Anti-tyrosinase, total phenolic content and antioxidant activity of selected Sudanese medicinal plants

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 b c

United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan Department of Chemistry, Faculty of Mathematics and Natural Sciences, and Biopharmaca Research Center, Bogor Agricultural University, Bogor, Indonesia Faculty of pharmacy, University of Science and Technology, P.O. Box 447, Omdurman, Sudan

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Article history: Received 22 May 2016 Received in revised form 17 September 2016 Accepted 13 December 2016 Available online 30 December 2016 Edited by AR Ndhlala Keywords: Sudanese medicinal plants Phenolic content FRAP Tyrosinase inhibition Acacia nilotica

a b s t r a c t The flora of Sudan is relatively rich in medicinal plants and represents an important component of traditional medicine. Fifty methanolic extracts of selected Sudanese medicinal plants were evaluated for their in vitro tyrosinase inhibitory effect, antioxidant activity and total phenolic content (TPC). The standard method of antioxidant evaluation, ferric reducing ability of plasma (FRAP), was employed to determine the antioxidant activity while the enzyme based tyrosinase inhibition was used for the anti-tyrosinase activity. Acacia nilotica (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 was used as a positive control. A. nilotica (bark) and Acacia seyal var. fistula (bark) exhibited good tyrosinase inhibitory activity using L-DOPA as substrate (IC50: 31.93, 36.32 μg/ml) compared to positive control (IC50: 37.63 μg/ml). The results revealed significant differences in TPC between plants extracts. The highest level of phenolic content was found in Terminalia brownii (bark; 46.02 μg GAE/mg) while the lowest was in Ziziphus spina–christi (fruits; 09.63 μg GAE/mg). The study indicated significant differences in total antioxidant 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 phenolic compounds. To the best of our knowledge, this is the first data presenting comprehensive data on antityrosinase, TPC, antioxidant activity of the Sudanese medicinal plants. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Tyrosinase (EC 1.14.18.1; PPO) is known to be a key enzyme in melanin biosynthesis and is widely distributed in plants and mammalian cells. Tyrosinase enzyme catalyzes two different reactions: the hydroxylation 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: [email protected] (A.M. Muddathir).

http://dx.doi.org/10.1016/j.sajb.2016.12.013 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

nevus, ephelis, post inflammatory state and melanoma of pregnancy (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 insecticide (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 production (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

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(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 antioxidants may reduce the hyperpigmentation (Ma et al., 2001). Total phenolic 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 residue 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 softening 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 melanogenesis, initially using a cell-free mushroom tyrosinase system, which has commonly been employed for the testing and screening of potential 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)-striazine (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 powdered 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 described 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 absorbance at 510 nm was determined using a micro plate reader. The percent inhibition of tyrosinase activity was calculated at the concentration 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) concentration, 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 positive 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 distributed 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 concentration of 125 and 500 μg/ml as well as IC50 values. The study revealed that 36 and 24% of extracts presented a good tyrosinase inhibitory activity which inhibited more than 50% inhibition for both

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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 specimen

Traditional uses and references

Ammi visnaga (L.) Lam.

Apiaceae

SD-OD-38

Carum carvi L. Hyphaene thebaica (L.) Mart. Aristolochia bracteolata Lam. Solenostemma argel (Delile) Hayne Xanthium brasilicum Vell. Balanites aegyptiaca (L.) Delile Kigelia africana (Lam.)Benth. Adansonia digitata L.

Apiaceae Arecaceae Aristolochiaceae Asclepiadaceae

SD-OD-48 SD-OD-41 SD-SH-04 SD-SH-23

It relaxes smooth muscles and lowers the urethral tonicity. A decoction is used to ease the passage of kidney calculus (Khalid et al., 2012). Antispasmodic, carminative, galactagogue and anthelmintic properties (Khalid et al., 2012). Against splenomegaly, alimentary system disorders and bacterial eye infections (El Ghazali et al., 2003). Analgesic, treatment of scorpion bite and malaria. It is also used anti-tumors (El-Tahir et al., 1999). The leaves are used as an antispasmodic, carminative and as an anti-diabetic (Kamel et al., 2000).

Asteraceae Balanitaceae

SD-SH-12 SD-SH-15

Bignoniaceae

SD-SH-21

Bombacaceae

SD-OD-27

Lepidium sativum L. Tamarindus indica L. Capparis decidus (Forssk.) Edgew. Maerua oblongifolia (Frossk.) A.Rich Combretum hartmannianum Schweinf. Guiera senegalensis J.F.Gmel Terminalia brownii Fresen. Terminalia laxiflora Engl. Ambrosia maritima L.

Brassicaceae Caesalpiniaceae Capparaceae

SD-KH-02 SD-SH-18 SD-SH-17

Used as a cold beverage and added to yogurt for treatment of diarrhea and amoebic dysentery (Mangan, 1988). Used as anti-asthmatic, anti-scorbutic, aperient, diuretic, galactagogue and stimulant (Adam et al., 2011). Fever, malaria and laxative (El Kamali and El-Khalifa, 1999). Jaundice, rheumatic arthritis and to treat swells (El Ghazali et al., 1997).

Capparaceae

SD-SH-45

Malaria (El Kamali and El-Khalifa, 1997).

Combretaceae

SD-KH-04

Combretaceae Combretaceae Combretaceae Composiate

SD-OD-40 SD-GF-02 SD-KH-03 SD-SH-03

Citrullus coloynthis (L.) Schrad. Abrus precatorius L.

Cucurbitaceae

SD-OD-05

Arthritis rheumatism, skin dryness, jaundice and bacterial diseases (El Ghazali et al., 1997; Al Magboul et al., 1988). Anti-pyretic, anti-diabetic , anti-hypertension and febrifuge (El Ghazali et al., 1994, 1997). Cough bronchitis and anti-rheumatic (El Ghazali et al., 1994, 1997). Cough treatments and fumigant (Musa et al., 2011). Urinary tract infections, elimination of kidney stones, anti-diabetic and anti-hypertensive (Mahmoud et al., 1999). Purgative and skin diseases (El Ghazali et al., 1997).

Leguminosae

SD-OD-22

Acacia nilotica (L.) Delile Acacia seyal var. seyal Del. Acacia seyal var. fistula (Schweinf.) Acacia tortilis (Forssk.) Hayne Parkinsonia aculeata L. Lawsonia inermis L. Abutilon pannosum (G.Forst.) Schltdl. Grewia tenax (Forssk) Fiori Hibiscus sabdariffa L. Khaya senegalensis (Desv.) A. Juss. Moringa oleifera lam. Polygonum glabrum Willd. Nigella sativa L. Ziziphus spina-christi (L.) Desf. Haplophyllum tuberculatum (Forssk.) A. Juss. Salvadora persica L.

Leguminosae Leguminosae Leguminosae

SD-OD-01 SD-GF-05 SD-GF-06

Leguminosae

SD-KH-07

Leguminosae Lythraceae Malvaceae

SD-SH-02 SD-SH-07 SD-SH-43

Malvaceae Malvaceae Meliaceae

SD-OD-42 SD-SH-47 SD-SH-14

Moringaceae Polygonaceae Ranunculaceae Rhamnaceae

SD-SH-08 SD-SH-A-03 SD-OD-16 SD-SH-06

Rutaceae

SD-KH-32

Salvadoraceae

SD-SH-09

Solanaceae Tamaricaceae

SD-SH-34 SD-OD-10

Gingivitis, malaria, liver swellings and HIV-1(El Kamali and Khalid, 1996; Hussein et al., 1999; Neuwinger, 2000). Applied to wounds and skin tumors as a dressing (El Kheir and Salih, 1980). Febrile, colds and hemorrhoids (El Ghazali et al., 1994; Neuwinger, 2000).

Zygophyllaceae

SD-OD-26

Muscular pains, antispasmodic and against heart burn (El Ghazali et al., 1997; El Ghazali, 1986).

Solanum dubium Fresen Tamarix nilotica (Ehrenb.) Bunge Fagonia cretica L.

Antiprotozoal (Nour et al., 2009). Venereal diseases, rheumatism, digestion problems, dysentery and bilharzias (Iwu, 1993; El Ghazali et al., 1997; Van Wyk et al., 1997). Stomach problem, cough, dysentery and venereal diseases (Neuwinger, 1996; El Ghazali et al., 1997).

Oral contraceptives, against chronic nephritis and diabetes (Manago and Alumanah, 2005; Garaniya and Bapodra, 2014). Diarrhea, cold, pharyngitis, used as anti-septic and tooth ache (Eldeen et al., 2010). Arthritis rheumatism and rheumatic fever (El Ghazali et al., 1997). Fumigant for rheumatic pain. It is also used to protect women from fever after childbirth and soften the skin (Khalid et al., 2012). Vermifuge, skin infections, oedema and allergic dermatitis (http://www.fao.org/ag/agp/agpc/doc/Gbase/DATA/Pf000139.htm). Fever, malaria, abortifacient and rheumatism (Orwa et al., 2009). Anti-pyretic, for treatment of urinary tract infections, skin diseases and alopecia (El Ghazali, 1986). Hepatoprotective, antiplasmodial and hypoglycemic activities (Cho-kang et al., 2007).

Malaria and iron deficiency anemia (Khalid et al., 2012). Hypertension, colds, antispasmodic and antimicrobial agent (Khalid et al., 2012). Tonic, fever, vermifuge, taenicide, emmenagogue, venereal diseases and antiprotozoal (Bever, 1986; Iwu, 1993; Kayser and Abreu, 2001). Antimicrobial, febrile, pulmonary diseases (Neuwinger, 2000; Anwar et al., 2007). Anthelmintic (Muddathir et al., 1987). Anti-inflammatory, allergies, eczema and malaria (Ahmed et al., 2010). Ulcers, gonorrhea, sore throat, chest pain, dysentery, venereal diseases and wounds (El Ghazali et al., 1997; Dafni et al., 2005). Used against diarrhea and spasms (Khalid et al., 2012).

substrates (L-tyrosine and L-DOPA, respectively). At the concentration of 500 μg/ml of extracts with L-tyrosine and L-DOPA substrates; Z. spinachristi (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 derivatives 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

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

Part used

Tyrosinase inhibition (%) L-Tyrosine

A. visnaga (L.) Lam. C. carvi L. H. thebaica (L.) Mart. A. bracteolata Lam. S. argel (Delile) Hayne X. brasilicum Vell. B. aegyptiaca (L.) Delile

K. africana (Lam.) Benth. A. digitata L. L. sativum L. T. indica L. C. decidus (Forssk.) Edgew. M. oblongifolia (Frossk.) A.Rich C. hartmannianum Schweinf. G. senegalensis J.F.Gmel T. brownii Fresen. T. laxiflora Engl. A. maritima L. C. coloynthis (L.) Schrad. A. precatorius L. A. nilotica (L.) Delile A. seyal var. seyal Del. A. seyal var. fistula (Schweinf.) A. tortilis (Forssk.) Hayne P. aculeata L. L. inermis L. A. pannosum (G.Forst.) Schltdl. G. tenax (Forssk) Fiori H. sabdariffa L. K. senegalensis (Desv.) A. Juss. M. oleifera lam. P. glabrum Willd. Nigella sativa L. Z. spina –christi (L.) Desf.

H. tuberculatum (Forssk.) A. Juss. S. persica L. S. dubium Fresen T. nilotica (Ehrenb.) Bunge F.cretica L. Kojic acid⁎⁎⁎

Fruits Fruits Fruits Aerial parts Leaves Leaves Leaves Bark Fruits Fruits Fruit puple Seeds Leaves Stems Stems Bark Wood Leaves Bark Wood Wood Aerial parts Dry fruits Seeds Pods Bark Bark Wood Bark Wood Bark Wood Leaves Leaves Leaves Fruits Flowers Bark Leaves Leaves Seeds Fruits Bark Leaves Aerial parts Leaves Stems Fruits Stems Aerial parts

Tyrosinase inhibition L-DOPA

L-Tyrosine

L-DOPA

125 μg/ml

500 μg/m

125 μg/ml

500 μg/ml

IC50 μg/ml⁎

IC50 μg/ml

18.55 –⁎⁎ 19.32 – – 08.86 – – 02.54 – 73.53 – 54.38 – – – 27.75 45.30 81.12 18.63 01.84 13.90 – – 96.39 94.20 84.03 91.80 91.49 – – 65.52 – 02.91 30.41 01.93 18.28 93.1 06.35 100 – – 72.77 – – 20.00 08.84 – 51.22 10.30 99.50

42.10 05.38 26.54 10.91 25.55 19.30 02.73 06.77 04.25 02.60 97.14 01.88 72.50 10.11 – 11.23 59.03 95.25 95.50 61.80 35.95 50.51 18.15 09.60 98.30 94.23 95.45 91.82 94.52 05.10 08.0 70.91 15.11 11.20 07.22 03.00 21.21 97.9 08.94 100 15.73 09.68 94.75 32.73 – 67.68 41.58 16.50 79.51 22.56 99.65

02.82 – 01.58 – – 14.30 – 06.10 – 02.4 59.20 – 37.65 – 09.4 12.46 13.86 22.86 51.70 08.35 – – – 26.14 64.7 70.9 69.77 38.50 68.74 – – 24.20 – 12.44 10.53 02.54 10.88 64.2 09.90 84.39 – – 42.41 – 05.50 11.86 14.01 – 30.52 – 83.54

28.90 06.93 08.40 13.76 30.32 27.23 16.02 14.71 06.00 13.7 77.62 – 49.79 – 18.0 12.70 43.88 80.89 70.18 32.35 21.41 27.82 12.20 69.40 81.9 84.5 83.06 48.91 81.80 04.40 – 33.65 12.20 12.78 18.72 02.80 12.23 85.7 12.13 91.75 17.64 – 77.30 24.43 14.42 46.57 32.83 12.73 53.00 16.12 96.40

nd nd nd nd nd nd nd nd nd nd 51.71 ± 4.27c nd 78.38 ± 9.69d nd nd nd 386.89 ± 11.29g 198.51 ± 14.18f 119.14 ± 9.39e 415.44 ± 21.70h nd 485.96 ± 11.96i nd nd 08.61 ± 0.94a 10.47 ± 1.42a 44.30 ± 3.97c 10.77 ± 0.35a 22.80 ± 2.70b nd nd 91. 51 ± 6.49d nd nd nd nd nd 20.87 ± 0.85b nd 40.89 ± 2.14c nd nd 51.57 ± 10.75c nd nd 356.87 ± 9.94g nd nd 118.19 ± 8.46e nd 10.02 ± 1.87a

nd nd nd nd nd nd nd nd nd nd 91.25 ± 5.97c nd nd nd nd nd nd 240.35 ± 10.17e 120.66 ± 10.22d nd nd nd nd 345.71 ± 13.49f 98.3 3± 3.79c 31.93 ± 1.60a 94.47 ± 2.39c nd 36.32 ± 4.12a nd nd nd nd nd nd nd nd 71.20 ± 11.20b nd 75.68 ± 0.72b nd nd 253.62 ± 11.11e nd nd nd nd nd 424.27 ± 29.30g nd 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 tyrosinase, 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 phenolics with ion reducing ability diminish the possibility of hydroxyl radical's formation path from superoxide anion radicals and additionally 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 Table 3 Total phenolic content and antioxidant capacity of selected Sudanese medicinal plants extracts. Botanical name

Part used

Total phenolic content FRAP □EC (μg GAE/mg •) (mM/mg)

A. visnaga (L.) Lam. C. carvi L. H. thebaica (L.) Mart. A.bracteolata Lam. S. argel (Delile) Hayne X. brasilicum Vell. B. aegyptiaca (L.) Delile

Fruits Fruits Fruits Aerial parts Leaves Leaves Leaves Bark Fruits Fruits Fruit puple Seeds Leaves Stems Stems

34.17 36.98 35.10 26.02 32.90 36.90 26.40 32.26 14.05 30.36 37.48 37.69 31.26 18.50 26.40

K. africana (Lam.) Benth. A. digitata L. L. sativum L. T. indica L. C. decidus (Forssk.) Edgew. M. oblongifolia (Frossk.) A.Rich C. hartmannianum Schweinf. Bark Wood G. senegalensis J.F.Gmel Leaves T. brownii Fresen. Bark Wood T. laxiflora Engl. Wood A. maritima L. Aerial parts C. coloynthis (L.) Schrad. Dry fruits A. precatorius L. Seeds A. nilotica (L.) Delile Pods Bark A. seyal var. seyal Del. Bark Wood A. seyal var. fistula Bark (Schweinf.) Wood A. tortilis (Forssk.) Hayne Bark Wood P. aculeata L. Leaves L. inermis L. Leaves A. pannosum (G.Forst.) Leaves Schltdl. G. tenax (Forssk) Fiori Fruits H. sabdariffa L. Flowers K. senegalensis (Desv.) A. Juss. Bark M. oleifera lam. Leaves P. glabrum Willd. Leaves N. sativa L. Seeds Z. spina–christi (L.) Desf. Fruits Bark Leaves H. tuberculatum (Forssk.) Aerial parts A. Juss. S. persica L. Leaves Stems S. dubium Fresen Fruits T. nilotica (Ehrenb.) Bunge Stems F. cretica L. Aerial parts Quercetin⁎ Ascorbic acid⁎ BHI⁎

0.97ab 0.55ab 0.61ab 0.58c 3.42b 1.33ab 0.81c 2.69 b 1.04d 0.68b 2.30ab 1.05ab 0.38b 0.96c 0.74c

0.37 ± 0.02m 0.35 ± 0.05n 0.57 ± 0.01k 0.40 ± 0.03l 0.28 ± 0.04o 0.88 ± 0.09j 0.29 ± 0.04o 0.65 ± 0.03jk nd 0.83 ± 0.02j 2.57 ± 0.17e 0.70 ± 0.03j 2.71 ± 0.06d 0.08 ± 0.01s 0.22 ± 0.02q

42.44 ± 3.51a 36.02 ± 1.77ab 40.50 ± 0.70a 46.02 ± 0.68a 42.19 ± 1.31a 41.11 ± 3.73a 37.80 ± 1.83a 22.80 ± 0.36c 39.50 ± 1.50a 44.44 ± 4.16a 45.06 ± 3.23a 41.40 ± 2.20a 35.65 ± 1.10ab 40.33 ± 1.81a 21.24 ± 2.04c 35.14 ± 1.71ab 36.97 ± 1.77ab 30.91 ± 0.67b 33.9 0± 1.41b 24.35 ± 0.55c

3.88 ± 0.03ab 2.75 ± 0.17d 3.65 ± 0.03b 3.94 ± 0.04a 3.93 ± 0.01a 3.99 ± 0.02a 0.78 ± 0.05j 0.07 ± 0.01s 3.46 ± 0.23c 3.97 ± 0.08a 3.96 ± 0.05a 3.93 ± 0.01a 2.97 ± 0.08d 3.49 ± 0.05c 0.29 ± 0.03o 2.62 ± 0.18e 2.36 ± 0.14f 0.45 ± 0.01k 1.74 ± 0.07h 0.22 ± 0.01q

26.5 4± 0.64c 35.80 ± 1.56ab 42.44 ± 6.62a 34.49 ± 1.30ab 43.70 ± 2.39a 24.11 ± 0.62c 09.63 ± 0.87e 42.53 ± 3.23a 35.85 ± 2.10ab 31.85 ± 2.73b

nd 1.03 ± 0.08i 3.93 ± 0.04a 0.70 ± 0.06j 3.88 ± 0.02ab 0.16 ± 0.03r nd 3.88 ± 0.03ab 1.19 ± 0.10i 0.28 ± 0.02o

25.14 34.70 42.25 35.52 30.09

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ±

1.17c 1.80ab 1.52a 2.08ab 2.03b

0.16 0.86 3.44 2.07 0.25 3.96 3.79 2.84

± ± ± ± ± ± ± ±

0.01r 0.06j 0.10c 0.11g 0.07p 0.11a 0.10b 0.03d

Means with different letters in the same column were significantly different at the level (p b 0.05); n = 3. nd: not detected. • (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

13

differences in total phenolic content between plants extracts. The highest phenolic concentrations (more than 37.80 μg GAE/mg) were found in: T. brownii (bark), A. nilotica (bark), A. nilotica (pods), P. glabrum, Z. spina-christi (bark), T. brownii (wood), C. hartmannianum (bark), K. senegalensis, S. dubium, A. seyal var. seyal (bark), T. laxiflora (wood), G. senegalensis, A. seyal var. fistula (bark), A. precatorius and A. maritima extracts. Our findingagrees with Cock (2015), and Sulaiman et al. (2014), who stated that Terminala and Acacia species were rich in plant phenolic compounds respectively. Comparatively the moderate phenolic concentration (between 37.69 and 30.00 μg GAE/mg) was found in : L. sativum, A. digitata, C. carvi, A. tortilis (wood), X. brasilicum, C. hartmannianum (wood), Z. Spina-christi (leaves), H. sabdariffa, A. seyal var. seyal (wood), T. indica, A. tortilis (bark), H. thebaica, S. persica (stems), M. oleifera, A. visnaga, L. inermis, S. argel, B. aegyptiaca (bark), H. tuberculatum, P. aculeate, K. africana and F. cretica extracts. G. tenax, B. aegyptiaca (leaves), M. oblongifolia, A.bracteolata, S. persica (leaves), A. pannosum, N. sativa, C. coloynthis, A. seyal var. fistula (wood) and C. decidus extracts, demonstrated relatively low level of phenolic content (between 26.54 to 18.50 μg GAE/mg). Whereas, B. aegyptiaca (fruits) and Z. spina-christi (fruits) revealed quite low phenolic level (14.05 and 9.63 μg GAE/mg respectively). Karou et al. (2011) found that aerial parts of B. aegyptiaca had low content of phenolics. Although phenolic compounds are a diverse and ubiquitous group of secondary metabolites in the plant kingdom, their distribution and concentration vary across and within species (Robards et al., 1999).

3.3. Antioxidant capacity The ferric reducing ability of plasma (FRAP) assay was used to evaluate the antioxidant potential of extracts of the selected Sudanese medicinal plants. The FRAP assay depends upon the reduction of ferric tripyridyltriazine [Fe (III)-TPTZ] to ferrous tripyridyltriazine [Fe (II)-TPTZ] at low pH. The [Fe (II)-TPTZ] complex has an intensive blue color and can be monitored at 590 nm. Ferric reducing ability of plasma assay is used in many studies because it is quick, simple to perform and reaction is reproducible and linearly related to the molar concentration of the antioxidant(s) present (Benzie and Strain, 1996). The reducing power property indicates that the antioxidant compounds are electron donors and can reduce the oxidized intermediates of the oxidative damage process, so that they can act as primary and secondary antioxidants (Yen and Chen, 1995). As shown in Table 3, there were significant differences in total antioxidant capacity between the plant species and the part used of the plant extracts. The FRAP values can be divided in four groups: very low FRAP (less than 2.0 mM/mg) n = 29; low FRAP (2.0– 2.62 mM/mg) n = 4; good FRAP (2.65–3.50 mM/mg) n = 6 and high FRAP (more than 3.50 mM/mg) n = 11. Quercetin, ascorbic acid and BHI as positive controls demonstrated antioxidant values of 3.96, 3.79 and 2.84 mM/mg respectively. The strongest antioxidant properties were observed on; T. laxiflora, A. nilotica (pods, bark), T. brownii (bark), A. seyal var. seyal (bark), K. senegalensis, T. brownii (wood), C. hartmannianum (bark), P. glabrum, Z. spina-christi (bark) 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

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A.M. Muddathir et al. / South African Journal of Botany 109 (2017) 9–15

terminate oxidation chain reactions by reducing the oxidized intermediates 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 components 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 related 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 tyrosinase 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 extracts with high EC value could be useful to reduce the hyperpigmention process.

4. Conclusion According to this study, a number of Sudanese medicinal plants revealed anti-tyrosinase activity, high phenolic content and antioxidant capacity. A. nilotica (pod-bark) expressed a higher potency very promising 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). References Adam, S., Shayma, S., Warda, A., 2011. In vitro antimicrobial assessment of Lepidium sativum L. seeds extracts. Asian Journal of Medical Sciences 3, 261–266. Ahmed, E.M., Nour, B.Y.M., Mohammed, Y.G., Khalid, H.S., 2010. Antiplasmodial activity of some medicinal plants used in Sudanese folk-medicine. Environmental Health Insights 4, 1–6. Ainsworth, E., Gillespie, K., 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nature Protocols 2, 875–887. Al Magboul, A.Z., Bashir, A.K., Salih, A.M., Farouk, A., Khalid, S.A., 1988. Antimicrobial activity of certain Sudanese plants used in flokloric medicine: screening for antibacterial activity (V). Fitoterapia 59, 57–62. Anwar, F., Latif, S., Ashraf, M., Gilani, A.H., 2007. Moringa oleifera: a food plant with multiple medicinal uses. Phytotherapy Research 21, 17–25. Baek, H., Rho, H., Yoo, J., Ahn, S., Lee, J., Lee, J., 2008. The inhibitory effect of new hydroxamic acid derivatives on melanogenesis. Bulletin of the Korean Chemical Society 29, 43–46. Batubara, I., Darusman, L., Mitsunaga, T., Rahminiwati, M., Djauhari, E., 2010. Potency of Indonesian medicinal plants as tyrosinase inhibitor and antioxidant agent. Journal of Biological Sciences 10, 138–144. Benzie, I., Strain, J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical Biochemistry 239, 70–76. Bever, B.O., 1986. Medicinal Plants in Tropical West Africa. Cambridge University Press, Cambridge, London, New.

Cho-Kang, J., Ali, H.A., Kim, J.B., Elamin, M.H., Ki, C., Kim, S.S., 2007. LC/PDA/ESI-MS flavonoid profiling, radical scavenging activity and antimicrobial activity of two Abitilon spp. The FASEB Journal 21, LB16. Cock, I.E., 2015. The medicinal properties and phytochemistry of plants of the genus Terminalia (Combretaceae). Inflammopharmacology 23, 203–229. Dafni, A., Levy, S., Lev, E., 2005. The ethnobotany of Christ's thorn jujube (Ziziphus spinachristi) in Israel. Journal of Ethnobiology and Ethnomedicine 1:8. http://dx.doi.org/10. 1186/1746-4269-1-8. El Ghazali, G., 1986. Medicinal Plants of Sudan Part I. Medicinal Plants of Erkawit. National Council for Research, Khartoum, Sudan. El Ghazali, G., El Tohami, M., Elegami, A., 1994. Medicinal Plants of the Sudan. Part III. Medicinal Plants of the White Nile Province. Khartoum University Press, Sudan. El Ghazali, G., El Tohami, M., Elegami, A., Abdalla, S., Mohammed, G., 1997. Medicinal Plants of the Sudan. Part IV. Medicinal Plants of Northern Kordofan. Omdurman Islamic University Press, Khartoum, Sudan. El Ghazali, G.E.B., Abdalla, W.E., Khalid, H.E., Khalafalla, M.M., Hamad, A.A., 2003. Medicinal Plants of the Sudan. Part V. Medicinal Plants of Ingassana Area. National Council for Research, Khartoum, Sudan. El Kamali, H.M., El-Khalifa, K.F., 1997. Treatment of malaria through herbal drugs in the Central Sudan. Fitoterapia 68, 527–528. El Kamali, H.H., El-Khalifa, K.F., 1999. Folk medicinal plants of riverside forests of the Southern Blue Nile district, Sudan. Fitoterapia 70, 493–497. El Kamali, H.M., Khalid, S.A., 1996. The most common herbal remedies in Central Sudan. Fitoterapia 57, 301–306. El Kheir, Y.M., Salih, M.H., 1980. Investigation of certain plants used in Sudanese folk medicine. Fitoterapia 51, 143–147. Eldeen, I.M., Van Heerden, F.R., Van Staden, J., 2010. In vitro biological activities of niloticane, a new bioactive cassane diterpene from the bark of Acacia nilotica subsp. Kraussiana. Journal of Ethnopharmacology 128, 555–560. El-Tahir, A., Satti, G., Khalid, S., 1999. Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Acacia nilotica. Phytotherapy Research 13, 474–478. FAO, Food and Agriculture Organization, Acacia tortilis (Forssk.) Hayne. Available at: http://www.fao.org/ag/agp/agpc/doc/Gbase/DATA/Pf000139.htm. Garaniya, N., Bapodra, A., 2014. Ethno botanical and phytopharmacological potential of Abrus precatorius L.: a review. Asian Pacific Journal of Tropical Biomedicine 4, S27–S34. Hassan, L.E., Ahamed, M., Majid, A., Baharetha, H., Muslim, N., Nassar, Z., 2014. Correlation of antiangiogenic, antioxidant and cytotoxic activities of some Sudanese medicinal plants with phenolic and flavonoid contents. BMC Complementary and Alternative Medicine. http://dx.doi.org/10.1186/1472-6882-14-406. Hussein, G., Miyashiro, H., Nakamura, N., Hattori, M., Kawahata, T., Otake, T., 1999. Inhibitory effects of Sudanese plant extracts on HIV-1 replication and HIV-1 protease. Phytotherapy Research 13, 31–36. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, Boca Raton, Florida, USA. Kadekaro, A., Kanto, H., Kavanagh, R., Abdel-Malek, Z., 2003. Significance of the melanocortin 1 receptor in regulating human melanocyte pigmentation, proliferation and survival. Annals of the New York Academy of Sciences 994, 359–365. Kähkönen, M., Hopia, A., Vuorela, H., Rauha, J., Pihlaja, K., Kujala, T., 1999. Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chemistry 47, 3954–3962. Kamel, M., Hassanin, H., Mohamed, M., Kassi, R., Yamasaki, K., 2000. Monoterpene and pregnane glucosides from Solenostemma argel. Phytochemistry 53, 937–940. Karou, S.D., Tchacondo, T., Ouattara, L., Anani, K., Savadogd, A., Agbonon, A., Attaia, M.B., De Souza, C., Sakly, M., Simpore, J., 2011. Antimicrobial, antiplasmodial, haemolytic and antioxidant activities of crude extracts from three selected Togolese medicinal plants. Asian Pacific Journal of Tropical Medicine 4, 808–813. Katalinic, V., Milos, M., Kulisic, T., Jukic, M., 2006. Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols. Food Chemistry 94, 550–557. Kayser, O., Abreu, P.M., 2001. Antileishmania and immunostimulating activities of two dimeric proanthocyanidins from Khaya senegalensis. Pharmaceutical Biology (Lisse, Netherlands) 39, 284–288. Khalid, S., Farouk, A., Geary, T.G., Jensen, J., 1986. Potential antimalarial candidates from African plants: an in vitro approach using Plasmodium falciparum. Journal of Ethnopharmacology 15, 201–209. Khalid, S., Yagi, S., Khristova, P., Duddeck, H., 1989. (+)-Catechin-5-galloyl Ester as a novel natural polyphenol from the bark of of Acacia nilotica of Sudanese origin. Planta Medica 55, 556–558. Khalid, H., Abdalla, W.E., Abdelgadir, H., Opatz, T., Efferth, T., 2012. Gems from traditional north-African medicine: medicinal and aromatic plants from Sudan. Natural Products and Bioprospecting 2, 92–103. Kim, D., Park, S., Kwon, S., Li, K., Youn, S., Park, K., 2004. (−)-Epigallocatechin-3-gallate and hinokitiol reduce melanin synthesis via decreased MITF production. Archives of Pharmaceutical Sciences and Research 27, 334–339. Kim, Y., Kang, K., Yokozawa, T., 2008. The anti-melanogenic effect of pycnogenol by its anti-oxidative actions. Food and Chemical Toxicology 46, 2466–2471. Lin, J.W., Chiang, H.M., Lin, Y.C., Wen, K.C., 2008. Natural products with skin-whitening effects. Journal of Food and Drug Analysis 16, 1–10. Ma, W., Wlaschek, M., Tantcheva-Poor, I., Schneider, L., Naderi, L., Razi, W., Schuller, J., Scharffetter-Kochanek, K., 2001. Chronological ageing and photoageing of the fibroblasts and the dermal connective tissue. Clinical and Experimental Dermatology 26, 592–599. Mahmoud, A.A., Ahmed, A.A., El Bassuony, A.A., 1999. A new chlorosesquiterpene lactone from Ambrosia maritime. Fitoterapia 70, 575–578. Malan, E., 1991. Derivatives of (+)-catechin-5-gallate from the bark of Acacia nilotica. Phytochemistry 30, 2737–2739.

A.M. Muddathir et al. / South African Journal of Botany 109 (2017) 9–15 Maldini, M., Montoro, P., Hamed, A., Mahalel, U., Oleszek, W., Stochmal, A., Piacente, S., 2011. Strong antioxidant phenolics from Acaia nilotica: profiling by ESI-MS and qualitative-quantitative determination by LC-ESI-MS. Journal of Pharmaceutical and Biomedical Analysis 56, 228–239. Manago, C.C., Alumanah, E.O., 2005. Antidiabetic effect chloroform-methanol extracts of Abrus precatorius seed. Journal of Applied Sciences and Environmental Management 9, 85–88. Mangan, J.L., 1988. Nutritional effects of tannins in animal feeds. Nutrition Research Reviews 1, 209–231. McEvily, A., Iyneger, R., Otwell, W., 1992. Inhibition of enzymatic browning in foods and beverages. Critical Reviews in Food Science and Nutrition 32, 253–273. Miyazawa, M., Tamura, N., 2007. Inhibitory compound of tyrosinase activity from sprout of Polygonum hydropiper L. (Benitade). Biological and Pharmaceutical Bulletin 30, 595–597. Momtaz, S., Lall, N., Basson, A., 2008. Inhibitory activities of mushroom tyrosine and DOPA oxidation by plant extracts. South African Journal of Botany 74, 577–582. Muddathir, A., Mitsunaga, T., 2013. Evaluation of anti-acne activity of selected Sudanese medicinal plants. Journal of Wood Science 59, 73–79. Muddathir, A.K., Balansard, G., David, T.P., Babadjamina, A., Yagoub, A.K., Julien, M.J., 1987. Anthelmintic properties of Polygonum glabrum. Journal of Pharmacy and Pharmacology 39, 296–300. Musa, S., Abdelrasool, F., Elsheikh, E., Ahmed, L., Mahmoud, A., Yagi, S., 2011. Ethnobotanical study of medicinal plants in the Blue Nile State, South-eastern Sudan. Journal of Medicinal Plant Research 5, 4287–4297. Neuwinger, D.H., 1996. African Ethnobotany Poisons and Drugs Chemistry (Germany). Chapman and Hall Gm bH, D-69469 Weinheim (Bundes Reublik Deutschland) 3-8261-0077-8. Neuwinger, D.H., 2000. African Traditional Medicine. A Dictionary of Plant Use and Applications. Med Pharm scientific Publisher, Stuttgart, Germany. Nour, A., Khalid, S., Kaiser, M., Brun, R., Abdalla, W., Schmidt, T., 2009. The antiprotozoal activity of sixteen Asteraceae species native to Sudan and bioactivity-guided isolation of Xanthanolides from Xanthium brasilicum. Planta Medica 75, 1363–1368. Orwa, C.A., Mutua, R., Kindt, R., Jamnadass, R., Simons, A.J., 2009. Agroforestry Tree Database: A Tree Reference and Selection Guide. Version 4.0, Nairobi, Kenya. Prasad, N., Divakar, S., Shivamurthy, G., Aradhya, S., 2005. Isolation of a free radical scavenging antioxidant from water spinach (Ipomoea aquatica Forsk). Journal of the Science of Food and Agriculture 85, 1461–1468. Rangkadilok, N., Sitthimonchai, S., Worasuttayangkurn, L., Mahidol, C., Ruchirawat, M., Satayavivad, J., 2007. Evaluation of free radical scavenging and antityrosinase

15

activities of standardized longan fruit extract. Food and Chemical Toxicology 45, 328–336. Robards, K., Prenzler, P., Tucker, G., Swatsitang, P., Glover, W., 1999. Phenolic compounds and their role in oxidative processes in fruits. Food Chemistry 66, 401–436. Robb, D., 1984. In: Lontie, R. (Ed.), “Tyrosinase” Copper Proteins and Copper Enzymes. CRC Press, Bora raton, Fla, USA, pp. 207–241. Salem, M., Davidorf, F., Abdel-Rahman, M., 2011. In vitro anti-uveal melanoma activity of phenolic compounds from the Egyptian medicinal plant Acacia nilotica. Fitoterapia 82, 1279–1284. Sánchez-Ferrer, A., Rodrígez-López, J., García-Carmona, F., 1995. Tyrosinase: a comprehensive review of its mechanism. Biochimica et Biophysica Acta 1247, 1–11. Sato, K., Toriyama, M., 2009. Depigmenting effect of catechins. Molecules 14, 4425–4432. Siddiqui, S., Patil, M., 2015. Assessment of antioxidant and cytotoxic activities of extracts of some Ziziphus species with identification of bioactive components. European Journal of Medicinal Plants 8, 202–213. Song, T., Chen, C., Yang, N., Fu, C., Chang, Y., Chen, C., 2009. The correlation of in vitro mushroom tyrosinase activity with cellular tyrosinase activity and melanin formation in melanoma cells A2058. Journal of Food and Drug Analysis 17, 156–162. Sulaiman, C.T., Gopalakrishnan, V.K., Balachandran, I., 2014. Phenolic compounds and antioxidant properties of selected Acacia species. Journal of Biologically Active Products from Nature 4, 316–324. Tachakittirungrod, S., Okonogi, S., Chowwanapoonpohn, S., 2007. Study on antioxidant activity of certain plants in Thailand: mechanism of antioxidant action of guava leaf extract. Food Chemistry 103, 381–388. Tomita, K., Oda, N., Ohbayashi, M., Kamei, H., Miyaki, T., Oki, T., 1990. A new screening method for melanin biosynthesis inhibitors using Streptomyces bikiniensis. Journal of Antibiotics (Tokyo) 43, 1601–1605. Van Wyk, B.E., Van Oudtshoorn, B., Gericke, N., 1997. Medicinal Plants of South Africa. first ed. Briza Publications, Pretoria, South Africa. Wang, C., Ng, C., Lin, H., Shyu, T., 2011. Free radical-scavenging and tyrosinase-inhibiting activities of extracts from sorghum distillery residue. Journal of Bioscience and Bioengineering 111, 554–556. Wood, J., Schallreuter, K., 1991. Studies on the reactions between human tyrosinase, superoxide anion, hydrogen peroxide and thiols. Biochimica et Biophysica Acta 1074, 378–385. Yasui, H., Sakurai, H., 2003. Age-dependent generation of reactive oxygen species in the skin of live hairless rats exposed to UVA light. Experimental Dermatology 12, 655–661. Yen, G., Chen, H., 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agricultural and Food Chemistry 43, 27–32.