Phytochemical screening of the leaves of Lophira lanceolata ...

Audu, et al, Phytochemical screening of the leaves of Lophira lanceolata (Ochanaceae) 

Phytochemical screening of the leaves of 

Lophira lanceolata (Ochanaceae) 

Sani Ali Audu 1,* , Ilyas Mohammed 2 , Haruna Abdul Kaita 2 

1 

Department of Pharmaceutical Chemistry, University of Maiduguri, Nigeria; 2 Department of Pharmaceutical and Medicinal 

Chemistry, Ahmadu Bello University, Zaria, Nigeria 

Received September 12, 2007 

Abstract 

Phytochemical screening of the ethanol extract of the leave of Lophira lanceolata indicated the presence of flavonoids, 

anthraquinones, carbohydrate, glycoside, phenols, saponin steroid, tannin, free reducing sugar. However alkaloid were 

absence in this species. The glycoside after hydrolysis afforded quercetin as the genin and a sugar was identified as glucose. 

[Life Science Journal. 2007; 4(4): 75 – 79] (ISSN: 1097 – 8135). 

Keywords: Lophira lanceolata; ash value; sugar; chromatogram 

1 Introduction 

While the forest is referred to as GOD’s own pharmacy 

(Treben, 1986) many medicinal plants are used in modern 

medicine where they occupy a very significance place as 

raw material for important drugs and plants used in traditional 

system of medicine in pharmaceutical houses are 

collected from wild sources (singh et al, 2003). Most 

drugs of plant origin used by medical practitioners are 

in the form of extract of the whole plant material or part 

of it. In view of this, local medicinal plants, which show 

suitable biological effect, could be standardized and similarly 

utilized. Some of the effects elaborated by extract 

of plants used in traditional medicine include antiviral, 

antitumor, antimicrobial, insecticide and central nervous 

system effect (Sofowora, 1982). 

During the past century, the extra ordinary results of 

research have unquestionably led to success at an exponential 

rate, which the practitioner of modern medicine 

now enjoys. However the exclusive use of this research 

oriented approach with little regard for knowledge acquired 

through the empirical method has served to delay 

the application of many potential benefits (Dastur, 1964). 

World health organization has estimated that perhaps 

about 80% of more than 4000 million people on earth rely 

chiefly on traditional medicine for their primary health 

*Corresponding author. Email: defencesani@yahoo.com 

care needs (Akerele, 1990), and also can safely be presumed 

that a major part of traditional therapy involve the 

use of plant extract or their active principles. Such treatments 

include the administration of infusion boiled parts, 

as some of the natural drugs are not usually in the form 

of tablet or pills (Oliver, 1960). Although the structure 

of some plant constituents are now known, many compounds 

that remain undiscovered in plant are beyond the 

imagination of scientist (Sievers, 1930). The chemical 

constituents in medicinal plant usually explain the rational 

for the use of the plants in traditional medicine (Fansworth, 

1984). 

2 Materials and Methods 

2.1 Extraction 

Dried and coarsely powdered leaves of Lophira lanceolata 

(350 g) were refluxed with petroleum ether (60 

– 80 ºC) for 10 hours. The extract was decanted off and 

fresh quantity of the petroleum ether was added again and 

refluxed for another 10 hours. 

The defatted leaves were completely dried and extracted 

with ethanol. The combine ethanol extracts where concentrated 

on water bath whereby a highly viscous greenish-brown 

mass was obtained. This was refluxed with 

petroleum ether (60 – 80 ºC), benzene and chloroform 

successively until the solvent in each case was almost 

colourless. The residue left behind was then treated with 

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hot water. The water insoluble portion was dissolved in 

ethanol and dried under reduced pressure. A solid brown 

residue (30.2 g) obtained respond to usual flavonoid colour 

tests was marked “A”. 

The aqueous solution was extracted with ethyl acetate. 

The process was repeated twice. The ethyl acetate extract 

where combined and the solvent was recovered under reduced 

pressure. The semi- solid residue was marked “A 1 ” 

and respond to usual flavonoidal test. 

2.2 Thin layer chromatographic examination of fraction 

“A” 

Thin layer chromatographic plate (5 × 20 cm) 0.5 mm 

thickness was prepared by usual method using silica gel 

G (E.Merck). The sample of 0.1% of “A” was dissolve in 

alcohol and spotted manually using a capillary tube. The 

plate was developed in BPF (benzene : pyridine : formic 

acid, 36 : 9 : 5) as solvent system. After development, on 

examination of the chromatogram under U.V. light and 

then sprayed with ferric chloride the presence of one spot 

with trailing was revealed. This was purified with column 

chromatography. 

2.3 Fraction “A 1 ” 

The ethanol solution of fraction “A 1 ” was subjected to 

paper chromatographic analysis using Whatmann paper 

No.1. In each solvent system, the chromatograms were 

developed for 10 hours. After drying in fume cupboard, 

the chromatograms were examined under U.V. light, 

which revealed the presence of two spots with trailing. 

2.4 Hydrolysis of fraction “A 1 ” 

10 mg of fraction “A 1 ” was dissolved in water and hydrolyzed 

by refluxing with 2 ml of 0.6 M hydrochloric 

acid. The hydrolysis appeared to be completed within a 

few munites, but the heating was continued for 2 hours to 

ensure complete hydrolysis. After leaving over-night, the 

aglycone was filtered, washed, dried at room temperature 

and marked fraction G. 

2.5 Chromatographic identification of the sugar 

The filtrate (from which the aglycone was removed) 

was neutralized with aqueous ammonia until it was neutral 

to litmus paper. It was concentrated under reduced 

pressure to a syrupy mass. This syrupy mass was chromatographed 

on whatmann paper No. 1 using n-Butanol 

: acetic acid : water (4 : 1 : 5) as solvent system with 

authentic (reference sugar) samples. The chromatogram 

after development was dried in a fume cupboard sprayed 

with aniline-H-phthalate reagent and then heated at 110ºC 

for 5 minutes. After development on examination the 

chromatogram shows the presence of a spots equivalent 

to R f values of authentic glucose were revealed. 

2.6 Preparation of authentic sugar 

The authentic sugar rhamnose, glucose, galactose, 

fructose, xylose and arabinose were prepared by dissolving 

small sample of the sugar in distilled water. 

2.7 Quantitative evaluation of the leave of Lophira lanceolata 

leaves. 

(a) Determination of total ash value 

(b) Determination of acid insoluble ash value 

(c) Determination of water-soluble ash value 

(d) Alcohol soluble extractive value 

(e) Water soluble extractive value (British Pharmacopoeia, 

1993). 

3 Results 

On defatting the powdered leave of the plant material 

with petroleum ether (60 – 80 ºC), the merc was extracted 

with ethanol. The ethanol extract was fractionated into 

fraction “A” and fraction “A 1 ” both of which gave usual 

colour test for flavonoid. 

3.1 Phytochemical screening 

The result of the phytochemical screening of ethanol 

(fraction “A”) (A), chloroform (B) and petroleum ether 

(C) extracts was summarized in Table 1. 

3.2 Determination of ash value of lophira lanceolata 

leaves 

Evaluation of crude powder leave of the plant Lophira 

lanceolata in Table 2. 

3.3 Colour test of fraction “A” 

The result of various colour reactions conducted on 

fraction “A” was summarized in Table 3. 

3.4 Colour test of fraction “A 1 ” 

The result of various colour reactions conducted on 

fraction “A 1 ” was summarize in Table 4. 

3.5 Thin layer chromatographic examination of fraction 

“A” in three different solvent 

BPF, 36 : 9 : 5; TPA, 10 : 1 : 1 and B : EA : A, 8 : 5 : 2 

gave a good chromatogram, Figure 1. 

3.6 Result of hydrolyzed extract G 

The aqueous sugar portion after hydrolysis and neutralization 

was chromatographed with reference authen- 

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Flavonoids 

(a) Ferric chloride test 

Table 1. The phytochemical screening results. 

Group Test Observation Inference 

Aqueous extract + 10% 

ferric chloride 

A green precipitate 

A (+++), B (+++), C (–) 

(b) Lead acetate test Aqueous extract + 10% lead acetate Buff coloured solution A (++), B (++), C (–) 

(c) NaOH test 

(d) Shinoda test 

Carbohydrate 

Ethanol extract + 10% NaOH + dil 

HCl 

Mg chip + alcoholic extract + few 

drops of conc. HCl 

Yellow solution turned colourless 

on addition of dil HCl 

Effeverscence dark brown solution 

turns red 

A (++), B (++), C (–) 

A (++), B (++), C (–) 

General test Extract + distilled water + H 2 SO 4 A dull violet precipitate A (+), B (+), C (–) 

Test for starch 

Barfoed’s test for sugar 

Reducing sugar 

Fehling’s test 

Combine reducing sugar 

Cardiac glycoside 

Steroidal nucleus Liebermann-Burchard 

test 

Salkowskii test 

Cardenolide 

Keller-Killiani test 

Saponin 

Aqueous extract + 5 ml 5% KOH 

solution 

Aqueous extract (filtrate) + 1 ml 

Barfoed’s reagent then heated in a 

beaker of boiling water 

Aqueous extract + 5 ml of equal 

volume of fehling solution A and B 

and boiled for 5 minutes 

Aqueous extract + dil HCl (heat) 

+NaOH + fehling A and B solution 

A cinary coloured solution 

A (++), B (+), C (+) 

A red precipitate A (++), B (+), C (+) 

Red precipitate A (+++), B (+++), C (+) 

Red precipitate A (+++), B (+++), C (+) 

Chloroformsolution of the plant + 

acetic anhydride + few drop of conc. A colour change from violet to blue A (+++), B (+++), C (+) 

H 2 SO 4 

Aqueous extract + 2 ml CHCl 3 + 

conc. H 2 SO 4 to form a lower layer 

A reddish brown colour at interphase 

A (+++), B (+++), C (+) 

Extract + pyridine + Sodium nitroprusside 

+ 20% NaOH Red colour fades to brownish yellow A (+++), B (+++), C (+) 

Extract 2 ml 3.5% FeCl 3 + glacial 

acetic acid + 2 ml conc. H 2 SO 4 

Reddish brown ring at interphase A (+++), B (+++), C (+) 

Frothing test Extract + distilled water was shaken Frothing persist on warning 

Tanins 

A (+), B (+), C (+) 

General test Ethanol extract H 2 SO 4 + 5% HCl A green solution A (+), B (+), C (+) 

Phlonatanins test Ethanol extract + 5 ml of 1% HCl Red precipitate A (+), B (+), C (+) 

Anthracene derivatives 

Alkaloids 

Ethanol extract + 5% HCl, filter, 

filtrate + CHCl 3 then 10% NH 3 

The aqueous was colourless A (+), B (+), C (+) 

General test Wagner reagent Yellow colour instead of brown ppt A (–), B (–), C (–) 

Morphine alkaloid 

Indole alkaloid 

Dragendoff’s reagent Red colour instead of red ppt A (–), B (–), C (–) 

Mayer’s reagent. Brown colour instead of buff ppt A (–), B (–), C (–) 

Radulescu test No colour change A (–), B (–), C (–) 

Extract + conc.H 2 SO 4 + potassium 

dichromate 

No colour change A (–), B (–), C (–) 

Quinoline alkaloid Thalleiqiune test No colour change A (–), B (–), C (–) 

Tropane alkaloid Vitali – Morin test No colour change A (–), B (–), C (–) 

Faintly: (+); Moderately: (++); Highly: (+++); Absent: (–). 

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Table 2. Evaluation of results 

Percentage (%) 

Total ash value content 9.5 

Acid insoluble ash value 6.8 

Water soluble ash value 6.4 

Alcohol soluble extractive value 2.4 

Water extractive value 4.9 

Table 4. Colour test of fraction “A 1 ” 

Reagent Colour produced Inference 

(1) Mg-HCl Orange ++ 

(2) Alc.Ferric Chloride Dark green + + + 

(3) Zn-HCl Red + + 

Table 3. Colour test of fraction “A” 

Reagent Colour produced Inference 

(1) Mg- HCl Orange ++ 

(2) Alc.Ferric Chloride Dark green + + + 

(3) Zn-HCl Red + + 

Figure 1. TLC chromatogram of fraction “A” in BPF, TPA and B: 

EA: A, respectively. 

tic sugar. The sample gave a spot with the same R f value The result of various solvent system used in developing 

solvent for the TLC on fraction “A” was summarized 

equivalent with that of an authentic (reference) glucose 

sample. 

in Table 6. 

3.7 Paper chromatography of hydrolyzed sugar 

Table 6. Solvent system development 

Technique: descending. 

Toluene : pyridine : acetic acid 10 : 1 : 1 

Paper: whatmann No 1. 

Toluene : ethanol 19 : 1 

Solvent system : n-butanol : acetate acid : water (4 : 1 

Benzene : ethyl acetate : acetic acid 8 : 5 : 2 

: 5) developed for 20 hours. 

Visualization: after drying in the fume cupboard, it was Ethyl acetate : formic acid : water 10 : 2 : 3 

sprayed with aniline-H-phthalate and heat at 110 ºC for 5 Chloroform : ethanol : water 1 : 2 : 1 

minutes (Figure 2 and Table 5). 

Ethyl acetate : methanol : water 10 : 14 : 1 

Methanol : acetic acid 9 : 1 

3.8 Paper chromatograpm of aglycone before hydrolysis 

Technique: descending. 

Paper: what mann No 1. 

Solvent system: n-butanol : acetate acid : water (4 : 1 : 

5) developed for 20 hours. 

Figure 2. Paper chromatogram of fraction “A” after hydrolysis. Visualization: after drying in the fume cupboard, it was 

sprayed with ferric chloride and heat at 110 ºC for 5 minutes 

(Figure 3 and Table 7). 

Table 5. R f value of spot in paper chromatogram 

of fraction “A” 

1 Hydrolyzed Portion B 

2 Arabinose 

3 Xylose 

4 Glucose 

5 Galactose 

6 Rhamnose 

Figure 3. Paper chromatogram of aglycone before hydrolysis. 

7 Fructose 

8 Free sugar A 1 

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Table 7. Solvent system for paper chromatogram of aglycone 

before hydrolysis of fraction “A” 

n-butanol : acetic acid : water 4 : 1 : 5 

Benzene : pyridine: n-butanol : water 1 : 3 : 5 : 3 

Benzene : ethyl acetate : acetic acid 8 : 5 : 2 

4 Discussion 

The plants ethanol extract was extracted with various 

organic solvents to afforded a dark brown mass 30.2 g 

The mass gave dark green colour with alcoholic ferric 

chloride, orange colour with Mg-HCl and red colour with 

Zn-HCl. This clearly indicates that this fraction contains 

flavone nucleus. The extract was treated with hot water 

and filtered. The hot water insoluble portion afforded a 

brown residue, which gave various colour tests for flavonoid 

and was marked “A” (Table 3). The hot water 

soluble portion was extracted with ethyl acetate to afford 

a dark brown mass which gave various colour test for flavonoid 

and was marked “A 1 ” (Table 4). 

Acid hydrolysis of fraction “A” yielded an aglycone. 

The fraction was co-chromatographed on Whatmann 

paper No.1, with two solvent systems, against authentic 

samples of aglycone (Table 7) and where identified as 

quercetin. 

The aqueous portion after neutralization gave a positive 

test for reducing sugars. This fraction was co-chromatographed 

on Whatmann paper No.1 with reference sugar 

sample in different solvent system for 18 hours (Figure 

2). The sugar portion gave the same R f value Table 5 with 

glucose in two different solvent system (Table 7). 

The quantitative evaluation of the dry powdered leave 

(Table 2) gave a total ash value 9.5%, acid insoluble ash 

value 6.8%, water soluble ash value 6.4%, alcohol soluble 

extractive value 2.4%, and water extractive value 4.9%. 

5 Conclusion 

It has been concluded that the leaf of Lophira lanceolata 

(Ochanaceae) showed the presence of flavonoids, 

anthraquinones, carbohydrate, glycoside, phenols, saponin 

steroid, tannin, and a sugars as glucose. 

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Phytochemical screening of the leaves of Lophira lanceolata ...

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