Pharmacognostical profiling on the root of Rauwolfia Serpentina

Abstract

Each medicinal plant and the specific plant part used as crude drug material contain active or major chemical constituents with a characteristic profile that can be used for chemical quality control and quality assurance. So the increasing demand for herbal medicines has inevitably led to maintaining the quality and purity of herbal raw materials and finished products. WHO acknowledged that Pharmacognostical standards should be proposed as a protocol for the authentication and quality assurance of herbal drugs. The important histological features of Rauwolfia serpentina are the cork, composed of alternating layers of broad and narrow cells giving a somewhat spongy and friable exterior to the drug; secondary cortex composed of several rows of parenchymatous cells, filled with starch grains and brown resin masses; secondary xylem consisting of vessels, tracheids, xylem parenchyma and wood fibres traversed by xylem rays, and showing annual rings; secondary phloem consisting of sieve cells, companion cells and parenchymatous cells containing starch grains, rosette and prismatic crystals of calcium oxalate and occasionally some brown resin masses. © 2014, International Journal of Pharmacognosy and Phytochemical Research. All rights reserved.

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International Journal of Pharmacognosy and Phytochemical Research 2014; 6(3): 612-616
ISSN: 0975-4873
*Author for correspondence: Email: asemosem@yahoo.com.
Research Article
Pharmacognostical Profiling on the Root of Rauwolfia Serpentina
*Rungsung W, Dutta S, Mondal DN, Ratha KK, Hazra J
National Research Institute for Ayurvedic Drug Development, CCRAS, 4-CN Block, Sector-V, Salt Lake, Kolkata-91.
Available Online: 1st September 2014
ABSTRACT
Each medicinal plant and the specific plant part used as crude drug material contain active or major chemical constituents
with a characteristic profile that can be used for chemical quality control and quality assurance. So the increasing demand
for herbal medicines has inevitably led to maintaining the quality and purity of herbal raw materials and finished products.
WHO acknowledged that Pharmacognostical standards should be proposed as a protocol for the authentication and quality
assurance of herbal drugs. The important histological features of Rauwolfia serpentina are the cork, composed of
alternating layers of broad and narrow cells giving a somewhat spongy and friable exterior to the drug; secondary cortex
composed of several rows of parenchymatous cells, filled with starch grains and brown resin masses; secondary xylem
consisting of vessels, tracheids, xylem parenchyma and wood fibres traversed by xylem rays, and showing annual rings;
secondary phloem consisting of sieve cells, companion cells and parenchymatous cells containing starch grains, rosette
and prismatic crystals of calcium oxalate and occasionally some brown resin masses.
INTRODUCTION
The increasing demand for herbal medicines, both in the
developing and developed countries, has inevitably led to
maintaining the quality and purity of herbal raw materials
and finished products. 1 WHO, therefore, acknowledged
that Pharmacognostical standards should be proposed as a
protocol for the authentication and quality assurance of
herbal drugs. Rauwolfia serpentina (Linn.) Benth. ex Kurz,
belonging to the family Apocynaceae, and popularly
known as India’s wonder drug plant, is an upright,
perennating, evergreen undershrub with tuberous roots
(Fig. 1). Leaves are simple, glabrous, lanceolate or obovate
and generally in whorls of three to four, crowding the
upper part of the stem; flowers white or violet-tinged and
borne on corymbose cymes; fruits tiny, oval, fleshy which
turn shiny purple-black when ripe.2,3,4,5 Mainly the roots of
the plant are used for various ailments like insomnia,
hypertension, insanity, epilepsy, intestinal disorders,
cardiac and liver diseases, hysteria, constipation and
schizophrenia. It is also anthelmintic, a tranquilizer and an
antidote against the bites of snakes and venomous
reptiles.6,7,8,9 The characteristic fluorescence patterns
emitted by the roots of Rauvolfia was studied under
daylight and ultraviolet radiation by Selvam and
Bandyopadhyay (2005).10 The principle alkaloid of
Rauwolfia serpentina is reserpine.11,12 Phyto-
pharmacognostical studies and quantitative determination
of reserpine have been carried out in different parts of
Rauwolfia species by Panda et al. (2012).13 The present
investigation was, therefore, undertaken to evaluate
various qualitative and quantitative parameters on the root
of Rauwolfia serpentina, the findings of which will be
helpful in setting standards for this medicinal plant.
MATERIALS AND METHODS
The plant material, collected at an appropriate stage of its
growth from NRIADD, Kolkata was authenticated through
detailed taxonomic study and then air-dried for
pharmacognostical study. Macroscopical study was
carried out with the naked eyes/aid of a magnifying lens to
determine the shape, size, texture, etc. as per requirement
of Indian Herbal Pharmacopoeia. Microscopical study was
performed by preparing a thin hand section of the rhizome,
cleared with chloral hydrate solution and stained as per the
standard protocol.14,15,16 The dried material was coarsely
powdered in a blender and subjected to various tests-
powder analysis was carried out with reference to the
presence or absence of particular diagnostic characters for
rapid and accurate determination of their identity
following the methods of Wallis (1999)17 and Trease and
Evans (2002)16; fluorescence analysis was carried out as
per the method advocated by Chase & Pratt (1949)18 and
Harborne (1973)19; and physico-chemical analyses such as
total ash, acid-insoluble ash, etc. on the basis of protocols
prescribed by WHO on Quality Control Methods for
Herbal Materials (2011)20 and Indian Pharmacopoeia
(2001)21. For chemical profiling of the plant, 100gm of the
dried powder was subjected to cold extraction with ethanol
and chloroform (1:1) for 7 days; the extract concentrated
and then carried out High Performance Thin Layer
Chromatography (HPTLC) following the method of Egon
Stahl (2005)22.
RESULTS
Macroscopic Analysis: Roots stout, hard, rough with
irregular longitudinal fissures on the surface, sub-
cylindrical to tapering, rather tortuous or curved, outer

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surface greyish-yellow to light brown and pale yellowish
white inside, odour indistinct and taste bitter. Rootlets
absent but few small circular root scars present. When
scraped, the bark separates readily from the wood. Fracture
short, irregular, the longer pieces readily breaking with a
snap. The transversely cut surface shows a finely radiating
xylem with clearly marked growth rings (Fig. 2).
Microscopic Analysis: Transverse section of root shows
alternating strata of suberized cork cells, the strata with
larger cells alternating with strata of markedly smaller
cells. The secondary cortex consists of several rows of
tangentially elongated to isodiametric parenchymatous
cells, being filled with starch grains and brown resin
masses. The secondary xylem represents the large bulk of
the root and composed of vessels, tracheids, xylem
parenchyma and xylem fibres traversed by xylem rays, and
showing one or more annual rings with a dense core of
wood at the center. Cambium is indistinct, narrow, dark
and wavering. The secondary phloem is relatively narrow
and consists of sieve cells, companion cells and
parenchymatous cells containing rosette and prismatic
crystals of calcium oxalate and occasionally some brown
resin masses in outer cells and phloem rays. Starch grains
mostly simple, but compound granules also occur.
Powdered-drug Analysis: Brownish to reddish grey in
color, slight odour and bitter taste; characterized by
spherical, simple, semi-compound and compound starch
grains; rosette and prismatic crystals of calcium oxalate;
brown resin masses; uniseriate medullary rays; elongated
cork cells; pitted vessels with simple perforation; lignified
and pitted tracheids; lignified xylem fibres, occurring
singly or in small groups; parenchymatous cells containing
starch grains and brown resin masses (Fig. 4).
Physico-chemical Analysis (Determination of Identity,
Purity and Strength): The physico-chemical analyses of
foreign matter, total ash, acid-insoluble ash, alcohol-
soluble extractive, water-soluble extractive and moisture
content taken in triplicate are shown below along with their
inferences (Table 1):
Fluorescence Analysis: The powdered drug on treating
with various reagents emitted the following fluorescence
properties or colours under UV chamber (Table 2):
HPTLC Profile of Extract:
Sample preparation: 50 grm of dried powder of the roots
of Rauwolfia serpentina (Sarpagandha) was subjected to
cold extraction with Ethanol Chloroform (1:1) for 7 days
and extract was filtered using filter paper (Whatman No
40). The whole extract was concentrated and used for the
Fig. 1: Habit of R. serpentina
Fig. 2: Root of R. serpentina
Fig. 3: T.S. of Root
Table 1: Physico-chemical Analysis
Material
Parameter
Value in %
Observation
I
Observation
I1
Observation
I11
Mean
Root powder
Foreign Matter
1.30
1.10
0.90
1.10
Total Ash
7.20
7.40
7.20
7.27
Acid-insoluble Ash
0.30
0.20
0.30
0.27
Alcohol-soluble
Extractive
8.10
8.00
7.80
7.97
Water-soluble Extractive
12.90
13.50
13.10
13.17
Moisture Content
(at 105°C)
10.60
11.10
10.80
10.83
Inference (mean of triplicate): Foreign Matter=1.10%; Total Ash=7.27%; Acid-insoluble Ash=0.27%; Alcohol-soluble
Extractive=7.97%; Water-soluble Extractive=13.17%; Moisture Content at 105°C=10.83%.

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HPTLC Profile.
Stationary Phase: Precoated (support on Aluminum
Sheets) Silica Gel Plate. Specification: TLC Silica Gel
60F254, Mfg. by Merck, 26.09.2011, Batch No.
1.05554.0007.
Mobile Phase: Toluene: Ethyl acetate: Methanol: Formic
acid (5.25:3.5: 1.25: 0.5) [GR grade solvent used , mfg. by
MERCK, India].
Sample application: Applied volume 5 µL as 9 mm band
and applied at 10 mm from the base of the plate. Plate size
10 X 10 cm.
Development: Developed up to 80 mm in CAMAG Twin
trough chamber, plate preconditioning (temp. 40°C and
relative average humidity 40%).
Derivatising Reagent: Dipped in 20% aqueous Sulphuric
acid and charred at 105°C for 10 minutes.
DISCUSSION
Each medicinal plant and the specific plant part used as
crude drug material contain active or major chemical
constituents with a characteristic profile that can be used
for chemical quality control and quality assurance. In the
present investigation, the macroscopic study reveals the
root to be hard, rough with irregular longitudinal fissures,
sub-cylindrical to tapering, rarely branched, greyish-
yellow to light brown outside and pale yellowish white
inside, indistinct odour and bitter taste. These gross
morphological characters of plant or plant part/s provide
the best basis for identification, and also reveal the
presence of contaminant or deterioration in a sample. The
microscopical or anatomical study gives a preliminary idea
about the nature and disposition of cells, tissues and cell
inclusions, and thus helps understand where the
Fig. 4: Powder Microscopy - a. Powdered root; b. Elongated cork cells; c. Parenchymatous cells containing starch
grains and brown resin masses; d. Tracheids and vessels along with medullary rays; e. Pitted vessel with simple
perforation; f. Lignified and pitted tracheid; g. Uniseriate medullary rays; h. Xylem fibre showing lumen; i. Group of
fibres; j. Rosette and prismatic crystals of calcium oxalate; k. Brown resin masses; l. Simple to compound starch grains.
Table 2: Fluorescence Analysis
Material
Solvent
Distinctive Colours Observed
Short UV (254nm)
Long UV (366nm)
Root powder
Water
+ ve greyish blue
+ ve whitish blue
Methanol
+ ve sky blue
+ ve whitish blue
Ethanol
+ ve sky blue
+ ve whitish blue
Ethyl Acetate
+ ve faint brownish
+ ve pale greyish
Chloroform
+ ve yellowish
+ ve creamish brown
Pet. Ether
Dark brownish
Faint greyish

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compounds of interest are located. Linear measurement of
cells and tissues further provides better diagnostic
characters for accurate identification. The internal
structure of root shows alternating strata of large and small
cork cells; secondary cortex composed of several rows of
parenchymatous cells, filled with starch grains and brown
resin masses; secondary xylem made up of vessels,
tracheids, xylem parenchyma and wood fibres traversed by
xylem rays, and showing annual rings; secondary phloem
consisting of sieve cells, companion cells and
parenchymatous cells containing starch grains, rosette and
prismatic crystals of calcium oxalate and occasionally
some brown resin masses. The cell contents of diagnostic
value as confirmed from the powder analysis are rosette
and prismatic crystals of calcium oxalate; brown resin
masses; spherical, simple and compound starch grains;
uniseriate medullary rays; elongated cork cells, up to 87
µm in length; pitted vessels with simple perforation, up to
345 µm in length and 58 µm in diameter; lignified and
pitted tracheids; lignified xylem fibres, occurring singly or
in small groups and parenchymatous cells containing
starch grains and brown resin masses. Fluorescence
analysis under the various reagents exhibited different
shades of colour, and thus helps in fulfilling the
inadequacy of physical and chemical methods for
identification of the crude drug. The physico-chemical
analysis is helpful in judging the identity and purity of the
crude drug even from the crushed or powdered form. Every
herb has a characteristic mineral content and
corresponding typical ash content. So, when plant drugs
are incinerated, they leave an inorganic ash which in the
case of many drugs varies within fairly wide limits, and
these values are of significance for the purpose of plant
drug evaluation. Inorganic ash includes both
‘physiological ash’ derived from the plant tissue itself, and
‘non- physiological ash’, which is the residue of
extraneous matter adhering to the plant surface. Hence, the
quantitative test of total ash helps in determining both
physiological and non-physiological ash. Acid-insoluble
indicates the amount of silica present, especially as sand
and siliceous earth; and the determination of extractive
matter reveals the amount of active constituents present.
Table 3: Rf Values of the three Plates
Observed at 254nm
Observed at 366nm
Observed at white light
Rf Values
Colour
Rf Values
Colour
Rf Values
Colour
0.09
0.12
0.21
0.36
0.41
0.47
0.58
Bluish grey
Sky blue
Light grey
Light grey
Very light grey
Very light grey
Very light grey
0.09
0.17
0.22
0.26
0.34
0.38
0.48
0.76
Blue
Faint blue
Bright blue
Sky blue
Sky blue
Light greenish
Light greenish
Faint grey
0.09
0.12
0.17
0.20
0.36
0.40
0.47
0.54
0.57
0.70
Grey
Deep grey
Grey
Grey
Grey
Grey
Grey
Deep grey
Grey
Grey
Plate 1
Plate 2
Plate 3
HPTLC Fingerprints observed at 254 nm (Plate 1), 366 nm (Plate 2) and white light (Plate 3)

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Checking moisture content helps reduce error in the
estimation of actual weight of the drug material. Low
moisture content is an indication of better stability against
degradation of product. And determination of foreign
matter enables to get the drug in pure form. HPTLC is a
valuable quality assessment tool for the identification and
quantification of chemical constituents present in plant
drugs. The retention factor (Rf) values obtained from it can
be used to identify compounds due to their uniqueness for
each compound. In the present study, the Rf values of
individual compounds appearing as spots vertically have
been noted (the less polar compounds moving higher up
the plates resulting in higher Rf values), which may thus be
used as a quality control profile for this drug.
ACKNOWLEDGEMENT
We, do hereby, express our deep sense of gratitude and
appreciation to the Director of NRIADD, Kolkata for
permitting us to undertake this research work, thus
enabling us to get the feel of doing the work and kept going
till completion.
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