chapter 3 chapter-3. theoretical analysis s.no name of the...
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35
CHAPTER 3
Chapter-3. Theoretical Analysis
S.No Name of the Sub-title Page No
3.1. Synthesis of Semisynthetic Derivatives 39
3.1.1 General procedure adopted for the synthesis of
hesperidin/ naringin derivatives
41
3.1.2. Instrumentation 42
3.1.2.1. Melting point determination 42
3.1.2.2. Solubility 42
3.1.2.3. Thin layer chromatography 42
3.1.2.4. FT-IR Spectroscopy 42
3.1.2.5. 1H NMR spectroscopy 43
3.1.2.6. Mass spectroscopy 43
3.2 In-vitro biological screening 43
3.2.1 Anti-bacterial activity 43
3.2.1.1 Culture medium 43
3.2.1.2. Composition of Nutrient agar medium 43
3.2.2. Antifungal Activity 44
3.2.3. Antioxidant Activity 45
3.2.3.1. Chemicals and Reagents 45
3.2.3.2 Preparation of standard solutions 46
3.2.3.3. Preparation of test compound solutions 46
3.2.3.4. Scavenging of ABTS radical cation 46
36
3.2.3.4.1 Preparation of ABTS solution 46
3.2.3.4.2. Standard graph of ascorbic acid for ABTS assay 46
3.2.3.5. Scavenging of DPPH 47
3.2.3.5.1. Preparation of DPPH solutions 47
3.2.3.5.2. Standard graph of ascorbic acid for DPPH assay 47
3.2.3.6. Scavenging of nitric oxide radical 47
3.2.3.6.1 Standard graph of ascorbic acid for nitric oxide
assay
48
3.2.4 Anthelmintic activity 48
3.3. In-silico studies 49
3.3.1. Energy minimization of protein and ligands 49
3.3.2. Protein preparation 49
3.3.3. Ligand preparation 49
3.3.4. Docking 50
3.4. In-Vivo Biological Activity 50
3.4.1 Acute Toxicity Studies 50
3.4.2 Anti-inflammatory studies 50
3.4.2.1 Materials 50
3.4.2.2 Animals 51
3.4.2.3 Vehicle 51
37
CHAPTER 3
Theoretical analysis:
The newly approved drugs mentioned in our previous chapter
were derived from medicinal plants and have been launched in the
market during the year 2000 – 2005. These new drugs have been
approved for the treatment of cancer, neurological diseases, infectious
diseases, cardiovascular and metabolic diseases, immunological,
inflammatory and related diseases, and genetic disorders, which
encompass many of the common human diseases. Besides new drugs
launched on the market from 2000 to the present, there are a variety
of new chemical entities from natural sources undergoing clinical
trials. Thus further research on these compounds at industrial,
governmental, and academic institutions is seen as vital for the
enhancement of human health.
Latest trends in drug discovery focuses a lot on the semi-
synthetic derivatives, synthesized from the intermediate or the final
lead compound in order to reduce the toxic side effects of synthetically
obtained organic compound or synthesize some complicated drugs like
vincristine, vinblastine, taxols etc., or to optimize the pharmacological
activity, ADME properties of a drug obtained from a natural origin.
Many drugs are available for treating the infections caused by
various bacteria’s, viruses, and fungus. The synthetically derived
compounds gets resistance for these pathogens, thus there is a need
of new molecules in future to treat the infections caused by these
resistant pathogens.
38
The present study was designed to synthesize some novel
derivatives of some selected compounds. The compounds selected for
the present study are hesperidin and naringin. The above mentioned
compound was reported for various pharmacological activities.
Various derivatives of the above mentioned compounds were designed
with a motto to optimize the pharmacological activity and should be
less expensive for synthesis of those compounds. The derivatives used
for the synthesis are flavanone glycosides and many literatures have
claimed that presence of sugar moiety the compound will not be
absorbed in GIT, when the drug passes the small intestine the
glycosidic linkages are broken by the intestinal flora and absorption
occurs, since to avoid hydrolysis of these sugar moieties the reaction
will be performed with much care. The selected molecules are
obtained from the waste food products so there will not be much
problem in the availability of the starting material.
The above mentioned compounds are used regularly in day to
day life and derived from natural product it could be safer than the
other synthetically derived compounds.
In the present investigation semisynthetic derivatives of
hesperidin and naringin were synthesized. These phytoconstituents
are consumed daily as food products. Even though these compounds
are safer but it is not used for their pharmacological activity due to
less potency. Thus the aims and objectives of the present investigation
as follows:
39
To synthesize semisynthetic derivatives of hesperidin and
naringin, this follows one or two step reactions.
To study the purity of the compounds.
To study the characterization of hesperidin and naringin
derivatives by AT-IR, 1HNMR, MASS spectral analysis
To perform SAR studies on following biological activities for
hesperidin and naringin derivatives.
Antibacterial activity
Antifungal activity
In-vitro antioxidant activity
Anthelmintic activity
Acute toxicity studies
Anti-inflammatory activity
3.1. Synthesis of semisynthetic derivatives
The chemicals, reagents and solvents used in the study were
purchased in their pure form from the manufacturers as mentioned in
Table No.3.1. The chemicals and reagents used for the study are of LR
grade whereas the standard compound like hesperidin and naringin
were obtained in AR grade. All the solvents used were of analytical
grade. All the solvents were pure and the newly obtained packs are
used for the present study.
40
Table.3.1: List of chemicals and their manufactures used for
synthesis
S.No Chemicals required Manufacturers
1 NaOH Sd. fine chem ltd.
2 Semicarbazide hydrochloride Sd. fine chem Ltd.
3 Sodium acetate Sd. fine chem Ltd.
4 Thiosemicarbazide Himedia laboratories Pvt.Ltd.
5 Benzoic Acid Himedia laboratories
Pvt.Ltd.
6 P-Nitro benzoic acid Himedia laboratories Pvt.Ltd.
7 2-Amino-4-chloro benzoic
acid.
Himedia laboratories
Pvt.Ltd.
8 3,5 dinitro-2-hydroxy Benzoic acid
SISCO R L Pvt.Ltd.
9 Petroleum Ether Merck Pvt. Ltd.
10 Methanol Sd. fine chem Ltd.
11 Acetic acid Moly chem Pvt. Ltd.
12 DMSO Merck Pvt. Ltd.
13 Iso-propanol Himedia Laboratories
Pvt.Ltd.
14 Ethyl alcohol SISCO R L Pvt.Ltd.
15 HCl Sd. fine chem Ltd
16 H2SO4 Merck Pvt. Ltd.
17 Hydrazine hydrate Moly chem Pvt. Ltd.
18 Chloroform Moly chem Pvt. Ltd.
19 Ethyl acetate Merck Pvt. Ltd.
20 Acetone Merck Pvt. Ltd.
21 Formic acid Sd. fine chem ltd
22 Anisaldehyde Sd. fine chem ltd
23 phenyl hydrazine
hydrochloride Sd. Fine chem ltd
24 p-hydroxy benzaldehyde Hi media
25 m-Nitro benzaldehyde Hi media
41
26 2,4 Dihydroxy benzaldehyde Hi media
27 2,5Dimethoxybenzaldehyde Hi media
28 Hesperidin Hi media
29 Naringin Hi media
3.1.1. General procedure adopted for the synthesis of hesperidin/
naringin derivatives.
Hesperidin and naringin both are flavonoidal glycosides
containing glycon portion and aglycone portion attached by an
–Olinkage. But from the literature survey the presence of
–O glycosidic linkages in flavonoidal glycosides will certainly avoid the
degradation of the aglycone portion in the stomach. The –O glycosidic
linkages will be hydrolyzed in small intestine by intestinal enzymes
and the aglycone portion will be absorbed from the small intestine. In
order to avoid the degradation of the aglycone portion the author has
designed very simple procedure for the synthesis of various novel
semisynthetic derivatives.
The procedure adopted in the present study is a simple
condensation reaction where the above mentioned compounds were
treated with various hydrazine, semicarbazone and hydrazide analogs
to form their respective hydrazones, carbazones with elimination of
one molecule of water. In order to avoid the hydrolysis of –O glycosidic
linkages strong hydrolyzing reagents like strong acids and bases were
avoided. The reaction was performed under very mild condition and
vigourous heating was also avoided.
42
3.1.2. Instrumentation:
All the melting points recorded in this thesis were determined in
open capillaries, using DBK Instruments melting point apparatus,
expressed in 0C and are uncorrected. The IR spectra of the
compounds were recorded on Bruker spectrophotometer using ATIR
technique expressed in cm-1. 1HNMR spectra were recorded on a 300
mhz NMR spectrometer (New) model AVANCE 11-300 Bruker
spectrophotometer using TMS as an internal standard and the values
are expressed in δ ppm. Mass spectra of the compounds were
recorded by gas chromatograph-triple sector mass spectrometer (GC-
MS) model AUTOSPEC-M, micromass, UK operating in the ESI mode
expressed in m/z.
3.1.2.1. Melting point determination:
The melting points of the synthesized derivatives were
determined by open capillary tube method and are uncorrected.
3.1.2.2. Solubility:
The solubility of the synthesized compounds were tested in
various solvents.
3.1.2.3. Thin layer chromatography:
Chromatography is an important technique to identify the
formation of new compounds and also to determine the purity of the
compound. The Rf value is characteristic for each compound(s).
3.1.2.4. FT-IR spectroscopy
The peaks in IR spectrum gives an idea about the probable
structure of the compound, IR region ranges between 4000-666cm-1.
43
The spectrum of this region corresponds to vibrational energy levels of
molecules.
3.1.2.5. 1H NMR spectroscopy
The proton NMR spectra enable us to know different chemical
and magnetic environments corresponding to protons in molecules.
3.1.2.6. Mass spectroscopy
Mass spectroscopy gives us the relative molecular masses
(molecular weights) with very high accuracy.
3.2. In-Vitro biological screening
3.2.1. Anti-bacterial activity:
3.2.1.1. Culture medium
Nutrient broth was used for the preparation of inoculum of the
bacteria and nutrient agar was used for the screening method.
3.2.1.2. Composition of Nutrient agar medium
Peptone 5.0 g
Sodium chloride 5.0 g
Beef extract 1.5 g
Yeast extracts 1.5 g
Agar 15.0 g
Distilled water up to 1000 ml
pH 7.4 ± 0.2
The test organisms were sub cultured using nutrient agar
medium. The tubes containing sterilized medium were inoculated with
respective bacterial strain. After incubation at 37 ±10C for 24 hours,
they were stored in refrigerator. The stock cultures were maintained.
44
Bacteria inoculum was prepared by transferring a loopful of stock
culture to nutrient broth (100 ml) in conical flasks (250ml). The flasks
were incubated at 37 0C ±10C for 48 hours before the experimentation.
Solution of the test compounds were prepared by dissolving 10mg
each in dimethyl formamide (10 ml, AR grade). A reference standard
for both gram positive and gram negative bacteria was made by
dissolving accurately weighed quantity of ampicillin sodium in sterile
distilled water, separately.
3.2.2. Antifungal activity:
The fungi employed for screening wereA.niger , A.flavus,
A.clavatus and C.albicans. The test organisms were sub-cultured
using potato-dextrose-agar medium. The tubes containing sterilized
medium were inoculated with test fungi and after incubation at 250C
for 48 hours, they were stored at 40C in refrigerator. The inoculum
was prepared by taking a loopful of stock culture to about 100 ml of
nutrient broth, in 250ml conical flasks. The flasks were incubated at
250C for 24 hours before use.
The solutions of test compounds were prepared by a similar
procedure described under the antibacterial activity. Reference
standard (1mg/ml conc.) was prepared by dissolving 10mg of
clotrimazole in 10ml of dimethyl formamide (AR grade). Further, the
dilution was made with dimethyl formamide itself to obtain a solution
of 100 g/ml concentration.
45
3.2.3. Antioxidant activity102
Free radical often attack DNA, protein molecules, enzymes and
cells leading to alterations in genetic material and cell proliferation 103
Reactive oxygen species in biological systems are related to free
radicals, even though there are non-radical compounds in reactive
oxygen species such as singlet oxygen and hydrogen peroxide 104. A
broad classification of reactive oxygen species is as follows.
Reactive species
Reactive nitrogen species Reactive oxygen species
●Nitric oxide
●Nitric dioxide (NO2˙) Oxygen centered radical Oxygen centered
non-radical ●Superoxide anion(˙O2) ●Hydrogen peroxide (H2O2)
●Hydroxyl radical (˙OH) ●Singlet oxygen (O2)
●Alkoxyl radical (RO˙)
●Peroxyl radical (ROO˙)
3.2.3.1. Chemicals and reagents
ABTS [2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid)]
diammonium salt, p-nitroso dimethyl aniline (p-NDA), ascorbic acid,
nitro blue tetrazolium (NBT), 2-Deoxy –D-ribose, 2,2-Diphenyl 1-picryl
hydrazyl solution(DPPH), sodium nitroprusside, naphthyl ethylene
diamine dihydrochloride (NEDD), sulphanilic acid.
46
3.2.3.2. Preparation of standard solutions
Required amount of ascorbic acid was accurately weighed and
dissolved in distilled water to prepare 1 mM stock solution. Solutions
of different concentrations of ascorbic acid 10 nM, 30 nM, 100 nM,
300 nM, 1 M, 3 M, 100 M, 300M, 1 mM were prepared from stock
solution.
3.2.3.3. Preparation of test compound solutions
Required amount of test compound was dissolved in methanol
and 1mM stock solution was prepared. Solutions of concentrations
ranging from 100 nM to 1 mM were prepared from the stock solution.
3.2.3.4. Scavenging of ABTS radical cation
ABTS reacts with potassium persulphate to generate ABTS
radical which is a bluish green colour chromogen. Antioxidant
scavenges these radicals and forms colourless solution. Decrease in
absorbance was measured at 734 nm 105,106.
3.2.3.4.1. Preparation of ABTS solution
ABTS (54.8 mg) was dissolved in 50 ml of distilled water to 2
mM concentration and potassium persulphate (17 mM) 0.3 ml was
added. The reaction mixture was left to stand at room temperature
overnight in dark before usage.
3.2.3.4.2 Standard graph of ascorbic acid for ABTS assay
0.16 ml of ABTS solution or and 1 ml of DMSO was added to 2.8
ml of ascorbic acid solution in a test tube wrapped with aluminium
foil and its absorbance was read out at 517 nm using UV-visible
47
double beam spectrophotometer. The results were plotted on a graph
and IC50 value was determined.
3.2.3.5. Scavenging of DPPH
The DPPH free radical is reduced to a corresponding hydrazine
when it reacts with hydrogen donors. The DPPH radical is purple in
colour and upon reaction with hydrogen donor’s changes to yellow in
colour. It is a discoloration assay, which is evaluated by the addition
of the antioxidant to a DPPH solution in ethanol or methanol and the
decrease in absorbance was measured.
3.2.3.5.1. Preparation of DPPH solutions
0.05 mM of DPPH was prepared by dissolving 19.71 mg of DPPH
in 100 ml of methanol. The solution was protected from sunlight to
prevent the oxidation of DPPH.
3.2.3.5.2. Standard graph of ascorbic acid for DPPH assay
0.2 ml of DPPH solution was added to 2.8 ml of ascorbic acid
solution in a test tube wrapped with aluminium foil and its
absorbance was read out at 517nm using UV-visible double beam
spectrophotometer. The results were plotted on a graph and IC50
value was determined.
3.2.3.6. Scavenging of nitric oxide radical
Aqueous sodium nitroprusside at physiological pH produces
nitric oxide, which interacts with oxygen to produce nitrite ions, which
can be estimated by the use of modified Griess Illosvay reaction107. In
the present investigation, Griess Illovay reagent is modified by using
naphthyl ethylene diamine dichloride (0.1% W/V) instead of
48
1-naphthylamine (5%). Nitrite ions react with griess reagent, which
forms a purple azo dye. In presence of test components, likely to be
scavengers, the amount of nitrites will decrease. The degree of
decrease in the formation purple azo dye will reflect the extent of
scavenging. The absorbance of the chromophore formed was
measured at 540 nm.
Sodium Nitroprusside NO (Nitric Oxide)
NO HNO3 + HNO2
Nitric Acid Nitrous Acid
3.2.3.6.1 Standard graph of ascorbic acid for nitric oxide assay.
6 ml of sodium nitroprusside (10 millimole, 4ml), Phosphate
Buffer Saline (PBS, pH 7.4) 1 ml was added to 2.8 ml of the ascorbic
acid solution. After incubating for 15 min at 25 0C, 1ml of sulphanilic
acid reagent was added, and kept aside for 5 mins. Add 1 ml of NEDD
to the above reaction mixture and mixed and allowed to stand for 30
min in diffused light. A pink colored chromophore was formed,
measure the absorbance at 540nm using Double beam UV-visible. The
absorbance was measured at 540nm. The results were plotted on a
graph and IC50 value was determined.
3.2.4. Anthelmintic activity
Anthelmintic activity of the synthesized compounds will be
performed on Indian adult earth worms (Pheretima postuma) of nearly
equal size 5-8 cm in length and 0.2-0.3cm in width.The standard drug
and test compounds were dissolved in minimum quantity of dimethyl
formamide (DMF) to get the desired concentration. The compounds
Dissolved O2/ Water
49
were evaluated for the time taken for complete paralysis and death of
earthworms. The mean lethal time for each test compound was
recorded and compared with standard drug. The time taken by worms
to become motionless was noted as paralysis time. To ascertain the
death of motionless worms, they were frequently applied with external
stimuli, which stimulate and induce movement in the worms, if alive.
3.3. In-silico studies108-110.
The docking studies will be performed between the ligands
(designed) and the protein (obtained from the RCSB protein data
bank) with a resolution more than 2.0 0A. The protein obtained and
the ligands will be subjected for energy minimization.
3.3.1. Energy minimization of protein and ligands
Energy minimization of protein and ligand is performed by using
hyperchem 8.0. 0.01k.Cal/Mol RMSgradient was used for convergence
using CHARMM- 27 force field in vacco conditions with polak-ribiere
as conjugate gradient minimizer.
3.3.2. Protein preparation
The protein structure in .pdb format was downloaded from PDB
with a resolution more than 2.0 0A. Water, hetero atoms and ligand
molecules were deleted and subjected for energy minimization using
the parameters mentioned above.
3.3.3. Ligand preparation
Ligands designed by using chemsketch were energy minimized
and saved in .mol format.
3.3.4. Docking
50
All the ligands designed along with a hesperidin and naringin
were docked with the protein using the software GOLD version 4.12.
The name of the amino acid residue which has highest interaction
with the ligand is set as active site residue. Radius of 10oA around the
selected amino acid was defined as binding site.
The Gold score function is a molecular mechanics–like function with
four terms:
GOLD Fitness = Shbext + 1.375 Svdwext + Shbint + Svdwint
Where Shbext - protein–ligand hydrogen-bond score.
Svdwext - protein-ligand vander waals score.
Shbint- intramolecular hydrogen bonds.
Svdwint intramolecular strain in the ligand.
The compound with highest binding affinity clearly indicates it
has more possibility to possess the activity. Thus only these
compounds will be subjected for in-vivo evaluation.
3.4. In-Vivo biological activity
3.4.1. Acute toxicity Studies111
In order to calculate the lethal dose and effective dose the
author has performed acute toxicity studies as per the standard
procedure approved by IAEC.
3.4.2. Anti-inflammatory studies
3.4.2.1. Materials:
Polypropylene cages with paddy husk, Plethysmograph,
Carrageenan, diclofenac sodium, Normal saline and water
51
3.4.2.2. Animals
All the experiments will be carried out using male, Wister rats
(150-200 g). Animals will be placed at random and allocated to
treatment groups in polypropylene cages with paddy husk as bedding.
Animals will be housed at a temperature of 24 ± 20C and relative
humidity of 30 - 70%. A 12:12 light: day cycle was followed. All
animals were allowed to free access to water and fed with standard
commercial rat chaw pallets. All the experimental procedures and
protocols used in this study were reviewed by the institutional animal
ethical committee.
3.4.2.3. Vehicle
Test compounds and diclofenac sodium were suspended in
0.5% w/v CMC and administered orally to animals. Carrageenan
diluted separately in normal saline and injected.