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ALKALOID EXTRACTION

METHOD

BERNA ELYA
PENDAHULUAN

Sejarahnya hampir setua peradaban manusia. Telah digunakan sebagai obat


dalam minuman, kedokteran, teh, tapel, dan racun selama 4.000 tahun. Tidak
ada usaha untuk isolasi komponen aktif dari ramuan tsb. hingga permulaan
abad ke-19
Pertama ditemukan adalah opium, getah kering Papaver somniferum.
Digunakan dalam obat selama berabad-abad dan sifat analegetik, narkotik
telah diketahui. Tahun 1803 Derosne mengisolasi alkaloida semi murni opium
diberi nama narkotina. Sertuner tahun 1805 meneliti lebih lanjut opium dan
berhasil mengisolasi morfin.
Tahun 1817 1820 di Lab. Pelletier dan Caventon di Fakultas Farmasi Paris,
melanjutkan penelitian alkaloida dan menemukan strikhnina, emetina, brusina,
piperina, kafein, kuinina, sinkhonina dan kolkhisina dalam waktu singkat.
Swedish pharmacist Scheele paved the way for isolating organic
plant acids; but it remained for a young German apothecary.
Friedrich Wilhelm Adam Sertumer, to
give the word opiums chief narcotic
principle, morphine; and to recognize
and prove the importance of a new
class of organic substances: alkaloids.
His first announcements challenged,
Sertumer in 1816 conducted a new
series of bold, starting experiments in
his apothecary shop in Einbeck,
including a series of physiologic tests
on himself and three young friends.
Recognition and followed, relocating
in an apothecary shop in Hameln,
Sertumer continued organic chemical
experimentation and discovery
throughout his life.
Caventou, Pelletier and Quinne
Taking their cue from Sertrner's alkaloidal
experiments, two French pharmacists, Messrs.
Pierre-Joseph Pelletier and Joseph-Bienaim
Caventou, isolated emetine from
ipecacuanha in 1817; strychnine and brucine
from nux vomica in 1818; then, in their
laboratory in the back of a Parisian
apothecary shop, they tackled the problem
that had baffled scientists for decades -
wresting the secrets of the Peruvian barks that
were so useful against malaria.
In 1820 Caventou and Pelletier announced
the methods for separation of quinine and
cinchonine from the cinchona barks;
prepared pure salts, had them tested
clinically, and set up manufacturing facilities.
Many other discoveries came from their
pharmacy-laboratory; high honors were
accorded them.
Tahun 1826 Pelletier dan Caventon menemukan koniina (penyebab
kematian Socrates). Koniina, alkaloida pertama ditentukan sifat (1870)
dan disintesis (1886).
Tahun 1884 ditemukan 25 alkaloida dari Cinchona sp. Penentuan
struktur alkaloida terhalang beberapa hal a.l kompleksitasnya, contoh
strikhnina ditemukan pertama Pelletier dan Caventon 1819, struktur
baru ditentukan Robinson dkk 1946 (140 tahun).
Tahun 1939 hampir 300 alkaloida telah diisolasi dan 200 telah
ditentukan struktur. Dalam seri Alkaloida yang diterbitkan pertama oleh
Manske pada 1950 memuat lebih 1000 alkaloida.
Dikenalnya teknik sistem analisis kromatografi preparatif dan instrumen
canggih maka penemuan alkaloida meningkat cepatnya.
Buku terbitan 1973 mencatat 4959 alkaloida dapat diisolasi dan 3293
ditentukan strukturnya. Perkembang Ilmu Pengetahuan dengan
penemuan berbagai macam kromatografi dan instrumen spektroskopi
dengan sistem komoputerisasi maka isolasi dan penentuan struktur
alkaloida sudah tidak terbilang lagi.
EKSTRAKSI
Keragaman golongan alkaloida pola ekstraksi dilakukan atas
dasar sifat basanya, dikenal 2 cara, yaitu :
pertama ekstraksi dengan air suasana asam
kedua ekstraksi pelarut organik suasana basa.
Ekstraksi awal alkaloida umum dilakukan dengan pelarut organik
suasana basa.
Beberapa alkaloida terdapat dalam biji, daun atau bagian
tumbuhan lain mengandung lilin (sangat nonpolar, mengganggu
proses ekstraksi) diawalemakkan dengan petroleum-eter.
Ekstrak petroleum eter dites alkaloida, kalau banyak alkaloida
tersari, atasi dengan membuat suasana asam (bentuk garam) larut
air, kemudian ekstraksi dengan peteroleum eter.
SAMPEL contoh bagan
ekstraksi alkaloida Yang
- CaO + Air dapat dikembangkan
- + Pelarut organik

AMPAS EKSTRAK
- Asamkan (HCl)
- + Air

Lap. Air Lap.Organik


- NH4OH / NaOH e
- + Pelarut organik

Lap. Air Lap.Organik ALKALOIDA


SAMPEL
Petroleum eter

Eks. Petroleum eter Residu


1. MeOH or EtOH 95%
2. Pemekatan
3. Partisi EtOAc, as.tartrat 2%

Lap. EtOAc
Alkaloida netral
Lap. Asam
Or basa lemah Tartrat 2%
Basakan NH3 or Na2CO3

EtOAc
Lar. Basa
Fraksi alkaloida
Berair
basa
Mungkin kandung
alk. quaterner
Satyajit D. Sarker (2006) Natural Products Isolation
Alkaloid drug with medium to high alkaloid contents (> 1%)

General method (Extraction method A)


Powdered drug (1g) is mixed thoroughly with 1 ml 10%
amonia solution or 10% Na2CO3 solution and then
extracted for 10 min with methanol under reflux.
The filtrate is the concentrated according to the total
alkaloid of the specific drug, so that 100 l con.tains 50-
100 l total alkaloid

Sabine Bladt, Eva M. Zgainski 1984. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer-Verlag
Berlin Heidelberg GmbH
Alkaloid drugs with low total alkaloids (<1 %)
Enrichment method, extraction Method B
Powdered drug (2 g) is ground in a mortar for about 1
min with 2 ml 10% ammonia solution and then
thoroughly mixed with 7 g basic aluminium oxide
(activity grade I).
This mixture is then packed loosely into a glass column
(diameter, 1.5 ml CHCl3 and the eluate is collected,
evaporated to 1 ml and used for TLC.

Sabine Bladt, Eva M. Zgainski 1984. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer-Verlag
Berlin Heidelberg GmbH
Alkaloid drugs with low total alkaloids (<1 %)
Sulphuric Acid, Extraction Method C
Powdered drug (0,4 2 g) is shaken for 15 min with 15 ml 0,1N
sulphuric acid and then filtered.
The filter is washed with 0,1 N sulphuric acid to a volume of 20 ml
filtrate; 1 ml concentrated ammonia is the added.
The mixture is shaken with two portions of 10 ml diethyl eter.
The ether is dried over anhydrous sodium sulphate, filtered and
evaporated to dryness and resulting residue dissolved in 0,5 ml
methanol.

Sabine Bladt, Eva M. Zgainski 1984. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer-Verlag Berlin
Heidelberg GmbH
Extraction

6 kg of dry weight
Extracted with methanol (CH3OH) several times
Exhaustive maceration technique
Methanol was evaporated using rotary evaporator
600 g of methanolic extract produced and applied
for the next fractionation process
Crude Alkaloid Fractionation
Air Dried Plant Material Acidified with HCl to pH 2

Extraction with MeOH Reduce N-oxides using


zinc powder
Dissolved in 0.5 M HCl
HCl
Basified to pH 9

Washed with CH2Cl2 Extraction with CH2Cl2


pH11
pH9
Zn
pH2
HCl

Basified to pH 9 CH2Cl2
Basified to pH 11

Extraction with CH2Cl2


Extraction with CH2Cl2
Basified to pH 11

Extraction with CH2Cl2


Crude Alkaloids Fractions
Fractions (mg)

A B C D E
Dried plant MeOH fraction
Weigh (g) (g) HCl pH9 pH11 pH9 pH11

6161.6 628.4 2815.5 984.2 211.1 357.6 197.7


Dragendorff reagent test -ve ++ve +ve +ve +ve

Fraction A: no alkaloids present


Alkaloids are presents in all other fractions
Fraction B is the alkaloids rich fraction
Fraction B applied for chromatographic
process
Melakukan isolasi dan purifikasi alkaloid
dari Corydalis yanhusuo W.T. Wang
dengan High Speed Counter-Current
Chromatography (HSCCC).
Dengan menggunakan metode ekstraksi
alkaloid metode A (metode umum)
M. Lebrini, et.al., 2011 (International Journal of Electrochemical
Science). Mengekstraksi alkaloid dengan cara; sampel daun kering
dibasakan dengan larutan NH4OH 5%. Larutan basa diekstraksi
dengan diklorometana (3x200ml).
Fraksi diklorometana dicuci dengan air (2x200 ml). Kemudian,
lapisan organik diekstraksi tiga kali dengan diencerkan HCl.
Fraksi berair gabungan dicuci dengan diklorometana (3x80 mL)
dan pH larutan berair disesuaikan dengan 9 dengan larutan
(NH4OH 25%).
Lapisan air ini diekstraksi dengan diklorometana (3x100 ml) dan
lapisan organik gabungan dicuci dengan air suling (2x100 ml),
dikeringkan dengan Na2SO4.
Geeta Singh dan Padma Kumar, 2012 (International
Journal of Pharmay and Pharmaceutical Sciences)
Alkaloids were extracted from different parts of the selected
plants by well established methods (Harborne. 1984) after
preliminary detection of alkaloids.
Finely powered sample (100g) of plant parts were extracted with
10% acetic acid in ethanol for 4 h.
Extracts were concentrated and were made alkaline by NH4OH.
Precipitate thus obtained was collected by centrifugation,
washed with 1% NH4OH, filtered, dried in vaccuo and weighed.
Extracts thus obtained were stored at 4C in air tight glass vials for
further use.
An amount of 5 kg of dried plant seeds was extracted with methanol applying
percolation method.
The obtained extract was evaporated under vacuum to give a viscose mass.
Then, an amount of 200 g of the extract was suspended in 600 mL of distilled water and
was partitioned sequentially with n-hexane (5 x 300 mL), carbon tetrachloride (5 x 300
mL), dichloromethane (5 x 300 mL), acidified chloroform (pH 3) (5 x 300 mL) and basified
chloroform (pH 9) (5 x 300 mL) at room temperature.
Totally, six major fractions were collected and concentrated under vacuum and stored at
-20oC until pharmacological tests.
Reimmel Kwame Adosraku, et.al., 2013 (Internasional Journal of
Pharmacy and Pharmaceutical Sciences)

120 g sampel direflux menggunakan pelarut etanol sebanyak 350


ml selama 3 jam. Setelah itu didinginkan dan disaring
Diulangi kembali dengan metode dan perlakuan yang sama.
Kemudian diuapkan menggunakan evaporator pada 70oC sampai
diperoleh konsentrat sekitar 25 ml, kemudian dipindahkan ke gelas
kimia.
Kemudian ditambah 50 ml KOH 10% dalam etanol, diaduk dan
didiamkan selama 2 jam. Kemudian larutan dituang dan
meninggalkan residu yang tidak larut dalam gelas.
Larutan supernatannya didiamkan selama 48 jam. Kemudian
terbentuk endapan kuning (piperin)
Dilakukan kristalisasi dalam aseton: heksan 3:2
Extraction and Isolation of the
Alkaloid
The dried bark (2.5 kg) of A. cuspidatum (Euphorbiaceae) were extracted with hexane (25.0
l) for 18 hours. The residual plant material was dried and left for 2 h after moistening with 25%
NH4OH.
Then, It macerated with CH2Cl2 (25.0 l) twice for 3-days. The extract CH2Cl2 was
concentrated to give residue (7,0 g) that was subjected to column chromatography on
silica gel (column dimension = 3 cm, length = 1 m, silica gel 60, 70230 mesh ASTM; Merck
7734) using CH2Cl2 gradually enriched with methanol to yield 58 fractions.
Fractions were then recombined on the basis of their TLC behavior to obtain 7 fractions.
Fractions 4 (98 mg), afforded an alkaloid identified as cuspidatin (1), using PTLC (Merck
KGaA silica gel 60 F254; CH2Cl2-MeOH; 9:1). Cuspidatinol (2) was obtained from fraction 5
(198 mg) by using PTLC (Merck KGaA silica gel 60 F254; CH2Cl2-MeOH; 8.5:1.5).