Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3737

Full Length Research Paper

Anti-bacterial activity of secondary metabolites from Chrysanthemum cinerariaefolium

Caroline J. Kosgei
  • Caroline J. Kosgei
  • Department of Biochemistry, Faculty of Science, Egerton University, P. O. Box 536-20115 Egerton, Kenya.
  • Google Scholar
Festus Tolo
  • Festus Tolo
  • Kenya Medical Research Institute, P. O. Box 54840-00200, Nairobi, Kenya.
  • Google Scholar
Josphat C. Matasyoh
  • Josphat C. Matasyoh
  • Department of Chemistry, Faculty of Science, Egerton University, P. O. Box 536-20115 Egerton, Kenya.
  • Google Scholar
Meshack Obonyo
  • Meshack Obonyo
  • Department of Biochemistry, Faculty of Science, Egerton University, P. O. Box 536-20115 Egerton, Kenya.
  • Google Scholar
Peter Mwitari
  • Peter Mwitari
  • Kenya Medical Research Institute, P. O. Box 54840-00200, Nairobi, Kenya.
  • Google Scholar
Lucia Keter
  • Lucia Keter
  • Kenya Medical Research Institute, P. O. Box 54840-00200, Nairobi, Kenya.
  • Google Scholar
Richard Korir
  • Richard Korir
  • Kenya Medical Research Institute, P. O. Box 54840-00200, Nairobi, Kenya.
  • Google Scholar
Beatrice Irungu
  • Beatrice Irungu
  • Kenya Medical Research Institute, P. O. Box 54840-00200, Nairobi, Kenya.
  • Google Scholar


  •  Received: 06 December 2019
  •  Accepted: 24 February 2020
  •  Published: 30 June 2021

 ABSTRACT

This study evaluated antibacterial activity of Chrysanthemum cinerariaefolium (pyrethrum) flower dichloromethane crude extract, fractions and isolated compounds; pyrethrin II, jasmolin I and cinerolone against methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Staphylococcus aureus and Shigella sonnei. The isolated compounds were obtained by carrying out column chromatography on dichloromethane extract and purifying the fractions using preparative High Perfomance Liquid Chromatography (HPLC). The structures of the isolated compounds were elucidated using 1D and 2D NMR. The bioactivity of crude extract, fractions and isolated compounds were determined using disc diffusion assay at a concentration of 100 mg/mL. The MIC and MBC were determined using microdilution method. The bioassay results showed that individually isolated compounds were not active on all the micro-organisms except Jasmolin I which showed slight activity on P. aeruginosa with 7.7±0.6 mm. There was significant difference in the activity of the isolated compounds as a mixture and the activity of individual compounds on MRSA, S. aureus, P. aeruginosa, with P= 0.01, P= 0.0002, P= 0.0007 respectively (α =0.05, Tukey’s test). Isolated compounds and isolated compounds as a mixture in a ratio of (1:1:1) were not active on S. sonnie. Those fractions and isolated compounds which caused inhibition zones of above 10 mm were subjected to MIC and MBC. The lowest MIC and MBC observed was for fraction 3 against MRSA which were 6.5 and 12.5 mg/mL respectively. The compound mixture had MIC and MBC of 25 and 50 mg/ml respectively against P. aeruginosa.

Key words: MIC (minimum inhibitory concentration), MBC (minimum bacteriostatic concentration), bioassay, Chrysanthemum cinerariaefolium, Jasmolin, Pyrethrin II, Cinerolone.


 INTRODUCTION

Microbial infections continue to be a growing concern in the  world  due  to   resistance   to   current   antimicrobial agents. According to estimates by Centre for Disease Control  and  Prevention  (CDC),  at  least  more  than 2.8 million antibiotic-resistant infections occur in the U.S. each year, and more than 35,000 people die as a result (CDC, 2019). In sub-Saharan Africa, 2.6 million babies required treatment for severe bacterial infection in the first month of life in 2012 (Anna et al., 2014). Bacteria are the causative agents of food borne illness globally in approximately 60% of cases requiring hospitalization (Sapkota et al., 2012).

Antibacterial agents currently used to treat bacterial infections work by inhibiting the growth of micro-organisms or kill them by interfering with cell wall synthesis and DNA replication (Senka et al., 2008). Regrettably, overuse and misuse of these antibacterial agents have led to the development of resistance by bacteria thus rendering these agents ineffective in treating infections associated with these micro-organisms (Davis and Davis, 2010). The concerns about the development of resistance to antimicrobial agents by bacteria drive efforts in bioprospecting for new novel compounds and formulations that can be used to target these resistant microorganisms. Plants remain the potential source of antimicrobial agents since time immemorial with about 60 to 90% of populations in the developing countries being reported to use plant-derived medicine as a traditional form of medicine against various bacterial infections (Alviano and Alviano, 2009). The potency of plant extracts against microbial agents is due to the presence of a variety phytochemicals in plants which include tannins, terpenoids, alkaloids, and flavonoids active against the microbial organisms (Talib and Mahasneh, 2010).

The pyrethrum plant, Chrysanthemum cinerariaefolium has a long history of use as an insecticide (Duchon et al., 2009). This is because the plant majorly produces pyrethrins that possess insecticidal properties besides other secondary metabolites. During the1980s and 1990s, Kenya was a global leader in pyrethrum production, contributing over 70% to the global market. The sub sector was a major foreign exchange contributor with earnings rising up to Ksh 2.1 billion in 1996 (Kariuki, 2013). A decline has been seen in the production of pyrethrum globally in the recent years with countries such as Kenya, Papua New Guinea (Corbett, 2015), Tanzania (UNIDO, 2001), Australia (Moslemi, 2017) and Rwanda (Jongschaap, 2018) being affected. This is attributed to introduction of low cost synthetic pesticides known as pyrethroids (Grdiša et al., 2009).

Besides insecticidal properties, the genus Chrysanthemum has been reported to possess other medicinal importance (Jung, 2009). For example, flowers of Chrysanthemum morifolium Ramat and its herbal infusions are used in the treatment of bacterial and viral infections,  sinusitis,  blood  pressure,  digestive,  skin problems,  influenza  virus  PR3,  leptospira,  HIV-1, human  colon  cancer, headache,  dizziness,  sore throat, hypertension,  flu,  cough  etc. (Yeasmin et al., 2016). Previous study  on  C.  cinerariaefolium  has  shown   that pyrethrin esters isolated from the plant inhibited multiple drug resistant (MDR) Mycobacterium tuberculosis at concentrations of 33 and 100 µg/ml (Rugutt et al., 1999).

To the best of the authors’ knowledge antibacterial activity of C. cinerariaefolium against (MRSA), Pseudomonas aeruginosa, Staphylococcus aureus and Shigella sonnei has not been ascertained. Therefore, finding new biomedical uses of crude extracts, fractions and isolated compounds from C. cinerariaefolium would help in curbing bacterial infections besides increasing the demand of pyrethrum; hence help in reviving and revamping the pyrethrum industry in Kenya and other countries that had suffered from upsurge of pyrethroids.


 MATERIALS AND METHODS

Collection of plant materials

Pyrethrum flowers were collected from local farmers in Elgeyo-marakwet County which is located at latitude 00 10' to 00 52" N, Longitude 350 25" to 350 45" E and altitude of 8389 m above sea level. The flowers were identified by a botanist Mr Patrick Mutiso and a voucher specimen (Tolo/Mwitari/Keter/002) was deposited at the Center for Traditional Medicine and Drugs Research (CTMDR) at Kenya Medical Research Institute (KEMRI), Nairobi.

Extraction of pyrethrum crude extracts

Air dried and ground plant material was extracted by repeated soaking (2 × 48 h) in a mixture of methanol and dichloromethane (1:1) at room temperature and evaporated to dryness under reduced pressure. Vacuum liquid chromatography (VLC) using solvents of  increasing polarity and silica gel (thin layer chromatography grade) was carried out to yield six VLC extracts as follows; VLC 1 (n-hexane), VLC 2 (1:1 n-hexane/dichloromethane), VLC 3 (dichloromethane), VLC 4 (1:1 dichloromethane/ethyl acetate), VLC 5 (ethyl acetate) and VLC 6  (methanol). The VLC extracts were then subjected to antibacterial activity against P. aeruginosa, MRSA, S. sonnie, and S. aureus. The most active extract was VLC 3 (dichloromethane extract) and was therefore subjected to column chromatography after a solvent system had been determined using thin layer chromatography (TLC).

Thin layer chromatography

The dichloromethane dry extract was first dissolved in dichloromethane solvent followed by spotting on 2×5 aluminum backed TLC plates using a capillary tube. The solvent mixture that gave optimum separation for the dichloromethane extract was acetone: petroleum ether (A: P) 5: 5.

Column chromatography

The dry dichloromethane extract was placed on top of silica in the column and the solvent system obtained from TLC analysis was then added to the reservoir attached to the column. The column was eluted gradually and the flow rates maintained at approximately 15 ml/5 min. A total of 20 fractions of equal volume were collected and TLC analysis of each fraction performed. Fractions with similar TLC patterns were pooled together resulting in 4 fractions as shown in Figure 1. After carrying out preliminary bioassay, fraction 4 was the most active fraction with a yield of  2.5 g.

The fraction was therefore subjected to preparative high performance liquid chromatography (HPLC) to obtain the pure compounds.

Preparative high performance liquid chromatography (HPLC)

Fraction 4 of dichloromethane extract was purified using preparative HPLC equipped with UV-vis detector with a stationery phase Grom-Sil 120 ODS-5 (250 × 20 mm; 10 μm; Grace Davison, Deerfield, IL, USA) column. The gradient applied was t0-t20 = 5-100% B, t21-t23 = 100% B and re-equilibration at 15% (B) till t25 with a flow rate of 15 mL/min. Solvents used were water and acetonitrile spiked with 0.1% HCOOH. The compounds were eluded at different times as follows, Compound 1 (0.028 g, tR 22.5 min), Compound 2 (0.6 g, tR 19 min) and Compound 3 (0.3 g, tR 12.5 min). The compounds were then divided into two portions; one portion of each compound was used for 1D and 2D high field NMR spectroscopy analysis while the other portions were subjected to assays against selected bacteria.

Nuclear magnetic resonance (NMR) spectroscopy

The 1H, 13C, DEPT, HSQC, COSY and HMBC NMR spectra were recorded on the Bruker Advance 500 MHz NMR spectrometer at the Technical University of Berlin, Germany. The readings were done in DMSO and chemical shifts assigned by comparison with the residue proton and carbon resonance of the solvent. Tetramethylsilane (TMS) was used as an internal standard and chemical shifts were given as δ (ppm). The structures were then simulated using ACD NMR manager program to obtain the chemical shifts of proton. The off- diagonal elements were used to identify the spin - spin coupling interactions in the 1H-1H COSY (Correlation spectroscopy). The proton-carbon connectivity, up to three bonds away, was identified using 1H-13C Heteronuclear Multiple Bond Correlation (HMBC) spectrum. The 1H-13C Heteronuclear Single Quantum Coherence (HSQC) spectrums were used to determine the connectivity of hydrogen to their respective carbon atoms.

Antibacterial bioassay

Test micro-organisms

Four bacterial strains methicillin-resistant Staphylococcus aureus (MRSA) (Clinical isolate), P. aeruginosa (ATCC 27853), S. aureus (ATCC 25923) and S. sonnei (ATCC 25931), were used for assay of antibacterial activities. The four micro-organisms were obtained from Kenya Medical Research Institute (KEMRI).

Disc diffusion assay

The disc diffusion method for antibacterial susceptibility testing was carried out according to NCCLS (2000). Mueller-hinton agar was prepared according to the manufacturer’s instruction and dispensed at 20 ml per plate in a Petri dish. Suspension of selected micro-organism was made in a sterile normal saline and adjusted to 0.5 Mc Farland standard (108 cfu/ml). Each labeled medium plate was then inoculated with P. aeruginosa, S. sonnie, S. aureus, and MRSA using a sterile cotton swab rolled in the suspension to streak the plate surface in a form that lawn growth can be observed. Dichloromethane crude extract, fractions, and isolated compounds equivalent to 100 mg/mL were applied to sterile paper discs (6 mm diameter) and the discs deposited on the surface of the inoculated agar plates and incubated for 24 h at 37°C. Mixture of the isolated compounds in a ratio of (1:1:1) at the same concentration of 100 mg/ml were also used in the bioassay. Zones of inhibition were measured in millimeter after 24 h of growth. The inhibition zones less than 10 mm in diameter were not considered for the antibacterial MIC and MBC analysis. For each extract, 3 replicates were assayed. The negative control used in this experiment was 1% dimethyl sulfoxide (DMSO) whereas 30 µg/disc chloramphenicol discs were used as the positive control. All tests were performed in triplicates.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The   MIC   was   determined  using   the  microdilution   method  as described by Clinical and Laboratory Standards Institute (2009). Serial two-fold dilutions of all the extracts were prepared with sterile saline in a 96-well microtiter plate, obtaining a concentration range from 100 to 6.5 mg/mL. It was followed by addition of 5 µL of P. aeruginosa, S. sonnei, MRSA and S. aureus suspension which were added to the wells containing the dilutions. Each dose was assayed in triplicate. Uninoculated wells containing sterile saline and extract were used as controls. After incubation for 24 h at 37°C, the samples were observed. MIC was recorded as the lowest concentration of each plant extract that inhibited the bacterial growth as detected by the absence of visual turbidity. To estimate the MBC, an aliquot of each well that did not show microbial growth in the prior tests was swabbed on the entire surface of Muller Hinton Agar plates and then incubated under the growth conditions described before. The lowest concentration that prevented the bacterial growth was registered as MBC.

Data analysis

Significant difference between means of zone of inhibition of the isolates by the extracts were determined using Tukey’s test (α =0.05)


 RESULTS AND DISCUSSION

Structure elucidation of the isolated compounds

The three isolated compounds, Compounds 1, 2, and 3 were subjected to 1D and 2D NMR spectroscopic studies such as 1H NMR, HMBC, COSY and DEPT NMR, as described in the methodology. This resulted in the elucidation of the compounds using spectral data summarized in Table 1.

The three isolated compounds in this study have been previously isolated and characterized (Bramwel et al., 1969; Crombie et al., 1969; Rugutt et al., 1999). The 1H NMR spectrum of compound 1 displayed signals at δH 4.98 (1H, dq, J = 8.1, 1.4 Hz), 1.96 (1H, dd, J = 8.1, 5.3 Hz) and δH 1.55 (1H, dd, J = 5.3Hz) assignable to the olefinic proton H-7, and the cyclopropane ring protons H-3 and H-1, respectively, deduced to be part of a cis-chrysanthemate moiety (Patenden et al.,1973). The protons for the two methyl groups attached to the cyclopropane ring resonated at δH 1.22 (3H, s) and 1.12 (3H, s). Additionally, the 1H NMR spectrum showed another signal at δH 5.65 (1H, dd, J = 18.6, 1.7 Hz), 2.82 (1H, dd, J = 18.6, 6.3 Hz) and δH 2.13 (1H, dd, J = 6.3, 1.7 Hz) corresponding to the cyclopentenone ring protons H-1', H-5a' and H-5b', respectively. Further, a doublet due to the exocyclic methylene protons was observed at δH 2.92 (2H, d, J = 7.4, H-7'), two sets of doublets of triplets due to the olefinic protons (designated H-8' and H-9') in a Z configuration based on the coupling constant (10.5 Hz), a methylene proton multiplet at δH 2.15 (2H, m, H-10'), and a saturated methyl triplet at δH 0.96 (3H, t, J = 7.5, H-11') for the protons in its side-chain (Patenden et al.,1973).

The 13C NMR, DEPT and HSQC analysis confirmed the presence of 21 carbons attributable to a carbonyl carbon at δC 203.1 (C-4'), an ester carbonyl  carbon  at  δ171.4 (C-4), four quaternary carbons at δC 164.8 (C-2'), δC 141.5 (C-3'), δC 134.5 (C-8) and δC 28.2 (C-2), six methine carbons at 132.4 (C-9'), δC 124.3 (C-8'), δC 120.9 (C-7), δC 72.9 (C-1'), δC 33.6 (C-1) and δC 32.2 (C-3), six methylene carbons at δC 41.5 (C-5'), δC 21.6 (C-10') and δC 20.7 (C-7'), and six methyl carbons at δC 25.3 (C-10), δC 20.1 (C-6), δC 20.0 (C-5), δC 18.1 (C-9), δC 13.9 (C-11') and δC 13.7 (C-6'). The HMBC cross-peaks from H-5 and H-6 methyl protons to C-1 and C-2 suggested the location of these methyl groups at C-2 while the attachment of the 2-methylprop-1-en-1-yl group to C-3 was suggested by the HMBC cross-peaks from H-7 to C-1. The pent-2-en-1-yl group was determined to be at C-2' based on the HMBC cross-peaks from H-7' to C-2', C-3', C-4' and those from H-8'to C-3'. From the above evidence and previous studies, the structure of compound 1 was  elucidated as (Z)-2-methyl-4-oxo-3-(pent-2-en-1-yl)cyclopent-2-en-1-yl2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropane-1-carboxylate, commonly known as jasmolin I (Bramwel et al., 1969). The structure, HMBC and 1H-1H COSY correlations for Compound 1 are shown in Figures 2 and 3 respectively. The 1H NMR and 13C NMR spectra of Compound 2 closely resembled those of compound 1 except for a few noted differences. First, there was a disappearance of the C-9 methyl carbon signal accompanied by the appearance of methyl ester carbon signals at δC 167.6 (C-9) and δC 52.1 (9-OCH3) attached to C-8 in compound 2 (Rugutt et al., 1999). The attachment of this group at C-8 was suggested by the HMBC cross-peaks from the olefinic proton H-7 to the ester carbonyl carbon at C-9. Second, there was the replacement of the pent-2-en-1-yl substituent in compound 1 with the penta-2,4-dien-1-yl substituent in 2 as evident from the doublet at δH 3.10 (2H, d, J = 7.7, H-7'), a doublet of triplet at δH 6.85 (1H, dt, J = 16.8, 10.8, H-8'), a triplet at δH 6.01 (1H, t, J = 10.8, H-9'), a multiplet at δH 5.36 (1H, m, H-10') and a set of two doublet of doublets for the two terminal olefinic protons at δH 5.27 (IH, dd, J = 16.8, 2.2, H-11a') and δH 5.20 (IH, dd, J = 10.2, 2.2, H-11b'). The direct bonding of proton to carbons was derived from the HSQC spectra and the structure of compound 2 was elucidated to be 2-methyl-4-oxo-3-((Z)-penta-2,4-dien-1-yl)cyclopent-2-en-1-yl-3-((E)-3-methoxy-2-methyl-3-oxoprop-1-en-1-yl)-2,2-dimethylcyclopropane-1-carboxylate, commonly known as pyrethrin II (Rugutt et al., 1999). The structure, HMBC and 1H-1H COSY correlations for Compound 2 are shown in Figures 4 and 5 respectively.

The 1H NMR spectrum of Compound 3 displayed signals at δH 4.52 (1H, d, J = 6.3 Hz), 2.63 (1H, dd, J = 18.1, 6.3 Hz) and δH 2.06 (1H, m) for the cyclopentenone ring protons H-4, H-5a and H-5b, respectively. Additionally, the 1H NMR spectrum also displayed signals for a doublet due to the exocyclic methylene protons at δH 2.90 (2H, d, J = 6.2, H-1'), two sets  of  multiplets  for  the olefinic protons at δH 5.27 (1H, m, H-2') and δH 5.27 (1H, m, H-3'), and  a  singlet  at  δH  1.03  (3H, s, H-4')  for  the but-2-en-1-yl substituent. A signal for methyl protons was also observed at δH 2.01 (3H, s, H-6).

The 13C NMR and HSQC data of Compound 3 exhibited 10 carbon signals which were classified as carbonyl carbon (δC 203.1, C-1), two quaternary  carbons  at  δC  170.9  (C-3)   and   δC 138.2 (C-2), three methine carbons at δC 131.9 (C-3'), δC 126.8 (C-2') and δC 69.9 (C-4), two methylene  carbons  at  δC  43.9 (C-5) and δC 21.4 (C-1'), and two methyl carbons at δC 22.8 (C-4') and δC 13.6 (C-6) (Crombie et al., 1969). The HMBC cross-peaks from H-5 to C-1/C-3/C-4 and from H-4 to C-2/C-3 revealed that the carbonyl group was at C-1 and the hydroxyl group was attached  to   C-4.  The  HMBC  cross-peaks  from  H-1' to C-1'/C-2'/C-3' were used to locate the but-2-en-1-yl substituent at C-2, while the HMBC cross-peaks from the methyl protons H-6 to C-2/C-3 suggested the attachment of this methyl group at C-3. Therefore, the compound was characterized as (Z)-2-(but-2-en-1-yl)-4-hydroxy-3-methylcyclopent-2-en-1-one, commonly known as cinerolone (Crombie et al., 1969). The structure, HMBC and 1H-1H COSY correlations for Compound 3 are shown in Figures 6 and 7 respectively.

Antibacterial activity of pyrethrum extracts and isolated compounds

Results of the  bioassay  against  the  selected  organism using disc diffusion assay are as shown in Table 2 and Figures 8 to 12. The values are the mean of three experiments ± S.D. Within a column, the inhibition zones of extracts sharing the same letter(s) were not significantly different while those with different letters are significantly different (α =0.05, Tukey’s test). The extracts which caused inhibition zone of above 10 mm mean ± S.D were subjected to MIC and MBC. These extracts were fraction 1, 3 and 4 subjected to MRSA and fraction 1, 2, 3, 4 and isolated compounds as a mixture (1:1:1) subjected to P. aeruginosa. The MIC for fraction 1, 3 and 4 against MRSA was 12.5, 6.5 and 12.5 mg/ml respectively while the MBC for fraction 1, 3 and 4 against MRSA was 25, 12.5 and 25 mg/mL respectively. The MIC for fraction 1, 2, 3, 4 and compound mixture against P. aeruginosa was 12.5, 25, 25, 25 and 25 mg/ml respectively while the MBC for fraction 1, 2, 3, 4 and compound mixture against P. aeruginosa were 25, 50, 50, 50 and 50 mg/ml, respectively.

In the current study, dichloromethane extract of C. cinerariaefolium  showed  some degree of activity against the selected bacteria; an indication that the plant has some bioactivity against both gram negative and gram positive bacteria. This concurs with a previous study which showed that large numbers of Chrysanthemum extracts were active against both gram-positive and gram-negative bacteria  (Sassi  et  al.,  2008).  It  is  also coinciding with previous studies which have shown that flowers of members of the Chrysanthemum genus (Asteraceae) possess phytochemicals that are of medicinal importance (Jung, 2009). For example, flowers of Chrysanthemum indicum have been used in folk medicine  for  the  treatment of several infectious disease such as pneumonia, colitis, stomatitis, cancer, fever, sore and hypertensive symptom (Jung, 2009).

Generally, the dichloromethane crude extract was more active against the gram positive bacteria, that is, MRSA and S. aureus than the gram negative bacteria that is, P. aeruginosa and S. sonnei. This is because gram-negative bacteria have  been  reported  to  be  less  susceptible  to crude extracts than the gram positive bacteria due to presence of cell membrane restricting the diffusion of compounds through its lipopolysaccharide layer (Perussi, 2007). The fractions and isolated compounds as a mixture in the ratio of (1:1:1) also showed some degree of bioactivity against all the selected micro-organsisms except S. sonnei. In contrast, to the  bioactivity  observed in the crude extracts, the fractions were more active on gram negative bacteria P. aeruginosa than both gram positive bacteria. This observation may be attributed to the fact that the amount of the active  components  in  the crude extract may have been diluted and fractionation may have increased their concentrations, hence reason for enhanced bioactivity in P. aeruginosa. There could also   be   possibility   of    antagonism    among    various antibacterial compounds in crude extracts when lumped together thus fraction may have reduced leading to enhanced activity observed in  P. aeruginosa (Kuete et al., 2011). 

Individually, the isolated compounds did not show any bioactivity against the selected microorganisms except Jasmolin I which showed some slight activity against P. aeruginosa.  When the three compounds were mixed together at the same concentration and ratio, there was increased bioactivity on the selected micro-organisms suggesting synergy. The activity observed in the compounds as a mixture could be due to cyclopropyl fragment ring in jasmolin I and pyrethrin II. These molecules have been reported to improve the overall activity of majority of biologically important molecules that contain them by acting as potent alkylation agent (Peterson, 2001). The cyclopropyl fragment is a versatile player that frequently appears in preclinical/clinical drug molecules (Tanaji, 2016). These molecules include quinolone antibiotics such as ciprofloxacin, clinofloxacin gemifloxacin and moxifloxacin. Other molecules that have the cyclopropyl fragment include tyrosine kinase inhibitor (4/lucitanib), HCV NS3/4A protease inhibitors, HIV-1 reverse transcriptase inhibitor (Lumacaftor), calcitriol, calcipotriol, amitifadine and etomide pro-drugs among others (Tanaji, 2016). In molecular structure-activity relationship studies of quinolone antibiotics it is evident that a cyclopropyl at position 1 of these quinolones are the optimal substituents, regardless of the changes made at other sites (Peterson, 2001).

The cyclopropane fragment has been reported to possess spectrum of biological properties ranging from enzyme inhibitions to insecticidal, antifungal, herbicidal, antimicrobial, antibiotic, antibacterial, antitumor and antiviral activities. Previous studies have shown that cyclopropane associated with fatty acids have been proven to have antifungal activity (Pohl et al., 2011). Another study showed cyclopropane associated with fatty acids from the marine bacterium labrenzia exhibited antimicrobial activity and it also activated orphan G-protein coupled receptor GPR84, which is vastly expressed on immune cells (Moghaddam et al., 2018).

Fraction 4 was more active than the other fractions on P. aeruginosa since it contained all the isolatedcompounds besides other biomolecules. The isolated compounds showed significant bioactivity against P. aeruginosa as a mixture in the ratio of (1:1:1). The proportion of the isolated compounds in fraction 4 could have been high before the compounds were fractionated. This therefore suggests synergy among the isolated compounds in fractions 4 together with other compounds present in this fraction.


 CONCLUSION

Results of this study show that pyrethrum crude extracts, fractions and isolated  compounds  possess  antibacterial activity against both gram negative and gram positive bacteria. To the best of the authors’ knowledge there is no previous work on antibacterial activity of C. cinerariaefolium on the selected bacteria; hence the findings will aid in exploitation of other uses of the plant besides aiding in the discovery of new antibacterial drugs.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



 REFERENCES

Anna CS, Hannah B, Alexander AM, Harish N, Rajiv B, Shamim AQ, Anita KZ, James AB, Simon NC, Joy EL (2014). Estimates of possible severe bacterial infection in neonates in sub-Saharan Africa, south Asia, and Latin America for 2012: A systematic review and meta-analysis. The Lancet Infectious Diseases 14:731-741.
Crossref

 

Alviano DS, Alviano CS (2009). Plant extracts: search for new alternatives to treat microbial diseases. Current Pharmceutical Biotechnology 10(1):106-121.
Crossref

 

Bramwel AF, Crombie L, Hemesley P, Pattenden G, Elliott M, Janes NF (1969). Nuclear magnetic resonance spectra of the natural pyrethrins and related compounds. Tetrahedron 25:1727-1741.
Crossref

 

CDC (2019). Antibiotic / Antimicrobial Resistance (AR / AMR).

View

 

Clinical and Laboratory Standards Institute (2009). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard 29:1-65.

 

Corbett P (2015). Pyrethrum processing in Papua New Guinea. Acta Horticulturae 1073:53-55.
Crossref

 

Crombie L, Hemesley P, Pattenden G (1969). Synthesis of ketols of the natural pyrethrins. Journal of the Chemical Society C: Organic 6:1016-1024.
Crossref

 

Davis J, Davis D (2010). Origins and evolution of antibiotic resistance, Microbiology and Molecular Biology Reviews 74:417-433.
Crossref

 

Duchon S, Bonnet J, Marcombe S, Zaim M, Corbel V (2009). Pyrethrum: A Mixture of Natural Pyrethrins Has Potential for Malaria Vector Control. Journal of Medical Entomology 46(3):516-522.
Crossref

 

Grdiša M, Carovi?-Stanko K, Kolak I, Šatovi? Z (2009). Morphological and biochemical diversity of Dalmatian pyrethrum (Tanacetum cinerariifolium) (Trevir.) Sch. Bip.). Agriculturae Conspectus Scientificus 74:73-80.

 

Jongschaap REE, Vos CH, de Jongsma MA (2018). Feasibility study plant extracts in Rwanda: Developing value chains in public private partnerships. Wageningen Research. 
Crossref

 

Jung EK (2009). Chemical composition and antimicrobial activity of the essential oil of Chrysanthemum indicum against oral bacteria. Journal of Bacteriology and Virology 19:61-69.
Crossref

 

Kariuki D (2013). Poverty Alleviation Through Pyrethrum Growing in Nakuru County Pyrethrum Value Chain-Kenya Agricultural Productivity Agribusiness Program (KAPAP). 

 

Kuete V, Kamga J, Sadjo LP, Ngameni B, Poumale HM, Ambassa P (2011). Antimicrobial activity of methanol extract, fractions and compounds from Ficus polita Vahl. (Moraceae). BMC Complementary and Alternative Medicine 11:1-6.
Crossref

 

Moghaddam JA, Dávila-Céspedes A, Kehraus S, Crüsemann M, Müller C E, König GM (2018). Cyclopropane-Containing Fatty Acids from the Marine Bacterium Labrenzia sp. 011 with Antimicrobial and GPR84 Activity. Marine Drugs 16(10):369.
Crossref

 

Moslemi A (2017). The pathology of pyrethrum yield-decline in Australia. PhD thesis.

View

 

NCCLS (2000). Performance standards for antimicrobial disk susceptibility tests. Approved standard, 7th ed. NCCLS document M2-A7. NCCLS, Wayne, Pa. Patenden G, Crombie L, Hemesley P (1973). The mass spectra of the pyrethrins and related compounds. Organic Mass Spectrometry 7:719-735.
Crossref

 

Perussi JR (2007). Photodynamic inactivation of microorganisms. Quim Nova 30:988-994.
Crossref

 

Peterson L (2001). Quinolone Molecular Structure?Activity Relationships: What We Have Learned about Improving Antimicrobial Activity pp. s181-s186 
Crossref

 

Pohl C, Kock J, Thibane V (2011). Antifungal free fatty acids: A review. In: Méndez-Vilas A., editor. Science Against Microbial Pathogens: Communicating Current Research and Technological Advances. Formatex Research Center; Badajoz, Spain, pp. 61-71.

 

Rugutt JK, Henry CW, Franzblau SG, Warner IM (1999). NMR and Molecular Mechanics Study of Pyrethrins I and II. Journal of Agricultural Food Chemistry 47:3402-3410.
Crossref

 

Sapkota R, Dasgupta R, Nancy RDS (2012). Antibacterial effects of plants extracts on human microbial pathogens and microbial limit tests. International Journal of Research in Pharmacy and Chemistry 2(4):926-936.

 

Sassi AS, Harzallah-Skhiri F, Bourgougnon N, Aouni M (2008). Antimicrobial activities of four Tunisian Chrysanthemum species. Indian Journal of Medical Research 127:183-192.

 

Senka D, Jagoda S, Blazenka K (2008). Antibiotic Resistance Mechanisms in Bacteria: Biochemical and Genetic Aspects. Food Technology and Biotechnology 46:11-21.

 

Talib WH, Mahasneh AM (2010). Antimicrobial, cytotoxicity and phytochemical screening of Jordanian plants used in traditional medicine. Molecules 15(3):1811-1824.
Crossref

 

Tanaji T (2016). The "Cyclopropyl Fragment "is a Versatile Player that Frequently Appears in Preclinical/Clinical Drug Molecules Journal Medicinal Chemistry 59 (19):8712-8756.
Crossref

 

UNIDO (2001). United Nations Industrial Development Organization workshop proceedings on Industrial utilization of pyerthrum held at Dar es Salaam, Tanzania on May 29th -30th 2000. 

 

Yeasmin D, Swarna RJ, Nasrin S, Parvez S, Alam MF (2016). Evaluation of antibacterial activity of three flower colours Chrysanthemum morifolium Ramat against multi-drug resistant human pathogenic bacteria. International Journal of Biosciences 9(2):78-87.
Crossref

 




          */?>