UJIAN AKHIR SEMESTER

COURSE: CHEMISTRY OF NATURAL MATERIALS

LECTURER: Dr. Syamsurizal, M.Si

TIME: 22-29 DECEMBER 2012

1. Explain the triterpenoid biosynthetic pathway, identify the factors that are very important to determine the quantity produced triterpenoid very much!

Answer : 

         Triterpenoid  is a compound that carbon skeleton derived from six isoprene units and reduced biosynthesis of acyclic hydrocarbons C-30, which skualena, these compounds are colorless, crystalline, high melting point and is optically active (Harborne, 1987).

Triterpenoid compounds can be divided into four groups, namely: real triterpenes, saponins, steroids and cardiac glycosides.

Triterpenoids is one class of compounds terpenoid natural ingredients.

Terpenoid biosynthetic pathway begins with three basic reactions, namely;

The first basic reaction : is a formation of active isoprene derived from acetic acid via the mevalonic acid pathway.

Acetic acid did Claisen type condensation produces Asetoasetil Ko-A after being activated by coenzyme A. then the compound will also ni aldol type condensation resulting branched carbon chains such findings in mevalonic acid pathway with acetyl Co-A.

The second reaction is the reaction joining two unit head and the tail of compounds that form terpenoida isoprene (mono-, di-, seskui-, Sester-and poly-)

The reaction continued after the formation of mevalonic acid is phosphorylated, elimination of phosphoric acid, and decarboxylation which then produces IPP (isopentenyl Pyrophosphate) which will then be berisomerasi with DMAPP (Dimethyl Allyl Pyrophosphate) with the help of isomerase enzymes to be joined from head to tail. This merger occurs because electrons attack the double bond of the isopentenyl Pyrophosphate carbon atom at the electron-deficient DMAPP and then generate geranil Pyrophosphate (GPP) in the presence of pyrophosphate ion exclusion. IPP and GPP units will combine to form famesil Pyrophosphate (FPP) with the same flow mechanism and is one seskuiterpenoida compounds.

Then diterpenoida compounds derived from geranil - geranil pyrophosphate (GGPP) with the same mekansme through unit PP and GPP.

The third is tail joining reaction of unit C-15 and C-20 which then produces triterpenoida and steroida.

Triterpenoida and tetraterpenoida derived from dimerization of C-15 and C-20 which is not a continuous polymerase unt C-5. Dimerization FPP into skualena which is the basis and source triterpenoda steroida.

Based triterpenoid biosynthetic pathway, it can be seen that the factor - an important factor that determines the quantity produced abnyak triterpenoids are:

• Merging head and tail isoprene unit and the tail of the C-15 and C-20 are produced triterpenoids and steroids.
• The addition of enzyme to be involved in triterpenoid biosynthesis pathway.
• Optimizing producing pathway by inhibiting triterpenoid work mevalonic acid pathway.
• Cyclization process squalen compounds that can form triterpenoids.

2. Describe the structure determination of flavonoids, specificity and intensity of absorption signal by using IR and NMR spectra. Give two examples of the two different structures!

Answer : 

Infrared spectroscopy is a method of observing the interaction of molecules with electromagnetic radiation whose wavelength is in the region 0.75 - 1,000 μm or wave number 13000-10 cm-1.

The infrared spectrum of a compound provides an overview of the functional group in an organic molecule.

For the identification of the IR spectrum, the first step is the identification of the main group (C = O, O - H, N - H, C - O, C = C, C = N, and NO) goal is to compare it with the literature that are known compounds.

Nuclear magnetic resonance spectroscopy (NMR) provides an overview of the types of atoms, number, and the hydrogen atom (1H NMR) and (carbon (13C NMR).

NMR spectroscopy is based on absorption of radio waves by certain nuclei in organic molecules, when the molecule is in a strong magnetic field.

For example, the flavonoid compounds:

Flavonoid Structure

Test physical properties of flavonoids conducted on the isolated compound in the form of solids.

Another important analysis using IR spectrophotometer to determine the functional groups in a compound, followed by GC-MS analysis of the spectra to determine the structure of these compounds. The results of the analysis with a spectrophotometer UV and IR showed that only f2, f4 and f9 which are isoflavones. Suspicion that the active compounds in the rhizome Intersection ireng are isoflavones, the identification of further structure only at fraction f2, f4 and f9.

IR spectrophotometer to determine the functional gugusgugus compounds are in fraction f2.

and for NMR spectrume of flavnoid compounds :

This was obtained as yellow crystals, 13 mg, R_f = 0.76, mp. 250 °C. UV–Vis λ_max in MeOH: (nm) 248, 281 sh, 307 sh 318 sh 362; (AlCl₃) 269 sh, 299, 319 sh, 423; (AlCl₃/HCl) 264, 296 sh, 320, 458; (NaOAc) 252 sh, 292, 331, 379; (NaOAc/H₃BO₃) 263 sh, 315, 381; (NaOMe) 252, 299, 331, 361, 410. IR (KBr), ν = 605, 700 (C–H, Ar), 1097 (C–O, ethe), 1512, 1570 (CC, Ar), 1604 (CO) and 3354 cm⁻¹ (OH). ¹H-NMR (DMSO-d₆), Fig. 1, δ = 6.89 (d, J = 8.8 Hz, 2H, H-6, H-8), 7.54 (dd, J = 2.2 Hz, J = 8.8 Hz, H5′, H-6′), 7.69 (d,J = 2.20 Hz, H-2′), 7.92 (d, J = 9.5 Hz, 1H, H-5), 10–14 ppm (broad singlet, 4-OH)

Figure 1. The ¹H-NMR chemical shift of flavonoid compound 1 in DMSO-d₆

Figure 2. The ¹³C-NMR chemical shift of flavonoid compound 1 in DMSO-d₆

Compound 1 substituted in position −7 as indicated by its UV–Vis spectra upon addition of diagnostic shift reagents (NaOAc). When we added boric acid (H₃BO₃) to methanolic sodium acetate bands 1 shifted (+19) nm indicating B-ring catechol system. However, flavonoids with 3′,4′ – hydroxylation pattern give two peaks in the UV–Vis spectrum band II has two peaks, the B-ring catechol moiety is probably located at C-3′ and C-4′. Further evidence in favour of the above structure accumulated from the mass spectrum (Fig. 4) where the molecular ion M⁺ 286.

Figure 4. Mass spectrum of flavonoid compound 1.

              

3. In isolation of alkoloid, in the early stages of acid or base required conditions. Explain the basis of the use of reagents, and give an example at least three kinds of alkoloid!

Answer :

Alkaloids are a class of compounds which mostly heterocyclic nitrogenous bases and contained in plants (but this does not exclude the compounds derived from animals).

Amino acids, peptides, proteins, nukleotid, nucleic acids, amino sugars, and antibiotics are usually not classified as alkaloids. And the same principle, which is a neutral compound biogenetics related alkaloids belong to this group.

Alkaloids are usually classified according to the origin of molecular similarity (precursors), based on the metabolic pathway (metabolic pathway) that is used to form a molecule. If the biosynthesis of an unknown alkaloids, alkaloid compounds are classified according to their names, including the name of the compound that does not contain nitrogen (due to their molecular structure present in the final product.

Example: opium alkaloid sometimes called "phenanthrenes"), or under the name of a plant or animal in which the compound was isolated. If after alkaloid was studied, an alkaloid modified classification according to the assessment, it usually takes the name-the-amine important biological striking in the process of synthesis.

Therefore, isolation of alkaloids can be drawn in the form of salts with alcohol in an acidic environment.

Alkaloid salts already in the base form and then withdrawn by the organic solvent.

Classification alkaloids are:

• Heterocyclic alkaloids
• Alkaloids with nitrogen and amine eksosiklik alifatis
• Alkaloid putreskin, spermidin, and sperin
• Peptide alkaloids
• Terpenes and steroidal alkaloids (Sudarmin, 1999)

Alkaloids consist of  carbon, hydrogen, nitrogen, and usually contains many alkaloids oksigen. Compounds contained in the roots, seeds, wood and leaves from plants  and also of animals.

Metabolisme of  alkaloid compounds is the result of plant and used as a backup for the synthesis of the plant alkaloid protein. Kegunaan is as protective of pests, plants and regulator amplifier hormones work. Alkaloids have physiological effects.

Source is a flowering plant alkaloid, angiosperms, animals, insects, marine organisms and microorganisms.  Included alkaloid plant families that are Liliaceae, solanaceae, Rubiaceae, and papaveraceae (Tobing, 1989).

4. Explain the relationship between the biosynthesis, isolation methods and the establishment of the structure of compounds of natural ingredients. Give an example?

Answer : 

Biosynthesis is the formation of a naturally occurring molecule in the cells of other molecules that are less complicated structure, through endeorganik reaction.

While the biosynthetic pathway can be defined as a sequence or a process in which consists of the stages of formation of simple compounds into complex compounds. Biosynthesis process will take place very complex, depending on the available enzymes that similar plants growing in different areas it is possible to have a certain metabolite formation paths are not identical (the phenomenon of "vikarias: Chemical-Ras).

The reason why the biosynthetic pathway to be studied are:

1. Precursors can turn into a more useful new compounds with the aid of cell suspension
2. Based biosynthesis, secondary metabolites can be fed with a precursor to a product faster with cell suspension cultures
3. Changing certain compounds into other compounds to replace the reaction with cell suspension cultures

How to determine the biosynthetic pathway in tissue culture are:

1. With the analysis of complex compounds that can be detected constituent building blocks that can lead us to the original compounds and biosynthesis pathways
2.  Labeling with radioisotopes.

Usefulness know biosynthetic pathway is able to perform derivatization. Once we know the biosynthesis pathway, and it branched biosynthesis pathway, we can do the blocking on one of the branches. With the blocking is so we can improve secondary metabolite biosynthetic pathways we want from us is not blocking.

Method engineering biosynthetic pathways :

1. With the addition of substrates, precursors, or enzymes that play a role.

One approach that can be used to increase the production of secondary metabolites in vitro culture is the addition of precursors. The addition of precursor into the culture medium to stimulate the activity of certain enzymes involved in the biosynthesis pathway, so as to increase the production of secondary metabolites.

One study conducted by Zakiah, et al (2003) showed that the addition of squalen as precursors to increase production in callus cultures Azadirahtin (Azadirachta indica A.Juss) with the addition of plant growth regulators 2,4-D and BAP. Addition skualen done after callus cell suspensions from 6 day provide azadirahtin levels in the cell while being increased. Giving azadirahtin done with 3 concentration is 10 μM, 100 μM and 1000 μM. The research proves that azdirahtin production increased rapidly on day 4 after the addition azadirahtin the 10-day-old culture with high levels of 0.0076 ± 0.006 g / g DM or increased to 85, 366% over azadirahtin content was highest in the control (0.041 g / g BK).

2. Activation of enzymes involved in the biosynthesis pathway.

3. Environmental factors engineering.

The addition of precursors intended to streamline the process of biosynthesis or in other words to increase the production of secondary metabolites. Besides the addition of precursors, giving "stress" in the culture can also affect the production of secondary metabolites. Events that may arise because of the treatment is likely to be the formation of new compounds that are not found in plant origin (de novo synthesis), but generally provide a favorable outcome. This type of "stress" are common, such as lack of water (draft), the lack of light, lack of nutrients (minerals), the temperature is above or below the optimum.

Generally in a growth phase, secondary metabolite biosynthesis takes place very slowly and often not started. After the growth phase ends, the phases of the production or secondary metabolite biosynthesis underway. The addition of precursors intended to streamline the process of biosynthesis or in other words to increase the production of secondary metabolites.

In order to increase the precursor biosynthesis pathway in the production of secondary metabolites:

1. To compound the desired amount can be increased by manipulating the media and with the addition of precursor compounds / precursors, stimulate the activity of certain enzymes involved in the biosynthesis pathway, thus increasing the production of secondary metabolites, such as the addition of skualen a significant effect in improving the content on the cell azadirahtin according to research conducted by Zakiah (2003)
2. Getting secondary metabolism which is a form of differentiation of plant cells
3. To obtain the content of secondary metabolites that are higher than its parent.

-' CHOLESTEROL :_

                 In humans, cholesterol itself can be synthesized in the body, which is in the liver, cortex, adrenal, skin, intestine, testis, stomach, muscle, adipose tissue, and brain. Approximately 17 percent of the dry weight of the brain composed of cholesterol. Thus, no cholesterol, brain structure might not formed.

                 Cholesterol is a product of the metabolism of typical animals. Cholesterol is found only in foods of animal origin such as meat, fish, eggs, milk, brain, and innards. Judging from their chemical structure, cholesterol is a steroid group, ie a substance which belongs to the class of lipids.

                 Although it is considered dangerous, but cholesterol is needed by the body. The average human need 1100 milligrams of cholesterol per day to maintain cell walls and other physiological functions. Of these 25-40 percent (200-300 mg) is normally derived from food and the rest is synthesized by the body.

                 If the amount of cholesterol in the body is less, the synthesis of cholesterol in the liver and intestine increased to meet the needs of other tissues and organs. Conversely, if the amount of cholesterol in the diet increased, the synthesis of cholesterol in the liver and intestine decreased.

                 Although the body can synthesize the cholesterol, the cholesterol that comes from food plays an important role because it is the major sterol in the human body. As well as components of cell surface and intracellular membranes.

                  Research on mice shows, if there is only 0.05 percent of the cholesterol in foods, 70-80 percent of the body's cholesterol is synthesized in the liver, small intestine, and adrenal glands. If the content of cholesterol in the diet up to 2 percent, the biosynthesis of cholesterol in the body decreased to 10-30 per cent.

                 Cholesterol is needed by the body such as in the synthesis of bile acids needed for digestion of fat or oil, the synthesis of vitamin D, and as a component of the cell membrane. Cholesterol has a very important role in the body because it is not only the membrane-forming cells, but also other common biosynthetic precursor, including sex hormone bile acids.

                Cholesterol is a precursor of bile acids expenditures which are synthesized in the liver and serves to absorb triglycerides (triasilogliserol) and fat soluble vitamins from food, as well as precursors of steroid hormones, estrogen and testosterone.

               Another role of cholesterol, which helps nerve cells to function. If no cholesterol, coordination of gestures and speech will be disrupted.

Cholesterol Synthesis

                Cholesterol is synthesized is part of the cholesterol in the body. Only a small fraction of cholesterol that comes from food.

From food: 300-750 mg

Synthesis of body / endogenous: 650-1000 mg

Cholesterol Biosynthesis

               Cholesterol is synthesized in the liver, adrenal cortex, intestine, skin, and aorta. The synthesis takes place in the cytosol and microsomes cell network.

Stages as follows:

1. The formation of acetyl CoA mevalonic

2. Formation of squalene from mevalonic

3. Change of squalene to lanosterol which then turned into cholesterol

Sequence of reactions:

1. Formation of acetyl CoA. Acetic acid molecule is activated to acetyl CoA by using energy derived from ATP and is catalyzed by the enzyme acetyl-CoA synthetase. Mg as a cofactor.

2. Two acetyl-CoA molecules condense to form asetoasetil CoA. Enzymes that work here are tiolase.

3. Asetoasetil CoA condenses with acetyl CoA molecules to form HMG CoA. The enzyme is HMG CoA synthetase catalyses. This process requires water and produce byproducts such as CoA-SH.

4. HMG CoA reductase is reduced by NADPH and with the assistance of H. The results will form mevalonic.

5. Mevalonic have 3 rows of reactions involving phosphorylation by 3 ATP and removes one carbon atom mevalonat. Isoprenoid units formed. Byproducts such as carbon dioxide and water.

6. Two units of isoprenoid pyrophosphate condense to form a 10-atom geranil C.

7. The isoprenoid molecules condense further to form farnesil geranil pyrophosphate pyrophosphate.

8. Two molecules combine to form squalene pyrophosphate farnesil with 30 atom C.

9. Ring closure to form lanosterol.

10. Lanosterol converted into a 14-desmetil lanosterol (loss of a methyl group) zimosterol → → → desmosterol cholesterol.

            Enzymes are important in the synthesis of cholesterol is HMG CoA reductase. This enzyme is inhibited by high-cholesterol foods and feedback inhibition. In extrahepatic tissues, HMG CoA reductase is inhibited by cholesterol in LDL. In the intestinal tissue, this enzyme is inhibited by bile acids. Thyroid hormone and insulin also played a role in the inhibition of these enzymes work.

http://josephinewidya.wordpress.com/2011/12/08/metabolisme-lipid-biosintesis-kolesterol/

TERPENE (GERANIOLS)

               Terpenes are a class of hydrocarbons produced by many plants and is mainly contained in the sap and cell vacuoles. In plants, class of terpene compounds and modifications, terpenoids, a secondary metabolite. Terpenes and terpenoids produced also by a number of animals, especially insects and some marine animals.

              Terpenes and terpenoids compiled many essential oils produced by plants. The content of essential oils affect the use of spice products, both as a condiment, as a perfume, as well as a medical, health, and accompanying rituals. Common names of this class of compounds is often taken from a volatile oil containing it. Furthermore, the name of the oil itself is taken from the name (Latin name) became the source when the plant was first identified. For example is citral, derived from oil extracted from orange (Citrus). Another example is eugenol, taken from the oil produced by cloves (Eugenia aromatica).

Terpenoids called isoprenoids. This is understandable because the framework constituent terpenes and terpenoids are isoprene (C5H8).

               Terpenes have a basic formula (C5H8) n, where n is the determinant of the type of terpene. Modified terpene (called terpenoids, meaning "similar to terpene") is a compound with a similar structure but can not be expressed with the basic formula. Both of these groups make up a lot of essential oils.

• Hemiterpena, n = 1, only isoprene
• Hemiterpenoid, for example prenol, isovalerat acid
• Monoterpena, n = 2, for example mircena, limonena, and ocimena
• Monoterpenoid, for example geraniol
• Sesquiterpene, n = 3, for example farnesen
• Seskuiterpenoid, eg farnesol, kurkumena, bisabolol
• Diterpena, n = 4, for example cembrena
• Diterpenoid, for example kafestol
• Triterpena, n = 6, for example skualena
• Triterpenoids, for example lanosterol, the basic material for steroid compounds
• Tetraterpena, n = 8, for example, is lycopene, carotenE
• Polyterpenes, n large, for example, is rubber and gutta-percha.           

>> GERANIOLS

GERANIOLS

             Geraniol is a primary alcohol found in geranium, lemongrass, lemon, and other oils. Used primarily in perfumery. 

Geraniol has more value as a substitute for oil produced from a rose oil produced from the leaves of lemongrass. 

            Lemongrass oil production in Indonesia is one of the largest in the world, this is because Indonesia is a good place for plants to grow lemongrass. Lemongrass oil isolated from leaves of fragrant lemongrass Java (Cymbopogon winterianus jowwit). During this time, lemongrass oil is exported to foreign countries in the form of pure lemongrass oil so that less bring in foreign exchange, so it is necessary to attempt to increase the value of lemongrass oil. This research will be conducted geraniol component separation of lemongrass oil.

             Geraniol has broad benefits in the field of cosmetics, animal husbandry and health
Geraniol found in geranium plants and citrus. This compound is insoluble in water, but soluble in common organic solvents and cyclization was more difficult than the other compounds.
Configuration of cis-to nerol and trans- to geraniol causes cyclization of geraniol to be more difficult for the cyclization.

 It smells pungent and often used as a perfume. Lemongrass oil can be isolated from the leaves of the lemongrass scented steam distillation method. Lemongrass oil distillate extracted with ether to separate it from the water. To improve the content geraniolnya, lemongrass oil hydrolyzed with NaOH in ethanol for 1 hour to hydrolyze geranil acetate into geraniol.Identification of compounds geraniol performed using gas chromatography-mass spectrometry (GC-MS). 

 Lemongrass scented leaf weighing 10 kg produces citronella oil as 42.5 mL (0.373%) with clear yellow shiny distinctive smell of lemongrass and has a refractive index of 1.4755. Data lemongrass oil GC chromatogram shows geraniol content as much as 65.34%. Geraniol enrichment NaOH solution in ethanol resulted in the hydrolysis reaction geranil acetate into geraniol which increases to 81.96% geraniol content. 

_" STEROID "_

       Steroids are organic compounds are not hydrolyzed fatty sterols can dihasil reduction reaction of terpene or skualena. Steroids are an important group of compounds with the basic structure sterana saturated with 17 carbon atoms and 4 rings.

Compounds which include derivatives of steroids, such as cholesterol, ergosterol, progesterone, and estrogen. In general, function as steroid hormones. Steroids have a basic structure consisting of 17 carbon atoms that make up the three cyclohexane rings and one cyclopentane ring. Different types of steroids are steroids that another one is on the functional group is bound by the four-ring oxidation and phase of each ring.  

       Some are anabolic steroids, such as testosterone, metandienon, nandrolone decanoate, 4-androstena-3 17-dione. Anabolic steroids can lead to a number of dangerous side effects, such as lowering the ratio of high density lipoprotein, which is useful for the heart, lowers low-density lipoprotein ratio, stimulation of prostate tumor, coagulation abnormalities and liver disorders, hair loss, thickening hair, acne and the incidence of male breast . In physiology, anabolic steroids can make a person aggressive.

       Steroids are made ​​up of several groups of compounds and grouping based on the physiological effects provided by each groups .

The group are sterols, bile acids, sex hormones, hormone adrenokortikoid, cardiac aglycone, and sapogenin.

In terms of molecular structure, the differences between the various groups steroidini determined by:

• Type substituents R 1, R 2and R 3, which bound to the carbon framework, as noted above
• The difference between the compound one another from certain suatukelompok determined by- carbon chain length of R 1
• functional groups present on the substituents R 1, R 2and R 3
•  The number and position of the oxygen functional groups and double bonds
• And the configuration of the asymmetric centers in the framework dasarkarbon it.

Some examples that represent each of the groups on the list of steroid diatastercntum 4.1.

Listing 4.1. Some natural steroid

Sterol :

ERGOSTREOL

STIGMASTEROL

Sex Hormones :

Sapogenin :

SARSAPOGENIN

               Another example of lipid types of steroids are sex hormones for men and womensuch as testosterone, estradiol and progesterone. The molecular structure and function can be seen in Table 14.8.
Table 14.8. Types of hormones and physiological function

           

          All steroids are made in cells with raw materials such as fatty sterols, either lanosterol in animals or function, or in the form sikloartenol in plants. Both types of fat on top is made of sterol cyclization squalena of triterpena. Cholesterol is a sterol other types of fat are common.

Biosynthesis Of Steroid

         Biogenetics Experiments show that steroids are derived from triterpenes in nature. Steroids found in animal tissue derived from triterpene lanosterol, while present in plant tissues derived from triterpenes sikloartrenol, after experiencing a series of triterpenes certain changes. Early stages of the biosynthesis of steroids are the same for all natural steroids, namely the conversion of acetic acid through mevalonic acid and skualen (a triterpene) to lanosterol or sikloartrenol.

            Biosynthesis of steroid hormones including testosterone begin with the transformation of cholesterol to pregnenolone. Setting steroid hormone biosynthesis mediated by an increase in cAMP-and Ca +2 intaselular melaluijalur inositol triphosphate. Stimulation of cAMP can be acute or chronic. Acute stimulation began delivery of cholesterol to the inner mitochondrial steroidogenic acute mediated regulatory (StAR), whereas chronic stimulation occurs during the conversion of cholesterol to pregnenolone. In this stage, the conversion process takes place in the mitochondria by putting side chain cleavage enzyme (scc). 

NADPH, oxygen and cytochrome P450 on a limited basis as needed. Unlike the protein hormone receptors, steroid receptors located in the cell cytoplasm or nucleus. At first hormone into cells by diffusion and bind to receptor proteins immediately spesifikdi in the cytoplasm. Streroid hormone receptors are inactive are in a heat shock protein 90 (hsp 90). In the event of ties between the hormone and the receptor, the hsp 90 becomes active and self meleaskan. Then the bonding hormone and the receptor will soon be heading to the nucleus. In the nucleus, the bonding hormone receptor complex affects the transcription factor koaktivator and thoroughly to produce an active transcriptional complex that would enhance gene expression and hormone effects sterid.