Indian Journal of Traditional Knowledge
Vol. 12 (4), October 2013, pp. 693-698
Pharmacognostic evaluation of stem, leaves and roots of
Merremia tridentata (L.) Hallier f.
Aron S, Maria Francis Jeffrey Bose N & Mehalingam P*
Research Department of Botany, VHN Senthikumara Nadar College (Autonomous), Virudhunagar– 626 001, Tamil Nadu, India
E-mail: [email protected]
Received 03.01.12, revised 24.06.13
Pharmacognostic investigation of leaf, stem and root of Merremia tridentata (L.) Hallier. f. was carried out to determine
its macromorphological, micromorphological, and chemomicromorphological profiles. The anatomy of the leaf reveals that
collateral vascular bundles, paracytic type of stomata, peltate type of glandular trichomes and druses calcium oxalates are
seen on the epidermis of the lamina. The stem shows the presence of bicollateral vascular cylinder, it consists of a few wide,
circular or angular vessels and xylem fibres. The root shows the presence of secondary phloem which is not distinct from the
cortex. The phloem rays are narrow and straight. Secondary xylem includes vessels, fibres and xylem rays. Preliminary
phytochemical analysis and HPTLC analysis was done along with fluorescence characteristics, extractive values,
quantitative estimation of ash values, acid insoluble ash, and water soluble ash which may serve as useful indices for the
correct identification of the powdered drug. Calcium oxalate crystals are abundant in the leaf and roots. This observation
would be immense value in the botanical identification and standardization of the drug in the crude form. This study would
be useful evidences for further investigations of this medicinal plant.
Keywords: Merremia tridentata, Pharmacognostic characterization, Microscopic characterization, Phytochemical analysis,
Fluorescence characters, Physico-chemical constants
IPC Int. Cl.8: A01D 9/07, A01D 23/06, A01D 1/68, A01D 4/02, A01D 4/54, A61K 36/00, A01D 20/19, A01D 20/26,
A01D 20/00
Merremia tridentata (L.) Hallier. f. belongs to the
family Convolvulaceae is a perennial, spreading
herb with thick root stock. It is distributed throughout
the India. M. tridentata is an important plant of
Indian Ayurvedic system of medicine which is used
in body pain, piles and toothache. In traditional
medicine system, this plant is used in swellings,
rheumatic affections, stiffness of the joints,
hemiplegia, urinary infections and general debility
apart from being a good laxative and astringent1.
In previous reports, M. tridentata had strong
wound healing, anti-inflammatory and anti-arthritic
activities2,3. The aerial parts of the M. tridentata
contain flavonoids, diosmetin, luteolin, and their 7-Οβ-D glucosides1. The acetone extract of root posses
high phenolic contents and rich potential of
antioxidant activity4. These plants are used as
toothache and dentifrice5. Despite the numerous
medicinal uses attributed to this plant, there are no
pharmacognostical reports on the leaves, stem and
——————
*Corresponding author
roots of this plant. Hence, the present work deals
with the morphological, anatomical evaluation,
physicochemical constants and preliminary phytochemical
screening which could serve as a valuable source of
information and provide suitable standards for the
further identification of this taxon.
Methodology
Collection of specimens
The leaf, stem and root of M. tridentata were
collected from Peraiyur village, Ramanathapuram
district, Tamil Nadu. Informed consent was obtained
from traditional herbal practitioners. The plant was
taxonomically identified by Dr. P. Jayaraman, Plant
Anatomy Research Centre, Chennai, Tamil Naud,
India. The voucher specimen (PARC/2011/259) was
deposited at Department of Botany VHNSN College,
Virudhunagar for future reference. Care was taken to
select health plants and normal organs. The required
samples of different organs were cut and removed
from the plant and fixed in FAA (Formalin (5 ml) +
Acetic acid (5ml) + 70% Ethyl alcohol (90 ml)). After
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INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12 (4), OCTOBER 2013
24 hrs of fixing, the specimens were dehydrated with
graded series of Tertiary- Butyl alcohol. Infiltration of
the specimens was carried by gradual addition of
paraffin wax (melting point 58-60° C) until TBA
solution attained its super saturation. The specimens
were cast into paraffin blocks.
Sectioning
The paraffin embedded specimens were sectioned
with the help of Rotary Microtome. The thickness of
the sections was 10-12 µm. Dewaxed sections were
stained with Toluidine blue. Wherever necessary,
the sections were also stained with Safranin and
Fast green. For studying the stomatal morphology,
venation pattern and trichome distribution, paradermal
sections (sections taken parallel to the surface of leaf)
were taken followed by clearing of leaf with 5%
Sodium hydroxide or epidermal peeling by partial
maceration by Jeffrey’s maceration fluid. Glycerine
mounted temporary preparations were made for
macerated/cleared materials. Powdered materials of
different parts were cleared with NaOH and mounted
in glycerine medium after staining. Different cell
component were studied and measured.
Photomicrographs
Microscopic descriptions of tissues were
supplemented with micrographs wherever necessary.
Photographs of different magnifications were taken
with Nikon lab photo 2 microscopic unit. For normal
observations bright field was used. For the study of
crystals, starch grains and lignified cells, polarized
light was employed. Since, these structures have
birefringence property under polarized light, they
appear bright against dark background. Magnifications
of the figures are indicated by the scale-bars.
HPTLC finger print profile
The HPTLC finger print profile of ethanol extracts
of M. tridentata leaves were performed on aluminium
plate pre-coated with silica gel 60 F254 of 0.2 mm
thickness as adsorbent and employing CAMAG
LINOMAT IV applicator. The mobile phase used
was ethyl acetate: hexane (6:4 v/v). The plate after
air drying was scanned using CAMAG TLC Scanner
II with WINCATS 4.05 version software at a
wavelength of UV 254 and using deuterium lamp9.
Results and discussion
(Figs. 1A–P)
Morphological/Macroscopic characters
Leaves-simple, spathulate, oblong to pandurate,
1.5-2.5 (3) × 0.7-1 cm, broader towards apex,
chartaceous, penninerved, glabrescent, base truncate,
hastate to auriculate, margin-entire, apex- acute, obtuse,
rarely retuse, mucronate, basal lobes-3 or 4, toothed;
petiole-0.3cm. Flower (s)-solitary, rarely cymose;
pedicel-1.5 cm. Calyx-lobes 5, subequal ovate, 6mm,
outer-3, acuminate, inner-2, acute. Corolla-cream to
yellowish with a purple throat, 1.5 cm-across, 1cm-long.
Stamens-5 to 7 mm; anthers 1.5 mm. Ovary-globose,
1mm; Style-6 mm; Stigma-capitate. Capsule-0.7 cmacross; seeds-glabrous (Fig. 1 A).
Microscopic characters
Physicochemical parameters of M. tridentata leaf,
stem and root powder were determined and reported
as total ash, water-soluble ash, acid-insoluble ash,
alcohol-soluble extractive, water-soluble extractive
and moisture content6.
The leaf is distinctly bilateral with reference to
the planoconvex midrib and dorsiventral lamina.
The midrib is flat on the adaxial side and semicircular
on the abaxial side. It is 180 µm thick and 240 µm
wide. It consists of fairly large circular epidermal
cells on the abaxial part and narrow tabular thin
walled cells on the adaxial side (Fig. 1 B). Inner to the
abaxial epidermis is seen 4-6 layers of spongy
mesophyll tissue forming a deep arc. The remaining
portion of the midrib ground tissue is parenchymatous
and the cells being large, angular and compact.
The vascular strand is small, collateral and broadly
conical in outline. It consists of a mass of xylem
elements, not arranged in rows; phloem occurs in thin
arc beneath xylem strand.
Preliminary phytochemical analysis
Lateral vein
Shaded dried and powdered plant samples were
successively extracted with ethanol, acetone and ethyl
acetate. The extracts were filtered and concentrated
using vacuum distillation. The different extracts were
subjected to qualitative tests for the identification of
various phytochemical constituents as per standard
procedure7,8.
The lateral vein is slightly thick. The epidermal
cells on the adaxial region of the lateral vein become
vertically oblong and papillate. There is a small
circular vascular strand with a few xylem elements
and phloem elements. The vascular strand is
surrounded by a single layer of parenchymatous
bundle sheath with adaxial extension.
Physico-chemical constants
ARON et al.: PHARMACOGNOSTIC STANDARDIZATION OF MERREMIA TRIDENTATA (L.) HALLIER. F.
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Fig.–1 A-P: A) Habit of Merremia tridentata L. B) TS of leaf through midrib and lamina, C) TS of lamina showing adaxial stoma, D)
Paradermal view of the epidermis showing stomata and glandular trichome, E) TS of lamina trichome-enlarged trichome on the abaxial
epidermis, F) Lamina-cleared to show the venation pattern, G) Vein termination with terminal tracheid, H) TS of thin stem entire view, I)
TS of thick root a sector enlarged, J) TS of thick root-secondary phloem, K) Druses in the mesophyll of the lamina, L) Druses in the
Phloem Parenchyma of the Root, M) Epidermal peeling of the stem, N) Non glandular epidermis peeling, O) Fibriform vessel elements, P)
Fibres
Abbreviations: AbE: Abaxial Epidermis, AdE: Adaxial Epidermis, Ads: Adaxial Stoma, Co: Cortex, Cr: Crystals, EC: Epidermal cells,
Ep: Epidermis, ETr: Epidermal (Non Glandular) Trichome, Fi: Fibres, GT: Ground Tissue, GTr: Glandular Trichome, IPh: Inner Phloem,
La: Lamina, MR: Midrib, MT: Mesophyll Tissue, OPh: Outer Phloem, Pe: Periderm, Ph: Phloem, PhR: Phloem Rays, Pi: Pith,
PM: Palisade Mesophyll, SC: Subsidiary Cells, SE: Sieve Elements, Sc: Sclerenchyma, SM: Spongy Mesophyll, SPh: Secondary Phloem,
St: Stomata, SX: Secondary Xylem, TTr: Terminal Tracheid, Ve: Vessel, VI: Vein-Islets, VS: Vascular Strand, VT: Vein Termination,
X: Xylem, XF: Xylem Fibres.
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INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12 (4), OCTOBER 2013
Lamina
The lamina is 100 µm thick. The adaxial and
abaxial epidermal layers have horizontally stretched,
thin walled tabular cells. Stomata are present on both
adaxial and abaxial sides (Fig. 1 C).The mesophyll
tissue is differentiated into adaxial band of palisade
cells and abaxial zone of spongy parenchyma.
Palisade layer consists of two layers of short
cylindrical cells. The spongy parenchyma cells are in
4-6 layers; they are small, spherical or lobed and are
loosely arranged with intercellular spaces.
Epidermal cells and stomata
As seen in paradermal sections, the epidermal cells
are fairly large, varying in shape and size with slightly
wavy anticlinal walls. Faint cuticular striations are
seen in dense and compact parallel lines on the
surface of the cells.
Stomata are dense and diffuse in distribution. They
are paracytic type; each stoma has two unequal
subsidiary cells lying parallel to the long axis of the
guard cells (Fig. 1 D). The guard cells are 40 µm long
and 35 µm wide.
Epidermal glandular trichomes
Subsessile, peltate type glandular trichomes are
often seen on the epidermis of the lamina. It has short,
less prominent stalk and spherical body. In surface
view the gland appears circular and plate like, consists
of about 8 triangular radiating cells; the cells have
dense darkly stained contents (Figs. 1 D& E). The
gland is 25 µm diameters.
Venation pattern
The veins are gradually thinner, the primary veins
being thicker, secondary and tertiary veins being
reduced in thickness. The vein-islets are well defined
by distinct vein-boundaries. The islets are variable in
shape and size. The vein-termination are invariably
seen in all islets. They are unbranched, branched once
or twice (Fig. 1 F). The terminatious are straight or
undulate. One or two short dilated terminal tracheids
are seen at the end of the termination (Fig. 1 G). The
tracheids have annular thickenings.
Stem
Young stem
Both young and mature stems were studied. The
young stem is five angled and five ridged (Fig. 1 H).
It is 1.8mm thick. The ridges are 200 µm long.
The young stem consists of a layer of epidermis
with circular cells. The subepidermal layer is
chlorenchymatous. The remaining portion of the
cortex is parenchymatous; there are 4-6 layers
of small circular parenchyma cells. The vascular
cylinder is circular, hollow and comprises mostly
primary xylem and phloem. There is a thin layer of
sclerenchyma cells along the outer boundary of the
vascular cylinder. Phloem is thin and continuous.
Xylem elements occur in regular radial rows; each
row consists of 3 or 4 xylem elements. The pith is
parenchymatous. Small nests of phloem elements are
seen around the pith portion. Secondary xylem is
thick, solid cylinder of wide scattered vessels with
sclerenchymatous ground tissue. The vessels in the
central portion are narrow; they are wider towards the
periphery.
Old stem
The old stem is also 5 angled with five thick short
ridges. The stem is 2.4 mm thick. It has well defined,
thick epidermal layer of rectangular or circular
cells with prominent cuticle. A single layer of
chlorenchyma cells occur inner to the epidermis. The
cortex is narrow consisting of about 4 layers of
parenchyma cells. The inner boundary of the cortex
consists of a ring of isolated sclerenchyma cells. The
vascular cylinder is bicollateral. It consists of outer
thin continuous layer of phloem and small groups
of isolated inner phloem or medullary phloem.
In between the phloem cylinders, the secondary
and primary xylem cylinder is present. The primary
xylem occurs in a regular ring of many radial rows
of 3 or 4 xylem elements. Secondary xylem occurs
outside the primary xylem. It consists of a few wide,
circular or angular vessels and xylem fibres (Fig. 1 I).
The vessels are 150-250 µm wide. Some of the pith
cells in the centre are disintegrated and forming an
irregular cavity.
Root
Thin root
The thin root is 1.9 mm thick. It has rough surface,
narrow periderm, wide cortex and thick, solid
vascular cylinder. The epidermis has been peeled off
leaving a narrow, less distinct periderm of about
6 layers of narrow tabular cells. The cortical zone
is fairly wide and includes up to the layers of
tangentially compressed parenchyma cells. Secondary
phloem is a narrow cylinder of small radial files
of cells.
ARON et al.: PHARMACOGNOSTIC STANDARDIZATION OF MERREMIA TRIDENTATA (L.) HALLIER. F.
Thick root
The thick root is 3.17 mm diameter. It consists
of thin, but distinct periderm, narrow cortex, wide
continuous secondary phloem and thick, dense solid
xylem cylinder. The surface of the root is covered by
dark disintegrated cells. The periderm consists of
four layers of tabular thin walled cells. The cortex is
narrow comprising three or four layers of compressed
cells. Secondary phloem is not distinct from the
cortex. The phloem consists of narrow compact
parallel lines of cells. The phloem rays are narrow
and straight. The sieve elements are rectangular in
sectional view and are arranged in regular radial lines
along with the companion cells and phloem
parenchyma (Fig. 1 J).
Secondary xylem is 900 µm thick. It includes
vessels, fibres and xylem rays. One or two growth
rings are seen in the xylem cylinder. The vessels are
semi-ring porous. The diameter of the vessels is
narrow in the centre and become wider at the end of a
growth. The vessels are circular to ovate or elliptical,
wide, thin walled and either solitary or in multiples
of two or three. The narrow vessels are 20 µm in
diameter. The wide vessels are 100 µm in diameter.
Xylem fibres are narrow, thick walled and lignified.
697
a) Glandular trichomes are peltate type, sub-sessile,
multicellular and discoid located in shallow
depression of the epidermis. The glandular
body consists of 8-12 triangular cells radiating
from a central point (Fig. 1 M).
b) Non-glandular
trichomes
are
unicellular,
unbranched and tapering into a pointed tip
(Fig. 1 N). They have thick lignified walls and
narrow lumen. The trichome is 250 µm long.
Fibres
Xylem fibres are abundant in the powder (Fig. 1 O).
They are very thin, long and thick walled and
lignified. Simple, circular pits are well developed on
their walls. The pits are in two vertical rows. The
fibres are 1.1 mm long and 15 µm thick.
Vessel elements
Unique types of vessel elements are quite abundant
in the powder. In length and thick, they look like
fibres. So, they are called fibriform-vessel elements.
They have multiseriate pits on the lateral walls.
The perforation on the end walls is simple oblique
or horizontal (Fig. 1 P). The vessel elements are
900 µm long.
Crystal distributions
Parenchyma cells
Calcium oxalate crystals are abundant in the leaf
and root. The crystals are druses or sphaero raphides.
In the leaf, the druses occur in the mesophyll tissue.
They are random in distribution (Fig. 1 K). In the
root, the druses are located in the parenchyma cells
of the secondary phloem (Fig. 1 L). The druses in
the leaf are larger and denser than those in the root.
The druses are 30-40 µm in diameter.
Wide, rectangular, thin walled parenchyma cells
are seen in vertical strands. They have wide, circular
simple pits. No cell inclusions are seen in the cells.
Powder Microscopy
Preliminary phytochemical screening and HPTLC profile
Powder preparation of the plant exhibited the
following component when examined under the
microscope.
In preliminary phytochemical screening, the
ethanol extract showed the presence of alkaloids,
Epidermal peeling
Fragments of epidermal peeling of the stem are
frequently seen in the powder. In surface view, the
peelings exhibit the epidermal cells and stomata. The
epidermal cells appear vertically aligned rectangular
thin walled cells. Stomata are abundant and they are
arranged in vertical paralleled rows. The stomata are
paracytic type with two vertically oblong parallel
subsidiary cells (Fig. 1 M).
Epidermal trichomes
Two types of epidermal trichomes are seen in
the powder
Physico-chemical/ Fluorescence studies
The results of physico- chemical analysis,
extractive values and fluorescence characters are
given in Tables 1 & 2.
Table 1—Physico-chemical constant of M. tridentate (L.)
Hallier. f.
Particulars
Total ash value
Water soluble ash value
Sulphated ash value
Acid insoluble ash value
Moisture content
Extractive values (Successive extraction)
a) Ethyl acetate
b) Acetone
c) Ethanol
d) Water
values % w/w
15.3
5.8
4.5
3.1
11.2
4
6
9
12
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INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12 (4), OCTOBER 2013
Table 2—Fluorescence analysis of M. tridentata (L.) Hallier. f.
Particulars of the treatment
Powder +Benzene
Powder + Ethyl acetate
Powder + 50% Aqueous ethanol
Powder +1M HCl
Powder +1 N NaOH Aqueous
Powder + 1N NaOH Alcoholic
Powder + Acetic acid
Powder + Nitric acid + Ammonia
Powder + Conc Nitric acid
Powder + 50% H2SO4
Day light
254nm
Brown
Green
Green
Brown
Brown
Green
Green
Yellow
Yellow
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
carbohydrates, amino acids, phytosterols, phenols,
flavonoids, tannins, saponins and quinones. The
ethyl acetate extract has tested positively for
phytosterols, flavonoids, saponins and quinones.
Acetone extract showed the presence of alkaloids,
phenols, phytosterols, flavonoids and tannins. All
the three extracts have phytosterols and flavonoids
compounds. These metabolites have been shown
to be responsible for various therapeutic activities
of medicinal plants10. The HPTLC profile of the
ethanol extract (20µl) of M. tridentata revealed 11
phytoconstituents at Rf value 0.03, 0.04, 0.20, 0.26,
0.31, 0.38, 0.50, 0.54, 0.56, 0.65 and 0.74.
The pharmacognostic characters and phytochemical
values reported in this work may play a major role in
setting some diagnostic indices for the identification
and preparation of a monograph of the plant, which
might broaden its pharmacological, botanical and
economical importance. With the help of this
referential information, a researcher can easily reject
the fake and adulterated plant products which are
deviated from the above mentioned characters
and select the correct herbal specimen for further
investigations.
Conclusion
Merremia tridentata has numerous uses in
traditional medicine to treat several ailments like
piles, body pain and toothache. Due to its wide
therapeutic importance it is worthwhile to standardize
it for use as drug. The present study reveals
standardization profile of drug M.tritentata, which
would be of immense value in botanical identification
and authentication of plant drug and may help us in
preventing its adulteration.
Acknowledgement
The authors are thankful to traditional herbal
practitioners for their willingness to share their
valuable knowledge and for providing ethnobotanical
information. Authors are sincerely thankful to
University Grants Commission, New Delhi for
providing financial assistance to carry out this work.
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