Phenolic Content and Antioxidant Activity of Selected Philippine

Traditional Medicinal Plants Used to Treat Gastro-intestinal

Ailments
Karissa Grace F. Mejiaa, Flordeliza C. de Verab
a
School of Chemical Engineering and Chemistry, Mapua Institute of Technology, Manila, Republic of Philippines
b
School of Chemical Engineering and Chemistry, Mapua Institute of Technology, Manila, Republic of Philippines

Abstract

Medicinal plants have been an important part of indigenous medical systems in the Philippines and frequent consumption of
these plants is associated with preventing and curing different types of diseases. This has been attributed to the presence of various
forms of antioxidants like polyphenol compounds including flavonoids. Six plants of the traditional medicine for gastrointestinal
disorders in the Philippines specifically Tsaang gubat, Banaba, Malunggay, Tubang-bakod, Sambong and Mangostan were analyzed
for their antioxidant activity, total polyphenol content (TPP), total flavonoid content (TFC) and antimicrobial assay. The TPP of the
extracts was determined spectrophotometrically by the Folin-Ciocalteau method. The TFC was obtained using the AlCl3 method
whereas the antioxidant activity was measured using the 2-2-diphenyl-1-picrylhydrazyl (DPPH) assay. The antimicrobial assay was
determined by the agar cup method using Escherichia coli and Staphylococcus aureus as the test organisms which are common causes
of diarrhea. Methanolic extracts of tsaang gubat exhibited the highest TPP, TFC, and DPPH Radical Scavenging Activity (RSA).
Sambong showed the lowest TPP and DPPH RSA, whereas tubang-bakod showed the lowest TFC. Most of the methanolic extracts
showed antimicrobial activity against Escherichia coli and had slightly inhibitory effects on Staphylococcus aureus.

Keywords: antimicrobial activity, antioxidant activity, flavonoid, gastro-intestinal ailments, polyphenols

I. Introduction Antioxidants are substances that can prevent or delay

the oxidative damage of lipids, proteins and nucleic acids by
Plants have formed the basis of sophisticated traditional
reactive oxygen species, which include reactive free radicals
medicine systems that have been in existence for thousands of
such as superoxide, hydroxyl, peroxyl, alkoxyl and non-radicals
years and which continue to provide mankind with new
such as hydrogen peroxide, hypochlorous, etc. They scavenge
remedies. Although some of the therapeutic properties attributed
radicals by inhibiting initiation and breaking chain propagation
to plants have proven to be erroneous, medicinal plant therapy is
or suppressing the formation of free radicals by binding to the
based on the empirical findings of hundreds and thousands of
metal ions, reducing hydrogen peroxide, and quenching
years (Gurub-Fakim, 2006). The interest in the study of
superoxide and singlet oxygen (Lim et. al, 2007). Antioxidant
medicinal plants as a source of pharmacologically active
sources like medicinal plants are used as herbal tea drinks, as
compounds has increased worldwide. It is recognized that in
body applications and for washing sores and wounds, are
some developing countries, where living conditions are crowded
medicinal for asthma, dysentery, skin diseases, cancer, wounds,
and hygiene is poor, plants are the main medicinal source to treat
boils, stomach troubles, colds and other pulmonary diseases.
infectious diseases and where diarrhea and dysentery caused by
There are fruits that help prevent such illnesses as diabetes, high
bacterial enteropathogens are among the main causes of
blood pressure, respiratory ailments and blood hemorrhage.
morbidity and mortality (Alanis et al., 2005). In the Philippines,
There are leaves used as medicinal application for wounds,
there is an astounding variety of medicinal plants that have been
broken bones and others. There are culinary herbs and spices
reported for their folkloric medicinal purposes but only a few
that also help prevent disease like gastrointestinal problems.
medicinal plants have been studied and documented for their
Studies on the antioxidant capacity and phenolic content
functions and uses. In recent years, interest has been shifted to
of Philippine medicinal plants are limited. There is a need for
antioxidants from various plants.
further studies on the properties of the medicinal plants to
*Corresponding author
validate their use in traditional medicine. There are a lot of
Email address: kismej@yahoo.com studies related to the common medicinal plants in other countries
but a different kind of environment would greatly affect the
growth of these plants as well as their benefits to humans. The
quantity and quality of phenolic phytochemicals present in fruits
and vegetables are significantly influenced by cultivar,
environment, soil type, and growing and storage conditions
(Singh et al., 2009).
The objective of the study was to determine the total
antioxidant activity, phenolic content, flavanoid content and
antimicrobial activity of selected common Philippine medicinal
plants that are intended for gastrointestinal disorders specifically
diarrhea, such as the leaves of queen's crape myrtle
(Lagerstroemia speciosa, banaba), drumstick (Moringa oleifera,
malunggay), physic nut (Jatropha curcas, tubang-bakod),
blumea camphor (Blumea balsamifera, sambong) and
mangosteen (Garcinia mangostana, mangostan).
The results of this study give scientific support to the
use of selected medicinal plants in the Philippines for the
treatment of gastrointestinal disorders such as diarrhea and
establish renewable sources of antioxidants from plants.
The analysis of the polyphenol content, flavonoid
content, antioxidant activity and antimicrobial activity used only
the leaves of the medicinal plant. The DPPH radical scavenging
method was used in determining the total antioxidant activity, the
Folin-Ciocalteu method for the total polyphenol content and the
AlCl3 method for total flavonoid content. Gallic acid was the
only standard used in this study. The results obtained were
compared to the positive control which is tsaang gubat that is
already an established drug of the Bureau of Food and Drugs an
approved by the Department of Health.
2. Experimental
Materials
The plants were collected and verified at the Bureau of
Plant Industry. Leaves of tsaang gubat, banaba, malunggay,
tubang-bakod, sambong and mangostan were used in the
experiment. The reagents used were gallic acid, 2,2-diphenyl-1-
picrylhydrazil (DPPH) solution and Folin-Ciocalteu reagent.
Staphylococcus aureus and Escherichia coli were used for
antimicrobial activity of the plant samples done at the Natural
Sciences Research Institute, University of the Philippines in
Diliman.
Preparation of gallic acid standards
Two hundred fifty milligrams of dry gallic acid were
dissolved in 1 ml of 95% ethanol and diluted to 500 ml with
distilled water to prepare 0.5 mg/ml stock standard solution of
gallic acid. The solution was stored in the freezer. Working
standards were prepared between 0.0 mg/ml and 1 mg/l by
diluting the stock solution with distilled water.
Sample preparation
Dried leaves were minced into the smallest pieces
possible before subjected to liquid extraction. Ten grams of the
minced samples were mixed with 100 ml methanol for 2 hours in
an orbital shaker. The heterogeneous solution was centrifuged
for 15 minutes. The extraction method was done in triplicate.
The supernatant collected was filtered and dried. The amount of
residue was determined in terms of weight (in mg). The residue
was dissolved in 100 ml methanol and analyzed for its total
polyphenol content, total flavonoid content and radical
scavenging activity. The solution was filtered before analysis.
DPPH radical scavenging method
A methanolic solution (0.1 ml) of sample extract was
added at various concentrations to 3.9 ml (0.025 g L-1) of DPPH
solution. The decrease in absorbance at 515 nm was determined
continuously every 1 minute with a Perkin-Elmer UV-Vis
Spectrophotometer until the reaction reached the plateau. The
percentage of DPPH radical scavenging was calculated using the
equation:
Initial absorbance − Final absorbance
% DPPH RSA = x 100
Initial absorbance
Total polyphenol content
The TPP content was determined by the Folin-Ciocalteu
method. Plant extracts (0.5 ml) or gallic acid standard solutions
were mixed with 2.5 ml of Folin-Ciocalteu reagent (FCR 1:10
dilution) and were left standing for 8 minutes at room
temperature to allow for the Folin-Ciocalteu reagent to react
completely with the oxidizable substances or phenolates. Two
milliliters of Na2CO3 (7.5% solution in water) were added to
destroy the residual agent. The absorbance was measured at 760
nm using a Lambda 40 UV-Vis Spectrophotometer (Perkin
Elmer, USA) after incubating at room temperature for 2 hours.
Results were expressed as milligrams of gallic acid equivalents
(GAE) per 100 g fresh weights.
Total flavonoid content
Total flavonoid content was measured by aluminum
chloride colorimetric assay. An aliquot (1ml) of stock solution
or standard solution of gallic acid in methanol (0 mg/ml to 100
mg/ml) was combined with 4 ml double-distilled water. The
amount of 0.3 ml 5% NaNO2 was added to the solution. After 5
minutes, 0.3 ml 10% AlCl3 was added and at the 6th minute, 2 ml
of 1 M NaOH was added and the volume adjusted to 10 ml with
double-distilled water. The solution was mixed well and the
absorbance was measured against a reagent blank at 510 nm.
Antimicrobial activity measurement
Staphylococcus aureus and Escherichia coli were used
for antimicrobial measurement of the plant samples at the
University of the Philippines - Natural Sciences Research
Institute. Microbial suspensions were prepared from 24-hour old
cultures of the bacteria. The suspending medium used was 0.1%
peptone water. One-tenth (0.1) ml aliquots of the bacteria were
transferred into pre-poured nutrient agar. Five ml of the
corresponding medium, melted and cooled to 45°C, was poured
onto the agar plate and swirled to distribute the inoculum evenly
on the agar surface. Three equidistant wells were made on the
agar plate using a cork borer (10 mm). Two hundred µl of the
sample were placed in each hole. The plates were incubated at
room temperature. Nutrient agar plates were observed after 24-
48 hours. The clearing zones were calculated. The antimicrobial
index (AI) was computed using the formula:
Diameter of clearing zone − diameter of well
AI =
Diameter of clearing zone
Statistical analysis
Data were analyzed using one-way analysis of variance
(ANOVA) test to calculate the significance of results in
Microsoft Excel 2007. A difference was considered to be
statistically significant when the P-value is less than 0.005 (*P <
0.005).
3. Results and discussion There are abundant polyphenols in each plant species
but relative amounts of flavonoids as shown in Figure 3.1,
Extraction yield of the medicinal plant samples
indicate that different classes of polyphenols, not just flavonoids,
The different medicinal plant varieties used in the study
might be present in the leaves of the selected plants.
contained levels of methanolic extracts ranging from 9.429 % of
Investigations by Igbinosa, et al., (2009) on the
banaba to 17.316 % of malunggay as shown in Table 1. Sultana
phytochemical screening of tubang-bakod stem bark extracts
et al. (2009), conducted a study on the effects of four extracting
revealed the presence of saponins, steroids, tannins, glycosides,
solvents [absolute ethanol, absolute methanol, aqueous ethanol
alkaloids and flavonoids. These compounds are known to be
(ethanol: water, 80:20 v/v) and aqueous methanol (methanol:
biologically active and therefore aid the antimicrobial activities
water, 80:20 v/v)] and two extraction techniques (shaking and
of tubang-bakod. These secondary metabolites exert
reflux) on the antioxidant activity of selected medicinal plants
antimicrobial activity through different mechanisms. Herbs that
including malunggay and achieved 9.61% to 17.9% yielded on
have tannins as their main components are astringent in nature
different extracting solvents. Solvent extraction is the most
and are used for treating intestinal disorders such as diarrhea and
frequently used technique for the isolation of plant antioxidant
dysentery. These observations therefore support the use of
compounds. However, the extract yields and resulting
tubang-bakod in herbal cure remedies.
antioxidant activities of the plant materials are strongly
dependent on the nature of extracting solvent, due to the presence
of different antioxidant compounds of varied chemical
characteristics and polarities that may or may not be soluble in a
particular solvent. Polar solvents are frequently employed for the
recovery of polyphenols from a plant matrix. The most suitable
of these solvents are (hot or cold) aqueous mixtures containing
ethanol, methanol, acetone, and ethyl acetate.

Table 1 Extraction Yield of the Different Medicinal Plants

Plant extracts Extraction yield
%
Tsaang gubat 17.038
Mangostan 15.284
Sambong 13.738
Tubang-bakod 11.711
Figure 1 Comparison of the TPP and TFC of the medicinal plants
Banaba 9.429
Malunggay 17.316
A phytochemical investigation was done by Fazitalun et
al., (2004) on the leaves of sambong by their chemical structures
Total polyphenol content and total flavonoid content
elucidated by means of elemental analyses and different
The total phenolic content and total flavonoid content of
spectroscopic methods, such as UV, IR, NMR and MS, which
the medicinal plants were expressed as mg GAE/100grams and
resulted in the isolation of 11 flavonoids (quercetin, rhamnetin,
are listed in Table 3.2. The phenolic content for the methanolic
luteolin, luteolin-7-methylether, L-ascorbic acid, blumeatin,
extracts of the medicinal plants ranged from 507.40 to 980.93 mg
butylated hydroxyanisole, 5,7,3',5'-tetrahydroxyflavanone,
GAE/100 g fresh weights. Tsaang gubat which is the positive
tamarixetin, butylated hydroxytoluene, α-tocopherol,
control had the highest polyphenol content followed by
dihydroquercetin-4'-methylether and dihydroquercetin-7,4'-
mangostan, malunggay, banaba and tubang-bakod. The lowest
dimethylether). The result indicates that flavonoid contents of
polyphenol content was achieved by sambong. Flavonoids
different solvent extracts of sambong leaves are responsible for
constitute the largest group of plant phenolics, accounting for
their antioxidant properties.
over half of the eight thousand naturally occurring phenolic
Phenolics are often extracted in higher amounts in more
compounds (Manach et al., 2009). In this study, the total
polar solvents such as aqueous methanol/ethanol as compared
flavonoid contents of the medicinal plants ranged from 13.80 to
with absolute methanol/ethanol (Sultana, et al., 2009).
475.81 mg GAE/100 g. Tsaang gubat had the highest flavonoid
Previous reports suggest that phenolic content increases
content and tubang-bakod had the lowest. Statistical analysis
with the increase in leaf-age and is lower in early stages of leaf
showed that the medicinal plants are significantly different
growth, gradually increasing with the maturity of leaves. It has
(*P<0.005), which affects the total phenolic and total flavonoid
also been reported that thermal processing conditions might
content.
result in the loss of natural antioxidants because heat may
Table 2 Total Polyphenol and Total Flavonoid Content of the Medicinal accelerate their oxidation and other degenerative reactions.
Plant Extracts Thus, heating temperature is of much consideration during
Plant extracts TPP TFC
processing.
mg GAE/100g mg GAE/100g
Tsaang gubat 980.93±5.9 475.81±8.3 DPPH radical scavenging activity
Mangostan 965.73±8.6 462.63±2.4 DPPH is a stable free radical with characteristic
Sambong 507.40±9.7 111.37±9.7 absorption at 515 nm and antioxidants react with DPPH and
Tubang-bakod 546.4±6.70 13.80±2.6
Banaba 865.00±5.6 26.30±8.3
convert it to 2,2-diphenyl-1-picrylhydrazine. The DPPH radical
Malunggay 885.07±5.9 279.50±5.8 has been widely used to test the free radical-scavenging ability of
various natural products and has been accepted as a model
compound for scavenging free radicals in lipids. In the DPPH activity against E. coli. Among all the extracts, tsaang gubat
radical-scavenging method, a compound with high antioxidant showed the highest antimicrobial activity against E. coli having a
potential effectively traps the radical, thereby preventing its diameter clearing zone of 25 mm and antimicrobial index of 1.5.
propagation and the resultant chain reaction. The degree of Malunggay did not show antimicrobial activity against E.coli and
discoloration from violet to yellow indicates the scavenging banaba showed only some thinning of E. coli. Mangostan,
potential of the antioxidant extract, which is due to the hydrogen malunggay and sambong had slightly inhibitory effects against S.
donating or radical scavenging ability (Verma, et al., 2009). aures, but banaba, tubang-bakod and tsaang-gubat did not inhibit
High % DPPH radical scavenging activity would imply a high the growth of S. aureus. Results in Table 4 showed that both
antioxidant activity. sambong and mangostan showed antimicrobial activity against
The DPPH radical scavenging of the methanolic Escherichia coli and Staphylococcus aureus. Medicinal plant
extracts on Table 3.3 showed that tsaang gubat had the highest extracts were significantly different (*P<0.005) on the
activity, which also had the highest polyphenol and flavonoid antimicrobial activity with respect to E. coli and S. aureus.
content followed by mangostan which also exhibited a high TPP
and TFC, but the correlation of the results should be studied Table 4 Antimicrobial Activity of the Methanolic Extracts of the Medicinal
Plants
further. The high polyphenolic contents of the methanolic
Escherichia coli Staphylococcus aureus
extracts could be responsible for the high antioxidant activity. UPCC1195 UPCC1143
Leong and Shui (2002) compared the total antioxidant capacity Diameter Diameter
of twenty-seven fruits available in the Singapore market, of of
including mangostan, using the ABTS and DPPH assays. clearing Antibacterial clearing Antibacterial
zone Index zone Index
Results showed that the mangostan extract had the eighth place
Tsaang gubat 25.0 1.5±0.00 0.0 0±0.00
in antioxidant efficiency. The DPPH radical scavenging activity Mangostan 20.0 1±0.00 16.0 0.6±0.17
of the six medicinal plants studied were significantly (*P < 0.005) Tubang-bakod 20.0 1±0.00 0.0 0±0.00
different from each other. Malunggay 0.0 0±0.00 11.3 0.1±0.06
Sambong 13.7 0.4±0.12 13.0 0.3±0.00
Table 3 DPPH Radical Scavenging Activity of the Medicinal Plant Extracts Banaba -(14) 0±0.00 0.0 0±0.00

Plant extracts % DPPH- RSA

Pedraza-Chaverri et al., (2008) isolated bioactive
Tsaang gubat 69.36 ±2.7 substances like xanthones from the fruits, pericarp and leaves
Mangostan 54.88 ±2.1 extracts of mangostan which demonstrated that the extracts have
Sambong 40.32 ±1.6 antioxidant, antitumoral, antiallergic, anti-inflammatory,
Tubang-bakod 41.36 ±3.1 antibacterial, and antiviral activities.
Banaba 42.13 ±2.9 Villaseñor, et al., (2004) reported that the triterpene
Malunggay 50.71 ±3.1
mixture, the major constituent isolated from tsaang gubat and
which turned out to be anti-diarrheal, was inactive against
It has been well established that free radical scavenging Escherichia coli and possessed moderate activities against
activity of plant extracts is mainly due to phenolic compounds. Staphylococcus aureus, Candida albicans, and Trichophyton
The weaker radical-quenching abilities observed for the mentagrophytes. The result is entirely different with the present
compounds in this test may be ascribed to their molecular study in which the extract of tsaang gubat is effective against
structures. Compounds having higher numbers of phenolic Escherichia coli and has no inhibition with Staphylococcus
hydroxyl groups exhibited a stronger free radical-scavenging aureus. In this study, tubang-bakod did not show activity against
activity, which is in keeping with the notion that phenolic E.coli and showed inhibition against S. aureus. The findings of
hydroxyl groups are able to donate hydrogens and those Lentz et al. (1998) were not in accordance with the results in
phenoxyl radicals, once formed, are stablilized by delocalization which tubang-bakod did not have antimicrobial activity against
of electron. E. coli and showed effectiveness on S. aureus. But in a study
Sultana, that agroclimatic locations and seasons have made by Adamu et al., (2005) tubang-bakod showed inhibition
profound effects on the antioxidant et al., (2009) concluded on both microorganisms.
activity of malunggay leaves. Antioxidant activity of samples Some considerations must be established for the study
from cold areas was relatively higher than those from temperate of the antimicrobial activity of plant extracts, essential oils and
regions. Antioxidant potential of malunggay leaves from the compounds isolated from them. This includes common
Pakistan was quite comparable or higher than literature values parameters, such as plant material, techniques employed, growth
for malunggay from other countries and some other potent medium and microorganisms tested. The solvent and the
antioxidants. This work shows that season and agroclimatic extraction system may both modify the final results. The most
locations have profound effect on the antioxidant activity of appropriate method would be that in which the extracts are the
malunggay leaves (Singh, et al., 2009). same as that used in folk medicine or phytotherapy, although in
the lab the use of methanol or ethanol extract is much more
Antimicrobial activity common. The pH of compounds in dilutions also modifies the
Escherichia coli and Staphylococcus aureus results, as can sometimes be observed when phenolic or
enteropathogens are the common causes of diarrhea and were carboxylic compounds are present in the extract. The
used as test organisms in this study. Thirty micrograms (30 ug) composition of the growth medium can also influence the
of chloramphenicol wereused as positive control having 6 mm in activity of the tested extracts or compounds (Rios & Recio,
diameter for S. aureus and 8 mm for E. coli. Mangostan, 2005).
tubang-bakod, sambong and tsaang gubat showed antimicrobial

Conclusion
The selected traditional medicinal plants demonstrated
abundant levels of polyphenols and it varies in their flavonoid
contents. Tsaang gubat leaves achieved the highest total
polyphenol, total flavonoid content and DPPH radical scavenging
activity. Mangostan leaves could be an alternative for Tsaang
gubat since it followed Tsaang gubat with the highest TPP, TFC
and DPPH RSA and it showed inhibition on Escherichia coli and
Staphylococcus aureus. The data gathered would certainly help
to ascertain the potency of the six tested medicinal plant leaves
as potential sources of natural antioxidants. Most of the
methanolic extracts showed antimicrobial activity against
Escherichia coli and had slightly inhibitory effects on
Staphylococcus aureus. This has to be studied further and some
general considerations must be considered such as the plant
material, techniques employed pH, extraction methods, solvents,
growth medium and microorganisms tested. The study of
traditional medicinal plants as antimicrobial agents is necessary
for gaining insight into the validity of their medicinal purposes.
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