Phytochemical study of an ethno medicinal plant Limnophila rugosa Roth. (Merr) (Scrophulariaceae) whole plant

Abstract

Limnophila rugosa Roth. (Merr) (Scrophulariaceae) is used as a botanical source of Bhringaraja by the traditional practitioners of Balangir and Baragarh district of Odisha. The present study was carried out to screen the preliminary phytochemical constituents of ethanol & aqueous extracts of the whole parts of L. rugosa including High Performance Thin Layer Chromatography. The extracts were subjected to various chemical tests in order to identify the main phytoconstituents of the plant. The study revealed that the ethanol extract contains glycosides, little amount of alkaloids and flavonoids where as the aqueous extract is rich in glycosides.

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Short Communication
Phytochemical study of an ethno medicinal plant Limnophila
rugosa Roth. (Merr) (Scrophulariaceae) whole plant
Rabinarayan Acharya, Riddhish Padiya, Esha D Patel, Harisha CR, Vinaya J Shukla
Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurveda University,
Jamnagar, India
Corresponding author email:drrnacharya@gmail.com
Abstract
Limnophila rugosa Roth. (Merr) (Scrophulariaceae) is used as a botanical source of
Bhringaraja by the traditional practitioners of Balangir and Baragarh district of Odisha. The
present study was carried out to screen the preliminary phytochemical constituents of ethanol
& aqueous extracts of the whole parts of L. rugosa including High Performance Thin Layer
Chromatography. The extracts were subjected to various chemical tests in order to identify the
main phytoconstituents of the plant. The study revealed that the ethanol extract contains
glycosides, little amount of alkaloids and flavonoids where as the aqueous extract is rich in
glycosides.
Key Words : Limnophila rugosa, Phytochemistry, Bhringaraja, Gandhamardana hills
Annals Ayurvedic Med. 2013:2(1-2)
Introduction
In India, plants are utilized as
therapeutic agents, since time immemorial,
in organized (Ayurveda, Siddha and
Unani) and unorganized (folk, tribal and
native) form. In these systems of medicines
the plant based drugs are described either
in Sanskrit or vernacular languages.
Classical texts of Ayurveda describes three
types (white, yellow and blue) of
Bhrinagaraja, a drug of herbal origin,
claimed to be use full in many disease
conditions
(1,2)
. The botanical identity of
white and yellow variety, based on their
flowers, has been confirmed as Eclipta
alba (L.) Hassk. (Asteraceae) and Wedelia
chinensis (Osbeck) Merrill. (Asteraceae)
respectively and has been extensively
studied regarding their pharmacognostical
and phytochemical charecters
(3)
. The
botanical identity of blue variety of
Bhringaraja is yet to be established. An
ethnomedicinal plant, Limnophila rugosa
Roth. Merr.(Scrophulariaceae), having
blue colour flowers is being known and
used as Bhringaraja by the traditional
practioners of surrounding areas of
Gandhamardan hill region of Odisha and
not reported for its preliminary
phytochemical characters except its
leaf
(4,5)
.
Hence, the present study has been
planned to evaluate the preliminary
phytochemical characters of the whole
plant.
Materials and Methods :
Collection and authentication of plant
material:

The plant known as Bhringaraja, growing
in Gandhamardana hill ranges, Balangir of
Odisha district of India
(6)
was identified as
Limnophila rugosa Roth. Merr. of family
Scrophulariaceae by studying the
morphological characters of various parts
of the plant and comparing them with the
various characters mentioned in various
floras
(6,7,8)
. A herbarium specimen was
prepared (Herbarium No. 6003) and was
stored in Pharmacognosy department, of
the institute, for further documentation.
The remaining plants were dried under the
shade and then were subjected for 60#
powdering for further study.
Physicochemical parameters:
Determination of loss on drying at 110º C,
total ash, acid insoluble ash, water and
alcohol soluble extractive values were
carried out by following various
parameters mentioned in Ayurvedic
Pharmacopoeia of India.
(9)
Preparation of extract
5 g of L. rugosa powder extracted with
methanol (100ml), keeping it for overnight
with initial occasional shaking up to 6 hrs,
and then set aside. After 24 hours it was
filtered and alcoholic extract was collected.
Similarly water extract were prepared and
collected.
(9)
Qualitative tests
Tests for Alkaloids, Flavonoid, Steroids,
Protein, Tannins, Carbohydrate,
Chlorophyll (Phase test) and Cyanogenetic
glycosides was carried out following
standard parameters.
(10)
Chromatographic study
Methanol extract of Limnophila rugosa
whole plant were used for the study. The
solvent system used was Chloroform :
Methanol : Acetic acid in the ratio (8:2:1).
The application mode was Camag Linomat
V and the development Chamber used was
Camag twin trough chamber. The plates used
were precoated silica gel
GF254
plates. The
chamber saturation duration, development
time and development distance was 30 min,
30 min and 8 cm respectively. The scanner
used was Camag scanner III. For detection
deuterium lamp and Tungsten Lamp were
used. Win cats software was used. After the
scanning done by the Camag Scanner III, the
area under the curve of the methanol extract
of Limnophila rugosa whole plant was
studied
11
.
HPTLC study was carried out by standard
method using Vanillin-Sulphuric acid as
Spray reagent.
Result and Discussion
The physico-chemical parameters of the
whole plant viz. foreign matter, loss on
drying, total ash, acid insoluble ash were
found to be nil, 5.9 % w/w, 9.00 % w/w
and 2.0 % w/w respectively. The
percentage of alcohol extractive was 0.150
% w/w and ether soluble extractive value
was 0.061 but the percentage of water
extractive was found to be significantly
higher i.e. 0.181 %.
For the detection of functional groups,
various chemical tests were performed
with aqueous and alcohol extract of the
sample. Functional groups like alkaloid,
tannin, triterpenoids (Steroid), flavonoid,
Protein, carbohydrate, glycosides and
phenols were found to be present in the
sample. (Table 1)
Detection of HPTLC plate after spraying
with Vanillin-Sulphuric acid reagent, 14
spots were detected at 366 nm & 18 spots
were detected at 254 nm in L. rugosa
whole plant methanol extract. (Figure 1)
(Table 2)
Table.1- Preliminary tests results
Sr. No Qualitative tests L. rugosa whole plant

1. Test for Alkaloids
1) Dragendorff's reagent +ve
2) Mayer's reagent +ve
3) Hager' reagent +ve
4) Wagner's reagent +ve
2. Test for Tannins +ve
3. Test for triterpenes (steroids)
Salkowski reaction +ve
4. Test for saponins -ve
5. Test for fixed oil -ve
6.
Tests for cynogenic
glycosides /
sugars Molisch's test
+ve
7. Test for flavonoids /
Shinoda's test +ve
8. Test for carbohydrates
1) Molish’s test +ve
2) Fehling's test -ve
3) Pentose sugar +ve
4) Hexose sugar +ve
5) Non reducing +ve
9. Test for phenols / neutral
FeCl
3
+ve
10. Test for amino acids -ve
11. Test for proteins
1) Conc. H
2
SO
4
+ve
2) CuSO
4
+ve
3) HgCl
2
+ve
4) Lead acetate +ve
5) Ammonium Sulphate +ve
11. Test for resin -ve
12. Test for gum -ve
Table.2- Results of HPTLC study for R
f
value under long and short UV
Track R
f
Value Long UV R
f
Value Short UV
Limnophila rugosa
whole plant methanol
extract
0.01, 0.12, 0.18, 0.21, 0.27,
0.30, 0.37, 0.40, 0.48, 0.54,
0.64, 0.73, 0.80, 0.90
0.01, 0.07, 0.10, 0.17, 0.21, 0.25,
0.30, 0.39, 0.41, 0.48, 0.52, 0.54,
0.60, 0.64, 0.67, 0.72, 0.81, 0.85
Conclusion:
Limnophila rugosa whole plant shows the
presence of different types of functional
groups like alkaloid, tannin, triterpenoids
(Steroid), cyanogenetic glycoside,
flavonoid, carbohydrate and protein. In
Chromatography, HPTLC method, after
spray with vanillin sulphuric acid reagent,

14 spots were detected under long UV and
18 spots were detected under short UV, at
different R
f,
, indicating presence of various
chemical groups in the plant sample.
References:
1. Gogte VM. Ayurvedic
Pharmacology & therapeutic uses
of medicinal plants
(Dravyagunavigyana), Bharatiya
Vidya Bhavan, Kulapati Munshi
marg, Mumbai (2000).pp 451-453.
2. Sharma SK, Chunekar KC.
Medicinal plants used in Ayurveda.
National academy of Ayurveda,
New Delhi, R.A.V. Publication
(1998).pp 83,195.
3. Anonymous. Quality standards of
Indian Medicinal Plants, Vol – I,
ICMR, New Delhi (2003).pp 226-
233.
4.
Acharya RN, Padiya RH, Patel ED,
Harisha CR, Shukla VJ
. Preliminary
phyto-chemical study on the leaf of
an ethno-medicinal plant
Limnophila rugosa Roth. (Merr.),
Ayurpharm Int J Ayur Alli Sci.
2012; 1(7): 138-143.
5. Acharya Rabinarayan, Padiya RH,
Patel ED, Harisha CR, Shukla VJ,
ChauhanMG. Pharmacognostical
evaluation of leaf of Limnophila
rugosa Roth. Merr.
(Scrophulariaceae), Global J Res.
Med. Plants & Indigen. Med.(
2012) 1(12): 705–711.
6. Saxena HO, Brahman M. The flora
of Orissa, vol. III, Regional
Research Laboratory, Orissa Forest
Development Corporation Ltd.,
Bhubaneswar, Orissa.(India).
(1995) pp 1237.
7. Hooker JD. The flora of British
India, Vol. IV, Bishen Singh
Mahendra Pal Singh, Dehradun,
India, (London) (1885) pp 394-395.
8. Haines HH. The Botany of Bihar
and Orissa, part III-IV. Bishen
Singh Mahendra Pal Singh,
Dehradun (India). (1988) pp 659.
9. Anonymous (1999), The
Ayurvedic Pharmacopeia of India,
edition 1
st
, Govt. of India. Ministry
of Health and Family welfare,
Department of I.S.M. & H., New
Delhi, Part-I, Volume –I; pp.143
10. Khandelwal KR. Practical
Pharmacognosy – Techniques and
Experiments. 9th ed.: Nirali
Prakashan, Pune. 2002. pp.24-29,
149-153.
11. Anonymous. Planner
chromatography, Modern Thin
Layer Chromatography.
Switzerland (1999) pp.2-16.