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Phyllanthus maderaspatensis
149
CHAPTER – 6
PHYLLANTHUS MADERASPATENSIS
6.1 Introduction
Phyllanthus maderaspatensis is commonly called as Kanocha in hindi while in english it
is known as Madras leaf flower. Phyllanthus has about 750-800 species, found in tropical
and sub-tropical regions. P. maderaspatensis, a traditional Indian herb is known from the
flora area from 19th-century collection from Hong Kong. It is also found in drier regions
as well. The Phyllanthus genus is a source of various plant chemicals secondary
metabolites like alkaloid, flavonoid, lignin, phenol, tannin and terpene. Many of the active
constituents contributed to the biologically active lignin, glycosides, flavonoids, alkaloids,
ellagilannins and phenyl propanoids that are found in the leaf, stem and roots of the plant.
Among the lipids were sterols and flavonols found in the plant (Annamalai, 2012).
6.2 Description of the Plant
The plant Phyllanthus maderaspatensis is herbaceous in nature. It is perennial but often
flowering takes place in the first year. The plant is 0.15-0.9 meters tall, monoecious in
nature and glabrous throughout. The stem is usually woody at the base with many
branches. The leaves are spiral with oval stipules measuring 1.5-2 mm. The petiole is very
short and the leaf blade varies from linear-lanceolate to obovate measuring 10 – 30 mm ×
2 - 7 mm. The leaves appear to be leathery, grayish green with rounded base and the apex
is short and acuminate. The leaf has lateral veins which are obscure. The inflorescences
are axillary fascicles with most axils are having one female flower, while 1 to 4 male
flowers are placed distally. The male flowers are pedicellate with 6 sepals that are
suborbicular with entire margins having 3 small and smooth stamens. The filaments of
stamens are connate at base and the anthers are dehiscent longitudinally. The female
flowers are also pedicellate with 6 sepals, suborbicular to subspatulate and entire margins;
which can be seen in berries as well. The ovary is globose 3-celled and smooth. There
are3 styles which are free. The berries is a capsule, oblate measuring 1.2 - 2 × 3 mm, olive
green in colour and has smooth texture. The seed is three-sided, 1.3 mm in size, light
Phyllanthus maderaspatensis
150
Figure. 6.1. Phyllanthus maderaspatensis
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Order : Euphorbiales
Family : Euphorbiaceae
Genus : Phyllanthus
Species : maderaspatensis
Phyllanthus maderaspatensis
151
brown in colour, and has rows of minute tubercles on back and sides (Chen, 2008).
The parts of the plant used in the present study are the seeds. Morphologically, the seed is
light brown, with rows of minute tubercles on back and sides. The seeds appear rounded
from distance but are three-sided on careful examination. The odour is very soft and mild.
The taste of the seeds is bad and carminative (Rani and Raju, 2014).
6.3 Geographical Distribution
Phyllanthus maderaspatensis is a native of Africa (Angola, Botswana, Cameroon, Cote
d’Ivoire, Egypt, Ethiopia, Ghana, Kenya, Madagascar, Mali, Mozambique, Namibia,
Niger, Nigeria, Senegal, Seychelles, Republic of South Africa, Sudan, Swaziland,
Tanzania, Togo, Uganda, and Zimbabwe) and Asia (Burma, India, Male Atoll, Saudi
Arabia, Sri Lanka, and Yemen). It has naturalized in Indonesia, the Philippines, and
Australia (NGRP, 2002; Chaudhary and Akram, 1987; Holm et. al., 1979; Hutchinson
et. al., 1958; Moody, 1989; Reed, 1977; Tackholm, 1974 and Wells et. al., 1986). The
eastern part of Europe which comes under the Russian Federation also inhabits the plant
(NGRP, 2002).
The P. maderaspatensis usually grows in deciduous wood land, wooded savanna and
grass land, on beaches and dunes, and also near streams and ponds in cultivated and
distributed localities, from sea level up to 1400 m altitude. The plant grows on almost all
kind of soils. Among all kinds of soil it likes heavy clay and alluvial soils of low altitude
river valleys on river banks and in flood plains. It is usually found in calcareous sites in
humid tropical areas (Annamalai, et. al., 2012).
6.4 Ethno-botanical Importance
Phyllanthus species are used since ancient times in folk medicine, particularly for treating
liver disorders and urinary tract infections (Calixto et. al., 1998). The extract of stems and
leaves of Phyllanthus maderaspatensis are used as a hepatoprotective agent (Asha et. al.,
2007), headache, bronchitis, ear ache, ophthalmia, griping, cough, ascites, incipient,
blindness, sores, ulcers, stomachache, inflammations, intestinal spasms, gonorrhoea,
anti-microbial and viral infections (Annamalai et. al., 2011 and Munshi et. al., 1993).
Traditional healers use the plant extract in treating fever and burns. The drug is reported
Phyllanthus maderaspatensis
152
for its chemoprotective (Munshi et. al., 1993), anti-edematic, anti-dysenterial, laxative,
carminative, diuretic and immunomodulatory effects (Sharma et. al., 2011).
The extracts prepared from the seeds of P. maderaspatensis are used for the treatment of
cough, ear ache, inflammations, intestinal spasms, sores, hard swellings, ulcers, stomach
ache. The decoctions prepared by the seed of P. maderaspatensis are also given in
gonorrhoea, internal inflammations dysentery are diarrhoea (Annamalai, et. al., 2012).
The extracts of P. maderaspatensis are rich in maderin, mucilage, essential oil and
β- Sitosterol, while the seeds also contain long chain fatty acids. It has deep yellow
coloured oil was extracted from the seeds of P. maderaspatensis. The de-fatted seed cake
contains mucilage, which yields galactose, arabinose, rhamnose and aldobionic acid
(Komuraiah et. al., 2009), niruriside (Schmelzer et. al., 2008), phyllanthin,
hypo-phyllanthin (Steward et. al., 1999) and cinnamoyl sucrose acetate (Qian-Cutrone
et. al., 1996).
The powder of dried plant material mixed with milk is known to treat jaundice, an herbal
medicine called Bhumyamalaki is sold which may be pure P. amarus or pure Phyllanthus
maderaspatensis L. or a mixture with P. fraternus (Gabriella, 2008).
The decoction of plant sap and leaves is full of emetic and purgative activities. In
Tanzania the whole plant is pounded and the solution applied for treatment of scabies. A
root decoction is taken to cure constipation, diarrhea, lack of appetite, intestinal pain,
menstrual problems, gastrointestinal disorders, testicular swelling, chest complaints and
snake bites. The gastrointestinal trouble in infants is usually treated by giving them a root
decoction of Phyllanthus maderaspatensis and Chamaecrista mimosoides. The plant sap
is used as nose drops to treat tooth ache. The ground leaves are usually rubbed on the skin
with lemon juice as treatment for rheumatism. In Niger the plant is used as an aphrodisiac.
In Somalia, P. maderaspatensis is considered poisonous (Schmelzer and Gurib-Fakir,
2008).
In India, P. maderaspatensis is widely used as an effective hepato-protective agent in the
indigenous systems of medicine and are considered bitter, astringent, stomachic, di-uretic,
febrifuge, de-obstruant and antiseptic. It is medicinally used to treat headache, bronchitis,
ear ache and ophthalmia (Rani and Raju, 2014). The powder from dried plant material
Phyllanthus maderaspatensis
153
mixed with milk is used to treat jaundice (Schmelzer and Gurib-Fakir, 2008).
In Kenya, the smoke from the burning plants is used to kill caterpillars in maize. The
ethanolic extract of P. maderaspatensis is a popular south Indian dietary supplement. It
has been studied for its chemoprotective property on adriamycin (ADR)-induced toxicity
and oxidative stress in mice (Bommu, et. al., 2008). The plant has been studied for
chemo-protective effect in modulating cisplatin-induced nephrotoxicity and genotoxicity
in Swiss albino mice (Chandrasekar, 2006).
In one of the study the methanolic extract of P. Maderaspatensis showed the presence of
tannins, triterpenoids, flavonoids, proteins and carbohydrates and also showed cytotoxic
potentials (Ravichandan 2012).
6.5 Determination of Extraction Yield of Seeds Extract (% yield)
The initial weight of 30 gms of the dried seeds was taken in 100 ml of methanol. In
methanolic extracts the percentage yield obtained in the seeds of Phyllanthus
maderaspatensis was 3.30 percent. The percentage yield of extracts of seeds of
P. maderaspatensis in methanol is given in Table 6.1.
S.No. Plant name
Weight of
dried
seeds
W0 (gm)
Weight of
empty
petri plate
W1 (gm)
Weight of
petri plate
with plant
extract
W2 (gm)
Percentage
yield (%)
1. Phyllanthus
maderaspatensis 30 gm 41.800gm 42.790 gm 3.3
Table No.6.1 The percentage yield of methanolic seed extracts of P.maderaspatensis,
extraction done by soaking dried plant material in methanol and extract
separation using distilling apparatus.
6.6 Total Phenolics Estimation of Seeds Extracts
The total phenolic content found in the seeds extract of Phyllanthus maderaspatensis was
estimated spectrophotometrically using the Folin-Ciocalteu Reagent at 765 nm.
A calibration curve was drawn using Gallic Acid which was used as standard. The level of
gallic acid in the methanolic extract of seeds extract was measured. The observed
Phyllanthus maderaspatensis
154
concentration was multiplied with dilution factor. The result was reported as Gallic Acid
Equivalent, (GAE) in mg/g of dry mass.
The gallic acid is a stable substance which is pure in nature and it is easily available. Since
this assay measures all phenolics, gallic acid is taken as standard in the study. The stability
of gallic acid standard solutions was also tested and it shows that it loses less than 5% of
their value over two weeks when refrigerated and kept tightly closed (Waterhouse, 1999
and 2009). The GAE for P.\maderaspatensis methanolic seed extract was estimated to be
8.48 GAE/g. The standard calibration curve is shown in Figure 6.2.
6.7 Tannins Estimation of Seeds Extracts
The total tannins content was also estimated spectrophotometrically at 765 nm using
Folin-Denis Reagent here tannic acid was used as standard. The total phenolic content
was expressed as mg/g tannic acid equivalents per gram, (TAE) expressed in mg/g of dry
mass using the following equation based on the calibration curve:
y = 0.002x + 0.98, R² = 0.979
Figure No. 6.2: Calibration curve for gallic acid for determining the phenolic
content.
y = 0.001x + 0.147R² = 0.979
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 100 200 300 400 500
Abso
rban
ce
Concentrtion of gallic acid (µg/ml)
GALLIC ACID STANDARD
Absorbance
Linear (Absorbance)
Phyllanthus maderaspatensis
155
Figure No. 6.3: Calibration curve for tannic acid for determining the
tannin content.
The experiment was replicated thrice and average data recorded for quality assurance. The
TAE for methanolic seed extract of P. maderaspatensis was estimated to be 2.37 TAE/g.
The standard calibration curve is shown in Figure 6.3.
6.8 Phyto-chemical Analysis of Seed Extract of Phyllanthus maderaspatensis
The phyto-chemical analysis involves the qualitative analysis of herbal plants. The
preliminary qualitative tests have been attempted in Phyllanthus maderaspatensis seeds to
find out the presence or absence of certain bio active compounds. The chemical tests were
carried out on the crude methanolic extract using standard procedures to identify the
active constituents.
The crude methanolic seed extract of Phyllanthus maderaspatensis were evaluated
qualitatively to analyze the presence of secondary metabolites. The secondary metabolites
present in the crude methanolic extract of seeds are anthraquinones, flavonoids,
phlobatannins, glycosides, saponins, tannins and terpenoids. The results of qualitative
phyto-chemical analysis are given in Table 6.2.
y = 0.002x + 0.98
R² = 0.979
0
0.5
1
1.5
2
2.5
0 100 200 300 400 500
Abso
rban
ce
Concentrtion of tannic acid (µg/ml)
TANNIC ACID STANDARD
Absorbance
Linear (Absorbance)
Phyllanthus maderaspatensis
156
The presence of alkaloid was analysed by using methanolic extract of seeds with
Wagner’s method. The presence of reddish brown coloured precipitate indicates the
presence of alkaloids. When the methanolic seeds extract of P. maderaspatensis was
evaluated using this assay, the methanolic extract showed the absence of alkaloids.
The Borntrager’s test was performed for the analysis of anthraquinones in the methanolic
seed extract. The formation of rose pink colour in plant extract confirmed the presence of
anthraquinones. The methanolic seeds extract when tested using this assay confirmed the
appearance of pink colour indicating the presence of anthraquinones.
The presence of flavonoids in the crude plant extract is determined quantitatively, the
appearance of yellow colour is the positive indication for the presence of flavonoids in
them. When the crude methanolic seeds extract of P. maderaspatensis was evaluated
using this test the appearance of yellow colour indicates the presence of flavonoids.
The phlobatannins presence was evaluated qualitatively by adding 1% of aqueous HCl in
boiled crude methanoilic extract of P. maderaspatensis seed, the presence of red colour
indicates a positive result. The crude methanolic extracts of P. maderaspatensis seed
showed the presence of red colour indicating the presence of phlobatannins.
The presence of glycosides in the P. maderaspatensis methanolic seeds extract evaluated
using the Fehling’s test. The brick red precipitate formation indicates the presence of
glycosides. P. maderaspatensis methanolic seeds extract showed the presence of brick red
precipitate thus confirming the presence of glycosides.
Similarly the presence of saponins in the plant extract evaluated using a frothing test. The
formation of froth confirmed the presence of saponins. The P. maderaspatensis seeds
extract showed the appearance of froth indicating the presence of saponins.
The P. maderaspatensis crude methanolic seed extract were also evaluated for the
presence of steroids by using the Salkwoski test. The change of colour from violet to blue
indicates a positive result. The crude methanolic seeds extract did not show the change in
colour indicating the absence of steroids.
The crude seed extract was further tested for the presence of tannins by using ferric
chloride test. The occurrence of blue-black precipitate indicates the presence of tannins.
Phyllanthus maderaspatensis
157
The P. maderaspatensis methanolic seeds extract showed the formation of blue black
precipitate thus confirming the presence of tannins.
Similarly, Salkowski test was also performed to evaluate the presence of terpenoids in
P. maderaspatensis crude methanolic seeds extract. The formation of reddish brown
colour indicates the presence of terpenoid. The seeds extract confirmed the presence of
terpenoids as the reddish brown colour appeared in it.
Table No. 6.2: Phyto-chemical analysis of seeds extract of Phyllanthus
maderaspatensis
6.9 Anti-oxidant Activities of Seeds Extract of Phyllanthus maderaspatensis
The anti-oxidant activity of crude methanolic seed extract of P. maderaspatensis was
determined in vitro using a number of assays such as super oxide scavenging activity by
alkaline DMSO method, DPPH free radical scavenging activity, nitric oxide free radical
scavenging activity, H2O2 radical scavenging activity and by total anti-oxidant capacity
method.
S.No. Active principle Phyto-chemical Analysis Result
1. Alkaloids Wagner’s Test -
2. Anthraquinones Borntrager’s Test +
3. Flavonoids Sodium Hydroxide (NaOH) Test +
4. Phlobatanins Hydrocloric Acid (HCl) Test +
5. Glycosides Fehling’s Test +
6. Saponins Frothing Test +
7. Steroids Salkwoski Test -
8. Tannins Ferric chloride (FeCl3) Test +
9. Terpenoids Salkwoski Test +
Phyllanthus maderaspatensis
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6.9.1 Scavenging of Superoxide Radical with the Alkaline DMSO (dimethyl
sulfoxide) Method
The superoxide radical scavenging assay, were studied in methanolic crude seeds
extracts at different concentrations ranging from 1.95 to 1000 µg/ml and
absorbance measured at the wavelength of 560 nm. The results are given as
percentage inhibition values of the extracts. The increase in percentage showed
stronger inhibition and highest scavenging activity of the plant extract.
The percentage inhibition values of seed extract of P. madacarpetensis were found
to range between 52.10 ± 0.00 and 1.15 ± 0.493 percent at the concentration of
1000 and 1.95 µg/ml respectively. Whereas the percentage inhibition values of
BHT were found to be 63.52 ± 0.020 and 6.63 ± 0.229 percent at the concentration
of 1000 and 1.95 µg/ml respectively. The percentage inhibition values of
P. madacarpetensis extracts along with standard (BHT) at different concentrations
are shown in Table 6.3 and 6.4 and Figure 6.4.
The methanolic extract of P. maderaspatensis scavenges super oxide radical and
thus inhibits formazan formation. The results obtained increase scavenging of
superoxide radicals in dose dependent manner due to the scavenging ability of the
P. maderaspatensis methanolic crude extract. The IC50 value was found to be
890.53 ± 6.10 μg/ml, whereas the IC50 value of BHT was found to be 792.49 ±
1.16 μg/ml.
6.9.2 Nitric Oxide Free Radical Scavenging Activity
The seed of P. maderaspatensis methanolic crude extracts were evaluated using
the nitric oxide free radical scavenging activity. The standard used for the study
was butylated hydroxytoluene (BHT). The methanolic seeds extract of
Phyllanthus. maderaspatensis showed significant scavenging activity, and the
percentage inhibition ranges between 90.61 ± 0.146 and 0.707 ± 0.305 percent, at
the concentration of 1000 µg/ml and 1.95 µg/ ml respectively, whereas the
percentage inhibition values of BHT were found to be 56.44 ± 0.113 and 1.90 ±
0.380 percent, at the concentration of 1000 µg/ml and 1.95 µg/ml respectively.
The nitric oxide radical scavenging activity values of the methanolic seed extracts
Phyllanthus maderaspatensis
159
along with standard (BHT) at different concentrations are given in Table 6.5 and
6.6 and Figure 6.5. The high percentage inhibition indicates high scavenging
activity of the plant extract. The IC50 value of P. maderaspatensis extract was
52.33 ± 0.30 μg/ml, whereas the IC50 value of BHT is 364.60 ± 3.51 μg/ml.
6.9.3 Scavenging of Radical with the H2O2 (Hydrogen peroxide) Method
The hydrogen peroxide is not a strong oxidizing agent. It can cause inactivation of
some enzymes directly, by oxidation of the thiol (-SH) groups. It can easily cross
cell membrane rapidly. Once reached inside the cell, H2O2 can possibly reacts with
Fe2+
and possibly Cu2+
to form hydroxyl radical. The formation of hydroxyl
radical is the initial step of the formation of many toxic effects (Miller et. al.,
1993). It is therefore very important and necessary for the cells to control the
production of hydrogen peroxide which was built up in vivo. The scavenging of
H2O2 attributes to their phenolic content which donate electrons to H2O2, thus was
neutralizing it to water (Halliwell and Gutteridge, 1985). The ability of the extract
to effectively scavenge hydrogen peroxide, determined according to the method
done by Ruch et. al. (1989), where they were compared with BHT. The
P. maderaspatensis extracts were capable of scavenging hydrogen peroxide in a
concentration dependent manner.
The methanolic seeds extract exhibited 66.00 ± 0.867 and 2.25 ± 0.604 percent
inhibition at the concentration of 1000 and 1.95 μg/ml respectively, by hydrogen
peroxide anti-oxidant method. On the other hand, using the same concentration of
butylated hydroxy toluene exhibited 77.03 ± 0.128 and 4.14 ± 0.128 percent
inhibition respectively, by hydrogen peroxide scavenging activity. The percentage
inhibition values of methanolic seed extracts and standard (BHT) have shown in
Table 6.7 and 6.8 and Figure 6.6. The IC50 value of P. maderaspatensis was found
to be 236.02 ± 4.681 μg/ml. whereas the IC50 value of BHT was found to be
26.16 ± 0.351 μg/ml.
6.9.4 Anti-oxidant Activity by DPPH (2, 2 – Diphenyl – 1- Picryl Hydrazyl)
Radical Scavenging Assay
The DPPH radical scavenging assay showed the ability of the extracts and the
standard to scavenge DPPH free radicals. The DPPH radical exists naturally in
Phyllanthus maderaspatensis
160
deep violet colour but when reacts with anti-oxidant it turn into a yellow coloured
di-phenyl picryl hydrazine. The degree of discoloration indicates the
radical-scavenging potential of the anti-oxidant (Tirzitis and Bartosz, 2010).
The results are shown as percentage inhibition values of the extracts at different
concentrations ranging from 1.95 to 1000 µg/ml. The crude methanolic extracts of
P. maderaspatensis seeds gave percent inhibition of 73.82 ± 0.390 and
1.14 ± 0.200 when tested at the concentration of 1000 and 1.95 µg/ml respectively,
of the plant extract which was compared with BHT, used as standard having
percent inhibition of 73.03 ± 0.128 and 12.59 ± 0.128, at the concentration of 1000
µg/ml and 1.95 µg/ml respectively. The DPPH radical scavenging activity values
of the methanolic extracts along with standard (BHT) have shown in Table 6.9 and
6.10 and Figure 6.7. The percentage inhibition indicates scavenging activity of the
plant extract. The IC50 value of P. maderaspatensis is 200 ± 2.19 μg/ml, whereas
the IC50 value of BHT was found to be 43.40 ± 1.307 μg/ml.
6.9.5 Total Anti-oxidant Capacity by Phosphomolybdenum Method
The total anti-oxidant capacity of the crude methanolic plant extracts and BHT
were determined by using the method of total anti-oxidant capacity by
phosphomolybdenum. The higher absorbance value indicates the greater
anti-oxidant activity. The total anti-oxidant capacity of plant extracts were
measured spectrophotometrically at 695 nm using phosphomolybdenum method,
which is based on the reduction of Mo (IV) to Mo (V) by the test sample and the
formation of green phosphate/Mo (V) compounds (Abbasi et. al., 2010). A high
absorbance value of the sample indicates its strong anti-oxidant activity. The total
anti-oxidant capacity may be contributed due to their chemical composition and
phenolic acid content.
The percentage inhibition values of the seed extracts were found to be
42.90 ± 0.171 and 0.18 ± 0.000 at the concentrations of 1000 and 1.95 µg/ml.
The percentage inhibition values of (standard) BHT were found to be
77.12 ± 0.322 and 20.10 ± 0.207 percent respectively, at the concentration of
1000 µg/ml and 1.95 µg/ml. The values of the methanolic extracts and standard
(BHT) of total anti-oxidant capacity by phosphomolybdenum method were
Phyllanthus maderaspatensis
161
given in Table 6.11 and 6.12 and Figure 6.8. The IC50 value of BHT was found
to be 124.25 ± 3.04 μg/ml.
6.10 IC50 Value of Different Anti-Oxidant Activity
The IC50 values of the methanolic extracts were calculated based on the results of different
anti-oxidant assay conducted such as DPPH, Alkaline DMSO, Nitric oxide scavenging
assay, total anti-oxidant assay and hydrogen peroxide method. The results are given below
in Table 6.13.
Table No. 6.3: The scavenging effect of methanolic seeds extract of
P. maderaspatensis by Alkaline DMSO method. The different
concentrations of extracts used from 1000 to 1.95 µg/ml. The
data represent the percentage alkaline DMSO inhibition.
Values are expressed as mean ± SD (n=3).
S.No. Plant conc. (μg/ml) P. maderaspatensis
(Absorbance)
Percent Inhibition
(%)
1. 1000 0.238 ± 0.000 52.10 ± 0.00
2. 500 0.198 ± 0.001 42.43 ± 0.29
3. 250 0.177 ± 0.001 35.59 ± 0.363
4. 125 0.154 ± 0.001 25.97 ± 0.480
5. 62.5 0.141 ± 0.001 19.14 ± 0.573
6. 31.25 0.130 ± 0.000 12.53 ± 0.386
7. 15.625 0.126 ± 0.001 9.52 ± 0.718
8. 7.8125 0.121 ± 0.000 6.29 ± 0.445
9. 3.906 0.117 ± 0.000 2.84 ± 0.476
10. 1.95 0.115 ± 0.000 1.15 ± 0.493
Phyllanthus maderaspatensis
162
Table No. 6.4: The scavenging effect of BHT by Alkaline DMSO method. The
different concentrations of extracts used from 1000 to 1.95
µg/ml. The data represent the percentage alkaline DMSO
inhibition. Values are expressed as mean ± SD (n=3).
Table No. 6.5: The nitric oxide radical scavenging activity of methanollic seeds
extract of P. maderaspatensis. The different concentrations of
extracts used from 1000 to 1.95 µg/ml. The data represent the
percentage nitric oxide inhibition. Values are expressed as mean
± SD (n=3).
S.No. Plant conc.
(μg/ml)
Butylated
Hydroxytoluene
(Absorbance)
Percent
Inhibition
(%)
1. 1000 1.041 ± 0.000 63.52 ± 0.020
2. 500 0.549 ± 0.000 30.82 ± 0.072
3. 250 0.532 ± 0.000 28.61 ± 0.077
4. 125 0.526 ± 0.001 27.75± 0.137
5. 62.5 0.488 ± 0.001 22.13± 0.159
6. 31.25 0.479 ± 0.001 20.66 ± 0.165
7. 15.625 0.461 ± 0.001 17.62 ± 0.206
8. 7.8125 0.435 ± 0.000 12.71 ± 0.115
9. 3.906 0.422 ± 0.001 9.95 ± 0.213
10. 1.95 0.407 ± 0.001 6.63 ± 0.229
S.No. Plant conc.
(μg/mi)
P. maderaspatensis
(Absorbance)
Percent
Inhibition
(%)
1. 1000 0.353 ± 0.001 90.61 ± 0.146
2. 500 0.06 ± 0.001 84.07 ± 0.244
3. 250 0.110 ± 0.001 70.79 ± 2.65
4. 125 0.122 ± 0.001 67.61 ± 0.269
5. 62.5 0.177 ± 0.000 53.00 ± 0.072
6. 31.25 0.211 ± 0.000 43.80 ± 0.273
7. 15.625 0.3 ± 0.001 20.17 ± 0.444
8. 7.8125 0.322 ± 0.01 14.51 ± 0.295
9. 3.906 0.35± 0.001 7.07 ± 0.158
10. 1.95 0.374 ± 0.000 0.70 ± 0.305
Phyllanthus maderaspatensis
163
Table No. 6.6: The nitric oxide radical scavenging activity of standard. The
different concentrations of standards used from 1000 to 1.95 µg/ml.
The standard used was butylated hydroxytoluene. The data represent
the percentage nitric oxide inhibition. Values are expressed as mean
± SD (n=3).
Table No. 6.7: The hydrogen peroxide radical scavenging activity of methanollic
seeds extract P. maderaspatensis. The different concentrations of
extracts used from 1000 to 1.95 µg/ml. The data represent the
percentage hydrogen peroxide inhibition. Values are expressed
as mean ± SD (n=3).
S.No. Plant conc. (μg/ml) Butylated Hydroxytoluene
(Absorbance)
Percent Inhibition
(%)
1. 1000 0.011 ± 0.000 56.44 ± 0.113
2. 500 0.121 ± 0.001 53.63 ± 0.4997
3. 250 0.138 ± 0.000 46.87 ± 0.111
4. 125 0.144 ± 0.001 44.82 ± 0.332
5. 62.5 0.151 ± 0.001 42.14 ± 0.604
6. 31.25 0.176 ± 0.001 32.56 ± 0.641
7. 15.625 0.203 ± 0.001 22.22 ± 0.681
8. 7.8125 0.212 ± 0.001 18.77 ± 0.352
9. 3.906 0.222 ± 0.001 14.94 ± 0.358
10. 1.95 0.257 ± 0.001 1.90 ± 0.380
S.No. Plant conc.
(μg/ml)
P. maderaspatensis
(Absorbance)
Percent Inhibition
(%)
1. 1000 0.085 ± 0.002 66.00 ± 0.867
2. 500 0.101 ± 0.001 59.76 ± 0.497
3. 250 0.121 ± 0.001 51.52 ± 0.575
4. 125 0.155 ± 0.001 38.24 ± 0.348
5. 62.5 0.183 ± 0.001 26.95 ± 0.326
6. 31.25 0.206 ± 0.001 17.92 ± 0.630
7. 15.625 0.214 ± 0.002 14.74 ± 1.136
8. 7.8125 0.221 ± 0.002 11.68 ± 1.112
9. 3.906 0.236 ± 0.002 5.84 ± 1.193
10. 1.95 0.245 ± 0.001 2.25 ± 0.604
Phyllanthus maderaspatensis
164
Table No. 6.8: The hydrogen peroxide radical scavenging activity of standard. The
different concentrations of standard used from 1000 to 1.95 µg/ml.
The standard used was butylated hydroxytoluene. The data represent
the percentage hydrogen peroxide inhibition. Values are expressed as
mean ± SD (n=3).
Table No. 6.9: The DPPH radical scavenging activity of methanollic seeds extract
P. maderaspatensis. The data represent the percentage inhibition
values. The different concentrations of extracts used from 1000 to
1.95 µg/ml. Values are expressed as mean ± SD (n=3).
S.No. Plant conc.
(μg/ml)
Butylated
hydroxytoluene
(Absorbance)
Percent Inhibition (%)
1. 1000 0.103 ± 0.005 77.03 ± 0.128
2. 500 0.119 ± 0.000 73.48 ± 0.128
3. 250 0.136 ± 0.000 69.70 ± 0.128
4. 125 0.167 ± 0.001 62.88 ± 0.222
5. 62.5 0.200 ± 0.001 55.55 ± 0.222
6. 31.25 0.217 ± 0.001 51.77 ± 0.222
7. 15.625 0.241 ± 0.000 46.37 ± 0.128
8. 7.8125 0.313 ± 0.001 30.37 ± 0.339
9. 3.906 0.380 ± 0.001 15.55 ± 0.222
10. 1.95 0.431 ± 0.000 4.14 ± 0.128
S.No. Plant conc. (μg/ml) P. maderaspatensis
(Absorbance)
Percent Inhibition
(%)
1. 1000 0.076 ± 0.001 73.82 ± 0.390
2. 500 0.109 ± 0.001 62.45 ± 0.282
3. 250 0.132 ± 0.000 54.42 ± 0.256
4. 125 0.164 ± 0.000 43.39 ± 0.273
5. 62.5 0.186 ± 0.002 35.70 ± 0.593
6. 31.25 0.203 ± 0.001 29.96 ± 0.396
7. 15.625 0.220 ± 0.000 23.99 ± 0.179
8. 7.8125 0.241± 0.000 16.87 ± 0.335
9. 3.906 0.261 ± 0.001 10.10 ± 0.506
10. 1.95 0.287 ± 0.001 1.14 ± 0.200
Phyllanthus maderaspatensis
165
Table No. 6.10: The DPPH radical scavenging activity standard. The different
concentrations of extracts used from 1000 to 1.95 µg/ml. The
standard used was butylated hydroxytoluene. Values are
expressed as mean ± SD (n=3).
Table No. 6.11: The total anti-oxidant activity by phosphomolybdenum method
of methanollic seeds extract of P. maderaspatensis. The
different concentrations of extracts used from 1000 to 1.95
µg/ml. The data represent the percentage inhibition values.
Values are expressed as mean ± SD (n=3).
S.No. Plant conc. (μg/ml) Butylated Hydroxytoluene
(Absorbance)
Percent Inhibition
(%)
1. 1000 0.121 ± 0.000 73.03 ± 0.128
2. 500 0.149 ± 0.000 66.81 ± 0.128
3. 250 0.194 ± 0.001 56.88 ± 0.222
4. 125 0.201 ± 0.000 55.25 ± 0.128
5. 62.5 0.213 ± 0.002 52.59 ± 0.462
6. 31.25 0.232 ± 0.000 48.44 ± 0.000
7. 15.625 0.248 ± 0.001 44.88 ± 0.222
8. 7.8125 0.287 ± 0.000 36.14 ± 0.128
9. 3.906 0.331 ± 0.000 26.29 ± 0.128
10. 1.95 0.393 ± 0.000 12.59 ± 0.128
S.No. Plant conc. (μg/ml) P. maderaspatensis
(Absorbance)
Percent Inhibition
(%)
1. 1000 0.301 ± 0.000 42.90 ± 0.171
2. 500 0.355 ± 0.000 32.74 ± 0.035
3. 250 0.377 ± 0.000 28.58 ± 0.162
4. 125 0.403 ± 0.000 23.59 ± 0.192
5. 62.5 0.422 ± 0.001 20.12 ± 0.121
6. 31.25 0.441 ± 0.000 16.52 ± 0.271
7. 15.625 0.455 ± 0.001 13.81 ± 0.176
8. 7.8125 0.487 ± 0.001 7.82 ± 0.280
9. 3.906 0.501 ± 0.001 5.17 ± 0.283
10. 1.95 0.527 ± 0.000 0.18 ± 0.000
Phyllanthus maderaspatensis
166
Table No. 6.12: Total Anti-oxidant Capacity of standard. The different concentrations of
extracts used from 1000 to 1.95 µg/ml. The standard used was
butylated hydroxytoluene. Values are expressed as mean ± SD (n=3).
Figure No. 6.4: Graphical representation of percent inhibition of methanolic extract of the
seeds of Phyllanthus maderaspatensis (PM) and butylated hydroxy toluene
(BHT) as standard by using Alkaline DMSO method.
0
10
20
30
40
50
60
70
1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95
Pe
rcen
tage
Inh
ibit
ion
(%)
Plant Concentration (µg/ml)
PM
BHT
S.No. Plant conc. (μg/ml) Butylated HydroxyTotuene
(Absorbance)
Percent Inhibition
(%)
1. 1000 0.099 ± 0.001 77.12 ± 0.322
2. 500 0.143 ± 0.001 66.92 ± 0.290
3. 250 0.176 ± 0.001 59.40 ± 0.365
4. 125 0.217 ± 0.001 50.03 ± 0.175
5. 62.5 0.234 ± 0.000 46.05 ± 0.061
6. 31.25 0.269 ± 0.000 38.06 ± 0.082
7. 15.625 0.300 ± 0.000 30.92 ± 0.091
8. 7.812 0.319 ± 0.001 26.47 ± 0.233
9. 3.906 0.337 ± 0.001 22.25 ± 0.347
10. 1.95 0.347 ± 0.001 20.10 ± 0.207
Phyllanthus maderaspatensis
167
Figure No. 6.6: Graphical representation of percent inhibition of methanolic extract of
the seeds of Phyllanthus maderaspatensis (PM) and butylated hydroxy
toluene (BHT) as standard by using hydrogen peroxide scavenging
method.
0102030405060708090
1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95
Per
cen
tage
Inh
ibit
ion
(%)
Plant Concentration (µg/ml)
PM
BHT
Figure No. 6.5: Graphical representation of percent inhibition of methanolic
extract ofthe seeds of Phyllanthus maderaspatensis (PM) and
butylated hydroxy toluene (BHT) as standard by using nitric
oxide radical scavenging activity.
0
10
20
30
40
50
60
70
80
90
100
1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95
Pe
rce
nta
ge In
hib
itio
n (%
)
Plant Concentration (µg/ml)
PM
BHT
Phyllanthus maderaspatensis
168
Figure No. 6.8: Graphical representation of percent inhibition of methanolic extract of
the seeds of Phyllanthus maderaspatensis (PM) and Butylated hydroxy
toluene (BHT) as standard by using total anti-oxidant capacity.
Figure No. 6.7: Graphical representation of percent inhibition of methanolic extract of
the seeds of Phyllanthus maderaspatensis (PM) and butylated
hydroxy toluene (BHT) as standard by using DPPH radical
scavenging activity.
0
10
20
30
40
50
60
70
80
90
1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95
Per
cen
tage
Inh
ibit
ion
(%)
Plant Concentration (µg/ml)
PM
BHT
0
10
20
30
40
50
60
70
80
1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95
Pe
rce
nta
ge In
hib
itio
n (%
)
Plant Concentration (µg/ml)
PM
BHT
Phyllanthus maderaspatensis
169
S.No. Test Performed IC50 value for
Seeds Extract
Butylated
Hydroxytoluene
1. Alkaline DMSO Method 890.53 ± 6.10* 792.49 ± 1.16*
2. DPPH Method 200.00 ± 2.19* 43.40 ± 1.307
3. H2O2 Method 236.06 ± 4.681 26.166 ± 0.351
4. Nitric Oxide Method 52.33 ± 0.30* 364.60 ± 3.510
5. Total Anti-oxidant Capacity
Method …. 124.25 ± 3.04
Table No. 6.13: IC50 value of different anti-oxidant activity of methanolic seeds
extract of P. maderaspatensis and standard. The standard used
was butylated hydroxytoluene (BHT). Unit for IC50 for all the
activities are µg/ml. Data are expressed as mean ± SD (n=3).
(* = P value: < 0.0001)
6.11 Thin Layer Chromatography
Thin layer chromatography (TLC) is a sophisticated method and a type of planar
chromatography used in the present study to identify the components in the Phyllanthus
maderaspatensis methanolic seed extract such as alkaloids, phenols, flavonoids, etc. The
separation depends on the relative affinity of compounds towards stationary and mobile
phase. The compounds travel under the influence of mobile phase (driven by capillary
action) over to the surface of the stationary phase. The compound with higher affinity
travels slowly in stationary phase while others travel faster. All the silica gel glass plates
contained a reference spot along with P. maderaspatensis methanolic seed extract. The
gallic acid and tannic acids were used as reference. The silica gel glass plates were
developed in an iodine chamber in the presence of iodine fumes. The yellow to purplish
pink colour spots or rockets were observed. The retention factor (Rf) of the unknown
compound is compared with Rf of the known compound (gallic acid and tannic acid). The
Rf is the retention factor, indicating how far the compound has travelled on the silica gel
plates.
The thin layer chromatogram was prepared by using 2 µg/ml of P. maderaspatensis
methanolic seed extract and standard compounds on silica gel plates. The Rf values of the
extract and standard were observed, calculated and compared. It was found that the
Phyllanthus maderaspatensis
170
chromatogram had been showing bands at the similar distances as that of the band of
tannic acid and gallic acid and their Rf values calculated were similar.
Rf value = Distance travelled by component
Distance travelled by solvent
The Rf value of P. maderaspatensis methanolic seed extract was observed to be 0.67. The
Rf value of gallic acid and tannic acid are 0.88 and 0.91 respectively. Thus, it indicates the
presence of phenolics present in the P. maderaspatensis methanolic seed extract. Apart
from these, other bands were also seen indicating the presence of other compounds as
well. The results are shown in Figure 6.9.
6.12 GC-MS Analysis of Phyllanthus maderaspatensis Methanolic Extract
The crude methanolic seeds extract of Phyllanthus maderaspatensis was characterized
using GC-MS analysis to evaluate the compounds present in it. The results of GC-MS
analysis showed that at least 54 compounds are present in methanolic extract of
P. maderaspatensis. The compounds were identified through mass spectrometry are
attached with GC. The mass spectra of these compounds were matched with those found
in the NIST05 and WILEY 8 spectral database. The fragmentation of major compound
found in was 9, 12-Octadecadien-1-ol with 55.65% (retention time: 15.634 min),
n-Hexadecanoic acid with 18.51% (retention time: 14.293 min) and (1S, 2E, 4S, 5R, 7E,
11E)-Cembra-2, 7, 11-trien-4, 5-diol (retention time: 16.012 min) 8.54%. The active
principles along with their retention time, area, area percent and compound name in the
methanolic extract of P. maderaspatensis. is given in Table 6.14. The chromatogram of
GC-MS is given in Figure 6.10.
Phyllanthus maderaspatensis
171
Figure No. 6.9: Thin Layered Chromatographic Analysis of Phyllanthus
maderaspatensis
Figure No. 6.10: Chromatogram of Phyllanthus maderaspatensis methanolic extract of
seeds.
P.M. T.A. G.A.
Phyllanthus maderaspatensis
172
Peak R Time Area Area% Name
1 3.599 4065035 0.15 2- Heptenal, (Z)-
2 4.113 3637136 0.14 1-[(Trimethylsilyl)oxy]propan-2-ol
3 4.425 1955799 0.07 2,4-Heptadienal, (E,E)-
4 5.958 835304 0.03 Undecane
5 6.142 648624 0.02 Butane-1,2,3,4-Tetraol
6 6.296 3047055 0.11 Glycerin
7 7.029 5711246 0.21 4H-Pyran-4-one, 2,3-dihydro-3, 5-
dihydroxy-6-methyl-
8 7.489 6638452 0.25 Octanoic acid
9 8.307 4215620 0.16 2-Decenal, (Z)-
10 8.700 3018599 0.11 2,4-Decadienal, (E,Z)-
11 8.996 7113423 0.26 2(10)- Pinen-3-one,
12 10.545 2335732 0.09 [1,1'-Bicyclopropyl]-2-octanoic acid,
2'-hexyl-, methyl ester
13 10.792 2438376 0.09 Phenol, 3,5-bis (1,1-dimethylethyl)-
14 11.376 3681631 0.14 3-Hexadecene
15 11.608 570485 0.02 Ledol
16 11.842 903082 0.03 Megastigmatrienone
17 12.075 1440269 0.05 Ar-tumerone
18 12.809 9789973 0.36 Tetradecanoic acid
19 13.140 862860 0.03 Isopropyl Tetradecanoate
20 13.492 3405217 0.13 1,2-Benzenedicarboxylic Acid Bis (2-
methylpropyl) Ester
21 13.713 489134 0.02 9-Hexadecenoic acid, methyl ester,
22 13.852 33935009 1.26 Hexadecanoic acid, methyl ester
23 14.293 497179925 18.51 n-Hexadecanoic acid
24 14.978 196591583 7.32 9,12-Octadecadienoic acid, methyl
ester
25 15.634 1494768500 55.65 9, 12-Octadecadien-1-ol
Phyllanthus maderaspatensis
173
26 16.012 229264654 8.54 (1S,2E,4S,5R,7E,11E)-Cembra-2,7,11-
trien-4,5-diol
27 16.540 4200990 0.16 9-Octadecenal, (Z)-
28 16.663 2808086 0.10 9-Octadecenamide, (Z)-
29 17.117 9493881 0.35 3-Cyclopentylpropionic acid, 2-
dimethylaminoethyl ester
30 17.258 3899434 0.15 Hexadecanal, 2-methyl-
31 19.726 2640308 0.10 Carpesterol dehydrate
32 20.134 3306089 0.12 Coumarine, 8-allyl-7-hydroxy-6-ethyl-
4-methyl-
33 20.423 10639426 0.40 Azulene, 1,2,3,3a,4,5,6,7-octahydro-
1,4-dimethyl-7-(1-methylethenyl)-
34 21.698 3271370 0.12 gamma.-Tocopherol
35 22.014 6752373 0.25
6S-2,3,8,8-
Tetramethyltricyclo[5.2.2.0(1,6)]undec-
2-ene
36 22.389 1785089 0.07 9,19-Cyclolanostan-3-ol, acetate,
(3.beta.)-
37 22.978 5295931 0.20 2H-Cyclopropa(G) Benzofuran,
4,5,5A,6,6A,6B-Hexahydro-4,4,6B
38 23.404 3351919 0.12
4-(2,2-Dimethyl-6-
methylenecyclohexylidene)- 3-methyl-
2-
39 24.509 3221150 0.12 Urs-12-en-28-oic acid, 3-hydroxy-,
methyl ester, (3.beta.)-
40 24.973 1411038 0.05 Ergosta-5,25-dien-3-ol, (3.beta.,24r)-
41 25.808 2212502 0.08
3Beta,21alpha-diacetoxy-18,22,22-
trimethyl-17,27,29,30-tetranor-c-
homoolean-
42 26.138 12247231 0.46 Gamma.-Sitosterol
43 26.714 1460541 0.05 Cholest-8(14)-ene-3,15-dione
44 27.055 9443923 0.35 4-(2,2-Dimethyl-6-Methylene-
Cyclohexylidene)- -3-Methyl-Butan-
45 27.795 42195542 1.57
6S-2,3,8,8-
Tetramethyltricyclo[5.2.2.0(1,6)]undec-
2-ene
46 29.092 5803884 0.22 Stigmast-4-en-3-one
Table No. 6.14: The peak results of Phyllanthus maderaspatensis methanolic seed
extract.
Phyllanthus maderaspatensis
174
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