IJNPR 4(4) 348

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 immunomodulatory, 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
_____________
*Correspondent author:
E-mail: drvshs@gmail.com
Phone: + 01438−228386
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
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.
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.
Macroscopic and microscopic studies
Organoleptic and macroscopic evaluation
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.
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).
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
Results
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 nonlignified 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
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
351
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
Table 1 Quantitative microscopy of E. neriifolia leaf
Variables
Stomatal number
Stomatal index
Epidermal cells
Vein islet number
Veinlet termination
number
Abaxial surface Value
Adaxial Surface
(in 1 mm2 area)
Value (in 1 mm2 area)
116±5 mm2
12.88%
784±10 mm2
33±5 mm2
16±5 mm2
52±5 mm2
11.60 %
396±10 mm2
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
INDIAN J NAT PROD RESOUR, DECEMBER 2013
352
Table 2 Florescence characteristic of the powder of E. neriifolia leaves in different means
Treatments with leaves powder
Powder as such
Distilled water
Picric acid
Glacial acetic acid
1N HCl
1N H2SO4
Conc. HNO3
Ferric Chloride (5%)
Iodine solution (5%)
Ammonia solution
1N NaOH
Potassium dichromate
HNO3 + NH3 solution
Methanol
Ethanol
Toluene
UV Short (254 nm)
UV Long (366 nm)
Light yellow
Yellow green
Light green
Yellow
Dark brown
Magenta
Yellow
Dark brown
Greenish yellow
Green
Green
Dark green
Yellow
Light Green
Light Green
Light Green
Light yellow
Fluorescent green
Fluorescent green
Light orange
Greyish
Magenta
Dark brown
Fluorescent green
Brown
Fluorescent green
Fluorescent green
Fluorescent green
Orangish
Light Grey
Fluorescent green
Yellow
Visible
Light green
Light green
Greenish yellow
Light yellow
Brownish yellow
Magenta
Yellow
Green
Yellow
Yellow green
Orangish
Brown
Orangish
Light Green
Light Green
Light Green
The colours mentioned in the table are based on the colour identification chart, Royal Botanica Garden, Edinburg (1969)
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
Table 3 Florescence characteristic of different extracts of
E. neriifolia leaves
Solvent
Pet-ether
Benzene
Chloroform
Ethyl Acetate
Ethanolic
Aqueous
Hydro-ethanolic
Under Ordinary light Under UV light (366 nm)
Yellowish green
Brownish green
Light green
Dark green
Dark brown
Brown
Dark brown
Fluorescent
Fluorescent blue
Light green
Greenish brown
Purplish Brown
Blackish brown
Dark brown
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,
SHARMA & PRACHETA: PHARMACOGNOSTICAL EVALUATION OF EUPHORBIA NERIIFOLIA
353
Table 4 Preliminary phyto-profile of extracts of E. neriifolia leaves in different organic solvents
Sequential
Direct
Ex.
S
PI
Extraction
Colour
Consistency
Nature
% Yield ±SD
HE
HE
PE
B
C
EA
E
Aq
0.0
2.7
4.1
4.4
5.2
9
Soxhletion
Maceration
Soxhletion
Soxhletion
Soxhletion
Soxhletion
Soxhletion
Dark Brown
Chocolate Brown
Florescent Green
Green
Green
Green
Brownish green
Dark Brown
Sticky
Sticky
Dry
Sticky
Sticky
Oily
Sticky
Dry
Semi solid
Solid
Solid
Solid
Solid
Solid
Semi solid
Solid
16.31±0.38
20.02±0.25
3.01±0.11
2.72 ±0.08
0.33±0.17
0.43±0.07
9.78±0.08
17.89±0.45
Maceration
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
Primary
Metabolites
Plant Constituents
Protein and
Amino acids
Carbohydrates
Fixed oils
and Fats
Alkaloids
Secondary Metabolites
Anthraquinones
Cardiac glycosides
Glycosides (Free sugar)
Flavonoids
Phenolic
Pholobatanins
Polyphenol
Saponins
Steroids
Tannins
Terpenoids
Gums &Mucilage
Test Performed
Millon’s test
Biuret test
Ninhydrin test
Xanthoproteinic test
Fehling’s test
Stain test
Soap test
Dragendroff's test
Mayer's test
Wagner's test
Borntrager's test
Killer Killani test
Baljet test
Legal's test
Shinoda test
Ammonia test
Lead acetate test
Alkaline reagent test
Ferric chloride test
Hydro chloride test
Folin-ciocalteau test
Frothing test
Olive oil test
Liebermann-Burchard's test
Ferric chloride test
Salowski test
Ruthenium Red test
HE
+
+
++
++
++
+
+++
++
+++
+++
+++
+++
++
+
+++
++
+++
++
+++
+
+++
+++
+++
+++
PE
+
+
+++
+
+++
++
+
++
++
+
+
++
+++
+++
+
E. neriifolia leaves extracts
B
C
EA
E
+
+
+
+
++
+
+
+
+
+++
++
++
++
++
++
+
++
+++
+
++
+
++
++
++
+
+
+
+
+
++
+
+
++
+++
++
+
++
++
++
+++
++
+
+
++
+++
+
+
+++
+
++
++
++
+++
+
+++
++
+++
+++
+++
+
+++
+
+
+
+++
+
+
++
+++
++
+
+
+++
++
+
++
+++
Aq
+
+
+
+
+
+
+
++
+
+
++
++
+
+
+++
+
+++
(+++) 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
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
INDIAN J NAT PROD RESOUR, DECEMBER 2013
354
Table 6 Thin layer chromatographic studies of different extracts of E. neriifolia
Extracts Solvents
HE
PE
B
C
EA
E
Aq
Mobile Phase
Ratio
n-B: AA: H2O
C:AA: H2O
n-B: AA: H2O
PE:C
B:M
n-B: AA: H2O
EA: M: H2O
n-B: AA: H2O
C: M
n-B: AA: H2O
EA: M: H2O
n-B: AA: H2O
C:E
C:E:H2O
n-B: AA: H2O
AA:HCl:H2O
2:2:6
3:2:5
7.5:3.75:0.5
3:2:5
0.5:9.5
9.5:0.5
4:1:5
7.5: 2: 4
7: 2: 4
3:2:5
9.5:0.5
2:2:6
7.5:1.25:0.5
4:1:7
2:2:6
8:3
8:2:1
4:2:4
10:1:3
Colour
Brown
Yellow Brown
Yellow green
Green, yellow
Yellow, grey
Yellow, grey
Light Yellow
Green, yellow, grey
Green, yellow
Brown
Light green
Grey, Yellow
Yellow, grey, brown
Brown
Brown
Brown
Brown, green
Brown
Brown
1Visible
1
2
2
4
3
2
3
3
3
3
2
5
4
2
2
4
1
1
1IC
2
2
2
5
5
3
6
5
3
7
2
7
5
3
2
8
2
1
TS
Rf values
1
2
2
2
5
5
5
6
5
3
7
2
7
5
2
2
8
2
1
0.74
0.21, 0.86
0.24, 0.62
0.11, 0.62
0.17, 0.33, 0.52, 0.60, 0.88
0.10, 0.40, 0.50, 0.60, 0.82
0.27, 0.31, 0.68, 0.83, 0.92
0.28, 0.38, 0.45, 0.67, 0.79, 0.90
0.40, 0.48, 0.38, 0.70, 0.90
0.81, 0.87, 0.94
0.20, 0.29, 0.40, 0.55, 0.74, 0.85, 0.95
0.75, 0.93
0.12, 0.23, 0.32, 0.43, 0.54, 0.72, 0.88
0.34, 0.48. 0.60. 0.74, 0.95
0.60, 0.79, 0.90
0.21, 0.91
0.04, 0.09, 0.13, 0.24, 0.37, 0.63, 0.90, 0.96
0.69, 0.96
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
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).
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
SHARMA & PRACHETA: PHARMACOGNOSTICAL EVALUATION OF EUPHORBIA NERIIFOLIA
355
Table 7 Chromatographic HPTLC of petroleum ether extract of E. neriifolia leaf
Samples
Quercetin
Rutin
Pet-ether
Mobile phase
B: M
9.5: 0.5
Max Rf
Max Height
Max %
Area
Area%
Assigned substances
0.88
0.47
0.18
0.25
0.27
0.35
0.38
0.57
0.76
0.80
0.83
0.95
98.2
114.3
60.7
36.0
28.1
51.3
53.3
121.5
12.3
17.0
17.1
20.5
34.98
58.25
11.77
6.99
5.44
9.95
10.33
23.57
2.38
3.30
3.32
3.97
1158.7
1055.1
907.0
282.9
331.0
922.4
1184.7
2868.0
294.1
189.0
323.1
687.3
38.93
55.20
7.53
2.35
2.75
7.66
9.84
23.81
2.44
1.57
2.68
5.71
Standard
Standard
Unknown
Unknown
Unknown
Unknown
Unknown
Rutin
Unknown
Unknown
Quercetin
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).
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 histoanatomical 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
356
INDIAN J NAT PROD RESOUR, DECEMBER 2013
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.
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