Antimicrobial and enzyme inhibitory assay of constituents of Lonicera lanceolata

The phytochemical study on the chloroform fraction of Lonicera lanceolata led to the first time isolation of four known compounds (1 to 4). The antimicrobial and enzyme inhibition assay of four compounds isolated from L. lanceolata are presented in this study. These compounds were screened against two human Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and four Gramnegative ones (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae and Klebsiella Pneumoniae) by performing Agar well diffusion method for antibacterial and antifungal activities. Minimum inhibitory concentration (MIC) was determined by Agar well dilution method. Minimum bactericidal concentration (MBC) was carried out by viable cell count method. Compound 1 showed maximum antimicrobial activities, while the other compounds also showed significant antimicrobial activities. In addition, the isolated compounds were assayed for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Moreover, the IC50 (50% inhibitory effect) values of compounds 1 and 2 against AChE were determined to be 1.99 and 1.75 μM, while the values obtained against BChE were 3.65 and 4.90 μM, respectively.

Microbial assay is a principal means of deterioration of foods and is frequently responsible for damage of quality and safety of the foods.The pathogenic and spoilage microorganisms in foods are increasing with the increase *Corresponding author.E-mail: kamran.gu@yahoo.com.Tel: +92 966 750359.Fax: +92 966 750250.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License in occurrence of food-borne diseases.Staphylococcus aureus is mostly responsible for post-operative wound infections, toxic shock syndrome, endocarditis, osteomyelitis and food poisoning (Mylotte et al., 1987).Listeria monocytogenes is mainly responsible for the harsh food-borne illness, listeriosis, which has been one of the rising zoonotic diseases during the last two decades (Farber et al., 2000).Escherichia coli is found in human intestines and is responsible for urinary tract infection, coleocystitis or septicaemia (Singh et al., 2000).

Chemicals and culture media
Thin layer chromatography (TLC) plates which are made up of aluminium (20 × 20, 0.6 mm thick) pre-coated with silica gel 60 F254 (20 × 20 cm, 0.2 mm thick; E. Merck, Darmstadt, Germany) were prepared in order to check the purity and isolation of the compounds.The support used in column chromatography (CC) was silica gel of 230 to 400 mesh (E.Merck, Darmstadt, Germany).Ceric sulphate and potassium permanganate solutions were applied as visualization reagents.UV spectra (λmax nm) were recorded on Shimadzu UV-2700 spectrophotometer (Shimadzu, Japan) in EtOH.Mass spectra were taken on Bruker TOF Mass Spectrometers (Billerica, USA) using electrospray ionisation (ESI). 1 H NMR and 13 C NMR spectra of the compounds were recorded on a Bruker DPX-400 NMR Spectrometer (Billerica, USA) (400 MHz for 1 H and 100 MHz for 13 C-NMR), using CDCl3 as solvents.

Plant
The plant materials of L. lanceolata were collected from Hazara Division, District Mansehra, in May 2012.The plant was identified by Professor Manzoor Ahmad, Plant Taxonomist on the basis of its morphology and the database present in the library at the Department of Botany, Government Degree College Abbotabad, Pakistan, where a voucher specimen were deposited in herbarium (Accession No. D-056).
Fraction D (6 g) was again introduced to a series of silica gel column chromatography eluting with n-hexane, n-hexanechloroform and chloroform with increasing polarity give compound 1 with 100% CHCl3 and to a preparative TLC using nhexane:chloroform (2:3) as solvent system and isolated compounds 2 and 3, respectively.
Fraction F (5 g) was again transferred over silica gel eluting with n-hexane, n-hexane:chloroform and chloroform in increasing order of polarity followed by preparative TLC eluted with n-hexane:CHCl3 (4:1) and isolated compound 4.

Microorganisms
To check the antibacterial activity of these compounds, two human Gram-positive bacteria and four Gram-negative bacteria, that is, E. coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa (clinical strain/PIMS), Enterobactercloacae (clinical strain/PIMS), S. aureus (MRSA, clinical strain/PIMS) and Micrococcus luteus (clinical strain/PIMS) were selected for the antibacterial test.Strains were gotten from Microbiology Research Laboratory, Beijing University of Chemical Technology (BUCT), China where their identification and characterization took place.These strains were placed on Agar slants at 4°C for antimicrobial screening.Microorganisms were kept in incubator overnight at 37°C in Mueller-Hinton Broth (Oxoid) at pH 7.2.Ofloxacin (10 μg) and ampicillin (10 μg) (Oxoid) were used as reference antibiotics (Table 1).

Screening for antibacterial activity by Agar well diffusion method
For determination of antibacterial activity of the isolated compounds, Agar well diffusion method (Hadacek et al., 2000) was carried out.All bacterial strains were first grown in Nutrient Broth at 37°C for 24 h incubated till turbidity became equal to McFarland 0.5 turbidity standard.Using a sterile swab, the inocula of the respective bacteria were streaked on to the Muller Hinton Agar (Oxoid) plates in order to make sure a uniform thick lawn of growth following incubation.Using sterile cork borer, wells of 5 mm in diameter were formed on to Nutrient Agar plates.The wells were filled with 100 μl of compounds 1 to 4 and the plates were then kept to stay for 2 h at 25°C.At last, the plates were incubated at 37°C for 24 h and the resultant diameters of zones of inhibition were measured carefully.

Agar dilution method
Agar well dilution protocol was followed to carry out MIC of  2002;WHO, 2002).After sterilizing, Muller Hinton Agar (oxoid) was allowed to cool to 50°C.With 1 ml of different concentrations of compound 1 in sterilized test tubes, about 19 ml of Muller Hinton Agar was mixed.After mixing carefully, this mixture was poured into pre-labelled sterile Petri dishes.Petri dishes having only growth media were prepared in the same way so as to serve for assessment with Petri plate containing compound.2000 to 0.156 μg/ml concentrations of the compound were used in this assay.The density of the suspensions of the respective microorganisms was adjusted to 0.5 McFarland turbidity standards.By using sterilized standard loop, these were inoculated onto the series of Agar plates.The incubation of these plates took place at 37°C for 24 h.The minimum concentration of the compound, which inhibited the growth of the respective organisms, was considered as MIC.The assay was carried out in triplicate.

Viable cell count method
For the determination of MBC of the compound 1, the viable cell count method was followed (Toda et al., 1989;Rodríguez-Tudela et al., 2003), and the results were shown as number of viable cells as a percentage of the control.

Screening for antifungal activity
By using sterilized standard loop the required amount of fungal strain was suspended in 2 ml of Sabauraud Dextrose Broth.This suspension was uniformly streaked on Petri plates having Sabauraud Dextrose Agar media by using sterilized cotton swabs.
Compounds were poured into wells using same technique for bacteria, but these were incubated at 25°C for 72 h, that is, 3 days.The plates were then examined for the presence of zones of inhibition and the results were measured and recorded.
Itraconazole was used as a standard or positive control which is a potent antifungal.
Butyrylthiocholine chloride and acetylthiocholine iodide were chosen as substrates to assay AChE and BChE, respectively.DTNB was used for the measurement of cholinesterase activity.0.2 mM DTNB in 62 mM sodium phosphate buffer (pH 8.0, 880 μl), test compounds solution (40 μl) and AChE or BChE solution (40 μl) were thoroughly mixed and incubated for 15 min at 25°C.The reaction was initiated by the addition of acetylthiocholine or butyrylthiocholine (40 μl), respectively.The hydrolysis of acetylthiocholine and butyrylthiocholine were monitored, when yellow 5-thio-2-nitrobenzoate anion was formed as a result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine, respectively, at a wavelength of 412 nm (15 min).All the reactions were carried out in triplicate in a BMS spectrophotometer (USA).The concentrations of test compounds that repressed the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) by 50% (IC50) were determined by monitoring the effect of increasing concentrations of these compounds in the assays on the inhibition values.The final DMSO concentration in the reaction mixture was 6% (Figure 1).

RESULTS
Compound 1 has high antibacterial activity as compared to other compounds as shown in Table 2. Thus, it was further considered for determination of MIC and MBC,  respectively.The MIC values ranged from 0.156 to >10 µg/ml for all tested strains, while the MBC values reported were many times higher than MIC (Table 3).The MBC value for K. pneumoniae was not detected.
In the same way, nearly similar pattern of defencelessness was reported against fungal strain Aspergillus niger.The widest zones of inhibition (maximum antifungal activity) were presented by Compounds 1 and 2. Compounds 3 have reasonable antifungal, while compound 4 has the lowest antifungal activity as shown in Table 4.
The interested of this study was to identify AChE and BChE inhibiting compounds from herbal medicinal plants, performed bioassay-guided search for AChE and BChE inhibitors from this medicinal plant.Compounds 1 to 4 isolated from L. lanceolata were tested against AChE and BChE, which show the most attention-grabbing target for drug treatment of neurone design and innovation of mechanism-based inhibitors for the degenerative disorders such as Alzheimer's disease (Zhang, 2004).The percentage of inhibition was first determined at 0.1 mM.Those compounds which showed greater than 50% enzyme inhibition, were consequently assayed for IC 50 (50% inhibitory effect) determination.Among the isolated compounds, compounds 2 and 3 showed the most effective inhibition activity against AChE and BChE as compared to standard drugs: allanzanthane and galanthamine in a dose dependent manner.The IC 50 values of compounds 2 and 3 against AChE were determined to be 1.99 and 1.75 μM, while against BChE, were measured as 3.65 and 4.90 μM, respectively.Compound 1 has no inhibition while compound 4 showed weak inhibition profile against AChE and BChE (Table 5).

DISCUSSION
The antimicrobial activities of four compounds isolated from chloroform fraction were tested against six bacteria species: E. coli, K. pneumoniae, P. aeruginosa, Enterobacter cloacae, S. aureus, and Micrococcus luteus.Nearly all compounds exhibited more or less antimicrobial activity against the test strains.Compound 1 exhibited the best activity against these bacteria.Besides that, compounds 2 and 3 have moderate and compound 4 have lowest activity against these bacteria.Nearly all the constituents from Lonicera lanceolata were primarily reported as active against the A. niger.Compounds 1 and 2 showed the highest antifungal activities, while compound 4 showed least antifungal activity.
The MIC of compound 1 was taken as 0.156 mg/ml.It is important that the MIC value is too high to be taken in susceptible ranges (Paul et al., 2006).The MBC value of compound 1 is many times higher than MIC.The antibacterial and antifungal assays were done by Agar well diffusion method.MIC was carried out by using Agar well dilution method, while MBC was performed by viable cell count method.The MBC values for K. pneumonia were not detected.
All the four compounds isolated from the chloroform fraction of L. lanceolata are polar and were primarily tested as antimicrobial reagents.Compounds 2 and 3 have the highest acetyl cholinesterase and butyryl cholinesterase inhibitory effects.This investigation is probably the first to exhibit the antimicrobial and enzyme inhibitory activities of compounds 1 to 4 of L. lanceolata, as a comprehensive literature review to the best of our knowledge; there is no information about the antimicrobial and enzyme inhibitory activities of these isolated compounds from this plant.

Conclusion
From the present studies, it is clear that four compounds isolated from chloroform fraction of L. lanceolata show prominent antimicrobial and enzyme inhibitory activities.Compound 1 exhibited the highest antibacterial activity.The MIC of compound 1 was taken as 0.156 mg/ml.These constituents of L. lanceolata can be used for enzyme inhibition and antimicrobial preparations.Therefore, other fractions, that is, n-hexane, ethyl acetate and n-butanol are further recommended for investigations to explore the potential medicinal compounds.

Table 1 .
Zone of inhibition of reference antibiotics.