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1
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.
2
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
3
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.
4
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.
5
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
16
9. Sample
10. Sulforic acid 2N
11. Wagner Reagent
5.3.Picture of Apparatus
Test Tube
VI. PROCEDURE
17
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
18
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
21
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:
22
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.
24
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:
25
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.
26
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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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/
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Nanno, M and R. Hägg. 1993."The Heraion at Samos" in Greek Sanctuaries: New
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http://www.sciencedirect.com/science?_ob=Article.htm
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