الفهرس 
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Abstract
Summary
Phytochemical and Biological Studies on Pulicaria incisa sub. candolleana
Family Asteraceae
P. incisa sub. candolleana is a wild plant that belongs to family Asteraceae (Compositeae),
tribe inuleae, genus Pulicaria. It was interesting to intensively investigate one of the neglected
wild plants since few reports were found in the literature concerning its phytochemical and
biological activities.
The present work is divided into four main parts:
1. Investigation of essential oil contents in the leaves and flowers of Pulicaria incisa sub.
candolleana & determination of their biological activities
2. Phytochemical investigation of P. incisa sub. candolleana ethanolic extract.
3. Biological investigation of the P. incisa sub. candolleana ethanolic extract.
4. Molecular docking study for the isolated compounds with COX-2 and PLA-2.
Part I: Investigation of Essential Oil Contents in the Leaves and Flowers of Pulicaria
incisa sub. candolleana & Determination of their Biological Activities
Investigation of essential oil contents in both leaves and flowers of P.incisa sub. candolleana
includes:
a. Extraction of leaves and flowers essential oil contents.
b. GC-MS analysis of essential oils of both leaves and flowers of Pulicaria incisa sub.
candolleana.
c. Evaluation of the cytotoxic activity of both oils against liver carcinoma cell line (HepG-2)
d. Determination of antimicrobial activity of both oils against different microorganisms.
The essential oils were isolated for the first time from freshly collected leaves or flowers
of P. incisa sub. candolleana by hydrodistillation in Clevenger’s apparatus. Both oils were
bright yellow in color and have a strong pleasant aromatic odor. Both oils were analyzed using
GC/MS technique where sixty eight components representing 84.29% of the oil were identified
from the flowers essential oil and fourty nine compounds representing 86.69% of the leaf oil
were also identified. The major compounds identified in both oils were carvotanacetone
followed by chrysanthenone. This result is in agreement with the results of other reports which
showed that carvotanacetone was also the major constituent in the essential oils isolated from
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P. jaubertii leaves from southern Saudi Arabia (98.59%) (Fawzy et al., 2013), P. jaubertii
flowers from South Yemen (93.5%) (Al-Fatimi et al., 2015), leaves of P. undulata from
yemen (91.4%), P. mauritanica (87.3%) (Cristofari et al., 2011) and P. jaubertii aerial parts
from Yemen (64.0%) (Al-gabr et al., 2012). Also P. undulata oil from Sudan was composed
of 55.9% carvotanacetone (El-Kamali et al., 2009).
The biological activities of the essential oils were investigated regarding the cytotoxic and
antimicrobial activities. The cytotoxic activity of both oils were tested against liver carcinoma
cell line HepG-2 using MTT assay and vinblastine as a reference drug where the leaf oil
showed higher cytotoxic activity with IC50 = 11.4 μg /mL compared with 37.4 μg /mL for the
flower oil.
Regarding the antimicrobial activity, it was determined for both leaf and flower oils
against different gram +ve, gram –ve bacteria and fungi.
The MICs values shown by the leaf oil varied between 3.9 and 15.63 μg/mL for the fungi, 1.95
and 3.9 μg/mL for the tested Gram-positive bacteria and 62.45 μg/mL for E. coli. The leaf oil
showed no activity against P. aeruginosa and C. albicans
While the MICs shown by the flowers oil varied between 62.5 and 125 μg/mL for the
fungi, 32.5 and 62.5 μg/mL for the Gram-positive bacteria, additionally, the flowers oil showed
no activity against C. albicans and the tested Gram-negative bacteria
from the observed results; the tested Gram-positive bacteria (S. pneumonia and B. subtilis)
and Geotricum candidum fungi were more sensitive to the leaf oil than the tested gramnegative
bacteria and the other tested fungi. In similar studies, essential oils from P. inuloides,
P. stephanocarpa and P. jaubertii showed antimicrobial activity against B. subtilis while the
oil of P. inuloides showed antimicrobial activity against S. pneumoniae.
Part II: Phytochemical Investigations of P. incisa sub. candolleana Ethanolic Extract
This chapter includes four main parts:
1. Preliminary phytochemical screening of the dried aerial parts.
2. Quantitative assay of total phenolic and flavonoids contents of Pulicaria incisa sub.
candolleana using colorimetric assay.
3. Identification of the main phenolic constituents using HPLC/MS.
4. Isolation and identification of chemical compounds.
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from the preliminary phytochemical screening of the dried aerial parts of P. incisa sub.
candolleana, it was found that the plant contains flavonoids, carbohydrates, tannins, saponins,
sterols/triterpenes and coumarins in considerable amounts while it contains neither alkaloids
nor anthraquinones. These results are similar to a previous study performed on the fruits of P.
incisa (Lam.) DC. sub. candolleana concerning the presence of flavonoids, tannins, ellagic
acid, saponins, sterols/terpenes and the absence of the alkaloids (Samba et al., 2016).
In this study the concentration of the phenolic contents of the ethanolic extract of P. incisa
sub. candolleana was quantitatively estimated for the first time to be 3.25 mg GAE/ g extract
and the flavonoids concentration was also estimated and expressed as quercetin equivalent for
the total aqueous ethanolic extract to be 30.372 mg/g.
There were no previous studies about the main phenolic content of P. incisa sub.
candolleana, therefore, a qualitative study of the phenolic compounds of P. incisa sub.
candolleana ethanolic extract were carried out by using HPLC/MS/MS analysis in both –ve
and +ve operative mode. Interpretation of the results and comparison with the available
literature allowed the tentative identification of thirteen compounds including seven phenolic
acid derivatives and seven flavonoids mainly of flavonol type (table 51).
Successive column chromatographic fractionation of the total ethanolic extract has been
performed.
As a result of this study 4 compounds were individually separated from different fractions and
their chemical structures were identified using different techniques; MS, 1H & 13C- NMR,
HSQC, HMBC and COSY
They are identified as:
1- Eugenol-1-O-β-glucoside
2- Chlorogenic acid (5-Caffeoylquinic acid)
3- 3, 5-Dicaffeoyl quinic acid
4- Quercetin- 3-O- β- glucoside
Although eugenol and chlorogenic acid was previously isolated from P. paludosa, this work
is the first isolation of Eugenol-1-O-β-glucoside and 3,5--dicaffeoyl quinic acid from genus
Pulicaria.
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This study was also the first isolation of chlorogenic acid, 3,5--dicaffeoyl quinic acid and
quercetin- 3-O- β- glucoside from P. incisa sub. candolleana.
Part III: Biological Investigations of the Pulicaria incisa sub. candolleana Ethanolic
Extract:
Different biological activities of the alcoholic extract of the aerial parts of P. incisa sub.
candolleana was investigated including the antimicrobial activity and in vivo hepatoprotective
effect.
The antimicrobial activity of the extract was tested against various gram +ve, gram –ve
bacteria and fungi. The results indicated that the total alcoholic extract of P. incisa sub.
candolleana exhibited the greatest antimicrobial activity against both Geotricum candidum
and Klebisella pneumoniae with MIC = 1.9 μg/mL followed by Aspergillus flavus and Bacillus
subtilis with MIC = 3.9 μg/mL then C. albicans and E. coli with MIC= 7.81 μg/mL. On the
contrary the extract showed a weak activity against Micrococcus luteus and S. aureus with
MIC of 62.5 and 15.63 μg/mL respectively and showed no activity against P. aeruginosa and
Vibrio cholera.
Concerning the in vivo assessment of the hepatoprotective effect, this work represents the
first in vivo study concerning the hepatoprotective effect of P. incisa sub. candolleana ethanolic
extract. Three doses of the extract were tested (100, 250 and 500 mg/kg) and their effect was
assessed by determining some biochemical parameters such as AST, ALT and ALP levels and
the hepatic tissue content of MDA, GSH and SOD activity, TNF-α and MPO activity in MTXinduced
hepatic dysfunction in rats. Their hepatoprotective effect was also assessed through a
histopathological examination of liver in MTX-induced hepatic dysfunction in rats as
mentioned in p. 113. Silymarin (100 mg/kg) as a reference hepatoprotective substance.
Injection of 20 mg/kg of MTX intraperitoneal to the rats had significantly increased the
serum levels of AST, ALT and ALP to 92.12, 116.32 and178.56 Unit/mL respectively when
compared to those of normal control group 8.87, 28.67 and 64.11 Unit/mL respectively. After
administration of the different doses of the alcoholic extract of P. incisa sub. candolleana (100,
250 mg/kg) a significant decrease in the levels of AST, ALT and ALP is noticed while after
administration of 500 mg/kg no significant decrease observed when compared to the MTX
control group.
Treatment of rats with the dose of 100, 250 mg/kg for 4 days resulted in significant
decrease in serum AST level by 62.74 % and 68.4%, serum ALT level by 64.57 % and 72.4%
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and serum ALP level to 50.63 % and 57.62 % of MTX control value respectively compared
with 82.5%, 71% and 60% for the AST, ALT and ALP respectively in silymarin control group.
While treatment of rats with the dose of 500 mg/kg of the alcoholic extract of P. incisa
sub. candolleana decreased the serum AST level by only 2.82 %, serum ALT by 15.87% and
serum ALP level by 5.54% of MTX control value respectively.
from the previous results, the administration of the total alcoholic extract of the leaves of
P. incisa sub. candolleana at a dose of 250 mg/kg appeared to have the best effect on decreasing
AST, ALT and ALP levels
Assessing hepatic tissue content of MDA, the normal control value was 78.22
nmol/g.tissue while exposure of rats to MTX resulted in significant increase in hepatic tissue
content of MDA to 184.87 nmol/g.tissue.
Treatment of rats with the dose of 100, 250 mg/kg of the alcoholic extract of P. incisa sub.
candolleana for 4 days resulted in significant decrease in hepatic tissue content of MDA by
43.38 % and 50.06% of MTX control value respectively. The effect of the dose of 250 mg/kg
of the alcoholic extract of P. incisa sub. candolleana was nearly similar to the effect of Silymarin
group (100 mg/kg) which showed a significant decrease in the hepatic tissue content of MDA
by 53.62%. On the contrary, treatment of rats with the dose of 500 mg/kg resulted in a small
decrease of hepatic tissue content of MDA by 8.09%.
Hepatic tissue content of GSH showed a normal control value of 366.22 mg/g.tissue and
hepatic tissue activity of SOD showed a normal value of 145.76 mg/g.tissue. Exposure of
rats to MTX resulted in a significant decrease in hepatic tissue content of GSH to 110.65
mg/g.tissue and hepatic tissue activity of SOD to 49.93 U/mg.tissue.
Treatment of rats with the dose of 100, 250 mg/kg of the alcoholic extract of the leaves
of P. incisa sub. candolleana for 4 days resulted in significant increase in hepatic tissue
content of GSH by 126.53%, and 174.24% respectively and a significant increase in hepatic
tissue activity of SOD by 115.44% and 151% compared to MTX control value respectively.
The effect of the dose 250 mg/kg of the alcoholic extract of P. incisa sub. candolleana was
nearly similar to silymarin group (100 mg/kg) where the hepatic tissue content of GSH and
SOD significantly increased by 200.38% and 179.45% respectively. When using the highest
dose (500 mg/kg) of the extract, the hepatic tissue content of GSH slightly increased to
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48.31% while the hepatic tissue activity of SOD slightly increased to 26.42% which indicates
non-significant improvement.
Evaluating the anti-inflammatory activity of the alcoholic extract, a significant decrease in
hepatic tissue content TNF-α by 64.88% and 67.91% when the rats treated with the dose of
100 and 250 mg/kg of the alcoholic extract of P. incisa sub. candolleana, respectively, while
the hepatic tissue activity of MPO was decreased by 68.95 and 77.77% respectively
The group treated with the dose (500 mg/kg) showed very little response where TNF-α were
decreased only by 31.22% and MPO was decreased by 28.53%.
For the doses of 100 and 250 mg/kg the results were very close to those obtained with the
silymarin treated group (100mg/kg) which decreased the TNF-α and MPO values to 70.85 and
81.68% respectively.
It is worthy to note that the rats treated with the highest dose (500 mg/kg) of P. incisa sub
candolleana extract showed the least improvement in their livers compared with the other
doses and least effectiveness in the all tested parameters while the doses of 100 and 250
mg/kg, the results were very close to that of silymarin concerning all tested parameters and
histopathological changes.
Part IV: Molecular Docking Study of the Isolated Phytoconstituents from P. incisa sub.
candolleana as Anti-inflammatory Drugs
In this research, a molecular docking study of the isolated compounds from P. incisa sub.
candolleana was carried out at Drug Design and Molecular Modeling Unit, October 6
University, using Molecular Operating Environment (MOE, 2014.09) software. The study was
carried on COX-2 (PDB ID: 4PH9) and PLA-2 (PDB ID: 1DCY) to correlate them with the
anti-inflammatory properties.
In general, the studied compounds showed higher docking scores with COX-2 than PLA-
2. Accordingly, they expected to produce higher inhibitory effect on COX-2 than PLA2
enzyme.
All of the studied compounds showed docking scores with COX-2 in the range of (-13.32
to -19.12 Kcal/mol) which means that all of them showed anti-inflammatory activity more
effectively than the co-crystallized ligand (ibuprofen) (-12.09 kcal/mol) where quercetin-3-O-
β-glucoside was the most active compound, while the least scoring was observed with eugenol-
1-O-β-glucoside (-13.32 kcal/mol).
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When the investigated compounds were tried to bind with the active sites of PLA-2, they
showed less docking scores (-4.01 to -5.68 Kcal/mol) than the co-crystallized ligand (indole
derivative) (-8.65 Kcal/mol) which in turn means that the compounds are less active than
indole derivative