IDENTIFICATION OF ANTHOCYANIN COMPOUNDS (ETHANOL EXTRACTOryza sativa L.

)AS ALTERNATIVE TREATMENTDHF

OFIDENTIFICATION OF ANTHOCYANINE AND SECONDARY METABOLIT FROM

ETHANOLIC EXTRACT OF BLACK(Oryza sativa L.

Glutinous RiceANTIDENATION SECONDARY FROM ETHANOLITE). METABOLITES FROM

EXTRACT ETHANOL BLACK GLUTINOUS RICE (Oryza sativa L.) IN UTILIZATION AS
COMPLEMENTARY MEDICINE TREATMENT OF DENGUE HAEMORRAHAGIC FEVER

KETUT AGUS ADRIANTA*

*Academy of Pharmacy Saraswati Denpasar, Jalan Cambodia No. 11A, Denpasar, Bali, Denpasar, Bali

Abstract : One of the natural ingredients that can be usedas a medicine is black glutinous rice which is thought
to contain anthocyanin compounds, this can be seen from the deep purple color it shows. This study aims to
determine the presence or absence of anthocyanin compounds, what types of anthocyanins are contained in
black glutinous rice. Black glutinous rice was extracted with 80% ethanol solvent which was acidified with 3%
citric acid. Identification of secondary metabolites was carried out by phytochemical screening method, while
the
identification of anthocyanin compounds was carried out by Thin Layer Chromatography using Forestal eluent
(concentrated HCL-acetic acid-water) in a ratio of 3:30:10 and BAA (n-butanol-acetic acid-water). with a ratio
of 4:1:5. The results showed that through a phytochemical screening test, it was found that black glutinous rice
was positive for secondary metabolites of alkaloids, flavonoids, tannins and triterpenoids. From the results of
analytical thin layer chromatography, black glutinous rice extract contains anthocyanin compounds. The
Forestal eluent did not produce a clear stain and the eluent was corrosive to the TLC plate, while the BAA
eluent produced a purple-red stain with an Rf value of 0.467. Based on the obtained Rf value, it was concluded
that black glutinous rice was positive for anthocyanin with the type of pelargonidin 3-glucoside.

Keywords: Rice black rice, secondary metabolites, phytochemical screening, thin layer chromatography

Abstract: One of the natural ingridients that can be used as medicine is the black Glutinous rice roomates is
Presumed to Contain anthocyanins compound, the which is seen from the dark purple color it shows. This
research has purpose to find out whether there is anthocyanin compound, what kind of anthocyanin that contains
in the black glutinous rice. The black glutinous rice is extracted with 90% ethanol solvent which was acidified
with 3% citrate acid. Identification of secondary metabolite compound is carried out by phytochemical
screening method, whereas identification of anthocyanin compound is carried out by Thin Layer
Chromatography using the eluent Forestal (HCL thick-acetate acid-water) with ratio of 3:30:10 and BAA (n -
buthanol-acetate acid-water) with a ratio of 4:1:5. Research result shows that through phytochemical screening
test it is obtained the result that the black glutinous rice is positively contains secondary metabolites of
alkaloids, flavonoids, tannins and triterpenoids. From the result of analytical thin layer chromatography, the
black glutinous rice extract contains the anthocyanin compound. In eluent Forestal there is no clear result of
stain and eluent corrosive to KLT plate whereas eluent BAA produced reddish purple colored stain with Rf
value for 0.467. Based on the Rf value obtained it is concluded that the black glutinous rice positively contains
anthocyanins of pelargonidin 3-glucoside type.

Keywords: Black glutinous rice, secondary metabolites, phytochemical screening, thin layer
chromatography
morbidity and mortality rate of DHF from year to
year which continues tofrom year to year.
INTRODUCTION increaseand occurs in all provinces in Indonesia. A
prominent clinical feature of DHF is the occurrence
Dengue hemorrhagic fever or commonly of blood plasma leakage. The healing process of
called Dengue Haemorrahagic Fever (DHF) is an DHF can be done by increasing platelet levels. Until
acute disease that is endemic and has a prevalence, now, the treatment of DHF is still supportive,
namely

Medicamento Scientific Journal•Vol.2 No.1•2016

 KETUT AGUS ADRIANTA. Scientific Journal of Medicamento 2(1) 2016; 17-22
overcomes plasma fluid loss due to increased
capillary vascular permeability.
Achmad and Wahono (2001) in Muharni et
al. (2013) stated that the group of tannins and
flavonoids can be an alternative treatment for
dengue fever. Rindiastuti and Tyasari (2008) in
Muharni (2011) stated that one of the natural
ingredients that can be used as an alternative is
Angkak. Wanti (2008) stated that the antioxidant
activity of Angkak from white, red and black rice
was highest in black rice.
From these data, researchers were
interested in identifying the content of anthocyanins
and other secondary metabolites found in black
glutinous rice as a preliminary study. The steps
carried out in this study were started from
phytochemical screening, extraction and then
continued with the thin layer chromatography test
method for the identification of anthocyanins.
This study aims to determine the presence
of anthocyanin compounds and secondary
metabolites contained in the ethanolic extract of
black glutinous rice.
MATERIALS AND METHODS
Research design. This research is a descriptive
laboratory research conducted in an analytical
manner using the TLC method and phytochemical
screening. The sampling technique used is
probability sampling with data collection
techniques in the form of color changes during the
reaction in the test tube and the Rf value of the stain
from the TLC test.
Ingredient. The material used in this research is
black glutinous rice (Oryza sativa L.) which grows
in the Jatiluwih, Tabanan area and chemicals
include ethanol 80% (Brataco), concentrated HCl,
glacial acetic acid, n-BuOH, aquadest, citric acid 3
%, Mg powder, Dragendorf and Mayer reagents,
amyl alcohol, chloral hydrate alcohol, FeCl3 1%,
anhydrous acetic acid and concentrated sulfuric
acid.
Method. The method or workflow of this research
is as follows:
1. Extraction
a. Weighed 1g of black glutinous rice powder and
put it in a porcelain cup. b. Maceration using 80%
ethanol solvent which is acidified with 3% citric
acid (Kristiana et al., 2012). The amount of
material and
the Scientific Journal of MedicamentoGlutinous Rice•Vol.2 No.1•2016
solvent mixture is 1:5 (w/v) (Ariviani, 2010).
Put the cup intotool, elmasonic and turn the
appliance at a temperature of 30oC for 3
minutes.
c. The mixture of black glutinous rice powder and
solvent was stirred using a stirring rod, then
macerated again in theapparatus elmasonic.
This stage is repeated up to 3 times.
d. The extract is filtered to separate the filtrate
from the macerate
e. The solvent in the filtrate was allowed to
evaporate by placing it in a fume hood for 10
minutes.
2. Phytochemical screening
a. Identification of alkaloids
Weigh 500 mg of simplicia powder, add 1
ml of 2 N hydrochloric acid and 9 ml of water,
heat on a water bath for 2 minutes, cool and
filter. The filtrate was divided into 2 parts of 3
ml each in a test tube. The first filtrate was
added 2 drops of Dragendorff's reagent then the
second filtrate was added 2 drops of Mayer's
reagent, observed. The results are positive when
a brown precipitate is formed when
Dragendorff's reagent is added and when
Mayer's reagent is added, a white or yellow
precipitate is formed which is soluble in
methanol (Suwarni et al., 2013).
b. Identification of flavonoids
0.5 g of simplicia powder was dissolved in
50 ml of water, heated for 5 minutes and
filtered. The remaining filtrate was used for the
next experiment. A total of 5 ml of the filtrate
was put into the tube and a little Mg powder was
added then added 1 ml of alcohol chlorhydrate
solution (a mixture of 37% HCl and 95%
ethanol with the same volume), added a few
drops of amyl alcohol, shaken vigorously, and
allowed to separate. There is a color in amyl
alcohol (red, yellow or orange) indicating the
presence of flavonoid compounds (Suwarni et
al., 2013).
c. Identification of tannins
Used filtrate obtained in test 5 ml filtrate
flavonoids drip with a few drops of a solution
ofFeCl3. 1% A positive result indicates the
green color violet on the addition ofFeCl3
1%(Suwarni et al., 2013).
d. Identification of saponins. The
filtrate from the flavonoid test was 10 ml of
the filtrate in a test tube, shaken vertically for
10 seconds. The results are said to be positive if
IDENTIFICATION OF ANTHOCYANIN COMPOUNDS ETHANOL EXTRACT OF BLACK(Oryza sativa L.) AS ALTERNATIVE TO TREATMENT OF DHF,
From the results of the elution on TLC,
stable foam is formed for 10 minutes, 1-10 cm the distance of the stain separation and the
high (Suwarni et al. ., 2013). e. Identification of distance traveled by the eluent were then
triterpenoids/steroids calculated to obtain the Rf value using the
A total of 50 mg of simplicia powder was formula in equation 2.1. After obtaining the Rf
placed in a porcelain dish and 15 drops of value of the stain, it was compared with the Rf
anhydrous acetic acid were added until it was value of anthocyanins in the literature.
completely submerged, left for about 15
minutes. Take the solution with a dropper and RESULTS AND DISCUSSION
put 6 drops in a test tube. Add 2-3 drops of
concentrated sulfuric acid. Positive results 1. Results of phytochemical screening
show a red orange or purple color
Phytochemical
(triterpenoids) or blue (steroids) (Sangi et al.,
screening was carried out qualitatively by
2008).
using a color reaction. The following are the
results of phytochemical screening:
3. Preparation of the eluent solution
a. Preparation of forestal eluent solution.
10 ml of solution was made by mixing
concentrated HCl-acetic acid-water in a ratio of
3:30:10 (Wahyuni, 2014). The eluent solution
is then put into the chamber and then the
chamber is closed. The aim of this treatment is
to make saturation go faster.
b. Preparation of BAA eluent solution. (a) (b)
10 ml of solution was made
by mixing n-butanol-acetic acid-water in a ratio
of 4:1:5 (Wahyuni, 2014). The eluent solution
is then put into the chamber and then the
chamber is closed. The aim of this treatment is
to make saturation go faster.

4. Separation by thin layer chromatography (c) (d)

(TLC)method
TLCusing a silica gel plate G60F254 with
a size of 9cm x 3cm. The mobile phase used to
separate the components was Forestal eluent =
concentrated HCl:acetic acid:water (3:30:10),
BAA = n-butanol:acetic acid:water (4:1:5).
Black glutinous rice extract was spotted at a
(e) (f)
distance of 2.5 cm from the bottom edge on a
Figure 1. Results of phytochemical
silica gel plate G60F254 with a capillary tube then
screening (a) alkaloid test with Dragendorf
dried in
reagent, (b) alkaloid test with Dragendorf
air and eluted for 5 cm. A positive reaction is
reagent, (c) )test
indicated by the presence of red to purple spots.
flavonoid, (d) tannin test, (e) saponin test and (f)
5. Data processing and analysis
triterpenoid/steroid test

Table.1 Phytochemical Screening Results

No Test Positive Reaction Observation(Theory) Observation Results Remarks

1 Alkaloid Test precipitate (Suwarni) , 2013). A brown precipitate is formed. (+)

with Dragendorf
reagent Chocolate

2 Alkaloid Test White or yellow precipitate soluble Produces a yellowish red (+)
with Mayer in methanol (Suwarni, 2013). solution with a
reagent slight cloudy precipitate.

3 Flavonoid Test Color in amalcohol (red, yellow or Produces a red solution. (+)
orange
 4 Tannin test The color is violet green with the
addition of FeCl3 (Suwarni,
2013).From the
5 Test of Saponin Foam is stable for 10 minutes with a
height of 1 to 10 cm (Suwarni, 2013)
No Test Positive Reaction Observation(Theory)
6 Triterpenoid / Color red orange or purple
Steroid Test (triterpenoids) or blue (steroids) (
Sangi, 2008).
in the test of alkaloids, both reagents and
mayer dragendorf produce precipitate the
corresponding literature so that it can be said posi
tif. According to Sangi et al. (2008), deposition
occurs due to ligand replacement. The nitrogen
atom which has a lone pair of electrons on the
alkaloids can replace the iodo ion in the reagent
used. Dragendorff's reagent contains bismuth
nitrate and potassium iodide in glacial acetic acid
solution (potassium tetraiodobismuthate(III)) while
Mayer's reagent contains potassium iodide and
mercury (potassium tetraiodomercurate(II)
chloride).
The flavonoid test produces a red solution
so it is said to be positive. According to Robinson
(1995) in Sangi et al. (2008), the resulting red color
indicates the presence of flavonoids as a result of
reduction by concentrated hydrochloric acid and
magnesium.
The tannin test showed a violet colored
solution, so it was said to be positive for tannins.
According to Harborne (1987) and Sangi et al.
(2008), chemically tannins are divided into two
groups, namely hydrolyzed tannins and condensed
tannins. Each group gave a different color reaction
to FeCI3 1%. At the time of addition, it is estimated
that FeC13 reacts with one of the hydroxyl groups
present in the tannin compound. The result of this
reaction is what gives rise to color. FeCIreagent3 is
widely used to identify phenolic compounds
including tannins. Harborne (1987) stated that the
Journal of Scientific MedicamentoGlutinous Rice•Vol.2 No.1•2016
tests carried out, the result is (+)
a violet colored solution.
tests carried out, the results (-)
obtained form
Observation Results Informatio
na
little foam that only lasts a
few seconds
From the tests results (+)
obtained that
showed orange-red colored
solution.
presence of tannins itself is closely related to
anthocyanins because in a condensed state, tannin
compounds are one part of anthocyanins, namely
proanthocyanidins.
The saponin test produces foam that only
lasts a few seconds, so it is said to be negative for
saponins. In Sangi et al. (2008) said that saponins
have glycosyls that function as polar groups and
steroid and triterpenoid groups as nonpolar groups.
Compounds that have polar and nonpolar groups
-are surface active so that when shaken with water,
saponins can form micelles. In the micelle structure,
the polar groups are facing outwards while the
nonpolar groups are facing inwards. This state is
what looks like foam.
The triterpenoid/steroid test showed a red-
orange color, so it was positive for triterpenoids. In
Sangi et al. (2008) argued that the color change is
based on the ability of compounds triterpenoids and
steroids forming color by H2SO4 concentrated in the
solvent acetic acid anhydride.
2. Results of stain separation using TLC
development with forestal eluent resulted in
poor stain separation . Elution using this eluent did
not get good stain separation results so that the Rf
value could not be calculated.
The expansion with BAA eluent resulted in
4 spots that were neatly separated, so that the Rf
value could be calculated.
IDENTIFICATION OF ANTHOCYANINE COMPOUNDS ETHANOL EXTRACT OF BLACK(Oryza sativa L.) AS ALTERNATIVE DHF TREATMENT
Table 2. Results of Color and Rf Value of Anthocyanin Identification by TLC
No. Separation of spots Rf value Rf value
(Theory)
1 Forestal eluent stain - -
2 Stain 1 BAA eluent 0.15 0.075
3 Stain 2 BAA eluent 0.15 0.192
4 Stain 3 BAA eluent 0.44 0.467
5 Spot 4 BAA eluent 0.6 0.6
The Rf value of the third spot on the BAA
eluent is 0.467, which is closest to the anthocyanin
Rf value in the literature for pelargonidin 3-
glucoside. In the literature, the standard Rf value of
pelargonidine 3-glucoside using BAA as eluent is
0.44. The color of the resulting stain is purple. In
Harborne (1987) it is said that the color of
anthosinins will follow the color of the type of
aglycone. The color of the pelargonidine aglycone
itself is red. The difference in color may also be
influenced by the bonding of the aglycone with the
glucoside of anthocyanins. The presence of
impurities during preparation, storage of materials
and tools and during extraction may also affect
color differences.
CONCLUSION
From the research that has been done, it can
be concluded that the tested black glutinous rice
(Oryza sativa L.) contains anthocyanin compounds
and other secondary metabolites. In the
phytochemical screening test the results were
positive containing alkaloids, flavonoids, tannins
and terpenoids. In identification using thin layer
chromatography method with the best eluent,
namely BAA, obtained an Rf value of 0.467 and
includes pelargonidin 3-glucoside anthocyanins.
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