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Pharmacognostical & Physicochemical Studies of Euphorbia Tithymaloides

(L.) Poit

Article in IOSR Journal of Pharmacy and Biological Sciences · March 2020

DOI: 10.9790/3008-1406011724

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IOSR Journal Of Pharmacy And Biological Sciences (IOSR-JPBS)
e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 14, Issue 6 Ser. I (Nov –Dec 2019), PP 17-24
www.Iosrjournals.Org

Pharmacognostical& Physicochemical Studiesof Euphorbia

Tithymaloides (L.) Poit
Ronit Poudel1*, Rajani Srivastava1,Adarsh Tripathi1
1
(Department of Pharmaceutical Sciences, SIHAS, Sam Higginbottom University of Agriculture, Technology &
Sciences, Prayagraj, Uttar Pradesh, India,211007)

Abstract:Euphorbia tithymaloides (L.) Poit (Euphorbiaceae) leaves are extensively used in traditional
medicine tocure asthma, mouth ulcers, persistent coughing, venereal troubles & ringworms. Besides this, it is
also known to possess antiprotozoal, mitogenic, anti-inflammatory, anti-plasmodial, anti-mycobacterial,
anthelminthic and antimicrobial activities. The present study is an attempt to provide detailed information on
pharmacognostical & physicochemical features of E. tithymaloides. Fresh plant was selected for macroscopical
and microscopical studies while air-dried powdered sample of whole plant was used for physicochemical
evaluation and powder microscopy. The macroscopical study of the plant showed that leaves are dark-green,
alternate, ovate in shape having entire margin, acute apex and attenuate base, root is externally smooth, light
brown tap root with few branches & numerous small lateral roots and has got a thin bark featured with few
cracks & fissures while stem is very long, non-woody , greenish & cylindrical in shape which has anisotomous
branching. Similarly, the microscopy of leaves revealed that it consists of single layered epidermis with cuticle
and trichomes, 2-3 layered collenchyma near the upper epidermis followed by 6-8 layered spongy parenchyma
and a centrally placed C-shaped bicollateral vascular bundles. Ash values, extractive values, loss on drying,
foaming index like physicochemical parameters were also found out. The results of the study can be used as
markers in the identification and standardization of this plant and also towards monograph development on the
plant.
Keywords: Euphorbia tithymaloides,Microscopy, Macroscopy, Pharmacognostical study,Physicochemical
study
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Date of Submission: 26-10-2019 Date of Acceptance: 11-11-2019
----------------------------------------------------------------------------------------------------------------------------- ----------

I. Introduction
Euphorbiatithymaloides (L.) Poit (Family: Euphorbiaceae), commonly known as devil‟s backbone, is
widely distributed throughout tropical and subtropical North America and Central America and some areas of
South Asia (India: Assam, Bihar, Gujarat, Madhya Pradesh, Maharashtra, Odisha, UP).[1] The soil must be sandy
&nutrient-rich, particularly in boron, copper, iron, manganese, molybdenum and zinc for its better growth. This
plant, which is often known by its old scientific name Pedilanthustithymaloides, is relatively intolerant of high
soil salinity levels.[2][3] As it has got a very impressive variegated foliage, it is mostly used as an ornamental
plant. The plant is 0.4 to 3m tall & 40-6cm wide whose fleshy tubular, zigzag stems produce thick, dark-green,
ovate leaves and peculiar beak shaped flowers. Leaves are sessile, glabrous and acuminate in shape having
length of about 1.4-3 inches while flowers whose color vary from bright white to pink are arranged in
dichotomous congested cyme fashion. Fruits are sulcate, deeply 3-lobed having sub globose grey-brown seeds.[4]
Although flowering in flushes year-round in warm tropical regions, it blooms most heavily in summer. When
flowering or chilly winter temperatures occur, the leaves may blush pink. An extended drought or winter cold
spell may cause the leaves to completely drop off.[5][6]
It is widely used in traditional medicine to treat asthma, persistent coughing and mouth ulcers. The
plant is also reported to possess various antioxidant principles like kaempferol 3-O-b-D-glucopyranoside-6‟-(3-
hydroxy-3-methylglutarate), quercitrin, isoquercitrin, scopoletin and other phytochemicals viz. steroids, tannins,
triterpenes, coumarins and saponins which have been shown to possess anti-diabetic, anti-viral, hemostatic, anti-
microbial, anti-helminthic and mitogenic activity.[7] Its sap has been traditionally used to treat callouses, ear
ache, insect stings, ringworm, skin cancer, toothache, umbilical hernias, and warts, though none has been
scientifically proven as effective.[8]

II. Materials and Methods

Chemicals and Instruments: Microscope, Camera Lucida, stage and eye piece micrometer, black chart paper,
blade, watch glass, slide, pipette, HCl, Phloroglucinol, Chloral hydrate, Ethanol, Chloroform & Sulfuric acid

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 Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.) Poit
Plant Collection: The fresh plant E. tithymaloides for quantitative microscopy and physicochemical studies was
procured from New Bridge Chouraha, Prayagraj with the help of local tribe and field botanist. The healthy
leaves of the plant were first collected and stored properly. They were then washed with water and dried in
sunlight for one hour and thereafter dried in shade. The dried leaves along with the dried plant were powdered
with the help of grinder and were passed through the sieve no 60. The pharmacognostical evaluation and
physicochemical studies of the fine powder and that of healthy leaves were then performed.
Macroscopical Studies: The morphological characters like condition, type, size, shape, apex, margin, venation,
base, petiole, surface, phyllotaxy, color, odor, taste of leaves, length, branching, color, shape, internodal length
of stem and type, shape, rootlets, odor, taste, presence or absence of bark of root of E. tithymaloides were
studied.
Microscopical Study: The fresh leaf collected from the plant was washed with clean water. A small part of leaf
from the midrib was then cut off with the help of blade. Then, a slice of a potato was cut into two parts not fully
(last end was united). The leaf section was placed in between the slices which was held with thumb and index
finger (Middle finger was kept down the potato slice). Then, the blade was driven parallel to the surface of the
index finger. The cut off sections were put in a watch glass containing mixture of phloroglucinol & HCl (1:1) &
then the better one was chosen for microscopical study. For the study of morphology of stomata, the surface of
the leaf was prepared.[9]
Quantitative Microscopy:
Stomatal Number: It is the average no of stomata per square mm of the epidermis of the leaf. First of all, a fresh
leaf collected from the plant was washed with clean water. The epidermis was then separated from the leaf by
breaking the leaf into pieces and placed carefully on a slide with the help of a brush along with 1-2 drops of
chloral hydrate. A square was then drawn on a drawing sheet. With the help of camera lucida, the stomata &
epidermal cells were traced in the square. The epidermal cells and the stomata outside the square were traced to
completion on two adjacent sides of the square, for counting purpose. Then the no of complete epidermal cells
and the stomata within the square and the cells that are more than half on two adjacent sides are counted and
stomatal no is calculated.[9]
Stomatal Index: It is calculated by using this formula:
I= S/E + S * 100
I= Stomatal Index, S=No of stomata per unit area, E= No of epidermal cells in the same area.
Powder Microscopy: The healthy plants were first collected and then stored properly. It was then dried under
sunlight for 4 hours and then at a shade for one whole day. The dried plant was then dried in a grinder and
passed through sieve no 60. The fine powder was then stained with phloroglucinol + HCl (1:1) and observed
under microscope .
Physicochemical Studies: The physicochemical parameters such as Total ash, Water Soluble Ash, Acid
insoluble Ash, Sulphated Ash, Solvent extractive value, Loss on drying, Foaming Index.[9][10][11]
Total Ash: 2g of air-dried powdered drug was accurately weighed and put into the tared silica crucible. The
crucible was supported on a pipe-clay triangle placed on a ring of retort stand. Then, it was heated with a burner,
using a flame about 2cm high and supporting the crucible about 7 cm above the flame heat till vapors almost
ceased to be evolved; then it was lowered and heated more strongly until all carbon was burnt off. It was then
cooled in a desiccator. The ash thus produced was weighed and the percentage of total ash with reference to the
air-dried sample of the crude drug was calculated.
Water Soluble Ash: The ash obtained while calculating total ash value was washed using 25 ml of distilled
water into 100ml beaker. It was boiled for 5 minutes and then filtered through an „ashless‟ filter paper. The
residue thus obtained was washed twice with hot water. The crucible was ignited, cooled and weighed. The filter
paper containing the residue was put into the crucible which was then heated gently until vapors ceased to be
evolved and then more strongly until all carbons has been removed. After the ignition, it was cooled in a
desiccator. The residue was weighed and water-soluble ash was calculated by subtracting the weight of residue
from weight of total ash. The % of water-soluble ash was then calculated with reference to air-dried powdered
drug.
Acid Insoluble Ash: First of all, the total ash was washed using 25ml of 2N HCl into 100ml beaker. It
was then boiled for 5 minutes and then filtered through an „ashless‟ filter paper. The residue thus obtained was
washed twice with hot water. The crucible was ignited, cooled and weighed. The filter paper containing the
residue was put into the crucible which was then heated gently until vapors ceased to be evolved and then more
strongly until all carbons have been removed. After the ignition, it was cooled in a desiccator. The residue was
weighed and acid- insoluble ash was calculated by subtracting the weight of residue from weight of total ash.
The % of acid insoluble ash was then calculated with reference to air-dried powdered drug.
Solvent Extractive Values:5gm of drug was macerated with 100ml of different solvents (90% alcohol-
alcohol soluble extractive, 90% chloroform water- water soluble extractive) in a dry 250 ml conical flask for
24hrs. It was shaken frequently during first 6 hrs. and allowed standing for 18hrs. Thereafter it was filtered.
25ml out of filtrate was evaporated to dryness on a water-bath. The drying was then completed in an oven at
DOI: 10.9790/3008-1406011724 www.iosrjournals.org 18 | Page
 Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.) Poit
100˚C. The weight was then measured and the percentage w/w of extractive was subsequently calculated with
reference to the air-dried drug.
Loss on drying: Firstly 3.0 gm of powdered drug was weighed accurately and put in a tarred porcelain
dish which was earlier dried at 105˚C using hot air ovens at constant weight. Using the difference in weight, the
percentage loss of drying with reference to the air-dried substance was calculated.
Foaming Index: About 1 gm of coarsely powdered drug was weighed accurately and transferred to
500ml conical flask containing 100ml of water maintained at moderate boiling at 80-90C for about 30 min.
After 30 min of boiling, it was cooled, filtered into a volumetric flask and sufficient water through the filter to
make the volume up to 100ml (V1). Clean 10 stopper test tubes were then taken and marked as 1, 2….,10. The
successive portions of 1ml ,2ml…..., 10ml drug was taken in separate tubes and then remaining volume was
adjusted with the liquid up to 10ml in each. After closing the tubes with stoppers, they were shaken for 15
seconds and allowed to stand for 15 min. Then the height was measured. If the height of the foam in each tube is
less than 1cm, the foaming index is less than 100(not significant). While, if the foam is more than 1cm height
after the dilution of plant material in the sixth tube, then corresponding number of the test tube was the index
sought. If the height of the foam in every tube is more than 1cm, the foaming index is more than 1000. In this
case, 10ml of the first decoction of the plant material needs to be measured and transferred to a 100ml
volumetric flask (V2) and volume is to be maintained up to 100ml and follow the same procedure. Foaming
Index was calculated by using this formula:
Foaming Index = 1000/a in case of V1;
Foaming Index = 1000 * 10/a in case of V2
Where, a= volume (ml) of decoction used for preparing the dilution in the tube where exactly 1cm or more foam
was observed.
III. Results & Discussion
Macroscopical Features:
i. Leaf: The information obtained from the macroscopical studies of the leaf (like type, base, margin,
apex, color, odor, etc.) are summarized in table no 1.

Fig. i): Upper Surface of the leaf

Fig. ii): Lower Surface of the leaf

Observation

Condition Fresh
Type Simple
Length: 4.4-7.4cm
Size
Width: 2.6 – 3.9cm
Shape Ovate to cordate
Margin Entire

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 Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.) Poit
Apex Acute
Base Attenuate
Venation Unicostate reticulate venation
Phyllotaxy Alternate
Surface Glabrous
Upper Surface: Dark Green
Color
Lower Surface: Light Green
Odor Slightly aromatic
Taste Mucilaginous
Extra Feature Very thick leaf with cuticle present in it.

Table No 1: Macroscopical Features of E. tithymaloides leaf

ii. Stem
The stem is about 28.3cm long and 1.6-3.1cm thick with branches different in both size/vigor and angle from the
main axis (anisotomous branching). It is non-woody, greenish in color and cylindrical in shape which sprouts
from woodycrown of the root. It has got distinct nodes and internodes (internodal length varies from 1.8cm-
3.9cm). The stem when broken exudes a caustic milky sap (latex) which gives mucilaginous taste. Its odor is
slightly aromatic.

Fig. iii): Stem of E.tithymaloides

iii. Root:
Root present in this plant is a tap root (having length of about 13.1cm) with few branches and numerous small
lateral roots that show horizontal downward growth. It is light brown in color. The woody crown present in the
top is durable in structure. The root is externally smooth with thin bark (easily gets peeled off exposing pale
yellow inner part) which is featured with few cracks and fissures on its surface. It is slightly aromatic in odor but
tasteless in taste.

Fig. iv): Root of E. tithymaloides

Microscopical Study: The microscopical studies of the leaf are summarized in the following points.

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 Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.) Poit
i. It consists of single layered upper and lower epidermis followed by 6-8 layers of spongy
parenchymatous cells. The cells are closely packed without any intercellular space.
ii. Presence of a thick cuticle and trichomes (both glandular and covering). 2-3 layers of collenchyma are
seen towards the upper epidermis.
iii. Anomocytic stomata are present in the epidermis of the leaf (Lower surface has comparatively higher
no of stomata than upper surface). The epidermal cells surrounding the subsidiary cells are thick walled and
irregular with walls straight at some part of the surface.
iv. Presence of C-shaped, centrally situated, bi-collateral vascular bundle (phloem-xylem-
phloem).Metaxylem is seen towards the lower epidermis side while protoxylem can be seen towards the upper
epidermis side. Pith is absent in the T.S.

Fig. v)T.S. of leaf of E. tithymaloides. (TR- Trichome, Col- collenchyma, U.E.- Upper Epidermis,
L.E.- Lower Epidermis, Cut-Cuticle, Xy- Xylem
Ph- Phloem, S.P. -Spongy Parenchyma)

Fig. vi) Surface preparation of leaf (upper surface) of E. tithymaloides.

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 Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.) Poit

Fig. vii) Surface preparation of leaf (lower surface) of E. tithymaloides.

Quantitative Microscopy of leaf: The quantitative microscopy of the leaf of E. tithymaloides helped us to
know the values of the following parameters.
Table No 2: Leaf Constants for E. tithymaloides leaf
Values
Leaf Constants (Lower Surface)
(Upper Surface)
Stomatal No 11.88 22.5
Stomatal Index 4.17 7.88

Powder Microscopy of Whole Plant: Powder of the plant is light brown in color, faint in odor and has got
slightly mucilaginous taste. Its microscopy revealed some diagnostic features like
i. Epidermal cells with numerous spongy parenchymatous cells.
ii. Numerous prismatic crystals of calcium oxalate (crystals are variable in size with smaller ones often
forming small aggregates).
iii. Very occasional covering trichomes (they are unicellular, thick-walled and conical in shape)
iv. Long, non-lignified fibres with slit-shaped pit. Some of them occur as associated with vessels. Numerous
lignified xylem tracheids (pink) and vessels are also seen.
v. The oil cells occur singly. They are very large, subspherical to ovoid in shape and have moderately
thickened walls. In uncleared mounts, cells are filled with oil and are yellow in color.

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 Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.) Poit

Fig. viii) Powder microscopy of whole plant of E. tithymaloides (A-E: CRY – Crystals of calcium oxalate, Fib-
Non-lignified fibre,Xy.Tr- Xylem tracheid along with vessel, C.Tr- Covering Trichome, O.cell- Oil cell)

Physicochemical Analysis:Estimation of ash value is a significant parameter for the detection of nature of
material, which is added to the drug for the purpose of adulteration, impurities & determination of authenticity,
quality & purity of test sample. The higher ash value (11.715%) of this plant indicates that carbonates,
phosphates, silicates & silica are present in higher amount.
The water-soluble extractive value (22.867%) indicates the presence of sugar, acids & inorganic compounds &
the alcohol-soluble extractive value (11.216%) indicates the presence of polar constituents like phenols,
steroids, glycosides & flavonoids. The determination of these values is primarily useful for the identification of
exhausted drug. Similarly, % moisture content (4.883%) indicates the percentage loss of volatile substances
along with water. Less moisture content is needed for prevention of chemical decomposition & microbial
contamination in the natural products.
The following table (Table No 3) shows the values of all the parameters obtained during physicochemical
studies of powder of whole plant of E. tithymaloides.

Analytical Parameter Values obtained (%w/w)

Total Ash 11.715%

Acid insoluble ash 4.12%
Water soluble ash 5.02%
Alcohol soluble extractive value 11.216%
Aqueous extractive value 22.867%
%moisture content 4.883%
Foaming Index Not significant
TableNo 3: Physicochemical Constants of Powder of Whole Plant of E. tithymaloides

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 Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.) Poit

IV. Conclusion
Thus, after a thorough investigation about the pharmacognostical and physicochemical profile of E.
tithymaloides, we were able to determine germane qualitative and quantitative information which helps to serve
as the standards to ascertain the identity and to determine the quality and purity of the plant material in future
studies .Besides these, these crucial information also serves as a basis for monograph development and as the
markers for the development of effective and non-toxic drug. As this plant has shown some promising features
regarding therapeutic uses and as there has been a large void in the network of its attainable data , there seems to
be a necessity for the involvement of researchers in its exhaustive study.

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Ronit Poudel." Pharmacognostical& Physicochemical Studiesof Euphorbia Tithymaloides (L.)

Poit." IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) 14.6 (2019): 17-24

DOI: 10.9790/3008-1406011724 www.iosrjournals.org 24 | Page

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