PHCOG J.

ORIGINAL ARTICLE

Preliminary Phytochemical and Pharmacognostical

Screening of the Ayurvedic Drug Hygrophila auriculata
(K. Schum) Heine
Mohammed Sarfaraj Hussain*1, Sheeba Fareed1, Mohammed Ali2
1

Faculty of Pharmacy, Integral University, Lucknow. 226026. India. 2Department of Pharmacognosy & Phytochemistry,
Faculty of Pharmacy Jamia Hamdard, New Delhi 110062. India.

ABSTRACT
The plant Hygrophila auriculata (K.Schum) Heine, (Acanthaceae), has been traditionally used for the treatment of
INAMMATIONPAINURINARYINFECTIONEDEMAGOUTANDASADIURETIC)TISDESCRIBEDINAYURVEDICLITERATUREAS)KSHURA
)KSHUGANDHAAND+OKILASHAHAVINGEYESLIKETHE+OKILAOR)NDIAN#UCKOO4HEPLANTISWIDELYDISTRIBUTEDTHROUGHOUT
)NDIA 3RI ,ANKA "URMA -ALAYSIA AND .EPAL &OLLOWING VARIOUS FOLK CLAIMS FOR CURE NUMEROUS DISEASES EFFORTS
HAVEBEENMADEBYRESEARCHERTOVERIFYTHEEFCACYOFTHEPLANTTHROUGHSCIENTICBIOLOGICALSCREENINGS4HEPLANTCONTAINS
SAPONINSALKALOIDSSTEROIDSTANNINSAVONOIDSANDTRITERPENOIDSARETHEMAINPHYTOCONSTITUENTS!SCRUTINYOFLITERATURE
REVEALED SOME NOTABLE PHARMACOLOGICAL ACTIVITIES LIKE ANTI NOCICEPTIVE ANTI TUMOR ANTIOXIDANT HEPATOPROTECTIVE
HYPOGLYCEMICHAEMATINICDIURETICSFREERADICALSCAVENGINGANTHELMINTICANTI INAMMATORYANTIPYRETICANABOLICAND
ANDROGENICACTIVITIES4HISSTUDYDEALSWITHTHEPRELIMINARYPHYTOCHEMICALSCREENINGANDDETAILEDPHARMACOGNOSTICAL
study of leaf, stem and root of Hygrophila auriculata+3CHUM (EINEWHICHINCLUDESMACROANDMICROSCOPICSTUDIES
DETERMINATION OF PHYSICO CHEMICAL PARAMETERS OF THE EXTRACT USING 4,# AND (04,# NGERPRINTING 4HE AQUEOUS
ALCOHOLIC PETROLEUM ETHER CHLOROFORM ETHYL ACETATE AND N BUTANOL FRACTIONS SEPARATED FROM THE ALCOHOLIC EXTRACT OF
Hygrophila auriculataWEREPREPAREDANDTHETOTALPHENOLICCONTENTWASESTIMATED0HYSICO CHEMICALSTUDIESREVEALED
ALCOHOLSOLUBLEEXTRACTIVEWW WATERSOLUBLEEXTRACTIVE TOTALASH ACIDINSOLUBLEASH WATER
SOLUBLEASHWW LOSSONDRYINGWW SWELLINGINDEXWW FOREIGNMATTERWW &LOURESCENCE
ANALYSISEXHIBITEDCONSIDERABLEVARIATIONANDPRELIMINARYPHYTOCHEMICALANALYSISREVEALEDTHEPRESENCEOFALKALOIDS
STEROIDSAVONOIDSTRITERPENOIDSTANNINSANDSAPONINS(04,#PROLEOFN BUTANOLFRACTIONREPRESENTPRESENCETHE
VARIOUS TYPES OF TRITERPENOIDS 4HESE STUDIES WILL PROVIDE REFERENTIAL INFORMATION FOR THE CORRECT IDENTICATION OF THE
crude drugs.
Key words: Hygrophila auriculata (K. Schum) Heine; Preliminary phytochemical screening; pharmacognostical
EVALUATION(04,#

INTRODUCTION
Search for eternal health and longevity and to seek remedy
to relieve discomfort prompted man to develop diverse
ways and means of health care. The early man explored his
immediate natural surroundings, tried many things like plants,
animals and minerals and developed a variety of therapeutic
agents. The knowledge gathered by generations was either
documented or passed on to the posterity and this practice
was generally termed as Traditional Medicine. The World
*Address for correspondence:
Mobile no: +91-9889902496
E-mail: sarfarajpharma@gmail.com
DOI: 10.5530/pj.2011.23.5

28

Health Organization (WHO) estimates that about 80% of

the population living in the developing countries relies almost
exclusively on traditional medicine for their primary health
care needs. In almost all the traditional medicines, the
medicinal plants play a major role and constitute the backbone
of the traditional medicines. The modern (Allopathic) system
of medicine is based on drastic cures through synthetic
drugs and chemical compounds. It is noteworthy that since
last twenty- ve years or so, a notable drop in the popularity
of this system is noticeable. This trend can be attributed
to the increasingly harmful side effects induced by some
of these synthetic drugs. There is a famous line that says,
One mans Aspirin is another mans peptic ulcer.[1] It is
now known that chronic ailments, which require long-term
treatments, are not always cured by allopathic drugs.
Moreover, the synthetic drugs and intermediatery chemicals
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Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

are extremely expensive. For these and other reasons synthetic

drugs are being widely replaced with the medicinal plants
which are non-polluting renewable resources and are the
only hope for sustainable supplies of cheaper medicines
for the worlds growing population. Plants are also appreciated
in pharmaceutical research as the major resource for new
medicine and a growing body of medical literature supports
the clinical ef cacy of herbal treatments. Today, about 40%
doctors, especially in India and in China (the Mystic Orient)
have reverted to increasing use of indigenous drugs and
natural medicines. Steadily, a sizeable section of scientists
in biological, biochemical and biomedicinal discipline have
embarked on research on medicinal plants, which are the
staple sources of many indigenous drugs.
Hygrophila auriculata (K. Schum)Heine (synonym: Asteracantha
longifolia Nees, Barleria auriculata schum, Barleria longifolia linn)
Acanthaceae, is a wild herb commonly found in moist places
on the banks of rivers, ditches and paddy elds throughout
India, Sri lanka, Burma, Malaysia, and Nepal. The plants are
described in the ayurvedic literature as Ikshura, Ikshagandha
and Kokilasha having eyes like the kokila or the Indian cuckoo.
It is classi ed in the ayurvedic system of medicine as
Seethaveryam, mathuravipaka and is used for the treatment
of a number of conditions including Premeham (diabetes)
and athisaram (dysentery).[2,3] A survey of the ethnobotanical
literature shows that the roots, seeds, and aerial parts of the
plant are widely used in the traditional system of medicine
for the treatment of jaundice, hepatic obstruction, rheumatism,
in ammation, pain, urinary infection, edema, gout, malaria,

and impotence and as an aphrodisiac.[4] The plant Hygrophila

auriculata (K. Schum) Heine has been reported to contain
avonoids (apigenin 7-O- glucuronide, apigenin 7-Oglucoside),[5] alkaloids (asteracanthine and asteracanthicine),[6]
aliphatic esters (25- oxo - hentricontyl acetate, methyl -8hexyltetracosanoate),[7] minerals (Fe, Cu, Co),[8] sterols
(Stimagsterol),[9] triterpenes (lupeol, hentricotane, betulin,
luteolin, luteolin -7-O- rutinosides)[10,11,7] and essential oils.[6]
Earlier scienti c investigation of Hygrophila auriculata
(K. Schum) Heine showed that the crude extract has antinociceptive,[12] antitumor,[13,14] antibacterial,[15,16] antioxidant,[17,18]
hepatoprotective,[19,20,21] hypoglycemic,[22] haematinic,[23]
diuretic,[24] anabolic and androgenic activities,[25] anthelmintic
activity,[26] anti-in ammatory and antipyretic activity.[27]
Hence, the present study was designed to study the detail
pharmacognostic study of the leaves, stem and roots including
macroscopy, microscopy, quantitative microscopy,
determination of various physicochemical parameters,
uorescence characters of powder, phytochemical screening
and TLC and HPTLC pro le of extracts, and determination
of total phenolic contents etc.

MATERIALS AND METHODS

Description of Hygrophila auriculata
(K. Schum) Heine

The plant is a sub shrub, usually growing in marshy places

along water courses. The stem is reddish brown and the
shoot has 8 leaves and six thorns at each node (Figure 1, 2).

Figure 1, 2: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Exomorphic features of the plant-Shoots
showing axillary flowers, thorns, and cluster leaves.
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29

Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

The leaves occur in whorls, the outer pair of leaves are

larger, lanceolate, scalerous, margins are minutely dentate,
subsessile, thorns strong straight or curved. Flowers occur
in axillary whorls, bract and bracteoles leafy. Calyx four
lobed, lobes unequal. Corolla, -5 petals gamopetalous,
unequally 2- lipped, middle lobe of the lower lip with yellow
palate; corolla purple coloured. Stamens - four, in two pair,
laments unequal; anthers divergent; ovary two celled; four
ovules in each cell. Fruit dehiscent capsule.
Collection of Plant materials

The fresh plants of Hygrophila auriculata (K. Schum) Heine

were collected from the eld area of Baryahi, Saharsa
District Bihar, India, in January 2009. The plant specimen
was authenticated by Dr. Anjani kumar Sinha, Principal,
M L T Saharsa College Saharsa, Saharsa, Bihar. A voucher
specimen no SHC 55/01/2009 has been deposited at
the herbarium, Department of Dept. of Botany, M L T
Saharsa College Saharsa- 852201. Care was taken to select
healthy plants and for normal organs. The required
samples of different organs were cut and removed from
the plant and xed in formalin acetic acid solution
(formalin: acetic acid: 70% ethyl alcohol in the ratio of
0.5:0.5:9). After 24 h of xation, the specimens were
dehydrated with graded series of tertiary-Butyl alcohol
as per the schedule given by Sass, 1940.[28] In ltration of
the specimens was carried by gradual addition of paraf n
wax (melting point 58-60 C) until thiobarbituric acid
solution attained super saturation. The specimens were
casted into paraf n blocks.
Preparation of sections

The paraf n embedded specimens were sectioned with

the help of a rotary microtome. 10-12 m thickness of
the sections was made. However, dewaxing of the sections
was done using customary procedure.[29] The sections
were later stained with toluidine blue as per the method
published by OBrien et al. (1964).[30] Since toluidine blue
is a polychromatic stain, the staining was remarkably good
and yielded varied cytochemical reactions. The dye rendered
pink color to the cellulose walls, blue to ligni ed cells, dark
green to suberin, violet to the mucilage, blue to the protein
bodies etc. However, where necessary, sections were also
stained with safranin, fast-green and iodine-potassium
iodide for starch. In studying the stomatal morphology,
venation pattern and trichomes distribution, paradermal
sections taken parallel to the surface of leaf were cleared
with 5% sodium hydroxide and epidermal peeling was
prepared through partial maceration by employing Jeffreys
maceration uid.[28] Cleared sections were then mounted
with glycerin for microscopical observation. Moreover,
powdered materials of different parts of the plant were
also cleared with sodium hydroxide and mounted in glycerin
medium after staining.
30

Photomicrographs

Microscopic descriptions of tissues are supplemented with

micrographs wherever necessary. Photomicrographs of
different magni cations were taken with Nikon Labphot
2 microscopic unit. For normal observation, a bright eld
microscopy was used and for the study of crystals, starch
grains and ligni ed cells, a polarized light was employed.
However, since these structures have birefringent property,
under polarized light they tend to appear bright against the
dark background. Magni cations of the gures are indicated
by the scale-bars. Descriptive terms of the anatomical
features are as per the standard anatomy books.[31]
Extraction and Fractionation

The air-dried plants of Hygrophila auriculata (K. Schum)

Heine were prepared as a coarse powder and 500 g of this
powdered material was macerated with distilled water and
95% w/v alcohol separately for 24 hrs and 72 hrs, respectively.
Then, the individual extracts were ltered through a muslin
cloth and the resultant ltrates were concentrated under
reduced pressure and vacuum dried. The yield of aqueous
extract was 43% w/w and that of the alcoholic extract was
29% w/w. The alcoholic extract was taken and suspended
in water and successively fractionated with petroleum ether,
chloroform, ethyl acetate and n-butanol.[32] The percentage
yield of the petroleum ether, chloroform, ethyl acetate
and n-butanol fractions was 5.78%, 3.93%, 2.09% and
1.19% w/w, respectively.
Quantitative investigation

Quantitative leaf microscopy to determine palisade ratio,

stomata number, stomata index, Vein-islet number and veinlet termination number were carried out on epidermal
strips.[31] Other parameters determined for the powdered
drug of Hygrophila auriculata (K.Schum) Heine were uorescent
analysis,[34,35] total ash, acid-insoluble ash, water-soluble ash,
alcohol (90% ethanol) and water soluble extractive values,
swelling indices and foreign matter.[34] Calibrated digital pH
meter was used to measure the pH of 1 and 10% aqueous
extracts and also loss on drying was noted.
Phytochemical screening and high performance
thin layer chromatography (HPTLC) fingerprinting

The preliminary phytochemical investigation of the aqueous

and other fractions of Hygrophila auriculata showed the
presence of avonoids, terpenoid saponins, and tannins as
the major phytoconstituents. In particular, the chemical
tests on the n-butanol fraction demonstrated the presence
of tepenoids. It has been shown that the plant Hygrophila
auriculata is unique with regard to its content of avonoids
and terpenoids.[35] A distinct chemopro le of the terpenoid
fraction of Hygrophila auriculata (K. Schum) Heine was
obtained using HPTLC. Precoated TLC plates of silica-gel
60 F254 (E. Merck, India), 0.2 mm thick, were used. Ten
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Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

microliters of the n-butanol fraction was spotted in the

form of a band using a CAMAG Linomat -V Automatic
Spotter (CAMAG, Switzerland) (Figure 14). The TLC pattern
of the n-butanol fraction was developed using n- hexane:
ethyl acetate (7:3) as a solvent system.[38,39,40] Then, the plates
were scanned in the CAMAG TLC Scanner-III and the
peaks were recorded at a wavelength of 366 nm. The TLC
and HPTLC ngerprinting studies of the n-butanol fraction
showed the presence of various terpenoids with their
respective Rf values (Table 1). The observed Rf values
from TLC agreed with the HPTLC ngerprints.

median vertical plane and 1Pm in horizontal plane. In the

adaxial part, the epidermis is prominent with squarish cells
and prominent cuticle. Beneath the epidermis occur about
three layers of small collenchyma cells. Further below the
collenchyma occur four or ve layers of wide thin walled
parenchyma cells. The abaxial part of the midrib has epidermis
similar to adaxial side. These may be one or two layers of

Determination of total phenolic contents

The total phenolic content was determined according to

the method described by Singleton (1999).[41] A suitable
aliquot of the TRF was placed in test tubes and made up
to 1 ml with distilled water. Then, 0.5 ml Folin-Ciocalteu
reagent (1:1 with water) and 2.5 ml sodium carbonate
solution (20%) were added sequentially to each tube. Then,
the tubes were vortexed for 2 min, kept in the dark for
40 min and the absorbance was recorded at 725 nm. The
amount of total phenolics was calculated as gallic acid
equivalents/mg of extract (Table 2).

RESULTS AND DISCUSSION

Anatomy of Leaf

The leaf is dorsiventral, smooth and even with fairly

prominent midrib (Figure 3). The midrib is Plano convex
in sectional view with at adaxial side and broadly semicircular
abaxial side (Figure 4). The midrib is 750 Pm along the

Figure 3: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Anatomy of the leaf - T.S of Leaf through
midrib with lamina.

Table 1: TLC profile of n-butanol fraction

of Hygrophila auriculata (K. Schum) Heine
S. No.
1
2
3
4
5

Solvent system
n- Hexane:Ethyl acetate
(7:3)

Rf value
of track-1

Rf value
of track-2

0.37
0.45
0.76
0.84
0.94

0.35
0.39
0.54
0.64
0.70

Table 2: Determination of total phenolic contents of

different extracts/fractions of Hygrophila auriculata
(K. Schum) Heine
Total phenolic contents
Extracts/ Fractions
Aqueous
Ethanolic
Pet ether
Chloroform
Ethyl acetate
n- Butanol

Phenolic contents (g/ml)

70.0
98.0
65.54
81.0
120.0
135.0

Pharmacognosy Journal | July 2011 | Vol 3 | Issue 23

Figure 4: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: T.S of Midrib enlarged.
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Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

collenchyma inner to the abaxial epidermis. The remaining

ground tissue consists of wide, compact, thin walled
parenchyma cells. The vascular bundle is single and elliptical
in cross - sectional view. It is 350 Pm horizontally and 150 Pm
vertically. It consists of 8-10, parallel rows of xylem elements
which are angular, thin walled and narrow. Phloem occurs
as a thin sheath along the abaxial side of the xylem. These
are two small, less prominent, circular accessory strands on
the adaxial part. They are circular with a cluster of xylem
elements and small nest of phloem elements.
Lamina

The lamina is uniform in thickness excepting the places

where lateral veins and veinlets are situated. The lateral
view is raised slightly on the adaxial and abaxial sides.
It consists of a small top-shaped collateral vascular
bundle surrounded by parenchymatous cells which extend
both adaxially and abaxially (Figure 5, 6). The lateral
vein is 400 Pm thick. The vein lets also project slightly
on the lower side. They have small cluster of xylem and
phloem with parenchymatous bundle sheath without
extensions.
The mesophyll is differentiated into adaxial zone of palisade
cells and abaxial spongy mesophyll tissue. The palisade
zone consists of a single row of thin pillar like cells which
are 100 Pm in height. The spongy mesophyll has four or
ve layers of small, lobed cells which are interlinked with
each other forming wide aeranchymatous tissue. The adaxial
epidermis is 30Pm thick. The abaxial epidermis is 20Pm
thick. Both of them are stomatiferous (Figure 7).

Cystolits

Calcium carbonate crystals of cystoliths are abundant in

the adaxial epidermis of the leaf (Figure 5 and 9). The
cystoliths are long, spindle shaped, straight or curved with
warty surface. They are 200 Pm long and 20 Pm thick. They
occur is specialized cells which are elongated and candle
like; these modi ed cells in which the cystoliths occur are
called lithocysts.
Stomata

Occur on both surfaces of the leaf. They are equal in

abundance and frequency in the upper and lower sides
(Figure 8, 9). The stomata frequency is 45-50/Pm2. The
stomata are predominantly diacytic type with two subsidiary
cells with their common walls at right angles to the stomatal
axis. The subsidiary cells may be equal or unequal in size.
The epidermal cells are variable in shape; they are narrowly
rectangular, slightly lobed or squarish. Their anticlinal
walls are fairly thick, straight or slightly undulate. Some of
the epidermal cells are elongated, narrow and canal like.
These lithocysts contain calcium carbonate cystoliths
(Figure 8). Glandular trichomes are occasionally seen in
the epidermis. They are circular in surface the cells darkly
staining (Figure 10).
Petiole

In cross-sectional view, the petiole is wide at and boat

shaped. The adaxial side is at and abaxial side is convex
(Figure 11). The petiole has lateral, thick, abaxially hanging
wings on either side. The vascular system consists of
a at wide main bundle and two wing bundles. The main

Figure 5, 6: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: T.S of lamina through lateral vein.

32

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Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

bundle is placed in the control part of the petiole. It is at

measuring 950 Pm long and 250 Pm thick (Figure 13). It
consists of several short, radial rows of xylem elements,
the xylem elements are 2 or 3 in each row; they are angular
to circular and thick walled (Figure 11). Phloem occurs
in smallest beneath the xylem strands. The wing bundle
are circular, nearly 150 Pm is diameter and have four or
ve short rows of xylem elements and a small patch of

Figure 7: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: T.S of Leaf margin.

Figure 8: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Epidermal morphology - Adaxial epidermis
with stomata and cystolith, under low magnification.

Pharmacognosy Journal | July 2011 | Vol 3 | Issue 23

phloem (Figure 12). The ground tissue is homogeneous

and parenchymatous. The cells are wide, circular, thin walled
and compact.
Anatomy of Stem

The stem is roughly four angled in sectional view with wide

arenchymatous cortex and four angled stele (Figure 14).
The epidermis is thin and less conspicuous. The outer

Figure 9: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Abaxial epidermis with stomata.

Figure 10: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Glandular trichomes and lythocyst enlarged.

33

Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

Figure 11: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Anatomy of the petiole - T.S of Petiole
ground plane.

Figure 13: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Central median portion vascular bundles
enlarged.

Figure 12: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Wing portion of the petiole enlarged.

cortex consists of four or ve layers of radially aligned,

small, compact squarish parenchyma cells. This zone is
uniformly 150 Pm wide. The inner cortex in quite wider
comprising of about ve rows wide, circular air chambers
formed by reticulate layers of narrow aerenchyma cells.
The stele has four semicircular thicker bundles placed at
four corners and two smaller bundles positioned opposite
to each other. The larger and smaller bundles are interlinked
by a thin cylinder of small compact, dense xylem elements.
The vascular bundles are collateral with dense xylem bers,
34

Figure 14: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Anatomy of the Stem - T.S of stem
ground plane.

widely separated radial rows xylem vessels and thin arc

of phloem. The pith is wide and parenchymatous. The pith
cells are circular less compact and thin walled.
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Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

Anatomy of Root

The root has intact, continuous rhizodermis (epidermis)

followed by two layers of tangentially oblong compact outer
cortex. The inner cortex is wide and aerenchymatous. Wide,
radially elongated air chambers are formed by thin, uniserate
partition laments, made up of thin walled parenchyma
cells. Some of the partition cells have thick walled, dilated
and squarish rectangular (Figure 15, 16). The vascular
cylinder has a thin endodermal layer and pericyclic layer.
ylem consists of ve exarch strands and a few wide angular
vessels in between the exarch strands. Phloem is in ve
small groups alternating with the primary xylem strands.
The central Part is narrow and parenchymatous (Figure 16).

vein terminations are long and slender, branched or

unbranched.
Abaxial epidermis fragments are seen which are
stomatiferous. The stomata are diacytic type. The epidermal
cells are small polygonal and thin walled; the walls are
straight (Figure 18).

Powder microscopy of Hygrophila auriculata

(K. Schum) Heine.

The macerated and powdered materials exhibited the

following components:
Organoleptic properties of powder drug

Colour - Brawnish green

Odour - Odourless
Taste - Tasteless
Small fragments of lamina shows the venation patterns
and vein lets with vein terminations (Figure 17). The
vein lets are prominent and form distinct vein-islets.
The Vein-islets are square shaped or rectangular. The
Figure 16: Macro- and micrscopical characters of the plant Hygrophila
auriculata (K. Schum) Heine: T.S of root entire view

Figure 15: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Anatomy of the root - T.S of root a sector
enlarged.
Pharmacognosy Journal | July 2011 | Vol 3 | Issue 23

Figure 17: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Leaf venation pattern showing vein islet
and vein termination.
35

Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

Cystoliths: Large, cylindrical cystoliths are abundant in

the mesophyll and epidermis. They are broad at one end
and narrow at the other end. The surface of the cystolith
is warty or spiny. The cystolith is 200 m long and 30 m
thick at the base (Figure 18).
Epidermal trichomes (Figure 19, 20). Covering types of
trichomes are frequently seen the powder. The trichomes

are multicellular, uniseriate, unbranched and smooth

walled. The marginal trichomes are slightly curved
(Figure 17) while surface trichomes are straight. The
trichomes are up to 1 cm long and 100 m broad at the
base. The powdered drug shows vessels element, xylem
bers and xylem parenchyma. Vessel elements are
long, cylindrical and thin walled (Figure 21). The vessel
elements are 250 m long and xylem bers may be wide

Figure 18: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Abaxial epidermis showing stomata and
cystolith

Figure 20: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Surface straight trichomes

Figure 19: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Marginal curved trichomes

Figure 21: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: One vessels element with tail.

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Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

and narrow
they are thick walled. The bers 450600 m in length (Figure 22).
Xylem parenchyma: Narrowly rectangular, thin walled
parenchyma cells are seen bundles. They are 140 m
long. They have no pits (Figure 22).
Quantitative investigation of Hygrophila auriculata
(K. Schum) Heine

The quantitative determination of some pharmacognostic

parameters is useful for setting standards for crude
drugs. The vein islet, and vein termination numbers and
the other parameters determined in the quantitative
microscopy, are relatively constant for plants and can be
used to differentiate closely related species. Quantitative
microscopy was performed using standard procedures
(Table 3). The uorescence analysis of the powdered drug
of Hygrophila auriculata (K. Schum) Heine in various solvents
and chemical reagents was performed under normal and
Ultra Violet (UV) light (Table 4). The physico-chemical
characters of powdered drug of Hygrophila auriculata (K.
Schum) Heine such as total alcohol soluble extractive, water
soluble extractive, ash value, acid insoluble ash, water-soluble

ash, loss on drying, swelling index, and foreign matter are

presented in table 5.
Phytochemical screening and high performance
thin layer chromatography (HPTLC) fingerprinting

The phytochemical and pharmacognostical investigation

of the plant Hygrophila auriculata (Schum) was carried out
with standard protocol. Earlier literature review of the plant
Hygrophila auriculata (K. Schum) Heine revealed that no work
has been carried out with regard to pharmacognostical
studies except for its taxonomical identi cations. The
extraction of plant material was carried (cold maceration)
out with water and 95% w/v alcohol. The yield of aqueous
extract is 37% w/w and alcoholic yield is 21% w/w. Both
aqueous and alcoholic extracts and different fractions
revealed that presence of various phytoconstituents like
avonoids, terpenoids saponins, and tannins as major
phytoconstituents (Table 6). It has been shown that the
plant Hygrophila species is unique for avonoids and terpenoids

Table 4: Flourescence analysis of Hygrophila

auriculata (K. Schum) Heine
Solvent used

Day Light

UV light
254 nm

366 nm

1N HCl
50% HCl
50% HNO3

Light green
Light green
Light brown

Green colour
Green colour
Dark green

50% H2SO4
1N NaOH

Slightly red
Light yellow

Slight green
Dark green

Alcoholic NaOH
Methanol
Benzene
Acetone

Green colour
Dark green
Slight buff
Fluorescent
green
Dark green
Green
White

1% KOH
Lead acetate

Light green
Light green
Yellow
Brownish
green
Light brown
Slight green
Brownish
yellow
Brown
White

Dark green
Dark green
Blackish
green
Light green
Moderate
green
Dark green
Slightly pink
Green
Transparent

Distilled water

Clear

Ethyl acetate
Chloroform
FeCl3

Figure 22: Macro- and micrscopical characters of the plant Hygrophila

auriculata (K. Schum) Heine: Fibres

Dark green
White
Green

Table 5: Physico-chemical analysis of Hygrophila

auriculata (K. Schum) Heine

Table 3: Quantitative microscopy Hygrophila

auriculata (schum) Heine
Parameters

Range

Palisade ratio
Stomatal number Upper surface
Stomatal number Lower surface
Stomatal index Upper surface
Stomatal index Lower surface
Vein- islet number
Veinlet termination number

14.45
21.78
24.26
15.4
25.4
27.4
29.2

Pharmacognosy Journal | July 2011 | Vol 3 | Issue 23

Green
Florescent
white
Green

Light yellow
Dark green
Green

Quantitative parameter

Values obtained (%) w/w

Alcohol soluble extractive

Water soluble extractive
Total ash
Acid insoluble ash
Water soluble ash
Loss on drying
Swelling index
Foreign matter

5.12
24.96
9.90
1.48
8.35
6.30
2.0
1.10

37

Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

contents. To fractionate the above compounds alcoholic

extract was fractionated with various solvents from petroleum
ether, chloroform, ethyl acetate and ethyl acetate insoluble
fraction was fractioned with n-butanol. Phytochemcial test
on n-butanol fraction showed that presence of terpenoids
and thus n-butanol fraction was labeled as terpenoid fraction.
Further TLC and HPTLC nger printing studies on

n-butanol fraction showed presence of various terpenoids

with their respective Rf values. The preliminary HPTLC
studies revealed that the solvent system n- hexane: ethyl
acetate (7:3) was ideal and gave well resolved sample peaks.
The spots of the chromatogram were visualized at 366 nm
(Figure 23) with a 500 k lter at Rf values of 0.07, 0.36,
0.50, 0.57, 0.73, 0.84, 0.94 and 0.97. The densitometric

Table 6: Phytochemical analysis of various extracts/fractions of Hygrophila auriculata (K. Schum) Heine
Phytoconstituents

Aqueous
extract

Alcohlic
extract

Pet-Ether
fraction

Chloroform
fraction

Ethyl acetate
fraction

n-butanol
fraction

Alkaloids
Steroids
Flavonoids
Terpenoids
Tannins
Protein
Carbohydrate
Saponins
Glycosides

+
+
+

+
+

+
+
+
+
+

+
+

+
+

Negative = Absent
+ Positive = Present

Figure 23: HPTLC Finger printing of n-butanol fraction where peaks (1-8) represent Presence of various terpenoids scanned at wavelength
366 nm.
Abbreviations: Abs- Abaxial side; GT- Ground Tissue; La- Lamina; LV- Lateral vein; MR- Midrib; Ade-Adaxial epidermis; Adab- Adaxial accessory
bundle; Col- Collenchyma; EP-Epidermis; Ph- Phloem; GT-Ground tissue; PM-Palisade mesophyll; X- Xylem; Abe- Abaxial epidermis; Ads-Adaxial
side, BS- Bundle sheath extension; LV-Lateral vein; Ph-Phloem; St- Stomata; SM-Spongy mesophyll; SC Subsidiary cells; Cy- Cystolith; GTrGlandular trichomes; LC- Lithocyst; GT- Ground tissue; VB- Vascular Bundles; W- Wing; WB- Wing bundles; AC- Arenchymatous inner cortex;
OC- Outer cortex; Pi-Pith; Co-cortex; SPh- Secondary Phloem; SX- Secondary Xylem; CO- Cortex, Ve- Vessels, XR- Xylem ray; SX- Secondary
xylem; LV- Lateral vein; VT- Vein termination; PP- Perforation plate, T- Tail; X P- Xylem Fibres, XF- Xylem fibres
38

Pharmacognosy Journal | July 2011 | Vol 3 | Issue 23

Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

scanning at 366 nm gives ve major spots with an area of

16.87, 20.46, 12.26, 22.33 and 31.40% at Rf value of, 0.57,
0.73, 0.84, 0.94 and 0.97 respectively.

5.

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Hussain MS, Nazeer Ahamed KFH, Ravichandiran V and Ansari MZH.

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Hygrophila auriculata (K.Schum) Heine root extract. J Ethanopharmacol.
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Fernando MR, Nalinie Wickramasinghe SMD, Thabrew MI, Ariyananda PL

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Hussain MS, Nazeer Ahamed KFH, Ansari MZH. Preliminary Studies on

Diuretic Effect of Hygrophila auriculata (Schum) Heine in Rats. Inter J of
Health Res. 2009; 2(1):59-64.

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Determination of total phenolic contents

These extracts and fractions were found to possess various

phenolic levels in table 2 ranging from 70 to 135 ( g/ml).
The highest concentration of total phenolics was present
in ethyl acetate and n-butanol fraction of Hygrophila auriculata
(K. Schum) Heine. The content of total phenolic compounds
in ethyl acetate fraction was 120 g/ml and n-butanol
fraction was 135 g/ml.

CONCLUSION
In conclusion, the pharmacognostic investigations on
physicochemical characteristics and uorescence analysis
shows that authentic botanical of this crude drug prevents
adulteration, substitution and has a crucial role in standardization
of crude drugs. The preliminary phytochemical screening
of the whole Hygrophila auriculata (K. Schum) Heine indicates
the presence of secondary metabolities, having an essential
role in medicine. The current report will also help researchers
and scientists design strategies for resolving cases of
misidenti cation of plant material. However advance
experimentation is under way in our laboratory to elucidate
the speci c mechanism of action of Hygrophila auriculata
(K. Schum) Heine.

ACKNOWLEDGEMENT
The authors are thankful to the Dr. K.F.H. Nazeer Ahmaed,
Assistant Professor, department of pharmacology, Vels
college of pharmacy, Chennai, for his assistance and
encouragement. We extend our sincere thanks to Dr. Anjani
kumar Sinha, Principal, M. L. T. Saharsa College Saharsa,
Saharsa, Bihar, for providing authenticated sample of
Hygrophila auriculata (K. Schum) Heine. We also extend our
sincere thanks to Mrs. Saba Ansari, research scholar, faculty
of pharmacy, Integral University, Lucknow, India, for
critically reading the manuscript and providing the valuable
suggestions.

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