Phytochemical, cytotoxic, antimicrobial and antioxidant studies of Moroccan Cladanthusmixtus extracts
Abstract
The antioxidant, antibacterial and cytotoxic activities of essential oil, aqueous and methanol extracts of the Moroccan Cladanthusmixtus (C. mixtus) collected from Bouznika (northwestern region)were studied and compared.Theessential oil was dominated by 2-methyl-2, trans-butenyl methacrylate(34%). It’shave a good antioxidant activity for DPPH (IC50=0.342±0.002mg/mL)and a strong antibacterial activity against all the microorganisms tested. The aqueous extract had a high amount of phenols, about 38.2±0.4 mg GAE/g. It has no inhibiting effect against the growth of bacterial strains tested. The HPLC analysis of methanolic extract of C. mixtus after acidic hydrolysis showedthe presence ofgallic,vanillic, caffeic and syringic acids, together withcatechin.The apigenin (4′, 5, 7,-trihydroxyflavone),not previously isolated from C. mixtus, was isolated and identified by means of liquidchromatography/mass spectrometryand NMR spectroscopy.Apigeninshowed a great cytotoxic activity towards the studied P3X63Ag8.653 myeloma (P3)Cell Line from mouseof BALB /c which are useful for researchintoboth cancer and immunology (IC50 = 17.9 µg/ml). The 7,4'-dipropargyl ether derivative of apigenin showed a lower cytotoxic activity than apigenin, which aimed us at elucidating the role of hydroxyl groups at positions C7and C4’on the cytotoxic activity.
Key words: Cladanthusmixtus; Antibacterial activity; Antioxidant activity;Cytotoxicactivity, Apigenin.
INTRODUCTION
The genus Anthemis (tribe anthemideae, family Asteraceae) is widely distributed in Western Europe, West, Southwest, and Central Asia, as well as in North Africa(Jahandiz & Maire 1934). This family is represented by 130 species, among them Cladanthusmixtus(L.)Chevall. (C.mixtus) [= Chamaemelummixtum(L.) All; Anthemismixta L.;Ormenismixta (L.) Dumort.]. Commonly referred to as Moroccan chamomile, it is an annual plant with fragrant yellow / white flowers and with 10-40 cm tall. In Morocco, it grows wildly along the Atlantic coast in sandy soils in the north (Quezel & Santana 1963),and it is commonly used for treatment of different ailments such as hepatic, gastric insufficiencies, anxiolytic and nervous breakdown in traditional medicine (Belakhdar 1997, Haddad et al 2003).
Different species of the Anthemis genus collected from several countries were studied and the extracts prepared from different parts of those chamomile seem to be a main source of natural products and active principles, particularly polyphenols, flavonoids and essential oils which have shown interesting biological properties(Carnat et al 2004, Srivastava et al 2010).
However, for the Moroccan species Cladanthus mixtus, the antioxidant and cytotoxic activities of its extracts has not been mentionedbefore. At present, the most commonly used antioxidants in food industry as preservatives for preventing or delaying the oxidation process are butylated hydroxyl anisole (BHA) and butylatedhydroxytoluene (BHT). Besides, the recent studies about BHA and BHT have mentioned their responsibility of liver damage and carcinogenesis (Biswas et al 2010). Therefore, the research of natural and safer antioxidants is increase (Albano & Miguel 2011, Srinivasan 2014).
On the other hand, especially in developing countries, infectious diseases still represent an important cause of mortality and morbidity among humans. The resistance by bacteria to the antibacterial drugs has increased and has now become a global concern (Kumar & Kamaraj. 2010). The antibacterial active principles isolated from higher plants such as the Asteraceae family particularly the chamomile species appear to be one of the important alternative approaches to contain antibiotic (Srivastava et al 2010).
This interest lies in the fact that green medicine is safe and reliable, compared to expensive synthetic drugs that have adverse effects.
The aim of the present work was subjected to phytochemical study and to evaluate the antioxidant, antibacterial and cytotoxic activities of C. mixtus extracts. We report also to the best of our knowledge, and for the first time the isolation, purification and characterization of apigenin from this species and its cytotoxic activity.
Further, to explore the importance of free hydroxyls in positions7and 4' in apigenin , they were converted to two propargyl groups to obtain a semi synthetic derivative of apeginin which was also studied for its cytotoxic activity.
MATERIAL AND METHODS
Chemicals
Ascorbic acid ,Butylatedhydroxytoluene(BHT) , 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-Azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and all polyphenol standards used in this study were purchased from Sigma-Aldrich (Germany). Folin-Ciocalteu reagent was obtained from DC PanreacQuimica SLU (Barcelona), Spain. The antibiotic Aminopenicillinic acid was obtained from Basingstoke, Hampshire, England.
HPLC grade acetonitrile, acetic acid and high purity grade ethanol and DMSO were purchased from Sigma-Aldrich (Steinheim, Germany).
Plant material
Dried aerial parts of wild Moroccan C. mixtus were harvested from Bouznika (northwestern region) in full flowering during 2011. The plant identification was done according to the flora of Morocco(Jahandiz & Maire 1934) and by Professor Moh Rejdali from the Department of Botany and Ecology at the Agronomic and Veterinary Institute, Hassan II, Rabat (Morocco). A voucher specimens number OM 26052011 have been deposited in the herbarium in Chemistry Department at the Faculty of Science Ain-chock, Hassan II University, Casablanca (Morocco).
Essential oil isolation and extracts preparation
– Essential oil preparation
The aerial parts of C. mixtus were hydro-distilled according to the method recommended in the European Pharmacopoeia, using a Clevenger-type apparatus for 3 hours (Council of Europe 2007). The essential oil (EO) was stored at 4–6 °C. The oil yield was expressed per weight of dried plant.
– Aqueous extract preparation
After hydrodistillation, the residual aqueous extract (AQE) was filtered and concentrated under vacuum at 70 ºC.
– Methanolic extract Preparation
The plant were macerated in methanol at room temperature for 48 h. The filtration and the evaporation of the solvent give us a residue which was re-dissolved in methanol to obtain the methanolic extract (ME).
Essential oil analysis
A gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS) were used to analyze the obtained essential oil. The details of oil analysis conditions were previously described (Elouaddari et al 2013).
Antioxidant activity
The determination of the antioxidant activity of the essential oil and the different types of extracts was performed by two methods, the DPPH (2,2-Diphenyl-1-picrylhydrazyl) and the ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid). Both techniques are based on the free radicals reduction by the sample. After the oxidation-reduction reaction, the violet color of DPPH and the blue ABTS, change the color to pale yellow and colorless respectively. A spectrophotometry technique was used for the quantification. The IC50 is one way to present the results of the antioxidant activity, which means the sample concentration that reduces oxidation to 50%. (Alam et al 2013, Magalhaes et al 2008).
– DPPH test
The DPPH method was evaluated according to Brand-Williams et al.(Brand-Williams et al 1995). In a vial of 1 ml, the ethanolic solution (25 µL) of each sample (essential oil and extracts) prepared at different concentrations was added to 975 µL DPPH ethanolic solution (60 After 30 min of reaction at room temperature, the absorbance were measured at 517 nm. The ethanol and DPPH solution were the negative control. The positive control was ascorbic acid. The tests were carried out in triplicate.
The calculate of percentage inhibition of the DPPH was performed using Eq. 1.
Eq. 1: Scavenging effect % = [(A0 – A1) / A0] * 100
– A0: the absorbance of the negative control
– A1: the absorbance of the sample.
The IC50 was obtained by plotting the inhibition percentage against concentrations of sample.
– ABTS•+ test
The ABTS•+ method was carried out as described by Dorman and Hiltunen (Dorman et al 2004). Thus, following to the reaction, of K2S2O8 (2.45 mM) with ABTS (7 mM) aqueous solution, in the dark and at room temperature during 16 h, we generate the radical ABTS•+ solution. Ethanol was added to the prepared solution to adjust the absorbance to 0.7.
Samples at different concentrations (10 l) were added to 990 l of ABTS•+. The absorbance was measured at 734 nm at time 0 (A0) and after 6 min (A1). The positive control was Butylatedhydroxytoluene (BHT). The tests were carried out in triplicate.
The percentage inhibition of the ABTS•+ was calculated using the same formula in Eq. 1.
– Total phenolic compounds
A Folin-Ciocalteau colorimetric method was used to determine the total phenol content in the samples (Singleton & Rossi 1965). Each sample solution (0.5 ml) was reacted for 4 min, with 2.5 ml of the Folin-Ciocalteau (diluted ten times). Then, 2 ml of Na2CO3 (75 mg/ml) aqueous solution were added to the reaction mixture. The absorbance was measured at 760 nm after 2 h incubation at room temperature. The reference standard was gallic acid. Total phenol content was expressed as mg gallic acid equivalents per gram plant extract (GAE)/g dry weight (dw). The determinations were done in triplicate.
– Total flavonoid compounds
The flavonoids content was measured according to a colorimetric assay reported by Adedapo et al.(Adedapo et al 2008). For the calibration curve, 1 ml of 2% AlCl3 methanol solution was added to 1 ml of quercetin standard solution which was prepared at different concentrations (0-25 g/ml). After incubation at room temperature, for 60 min, the absorbance at 420 nm was measured. The same procedure was applied to the extracts solutions (0.1-0.5 g/ml). The analyses were done in triplicate. Total flavonoid percentage was expressed as mg quercetin equivalent per gram plant extract (QuE)/g (dw).
HPLC analysis
– Hydrolysis of glycosides to aglycons
The methanolic extract was evaporated at 60 °C and then redissolved with a mixture of water-acetonitrile (88-12). The solution was filtered by a column containing the residue of exhausted chamomile to eliminate chlorophyll and lipophilic compounds. This technique was developed in our laboratory and revealed efficient to get rid of chlorophyll, beta carotene and other lipophilic constituents.We then proceeded to a treatment in acidic medium to hydrolyze glycosidic linkages (Engida et al 2013). To 20 mL of extract, 5 ml of 6N HCl and 12.5 mg of hydroquinone (HQ) were added and the mixture was heated at 80 °C for 1 h. HQ is used as an antioxidant to prevent degradation of flavonoids. After cooling, a volume of 25 mL of distilled water is added to the mixture, followed by filtration and analysis of the filtrate by HPLC.
– Polyphenol aglycons analysis
Qualitative analysis of standard phenolic compounds in the methanol extracts of C. mixtus was performed using high performance liquid chromatograph (HPLC) type JASCO PU-1580 equipped with a JASCO875 UV/Vis detector and the Azur data processing station 3.0.3.0 version.
The compounds were eluted from a RP-C18 column with an isocratic elution of mobile phase water, acetonitrile, acetic acid (88, 12, 1, respectively) and monitored at 280 nm. The injection volume of all samples and the mixture of polyphenol standards was 20 μL. The flow rate was 1 mL/min. The polyphenol standards (10-30 mg/L) used were: caffeic acid, ferulic acid, rutin, vanillic acid, vanillin, catechin, gallic acid and syringic acid. The flavonoid glycoside rutin was used as a standard because it was reported that its deglycosylation depends on the reaction conditions(Wang et al 2011) . The identification of compounds in the extract was accomplished by comparison of their retention times with those of pure standards.
Antibacterial test
– Microorganisms
Gram-positive (Staphylococcus aureus ATCC25923, Enterococcus faecalis, Bacillus cereus, Streptococcus A and Listeria monocytogene) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Salmonella typhi) bacteria and Candida albicans were used for antimicrobial activity studies. The microbial strains were obtained from the Laboratory of Microbiology, Pharmacology, Biotechnology and Environment at the department of biology, Faculty of Science Ain-Chock, Hassan II University, Casablanca, Morocco.
– Antibacterial activity assay
The Agar well diffusion method was used to evaluate the antibacterial activity of essential oil and extracts against different bacteria as described by Okeke et al.(Okeke et al 2001). Each pure isolated bacterium was sub-cultured for 24 h, in nutrient broth at 37ºC. 100 L of each the tested bacterium (about 106CFU/mL, standardized by 0.5 Mac-Farland) was spread on to a sterile Muller-Hinton Agar plate so as to achieve a confluent growth. A sterile cork borer of diameter 5.0 mm was used to bore wells in the dry Agar plates, in which the extract (50 μL) was introduced.
The negative controls were methanol and sterile water, while the antibiotic 6-AminoPenicillinic acid (6-APA) was the positive control. Each experiment was carried out in duplicate.
Cytotoxicity
– Cell culture
The cell line used is the P3X63Ag8.653 myeloma (P3) of BALB /c mice. These are tumor cells that are actively multiplying by escaping apoptosis or programmed cell death.
Before use, the cells were thawed and then cultured at a concentration of 106 cells / ml in culture medium RPMI 1640 (Roswell Park Memorial Institute medium) supplemented with 4.2 g / l streptomycin, 26.3 g / l penicillin and 10% fetal calf serum.
– MTT test
The colorimetric MTT (microculturetetrazolium) is an in vitro assay measuring the overall growth of a cell population. The cell line used in this study has been incubated for 48 h in 96-well plates (40 000 cells /well) then the substance which it is desired to measure the effect on cell growth was added.
After 72 h of incubation the evaluation of the cellular proliferation is done when the living cells reduce MTT, from yellow color, to its metabolite blue formazan (purple). The intensity of the violet color measured quantitatively by spectrophotometry using a microplate reader at a wavelength of 490 nm (with a reference of 630 nm) is directly proportional to the number of living cells.
Each experimental condition was analyzed in triplicate with several concentrations.
Extraction and isolation
1.5Kg of dried and ground plant was subjected to maceration in acetone at room temperature for one week. After filtration, the plant residue was re-extracted twice. The two filtrates obtained were evaporated using a rotary evaporator.
To the crude extract, 500 ml of distilled water-ethanol (70-30) were added. After vigorous stirring, the mixture was filtered using a column containing the residue of exhausted chamomile to eliminate chlorophyll and other lipophilic compounds.
The hydroalcoholic solution thus obtained is evaporated to remove the maximum amount of ethanol and then subjected to liquid-liquid extraction with hexane, dichloromethane and ethyl acetate, respectively.
The dichloromethane extract (3.5g) was subjected to separation by column chromatography (CC) on silicagel eluted with hexane-EtOAc with increasing polarity (100:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:100). Fractions having similar TLC profile have been combined to yield four fractions AI-AIV. Column chromatography on silica gel of fraction AIII eluting with hexane-EtOAc (100-0 to 50-50) afforded sub-fraction AIII/2,which was subjected to further purification on column chromatography eluting with hexane-AcOEt-MeOH (57-40 -3) to yield 140 mg of product 1(apigenin).
Semi-synthesis of compound 2
Compound 1 (20 mg, 0.074mmol) was added under stirring to a mixture of propargyl bromide (25 µl; 0.33mmol),K2CO3 (10.23 mg;0.074mmol) and DMF (0.5 ml) at room temperature. After stirring for 24 hours, dichloromethane (10 ml) was added and the resulting suspension filtered to remove excess base. The filtrate was then washed with water (2x5ml), dried over anhydrous Na2SO4 and concentrated under reduced pressure. Purification through a short column of silica gel eluting with hexane-AcOEt (7: 3) yielded 16 mg of compound 2 (scheme 1).
Scheme 1. Semi-synthetic derivative of apigenin.
Statistical analysis
The comparison of more than two means was performed using with the SPSS Statistics software according a one-way analysis of variance (ANOVA). A difference was considered statistically significant when p <0.05.
Results and discussion
Essential oil composition
The chemical composition of the essential oil has already been reported previously by our group (Elouaddari et al 2014).As mentioned,2-methyl-2, trans-butenylmethacrylate and ar-curcumene were the main constituents representing almost 50 % of the total composition of the C. mixtus essential oil collected from Bouznika region (Table 1).
Table 1.Percentage of the main constituents of the essential oil isolated from C.mixtus collected from Bouznika region
Component Percentage
2-Hydroxy-2-methyl-3-butenyl methacrylate 1.7
2-Methyl-2-trans-butenyl methacrylate 34.0
-Campholenal 0.1
Camphor 0.5
trans-Pinocarveol 4.1
Pinocarvone 5.6
ar-Curcumene 13.6
-Caryophylleneoxide 1.5
Determination of total flavonoid and phenolic contents
High levels of phenolic content in terms of gallic acid equivalent were found in the aqueous extracts of the aerial parts of C. mixtus(38.2 mg GAE /g) comparing to methanolic extract (19.5 mg GAE /g).Conversely, higher flavonoid content was found in the methanolic extract (3.2mg QuE/gvs. 2.7 mg QuE/g in aqueous extract).
Table 2: Concentration of total phenols and flavonoid in methanolic and aqueous extracts of C.mixtus .L. Chevall of Morocco
Type of extract Total phenolics
mg of GAE/g extract Total flavonoid
mg of QE/g extract
Methanolic 19.5±0.4 3.2±0.01
Aqueous 38.2±0.4 2.7±0.1
Results are given as mean±standard deviation of three different experiments..
GAE: gallic acid equivalents; QE: quercetin equivalents
Qualitative analysis of flavonoids by HPLC
The nature of flavonoid and phenolic compounds present in the methanolic extract of C. mixtus using HPLC after acidic hydrolysiswas studied. By matching the retention times against those of standards (figure 1), the identified phenolic compounds were:gallic acid, catechin, vanillic acid, caffeicacid and syringic acid (figure 2).
Figure 1. HPLC Chromatogram of nine available polyphenol standards monitored at 285 nm
Figure 2. HPLC chromatogram of the methanolic extract of C.mixtus
Antioxidant activity of the extracts
– Essential oil
The essential oil of C. mixtusfromBouznika region showedan interesting antioxidant activity inDPPH test (IC50 = 0.342 ± 0.002 mg/mL) and average activity for ABTS test (IC50 = 2.030 ± 0.006 mg/mL) (table 3, Fig. 1). From these results, it appears that the essential oil of C.mixtus has satisfactory antioxidant activity but it is less effective than ascorbic acid (IC50 = 0.1 mg/mL) and BHT (IC50 = 0.004). This activity is likely due to the main components of the essential oil2-methyl-2-trans-butenyl methacrylate and ar-curcumene. These compounds are known for their high antioxidant activity. Curcumene is the maincompound of Acaliphahispidaand showed a high antioxidant activity (Onocha et al 2011, Vishnupriya.M et al 2012, Younes 2012).It has to be noted that minor compounds can also contribute to the antioxidant activity by interacting synergistically to create an effective system against free radicals (Lu & Foo 2001, Singh et al 2006).
Furthermore, the antioxidant activity of a pure product may change once introduced into a mixture. The complexity of essential oils chemical composition varies depending on several factors. The variably of these factors must be taken into account to replace synthetic compounds by natural antioxidants. In this case, additional work is needed to accurately assess the potential and limits of the essential oils of C. mixtus.
– Methanolic and Aqueous Extracts
The results of the antiradical activity of methanol and water extracts and reference molecules (ascorbic acid and BHT) are shown in figure 3 and Table 3.
The results of these tests show that the water and methanol extracts of C.mixtus present an average activity with IC50 = (1.933 ± 0.048 and2.017 ± 0.073, respectively) for the DPPH test and IC50 = (2.103 ± 0.023 and 3.473 ± 0.096, respectively) for the ABTS test. However, the IC50 values found for these samples are higher compared to references.
We are seeing an average correlation between the anti-free radical activity and the quantity of flavonoids in the extracts tested (r = 0.61 p <0.05), and low correlation with the amount of total polyphenols (r = 0.36 p <0.05). Hinneburg et al.(Hinneburg et al 2006) studying several plant families, reported no correlation between the content of total polyhenols and anti-free radical activity. However, Dorman et al.(Dorman et al 2003) reported a higher correlation between antioxidant activity and polyphenols in aqueous extracts of plants of the Lamiaceae family. Therefore, the antioxidant activity cannot be explained solely on the basis of the content of phenolic compounds and can be attributed to the presence of other molecules such as pigments and acids. In addition, it has been mentioned that the antioxidant activity of phenolic compounds is primarily determined by their structure. Aglycones are generally more effective than the corresponding glycosides. For instance, luteolin and quercetin aglycones are more active than the 3-, 4- and 7-O-corresponding glycosides (Heim et al 2002, Satué-Gracia et al 1997).
Table 3: Antioxidant activities of essential oil, methanolic and aqueous extracts of C.mixtus
Type of extrat DPPH (Ic50 mg/ml) ABTS (IC50 mg/ml)
Methanolic 1.933 ± 0.048 2.017± 0.073
Aqueous 2.103± 0.023 3.473± 0.096
Essential oil 0.342±0.002 2.030±0.006
BHT nd 0.004±0.002
Ascorbic acid 0.1± 0.004 nd
Results are expressed as mean ± standard deviation of three different experiments.
nd: not determined
Figure 3: Antioxidant activities of essential oil, methanolic and aqueous extracts of C.mixtus
Antibacterial test
– Essential oil
The antibacterial activity of the essential oil and aqueous and methanol extracts of C.mixtus are gathered in Table 4.
The essential oil of C. mixtus showed a strong antibacterial activity against all the microorganisms tested. We observed also that the inhibition zone diameter was particularly higher than the antibiotic (6-APA) for B.cereus, P.aeruginosaandC.albicans.
This activity is linked to the nature of its main constituents. Indeed, the essential oil of Bouznika region is rich in oxygenated compounds (49.5%). This seems consistent with the results previously described by Radailliet al. (2016)(Radaelli et al 2016) who showed that oxygenated monoterpenes exhibit high antimicrobial activity, while the hydrocarbon derivatives show a lower activity due to their lipophilic nature which limits their diffusion across the bacterial cell membrane. Griffin et al.(Griffin et al 2000) showed that hydrocarbons structures is responsible of their low activity because they are limited to establish hydrogen bonds due to their very low solubility in water.
However, a synergistic effect between minor and major compounds of the essential oil of this species can explain the activity.
Our results are comparable to those reported by Satrani et al.(Satrani et al 2007)in which they mentioned that the Moroccan essential oil of C. mixtus from Kenitra region have a good activity against S.aureus, B.subtilis, E.coli and Micrococcus luteus. Darriet et al.(Darriet et al 2012),while working on the antibacterial activity of the essential oil of Chamaemelum mixtum from Corsica against eight strains, mentioned that the oil had a strong antimicrobial activity effect against E.faecalis, Klebsiellapneumoniae, S.aureus, Citrobacterfrendii, , L.monocytogenes and E.coli.
– Methanolic and Aqueous Extracts
The results of the antibacterial activity of the extracts indicate that the aqueous extract of C.mixtus has no inhibiting effect against the growth of bacterial strains tested. Several studies have also reported that the aqueous part of the extracts of different plants from the family Asteraceae showed no antibacterial activity (Candan et al 2003, Sökmen et al 2004). We also found that the methanolic extract of C. mixtus showed moderate antibacterial activity.
Many studies reported that the compounds responsible for the antibacterial action seem likely to be the main compounds of the moderately polar fraction of plant extracts. Some reports have found that the less polar phenolic compounds are apparently the ones responsible for antibacterial activities. These compounds are extracted by average polarity solvents, such as dichloromethane (Fernández-López et al 2005). This could explain the absence of antibacterial activity of the water extract and the modest activity of the methanol extract, since they are highly polar solvents.
Table 4: Antibacterial activities of the essential oil and methanolic and aqueous extracts of C.mixtus
Microorganisms Inhibition zone diameter ( mm)
EO
AQ
ME 6-APA Negative control
Methanol Water
Escherichia coli 14 na na 30 na na
Staphylococcus aureus 25 na 12 40 na na
Salmonella typhi 19 na 11 32 na na
Candida albicans 29 na 13 24 na na
Enterococcus faecalis 16 na 1 22 na na
Streptocoques A 11 na na 24 na na
Pseudomonas aeruginosa 11 na na na na na
Bacillus cereus 27 na 15 16 na na
Listeria monocytogene 12 na 1 27 na na
na: not active.
EO: essential oil; AQE: aqueous extract; ME: methanolic extract
6-APA: Aminopenicillinic acid
Cytotoxic activity
The IC50 of the inhibitory activity on cell growth of the methanol extract and the essential oil of C. mixtus assessed using the MTT assay on the cancer cell line P3 are shown in Table 6.
The essential oil of Bouznika region has good activity with an IC50 = 85,27μg / ml greater than that of the methanol extract (IC50 = 125.5 g/ml). One study showed that the high toxicity of the essential oil has been attributed to the existence of highly hydrophobic components of low molecular weight. These compounds could easily pass through and / or interact with the membrane to cause a loss of structural integrity. The enhanced permeability of ions can cause cell death(Gaunt et al 2005) . In addition, the allocation of the cytotoxic activity of essential oils is due to the synergism between the active compounds of these oils (Yang et al 2010). For example, borneol, camphor and caryophyllene inhibit the proliferation of cancer cells by stimulating apoptosis (El-Sawi et al 2007). Benbaceet al.(Benbace et al 2012) reported that the methanolic extract of Ormenis mixta from the region of Sidi Boughaba Mahdia (western region) showed low inhibitory activity on the proliferation of cancer cells SiHa and HeLa with an IC50 = 383μg / ml and 311μg / ml, respectively
Identification and cytotoxic activities of the major isolated polyphenol
Repetitive column chromatography separation and crystallization helped to isolate from the complex mixture, a yellow powder, which was the major phenolic compound. Its mass spectral data showed [M+H]+ ion at m/z 271 corresponding to C15H10O5. The 1H NMR spectrum showed the existence of two meta coupled aromatic doublets at δ 6.16 and 6.45 ppm (J= 2Hz) , two doublet of doublets at δ 6.88 and 7.88 ppm (J=7.9 Hz) and a singlet at δ 6.73 ppm . These signals are those of the flavone apigenin (fig. 3). The presence of an unsaturated carbonyl carbon, seven quaternary carbons, five methine carbons, and twelve aromatic carbons in the13C NMR confirm this assignment. All the 13C and 1H signals were assigned on the basis of HMBC and HMQC correlations (table 5). The structure was achieved using HMBC and COSY correlations as shown in Fig. 3.
Fig. 3. COSY and HMBC correlation of apigenin
– Etherification of C7-OH and C4’-OH
In order to synthesize compounds capable of exhibiting interesting biological activities, we have carried out an alkylation reaction of apigenin. We have opted for the incorporation of an acetylene group because of the broad spectrum of biological activity represented by alkynes, such as anti-cancer, antitumor, antibacterial and antifungal activities (Shi Shun & Tykwinski 2006).
Excess propargyl bromide was left to react with apigenin in DMF at room temperature for24hours, after which time the resulting product was purified by column chromatography on silica gel.
The characteristic absorption bands in the IR spectrum of compound 2 are at 3287.1 cm-1 (C-H of terminal alkyne), 3187.5 cm-1 (OH),2166.6 cm-1 ( ), and1663.2 cm-1 (C = O). 13C NMR showed the appearance of six carbons (two secondary carbons at 56.08 and 55.85 ppm, two tertiary at 76.71 ppm and twoquaternary at 76.94 ppm).1H NMR showed the disappearance of two hydroxyl groups at 10.2 ppm, which confirms that two propargylgroups are indeed bonded with the oxygen atoms in positions 4 'and 7(Figure 4).
Figure 4: apigenin 7,4'-dipropargyl ether
Table 5: Assignment of carbons of apigenin and its 7,4'-dipropargyl etherderivative.
Apigenin apigenin 7,4'-dipropargyl ether
Intégration RMN 1H
δ ppm RM N 13C
δ ppm RMN 1H
δ ppm RM N 13C
δ ppm
6 1H 6.16 94.17 6.40 93.12
8 1H 6.45 98.97 6.76 98.98
3 1H 6.74 102.98 6.73 104.15
10 104.13 – 105.58
3’, 5’ 2H 6.88 116.1 7.20 115.44
1’ 121.84 – 124.11
2’, 6’ 2H 7.88 128.31 8.06 128.24
9 157.87 – 157.64
4’ 161.69 – 160.8
7 162.33 – 162.20
2 164.24 – 163.55
5 164.73 – 164.09
4 182.17 – 182.85
a 4H 4.93 55.85
56.08
b 78.23
77.94
c 2H 3.12 77.04
76.75
H (2xOH) 10.2
H (OH) 12.9
– Cytotoxic activity of apigenin 1 and its derivative 2
Apigenin showed a great cytotoxic activity vis-a-vis the studied cells (IC50 = 17.9 µg/ml) (Table 6). Indeed, according to the literature, it has been demonstrated that apigenin shows various biological activities such as anti-mutagenic and anti-proliferative activity in several cancerous cell lines (Hussain et al 2010). This anticancer ability could be explained by several potential mechanisms. On the one hand, it has been suggested that apigenin could significantly inhibit NFkB, which plays an essential role in the regulation of cell proliferation, cell cycle arrest and induction of apoptosis(Hastak et al 2003). Furthermore, Chan et al.(Chan et al 2012) have reported that apigenin induces apoptosis via the TNF- and Bcl-2 pathway. While Garnet-Payrastreet al.(Gamet-Payrastre et al 2000)suggest the possibility of induction of apoptosis by the stimulation of the expression of pro-apoptotic protein p53.
The dipropargyloxy derivative of apigenin (IC50 = 80 µg/ml) showed a much lower activity than that of apigenin. This brings us back to link the high activity of apigenin to the presence of one or two free hydroxyls in positions7and 4'. These results are in accordance with the literature. Yasmine et al.(Touil et al 2009) reported that the cytotoxicity of flavonoids increases with the presence in position 4' of a hydroxyl group. Ueda et al. (Ueda et al 2004) confirmed that the cytotoxic activity is easily influenced by the position of the hydroxyl group, and they reported that inhibition of the production of TNF- is done by flavones with hydroxyl groups in position 5, 7 and 4'. A rational and well defined structural modification is required to improve the cytotoxicity.
Table 6: Cytotoxic activity of C. mixtus
IC50 µg/ml
Cell line EO ME Apigenin1 apigenin 7,4'-dipropargyl ether 2
P3/X63 85,27 125,5 17,9 88
EO: essential oil; ME: methanolic extract
Conclusion
This study shows that the essential oil of C. mixtus aerial parts possesses a good antibacterial activity which justifies its use in traditional medicine. Furthermore, evaluation of the antioxidant capacity of essential oil, methanolic and aqueous extracts of this plant has also provided interesting results. Thus, we consider that the good antimicrobial and antioxidant proprieties shown and observed by C. mixtus can provide opportunities to explore it as a source of antimicrobial drugs and a preservative. Meanwhile, beside apigenin, which shows a great cytotoxic activity, further chemical and pharmacological investigation should be carried out so as to search other natural active molecules in those extracts.
References:
Adedapo A, Jimoh F, Koduru S, Afolayan A, Masika P(2008). Antibacterial and antioxidant properties of the methanol extracts of the leaves and stems of Calpurnia aurea. Complement.Altern. Med. 8: 53
Alam MN, Bristi NJ, Rafiquzzaman M (2013). Review on in vivo and in vitro methods evaluation of antioxidant activity. Saud. Pharm. J. 21: 143-52
Albano SM, Miguel MG(2011). Biological activities of extracts of plants grown in Portugal. Ind. Crops Prod.33: 338-43
Belakhdar J. 1997. Médecine arabe ancienne et savoirs populaires, la pharmacopée marocaine traditionnelle. 201
Benbace L, Merghoub N, Btaouri HE, Gmouh S, Attaleb M, et al. (2012). Antiproliferative Effect and Induction of Apoptosis by Inula viscosa L. and Retama monosperma L. Extracts in Human Cervical Cancer Cells Topics on Cervical Cancer With an Advocacy for Prevention ISBN: 978-953-51-0183-3
Biswas M, Haldar PK, Ghosh AK (2010). Antioxidant and free-radical-scavenging effects of fruits of Dregea volubilis. J. Nat. Sci. Biol. Med. 1: 29-34
Brand-Williams W, Cuvelier ME, Berset C(1995). Use of a free radical method to evaluate antioxidant activity. Food Sci. Technol. 28: 25-30
Candan F, Unlu M, Tepe B, Daferera D, Polissiou M, et al. (2003). Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae). J. Ethnopharmacol. 87: 215-20
Carnat A, Carnat AP, Fraisse D, Ricoux L, Lamaison JL (2004). The aromatic and polyphenolic composition of Roman camomile tea. Fitoterapia 75: 32-8
Chan LP, Chou TH, Ding HY, Chen PR, Chiang FY, et al. (2012). Apigenin induces apoptosis via tumor necrosis factor receptor- and Bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatin. Biochim. biophys. acta 1820: 1081-91
CouncilofEurope (2007). European Directorate for the Quality of Medicines. European Pharmacopoeia. Strasbourg
Darriet F, Bendahou M, Costa J, Muselli A(2012). Chemical compositions of the essential oils of the aerial parts of Chamaemelum mixtum (L.) Alloni. J. agric. food chem. 60: 1494-502
Dorman HJ, Bachmayer O, Kosar M, Hiltunen R (2004). Antioxidant properties of aqueous extracts from selected lamiaceae species grown in Turkey. J. agric. food chem.52: 762-70
Dorman HJ, Kosar M, Kahlos K, Holm Y, Hiltunen R (2003). Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars. J. agric. food chem. 51: 4563-9
El-Sawi SA, Motawae HM, Ali AM(2007). Chemical Composition, Cytotoxic Activity and Antimicrobial Activity of Essential Oils of Leaves and Berries of Juniperus Phoenicea L. Grown in Egypt.Afr. J. Tradit. Complement. Altern. Med. 4: 417-26
Elouaddari A, El Amrani A, Eddine JJ, Correia AID, Barroso JG, et al. (2013). Yield and chemical composition of the essential oil of Moroccan chamomile [Cladanthus mixtus(L.) Chevall.] growing wild at different sites in Morocco. Flavour Frag. J. 28: 360-66
Elouaddari A, El Amrani A, JamalEddine J (2014). Intraspecific variability of the essential oil of Cladanthus mixtus from Morocco. Nat. Prod. Commun. 9: 133-6
Engida AM, Kasim NS, Tsigie YA, Ismadji S, Huynh LH, Ju YH(2013). Extraction, identification and quantitative HPLC analysis of flavonoids from sarang semut (Myrmecodia pendan). Ind. Crops Prod. 41: 392-96
Fernández-López J, Zhi N, Aleson-Carbonell L, Pérez-Alvarez JA, Kuri V (2005). Antioxidant and antibacterial activities of natural extracts: application in beef meatballs. Meat. Sci. 69: 371-80
Gamet-Payrastre L, Li P, Lumeau S, Cassar G, Dupont MA, et al. (2000). Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer.cells. Cancer. Res. 60: 1426-33
Gaunt LF, Higgins SC, Hughes JF (2005). Interaction of air ions and bactericidal vapours to control micro-organisms.J. appl. microbiol. 99: 1324-9
Griffin SG, Markham JL, Leach DN (2000). An Agar Dilution Method for the Determination of the Minimum Inhibitory Concentration of Essential Oils.J. Essent. Oil Res.12: 249-55
Haddad PS, Depot M, Settaf A, Chabli A, Cherrah Y (2003). Comparative study on the medicinal plants most recommended by traditional practitioners in Morocco and Canada. J. Herbs. Spices Med. Plants 10: 25-45
Hastak K, Gupta S, Ahmad N, Agarwal MK, Agarwal ML, Mukhtar H(2003). Role of p53 and NF-kappaB in epigallocatechin-3-gallate-induced apoptosis of LNCaP cells. Oncogene 22: 4851-9
Heim KE, Tagliaferro AR, Bobilya DJ (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J. Nutr. Biochem. 13: 572-84
Hinneburg I, Damien Dorman HJ, Hiltunen R (2006). Antioxidant activities of extracts from selected culinary herbs and spices. Food Chem. 97: 122-29
Hussain AR, Khan AS, Ahmed SO, Ahmed M, Platanias LC, et al (2010). Apigenin induces apoptosis via downregulation of S-phase kinase-associated protein 2-mediated induction of p27Kip1 in primary effusion lymphoma cells. Cell prolif. 43: 170-83
Jahandiz E, Maire R (1934). Catalogue des Plantes du Maroc. Algiers: Imprimerie inerva.
Kumar SS, Kamaraj. M (2010). analys of phytochemical constituents and antimicrobial activities of cucumis anguria. against clinical pathogens.Am. Eurasian J. Agric. Environ. Sci. 7: 176
Lu Y, Foo LY (2001). Antioxidant activities of polyphenols from sage (Salvia officinalis). Food Chem.75: 197–202
Magalhaes LM, Segundo MA, Reis S, Lima JL(2008). Methodological aspects about in vitro evaluation of antioxidant properties. Anal. chim. acta 613: 1-19
Okeke MI, Iroegbu CU, Eze EN, Okoli AS, Esimone CO (2001). Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity. J. Ethnopharmacol.78: 119-27
Onocha PA, Oloyede GK, Afolabi QO (2011). Chemical Composition, Cytotoxicity and Antioxidant Activity of Essential Oils of Acalypha hispida Flowers. Int. J. Pharm. 7: 144-48
Quezel P, Santana S (1963). Nouvelle Flore de l’Algérie et des Régions Désertique Méridionales Editions National Centre ofScientific Research, Paris, II, 977.
Radaelli M, da Silva BP, Weidlich L, Hoehne L, Flach A, et al. (2016). Antimicrobial activities of six essential oils commonly used as condiments in Brazil against Clostridium perfringens. Braz. J.Microbiol.47: 424-30
Satrani B, Ghanmi M, Farah A, Aafi A, Fougrach H, et al (2007). Composition chimique et activité antimicrobienne de l’huile essentielle de cladanthus mixtus.Bull. Soc. Pharm. Bordeaux 146: 85-96
Satué-Gracia MT, Heinonen M, Frankel EN (1997). Anthocyanins as Antioxidants on Human Low-Density Lipoprotein and Lecithin−Liposome Systems. J. agric. food chem. 45: 3362-67
Shi Shun AL, Tykwinski RR (2006). Synthesis of naturally occurring polyynes. Angew. Chem. Int. Ed. 45: 1034-57
Singh G, Marimuthu P, de Heluani CS, Catalan CA(2006). Antioxidant and biocidal activities of Carum nigrum (seed) essential oil, oleoresin, and their selected components. J. agric. food chem. 54: 174-81
Singleton VL, Rossi JA (1965). Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am. J. Enol. Vitic. 16: 144-58
Sökmen A, Sökmen M, Daferera D, Polissiou M, Candan F, et al(2004). The in vitro antioxidant and antimicrobial activities of the essential oil and methanol extracts of Achillea biebersteini Afan. (Asteraceae). Phytother. Res. 18: 451-56
Srinivasan K (2014). Antioxidant potential of spices and their active constituents. Crit. rev. food Sci. Nut. 54: 352-72
Srivastava JK, Shankar E, Gupta S (2010). Chamomile: A herbal medicine of the past with bright future. Mol. Med.Rep. 3: 895-901
Touil YS, Fellous A, Scherman D, Chabot GG(2009). Flavonoid-induced morphological modifications of endothelial cells through microtubule stabilization. Nutr. cancer 61: 310-21
Ueda H, Yamazaki C, Yamazaki M (2004). A hydroxyl group of flavonoids affects oral anti-inflammatory activity and inhibition of systemic tumor necrosis factor-alpha production. Biosci. biotechnol. biochem. 68: 119-25
Vishnupriya.M, Nishaa.S, Sasikumar.J.M, Darsini.D TP. 2012. Chemical Composition And Antioxidant Activity Of Essential Oil From Curcuma Amada Roxb. Int. Res. J. Pharm.3: 99-103
Wang J, Zhao LL, Sun GX, Liang Y, Wu FA, et al. (2011). A comparison of acidic and enzymatic hydrolysis of rutin. Afr. J. Biotechnol. 10: 1460-66
Yang Y, Yue Y, Runwei Y, Guolin Z (2010). Cytotoxic, apoptotic and antioxidant activity of the essential oil of Amomum tsao-ko. Bioresour. Technol. 101: 4205-11
Younes K. 2012. Chemical composition, antibacterial and antioxidant activities of a new essential oil chemotype of Algerian Artemisia arborescens L. Afr. J. Pharm. Pharmacol. 6: 2912-21