Chemical Composition, Antioxidant, Anti-Inflammatory and Anti-Proliferative Activities of Essential Oils of Plants from Burkina Faso

This research highlights the chemical composition, antioxidant, anti-inflammatory and anti-proliferative activities of essential oils from leaves of Ocimum basilicum, Ocimum americanum, Hyptis spicigera, Lippia multiflora, Ageratum conyzoides, Eucalyptus camaldulensis and Zingiber officinale. Essential oils were analyzed by gas chromatography–mass spectrometry and gas chromatography–flame ionization detector. Major constituents were α-terpineol (59.78%) and β-caryophyllene (10.54%) for Ocimum basilicum; 1, 8-cineol (31.22%), camphor (12.730%), α-pinene (6.87%) and trans α-bergamotene (5.32%) for Ocimum americanum; β-caryophyllene (21%), α-pinene (20.11%), sabinene (10.26%), β-pinene (9.22%) and α-phellandrene (7.03%) for Hyptis spicigera; p-cymene (25.27%), β-caryophyllene (12.70%), thymol (11.88), γ-terpinene (9.17%) and thymyle acetate (7.64%) for Lippia multiflora; precocene (82.10%)for Ageratum conyzoides; eucalyptol (59.55%), α-pinene (9.17%) and limonene (8.76%) for Eucalyptus camaldulensis; arcurcumene (16.67%), camphene (12.70%), zingiberene (8.40%), β-bisabolene (7.83%) and β-sesquiphellandrène (5.34%) for Zingiber officinale. Antioxidant activities were examined using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. O. basilicum and L. multiflora exhibited the highest antioxidant activity in DPPH and ABTS tests, respectively. Anti-inflammatory properties were evaluated by measuring the inhibition of lipoxygenase activity and essential oil of Z. officinale was the most active. Anti-proliferative effect was assayed by the measurement of MTT on LNCaP and PC-3 prostate cancer cell lines, and SF-763 and SF-767 glioblastoma cell lines. Essential oils from A. conyzoides and L. multiflora were the most active on LNCaP and PC-3 cell lines, respectively. The SF-767 glioblastoma cell line was the most sensitive to O. basilicum and L. multiflora EOs while essential oil of A. conyzoides showed the highest activity on SF-763 cells. Altogether these results justify the use of these plants in traditional medicine in Burkina Faso and open a new field of investigation in the characterization of the molecules involved in anti-proliferative processes.


Introduction
Representing 7.6 million deaths worldwide, or approximately 13% of deaths, cancer is the second cause of mortality [1]. Cancer is nowadays case of major death in the world particularly in the low income countries and middle-incomes. Those numbers of cancer mortality could increase by 50% to reach 15 million by 2030 worldwide [1]. In Africa, in 2008, 682,000 people have been affected by cancer. The mortality was 572,402 during the same period. Epidemiological studies provide 1.2 million new cases of cancer in Africa by 2030 with more than 970,000 dead if adequate preventive measures are not taken quickly. The most common types of cancer in Africa are women cervical cancer of the uterus, breast and primary liver cancers, and men prostate cancer and Kaposi's sarcoma; even though epidemiological data regarding cancer in Sub-Saharan Africa are scarce. In Burkina Faso, the few statistics show that the annual incidence of cancer from January 1986 to December 2006 in the three laboratories of anatomy and pathological cytology of Ouagadougou was 200 cases per year [2]. This extremely low rate was mainly explained by the very low participation of the population in screening of cancers. The most frequent cancers for men are skin cancer (11.37%), followed by non-Hodgkin lymphoma (9.80%) and prostate cancer (9.69%) while breast (23.81%) and uterine neck cancers (22.99%) are the most frequent for women [2].
Plants are a potential source of drug discovery and development of cancer chemoprevention [3,4]. They could thus provide a hope for finding anticancer molecules available and efficient for the treatment of persons with cancer. In fact, many cytotoxic molecules, which are of plant origin, are widely used in chemotherapy [5]. Burkina Faso is a country with many unknown plant whose compounds could be used in medicine [6]. Essential oils (EOs) are volatile, complex compounds characterized by a strong odor and are formed by aromatic plants as secondary metabolites. They have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, anticancer, antioxidant, antidiabetic, cardiovascular, and cosmetic and food applications [7,8]. Among the various plants with putative pharmacological properties, Ocimum basilicum Linnaeus, Ocimum americanum Linnaeus, Hyptis spicigera Lamarck, Lippia multiflora Moldenk, Ageratum conyzoides Linnaeus, Eucalyptus camaldulensis Dehnhardt and Zingiber officinale Roscoe are common in Burkina Faso. Leaves and leaf stems of these plants are widely used in traditional medicine in Burkina Faso among others to treat angina, swellings, wounds, scorpion and snake bites, malaria, hemorrhoids, rheumatism, fevers, nervous dyspepsia, rheumatism, uterus diseases, jaundice [9] and are also used as antioxidant, antiinflammatory and fungicide [9]. The plants used in the treatment of certain inflammatory and oxidative diseases may have anticancer effects. Indeed, there is a relationship between the production of reactive oxygen species (ROS) to the origin of oxidation and inflammation that can lead to cancer [10]. In fact, initial experiments on the role of ROS in tumor initiation have assumed that oxidative stress acts as a DNA-damaging agent, effectively increasing the mutation rate within cells and thus promoting oncogenic transformation [10]. However, recent studies have revealed that in addition to inducing genomic instability, ROS can specifically activate signaling pathways and thus contribute to tumor development through the regulation of cellular proliferation, angiogenesis, and metastasis [11]. Chronic inflammation has been linked to various steps involved in carcinogenesis, including cellular transformation, promotion, survival, proliferation, invasion, angiogenesis, and metastasis [12,13]. Several human chronic disease states including cancer have been associated with oxidative stress produced through either an increased free radical generation and/or a decreased antioxidant level in the target cells and tissues [14,15].
The aim of the present study was to investigate chemical compositions and chemotypes, antioxidant, anti-inflammatory and antiproliferative activities of EOs from these seven plants of Burkina Faso in vitro as well as on cell cultures.

Essential oils (EOs)
Fractions of each dried plant material (200 g) were submitted to hydrodistillation using an alembic/Clevenger-type apparatus for 3 h as previously described [41]. Anhydrous sodium sulfate was used to remove water after extraction. EOs were stored in airtight containers in a refrigerator at 4uC until GC-FID and GC/MS analyses and biological tests. EOs were diluted in hexane (1/30, v/ v) for GC/FID analysis.

Gas chromatography-mass spectrometry (GC/MS) analysis
EOs were analyzed on an Agilent gas chromatograph Model 7890, coupled to a Agilent MS model 5975, equipped with a DB5 MS column (20 m60.20 mm, 0.20 mm film thickness), programming from 50uC (5 min) to 300uC at 8uC/min, 5 min hold. Helium was used as carrier gas (average flow 1.0 ml/min). Oven temperature program was from 50uC (3.2 min) to 300uC at 8uC/ min, 5 min post run at 300uC. Sample (1 mL) was injected in split mode (1:150); injector and detector temperature were at 250uC and 280uC respectively. The MS working in electron impact mode at 70 eV; electron multiplier, 1500 V; ion source temperature, 230uC; mass spectra data were acquired in the scan mode in m/z range 33-450.

Identification of components
Oil constituents were identified by comparison of their retention indices with those of the literature, determined in relation to a homologous series of n-alkanes (C8-C32) under the same operating conditions. Further identification was made by comparison of their mass spectra with those stored in NIST library [42] or with mass spectra from literature [43]. Component relative percentages were calculated based on GC peak areas without using correction factors. The major identified compounds are indicated on Figure 1.
DPPH radical scavenging assay DPPH (Sigma-Aldrich, L'Ile d'Abeau, France) radical scavenging activity was measured as described by Velasquez [20]. Briefly, 0.5 mL of EO (8 mg/mL in methanol) was added to 1 mL of DPPH solution (20 mg/mL in methanol) freshly prepared. After shaking, the mixture was incubated for 15 min in darkness at room temperature and then absorbance was measured at 517 nm against a blank (mixture without EO). Quercetin (Sigma-Aldrich) was used as positive control. Inhibition percentage of free DPPH radicals (I %) was calculated following the formula: I (%) = (12A Sample/A Blank)6100, A blank and A sample being the absorbance of the blank and sample reactions respectively.

ABTS + radical cation decolorisation assay
The spectrophotometric analysis of ABTS + scavenging activity was determined according to the method Re et al. [44]. Preparation of ABTS +. solution was done by dissolving 10 mg of ABTS in 2.6 ml of distilled water. Then, 1.7212 mg of potassium persulfate was added and the mixture is kept in the dark at room temperature for a maximum of 12 hours. The mixture was then diluted with ethanol in order to obtain an absorbance of 0.7060.02 to 734 nm. In 96-well plates, 50 ml of test sample solution of essential oil (8 mg/mL) were added to 200 ml of ABTS + solution freshly prepared. The same process was performed for quercetin (1 mg/mL) used as a positive control. Wholes are protected from light for 15 min and the concentration is read at 734 nm in a spectrophotometer against a standard curve with 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Sigma-Aldrich). Concentration compounds having a reducing effect on the radical cation ABTS + (antioxidant) is expressed in micromoles Trolox equivalent per gram of EO (mmolET/g) using the following formula: C = (c6D)/Ci. C, concentration of antioxidant compounds in mMET/g; c, concentration of sample read; D, dilution factor; Ci, concentration of stock solution.

Anti-inflammatory capacity
Lipoxygenase (EC 1.13.11.12) type I-B inhibiting activity was essayed spectrophotometricaly as described by [45] with minor modifications. Briefly 100 mL of the enzyme solution (at the final concentration of 200 U/mL) was prepared in boric acid buffer (0.2 M; pH 9.0), mixed with 25 mL of extract solution (8 mg/mL in boric acid buffer) and then incubated at room temperature for 3 min. Reaction was subsequently initiated by the addition of substrate solution (linoleic acid, 250 mM), and the velocity was recorded for 90 seconds at 234 nm. Boric acid buffer was used as negative control (activity of enzyme without extract solution). The percentage of lipoxygenase inhibition was calculated according to the following equation: Inhibition (%) = (Vo control 2Vo sample ) 6100/Vo control . Vo control is the activity of enzyme in absence of extract solution, and Vo sample is the activity of the enzyme in the presence of the analyzed EOs.

Cell culture
Two human prostate cancer cell lines were used: LNCaP, an androgen responsive cell line with a low metastatic potential derived from a lymph node metastasis [46], and PC-3, an androgen insensitive cell line with a high metastatic potential derived from bone metastasis [47]. Two human glioblastoma cell lines with disparate radiation sensitivity were used, SF-763 and SF-767. Cells were grown at 37uC in a humidified chamber with 5% CO 2 as monolayer adherent cultures in 75 cm 2 tissue culture

Cytotoxicity assay
Stock solution of 10 ml/ml of EOs was prepared in 1% DMSO (Sigma-Aldrich) in complete medium. Global cell growth was assessed using the colorimetric MTT (3[4,5-dimethylthiazol-2-yl]diphenyltetrazolium bromide) assay (Sigma-Aldrich). Cells were incubated for 24 hours in 96-well plates (50,000 cells/ml) before incubation with EOs. Cell proliferation test is based on the ability of living cells to reduce MTT (yellow) into its metabolite blue formazan (violet). After 72 h incubation with or without EO, the number of living cells is directly proportional to the intensity of the violet color measured quantitatively by spectrophotometry using a microplate reader type Bio-Rad 11885 at 490 nm. Each experimental condition was analyzed in quadruplicate, with three experiments for each EO. Growth inhibition was calculated as follow: % growth inhibition = 1002(OD test sample 2OD blank / OD control 2OD blank )6100.

Statistical analysis
All in vitro experiments were done in quadruplicate and each data point represents the average of at least three independent experiments. All data are reported as the mean 6 SD. Data were analyzed by 1-way analysis of variance followed by the Tukey multiple-comparison test. Analyses were done by using XLSTAT7.1 software. A P value less than 0.05 was used as the criterion for statistical significance.

Results and Discussion
Composition analysis of the various essential oils  Table 1. Briefly, EOs mainly contain a complex mixture of monoterpene hydrocarbons, sesquiterpene hydrocarbons and oxygen containing mono-and sesquiterpenes (Figure 1). Monoterpenes hydrocarbons were dominant in the EOs of E. camaldulensis, L. multiflora, H. spicigera and O. americanum. Sesquiterpene hydrocarbons were the most abundant in the EO of Z. officinale. Oxygenated terpenes were the most dominant constituents of the EOs of A. conyzoides and O. basilicum. The EO of L. multiflora and O. americanum also contained relative percentage of oxygenated terpenes, 25.10% and 23.94%, respectively.
A total of 29 compounds were identified in the EO of O. basilicum (Figure 2A). Two major compounds were identified: aterpineol (59.78%) and b-caryophyllene (10.54%). The minor compounds were a-humulene  (7) bergamotene chemotypes [48]. The terpineol a/b caryophyllene chemotype of the EO in leaves of O. basilicum of our analysis (Table 1) has not been reported yet. This is not surprising as the composition of EO depends on genetics, age, season and/or environment of the plant [49].

Antioxidant activity of the essential oils
An antioxidant can be defined as any substance capable of competing with other oxidizable substrates at relatively low concentrations and delay or prevent the oxidation of these substrates. The DPPH radical-scavenging activities of the seven EOs and of references are shown in Table 2. EO of O. basilicum showed the best ability to scavenge DPPH + radical created in vitro with a percentage of inhibition of 55.67% for a concentration of 8 mg/mL while L. multiflora exhibited the highest capability to reduce ABTS + (1.02 mmolET/g) followed by O. basilicum. Besides, E. camaldulensis, L. multiflora and H. spicigera respectively also   presented interesting antioxidant activities. For some EOs, results differed between DPPH and ABTS methods. These variations may be explained by the mechanisms involved in the radical antioxidant reactions. Indeed, antioxidant activity of the tested compounds depended on the stressing agent used and the mechanism of action of the antioxidant [56]. Other factors, such as stereo-selectivity of the radicals or the solubility of EOs in different testing systems, may also affect the capacity of individual EO to react and quench different radicals [57]. In this sense, Del Castillo et al. [58] analyzed coffee brews from several roasting processes and reported higher responses by the ABTS test in aqueous vs. ethanol dilution. They attributed the difference to the fact that some components, making an important contribution to the antioxidant activity of the aqueous dilutions, were not soluble in ethanol. Probably the fact that the DPPH method was developed in methanol media was responsible for the lower response found. Likewise, Wang and Jiao [59] evaluated the radical scavenging capacity of berry crops using superoxide radicals, hydroxyl radicals, and other reactive oxygen species. The berry crop that had a greater scavenging activity against superoxide radicals did not necessarily have a higher activity to quench hydroxyl radicals [59]. In our study, the major compounds of EO of O. Basilicum are alcohol monoterpenes while the major compounds of EO of L. multiflora are carbohydrate monoterpenes; this difference of chemical functions of compounds in these two EOs could hence explain the activity differences between the two methods.
In addition monoterpenes found in these EOs may act as antioxidant agents. Previous studied showed that oxygenated monoterpenes, such as thymol, carvacrol and a-terpineol, were mainly responsible for the antioxidant potential of the plant oils which contain them [60,61]. The monoterpene b-caryophyllene also possesses a free radical scavenging activity using the DPPH assay [62].

Anti-inflammatory properties of the essential oils
Chronic inflammation increases the risk for various cancers, indicating that eliminating inflammation may represent a valid strategy for cancer prevention and therapy. For that purpose we evaluated the anti-inflammatory properties of EOs we isolated. Among the seven plants, Z. officinale presented the highest antiinflammatory activity indeed a 50.9% of inhibition of lipoxygenase at 0.4 mg/ml while this inhibition was complete (100%) at 8 mg/ ml (Table 3). Z. officinale activity (50.9% of inhibition at 0.4 mg/   (Table 3) were active at 8 mg/ml and no significant effect was observed at 0.4 mg/ml.. These results are in agreement with several studies of anti-inflammatory abilities of sesquiterpenes compounds [63]. Lipoxygenases and their active metabolites (HPETE and HETE) are involved in many human cancers [64]. Pidgeon et al. [65] have shown the involvement of 15-lipoxygenase in the development of breast, prostate, colorectal cancers where it is over expressed [65]. Moreover, the 5-LOX mRNA and/or its activating protein FLAP are over expressed in prostate cancer [66].

Anti-proliferative effects of the essential oils
To best of our knowledge, this is the first report on the antiproliferative activity of these EOs. To test such effects, we chose two types of cancer cell lines. Prostate cancer cell lines, LNCaP and PC3, represent the paradigm of the prostate tumors with androgen sensitivity and androgen resistance, respectively; besides, this cancer has its incidence increasing in Westernized countries. Glioblastoma is recognized as the most common and lethal form of central nervous system cancer. Currently used surgical techniques, chemotherapeutic agents, and radiotherapy strategies have done very little in extending the life expectancies of patients diagnosed with this tumor. SF-767 and SF-763 cell lines are radiotherapy resistant; in SF-767 cells, STAT3 pathway is activated while in SF-763, both STAT3 and AKT pathways are constitutively activated.
O. basilicum, Z. officinale, L. multiflora and A. conyzoides have shown anti-proliferative activity on both LNCaP and PC-3 cell lines of prostate cancer and both SF-767 and SF-763 cell lines of glioblastoma (Table 4). Conversely, EOs from E. camaldulensis, O. americanum and H. spicigera did not present any antiproliferative activity even at a maximum concentration of 8 mg/ml (Table 4). A. conyzoides (IC 50 0.35 mg/mL) and Z. officinale (IC 50 0.38 mg/ mL) have the highest antiproliferative activity on LNCaP cell line. EOs of L. multiflora and O. basilicum were the most active on PC-3 cell line (IC 50 0.30 mg/mL) and SF-767 cell line with (IC 50 0.30 mg/mL). Monoterpenes prevent the carcinogenesis process at both initiation and promotion/progression stages [67]. Monoterpenes are effective in treating early and advanced cancers. In fact, monoterpene pirillyl alcohol has been described to have an anti-proliferative activity on glioblastoma by inhibiting the Na/K-ATPase pump [68]. Other monoterpenes such as limonene have been shown to prevent mammary, liver, lung, and other cancers [67]. Thus, the high content of monoterpenes of these essential oils could explain their antiproliferative activity. In our study, all antiproliferative effects of the tested EOs are dose- (Figure 3) and time- (Figure 4) dependent.
All EOs with an anti-proliferative activity (Table 4) are also antioxidant ( Table 2) and show anti-inflammatory properties (Table 3). Even though there is a relationship between these three activities, the various mechanisms involved for each EO could explain why the variation of these effects. Indeed reactive oxygen species may interact with and modify cellular protein, lipid, and DNA, which results in altered target cell function. The accumulation of oxidative damage has been implicated in both acute and chronic cell injury including possible participation in the formation of cancer [69]. A link between inflammation and cancer has long been suspected, but its molecular nature remained still to be defined [70]. Chronic infection and consecutive inflammation may directly affect the cells that eventually become transformed as well as exert indirect effects on the tumor cell through surrounding cells [70].
EOs of O. americanum, H. spicigera and E. camaldulensis have shown antioxidant activity and anti-inflammatory activity but no antiproliferative activity. The Eucalyptol or 1, 8-cineol, the major compound of essential oil of O. americanum and E. camaldulensis has also not effect on LNCaP and PC-3 cell lines of prostate cancer, and the SF-767 and 763 cell lines of glioblastoma to a maximum of 1 mg/mL concentration tested ( Figure 5). However, Murata et al. showed that 1, 8-cineole induced specific apoptosis, not necrosis, in human colon cancer cell lines HCT116 and RKO [71]. Each type of cancer involves a specific signaling pathway. Moreover an anticancer compound does not necessarily treat all types of cancer; such is the case of a-pinene ( Figure 5). What may explain the fact that the 1, 8 cineole is inactive on prostate cancer and glioblastoma. Accordingly the results of our work show that a-pinene, the major compound of H. spicigera, showed antiproliferative activity on LNCaP and PC-3 cell lines of prostate cancer with IC 50 of 0.37 mg/ml and 0.82 mg/ml, respectively. The combination of a-pinene and the essential oil of H. spicigera (50:50, v:v) resulted in a decreased of this activity ( Figure 5). This significant decrease in the activity of combinations of a-pinene with EO of H. spicigera leads to an antagonistic effect between apinene and the essential oil of H. spicigera. This antagonistic effect could justify the inactivity of EO from H. spicigera alone (Figure 3). On SF-763 cells IC 50 of a-pinene is 0.43 mg/ml while no significant anti-proliferative activity was observed on SF-767 cells ( Figure 5). The main difference between these two cell lines regards the survival pathways: indeed STAT3 pathway is activated in SF-767 cells, while both Akt and STAT3 pathways are induced in SF-763 cells. These differences were hypothesized to explain chemoresistance [72] and could also be linked to the differential response to a-pinene.
In conclusion we have evaluated for the first time the chemical composition, ability to scavenge free radicals, anti-inflammatory activity by inhibition of lipoxygenase, and anti-proliferative activity on various cancer cell lines of EOs from plants of Burkina Faso. EOs enriched in sequiterpenes presented the highest antiinflammatory activity while those enriched in monoterpenes and oxygenated terpenes showed highest anti-proliferative characteristics. This work therefore provides a scientific basis for the local use of these plants and also a tool promoting the use therapeutic benefits of EOs from Burkina Faso.