4.1 Leaf morphology of male and female adult treesshodhganga.inflibnet.ac.in/bitstream/10603/72718/1/10... · 2018. 7. 8. · 4.1 Leaf morphology of male and female adult trees The

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.

Chapter 4 Results


Figure 4.1: Leaves of female tree of P. ciliata

Figure 4.2: Leaves of male tree of P. ciliata

Chapter 4 Results


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/

Chapter 4 Results


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)

Chapter 4 Results


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 %

Chapter 4 Results


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

Chapter 4 Results


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

Chapter 4 Results


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

Chapter 4 Results


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.

Chapter 4 Results


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

Chapter 4 Results


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).

Chapter 4 Results


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.

Chapter 4 Results


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.

Chapter 4 Results


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.

Chapter 4 Results


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 (++)

Chapter 4 Results


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).

Chapter 4 Results


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

Chapter 4 Results


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

Chapter 4 Results


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

[email protected]

Chapter 4 Results


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

[email protected]

Chapter 4 Results


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


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


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


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


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


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


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


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).


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


June


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).


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


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


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


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).


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


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).


(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


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


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


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


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


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


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


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


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


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


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


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



of February.


the bark of P. ciliata (female tree) in the month of February.

Chapter 4 Results



February.


the bark of P. ciliata (male tree) in the month of February.

Chapter 4 Results



of March


the bark of P. ciliata (female tree) in the month of March.

Chapter 4 Results



March


the bark of P. ciliata (male tree) in the month of March.

Chapter 4 Results



of April.


the bark of P. ciliata (female tree) in the month of April.

Chapter 4 Results



April.


the bark of P. ciliata (male tree) in the month of April.

Chapter 4 Results



of May.


the bark of P. ciliata (female tree) in the month of May.

Chapter 4 Results



May.


the bark of P. ciliata (male tree) in the month of May.

Chapter 4 Results



of June.


the bark of P. ciliata (female tree) in the month of June.

Chapter 4 Results



June.


the bark of P. ciliata (male tree) in the month of June.

Chapter 4 Results



of July.


the bark of P. ciliata (female tree) in the month of July.

Chapter 4 Results



July.


the bark of P. ciliata (male tree) in the month of July.

Chapter 4 Results



of August.


the bark of P. ciliata (female tree) in the month of August.

Chapter 4 Results



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



of September.


the bark of P. ciliata (male tree) in the month of September.

Chapter 4 Results



September.


the bark of P. ciliata (male tree) in the month of September.

Chapter 4 Results



of October.


the bark of P. ciliata (female tree) in the month of October.

Chapter 4 Results



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



of November.


the bark of P. ciliata (female tree) in the month of November.

Chapter 4 Results



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



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



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


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


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


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)

Documents

4.1 Leaf morphology of male and female adult treesshodhganga.inflibnet.ac.in/bitstream/10603/72718/1/10... · 2018. 7. 8. · 4.1 Leaf morphology of male and female adult trees The