By Wahua, C; James, EN (2024). Greener Journal of Biological Sciences, 14(1): 1-6.
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Greener Journal of Biological Sciences
Vol. 14(1), pp. 1-6, 2024
ISSN: 2276-7762
Copyright ©2024, Creative Commons Attribution 4.0 International.
https://gjournals.org/GJBS
Department of Plant Science and Biotechnology, Faculty of Science,
University of Port Harcourt, Choba, P.M.B. 5323, Nigeria.
Article No.: 011224004
Type: Research
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The research was set to study the anato-phytochemical characteristics of Annona muricata L. of Annonaceae. a perennial small tree, readily distinguished by its large edible aggregate berry fruits derived with delicious whitish pulp, introduced into the Niger Delta and commonly known as sour sop. Plant sample was collected fresh within the University of Port Harcourt Campus, (4053130.1211North, and 6055139.611East). These were fixed in formaldehyde, glacial acetic acid, 70% alcohol in the ratio of 1:1:18, dehydrated in alcohol solutions of 50 %, 70 %, 90 %, absolute and sectioned, stained in 2 % aqueous solution of Safranin O, counter stained in Alcian blue, mounted in glycerine, The phytochemical analysis, in Stage 1, 0.1 g of the leaves were weighed into a conical flask of 250 ml capacity, 3 g of digestion catalyst was placed into the flask and 20 ml conc. Suphuric acid added and heated to digest. Color change observed from black to sky-blue, cooled to room temperature and then diluted to 100 ml with distilled water. In Stage 2, 20 ml diluted digest was measured into a distillation flask and held in place on hot plate was attached to a Liebig condenser connected to a receiver containing 10 ml of 2 % boric acid indicator. 40 ml NaOH was injected into the digest, and heated to boiling and the distilled ammonia gas via the condenser into the receiver beaker. The color of the boric acid change from purple to green as ammonia distillate was introduced into the boric acid. In Stage 3, the distillate was titrated with standard 0.1 N HCl solution back to purple from greenish. The volume of HCl added to effect this change was recorded as titre value. Results of anatomy revealed unilacunar node with 2 lateral leaf traces and 1 leaf trace median. In the phytochemistry, alkaloids, tannins, saponins, phenolics and flavonoids in both fruits and leaves were shown to be present while cardiac glycosides was present in the leaves but absent in the fruit. This information would assist for further delimitation of the species.
Published: 08/02/2024
Dr. Chika Wahua
E-mail: chika.wahua@ uniport.edu.ng
Phone: +2348064043448
Keywords: Anatomy, phytochemical, Annona muricata, Annonaceae
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.
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.
Plant sample was collected fresh within the University of Port Harcourt Campus, (4053130.1211North, and 6055139.611East). Plate 1.
Plate 1: Annona muricata L. Arrows revealed flower and small fruit.
Anatomical study showcased one layered epidermis. The hypodermis is made of 2 to 3 rolls of collenchyma, the general cortex has 7 to 10 rolls of parenchyma and the pith is made of parenchyma. The node is unilacunar with 2 lateral leaf traces and 1 leaf trace median. Plates 3 a, b. c, d and e.
Plate 2a: Stem anatomy of Annona muricata (T.S.); 2b: Nodal anatomy of A. muricata T.S.). 2c: Root anatomy (T.S.); 2d: Mid rib of A. muricata (T.S.). Node is unilacunar. Pa- parenchyma, Co – collenchyma. Pi – pith, vb – vascular bundle.
The phytochemical analysis revealed the presence of alkaloids, tannins, saponins, flavonoids, while cardiac glycosides was observed absent in fruits but present in the foliar organ, the leaf. Table 2.
Table 2: Qualitative Phytochemical Analysis of leaves and fruit of Annona muricata L.
Phytochemical contents
Key: +ve represents Presence while –ve absence.
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).
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.
The effort of Whuatorhe, Promise Edesiri, who assisted in some of the laboratory work is immensely commended.
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