Daldinia Concentrica

Welcome To My Presentation on-
In vivo analgesic, CNS depressant activity in Swiss albino mice, chemical and in vitro
antimicrobial screening of ethanolic extract from
Daldinia concentrica ( Xylariaceae )
Presented By-
Salma Sultana Rashma
I.D.- 13609015
Department of Pharmacy
University of Chittagong
Contents
 Introduction
 Aim & Objective of the work
 Fungus Preview
 Ethanolic Extract Preparation
 Study Protocol
 Experimental Animal
 Experimental Design
 Preparation of Test Sample
 Evaluation of Analgesic Activity
 Evaluation of CNS depressant Activity
 Chemical Screening
 Antimicrobial Screening
 Conclusion
Introduction
 The role of modern technology in human civilization is expanding every day. However,
humans still face, and will continue to face, three basic problems: shortage of food,
pollution of the environment, and diminishing quality of health. Macro fungi not only
can convert the huge lignocellulosic biomass waste into human food, but also can
produce notable medicinal/nutriceutical products that have many health benefits.
 Fungus have been used in health care for treating simple and age old common
diseases like skin diseases to present day complex and pandemic disease like AIDS.
They are reputed to cure epilepsy, wounds, skin diseases, heart ailments, rheumatoid
arthritis, diarrhea, dysentery, cold, anesthesia, liver diseases, gall bladder diseases
and used as vermicides.
Aim & Objective of the work
 Discover promising newer medicinal activities of the fungus.
 Study different pharmacological activities of fungus extract and

fractions.
 Ultimately rationalize the traditional use of the selected fungus.

Fungus Preview
 Scientific Name: Daldinia concentrica (Bolton) Ces. & De Not.
 Common Names: King Alfred's cake
Cramp ball
Coal fungus
 Family: Xylariaceae
Figure: Daldinia concentrica

Ethanolic Extract Preparation
 Extraction of the powdered fruit bodies of the fungus Daldinia concentrica
with ethanol for fifteen days.
 Filtration of the crude ethanolic extract by using the Markin cotton cloth and
subsequently through the filter paper and solvent evaporation with water
bath.
Study Protocol
The study protocol consists of the following steps:
 Pharmacological investigation
1. Cold extraction of the powdered fruit bodies of Daldinia concentrica.
2. Evaluation of peripheral analgesic effect by acetic acid induced writhing

method.
3. Evaluation of CNS depressant activity by Hole Cross method.
4. Chemical screening for the bioactive compounds.
5. Evaluation of anti-microbial effect by Disc Diffusion method.

Experimental Animal
Experimental Design
20 mices were randomly selected
They were divided into four groups (Group-I, Group-II, Group-III, Group-IV)
Each group was consisted of 5 mice
The four groups received Control, Standard, 200mg/kg & 400mg/kg of test
samples respectively
Preparation of Test Sample
100 mg of extract were weighed in two micro vials
Triturated by Tween-80 (Suspending agent)
After proper mixing normal saline was added
The final volume was made up to 4ml & 2ml for 400mg/kg & 200mg/kg of body weight of sample
respectively
Then vortex was done to stabilize the solution

Evaluation of Analgesic Activity
 Method: Acetic acid induced writhing method

 Preparation of Standard Sample:
For the preparation of Aceclofenac at the dose of 50 mg/kg-body weight, 25 mg
of Aceclofenac was taken and a suspension of 3.0 ml was made.
 Preparation of Acetic Acid Solution:
For the preparation of acetic acid solution, 0.7 ml acetic acid was diluted with
100ml distilled water.
Figure: Oral Administration

Continued
Results & Discussion

Analgesic activity of ethanolic extract of D. concentrica. EE= Ethanolic extract* indicates
considerable peripheral analgesic activity.
Continued
 Results & Discussion:
 The ethanolic extract of D. concentrica dose dependently induced a

significant (P<0.001) decrease in the number of writhes with 46.11% and
57.36% of inhibition at the dose of 200 and 400 mg/kg body weight,
respectively when compared to the control untreated group which was
comparable to that of the standard drug aceclofenac sodium (60.68 %
inhibition, P<0.001).
 So, it can be said that the extract of Daldinia concentrica has

significant analgesic activity.
Evaluation of CNS depressant activity
 Method: Hole Cross Method

 Preparation of positive control:
For the preparation of positive control standard drug diazepam (10 mg/kg)
was taken and a suspension of 3ml was made.
 Procedure:
 The animals were divided into control, positive control, and test groups containing five
mice each. The test groups received extracts at the doses of 200 mg/kg and 400 mg/kg
body weight orally whereas the vehicle control and positive control groups received
vehicle (1% Tween 80 in saline) and the standard drug diazepam (10 mg/kg) respectively.
 The number of passage of a mouse through the hole from one chamber to other was
counted for a period of 3 min at 0, 30, 60, 90 and 120 minute after oral administration
of the test drugs and the standard.
Continued
Result & Discussion:
Figure: Hole Cross Test of D. concentrica

Continued
Results & Discussion:
 For the study 200mg/kg and 400mg/kg doses were

administered in the mice orally. In comparison to the
control group, a significant decrease in locomotor activity
was observed to the test group. Locomotor activity
considered as an increase in alertness and decrease in
locomotor activity indicated the sedative effect. Extracts
of Daldinia concentrica decreased locomotor activity
indicates its CNS depressant activity.
Chemical Screening of Daldinia concentrica
 Chemical/reagents necessary for group identification
Reagents Composition
Fehling`s solution A 34.64gm copper sulphate in 500ml distilled water
Fehling`s solution B 173gm potassium sodium tartrate and 50gm
sodium hydroxide in 500 ml distilled water
Salkowski reagent Choloroform and few drops of concentrated sulfuric
acid
5% ferric chloride solution 5 gm ferric chloride in 100ml distilled water
10% potassium dichromate solution 10 gm potassium dichromate in 100 ml distilled
water
Keller-killiani reagent 0.5 ml glacial acetic acid, few drops of ferric chloride
and conc. Sulfuric acid
Wagner`s reagent 2 gm iodine and 6 gm potassium iodide in 100 ml
distilled water
Continued
Observations & Results:
Secondary Metabolite Test Name Result
Alkaloids Wagner’s Test ++

Glycosides General Test ++
Cardiac Glycoside Baljet’s Test --
Triterpenes Salkowsky Test ++
Carbohydrates Molisch Test --
Flavonoids General Test ++
Steroids Libermann- ++
Burchard’s Test
Tannins Ferric Chloride ++
Test
Saponins Frothing Test --
Continued
 Discussion:
 Analysis of the extract of Daldinia concentrica showed that it contains

alkaloids, flavonoids, glycosides, triterpenes and steroids based on the
procedure used. This would probably contribute to addressing the
major challenge of present day wide-spread antimicrobial resistance.
Antimicrobial Screening
Method: Disc Diffusion Method

Preparation of Test Sample:
The test sample was prepared by dissolving the extract of the sample in DMSO. The
concentration of 20μl/disc was used. The solution was applied separately on sterile
paper discs and left for sufficient time for complete evaporation.
Preparation of Discs:
 The types of discs were prepared for antibacterial screening are:
a) Sample discs
b) Standard discs
c) Blank disc
Continued
Composition of nutrient agar media (1000ml)
Ingredients Amounts
Beef extracts 3.0gm

Peptone 5.0gm
Sodium chloride 0.5 gm
Becto agar 16.0 gm
Distilled water qs. to 1000ml
Continued
 Preparations of the test plates:
 The test organism was transferred from the subculture to the test tube containing
20ml autoclaved media with the help of an inoculating loop in an aseptic area.
 The test tube was shaken by rotation to get a uniform suspension of the organism.
 The bacterial suspensions were immediately transferred to the sterile petri dishes in
an aseptic area.
 The Petri dishes were rotated several times, clockwise and then anticlockwise, to
assure homogenous distribution of the test organisms. This media were poured into
Petri dishes in such a way as to give a uniform depth of approximately 4mm.
 After the medium has cooled to room temperature; it was stored in a refrigerator
(4ºC).
Continued
Test organisms:
No Name of the Bacteria

1 Staphylococcus aureus
2 Staphylococcus albonii
3 Bacillus subtilis
4 Eschericia coli
5 Klebsiella pneumonia
6 Pseudomonas aeruginosa
7 Acinetobacter baumanii
Continued
Results of antibacterial assay:
SI Name of the bacteria Sample Extract Standard

Concentration (µl/disc) Kanamycin
Concentration
(µl/disc)
20µl/disc 30 µl/disc
B 01 Staphylococcus aureus 6 32
B 02 Staphylococcus albonii 0 29
B 03 Bacillus subtilis 14 27
B 04 Eschericia coli 0 28
B 05 Klebsiella pneumonia 0 30
B 06 Pseudomonas aeruginosa 10 34
B 07 Acinetobacter baumanii 0 31
Continued
 Discussion:
 The ethanolic extract of Daldinia concentrica shows antimicrobial

effect against Staphylococcus aureus, Bacillus subtilis & Pseudomonas
aeruginosa. This extract does not show antimicrobial effect against the
remaining four bacterial species at 20µl/disc. More elaborate study is
required to confirm its accurate antimicrobial properties.
Conclusion
 The result of the mycological investigation showed that the fungus has potential & significant
analgesic & CNS depressant activity.
 It also showed that the fungus has potential antimicrobial activity against specific bacteria.
 The fungus contains alkaloids, triterpenoids, glycosides, steroids which has made it chemically
significant and biologically important drug candidate.
Thank You

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Welcome To My Presentation on-

 Discover promising newer medicinal activities of the fungus.

 Study different pharmacological activities of fungus extract and

 Ultimately rationalize the traditional use of the selected fungus.

Figure: Daldinia concentrica

The study protocol consists of the following steps:

1. Cold extraction of the powdered fruit bodies of Daldinia concentrica.

2. Evaluation of peripheral analgesic effect by acetic acid induced writhing

3. Evaluation of CNS depressant activity by Hole Cross method.

4. Chemical screening for the bioactive compounds.

5. Evaluation of anti-microbial effect by Disc Diffusion method.

Each group was consisted of 5 mice

Triturated by Tween-80 (Suspending agent)

After proper mixing normal saline was added

Then vortex was done to stabilize the solution

 Method: Acetic acid induced writhing method

Figure: Oral Administration

Results & Discussion

 Results & Discussion:

 The ethanolic extract of D. concentrica dose dependently induced a

 So, it can be said that the extract of Daldinia concentrica has

 Method: Hole Cross Method

Figure: Hole Cross Test of D. concentrica

Results & Discussion:

 For the study 200mg/kg and 400mg/kg doses were

Alkaloids Wagner’s Test ++

 Analysis of the extract of Daldinia concentrica showed that it contains

Method: Disc Diffusion Method

Composition of nutrient agar media (1000ml)

Beef extracts 3.0gm

No Name of the Bacteria

Results of antibacterial assay:

SI Name of the bacteria Sample Extract Standard

 The ethanolic extract of Daldinia concentrica shows antimicrobial

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