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Clerodendrum splendens: A Potential Source of Antimicrobials.

Byline: Muhammad Ajaib Farah Boota Khalid Mohammed Khan Shahnaz Perveen and Shazia Shah

Summary: The antimicrobial status of the bark and leaves of Clerodendrum splendens was evaluated.The inquisition had provided that the leaf and bark of the plant had exhibited maximum antimicrobial potential. The antibacterial activity was significant against S. aureus with the maximum effectiveness of 71 0.6 mm displayed by the petroleum ether extracts of bark. Moreover all the petroleum ether extracts exhibited maximum inhibitory effects against all the bacterial strains. The Gram negative strains i.e. K. pneumoniae P. aeruginosa and E. coli had provided good to satisfactory results within the range of 20 0.8 mm to 69 0.9 mm. Excellent activities were exhibited by leaf and bark extracts against the fungal pathogens with the maximum potential displayed by the petroleum ether extracts of leaf i.e. 57 0.5 mm against A. oryzae. The MIC assays were carried out to further authenticate the results obtained by zones of inhibitions.

The MIC potential exhibited by Gram negative bacteria for K. pneumoniae was evaluated to be greater than 0.032 mg/mL while for the S. aureus was roughly evaluated to be greater than 0.088. Moreover the MIC activity displayed against other Gram negative bacterial strains was reported greater than 0.112 0.7 mg/mL for E. coli and greater than 0.059 1.0 mg/mL for P. aeruginosa. The MIC effectiveness for the fungal strain of A. niger and A. oryzae was evaluated to be 0.118 0.7 mg/mL.

Key words: Clerodendrum splendens antibacterial antifungal MIC.

Introduction

The studies on phytochemical pharmacological antibacterial and antifungal activities of plant extracts present an alternative source of medicines. The plant-derived material such as digitonins rutine papain morphine codeine centonine and many other exhibit a broad range of pharmacological activity [1]. Plants are used medicinally in different countries as a source of many potent and powerful drugs. Considering the vast potential of plants as source for antimicrobial drugs with reference to antibacterial and antifungal agents a number of investigations have done to screen the local flora for antibacterial and antifungal activity [2].

An increase in antibiotic resistant bacteria is threatening world population with the recurrence of infectious diseases that were once thought to be under control at best in developed countries. Methicillin resistant Staphylococcus aureus (MRSA) has gained much attention in the last decade as the MRSA is a major cause of hospital acquired AY-lactam antibiotic is the preferred drug against the S. aureus infections. S. aureus has developed resistance against the AY-lactam antibiotics due to the production of chromosomal or plasmid mediated AY-lactamases. Therefore concerted efforts are to be made to identify antimicrobial materials from natural products and traditional medicines [3].

Skin hair and subcutaneous tissues in human and animal are subjected to infection by several organisms mainly fungi named dermatophytes and cause dermatophytoses [4]. Different treatments have been recommended to control dermatophytes. In general pharmacological treatment option includes antifungal agents but recently the use of some natural plant products has been emerged to inhibit the fungal activities [5]. These natural plants involve garlic lemon grass datura kiker ginger black seed neem basil eucalyptus and alfalfa [6]. They are safe to human and the ecosystem than the chemical antifungal compounds and can easily be used for the public who used them for thousands of years to enhance flavor and aroma of foods as well as its economic value [7]. The selection of the microorganisms for antibacterial and antifungal evaluation in this study was based on their known pathogenic effects in both human and animals with emphasis on diarrheal pathogens.

Pathogenic E. coli is a Gram negative bacterium and has been causing in diseases such as diarrhea hemorrhagic colitis hemolytic uremic syndrome and thrombocytopenic purpura. Pseudomonas aeruginosa is a Gram negative bacterium that causes diseases such as mastitis abortions and upper respiratory complications. Staphylococcus aureus is a Gram positive cocci and one of the prominent microbe causing skin infection such as boils abscesses carbuncles sepsis of wounds and it also produces toxins such as vomiting. Aspergillus niger and A. oryzae both are pathogenic fungal strains and are associated with certain skin disorders [8].

Clerodendrum splendens G. Don belongs to family verbenaceae is an evergreen densely branched climber or sarmentose vine or running shrub up to 5 m in hight. It is the native to Western Africa and is widely distributed in west and central Africa. It is commonly called as bleeding heart vine flaming glorybower and pagoda flower. It is cultivated in the gardens for ornamental purposes and for its medicinal value as well [9]. The plant is used to treat yellow fever and panacea of witchcraft.

The family Verbenaceae includes 75 genera and 2500 species chiefly of tropical and subtropical regions. Genus Clerodendrum contains a lot of species that are included in flora of Pakistan having ethnopharmacological importance used in chest troubles syphilitic rheumatism stomach troubles and swellings in cattle. The essential oil of this genus used for taxonomic treatment used as a febrifuge and tonic and root as a cure for scorpion and snake bite. The decoction is given in tetanus rheumatism and malaria. As a lot of work has been done on the members of Verbenaceae family thus this species is selected to investigate the possible antimicrobials potentials [10].

Results and Discussion

The investigation of the antimicrobial potential of the plant C. splendens was carried out on leaf and bark. The respective extracts were prepared by maceration procedure due its cost effectiveness and good extract yield in comparison to other techniques. Their percentage extraction yield was calculated before subjecting them to further analysis to provide the glimpse of the economical perspective of the whole procedure. The highest yield thus obtained from the leaves of C. splendens was 6.81%. The yield of petroleum ether extract in the range of 1- 2.60% chloroform extracts yield in the range of 4- 6.28% and methanol extracts yield in the range of 4.86-6.81% (Table-1).

Table-1: Percent extraction yield of the leaf and bark of C. splendens

###%extraction yield###

Plant Part

###Petroleum ether###chloroform###Methanol###Aqueous

Leaf###1.08###4.36###6.81###5.70

Bark###2.58###6.28###4.86###5.96

Antimicrobial potential of plant extracts was observed by calculating the inhibition zones produced by plant extract against bacterial and fungal strains. The crude extract of plant in different polar and non-polar solvents restricted the growth of various bacterial and fungal strains. The standard discs were used against microorganisms to make a comparison between the zones of inhibition produced by the commercially available discs and plant extract against four different bacterial strains (Gram-positive bacteria) S. aureus and (Gram-negative bacteria) P. aeruginosa E. coli and K. pneumoniae. Four antifungal standard discs were used against the fungal strains i.e. A. niger and A. oryzae. The standard discs were used to determine the susceptibility of the microbial specimens employed as the test organisms (Table-2 3). The negative control was established by evaluating the solvents in which the extracts were prepared against the bacterial strains used as test organisms.

Thus for fungal strains the standard discs were also used to compare the experimental to the control as in the following Table-3.

Table-2: Zone of Inhibition (mm) produced by the bacterial strains against Standard antibiotic discs.

Antibiotic Standard disc Conc. g Bacterial Strains

Amikacin###30

###E. coli

Azithromycin###15###

Erythromycin###15

Azithromycin###15

###S. aureus

Cephradine###10###P. aeruginosa

Meropeneme###10###K. pneumonia

Table-3: Zone of Inhibition (mm) of the fungal strains against the Standard antifungal discs.

Antifungal standard disc###Conc.

Fungivin###100###15 3.4

Grisofluvin###100###23 1.5

Nystatin###100###24 2.0

Tezole###100###22 0.5

However all the extracts had exhibited some activity against both types of strains investigated with their potential ranging from 16 0.2 mm to 71 0.6 mm. The broad spectrum of potential was displayed by the extracts of the C. splendens against the fungal strains. Some of the extracts had documented tremendous potential against the mycotic agents including leaf and bark extracts macerated in petroleum ether possessing 26 1.0 mm to 71 0.6 mm zones respectively among the others like chloroform extract had also exhibited antifungal potential methanol and water extracts had exhibited satisfactory to weak activity. Therefore this study suggests that methanolic extracts of screened plants would be helpful in treating diseases in plants caused by A. niger.

The petroleum ether extracts exhibited maximum zone of inhibition against Gram-positive bacteria S. aureus within the range of 51 0.3 mm to 71 0.6 mm (Table-4). The maximum potential was provided by the extracts of bark. The standard discs i.e. erythromycin and azythromycin were used to compare the results of the petroleum ether extract but the standard discs showed less inhibition as compared to the plant extracts (Fig. 1). Overall the petroleum ether extracts of C. splendens showed highest resistance against all the tested bacterial strains. The zones measured against K. pneumoniae were greater than the standard disc

The chloroform extracts had displayed maximum antibacterial potential as compared to the other extracts including water and methanol. The significant zones exhibited by chloroform extracts against K. pneumoniae and were compared with the standard disc meropeneme. The results obtained by standard disc mostly were similar to the plant extracts.

The aqueous extracts showed poor results as the inhibition exhibited by these extracts was comparatively very much less as compared to the other solvents. Against all the bacterial strains less activity was observed by water extracts.

The extracts had demonstrated more potential against the Gram-positive bacteria i.e. S. aureus and among Gram negative bacteria K. pneumoniae had exhibited higher potential as compared to P. aeruginosa and E. coli (Fig. 3 4). Conclusively S. aureus and K. pneumoniae had documented maximum resistance against the extracts of the particular plant material. As far as antimycotic potential was concerned the A. oryzae had offered more resistance to the plant extract than A. niger.

Table-4: Zone of inhibition exhibited by different parts of C. splendens against bacterial test organisms.

###Zone of Inhibition (mm)

###Plant Part###Solvents

###E. coli###S. aureus###P. aeruginosa###K. pneumoniae

###Petroleum ether###53 0.3###51 0.3###49 0.3###69 1.1

###Chloroform###20 0.5###29 0.5###24 0.4###47 0.3

###Leaf

###Methanol###25 1.2###20 0.5###19 0.2###23 0.2

###Aqueous###17 0.5###21 0.8###22 0.2###16 0.2

###Petroleum ether###26 1.0###71 0.6###61 0.3###63 0.2

###Chloroform###39 0.3###35 0.9###30 0.5###51 0.3

###Bark

###Methanol###29 0.7###23 1.0###26 1.0###39 0.9

###Aqueous###22 0.8###25 0.3###23 0.5###19 0.9

This trend of the antifungal action had exhibited by the extracts of C. splendens was somewhat similar in the antibacterial potency of the plant. The petroleum ether extracts of leaf showed maximum effectiveness against the A. oryzae while less activity was observed with the water extracts. The effectiveness of the extracts in this regard can be documented as petroleum ether greater than chloroform greater than methanol greater than aqueous. These results were similar with the results of the standard disc tezole that was used for the comparison (Table-5). Almost the same trend has been exhibited by the plant extract against the both fungal strains A. niger and A. oryzae

Table-5: Zone of inhibition exhibited by different parts of C. splendens against bacterial test organisms.

Plant Part###Solvents

###A. niger###A. oryzae

###Petroleum ether###48 1.4###57 0.6

###Chloroform###24 0.9###25 0.7

Leaf

###Methanol###18 1 .5###23 0.9

###Aqueous###13 0.6###10 0.6

###Petroleum ether###49 2.0###55 0.9

###Chloroform###29 1.7###32 0.8

Bark

###Methanol###17 0.8###19 0.8

###Aqueous###15 0###12 0.4

The further elaboration towards the evaluation of the antibacterial potential of the C. splendens was provided by adopting Microbroth dilution assay to evaluate MIC value. The methanolic extracts were only subjected to MIC assay because of its potential for the consistent extraction of constituents possessing antibacterial potential. The MIC values of the methanolic extracts of the plant under investigation range from 0.121 0.6 to 0.032

In addition antifungal MIC values of the methanolic extracts of C. splendens were evaluated and found to be in the range of 0.119 0.6 to 0.179 0.9 mg/mL. The MIC potential exhibited by Gram negative bacteria for K. pneumoniae was evaluated to be greater than 0.032 mg/mL while for the Gram negative S. aureus was roughly evaluated to be greater than 0.088. Moreover the MIC activity displayed against other Gram negative bacterial strains was reported greater than 0.112 0.7 mg/mL for E. coli and greater than 0.059 1.0 mg/mL for P. aeruginosa. The potential of the plant against the bacterial strains was K. pneumoniae greater than P. aeruginosa greater than S. aureus greater than E. coli

The MIC effectiveness for the fungal strain of A. niger and A. oryzae was evaluated. The bark extract had exhibited maximum potential against A.

niger that was as 0.118 mg/mL (at 0.9 mg/mL) and for A. oryzae it was evaluated as 0.179 mg/mL (at 0.9 mg/mL). Thus it was clearly demonstrated that the potential of the plant against fungal strains was more significant in A. niger as compared to A. oryzae (Fig. 12).

The MIC results for the estimation of the antibacterial potential were in accordance with the results obtained from the evaluation of zone of inhibition. However some of the results had shown marked deviation. The underlying cause might be the impact of temperature and the nature of medium on the susceptibility of bacteria as the MIC was carried out in the liquid medium while the zone of inhibition was married in the solid medium.

The results obtained by the antimicrobial investigation of C. splendens showed that this plant has significant medicinal value.

Experimental

Plant Material

The plant C. splendens was collected from the Botanic garden of the Government College University Mall Road Lahore. Plant specimen was identified with the authenticated voucher no. GC. Herb. Bot. 988 and deposited to Dr. Sultan Ahmad herbarium Department of Botany Government College University Lahore Pakistan.

Test Organisms

The bacterial test organisms utilized for investigation i.e. S. aureus E. coli K. pneumonia and P. aeruginosa were obtained and identified from Pathology laboratory of mayo hospital and PCSR laboratory Lahore. The fungal strains used for the evaluation of the antifungal potential were A. niger and A. oryzae obtained from the Institute of industrial biotechnology and microbiology Government college university Lahore.

Methodology Adopted

The plant materials of C. splendens was primarily distributed into its constituents such as leaf and bark and then shade dried at room temperature for 20-30 days. Eventually the dehydrated plant material was powdered and macerated.

Maceration of the Plant Material

During the static-state maceration setup taken into consideration the evaluated quantity of the delicately crumbled plant material was uniformly positioned in impenetrable glass container and was plunged in the solvent. The glass container was subsequently positioned aside with repeated agitation at the room temperature for approximately 7 days. Following the achievement of the time interval the ingredients present within the impermeable glass container were subjected to filtration via filter paper. The residue of the plant material left behind was air- dried for about 20 minutes and was then executed once more to the maceration with another fluent. The solvents employed for maceration include petroleum ether (P.E.) chloroform (Chl.) methanol (MeOH) and water (Aq.). The fluent (menstruum) were applied in agreement with their polarity gradient with maceration scheduled from non-polar solvents to polar solvents.

Ultimately the extracts obtained was concentrated using rotary evaporator for the petroleum ether chloroform and methanol extracts (at low pressure and temperature roughly up to 40 C). The concentrated extracts were afterwards stored at 20 C.

Preparation of Plant Sample

0.2 g of the macerate was transferred to the separate glass vial and the ultimate volume was brought to 20 mL with the solvent in which the extract was macerated. The glass vials enclosing the mg/mL concentration were finally stored at 20 C.

Preparation of Medium

The Gram positive and Gram negative bacteria used for investigation of the antimicrobial potential of the plant extracts was cultured on the nutrient agar medium. The medium was prepared according to Cruick-Shank et al. [11]. The nutrient broth was prepared for the preliminary inoculation of the bacterial specimens.

The fungal specimens were cultured on PDA medium prepared as mentioned by Johansen (1940) [12].

Preparation of Medium Plates

The 15 mL of medium was allocated to every autoclaved Petri plate and the plates were preserved with the cling film and were stored in the uncontaminated location at room temperature until consumption.

Preparation of Inoculums

For Bacterial Strains

The 20 mL of autoclaved nutrient broth was poured into each of the test tube and the loopful of the bacterial strain was introduced in it under aseptic conditions. The test tubes were appropriately labeled and were placed in the incubator at 35 2 C for about 24 h.

For Fungal Strains

The autoclaved Petri plate containing PDA medium was streaked with the fungal strain following the aseptic circumstances. Afterwards the Petri plates containing the inocula were sealed with cling film and were placed for 48 h in the incubator at 25 2C.

Adjusting the Turbidity of the Inocula

For Bacterial Inoculum

The turbidity of the bacterial inoculum was acclimatized to 1.5A-108 colony-forming unit (CFU)/mL by contemplating it with McFarland 0.5 Barium chloride turbidity standard.

For Fungal Strains

4-5 colonies from 48 h fungal culture dish were conveyed to 10 mL saline solution i.e. 0.9 % NaCl solution and its consistency was homogenized by employing 0.5 McFarland density standards within the series of 1A-103 to 5A-103 CFU/mL.

Agar Well Diffusion Method

The agar well diffusion technique engaged for the investigation was modified by W. M. M. Kirby and his coworkers at the University Of Washington School Of Medicine in 1956. To set up the analysis plates primarily the inoculum was extended homogeneously on the plane of the solidified medium with the assistance of disinfected cotton swab. Ultimately the well is prepared by cork borer no. 4 in the center of the petri plate containing medium and 1 mL plant extract was replenished in the hole by means of pipette. The lid of the plate was positioned on it and the plate is preserved with cling film. Each plate was aptly labeled and the trial was performed in triplicates for the accurate results.

The prepared plates were incubated at 37 2 C for 24 h afterward the plates were placed in the incubator at 25 2 C for 48 h. The standard discs were employed against the microbial specimens as positive control and the solvents utilized during maceration were provided as the negative control.

Measurement of Zone of Inhibition

After incubation the diameter of zone was measured in mm by means of ruler. To verify the reading the flipside of the Petri plate was apprehended few inches away and the diameter were measured which was discernible to the unaided eye. Growth up to the boundary of the disk was notified as a zone of 0 mm.

Estimation of Minimum Inhibitory Concentration (MIC)

The methanolic extracts of leaf and bark of Clerodendrum splendens were employed for the determination of minimum inhibitory concentration (MIC).

Preparation of Plant Sample

The concentration of the plant extract prepared for MIC was 10 20 30 40 50 60 70 80 90 and 100 mg/mL.

MIC assay

The MIC assessment of C. splendens was carried out by Murray et al. [13] with trivial modifications. Two series were prepared each having 10 test tubes corresponding to the varying consistency of the plant extract i.e. 10-100 mg/mL for a single activity. The one test tube series was serving as control while the other series of the test tubes was allocated for the experimental purpose. The single experimental dilution was prepared by adding 1 L of the microbial suspension from the calibrated inoculum 1 L of the corresponding methanolic plant dilution stock solution 2.5 L medium i.e. nutrient broth for the bacterial strains and malt extract for the fungal strains and then bringing the final volume to 3 mL by the supplementation of additional double distilled water. In the similar lines all the series of the test tubes were prepared i.e.

by keeping the volume of the medium and inoculum constant and varying the concentration of the plant extract keeping the amount poured in he assessment test tube constant. The control had differed from the experimental series in that the test tubes lack the microbial inoculum.

The prepared test tubes were lodged in the incubator at 37 2 C for 24 h when the antibacterial investigation was carried while in case of estimation of antifungal activity the test tubes were placed in the incubator at 25 2 C for 48 h and the readings were documented by means of the spectrophotometer at the absorbance of 595 nm.

Conclusion

The results obtained by the antimicrobial investigation of Clerodendrum splendens showed that this plant has significant medicinal value. This plant exhibited antimicrobial properties and it can be used to kill certain resistant bacteria as the maximum zone of inhibition that had recorded by the investigation is 71 mm against the S. aureus macerated in petroleum ether so it can be used to treat bacterial infections. The antifungal activity had also recorded high in the petroleum ether extracts. This trend revealed that the plant C. splendens having more non-polar compounds as fats and oils that are very much soluble in non-polar solvent like petroleum ether. Phenolic compounds inhibit microorganisms probably due to iron deprivation and forming hydrogen bonds with vital proteins like microbial enzymes. Infections related to fungi have been increasing day by day and there is need to find out more effective medicines.

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Publication:Journal of the Chemical Society of Pakistan
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Date:Aug 31, 2014
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