Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 40-43
Published Online November 2013 (http://www.scirp.org/journal/jcdsa)
http://dx.doi.org/10.4236/jcdsa.2013.33A2010
Open Access JCDSA
Melanin Biosynthesis Inhibitory Activity of Compounds
Isolated from Unused Parts of Ammi visinaga
Ahmed Ashour1,2, Saleh El-Sharkawy2,3, Mohamed Amer2, Fatma Abdel Bar2, Ryuichiro Kondo1,
Kuniyoshi Shimizu1*
1Department of Agro-Environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan; 2Department of Pharma-
cognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt; 3Department of Pharmacognosy, Faculty of Pharmacy, Delta
University for Science and Technology, Mansoura, Egypt.
Received October 17th, 2013; revised November 10th, 2013; accepted November 17th, 2013
Copyright © 2013 Ahmed Ashour et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Ten compounds have been isolated from the unused parts of Ammi visinaga. The isolated compounds were identified as
tetracosanoic acid (1), β-sitosterol (2), visnadine (3), khellin (4), β-sitosterol glucoside (5), norkhellol (6), khellol (7),
rhamnazin (8), cimifugin (9), and cis-khellactone-3’-β-glucopyranoside (10). The chemical structures of these com-
pounds were elucidated based on spectroscopic data (NMR, UV, MS and IR spectra). This is the first report on the
identification of tetracosanoic acid (1), norkhellol (6) and cimifugin (9) in the Ammi genus. The melanin biosynthesis
inhibitory activities of khellin (4), khellol (7), visnadine (3), cimifugin (9), β-sitosterol (2) and β-sitosterol glucoside (5)
were evaluated. Khellin (4) exhibited a potent melanin inhibitory effect compared to arbutin with less toxicity.
Keywords: Ammi visinaga; Khellin; Melanin
1. Introduction
Natural products derived from plant sources have been
used extensively in traditional medicine for treatment of
a myriad of diseases including various types of cancers
[1]. Further evidence of the importance of natural prod-
ucts is provided by the fact that close to half of the best
selling pharmaceuticals in 1991 were either natural prod-
ucts or their derivatives [2].
A. visinaga is a perennial herb widely distributed in
the Mediterranean area. Among Egyptians people, it is
called Khilla, Chellah or Kella, while in Europe the plant
has often been referred to as the Toothpick Herb or
Bishop’s weed [3]. Turkish people referred to this plant
as “disotu”, “kilir”, and “hiltan” [4].
A. visinaga extracts have demonstrated to have a broad
range of therapeutic effects such as antihyperglycemic
[5], vasodilator effect [6], anti-inflammatory [7], and in-
hibition of oxalate nephrlithiasis [8].
Previous phytochemical studies on Ammi genus have
reported the presence of naphthoquinones, naphtopyranes,
steroid, triterpene and flavanoid [4,9-11]. Mostly these
phytochemical studies were done on A. visinaga fruit
which is the official part of the plant, however little phy-
tochemical studies were found concerning other parts of
the plant, so this research was conducted to isolate the
chemical constituents of the unused parts of A. visinaga
and evaluate its potential use in pharmacy and medicine.
2. Material and Methods
2.1. Reagents
NaOH and DMSO were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). The 3-(4,5-
dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) from Sigma (St. Louis, MO), EMEM from Nissui
Chemical Co (Osaka, Japan). Other chemicals are of the
highest grade commercially available.
2.2. Plant Material
A visinaga waste represented by all the aerial parts ex-
cept the fruit was collected in May 2011 from crops
grown at Faculty of Pharmacy fields. The plant was iden-
tified by Prof. Ibrahim Mashaly, Systematic Botany De-
*Corresponding author.

Melanin Biosynthesis Inhibitory Activity of Compounds Isolated from Unused Parts of Ammi visinaga 41
partment, Faculty of Sciences, Mansoura University. A
voucher specimen (No. 865) is kept in Pharmacognosy
Department, Faculty of Pharmacy, Mansoura University.
2.3. Extraction and Isolation Procedures
Dried powdered plant (2.5 kg) was percolated with
MeOH till exhaustion at room temperature. The com-
bined extracts were collected and evaporated to dryness
under reduced pressure at 40˚C. The residue, 273 g, was
suspended in distilled water and extracted successively
with pet. ether, methylene chloride and EtOAc. The dif-
ferent extracts were evaporated under reduced pressure to
obtain pet. ether fraction (fraction A, 48 g), methylene
chloride fraction (fraction B, 50 g) and EtOAc fraction
(fraction C, 10 g).
2.4. Isolation of Compounds
Fraction A was dissolved totally in the smallest volume
of methylene chloride (75 mL) and then mixed well with
about 30 g silica gel and left at room temperature to dry
and applied onto the top of a silica gel packed glass
column (65 × 4.5 cm, 420 g), previously packed in pe-
troleum ether. Elution started with petroleum ether (2 L),
then using petroleum ether/ethyl acetate [2%/98% (7 L),
3/97 (4 L), 5/95 (7.5 L), 9/91 (1.5 L), 11/89 (1.5 L),
15/85 (1.5 L), 20/80 (3 L), 25/75 (0.5 L), 30/70 (3.5 L),
35/65 (6 L), 40/60 (1 L), 60/40 (4 L)] and finally washed
with 100% ethyl acetate. Effluents, 200 mL fraction each,
were separately concentrated, monitored by TLC plates
in solvent system 5% - 40% v/v petroleum ether/ethyl
acetate, and the developed TLC were heated after spray-
ing with vanillin/sulfuric acid spray reagent. Similar frac-
tions were pooled in order to be subjected for further
chromatographic separation and purification. These frac-
tions are purified by chromatographic and repeated crys-
tallization methods to afford tetracosanoic acid (1, 12
mg), β-sitosterol (2, 750 mg), visnadine (3, 500 mg),
khellin (4, 800 mg) and β-sitosterol glucoside (5, 270
mg).
Fraction B was dissolved totally in the smallest vol-
ume of methanol and then mixed well with about 30 g
silica gel and left at room temperature to dry and applied
onto the top of a silica gel packed glass column (70 × 4.5
cm, 450 g), previously packed in petroleum ether and
developed by gradient elution using petroleum ether/
ethyl acetate [2%/98% (1.5 L), 5/95 (2 L), 10/90 (2 L),
20/80 (7 L), 25/75 (1 L), 30/70 (8 L), 35/65 (2 L), 40/60
(1.5 L), 50/50 (6 L), 60/40 (3 L), 80/20 (0.5 L), 100/0 (4
L)], then the elution was continued using methanol/ethyl
acetate [5%/95% (4 L), 20/80 (1 L), 35/65 (1 L)] and
finally washed with 100% methanol. The effluents, 200
mL fraction each, were separately concentrated, moni-
tored by TLC plates in solvent system 20% - 90% ethyl
acetate/petroleum ether, and the developed TLC were
heated with vanillin/sulfuric spray reagent. Similar frac-
tions were pooled, concentrated and subjected to chro-
matographic separation and purification. These fractions
are purified by chromatographic and repeated crystalliza-
tion methods to afford norkhellol (6, 7 mg), khellol (7, 14
mg), rhamnazin (8, 6 mg), cimifugin (9, 9 mg) and
cis-khellactone-3’-β-glucopyranoside (10, 9 mg).
Fraction C was dissolved totally in the smallest vol-
ume of methanol and then mixed well with about 9 g
silica gel and left at room temperature to dry and applied
onto the top of a silica gel packed glass column (60 × 4.5
cm, 325 g), previously packed in petroleum ether and
developed by gradient elution using petroleum ether/
ethyl acetate [10%/90% (2.5 L), 15/85 (2 L), 25/75 (3 L),
30/70 (2 L), 35/65 (3 L), 40/60 (1 L), 50/50 (2 L), 60/40
(2 L), 70/30 (1 L), 80/20 (1 L), 90/10 (1 L), 100/0 (0.5
L)], then the elution was continued using methanol/ethyl
acetate [5%/95% (0.5 L), 10/90 (2 L), 30/70 (1.5 L),
50/50 (2 L), 70/30 (1 L)] and finally washed with 100%
methanol. The effluents, 200 mL fraction each, were
separately concentrated, monitored by TLC plates in
solvent system 30% - 90% ethyl acetate/petroleum ether,
and the developed TLC were heated with vanillin/sulfu-
ric spray reagent. Similar fractions were pooled, concen-
trated and subjected to chromatographic separation and
purification. These fractions are purified by chroma-
tographic and repeated crystallization methods to afford
visnadine (3, 14 mg), khellol (7, 2 mg) which were pre-
viously isolated from other fractions.
2.5. Cell Line
A mouse melanoma cell line, B16, was obtained from
RIKEN Cell Bank. The cells were maintained in EMEM
supplemented with 10% (v/v) fetal bovine serum (FBS)
and 0.09 mg/mL theophylline. The cells were incubated
at 37˚C in a humidified atmosphere of 5% CO2.
2.6. B16 Melanoma Cell Line Assay
This assay was determined as described previously [12].
The cells were placed in two plates of 24-well plastic
culture plates (one plate for determining melanin and the
other for cell viability) at a density of 1 × 105 cells/well
and incubated for 24 h in media prior to being treated
with the samples. After 24 h, the media were replaced
with 998 µL of fresh media and 2 µL of the test sample
at maximum solubility (n = 3). At the same time, nega-
tive control (2 µL DMSO) and positive control; Arbutin
at concentration 50 mg/mL in DMSO were tested. The
cells were incubated for an additional 48 h, and then the
medium was replaced with fresh medium containing each
sample. After 24 h, the remaining adherent cells were
assayed. To determine the melanin content (for one plate)
Open Access JCDSA

Melanin Biosynthesis Inhibitory Activity of Compounds Isolated from Unused Parts of Ammi visinaga
Open Access JCDSA
42
Biological Activity after removing the medium and washing the cells with
PBS, the cell pellet was dissolved in 1.0 mL of 1 N
NaOH. After overnight keeping in dark, the crude cell
extracts were assayed by using a microplate reader at 405
nm to determine the melanin content. The results from
the cells treated with the test samples were analyzed as a
percentage of the results from the control culture. On the
other hand, cell viability was determined by using MTT
assay which provides a quantitative measure of the num-
ber of viable cells by determining the amount of forma-
zan crystals produced by metabolic activity in treated
versus control cells. So, for the other well plate, 50 µL of
MTT reagent in PBS (5 mg/mL) was added to each well.
The plates were incubated in a humidified atmosphere of
5% of CO2 at 37˚C for 4 h. After the medium was re-
moved, 1.0 mL isopropyl alcohol (containing 0.04 N HCl)
was added, and the absorbance was measured at 570 nm
after overnight keeping in dark.
In ancient Egypt, vitiligo lesions were treated with ex-
tracts of the Ammi genus plant followed by exposure to
the sun whereby UVA irradiations are given 2 h after
administration of 8-methoxypsoralen, a photosensitizer
[19]. In present study, we evaluate anti melanogenesis
property of β-sitosterol (2), visnadine (3), khellin (4),
β-sitosterol glucoside (5), khellol (7) and cimifugin (9).
The yield of other compounds is very low having no fur-
ther amounts for biological evaluation. These compounds
were assayed by using B16 melanoma cells in order to
evaluate the inhibition of melanin formation and cell
viability at their maximum solubility. For khellin and
β-sitosterol, the maximum solubility was 20 μg/mL while
for other compounds the maximum solubility was 40
μg/mL. In Figure 2, the inhibition of these compounds
on melanin formation in B16 melenoma cells was shown
at various concentrations.
Taking into consideration of the cytotoxicity to cell
lines, the most active compound exhibiting melanin syn-
thesis inhibition (~37%) and at the same time with low
cytotoxicity (~1%) was khellin (4) at 20 µg/mL, followed
by khellol (7) (~28% inhibition) but with a toxic effect to
some extent (~20% cytotoxicity). Visnadine (3) at con-
centration of 40 and 20 μg/mL showed cytotoxicity on
B16 melenoma cells rather than melanin formation inhi-
bition, but at a concentration of 10 μg/mL, it showed
about 8% melanin inhibition with less toxicity. Also ci-
3. Results and Discussion
Ten compounds have been isolated from the aerial parts
(except fruit) of A. visinaga. They were identified by
comparative study to those cited in the literature [13-18].
These compounds (Figure 1) are tetracosanoic acid (1),
β-sitosterol (2), visnadine (3), khelllin (4), β-sitosterol
glucoside (5), norkhellol (6), khellol (7), rhmnazin (8),
cimifugin (9) and cis-khellactone-3’-β-glucopyranoside
(10).
COOH
HO H
H
H
H
12 4 35
O
HO
HO OH
OH
H
H
H
H
O
O OO
OCOCH3
OCOCHCH3CH2CH3
OO
O
OCH3
OCH3
H3C
76 89 10
OO
OOH
HOH2CO O
OOCH3
HOH2COO
OOCH3
HOH2C
CH3
OH
OH3CO
OH O
OH
O OO
H3C
H3CO
OH
O
HO
HO
OH
OH
CH3
OH
OCH3
Figure 1. Chemical structures of isolated compounds.
Figure 2. Chemical structures of isolated compounds.

Melanin Biosynthesis Inhibitory Activity of Compounds Isolated from Unused Parts of Ammi visinaga 43
mifugin (9) at concentration of 40 μg/mL showed cyto-
toxicity on B16 melenoma cells rather than melanin for-
mation inhibition; β-sitosterol (2) and β-sitosterol gluco-
side (5) had no effect on melanin inhibition.
Previously, khellin was reported to have some biologi-
cal effects such as smooth muscle relaxant [20] and pre-
vention of stone formation associated with hyperoxaluria
[8], while in our study we concluded that khellin (4) is a
promising compound which could be useful for treating
hyperpigmentation, as a skin-whitening agent with less
toxicity even than the standard drug arbutin. To the best
of our knowledge, we obtained this compound from the
waste of A. visinaga which caused a serious problem so
we can get a great beneficial from these waste.
4. Acknowledgements
The Egyptian Government is acknowledged for the fel-
lowship support to Ahmed Adel Ashour. We are thankful
to Dr. Miyamoto for optical rotation measurement.
REFERENCES
[1] M. S. Butler, “The Role of Natural Product Chemistry in
Drug Discovery,” Journal of Natural Product, Vol. 67,
No. 12, 2004, pp. 2141-2153.
http://dx.doi.org/10.1021/np040106y
[2] G. M. Cragg, D. J. Newman and K. M. Snader, “Natural
Products in Drug Discovery and Development,” Journal
of Natural Product, Vol. 60, No. 1, 1997, pp. 52-60.
http://dx.doi.org/10.1021/np9604893
[3] F. K. El-Fiky, R. P. Remmel and E. J. Staba, “Ammi
Visinaga: Somatic Embryo Induction and Furanochrom-
mone Production in Embryos, Seedlings and Plants,”
Planta Medica, Vol. 55, No. 5, 1989, pp. 446-451.
http://dx.doi.org/10.1055/s-2006-962061
[4] K. Günaydin and N. Beyazit, “The Chemical Investiga-
tion on the Ripe Fruits of Ammi visinaga (Lam.) Lamarck
Growing in Turkey,” Natural Product Research, Vol. 18,
No. 2, 2004, pp. 169-175.
http://dx.doi.org/10.1080/14786410310001608091
[5] H. Jouad, M. Maghrani and M. Eddouks, “Hypoglycemic
Effect of Aqueous Extract of Ammi visnaga in Normal
and Streptozotocin-Induced Diabetic Rats,” Journal of
Herbal Pharmcotherapy, Vol. 2, No. 4, 2002, pp. 19-29.
http://dx.doi.org/10.1080/J157v02n04_03
[6] J. Duarte, F. Perez-Vizcaino, A. Torres, A. Zarzuelo, J.
Jimenez and J. Tamargo, “Vasodilator Effects of Vis-
nagin in Isolated Rat Vascular Smooth Muscle,” Euro-
pean Journal of Pharmacology, Vol. 286, No. 2, 1995, pp.
115-122.
http://dx.doi.org/10.1016/0014-2999(95)00418-K
[7] J. Lee, J. Jung, S. Park, S. Park, Y. Sim, S. Kim, T. Ha
and H. Suh, “Anti-Inflammatory Effect of Visnagin in
Lipopolysaccharide-Stimulated BV-2 Microglial Cells,”
Archives of Pharmacal Research, Vol. 33, No. 11, 2010,
pp. 1843-1850.
http://dx.doi.org/10.1007/s12272-010-1117-1
[8] P. Vanachayangkul, K. Byer, S. Khan and V. Butterweck,
“An Aqueous Extract of Ammi visnaga Fruits and Its
Constituents Khellin and Visnagin Prevent Cell Damage
Caused by Oxalate in Renal Epithelial Cells,” Phytomedi-
cine, Vol. 17, No. 8-9, 2010, pp. 653-658.
http://dx.doi.org/10.1016/j.phymed.2009.10.011
[9] M. Antunes, S. Pina Dos Santos and F. Brito, “Coumarins
from Ammi huntii,” Colloques—Institut National de la
Recherche Agronomique, Vol. 69, 1995, pp. 305-306.
[10] Y. Selim and N. Ouf, “Anti-Inflammatory New Coumarin
from the Ammi majus L.,” Organic and Medicinal Chem-
istry Letter, Vol. 2, 2012, pp. 1-4.
http://dx.doi.org/10.1186/2191-2858-2-1
[11] R. Bencheraiet, H. Kherrab, A. Kabouche, Z. Kabouche
and M. Jay, “Flavonols and Antioxidant Activity of Ammi
visnaga L. (Apiaceae),” Records of Natural Products,
Vol. 5, No. 1, 2011, pp. 52-55.
[12] E. T. Arung, K. Shimizu and R. Kondo, “Structure Activ-
ity Relationship of Phenyl Substituted Polyphenols from
Artocarpus heterophyllus as Inhibitors of Melanin Bio-
synthesis in Cultured Melanoma Cell,” Chemistry & Bio-
diversity, Vol. 4, No. 9, 2007, pp. 2166-2171.
http://dx.doi.org/10.1002/cbdv.200790173
[13] M. M. Amer, S. H. El-Sharkawy, F. M. Abdel Bar and A.
A. Ashour, “Phytochemical Investigation of Unused Parts
of Hibiscus sabdariffa,” Journal of American Science,
Vol. 8, No. 12, 2012, pp. 29-35.
[14] Y. Ikeshiro, I. Mase and Y. Tomita, “Dihydropyrano-
coumarins from Roots of Peucedanum japonicum,” Phy-
tochemistry, Vol. 31, No. 12, 1992, pp. 4303-4306.
http://dx.doi.org/10.1016/0031-9422(92)80463-O
[15] A. Abou-Mustafa, M. Saleh, A. Elgamal, M. Shalaby and
H. Duddeck, “A Further Contribution to the γ-Pyrone Con-
stituents of Ammi visnaga Fruits,” Planta Medica, Vol. 56,
No. 1, 1990, p. 134.
http://dx.doi.org/10.1055/s-2006-960912
[16] P. Agrawal, R. Thakur and M. Bansal, “Carbon-13 NMR
of Flavonoids,” Elsevier Science Publishing Company
Inc., New York, 1989.
[17] H. Sonnenberg, M. Kaloga, N. Eisenbach and K. Froem-
ming, “Isolation and Characterization of an Angular-Type
Dihydropyranocoumaringlycoside from the Fruits of Ammi
visnaga (L.) LAM. (Apiaceae),” Zeitschrift fur Naturfor-
schung C: Journal of Biosciences, Vol. 50, No. 9-10,
1995, pp. 729-731.
[18] P. Cao, X. Pu, S. Peng, X. Zhang and L. Ding, “Chemical
Constituents from Cimicifuga foetida,” Journal of Asian
Natural Products Research, Vol. 7, No. 2, 2005, pp. 145-
149. http://dx.doi.org/10.1080/1028602042000204081
[19] R. Roelandts, “Photo (chemo) Therapy for Vitiligo,” Photo-
dermatology, Vol. 19, No. 1, 2003, pp. 1-4.
http://dx.doi.org/10.1034/j.1600-0781.2003.00003.x
[20] R. Kory, A. Townes, R. Mabe, E. Dorris and G. Meneely,
“Drug Evaluation in Angina Pectoris: Khellin, Heparin,
Peritrate,” American Heart Journal, Vol. 50, No. 2, 1955,
pp. 308-321.
http://dx.doi.org/10.1016/0002-8703(55)90326-4
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