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PRELIMINARY INVESTIGATION
OF THROMBOLYTIC ACTIVITY OF
CRUDE METHANOLIC EXTRACT
AND SILVER NANO PARTICLE
OBTAINED FROM
DRYNARIA QUERCIFOLIA

Guided by Submitted By
Mrs.S.CHITRA,M Sc.,M.Phil., R.DEEPA
(16CAB1006)
Assistant Professor MASTER OF PHILOSOPHY IN BIOCHEMISTRY
M.G.R COLLEGE
DEPARTMENT OF BIOCHEMISTRY Dr.M.G.R Nagar,
M.G.R COLLEGE Hosur-635 109
Hosur-635 109
 INTRODUCTION
 Nanotechnique is the science which deals in materials in the range of 1 to 100 nm.

 These nanoparticles have multiple advantages in various fields such as electronics, cosmetics, coatings,

packaging, and biotechnology.

 Due to their optical properties the colloidal solution of metal nanoparticle is transparent, thus they are useful

in cosmetics, coatings, and packaging.

 Among nanoparticles, silver nanoparticle has wide application in industry and medicine due to its

antimicrobial, antifungal, and anti-parasitic characters. Because of their wide applications beneficial to

humans there is a need to develop rapid and reliable experimental protocols for the production of silver

nanoparticles.

 The production of silver nanoparticles using biological entities is gaining momentum as; biological methods

are providing, nontoxic and environmentally acceptable “green chemistry” procedures.

 SILVER NANOPARTICLES
 Among the several noble metal nanoparticles, AgNPs
have attracted special attention due to their unique
properties like electrical conductivity, chemical stability,
catalytic and antimicrobial activities (Vijay Kumar et al.,
2014).
 THROMBOLYTIC ACTIVITY
 Thrombolysis is the breakdown (lysis) of blood clots by pharmacological means, and commonly called clot

busting.

 It works by stimulating secondary fibrinolysis by plasmin through infusion of analogs of tissue plasminogen

activator (tPA), the protein that normally activates plasmin. Some commonly used thrombolytics are: Streptokinase,

Urokinase.

 Formation of blood clots lies at the basis of a number of serious diseases. By breaking down the clot, the disease

process can be arrested, or the complications reduced. While other anticoagulants prevent the "growth" of a clot,

thrombolytic agents actively reduce the size of the clot (Sardar M 2005) Most thrombolytic agents work by

activating the enzyme plasminogen, which clears the cross linked fibrin mesh .

 This makes the clot soluble and subject to further proteolysis by other enzymes, and restores blood flow over

occluded blood vessels.

DESCRIPTION OF THE PLANT
Scientific classification

Kingdom : Plantae

Division : Pteridophyta

Class : Polypodiopsida/Pterdopsida

Order : Polypodiales

(unranked) : Eupolypods I

Family : Polypodiaceae

Tribe : Drynarieae

Genus Drynaria

Species : D. quercifolia

Binomial name

Drynaria quercifolia
Drynaria quercifolia
 AIM AND OBJECTIVES
 To prepare crude methanolic extract of Drynaria
quercifolia plant.
 To perform phytochemical analysis of the crude
methanolic extract of Drynaria quercifolia plant.
 To synthesis of silver nano particles from the methanolic
extract.
 To evaluate antibacterial activity and thrombolytic activity
of Drynaria quercifolia extract and silver nanoparticles.
 MATERIALS AND METHODS
Selection and collection of medicinal plant

The Drynaria quercifolia linn was collected from

Yercaud, Salem district, Tamil Nadu, India.

The collected samples were carefully kept in polythene bags.

The plant was identified by Dr.G.Prabakaran,
M.Sc,PhD.,Assistant professor, Department of Botany, Govt
Arts College, Dharmapuri, Tamilnadu, India.
 Preparation of plant extracts
Aqueous extract

10g powder of Drynaria quercifolia was macerated in pestle and

mortar with 100 ml distilled water at room temperature and then filtered using
muslin cloth. Filtrate obtained was subsequently passed through Whattman's No.
1 Filter paper under aseptic conditions and the filtrate was collected in fresh
sterilized glass tubes and used within 24h for evaluation of antibacterial activity.

Solvent Extraction

10g of powder of Drynaria quercifolia were mixed with 100 ml of

(methanol) in Soxhlet apparatus. The mixture thus obtained was filtered through
muslin cloth and subsequently passed through Whattman's No. 1 Filter paper.
The filtrate was concentrated by evaporation of solvent at room temperature.
Extracts were stored at 4°C and were used for phytochemical activity and
antibacterial activity studies.
 THROMBOLYTIC STUDY
 Experiments for clot lysis were carried as reported earlier (Prasad et al.,., 2007).

 Venous blood was drawn from healthy volunteers (n = 10) and transferred in different pre-weighed sterile alpine
tube (500 μl/tube) and incubated at 37°C for 45 minutes. After clot formation, serum was completely removed.

 Each tube having clot was again weighed to determine the clot weight.

 Each alpine tube containing clot was properly labeled and 100 μl of plant extract was added to the tubes. As a
positive control, 100 μl of SK and as a negative non thrombolytic control, 100 μl of distilled water were separately
added to the numbered control tubes.

 All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, fluid
obtained was removed and tubes were again weighed to observe the difference in weight after clot disruption.

 Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis.

 The test was repeated three times.

 RESULTS AND DISCUSSIONS
Drynaria quercifolia is distributed throughout India, in the wet regions in
the plains growing on tree trunks, rock surfaces and old walls. Rhizome and
roots are slightly bitter in taste. Rhizome is used in the treatment of typhoid and
hectic fever, dyspepsia, cough and phthesis (Nayar, 1959).
Rhizome of Drynaria quercifolia showed higher potential of therapeutic
activity compared to its fronds and therefore rhizome of the plant material was
selected as the active plant part. About 1 kg of shade-dried and powdered
rhizome of D. quercifolia was extracted with methanol (Reichardt, 1988) using
Soxhlet Apparatus. Each extract was concentrated by rotary evaporator.
These extracts were stored at 4°C until the completion of experiment. The crude
extract was dissolved in the respective solvent to obtain a sample concentration
of 50 mg/ml (Eloff, 1998)
phytochemical analysis
Methanol
s.no PhytoChemical
extract

1 Alkaloids +

2 Carbohydrates +

3 Saponins +

4 Glycosides +

5 Proteins& amino acids +

6 Phytosterol +

7 Phenolic compounds +

8 Flavonoids +

9 Terpenoids +

10 Tannins +
ANTI BACTERIAL ACTIVITY
OF METHANOL EXTRACTS

Staphylococcus aureus Salmonella sp

E.coli
SEM image of silver nano particals
synthesized from D.quercifolia
 Thrombolytic activity

Blood sample + Sk (positive control) Blood sample + D.Water (Negative control)

Blood sample + ME Blood sample + SNP

 Initial Wt of
Wt of clot Percentage
Content Wt Reduced
S.No (mg) (mg)
(mg) Clot (mg)
Blood
1 sample + 1.81 0.49 1.32 37.1
Sk
Blood
2 Sample + 1.81 0.02 1.79 1.81
D.Water
Blood
3 Sample + 1.86 0.05 1.48 33.7
ME
Blood
4 Sample + 1.86 0.43 1.43 30.0
SNP
 SUMMARY AND CONCLUSION
 Recently blood clot formation has been a severe problem of blood circulation.

 Thrombus or embolus hinders the blood flow by blocking the blood vessel therefore depriving tissues of
normal blood flow and oxygen.

 These consequence yield necrosis of the tissue in that area. Thrombin formed blood clot from fibrinogen
and is lysed by plasmin, which is activated from plasminogen by tissue plasminogen activator (tPA).

 The purpose of a fibrinolytic drug is to dissolve thrombin in acutely occluded coronary arteries thereby to
restore blood supply to ischemic myocardium, to limit necrosis and to improve prognosis.

 For the treatment of myocardial infarction, many thrombolytic agents are used. Among them,
streptokinase is remarkable and widely used.

 Moreover, Tissue-type Plasminogen activator is more effective and safer than either urokinase or
streptokinase type activators. It is noted that all available thrombolytic agents still have significant
deficiencies, including the necessity of large doses to be maximally effective, limited fibrin specificity
and a significant associated bleeding tendency.

 Therefore, steps are taken to develop improved recombinant variants of these drugs in order to minimize
deficiencies of the available thrombolytic drugs.
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Thank you