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LAPORAN PRAKTIKUMKIMIA ORGANIK II

Identification SecondaryMetabolite 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. PRINCIPLES

1. 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

Which : distribution coeficient

organic phase concentration

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.

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2. Alkaloid Test

Alkaloid complexs precipitation reaction there not soluble in heavy metal

reagent.

3. Liebermann-Buchard Test

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

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3. Liebermann-Buchard Test

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

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.

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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.

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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)

Kaempferol (Sambucus nigra, Cassia senna, Equisetum arvense, Lamium album,

Polygonum bistorta).

Myricetin ().

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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.

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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.

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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.

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.

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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.

Example of a BIOLOGICALLY ACTIVE ISOFLAVONOID

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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

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:

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Colchicine is an example of a proto-alkaloid.

Pseudo-alkaloids can be derived from;

Terpenoids or

Purines

TRUE ALKALOIDS

The basic unit in the biogenesis of the true alkaloids are AMINO ACIDS. The non-

nitrogen 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.

o Dragendorff's reagent (solution of potassium bismuth iodide) gives

orange coloured precipitate with alkaloids.

o Caffeine, a purine derivative, does not precipitate like most alkaloids.

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4. Alkaloids are basic - they form water soluble salts. Most alkaloids are well-

defined crystalline substances which unite with acids to form salts. In plants,

they may exist

o in the free state,

o as salts or

o 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).

(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

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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

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

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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).

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

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9. Sample

10. Sulforic acid 2N

11. Wagner Reagent

5.3.Picture of Apparatus

Test Tube

VI. PROCEDURE

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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 and watch closely

precipitation formed.

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 Physic Properties Chemical Properies

Water H2O Cair tak bewarna Not Reactive

Mp = 0oC Stable

Bp = 100oC Polar Solvent

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Ρ= 1 g/mL Have Hidrogen Bond

Ethanol TL= -117,3oC Volatile

CH3CH2OH Bp =78,3oC Flamable

Mr= 46

Chloride Acid

Mr= 36,5 Soluble in waterHCL

Sulforic Acid Mr= 99,08 Strong Acid

H2SO4 Mp = 10.36oC Soluble in water

  Bp=338oC Corrosive

7.2 Tabel Pengamatan

Subtance Alkaloid Test Flavonoid

Test

Steroid Triterpenoid Saponin

Hager Mayer Wagner D.droff Test Test Test

Sample - - + - - - + +

Subtance Treatment Result

Sample Finely pounded Finely poundes leave

-Alkaloid Test-

Finely poundes leave Extraction with MTC ammoniakal

Ekstrak MTC Filter with cotton MTC extract dan residu

Placed into 2 test tube MTC extract in each test

tube

1st test tube Added sulfuic acid 1:1 there 2 phase:

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upper = acid phase ; lower

=MTC

added wegner tester positive

added meyer tester Negative

added dregendroff tester Negative

2nd terst tube added hager tester Negative

-Steroid,Triterpen dan Saponin Test -

Finely poundes leave Extraction with ethanol there 2 phase:

Warmed and filtered upper = acid extract ; lower

=Residu

Ethanolic Extract Added into vapor cup

Vapor solvent

Cooled

Added Ether

Ether Extract Added into test plat

Added L-B reagent Positive

Residu Added hot water

Put into test tube foam= saponin positive

Shaked

-Flavanoid Test-

Finely poundes leave Extraction with methanol

Filtered with cotton there 2 phase:

upper = acid extract ; lower

=Residu

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Extract Added HCl 2N

  Added Mg powder Orange Solution

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 fitohimičeskie. 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

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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.

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:

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Mayer’s reagent

Wagner’s reagent

Dragendorff’s reagent

Alkaloid test results on samples positive to say because of the four reagents

were added, there are positive that with the reagent Wagner.

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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.

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.

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.

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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.

To test and streoid triterpenoid content in leaf samples, ether extract added reagent-

Buchard 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 π electron transition πΔ and n

to πΔ 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:

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O

O

O

H2SO4+

HO

O

OH-O

O

+

O

O

-H++ CH3COOH + H2SO4 + CH3COOH

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.

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Friedli, G.L.1996.Alkaloid.

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Hartoyo, D. 2010. CULTIVATION LONG BEANS Vigna spp.

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Nanno, M and R. Hägg. 1993."The Heraion at Samos" in Greek Sanctuaries: New

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