Melanin Biosynthesis Inhibitory Activity of Compounds Isolated from Unused Parts of <i>Ammi visinaga</i>

doi:10.4236/jcdsa.2013.33A2010

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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

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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.

Email: *[email protected]

Received October 17th, 2013; revised November 10th, 2013; accepted November 17th, 2013

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)

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Melanin Biosynthesis Inhibitory Activity of Compounds Isolated from Unused Parts of Ammi visinaga

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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

12 4

HO OH

O OO

OCOCH3

OCOCHCH3CH2CH3

OCH3

H3C

76 89 10

OOH

HOH2CO O

OOCH3

HOH2COO

OOCH3

HOH2C

CH3

OH3CO

OH O

O OO

H3C

H3CO

CH3

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.

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