Article Wjpps 1559285511 (Harrizul Rivai)

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Rivai et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 7.421
Volume 8, Issue 6, 149-167 Research Article ISSN 2278 – 4357
QUALITATIVE AND QUANTITATIVE ANALYSIS OF THE CONTENT

OF CHEMICAL COMPOUNDS FROM EXTRACTS OF HEXANE,
ACETONE, ETHANOL, AND WATER FROM AVOCADO LEAVES
(Persea americana Mill)
Harrizul Rivai1*, Liza Asbari Hasni2 and Zulharmita Zulharmita2
1
Faculty of Pharmacy, Andalas University, Limau Manih Campus, Padang 25163, Indonesia.
2
School of Pharmaceutical Science (STIFARM), Jl. Kurao Pagang, Siteba, Padang 25143,
Indonesia.
Article Received on
ABSTRACT
16 April 2019, Research on the qualitative and quantitative analysis of the content of
Revised on 06 May 2019,
Accepted on 27 May 2019 chemical compounds has been carried out on extracts of hexane,
DOI: 10.20959/wjpps20196-14049 acetone, ethanol, and water from avocado leaves (Persea americana
Mill). The purpose of this study was to determine the content and
*Corresponding Author levels of the chemical compounds contained in each extract. Extraction
Dr. Harrizul Rivai was carried out using two methods, namely maceration method for
Faculty of Pharmacy, hexane, acetone, ethanol and infusion methods for water solvents. The
Andalas University, Limau
results of the qualitative analysis showed that the hexane extract
Manih Campus, Padang
contained flavonoids and alkaloids. Acetone extract contains
25163, Indonesia.
flavonoids, phenols, tannins, and alkaloids. Ethanol extract contains
flavonoids, phenols, tannins, alkaloids, and saponins. Water extract contains flavonoids,
phenols, tannins, and alkaloids. The results of the quantitative analysis showed that the levels
of flavonoids in hexane, acetone, ethanol, and water extracts were 0.6%, 0.3%, 0.3%, and
0.5% respectively. The levels of phenol compounds in acetone, ethanol, and water extract
were 5.1%, 3.9%, and 3.7%, respectively. The levels of tannins in acetone, ethanol, and water
extracts were 0.3%, 0.2%, and 0.1% respectively. The levels of alkaloid compounds in
hexane, acetone, ethanol, and water extract were 0.1%, 13.6%, 15.6%; and 5.5%. The
saponin content in ethanol extract was 3.3%.
KEYWORDS: Analysis qualitative, analysis quantitative, avocado leaves, extraction,

gravimetry, spectrophotometry.
www.wjpps.com Vol 8, Issue 6, 2019. 149

INTRODUCTION
Avocado (Persea americana Mill) (Figure 1) is one of the plants that has benefits as
traditional medicine. Almost all parts of this plant have properties as a source of medicines,
including avocado leaves which can be used as herbs for the treatment of dyslipidemia.[1]
Therefore, it is necessary to determine the content of the chemical compounds found in
avocado leaves.
Figure 1: Trees and stems of avocados.
Avocado leaves have several chemical compounds that are very beneficial for health. Based
on previous research, Kamagate et al.[2] in the phytochemical screening test on methanol
extract of avocado leaves have proven the presence of saponin compounds, polyphenols,
flavonoids, alkaloids, sterols or polyterpenoids, and coumarin. Also, in the water extract of
avocado leaf, tannin content was found. Boadi et al.[3] have also identified the presence of
glycosides, alkaloids, tannins, saponins, flavonoids, terpenoids, and steroids from extracts of
methanol, chloroform, ethyl acetate, and petroleum ether from avocado leaves extracted by
the soxhletation method.
Quantitative analysis of the levels of total polyphenol compounds from each water extract,
ethanol extract, and methanol extract of avocado leaves was carried out using the colorimetric
method using Folin-Ciocalteu reagent. The results showed that the total polyphenol level
(gallic acid equivalent) was 2707.3 ± 155.4 µg/g for water extract, 2952.7 ± 166.0 µg/g for
ethanol extract, and 1873.1 ± 63.5 µg/g for methanol extract. The total flavonoid compounds
were determined by spectrophotometric methods and showed results of 0.543 ± 0.007%,
0.582 ± 0.012%, and 0.474 ± 0.007% from each water extract, ethanol extract, and methanol
extract of avocado leaves.[2]

The aqueous extract of Persea americana leaves produced a dose-dependent inhibition of

both phases of formalin pain test in mice, a reduction in mouse writhing induced by acetic
acid and an elevation of pain threshold in the hot plate test in mice. The extract also produced
a dose-dependent inhibition of carrageenan-induced rat paw edema. The results obtained
indicate that the extract possesses analgesic and anti-inflammatory effects.[4]
The leaves of avocado have been popularly used in the treatment of diabetes in countries in
Latin America and Africa. Aim of the study is to investigate the hypoglycaemic properties
and to determine the molecular mechanism by which the hydroalcoholic extract of the leaves
of avocado reduce blood glucose levels in streptozotocin (STZ)-induced diabetes in
rats via the enzymatic pathway of protein kinase B (PKB/Akt). The hydroalcoholic extract of
the leaves of avocado (0.15 and 0.3 g/kg/day), vehicle and metformin (0.5 g/kg/day) were
administered orally to STZ-diabetic rats (n = 7/group) for four weeks. Changes in body
weight, food and water intake, fasting glucose levels, and oral glucose tolerance were
evaluated. Phosphorylation and the expression of PKB in the liver and soleus muscle were
determined by Western blot. The hydroalcoholic extract of the leaves of avocado reduced
blood glucose levels and improved the metabolic state of the animals. Additionally, PKB
activation was observed in the liver and skeletal muscle of treated rats when compared with
untreated rats. The results indicate that the hydroalcoholic extract of the leaves of
avocado has anti-diabetic properties and possibly acts to regulate glucose uptake in liver and
muscles by way of PKB/Akt activation, restoring the intracellular energy balance.[5]
There are three primary races or groups of avocado: Mexican, Guatemalan, and West Indian
named for the areas where they were originally cultivated. The plant is used in traditional
medicine for the treatment of various ailments, such as menorrhagia, hypertension, stomach
ache, bronchitis, diarrhea, and diabetes. Peptone, b-galactoside, glycosylated abscisic acid,
alkaloids, cellulose, polyglactin urease, polyuronides, cytochrome P-450, and volatile oils are
reported to be present in this plant. Biotechnology approaches show that modified MS
medium supplemented with 1.0 mg benzyladenine/L, 0.1 mg Indole Butyric Acid/L, 0.1 mg
Gibberellic Acid 3/L was optimum for adventitious shoot development.[6]
Based on the description above, there has never been a qualitative and quantitative analysis of
the content of chemical compounds from extracts of hexane, acetone, ethanol, and water from
avocado leaves. Therefore, the authors are interested in researching the chemical content of
extracts of hexane, acetone, ethanol, and water from avocado leaves. This study aims to

determine what groups of chemical compounds are and what the levels of each group of
chemicals are in extracts of hexane, acetone, ethanol, and water from avocado leaves.
MATERIALS AND METHODS

Equipment
The equipment used in this study was the Ultraviolet-Visible double beam spectrophotometer
(Shimadzu 1800), sonicator (Branson 1800), rotary vaporizer (IKA), oven (Memmert),
ultraviolet light (Camag), maceration container (dark bottle), analytic scales (Precisa), and
laboratory equipment that is common for chemical analysis.
Chemicals
The material used in this research was dried avocado simplicia powder (Persea americana
Mill) purchased from PT Temu Kencono, hexane (EMSURE), acetone (Merck), ethanol
(EMSURE), distilled water (CV NOVALINDO), ammonia (EMSURE), TLC silica gel 60
F254 plate 20 × 20 cm (Merck), quercetin (Sigma), gallic acid (Sigma), catechins (Sigma), and
all reagents purchased from Merck: aluminum chloride, chloroform, methanol, ethyl acetate,
hydrochloric acid, α-naphthol, sulfuric acid, rhodamine B, zinc powder, magnesium powder,
boric acid powder, oxalic acid powder, iron (III) chloride, vanillin, gelatin, iodine, potassium
iodide, mercury (II) chloride, bismuth nitrate, nitric acid, ammonium molybdate, ether,
potassium permanganate, anhydride acetic acid, sodium hydroxide, n-butanol, diethyl ether,
pH paper.
PROCEDURES
Sample Preparation
Dry simplicia powder of avocado leaves (Persea americana Mill) was purchased from PT
Temu Kencono which is located at Jalan Koesbidjono Tjondrowibowo, Sumurejo Village,
RT / RW 01/03, Gunungpati, Semarang, Indonesia.
Standardization of avocado leaf simplicia

Standardization of avocado leaf simplicia was carried out by Indonesian Herbal
Pharmacopoeia Supplement I.[7]
Preparation of avocado leaf extract

Preparation of hexane, acetone, and ethanol extracts. Fifty grams of dry powder simplicia of
avocado leaves were put into the macerator, 500 mL of solvent was added (ratio 1:10 w / v).

The mixture is soaked for the first 6 hours while stirring occasionally, then let stand for 18
hours. Macerates are separated by filtration using flannel cloth. This extraction process is
repeated twice using the same type and amount of solvents. All macerates were collected,
then applied with a rotary evaporator at a temperature below ± 40ºC so that the liquid extract
of 500 mL was recovered.
Furthermore, the preparation of avocado leaf infusion was done by weighing as much as 50
grams of avocado simplicia and put in an infusion pan and added with 500 mL of distilled
water, then heated in a water bath for 15-20 minutes at 98ºC, then filtered using flannel cloth.
In the end, the infusion volume is sufficient to obtain 500 mL of avocado leaf extract.
Qualitative analysis of avocado leaf extract

Qualitative analysis of avocado leaf extract was carried out according to the method
described in Materia Medika Indonesia[8] and Hanani.[9]
Quantitative analysis of avocado leaf extract

1. Determination of total flavonoid levels
Determination of total flavonoid levels was carried out according to the method described in
the Indonesian Herbal Pharmacopoeia Supplement I.[7]
2. Determination of total phenol levels

Determination of total phenol levels was carried out according to the method described in the
Indonesian Herbal Pharmacopoeia Supplement I.[7]
3. Determination of total tannin levels

Determination of total tannin levels was carried out according to the method described in the
Indonesian Herbal Pharmacopoeia Supplement I.[7]
4. Determination of total saponin levels

Twenty mL of liquid extract was added with 100 mL of 20% ethanol in water, heated over a
water bath for 4 hours at 55°C while stirring occasionally, then filtering. The rest is returned
with 200 mL of 20% ethanol in water, filtered. The two extracts were combined, then
evaporated to 40 mL above the water bath at 90°C. Then the extract is put into a 250 mL
separating funnel plus 20 mL diethyl ether, then shaken vigorously. The water layer is stored,
the remaining diethyl ether layer is added with 20 mL diethyl ether, and shaken. Repeat until
the layer of water is clear. The water layer is combined, then 60 mL of n-butanol is added,

washed with 10 mL of 5% sodium chloride. The butanol layer was taken, then evaporated to
dryness at 60 °C. The remaining on drying was calculated as total saponin.[10]
RESULTS
Standardization of avocado leaf simplicia
Microscopic examination
Microscopic examination revealed the presence of upper epidermis and leaf bone, lower
epidermis with anisositic type stomata, closed hair and crystal idioblast, lower epidermis,
mesophyll and oil glands, and mesophyll, oil cells, and transporting files (Figure 2).
Thin layer chromatography pattern of avocado leaf simplicia

Rf values obtained from thin layer chromatography profiles of avocado leaf simplicia are as
follows: Comparative Rf (Quercetin) = 0.35; Rf 1 = 0.10; Rf 2 = 0.20; Rf 3 = 0.34; Rf 4 =
0.54; Rf 5 = 0.64; Rf 6 = 0.80 (Figure 3).
Upper epidermis and leaf bone Lower epidermis with anisocitic type
stomata
Hair cover and crystal idioblasts Upper epidermis, mesophyll, and oil
glands

Mesophyll, oil cell, and transport file.

Figure 2: Microscopic photo of simplicia of avocado leaves.
S Q
Figure 3: Thin layer chromatography profile of avocado leaf simplicia.
Information
S = Avocado leaf sample
Q = Comparison of quercetin
Stationary phase: Silica gel F254
Mobile phase: Chloroform-methanol-water (80: 12: 2)
Detection: Aluminum chloride and UV366
Loss on drying
The result is 8.6431% ± 0.0299% which meets the standard found in Indonesian Herbal
Pharmacopoeia Supplement I[7], where the shrinkage value of drying is not more than 10%.

 Total ash content

The results are 3.3164% ± 0.0040%, which meets the standards contained in the Indonesian
Herbal Pharmacopoeia Supplement I[7], where the total ash content value is not more than
4.2%.
 Levels of insoluble acid ash

The results are 0.4011% ± 0.1358%, which meets the standard found in Indonesian Herbal
Pharmacopoeia Supplement I[7], where the value of insoluble acid ash is not more than 1.1%.
 Water-soluble extract levels

The result is 22,3791% ± 0,0221%, which meets the standard found in Indonesian Herbal
Pharmacopoeia Supplement I[7], where the value of water-soluble extract content is not less
than 20.2%.
 Levels of soluble extract in ethanol

The result is 19.7912% ± 0.1109% that meets the standard found in Indonesian Herbal
Pharmacopoeia Supplement I[7], where the value of ethanol soluble extract content is not less
than 18.9%.
Qualitative analysis
The results of the qualitative analysis of hexane, acetone, ethanol, and water extract from
avocado leaves are shown in Table 1.

Table 1: Qualitative test results from extracts of hexane, acetone, ethanol, and water
from avocado leaf simplicia.
Chemical Extract of
Reagent
Content Hexane Acetone Ethanol Water
Asam Amino 0.1% Ninhydrin-acetone (-) (-) (-) (-)
Fehling
(-) (-) (-) (-)
Carbohydrate (A & B)
Molisch (-) (-) (-) (-)
25% sulfuric acid (-) (-) (-) (-)
Fatty acid
Rhodamin B (-) (-) (-) (-)
Zinc powder + (negative 3- (negative 3- (negative 3-
(negative 3-flavonol
concentrated hydrochloric flavonol flavonol flavonol
glycosides)
acid glycosides) glycosides) glycosides)
Mg powder + (+) (Contains
Flavonoids concentrated hydrochloric (-) (-) (-) flavones, chalcones
acid and auron)
Samples + acetone +
boric acid + oxalic acid + (+) (+) (+) (-)
ether, observe UV 366
Iron (III) chloride
(-) (+) (+) (+)
solution
Vanilin - concentrated
Phenolic (-) (+) (+) (+)
sulfuric acid
compounds
Vanilin - concentrated
(-) (+) (+) (+)
hydrochloric acid
Folin Ciocalteu (-) (+) (+) (+)
Iron (III) chloride
(-) (+) (+) (+)
Tannins solution
Gelatine solution (-) (+) (+) (+)
Bouchardat (-) (+) (+) (+)
Mayer (-) (+) (+) (-)
Alkaloids Wagner (-) (+) (+) (+)
Dragendroff (-) (+) (+) (-)
Hager (-) (+) (+) (-)
Samples + 2N
Anthraquinone hydrochloric acid, heated (-) (-) (-) (-)
+ ether + ammonia 6N
Essential oil Potassium permanganate (-) (-) (-) (-)
Acetic anhydride (-) (-) (-) (-)
Saponin Water (-) (-) (+) (-)
Cardiac Baljet (-) (-) (-) (-)
Glycosides Keller – Killiani (-) (-) (-) (-)
Quantitative analysis
The results of the determination of secondary metabolites in extracts from hexane, acetone,
ethanol, and water from avocado leaves are shown in Table 2.

Table 2: Levels of secondary metabolites in extracts from hexane, acetone, ethanol, and
water from avocado leaves.
Secondary metabolites
Extract type Flavonoids Phenolics Tannins Alkaloids Saponin
(%) (%) (%) (%) (%)
Hexane 0.6 - - 0.1 -
Acetone 0.3 5.1 0/3 1.36 -
Ethanol 0.3 3.9 0.2 1.56 3.3
Water 0.5 3.7 0.1 0.55 -
DISCUSSION
Simplicia
Samples that have been obtained are then tested for simplicia which aims to obtain good
quality simplicia, which includes: Microscopic examination, thin layer chromatography
patterns, shrinkage drying, total ash content, levels of insoluble acid ash, water-soluble
extract content, and level of the soluble extract in ethanol. These results meet the standards
contained in Indonesian Herbal Pharmacopoeia.[7]
Preparation of extracts
Samples that have met the standardization requirements for simplicia are then weighed as
much as 50 grams using analytical scales to be used as liquid extracts. The extract is made by
maceration by soaking the simplicia in a solvent at room temperature so that damage or
degradation of metabolites can be minimized. At maceration, the concentration equilibrium
occurs so that it is necessary to replace the solvent repeatedly and with shaking.[9]
The weighed simplicia was put in a dark glass bottle, added 500 mL hexane solvent, soaked
for 6 hours while stirring occasionally and left for 18 hours. Then the mixture was filtered
and repeated two times with the same type and amount of solvent. Then the maserate is
collected and then evaporated with a rotary evaporator at a temperature of ± 40ºC. This
evaporation aims to vaporize the solvent so that a liquid extract is obtained. The same is done
for acetone and ethanol solvents.
For water solvents, the avocado leaf extract is made using the infusion method. The infusion
method is an extraction method using water solvents, at a temperature of 96-98 ºC for 15-20
minutes (calculated after the temperature of 96 ºC is reached).[9] Simplicia is weighed as
much as 50 grams and then put in an infusion pan plus water solvent, then put in a water bath,
heated for 15-20 minutes at 98ºC, then filtered with a flannel cloth to obtain water extract.

Qualitative analysis
After obtaining the liquid extract from hexane, acetone, ethanol, and water from avocado
leaves, the compound content of each liquid extract was then analyzed. Analysis of
compound content was carried out to identify the content of primary metabolites and
secondary metabolites present in plants with a qualitative phytochemical test method. There
are eleven metabolite tests carried out, including testing of amino acids, carbohydrates, fatty
acids, flavonoids, alkaloids, phenols, tannins, anthraquinones, essential oils, saponins, and
cardiac glycosides (Table 1).
Amino acid tests that have been carried out on hexane, acetone, ethanol, and water extract
from avocado leaves with a solution of 0.1% ninhydrin-acetone gave negative results to the
extract. There is no purple to a grayish blue appearance on the extract which shows the
absence of amino acids. Yellow does not appear on the extract, which indicates the absence
of proline amino acids. This experiment proves that the absence of amino acids in all four
avocado leaf extracts.
The carbohydrate test carried out on hexane, acetone, ethanol, and water from avocado leaves
with Fehling's solution gave negative results to the extract. There is no appearance of red
brick copper paste on the extract, which shows the absence of carbohydrates. Then the tests
carried out with the Molish reaction also gave negative results on hexane, acetone, ethanol,
and water from avocado leaves. The purple ring does not appear on both fluid boundaries
when the addition of sulfuric acid drop by drop through the tube wall. This experiment proves
that the absence of carbohydrates in all four avocado leaf extracts.
The fatty acid test carried out on hexane extract, acetone, ethanol, and water from avocado
leaves with 25% sulfuric acid solution gave negative results to the extract. The absence of
light brown color on the extract showed no fatty acids, and red and bright red also did not
appear on the extract, which showed the absence of glycolipid and sulfolipid fatty acids. Then
the fatty acid test was also carried out with a 0.5% rhodamine B solution in ethanol, which
gave negative results to hexane, acetone, ethanol, and water from avocado leaves.
Flavonoid tests that have been carried out on hexane, acetone, ethanol, and avocado leaves
using the methods stated in Materia Medika Indonesia.[8] Method 1 gives negative results on
hexane, acetone, ethanol, and water from avocado leaves. Intense red does not appear in the
extract, which shows the absence of flavonoids of 3-flavonol glycosides. Method II, with the

addition of magnesium and hydrochloric powder in flavonoid testing, will cause the reduction
of existing flavonoid compounds to give rise to a red reaction, which is a feature of
flavonoids. However, in this test, magnesium powder did not give a reduction reaction to
flavonoid compounds so that the test solution did not provide color changes to the extract
which showed no flavonoids. However, in this test, an orange color appears on the extract of
water from avocado leaves, which gives a positive result. These results indicate the presence
of flavonoids of flavones, chalcones, and aurones. However, the hexane, acetone, and ethanol
extract from avocado leaves did not appear in orange, which showed no flavonoids of
flavones, chalcone, and aurone types. Method III gives positive results on hexane extract,
acetone, and ethanol from avocado leaves. In the solution of avocado leaf extract,
fluorescence appears when observed with an ultraviolet light wavelength of 366 nm, which
indicates the presence of flavonoids. However, the water extract from avocado leaves did not
fluoresce, indicating the absence of flavonoids.
Phenol test with iron (III) chloride solution gave positive results on acetone, ethanol, and
water from avocado leaves. Color changes occur because of the presence of hydroxyl groups
in phenol compounds extracted with acetone, ethanol, and water solvents. The appearance of
green to blue-black on the extract indicates the presence of phenol. However, the hexane
extract from avocado leaves does not appear green to black blue, which shows the absence of
phenol.
Phenol test with color reagent using vanillin solution in concentrated sulfuric acid gave
positive results on acetone, ethanol, and water from avocado leaves. The color change in the
extract indicates the presence of phenol. However, there was no change in the hexane extract
from avocado leaves that showed no phenol.
Phenol testing with vanillin solution in concentrated hydrochloric acid gave positive results
on acetone, ethanol, and water extracts of avocado leaves. The color change in the extract
with this reagent shows the presence of phenol. However, there was no change in the hexane
extract from avocado leaves that showed no phenol.
Phenol test with Folin-Ciocalteu reaction gave positive results on acetone, ethanol, and water
from avocado leaves. The color change in the extract with this reagent shows the presence of
phenol. However, there was no color change in hexane extract that showed the absence of
phenol.

The tannin test using iron (III) chloride salt solution gave positive results to acetone, ethanol,
and water from avocado leaves. Color changes occur due to the presence of hydroxyl groups
present in tannin compounds so that blue-colored deposits appear showing the presence of
gallotannin and ellagitannin. However, hexane extract does not appear in blue or brown-
brown color deposits showing no gallotannin and ellagitannin or condensed tannins.
Test of tannin with 1% gelatin solution in 10% sodium hydroxide gave positive results on
acetone, ethanol, and water from avocado leaves. The appearance of deposits in the extract
shows the presence of tannins. However, hexane extract does not cause deposits with these
reagents, which show the absence of tannins.
The alkaloid test using the Bouchardat reaction gave positive results on acetone extract,
ethanol extract, and extracts of water from avocado leaves. The appearance of deposits in the
extract indicates the presence of alkaloids. However, in hexane extract, there is no precipitate
which shows the absence of alkaloids. The alkaloid test with Mayer reagent gave positive
results on acetone extract and ethanol from avocado leaves. The nitrogen in the alkaloid will
react with potassium metal ions from potassium tetraiodomercurate(II) to form a potassium-
alkaloid complex that settles indicating the presence of alkaloids. However, none of the
hexane extracts and water from avocado leaves appeared which showed no alkaloids. The
alkaloid test with Wagner reagent gave positive results on acetone, ethanol, and water from
avocado leaves. Potassium metal ions will form covalent bonds with nitrogen in the alkaloid
to form a potassium-alkaloid complex that settles indicating the presence of alkaloids.
However, no water deposits from avocado leaves appeared, which showed no alkaloids.
Alkaloid test with Dragendroff reagent gave positive results on acetone and ethanol extract
from avocado leaves. Nitrogen is used to form coordinate covalent bonds with potassium ions
to form orange deposits in extracts which indicate the presence of alkaloids. However, none
of the hexane extracts and water from avocado leaves appeared which showed no alkaloids.
The alkaloid test with Hager reagent gave positive results on acetone and ethanol extract. The
appearance of deposits in extracts shows the presence of alkaloids. However, there were no
sediments in hexane extract and water from avocado leaves, which showed no alkaloids.
Anthraquinone testing using 2 N hydrochloric acid gave negative results to all four avocado
leaf extracts. No appearance of red on all four avocado leaf extracts proves that there is no
anthraquinone,

Test of essential oil using potassium permanganate solution gave negative results on all four
avocado leaf extracts. The color of potassium permanganate does not turn pale or disappear
when added to avocado leaf extract, which shows no essential oil in the four extracts. The
essential oil test was also carried out with acetic anhydride, which gave negative results to all
four avocado leaf extracts. There was no blue-green on the four avocado leaf extracts which
showed no essential oil. This test proves that the absence of essential oils in the four avocado
leaf extracts.
Saponin test by doing the froth test (foam test) gave positive results on the ethanol extract of
avocado leaves. The formation of a stable froth as high as 1.5 cm for 10 minutes after adding
2 N hydrochloric acid showed the presence of saponins. Whereas in hexane, acetone, and
water extracts, there was no solid foam which showed no saponin.
Test of cardiac glycosides using the Baljet reaction gave negative results on all four avocado
leaf extracts. No appearance of orange color on all four avocado leaf extracts showed no
cardiac glycosides of the type cardenolide aglycone. The cardiac glycoside test was also
carried out with the Keller-Kiliani reaction, which gave negative results to all four avocado
leaf extracts. There was no blue-green ring on the four avocado leaf extracts which showed
no cardiac glycosides. This test proves that the absence of cardiac glycosides in all four
avocado leaf extracts.
Quantitative analysis
Determination of total flavonoid content from hexane, acetone, ethanol, and water extract
from avocado leaves was carried out using the aluminum chloride colorimetric method by
Ultraviolet-Visible spectrophotometry, which was calculated as quercetin. The principle of
determining the levels of flavonoids by the method of aluminum chloride is a measurement
based on the formation of a colored complex between aluminum chloride with a keto group
on C-4 atoms and hydroxy groups on C-3 or C-5 atoms on flavonoids.
Aluminum chloride is used to provide a batochromic effect by shifting towards a longer

wavelength, thus changing quercetin wavelengths to a range of visible wavelengths. Also,
aluminum chloride also produces a hyperchromic effect that causes an increase in the
intensity of quercetin solutions. The maximum wavelength generated from the quercetin
measurement is 440 nm (Figure 4).

Figure 4: Spectrum of 60 ppm quercetin solution in 80% ethanol with aluminum

chloride reagent.
Based on the calibration curve in Figure 5, the regression equation is y = 0.007x + 0.111,
with the correlation coefficient R = 0.999. The total flavonoid content in each extract was
0.6% in hexane extract, 0.3% in acetone extract, 0.3% in ethanol extract, and 0.5% in water
extract (Table 2).
Figure 5: Calibration curve of quercetin solution in 80% ethanol with aluminum

chloride reagent at a maximum wavelength of 440 nm.
Determination of total phenol levels was carried out using Ultraviolet-Visible

spectrophotometry using the Folin-Ciocalteau method. Folin-Ciocalteu reagent is used
because phenolic compounds can react with this reagent to form a blue solution that can be
measured absorbance at a maximum wavelength of 749 nm (Figure 6).

Figure 6: Spectrum of 50 ppm gallic acid solution in methanol with reagent Folin
Ciocalteu and sodium hydroxide.
Phenolic compounds react with Folin-Ciocalteu reagents only in alkaline conditions so that
protons dissociate in phenolic compounds into phenolic ions. A 1% sodium hydroxide
solution is used to make alkaline conditions. For total phenol content, gallic acid is used as a
comparison.
Based on the calibration curve in Figure 7, the regression equation obtained is y = 0.010x -
0.030 with the correlation coefficient R = 0.999. The total phenol content in each extract was
5.1% in acetone extract, 3.9% in ethanol extract, and 3.7% in water extract (Table 2).
Figure 7: Calibration curve of gallic acid solution in methanol with the reagent of Folin
Ciocalteu and sodium hydroxide at a maximum wavelength of 749 nm.

Determination of total tannin was carried out using Ultraviolet-Visible spectrophotometry

with a comparison of catechins. The diluted extract is inserted into the sonicator tool for 5
minutes. The measurement results of the absorption of the comparison solution at a maximum
wavelength of 280 nm were 0.513 (Figure 8).
Figure 8: The spectrum of the solution for catechins in ethyl acetate.
The measurement results of the absorbance of acetone extract were 0.830, and the tannin
level was 0.3%. The absorbance of the ethanol extract is 0.489, and the tannin content is
0.2%. The absorbance of the water extract is 0.138, and the tannin content is 0.1% (Table 2).
Determination of total alkaloid levels was carried out by the gravimetric method. Gravimetric
method is one method of determining quantitatively by measuring the weight of components
in a pure state after going through the separation process. Ammonia is used to release
alkaloid bonds with the acid so that the alkaloid is back in a free state. Two layers will be
formed, namely the acid layer and the chloroform layer. Ammonia will react with
hydrochloric acid and form salts that are soluble in water while alkaloids return in basic form
and are insoluble in water but readily soluble in chloroform. Free alkaloids can be extracted
with chloroform, resulting in chloroform extract, which is a total alkaloid. The total alkaloid
content of hexane extract is 0.1%, acetone extract is 1.36%, ethanol extract is 1.56%, and
water extract is 0.55 % (Table 2).
Determination of saponin levels was carried out by the gravimetric method. The gravimetric
method is one method of determining qualitatively by measuring the weight of components in

a pure state after going through the separation process. Extract was extracted with 20%
ethanol in water. Ethanol is a universal solvent that can attract most of the chemical
compounds, while water is used to attract saponins because saponins are readily soluble in
water and insoluble in ether. The results of determining the total saponin content in ethanol
extract were 3.3% (Table 2).
CONCLUSION
The chemical compounds of hexane extract from avocado leaves are flavonoids and
alkaloids. The chemical compounds of acetone extract are flavonoids, phenols, tannins, and
alkaloids. The chemical compounds of ethanol extract are flavonoids, phenols, tannins,
alkaloids, and saponins. The chemical compounds of water extract are flavonoids, phenols,
tannins, and alkaloids. The total flavonoid content in hexane extract from avocado leaves was
0.6%, the acetone extract was 0.3%, the ethanol extract was 0.3%, and the water extract was
0.5%. Total phenol content in acetone extract was 5.1%, ethanol extract was 3.9%, and water
extract was 3.7%. The total tannin content in acetone extract was 0.3%, ethanol extract was
0.2%, and water extract was 0.1%. The total alkaloid content in hexane extract was 0.1%,
acetone extract 1.36%, ethanol extract 1.56%, and water extract 0.55%. The total saponin
content in ethanol extract was 3.3%.
ACKNOWLEDGMENTS
The authors express their gratitude to the Dean of the Faculty of Pharmacy, Andalas
University and Head of the School of Pharmaceutical Science (STIFARM), Padang for their
assistance in using laboratory equipment in this study.
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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Rivai et al. World Journal of Pharmacy and Pharmaceutical Sciences

Volume 8, Issue 6, 149-167 Research Article ISSN 2278 – 4357

QUALITATIVE AND QUANTITATIVE ANALYSIS OF THE CONTENT

Harrizul Rivai1*, Liza Asbari Hasni2 and Zulharmita Zulharmita2

KEYWORDS: Analysis qualitative, analysis quantitative, avocado leaves, extraction,

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Figure 1: Trees and stems of avocados.

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The aqueous extract of Persea americana leaves produced a dose-dependent inhibition of

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MATERIALS AND METHODS

Standardization of avocado leaf simplicia

Preparation of avocado leaf extract

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Qualitative analysis of avocado leaf extract

Quantitative analysis of avocado leaf extract

2. Determination of total phenol levels

3. Determination of total tannin levels

4. Determination of total saponin levels

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Thin layer chromatography pattern of avocado leaf simplicia

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Mesophyll, oil cell, and transport file.

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 Total ash content

 Levels of insoluble acid ash

 Water-soluble extract levels

 Levels of soluble extract in ethanol

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Aluminum chloride is used to provide a batochromic effect by shifting towards a longer

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Figure 4: Spectrum of 60 ppm quercetin solution in 80% ethanol with aluminum

Figure 5: Calibration curve of quercetin solution in 80% ethanol with aluminum

Determination of total phenol levels was carried out using Ultraviolet-Visible

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Determination of total tannin was carried out using Ultraviolet-Visible spectrophotometry

Figure 8: The spectrum of the solution for catechins in ethyl acetate.

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