Chapter 4

Phytochemical analyses of the extracts

4.1 INTRODUCTION

The phytochemicals present in various plants are responsible for medicinal properties of the
plants. These phytochemicals are the secondary metabolites of the plants, which play a significant
role in the treatment of various diseases like cancer, diabetes and coronary heart disease and
various ailments related to oxidative stress (Boyer and Liu, 2004; Saxena et al., 2013).
Alkaloids, glycosides, flavonoids, phenolics, saponins, tannins, terpenes, anthraquinones,
essential oils and steroids are some phytochemicals present in plant which are responsible for the
therapeutic value of the plant. Medicinal properties of the plant are due to either a single
compound or synergistic effect of various compounds present in crude extract of plants
(Rasoanaivo et al., 2011; Karna et al., 2012; Solowey et al., 2014).
There are many drugs which are derived from plants. Most of the plant derived popular drugs are
phenolics and alkaloid e.g. morphine (an alkaloid), salicylic acid or aspirin (a type of phenolic
acid), digitoxin (a cardiac glycoside), quinine (an alkaloid) and forskolin (an alkaloid), etc.
(Balunas and Kinghorn, 2005; Salim et al., 2008; Veeresham, 2012). Quantitative and qualitatative
analyses of phytochemical involve various techniques, such as High performance liquid
chromatography (HPLC), High performance thin layer chromatography (HPTLC), Gas
chromatography-Mass spectrometery (GC-MS) and spectrophotometer based techniques
(Khoddami et al., 2013). High Performance Thin Layer Chromatography (HPTLC) is a powerful
analytical tool in analyzing qualitatively and quantitatively phytochemicals present in the plant
extract (Andola and Purohit, 2010). HPTLC is more advanced form of TLC and is far better than
TLC in accuracy, reproducibility and documentation of data (Attimarad et al., 2011). HPTLC
evaluates the data with variety of parameters (Siddiqui et al., 2013). GC-MS is a combination of
two different analytical techniques Gas chromatography and Mass spectrometry to identify
different substances of a sample (Hussain and Maqbool, 2014).Technique is suitable for analysis of
active constituents of the plant with low polarity, low boiling point, and constituents which get
easily vaporized. Technique has its advantage due to high resolution, sensitivity and good
reproducibility and low cost (Xue et al., 2015). GC-MS relies on separation compounds based on

45
 the difference in their distributions coefficients (Sermakkani and Thangpandian, 2012).Gas
chromatography (GC) portion separates compounds on the bases of their volatility with flowing
inert gas, which act as carrier in the stationary phase, and spectra of the compounds are identified
by MS (Mass Spectrometer). MS identify chemicals according to ratio of their mass and charge
(Hussain and Maqbool, 2014). This mass to charge ratio is compared with the standard database of
the samples, National Institute Standard and Technology (NIST), which have more than 62,000
patterns (Sermakkani and Thangpandian, 2012).
In the present study, leaf extracts, prepared through pressurized liquid extraction of E. sphaericus
and P. wallichiana were subjected to phytochemical analyses using modern methods.

4.2 METHODOLOGY
Concentrations of different plant extracts used in present study was 1 mg ml-1 (which is equivalent to
a). 10 mg ml-1 dried leaf of E. sphaericus for EM, b). 11.26 mg ml-1 of dried leaf of E. sphaericus
for EA, c) 18.38 mg of dried leaf of P. wallichiana for PWM, and d). 11.76 mg ml-1 of dried leaf of
P. wallichiana for PWA).
4.2.1 Preliminary phytochemical analysis
Preliminary qualitative analyses of phytoconstituents were done with the help of spectrophotometric
based colorimetric assays (Dubey et al., 2014). Extract ID used as given in table 3.2.
Tests for carbohydrates
The following tests were performed for the presence of carbohydrates.
a) Fehling’s test: One ml of Fehling’s solution A and 1ml of Fehling’s solution B were mixed and
boiled for 1 minute. Equal volumes of extract (2ml) were added and heated in the water bath for 5-
10 minutes for observing red precipitate. Glucose was used as positive control.
b) Benedict’s test: Equal volumes (1ml) each of Benedict’s reagent and plant extract were mixed
and heated in boiling water bath for 5 minutes. The reducing sugars were analyzed by the presence
of green, yellow or red precipitate depending on the amount of the sugar. Glucose was used as
positive control.
c) Barfoed’s reagent test: Equal volumes, 1 ml each of the reagent and the plant extract were added
and heated in the water bath for 1-2 min. Mixture was then cooled to observe red precipitate for the
presence of the carbohydrates. Glucose was used as positive control.

46
Tests for proteins
a) Biuret test: To the 3 ml of plant extract, few drops of 4% NaOH and few drops of 1% CuSO 4
were added and the solution was observed for pink or violet colour development.
b) Xanthoprotein test: One ml concentrated H2SO4 was mixed with 3 ml of plant extract and the
white precipitate was observed for the confirmation of proteins.
c) Precipitation test: Two ml of plant extract was added to 3-4 drops of 5% lead acetate and 5%
CuSO4 and the reaction mixture was observed for the white precipitate to confirm the proteins.
d) Ninhydrin test: Three ml plant extract was mixed with 3-4 drops of 5% Ninhydrin solution.
Mixture was heated in boiling water bath for 10 minutes and the appearance of purple or bluish
colour was observed.
Test for phenols
a) Ferric chloride test: Two ml of extract was mixed with few drops of 5% FeCl3 reagent and
the mixture was observed for the formation of deep blue-black colour.
b) Lead acetate test: The few drops of 10% lead acetate solution were added to 2 ml of the
extract and the mixture was observed for formation of a white precipitate.
Test for flavonoids
a) Aqueous sodium hydroxide test: Few drops of 1N aqueous NaOH solution were mixed with
2 ml of extracts. The mixture was observed for the formation of yellow-orange colouration.
b) Sulphuric acid test: One ml of extract was treated with few drops of H2SO4 and mixture was
observed for formation of orange colour.
Test for saponin glycosides
a) Foam test: Two ml of plant extract was taken in a test tube and shaked, after formation of the
foam it was left stable for 15 min. If foam exists, it conform the presence of saponin glycosides.
4.2.2 Determination of total phenolic content
Total phenolic content (TPC) was analyzed using Folin-Ciocalteu method following,
Singleton and Rossi, 1965 and Cheng et al., 2003. One fifty microleter of the plant extract was
mixed with 150 µl Folin-Ciocalteu reagent (FCR) 0.25 N and 2400 µl water was added to it. This
mixture kept for incubation for 3 min in dark, afterwards, 300 µl of 1N Na2CO3 was added to it.
This mixture was thoroughly mixed and kept for incubation for 2 hr. The O.D. was taken at 765
nm. Different dilutions of gallic acid (0.01 mg ml-1, 0.02 mg ml-1, 0.04 mg ml-1, 0.08 mg ml-1 and

47
0.1 mg ml-1) were used as a standard for drawing the calibration curve. The result was expressed as
mg gm-1 gallic acid equivalent (GAE)*.
Formula used for calculation-
*GAE= X.V/M
-1
[X: concentration of extract (mg ml ), V: volume of extract (ml), M: weight of extract (gm)]
4.2.3 Determination of total flavonoid content
Total flavonoid content (tfc) was analyzed according to the method given by Singleton and
Rossi, 1965; Cheng et al., 2003. One ml of the extract was mixed with 2 ml of distilled water and
then 0.15 ml of NaNo2 (5%) was mixed in it and incubated for 6 min. After incubation, 0.15 ml of
AlCl3 solution (10%) was added and the mixture was allowed to stand for next 15 min before
taking the O.D. at 510 nm. Different dilutions of rutin (0.01 mg ml-1, 0.02 mg ml-1, 0.04 mg ml-1,
0.08 mg ml-1 and 0.1 mg ml-1) were used as a standard for drawing the calibration curve. The result
was expressed as mg gm-1 rutin equivalent (RE)*.
Formula used for calculation-
*RE = X.V/M
(Where X: concentration of extract (mg ml-1), V: volume of extract (ml), M: weight of extract
(gm)
4.2.4 Analysis by High Performance Thin Layer Chromatography (HPTLC)
HPTLC analysis of phytochemicals was performed following Menon et al., 2012.
Flavonoids (hesperidin and quercetin), phenolic (gallic acid) and vitamin C (ascorbic acid) were
used as a standard for HPTLC analysis.
Preparation of sample solution for HPTLC
Twenty mg lyophilized extract was dissolved in 1ml methanol. Insoluble extract was removed and
the effective concentration is given in table 4.1, depending upon the solubility of different extracts.
Preparation of standards solution for HPTLC study
A standard mixture, consisting 200µl each of gallic acid, ascorbic acid, hesperidin and quercetin,
was prepared from a stock solution of 1 mg ml-1 in methanol of each standard. All standards and
solvents used in this analysis were purchased from sigma Aldrich.

48
 Table 4.1 Concentrations of the extracts used for HPTLC analysis.
S. No. Sample ID Concentration of sample solution (mg ml-1)

1. EA 9.67

2. EM 11.62

3. PWA 12.51

4. PWM 14.91

[Link] Development of HPTLC technique

Ten µl of each sample was spotted in the form of bands with CAMAG microliter syringe on a
0.2 mm thick pre-coated silica gel 60 F254 HPTLC plate using CAMAG LINOMAT 4 Applicator.
Bands of 6 mm width were applied using the automatic LC sampler 4 (ATS4, CAMAG, Muttenz,
Switzerland) with a track distance of 8 mm. The spots were dried in a current of air. The mobile
phase used was ethyl acetate: dichloromethane: glacial acetic acid: formic acid: methanol in the
ratio of (5/5/0.5/0.5/1 v/v). Then plate was placed inside the chamber and developed until the solvent
front had travelled at a distance of 150 mm above the base of the plate. Afterwards, plate was
removed from the chamber and dried in a current of air. Detection and quantification was performed
with CAMAG Scanner III at a wavelength of 254 nm, with following conditions:
1. Slit dimension 4.00 mm X 0.30 mm.
2. Scanning speed 20 mm per second.
3. Data resolution 100 m step-1.
UV absorption spectrum of each standard was overlaid with the corresponding band on the
basis of retention factor (Rf values) in the sample track. For quantification, Rf and area under curve
(AUC) were analyzed with winCATS Planar Chromatography Manager software. Calibration curve
was plotted for all standards using five dilutions of the standards (200 ng, 400 ng, 800 ng, 2000 ng,
4000 ng). Gallic acid, hesperidin and ascorbic acid quantified together and quercetin were quantified
separately in E. sphaericus extract, as it was not detected in first plate.
[Link] Derivatization for the presence of antioxidants
Derivatization was done in order to show the presence of antioxidants in the respective
samples. After developing complete chromatogram of standard and extracts on TLC plate, it was

49
dipped in DPPH solution concenbtration (24 mg ml-1) (Satam et al., 2013). Plates were observed
under 254 nm and 366 nm wavelentgh for the conversion of white to yellow zones in the samples.
4.2.5 Analysis by Gas Chromatography-Mass Spectrometery (GC-MS)
Methanolic extracts of E. sphaericus and P. wallichiana (EM and PWM) were analyzed
through GC-MS (Fiehn, 2002; Das et al., 2014). The sample was prepared by dissolving lyophilized
methanolic extract of E. sphaericus and P. wallichiana in methanol at a concentration of 1 mg ml-1.
One µl of each sample was injected in GC-MS system for the analysis of possible active
phytoconstituents. GC-MS analysis was performed at University Science Instrumentation Centre,
AIRF, Jawaharlal Nehru University, Delhi in GCMS QP2010 Plus (Shimadzu, Kyoto, Japan) ultra
system, which was interfaced with a mass spectrometer. Following condition were employed during
the operation of instrument. The GC-MS system had a elite-1 fused silica capillary column, having a
dimension of 30 × 0.25 mm Id × 0.25 μm df, composed of 5% diphenyl 95% dimethyl poly siloxane,
operated in the electron impact mode with ionization energy of 70Ev. Helium gas (99.999%) was
-1
used as a carrier gas at a constant flow rate of 1 ml min with an injection volume of 2 µl and
having split ration 10:1. Scan interval time was 3 minutes and fragment size range was 40-500
Daltons. Total GC running time was 23 minutes. Average peak area was compared with total peak
area for getting relative % amount of each component.
[Link] Indication of the compounds
Mass spectra were interpreted with the help of NIST library. Spectrum of unknown was
compared with the databases of spectrum of known present in NIST library. Name, Molecular
weight and structure of the components of the test material were ascertained.
4.2.6 Statistical Analysis
Experiment was repeated at least in triplicates. Mean values along with standard deviation
are used for plotting the graphs. Significant differences between two data series are calculated
through student’s T test. P value ˂ 0.05 are taken as significant and are represented in the figure *.

4.3 RESULT
4.3.1 Preliminary phytochemical analysis
Preliminary qualitative analysis of the both the plant extracts have revealed the presence of
carbohydrate, proteins, phenols, flavonoids in aqueous and methanolic extracts (table 4.2). Saponin

50
glycosides were present in aqueous extracts of both the plants, but were found absent in methanolic
extracts of both the plants (table 4.2).
Table 4.2 Preliminary qualitative analysis of the plant extract.
Phytochemicals EA EM PWA PWM
Carbohydrates + + + +
Proteins + + + +
Phenols + + + +
Flavonoids + + + +
Saponin Glycosides + - + -
+ = Presensce of phytochemicals - = Absensce of phytochemicals

4.3.1 a) Total phenolic content

Different concentrations of gallic acid (table 4.3) were used to plot standard calibration curve for
TPC (linear equation, Y= 4.096 X + 0.049 and regression coefficient, R2 = 0.988) (table 4.4, figure
4.1a). The TPC values of all extracts are shown in figure 4.1. Values of TPC of the extracts (based
upon mean values of O.D. 765 nm) are shown in figure 4.1 (b) & table 4.4. The E. sphaericus
extracts (TPC of EA 54.333 ± 0.577 mg gm-1 GAE & TPC of EM 94.666 ± 4.040 mg gm-1 GAE
have shown the higher TPC than that of P. wallichiana extracts (TPC of PWA 45.000 ± 1.000 mg
gm-1 GAE & PWM 26.776 ± 2.260 mg gm-1 GAE ).TPC of EM was the highest among all i.e.
(94.66 ± 4.040 mg gm-1 GAE). Two extracts of same plants have shown significant differences in
the TPC value.
Table 4.3 Total Phenolic content assay for standard, gallic acid.

Gallic acid (mg ml-1) Mean value of O.D. 765 nm ± SD

0.01 0.089 ± 0.006

0.02 0.125 ± 0.004

0.04 0.216 ± 0.004

0.08 0.404 ±0.005

51
 0.10 0.438 ± 0.043

Table 4.4 O.D. and quantitative analysis of total phenolic content in different solvent extracts of E.
sphaericus and P. wallichiana.

Plant extracts Mean value of O.D. 765 nm ± SD Total phenolic content

( mg gm-1 GAE ) ± SD

EA 0.276 ± 0.007 54.333 ± 0.577

EM 0.438 ± 0.016 94.666 ± 4.040

PWA 0.236 ± 0.004 45.000 ± 1.000

PWM 0.160 ± 0.007 26.776 ± 2.260

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a)

b)
Figure 4.1 Analysis of total phenolic content of the extracts through TPC assay. a) Standard graph
of gallic acid, plotted between O. D. at 765 nm at Y-axis and gallic acid concentrations at X-axis.
b) TPC of different extract of E. sphaericus and P. wallichiana. *P value<0.05.

53
4.3.1 b) Total flavonoid content
Different dilutions of rutin (table 4.5) were used to plot standard calibration curve. For TFC
calculation linear equation was derived (Y=1.216 X + 0.04) with a regression coefficient, R2 =
0.997 (table 4.6 and figure 4.2 a). Total flavonoid contents of different extracts of the plants are
shown in figure: 4.2 b. TFC was found maximum for the methanolic extract of the leaves of E.
sphaericus (172.33 ± 3.21 mg gm-1 RE), followed by aqueous extract (65.33 ± 2.08 mg gm-1 RE)
(table 4.6).
TPC value of PWA & PWM were reported 86.666 ± 2.081 mg gm-1 RE & 39.666 ± 2.516 mg gm-1
RE respectively. PWA has shown higher TFC than that of EA, whereas EM has shown higher TFC
value than that of PWM (table 4.6).

Table 4.5 Total flavonoid content assay for standard, rutin.

Rutin (mg ml-1) Mean value of O.D. 510 nm ± SD

0.01 0.054 ± 0.004

0.02 0.062 ± 0.005

0.04 0.088 ± 0.005

0.08 0.140 ± 0.004

0.10 0.160 ± 0.006

54
Table 4.6 O. D. and quantitative analysis of total flavonoid content in different solvent extracts of
E. sphaericus and P. wallichiana.

Plant extracts Mean value of O. D. 510 nm ± SD Total flavonoid content

(RE mg g-1 of extract ) ± SD

EA 0.120±0.002 65.333±2.081

EM 0.258±0.012 172.333±3.214

PWA 0.146±0.002 86.666±2.081

PWM 0.088±0.003 39.666±2.516

(O. D. Optical density)

55
a)

b)
Figure 4.2 Analysis of total flavonoid content of the extracts through TFC assay. a) Standard
graph of rutin, plotted between O.D. at 510 nm at Y-axis and rutin concentrations at X-axis. b)
TFC of different extract of E. sphaericus and P. wallichiana. *P value<0.05.

56
 4.3.2 Analysis by High performance Thin Layer Chromatography (HPTLC)
The analyses of selected phenolics were done with the help of HPTLC. A good resolution of
the bands was obtained in the solvent, ethyl acetate: dichloromethane: glacial acetic acid: formic acid:
methanol (5:5:0.5:0.5:1 v/v). The HPTLC method was validated for specificity and linearity. Linear
regression equation and correlation coefficient for each standard were estimated by five point
calibration curve (figure 4.3, table 4.7). Correlation coefficient varied from 0.990 to 0.999, suggesting
a good linearity response for the developed method. The optimized solvent system (mobile phase)
gave the best resolution, with symmetric and reproducible peaks confirmed by specific Rf values for
each standard (ascorbic acid: 0.31, gallic acid: 0.74, hesperidin: 0.20, and quercetin: 0.87) (table 4.7).
The plates were visualized under UV light at 254 nm and 366 nm for generating fingerprints of
phytoconstituents (figure 4.4). The identity of the compounds in the bands was confirmed by
comparing the Rf values of the bands in samples with that of standards in the chromatograms (figure
4.4).

Table 4.7 Linear regression and Rf of standards used in HPTLC analysis.

Standards Rf Linear regression Correlation coefficient (R2)

equation via area

Ascorbic Acid 0.31 Y = -1569.00 + 5.797 X 0.994

Gallic Acid 0.74 Y = 797.50 + 4.566 X 0.995
Hesperidin 0.20 Y = 251.20 + 0.997 X 0.999
Quercetin 0.87 Y = - 457.20 + 4.571 X 0.990

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 Figure 4.3 Calibration curve of a) ascorbic acid, b) hesperidin, c) gallic acid, and d) quercetin.

Densiometric chromatogram indicated the presence of ascorbic acid, gallic acid, hesperidin
and quercetin in different concentrations in the aqueous and methanolic extracts of E. sphaericus and
P. wallichiana (figure 4.5). Aqueous and methanolic extracts of E. sphaericus as well as P.
wallichiana have shown the presence of ascorbic acid. Methanolic extract of E. sphaericus has
shown the highest amount of ascorbic acid (21.01 mg gm-1 of the extract), followed by aqueous
extract of E. sphaericus (15.53 mg gm-1 of the extract). Aqueous and methanolic both extracts of P.

58
wallichiana have shown the presence of ascorbic acid but at relatively lower concentrations (PWA
5.85 mg gm-1 of the extract & PWM 5.18 mg gm-1 of the extract) (Table 4.8).
Quercetin has shown its presence in aqueous and methanolic both extracts of E. sphaericus
and P. wallichiana. EM has shown the highest amount of quercetin i.e.7.70 mg gm-1 of the extract,
followed by EA, 3.61 mg gm-1 of the extract. Quercetin was found in PWA & PWM in the
concentrations of 3.50 mg gm-1 and 1.04 mg gm-1 of the extracts, respectively.
Maximum amount of gallic acid was present in the EA (4.11 mg gm-1 of the extract),
followed by the EM (1.19 mg gm-1 of the extract). Gallic acid was not detected in aqueous extract
of P. wallichiana, whereas in methanolic extract, it was present below detection limit (table 4.8).
EM has shown the maximum amount of hesperidin (7.67 mg gm-1 of the extract), followed
by EA (2.61 mg gm-1 of the extract). Aqueous extract of P. wallichiana aqueous did not show the
presence of hesperidin, but in the methanolic extract it was present below detection limit (from
table 4.8). Thus, HPTLC analyses have revealed the presensce of good amount of above mentioned
phenolics in the different extracts of E. sphaericus & P. wallichiana.

Table 4.8 Quantification of ascorbic acid, quercetin, gallic acid, and hesperidin in extracts.
Phytochemicals Concentration Concentration Concentration Concentration of
of standards in of standards in of standards in standards in
EA (mg gm-1) EM (mg gm-1) PWA (mg gm-1 PWM (mg gm-1)

Ascorbic acid 15.53 21.01 5.85 5.18

Quercetin 3.61 7.70 3.50 1.04
Gallic Acid 4.11 1.19 #ND *BDL
Hesperidin 2.61 7.67 #ND *BDL

(#ND – Not Detected *BDL- Below Detection

59
 a) b)

Figure 4.4 HPTLC Chromatograms. a) HPTLC Chromatogram at 254 nm; b) HPTLC

Chromatogram at 366 nm. Q: Quercetin, GA: Gallic Acid, AA: Ascorbic acid, H: Hesperidin, EA:
Aqoueous extract of E. sphaericus, EM: Methanolic extract of E. sphaericus, PWA: Aqueous
extract of P. wallichiana, PWM: Methanolic extract of P. wallichiana, S1, S2, S3, S4, S5: Different
dilutions of standards (S1: 200 ng, S2: 400 ng, S3: 800 ng, S4: 2000 ng, S5:4000 ng).

60
 Rf

Figure 4.5 Densiometric chromatogram of plant extracts used. a) & e)Densiometric

chromatogram of all standard used (AA:ascorbic acid, GA: gallic acid, Q:quercetin,
HESP:hesperidin); b)Densiometric chromatogram of aqueous extract of E. Sphaericus for
61
ascorbic acid, gallic acid, hesperidin; c) Densiometric chromatogram of aqueous extract of E.
sphaericus for quercetin; d) Densiometric chromatogram of aqueous extract of P. wallichiana for
ascorbic acid and quercetin; f) Densiometric chromatogram of methanolic extract of E. sphaericus
for ascorbic acid, gallic acid and hesperidin; g) Densiometric chromatogram of methanolic
extract of E. sphaericus for quercetin; h) Methanolic extract of P. wallichiana for ascorbic acid,
gallic acid, quercetin, hesperidin.

4.3.3 Derivatization analysis

Derivatization results confirmed the presence of antioxidants as a result of change in colour
of resolved bands of extracts from white to yellow. Lanes of the extracts have shown yellow spots
after derivatization, which confirm the presence of antioxidants in the extracts (figure 4.6).

a) b)
Figure 4.6 Derivatization of bands on HPTLC plate. a) Images of plates before derivatization; b)
Images of plates after derivatization in 24%DPPH. Q: Quercetin, GA: Gallic Acid, AA: Ascorbic
acid, H: Hesperidin, EA: Aqoueous extract of E. sphaericus, EM: Methanolic extract of E.
sphaericus, PWA: Aqueous extract of P. wallichiana, PWM: Methanolic extract of P. wallichiana,
S1, S2, S3, S4, S5: Different dilutions of standards used (S1: 200 ng, S2: 400 ng, S3: 800 ng, S4:
2000 ng, S5:4000 ng).

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 4.3.4 Analysis by Gas Chromatography-Mass Spectrometery (GC-MS)
Phytoconstituents of the EM and PWM were analyzed through GC-MS also. Unknown
compounds were determined by comparing the ion chromatogram spectra of the unknown
compouns with the spectra of available compounds in the database of the library of National
Institute of Standard and Technology (NIST Library) on the basis of retention time (RT),
molecular mass & peak area.
The total ion chromatogram of EM has shown that the retention time of different
compounds ranged from 3.157 to 36.68 minutes (figure 4.7).
Linoleyl alcohol, stigmost-5-en-3-ol, pentadecanoic acid and methyloctadeca-9, 12 deonate
were the compounds found with high peak area per cent in the methanolic extract of leaves of E.
sphaericus (table 4.9 and figure 4.8).
Relaive Abundance

Retention time (minutes)

63
Figure 4.7 Total Ion chromatogram (TIC) of methanolic extract (EM) of the leaves of E.
sphaericus.

a)

b)

c)

d)

Figure 4.8 Standard molecular spectra of a) linoleyl alcohol, b) Stigmast-5-En-3-ol, c)

Methyloctadeca-9-12-deonate and d) Pentadecanoic acid compounds.

64
GC-MS analyses have indicated the presensce of 53 compounds in the EM (table 4.9).

Table 4.9 Total compounds reported in the methanolic extracts of E. sphaericus through GC-
MS analysis.
Molecular
Peak Retention Time Area% Name
Fomula
1 3.157 1.84 Di-glyceraldehyde dimer
C6H12O6
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-
2 4.467 1.01
3-one C6H8O4
3 4.743 2.86 2H-pyran-2,6(3H)-dione
C5H4O3
4 5.301 0.90 D-Limonene
C10H16
1,6-Octadien-3-ol, 3,7-dimethyl-(linalyl
5 6.476 1.79
propionate) C13H22O2
6 6.796 0.94 Benzeneethanol
C8H12O
2,3-Dihydro-3,5-dihydroxy-6-methyl-4h-
7 7.302 2.84
pyran-4-one C6H8O4
8 7.600 0.36 Acetic acid( phenylmethyl ester)
C9H10O2
9 7.870 0.42 Terpinen-4-ol
C10H18O
10 8.084 2.95 Dodecane
C12H26
11 8.565 2.25 6-Octen-1-ol, 3,7-dimethyl- (Citronellol)
C10H20O
2,3-Dihydroxypropyl acetate
12 8.864 0.70 C5H10O4

13 8.955 1.03 3-Methyl-2-butenoic acid, decyl ester

C15H28O2
14 9.348 0.56 2-Propenal, 3-phenyl- (Cinnamaldehyde)
C9H8O
Phenol, 5-methyl-2-(1-methylethyl)-
15 9.587 0.36
(Thymol) C10H14O
1,6-Octadien-3-ol, 3,7-dimethyl-, formate
16 9.701 0.55
(Linalool) C11H18O2
17 10.383 0.65 Undecane, 4,4-dimethyl-
C13H28
2,6-Octadien-1-ol, 3,7-dimethyl-, acetate, 1
18 10.572 0.35
(Z)- C12H20O2
19 10.835 2.83 Geranyl acetate
C12H20O2
20 11.023 3.98 Tetradecane
65
 C14H30
Bicyclo[7.2.0]undec-4-ene, 4,11,11-
21 11.522 0.48
trimethyl-(Caryophyllene) C15H24
7-Isopropenyl-4a-methyl-1-
22 12.409 0.54
methylenedeca(β-selinene) C15H24
23 12.710 0.80 4-Cyclopropylmethylbenzonitrile
C11H11N
24 12.979 0.59 Nonadecane, 9-methyl-
C20H42
25 13.207 0.33 2-Bromotetradecane
C14H29Br
26 13.554 3.13 Heptadecane
C17H36
1,2-Benzenedicarboxylic acid, diethyl
27 13.638 2.31
ester (di-ethyl phthalate) C12H14O4
1,3,4,5-Tetrahydroxy-cyclohexanecarboxy
28 13.856 3.56
(quinic acid) C7H12O6
Methyl (3-oxo-2-
29 14.320 0.76 pentylcyclopentyl)acetate
C13H22O3
(dihydrojasmonate)
1-Naphthalenol, 1,2,3,4,4a,7,8,8a- C15H26O
30 14.439 0.67
octahydro-
1-(4-isopropylphenyl)-2-methylpropyl
31 14.562 1.00 C15H22O2
acetate
3-Methyl-4-(2,6,6-trimethyl-1-
32 14.922 0.30
cyclohexen-1-yl C14H22O
33 15.277 0.24 Pentadecane, 8-hexyl-
C21H44
34 15.415 0.82 Octanal, 2-(phenylmethylene)-
C15H20O
Benzoic acid, phenylmethyl ester (benzyl
35 15.663 0.23
benzoate) C14H12O2
36 15.797 2.51 Tricosane
C23H48
37 15.933 0.46 9-Octadecenoic acid (Z)- (oleic acid)
C18H34O2
2,6,10-Trimethyl,14-ethylene-14-
38 16.239 1.42
pentadecne C20H38
3,7,11,15-Tetramethyl-2-hexadecen-1-ol
39 16.687 0.66
(phytol) C20H40O
40 17.293 0.67 1,4-Dioxacyclohexadecane-5,16-dione
C14H24O4
Methyl Ester of 3-(3,5-di-tert-butyl-4-
41 17.403 0.93
hydroxyphenyl-propionic acid C18H28O3
42 17.482 7.40 Pentadecanoic acid
C15H30O2

66
 1,2-Benzenedicarboxylic acid, dibutyl
43 17.608 2.25
ester (Di butyl pthalate) C16H22O4
44 17.828 0.36 Eicosane
C20H42
2-hexadecen-1-ol, 3,7,11,15-tetramethyl-,
45 18.965 0.83
[r-[(phytol) C20H40O
46 19.219 12.30 9,12-Octadecadien-1-ol (Linoleyl alcohol)
C18H34O
47 19.376 2.79 Octadecanoic acid (Stearic acid)
C18H36O2
Methyl octadeca-9,12-dienoate (methyl
48 19.592 5.56
lineolate) C19H34O2
9,12-Octadecadienoic acid (Z,Z)- (linoleic
49 19.829 0.41
acid) C18H32O2
50 19.952 3.07 9,12-Octadecadienoic acid (Z,Z)-
C18H32O2
51 21.165 0.48 1-Phenanthrenecarboxylic acid, 7-ethenyl C21H32O2
2,5,7,8-Tetramethyl-2-(4,8,12-
52 32.338 1.92
trimethyltridecyl-3 (vit E, α-tocopherol) C33H54O5
53 36.682 11.04 Stigmast-5-en-3-Ol
C29H50O
Total
100

Methanolic extract of the needles of P. wallichiana was reported to contain around 38

compounds in the GC-MS analysis (figure 4.9 and table 4.10). According to the total ion
chromatogram retention time of different compounds ranged from 3.12-36.6 minutes (figure 4.9).
Momeinositol, 9, 12-octadecanoic acid, pentadecanoic acid, dibutyl phthalate were the compounds
revealed in the GC-MS analysis with the high peak area per cent (figure 4.10).
This is the first report on the GC-MS analyses of E. sphaericus & P. wallichiana, which revealed
the presensce of various compounds of industrial and pharmaceutical importance.

67
ce
Relative abundance

Time (minutes)

Figure 4.9 Total Ion chromatogram (TIC) of methanolic extract (PWM) of the needles of P.
Wallichiana.

68
a)

b)

c)

d)
Figure 4.10 Standard molecular spectra of a) momeinositol b) pentadecanoic acid c) octadecanoic
acid compounds d) Dibutyl phthalate compounds.

69
Table 4.10 Total compounds reported in the methanolic extract of needles of P. wallichiana.

Peak Retention Area % Name Molecular Formula

Time
1 3.127 1.38 Dl-Glyceraldehyde dimer C6H12O6

2 4.275 1.64 1,2,3-Propanetriol C3H8O3

(Glycerol)
3 4.676 0.57 Octane, 2,4,6-trimethyl- C11H24

4 7.289 2.07 2,3-Dihydro-3,5-dihydroxy-6- C6H8O4

methyl-4h-pyran-4-one
5 7.567 0.59 Benzoic acid C7H6O2

6 8.072 0.94 Dodecane C12H26

7 8.840 0.65 1,2,3-Propanetriol, 1-acetate C5H10O4

8 11.017 1.44 Tetradecane C14H30

9 13.552 0.82 Hexadecane C16H34

10 13.854 0.95 1,3,4,5-Tetrahydroxy- C7H12O6

cyclohexanecarboxy(quinic Acid)
11 13.991 0.83 Ethyl-alpha.-d-glucopyranoside C8H16O6

12 15.012 43.27 Mome Inositol C7H14O6

13 15.408 0.56 10-Methoxy-nb-alpha.- C21H29N2O2

methylcorynantheol.
14 15.798 0.53 Nonadecane C19H40

15 15.936 0.33 (1-Butyloctyl) C18H30

Benzene
16 17.125 0.16 Hexadecanoic acid, methyl ester C17H34O2
(Palmitic acid)
17 17.294 0.19 1,4-Dioxacyclohexadecane-5,16- C14H24O4
dione
18 17.403 0.35 Benzenepropanoic acid, 3,5-bis C24H40O3
(1,1-dimethylethyl)-4-hydro
19 17.478 4.12 Pentadecanoic acid C15H30O2

20 17.603 3.34 Dibutyl phthalate C16H22O4

70
 21 17.826 0.14 Eicosane C20H42
(Icosane)
22 18.778 0.22 3,7-Dihydroxy-3-phenyl-4- C15H12O4
chromanone
23 19.143 1.23 9,12-Octadecadienoic Acid (Z,Z)- C18H32O2

24 19.217 12.52 9,12-Octadecadienoic Acid C18H32O2

(Lionolelaidic acid )
25 19.372 1.41 Octadecanoic acid (Stearic acid) C18H36O2

26 19.581 5.03 9,12-Octadecadienoic acid (Z, Z)- C18H32O2

27 19.814 0.82 9,12-Octadecadienoic acid (Z, Z)- C18H32O2

28 19.941 3.96 9,12-Octadecadienoic acid (Z, Z)- C18H32O2

29 20.970 0.39 Ricinoleic acid C18H34o3

30 21.076 0.36 1-Methyl-4-methylene-2-(2- C15H34

methyl-1-propen
31 21.361 2.42 2,4a,8,8-Tetramethyl-decahydro- C15H26O
cycloprop(viridiflorol)
32 21.681 1.45 1,4,4-Trimethyl-8-methylene-1,5- C15H24
cycloundecaniene
33 22.061 0.49 1-Phenanthrenecarboxylic acid, 7- C21H32O2
ethenyl-1,2,3,4,4a,4b,5,6,
34 22.534 1.44 Dehydroabietic acid C20H28O2

35 26.459 1.25 Acetate, [6-(acetyloxy)-5,5,8a- C19H30O4

trimethyl-2-methyleneperhydro-1-
napthalenyl]Methyl ester
36 27.178 0.81 1-Phenanthrenecarboxylic acid, 7- C21H32O2
ethenyl (leopimaric acid)
37 31.518 0.54 10-Nonadecanol C19H40O

38 36.663 0.78 Stigmast-5-En-3-Ol, ([Link].)-(Β C29H50O

sitosterol )
Total: 100.00

4.4 DISCUSSION
Medicinal properties of E. sphaericus have been reported in various reports. The plant is well
known for its ethnopharmacological importance (Singh et al., 2010; Joshi and Jain, 2014). P.

71
wallichiana is known for its timber quality and medicinal properties (Jain et al., 2008). In the
present study, phytochemical analyses of extracts (aqueous and methanolic) of E. sphaericus and
P. wallichiana have been performed by TPC, TFC, HPTLC and GC-MS. The preliminary
qualitative phytochemical analyses have revealed the presence of phenols, flavonoids and saponin
glycosides in the extracts of E. sphaericus and P. wallichiana (table 4.2). Saponin glycosides were
present in aqueous extract of both the extract but in methanolic extract they were absent. Saponins
were also not reported in a previous study on methanolic seed extract of E. sphaericus (Tripathi et
al., 2015).
Total Phenolic content (TPC) and total flavonoid content (TFC) were quantified to measure
the redox potential (an important antioxidant property) of different extracts of both the [Link]
and TFC values were found higher in the extracts of E. sphaericus, when compared to that of
extracts of P. wallichiana (table 4.4, table 4.6). According to a previous study, TPC in ethanolic
extract of leaves of E. sphaericus was 56.79 ± 1.6 mg gm-1 GAE (Kumar et al., 2008), which is
lower than the TPC value of methanolic extract, reported in present study (94.66 ± 4.04 mg gm-1
GAE). Similarly, TFC of methanolic extract of leaves of E. sphaericus was found to be 172.33 ±
1.21 mg gm-1 RE (table 4.6) which was much higher than TFC value of ethanolic extract of leaves
of E. sphaericus (18.58 ± 0.3 mg gm-1 RE), reported in a previous study (Kumar et al., 2008).
Maimoona et al., (2011), did not report TPC in aqueous extract of needles of P. wallichiana, but in
the present study significant amount of phenolics have been reported (table 4.4). Differences in
solvent, plant parts and technique, used for extraction are responsible for these variations in
phenolic and flavonoids contents (Khoddami et al., 2013; Pietrzak et al, 2014). TPC & TFC values
have indicated the presensce of good amount of phenolics in the extracts prepared in present study
using PLE.
Different extracts of E. sphaericus and P. wallichiana were analyzed through HPTLC for
the presence of selected phytochemicals. E. sphaericus leaf extracts have shown the presence of
ascorbic acid, quercetin, gallic acid and hesperidin, whereas P. wallichiana needles extracts
showed the presence of ascorbic acid and quercetin (gallic acid and hesperidin was found to be
absent or below detection limit) (table, 4.8). Quercetin, the most studied and most distributed
flavonoid present in food, belongs to flavonol groups and is helpful in the treatment of various
diseases such as eye diseases, allergic disorders, arthritis, cancers and other (Lakhanpal and Rai,
2007). In a previous study, the quercetin (hyperoside type) was reported in methanolic seed extract

72
of E. sphaericus, ranging from 3.1 to 5 mg gm-1 (Awan, 2013). Presensce of quercetin in ethanolic
seed extract of E. sphaericus was also reported by Singh et al., 2013.
In a study, HPLC analysis of methanolic extract of needle and bark of P. wallichiana has
shown the presence of quercetin, which was absent in the aqueous extracts (Naeem et al., 2010).
Ascorbic acid or vitamin C is a well known antioxidant, which plays an important role in
human health. Biochemical role associated with vitamin C is the activation of certain enzymes,
collagen biosynthesis, hormonal activation, antioxidant, detoxification of histamine, phagocytic
functions of leukocytes, formation of nitrosamine, and proline hydroxylation (Walingo, 2005). It
also plays major role in controlling various diseases like cancer, blood pressure, low immunity,
drug metabolism and urinary hydroxyproline excretion (Lupulescu, 1994). The extract prepared by
PLE revealed the presence of high amount of ascorbic acid in the aqueous and methanolic extracts
of leaves of E. sphaericus and P. wallichiana.
Gallic acid, a polyphenol, has been reported for its anticancer, antioxidant and
antimicrobial properties (Verma et al., 2013; Nayeem et al., 2016). E. sphaericus extracts has
shown the presence of gallic acid and hesperidin, whereas these were found either absent or below
detection limit in the extracts of P. wallichiana.
Hesperidin is a flavonoid known to be used as anticancer, antioxidant and anti-
inflammatory agent. Citrus fruits are the good sources of hesperidin (Ahmadi et al., 2008; Parhiz et
al., 2015). To the best of our knowledge this is the first report of the quantification of gallic acid,
ascorbic acid and hesperidin in both the plants through HPTLC. Method of extraction, PLE may be
responsible for the good amount of phenolics & flavonoids in the extracts.
GC-MS was also applied for the analysis of phytochemicals in the extracts EM & PWM.
GC-MS analyses revealed the presence of several bioactive compounds of therapeutic and
industrial importance in the methanolic extract of leaves of E. sphaericus and in the needles of P.
wallichiana. Therapeutic and industrial importances of some compounds are discussed below.
Compound, 9-12-octadecadien-1-ol (linoleyl alcohol) has been reported for showing anti-
inflammatory, acne reductive and moisture retaining properties, which make it most applicable
products for cosmetic industry (Diezel et al., 1993; Letawe et al., 1998, Darmstadt et al., 2002).
Stigmast-5-en-3-ol has become a major interest of researchers due to its antidiabetic potential, as it
has shown properties similar to insulin and also activates transport of glucose. It also reduces
benign prostatic hyperplasia and blood cholesterol level (Wilt et al., 2000; Rudkowska et al., 2008;

73
Kim et al., 2012; Das et al., 2014). Studies have analyzed pentadecanoic acid as a marker for milk
fat consumption (Smedmen et al., 1999). D-Limonene was also detected in E. sphaericus
methanolic extract (0.9%), which is reported as a chemo-preventive and chemotherapeutic agent
(Crowell, 1999). Thymol is reported for antitumor properties (Andersen, 2006)
Extracts were also reported for the presence of compounds of industrial importance, which
are discussed below. One compound, 2,4-Dihydroxy-2,5-dimethyl-3(2h)-furan-3-one is used as
flavoring and sweetening agent, as it has aroma of strawberry (Ulrich et al., 1995). 4-Terpineol has
been reported for its anti-inflammatory, antibacterial and antifungal effect, and also considered the
primary active ingredient of tea tree oil (Hart et al., 2000; Hammer et al., 2012). Citronellol used
as mosquito repellent and mite attractant (Taylor and Schreck, 1985). Cinnamaldehyde has been
reported to be used as flavoring agent in chewing gum, ice cream, candy, and beverages
(Fahlbusch et al., 2003). It is also used as fungicide and insecticide (Cheng et al., 2008). Thymol
has been reported for its biocidal properties, having strong attributes with antimicrobial properties
and fungicide properties (Ahmad et al., 2010; Palaniappan and Holly, 2010). Linalool has been
reported as perfumery agent and insecticide (Ahmed et al., 2000). Geranyl acetate is used as
flavoring agent. Caryophyllene has been reported for its, anti-inflammatory, antinociceptive,
neuroprotective, anxiolytic and antidepressant properties (Gertsch et al., 2008; Guimaraes-Santos
et al., 2012; Katsuyama et al., 2013). Diethyl phthalate is often used to bind cosmetics and
fragrances (Api, 2001). Dihydrojasmonate is used as perfumery agent (Leffingwell, 2001). Benzyl
benzoate used as topical agent and insects repellent, it also used in the treatment of asthma and
cough due to its vasodilating effects (Shaikh, 2005; Bruhne and Wright, 2007; Burns, 2010).
Tricosane is a higher alkane, used in the formation of lubricating oil and fuel oil (Weast, 1982).
Oleic acid is used as emulsifying agent, as emollient and as solublizing agent in various aerosol
products (Smolinske, 1992; Carrasco, 2009). Pentadecane has been reported for its anti-microbial
activity (Mihailovic et al., 2011).
Phytol is used in the fragrance industry and used in cosmetics, shampoos, toilet soaps,
household cleaners, and detergents (Mc Ginty et al., 2010). Methyl Ester of 3-(3,5-Di-tert-butyl-4-
hydroxyphenyl-propionic acid has been reported for having good antioxidant potential (Zeinalov et
al., 1998). Linoleic acid has been reported for its anti-inflammatory, acne reductive, and moisture
retentive properties. It also used in the preparations of paint and varnishes (Diezel et al., 1993;

74
Letawe et al., 1998; Darmstadt et al., 2002). Vitamin E or α- tocopherol is used as antioxidant and
in the treatment of atherosclerosis (Villacorta et al., 2003; Traber et al., 2011).
A previous study, on essential oil’s GC-MS analysis of P. wallichiana, has shown presence
β-pinene and α-pinene, which has been reported for their antibacterial and cytotoxic activities (Dar
et al., 2012; Wang et al., 2012). Momeinositol have been reported for showing its antialopecic,
anti-cirrhotic, anti-neuropathic, cholesterolytic, lipotropic and as a sweetening agent (Das et al.,
2014). Pentadecanoic acid also reported in E. sphaericus, naturally which is very rare and present
only in cow’s milk (Smedmen et al., 1999).
Beside these compounds presence of other compounds were also indicated through GC-MS
analysis of P. wallichiana needles methanolic extracts e.g. glycerol which is used as solvent and
sweetening agent used food preservative and in the preparation of soap (Leffingwell et al., 1945).
Palmitic acid, which is used in soap preparation, cosmetics and as releasing agents. Ricinoleic acid,
which have the anti-inflammatory and analgesic properties (Vieria et al., 2000). Dehydroabietic
acid reduces glucose and insulin level in plasma of mice and useful for treating obesity related
disease (Kang et al., 2009).
Some compounds were found present in both plant extracts (table 4.11).
Table 4.11 Compounds indicated their presensce through GC-MS analysis in methanolic extracts
of both of the plant extracts (E. sphaericus and P. wallichiana).

S. Compounds Application/ Biological properties Reference

No

1. 2,3-Dihydro-3,5- As antioxidant Yu et al.,

Dihydroxy-6-Methyl-4h- 2013.
Pyran-4-one
2. Dodecane As solvent, distillation chaser, and scintillator Rydberg,
component and also as diluents 2004.
3. Quinic acid As astringent and starting material of tamiflu, Santos, 2011.
medicine used in the treatment of influenza A
and B virus.
4. Dibutyl thalate As plasticizer and additive of printing inks. David, 2006.
5. Stearic acid In preparation of soap, shampoo and Tseng et al.,
detergents and as lubricants. 1999.
Both plant extracts have shown the presensce of high amount of phenols and flavonoids of
medicinal and industrial value. Therefore these extracts can be can be used as good source of
various products of commercial and therapeutic importance.

75