*Corresponding Author:
T. A. Aburjai
Faculty of Pharmacy, Jordan University of Science and Technology, Irbid-Jordan
E-mail: [email protected]

W. Alshaer
Cell Therapy Center, The University of Jordan, Amman-Jordan
Date of Submission 11 July 2019
Date of Revision 07 October 2019
Date of Acceptance 24 December 2019
Indian J Pharm Sci 2020;82(1):179-184  

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

Abstract

In the current study, the compositions of the essential oil obtained from leaves of Ocimum basilicum ‘Cinnamon’ grown in Jordan in a greenhouse was analysed. The antitumor activity of the essential oil was examined against three different cancer cell lines including MDA-MB-231, MCF7 and U-87 MG. The hydrodistillation method was used to extract the essential oil from Ocimum basilicum and the chemical components were analyzed and identified using gas chromatography-mass spectroscopy. The dry weight yield of essential oil was 0.50 % (w/w). Thirty-one components representing 97.80 % of the essential oil were identified. The main chemical components revealed the presence of linalool, eugenol, eucalyptol, hinesol, trans-α-bergamotene and γ-cadinene as the major constituents. In conclusion, the essential oil extracted from greenhouse cultivated Ocimum basilicum European chemotype showed potent antitumor activity.

Keywords

Basil, Ocimum basilicum, essential oils, greenhouse cultivation, antitumor, cancer

Basil (Ocimum basilicum L.) is a culinary herb of the family Lamiaceae and considered as one of the most aromatic plants grow widely at different places around the world, especially in Asia, North America, and Europe[1,2]. Basil is used for many applications in the pharmaceutical and food industry[3]. Traditionally, sweet basil has been used as a medicinal plant in the treatment of kidney malfunction, worms, cough, diarrhea and headache[1,4-8]. Moreover, basil contains a group of phenolic compounds that impart a good antioxidant effect[5,9-11]. O. basilicum is a popular culinary herb and a source of essential oils extracted from leaves and flowering tops that are used in flavoring foods, dental and oral products and fragrances[4].

Essential oil of basil primarily consists of monoterpenes, sesquiterpenes and phenylpropanoids with oxygenated derivatives[1,3,12,13]. There are important differences among basil species including the variation in the chemical compositions and the essential oil content and components. In addition to the species, such variation may be linked to the growing and cultivation environment that varies among different locations[1,14]. There are two main types of basil oil commonly used in the market, the reunion type which is mostly composed estragole (80 %) and (ii) the European type usually obtained from France, Italy, Egypt, and South Africa, which is mostly composed of linalool (35-50 %) and estragole (15-25 %)[15].

Cancer is a heterogeneous group of diseases characterized by the uncontrolled growth of cells that escape from body defenses and many times from the conventional antitumor drugs. Cancer is a leading cause of morbidity and mortality worldwide. In 2018, 18.1 million new cancer cases were diagnosed and 9.6 million cancer-related deaths were reported[16,17]. The development of new therapeutics and therapeutic systems is a continuous battle for successful and effective cancer treatment. Tumor cells are heterogeneous and can develop intrinsic or acquired multidrug resistance mechanisms including increased drug efflux, drug inactivation, drug target alteration, increased DNA damage repair, epigenetics, cell death inhibition and epithelial-mesenchymal transition[18,19]. Plants are considered a rich source of natural compounds that can be used for the discovery of anticancer agents. Around 50 % of conventional anticancer chemotherapeutics are originated from plants. One important example is paclitaxel which is widely used for the treatment of different types of cancers, including breast, ovarian and pancreatic cancer[20-22]. Essential oils are aromatic secondary metabolites of plants that possess wide range bioactivities with pharmacological interests including anticancer activity[23,24]. Consequently, adding more therapeutic options for cancer prevention.

It appears based on the literature search, no studies were reported in which the composition of the essential oil of O. basilicum grown in Jordan was investigated. Therefore, this study was taken up to carry out a detailed chemical investigation of the essential oil composition of the chemotypes of O. basilicum grown in Jordan. Moreover, this study aimed to investigate the anticancer activity of essential oil of O. basilicum grown in a greenhouse.

Seeds of O. basilicum ‘cinnamon’ were collected from a mother plant grown in the botanical garden of Faculty of Agriculture, The University of Jordan (UJ)-Amman, seeded and transplanted for cultivation under controlled greenhouse conditions; 25° and 40-60 % relative humidity with conventional cultivation practices of irrigation and fertilization (20-20-20-TE). Basil leaves were collected when the height of the plant reached 20-25 cm. Plant material was taxonomically identified by direct comparison with an authenticated sample at the herbarium of the Faculty of Science, Department of Biology, the University of Jordan. A voucher specimen of plant material was deposited at the School of Pharmacy, JU (GH-B-18, for greenhouse). N-hexane (GC grade) was purchased from Tedia (USA), A hydrocarbon mixture of n-alkanes (C8-C20) was purchased from Fluka (Switzerland).

Plant material (500 g) was collected as mentioned above and dried at room temperature in a shaded and ventilated area. Dried plant material (100 g) was hydrodistilled for 3 h using the Clevenger apparatus (JSGW, India). The obtained essential oil was separated, dried over anhydrous sodium sulfate (Analar, England) and stored in a sealed vial in the refrigerator. The yield of oil was calculated as percent weight by weight (w/w %) of the dry plant material.

A 5 μl aliquot of oil samples diluted with 1 ml of GCgrade n-hexane and then 1 μl samples of the diluted oil were injected into the GC-MS systems for analysis. From the diluted oil sample prepared above, 1 μl aliquot was injected using an automated injector into the GCMS system for analysis using a variant chrompack CP- 3800 GC/MS/MS-200 (Satum, Netherlands) equipped with split-splitless injector and DB-5 capillary column (5 % diphenyl 95 % dimethyl polysiloxane, 30 m×0.25 mm ID, 0.25 μm film thickness). The injector temperature was set at 250° with a split ratio of 1:30. A linear temperature program was used to separate the different oil components. The column temperature was held constant at 60° for 1 min. The temperature was then increased to 250°, at a rate of 3°/min and then was held constant at 250° for 2 min, with a total runtime of about 66 min. The flow rate of the carrier gas (helium) was 1 ml/min. Each sample was analyzed twice. The mass detector was set to scan ions between 40-400 m/z using full scan mode and electron impact (El, 70 eV). A hydrocarbon mixture of n-alkanes (C8-C20) was analyzed separately by GC-MS using the same column (DB-5) and under the same chromatographic conditions. Linear retention index (Arithmetic-Kovats index) was calculated for each component (each peak) separated by GC-MS using the value of its retention time and the retention times of the reference n-alkanes applying Van Den Dool Eqn.[25]. The constituents of the essential oils were identified using their recorded mass spectra and the built-in computerized matching with WILEY, NIST and ADAMS libraries, and by comparing their calculated arithmetic indices with reported values in the literature[26]. Identification of linalool, eugenol, and eucalyptol was further confirmed by co-chromatography with authentic standards (Sigma-Aldrich) under the same chromatographic conditions mentioned above.

GC-FID analysis was also carried out to obtain qualitative complementary results to those obtained by GC-MS as well as to obtain quantitative measures of the identified compounds. GC-FID was carried out on a ThermoQuest gas chromatograph coupled to an FID detector equipped with a split-splitless injector (split ratio 1:30) and HP-5 capillary column (Crosslinked 5 % PH ME Silicone, 30 m×0.32 mm×0.25 μm film thickness). One microlitre aliquots were autoinjected into the GC-FID system and analyzed using the same linear temperature program applied in the GCMS analysis above. The carrier gas was nitrogen (N2 99.99 %) and the flow rate was 1 ml/min. Each sample was analyzed triplicate. Essential oil constituents were identified by calculating the linear retention index of each constituent relative to (C8-C20) n-alkanes mixture (which was analyzed separately by GC-FID using the same column) and applying Van Den Dool Eqn.[25]. To identify the constituents, the calculated arithmetic indices were then compared with reported values in the literature[26]. Constituents quantification was achieved by dividing the peak area of each constituent by the total peak area of all oil constituents and multiplying the result by 100.

U-87 MG (glioblastoma cell line), MCF7 (ER+ breast cancer) and MDA-MB-231 (triple negative breast cancer cell line), were obtained from American Type Culture Collection (ATCC®; USA). U-87 MG was cultured in Eagle’s minimum essential medium (EMEM, ATCC®, USA), containing 1 mM L-glutamine, 100 U/ml penicillin-streptomycin and 10 % fetal bovine serum (Gibco, USA). MCF7 was maintained in Roswell Park Memorial Institute 1640 medium (RPMI; Euroclone, UK) supplemented with 1 mM L-glutamine, 100 U/ml penicillin-streptomycin and 10 % fetal bovine serum. MDA-MB-231 was cultured in minimum essential medium (MEM, Euroclone, UK) supplemented with 1 mM L-glutamine, 100 U/ml penicillin-streptomycin and 10 % fetal bovine serum. Cells were grown at 37° in 5 % CO2 atmosphere.

U-87 MG, MCF7 and MDA-MB-231 were seeded in 96-well plates at 7000 cells/well in 0.1 ml in complete EMEM, RPMI and MEM consequently. After 24 h, cells were treated and incubated with different concentrations of basil extract for 72 h at 37°. Afterward, the old media was aspirated and 100 μl of fresh medium and 15 μl of MTT reagent was added to each well and incubated for 4 h to allow the formation of formazan crystals. After removing all media from the wells, 50 μl of dimethyl sulfoxide (DMSO) was added to each well and mixed carefully. The absorbance values were measured using a microplate reader (GloMax®-Multi+ Detection System) at 570 nm to determine relative viabilities. Finally, data were analyzed by GraphPad Prism 6.

The results obtained revealed the presence of 31 components of the essential oil of O. basilicum based on GC-MS analysis (Figure 1). The identified compounds are listed in Table 1 in elution order from the DB-5 column, along with the percent composition of each component, its KI and class. The major constituents of the essential oil of O. basilicum were linalool (36 ±2.6 %), eugenol (14.2±3.4 %), eucalyptol (11.4±2.2), trans-α-bergamotene (9.0±0.9 %), hinesol (3.2±0.1 %), and γ-cadinene (3.1±0.1 %) summarized in figure 2A.

ijpsonline-chromatographic-components

Figure 1: GC-MS chromatographic profile of essential oil components of O. basilicum 'Cinnamon'

ijpsonline-components-class

Figure 2: Major components and class
(A) Comparison of the % contents of major components identified in the essential oil of O. basilicum cinnamon leaves, (B) comparison of the % contents of chemical class group identified in the essential oil of O. basilicum cinnamon leaves

Compound name Formula Mol weight (g/mol) KI* cal. KI** lit. % content GH
(Avg±SD)
5 µl oil/1 ml hexane
Chemical class
α-Pinene C10H16 136.24 934 932 0.28±0.02 Monoterpene hydrocarbon
Sabinene C10H16 136.23 973 969 0.31±0.05 Monoterpene hydrocarbon
β-Pinene C10H16 136.24 980 974 0.88±0.21 Monoterpene hydrocarbon
Myrcene C10H16 136.24 988 988 0.44±0.13 Monoterpene hydrocarbon
Limonene C10H16 136.24 1030 1024 0.30±0.09 Monoterpene hydrocarbon
Eucalyptol (1,8-cineol) C10H18O 154.25 1034 1026 11.36±2.23 Oxygenated monoterpene
Terpinolene C10H16 136.24 1087 1086 0.26±0.05 Monoterpene hydrocarbon
Linalool C10H18O 154.25 1101 1095 36.26±2.58 Oxygenated monoterpene
Trans-sabinenehydrate C10H18O 154.25 1105 1098 0.47± 0.02 Monoterpene hydrocarbon
Camphor C10H16O 152.24 1150 1141 1.61±0.09 Oxygenated monoterpene
δ-Terpineol C10H18O 154.25 1172 1162 0.26±0.02 Oxygenated monoterpene
Terpinen-4-ol C10H18O 154.25 1182 1174 0.24±0.00 Oxygenated monoterpene
α-Terpineol C10H18O 154.25 1197 1186 1.91±0.07 Oxygenated monoterpene
Bornyl acetate C12H20O2 196.29 1286 1287 1.11±0.02 Miscellaneous
Eugenol C10H12O2 164.20 1355 1356 14.2±3.36 phenolic compound
β-Elemene C15H24 204.36 1391 1389 2.78±0.02 Sesquiterpene hydrocarbon
Methyl eugenol C11H14O2 178.23 1400 1403 2.1±0.00 Phenolic compound
Trans-α-bergamotene C15H24 204.35 1434 1432 9.00±0.86 Sesquiterpene hydrocarbon
α-Guaiene C15H24 204.36 1438 1437 0.41±0.15 Sesquiterpene hydrocarbon
Trans-β-farnesene C15H24 204.36 1452 1454 1.00±0.08 Sesquiterpene hydrocarbon
α-Humulene C15H24 204.36 1458 1452 0.59±0.02 Sesquiterpene hydrocarbon
Cis-murrola-4(14),5-diene C15H24 204.36 1464 1465 0.86±0.04 Sesquiterpene hydrocarbon
Germacrene D C15H24 204.36 1483 1484 2.01±0.16 Sesquiterpene hydrocarbon
Cis-β-guaiene C15H24 204.36 1491 1492 0.29±0.01 Sesquiterpene hydrocarbon
Valencene C15H24 204.36 1498 1496 0.99±0.04 Sesquiterpene hydrocarbon
Trans-β-guaiene C15H24 204.36 1504 1502 1.31±0.08 Sesquiterpene hydrocarbon
Cis-α-bisabolene C15H24 204.35 1509 1506 0.37±0.05 Sesquiterpene hydrocarbon
γ-cadinene C15H24 204.36 1515 1513 3.2±0.11 Sesquiterpene hydrocarbon
Trans-calamenene C15H22 202.34 1522 1521 0.28±0.00 Sesquiterpene hydrocarbon
β-Sesquiphellandrene C15H24 204.35 1524 1521 0.60±0.19 Sesquiterpene hydrocarbon
Hinesol C15H26O 222.37 1645 1640 3.17±0.07 Oxygenated sesquiterpene

Table 1: Composition of the essential oil of O. basilicum

The chemical components of sweet basil have been described in several reports. For example, Kruger et al. reported the major components of 270 sweet basil accessions[27]. The major constituents were found to be linalool, methyl chavicol, citral, 1,8-cineole, camphor, thymol, methyl cinnamate, eugenol, methyl eugenol, methyl isoeugenol and elemicin[27]. Moreover, Marotti et al. have reported the major oil constituents of different basil chemotypes such as in the Italian basil type linalool and methyl chavicol, in the tropical basil chemotype, methyl cinnamate and in the basil chemotype grown in North Africa, Russia, Eastern Europe and parts of Asia was eugenol[28].

The major components of the essential oil can be classified into 5 major classes including oxygenated monoterpenes (51.7±4.8), sesquiterpene hydrocarbons (23±1.4), phenolic compounds (16.3±3.3), oxygenated sesquiterpenes (2.6±1.1) and monoterpene hydrocarbons (3.2±0.1). These major components represent 97.1 % of the essential oil (Figure 2B).

After analyzing the major components and chemical classes of the O. basilicum essential oil, an attempt was made to investigate the anticancer effect against known common and invasive cancers. For this purpose, 3 cancer cell lines were chosen for representing two types of cancers, including a triple negative breast cancer cell line (MDA-MB-231), ER+ breast cancer (MCF7), and the glioblastoma (U-87 MG). The cells were treated with different concentrations of essential oil for 72 h followed by the detection of viable cells using MTT assay. The inhibitory concentrations 50 (IC50) were calculated based on the percent remaining viable cells of 3 independent experiments. The IC50 values were 432.3±32.2 μg/ml on MDA-MB-231, 320.4±23.2 μg/ml on MCF7 and 431.2±15.3 μg/ml on U-87 MG as shown in figure 3. These results agree with previously reported results. Linalool, eugenol and eucalyptol are the major components of O. basilicum essential oil, which have been shown to exhibit anticancer effect against different types of cancers such as leukemia, lymphoma, breast, colorectal, bone sarcoma, hepatic, gastric, lung and melanoma[14,29-31]. Interestingly, linalool was reported to potentiate doxorubicin on both MCF7 breast cancer and the doxorubicin-resistant MCF7/Adr cells through inducing apoptosis[32]. Different mechanisms have been described for such anticancer effects, including the induction of DNA repair, apoptosis, cell cycle arrest, detoxification and antioxidant enzymes[29-31].

ijpsonline-cancer-cell

Figure 3: Effect of O. basilicum essential oil on viability of cancer cell lines
Dose response curves to determine IC50 of O. basilicum essential oil on A. U-87 MG, B. MDA-MB-231 and C. MCF7 cell lines

To the best of our knowledge, this is the first report on the essential oil composition of O. basilicum grown in Jordan and its chemotype. The main essential oil components of O. basilicum were linalool/eugenol, suggested the linalool chemotype essential oil of O. basilicum European chemotype. In the literature, there is a low amount of data describing the anticancer effect of O. basilicum essential oil on triple-negative breast cancer cell line (MDA-MB-231), ER+ breast cancer (MCF7), and the glioblastoma (U-87 MG). The findings of this study revealed the promising anticancer effect of O. basilicum essential oil cultivated in Jordan.

Conflict of interest:

The authors declare no conflict of interest.

Acknowledgment:

The authors are grateful to the Deanship of Scientific research, University of Jordan, Amman – Jordan for supporting this work (Fund No. 2086). Authors thank Prof, Dr. Dawoud Al-Issawi, Plant taxonomist, Department of Biology, Faculty of Science, JU for authenticating the plant material.

References