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: saiqa.pharmacy@pu.edu.pk

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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Table 3 — Preliminary phytochemical analysis of whole herb,

M. pumalis

S. No. Group Name of tests Inferences

1 Proteins Millon’s test

Ninhydrin test

Biuret test

Xanthoproteic test

+ +

–

+ +

–

2 Carbohydrates Barfoed’s test

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Benedict’s test

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Legal’s test

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Table 4 — Extractive values of whole herb, M. pumilus

S. No. Parameters Percentage yield ± SEM

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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