Chemical composition , antibacterial activity and chromosome number of Algerian populations of two chrysanthemum species

Takia Lograda, Messaoud Ramdani, Pierre Chalard, Gilles Figueredo, Hafsa Silini and Meriem Kenoufi Laboratory of Natural Resource Valorisation, Sciences Faculty, Ferhat Abbas University, 19000 Setif, Algeria. Clermont Université, Université Blaise Pascal, BP 10448, F-63000 Clermont Ferrand. LEXVA Analytique, 460 rue du Montant, 63110 Beaumont, France. 4 LEXVA Analytique, 460 rue du Montant, 63110 Beaumont, France. 5 Laboratory of Applied Microbiology, Sciences Faculty, Ferhat Abbas University, 19000 Setif, Algeria.


INTRODUCTION
The genus Chrysanthemum, golden flower in Greek, belongs to the Asteraceae family; it includes about 300 species (Kumar et al., 2005).The chrysanthemum is distributed in two main centres, one in the Mediterranean area, the other in China and Japan (Dowrick, 1952).In Algeria, the genus includes 20 species with 8 endemic (Quézel et Santa, 1963).Chrysanthemum coronarium L. is an annual herbaceous weed widely distributed in the Mediterranean region, Japan, China and The Philippines (Sanchez-Monge, 1991) and it has big capitula, usually bicolored white and yellow.The species is an ornamental plant.The variety 'spatiosum', is appreciated as a Chinese vegetable.Other uses of this species have been reported in the literature (Sulas and Caredda, 1997;Tada and Chiba, 1984;Alvarez-Castellanos and Pascual-Villalobos, 2003).Chrysanthemum fontanesii (B.& R.) (Q.& S.) is a perennial plant of 100-150 cm long, very rower suffrutescent at the base, the stems are not hairy.The Leaves, sessile amplexicaul are strongly toothed.The achenes are all similar, bald, without pappus.It is an endemic of North Africa, located in the Tell of Algiers and Constantine (Quézel et Santa, 1963).In Chinese medicine, C. morifolium is widely used as a dietary supplement or herbal tea (Chu et al., 2004;Lai et al., 2007) and has an antihepatotoxic and antigenotoxic effect (Lee et al., 2011); it exhibit an allelopathic activity (Beninger and Hall, 2005).In Korea, China and Japan C. indicum is used to treat infectious diseases and disorders of hypertension (Shunying et al., 2005;Lee et al., 2009).It has anti-inflammatory, immunomodulatory humoral and cellular, and mononuclear phagocytic activities (Cheng et al., 2005;Su et al., 2011).The essential oils of C. indicum, C. parthenium, C. trifurcatum and C. viscidehirtum have a significant antibacterial activity (Ren et al., 1999 ;Khallouki et al., 2000 ;Shafaghat et al., 2010 ;Aridogan et al., 2001 ;Ben Sass et al., 2008).The essential oil of C. balsamita is an antioxidant (Pukalskas et al., 2010).The flowers of C. cinerariaefolium and C. macrotum have insecticidal and herbicide effects (Kumar et al., 2005;Haouas et al., 2011).Chrysanthemum coronarium has medicinal properties; the leaves are expectorant and stomachic, while the flowers are stomachic (Bar-Eyal et al., 2006), it is used against constipation (Song et al., 2003), and effective in the fight against nematodes and protects plants against caterpillars (Wood et al., 2010).
The extract of C. coronarium showed strong and selective allelopathic activity against weeds (Hosni et al., 2013;Alvarez-Castellanos et al., 2001).The chemical composition of essential oils of C.coronarium is highly variable.The major compounds are α-pinene, the camphor, β-pinene, transchrysanthenyl acetate, trans-chrysanthenyl isovalerate, cischrysanthenyl acetate, camphene and myrcene (Alvarez-Castellanos et al., 2001;Senatore et al., 2004;Basta et al., 2007).The butanol extract of the leaves of Chrysanthemum fantanesii additioned with vitamin E and C reduces birth defects and oxidative damage in mice (Amrani et al., 2012); it is the free radical scavengers and powerful antioxidants and has an average antibacterial activity (Ben Aissa, 2011).The phytochemical study of C. fontanesii unveiled biodiversity of secondary metabolites on four families': flavonoids, terpenes, phenolic acids and coumarins (Ben Aissa et al., 2011).The genus Chrysanthemum has been many karyological studies (Abd El-Twab et al., 2008 ;Lee et al., 2002 ;Kondo et al., 2010 ;Zhmyleva et al., 2006 ;Garcia et al., 2004); the chromosomes counting are focused on the Asian species, while studies on Mediterranean species are old and fragmentary or nonexistent in Algeria (Ramdani, 1993).The base chromosome number in the genus is x = 9 (Dowrick, 1952;Zhao et al., 2009;Pellicer et al., 2007;Inceer and Hayirlioglu-Ayaz, 2007).The Chrysanthemum diploid taxa are distributed mainly in Central Asia, while the tetraploid and hexaploid taxa are located in the Mediterranean, Europe, and Asia.In Japan and Taiwan the octaploid taxa and decaploide are found (Zhao et al., 2009).Chrysanthemum shows a polyploid series of 2x to 22x, but the most species are diploid.The presence of polyploid taxa is very marked; the artioploides have a significant presence, while perissoploid are poorly represented.The highest number of chromosome was observed in Chrysanthemum lacustre with 2n = 198 (Dowrick, 1952;Natarajan, 1964).
To the best of our knowledge, the chemical composition of essential oil of Chrysanthemum fontanesii has not been studied yet, as well as its antimicrobial properties.The aim of this work was to investigate the chemical composition, antibacterial of essential oil and chromosome numbers from C. fontanesii and C. coronarium growing in Algeria.

Plant material
Chrysanthumum Fontanesii and C. coronarium are collected from two localities in eastern Algeria, Aouana (Jijel and Aouakas (Bedjaia) respectively.Aerial parts were collected during the flowering stage in June 2012.The air dried materials were subjected to hydro-distillation for 3h using a Clevenger apparatus type.Voucher specimens were deposited in the herbarium of the Department of Ecology and Biology, Setif University, Algeria.The oil obtained was collected and dried over anhydrous sodium sulphate and stored in screw capped glass vials in a refrigerator at 4-5°C prior to analysis.Yield based on dried weight of the samples was calculated.

Essential oil analysis
The essential oils were analysed on a Hewlett-Packard gas chromatograph Model 5890, coupled to a Hewlett-Packard model 5971, equipped with a DB5 MS column (30 m X 0.25 mm; 0.25 μm), programming from 50°C (5 min) to 300°C at 5°C/min, with a 5 min hold.Helium was used as the carrier gas (1.0 mL/min); injection in split mode (1:30); injector and detector temperatures, 250 and 280°C, respectively.The mass spectrometer worked in EI mode at 70 eV; electron multiplier, 2500 V; ion source temperature, 180°C; MS data were acquired in the scan mode in the m/z range 33-450.The identification of the components was based on comparison of their mass spectra with those of NIST mass spectral library (Masada, 1996;NIST, 2002) and those described by Adams, as well as on comparison of their retention indices either with those of authentic compounds or with literature values (Adams, 2001).

Antibacterial Activity
The antimicrobial activities of the essential oils were evaluated against both Gram positive (Enterobacter cloacae ATCC 13047, MRSA (Methicillin-resistant Staphylococcus aureus), Staphylococcus aureus ATCC 25923) and six Gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas syringae, Salmonella sp, Serratia liquefaciens ATCC 27592, Serratia marcescens ATCC 14756, Shigella sp).The bacterial inoculums was prepared from overnight broth culture in physiological saline (0.8 % of NaCl) in order to obtain an optical density ranging from 0.08-01 at 625 nm.Muller-Hinton agar (MH agar) and MH agar supplemented with 5 % sheep blood for fastidious bacteria were poured in Petri dishes, solidified and surface dried before inoculation.Sterile discs (6 mm Φ) were placed on inoculated agars, by test bacteria, filled with 10 μl of mother solution and diluted essential oil (1:1, 1:2, 1:4, and 1:8 v:v of DMSO).DMSO was used as negative control.Bacterial growth inhibition was determined as the diameter of the inhibition zones around the discs.All tests were performed in triplicate.Then, Petri dishes were incubated at 37°C during 18 to 24h aerobically (Bacteria).After incubation, inhibition zone diameters were measured and documented.

Caryology
For karyotypic analysis, the squashing method is used.The root-tip meristems of from germinating seeds were usually used for chromosome preparations; only the root-tips of C. fontanesii were taken from wild plants in their natural habitat.A pre-treatment at room temperature for 1.5 hours was usually applied before fixation of the root-tips, in a 0.05% water solution of colchicine.After fixation in a cold mixture of ethanol acetic acid (3:1), the root-tips were stored in cold 70° ethanol until used.The following procedure involved the maceration in 45% acetic acid for 15 min.Staining of chromosomes is made of emerging root-tips in acetic orcein with heating for one minute.Cutting off the meristems and squashing them in a drop of orcein.

Result
The hydrodistillation of the essential oil of Chrysanthemum fontanesii and C. coronarium gave a viscous liquid with a color blue and yellow, respectively.The yield of essential oil of our samples is 0.1% for C. fontanesii and 0.06% for C. coronarium.The analysis and identification of the components of the essential oil of both species was performed using the (GC-MS).The compounds identified in these oils and their relative abundances are presented in order of their appearance.
The results show that the essential oil of C. coronarium has low activity against Staphylococcus aureus, while its activity against Salmonella sp and Shigella sp is present only with pure oil, the dilutions have no effect on bacteria mentioned.The remains of the tested bacteria are resistant to C. coronarium oil.The concentrated essential oil of C. fontanesii generates moderate activity against bacteria (Staphylococcus aureus, MRSA, Shigella sp and Serratia liquifaciens).With dilutions ¼ and 1/8 only Staphylococcus aureus is sensitive.The rest of the bacteria are resistant to all dilution tested.Based on the results obtained, the essential oil of C. fontanesii has a low activity compared to the essential oil of C. coronarium.
The observation of metaphase plates of Chrysanthemum fontanesii and C. coronarium, allowed us to observe a diploid chromosome number 2n = 2x = 18 and a tetraploid chromosome number 2n = 4x = 36, respectively; with a basic chromosome number x = 9 (Figure 1).

DISCUSSION
Generally yields essential oil of the genus Chrysanthemum are low.The oil yield was 0.055% for C. trifurcatum (Ben Sass et al., 2008) and 0.23% for C. indicum (Shunying et al., 2005).Compared with other herbs, (1 to 2.5%) for Rosmarinus and (2 to 2.75%) for Thymus (Edward et al., 1987), the yield of C. fontanesii and C. coronarium are very low.
The essential oils of C. coronarium from Greece contain the major compounds, the Camphor, trans-Chrysanthenyl acetate, cis-Chrysanthenyl acetate and the β-pinene oxide (Basta et al., 2007).The oils of C. coronarium collected in the regions of Italy have almost the same major compounds (Trans-Tonghaosu, transchrysanthenyl acetate, cis-chrysanthenyl and camphor) (Senatore et al., 2004).
The chemical profile of the essential oil of C. coronarium (of the region Aouakas, Algeria) is different from those reported by other authors.Our sample contains high percentages of 1,1-Difluror-tetramethylcyclopropane, camphor and santolina triene, these components are absent in populations of Greece and Italy, while the trans-chrysanthenyl acetate, cis-chrysanthenyl acetate, the β-Pinene oxide and trans-Tonghaosu are absent in the sample of Algeria.
The bacteriological results of C. coronarium are similar to those in the literature.The essential oils of C. coronarium of Italy have no activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa (Senatore et al., 2004).
Our karyological result of C. coronarium is similar to those found by (Dowrick, 1952) with 2n = 36, and also reported a diploid with 2n = 18 in the Mediterranean.The Turkey and Egypt populations of C. coronarium have diploid with 2n = 18 (Abd El-Twab et al., 2008;Inceer and Hayirlioglu-Ayaz, 2007).The same result with 2n = 2x = 18 is reported by (Ramdani, 1993) in the population of Guerrouch (Jijel) for C. fontanesii.The basic chromosome number of C. coronarium and C. fontanesii is x = 9.This number is most common in the genus Chrysanthemum, in the tribe Anthemideae well as in the family Asteraceae, it is the ancestral basic number (Bremer, 1994).The diploid and tetraploid taxa of the Chrysanthemum are distributed mainly in Mediterranean and Europe (Dowrick, 1952), which is consistent with our results.

CONCLUSION
The results of the analysis show that the chemical composition of essential oils of C. coronarium differs from those of C. fontanesii.The testing of the antibacterial activity of essential oils of C. coronarium and C. fontanesii by the method of record shows that the oils of both species have a very low antibacterial activity.The karyological study of two species, C. coronarium and C. fontanesii , based on chromosome counting, allows us to determine two chromosome numbers, a tetraploid with 2n = 4x = 36 for C. coronarium and a diploid with 2n = 2x = 18 for C. fontanesii, with a basic chromosome number x = 9.

Table . 1
: Chemical composition of essential oil of Chrysanthemum fontanesii.

Table . 2
: Chemical composition of essential oil of Chrysanthemum coronarium.