INTRODUCTION:
India
a leading country in Asia having traditional knowledge in the use of plant
species and have higher plant species
around 17000 species of which 7500 are medicinal plants1. Bauhinia purpurea Linn. (Caesalpinaceae)
is an ornamental, medium-sized deciduous tree2 have 250 species
and 16 genera present in subtropical
India, North - South America, Nepal, Australia, Africa, United Kingdom. In
India alone about 15 species are
available in tropical regions and found throughout India like Punjab, central,
south India, Assam, Sikkim, Chotanagpur as well as in sub Himalayan tract and outer
Himalaya’s upto1300 meters and also in
Western peninsula, Kumaon pakisthan, Nepal, Bhutan, Burma, Myanmar, China.
The
healing of disease begins with the use of herbs3 as Indian medicinal
plants are richest source of antioxidants 3 able to put a stop
to/delay different diseased states.4 Stem bark of B. purpurea in Malays known as pokok
tapak kerbau was used by Indian, Sri Lankan, Pakistani community to treat
ulcer, wound, glandular swelling, stomach tumor, antidote to poison,
anastringent to treat diarrhea, antihelminths, leprosy, menstrual disorders, disorders
of the rectum. According to reports Bauhinia contain steroidal glycosides,
terpenoids, lactones, flavonoids.5 Bauhinia purpurea reported to exhibit various pharmacological activities
such as, anti-oxidant activity, hepatoprotective activity, hypoglycaemic
activity, antiproliferative, anti-inflammatory activity.6,7,8 Hence,
the present study was undertaken to assess the nutrients, antioxidants,
secondary metabolites, qualitative analysis.
Bauhinia purpurea Linn. stem
MATERIALS AND
METHODS:
Sample Collection:
The
Bauhinia purpurea stem was collected
from Navodaya academy senior
secondary school located at Namakkal District, Namakkal, Tamil Nadu, India,
during the month of January - February 2015. The collected stems were cleaned
thoroughly and dried under the shade. Once the drying process is complete, the
dried stems were ground to powder using blender for further use. The plant Bauhinia purpurea Linn was identified
with the help of articles collected from online sources.
Aqueous Extract Preparation
Aqueous
extract was prepared by dissolving
15g of powdered Bauhinia purpurea 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 water
extract was filtered through filter paper was used for qualitative analysis and
0.1ml was used for the Quantitative analysis. All Quantitative experiment was
performed three times. The percent yield of the sample studied was assessed by
taking 1g of dried samples and
dissolved in 10ml of distilled water and the extract was prepared, and
then poured in to a petridish, allowed to dry, then the dry powder was taken
out by scrapping and weight 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:
Qualitative
analysis was done for the presence of alkaloid, flavonoid, Saponin,
anthroquinone, terpenoids, steroids, glycoside.9,10
Test
for Alkaloids:
This test was performed by adding few drops of saturated
solution of picric acid to a drop of extract. Positive test shows the presence
of yellow color precipitation.
Test
for Flavanoids:
To a drop of extract added magnesium turnings followed by 1/2
drops of concentrated hydrochloric acid. Positive result gives red color.
Test for Saponins:
Foams produces when extract
was mixed well with water (vigorous
shaking).
Test for free Anthraquinones:
5
ml of chloroform was added to the powdered sample and filtered after shaking it for 5 mins. To this add equal
volume of 10% ammonia solution. Positive result shows the appearance of bright
pink colour in the aqueous layer.
Test for Steroids and Terpenoids:
Dissolve
a small portion of extract in 1ml chloroform and then filtered. The filtrate
was kept on ice, to this 1 ml of acetic acid and few drops of conc. sulphuric
acid was added. The appearance of a pink
color indicates the presence of terpenoids. The appearance of blue color
indicates the presence of steroids. A mixture of pink and blue color indicates
the presence of both.
Test
for Glycosides:
Powered sample was boiled
with 1.0 ml of sulfuric acid in a test tube, filtered while hot and then
cooled. Then equal volume of chloroform was added.10ml ammonia was added to the
separated chloroform layer of mixture. The presence of reddish brown
precipitate in the filtrate shows positive result.
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 extract
(0.1ml) were mixed with folinciocalteau 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.11,12
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.13
Determination of antioxidant activities:
Reducing
power assay, Total antioxidant assay, Nitric oxide scavenging assay, Metal
chelating activities were performed.
Reducing Power Assay:
Aqueous
extract 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.14
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.15
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.16
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.17
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.
Statistical tool:
Each
experiments were 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 of Phytochemicals present in Bauhinia purpurea stem
is shown in Table.1
Table.1 Phytochemicals in aqueous extract
of Bauhinia purpurea stem
|
S.No |
Name of the test |
Results |
|
|
leaf |
stem |
||
|
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 |
+++ ++ |
+++ +++ |
|
9. |
Test for fixed oil a)Copper sulphate test |
+++ |
+++ |
+Slight
changes, ++ Moderate, +++ Stronger reactions
The
results of phytochemical analysis Bauhinia
purpurea stem
is shown in Table. 1. The results were found to be positive for stem. The
result obtained might be because of
the stage of the stem at which it
was collected. The obtained results show alkaloid, flavonoid, saponin,
anthroquinone, glycosides, protein, fat, tannins etc.