anti oxidant best ppt for reference

1. “Biogenic synthesis of silver nanoparticles and its Pharmacotherapeutic
Applications using aqueous extract of Lepidium Sativum (Garden Cress
Seeds)”
Name of Research Scholar- P.Sree Vennela Rao
Supervisor- Prof.B.Veeresh
Scholarship- National Fellowship
Registration Num- 9009-12-891-003
Presentation- PROGRESS SEMINAR
Dept of Pharmacy,
University College of Technology,
Osmania University,Hyderabad,500007.

2. Overview of Presentation
1.Introduction and Literature review
2.Objectives
I) Synthesis and characterization of silver nanoparticles.
II) Anti bacterial, Anti oxidant and Anti inflammatory activities.
III) Anti cancer activity (in vitro)
IV) Anti inflammatory/Wound healing activity (in vivo).
3.Methodology
4.Expected outcome
5.Summary and Discussion.
6.Limitations and Future prospects
7.Publications
6.Certifications

3. INTRODUCTION&
LITERATURE REVIEW

4. Nanoparticles…
• A nanoparticle is a small particle that ranges between 1 to 100
nanometers in size. Undetectable by the human eye, nanoparticles can
exhibit significantly different physical and chemical properties to their
larger material counterparts.
• Nanoparticles have large surface area, which gives them more
pronounced effects.
Particle Type Diameter Size Range
Coarse particles 2500 to 10,000 nm
Fine particles/Dust 100 to 2500 nm
Nano particles 1 to 100 nm
Atoms and small molecules 0.1 nm

5. Nano particle and its Applications..

6. Types of nanoparticles…
• Sea spray
• Mineral composites
• Volcanic ash
• virus
NATURAL/
NATURE
• Cooking smoke
• Diesel exhaust
• Welding fumes
• Industrial effluents
• Sand blasting
INCIDENTAL/
MANMADE
• Metals (Ag,Au,Zn,Cu)
• Quantum dots
• Nanotubes/bulkyballs
(60 atoms)
• Suncreen pigments
• Nanocapsules/nanorods
ENGINEERED/
MANFACTURED

8. Applications of AgNP…

9. Nanoparticles Synthesis

10. Silver nanoparticles Specialty
• A great number of scientists have focused on silver nano-particles, which
are applied the most widely due to their well-known antibacterial and
antifungal effects, as well as their plasmonic (biosensors) and opto-
electrical properties (molecular diagnostics) (Pokhrel and Dubey 2013)
• Anti microbial assessment.
• Toxicity evaluation.
• Photo thermal ablation.
• Therapeutic efficacy.
• Anti cancerous activity.
• Wound healing capacity.

11. Why Green synthesis..?
• The growing production and exploitation of metal nanoparticles raise
concerns about their release to the atmosphere, as such release may have a
negative impact on the environment, and thus also on human health
(Cvjetko et al. 2018).
Biologically-prepared NPs -
• high yield, solubility, and stability
• simple, rapid, non-toxic, dependable, Eco friendly,Economical
• produce well-defined size and morphology useful for translational research.
• In the end, a green approach for the synthesis of NPs shows promising
outcome for future research.

12. LEPIDIUM SATIVUM (GARDEN CRESS)
Taxonomic classification
Kingdom: Plantae
Division: Tracheophyta
Class: Magnoliopsida,
Order: Brassicales,
Family: Brassicaceae
Genus: Lepidium, Species: Lepidium sativum [8].
OTHER NAMES-Garden Cress (English),Chandrashoor (Sanskrit),Adityalu or Aadhalu (Telugu).
DISTRIBUTION-It is distributed in Africa (Egypt, Ethiopia and Kenya), Asia (Kuwait, Oman, Saudi Arabia, United
Arab Emirates, Yemen, Afghanistan, Iran, Iraq, Palestine, Jordan, Lebanon, Syria, Turkey, Pakistan, China, Japan,
India).

13. Introduction to Lepidium Sativum Seeds
• Herbal medicine is the oldest form of healthcare known to mankind. Herbs had been used by
all cultures throughout history. Plants are a valuable source of a wide range of secondary
metabolites, which are used as pharmaceuticals, agrochemicals, flavours, fragrances, colours,
biopesticides and food additives .
• The phytochemical analysis of Lepidium sativum showed that it contained cardiac glycoside,
alkaloids, phenolic, flavonoids, cardiotonic glycosides, coumarins, glucosinolates,
carbohydrates, proteins and amino-acids, mucilage, resins, saponins, sterols, tannins, volatile
oils, triterpene, sinapic acid and uric acid.

14. Phytochemical screening of LS
Constituents Petroleum Chloroform Alchohol Aqueous
Alkaloids - + + +
carbohydrates - - - +
Phenolic
compounds
- + + -
Flavonoid - + + +
Protiens and amino
acids
- - + +
Saponins - - + +
Mucilage - - + -
Resins + + + -
Lipids/fats + - - -

15. Physiochemical characteristics of Lepidium Seeds -100g
physio chemical g/100g
Moisture 5.30± 0.08
Ash 4.25 ± 0.05
Protein 22.83 ± 0.72
Total fat 25.61 ± 1.04
Insoluble dietary fiber 5.68 ± 0.46
Soluble dietary fiber 1.59 ± 0.23
Total Dietary fiber 7.27 ± 0.60
Carbohydrate 34.67 ± 0.88
Energy
K.Cal
474.23

16. Water soluble Vitamins on fresh weight basis mg/100g
Sample
name
Ribof
lavin
(B2)
Niacin
(B3)
Pantoth
enic
acid
(B5)
Pyridox
ine (B6)
Folic
acid
(B9)
Vit-C
lepidium
seeds
0.28
±
0.01
0.97 ±
0.03
0.286 ±
0.01
-- 0.116
± 0.01
63 ±
5.26

17. GC-MS report presence of alkaloids of
Lepidium Sativum Seeds
• eicosatetraenoate (10.24%),
• guanosine (9.29%),
• dodecanamide, n-(2-hydroxyethyl) (7.48%),
• hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (7.13%),
• 1-(1-adamantyl)-3-(1-piperidinyl)-1-propanone (6%),
• hexadecanoic acid (5.33%),
• 3-butylindolizidine (4.80%),
• 9,12-octadecadienoic acid (Z,Z)-,
• 2-hydroxy-1-hydroxymethyl (4.79%),
• 3-methyl alpha.-d-glucopyranoside (1.81%),
• stigmast-5-en-3-ol, (3. beta.) (3.58%),
• soyasapogenol B (1.15%),
• stigmasterol (1.07%),
• fucosterol (3.29%),
• gamma-tocopherol (5.04%)
• squalene (3.44%)

18. OBJECTIVE 1/CHAPTER 1
I) SYNTHESIS AND CHARACTERIZATION OF
SILVER NANO PARTICLES USING LEPIDIUM
SATIVUM.

19. Methodology
Preparation of 1% aqueous extract and 0.1mM AgNo3
• Seeds have been procured from local market identified and certified by
dept of botany Osmania university
• 100 gms of ls seeds coarsely grounded and from that 1 gram of
powder+100 ml distilled water is boiled at 80 c for 20 mins.
• This solution is filtered using Whatman filter NO 1 paper and aqueous
extract is collected the Final obtained solution was 50 ml.
• 1mM AgNo3 (0.168 mg) in 250 ml Milli Q water kept aside for
further evaluation.

20. Green synthesis of Silver Nanoparticles
40 ml of 1% LS extract
at pH 4 +10 ml of
0.1mM AgNO3 (1:4v/v)
Left at room
temperature 10
min-1hr
Nanoparticle
formed pale yellow
to brown colour
Nanoparticle extract mq water Agno3

21. Characterisation of Lepidium Sativum Silver
Nanoparticles (LsAgNp)
1. Uv-(Silver Nano Particle formation peak)
2. Ftir-(identification of functional groups)
3. Zeta potential-(charge)
4. Sem-(shape of nanoparticle)

22. Ultraviolet Spectrophotometry-404nM
Silver Nanoparticle formation was by
peak at 404 nM

23. FTIR Spectrum of silver nanoparticles
Band at 3728 cm-1 can be
associated with OH amide
vibrations.
• The bands at 2924 cm-1 and
794 cm-1 are CH amide groups
which confirms the presence
of alkenes
• The finger print regions at 765
and 588 are alkyl halides.
• The reduction,capping and
stabilization of nanoparticles
are involved in these bioactive
molecules

24. Scanning Electron Microscopes
The AgNp are polymorphic showing triangular, hexagonal, deformed
spherical.

25. Zeta potential is a measure of the effective electric charge on the nanoparticle surface. The
magnitude of the zeta potential provides information about particle stability.
PeakNo. Zeta
Potential
ElectrophoreticMobili
ty
1
-31.2 mV
-0.000242cm2/Vs
2 ---mV ---cm2/Vs
3 ---mV ---cm2/Vs
Zeta Potential(Mean) :-31.2mV
ElectrophoreticMobilitymean :-0.000242cm2/Vs

26. Results
• Synthesis of Nanoparticle-Sucessfully the silver nano particles are synthesized from Lepidium
sativum aqueous extract from Pale yellowish solution to dark brown solution.
• UV-Vis -Further to confirm AgNp through Uv vis spectrophotometer confirmed and the peak was
observed at 404 nm
• FTIR-The bands observed at 2924 cm-1 and 794 cm-1 are CH amide groups which confirms the
presence of alkenes,The finger print regions at 765 cm-1 and 588cm-1 are alkyl halides.The
reduction, capping and stabilization of nanoparticles are involved in these bioactive molecules.
• SCANNING ELECTRON MICROSCOPIC -The AgNp are polymorphic showing triangular,
hexagonal, deformed spherical.
• ZETA POTENTIAL -The small distribution of particulate size with z-average value of 40.5 nm
and low index of Polydistributor 0.31 is evident from of the Lepidium sativum seed extract
AgNPS.

27. Objective II
Anti Bacterial Activity of Lepidium
Sativum Silver Nanoparticles

28. Methodology
MEDIA PREPARATION:
1. 28 g of NA powder was Suspend in 1L of distilled water
in a 2 L conical flask.
2. The suspended NA mixture was digested and stirred to
completely dissolve.
3. It was autoclaved at 1210 c / 15 min.
4. When the flask was autoclaved it was permitted to cool
but not solidify
5. The medium solidified NA media was poured 25ml into
each petri plate, leaving the plates on the clean, uniform
surface at room temperature until the agar was solidified.
6. After the petri plates were solidified completely, these
were stored for further use.
Nutrient Agar Composition
Peptic digest of animal
tissue
5.000
Sodium chloride 5.000
Beef extract 1.500
Yeast extract 1.500
Agar 15.000
Final pH ( at 25°C) 7.4±0.2
Standard drug -OFLAXACIN

29. 1 2 3
4 5 6
Gram Positive
Bacteria
1. Esterisia coli
2. Bacillus subtills
3. Klebsicella
pnenmoniae
4. Bacillus
stearothermoph
ilis
5. Staphylococus
aureus
6. Pseudomonas
aeruginosa

30. 7 8 9
10 11 12
Gram Negative
Bacteria
7.Salmonella para
typhi
8.Salmonella typhi
9. Micrococcus
luteus
10.Proteus
vulgaris
11.Bacillus
megaterium
12. Bacillus cereus

31. Anti Bacterial Activity studies
Test Organisms Lepidium Silver
nanoparticles
AgNo3 Standard DW Extract
Zone of inhibition in mm
Salmonella typhi 23 18 09 ND ND
Salmonella Para typhi 24 16 20 ND ND
Staphylococcus aureus 19 10 06 ND ND
Proteus vulgaris 25 15 17 ND ND
Pseudomonas aeruginosa 21 16 30 ND ND
Micrococcus luteus 26 17 10 ND ND
klebsiella pneumoniae 21 09 22 ND ND
E.Coli 29 11 06 ND ND
Bacillus stearothermophilus 30 09 07 ND ND
Bacillus subtilis 31 18 07 ND ND
Bacillus megaterium 19 19 08 ND ND

32. Observations
1. The anti bacterial studies of LsAgNp indicate that the zone of
inhibition is more for Bacillus stereothermphilus (30), Bacillus
subtilis (31).
2. These above bacteria causes ANTHRAX bacteremia, endocarditis,
pneumonia, and septicemia.
3. Hence the LsAgNp may be used for treatment of anti microbial
resistant therapy providing maximum therapeutic effect with
minimal side effect.
4. The results confirm the potential uses of LS-AgNP as broad-
spectrum microbicides, disinfectants, and wound care products.

33. LsAgNP as Antimicrobial resistant treatment

34. Objective II-Anti Oxidant Activity Studies
• DPPH ASSAY
• NITRIC OXIDE
• ANTI OXIDANT REDUCING POWER ASSAY

35. DPPH ASSAY
METHOD
• DPPH-2,2-Diphenyl-1-Picrylhydrazyl (crystalline purple
compound,stable free radical,light and heat sensitive)
• Standard-Ascorbic acid
• Principle -DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free
radical method is an antioxidant assay based on electron-transfer that
produces a violet solution in ethanol . This free radical, stable at room
temperature, is reduced in the presence of an antioxidant molecule,
giving rise to colorless ethanol solution.

36. Procedure of DPPH Assay
1.Various concentrations of aqueous extract and standard (Ascorbic Acid) are been taken from (20 ug -100ug) and made
up to 3ml with methanol in test tubes.
2.Further 1 ml of 0.004% DPPH is been added and left at dark for 30mins and absorbance is calculated at 517 nm using
The below formula.
• The DPPH radical scavenging activity increased dependently on concentration
• Purple DPPH was converted to yellow DPPH
• The highest Inhibition percentage of lepidium sativum silver nanoparticles was 53.29±0.64 % in 100µg/ml
and the extract with 49.01±0.64%, while the standard ascorbic acid activity was 53.54± 0.20%.

37. Anti-inflammatory activity by bovine serum albumin denaturation
of lepidium sativum Silver nanoparticles.
0
20
40
60
80
100
120
1 2 3 4 5
indomethacin lsnp lsae
Concentr
ation
μg/ml
Indometh
acin
lepidium
sativum
seed
aqueous
mediated
silver
nanoparticl
es (LSNP)
lepidium sativum
seed aqueous
extracts (LSAE)
25 71.4 67 61
50 81.5 70.3 67.7
100 92.7 75.2 72.9
150 94.8 80.1 76.8
200 95.9 84.5 81.4

38. Results -Anti oxidant (DPPH,Nitric oxide,FRAP)
and Anti inflammatory (BSA Denaturation)
•Anti oxidant activity studies –
conducted by DPPH,NO,FRAP shows a dose dependent anti
oxidant activity with standard taken as ASCORBIC ACID
• Anti Inflammatory activity studies-
Conducted by Bovine Serum Albumin Denaturation method shows
dose dependent anti inflammatory activity with standard taken as
INDOMETHACIN.

39. Objective III (in-vitro)
ANTICANCER ACTIVITY STUDIES –MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)
ASSAY

40. Method –MTT stock solution
• MTT stock solution: Dissolve in the 10 mL phosphate buffer solution, 500 mg MTT powder. Stir the solution in the dark
place for around 1 hr with a magnet stirrer. Filter the sterilized solution with a 0.22 mm filter (Millipore, Ireland), then hold
at -20 ° C (van Meerloo et al., 2011) with 10-ml aliquots (50 mg / mL). On the day of the experiment dilution must produce
the working solution (5 mg / mL).
• ANTICANCER ACTIVITY (MTT ASSAY)
 Cells were seeded at a density of 1/104 cells/well in a 96-well flat-bottom microtiter plate and allowed to stay in a CO2
incubator for 24 hours at 37 ° C.
 Following 24 hrs of incubation, the media was replaced by a fresh medium.
 Cells were then treated in a CO2 incubator with different concentrations of the target compound for 24 hours at 37 ° C
 The culture media was replaced with a fresh medium after 24 hrs of incubation.
 10 μL of MTT working solution (5 mg / mL in phosphate buffer solution) was subsequently applied to each well and the
plate was incubated in a CO2 incubator for 4 hrs at 37 ° C.
 The formed formazan crystals were then solubilized by adding 50 μL DMSO per pool at 37 ° C for 30 min at a CO2
incubator.
 Eventually, with the ELISA plate reader at 570 nm, the pressure of the dissolved formazan crystals (purple colour) was
quantified.

41. Schematic Representation of MTT ASSAY

42. Cell viability
S.No Test Compound
Cell
Line Concentration
treated to cells
1 Untreated HeLa No treatment
2 Cisplatin HeLa 15ug/ml
3 Blank -
Only Media
without cells
4
lepidium
sativum seed
mediated Silver
nanoparticles.
HeLa
6.25,12.5,25,50
,100µg/ml 0
10
20
30
40
50
60
70
80
90
100
UNTREATED STD 6.25 12.5 25 50 100
%
of
Cell
Viability
Drug Concentration (ug/ml)
Comparative % of Cell viability of Silver NPs on HeLa cells
Graph : % cell viability values of SILVER NPS on HeLa cells

43. 100 ug/ml Control Untreated
6.25 µg/ml 12.5ug/ml 25 ug/ml 50ug/ml

44. OVERALL CONCLUSIONS ..
• An successful eco-friendly and inexpensive method has been achieved for the
green synthesis of silver nanoparticles (AgNPs) by Lepidium sativum seed extract.
• Biosynthesized AgNPs is spherical in form, by antibacterial activity studies the
nanoparticle shows more inhibition on gram positive bacteria
• DPPH antioxidant studies shows more reducing power with increase in
concentration of AgNp extract.
• lepidium sativum seed mediated Silver nanoparticles as anticancer in nature on
HELA cells due to its lower IC50 values with 13.98 suggest that the lepidium
sativum seed mediated Silver nanoparticles is anticancer in nature and exhibiting
cytotoxic potency against Human cervical cancer cells (HeLa)
• This green biosynthesis approach is a non-toxic alternative to conventional
chemical and physical approaches and will be suitable for large-scale biological
processing and prospective therapies.

45. OBJECTIVE IV-(IN-VIVO)
• ANTI INFLAMMATORY AND WOUND HEALING IN ANIMAL MODEL
BENCH WORK COMPLETED BUT AWAITING FOR COMPILATION AND
CALCULATIONS.

47. UGC CARE LIST IOSR-Journal of Pharmacy
and Biological Sciences

48. Other publications (2014-2021)
1. Sree Vennela Rao1 and Veeresh1, “Biogenic synthesis and characterization of silver nanoparticles using aqueous extract
of Lepidium sativum assessing its antibacterial and antioxidant properties” IOSR Journal of Pharmacy and Biological
sciences (IOSR-JPBS),e-ISSN:2278-3008,Volume 16,Issue 1 Ser.II (jan.-feb.2021) pp (46-56).
2. P Amruth Rao1,Sree Vennela Rao2,Kavanshri N P,Devi CH V R, “Effect of probiotic curd (Indian dahi) Supplementation
in delaying the progression of prediabetes to diabetics Mellius-a pilot study, “International Journal of Diabetes and
Metabolic disorders-2021” ,ISSN 2475-5451,Vol 6.Issue 1,pg 136-139.
3. Amruth rao P,Shivaprakash M ,Madhavi G ,Sree Vennela Rao , MD.Khaleel Pasha , V Srinivas . R. Hemalatha. “Effect of
probiotic curd on cardiovascular disease (CVD) risk factors and body mass composition in People living with
HIV(PLWH)” - A pilot study Clinical Division,NIN, Hyderabad, India; BMC Infectious Diseases 2020, 20(Suppl
1):ISSHID.
4. Sree Vennela1*, Neharika Reddy, Rani Samyuktha, Poornachandar.G, T.Tamilanban, Venkataramana Devi.P “Therapeutic
Efficacy and Safety Profile of Tolvaptan Administered In Hyponatremia Patients” American Journal of Pharmatech
Research 2013 Oct issue 5.
5. Poorna chander G, Sree Vennela.P, Dr.Ramana Devi, Srinivas Naik.B “Invitro screening for different medicinal plants”
Journal of Phamacy and Biological Sciences” ISSN -2278-3008,volume 9,issue 2 ver II (mar-apr.2014).pg 68-79.
6. Amruth Rao P.*1 , Sree Vennela Rao P.3 , Khaleel Pasha MD.2 , Srinivas V.2 and Hemalatha R. “A study on
the effect of probiotic curd supplementation on HIV (PLWH) undergoing anti retroviral therapy (ART)” World Journal of
Pharmaceutical Research, ISSN 2277– 7105, Volume 10, Issue 5, 1492-1497,Apr 2021,pg 1492-1497.

50. THANK YOU…