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Md Shamsuddin Sultan Khan et al /2014
Volume 4 Issue 4 [2014] 99
Screening of BADH Activity of Borreria articularies (Linn.) for the
Inhibition of P. aeruginosa
Md Shamsuddin Sultan Khan*1, Abdullah Al Mamun2, Shamima Parvin3, Md. Ershad Alam2
and Md. Ahsan Habib2
1EMAN Research and Testing Laboratory, The School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM,
Penang, Malaysia.
2Department of Pharmacy, Bangladesh University, Mohammodpur, Dhaka
3Stamford University, Dhaka, Bangladesh.
*Correspondence Info:
Md Shamsuddin Sultan Khan
EMAN Research and Testing Laboratory,
The School of Pharmaceutical Sciences,
Universiti Sains Malaysia, 11800 USM, Penang, Malaysia.
E-Mail: jupitex@gmail.com
1. Introduction
Borreria articularis Linn. is a creeping branched annual evergreen herb in South East Asia. It is useful in treating fever, bladder
stones, sores, wounds, headache, constipation, dental problems, fevers, diarrhea, head cold, hemorrhoids and conjunctivitis, gallstones, vitiated
vata, pitta, arthritis, pain, muscle ache, edema, trauma, indigestion, colic, skin disease, menorrhagia and leucorrhea1,2,3. Plant principally contains
alkaloids, glycosides, sterols, D-mannitol and ursolic acid. The plant contains sitosterol, ursolic acid and d-mannitol. Seeds contain isorhamnetin4.
The chloroform extract of the aerial parts and roots of Borreria articularis contains triterpene, 3-α acetoxy-oleana-12-en-29-oic acid along with
β-amyrin. Microorganisms are major health concern although the current development of vaccination and chemotherapy. To identify a new
antibacterial agents, it is not sufficient to study microbial growth inhibition but also important to study mode of action in pathogenic
microorganisms such as effects on bacterial cell membranes and metabolic enzymes. Thus, plant compounds need to test after positive screening
for microbial growth inhibition. Microorganisms can be inhibited by many possible target enzymes. The gram negative bacterium Pseudomonas
aeruginosa is an important pathogen of plants and animals. Betaine aldehyde dehydrogenase (BADH) is a target enzyme for inhibition of P.
aeruginosa growth. BADH catalyzes the synthesis of glycine betaine to inhibit P. aeruginosa in osmotic stress presented in the infected human
tissues5. Glycine betaine may also protect intracellular enzymes and organelles against the adverse effects of increases in intracell ular ionic
strength or temperature6,7,8.
Alkaloids are heterocyclic nitrogen compounds characterized by different antimicrobial activities. Plant-derived compounds are
mostly secondary plant metabolites traditionally used for medicinal purposes. Activity the medicinal plants are varied due to species, topography
and climate and categories of active principles9,10 chemical compositions and its proportion in the crude extract modifies their antimicrobial
activity. Some main categories of phytochemicals extracted from medicinal plants are examined to evaluate their antibacterial cellular activity. In
this research work we investigated the scientific basis of the traditional uses of the plant for infection.
International Journal of Phytopharmacy Research Article
Vol. 4 (4), pp.99-104, Jul-Aug 2014
ISSN: 2277-2928 (Online)
Journal DOI:10.7439/ijpp
©Scholar Science Journals
www.ssjournals.com
Abstract
Purposes: The present study was designed to investigate the antibacterial activities of the Ethanol and methanol extracts of the leaves of
the plant Borreria articularies (Linn.) effects on microbial growth inhibition in vitro, microbial cells in vivo and molecular enzyme
(BADH) targets in vitro.
Methods: The preliminary phytochemicals of the extracts was determined by the standard methods and aliquoted with Thin Layer
Chromatography (TLC) and stored at 2-4oC. fluorescein diacetate (FDA) and ethidium bromide (EB) live-dead cell viability test for
distinguishing the membrane active phytochemicals of the plant extract. Betaine aldehyde dehydrogenase (BADH) activity was assessed
by spectrophotometer. Alkaloids, glycosides, steroids, gums, saponin and reducing sugar were found in extracts.
Results: The results of the disc diffusion indicated that the crude extracts were able to inhibit the growth of bacteria within a
concentration range of 0.5 to 2.0 mg/mL. At a similar concentration range (0.5 to 2.0 mg/mL) the extract inhibited the growth of 90.12%
of the tested microorganisms. Bacterial cell viability was found minor in the phytochemicals of crude extract. Also, constituents of crude
extract inhibited the BADH activity to protect the adaptation in stress environment of the bacteria.
Conclusion: Results of the present study showed the possible use of the studied plants extracts in the control of bacterial infections.
Keywords: Antibacterial activities, Borreria articularies Linn., Methanol, Ethanol, Extract, Phytochemicals, BADH, Osmoprotectant,
Live-dead cell
Md Shamsuddin Sultan Khan et al /2014
Volume 4 Issue 4 [2014] 100
2. Material and Methods
2.1 Materials
2.1.1 Plant: Borreria articularis (Linn.), Bengali name Bonkodu, was collected from Gonoshasthaya kendra Medicinal Plant Garden,
Bangladesh. The authenticity of the plant specimen has been confirmed by Bangladesh National Herbarium, Mirpur, Dhaka, Accession number:
32577.
2.1.2 Chemicals and biochemicals: Methanol and Ethanol were bought from Merck, Germany. Mayer’s reagent, Dragendroff’s reagent,
Fehling’s solution A, Fehling’s solution B, Be nedicts reagent, and Molisch reagent were prepeared in the laboratory. fluoresc ein diacetate (FDA)
and ethidium bromide (EB) were purchased from Sigma, USA. Hanks Balanced Salt Solution (HBSS) was bought from Gibco, USA. Dubos
Liquid Medium (DLM) and Dubos Agar Medium (DAM) were provided by Difco, USA. Betaine aldehyde chloride, glycine betaine (free base),
choline chloride, NAD(P)1, NADPH, dithiothreitol (DTT), Tris were obtained from Sigma, USA. EDTA and glycerol were from Merck KGaA,
Germany. Ampholines and nitrocellulose membranes were from Bio-Rad (Hercules, Calif.), and Immobilon-PSQ membrane was from Millipore
(Bedford, Mass.). 3-Dimethylsulfoniopropionaldehyde was collected from USA. g-Aminobutyraldehyde (diethylacetal form) was from Aldrich.
g-Aminobutyraldehyde chloride was freshly prepared from the diethylacetal form by the procedure described by Flores and Filner11. Materials for
column chromatography were purchased from Shimadzu, Japan. All other chemicals were from standard suppliers in analytical grade.
2.1.3 Organism: Staphylococcus aureus and Candida albicans were used as Gram-positive and Escherichia coli was used as Gram-negative
microorganism. Nutrient Agar (NA) Medium (pH, 7.4 ± 0.2) and Sabeuraud Dextrose Agar Medium (SAD, pH at 25oC, 5.6 ± 0.2) were prepared
in the laboratory for culture preparation. Mycobacterium smegmatis ATCC 607 and P. aeruginosa PAO1 were collected from ICDDR’B.
2.2 Method
2.2.1 Plant Extraction: The plant samples were primarily dried in the shadow and finally dried in an oven at 40oC temperature. The dried
samples were ground to coarse powder with a mechanical grinder and then stored in a plastic container. 500g of the grounded plant was extracted
by maceration with methanol and ethanol for 3 days at room temperature. The extracts were then filtered and concentrated by Rota-vapor at 40oC
to give the crude extracts. All extracts were kept at 4°C until further investigations.
2.2.2 Preliminary phytochemical screening: The secondary metabolites such as alkaloids, Glycosides, Steroids, gums, reducing sugars,
Tannins, Flavonoids, Saponins were screened using 10% (w/v) according to the common phytochemical methods described by Harbone12.
2.2.3 Separation of phytochemical: The biologically active phytoconstituents were separated from the crude extracts by the column
Chromatography and Thin layer chromatography. The column was packed with silica gel (Merk- 60, mesh 120). Slurry of silica gel in n-hexane
was added into a glass column with the length and diameter of 55 cm and 1.1 cm respectively. When sufficient height of the ad sorbent bed was
obtained, a few hundred milliliter of n-hexane was run through the column for proper packing of the column. The sample was prepared by
adsorbing 750 mg of n-hexane soluble fraction onto silica gel (Merk 60, mesh 120), allowed to dry and subsequently applied on top of the
adsorbent layer. The column was eluted with n-hexane, ethyl acetate and methanol at different ratio. Solvent systems were used as mobile phases.
Total 20 fractions each of 10 ml were collected. The column was eluted gradually with order of polarity of solvents from non polar to high polar.
The individual eluents were examined by TLC.
Standard size TLC plates were cut into small pieces as Length – 7 cm, Width – 2.5 cm, Labeling space – 1 cm from both upper &
lower side. The plant extract was used for chromatographic separation by TLC method. TLC plates in Iodine chamber showed a significant
results in 9:1, 8:2, 7:3 and 6:4 mobile phase solvent ratio (n-hexane : ethyl acetate) and found spots in TLC with Rf values 0.63, 0.68, 0.66, 0.65
and indicated different verities of compounds (Figure 1).
Figure 1: TLC plate after placing in Iodine Chamber, Extract – methanolic, Solvent system –n-hexane : ethylacetate (8 : 2),
Rf Value - 0.63, 0.68, 0.66, 0.65
2.2.4 Disc Diffusion antimicrobial Screening: Nutrient Agar (NA) Media (plant and tube) was prepared in Lab. The pH of the media was
adjusted using NaOH/ HCl solution. The dissolved material was transferred to larger conical flask plugged by cotton and aluminum foil. The
flask was autoclaved for sterilization at 121oC for 15min. After autoclaving the media was cooled about 45oC and it was poured on Petridis
(20ml per plate) and in test tube for marking solid plate and slant medium. The Sabeuraud Dextrose Agar Medium (SAD) medium was prepared
with same process. Test cultures were prepared from pure culture, by transferring the organism to sterile NA and SDA media with the help of a
sterile inoculation loop inside laminar airflow chamber in aseptic condition. The inoculated mediums were then i ncubated at 37oC for 24hrs for
growth of the organisms. All of the petridishes were sterilized at 180oC for 24 hrs. NA and SDA medium were taken into the petridishes. The
plate was placed in the refrigerator for solidification. NA media were taken by sterile loop. Organisms were spread over the plate. Sterilized
Pasteur pipette were taken and three holes were made. The plates were marked properly by name of the organism in one side and marking the
position of the sample and standard disk on the other side. About 100µl of prepared sample solution was poured into each of the plate thru hole.
The plates were then carefully stored in incubator for the development of the zone. Test samples were prepared by dissolving 50 mg of the
ethanol and methanol extracts in 2 ml of respective solvents to make 1 mg/disc. All the plates (sample, standard and control) were incubated at a
temperature of 25±2oC for 5 days. The radial growth of fungal colony was measured with a transparent scale in mm and the percentage of
inhibition of mycelial growth was calculated using the following equation:
Here, I = Percentage of inhibition, C = Diameter of fungal colony in control, T = Diameter of fungal colony in treatment
100
C
TC
I
Md Shamsuddin Sultan Khan et al /2014
Volume 4 Issue 4 [2014] 101
2.2.5 Cell Viability antimicrobial Screening: Mycobacterium smegmatis ATCC 607 was inoculated on Lowenstein-Jensen Medium and
incubated at 37°C for 72 hr. The stock slants were stored at 4°C for later use. Dubos Liquid Medium (DLM) with 0.4% glycerol was used for
broth cultures after inoculation with the appropriate organism from a stock culture and incubation at 37°C on a rotary shaker at 180rpm. Dubos
Agar Medium (DAM) was used for plating using same process of DLM except for the addition of 1.5% agar. Single-cell suspensions were
prepared to ensure reliable plate and total counts to prevent the clump of organism in liquid medium for their lipophilic nature to the cell walls.
DLM culture (2.0 ml aliquot) was centrifuged at room temperature for 15 min. Hanks Balanced Salt Solution (HBSS) with 0.05%Tw een-80 was
used for decantation of supernatant fluid and resuspension of cell pellet. Single-cell suspension containing 106 to 107 cells per milliliter was
filtered through a 5 µm membrane filter using 10 ml syringe. All viability tests were performed using single-cell suspensions.
The fluorescein diacetate (FDA) and ethidium bromide (EB) fluorescent viability test was used to find living and dead cells. A stock
solution of FDA (Sigma, U.S.A.) was prepared by dissolving 100 mg of FDA in 20 ml of acetone to give a final concentration of 5 mg per ml. A
stock solution of EB (Sigma, U.S.A.) was prepared by dissolving 20 mg of EB in 10 ml of HBSS, pH 7.4, containing 0.05% Tween-80 to give a
final concentration of 2 mg per ml. One milliliter volumes of FDA and EB were distributed to 13 x 100 mm screw cap test tubes and stored at -
20°C in a dark place. These solutions were stable for two years and stored for working solution. A fresh working solution of FDA was prepared
daily by diluting the FDA stock solution 1: 10 in acetone to get 500 µg per ml. A 0.02 ml volume of the diluted FDA solution was added to 5.0
ml of HBSS with 0.05% Tween-80 to make 2 µg per ml. A 0.01 ml volume of EB stock solution was then added to the same 5.0 ml of HBSS,
containing the FDA, giving a final EB concentration of 4 µg per ml.
A 0.5ml volume of the FDA-EB working solution and phytochemicals of crude extract were added to a 1.0 ml aliquot of single-cell
suspension and incubated for 1 min at room temperature. After preparing the wet mount and sealing to prevent evaporation, the suspension was
observed in 950x Leitz Dialux microscope. Cells were counted in replicate and differentiated on the basis of their color. Gre en cells were
considered to be live and red-stained cells were considered to be dead. Percent viability was calculated by dividing the number of green cells by
the total number of cells observed and multiplying by 100.
Viable counts were performed in Neubauer-Hausser chamber. The inoculum was evenly distributed on the agar surface with a flamed
L-shaped glass rod. Inoculated plates were incubated at 37°C for three days before plate counts were made. 1 ml volume of the original single -
cell suspension was autoclaved at 121°C for 10 min to kill the bacteria. 0.5 ml volume of the FDA-EB working solution and phytochemicals of
crude extract were added to the killed single-cell suspension and both chambers of the hemacytometer filled with stained-cell suspension.
Bacterial counts were performed under incident UV illumination at 400x magnification after 10-min incubation at room temperature.
2.2.6 Osmoprotection by exogenous glycine betaine: Glycine betaine can protect plants from salt and freezing stress13,14. To assess the
feasibility of glycine betaine as an osmoprotectant in Borreria articularis Linn., exogenously supplied glycine betaine was tested as prevention of
hydroponically growing Borreria articularis Linn. in salt stress. To measure the effect of salt on plant growth we compared the 3-week-old
grown plant in the presence or absence of 0.2 M NaCI.
Plants grown in the absence of NaCI reached an average height of 5.52 cm, plants grown in the presence of NaCI only reached a
height of 2.91 cm. This inhibitory effect of NaCI could be improved by addition of glycine betaine. Supply of glycine betaine, with a 20 mM final
concentration, to the growth medium together with NaCI resulted in plants with an average height of 5.21. These preliminary results indicated
that exogenous glycine betaine can protect uncontaminated Borreria articularis Linn. exposed to inhibitory concentrations of NaCI and
recommend that introduction of the betaine biosynthetic pathway to Borreria articularis Linn. could result in enhanced stress tolerance.
2.2.7 BADH Enzyme target antimicrobial Screening: P. aeruginosa PAO1 was provided by ICDDR’B was used in this experiment. Cells were
grown aerobically at 37°C in liquid media. The basal medium was used to grow the cells as the M63 minimal medium. 20 mM choline was used
as carbon and nitrogen source for maximum induction of BADH. The medium (1.8 litre) was inoculated with a seed culture (36 ml) in the log
phase and was grown on a gyratory shaker (150 rpm) at 37°C until the stationary phase was reached. The cells from 1.8 liters of culture medium
were harvested by centrifugation at 3,000X 3 g for 10 min and then resuspended in 90 ml of 50 mM potassium phosphate buffer (pH 6.5)
containing 0.1 mM EDTA and 20 mM b-mercaptoethanol (buffer A).
The purification process was conducted at 4°C. The disintegrated resuspended cells by sonic oscillation (90 s at 60 W) in a Branson
Sonifier Cell Disruptor were centrifuged at 14500x 3 g for 30 min. 20% (w/v) sucrose (buffer B) was placed into the supernatant (cell extract)
and then applied to a Q-Sepharose Fast Flow column (1.8 X 6.5 cm) equilibrated with buffer B. The column was washed with the buffer B and
the enzyme was eluted with 110 ml of a linear salt gradient of 0 to 250 mM KCl in buffer B at a flow rate of 1 ml/min. Fractions with enzyme
activity were collected and the pH was adjusted to 6.0 with diluted HCl. after pH adjustment, the enzyme was applied to a 29, 59-ADP–
Sepharose column (1.1 X 3.5 cm) equilibrated with buffer C (10 mM potassium phosphate with pH 6.0 containing 5 mM DTT, 20% (w/v)
Alkaloids, Glycoside, Reducing sugar, Gums and Saponin of plant extract, 20% (w/v) sucrose, 0.1 mM EDTA, and 25 mM KCl). After washing
with buffer C (50 ml), the enzyme was eluted at a flow rate of 1 ml/min with 85 ml of a linear pH gradient, from 6.0 to 8.0 of buffer C. The
enzyme eluted at pH 6. Fractions with enzyme activity were collected, aliquoted, and stored at 220°C.
During the purification procedure, the BADH activity was assayed spectrophotometrically by monitoring the absorbance at 340 nm
(NADPH formation) in a mixture (0.5 ml) consisting of 1.0 mM betaine aldehyde and 0.3 mM NADP1 in a 100 mM potassium phosphate buffer,
pH 8.0 (standard assay). Shimadzu spectrophotometer (Japan) equipped was used at 30°C in 1.0-cm-path-length cuvettes for the assays. All
assays were initiated by addition of the enzyme. The initial rate of betaine aldehyde oxidation was proportional to the enzyme concentration over
a range of 0.06 to 2.3 mg of protein per ml of reaction mixture. Each determination was performed twice. One unit of activity is defined as the
amount of enzyme that catalyzes the formation of 1 mmol of NADPH per min in our standard assay.
3. Results and Discussion
3.1 Phytochemical group Analysis: Phytochemical study showed the presence of alkaloids, glycosides, steroids, gums, saponin and reducing
sugar were presented in the two extracts and found absence of other components (Table 1).
Table 1: Phytochemical composition of the Borreria articularies (Linn.)
Secondary metabolites
Extract
Methanol
Ethanol
Alkaloid
+
+
Glycoside
+
+
Steroids
+
±
Gums
±
±
Reducing sugar
+
+
Tannins
-
-
Flavonoids
-
-
Saponin
-
±
(+) = Presence, (-) = Absence, (±) = medium
Md Shamsuddin Sultan Khan et al /2014
Volume 4 Issue 4 [2014] 102
3.2 Disc Diffusion assay: Extracts were tested for antibacterial activities on gram positive (Staphylococcus aureus) and gram-negative
(Escherichia coli) bacteria using disc diffusion method. 1 mg of the extracts was applied in each disc. A standard antibiotic disc of Amoxicillin (30
µg/disc) was used for comparison of antibacterial activity. The antibacterial activities of the extracts were shown in table 2. Methanol extract of
the plant showed low-level activity on the test organisms.
Table 2: In-Vitro antibacterial activities of Borreria articularis at 2 days
Organism
Zones of inhibition (mm)
Percent inhibition
Methanol extract (1mg/disc)
Ethyl Alcohol extract (1mg/disc)
Amoxicillin (30µg/disc)
Staphylococcus aureus
1.3
Nil
2.8
53.57%
Escherichia coli
1.5
Nil
2.8
46.43%
3.3 In Vitro Antifungal screening in yeast: The extracts were tested for the antifungal properties on human pathogenic yeast (Candida
albicans). Clotrimazole (30µg/disc) was used as Standard. Results obtained from the screening were shown in the following table 3. The extracts
did not show antifungal activities.
Table 3: In Vitro Antifungal activities of Borreria articularis Linn. in the yeast
Name of the yeast
Methanol (1mg/disc)
Ethanol (1mg/disc)
Clotrimazole (30µg/disc)
Candida albicans
No activity
No activity
Strong activity
3.4 BADH activity assay: Growth of P. aeruginosa on choline showed an induction of significant intracellular levels of BADH activity (around
1 U per mg of protein in the cell extract), which was completely absent in cells grown in the standard glucose medium and in the plant
phytochemicals. This may be due to induction of the enzyme by choline in Pseudomonas15 and other bacteria16,17. Inhibition of BADH in the
phyto constituents may be for the blocking choline degradation, abolishing synthesis of the osmoprotectant glycine betaine, or accumulating the
BADH substrate, betaine aldehyde18,19,20. The presence of glycine betaine or choline greatly increases the ability of P. aeruginosa to thrive under
osmotic stress20, 21, 22. Also, the virulence of P. aeruginosa is associated with its ability to adapt to osmotic stress23 and with the expression of
phospholipase C24, produced from bacteria to produce choline from phosphatidylcholine. P. aeruginosa produces glycine betaine through BADH-
catalyzed reaction in the presence of NADH and NADPH when the bacterium is growing in choline or choline precursors25. In this regard BADH
might be a key enzyme to grow the pathogen. BADH activity is critical for the growth of P. aeruginosa in infection. BADH metabolic role is the
synthesis of the osmoprotectant, such as E. coli26, Bacillus subtilis27, amaranth leaves28, and porcine kidney29.
The activity BADH was determined in the presence of NaCl, NH4Cl, glucose, choline, alkaloids, glycosides, reducing sugar, gum and
saponin (Table 4). The activity was reduced in the phytochemicals. The significant amount of BADH activity in P. aeruginosa cells grown on
glucose plus choline, the finding that BADH activity is tolerant to salt that is also varied and found in earlier study25. It suggests may play an
important physiological role in the infection sites of human and plant cells thru glycine betaine synthesis in vivo.
Table 4: Effect of methanol-ethanol extract of plant, glucose, choline and salt on the expression of BADH activity in P. aeruginosa
Culture conditions
BADH activity (mU/mg protein)
11.1 mM glucose/15 mM NH4Cl
8.0
11.1 mM glucose/15 mM NH4Cl/0.4 M NaCl
11.0
Alkaloids/15 mM NH4Cl
15.0
Alkaloids /15 mM NH4Cl/0.4 M NaCl
22.0
Alkaloids /11.1 mM glucose
5.0
Alkaloids/20 mM choline
27.0
Alkaloids/20 mM choline/0.4 M NaCl
30.0
Glycoside/15 mM NH4Cl
12.0
Glycoside/15 mM NH4Cl/0.4 M NaCl
18.0
Glycoside /11.1 mM glucose
12.0
Glycoside/20 mM choline
10.0
Glycoside/20 mM choline/0.4 M NaCl
15.0
Reducing sugar/15 mM NH4Cl
16.0
Reducing sugar/15 mM NH4Cl/0.4 M NaCl
12.0
Reducing sugar /11.1 mM glucose
12.0
Reducing sugar/20 mM choline
13.0
Reducing sugar/20 mM choline/0.4 M NaCl
13.0
Gums/15 mM NH4Cl
8.0
Gums/15 mM NH4Cl/0.4 M NaCl
8.5
Gums /11.1 mM glucose
10.0
Gums/20 mM choline
11.0
Gums/20 mM choline/0.4 M NaCl
9.0
Saponin/15 mM NH4Cl
13.0
Saponin/15 mM NH4Cl/0.4 M NaCl
13.0
Saponin /11.1 mM glucose
10.0
Saponin/20 mM choline
9.0
Saponin/20 mM choline/0.4 M NaCl
14.0
20 mM choline
600
20 mM choline/0.4 M NaCl
302
20 mM choline/11.1 mM glucose
150
20 mM choline/11.1 mM glucose/0.4 M NaCl
170
3.5 Viability assay
A suspension of M. smegmatis was subdivided into 1.0 ml aliquots and exposed to FDA, EB, alkaloids, glycosides, reducing sugar,
gum and saponin singly and in combination to determine effect in viability of the organisms during the staining procedure. Af ter various periods
of exposure, a 0.1 ml volume of cell suspension was removed from each tube, diluted in HBSS and plated on DAM, and plate counts were
performed. FDA, EB had no effect upon the viability of the cells during an incubation period of up to 6 hr at room temperature (Table 5).
Alkaloids, glycosides, reducing sugar, gum and saponin showed inhibition of most bacteria in the media (Table 5).
Md Shamsuddin Sultan Khan et al /2014
Volume 4 Issue 4 [2014] 103
Table 5: Effect of phytochemicals of crude extract, fluorescein diacetate (FDA) and ethidium bromide (EB) on the viability of
Mycobacterium smegmatis as determined by plate counts
Time (hr)
No of bacteria (x10) per ml of HBSS containing
Normal
FDA
EB
FDA-EB
Alkaloids
Glycosides
Reducing sugar
Gums
Saponin
0
2
2
3
2
1
0
0
1
0
3
3
3
3
2
0
0
0
1
1
6
2
3
3
2
0
0
0
1
1
Mycobacterial viability was compared to standard bacteriological plate counts using 18 hr to 27 hr DLM cultures at 37°C. A high
degree of correlation (p < 0.005) was found between the two methods with an average difference of 20%. Higher percent viability may be due to
a small amount of cell reclumping after single-cell suspensions preparation (Table 6). Two or more clumped cells may be grown in plating into a
single colony and may give lower plate counts. The adherence of bacteria to the L-shaped spreading rod is the reason for the lower counts.
Table 6: comparison of Mycobacterium smegmatis cell viability in plating, FDA-EB and phytochemicals of crude extract
Organism
Culture age
(hour)
Percent viability determined
Plating
FDA-EB
Alkaloids
Glycosides
Reducing sugar
Gums
Saponin
Mycobacterium
smegmatis
22
95
95
85
89
90
90
85
25
80
92
87
85
90
90
85
27
80
92
85
88
89
87
89
After experiments, it was found that some cells were dual stained. These cells were characterized as thickness variable with green. It
indicated acetyl esterase activity and diffusion to red-orange nuclear area as penetration of EB to confirm the cells with damaged cell membrane.
4. Conclusion
Taken together, Borreria articularies Linn. showed its antibacterial activities in the bacterial cell and enzyme. The TLC separated
Phytochemicals of alkaloids, gl ycosides, reducing sugar, gum and saponin were isolated (Figure 2) to justify the antibacterial effects of these
compounds. These compounds showed antibacterial activity in many published study, too so that according to results of this study, it can be
targeted to discover a new antibiotic. On the other hand crude extracts did not show any anti-yeast properties due to absence of BADH enzyme.
Further study is required to find the reason of this effect of Borreria articularies Linn.
Figure 2: Aliquoted phytochemicals of alkaloids, glycosides, gum and saponin in column chromatography
Acknowledgement
We are thankful to Dr. Kohinur Bagum and Ms Sanjida Haque, Department of Pharmacy, Bangladesh University. We are especially
grateful to ICDDR’B and Department of Microbiology both at Dhaka University and University of Asia Pacific.
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