Secondary Metabolite Screening and Brine Shrimp Lethality Assay of Cinnamomum mercadoi Vidal Leaf Crude Methanolic Extract

THESIS IT

YEAR 6

April 25, 2017

Cinnamon

Our Heavenly Father, the source of love and wisdom,

we praise and thank You for the countless gifts

Cinnamomum mercadoi

Brine Shrimp Lethality Assay

Importance:

– Determines the cytotoxic effects of different compounds from plants or other materials on Artemia franciscana.

You generously shower upon us.

We thank You for the strength, the will, and the inspiration to work on this research.

Aside from the educational competence we gained,

we learned the value of perseverance, cooperation and diligence.

Medicinal Values

• Betterment of digestion
• Headaches
• Rheumatism
• Stomach troubles
• Tuberculosis

We humbly offer You then this research work.

Let it be an expression of our desire to grow not only in knowledge, but more in wisdom.

Secondary Metabolite Screening

May we imitate the examples of

St. John Baptist de La Salle

Who in all humility, remained faithful and

attributed everything to You.

AMEN.

St. John Baptist de La Salle. Pray for us.

Live Jesus in our hearts.

Forever

Importance:

– For the separation and isolation of the active principle from plant sources.

Number of Dead Nauplii and Percent Mortality in Different Dilution Concentration of

C. mercadoi Leaf Crude Methanolic Extract

Linear Regression

C. mercadoi Leaf Crude Methanolic Extract Level of Cytotoxicity

Brine Shrimp Lethality Assay

Conclusion

Secondary Metabolite Screening on C. mercadoi Leaves

Legend: “–“ is negative, “+” is slightly positive, “++” is positive, “+++” is highly positive.

Recommendation

Secondary Metabolite Screening

1. Further study on the plant parts is recommended

such as a study on the roots and the stem.

2. A comparison between the results of the study

using the leaves and the bark of C. mercadoi.

3. A cytotoxicity assay on higher test organisms, such

as mice/rats, using the leaf extracts of C. mercadoi.

RESULTS AND DISCUSSION

• Secondary metabolite screening

– No reaction / (absent)

+++ High-Intense color change / (highly positive)

++ Average-Moderate color change / (positive)

+ Low-Faint color change / (slightly positive)

• Brine shrimp lethality assay

> Mortality rate

SECONDARY METABOLITE SCREENING AND BRINE SHRIMP LETHALITY ASSAY OF Cinnamomum mercadoi Vidal LEAF CRUDE METHANOLIC EXTRACT

DATA GATHERING

Brine shrimp lethality assay

> Mortality rate

MmP Percent mortality of individuals in time T [%]

Nd Number of dead individuals

Ni Initial number of living individuals placed into every

concentration in the beginning of the experiment

Statistical Analysis

• Linear regression analysis
• Chi-square (χ2) test

METHODOLOGY

Research Design

• Descriptive–experimental

Research Procedure

Research Setting

Plant Collection & Identification

Preparation & Extraction of Leaves

• Collection: Mts. Palay Palay
• Experimentation:

– Chemistry Research Laboratory

– Biological Sciences Research Laboratory

Brine Shrimp Lethality Assay

ASW:

- 1L Distilled Water

- NaCl

Secondary Metabolite Screening

• Alkaloids
• Flavonoids
• Terpenoids
• Tannins
• Saponins
• Cardiac Glycosides
• Anthraquinones

Brine Shrimp Lethality Assay

• Hatching of Brine Shrimp Eggs
• Lethality Assay

Secondary Metabolite Screening

Alkaloids

Cardiac glycoside

Plant Collection & Identification

Tannins

3mL Hager's Ragent + 1mL extract

= Yellow coloration when Hager's reagent is dropped

Preparation & Extraction of Leaves

0.5mL extract + 5mL distilled water + 2mL glacial acetic acid + 1 drop 2% Ferric chloride

= Brown ring formed in interface

ground

Flavonoids

0.5mL extract + 2-5 drops 1% Aluminum chloride

= Yellow coloration that disappears instantly

Anthraquinones

Saponins

Terpenoids

5mL extract + 2mL hexane + 3mL conc. sulfuric acid

= From violet to blue/green

Serial Dilution

- End-

Thank you!

Adedoja, Maria Emilia Adenike V.

dela Cruz, Natazsha Ann Mari V.

April 25, 2017

SCOPE AND LIMITATION

BACKGROUND OF THE STUDY

SIGNIFICANCE OF THE STUDY

• Pharmaceutical Companies
• Pharmaceutical Researchers
• Ethnobotanists
• Student Researchers

BACKGROUND OF THE STUDY

FRAMEWORK

Cinnamomum mercadoi leaf extract

Secondary metabolite Screening and Brine Shrimp Lethality Assay

STATEMENT OF THE PROBLEM

1. What were the secondary metabolites present in

the methanolic crude leaf extract of C. mercadoi using secondary metabolite screening?

2. Did the leaf crude methanolic extract of C. mercadoi

exhibit cytotoxic effects when tested against Artemia franciscana (brine shrimp) in varying dilutions?

3. Was there significant difference in the 80 ppt, 8 ppt

and 0.8 ppt serial dilution?

Hypothesis

1. The secondary metabolites of alkaloids,

flavonoids, terpenoids, tannins, saponins, cardiac glycosides and anthraquinones are not present.

2. Leaf crude methanolic extract of C. mercadoi

did not exhibit cytotoxic effects againts brine shrimp in varying dilutions.

3. There is no significant difference in the 80 ppt,

8 ppt and 0.8 ppt concentrations.