1. INTRODUCTION:
Clinical use of antibiotics and
chemotherapeutics occasionally results human natural system hazards by
inhibiting protein synthesis or by misbalancing of natural flora in associated
with adverse effects like inflammation, immune-suppression, hypersensitivity,
weakness, diarrhoea and many more (Ahmad et al.,1998). Therefore
traditional folklore plant extracts have been a major concern for experimental
development by the modern Bio-science and proposed from the ancient time to be
used as antimicrobial therapeutics (Del Campo et al., 2000). Many
defined or undefined human diseases has been proven to be cured by the active
constituents of Medicinal plants (Stary and Hans, 1998). In search of the
scientific basis of the uses of medicinal plants many researchers invested
their interests. (Ghosh et al., 2007) studied the antimicrobial
performance of a number of traditional medicinal plants viz. antiba
Terminallia chebula, Terminallia bellerica,, Punica granatum, Lawsonia alba
and Mikaniamicr anthacterial showed effect on Staphylococcus aureus,
Bacillus subtilis, Escherichia coli, Proteus vulgaris and Enterobacter
aerogenes. Our experiments were carried out with one of these useful
medicinal plant Phallyanthus emlica L. The medium-sized deciduous tree
belongs to the plant family Phyllanthaceae. It is also named as Emblica
officinalis, Aonla, Aola, Amalaki, Dhatry, Amla and Indian Gooseberry. The high
nutrition value and enrichment with vitamin C, minerals (such as calcium,
phosphorus, iron etc.), amino acids, carotene, thiamine, riboflavin, and
niacin, other flavonoids and fibre contain makes Amla attractive for scientific
research (Srinivasan 1994, D.A Dhale 2012). Exhaustive survey reports proved
that the plant is a standard remedy for different types of ailments like emetic
and purgative (Lemmenset al.,1999).
Many varieties of the species, belonging to
the genus Phyllanthus, have been investigated phytochemically and
pharmaceutically and different molecules have been isolated and identified. Our
study is based on a systematic screening of leaf and stem berk of Phallyanthus
emlica L. and study effect on five different strains of each of gram-positive
bacteria Staphylococcus aureus (e-145, ML-366, ML-15, ML-276, ML-145)
and gram negative bacteria Salmonella typhi (A-2467, e-3404, E-145,
B-111, NCTC-14) by mean of MIC. Both of the bacteria show faster resisting
evolution which is a great threat on medical science (Chin et al., 2002;
Benoit et al., 2003; Abdullah et al., 2005and Chambers et al.,
1997). Astonishingly, the bacteria showed strain to strain MIC level
variation. The study also focuses on the potentiality of the phytochemical
extracts of leaf and bark of Amla for futuretherapeutic use.
2. MATERIAL AND METHODS:
2.1. Collection and identification of the plant
material:
Phyllanthus emblica L. (synonym: Emblica officinalis Gaertn.) was collected from the permanent site of
Indian Botanical Garden and certified by Central National Herbarium, office of
the Scientist – ‘D’, Botanical Survey of India, Ministry of Environment and
Forest, Howrah. CNH/TECH.II/2014/85/185 is the reference number of the
herbarium.In all experiments, fresh plant materials were started for processing
within 1-2 hours of collection and the extractions were done by cold maceration
technique for in this study.
2.2. Sample of plant material extraction
The protocol followed by Vogel et al., 988 was
followed for the extraction preparation. Each of the plant materials was
grinded and 10g of each was added to 100ml of distilled water and ethanol in
order to obtain water or Ethanol extract (100mg/ml). This crude extraction was
done at room temperature. After three days, mixture was filtered with 20 mesh
cheese cloth and was then further purified by Millipore membrane filter of
0.45μm pore-size and filtrate was evaporated.The sterile extract obtained
was stored in sterile capped bottles and refrigerated at 4şC until when required
for use.
2.3. Phytochemical screening
Preliminary analysis of chemical
composition of extracts was conducted following the standardized methods of
Sofoworaet al.,1993, Brain and Turner et al.,1975, Ushie and
Adamu et al.,2010 and Ushieet al.,2012, Trease and
Evan et al.,1989 to identify the presence of Secondary Metabolites
(Alkaloids, Anthraquinones, Flavonoids, Tannins, Saponins, Glycosides, Cardiac
glycosides, Terpenes) and to detect the presence of acidic compound, amino
acid, protein carbohydrate, alurone grains, cellulose, lignin; the experiments
suggested in Pharmacognosy Paperback was followed.
2.3.1. Test for acidic compound
Sodium bicarbonate was added to the
Alcoholic extracts of both of the extract in 1:1 ratio.
2.3.2. Test for alurone grain
To the sample few drops of alcoholic
trinitrophenol was added.
2.3.3. Test for Alkaloids
0.5 g of the extract was mixed with 2 (M)
aqueous hydrochloric acid (5.0 ml) with continuous stirring on a steam bath. In
a separate experiment 1.0 ml of the filtrate were added with a few drops of
Mayer’s reagent, Drangendoff’s reagent and Wagner s reagent. The resulting
solution was observed for colour changes.
2.3.4. Test for amino acid
Amino acid identification was done by
millon’s test.To the test solution about (2ml) of Millon’s reagent was added
and observed for colour change.
2.3.5. Test for carbohydrate
To the test solution few drops alcoholic
alpha naphthal was added, then few drops of concentrated sulphuric acid was
added through sides of test tube and observed for colour change.
2.3.6. Test for cellulose
To the sample extract, 0.1 (M) iodine
solution and sulphuric acid was added and observed for colour change.
2.3.7. Test for lignin
Saffranine solution was mixed to the
extract and observed for colour change.
2.3.8. Test for Volatile oil
To 1ml of extract Sudan III solution was
added and colour change was observed.
2.3.9. Test for Fixed oil
5 drops of sample with 1ml of 1% copper
sulphate solution was given.Then 10 % sodium hydroxide solution was mixed and
the colour change was noticed.
2.3.10. Test for Tannins
0.5 g of each of the plant extracts was
treated with distilled water (100 ml) and boiled for 5 minutes then cooled and
2.0 ml of the solution (filtrate) was mixed with a few drops of ferric chloride
was added. The colour change was to notice.
2.3.11. Test for Glycosides
A small part of each of the plant extracts
was taken in two separate test tubes and 0.1 M H2SO4 was
added to one and distilled water (5.0 ml) added to the other. Both of the test
tubes were heated for 45 minutes in water bath. The solutions were cooled and
alkalised with a solution of 2 (M)NaOH. Fehling solutions (5.0 ml) were added
in ratio1:1 to the two test tubes and waited for 3 minutes. The changes in
reaction were observed and recorded.
2.3.12. Test for Saponins (lipid test)
To determine the presence of saponins, the
emulsion test was followed. A small portion of each of the plant extracts was
added to distilled water (20 ml), boiled and filtered and the filtrate used for
the test. Two drops of olive oil was added to the frothing solution and shaken
vigorously. Any change in the result was observed.
2.3.13. Test for Anthraquinones
0.5 g of each of the plant extracts were
mixed and shaken with benzene (2.0 ml), and then 10% ammonia solution (4.0 ml)
was added. The consequent mixture was shaken and the reaction was observed.
2.3.14. Test for Flavonoids
Lead Acetate Test: 0.5g of each extract
dissolved in 5 ml of distilled water and mixed with 10 % of lead acetate
solution (1.0 ml). The colour formation was noted.
Iron (III) chloride. To a portion of 0.5 g
of the extract in water, two drops of iron (III) chloride was added and noted
for colour change.
2.3.15. Test for Terpenoids (Salkowski
test)
A solution of each of the extract was taken
in 0.5 g and dissolved in 2.0 ml of chloroform and concentrated H2SO4.
The presence of terpenes in the sample was detected as the colour changes.
2.3.16. Test for Waxes
To the test solutions, alcoholic alkali
solution was mixed with stirring and observed for any change.
2.3.17. Test for Starch
To a portion of the aqueous extract of both
plant extracts, weak aqueous iodine solution was added drop wise and the change
of the colouration was under observation.
2.3.18. Test for Protein
Small part of both of the extracts were
heated in a boiling water bath (100 ̊ C) for 10 minutes and noticed for
any coagulation.
2.4. Thin layer chromatography for identification of
Flavonoids:
For checking impurities, separating and for
identifying organic molecules by TLC (Fried and Sherma 1994) was done.
2.4.1. Preparation and activation of TLC Plates:
The slurry, which was a mixture of stationary phase
and water, was prepared by using the ratio 1:2 and 1:5 as required given in the
table below. After preparing the slurry, the TLC plates were prepared by using
the spreading technique.
2.4.2. Spreading technique:
The glass rod was used. The glass plates were staked
on the base plate. The slurry after preparation was poured over the plates and
the glass rod was allowed to roll over it. The thickness of the adsorbent layer
should be 0.25 – 2mm.After setting, the plates were activated by keeping it in
the oven for 1hr at 100şC – 120şC.
2.4.3. Application of sample Developing TLC chamber:
2-5µl of 1% solution test sample was spotted by
capillary tube on the plates. The spot was kept (2cm) above the base of the
plate. Now the TLC chamber was followed to develop by letting the solvent run
against the gravity for 30 minutes.
2.4.4. Detection:
Detection was under UV chamber at 254 nm.
2.4.5. Determination of Rf value:
The Rf value was calculated for identifying the spots
in qualitative analysis.It is the ratio of distance travelled by the solute to
distance travelled by the solvent front.
2.4.6. TLC experiments on different mobile phases for
flavonoids:
Maintaining the stationary phase as Silica gel 60F254
with different mobile phases viz. given in the table:
Table 1: Diferent ratios of mobile phases
|
STATIONARY
PHASE
|
MOBILE
PHASES
|
|
Silicagel
60F254
|
Ethyl
acetate : formic acid : glacial acetic acid : water(100:11:11:27)
|
|
Chloroform
: acetone : formic acid (75:16.5:8.5),
|
|
Chloroform
: ethyl acetate(60:40)
|
|
Benzene
: pyridine: formic acid (72:18:10),
|
|
Toluene
: ethyl formate : formic acid (50:40:10
|
|
n-Butanol:glacial
acetic acid : water (40:10:50)
|
2.5. Determination of total ash value and acid soluble
ash value:
·
Total ash value
A silica or platinum crucible was heated to red hot
for 30 minutes, allowed to cool in a desiccator and weighed. Unless otherwise
specified in the individual monograph, weigh accurately about 1gm of the
substance being examined and evenly distributed it in the crucible. Drying it
at 100̊C to 105̊C for 1hr and was ignited to constant weight in a
muffle furnace at 600̊C. The crucible was cooled in a desiccator after
each ignition. Now the percentage of ash was calculated with reference to the
air dried substance.
·
Acid insoluble ash
value
The ash was boiled with 25 ml of 2(M)HCl acid for
5minutes and the insoluble matter was collected in a gooch crucible and washed
with hot water, ignited, cooled in a desiccator and weighed.The percentage of
acid insoluble ash was calculated.
2.6. Determination of extractive values:
·
Water soluble
extractive value
5gm of the air dried powdered leaf and stem bark macerated
with 100ml of water closed flask for 24hrs, shaking was done frequently during
the first 6 hrs and allowed to stand for 18hrs. 25ml of filtrate was evaporated
to dry in a tared flat bottom shallow dish at 105̊C and weighed. The
percentage of water soluble extractive value was calculated.
·
Ethanol soluble
extractive value
5gm of the air dried plant material, coarsely powdered
have to be macerated with 100ml of ethanol of a specified strength in a closed
flask for 24hrs, shaking frequently during the first 6 hrs and allowing to
stand for 18hrs.Therefore filter rapidly taking precautions against loss of
water, 25ml of filtrate was evaporated in a tared flat bottom shallow dish at
105̊C and weighed. The percentage of ethanol soluble extractive value was
calculated.
2.7. FTIR analysis of the extracted sample
The Fourier Transport Infrared Spectroscopy was done
for structural elucidation and identification of the functional group. The wave
number and the relative strength were observed.The functional groups and their
corresponding wavenumbers noted.
2.8. Microbial inhibitory potentiality test
2.8.1. Test Organisms
The pure clinical isolates of the five
strains of Staphylococcus aureus, e-145, ML-366, ML-15, ML-276, ML-145
and another five strains of salmonella typhi, A-2467, e-3404, E-145,
B-111, NCTC-14 were obtained from Bengal School of Technology (a college of
Pharmacy), Sugandha, Delhi road, Hooghly under West Bengal University of
Technology, Bidhannagar, india. All the isolates were checked for purity and
cultured in nutrient broth(Hi-media, M002) at 37 ̊ C and then maintained
on Nutrient agar slant at 40 ̊ C in the refrigerator until required for
use.
2.8.2. Preparation of the Media
Two media, Nutrient agar (NA) and Nutrient broth (NB)
both of Hi-media, M002 were prepared in order to evaluate the MIC and Bacterial
inoculums preparation. The media was prepared by dissolving 28 g of NA in 1 L
of distilled water on the other hand 50 g of Nutrient broth was dissolved in 1
L of distilled water .They were autoclaved at 121şC for 20 mins and
subsequently allowed to cool to about 45şC (temperature at which the agars
remains molten) and poured in plate (Petri dishes) allowed for gel or
solidification and broth is used for inoculums preparation.
2.8.3. Standardization of Inoculums
10 test organisms were sub-cultured in nutrient broth
using a wire loop (done aseptically) and incubated for 24 hr at 37şC in an
incubator. The growth of the micro-organisms in the broth by the turbidity
produced was adjusted to match 0.5 McFarland standards (108cfu/ml).
2.8.4. Inoculation of the Plates and
Application of the Extracts
The nutrient agar plates were inoculated with small
volume (0.05 ml to 0.10ml) of liquid sub-cultured nutrient broth inoculums with
a “spreader” in such a way that the surface of the agar in the plates were
covered with bacteria and thus total eleven plates were arranged for ten
bacterial strains and one control. The plant extracts were diluted 103
fold using dilution method for 10mg, 25mg, 50mg, 100mg, 150mg, 250mg and 300mg
doses with appropriate labels. In wells (hole) diluted plant extract was added.
Ethanol was also added in a well as control. The inoculated plates were kept in
the incubator at 37şC for about an hour to allow the extracts diffuse into the
agar. The NA (nutrient agar) was again aerobically incubated at 37ş C for 23
hr. The growth was observed.
3. RESULT AND DISCUSSION:
3.1. Phytochemical screening
Plant material screening was done by mean of sieving
and separating the hard polymerised materials (like lignin, cellulose, etc.),
thus mainly the ethanol dissolvable materials were screened as shown in Table
2. The both extracts responded about in similar way is all the experiments. The
flavonoid content was further screened by TLC method. The phytochemical extract
of ethno medical plant P. emblica was proved worthy onto the traditional
uses for anti- inflammatory action (Summanen et al.,1999).
The extract when applied on the S. typhi, the
bark extract was seen to be more potent as antimicrobial compound as 25µg/ml concentration prevented growth in
most of the cases and 50µg/ml concentration was effective in case of E- 3404
strain. The leaf extract effectively inhibited the growth in a concentration of
100µg/ml but 250µg/ml for the strain A- 2467. The antibiotics resistance to an antimicrobial agent
was almost impossible when application is done in a repeated manner (Ekhaise
and Okoruwa et al., 2001). Therefore, phytochemical extract of these
types of folklore plants could be significant ethno botanically for the use of
these antimicrobial purposes.
4. CONCLUSION:
The preliminary phytochemical studies revealed the
presence of flavonoids, tannins and fixed oil in ethanolic extract of the
leaves and bark obtained from Phyllanthus emblica. Bioactive component
present therein was separated out in TLC plate. Where mostly we found different
types of flavonoids. The study also showed antimicrobial activity of ethanolic
plant extract of Phyllanthus emblica against many strains of both gram
positive and gram negative bacteria and thus it makes this plant worthy to be
used as antimicrobial drug undoubtedly . But for which specific component it is
showing its antimicrobial effect is not revealed hence in future, science we
could focus on this unknown component. The results also indicated that
scientific studies carried out on medicinal plants having traditional claims of
effectiveness might warrant fruitful results.
5. ACKNOWLEDGEMENT:
The authors are grateful to the authorities of Bengal
School of Technology, College of Pharmacy, Chinsurah, West Bengal and Indian
Statistical Institute, Kolkata.
6. CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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