International Journal of Pharmaceutical and Biological Archive

1. *Corresponding Author: Dr. Anita Joshi, Email: Anitajoshi1980@gmail.com
ISSN 0976 – 3333
RESEARCH ARTICLE
ICLE
Available Online at www.ijpba.info
International Journal of Pharmaceutical & Biological Archives 2017; 8(3):40-43
In vivo and in vitro comparative Study of primary metabolites of Boerhaavia diffusa
(L)
*Dr. Anita Joshi, Dr. Poonam Dhawan, and Shilpi Damor
* Department of Botany, Poddar International College, Mansarovar Jaipur, India
Received 13 July 2016; Revised 11 Feb 2017; Accepted 27 May 2017
ABSTRACT
Boerhaavia diffusa belonging to family Nyctaginaceae. It is an important medicinal herb. It contains
Primary metabolites and secondary metabolites viz., carbohydrates, lipids, alkaloids, flavonoids, essential
oils, sterols, lignin and tannins .In this investigations various primary metabolites were observed from
Leaf, Stem, and Callus of Boerhaavia diffusa. Protein contents were maximum in Callus and minimum in
stem. Carbohydrates contents were maximum in leaf and minimum in Stem. Lipid was observed
maximum in leaf and minimum in callus sample.
Key word- Primary metabolites, Alkaloids, Favonoids, Protein, Sterols, Lipid and Carbohydrates.
INTRODUCTION
Boerhaavia diffusa L. is perennial creeping herb
emerging in rainy season throughout the country
(figure-1).
Fig. 1: Boerhaavia diffusa
Leaves and roots of the plant are used
medicinally. (Mahesh et al, 2012). The roots and
seeds are added to cereals, pancakes, and other
foodstuffs. The entire plant including the roots is
used as vegetable. In many parts of India tribals
cook Boerhaavia leaves as vegetables (Jana, 2007,
Aslam, 1996). Punarnava is mainly used to treat
accumulation of fluids (Oedematous conditions)
in the body. It is also used in the treatment of
anemia and liver diseases as recommended
by Indian Ayurveda. It has been identified to act
as a diuretic, anti inflammatory and
hepatoprotective agent (Deepti et al., 2013). The
rapidly increasing diabetes mellitus is becoming a
serious threat to human health. B. diffusa has
potent anti-diabetic activity (Nayak and
Thirunavoukkarasu, 2016). Roots have a
numerous medicinal properties in treatment of
abdominal pain, antistress, antidiabetic and as
antioxidant in different models (Sumanth and
Mustafa, 2007). The leaves are very useful in
opthalmia and in joint pains. Miralles et al.
reported 15 amino acids (6 essential) in the whole
plant and 14 amino acids (7 essential) in the roots
. The plant contains a large number of such
compounds as flavonoids, alkaloids, steroids,
lipids, lignins, carbohydrates, boerhavin and
boerhavic acid, proteins, and glycoproteins
(Humayun Riaz et al.,2014 and Santhosha et
al.,2011). This study suggests that plant parts
having rich primary metabolites can be used
industrially as raw materials having commercial
importance. These primary metabolites could be
further used for biosynthesis of secondary
metabolites or bioactive compounds. Results
obtained show the great interest in plant
pharmaceuticals.
Explants source
B.diffusa explants were collected from the
Poddar college campus. The explants must be
thoroughly surface sterilized before inoculating on
culture medium. Leaves of B.diffusa washed
under running tap water for 30 min, and then with
Tepol for callus induction. The explants were then
disinfected using 0.1 %( w/v) HgCl2 (Hi-media)
for 2 min (Neeta et al., 2002). There after the
segments were washed 5-7 times with sterile
distilled water.

2. Joshi Anita et al. In vivo and in vitro comparative Study of primary metabolites of Boerhaavia diffusa (L)
© 2010, IJPBA. All Rights Reserved 41
Culture media preparation
Murashige and Skoog’s (MS) medium
supplemented with sucrose (3%), agar (0.8%) and
different concentrations of auxin and cytokines
were prepared for callus induction. The pH of
media was adjusted to 5.8 and sterilized by
autoclave. The media was then poured in Flask.
Explants were implanted on the media and were
maintained for data analysis. The experiment was
set in triplicate. Stock callus was obtained from
leaf explants on Ms Medium supplement with 2,
4-D (5.0mg/I) +BAP (0.5mg/I).
PRIMARY METABOLITES
Protein (Lowry method 1951) –
Different dilutions of BSA solutions were
prepared along with alkaline copper sulphate and
the solution is incubated at room temperature for
10 min. Then reagent folin -ciocalteau solution
was added and incubation was done for 30 min.
Finally the OD was taken at 660 nm. The
absorbance of unknown sample was checked and
concentration of the unknown sample was plotted.
Carbohydrate (Anthrone method) –
100 mg of sample was Weighted and maceratedin
80%ethanol. The volume was made upto 100 ml
and entrifuged.supernatant was collected.
Anthrone reagent was added and heated for about
8 min in boiling water bath. The green colour
solution so formed was checked for absorbance at
630nm concentration of the unknown sample was
plotted.
Lipid (Bligh and Dyer, 1959) -
Weigh 50 mg plant material in a centrifuge tub
and add deionized water. Add 2 ml. Methanol and
1 ml. chloroform. Mix thoroughly on a vortex.
Add 1 ml. deionized water and 1ml chloroform.
Mix again on a vortex. Centrifuse for 10 minutes
at 4500 rpm. Now there is a two-phase extract
water/methanol above, chloroform below,
separated by a dense protein layer. Remove the
chloroform with a Pasteur pipette. Extract the
water/methanol two more times with 1ml
chloroform. Remove the chloroform after
centrifugation at 4500 rpm. Evaporate the
chloroform of the extract. After removal of the
chloroform the lipid fraction must be dried in
stove at 700
C for 24 hours. Lipids are determined
gravimetrically by weighing the centrifuge tube
with lipid fraction.
RESULT AND DISCUSSION
Protein –During the present investigation, an
increased level of protein was seen in callus
(Graph-1). Dhawan et al. (2016) also found to be
higher amount of protein in callus. Molina et al.,
(2006) reported that sunflower storage proteins
transported to a special storage compartments in
seed. Rapid production of milligram quantities of
protein in a batch cell –free synthesis system has
been reported (kim et al., 2006).
Graph-1
PROTEIN
Carbohydrate- During the present investigation
highest amount of carbohydrate content was
recorded in leaf which gradually declined in callus
and stem (Graph-2). In contrast to this Yadav et
al., (1996) found that callus culture exhibited
higher sugar contents compared to explants.
Baxter et al. (2005) reported fruit carbohydrate
metabolism in a introgression line of tomato with
increase food solids.
Graph-2
CARBOHYDRATE
Lipid - In our present investigation highest
amount of lipid content was seen in leaf, lower in
stem and callus (Graph-3). Poorter & Villar
(1997) found that leaf material have higher lipid
contents compared to Stem and callus.
Graph-3
LIPID
0
0.5
1
1.5
Leaf Stem Callus
Protein
mg/mg
fresh
weight
0
0.5
1
1.5
Leaf Stem Callus
Carbohydrate
mg/mg
fresh
weight
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3. Joshi Anita et al. In vivo and in vitro comparative Study of primary metabolites of Boerhaavia diffusa (L)
© 2010, IJPBA. All Rights Reserved 42
ACKNOWLEDGEMENT –
The author are thankful for the financial support
provided by Department of UGC and are also
grateful to the Chairperson Mr. Anand Poddar and
Director Mrs. Roopal Poddar, Poddar
International College, Mansarovar, Jaipur for
providing facilities for research work.
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4. Joshi Anita et al. In vivo and in vitro comparative Study of primary metabolites of Boerhaavia diffusa (L)
© 2010, IJPBA. All Rights Reserved 43
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