Research Article
Phytochemical and Antimicrobial Activity of African Star Apple [Chrysophllum albidum]
https://doi.org/10.21203/rs.3.rs-5345688/v1
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The quantitative phytochemical analysis of Chrysophyllum albidum cotyledon crude extract indicated that it was rich in phenolics, oxalates, saponins, flavonoids, terpenoids, Phylates and tannins. The result of analysis indicates that ascorbic acid content was high compared to Niacin, riboflavin and thiamine.
The result of the antimicrobial analysis indicates that Escherichia coli was significantly susceptible to all the extracts compared to other bacteria studied.
Phytochemical
antimicrobial
African Star Apple
Chrysophllum albidum
Chrysophyllum albidum belongs to the family Sapotaceae which contains a number of fruit trees like C. cainito, C. welwitschii, C. delvoyi, C. pruniforme and others; all indigenous to tropical Africa [Hutchinson and Dalziel, 1963]. The genus Chrysophyllum is an evergreen tropical tree. Chrysophyllum albidum is a tall straight tree from 30 m to 60 m under favourable site conditions. The bole is sometimes long and straight but often branched, deeply fluted occasionally with small buttresses (about 30 cm high) at the base. The crown is usually dense [Katende et al., 1995]. The bark is thin, pale grey-brown or pale brownish-green, while the pale brown slash exudes copious white gummy latex, a characteristic of the Sapotaceae family. It has a network of zigzag fissures; twigs grooved [Irvine, 1961]. It is a timber species. The wood is brownish-white, soft, coarse and open grain; very perishable in contact with the ground. It is easy to saw and plane, nails well, and takes a fine polish [Opeke, 1982]. The tree commonly flowers from April to June annually [Irvine, 1961]. Okafor [Okafor, 1981] however noted that in parts of Anambra, Ebonyi and Enugu states (all in Nigeria) flowering occurs at various times such as January-February; April-June and September. This could be an attribute to be exploited in pollen collection [Bada, 1997].
Collection and Identification of Plant materials:
Ripe and fresh Chrysophyllum albidum fruits used for this study were purchased from Utako Market, in Abuja, Nigeria. The fruit were identified and authenticated at the Herbarium of the Department of Biological Sciences, University of Abuja, Nigeria.
Preparation of Plant Materials:
The sample was prepared according to the methods described by Anibijuwon and Udeze [2009]. The fruit samples were washed and the seeds removed from the fruits and air dried to constant weight. The dried seeds were deshelled and the cotyledon pounded into smaller granules using laboratory Mortar and pestle. The blender was used to pulverize the pounded cotyledon. The pulverized cotyledon sample was kept in an air tight container.
Preparation of The Plant Extract:
The method described by Arekemase et al., [2011] and Akin-Osanaiye et al.,[2018] were used. Pulverized plant samples of known quantity (500g) was extracted successively with different solvents of varying polarity ranging from hexane, ethyl acetate, ethanol and water using soxhlet extraction method.. The crude extracts was filtered and concentrated in a rotary evaporator at 40ᵒC. The extracts was dried to completion in a hot air- oven at 40ᵒC, kept in Mc- carthny bottles and refrigerated at 2-4 ᵒC for further use. The crude extracts were weighed and yielded 7.44g, 1.37g, 16.06g and 65.44g for hexane, ethylacetate, ethanol and water extracts respectively.
Quantitative Analysis of Phytochemical Constituents:
The quantitative phytochemical analysis was carried out in the Department of chemistry SHEDAScience and Technology Complex Gwagwalada Abuja. Each sample (pulverized seed cotyledon of C. albidum fruits) was analysed in triplicate. This was carried out according to the method of AOAC [1990]
Quantitative Determination of Alkaloid:
The alkaloid content was determined gravimetrically. Five grams of the sample were weighed into a 250 ml beaker and 200ml of 20% acetic acid in ethanol was added and covered to stand for 4 h. This was filtered and the extract was concentrated using a water-bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitate was collected by filtration using Whatman filter paper No. 4 (125 mm) and weighed (Obadoni and Ochuko, [108].
Quantitative Determination of Saponin:
Saponin content was determined using the method described by [AOAC, 2000]. Twenty grams (20 g) of each ground samples were dispersed in 200 ml of 20% ethanol. The suspension was heated over a hot water bath for 4 h with continuous stirring at about 55oC. The mixture was filtered and the residue re-extracted with another 200 ml of 20% ethanol.
The combined extracts were reduced to 40 ml over water bath at about 90oC. The concentrate was transferred into a 250 ml separator funnel and 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 60 ml of n-butanol was added. The mixture of n-butanol and extracts was washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath at about 90 oC. The samples were dried in an oven at 100oC until a constant weight was obtained. The saponin content was calculated in percentage [badoni and Ochuko, 2001].
Quantitative Determination of Tannin:
Tannin content was determined going by the method described by Van-Burden and Robinson [Van-Burden and Robinson, 2009]. Five hundred milligrams of the sample was weighed into 100 ml plastic bottle. 50 ml of distilled water was added and shaken for 1 h in a mechanical shaker. This was filtered into a 50 ml volumetric flask and made up to the mark. Then 5 ml of the filtrate was pipette out into a tube and mixed with 3 ml of 0.1M FeCl3 in 0.1 N HCl and 0.008M potassium ferrocyanide. The absorbance was measured in a spectrophotometer at 120 nm wavelength, within 10 min. A blank sample was prepared and the colour also developed and read at the same wavelength. A standard was prepared using tannin acid to get 100 ppm and measured [Van-Burden and Robinson, 2009].
Quantitative Determination of Flavonoid:
Flavonoid content was determined using the method described by Boham and Kocipai [2001]. Ten grams of the ground samples were extracted repeatedly with 300 ml of methanol: water (80:20) at room temperature. The whole solution was filtered through Whatman filter paper No. 42 (125 mm). The filtrate was later transferred into a crucible and evaporated to dryness over a water bath and weighed.
Quantitative Determination of Total Phenols:
Total phenols were determined using the method described by [AOAC, 2000]. For the extraction of phenolic component, the fat free sample was boiled with 50 ml of ether for 15 minutes. 5 ml of the extract was pipette into a 50 ml volumetric flask, then 10 ml of distilled water was added. 2 ml of ammonium hydroxide solution and 5 ml of concentrated amyl alcohol were also added. The samples were made up to mark and left to react for 30 min for colour development. The absorbance of the solution was read using a spectrophotometer at 505 nm wavelengths.
Phytate Determination:
The Oberlese spectrophotometer method described by Ojinnaka [1994] was used. Two gram of the sample was extracted by acid hydrolysis, cold maceration and 0.05 M standard phytate solution was prepared by weighing and dissolution. Also, 0.5 mL of the extract and 1 mL of standard phytate solution were put in separate test tubes, treated with 1mL ferric ammonium sulphate solution, corked with stoppers and boiled in a water bath for 30 min. These were then cooled to 25 ºC in ice and 2 mL 2, 2-Bipyrimidine solution was added to each tube, mixed well and their respective absorbance measured with a UV-spectrophotometer at 519 nm.
The phytate content of the sample was calculated by:
Percentage phytate =
Where; W, Au, As, C, Vt and Va are weight of sample, absorbance of sample, absorbance of standard phytate solution, concentration of standard phytate (mg/mL), total extract volume and volume of extract used respectively.
Oxalate Determination:
The permanganate titration method described by Ojinnaka et al. [2013] was used. A 2g portion of the sample was suspended in 100 mL distilled water and 5 mL 6 M HCL was added. The mixture was digested by heating at 100oc for one hour, then cooled and filtered. The PH was adjusted to 4.5 by drop wise addition of concentrated aqueous ammonia solution before heating to 90 oC in a water bath. This was then cooled and filtered. The filtrate was again heated to 90oC and 10ml of 5% CaCl2 solution added with constant stirring and then allowed to cool and stored overnight in a refrigerator at 5oC. The mixture was then centrifuged at 300xg for 5 minutes. The supernatant was decanted and the precipitate dissolved in 10 mL 20% H2SO4. The solution was made up to 100 mL with distilled water and titrated against 0.05 M KMnO4 solution to a faint pink colour which persisted for30 seconds. The oxalate content is given by the relationship that 1 mL of 0.05M KMnO4 solution corresponds to 0.00225 g oxalate [Ojinnaka et al., 2013].
The formula below was then used to calculate the oxalate content:
Percentage oxalate =
Where, W represents the weight of sample use
Vitamin Analysis:
The quantitative vitamin analysis was carried out in the Department of chemistry SHEDAScience and Technology Complex Gwagwalada Abuja. Each sample (pulverized seed cotyledon of C. albidum fruits) was analysed in triplicate. This was carried out according to the method of [105]
Determination of thiamin (Vitamin B1):
5 g of the sample were homogenized with ethanolic sodium hydroxide (50 ml). It was filtered into a 100 ml flask. 10 ml of the filtrate was pipette and the colour developed by addition of 10 ml of potassium dichromate and read at 360 nm. A blank sample was prepared and the colour also developed and read at the same wavelength.
Determination of riboflavin (Vitamin B2):
5 g of the sample was extracted with 100 ml of 50% ethanol solution and shaken for 1 h. This was filtered into a 100 ml flask; 10 ml of the extract was pipette into 50 ml volumetric flask. 10 ml of 5% potassium permanganate and 10 ml of 30% H2O2 were added and allowed to stand over a hot water bath for about 30 min. 2 ml of 40% sodium sulphate was added. This was made up to 50 ml mark and the absorbance measured at 510 nm in a spectrophotometer.
Determination of niacin (Vitamin B3):
5 g of the sample was treated with 50 ml of 1 N sulphuric acid and shaken for 30 min. 3 drops of ammonia solution were added to the sample and filtered. 10 ml of the filtrate was pipette into a 50 ml volumetric flask and 5 ml potassium cyanide was added. This was acidified with 5 ml of 0.02 N H2SO4 and absorbance measured in the spectrophotometer at 470 nm wavelengths.
Determination of ascorbic acid (vitamin C):
Vitamin C content was determined by UV-spectrophotometry as described by Rahman et al. [2007]. One gram (1g) each of sample was weighed into a test tube. One millilitre ascorbic acid stock was pipetted into a separate test tube as a standard. One millilitre trichloroacetic acid (TCA) solution was placed in another test tube to serve as blank. Ten millilitre TCA solutions were added to the sample tubes. One millilitre dinitrophenyl hydrazine-thiourea-copper sulphate (DTCS) reagent was added to all the tubes and caped. The tubes was incubated in a water bath at 37oC for 3 hours, removed from the water bath and chilled for 10 min in an ice bath while shaking slowly and 2 ml of cold 12 M H2SO4 was added to all the test tubes. The absorbance of standard and test samples was read at 520 nm. The result was calculated as follows:
Vitamin C (g/100g) = x x 1000
Antimicrobial screening:
Pathogenic clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Candida albicans were collected from Ritchez medical laboratory, Maitama, Abuja, Nigeria
The quantitative phytochemical analysis of Chrysophyllum albidum cotyledon crude extract indicated that it was rich in phenolics, oxalates, saponins, flavonoids, terpenoids, Phylates and tannins. The result of the analysis (Table 1) indicated that the extract had high quantity of oxalates, flavonoids and terpenoids. Ajayi and Ifedi [2015] reported the presence of saponin, flavonoids and alkaloids in Chrysophyllum albidum seed powder. The phytochemical quantities observed in this study were lower than those reported by Ibrahim et al.[2017].
Ibrahim et al. [2017] studied the quantitative phytochemical constituents of Chrysophyllum albidum seed pericarp, fruit pulp and fruit skin. The authors reported that the seed pericarp had a significantly higher alkaloids, tannins flavonoids and vitamin C compared to the fruit pericarp and fruit skin. The vitamin analysis of C. albidum is shown in Table 2.
The result of analysis indicates that ascorbic acid content was high compared to Niacin, riboflavin and thiamine. This is in agreement with findings of Ibrahim et al. [2017] who reported high ascorbic acid content of the seed pericarp of C. albidum
The antimicrobial analysis for cotyledon extracts showed no activity at concentrations lower than 160 mg/ml. The MIC as shown in Table 3 for different extracts was at a concentration of 160 mg/ml.
The result of the antimicrobial analysis indicates that Escherichia coli was significantly susceptible to all the extracts compared to other bacteria studied. The bacterium (E. coli) was highly susceptible to the aqueous extract (p<0.05). Pseudomonas aeruginosa was only susceptible to ethanol and ethyl acetate extracts. Staphylococcus aureus was highly susceptible to hexane and ethanol extracts. Bacillus spp. and Candida albicans had the least susceptibility to the test extracts. This result is in agreement with findings of Akin-Osanaiye et al. [2018].
Akin-Osanaiye et al. [2018] studied the antibacterial efficacy of extract of C. albidum. The authors reported that C. albidum extract successfully inhibited the growth of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. Their study indicated that E. coli, Staphylococcus aureus and P. aeruginosa was susceptible to ethanol extract at all concentrations studied. The antimicrobial analysis indicated that Bacillus spp. had poor susceptibility to the extracts of C. albidum. This result is in agreement with the findings of Oputah et al. [2016]. The authors reported that all test bacteria were susceptible to ethanol seed extract of C. albidum except Bacillus cereus.
Oputah et al. [2016] also reported that Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris exhibited significant susceptibility to ethanol extract of C. albidum.
The authors declare no competing interests.