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Screening for melanogenesis-controlled agents using Sudanese medicinal plants and identification of active compounds in the methanol extract of Terminalia brownii bark


We isolated and identified compounds in medicinal plant extracts that could control melanogenesis. Sudanese medicinal plants were extracted with methanol (MeOH) and 50 % ethanol (EtOH)/water, yielding 104 extracts that were screened for melanogenic activity using B16 melanoma cells. The MeOH extract of Terminalia brownii bark dose-dependently enhanced intracellular and extracellular melanogenesis, with no cytotoxicity. Furthermore, we isolated and identified the components in T. brownii MeOH extract. Gallic acid (1), α,β-punicalagin (2), α,β-terchebulin (3), ellagic acid 4-O-α-l-rhamnopyranoside (4), ellagic acid (5), and 3,4,3′-tri-O-methylellagic acid (6) were isolated by chromatography and identified using nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization (MALDI) or ultra-performance liquid chromatography–time-of-flight mass spectrometry (UPLC–TOFMS), and ultraviolet (UV) spectroscopy data. Among the isolated compounds, 2, 3, 5, and 6 enhanced melanogenesis. Furthermore, compound 1 inhibited intracellular and extracellular melanogenesis with no cytotoxicity.


Melanin, a pigment that is synthesized from tyrosine by tyrosinase-mediated enzymatic oxidation, is widely distributed in the body surface, retina, nigra of the brain, and adrenal medullae. The primary function of melanin is to prevent skin cancer by protecting cells from ultraviolet (UV) rays [1, 2]. However, melanin production sometimes causes problems for beauty and health. For instance, decreased melanin production due to aging or stress causes gray hair. Furthermore, excess melanin production causes sunburn and mottle. Thus, control of melanogenesis is strongly desired. Melanin is synthesized in unique cells, called melanocytes, using l-tyrosine as the starting material. The key enzyme in melanin biosynthesis is tyrosinase, which contains copper and catalyzes two reactions [3]. The first step of melanin biosynthesis is the hydroxylation of l-tyrosine to l-DOPA, which is followed by the oxidation of l-DOPA to l-DOPA quinine, which produces the red-orange coloration of pheomelanin and blackish brown coloration of eumelanin [4].

Melanosomes are transported in melanocytes and transferred to keratinocytes. Interestingly, extracellular melanin elicits the skin pigmentation. Nevertheless, most reports evaluating compounds that modulate melanogenesis only determine changes in intracellular melanin. In this paper, we determined both extracellular and intracellular melanin content to identify compounds in Sudanese medicinal plants that regulate melanogenesis.

Sudanese medicinal plants have immunomodulatory [5], anti-bacterial [6, 7], and anti-malarial activity [8]. However, the effect of Sudanese medicinal plant extracts on melanogenesis remains unclear. Here we screened 104 extracts from Sudanese medicinal plants to evaluate their effects on melanogenesis. Furthermore, the active compounds in the methanol (MeOH) extracts of Terminalia brownii Fresen (Combretacae) bark, which is widely used in traditional medicine to treat bacterial, fungal, and viral infections [9], were isolated and identified. We found that these compounds modulate melanogenesis.

Materials and methods

General experimental procedures

1H and 13C nuclear magnetic resonance (NMR) spectra were recorded in methanol-d 4 or dimethyl sulfoxide (DMSO)-d 6 with a JEOL EC600 MHz NMR (Tokyo, Japan). Ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS, WatersXevo™ QTof MS, Waters, Milford, MA, USA) was performed using a C18 column (2.1 × 100 mm, Waters). The UPLC–TOFMS data were collected in negative ionization mode. The capillary voltage was 3.0 kV. Cone and desolvation gas flow rates were set at 50 and 1000 L/h, respectively. The source and desolvation temperatures were 150 and 500 °C, respectively. Matrix-assisted laser desorption/ionization (MALDI)-TOFMS spectra were measured on an SHIMADZU AXIMA-Resonance spectrometer (Kyoto, Japan) equipped with a nitrogen laser (λ = 337 nm). The samples were mixed with matrices [2,5-dihydroxy benzoic acid (DHB) in 30 % acetonitrile, 10 mg/ml] and loaded onto a 384-well MALDI sample plate. Preparative high-performance liquid chromatography (HPLC, SHIMADZU LC-6AD) was performed using an Inertsil ODS-3 column (20 × 250 mm, GL Sciences Inc., Tokyo, Japan). Analytical HPLC (SHIMAZU SIL-20A) was performed using an Inertsil ODS-3V column (4.6 × 250 mm, GL Sciences Inc., Tokyo, Japan). Commercially available products were purchased from Wako Chemical (Osaka, Japan). UV spectra were recorded on a Shimadzu SPD-M20A diode array detector.


Plants were collected from Khartoum and Gadarif states in Sudan in March 2011. Voucher specimens are deposited in the Horticultural Laboratory, Department of Horticulture, Faculty of Agriculture, University of Khartoum. A list of voucher numbers and ethnomedical uses of the investigated species are shown in Table 1.

Table 1 The intracellular melanogenesis activity and cell viability of extracts (100 μg/ml) from Sudanese medicinal plants

Extraction of plant materials

Extraction of plant materials were performed as previously described [6].

Isolation of compounds from T. brownii MeOH extract

T. brownii bark MeOH extract was separated with medium pressure chromatography eluting [ODS-25 (40 × 200 mm) water:MeOH = 95:5 (30 min), 80:20 (60 min), 70:30 (90 min), 40:60 (120 min), 0:100 (150 min), flow rate: 5 ml/min] to obtain fractions (Fr) 1, 2, 3, and 4. Fr. 1 was separated by Sephadex LH-20 gel column chromatography, eluting [LH20 (30 × 430 mm) MeOH 100 % (3 h), acetone:water 70:30 (3 h), flow rate: 0.5 ml/min] to obtain Fr. 1–1, 1–2, 1–3, and 1–4. Compound 1 was isolated from Fr.1–1, 2 was isolated from Fr.1–3, 3 was isolated from Fr.1–4, and 4, 5, and 6 were isolated from Fr. 2, 3, and 4, respectively using preparative HPLC [Colum: ODS-3 (20 × 250 mm), flow rate: 9 ml/min, wavelength: 254 nm, gradient program: MeOH:0.05 % TFA aqueous solution = 20:80 (0 min), 20:80 (20 min), 50:50 (50 min), 100/0 (60 min)]. The yields of 16 were 8.1, 4.3, 2.6, 4.9, 2.8, and 1.2 mg, respectively, against 291 g of T. brownii bark powder. The purity was confirmed using analytical HPLC [Colum: ODS-3V (20 × 250 mm), flow rate: 1 ml/min, wavelength: 256 nm, gradient program: MeOH: 0.05 % TFA aqueous solution = 5:95 (0 min), 10:90 (5–10 min), 20:80 (10–20 min), 50:50 (35 min), 100:0 (45–55 min)].

Identification of isolated compounds from T. brownii bark MeOH extracts

The isolated compounds from T. brownii bark MeOH extract were identified using the NMR, MALDI or UPLC-TOFMS, and UV spectrometry data. Gallic acid (1): white powder; UV λ max: 195, 213, 270 nm; UPLC-TOFMS: m/z 169.011 [M-1]. α,β-Punicalagin (2): pale green powder; UV λ max: 212, 253, 372 nm; MALDI-TOFMS: m/z 1107.900 [M + Na]+. α,β-Terchebulin (3): pale green powder; UV λ max: 200, 253, 379 nm; MALDI-TOFMS: m/z 1107.878 [M + Na]+. Ellagic acid 4-O-α-l-rhamnopyranoside (4): pale yellow powder; UV λ max: 210, 253, 360 nm; UPLC-TOFMS ES: m/z 447.059 [M-H]. Ellagic acid (5): pale yellow powder; UV λ max: 211, 253, 367 nm; UPLC-TOFMS ES: m/z 301.0381 [M-H]. 3,4,3′-tri-O-methylellagic acid (6): pale yellow powder; UV λ max: 199, 247, 372 nm; UPLC-TOFMS: m/z 343.0429 [M-H]. NMR data for these compounds were completely the same to previous reports [1017].

Tyrosinase activity assay

Tyrosinase activity was assayed based on protocols by Yamauchi et al. [18]. The sample (60 μl) was placed in a 96-well plate. Mushroom tyrosinase (30 μl, 333 U/ml in 50 mM phosphate buffer, pH 6.5) and substrate [110 μl l-tyrosine (2 mM) or l-DOPA (2 mM)] were added. After incubation at 37 °C for 30 min, the absorbance at 510 nm was measured using a microplate reader. Each experiment was repeated twice. The tyrosinase activity was expressed as a percentage of the control treated with the solvent DMSO/water, without samples.

Cell culture

Murine melanoma B16-F0 cells (DS Pharma Biomedical, Osaka, Japan) were grown in Dulbecco’s modified Eagle’s medium (DMEM) without phenol red, supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin. Cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO2.

Measurement of cellular melanin content

Confluent B16 melanoma cells were rinsed in phosphate-buffered saline (PBS) and removed using 0.25 % trypsin/EDTA. Cells were placed in 24-well plates (0.5 × 105 cells/well) and allowed to adhere at 37 °C for 24 h. After adding samples, cells were incubated for 72 h, and 150 μl medium was placed in a 96-well plate. The absorbance of the medium was measured at 510 nm with a microplate reader (Biotec. Immuno Mini NJ-2300, Tokyo, Japan). The cells were washed with PBS, lysed in 600 μl NaOH (1 M), and heated for 15 min at 100 °C to solubilize the melanin. An aliquot of the resulting lysate (250 μl) was placed in a 96-well microplate, and the absorbance was measured at 405 nm with a microplate reader. Each experiment was repeated twice. Melanin production was expressed as a percentage of the control cells treated with the solvent DMSO/water without sample.

Cell viability

Cell viability was measured according to a previously reported method [19], using a micro-culture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) technique. Cells (0.5 × 105 per well) were grown in 24-well plates. After incubation, 50 μl MTT reagent (5 mg/ml) was added to each well. The plates were incubated in a humidified atmosphere at 37 °C for 4 h. After the medium was removed, 1.0 ml isopropyl alcohol containing 0.04 N HCl was added to the plate. An aliquot (150 μl) was placed in a 96-well plate, and the absorbance was measured at 590 nm with a microplate reader. Each experiment was repeated twice. The cell viability was expressed as a percentage of the control cells treated with the solvent DMSO/water without sample.

Statistical analysis

All data in Table 2 were expressed as the mean ± SD. Differences were examined for statistical significance using the Student’s t test. The data in Table 1 were expressed as the mean.

Table 2 Tyrosinase activity, cell viability and intracellular and extracellular melanogenesis activity of Terminalia brownii bark MeOH extract and its isolated compounds [gallic acid (1), α,β-punicalagin (2), α,β-terchebulin (3), ellagic acid 4-O-α-l-rhamnopyranoside (4), ellagic acid (5), 3,4,3′-tri-O-methylellagic acid (6)] using B16 melanoma cells

Result and discussion

Screening of Sudanese medicinal plant extracts

Sudanese medicinal plants were extracted with MeOH and 50 % EtOH. The effect of the 104 obtained extracts on melanogenesis was evaluated in B16 melanoma cells. As shown in Table 1, MeOH extracts of Xanthium brasilicum leaves, Guiera senegalensis J. F. Gmel, Balanites aegyptiaca bark, T. brownii bark, and Haplophyllum tuberculatum aerial parts enhanced melanogenesis by 207, 179, 163, 211, and 187 %, respectively. Among these, MeOH extracts from T. brownii bark had the greatest effect on intracellular melanogenesis, with no observed cytotoxicity. These result indicated that MeOH extracts of T. brownii bark should be further investigated.

Isolation and identification T. brownii bark MeOH extract compounds

Compounds 16 were isolated from T. brownii by a series of chromatographic experiments, and were identified as gallic acid (1), punicalagin (2), terchebulin (3), ellagic acid 4-O-α-l-rhamnopyranoside (4), ellagic acid (5), and 3,4,3′-tri-O-methylellagic acid (6) by comparing the NMR, MS, and UV spectrum data (Fig. 1) [1017]. Among the isolated compounds, 2 and 3 were identified as α/β(3/2)-punicalagin (2) and α/β(3/2)-terchebulin (3). These compounds existed as an equilibrium mixture of the α- and β-forms.

Fig. 1

Structures of isolated compounds from Terminalia brownii MeOH extract

Effects of isolated compounds on melanogenesis and tyrosinase activity

The effect of the MeOH extract from T. brownii bark at 200–50 μg/ml and isolated compounds on intracellular and extracellular melanogenesis and cell viability was evaluated (Table 2). Compound 2 dose-dependently enhanced intracellular melanogenesis; however, it was cytotoxic at 46 and 92 μM. In contrast, compound 3 dose-dependently enhanced extracellular melanogenesis, with less cytotoxicity than compound 2, even though these compounds had similar chemical structures. The structure differed between the two compounds at the D2 position. In the case of compound 2, C-D2 was attached to C-5, forming a C–C bond. In contrast, in compound 3, C-D2 and C-4 were bound via an oxygen, which has higher flexibility than a C–C bond. The difference in flexibility between compounds may cause the difference in cell viability and melanogenesis.

Compounds 4, 5, 6 were ellagic acid and its derivatives. Compound 4, an ellagic acid rhamnoside, had no effect on melanogenesis, whereas 5 and 6 stimulated melanogenesis. Compound 5 dose-dependently increased intracellular melanogenesis with no cytotoxicity. Furthermore, it exhibited higher activity than theophylline, which is known to stimulate melanogenesis. However, its effect on extracellular melanogenesis was less than theophylline. Compound 6, a methylated form of 5, induced cell proliferation. Intracellular melanin levels were increased in parallel with the number of melanoma cells.

Among the isolated compounds from T. brownii bark MeOH extract, compound 1 inhibited melanogenesis. In particular, extracellular melanogenesis following treatment with 100, 50, or 25 μM compound 1 was 16, 13, and 12 % of the control, respectively. This inhibitor activity was greater than arbutin, a known inhibitor of melanogenesis. Compound 1 had a lesser effect on intracellular melanogenesis, which was inhibited approximately 70 % at the same concentrations. Skin surface pigmentation is regulated by extracellular melanin. Thus, compound 1 may be a beneficial lead compound to develop whitening agents.

The effects of the isolated compounds on tyrosinase activity using l-tyrosine and l-DOPA as substrates are shown in Table 2. Compounds 2, 3, 5, and 6 stimulated melanogenesis, but did not stimulate tyrosinase activity at 200, 100, and 50 μM. Furthermore, compound 1 did not inhibit tyrosinase, despite its ability to inhibit extracellular melanogenesis in B16 melanoma cells. Thus, the effects of compounds 1, 2, 3, 5, and 6 on melanogenesis is likely not due to tyrosinase activity. Rather, they may affect tyrosinase expression. Microphthalmia-associated transcription factor (MITF), which upregulates the expression of tyrosinase, is of interest in the study of melanogenesis [2123]. The effects of compounds 1, 2, 3, 5, and 6 on melanogenesis may be associated with the expression or activity of MITF. Furthermore, compounds 1 and 3, which had a strong effect on extracellular melanin content, may alter melanin transport in B16 melanoma cells. Elucidation of the precise mechanisms of these compounds is necessary. Nevertheless, a fundamental investigation of medicinal plant extracts and their components, and their effects on intracellular and extracellular melanin content, was achieved in this study.


Our B16 melanoma cell screen revealed that the MeOH extract of T. brownii bark showed the greatest enhancement in melanogenesis among 104 extracts of Sudanese medicinal plants, with no cytotoxicity. We isolated and identified 6 compounds, and determined their effects on intracellular and extracellular melanogenesis and tyrosinase activity. Among the isolated compounds, 2, 5, and 6 enhanced intracellular melanogenesis, whereas compound 3 enhanced extracellular melanogenesis. In contrast, compound 1 decreased both extracellular and intracellular melanin content, with a stronger effect on extracellular melanin.


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Correspondence to Tohru Mitsunaga.

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Yamauchi, K., Mitsunaga, T. & Muddathir, A.M. Screening for melanogenesis-controlled agents using Sudanese medicinal plants and identification of active compounds in the methanol extract of Terminalia brownii bark. J Wood Sci 62, 285–293 (2016).

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  • Sudanese medicinal plants
  • B16 melanoma cell
  • Terminalia brownii
  • Tyrosinase