The antioxidant properties of the Philippine medicinal plants Cassia sophera Linn., Derris elliptica Benth, Ficus minahassea Tesym. and De Vr., Leea aculeata Blume and Leucosyke capitellata Wedd

Full text

AAB Bioflux, 2015, Volume 7, Issue 3.
http://www.aab.bioflux.com.ro 150
AAB BIOFLUX
Advances in Agriculture & Botanics-
International Journal of the Bioflux Society
The antioxidant properties of the Philippine
medicinal plants Cassia sophera Linn.,
Derris elliptica Benth, Ficus minahassea Tesym.
and De Vr., Leea aculeata Blume and
Leucosyke capitellata Wedd
Mylene M. Uy, Marvelous G. L. Villazorda
Department of Chemistry, Mindanao State University - Iligan Institute of Technology,
Iligan City, Philippines. Corresponding author: M. M. Uy, mylene603@yahoo.com
Abstract. The search for natural antioxidants has received much attention and efforts have been made
to identify and validate plant sources of these natural products. In this study, the antioxidant activities of
the decoction, crude ethanol and 50:50 ethanol-water extracts from the leaves of Cassia sophera Linn.,
Derris elliptica Benth, Ficus minahassea Tesym. & De Vr., Leea aculeata Blume and Leucosyke capitellata
Wedd were determined. The results obtained from the in vitro antioxidant screening of the plant leaf
extracts using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method, total phenolic contents
by Folin-Ciocalteu method and phosphomolybdenum method provide good correlation coefficient (r =
0.83288). The highly ranked plant species in all methods used are from the decoction, crude ethanol and
50:50 ethanol-water extracts of the plant L. capitellata and L. aculeate with interestingly comparable
antioxidant profiles with those of the reference standards ascorbic acid and butylated hydroxytoluene
(BHT). Results indicate that natural antioxidants and free radical scavengers can be obtained from these
species.
Key Words: Natural products, free radical scavengers, total phenolic content, phosphomolybdenum
method, folkloric remedies.
Introduction. Plants are known to be rich in biologically active substances, many of
which exhibit outstanding antioxidant activity which consequently may become a
potential source of herbal drugs and supplements (Sati et al 2010; Brewer 2011; Kasote
et al 2015). In the Philippines, plants with medicinal values are gaining popularity and
getting recognition. To advance knowledge on specific plant chemical constituents,
assays such as antioxidant screenings are being conducted to determine the actual value
of folkloric remedies.
Cassia sophera Linn. is a plant belonging to the family Caesalpiniaceae. The juice
of the plant’s leaves are used for ringworm, externally used for washing syphilitic sores
and dropped into ears invaded by insects (Aminabee & Lakshmana Rao 2012). Internally,
it is used as expectorant for coughs while the decoction of the whole plant is used as
expectorant in acute bronchitis (Hudaib et al 2008). The roots are used for snake bites
(Hudaib et al 2008). Derris elliptica Benth is a member of a Fabaceae (pea) family. It has
been used as a fish poison and as an effective pesticide (Starr et al 1999). Scrap of the
root with a little opium is an abortifacient while infusion or decoction of roots with
coconut oil may be applied to itchy lesions (Khan et al 2006). A plaster of the root is used
for abscesses and leprosy (Wiwattanapatapee et al 2009). Ficus minahassea Tesym. & De
Vr. belongs to the Moraceae/Ficeae family. The bark decoction has astringent properties
(Koh et al 2009). The decoction of boiled roots in water is administered three times daily
to enhance milk production in lactating mother; also for relief muscle pains or for fatigue
in women (Olowa et al 2012). Leea aculeata Blume is a member of Vitaceae family, a
species in the genus Leea. It is used in aid of fever, after childbirth, headache and used
for poulticing (http://www.asianplant.net/Vitaceae/Leea_indica.htm). Leucosyke capitellata

AAB Bioflux, 2015, Volume 7, Issue 3.
http://www.aab.bioflux.com.ro 151
(Prior) Wedd belongs to the Urticaceae family. Its root extract is a cure for phthisis,
cough, headache and gastralgia (http://www.stuartxchange.com/Alagasi.html). This
plant is believed to have potential to treat diabetes (Ahmad & Ismail 2003) with its leaf
extracts which possess antihyperglycaemic effect evidenced in diabetic rats (Ling 2008).
Antioxidants have been shown to inhibit the propagation of free radical reactions,
protect the human body from diseases like heart disease, stroke, arteriosclerosis,
diabetes, and cancer (Tanizawa et al 1992; Hertog et al 1993; Duh 1998). Some studies
showed that people with low intakes of antioxidant-rich fruits and vegetables were at
greater risk for developing these chronic conditions (Pyrzynska & Pekal 2013). Plants are
known to be rich in biologically active substances, many of which exhibit antioxidant
activity (Krishnaiah et al 2011). Antioxidant activity screening provides information on
potential plant material having specific and effective antioxidant properties. These plants
can then be used to maintain cellular health (Gulcin 2012).
This work evaluated the antioxidant activities of the ethanol, 50:50 ethanol:water
and water extracts of the leaves of the five plants of interest with the aim of bringing
forward the continuing search for naturally-occurring antioxidants in plants.
Material and Method
Plant materials. Plant leaf samples were collected from as follows: C. sophera (Binuni,
Demologan Bacolod Lanao del Norte, Philippines), D. elliptica (Pangangan, Bacolod,
Lanao del Norte, Philippines), F. minahassea (Merila, Barangay Ubaldo Laya, Iligan City,
Philippines), L. aculeata (Kibanggay Mount Kitanglad, Bukidnon, Philippines) and L.
capitellata (Pandanan, Sultan Naga Dimaporo, Lanao del Norte, Philippines). The
collected plant samples were authenticated and identified by Tabaranza A. E., a
taxonomist from the Department of Biology, MSU-IIT, Iligan City, Philippines. These were
thoroughly cleaned, air-dried for two or more weeks, and then homogenized into fine
powder.
Plant extraction. The ground leaves (1 kg) of each plant sample were soaked
separately with absolute ethanol (about 2.5 L) and 50:50 (1L:1L) ethanol-water for
three days. The solution was filtered, concentrated under a rotary evaporator at ambient
temperature and freeze-dried to yield the crude ethanol and aqueous extracts. The
decoctions were prepared by boiling small pieces of fresh leaves (1 kg) of the plant
samples for 15 minutes with sufficient amount of distilled water (1 kg : 2 L), cooled,
filtered and freeze-dried.
DPPH radical scavenging assay. The DPPH radical scavenging activity of each of the
test plant extracts was examined by comparison with that of known antioxidant butylated
hydroxytoluene (BHT) as described by Lee & Shibamoto (2001). Four concentrations (25,
50, 100, and 500 ppm) were added with 3 mL of methanolic solution of DPPH (0.1 mM).
The solution was shaken vigorously and then allowed to stand at room temperature for
one hour. Three trials were done for each plant extract. Absorbance was measured at 517
nm against methanol as a blank in the spectrophotometer. The effective concentration of
sample required to scavenge DPPH radical by 50% (EC50) was obtained by linear
regression analysis of dose-response curve plotting between percent inhibition and
concentration. The percent of DPPH discoloration of the samples was calculated according
to the formula:
100
A
A -A
activity lAntiradica
control
samplecontrol x
Total antioxidant activity assay. The total antioxidant activity of the extracts was
evaluated by the phosphomolybdenum method as described by Prieto et al (1999).
Briefly, 0.3 mL extract solution at 200 µg/mL was added with 3.0 mL reagent solution
(6M H2SO4, 28 mM sodium phosphate, 4 mM ammonium molybdate), incubated at 95oC

AAB Bioflux, 2015, Volume 7, Issue 3.
http://www.aab.bioflux.com.ro 152
for 90 minutes and cooled to room temperature. Its absorbance was read at 695 nm
using a spectrophotometer. Three trials were done for each test extract. The antioxidant
activity of the test plant extracts were expressed as ascorbic acid equivalents (AAE) and
butylated hydroxytoluene equivalents (BHTE) using their corresponding calibration
curves.
Total phenolics content assay. The total phenolic content of each sample was
determined by combining 0.1 mL (0.5 µg/mL) of the sample with 2.8 mL of 10% Na2CO3
and 0.1 mL of 2N Folin-Ciocalteu reagent as described in Makkar et al (1993). After 40
minutes, absorbance at 725 nm was measured. Total phenolics was determined as gallic
acid equivalence (GAE) in milligrams per gram of sample by computing from a standard
calibration curve constructed for different concentrations of gallic acid (25-200 mg g-1).
Results and Discussion
DPPH radical scavenging assay. The DPPH radical scavenging activities of the plant
extracts were examined by comparison with that of a known antioxidant BHT. The radical
scavenging capacity resulted in a color change from purple to yellow, which was
measured spectrophotometrically. The disappearance of the purple color was monitored
after 1 h at 517 nm. The plant extracts’ scavenging activities against the DPPH radical are
shown in Table 1.
Table 1
DPPH radical-scavenging activities of the plant leaf extracts at various concentrations
Antiradical activity, %*
Plant/
Standard Extracts Code 25
ppm 50
ppm 100
ppm 500
ppm EC50, ppm
Decoction CsD 7.27 7.70 9.53 22.22 >500.00
Ethanol CsE 0.00 0.00 0.49 11.11 >500.00
C. sophera EtOH:H2O CsA 0.00 0.00 2.00 19.61 > 500.00
Decoction DeD 12.18 19.71 37.54
91.72 229.85
Ethanol DeE 0.00 1.54 5.51 30.79 >500.00
D. elliptica EtOH:H2O DeA 2.11 6.66 14.55
71.60 350.05
Decoction FmD 12.33 12.54 17.63
39.47 >500.00
Ethanol FmE 1.61 3.72 5.51 30.79 >500.00
F. minahassea
EtOH:H2O FmA 2.11 6.66 14.55
71.60 426.71
Decoction LaD 22.22 34.92 59.77
93.40 149.02
Ethanol LaE 17.53 27.33 53.92
93.80 181.61
L. aculeata EtOH:H2O LaA 29.86 51.95 83.15
92.40 40.45
Decoction LcD 6.88 8.81 17.31
64.18 445.84
Ethanol LcE 0.00 0.00 0.14 14.44 >500.00
L. capitellata EtOH:H2O LcA 45.93 47.29 51.95
90.61 74.95
BHT - - 18.78 39.29 68.41
93.80 128.97
* - mean of triplicate analysis.
The results show that the radical-scavenging activities of all the extracts as well as those
of the positive control were concentration-dependent. At the lowest concentration tested,
the EtOH:H2O extract of L. capitellata exhibited a higher activity than that of BHT.
Furthermore, four plant extracts showed similar radical-scavenging potency as the
control at 500-ppm concentration. These are the decoction of D. elliptica, the EtOH:H2O
extract of L. capitellata and all the extracts of L. aculeata. Accordingly, the effective
median concentration (EC50) values of two of these extracts are even lower than that of
BHT. In fact, the results indicate that the EtOH:H2O extract of L. capitellata has a

AAB Bioflux, 2015, Volume 7, Issue 3.
http://www.aab.bioflux.com.ro 153
radical-scavenging activity that is three times more potent than that of the known
antioxidant BHT.
Total antioxidant activity assay. The phosphomolybdenum method usually detects
antioxidants such as ascorbic acid, some phenolics, tocopherols and carotenoids. The
total antioxidant capacity of the plant extracts was measured spectrophotometrically at
695 nm and is based on the reduction of Mo (VI)–Mo (V) by the antioxidants with
subsequent formation of a green phosphate/Mo (V) complex at acid pH (Brighente et al
2007). As illustrated in Figures 1 & 2, plant extracts with high hydrophilic antioxidant
contents consistently exhibited relatively high lipophilic antioxidant contents as indicated
by their AAE and BHTE values, respectively.
Figure 1. Total antioxidant capacities of the leaf extracts at 200-ppm concentration
expressed as ascorbic acid equivalents (AAE) arranged in decreasing order.
Figure 2. Total antioxidant capacities of the leaf extracts at 200-ppm concentration
expressed as butylated hydroxytoluene equivalents (BHTE) arranged in decreasing order.

AAB Bioflux, 2015, Volume 7, Issue 3.
http://www.aab.bioflux.com.ro 154
In general, all plant extracts have higher BHTE values than AAE values indicating that
they possess more lipophilic antioxidant than hydrophilic antioxidants. Moreover, the top
five plant extracts in terms of total antioxidant capacities expressed both as AAE and
BHTE values are the ethanol extract of L. aculeata, LaE (69 AAE , 120 BHTE), the
EtOH:H2O extract of L. capitellata, LcA (66 AAE, 115 BHTE), the decoction of L. aculeata,
LaD (60 AAE, 104 BHTE), the ethanol extract of D. elliptica, DeE (60 AAE, 103 BHTE) and
the ethanol extract of L. capitellata, LcE (55 AAE, 96 BHTE).
Total phenolics content assay. It has long been recognized that phenolic compounds
have strong ability to scavenge radicals, thereby protecting cells against the detrimental
effects of reactive oxygen species (ROS) (Hogan et al 2009). The plant extracts were
subjected to determination of the total phenolics content measured spectrophotometrically
and expressed as Gallic Acid Equivalence (GAE, mg gallic acid/g sample). The antioxidant
properties of plant extracts can be attributed to their phenolic compounds (Soong &
Barlow 2004; Ismail et al 2004; Song et al 2010; de Oliveira et al 2012). Among the
plant extracts tested, the top five highest values were shown by the decoction of L.
aculeata (LaD, 422.76 GAE), the EtOH:H2O extract of L. capitellata (LcA, 335.62 GAE),
the decoction of L. capitellata (LcD, 278.00 GAE), the ethanol extract of L. aculeata
(233.71 GAE) and the EtOH:H2O extract of F. minahassea (FmA, 215.62 GAE). The
presence of phenolic compounds is reflected by the type of plant extracts having the
highest GAE values; the polar extracts contain more of these compounds since such
compounds are also relatively polar. Plant leaf extracts having more phenolics content is
generally believed to show good antioxidant activity (Brighente et al 2007). It can be
stated that phenolics content of the plant may be a good indicator of its antioxidant
capacity (Chanda & Dave 2009).
Conclusions. Linear correlations between the DPPH radical scavenging capacity, total
phenolic content and the total antioxidant activity of the studied five plant leaf extracts
emphasize that these in vitro assays are convenient and reliable for the determination of
the antioxidant profile of plant leaf extracts. The quantification of phenols and total
antioxidant activity was based on the standard curve generated by the use of GAE (R2 =
0.9939), BHTE with R2 = 0.9999 and AAE with R2 = 1, respectively. Among the five
plants of interest, the highly ranked antioxidant profiles of plant species L. capitellata and
L. aculeata were noteworthy. The antioxidant results obtained for the remarkable plant L.
aculeata may be a significant factor for its folkloric uses as poulticing and its aid for fever
(http://www.asianplant.net/Vitaceae/Leea_indica.htm). A recent study has established
the presence of terpenoids, cardiac glycosides and flavonoids in the leaves of L. aculeata
(Lagunay & Uy 2015) which could have contributed to these significant antioxidant
properties. The antioxidant activity result obtained for L. capitellata is in agreement with
the studies conducted by Ling (2008) wherein leaf extracts were evaluated to have high
total phenolics content and antioxidant activity values.
In vitro assays can only rank antioxidant activities for their particular reaction
systems and their relevance to in vivo health protective activities is uncertain (Niki 2011;
Apak et al 2013). Thus, it is recommended to include at least one in vivo assay that has
biological relevance. Further purification and isolation of the noteworthy plants in terms
of antioxidant activities specifically the L. capitellata and L. aculeata is also highly
recommended.
Acknowledgements. The authors are grateful to the Science Education Institute and
Philippine Council for Health Research and Development of the Department of Science
and Technology of the Philippine government for the financial and technical support
provided for the duration of the study.

AAB Bioflux, 2015, Volume 7, Issue 3.
http://www.aab.bioflux.com.ro 155
References
Ahmad F. B., Ismail G., 2003 Medicinal plants used by Kadazandusun communities
around Crocker range. ASEAN Review of Biodiversity and Environmental
Conservation (ARBEC), pp. 1-10.
Aminabee S. K., Lakshmana Rao A., 2012 A plant review of Cassia sophera Linn.
International Journal of Pharmaceutical, Chemical and Biological Sciences
2(3):408-414.
Apak R., Gorinstein S., Böhm V., Schaich K. M., Özyürek M., Güçlü K., 2013 Methods of
measurement and evaluation of natural antioxidant capacity/activity (IUPAC
Technical Report). Pure Appl Chem 85(5):957-998.
Brewer M. S., 2011 Natural antioxidants: sources, compounds, mechanisms of action,
and potential applications. Compr Rev Food Sci Food Saf 10(4):221-247.
Brighente I. M. C., Dias M., Verdi L. G., Pizzolatti M. G., 2007 Antioxidant activity and
total phenolic content of some Brazilian species. Pharm Biol 45:156-161.
Chanda S., Dave R., 2009 In vitro models for antioxidant activity evaluation and some
medicinal plants possessing antioxidant properties: An overview. Afr J Microbiol Res
3(13):981-996.
de Oliveira A. M. F., Pinheiro L. S., Pereira C. K. S., Matias W. N., Gomes R. A., Chaves
O. S., de Souza M. F. D., de Almeida R. N., de Assis T. S., 2012 Total phenolic
content and antioxidant activity of some Malvaceae family species. Antioxidants
1:33-43.
Duh P.-D., 1998 Antioxidant activity of burdock (Arctium lappa Linné): It’s scavenging
effect on free- radical and active oxygen. J Am Oil Chem Soc 75(4):455-463.
Gulcin I., 2012 Antioxidant activity of food constituents: an overview. Arch Toxicol
86:345–391.
Hertog M. G. L., Feskens E. J. M., Hollman P. C. H., Katan M. B., Kromhout D., 1993
Dietary antioxidant flavonoids and risk of coronary heart disease: the Zupthen
Elderly Study. Lancet 342(8878):1007-1014.
Hogan S., Zhang L., Li J., Zoecklein B., Zhou K., 2009 Antioxidant properties and
bioactive components of Norton (Vitis aestivallis) and Cabernet Franc (Vitis vinifera)
wine grapes. LWT-Food Sci Technol 42(7):1269-1274.
Hudaib M., Mohammad M., Bustanji Y., Tayyem R., Yousef M., Aburjaie M., Aburjai T.,
2008 Ethnopharmacological survey of medicinal plants in Jordan, Mujibnature
reserve and surrounding area. J Ethnopharmacol120:63-71.
Ismail A., Marjan Z. M., Foong C. W., 2004 Total antioxidant activity and phenolic content
in selected vegetables. Food Chem 87(4):581–586.
Kasote D. M., Katyare S. S., Hegde M. V., Bae H., 2015 Significance of antioxidant
potential of plants and its relevance to therapeutic applications. Int J Biol Sci
11(8):982-991.
Khan M. R., Omoloso A. D., Barewai Y., 2006 Antimicrobial activity of the Derris elliptica,
Derris indica and Derris trifoliate extractives. Fitoterapia 77(4):327-330.
Koh H. L., Chua T. K., Tan C. H., 2009 A guide to medicinal plants. An illustrated,
scientific and medicinal approach. World Scientific Publishing Co. Pte. Ltd.,
Singapore, 79 pp.
Krishnaiah D., Sarbatly R., Nithyanandam R., 2011 A review of the antioxidant potential
of medicinal plant species. Food and Bioproducts Processing 89(3):217-233.
Lagunay R. A. E., Uy M. M., 2015 Evaluation of the phytochemical constituents of the
leaves of Ficus minahassae Tesym & De Vr., Casuarina equisetifolia Linn.,
Leucosyke capitellata (Pior) Wedd., Cassia sophera Linn., Derris elliptica Benth.,
Cyperus brevifolius (Rottb.) Hassk., Piper abbreviatum Opiz., Ixora chinensis Lam.,
Leea aculeata Blume, and Drymoglossum piloselloides Linn. ABB Bioflux 7(1):51-58.
Lee K. G., Shibamoto T., 2001 Antioxidant property of aroma extract isolated from clove
buds [Syzygium aromaticum (L.) Merr. et Perry]. Food Chem 74:443-448.
Ling L., 2008 Evaluation of anti-hyperglycemic effect of Leukosyke capitellata leaf in
normal and streptozotocin - induced diabetic rats. BSc Thesis, Conservation Biology
Programme, School of Science and Technology, University Malaysia Sabah.

AAB Bioflux, 2015, Volume 7, Issue 3.
http://www.aab.bioflux.com.ro 156
Makkar H. P. S., Blummel M., Borrowy N. K., Becker K., 1993 Gravimetric determination
of tannins and their correlation with chemical and protein precipitation methods. J
Sci Food Agric 61:161-165.
Niki E., 2011 Do free radicals play causal role in atherosclerosis? Low density lipoprotein
oxidation and vitamin E revisited. J Clin Biochem Nutr 48(1):3–7.
Olowa L. F., Torres M. A. J., Aranico A. C., Demayo C. G., 2012 Medicinal plants used by
the Higaonon tribe of Rogongon, Iligan City, Mindanao, Philippines. Advances in
Environmental Biology 6(4):1442-1447.
Prieto P., Pineda M., Aguilar M., 1999 Spectrophotometric quantitation of antioxidant
capacity through the formation of a phosphomolybdenum complex: specific
application to the determination of vitamin E. Anal Biochem 269(2):337–341.
Pyrzynska K., Pekal A., 2013 Application of free radical diphenylpicrylhydrazyl (DPPH) to
estimate theantioxidant capacity of food samples. Anal Methods 5:4288-4295.
Sati S. C., Sati N., Rawat U., Sati O. P., 2010 Medicinal plants as a source of
antioxidants. Research Journal of Phytochemistry 4(4):213-224.
Soong Y., Barlow P. J., 2004 Antioxidant activity and phenolic content of selected fruit
seeds. Food Chem 88(3):411–417.
Song F., Gan R., Zhang Y., Xiao Q., Kuang L., Li H., 2010 Total phenolic contents and
antioxidant capacities of selected Chinese medicinal plants. Int J Mol Sci
11(6):2362-2372.
Starr F., Martz K., Loope L., 1999 Poison vine (Derris elliptica): An alien plant report.
United States Geological Survey Biological Resources Division, USA.
Tanizawa H., Ohkawa Y., Takino Y., Miyase T., Ueno A., Kageyama T., Hara S., 1992
Studies on natural antioxidants in citrus species I. Determination of antioxidative
activities of citrus fruits. Chem Pharm Bull (40):1940-1942.
Wiwattanapatapee R., Sae-Yun A., Petcharat J., Ovatlarnporn C., Itharat A., 2009
Development and evaluation of granule and emulsifiable concentrate formulations
containing Derris elliptica extract for crop pest control. J Agric Food Chem
57(23):11234-11241.
*** http://www.stuartxchange.org/Alagasi.html (Accessed 1.10.2014)
*** http://www.asianplant.net/Vitaceae/Leea_indica.htm (Accessed 1.10.2014)
Received: 28 July 2015. Accepted: 29 September 2015. Published online: 09 October 2015.
Authors:
Mylene Mondarte Uy, Mindanao State University - Iligan Institute of Technology, Department of Chemistry,
Philippines, Iligan City, 9200, Andres Bonifacio Avenue, e-mail: mylene603@yahoo.com
Marvelous Grace Leyson Villazorda, Mindanao State University - Iligan Institute of Technology, Department of
Chemistry, Philippines, Iligan City, 9200, Andres Bonifacio Avenue, e-mail: villazordamarvelous@gmail.com
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.
How to cite this article:
Uy M. M., Villazorda M. G. L., 2015 The antioxidant properties of the Philippine medicinal plants Cassia sophera
Linn., Derris elliptica Benth, Ficus minahassea Tesym. and De Vr., Leea aculeata Blume and Leucosyke
capitellata Wedd. AAB Bioflux 7(3):150-156.