Comprehensive study on pharmacognostic, physico and phytochemical evaluation of Terminalia arjuna Roxb. stem bark

~ 186 ~
Journal of Pharmacognosy and Phytochemistry 2015; 4(3): 186-193
E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2015; 4(3): 186-193
Received: 13-07-2015
Accepted: 14-08-2015
Gunjan M Chaudhari
Department of Biochemistry,
Moolji Jaitha College,
Jilha peth, Jalgaon-425002
(M.S.), India.
Raghunath T Mahajan
Post Graduate College of Science,
Technology and Research,
Moolji Jaitha College Campus,
(M.S.) India.
Correspondence
Gunjan M Chaudhari
Department of Biochemistry,
Moolji Jaitha College,
(M.S.), India.
Email:
gmchaudhari9@gmail.com
Comprehensive study on pharmacognostic, physico and
phytochemical evaluation of Terminalia arjuna Roxb.
stem bark
Gunjan M Chaudhari, Raghunath T Mahajan
Abstract
Terminalia arjuna Roxb. (Family-Combretaceae) is commonly known as Arjun tree and valued for its
medicinal uses. In the present investigation, the detailed pharmacognostic study of T. arjuna stem bark
(TASB) is carried out to lay down the standards which could be useful in forthcoming experimental
studies. The study includes microscopy, proximate analysis and physicochemical evaluation. Atomic
absorption spectroscopic analysis of TASB revealed the presence of heavy metals within the
recommended safety range. Qualitative phytochemical analysis of the methanolic extract of TASB
(MeOH-TASB) confirmed the presence of alkaloids, glycosides, tannins, flavonoids, phenols, saponins
and steroids in the extract. The UV-Vis and FTIR spectroscopic analysis of MeOH-TASB indicated the
presence of aromatic phytoconstituents as predominant ingredient of the extract. The chemical
fingerprinting of MeOH-TASB was performed by using TLC and GC-MS analysis which showed
bands/peaks of different phytoconstituents. Findings of this study provide reference information on
TASB for its quality evaluation.
Keywords: Terminalia arjuna; Pharmacognosy; Phytochemical screening; Fluorescent analysis;
Spectroscopic evaluation; Chemical fingerprinting.
Introduction
The plant kingdom is a treasure house of potential drugs and in the last few decades there has
been an exponential growth in the field of herbal medicine. It is getting popularize in
developing and developed countries owing to its natural origin and lesser side effects [1]
.
According to World Health Organization (WHO) more than 80% of the world's population
relies on herbal medicine for their primary healthcare needs [2]
. Herbal formulation involves
use of fresh or dried plant part. Thus appropriate identification of the plant material is very
much necessary. Proper identification of the starting material is an essential prerequisite to
ensure quality, safety and efficacy [3]
. In herbal technology, pharmacognostic studies play
important role as it provides standardization parameters which will help to prevent
adulterations in the original plant material and ensures plant identity. This information will
help in authentication of the plants and ensures reproducible quality of herbal products which
will result in safety use and retaining effectiveness of natural products [4]
.
The plant Terminalia arjuna Roxb. Commonly known as Arjuna, a common tree for its
important phyto constituents belongs to the family combretaceae. It has been grown in most
parts of India and used in Ayurvedic formulations since ancient times. The plant parts such as
stem bark, leaves and fruits of T. arjuna are used in indigenous system of medicine for
different ailments. The bark powder has been found to possess cardioprotective properties,
anti-ischemic, antioxidant action [5]
, hypercholesterolemia effect [6]
, fungicidal and
antibacterial [7]
, antimicrobial [8]
, Anti-inflammatory, immunomodulatory and antinociceptive
activity [9]
, It is also useful to cure obesity, hypertension and hyperglycemia [10]
. The higher
antioxidant potential of T. arjuna stem bark is due to the presence of higher amount of
phenolic and flavonoids [11]
. The T. arjuna based phytochemicals are considered as one of the
best heart tonic [12]
therefore, it can be used on daily bases as tonic for healthy cardiovascular
system.
An ethano pharmacological importance and increasing demand of T. arjuna stem bark in
several herbal formulations may leads to adulteration. Therefore, the main objective of this
study is validation of earlier information on pharmacognostic studies of T. arjuna stem bark
(TASB) and to supplement some information with regards to its identification, characterization
and standardization to ensure the quality and purity of TASB material.

~ 187 ~
Journal of Pharmacognosy and Phytochemistry
In this point of view, work has been carried out to evaluate
some pharmacognostic, physicochemical and phytochemical
characteristics of TASB followed by determination of volatile
organic constituents by GS-MS analysis.
Materials and methods
Plant material
Dried parts of stem bark of T. arjuna were collected in the
month of September 2013 from Whaghur Dam, District -
Jalgaon (MS), India. The plant material was identified from
the expert taxonomist. The pieces of stem bark of T. arjuna
were washed thoroughly with tap water, shade dried, ground
into fine powder and stored in air tight bottles.
Macroscopic and Microscopic studies
Macroscopic studies such as morphological observations of
TASB were performed under magnifying lens. In order to
study microscopic features, free hand section of stem bark was
taken and stained by phloroglucinol (0.2% in water w/v) and
iodine (0.2% in water w/v) to confirm its lignification and
presence of starch grains respectively. Microscopic analysis of
TASB fine powder was also performed in the similar manner
to record the specific diagnostic characteristics.
Fluorescence analysis
Fluorescence study of TASB powder was performed as per
reported standard procedures [13]
. A small quantity of the stem
powder was added in few ml of freshly prepared reagent
solution, mixed by gentle shaking the tube and waited for few
minutes. Then the colors of the mixtures were observed in
visible light, and long ultra violet radiations (365 nm) in the
UV chamber.
Physicochemical analysis
The physicochemical analysis of the crude powder TASB was
carried out as per WHO (2002) guidelines [14]
. The parameters
analyzed were loss on drying, total ash, alcohol soluble
extractive and water soluble extractive.
Atomic absorption spectroscopy
TSBA powder was subjected to elemental analysis according
to standard procedure involving use of atomic absorption
spectrophotometer [15]
. In brief, 2 gm of dry TASB powder was
digested in 40 ml of aquaregia solution [HNO3: H2SO4 (3:1
v/v)] by COD digester at 150 ºC for 90 minutes. Then digested
solution was filtered through Whatman filter paper No. 1 and
diluted up to 40 ml using 1% nitric acid solution. The minerals
[calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu),
iron (Fe), lead (Pb), magnesium (Mg), manganese (Mn),
potassium (K), sodium (Na), and zinc (Zn)] contents of the
digested solution was determined using Atomic Absorption
Spectrophotometer (Model: S2, Make: Thermo, USA) with
SOLAAR software.
Preparation of methanolic extract of TASB
Methanolic extract of TASB (ME) was prepared by using
Soxhlet extraction method. Thirty gram of finely ground plant
part powder was placed in porous bag made of muslin cloth,
which was loaded into the main chamber of the Soxhlet
.
extractor. The extraction was carried out with methanol in 1:10
powder to solvent ratio at temperature 65 ºC for 8 h. The
extract was filtered through Whatman filter paper No. 1 and
the filtrate was stored in the refrigerator at 4 ºC until its further
use.
Qualitative phytochemical analysis
The ME was subjected to qualitative phytochemical analysis
[16]
to detect the presence of carbohydrate, amino acids, cardiac
glycosides, alkaloids, saponins, phenols, flavonoids, steroids,
terpenoids, tannins, anthraquinones, quinones, fats and volatile
oils.
Thin layer chromatography fingerprinting
TLC fingerprinting of ME was performed using aluminium
plates precoated with silica gel 60 (F-254) having 0.2 mm
thickness (Merck, Mumbai, India). TLC plates were activated
by preheating in oven at 60 ºC. About 10-20 μL of ME was
loaded on TLC plate and the chromatogram was developed by
using solvent system consisting of toluene: ethyl acetate:
formic acid: methanol (10:3:1:2 v/v/v/v) as mobile phase to
separate phytoconstituents. The developed plate was dried
under hot air. Then 10% H2SO4 in ice cold methanol was
sprayed on the surface of plate followed by heating at 110 ºC
for 5 min for derivatization of phytoconstituents. The
derivatized TLC plate was observed under visible light and the
Rf value of the bands were recorded.
GC– MS analysis
The GC-MS analysis of ME of TASB was carried out by using
a Gas Chromatograph (TRACE 1300, Thermo Scientific,
USA) equipped with Triple Quadrupole Mass Spectrometer
(TSQ 8000, Thermo Scientific- USA) equipment.
Experimental conditions of GC-MS system were as follows:
TG 5-MS capillary standard non-polar column (dimension: 30
m, ID: 0.25 mm, film: 0.25 µm) was used. The flow rate of
mobile phase (carrier gas: He, 99.999%) was set at 1.0 ml/min
and the injection volume was 1 µL. An injector and ion source
temperature were 250 and 280 ºC respectively. Oven
temperature program was 60 ºC hold for 2 min to 250 ºC at the
rate of 15 ºC/min and hold it for 15 min. Samples which
dissolved in methanol were run fully at a range of 50-1000
m/z. The mass spectra detected in 26 min.
The name, molecular weight and nature/structure of the
components of the ME were confirmed using computer
searches on the database of National Institute Standard and
Technology (NIST) Ver. 11 MS data library and comparing
the spectrum obtained through GC-MS with the spectrum of
the known components stored in the NIST library.
Results and discussion
T. arjuna is a large, deciduous tree, growing to a height of 60-
90 feet with a spreading crown and dropping branches (Fig 1-
A). T. arjuna has a buttressed huge trunk of diameter about 3-
8 feet (Fig. 1-B). The outermost layer of stem of T. arjuna is
usually known as stem bark which has wide applications in
ayurvedic medicine. The present investigation was mainly
focused on pharmacognostic standardizations and
phytochemical evaluations of TASB and its methanolic
extract.

~ 188 ~
Fig 1: Terminalia arjuna Roxb. A- Whole tree, B- Trunk, C- Stem
park pieces, D- Stem bark powder.
Macroscopic study of TASB
The bark is grayish and smooth externally, internally brown or
red coloured and smooth. The shape of bark was flat and
curved (Fig. 1-C). The transversely cut bark showed brownish
surface, fracture, short in inner and laminated in outer part.
The powder of TASB was light brown (Fig. 1-D) with
astringent taste and no odour.
Microscopic study of TASB section
Microscopic evaluation is one of the cost effective and
simplest methods for the correct identification of the plant
material. Transverse section of fresh stem bark showed typical
anatomical characters. It had outer cork region composed of
uniformly arranged several layers of small, tangentially
elongated cells (Fig. 2-A). The region of cortex, composed of
thin walled, more or less brick shaped parenchymatous cells
containing cluster crystals of calcium oxalate and few groups
of sclerenchymatous pericyclic fibres with scattered
arrangement were observed below the cork region (Fig. 2-B).
The innermost region showed presence of secondary phloem
consisting of thin walled, polygonal cells with wavy walls
containing cluster crystals of calcium oxalate and pigmented
cells (Fig. 2-C).
Fig 2: Microscopic photo of T. arjuna stem bark, A- outer region, B-
inner region C- magnified inner region
Microscopy of Powder
The unstained TASB powder under light microscope at 10x
magnification showed the presence of reddish-brown particles
of irregular shape, fragments of cork cells, uniseriate phloem
rays, fibres and a number of white colored rosette crystals of
calcium oxalate (Fig. 3-A). After staining by 0.2%
phloroglucinol pink colored fibres appeared (Fig. 3-B), this
confirmed the presence of lignified material [13]
.
After staining by 0.2% iodine solution, violet colored granules
with elliptic or round to oval shape having 2-3 components
with concentric striations were also observed (Fig. 3-C) which
confirmed the starch material [13]
. This microscopic
observation of powder could be useful in identification of
TASB material.
Fig 3: Microscopic photo of Powder of T. arjuna stem bark powder,
A- without stain, B- stained with 0.2 % phloroglucinol, C- stained
with 0.2 % iodine
Physiochemical characters of T arjuna stem bark
Loss on drying at 105 ºC, pH, total ash content are the major
factors responsible for the stability of herbal plant material.
The results of physiochemical characterization of TASB are
given in the table 1. Usually low moisture content is desirable
for higher stability of drugs [17]
. In the present investigation,
TASB showed low moisture content (Table 1) which is laying
between recommended levels 8% and 14% [18]
. The pH of the
TASB was in the acidic region. It had higher ash value
reflecting the presence of carbonate, phosphate, oxides,
silicates and silica material in higher amount [19]
. The TASB
had higher alcohol soluble and water soluble extractive value
indicating the presence of highly polar chemical constituents
such as phenolic compound, flavanoids, proteins,
carbohydrates etc. [4]
.
The results of macroscopy, microscopy and physiochemical
analysis are in accordance with Ayurvedic Pharmacopoeia of
India [20]
.
Table 1: Physicochemical parameters of T. arjuna stem bark
Sr. No. Test Results
1 Moisture content 10.19 ± 1.30a
2 pH 4.13 ± 0.06b
3 Total Ash 17.49 ± 1.31a
4 Water-soluble extractive 20.50 ± 0.66a
5 Alcohol soluble extractive 28.48 ± 3.71a
a
Values are expressed in percentage; b
Value is expressed as pH
value; All results are given as Mean ± standard deviation (n=3).
Fluorescent evaluations of TASB powder
Fluorescence study is an essential parameter for first line
standardization of crude drug. The changes in color and
fluorescent analysis of TASB powder with different
reagents/solvents under visible and UV light respectively were
tabulated in Table 2. Under visible light the TASB powder
appeared red, dark brown to yellowish brown in most of the
reagents and faint yellow in ethyl acetate. Under long UV light
the TASB powder appeared black in most of the reagents and
with few reagents it appears as dark brown to greenish brown.

~ 189 ~
Table 2: Fluorescence analysis of TASB powder under visible and
UV light after treated with different reagents/solvents
Treatment Visible light UV light
Powder Brown Greenish brown
Powder + water Red Black
Powder + 1N NaOH Red Blackish brown
Powder + 1N NaOH (alc.) Yellowish brown Black
Powder + Ammonia Dark red Black
Powder + 50 % HCl Yellowish Red Black
Powder + 50 % H2SO4 Dark brown/black Black
Powder + 50% HNO3 Brownish red Black
Powder + FeCl3 Greenish yellow Greenish black
Powder + Methanol Dark red Black
Powder + Ethanol Red Dark brown
Powder + Ethyl acetate Faint yellow Black
Powder + Benzene cream Black
Powder + Petroleum ether Colourless Black
Atomic absorption spectroscopy analysis
The Atomic Absorption Spectrometric analysis of the TASB
(Table 3) showed the presence of various essential (Calcium,
Chromium, Cobalt, Copper, Iron, Manganese, Nickel and Zinc
etc.) as well as non-essential (Arsenic, Cadmium, Lead and
Mercury etc.) elements. The concentrations of all these
elements are within the permissible limits set by WHO 1996
and 2005 [21, 22]
. The result can assure the usefulness of these
elements in the physiological administration of crude drug
since some of the elements were of health benefits and
similarly within recommended safe limits.
Table 3: Heavy metal analysis of TASB by Atomic absorption
spectroscopy
Sr.
No.
Metal in plant
material
Concentration of elements (mg/kg)
Permissible limits of metals in
plant material (WHO 1996 &
2005) [21, 22]
In this
study
1 Arsenic (As) 5 0.066
2 Cadmium (Cd) 0.3 0.296
3 Calcium (Ca) NA 86.274
4
Chromium
(Cr)
1.5 0.826
5 Cobalt (Co) NA 2.078
6 Copper (Cu) 10 9.534
7 Iron (Fe) 20 16.036
8 Lead (Pb) 10 0.124
9
Manganese
(Mn)
200 9.154
10 Mercury (Hg) 0.2 0.092
11 Nickel (Ni) 1.5 0.592
12 Zinc (Zn) 50 8.698
NA-data not available
Phytochemical analysis
The phytochemical profiling of methanolic extract of TASB
showed the presence of carbohydrate, amino acid, cardiac
glycosides, alkaloids, saponins, phenols, flavonoids, steroids,
terpenoids and lignins as presented in Table 4. This serves as
an important tool for the quality assurance of plant for future
studies.
Table 4: Phytochemical analysis of methanolic extract of TASB
Sr.
No.
Phytoconstituents Test Results
1 Carbohydrate
Molisch's Test ++
Fehling's test ++
Test for starch -
Test for cellulose -
2
Amino acid and
Protein
Millon's test ++
Biuret test -
Ninhydrin test -
3 Cardiac glycoside
Keller Kelliani’s test +++
Legal's test +++
4 Alkaloid
Mayer's test +
Dragendroff's test +
Wagner's test +
5 Saponin Foam test +
6 Phenol
Ferric chloride test +++
Phosphomolybdic acid
test
+++
7 Flavonoid
Shinoda test ++
Alkaline reagent test -
Ammonium Test ++
Aluminium Chloride
Test
+++
Lead acetate test +++
Pew test ++
8 Steroid Salkowski Test +
9 Terpenoid Salkowski Test ++
10 Tannin Braymer’s test ++
11 Anthraquinone Borntrager's test -
12 Quinone Hydrochloric acid test -
13 Fats and volatile oil Precipitation test -
14 Lignin Spot test +
- Not detected; + slightly positive reaction; ++ Positive reaction; and
+++ Strong positive reaction
UV-VIS spectroscopic analysis
The UV-VIS spectrum of this plant extracts has absorption
bands at 226, 275, broad peak at 350- 375, and minor peak at
487 nm (Fig. 4). These absorption bands are characteristic for
flavonoids and its derivatives. The flavonoids spectra typically
consist of two absorption maxima in the ranges 240-285 nm
(band II) and 300-380 nm (band I) [23]
. The precise position
and relative intensities of recorded maxima in the spectrum
indicate that the flavonoid is a predominant ingredient of
TASB methanolic extract.
Fig 4: UV-VIS spectrum of TASB methanolic extract

~ 190 ~
FTIR Spectroscopic analysis
The FTIR spectrum of TASB methanolic extract showed
presence of various functional groups of the active
components based on the peaks values in the region of IR
radiation (Fig. 5). The peak at 3334.7 (O-H stretch) phenol,
2931.3 (C-H stretch) alkanes, 1724.06 (C=O stretch)
carboxylic acids, 1613.84 (N-H bend) primary amines,
1520.98 and 1445.39, (C-C stretch in ring), aromatics, 1351.86
(N-O symmetric stretch), nitro compounds, 1250–1020 (C-N
stretch) which may be due to the presence of aliphatic amines.
The results of FTIR analysis confirmed the presence of
alcohol, phenol, alkanes, Carboxylic acids, primary amines,
aromatic compound, nitro compounds, and aliphatic amines.
Fig 5: FTIR spectrum of TASB methanolic extract
Thin layer chromatography fingerprinting
The TLC chromatogram of crude methanolic extract of TASB
(Fig. 6) showed eleven major bands having Rf values of
different spots are: 1 (0.11), 2 (0.27), 3 (0.33), 4 (0.47), 5
(0.59), 6 (0.59), 7 (0.64), 8 (0.76), 9 (0.80), 10 (0.86) and 11
(0.92).
The various bands in TLC of crude methanolic extract of
TASB showed the presence of different phyto constituents
with characteristic fingerprints. This TLC fingerprint of this
extract could be used to check quality and purity of this drug
even at very low concentration.
Fig 6: TLC fingerprinting of T. arjuna stem bark methanolic extract
GC-MS analysis
Gas Chromatography-Mass spectrometry (GC-MS) is one of
the best techniques to identify the constituents of volatile
matter, long chain, branched chain hydrocarbons, alcohols
acids and esters, etc. GC-MS analysis of the methanolic extract
of TASB revealed the presence of 10 major compounds (Fig
7). The retention time, name, molecular formula, molecular
weight, peak area and nature of the compounds are given in
Table 5. The individual mass fragments of these ten major
compounds are presented in Fig. 8 (I) and (II).
Fig 7: Gas chromatogram spectrum of T. arjuna stem bark

~ 191 ~
Table 5: Phyto components detected in the methanolic extract of of T. arjuna stem bark by GC-MS analysis
Sr.
No.
RT Name of compound
Molecular
formula
MW
Peak area
%
Nature of the
compound
1 3.03 1-Methoxyhexane C7H16O 116.20 4.37 Aliphatic hydrocarbon
2 4.50 9-Oximino 2,7-diethoxyfluorene C17H17NO3 283.32 1.63 Aromatic hydrocarbon
3 7.30 Heptadecane, 9-hexyl C23H48 324.62 3.09 Alkane hydrocarbon
4 11.65 9,10 Secocholesta 5,7,10(19) triene 3,24,25 triol C27H44O3 416.63 1.85 Steroid
5 12.01 Butanoic acid, 2,3 dihydroxypropyl ester C7H14O4 162.18 5.63 Ester
6 12.53 2-Naphthalene methanol C15H26O 222.36 10.93 Alcohol
7 13.91
9,12,15-Octadecatrienoic
acid, methyl ester, (Z,Z,Z)
C27H52O4Si2 496.87 2.34 Fatty ester
8 15.54 3-Hydroxyspirost-8-en-11-one C27H40O4 428.60 5.40 Steroidal saponins
9 16.20 Protriptyline C19H21N 263.37 10.15 Secondary amines
10 20.02
Olean-12-ene-3,15,16,21,22,28-hexol,
(3α,15α,16α,21α,22α)
C30H50O6 506.71 2.40 Saponin
Fig 8(I): The individual mass fragments of some major compounds from methanolic extract of T. arjuna stem bark detected by GC-MS analysis

~ 192 ~
Fig 8(II): The individual mass fragments of some major compounds from methanolic extract of T. arjuna stem bark detected by GC-MS
analysis
Conclusion
The present study provides various resourceful information in
relation to pharmacognostic identification of T. arjuna stem
bark. Furthermore, information regarding physicochemical
characteristics of stem bark and nature of chemical
constituents present in them would also be useful for
standardization of herbal drug material TASB and enrichment
of Ayurvedic Pharmacopeia. TLC fingerprinting and GC-MS
analysis could also help to identify and isolate important
phyto-constituents. Thus, this type of study may give
information on nature of active principles present in the
medicinal plants and to identify the plants from their
adulterants using isolated compounds as marker. These
findings could be helpful in identification, authentication and
standardization of TASB and its methanolic extract. It would
also help scientists to utilize such needful information
regarding the plant material identity and characteristics in
building new polyherbal formulations.
Acknowledgement
GMC is thankful to UGC, New Delhi, India for providing SRF
on major research project. RTM and GMC are thankful to
Management and Principal of Moolji Jaitha College, Jalgaon
for encouragement and providing necessary facilities for
research work. Both authors are grateful to Mr. Sandeep N.
Patil and Dr. Bhushan L. Chaudhari, School of life sciences,
North Maharashtra University, Jalgaon for giving critical
comments and suggestion. Authors are thankful to SAIF-CIL
Punjab University, Chandigarh for carrying out GC-MS
analysis of the sample. Authors wish to thank School of Life
Sciences, North Maharashtra University, Jalgaon for providing
FTIR and AAS facility.
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