ISSN: 2279 - 0594

Journal of Biomedical and Pharmaceutical Research

Available Online at www.jbpr.in
CODEN: - JBPRAU (Source: - American Chemical Society)
Index Copernicus Value: 63.24
PubMed (National Library of Medicine): ID: (101671502)
Volume 6, Issue 1: January-February: 2017, 23-31

Research Article
ISOLATION AND CHARACTERIZATION OF A FLAVONOID FROM ETHANOLIC EXTRACT OF
ALTERNANTHERA SESSILIS LINN.
Mrinmay Das*1, Ashok Kumar D2, Jyotirmoy Deb3and Durga Srinivasa Rao3
1
PRIST University, Vallam, Thanjavur, Tamilnadu-613403
2
Department of Pharmacy, Pratistha Institute of Pharmaceutical Sciences, Suryapet Dist, Andhra Pradesh-508214.
3
Department of Pharmacy, S. Chaavan College of Pharmacy, Jangalakandriga (Vi), Nellore Dist, Andhra Pradesh-524346.
Received 20 Nov. 2016; Accepted 02 Jan. 2017

ABSTRACT
This study was performed to isolate and characterize the flavonoid present in Alternanthera sessilis Linn.
Direct soxhlet extraction process was adopted for extraction by using 95% ethanol and vacuum evaporator
was used for drying the extract. The concentrated ethanolic fraction was subjected to thin layer
chromatography and column chromatography for isolation. The isolated compound was identified as a
flavonoid by confirming the standard flavonoid tests: viz. Shinoda’s test. The Rf value of isolated flavonoid
was calculate and different physical tests were also performed to find out its physical characteristics.
Characterization of isolated flavonoid was done by FTIR, 1H NMR and MASS. The IR spectrum indicated the
presence of hydroxyl and carbonyl functions. 13C NMR signal indicated the presence of hydroxyl group and
unsaturated keto function and four methoxy group at different position of flavones skeleton. The 1H-NMR
further showed the presence of three hydroxyl, four methoxy groups and three methine groups. On the
basis of chemical and spectral analysis the structure was elucidated as 3´, 3, 6, 7-tetramethoxy - 4´, 5, 8-
trihydroxy flavones.
Keywords: Flavonoids, TLC, Column Chromatography, FTIR, NMR, MASS.
when herbal therapies use with various other
INTRODUCTION:
traditional remedies in America. Integrative
The bioactive compounds are mostly plant medicine came into being when the alternative
secondary metabolites, which become medicine medicine, mainly the aforementioned traditional
after processing to pure compounds; some are and folk medicines used worldwide, with
very useful dietary supplements, and many useful conventional medicine (Western medicine). In
commercial products. Further modification of the recent years, the popularity of complementary
active compounds lead to enhance the biological medicine has increased.
profiles and a large number of such compounds
Flavonoids are secondary metabolites
which are approved or undergoing clinical trials for
Characterized by flavan nucleus [3] and a C6-C3-C6
clinical uses against different diseases like
carbon skeleton. These are group of structurally
pulmonary diseases, cancer, HIV/AIDS, malaria,
related compounds with a chromane - type
Alzheimer’s and other diseases [1, 2]. Crude herbs
Skeleton having phenyl substituent in C2 - C3
are used as drugs in different country of the world
position. The flavonoids belong to one of the most
and therefore it take a basic part of many
bioactive compounds which naturally exist in the
traditional medicines worldwide. In Asia,
plant kingdom. Till now, over 8000 varieties of
traditional Chinese medicine (TCM), Korean
flavonoids have been identified [4]. Different
Chinese medicine, Japanese Chinese medicine
naturally occurring flavonoids have been described
(kampo), ayurvedic medicine (India) and jamu
and sub-categorized into flavones, flavans,
(Indonesia), phytotherapy and homeopathy in
flavanones, isoflavonoids, chalcones, aurones and
Europe, alternative medicines are typically named
anthocyanidines. These flavonoids have

*Corresponding author: Mrinmay Das | E-mail:

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 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

remarkable biological activities, including Alternanthera sessilis Linn. (Amaranthaceae) is an

inhibitory effects on enzymes, modulatory effect annual or perennial prostate herb with several
on some cell types and protection against allergies, spreading branches, bearing short petioled simple
antiviral, anti-malarial, anti inflammatory and anti- leaves and small white flowers, found throughout
carcinogenic properties. A number of flavones, the hotter part of India, ascending to an altitude of
flavonols, flavanones, and isoflavones, as well as 1200m [12]. The plant spreads by seeds, which are
some of their methoxy, isoprenyl, and acylated wind and water-dispersed and by rooting at stem
derivatives, show antibacterial activity [5]. nodes. Young shoots and leaves are eaten as a
Flavonoids are major components of medicinal vegetable in Southeast Asia [13]. It is a weed of
plants and have been used in traditional medicine rice throughout tropical regions and of other
around the world. cereal crops, sugarcane and bananas. Although it is
a weed, it has many utilities. The leaves were used
Flavonoids are phenolic compounds which are
in eye diseases, cuts, wounds and antidote to
widely distributed in plants, and have been
snake bite; skin diseases [14].
reported to exert multiple biological effects,
including antioxidant, free radicals scavenging It is also reported about the wound healing
abilities anti- inflammatory and anti-carcinogenic property of Alternanthera sessilis Linn. [15]. The
activity [6-8]. degenerative and necrotic changes in the liver and
kidney in Swiss mice, caused by oral administration
The process of separation of the individual
of water extract of A. sessilis in high doses through
components of a mixture based on their relative
histopathological test were revealed [16].
affinities towards stationary and mobile phases is
called as chromatography. The identification, MATERIALS AND METHODS:
separation and purification of plant constituents Plant material
are mainly carried out using one or a combination
of chromatographic techniques. The plant was identified by the Botanist of VR
College, Nellore, Andhra Pradesh. After
The IR region is divided into three regions: the authentication the fresh aerial parts were
near, mid, and far IR. Infrared radiation is collected from rural belt of Jangalakandriga village,
absorbed by organic molecules and converted into Nellore, Andhra Pradesh. The plants were washed
energy of molecular vibration. The wave numbers properly, shade dried and then milled to coarse
(sometimes referred to as frequencies) at which an powder by a mechanical grinder. The crude
organic molecule absorbs radiation give powder drug was kept in air tight container for
information on functional groups present in the further use.
molecule [9].
Preparation of extract
Nuclear magnetic resonance, induces changes in
the magnetic properties of certain atomic nuclei, The powdered plant material was defatted with
notably that of hydrogen. NMR spectroscopy is petroleum ether (60-80°C) and then extracted
used to investigate the properties of organic with 95% ethanol using Soxhlet apparatus. The
molecules and provide detailed information about solvent was removed under reduced pressure,
the structure, dynamics, reaction state and which gave a greenish-black coloured sticky
chemical environment of molecules [10]. residue (yield- 14.8% w/w on dried material basis).
Preliminary phytochemical screening [17] of the
Mass spectrometry is a powerful analytical extract gave positive tests for presence of
technique used to quantify known materials, to alkaloids, flavonoids, triterpenoids, glycosides,
identify unknown compounds within a sample, and tannins, amino acids and saponins.
to elucidate the structure and chemical properties
of different molecules. The complete process Identification of phytoconstituents by TLC
involves the conversion of the sample into gaseous In thin layer chromatography technique the
ions, with or without fragmentation, which are extract was dissolved in solvent and mixed
then characterized by their mass to charge ratios thoroughly. The mixture was then used for
(m/z) and relative abundances [11]. spotting in the TLC plates. The plates are prepared

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 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

by using adsorbent like Silica gel G. A fine capillary the solvent by using rotary vacuum evaporator and
had been used for spotting. The spot was done on tested for the components by using Thin Layer
the TLC plate near about 1cm above from the Chromatography. TLC spot was identified by
bottom of the plate. The plate was then dried and spraying 5% w/v alcoholic solution of H2SO4 as a
kept in developing chamber containing suitable spraying reagent. The sprayed plates were heated
solvent systems. After a proper running period the at 100°C for 5-10 min and the numbers of
plate(s) were removed and dried in the air and constituents present in the each fraction were
spraying reagent was used to locate the spot. The found.
Rf value was calculated. Different solvents were
Isolation of phytoconstituents from EEAS:
used in different ratios and TLC had been carried
out to confirm the presence of different mixtures The Chloroform-Ethanol 60:40 ratios gives the
of phytoconstituents in the extract [18-20]. fractions 102 to 107, were found to be similar and
showed a single spot. Thus they were mixed and
recrystallized from ethanol as colourless powder
which shown a melting point of 178-1800C (104
Separation of Phytoconstituents by Column mg). It light dark green colour with ferric chloride,
Chromatography pink colour in Shinoda’s test suggesting that it was
In this technique the stationary phase is solid and a flavone. The isolated compound was designated
the mobile phase is liquid. The separation takes as EEAS-I. The physical characteristic of the
place when the component of two or more compound was tabulated in Table 2. The TLC
compound mixture is more strongly adsorbed than solvent system and Rf value of the isolated
the other by the solid stationary phase. The compound EEAS-I is given in the Table 3.
isolation of active constituent was performed by CHARACTERIZATION OF ISOLATED COMPOUND:
using the Column chromatography technique [21].
IR spectrum of EEAS-I
The adsorbent was dissolved in chloroform to
make slurry poured in to a column up to ¾th level. The IR region is divided into three regions: the
The solvent were continuously run to get proper near, mid, and far IR. The mid IR region is of
packing. Then the sample was packed as slurry greatest practical use to the organic chemist. This
with the same solvent [22]. Mobile phase was is the region of wavelengths between 3 x 10–4 and
poured on the column bed to make the column to 3 x 10–3 cm. In wave numbers, the mid IR range is
settle properly. Then sample was mixed with 4000–400 cm–1. Infrared radiation is absorbed by
chloroform and poured in to the column. Different organic molecules and converted into energy of
solvent systems of n-Hexane, Benzene, Chloroform molecular vibration.
and Ethanol in different ratios were used for the The IR spectrum of isolated compound EEAS-1 had
elution of phytoconstituents. The fractions of 200 shown absorption bands at 3608.9 to 3315.7 (O-H,
ml were collected each time. Detection of the free hydroxyl group), 2953.1 (Cyclic C-H,
component was done by monitoring each fraction stretching), 2866.3 (Ali- C-H, stretching), 1660.7
by TLC. The fraction details are tabulated in Table (C=O, stretching), 1500.0-1400.3 (C-C, ring
1. stretching), 1284.6-1193.8 (C-C, ring stretching),
Confirmation of constituents by using Thin Layer 1114.8-997.2 (O-H, out of plane bend).The FT-IR
Chromatography spectrum of EEAS-I had shown in Fig: 1. The
spectral data of the compound EEAS-I and their
The fractions were collected and the residue of functional group assignments were tabulated in
fraction was obtained each time by evaporating Table 4.

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 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

Fig 1: FT-IR Spectrum of Isolated Compound EEAS-I

Nuclear Magnetic Resonance Study carbon present in the sugar moiety and exhibits
carbon resonance signal extending over 200 ppm.
Nuclear magnetic resonance, induces changes in
1
the magnetic properties of certain atomic nuclei, H-NMR spectrum of EEAS-I
notably that of hydrogen. Hydrogen atoms in
The 1H-NMR spectrum of the isolated compound
different environments can be detected, counted
EEAS-I had displayed the characteristic signals at
and analyzed for structure determination.
δH 7.80 (H-2´, s), 7.30 (H-5´, d), 6.67 (OH-4´, s), 5.89
13
C NMR spectroscopy is the most powerful and in (OH- 5, s), 4.29 (OH-8, s), 4.17 (OCH3-3, s), 3.85
dispensable technique provide information about (OCH3-3´, s), 3.14 (OCH3-6, s), 2.73 (OCH3-7, d). The
1
intricate nature of the carbon skeleton of a H-NMR spectrum of EEAS-I had shown in Fig 2 and
compound such as, the total number of carbon, data were tabulated in Table 5.
number of oxygenated carbons and the number of

Fig 2: 1H-NMR spectrum of isolated compound EEAS-I

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 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

13
C NMR spectrum of EEAS-I presence of carbons due to the flavones skeleton.
The hydroxylated C-2, C-3, C-4, C-5, C-6, C-7 and C-
The 13C-NMR spectrum of isolated compound
8 resonate at δ ppm. The 13C-NMR spectrum had
EEAS-I had shown the characteristic signals at δH 2-
shown in Fig: 3 and the spectral data of EEAS-I and
77.37, 3-126.99, 4-123.17, 5-135.55, 6-140.11, 7-
corresponding signal assignments were tabulated
145.84, 8-105.20, 3´-148.77, 1´-100.62, 2´-77.00,
in Table 6.
5´-76.57, 6´-64.21.The carbon signals indicated the

Fig 3: 13C-NMR spectrum of isolated compound EEAS-I

MASS spectrum of EEAS-I The mass data of isolated compound EEAS-I had
shown the m/z = 390 indicative of C19H18O9, m/z =
Mass spectrometry is a powerful analytical
358 indicative of C18H14O8, m/z = 334 indicative of
technique used to quantify known materials, to
C16H14O8, m/z = 304 indicative of C15H12O7, m/z =
identify unknown compounds within a sample, and
212 indicative of C9H8O6, m/z = 198 indicative of
to elucidate the structure and chemical properties
C5H6O2, m/z = 42 indicative of C2H2O. The mass
of different molecules. The complete process
data were in decreased sequence due to the
involves the conversion of the sample into gaseous
absence of different parts on the compounds. The
ions, with or without fragmentation, which are
MASS spectrum of isolated compound EEAS-I had
then characterized by their mass to charge ratios
shown in Fig. 4 and the EI-MS spectrum of EEAS-I
(m/z) and relative abundances.
exhibited the molecular ion peak at m/z 390.

Fig 4: MASS spectrum of isolated compound EEAS-I

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 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

Structure of Isolated Compound

Fig: 5: 3´, 3, 6, 7-tetramethoxy - 4´, 5, 8-trihydroxy flavones

RESULT:
Table: 1 Chromatographic fractions of ethanolic extract of Alternanthera sessilis Linn.

Sl. No. Elution Composition Fractions Compounds

1. n-Hexane 1-5 Oily
2. n-Hexane: Benzene (95:5) 6-11 Waxy
3. n-Hexane: Benzene (90:10) 12-20 Waxy
4. n-Hexane: Benzene (50:50) 21-28 Waxy
5. n-Hexane: Benzene (40:60) 29-39 Waxy
6. Benzene 40-45 Intractable gum
7. Benzene: Chloroform (95:5) 46-49 Intractable gum
8. Benzene: Chloroform (85:15) 50-54 Intractable gum
9. Benzene: Chloroform (65:35) 61-66 Intractable gum
10. Benzene: Chloroform (50:50) 67-72 Intractable gum
11. Benzene: Chloroform (30:70) 73-80 Intractable gum
12. Chloroform 81-85 Intractable gum
13. Chloroform: ethanol (97:3) 86-89 Intractable gum
14. Chloroform: Ethanol (95:5) 90-96 Intractable gum
15. Chloroform: Ethanol (90:10) 97-101 Intractable gum
16. Chloroform: Ethanol (60:40) 102-107 EEAS-I
17. Chloroform: Ethanol (50:50) 108-116 Intractable gum
18. Chloroform: Ethanol (20:80) 117-122 Intractable gum
19. Ethanol 123-127 Intractable gum

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 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

Table 2: Properties of isolated compound EEAS-I

Sl. No. Property Observation

1. Appearance Colourless Powder
2. Melting Point 178 – 1800C
3. Solubility Ethanol, Methanol, Chloroform

Table: 3 Rf value of the isolated compound EEAS-I

Sl. No. TLC Solvent System Rf Value

1. n-Hexane : Diethyl ether = (1 : 1) 0.47

Table 4: FT-IR spectral data of compound EEAS-I

Sl. No. Wave Number (cm-1) Type of Vibration Functional group assigned
1. 3608.9 – 3315.7 O-H Free hydroxyl group

2. 2953.1 Cyclic C - H, str Aromatic Hydrocarbon

3. 2866.3 C - H, str Aliphatic Hydrocarbon

4. 1660.7 C = O, str Ketone

5. 1500.0 – 1400.3 C = C, ring stretch Aromatic Nuclei

6. 1248.6 – 1193.8 C – C, str Aliphatic Hydrocarbon

Table 5: 1H-NMR spectral data of compound EEAS-I

Sl. No. Chemical shift value (δ ppm) Signal Assignment - H

1. 7.80 2´ - H, s

2. 7.30 5´ - H, d

3. 6.67 4´ - OH, s

4. 5.89 5 - OH, S

5. 4.29 8 - OH, s

6. 4.17 3 - OCH3, s

7. 3.85 3´ - OCH3, s

8. 3.14 6 - OCH3, S

9. 2.37 7 - OCH3, d

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 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

Table 6: 13C-NMR spectral data of isolated compound EEAS-I

Sl. No. Chemical shift value (δ ppm) Signal Assignment - C

1. 77.37 C-2
2. 126.99 C-3
3. 123.17 C-4
4. 135.55 C-5
5. 140.11 C-6
6. 145.84 C-7
7. 105.20 C-8
8. 148.77 C-3´
9. 100.62 C-1´
10. 77.00 C-2´
11. 76.57 C-5´
12. 64.21 C-6´

DISCUSSION: flavonoids. The structures of isolated compound

EEAS-I was elucidated by IR, NMR and Mass
Thin layer chromatography was the first attempt
spectroscopy.
taken to find out the presence phytoconstituents
presents in the extracts. TLC was performed by The Compound EEAS-I was isolated and its
using stationary phase as silica gel G and mobile molecular formula was determined as C19H18O9
phase as n-hexane : diethyl ether in the ratio of 1: (m/z = 390 (100) [M+]). The structures of the
1 for ethanol extract of Alternanthera sessilis Linn. flavone were identified on the basis of extensive
The TLC plate showed mixture of compounds with spectroscopic data analysis and by comparison of
yellow and brownish yellow colour spots. This was their spectral data with those reported in the
separated by column chromatography. literature. The IR spectrum indicated the presence
of hydroxyl (3608.9 cm-1) and carbonyl functions
The ethanolic extract of Alternanthera sessilis Linn.
(1660.7 cm-1). The occurrence of a flavone
was packed on column chromatography with silica
skeleton in the molecule could be easily deduced
gel G 60-120 mesh size and mobile phase was
from the 1H-NMR and 13C-NMR spectrums. From
eluted as per increasing polarity. The fractions
the above mentioned data of 13C NMR signal
were collected and tested for the components by
indicated the presence of hydroxyl group at C-5, C-
using Thin Layer Chromatography. TLC spot was
8 and C-4´and unsaturated keto function. The 13C
identified by spraying 5% w/v alcoholic solution of
NMR signal also reported the presence of four
H2SO4 as a spraying reagent. The sprayed plates
methoxy group at different position of flavones
were heated at 100°C for 5-10 min and the
skeleton. The 13C-NMR signal of different location
numbers of constituents present in the each
of carbon for functional group was confirmed by
fraction were found. The Chloroform-Ethanol
the signal of were confirmed spectra of 1H-NMR.
60:40 ratios gives the fractions 102 to 107, were
The 1H-NMR further showed the presence of three
found to be similar and showed a single spot. Thus
hydroxyl, four methoxy groups and three methine
they were mixed and recrystallized from ethanol
groups. The compound characterized as 3´, 3, 6, 7-
as colourless powder which shown a melting point
tetramethoxy - 4´, 5, 8-trihydroxy flavones.
of 178-1800C (104 mg). It light dark green colour
with ferric chloride, pink colour in Shinoda’s test REFERENCES:
suggesting that it was a flavone. The isolated 1. Butler MS. The role of natural product chemistry in
compound designated as EEAS-I. drug discovery. J Nat Prod. 2004; 67: 2141-2153.
The isolated compound EEAS-I had shown the 2. Newman DJ, Cragg GM, Snader KM. Natural
positive response to the Shinoda’s test for products as sources of new drugs over the period
1981-2002. J Nat Prod. 2003; 66: 1022 – 1037.
© 2017 All Rights Reserved. CODEN (USA): JBPRAU
30
 Mrinmay Das et al., Journal of Biomedical and Pharmaceutical Research

3. Heim KE, Tagliaferro AR, Bobliya DJ. Flavonoids 13. Scher J. Federal Noxious Weed disseminates of
antioxidants: Chemistry, metabolism and structure the U.S. Center for Plant Health Science and
- activity relationships. The Journal of Nutritional Technology, Plant Protection and Quarantine,
Biochemistry. 2002; 13: 572 - 584. Animal and Plant Health Inspection Service,
4. De Groot H, Raven U. Tissue injury by reactive
U.S. Department of Agriculture. 2004; 291-
oxygen species and the protective effects of
Flavonoids. Fundam Clin Pharma Col. 1998; 12: 249
300.
- 255. 14. Gupta A, Indian Medicinal Plants. ICMR, New
5. Harborne JB, Williams CA. Advances in flavonoids Delhi: 151-157 (2004).
research since 1992. Phytochemistry. 2000; 55: 481 15. Paridhavi Sunil SJ, Nitin A, Patil MB, Chimkode
- 504. R, Tripathi A. International Journal of green
6. Wei H, Tye L, Bresnick E, Birt DF. Inhibitory effect of pharmacy. 2008; 2: 141-144.
apigenin, plant flavonoids on epidermal ornithine 16. Gayathri BM, Balasuriya K, Gunawardena
decarboxylase skin tumor promotion in mice. GSPS, Rajapakse RPVJ, Dharmaratne HRW.
Cancer Res. 1990; 50: 499 - 502. Research Communications Current Science.
7. Baba S, Osakabe N, Kato Y, Natsume M, Yasuda A,
2006; 91(10): 1517-1520.
Kido T, Fukuda K, Muto Y, Konda K. Continuous
intake of polyphenolic compounds containing
17. Evans WC, Trease GE. Pharmacognosy, 12th
cocoa powder reduces LDL – oxidative ed., Balliere Tindall: London: 735 (1983).
susceptibility and has beneficial effects on plasma 18. Srivastava VK, Srivastava KK. Introduction to
HDL - cholesterol concentration in human. Am J Chromatography: Theory and Practices, S. Chand
Clin Nutr. 2007; 85: 709 - 717. and Company Ltd., New Delhi: 46-58, 68-70 (2007).
8. Deendayal P, Sanjeev S, Sanjay G. Apigenin and 19. Patania VB, Analytical Chromatography. Campud
cancer chemoprevention: Progress, potential and Book International, New Delhi: 38-48, 105-133
promise (review). Int J Oncol. 2007; 30: 233 - 245. (2002).
9. Skoog, Holler, Nieman, Principles of Instrumental 20. Chatwal GR., Anand SK. Instrumental methods of
th
th
Analysis. 5 Edn, Michigan: Thomson book: 480- Chemical Analysis. 5 Edn. Himalaya Publishing
503, (2004). House Pvt. Ltd., New Delhi: 78, 220, 296 (2005).
10. Frank S, Handbook of Instrumental Techniques for 21. Khadijeh G, Hassan A, Maryam G, Elham NK, Sd
Analytical Chemistry. 2nd Edn. Vol 4, Pearson Hassan N, Ata K. Column Chromatography: A Facile
Education Pvt. Ltd, New Delhi: 236-257, (2006). and inexpensive procedure to purify the red dopant
11. Robert M, Silverstein S, Francies X, Spectrometric DCJ applied for OLEDs. Adv Mat Phy Chem. 2011; 1:
Identification of Organic Compounds. 6th Edn, Vol 9, 91-93.
London: John Wiley and sons: 3-5, 144, (2002). 22. Ochtavia PS, Titik T. Isolation and identification of
12. The Wealth of India. Raw Materials. Vol1 flavonoid compound ethyl acetate fraction
extracted from the rhizomes finger roots of
(Revised), New Delhi: CSIR: 318-319, (1985).
Boesenbergia pandurata. Indo J Chem. 2006; 6(2):
219 – 223.

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