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Comparative Study on Chemical Composition and Antioxidant Activity of

Annona Muricata Plant Parts Cultivated in Covenant University, Ota, Ogun
State, Nigeria

Article · July 2018

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ISSN: 2347-467X, Vol. 06, No. (3) 2018, Pg.

Current Research in Nutrition and Food Science

Journal Website:www.foodandnutritionjournal.org

Comparative Study on Chemical Composition and Antioxidant

Activity of Annona Muricata Plant Parts Cultivated in
Covenant University, Ota, Ogun State, Nigeria
Omotosho Omolola Elizabeth*, Iheagwam Franklyn Nonso,
Noiki Ifeoluwa Adebola and Omini Joy John

Department of Biochemistry, College of Science and Technology, Covenant University, Km 10, Idiroko
Road, Canaanland, P.M.B. 1023, Ota, Ogun State, Nigeria.

Abstract
Annona muricata plant parts possess a broad range of medicinal and
biological properties. This research compared the chemical composition Article History
and antioxidant properties of Annona muricata parts. Proximate, mineral,
total phenol and total flavonoid content as well as in vitro antioxidant activity Received: 19 September
2017
were examined. Results revealed the leaves contained significantly (p<0.05) Accepted: 10 July 2018
higher composition of moisture (8.69±0.22 %), ash (4.60±0.02 %), protein
(14.53±0.11 %), crude fat (10.28±0.03 %), chromium (0.38±0.05 mg/100g), Keywords
nickel (1.75±0.04 mg/100g), total phenol (1.01±0.03 mg pyrocatechol/mL)
Annona muricata,
and total flavonoid (1.12±0.03 mgGAE/mL) compared to the respective Proximate analysis,
values for root. Carbohydrate (9.29±0.24 %), lead (0.13±0.02 mg/100g) Mineral content,
and cobalt (1.93±0.02 mg/100g) composition was significantly lower Antioxidant,
Flavonoid,
(p<0.05) in the leaves compared to the respective compositions in the root. Scavenging activity.
The leaf and root extract exhibited a concentration-dependent increase in
hydroxyl radical scavenging activity with no observable (p<0.05) difference
in their EC50 value. This study suggests the leaves of A. muricata found in
Covenant University had better chemical composition when compared to the
root. Nonetheless, these plant parts may be further exploited for not only
their nutritive composition and mineral content but also a natural source of
antioxidant agents.

Introduction functional foods and for treatment and management

Plants are a rich bio resource of natural products of diseases.1 Despite the availability of various
and phytoconstituents which make them potent as varieties of synthetic drugs which are highly effective

CONTACT Omotosho Omolola Elizabeth [email protected] Department of Biochemistry, College of

Science and Technology, Covenant University, Km 10, Idiroko Road, Canaanland, P.M.B. 1023, Ota, Ogun State, Nigeria.

© 2018 The Author(s). Published by Enviro Research Publishers.

This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY).
Doi:
2

in curing various diseases, there are people who Materials and Methods
still prefer using traditional folk medicines and Sample Collection and Preparation
therapy as a result of their less harmful effects. The leaves and roots of Annona muricata were
There is a wide diversity of compounds found in obtained from Covenant University farm, Canaan
plants, especially secondary plant metabolites, land, Ota, Ogun State, Nigeria in December 2013
identified and isolated from plants.2 Various studies and identified. Samples were dried for two weeks at
have shown that these compounds exhibit various room temperature and pulverised. Dried leaf and root
properties such as anticancer, antibacterial, powder (50g) was packed into a Soxhlet apparatus
analgesic, anti-inflammatory, antitumor and antiviral. and macerated with 250mL methanol at 60–65°C
Their ethnomedicinal use by the locals, suggests for 3–4h. The extract was filtered through Whatman
easy access which makes them an easily obtainable filter paper No. 1, and the filtrate was concentrated
source of treatment.3 Annona muricata L. commonly under reduced pressure at 40°C using a rotary
known as soursop, graviola, guanabana and sirsak evaporator.
which belongs to the Annonaceae family, is found
in tropical and subtropical regions such as South Chemicals and Reagents
and Central America, Asia and Africa including Sodium Carbonate (Na 2CO 3), Sodium Nitrate
Nigeria.1 Soursop is the most versatile fruit from the (NaNO3), Nitric Acid (HNO3), Aluminium Chloride
Annonaceae family utilised in industrial processes (NaOH) and Hydrogen Peroxide (H 2O 2), were
due to its low oxidising rate as well as a huge retrieval purchased from Merck, Germany. All other chemicals
of fruit pulp during processing. The fruit is 15-30 cm used were of analytical grade.
long with an oval irregular shape and sparse soft green
curved spines. It has a white cotton like mesocarp Proximate Analysis
which is fibrous, contains shiny black seeds, a Leaves and root of A. muricata were pulverised
sour taste, pleasing flavour and aroma.4 Extensive to uniform size and analysed for moisture, crude
phytochemical evaluations carried out on Annona protein, crude fat, ash, and crude fibre composition
muricata L. different parts (leaves, roots, fruits, according to the standard method of AOAC
stem, bark and seeds) have shown the presence 15.950.01, 15.976.05, 15.920.39, 15.955.03 and
of several bioactive compounds such as alkaloids, 15.962.09, respectively as described below.14
megastigmanes, flavonol triglycosides, phenolics,
cyclopeptides, essential oils and annonaceous Determination of Moisture Content
acetogenin compounds. Major minerals such as K, Dry crucible in the oven and cool in the desiccator
Ca, Na, Cu, Fe and Mg have been reported to be for 15 minutes. Weigh empty petri dish to get (W1),
present5, which makes them ideal dietary sources of Weigh in 1.5 g (liquid samples) or 2 g (solid samples)
electrolytes, essential nutrients and elements which into the petri dish and record as (W2) Put in oven for
are utilised in various biological processes.4 The 6-12 hrs at 105 oC. Remove and cool in desiccators.
fruit is used to produce juice, candy and sherbets.6. Weigh after cooling to get (W3)
The leaves and seeds are known to possess anti-
arthritic7, anti-cancer8, anticonvulsant9, antidiabetic Calculation
and hypolipidemic10, anti-inflammatory and anti- % Moisture=(W2-W3)/(W2-W1) × 100
nociceptive11, antioxidant12, antihypertensive13 and
antiparasitic14 activities. The roots are known for W1 = weight of empty crucible.
their antihelmintic, antiphlogistic14, antiparasitic and W2 = weight of sample and crucible.
pesticidal properties.6 Despite a broad study on W3 = weight of after drying in the oven
these plant parts, there is a paucity of information on
the comparative chemical composition of the plant Determination of Crude Protein
parts. Hence, the current study was carried out to 0.5-1 g of dried samples was taken in digestion flask.
compare the proximate composition, mineral content 10-15 mL of concentrated H2SO4 and 8 g of digestion
and in vitro antioxidant activity of Annona muricata mixture i.e. K2SO4:CuSO4 (8:1) was added to the
leaves and roots found in Covenant University, Ota, sample. The flask was swirled in order to mix the
Nigeria. contents thoroughly then placed on heater to start
3

digestion till the mixture became clear (blue green in was placed in muffle furnace at 550 oC for 2-4 h. The
colour). After 2 hrs the digest was cooled, transferred appearances of grey white ash indicated complete
to 100 mL volumetric flask and the volume was made oxidation of all organic matter in the sample. After
up to mark by the addition of distilled water. 10 mL of ashing furnace was switched off. The crucible was
digest was introduced in the distillation tube then 10 cooled and weighed (W3).
mL of 0.5 N NaOH was gradually added. Distillation
was continued for at least 10 min and NH3 produced Calculation
was collected as NH4OH in a conical flask containing % Ash=(W3-W2)/(W2-W1)×100
20 mL of 4% boric acid solution with few drops of
modified methyl red indicator. The distillate was then W1 = weight of empty crucible.
titrated against standard 0.1 N HCl solution till the W2 = weight of sample and crucible.
appearance of pink colour. A blank was also run W3 = weight of after drying in the oven
through all steps as above.
Determination of Crude Fibre
Calculations 0.153 g of sample was weighed (W0) and transferred
% Crude Protein=6.25x×%N (x=correction factor) to porous crucible. 150 mL of preheated H2SO4
solution and some drops of foam-suppresser was
%N=((S-B)×N×0.014×D×100)/(weight of added to each column. Sample was boiled and left
sample×V) for 30 mins. Valves were opened for drainage of acid
and rinsed with distilled water thrice to completely
S = Sample titration reading ensure the removal of acid from sample. The same
B = Blank titration reading procedure was used for alkali digestion by using
N = Normality of HCl KOH instead of H2SO4. Sample was oven dried
D = Dilution of sample after digestion at 150oC for 1 hr, then cooled in a desiccator and
V = Volume taken for distillation weighed (W1). The sample crucibles were kept in
0.014 = Milli equivalent weight of Nitrogen. muffle furnace at 55oC for 3-4hrs and later cooled
in a desiccator and weighed again (W2).
Determination of Crude Fat
1 g of moisture free sample was wrapped in filter Calculations
paper, placed in fat free thimble and then introduced
in the extraction tube. The receiving beaker was filled % Crude fibre=(W1-W2)/W0×100
with petroleum ether and fitted into the apparatus.
After 4-6 siphoning, ether was allowed to evaporate Determination of Carbohydrate
and beaker was removed before last siphoning. Carbohydrate was calculated by difference after
Extract was transferred into a clean glass dish with analysis of all the other items method in the
ether washing and evaporated ether on water bath. proximate analysis, i.e. 100 - (moisture + crude
Dish was placed in an oven at 105oC for 2 hrs and protein + crude fat + crude fibre + ash).
cooled in a desiccator.
Mineral Determination
Calculation The official method of AOAC15 was adopted for the
% Crude fat=(weight of ether sample×100)/(weight mineral analysis of the samples: 1 g of the sample
of sample) was weighed in a Vycor dish, dried for 1hr at
150oC in air forced oven and then ashed overnight
Determination of Ash (16h) at 550oC before cooling in a desiccator. One
For the determination of ash, clean empty crucible mL of HNO3 was added to dissolve the ash. The
was placed in a muffle furnace at 600oC for an sample is then transferred to a 250 mL volumetric
hour, cooled in desiccator and then weight of empty flask and made up to volume with H2O. Sodium
crucible was noted (W1). One gram of each of (Na) and potassium (K) levels of the samples were
sample was taken in crucible (W2). Then the crucible ascertained using a flame emission photometer
4

with NaCl and KCl as standards. All other metals A test is the absorbance in the presence of the
were determined by atomic absorption spectrometry sample extract.
(AAS) method.16
Statistical Analysis
Total Phenol Quantification Results were expressed as mean ± standard error of
Total phenolic content of the extracts was analysed mean (SEM) of triplicate values. Statistical analysis
using the Folin-Ciocalteau reagent method described was performed by one-way ANOVA followed by
by Lee et al.,17, with slight modification. 10 μL of Duncan test as post hoc. IBM SPSS statistics 23
the extract was added to 600 mL of distilled water was used and a probability (p) value < 0.05 was
followed by 50 μL of 10% of Folin-Ciocalteau reagent. considered to be statistically significant.
150 μL of 7% Na2CO3 was added and vortexed.
The mixture was left at room temperature for 8 Result
mins before 190 μL of water was added and kept at Proximate Composition
room temperature for 2 hrs. Absorbance was read at The proximate composition as reported in table 1 on
765 nm. Total phenolic content was calculated from a dry weight basis showed there was a significantly
the calibration curve, and the results were expressed (p<0.05) higher composition of moisture, ash,
as mg of pyrocathecol equivalent per mL. crude fat and protein in the leaves (8.69±0.22 %,
4.60±0.02 %, 10.28±0.03 % and 14.53±0.11 %)
Total Flavonoid Quantification than that of the roots (2.40±0.03 %, 1.20±0.06 %,
The total flavonoid content of the crude extract was 6.46±0.04 % and 7.53±0.11 %). Carbohydrate was
determined by the aluminium chloride colourimetric significantly (p<0.05) higher in the root (27.23±
method described by Baba and Malik18, with slight 0.14 %) than the leaves (9.29±0.24 %).However,
modification. Distilled water (490 μL) was added to the crude fibre composition of leaves (52.63±
10 μL of extract, 30 μL of 5% Sodium Nitrate, 30 μL 0.36 %) was not significantly (p<0.05) different from
of 10 % AlCl and incubated at room temperature that of the roots (55.19±0.02 %).
for 5 mins. One M NaOH followed by 240 μl of H2O
was added and vortexed thoroughly. Absorbance
was read at 510 nm. The total flavonoid content Table 1: Proximate analysis of A. muricata
was calculated from a calibration curve, and the leaves and roots
result was expressed as mg gallic acid equivalent
per mL. SAMPLE LEAF ROOT

Hydrogen Peroxide Scavenging Activity Moisture (%) 8.69±0.22a 2.40±0.03b

The ability of extract to scavenge H 2 O 2 was Ash (%) 4.60±0.02a 1.20±0.06b
determined according to the method of Sharma Crude Fats (%) 10.28±0.03a 6.46±0.04b
et al., 19 with slight modification. Hundred μL Crude Fibre (%) 52.63±0.36a 55.19±0.22a
of extract was incubated with 0.6 mL of H 2O 2 Protein (%) 14.53±0.11a 7.53±0.11b
(40mM in a phosphate buffer, 0.1M pH 7.4) in Carbohydrate (%) 9.29±0.24a 27.23±0.14b
dark for 10 mins. A negative control was set up in
parallel with entire reagent except for either extract Values are expressed as mean ± SEM of 3 replicates.
or standard. The absorbance of H2O2 at 230 nm ab
Values with different superscript on a row are
was determined against a blank solution containing significantly different (p<0.05)
phosphate buffer.
Mineral Composition
Calculation Mineral content as revealed in table 2 revealed
Scavenging ability on hydroxyl radicals (%)= chromium and nickel were significantly (p<0.05)
(Acontrol-Atest /Acontrol )×100 h i g h e r i n l e ave s ( 0 . 3 8 ± 0 . 0 5 , 1 . 7 5 ± 0 . 0 4
mg/100g) compared to the root (0.30±0.05,
Where 1.25±0.03 mg/100g) respectively. Lead and cobalt
Acontrol is the absorbance of the control reaction content was significantly (p<0.05) higher in roots
5

(0.23±0.05, 2.80±0.04 mg/100g) compared to the the amount of zinc, cadmium, copper, magnesium,
leaves (0.13±0.02, 1.93±0.02 mg/100g) respectively. sodium, calcium, potassium and iron detected in the
There was no significant (p<0.05) difference between leaves and root.

Table 2: Mineral content of A. muricata leaves and roots

SAMPLE LEAF ROOT

Zinc (mg/100g) 8.70±0.29a 8.40±0.18a

Cadmium (mg/100g) 5.23±0.09a 5.50±0.10a
Chromium (mg/100g) 0.38±0.05a 0.30±0.05b
Lead (mg/100g) 0.13±0.02a 0.23±0.05b
Copper (mg/100g) 7.35±0.11a 7.80±0.09a
Nickel (mg/100g) 1.75±0.04a 1.25±0.03b
Cobalt (mg/100g) 1.93±0.02a 2.80±0.04b
Magnesium (mg/100g) 30.73±0.21a 32.80±0.27a
Sodium (mg/100g) 49.88±0.12a 51.28±0.20a
Calcium (mg/100g) 155.03±0.39a 151.30±0.57a
Potassium (mg/100g) 23.95±0.19a 24.88±0.44a
Iron (mg/100g) 37.55±0.23a 38.55±0.88a

Values are expressed as mean ± SEM of 3 replicates. abValues with

different superscript on a row are significantly different (p<0.05)

Total Phenolic and Flavonoid Quantification root (0.74±0.02 mg pyrocatechol/mL, 0.25±0.01

Results recorded in table 3 show total phenolic mg GAE/mL). There was no significant (p<0.05)
and flavonoid content was significantly (p<0.05) difference in the EC50 value for hydroxyl radical
higher in the leaves (1.01±0.03 mg pyrocatechol/ scavenging activity of the leaf (2.92±0.02) and root
mL, 1.12±0.03 mg GAE/mL) compared with the extract (2.65±0.03).

Table 3: Total phenolic, flavonoid composition and EC50 values for

hydroxyl radical scavenging activity

SAMPLE Leaves Roots

Total Phenol (mgpyrocatechol/mL) 1.01±0.03a 0.74±0.02b

Total Flavonoid (mgGAE/mL) 1.12±0.03a 0.25±0.01b
EC50 2.92±0.02a 2.65±0.03a

Values are expressed as mean ± SEM of 3 replicates. abValues with

different superscript on a row are significantly different (p<0.05)

Hydroxyl Radical Scavenging Activity There was, however, a slight scavenging activity
A concentration-dependent increase in hydroxyl decrease in the highest concentration of the leaf
radical scavenging activity of A. muricata leaf and extract.
root methanolic extract was depicted in figure 1.
6

Fig. 1: Hydroxyl radical scavenging activity of methanolic extract of leaves and roots of A.
muricata. Values are expressed as mean ± SEM of 3 replicates

Discussion makes it highly susceptible to microbial attack

The results from the proximate composition of during storage.25 The high crude fat, protein and
the leaves and root of A. muricata further show ash contents of the leaf suggest that they could
their nutritive and medicinal properties. Cellular be used as a better source of plant fat, protein
metabolism thrives on and requires energy provided and minerals in feed supplementation. Fat is an
by carbohydrate to run continuously.20 Dietary essential macromolecule needed for energy and
fibre improves motility in the digestive system and absorption of fat-soluble vitamins.26 Proteins are
plays a role in cardiovascular diseases and cancer essential components of diets required for energy
prevention.21 The amount of crude fibre present in generation, maintenance of body tissues and
the leaf and root of A. muricata in this study, were synthesis of enzymes, hormones as well as other
very high compared to that of seed previously substances required for healthy functioning.27,28
reported. However, the inverse was the case for The crude protein content of the leaves and roots
the carbohydrate content as it was higher in the shows A. muricata may be used as a natural protein
seed.22 The high carbohydrate and fibre content of supplement for animals. The protein and fat content
the root shows that it can be used to aid digestion of the leaves are higher than the 12.5% and 1.49%
and as a source of energy.23 The moisture content reported for Nauclea latifolia leaf but lower than
of the leaves suggests it may be a better source of the reported protein (27.74% and 20.72%) content
hydration, but nonetheless, it may be more prone to for Vitex doniana and Moringa oleifera leaves
microbial attack during storage than the roots. This respectively.29 The reported high fat content of
indicates that the leaf may have reduced shelf life the leaves and root were in line with the reported
which corresponds with a study on Costus afer24 values (8.3 – 27.0%) of leafy vegetables.29 It is also
as a high moisture content of any biological matter corroborated by the findings of Agu and Okolie30 on
7

the leaf and root extracts which may be as a result dependent increase in the scavenging capacity of the
of the high presence of annonaceous acetogenins leaves and roots may be attributed to the presence of
which are long chain fatty acids derivative. Minerals flavonoids and phenolics in the methanolic extracts,
play a massive role in metabolic pathways, disease as they are known to be soluble in polar solvents.24
prevention and management. Their presence in These plant secondary metabolites are known to
A. muricata leaves and roots may be the reason possess potent antioxidant capacity which may be
for the folkloric use in treatment and management as a result of the presence of the hydroxyl groups
of diseases.24 High concentration of minerals was present in their ring structures. These groups are
observed to be present in the leaves and root of effective in scavenging of reactive oxygen species by
A. muricata with calcium, sodium, iron, magnesium donating and accepting electrons with free radicals
and potassium being the most abundant. Calcium is thereby quenching them.36,41 The findings agree
essential for healthy bones, teeth, blood, muscles31, with the studies carried out by Agu and Okolie30,
as well as absorption of dietary vitamin B, for the Kalidindi et al.,42 and Mariod et al.,43 attributing the
synthesis of the neurotransmitter acetylcholine and antioxidant properties of their plants to the phenol
immune response. 32 Magnesium, potassium as and flavonoid content.
well as calcium are involved in enzyme synthesis,
cofactors for enzyme activation, biological structure Conclusion
promoter and optimal physiological function.33,34 This study suggests the leaves of A. muricata have
Sodium is required for optimal acid-base balance, more phenolic and flavonoid contents compared to
maintenance of osmotic pressure and cellular the root. Nonetheless, these plant parts are a rich
homeostasis.35 Iron is important for animal survival source of nutrients and can be capitalised for feed
as it plays a role in respiration, DNA synthesis and supplementation, the potential to supply sufficient
blood functioning.36 Chromium prevents diabetes amount of minerals for consumers and microbial
by being directly involved in insulin production and media for microorganisms. Besides their nutritional
function.37 All other minerals present in the leaves and mineral value, they possess antioxidant activity,
and root play a part in the optimal function of thus may be utilised as an alternative natural source
various physiological and biochemical processes.38 of antioxidants.
Interestingly results of mineral content analysis of the
leaves were in contrast with that of Usunomena and Acknowledgement
Paulinus39 except chromium which had almost similar The authors are grateful to Covenant University
value. This observation, may be as a result of the Centre for Research and Discovery, Ota, Ogun
soil location, where this plant was cultivated, as well State for providing a platform for the publication of
as the environmental practices of the surrounding the research work.
populace. Phenolic substances are abundant low
molecular weight bioactive compounds in plants Conflicts of interest
which have various health benefits.40 The dose- No conflict of Interest.

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