INTRODUCTION:
Antiseptics are antimicrobial substances that are
applied to living tissue/skin to reduce the possibility of infection, sepsis,
or putrefaction. Antiseptics are generally distinguished from antibiotics by
the latter´s ability to be transported through the lymphatic system to destroy
bacteria within the body, and from disinfectants, which destroy microorganism found
on nonliving objects.
The
new antimicrobial skin preparations useable to disinfect a surgical site for
surgery. The antimicrobial skin-preparation formula includes povidone-iodine and
extract of Azadirachta indica (neem).
The
new pre-operative skin-preparation quickly and effectively kills microorganisms
when applied to the surgical site. The skin-preparation continues to
effectively inhibit microorganism growth in the applied area over an extended
period of time. The role of the newly developed formulations of povidone-iodine
preparations, its antimicrobial activity, the possibility of impairing the wound
healing process, the role of povidone-iodine in the problem of growing
resistance against antibiotics and antiseptics.
Poly
vinyl pyrrolidone–iodine (Povidone-iodine) is a widely used antiseptic
introduced by Shelanski and Shelanski (1) in 1956. The preparations of poly
vinyl pyrrolidone-iodine commercially available are povidone-iodine solution,
scrub, ointment, and foam; of these, the solution is most commonly used. The
10% povidone-iodine solution contains 90%water, 8.5% polyvinylpyrrolidone, 1% available
iodine and iodide (2). poly vinyl pyrrolidone is a similar to dextran. The
molecular weight of the poly vinyl pyrrolidone utilized in poly vinyl
pyrrolidone-iodine solutions ranges from 10 to 40000 daltons (3). Micro-organism
on the skin can be transient or resident. Transient microorganisms lie on the
surface of the skin, while resident microorganisms are found at deeper site of
the skin (4-7).
Some 30 years after the synthesis of povidone-iodine,
a paradox in the behaviour of 10% solutions was reported: its antibacterial
action increased with the degree of dilution (low concentrations, i.e. 0.1% to
1%, were more rapidly bactericidal than a full-strength, i.e. 10%, solution)
(8). One hypothesis is that the concentration of “free” iodine significantly contributes
to the bactericidal activity of povidone-iodine solution: dilution of povidone iodine
results in weakening of the iodine linkage to the carrier polymer with a
concomitant increase in the amount of elemental (free) iodine in solution (9,10).
Poly vinyl-pyrrolidone-iodine newer vehicles include
PVP-I Solution, PVP-I Ointment, PVP-I Cream (11) and PVP-I Gel Alcohol (12).
Calfee found no significant difference in skin disinfection among 10% PVP-I,
70% isopropyl alcohol, tincture of iodine and PVP-I with 70% ethyl alcohol
(Persist), although there was some evidence suggesting greater efficacy among
the alcohol-containing antiseptics (13). This was confirmed in another study by
Arata et al. (14). Wiping aqueous PVP-I 10% off after 30 seconds of application
did not show a significant difference in the reduction from baseline counts of
skin flora at 5, 30, 60 and 120 min.
Azadirachta indica (Meliaceae) commonly known as neem is native of India and
naturalized in most of tropical and subtropical countries is of great medicinal
value and distributed wide spread in the world. Other compounds that have a
biological activity are salannin, volatile oils, meliantriol and nimbin (15,16)
Jacobson et al., 1990. It is used as anticancer agent and it has hepato-renal protective
activity and hypolipidemic effects ((C) Fitoterapia part I and part II). Medicinal
plants have been found useful in the cure of a number of diseases including
bacterial diseases. Medicinal plants are a rich source of antimicrobial agents
(17-20) Mahesh and Satish et al., 2008). Almost every part of the tree is
bitter and finds application in indigenous medicine.
EXPERIMENTAL
METHODS:
Preparation
of PVP-i:
Weight
around 2g of citric acid poured into 2 neck round bottomed flask. Then 40g of
Iso propyl alcohol added slowly with constant stirring. Then 4g of Iodine is
added. Above the mixture stirring for 12 hrs. The prepared paste is dried in vacuum
oven for 1 hr at pressure 50 and temperature also 50oc. Then the
crude sample is crushed into fine powder and sieved using 1200 mesh cloth.
PREPARATION OF LEAF EXTRACT:
20-30grams of fresh leaves were boiled with 200mL of
solvent for 1 hour. The extract was filtered using Whatmann filter paper No. 1
and then concentrated in vacuum at 40°-50°C using a rotary evaporator.
Evaporation of solvent in the rotary evaporator affords a crude extract of the
soluble components and these extracts were subjected to the qualitative
phytochemical analysis and antibacterial
RESULT AND DISCUSSION:
The findings of the preliminary Phytochemical
investigations and the results of antibacterial activity were depicted in the respective
Tables. The preliminary phytochemical tests performed were of qualitative type
and from the phytochemical investigations it was observed that alkaloids, tannins, flavonoids, terpenoids, saponins Glycoside
and compounds reducing were present in the extracts. The ethanol, chloroform and
aqueous extract showed considerable activity against Salmonella typhii, The
ethanol extract was more active than the standard against Salmonella typhii.
Previous study conducted by (Ben Gueddeur et al., 2002) (21) suggests that the
essential oil of O. majorana posses antibacterial activity.
The work conducted by (Farooqi and Sreeramu, 2004) (22)
reveals that the leaves of marjoram have antibacterial activity against Escherichia
Coli, Pseudomonas aeroginosa, Staphylococcus aureus and Salmonella
typhii. similarly antibacterial activity of ethanol, chloroform and water extract
of Marrubium vulgare, was further assessed against, Salmonella typhii,
Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, were
recorded (Al-Bakriet al., 2006) (23). The presence of these phytochemical
components may be responsible for the observed antimicrobial activity of the plant
leaf extract. This findings conforms to there port of (Anyanwu and Dawet, 2005)
(24) in which similar constituents was found to exhibits anti protozoal and
antibacterial activities. Flavonoid has also been reported to have greater
potential benefit to human Health (Jouad et al., 2001 (25).
Imaran Khan et al., 2010 (26) studied that
phytochemical analysis of Azadirachta indica leaves by using different solvent
such as Petroleum ether, chloroform, methanol show the presence of triterpenes,
glycosides and fatty acids. Other phytochemicals studied in this analysis were absent
in all extract of leaves. Antibacterial activity of Azadirachta indica was
analyzed by previous workers showed that the chloroform extract of leaves
possess significant activity, than petroleum ether and methanol extracts. Early
studies proved ethanol as the most efficient solvent for extracting broad spectrum
of antibacterial compounds from plants. Himal Paudel Chhetri et al., 2008 (27)
reported that the ethanolic extract of Azadirachta indica whole plant
shows presence of flavonoids and tannins only. Similarly the extract of Azadirachta
indica is active against E. coli followed by Staphylococcus aureus. Earlier
observation done by (Srinivasan et al. 2001) (28) also showed the antifungal and
antibacterial activity of A. Indica.
UV SPECTRAL STUDIES:
The
fine structure of the UV spectra of PVP in water at relatively high
concentration is entirely reproducible. Very few polymers exhibit fine
structure in UV spectra unless the chain unit is of sufficiently complicated
structure on dilution of the PVP solution with water the spectrum is shifted to
shorter wavelength and below a weight concentration of about 0.002% and maximum
as apparently shifted to a wavelength below 210mµ. We have followed the polymerisation
of PVP as a function of time by absorbing the decrease in absorption at
235mµ.The polymerisation was carried adding to a 20% of aqueous solution of PVP-I.
It was found that the weight of polymer used is close to that calculated from
the decrease in absorption. Molecular iodine as an absorption in the near UV which
is considerably ultered in the presence of PVP. The alternation in absorption
of molecular iodine at 290mµ as a function of PVP concentration is illustrated
in absorption at 290mµ of the solution to that of the same concentration of
iodine alone is determined. PVP-I Shows high absorbance at 300nm and slight absorbance
at 365nm, PVP-I+NEEM there is only slight absorbance at 290nm and 360nm shows
less absorbance compared with PVP and NEEM there is only slight absorbance at
290nm and 360nm shows less absorbance compared with PVP-I+NEEM.
Fig.1 UV Spectra
IR
SPECTRA FOR PVP-I:
The
iodine loss of aqueous solutions of the polyvinylpyrrolidone-iodine was 18-20 %
or 2-4% and was independent of the preparation method. Apparently even
proportion is introduced or formed during heating readily found the pyrrolidone
group carbonyl group needed for complexation due to the large molar excess of
polypyrrolidone monomer units. Even without detailed knowledge of the origin of
the adsorption, it is apparent that the IR spectra of the model substances
agree with the polyvinyl pyrollidone spectrum. Therefore the frequency and
intensity of the vibration of the CN , NC=O and carbonyl group and the ring are
largely independent of the alkyl side chain.
Fig.2
IR Spectra for PVP-I
Fig.3
IR Spectra for PVP-I with Neem
The
bonds in the complexes of the model substances and in the corresponding
polyvinylpyrrolidone adduct must be least very similar. This view may be
confirmed by comparing IR spectra of model substances with those of
polyvinylpyrrolidone. Hydrogen iodide shows peak at 567 to 815 cm-1 NC=O
shows peak at 1653 cm-1, five membered ring shows peak at 1745 cm-1
C-H bending shows peak at 1375 to 1464 cm-1 and C-H stretching
shows peak at 2953 cm-1 and OH shows peak at 3443 cm-1
PVP-I
with Neem:
Hydrogen
iodide shows peak at 567cm-1 to 815 cm-1 NC=O
shows peak at 1653, five membered ring is absent, C-H bending shows peak at
1375cm-1 to 1464 cm-1 and C-H stretching shows peak at
295cm-1 and OH shows peak at 3443cm-1. From the graph we
came to know that in the binding of PVP-I with Azadirachta indica all
the functional group of PVP-I is existing in the mixture.
PHYTOCHEMICAL ANALYSIS:
The extracts were analyzed by the following procedures
to test for the presence of the alkaloids, saponins, tannins, Terpenoids,
flavonoids, glycosides, volatile oils and reducing sugars
SAPONIN:
Saponins were detected using the froth test. 1g of the
sample was weighed into a conical flask in which 10ml of sterile distilled water
was added and boiled for 5 minutes. The mixture was filtered and 2.5ml of the
filtrate was added to 10ml of sterile distilled water in a test tube. The test
tube was stoppered and shaken vigorously for about 30 seconds. It was then
allowed to stand for half an hour. Honeycomb froth indicated the presence of
saponins.
TANNIN:
To a portion of the extract diluted with water, 3-4
drops of 10% ferric chloride solution is added. A blue color is observed for
gallic tannins and green color indicates for catecholic tannins
REDUCING SUGAR:
To 0.5ml of plant extracts, 1ml of water and 5-8 drops
of Fehling’s solution was added and heated over water bath. Brick red
precipitate indicates the presence of reducing sugar
ALKALOIDS:
2ml of extract was measured in a test tube to which
picric acid solution was added. An orange coloration indicate the presence of
alkaloids.
FLAVONOIDS:
4ml of extract solution was treated with 1.5ml of 50%
methanol solution. The solution was warmed and metal magnesium was added. To
this solution, 5-6 drops of concentrated hydrochloric acid was added and red
color was observed for flavonoids and orange color for flavones
ETHANOL EXTRACT:
Azadirachta indica leaves (100 g) were ground into fine powder ((M) Himal Pauel Chhetri
et al., 2008) (24) using a stainless-steel grinder, and deep in100% ethanol
(200 mL) for overnight. The ethanol fraction was separated using sterile muslin
cloth and filter through sterile Whatman filter paper (no. 02). The filtered
extract was concentrated by a rotary film evaporator.
The phytochemical analysis of plant extracts using
Acetone, Ethanol and Methanol was showed in Table - 1. From the phytochemical
analysis catecholic reducing sugar were found in Azadirachta indica in
the solvents such as Acetone, Ethanol and Methanol. The Ethanol extract of Azadirachta
indica showed the presence of flavonoids, saponins, tannin, reducing sugar
were found in presence of Ethanol extract. Reducing sugar, glycosides were
observed only in Acetone extract of Azadirachta indica. In plant all
extracts found glycosides except in Ethanol extract of Azadirachta indica.
Saponin were observed in the Acetone and Ethanol extract of Azadirachta
indica. Terpenoids were observed only Methanol extract of Azadirachta
indica. The Acetone, Ethanol and Methanol all extract of Azadirachta
indica showed the absences of alkaloid and volatile oil.
Determination of anti bacterial activity:
The antibacterial activity of the leaf extracts was determined
using agar well diffusion method by following the known procedure. Nutrient
agar was inoculated with the given microorganisms by spreading the bacterial
inoculums on the media. Wells were punched in the agar and filled with plant
extracts. Control wells containing neat solvents (negative control) were also
run parallel in the same plate. The plates were incubated at 37°C for 18 hours
and the antibacterial activity was assessed by measuring the diameter of the zone
of inhibition.
Table-2: Antibacterial activity of Acetone, Ethanol,
Methanol Extract of Azadirachta indica medicinal plants against human
pathogens
|
Plants
|
Extracts
|
Escherichia Coli
|
Pseudomonas aeruginosa
|
Staphylococcus aureus
|
Salmonella typhi
|
|
Zone of inhibition (mm)
|
|
Azadirachta indica
|
Acetone
|
17
|
15
|
18
|
16
|
|
Methanol
|
16
|
17
|
12
|
20
|
|
Ethanol
|
24
|
20
|
19
|
30
|
Table-2 showed the antibacterial activity of Ethanol
extract of Azadirachta indica showed maximum zone of inhibition (30mm)
against Salmonella typhii, followed by Escherichia coli (24mm), Pseudomonas
aeruginosa (20mm), Staphylococcus aureus (19mm)
The antibacterial activity of Acetone extract of Azadirachta
indica showed maximum zone of inhibition (18mm) against Staphylococcus
aureus, followed by Escherichia Coli (17mm), Salmonella typhii (16mm)
and Pseudomonas aeruginosa (15mm). The antibacterial activity of
Methanol extract of Azadirachta indica showed maximum zone of inhibition
(20mm) against Salmonella typhi, followed by Pseudomonas aeroginosa
(17mm), Escherichia coli (16mm) and Staphylococcus aureus (12mm).
Minimum
Inhibition Concentration Test In Evaluation Of Anti-Microbial Efficacy
An
antimicrobial skin-preparation according to the present invention was tested
for anti microbial efficacy with the challenge gram positive and gram negative
bacteria with resistance to various antibiotics. The minimum inhibition
concentration test was also conducted to evaluate the long term effectiveness
at killing microorganism.
· Staphylococcus aureus (ATTCC#6538) Gram positive
bacteria
· Streptococcus pneumonia (ATCC#35088)
· 
Escherichia coli (ATCC#11229) Gram negative bacteria
· Pseudomonas aeruginosa(ATCC#27853)
Further
the polyvinylpyrrolidone-iodine and polyvinylpyrrolidone-iodine Azadirachta
indica by using anti-bacterial activity. Two strains including Gram
negative E. coli, and Pseudomonas aeruginosa and Gram-positive Streptococcus
pneumonia and Staphylococcus aureus were selected for antibacterial tests
because they are usually associated with the medical-associated infections. The
comparative Anti-bacterial property was investigated by calculating
anti-bacterial ratios based on the numbers of bacteria colonies incubated with
different dosages .The equivalent volumes of each of the bacterial cultures was
added into each of the testing products (PVP-I and Azadirachta indica)
and a series of 1:1 dilution of the mix made with sterile saline. The mixed
materials were incubated for 24hrs to observe the microbial growth.
The
electronic microscopic images was showed that the present invention (PVP-I and Azadirachta
indica) produced similar antimicrobial inhibition activity to the control.
The concentration of (PVP-I and Azadirachta indica) control that limit
microbial growth to a specified amount over a 24hrs period.
Fig.4
Bacterial culture
Fig.5
growth of microbial
Fig.6
PVP-I inhibit the growth of microbials
Fig.7
Azadirachta inhibit the growth of microbials
Fig.8
PVP-I and Azadirachta indica inhibit the growth of microbials
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