Antimicrobial combination effects between curcuminoids and ampicillin against standard and clinical strains of staphylococcus aureus

Antimicrobials combination therapy has been used to treat infections for decades, with the goal of achieving synergistic effects, producing wider spectrums of coverage and minimizing any toxicity effects of conventional antimicrobial agents. Plant metabolites are among the suitable candidates to be used as antimicrobial and synergistic agents, which can help mankind to curb the evolution of drug-resistant strains of microbes. S. aureus infections are commonly treated with the penicillin group antibiotics such as ampicillin. However, S. aureus resistance to these antibiotics keeps on increasing and the therapy produce serious side effects, such as hypersensitivity or anaphylactoid reactions. Curcuminoids, which are responsible for the yellow colour of Curcuma longa L. or turmeric rhizomes possess bio-protective properties, which include promising antimicrobial activity with very low incidence of toxicity. For these reasons, our research effort turned to the antimicrobial combination study between ampicillin and curcuminoids in a view to enhance antimicrobial efficacy and developing safer drugs. In this study, the antimicrobial activity of curcuminoids mixture, which was fractionated from the dichloromethane extract of turmeric rhizomes was determined alone and in combination with ampicillin. The activity was tested against the standard strain of Staphylococcus aureus ATCC 25923 and the clinical isolate, which was obtained from patient diagnosed as having S. aureus infection at Hospital Tuanku Ampuan Afzan (HTAA), Kuantan. The minimum inhibitory concentration (MIC) for antimicrobial activity of the curcuminoids fraction and ampicillin, alone and in combination were evaluated by means of broth microdilution assay and chequerboard assay, respectively. The curcuminoid constituents of the fraction were analysed by TLC co-chromatography with authentic samples. TLC Agar Overlay Bioautographic assay was performed to screen the responsible curcuminoids for the antimicrobial activity of the fraction against both strains.The antimicrobial activity studies showed that the combination of ampicillin with curcuminoids fraction is likely to reduce the MIC of ampicillin compared with when tested alone against both strains of S. aureus. The results highlighted the occurrence of a pronounced synergism between 312.50 µg/ml of curcuminoids fraction and 1.56 µg/ml of ampicillin against the clinical strain with Fractional Inhibitory Concentration Index (FICI) of 0.25. These curcuminoids-drug combination augmented the antimicrobial activity of both ampicillin and curcuminoids fraction eight times compared with when tested alone. TLC profile of the curcuminoids fraction showed three yellow phenolic pigments, namely curcumin, demethoxycurcumin and bisdemethoxycurcumin, with the Rf values of 0.66, 0.46 and 0.31, respectively in dichloromethane(18):ethyl acetate(1) solvent system. TLC bioautographic assay has revealed these curcuminoids as the responsible compounds in the fraction that act synergistically with ampicillin. The finding suggests that curcumin, demethoxycurcumin and bisdemethoxycurcumin are promising synergistic agents for antimicrobial combination therapy with ampicillin. The synergistic combination is found useful in combating S. aureus resistance towards ampicillin and minimizing the undesired effects of the antibiotic

Bioautographic screening for natural quinolone antimicrobial agents from Glycosmis pentaphylla (Retz.) DC., Ruta angustifolia (L.) Pers. and Lunasia amara Blanco

Background The quinoline alkaloids are a group of alkaloids with diverse structural types. The 4-oxygenated quinolone and 2-substituted 4-quinolone are the types of quinoline alkaloids that possess the chromophore structures which are similar to the pharmacophore of the conventional 4-quinolone antimicrobial agents. Therefore it is presumed that these types of natural quinoline alkaloids could also share the same activity as the conventional quinolone antimicrobial agents. Although these conventional agents demonstrate an excellent treatment against so many infectious diseases, the emergence of quinolone resistance against some microbes has been a disturbing feature of microbial infection (Shigemura, et al., 2003). Objectives This study aims primarily to search for naturally occurring quinolone antimicrobial agents from three excellent plant sources of quinolone alkaloids, which are Glycosmis pentaphylla (Retz.) DC., Ruta angustifolia (L.) Pers. and Lunasia amara Blanco. It is aimed that the natural alkaloids will provide new structural types of quinolone antimicrobial agents in view to overcome microbial resistance towards the conventional agents. Methods The plant materials, which include the leaves and root barks of G. pentaphylla, and the leaves of R. angustifolia and L. amara were dried and powdered and subjected to extraction. Acid base extraction was employed following the continuous extraction by hexane and acetone to furnish the crude alkaloidal extracts. The extract was then fractionated by column chromatography. The fractions yielding major alkaloidal compounds were selected to be screened by using TLC Agar Overlay Bioautographic Assay for antimicrobial active quinolone alkaloids against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Candida albicans ATCC 90028. Results The column chromatography on the crude alkaloidal extracts has afforded two fractions containing major alkaloids from G. pentaphylla and one fraction each from R. angustifolia and L. amara. TLC Agar Overlay Bioautographic screening for antimicrobial active alkaloids has revealed four antimicrobial active alkaloids. Two alkaloids, labeled as GP-3 and RA-3 which were active against S. aureus and C. albicans have been detected from the stem bark fraction of G. pentaphylla and the leaf fraction of R. angustifolia. Two major alkaloids such as GP-4 from the leaf fraction of G. pentaphylla and LA-4 from L. amara leaf fraction were found to be active against both S. aureus and E. coli. Conclusion The reported results of all three plant species therefore proved to have high potential in providing new structural types of lead compounds for synthesizing new drugs to overcome microbial resistance towards conventional quinolone antimicrobial agents

Photo activated bacterial DNA-binding active Rutaceous alkaloids from Glycosmis pentaphylla (Retz.) DC. and Ruta angustifolia (L.) Pers.

Arborine, a quinazoline and graveoline, a 4-quinolone are the antimicrobial active Rutaceous alkaloids isolated from Glycosmis pentaphylla and Ruta angustifolia, respectively. Both alkaloids possess chromophore with a few structural similarities with the pharmacophore of 4-quinolone antimicrobial agent. Their bacterial DNA-binding activity was assessed by photo activated DNA binding assay and agarose gel electrophoresis. Ten different restriction enzymes which recognise and cleave DNA in a sequence-specific manner were competed by the alkaloids. The DNA binding activity was detected as inhibition of the enzymatic restriction resulting in the detection of uncleaved DNA fragments of the original size. Arborine showed inhibitory activity against the restriction enzymes EcoR I, Pae I and Dra I with the highest intensity of inhibition for Dra I which have 5’-TpA sequence. Graveoline was active against EcoR I, Dra I and Pst I. Ciprofloxacin, the second generation of quinolone antimicrobial agent only the inhibitor of Kpn I and Pst I. The photo activated bacterial DNA-binding activity was in the sequence of arborine>graveoline>ciprofloxacin. This finding revealed the potential of arborine and graveoline as lead compounds for future development of quinolone antimicrobial agents in resolving the antibiotic resistance cases which are globally common nowadays in clinical setting

Graveoline from Ruta angustifolia (L.) Pers. and its antimicrobial synergistic potential in erythromycin or vancomycin combinations = Graveolin daripada Ruta angustifolia (L.) Pers. dan potensi sinergistik antimikrobnya dalam gabungan erithromicin atau vancomicin

Ruta angustifolia (L.) Pers. is a Rutaceous species which contains various anthranilic acid derived alkaloids including the bioactive quinolones. This study is aimed at identifying the antimicrobial active alkaloids of R. angustifolia and evaluating their potential as synergistic enhancers in alkaloid-antibiotic combinations. Antimicrobial bioautography- guided isolation of alkaloidal fractions of R. angustifolia leaves has led to the identification of 2,3-dimethoxy-1-hydroxy- 10-methylacridone [arborinine]; and 4,7,8-trimethoxyfuro[2,3-b]quinoline [skimmianine]; together with the major active alkaloid, 1-methyl-2-[3’,4’-methylenedioxyphenyl]-4-quinolone [graveoline]. Graveoline showed Minimum Inhibitory Concentration (MIC) values ranging from 500 to 1000 μg/mL against Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922. Checkerboard assay for antimicrobial combination effects between graveoline with either erythromycin or vancomycin showed enhancement of the antimicrobial activity of both antibiotics with Fractional Inhibitory Concentration Indices (FICI) ranged from 0.37 to 1.50. Synergistic effect with FICI of 0.37 was observed for graveoline-erythromycin combination against S. aureus compared to FICI of 1.00 for ciprofloxacin-erythromycin additive effect. Graveoline was a potential candidate for antimicrobial combination agent especially against S. aureus. The result supports the idea of using plant metabolites as antimicrobial synergistic agents. ********************************************************* Ruta angustifolia (L.) Pers. adalah spesies Rutaceae yang mengandungi pelbagai alkaloid yang hasilkan daripada asid anthranilik seperti alkaloid quinolon. Kajian ini bertujuan untuk mengenal pasti alkaloid aktif daripada R. angustifolia dan potensinya sebagai penggalak kesan sinergi bersama antibiotik terpilih. Pemencilan alkaloid aktif berpandukan bioautografi antimikrob daripada pecahan ekstrak daun R. angustifolia berjaya mengenal pasti 2,3-dimetoksi-1-hidroksi-10-metilakridon [arborinin]; dan 4,7,8-trimetoksifuro[2,3-b]quinolin [skimmianin]; dan akaloid aktif utama 1-metil-2-[3’,4’-metilenedioksifenil]-4-quinolon [graveolin]. Graveolin menunjukkan Kepekatan Perencatan Minima (MIC) pada julat antara 500 dan 1000 μg/mL terhadap Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 dan Escherichia coli ATCC 25922. Gabungan antara graveolin dan erithromicin atau vankomicin yang dinilai melalui Assay Checkerboard menunjukkan bahawa graveolin meningkatkan aktiviti kedua-dua antibiotik dengan Indeks Pecahan Kepekatan Perencatan (FICI) antara 0.37 dan 1.50. Kesan sinergi dengan nilai FICI 0.37 ditunjukkan oleh gabungan graveolin-erithromicin terhadap S. aureus berbanding kesan tambahan dengan nilai FICI 1.00 oleh gabungan ciprofloxacin-erithromicin. Graveolin mempunyai potensi sebagai agen gabungan antimikrob terutama terhadap S. aureus. Keputusan kajian ini menyokong penggunaan metabolit daripada tumbuhan sebagai agen sinergi antimikrob

Bioautography, combination effects and photo-activated enzymatic restriction inhibitory activity of antimicrobial alkaloids from Glycosmis pentaphylla (Retz.) DC.

Glycosmis pentaphylla (Retz.) DC., locally known as nerapan, has long been used in Asian countries as a traditional remedy for ailments attributed to microbial infections. This study aims to isolate antimicrobial alkaloids from G. pentaphylla, to determine their combination effects with selected antimicrobial agents and to screen for their photoactivated enzymatic restriction inhibitory activity. Bioautography-guided isolation of antimicrobial alkaloids was performed by using column chromatography with Staphylococcus aureus, Escherichia coli, and Candida albicans as the indicator microbes. The antimicrobial effects of the alkaloids combined with selected antimicrobial agents, namely, ciprofloxacin, erythromycin, vancomycin, and ketoconazole, were determined by using a checkerboard assay. Photoactivated enzymatic restriction inhibitory activity was assessed by using agarose gel electrophoresis. Two antimicrobial active alkaloids were isolated and identified as arborinine and arborine. The antimicrobial activity of arborinine and arborine was determined to be in the range of 250 μg/ml and 1000 μg/ml. Partial synergy was observed for all arborine-antibiotics and arborinine-ketoconazole interactions against S. aureus and C. albicans, respectively. Arborine was relatively the strongest photoactivated enzymatic restriction inhibitor, particularly against EcoRI, PstI, and SalI. The results obtained are promising and encourage further research on the alkaloids as potential antimicrobial-enhancing agents

Bioassay-guided isolation of antimicrobial active alkaloids from Ruta angustifolia (L.) Pers

Plants of the Rutaceae family are sources of active alkaloids with high potential to defeat microbial infectious diseases and overcome microbial resistance towards antibiotic. The anthranilic acid derived alkaloid especially quinoline, furoquinoline, acridone and 4-quinolone alkaloids are the common types of antimicrobial active alkaloids found in Rutaceae. In this study bioautography-directed fractionation of the crude alkaloidal extract from Ruta angustifolia (L.) Pers. was carried out to focus on the isolation of the antimicrobial active alkaloids. The isolated alkaloids were further identified by spectroscopic method of NMR. The activity of each isolated antimicrobial active alkaloid was quantified by using broth microdilution assay for Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 25933, Pseudomonas aeruginosa ATCC 27853, Helicobacter pylori ATCC 43504, Enterococcus faecalis ATCC 29212 and Candida albicans ATCC 90028. Two promising antimicrobial alkaloids which are an acridone, arborinine and a 4-quinolone, graveoline were isolated. Arborinine showed an activity against S. aureus, E. coli, P. aeruginosa and C. albicans while graveoline exhibits an activity against all of the bacteria except E. faecalis. MIC values for the two alkaloids against the susceptible microbes ranged between 0.01 mg/mL and 0.025 mg/mL while the MBC values were more than 0.01 mg/mL. These results suggest that the two compounds could be considered as a source of antimicrobial agent which might be studied in more details

Arborine, a quinazolin-4-one antimicrobial alkaloid from Glycosmis pentaphylla (Retz.) DC

Quinazolin-4-one is a type of alkaloid that possesses a chromophore with some structural similarities to that of the pharmacophore of the conventional 4-quinolone antimicrobial agents. Therefore it is presumed that this type of natural quinazoline alkaloid could also share the same activity as the conventional quinolone agents. These characteristics found a great importance in view to find new structural type in overcoming the emergence of quinolone resistance against some microbes which has been a disturbing feature of microbial infections. In this study the leaves of Glycosmis pentaphylla (Retz.) DC., a small tree or shrub from the genus Glycosmis of the Rutaceae family were extracted following acid base extraction method to furnish crude alkaloidal extract. The extract was then fractionated by column chromatography and each fraction was screened by using TLC Agar Overlay Bioautographic Assay for antimicrobial active alkaloids against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Candida albicans ATCC 90028. The active fraction was re-column chromatographed for the isolation of the active alkaloids. This bioassay-guided isolation has afforded arborine or 2-benzyl-1-methylquinazol-4-one as the active principle. Arborine is a simple substituted quinazolin-4-one which is characteristic alkaloid of G. pentaphylla leaves. Quantitative antimicrobial activity was determined by means of the minimum inhibitory concentration (MIC) and the minimum bacteriocidal concentration (MBC) using broth microdilution assay. The MIC value of arborine against the tested microbes ranged between 2000 µg/ml and 500 µg/ml whereas the MBC values against all microbes are above 2000 µg/ml. The results suggested that arborine may provide a new structural type for synthesizing new quinolone agent and also a natural candidate for antimicrobial combination study

Graveoline from Ruta angustifolia (L.) Pers. and its antimicrobial synergistic potential in erythromycin or vancomycin combinations

Ruta angustifolia (L.) Pers. is a Rutaceous species which contains various anthranilic acid derived alkaloids including the bioactive quinolones. This study is aimed at identifying the antimicrobial active alkaloids of R. angustifolia and evaluating their potential as synergistic enhancers in alkaloid-antibiotic combinations. Antimicrobial bioautography-guided isolation of alkaloidal fractions of R. angustifolia leaves has led to the identification of 2,3-dimethoxy-1-hydroxy-10-methylacridone [arborinine]; and 4,7,8-trimethoxyfuro[2,3-b]quinoline [skimmianine]; together with the major active alkaloid, 1-methyl-2-[3’,4’-methylenedioxyphenyl]-4-quinolone [graveoline]. Graveoline showed Minimum Inhibitory Concentration (MIC) values ranging from 500 to 1000 μg/mL against Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922. Checkerboard assay for antimicrobial combination effects between graveoline with either erythromycin or vancomycin showed enhancement of the antimicrobial activity of both antibiotics with Fractional Inhibitory Concentration Indices (FICI) ranged from 0.37 to 1.50. Synergistic effect with FICI of 0.37 was observed for graveoline-erythromycin combination against S. aureus compared to FICI of 1.00 for ciprofloxacin-erythromycin additive effect. Graveoline was a potential candidate for antimicrobial combination agent especially against S. aureus. The result supports the idea of using plant metabolites as antimicrobial synergistic agents

Graveoline, an antimicrobial activ alkaloid from Ruta angustifolia (L.) Pers. and its in vitro antimicrobial combination effects with either erythromycin or vancomycin

Ruta angustifolia (L.) Pers. is a Rutaceous species which contains various anthranilic acid derived alkaloids including the bioactive quinolones. This study is aimed at identifying the antimicrobial active natural quinolones of R. angustifolia and evaluating their potential as synergistic enhancers in alkaloid-antibiotic combinations. Antimicrobial bioautography-guided isolation of alkaloidal fractions of R. angustifolia leaves has led to the identification of 1-methyl-2-[3’,4’-methylenedioxyphenyl]-4-quinolone [graveoline], (III) as the major antimicrobial active alkaloid together with 2,3-dimethoxy-1-hydroxy-10-methylacridone [arborinine], (I) and 4,7,8-trimethoxyfuro [2,3-b]quinoline [skimmianine], (II). Alkaloid III and I showed Minimum Inhibitory Concentration (MIC) values ranging from 250 µg/ml to 1000 µg/ml against Staphylococcus aureus ATCC 25923, Enterococcus fecalis ATCC 29212 and Escherichia coli ATCC 25922. Checkerboard assay for antimicrobial combination effects between alkaloid III with either erythromycin or vancomycin showed enhancement of the antimicrobial activity of both antibiotics with Fractional Inhibitory Concentration Index (FICI) ranged from 0.37 to 1.50. Synergistic effect with FICI of 0.37 was observed for III-erythromycin combination against S. aureus compared to FICI of 1.00 for ciprofloxacin-erythromycin indifference effect. Alkaloid III was a potential candidate for antimicrobial combination agent especially against S. aureus which supports the idea of using plant metabolites as antimicrobial synergistic agents

Bioautographic profile as standard reference for qualitative analysis of the efficacy of herbs as antiseptic and antioxidant

This study was designed to develop a standard reference for qualitative analysis of the efficacy of herbs as antiseptic and antioxidant. Its main aim was to validate the authenticity of the herbal materials and quality control of their pharmaceutical herbal preparations. A method combining bioautographic assay and thin layer chromatography (TLC) analysis was employed to detect the antioxidant and antiseptic biomarkers and to obtain the TLC profile, respectively. The herbal extracts were subjected to TLC analysis on silica gel plates and each separated compound was visualized by various detection methods. Extracts of three selected herbs were studied and they were Piper betle L. (leaves), Ficus deltoidea Jack (leaves) and Ruta angustifolia Pers. (aerial parts). Antiseptic biomarkers were detected by using bioautographic agar overlay assay against selected standard bacterial and fungal strains, namely Staphylococcus aureus, Escherichia coli, Candida albicans, Microsporum cannis and Trichophyton rubrum. Antioxidant biomarkers were examined using TLC and DPPH staining assay. The complete bioautographic profile which included the antioxidant and antiseptic biomarkers with their TLC characteristics could be used as standard reference for qualitative analysis on the efficacy of these herbs and their herbal preparations. This method would also be applicable to other herbs for efficacy analysis