Phyllanthus amarus has a old history in the traditional medicine in

every country. It is used in the treatment of diabetic, liver problems

kidney and gol bladder problems. The lignin phyllanthin is the main
therapeutic active components of P. amarus. Its shown to posses anti-
hepatitis B antigen activity.

On the basis of chapter III phyllanthin and hypophyllanthin are

isolated from the leaves of Phyllanthus amarus.

In the present study correlation could be established between hydro

ethanol. Extract and blood sugar level in a diabetic albino mice.

According to Brunzel et al., (1985) hypertriglyceridemia with therapy

with either insulin or sulfonylurea

On the basis chapter extract may be categorized, though one may

next conformed by these primary studies. It is however very indicative
of differences in concentration of constituents in different extracts.
Further studies are needed to know about them fully. U-V spectroscopy
would be helpful in identifying the λ-max point, while IR
spectrophotometry may be helpful in identifying the constituents of the
extracts in term of their qualitative and quantities differences (Eglinton
1970)

Variations have been found in infra-specific level in many plant

Wakhloo(1963) found variations in the plant Rauwalfia serpentine.
It has also been found that a medicinal plant shows an average content in
the center of the area of growing the content becoming variable away
from the centre.

From recent study, it is evident that different types of terpenes are

present in Phyllanthus amarus. Mixture of several compounds posses
problem during separation and careful separation procedures are thus
needed to achieve the objective in full totality. Therefore combination of
spectrophotometry and complicated chromatographic separation would
be most useful. However, it is beyond doubt that quantitative differences
in phytochemical substances exist which however do not neglate the
possibility of qualitative differences. During this study it was noted
higher dose if extract is highly effective to decrease the sugar level in
albino mice.

Change in blood glucose level and body wt in normal, diabetic and

on treatment of diabetic albino mice with Phyllanthus amarus extracts.

The administration of Phylanthus amarus ethanolic leaf extract

showed significantly (p<0.05) increased activity of hexokinase in
diabetic albino mice when compared to that of diabetic control group.

The experiment showed the P. amarus extract cause significant

(p<0.05) reduction in the blood glucose level in diabetic albino mice .
 Phyllanthus genus in world folk medicine is used consequently,
substantial chemical and pharmacological (preclinical and clinical)
studies are available in the literature, one reason for this is certainly the
fact that Phyllanthus amarus is so widely distributed, bothd in tropical
and sub-tropical countries. Phyllanthus amarus has been calimed in folk
medicine to present beneficial therapeutic effects in the treatment of
genitourinary infections and hepatitis B virus, in the management of
airway diseases, besides its reported use as an anti-diabetic.

In earlier studies it was reported that the extract obtained form the
leaves, stem and roots of Phyllanthus amarus show antispamaidic
properties when assessed in several smooth muscles in guinea pigilcum
and urinary bladder, rat uterus, ureter, and vascular smooth muscles
from dogs the ether extract obtained from Phyllanthus amarus was
found to be the most active as an antispasmodic, being about 6 to 26 fold
more effective in causing antispasmodic actions in vascular and
nonvascular preparations than other extracts (Nakarmara et al. 2010).
More recently using HPLC analysis, it was reported that the aqueous
extract of Phyllanthus amarus contain more than 50 compounds. The
majority of these compounds have been isolated and characterized
chemically by other groups. The chloroform extract of Phyllanthus
amarus obtained either by partition or by extraction 25.200mg/1000ml
produced concentration dependent rightward displacement associated
with inhibiting of maximal contractions for bradkymin,histamine, and
acetylcholine when assessed in guniea pig and for bradykin and
acetylcholine in rat uterus in vitro. In addition, the extract obtained by
partition 25-100mg/1000ml caused graded and significant inhibition of
twitches induced by transmural electric stimulation in nonvascular
smooth muscles, while the fraction obtained through polar solvent had a
much smaller inhibitory effect.

Power product:- Healthy leafs are collected in different localities

then, wash hand well dried sun temp and grind with grinder mixture,
then make powder with the help of mixer-machine. The harvested fresh
leave were sun dried and ground into a fine powder.

About 1000g of the powder leaves was extracted in one ltr. of cold
sterile distilled water maintained on a mechanical shaker. The aqueous
extract was filtrated with No.1 Whitman Millipore filter paper and
concentrated to dryness with a rotary evaporator at 50±5c and
lyophilized. A yielded freeze-dried material of approximately 40g was
obtained. The freeze-dried sample was stored in a cool dry place until
ready for use store at 28°c in incubator for further studies.

Phytochemical Screening of Phyllanthus amarus.

The phytochemical components of Phyllanthus amarus leaves

were screened using standard method described by Harbrone. The leaf
extract was screened for alakloids, flavonoids, saponins, tannins,
steroids, lignins, glycosides, terpeniods, polyphenols, coumarins and
reins. The color intensity of the precopiatate formed was used as
analytical response to these tests. The result for the phytochemical
screening of Phyllanthus amarus as shown in Table 4.1 revealed the
presence of alkaloids, saponins, glycosides, tannins, steroids, flavonoids
and carbohydrates.

Phytochemical Screening of the Extracts

Table 4.1 Result of phytochemical screening of the extract of

Phyllanthus amarus.

SAMPLES
TEST
1 2 3

ALKALOIDS + + ++

SAPONINS + ++ +

GLYCOSIDES + + +

TANNINS ++ ++ ++

STERIODS AND
+ + +
TERPENES

FLAVONOIDS + + +
CARBOHYDRAIC + ++ +

+ = LOW ++= MODERATE +++= HIGH

The components detected have been shown to exhibit various

therapeutic properties such as the astringent actions of tannins, anti-
inflammatory and anti-allergic effects of flavonoids. Some
carbohydrates, flavonoids, steroids and alkaloids have been shown to
exhibit anti-diabetic action (Trease and Evans,2001). The detection of
most of these plant constituents in Phyllanthus amarus have suggested
the possibility of using the plant in the treatment and management of
many aliments including noninsulin dependent diabeties mellitus.
Saponins inhibit sodium efflux by blocking the entrance of the sodium
ions into the cell hence activating sodium calcium anti-porter producing
elevated cytosolic calcium which strengthens the contractions of heart
muscles and thus reducing congestive heart failure.

Table 4.2: Compounds found in whole plant body Phyllanthus

Amarus.

Collected from three different locations of Patna (Bihar)

SPECIMEN CLASS COMPOUND

Phyllanthus Amarus. ALKALOID 4-METHOXY-NOR-

 SECURININE
NIRURINE

ENT-NORSECURININE

GALLIC ACID
BENZENOID
CORILAGIN

ELLAGIC ACID
COUMARIN ETHYL BREVIFOLIN
CARBOXYLATE

QUERCETIN

RUTIN

ASTRAGALIN
QUERCITRIN

FLAVONOID ISOQUERCITRIN
KAEMPFEROI-4’-

RHAMNOPYRANOSIDE
ERIDICTYOL-7-
RHAMNOPYRANOSIDE
TISETIN-4-O-
GLUCOSIDE NIRURIN

PHYLLANTHIN
HYPOPHYLLANTHIN
NIRANTHIN
NIRTETRALIN
LIGNAN
PHYLTETRALIN
HINOKININ

ISOLINTETRALIN
 RICINOLEIC ACID
LIPID
PHYLLESTER

PHYTALLATE ESTRADIOL

Β-SITOSTEROL
STEROL ISOPROPYL-24-
CHOLESTEROL

TANNIN GERANIIN

LUPEOL ACETATE

LUPEOL

3,7,11,15,19,23-
HEXAMETHYL-2Z.6Z

TRITERPENE 10Z,14E,18E,22E-
TETRACOSHENEN-1-ol

PHYLLANTHENOL
PHYLLANTHENONE
PHYLLANTHEOL

Table 4.3: CHEMICAL CONSTITUENTS PRESENT IN DIFFERENT

PART OF PLANT

LEAF ROOT SHOOT SEED

 1. 4- 1. CATECHIN 1. 24- 1. NIRTETRAL
HYDROXY- 2. GALLOCATEC ISOPROPYL IN
LINTETRAL HIN CHOLESTRO 2. 4-
IN 3. EPICATECHIN L, AER METHOXY-
2. 2- 4. EPIGALLOCAT 2. DOTRIACON NOR-
3DIMENTH EEHN TANOIC SECURININ
OPEXY-ISO- 5. GALLIC ACID ACID E,
LINTERTRA 6. EPIGALLOCAT 3. NIRPHYLLI 3. RUTIN
LIN ECHIN-3- N, 4. PHYLLANT
3. ASTRAGAL OGALLA 4. NIRURINE HINE
IN 7. ERIODICTYOL 5. PHYLLANT 5. PHYLLOCH
4. BETA -7-O-ALPHA-L HENOL RYSINE
SITOSTERO 8. FISETIN-41-O- 6. PHYLLANT 6. QUERCETIN
L ALPHA-L- HEOL
5. DEMETHYL RHAMONISID 7. PHYLLESTE
ENEDIOXY E R
NIRANTHIN 9. LUPEOLACET 8. PHYLLINUR
6. HYDROXY ATE IN
NIRANTHIN 10.LUPEL 9. PHYLLTETR
7. HYPOPHYT 11.NOR- IN
HANTHIN SECURININE 10.TRIACONTA
8. ISO- N-1-AL
QUERCITIN 11.TRIANCONT
9. LIIMANTHI AN-1-ol,
N
10.LINTETRAL
 IN
11.NIRANTHIN
12.QUERDTRI
N
13.SALICYLIC
ACID
METHYL
ESTER
14.SECO-4-
HYDROXY-
LINTETRAL
IN

Pharmacological screening revealed that phyllanthin is a

hepatoprotective, antioxidants, antihyperuricemic, antimicrobial,
antigenotoxic, anti-inflammatory and vasorelaxant compound. Very few
studies have been reported on the extraction and anaylsis of Phyllanthin.
For phyllanthin extraction form Phyllanthus amarus numerous methods
have been reported: maceration, hot percolation, cold percolation and
ultra-sonication with subsequent analysis by HPTLC, HPLC, HPLC-
MS. It is a well known fact that conventional solvent extraction methods
are tedious and time consuming. Moreover, these processes may lead to
thermal, oxidative and photo decomposition of active phytoconstiuents.
 Fig: Structure of phyllanthin

Moisture content:

The proximate analysis showed the moisture content of

Phyllanthus amarus to be 69.96 ± 0.05% (wet weight). This result
indicated that the shelf life of this plant while fresh is low and long
storage lead to spoilage due to its susceptibility of microbial attacks.
This justifies the practice of storage in dry form by users. Moisture
content is one of the most important and widely used measurements in
the processing, preservation and storage of foods and drugs.

Table 4 .4

Proximate Composition (% dry matter)

Component Concentration (%)

Moisture (wet weight) 69.96 ± 0.05

Ash content 11.20 ± 0.17

Crude fibre 6.95 ± 0.03

Crude protein 10.50 ± 0.15

Lipids 6.07 ± 0.03

Carbohydrate 65.28 ± 0.04

Caloric value (kcal/100g) 357.75 ± 0.03

Ash Content

The result obtained for ash was 11.20 + 0.17% (dry matter). This
result is comparable with those reported by (Etukudo, 2003) for some
other plants and plant parts. The ash content reflects the mineral content.

Crude Fibre
 The value of crude fibre obtained for Phyllanthus amarus was 6.95
± 10.03% (dry matter). Crude fibre in foods or plants is an indication of
the presence of non digestible carbohydrate and lignin. The low value
obtained for Phyllanthus amarus is considered appropriate, studies have
shown that crude fibre aids in reducing peaks of blood glucose
following a meal due to delayed gastric emptying. The low crude fibre
content of this plant is advantageous in absorption of glucose and fat.
Although crude fibre enhance digestibility, its presence in high levels
can cause intestinal irritation, lower digestibility and decreased nutrient
utilization (Harbone, 1973).

Crude Protein

The crude protein in Phyllanthus amarus was 10.50 ± 0.15% (dry

matter). Hence the plant is a moderate source of protein, suggested that
problem from plant sources have lower quantity, but their combination
with many other sources of protein such as animal protein may result in
equivalent nutritional value. The recommended dietary allowance
(RDA) for protein is 56g for individual weighing 70kg and 46kg for
adult weighing 50kg. Children may consume 2g/kg/day (Calixto et al,
1998).

Crude Lipids
 The crude lipids content was 6.07 ± 0.03% (dry matter). Many
body functions depend on lipids. Lipids provide excellent source of
energy and enhance transport of fat soluble vitamins, insulate and
protect internal tissues and enhance transport of fat soluble vitamins,
insulate and protect internal tissues and contribute to vital cell processes.
It has been suggested that enough lipid (fat) be included in the diet to
account for at least 20 – 25% of the total caloric intake.

Carbohydrate Content

The carbohydrate content of Phyllanthus amarus was 65.28 ± 0.02

(dry matter). This carbohydrate may be one of the contributing factors
for the efficacy of Phyllanthus amarus as an anti-diabetic agent. Pemela
et al., observed that some carbohydrate containing foods have a rapid
rise followed by a slow decline in blood glucose concentration. The
RBA for carbohydrate is 30g/kg.

Caloric value

The caloric value of Phyllanthus amarus was 357.75 ± 0.03

kcal/100g dry matter. An average person requires 2000-3000 kcal/day.
The value obtained shows that Phyllanthus amarus could contribute to
energy requirement of its consumers.

In order to analyse the effects of Phyllanthus amarus extracts on

hepatitis C replication, the viral copies per cell was calculated for HCV-
infected cells with treatment and without treatment. Because we found
that the number of cells changed due to the extract, the viral load per
cell was a more useful indicator to compare samples than the viral load
alone. Based on the percent change in the viral copies between treated
and untreated cells, it was possible to identify the most effective
concentrations for Extracts 1 to 3 and compare their effect with that of
standard interferon. This percent change is also referred to as the percent
suppression.

The suppression of HCV over time does not change very much for
extract 1 at 0.001mg/1000ml and extract 2 at 0.001mg/100 mg.
suppression for extract 1 at 0.001mg/1000ml ranges from 67.4% to
79.4% from 24 to 96 hours, and while Extract 2 at 0.001 mg/1000ml
experiences a wider range from 67.0-88.5%, it is not much larger. Other
extracts, however, show different behaviour. Extract 3 at 1mg/1000ml
exhibit strong suppression early that does not last over time, but the
slope of this decline is different between these two extracts. 3
experiences a slower initial decline, changing from 70.3% to 66.7%
suppression from 24 to 48 hours. However, by 96 hours, the suppression
effect dropped by half. Extract 3 exhibits the strongest suppression of
HCV viral copies per cell, but the effect drops off more quickly.

At 24 hours. Extract 1, Extract 2, and Extract 3 do not demonstrate

significant changes in HCV suppression among concentrations. The
suppression for these extracts narrowly ranges from 62.7%-82.7%
consistently across these extract, which may indicate that a wider range
of concentrations may be need to have been tested in order for a
behaviour pattern to be more clearly qualified.

This could be an indication of solubility issues, a fluke, or a error

in the instrument while reading this time point. However, as
concentrations decreased, activity returned in a dose-dependent manner.

At 48 hours, in general, the effect across concentrations of all

extracts is between 53.9%-87.2%. There seems to be little variation in
the activity across concentrations, and although extracts do experience
dips in activity across concentrations at certain points the error bars
overlab too closely to be able to say a discernible difference exists
between samples. Extract 1 dips in its effect on HCV viral suppression
at 0.01mg/1000ml, which like the extracts at 24 hours, may be an
indication of solubility problems.

At 96 hours, across concentrations of each extract, we begin to see

the behaviour of the extract more clearly elucidated over time. For
Extract 1, in general, the suppression had not changed significantly from
48 hours or 24 hours, staying within approximately 60-80% suppression.
This may indicate that the effect may be longer-lasting at 0.01
mg/1000ml and 0.1 mg/1000ml for Extract 1, there is a dip in the
effectiveness of the extract. This may be an indication of solubility
issues, a fluke, or an error in the instrument while reading this time
point, since as concentrations increased, activity returned in a dose-
dependent manner. For Extract 2, similar behaviour occurs at 1µg/mL,
but it exhibits the opposite behaviour as Extract 1. As concentrations
decreased, the suppression increased again in a dose-dependent manner.
For Extracts 3, the suppression of HCV was strong initially, but over
time by 96 hours, the effect was not sustained. At 96 hours, this extract
was the weakest of all three extract to suppress the number of HCV viral
copies per cell.

Few different assays were performed to determine the

antioxidative power of Phyllanthus amarus leaf aqueous extract (20-
300mg/1000ml) as described below.
In each of these assays, ascorbic acid (20-300mg/1000ml) was used as
a reference substrate. The ability of extract to scavenge or inhibit free
radicals was expressed as percentage inhibition and was calculated using
the
following formula:

𝐴0−𝐴1
% of Inhibition = 𝑋100
𝐴0

Where A0 is absorbance of thequ control group (without plant extract)

and A1 is absorbance of Phyllanthus amarus extract. All determinations
were carried out in absorbance of triplicate.
DPPH RADICAL SCAVENGING ACTIVITY

DPPH radical scavenging activity of Phyllanthus amarus leaf aqueous

extract was determmed according to the method by Katalinic et al., In
brief, 0.5 ml of 0.1 mM DPPH solution was prepared in methanol just
before use. 1.0ml of P. niruri leaf aqueous extract was added at different
concentrations (20-300mg/1000ml ) to DPPH solution. Double distilled
H20 was used in the control group instead of samples, with the same
procedures applied. The abihty of the substrateto reduce the stable
radical DPPH from deep purple to yellow-coloured
digihenyipicryihydrazine indicates its antioxidative potentiai. The
mixture was shaken vigorously and left to stand for 30 min in the dark,
and absorbance was measured at 517/530nm using a spectrophotometer
(UV-1700). Lower absorbance at 517/520nm represents higher DPPH
scavenging activity.

SUPEROXIDE RADICAL SCAVENGING ACTIVITY

Measurement of superoxide radical scavenging activity of P. niruri leaf

extract followed the method by Xiang and Ning. In brief, superoxide
anions were generated in nonenzymatic phenazine
methosulfatenicotinamide adenine dinucieotide (PMS-NADH) system
through the reaction of PMS, NADH and oxygen. It was assayed by the
reduction of NBT in the presence of different concentrations (20-
300mg/1000ml of the extract). The reaction was initiated by
adding 0.75 mL of PMS (120 μM) to the mixture. The absorbance was
measured at 560 nm by using a spectrophotometer (UV-l700) following
S-minute incubation at room temperature.

HYDROXYL RADICAL SCAVENGING ACTIVITY

The hydroxyl radical scavenging activity of Phyllanthus amarus leaf

extract was measured according to a modified method by Eswar Kumar
et al., The reaction mixture contained 60 μl. of 1.0 mM FeCl2, 90 μl of l
mM 1, 10-phenanthroline, 2.4 ml. of 0.2 M phosphate buffer
(pH 7.8), 350 μl of 0.17M hydrogen peroxide (H2D2). Phytochemical
studies carried out on these plants isolate and characterize great classes
of compounds, including alkaloids, flavonoids lignans.

Total bilirubin

In normal control chicks, the total bilirubin showed their level as 0.25 
0.02 (mgms%). Intoxication of paracetamol caused a significant
elevation of levels 0.35  0.0621 U/ml) when compared to control
chicks. The total bilirubin level was restorated normal levels on the
administration of silymarin at a dose of 100 mg/kg and the total bilirubin
level was 0.28  0.04. The total bilirubin levels in treated birds with
Phyllanthus amarus 0.30  0.00. It was found that the maximum
reduction in the level of total bilirubin was observed in the animals
administered with leaf sample and the minimum was in the animal
treated with root extracts.

Aspartate aminotransferase (AST) showed their level in control

chicks 64.30  4.41 U/ml. Intoxication of paracetamol caused a
significant elevation of this enzyme level (88.11  2.01 U/ml) when
compared to control birds. There was significant restoration of these
enzyme levels on administration of the silymarin at a dose of 100 mg/kg
and the activity of serum glutamate oxaloaccetate transaminase was
70.01  1.33. serum glutamate oxaloacetate transaminase levels of
chicks administered with Phyllanthus amarus remained 65.23  1.73.
maximum reduction of SGOT activity was observed with plant leaves
and the minimum was in the animals treated with stem extracts.

ALT

Alanine aminotransferase (ALT) or serum glutamate pyruvate

transaminase (SGPT) showed level in control birds as 10.61  1.01
U/ml. Paracetamol intoxicated birds had their SGPT level as 17.31 
2.81. There was a restoration in the enzyme levels on administration of
the silymarin at a dose of 100 mg/kg and the serum glutamate pyruvate
transaminase level was 13.15  3.01. SGPT levels of Phyllanthus
amarus stem extract treated animals were 13.56  1.07 and 12.93  1.77
respectively. Maximum reduction of Alanine aminotransferase activity
was observed with leaf sample and the minimum was in the animals
treated with samples of root.

Alkaline Phosphatase

The normal control chicks showed their enzyme level as 51.07 

4.06. Deseased group, which received normal saline and paracetamol
showed increase in the respective liver enzyme activities with a value of
70.15  9.97 U/ml when compared to control birds. There was a
restoration of these enzyme levels on administration of the silymarin at a
dose of 100 mg/kg and the alkaline phosphatase level was 60.49  7.72.
the alkaline phospatase levels of the treated birds with 55.00  4.03 root,
stem, leaves and fruit were 55.004.03, 56.00  3.81, 57.31  3.31 and
51.00  4.97 respectively. Maximum reduction of alkaline phosphatase
activity was observed with leaf extract and the minimum was in the
animals treated with stem extract.

Table 4.5

Experiments shows (Step IInd)

Table 4.5: Effect of different doses of aqueous extract act

hydroethanol extract of Phyllanthus amarus on blood glucose level.

Blood glucose level mg/dl

 Group Day 1 Day 7 Day 15

Control 90.62  3.41 84.25  5.9 81.75  3.37

Diabetic 328  32.56 316  37.23 310.5  34.27

control

Aqueous 327.14  33 325.28  38 210.28  39

Extract
500mg/kg

Aqueous 345.14  223.71  47 210.5  35.6

extract 1000 35.30
mg/kg

Genomic DNA was extracted from root, patal and leaf of Phyllanthus
amarus with a view to loop into wheather persistant aneuromaty induces
any kind of genomic DNA imbalance presents the result of
electrophoresis of genomic DNA from root and leaf respectively
protocol of genomic DNA extraction has been discussed earlier in this
reaction.

It present the electrophoresis results of restrict digested genomic

DNA of Phyllanthus amarus.
 The run of 1kb ladder A – 2, Eco R1 digest of Petal DNA Bam H1
digest of Petal DNA.

The observation shows 1kb ladder while B – 2 presents fragile root

genomic DNA. Eco R1 digests petal DNA shows two bands while Bam
H1 digests of petal DNA shows two bands. Treatment of root DNA by
restriction enzymes resulted into non distinguishable bands and
apparently continuous flow of DNA. Genomic DNA untreated with any
band separation except the main group and all the four lanes had mass to
genomic DNA separated to the same level. It proves that there has been
no difference in total DNA contents of the plant by apparent increase in
few chromosomes no. due to endo-duplication.

Genomic DNA separation involves extraction of DNA from

hundred of the cells having same amount of DNA approximately and
that is why, there would be no discernible difference in the amount of
separated DNA through gel electrophoresis. To investigate into minor
genomic DNA difference from cell to cell, other advance technique
would be required difference from cell to cell, other advance technique
would be required.

In the present study simple electrophoretic separation of genomic DNA

has been observed.

Bam H1 could destabilise DNA due to additional chromosome

having DNA length that had restriction site. Additional cuts lead to
destabilization and free flow could not be observed in case of DNA
isolated from root and leaf.

In the present study, simple electrophoresis separation of genomic

DNA has been observed.

Bam H1 could destabilize DNA due to additional chromosome

having DNA length that had restriction site. Additional cuts lead to
destabilization and free flow of root DNA. Such free flow could not be
observed in case of DNA isolated from root and leaf.

Restriction digestion and electrophoresis of genomic DNA