Phytochemical and Antioxidants Screening of Chrysophyllum albidum, Mezoneuron benthamianum, Phyllanthus muellerianus And Acalypha fimbriata

Qualitative and quantitative phytochemical screening of Chrysophyllum albidum, Mezoneuron benthamianum, Phyllanthus muellerianus and Acalypha fimbriata. All the four plants contained alkaloids, tannin and flavonoids. Steroids and anthraquinone were present in C. albidum, M. benthamianum, A. fimbriata. Saponin was present in C. albidum, M. benthamianum and P. muellerianus. Terpenoids were found in C. albidum and A. fimbriata. Cardiac glycosides was present in C. albidum, P. muellerianus and A. fimbriata. The antioxidant activities of the plants were evaluated using DPPH free radical scavenging activity, Nitric oxide scavenging activity and Reducing power activity. Chrysophyllum albidum had the lowest calculated IC50 for DPPH and NO assessment, 0.913 mg/ml and 117.818 μg/ml respectively. C. albidum also showed the highest value of total antioxidant capacity, 70.36. Acalypha fimbriata had the lowest IC50 for Reducing power potential,11.007 μg/ml but a total antioxidant capacity of 48.9. The Iron II chelation ability of the plants were dose dependent with Acalypha fimbriata showing the closest potential to the standard EDTA and Chrysophyllum albidum showing the least potential. The research has shown that the phytoconstituents and the antioxidant properties of these medicinal plants would be responsible for the therapeutic claims of the plants.


Introduction
Medicinal plants are of great importance in drug development and humans have used them for different diseases from the beginning of human history (Rahman et al, 2017).The medicinal power of plants lies in phytochemical constituents that cause definite pharmacological actions on the human body (Akinmoladun et al, 2007). It is believed that crude extract from medicinal plants are more biologically active than isolated compounds due to their synergistic effects (Jana and Shekhawat, 2010). The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic compounds (Doss, 2009). Majority of phytochemicals have been known to bear valuable therapeutic activities such as insecticides (Kambu et al, 1982 ) It can be formed in living organisms by both endogenously (respiration, peroxisomes stimulation of polymorphonuclear leucocytes and macrophages) and exogenously (ionizing radiation, tobacco smoke, pollutants, pesticides and organic solvents) .These free radicals are produced by our body to stabilize the body's natural function. The excess amount of free radical could cause oxidative cell and tissue damage (Sen et al, 2010). It can also cause oxidative damage to proteins, lipids and DNA and chronic diseases such as cancer, diabetes, aging and other degenerative diseases in humans (Aiyegoro and Okoh, 2010). Antioxidants are capable of stabilizing, or deactivating, free radicals before they attack cells. Antioxidants are absolutely critical for maintaining optimal cellular and systemic health and wellbeing ( Van et al, 2000).
Iron overload is the excess iron in the body. The body has limited capacity to excrete excess iron. Though iron deficiency can cause anemia, its excess in the body increases the body susceptibility to infection and promotes free radical tissue damage. Iron overload impairs host immune defence mechanism, promotes replication and growth of pathogens. Thus removal of excess iron during infection in iron overload host may be beneficial by restoring the body immunity and denying the pathogen of excess iron for its replication and growth (Cronje and Bornman, 2005).
Chrysophyllum albidum, African star apple, belongs to the plant family Sapotaceae. The plant is a lowland rain forest tree species that grows up to 25 to 37 m in height at maturity with a girth varying from 1. Aqueous extract of Mezoneuron benthamianum had significant vasorelaxing, antioxidant and aphrodiasiac properties (Zamble et al, 2008). An infusion of the dried roots of the plant is drunk or used as a bath against general malaise. Gallic acid and gallic acid derivatives have been isolated from the leaves of Mezoneuron benthamianum (Binutu and Cordell, 2000). A decoction of roots, bark and leaves is used to cure urethral discharge (Schmeizer et al, 2008). Traditionally, Mezoneuron benthamianum is used in management of erectile dysfunction, dysentery, urethral discharges, skin diseases and wounds (Akosua et al, 2011). Gallic acid and its methyl ester (methylgallate) inhibit the growth of both Gramnegative and Gram-negative bacteria, but this other gallate derivatives only suppress Gram-positive bacteria (Osho, 2014).
Phyllanthus muellerianus is widely used to treat intestinal troubles. An infusion of the young shoots is taken to treat severe dysentery (Schmelzer et al, 2008). Phthalates compounds have been isolated from Phyllanthus muellerianus (Euphorbiaceae) (Saleem et al, 2009). Decoction of Acalypha fimbriata is used as laxatives (Kola et al, 2008). The leaves of acalypha fimbriata are used in ashma, rheumatism, syphilis, ulcers and also as antihelmintic, antimicrobial and antifungal in Nigeria (Odugbemi, 2008). The aim of this research is to study the phytochemical constituents and the antioxidant potential of these four medicinal plants of which their anti-mycobacterium tuberculosis had been earlier reported.

MATERIALS AND METHODS Collection of Plant Samples:
Batches of Chrysophyllum albidum fruits, Phyllanthus muellerianus and Acalypha fimbriata were purchased in Mushin market in Lagos State, while Mezoneuron benthamianum plant was obtained from Iberekodo market in Ogun State, Nigeria.

Phytochemical Screening:
Qualitative and quantitative phytochemical screening of the plants was carried out according to Sofoluwe

Test for alkaloids:
A 5 mg sample of the extract dissolved in3 ml of acidified ethanol was warmed slightly and then filtered. Few drops of Mayerʼs reagent and 1 ml of Dragendroffʼs reagent were added to 1 ml of the filtrate and turbidity was observed.
Test for Saponins: 5 ml of the extract solution was shaken vigorously for a stable persistent froth. The frothing was mixed with olive oil and was shaken vigorously. The formation of emulsion indicated the presence of saponins in the samples.
Test for Tannins: 0.25 g of various solvent extract was dissolved in 10 ml distilled water and filtered. 1% aqueous Iron chloride (FeCl 3 ) solution was added to the filtrate. The appearance of intense green, purple, blue or black colour indicated the presence of tannins in the test sample.

Test for Phlobatannins:
Two millilitres (2 mL) of the aqueous solution of the extract were added into 1% aqueous hydrochloric acid and was then boiled with the help of Hot plate stirrer. Formation of red colored precipitate confirmed a positive result.

Test for Anthraquinones:
One gram (1 g) of the powdered seed 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 as indicative of the presence of Anthraquinones. .

Test for Terpenoids (Salkowski test):
5 ml of solvent extract was mixed in 2 ml of chloroform and 3 ml of concentrated H 2 SO 4 was carefully added. A layer of the reddish brown colouration was formed at the interface thus indicating a positive result for the presence of terpenoids.
Test for Flavonoids: 0.5 g of various extract was shaken with petroleum ether to remove the fatty materials (lipid layer). The defatted residue was dissolved in 20 ml of 80% ethanol and filtered. The filtrate was used for the following tests: (a) 3 ml of the filtrate was mixed with 4 ml of 1% aluminium chloride in methanol. Formation of yellow colour indicated the presence of flavonols, flavones and chalcones. (b) 3 ml of the filtrate was mixed with 4 ml of 1% potassium hydroxide. A dark yellow colour indicated the presence of Flavonoids. (c) 5 ml of the dilute ammonia solution was added to the portion of the aqueous filtrate of each plant extract followed by the addition of concentrated H 2 SO 4 . The appearance of the yellow colouration indicated the presence of flavonoids.

Test for steroids:
A 5 ml sample of the extract was added to 2 ml acetic anhydride and 2 ml H 2 SO 4 . The colour change from violet to blue or green in some samples indicated the presence of steroids.

Test for Cardiac glycosides (Keller-Killani test):
1ml of the extracts were dissolved in 1ml of glacial acetic acid and cooled, after cooling, 2-3 drops of ferric chloride was added. To this solution 2ml of conc. sulphuric acid was added carefully along the walls of the test tube. Reddish brown ring was formed at the interface which indicated the presence of deoxysugar of cardenoloides.

Estimation of alkaloids:
200 cm 3 of 10% acetic acid in ethanol was added to each plant powder sample (2.50 g) in a 250 cm 3 beaker and allowed to stand for 4 hours. This was filtered and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added dropwise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed and the percentage of alkaloid is expressed mathematically as % Alkaloid = Weight of alkaloid x 100 Weight of sample  ) were added. Two-hour reflux of the mixture was carried out, cooled, and diluted to 500 cm 3 with distilled water. One gram of each plant powder in a conical flask was added to 100 cm 3 of distilled water. This was boiled gently for 1 hour on an electric hot plate and filtered using number 42 (125 mm) Whatman filter paper in a 100 cm 3 volumetric flask. Addition of 5.0 cm 3 Folin-Denis reagent and 10 cm 3 of saturated Na 2 CO 3 solution into 50 cm 3 of distilled water and 10 cm 3 of diluted extract (aliquot volume) was carried out after being pipetted into a 100 cm 3 conical flask for colour development. The solution was allowed to stand for 30 minutes in a water bath at a temperature of 25°C after thorough agitation. With the aid of a Spectrum Lab 23A spectrophotometer optical density was measured at 700 nm and compared on a standard tannic acid curve. Dissolution of 0.20 g of tannic acid in distilled water and dilution to 200 cm 3 mark (1 mg/cm 3 ) were used to obtain tannic standard curve. Varying concentrations (0.2-1.0 mg/cm 3 ) of the standard tannic acid solution were pipetted into five different test tubes to which Folin-Denis reagent (5 cm 3 ) and saturated Na 2 CO 3 (10 cm 3 ) solution were added and made up to the 100 cm 3 mark with distilled water. The solution was left to stand for 30 minutes in a water bath at 25°C. Optical density was ascertained at 700 nm with the aid of a Spectrum Lab 23A spectrophotometer. Optical density (absorbance) versus tannic acid concentration was plotted. The following formula was used in the calculation: where C is concentration of tannic acid read off the graph Estimation of Steroids: 1 ml of text extract of steroid solution was transferred into 10 ml volumetric flask. Sulphuric acid (4N, 2 ml) and iron (III) chloride (0.5% w/v, 2 ml) were added followed by potassium hexacyanoferrate (III) solution (0.5% w/v, 0.5 ml). The mixture was heated on a water bath maintained at 70 ±2 o C for 30 minutes with occasional shaking and was diluted to the mark with distilled water. The absorbance was measured at 780 nm against the reagent blank (Mahdu et al, 2016)

Estimation of Phenols:
The determination of total phenolics based on Folin-Ciocalteu reagent assay. An aliquot (1ml) of extracts and standard solution of Gallic acid (100 mg/ml) was added to 25 ml volumetric flask, containing 9 ml distilled water. The distilled water itself was used as blank. One ml of Folin-Ciocalteu reagent was added to the mixture and shaken. After 5 min, 10 ml of 7% Na 2 CO 3 solution was added to the mixture. The solution was diluted to volume (25 ml) with distilled water and mixed. After incubation for 90 min at room temperature, the absorbance against prepared reagent blank was determined at 750 nm with an UV-Vis Spectrophotometer. The total phenolic content of root extracts expressed as mg Gallic acid equivalents (GAE)/100 G fresh weights

Estimation of terpenoids:
About 2 g of the plant powder was weighed and soaked in 50 ml of 95 % ethanol for 24 h. The extract was filtered and the filtrate was extracted with petroleum ether (60 to 80 o C) and concentrated to dryness. The dried ether extract was treated as total terpenoids (Ferguson, 1956).
Estimation of Cardiac Glycosides ( using Muhammad and Abubakar, 2016) 8 ml of plant extract was transferred to 100 ml volumetric flask and 60 ml of H 2 O and 12.5 % of lead acetate were added, mixed and filtered. 50 ml of the filtrate was transferred into another 100ml flask and 8 ml of 47 % Na 2 HPO 4 were added to precipitate excess Pb 2+ ion. This was mixed and completed to volume with water. The mixture was filtered twice through same filter paper to remove excess. 10 ml of purified filtrate was transferred into clean Erlenmeyer flask and treated with 10 ml Balfet reagent. A blank titration was carried out using 10 ml distilled water and 10 ml Balfet reagent. This was allowed to stand for one hour for complete colour development. The colour intensity was measured colororimetrically at 495 nm. Calculation % of total glycosides = A x 100 g % 77 Where A = Absorbance DPPH radical scavenging assay: DPPH (2, 2-diphenyl picryl hydrazyl) is a commercially available stable free radical, which is purple in colour. The antioxidant molecules present in the herbal extracts, when incubated, react with DPPH and convert it into di-phenyl hydrazine, which isyellow in colour. The degree of discoloration of purple to yellow was measured at 520 nm, which is a measure of scavenging potential of plant extracts. 10 µl of plant extract was added to 100 µl of DPPH solution (0.2mM DPPH in methanol) in a microtitre plate. The reaction mixture was incubated at 25 0 C for 5 minutes, after that the absorbance was measured at 520 nm. The DPPH with corresponding solvents (without plant material) serves as the control. The methanol with respective plant extracts serves as blank. The DPPH radical scavenging activity of the plant extract was calculated as the percentage inhibition.

NITRIC OXIDE Scavenging Activity:
NO. generated from sodium nitroprusside (SNP) was measured according to the method of Marcocci et al. (1994). Briefly, the reaction mixture (5.0 ml) containing SNP (5 mM) in phosphatebuffered saline (pH 7.3), with or without the plant extract at different concentrations, was incubated at 25˚C for 180 min in front of a visible polychromatic light source (25W tungsten lamp). The NO. radical thus generated interacted with oxygen to produce the nitrite ion (NO. ) which was assayed at 30 min intervals by mixing 1.0 ml of incubation mixture with an equal amount of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediaminedihydrochloride).
The absorbance of the chromophore (purple azo dye) formed during the diazotisation of nitrite ions with sulphanilamide and subsequent coupling with naphthylethylenediaminedihydrochloride was measured at 546 nm. The nitrite generated in the presence or absence of the plant extract was estimated using a standard curve based on sodium nitrite solutions of known concentrations. Each experiment was carried out at least three times and the data presented as an average of three independent determinations.

Reducing power assay:
The sample extracts of the plants were put in 1 ml of phosphate buffer in a test tube and 5 ml of 0.2 M phosphate buffer, pH 6.6, was added. To this, 5 ml of 1 % potassium ferricyanide solution was added. The mixture was then incubated at 50 o C for 20 min. 5 ml of 10 % TCA was added after the incubation and the content was centrifuged at 1000 rpm for 10 min. The upper layer of the supernatant (5 ml) was mixed with 5 ml of distilled water. 1 ml of ferric chloride was then added and vortexed. Then, the absorbance of the reaction mixture was read spectrophotometrically at 700 nm against water blank (Oyaizu, 1986).

Metal Chelating Activity:
The chelating ability of the plants extracts was examined using Dinis et al, 1994. 50 µl of 2 mM FeCl 2 was added to 1 ml of different concentration of the extract (2, 3, 4, 5 mg/ml). The reaction was initiated by the addition of 0.2 ml of 5 mM ferrozine solution. The mixture was vigorously shaken and left to stand at room temperature for 10 minutes. The absorbance of the solution was thereafter measured at 562 nm. The percentage inhibition of ferrozine-Fe 2+ complex formation was calculated as [(A o -A s )/A s ] x 100, where A o was the absorbance of the control and A s was the absorbance of the extract/standard. Na 2 EDTA was used as positive control.

RESULTS AND DISCUSSION:
Evidence from laboratory studies show that phytochemicals have therapeutic effect against some severe disorders. In this study, qualitative and quantitative phytochemical screening of Chrysophyllum albidum, Mezoneuron benthamianum, Phyllanthus muellerianus and Acalypha fimbriata were investigated. Table 1 shows the result of the phytochemical screening.     The DPPH free radical scavenging, Nitric Oxide scavenging, Reducing power potential and Iron II chelation ability of the four plants were assessed. The results of the antioxidant activities were shown graphically in Figures 5-9. The IC 50 values calculated were shown in Table 2.   Both Chrysophyllum albidum and Acalypha fimbriata have IC 50 values lower than that of the standard ascorbic acid in DPPH scavenging activity as shown in Table 3. Also observed from the table 3, the IC 50 values of Reducing Power of Acalypha fimbriata, Chrysophyllum albidum and Mezoneuron benthamianum are lower than that of the ascorbic acid. Figure 12 shows Chrysophyllum albidum has the highest total antioxidant capacity (70. 36 Figure 9 showed the Fe 2+ chelation ability of the plants extracts to be concentration dependent. EDTA was used as standard. From figure 12, the values of the total flavonoids of the plants were 6.72 for A. fimbriata, 4.085 for P. muellerianus, 2.777 for M. benthamianum and 1.003 for Chrysophyllum albidum. The Fe 2+ chelation ability of the extracts showed the chelating ability of A.fimbriata > P. muellerianus > M. benthamianum > C. albidum. Thus there is a direct relationship between the flavonoid content and chelation potential of the plants. Acalypha fimbriata has the lowest IC 50 value for reducing power (11.007 µg/ml) and the highest concentration dependent Fe 2+ chelation ability. Excess Iron is stored as Fe 3+ in Ferritin and iron overload sustains for a long period if the stored iron is not getting reduced and released for metal chelating drugs (Sarkar et al, 2012). Thus A. fimbriata with significant ability to reduce Fe 3+ and good Fe 2+ chelation potential seems a promising medicinal plant with a flavonoid that could be of health benefit to iron overload infected people. In general, the selected medicinal plants, C. albidum, M. bethamianum, P. muellerianus and A. fimbriata would possess several health benefits by virtue of their antioxidant properties.

Conclusion
This research work showed that plants contained phytochemicals that could be responsible for their medicinal values. The four medicinal plants showed strong antioxidant activity with Chrysophyllum albidum having the highest total antioxidant capacity. Acalypha fimbriata had the lowest IC 50 value for reduction potential and the highest dose dependent Iron II chelation ability. These properties of Acalypha fimbriata could be due to the presence of flavonoids in the plants. Isolation and identification of the bioactive compounds in the plants would help more in their ethnomedicinal uses.

Conflict of Interest
The authors declare no conflict of interest.