ISSN 0974-3618 www.rjptonline.org
RESEARCH ARTICLE
Screening of
Active Pharmaceutical Ingredients using Methanolic Leaf Extract of C. papaya L. against Hospital Acquired MDR
Uropathogens
Akhilesh Upgade*
and P Prabhakaran
1Centre for Research and Development, PRIST
University, Thanjavur, Tamilnadu
2Department of Microbiology, Marudupandiyar
College, Vallam Thanjavur, Tamilnadu
*Corresponding Author E-mail: akhiupgade@gmail.com
ABSTRACT:
Emergence of specific infections associated
with hospitals is special concern as per the recent reviews. As far as, there
is uncontrolled population growth responsible for such a conditions. Managing
diseases in tertiary care hospital including Government hospital, Private
hospitals, mother care homes etc is tedious task. Nosocomial infections caused by the gram
negative organisms to the admitted patients of all group occurs. Urine tract
infection is one of the most threaten condition those admitted not only that
they are multidrug resistant bugs which can difficult to manage. Hence, this
study involves medicinally important plant to evaluate the antibacterial effect
of plant on selected UTI causing bacteria like Pseudomonas aeruginosa, Klebsiella
pneumoniae and E.coli. Traditional methods have been employed to
check activity of methanolic extract C.
papaya. The multi benefit plant in
ancient literature describes about seeds and fruit effectively hence leaves has
taken into accounts to accelerate scope of research. In results, the extract shows different
antibacterial activity which was confirmed by measuring zone of inhibition as
compare to standard antibiotic. E.coli
and K. pneumonia was found most sensitive against plant extract and
found more effective than antibiotic like Cef: Cefpodoxime, Chlor:
Chloramphenicol, Ceftri : Ceftriaxone i.e. 27mm and 30 mm
KEYWORDS: C. papaya, Uropathogens, Nosocomial
infections, MDR, UPEc.
INTRODUCTION:
One
of the most deadly infection occurs in India in both the sexes is Urine tract
infection. Uropathogens are the common bacteria available in cases
infections. E.coli is the most prevalent in India about 68% followed by the Klebsiella pneumoniae and Pseudomonas aeruginosa 50 % and 40 %
respectively. Root cause is misdiagnosis.
Symptoms are look like a regular fever such as chilling, fever, slight
pain in stomach; itching etc. due to
this patient suffers from misdiagnosis and undergoes various clinical trials.
And this leads to antibiotic resistance. (1-3) Most of the organisms are now
resistant against second and third generation antibiotics such as cephalosporin
and amiloglycosides etc. Extended
spectrum β-lactamase (ESBL) producing organisms pose a major problem for
clinical therapeutics.(4) So, there is a need to develop a new herbal
formulation which is as competitive to such antibiotics. Present study designed
to evaluate and formulate new herbal formulation using plant C. papaya leaves. Papaya is a large
perennial herb with a rapid growth rate.
The
plants are usually short-lived with various patterns of fruits for long
periods. It can be defers from geographical area. Elizabeth in 1994 reported
that C. papaya leaves are better antiseptic agents.(5). In ancient
literature, Carica papaya was mentioned as basket of
benefit because of its pharmacological activities. Yet, community using the plant for various
ailments like digestion, constipation, gums, fever, pain, infection as
antiseptics, warts, sinusitis, eczema, cutaneous tubercles and hardness of the
skin etc. still there are various
studies reported the activities but very few mentioned the active
pharmaceutical ingredients in leaves (API). Hence this study reports and
hypothesized ingredients which may responsible for the antibacterial action of
API from methanolic extract of C. papaya.
MATERIALS AND METHODS:
Plant
Materials:
The
plant materials which include fresh green pawpaw leaves, only were directly
drawn from the plant from Thanjavur region at down south part of India.
Climatic conditions were noted and taken into accounts.
Extraction of Plant Leaves:
Two
hundred grams of dried leaf powder was used for traditional Soxhlets extraction
method. 1.5 liters of methanol used as a solvent to get most of the dissolved
compounds from plant materials. About 30 cycles were performed for a week and
sample was subjected to evaporate at 4OC. (5)
Identification of
API’s:
Methanolic leaf extract was then collected and subjected to Gas
Chromatography and Mass spectroscopy analysis (GCMS), to identify the compounds
present and their mass. Perkin-Elmer GC Clarus 500 system and Gas Chromatograph
interfaced to a Mass spectrometer (GC-MS) equipped with a Elite-I, fused silica
capillary column (30mm X 0.25mm 1D X 1μ Mdf, composed of 100% Dimethyl
poly siloxane) was employed for analysis.(6) Data obtained from NCI Library and
analysis noted.
Qualitative
Analysis of Plant Leaf:
Various qualitative analysis test were
performed for screening of Alkaloids flavenoid saponins, tannins, phenolic,
steroid compounds present in leaves of selected plant material using traditional
methods of (7,8)
Test Organisms and
Isolation:
Total 25 urine samples were collected from
different private clinics out patients departments from different areas like
Andhra Pradesh and Maharashtra. Standard operating protocol (SOP) was used to
process the sample and was stored at 4-50C. Basic identification tests
were performed using biochemical routine analysis and isolates were confirmed.
To ensure the correctness, MTCC cultures were also used to compare. Out of 25
samples, 9 were E. coli, 3 Klebsiella pneumoniae and 2 were Pseudomonas aeruginosa. One of each was used to confirm. Finally only
MTCC cultures were used to prepare inoculum.
Inoculums
Preparation:
Primary isolation was initiated by nutrient
agar plating and subsequently transferred to selective media like Pseudomonas
Isolation Agar, MacConkey Agar and EMB agar for better isolation. Using sterile
inoculation loop colonies of the test organism are transferred to 5ml of
sterile nutrient broth and incubated at 37°C overnight for 24hrs. Then this
bacterial culture were suspended in saline solution (0.85%Nacl) and adjusted to
a turbidity of 0.5 Mac Farland standards (108cfu/ml). This
suspension was used for preliminary screening of anti bacterial activity
Antibacterial Activity
of Plant Extract:
An altered agar well diffusion
method of Perez et al. (1990) (9) was employed. Selected medium were
inoculated. 6mm wells were punched on solidified media and filled with 25μL of the plants extracts and blanks (methanol). The
concentration of the extracts set to 25 μg / ml. The test was carried out by triplicate
method. The plates were incubated at 37 ± 2°C for 24- 48 h. The antimicrobial
activity was calculated in mm. The antibiotics were used as references were
selected on the basis of survey reported in 2014 in India. Hence only highly
sensitive antibiotics applied to cut the time.
RESULTS:
Standard procedures were used to test the
organisms. Plant extraction was done using the most suitable medium methanol to
get maximum yield of antimicrobial compounds present in the plant extract. Phytochemical analysis was also done and
supported by GCMS data library to ensure compounds in leaf. Shown in table 2 and
3. Antimicrobial testing’s were performed with well diffusion method using
standard antibiotics and were compared with the methanolic extract at unique
concentration various zones were obtained. E.coli
and K. pneumoniae were most sensitive
towards the selected leaf extract as compared to Pseudomonas aeruginosa sp. Shown in table 4.
Table 3.
Phytochemical screening of C.papaya
Leaf extract
|
Phytochemicals
|
Test
methods used
|
Methanolic extract
|
|
Saponins
|
Chloroforms test
|
+
|
|
Phytosterols
|
Libermanns method
|
+
|
|
Alkaloids
|
Dragendroffs test
|
-
|
|
Glycosides
|
Molish test
|
+
|
|
Tannins
|
Ciulci’s
|
+
|
|
Phenolic compounds
|
Ferric chloride test
|
+
|
|
Terpenoids
|
Sofowora’s
|
-
|
|
Flavenoids
|
Sofowora’s
|
+
|
+ : Present , - : Absent
Table 4. Zone of inhibition (mm) formed by isolates in
response to Carica papaya leaf extracts and antibiotics
|
Concentrations
of Extract
|
P. aeroginosa
|
E.coli
|
K. pneumoniae
|
|
Cefpodoxime
|
33 mm
|
R +
|
R+
|
|
Chloramphenicol,
|
R+
|
R+
|
20 mm
|
|
Ceftriaxone
|
R+
|
30 mm
|
19 mm
|
|
Methanolic leaf extract 100
|
25 mm
|
30 mm
|
27 mm
|
R+ : Resistance
DISCUSSION:
Urine tract infection caused by
uropathogens is challenge for drug development scientists. Specially, in case
of UPEC UTI caused by E.coli, there
are various MDR strains available in India, almost all the antibiotics got fail
to stop the virulence of this pathogens. With this, other like Pseudomonas aeruginosa , Klebsiella pneumoniae adding their role
and making more complications. Constant death and mortality ration rise is due
to resistant and delay in diagnosis.
Side effects are another health threat on one end. Trail and errors on
patients due to lack of pathology test also leads to increase in drug
resistance.
According to Niranjan et al 2014, every
urine sample contains 56-60% E.coli,
15% Klebsiella pneumoniae and 8-10 % Pseudomonas
aeruginosa sp. It is also declared that, In India various studies reported
UPEC has 85-89% resistance occurs since 2002-2013. (10) Pseudomonas aeruginosa and Klebsiella
pneumoniae also plays a crucial role, Rajat Rakesh et.al reported 48 % of
UTI caused by the Nosocomial infection, hospital acquired, ventilator,
catheters etc associated. Among all, 60% strains found resistant to
antibiotics.(11)
Herbal drug development using this plant
has a tremendous scope, several reports discussed this issue regarding the
antibacterial activity, antimicrobial activity of leaf, seed, stem bark extract
of C. papaya plant. But still, there is lack of evidence that what are
the exact compounds present in plant which plays the role reported by others.
Hence this study focuses on the active pharmaceutical ingredients present in C. papaya leaf and could plays role
against MDR bacteria. GCMS data shows 6 antimicrobial compounds occurs in this
leaves. According to O. Victor Njoku
and Chidi Obi alkaloids or phenolics
groups present in the plants shows
better antibacterial activity in presence of tannins.(12,13,14) With this our
study also confirms tannins and phenolics like 2- Methoxy-4-vinylphenol,
4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol and N-Aminomorpholine and also shows
antibacterial antioxidant properties.
A result of antibacterial test and
comparison with standard antibiotics give a strong support to this study and
allows developing more potent antibiotics from this plant.
This is the primary study which ensures the
presence of compounds which inhibits bacterial growth. But still various other
compounds yet to be discussed and we are trying to develop a list of compounds
playing role in various activity of this plant. This compounds requires more
validation and pharmacological studies to designate as a drug in future
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