LAPORAN PRAKTIKUM KIMIA ORGANIK II

Identification Secondary Metabolite Long Bean (Vigna sinensis (L.))

Disusun oleh:

ANDI AGRAH A DJ 140210080039

JURUSAN KIMIA FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM UNIVERSITAS PADJADJARAN 2011

Identification Secondary Metabolite from Long Bean (Vigna sinensis(L.) )

I.

PURPOSES 1. Identify alkaloid compound from long bean (Vigna sinensis (L.)) with Hager test, Wagner test, Mayer test dan uji Dregendorf test. 2. Test have triterpena,steroid and saponin in long bean (Vigna sinensis (L.)) with Liebermann-Burchard test.

II. 1.

PRINCIPLES Extraction a. Nernst Distribution Law Into two solvent which are immeable each other added with a third material, so it distributed into each solvent with a constant certain
KD = Co Ca

Which

K D = distribution coeficient
C o = organic phase concentration C a = water phase concentration

b.Like Dissolve Like A compound is easier to soluble in a solvent that has the relatively same polarity with the compound it self.

2. Alkaloid Test Alkaloid complexs precipitation reaction there not soluble in heavy metal reagent. 3. Liebermann-Buchard Test 3

Colour form reaction because have chromopore function in a compound. 4. Saponin Test Foam form reaction because shaked with water. 5. Hidrolisis Decompotition reaction a compound with water help. 6. Sublimasi Purity process which a solid phase change into gas form wihout melt first.

III.

REACTION

1. Mayer Reaction Alkaloid + H2SO4 + Mayer Precipitation R3N + H2SO4 R3NH+ + H2SO4 R3NH+ + Mayer R = NHq 2. Wagner Reaction
O

NaCH3COO Butanol

3. Liebermann-Buchard Test

O O O

HSO4
O OH O

CH3COOH
H O

H+

H2SO4

O O

IV.

THEORY Phytochemicals - Phytochemicals is simply a word that means plant

chemicals. Once, researchers attributed the health promoting affects of plants to their numerous vitamins, minerals and fibers. More recently, however, researchers have discovered that the many other chemical compounds in plants also provide benefits to humans when consumed. Phytochemicals provide plants with protection from the environmental challenges they face, such as ultraviolet light. When we consume plants rich in phytochemicals, they seem to protect us as well. Some researchers estimate that up to 40,000 different phytochemicals will someday be fully catalogued 5

and understood. Polyphenols are a class of phytochemicals that are particularly rich in antioxidants and plentiful in Pomegranate Juice (Wonderful.2008). The flavonoids are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon chain.

The skeleton above, can be represented as the C6 - C3 - C6 system. Flavonoids constitute one of the most characteristic classes of compounds in higher plants. Many flavonoids are easily recognised as flower pigments in most angiosperm families (flowering plants). However, their occurence is not restricted to flowers but include all parts of the plant. The chemical structure of flavonoids are based on a C15 skeleton with a CHROMANE ring bearing a second aromatic ring B in position 2, 3 or 4.

In a few cases, the six-membered heterocyclic ring C occurs in an isomeric open form or is replaced by a five - membered ring.

AURONES (2-benzyl-coumarone) The oxygen bridge involving the central carbon atom (C2) of the 3C - chain occurs in a rather limited number of cases, where the resulting heterocyclic is of the FURAN type. Various subgroups of flavonoids are classified according to the substitution patterns of ring C. Both the oxidation state of the heterocyclic ring and the position of ring B are important in the classification. Examples of the 6 major subgroups are: 1. Chalcones

2. Flavone (generally in herbaceous families, e.g. Labiatae, Umbelliferae, Compositae). Apigenin (Apium graveolens, Petroselinum crispum). Luteolin (Equisetum arvense)

3. Flavonol (generally in woody angiosperms) Quercitol (Ruta graveolens, Fagopyrum esculentum, Sambucus nigra) 7

Kaempferol (Sambucus nigra, Cassia senna, Equisetum arvense, Lamium album, Polygonum bistorta). Myricetin ().

4. Flavanone

5. Anthocyanins

6. Isoflavonoids

Most of these (flavanones, flavones, flavonols, and anthocyanins) bear ring B in position 2 of the heterocyclic ring. In isoflavonoids, ring B occupies position 3. A group of chromane derivatives with ring B in position 4 (4-phenyl-coumarins = NEOFLAVONOIDS) is shown below.

The Isoflavonoids and the Neoflavonoids can be regarded as ABNORMAL FLAVONOIDS. CHALCONE Chalcone is derived from three acetates and cinnamic acid as shown below.

ANTHOCYANIDIN

Anthocyanidin is an extended conjugation made up of the aglycone of the glycoside anthocyanins. Next to chlorophyll, anthocyanins are the most important group of plant pigments visible to the human eye.

The anthocyanodins constitute a large family of differently coloured compounds and occur in countless mixtures in practically all parts of most higher plants. They are of great economic importance as fruit pigments and thus are used to colour fruit juices, wine and some beverages. The anthocyanidins in Hydrangea, colours it RED in acid soil and BLUE in alkali soil.

They will chelate with metal ions like Ca2+ and Mg2+ under alkali conditions. This extends the conjugation as shown below. 10

ISOFLAVONOIDS
In contrast to most other flavonoids, isoflavonoids have a rather limited taxonomic distribution, mainly within the Leguminosae. Most of our knowledge about the biosynthesis of isoflavonoids originates from studies with radioactive isotopes, by feeding labelled 14C cinnamates. The isoflavonoids are all colourless. It has been established that acetate gives rise to ring A and that phenylalamine, cinnamate and cinnamate derivatives are incorporated into ring B and C-2, -3, and -4 of the heterocyclic ring.

Since chalcones and flavanones are efficient precursors of isoflavonoids, the required aryl migration of ring B from the former 2 or beta position to the 3 or alpha position of the phenylpropanoid precursor must take place after formation of the basic C15 skeleton.

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Example of a BIOLOGICALLY ACTIVE ISOFLAVONOID

Rotenone comes from Derris root and Lonchocarpus species leaf (Family: Leguminosae) It is an insecticide and also used as a fish poison. * (blue): carbons derived from methionine. (red): carbons derived from PRENYL (isoprenoid). Biochemical pathway to the formation of rotenone.

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Six rotenoid esters occur naturally and are isolated from the plant Derris eliptica found in Southeast Asia or from the plant Lonchocarpus utilis or L. urucu native to South America. Rotenone is the most potent. It is unstable in light and heat and almost all toxicity can be lost after two to three days during the summer. It is very toxic to fish, one of its main uses by native people over the centuries being to paralyze fish for capture and consumption. Crystalline rotenone has an acute oral LD50 of 60, 132 and 3000mg/kg 13

for guinea pigs, rats, and rabbits (Matsumura, 1985). Because the toxicity of derris powders exceeds that of the equivalent content of rotenone, it is obvious that the other esters in crude preparations have significant biologic activity. Acute poisoning in animals is characterized by an initial respiratory stimulation followed by respiratory depression, ataxia, convulsions, and death by respiratory arrest (Shimkin and Anderson, 1936). The anesthetic-like action on nerves appears to be related to the ability of rotenone to block electron transport in mitochondria by inhibiting oxidation linked to NADH2, this resulting in nerve conduction blockade (O'Brien, 1967; Corbett, 1974). The estimated fatal oral dose for a 70kg man is of the order of 10 to 100g. Rotenone has been used topically for treatment of head lice, sacbies, and other ectoparasites, but the dust is highly irritating to the eyes (conjunctivitis), the skin (dermatitis), and to the upper respiratory tract (rhinitis) and throat (pharyngitis) (Friedli,1996).

There are three main types of alkaloids:

Colchicine is an example of a proto-alkaloid. Pseudo-alkaloids can be derived from;

Terpenoids or Purines 14

TRUE ALKALOIDS The basic unit in the biogenesis of the true alkaloids are AMINO ACIDS. The nonnitrogen containing rings or side chains are derived from TERPENE units and / or ACETATE, while METHIONINE is responsible for the addition of methyl groups to nitrogen atoms. Alkaloids are highly reactive substances with biological activity in low doses. DEFINITION 1. Contains nitrogen - usually derived from an amino acid. 2. Bitter tasting, generally white solids (exception - nicotine is a brown liquid). 3. They give a precipitate with heavy metal iodides.
o

Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer's reagent (potassiomercuric iodide solution). Cream coloured precipitate. Dragendorff's reagent (solution of potassium bismuth iodide) gives orange coloured precipitate with alkaloids. Caffeine, a purine derivative, does not precipitate like most alkaloids.

4. Alkaloids are basic - they form water soluble salts. Most alkaloids are welldefined crystalline substances which unite with acids to form salts. In plants, they may exist
o o o

in the free state, as salts or as N-oxides.

5. Occur in a limited number of plants. Nucleic acid exists in all plants, whereas, morphine exists in only one plant species. Alkaloids can be classified;

in terms of their BIOLOGICAL activity, CHEMICAL structure (nucleus containing nitrogen), BIOSYNTHETIC pathway (the way they are produced in the plant).

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(Friedli,1996). Long Beans are mainly a warm-season crop and will survive extreme humidity and heat. It can be planted in a wide range of climatic conditions but is very sensitive to cold temperatures. Propagation is through direct seeding or transplanting. They prefer a light, well-drained soil with a pH of 5.5 to 6.8, enriched with organic matter, such as compost or dried manure.Plant the seeds 1-2 inches deep in warm soil. Germination will take place within 6 to 10 days. Soak the seeds in water before sowing, for better germination. Transplants should be done in such a way as to avoid disturbance to the root system. The plants should be placed 2 to 3 feet in rows and the distance between rows should be 4 to 6 feet apart on raised beds or ridges. Dwarf growing forms can be planted much more densely. Place poles and give wire or twine supports in rows. Extra fertilization is not essential as long as you have enriched the soil before planting. But the plant needs heavy watering at regular intervals. Flowering will occur 5 weeks after sowing. Fruits will grow from open flower to suitable length in about 10-12 days. Pick the pods at the tender stage at maximum length, before the seeds mature or swell. Yard long beans may grow up to 24inch long, but it is better to pick them at 12 to 18 inches. While picking, cut the pods with a sharp knife to minimize plant damage, thus maximizing harvest. Leave the pods to reach full maturity, allowing them to dry on plant, if the pods have to be reserved for seeds. The pods will break open and the seeds can be collected. Long beans often appear in our dishes daily. Besides being easily obtained, long beans can also be processed into a wide range of cuisine. Besides being easily cooked, can be pan-fried or made part of other dishes. Long beans also have a variety of benefits that are good for health. Long bean or vignasinensis, easily found in fields, in gardens, courtyard houses, in fields or other crops as a distraction. The treatment is

16

easy, making this one easy plants planted. On young bean when eaten crisp and delicious raw engulfed.

Scientific classification of plants Long Beans Klasifikasi Kingdom: Plantae (Tumbuhan) Subkingdom: Tracheobionta (Tumbuhan berpembuluh) Super Divisi: Spermatophyta (Menghasilkan biji) Divisi: Magnoliophyta (Tumbuhan berbunga) Kelas: Magnoliopsida (berkeping dua / dikotil) Sub Kelas: Rosidae Ordo: Fabales Famili: Fabaceae (suku polong-polongan) Genus: Vigna Spesies: Vigna sinensis (L.) Savi Ex Has

(Hartoyo, 2010) In some countries, long beans are used to treat rheumatism, arthritis, and urinary tract disorders. This nutritious vegetables to keep the skin from acne problems, help the recovery of burns, bullet urine, diarrhea, eczema, kidney disorders, itching, etc (Mazenchips, 2008) Benefits: contain beta-carotene, chlorophyll, vitamins B1 and B2, fiber and pectin. Vegetables are useful for controlling blood sugar levels, hypertension, minimize the risk of stroke and heart attack, improve the functioning of the digestive organs, reducing the risk of cancer and helps fight constipation. Also has diuretic properties (peluruh urine) levels are (Mazenchips, 2008).

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V.

APPARATUS AND MATERIALS 5.1. Apparatus 1. Bunsen 2. Mortar and alu 3. Pipette 4. Test plat 5. Test tube 6. Vapor cup 5.2.Material 1. Anhidrida Acetat 2. Ethanol 3. Chloric acid 2N 4. Cloroform 5. Cloroform ammoniacal 6. Dregendroff Reagent 7. Hagger Reagent 8. Mayer Reagent 9. Sample 10.Sulforic acid 2N 11.Wagner Reagent

5.3.Picture of Apparatus

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Test Tube VI. PROCEDURE 6.1 Alkaloid Test A 3-5 gram sample pounded with mortir then finely poundes leave placed into test tube and extracted with chloroform amoniacal, after extracted, sample filtered with cotton. Residu throw away, extract put into 2 test tube(test tube A and B). In test tube A, added sulfuric acid into test tube, after added, formed two phase in test tube. Acid phase (upper phase) separated into 3 different test tube.First test tube added Myer reactant, second test tube added Wagner reactant, Third test tube adeed Dragendorf reactant and watch closely precipitation formed. In test tube B, adeed Hager reactant and watch closely precipitation formed. 6.2 Flavonoid Test A 2 drop long bean (Vigna Sinensis (L.)) leave extract adde sulfuric acid 3-5 drop. Then added to Maagnesium powder intotest tube precipitation formed. 19 and watch closely

6.3 Steroid, triterpenoid, dan Saponin Test A 3-5 gram sample pounded with mortir then finely poundes leave placed into test tube and extracted with ethanol 1:1. After have been extracted with ethanol, sample warmed and filtered. Residu throw away dan and formed ethanolic extract put into vapor cup then vapor cup warmed to throw away the solution. After all the solution vapor, added ether into vapor cup. Take ether extract and put into drop plat then adde anhidrided acetat and sulfuric acid (2:1) as Liebermann buchard reagent. And watch closely colour changing (if positive, steroid green or blue coloured, if positive, triterpenoid red,purple, or brown coloured). Residu that formed take and put into test tube , added warmed water and shaked, if have a saponin, foam formed in test tube. VII. DATA PENGAMATAN

7.1. Physical Properties and Chemical Properties Subtance Water H2O Physic Properties Cair tak bewarna Mp = 0oC Bp = 100 C = 1 g/mL Ethanol CH3CH2OH Chloride Acid HCL Sulforic Acid H2SO4 TL= -117,3oC Bp =78,3oC Mr= 46 Mr= 36,5 Mr= 99,08 Mp = 10.36oC Bp=338oC 7.2 Tabel Pengamatan Subtance Alkaloid Test Hager Mayer Wagner D.droff + Flavonoid Steroid Test Sample Test Triterpenoi d Test + Saponin Test +
o

Chemical Properies Not Reactive Stable Polar Solvent Have Hidrogen Bond Volatile Flamable

Soluble in water Strong Acid Soluble in water Corrosive

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Subtance Sample

Treatment Finely pounded

Result Finely poundes leave

-Alkaloid TestFinely poundes leave Ekstrak MTC

Extraction with MTC ammoniakal Filter with cotton Placed into 2 test tube Added sulfuic acid 1:1

MTC extract dan residu MTC extract in each test tube there 2 phase: upper = acid phase ; lower =MTC positive Negative Negative Negative there 2 phase: upper = acid extract ; lower =Residu

1st test tube

added wegner tester added meyer tester added dregendroff tester 2nd terst tube added hager tester -Steroid,Triterpen dan Saponin Test Extraction with ethanol Finely poundes leave Warmed and filtered

Ethanolic Extract

Ether Extract Residu

Added into vapor cup Vapor solvent Cooled Added Ether Added into test plat Added L-B reagent Added hot water Put into test tube Shaked Extraction with methanol Filtered with cotton

Positive foam= saponin positive

-Flavanoid TestFinely poundes leave

there 2 phase: upper = acid extract ; lower =Residu

Extract

Added HCl 2N Added Mg powder

Orange Solution

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VIII. DISCUSSION secondary metabolites that are found mainly in plants is a source of chemicals which will never end. secondary metabolites are compounds that can be used in the interests of medicine and industry. Therefore, the isolation and the development of secondary metabolites is very useful. This experiment aims to determine the content of alkaloids from the leaves of the plants of Vigna sinensis l. Hager, of Richard Wagner, Mayer and Dragendroff test, the test to detect the presence of flavonoids by the reaction between chloride magnesium and acid from the leaves of the plants of Vigna sinensis l. and the proof of the presence of triterpenes and steroidal saponins from the leaves of the plants of Vigna sinensis l. Liebermann-Burchard test. The first thing to do in testing a collection of plant fitohimieskie. Tests using a sample of Fresh Greens is designed to avoid damage to the plant cell tissue. This tissue can lead to loss or damage of active compounds in the plant due to heat or plant quiet too long feared the active connections will be damaged as a result of enzyme or water contained in plants, characterized by a discolouration (dry or dry). The phytochemical testing to determine the contents of the secondary metabolites (alkaloids, saponins, steroids and triterpenovye), Vigna Sinensis plant leaf samples l. dipotong-split into pieces and then banged into powder, so that the walls of plant cell open so that the secondary metabolites and easier access to more easily extract. The samples that have been tested with the alkaloids of the test of accuracy. Although 4.3 sample Gram is then put into a test tube and chloroform insert up submerged amoniakal, then, are shaken with a stirring bar, then putting cotton to separate the extract with the residue, the addition of the function amoniakal chloroform to release the alkaloid of the form of salts. In general, the compounds of alkaloids in the plants are not in a free State, but bonded complex partial Ionic bond with an organic acid. In addition to amoniakal chloroform (with a strong basicity of alkaloids compounds), organic acids is obliged by the amoniakal chloroform to alkaloids-free compounds. 22

Amoniakal chloroform extract which has been separated with cotton, put into four different tubes, the first tube directly added to the reagent Hager (pikrat acid), shaken for a moment and see the results. This does not arise yellow precipitate, showed negative results. Then the remaining three tubes coupled with 2N sulfuric acid to bind to return to the alkaloid alkaloid salt for the reagent to react with heavy metals and complex shapes that are not soluble inorganic salt, and returns an alkaloid extracted into shape so that the salt alkaloid to be separated with other components besides plant cells that take part dispersing phase was extracted with acid. This aims to prevent other components in addition to participating mengendapnya alkaloids which would produce less accurate test. Because the polarity and density differences between amoniakal chloroform and sulfuric acid will be formed in two stages, the acid will be in the top while the chloroform phase under amoniakal. In the phase separated by using a pipette, and the remaining acid phase reactants added heavy metals. Containing alkaloids characterised by deposition. This happens because the alkaloid compounds containing nitrogen atoms that has lone pair. These free electrons will be donated to the heavy metal atoms to form complex compounds with groups that contain atoms of nitrogen such as ligannya. This complex compound is insoluble (precipitated) and provide color according to the reagent used. The reagent of Wagner is will precipitate held accountable Brown, the reagent Dragendorf will precipitate held accountable the Orange and the reagent Mayer is will precipitate held accountable the white of the four reagents that generate positive results in Mayer, Wagner and Dragendorff reagent. reactions that occur: Mayers reagent

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Wagners reagent
CH3 OCH3 CH3

N2OH CH3 CH3

I2, KI
CH3 CH3

OCH3

CH3

OCH3

CH3

Na CH3COOH
CH3 CH3

Toluena ROH
CH3 CH3

+ NH3

Dragendorffs reagent

Alkaloid test results on samples positive to say because of the four reagents were added, there are positive that with the reagent Wagner. This is done following test to indicate the presence or absence of flavonoids in the sample, samples, as elaborated by 3.1, weighed, and then extracted with methanol. 24

Extraction of flavonoids from the plant can be done using polar solvents. Flavonoids are polar compounds, because it is the number of hydroxyl groups. Therefore, flavonoids are generally soluble in polar solvents such as methanol. Methanol serves as the liberator of flavonoids from the form of salt, then added 2N sulfuric acid, sulfuric acid serves to protonation to form a salt flavonoid flavonoids. After the magnesium powder is added. A positive result is indicated by changes in the color orange solution. It turned out that the samples did not show positive results, this indicates that the sample does not mengandeung leaf flavonoids. The reaction can be seen from the following reaction:

Next is the testing of steroids, triterpenoids and saponins, after leaf samples weighed 4.3 g, added hot ethanol. Ethanol solvent used because ethanol has two groups, namely the polar groups on alcohol and nonpolar groups in the hydrocarbon. CH3 - CH2 - OH Gugus non polar Gugus polar

Steroids and triterpenoids are relatively non-polar, saponins tend bersifar polar. Through the use of ethanol, all these compounds can be removed. The use of hot ethanol increases the solubility of the compounds Suatau so I hope that it will be all the steroids, triterpenoids and saponins contained in plant extract in ethanol. Etanolik Solvents then evaporated and then diluted with ether to attract nonpolar components in the dried extract in accordance with the principle of like dissolves like. 25

To test and streoid triterpenoid content in leaf samples, ether extract added reagentBuchard Lieberman (LB), which is a mixture of acetic acid Anhydride with concentrated sulfuric acid (2:1). positive indications of steroid marked with blue or green discoloration. Blue or green color that is not absorbed, but complementary color. The color orange is so engrossed steroids are known to absorb at a wavelength of 585-647 nm. While on a positive indication triterpenoid marked with color change to red, purple or brown. The color green is absorbed by the triterpenoid with a wavelength of 491-570 nm. -OH in triterpenoid will experience a shift in wavelength that is absorbed so that the resulting color is different. So red, purple or brown are complementary colors. Color formation reaction can occur because the chromophore group (unsaturated group) caused by the absorption of certain wavelengths by organic compounds. Organic compounds with large conjugation absorb certain wavelengths due to the electron transition and n to so that the color that is absorbed is not a color that looked but complementary color. If the sample contains triterpenoids and steroids as well as the first color that appears and then followed with a color from the color triterpenoid steroids. This is because the wavelength is absorbed by the triterpenoid the longer mean lower energy so that it will appear first. The results showed tebentuknya brown color indicates that the positive samples have triterpenoid, but because it does not appear wana green or blue, indicating that leaf samples did not contain steroids.

Lieberman buchard reaction:

O O

H2SO4

+ + CH3COOH H+

HO O
H-O

+
O

H2SO4 + CH3COOH

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Residues that do not dissolve when the addition of ether, added to hot distilled water to test the saponin. The presence of saponin is characterized by the onset of foam after shaking with distilled water and foam constant heat for 15 minutes. Foam was formed because of air bubbles trapped in the solution. Saponin is a substance that has a surface-active compounds and the like soap so that recognition can be done easily degan. Following reaction:

Saponin is a ampifilik polar lipid components (a group of hydrophilic and hydrophobic groups). In liquid systems, liquid lipid to form micelles spontaneously spread to the change found is the alteration found is phyllic tails that intersect with liquid media. Micelles may contain thousands of lipid molecules. Lipids form a liquid layer with a thickness of one molecule of a single layer. In this system, the hydrocarbon tail is open, so avoid the water and the hydrophilic layer extends into the polar water, the system is called a foam place. The results showed no foam indicates that the sample does not contain saponins.

IX.

CONCLUSION
1.

The contents of alkaloids from the leaves of the plant Annona squamosa L. test known as Hager (-), Wagner (+), Mayer (-), and Dragendorff (-).

2.

The content of flavonoid (-) can be determined by reacting the acid chloride and magnesium from the leaves of plants Vigna Sinensis L.

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3.

Contents triterpenoid (+), saponin (+), and steroid (-) can be known handle links with Liebermann-Burchard test of plant leaves Vigna Sinensis L.

REFERENCES

Friedli, G.L.1996. Flavanoids. http://www.friedli.com/herbs/phytochem/flavonoids.html#phyto_tutorial.html Friedli, G.L.1996.Alkaloid. http://www.friedli.com/herbs/phytochem/alkaloids/alkaloid1.html Hartoyo, D. 2010. CULTIVATION LONG BEANS Vigna spp.

http://htysite.co.tv/budidaya%20kacang%20panjang%20ig.htm

Mazenchips.2008.ManfaatKacangPanjang.http://www.masenchip.blogspot.com/manf aat-kacang-panjang.htm Nanno, M and R. Hgg. 1993."The Heraion at Samos" in Greek Sanctuaries: New Approaches. name=Pomegranate Shellards. 2010. Identification of alkaloid-containing micro drugs by thin-layer distillation. http://www.mamaherb.com/Ingredients/View.aspx?

chromatography

after

steam

http://www.sciencedirect.com/science?_ob=Article.htm 28

Seeram NP, Lee R, Heber D .2004. "Bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate (Punica granatum L.) juice". Clin Chim Acta 348 (1-2): 638. Staples, Danny, Ruck, Carl A. P. 1994.The world of classical myth: gods and goddesses, heroines and heroes. Durham, N.C: Carolina Academic Press. Wonderful, P.2008. POM Glossary.http://www.longbean.com/glossary.html

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