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EKSTRAKSI DAN ISOLASI

HAMSIDAR HASAN

PENDAHULUAN

Tumbuhan sebagai sumber bahan obat. Perkembangan teknologi.

Tahapan pengolahan bahan baku


- Ekstraksi - Isolasi - Penentuan struktur kimia

EKSTRAKSI KANDUNGAN KIMIA

DEFENISI
EKSTRAKSI: Proses penyarian zat-zat berkhasiat atau zat aktif dari bagian tanaman obat, hewan atau mineral.

Cairan Penyari

Sel

ekstrak

Target of the extraction

An unknown bioactive compound. A known compound present in an organism. A group of compounds within an organism that are structurally related. All secondary metabolites produced by one natural source that are not produced by a different control source, e.g., two species of the same genus or the same species grown under different conditions. Identification of all secondary metabolites present in an organism for chemical fingerprinting or metabolomics study.

METODA EKSTRAKSI & PEMURNIAN


1. Senyawa yang akan diekstraksi

Dapat mengikuti prosedur yang telah dipublikasi Dengan modifikasi sesuai dengan kebutuhan atau mengikuti persyaratan-persyaratan Prosedur dapat dilakukan mengikuti klas senyawa aktif/senyawa interest Diikuti dengan uji kromatografi

2. Species dari bahan material

3. Pemakaian di masyarakat (Ethnopharmacology)


Disesuaikan dengan penggunaan di masyarakat yang pada umumnya dilakukan dengan merebus

4 Belum pernah dilaporkan sebelumnya

Dilakukan berdasarkan hasil skrining (random ataupun selektif) aktivitas Pada umumnya hanya satu atau dua macam ekstrak untuk setiap species yang berbeda kepolaranya

Sifat-sifat senyawa yang diekstraksi:


1. Polaritas Senyawa aktifnya telah atau belum diketahui, prinsip kerja adalah Like dissolves like, Pelarut polar akan melarutkan senyawa polar 2. Effek PH Senyawa yang terionisasi harus menjadi pertimbangan Kelarutan senyawa dalam temperatur tinggi akan naik karena solvent

lebih dapat penetrasi pada jaringan tanaman Senyawa Phenol, & asam organik - asam Untuk mendapatkan hasil optimum, PH harus diatur Contoh : Alkaloida --- basa/alkalis Walau demikian, harus dilakukan hati-hati karena adanya ester atau glikosida dapat pecah karena pengaturan pH

3. Thermostabilitas Adanya senyawa yang kurang stabil, kemungkinan terjadi Artefacts

Sifat-sifat
3. Reaktivitas Beberapa solvents cukup reaktif, sehingga mudah terbentuk artefacts. Mis: solvents dengan gugus

C=O (aseton, metiletilketon dapat bereaksi dengan senyawa nukleofil; metanol, etanol dapat menyebabkan alkilasi

4. Harga /Cost Digunakan solvent dengan kemampuan hampir

sama dengan harga lebih murah Petroleum eter lebih murah dibanding n-heksana, keduanya mempunyai kemampuan sama

Sifat-sifat solvent yang digunakan:


1. Volatility, Flammability dan Boiling point Boiling point memberi gambaran kemudahan diuapkan

dengan sedikit pemanasan Tetapi semakin mudah menguap, semakin dibutuhkan penangan lebih serius Penggunaan eter, dihindari (flammable & peroxides)

2. Toksisitas Sifat toksisitas terhadap operator/pelaku Kloroform & eter menyebabkan depresi pernafasan serta

anestesia Acetonitrile & metanol- beracun Karbontetraklorida - hepatotoksik Benzen -- karsinogenik Beberapa solvents defatting skin dermatologik Kontak dengan solvent harus maksimal dihindari

Jenis ekstrak yang akan digunakan:


Jenis ekstrak yang akan digunakan turut menentukan metoda ekstraksi. Pada penggunaan ekstrak dimaksudkan untuk makanan & obat, terdapat batasan sisa solvent yang tergantung pada sifat ketoksikan residu. Ekstrak yang digunakan untuk bioassay, kriteria khusus harus diperhatikan karena pada umumnya bioassay dilaksanakan dalam aquaeous media. Salah satu alternatif digunakan DMSO untuk melarutkan ekstrak yang non polar.

Solvent recycling:

Solvent recycling sangat penting dalam pertimbangan lingkungan (ekologi) dan ekonomi Recovery dan penggunaan non azeotrop solvent (mis. Kloroform: metanol 1:1 v/v) baik, tetapi pemisahan menjadi komponennya sulit & mahal Sehingga lebih disukai solvent tunggal

EKSTRAKSI KANDUNGAN KIMIA

METODE EKSTRAKSI
1. Maserasi 2. Perkolasi 3. Refluks 4. Soxhlet 5. Destilasi Uap Air 6. Ultrasound-assisted solvent extraction 7. Pressured-solvent extraction 8. Supercritical Fluid Extraction

Prosesnya Tersarinya Senyawa Aktif : 1. Tidak Berkesinambungan : - Maserasi - Destilasi uap air 2. Berkesinambungan : - Perkolasi - Soxhletasi - Reflux

Didasarkan Pada Suhu 1. Penyarian Panas - Destilasi Uap Air (Steam Destillation) - Reflux - Soxhlet 2. Penyarian Dingin - Maserasi - Perkolasi - Soxhlet

1 2

CONDENSORS

8 7

6
4 3 2 1 11 5 6 7 8 9 3 2 1 10 4 5 6 7 8 9

CONDENSORS

11 1

10

8 2

4
5

12
4 3 2 5 6 7 8
1 11 10

4 9 3 2 1

9
6

7 8 9

10

Ultrasound-assisted solvent extraction

This is a modified maceration method where the extraction is facilitated by the use of ultrasound (high-frequency pulses, 20 kHz). Ultrasound is used to induce a mechanical stress on the cells through the production of cavitations in the sample. The cellular breakdown increases the solubilization of metabolites in the solvent and improves extraction yields. The efficiency of the extraction depends on the instrument frequency, and length and temperature of sonication. Ultrasonification is rarely applied to large-scale extraction; it is mostly used for the initial extraction of a small amount of material. It is commonly applied to facilitate the extraction of intracellular metabolites from plant cell cultures

PRESSURIZED SOLVENT EXTRACTION

Pressurized solvent extraction, also called accelerated solvent extraction, employs temperatures that are higher than those used in other methods of extraction, and requires high pressures to maintain the solvent in a liquid state at high temperatures. It is best suited for the rapid and reproducible initial extraction of a number of samples. An additional advantage is that the technique can be programmable, which will offer increased reproducibility. However, variable factors, e.g., the optimal extraction temperature, extraction time, and most suitable solvent, have to be determinedfor each sample.

Supercritical Fluid Extraction

Supercritical fluids (SCFs) are increasingly replacing organic solvents, e.g., n-hexane, dichloromethane, chloroform, and so on, that are conventionally used in industrial extraction, purification, and recrystallization operations because of regulatory and environmental pressures on hydrocarbon and ozone-depleting emissions. In natural product extraction and isolation, supercritical fluid extraction (SFE), especially that employing supercritical CO2, has become the method of choice. Sophisticated modern technologies allow precise regulation of changes in temperature and pressure, and thus manipulation of solvating property of the SCF, which helps the extraction of natural products of a wide range of polarities. The critical point of a pure substance is defined as the highest temperature and pressure at which the substance can exist in vaporliquid equilibrium. At temperatures and pressures above this point, a single homogeneous fluid is formed, which is known as supercritical fluid (SCF). SCF is heavy like liquid but has the penetration power of gas

Principle of Solvent-Free Extraction Process: A Typical Supercritical CO2 System


Liquid CO2 is forced into supercritical state by regulating its temperature and pressure. Supercritical CO2 has solvent power and extracts predominantly lipophilic and volatile compounds. Gaseous CO2 returns to CO2 tank. After a full round, the new extraction starts with circulating CO2.

The main advantages of using SCFs

inexpensive, contaminant-free, selectively controllable, and less costly to dispose safely than organic solvents. Oxidative and thermal degradation of active compounds is much less likely in SFE than inconventional solvent extraction and steam distillation methods. SCFs can have solvating powers similar to organic solvents, but with higher diffusivities, lower viscosity, and lower surface tension

Important Factors in SFE Method Development

The solubility of the target compound(s) in supercritical CO2 or other SCF has to be determined. The effect of cosolvents on the solubility of the target compound(s) needs to be determined. The effect of matrix, either has the analyte lying on its surface (adsorbed), or the analyte is entrained in the matrix (absorbed), has to be considered carefully. The solvating power of SCF is proportional to its density, which can be affected by any temperature change for any given pressure. Therefore, strict temperature control has to be in place. The partition coefficient of the analyte between CO2 and the matrix, which is often affected by the flow rate, has to be considered. Higher flow rates and longer extraction time may be necessary to sweep the analyte out of the extraction chamber. Lower flow rates may be applied if the kinetics of the system are slow. Careful consideration has to be given in choosing appropriate modifiers

SFE of Taxol From Pacific Yew Tree

Taxol, one of the most commercially successful and effective anticancer natural product drugs, is a complex diterpene isolated from the Pacific yew tree (Taxus brevifolia). The SFE protocol for the extraction of Taxol from the bark was introduced by Georgia Tech, Athens, GA About 50% of the Taxol present in the bark was selectively extracted using a CO2 EtOH mixture as opposed to 25% extraction with supercritical CO2 alone.

ISOLASI KANDUNGAN KIMIA

DEFENISI
ISOLASI: Suatu metode untuk menarik dan memisahmisahkan kandungan kimia dari tumbuhan dengan menggunakan pelarut tertentu.

Kromatografi
Suatu teknik pemisahan komponen dari suatu campuran menggunakan prinsip perbedaan distribusi komponen tsb dalam 2 fase, fase gerak dan fase diam.

Analisis
Pemisahan

Identifikasi Kemurnian Kuantifikasi

Campuran

Komponen

Proses elusi senyawa

Klasifikasi Kromatografi
Berdasarkan fasa geraknya: Liquid Chromatography Gas Chromatography Berdasarkan fasa diamnya (interaksi komponen dengan fasa diam): Partition Chromatography Adsorption Chromatography

Fasa diam yg digunakan:

silica gel - silicon dioxide (SiO2)x


(a common, inexpensive stationary phase) O O O | | | -O-Si-O-Si-O-Si-O-H | | | These exposed OH units O O O give silica gel a | | | relatively polar surface. -O-Si-O-Si-O-Si-O-H | | | O O O bulk (SiO2)x surface

4. VISUALISASI BERCAK

Langsung / mata telanjang

Lampu UV
Uap iodin Reagen penyemprot Media bakteri

Spray Reagents for Natural Product TLC Visualization

5. INTERPRETASI HASIL Tentukan Retention factors(Rf) masing2 bercak. Rf =


distance spot ___________ has moved ____________ distance solvent has moved

X Y

Rf =

distance spot has moved _______________________ distance solvent has moved

Z Y
Z Z

Rf =

distance spot has moved _______________________ distance solvent has moved

T Y

Troubleshooting KLT
Bercak tidak membulat (mbleber)

Sampel terlalu pekat. Kembangkan lagi KLT setelah sampel diencerkan. Sampel terlalu banyak mengandung komponen. Perlu dilakukan partisi terhadap sampel. Tidak nampak bercak Sampel terlalu encer. Pekatkan sampel, atau tambahkan volume sampel yang ditotolkan. Beberapa senyawa memang tidak menunjukkan pemadaman di bawah lampu UV. Pakailah reagen semprot untuk menampakan bercak (biasanya uap iodin atau serium sulfat)

Troubleshooting KLT
Garis batas atas (akhir eluen) tidak rata

Chamber tidak/kurang jenuh eluen (penjenuhan kurang optimum). Pemasangan plat dalam chamber tidak pas (miring).
Bercak berekor

Senyawa mengandung gugus yang bersifat asam atau basa kuat (amina atau asam karboksilat). Tambahkan beberapa tetes NH4OH (amina) atau asam asetat (asam karboksilat) pada eluen.

Aplikasi KLT
ANALISIS KUALITATIF

Bercak dibandingkan Rf-nya dengan baku. Menggunakan reagen penyemprot untuk menentukan golongan senyawa (dragendorf, lieberman-burchat, AlCl3, dll).
ANALISIS KUANTITATIF Perlu beberapa totolan larutan baku (yg berbeda konsentrasi/volume penotolannya) untuk membuat kurva baku. Bercak dianalisis densitasnya dengan densitometer untuk kuantifikasi.

Preparative Thin-Layer Chromatography (PTLC)


Advantages: 1. Cost effective compared to the instrumentation required, for example, HPLC or CCC. 2. A simple technique that requires little training or knowledge of chromatography to be used. 3. An analytical method may be easily scaled up to a preparative method. 4. Ability to isolate natural products quickly in the milligram to gram range. 5. Flexibility of solvent and stationary phase choice, i.e., the solvent system can be changed quickly during a run. 6. The separation can be optimized readily for one component, i.e., it is relatively easy to zero in on a particular product. 7. Methods are quickly developed. 8. A large number of samples can be analyzed or separated simultaneously.

Disadvantages: 1. Poor control of detection when compared to HPLC. 2. Poor control of elution compared to HPLC. 3. Loading and speed are poor compared to VLC. 4. Multiple development methods to isolate grams of material may be time consuming. 5. Restricted to simple sorbents, such as silica, alumina, cellulose, and RP-2.

Centrifugal Preparative Thin-Layer Chromatography (CPTLC)

COLUMN CHROMATOGRAPHY

Hyphenated Techniques
The technique developed from the coupling of a separation technique and an on-line spectroscopic detection technology.

Hirschfeld introduced the term hyphenation to refer to the on-line combination of a separation technique and one or more spectroscopic detection techniques. This technique, developed from a marriage of a separation technique and a spectroscopic detection technique.

EKSTRAKSI UNTUK TUJUAN SKRINING AKTIVITAS BIOLOGI/BIOASSAY

Penting pada pengerjaan skrining bahan dalam jumlah besar (HTS), yang sifat senyawa aktifnya belum diketahui Salah satu alternatif adalah mengekstraksi dengan solvent-solvent dengan perbedaan polaritas, sehingga diperoleh 4 ekstrak dari 1 bahan. Tetapi umumnya disukai hanya 2 ekstrak saja yang mempunyai jarak polaritas berbeda (mis. N-heksana/pet. Eter kemudian dengan 70% etanol)

Techniques for Detection of Phytochemical Groups in Extracts


Alkaloids

Mayer reagent Solution I: dissolve 1.36 g HgCl2 in 60mL water. Solution II: dissolve 5 g KI in 10mL water. Procedure: combine the two solutions and dilute with water to 100mL. Add a few drops to an acidified extract solution (diluted HCl or H2SO4), and if alkaloids are present, a white to yellowish precipitate will appear. Care should be taken not to agitate the test system, because the precipitate may be redissolved. Dragendorff reagent Solution I: dissolve 8.0 g bismuth subnitrate [Bi(NO3)3. H2O] in 30% w/v HNO3. Solution II: dissolve 27.2 g KI in 50mL water. Procedure: combine the solutions and let stand for 24 h, filter, and dilute to 100mL with deionized water. In acid solutions, an orange-brownish precipitate will appear. The alkaloids may be recovered by treatment with Na2CO3 and subsequent extraction with diethyl ether. This reaction may also be performed on a filter paper or on a TLC plate by adding a drop of the reagent onto a spot of the sample. Wagner reagent Solution: dissolve 1.27 g I2 (sublimed) and 2 g KI in 20mL water, and make up with water to 100 mL. Procedure: a brown precipitate in acidic solutions suggests the presence of alkaloids. Ammonium reineckate Solution: add 0.2 g hydroxylamine to a saturated solution of 4% ammonium reineckate {NH4[Cr(NH3)2(SCN)4].H2O}, and acidify with dilute HCl. Procedure: when added to extracts, a pink precipitate will appear if alkaloids are present. The precipitate is soluble in 50% acetone, which may also be used for compound recrystallization.

Sesquiterpene Lactones

Kedde reagent Solution I: dissolve 2% of 3,5-dinitrobenzoic acid in MeOH. Solution II: 5.7% aqueous KOH. Procedure: add one drop of each solution to 0.20.4mL of the sample solution, and a bluish to purple color will appear within 5 min. The solution should not contain acetone, which gives a deep bluish color. Baljet reagent Solution I: dissolve 1 g picric acid in 100mL EtOH. Solution II: 10 g NaOH in 100mL water. Procedure: combine solutions I and II (1:1) before use and add two to three drops to 23 mg of sample; a positive reaction is indicated by an orange to deep red color.

Flavonoids

Shinoda test Procedure: to an alcoholic solution of the sample, add magnesium powder and a few drops of concentrated HCl. Before adding the acid, it is advisable to add t-butyl alcohol to avoid accidents from a violent reaction; the colored compounds will dissolve into the upper phase. Flavones, flavonols, the corresponding 2,3-dihydro derivatives, and xanthones produce orange, pink, red to purple colors with this test. By using zinc instead of magnesium, only flavanonols give a deepred to magenta color; flavanones and flavonols will give weak pink to magenta colors, or no color at all. Sulfuric acid Procedure: flavones and flavonols dissolve into concentrated H2SO4, producing a deep yellow colored solution. Chalcones and aurones produce red or red-bluish solutions. Flavanones give orange to red colors.

Other Polyphenols

Ferric chloride Solution: dissolve 5% (w/v) FeCl3 in water or EtOH. Addition of several drops of the solution to an extract produces a blue, blueblack, or blue-green color reaction in the presence of polyphenols. This is not a specific reagent for tannins, as other phenolic compounds will also give a positive result.

Gelatin-salt test Procedure: for the detection of tannins in solution, dissolve 10 mg of an extract in 6mL of hot deionized, distilled water (filtering if necessary), and the solution is divided between three test tubes. To the first is added a 1% solution of NaCl, to the second is added a 1%-NaCl and 5%-gelatin solution, and to the third is added a FeCl3 solution. Formation of a precipitate in the second treatment suggests the presence of tannins, and a positive response after addition of FeCl3 to the third portion supports this inference.

Sterols

LiebermannBurchard test Solution: combine 1mL acetic anhydride and 1mL CHCl3, and cool to 0C, and add one drop concentrated H2SO4. Procedure: when the sample is added, either in the solid form or in solution in CHCl3, blue, green, red, or orange colors that change with time will indicate a positive reaction; a blue-greenish color in particular is observed for sterols, with maximum intensity in 1530 min. (This test is also applicable for certain classes of unsaturated triterpenoids.) Salkowski reaction Procedure: dissolve 12 mg of the sample in 1mL CHCl3 and add 1mL concentrated H2SO4, forming two phases, with a red color indicating the presence of sterols.

Saponins

When shaken, an aqueous solution of a saponin-containing sample produces foam, which is stable for 15 min or more. An additional test for saponins makes use of their tendency to hemolyze red blood cells (20,58), although this tendency may be inhibited by the presence of tannins in the extract, presumably because tannins crosslink surface proteins, thereby reducing the cells susceptibility to lysis

METODA EKSTRAKSI UNTUK KLAS SENYAWA TERTENTU


Salah satu faktor penentu metoda ekstraksi adalah tipe senyawa yang akan diekstrak. Dibawah ini beberapa metoda ekstraksi tipe senyawa tersebut:

Dapat pula dengan kloroform, metanol, eter, etanol; tetapi senyawa lain akan ikut terekstraksi

MINYAK, LILIN, LEMAK Minyak (cair) sedang lilin (waxes) dan lemak bentuk padat Pet. Eter, n-heksana baik untuk mengekstraksi minyak, lilin, lemak Tipe senyawa ini sering mengganggu proses partisi dan fraksinasi, sehingga sering dipisahkan dulu MINYAK MENGUAP komponen penyusunnya mono & seskui terpene serta senyawa fenolik Dapat disari dengan pet. Eter, tetapi lilin, waxes sering ikut; oleh karena itu lebih tepat dilakukan dengan kloroform Dapat dipisahkan dengan distillasi uap

KAROTENOIDA

Pada umumnya tetraterpenoida (40 karbon), dapat dibagi 2 : hidrokarbon dan teroksigenasi Hidrokarbon-non polar, sehingga dapat diekstraksi dengan pet. Eter; sedang yang teroksigenasi umum mempunyai gugus OH, C=O, aldehid, epoksid dsb. sehingga menjadi lebih polar dan dapat diekstraksi dengan etanol dan juga dengan kloroform

ALKALOID
The literature of alkaloids can conveniently be divided into five sections, dealing with
(1) The occurrence and distribution of these substances in plants ; (2) Biogenesis, or the methods by which alkaloids are produced in the course of plant metabolism ; (8) Analysis, ranging from the commercial and industrial estimation of particular alkaloids to the separation, purification and description of the individual components of the natural mixture of alkaloids, which normally occurs in plants ; (4) Determination of structure ; and (5) Pharmacological action.

ALKALOIDA

Berisi 1 atau lebih atom N; bersifat basa Bentuk basa bebas larut dalam pelarut organik, sebagai bentuk garam larut dalam air Beberapa hal khusus yang perlu diperhatikan:

Pada PH rendah, ester-ester dapat terhidrolisis Amonia dapat bereaksi dengan senyawa organik membentuk suatu artefak Adanya senyawa fenolik pada ekstraksi asam-basa dapat menyebabkan kurang larut dalam pelarut organik Adanya tanin-tanin, dapat dihilangkan dengan penambahan kalsium hidroksida untuk mengendapkan tannin, sehingga ekstraksi alkaloid dapat dilanjutkan (Qunine tannate)

Analisis Alkaloid

During the isolation process, if the activity is lost or reduced to a significant level, the possible reasons could be as follows:
1. The active compound has been retained in the column. 2. The active compound is unstable in the conditions used in the isolation process. 3. The extract solution may not have been prepared in a solvent that is compatible with the mobile phase, so that a large proportion of the active components precipitated out when loading on to the column. 4. Most of the active component(s) spread across a wide range of fractions, causing undetectable amounts of component(s) present in the fractions. 5. The activity of the extract is probably because of the presence of synergy among a number of compounds, which, when separated, are not active individually.

Purification by Solvent Extraction Using Partition Coefficient

One such separation technique is the solvent partitioning method, which usually involves the use of two immiscible solvents in a separating funnel. In this method, compounds are distributed in two solvents according to their different partition coefficients. This technique is highly effective as the first step of the fairly largescale separation of compounds from crude natural product extracts.

Crystallization as a Separation Method


assuming we have a product comprising target component A mixed in with impurities B and C: 1. A sample of the mixture is dissolved in a hot solvent the solvent is chosen such that B and C are soluble at any temperature reached in the crystallization, while component A is not. 2. Cooling yields a crop of A, separated from components B and C. 3. Steps 1 and 2 are repeated, using fresh solvent each time, until the required degree of separation is achieved (note that one crystallization step from a mixture of compounds does not guarantee a chemically pure crystal product).

Examples of Purification of Natural Products by Crystallization

Crude solanine, extracted from the potato plant, is purified by dissolving in boiling methanol, filtering, and concentrating until the alkaloid crystallizes out. Naringin is isolated from grapefruit peel by extracting into hot water, filtering through celite, and concentrating the filtrate to the extent that naringin crystallizes at fridge temperatures as the octahydrate (melting point83C). Recrystallization from isopropanol (100mL to 8.6 g naringin) yields the dihydrate (melting point 171C). The di- and octahydrate compounds are examples of crystalline solvates. Piperine is extracted from powdered black pepper with 95% ethanol. The extract is filtered, concentrated, 10% alcoholic KOH added, and the residue formed is discarded. The solution is then left overnight to yield yellow needles of piperine. Capsanthin is isolated from red pepper or paprika. A 20mL volume of concentrated ether extract diluted with 60mL petroleum and left to stand for 24 h in a fridge produces crystals of almost pure capsanthin. Salicin is extracted from willow bark into hot water. The solution is filtered and concentrated and the tannin removed by treatment with lead acetate; further concentration and cooling yields salicin crystals. It is also worth highlighting the potential use of derivatives in fractional crystallizations, for example, picrates of alkaloids and osazones of sugars.

PENENTUAN STRUKTUR KIMIA

METODE
1. Kimia 2. Instrument/Spectroscopy - Infra Red Spectroscopy - Mass Spectroscopy - Low Mass Spectroscopy - High Mass - Nuclear Magnetic Resonance Spectroscopy - 1H- dan 13C-NMR - 2D NMR