Indian Journal of Traditional Knowledge
Vol. 17 (1), January 2018, pp 106 - 112.
Mazus pumilus (Burm. f.) Steenis; Pharmacognosy
Saiqa Ishtiaq1*, Uzma Hanif
2 & Muhammad Shaharyar Khan Afridi
1
1Punjab University College of Pharmacy, University of the Punjab, Allama Iqbal Campus-54000,
Lahore, Pakistan; 2Department of Botany, Government College University, Lahore, Pakistan
Email: [email protected]
Received 07 February 2017; revised 13 April 2017
Mazus pumilus (Burm. f.) Steenis, is a well-known traditional medicinal plant belonging to family Mazaceae. The
research work is about the pharmacognostic standardization of M. pumilus which includes; macroscopic features and
microscopic evaluation of leaf, stem and roots. TS of leaf, stem and root showed the arrangement of the different cells.
Histochemistry of TS of leaf, stem and root gave distinctive results with conc. HCl, phloroglucinol, ferric chloride, iodine
solution and Sudan III which indicated the presence of Ca+2 oxalate crystals, lignin, tannins, starch and oil cells,
respectively. Powder study of leaf depicted the presence of fibres, epidermal cells, resinous matter and vessels. The
powdered study of stem showed collenchyma, vessels, fibers, cortex cells with tracheids, and helical vessels. While, root
powder contained pithed vessels, cork cells, parenchyma and phelloderm. The quantitative analysis of TS of leaf was also
performed for the establishment of leaf constants. In fluorescence analysis of herb, different colors were observed under
ordinary light, short and high wavelength UV light. Phytochemical analysis of the methanolic extract of whole herb
confirmed the presence of alkaloids, glycosides, flavonoids, saponins, sterols, triterpenoids, carbohydrates, proteins and
tannins. All these results will help in identification, confirmation and quality characterization besides, laying down the
pharmacopoeial standards for M. pumilus.
Keywords: Mazus pumilus, Mazus japonicas, Histochemistry, Pharmacognostic, Phytochemical, Pharmacognosy
IPC Int. cl.8: A61K 36/00, C07, C08, C12, A61K 38/00, A61K 39/00
Plant based medicines are now popularized
worldwide and therefore there is a great demand of
medicinal plants for herbal medicine system. To meet
the need for this purpose the standardization of these
plant materials is very important1. Mazus is a genus of
low growing perennial plants consisting of around 30
species. This genus is generally found in damp
habitats in lowland or mountain regions of China,
Japan, South East Asia, Australia and New Zealand2.
Mazus pumilus (Burm. f.) Steens syn. Mazus japonius
(Thunb.) Kuntze syn. Mazus rugosus Lour.
is
commonly occurring glabrous annual hairy herb in
Punjab region of Pakistan3,4
. M. pumilus is used as a
traditional medicine in different areas of its
geographical distribution by the local natives. The
plant has been used as a whole as well as by parts,
specifically leaves5. Ethnomedicinally, the whole
plant has been employed as a remedy for epilepsy6.
The infusion of the whole plant is consumed as
tonic2,3
. Similarly, whole herb is utilized as an
antifebrile, emmenagogue and aperitive7,8
. Certain
species of the genus are edible9. In an ethnobotanical
survey, it is found to be used as fodder for cattle10
.
The herb also possesses antibacterial and antifungal
action11
. Modern researches have proven that various
leaf extracts of M. pumilus has high potential of
anticancer activity on human cancerous cell lines12
.
The juice of the herb is used in the cure of
typhoid13,14
. Due to its good antioxidant potential, the
plant material has shown cardioprotective effect.
Recent phytochemical investigations of M. pumilus
have revealed that it is enrich with saponins as a
major class of secondary metabolites15,16
.
In spite of a lot of attention to therapeutic effects;
there isn’t any detailed pharmacognostic data
available on structural morphology and other
physicochemical standards, as it is required for the
identification and quality standardization of the plant.
Therefore, an extensive anatomical, physicochemical
and phytochemical screening is required that will be
helpful to avoid any ambiguity17
. Morphological
results are helpful in explanation of an exclusive drug
with a major focus on quantitative and qualitative
microscopy18
. Therefore, the present research ___________
*Corresponding author
ISHTIAQ et al.: PHARMACOGNOSTIC STANDARDIZATION OF MAZUS PUMILUS (BURM. F.) STEENIS
107
comprises of anatomical, structural and histochemical
evaluations of the leaf, stem and root, along with the
whole herb assessment of physicochemical parameters
(ash values, extractive values), fluorescence analysis and
preliminary phytochemical properties.
Methodology
Plant material
The plant was collected from botanical garden of
Government College University Lahore Pakistan, and
was authenticated by Dr. Uzma Hanif, Assistant
Professor, Department of Botany, Government
College University Lahore Pakistan. A specimen of
plant was deposited in herbarium of Government
College University, Lahore Pakistan, under voucher
No: GC. Herb. Bot. 2270. All plant parts were dried
under shade, were powdered and preserved in brown
containers at dry place.
Pharmacognostic evaluations
Macroscopic evaluation
All macroscopic evaluations were carried out on 5
samples of each part. The taxonomical description
was made according to the related articles and the
data given in books19
.
Microscopic evaluation Fresh leaves, stem and root were immediately fixed
in formalin: acetic acid: 70 % alcohol (5:5:90) for 24
h. TS of leaf, stem and root were made by commonly
used blade and razor method. Sections were stained
with safranin and fast green dye20
.
Histochemistry
The sections of leaf, stem and roots were taken and
they were treated with various chemicals to note the
chemical reaction and color change. Inferences were
made to evaluate the particular reaction for
histochemical analysis in the plant tissues used,
accordingly. Phlorogucinol, concentrated HCl, Iodine
solution, Ferric chloride and Sudan III solution were
used to locate the presence of Ca+2
oxalate crystals,
lignin, starch, tannins and oil globules, respectively.
After treatment with particular reagent the sections
were observed under light microscope and their
photographs were taken using digital camera21,22
.
Powder study Chloral hydrate (75 %) solution was used as
clearing reagent prior to observation of powder drug
using microscope. Slides of powdered leaves, stem
and roots were prepared according to the prescribed
procedures. Photographs were captured through the
microscope with digital camera23
.
Quantitative evaluation Microscopic examination is employed for the
quantitative evaluation and for this purpose some
specific histological features including, stomatal
index, vein-islet and vein termination number and
palisade ratio were noted24,25
.
Fluorescence analysis Ultraviolet fluorescence analysis of whole herb was
carried out by treating them with different reagents
and was observed in ordinary light and UV light
(short wavelength of 254 nm and long wavelength of
366 nm)26
.
Preliminary phytochemical analysis
The preliminary phytochemical analysis of whole
herb was carried out according to the standard
procedures27
.
Physicochemical analysis For the purpose of physicochemical analysis, whole
herb was powdered. The powdered samples were
subjected to analysis for their extractive values in
chloroform, ethyl acetate and n-butanol, along with total
ash, water soluble ash and acid insoluble ash values28,29
.
Results
Macroscopic evaluation
Mazus pumilus is an annual herb growing to 20 cm.
It is a branched herb, average 7 cm tall; stems hispid.
Lower leaves are opposite and closely packed, to 2 x
1.1 cm, obovate, obtuse, crenate to serrate, upper ones
alternate, smaller.
Microscopic evaluation TS of Leaf showing vascular bundles with xylem
and phloem tissue in the central area, which are
surrounded by the circular cells of collenchyma, with
an outer boundary of epidermal cells (labelled as a,
b&d in Fig. 1A). Trichomes were observed on the
surface of epidermal cells (labelled as c and a in Figs
1A&B). Abundant stomata were seen in the lower
epidermis (Fig. 1C).
Vascular bundles were observed around the central
pithy portion of the stem TS. Outer to the vascular
INDIAN J TRADIT KNOWLE, VOL. 17, NO.1, JANUARY 2018
108
bundles, cortex cells were observed with outer layer of
epidermal cells of stem (labelled as a, b, c & d in Fig.2).
The TS of root was almost elliptical in shape and
showed marginal epidermal cells, inner cortex,
pericycle and rounded pith cells (labelled as a, b, c &
d in Fig. 3).
Histochemical analysis of TS Histochemical analysis of the leaf, stem and root
was carried out. The transverse sections were exposed
to conc. HCl, phlorogucinol, ferric chloride, iodine
solution and Sudan III. The histochemistry of leaf
showed the presence of Ca+2
oxalate crystals, tannins,
and lignins as it gave effervescence with conc. HCl,
megnata coloration with phlorogucinol and black
coloration with ferric chloride solution. However, it
didn’t show any change when exposed to iodine
solution and Sudan III, which confirms the absence of
starch granules and oil globules.
Histochemistry of stem demonstrated the
presence of Ca+2
oxalate crystals, tannins, lignins
and oil globules, as an effervescent response was
observed conc. HCl, megnata coloration when
treated with phlorogucinol, greenish black
coloration in case of ferric chloride solution. While,
the presence of oil globules were confirmed by the
treatment with Sudan III, when the globules turned
pinkish red. The presence of aleurone and oil cells
was also confirmed in powder microscopy of stem.
However, it didn’t show any change when exposed
to iodine solution which confirms the absence of
starch granules in the stem.
On the other hand, transverse sections of root
unveiled the presence of Ca+2
oxalate crystals,
tannins, lignins and starch granules. Production of
tiny bubbles which were seen on the slide under the
microscope, megnata coloration of the section and
dark coloration of the of the cells were clear signs
of the presence of Ca+2
oxalate crystals, tannins and
lignins, respectively. In addition to that, on
exposure to iodine solution, blue shade appeared
within the slide in certain cells. This proved the
presence of starch granules in the root. It was
further verified in the powder microscopy of root.
Unlike stem, the TS of root is devoid of oil cells.
Powder microscopy
The powder microscopy of leaf shows; reticulate
vessels with inter costal region in which Ca+2
oxalate
crystals were distributed, thin asymmetric elongated
fibers with pointed edges, boarder pitted grouped
vessels, vein islets with brownish resinous matter
associated with it and brick shaped rectangular
epidermis cells (Fig. 4).
Fig. 1 — Microscopy of the leaf of M. pumilus
Fig. 3 — T.S. of root of M. pumilus showing a: Epidermis; b:
Cortex; c: Pericycle; d: Pith.
Fig. 2 — T.S. of stem of M. pumilus m; a: Vascular bundle with
xylem and phloem; b: Pith; c: Cortex; d: Epidermal cells
ISHTIAQ et al.: PHARMACOGNOSTIC STANDARDIZATION OF MAZUS PUMILUS (BURM. F.) STEENIS
109
The powder of stem highlighted the thin
polygonal cortex cells, group of pithed vessels,
collenchyma, irregular shaped Ca+2
oxalate crystals,
collenchyma, elongated fibers of xylem, i.e.
tracheids, aleurone and oil cells with round to
circular dispersed oil globules, flexible and twisted
helical vessels and thin fibers with visible lumen
and pointed edges (Fig. 5).
The powder microscopy of root revealed the
presence boarded pitted vessels that were fused into
each other, cells of phelloderm–a part of periderm
with crisscross network, brick shaped quadrilateral
secondary phloem tissues with Ca+2
oxalate crystals,
dense walled either penta or hexagonal cork cells
(phellem), small irregular shaped parenchyma cells
with starch granules in small aggregates, and thick
walled polygonal pith cells (Fig. 6).
Quantitative evaluation The quantitative evaluation helped in establishing leaf
constants including; palisade ratio (7.0 ± 0.31), stomatal
index (11.35 ± 0.41), vein-islet number (11.0 ± 0.55)
and vein termination number (15.5 ± 0.76) (Table 1).
Fluorescence analysis The fluorescence analysis of whole herb revealed
various colors of the extracts under ordinary light, Short
wavelength (254 nm) UV light, and Long wavelength
(366 nm) UV light indicating the presence of fluorescent
compounds in the methanolic extract (Table 2).
Preliminary phytochemical analysis The phytochemical screening of whole herb mainly
revealed the presence of terpenoids, sterols,
glycosides, flavonoids, alkaloids, carbohydrates,
tannins, saponins, and proteins (Table 3).
Physicochemical constants The extractive value of whole herb in ethyl acetate
was high, followed by n-butanol. The chloroform
extractive value was the lowest as compare to other
solvents (Table 4). The ash values of whole herb
showed high content of total ash and water-soluble
ash followed by acid insoluble ash (Table 5).
Discussion Herbal drug’s purity, safety, identity, and quality
can be maintained by means of standardization which
Fig. 4 — Powder microscopy of the leaf of M. pumilus
A: a. reticulate vessels, b. inter costal region, c. Ca+2 oxalate
crystals; B: group of vessels; C: epidermal cells; D: Vein islet
with resinous matter; E: Fiber
Fig. 5 — Powder microscopy of the stem of M. pumilus
A: Cortex; B: Aleurone and oil cells; C: Helical vessels of stem;
D: Collenchyma tissues in longitudinal view; E: Ca+2 oxalate
crystals; F: Single vessel with piths; G: Tracheid; H: Fiber
Fig. 6 — Powder microscopy of the root of M. pumilus
A: Group of bordered pithed vessels; B: Parenchyma with starch
granules; C: Pith cells; D: Phelloderm; E: Secondary phloem with
Ca+2 oxalate crystals; F: Thick walled cork cells
INDIAN J TRADIT KNOWLE, VOL. 17, NO.1, JANUARY 2018
110
involve both qualitative and quantitative parameters.
Among qualitative parameters microscopy is simple
and economical measure to identify source material.
On the other hand, the quantitative parameters involve
determination of stomatal index, vein islet, vein
termination number and palisade ratio. By these
values the purity of drug can be illustrated. Literature
revealed that both qualitative and quantitative studies
are important for identification of raw material during
drug manufacturing30,31
.
The transverse section of leaf showed the basic
profile of botanical anatomy showing the arrangement
of vascular bundles, collenchyma tissues, trichome
and epidermis. The abundant stomata were also
observed in the lower epidermis (Fig. 1). The TS of
stem vascular bundles arranged in circular form along
with pith cells, cortex and epidermis (Fig. 2). The TS
of root also disclosed the presence of cortex, pith,
epidermis and pericycle in the middle (Fig. 3).
Histochemical assessment of transverse sections
were carried out to acquire a realistic image on
preparatory scale at cellular level utilizing conc.
HCl, phloroglucinol, ferric chloride solution, iodine
solution, and Sudan III, which demonstrated the
existence of tannins, lignin and Ca+2
oxalate
crystals in the leaf, stem and root. Effervescent
response of all parts to conc. HCl indicted the
presence of Ca+2
oxalate crystals. Ferric chloride
solution determined the existence of tannins in all
parts by showing blackish coloration on exposure,
and megnata coloration due to the application of
phlorogucinol demonstrated presence of lignins.
Occurrence of oil globules in the pith cells of stem
was confirmed, as they turned pinkish red in color
on exposure of Sudan III dye, though results were
negative with leaf and root indicating they are
devoid of oil cells. Similarly, the iodine solution
gave positive result for starch granules in root,
when its exposure turned them blue. While, it gave
negative results for leaf and stem.
The powdered microscopy of the leaf of the herb
depicted the reticulate vessels, with inter costal region
among them, along with that thick walled vessels,
epidermal cells, thin irregular shaped fibers (Fig. 4).
The powdered micrographs of stem showed the
helical vessels, crystals of Ca+2
oxalate, aleurone with
oil forming cells. The compound and single vessels
with piths were also visible. Besides that, the basic
structures including cortex, collenchyma tissues, thin
fibers pointing at the edges, and tracheids (Fig. 5).
The powder study of root revealed bordered pithed
vessels, pith cells, secondary phloem tissues with Ca+2
crystals. In addition to them, thick and thin cork cells
were also observed. Phelloderm (modified cork cells)
was also seen (Fig. 6).
The quantitative evaluation of TS of leaf helped in
the establishment of leaf constants that proved to be a
helpful tool to aid in the authentication and
confirmation process of this therapeutically important
drug (Table 1). The fluorescence analysis is an
appreciated, easy and direct method for the
identification of fluorescent compounds. Different
compounds give fluorescence when they are exposed
to short and long wavelength UV light32
. The powder
of whole herb gave various colors when observed
under ordinary daylight, in short and long wavelength
of UV light Table 2. The preliminary phytochemical
screening of methanolic extract of M. pumilus whole
Table 2 — Fluorescence analysis of whole herb, M. pumalis
S. No. Chemicals
Observations
Ordinary light Short wave length
245nm
Long wave length
366nm
1 Water Light green Purplish blue Dark green
2 Chloroform Dark green Reddish Yellowish green
3 Aniline Dark red Brick red Dark green
4 Powder Light green Bluish green Light green
5 66% H2SO4 Brownish green Light green Dark green
6 50% H2SO4 Pale yellow Brownish black Dark green
7 50% HNO3 Lemon yellow Brownish green Dark green
8 5% NaOH Light green Brownish black Dark green
9 5% FeCl3 Brown Dark grey Dark green
10 10% KOH Yellowish green Reddish green Dark green
Table 1 — Leaf constants of leaves of M. pumilus
Leaf constants Range Mean ± SEM
Palisade ratio 6 – 8 7.0 ± 0.31
Stomatal index of lower epidermis 10.4 – 12.3 11.35 ± 0.41
Vein-islet number 9.5 – 12.5 11.0 ± 0.55
Vein termination number 13.2 – 17.8 15.5 ± 0.76
ISHTIAQ et al.: PHARMACOGNOSTIC STANDARDIZATION OF MAZUS PUMILUS (BURM. F.) STEENIS
111
herb, revealed the presence of various important
active constituents; carbohydrates, proteins, saponins,
alkaloids, glycosides, flavonoids, lipids, and
terpinoids (Table 3).
The extractive values are an integral part of
physicochemical analysis and these quantitative
values are determined for further standardization of
the plant powder. These values give a clear idea about
the yields of the extract in different solvents33
. The
evaluation was performed on whole herb with
chloroform, ethyl acetate and n-butanol and the
results are tabulated in Table 4. The ash values for the
powdered plant are calculated to figure out the
amount of siliceous material left over in the residue
and to determine the extraneous material adhered to
the plant while its collection34
. The ash values of
whole herb were carried out and the results are
presented in Table 5.
Traditional significance of study
The herb has been used as a traditional medicine.
This standardization approach will invoke the
scientists to work further for the assessment of the
distinctive chemical constituents present in its
extract that are rendered responsible for its
biological activities and traditional uses. Their
chemistry and structural elucidation will assist in
describing the mechanism of their actions. Besides that,
these constraints will help in the identification of the
correct herb while collection and minimize the chances
of adulteration, substitution or sophistication.
Conclusion The study of pharmacognostical features had shown
the standards, which will be useful for the recognition of
its distinctiveness and genuineness. Physicochemical
parameters like ash values, and extractive values are all
indicators of the quality of material. These anatomical
perspectives, pharmacognostical evaluations and
physicochemical characterization, carried out in this
research work can be utilized as a reference for
authenticity of this plant material. Declaration of interest
We declare that we have no conflict of interest.
Acknowledgement The research work was funded by the Punjab
University College of Pharmacy, University of the
Punjab, Lahore Pakistan.
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M. pumalis
S. No. Group Name of tests Inferences
1 Proteins Millon’s test
Ninhydrin test
Biuret test
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+ +
–
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–
2 Carbohydrates Barfoed’s test
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Legal’s test
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1. Chloroform 1.65 ± 0.131
2. Ethyl Acetate 8.87 ± 0.191
3. n-butanol 5.40 ± 0.263
Table 5 — Ash values of whole herb, M. pumilus
S. No. Parameters Values % (w/w)
1 Total ash 15.15 ± 0.028
2 Acid insoluble ash 2.326 ± 0.116
3 Water soluble ash 15.233 ± 0.044
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