This and without skin using various solvents like

This study was designed to test the
antimicrobial activity of various solvent extracts of A. sativum and gomutra. The antimicrobial activity for the extracts
was observed against the common wound infection causing bacterial strains Staphylococcus aureus and Pseudomonas sp and fungal strains Aspergillus niger and Penicillium sp.

The antimicrobial activity was tested by agar
well diffusion method. The A. sativum
extract  was prepared with and without
skin using various solvents like ethanol, methanol and distilled water. Based
on the different solvents used for extraction the extracts were labeled as GJA,
GJM, GJE, GJWSA, GJWSM and GJWSE. Almost all the solvent extracts exhibited antibacterial and antifungal
activity against the tested organism, when compared to other solvents aqueous
extract is more effective. Also those extracts with skin showed increased zone
of inhibition.

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Preliminary phytochemical analysis of the
plants constituents were assessed by using qualitative methods. Test was
conducted for the following active components: alkaloids, flavanoids, steroids,
phlobatannin, cardiac glycosides, phenols, saponins, volatile oil and
glycosides.

FTIR analysis was done to find out different
functional groups present in the extracts. HPLC and GCMS were also done. Also MIC value of the various solvent extracts of A. sativum was determined. The MIC value
for GJA, GJWSA, GJE and GJWSE against Pseudomonas
sp is 1, 10, 10 and 1 mg/ml respectively. The MIC for GJA, GJWSA, GJE and
GJWSE against S. aureus is 100, 100,
100 and 1 mg/ml respectively.

 

 

 

 

 

 

 

1.0 INTRODUCTION

 

The frequency of life-threatening infections caused
by pathogenic microorganisms has increased worldwide and is becoming an
important cause of morbidity and mortality in immune compromised patients in
developing countries (Al-Bari et al., 2006). The increasing prevalence of
multi-drug resistant strains of bacteria and the recent appearance of strains
with reduced susceptibility to antibiotics raised the specter of ‘untreatable’
bacterial infections and adds urgency to search for new infection-fighting
strategies (Zy et al., 2005; Rojas et al., 2006).

 

Plant products are the cheapest and safer
alternative sources of antimicrobials (Pretorius and Watt, 2001; Sharif and
Banik, 2006; Doughari et al., 2007) as there are no side effects. For a long
time, plants have been an important source of natural products for human
health. The antimicrobial properties of plants have been investigated by a
number of studies worldwide and many of them have been used as therapeutic
alternatives because of their antimicrobial properties (Adriana et al., 2007).
Plants have many antimicrobial properties especially in secondary metabolites
such as alkaloids, phenolic compounds, etc.

 

Some plants are known to possess medicinal
properties because they contain active substances that cause certain reactions,
from relenting to the cure of diseases, on humans (Silva Junior et al., 1994).
Knowledge on medicinal plants sometimes means the only therapeutic resource of
some communities and ethnic groups (Di Stasi, 1996); and their use, especially
in South America, contributes significantly to primary health care (Holetz et
al., 2002).

 

1.1 Allium
sativum

 

A.
sativum is commonly known
as garlic. It is a species in the onion genus “Allium”. Its close relatives include the onion, shallot, leek,
chive and rakkyo (Block, 2010). The plant is a member of the Liliaceae family
and one of the most popular herbs used worldwide to reduce various risk factors
associated with several diseases (Thomson et al., 2007). Also, it is a bulbous
perennial food plant of the family Alliaceae which give it a botanical name
known as Allium sativum (which comes
from old English genlac meaning “spear lack”). When crushed, A. sativum yield allicin, an antibiotic
and antifungal compound (phytoncide). It has been claimed that it can be used
as home remedy to help speed recovery from strep throat or other minor ailments
because of its antibiotic properties.

 

1.2 Indian cow and gomutra

 

Cow is the most
valuable animal in all Veda and it is called mother of all. The vital products
we get from cow: urine, dung, milk, ghee and curd. These five ingredients are
called as panchagawya. Gomutra has found therapeutic applications since
days of yore. Gomutra is consumed by the majority of the rural population as a
traditional remedy in almost the whole Indian continent (Jerald et al., 2008). Gomutra based preparations are
able to counter viral, microbial, and fungal ailments. These potions promote
powerful antimicrobial, antiviral, anti allergic, and antioxidant activity (Piyush M, 2005).
There are many research mainly centered on the exploration of the antimicrobial
powers of gomutra and also its phyto chemical properties (Shivkumar et al.,
2011).

 

The present study was designed to test the
antimicrobial activity of various solvent extracts of A. sativum and gomutra. The antimicrobial activity for the extracts
was observed against the common wound infection causing bacterial strains Staphylococcus aureus and Pseudomonas sp and fungal strains Aspergillus niger and Penicillium sp.

2.0 MATERIALS AND METHODS

 

2.1 Collection of A. sativum

The
garlic (A. sativum) used for the
study was purchased from the local market of Vellore District, Tamil Nadu,
India.

 

2.2 Preparation of A. sativum extract

2.2.1 Aqueous extract

 20g of
raw A. sativum without skin was
ground to paste and the volume was made up to 100ml with sterile distilled
water and allowed to stand for 72 hrs. Then the residue was filtered by Whatman
No 1. filter paper. Then the extract was stored at 4°C till it was used.

20g of raw A. sativum with skin was ground to paste and the volume was made up
to 100ml with sterile distilled water and allowed to stand for 72 hrs. Then the
residue was filtered by Whatman No 1. filter paper. Then the extract was stored
at 4°C till it was used.

2.2.2
Ethanol extracts

20g of raw A. sativum without skin was ground to paste and the volume was made
up to 100ml with ethanol and allowed to stand for 72 hrs. Then the residue was
filtered by Whatman No 1. filter paper. Then the extract was stored at 4°C till
it was used.

20g of raw A. sativum with skin was ground to paste and the volume was made up
to 100ml with ethanol and allowed to stand for 72 hrs. Then the residue was
filtered by Whatman No 1. filter paper. Then the extract was stored at 4°C till
it was used.

2.2.3 Methanol extracts

20g of raw A. sativum without skin was ground to paste and the volume was made
up to 100ml with methanol and allowed to stand for 72 hrs. Then the residue was
filtered by Whatman No 1. filter paper. Then the extract was stored at 4°C till
it was used.

20g of raw A. sativum with skin was ground to paste and the volume was made up
to 100ml with methanol and allowed to stand for 72 hrs. Then the residue was
filtered by Whatman No 1. filter paper. Then the extract was stored at 4°C till
it was used.

2.3
Procurement of gomutra and analysis

Fresh gomutra was collected in sterile screw
cap bottles and brought to the laboratory for testing. It was filtered through
Whatman filter paper before being subjected to further testing. The gomutra was
sent to a laboratory at Kanchipuram for analysis to confirm the absence of
bacteria and fungi. 

2.4
Labeling of the samples

The various solvent A. sativum juice extract and gomutra is labeled as shown in Table 1
throughout the study.

Table
1: Labeling of the samples

Extract/ Sample name

Labeling

Aqueous extract of A. sativum without skin

GJA

Methanolic extract of A. sativum without skin

GJM

Ethanolic extract of A. sativum without skin

GJE

Aqueous extract of A. sativum with skin

GJWSA

Methanolic extract of A. sativum with skin

GJWSM

Ethanolic extract of A. sativum with skin

GJWSE

 

2.5 Phytochemical screening

 To confirm the presence or absence of the
following plant secondary metabolites phytochemical screening were carried
out:  phenols, alkaloids, steroids,
cardiac glycosides, flavonoids, saponins, phlobatannins, volatile oil and
glycosides.

2.5.1 Test for Phenols

 Equal volumes of each extract and ferric
chloride solution (which is prepared by dissolving 1352g of FeCl3.6H2O
in distilled water containing 20 ml of concentrated HCl dilute to 1 liter) are
added together. A deep bluish green precipitate indicates the presence of
phenol.

2.5.2 Test for Alkaloids

 To each extract was added to 1%
aqueous HCl over water bath and filtered. The filtrate was treated with (2g of
Iodine in 6g of Potassium iodide in 100 ml of distilled water). Formation brown
or reddish brown precipitate indicates presence of alkaloids.

2.5.3 Test for Steroids

Into each extract was added to 2ml acetic anhydride and 2ml H2SO4.
Color change from violet to blue or green indicates the presence of steroids.

2.5.4 Saponins

1g each extract was boiled with 5ml distilled water and filtered. 3ml
distilled water was added to the filtrate and shaken vigorously for 5 minutes.
Persistent frothing on warming indicates the presence of Saponins.

2.5.5 Cardiac glycosides

 The extract was treated with 2ml
glacial acetic acid with a drop of Ferric Chloride solution and underplayed
with 1ml H2SO4. A browning at the interface indicates the
presence of cardiac glycosides.

2.5.6 Phlobatannins

  A few drops of 1% HCl were added
to 1ml of test extract and was boiled. A reddish precipitates indicate the
presence of phlobatannins.

2.5.7 Flavonoids

5ml Ammonium solution was added to the aqueous filtrate of extract and
then a few drops of concentrated H2SO4. Yellow coloration
indicates the presence of Flavonoids.

2.5.8 Volatile oil

 A small quantity of the test
extract was shaken with dilute NaOH and 0.1ml HCl. The formation of a white
precipitate indicates a positive result.

2.5.9 Glycosides

 A small amount of the alcoholic
extract was taken in 1mlof water in a test tube and a few drops of aqueous NaOH
were added. A yellow coloration indicates the presence of glycoside.

2.6 Maintenance of pure culture

2.6.1 Test organism

Test organisms were collected from MTCC and an antibacterial activity of
various extracts of A. sativum and
gomutra was tested against Pseudomonas sp
and S. aureus.  

2.6.2 Standardization of broth culture

 To the test tube containing 10ml
of nutrient broth, 0.1ml of the bacterial inoculum was added and mixed for
homogeneity and was incubated for 3 hrs. Then the broth cultures were estimated
for the presence of l08 cfu/ml of bacterial inoculums.
Standardization of broth culture was done by comparing the turbidity to that of
0.5 McFarland turbidity standards.

 

2.7 Agar well
diffusion method

The antibacterial activity of the aqueous, ethanol
and methanol extracts of A. sativum and the synergistic effect of
aqueous, ethanol and methanol extracts of A. sativum with gomutra was
evaluated by agar well diffusion method. The Muller-Hinton
agar was prepared and poured into sterile petri dishes and allowed to solidify.
The test organism was inoculated onto the sterile agar plates. Four wells of
6mm in diameter each were aseptically bored using a sterile cork borer on each
agar plates. 100µl of the different extracts of A. sativum and gomutra were added to each well. Also 100 µl of A. sativum with gomutra (50 µl + 50 µl) was added to a well. The effect of solvent on the bacteria was also
checked by adding the respective solvent on one well. The plates were
then incubated at 37°C for 18-24 hrs and room temperature for 3-4 days for
bacteria and fungi respectively. Effects of the extracts were assessed by
measuring the diameters of zones of inhibition.

 

2.8 FTIR spectral analysis

 

The
FTIR spectra were performed and recorded with a Fourier transform infrared
spectroscopy. The infrared radiation is propagated through the sample to obtain
the corresponding spectrum, which was averaged from several data acquisitions.
FTIR spectra were acquired in the wave number range of 500-4000 cm-1.

2.9 HPLC analysis

 

The HPLC analysis was done with aqueous and ethanolic
extracts of A. sativum both with and
without skin. The
extracts were sent to VIT University, Vellore for HPLC analysis with HPLC model 1525. The flow rate was 0.5 ml/min. The
injected volume was 20µl and the UV detector was set at 254 nm.

2.10 GCMS analysis

The bioactive compound was identified
in ethanolic extracts of A. sativum.
The extracts were sent to VIT University, Vellore for GC-MS analysis and results
with identity of compound were collected.

2.11 Calculation of minimal inhibitory
concentration (MIC) by broth dilution method

Serial dilutions of aqueous and
ethanolic extract of A. sativum with
and without skin (GJA, GJWSA, GJE and GJWSE) were prepared in Mueller-Hinton
broth with 1ml of standard inoculums of the microorganisms. To the first tube
1mg/ml of A. sativum juice was added
and serially diluted.  A tube of the
growth medium without A. sativum
juice, served as a growth control. An uninoculated tube of the medium was
incubated to serve as a negative growth control. After 24 hrs of incubation,
the tubes were examined for turbidity, indicating growth of the microorganisms.
The lowest concentration of the garlic extracts that inhibits growth of the
organism was designated the minimum inhibitory concentration (MIC) (Forbes et
al., 1998).