4 RESULTS AND DISCUSSION

The plant Chrysophyllum cainito concentrated methanolic extract was black-green in color. Test for phytochemical screening for presence of alkaloids was conducted first, a positive result will show an orange and white precipitate formation on both general test and primary assay of the two reagents namely Dragendorff’s and Mayer’s reagent. The plant extract gave negative results of no precipitate formation on both.

Mayer's Reagent contain potassium mercuric iodide solution, gives white precipitate with the presence of a heavy metal, mercury, yield double salts. Dragendorff's reagent contain potassium bismuth iodide that gives orange precipitate with presence of a heavy metal atom.

Fig. 4.1 Results of General Fig. 4.2 Results of Primary AssayTest of Alkaloids of Alkaloids

Phytochemical screening for Saponin was tested using Froth Test and Capillary Test. The plant extract gave a positive result of “honeycomb” froth greater than two centimeters that persisted for more than ten minutes which indicates presence of detergent property. It also gave a positive result on Capillary test having a ten milimeter height of water and four milimiter height on the extract. Plant extract is positive for Saponin.

Saponins in plant materials can cause persistent foam when the aqueous solution is agitated, the foam formed on the water surface indicates presence of saponin. Lowering surface tension of water is also one of the characteristics of saponin which is the principle involved in capillary test.

Fig.4.3 Froth Test of Saponin Fig. 4.4 Capillary Test of Saponin

Test for Cardiac Glycoside was conducted using Keller-Killani,Kedde’s reagent and Liebermann-Burchard. Keller-Killani gave a negative result of a solution with brown ring at the middle which means absence of deoxysugars. Kedde’s reagent also produced a negative result of a reddish-brown solution with oil globule at the center, indicates absence of lactone ring, and finally Liebermann-Burchard with no discoloration or a clear colorless solution, denotes absence of aglycone. The group confirmed the plant extract does not contain cardenolides.

Keller killani is a solution of anhydrous acetic acid, ferric chloride and sulfuric acid it isolates glycosides which gives a reddish brown color that may turn blue or purple.

Keddes reagent is a solution of 3,5-dinitrobenzoic acid and NaOH, is used for detecting the presence of a lactone ring of cardenolides in the cardiac glycosides.

Libermann-Burchard is a solution of acetic anhydride and concentrated sulfuric acid allows detecting steroidal nucleus of cardiac glycosides

Fig. 4.5 Keller-Killani’s Test Fig 4.6 Kedde’s Test Fig.4.7 Liebermann-Burchard

Phytochemical screening for Anthraquinones was tested using Borntrager and Modified Borntrager test. The plant extract did not give a red and pink coloration which denotes negative for anthraquinones and anthraquinone glycoside.

Borntrager's test is a solution by adding ammonia to the benzene extract used for the presence of anthraquinone, the solution is shaked that gives a red coloration on the lower ammoniacal layer. Anthraquinones, in some cases may not respond due to its reduced form so modified borntrager will be used.

Modified Borntrager makes use of a solution made by adding ammonia to the acidified benzene extract. The solution is also subjected to shaking that will give a pink coloration.

Fig4.8 Borntrager’s Test for Anthraquinones Fig4.9 Modified Borntrager’s test for Anthraquinones

Bate-Smith and Metcalf and Wilstatter “cyanidin” test were used to conduct the Phytochemical screening for flavonoids. Bate-Smith and Metcalf gave a negative result of yellow solution which indicates absence of Leucoanthocyanin, and also a negative result for Wilstatter “cyanidin” that formed an oily layer which means absence of y-benzopyrone. Plant sample is negative for Flavonoids.

In the Bate-Smith and Metcalf’s test, the acidification of the extract identifies the presence of the leucoanthocyanin structure which will yield to a strong red or violet color in the solution. While in the Wilstater “Cyanidin” test, the acidification and reduction of flavonoids identifies the presence of ϒ-benzopyrone. When redox reaction is exhibited, a change in color ranging from orange to red to crimson, and magenta, occasionally to green or blue indicates a positive result

Fig 4.10 (B)Bate-Smith and Metcalf and (C) Wilstatter “Cyanidin” test for Flavonoids

Gelatin and Ferric Chloride test were used as Phytochemical screening for Tannins. The plant sample gave a positive result of forming a gelatin precipitate which indicates positive for Tannins. Ferric Chloride test was used to differentiate hydrolyzable from condensed tannins, the result was a brownish green solution which confirms that it is a condensed tannin.

In the Ferric Chloride test, the hydrolyzable and condensed tannins present in the plant extract are detected by adding of ferric chloride to yield a color reaction. This test differentiates the hydrolysable and condensed tannins. The Blue-black coloration indicates the presence of hydrolyzable tannins while the Brownish-green is for the condensed tannins. Another test that was performed is the Gelatin test. Tannins usually cause proteins to precipitate. Therefore, the formation of a gelatin precipitate in the solution indicates the presence of tannins since gelatin is a mixture of peptide and proteins.

Guignard’s Test was conducted in Phytochemical screening for cyanogenic glycosides or cyanophore. It failed to give an appearance of various shades of red on the pictrate paper which makes it negative for cyanophore.

Cyanogenic Glycosides In the Guignard’s test, a positive result would be the liberation of HCN gas from the extract, turning the Sodium Picrate paper from its original yellow color to brick red. The change in color is due to the formation of sodium isopurpurate.

Fig 4.11 Test for Tannins Fig 4.12 Guignard’s Test for Cyanophore

Gelatin Test

Ferric Chloride Test

Fractionation made use of DCM, hexane and water extracts for Thin Layer Chromatography (TLC) Assay ,three chambers namely A,B and C each containing different tests and observed under UV light . Chamber A with solvents of DCM, Acetic acid and water (5:4:1) that was subjected to Kedde’s reagent did not result to blue/red violet coloration, hexane giving an Rf value of 0.4583. Chamber B with solvents of water :DCM (5:1) and was subjected to Dragendorff’s reagent also failed to give a positive result with hexane giving an Rf value of 0.5625 which indicates absence of alkaloids. Chamber C with methanol:water solvents subjected to Potassium Ferricyanide or “Ferric Chloride” also gave a negative result, water having an Rf value of 0.7292.

Fig.4.13 Thin Layer Chromatography results

Thin Layer Chromatography was conducted for the detection of cardenolides, alkaloids and phenolic compounds in the plant sample. Results may have varied from the Phytochemical screening of Tannins having a positive result when TLC gave a negative result, this can be due to some error in extraction or the reagents used since the experiment were conducted on different dates.

Table 4.1 Summary of results of Phytochemical screening of Chrysophyllum cainito.

Constituent Name of Test Positive result Experimental result

Inference

General TestAlkaloids Mayer’s Test White precipitation No white

precipitationNegative for alkaloids

Dragendorff’s test

Orange precipitation

No orange precipitaion

Negative for alkaloids

Primary AssayMayer’s Test White precipitation No white

precipitationNegative for alkaloids

Dragendorff’s test

Orange precipitation

No orange precipitaion

Negative for alkaloids

Saponin Froth Test >2cm honeycomb froth that persisted

2cm honeycomb froth that

Presence of detergent property

Chamber A

Chamber B

Chamber C

for more than 10 minutes

persisted for more than 10 minutes

Capillary Test <1/2 level of water 4mm height of extract

Positive for saponins

Cardiac Glycosides

Keller-Killani Reddish brown which may turn blue or purple

With brown ring at middle

absence of deoxysugar

Liebermann Burchard

Blue to green, red pink, purple or violet coloration

No discoloration absence of aglycone

Kedde’s Test Blue-violet color Reddish brown solution with oil globule at the center

Absence of lactone ring

Anthraquinones Borntrager’s test

Red coloration No red coloration Absence of anthraquinone

Modified Borntrager’s test

Pink coloration No pink coloration Absence of anthraquinone glycosides

Flavonoids Bate Smith & Metcalf

Strong red or violet coloration

Yellow solution Absence of leucoanthocyanin

Wilstatter “cyanidin”

Orange to red to crimson

Formed an oily layer

Absence of y-benzopyrone

Tannins Gelatin Test Formation of jelly precipitate

With gelatin precipate

Positive for Tannins

Ferric Chloride test

Blue black- hydrolyzable

Brownish green- condensed tannin

Brownish green solution

Condensed tannin

Cyanogenic Glycosides

Guignard’s Test Various shades of red

No various shades of red

Absence of cyanophore

Table 4.2 Summary of results of Thin Layer Chromatography test

Solvent System Spray Reagent + result Experiment result Rf Value Inference

DCM : Acetic acid : water

Kedde’s reagent

Blue to red violet

Hexane: negative 0.4583 Hexane, DCM & water do not contain cardenolides

DCM: negativeWater : negative

Hexane: negative 0.5625 Hexane, DCM &

DCM:water Dragendorff’s reagent

Brownish orange

water do not contain alkaloids

DCM: negativeWater : negative

Methanol:water

Potassium ferricyanide or Ferric Chloride

Blue precipitate

Hexane: negative Hexane, DCM & water do not contain Phenolic groups

DCM: negativeWater : negative 0.7292