21

International Journal of Advanced Scientific and Technical Research Issue 4 volume 2, March-April 2014
Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954

R S. Publication, rspublicationhouse@gmail.com Page 254

S Sc co op po ol le et ti in n, , a a c co ou um ma ar ri in n d de er ri iv va at ti iv ve e c co om mp po ou un nd d i is so ol la at te ed d f fr ro om m h hy yd dr ro o- -a al lc co oh ho ol li ic c
e ex xt tr ra ac ct t o of f p pl la an nt t A Ar rg gy yr re ei ia a n ne er rv vo os sa a ( (B Bu ur rm m. . F F. .) )
* *N Ne ee el lu u S So oo od d
1 1
, , D De ee ep pa ak k P Pr ra ad dh ha an n
2 2
, , P Pr ra at ti iv va a B Bi is sw wa as sr ro oy y
3 3

* *1 1
D De ep pa ar rt tm me en nt t o of f B Bo ot ta an ny y, , F Fa ac cu ul lt ty y o of f L Li if fe e S Sc ci ie en nc ce es s, , K Ku ur ru uk ks sh he et tr ra a U Un ni iv ve er rs si it ty y, , K Ku ur ru uk ks sh he et tr ra a, , 1 13 36 61 11 19 9
2 2
C Co ol ll le eg ge e o of f P Ph ha ar rm ma ac cy y, , P Pt t. . B B. . D D. . S Sh ha ar rm ma a U Un ni iv ve er rs si it ty y o of f H He ea al lt th h S Sc ci ie en nc ce es s, , R Ro oh ht ta ak k, , 1 12 24 40 00 01 1
3 3
R Ro ol la an nd d I In ns st ti it tu ut te e o of f P Ph ha ar rm ma ac ce eu ut ti ic ca al l S Sc ci ie en nc ce es s, , B Be er rh ha am mp pu ur r, , O Od di is sh ha a. .

Abstract: Argyreia nervosa Burm. F. (Syn. Argyreia speciosa) is a perennial climbing vine that
is native to the India, Africa, subcontinent Hawaii and Caribbean countries. It is commonly
known as Vridha daraka in sanskrit. This herb occupies a significant position in Ayurveda and
is known to possess phytoconstituents like alkaloids, carbohydrates, tannins, amber-colored
resins, sterols and saponins. This herb is better known for its hallucinogenic properties.
Pharmacologically this herb shows antimicrobial, analgesic, anti-inflammatory, aphrodisiac,
antiulcer, immunomodulatory, hypoglycemic, anticonvulsant, hepatoprotective, anti
inflammatory and nootropic effects. The current work is projected towards the preliminary
phytochemical screening, purification, identification, characterization of molecule isolated from
hydro alcoholic extract of plant Argyreia nervosa. The dried powder of the stem was subjected to
extraction with aqueous, hydro alcoholic and ethanolic solvents by cold maceration method.
Each extract was concentrated by thin film rotary evaporator yielding 9.75, 7.05 and 3.75% w/w
respectively. The isolated scopoletin was obtained by the column chromatography using
chloroform-methanol gradient and recrystallized it from ice cold methanol. The pure scopoletin
obtained was yellow, needle shaped crystals having melting point 208.2
o
C and R
f
value 0.573.
The spectral analysis of the scopoletin was carried out using Shimadzu- 1700 UV-Vis
spectrophotometer, IR model Bruker, (KBr pellet method) and Mass spectroscopy, Bruker
Daltonics (Esquire 3000). NMR (
1
H and
13
C) studies are carried out at 200 MHz and 500 MHz
using models Dpx-Bruker NMR spectrometer.

Key words: Argyreia nervosa Burm. F., preliminary phytochemical screening, scopoletin.


Introduction
Mankind has always been interested in naturally occurring compounds obtained from microbial,
plant and animal sources. Recently natural product chemistry has undergone explosive growth
due to advanced isolation techniques, spectroscopic and chromatographic methods
1, 2
. Lot of
efforts have been made for the isolation of bio-active molecules, standardization of herbal
extracts and their use in dietary supplements
3
. Argyreia nervosa (family: Convolvulaceae)
4-5
is a
perennial climbing vine that is native to India, Africa, subcontinent Hawaii and Caribbean
countries
6-9
. Traditionally, roots of this herb are used in treatment of ulcers, cough, bronchitis,
hemorrhoids, obesity, diabetes, anemia, tuberculosis and arthritis
10-12
. From the ancient time,
leaves of this plant have been used by Rajasthani tribes to prevent conception
13
. This herb is
known for possess nootropic
14
, antipyretic
15
, hypoglycemic
16
, aphrodisiac
17
, analgestic
18
,
anticonvulsant
19
, hepato-protective
20
, spasmolytic
21
and anti-inflammatory activities
22
. The
phyto-chemical analysis has revealed the presence of triterpenoids, flavanoids, steroids and
lipids
23-27
. Roots of this herb have immunomodulatory activity against the myelo-suppressive
effects induced by cyclophosphamide
28
. Present work represents development of a simple
method for isolation of scopoletin and its characterization by various analytical methods such as
UV-VIS, IR spectrophotometers, NMR (
1
H and
13
C) and LC-MS spectrometers.

Materials and methods
Collection of plant material
The stem parts of Argyreia nervosa were collected from Jammu region of India. The plant was
authenticated by taxonomist Dr. S. N. Sharma, Department of Taxonomy, I.I.I.M, Jammu, India,
having accession no-21189. The stems were shade dried until they were free from the moisture
and subjected to physical evaluation with different parameters such as nature, odor, color, taste,
size, shape, width and length. The air dried stem was pulverized to coarse powder and kept in
dessicator.

Physicochemical parameters:
Determination of ethanolic extractive value
400 G of the coarse powder was macerated, with 2.4 L of ethanol in a closed flask for 24 Hr by
occasional shaking. After 24 Hr, the liquid extract was filtered out by taking precaution against


loss of solvent. The extracting solvent was evaporated in a tared flat-bottomed shallow dish, at
105
0
C. The ethanolic extractive value was calculated with reference to the air dried drug.

Determination of water-soluble extractive value
400 G of the coarse powder was macerated, with 2.4 L of distilled water in a closed flask for 24
Hr by occasional shaking. After 24 Hr, the liquid extract was filtered out. The extracting solvent
was subjected to freeze drying in order to remove the solvent. The water-soluble extractive value
was calculated with reference to the air dried drug.

Determination of hydro-alcoholic (1:1) extractive value
400 G of the coarse powder was macerated, with 2.4 L solvent - ethanol: distilled water (1:1) in a
closed flask for 24 Hr by occasional shaking. After 24 Hr, the liquid extract was filtered out by
taking precaution against loss of solvent. The extracting solvent was evaporated in a tared flat-
bottomed shallow dish, at 105
0
C. The hydro-alcoholic extractive value was calculated with
reference to the air dried drug.

Loss on Drying (LOD)
2 G of powder was weighed accurately in a petridish and kept in hot-air oven, maintained at
110
0
C for 4 Hr. The petridish was allowed to cool in desiccator and the loss in weight of the
sample was recorded. The procedure was repeated till constant weight was obtained.

Determination of ash value
2 G of crude powder was weighed accurately in a previously ignited and weighed platinum
crucible. The crucible was ignited at 650
0
C for 6 Hr. The ash colored residue left in the crucible
was allowed to cool in dessicator and the total ash value was calculated with reference to air
dried drug.

Determination of acid-insoluble ash
1 G ash was boiled with 25ml of dilute hydrochloric acid for 10-15 minutes. The solution was
filtered by using an ash less filter paper, the filter paper was washed 3-4 times with hot water.


The residue left over the filter paper was weighed accurately and the percentage of acid insoluble
ash was calculated with reference to the air-dried drug.

Determination of water-soluble ash
1 G ash was boiled with 25ml of distilled water for 10-15 minutes. The solution was filtered by
using an ash less filter paper, the filter paper was washed 3-4 times with hot water. The residue
left over the filter paper was weighed accurately. The quantity of water soluble ash was
determined by subtracting the residue left over the ash less filter paper from the total weight of
the ash taken. Water soluble ash was calculated with reference to the air dried drug.

Preliminary phytochemical screening
The aqueous, ethanolic and hydro-alcoholic extracts of plant Argyreia nervosa were subjected to
preliminary phytochemical screening using approved standard protocol
29
.

Extraction procedure
500 G of air dried coarse powder was extracted with cold maceration method using 2.5 L of 50%
hydro alcoholic solvent 5 times. The liquid extract was filtered through muslin cloth and
concentrated under reduced pressure in a thin film evaporator.

Isolation and purification of phyto-constituents from hydro-alcoholic extract

A suitable glass column of dimensions 45 CM length, 4.2 CM outer diameter and 3.8 CM inner
diameter was considered for isolation. 20 G of the hydro-alcoholic extract was loaded and eluted
with chloroform-methanol gradient. Fractions of 25 ML each were collected in conical flasks.
All fractions were monitored with TLC. Fractions having identical R
f
value were pooled
together. Fraction nos. 145-186 showed single spot in the TLC (Fig. no. 1) and recrystallized
from ice cold methanol.

Development of mobile phase for TLC
TLC was developed in 5% v/v solution of methanol in chloroform. The plates were visualized
using vanillin-boric acid reagent.


Physical properties of isolated compound:
Test for solubility
A small amounts of compound were taken in clean test tubes and dissolved in different solvents
viz. distilled water, methanol, ethanol, acetonitrile and chloroform.

Determination of melting point
A few crystals of pure isolated compound were taken in a melting point capillary tube and the
tube was tapped until the compound settled down to the bottom. Then the capillary was inserted
in sample space provided in the melting point apparatus. The melting point of the compound was
determined and is shown in table no. 4.

Spectral analysis and structural elucidation of the pure isolated compound:
The pure isolated compound was subjected to chemical characterization by using modern
analytical techniques such as UV-VIS, IR spectrophotometery, NMR (
1
H and
13
C) and LC-MS
spectrometery.

UV-VI S spectral analysis
The pure isolated crystalline compound was dissolved in methanol and the UV absorption
spectrum was recorded between 400-200nm using Shimadzu-1700 UV-Vis spectrophotometer.

I R spectral analysis
Infra Red analysis of pure isolated compound was performed by IR Bruker model, Vector 32
using KBr pellet method. 1 MG of pure sample was thoroughly triturated with 1-2 G of KBr in a
mortar. Then the whole mass was transferred to a die and compressed by means of hydraulic
pressure to form a thin pellet of thickness 0.5 MM and 3 MM diameter and taken for IR study.

NMR spectral analysis
The pure crystalline isolated compound was individually subjected to
1
H and
13
C NMR studies
using instrument model Dpx Bruker and Avance at 200 MHz and 500 MHz. 0.2 MG of
compound was dissolved in CDCl
3
and considered for NMR study. For
13
C NMR analysis 2 MG
compound was taken.


LC-MS method
The mass of the isolated pure compound was determined using LC-MS Model, Agilent 1100-
Bruker Daltonics (Esquire 3000). A suitable mobile phase system was developed with water
(0.1% v/v formic acid) as solvent-A: Acetonitrile as solvent-B. 1 MG of compound was
dissolved in 10 ML of HPLC grade methanol to yield 100 PPM. The solution was sonicated for
15 minutes, followed by filtration through 0.2 m membrane.

Results and Discussion
The physico-chemical analysis of the coarse powder is shown in table no. 1. The preliminary
results of photo-chemical analysis of the different extracts are shown in table no. 2. The TLC
plate of the pure compound is shown in figure no. 1. The purified compound was characterized
by different modern analytical techniques like UV Vis spectrophotometry (table no. 3 and figure
no. 2), IR spectrophotometry,
1
H NMR,
13
C NMR and LC-MS spectrometry (figure no. 3).
Characterization summary of pure compound is reported in table no. 4.

Table no. 1: Physico-chemical analysis of the stem powder obtained from Argyreia nervosa

Sl. No Parameters Values (% w/w)
1. Loss on drying 4.57
2. Ash values
a. Total ash 4.31
b. Acid insoluble 1.68
c. Water soluble ash 3.93
d. Sulfated ash 11.87
3. Extractive values
a. Ethanolic extract 3.75
b. Hydro-alcoholic extract 7.55
c. Aqueous extract 9.29



Table no. 2: Preliminary phytochemical screening of different extracts of Argyreia nervosa

Sl.
No.
Tests Ethanolic
extract
Aqueous
extract
Hydro-alcoholic
extract
1. Alkaloids
a. Dragendroffs test + + ++
b. Mayers test + + ++
c. Hagers test + + ++
d. Wagners test + + ++
2. Carbohydrates
a. Molishs test + + +
b. Fehling solution test + + +
c. Benedicts test + + +
d. Caramelisation + + +
3. Gums/Mucilage
a. Water - - -
b. Alcohol - - -
4. Tannins
a. Ferric chloride test + +++ ++
b. Alkaline reagent test + +++ ++
c. Gelatin test + +++ ++
5. Flavonoids
a. Lead acetate test - ++ +
b. Shinoda test - ++ +
c. Zinc hydrochloride test - ++ +
6. Saponins
a. Foam Test + + +
b. Lead acetate test + + +
7. Sterols
a. Salowaski test ++ + +
b. Libberman Burchad
test
++ + +
8. Coumarin
a. UV Fluorescence test + + +
9. Amino acids
a. Millons test - - -
b. Ninhydrin test - - -
(+): Present, (++): mild strong, (+++): highly strong, (-): absent



Figure no. 1: TLC plate of the isolated compound, scopoletin.

Spectral analysis and structural elucidation of the isolated compound
Table no. 3: Absorbance at different wavelength of isolated compound at concentration of
10g/ml

I nterpretation of isolated compound on the basis of infra red spectra
In infra red spectrum, the region 900-700 cm
-1
exhibited sharp bands which indicated for
aromatic C-H stretching and also satisfied the presence of aromatic ring in the
1
H NMR by
giving peak at 7.65 . This indicates that the unsymmetrical pattern of aromatic ring. A sharp
band near 3029 cm
-1
was the characteristic band of =C-H stretching for aromaticity. A sharp
band at 740 cm
-1
indicated that the aromatic ring is di substituted. A conformational broad band
appears near 3422 cm
-1
for intra molecular hydrogen bonding O-H (aromatic) and may be ortho
substituted. The peak occurs in the region of 1705 cm
-1
indicates a C=O stretching. It is further
confirmed by UV analysis that peak at 345nm is due to n- * transition.

Wavelength (nm)

Absorbance
345 1.131
296 0.339
248 0.478
204 2.312
Compound


Figure no. 2: UV spectrum of compound,
max
: 345nm (peak).
I nterpretation of isolated compound by
1
H NMR
H
3
= 7.26, H
4
= 7.72, H
5
= 6.9, H
8
= 6.8, OCH
3
= 3.9, OH =6.2. From
1
H NMR spectrum,
it is clear that there are 6 types of protons in the ratio of 1:2:1:1:1:2, hence total eight number of
protons may be present in the isolated compound. One proton singlet at 6.2 results for the intra
molecular hydrogen bonding, probably it speaks of the presence of O-H (aromatic) group and it
may be ortho substituted which is confirmed by IR-spectrum giving broad band near 3422 CM
-1
.
The intense singlet peak near 3.88 in
1
H NMR indicates the presence of three protons
containing -OCH
3
.

I nterpretation of isolated compound by
13
C NMR
In
13
C NMR, 10 decoupled peaks indicate that there may be presence of 10 carbon atoms: C
2
=
167.013, C
3
= 116.447, C
4
= 154.376, C
5
= 112.442, C
6
= 155.344, C
7
= 148.635, C
8
=
107.566, C
9
= 149.715, C
10
= 115.475 and OCH
3
= 60.
Wavelength (nm)
Abs.
Compound
Solvent


I nterpretation of isolated compound by LC-MS
MS Condition: Source: ESI, Nebuliser: 35 psi, Dry gas: 12 L/M, Dry temperature: 320
0
C, Target
mass: 178 m/z, Ion polarity: -ve, ICC target: 8000, Maximum accelerating time: 200 MS, Scan
range: 50-700 m/z, Average: 5

Confirmation of the isolated compound by specific chemical tests and UV fluorescence
analysis
A small amount of isolated compound was taken in a clean test tube and dissolved in alcohol. A
few drops of sodium hydroxide solution were added to it. The solution was spotted on a filter
paper and observed under UV light. Yellow fluorescence was observed which confirmed the
presence of coumarin derivative. After interpretation of all data obtained from UV, IR
spectroscopy,
1
H NMR,
13
C NMR, LC-MS spectrometry and the chemical test, it was confirmed
that the isolated compound is coumarin derivative and it is Scopoletin (Figure no. 4).

Figure no. 3: LC-MS spectra of the isolated compound.



O O HO
H
3
CO

Figure no 4: Chemical structure of scopoletin), IUPAC name: (7-Hydroxy, 6-methoxy coumarin)
Table no. 4: Different chemical characterization parameters (scopoletin)
Color of crystal Yellow
Shape Needle shaped crystal
Melting point 208.2
0
C
TLC R
f
value = 0.573
Yield 0.135 %w/w
Solubility
Distilled water
Methanol
Ethanol
Acetonitrile
Chloroform

soluble
Freely soluble
Freely soluble
soluble
soluble
UV-Vis Spectra (
max
)

345nm
IR spectra
900-700 cm
-1
: C-H stretching, 3029 cm
-1
:

=C H stretching,
3422 cm
-1
: O-H (aromatic) group
1
H NMR
H
3
= 7.26, H
4
= 7.72, H
5
= 6.9, H
8
= 6.8, OCH
3
= 3.9,
OH =6.2
13
C NMR
C
2
= 167.013, C
3
= 116.447, C
4
= 154.376, C
5
= 112.442,
C
6
= 155.344, C
7
= 148.635, C
8
= 107.566, C
9
= 149.715,
C
10
= 115.475, OCH
3
= 60
LC-MS Rt= 33.4 min, m/z = 190.0498 [M + H]
+

Conclusions
The current study concludes preliminary photochemical analysis of different extracts of Argyreia
nervosa stem that highlighted the presence of phyto-constituents such as alkaloids,
carbohydrates, flavonoids, sterols, saponins and tannins. A simple method was devloped for


isolation of scopoletin, a coumarin derivative. The isolated pure compound (scopoletin) was
characterized with modern analytical techniques such as UV-Vis Spectra (
max
), IR spectra,
1
H
NMR,
13
C NMR and LC-MS. Today ethno-medicinal studies amalgamated with natural product
chemistry have gained attention as the duo brings known and unknown medicinal virtues of plant
origin to light. These investigations need phytochemical evaluation on modern scientific lines.
Such isolations, analyses and pharmacological characterizations of bio-active constituents will
provide valuable information for further investigations and development of newer potent drugs.
Conflict of interests: The authors report no conflict of interests.
Acknowledgement: Authors thankfully acknowledge the contribution of Dr. K. A. Suri, Ex-
Directors grade scientist, IIIM, Jammu, India in spectral studies.
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International Journal of Advanced Scientific and Technical Research Issue 4 volume 2, March-April 2014

Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954

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