Journal of Medicinal Plants Research Vol. 6(12), pp.

2289-2294, 30 March, 2012

Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.201
ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Phytochemicaland proximate analyses and thin layer

chromatography fingerprinting of the aerial part of
Chenopodium ambrosioides Linn. (Chenopodiaceae)
Okhale, Samuel Ehiabhi1*, Egharevba, Henry Omoregie1, Ona, Eneyi Comfort1,2 and
Kunle, Oluyemisi Folashade1,3
1
Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and
Development (NIPRD), Idu Industrial Area, Idu, P. M. B. 21 Garki, Abuja, Nigeria.
2
Department of Chemistry, Ahmadu Bello University, Zaria, Nigeria.
3
Department of Pharmacognosy, Faculty of Pharmacy, University of Jos, Nigeria.
Accepted 1 February, 2012

The aerial part of Chenopodium ambrosioides L., reputable for the treatment of malaria and diabetes in
Nigeria, was qualitatively screened for the presence of secondary metabolites using standard methods.
Some proximate parameters were also determined. The result of the phytochemical screening revealed
the presence of alkaloids, tannins, saponins, flavonoids, terpenes, sterols, cardenolide aglycone,
volatile oils and carbohydrates. The proximate analysis revealed moisture content of 10.90%, total ash
value of 14.65%, acid-insoluble ash value of 3.05%, water-soluble ash of 6.25%, water-soluble extractive
value of 3.18% and alcohol-soluble extractive value of 13.20%. The thin layer chromatography
fingerprinting and phytochemical screening revealed several chemical components, which could be
isolated from the plant. This study shows that C. ambrosioides is a potential drug plant considering the
rich phytochemical and proximate pharmacognostic profile of the aerial part, hence its folkloric uses.
The result of this study is the first of its kind on the Nigerian species of this plant drug, and is
informative for standardization and monograph development of this herbal plant.

Key words: Chenopodium ambrosioides, secondary metabolites, thin layer chromatography, pharmacognostic
analysis, antimalaria, Mexican tea, ash value.

INTRODUCTION

Chenopodium ambrosioides L. also known as epazote,
Mexican tea, and wormseed, belongs to the family
Chenopodiaceae. The species is wide-spread and
originates from tropical America (Munz, 1975). C.
ambrosioides is an annual or short-lived perennial herb
that grows to over 1 m high, with aromatic glandular
hairs. It has a strong rank smell when bruised and the
leaves have a pungent smell (Burkill, 1985).
Chenopodium species have a wide variety of medicinal
properties (Burkill, 1985; Tapondjou et al., 2002). In
*Corresponding author. E-mail: samokhale@yahoo.com.
Spain, the dry or fresh aerial part of C. ambrosioides is
boiled in water and drunk after meals as digestifs,
stomachics, and in some cases as hemostatic, stimulant,
laxative and antidiarrhoeic. It is also used to reduce blood
pressure and to treat colds and fibroids (Filipoy, 1994). It
has been used for centuries as condiment, traditional
antihelmintic and antimalarial (Ruffa et al., 2002). The
plant is sometimes cultivated principally for medicinal
uses in West Africa where the leaves are added as
flavorings to soup, pounded leaves are applied to sores,
or to swellings on the body and to areas of pain. The
aromatic smell is inhaled for headache. The plant is used
for the treatment of diabetes. In Nigeria, the whole plant
is pounded and eaten as a laxative, and its infusion is
2290 J. Med. Plants Res.
used as febrifuge, as well as to treat coughs and
tuberculosis (Burkill, 1985). A decoction of the aerial part
is used in Northern Nigeria as an antimalarial.
In view of the enormous bioactivity profile and the
documented ethnomedicinal uses of C. ambrosioides,
this study aims to establish its phytochemical,
pharmacognostic and thin layer chromatographic
characteristics that could be useful for standardization
and monograph development of this potential drug plant.
MATERIALS AND METHODS
All chemicals used were of analytical reagent grade (Sigma) and
used as supplied.
Plant materials and processing
Plant material was collected by Mallam Muazzam Ibrahim from
Chaza in Suleja, Niger state, Nigeria. The plant was identified and
authenticated at the Herbarium of the National Institute for
Pharmaceutical Research and Development, Abuja, Nigeria. The
aerial part of the plant was air-dried for three weeks at room
temperature, pulverized and used immediately for analyses.
Phytochemical screening
The phytochemical and proximate analyses of the powdered aerial
part of the plant were performed using the methods described by
Harborne (1998), MHFW (1999), Evans (2002) and Sofowora
(2008). The analyses were carried out as thus explained.
Test for cardenolide aglycone
0.5 g of the powdered sample was boiled with 10 ml of 95% alcohol
for 2 min. The mixture was filtered; the filtrate was allowed to cool
and diluted with water to 10 ml. Three drops of a saturated solution
of lead sub-acetate were added and filtered thoroughly. The filtrate
was treated with 1 ml of 2% solution of 3, 5-dintrobenzoic acid in
95% alcohol and the solution basified with 5% sodium hydroxide. A
purple-blue colour indicates the presence of free or combined
cardenolide aglycone (Sofowora, 2008).
Test for terpenes and sterols
5 g of the powdered sample was extracted by maceration with 50
ml of chloroform. The extract was filtered and evaporated to
dryness on water bath. The residue was dissolved in 10 ml of
anhydrous chloroform and filtered. The filtrate was divided into two
equal portions and used for the following tests:
Lieberman-Burchard test for terpenes: To the first portion of the
chloroform solution was added to 1 ml of acetic anhydride and
shaken. Then 1 ml of concentrated sulphuric acid was added down
the wall of the test tube to form a layer underneath. The formation
of a reddish-violet colour at the lower layer indicates the presence
of terpenes (Sofowora, 2008).
Salkowskis test for sterols: The second portion of the chloroform
solution was carefully mixed with 2 ml of concentrated sulphuric
acid so that the sulphuric acid formed a lower layer. A reddish-
brown colour at the interface indicates the presence of a steroidal
ring.
Test for saponins
0.5 g of the powdered plant material was added to 5 ml of 95%
ethanol and boiled for 2 min; filtered into a test tube and diluted to
10 ml with distilled water. The test tube was shaken vigorously for 1
min. The formation of a persisting honey comb indicates the
presence of saponins.
Test for tannins and phlobatannins
Ferric chloride test for tannins: 10 ml of distilled water was added
to 1 g of the powdered sample in a test tube and boiled for 3 min in
a water bath. The mixture was allowed to cool and then filtered with
Whatman No.1 filter paper. 1 ml of the filtrate was diluted with 4 ml
of distilled water and few drops of 10% ferric chloride were added.
Instant formation of blue-black or green coloured solution indicates
the presence of tannins (Evans, 2002).
Test for Phlobatannins: 10 ml of distilled water was added to 1g of
the powdered sample in a test tube and boiled for 3 min in a water
bath. The mixture was allowed to cool and then filtered with
Whatman No. 1 filter paper. 1 ml of the filtrate was boiled with equal
volume of 1% v/v hydrochloric acid. The formation of a red
precipitate indicates the presence of phlobatannins (Evans, 2002).
Test for anthraquinone (Borntragers tests)
0.5 g of the powdered sample was poured into a dried test tube and
10 ml of chloroform added. The mixture was shaken for 5 min and
filtered. The filtrate was shaken with 10% w/v potassium hydroxide
solution. A bright pink-red colour in the upper aqueous layer
indicates the presence of free anthraquinones (Evans, 2002).
Test for balsams
1 g of the powdered sample in a test tube was extracted with 10 ml
of 90% v/v ethanol and filtered. Two drops of 10% (w/v) alcoholic
ferric chloride solution was added to 5 ml 5 of the filtrate. Formation
of a dark green coloured solution indicates the presence of
balsams.
Tests for resins
0.5 g of the pulverized plant was extracted with 15 ml petroleum
ether and filtered. 5 ml of the filtrate was dispensed into a test tube
and shaken vigorously with equal volume of copper acetate solution
TS. The mixture was allowed to stand for a few minutes. Formation
of a green coloured solution indicates the presence of resins.
Test for alkaloids
3 g of the powdered sample was macerated with 50 ml of methanol
at room temperature and filtrate evaporated to dryness on a hot
water bath without overheating. 10 ml of 1% v/v HCL was added to
the residue. The solution was divided equally into five test tubes,
and two drops of the following reagents were added to the
respective test tubes: Mayers reagent (potassium mercuric iodide
 Okhale et al. 2291

Table 1. Result of phytochemical screening of the aerial Extraction and thin layer chromatographic fingerprinting
part of C. ambrosioides.
Successive extraction of 1 g powdered aerial part was carried out
Phytochemical constituents Result with n-hexane (10 ml 2); ethyl acetate (10 ml 2); and methanol
(10 ml 2) at room temperature (27 to 30C) for 24 h. The extract
Cardenolide aglycone + from each solvent was vacuum filtered with Whatman No. 1 filter
Terpenes + paper, and the filtrates evaporated to dryness in the fume hood at
Sterols + room temperature. The yield for each extract was determined. Then
0.05 g each of the extracts was reconstituted in 5 ml of their
Saponins +
respective solvent of extraction and spotted on glass TLC plate
Tannins + precoated with silica gel 60. The plate was previously activated at
Anthraquinones ND 105C for 2 h. The plates were developed using a mobile phase
Balsams ND comprising n-hexane and ethyl acetate (3: 2). The plates were
observed in daylight and under UV at 365 nm. Visible spot were
Resins ND
marked. The plates were then sprayed with a solution of 1% (w/v)
Alkaloids + vanillin in sulphuric acid and heated in oven at 110C for 3 min. The
Phlobatannins ND coloured spots revealed after spraying were marked. The
Flavonoids + retardation factors (Rf) of all components detected were then
computed.
Phenols +
Volatile oil +
+: Detected; ND: not detected. RESULTS AND DISCUSSION

solution); Dragendorffs reagent - (potassium bismuth iodide
solution); Wagners reagent (solution of iodine in potassium
iodide); Hagers reagent (a saturated solution of picric acid); and
10% tannic acid solution. The formation of amorphous or crystalline
precipitates or coloured precipitate in at least 3 or all of these tests
indicates the presence of alkaloids.
Test for flavonoids
Lead acetate test: 5 g of the powdered sample was detanned by
wetting it with acetone, and the acetone was completely evaporated
on a hot water bath. The residue was extracted with 20 ml of warm
distilled water and filtered. 5 ml of the filtrate in a test tube was
added two drops of 10% (w/v) lead acetate solution. Formation of a
coloured precipitate indicates the presence of flavonoids.
Sodium hydroxide test: To 5 ml of the filtrate from above equal
volume of 10% (w/v) sodium hydroxide solution was added.
Formation of yellow coloured solution indicates the presence of
flavonoids.
Test for volatile oil
0.5 g of powdered sample was shaken with 1 ml of 0.1 M sodium
hydroxide solution and 1% aqueous hydrochloric acid. The
formation of a white precipitate indicates the presence of volatile oil.
Determination of water-soluble ash
Water soluble ash was determined as reported in MHFW (1999),
with slight modification. Briefly, total ash was determined. The total
ash was boiled for 5 min with 25 ml of distilled water; the insoluble
matter was collect on an ashless filter paper, washed with hot
distilled water, and ignited for 15 minutes at a temperature not
exceeding 450C. The weight of the insoluble matter was
subtracted from the weight of the total ash; the difference in weight
represents the water-soluble ash. The percentage of the water-
soluble ash was calculated with reference to the air-dried powdered
plant sample.
Phytochemical screening
The results of the phytochemical screening of the aerial
part of Chenopodium ambrosioides collected from
Northern Nigeria are presented in Table 1. Nine major
classes of secondary metabolites were detected, namely
cardenolide aglycone, terpenes, sterols, saponins,
tannins, alkaloids, flavonoids, phenols and volatile oil.
C. ambrosioides has been shown to exhibit antifungal,
antihelminthic, anticatarrhal, antibacterial, antiviral,
insecticidal, nematicidal and allelopathic activities (Verma
et al., 1983; Dubeyand Kishore, 1987; Peterson et al.,
1989; Begum et al., 1993; Kishore et al., 1993; Hegazy
and Farrag, 2007; Valery et al., 2008). The antihelminthic
and anticatarrhal activities have been attributed to the
presence of some bioactive compounds shown in Figure
1 such as ascaridole (1), isoascaridole (2), -terpinene
(3) and ascaridole glycol (4) (Valery et al., 2008).
Ascaridole has been reported to possess sedative, pain-
relieving and antifungal properties (Okuyama et al., 1993;
Pare et al., 1993). It has also been reported to exhibit
antimalarial and antiparasitic activities. Pollack et al.
(1990) reported its inhibition of the in vitro development of
Plasmodium falciparum, while its activities against
Trypanosoma cruzi and Leishmania amazonensis were
reported by Kiuchi et al. (2002) and Monzote et al.
(2006), respectively. Ascaridole was also reported to
exhibit in vitro activity against different tumor cell lines
(CCRF-CEM, HL60, and MDA-MB-231) (Valery et al.,
2008).
Our finding is in agreement with previously reported
work of Hegazy and Farrag, (2007), who reported the
presence of sterols and terpenes in the Egyptian species,
and Onocha et al. (1999), who reported the presence of
essential oil in Chenopodium ambrosioides collected from
Western Nigeria. Several other secondary metabolites
have been previously isolated from Chenopodium
2292 J. Med. Plants Res.

O
O

O
1
2

OH

O
OH

4
3
Figure 1. Chemical structures of some bioactive
compounds of Chenopodium ambrosioides.

Table 2. Result of proximate analysis of the aerial part of C.

ambrosioides.

Parameter Values (%)*

Moisture content 10.90 0.05
Total ash value 14.65 0.3
Acid-insoluble ash value 3.05 0.05
Water-soluble ash value 6.25 0.02
Alcohol-soluble extractive value 13.20 0.06
Water-soluble extractive value 3.18 0.03
* Each value in the table was obtained by calculating the average of
three experiments SEM.

ambrosioides, such as alkaloids (Haseeb et al., 1978),
saponins (Gupta and Behari, 1972) and flavonoids (Jain
et al., 1990).
Proximate analysis
The proximate analysis (Table 2) revealed moisture
content of 10.90%, which is within the acceptable limits of
about 6 to 15% for most vegetable drugs (Kunle, 2000).
Low moisture content reduces errors in the estimation of
the actual weight of drug material, reduces components
hydrolysis by reducing the activities of hydrolytic
enzymes which may destroy the active components, and
also reduces the proliferation of microbial colonies and
therefore minimize the chance of spoilage due to
microbial attack (Shellard, 1958). Total ash value of
14.65%, though high, is within range given for some
official drugs such as citrus leaf (7.0%), neem leaf
(11.6%) and atropa leaf (16%) (Kunle, 2000). The total
ash value is a diagnostic purity index. It represents the
physiological ash and non-physiological ash.
Physiological ash is the ash inherent in the plant due to
biochemical processes and the non-physiological is
contaminants from the environment. These may be
carbonates, phosphates, nitrates, sulphates, chlorides
 Okhale et al. 2293

Table 3. Result of thin layer chromatography fingerprint of n- chromatogram showed eight spots and seven spots for
hexane extract of aerial part of C. ambrosioides. the hexane and ethyl acetate extracts, respectively.
Spot Rf value Color/visualization
1 0.15 Pink/vanillin spray Conclusion
2 0.31 Purple/vanillin spray
3 0.49 Purple/vanillin spray Nigerian species of C. ambrosioides is rich in
4 0.65 Pink/vanillin spray phytochemicals, some of which may be attributed to its
5 0.70 Light green/daylight; pink/UV 365 nm ethnomedicinal uses for management of coughs,
6 0.81 Green/daylight; pink/UV 365 nm tuberculosis, diabetes, and as antimalarial. The presence
7 0.89 Deep pink/vanillin spray of some secondary metabolites like alkaloids, flavonoids,
8 0.93 Deep pink/vanillin spray saponins, tannins, phenols, terpenes and sterols, all of
which have been reported to exhibit physiological
activities in man, animals and microorganisms, suggests
that the plant may be use as a potent vegetable drug.
Table 4. Thin layer chromatography fingerprint of ethyl acetate Some phytochemicals are used in the pharmaceutical
extract of aerial part of C. ambrosioides with Rf values. industry for the production of various drugs. Examples
include the quinine analogues, nicotine, taxol, artemisinin
Spot Rf value Color/visualization and cocaine (Evans, 2002). Flavonoids and saponins
1 0.05 Green/daylight have been reported to possess anti-oxidants, anti-
2 0.50 Purple/vanillin spray inflammatory and hypoglycemic activities, and are used
3 0.56 Purple/vanillin spray
as anti-microbial, anti-cancer and anti-allergic remedies.
Saponins and cardiac glycosides have also been
4 0.65 Pink/vanillin spray
reported as antifungal as well as cardiotonics (Kunle and
5 0.70 Light green/daylight; pink/UV 365 nm
Egharevba, 2009). Some tannins had been reported as
6 0.81 Green/daylight; pink/UV 365 nm anti-viral and anti-tumor agents as well as diuretics.
7 0.93 Deep pink/vanillin spray Terpenes like the mono-, sesqui- and triterpenes, and
sterols had been reported to exhibit various biological
activities in animals and microorganisms. Some of which
include, anti-inflamatory, anti-microbial and hormonal
and silicates of various metals which were taken up from activities. Some steroidal compounds have been reported
the soil (Kunle, 2000). The non-physiological ash to exhibited anti-diabetic properties (Evans, 2002).
component of the total ash could be reduced by rinsing
the fresh plant material several times in clean water
before drying and processing for medicinal uses. The ACKNOWLEDGEMENT
acid-insoluble ash value measures the amount of silica,
especially siliceous earth, present in the drug plant The authors wish to thank Muazzam Ibrahim Wudil for
(Kunle, 2000). The physiological ash gets dissolved in the providing ethnomedicinal uses of C. ambrosioides in
dilute acid while some of the non-physiological ash Northern Nigeria.
remains undissolved (Shellard, 1958). The value of
3.05% which was obtained in this work is within range
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