Research article
Studies on Antimicrobial Potentials of Aerva lanata Fractions
B M DINNIMATH*1 AND S S JALALPURE2
*1,2 KLEU’S college of pharmacy, Nehrunagar, Belgaum-590010
Studies on different fractions of Aerva lanata extract for Antimicrobial potency
*” Address For correspondence”
B M Dinnimath
Ph No-9632045496
basavaraj_dm@yahoo.co.in
1
ABSTRACT
The aim of this study is to test the efficacy of different fractions of Aerva lanata
extract for their antimicrobial activity and analyze the influence of fractions on
the virulent factors of microorganisms. The whole plant Aerva lanata
( L) Juss.Ex. Schlt, commonly known as Pashanabheda
physicians
for various
diseased conditions traditionally.
used by various Ayurvedic
As this plant is widely
available in western ghats and no scientific reports on the antimicrobial potency of the
plant fraction/s reported till date, we have conducted this study with an objective that the
results will enlarge the existing knowledge. In this study the whole plant was subjected
for extraction with hydroalcohol followed by fractionation with different solvents based
on their polarities. Urolithiasis is found to be one among the top five critical conditions
responsible for urinary tract infections worldwide and as this plant is used as
Pashanabheda traditionally, we are aimed at identifying the antimicrobial potency of this
plant which shall help to substantiate the traditional claims of this plant in nurolithiasis
treatment. We found that different fractions of this plant have produced promising
antimicrobial results against E.coli, Bacillus sp, S.aureus. Proteus sp.
Keywords: Aerva lanata , antimicrobial activity, Pashanabheda, urinary tract infections
.
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Introduction
Plants are used as medicine since time immemorial. India is a rich source of
medicinal plants. They are widely used in ancient systems of medicine. It is
reported that, two or three antibiotics that are launched every year are derived
from microorganisms . After a downturn in that pace in recent decades, the pace is
again quickening as scientists realize the effective lifespan of any antibiotic is
limited . New medicines have to be discovered with traditional, empirical and
molecular approaches. Plants have limited less ability to synthesize secondary
metabolites of which 12000 have been isolated, a number estimated to be less
than 10% of the total[1-5].
Urolithiasis also known as crystalluria, is the condition where urinary calculi are
formed in the urinary tract. As per WHO it is the 3rd major disorder of urinary
tract and it affects 4-8% of population
in
UK, 15% in US, 20% in Gulf
countries and 11% in India. Complications of stone disease may result in severe
infection, renal failure or in rare cases even death [6-7].
This plant Aerva lanata Linn. (Amaranthaceae) is a herbaceous perennial weed
growing wild in the tropical regions and western ghats of India. Aerva lanata has
been claimed to be useful as diuretic, anthelmintic, antidiabetic, expectorant and
hepatoprotective, Antimicrobial and cytotoxicity activity , urolithiasis
and anti-
inflammatory activity of Aerva lanata has been reported[8-9].
This plant is commonly used in Ayurvedic medicines and recommended in texts for
various diseases. The plant is astringent, bitter, cooling, emollient,vermiuge,supparative,
diuretic and lithontriptic. It is useful to treat boils, cephalalgia, cough, strangury and
lithiasis . The plant has useful
nephroprotective
activity,
immunomodulatory effect,
diuretic
medicinal value, the extract is proved for
effect,
cytotoxicity
and
antioxidant,
diuretic effect, anti-inflammatory effect, antimicrobial
activity, hepatoprotective activity, antihyperglycemic effect[10-13].
Although this plant is used traditionally by Ayurvedic physicians, however, there is no
report on the antimicrobial potency of fractions of Aerva lanata extract and their
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efficacy against various microbes in urinary tract infection. Hence we have selected this
plant from western ghats of Khanapur region for study. In this present study the
hydroalcoholic(70-30) extract (to increase the solubility of different constituents of the
plant) of the plant is fractionated and studied for its antimicrobial activity.
Materials and Methods
Media Composition for the antimicrobial activity:

Thioglycollate Medium with Hemin and Vit K (For 1 litre)

Tryptose

Yeast extract: 10gms

Sodium thioglycollate: 0.50gms

Sodium chloride: 2.5gms

L-cystine HCL: 0.5gms

Sod. Bicarbonate: 0.40gms

Resazurine: 0.001gms

Hemin: 0.005gms

Vit K : 0.0005gms

Agar: 0.75gms
: 15gms
The microorganisms used for the study and their strain no (ATCC No) are
E.Coli (ATCC no – 25922), S. aureus ( ATCC no – 12598), Proteus sp (ATCC no
– 49565) and Bacillus sp (ATCC no- 6051). The standard drug used was
Ciprofloxacin (10µg).
Extraction and Fractionation [14-16]
The whole plant Aerva lanata is procured from western ghats region of Khanapur
taluka for our study. It’s a herb widely available in this region. The plant is
authenticated by taxonomist
Dr. Harsha Hedge by RMRC, Belgaum (
specification no-RMRC-507). The whole plant is subjected to shade drying and
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then it was ground to coarse powder. The powdered dry material was used for the
extraction.
Dried and powdered plant material (200 gm) was initially defatted with pet ether
(40-600C) for 2-4 hrs and the defatted material was subjected to successive hot
extraction in Soxhlet apparatus with ethanol (80%). After extraction , the solvent
(ethanol-80%) was subjected to vacuum distillation under reduced pressure
(BUCHI ROTAVAPOUR), the extract was concentrated to yield semisolid
residue which is sealed and stored at 40C. This has yielded 24.5% petroleum
ether fraction of 10g (10%) dried hydroalcoholic extract.
Aqueous extract was prepared by dissolving the dried ethanolic extract in distilled water
and the aqueous extract was partitioned with different solvents of varying polarities
(dichloromethane, ethyl acetate and n-butanol ) in a separating funnel with vigorous
shaking . The organic fractions were purified by washing with distilled water treated
with sodium sulphite overnight to remove moisture. These fractions were subjected to
qualitative tests and chromatographic analysis.
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Collection of the plant
Extraction
pet ether
Hydroalcohol
Fractionation
Dichloro methane
fraction I
Ethyl acetate
fraction II
n-butanol
fraction III
Determination of MIC (For Anaerobes)[19-20]
Dilutions (total 9) of each drug have to be prepared with Thioglycollate broth for
MIC. In the initial tube 20 ml of drug was added into the 380 ml of Thioglycollate
broth. For dilutions 200 ml of Thioglycollate broth was added into the next 9
tubes separately. Then from the initial tube 200 ml was transferred to the first tube
containing 200 ml of Thioglycollate broth. This was considered as 10-1 dilution.
From 10-1 diluted tube 200ml was transferred to second tube to make 10-2
dilution. The serial dilution was repeated up to 10-9 dilution for each drug. From
the maintained stock cultures of required organisms, 5 ml was taken and added
into 2 ml of Thioglycollate broth. In each serially diluted tube 200 ml of above
culture suspension was added. The tubes were incubated for 48-72 hours in
anaerobic jar at 37°C and observed for turbidity.
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Results and Discussion
The ethyl acetate and n-butanol fractions of Aerva lanata were taken for their
antimicrobial activity study against S.aureus , E.coli , Bacillus sp and Proteus sp
as these are found to be responsible for Urinary Tract Infections and also
becoming resistant to many antibiotics used for UTI. There are reports that plant
extract has shown antimicrobial activity against these microbes and this has
aroused interest in conducting the present study which may be useful to increase
the present knowledge. As urinary calculi are also found to be responsible for
UTIs, this plant which is used as Pashanabheda was studied for antibacterial
activity which may be an additional effect associated with its Antiurolithiatic
potency.
The fractions were dissolved in Thioglycollate broth and 9 dilutions were
prepared for MIC which was identified with the help of turbidity produced in the
tubes . Based on this turbidity fractions were identified as sensitive (S) or resistant
(R). The MIC values for the fractions were calculated (table no 2).
Antimicrobial properties of medicinal plants are being increasingly reported from
different parts of the world. The World Health Organization estimates that plant
extract or their active constituents are used as folk medicine in traditional
therapies of 80% of the world's population. It is found that the activity
(sensitivity) is absent in the fractions as the dilution in reduced. In the present
work, n-butanol and ethyl acetate fractions from Aerva lanata hydroalcoholic
extract are tested for their antimicrobial activity. Ethylacetate fraction has shown
sensitivity against E.coli ( 2 µg/ml) at 100 µg/ml dilution and n-butanol has
failed to show sensitivity against E.coli. Ethylacetate fraction has produced
positive results against Bacillus sp at 100 and 50 µg/ml dilutions whereas nbutanol has shown positive result against Bacillus sp at 100 µg/ml dilution only.
Ethyl acetate fraction has shown positive results against S.aureus at 100, 50 and
25 µg/ml dilution and n-butanol fraction has shown positive results against
S.aureus at 100 µg/ml dilution only. Ethyl acetate and
n-butanol fractions have
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produced positive results against Proteus sp at 100 µg/ml only. All these results
produced by fractions are compared against Std drug Ciprofloxacin (10µg). All
the results are shown in table no-1.
From the above results it is evident that both the fractions of Aerva lanata have
shown moderate activity against the microorganisms used for the test compared
with std drug Ciprofloxacin. The fractions have shown sensitivity at higher
dilutions like 100, 50 and 25 and resistance at other dilutions as shown in the table
no-1. Since the fractions have shown sensitivity against the microorganisms , they
may become successful in combating the infection due to these microorganisms
associated with kidney stones in urinary tract, which would be beneficiary effect
and an added advantage for the patient suffering from Urolithiasis. As these two
fractions found to contain polyphenolic compounds like flavanoids, terpenoids
and saponins, the results gives us an indication that these compounds may be
responsible for the reported antimicrobial activity. Perhaps further study is needed
to establish this fact.
Conclusion
The whole plant Aerva lanata was subjected to extraction with hydroalcohol
followed by fractionation with n-butanol and ethylacetate. The fractions were
subjected to analysis of antimicrobial potency against E.coli, Bacillus sp, Proteus
sp and S.aureus at different dilutions. The std drug used was Ciprofloxacin. The
tested fractions of the plant extract have shown moderate activity (sensitivity)
against the microbes at 100, 75 and 50 µg/ml as compared with the std drug
Ciprofloxacin.
The objective of this study was to know the probable potency of Aerva lanata as
antimicrobial drug in urinary tract infections as this plant is used as
Pashanabheda in Urolithiasis and this condition is one of the causes of UTIs.
Hence the plant fractions were subjected to antimicrobial analysis against the
selected microorganisms. The results have shown moderate potency of the plant
fractions as antimicrobial drug which may be an associated activity in case of
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Urolithiasis. Although the present results shows the possibility of curative
treatment by Aerva lanata in UTIs, further studies are needed to confirm the
potency of the plant and its constituents (polyphenolic compounds) as
antimicrobial drug.
Acknowledgement
Authors thank KLE University and Principal, KLEU’S College of Pharmacy, Belgaum
for carrying out this research work.
References
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Table-1 Results of Antibacterial activities of Aerva lanata fractions at different
dilutions
Fraction
Microorganism
Ethylacet
ate
nButanol
E.coli
100
S
50
R
25
R
12.5
R
6.25
R
3.12
R
1.6
R
0.8
R
0.4
R
0.2
R
E.coli
R
R
R
R
R
R
R
R
R
R
Ethylacet
ate
Bacillus sp
S
S
R
R
R
R
R
R
R
R
nButanol
Bacillus sp
S
R
R
R
R
R
R
R
R
R
Ethylacet
ate
S.aureus
S
S
S
R
R
R
R
R
R
R
nButanol
S.aureus
S
R
R
R
R
R
R
R
R
R
Ethylacet
ate
Proteus sp
S
R
R
R
R
R
R
R
R
R
nButanol
Proteus sp
S
R
R
R
R
R
R
R
R
R
Note: S-Sensitive
R-Resistant
Standard drug- Ciprofloxacin
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Table no-2
Fraction
Results of Minimum Inhibitory Concentration of Fractions
Microorganism
Dilutions and MIC ( mm)
100
50
25
7
n
S.aureus
9
8
Butanol
E.coli
8
7
9
7
Proteus sp
8
8
Ethyl
S.aureus
8
7
6
acetate
E.coli
9
7
6
Bacillus sp
9
6
5
Proteus sp
8
7
6
Bacillus sp
6
7
6
Values for MIC of std drug Ciprofloxacin (10µg):
S.aureus -16 mm
E.coli
- 18 mm
Bacillus sp - 19 mm
Proteus sp - 15 mm
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Pictures of Antimicrobial activity study of Aerva lanata fractions
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