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Morphoanatomical and physicochemicalstandardization of Casuarina equisetifoliaL. stem bark

Dinesh Kumar a,*, Ajay Kumar a, Om Prakash b

a Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, IndiabManav Bharti University, Solan, H.P., India

a r t i c l e i n f o

Article history:

Received 22 December 2013

Received in revised form

16 February 2014

Accepted 17 February 2014

Available online 5 March 2014

Keywords:

Casuarina equisetifolia

Morphoanatomical

Physicochemical analysis

Standardization

* Corresponding author. Division of PharmacKurukshetra 136119, Haryana, India. Tel.: þ9

E-mail address: dineshbarbola@yahoo.co

Peer review under the responsibility of Ben

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http://dx.doi.org/10.1016/j.bjbas.2014.02.0042314-8535/Copyright 2014, Beni-Suef Univers

a b s t r a c t

Objective: The present work was carried out to perform the morphoanatomical and phys-

icochemical evaluation of Casuarina equisetifolia L. stem bark.

Method: The pharmacognostic studies were carried out in terms of organoleptic, macro-

scopic, microscopic, fluorescence analysis and physicochemical parameters.

Results: The bark consists of channelled, curved, slightly quilled, usually 0.2e0.8 cm thick,

lenticellate pieces with outer surface ash-grey to greyish-brown and internal surface light

yellow to deep dirty brown coloured having no odour and astringent taste. The main

microscopic characteristics of the bark include phellem (2e5 or more layers of cork),

phellogen (3e5 layered) followed by 12e18 layered phelloderm. Other important micro-

scopic components observed include phloem parenchyma, phloem fibre and stone cells.

Stem bark powder showed thick walled oval to polygonal cork cells, hexagonal phelloderm

cells, rectangular thin walled cortex cells, thick walled elongated phloem fibres, lignified

stone cells and rhomboidal crystals of calcium oxalate. Further, physicochemical analysis

of the bark power showed loss on drying, total ash, water soluble ash, acid insoluble ash

and sulphated ash as 2.3, 5.0, 1.6, 0.7 and 5.7% w/w respectively. The alcohol and water

soluble extractives values of the stem bark were 17.10 and 11.0% w/w respectively.

Conclusion: Various pharmacognostic characters observed in this study helps in botanical

identification and standardization of C. equisetifolia L. in crude form.

Copyright 2014, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved.

ognsoy and Phytochemistry, Institute of Pharmaceutical Sciences, Kurukshetra University,1 1744 239617, þ91 9466772500; fax: þ91 1744 238277..in (D. Kumar).

i-Suef University

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b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 3 2e3 6 33

1. Introduction

Casuarina equisetifolia L. synonym Casuarina litorea L. (family:

Casuarinaceae) popularly known as ‘Junglisaru ’ in Hindi and

‘She-oak or Horse tail or Whistle pine’ in English, is an exotic

species to India and native to South-East Asia, Australia and

Polynesia (Parrotta, 1993; Pinyopusarerk and House, 1993). It is

a large erect evergreen tree up to 50 m height with drooping

branches and needle-like branchlets and commonly found

along the coast on beaches, rocky coasts, limestone out-

croppings, dry hillsides and open forests in India, Sri Lanka

and Australia (Han, 1998). In India, it is cultivated in coastal

regions from Gujarat to Orissa, West Bengal and

Andaman (Anonymous, 1992).

The plant is traditionally used to treat constipation, cough,

diabetes, diarrhoea, dysentery, gonorrhea, nervous disorders,

stomach acne, throat infections and ulcer (Weiner, 1971;

Whistler, 1992; Prajapati et al., 2003). The plant is reported to

possess various biological activities like antiasthmatic

(Karimulla and Kumar, 2011), antimicrobial (Parekh et al.,

1971), antioxidant (Chopra et al., 1956; Prakash et al., 2007;

Zhang et al., 2010), antifungal (Han, 1998), hepatoprotective

(Ahsan et al., 2009), nitrogen fixation (Li-Hua et al., 2009),

antidiarrhoeal (Kumar, 2011), antidiabetic, antihyperlipidemic

(Sriram, 2011), antiulcerogenic (Shalini and Kumar, 2011).

Phytoconstituents reported in C. equisetifolia include b-sitos-

terol, campesterol, stigmasterol, cholesterol, cholest-5-en-3-

b-ol derivatives, casuarine, catechin, citrulline, cupressu-

flavone, epicatechin, gallicin, gentisic acid, isoquercitrin,

juglanin, kaempferol, proanthocyanidins, rutin, trifolin

(Cambie and Ash, 1994; Nash et al., 1994).

A review of literature revealed that no pharmacognostic

standards have been recorded for this crude drug. Owing to its

ethnopharmacological importance, the present investigation

has been undertaken with an objective to establish mor-

phoanatomical and physicochemical standards for stem bark

of C. equisetifolia so that authentic plant material could be

explored for its therapeutic claim.

2. Materials and methods

2.1. Chemicals

All the chemicals used in the study were of analytical grade

and were obtained from Rankem Limited India and Hi-Media

laboratories, Mumbai, India.

2.2. Procurement of plant materials

Stem bark of the plant was collected from the campus of

Kurukshetra University, Kurukshetra during May 2011 and

authenticated by Dr. H.B. Singh, NISCAIR (No: NISCAIR/RHMD/

Consult/-2012-13/2105/112).

2.3. Morphological evaluation

Various organoleptic and morphological characters of C.

equisetifolia stem bark like colour, shape, size, taste, odour,

fracture and configuration etc. were studied (Khatoon et al.,

2009).

2.4. Anatomical evaluation

Anatomy allows one to discover convincing diagnostic char-

acters for a specific plant species (of crucial importance in

quality control), and it also allows one to observe the distri-

bution of compounds in the plant matrix. In microscopic

evaluation, studies were conducted on both grounds qualita-

tively and quantitatively. The model of microscope used for

study of different characters was SKC-400, Suswox Optik,

Sudheer Scientific Works, India.

2.5. Qualitative microscopy

In this study, transverse sections of stem bark were studied

under photomicrograph. Staining reagents (such as

phloroglucinol-HCl) were used as per standard procedures

(Kokate, 2010; Bisth et al., 2011). The various identifying fea-

tures of the drug were studied with or without staining and

recorded.

2.6. Stem bark microscopy

The stem bark was dipped in a test tube containing sufficient

water and was boiled for few minutes. The softened bark was

transversally sliced into fine sections which were subjected to

staining reagent 0.1% w/v phloroglucinol followed by

concentrated conc. hydrochloric acid. The stained sections

were observed under microscope (Ahmed and Urooj, 2011;

Bhide et al., 2011). Different layers of cells and identifying

characters were observed and thereafter, photomicrography

was done.

2.7. Powder microscopy

The dried stem bark was powdered and studied under mi-

croscope. Different staining reagents (such as iodine for

detection of starch grains and phloroglucinol for detection of

lignified components) were used. To a little quantity of stem

bark powder taken over a microscopic slide, 1e2 drops of

0.1% w/v phloroglucinol solution and a drop of concentrated

hydrochloric acid were added and covered with a cover slip.

The slide preparation was mounted in glycerol and examined

under microscope (Khandelwal, 2011). The characteristic

structures and cell components were observed and their

photographs were taken using photomicrography.

2.8. Fluorescence analysis

Fluorescence study of stem bark powderwas performed as per

reported standard procedure (Kokashi et al., 1958). A small

quantity of the bark powder was placed on a grease free clean

microscopic slide and 1e2 drops of the freshly prepared re-

agent solution were added, mixed by gentle tilting the slide

and waited for 1e2 min. Then the slide was placed inside the

UV chamber and observed in visible light, short (254 nm) and

long (365 nm) ultraviolet radiations. The colours observed by

b e n i - s u e f un i v e r s i t y j o u rn a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 3 2e3 634

application of different reagents in different radiations were

recorded.

2.9. Physicochemical analysis

In this study, air dried material was used for quantitative

determination of physiochemical values like loss on drying,

total ash, acid insoluble ash, water soluble ash, sulphated ash

values and extractive values were determined as per reported

method (WHO, 1992).

Fig. 2 e T.S. of C. equisetifolia Linn. stem bark. Cr e cork

(Phellem), Pg e Phellogen (Cork cambium), Pd e Phelloderm

(secondary cortex), Pf e Phloem fibre, Pp e Phloem

parenchyma, Sc e Stone cells.

3. Results

3.1. Macroscopic study of stem bark

Morphological examination of the stem bark (Fig. 1) shows

that the bark consists of channelled, curved, slightly quilled,

usually 0.2e0.8 cm thick, lenticellate pieces which are smooth

on small trunks, becoming rough and thick furrowed on older

trees. Outer surface of the bark is ash-grey to greyish-brown

and internal surface light yellow to deep dirty brown col-

oured with short fracture, odourless and astringent taste.

3.2. Microscopic study of stem bark

Transverse section study of the stem bark (Fig. 2) depicts that

bark shows 2e5 layers of cork (phellem), consisting of polyg-

onal thick-walled parenchymatous cells filled with reddish-

brown content. Phellogen (cork cambium) is 3e5 layered

thick having polygonal and tangentially elongated thin-walled

parenchymatous cells. Secondary cortex (phelloderm) con-

sists of 12e18 layers having oval to polygonal, tangentially

elongated thin-walled parenchymatous cells. Beneath sec-

ondary cortex, a large group of oval to elongated stone cells

arranged in a tangential manner forming a continuous or

discontinuous band. Secondary phloem is composed of

phloem parenchyma, phloem fibres and stone cells

Fig. 1 e Morphological views of C. equisetifolia stem bark.

alternating with lignified stone cells. Lignified fibres are small

thin-walled polygonal cells present in cork. A few stone cells

are also found scattered in secondary cortex as in secondary

phloem.

3.3. Powder study

Stem bark powder appears brownish showing thick walled

oval to polygonal cork cells; hexagonal phelloderm cells;

rectangular thin walled cortex cells; thick walled elongated

phloem fibres; lignified stone cells and rhomboidal crystals of

calcium oxalate. Powder characteristics of the bark have been

shown in Fig. 3.

3.4. Fluorescence analysis

The fluorescence characteristics of the stem bark powderwith

different chemical reagents are summarized in Table 1.

3.5. Physicochemical analysis

In this study, various physicochemical parameters like loss on

drying, total ash, acid insoluble ash, water soluble ash,

sulphated ash and extractive values were determined in

triplicate as mentioned in Table 2.

4. Discussion

Owing to the wide use of herbal drugs in traditional medi-

cines, standardization becomes an important measure for

ensuring quality, purity and authenticity of the crude drugs.

Fig. 3 e Powder characteristics of C. equisetifolia stem bark.

Table 2e Physicochemical analysis of C. equisetifolia stembark.

Parameters Value obtained on dryweight basis (% w/w)a

Loss on drying 2.3 � 0.42

Total ash value 5.0 � 0.21

Acid insoluble ash value 0.7 � 0.28

Water soluble ash 1.6 � 0.35

Sulphated ash 5.7 � 0.53

Petroleum ether soluble extractive 1.4 � 0.16

Chloroform soluble extractive 2.1 � 0.08

Methanol soluble extractive 17.10 � 0.39

Water soluble extractive 11.0 � 0.48

a Average of three reading � SEM.

b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 3 2e3 6 35

First step in this regard is the authentification of plant species.

For this purpose, morphological and anatomical analysis is

one of the simplest and cheapest methods to start with

establishing the correct identification of the source materials

(Nirmal et al., 2012; Kumar et al., 2012). As there is no phar-

macognostic work recorded on this medicinally potent plant,

the present work was undertaken to lay down the standards

which could be useful for establishing its authenticity. The

present study reports the morphoanatomical and physico-

chemical characteristics of C. equisetifolia L. stem bark. Phys-

icochemical studies of stem bark acts as a reliable tool for

detecting adulteration (Evans, 2009; Zhao et al., 2011; Raj and

Radhamany, 2012).

Studies of physicochemical constants can serve as a valu-

able source of information which is usually helpful in evalu-

ation of purity and quality of a crude drug. The extractive

values give an idea about the chemical constitution of the

drug (Kumar et al., 2012). In the present study, the extractive

value of methanol was maximum followed by water. The ash

values determine the earthy matter or inorganic composition

and other impurities present along with the drug. Fluores-

cence characteristics are an alternative rapid method for

resolution of doubtful specimen.When physical and chemical

methods are inadequate, the plant material may be identified

and differentiated form their adulterants on the basis of

fluorescence characteristics (Kumar et al., 2012). Fluorescence

is the phenomenon exhibited by various chemical constitu-

ents present in the plant material. Some constituents show

Table 1 e Fluorescence analysis of C. equisetifolia stem bark po

Treatment Visible light

Shor

Powder Yellowish brown

Powder þ 1N NaOH (aq.) Light brown

Powder þ 1N NaOH (alc.) Light brown

Powder þ Ammonia Brown

Powder þ Picric acid Yellowish brown

Powder þ Pet. ether Light brown

Powder þ 50% HCl Light brown

Powder þ 50% H2SO4 Pinkish Brown

fluorescence in the visible range in daylight. The ultraviolet

light produces fluorescence in many natural products (e.g.

alkaloids like berberine), which do not visibly fluoresce in

daylight. If the substances themselves are not fluorescent,

they may often be converted into fluorescent derivatives or

decomposition products by applying different reagents.

Hence, some crude drugs are often assessed qualitatively in

this way and it is an important parameter for pharmacog-

nostical evaluation of crude drugs. Adulteration of the

genuine raw material is the main cause of degradation of

desired therapeutic effect of plant species used in various

traditional systems of medicine. Thus, industries could utilize

wder.

Under UV light

t wavelength (254 nm) Long wavelength (365 nm)

Light brown Brown

Dark brown Brownish black

Dark green Black

Green Black

Green Dark brown

Light green Orange

Green Black

Greenish brown Brownish

b e n i - s u e f un i v e r s i t y j o u rn a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 3 2e3 636

the scientific background for identification of raw material

and this work is not only beneficial to the industries but also

enhance the credibility of Indigenous System of Medicine

(Chumbhale and Upasani, 2012).

In the present paper, the morphoanatomical and physi-

cochemical studies of the stem bark material help to deter-

mine the quality and purity of the C. equisetifolia L. Thus, the

above finding will serve in the development of pharmaco-

poeial standards for the future studies.

Acknowledgement

The authors express sincere thanks to UGC, New Delhi (39-

955/2010 SR) for financially supporting the study.

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