Toxicity of Steroid Compound in n-Buthanol

Fraction Isolated by Thin Layer Chromatoghraphy

from Microalgae Chlorella sp.

PRESENTED BY Hasan Ali Mahbubi - (NIM. 16630062)

Supervisor I : A. Ghanaim Fasya, Examiner : Elok Kamilah Hayati,
Examiner : Ahmad Hanapi, M.Sc
M.Si M.Si
Department of Chemistry
Faculty of Science and Technology
State Islamic University Maulana Malik Ibrahim Malang
2019
Outline of presentation
Background of The
Research Aim Of The
Research Scope of The
Research
Methodol
ogy

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Background of The Research

َ ‫ض َح ََل اًل‬
‫ط ِيباا َو ًَل‬ ِ ‫اس ُكلُوا ِم َّما ِفي ْال َ ْر‬ ُ َّ‫يَا أَيُّ َها الن‬
َ ْ ‫ان ۚ ِإنَّ ُهُ لَ ُك‬
‫دو مُو ُم ِبين‬ ِ ‫ط‬َ ‫ي‬
ْ َّ
‫ش‬ ‫ال‬ ‫ت‬
ِ ‫ا‬ ‫و‬ ُ
َ ‫تَت َّ ِبعُوا ُخ‬
‫ط‬
“O mankind, eat from whatever is on earth [that is] lawful and good and do
not follow the footsteps of Satan. Indeed, he is to you a clear enemy.”

Al-Quran
Al-Baqarah : 168

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Background of The Research
 Phytosterols The Advantages
 Anti-inflamantory Effects
 Induction of Apopotis
in Cancer Cells
 Anti-oxidant
 Immunomodulatory
 Anti-hypercholesterolemic
 Antidiabetic
 Anticancer
(Caroprese, 2012)
(Bolaños-Carrillo, 2015)
(Maghfiroh, 2019)
(Caroprese, 2012)
(Rasmussen, 2008)
(Lee, 2004)
(Sheu, 1999)

Basic Structure of Phytosterols

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Background of The Research
 Chlorella sp.
What is Chlorella sp. ?
Is a single-celled, green freshwater algae, with
contain more phytosterols compound, when
compared to plant sterols
(Andrade, 2018)

Chlorella sp.
Superkingdom : Eukaryota
Classification
Kingdom : Viridiplantae
Phylum : Chlorophyta
Class : Trebouxiophyceae
Order : Chlorellales
Familly : Chlorellaceae
Genus : Chlorella
(NCBI, 2018)
The Advantages of
 Affordable
Chlorella sp.
 Easy to cultivate and fast growth Chlorella sp.
 High sterols producer

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Background of The Research
 Chlorella sp.
Others benefit of Chlorella
Chlorella sp. is the most prominent
sp.
microalgae due to its high production of:
• Long-Chain Polyunsaturated Fatty Acids
• Phenolic Compounds
• Volatile Compounds (carbonyls, alcohols,
aldehydes, esters,terpenes)
• Sterols (phytosterol, brassicasterol,
ergostenol, poriferasterol and
clionasterol)
• Protein, Amino Acids (lysine, leucine,
isoleucine, tryptophan and valine)
• Vitamins (vitamin A (beta-carotene),
vitamin C, vitamin E, B1, B2, B3, B5)
• Polysaccharides
• Pigments and Food Chlorella sp.

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Background of The Research
 Phytosterols in Chlorella
sp.

Ergosterol brassicasterol poriferasterol

“is the primary sterol in Chlorella
(Patterson, 1974) (Patterson, 1974)
species”
(Patterson, 1969)

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Background of The Research
 Urgency of Toxicity test

- Toxicity test is used to know the ability of

posions can damage inside the body and
also can be correlated with cytotoxicity
activity

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Background of The Research
 Lethal Concentration 50 (LC50) in ppm

>
500- 100-
100 1000 500
0-100
0
Non Toxic Less Toxic Mid Toxic High Toxic

(Clarkson, 2004 in Hamidi, 2014)

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 Background of The Research
 Brine Shrimp Lethal Test
Using Artemia salina Leach

The Advantages
 Affordable
 Easy to examine
 Fast growth
 No specific laboratory needed
 Trusted result
 Far from ethical issues

(Kristianti, 2006)

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Background of The Research
 Previous research

LC50 values of various fraction sterol compound

Fraction LC50 (ppm) T-SHIRTS
Ethyl Acetate 14,9625 Shofiyah, 2016 – Chlorella

Petroleum Ether 19,6889 Miniati, 2016 – Chlorella

Ethyl Acetate 30,8615

Chloroform 34,6766 Khasanah, 2016 – Hydrilla

n-buthanol 39,1573

1-buthanol 70,32 Afif, 2015 – Red Algae

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Background of The Research
 Previous research

The result of spots showed on TLC test with various eluent concentration
Various Eluent Spots Refference
(n-hexane: Ethyl Acetate)
3,75 : 1,25 14
4,00 : 1,00 13
Fikri, 2019- Chlorella sp.
4,25 : 0,75 11
4,50 : 0,50 10

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AIM of THE RESEARCH
To determine the level of toxicity of the steroid
compound n-butanol fraction hydrolysis of methanol
extracts of microalgae Chlorella sp.

To find out the results of the identification of steroid

compounds from TLC n-butanol fraction hydrolysis of
methanol extracts of microalgae Chlorella sp.

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SCOPE OF THE RESEARCH The Hydrolysis of concentrate
methanol extract using HCl 2N as
catalyst and then Partitioned
using n-buthanol
The cultivation of Microalgae
Chlorella sp using bean sprout
extract medium (4%)
Toxicity test for steroid compound
using BSLT method by Artemia
salina Leach

Extraction active compound using

methanol solvent
Identification steroid compound
using UV-Vis Spectrophotometer,
FTIR spectrophotometer, and LC-
MS/MS

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METHODOLOGY
 Time and place

 January – April
2020

 Organic Chemistry Laboratory,

Department of Chemistry, Maulana
Malik Ibrahim State Islamic
University of Malang

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METHODOLOGY
 Steps of the research

Partition And
Sample Phytochemical
Preparatio test Toxicity Test
n

Cultivation Extraction and PREPARATIVE identification

Hydrolysis TLC

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METHODOLOGY
 Step of the research -
Cultivation

Chlorella sp. bean sprout

isolate extract medium
4%

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METHODOLOGY
 Step of the research – Sample
Preparation

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METHODOLOGY
 Step of the research –
extraction

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METHODOLOGY
 Step of the research –
Hydrolysis
HCl 2 N NaHCO3

Stirrer

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METHODOLOGY
 Step of the research – partition

n-buthanol
Hydrolysate

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METHODOLOGY
 Step of the research –
Phytochemical test
Liebermen Burchard
Reagent

Add Lieberman burchard

reagent and observe the
color changes

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 METHODOLOGY
 Step of the research –
Preparative TLC

Fraction n-
buthanol of
methanol
extract
Eluen n-hexane : Ethyl acetate
(3,75 : 1,25)

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METHODOLOGY
 Step of the research – Toxicity
Test

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METHODOLOGY
 Identification

UV-VIS FTIR
LC-MS/MS
Spectrophotometer Spectrophotometer

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THANK YOU FOR
YOUR TIME