Comparative Morphology of the Leaf Epidermis in Six Citrus Species ...
4.1 Leaf morphology of male and female adult...
Transcript of 4.1 Leaf morphology of male and female adult...
Chapter 4 Results
School of Biological and Environmental Sciences 55
For effective hybridization programs and afforestation purposes, it is important to
identify the superior phenotype or an accurate provenance (Rajora, 1988). The
identification of male and female trees at seedling stage would help in mass clonal breeding
or hybridization programs. The differences based on the morphological, molecular and
biochemical characters, presented in this chapter, in the form of theory, tabulated data,
pictures and graphs include the following heads.
4.1 Morphological differences between leaves of male and female adult trees
4.2 Markers for gender identification at seedling stage
4.3 Phytochemical analysis of leaves, buds and bark
4.4 Monthly variations and quantification of two phenolic glycosides
4.1 Leaf morphology of male and female adult trees
The morphological features of male and female trees of P. ciliata based on the
quantitative and qualitative characters are presented under two separate heads.
4.1.1 Qualitative characters
Table 4.1 summarizes the morphological shape of base and tip of leaf blade, sinus
with petiole, pubescence on the lower surface of the leaf blade, leaf margin and color of the
blade. The differences between female and male trees were seen in shape of the leaf blade
being deltoid- cordate in females and deltoid-ovate in males. Similarly the shape of the
sinus with petiole was deep in females while shallow in males and serration of the leaf
blade margin was sparsely serrate in females and densely serrate in males (Figs. 4.1 & 4.2).
These differences are not well marked at the juvenile stage.
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Figure 4.1: Leaves of female tree of P. ciliata
Figure 4.2: Leaves of male tree of P. ciliata
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S. No. Characters of leaf Females Males
1 General shape of base of leaf blade Deltoid-cordate Deltoid-ovate
2 Shape of sinus with petiole Deep Shallow
3. General shape of tip of leaf blade Acute Acute
4 Pubescence on lower surface of leaf
blade
On whole surface On whole surface
5 Serration of the edge of leaf blade
(leaf margin)
Sparsely serrate Densely serrate
6 Color of leaf blade Green on upper
surface and whitish
on lower surface
Green on upper
surface and whitish
on lower surface
Table 4.1: Qualitative characters of female and male trees of P. ciliata
4.1.2 Quantitative characters
Different quantitative characters studied were leaf area, lamina length/width (L/W)
percent, petiole length/ nerve length (P/N) percent, perimeter, aspect ratio and shape factor
(Table 4.2). Males in general exceeded the females in all characters enumerated in this
study except the lamina length/ width percent where in males (127.13) it was marginally
lower than the females (128.30) (Fig. 4.4). The values of average leaf area (Fig. 4.3), petiole
length/ nerve length percent (Fig. 4.5), perimeter (Fig. 4.6), aspect ratio (Fig. 4.7) and shape
factors (4.8) in male trees were 92.27, 71.59, 36.72, 0.80 and 0.85, while in female trees
these came to 86.12, 70.59, 35.58, 0.78 and 0.21, respectively. These differences at
quantitative level were observed in mature trees.
4.1.3 Tukey’s multiple comparison test
Tukey’s multiple comparison test was applied to compare the significance level of
difference in leaf area between males vs males, males vs females and between males vs
females (Table 4.3). A non-significant difference was observed between male 1 vs male 4,
female 3 vs female 4, male 1 vs female 1 and female 4 vs female 2, 3 and 4, whereas other
combination gave significant difference. The overall comparison of leaf area, leaf length/
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width percent and leaf petiole/ middle nerve length between male and female also showed
non- significant difference, but the comparison of shape factor between males and females
showed significant results (Table 4.4).
Table 4.2: Quantitative characters of male and female P. ciliata trees
S. No. Species/ Leaf Characters P. ciliata
Mean (SD)
Females Males
1. Leaf Area (cm2) 86.12 (17.73)
92.27 (34.01)
2. Lamina Length
Widh× 100
128.30 (3.11) 127.13 (10.99)
3. Petiole Length
Nerve Length× 100
70.60 (7.54) 71.59 (5.60)
4. Perimeter 35.58 (5.50)
36.72 (8.32)
5. Aspect Ratio 0.78 (0.13)
0.80 (0.13)
6. Shape factor 0.21 (0.03)
0.85 (0.01)
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Figure 4.3: Leaf area (cm2) (mean average value) variation in the male and female trees of
P. ciliata
Figure 4.4: Variation in the mean average Lamina / width (%) in the male and female trees
of P. ciliata
Mal
es
Femal
es
0
20
40
60
80
Lea
f p
etio
le/m
idd
le n
erv
e le
ng
th %
Figure 4.5: Variation in the mean average leaf petiole / middle nerve length (%) in the male
and female trees
Mal
es
Femal
es
0
50
100
150
Lea
f are
a
(cm
2)
Mal
es
Femal
es
0
50
100
150
Lea
f le
ng
th/
wid
th %
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Mal
es
Femal
es
0
10
20
30
40
50
Le
af
Pe
rim
ete
r
Figure 4.6: Variation in the mean average leaf perimeter (cm) of male and female trees of
P. ciliata
Mal
es
Femal
es
0.0
0.2
0.4
0.6
0.8
1.0
Asp
ect
ra
tio
Figure 4.7: Variation in the aspect ratio of male and female trees of P. ciliata
Mal
es
Femal
es
0.0
0.2
0.4
0.6
0.8
1.0
Genotypes of P. ciliata
Sh
ap
e fa
cto
r
Figure. 4.8: Variation in the mean average shape factor of male and female trees of P.
ciliata
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Table 4.3: Tukey’s multiple comparison tests between male and female trees of P. ciliata in
different pair combinations.
Tukey’s Multiple
comparison test
Mean Difference
t value P value Significant
Male 1 Vs Male 2 50.35 ± 8.05 6.29 < 0.0001 ***
Male 1 Vs Male 3 -31.20 ± 9.37 3.33 0.0015 **
Male 1Vs Male 4 16.98 ± 10.59 1.60 0.1142 ns
Male 2 Vs Male 3 -81.55 ± 6.64 12.27 0.0001 ***
Male 2 Vs Male 4 -33.37 ± 8.28 4.03 0.0020 ***
Male 3 Vs Male 4 48.18 ± 9.60 5.02 < 0.0001 ***
Female 1 Vs Female 2 42.39 ± 5.66 7.49 < 0.0001 ***
Female 1 Vs Female 3 26.20 ± 5.90 4.44 <0.0001 ***
Female 1 Vs Female 4 28.93 ± 7.05 4.10 0.0001 ***
Female 2 Vs Female 3 -16.18 ± 4.61 3.51 0.0009 ***
Female 2 Vs Female 4 -13.46 ± 6.01 2.24 0.0290 *
Female 3 Vs Female 4 2.73 ± 6.24 0.44 0.6640 ns
Male 1 Vs Female 1 -9.20 ± 8.75 1.05 0.2974 ns
Male 1 Vs Female 2 33.18 ± 7.94 4.18 <0.0001 ***
Male 1 Vs Female 3 17.00 ± 8.12 2.10 0.0406 *
Male 1 Vs Female 4 19.73 ± 8.98 2.19 0.0321 *
Male 2 Vs Female 1 -59.55 ± 5.75 10.36 <0.0001 ***
Male 2 Vs Female 2 -17.17 ± 4.41 3.89 0.0003 ***
Male 2 Vs Female 3 -33.35 ± 4.73 7.06 <0.0001 ***
Male 2 Vs Female 4 -30.62 ± 6.09 5.02 <0.0001 ***
Male 3 Vs Female 1 22.00 ± 7.53 2.92 0.0050 **
Male 3 Vs Female 2 64.38 ± 6.57 9.81 <0.0001 ***
Male 3 Vs Female 3 48.20 ± 6.78 7.11 <0.0001 ***
Male 3 Vs Female 4 50.93 ± 7.79 6.53 <0.0001 ***
Male 4 Vs Female 1 -26.18 ± 9.03 2.91 0.0051 **
Male 4 Vs Female 2 16.20 ± 8.21 1.97 0.9981 ns
Male 4 Vs Female 3 0.02 ± 8.39 0.01 0.9981 ns
Male 4 Vs Female 4 2.75 ± 9.23 0.30 0.7671 ns
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Table 4.4: Tukey’s multiple comparison tests between leaf area, leaf length/width (%), leaf
petiole/ middle nerve length (%), perimeter of leaf, aspect ratio and shape factor of male and
female trees of P. ciliata.
Tukey’s
comparison
multiple test
Mean Difference t value P value Significant
Leaf area (cm2)
Male vs Females 6.14 ± 19.18 0.32 0.76 ns
𝐋𝐚𝐦𝐢𝐧𝐚 𝐋𝐞𝐧𝐠𝐭𝐡
𝐖𝐢𝐝𝐡× 𝟏𝟎𝟎
Males vs females -1.17± 5.77 0.20 0.85 ns
𝐏𝐞𝐭𝐢𝐨𝐥𝐞 𝐋𝐞𝐧𝐠𝐭𝐡
𝐍𝐞𝐫𝐯𝐞 𝐋𝐞𝐧𝐠𝐭𝐡× 𝟏𝟎𝟎
Males vs females 0.99±4.70 0.21 0.84 ns
Perimeter of leaf
Males vs Females 1.14 ± 4.31 0.26 0.80 ns
Aspect ratio
Males vs Females 0.02±0.03 0.73 0.49 ns
Shape factor
Males vs Females 0.64±0.01 56.19 <0.0001 ***
*P<0.05. **P<0.01, ***P<0.0001, ns- Non-significant (P>0.05)
4.2 Markers for gender identification at seedling stage
Two molecular markers (RAPD and isozyme analysis) were used for the purpose
for identification of sex at the seedling stage. The results obtained by these two markers are
presented in two separate heads.
4.2.1 RAPD analysis
In the present study the genomic DNA was successfully isolated using the modified
CTAB protocol given by Porebski et al. (1997) for the isolation of genomic DNA from
plants containing high polysaccharides and polyphenolic components. It is evident from
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Fig. 4.9 that the genomic DNA isolated by modifying CTAB method showed clear bands
on an agarose gel. Young fresh leaves gave good quality and quantity of DNA instead of
stored mature leaves, which were difficult to use for DNA isolation purposes because of
the accumulation of excess polyphenolic compounds which were difficult to remove and
affected the quality of isolated genomic DNA for PCR amplification.
Genetic differences among four staminate and four pistillate trees were investigated
using ten RAPD primers. Isolated genomic DNA samples extracted from the young fresh
leaves were amplified with ten decamer random primers. All primers showed successful
amplification of RAPD bands from all trees which were clear and easy to interpret (Table
4.6). The primers were selected also based upon their percentage of polymorphism (more
than 60 %) (Table 4.5). Primers with different oligonucleotide sequences generated
amplification fragments different in number and size. A total number of approximately 75
bands were produced from 10 decamer primers. The number of fragments generated per
primer varied between 4 and 14 (Table 4.6). All primers gave similar pattern and bands
ranged in molecular size between 100 and 1500 bp approximately. The dominant scorable
bands were calculated, but the weak bands were excluded.
Out of ten RAPD primers, one primer (OPK-20) gave significant difference
between male and female trees (Appendix 1 & 2). Total amplification products amplified
by OPK-20 primer were seven out of which three were unique bands, i.e. present only in
female trees and four were monomorphic, i.e. present in both male and female trees (Fig.
4.10). OPK-20 primer produced female specific three unique amplification products of
different sizes, i.e. 400 bp, 500 bp and 800 bp from female trees, but failed to do so in the
four male trees. This DNA marker was found to be reproducible. The experiment was
repeated twice with both male and female DNA samples in order to check the reliability
and stability of the marker. OPK-20 was found completely linked to the female sex.
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Table 4.5: The sequence, GC % and annealing temperature of primers
Table 4.6: Total number of male and female specific PCR products and total number of bands
present in male and female P. ciliata trees.
Primer
Showing
Polymorphism
Total number of
PCR product per
genotype
Number of PCR
product specific for
genotype
Approximate
size of the M/F
specific DNA
band
M F M F
OPK-20 4 7 - 3 400 bp, 500 bp,
800 bp
OPE-04 7 7 - - -
OPG-06 9 9 - - -
OPG-17 4 4 - - -
OPG-02 9 9 - - -
OPC-11 14 14 - - -
OPA-10 6 6 - - -
OPG-05 5 5 - - -
OPH-05 4 4 - - -
OPC-19 10 10 - - -
Primer code Sequence GC % Annealing
Tm ºC/Sec
OPA-10 5ꞌ-GTGATCGCAG-3ꞌ 60 32/60
OPC-19 5ꞌ-GTTGCCAGCC-3ꞌ 70 34/60
OPE-04 5ꞌ-AAGACCCCTC-3ꞌ 60 32/60
OPG-02 5ꞌ-GGCACTGAGG-3ꞌ 70 34/60
OPH-05 5ꞌ-AGTCGTCCCC-3ꞌ 70 34/60
OPK-20 5ꞌ-GTGTCGCGAG-3ꞌ 70 34/60
OPG-06 5ꞌ-GTGCCTAACC-3ꞌ 60 32/60
OPC-11 5ꞌ-AAAGCTGCGG-3ꞌ 60 32/60
OPG-05 5ꞌ-CTGAGACGGA-3ꞌ 60 32/60
OPG-17 5ꞌ-ACGACCGACA-3ꞌ 60 32/60
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Figure 4.9: Agarose gel showing bands of genomic DNA isolated from young fresh leaves of
male and female P. ciliata trees using modified CTAB method.
Figure 4.10: RAPD profile generated by primer OPK-20 using fresh young leaves genomic
DNA (L represents the molecular size 100 bp marker; F represents the female and M
represents the male tree samples. Primer OPK-20 produced three amplification products of
different sizes i.e., 400 bp, 500 bp and 800 bp from female plants but failed to do so in the four
male plants).
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4.2.2 Isozyme analysis
In the present investigation, four male and four female samples of P. ciliata were
examined with seven enzyme systems, viz. peroxidase (PER), esterase (EST), catalase
(CAT), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), acid phosphatase
(APH) and ribulose bisphosphate carboxylase (RUBISCO). Out of these, only two enzymes
(peroxidase and esterase) showed differentiation between male and female trees.
Peroxidase enzyme system produced three anodal bands PER-1, PER-II and PER-
III at the position of Rm values 0.10, 0.20 and 0.29, respectively. The two slower moving
bands at RM values of 0.10 and 0.20 were found to be monomorphic, since they were
observed in all the male and female trees, whereas the third band (PER-III) at RM value of
0.29 was observed only in four female trees (Fig. 4.11 & Appendix 3). This band was found
to be specific for female trees.
In case of esterase enzyme (EST) system for the male and female trees, five
differentiating bands EST-I, EST-II, EST-III, EST-IV and EST-V were observed at the
anodal position of Rm value 0.06, 0.11, 0.15, 0.27 and 0.30 respectively. The fastest
moving band (EST-V) at the position of Rm values 0.30 was present only in male trees,
whereas slow moving very light band (EST-II) at the position of Rm 0.11 was only present
in female trees. Both bands were identified as male and female specific bands (Fig. 4.12 &
Appendix 4). Thus, two female specific sex markers and male specific marker were
identified, which could successfully differentiate between the staminate and pistillate trees
of P. ciliata.
Other enzymes like alcohol dehydrogenase, ribulose bisphosphate carboxilase, and
catalase showed their activity in both male and female trees but could not be used for gender
differentiation in P. ciliata. Even in repeated attempts, malate dehydrogenase and acid
phosphatase enzyme systems showed no activity in any of the male and female trees.
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Figure 4.11: Zymogram showing peroxidase enzyme pattern in four male and four female
trees of P. ciliata
Figure 4.12: Zymogram showing the esterase enzyme pattern of four male and four female
trees of P. ciliata.
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4.3 Phytochemical analysis of buds, leaves and bark
4.3.1 Preliminary phytochemical screening
The leaves, buds and bark were collected from the trees (sample site) in the month of
August, dried in the vacuum oven and crushed using a grinder. The methanolic and aqueous
extract were prepared and used for further phytochemical screening.
The results of phytochemical screening are shown in Table 4.7. Results showed that
the P. ciliata plant was rich in carbohydrates, proteins, phenolic glycosides, tannins and
gum and mucilages. The presence of carbohydrates, saponins, tannins, proteins and amino
acids were not obtained to be uniform in both solvents. Proteins and amino acids were
found to be absent in bark extract, whereas steroids were observed only in methanolic
extracts of bark. Foam test showed positive test only with both aqueous extract and
methanolic extract of bark. On the other hand, this test showed negative results with leaf
and bud methanolic extracts. Gum and mucilages were observed in only bud extracts and
found to be absent in bark and leaves. Both methanolic and aqueous of leaves, bud and bark
showed positive results for carbohydrates, flavonoids glycosides, phenols and tannins.
Saponins were absent in methanolic extract of leaf and buds, but found to be positive in
methanolic extract of bark and aqueous extract of all plant parts. There was also the
difference in the concentration of different chemical constituents found in leaves, buds and
bark extract. Phenols were densely colored for leaf extract as compared to diluted in others
phytochemicals analyzed. Phenolic glycosides were found to be more in bark and buds.
Flavonoids are totally absent in the both methanolic and aqueous extract of leaf, buds and
bark. This combination of the phytochemicals creates the possibility of their use in
medicinal application.
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Table 4.7: Phytochemical screening of methanolic and phosphate buffer extract of P. ciliata
(leaves, buds and bark)
Plant constituent
test/ reagent used
Plant extracts
Methanolic extract Aqueous extract
Bud
(M/F)
Leaf
(M/F)
Bark
(M/F)
Bud
(M/F)
Leaf
(M/F)
Bark
(M/F)
1. Test for alkaloids
Wagner test + + + + + +
2. Test for carbohydrate
b. Fehling test
d. Benedict’s test +
+
+
+
+
+
+
+
+
+
+
+
3. Test for phenolic compounds & tannins
a. FeCl3 test
b. Lead acetate test
c. Blue litmus test
d. Gelatin test
++
+
+
-
++
+
+
-
++
+
+
-
+
+
-
+
+
+
-
+
+
-
-
+
4. Tests for proteins & amino acids
a. Biuret test
b. Ninhydrin test
c. Keller-Killiani test
+
+
+
+
+
+
-
-
-
+
+
+
+
+
+
-
-
-
5.Tests for flavonoids and flavanol glycosides
a. Sinodha test
b. Zink dust test
c. Akaline reagent
test
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
6. Tests for saponins
a. Foam test - - + + + +
7. Test for glycosides and steroids
- - + - - +
8. Test for gum and mucilages
+ - - + - -
Positive (+); negative (-); densely positive (++)
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4.4 Gender related variation in two phenolic glycosides synthesis
Gender related differences in phenolic glycosides synthesis was observed from the
bark and buds of male and female P. ciliata trees. Two phenolic glycosides (salicin and
populin) were studied from the bark and buds of male and female trees of P. ciliata. The
monthly variation in the concentration of salicin and populin was analyzed by HPLC and
their quantity was detected. For this, salicin was purchased from the market and populin
was chemically synthesized in the laboratory from salicin as standard.
4.4.1 Characterization of synthesized populin
Synthesized sample showed needle like white colored crystals (molecular formula-
C20H22O8, m.p.-179ºC lit. 180ºC; Richtmyer and Yeakel, 1934) with light sweetish taste.
The confirmation of the compound was also done with conc. H2SO4 which produced light
pinkish color. The physical characterization was proceeded with IR (Fig. 4.13), 1H NMR
(Fig. 4.14) and 13C NMR (Fig. 4.15) (Burker Advance II 400 NMR spectrophotometer
SAIF, Panjab University, Chandigarh).
4.4.1.1 FTIR results
FTIR results are presented in Fig. 4.13. In IR, the values 3376 cm-1 and 3294 cm-1
correspond to stretching frequencies of O-H groups. The spectrum also shows stretching
frequencies at 2933 cm-1 and 2884 cm-1 due to asymmetrical and symmetrical stretching of
C-H and 1050-1250 cm-1 due to C-O stretchings, respectively. The peaks at 1450, 1500-
1650 cm-1 are attributed to the presence of aromatic ring C-C stretchings. Additionally, the
FTIR spectrum of populin has an intense, sharp peak at 1719 cm-1 which is attributed to the
presence of a -COOR group in contrast to the salicin FTIR (Appendix 5).
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Figure 4.13: FTIR of synthesized populin
`4.4.1.2 1H NMR results
1H NMR results are shown in Fig. 4.15. Populin was synthesized from salicin.
Structurally salicin is composed of two main parts, one is salicyl alcohol (aromatic part)
and other is sugar part. Therefore, signals obtained by the 1H NMR spectroscopy should lie
in two regions- first one is the aromatic proton that is much down-field i.e. 6-8 ppm.
Another part should give the values typical of oxygenated and other aliphatic protons and
should be in the up-field. All values are presented in table 4.8.
Figure 4.14: Chemical structure of populin
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Table 4.8: Different signals of 1H NMR
The 1H NMR of the salicin is giving us exact detail as expected (values in δ; DMSO; TMS= 0)
S. No. Proton (attached H to C) Shift (ppm) H1s Type J (Hz)
1. 2 7.13 1 d 7.32
2. 3 7.23 1 T 8.20
3. 4 6.99 1 T 7.32
4. 5 7.30 1 d 7.45
5. 7 4.43, 4.74 2 d Germinal
coupling
6. 8 4.77 1 d
7. 9 3.40 1 T
8. 10 3.30 1 T
9. 11 3.46 1 T
10. 12 3.80 1 d/T 11.24
11. 13 3.50 2 d
The major difference between salicin and populin 1H NMR is clearly understandable.
Populin shows extra signals for benzoate five protons to confirm the successful reaction
as given below:
S.
No.
Proton (attached
H to C)
Shift
(ppm)
1Hs Type J
(Hz)
1. 2ꞌ 8.01 1 d 7.36
2. 3ꞌ 7.51 1 T 7.70
3. 4ꞌ 7.64 1 T 7.30
4. 5ꞌ 7.51 1 T 7.70
5. 6ꞌ 8.01 1 d 7.36
Besides, the significant observation, the chemical shift of C-7ꞌ methylene protons of
sugar moiety shows characteristic peak shift from 3.35 and 3.66 to 4.35 and 4.67 as
expected shift of 1 ppm by the ester formation. Rest of the signal was more or less
the same as that of the salicin (Appendix 6).
Sugar
proton
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School of Biological and Environmental Sciences 73
Figure 4.15: 1H NMR of populin
4.4.1.3 13C NMR results
13C NMR results are shown in Fig. 4.17. 13C NMR spectroscopy supports populin
stretching as depicted by 13C NMR results with characteristic peaks of salicylic alcohol
units between 58 to 155 ppm (C-1, 154.88; C-2, 132.91; C-3, 129.65; C-4, 121.87; C-5,
128.26; C-6, 114.90; C-7, 58.89). The intense peak at 58.89 ppm is the characteristics of
primary alcohol present in salicylic alcohol. The peaks for sugar unit of populin appear
between 58 to 101 ppm (C-1ꞌ, 101.54; C-2ꞌ, 73.21; C-3ꞌ, 76.29; C-4ꞌ, 70.11; C-5ꞌ, 76.29; C-
6ꞌ, 66.09), where anomeric carbon (C-1ꞌ) in sugar unit is evaluated at 101.54 ppm.
Additionally, populin also shows extra signals in aromatic region for benzoate five protons
at substituent C-6ꞌꞌ of sugar unit between 114 to 165 ppm. The peak at 165.37 ppm is
associated for ester linkage between benzoic acid and sugar unit of populin. The detail of
salicin 13C NMR are shown in Appendix 7.
11 10 9 8 7 6 5 4 3 2 1 0 ppm
0.0000
1.2461
2.5465
2.5505
3.3500
3.3706
3.3967
3.4196
3.4398
3.4593
3.4804
3.7298
3.7486
3.7677
4.3332
4.3521
4.3625
4.3815
4.4259
4.4601
4.6717
4.6757
4.7008
4.7350
4.8084
4.8264
6.9327
6.9508
6.9681
6.9878
6.9922
7.0063
7.0105
7.0651
7.0844
7.3082
7.3124
7.3262
7.3299
7.4934
7.5126
7.5319
7.6225
7.6409
7.6594
8.0082
8.0266
8.0877
3.47
1.42
1.34
1.24
2.21
1.12
2.00
1.02
1.02
2.04
1.01
2.00
Current Data Parameters
NAME Oct23-2013
EXPNO 50
PROCNO 1
F2 - Acquisition Parameters
Date_ 20131023
Time 15.24
INSTRUM spect
PROBHD 5 mm PABBO BB-
PULPROG zg30
TD 65536
SOLVENT DMSO
NS 8
DS 2
SWH 12019.230 Hz
FIDRES 0.183399 Hz
AQ 2.7263477 sec
RG 287
DW 41.600 usec
DE 6.00 usec
TE 295.9 K
D1 1.00000000 sec
TD0 1
======== CHANNEL f1 ========
NUC1 1H
P1 10.90 usec
PL1 -3.00 dB
SFO1 400.1324710 MHz
F2 - Processing parameters
SI 32768
SF 400.1299852 MHz
WDW EM
SSB 0
LB 0.30 Hz
GB 0
PC 1.00
PC-2PBRUKER
AVANCE II 400 NMR
Spectrometer
SAIFPanjab University
Chandigarh
Chapter 4 Results
School of Biological and Environmental Sciences 74
Fig. 4.16: Chemical structure of populin
Figure 4.17: 13C NMR of populin
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
38.94
39.15
39.36
39.57
39.77
39.98
40.19
58.89
64.09
70.11
73.21
73.91
76.29
78.17
78.50
78.70
78.83
101.54
114.90
121.87
127.54
127.77
128.26
129.07
129.65
131.37
132.91
154.88
165.37
Current Data Parameters
NAME Oct23-2013
EXPNO 51
PROCNO 1
F2 - Acquisition Parameters
Date_ 20131023
Time 17.17
INSTRUM spect
PROBHD 5 mm PABBO BB-
PULPROG zgpg30
TD 65536
SOLVENT DMSO
NS 512
DS 4
SWH 29761.904 Hz
FIDRES 0.454131 Hz
AQ 1.1010548 sec
RG 724
DW 16.800 usec
DE 6.00 usec
TE 296.6 K
D1 2.00000000 sec
d11 0.03000000 sec
DELTA 1.89999998 sec
TD0 1
======== CHANNEL f1 ========
NUC1 13C
P1 9.60 usec
PL1 -2.00 dB
SFO1 100.6228298 MHz
======== CHANNEL f2 ========
CPDPRG2 waltz16
NUC2 1H
PCPD2 80.00 usec
PL2 -3.00 dB
PL12 14.31 dB
PL13 18.00 dB
SFO2 400.1316005 MHz
F2 - Processing parameters
SI 32768
SF 100.6128193 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40
PC-2P BRUKER
AVANCE II 400 NMR
Spectrometer
SAIF
Panjab University
Chandigarh
Chapter 4 Results
School of Biological and Environmental Sciences 75
4.4.2 Thin layer chromatography of methanolic extract of bark
Phenolic glycosides were present only in the methanolic extract of the buds, bark
and leaves (Table 4.7). For the detection of the presence of salicin and populin in the
methanolic extract of bark, TLC technique was used. Different solvent systems were tried
for the separation of salicin and populin from the bark extract containing mixture of
compounds and the best results were obtained with two solvent systems, i.e. methanol:
water (70:30) and ethyl acetate: methanol (70:30) observed under UV (Fig. 4.18). Different
spots were generated on silica gel plate with different Rf values and the identification of
compounds was done by comparing the extract with standards (salicin and populin) (Fig.
4.18). The Rf value with different solvent systems for salicin and populin are shown in
Table 4.9. These solvent systems can be used to separate the salicin and populin from the
plant parts at industrial level.
Table 4.9: Rf values of salicin and populin in two different solvent systems.
S. No. Solvent system Ratio Rf value
Salicin Populin
1. Methanol: water 70:30 0.52 0.84
2. Ethylacetate: methanol 70:30 0.48-0.68 0.74-0.83
Figure 4.18: TLC plate showing bands of phenolic glycosides in bark of P. ciliata methanolic
extract (Sa) with standard populin (P), salicin (S) in solvent system ethyl acetate: methanol
(70:30) (a); methanol: water (70:30) (b).
Salicin Populin
P Sa S P Sa S
Chapter 4 Results
School of Biological and Environmental Sciences 76
4.4.3 Phenolic glycosides (salicin and populin) quantification
Validation and quantification of chromatographic conditions for RP-HPLC method
was performed for the estimation of two phenolic glycosides salicin (Table 4.10) and
populin (Table 4.11). Among all tried experiments, the mobile phase combination of
acetonitrile with 0.10 % formic acid and water of HPLC grade in the ratio of 40:60 v/v with
RP-HPLC flow rate of 0.50 ml/min was found to be most suitable. Best resolution and
sensitivity of the method was obtained for salicin and populin at 254 nm (Fig. 4.19 & 4.20).
A calibrated curve of salicin and populin was prepared separately using different
concentration (1, 2, 3, 4 and 5 µg/ml) of pure salicin (Fig. 4.21) and populin (Fig. 4.22).
Typical chromatogram with optimized condition gave sharp and symmetric peak with
specific retention time of 12.13 ± 0.01 minutes for salicin (Fig. 4.23), whereas for populin
it was 21.23 ± 0.19 (Fig. 4.23).
Figure 4.19: Chromatogram of standard salicin showing peak with Rt- 12 at 254 nm.
Figure 4.20: Chromatogram of standard populin showing peak with Rt -21 at 254 nm.
Chapter 4 Results
School of Biological and Environmental Sciences 77
Table 4.10: Validation and optimization of HPLC for salicin
Table 4.11: Validation and optimization of HPLC for populin
Sr. No. Validation parameter Results
1 Linearity range 4.00-5.00 ppm
2
Regression equation Slope 210752
Intercept 137050
3 Regression coefficient 0.99
4
Precision % RSD first day 0.07
% RSD second
day
0.06
5 Accuracy (% recovery) Mean 100.20
SD 2.82
% RSD 2.81
6 LOD (Limits of detection) 0.02
7 LOQ (Limits of quantification) 0.06
Sr. No. Validation parameter Results
1 Linearity range 1.00-5.00 ppm
2
Regression equation Slope 65213
Intercept 82133
3 Regression coefficient 0.99
4
Precision %RSD first day 0.45
% RSD second day 0.41
5 Accuracy (% recovery) Mean 101.28
SD 2.41
% RSD 2.38
6 LOD (Limits of detection) 0.05
7 LOQ (Limits of quantification) 0.15
Chapter 4 Results
School of Biological and Environmental Sciences 78
Figure 4.21: Calibration curve of salicin for the estimation of salicin content from bark of P.
ciliata tree.
Figure 4.22: Calibration curve of populin for the estimation of populin content from bark of
P. ciliata tree.
y = 65213x + 82133R² = 0.9965
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
0 1 2 3 4 5 6
Are
a (
mA
U)
Concentration of Salicin (µg/ml)
y = 210752x + 137050R² = 0.998
0
200000
400000
600000
800000
1000000
1200000
1400000
0 1 2 3 4 5 6
Are
a (
mA
U)
Concentration of Populin (µg/ml)
Chapter 4 Results
School of Biological and Environmental Sciences 79
Figure 4.23: Chromatograms of pure salicin and populin at different concentration, i.e. 1
µg/ml (a), 2 µg/ml (b), 3 µg/ml (c), 4 µg/ml (d) and 5 µg/ml (e).
Chapter 4 Results
School of Biological and Environmental Sciences 80
4.4.3.1 Monthly variation in populin and salicin content
The bark of female and male trees of P. ciliata in each month, i.e. from January to
December were collected and analyzed for two phenolic glycosides (salicin and populin)
content. Methanolic extracts of bark of both trees showed an almost similar pattern of two
peaks. The chromatogram was characterized by the dominant salicin and populin peaks of
standard at approximately Rt = 12 and 21 minutes respectively (Table 4.12).
Table 4.12: Retention time of the main peaks
Peaks Retention time (minutes) Compound
Salicin (S) 11.91 – 12.15 Salicin
Populin (P) 20.47 – 21.50 Populin
The salicin and populin content were measured in both male and female trees
quantitatively at each month of one year (2013) from January to December. The month
wise description of salicin and populin content variation in both male and female trees is
given below:
January
In female tree, the chromatogram was characterized by the presence of three
dominant sharp peaks and five short peaks (Fig. 4.30). Peaks of salicin and populin are
indicated with S1F at Rt = 12.10 and P1F at Rt = 21.13, respectively (Table 4.13 & 4.16; Fig.
4.30). The salicin content was found to be 1.17 mgg-1DW, whereas the populin content was
found to be 0.0005 mgg-1DW which was much less than salicin content (Table 4.15; Fig.
4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of three sharp
peaks in which two were unknown. The peak of salicin is indicated by S1M at Rt = 12.05
and populin is represented by P1M at Rt = 21.09 (Fig. 4.31; Tables 4.14 & 4.17). The salicin
content was found to be 2.32 mgg-1DW, whereas populin content was observed 0.031 mgg-
1DW (Table 4.15; Fig. 4.25 & 4.27).
Chapter 4 Results
School of Biological and Environmental Sciences 81
February
In female tree, the chromatogram was characterized by the presence of only two
dominant sharp peaks (one known) and six very short peaks of which five were unknown
(Fig. 4.32). Peaks of salicin and populin contents are indicated with S2F at Rt = 11.98 and
P2F at Rt = 20.93, respectively (Tables 4.13 & 4.18; Fig. 4.32). The salicin content was 1.23
mgg-1DW and populin content was found to be 0.009 mgg-1DW. In this month salicin
content has increased about 5.00 %, whereas populin content was decreased ca. 49 % as
compared to month of January (Table 4.15; Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of two sharp (one
known) and three small peaks (three unknown). The peak of salicin is indicated by S2M at
Rt = 12.03 and populin is represented by P2M at Rt = 20.85 (Fig. 4.33; Tables 4.14 & 4.19).
The salicin content was found to be 1.52 mgg-1DW, whereas populin content was observed
0.011 mgg-1DW. In this month both salicin and populin content was seen to be decreased
ca. 34 % and 65 %, respectively as compared to January (Table 4.15; Fig. 4.25 & 4.27).
March
The chromatogram of bark of female tree was characterized by the presence of three
unknown and one known dominant sharp peaks and another five to six short peaks, of
which only one was known (Fig. 4.34). Peaks of salicin and populin are indicated with S3F
at Rt =11.99 and P3F at Rt = 20.93 respectively (Tables 4.13 & 4.20; Fig. 4.34). The salicin
content was found to be 1.816 mgg-1DW and populin content was found to be 0.012 mgg-
1DW. In this month both salicin and populin contents increased to 32 % and 20 % when
compared to the month of February (Table 4.15; Fig. 4.24 & 4.26).
The chromatogram of male tree was characterized by the presence of three sharp
and seven small peaks, of these only two were known. The peak of salicin is marked by
S3M at Rt = 11.84 and populin is represented by P3M at Rt = 21.39 (Fig. 4.35; Tables 4.14
& 4.21). The observed salicin content was 2.46 mgg-1DW, whereas populin content was
0.016 mgg-1DW. In this month both salicin and populin contents were analyzed to be
increased to 62 % and 45 %, respectively as compared to last month (Table 4.15; Fig. 4.25
& 4.27).
Chapter 4 Results
School of Biological and Environmental Sciences 82
April
In female tree, the chromatogram was characterized by the presence of six unknown
and two known peaks (Fig.4.36). Peaks of salicin and populin are indicated with S4F at Rt
= 11.91 and P4F at Rt = 21.01, respectively (Tables 4.13 & 4.22; Fig. 4.36). The salicin
content was found to be 2.81 mgg-1DW and populin content was found to be 0.004 mgg-
1DW. In this month, salicin content was estimated to have increased to 35 % and populin
content was found to be decreased to 63 % as compare to month of March (Table 4.15; Fig.
4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of five sharp and
four small peaks of which only two were known. The peak of salicin is indicated by S4M at
Rt = 11.74 and populin is represented by P4M at Rt = 20.67 (Fig. 4.37; Tables 4.14 & 4.23).
The salicin content was observed to be 4.37 mgg-1DW, whereas populin content was 0.050
mgg-1DW. In this month both salicin and populin content was found to be increased to 77
% and 221 %, respectively as compared to the month of March (Table 4.15; Fig. 4.25 &
4.27).
May
In female tree, the chromatogram was characterized by the presence of two
dominant sharp peaks and two to three very short peaks (Fig. 4.38). Peaks of salicin and
populin are indicated with S5F at Rt = 11.97 and P5F at Rt = 20.89, respectively (Tables 4.13
& 4.24; Fig. 4.38). The salicin content was found to be 1.51 mgg-1DW and populin content
was found to be 0.007 mgg-1DW. In this month both salicin content was observed to
decreased to 46 % and populin content came down to 53 % as compared to month of April
(Table 4.15; Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of three sharp
and seven small peaks of which only one peak was known. The peak of salicin is indicated
by S3M at Rt = 11.97, whereas the populin peak P3M at Rt = 20.89 was absent (Fig. 4.39;
Tables 4.14 & 4.25). The observed salicin content was 1.93 mgg-1DW, whereas populin
content was 0. In this month both salicin and populin content was reduced to 55 % and 100
% respectively as compared to the month of April (Table 4.15; Fig. 4.25 & 4.27).
Chapter 4 Results
School of Biological and Environmental Sciences 83
June
In female tree, the chromatogram was characterized by the presence of two
dominant sharp peaks of which one known and five unknown minute peaks (Fig. 4.40).
Peak of salicin is indicated with S6F at Rt = 11.91, whereas populin peak at P6F at Rt = 20.89
was absent (Tables 4.13 & 4.26; Fig. 4.40). The salicin content was found to be 1.08 mgg-
1DW. In this month both salicin and populin content was found to have decreased to 28 %
and 100 % respectively as compared to month of the May (Table 4.15; Fig.4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of three sharp
and seven short peaks embodying only two were known peaks. The peak of salicin is
indicated by S6M at Rt = 11.99, whereas the populin peak P6M at Rt = 20.95 was very small
(Fig. 4.41; Tables 4.14 & 4.27). The salicin content came to be 1.07 mgg-1DW, whereas
populin content was found to be increased, i.e. 0.004 mgg-1DW. In this month both salicin
and populin contents were found to have increased as compared to the May (Table 4.15;
Fig. 4.25 & 4.27).
July
In female tree, the chromatogram was characterized by the presence of two
dominant sharp peaks of which one is known and two to three unknown minute peaks (Fig.
4.42). Peaks of salicin and populin are indicated with S7F at Rt = 12.00 and P7F at Rt = 21.14
respectively (Tables 4.13 & 4.28; Fig. 4.42). The salicin content was found to be 0.3266
mgg-1DW and populin content was found to have 0.0001 mgg-1DW. In this month, salicin
content decreased, whereas populin content increased in comparison to June (Table 4.15;
Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of two sharp and
six small peaks encompassing only one known peak. The peak of salicin is indicated by
S7M at Rt = 12.07, whereas the populin peak P7M at Rt = 21.11 was absent (Fig. 4.43; Tables
4.14 & 4.29). The salicin content was observed to be 0.71 mgg-1DW. In this month salicin
content came down to 33 % (Table 4.15; Fig. 4.25 & 4.27).
August
The female tree chromatogram was characterized by the presence of three dominant
sharp peaks while no small peaks were observed (Fig. 4.44). Peaks of salicin is indicated
Chapter 4 Results
School of Biological and Environmental Sciences 84
with S8F at Rt = 11.97, whereas populin peak P8F at Rt = 21.25 was absent (Tables 4.13 &
4.30; Fig. 4.44). The salicin content was found to be 0.26 mgg-1DW and populin content
came to 0. In this month both salicin and populin contents showed a declining trend (Table
4.15; Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of two sharp and
two small peaks. The peak of salicin is indicated by S8M at Rt = 12.03, whereas the populin
peak P8M at Rt = 21.38 was again absent like female tree chromatogram (Fig. 4.45; Tables
4.14 & 4.31). The salicin content was found to be 0.53 mgg-1DW, whereas populin content
was nil. In this month salicin content decreased as compared to July (Table 4.15; Fig. 4.25
& 4.27).
September
In female tree, the chromatogram was characterized by the presence of three
dominant sharp peaks with one was known peak. No small peaks were noticed (Fig. 4.46).
Peaks of salicin is indicated with S9F at Rt = 12.15 (Tables 4.13 & 4.32; Fig. 4.46). The
salicin content was found to be 0.17 mgg-1DW and populin content was found to be 0. In
this month salicin content was decreased to 35 % and populin content was unchanged,
similar to previous month (Table 4.15; Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of three sharp
and two to three small peaks. Only populin and salicin were known. The salicin and populin
peaks are represented by S9M at Rt = 12.09 and P9M at Rt = 20.83 respectively in
chromatogram (Fig. 4.47; Tables 4.14 & 4.33). The salicin content was found to 1.0017
mgg-1DW, whereas populin content came to 0.012 mgg-1DW. Both salicin and populin
content was found to have increased to 88 % and 100 % respectively as compared to August
(Table 4.15; Fig. 4.25 & 4.27).
October
In female tree, the chromatogram was characterized by the presence of only three
dominant sharp peaks, including one known peak (Fig. 4.48). Peaks of salicin and populin
are indicated with S10F at Rt = 11.97 and P10F at Rt = 21.25 respectively (Tables 4.13 &
4.34 and Fig. 4.48). The salicin content was found to be 0.189 mgg-1DW and populin
Chapter 4 Results
School of Biological and Environmental Sciences 85
content again came to 0. The salicin content was found to be increased ca 10 %, whereas
populin content was totally nil (Table 4.15; Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of three sharp
(two known) and no small peak. The peak of salicin is indicated by S10M at Rt = 12.10,
whereas the populin peak P10M at Rt = 20.67 was found to be absent (Fig. 4.49; Tables 4.14
& 4.35). The salicin content was observed to be 3.90 mgg-1DW, whereas populin content
was found to be 0.021 mgg-1DW. Both salicin and populin contents were found to have
increased about 289 % and 76 % respectively as compared to last month (Table 4.15; Fig.
4.25 & 4.27).
November
In female tree, the chromatogram was characterized by the presence of three
dominant sharp peaks with one known peak. Small peaks were not found (Fig. 4.50). Peaks
of salicin is indicated with S11F at Rt =12.00 and poplin peak P11F at Rt = 20.90 was not
observed (Tables 4.13 & 4.36 and Fig. 4.50). The salicin content was found to be 0.37 mgg-
1DW. In this month salicin content was increased to ca 96 % (Table 4.15; Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of four sharp
peaks, of these two were known. The peak of salicin is indicated by S11M at Rt = 12.05,
whereas the populin peak is represented by P11M at Rt = 20.83 (Fig. 4.51; Table 4.14 &
4.37). The calculated salicin and populin content were found to be 0.81 mgg-1DW and 0.008
mgg-1DW respectively. In this month salicin content was decreased ca. 79 % and populin
content was ca. 64 % as compared to the October month (Tables 4.15; Fig. 4.25 & 4.27).
December
The female tree chromatogram was characterized by the presence of three dominant
sharp peaks of which only salicin peak is known (Fig. 4.52) and represented with S12F at
Rt = 11.98, whereas populin peak P12F at Rt = 21.01 was not detected (Tables 4.13 & 4.38;
Fig. 4.52). The salicin content was found to be 0.61 mgg-1DW and populin content was
found to be 0. In this month, salicin content was found to be increased about 64 % (Table
4.15; Fig. 4.24 & 4.26).
In male tree, the chromatogram was characterized by the presence of four sharp
peaks (one known). Only one known small peak was analyzed. The peak of salicin is
Chapter 4 Results
School of Biological and Environmental Sciences 86
indicated by S12M at Rt = 12.05, whereas the populin peak is represented with P12M at Rt =
20.83 (Fig. 4.53; Tables 4.14 & 4.39). The salicin content was observed to be 1.36 mgg-
1DW, whereas populin content was found to be 0.020 mgg-1DW. In this month both salicin
and populin contents were found to have increased about 67 % and 168 % respectively as
compared to the month of November (Table 4.15; Fig. 4.25 & 4.27).
4.4.3.2 Comparison between salicin and populin contents in both sexes (male and
female) of P. ciliata
Salicin content: The monthly status between male and female trees regarding
salicin content is shown in Fig. 4.28 and Table 4.15. Starting from September to April
salicin content was found to have increased and then from April to August it declined. This
trend was observed equally in both male and female trees. The trees produced maximum
content of salicin in the April, whereas minimum was observed in September in female and
August in male tree. The major difference between the two trees was observed in October
where salicin content in females was maximum ca. 3.90 mgg-1 DW, whereas in males, it
was minimum ca. 0.19 mgg-1DW. This difference in the two sex related well marked while
in remains the variation pattern was almost similar. But male tree bark has more salicin
content in all month as compared to female tree bark.
Populin content: The monthly analysis of populin content is shown in Fig. 4.29
and Table 4.15. However the male and female trees revealed differences in populin content,
being higher in male tree than the female like the trend observed in salicin content. In male
tree it was maximum from September to April (0.050 mgg-1DW). The lowest concentration
of populin was observed in June, while in remaining it was totally absent. On the other
hand, in female tree spreading over January to May the trend was positive while in another
months it was absent. In July populin production was insignificant (0.0001 mgg-1DW). The
maximum concentration of populin in female tree was observed in March (0.012 mgg-
1DW). This difference was found to be very much prominent in both the trees. However,
in male tree populin content was also observed from September onward till December and
also in June. In females populin content was found to be totally absent. Monthly variation
of populin content in both trees also revealed the similar pattern only from January to April
while in other months pattern was dissimilar.
Chapter 4 Results
School of Biological and Environmental Sciences 87
The comparison of salicin and populin content revealed that the concentration of
salicin and populin content was higher in males than in females. Salicin production in
general, was higher than the populin in both sexes of the trees.
4.4.4. Quantification of salicin and populin from buds and their comparison with bark
samples
In female tree buds, the chromatogram was characterized by the presence of four
dominant sharp peaks and three small peaks (Fig. 4.54). Whereas male tree buds were
characterized by the presence of four sharp and three small peaks (Fig. 4.55). The salicin
(S) and populin (P) content were observed 2.45 mgg-1DW and 1.60 mgg-1DW from female
while in males, they were seen 2.80 mgg-1DW and 2.00 mgg-1DW, respectively.
The comparison of salicin and populin content from buds and bark revealed that the
concentration of both phenolic glycosides was highest in buds as compared to bark of both
male and female trees. Similar to bark samples, male buds also have higher concentrations
of salicin and populin than females.
It was interesting to observe a significant larger peak, which was identified by LC-
MS studies using a solvent system (acetonitrile with 0.10 % formic acid and distilled water,
HPLC grade) as cinnamoyl- salicin M+ at 463 (M-H+HCOOH + H i.e. 416 + 46 + 1) (Fig.
4.56). The position of location of salicinnate could not be ascertained due to the absence of
NMR of the pure compound. The presence of this molecule appears to be new to literature
as no such molecule has been reported so far. It may be again mentioned that its presence
is significant and remain constant in flowering season of the year.
Chapter 4 Results
School of Biological and Environmental Sciences 88
Figure 4.24: Monthly Variation in concentration of salicin content in female tree.
Figure 4.25: Monthly variation in concentration of salicin content in male tree.
Sept. to Feb- Leaf sheading and budding
March to April- Flowering
May- Pollination
June-July- Fruit maturation
0.17
0.19
0.37
0.61
1.17
1.23
1.81
2.81
1.51
1.09
0.33
0.26
0 0.5 1 1.5 2 2.5 3
September
October
Nomber
December
January
February
March
April
May
June
July
August
Salicin content (mgg-1DW)
Mo
nth
va
ria
tio
n in
fem
ale
tre
e
62.61
243.77
57.96
85.14
116.20
75.98
154.40
272.98
137.63
53.53
44.35
44.27
0 50 100 150 200 250 300
September
October
November
December
January
Ferbruary
March
April
May
June
July
August
Salicin content (mgg-1DW)
Mon
thly
vari
ati
on
in
male
tre
e
Chapter 4 Results
School of Biological and Environmental Sciences 89
Figure 4.26: Monthly variation in concentration of populin content in female tree.
Figure 4.27: Monthly variation in concentration of populin content in male tree.
Sept. to Feb- Leaf sheading and budding
March to April- Flowering
May- Pollination
June-July- Fruit maturation
0
0
0
0
0.0005
0.009
0.012
0.004
0.007
0
0.0001
0
0 0.002 0.004 0.006 0.008 0.01 0.012 0.014
September
October
Nomber
December
January
February
March
April
May
June
July
August
Populin content (mgg-1DW)
Mo
nth
ly v
ari
ati
on
in
fem
ale
tre
e
0.012
0.021
0.008
0.020
0.031
0.011
0.016
0.050
0
0.004
0
0
0 0.01 0.02 0.03 0.04 0.05 0.06
September
October
November
December
January
Ferbruary
March
April
May
June
July
August
Populin content (mgg-1DW)
Mon
thly
vari
ati
on
in
male
tre
e
Chapter 4 Results
School of Biological and Environmental Sciences 90
Figure 4.28: Comparison in monthly variation in concentration of salicin content in both male
and female trees of P. ciliata.
Figure 4.29: Comparison in monthly variation in concentration of populin content in both
male and female trees of P. ciliata.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5S
ali
cin
con
ten
t (m
gg
-1D
W)
Trees of P. ciliata in each month
Female Male
Chapter 4 Results
School of Biological and Environmental Sciences 91
Table 4.13: Characteristic of salicin (SF) and populin (PF) chromatogram in every month
from January to December of P. ciliata (female tree)
Month Peak Retention time
(min.)
Peak Area
(%)
Peak Height
(%)
January
S1F 12.10 49.31 29.78
P1F 21.13 1.45 1.91
February
S2F 11.98 40.29 23.88
P2F 20.93 0.33 0.41
March
S3F 11.99 44.86 25.55
P3F 20.93 1.75 2.25
April
S4F 11.70 60.36 35.40
P4F 20.47 0.93 1.17
May
S5F 11.91 43.11 34.23
P5F 21.01 0.39 0.74
June S6F 11.91 48.50 34.66`
P6F 20.89 0.47 0.82
July S7F 12.01 40.85 26.46
P7F 21.14 0.38 0.37
August S8F 11.97 5.42 2.16
P8F 21.25 0.34 0.36
September S9F 12.15 21.57 19.91
P9F 21.51 0.32 0.34
October S10F 11.97 5.42 2.16
P10F 21.25 0.34 0.36
November S11F 12.01 6.10 3.06
S11F 20.90 0.55 0.44
December S12F 11.98 9.29 15.19
P12F 21.01 0.39 0.33
Chapter 4 Results
School of Biological and Environmental Sciences 92
Table 4.14: Characteristic of salicin (SM) and populin (PM) chromatogram in every month
from January to December of P. ciliata (male tree)
Month Peak Retention time
(min.)
Peak Area
(%)
Peak Height
(%)
January
S1M 12.05 32.38 19.79
P1M 21.09 1.95 1.72
February
S2M 12.03 45.54 25.67
P2M 20.85 0.23 0.23
March
S3M 11.84 27.24 16.82
P3M 21.39 2.63 2.26
April
S4M 11.74 29.48 20.80
P4M 20.67 1.32 1.15
May
S5M 11.97 48.77 35.08
P5M 20.89 0.28 0.41
June S6M 11.99 67.14 44.49
P6M 20.95 3.44 4.72
July S7M 12.07 38.36 23.30
P7M 21.11 0.48 0.57
August S8M 12.03 44.02 25.89
P8M 21.38 0.17 0.33
September S9M 12.09 17.02 15.72
P9M 20.83 1.21 1.24
October S10M 12.10 29.09 23.74
P10M 20.67 0.76 0.97
November S11M 12.05 20.63 12.43
S11M 20.83 1.34 1.14
December S12M 12.05 58.82 35.45
P12M 20.83 1.54 2.01
Chapter 4 Results
School of Biological and Environmental Sciences 93
Table 4.15: Concentration of salicin and populin content (mgg-1DW) in each month of one
year in male and female tree.
Month
Trees of P. ciliata
Female Male
Salicin
(mgg-1DW)
Populin
(mgg-1DW)
Salicin
(mgg-1DW)
Populin
(mgg-1DW)
January 1.17 0.0005 2.32 0.031
February 1.23 0.009 1.52 0.011
March 1.82 0.012 2.46 0.016
April 2.81 0.004 4.37 0.050
May 1.51 0.007 1.93 0
June 1.09 0 1.07 0
July 0.33 0.0001 0.71 0
August 0.26 0 0.53 0
September 0.17 0 1.00 0.012
October 0.19 0 3.90 0.021
November 0.37 0 0.81 0.008
December 0.61 0 1.36 0.020
Chapter 4 Results
School of Biological and Environmental Sciences 94
Table 4.16: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of January.
Figure 4.30: HPLC chromatogram of phenolic glycosides (salicin ‘S1F’ and populin ‘P1F’) in
the bark of P. ciliata (female tree) in the month of January.
Chapter 4 Results
School of Biological and Environmental Sciences 95
Table 4.17: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
January.
Figure 4.31: HPLC chromatogram of phenolic glycosides (salicin ‘S1M’ and populin ‘P1M’) in
the bark of P. ciliata (male tree) in the month of January.
Chapter 4 Results
School of Biological and Environmental Sciences 96
Table 4.18: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of February.
Figure 4.32: HPLC chromatogram of phenolic glycosides (salicin ‘S2F’ and populin ‘P2F’) in
the bark of P. ciliata (female tree) in the month of February.
Chapter 4 Results
School of Biological and Environmental Sciences 97
Table 4.19: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
February.
Figure 4.33: HPLC chromatogram of phenolic glycosides (salicin ‘S2M’ and populin ‘P2M’) in
the bark of P. ciliata (male tree) in the month of February.
Chapter 4 Results
School of Biological and Environmental Sciences 98
Table 4.20: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of March
Figure 4.34: HPLC chromatogram of phenolic glycosides (salicin ‘S3F’ and populin ‘P3F’) in
the bark of P. ciliata (female tree) in the month of March.
Chapter 4 Results
School of Biological and Environmental Sciences 99
Table 4.21: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
March
Figure 4.35: HPLC chromatogram of phenolic glycosides (salicin ‘S3M’ and populin ‘P3M’) in
the bark of P. ciliata (male tree) in the month of March.
Chapter 4 Results
School of Biological and Environmental Sciences 100
Table 4.22: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of April.
Figure 4.36: HPLC chromatogram of phenolic glycosides (salicin ‘S4F’ and populin ‘P4F’) in
the bark of P. ciliata (female tree) in the month of April.
Chapter 4 Results
School of Biological and Environmental Sciences 101
Table 4.23: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
April.
Figure 4.37: HPLC chromatogram of phenolic glycosides (salicin ‘S4M’ and populin ‘P4M’) in
the bark of P. ciliata (male tree) in the month of April.
Chapter 4 Results
School of Biological and Environmental Sciences 102
Table 4.24: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of May.
Figure 4.38: HPLC chromatogram of phenolic glycosides (salicin ‘S5F’ and populin ‘P5F’) in
the bark of P. ciliata (female tree) in the month of May.
Chapter 4 Results
School of Biological and Environmental Sciences 103
Table 4.25: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
May.
Figure 4.39: HPLC chromatogram of phenolic glycosides (salicin ‘S5M’ and populin ‘P5M’) in
the bark of P. ciliata (male tree) in the month of May.
Chapter 4 Results
School of Biological and Environmental Sciences 104
Table 4.26: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of June.
Figure 4.40: HPLC chromatogram of phenolic glycosides (salicin ‘S6F’ and populin ‘P6F’) in
the bark of P. ciliata (female tree) in the month of June.
Chapter 4 Results
School of Biological and Environmental Sciences 105
Table 4.27: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
June.
Figure 4.41: HPLC chromatogram of phenolic glycosides (salicin ‘S6M’ and populin ‘P6M’) in
the bark of P. ciliata (male tree) in the month of June.
Chapter 4 Results
School of Biological and Environmental Sciences 106
Table 4.28: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of July.
Figure 4.42: HPLC chromatogram of phenolic glycosides (salicin ‘S7F’ and populin ‘P7F’) in
the bark of P. ciliata (female tree) in the month of July.
Chapter 4 Results
School of Biological and Environmental Sciences 107
Table 4.29: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
July.
Figure 4.43: HPLC chromatogram of phenolic glycosides (salicin ‘S7M’ and populin ‘P7M’) in
the bark of P. ciliata (male tree) in the month of July.
Chapter 4 Results
School of Biological and Environmental Sciences 108
Table 4.30: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of August.
Figure 4.44: HPLC chromatogram of phenolic glycosides (salicin ‘S8F’ and populin ‘P8F’) in
the bark of P. ciliata (female tree) in the month of August.
Chapter 4 Results
School of Biological and Environmental Sciences 109
Table 4.31: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
August.
Figure 4.45: HPLC chromatogram of phenolic glycosides (salicin ‘8M’ and populin ‘P8M’) in
the bark of P. ciliata (male tree) in the month of August.
Chapter 4 Results
School of Biological and Environmental Sciences 110
Table 4.32: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of September.
Figure 4.46: HPLC chromatogram of phenolic glycosides (salicin ‘S9F’ and populin ‘P9F’) in
the bark of P. ciliata (male tree) in the month of September.
Chapter 4 Results
School of Biological and Environmental Sciences 111
Table 4.33: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
September.
Figure 4.47: HPLC chromatogram of phenolic glycosides (salicin ‘S9M’ and populin ‘P9M’) in
the bark of P. ciliata (male tree) in the month of September.
Chapter 4 Results
School of Biological and Environmental Sciences 112
Table 4.34: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of October.
Figure 4.48: HPLC chromatogram of phenolic glycosides (salicin ‘S10F’ and populin ‘P10F’) in
the bark of P. ciliata (female tree) in the month of October.
Chapter 4 Results
School of Biological and Environmental Sciences 113
Table 4.35: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
October.
Figure 4.49: HPLC chromatogram of phenolic glycosides (salicin ‘S10M’and populin ‘P10M’) in
the bark of P. ciliata (male tree) in the month of October.
Chapter 4 Results
School of Biological and Environmental Sciences 114
Table 4.36: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of November.
Figure 4.50: HPLC chromatogram of phenolic glycosides (salicin ‘S11F’ and populin ‘P11F’) in
the bark of P. ciliata (female tree) in the month of November.
Chapter 4 Results
School of Biological and Environmental Sciences 115
Table 4.37: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
November.
Figure 4.51: HPLC chromatogram of phenolic glycosides (salicin ‘S11M’ and populin ‘P11M’)
in the bark of P. ciliata (male tree) in the month of November.
Chapter 4 Results
School of Biological and Environmental Sciences 116
Table 4.38: Characterization of HPLC chromatogram of P. ciliata (female tree) in the month
of December.
Figure 4.52: HPLC chromatogram of phenolic glycosides (salicin ‘S12F’ and populin ‘P12F’)
in the bark of P. ciliata (female tree) in the month of December.
Chapter 4 Results
School of Biological and Environmental Sciences 117
Table 4.39: Characterization of HPLC chromatogram of P. ciliata (male tree) in the month of
December.
Figure 4.53: HPLC chromatogram of phenolic glycosides (salicin ‘S12M’ and populin ‘P12M’)
in the bark of P. ciliata (male tree) in the month of December.
Chapter 4 Results
School of Biological and Environmental Sciences 118
Table 4.40: Characterization of HPLC chromatogram of P. ciliata (female tree buds) in the
month of January.
Figure 4.54: HPLC chromatogram of phenolic glycosides (salicin ‘S’ and populin ‘P’) in the
buds of P. ciliata (female tree) in the month of January.
Chapter 4 Results
School of Biological and Environmental Sciences 119
Table 4.41: Characterization of HPLC chromatogram of P. ciliata (male tree buds) in the
month of January.
Figure 4.55: HPLC chromatogram of phenolic glycosides (salicin ‘S’ and populin ‘P’) in the
buds of P. ciliata (male tree) in the month of January.
Chapter 4 Results
School of Biological and Environmental Sciences 120
Figure 4.56: LC-MS of buds extract
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100
AMITA PC-1 FA 149 (1.572) Cm (137:174) 1: TOF MS ES+ 1.40e3463.1
1402
442.2618
104.1415 177.1
387
107.1352 163.0
298
147.148
325.1284
309.1115
181.194 267.1
84
256.378
195.170
213.169
249.145
295.158
340.3136
339.250
381.198365.1
69340.8
52441.2
50
447.2442
464.1212
465.2128
479.282
491.3;43
Cinnamoyl- salicin M+
at 463 (M-
H+HCOOH + H i.e. 416 + 46 + 1)