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Highlights in BioScience             

ISSN: 2682-4043 

DOI:10.36462/H.BioSci.20216                                                                                                          
 

 

Highlights in BioScience                                                                                                                                                                       August 2020| Volume 3 

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Research Article 

 

 

Open Access 

 
 

 

 

 

 

1Department of Biological Sciences, Wesley 

University Ondo, Nigeria 

2Department_of_Biochemistry,_Federal 

University of Technology Akure, Nigeria 

 

 

Contacts of Authors  

 

* To whom correspondence should be 

addressed: Oluwasegun Victor 

Omotoyinbo 

 

Citation: Omotoyinbo OV, Awojulu EO and 

Sanni DM (2020). Phytochemical screening, 

antioxidant and tyrosinase inhibitory studies of 

methanol leaf extracts of two tomato varieties. 

Highlights in BioScience Volume 3. Article ID 

20216. dio:10.36462/H.BioSci.20216 

 

Received:  June 8, 2020 

 

Accepted:  July 18, 2020 

 

Published: August 18, 2020 

 
 

Copyright: © 2020 Omotoyinbo et al. This is 

an open access article distributed under the 

terms of the Creative Commons Attribution 

License, which permits unrestricted use, 

distribution, and reproduction in any medium, 

provided the original author and source are 

credited. 

 

 

Data Availability Statement: All relevant data 

are within the paper and supplementary 

materials 

 

 

Funding: The authors have no support or 

funding to report. 

 

 

Competing interests: The authors declare that 

they have no competing interests. 

Phytochemical screening, antioxidant and tyrosinase 

inhibitory studies of methanol leaf extracts of two tomato 

varieties 

 
Oluwasegun Victor Omotoyinbo

1
, Emmanuel Olumide Awojulu

2
 and David 

Morakinyo Sanni
2
*  

 

Abstract 

       This study evaluated the phytochemical compositions, antioxidant 

properties, chlorophyll content and anti-tyrosinase activity of methanol 

leaf extracts of two tomato varieties, Lycopersicon esculentum (var. Eva 

F1) and Lycopersicon esculentum Mill (var. Hausa). The dried pulverized 

of the plant’s leaves were extracted by decoction and mild agitation. 

Phytochemicals such as flavonoids, tannin, glycoside, saponin, terpenoid 

and anthraquinone were present in the extracts of both varieties 

examined, while alkaloid and phlobatannin were confirmed absent in the 

extracts. The presence of steroid was observed in var. Eva F1 but absent 

in var. Hausa. Total phenolic content (TPC) and total flavonoid content 

(TFC) of var. Eva F1 were 505.9 ± 2.61 mg GAE/ge, and 35.5 ± 1.64 mg 

RE/ge, while var. Hausa recorded a TPC and TFC value of 344.3 ± 2.01 

and 7.8 ± 0.15 mg RE/ge respectively. The chlorophyll content of the 

extracts were 6.6 ± 0.02 mg/ge (chlorophyll a), 5.7 ± 0.05 mg/ge 

(chlorophyll b) and 12.6 ± 0.14 mg/ge (total chlorophyll content) for Eva 

F1 variety, while the chlorophyll contents for var. Hausa were 7.6 ± 0.32 

mg.ge (chlorophyll a), 5.6 ± 0.06 mg/ge (chlorophyll b) and 13.7 ± 0.14 

mg/ge. Eva F1 and Hausa showed percentage inhibition of 76.3 % and 

61.2 % at 400 μg/mL. The IC50 value of var. Eva F1 and var. Hausa were 

110 μg/mL and 160 μg/mL. The inhibition constant (KI) of var. Eva F1 

and var. Hausa, were 0.006 and 0.016 μg/mL, respectively, and both 

extracts showed partial competitive inhibition. Hence, this confirms the 

phytoprotective and tyrosinase inhibitory properties of tomato plant 

leaves.  

 

Keywords: Tyrosinase, Inhibition, kinetics, phytochemicals, antioxidants, 

Lycopersicon esculentum. 

 

Introduction  

Tyrosinase (EC 1.14.18.1) is a metalloenzyme that catalyzes the rate-

limiting reactions that are important to melanogenesis. It is classified as an 

oxidase and involves two distinct reactions, the hydroxylation of a monophenol, 

and conversion of an o-diphenol to the corresponding o-quinone. The oxidative 

polymerization of dopaquinone derivatives gives rise to melanin [1]. Melanin is a 

biopolymer synthesized by melanocyte, within specialized organelles called 

melanosomes. It is important to prevent UV-induced skin damage by absorbing 

UV sunlight and removing reactive oxygen species [2]. Although tyrosinase is a 

relatively important enzyme, abnormal biosynthesis or distribution of melanin 

may result in several dermatological disorders, such as age-spots, lentigines, 

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Omotoyinbo et al., 2020                                                          Phytochemical, antioxidant and antityrosinase studies of MeOH leaf extracts of tomato varieties 

 

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melasma, and inflammatory hyperpigmentation. Due to the 

rate-limiting step of tyrosinase in the melanin biosynthesis 

pathway, inhibition of this enzyme have become 

increasingly important and productive in cosmetics and 

pharmaceutical industries, where it is used as potent skin-

lightening agents for treating skin pigmentation disorders 

[3,4], and most of the inhibitors characterized from 

literature are either synthetic compounds or bioactives’ 

obtained from medicinal plants such as polyphenols, 

flavonoids, aldehydes and their derivatives [5]. 

Lycopersicon esculentum (tomato plant) comes from 

the Solanaceae family and is a good source of phenolic 

compounds, pigments, antioxidants, and other nutrients 

when consumed [6]. The extract of tomato leaves has been 

reported to exhibits antimicrobial and antioxidant properties 

at a ratio higher than in other parts of the plant [7]. Tomato 

leaf extract has also been used in much traditional 

preparation of skin-toning mixtures, while some evidence 

indicates that it may be a valuable bioactive source, and 

would seem to be applicable in both medical fields and food 

industry [7,8].  

In this study, the methanol leaf extracts from two 

different cultivars of tomato (Lycopersicon esculentum (var. 

Eva F1) and Lycopersicon esculentum Mill. (var. Hausa) 

were screened for the presence of Phytochemicals and some 

antioxidants, while the antityrosinase potential of the 

extracts was evaluated on mushroom tyrosinase. 

Materials and Methods 

Reagents: Mushroom tyrosinase (1.14.18.1), kojic acid, 

sodium carbonate (Na2CO3), aluminium trichloride (AlCl3), 

sodium nitrite (NaNO2), were obtained from Merck 

(Sternheim USA). All other chemicals and reagents used 

were of analytical grade. 

Plant material: Leaves of Lycopersicon esculentum (var. 

Eva F1) (Figure 1a, b) and Lycopersicon esculentum Mill. 

(var. Hausa)  (Figure 1c) were collected at the Federal 

University of Technology, Akure greenhouse farm and 

Muyiwa Oni Avenue, Akure, Ondo state, respectively. 

Identification and authentication of samples were carried 

out at the department of Crop, Soil and Pest Management, 

Federal University of Technology, Akure. 

Extraction: The leaves were subjected to drying under a 

shade at room temperature for 21 days. The dried materials 

were then pulverized using Kanchan International Blender 

(China). 90 g of each powdered material was then extracted 

by decoction at 50 
o
C for a period of 5 hours using 

methanol as an extraction solvent. Afterwards, the samples 

were filtered using a muslin cloth, and subsequently with 

Whatman filter paper. The residue obtained after filtration 

was extracted to exhaustion by methanol (absolute). The 

extraction was repeated three times and filtrates obtained in 

each stage were combined and concentrated using a rotary 

evaporator extractor. Finally, the extracts were air-dried at 

ambient temperature.   

 
Figure 1: Photograph of L. esculentum Mill. (var. Hausa) 

(a) Leaves (b) Fruits and; (c) Photograph of L. esculentum 

(var. Eva F1) leaves and fruits. 

 

Phytochemical screening: Screening of the phytochemical 

constituents in the leaf extract was carried out to identify 

the constituents using standard phytochemical methods 

[9,10]. 

Test for saponin: This test was carried out by dissolving 10 

mg of extract in water inside a test-tube and the mixture was 

agitated. Frothing, which persisted on warming was taken as 

preliminary evidence for the presence of saponin in the leaf 

extract. 

Test for tannin: The extract (10 mg) was dissolved in 5 ml 

of distilled water and then filtered. Thereafter, 1 ml of ferric 

chloride was added to the filtrate. A blue-black precipitate 

indicated the presence of tannin. 

Test of phlobatannin: The solution of the extract in 

methanol (0.1 mg/ml) was boiled with 1% of hydrochloric 

acid. Deposition of red precipitate indicated the presence of 

Phlobatanin in the extract. 

Test for anthraquinone: About 5 ml of 0.1 mg/ml aqueous 

solution of the extract was dissolved in 10 ml of benzene 

solution, the mixture was filtered and 10% ammonia 

solution was added to the filtrate and then shaken. The 

formation of an amber coloured solution in the ammonia 

lower phase confirmed the presence of anthraquinone in the 

extract. 

Test for glycoside: The extracts (10 mg) were dissolved in 

2 ml of pyridine with 5 drops of 2% sodium nitroprusside 

and 20% sodium hydroxide added. A yellow colouration at 

the interface of the mixture confirmed the presence of 

glycoside. 



 

 

Omotoyinbo et al., 2020                                                          Phytochemical, antioxidant and antityrosinase studies of MeOH leaf extracts of tomato varieties 

 

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Test for steroids: Exactly 20 ml of ethanoic anhydride 

were added to 0.5 g of the extract and then filtered before 

adding 2 ml of concentrated H2SO4 to the filtrate. The 

presence of steroids was confirmed with a colour change of 

the solution from violet to blue or green. 

Test for terpenoid: Exactly 5 ml of 0.1 mg/ml methanol 

solution of the extracts was mixed with 2 ml chloroform; 

afterwards another 3 ml of concentrated H2SO4 was 

carefully added to the solution. The formation of a russet 

colour at the interface of the mixture confirmed the 

presence of terpenoids. 

Test for alkaloid: Exactly 5ml of 1 % aqueous HCl was 

added to 0.5 g of the extract and placed in a steam water 

bath while stirring for 2 minutes. The solution was then 

filtered and 1 ml of the filtrate was treated with a few drops 

of dragendorf reagent, blue-black turbidity was taken as 

preliminary evidence for the presence of alkaloid. 

 

Antioxidant properties 

Total phenolic content: This was determined according to 

the method of Harborne and Williams [11], with slight 

modification. Exactly 0.1 ml of 0.1 mg/ml of the extracts 

was oxidized with 0.5 ml of 10 % folin-ciocalteu’s phenol 

reagent (v/v) and neutralized by 2.5 ml of 7.5% sodium 

carbonate. The reaction mixture for each cultivar extract 

was incubated for 1 hour at room temperature and the 

absorbance was measured at 700 nm using the same mixture 

without the sample as blank. The total phenolic content was 

expressed as mg Gallic Acid Equivalent of extract (mg 

GAE/g of extract). 

Total flavonoid content: This was determined according to 

the method of Bao et al. [12] with a slight modification. In 

a test tube, 0.3 ml of 5 % NaNO2 was added to 0.1 ml of 0.1 

mg/ml extract and the mixture was then incubated at room 

temperature for 6 minutes. After incubation, 0.6 ml of AlCl3 

(10%) was added and the mixture was further incubated for 

5 minutes, then 2 ml of NaOH (1 M) was finally added to 

the reaction mixture and the absorbance was read at 510 nm 

using the same mixture without the extract as blank. The 

result was expressed as mg Rutin equivalent/g of extract 

(mg RE/ge). 

Chlorophyll content: The chlorophyll content of the 

extracts was determined according to the method of 

Lichtenthaler and Babani [13]. Exactly 0.2 g sample was 

extracted exhaustively with 80% acetone until the green 

pigments disappeared. The final mixture was then filtered, 

and the supernatant combined. The absorbance of the 

supernatant was measured at 663, 652, 645 and 470 nm 

using a UV-VIS spectrophotometer. The chlorophyll 

contents were expressed as mg/g of extract (ge) according 

to the following equations: 

Chlorophyll a = (12.7 × A663 – 2.7 × A645). 

Chlorophyll b = (22.9 × A645 – 2.7 × A663). 

Total chlorophyll = (27.8 × A652).      

Assay of tyrosinase inhibitory activity: Tyrosinase inh-

ibitory activity was measured according to the procedure 

described by Liu et al. [14]. Exactly 50 U/ml of tyrosinase 

was prepared in 50mM sodium phosphate buffer (pH 6.8), 

0.35 ml of the leaf extract of various concentrations (3.1, 

6.2, 12.5, 25, 50, 100, 200 and 400 μg/ml) was added to 

0.15ml of tyrosinase respectively, and the mixture was 

incubated for 10 minutes at room temperature. Then, 0.55 

ml of 12 Mm L-DOPA was added to the existing mixture 

and further incubated at room temperature for 20 minutes. 

The reaction mixture without the leaves extract served as 

control and the change in absorbance was measured at 492 

nm against the blank. Kojic acid was used as a standard and 

all tests were performed in triplicate, and the percentage 

inhibition of tyrosinase activity was calculated using the 

formula: 

Tyrosinase inhibition = 
Control

SampleControl

Abs

AbsAbs 
× 100 

Results 

The result of phytochemical screening of the methanol 

leaf extracts of Lycopersicon esculentum (var. Eva F1) and 

Lycopersicon esculentum Mill (var. Hausa) (Table 1) 

shows that the methanol leaf extracts contain saponin, 

flavonoids, anthraquinone, tannin, steroid, glycoside, and 

terpenoids, while alkaloid and phlobatanin were confirmed 

absent, however steroid content was observed in var. Eva 

F1 but absent in var. Hausa. 

 

Table 1: Phytochemical constituents of methanol extract of 

var. Eva F1 and var. Hausa. 

Tomato variety 
Phytochemical constituents 

Var. Eva F1 Var. Hausa 

Flavonoid + + 

Tannin + + 

Glycoside + - 

Alkaloid - - 

Steroid + - 

Saponin + + 

Phlobatanin - - 

Terpenoid + + 

Anthraquinone + + 

*Note: the symbol – indicates the absence of the 

phytochemical under investigation (hence negative), while 

+ indicates the presence of the phytochemical under 

investigation (hence positive).  

Var. Eva F1 means Eva F1 variety, while Var. Hausa means 

Hausa variety. 
 

Total phenolics and flavonoids content of the 

methanol extract of the respective cultivars (Table 2) 



 

 

Omotoyinbo et al., 2020                                                          Phytochemical, antioxidant and antityrosinase studies of MeOH leaf extracts of tomato varieties 

 

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reveals that the total phenolic content of the extracts was 

expressed as gallic acid equivalent (GAE) were 505.96 ± 

2.57 and 344.26 ± 2.02 mg GAE/ge, while the total 

flavonoid content expressed as rutin equivalent (RE) was 

35.55 ± 1.66 and 7.78 ± 0.15 for Eva F1 variety and Hausa 

variety, respectively. 

 

Table 2: Total phenol and total flavonoids content of 

extract of tomato leaf; var. Eva F1 and var. Hausa. 

Tomato variety TPC (mg GAE/ge) TFC (mg RE/ge) 

Var. Eva F1 505.96 ± 2.57 35.55 ± 1.66 

Var. Hausa 344.26 ± 2.02 7.78 ± 0.15  

Values are expressed as mean ± standard deviation (n=3). 

Note: GAE- Gallic acid equivalent; RE- Rutin equivalent. 
 

The two tomato varieties varied in the chlorophyll 

contents (Table 3), such that for var. Eva F1 had values of 

6.63 ± 0.02, 5.76 ± 0.05 and 12.57 ± 0.14 mg/ge for 

chlorophyll a, chlorophyll b and total chlorophyll content, 

respectively, var. Hausa had 7.63 ± 0.32, 5.63 ± 0.06 and 

13.67 ± 0.14 mg/ge. The analysis revealed that all samples 

had a non-zero standard deviation from the mean values. 

 

Table 3: Chlorophyll content of extract of tomato leaf; var. 

Eva F1 and var. Hausa. 

Tomato 

variety 

Chlorophy

ll a (mg/ge) 

Chlorophy 

ll b (mg/ge) 

Total chlorophy 

ll (mg/ge) 

Var. Eva F1 6.63 ± 0.02 5.76 ± 0.05 12.57 ± 0.10  

Var. Hausa 7.63 ± 0.32 5.63 ± 0.06 13.67 ± 0.14 

Values are expressed as mean ± standard deviation (n=3). 

Note: mg/ge refers to mg/g of extract. 

 

Inhibitory concentration at 50% (IC50) values of the 

extracts compared with kojic acid (the standard inhibitor) is 

represented in (Table 4).  

 

Table 4: Michaelis-Menten parameter (Ki), and IC50 values 

of inhibitors. 

Tomato variety 
IC50 

(µg/ml) 

KI Value  

(µg/ml)  
Type of inhibition 

L. esculentum 

(var. Eva F1) 
65 0.006 Partial competitive 

L. Esculentum 

(var. Hausa) 
198 0.016 Partial competitive 

IC50 value of kojic acid (positive control) is 25 µg/ml. 

The extracts had an inhibitory effect on tyrosinase in a 

dose-dependent manner (Figure 2 and 3). The percentage 

inhibition of tyrosinase at the maximum test concentration 

(400 μg/ml) for extracts were 76.30% and 61.24% for Eva 

F1 and Hausa varieties, respectively while kojic acid had a 

higher inhibitory power of 80.57%. Further kinetic studies 

revealed (Figure 4 and 5) the inhibition type of both 

extracts as competitive inhibition and the inhibition 

constant (KI) for Eva F1 and Hausa varieties were 0.006 

and 0.016 μg/mL, respectively.  

 
Figure 2: The percentage inhibition curve of L. esculentum 

(var. Eva F1) and kojic acid. 

 

 
Figure 3: The percentage inhibition curve of L. esculentum 

Mill. (var. Hausa) and kojic acid. 

 

Discussion 
In this study, the phytochemical analysis of the 

methanol leaf extracts of var. Eva F1 and var. Hausa 

revealed a single variation in plant secondary metabolites 

examined. It was observed that while the analysis of both 

varieties of methanol leaves extract confirmed the presence 

of flavonoids, tannin, glycoside, saponin, terpenoid and 

anthraquinone, and the absence of alkaloid and phlobatanin, 

var. Eva F1 contains steroid while var. Hausa extract did 

not contain the steroids. Manosroi et al. [15] reported that 

phytochemicals found in plant extract depend on the nature 

of the plant, the solvent system, temperature and time used 

in the extraction process. The variation in steroid 

composition could be a result of the mode of cultivation of 

individual plants, however, the result obtained showed 

greater similarity in the phytochemical constituent of the 

extracts of both varieties, and could be an indication of 

some sort of genetic resemblance.  



 

 

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Figure 4: Kinetic properties of L. esculentum (var. Eva F1) (A) Lineweaver-Burk plot for the inhibition of var. Eva F1, (B) 

Re-plot of the slope of the Lineweaver-burk plot versus inhibitor concentration, (C) 1/ΔSlope versus 1/[I]. 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5: Kinetic properties of L. esculentum Mill (var. Hausa) (A) Lineweaver-Burk plot for the inhibition of var. Hausa (B) 

Replot of the slope of  the Lineweaver-burk plot versus inhibitor concentration (C) 1/ΔSlope versus 1/[I]. 

 

A 

B 

C 

A B 

C 



 

 

Omotoyinbo et al., 2020                                                          Phytochemical, antioxidant and antityrosinase studies of MeOH leaf extracts of tomato varieties 

 

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The observed high TPC and TFC indicate that var. 

Eva F1 has the best antioxidant properties compared to var. 

Hausa. Silva-Beltran et al. [7] reported that high phenolic 

content in tomato plants could be as a result of their varied 

phenolic content and its derivatives, which are essential for 

the plant’s growth and reproduction. Phenolic content 

present in tomato plants majorly consist of hydrox-

ycinnamic acid and flavonoids and are located mainly in 

their leaves. The variation observed in TPC and TFC in the 

two tomato varieties has been affirmed by Silva-Beltran et 

al. [7], who reported that the concentration of flavonoids 

varied among the plant’s cultivars. 

Results obtained in this study showed that var. Hausa 

had the highest content of chlorophyll a, while var. Eva F1 

had the highest content of chlorophyll b. However, var. 

Hausa had the highest total chlorophyll content (TCC). The 

content of chlorophylls observed in this study was found to 

be higher than those observed by Silva-Beltran et al. [7] for 

Pitenza and Floradade variety of tomato plants. Lumpkin 

[16] also reported that the chlorophyll content of tomato 

plants is strongly influenced by the incidence of light and its 

concentration increases with exposure to light. Tomato 

plants synthesize metabolites and pigments such as 

chlorophyll and carotenoids, that beneficially contributes to 

consumers nutrition and health [7], while the work of Choi 

et al [8] shows that these Phytochemicals help prevent 

photooxidation are strongly influenced by the maturity of 

the plant. In the same light, chlorophyll shows potent 

antioxidant activity, and it has been suggested that 

chlorophyll reduces free radicals, acting as a hydrogen ion 

donor to break the chain reaction resulting to cellular 

oxidation [17]. The methanol leaf extracts of var. Eva F1 

and Hausa inhibited mushroom tyrosinase in a dose-

dependent manner. Eva F1 variety revealed higher 

tyrosinase inhibitory activity of 76.3% at the highest 

concentration considered (400µg/mL) compared with 

61.2% in Hausa variety. Zaveri and Patel [18] reported a 

percentage inhibition of 58% in tomato fruit (at the highest 

inhibitor concentration considered), which is lower than the 

value obtained in the present study.  This is quite 

reasonable, as secondary metabolites are primarily, 

concentrated in leaf section of plants to serve a defensive 

role. The IC50 values obtained for both extracts in this study 

were higher compared to those obtained by Zaveri and Patel 

[18] for curry tree and tomato fruit, which could be as a 

result of the difference in the enzyme units utilized. 

However, kojic acid (standard inhibitor) recorded an IC50 of 

25 μg/mL. This result points out Eva F1 variety as the most 

potent tyrosinase inhibitor among the two cultivars were 

evaluated since low IC50 values imply that a smaller amount 

of the extract is needed to reduce the activity of mushroom 

tyrosinase by half. Kinetic data analysis using Lineweaver-

Burk plot, which is also called the double reciprocal plot 

reveals that the inhibitory mechanism of both methanol leaf 

extracts on mushroom tyrosinase was competitive, viz 

partial competitive inhibition. This correlates with Yang 

and Quyang [19] conclusion for Olea leaf extract analysed 

by Lineweaver-Burk plot. Furthermore, L. esculentum var. 

Eva F1 had the lowest inhibition constant (KI) value of 

0.006 μg/mL while var. Hausa had KI value of 0.016 

μg/mL. This implies that methanol leaf extract of Eva F1 

variety has a higher affinity for the active site of mushroom 

tyrosinase than that of Hausa variety. Also, the KI value 

obtained in this study is considerably lower than that 

obtained by Yang and Quyang [19] for Olea europa leaf 

extract, which had a KI value of 0.226 mg/mL. 

Conclusion 

Although the two varieties of tomato leaf methanol 

extract showed the presence of most phytochemical 

evaluated, both varieties tests were negative for alkaloid 

and phlobatannin content, while var. Hausa also lacked any 

steroid content. The highest TPC, and TFC were observed 

for var. Eva F1, while var. Hausa had the highest total 

chlorophyll content. Eva F1 variety had the best tyrosinase 

inhibitory activity at the peak concentration considered. 

This study therefore further establishes the skin whitening 

and antioxidant properties of tomato leaves as traditionally 

used in some cultures. 

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