Isolation, characterization and antimicrobial evaluation of a novel compound N-octacosan 7β ol, from Fumaria parviflora Lam
© Jameel et al.; licensee BioMed Central Ltd. 2014
Received: 13 November 2013
Accepted: 5 March 2014
Published: 12 March 2014
Fumaria parviflora Lam. (Fumaraceae) is widely used in traditional as well as folkloric system of medicine from ancient. It is commonly known as ‘Pitpapra’ or ‘Shahtrah’ in Indian traditional system of medicine and used for treating numerous ailments like diarrhea, fever, influenza, blood purifier and other complications. The object of the present study was to evaluate the Antileishmanial, antibacterial, antifungal and cytotoxic potential of isolated compound.
Methanolic extract of whole plant of Fumaria parviflora was dried under reduced pressure to obtain a dark brown residue which was adsorbed on silica gel column grade (60–120 mesh) to obtain a slurry and chromatographed over silica gel loaded column in petroleum ether – chloroform (3:1, 1:1 and 1:3 v/v). The in vitro antileishmanial evaluation of isolated compound against Leishmania donovani promastigotes was investigated by growth kinetics assay, reversibility assay, analysis of cellular morphology, adverse toxicity and determination of 50% growth inhibitory concentration (GI50). Disc diffusion and broth micro dilution methods were used to study the antibacterial (Gram + Staphylococcus epidermidis and Bacillus subtilis; Gram - Escherichia coli and Salmonella typhimurium) and antifungal (Candida albicans and Aspergillus niger) potential in vitro.
Structure elucidation by spectral data analysis revealed a novel compound, n-octacosan-7β-ol (OC), yield (0.471%), having significant antimicrobial activity against Leishmania donovani promastigotes, Staphylococcus epidermidis, Escherichia coli, Candida albicans and Aspergillus niger in vitro with GI50 = 5.35, MIC 250, MIC 250 and MFC 500 and MIC 250 μg ml-1 respectively. The isolated compound did not show adverse effect against mammalian macrophages.
The available evidence of compound suggested that it may be used as antimicrobial agent in future and may provide new platform for drug discovery programmes for leishmaniasis.
KeywordsFumaria parviflora Homaira Shahtrah Leishmanial potential Novel Isolation
The traditional medicine has been a vibrant, innovation-driven highly successful component of global industry and confluence of spectacular advances in chemistry, molecular biology, genomics and the cognate fields of spectroscopy, chromatography and crystallography led to the new findings and development of numerous novel curative agents for the treatment of a wide spectrum of ailments. They have formed the basis of sophisticated traditional medicine systems among which Ayurvedic, Unani and Chinese systems have given ascent to crucial unmatched lead molecules still in use today. The number of higher medicinal plant species on this planet is estimated at 250,000 but of these only about 6% have been screened for biological activity, 15% have been evaluated phytochemically and only about 0.75% herbal drugs have been studied in clinical trials. So the search for new molecules nowadays has taken a faintly different route where the science of ethanomedicine is being used as a guide to lead the chemist towards different sources and classes of molecules.
Fumaria parviflora Lam. is widely used in traditional and as well as folkloric system of medicine named as “fumitory, earth smoke, beggary, fumus, vapor and fumitory or wax dolls” in English. It is locally known as ‘Pitpapra’ or ‘Shahtrah’ in India, “Homaira” in Saudi Arabia. Phytochemical investigation of this medicinal plant revealed the presence of several alkaloids[9, 10], and shown to possess pharmacological activities like antipyretic, hepatoprotective, hypoglycemic, antidiarrheal, antispasmodic, bronchodilator, anthelmintic, antieczema and nematocidal activities.
Visceral leishmaniasis is an infectious disease caused by protozoan parasite, leishmania donovani which is considered as a major public health trouble in developing countries. Primary health care systems are not always significant to use the herbal medicines in common practice because very few studies were carried out to investigate the potential use of these medicinal plants in treatment of parasitic diseases. The chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing severe concerns of toxicity, high cost, and drug resistance. In view of that concern an integrated approach towards the discovery and development of novel chemical entity with ethanopharmacological implications for promotion and development of leads for leishmaniasis as well as traditional medicine, remains a significant hope in the current, target-rich scenario which may offer unprecedented diversity in structures and bioactivity. So isolation, characterization and antimicrobial evaluation has been aimed for novel bioactive compound by chromatographic techniques, and bacterial strain (Gram + Staphylococcus epidermidis and Bacillus subtilis; Gram - Escherichia coli and Salmonella typhimurium), fungal strain (Candida albicans and Aspergillus niger) and Leishmania donovani promastigotes.
M199 medium, RPMI 1640 medium were obtained from Sigma-Aldrich, fetal bovine serum (FBS) from Gibco- BRL, DMSO from SRL and methanol from Merck. All other chemicals were from Sigma-Aldrich unless otherwise stated. Melting point was determined on a Perfit apparatus without correction. The IR spectrum was measured in KBr pellet on a Bio-Red FT-IR spectrometer. UV spectrum was obtained in methanol with a Lambda Bio 20 spectrometer, 1H (400 MHz), 13C (100 MHz), NMR spectra were recorded on Bruker spectrospin spectrometer. CDCl3 (sigma-Aldrich) were used as solvent and TMS as an internal standard. ESI- MS analysis was performed on a Synapt Mass spectrometer (Waters) equipped with direct inlet probe system. Column chromatography separations were carried out on column grade silica gel (Merck, 60–120 mesh). Precoated silica gel plates (Merck, Silica gel 60 F254) were used for analytical thin layer chromatography (TLC) visualized by exposure to iodine and UV radiations. Microbial strains used for the antimicrobial evaluation, a panel which included laboratory control bacterial strains (Gram + Staphylococcus epidermidis and Bacillus subtilis; Gram - Escherichia coli and Salmonella typhimurium) fungal strains (Candida albicans and Aspergillus niger), and Leishmania donovani promastigote. Streptomycin, amphotericin B and DMSO analytical grade used for positive and negative control respectively.
The Fumaria parviflora Lam. (whole plant) was collected from the herbal garden of Jamia Hamdard, New Delhi and identified by Prof. Javed Ahmad, Incharge herbal garden. A specimen voucher of the drug was deposited to the Phytochemistry Research Lab (PRL) with a reference number PRL-JH/2011/05.
L. donovani strain AG83 was a kind gift from Dr. Nahid Ali (IICB, Kolkata, India) and was maintained in vivo in BALB/c mice. Promastigotes were maintained in medium M199 supplemented with 100 U ml-1sodium penicillin G and 100 μg ml streptomycin1 sulfate (incomplete medium) and 10% heat-inactivated FBS at 22°C, and subcultured every 72 h in the same medium at a mean density of 2 × 106 cells/ml.
Preparation of crude extract and isolation
The dried F. parviflora Lam. whole plant (1.25 kg) was coarsely powdered and extracted with methanol for 72 h using a Soxhlet extractor. The extract was dried under reduced pressure to obtain a dark brown residue (190 g). The residue (100 g) was dissolved in minimum amount of methanol and adsorbed on silica gel column grade (60–120 mesh) to obtain slurry. The slurry was dried in air and chromatographed over silica gel column loaded in petroleum ether. The column was eluted with petroleum ether and petroleum ether –chloroform (3:1, 1:1, 1:3 v/v) mixtures.
General procedure for antimicrobial evaluation
Agar disc diffusion method and broth microdilution method were employed for the determination of antibacterial and antifungal activities. The minimum inhibitory concentration (MIC), minimum bactericidal (MBC), minimum fungicidal concentration (MFC) and the inhibition zone of the OC against the test microorganisms were determined by the broth microdilution method. The MIC, MBC, MFC and the inhibition zone of the n-octacosan-7β-ol (OC) were also determined in parallel experiments in order to control the sensitivity of the test microorganisms.
A suspension of the bacterial strain (Gram+Staphylococcus epidermidis and Bacillus subtilis; Gram - Escherichia coli and Salmonella typhimurium), fungal strain (Candida albicans and Aspergillus niger) (0.1 ml of 105 CFU ml-1) was spread on the solid media plates. Nutrient agar and Czapek Dox Agar sterilized in a flask and cooled to 45~ 50°C were distributed to sterilized Petri dishes with a diameter of 9 cm (15 ml). The filter paper discs (6 mm in diameter) were individually impregnated with 50 μl (1000 μg ml-1) of the OC and then placed onto the agar plates which had previously been inoculated with the tested microorganisms. The plates were inoculated with bacterial strains incubated at 37°C for 24 h and at 28°C for 48 h for the fungal strains. The diameters were measured in millimeters. Streptomycin (10 μg/disk) was used as a positive control for bacteria, amphotericin B (5 μg/disk) as a positive control for fungi, and DMSO was used as the negative control. All assays were done in duplicate. A broth microdilution method was used to determine the MIC, MBC and MFC according to the National Committee for Clinical Laboratory Standards. All tests were performed in Nutrient broth and Czapek-Dox broth supplemented with ethanol at final concentration of 0.5% (v/v) for all microorganisms. Two folds serial dilutions of the OC were prepared in a 96-well microtiter plate ranged from 1000 μg ml-1 to 1.038 μg ml-1. Overnight broth cultures of each strain were prepared and the final concentration in each well was adjusted to 105 CFU ml-1for bacterial strains and fungal strains. 96-well microtiter plate injected with fungal strain was incubated at 28°C for 48 h, and the bacteria were incubated at 37°C for 24 h. The MIC is defined as the lowest concentration of the OC at which the microorganism does not demonstrate visible growth. To determine MBC and MFC, 10 μl broth was taken from each well and inoculated in Nutrient for 24 h at 37°C for bacteria or in Czapek Dox Agar for 48 h at 28°C for the fungi. The MBC is defined as the lowest concentration of the OC at which inoculated bacteria were completely killed. The MFC is defined as the lowest concentration of the OC at which inoculated fungi were completely killed. All determinations were performed in duplicate and two growth controls consisting of Nutrient Agar and Czapek Dox agar medium were included. The streptomycin and amphotericin B served as a positive control.
Growth kinetics assay
Promastigotes of L. donovani strain MHOM/IN/83/AG83 (2 × 106 cells ml-1) were incubated in the presence of OC in M199 containing 10% FBS (complete medium) at a concentration of 100 μg ml-1. Pentamidine (100 μg ml-1) served as the reference antileishmanial drug, while 0.2% DMSO, which represented the highest concentration in the test compound, was used as a solvent control. Parasites in medium alone were taken as control. Viable parasites were enumerated for 7 days using a phase-contrast microscope under a 40X objective.
Growth reversibility assay
To confirm the leishmanicidal effect of OC, treated and untreated parasites after 7 days of incubation were washed twice with incomplete medium and finally resuspended in complete medium and cultured at 22°C for a further 96 h. The viability of the parasites was ascertained microscopically.
Analysis of cellular morphology
Morphology of the promastigotes was evaluated after 96 h of treatment with the test OC, pentamidine and or 0.2% DMSO and photomicrographs were taken at 400x magnification under phase-contrast microscope.
Determination of GI50
To determine the GI50 (concentration of compound that inhibited growth of parasites by 50%), promastigotes at a density of 2 × 106 cells ml-1 were incubated in triplicate with or without OC at serial three fold dilutions starting from 100 μg ml-1 for 96 h. Pentamidine served as the reference drug.
Cytotoxicity to mammalian cells
Macrophages were collected by peritoneal lavage from starch-stimulated mice. Peritoneal cells were collected in RPMI 1640 medium (incomplete), pelleted by centrifugation at 800 x g for 10 min at 4°C, washed twice and finally resuspended in complete medium. Macrophages at a cell density of 1 × 106 cells ml-1 were incubated with OC at varying concentrations for 48 h in a CO2 incubator (5% CO2, 37°C). Pentamidine served as reference drug and 0.2% DMSO as solvent control. Macrophages without any treatment were taken as control. Cells were observed under phase-contrast microscope and viability was ascertained after trypan blue staining.
Structural elucidation of isolated compound
Elution of the column with ratio of petroleum ether-chloroform afforded star-shaped colorless of OC, recrystallized from chloroform-methanol (1:1), 3.1 g (0.471% yield); Rf 0.54 ± 0.02 (petroleum ether-chloroform, 1:1 v/v), m.p. 74–75°C; UV λ max (MeOH); 209 nm (log ϵ 3.6); IR λ max (KBr); 3318, 2954, 2848, 1465, 1378, 1236, 1134, 857, 721 cm−1; 1H NMR (CDCl3); δ 3.67 (1H, brm, w1/2 = 14.1 Hz, H-7α), 2.36 (2H, m H2 -6), 2.14 (2H m, H2-8), 1.70 (4H, m, 2xCH2), 1.51 (8H, m, 4x CH2), 1.34 (34H, 17x CH2), 0.98 (3H, t, J= 6.0, Me-1), 0.95 (3H, t, J= 6.8, Me-28); 13C NMR (CDCl3); δ 72.06 (C-7), 37.50 (CH2), 31.95 (CH2), 31.91 (CH2), 29.73 (6x CH2), 29.67 (7x CH2), 29.61 (5x CH2), 29.40 (CH2), 29.36 (CH2), 25.68 (CH2), 22.72 (CH2), 14.16 (Me-1, Me-28). +ve ESI-MS m/z (rel. int.); 411 [M+1]+ (9.6), (C28H59O), 325(100).
Antibacterial and antifungal study
Antibacterial and antifungal activities of OC as inhibition zones (mm)
Staphylococcus epidermidis (Gram +)
Bacillus subtilis (Gram +)
Escherichia coli (Gram -)
Salmonella typhimurium (Gram -)
Minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and minimum fungicidal concentrations (MFC) of OC
Amphotericin B (μg/ml)
In vitro antileishmanial activity by growth kinetics assay
Alteration of cellular morphology
Growth reversibility assay
Evaluation of the GI50 against promastigotes
Cytotoxicity on mammalian macrophages
Natural products compounds have been the mainstay of several drug discoveries since the early days of the antibiotic era due to challenge posed by antibiotic resistance. Discovery and development of a new leads of antimicrobial drugs as well as many others could be better explained by reverse pharmacology which is an evolutionary process, based on traditional knowledge which facilitates not only to search experimental database for many novel leads but also minimize time, money and toxicity since several years. VL is considered as an opportunistic infection among immunocompromised patients and mainly treated with toxic pentavalent antimonials, second line drugs such as amphotericin B and pentamidine. Currently there is no vaccine and resistance to antimonial chemotherapy, coupled with its high cost, toxicity and parenteral route of administration, is a matter of great concern in endemic regions of developing countries. Due to severe adverse and toxic profile of synthetic molecules, it is essential requirement for routine screening for safer bioactive compounds from medicinal, aromatic and food plants. Hence there could be no doubt to search for novel antimicrobial agent having antileishmanial potency is one of the core challenges in the current drug discovery programme and this worldwide problem. Previously reports exhibited as anthelmintic and nematocidal activity from extract of F. parviflora but still no report of such activity by novel isolated molecule as n-octacosan-7β-ol, OC (0.471% yield) may be a new hope for this widespread problem.
The authors are very thank full to Central Council for Research in Unani Medicine, New Delhi, India and Jamia Hamdard University, New Delhi, India, for providing research and instrumental facility such as NMR, FT-IR and LC-MS.
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