1. In vitro conservation of Centella asiatica (Linn.)Urban.
and Bacopa monnieri (Linn.): Analysis for Antioxidant
and Antifungal activity.
Sukanya Kulkarni1
, Sugandhika Kumari3
, Abhijeet Purude2,
Jayant Londhe1
, S. S Sheelavantmath 1
1
Department of Biotechnology, 2
Department of Chemistry Sinhgad College of Science, Ambegaon
(BK), Pune 411041, 3
Hettigoda Industries (pvt)Ltd, H. T. 34 , Hospital road, Hasalaka , Sri Lanka
Email: sumanss.scos@sinhgad.edu Contact Number: 9527840941
Abstract-
In the recent years, people are much conscious about health and for the same herbal medicines are
been used preferably. Now a days isolation of herbal medicines became a crucial task because of distinction of
the respective plants. For the same conservation of plants in vitro through clonal propagation is a optimistic
solution. Through the same context, in the present protocol in vitro initiation of multiple shoots from leaf and
nodal explants of species Centella asiatica and Bacopa monnieri is carried out. These plants produce
antioxidant compounds to neutralize Reactive Oxygen Species (ROS) in body through essential oils.
Index Terms- Centella asiatica, Bacopa monnieri, Brahmi, invitro cultivation, Antioxidant property, Oil
Extraction.
1. INTRODUCTION
Centella asiatica & Bacopa monnieri are commonly
called as Bramhi. Both of them claimed to possess a
wide range of pharmacological properties.
Micropropagation of medicinal plants has many
objectives like they can be grown throughout the year
indepent of season by specific protocol in vitro and
plant seedlings can be isolated without seasonal
variation . Huge number of plants can be grown in
shorter area requirements. Nodal explants of Bacopa
monnieri and were propagated in vitro using liquid
shake cultures [7] [9]
. In the present method rooting of
micro shoots have been obtained in MS medium with
IAA, IBA, and NAA which are used separately or in
combinations. Maximum root induction was observed
with MS + IAA [5] [6]
The crude methanol and petroleum ether extract of
Centella asiatica is studied to evaluate its antioxidant
activity, antioxidant radical scavenging activity via
DPPH and FRAPS assays, antifungal activities is
checked against Aspargillus niger. Further the fat
content of the sample (oil) is carried out via
saponification and it is found to be in the satisfactory
range.
2. MATERIALS & METHODS
Plant Material: Nodal explants and leaves of Bacopa
monnieri and Centella asiatica were collected from
Ayurvedic Rasashala, Pune. MS Medium is prepared
using micro elements, macro elements, salts and
organic supplements. Fresh explants such as nodal
parts shoot tips and leaves were surface sterilized
using 0.1% HgCl2 and 2% NaOCl. Explants were
washed under tap water for 20 min and with 2-3 drops
of laboline for 3 min followed by brief washing with
distilled water.[5][6]
,
2.1. Medium and Culture Conditions:
Murashige and Skoog’s (MS) medium [12]
containing
3 %( w/v) sucrose was used in all experiments. The
pH of the medium was adjusted to 5.8 prior to the
addition of 0.8 % (w/v) agar. The medium was
autoclaved at pressure 15 psi for 20 min. [6]
. All the
cultures were incubated in a culture room maintained
at 25+ 2o
C where 12h photoperiod provided through
white fluorescent light. [6]
2.2. Preparation of Extracts
The aerial parts of Centella asiatica was cleaned
with deionised water and dried in shade and
pulverized into fine powdered substance by grinding
machine. Each 20 gram of powder of Centella
asiatica was weighted with the electric balance and
transferred to three separate 100ml conical flasks.
Then each 40ml of petroleum ether, n-hexane and
methanol respectively added to flasks. The conical
flasks were closed by parafilm and placed at shaker
for 24 hours. The crude extracts were then filtered by
Whatmann filter paper No.1 Extracts were prepared

2. by making following concentrations and named as
sample 1, sample 2, sample 3 and sample 4
accordingly as show in table 2.1
3. Assays:
3.1 DPPH Assay for Free Radical Scavenging
Activity:
The DPPH molecule is a stable-free radical
by virtue of the delocalization of the spare electron
over the molecule; this delocalization produces a deep
violet color, characterized by an absorption band in
ethanol or methanol solution centered at about 517
nm. When a solution of DPPH is mixed with that of a
substance, that can donate a hydrogen atom, this gives
rise to the reduced form (Diphenylpicrylhydrazine),
with the loss of the violet color. The optical density
change at 517 nm was measured 30 min later by a
spectrophotometer. A blank was used to remove the
influence of the color of the sample. An ethanolic
solution of DPPH was used as negative control or also
called as blank. The radical scavenging activity was
determined by the 2, 2-diphenyl-2-picrylhydrazyl
hydrate (DPPH) method.
3.2 Ferric Reducing Antioxidant Property /
Ferric Reducing Ability of Plasma. (FRAP) Assay:
FRAP reagent is prepared using TPTZ
(2, 4, 6 Tripyridyltrizine) and taken 1ml for each 50ul
of sample. Incubation period of 10min at 37oC is
given Spectrophotometer at 595nm (UV-VIS)
3.3 Antifungal Activity against organism
Aspargillus niger:
The in vitro antimicrobial activities of the
test samples were carried out by well diffusion
method [14]. In this method, Potato Dextrose Agar
was used as culture media and the discs were placed
aseptically over the bacterial culture on Potato
Dextrose agar plates. Cups cut in medium using
sterile cork borer about 8 mm in diameter are done for
fungal culture. Then, standard dilution of the
petroleum ether, Methanol, n-hexane extracts were
placed in appropriate position on the plate. After
incubation at 37°C for 24 hours, the zone of inhibition
around the discs was measured by millimeter scale.
Discs were impregnated with each treatment and
control was assayed on duplicate agar medium plate.
The experiment was replicated two times to confirm
the reproducible result.
4. Saponification Value of Fat:
On refluxing with Alkali, glycerol esters of
fatty acids get hydrolyzed to give glycerol and
potassium salt of fatty acids (Soaps). Saponification
value is the number of milligrams of potassium
hydroxide required to neutralize fatty acids resulting
from complete hydrolysis of one gram fat. The
Saponification value gives an indication of the nature
of fatty acids in the fat. Since longer carbohydrate
chain, lesser the acid liberated per gram of fat
hydrolyzed.
Saponification Value = X mg of KOH consumed by
1g of fat.
Weight of KOH = Normality of KOH × Equivalent
Weight × Volume of KOH in liters.
Volume of KOH consumed by 1g of Fat =
[Blank – Test] ml
Table and Calculation:
Calculation:
N1 V1 = N2 V2
0.5 × 100 = N2× X
X = 4.3478
Where X is Saponification Value
Sample
Name
Blank Blank -
Test
Saponification
No.
(X)
Sample 1 32 32 – 22 10
Sample 2 32 32 – 21 11
Sample 3 32 32 - 23 09
Sample 4 32 32 - 24 08
4. Results and Discussion:
4.1 Results for DPPH Assay:
Name of
the
sample
Amount
Of Brahmi
Powder
Name &
Amount Of
Solvent
Amount of
Oil
Extracted
(Approx.)
Sample 1 50 gm 100ml (n
hexane)
8-10ml
Sample 2 100 gm 100ml (n
hexane)
8-10ml
Sample 3 50 gm 100ml
(Methanol)
8-10ml
Sample 4 100 gm 100ml
(Methanol)
8-10ml
Table 2.1: Extracts concentration
0
50
100
150
Sample 1
Sample 2
Sample 3
Sample 4
On X-Axis Concentration and on Y-
Axis Scavenging Effect

3. 5. Figures, Tables and Photographs:
Fig 1: 1) Nodal culture of B. monnieri 2) Nodal
explant developing into callus with multiple shoot
buds 3) shoot inition after 2 weeks 4) growing shoot
bud 5) micropropagated shoots on MS medium with
0.5 mg/l IAA 6) multiple shoot induction as effect of
IAA 7) rooting of B. monnieri 8) Root development
after 3 weeks 8) exposing plant to greenhouse.
Discussion:
Earlier studies available on Bacopa monnieri showed
plant regeneration through nodal explants with IAA,
BAP and kinetin [17] [18
. The nodal explants implanted
on MS medium showed multiple shoot and root
induction within 2-3 weeks of incubation. Shoot
regeneration which is potential of IAA has been
showed by Bacopa monnieri with nodal explants.
MS basal medium supplemented with IAA including
leaf as explants significantly showed shoot induction
after 3 weeks of incubation. n-hexane extract showed
higher activity for ferric ions where as methanolic
extract showed lesser activity. If flavonoids content is
higher methanolic extract will show higher results
against ferric ions and if the oil content is higher n-
hexane extract will show higher results. Antifungal
assay was performed against Aspargillus niger, a
fungus. Samples were poured in wells made on potato
dextrose agar plate
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
sample 1 sample 2 sample 3 sample 4
Absorbance
Samples
0.01%
0.10%
1%
Fig 2: FRAP assay samples concentration against
absorbance.
.
Fig 3: Zone of inhibition for all samples varying in
concentration 0.01% , 0.1% and 1% left to right
respectively.
Acknowledgments
Authors thank Dr. M. P. Ghatule, Principal Sinhgad
College of Science for providing all the necessary
laboratory facilities.
1 2 3
4 5 6
7 8 9

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[*Corresponding author]
The Antiseptic 2004; 101(9), 368-372