Pharmacognostic study of the root of Justicia gendarussa Burm

Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
Regular articles
Pharmacognostic study of the root of Justicia
gendarussa Burm
Patel Sonal, Zaveri Maitreyi *
Department of Pharmacognosy, K.B. Institute of Pharmaceutical Education and Research,
Sector-23, GH-6, Gandhinagar 382023, Gujarat, India
Abstract
The recent global resurgence of interest in herbal medicines has led to an increase in the demand for them. Commercialization
of the manufacture of these medicines to meet this increasing demand has resulted in a decline in their quality, primarily due to
a lack of adequate regulations governing this sector of medicine. There is now a need to develop a systematic approach for the
authentication of herbal plants and to develop well-designed methodologies for their standardization. Therefore, the present
paper deals with the standardization of a well-known folklore remedy for chronic rheumatism using Justicia gendarussa Burm.
The standardization is carried out from a pharmacognostic point of view involving phytochemical studies, physico-chemical
studies, a qualitative study of metabolites using TLC and a preliminary HPTLC analysis of different extracts of the roots of J.
gendarussa based on WHO guidelines. Hence, the present investigation was undertaken to investigate the standardization of J.
gendarussa.
Key words: Justicia gendarussa, Standardization, HPTLC analysis.
1. Introduction
India has a rich source of traditional medicines.
The crude drugs are always cheap and available
in abundance, with negligible side effects and are
frequently prescribed to patients of all age groups.
The multiple therapeutic actions and uses of these
drugs are described in detail in classical literature on
indigenous medicines in many books on medicinal
plants. Various species of genus Justicia are used
traditionally for a wide variety of ethnomedicinal
purposes. Justicia gendarussa belongs to the family
* Author to whom correspondence should be addressed. Address:
Department of Pharmacognosy, K.B. Institute of Pharmaceutical
Education and Research, Sector-23, GH-6, Gandhinagar 382023,
Gujarat, India; Tel.: 9898214158 ; E-mail: khandharmaitreyi@yahoo.
com. khandharmaitreyi@gmail.com
Received: 2010-08-29 Accepted: 2010-12-02
Acanthaceae and is commonly known as Nili-
Nirgundi [1]. The present plant is up to one meter
in height and found in tropical and subtropical parts
of Asia and in India in seashore areas like Valsad
and Surat and in hilly areas like the Khasi hills,
Pavagarh. The herb of J. gendarussa is cultivated in
Indian gardens for its attractive foliage and flowers
[2, 3]. The flowers of J. gendarussa are white with
a purplish tinge when fresh. Ethnobotanically, a
decoction of the root of J. gendarussa is a popular
remedy for chronic rheumatism [5, 6, 7]. The roots
of this plant are also used to treat fever, cough,
jaundice, thrush, arthritis, cephalgia, hemiplegia,
facial paralysis, otalgia, hemicrania, bronchitis
and liver disorders [8, 9]. A literature survey and
screening of the published scientific data shows that
large number of indigenous drugs have already been
subjected to botanical and chemical investigations.
However, a systematic standardization including
61

Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
pharmacognostic and physico-chemical studies is
still lacking. Accordingly, the present investigation
of J. gendarussa was carried out to establish
botanical, chemical, and analytical tools which
would help in crude drug identification as well as in
checking for any adulteration. In addition, this study
will greatly help in quality assurance investigations
of the finished products involving herbal drugs.
2. Materials and methods
The roots of Justicia gendarussa were collected
from Anand farm and nursery Gandhinagar, Gujarat,
India. The plant was authenticated by a taxonomist
at the Department of Botany, School of Science,
Gandhinagar, Gujarat, India. A voucher specimen
is kept at the Department of Pharmacognosy K.B.
Institute of Pharmaceutical Education and Research,
Gandhinagar, Gujarat. A fresh sample of the roots
was subjected to morphological and microscopical
studies and after the dried roots were powdered and
passed through a 60 mesh sieve and then stored
in an airtight container for further use. Macerates
were prepared by the Schulz maceration method [7].
Photomicrographs were obtained for histological
observation (Labomed). A powder of the dried roots
was used for chemical analysis. Physicochemical
studies [9, 10] of the powdered drug, such as
determination of the ash value, extractive value, loss
on drying and crude fiber content were performed
according to the WHO guidelines. An extract of
the dried root powder of Justicia gendarussa was
prepared by using a soxhlet extractor involving
successive extractions with increasingly polar
solvents, petroleum ether, methanol and water. The
dried extracts were then stored in airtight containers
until required. These three extracts were analyzed for
the presence of alkaloids [11], flavanoids [12, 13],
saponins [14, 15], carbohydrates [16], steroids [17],
triterpenoids [18], carotenoids, amino acids, tannins
[19, 20], phenolics [21, 22], coumarins [23, 24] and
anthraquinones [25] using standard procedures.
2.1. Quantitative Standards
2.1.1. Determination of phenolics [26, 27]
To 1 ml of the methanolic extract of the roots
of J. gendarussa, 10 ml distilled water and 1.5 ml
diluted (1:2) Folin ciocalteu reagent were added and
the mixture was allowed to stand for 5 min. After
adding 4 ml 20 % w/v Na 2 CO 3 solution, the final
volume was adjusted to 25 ml using distilled water.
The absorbance was measured at 765 nm at intervals
of 30 min up to 2 h using distilled water as a blank.
The total phenol content was measured using
following formula:
C = (A×282.6) - 8.451
where, A= Absorbance.
2.1.2. Determination of saponins [28]
According to the results obtained from the
foaming test, the foaming index of the roots of J.
gendarussa was high. Further studies were carried
out to estimate the total saponin content. The
decoction was poured into 10 stoppered test-tubes
(height 16 cm, diameter 16 mm) in successive
portions of 1 ml, 2 ml, 3ml. up to 10 ml, and the
volume of liquid in each tube was made up with
water to 10 ml. The tubes were stoppered and shaken
for 15 sec, at two shakes per second. After standing
for 15 min, the height of the foam was measured and
the froth index was calculated.
2.1.3. Development of a preliminary HPTLC method
for the analysis of different extracts of the roots of
Justicia gendarussa
An accurate and sensitive preliminary HPTLC
method was developed for the analysis of different
extracts of the roots of J. gendarussa with the help
of a microliter syringe. Standard or sample solutions
of appropriate volume were applied to TLC plate
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Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
using a semiautomatic spotter (Camag Linomat V).
Triplicate samples of different root extracts of J.
gendarussa were spotted in the form of bands of
width 6 mm with a Camag 100 microlitre sample
syringe (Hamilton, Bonaduz, Switzerland) on to
silica gel precoated aluminum 60 F 254 , plates (20
cm×10 cm) 0.2 mm thick (E. Merck, Darmstadt,
Germany), using a Camag Linomat V (Switzerland)
sample applicator. The plates were prewashed with
methanol and activated at 110 ºC for 5 min prior to
chromatography. A constant application rate of 0.1
L/s was employed and space between bands was 5
mm. The slit dimension was kept at 5 mm×0.45 mm
and a 10 mm/sec scanning speed was employed.
The monochromator bandwidth was set at 20 nm,
and each track was scanned three times and a
baseline correction was used. The mobile phase
was toluene: methanol: formic acid (9:4.5:0.6)
for the petroleum ether extract and toluene: ethyl
acetate: formic acid (5:4:1) for the hydrolysed
methanolic extract and water extract and 15 ml of
mobile phase was used for each chromatographic
separation. Linear ascending development was
carried out in 20 cm×10 cm twin trough glass
chambers (Camag, Muttenz, Switzerland) saturated
with mobile phase. The optimum saturation time for
the mobile phase was 30 min at room temperature
(25 ± 2 ºC) and a relative humidity of 60 % ± 5 %.
The length of the chromatographicrun was 8 cm.
After development, the TLC plates were dried in a
current of air with the help of an air dryer in a well
ventilated wooden chamber. The flow rate in the
laboratory was maintained unidirectional (laminar
flow, towards the exhaust system). Densitometric
scanning was performed on a Camag TLC scanner
V in the reflectance absorbance mode at 254, 366
and 524 nm and operated using CATS software (V
3.15, Camag). The source of radiation used was a
deuterium lamp emitting a continuous UV spectrum
between 190 and 400 nm. The concentrations of the
Fig. 1. Morphology of J. gendarussa root
Fig. 1 Morphology of J gendarussa root
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Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
Fig. 2. T. S. of root of J. gendarussa (10×)
Fig. 2 T. S. of root of J. gendarussa (10X)
Fig. 2 T. S. of root of J. gendarussa (10X)
Fig. 3. T. S. of J. gendarussa root (45×)
Fig. 3 T. S. of Fig. J. 3 gendarussa T. S. of J. root gendarussa (45X)
root (45X)
Fig. T. S. of Fig. J. 3 gendarussa T. S. of J. root gendarussa (45X) root (45X)
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Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
Table 1. Physico-chemical parameters of root of J. gendarussa
Sr No. Quality parameters Samples (%, w/w )
1 Ash value
a. Total ash value 10.50 ± 0.55
b. Acid insoluble ash 1.89 ± 0.78
c. water soluble ash 3.21 ± 0.63
2 Extractive value
a. Water soluble extractive 12.0 ± 0.35
b. Alcohol Soluble extractive 9.10 ± 0.51
c. Acetone soluble extractive 3.40 ± 0.10
d. Chloroform extractive 2.70 ± 0.68
e. Petroleum ether extractive 2.00 ± 0.23
3 Loss on drying 7.00 ± 0.14
Standard deviation (SD) = ± SD , Number of readings (n) = 3
Table 2. Phyto-chemical screening of different extracts of roots of J. gendarussa
Phytoconstituents Tests Petroleum ether
extract
Methanolic
extract
Water extract
Alkaloid Dragondroff’s reagent test -ve +ve +ve
Flavonoid
Shinoda test
Fluroscence test
Phenolics With FeCl 3
With Folin ciocalteu reagent
Carbohydrates
Molisch’s test
Fehling test
Steroids and triterpenoids Liberman burchard test
Salkowski reaction
Carotenoids
Antimony trichloride
Sulphuric acid
Hydrochloric acid
Tannins
Test with gelatin
Test with lead acetate
Saponins
Froth test
Haemolytic zone test
Coumarins
With ammonia
With hydroxylamine hydrochloride
Anthraquinone glycoside
Borntrager’s test
Modified borntrager’s test
-ve
-ve
-ve
-ve
-ve
-ve
+ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
+ve
+ve
+ve
+ve
+ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
+ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
+ve
+ve
+ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
+ve
+ve
-ve
-ve
-ve
-ve
chromatographed compounds were determined from
the intensity of the diffused light. The plate with
the petroleum ether extract plate was sprayed with
anisaldehyde sulphuric acid reagent while the plates
with the methanolic and water extracts plates were
sprayed with phenol reagent.
Standard preparation: A solution of 1mg/ml
lupeol and β-sitosterol was prepared in petroleum
ether.
Sample preparation: A 1 mg/ml solution
of the petroleum ether extract of J. gendarussa
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Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
Fig. 4 Powder study of root of J. gendarussa.
Table 3. Estimation of phytoconstituents in root of J.
gendarussa
Sr.
Phytoconstituents
No.
1 Phenolics
(methanolic)
2 Saponins Foaming
Index
(water extract)
Value
1.02 % ± 0.02 % (w/w )
111.00 ± 0.00
Standard deviation (SD) = ± SD , Number of readings (n) = 3
Fig. 4. Powder study of root of J. gendarussa
Fig. 4 Powder study of root of J. gendarussa.
Fig. Powder study of root of J. gendarussa.
was prepared in petroleum ether, while a 1 mg/ml
solution of the hydrolyzed methanolic extract and
hydrolyzed water extract of J. gendarussa was
prepared in chloroform.
Preparation of the hydrolyzed methanolic
extract and hydrolyzed water extract: 1 g of the
methanolic extract of the roots of J.gendarussa and
1 g of the water extract of the roots of J.gendarussa
were refluxed separately with 1 N hydrochloric acid
66

HPTLC plate scanned at at 459 nm
Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
HPTLC plate scanned at 459 nm
HPTLC plate scaned at 459 nm
R ff
Relative
area (%)
0.58 R f
Relative
14.54
area (%)
0.70
0.58 11.70
14.54
R 0.81 f
0.70 12.27
11.70
Relative area
(%)
0.58 14.54
0.70 11.70
0.81 12.27
0.81 12.27
Plate scanned at at 366 nm
HPTLC plate scaned at 366 nm
Plate scanned at 366 nm
0AU
700
600
500
400
300
200
100
0.00 0-10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 R f
Fig. 5. HPTLC analysis of methanolic extract of root of J .gendarussa
R ff
Relative
area (%)
Relative
0.31 R f
14.99
Relative area (%) area
R f
(%)
0.56 8.27
0.31 0.58 14.54 14.99
11.70
0.70 38.97
0.56 0.81 12.27 8.27
0.87 16.06
0.70 38.97
0.87 16.06
(HCl) on a water bath for 30 min. Then, the acidic
extract was separated using chloroform and the
chloroform layer was collected, concentrated and
used for sample preparation for HPTLC analysis.
Fig. 5 HPTLC analysis of of methanolic extract of of root of of J.gendarussa
Fig. 5 HPTLC analysis of methanolic extract 3.2. Microscopy of root of J.gendarussa
3. Results
3.1. Macroscopy
The shade-dried intact roots of Justicia
gendarussa (Fig. 1) are 10-20 cm long with a
diameter of 0.1 to 0.5 cm. The outer surface is
grayish white in colour with nodules, while the outer
surface has longitudinal wrinkles, transverse cracks
and root scars. The wood is smooth and yellowish
white in colour.
The transverse section of the J. gendarussa roots
appears almost circular.
Cork cell (CK): The cork of J.gendarussa
consists of 2-3 layers. The cells are radially
elongated, tubular and compactly arranged.
67

HPTLC Plate scanned at at 254 nm
HPTLC Plate scanned at 254 nm
HPTLC plate scaned at 254 nm
Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
[mm]
HPTLC Plate scanned at at 366 nm
HPTLC plate scaned at 365 nm
AU
HPTLC Plate scanned at 366 nm
200.0
150.0
800.0
600.0
100.0
400.0
50.0
200.0
0.0
0.0
0.00 0-10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 R f
1000.0
800.0
600.0
400.0
200.0
0.0
AU
R f
Relative
area (%)
0.58 14.54
0.70 11.70
0.81 12.27
R f
Relative
area (%)
0.09 24.61
0.16 46.84
0.29 11.64
0.46 10.29
Fig. 6. HPTLC analysis of petroleum ether extract of root of J. gendarussa
Cortex (CT): The major part of the section
is made of the cortex. The outer 2-3 layers are
compact. The cells are spherical to oval, thin
walled and a few smaller intercellular spaces are
present. The rest of the region comprises large air
chambers, separated from one another by thin walled
parenchymatous tissues called aerenchyma, arranged
in uniseriate rows. The air chambers are large and
occur toward the periphery and their size decreases
gradually towards the centre. Stone cells are also
present singly or in groups.
Endodermis: A distinct, single layer of
endodermis separates the cortical region from
the vascular region and there is a reticulate
parenchymatous cell which is lignified.
Fig. 6 HPTLC analysis of of petroleum ether extract of of root of of J. J. gendarussa
Vascular tissue: The vascular bundle is
collateral and conjoint. The phloem (Ph) region has
phloem collenchymatous cells and phloem fibers
which are in groups. The xylem (Xy) has xylem
vessels, tracheids, xylem fibres and medullary rays.
The medullary rays are uni-seriate to bi- or triseriate
and the central region has lignified xylem
parenchymatous cells and pith is also present which
shows the characteristics of hydrophytes as shown in
Fig. 2-4.
Fig. 6 HPTLC analysis of petroleum ether extract of root of J. gendarussa
68

HPTLC HPTLC plate plate scanned plate scanned at 524 at at nm 524 nm nm
Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
HPTLC plate scaned at 524 nm
R f
Relative
area (%)
0.14 6.80
0.26 4.65
0.35 2.45
0.50 16.43
0.77 21.44
0.81 8.24
0.91 33.85
Co- Co- chromatography of with lupeol and β – sitosterol
Co-chromatography of with of with lupeol lupeol and β-sitosterol and β – sitosterol
Co- chromatography of with lupeol and β – sitosterol
Fig. 7. HPTLC analysis of petroleum ether extract of root of J. gendarussa.
The physico-chemical standard parameters, like
ash values, extractive values and loss on drying, are
shown in Table 1.
Phytochemical screening showed the presence
of phenolics, steroids, triterpenoids, carotenoids, and
Fig saponins . Fig 7 HPTLC . in 7 different HPTLC analysis extract analysis as of shown petroleum of in petroleum Table 2. ether ether extract extract of root of of root J. of gendarussa J. gendarussa
The methanolic extract of the roots of J.
gendarussa contain large amounts of phenolics
while the water extract of the roots of J.gendarussa
contain less saponins as shown in Table 3.
The extracts were standardized using HPTLC
analysis. The HPTLC chromatogram of the
petroleum ether extract of the roots of J. gendarussa
contained 9 peaks with R f values of 0.14, 0.26, 0.35,
0.50, 0.77, 0.81, and 0.91 along with lupeol (0.50 R f )
and β-sitosterol (0.35 R f ) after derivatization with
anisaldehyde sulphuric acid and the methanolic
extract of the roots showed 4 peaks at R f values of
0.31, 0.56, 0.70, and 0.87 and the water extract of
the roots showed 6 peaks at R f values of 0.12, 0.25,
0.40, 0.58, 0.63, and 0.89 at 366 nm as shown in
Fig . 7 HPTLC analysis of petroleum ether extract of root of J. gendarussa
Fig. 5-8.
69

HPTLC plate scanned at 254 nm
HPTLC plate scanned at 254 nm
HPTLC plate scanned at 254 nm
HPTLC plate scaned at 254 nm
Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
R f Area (%)
0.29 13.76
0.55 14.41
0.78 13.05
0.89 22.45
HPTLC HPTLC plate plate scanned at at 366nm
HPTLC plate scanned at 366nm
HPTLC plate scaned at 366 nm
AU
AU
400.0
350.0
300.0
250.0
200.0
150.0
100.0
50.0
0.0
0.00 0-10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90
R f
Max R f Area (%)
0.12 4.64
0.25 10.78
0.40 8.99
0.58 7.65
0.63 12.96
0.89 29.77
R f
Fig. 8. HPTLC analysis of water extract of root of J. gendarussa
4. Discussion
The above studies provide information about
the identification, chemical constituents and
physicochemical characters which may be useful
for pharmacognostic studies and standardization
of herbal drugs used currently in folk medicine.
They also provide therapeutic diagnostic tools for
scientists who wish to evaluate herbal medicines
obtained from indigenous resources.
Morphological study of the roots of J.
gendarussa shows that they are nodular. The
microscopic study shows the presence of lacunas
in the cortex and with a pith that is characteristics
of hydrophyte plants which supports the fact that
this plant is widely grown in coastal areas. The
identifying characteristic of the root is the reticulated
endodermis. The phytochemical study revealed
the presence of phenols, saponins, triterpenoids
and steroids in different extracts of the roots of J.
Fig. Fig.
8 HPTLC
8 HPTLC
analysis analysis
of of of
water water
extract extract
of of of
root root
of of of
J. J. J.
gendarussa
gendarussa
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Pharmacognostic study of the root of Justicia gendarussa Burm / Asian Journal of Traditional
Medicines, 2011, 6 (2)
gendarussa. Thus, these preliminary phytochemical
tests will be helpful in finding chemical constituents
in the plant material that may lead to their
quantitative estimation and also in locating the
source of pharmacologically active chemical
compounds. This study is the first report of the
HPTLC profile of the roots of J. gendarussa, which
identifies components that are useful for quality
evaluation and drug standardization. It also confirms
the presence of lupeol and β-sitosterol as chemical
markers in the petroleum ether extract of the present
plant.
Therefore, the investigation of the pharmacognostic
characteristics and phytochemical screening of J.
gendarussa provide useful information, which may
help in authenticating genuine plants and the nature of
the phytoconstituents present.
The present study is the first report of the
standardization of Justicia gendarussa root.
Acknowledgement
We are very thankful to GUJCOST for financial
assistance for a period of two years as a Minor
Research Project scheme in the year starting May-
2010.
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