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A. Title Experiment : Phytochemical Test on Curcuma
Zanthorriza
B. Day/Date of Experiment : Tuesday/ October, 21th
2014
C.
Done : Tuesday/ October, 21th2014
D. Purpose :
1.
Selecting required tools needed based on experiment conducted
2. Selecting required materials needed based on experiment conducted
3. Identifying chemical component of plant from terpenoids, steroids,
phenolic (antraquinon, tannin, and phenol), flavonoids, and alkaloids
E. Basic Theory
Curcuma Xanthorr izaor Curcuma Zanthorr iza(Temu Lawak)
Synonym
Curcuma javanica, Curcuma zedoaria,
Javanese turmeric
.
Common name
Curcumine, ged, haridra, indian saffron,
java turmeric, jiang huang, shu gu jiang
huang, temu lawak, tumulawak.
Family
Zingiberaceae (Ginger family).
Temulawak rhizome and
Temulawak dlower
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Overview
Temulawak, originally from Indonesia, can grow up to 8 feet tall. The
flower is yellow. The large leaves stem from the root and the large
rhizome of the plant contains herbal qualities. Cultivation has spread to
other countries including Surinam.
Java - turmeric is a mild spice used in
several drinks to give these flavor and color
(yellow). It is also used for seasoning food
in the Indonesian cuisine; and used
extensively in India (in curry).
It has an aromatic, pungent odor and a bitter
taste.
Medicinal Applications
Interesting is that the rhizome is used medicinally; it has liver protection
properties.
Usefulness
-
The active ingredients (anti-oxidant and anti-edemic) are the
curcuminoids (curcumin, dicinnamoyl - methane, bis - demethoxy -
curcumin, demethoxy curcumin) encourage bile and prevent the
formation of gallstones.
- It also has essential oils, cinnamaldehyde and starch / carbohydrate.
The rhizomes have anti - viral and anti - inflammation properties
(Hepatitis B and C).
- Used against acne (inhibits bacterial growth); normalize digestion.It
increases breast milk production.
-
Can be used in the treatment of inflammatory bowel disease; effective
the for long-term maintenance therapy in patients with ulcerative
colitis; urinary tract infection (UTI).
-
Decreases cholesterol levels in blood and liver.
http://start%28%27view-kunirpowder02.jpg%27%29/ -
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- A tincturecan be used against, among other applications,
high cholesteroland blood triglycerides.
-
Temu lawak can also be used as a spice for food coloring.
Hardness: USDA zone 9 B - 11.
Propagation: Rhizome and bulb.
Culture:Full sun, sandy loam soil, plant in frost free areas, can also be
grown in a pot or as a garden plant.
In the following experiment chemical component of Curcuma
Xanthorrizawould be identified using Phytochemical Test. The series of
test would discover whether Curcuma Xanthorriza contains terpenoids,
streroids, phenolic, flavonoids and alkaloids group..
Phytochemical Test
The subject of phytochemistry or plant chemistry has developed in
recent years as adistinct discipline, somewhere in between natural product
orgnic chemistry and plant biochemistry and is closely related to both. It is
concerned with the enourmous variety of organic substances that are
elaborated and accumuated by plants and deals with the chemical
structures of these substances, their biosynthesis, turnover and
metabolism, their natural distributuon and their biological function.
In all these operations, methods are needed for separation,
purification and identification of the many different constituents present in
plants. Thus, advances in our understanding of phytochemistry are directlyrelated to the successful exploitation of known techniques, and the
continuing development of new techniques to solve outstanding problems
as they appear. One of the challenges of phytochemistry is to carry out all
the above operations on vanishingly small amounts of material.
Frequently, the solution of a biological problem in, say, plant growth
regulation, in the biochemistry of plant-animal interactions, or in
understanding the origin of fossil plants depends on identifying a range of
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complex chemical structures which may only be available for study in
microgram amounts.
Phytochemical progress has been aided enormously by the
development of rapid and accurate methods of screening plants for
particular chemicals and the emphasis in this book is inevitably on
chromatographic techniques. These procedures have shown that many
substances originally thought to be rather rare in occurrence are of almost
universal distribution in the plant kingdom. The importance of continuing
surveys of plants for biologically active substances needs no stressing.
Certainly, methods of preliminary detection of particular classes of
compound are discussed in some detail in the following chapters.
Although the term 'plant' is used here to refer to the plant kingdom
as a whole, there is some emphasis on higher plants and methods of
analysis Introduction 3 for micro-organisms are not dealt with in any
special detail. As a general rule, methods used with higher plants for
identifying alkaloids, amino acids, quinones and terpenoids can be applied
directly to microbial systems. In many cases, isolation is much easier,
since contaminating substances such as the tannins and the chlorophylls
are usually absent. In a few cases, it may be more difficult, due to the
resilience of the microbial cell wall and the need to use mechanical
disruption to free some of the substances present.
Methods of Extraction and Isolation
The plant material ideally, fresh plant tissues should be used for
phytochemical analysis and the material should be plunged into boilingalcohol within minutes of its collection. Sometimes, the plant under study
is not at hand and material may have to be supplied by a collector living in
another continent. In such cases, freshly picked tissue, stored dry in a
plastic bag, will usually remain in good condition for analysis during the
several days required for transport by airmail. Alternatively, plants may be
dried before extraction. If this is done, it is essential that the drying
operation is carried out under controlled conditions to avoid too many
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chemical changes occurring. It should be dried as quickly as possible,
without using high temperatures, preferably in a good air draft. Once
thoroughly dried, plants can be stored before analysis for long periods of
time. Indeed, analyses for flavonoids, alkaloids, quinones and terpenoids
have been successfully carried out on herbarium plant tissue dating back
many years.
In phytochemical analysis, the botanical identity of the plants
studied must be authenticated by an acknowledged authority at some stage
in the investigation. So many mistakes over plant identity have occurred in
the past that it is essential to authenticate the material whenever reporting
new substances from plants or even known substances from new plant
sources. The identity of the material should either be beyond question (e.g.
a common species collected in the expected habitat by a field botanist) or
it should be possible for the identity to be established by a taxonomic
expert. For these reasons, it is now common practice in phytochemical
research to deposit a voucher specimen of a plant examined in a
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recognized herbarium, so that future reference can be made to the plant
studied if this becomes necessary.
Extraction
The precise mode of extraction naturally depends on the texture
and water content of the plant material being extracted and on the type of
substance that is being isolated. In general, it is desirable to 'kill' the plant
tissue, i.e. prevent enzymic oxidation or hydrolysis occurring, and
plunging fresh leaf or flower tissue, suitably cut up where necessary, into
boiling ethanol is a good way of achieving this end.
Alcohol, in any case, is a good all-purpose solvent for preliminary
extraction. Subsequently, the material can be macerated in a blender and
filtered but this is only really necessary if exhaustive extraction is being
attempted. When isolating substances from green tissue, the success of the
extraction with alcohol is directly related to the extent that chlorophyll is
removed into the solvent and when the tissue debris, on repeated
extraction, is completely free of green colour, it can be assumed that all
the low molecular weight compounds have been extracted.
The classical chemical procedure for obtaining organic constituents
from dried plant tissue (heartwood, dried seeds, root, leaf) is to
continuously extract powdered material in a Soxhlet apparatus with a
range of solvents, starting in tum with ether, petroleum and chloroform (to
separate lipids and terpenoids) and then using alcohol and ethyl acetate
(for more polar compounds). This method is useful when working on the
gram scale. However, one rarely achieves complete separation ofconstituents and the same compounds may be recovered (in varying
proportions) in several fractions. The extract obtained is clarified by
filtration through celite on a water pump and is then concentrated in
vacuo. This is now usually carried out in a rotary evaporator, which will
concentrate bulky solutions down to small volumes, without bumping, at
temperatures between 30 and 40C.
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In most cases, mixtures of substances will be present in the crystals
and it will then be necessary to redissolve them in a suitable solvent and
separate the constituents by chromatography. Many compounds also
remain in the mother liquor and these will also be subjected to
chromatographic fractionation. As a standard precaution against loss of
material, concentrated extracts should be stored in the refrigerator and a
trace of toluene added to prevent fungal growth. When investigating the
complete phytochemical profile of a given plant species, fractionation of a
crude extract is desirable in order to separate the main classes of
constituent from each other, prior to chromatographic analysis.
The Phenolic
The term phenolic compound
embraces a wide range of plant
substances which possess in
common an aromatic ring
bearing one or more hydroxyl
substituents. Phenolic substances
tend to be water-soluble, since
they most frequently occur
combined with sugar as
glycosides and they are usually
located in the cell vacuole.
Among the natural phenolic compounds, of which several thousand
structures are known, the flavonoids form the largest group but simplemonocyclic phenols, phenylpropanoids and phenolic quinones all exist in
considerable numbers.
Several important groups of polymeric materials in plants - the
lignins, melanins and tannins - are polyphenolic and occasional phenolic
units are encountered in proteins, alkaloids and among the terpenoids.
While the function of some classes of phenolic compound are well
established (e.g. the lignins as structural material of the cell wall; the
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anthocyanins as flower pigments), the purpose of other classes is still a
matter of speculation. Flavonols, for example, appear to be important in
regulating control of growth in the pea plant (Galston, 1969) and their
adverse effects on insect feeding (Isman and Duffey, 1981) have indicated
that they may be natural resistance factors; neither of these functions,
however, has yet been firmly established.
Flavonoid Pigments
The flavonoids are all structurally derived from the parent
substance flavone, which occurs as a white mealy farina on Primula
plants, and all share a number of properties in common. Some ten classes
of flavonoid are recognized (Table 2.7) and these different classes will be
considered in more detail in subsequent sections. In this section, general
procedures of identification are discussed and methods for distinguishing
the different classes are outlined. Flavonoids are mainly water-soluble
compounds. They can be extracted with 70% ethanol and remain in the
aqueous layer, following partition of this extract with petroleum ether.
Flavonoids are phenolic and hence change in colour when treated
with base or with ammonia; thus they are easily detected on
chromatograms or in solution. Flavonoids contain conjugated aromatic
systems and thus show intense absorption bands in the UV and visible
regions of the spectrum. Finally, flavonoids are generally present in plants
bound to sugar as glycosides and anyone flavonoid aglycone may occur in
a single plant in several glycosidic combinations.
For this reason, when analysing flavonoids, it is usually better toexamine the aglycones present in hydrolysed plant extracts before
considering the complexity of glycosides that may be present in the
original extract. Flavonoids are present in all vascular plants but some
classes are more widely distributed than other; while flavones and
flavonols are universal, isoflavones and biflavonyls are found in only a
few plant families. Detailed references to the natural distribution of the
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flavonoids may be found in Harborne (1967a), Harborne et al. (1975),
Harborne and Mabry (1982) and Harborne (1988, 1994).
Preliminary classification, Flavonoids are present in plants as
mixtures and it is very rare to find only a single flavonoid component in a
plant tissue. In addition, there are often mixtures of different flavonoid
classes. The coloured anthocyanins in flower petals are almost invariably
accompanied by colourles flavones or flavonols and recent research has
established that the flavones are important co-pigments, being essential for
the full expression of anthocyanin colour in floral tissues. Mixtures of
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anthocyanins are also the rule, particularly in the flowers of ornamental
plants, and anyone flower tissue may contain up to ten different pigments.
Tannins
Tannins occur widely in vascular plants, their occurrence in the
angiosperms being particularly associated with woody tissues. By
definition, they have the ability to react with protein, forming stable
waterinsoluble co-polymers. Industrially, tannins are substances of plant
origin which because of their ability to cross-link with protein are capable
of transforming raw animal skins into leather. In the plant cell, the tannins
are located separately from the proteins and enzymes of the cytoplasm but
when tissue is damaged, e.g. when animals feed, the tanning reaction may
occur, making the protein less accessible to the digestive juices of the
animal.
Plant tissues high in tannin are, in fact, largely avoided by most
feeders, because of the astringent taste they impart. One of the major
functions of tannins in plants is thought to be as a barrier to herbivory.
Chemically, there are two main types of tannin, which are distributed
unevenly throughout the plant kingdom.
The condensed tannins occur almost universally in ferns and
gymnosperms and are widespread among the angiosperms, especially in
woody species. By contrast, hydrolysable tannins are limited to
dicotyledonous plants and here are only found in a relatively few families.
Both types of tannin, however, can occur together in the same plant, as
they do in oak bark and leaf. Condensed tannins or flavolans can beregarded as being formed biosynthetically by the condensation of single
catechins (or gallocatechins) to form dimers and then higher oligomers,
with carboncarbon bonds linking one flavan unit to the next by a 4-8 or 6-
8 link. Most flavolans have between two and twenty flavan units. The
name proanthocyanidin is used alternatively for condensed tannins
because on treatment with hot acid, some of the carbon--earbon linking
bonds are
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plant are broken and anthocyanidin monomers are released.
Characterization thus involves the identification of the
anthocyanidin or anthocyanidins so released. Hydrolysable tannins are
mainly of two classes, the simplest being the galloylglucose depside, in
which a glucose core is surrounded by five or more galloyl ester groups.
A second type occurs where the core molecule is a dimer of gallic acid,
namely hexahydroxydiphenic acid, again with glucose attachment; on
hydrolysis, these ellagitannins yield ellagic acid. Within these two classes
it is possible to further subdivide the known compounds according to their
biogenetic origin (Haddock et al., 1982). The chemical elucidation of
tannins has been difficult to accomplish and it is only within the last
decade that some of the structural complexities have been fully
appreciated. There are, for example, significant stereochemical differences
between the proanthocyanidins of monocotyledonous and dicotyledonous
plants (Ellis et al., 1983). Detailed accounts of plant tannins include those
of Swain (1979) and Haslam (1989).
Triterpenoids and Steroids
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Triterpenoids are
compounds with a
carbon skeleton
based on six
isoprene units
which are derived
biosynthetically
from the acyclic
C30 hydrocarbon,
squalene. They have
relatively complex
cyclic structures,
most being either
alcohols, aldehydes
or carboxylic acids.
They are colourless,
crystalline, often high melting, optically active substances, which are
generally difficult to characterize because of their lack of chemical
reactivity.
A widely used test is the Liebermann-Burchard reaction (acetic
anhydride- conc.H2S04), which produces a blue-green colour with most
triterpenes and sterols. Triterpenoids can be divided into at least four
groups of compounds: true triterpenes, steroids, saponins and cardiac
glycosides. The latter two groups are essentially triterpenes or steroids
which occur mainly as glycosides. There are also the steroidal alkaloids,but these are covered in this book along with the other alkaloids.
Many triterpenes are known in plants and new ones are regularly being
discovered and characterized (Connolly and Hill, 1991). So far, only a few
are known to be of widespread distribution. This is true of the pentacyclic
triterpenes a- and ~-amyrin and the derived acids, ursolic and oleanolic
acids (see Fig. 3.8). These and related compounds occur especially in the
waxy coatings of leaves and on fruits such as apple and pear and they may
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serve a protective function in repelling insect and microbial attack.
Triterpenes are also found in resins and barks of trees and in latex
(Euphorbia, Hevea, etc.). Certain triterpenes are notable for their taste
properties, particularly their bitterness. Limonin, the lipid-soluble bitter
principle of Citrus fruits, is a case in point. It belongs to a series of
pentacyclic triterpenes which are bitter, known as limonoids and
quassinoids. They occur principally in the Rutaceae, Meliaceae and
Simaroubaceae and are also of chemotaxonomic interest (Waterman and
Grundon, 1983).
Another group of bitter triterpenes are the cucurbitacins, confined
mainly to the seed of various Cucurbitaceae but detected also in several
other families including the Cruciferae (Curtis and Meade, 1971). Sterols
and triterpenes are based on the cyclopentane perhydrophenanthrene ring
system. At one time, sterols were mainly considered to be animal
substances (as sex hormones, bile acids, etc.) but in recent years, an
increasing number of such compounds have been detected in plant tissues.
Indeed, three so-called 'phytosterols' are probably ubiquitous in occurrence
in higher plants: sitosterol (formerly known as B-sitosterol), stigmasterol
and campesterol. These common sterols occur both free and as simple
glucosides. A less common plant sterol is aspinasterol, an isomer of
stigmasterol found in spinach, alfalfa and senega root. Certain sterols are
confined to lower plants; one example is ergosterol, found in yeast and
many fungi.
Others occur mainly in lower plants but also appear occasionally in
higher plants, e.g. fucosterol, the main steroid of many brown algae andalso detected in the coconut. Phytosterols are structurally distinct from
animal sterols, so that recent discoveries of certain animal sterols in plant
tissues are most intriguing. One of the most remarkable is of the animal
estrogen, estrone, in date palm seed and pollen (Bennett et al., 1966).
Pomegranate seed is another source, but the amounts present are very low;
according to Dean et al. (1971), there is only 41lg estrone kg-I tissue. Less
remarkable, perhaps, is the detection of cholesterol as a trace constituent in
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several higher plants, including date palm, and in a number of red algae
(Gibbons et al., 1967). Finally, the occurrence of insect moulting
hormones, the ecdysones, in plants must be mentioned, since they provide
a fascinating insight into the way plants may have evolved in order to
protect themselves from insect predation. Ecdysones were discovered in
plants in 1966 (Nakanishi et al., 1966) and have subsequently been found
in a range of plant tissues with high concentrations being present in a
number of ferns (e.g. bracken, Pteridium aquilinum) and gymnosperms
(e.g.Podocarpus nakaii).
Saponins are glycosides of both triterpenes and sterols and have
been detected in over seventy families of plants (Hostettmann and Marston
1995). They are surface-active agents with soap-like properties and can be
detected by their ability to cause foaming and to haemolyse blood cells.
The search in plants for saponins has been stimulated by the need for
readily accessible sources of sapogenins which can be converted in the
laboratory to animal sterols of therapeutic importance (e.g. cortisone,
contraceptive estrogens, etc.). Compounds that have so been used include
hecogenin from Agave and yamogenin from Dioscorea species. Saponins
are also of economic interest because of their occasional toxicity to cattle
(e.g. saponins of alfalfa) or their sweet taste (e.g. glycyrrhizin of liquorice
root). The glycosidic patterns of the saponins are often complex; many
have as many as five sugar units attached and glucuronic acid is a common
component.
The last group of triterpenoids to be considered are the cardiac
glycosides or cardenolides; here again there are many known substances,with complex mixtures occurring together in the same plant. A typical
cardiac glycoside is oleandrin, the toxin from the leaves of the oleander,
Nerium oleander, Apocynaceae. An unusual structural feature of oleandrin
(see Fig. 3.8) and many other cardenolides is the presence of special sugar
substituents, sugars indeed which are not found elsewhere in the plant
Triterpenoids and steroids 133 kingdom. Most cardiac glycosides are toxic
and many have pharmacological activity, especially as their name implies
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on the heart. Rich sources are members of the Scrophulariaceae, Digitalis,
Apocynaceae, Nerium, Moraceae and Asclepiadaceae, Asclepias. Special
interest has been taken in the cardiac glycosides ofAsclepias,because they
are absorbed by Monarch butterflies feeding on these plants and are then
used by the butterflies as a protection from predation by blue Jays
(Rothschild, 1972). The butterfly is unharmed by these toxins, which on
the other hand act as a violent emetic to the bird.
Alkaloids
The identification using Culvenor-Fitzgerald. Reagent Meyer gives
peach, reagent Wagner gives brown, reagent dragendorff give white
precipitate. The alkaloids, of which some 10000 are known, comprise the
largest single class of secondary plant substances. There is no one
definition of the term 'alkaloid' which is completely satisfactory, but
alkaloids generally include 'those basic substances which contain one or
more nitrogen atoms, usually in combination as part of a cyclic system'.
Alkaloids are often toxic to man and many have dramatic physiological
activities; hence 204 Nitrogen compounds their wide use in medicine.
They are usually colourless, often optically active substances; most are
crystalline but a few (e.g. nicotine) are liquids at room temperatures. A
simple but by no means infallible test for alkaloids in fresh leaf or fruit
material is the bitter taste they often impart to the tongue.
The alkaloid quinine, for example, is one of the bitterest substances
known and is significantly bitter at a molar concentration of 1x 10-5. The
most common precursors of alkaloids are amino acids, although thebiosynthesis of most alkaloids is more complex than this statement
suggests. Chemically, alkaloids are a very heterogeneous group, ranging
from simple compounds like coniine, the major alkaloid of hemlock,
Conium maculatum, to the pentacyclic structure of strychnine, the toxin of
the Strychnos bark. Plant amines (e.g. mescaline) and purine and
pyrimidine bases (e.g. caffeine) are sometimes loosely included in the
general term alkaloid.
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Many alkaloids are terpenoid in nature and some (e.g. solanine,
thesteroidal alkaloid of the potato, Solanum tuberosum) are best
considered from the biosynthetic point of view as modified terpenoids.
Others are mainly aromatic compounds (e.g. colchicine the tropolone
alkaloid of autumn crocus bulbs), containing their basic group as a side-
chain attachment. A considerable number of alkaloids are specific to one
family or to a few related plants. Hence, the names of alkaloid types are
often derived from the plant source, e.g. the Atropa or tropane alkaloids
and so on.
To illustrate their natural occurrence, some of the more common
alkaloids are mentioned (Table 5.5) in relation to their occurrence in
particular families. This table shows only those families in which over
fifty alkaloid structures have been detected (Hegnauer, 1967). These are
the angiosperm families which are particularly rich in these bases, but it
should be borne in mind that alkaloid distribution is very uneven and many
families lack them altogether.
Alkaloids are generally absent or infrequent in the gymnosperms,
ferns, mosses and lower plants. The structures of some of the more
common alkaloids are illustrated in Fig. 5.5. The functions of alkaloids in
plants are still largely obscure, although individual substances have been
reported to be involved as growth regulators or as insect repellents or
attractants. The theory that they act as a form of nitrogen storage in the
plant is not
now
generallyaccepted.
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F. Tools and Materials
Tools Materials
Blender Curcumin
Ohauss balance concentrated HCl
Beaker glass 100 ml concentrated H2SO42N
Beaker glass 300 ml FeCl31%
Test tube CH3Cl
Pipette NH3
Threepod Mg plate
Drop plat methanol 60-80%
Spatula ethanol 70%
Funnel Aquadest
Reagent:
Lieberman-Burchard
Meyer
Wagner
Dragendorf
G.
Procedure
1. Methanol Curcuma Extract Preparation
Fresh Curcuma
-peeled out the skin
- sliced, dried up
-blended
Dried Curcuma Powder
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2. Identification of Alkaloids using Culvener-Fitzgerald (Harborne,
1987)
5 g Dried Curcuma
Powder
-
Put into beaker glass 100 ml-Added 15 ml methanol 60-80%
heated until left 10 ml
-
Filtered using buchner funnel
FiltrateResidue
-Concentrated in
bath water
-
Used as sample
1 ml sample
-Added 1 ml CH3Cl
and 1 ml ammonia
-Put into test tube,
heated in bath water
FiltrateResidue
-
Divided into three testtube
Filtrate 1 Filtrate 2 Filtrate 3
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3. Identification of Flavonoids (Harborne, 1987)
-Added 3 drops of
HCl 2N, shaken
-Let a minute until
forms layer
-
Put the upper layer
-Added 3 drops of
HCl 2N, shaken
-Let a minute until
forms layer
-
Put the upper layer
-Added 3 drops of
HCl 2N, shaken
-
Let a minute until
forms layer
-
Put the upper layer
Upper layer
-Tested with Mayer
reagent
Peachprecipitate
Upper layer
-Tested with
Wagner reagent
Brownprecipitate
Upper layer
-Tested with
Dragendorff reagent
Whiteprecipitate
1 ml sample + 3 ml ethanol 70%
-
Shaken
-
Heated, shaked
-Filtered
FiltrateResidue
-Added 0,1 g of Mg
and two drops of
concentrated HCl 2N
Red color in ethanol layer: (+)
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4. Identification of Saponin (Harborne, 1987)
5.
Identification of Steroids
6. Identification of Triterpenoid
1 ml sample
-Boiled in 10 ml water
in bath water
Filtrate
-Shaken let in 15
minute, observed
Foam formed (+)
1 ml sample
-Added with 3 ml ethanol 70%, 2m l
conc, HCl and 2 m CH3COOH
Liebermann-Burchard
Color changes: Violetbluegreen
steroids (+)
1 ml sample
-Added with 2 ml CH3Cl and 3 ml
concentrated H2SO4
Color changes: sorrel in between layer
Terpenoids (+)
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7. Identification of Tannin
H. Result of Experiment
1 ml sample
-Boiled in bath ater
using 20 ml water
FiltrateResidue
-
Added 2-3 drops of
FeCl31%
Greenish brown/dark blue: tannin (+)
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I. Analysis
ANALYSIS AND DISCUSSION
Phytochemical test is one important step in the effort to uncover the
potential resources of herbs. Phytochemical the results of the analysis could give
clues about the existence of chemical component ( a compound ) type of class
alkaloid, flavonoid, saponin, steroids, triterpenoid, and tannin.Based on
experiment phytochemical on test extract rhizome curcuma ( curcuma
zanthorrhiza ) which aims to identify chemical component of herbs of terpenoid,
group steroids, phenolic ( anthraquinone, tannin, and phenol ) flavonoid, and an
alkaloid contained in extracts rhizome curcuma.
1.
The Preparation Of An Extract Methanol Curcuma Rhizomes
The first thing done is preparing an extract of methanol rhizomes curcuma.
Rhizomes curcuma fresh are cleaned and flayed then dried at rooms that is not
exposed to the sun, milled to get the pollen that is dry as many as five grams of
rhizomes curcuma is inserted into a beaker glass 100 mL to extracted by
immersing the powder into 15 mL of methanol 60- 80 %, and heated to speed up
the process of extracting then filtered with filters buncher.
Extract + CH3OH Yellow sample of concentrated
Filtrat produced then evaporated in water bath to produce an extract viscous. An
extract of methanol in yellow rhizomes curcuma concentrated, this is a sample for
test phytochemical on rhizomes curcuma.
2. Identification Alkaloid With Method Culvenor Fitzgerald (Harborne,
1987)
For identification an alkaloid, as many as 1 mL sample mixed with 1 mL
of chloroform and 1 mL ammoniac formed 2 layers. On the top layer of dark
sorrel colored and the lower layer of orange. Then heated above water bath then
shaken and distilled, filtrat resulting colored orange light on the top layer, and in
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the lower layer of dark orange, then the top layer been divided into three part is
equally the flattened and incorporated into test tube. On tube A B C added H2SO4
2N then shaken and ignored a few minutes. On tube A then tested with reagent
meyer ( colorless ) and produced the sediment peach-colored, it can be inferred
sample positive ( + ) contain the alkaloid.
Extraction by the addition of chloroform aims to sever ties between an
acid and an alkaloid bound in tannin ionic where atoms n of an alkaloid with a
hydroxyl group bonded mutual stable genolik of the acid of tannin. On the dotted
ties with this an alkaloid shall be set free while sour tannin will be bound by
chloroform. The addition of sulphuric acid 2N it serves to bind back an alkaloid
being salt an alkaloid to be able to react with reagent heavy metal that is a specific
for an alkaloid that produces complex inorganic salts that are not soluble so as to
separate with metabolic secondary.
The addition of sulphuric acid 2N resulting in a solution of formed into
two phases because of the different levels of polarity between phases of aqueous
the polar and chloroform that relative less polar. A salt of alkaloids will dissolve
in the top layer, while a layer of chloroform was in the lining of the bottombecause it has a mass of larger kinds.While shake with strong aims to dissolve
those compounds of every layers precisely and perfect. Reagent meyer aims to
detect an alkaloid, where reagent this bind with the alkaloid through coordinate
bonds between the atoms N alkaloids and Hg reagent meyer so as to produce
complex compounds of mercury with which nonpolar settles peach- colored. The
reaction by the experiment an alkaloid with reagent meyer is:
N +K2[HgI4] NK+ +
K2[HgI4]
In tube B was tested with reagents obtained Waghner greenish brown solution and
a brown precipitate that indicate (+) test alkaloids. The reaction on the reagent test
Waghner alkaloids are:
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N + KI + I2 NK+ + I3-
In tube C was tested with reagent Dragendorf light brown color and a no
precipitate is formed, that indicate (-) alkaloid test. The reaction on the reagent
test Dragendorf alkaloids are:
N + K[BiI4] NK+ + K[BiI4]
-
3.
Identification Flavonoid (Harborne, 1987)
In flavonoid test sample of 1 ml mixed with 3 mL of 70% ethanol solution
and then heated and filtered to separate the filtrate and the residue produces a red
colored solution. The filtrate plus the Mg metal, then add 2 drops of concentrated
HCl to detect the presence of flavonoid compounds will react with Mg, after the
addition of HCl, the solution turned into a brownish red. The addition of Mg metal
and HCl to detect the presence of flavonoid compounds which will react with Mg
flavonoids after the addition of concentrated hydrochloric acid with a red color
change occurs because of light absorption of flavonoids undergo changes toward
larger wavelengths due to the reduction reaction by HCl.
A compound of phenol is a compound having a ring aromatic and
containing one or two of hydroxyl groups. A compound of phenol tending readily
soluble in water because generally bonded with sugar as glycosides and usuallycontained in vacuoles of cells so that a rhizome and fronds curcuma positive there
are flavonoid. The activity of phenolic compounds in plants are many and very
diverse. Flavonoids are good reducing compound, these compounds inhibit many
oxidation reactions either in enzimatis or non enzimatis. Flavonoids act as a good
reservoir of free radical and superoxide lipid membrane so that it can protect
against the damaging reaction.
4.
Identification Saponin (Harborne, 1987)
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For the identification of saponin, 1 ml sample with 10 mL of water boil in
a water bath produces orange color in the solution. After the filtrate was shaken
and allowed to stand for 15 minutes. Formed stable foam indicates positit (+) to
test for saponins saponins glikosid similar properties that have the characteristics
of skill frothy when shaken akuos solution. Saponins are components that are
ampifilik polar lipids (having hydrophilic groups and hydrophobic groups). In
liquid systems, liquid lipids spontaneously form micelles dispersed with phyllic
tails that intersect with the liquid medium. Micelles may contain thousands of
lipid molecules. Liquid lipids form a layer with a thickness of one molecule is a
single layer. In such systems, the hydrocarbon tail open so away from water and a
hydrophilic layer that extends to the water is polar, the system is called premises
foam.
Saponin is a compound of active a strong surface, give rise to foam if
shaken with water and at concentrations that are low-growing often causing
hemolysis red blood cells. Saponin in a solution very dilute, can be as a fish
poison besides saponin also reported potentially to as antimicrobial, can be used
as raw materials for the synthesis of steroid hormones. Two kinds of saponin
which was known that is a glycoside triterpenoid alcohol and glycosides structure
steroid. Aglikon called sapogenin, obtained by the hydrolysis in an acid or using
an enzyme ( robinson, 1995 ).Rhizomes curcuma containing saponin rhizomes
that is only because there are active compounds called curcumin.
5. Identification Steroids (Harborne, 1987)
On Steroids test. Samples plus 3 mL of 70% ethanol and then added 2 mL
of concentrated H2SO4and 2 mL of acetic acid and then shaken and let stand 15
minutes. The function of acetic acid is to form an acetyl derivative of the steroid.
While the function is to reduce acetyl H2SO4formed a positive test if formed blue
solution, but in this experiment formed a yellow color that indicates a negative
steroid.
6.
Identification Triterpenoid (Harborne, 1987)
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Samples added 2 mL of chloroform and 3 mL H2SO4 concentrared. The
function of chloroform is to dissolve triterpenoid that it is readily soluble in
organic solvents. Function H2SO4is to reduce tripenoid that which produces color
sorrel. This exhibiting positive ( + ) the existence of triterpenoid. Triterpenoid is a
component of plants which have the odor and can be isolated from vegetable
matter by the distillation of as a volatile oil. Triterpenoid consists of a frame with
3 cyclic 6 who joined with the cyclic 5 or 4 cyclic 6 that has a cluster of on cyclic
certain ( Lenny, 2006 ).
A compound triterpenoid this test to see whether extract compound
containing triterpenoid. A compound triterpenoid will experience dehydration by
the addition of a strong acid and forms salts gave some reaction of color. A
solvent chloroform can dissolve this compound because soluble in either in
chloroform and not containing in the molecule of water. The addition of acetic
acid anhydrous will be turned into acetic acid before the reaction takes place and
are not formed a derivative of acetyl. Triterpenoid respond establishment of a
brownish color when this compound was detected of concentrated sulphuric acid
through its walls meanwhile, will produce bluish green color ( Robinson, 1995 ).
7. Identification Taninns (Edeoga et all., 2005)
Samples of 1 mL boiled with 20 mL of water and then filtered. The
resulting filtrate was added a few drops (2-3 drops) of 1% FeCl3formed brownish
black color in the solution. This indicates a negative (-) test tannins.
Test phytochemical use FeCl3to know whether an extract of brutes having
a cluster of fenol. Tanin is a compound polifenol. Bluish green color formed at
extract after added FeCl3because tannin form compound complek with FeCl3. As
a compound of phenol other tannin produces a bluish green with iron ( iii )
chloride ( Robinson, 1995 ).
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The formation of color the occurrence of this because the establishment of
complex compounds between the metals Fe and tannin. Complex compounds
formed because of a covalent bond coordination between ion or atom of metal
with the atoms nonmetals ( Effendy, 2007 ). Tendency Fe in the formation of
complex compounds can fasten 6 pairs of electrons free. Ion Fe3+ in the
formation of complex compounds form will hybridization d2sp3, so will unfilled
by 6 pairs of free electrons atoms O ( Effendy, 2007 ).
The tannin stability can be achieved if repulsion between ligan on 3 tannin
minimum.This happens if 3 tannin the position taken away. The result of a
rhizome curcuma negative because does not occur the formation of the compound
colors complex between the metals Fe and tannin. Complex compounds formed
due to a covalent bond coordination between an ion or atom of metal with an atom
the nonmetals.
FeCl3 Fe3++3Cl-
HO
OH
OH
HO
OH
+Fe3+ OH
OH
HO
OH
+Fe(OH)3
black
J. Conclusion
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Reference
Anonymous. 2014. Curcuma XanthorrizaTemulawak.
http://www.tropilab.com/tumulawak.html (acessed on 25 October 2014 at 9.21am)
Harborne, J.B. 1998.Phytochemical Methods A Guide to Modern Techniques of
Plant Analysis Third Edition. London: Chapman & Hall (ebook)
Philipson, J. David. 2000.Phytochemistry and medicinal plants. Phytochemistry
56 (2001) 237243 (online http://www.elsevier.comaccessed 25 October 2014 at
10 am)
Robinson, T. 1963.The Organic Constituents of Higher Plants: Their Chemistry
and Interrelationships. New York:Minneapolis, Burgess Pub. Co (ebook)
Tim Dosen Kimia Organik. 2014.Penuntun Praktikum Kimia Organik 2.
Surabaya : UNESAPRESS.
http://www.elsevier.com/http://www.elsevier.com/http://www.elsevier.com/http://www.elsevier.com/ -
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Question and Answer
1.
Write each reaction in phytochemical test!
2.
Write basic structure of each steroids, , triterpenoids, tannin, saponin,flavonoids, and alkaloids group.
3.
Mention flavonoids compound that contained in curcuma rhizome based
on literature.
4. Mention function and benefits of curcuma rhizome for human life.
Answer
1.
Reaction
Phytochemical
test
Reagent Reaction
Identification of
alkaloids
Mayer
N +K2[HgI4]
NK+ +K2[HgI4]
Wagner
N + KI + I2
NK+ + I3-
Dragendorff
N +K[BiI4]
NK+ + K[BiI4]
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Identification of
flavonoids
Mg+conc.H
Cl+ethanol
Identification of
saponin
Identification of
steroids
Liebermann
-Burchard
Identification of
triterpenoids
Liebermann
-Burchard
Identification of
tannin
FeCl31% FeCl3 Fe3++3Cl-
HO
OH
OH
HO
OH
+Fe3+
OH
OH
HO
OH
+Fe(OH)3
black
2. Basic structure
Alkaloids Contain N in heterocyclic ring
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Flavonoids
(Robinson, 1963)
Saponin
Steroids
(Robinson, 1963)
Triterpenoids
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- Used against acne (inhibits bacterial growth); normalize digestion.It
increases breast milk production.
-
Can be used in the treatment of inflammatory bowel disease; effective
the for long-term maintenance therapy in patients with ulcerative
colitis; urinary tract infection (UTI).
-
Decreases cholesterol levels in blood and liver.
- A tincturecan be used against, among other applications,
high cholesteroland blood triglycerides.
-
Temu lawak can also be used as a spice for food coloring.