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Phytochemistry and Antioxidant Activity of Irvingia gabonensis (Bush mango) Seed Sample

Phytochemistry and antioxidant activity of Irvingia gabonensis (Bush mango) seed sample were evaluated. Dried milled seeds of I.gabonensis popularly known as “ogbono” passed through phytochemical screening, Gas chromatography-mass spectrometry (GC-MS) analysis and antioxidant studies. Tannins, saponins, flavonoids, alkaloids, steroids, and reducing sugars were among the phytochemicals found present after screening. About 20 constituents were revealed by the GC-MS analysis out of which 1,3-O-Benzylidine glyceryl-2-myristate had the highest molecular weight, 2-Undecanone had the highest peak area of 45.39% whereas 3-Dibenzofuranamine had the highest retention time. Some of these constituents could have one or two applications in medicinal, food or cosmetic industries. I.gabonensis seeds showed better antioxidant activity against the Original Research Article Sunday et al.; AJBGMB, 7(4): 25-34, 2021; Article no.AJBGMB.63380 26 control at concentrations considered in this study. Some of these constituents as well could be behind the observed antioxidant activity. This study has evaluated the phytochemistry and antioxidant activity of I.gabonensis (Bush mango) seed sample.


INTRODUCTION
Plants have contributed immense to humans and humanity [1][2][3][4][5][6]. The benefits of plants to humans cannot be quantified and are as old as mankind on earth [7][8]. The use of plant to salvage some of the problems of humans could date to creation. Apart from food production [9-19], plants are put to different uses by man [7,[20][21][22][23][24][25][26][27]. The medicinal aspect of plants took a dimension after the Alma-Ata declaration in 1978, where the use of plant in primary healthcare was publicly accepted [7,28]. Since then, efforts are being made to discover plants and plant products with medicinal potentials. Studies began to see edible portion of plants beyond serving as food only.
The potency of plants has also been extended to their ability to quench some biomolecules that are associated with stress in living organisms [57][58][59][60]. Oxidative stress as found in living organisms has been described as a situation that rises when there is excessive production of reactive oxygen species (ROS) against the defense mechanisms. The known defense mechanisms include group of complex antioxidant systems. Antioxidants counter the activities of reactive oxygen species. They include the major cellular redox buffers ascorbate (AsA) and glutathione (γ-glutamylcysteinyl-glycine, GSH) as well as tocopherol, carotenoids, and phenolic compounds; and enzymatic components such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), enzymes of ascorbate-glutathione (AsA-GSH) cycle ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) [61][62][63][64][65][66].
Irvingia gabonensis, a species of African tress could be among the plants with phyto and bioactivity of constituents that can as well quench some biomolecules associated with stress in living organisms. I.gabonensis is commonly known as bush mango, African mango, or wild mango [67]. It bears edible fruit that looks like mango fruit. The fruits also bear seeds popularly known as "ogbono" in Southern Nigeria. "ogbono" is referenced because of its usefulness as a thickener in the preparation a soup known as "ogbono soup", a particular type of draw soup. I.gabonensis is a deep soil plant, which survives in sandy loam soil of acidic pH [68]. It could be propagated by grafting, budding, air layering, cutting, marcoting or seeds. The tree can grow up to 40 m in height and 1 m in diameter. The leaves bear slightly obovate, dark green coloration and elliptic shape and can measure up to 5 to 15 x 2.5 to 6 cm. The Igbos of Southeastern Nigeria call the fruit of I.gabonensis "ugiri" and the seed "ogbono" [67][68][69][70]. The fruit is also known as "ogwi" by the Benin people; "biri" or "goronor" by the Hausa people; and "oro", "apon" or "aapon" by the Yoruba people. The high glucose lowering potency [71][72], weight reducing ability [71], analgesic properties [73], serum cholesterol level regulation [71], antidysentery potency, antibacterial and antifungal properties [74] as well as anticonstipation, anti-indigestion, astringent [75] and the industrial uses of I. gabonensis have been reported by different authors. The stem has also been implicated in dental care as chewing stick for cleaning of teeth [67].
There is need to ascertain the constituents that could be linked to the aforementioned medicinal properties of I.gabonensis. The present study looked into this area and evaluated the Phytochemistry and antioxidant activity of I.gabonensis (Bush mango) with its seeds.

Sample Collection and Identification
The I.gabonensis fruits used were plucked from the tree found in a farm in Umunchi village, Isiala Mbano LGA of Imo State. The fruits were properly identified by a Botanist in the Department of Plant Science and Biotechnology, Imo State University, Owerri, Nigeria. The fruits were peeled to obtain the nuts. The nuts were forcefully cracked open to expose the seeds, which were harvested from the nuts. The harvested seeds were dried in the sun for few days and were milled into powder for further studies.

Preparation of Aqueous Extract
Ten gram of the milled seeds was extracted by maceration in 50 ml of water for 3 days with frequent agitation at a speed of 280 rpm at 28°C in dark. Between extractions, the sample was centrifuged for 10 min with 2000 rpm. The combined supernatants were collected, filtered through Whatman No. 1 filter paper and concentrated in vacuum. It was kept in a vacuum desiccator for complete removal of solvent. The yield extract was thus used for some of phytochemical screening, GCMS analysis and assessment of antioxidant activity.

Qualitative Phytochemical Determinations 2.3.1 Test for tannins
To 1 mL of the extract, equal volume of bromine water was added. The formation of a greenish to red precipitate was taken as the presence of tannins,

Test for saponins
One mL of the extract was boiled with 5 mL of distilled water for 5 min. and decanted while hot. 4 mL of distilled water was added to 1 mL of the filtrate before it was shaken vigorously for observation of stable froth on standing.

Test for flavonoids
0.5 g of the extract was added, in a test tube and 10 ml of distilled water, 5 mL of dilute ammonia solution were added to a portion of the aqueous filtrate of the extract followed by addition of 1 mL concentrated H2S04. Indication of yellow color shows the presence of flavonoid in each extract.

Test for alkaloids
One (1) mL each of the extract was shaken with 5 mL of 2% HCl on a steam bath and then filtered. To I mL of the filtrate, Wagner`s reagent (iodine in potassium -iodide solution) was added and reddish brown precipitates was observed for positive result.

Test for steroids
Half (0.5 g) gram of the extract was dissolved in 10 mL anhydrous chloroform and filtered. The filtrate was divided into two equal portions for the following tests. The first portion of the solution above was mixed with one mL of acetic anhydride followed by the addition of 1 mL of concentrated sulphuric acid down the side of the test tube to form a layer underneath. The test tube was observed for green colouration as indicative of steroids.

Test for terpernoids
One gram of seed sample was shaken in a test tube with 10 mL of methanol, and then filtered. 5 mL extract was then mixed with 2 mL of chloroform and 3 mL of sulphuric acid was added. Formation of reddish brown color indicates the presence of terpenoids in the selected plants.

Cardiac glycosides
One mL of the seed extract was dissolved in 2 mL of chloroform in a test tube. 1 mL conc. H2SO4 was carefully added to the test tubes through the side and was observed for a red or reddish brown colouration at the interphase, which indicates positive result.

Test for phlobatannins
One percent aqueous hydrochloric acid was added to the seed extract in a test tube (about 2 mL), and then boiled with the help of Hot plate stirrer. Formation of red coloured precipitate confirmed a positive result.

Test for phenolic compounds
To 2 mL of the seed extract, 1% FeCl3 was added and observation was made for blue, violet, purple, green or red-brown colour.

Test for proteins
Five drops of 1% hydrated copper sulphate was added to 2 mL the seed extract in a test tubes. Two mL of 40% NaOH was also added, and the test tube was shaken vigorously to mix the content and presence of purple colouration indicated the presence of proteins.

Test for reducing sugars
One mL of ethanol was mixed with 2 mL each of the plant extract, after which 1 mL each of Fehling solution A and B were added to the test tubes. The test tubes were heated to boiling while observation was made for presence of reddish brown colouration which indicates positive results.

Test for anthroquinones
One gram of the seed extract was placed in a dry test tube and 20 mL of chloroform was added. This was heated in steam bath for 5 min. The extract was filtered while hot and allowed to cool.
To the filtrate was added with an equal volume of 10% ammonia solution. This was shaken and the upper aqueous layer was observed for bright pink colouration, which for the presence of anthraquinones. This was repeated with all the plant samples.

GC-MS Analysis of the Extracts
GC-MS analysis of the aqueous extracts was carried out using AOC-20i auto sampler and gas chromatograph interface to a mass spectrometer (GC-MS) instrument. Employing the following conditions; column Elite-1 fused silica capillary column (30 mm×0.25 mm ID×1Μm df, composed of 100% Dimethyl poly siloxane), operating in electron impact mode at 70 Ev; helium (99.999%) was used as carrier gas at a constant flow of 1 ml/ min, and an injection volume of 0.5μl, Split ratio of 10:1), with injector temperature 250°C; and ion-source temperature 280°C. The oven temperature was programmed from 110°C (Isothermal for 2 min), with an increase of 10°C/min to 200°C, then 5°C/min to 280°C, ending with a 9 mins isothermal at 280°C. Mass spectra were taken at 70 Ev; a scan interval of 0.5 seconds and fragments from 45 to 450Da. Total GC running time was 36 mins. The plant extract was dissolved in aqueous and filtered with polymeric solid phase extraction (SPE) column and analyzed in GC-MS for different components. Interpretation of mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62 000 patterns. The spectrum of the unknown components was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test were ascertained.

RESULTS AND DISCUSSION
Phytochemical screening of I.gabonensis as represented in Table 1 showed the presence of tannins, saponins, flavonoids, alkaloids, steroids, cardiac glycosides, phlobatannins, proteins, and reducing sugars. These phytochemicals become important when their effects are considered in living organisms. The astringency of tannins [35,41], foaming and bitter tastes of saponins [44][45], antibacterial and anti-inflammatory property of flavonoids [46][47][53][54], stimulant and analgesic property of alkaloids [77], hormonal nucleus role of steroids [41], the beneficial nature of cardiac glycosides to the heart at low concentration[ ], the antiinflammatory, analgesic and wound healing properties of phlobatannins [54,59], tissues replacement ability of proteins and energy production potency of sugars [41] have all been reported. Tannins, saponins, steroids, and proteins were present in high concentrations. Flavonoids, alkaloids, cardiac glycosides, phlobatannins, and reducing sugars were present in moderate concentrations whereas terpernoids, phenolic compounds and anthroquinones were absent in I.gabonensis milled seed.
Some of these constituents could have one or two applications in medicinal, food or cosmetic industries. Glycerine is used as a hyperosmotic, osmotic diuretic, and ophthalmic agent. Glycerin has many other uses in the agricultural, food and pharmaceutical industry. 2-Undecanone is an insect and tick repellant. 3,4-Furandiol, tetrahydro-, trans-is an excellent manufacturer of organic compound. 2-Tridecanone is a nonalkaloid and has insecticidal potency. Dodecanoic acid, methyl ester, is a fatty acid of methyl and dodecanoate esters, which has a metabolite role. Dodecanoic acid, a considered lauric acid, can be applied in soaps, cosmetics, resin and wetting agents. Tetradecanoic acid, Antioxidant activity as presented in Fig. 1, showed percentage inhibitions of I.gabonensis taken at the 10, 15, 30 and 40 mg/L concentrations as 20.22%, 28.09%, 34.83%, and 52.81%. These inhibitions were against 1.12%, 15.73%, 23.60% and 40.45% observed for the control. In generality, I.gabonensis showed higher antioxidant activity than the control in the present study. Plants with antioxidant activity have been reported by different authors [58][59][60][61][62][63][64][65][66].

CONCLUSION
Tannins, saponins, flavonoids, alkaloids, steroids, cardiac glycosides, phlobatannins, proteins, and reducing sugars were found in I.gabonensis. The GC-MS analysis revealed a total of 20 constituents of either medicinal, food or cosmetic importance. Some of these constituents could also be behind the antioxidant activity of the plant. This study has revealed the Phytochemistry and antioxidant activity of Irvingia gabonensis (Bush mango) seed sample.