INTRODUCTION
Annona muricata is a species of the
genus Annona in the family Annonaceae, these fruits contain sweet whitish pulp
lavishly consumed because of its palatability. The habit consists of trees,
shrubs or rarely Lianas and is believed to have about 108 genera and 2,400 species
(Chukwuka et al.,
2011) commonly called custard apple family or graviola
family and sour sop.
It is well cultivated in Africa, mainly in the lowlands of Eastern and
Western Africa, temperate and tropical Asia, Australia, North America, South
Central Pacific Islands, the Caribbean and Mesoamerica (USDA-ARS, 2013). Annona muricata is an evergreen quick growing tree and could
reach up to 28 ft to 30 ft
(8 m to 9 m) in height and a hermaphrodite producing flowers singly or in
clusters (Paull and Daurte,
2012). Its environment requires a warm
and humid tropical climate, well drained and loose, farm rich, deep loamy soil with
pH range of 5 to 6.5, and very intolerant to waterlogged soil and can be
stunted or killed by cold spells or light frost as it’s a shallow rooted tree (Kooesriharti, 1991; Orwa et al.,
2009). The fruits are heart shaped to oval dark green in color. It can be propagated by micro propagation
with its root stock, vegetative or clonally, in particular through various
budding and grafting methods on seedling stocks, however, the species is
commonly raised from seeds (Morton, 1987). It has been observed that certain
species in the genus Annona are
confused for others, Annona muricata is
sometimes erroneously regarded as Annona glabra and Annona montana by some growers (Pinto
et al., 2005).
A. muricata tree grows at
altitudes below 1200 m above sea level, at a relative humidity of 60 %–80 %, a
temperature ranges of 25–28 °C, and with more than 1500 mm of annual rainfall (Wagner
et al., 1990). A. muricata is evergreen and blooms, bearing fruits
almost throughout the year (Wagner et al., 1990).
Molecular phylogenetic investigations on the
species proved helpful (Doyle and Thomas, 1996; Mols
et al., 2004; Pirie et al., 2006; Couvreue et al.,
2012). Investigation into the foliar epidermal features and petiole anatomy of A. muricata
are shown by Folorunso (2014).
Plants are sources of
natural ingredients that are widely used as medicines. The compounds present in
plants are the reasons for their activities against various diseases, and
studies have shown some of these active compounds in plants and their
pharmacological responses to respective diseases are determined and confirmed
through researches, Moghadamtousi et al. (2015). Phytochemicals accumulate in
different parts of the plant tissues: roots, stems, leaves, flowers, fruits and
seeds (Costa et al., 1999). The presence of saponins, condensed tannins, glycosides and trace
amounts of flavonoids contribute immensely to the bioactivity of A. muricata in
combating various diseases as it possesses different properties such as
anti-oxidant activity (Adewole et al., 2009) as well
as hepatoprotective effect and antibacterial agents (Chukwuka et al., 2011). The plant is used as a traditional
medicine for skin disease, respiratory disease, fever, bacterial infections,
diabetes, hypertension, and cancer (Stevens, 2022). Preliminary phytochemical
analysis revealed the presence of secondary metabolites like tannins, steroid,
cardiac glycosides, etc. were present in trace amounts in the leaves of A. muricata (Pathak et al., 2010). The seeds combat parasitic
infections; the fruit is used for the treatment of arthritis, nervous
disorders, and diarrhea; and the leaves are used to treat cystitis, headaches,
insomnia, and cancer (Wélé et al., 2004). Other phytochemical analysis of
the n-butanol leaf extract of A. muricata revealed the presence of flavonoids, terpenoids, tannins, cardiac glycosides and reducing sugars.
Whereas, the extract showcased the absence of saponins,
steroids, phlobatannins, oil and anthraquinones
(Kumar et al., 2012). Phytoconstituents in the leaves of A. muricata revealed an alkaloidal principle, named 6-Hydroxyundulatine and other
alkaloids (Vimala et al., 2012). The main active
components of A. muricata are acetogenin,
alkaloids, and flavonoids (Coria-Téllez et al., 2018).
Analysis of the compounds in A.
muricata leaf extract showed secondary
metabolites such as flavonoids, terpenoids, saponins, coumarins, lactones, anthraquinones, glycosides, tannins, and phytosterols (Gavamukulya et
al., 2014). A. muricata leaves are used to treat headaches,
insomnia, cystitis, and cancer, the seeds are used to treat parasitic
infections (Moghadamtousi et al., 2015),
and the fruit is used to treat diarrhea and neuralgia, eliminate worms and
parasites, increase milk production in lactating women, and reduce fever (Pieme et al., 2014). In South America, A. muricata
fruit juice is used to treat many diseases, such as heart and liver disease,
and has antidiarrheal and antiparasitic effects (Shaw et al., 2012). The fruit flesh is used to
increase breast milk production after childbirth and treat rheumatism,
arthritic pain, fever, neuralgia, dysentery, heart and liver diseases, and skin
rashes, and it has antidiarrheal, antimalarial, antiparasitic,
and anthelmintic properties (Moghadamtousi et al., 2015, Hajdu et al., 2012). Presently, there may not be
many research publications on phytochemical screening of A. muricata L. leaves and on their
antimicrobial activity against Gram-positive and Gram-negative bacteria (Obiazi et al.,
2018).
The need to add more information to the
existing taxonomic relevance has created the interest to investigate the anato-phytochemical characteristics of Annona muricata L. of Annonaceae.
It is envisaged that the study would be an important resource to this effect.
MATERIALS
AND METHODS
Geographic Location
Plant sample was collected fresh within the University of Port
Harcourt Campus, (4053130.1211North, and 6055139.611East).
Anatomical Study
Annona muricata L. stems, leaves, petioles, flowers, fruits
and roots harvested for the study, were fixed in FAA prepared in the ratio of
1:1:18 of 40 % formaldehyde, glacial acetic acid and 70 % alcohol for 2 to 48
hours following the methods of Johansen (1940) modified; Free hand sections
were done as described by Wahua (2020). Slides with
good sections were placed on the stage, viewed and photo-micro graphed using
Leica WILD MPS 52 microscope camera on Leitz Dra plan microscope.
Phytochemical Study
The leaves of the Annona muricata were sun dried for 72 hours and
later weighed. Fifty grams (50 g) of the dried leaves were macerated in 96 %
ethanol with a pestle and a mortar. The extract was filtered and then
evaporated to dryness using a rotary evaporator set at 450 C.
Residue yields were noted and a portion used for the phytochemical
investigation.
Test for alkaloids
This involved using
0.5 g of the plant extract, stirred with 5 ml of 1 % aqueous hydrochloric acid
on a water bath; 1ml of the filtrate was treated with few drops of Mayer’s
reagent and a second 1 ml portion was treated in same way with Dragendorff’s reagent. The third 1 ml was treated with
Wagner’s reagent. Turbidity or precipitation with these reagents was taken as
preliminary evidence for the presence of alkaloids (Harborne,
1973; Trease and Evans 1989). A modified thin-layer
chromatography (TLC) method as described by (Farnsworth, 1962) was used. One
gram (1 g) of the extract was treated with 40 % calcium hydroxide solution
until the extract was distinctly alkaline to litmus paper, and then treated
twice with 10 ml of chloroform. The extracts were combined and concentrated to
5 ml. The chloroform extract was spotted on thin-layer plates. Four different
solvent systems were used to develop each plant extract. The presence of
alkaloids in the developed chromatograms was detected by spraying the
chromatograms with freshly prepared Dragendorff’s
spray reagent. A positive reaction on the chromatograms (indicated by an orange
or darker colored spot against a pale yellow background) was used as
confirmatory evidence for the presence of alkaloid.
Test for flavonoids
Shinoda reduction test: 5 g
of the pulverized sample was boiled in 5 ml of distilled water for 5 minutes on
water bath and filtered while hot. Magnesium (Mg) was added to the filtrate and
few drops of conc.H2SO4 were carefully introduced into
the mixture. The formation of orange, red, crimson or magenta was taken as
evidence of preliminary presence of flavonoid.
Lead acetate test: 5 g of pulverized sample was
boiled in 5 ml of distilled water for 5 minutes in water bath and filtered
while hot. 2 ml of 10 % lead acetate was added to the filtrate and observed.
Yellow precipitate indicated presence of flavonoids.
Test for tannins
Ferric chloride test
(FeCl3)
5 g of the prepared sample was boiled in 5 mls
of distilled water for 5 minutes on water bath. This was filtered
while hot. 1ml of 5 % FeCl3 was added to the filtrate and observed.
Blue-black, green or blue-green precipitate was taken as tannins present in the
sample (Trease and Evans 1989).
Test for cardiac
glycosides
Lieberman’s test
0.5 g of the extract
was dissolved in 2 ml of acetic anhydride and cooled in ice. One milliliter (1 ml)
of Sulphuric acid was added in drops until a color
change from violet to blue to green indicating that steroidal aglycones were present in the extract (Shoppe, 1964).
Test for Saponins
Frothing tests:
Preliminary following
the method described by (Wall, 1952) was observed. The ability of saponins to produce frothing in aqueous solution and to haemolyse red blood cells was observed as screening test
for saponins. 0.5 g of the plant extract was shaken
with water in a test tube. Frothing which continued on warming was taken as
preliminary evidence that saponins were present in
the sample. The disc was then washed in ether, dried and placed on a 7 % blood
agar. Complete haemolysis of red blood cells around
the disc after about 6 hours was taken as further evidence of the presence saponins in sample.
RESULTS
Plant sample was collected fresh within the University of Port
Harcourt Campus, (4053130.1211North, and 6055139.611East).
Plate 1.
DISCUSSION
Phytochemicals
accumulate in different parts of the plant tissues (Costa et al., 1999).
Preliminary phytochemical studies using the fruits and leaves of A. muricata
showed presence of tannins, cardiac glycosides etc. in trace amount in leaves
of A muricata
(Pathak et al., 2010). Other parts of plant showcased
presence of tannins, alkaloids and others, such as alkaloidal principle, 6-Hydroxyundulatine
(Vimala et al., 2012). The presence of saponins, condensed tannins, glycosides and trace
amounts of flavonoids contribute immensely to the bioactivity of A. muricata in
combating various diseases as it possesses different properties such as
anti-oxidant activity (Adewole et al., 2009) as well
as hepatoprotective effect and antibacterial agents (Chukwuka et al., 2011).
CONCLUSION
Annona muricata L. has not so much
work in anatomy especially as it has to do with nodal anatomy. A very clear
background of types of the qualitative phytochemical analysis is contributory
information made addition to other existing knowledge on the plant. More
research findings in the DNA barcode is recommended.
ACKNOWLEDGEMENT
The
effort of Whuatorhe, Promise Edesiri,
who assisted in some of the laboratory work is immensely commended.
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