PHARMACY/CHEMISTRY

Phytochemical Screening of Five Folkloric

Plants Used by Indigenous Tribes in Mindanao
Florie L. Casalan; Joyce Mae Veronne R. Ayo;
Mabelliza E. Bebillo; Jeanie Rose Fano

Abstract

Plant is widely used as a source of medicines by some Mindanaoan

tribes. The researchers aimed to study five folkloric plants used by
five different tribes to treat different illnesses and sickness. These
plants were collected and selected based on the interview with the
traditional healers of Manobo tribe of Kimagting, Kalilangan Bukidnon,
Matigsalog tribe of Kimanait, Kalilangan Bukidnon, Talaandig tribe of
Pangantucan Bukidnon, Maranao tribe of Kalilangan Bukidnon and the
Higanon tribe Ulayan, Kalilangan Bukidnon. The plants were identified
as Amor-seco (Bidens pilosa), Anuang (Kyllinga monocephala), Bila-
bila (Eleusine indica), Bugang (Pennisetum purpureum), and Gemilina
(Gmelina arborea). The said plants were tested on its alkaloids,
cardenolides and bufadienolides, anthraquinones, flavonoids, saponins
and tannins. Field test was done in Bukidnon and remaining tests in
University of the Immaculate Conception. Results showedabsence of
alkaloid while saponins were only present in K. monocephala and P.
purpureum. Steroids were detected to all plants except E. indica and K.
monocephala. While tannins were also detected in all plants except for
K. monocephala and B. pilosa. Flavonoids were shown to be positive in
B. pilosa and G. arborea. All were negative in anthraquinones.

Keywords: Folkloric plants; Mindanaoan Lumads, Phytochemical

Screening

106•Root Gatherers University of the Immaculate Conception

 PHARMACY/CHEMISTRY

Introduction

Medicinal plants as re-emerging health aid has been fuelled by

the rising costs of prescription drugs in the maintenance of personal
health and well-being and the bioprospecting of new plant-derived
drugs. Several issues as well as range of interests and activities in a
number of countries are dealt with. Based on current research and
financial investments, medicinal plants will seemingly continue to play
an important role as a health aid (DaSilva, 1999).

One of the most striking differences between traditional and

modern medicines is the legal protection given to knowledge. Traditional
practitioners have historically shared their knowledge and experience
freely – defining ‘open-access’ before the term even existed. Modern
medicine, on the other hand, has stringent intellectual property laws
and a highly evolved patenting system used to protect knowledge about
drugs or medical techniques (Shetty, 2010). The practice of traditional
medicine is widespread in China, India, Japan, Pakistan, Sri Lanka and
Thailand. In China about 40% of the total medicinal consumption is
attributed to traditional tribal medicines. In Thailand, herbal medicines
make use of legumes encountered in the Caesalpiniaceae, the Fabaceae,
and the Mimosaceae. And, in Japan, herbal medicinal preparations are
more in demand than mainstream pharmaceutical products (DaSilva,
1999).

In most cities in the Philippines today you’ll find gleaming

hospitals with all the accouterments of modern medicine. Yet venture
into the countryside and many people rely upon indigenous medicines
passed down from generation to generation, as well as other alternative
therapies and faith healing. People in the Philippines embraced both for
health and healing (Grunert, 1998).

This awareness has led the researchers to study five medicinal

plants being used by some Mindanaoan Lumads namely: “Amor-seco”
(Bidens pilosa) by the Talaandig Tribe, “Anuang” (Kyllinga monocephala)
by the Higanon tribe, “Bila- bila” (Eleucine indica) by the Manobo

University of the Immaculate Conception Root Gatherers•107

PHARMACY/CHEMISTRY

tribe, “Bugang” (Pennisetum purpureum) by the Matigsalog Tribe and

“Gemilina” (Gmelina arborea) by the Maranao tribe. The researchers
aimed to screen the phytochemical properties from the plants.

MATERIALS AND METHODS

The researchers made use of descriptive research design for the

phytochemical of Amor-seco, Anuang, Bila- bila, Bugang, and Gemilina.
The researchers used the crude leaf extract of five medicinal plants.
There were three replicates for every trial.

Leaves of Bidens pilosa, Kyllinga monocephala, Eleusine indica,

Pennisetum purpureum, and Gmelina arborea were taken and collected
randomly from Kalilangan, Bukidnon. For the confirmatory testing,
experimentation was done at the analytical chemistry laboratory of the
University of the Immaculate Conception-Main Campus while the field
testing was done at Purok 2, Central PoblacionKalilangan, Bukidnon.

Collection and preparation of

Plant Material for Extraction

The leaves of each five medicinal plants were collected at

Kalilangan, Bukidnon and washed with running water. Finally, garbling
was done to remove extraneous matter.

Preparation of Plant Extracts

The fresh leaves were cut, weighed to 500 grams, and added with
sufficient amount of 95% ethyl alcohol to completely submerge the plant
material. After 48 hours, filtration, evaporation, and concentration of
the extract were conducted. The concentrated extract was stored in a
tightly stoppered amber bottle container in a cold dry place.

108•Root Gatherers University of the Immaculate Conception

 PHARMACY/CHEMISTRY

Phytochemical Screening

Alkaloid Testing

Laboratory Methods for Alkaloid Analysis

Preliminary Test

An aliquot portion of the extracted plant material at 20 g was taken

from the stock plant extract and evaporated to a syrupy consistency
over a steam bath. Five mL of 2M HCl was added, heated with stirring
for about five minutes and allowed to cool.

About 0.5 g NaCl was then added, stirred and filtered, and residue
was washed with enough 2M HCl to bring the filtrate to a volume of
5 mL. One milliliter of the filtrate was taken and tested with two to
three drops of Dragendorff’s reagent. Another 1 mL of the filtrate was
taken and tested with two to three drops of Mayer’s reagent. A positive
result was indicated by an orange precipitate with Dragendorff’s and a
white precipitate with Mayer’s test. It was preformed into three trials
(Guevara et al., 2005).

Confirmatory Test

The remaining 3 mL of the filtrate obtained from the latter method

was taken and added with enough 28% ammonia until the solution was
alkaline to litmus paper. The alkaline solution was cautiously extracted
three times with 10 mL portion of chloroform, and the chloroform
extracts were combined and evaporated to dryness under the fumehood
and over a steam bath. Five mL of 2M HCl was used to take up the
residue, stirred over the steam bath for about 2 minutes, and allowed
to cool. The filtrate was filtered and divided into two portions. One was
tested with Dragendorff’s reagent and the other with Mayer’s reagent.
It was performed into three trials (Guevara et al., 2005).

University of the Immaculate Conception Root Gatherers•109

PHARMACY/CHEMISTRY

Saponins

Capillary Method

Three capillary tubes were loaded with the extract used in the froth
test to a height of 10 mm. The fourth tube was loaded with distilled
water. All tubes were kept in a vertical position and allowed the liquid
to flow freely. Then, the levels of the liquid in each tube were compared.
When the level of the plant extract in the tube was half or less than that
of water, the presence of saponins was inferred (Guevara et al., 2005).

Cardenolides and Bufadenolides

Keller-Kiliani Test

A volume of the extracted plant material at 10 g was taken,

evaporated to incipient dryness over a water bath, and allowed to cool at
room temperature. The residue was defatted by triturating with hexane
and the hexane extract was decanted off and repeated the treatment
until most of the colored pigments were removed. The hexane extract
was then discarded.

The defatted residue was warmed to remove the residual hexane,

added with three ml Ferric chloridereagent, stirred and transferred the
mixture to a test tube. With the test tube in an inclined position, 1 mL
of concentrated sulfuric acid was cautiously added, letting the acid run
along the sides of the tube.

The mixture was allowed to stand and observed for any coloration
at the interface. A reddish-brown color, which may turn to blue or
purple, was used to observe the presence of 2-deoxysugars. This was
performed into three trials (Guevara et al., 2005).

110•Root Gatherers University of the Immaculate Conception

 PHARMACY/CHEMISTRY

Kedde Test

An equivalent of 2 g plant material of each plant crude extract

was taken. Two mL of chloroform was cautiously added and mixed
well. The lower chloroformlayer was taken and treated with four drops
of Kedde’s reagent and a blue violet color was used to indicate the
presence of Cardenolides and Bufadienolides.

For positive standard, two mL of 0.025% digitoxin in methanol

was treated as in the above procedure. It was performed into three
trials (Guevara et al., 2005).

Flavonoids

Preparation of the Plant Extract

About 10 g of plant material was taken, evaporated to incipient

dryness over a water bath, and cooled to room temperature. The
residue was defatted by treating with hexane until the extracts was
almost colorless. The hexane extract was discarded afterwards.

The residue was taken up with 10 mL of 80% ethyl alcohol. It was

then filtered and divided the filtrate into four test tubes. One portion
was taken as control (Guevara et al., 2005).

Bate- Smith and Metcalf Test for Leucoanthocyanins

One portion of the preparation was treated with 0.5 ml

concentrated HCl and observed for any color change. It was warmed for
15 minutes in a water bath. Observation for further color change within
an hour was done. A strong red or violet color was used to indicate
leucoanthocyanins. This was performed in three trials (Guevara et al.,
2005).

University of the Immaculate Conception Root Gatherers•111

PHARMACY/CHEMISTRY

Wilstater Cyanidin Test

Portion was treated with 0.5 ml concentrated HCl. About 3-4 pieces
of magnesium turnings were added and observed for any color change
within 10 minutes. When definite coloration occured, diluted with an
equal amount of water and added with 1 mL octyl alcohol. Then shaken
well and allowed to stand. The color was noted in each layer.

Tannins and Polyphenolic Compounds

Screening Methods

Preparation of the Plant Extract

An equivalent of 10 g plant material of the plant extract was taken

and evaporated to incipient dryness over a water bath. The residue was
extracted with 20 milliliters of hot distilled water. Five drops of 10%
NaCl was added, filtered and divided the filtrate into three test tubes.
One portion was taken as control and an aqueous solution of tannic
acid was used as reference standard (Guevara et al., 2005).

Gelatin Test

One portion of the filtrate was treated with three drops of gelatin-
salt solution. Likewise to the tannic acid was done. Formation of
a precipitate indicated the presence of tannins. The researcher then
compared it with the control (Guevara et al., 2005).

Ferric Chloride Test

Another portion of the filtrate was treated with three drops of ferric
chloride solution. Likewise to the tannic acid was done. A blue-black
color was observed to indicate the presence of hydrolysable tannins,
while a brownish green color for the presence of condensed tannins.
The result was compared with the control. This was performed into
three trials (Guevara et al., 2005).

112•Root Gatherers University of the Immaculate Conception

 PHARMACY/CHEMISTRY

Anthraquinones

Test tube Screening Methods

Borntrager’s Test

An equivalent of one gram of plant material of the each plant crude

extract was taken and evaporated o incipient dryness over a water bath.
The residue was taken up with ten milliliters of distilled water and
filtered. The researchers extracted the filtrate twice with 5 mL portions
of benzene and divided the benzene extracts into two portions. One
portion was reserved as control.

The other portion was treated with five milliliters of ammonia

solution and shake well. A red coloration in the lower alkaline layer was
indicated for the presence of anthraquinones (Guevara et al., 2005).

Results and Discussions

Table 1 shows the results of the phytochemical screening. Alkaloids

were not detected in any of the plants in the preliminary screening for
both in laboratory and field tests, in which there is no precipitate form
upon the addition of Mayer’s and Dragendorff’s reagent on the plant
extracts.

The presence of saponins was detected in Anuang and Bugang for

both laboratory and field tests. In the capillary method, these plants
have shown a positive result, in which the levels of the plant extracts
were half and less than that of water. This is due to the ability of the
saponins to lower the surface tension of the water. Thus, the presence
of saponins in Anuang and Bugang extracts were observed.

University of the Immaculate Conception Root Gatherers•113

PHARMACY/CHEMISTRY

Table 4.1 Phytochemical Screening

Phytochemical Screening Plants Field Laboratory
PT CT PT CT
Amor-seco – – – –
Anuang – – – –
Alkaloids Bila-bila – – – –
Bugang – – – –
Gemilina – – – –
CP
Amor-seco – –
Anuang + +
Saponins Bila-bila – –
Bugang + +
Gemilina – –
KK KT KK KT
Amor-seco + –
Anuang – –
Steroids Bila-bila – – Not Performed
Bugang + –
Gemilina + –
BM WC BM WC
Amor-seco + –
Anuang – –
Flavonoids Bila-bila – – Not Performed
Bugang – –
Gemilina + –
GT FC GT FC
Amor- seco – + – +
Anuang – – – –
Tannins Bila- bila + + + +
Bugang + + + +
Gemilina + + + +
BT
Amor- seco –
Anuang –
Anthraquinones Bila- bila – Not Performed
Bugang –
Gemilina –
PT = Preliminary test CP = Capillary test BM = Bate Smith & Metcalf tests GT = Gelatin test
CT = Confirmatory test KK = Keller Kiliani test WC = WilstaterCyanidin test FT = Ferric Chloride test
BT = Borntrager’s test

114•Root Gatherers University of the Immaculate Conception

 PHARMACY/CHEMISTRY

Since it is positive to saponins, these plants have the ability to lower

cholesterol, strengthen the immune system and fight off pathogens that
can cause chronic arthritic pain and low grade inflammation (Cheman,
2010).

Furthermore, the test for Cardenolides and Bufadienolides in field

testing revealed that Amor-seco, Bugang and Gemilina were positive
using the Keller-Killiani test and so these plants has a dramatic effect
on the heart muscle.

The result observed the formation of the reddish-brown color;

specifically it indicates the presence of deoxysugar of Cardenolides and
Bufadienolide.

However, none among the plant extracts produced a blue-violet

coloration in Kedde test. Thus, there was an absence of unsaturated
lactone containing Cardenolides and Bufadienolides in the five selected
folkloric plants.

In flavonoid screening, which is being performed in the field, the

positive result was found in amor-seco and gemilina, which shows a
strong red coloration in the Bate-Smith and Metcalf Test. This test
identifies the leucoanthocyanins. However, there was no formation
of violet color or change in coloration of the five plant extracts in
Wilstater Cyanidin tests. Amor- seco and gmilina were then found to
have antiviral, antifungal, anti-inflammatory and cytotoxic activities.

The presence of tannins and polyphenolic compounds were

detected in bila- bila, bugang and gemilina in both laboratory and field
tests. The plant extracts were indicated as positive since the formation
of precipitates in gelatin test was seen. Presence of tannins was also
confirmed in Ferric Chloride test of bila-bila, bugang and gmilina.
The Ferric Chloride test determined the specific tannins present in the
plant. Bila-bila and gemilina formed blue-black coloration therefore
positive for hydrolysable tannins whereas gemilina formed a brownish
green coloration indicating the presence of condensed tannins. Bila-

University of the Immaculate Conception Root Gatherers•115

PHARMACY/CHEMISTRY

bila, bugang and gemilina were then found to have an antidiarrheal,

hemostatic, and antihemmorhoidal activity since it was found positive
to tannins.

Table 1 also shows that none from the five folkloric plants contain
Anthraquinones because there was no formation of red color. This test
was performed in the field.

Conclusions

The researchers screened all of the five medicinal plant extracts

used as medicines by tribes, identified their secondary metabolites
through field test and laboratory testing. But the laboratory test was
only limited to alkaloids, saponins, and tannins. Such metabolites
found in the field test are: Saponins in Kyllinga monocephala and
Pennisetum purpureum; steroids in Bidens pilosa, Pennisetum purpureum
and Gmelina arborea; flavonoids in Bidens pilosa and Gmelina arborea;
and tannins in Eleusine indica, Pennisetum purpureum and Gmelina
arborea. While in the laboratory test the secondary metabolites found
are: saponins in Eleusine indica and Pennisetum purpureum; and tannins
in Eleusine indica, Pennisetum purpureum and Gmelina arborea.

116•Root Gatherers University of the Immaculate Conception

 PHARMACY/CHEMISTRY

Recommendations

The researchers saw the need to propose the following

recommendations. These are enumerated below:

1. To perform for the presence of steroids, flavonoids,

anthraquinones and glycosides in “Amor-seco” (Bidens pilosa),
“Anuang” (Kyllinga monocephala), “Bila-bila” (Kyllinga
monocephala), “Bugang” (Pennisetum purpureum) and
“Gemilina” (Gmelina arborea).

2. To determine the quantitative content of each of the identified

bioactive constituents through chromatographic screening of
the plant ethanolic extracts.

3. To discover the potential value of tannins containing plants

like Pennisetum purpureum and Gmelina arborea since they are
known to be cytotoxic/antineoplastic and astringent.

4. To conduct investigation into the antimicrobial activity of the

plant ethanolic extracts against clinical isolates.

5. To conduct antimicrobial tests using the plant ethanolic extracts

such as minimum bactericidal and fungicidal concentration
and comparison of antimicrobial activity of the plan ethanolic
extracts with the standard antibiotic.

6. To explore and isolate saponins in Kyllinga monocephala and

Pennisetum purpureum and conduct pharmacological screening
like anti-inflammatory activity since saponins according to
Guevara et al. (2005) they are a great importance because of
their relationship to such compounds as sex hormones and
cortisone.

University of the Immaculate Conception Root Gatherers•117

PHARMACY/CHEMISTRY

REFERENCES

Aniszewski, T. 2007. Alkaloids-Secrets of life. Alkaloid

Chemistry, biological significance, Applications and Ecological
Role. Oxford OX5 IGB, UK (Copyright 1st Edition).

Balick, Michael J. and Paul A. Cox. 1996. Plants, People,

and Culture: The science of ethnobotany. Scientific American
Library. Reprint edition in 2005 by the American Botanical
Council, Austin TX.

Chadwick, Derek J. and J. Marsh, eds. 1994. Ethnobotany

and the search for New Drugs. Ciba Foundation Symposium
185. John Wiley and Sons.

Che Man, Nuha. 2010. Phytochemical analysis of the leaves of

Chromolaenaodorata (Asteraceae) / NuhaChe Man. Retrieved
from http://eprints.ptar.uitm.edu.my/2360/

Da silva, L. 1999. Medicinal plants: a re-emerging health aid

Retrieved from http://www.ejbiotechnology.info

Delgado, Jaime, et al. 1991. Organic Medicinal and

Pharmaceutical Chemistry Ninth Edition. Philadelphia, WA.
Lippincott Company, (1991)

Evans, W.C. 2002. Trease and EvancePharmacognosy. 15th

Edition. University of Nottingham, Nottingham, USA.

Guevara, B., et al. (2005). A guide to plant screening:

phytochemical and Biological (p.67-98). Manila: Research
Center for Natural Sciences

Kubmarawa, GA Ajoku, NM Enwerem, DA Okorie. 2007.

Retrieved from http://www.ajol.info

118•Root Gatherers University of the Immaculate Conception

 PHARMACY/CHEMISTRY

Lumads. 2010. Retrieved from http://en.wikipedia.org/wiki/Lumad

Matigsalog. 2010. Retrieved from http://en.wikipilipinas.org/

matigsalog

Phillipine Alternative Medicines. 2010. Retrieved from

www.stuartXchange.com

Philippine Herbal Medicine. 2008. Retrieved from www.

philippineherbalmedicine.org

Quisumbing, Eduardo. 1978. Medicinal Plants of the

Philippines. Quezon City: JMC Press Inc. 1978.

Paterson, RT. 1994. Use of Trees by Livestock: Erythrina.

Retrieved from http://www.smallstock.info/research/reports/
R5732/NR08UE/B1701_8.HTM

Rasul, et al. 1989. Preliminary Phytochemical Screening of

Four Common Plants of family caesalpimaceae.Retrieved from
http://www.pjps.pk/CD-PJPS-2-1-89/Paper-9.pdf

Tannins. 2010. Retrieved from http://en.wikipedia.org/wiki/Tannin

Taylor, et al., 2009. Cane toad habitat potential, likely points of

entry, and potential impacts on native wildlife. Retrieved from
brg.cma.nsw.gov.au/uploads/CaneToadFinalReport_comp.pdf

The Higaonon Tribe of Mindanao. 2003. Retrieved from

http://www.unahi.org/higaonon-tribe.htm

Togon, A. S. &Ayuban, J. 1998. Indigenous Medicinal Plants

and Practices of Ten Ethnic Tribes in Mindanao.

Tyler, Varro, et al. 1998. Pharmacognosy. 9th Edition.

Philadelphia: JMC Press Inc.

University of the Immaculate Conception Root Gatherers•119