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RESULTS
Results
58
4. RESULTS
4.1 Antianxiety activity of various extracts of A. farinosa, C. behen, E. ganitrus
and G. superba
The four extracts viz. petroleum ether, chloroform, ethanol and water, of
A. farinosa, C. behen, E. ganitrus and G. superba were screened for antianxiety activity
in mice using EPM apparatus at various dose levels – 50, 100, 200, 400 or 800 mg/kg p.o.
The mean number of entries and time spent by the mice in open arms are shown in tables
5-8 and figures 5-8, respectively.
Table 5: Results of antianxiety activity evaluation of various extracts of A. farinosa
roots using EPM apparatus
Treatment Dose (mg/kg) Meann
number of entries ± S.E.M.
Meann time
b
(sec) ± S.E.M.
Control Vehicle 4.6 ± 0.58a 3.39 ± 0.42
a
Diazepam 2.0 19.6 ± 0.81* 14.16 ± 0.40*
Petroleum ether extract
50
100
200
400
800
4.0 ± 0.45a
4.8 ± 0.37a
5.4 ± 0.51a
4.6 ± 0.40a
5.8 ± 0.37a
2.87 ± 0.44a
3.69 ± 0.35a
4.00 ± 0.38a
3.72 ± 0.47a
4.09 ± 0.44a
Chloroform extract
50
100
200
400
800
4.8 ± 0.51a
5.2 ± 0.45a
6.4 ± 0.25a
5.8 ± 0.58a
6.2 ± 0.20a
3.43 ± 0.42a
3.99 ± 0.50a
4.47 ± 0.57a
4.36 ± 0.54a
4.47 ± 0.32a
Ethanol extract
50
100
200
400
800
4.2 ± 0.32a
6.2 ± 0.45a
5.8 ± 0.66a
6.8 ± 0.58a
6.8 ± 0.86a
2.43 ± 0.37a
4.22 ± 0.39a
4.13 ± 0.55a
4.82 ± 0.32a
4.80 ± 0.59a
Water extract
50
100
200
400
800
4.8 ± 0.37a
5.2 ± 0.24a
6.0 ± 0.51a
4.8 ± 0.60a
6.8 ± 0.37a
3.48 ± 0.41a
3.56 ± 0.19a
4.08 ± 0.54a
3.59 ± 0.25a
5.40 ± 0.13a
n = 5; b = Average time each animal spent in open arms = total duration in open arms/number of entries in
open arms; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way ANOVA followed by Studentized Tukey’s
test.
Results
59
A
B
Figure 5: Relative anxiolytic activity profile of A: mean number of entries B: mean time (sec) spent in
open arms by mice treated with various extracts of A. farinosa roots using EPM apparatus. The
data is expressed as mean ± S.E.M.; n = 5; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way
ANOVA followed by Studentized Tukey’s test.
Results
60
Table 6: Results of antianxiety activity evaluation of various extracts of C. behen
roots using EPM apparatus.
Treatment Dose
(mg/kg)
Meann
number of
entries ± S.E.M.
Meann time
b
(sec) ± S.E.M.
Control Vehicle 4.8 ± 0.24a 3.56 ± 0.19
a
Diazepam 2.0 17.2 ± 0.58* 12.15 ± 0.60*
Petroleum ether extract
50
100
200
400
800
4.2 ± 0.37a
4.0 ± 0.31a
4.2 ± 0.24a
4.4 ± 0.24a
4.6 ± 0.37a
3.16 ± 0.28a
2.82 ± 0.11a
2.93 ± 0.17a
3.56 ± 0.19a
3.18 ± 0.28a
Chloroform extract
50
100
200
400
800
4.6 ± 0.37a
4.8 ± 0.37a
4.8 ± 0.31a
4.6 ± 0.37a
4.4 ± 0.40a
3.20 ± 0.28a
3.24 ± 0.25a
3.53 ± 0.17a
3.48 ± 0.25a
3.46 ± 0.27a
Ethanol extract
50
100
200
400
800
9.6 ± 0.51a*
10.4 ± 0.40*
11.6 ± 0.40a*
12.8 ± 0.18*
10.0 ± 0.31a*
7.01 ± 0.61a*
8.90 ± 0.14a*
9.12 ± 0.14*
10.3 ± 0.10*
8.03 ± 0.11a*
Water extract
50
100
200
400
800
4.0 ± 0.31a
5.4 ± 0.24a
5.2 ± 0.20a
5.0 ± 0.31a
4.8 ± 0.37a
2.83 ± 0.24a
4.1 ± 0.16a
3.9 ± 0.12a
3.6 ± 0.42a
3.1 ± 0.33a
n = 5; b = Average time each animal spent in open arms = total duration in open arms/number of entries in
open arms; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way ANOVA followed by Studentized Tukey’s
test.
Results
61
A
B
Figure 6: Relative anxiolytic activity profile of A: mean number of entries B: mean time (sec) spent in
open arms by mice treated with various extracts of C. behen roots using EPM apparatus. The
data is expressed as mean ± S.E.M.; n = 5; *P<0.05 vs. control; aP<0.05 vs. diazepam; one
way ANOVA followed by Studentized Tukey’s test.
Results
62
Table 7: Results of antianxiety activity evaluation of various extracts of
E. ganitrus beads using EPM apparatus.
Treatment Dose
(mg/kg)
Meann
number of
entries ± S.E.M.
Meann time
b
(sec) ± S.E.M.
Control Vehicle 4.6 ± 0.24a 3.43 ± 0.16
a
Diazepam 2.0 18.8 ± 0.37* 12.97 ± 0.28*
Petroleum ether extract
50
100
200
400
800
4.0 ± 0.31a
3.6 ± 0.20a
3.8 ± 0.37a
4.2 ± 0.37a
4.0 ± 0.58a
3.36± 0.32a
2.82 ± 0.11a
2.97 ± 0.15a
3.26 ± 0.21a
3.21 ± 0.28a
Chloroform extract
50
100
200
400
800
8.4 ± 0.22a*
8.8 ± 0.28a*
9.6 ± 0.34a*
12.0 ± 0.51*
10.0 ± 0.54a*
6.2 ± 0.48a
6.7 ± 0.28a
7.4 ± 0.10a*
9.95 ± 0.11a*
8.02 ± 0.17a*
Ethanol extract
50
100
200
400
800
11.2 ± 0.53a*
12.0 ± 0.34*
17.4 ± 0.49*
16.6 ± 0.81*
10.2 ± 0.31a*
8.8 ± 0.60*
9.7 ± 0.59*
12.08 ± 0.12*
11.87 ± 0.56*
8.0 ± 0.11a*
Water extract
50
100
200
400
800
4.2 ± 0.24a
4.0 ± 0.37a
4.6 ± 0.24a
5.0 ± 0.31a
5.6 ± 0.37a
3.23 ± 0.14a
3.16 ± 0.18a
3.66 ± 0.14a
3.99 ± 0.10a
4.13 ± 0.11a
n = 5; b = Average time each animal spent in open arms = total duration in open arms/number of entries in
open arms; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way ANOVA followed by Studentized Tukey’s
test.
Results
63
A
B
Figure 7: Relative anxiolytic activity profile of A: mean number of entries B: mean time (sec) spent
in open arms by mice treated with various extracts of E. ganitrus beads using EPM
apparatus. The data is expressed as mean ± S.E.M.; n = 5; *P<0.05 vs. control; aP<0.05 vs.
diazepam; one way ANOVA followed by Studentized Tukey’s test.
Results
64
Table 8: Results of antianxiety activity evaluation of various extracts of
G. superba roots using EPM apparatus.
Treatment Dose
(mg/kg)
Meann
number of
entries ± S.E.M.
Meann time
b
(sec) ± S.E.M.
Control Vehicle 4.4 ± 0.51a 3.18 ± 0.22
a
Diazepam 2.0 18.0 ± 0.58* 12.33 ± 0.88*
Petroleum ether extract
50
100
200
400
800
3.8 ± 0.49a
4.0 ± 0.32a
4.0 ± 0.51a
4.2 ± 0.58a
3.8 ± 0.60a
2.28 ± 0.58a
3.25 ± 0.22a
3.06 ± 0.26a
3.55 ± 0.13a
2.59 ± 0.25a
Chloroform extract
50
100
200
400
800
4.0 ± 0.32a
4.2 ± 0.37a
4.4 ± 0.49a
4.2 ± 0.37a
4.6 ± 0.63a
2.80 ± 0.21a
3.12 ± 0.29a
3.40 ± 0.13a
3.36 ± 0.17a
3.53 ± 0.28a
Ethanol extract
50
100
200
400
800
4.0 ± 0.45a
4.0 ± 0.45a
4.4 ± 0.37a
4.6 ± 0.45a
4.4 ± 0.49a
3.00 ± 0.21a
2.92 ± 0.17a
3.44 ± 0.19a
3.62 ± 0.25a
3.43 ± 0.39a
Water extract
50
100
200
400
800
4.0 ± 0.40a
4.2 ± 0.40a
4.2 ± 0.45a
4.6 ± 0.58a
4.2 ± 0.51a
3.00 ± 0.19a
3.28 ± 0.29a
3.24 ± 0.40a
3.35 ± 0.33a
3.87 ± 0.18a
n = 5; b = Average time each animal spent in open arms = total duration in open arms/number of entries in
open arms; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way ANOVA followed by Studentized Tukey’s
test.
Results
65
A
B
Figure 8: Relative anxiolytic activity profile of A: mean number of entries B: mean time (sec) spent in
open arms by mice treated with various extracts of G. superba roots using EPM apparatus. The
data is expressed as mean ± S.E.M.; n = 5; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way
ANOVA followed by Studentized Tukey’s test.
Results
66
4.2 Acute toxicity study of ethanol extract of E. ganitrus beads
In acute toxicity study, there was no change in motor activity and gross behavior
of mice during 24 h of observation, and the plant extract was found to be safe up to 5
g/kg body weight, p.o.
4.3 Microscopic studies of E. ganitrus beads
4.3.1 Study of sections
Plate 9 shows representative photomicrographs of transverse section of E.
ganitrus bead. Transverse section of the seed is shown in plate 10. Longitudinal section
of the seeds is shown in plate 11. Cell inclusions are shown in plates 12 and 13.
A
B
Plate 9: Representative photomicrographs of T.S. of E. ganitrus beads showing A: seeds (S) and stony
endocarp (SE). B: T.S. outer portion enlarged, showing epidermis (Ep), parenchymatous
mesocarp (PMCc), sclereids (SC) and tanniniferous cells (TC).
Results
67
A
B
Plate 10: Representative photomicrographs of T.S. of E. ganitrus seeds. A: a section of upper
portion. B: a section of the seed through middle part. (En- endosperm; Fu- funicle;
ISC- inner seed coat; Osc- outer seed coat; SC- seed coat; Scl- sclereids).
Plate 11: Representative photomicrographs of L.S. of E. ganitrus seed showing endosperm (En),
funicle (Fu) and seed coat (SC)
Results
68
Plate 12: Representative photomicrographs of seed coat of E. ganitrus seeds showing crystals (Cr)
and sclereids (Scl).
Plate 13: Representative photomicrographs showing calcium oxalate clusters (Cr) in the
endosperm cells (Enc) of E. ganitrus seeds.
Results
69
4.3.2 Powder microscopy
Representative photomicrographs of powdered E. ganitrus beads are shown in
plates 14 (A - C).
A
B
C
Plate 14: Representative photomicrographs of E. ganitrus beads A: showing parenchymatous mesocarp
(Pa) and sclereids (ScI). B: a single mass of sclereid (SCM). C: calcium oxalate crystals (Cr)
under polarized light.
Results
70
4.4 Results of determination of various physico-chemical parameters of E. ganitrus beads
Table 9 shows results of various physico-chemical parameters viz. moisture
content, ash values and extractive values of E. ganitrus beads. Results of toxic residues,
heavy metals, pesticide residues and microbial determinations are shown in tables 10 and
11.
Table 9: Mean values of various physico-chemical parameters of E. ganitrus beads.
Parameter Meann value (%)
Moisture content 9.70
Total ash* 4.55
Acid insoluble ash* 1.50
Water soluble ash* 0.96
Pet ether soluble extractive value* 1.32
Ethanol soluble extractive values* 20.44
Water Soluble extractive value* 18.10
n=3 *dry weight basis
Table 10: Aflatoxins, heavy metals, arsenic and pesticide residues content in
E. ganitrus beads.
Parameters Observation
Limits (As prescibed by WHO and IHS)
Aflatoxins B1 B2 G1 G2 Total
Not detected Not detected Not detected Not detected Not detected
≤5 μg/kg
Should be absent
Should be absent
Should be absent
20μg/kg
Heavy metals Lead Mercury Cadmium Arsenic
Complies Complies Complies Complies
10 ppm
0.2 ppm
0.3 ppm
5 ppm
Pesticides Aldrin, azinphos-methyl, chlordane, cypermethrin, DDT, Endrin, heptachlor, lindane, melathion, parathion, pyrethrins
Not detected
0.5, 1.0, 0.05, 1.0, 1.0, 0.05, 0.05, 0.6, 1.0, 0.5, 3.0, respectively.
Results
71
Table 11: Microbial content in E. ganitrus beads.
4.5 Phytochemical screening of various extracts of E. ganitrus beads
Table 12 shows the yield of various extracts of E. ganitrus beads. Results of
phytochemical screening of the extracts are reported in table 13.
Table 12: Yield of various extracts of E. ganitrus beads
Extract Yield (% w/w) Colour
Petroleum ether
Chloroform
Ethanol
Water
1.65
4.58
15.38
10.13
Cream
Brownish black
Reddish brown
Dark brown
Microbes Observation Limit (As prescribed by
WHO and IHS)
Total bacterial count 246 cfu/10g NMT 1000 cfu/g
Total fungal count Nil cfu/10g NMT 100 cfu/g
Salmonella Absent Should be absent
Escherichia coli Absent Should be absent
Pseudomonas auroginosa Absent Should be absent
Staphylococcus aureus Absent Should be absent
Clostridium botulinum Absent Should be absent
Clostridium perfringens Absent Should be absent
Clostridium tetani Absent Should be absent
Salmonella typhimurium Absent Should be absent
Results
72
Table 13: Results of phytochemical evaluation of various extracts of E. ganitrus
beads.
Class of
phytoconstituents
Petroleum
ether extract
Chloroform
extract
Ethanol
extract
Water
extract
Alkaloids - + + -
Anthraquinone
glycosides
- - - -
Cyanogenic glycosides - - - -
Cardiac glycosides - - - -
Steroids/Triterpenoids +/- -/- -/- -/-
Saponins - - - +
Flavonoids - - ++ -
Coumarins - - - -
Tannins - - ++ +
Carbohydrates - - + +
Proteins - - + +
+: present, - : absent
4.6 TLC fingerprint profiles of various extracts of E. ganitrus beads
Various mobile phases which were employed to develop thin layer
chromatograms of petroleum ether, chloroform and methanol extracts are summarized in
tables 14-16, respectively. Results of thin layer chromatography have been shown in table
17.
Table 14: Mobile phases employed for TLC of petroleum ether extract of E. ganitrus
beads.
S. No. Mobile Phase Proportions
1 Hexane : Chloroform 19 : 1
2 Hexane : Chloroform 9 : 1
3 Hexane : Chloroform 17 : 3
4 Hexane : Chloroform 4 : 1
5 Petroleum ether : Toluene 9 : 1
6 Petroleum ether : Chloroform 9 : 1
7 Petroleum ether : Chloroform 4 : 1
8 Petroleum ether : Chloroform 3 : 2
9 Petroleum ether : Chloroform 1 : 1
10 Hexane : Dichloromethane 9 : 1
11 Hexane : Dichloromethane 4 : 1
12 Hexane : Dichloromethane 3 : 2
13* Hexane : Dichloromethane 1 : 1
*optimum resolution
Results
73
Table 15: Mobile phases employed for TLC of chloroform extract of E. ganitrus
beads.
S. No. Mobile Phase Proportions
1 Chloroform: Methanol 19 : 1
2 Chloroform: Methanol 9 : 1
3 Chloroform: Methanol 17 : 3
4 Toluene: Ethyl acetate 9 : 1
5 Toluene: Ethyl acetate 17 : 3
6 Toluene: Ethyl acetate : Acetone 8 : 1 : 1
7 Toluene: Ethyl acetate : Acetone 7 : 2 : 1
8 Toluene: Dichloromethane : Methanol 5 : 4 : 1
9 Chloroform: Dichloromethane : Ethyl acetate 5 : 3 : 2
10 Toluene: Ethyl acetate : Glacial acetic acid 17 : 2 : 1
11 Toluene: Glacial acetic acid 19 : 1
12* Toluene: Ethyl acetate : Glacial acetic acid 18 : 1 : 1
*optimum resolution
Table 16: Mobile phases employed for TLC of ethanol extract of E. ganitrus beads.
S. No. Mobile Phase Proportions
1 Chloroform : Methanol 9 : 1
2 Chloroform : Methanol 17 : 3
3 Toluene : Ethyl acetate : Glacial acetic acid 17 : 2 : 1
4 Toluene : Ethyl acetate : Glacial acetic acid 7 : 2 : 1
5 Toluene : Ethyl acetate : Glacial acetic acid 5 : 3 : 2
6 Ethyl acetate : Butanol : Glacial acetic acid 4 : 5 : 1
7 Ethyl acetate : Butanol : Glacial acetic acid 3 : 6 : 1
8 Ethyl acetate : Butanol : Glacial acetic acid 2 : 7 : 1
9 Dimethyl ketone : Butanol : Glacial acetic acid 5 : 4 : 1
10 Dimethyl ketone : Butanol : Glacial acetic acid 4 : 5 : 1
11 Dimethyl ketone : Butanol : Glacial acetic acid 3 : 6 : 1
12 Dimethyl ketone : Butanol : Glacial acetic acid 2 : 7 : 1
13 Propanol : Water 4 : 1
14 Propanol :Water 3 : 2
15**
Butane-2-one : Ethyl acetate : Glacial acetic acid 4 : 5 : 1
*optimum resolution
Results
74
A B C
Plate 15: Representative photographs of thin layer chromatograms of A: petroleum ether
(mobile phase hexane:dichloromethane 1:1) B: chloroform (mobile phase
toluene:ethylacetate:glacial acetic acid 18:1:1) and C: ethanol extract of E. ganitrus
beads (But-2-one:Ethyl acetate:Glacial acetic acid (4:5:1). Spots were visualized by
spraying with 0.5% anisaldehyde followed by heating for 2 min at 105ºC.
Table 17: Results of thin layer chromatography of various extracts of E. ganitrus
beads.
Extract Mobile phase Number of spots*
Petroleum ether Hexane : Dichloromethane
(1:1)
Four spots
Rf
values – 0.06, 0.17, 0.26
and 0.30
Chloroform Toluene : Ethylacetate :
Glacial acetic acid
(18:1:1)
Eight spots
Rf values – 0.09, 0.19, 0.25,
0.33, 0.54, 0.60, 0.78 and 0.88
Ethanol But-2-one : Ethyl acetate :
Glacial acetic acid
(4:5:1)
Eleven spots
Rf values – 0.09, 0.13, 0.19,
0.25, 0.31, 0.40, 0.48, 0.52,
0.69, 0.83 and 0.96
*Spots were visualized by spraying with 0.5% anisaldehyde followed by heating for 2 min at 105ºC.
Results
75
4.7 Antianxiety activity-guided fractionation of ethanol extract of E. ganitrus
beads
Results of column chromatography of ethanol extract are shown in table 18. A
total of eight fractions (F1-F8) were obtained. Mean number of entries and the mean time
spent by the mice in open arms after oral administration of various fractions (F1-F8) of
ethanol extract, have been shown in table 19. Column chromatography of fraction F5
yielded seven subfractions (F5.1-F5.7, table 20). Mean number of entries and the mean time
spent by the mice in open arms after oral administration of various sub-fractions (F5.1-
F5.7) have been shown in table 21. Repeated preparative TLC of F5.4 yielded two pure
isolates BS1 (88 mg) and BS2 (16.0 mg). Anxiolytic activity of BS1 and BS2, isolated
from F5.4, has been shown in table 22.
Table 18: Fractionation of bioactive ethanol extract of E. ganitrus beads using
column chromatography
Fraction Eluant Yield (g)
F1 CHCl3 10.85
F2 CHCl3
CHCl3 + MeOH (49 : 1)
CHCl3 + MeOH (19 : 1)
6.20
F3 CHCl3 + MeOH (9 : 1) 18.50
F4 CHCl3 + MeOH (4 : 1) 26.65
F5 CHCl3 + MeOH (4 : 1)
CHCl3 + MeOH (1 : 1)
135.85
F6 CHCl3 + MeOH (1 : 1) 49.50
F7 MeOH 53.30
F8 MeOH + Acetonitrile (1 : 1) 31.20
Results
76
Table 19: Results of antianxiety activity evaluation of various fractions obtained
from ethanol extract of E. ganitrus beads using EPM apparatus.
Treatment Dose (mg/kg) Meann
number of entries ± S.E.M.
Meann time
b (sec)
± S.E.M.
Control Vehicle 4.6 ± 0.51a 3.18 ± 0.22
a
Diazepam 2.0 17.2 ± 0.58* 12.25 ± 0.60*
F1
25
50
75
100
4.0±0.24a
4.2±0.21a
4.3±0.12a
4.2±0.22a
2.42±0.27a
2.80±0.12a
3.06±0.12a
3.06±0.27a
F2
25
50
75
100
4.8±0.27a
4.5±0.37a
4.3±0.22a
4.2±0.27a
3.36±0.27a
3.23±0.35a
3.13±0.25a
3.06±0.12a
F3
25
50
75
100
4.2±0.20a
4.5±0.19a
4.2±0.24a
4.4±0.18a
2.99±0.22a
3.39±0.19a
3.03±0.41a
3.19±0.22a
F4
25
50
75
100
4.4±0.20a
4.2±0.17a
3.9±0.24a
4.4±0.23a
3.53±0.16a
3.06±0.17a
2.79±0.20a
3.19±0.20a
F5
25
50
75
100
8.4±0.24a*
9.9±0.20a*
16.4±0.31*
6.80±0.37a
5.27±0.08a*
6.29±0.10a*
11.23±0.03*
4.33±0.11a
F6
25
50
75
100
4.2±0.24a
4.1±0.31a
3.8±0.20a
4.4±0.24a
3.29±0.13a
3.59±0.11a
3.13±0.08a
3.94±0.11a
F7
25
50
75
100
4.2±0.22a
4.0±0.24a
3.8±0.37a
3.9±0.18a
3.43±0.41a
3.20±0.12a
3.01±0.18a
3.13±0.16a
F8
25
50
75
100
3.9±0.30a
4.0±0.32a
4.2±0.31a
4.0±0.29a
3.15±0.15a
3.26±0.11a
3.36±0.11a
3.24±0.16a
n = 5; b = Average time each animal spent in open arms = total duration in open arms/number of entries in
open arms; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way ANOVA followed by Studentized Tukey’s
test.
Results
77
Table 20: Fractionation of F5 using column chromatography
Fraction Eluant Yield (g)
F5.1 CHCl3 5.90
F5.2 CHCl3 CHCl3 + MeOH (49 : 1)
2.59
F5.3 CHCl3 + MeOH (49 : 1) CHCl3 + MeOH (19 : 1)
9.88
F5.4 CHCl3 + MeOH (19 : 1) CHCl3 + MeOH (9 : 1)
33.84
F5.5 CHCl3 + MeOH (9 : 1) CHCl3 + MeOH (17 : 3)
9.55
F5.6 CHCl3 + MeOH (17 : 3) CHCl3 + MeOH (4 : 1)
10.25
F5.7 CHCl3 + MeOH (4 : 1) CHCl3 + MeOH (1 : 1)
19.20
Table 21: Results of antianxiety activity evaluation of various sub-fractions obtained from F5 using EPM apparatus.
Treatment Dose (mg/kg)
Meann
number of entries ± S.E.M.
Meann time
b
(sec) ± S.E.M.
Control Vehicle 4.8 ± 0.51a 3.20 ± 0.22
a
Diazepam 2.0 16.9 ± 0.48* 12.15 ± 0.30*
F5.1
10 20 30
4.2±0.12a
4.4±0.20a
4.8±0.21a
2.76±0.17a
2.76±0.16a
3.26±0.12a
F5.2
10 20 30
4.2±0.20a
5.0±0.24a
4.8±0.22a
2.60±0.17a
3.40±0.18a
3.19±0.13a
F5.3
10 20 30
5.0±0.31a
5.2±0.20a
5.0±0.44a
3.49±0.16a
3.38±0.12a
3.41±0.05a
F5.4
10 20 30
8.4±0.24a*
9.0±0.31a*
16.4±0.37*
5.32±0.05a*
6.27±0.10a*
11.87±0.10*
F5.5
10 20 30
4.8±0.24a
4.8±0.20a
4.2±0.37a
3.16±0.19a
3.36±0.09a
2.99±0.23a
F5.6
10 20 30
4.6±0.24a
4.4±0.40a
4.6±0.39a
3.29±0.13a
3.26±0.12a
3.26±0.12a
F5.7
10 20 30
4.4±0.24a
4.6±0.20a
4.4±0.40a
3.03±0.13a
3.30±0.12a
3.16±0.10a
n = 5; b = Average time each animal spent in open arms = total duration in open arms/number of entries in
open arms; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way ANOVA followed by Studentized Tukey’s
test.
Results
78
Table 22: Results of antianxiety activity evaluation of BS1 and BS2 isolated from
F5.4 using EPM apparatus.
Treatment Dose
(mg/kg)
Meann
number of
entries ± S.E.M.
Meann time
b
(sec.) ± S.E.M.
Control Vehicle 5.0±0.40a 3.33±0.29
a
Diazepam 2.0 18.6±0.93* 12.13±0.66*
BS1
10
15
20
25
5.2±0.24a
6.0±0.31a
8.0±0.31a*
17.0±0.40*
3.41±0.07a
4.98±0.25a
5.84±0.23a*
12.07±0.06*
BS2
10
15
20
25
5.2±0.31a
5.4±0.20a
5.4±0.24a
5.8±0.20a
3.41±0.07a
3.36±0.11a
3.29±0.13a
3.56±0.06a
n = 5; b = Average time each animal spent in open arms = total duration in open arms/number of entries in
open arms; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way ANOVA followed by Studentized Tukey’s
test.
4.8 Characterization of bioactive constituent of E. ganitrus beads
The bioactive constituent BS1 was subjected to FT-IR, 1H NMR,
13C NMR and
mass spectroscopy (Figure 9). Results of characterization of BS1 are presented below.
BS1
Melting point: 311 – 315°C
UV λmax (EtOH): 258, 372 nm
IR(KBr) (cm-1
): 3410.1, 1622.1, 1521.8, 1382.3, 1319.0, 1262.8, 1168.4, 1005.8,
861.2 and 818.2 cm–1
.
1H NMR (DMSO) dH =12.4 (s, 1H, -OH), 10.8 (s, 1H, -OH), 9.60 (s, 1H, -OH),
9.38 (s, 1H, -OH), 9.31 (s, 1H, -OH), 7.67 (d, 1H, J = 2 Hz, C2'-H), 7.54 (dd, 1H, J = 8.4
and 2 Hz, C6'-H), 6.88 (d, 1H, J = 2 Hz, C5-H), 6.40 (d, 1H, J = 2 Hz, C6-H), 6.10 (d,
1H, J = 2.0 Hz, C8-H).
Results
79
A
B
9095100105110115120125130135140145150155160165170 ppm
93.1
0
98.0
2
102.
94
114.
8711
5.22
119.
81
122.
06
135.
63
144.
68
147.
27
156.
07
160.
67
163.
65
C
Figure 9: A:
1H NMR spectra of BS1 B:
13CNMR spectra of BS1 C: Mass-HRMS spectra of BS1
Results
80
13CNMR spectra of BS1(DMSO) dC =176.2 (C=O), 163.9 (C-OH), 160.7 (C-OH),
156.1(C-OH), 147.7 (CH-aromatic), 146.2 (C-OH), 145.5 (CH-aromatic), 135.7 (CH-
aromatic), 126.2 (CH-aromatic), 123.2 (q-aromatic), 122.8 (q-aromatic), 115.0 (q-
aromatic), 103.0 (CH-aromatic), 98.2 (C-OH), 94.2 (q-aromatic)
Mass-HRMS (micro TOF-Q II 10356)-m/s Analytical calculated (C15H10O7) –
303.0499 and found 303.0493
BS1: 3,3',4',5,7-Pentahydroxyflavone (Quercetin)
4.9 Quantitative determination of bioactive constituent (BSI) in E. ganitrus beads
using TLC densitometric method Figure 10 shows standard plot of BSI. Figures 11 and 12 show TL chromatogram
of ethanol extract of E. ganitrus and that of acid hydrolyzed ethanol extract of E.
ganitrus. Table 23 shows BS1 content in E. ganitrus as determined by TLC densitometry.
Results of intra-day and inter-day precision, and recovery studies of BS1 are shown in tables
24 and 25, respectively. Results of method validation parameters are shown in table 26.
Figure 10: Standard curve of absorbance against amount of BS1.
Results
81
Figure 11: TL densitometric chromatogram of ethanol extract of E. ganitrus beads
Figure 12: TL densitometric chromatogram of acid hydrolyzed ethanol extract of
E. ganitrus beads.
Results
82
Table 23: BS1 content in E. ganitrus beads as determined by TLC densitometry.
Method BSI content (% w/w) (Mean
n ± S.D.)
Direct extraction with ethanol 0.03± 0.001
After acid hydrolysis of ethanol extract 0.11± 0.001
n = 3
Table 24: Intra-day and Inter-day precision of BS1.
Marker Concentration (ng/spot)
Intra-day precision
(% RSD)
Inter-day precision
(% RSD)
BS1 200 0.48 0.41
400 0.53 0.65
800 0.52 0.56
Table 25: Recovery study of BS1.
Marker Amount of marker present
(µg)
Amount of
marker added (µg)
Amount of marker found (µg)
(Mean ± S.D.)
Recovery (%)
Average recovery
(Mean ± S.D.)
BS1
400 200 591.2 ± 5.64 98.53 98.32 ± 0.1
400 400 786.2 ± 6.22 98.28
400 500 883.4 ± 7.84 98.16
Results
83
Table 26: Method validation parameters for the quantification of BS1 by TLC densitometric method.
Parameter Observations
Instrumental precision (% RSD, n=7) 0.39
Repeatability (% RSD, n=5) 0.33
Accuracy (Average percent recovery; Mean ± S.D.) 98.32 ± 0.1
Limit of detection (ng) 60
Limit of quantitation (ng) 140
Linearity range (ng/spot) 200-1600
Linearity (Correlation coefficient) 0.994
Specificity Specific
4.10 Characterization of synthetic analogues
The synthesized compounds 4a,e and 6d,e were subjected to 1H NMR and
13C
NMR spectroscopy (Figures 13-19). The obtained data is given below.
4a.
1H NMR (400 MHz, CDCl3): d 10.3 (s, 1H, -CHO), 8.55 (s, 1H, C2-H), 8.30 (dd,
1H, J = 7.6 and 1.56 Hz, C5-H), 7.78-7.73 (m, 1H, C6-H), 7.56-7.47 (m, 2H, C7-H and
C8-H)
13C NMR (100 MHz, CDCl3): d 188.6 (C=O, Aldehydic), 176.0 (C4), 160.6 (C2), 156.2
(C8a), 134.8 (C5), 126.6 (C7), 126.1 (C6), 125.3(C3), 120.3 (C4a), 118.9 (C8).
O
O O
4a
H
Results
84
4b.
1H NMR (400 MHz, DMSO): d 11.09 (s, 1H, -OH), 10.1 (s, 1H, CHO), 8.79 (s,
1H, C2-H), 8.0 (d, 1H, J = 8.8 Hz, C5-H), 7.01 (dd, 1H, J = 8.8 and 2.2 Hz, C6-H), 6.90
(d, 1H, J = 2.0 Hz, C8-H).
13C NMR (400 MHz, DMSO): d 188.2 (C=O, Aldehydic), 174.0 (C-4), 163.5 (C-
7), 161.1 (C-2), 157.4 (C-8a), 126.8 (C-5), 119.5 (C-3), 116.9 (C-4a), 115.8 (C-6), 102.9
(C-8).
O
O O
4b
HO
H
4c.
1H NMR (400 MHz, CDCl3): d 10.3 (s, 1H, CHO), 8.53 (s, 1H, C2-H), 8.08 (br s,
1H, C5-H), 7.56 (dd, 1H, J = 8.4 and 2.0 Hz, C7-H), 7.43 (d, 1H, J = 8.4 Hz, C8-H), 2.51
(s, 3H, CH3).
13C NMR (100 MHz, CDCl3): d 188.8 (C=O, Aldehydic), 176.1 (C-4), 160.6 (C-
2), 154.0 (C-8a), 136.9 (C-6), 136.0 (C-5), 125.5 (C-7), 124.9 (C-3), 120.2 (C-4a), 118.4
(C-8), 21.0 (CH3).
O
O O
4c
H3CH
Results
85
4d.
1H NMR (400 MHz, CDCl3): d 10.2 (s, 1H, -CHO), 8.80 (s, 1H, C2-H), 8.10 (d,
1H, J = 2.6 Hz, C5-H), 7.79 (dd, 1H, J = 8.6 and 2.4 Hz, C7-H), 7.67 (d, 1H, J = 8.6 Hz
C8-H).
13C NMR (100 MHz, CDCl3): d 187.4 (C=O, Aldehydic), 173.8 (C-4), 162.2 (C-
2), 154.0 (C-8a), 134.6 (C-5), 131.6 (C-6), 125.7 (C-3), 124.4 (C-7), 120.6 (C-4a), 119.7
(C-8).
O
O O
4d
HCl
4e.
1H NMR (400 MHz, CDCl3): d 10.3 (s, 1H, -CHO), 8.59 (s, 1H, C2-H), 8.15 (d,
1H, J = 2.4 Hz, C5-H), 7.80 (d, 1H, J = 2.4 Hz, C7-H).
13C NMR (CDCl3): d 187.6 (C-4), 174.2 (-C=O), 160.4 (C-2), 150.6 (C-8a), 135.0
(C-5), 132.6 (C-6), 127.2 (C-8), 125.1 (C-4a), 124.2 (C-7), 120.2 (C-3).
O
O O
4e
Cl
Cl
H
Results
86
6d.
1H NMR (400 MHz, CDCl3): d 12.4 (s, 1H, NH), 10.2 (s, 1H, CHO), 8.18 (d, 1H,
J = 2.4 Hz, C5-H), 7.56 (dd, 1H, J = 8.8 & 2.4 Hz, C7-H), 7.50-7.45 (m, 4H, Ar-H), 7.37-
7.26 (m, 2H, Ar-H).
13C NMR (CDCl3): d 189.7 (CHO), 174.59 (C-4), 162.00 (C-2), 151.4 (C-8a),
133.8 (C-7), 131.9 (CH), 130.2 (q), 129.5 (CH), 128.3 (CH), 126.9 (CH), 124.1 (CH),
123.1 (C-4a), 118.5 (C-8), 99.3 (C-3)
O
O
H
O
6d
Cl
NH
6e.
1H NMR (400 MHz, CDCl3): d 12.51 (s, 1H, -NH), 10.2 (s, 1H, -CHO), 8.12 (d,
1H, J = 2.4 Hz, C5-H), 7.68 (d, 1H, J = 2.4 Hz, C7-H), 7.61 (d, 2H, J = 8.0 Hz, Ar-Hs),
7.49-7.44 (m, 2H, Ar-Hs), 7.38-7.29 (m, 1H, Ar-Hs).
13C NMR (CDCl3): d 189.0 (C=O, Aldehydic), 173.8 (C-4), 161.4 (C-2), 147.8 (q-
Ar), 134.4 (q-Ar), 133.8 (CH-Ar), 131.8 (q-Ar), 129.5 (CH-Ar), 126.9 (CH-Ar), 126.2 (q-
Ar), 124.5 (CH-Ar), 123.0 (q-Ar), 122.6 (CH-Ar), 99.1 (C-3)
O
O O
6e
Cl
NH
Cl
H
Results
87
A
13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm
-0.0
078
0.00
00
1.78
93
2.17
47
2.64
02
6.90
746.
9647
6.96
676.
9854
7.27
597.
4756
7.49
527.
4975
7.51
537.
5355
7.53
937.
5608
7.73
077.
7346
7.74
527.
7495
7.76
357.
7669
7.77
057.
7844
7.78
858.
2941
8.29
818.
3140
8.31
798.
5565
10.3
931
12.1
092
12.2
587
0.25
2.27
1.18
1.04
1.02
1.00
0.09
CHR-I
B
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
0.00
76.7
377
.05
77.3
7
118.
6411
8.94
120.
3212
5.32
126.
1812
6.66
130.
7213
4.85
136.
48
156.
21
160.
64
176.
00
188.
63
Current Data ParametersNAME Dec19-2012EXPNO 481PROCNO 1
F2 - Acquisition ParametersDate_ 20121219Time 21.07INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT CDCl3NS 512DS 4SWH 29761.904 HzFIDRES 0.454131 HzAQ 1.1010548 secRG 1030DW 16.800 usecDE 6.00 usecTE 296.1 KD1 2.00000000 secd11 0.03000000 secDELTA 1.89999998 secTD0 1
======== CHANNEL f1 ========NUC1 13CP1 9.60 usecPL1 -2.00 dBSFO1 100.6228298 MHz
======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -3.00 dBPL12 14.31 dBPL13 18.00 dBSFO2 400.1316005 MHz
F2 - Processing parametersSI 32768SF 100.6127690 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40
CHR-I
Figure 13: A: 1H NMR spectra of 4a B:
13CNMR spectra of 4a
Results
88
A
B
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
38.9
739
.17
39.3
839
.59
39.8
040
.01
40.2
2
77.8
778
.20
78.5
3
102.
3810
2.93
115.
8411
6.94
119.
45
126.
79
157.
4216
1.13
163.
49
174.
27
188.
18
Figure 14: A: 1H NMR spectra of 4b B:
13CNMR spectra of 4b
Results
89
A
B
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
21.0
3
76.7
677
.08
77.4
0
118.
3712
0.15
124.
9312
5.52
136.
0113
6.95
154.
48
160.
60
176.
11
188.
82
Figure 15: A: 1H NMR spectra of 4c B:
13CNMR spectra of 4c
Results
90
A
11 10 9 8 7 6 5 4 3 2 1 0 ppm
0.00
00
2.56
442.
5689
2.57
342.
6226
2.65
362.
7789
3.78
65
7.66
507.
6873
7.78
557.
7921
7.80
797.
8144
8.00
838.
0970
8.10
358.
8053
10.2
041
2.09
1.24
1.11
1.00
1.02
1.02
CHR-II
B
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
38.9
639
.17
39.3
739
.58
39.7
940
.00
40.2
1
77.9
878
.31
78.6
4
119.
7512
0.64
124.
4712
5.76
131.
6213
4.66
154.
02
162.
30
173.
81
187.
42
Current Data ParametersNAME Dec19-2012EXPNO 491PROCNO 1
F2 - Acquisition ParametersDate_ 20121219Time 21.43INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT DMSONS 512DS 4SWH 29761.904 HzFIDRES 0.454131 HzAQ 1.1010548 secRG 1030DW 16.800 usecDE 6.00 usecTE 296.0 KD1 2.00000000 secd11 0.03000000 secDELTA 1.89999998 secTD0 1
======== CHANNEL f1 ========NUC1 13CP1 9.60 usecPL1 -2.00 dBSFO1 100.6228298 MHz
======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -3.00 dBPL12 14.31 dBPL13 18.00 dBSFO2 400.1316005 MHz
F2 - Processing parametersSI 32768SF 100.6128193 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40
CHR-II
Figure 16: A: 1H NMR spectra of 4d B:
13CNMR spectra of 4d
Results
91
A
B
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
76.7
377
.05
77.3
7
120.
2612
4.26
125.
1112
7.25
132.
6013
5.01
150.
61
160.
46
174.
26
187.
68
Figure 17: A: 1H NMR spectra of 4e B:
13CNMR spectra of 4e
Results
92
A
13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm
0.00
00
1.74
89
2.17
42
7.26
127.
2686
7.28
327.
3167
7.32
307.
3307
7.33
307.
3380
7.34
487.
3533
7.35
777.
3727
7.45
087.
4566
7.46
617.
4730
7.48
087.
4872
7.49
667.
5026
7.55
727.
5638
7.57
927.
5857
8.18
008.
1865
10.2
835
12.4
457
1.27
2.00
4.40
1.12
1.02
1.03
1.03
BL-2
B
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
0.00
76.7
377
.05
77.3
7
99.3
7
118.
5012
3.16
124.
1212
5.86
126.
9712
8.32
129.
5913
0.24
131.
9613
3.87
134.
50
151.
46
162.
00
174.
59
189.
73
Figure 18: A: 1H NMR spectra of 6d B:
13CNMR spectra of 6d
Results
93
A
B
2030405060708090100110120130140150160170180190200210 ppm
76.7
377
.05
77.3
7
99.1
3
121.
1712
2.74
123.
0112
4.54
125.
2512
6.38
126.
9712
9.03
129.
3912
9.58
131.
8413
3.88
134.
4114
7.82
161.
38
173.
80
189.
71
Figure 19: A: 1H NMR spectra of 6e B:
13CNMR spectra of 6e
Results
94
4.11 Antianxiety activity of Synthetic analogues 4a-e and 6d,e
Results of antianxiety activity screening of synthetic analogues 4a,e and 6d,e
using EPM apparatus, are shown in table 27 and figure 20.
Table 27: Results of antianxiety activity evaluation of synthetic analogues using
EPM apparatus.
Treatment Dose
(mg/kg)
Meann
number of
entries ± S.E.M.
Meann time
b
(sec) ± S.E.M.
Control Vehicle 5.0 ± 0.51a 3.18 ± 0.22
a
Diazepam 2.0 18.0 ± 0.93* 12.13 ± 0.66*
4a
5.0
10.0
15.0
20.0
4.2±0.37a
4.4±0.37a
4.8±0.24a
4.4±0.24a
2.40±0.24a
2.93±0.06a
3.10±0.10a
3.00±0.00a
4b
5.0
10.0
15.0
20.0
5.8±0.24a
6.0±0.24a
8.6±0.20a*
9.2±0.37a*
3.74±0.19a
3.94±0.23a
5.15±0.06a*
6.17±0.07a*
4c
5.0
10.0
15.0
20.0
6.4±0.24a
6.8±0.24a
9.2±0.48a*
11.6±0.68a*
4.08±0.11a
4.34±0.04a
6.18±0.33a*
7.6 ± 0.34a*
4d
5.0
10.0
15.0
20.0
6.8±0.60a
8.1±0.79a
9.9 ± 0.59a*
9.3 ± 0.76a*
4.9±0.47a
5.0±0.28a*
7.2±0.39a*
6.5±0.89a*
4e
5.0
10.0
15.0
20.0
7.1±0.60a
8.5±0.85a
10.1±0.70a*
9.5±0.96a*
5.1±0.21a
5.9±0.46a*
7.8±0.31*
6.4±0.39a*
6d
5.0
10.0
15.0
20.0
7.8±0.31a
16.0±0.24*
12.4±0.34*
10.8±0.34a*
5.09±0.05a
11.2±0.23*
9.9±0.59*
7.2±0.39*
6e
5.0
10.0
15.0
20.0
6.8±0.20a
9.0±0.20a*
14.0±0.74*
8.8±0.24a*
4.01±0.09a
6.19±0.04a*
9.9±0.44*
5.31±0.09a*
n = 5; b = Average time each animal spends in open arms = total duration in open arms/number of entries
in open arms; *P<0.05 vs. control; aP<0.05 vs. standard; one way ANOVA followed by Studentized
Tukey’s test.
Results
95
A
B
Figure 20: Relative anxiolytic activity profile of A: mean number of entries B: mean time (sec) spent in
open arms by mice treated with synthetic analogues (4a-d, 6d,e) using EPM apparatus. The
data is expressed as mean ± S.E.M.; n = 5; *P<0.05 vs. control; aP<0.05 vs. diazepam; one way
ANOVA followed by Studentized Tukey’s test.
Results
96
4.12 Antianxiety activity of BS1 and synthetic analogue compound 6d
Table 28 and figure 21 show the results of antianxiety activity of BS1 and
synthetic analogue compound 6d using light and dark model, whereas those obtained
using mirrored chamber are shown in figure 22.
4.12.1 Light/dark test
Table 28: Results of antianxiety activity of BS1 isolated from E. ganitrus and
synthetic analogues compound 6d using light and dark model in mice.
Treatment Time spent in light
box
Time spent in dark
box
Transfer Latency
Control 34.6± 0.51a
262.4 ± 0.81a
103.6± 0.74a
Diazepam 204.2± 0.73*
94.8± 0.58*
25.6± 0.74*
BS1 (25 mg/kg) 195.6± 0.51a* 103.4± 0.67
a*
33.6± 0.51a*
Compound 6d
(10 mg/kg)
188.2±0.80a* 106.0±0.70
a* 35.8±0.58
a*
n = 5; Values are expressed as mean ± SEM; one way ANOVA followed by Studentized Tukey’s test.
*P<0.05 vs. control; aP<0.05 vs. diazepam;
Results of antianxiety activity of BS1 and synthetic analogue compound 6d using
light/dark test have been shown in figure 7.
Figure 21: Relative anxiolytic activity profile of BS1 and synthetic analogue compound 6d using
light/dark model. The data is expressed as mean ± S.E.M.; n = 5; *P<0.05 vs. control; aP<0.05
vs. diazepam; one way ANOVA followed by Studentized Tukey’s test.
Results
97
4.12.2 Mirrored chamber test
Results of antianxiety activity of BS1 and synthetic analogues compound 6d using
mirrored chamber test have been shown in figure 22.
A
B
C
Figure 22: Relative anxiolytic activity profile of BS1 and synthetic analogue compound 6d using mirrored
chamber test (A: transfer latency, B: number of entries in mirrored chamber, C: time spent in
mirrored chamber). The data is expressed as Mean ± S.E.M.; n = 5; *P<0.05 vs. control; aP<0.05 vs. diazepam; ANOVA followed by Studentized Tukey’s test.
