it forms two H-bonds with 4 J. A. Duke, M. J. Bogenschutz-Godwin, J. DuCellier and His222 and Phe241, in addition to a π-donor hydrogen bond P.-A. K. Duke, Handbook of Medicinal Spices, CRC Press, with Thr245 with the latter (Fig. 7C). However, the interaction Boca Raton, London, New York, Washington, D.C., 2003. of chavicol within the active sites of the examined proteins 5 B. Contreras, Chemical composition of essential oil of could be interpreted by virtue of the formation of one π-alkyl genus Pimenta (Myrtaceae): Review, in Potential essential bond with Arg503 and one H-bond with Lys505 at the TOP-2 oils, BoD – Books on Demand, 2018, pp. 21–39. binding site, in addition to the formation of two π-alkyl bonds 6 P. S. Rao, S. Navinchandra and K. Jayaveera, An important with Leu134 and Ile10 with the CDK-2 active center. Moreover, spice, Pimenta dioica (Linn.)Merill: A Review, Int. Curr. it forms one H-bond with Met253, one π-alkyl bond with Pharm. J., 2012, 1, 221–225. Leu247, and one π–π bond with Phe252 at the MMP-13 7 T. K. Lim, Edible Medicinal and Non-Medicinal Plants, binding center (Fig. S1†). Thus, it can be concluded that the Fruits, Springer Netherlands, 2012, vol. 3. cytotoxic effects of the tested essential oils are mainly attribu- 8 E. A. Aboutabl, S. H. Tadros and A. M. A. El-Azzouny, ted to its main components, such as o-cymene, eugenol and Composition and antimicrobial activity of volatiles from chavicol, which showed the highest fitting score in the mole- the leaf, flower and fruit of Pimenta officinalis (Berg.) L. and cular docking experiment, in addition to the synergistic effect Pimenta racemosa (Mill) J.W. Moore cultivated in Egypt, of the various oil components, such as eucalyptol, linalool, Egypt. Pharm. J., 2003, 1,45–54. α-terpineol, terpinen-4-ol, β-myrcene, and geraniol.35 9 H. A. El-gizawy, Y. O. Mosaad, N. A. E. Gobba and M. A. Hussein, Chemical composition of the essential oil of the leaves of Pimenta diocia (L.) Merr.and Pimenta race- Conclusions mosa (Mill.) cultivated in Egypt, Int. J. Phytomed., 2018, 10, 226–234. ff The results of this study clarify the di erences between the 10 A. Al-Gendy, F. Moharram and M. Zarka, Chemical compo- chemical compositions of both P. dioica and P. racemosa essen- sition, antioxidant, cytotoxic and antimicrobial activities of ff ff tial oils using di erent harvesting season and di erent organs. Pimenta racemosa (Mill.) J.W. Moore flower essential oil, Eighty-nine compounds were identified in all samples, in J. Pharmacogn. Phytochem., 2017, 6, 312–319. which the oxygenated monoterpene represented by eugenol 11 R. Labib, F. Youssef, M. Ashour, M. Abdel-Daim and was the major constituent. Chemometric analysis was success- S. Ross, Chemical composition of Pinus roxburghii bark fully applied, discriminating both leaves and stems of volatile oil and validation of its anti-inflammatory activity P. racemosa in various seasons. However, P. dioica was partially using molecular modelling and bleomycin-induced inflam- Published on 26 April 2021. Downloaded 7/9/2021 2:15:43 PM. ff segregated. 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