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International Conference on Multidisciplinary Research & Practice
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Phytochemical Analysis and In-Vitro Antibacterial
Activity of Kalanchoe Pinnata against Human
Pathogens Isolated from UTI
Dholaria M. D.#, Desai P. V.*
#
Department of Medical Technology, Naran Lala College of Professional and Applied Sciences, India
*Department of Biosciences, Veer Narmad Siuth Gujarat University, India
Fig. 1 Kalanchoe pinnata
Abstract- Various solvent extract of leaves of Kalanchoe
pinnata were analyzed for phytochemicals and in vitro
antibacterial activity against selected species isolated from
urine samples. The phytochemical analysis revealed that the
plant extract contains the important phyto-constituents like
tannin, flavonoid, carbohydrate, protein, terpanoid and
cardiac glycoside indicating its potential activity for
treatment of Urinary Tract Infection (UTIs). To provide a
scientific basis to treat UTIs, the extracts of leaves of
Kalanchoe pinnata were subjected to antibacterial activity
against selected bacterial species by well diffusion assay.
MIC values were also determined to find minimum effective
concentration. All the extracts show good antibacterial
activity against highly resistant UTI isolates.
Key words: Kalanchoe pinnata, anti-bacterial activity,
phytochemical activity, minimum inhibitory concentration,
Urinary Tract Infection (UTI).
I.
II. MATERIAL AND METHODS
A. Plant Material
INTRODUCTION
Department of Biosciences, Veer Narmad South Gujarat
University, Surat, Gujarat. Leaves of the plant was
washed with running water, dried in shade at room
temperature, ground to powder and stored in air tight bag
in dry at low temperature.
U
rinary Tract Infections (UTIs) are the most
widespread microbial disease in India as per WHO.
The common pathogenic bacteria causes UTI are
Escherichia coli, Klebsiella pneumoniae, Haemophilus
influenza, Streptococcus pneumonia and Proteus vulgaris
are the major causative agents of nosocomial infections
[1]. Many of the antibiotics and synthetic drugs become
sensitive and show many undesirable side effects. So
natural altrnative must be selected as they are more safe in
biological system [2].
B. Extraction
The powdered sample was used for extraction using
absolute methanol, ethanol and 50% methanol as a solvent
by Soxhlet apparatus. After removing pigments, the
solvent was evaporated and the residue was dissolved in
DMSO. The % yield of different solvent extract was
calculated using following formula,
Plants and herbal medicines have important position in
modern medicine, due to their chemical and medicinal
contents found in natural form. They contain various
secondary metabolites which work together and show
wide range of antibacterial activities. Microorganisms
may get mutated and become resistant to many antibiotics
and so it generates a global health problem. These
inspired scientists to search out new natural alternative to
treat diseases [3].
% yield = Weight of final extract × 100 / weight of
powdered sample [5].
C. Isolation and Selection of Bacterial species
Urine samples of the patient suffering from UTI were
collected from Bhanumati laboratory and Parsi hospital at
Navsari, Gujarat. The isolated bacterial species were
identified
by
biochemical
tests
[6].
From all the identified causative agents, highly resistant
species were selected by performing antibiotic
susceptibility test against 11 different antibiotics that are
Ampicillin (AS), Co-Trimoxazole (BA), Cefataxime
(CF),
Piperacillin(PC),
Chloramphenicol
(CH),
Ciprofloxacin (RC), Tetracyclin (TE), Ceftizoxime (CI),
Ofloxacin (ZN), Gentamycin (GM), Amikacin (AK).
Kalanchoe pinnata is also known as Bryophyllum
pinnatum, Patharkuchi, Air plant, Life plant, Mirecal
Leaf, Goethe plant< tht Katakataka and also called
“Wonder of the World”.The genus was first described by
the botanist Michel Adanson in 1763.The main
phytoconstitutes found in Kalanchoe are alkaloids,
triterpeness, glycosides flavonoids, steroids, bryophyllin,
palmitic acid, oxalic acid etc. [4].
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International Conference on Multidisciplinary Research & Practice
D. Phytochemical Evaluation
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Proteus vulgaris and gram positive Staphylococcus
aureus were isolated. Out of 50 isolates,36 Escherichia
coli, 10 Klebsiella pneumoniae, 2 Pseudomonas
aeruginosa, 1 Proteus vulgaris and 1 Staphylococcus
aureus were obtained. Frequency of microorganisms to
cause UTI is given in figure 2.
Preliminary phytovhemical analysis was carried out using
standard protocol for determinationis phytoconstituents :
alkloids, tannins, saponin, aonthocyanide, phenolic
flavonoids, flavonoids, carbohydrate, protein, terpenoids,
cardiac glycoside and phlobatannins as directed by
references [7], [8], [9] and [10].
Fig. 2 Frequency of microorganisms in UTI
E. Anti-bacterial Assay
PV PA SA
4% 2% 2%
Determination of anti-bacterial activity was performed by
agar well diffusion assay against all selected bacterial
species [11]. Diameter of zone of inhibition was measured
in mm and Minimum Inhibitory Concentration (MIC) was
also determined using 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64
dilutions.
KP
20%
III. RESULTS AND DISCUSSION
EC
72%
A. Percentage Yield
The highest yield was obtained with 50% methanol
extract (33.7 mg/ml) followed by methanol extract (28
mg/ml) while lowest yield was obtained with ethanol
solvent (24.3 mg/ml).
B. Isolation and Selection of Bacterial species
All the isolates were tested for antibiotic susceptibility
and highly resistant specieses were selected. The results
of antibiotic susceptibility test of selected species were
given in Table 1.
50 urine samples were collected from different
laboratories of Navsari, Gujarat. Gram negative bacteria
isolated from urine sample were Escherichia coli
Klebsiella pneumoniae, Pseudomonas aeruginosa,
TABLE I
Antibiotic Susceptibility Test of Urine Isolates
No.
Name of
Antibiotic
Name of Organism
Escherichia
Klebsiella
Proteus
Pseudomonas
Staphylococcus
coli
pneumonia
vulgaris
aeruginosa
aureus
1.
Ampicillin
S
R
S
R
S
2.
Co-Trimoxazole
R
R
R
R
R
3.
Cefataxime
R
R
R
R
R
4.
Piperacillin
R
R
R
R
R
5.
Chloramphenicol
R
R
S
S
S
6.
Ciprofloxacin
R
R
R
R
R
7.
Tetracyclin
R
R
S
R
R
8.
Ceftizoxime
R
R
R
R
R
9.
Ofloxacine
R
R
R
R
R
10.
Gentamycin
R
R
S
S
S
11.
Amikacin
S
R
S
S
S
S= Sensitive and R=Resistant
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C. Anti-bacterial Assay
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activity might be due to t be due to presence of bioactive
compounds in plant extracts.
MIC of the plant extracts was determined by well
diffusion method by using different dilutions and results
of MIC was given in Table 2. Lowest MIC (0.303
mg/100µl) was shown by ethanol extract of Kalanchoe
pinnata against all the bacterial species except
Staphylococcus aureus. Lowest MIC value of methanol
extract was shown against Escherichia coli, Proteus
vulgaris and Pseudomonas aeruginosa and 50% methanol
extract against all except Staphylococcus aureus.
All the extracts were tested for the antibacterial activity
against all the selected highly resistant bacterial species.
Zone of inhibition was measured in mm which is given in
Figure 3. Methanol extract showed maximum activity
against all selected isolates except Pseudomonas
aeruginosa while maximum activity against Pseudomonas
aeruginosa was shown by ethanol extract. Formation of
clear zone of inhibition surrounding the well clearly
showed that all the extracts of Kalanchoe pinnata were
effective on all the selected isolates. This antibacterial
Fig. 3 Antibacterial activity of Kalanchoe pinnata extract
Diameter of Zone of
Inhibition
25
20
15
METHANOL
10
ETHANOL
5
50% ETHANOL
0
EC
KP
PV
PA
SA
Organism
EC= Escherichia coli, KP= Klebsiella pneumoniae, PV= Proteus vulgaris, PA= Pseudomonas aeruginosa, SA= Staphylicoccus aureus
TABLE II
MIC in mg/100µl of Kalanchoe pinnata extract
Plant extract
MIC in mg/100µl
Escherichia coli
Klebsiella
Proteus
Pseudomonas
Staphylococcus
pneumoniae
vulgaris
aeruginosa
aureus
Methanol
0.35
0.7
0.35
0.35
0.7
Ethanol
0.303
0.607
0.303
0.303
1.215
50% Methanol
1.685
1.685
1.685
1.685
3.73
D. Phytochemical Evaluation
Pseudomonas aeruginosa. Kalanchoe pinnata showed
presence of various ptytoconstituents which might be
effective to inhibit microbial growth.These results suggest
that solvent extracts of this plant may be a good source of
natural treatment of UTI.
The phytochemical analysis reveals presence of tannin,
flavonoid, carbohydrate, protein, terpanoid and cardiac
glycoside.Their presence in extract indicate its
effectiveness in inhibition of microbial growth.
ACKNOWLEDGEMENT
IV. CONCLUSION
The author wish to thank the Management, Director and
Department of Medical Technology of Naran Lala
College of Professional and Applied sciences.
The present study showed that all the plant extracts of
Kalanchoe pinnata had successfully inhibited all the
selected isolates of UTIs. .Ethanol extract showed least
MIC value against Escherichia coli, Proteus vulgaris and
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[6]. Holt, J.G., Kreig, N,R., Sneath, P.H.A., Staley, J.T. amd Williams,
S.T., (1994). In: Bergy’s Manual of DeterminativeBacteriology,
ninth ed. Williams and WilkinsPub., MD, USA.
[7]. Trease,
G.E.,
Evans
W.C.,(1978).
Pharmacognosy.11 th
ed.BrailliarTiridel andMacmillian Publishers, London.
[8]. Harborne, J.B. and Harborne, A.J.,(1998). Phytochemical methods:
Aguide to Modern Techniques of
Plant analysis. Kluwer
Academic Publishers, London, UK.
[9]. Kaur, G.J. and Arora, D.S., (2009). Antibacterial and
Phytochemical Screeningof Anethum graveolens, Foeniculum
vulgare and Trechyspermum ammi: BCM Complement Altern Med
9: 30.
[10]. Kumar, K.A., Narayani,M., Subanthini, A. andJaykumar, M.,
920110. Antibacterial Activity and Phytochemical Analysis of
Citrus fruit peels Utilization of Fruit waste: Int J Environ Sci Tecj,
3: 5415-5421.
[11]. Ettebong, E. andNwafor P., (2009). In Vitro Antibacterial
Activities of extracts of Carpolobia lutea root: Pak J Pharm Sci,
22(3): 335-338.
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