INTRODUCTION:
Parthenium hysterophorus L. (Asteraceae). Parthenin from Parthenium hyterophorus exhibited
anti-malarial, anti-inflammatory activity.1,2 Parthenium i.e.,
P. argentatum A. Gray are called as petroplants. Stem of Parthenium
argentatum plant gives rubber, a substitute for Hevea in the
production of tyres, foot wear, belting,
hose. 10% rubber was produced from two year old young plant, which can be
increased upto 300% by using stimulants. Parthenium
hysterophorus biomass forms a suitable substrate for energy source in
future in an attempt to find alternate energy source. Apart from these, it is
also finding its suitability in biomanure, pesticide production, bioremediation
of toxic chemicals, nanomedicine. Keeping all its significant, beneficial
aspects an initiative was taken to utilize the underutilized weed and also to
study the phytochemicals qualitatively and phytonutrients, secondary
metabolites, antioxidant activity by means of quantitative analysis in the
shade dried root, stem samples in order to study the properties contained in it
.
MATERIALS AND METHODS:
Sample
collection:
The sample Parthenium hysterophorus root, stem was collected from Periyar
University campus, Salem, Tamil Nadu, India. The collected leaves were cleaned
thoroughly allowed to shade dry and ground to powder using blender for further
use. The plant
was authenticated by Dr. A. Balasubramanian. The authentication number was
AUT/PUS/069 dated 17/12/2014.
Aqueous
extract preparation:
Aqueous extract was prepared by
dissolving 15g of powdered Parthenium hysterophorus root, stem in 200ml of distilled water. The mixture was
heated on a hot plate with continuous stirring at 30-40°C for 20minutes. Then
the filtered water extract was used for qualitative analysis. The percent yield
of the sample studied was assessed by taking 1g of dried samples and dissolved
in 10ml of distilled water then the prepared extract was poured in to a
petridish, allowed to dry, then the dry powder was taken out by scrapping and
weighed for dry weight. The dry powder obtained was used for the behavior of
drugs powder with different chemical reagents, fluorescence analysis. This
gives percent yield.
Phytochemical
analysis:
The extract was tested for the presence of
bioactive compounds by adopting standard procedures[3,4] fluoresence
analysis [5], behaviour of drugs powder with different chemical
reagents [6].
Test
for carbohydrate:
Molisch’s test: To the extract added few
drops of alcoholic alpha napthol solution, few drops of concentrated Sulphuric
acid along the sides of test tube.
Positive result gives purple or violet colored ring at the junction.
Fehlings test: To the extract added equal
amount of Fehlings A and B solution, heat the tubes in a boiling water bath.
Brick red precipitation of cuprous oxide is formed, if reducing sugar is
present.
Benedicts test: To the extract add
Benedicts reagent, the tubes were heated in a boiling water bath. Red
precipitation indicates positive result.
Test
for alkaloids:
Wagners test: To the extract add few drops
of iodine solution in potassium iodide. Reddish brown precipitate shows
positive result.
Hagers test: To the extract add few drops
of saturated solution of picric acid. Yellow colour precipitation signifies
positive result.
Test
for steroids and sterols:
Libermann-Burchard test: To the extract
add 2ml chloroform, 10 drops of acetic anhydride, 2 drops of concentrated
sulphuric acid. Bluish red to cherry red colour in chloroform layer shows
positive result.
Salwoski test: To the extract add few
drops of chloroform, concentrated
sulphuric acid. Bluish red to cherry red colour.
Test
for Glycosides:
Legal test: To the extract added pyridine,
sodium nitroprusside. Positive result shows pink red colour.
Baljet test: To the extract add picric
acid. Appearance of orange color signifies positive result.
Test
for saponins:
Foaming test: Foams produces when the
extract is shaked with water.
Test
for flavonoids:
Shinoda test: To the extract added
magnesium turnings, 1-2 drops of concentrated hydrochloric acid. Appearance
of red color indicates positive result.
Zinc hydrochloride test: To the extract
added zinc dust, 1-2 drops of concentrated hydrochloric acid. Appearance of red color indicates positive
result.
Test
for tannin and phenolic compounds:
Ferric chloride test: To the extract add
ferric chloride. Formation of greenish black colour shows positive result.
Potassium dichromate test: To the extract
add potassium dichromate solution. Positive result is confirmed by a formation
of brown precipitate.
Gelatin test: To the extract add 1%
gelatin solution containing 10% sodium chloride gives white precipitation.
Test
for protein and amino acids:
Biuret test: To the extract added 4%
sodium hydroxide, few drops of 15% copper sulphate gives purple colour.
Ninhydrin test: Bluish violet colour forms
when a solution of ninhydrin and extract mixture was heated.
Heat test: Protein coagulation shows
positive result when test solution is heated on a boiling water bath.
Test
for fixed oil:
Copper sulphate test: Blue colour forms
when the extract is mixed with 1ml of 1% copper sulphate and 10% sodium hydroxide.
Quantitative
analysis of phytonutrients:
Total carbohydrates [7], proteins
[8], aminoacids [9]
were performed according to the standard prescribed methods.
Estimation
of carbohydrate:
The total carbohydrate was estimated by
anthrone method. 1mg of Parthenium
hysterophorus root, stem powder
was hydrolysed to simple sugars by keeping it in a boiling water bath for three
hours with 5ml of 2.5N HCl and cool to room temperature. After neutralizing,
the contents were centrifuged and 0.1 ml of supernatant was used for the
analysis. To the sample add 4ml of anthrone reagent and the contents were
heated in a boiling water bath for 8 minutes. The tubes were cooled and read at
630nm using spectrophotometer Shimadzu Model - UV 1800. The standards were
developed with glucose. Standard graph plotted was used to find out
concentration of glucose present in unknown/ sample.
Estimation
of Protein:
The total protein was estimated by Lowry’s
method. To 0.1ml of extract added 2ml of alkaline copper reagent, mixed well
and incubated for 10minutes. After the incubation period 0.2ml of folin ciocalteau
reagent (diluted in the ratio of 1: 2) was added and allowed for 30minutes
incubation, then read at 660nm using spectrophotometer Shimadzu - Model UV
1800. The standards were developed with Bovine serum albumin. Standard graph
plotted was used to find out concentration of protein present in
unknown/sample.
Estimation
of Aminoacids:
The amino acid was estimated by Ninhydrin
method. To 0.1 ml of sample added 1 ml of ninhydrin solution dissolved in
ethanol. Cover the test tube with a piece of paraffin film to avoid the loss of
solvent due to evaporation. With gentle stirring, react at 80-100ºC for 4-7
minutes. Cool the test tubes and the colour developed was read at 570nm.
Tyrosine was used for developing standards.
Determination
of secondary metabolites:
The phenol and flavonoid content of
aqueous extract was analysed.
Determination
of total phenol content:
Total phenolic content were determined by
Folil-ciocalteau method. The extracts (0.1ml) were mixed with folinl-ciocalteau
reagent (5ml, 1:10 diluted with distilled water) for 5min and aqueous NaCo3
(4ml, 1M) were added. The mixture was allowed to stand for 15min and the
phenols were determined by colorimetric method at 765nm. The standard curve was
prepared. Total phenol values are expressed in terms of gallic acid equivalent
(mg/g of dry mass), which is a common reference compound. 10,11
Estimation
of flavonoids:
The aluminium chloride method was used for
the determination of the total flavonoid content. Extract solution were taken
and to this 0.1ml of 1M potassium acetate, 0.1ml of AlCl3 (10%),
2.8ml distilled water were added sequentially. The test solution was vigorously
shaken. Absorbance at 415 nm was
recorded after 30min of incubation. A standard calibration plot was generated
using known concentration of quercetin. The concentration of flavonoid in the
test samples were calculated from the calibration plot and expressed as mg
quercetin equivalent/g of sample.12
Determination
of antioxidant activities:
Reducing power assay, Total antioxidant
assay, Nitric oxide scavenging assay, Metal chelating activities were
performed.
Reducing
power assay:
Aqueous extracts was mixed with phosphate
buffer (2.5ml, 0.2M, PH 6.6) and potassium ferricyanide (2.5ml, 1%).
The mixture was incubated at 50°c for 20min. 1.0 ml of Trichloro acetic acid
(10%) was added to stop the reaction, which was then centrifuged at 3000rpm for
10min. The upper layer of solution (1.5ml) was mixed with distilled water
(1.5ml) and FeCl3 (0.1ml, 0.1%)
after mixing, the contents were
incubated for 10min and the absorbance was measured at 700nm. Increased
absorbance of the reaction mixture indicated increased reducing power. Ascorbic
acid was used as a positive control.13
Total
antioxidant capacity:
Total antioxidant capacity by phosphomolybdenum method assay is based on the reduction of Mo
(V1) to Mo (V) by the sample analyte and the subsequent formation of green
phosphate/Mo (V) complex at acidic pH by adding 4ml reagent solution containing
0.6M Sulphuric acid, 28mM Sodium phosphate, 4mM Ammonium molybdate. The tubes
were incubated in water bath at 95°C for 90 minutes. After the samples had been
cooled to RT, the absorbance of mixture was measured at 695nm against blank.
The phosphomolybdenum method is quantitative, since, the total antioxidant
activity is expressed as the number of equivalents of ascorbic acid.14
Nitric
oxide scavenging activity:
This procedure is based on the principle
that, sodium nitroprusside in aqueous solution, at physiological pH
spontaneously generates nitric oxide which interacts with oxygen to produce
nitrite ions that can be estimated using Griess reagent. Scavengers of nitric
oxide compete with oxygen, leading to reduced production of nitrite ions. For
the experiment, sodium nitroprusside (10mM), in phosphate buffered saline, was
mixed with extract and incubated at room temperature for 150min. After the
incubation period, 0.5ml of Griess reagent was added. The absorbance of the
chromophore formed was read at 546nm.
Ascorbic acid was used as a positive control.15
Metal
chelating activity:
Add extract (0.1ml) to a solution of 2mM
FeCl2 (0.05ml). The reaction
was initiated by the addition of 5mM
Ferrozine (160µl), the mixture was shaken vigorously and left standing at room
temperature for 10min. Absorbance of the solution was then measured
spectrophotometrically at 562nm. Standard curve was plotted using ascorbic
acid. Distilled water (1.6ml) instead of
sample solution was used as a control.
Distilled water (160µl) instead of ferrozine was used as a blank, which
is used for error correction because of unequal color of sample solution.16
For all estimations, readings were taken
using UV- Visible spectrophotometer- Shimadzu, Japan make. Model UV 1800.
Standard graph were plotted for all experiments using their respective
standards and samples were plotted against standard by taking concentration in X axis and OD in Y
axis.
Table.1 Qualitative analysis of phytochemicals in Aqueous Parthenium hysterophorus root, stem extract
|
S.No |
Name of the test |
Results |
|
1. |
Test for carbohydrate a)Molisch’s test b)Fehlings test c)Benedicts test |
+++ +++ +++ |
|
2. |
Test for alkaloids a)Wagners test b)Hagers test |
+++ ++ |
|
3. |
Test for steroids and
sterols a)Libermann - Burchard test b)Salwoski test |
+++ ++ |
|
4. |
Test for Glycosides a)Legal test b)Baljet test |
+++ ++ |
|
5. |
Test for saponins Saponin test |
+++ |
|
6. |
Test for flavonoids a)Shinoda test b)Zinc hydrochloride test |
++ ++ |
|
7. |
Test for tannin and
phenolic compounds a)Ferric chloride test b)Potassium dichromate test c)Gelatin test |
++ ++ ++ |
|
8. |
Test for protein and amino
acids a)Biuret test b)Ninhydrin test c)Heat test |
+++ ++ + |
|
9. |
Test for fixed oil a)Copper sulphate test |
+++ |
+ Slight changes,
++ Moderate, +++ Stronger reactions
STATISTICAL
TOOL:
Each experiment was carried out in
triplicate and the results are given as the Mean ± Standard deviation. The Mean
and Standard deviation (S) was calculated by using the following formula: Mean = Sum of x values / n (Number of
values), 
RESULTS AND DISCUSSION:
The results obtained are shown in Table 1
to Table 7. Phytochemicals assessed qualitatively are expressed in Table.1. The
yield calculated was tabulated in Table.2. The results obtained for the
analysis of root, stem powder with
different chemical reagents are shown in Table.3 & 4. The fluorescence
analysis results are depicted in Table.5. Table.6 shows the results of
nutrients analysed. Table.7 illustrates the results of secondary metabolites
and antioxidant activities.
Phytochemical analysis:
The results of qualitative phytochemical
analysis are shown in Table.1.
Results showed positive result for
carbohydrate, alkaloids, steroids, sterols, glycosides, saponin, flavonoid,
tannin, phenolic compound, ninhydrin, aminoacids, oil. (Table.1)
Table.2 Percentage
yield of Parthenium hysterophorus root, stem aqueous extract
|
S.No |
Name of the powder |
Recovery(%) |
|
1. |
Parthenium hysterophorus root |
40% |
|
2 |
Parthenium hysterophorus stem |
20% |
The percentage recovery of the aqueous
extract obtained was calculated and expressed in Table 2. The percent recovery
was found to be 40% and 20% with respect to Parthenium
hysterophorus root, Parthenium
hysterophorus stem.
Analysis
of Parthenium hysterophorus root, stem powder for its behavior
The results obtained for the analysis of
leaf powder with different chemical reagents are shown in Table.3.
Table 3 shows the results of
behaviour of Parthenium hysterophorus root powder with different chemical
reagents. The behaviour of Parthenium hysterophorus root powder with different chemicals showed
positive result for alkaloids, steroids, anthroquinone, flavonoid and protein.