South African Journal of Botany 109 (2017) 9–15

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Anti-tyrosinase, total phenolic content and antioxidant activity of

selected Sudanese medicinal plants
A.M. Muddathir a,⁎, K. Yamauchi a, I. Batubara b, E.A.M. Mohieldin c, T. Mitsunaga a
a
United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
b
Department of Chemistry, Faculty of Mathematics and Natural Sciences, and Biopharmaca Research Center, Bogor Agricultural University, Bogor, Indonesia
c
Faculty of pharmacy, University of Science and Technology, P.O. Box 447, Omdurman, Sudan

a r t i c l e i n f o a b s t r a c t

Article history: The flora of Sudan is relatively rich in medicinal plants and represents an important component of traditional
Received 22 May 2016 medicine. Fifty methanolic extracts of selected Sudanese medicinal plants were evaluated for their in vitro
Received in revised form 17 September 2016 tyrosinase inhibitory effect, antioxidant activity and total phenolic content (TPC). The standard method of
Accepted 13 December 2016
antioxidant evaluation, ferric reducing ability of plasma (FRAP), was employed to determine the antioxidant
Available online xxxx
activity while the enzyme based tyrosinase inhibition was used for the anti-tyrosinase activity. Acacia nilotica
Edited by AR Ndhlala (pods, bark) and Acacia seyal var. seyal (wood) demonstrated comparable anti-tyrosinase inhibitory activity
using L-tyrosine as substrate (08.61, 10.47 and 10.77 μg/ml respectively) to Kojic acid (10.02 μg/ml) which
Keywords: was used as a positive control. A. nilotica (bark) and Acacia seyal var. fistula (bark) exhibited good tyrosinase
Sudanese medicinal plants inhibitory activity using L-DOPA as substrate (IC50: 31.93, 36.32 μg/ml) compared to positive control
Phenolic content (IC50: 37.63 μg/ml). The results revealed significant differences in TPC between plants extracts. The highest
FRAP level of phenolic content was found in Terminalia brownii (bark; 46.02 μg GAE/mg) while the lowest was in
Tyrosinase inhibition Ziziphus spina–christi (fruits; 09.63 μg GAE/mg). The study indicated significant differences in total antioxidant
Acacia nilotica
capacity between the extracts. Terminalia laxiflora (wood), A. nilotica (pods, bark), T. brownii (bark), A. seyal
var. seyal (bark), Khaya senegalensis (bark), T. brownii (wood) Combretum hartmannianum (bark), Polygonum
glabrum (leaves), Z. Spina-christi (bark) and Guiera senegalensis (leaves) extracts displayed the high antioxidant
equivalent concentration (EC) values. A. nilotica (pods, bark) expressed promising activity that warrant further
research since it has high tyrosinase inhibitory activity, antioxidant activity and could be a good source of pheno-
lic compounds. To the best of our knowledge, this is the first data presenting comprehensive data on anti-
tyrosinase, TPC, antioxidant activity of the Sudanese medicinal plants.
© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction nevus, ephelis, post inflammatory state and melanoma of pregnancy

Tyrosinase (EC 1.14.18.1; PPO) is known to be a key enzyme in mel-
anin biosynthesis and is widely distributed in plants and mammalian
cells. Tyrosinase enzyme catalyzes two different reactions: the hy-
droxylation of monophenols to o-diphenols (monophenolase activity)
and the oxidation of o-diphenols to o-quinones (diphenolase activity).
o-Quinone is transformed into melanin in a series of non-enzymatic
reaction (Sánchez-Ferrer et al., 1995). Melanogenesis is a physiological
process resulting in the synthesis of melanin pigments, which plays a
crucial protective role against skin photocarcinogenesis. In humans
and other mammals, the biosynthesis of melanin takes place in a lineage
of cells known as melanocytes, which contain the enzyme tyrosinase
(Robb, 1984). Melanin synthesis inhibitors are topically used for
treating such localized hyperpigmentation in humans as lentigo,
⁎ Corresponding author at: Department of Horticulture, Faculty of Agriculture,
University of Khartoum, Khartoum North, 11334 Shambat, Sudan.
E-mail address: a_muddathir@yahoo.com (A.M. Muddathir).
(Tomita et al., 1990). Melanin formation is considered to be deleterious
to the color quality of plant-derived food. Prevention of this browning
reaction has always been a challenge to food scientists (McEvily et al.,
1992). Tyrosinase is also one of the most important key enzymes in
the insect molting process therefore it could be used as alternative in-
secticide (Miyazawa and Tamura, 2007). Therefore the inhibitors of
this enzyme may lead to novel skin whitening agents, anti-browning
substances or compounds for insect control. Recently applications of
tyrosinase-inhibiting agents are increasingly used in cosmetic products
for maintaining skin whiteness (Kadekaro et al., 2003). Plants and their
extracts are inexpensive and rich resources of active compounds that
can be utilized to inhibit tyrosinase activity as well as melanin produc-
tion (Momtaz et al., 2008).
The idea behind using antioxidants for skin-lightening activities lies
in the hypothesis that the oxidative effect of UV-irradiation contributes
to activation of melanogenesis. UV irradiation can produce reactive
oxygen species (ROS) in the skin that may induce melanogenesis by
activating tyrosinase as the enzyme prefers superoxide anion radical

http://dx.doi.org/10.1016/j.sajb.2016.12.013
0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
10 A.M. Muddathir et al. / South African Journal of Botany 109 (2017) 9–15
(Wood and Schallreuter, 1991). Additionally these ROS enhance the
damage of DNA and may induce the proliferation of melanocytes
(Yasui and Sakurai, 2003). Therefore ROS scavenger such as antioxi-
dants may reduce the hyperpigmentation (Ma et al., 2001). Total phe-
nolic and flavonoid contents were significantly correlated with free
radical-scavenging and tyrosinase-inhibiting activities. Thus, the strong
free radical-scavenging and tyrosinase-inhibiting properties increased
proportionally with the level of antioxidants in Sorghum distillery resi-
due extracts (Wang et al., 2011).
Medicinal plants represent an important component of traditional
medicine in the world including in Sudan. The flora of Sudan is relatively
rich in medicinal plants corresponding to a wide range of ecological
habitats and vegetation zones of the country (Khalid et al., 1986). Due
to the rich plant diversity existing in Sudan, it is very encouraging to
explore the potential of Sudanese plants for cosmaceutical purposes.
Despite few of these medicinal plants used for skin decoration and soft-
ening the authors decided to investigate the ability of some Sudanese
medicinal plants as skin whitening which it could be useful for
cosmaceutical industry. The ability of different extracts of Sudanese
medicinal plants to act as a skin-lightening agents was tested as
their ability to inhibit tyrosinase, the rate limiting enzyme in melano-
genesis, initially using a cell-free mushroom tyrosinase system, which
has commonly been employed for the testing and screening of poten-
tial skin-lightening agents (Song et al., 2009). In this study, fifty
methanolic extracts of Sudanese medicinal plants were evaluated for
their anti-tyrosinase, total phenolic content and antioxidant properties
in order to find the most potential plant extracts for the skin-lightening
agent.
2. Materials and methods
2.1. Plant materials
The plant materials which have medicinal values were randomly
selected from Khartoum and Gadarif states of Sudan in March 2011.
Identification of the plant materials was done at the University of
Khartoum, Faculty of Agriculture and Faculty of Forest. Authentication
voucher specimens are deposited in the Horticultural Laboratory,
Department of Horticulture, Faculty of Agriculture, University of
Khartoum.
2.2. Chemicals and reagents
Dimethylsulfoxide (DMSO), iron (III) chloride hexahydrate, Folin–
Ciocalteau, L-tyrosine and L-dihydroxyphenylalanine (DOPA) were
purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Mushroom tyrosinase (2870 units/mg), and 2,4,6-Tris (2pyridyl)-s-
triazine (TPTZ) were purchased from Sigma. Quercetin, butylated
hydroxytoluene, ascorbic acid and gallic acid were purchased from
Naka Lai Tesque, Inc. (Kyoto, Japan). Other chemicals were of the highest
grade commercially available.
2.3. Extract preparation
Plant materials were shade dried at room temperature and pow-
dered before extraction; they were each extracted three times with
absolute methanol (ratio of 1 g sample: 10 ml solvent) for 12 h three
times. The extracts were filtered and then the solvent was removed
under vacuum using rotary evaporator. All extracts were stored at 4 °C
prior to analysis.
2.4. Inhibition of tyrosinase activity
The tyrosinase inhibitory activity was performed by the method de-
scribed by Batubara et al. (2010). Briefly, the sample (70 μl) was added
in 96-well plate. Tyrosinase (30 μl, 333 unit/ml in phosphate buffer
50 mM, pH 6.5) and 110 μl of substrates (L-tyrosine 2 mM or L-DOPA
12 mM) were added. After incubation at 37 °C for 30 min, the absor-
bance at 510 nm was determined using a micro plate reader. The
percent inhibition of tyrosinase activity was calculated at the concentra-
tion of 125 and 500 μg/ml. The extracts showed activities up to 50%
inhibition of the enzyme were expressed as (IC50). Kojic acid was used
as a positive control.
2.5. Determination of total phenolic content
The total phenolics assay was performed as described previously by
Ainsworth and Gillespie (2007). Plant extract was dissolved in 50%
methanol and 100 μL was transferred into test tubes, followed by
200 μL 1 N Folin Ciocalteau reagent 10% (v/v). Then they were mixed
with 800 μl of sodium carbonate (700 mM) and maintained at room
temperature for 2 h. Two hundred microlitres of samples from the
assay tube was transfer to 96-well microplate and read the absorbance
at 765 nm using microplate reader. Total phenolic concentrations were
expressed as microgram of gallic acid equivalents (GAE).
2.6. Antioxidant capacity
The total antioxidant potential of extracts was determined by
using a ferric reducing ability of plasma (FRAP) assay described by
Tachakittirungrod et al. (2007). Briefly, the FRAP reagent was freshly
prepared. The extracts were dissolved in ethanol to a concentration of
1 mg/ml. An aliquot of 20 μL test of solution was mixed with 180 μL of
FRAP reagent. The absorption of the reaction mixture was measured at
590 nm by a microplate reader. Ethanolic solutions of known Fe (II) con-
centration, in the range of 50–500 μM (FeSO4), were used as calibration
curve. The reducing power was expressed as equivalent concentration
(EC). This EC was defined as the concentration of antioxidant having
a ferric reducing ability equivalent to that of 1 mM FeSO4. Quercetin,
ascorbic acid and butylated hydroxytoluene (BHT) were used as posi-
tive controls.
2.7. Statistical analysis
The IC50 values of tyrosinase inhibitory, total phenolic content and
antioxidant activities were expressed as the mean (mean ± standard
deviation). The significant differences between extracts and positive
controls were assessed by one-way analysis of variance (ANOVA)
followed by pairwise comparison of the mean with positive control
using Tukey's multiple comparison test. Values were determined to be
significant when p was less than 0.05 (p b 0.05).
3. Results and discussion
Table 1 summarizes the botanical name, family, voucher specimen
and summary of traditional uses of the investigated plant species.
Within these selected Sudanese medicinal plants Lawsonia inermis,
Combretum hartmannianum, Acacia seyal var. fistula, Solanum dubium,
Citrullus coloynthis and Acacia tortilis are used for preventing the dryness
and bacterial infection of the skin in addition to other uses. The 50 plant
extracts used in this research belong to 39 plant species which are dis-
tributed among 27 families from different plant parts.
3.1. Tyrosinase inhibitory activity
L-tyrosine and L-DOPA were used as the substrate to determine the
monophenolase and diphenolase activities of mushroom tyrosinase.
The tyrosinase inhibitory activities of all extracts are presented in
Table 2 as percentage of L-tyrosine and L-DOPA inhibition at the con-
centration of 125 and 500 μg/ml as well as IC50 values. The study re-
vealed that 36 and 24% of extracts presented a good tyrosinase
inhibitory activity which inhibited more than 50% inhibition for both
 A.M. Muddathir et al. / South African Journal of Botany 109 (2017) 9–15 11

Table 1
Botanical name/authority, family, voucher specimen, traditional uses and references of selected Sudanese medicinal plants used in traditional medicine.

Botanical name Family Voucher Traditional uses and references

specimen

Ammi visnaga (L.) Lam. Apiaceae SD-OD-38 It relaxes smooth muscles and lowers the urethral tonicity. A decoction is used to ease the passage of
kidney calculus (Khalid et al., 2012).
Carum carvi L. Apiaceae SD-OD-48 Antispasmodic, carminative, galactagogue and anthelmintic properties (Khalid et al., 2012).
Hyphaene thebaica (L.) Mart. Arecaceae SD-OD-41 Against splenomegaly, alimentary system disorders and bacterial eye infections (El Ghazali et al., 2003).
Aristolochia bracteolata Lam. Aristolochiaceae SD-SH-04 Analgesic, treatment of scorpion bite and malaria. It is also used anti-tumors (El-Tahir et al., 1999).
Solenostemma argel (Delile) Asclepiadaceae SD-SH-23 The leaves are used as an antispasmodic, carminative and as an anti-diabetic (Kamel et al., 2000).
Hayne
Xanthium brasilicum Vell. Asteraceae SD-SH-12 Antiprotozoal (Nour et al., 2009).
Balanites aegyptiaca (L.) Balanitaceae SD-SH-15 Venereal diseases, rheumatism, digestion problems, dysentery and bilharzias (Iwu, 1993; El Ghazali
Delile et al., 1997; Van Wyk et al., 1997).
Kigelia africana Bignoniaceae SD-SH-21 Stomach problem, cough, dysentery and venereal diseases (Neuwinger, 1996; El Ghazali et al., 1997).
(Lam.)Benth.
Adansonia digitata L. Bombacaceae SD-OD-27 Used as a cold beverage and added to yogurt for treatment of diarrhea and amoebic dysentery
(Mangan, 1988).
Lepidium sativum L. Brassicaceae SD-KH-02 Used as anti-asthmatic, anti-scorbutic, aperient, diuretic, galactagogue and stimulant (Adam et al., 2011).
Tamarindus indica L. Caesalpiniaceae SD-SH-18 Fever, malaria and laxative (El Kamali and El-Khalifa, 1999).
Capparis decidus (Forssk.) Capparaceae SD-SH-17 Jaundice, rheumatic arthritis and to treat swells (El Ghazali et al., 1997).
Edgew.
Maerua oblongifolia Capparaceae SD-SH-45 Malaria (El Kamali and El-Khalifa, 1997).
(Frossk.)
A.Rich
Combretum hartmannianum Combretaceae SD-KH-04 Arthritis rheumatism, skin dryness, jaundice and bacterial diseases (El Ghazali et al., 1997;
Schweinf. Al Magboul et al., 1988).
Guiera senegalensis J.F.Gmel Combretaceae SD-OD-40 Anti-pyretic, anti-diabetic , anti-hypertension and febrifuge (El Ghazali et al., 1994, 1997).
Terminalia brownii Fresen. Combretaceae SD-GF-02 Cough bronchitis and anti-rheumatic (El Ghazali et al., 1994, 1997).
Terminalia laxiflora Engl. Combretaceae SD-KH-03 Cough treatments and fumigant (Musa et al., 2011).
Ambrosia maritima L. Composiate SD-SH-03 Urinary tract infections, elimination of kidney stones, anti-diabetic and anti-hypertensive (Mahmoud
et al., 1999).
Citrullus coloynthis (L.) Cucurbitaceae SD-OD-05 Purgative and skin diseases (El Ghazali et al., 1997).
Schrad.
Abrus precatorius L. Leguminosae SD-OD-22 Oral contraceptives, against chronic nephritis and diabetes (Manago and Alumanah, 2005; Garaniya
and Bapodra, 2014).
Acacia nilotica (L.) Delile Leguminosae SD-OD-01 Diarrhea, cold, pharyngitis, used as anti-septic and tooth ache (Eldeen et al., 2010).
Acacia seyal var. seyal Del. Leguminosae SD-GF-05 Arthritis rheumatism and rheumatic fever (El Ghazali et al., 1997).
Acacia seyal var. fistula Leguminosae SD-GF-06 Fumigant for rheumatic pain. It is also used to protect women from fever after childbirth and soften
(Schweinf.) the skin (Khalid et al., 2012).
Acacia tortilis (Forssk.) Leguminosae SD-KH-07 Vermifuge, skin infections, oedema and allergic dermatitis
Hayne (http://www.fao.org/ag/agp/agpc/doc/Gbase/DATA/Pf000139.htm).
Parkinsonia aculeata L. Leguminosae SD-SH-02 Fever, malaria, abortifacient and rheumatism (Orwa et al., 2009).
Lawsonia inermis L. Lythraceae SD-SH-07 Anti-pyretic, for treatment of urinary tract infections, skin diseases and alopecia (El Ghazali, 1986).
Abutilon pannosum Malvaceae SD-SH-43 Hepatoprotective, antiplasmodial and hypoglycemic activities (Cho-kang et al., 2007).
(G.Forst.)
Schltdl.
Grewia tenax (Forssk) Fiori Malvaceae SD-OD-42 Malaria and iron deficiency anemia (Khalid et al., 2012).
Hibiscus sabdariffa L. Malvaceae SD-SH-47 Hypertension, colds, antispasmodic and antimicrobial agent (Khalid et al., 2012).
Khaya senegalensis (Desv.) A. Meliaceae SD-SH-14 Tonic, fever, vermifuge, taenicide, emmenagogue, venereal diseases and antiprotozoal (Bever, 1986;
Juss. Iwu, 1993; Kayser and Abreu, 2001).
Moringa oleifera lam. Moringaceae SD-SH-08 Antimicrobial, febrile, pulmonary diseases (Neuwinger, 2000; Anwar et al., 2007).
Polygonum glabrum Willd. Polygonaceae SD-SH-A-03 Anthelmintic (Muddathir et al., 1987).
Nigella sativa L. Ranunculaceae SD-OD-16 Anti-inflammatory, allergies, eczema and malaria (Ahmed et al., 2010).
Ziziphus spina-christi (L.) Rhamnaceae SD-SH-06 Ulcers, gonorrhea, sore throat, chest pain, dysentery, venereal diseases and wounds (El Ghazali et al.,
Desf. 1997; Dafni et al., 2005).
Haplophyllum tuberculatum Rutaceae SD-KH-32 Used against diarrhea and spasms (Khalid et al., 2012).
(Forssk.) A. Juss.
Salvadora persica L. Salvadoraceae SD-SH-09 Gingivitis, malaria, liver swellings and HIV-1(El Kamali and Khalid, 1996; Hussein et al., 1999;
Neuwinger, 2000).
Solanum dubium Fresen Solanaceae SD-SH-34 Applied to wounds and skin tumors as a dressing (El Kheir and Salih, 1980).
Tamarix nilotica (Ehrenb.) Tamaricaceae SD-OD-10 Febrile, colds and hemorrhoids (El Ghazali et al., 1994; Neuwinger, 2000).
Bunge
Fagonia cretica L. Zygophyllaceae SD-OD-26 Muscular pains, antispasmodic and against heart burn (El Ghazali et al., 1997; El Ghazali, 1986).

substrates (L-tyrosine and L-DOPA, respectively). At the concentration
of 500 μg/ml of extracts with L-tyrosine and L-DOPA substrates; Z. spina-
christi (bark), A. digitata , A. nilotica (pods and bark), K. senegalensis,
G. senegalensis, T. brownii (bark), A. seyal var. seyal (bark), A. seyal var.
fistula (bark) and P. glabrum showed inhibition levels more than 70%. In
addition to that Kojic acid which was used as a positive control showed
an inhibition level of 99.65 and 94.40% to L-tyrosine and L-DOPA,
respectively.
The IC50 values of A. nilotica (pods- bark), A. seyal var. seyal (wood)
and kojic acid (positive control) demonstrated significantly lower IC50
for L-tyrosine substrate inhibition (8.61, 10.47, 10.77 and 10.02 μg/ml
respectively) than other extracts. Moreover A. nilotica (bark), A. seyal
var. fistula (bark) and positive control exhibited the lowest IC50 for
L. DOPA substrate inhibition (31.93, 36.32 and 37.63 μg/ml respectively)
and it's significantly different from other extracts.
Maldini et al. (2011) reported the isolation of compounds from
A. nilotica pods including: galloylated catechin and gallocatechin deriv-
atives along with galloylated glucose derivatives. Also previous studies
of the bark of A. nilotica resulted in the separation of gallic acid, catechin
5-galloylester (Khalid et al., 1989) and galloylated derivatives of
12 A.M. Muddathir et al. / South African Journal of Botany 109 (2017) 9–15

Table 2
Tyrosinase inhibitory activities of selected Sudanese medicinal plants extracts.

Botanical name Part used Tyrosinase inhibition (%) Tyrosinase inhibition

L-Tyrosine L-DOPA L-Tyrosine L-DOPA

125 μg/ml 500 μg/m 125 μg/ml 500 μg/ml IC50 μg/ml⁎ IC50 μg/ml

A. visnaga (L.) Lam. Fruits 18.55 42.10 02.82 28.90 nd nd

C. carvi L. Fruits –⁎⁎ 05.38 – 06.93 nd nd
H. thebaica (L.) Mart. Fruits 19.32 26.54 01.58 08.40 nd nd
A. bracteolata Lam. Aerial parts – 10.91 – 13.76 nd nd
S. argel (Delile) Hayne Leaves – 25.55 – 30.32 nd nd
X. brasilicum Vell. Leaves 08.86 19.30 14.30 27.23 nd nd
B. aegyptiaca (L.) Delile Leaves – 02.73 – 16.02 nd nd
Bark – 06.77 06.10 14.71 nd nd
Fruits 02.54 04.25 – 06.00 nd nd
K. africana (Lam.) Benth. Fruits – 02.60 02.4 13.7 nd nd
A. digitata L. Fruit puple 73.53 97.14 59.20 77.62 51.71 ± 4.27c 91.25 ± 5.97c
L. sativum L. Seeds – 01.88 – – nd nd
T. indica L. Leaves 54.38 72.50 37.65 49.79 78.38 ± 9.69d nd
C. decidus (Forssk.) Edgew. Stems – 10.11 – – nd nd
M. oblongifolia (Frossk.) A.Rich Stems – – 09.4 18.0 nd nd
C. hartmannianum Schweinf. Bark – 11.23 12.46 12.70 nd nd
G. senegalensis J.F.Gmel Wood 27.75 59.03 13.86 43.88 386.89 ± 11.29g nd
Leaves 45.30 95.25 22.86 80.89 198.51 ± 14.18f 240.35 ± 10.17e
T. brownii Fresen. Bark 81.12 95.50 51.70 70.18 119.14 ± 9.39e 120.66 ± 10.22d
Wood 18.63 61.80 08.35 32.35 415.44 ± 21.70h nd
T. laxiflora Engl. Wood 01.84 35.95 – 21.41 nd nd
A. maritima L. Aerial parts 13.90 50.51 – 27.82 485.96 ± 11.96i nd
C. coloynthis (L.) Schrad. Dry fruits – 18.15 – 12.20 nd nd
A. precatorius L. Seeds – 09.60 26.14 69.40 nd 345.71 ± 13.49f
A. nilotica (L.) Delile Pods 96.39 98.30 64.7 81.9 08.61 ± 0.94a 98.3 3± 3.79c
Bark 94.20 94.23 70.9 84.5 10.47 ± 1.42a 31.93 ± 1.60a
A. seyal var. seyal Del. Bark 84.03 95.45 69.77 83.06 44.30 ± 3.97c 94.47 ± 2.39c
Wood 91.80 91.82 38.50 48.91 10.77 ± 0.35a nd
A. seyal var. fistula (Schweinf.) Bark 91.49 94.52 68.74 81.80 22.80 ± 2.70b 36.32 ± 4.12a
Wood – 05.10 – 04.40 nd nd
A. tortilis (Forssk.) Hayne Bark – 08.0 – – nd nd
Wood 65.52 70.91 24.20 33.65 91. 51 ± 6.49d nd
P. aculeata L. Leaves – 15.11 – 12.20 nd nd
L. inermis L. Leaves 02.91 11.20 12.44 12.78 nd nd
A. pannosum (G.Forst.) Schltdl. Leaves 30.41 07.22 10.53 18.72 nd nd
G. tenax (Forssk) Fiori Fruits 01.93 03.00 02.54 02.80 nd nd
H. sabdariffa L. Flowers 18.28 21.21 10.88 12.23 nd nd
K. senegalensis (Desv.) A. Juss. Bark 93.1 97.9 64.2 85.7 20.87 ± 0.85b 71.20 ± 11.20b
M. oleifera lam. Leaves 06.35 08.94 09.90 12.13 nd nd
P. glabrum Willd. Leaves 100 100 84.39 91.75 40.89 ± 2.14c 75.68 ± 0.72b
Nigella sativa L. Seeds – 15.73 – 17.64 nd nd
Z. spina –christi (L.) Desf. Fruits – 09.68 – – nd nd
Bark 72.77 94.75 42.41 77.30 51.57 ± 10.75c 253.62 ± 11.11e
Leaves – 32.73 – 24.43 nd nd
H. tuberculatum (Forssk.) A. Juss. Aerial parts – – 05.50 14.42 nd nd
S. persica L. Leaves 20.00 67.68 11.86 46.57 356.87 ± 9.94g nd
Stems 08.84 41.58 14.01 32.83 nd nd
S. dubium Fresen Fruits – 16.50 – 12.73 nd nd
T. nilotica (Ehrenb.) Bunge Stems 51.22 79.51 30.52 53.00 118.19 ± 8.46e 424.27 ± 29.30g
F.cretica L. Aerial parts 10.30 22.56 – 16.12 nd nd
Kojic acid⁎⁎⁎ 99.50 99.65 83.54 96.40 10.02 ± 1.87a 37.63 ± 2.60a

nd: Not detected.

Means with different letters in the same column were significantly different at the level (p b 0.05); n = 3.
⁎ IC50-Half minimal inhibitory concentration.
⁎⁎ No inhibition was detected.
⁎⁎⁎ Positive control.

catechin 5-O-gallate (Malan, 1991). Salem et al. (2011) demonstrate the
isolation of gallocatechin 5-O-gallate compound from A. nilotica pods
exhibiting in vitro activity and selectivity towards uveal melanoma
cell lines comparable to that of the known compound epigallocatechin
gallate (EGCG) from green tea. EGCG reported as potential whitening
agent by reducing melanin production in mouse melanoma cells (Kim
et al., 2004). Sato and Toriyama (2009) clarified that the catechin
group inhibited melanin synthesis in B16 melanoma cells through
inhibiting melanogenic protein, namely tyrosinase. Also he suggested
the catechin group as a candidate for being anti-melanogenic agent
and that it might be effective in hyperpigmentation disorders.
Therefore the in vitro activity of A. nilotica pods and bark against tyrosi-
nase, it may be due to the catechin derivatives compounds.
3.2. Total phenolic content
Polyphenolic compounds are commonly found in both edible and
inedible plants and they have been reported to have multiple biological
effects (Kähkönen et al., 1999). Lin et al. (2008) mentioned that pheno-
lics with ion reducing ability diminish the possibility of hydroxyl
radical's formation path from superoxide anion radicals and addition-
ally inhibit enzymes due to their abilities to chelate copper at the active
 A.M. Muddathir et al. / South African Journal of Botany 109 (2017) 9–15 13

Table 3 differences in total phenolic content between plants extracts. The

Total phenolic content and antioxidant capacity of selected Sudanese medicinal plants highest phenolic concentrations (more than 37.80 μg GAE/mg) were
extracts.
found in: T. brownii (bark), A. nilotica (bark), A. nilotica (pods),
Botanical name Part used Total phenolic content FRAP □EC P. glabrum, Z. spina-christi (bark), T. brownii (wood), C. hartmannianum
(μg GAE/mg •) (mM/mg) (bark), K. senegalensis, S. dubium, A. seyal var. seyal (bark), T. laxiflora
A. visnaga (L.) Lam. Fruits 34.17 ± 0.97ab 0.37 ± 0.02m (wood), G. senegalensis, A. seyal var. fistula (bark), A. precatorius
C. carvi L. Fruits 36.98 ± 0.55ab 0.35 ± 0.05n and A. maritima extracts. Our findingagrees with Cock (2015),
H. thebaica (L.) Mart. Fruits 35.10 ± 0.61ab 0.57 ± 0.01k
and Sulaiman et al. (2014), who stated that Terminala and Acacia
A.bracteolata Lam. Aerial parts 26.02 ± 0.58c 0.40 ± 0.03l
S. argel (Delile) Hayne Leaves 32.90 ± 3.42b 0.28 ± 0.04o species were rich in plant phenolic compounds respectively. Compara-
X. brasilicum Vell. Leaves 36.90 ± 1.33ab 0.88 ± 0.09j tively the moderate phenolic concentration (between 37.69 and
B. aegyptiaca (L.) Delile Leaves 26.40 ± 0.81c 0.29 ± 0.04o 30.00 μg GAE/mg) was found in : L. sativum, A. digitata, C. carvi,
Bark 32.26 ± 2.69 b 0.65 ± 0.03jk A. tortilis (wood), X. brasilicum, C. hartmannianum (wood), Z. Spina-christi
Fruits 14.05 ± 1.04d nd
K. africana (Lam.) Benth. Fruits 30.36 ± 0.68b 0.83 ± 0.02j
(leaves), H. sabdariffa, A. seyal var. seyal (wood), T. indica, A. tortilis (bark),
A. digitata L. Fruit puple 37.48 ± 2.30ab 2.57 ± 0.17e H. thebaica, S. persica (stems), M. oleifera, A. visnaga, L. inermis, S. argel,
L. sativum L. Seeds 37.69 ± 1.05ab 0.70 ± 0.03j B. aegyptiaca (bark), H. tuberculatum, P. aculeate, K. africana and
T. indica L. Leaves 31.26 ± 0.38b 2.71 ± 0.06d F. cretica extracts.
C. decidus (Forssk.) Edgew. Stems 18.50 ± 0.96c 0.08 ± 0.01s
G. tenax, B. aegyptiaca (leaves), M. oblongifolia, A.bracteolata,
M. oblongifolia (Frossk.) Stems 26.40 ± 0.74c 0.22 ± 0.02q
A.Rich S. persica (leaves), A. pannosum, N. sativa, C. coloynthis, A. seyal var. fistu-
C. hartmannianum Schweinf. Bark 42.44 ± 3.51a 3.88 ± 0.03ab la (wood) and C. decidus extracts, demonstrated relatively low level of
Wood 36.02 ± 1.77ab 2.75 ± 0.17d phenolic content (between 26.54 to 18.50 μg GAE/mg). Whereas,
G. senegalensis J.F.Gmel Leaves 40.50 ± 0.70a 3.65 ± 0.03b B. aegyptiaca (fruits) and Z. spina-christi (fruits) revealed quite low phe-
T. brownii Fresen. Bark 46.02 ± 0.68a 3.94 ± 0.04a
nolic level (14.05 and 9.63 μg GAE/mg respectively). Karou et al. (2011)
Wood 42.19 ± 1.31a 3.93 ± 0.01a
T. laxiflora Engl. Wood 41.11 ± 3.73a 3.99 ± 0.02a found that aerial parts of B. aegyptiaca had low content of phenolics.
A. maritima L. Aerial parts 37.80 ± 1.83a 0.78 ± 0.05j Although phenolic compounds are a diverse and ubiquitous group of
C. coloynthis (L.) Schrad. Dry fruits 22.80 ± 0.36c 0.07 ± 0.01s secondary metabolites in the plant kingdom, their distribution and con-
A. precatorius L. Seeds 39.50 ± 1.50a 3.46 ± 0.23c
centration vary across and within species (Robards et al., 1999).
A. nilotica (L.) Delile Pods 44.44 ± 4.16a 3.97 ± 0.08a
Bark 45.06 ± 3.23a 3.96 ± 0.05a
A. seyal var. seyal Del. Bark 41.40 ± 2.20a 3.93 ± 0.01a
Wood 35.65 ± 1.10ab 2.97 ± 0.08d 3.3. Antioxidant capacity
A. seyal var. fistula Bark 40.33 ± 1.81a 3.49 ± 0.05c
(Schweinf.) Wood 21.24 ± 2.04c 0.29 ± 0.03o
The ferric reducing ability of plasma (FRAP) assay was used to
A. tortilis (Forssk.) Hayne Bark 35.14 ± 1.71ab 2.62 ± 0.18e
Wood 36.97 ± 1.77ab 2.36 ± 0.14f evaluate the antioxidant potential of extracts of the selected Sudanese
P. aculeata L. Leaves 30.91 ± 0.67b 0.45 ± 0.01k medicinal plants. The FRAP assay depends upon the reduction of
L. inermis L. Leaves 33.9 0± 1.41b 1.74 ± 0.07h ferric tripyridyltriazine [Fe (III)-TPTZ] to ferrous tripyridyltriazine
A. pannosum (G.Forst.) Leaves 24.35 ± 0.55c 0.22 ± 0.01q
[Fe (II)-TPTZ] at low pH. The [Fe (II)-TPTZ] complex has an intensive
Schltdl.
G. tenax (Forssk) Fiori Fruits 26.5 4± 0.64c nd
blue color and can be monitored at 590 nm. Ferric reducing ability
H. sabdariffa L. Flowers 35.80 ± 1.56ab 1.03 ± 0.08i of plasma assay is used in many studies because it is quick, simple to
K. senegalensis (Desv.) A. Juss. Bark 42.44 ± 6.62a 3.93 ± 0.04a perform and reaction is reproducible and linearly related to the molar
M. oleifera lam. Leaves 34.49 ± 1.30ab 0.70 ± 0.06j concentration of the antioxidant(s) present (Benzie and Strain, 1996).
P. glabrum Willd. Leaves 43.70 ± 2.39a 3.88 ± 0.02ab
The reducing power property indicates that the antioxidant compounds
N. sativa L. Seeds 24.11 ± 0.62c 0.16 ± 0.03r
Z. spina–christi (L.) Desf. Fruits 09.63 ± 0.87e nd are electron donors and can reduce the oxidized intermediates of the
Bark 42.53 ± 3.23a 3.88 ± 0.03ab oxidative damage process, so that they can act as primary and second-
Leaves 35.85 ± 2.10ab 1.19 ± 0.10i ary antioxidants (Yen and Chen, 1995).
H. tuberculatum (Forssk.) Aerial parts 31.85 ± 2.73b 0.28 ± 0.02o
As shown in Table 3, there were significant differences in total
A. Juss.
S. persica L. Leaves 25.14 ± 1.17c 0.16 ± 0.01r
antioxidant capacity between the plant species and the part used
Stems 34.70 ± 1.80ab 0.86 ± 0.06j of the plant extracts. The FRAP values can be divided in four groups:
S. dubium Fresen Fruits 42.25 ± 1.52a 3.44 ± 0.10c very low FRAP (less than 2.0 mM/mg) n = 29; low FRAP (2.0–
T. nilotica (Ehrenb.) Bunge Stems 35.52 ± 2.08ab 2.07 ± 0.11g 2.62 mM/mg) n = 4; good FRAP (2.65–3.50 mM/mg) n = 6 and high
F. cretica L. Aerial parts 30.09 ± 2.03b 0.25 ± 0.07p
FRAP (more than 3.50 mM/mg) n = 11. Quercetin, ascorbic acid
Quercetin⁎ 3.96 ± 0.11a
Ascorbic acid⁎ 3.79 ± 0.10b and BHI as positive controls demonstrated antioxidant values of
BHI⁎ 2.84 ± 0.03d 3.96, 3.79 and 2.84 mM/mg respectively. The strongest antioxidant
Means with different letters in the same column were significantly different at the level
properties were observed on; T. laxiflora, A. nilotica (pods, bark),
(p b 0.05); n = 3. T. brownii (bark), A. seyal var. seyal (bark), K. senegalensis, T. brownii
nd: not detected. (wood), C. hartmannianum (bark), P. glabrum, Z. spina-christi (bark)
•

□
(μg GAE/mg)-μg of gallic acid equivalent per mg dry weight of plant extract.
Ferric reducing ability of plasma; EC: equivalent concentration; (mM/mg)- mM Fe2+
per mg dry weight of plant extract.
⁎ Positive controls.
site. Owing to this polyphenols has attracted researchers to topical skin
applications uses. The amount of total phenolic content was measured
by Folin–Ciocalteu method. The phenolic content varied widely in our
tested extracts and ranged from 46.02 to 09.63 μg GAE/mg (Table 3).
The highest level of phenolic content was found in T. brownii (bark),
while the lowest was in Z. spina–christi (fruits). There were significant
and G. senegalensis extracts. This result is similar to the findings of
Muddathir and Mitsunaga (2013), Siddiqui and Patil (2015) and
Hassan et al. (2014). They demonstrated that, A. nilotica (pod, bark),
T. laxiflora (wood), C. hartmannianum (bark), K. senegalensis, A. seyal
var. seyal (bark), G. senegalensi and Z. spp. (bark) showed an excellent
antioxidant activities when measured by 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging assay. Similarly, high antioxidant activities
measured by trolox equivalent antioxidant content (TEAC) assay have
also been reported for the genus Acacia and Terminalia which seem to
be correlated with their phenolic contents (Maldini et al., 2011; Cock,
2015). The extracts showed high EC values, it could be considered that
compounds in these extracts were good electron donors and could
14 A.M. Muddathir et al. / South African Journal of Botany 109 (2017) 9–15
terminate oxidation chain reactions by reducing the oxidized interme-
diates into the stable form.
It is worth to be mentioned that a significant relationship is observed
between (r = 0.944) total phenolic content and antioxidant compo-
nents of the extracts that showed potent anti-tyrosinase activity
(Table 2). This result is in agreement with Katalinic et al. (2006) who
confirmed a significant linear correlation between total phenolic and re-
lated FRAP of medicinal plant extracts. The greater number of hydroxyl
groups in the phenolics, the higher antioxidant activity (Prasad et al.,
2005; Rangkadilok et al., 2007). The inhibition of tyrosinase activity
might depend on the hydroxyl groups of the phenolic compounds of
the extracts that could form a hydrogen bond to a site of the enzyme,
leading to a lower enzymatic activity. Some tyrosinase inhibitors act
through hydroxyl groups that bind to the active site on tyrosinase,
resulting in steric hindrance or changed conformation. The antioxidant
activity mechanism may also be one of the important reasons for tyros-
inase inhibition activity (Baek et al., 2008; Kim et al., 2008).
Ma et al. (2001) suggested that ROS scavenger such as antioxidants
may reduce the hyperpigmentation, therefore we proposed that the ex-
tracts with high EC value could be useful to reduce the hyperpigmention
process.
4. Conclusion
According to this study, a number of Sudanese medicinal plants re-
vealed anti-tyrosinase activity, high phenolic content and antioxidant
capacity. A. nilotica (pod-bark) expressed a higher potency very prom-
ising for further research; since it has high tyrosinase inhibitory activity,
antioxidant activity and a good phenolic content. Consequently these
findings could be applied in the industry for the obtainment of natural
antioxidants and tyrosinase inhibitors. However, the results obtained
need further investigation in pigment cell assays and in clinical studies.
Acknowledgments
The authors wish to thank Ms. Noah Mohamed from the Ministry
of Agriculture and Forestry (Gadarif State), Mrs. Hamza Tag EL-Sir,
Botanist University of Khartoum, Faculty of Agriculture, Department
of Botany (Herbarium) and Dr. Ashraf Mohamed from the Faculty of
Forestry, University of Khartoum, Sudan. They are warmly thanked for
their assistance in the plants identification and authentications. This
project was supported financially by the Ministry of Education, Culture,
Sports, Science and Technology, Japan (MEXT Grant).
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