HAZARA UNIVERSITY MANSEHRA, PAKISTAN

DEPERTMENT OF BOTANY

SYNOPSIS FOR Ph.D DEGREE IN BOTANY

Chemical characterization and Biological evaluation of Silver Nano

particles synthesized from selected medicinal plants.
Name of Student: Muhammad Nisar Ul Haq
Father’s Name: Israr Ul Haq
Roll No: 49386
Registration No: 17-PHD-BOT-F-HU-1
Date of Admission: Fall 2017
Date of Initiation: October 2017
Probable Duration: 2017-2020

SYNOPSIS COMMITTEE

Supervisor Prof Dr. Manzoor Hussain …………………...

Chairman, Department of Botany Signature

Member Dr. Zafar Iqbal …………………

Assistant Professor Signature

Member Dr. Niaz Ali …………………...

Assistant Professor Signature

…………………...
Chairman
Department of Botany
Chemical characterization and Biological evaluation of Silver Nano
particles synthesized from selected medicinal plants.

RESEARCH SUPERVISOR Prof DR Manzoor Husain

Chairman department of Botany
Hazara University Mansehra

CO – SUPERVISOR Assistant Prof DR Rahmat Ali Khan

Chairman Department of Biotechnology
UST Bannu

DEPARTMENT OF BOTANY
HAZARA UNIVERSITY MANSEHRA
2019
Biological and Chemical characterization of nanoparticles synthesized
from selected medicinal plants

Introduction

Nanotechnology is an important field of modern research dealing with synthesis,

strategy and manipulation of particle’s structure ranging from approximately 1 to
100 nm in size. Within this size range all the properties (chemical, physical and
biological) changes in fundamental ways of both individual atoms/molecules and their
corresponding bulk. Novel applications of nanoparticles and nanomaterial’s are
growing rapidly on various fronts due to their completely new or enhanced properties
based on size, their distribution and morphology. It is swiftly gaining renovation in a
large number of fields such as health care, cosmetics, biomedical, food and feed, drug-
gene delivery, environment, health, mechanics, optics, chemical industries, electronics,
space industries, energy science, catalysis, light emitters, single electron transistors,
nonlinear optical devices and photo-electrochemical applications. Tremendous growth
in these expanding technologies had opened applied frontiers and novel fundamentals.
This includes the production of nan scale materials afterwards in investigation or
utilization of their mysterious physicochemical and optoelectronic
properties (Korbekandi and Iravani, 2012, Khalil et al., 2013, Kaviya and Viswanathan,
2011 ).

The nanoparticles used for all the aforesaid purposes, the metallic nanoparticles
considered as the most promising as they contain remarkable antibacterial properties
due to their large surface area to volume ratio, which is of interest for researchers due to
the growing microbial resistance against metal ions, antibiotics and the development of
resistant strains (Khalil et al., 2013). Among the all noble metal nanoparticles, silver
nanoparticle are an arch product from the field of nanotechnology which has gained
boundless interests because of their unique properties such as chemical stability, good
conductivity, catalytic and most important antibacterial, anti-viral, antifungal in
addition to anti-inflammatory activities which can be incorporated into composite
fibers, cryogenic superconducting materials, cosmetic products, food industry and
electronic components (Ahmad et al., 2013, Klaus-Joerger et al., 2001 ). For biomedical
applications; being added to wound dressings, topical creams, antiseptic sprays and
fabrics, silver functions’ as an antiseptic and displays a broad biocidal effect against
microorganisms through the disruption of their unicellular membrane thus disturbing
their enzymatic activities.

Synthesis of silver nanoparticles is of much interest to the scientific community because

of their wide range of applications. These silver nanoparticles are being successfully
used in the cancer diagnosis and treatment as well (Popescu et al., 2010, Baruwati et al.,
2009). Generally, nanoparticles are prepared by a variety of chemical and physical
methods which are quite expensive and potentially hazardous to the environment
which involve use of toxic and perilous chemicals that are responsible for various
biological risks. The development of biologically-inspired experimental processes for
the syntheses of nanoparticles is evolving into an important branch of nanotechnology.
Generally there are two approaches which are involved in the syntheses of silver
nanoparticles, either from “top to bottom” approach or a “bottom to up” approach .In
bottom to top approach, nanoparticles can be synthesized using chemical and biological
methods by self-assemble of atoms to new nuclei which grow into a particle of nan scale
while in top to bottom approach, suitable bulk material break down into fine particles
by size reduction with various lithographic techniques e.g. grinding, milling, sputtering
and thermal/laser ablation.
Aims and objective
The investigation will be carried out to achieve the following objectives.

1. Synthesis of Nanoparticles from selected medicinal plants

2. Chemical characterization of the nanoparticles
3. Biological evaluation of the nanoparticles
Material and methods:

Extraction:
Extraction of selected medicinal plants material will be carried by simple maceration
process. Powdered medicinal plants will be soaked in methanol and will be blended for
vigorous shaking and mixing. The poorly homogenized mixture will be kept at room
temperature (25±2) oC for 3 weeks. Then maximum solvent will be separated from the
mixture. Filtrate will be filtered twice initially using the ordinary filter paper and then
Whatman filter paper # 1. Solvent will be completely evaporated by rotary evaporator
and the gummy extract will be put in separate bottles and labeled. The residues will be
again soaked in their respective recovered methanolic solvent for another two weeks
and the process will be repeated with three washings for each plant extract. After
dryness the extract will be weighed.
Green synthesis:
The use of plants as the production assembly of silver nanoparticles has drawn
attention, because of its rapid, eco-friendly, non-pathogenic, economical protocol and
providing a single step technique for the biosynthetic processes. The reduction and
stabilization of silver ions by combination of biomolecules such as proteins, amino
acids, enzymes, polysaccharides, alkaloids, tannins, phenolics, saponins, terpinoids and
vitamins which are already established in the plant extracts having medicinal values
and are environmental benign, yet chemically complex structures (Kulkarni and
Muddapur, 2014). A large number of plants are reported to facilitate silver
nanoparticles syntheses are mentioned and are discussed briefly in the presented
review. The protocol for the nanoparticle syntheses involves: the collection of the part of
plant of interest from the available sites was done and then it was washed thoroughly
twice/thrice with tap water to remove both epiphytes and necrotic plants; followed
with sterile distilled water to remove associated debris if any. These; clean and fresh
sources are shade-dried for 10–15 days and then powdered using domestic blender. For
the plant broth preparation, around 10 g of the dried powder is boiled with 100 mL of
deionized distilled water (hot percolation method). The resulting infusion is then
filtered thoroughly until no insoluble material appeared in the broth. To 10−3 M
AgNO3 solution, on addition of few mL of plant extract follow the reduction of pure Ag
(I) ions to Ag (0) which can be monitored by measuring the UV–visible spectra of the
solution at regular intervals (Sahayaraj and Rajesh, 2011)

Chemical characterization:

XRD:

X-ray powder diffraction (XRD) is a rapid analytical technique primarily used for phase
identification of a crystalline material and can provide information on unit cell
dimensions. The analyzed material is finely ground, homogenized, and average bulk
composition is determined.

UV-Vis:

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-

Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in
the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent
ranges. The absorption or reflectance in the visible range directly affects the
perceived color of the chemicals involved. In this region of the electromagnetic
spectrum, atoms and molecules undergo electronic transitions. Absorption
spectroscopy is complementary to fluorescence spectroscopy, in that fluorescence deals
with transitions from the excited state to the ground state, while absorption measures
transitions from the ground state to the excited state (Skoog et al., 2007)

SEM:

A scanning electron microscope (SEM) is a type of electron microscope that produces

images of a sample by scanning the surface with a focused beam of electrons. The
electrons interact with atoms in the sample, producing various signals that contain
information about the sample's surface topography and composition (Stokes and
Debbie, 2008)
TEM:

Transmission electron microscopy (TEM, also sometimes conventional transmission

electron microscopy or CTEM) is a microscopy technique in which a beam
of electrons is transmitted through a specimen to form an image. The specimen is most
often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is
formed from the interaction of the electrons with the sample as the beam is transmitted
through the specimen. The image is then magnified and focused onto an imaging
device, such as a fluorescent screen, a layer of photographic film, or a sensor such as
a charge-coupled device (nobelprize.org).

FTIR:

Fourier-transform infrared spectroscopy (FTIR) is a technique used to obtain

an infrared spectrum of absorption or emission of a solid, liquid or gas. An FTIR
spectrometer simultaneously collects high-spectral-resolution data over a wide spectral
range (Griffiths and de Hasseth, 2007)

Biological evaluation:
In-vitro activities:
Antibacterial Assay:
Agar diffusion method (Well diffusion method) will be used for determination of
antibacterial activity (Leven, 1979).
Antifungal Assay:
The Agar tube dilution method will be used for antifungal activity as reported by
Choudhary et al. (1995).
Antioxidant Activity (DPPH Free Radical Scavenging Assay):
The scavenging activity of DPPH free radicals by different test samples will be
determined according to the method reported by Brand-Williams et al. (1995).
Acetyl cholinesterase inhibition assay (in vitro):
Acetyl cholinesterase inhibition activity will be determined using the substrate
analogue acetylthiocholine iodide, which is converted to thiocholine. The reaction of
thiocholine with the chromogenic substrate dithionitrobenzoic acid (DTNB) leads to the
formation of a yellow anion, nitrobenzoic acid, which absorbs strongly at 412 nm. The
acetyl cholinesterase inhibitory activity of the plant extracts was evaluated as the
method previously described by Rahman and Choudhary, 2001.

Citotoxicity assay against the brine shrimp larvae:

Cytotoxicity of the extracts will be evaluated using brine shrimp lethality assay (BSL) as
described by Meyer, Ferrigni, and Putnam (1982). Brine shrimp eggs (Artemia salina
leach).

Antidiabetic activities:

Inhibition of yeast alpha-glucosidase activity.The enzyme inhibition activity against

yeast α- glucosidase (Sigma-Aldrich, USA) will be determined using 1 mM of p-
nitrophenyl-α-D-glucopyranoside (PNPG) as substrate according to Wan et al.,
2013.with modifications.
Invivo activities:
Effect of nano particles on diabetic rats synthesized from selected medicinal plants:
Experimental animals
Both male and female albino Wistar rats will be procured from Institutional
Animal House, Quaid-e- Azam University, Islamabad, Pakistan, for the study. In steel
cages rats will be kept at room temperature with a 12 hr light/dark cycle and
acclimatized to the laboratory conditions for 7 days. Ethical board of University
approved the study protocol. All these rats will be equally divided into two
experiments for in vivo evaluation of both nano particles synthesized from selected
medicinal plants against alloxan induced diabetes and toxicity in various tissues.
Acute toxicity testing
Studies will be performed in order to check the toxic effects of the extracts. Rats
will be used for this purpose. The animals were fasted overnight, only water was given,
after which to the respective groups the extract will be administered orally at the dose
level of 2000 mg/kg body weight by gastric cannula and observed the groups
continuously for 24 hrs for behavioral, neurological and autonomic profiles, and then at
24 hrs and 72 hrs for any lethality. Observed the animals for further toxic symptoms for
21 days. According to the guidelines if the observed mortality is in 2 or 3 animals, then
the given dose will be a toxic dose. If the observed mortality will be in only one animal,
then again the same dose will be given to confirm the toxic dose. If the mortality would
not observed at all, the plant extract will be considered as non-toxic. Alternatively, the
toxicity test will be started with a dose of 100 mg/kg body weight and repeated for
further other doses such as 250, 500, 1000 and finally 2000 mg/kg body weight (Wang et
al., 2007)
Effect of nano particles synthesized from selected medicinal plants on physiological
and biochemical parameters
The experiments will be designed to evaluate pharmacological effects of nano
particles synthesized from selected medicinal plants against the diabetes induced with
alloxan in Albino rats.
Alloxan induced albino rats’ model for diabetes
1. Healthy Wistar strain albino rats will be selected and randomly divided into six
groups with six animals in each group serving as group 1 = normal, group 2= diabetic
control, group 3 = reference control i.e. standard drug (glibenclamide, 10 mg/kg b. wt.),
group 4 = diabetic group given 200 mg/kg b. wt of the extract , group 5 = diabetic
group given 400 mg/kg b. wt of the extract and group 6 = group given 200 mg/kg b.
wt of the extract alone (Sharma et al., 2010)
2. An identification mark will be given to the rats of each group using picric acid as dye.
Each rat of a specific group will be marked with a serial numbers that is from 1 to 6 and
each specific group will be kept in a separate steel cage. Each rat will be weighed and
the doses will be calculated accordingly (Sharma et al., 2010) .
3. Diabetes will be induced in each group using freshly prepared solution of alloxan
Monohydrate dissolved in normal saline (0.9 % w/v of NaCl) except the group 1, which
served as normal control. For inducing diabetes the rats will be kept on fasting for 12
hours and diabetes will be induced by giving a single intraperitoneal injection of
alloxan monohydrate (150 mg/kg b. wt). To prevent fatal hypoglycemia due to massive
pancreatic insulin release, the rats will be provided with 20% glucose solution after six
hours through gastric cannula and supplied 5% glucose in water bottles in their cages
for next 24 hours (Sharma et al., 2010) .
4. The fasting blood glucose level will be measured after 48 h of alloxan injection.
Blood glucose levels (mg/dl) will be determined using an Accu- Check active (Roche
Diagnostic GmbH, Germany), based on the glucose oxidase method. From tip of the tail
blood samples will be collected at the defined intervals of time. The rats having fasting
blood glucose level above 200mg/dl will be considered as diabetic 11, 12 and these
diabetes induced animals will be selected for further experimentation (Sharma et al.,
2010) .
5. Rats will be randomly allotted into 6 groups of six animal (n=6) each. Group 1 -
Served as normal control and received simple drinking water. Group 2 - Served as
diabetic control and also received simple drinking water. Group 3 - Diabetic animals
treated with standard drug glibenclamide at a dose of 10 mg/kg of b. wt/day. by the 18
No. gastric cannula. All the groups will be given respective treatment daily for 21 days.
Group 4 - Diabetic animals treated orally with extract at a dose of 200 mg/kg b.wt./day.
Group 5 - Diabetic animals treated orally with extract at a dose of 400 mg/kg b. wt.
/day. Group 6 – Non-diabetic animals treated orally with extract at a dose of 200
mg/kg b. wt. /day alone.
The drug treatment will be carried out every day with the help of 18 No. gastric
cannula. for a period of 21st days. Blood sugar will be determined before treatment and
on 7th day, 14th day and on 21st. of drug treatment. Body weight will be determined on
the same days (Sharma et al., 2010),
6. Blood will be tested on the 0th day, means the day on which the dosing will be
started, 7th day and 14th day and 21st day. From tip of the tail blood samples will be
collected at the defined intervals of time.
7. Physiological parameters like body weight will be monitored during the
experimentation. In order to check the effect of the extracts on the weight of the rats will
be recorded prior to the administration of the extracts, on 7 th day, on 14th day and at the
end of the study i.e. on the 21st day.
8. For estimation of Glucose and other clinical parameters: The serum from the blood
will be separated as under:
In glass tubes sample will be collected and left for 1 hr at 35°C for clotting.
Using a glass pasteur carefully the clot will be loosened from the sides of the tube.
The serum will be centrifuged at 4000 rpm for 10 min at room temperature.
The serum will be collected from the clot into a clean tube by gently pipetting off using
a micropipette.
The serum will be labeled with the animal number & group number and the estimations
will be made. The serum glucose level; the enzymes ALT and ALP, and Bilirubin level,
the lipid profile (Total cholesterol, Triglyceride level LDL, and HDL,) and Kidney
profile (Urea and Creatinine) will be determined enzymatically on Micro lab
autoanalyser.
After taking blood the organs (liver, lungs and kidneys) will be removed in ice cold
saline will be washed, dried with blotting paper and into two portions the tissues will
be divided.
1. From all the organs the parts will be cut off and for histology stored in fixative sera.
2. The tissue second part will be kept in normal saline solution and stored at -20 °C for
further studies (Sharma et al., 2010).
Oral Glucose tolerance test
The oral glucose tolerance test will be performed in overnight fasted (18hr) normal
rats as per Bonner, 1988. Healthy rats will be randomly selected and distributed into six
groups (n=6). One of those groups will be administered distilled water and the rest five
groups were given orally extract of Acacia modesta leaves and extracts of
Chenopodium murale (200 mg/kg bw., and 400mg/kg b.w., respectively) and
glibenclamide (10mg/kg bw.). Glucose (3g/kg b.w.) will be fed 1 hr after the treatment
of extracts and glibenclamide. Blood will be collected from the vein of the tail at 0, 30, 90
and 150 min administration of glucose and levels of glucose will be calculated using
Accucheck blood glucose monitoring kit (Harris et al., 1998)
Effect of nanoparticles on normoglycemic rats:
1. Healthy Wistar strain albino rats will be randomly selected and divided into
different groups with six animals in each group serving as group ‘A’ = normal control,
group ‘B’= diabetic control, group ‘C’ = gliben clamide 10 mg/kg b. wt.; group ‘D’ =
Extract, 200 mg/kg b. wt; group ‘E’ = Extract, 400mg/kg b. wt.; 'F' = Extract 200 mg/kg
b. wt. alone (Qinna & Badwan, 2015).
2. An identification mark will be given to the rats of each group using picric acid.
3. The level of blood glucose of the rats will be measured after overnight fasting.
4. Group ‘A’ will be fed with simple drinking water which served as normal control and
rest of the groups will be fed with the respective extracts, mentioned above, orally
following standard methodology. All the groups will be given respective treatments
daily for 21 days.
5. Blood will be collected again on the 7th day and 14th day and 21st day of dosing,
through the retro orbital sinus of the rats (Qinna & Badwan, 2015).
6. The serum from the blood will be separated and labeled with the animal number. The
estimation of glucose level was measured enzymatically on an autoanalyser (Qinna &
Badwan, 2015).
Histopathological studies
At the end of the study i.e. on 21th day the rats will be sacrificed and the tissues
(Kidneys, liver and lungs) will be collected. The histopathological process will be
carried out in the lab of Islamabad and Bannu, (Tissue fixation, Processing and
embedding)]. The rats will be anesthetized by using diethyl ether. Using large scissors a
cut will be made laterally from just below the rib cage and extended fully to sides.
Again using scissors another cut will be made to cut the diaphragm fully on the border
of the rib (from centre to sides). Using hemostats fasten to hold, the chest cavity will be
opened. Then tissues will be removed in a systematic fashion and a requisite tissue
sample will be taken for the histology. The tissue sample will be kept in 10% formalin
for overnight. Then the sample will be dehydrated in graded alcohol. Isopropyl alcohol
will be used. Dehydration will be done by passing the tissue through increasing
concentration of alcohol. 70% alcohol for 1 hour, 90% alcohol for 1 hour, and 100%
alcohol for 1 hour in an automatic tissue processor. After that it will be cleaned in
xylene. Dehydrated tissues will be kept in a jar of alcohol xylene mixture for 30 min and
then subjected to 2 changes of xylene of 30 min each. The tissue samples will be then
impregnated in paraffin wax (58-60 0C). After clearing, the tissue pieces will be
subjected to 2 changes of melted paraffin wax of 2 hours each. Then paraffin blocks will
be made in which samples will be embedded and allowed to cool. Hardened blocks will
be taken for sectioning and staining as per the standard procedure of Section cutting
and staining. All the tissue blocks will be kept in freezer for 20-30 min before cutting.
This process hardened the tissue blocks. Using automated microtome tissue samples
will be cut into sections of 5μ thickness and with the help of forceps placed in the tissue
flotation bath which will be kept at 400C. Then the sections will be lifted on slide and
kept in a slide tray. This slide tray will be kept in oven at 600C for 5 min. The slide tray
will be then removed outside and let to cool. This process fixed the tissue section to the
slide. Then again the slide tray will be kept in oven for next 5 min. The hot slide will be
removed from the oven and placed in slide rack. The slide rack will be dipped in a
beaker containing xylene; the same will be repeated in another beaker containing
xylene. The slides will be then subjected to staining using hematoxylin and eosin. The
slide rack will be passed through the decreasing concentration of alcohol. 100% alcohol
1 dip, 90% alcohol 1 dip, 80% alcohol 1 dip, 70% alcohol 1 dip and then dipped in a
beaker containing tap water. After that the slide rack was immersed in hematoxylin jar
for 15-20 min and then again dipped the slide rack in a beaker containing tap water. The
slides will be decolorized by a single dip in 1% acid alcohol and then dipped the rack in
a beaker containing tap water. The slide rack will be dipped in tap water (3-5 dips) for
bluing and the slide rack was then dipped in a beaker containing tap water. The slides
will be counter stained by a dip in Eosin for 1 minute and again the slides will be
dipped in a beaker containing tap water. Then the slides will be subjected to a single dip
in 100% alcohol, 90% alcohol, 80% alcohol, 70% alcohol and in xylene. The extra stain
will be cleaned with gauge dipped in xylene. The stained sections will be mounted
using DPX and covered with the cover slip. The slides will be labeled with self-sticking
labels. The slides will be then observed under microscope and photographed in order to
check the effect of the extracts on the tissues and the slides will be then kept safely
(Murayama et al., 1998)
Biochemical investigations
To study the pharmacological activity of nanoparticles against the Alloxan
induced diabetic rats following assays will be carried out as reported by (Raut &
Gaikwad, 2006).
Biochemical analysis of serum
Estimation of total protein, albumin, urea, and creatinine will be carried out with
different kits (Ahmed & Fatani, 2005).
Estimation of total protein
Principle
In serum total protein will be estimated from a colored complex in the presence of salt
of copper in alkaline solution through kit of AMP Diagnostics Company biuret method
(Vessal et al., 2003).
Procedure of assay
1 ml of reagent (potassium iodide, copper sulphate, sodium hydroxide and potassium
tartrate) will be mixed with 10 µl standard (albumin) or sample, for blank 10 µl distilled
water and after 10 min of incubation take the absorbance at 550 nm.Through formula
total protein will be calculated; (Absorbance of sample / Absorbance of standard) × n
Where n is concentration of standard. (Vessal et al., 2003).
Urea determination
Principle
Determination of urea will be carried out by purchasing the kit from AMP
Diagnostics Company.
Urea + H2O urease 2NH3 + CO2
2 NADH + 2 α-ketoglutarate + 2NH4+ GIDH 2L-glutamate + 2NAD+ + 2H2O
Procedure of assay
1 ml reagent (Tris buffer, α-ketoglutarate, urease, GIDH and ADP) will be mixed with
10 µl blood and standard and variation in OD (340 nm) between 30 sec and 90 sec will
be recorded. Through the formula urea was calculated; (OD of sample / OD of
standard) × n
Where n is concentration of standard (Mansour et al., 2002).
Creatinine determination
Principle
The rat of formation of a colored complex between alkaline pirate and creatinine will
be measured. Creatinine was determined using kit from AMP Diagnostics Company
(Mansour et al., 2002).
Procedure of assay
1 ml working reagent (R) (picric acid) will be mixed with 100 µl sample or standard, and
absorbance was recorded at 500 nm after 25 sec. Exactly 2 min after the first reading
(A1), 2nd reading (A2) will be recorded. Through the formula creatinine will be
calculated;
(A2-A1 sample / A2-A1 standard) × n
Serum bilirubin determination
Principle
Sulfanilic acid reacts with sodium nitrate to form diazotized sulfanilic acid. In the
presence of DMSO total bilirubin reacts with diazotized sulfanilic acid to form
azobilirubin. In the presence of DMSO only direct bilirubin reacts to give azobilirubin
using kit from AMP Diagnostics Company (Mansour et al., 2002).
Procedure of assay
1.5 ml working reagent (R1) (sulfanilic acid, DMSO, HCl), will be mixed with 0.5
ml working reagent (R2) with 100 µl sample or calibrator, 100 µl sample without R2 for
sample blank and OD (555 nm) will be noted after 5 min incubation at 37 °C
Through the formula Serum total bilirubin will be calculated;
(OD of sample - OD of sample blank) × F
Where F is factor of standard concentration

Serum cholesterol determination

The cholesterol quantitative determination will be done by using kit from AMP
Diagnostics Company.
Principle
In the sample cholesterol forms colored complex, according to the following reactions:
Cholesterol esters + H2O cholesterol esterase Cholesterol + fatty acids
Cholesterol + O2 cholesterol oxidase 4-Cholestenona + H2O2
2H2O2 + Phenol + 4-Aminoantipyrine peroxidase Quinonimine + 4H2O2
Procedure of assay
At 37ºC temperature the assay will be done with 505 nm wavelength in cm
optical path. 10 µl of standard or sample will be mixed with I ml reagent. The sample
absorbance will be noted against the blank within 60 minutes (Vessal et al., 2003).
Through the formula serum cholesterol will be calculated; (OD of sample / OD of
standard) × n
Where n is the concentration of standard

High density lipoprotein (HDL)

The determination of HDL was done by kit from AMP diagnostic company. Antihuman
β-lipoprotein antibody in R1 binds to lipoproteins. The antigen-antibody complexes
formed with the addition of R2. CHE and cholesterol oxidase in R2 only react with
HDL. HDL gives blue color with hydrogen peroxide produced by the enzymatic
reactions. HDL will be measured at 593 nm (Xu et al., 2008)

Procedure of assay
The whole procedure was performed at 37ºC temperature at 600 nm wavelength with 1
cm optical path against the blank reagent. 2.5 µl of sample will be mixed with the 2.5 µl
calibrator and after 5 min OD was measured then R2 of 60 µl was added and mixed.
After incubation for 5 min again the absorbance will be checked.
Calculation
(OD2-OD1) sample / (OD2-OD1) Calibrator × n
n=Calibrator concentration
Calibration
AMP HDL-Calibrator (BR9812) used for calibration.

Determination of low density lipoprotein (LDL)-Cholesterol

By the formula LDL will be calculated:
LDL= TG/5+ HDL– cholesterol

Triglycerides determination
Procedure of assay
1 ml of the reagent (Mg+2, Potassium ferrocynate, Glycerol kinase, ATP) mixed with 10
µl distilled water, standard and sample used as blank, standard and sample, incubate at
37 0C for 10 min and measured OD at 500 nm (Vessal et al., 2003).
Calculation
OD of sample/OD of standar×n
Where n is the concentration of standard.
Alanine transaminase (ALT)
The photometric method will be used for the determination of ALT (Vessal et al., 2003).
Reagents
Reagent a, 5x 80ml reagent solution
Reagent b, 1x 100ml start reagent
Reaction solution
Mix reagent a and reagent b at a ratio of 4‫׃‬1, e. g 5ml start reagent plus 20ml of
reaction solution.
Procedure
By the Microlab Chemistry analyzer the reaction will be automatically performed
under the feed program in computer. The temperature will be 37°C for the reaction at
340 nm wavelength with 1cm optical path against air blank.
The enzyme activity calculations
By using the formula the enzyme activity was calculated as unit per litter (U/L).
Activity of enzyme = (ΔA/min) x F
Where (ΔA/min) is sample average absorbance per min, while F is a wave length
specific factor. It is 1746 for 340 nm wavelength.
Alkaline phosphatase (ALP)
According to the recommendation of German society of clinical chemistry alkaline
phosphatase enzyme will be determined by Microlab Spectrophotometer systems
(Mansour et al., 2002).
Principle
Para – Nitro phenyl phosphate + H2 O ALP Phosphate + para-nitrophenol
Reagents (R)
R1‫ ׃‬Diethanolamine (pH 9.8) 1.25 mM
R2‫ ׃‬p-Nitrophenylphosphate 50 mM
Reaction solution
Reagent R1 and reagent R2 will be mixed (4:1) to obtain a mono reagent. From light
mono reagent was protected.
Procedure of assay
The reaction will be automatically performed by the Microlab Chemistry
analyzer under the feed program in computer. The temperature will be 37°C for the
reaction at 405 nm (400 nm-420 nm) with 1 cm optical path against air blank. 20 µl of
sample will be mixed with mono reagent (1000 µl) and OD will be recorded after 1, 2
and 3 min.
Calculation
ALP activity (U/L) Δ= (ΔA/min × F)
ΔA/min = absorbance per min and F = factor (for ALP it is 2757 at 405 nm)
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