Indian Journal of Natural Products and Resources

Vol. 4 (4), December 2013, pp. 348-357

Microscopic studies and preliminary pharmacognostical evaluation of

Euphorbia neriifolia L. leaves

Veena Sharma* and Pracheta

Department of Bioscience and Biotechnology, Banasthali University,

Banasthali- 304022, Rajasthan, India

Received 14 September 2012; Accepted 11 February 2013

Euphorbia neriifolia Linn. (Euphorbiaceae) is commonly known as Indian Spurge Tree in English, and Sehundah in

Ayurveda. Its leaves are traditionally used for treatment of various diseases like inflammation, fever, asthma, cough,

wounds, ulcers, cancers and diabetes. In the present study an attempt has been made to highlight this folk herbal medicine

which will assist in the identification of fresh as well as dried crude samples of leaves anatomically and

pharmacognostically. The present study also deals with macroscopic, microscopic, fluorescence, phytochemical

characteristics and other WHO recommended methods for standardization of leaf powder. Phytochemical screening and

chromatographic studies help in determining the antioxidant potential and predominant classes of active ingredients

contribute to the activity. These studies will provide referential information for correct identification and help in checking

adulteration in market samples used in the preparation of various herbal medicines. These observations will also be helpful

in differentiating the leaves of this species from closely related species of same genus and family.

Keywords: Euphorbia neriifolia, Microscopic, Pharmacognostic, Phytochemical Screening, Standardization, Chromatography.

IPC code; Int. cl. (2011.01)−A61K 36/00.

Introduction

An authentication and quality assessment of herbal

material deals with the pharmacognosy that is based on

macroscopic and microscopic characters1. A big quantum

of research works in the area of authentication of the

correct plant source has been undertaken to provide means

of differentiation among many available plant sources2.

Euphorbia neriifolia L. (Euphorbiaceae) is a widely

distributed large succulent shrub or small tree up to 7.5 m

in height, with stipular thorns and found throughout the

Deccan Peninsula of India. It is commonly known as

Sehundah (Ayurveda), Ilachevikalli (Siddha), Indian

Spurge Tree, Oleander Spurge and Hedge Euphorbia3-4

.

Traditionally it is used as antibacterial, antifungal, antiviral,

antiparasitic, antiarthritic, antidiabetic, anticonvulsant,

antioxidant, analgesic, wound healing and immuno-

modulatory, radioprotective, spasmodic, aphrodisiac,

anticancer, purgative and limit diseases caused by Tiba

(indigestion and badkhara and Tipa) and diuretic properties

due to the presence of phytoconstituents4-10

. The efficacy of

this plant in the treatment of various diseases necessitated

the present work in order to identify the classes of natural

products present in the leaf of the E. neriifolia. For

standardization and quality assurance purpose, the

following three attributes must be verified: authenticity,

purity and assay11

. Literature survey did not provide

sufficient information about pharmacognostical studies of

this plant. The current work aims to contribute in solving

the problems of controversial drugs prevalent in Ayurveda

besides helping in laying down pharmacopoeial standards.

Therefore, keeping above view in mind various

macroscopic, microscopic and pharmacognostical studies

on fresh and dried leaves of E. neriifolia were carried out in

present study.

Materials and Methods

Procurement and authentication

Euphorbia neriifolia leaves were collected from

Medicinal garden of Banasthali University, Banasthali,

Rajasthan, India, in the month of September-November

2009 and authenticated by Botanist of Krishi Vigyan

Kendra, Banasthali University, Banasthali.

Preparation of leaves extracts

Shade dried powder was extracted by macerating 50 g

in 250 mL of ethanol (70% v/v) for one week with

occasional stirring. Coarse powder (50 g) was also

defatted in 250 mL of ethanol (70% v/v) by using Soxhlet

_____________

*Correspondent author:

E-mail: drvshs@gmail.com

Phone: + 01438−228386

SHARMA & PRACHETA: PHARMACOGNOSTICAL EVALUATION OF EUPHORBIA NERIIFOLIA

349

apparatus. Successive extraction with different solvents in

their ascending order of polarity was also carried out

using Soxhlet apparatus. The macerated and Soxhleted

mixture was filtered, evaporated and stored at 4°C in air

tight container and was used for further studies12

.

Macroscopic and microscopic studies

characters were examined according to Brain and

Turner13

. Quantitative microscopy include surface

constants like stomatal number, stomatal index, veins,

vein-islets and vein terminations were studied by

using camera lucida. Various diagnostic characters of

fresh leaves and leaf powder were studied by

microscopic analyses with or without staining14-17

.

Pharmacognostical analysis

Pharmacognostical values such as the foreign

organic matter, percentage of total ash value, acid

insoluble & water soluble ash value, moisture content

and extractive values were performed according to the

WHO guidelines on quality control methods for

medicinal plant materials18-21

. Chemical test and

fluorescence analysis15, 19,22,23

were also studied.

Preliminary phytochemical screening

Preliminary qualitative phytochemical screening of

all the extracts for the detection of various active

ingredients was carried out by using standard

conventional procedures13,23,24

.

Chromatographic fingerprinting

Thin Layer Chromatography (TLC) of all the extracts

was carried out in various solvents at 30°C using Silica gel

G as adsorbent and the Rf values were determined. The

selected mobile phase was used for the high performance

thin layer chromatography (HPTLC) densitometry analysis

different extracts of EN were also performed25

.

Results

Organoleptic and macroscopic evaluation

Leaves of E. neriifolia are succulent, deciduous,

terminal on branches, fleshy, stipular thorns in pairs,

3-5 mm long. Macroscopically, the fresh young leaves

are simple, dark green in colour having leathery texture,

cuneate shaped, apex sub-acute and base acute, margins

entire (even, smooth throughout) without toothing,

surface glabrous, venation reticulate and average leaf

size 8-24 ± 2 cm (length) and 4-8 ± 2 cm (breadth) &

1.3±0.2 mm (thickness) with pointed and acute tip. The

length of reduced petiole 1.5-2.5 cm (Plate 1).

Powder microscopy

The fine powder was mounted in glycerin and stained

with iodine, phlorogucinol + conc. HCL and Sudan III.

Observed features revealed that the leaf powder contains

numerous idioblastic, rosette, square, prismatic and

acicular shaped calcium oxalate crystals and starch grains,

both simple and compound. The powder also showed the

presence of well arranged annular vessels, anomocytic

stomata, unicerrate multicellular trichome with blunt tip,

epidermal cells, spongy parenchyma, xylem parenchyma,

vittae- volatile contain schzogenous cells, polyhedral or

sharp angles typed starch grains and lignified xylem fibers

(Plates 2-6). After treatment with HCL calcium oxalate

crystals changed into needle shaped crystals (Plate 2).

Plate 1 (a-c)- Organoleptic and morphological features of E. neriifolia leaves

Plate 2 (a-e)- Different types of calcium oxalate crystals present in powder of E. neriifolia leaves

INDIAN J NAT PROD RESOUR, DECEMBER 2013

350

Leaf microscopy

Transverse section of leaf showed the single

layered thick rectangular or tubular adaxial epidermal

cells. Single to double layered abaxial epidermis with

circular to rectangular epidermal cells (Plate 7 a-c).

Mesophyll tissue was differentiated into two to three

layered adaxial zones of radially elongated palisade cells

and wider abaxial spongy mesophyll cells revealed the

differentiated dorsiventral lamina (Plate 7d). The spongy

mesophyll had wide air-chambers and partition filaments

formed by lobed and interconnected 6-9 layered spongy

parenchyma cells. Midrib region slightly raised on the

abaxial side whereas broadly semi-circular on the adaxial

side. Midrib composed of epidermis, collenchyma and

spongy parenchyma cells (Plate 7b). In certain regions of

epidermis, five celled unicereate trichomes with blunted

tip (Plate 7a) and glandular trichomes with bicellular head

(Plate 7 c) were embedded.

Vascular bundles (VB) prominent towards the ventral

side and covered with endodermis. The vascular bundles

consist lignified xylem (pink colour) whereas non-

lignified phloem (Plate 8a). Parenchymatous cells of leaf

Plate 3 & 4 (a-e)- Different types of trichomes, Anisocytic stomata present in powder of E. neriifolia leaves

Plate 5 & 6 (a-d)- Annular xylem trachieds, lignified fibre & vessels, - Xylem parenchyma cells, spongy parenchymatous cells seen in

the powder of E. neriifolia leaves

Plate 7 & 8 (a-d)- Transverse sections of E. neriifolia leaf, a) UE with unicellular trichomes, b) LE with Col & mesophyll SP, c)

UE with glandular trichomes (GT), d) Palisade parenchyma (PalP); 8a a.Vascular bundle (VB), b. Transfusion tissue

SHARMA & PRACHETA: PHARMACOGNOSTICAL EVALUATION OF EUPHORBIA NERIIFOLIA

351

showed the presence of transfusion tissue after stained

with acid (Plate 8b). Between loosely arranged spongy

cells rosette, octahedral and thick calcium oxalate

crystals were seen (Plate 9 a). These crystals bearing

cells were distinct from the neighboring mesophyll cells

were called idioblasts. Direct stained with potassium

iodide the spongy parenchyma showed the presence of

starch granules (Plate 9b). The spongy mesophyll had

wide air-chambers and partition filaments formed by

lobed and interconnected 6-9 layered spongy

parenchyma cells (Plate 9 c).

E. neriifolia leaf surface showed the anomocytic

type of stomata that were covered with guard cells

surrounded by 2-3 subsidiary cells followed by

polygonal epidermal layers (Plate 10b). Adaxial

surface contains more stomata in comparison to abaxial

surface of leaf (Plate 10 a-c). Some covering trichomes

with collapsed cell were also seen in upper stomata

(Plate 10b). Leaf surface also showed the presence of

veins, vein islets and vein terminations (Plate 10d). The

primary and secondary veins branched profusely and

gave rise to ultimate veinlets. The vein islets were

distinct, small and squarish or rectangular. Each vein

islet had one or two vein terminations and filled with

areoles. Leaf constants such as stomatal number,

stomatal index, veinlet terminations and vein-islet

number are measured and shown in Table 1.

Pharmacognostical analysis

Foreign organic matter recorded in the powdered

plant material was 0.87±0.03% and the percentage of

extractive values of E. neriifolia was found to be

14.32±0.04 in alcohol (ethanol) and 26.31±0.12 in

Plate 9 (a-c)-TS of E. neriifolia leaf a) Idioblastic calcium oxalate crystals & simple starch granules, b) Polyhedral sharp angles type

starch granules, c) Spongy parenchyma

Plate 10 (a-c)- Upper & lower stomata (10 X & 40X), d) Vein-islet (VL) & vein termination (VT) at 40X; SC: subsidiary cells,

GC: guard cells; S; stomata; EC: epidermal cells

Table 1 Quantitative microscopy of E. neriifolia leaf

Variables Abaxial surface Value

(in 1 mm2 area)

Adaxial Surface

Value (in 1 mm2 area)

Stomatal number 116±5 mm2 52±5 mm2

Stomatal index 12.88% 11.60 %

Epidermal cells 784±10 mm2 396±10 mm2

Vein islet number 33±5 mm2

Veinlet termination

number

16±5 mm2

INDIAN J NAT PROD RESOUR, DECEMBER 2013

352

water. The percentage of weight loss on drying or

moisture content at 105ºC of EN was found to be

3.45± 0.09, respectively whereas the pH of drug was

found to be slightly acidic and the values being

6.4± 0.2 in 1% and 5.8± 0.04 in 10% aqueous

solution. Physical state of dry ash appeared as fine

powder and the sample ash as greyish white colour.

The taste of the ash was found to be pungent and the

amount of total, acid insoluble and water soluble ash

of EN were found to be 7.36±0.07, 0.82±0.04, and

4.54±0.11%, respectively.

The behaviour of E. neriifolia leaves powder upon

treatment with different chemical reagents showed

creamy colour when powder was as such; light yellow

brown colour with distilled water; greenish yellow

with picric acid; yellow brown with glacial acetic

acid; radish brown with 1N HCl and 1N H2SO4;

yellow brown with Conc. HNO3; grey with ferric

chloride (5%); greenish brown with iodine solution

(5%); olive green with ammonia solution; golden

brown with 1N NaOH; dark brown with potassium

dichromate; yellowish orange with HNO3 + NH3

solution; pale green with methanol and ethanol; yellow

green colour with toluene. Likewise the fluorescence

characteristics of powdered leaves after treatment with

different reagents emitted various colour radiations

under ultraviolet light (Table 2). The fluorescence

characteristics of different extracts of leaf was also

studied under ordinary and UV light (366 nm), wherein

the leaf extracts showed the visibility of varying

colours which are tabulated in the Table 3.

The preliminary phyto–profiling for the leaves

extracts was carried out and the consistency was

found to be sticky, dry and oily. The percentage yield

(w/w) of the extracts were also analysed (Table 4),

and the highest yield was found to be in aqueous

extract (17.89%) in sequential soxhletion method and

in hydro-ethanolic extract (20.02 %) in direct method.

Preliminary phytochemical screening

The results of preliminary phytochemical

screenings of different extracts of leaves mainly

revealed the presence of proteins, alkaloids,

glycosides, flavonoids, phenolics, saponins and

terpenoids in appreciable, moderate and trace amount

(Table 5). Our results also revealed that the plant

possesses proteins and amino acids in negligible

amount. This indicates that, the presence of secondary

metabolites may have suppressed the activity of

proteins. In addition, the solvent might have also

denatured the proteins because of which it is detected

in fewer amounts in all extracts.

Chromatographic analysis

The resolution of different kinds of chemical

components and the marker compound has been

separated using TLC and HPTLC with the standards,

Table 2 Florescence characteristic of the powder of E. neriifolia leaves in different means

Treatments with leaves powder UV Short (254 nm) UV Long (366 nm) Visible

Powder as such Light yellow Light yellow Light green

Distilled water Yellow green Fluorescent green Light green

Picric acid Light green Fluorescent green Greenish yellow

Glacial acetic acid Yellow Light orange Light yellow

1N HCl Dark brown Greyish Brownish yellow

1N H2SO4 Magenta Magenta Magenta

Conc. HNO3 Yellow Dark brown Yellow

Ferric Chloride (5%) Dark brown Fluorescent green Green

Iodine solution (5%) Greenish yellow Brown Yellow

Ammonia solution Green Fluorescent green Yellow green

1N NaOH Green Fluorescent green Orangish

Potassium dichromate Dark green Fluorescent green Brown

HNO3 + NH3 solution Yellow Orangish Orangish

Methanol Light Green Light Grey Light Green

Ethanol Light Green Fluorescent green Light Green

Toluene Light Green Yellow Light Green

The colours mentioned in the table are based on the colour identification chart, Royal Botanica Garden, Edinburg (1969)

Table 3 Florescence characteristic of different extracts of

E. neriifolia leaves

Solvent Under Ordinary light Under UV light (366 nm)

Pet-ether Yellowish green Fluorescent

Benzene Brownish green Fluorescent blue

Chloroform Light green Light green

Ethyl Acetate Dark green Greenish brown

Ethanolic Dark brown Purplish Brown

Aqueous Brown Blackish brown

Hydro-ethanolic Dark brown Dark brown

SHARMA & PRACHETA: PHARMACOGNOSTICAL EVALUATION OF EUPHORBIA NERIIFOLIA

353

and the Rf values were calculated in order to

standardize the drug for its identity, purity and

strength. TLC fingerprinting of different extracts of

E. neriifolia leaf was done and phytoconstituents were

separated in eight different mobile phase of varying

polarity. Among all, chloroform: methanol (9.5: 0.5)

mobile phase was suitable for chloroform extract,

ethylacetate: methanol: water (7.5: 1.25: 0.5) for ethyl

acetate extract and chloroform: ethanol: water (8:2:1)

and n-butanol: acetic acid: water (4: 1: 7) were found

to be most appropriate solvent system for separation

of flavonoid phytoconstituents for ethanol extract.

Iodine vapours were used as a developer. TLC studies

of chloroform and ethyl acetate extract revealed

7 spots whereas ethanol extract revealed 8 spots at

varying Rf values depicted in Table 6 and Plate 11.

The n-butanol: methanol (9.5: 0.5) showed good

separation of the flavonoids from petroleum ether

extract of E. neriifolia leaves. The petroleum ether

extract displayed the presence of 10 types of

Table 4 Preliminary phyto-profile of extracts of E. neriifolia leaves in different organic solvents

Ex. S PI Extraction Colour Consistency Nature % Yield ±SD

HE - Soxhletion Dark Brown Sticky Semi solid 16.31±0.38

Dir

ect

HE - Maceration Chocolate Brown Sticky Solid 20.02±0.25

PE 0.0 Soxhletion Florescent Green Dry Solid 3.01±0.11

B 2.7 Soxhletion Green Sticky Solid 2.72 ±0.08

C 4.1 Soxhletion Green Sticky Solid 0.33±0.17

EA 4.4 Soxhletion Green Oily Solid 0.43±0.07

E 5.2 Soxhletion Brownish green Sticky Semi solid 9.78±0.08

Seq

uen

tial

Aq 9 Maceration Dark Brown Dry Solid 17.89±0.45

Ex.: Extract; S: Solvent; PI: Polarity index; HE: Hydro-ethanolic; PE: Petroleum-ether; B: Benzene; C: Chloroform; EA: Ethyl-acetate

E: Ethanolic; Aq: Aqueous

Table 5 Qualitative test for various organic substances in the extracts of E. neriifolia leaves

E. neriifolia leaves extracts

Plant Constituents Test Performed HE PE B C EA E Aq

Protein and Millon’s test - + - + + - +

Biuret test + + + + ++ + -

Amino acids Ninhydrin test - - - - - - -

Xanthoproteinic test + - + - + + +

Carbohydrates Fehling’s test ++ - - +++ - ++ -

Fixed oils Stain test ++ +++ ++ ++ ++ ++ +

Pri

mar

y

Met

abo

lite

s

and Fats Soap test ++ + - - + ++ -

Alkaloids Dragendroff's test + +++ +++ + ++ + +

Mayer's test +++ ++ ++ ++ - ++ -

Wagner's test ++ + - + + + -

Anthraquinones Borntrager's test +++ - - + + ++ +

Cardiac glycosides Killer Killani test +++ ++ + + ++ +++ +

Baljet test +++ ++ - ++ + ++ +

Glycosides (Free sugar) Legal's test +++ - ++ ++ +++ ++ ++

Flavonoids Shinoda test ++ - - + + ++ +

Ammonia test + - +++ + + +++ +

Lead acetate test +++ - - + ++ ++ ++

Alkaline reagent test ++ - - - ++ +++ ++

Phenolic Ferric chloride test +++ - - - + +++ +

Pholobatanins Hydro chloride test ++ - - - - ++ +

Polyphenol Folin-ciocalteau test +++ + - - +++ +++ +++

Saponins Frothing test - + +++ + - - -

Olive oil test + ++ +++ + + + -

Steroids Liebermann-Burchard's test +++ +++ +++ + - + -

Tannins Ferric chloride test +++ - - - ++ +++ +

Terpenoids Salowski test +++ +++ ++ + + +++ -

Sec

on

dar

y M

etab

oli

tes

Gums &Mucilage Ruthenium Red test +++ + ++ + ++ +++ +++

(+++) appreciable amount; (++) moderate amount; (+) trace amount; (-) completely absent. HE: Hydro-ethanolic extract; PE: Pet-ether

extract; B: Benzene extract; C: Chloroform extract; EA: Ethyl-acetate extract; E: Ethanol extract; Aq: Aqueous extract

INDIAN J NAT PROD RESOUR, DECEMBER 2013

354

flavonoids with 10 different Rf values ranging from 0.18

to 0.95 (Table 7). The chromatogram was observed in

UV chamber at 254 nm in absorbance and at 366 nm in

fluorescence modes (Plate 12). The characteristics

pattern of petroleum ether extract showed fine separated

pattern of bands and served as idiosyncratic fingerprint

for qualitative evaluation of leaf.

Discussion

Today sophisticated modern research tools for

evaluation of the plant drugs are available but

microscopic method is still one of the simplest and

cheapest methods to establish the correct identity of

the source materials26

. Fluorescence is an important

phenomenon exhibited by various chemical

constituents present in plant material. If the

substances themselves are not fluorescent, they may

often be converted into fluorescent derivatives by

applying different reagents hence some crude drugs

are often assessed qualitatively in this way and it is an

important parameter of pharmacognostical

evaluation27

.

According to the World Health Organization

(WHO, 1998)21

, the macroscopic and microscopic

description of a plant is the first step to establish the

identity and the degree of purity of such materials and

Plate 11 (a-f)- Thin layer chromatogram of HEEN and sequential extracts of E. neriifolia leaves a) Hydro-ethanol extract- n-B: AA:

H2O (3: 2: 5); b) Pet-ether extract- B: M (9.5:0.5); c) Benzene extract-EA: M: H2O (7.5: 2: 4); d) Chloroform extract-C: M (9.5: 0.5); E)

Ethyl acetate extract- EA: M: H2O (7.5: 1.25: 0.5); f) Ethanol extract-C: E: H2O (8:2:1).

Table 6 Thin layer chromatographic studies of different extracts of E. neriifolia

Mobile Phase

Extracts Solvents Ratio Colour

Visible IC

TS Rf values

HE n-B: AA: H2O 2:2:6 Brown 1 1 1 0.74

3:2:5 Yellow Brown 1 2 2 0.21, 0.86

C:AA: H2O 7.5:3.75:0.5 Yellow green 2 2 2 0.24, 0.62

PE n-B: AA: H2O 3:2:5 Green, yellow 2 2 2 0.11, 0.62

PE:C 0.5:9.5 Yellow, grey 4 5 5 0.17, 0.33, 0.52, 0.60, 0.88

B:M 9.5:0.5 Yellow, grey 3 5 5 0.10, 0.40, 0.50, 0.60, 0.82

B n-B: AA: H2O 4:1:5 Light Yellow 2 3 5 0.27, 0.31, 0.68, 0.83, 0.92

EA: M: H2O 7.5: 2: 4 Green, yellow, grey 3 6 6 0.28, 0.38, 0.45, 0.67, 0.79, 0.90

7: 2: 4 Green, yellow 3 5 5 0.40, 0.48, 0.38, 0.70, 0.90

C n-B: AA: H2O 3:2:5 Brown 3 3 3 0.81, 0.87, 0.94

C: M 9.5:0.5 Light green 3 7 7 0.20, 0.29, 0.40, 0.55, 0.74, 0.85, 0.95

EA n-B: AA: H2O 2:2:6 Grey, Yellow 2 2 2 0.75, 0.93

EA: M: H2O 7.5:1.25:0.5 Yellow, grey, brown 5 7 7 0.12, 0.23, 0.32, 0.43, 0.54, 0.72, 0.88

E n-B: AA: H2O 4:1:7 Brown 4 5 5 0.34, 0.48. 0.60. 0.74, 0.95

2:2:6 Brown 2 3 2 0.60, 0.79, 0.90

C:E 8:3 Brown 2 2 2 0.21, 0.91

C:E:H2O 8:2:1 Brown, green 4 8 8 0.04, 0.09, 0.13, 0.24, 0.37, 0.63, 0.90, 0.96

Aq n-B: AA: H2O 4:2:4 Brown 1 2 2 0.69, 0.96

AA:HCl:H2O 10:1:3 Brown 1 1 1 0.86

O: Ordinary; n-B: n-Butanol; AA: Acetic acid; H2O: Water; EA: Ethyl acetae; M: Methanol; C: chloroform; IC: Iodine Chamber;

TS: total number of spots

SHARMA & PRACHETA: PHARMACOGNOSTICAL EVALUATION OF EUPHORBIA NERIIFOLIA

355

should be carried out before any tests are undertaken

(Neeli et al, 2008). Macroscopical evaluation is a

qualitative evaluation based on the study of

morphological and sensory profiles of drugs and serve

as diagnostic parameters. In the present study entire

leaf margin and anisocytic type stomata is noticed in

E. neriifolia leaf. The microscopical studies of the

transverse sections showed presence of unicellular

blunted and glandular trichomes. Numbers of leaf

measurements which included stomatal number, vein

islet number, veinlet termination number and stomatal

Index were used to study microscopic features

(Evans, 1998). The present macroscopic and histo-

anatomical observations of leaves thus provide useful

information for quality control parameters for the

crude drug. Powder, quantitative and fluorescence

standards provide valuable information to substantiate

and authenticate the phytomedicine.

Estimation of ash values is also a significant

parameter for the detection of nature of material,

which is added to the drug for the purpose of

adulteration, impurities and determination of

authenticity, quality and purity of test sample28

. The

ash values usually represent the inorganic salts

present in the drug/test sample and is the residue

remaining after incineration21

. Total ash values of

leaves indicate the inorganic composition or earthy

materials and other impurities present along with the

plant material29

. The total ash value was relatively

higher which may be due to high content of

carbonates, phosphates, silicates and silica.

Extractive values determination were primarily

useful for the identification of exhausted drugs. The

amount of the extract that drug yields in a solvent is

often an approximate measure of the amount of

certain constituents that the drug contains. The water

soluble extractive value was indicating the presence

of sugar, acids and inorganic compounds and the

alcohol soluble extractive values indicate the presence

of polar constituents like phenols, steroids, glycosides

and flavonoids.

Determination of the moisture content of the drugs

used in Ayurvedic system of medicine is very

important. The higher or lower percentage shows that

the drug was resorted in humid, wet or dry climate.

Excessive moisture may favour the growth of fungal

Table 7 Chromatographic HPTLC of petroleum ether extract of E. neriifolia leaf

Samples Mobile phase Max Rf Max Height Max % Area Area% Assigned substances

Quercetin 0.88 98.2 34.98 1158.7 38.93 Standard

Rutin 0.47 114.3 58.25 1055.1 55.20 Standard

0.18 60.7 11.77 907.0 7.53 Unknown

0.25 36.0 6.99 282.9 2.35 Unknown

0.27 28.1 5.44 331.0 2.75 Unknown

0.35 51.3 9.95 922.4 7.66 Unknown

0.38 53.3 10.33 1184.7 9.84 Unknown

0.57 121.5 23.57 2868.0 23.81 Rutin

0.76 12.3 2.38 294.1 2.44 Unknown

0.80 17.0 3.30 189.0 1.57 Unknown

0.83 17.1 3.32 323.1 2.68 Quercetin

Pet-ether

B: M

9.5: 0.5

0.95 20.5 3.97 687.3 5.71 Unknown

Plate 12 (a-d)- HPTLC chromatogram of Petroleum-ether extract of E. neriifolia: a) under UV 254 nm b) Under UV 366 nm; c)

After derivatization ; and d) Peak densitogram spectral display (B: M; 9.5: 0.5).

INDIAN J NAT PROD RESOUR, DECEMBER 2013

356

or may cause other micro-organic contamination that

may results in the deterioration of drug. Percentage of

loss of weight on drying indicates the loss of volatile

substances along with water, which is determined by

subtracting the moisture content of the powder drug

from the loss of weight on drying. So, the loss on

drying percentage was also determined. The less value

of moisture content could prevent bacterial, fungal or

yeast growth21,30

. The pH value of powder drug is also

an important parameter. The drugs in the opposite pH

are unionized and absorbed rapidly from stomach.

The micro-chemical tests of the drug were carried

out with different concentrated mineral acids. The

colours produced by these reagents represent the

presence of active constituents. The fluorescence

character of any powdered character of any powdered

drug is very distinctive and helpful distinguishing

features for the determination of a drug. The analysis

of powdered drug under ultraviolet light establishes

the colour of the drug, as such and after treatment

with different reagents.

The presence of phytochemicals in all extracts of

leaves of this species is known to be useful in the

treatment of inflamed or ulcerated tissues and they

have remarkable activity in cancer prevention6-8

.

Flavonoids serve as health promoting compound as a

results of its anion radicals31

and could prevent the

accumulation of DNA damage induced by UV

radiation. These observations support the usefulness

of this plant in folklore remedies in the treatment of

stress related ailments and as a wound healing

properties33

. The plant extract was also positive for

steroids which are very important compounds

especially due to their relationship with compounds

such as sex hormone34

. Secondary metabolites

observed in this plant may be responsible for various

pharmacological effects5-11

.

TLC and HPTLC methods for detection and

quantification of active ingredients present in

E. neriifolia have not been reported in literature. TLC

had been developed in the present work for the

separation and determination of constituents of HEEN

and sequential extracts of E. neriifolia but the

densitometric HPTLC had been developed for the

separation and determination of flavonoids from

petroleum ether extract of E. neriifolia. Screening

results clearly depicts that all extracts contain wide

range of active ingredients. All mobile phases used

for the chromatography were good and suitable for

the extraction of flavonoids from plant extracts12

.

TLC and HPTLC results of present study firmly

depicted that this plants contains a wide range of

flavonoids and that’s the reason behind its excellent

pharmacological properties5-10,35,36

.

Conclusion

No detailed standardized work has been reported in

literature for this plant. Leaves powder subjected for

microscopic, pharmacognostical and preliminary

phytochemical analysis provides relevant information

which may be helpful in authentication of the crude

drug and check adulteration for quality control of raw

material. Chromatographic analysis showed the

presence of flavonoid in extracts. The

pharmacognostic parameters observed in present

study, being reported for the first time adds to the

existing knowledge of E. neriifolia and be quite useful

for identification, standardization, development and

preparation of crude drug’s formulation and inclusion

in various pharmacopoeias for treating various

ailments. The current observation will also be helpful

in differentiating the leaves of this species from

closely related species of same genus and family.

Acknowledgements The authors are grateful to University Grants

Commission (UGC) for providing financial assistance

and to authorities of Banasthali University, Rajasthan

for providing necessary facilities to carry out the

present study. We also acknowledge Mr. Pankaj

Kumar Jain, Assistant professor, Department of

Pharmacy, Banasthali University for his valuable help

and guidance to conduct this research work.

References 1 Heinrich M, Ethnobotany and its role in drug development,

Phytother Res, 2000, 14, 479.

2 Padashetty SA and Mishra S H, Phytochemicals and

pharmacognostical parameters for standardization of

Tricholepis glaberrima: A medicinal herb, J Med Arom Pl

Ski, 2008, 30(4), 381.

3 Chatterjee A and Pakrashi S C, The Treatise on Indian

Medicinal Plants (Council of Scientific and Industrial

Research, New Delhi), 1994, 3.

4 Sharma V, Janmeda P and Singh L, A review on Euphorbia

neriifolia (Sehund), Spatulla DD, 2011, 1(2), 107-111.

5 Sharma V, Pracheta, Paliwal R, Singh L, Sharma V and

Sharma SH, Anticarcinogenic potential of Euphorbia

neriifolia leaves against N-Nitrosodiethylamine-induced

nephrotoxicity in mice, Biochem Cell Arch, 2011, 11(2),

393-398.

6 Pracheta, Sharma V, Paliwal R, Sharma S, Singh L, Janmeda

B S, Savita, Yadav S and Sharma S, Chemo-protective

activity of hydro-ethanolic extract of Euphorbia neriifolia

SHARMA & PRACHETA: PHARMACOGNOSTICAL EVALUATION OF EUPHORBIA NERIIFOLIA

357

Linn. leaves against DENA-induced liver carcinogenesis in

mice, Biol & Med, 2011, 3(2) Special Issue, 36-44.

7 Pracheta, Sharma V, Paliwal R and Sharma S, In vitro free

radical scavenging and antioxidant potential of ethanolic

extract of Euphorbia neriifolia Linn., Int J Pharm Pharm

Sci, 2011, 3(1), 238-242.

8 Pracheta, Sharma V, Paliwal R and Sharma S, Preliminary

phytochemical screening and in vitro antioxidant potential

of hydro-ethanolic extract of Euphorbia neriifolia Linn., Int

J Pharm Tech Res, 2011, 3(1), 124-132.

9 Janmeda P, Sharma V, Singh L, Paliwal R, Sharma S,

Sachdev Yadav and Shatruhan Sharma, Chemopreventive

effect of hydro-ethanolic extract of Euphorbia neriifolia

leaves against DENA-induced renal carcinogenesis in mice,

Asian Pacific J Cancer Prev, 2011, 12(3), 677-683.

10 Sharma V, Pracheta, Paliwal R, Singh L and Sharma C,

Elucidation of analgesic activity of hydroethanolic extract of

Euphorbia neriifolia leaves in Swiss albino mice, J Plant

Develop Sci, 2012, 4(2), 183-189.

11 Torey A, Sasidharan S, Yeng C and Latha LY,

Standardization of Cassia spectabilis with respect to

authenticity, assay and chemical constituent analysis,

Molecules, 2010, 15, 3411-3420.

12 Harborne JB, Phytochemical Methods, A Guide to Modern

Techniques of Plant Analysis, 3rd edn, Springer (India) Pvt.

Ltd., New Delhi, 1998, 124.

13 Brain K R and Turner T D, The practical evaluation of

phytopharmaceuticals, Wright- Scientechnica, Bristol, 1975,

4-9, 36.

14 Evans W C, Pharmacognosy, 15th edn, E Dinburg, London,

2002, 475, 101-105.

15 Kokate CK, Practical Pharmacognosy, 1st edn, Vallabh

Prakashan, New Delhi, 1986, 111.

16 Goyal RK and Shah BS, Practicals in Pharmacognosy, Nirali

Prakashan, Pune, 2005, 128-155.

17 Jain VK, B Jain and Saha D, Pharmacognostical and

physicochemical evaluation of Pterospermum suberifolium

Linn. leaves, Int J Pharma Res Develop, 2011, 2(11), 89-104.

18 Raghunathan, Pharmacopoeia standards for Ayurvedic

formulations, Central Council for Research in Indian

Medicine & Homeopathy, New Delhi, 1976.

19 Usha S, Pannine J and Sharma HP, Pharmacognostic studies

on Artemisia scoparia Waldst and Kit, Proc Indian Acad Sci

(Plant science), 1984, 93, 151-164.

20 WHO/QCMMPM, Quality control methods for medicinal

plant material, organisation Mondiale De Sante, Geneva,

1992, 22-23

21 Anonymous, World Health Organization (WHO), Quality

control methods for medicinal plant materials, WHO,

Geneva, 1998.

22 Anonymous, Guidelines for the Assessment of Herbal

Medicines, World Health Organization, Geneva, 1998, 8-9,

28-29, 30, 31-33.

23 Chase CR and Pratt RJ, Florescence of powdered vegetable

drugs with particular reference to development of system of

identification, J Am Pharm Assoc, 1949, 38, 324-331.

24 Harborne J B, Phytochemistry, Academic Press, London,

1993, 89-131.

25 Trease GE and Evans WC, Pharmacology, 15th edn, Saunders

Publishers, London, 2002, 42-44, 221-229, 246-249, 303-

306, 331-332, 391-393.

26 Pavanasasivam G and Suktanbawa MU, Flavonoids of

some Dilleniaceae species, Phytochemistry 1975, 14,

1127-1128.

27 Janchen D and Issaq HJ, Modern thin layer chromatography:

advances and perspectives, J Liquid Chromatogr, 1988, 11,

1941-1965.

28 Wallis TE, Practical Pharmacognosy, 5th edn, J and A

Churchill Ltd., London, 1984.

29 Ansari SH, Essentials of Pharmacognosy, 1st edn, Birla

Publications Pvt. Ltd., New Delhi, 2006.

30 Kokate CK, Purohit AP and Gokhale SB, Practical

Pharmacognosy, 4th edn, Vallabh Prakashan, New Delhi,

2006, 107-108.

31 Norskhydro AS and Kiellant S, W.O. 9321925 (ICI A61K

31/56), 1993, l, 921666.

32 Li H, Wang Z and Liu Y, Review in the studies on tannins

activity of cancer prevention and anticancer, Zhong-Yao-Cai,

2003, 26(6), 444-448.

33 Ilyas M and Perveen M, A novel triterpene (neriifolione): A

potent anti-inflammatory and antiarthritic agent from

Euphorbia neriifolia, Humdard Med, 2003, XLVI(2),

97-102.

34 Rasik AM, Shukla A, Patnaik BN and Srivastava DK,

Wound healing activity of latex of Euphorbia neriifolia,

Indian J Pharm, 1996, 28, 107-109.

35 Sharma DK, Bioprospecting for drug research and

functional foods for the prevention of diseases-role of

flavonoids in drug development, J Sci Indust Res, 2006, 65,

391-401.

36 Sharma V, Janmeda P, Paliwal R and Sharma SH,

Anti-hepatotoxic activity of Euphorbia neriifolia extract

against N-nitrosodiethylamine-induced hepatocarcinogenesis

in mice, J Chinese Integr Med, 2012, 10, 1303-1309.