Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

COMPARATIVE STUDY ON THE PHYTOCHEMICAL, PHENOLIC, AND ANTIOXIDANT

PROFILES OF THE LEAF, ROOT, AND STEM BARKS OF TERMINALIA GLAUCESCENS
(PLANCH.EX BENTH)
Ukwueze, Stanley Ejike and Ekpemogu, Doris Uchenna.
Department of pharmaceutical and medicinal chemistry, Faculty of pharmaceutical Sciences, University of Port Harcourt,Port
Harcourt, Nigeria.
ARTICLE INFO
ABSTRACT
The aim of the present study was to evaluate and compare the relative contributions of
Article history
Received 20/11/2014
different polyphenols to the antioxidants activities of different parts of the plant Terminalia
Available online
glaucescens. Preliminary phytochemical screening of the crude extracts and solvent fractions
25/12/2014
of the root, stem and leaves of the plant was carried out using standard procedures.
Quantitative determination of the total phenolic and flavonoid contents (TPC and TFC) of the
test materials were carried out using Folin Ciocalteu and aluminum chloride colorimetric
methods respectively. The in-vitro antioxidant activity was investigated using in-vitro
Keywords
Terminalia Glaucescens,
antioxidant model; 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay. The results showed that the
Total Phenolic Content,
TPC of the methanol fraction of the leaves (96.500.25 mg GAE/g extract) were significantly
Total Flavonoid Content,
higher than the other fractions. Flavonoids were found more in the leaf than those from other
Antioxidant Activity.
plant parts with the exception of the ethyl acetate fraction from the stem which showed a
slightly higher content. Phytochemical screening of T. glaucescens parts revealed the
presence of anthraquinones, cardiac glycosides, flavonoids, saponins, phlobatannins and
carbohydrates at varying degrees. The IC50 value based on the DPPH of methanol extract of
the leaves (2.971.0 mg/ml) was the lowest when compared to other plant parts or solvent
fractions suggesting potentially better antioxidant properties. A marginally positive
correlation was found between TPC and IC50 values for DPPH and TFC. These data therefore
revealed that the alcoholic extract of the leaves of Terminalia glaucescens should be
preferentially utilized for health conditions where the antioxidant activity of the plant may be
desirable.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
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Please cite this article in press as UKWUEZE & EKPEMOGU et al. Comparative Study on the Phytochemical, Phenolic, and
Antioxidant Profiles of the Leaf, Root, and Stem Barks of Terminalia glaucescens (planch.ex benth. Indo American Journal of
Pharm Research.2014:4(12).

5801

Corresponding author
UKWUEZE & EKPEMOGU
Department of pharmaceutical Chemistry,
Faculty of Pharmaceutical Sciences,
University of Port Harcourt,
Choba, Rivers State, Nigeria.
stanley.ukwueze@uniport.edu.ng;
stanejike@yahoo.com
+2347031044254

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INTRODUCTION
Antioxidants are both natural and synthetic compounds, capable of scavenging free radicals and inhibiting oxidative
processes caused by free radicals. [1] Antioxidants scavenge species that initiate peroxidation, chelating metal ions such that they are
unable to generate reactive species or decompose lipid peroxides, quenching O2 and preventing formation of peroxides leading to
breakage of the auto-oxidative chain reaction, and/or reducing localized O2 concentrations[2]. Plants synthesize a vast range of organic
compounds that are traditionally classified as primary and secondary metabolites. Phenolic secondary metabolites have been
associated with beneficial health effects which have been attributed to their antioxidant activity [3, 4, 5]. They could be a major
determinant of antioxidant potentials of many foods [4], and could therefore be natural sources of antioxidants. Plant polyphenols are
known to have multi-functional properties such as reducing agents, hydrogen donating antioxidants and singlet oxygen quenchers.
Flavonoids and their derivatives are the largest and most important group of polyphenols. Their most important property is the
capacity to act as antioxidants protecting the body against reactive oxygen species.
Terminalia glaucescens is a locally abundant plant found in tropical savannah regions. The tree grows up to a height of 20
metres with a deeply fissured dark bark that rapidly turns darker. Its bark is used in southern Nigeria as chewing stick. The rootdecoction is taken in Ivory Coast for diarrheas and dysenteries while the powdered root is applied to gums for toothache. The plant has
been reported to have potential for oral infection treatment [6], employed in local dental hygiene [7], and found to show impressive
activity against broad spectrum organisms [8].
MATERIALS AND METHOD:
Plant material
The leaves, stem and root barks of the plant Terminalia glaucescens were collected from a town Iju in Ondo State, Nigeria.
The plant was authenticated at the Forest Research Institute of Nigeria (FRIN), Ibadan where voucher specimen was deposited and
voucher number FHI 109741 allocated.
Reagents Used
Analytical grade solvents such as methanol, chloroform, n-hexane, and ethyl acetate (all sourced from Sigma-Aldrivir,
Germany) were used. Freshly prepared laboratory standard reagents were also employed.
Extraction of the Plant Material
One hundred gram (100g) of the pulverized dried leaves, stem and root barks of the plant were each defatted by maceration
with (1.5L) of n-hexane for 72 hours. The filtrates were collected and concentrated in vacuo using a rotary evaporator. The resulting
concentrates were then dried on a water bath at 40c, while the marcs were air dried. The dried marcs (95.0g, 92.5g and 84.5g of the
leaf, stem and root extracts respectively) were each continuously extracted with methanol (1L) using Soxhlet extractor for 48hours,
after which the filtrates were collected, concentrated in vacuo and dried as above.
Fractionation Method
5.3, 6.5 and 7.4g (leaf, stem and root respectively) of the methanol extracts obtained from previous extractions were
separately adsorbed on silica gel (60-120 mesh, 250g), packed in a glass column (3.5 X 75cm) with the bed of 30cm height and eluted
in succession with 2.5L and 1.5L of chloroform and ethyl acetate respectively to obtain the chloroform and ethyl acetate fractions.
Aliquots of 25ml were collected and monitored with TLC and phytochemical reactions. Aliquots containing mixtures of chloroform
and ethyl acetate eluents were discarded while similar solvent fractions were combined, concentrated in vacuo and air-dried. The
extracts/fractions (n-hexane, methanol, chloroform and ethyl acetate of the plant parts) were weighed, stored in air-tight containers and
preserved in the refrigerator for subsequent use.

IC50 was used to determine the level of antioxidant activity.

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Dpph Radical Scavenging Activity

The DPPH scavenging activity of the leaf, stem, and root extracts was measured by colorimetric method [10]. In brief, an
aliquot of 80 L of sample solution at different concentrations (0.125 -2.00 mg/mL) was mixed with 80 L of DPPH solution (0.3
mmol/L in methanol). The reaction mixture was incubated for 30 minutes in the dark at room temperature. The absorbance of the
resulting solution was measured at 517nm with a spectrophotometer (Jenway spectrophometer 6405-uv/vis). Controls were prepared
in a similar way as for the test group except for the replacement of the test sample with the corresponding extraction solvent. Ascorbic
acid was used as the standard while all determinations were performed in triplicate. The radical scavenging capacity of the tested
samples was measured using the equation:

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Phytochemical Studies
Phytochemical tests were carried out according to the procedures outlined by Harborne [9] to detect the presence of anthraquinones,
alkaloids, tannins, glycosides, reducing sugars, flavonoids and saponins.

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Determination of Total Phenolic Content

Total phenolic contents (TPC) were quantified and expressed as Gallic acid equivalents (GAE) according to a method
proposed by Singleton et al. (1999)[11], using the Folin- Ciocalteu reagent. 1ml of standard solution of concentrations 0.01, 0.02, 0.03,
0.04 and 0.05 mg/ml of gallic acid was prepared in methanol. Concentrations of 0.1 and 1mg/ml of plant extract were also prepared in
methanol and 0.5ml of each sample was introduced into test tubes and mixed with 2.5ml of a 10-fold dilute Folin- Ciocalteu reagent
and 2ml of 7.5% sodium carbonate. The tubes were covered with parafilm and allowed to stand for 30 minutes at room temperature
before the absorbance was read at 760nm using a spectrophotometer. Gallic acid was used as a standard and the total phenolics were
expressed as mg/g gallic acid equivalents (GAE) using the formula:
C=C1 V/M.
Where;
C =Total content of phenolic compounds in mg/g in GAE;
C = concentration of gallic acid established from the calibration curve in mg/ml;
1

V= volume of extract in ml;

M= weight of plant extract in g.
Determination of Total Flavonoid Content (TFC) Using Aluminum Chloride Colorimetric Method.
The TFC was determined according to the method described by Mustofa et al. (2000)[12]. Quercetin was used to make the
calibration curve. 10 mg of quercetin was dissolved in methanol and then diluted to 6.25, 12.5, 25, 50, 80, and 100 g/ml. A
calibration curve was made by measuring the absorbance of the dilutions at 415 nm (max of quercetin) with a Jen-way 6405-uv/vis
spectrophotometer.
In brief, a volume of 0.5 mL of 2% AlCl 3 in methanol solution was added to 0.5mL of different plant extracts. After 1hr incubation at
room temperature, the absorbance was measured at 415nm. Appearance of yellow color indicated the presence of flavonoids. The
extracts/fractions were evaluated at a final concentration of 0.1g/mL. Total flavonoid contents were calculated as quercetin equivalent
(mg/g) using the formula:
TFC = R x D.F x V x 100 W
Where:
R - Result obtained from the standard curve
D.F - Dilution factor
V - Volume of stock Solution 100 - For 100 g dried plant
W - Weight of plant used in the experiment
Determination of Total Flavonols
Total flavonols in the plant extracts were estimated by standard method [13]. Briefly, 1.0mL of 2% AlCl3 in methanol and
1.5mL sodium acetate (50g/L) solutions were added in 0.10mL of leaves, stem, and root extract solutions. The absorption at 440nm
was monitored after 2.5hr of incubation at 20C. The sample extracts were evaluated at a final concentration of 0.1mg/mL. Total
flavonol content was calculated as quercetin equivalent (mg/g) using the same formula as that of total flavonoid.
RESULTS AND DISCUSSIONS
The results of the extraction and fractionation procedures are as presented in Table 1.
Table 1: Yield of Various Plant Part Extracts and Solvent Fractions.

Root
1.47 (1.47)
0.26 (0.31)
2.45 (2.99)
7.43 (10.18)

The phytochemical screening of the different solvent fractions showed the presence of the tested constituents in varying
degrees (Table 2). With the exception of alkaloids, all other phyto-constituents tested for were present in the leaves, stem, and root of
the ethyl acetate fractions. The result of phytochemical screening for methanol fraction of the plant showed similar result with that of
ethyl acetate except that the methanol fraction of stem showed the presence of phlabotannins. For n-hexane extract, it was observed
that flavonoids, phlabotannins and saponins were absent in the leaves, stem and root while carbohydrate and tannins were found
present in the leaf and root. The root and stem contained cardiac glycosides while reducing sugars were found in abundance in the
leaves.

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Yield [g (% in parenthesis)]
Leaves
Stem
1.47 (1.47) 2.44 (2.44)
0.71 (0.75) 0.63 (0.68)
4.0 (4.3)
1.14 (1.27)
5.32 (5.91) 6.9 (8.73)

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Solvent
n-hexane
Chloroform
Ethyl acetate
Methanol

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Table 2: Comparison of phytochemical constituents present in the pulverized parts (leaves, stem and root) and various solvent
extract of Terminalia glaucescens.
Chemical
class

Plant part

Alkaloids

Anthraquinones

Carbohydrate

Flavonoids

Reducing sugar

Saponins

Tannins
Cardiac glycoside

Phlabotannis

Leaves
Stem
Root
Leaves
Stem
Root
Leaves
Stem
Root
Leaves
Stem
Root
Leaves
Stem
Root
Leaves
Stem
Root
Leaves
Stem
Root
Leaves
Stem
Root
Leaves
Stem
Root

Crude Powder

n-hexane extract

+
+
+

+
+
+
+
+
+
+
-

+
+
+
+
+
+
+
+
+
+

+
+

+
+
-

Chloroform
extract
+
+
+
+
+
+
+
-

Ethyl acetate
extract
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Methanol
Extract
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Key: + = present; - = absent

It is well known that plant polyphenols are widely distributed in the plant kingdom and that they are sometimes present in
surprisingly high concentrations [14]. Plant phenolics are a major group of compounds acting as primary antioxidants and free radical
scavengers. They are characterized by the presence of several phenol groups and act by donating a hydrogen atom or an electron
which makes them very reactive in neutralizing free radicals and chelating metal ions in aqueous solutions [15]. The result of the
present study equally showed that phenolic compounds were the major compounds with antioxidant activity and this could be
attributed to their redox properties [16]. Quantitative analysis of total phenolic content (TPC) of various crude extracts of Terminalia
glaucescens showed that the methanol extract of leaf (97.00mgGAE/g) had the highest concentration while that of n-hexane extract of
root (25.00mgGAE/g) had the least amount (Table 3 and Figure 1).
Table 3: Total Phenolic Content of different solvent extract in different plant parts.

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The results showed that TPC increases as the concentration of the extract increases. It also increases as the polarity of the
extracting solvent increases. The TPC of the methanol leaf fraction was thus higher than that of the stem fraction while that of the root
was the least.

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Total phenolic content ( GAE equivalent mg/g dry material)

Solvent
Leaves
Stem
Root
n-hexane
36 .000.02
26.000.00 25.000.00
Chloroform
61.000.05
34.500.35 32.000.01
Ethyl acetate
93.000.02
44.500.11 61.000.05
Methanol
96.500.25
88.500.10 88.000.06

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Figure 1: Standard curve for gallic acid.

Flavonoids and flavonols are regarded as another of the most widely spread groups of natural constituents found in the plant.
It has been recognized that flavonoids show antioxidant activity through scavenging or chelating process [17]. Flavonoids as
antioxidants interfere with the biochemical pathways which are involved in generation of free radical (ROS), quench them, chelate
them and make them redox inactive [18]. The result of the present study indicated that the quantity of flavonoid and flavonols varied
from one part of the plant to another (Tables 4 & 5).
Table 4: Total Flavonoid Content of different Solvent Extract of different Plant Parts.
Total flavonoid content [quercetin equivalent per 100g of dry weight (% TFC in grams)]
Solvent
Leaves
Stem
Root
n-hexane
0.474
0.452
0.467
Chloroform
0.463
0.474
0.433
Ethyl acetate
0.519
0.563
0.493
Methanol
0.526
0.422
0.485

Although it was observed that flavonoids were found to be highest in the ethyl acetate fraction of stem (as against that of the
leaf extracts) but this can be due to the fact that the plant parts were harvested during the flowering season. Thus, most of the
flavonoid and phenols must have been used up during the flowering process thus making it excessively accumulated in the stem.

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Figure 2: Standard curve for Quercetin.

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Highest amounts of flavonoid were observed in the ethyl acetate fraction of stem (0.56%), methanol fraction of leaves
(0.53%) and ethyl acetate fraction of leaves (0.52%). Other fractions contained high to moderate amounts of flavonoid (Table 4 and
Figure 2).

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Table 5: Total Flavonol Content of different Solvent Extracts of various Plant Parts.
Total flavonol content [quercetin equivalent per 100g of dry weight (% TFC in grams)]
Solvent
Root
Stem
Leaves
n- hexane
0.559
0.655
0.619
Chloroform
0.563
0.548
0.848
Ethyl acetate
0.804
1.289
0.999
Methanol
0.633
0.685
1.011

Antioxidants on interaction with DPPH transfer electron or hydrogen atom to 1, 1, biphenyl -2- picrylhydrazine and the
degree of discoloration indicates scavenging activity of the drug. The reduction capacity of DPPH radical is determined by the
decrease in its absorbance at 517nm induced by antioxidants. In this study, DPPH free radical scavenging activity of the crude extracts
of plant parts and their different soluble fractions were found to increase with the increase in concentration of the fractions. Analysis
of the various solvent fractions revealed that they all had antioxidant activity as compared to the reference ascorbic acid (Table 6
&Figure 3).

FIGURE 3: Standard curve for ascorbic acid.

The methanol fraction of leaves showed the highest free radical scavenging activity with IC 50 value of 2.97mg/ml. At the
same time the ethyl acetate-soluble, chloroform and n-hexane fractions of the different parts also exhibited moderate antioxidant
potential. The high radical scavenging potential of the methanol fraction of the leaves may be attributed to the presence of highest
amount of phenolic and flavonoid contents that can provide the necessary component as radical scavengers. This supported our earlier
observation that the free radical scavenging activity of antioxidant components is very much associated with their phenolic and
flavonoids contents.
Table 6: Comparative DPPH radical scavenging activity of different extracts/fractions from the leaf, stem and root of
Terminalia glaucescens.

Conc.
(mg/ml)
0.125
0.25
0.50
1.00
2.00
IC50

methanol

Percentage (%) Inhibition

Ethyl acetate
chloroform

n-hexane

11.67a, 5.19 b, 7.79 c

14.29 a, 10.38 b, 9.09 c
25.97 a, 14.27 b, 11.69 c
27.27 a, 22.07 b, 15.58 c
36.37 a, 24.67 b, 16.87 c
2.97 a, 4.37 b, 8.68 c

10.39 a, 7.79 b, 20.78 c

19.48 a, 9.09 b, 23.38 c
23.38 a, 10.39 b, 24.68 c
27.27 a, 12.99 b, 25.97 c
33.77 a, 18.18 b, 27.27 c
3.39 a, 7.90 b, 9.68 c

3.89 a, 9.09 b, 1.29 c

5.19 a, 14.29 b, 3.89 c
7.79 a, 15.58 b, 5.19 c
12.99 a, 16.88 b, 7.79 c
15.58 a, 19.48 b, 10.37 c
7.28 a, 8.92 b, 10.89 c

12.98 a, 6.49 b, 2.56 c

9.09 a, 9.10 b, 3.89 c
10.39 a, 10.29 b, 7.77 c
11.68 a, 14.47 b, 10.37 c
24.68 a, 19.47 b, 12.97 c
4.45 a, 6.69 b, 8.77 c

Ascorbic
acid
10.39
11.69
14.29
25.97
27.27
4.17

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CONCLUSION
Terminalia glaucescens is one of the several medicinal plants used traditionally for various ailments in the tropics. The
present study showed that the leaf, stem and root barks of the plant possess significant amount of phenols and flavonoids with highest
amounts found in the methanol fraction of the leaves. This makes the plant an abundant source of natural antioxidants and also a great
therapeutic agent for the prevention and management of free radical-mediated diseases. However, we can conclude that the alcoholic
extract of the leaves of Terminalia glaucescens should be preferentially utilized for health conditions where the antioxidant activity of
the plant may be desirable.

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Key: a = leaf extract/fraction; b = stem extract/fraction; c = root extract/fraction

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Authors Statement
Competing Interests
The authors declare no conflict of interest.
REFERENCES
1. Sies H. Oxidative stress: Oxidants and antioxidants. Experimental physiology 1997; 82 (2), 291-5.
2. Davies KJ. Oxidative stress: the paradox of aerobic life. Biochemical Society Symposia 1995; 61, 1-31.
3. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease:
the Zutphen Elderly Study, Lancet 1993; 342, 1007-1011.
4. Parr AJ, Bolwell GP. Phenols in the plant and in man. The potential for possible nutritional enhancement of the diet by modifying
the phenols content or profile. J Sci Food Agric 2000; 80, 985-1012.
5. Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships, J Nutr
Biochem, 2002; 13, 572-584.
6. Siddhuraju P, Becker K. Antioxidant properties of various solvent extracts of total phenolic constituents from three different
agroclimatic origins of drumstick tree (Moringa oleifera Lam.) leaves. J Agric Food Chem 2003; 51, 2144-2155.
7. Iqbal S, Bhanger MI. Effect of season and production location on antioxidant activity of Moringa oleifera leaves grown in
Pakistan. J Food Compos Anal 2006; 19, 544551.
8. Van Wyk BE, Wink M. Medicinal Plants of the World. Pretoria, South Africa: Briza publications; 2004.
9. Harborne JB. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 3rd ed. London: Chapman and Hall;
1998.
10. Ogundiya MO, Okunade MB, Kolapo AL. Antimicrobial activities of some Nigerian chewing sticks. Ethnobotanical Leaflets
2006; 2006 (1) article 28.
11. Ingabire G, Koumaglo HK, De Souza C, Dotse CK, Anani K, Kabera J, et al. Antimicrobial activity and preliminary
phytochemical screening of Turraea heterophylla and Terminalia glaucescens used in Togo ethnomedicine to treat common
infections. Planta Med 2007; 73 (9) DOI: 10.1055/s-2007-986996.
12. Mustofa M, Valentin A, Benoit-Vical F, Pelissier Y, Kone-Bamba D, Mallie M. Antiplasmodial activity of plants used in west
African traditional medicine. J Ethnopharmacol 2000; 73, 145-151.
13. Farvin FHS, Jacobsen C. Phenolic compounds and antioxidant activities of selected specie of seaweeds from Danish coast. Food
Chem 2013; 130 (2), 394-403.
14. Akbay P, Basaran AA, Undeger U, Basaran N. In vitro immunomodulatory activity of flavonoid glycosides from Urtica dioica L.
Phytother Res 2003; 17, 34-37.
15. Kaufman PB, Cseke LJ, Warber S, Duke JA, Brielmann HL. Natural Products from Plants. Boca Raton, FL: CRC Press; 1999.
16. Kumaran A, Joel Karunakaran R. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. LWTFood Sci Tech 2007; 40, 344-352.
17. Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, van Norren K, van Leeuwen PA. Flavonoids: a review of probable
mechanisms of action and potential applications. Am J Clin Nutr 2001; 74, 418-25.
18. De Queiroz Siqueira CF, Cabral DLV, Sobrinho TJdSP, de Amorim ELC, de Melo JG, Araujo TAdS, et al. Levels of tannis and
flavonoids in medicinal plants: evaluating bioprospecting strategies. Evid Based Complement Alternat Med 2012; 2012, 434782.

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