Bioactivity Evaluation and Phytochemical Characterization of Pajanelia longifolia
(Willd.) K. Schuman (Bignoniaceae)
Chapter 4
—IP
- ' • " I»""H"l»i
:$-
\i
Results
23
Results
BIO ACTIVITY EVALUATION OF CRUDE EXTRACTS
The crude extract of the leaves and bark were studied for the analysis of
anti-microbial activity against various pathogenic micro-organisms and
hepatoprotective activity.
4.1. Anti-Microbial Activity of Crude Extracts
The crude extracts of leaves and bark of Pajanelia longifolia were
investigated for its therapeutic efficacy against pathogenic microorganisms viz., Klebsiella ,sp.. Streptococcus sp., Staphyllococcous sp.,
Candida sp.. Salmonella sp.. Bacillus sp., E. coli and Proteus sp. The extract
showed anti-microbial activity (Table 4.1).
The ethyl acetate and 70% ethanolic extracts of the leaves and bark were
most active against the micro-organisms studied. A good degree of
activity was recorded for ethyl acetate as compared to 70% ethanol
extract and petroleum ether extract. Ethyl acetate of leaves showed high
activity against Staphylococcus sp. and Streptococcus sp. However, the
activity of the ethyl acetate extract for all other micro-organisms
remained quite similar during the study. The petroleum ether extracts
of the leaves did not have any significant antimicrobial activity.
24
Table 4.1 Anti-Microbial Activity of Crude Extracts of Pajanelia
longifoUa
Name of the
Microorganisms
Zone of Inhibition (mm)
Leaf
Bark
Petroleum
Ether
Not
Measurable
Ethyl
Acetate
70%
Ethanol
Petroleum
Ether
Ethyl
Acetate
70%
Ethanol
27
12
7
12
18
8
31
16
8
11
Candida sp.
7
18
12
8
12
E. coli
Not
Measurable
Not
Measurable
Not
Measurable
Not
Measurable
Not
Measurable
14
9
11
16
11
7
8
8
15
11
14
11
11
9
Not
Measurable
Nil
13
15
Not
Measurable
Not
Measurable
Not
Measurable
Not
Measurable
Not
Measurable
Not
Measurable
Not
Measurable
15
12
Staphylococcus
sp.
Streptococcus sp.
Bacillus sp.
Proteus sp.
Klebsiella sp.
Salmonella sp.
The bark extracts also showed anti-microbial activity for both ethyl
acetate and 70% ethanolic extracts. This finding correlates with the
traditional medicinal use, where leaves are used in skin infections like
rashes, boils, nail decay, etc. Interestingly, the acetone and methanolic
extracts of both leaves and bark did not exhibit significant antimicrobial activity (Plate 1 and 2).
The effect
Amikacin,
of the standard
Gentamycin,
antibiotics viz..
Norfloxacin,
Nystatin,
Ciprofloxacin,
Penicillin
and
Streptomycin were studied (Table 4. 2 and Figure 4.1- 4.2). As shown in
the figure, most of the standard antibiotics used have significant effect
on the growth of the micro-organisms.
Plate 1. Analysis of antimicrobial activity of crude extracts of leaves of Pajanelia longifolia (Willd). K.
Schuman showing the zone of inhibition.
(A-B): Zone of inhibition of ethyl acetate and 70% (v/v) ethanolic extracts of lea\es and standard antibiotics penicillin,
amikacin, gcntamycin and streptomycin against Streptococcous sp. and StaphyUococcous sp. (C-E). Zone of inhibition of
petroleum ethar, ethyl acetate and 70% (vAr) ethanolic extracts of leaves against Proteus sp., Klebsiella sp. and Salmonella
sp. (F-G): Zone of inhibition ofpetroleum ether, ethyl acetate and 70% (v/v) ethanolic extracts ofBacillus sp. and E coli. (H):
Zone of inhibition of peuoleum eiher, ethyl aceiale and 70% (v/v) ethanolic extracts ofhtikStreptococcuos sp. (I-K): Zone
of inhibition ofpetroleum ether, ethyl acetate and 70% (v/v) ethanolic extracts of baric against Staphyllococcous sp., Proteus
sp., Klebsiella sp.
Plate 2. Analysis of antimicrobial activity of crude extracts of leaves and bark of Pajanelia
longifolia (Willd.) K. Schuman showing zone of inhibition
(A - C): Zone of inhibition of petroleum ether, ethyl acetate and 70% (v/v) ethanolic extract of bark against Candida sp .,
Salmonella spp. and Bacillus sp . (D-F): Zone of inhibition of methanolic extract of leaves against Staphyllococcous sp .
Streptococcous spp. and Candida sp . showing no significant effect. (G): Zone of inhibition of ethyl acetate, acetone and
methanolic extract of bark Bacillus sp . (H-I): Zone of inhibition of standard antibiotics.
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Fig. 4.1. Zone of inhibition exerted by standard antibiotics on microorganisms. The data presented are mean of three separate experiments.
C Ncrflcxacin nNystatin • Penicillin • Spreptomydn
fi li HI
J"
v>3>
J^
^^
,•>"
,<f
./'
.^'
<>
,-f
Miao-oiyiiiifsitis
Fig. 4.2. Zone of inhibition exerted by standard antibiotics on microorganisms. The data presented are mean of three separate experiments.
4.2. Hepatoprotective Activity of Crude Extracts
The results on the hepatoprotective effect of the crude bark extract of
Pajanelia longifolia on CCU - intoxicated albino mice are shown in Figure
4.3 - 4.6. The serum bilirubin content was increased to 6.92 mg/dl,
while it was recorded to be 1.28 mg/dl in control. When treated with
Silymarin, there was drastic decline in the bilirubin content (3.73
n
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26
mg/dl). The effects of the crude extracts were also significant. The ethyl
acetate crude extract of the bark was most effective in comparison to
petroleum ether and 70% (v/v) ethanolic extract of the bark. The
bilirubin content was reduced to 3.38 m g / d l and 4.58mg/dl in group
treated with ethyl acetate and 70% (v/v) ethanol respectively. The
petroleum ether extract have no significant effect with bilirubin content
of 6.67 m g / d l .
c> 6
S
2-
Cwrtrol
CCI4
Silyimirin*
Ca,
'4
PE*CCL
4
'
EA*CCI^
4
EtOH*CCI
4
Treatment Forniiilatioiis
Fig. 4.3. Effect of bark extracts of Pajanelia longifolia (Willd.) K.
Schumann on the Serum Bilirubin content in CCU induced
Hepatotoxicity in mice. The data presented are mean of three separate
experiments + SE. (PE, EA and EtOH stand for Petroleum ether, ethyl
acetate and 70% ethanolic extracts respectively).
The Serum Alkaline Phosphatase (SALP) level (Fig. 4.4) in control
mice was recorded to be 127.52 U/L, which increased to 183.88 U / L
upon CCI4 treatment. The effect of the standard drug Silymarin was
visible with SALP level of 141.84 U/L. The effect of the crude extracts.
27
however, not significant. The SALP level increased to 219.12,197.22 and
212.82 U/L for petroleum ether, ethyl acetate and 70% (v/v) ethanolic
extracts of the bark respectively.
S- 250
I 200
llllll
EA
EtOH
ccr,
cci^
Ti«atmeiit Fomnilatioiis
Fig. 4.4. Effect of bark extracts of Pajanelia longifolia (Willd.) K.
Schumann on the Serum Alkaline Phosphatase Level in CCU induced
Hepatotoxicity in mice. The data presented are mean of three separate
experiments + SE. (PE, EA and EtOH stand for Petroleum ether, ethyl
acetate and 70% ethanolic extracts respectively).
The SGPT level (Fig. 4.5) showed remarkable increase after CCI4
treatment (281.86 U/L). The level came down to 86.73 U/L upon
Silymarin treatment. The effect of the crude extract was evident in case
of the ethyl acetate bark extracts, where the SGPT level decreased to
151.36 U/L. The petroleum ether extract was not effective in reducing
the SGPT level (271.11 U/L). The SGPT level was observed to be 187.29
U/L in case 70% ethanolic extract of the bark.
28
«? 200
IB
jT 150
CL
O
</> 100
Control
CCI4
Sllyimiiln*
CCI^
PE + CCI.
4
EA + CCI.
4
EtOH+CCI.
4
Treatinoiit Formulations
Fig. 4.5. Effect of bark extracts of Pajanelia longifolia (Willd.) K.
Schumann on the SGPT Level in CCU induced Hepatotoxicity in mice.
The data presented are mean of three separate experiments + SE. (PE,
EA and EtOH stand for Petroleum ether, ethyl acetate and 70%
ethanolic extracts respectively).
In SGOT level (Fig. 4.6) also increased significantly to 321.26 U/L after
CCI4 treatment, while under control conditions the SGOT level was
recorded to be 87.29 U/L. Upon Silymarin treatment, the SGOT level
came down only to 297.17 U/L, while in case of ethyl acetate extracted
bark the level was significantly reduced to 171.21 U/L. The petroleum
ether and 70% ethanolic extracts showed the SGOT level to be 343.21
U/L and 297.33 U/L respectively.
29
400
350300^
260
^
o
O
200
150
100
50-j
Coifiol
mil
CCI4
Silymariii«
CCI^
PE»ca.
4
EA*CCI.
4
EtOH*CCI.
4
Tr«i)tinent Formulations
Fig. 4.6. Effect of bark extracts of Pajanelia longifolia (Willd.) K.
Schumann on the SGOT Level in CCU induced Hepatotoxicity in mice.
The data presented are mean of three separate experiments + SE. (PE,
EA and EtOH stand for Petroleum ether, ethyl acetate and 70%
ethanolic extracts respectively).
The biochemical studies on various biochemical parameters like Serum
Bilirubin, SALP, SGPT and SGOT showed that the ethyl acetate extract
of the bark was highly effective in providing protection against hepatic
damage and infection.
30
Phytochemical Characterization of Pajanelia longifolia
(Willd.) K. Schuman
5.1. Qualitative Colour Test
The leaves and bark extract of Pajanelia longifolia was studied for
isolation and characterization of natural products. The crude extract of
the leaves and barks were studied for presence of secondary metabolites
qualitatively (Table 5.1). The petroleum ether leaf extract revealed the
presence of alkaloid, steroid and tannin, however, saponin and
flavonoid were absent. Steroid, tannin and terpenoid was detected in
bark only.
In the 70% ethanolic extracts of leaf, significant presence of all the
secondary metabolites were observed. Alkaloid, steroid, terpenoid,
tannin and flavonoid were present in leaves except saponin. In the 70%
ethanolic extracts of the bark alkaloid, steroid, terpenoid and tannin
were detected. The ethyl acetate extracts of leaves showed the presence
of alkaloid, steroid, tannin and terpenoid. In the bark extracts only
alkaloid, tannin and flavonoid were detected.
31
Table 5.1. Qualitative colour test for detection of secondary
metabolites of leaf and bark extracts of Pajanelia longifolia (Willd.)
K. Schuman.
Plant
Part
Leaf
Bark
Alkaloid
Secondary Metabolites
Steroid Tannin Terpenoid Saponin Flavonoid
Petroleum Ether
+
-
+
+
+
+
+
+
-
-
-
-
+
-
-
-
-
+
-
+
+
Ethyl Acetate
Leaf
Bark
+
+
+
-
+
+
70% (v/v) Ethanolic Extract
Leaf
Bark
+
+
+
+
+
-
+
+
-
(+, Present; -, absent)
5.2. Thin Layer Chromatography
Plate 3 shows the thin layer chromatography (TLC) fingerprints of
various crude extracts of leaves and bark of Pajanelia longifolia. As
shown in the figure, TLC fingerprints of the ethyl acetate extracts of
leaves showed highest number of isolable metabolites in comparison
with the petroleum ether and 70% (v/v) ethanolic extracts. In case of
bark, the results of the TLC fingerprints were quite similar. The crude
fractions eluted with various combination of petroleum ether and ethyl
acetate (10:0, 9.8:0.2, 9.5:0.5; 9:1, etc.) had no significant change when
eluted with n Hexane and ethyl acetate (10:0, 9.8:0.2, 9.5:0.5; 9:1, etc.).
Plate 3. Thin Layer Chromatography (TLC) fingerprints of \arious crude extracts of leaves and hark of
Pajanclia l()n<j;ifo/i(i (Willd.) K. Schuman.
(A) shows ihfc' TLC" ringerprim of Ethyl acetate exlracl ofleuNes. TLC was done o\er Silica gel G TLC plates eluted with
petroleum etlior and elliyl aeelate (9:1); (13) shows ilie TLC linger print oI'L'lliyl acetate extract of leaves eluled with //liexane
and ethyl acetate (9.8:0.2).
(C-D) shows the TLC linger prints of 70% (\/v) ethanolic and petroleum ether exlracl ofleaves. TLC was done o\er Silica gel
G TLC plates eluled with peiroeium ether and ethyl acetate.
(E - 11) shows the TLC fnigerprinls ofpetroleum ether, ethyl acetate, 70% (\/\) ethanolic and 100% ethanolic extract olThe bark.
TLC was done over Silica ge! G TLC plates eluted with petroleum ether and eihyl acetate.
32
The extracts were then further subjected to gas chromatography for
identification of number of isolable fractions.
5.2. Gas Chromatography of Crude Extracts
The gas chromatography of analysis of petroleum ether extract of leaves
of Pajanelia longifolia showed the presence of various isolable fractions
(Figure 5.1)
100-1
3.25
5.25
7.25
9.25
11.25
13.25
15.25
Fig. 5.1. Gas chromatogram of petroleum ether extract of leaves of
Pajanelia longifolia showing ten major peaks at different retention time.
The gas chromatogram analysis showed the presence of various
fractions in the petroleum ether crude leaf extract of Pajanelia longifolia.
Ten major peaks were observed at retention time of 8.98, 10.40, 11.74,
12.44, 13.01, 13.55, 14.21, 14.59, 15.35 and 15.99 min. However, the
analysis of Mass library against these peaks did not reveal any
important isolable metabolite to be present.
As shown in Figure 5.2, the ethyl acetate extract of Pajanelia longifolia
showed eleven major peaks in the chromatogram at retention time of
33
7.43, 8.40, 8.93, 9.11, 9.95,10.36,11.00,11.38,11.70,12.99 and 14.22 min.
The 70% (v/v) ethanolic extract of the leaves showed six major peaks at
retention time of 9.95, 10.23, 10.36, 10.49, 10.58 and 10.65 min (Figure
5.3).
S H U V A S I S H LEAF 3
Scan EI+
TIC
1.35eS
100-1
8.93
57
11.70
57
%
9.11 9 86
67
57
^•^3
57
8.40
43
11 00 11.38
8 3 , 67
1422
57
12.99
32
Time
3.25
7.25
5.25
9.25
11.25
13.25
15.25
Fig. 5.2. Gas chromatogram of ethyl acetate extract of leaves Pajanelia
longifolia showing eleven major peaks at different retention time.
scan ti+
TIC
1.35e5
10.36;57
h
lOOn
I 1
1 i
%-
1
9.95
57
- _ / \
0
,
1 1 .
1 , 1 1 1 , 1 1 , 1 1 1 1
9.94
I
10.23
55
— , - , - , .
10.04
. . .
, .
.
.
10.14
1
. .
. . .
10.24
JL
10.34
10.44
10.49
57
10.58
57
10.66
138
,,,,,,
10.54
10.64
_..., Time
10.74
Fig. 5.3. Gas chromatogram of 70% ethanolic extract of leaves of
Pajanelia longifolia showing eleven major peaks at different
retention time.
As shown in Figure 5.4, the ethyl acetate extract of bark of Pajanelia
longifolia subjected to gas chromatography showed the presence of six
major peaks at retention time of 10.39, 11.72, 13.00, 13.01, 14.22 and
34
15.39 min. The similarity of the retention time of two fractions at 13.00
and 13.01 might be due to chemical and structural similarity of the two
components.
, l-MAY-2008 + 00^822
Stiuvasish E1 KR+Sm (SG, 4)6): Sb (2.89.00); KR+Sm (SG, 4)6)
Scan EH
TC
2.12e7
1300:32
lOOi
2.65
32
11.72
32
15,38
32
14.22
32
ia3s
32
0 '' 111 ^H111111111111 iw7»»»»7TWTT^TTT7^^TiTwwTTTWT^TWTTTW7wTTri^Ti) I PI I ["i I'l iT»»n^»»T»|»w<iT"*T^*n'*^'T*^'*f'***T*'*'T*'''*T** Time
325
4 25
5.25
8.25
7.25
fl.25
9.25
10.25
11.25
12.25
1325
1425
15.26
Fig. 5.4. Gas chromatogram of ethyl acetate extract of bark of
Pajanelia longifolia showing six major peaks at different retention
time.
The gas chromatogram, of the 70% (v/v) ethanolic extract revealed
some interesting data (Figure 5.5).
Shuvasish E t O H 2
%
2.56
32
b e a n CI+
TIC
g.19e^
10.17;43
100-1
5.59
03
^ssiai
11.19
32
6.43
32
8.16„
7.58 ^ 8 . 4 4
11.73
32
9.22
32 9.94
14.19
32
12.05
32
32
Xijjj'^—L_Xj
12.99
57
13.74
32
15.33
32
15.94
32
ILJ—
• Time
3.25
5.25
7.25
9.25
11.25
13.25
15.25
5.5. Gas chromatogram of 70% (v/v) ethanolic extract of bark of
Pajanelia longifolia showing six major peaks at different retention
time.
35
As shown above, a total of twenty one different peaks were observed in
the gas chromatogram. The peaks were observed at retention time 3.50,
3.80, 4.35, 4.80, 4.86, 5.59, 6.43, 7.58, 8.16, 8.44, 9.22, 9.94, 10.17, 11.19,
11.73, 12.05, 12.99, 13.74, 14.19, 15.33 and 15.94 min. These data
indicated that the extracts contained large number of isolable
metabolites. Basing on the data, the extracts were further subjected to
column chromatography (CC) for isolation of pure compounds.
5.3. Elucidation of structure of compounds
Four compounds (coded as Compound 1, 2, 3 and 4) were isolated from
ethyl acetate and 70% (v/v) ethanolic extract of leaves and bark. The
chemical structures of the compounds have been elucidated from
spectra obtained from Infra red spectroscopy, gas chromatography, ^H
and
13C Nuclear Magnetic Resonance Spectroscopy and Mass
Spectroscopy.
5.3.1. Elucidation of structure of Compound 1
Compound 1 was isolated as wine red semi solid compound. The
material produced a single peak in GC chromatogram.
Compound 1 exhibited molecular ion peak at m/z 354 in the electron
impact mass spectra obtained from GC-MS. The compound exhibited a
36
series of peaks at m / z 31, 43, 57, 71, 85, 99, which are characteristics of
hydrocarbon
fragment
(comparing
with
eicosane,
2,6,10,14-
tetramethylheptadecane, from the NIST Hit List). From the MS and the
elemental analysis the molecular formula of the compound was found
to be C21H38O4 and hence the compound is having 3(three) double bond
equivalents (DBE). The even fragmentations at m / z 112 and 126
(instead of 113 and 126 respectively in straight chain hydrocarbon
moiety) indicate that a ring might be there. The compound is nonaromatic as per ^HNMR and MS.
, ZA-AfR-Ziim + »l-Mi:
P11831 (11.733)
2.1«
i
I 43
^^i..)^!^^
«7
V,
' K . /'..^^,
^1^ }^., '"f ...ilSS 169 y f 190198
219 235
252
^
,
J^
Figure. 5.6. Mass Spectra of Compound 1.
The FT-IR spectrum (Figure 5.7) of the compound exhibited band at
about 2956 cm-i due to v(C-H) asymmetric stretching of methyl group.
The peak at 2916 cm-i is due to v(C-H) asymmetric stretching of
methylene moiety (>CH2). The band at 1737 cm-i is due to v(C=0) of
esteric group. The band at 1653 cm-i is attributable to v(C=C). The peak
37
at 1172 cm-^ and at about 1166 cm-i is attributable to 0-C(=0)-C
stretching of ester and
3750
PAJALI
3450
3150
C-O-C stretching of etheric group.
2850
2550
2250
1950
1650
1350
1050 900 750 600 450
I/cm
Figure. 5.7. FT-IR Spectra of Compound 1.
In this compound, the vinyl protons H3 and H4 exhibited peak at 6 5.54.
The broad peak at 6 5.12 is attributable to the protons HIO. The doublet
at 6 4.58 is due to the protons H2. The peak at 6 2.9 is attributable to HI.
The protons H6 and H7 appear as triplet at 6 2.40. The protons at H18
appear as multiplet at 6 0.98. Other protons exhibited peaks in region of
61.00-2.50 (Figure 5.8).
38
111 P - l .
CDCL3.
!_:.• '_• ' ' I ihii
\\l/
.2
8.0
7.6
7.0
e.S
S, O
2.S
2.2
4.
^rv
6.0
6.6
p-i,
\\1//
4.B
6.0
4.6
ft
4.0
ppn-
3.6
3.0
2.6
2.0
1.6
1.0
0.6
0.0
ppm
=nci.s, ( . 13 . oe.
\\i// W 1 ';/
2.1
2.0
I.B
1.B
1.7
1.6
1.B
1.«
1.3
1.2
1.1
1.0
O.B
•1
I
I'
O.B
0.7
O.B
B.S
Figure 5.8 Contd.
ppm
39
13C P - i , CDCL3, A/l7/?XiO(*.
DRW, SftI F NEKi;
ot>o<MO"^ooncMOr^i-«r«j"ri-»rsi--ivoa>fnr-
iT)
^r
•
oJLL
iHLiiiiiUi
"I
180
I
I"
170
160
160
J
•iMMMMMMMtWil
'
1
1
1
1
I
140
130
120
110
100
90
liiiilnLiiiilliil
-p..,-r-..,j«™.,—r"""-i
80
70
60
I
60
40
T
1
1
30
20
10
t
13C P-1, CDCL3, 4/12/2008, DBM, SAIF HEUU
\V/\^I(^/W/\IW,
IWW |niMli^^nlW>)M»W>iHfi»\l<Hl»>l
1^*111 tw\rn»»iAin
«iniviniM^
58
57
56
55
54
53
5?
5l
50
49
mimiw>ii^>iiii>irmiiiiii *mJ'im»,^,tJ)XX'.•.^'AJ^*'J^
IW«ii"I <*»i«Li
4fl
47
46
45
44
43
42
41
40
39
36
37
36
35
34
33
Figure 5.8 Contd.
32
—
pptn
ppm
40
13C P-1, CDCL3, 4/12/2008, DRW, SAIK NF.HU
•~iOC)
inoD.-ivDOD'^iri^Hotrio
•-tc-jcDiriir)
0^
iij^#i^iif>^i^)i>)(f|pi»(l
— I
.
144
1
1
142
140
.
p
•
138
,
. — . — I .
,
136
134
132
.
.
,
130
.
,
128
.
^
,
126
124
^
,
122
120
.
1
.
,
118 ppm
13C P-1, CDCL3, 4/12/2P08, DRW, SAIF NEHU
¥i\ii\\\mi////\m
y
W^_JJ^J^iJJlJJiLiJL~a.wJ^i^
31
30
29
20
27
26
25
2fl
23
22
21
20
19
ifWi*^
18
1?
16
15
Figure 5.8. ^H and " C NMR Spectra of Compound 1
H
13
pptr.
41
1
O
H
2
W
5
H
^
4
6
O
\
18
8
V
7
15
13
//
16
14
11
12
The important ElMS fragmentations of the compound are presented
below:
C21H38O4
+
[C21H38O4J
2^
Molecular ion
m/z = 354
\
31
1
85: 99:
v
/
/
:0-
;71 157
112
;i98
169
126
43
42
Compiling all tlie date the structure of the compound proposed is as
follows
—O
4-isopentylcyclohexyl 4-(5-methoxypent-3-enyloxy)butanoate
A generic name Longifolate was given depending upon the chemical
nature of the isolated compound.
5.3.2. Elucidation of structure of Compound!
Compound 2 was isolated as yellowish amorphous compound. The
material produced single peak in the GC chromatogram (Figure 5.9).
. lS-MAY-20081-03:26:34
SHUVAStSN BARK 1P
100
3.35
Sc*n£i*
4.25
5.29
6.25
7.25
825
9.26
1025
nJ5
12.25
13.2S
14.25
Figure 5.9. GC Chromatogram of Compound 2
1575
43
Compound 2 exhibited molecular ion peak at m/z 271.9 in the high
resolution mass spectra. The peak was associated with peaks at m/z
272.9 (18% of the molecular ion peak) indicating that it was the peak
corresponding to [M+1] isotopic radical cation. The compound
exhibited peak at m / z 270.9 corresponding to the deprotonated ion
peak. This peak in turn provides an indication of protic functional
group in the compound. As the compound does not seem to contain
nitrogen as even number of nitrogen atoms (according to Nitrogen
Rule) could not be justified in the MS fragmentation or from the ^H
NMR spectrum of the compound. The peak at m/z 254.9 was due to the
removal of OH moiety [M-17]. Thus, the molecular formula of the
compound seems to be C17H20O3 and hence the compound is having
8(eight) double bond equivalents (DBE). The absence of peak
corresponding to [M-18] indicates that the molecular structure of the
compound is such, from where H2O molecule cannot be eliminated
easily. The peaks at m / z 11 and m / z 91 ascertain that the compound
contains benzene moiety.
44
18-MAY-iO08 + a>dK:34
SKUVASrSH BARK I P 1041 (7.780)
deanEl*
1C0-,
-,—,11 ....Uil.'ii, h | l . i . f : . . . , i l .
25
35 I
45
6$
n?!'^j!,';!.
75
es
95
105
115
125
135
145
1SS
IBS
175 185
105
205
215
tii
Figure 5.10. Mass Spectra of Compound 2
The FT-IR spectrum of the compound exhibited band at about 2964 cm-^
due to v(C-H) asymmetric stretching of methyl group. The peak at 2904
cm-i is due to v(C-H) asymmetric stretching of methylene moiety
(>CH2). The band at 1683 cm-i is due to v(C=0) of conjugated carbonyl
group. The carbonyl group seems to be a conjugated ketonic one as no
band at 2830-2695 cm-^ is observed (for aldehyde). The band at 1653 cm1 is due to conjugated cyclic v(C=C). The band with moderate intensity
centered at about 759 cm-i corresponds to C-H bending in 1,3disubstituted benzene ring. The peak at 1166 cm-^ is attributable to C-OC stretching of ether.
45
Figure 5.11. FT-IR Spectra of Compound 2
The proton at H12 exhibited at 6 4.35. The peaks at 5 7.42 and 6 6.85 are
attributable to the protons H l l and HIO respectively. These peaks
appeared as doublets due to their mutual spin-spin splitting. The
doublet-of-doublet (dd) peak appearing at 6 6.28 is attributable to H3.
The splitting is due to ortho and meta spin coupling of the aromatic
protons. Due to the similar reasons, H4, H2, H5 exhibited peaks at 66.63
(dd), 67.54 (m) and 6 7.90 towards downfield respectively. The proton
H15 appeared as a singlet at 66.49. The downfield shift of this proton
seems to be due to its acidic nature (calculated 69.0), which in turn
explains the unexpected high relative intensity of the
[M-1] peak in
the mass spectrum. The protons H6 and H8 appears at 64.24 (m) and at
46
63.92(m) respectively. The methyl protons HI, H7, H9 appear in 60.92.0 (Figure 5.12)
IH. ETOH PARK, CDCL3-f MeODd4, 25.11.08, DiW, EAIF, NEhU, WATER SUPPRESSED
o ^ o ^ m t ^ i O t O t O ' ^ ^ ^
>m«nMrjrjc^T
ail
SPSS^KSSS
i
w my/^
SSSiSsHSl SS£;
sssas »aa
w w v w//
W W
riA,.d?>U
:
IsrV^TaT
-
^ O O O O
O
O
ww
^
ppn
Figure 5.12 con id
47
l-iQ E T 0 8 BARK> H«00d4-f^nSCLd . Z S / l l / 2 0 0 e »
DKH, SAIF NDHU
>eorn<sirjtvioqa>fM<ocJ'*><»o^«H"»'r-n-r'its)iO'^c*ior*-'wu>
> .
.
.
.
.
'.
.
-
-
•
•
ro 0\ <0 CH ^f *
dt r - \ 0 ig* O (
r - p * M j ' - i ' 0 D t n c s j f ^ r * * O ' » w £ > I— »0, y^ <« c j I
•
> 00 09 O Ch (
• ^ «« m <M t
Ji3S5S^SS22
IU
!l
m
It*.a
1. il
XM.c
1
f^l|,lJ,..a.
— 1 —
ISO
«0
WO
wtft
— I —
—I—
40
—I—
00
100
MWttaML
miatmnlmmm
«0
—I—
man
cn a (^ -v \p
m m a>
m at m
OD U) V r ^
«»• CSJ OCT>CO
r~ vo TT
^^1,
I
>||^<|^^^|PH|H»i^|»
HilPWWllf'W'l'P^l^<>>»(W>4p'»<^b
1
1
116 l i s
1
1
1
1
1
I
I
I
I
|... ...I |..— • J
1
1—'—I
114 113 112 111 110 10S 108 107 106 105 104 103 102 101 100
I
I
99
98
I ' '—I
97
96
Figure 5.12 contd
r
I
95
94
I
ppm
48
13C ETOH BARK. MvOOdi-* axi3
ao
as
CO
tn
CM
m
25/11/2000,
00
en
rH
eg
f^
o
n
c m
r-l O
<N eg
n n
CD
UKW, S A i t UCUD
<7N
(St
o
j<4>iwitM4p*wwii>(iiiM^uJti»w>i>w»^»^fc
—T
4M
•
1
««ft
•
!
i«»
^-^,
Me
•
1
M4
•
1
1S2
•
1
160
• ' ! • ' • '
itZ
131
«»
W#»A*wfwi|ivf*Ww#Miwf^
• • ! ' ' •
130
129
1Z8
Figure 5.12. ^H and i^c NMR Spectra of Compound 2
The important EIMS fragmentations of the compound are presented
below:
t-H
\ 0 VD
CS CJ OJ
Uvrt*
-—T
14«
^ r' 1—
oo •-H cc
P^ fO <N)
"T—
127
I
ppm
49
C17H20O3
-29
[C17H20O3J
[C,7H,903]®+
Molecular ion
m/z = 272
m/z = 271
H'
Compiling all the date the structure of the compound proposed is as
follows:
3-(4-hydroxycyclopenta-l,3-dienyloxy)-2-methyl-l-m-tolylbutan-l-one
A generic name was given to the compound as Pajanal depending upon
the chemical nature of the compound.
50
5.3.3. Elucidation of structure of Compound 3
Compound 3 was isolated as dark yellow semi solid compound. The
produced a single peak in the GC chromatogram (Figure 5.13).
s o Sfc» ( O . e e . O O >; M R - ^ S m < S O . 4 x 2 >
100 T
5 - I V O V - 2 0 0 S -•- 0 2 : 3 5 : 3 5
Scan E l *
2T-01:1<I7
TIC
1 .83o8
I
Figure 5.13. GC Chromatogram of Compound 3
Compound 3 exhibited molecular ion peak at m/z 148.1 in the high
resolution mass spectra. The peak was associated with peaks at m/z
149.1(17% of the molecular ion peak) and m/z 150.1(1.0% of the
molecular ion peak) indicating that they were corresponding to [M+1]
and [M+2] isotopic radical cation peaks. The compound exhibited peak
51
at m / z 147.1 corresponding to the [M-H] ion peak. This peak in turn
provides an indication of protic functional group in the compound.
The peak at m/z 254.9 is due to the removal of OH moiety [M-17]. From
the elemental analysis and the mass spectrum, the molecular formula of
the compound was found to be C10H12O and hence the compound is
having 5 (five) double bond equivalents (DBE). The absence of peak
corresponding to [M-18] indicates that the molecular structure of the
compound is such, from where H2O molecule cannot be eliminated. The
peaks at m / z 11 ascertain that the compound contains phenyl moiety.
52
S5 4926 (27.005) Rf (7,3.000)
147.1
148.1
100
77.0
%-i
32.0
131.1
76.0
51 .0
102.1
92.0
149.1
119.0
150.1 ^g^^ 218.4 234.9
I
15.0
I'V""!'
i UI •i " r'
65.0
MlLl
iKu
115.0
•Ji.
!''.'fM '<
165.0
215.0
265.0
Figure 5.14. Mass Spectra of Compound 3
The FT-IR spectrum of the compound exhibited band at about 2966 cm-^
due to v(C-H) asymmetric stretching of methyl group. The peak at 2904
cm-i is due to v(C-H) asymmetric stretching of methylene moiety
(>CH2). The peak at 3049 cm-i is attributable to the aromatic C-H
stretching. The band at 1699 cm-^ is due to v(C=0) of aromatic aldehyde
group. The band at 1622 cm-^ is due to ring (C=C) stretching (Figure
5.15).
53
I I 1 [ I I I I [ I I I I I I I I I I 1 I I I I I I I 1 ; 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 1 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 1 1 [ I I I I I I I ' I I I I I I I I I I I I I I I I I '
3730
3450
3150
2850
2550
2250
1950
1650
1350
1050 900 750
SS
Figure 5.15. FT-IR Spectra of Compound 3
The iRNMR spectral pattern of the compound in the downfield region
of 6 6.0 to 6 8.0 provide clue about o-substituted aromatic ring system.
The doublet at 6 IHl is attributable to the proton HI. The double-ofdoublet (dd) at 67.55 is assignable to H3 which para to the higWy
electron withdrawing aldehyde group. The protons H2 and H4 appear
as doublet at 66.46 and as dd at 67.41 respectively. H4 appeared at more
downfield compared to H2 probably due to the presence of alkyl
moiety ortho to it. The protons H5, H6, H7 appears in their usual region
of 6 2.36 (triplet), 6 1.23 and 6 0.86 (triplet) respectively (Figure 5.16).
600
450
1/cm
54
111 fl-S, CDCLS. 2G. I I . o n .
^\I/^M\^/1\^^
8.S
a.O
7.6
7.0
6.6
e.O
6.6
6.0
13C S - 5 , CDCL3 , 2 6 / 1 1 / 2 0 0 8 ,
n
o r-j ey^
«x>
I^
>
r
f a\ r^
CD CO ao
m CM CM
o
4.6
170
' •!
160
150
3.0
2.6
2.0
1.6
1.0
0.6
O.O
m —t o
m C3 r-r- r
\ ^^
«|MMMMI«l«J«MMnM«MMM^
•MiN^MMmwiiiMira
mtttmmmmmmmmm
I
3.6
DRW, S M F NEHU
\ /
••••I
4.0
I
I
I
I
140
130
120
110
I'-—
100
I
I
I
I
90
80
70
60
V"-
SO
I
40
•• • I
30
I
I
20
10
Figure 5.16. iR and i^C NMR Spectra of Compound 3
I
r
0 ppm
55
Aromatic aldehydes are often characterized by a large molecular ion
peak and by an
[M-1] peak (Ar-CO^) that is always large and may be
larger than the molecular ion peak. The presence of aldehyde is
supported by the presence of [M-1-28] peak. The presence of peak at
m/z n
is attributable to the presence of phenyl moiety in this
compound, which in turn eliminates C2H2 to give the C4H3* ion ( m / z
51). The absence of recognizable peaks due to [M-18] (loss of water) and
at m / z 29 (CHO+) further affirms that the aldehyde group is attached to
the phenyl unit.
The important EIMS fragmentations of the compound are presented
below:
^
CinHi-jO
•
r
1*+
[c,oHi20]
Molecular ion
m/z =148.1
•
r
1®
[c,oH,,0]+
m/z =147.1
•
H
56
n
•®
J
\
/
\
m/z=148.1
-H
ne
iO
\
/
\
m/z= 147.1
-CO
m/z=119.0
Compiling all the date the structure of the compound proposed is as
follows
I
\
/
H
2-propylbenzaldehyde
57
5.3.4. Elucidation of structure of Compound 4
Compound 4 was isolated a wine red semi solid material. The material
showed single peak in the GC chromatogram (Figure 5.17).
rtii«#i*»l»r2.M(iO).8Ba.«9«JirR.»n(Sa4je)
0 ' ' - | -
• > • ! I• I I I
sis
'
I
IS
Ta
ias
^'^^
?5
BJS
>M
laiiKiO
ICM
I I On
njs
«»
tjjs
wis
'««
Figure. 5.17. GC Chromatogram of Compound 4
The compound was isolated by column chromatography. The FT -IR
spectra of the compound showed characteristic bands for O-H
stretching (3365 cm-^). The peak at 3197.74 cm-i was due to the presence
of benzene ring. The peaks at 2954, 2923, 2854 cm-^ is attributed to the
methyl and methylene C-H stretching. The peak at 1747 cm-i was due to
the presence of -COO stretching of esters. The peaks at 1456 and 1379
cm-i was due to the presence of Asymmetric Binding of Methyl Group
58
and Symmetric binding of Methyl Group (and / or Metylene Group)
respectively.
.%T
itc
5000
1500
cm-1
1000
Figure. 5.18. FT-IR Spectra of Compound 4
The Mass Spectral Library studied during the study showed that the
compound has some similarity with 2-(4,5-dihydro-3-methyl-5-oxo-lphenyl-4-pyrazolyl)-5-nitrobnzoic acid. Moreover, the FT-IR spectra
also indicated the present of various functional groups similar to those
present
in
2-(4,5-dihydro-3-methyl-5-oxo-l-phenyl-4-pyrazolyl)-5-
nitrobnzoic acid with m / z 447.00.
59
SlllMJisM 37 (5£87) Cm (2: ;S6)
100,
" '
399
31,0
14.0
38,03893 3661
i^T-^
64 0
114 0
214 0
284J1
314.0
415B
447i
' ' ' • I ' ' ' ' I ' • I
' ' I ' I ' ' I
164 0
3&I.D
414 0
4641;
I '
:i4.0
Fi^;ure. 5.19. Mass Spectra of Compound 4
/CH3
o^^^^OH
N:
2-(4,5-dihydro-3-methyl-5-oxo-l-phenyl-4-pyrazolyl)-5-nitrobnzoic
acid
60
Colour:
State;
Molecular Wt:
Chemical Formula
Wine Red
Semi-solid
354
C-i.H^jO
-1
jo
4-isopentylcycIohexyl 4-(5-methoxypenl-3-enyloxy) butanoale
Colour:
State:
Molecular Wt:
Chemical Formula
Yellowish
Amorphus
271.9
C.H O ,
Colour:
State:
Molecular Wt:
Chemical Formula
Dark yellow
Semi-solid
148.1
C H O
3-(4-hydroxycyclopenta-1.3-dienyloxy)-2-methyl-1 -m-tolylbutan-1 -one
2-propylbenzaldehyde
3
0~^
^OH
2-(4,5-dihydro-3-inethyl-5-oxo-l-phcnyl-4-pyrazolyl)-5-nitrobn7.oic acid
Colour:
State:
Molecular Wt:
Wine red
Semi soild
447.00
61
In silico Analysis of Isolated Compounds
6.1. Molecular properties and drug - likeliness of isolated compounds
The molecular properties and dug likeliness of the isolated compounds
were studied with online server http://www.molsoft.org. As shown in
Plate 4, compound 1 showed highest drug likeliness score as compared
to compound 2 and compound 4, which indicate that compound 1 is a
good drug candidate.
6.2. Pharmacophore modeling of isolated compounds
The pharmacophore mapping of the isolated compounds are shown in
Plate 5-7. In accordance with the Lipinski's Rule, which states that, in
general, an orally active drug has no more than one violation of the
following criteria:
(a) Not more than five hydrogen bond donors (nitrogen or oxygen
atom with one or more hydrogen atom).
(b) Not more than ten hydrogen bond acceptor (nitrogen or oxygen
atom).
(c) Molecular weight under 500 Dalton
(d) An octanol-water partition coefficient log P of less than 5.
62
Considering the Lipinski's Rule, Compound 1 has four hydrogen bond
acceptor (oxygen atoms) and hydrophobic in nature. The compound
showed octanol - water coefficient (Log P) of 4.33 and molecular weight
of 354 Daltonji. Compound 2 has three hydrogen bond acceptors
(oxygen atoms) and one hydrogen bond donor (OH atom). The
compound showed hydrophobicity with Log P value of 4.61 and
molecular weight of 271.9 Daltons. Compound 4 on the other hand
showed good drug character. The compound is hydrophobic with six
hydrogen bond acceptor (oxygen and nitrogen atoms), two aromatic
rings and positive and negative ionization. The compound showed Log
P of 1.98. This indicates that the isolated compounds bear characters to
be a orally active drug. The data satisfies Lipinski's Rule, which is
prerequisite for a compound to be drug.
The compound 1 and Compound 4 were than docked with two
different receptors viz., RNA-dependent RNA polymerase genotype 2a
of hepatitis C virus and DNA polymerases from £. coli (Plate 8 -9) The
analysis showed no positive interactions of the compounds with these
receptors. This indicates that, though the isolated compounds have
good drug - likeliness properties, proper receptor could not be found
where it can bound to inhibit its function.
Drugs
Non-drugs
Your compound
Compound 1
Molecular Properties and Drug-likeness
Number of HBA: 4
Number of 11BD:0
MolLogP: 4.33
MolLogS : -4.17 (in Log(moles/L)) 23.86 (in mg/L)
MolPSA: 38.07 A2
MoiVol: 408.67 A3
Number of stereo centers; 0
-6.00
6.00
-4.00
Drugs
Non-drugs
Your compound
Compound 2
Molecular Properties and Drug-likeness
NumberotlIBA;2
Number of HBD: 1
MolLogP;4.61
MolLogS : -3.65 (in Log(moles/L)) 62.07 (in mg/L)
MolPSA: 23.88 A2
MolVoJ;303.19A3
Number of stereo centers: 4
-2.00
-6.00
Drugs
Non-drugs
Your compound
Compound 4
Molecular Properties and Drug-likeness
Number of HBA: 6
Number of 11BD:0
MolLogP: 1.98
MolLogS : -4.42 (in Log(moles/L)) 12.89 (in mg/L)
MolPSA: 90.76 A2
MolVol:315.18A3
Number of stereo centers: 1
-6.00
-4.00
-2.00
4.00
6.00
Plate 4. Molecular properties and drug-likeliness of Compound 1, 2 and 4
HBA
HBA
HBA
HBA
H
Plate 5. Pharmacophore mapping of Compound 1. The red and yellow contours represents hydrogen bond
acceptors (HBD) and hydrophobic feature. (HBA: Hydrogen Bond Donor; H: Hydrophobic)
iHBAI
H
i^El
Plate 6. Pharmacophore mapping of Compound 2. The red, green, blue and yellow contours represents
hydrogen bond acceptors (HBA), hydrogen bond acceptor (HBA), aromatic ring and hydrophobic feature.
(HBD: Hydrogen Bond Donor; HBA: Hydrogen bond acceptor; AR: Aromatic ring, H: Hydrophobic)
Plate 7. Pharmacophore mapping of Compound 4. The red, green, blue and yellow contours represents
hydrogen bond acceptors (HBA), hydrogen bond acceptor (HBA), aromatic ring and hydrophobic feature.
(HBD: Hydrogen Bond Donor; HBA: Hydrogen bond acceptor; AR: Aromatic ring, H: Hydrophobic; PI:
Positive Ionization; NI: Negative Ionization)
Plate 8. Structure of replicative and translesion DNA polymerases of E. coli. (B). Three
dimensional structure of 4-isopentyicyclohexyl-4-(5-methoxypent-3-enyloxy) butanoate isolated
from crude leaf extracts extracted with ethyl acetate oiPajanelia longifolia (Willd.) K. Schuman
Plate 9. Docking results, showing negative binding affinity of of 4-isopentylcyclohexyl-4-(5methoxypent-3-enyloxy) butanoate with replicative and translesion DNA polymerases of E.
coli. . The result was generated using AutoDock 4.0.
Plate 10. (A). Crystal Structure of RNA-dependent RNA polymerase genotype 2a of
hepatitis C virus expressed in E. coli. (B). Three dimensional structure of 2-(4,5-dihydro3-methyl-5-oxo-l-phenyl-4-pyrazolyl)-5-nitrobnzoic acid isolated from crude bark extract
Pajanelia longifolia (Willd.) K. Schuman, extracted with ethyl acetate.
Plate 11. Docking results, showing negative binding affinity of 2-(4,5-dihydro-3-methyl-5-oxo-l-phenyl4-pyrazoiyl)-5-nitrobnzoic acid with RNA-dependent RNA polymerase genotype 2a of hepatitis C virus.
The result was generated using AutoDock 4.0.