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Phyllanthus maderaspatensis

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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


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Figure. 6.1. Phyllanthus maderaspatensis

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Euphorbiales

Family : Euphorbiaceae

Genus : Phyllanthus

Species : maderaspatensis


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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


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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


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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

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bommu%20P%22%5BAuthor%5D


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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)


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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)


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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.


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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 +


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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


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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


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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


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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


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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


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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


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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


(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


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


(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


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


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

(%)


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 (%

)


PM

BHT


168


the seeds of Phyllanthus maderaspatensis (PM) and Butylated hydroxy

toluene (BHT) as standard by using total anti-oxidant capacity.


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

(%)


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 (%

)


PM

BHT


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


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.


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.


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


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.


174

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