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Chapter 9:

Isolation of fractions EJ-1 and FA-1 from


the leaf extract of Euryajaponica Thunb.
and Ficus auriculata Lour.
and
The structural elucidation of EJ-1
fraction from Euryajaponica Thunb.
CHAPTER: 9

ISOLATION OF FRACTIONS EJ-1 & FA-1 FROM THE LEAF EXTRACT OF


EURYA JAPONICA THUNB. AND FICUSAURICULATA LOUR.

AND

STRUCTURAL ELUCIDATION OF EJ-1 FRACTION OF EURYA JAPONICA


THUNB.

9.1 Introduction:
The evaluation of various plant products according to their traditional uses and
medicinal value based on their therapeutic efficacy leads to the discovery of newer
and recent drugs for treating various ailments. This fact forms the basis for the
development of new drugs from various plant sources (Garima et al, 2011).The
phytoconstituents present in plants confer specific characteristics and properties to the
plants. Therefore, the analysis of these constituents would help in determining various
biological activities of plants. A variety of techniques can be used to determine and
estimate the presence of such phytoconstituents in medicinal plants. Chromatography
and spectroscopic techniques are the most useful and popular tools used for this
purpose.
9.1.1 Chromatography:
Chromatography in its various forms is perhaps the most important known method of
chemical analysis of mixtures. Thin layer chromatography is a simple technique that
is used to separate mixtures into the individual components of the mixture (James,
1993). Chromatography is a method used to separate mixtures of substances into their
components (Upadhyay et al, 2006). Chromatography is the separation of two or
more compounds or ions by the distribution between two phases, one which is moving
and the other which is stationary. These two phases can be solid-liquid, liquid-liquid
or gas-liquid phase (Fried and Sherma, 1999). The first detailed description of
Chromatography is generally credited to Micheal Tswett, a Russian biochemist, who
separated chlorophyll from a mixture of plant pigments in 1906.

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9.1.2 Thin layer chromatography:
Thin layer chromatography (TLC) is a simple chromatography technique used to
separate mixtures (Laurence et.al.,]999). Thin-layer chromatography or TLC is a
solid-liquid form of chromatography where the stationary phase is normally a polar
absorbent and the mobile phase can be a single solvent or combination of solvents.
TLC is a quick, inexpensive micro- scale technique that can be used:

• To determine the number of components in a mixture.


• To determine the identity of two substances.
• To monitor the progress of a reaction.
• To determine the effectiveness of a purification.
• To determine the appropriate conditions for a column chromatographic
separation.
• To monitor column chromatography.

9.1.3 Column chromatography:


Column chromatography is similar to TLC in that a mixture of compounds can be
separated by being carried by a mobile phase through a stationary phase. However,
instead of just giving us information about the mixture, column chromatography is
used to actually separate larger amounts of the compounds.
The technique of TLC was useful in determining the type and number of ingredients
in the mixture, but it was not helpful for collecting the separated components. One
could only separate and visualize the spots. However, in column chromatography it
allows us to separate and collect the compounds individually and TLC is being used
to monitor the effectiveness of this separation.
9.1.4 Preparative TLC:
In the study for isolation and purification of compounds obtained from medicinal
plants, separation by column chromatography alone is a difficult task, therefore
preparative TLC is employed for further purification and isolation of the compounds
collected by column chromatography. Larger TLC plates, called a Preparative Plates
(PTLC), is used for separations of milligram quantities of materials. PTLC is of
great utility in difficult separations of small amounts of material , such as
extracts of natural products and separation of diastereomers (www.http/Zcarbon.
indstate.edu.).

95
In the present experiment, TLC profiling, Column Chromatography and preparative
TLC were used to isolated and purify compounds collected from the leaf extracts of
Eurya japonica Thunb. and Ficus auriculata Lour. The isolated compounds were
analyzed for spectral studies namely IR, ' H & '^C NMR and GC-MS for structural
elucidation.

96
9.2 Materials and methods:
9.2.1 Preparation of extract:
The leaves of Eurya japonica Thunb. and Ficus auriculata Lour, plants are shade
dried and powdered . Powdered sample of lOOg leaf was weighed and subjected to
soxhlation with petroleum ether for 72 hrs. Similarly, the procedure was repeated with
ethyl acetate, acetone, methanol and ethanol as solvents, using lOOg of the fresh
ground sample, for each extraction .The solvents were distilled off at lower
temperature under reduced pressure in the rotary evaporator and concentrated to
dryness.
9.2.2 TLC profiling:
For each of the five extracts, 4 different solvent systems were used as developing
systems. These were petroleum ether (P.E) 100 %, chloroform (C) 100%, petroleum
ether .ethyl acetate (P.E :E.A,)7;3%, and petroleum ether ; ethyl acetate (P.E ;
E.A)9:1%.
9.2.2a Visualization of thechromatogram:
If the substance being chromatographed is coloured then it is possible to detect the
components visually. Colorless substances can be detected by placing the dry TLC
plates in ajar containing a few crystals of iodine (Figure 9.2.2a). The iodine vapour
will be preferentially adsorbed by the substances on the plate and they will appears as
brown spots on a lighter- coloured background. The plate is removed from the jar and
the outline of the spots traced lightly with pencil because the iodine will soon
evaporate (Knneth,1989).
Cmt'nMUit-

solvent front
^ >.

distance
dist travelled by solvent front
spot | < 7 K

distance ravelled by
substanc(
^ 4' spot origin
1
Hall Csniiic Into
Salnnt Vajnr

Figure 9.2.2a: Functional schematics of a) Ascending development chamber for TLC


and b) TLC plate showing distances travelled by the spot and the solvent when the
solvent front has nearly reached the top of the adsorbent.

97
9.2.2b The Rf Values:
The behavior of an individual compound in TLC is characterized by a quantity
known as Rf and is expressed as a decimal fraction. The Rf is calculated
by dividing the distance the compound traveled from the original position by
the distance the solvent traveled from the original position (the solvent front).

The Rf (retention factor) value for each spots developed is then worked out using the
formula:
Q distance travelled by component
rif =
distance travelled by solvent

The distance a compound travels indicates that compound's physical


character!stics.The greater the similarity to the mobile phase, the further it will be
pulled up through the stationary particles on the TLC plate.
9.2.3 Chromatography of methanol extract from \ea( of Eurya japonica Thunb.
and Fie us auric ulaia Lour.:
In this experiment, the shade dried powdered leaves of Eurya japonica Thunb. and
Ficus auriculata Lour, were defatted with petroleum ether for 24 hours followed by
methanol extraction for 48 hours. Column chromatography of methanol leaf extracts
of Eurya japonica Thunb. and Ficus auriculata Lour, were conducted using silica gel
(Mesh 60-120) that was packed by wet packing method in petroleum ether (Mc
tony,2006). The column was then eluted with different solvents (Petroleum ether.
Petroleum ether: Ethyl acetate ratio) in increasing order of polarity. The fractions
were collected and marked. The marked fractions were subjected to thin layer
chromatography to check homogeneity of various fractions. Similar fractions were
pooled together. Eluates with mixed fractions were further purified using preparative
TLC. Spots were identified and recovered by scraping the adsorbent off the plate (or
cutting out the spots if the supporting material can be cut) and extracting the
substance from the adsorbent using methanol/ Chloroform. Then it was centrifuged at
3000 RPM, room temperature for 5 minutes for 2/3 times. The solvent was filtered
and dried. The dried fraction collected was analysed for spectral studies namely IR,
'H & '^C NMR and GC/LC MS for structural elucidation (Bersa, 1998; Belisle et al,
Dittmerand Lester, Slayden and Barry, 2001).

98
9.3: Results and discussion:
9.3.1: TLC profiling:
TLC profiling of the leaf extracts of Eurya japonica on different solvents shows the
presence of different spots (Figure 9.3.1). Various phytochemicals give different Rf
values in different solvent system. This number can be calculated for each spot
observed on a TLC plate. Essentially it describes the distance traveled by the
individual components. If two spots travel the same distance or have the same R/
value then it might be concluded that the two components are the same compound.
In petroleum ether extract, 100% PE (petroleum ether) has shown the presence of 1
spot ,100% C (chloroform) has shown the maximum number of spots with the
presence of 5 spots, 7:3 % PE-EA has shown the presence of 4 spots and 9:1%PE-EA
has shown the presence of 3 spots. Their Rf values have been compared and presented
in the table 9.3.1.
In ethyl acetate extract, 100% PE (petroleum ether) has shown the presence of 1 spot,
100% C (chloroform) has shown the presence of 3 spots ,7:3 % PE-EA has shown the
maximum number of spots with the presence of 6 spots and 9:1%PE-EA shows the
presence of 4 spots. Their Rf values have been compared and presented in the table
9.3.1.
In acetone extract, 100% PE (petroleum ether) has shown the presence of 3 spots,
100% C (chloroform) has shown the presence of 5 spots, 7:3 % PE-EA has shown the
maximum number of spots with the presence of 6 spots and 9:1%PE-EA has shown
the presence of 5 spots. Their Rf values have been compared and presented in the
table 9.3.1.
In methanol extract, 100% PE (petroleum ether) has shown the presence of 2 spots,
100% C (chloroform) and 9:1% PE-EA has shown the maximum number of spots
with the presence of 4 spots and 7:3 % PE-EA has shown the presence of 3
spots.Their Rf values have been compared and presented in the table 9.3.1.
In ethanol extract, 100% PE (petroleum ether) has shown the presence of 2 spots,
100% C(chloroform) has shown the presence of 3 spots, 7:3 % PE-EA has shown the
maximum number of spots with the presence of 4 spots and 9:1%PE-EA has shown
the presence of 3 spots. Their Rf values have been compared and presented in the
table 9.3.1.

99
Similarly, TLC profiling of the leaf extracts of Ficus auriculata on different solvents
have also shown the presence of different spots. Various phytochemicals give
different Rf values in different solvent system (Figure 9.3.1).
In petroleum ether extract, 100% PE (petroleum ether) has shown the presence of 1
spot, 100% C (chloroform) and 9:1%PE-EA has shown the maximum number of
spots with the presence of 3 spots,7:3 % PE-EA has shown the presence of 2 spots.
Their Rf values have been compared and presented in the table 9.3.2.
In ethyl acetate extract, 100% PE (petroleum ether) has shown the presence of 3 spot
,100% C (chloroform) has shown the maximum number of spots with the presence of
5 spots ,7:3 % PE-EA has shown the presence of 3 spots and 9:1%PE-EA has show
the presence of 3 spots. Their Rf values have been compared and presented in the
table 9.3.2.
In acetone extract , 100% PE (petroleum ether) has shown the presence of 3 spot
,100% C (chloroform) and 9:1%PE-EA has shown the maximum number of spots
with the presence of 4 spots each on the plates, 7:3 % PE-EA has shown the presence
of 3 spots. Their Rf values have been compared and presented in the table 9.3.2.
In methanol extract, 100% PE (petroleum ether) has show the presence of 3 spot,
100% C (chloroform) has shown the maximum number of spots with the presence of
5 spots, 7:3 % PE-EA and 9:1%)PE-EA has shown the presence of 4 spots. Their Rf
values have been compared and presented in the table 9.3.2.
In ethanol extract, 100% PE (petroleum ether) has shown the presence of 2 spot,
100% C (chloroform) has shown the presence of 2 spots, 7:3 % PE-EA and 9:1% PE-
EA has shown the maximum number of spots with the presence of 4 spots each on the
plates. Their Rf values have been compared and presented in the table 9.3.2.
TLC profiling of the two leaf extracts in different solvents has given an impressive
results directing towards the presence of number of phytochemicals. Various
phytochemicals gave different Rf values in different solvent system. This variation in
Rf values of the presence of phytochemicals provides a very important clue in
understanding their polarity and also helps in the selection of appropriate solvent
system for separation of pure compounds by column chromatography. Compounds
showing high Rf value in less polar solvent system is suppose to have low polarity and
with less Rf value is suppose to have high polarity. Mixture of the solvents with
variable polarity in different ratio can be used for the separation of pure coumpounds
from plant extracts. The selection of appropriate solvent system for a particular plant
100
extract can only be achieved by analysing the Rf values of the compounds in different
solvent system.
In the present work, TLC profiling of the leaf extracts of Eurya japonica Thunb. and
Ficus auriculata Lour, in different solvent system indicated the presence of diverse
type of phytochemicals in the selected plants. Different Rf values of the compounds
also reflects on the idea about the polarity. This information will help in the selection
of appropriate solvent system for further separation of compound from these two plant
extracts. From the present result methanol extract of both the plants Eurya japonica
Thunb. and Ficus auriculata Lour, were chosen for further isolation of compounds by
column chromatography.

101
Table 9.3.1: TLC profiling and measurement of retention factor (Rf) of Eurya
japonica Thunb. leaf extract in different solvent system:

Eurya Developing solvent system


japonica 100% P.E 100% C 7:3 (PE-EA) 9:1 (PE-EA)
Thunb. No. R/value No. of R/value No. R/value No. R/value
extract: of spots of of
spots spots spots
Petroleum 1 0.33±0.03 5 0.06±0.01 4 0.1810.07 3. 0.1310.02
ether 0.24±0.04 0.2910.06 0.2610.04
extract 0.50±0.05 0.5410.05 0.9410.02
0.75±0.05 0.9310.03
0.94±0.02
Ethyl 1 0.56±0.11 3 0.13±0.03 6 0.13+0.03 4 0.2310.11
acetate 0.42±0.08 0.2210.04 0.3710.05
extract 0.90±0.03 0.29+0.05 0.7310.00
0.4710.10 0.9210.05
0.6510.06
0.8910.05
Acetone 3 0.06±0.01 5 0.08±0.03 6 0.1210.04 5 0.0710.01
extract 0.14±0.01 0.1110.03 0.4110.05 0.5610.16
0:26±0.06 0.19±0.03 0.5010.08 0.6410.15
0.46±0.11 0.60+0.06 0.9010.06
0.9510.01 0.8710.00 0.9810.00
0.9610.00

Methanol 2 0.13±0.01 4 0.14+0.00 3 0.2010.07 4 0.1610.02


extract 0.24±0.01 0.24+0.01 0.3110.03 0.3710.01
0.57+0.02 0.4410.01 0.6310.04
0.95+0.00 0.9310.01
Ethanol 2 0.09±0.02 3 0.0610.01 4 0.08+0.00 3 0.0510.01
extract 0.14±0.01 0.3210.19 0.1610.01 0.2010.07
0.4810.16 0.3810.04 0.2610.05
0.5010.02

Results are expressed as mean ISD (n=3)

102
Table 9.3.2: TLC profiling and measurement of retention factor (Rf) of Ficus
auriculata Lour, leaf extract in different solvent system .•

Ficus Developing solvent system

auriculata 100% P.E 100% C 7:3 (PE-EA) 9:1 (PE-EA)

Lour.extract : No. R/value No. R/value No. R/value No. R/value

of of of of

spots spots spots spots

Petroleum 1 0.24±0.02 3 0.3310.01 2 0.4410.16 3 0.2510.06

ether extract 0.5910.03 0.8810.03 0.5210.05

0.8510.05 ' 0.7610.05

Ethyl acetate 3 0.15±0.07 5 0.2010.02 3 0.6410.02 3 0.3110.14

extract 0.53±0.12 0.2510.00 0.7010.02 0.3910.09

0.96±0.02 0.3310.00 0.7610.02 0.9510.01

0.5110.05

0.8410.07

Acetone 3 0.12±0.03 4 0.1710.02 3 0.4910.02 4 0.1810.02

extract 0.26±0.13 0.4510.09 0.7810.03 0.2510.01

0.35±0.12 0.7310.03 0.9010.03 0.3310.03

0.9510.01 0.9410.01

Methanol 3 0.1610.05 5 0.1410.01 4 0.1310.04 4 0.1610.00

extract 0.2510.07 0.3210.02 0.5010.01 0.3010.01

0.3310.12 0.5810.05 0.6410.02 0.5410.02

0.76+0.05 0.7510.00 0.8210.01

0.8810.03

Ethanol 2 0.1410.02 2 0.5710.15 4 0.5310.06 4 0.4210.01

extract 0.2510.01 0.6510.14 0.5910.03 0.4910.02

0.6810.03 0.7510.03

0.7710.04 0.7910.04

Results are expressed as mean ISD (n=3)

103
Photo Plate 9.3.1 : TLC profiling plates oiEurya japonica Thunb;

DIlDlJ
HD
Figure 9.3.1: A) Acetone extract at P.E:E.A (7:3). B) Acetone extract at 100% P.E.
C) Ethyl acetate extract at P.E:E.A (7:3). D) Methanol extract at P.E:E.A (7:3).
E) Petroleum ether extract at P.E:E.A (7:3). F) Ethanol extract at 100%C.

Photo Plate 9.3.2: TLC profiling plates of Ficus auriculata Lour:

Figure 9.3. 2: A. Methanol extract at P.E:E.A (7:3). B. Ethyl acetate extract at 100%
P.E. C. Acetone extract at 100%C. D. Methanol extract at P.E:E.A (9:1).

104
9.3.2: Column chromatography:
Column chromatography of the methanol leaf extracts of Eurya japonica Thunb and
Ficus auriculata Lour has helped to collect some fractions. These fractions were
subjected to Preparative TLC.

Table 9.3.3: Column chromatography fractions of leaf extract of the plants under
study:
Eluent Fraction Remark
Eurya japonica Thunb.
P.E:E.A(9:1) 1 (EJ-1) Dark green colour
Ficus auriculata Lour.
P.E:E.A(9:1) 1 (FA-1) Dark green color

The Preparative TLC of the fraction (Figure 9.3.3) of Eurya japonica Thunb.
collected 1 distinct spot that were isolated and collected separately in a tube and
marked as EJ-1. The Preparative TLC of the fraction (Figure 9.3.4) of Ficus
auriculata Lour, collected 1 distinct spot was isolated and collected separately in a
tube and marked as FA-1. The isolated fractions (EJ-1 & FA-1) were analyzed for
spectral studies namely IR, ' H & '^C NMR and GC MS for structural elucidation

105
EJ-1

Figure 9.3.3: Preparative TLC of the methanolic leaf extract of Eutya japonica
Thunb.

--n

*• FA-l

Figure 9.3.4: Preparative TLC of the methanolic leaf extract of Ficus auriculata Lour.

106
9.3.3: Spectral analysis and structural elucidation of the isolated compound
(EJ-1) from the methanol leaf extract of Euryajaponica Thunb.:

100-

95-

90-

35-

so-

70-

65-
l l l l l l l l l l l l l l l l l l l T i l l It
I' ' ' ' I'' ' 'I ' ' ' ' I
3500 3250 3000 :750 2500 2250 2000 1750 1500 1 2 5 0 1 0 0 0 750 500
l.cm

Figure 9.3.5: FT-IR spectrum of the isolated compound (EJ-1) from the methanol leaf
extract of Euryajaponica Thunb.

Table 9.3.4: Assignment of functional group corresponding to the peaks obtained


from FT-IR of the isolated compound (EJ-1) from the methanol leaf extract of Eurya
japonica Thunb.:

Wave number (cm ) Characteristic peaks

3194 Yo-H Stretch of-O-H


Y c-H Stretch of Methyl and methylene moiety (-C-H)
2955 and 2928
Yc=o Stretch of Ketonic group (-C=0)
1666
Yc-H Stretch of Aldehydic group
2721 0
II
-C-H

107
The analytical GC spectrum of the compound (Figure 9.3.6) exhibited one major peak
at 28.51 min. when eluted with helium as carrier gas. Trace of methanol solvent
accompanying the extract was eluted off with the carrier gas during the initial phase
of GC (before 12 minitues) of the experimental analysis.

Qudlltative/Quantitative Report

RIe: C.ITURBOIil'^SSlOFN.PRaDatalEJ.raw
k0M. 30-NOV-1310:56:15AM Printed: 04-Oec-13 02:41 PM
Osscripto GBPIC
GdSMetiiod: 60:40-300 AT 10 RAMRnith MS: # 3 0 0 AT 10 RAIf.EXP Page lot 2
Sample ID: 6CMS#}64^BOH-APL Vial Number 1

Scan EI+
TIC
tODi

4fMMM

I I I I I M I Mil I i | i n i | i i i i | i i i i i i i i i | i i i i i i i i i | i i i i | i i i i | i i i i | i i i i | i i i i | M i i | i i i i | i i i i | i i M | i i i i m i i | i M i | i i i i | i i i i | i Tme
TJTTTTl

12,03 16.00 ISJOO 20.00 22.00 2400 26.00 28.W ^.OO 32.00 34.09 38.00

Figure 9.3.6: Mass spectrum of the isolated compound (EJ-1) from the methanol leaf
extract of Eurya japonic a Thunb.

108
CKL3, c-i/ii/is, £*.:?, : E H T ;

i rv '/I --' J^ ^ t -^ •;? irt «' i?^ f^ f-j •f \^ cr •? c"


s5H?i;5is5sHvi > f 1 o o cv r- r- in f-i o oc- O c^
BRUKER
^i^S=^

lllJjLX^^
-T—•
11
r~
10
)(WHH V y>rirt r
I ' r
6
~T
4 3
r
0 ppm

Figure 9.3.7a: H NMR spectra of isolated compound (EJ-1) from the leaf extract of
Euryajaponica Thunb.

-1,3, 0«. i l , ' I 3 . SAir, IJEHU

\\// \' \/ W / 1/

'I ft

i!
M A v,—r'''v.^-,
•'\

T 1 ^ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 "\ 1 1 1 1 1 1 1 1 \
!.7 e.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 75 7.< 7.3 7.2 7.1 7.0 8.8 8.8 8.7 €.6 6.5 6.4 6.3 62 6.1 ppm

Figure 9.3.7b: H NMR spectra of the isolated compound (EJ-1) from the leaf extract
o^Euryajaponica Thunb.

109
Table 9.3.5: Assignment of corresponding proton units to the peaks obtained from
'H N M R spectrum of the isolated compound (EJ-1) from methanol leaf extract of
Euryajaponica Thunb.

'H NMR spectrum

Peaks (5 ppm) Corresponding peaks assignment to


protons
3-5 Sugar moiety protons
Doublet at 9.49 Aldehydic proton (H-1)
5.1 Oliphenic proton (H-7)
6-9 Proton present over the substituted
aromatic system (H-14-20)
Two doublet of doublets at 6.25 and 6.26 Proton (H-16, H-17)
7.25 and 8.55 H-15andH-18
9.35 H- bonded phenolic -OH (H-19)
0.87 Methyl proton (H-6)
1.2 to 2.5 Proton H-2,H-3 and H-5

\'

H 0 H 19
18

HO"
"^
HO .x^-
10 fTllSOM 12 13 15

Figure 9.3.7c: Assignment of the protons corresponding to the peaks obtained from
'H NMR of isolated compound obtained from the methanol leaf extract of Eurya
japonica Thunb.

Assignment of corresponding proton unit to the peak obtained from 'H NMR
spectrum of the isolated compound (EJ-1) from methanol leaf extract of Eurya
japonica Thunb. is showed in Table 9.3.5. The ' H NMR spectrum of the compound
(Figure 9.3.7a & b) exhibited characteristic peak for the presence of sugar moiety in 8

110
3-5 ppm. The doublet at 5 9.49ppm is due to the aldehydic proton present in the
compound. The oliphenic proton (H-7) appears as a singlet at 5 5.1 ppm. The peak
from 8 6-9 ppm corresponds to the proton present over the substituted aromatic
system. The two doublet of doublets at 5 6.25 ppm and 8 6.26 ppm corresponds to the
proton (H-16, H-17). The peak at 8 7.25 ppm and 8 8.55 ppm attributed to H-15 and
H-18 respectively. The H- bonded phenolic -OH (H-19) resonated at 8 9.35 ppm. The
presence of strong H- bonding forming a stable six member ring like structure is
responsible for the down field resonance of this proton. The methyl proton (H-6)
appears at 8 0.87 ppm whereas the proton H-2, H-3 and H-5 resonated as multiplate
between 8 1.2 ppm to 8 2.5 ppm.

:sc E--1, CDCLS, 04/11/1; SkZT, fEKU

' r- CD O c^ i^i «-i vft r : —1 r-* 'V (M a> -ii o nj T CO T r-i I


-. u. « r- >P Ti i ; X r j T •ri "J >•;> •-* i'- csOTi«'i -i; -c w r
<-i f". O ^ M> r* e; u -1 r- o <N *•)? r- 'ii U' •-* (M ii> (?t C*^ :- v '.
t f-t o r* m r- r- o T H ^^; 'H o c>i '?•. r- 'D (N r-j '-1 f-1 c? c^ r- ^
• w 01 Ci f- r- r* 'ii 'L" m ifi to (^ r^i r*i n r; fn n n r> M n f
^ ^ c ^ - ^ ^ ^

— I — • — I — ' — I — • — I — • — I — • — I —
—r- —I • 1 •
220 200 180 160 110 120 100 60 40 20 0 ppm
80

1^

Figure 9.3.7d: C NMR spectra of the isolated compound (EJ-1) from the leaf extract
of Euryajaponica Thunb.

Ill
Table 9.3.6: Assignment of corresponding carbon units to the peaks obtained from
'^C NMR spectrum of the isolated compound (EJ-1) from methanol leaf extract of
Eurya japonica Thunb.

'^C NMR spectrum

Peaks (5 ppm) Corresponding peaks assigment to carbons

190 Aldehyde carbon (C-1)


136 Oliphenic carbon (C-4)
112 C-7.
93.11 Aromatic carbon (C-16)
97.52 Aromatic carbon (C-17)
104.42 Aromatic carbon (C-15)
117 Aromatic carbon (C-18)
122.79 Aromatic carbon (C-14)
129.05 Aromatic carbon (C-20)
50-70 Five carbon of the sugar unit
24.96 Carbon C-2
29.69 Carbon C-3
23.07 Carbon C-6
36.62 Carbon C-13
31.61 Carbon C-5

19
18
H-^ --r--^ --,17

'O-
15
13 15

Figure 9.3.7e: Assignment of the carbons corresponding to the peaks obtained from
'^C NMR of isolated compound obtained from the methanol leaf extract of Eurya
japonica Thunb.

13/
Assignment of corresponding carbon unit to the peak obtained from C NMR
spectrum of the isolated compound (EJ-1) from methanol leaf extract of Eurya
13r
japonica Thunb. is showed in Table 9.3.6. The C NMR spectrum (Figure 9.3.7d)

112
provided the evidence for the presence of carbonyl group.The aldehyde carbon C-1
resonated at 8 190 ppm. The peak at 5 136 ppm corresponda to C-4 which is and
oliphenic carbon. Similarly the peak at 5 112 ppm is attributable at C-7. The aromatic
carbon resonated at 5 93.11 ppm (C-16), 5 97.52 ppm (C-17), 6 104.42 ppm (C-15),
5 117 ppm (C-18), 6 122.79 ppm (C-14) and 5 129.05 ppm (C-20) respectively. The
five peaks from 8 50- 70 ppm is attributable to the five carbon of the sugar unit. The
carbon C-2, C-3, C-6, C-13 and C-5 appears at 8 24.96 ppm, 8 29.69 ppm, 8 23.07
ppm, 8 36.62 ppm and 8 31.61 ppm respectively.

Keeping these values in concern (IR, 'H & '^C MNR and GC-MS) the tentative
structure of the compound is elucidated to be as follows:

Figure 9.3.7f: The structure of 6-(2-hydroxybenzyloxy)-3,4,5-trihydroxy-


tetrahydro-2H-pyran-2-yl)methoxy)-3-methylpent-4-enal isolated from the leaf
of Eurya japonica Thunb.

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9.4 Conclusion:
From the above spectral analysis, the compound isolated from the leaf extract of
Eurya japonica Thunb. was found to be 6-(2-hydroxybenzyIoxy)-3,4,5-trihydroxy-
tetrahydro-2H-pyran-2-yl)methoxy)-3-methyIpent-4-enal.
Further research work is necessary for optimization of bioactivity and to know the
potency as antidiabetic property of the compound.
However due to the presence of some impurities in the isolated fi^ction (FA-1) of
Ficus auriculata Lour, the spectra (IR, GC-MS and ' H & '^C MNR) could not reveal
any probable structure of the isolated fraction. It transpired that the fraction is a
mixture of similar group of compounds which eluted together with column
chromatography and which could not be separated on TLC. Therefore, further
isolation of the extract of Ficus auriculata Lour, will help to corroborate the
antidiabteic activity of the extract and determination of bioactivity of the isolated
fraction.

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