Effect of polyphenols from Vicia faba L on lipase activity and melanogenesis

Article abstract of DOI:10.1080/14786419.2017.1359169

Two new flavonoid glycosides, kaempferol 3-O-α-L-rhamnopyranosyl (1→6) (3′′-acetyl)-β-D-galactopyranoside 1 and kaempferol 3-O-α-L-arabinopyranosyl-5-O-α-L-rhamnopyranoside 2, along with six known ones 3–8 were isolated from the flowers of Vicia faba L. (Fabaceae). Methanol extract and the isolated compounds were tested against lipase and melanogenesis inhibition activities and resulted in that compound 2 showed 53 and 77% lipase inhibition activity in concentrations of 400 and 800?μg/mL, respectively. For melanogenesis, compounds 2, 3 and 4 exhibited potent melanogenesis inhibition activity where the melanin content in melanoma cells was decreased to be about 57.5, 56 and 61%, respectively, with no obvious melanocytotoxicity. The rest of compounds showed weak to moderate activity. The results of melanogenesis inhibition activity of this study suggested the potential use of Vicia faba flowers as a skin-whitening agent and reveal the flowers to be a rich source of important phytochemicals with antilipase and melanogenesis inhibitory activity.

Full text of DOI:10.1080/14786419.2017.1359169

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Effect of polyphenols from Vicia faba L on lipase
activity and melanogenesis
Ahmed E. Allam, Alaa M. Nafady, Toshinori Nakagawa, Naomichi Takemoto
& Kuniyoshi Shimizu
To cite this article: Ahmed E. Allam, Alaa M. Nafady, Toshinori Nakagawa, Naomichi Takemoto
& Kuniyoshi Shimizu (2017): Effect of polyphenols from Vicia faba L on lipase activity and
melanogenesis, Natural Product Research, DOI: 10.1080/14786419.2017.1359169
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Date: 03 August 2017, At: 17:03

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1359169
Effect of polyphenols from Vicia faba L on lipase activity and
melanogenesis
Ahmed E. Allama,b, Alaa M. Nafadyb, Toshinori Nakagawaa, Naomichi Takemotoa and
Kuniyoshi Shimizua
adivision of systematic Forest and Forest Products sciences, department of agro-environmental sciences,
Graduate school of Bioresource and Bioenvironmental sciences, Kyushu university, Fukuoka, Japan; bFaculty
of Pharmacy, department of Pharmacognosy, al-azhar university, assiut, egypt
ABSTRACT
ARTICLE HISTORY
received 28 March 2017
accepted 2 July 2017
Two new flavonoid glycosides, kaempferol 3-O-α-L-rhamnopyranosyl
(1→6) (3′′-acetyl)-β-D-galactopyranoside 1 and kaempferol 3-O-α-L-
arabinopyranosyl-5-O-α-L-rhamnopyranoside 2, along with six known
ones 3–8 were isolated from the flowers of Vicia faba L. (Fabaceae).
Methanol extract and the isolated compounds were tested against
lipase and melanogenesis inhibition activities and resulted in
that compound 2 showed 53 and 77% lipase inhibition activity in
concentrations of 400 and 800 μg/mL, respectively. For melanogenesis,
compounds 2, 3 and 4 exhibited potent melanogenesis inhibition
activity where the melanin content in melanoma cells was decreased
to be about 57.5, 56 and 61%, respectively, with no obvious
melanocytotoxicity. The rest of compounds showed weak to moderate
activity. The results of melanogenesis inhibition activity of this study
suggested the potential use of Vicia faba flowers as a skin-whitening
agent and reveal the flowers to be a rich source of important
phytochemicals with antilipase and melanogenesis inhibitory activity.
KEYWORDS
Vicia faba; flavonoid
glycosides; lipase and
melanogenesis inhibition
activities
CONTACT Kuniyoshi shimizu
shimizu@agr.kyushu-u.ac.jp
this research was conducted at Kyushu university.
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1359169.
© 2017 Informa uK limited, trading as taylor & Francis Group

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A. E. ALLAM ET AL.
1. Introduction
Vicia faba L. (broad bean) is a legume belonging to the plant family Fabaceae. It is an impor-
tant winter crop in Mediterranean areas and is mostly a spring crop in other regions of Europe
and South America and is one of the major plant food item for the Nile River populations.
(Amarowicz and Pegg 2008). It is used in folk medicine as antihyperlipidimic to control
cholesterol (Rabey et al. 1993; Mulvihill and Huff 2010; Bouchenak and Lamri-Senhadji 2013).
For this reason, we were encouraged to search for bioactive compounds that have antilipase
activity. The hydrolysis of triacylglycerols, and thus, its movement from the intestinal lumen
into the body is stopped or minimised, the prevalence of obesity can be reduced (Han et al.
2005; Sharma et al. 2005). Also, in an effort to find a new whitening agent, we examined Vicia
faba flowers and their isolated compounds with the aim of developing effective treatments
against hyperpigmentation which occurs when an excess of melanin deposits in the skin.
Common forms of hyperpigmentation are melasma, lentigo and age spots, (Briganti et al.
2003; Lim et al. 2009; Yamasaki et al. 2015). This study is the first of its kind to evaluate the
ability of Vicia faba flowers and its isolated compounds to inhibit both lipase enzyme and
melanogenesis in B16-F10 melanoma cell line.
2. Results and discussion
2.1. Structure elucidation of 1 and 2
Chemical study of the ethyl acetate fraction of V. faba afforded eight compounds (1–8)
(Figure 1) of which six were identified by comparing their physicochemical and spectroscopic
data with those reported in the literature as kaempferol 7-O-α-L-rhamnopyranoside 3, (Veit
and Pauli 1999), kaempferol 3-O-α-L-arabinopyranosyl-7-O-α-L-rhamnopyranoside
4
(Lawrence et al. 2003), kaempfrerol 3-O-rutinoside 5 (Petpiroon et al. 2015), kaempferol
3-O-α-L-rhamnopyranosyl-(1→3)-β-d-galactopyranosyl-7-O-α-L-rhamnopyranoside 6, (Xu
et al. 2009), kaempferol-3-O-β-d-galactopyranosyl-7-O-α-L-rhamnopyranoside 7, (Keyume
et al. 2006) and kaempferol 3-O-α-L-rhamnopyranosyl (1→6)-β-d-galactopyranosyl-7-O-α-
L-rhamnopyranoside 8. (Jiaju et al. 2011).
Compound 1 was obtained as a yellow amorphous powder. The HR-TOF–MS spectrum
showed a quasi-molecular ion peak at m/z 637.1245 [M + H]+, calculated as (636.17) in
Figure 1. structures of compounds 1–8.

NATURAL PRODUCT RESEARCH
3
accordance with the molecular formula C29H32O16. It exhibited (UV) absorption at 230 and
254 nm. The structure of 1 was elucidated by 1-D and 2-D NMR spectroscopy, including 1H,
13C and HMBC experiments, as well as HR-LC-TOF-MS. The 1H NMR spectrum of 1 indicated
the presence of a kaempferol moiety, two sugar moieties in addition to the presence of an
acetyl moiety where a pair of doublets each is equivalent to two protons at δH 8.06, J = 8.9 Hz.
(H-2′, H-6′) and at δH 6.89, J = 8.9 Hz. (H-3′, H-5′) is present, which are two features charac-
teristic of a flavonol with phenolic group 4′. The spectrum showed also a signal at δH 1.90,
3H, suggesting the presence of an acetyl moiety. Analysis of chemical shifts and coupling
constants in 1H spectrum are in accordance with suggestion that the sugar residue is
galactopyranosyl (Lambert et al. 1998). The anomeric protons showed characteristic doublets
in the 1H NMR spectrum at δH 5.12 for galactose with a doublet splitting of 7.2 Hz, indicating
its β-configuration, and at δH 4.51 for rhamnose, with a doublet splitting of 1.2 Hz indicating
its α-configuration (Markham et al. 1978; Hasegawa et al. 2008). Identification of the sugar
residues was supported by GC/MS analysis of the alditole acetates obtained by mild acid
hydrolysis (Leontein et al. 1978) as galactose and rhamnose. The 13C NMR spectrum was in
agreement with 3-substituted kaempferol moiety. Long-range correlations were observed
in HMBC between the anomeric proton of galactose (δH 5.12) and the C-3 of flavonol (δC
135.5), confirming that the galactose was connected at this site, and between the anomeric
proton of rhamnose (δH 4.51) and the C-6 of galactose (δC 68.6), verifying that rhamnose was
connected at that site. Another HMBC correlation was also observed between H-3 of galac-
tose (δH 3.53) and the carbonyl residue of the acetate moiety, confirming the site of its
attachment. In addition to HMBC data, the sugar sequence was also confirmed to be (1→6)
linkage from the NOESY experiment where the presence of inter-glycosidic NOE from the
anomeric proton of rhamnose moiety (δH 4.51) to both H-6 of galactose (δH 3.80 and 3.42),
which provides a very powerful means for determining the sugar sequence (Atta-ur-Rahman
2002). Also, 13C NMR downfield shift of C-6 of galactose residue to δC 68.6 confirmed the
site of attachment to be at C-6 of galactose moiety where in the C-6 free analogue, C-6 usually
at δC 61–62 (Markham et al. 1978).
Compound 2 was obtained as a yellow amorphous. The HR-LC-TOF MS spectrum showed
a quasi-molecular ion peak at m/z 565.1508 [M + H]+, calculated as (564.15), in accordance
with the molecular formula C26H28O14. It exhibited (UV) absorption at 230 and 280 nm. The
1H-NMR spectrum of 2 was similar in its general features to that of 1 except for the presence
of two 6-deoxy-hexose moieties attached to kaempferol; this was confirmed from the 13C
NMR spectrum, where the presence of eight methine carbon signals in addition to two
anomeric carbon signals at δC 101.1 and 98.3, and one methyl carbon signal at δC 17.9, sug-
gested the presence of two pentose moieties. One was confirmed to be rhamnose from both
1H and 13C NMR values at δH (5.55, brs., H-1′′′, 1.25, 3H, d, J = 6.0, H-6′′′) and at δC (98.3, C-1′′′,
17.9, C-6′′′), respectively. The other 6-deoxy-hexose moiety was confirmed from 1H and 13
C
NMR values to be β-D-arabinopyranosyl, where C-1′′, C-2′′, C-3′′ and C-4′′ of 2 were at δC
101.1, 71.5, 70.0 and 70.7, respectively, while in the α-L-arabinofuranosyl analogue, these
values were shifted downward to about δC 107, 82, 76 and 85, respectively (Zhu et al. 2013).
Identification of the sugar residues was supported by GC/MS analysis obtained by acid hydrol-
ysis as arabinose and rhamnose. Long-range correlations were observed in HMBC between
the anomeric proton of arabinose (δH 5.35) and the C-3 of flavonol (δC 133.8), confirming
that it was the site of connection, and between the anomeric proton of rhamnose (δH 5.55)
and the C-5 of flavonol (δC 160.8), clarifying that rhamnose was connected at that site.

4
A. E. ALLAM ET AL.
2.2. Anti-lipase assay
Lipase inhibition activity of both methanol extract and the isolated compounds from V. faba
L. flowers was shown in (Table 1). The results showed that methanol extract and compound
2 exhibit 66 and 81% for methanol extract, and 53 and 77% inhibition activity for compound
2 in concentrations of 400 and 800 μg/mL, respectively.
2.3. Melanogenesis inhibition assay
After establishing the structures, compounds (1–8) were investigated using B16-F10 melanoma
cells to evaluate the inhibition of melanogenesis and cell viability at a final concentration of
20 μM using arbutin as a positive control at the same concentration. The effect of the com-
pounds on cell viability refers to the cytotoxic effect of these compounds on melanocytes
(melanocytotoxicity). The results of the assay are shown in (Table 2). Referring to cytotoxicity,
the most active compounds with potent melanin synthesis inhibition and no obvious cytotox-
icity were compounds 2, 3 and 4 where the melanin content in melanoma cells was decreased
to be about 57.5, 56 and 61%, respectively at a final concentration of 20 μM. Furthermore, these
compounds are being safe to melanocytes reflected by the cell viability about 98, 102 and
100%, respectively, using MTT assay. For compound 2 and 3, their inhibition activity was nearly
Table 1. results of the in vitro assays for lipase inhibition activities of both methanol extract and isolated
compounds of V. faba flowers.
Lipase inhibition %
Compound
Concentration (400 μg/mL)
Concentration (800 μg/mL)
1
33.60 2.30
52.98 1.04
12.52 1.67
4.78 1.80
1.96 1.67
7.09 0.93
15.53 2.07
5.37 3.03
66.2 2.41
42.26 3.41
77.06 1.25
22.77 2.08
12.13 2.70
9.29 0.80
13.60 1.93
32.23 0.60
8.37 2.49
81.18 1.68
2
3
4
5
6
7
8
Methanol extract
Positive control (Orlistat) (0.22 μg/ml)
95.94 1.16
Note: data are shown as the mean sd (n = 3).
Table 2. effects of the methanol extract and the isolated compounds on melanogenesis and cell prolif-
eration of B16-F10 melanoma cells.
Compound
Melanin content %
61.85 7.75
Cell viability %
84.80 26.75
1
2
57.54 6.80
56.17 0.26
61.09 4.54
74.90 24.07
71.87 27.82
89.47 0.85
98.12 7.39
38.65 5.79
55.40 3.77
54.80 3.46%
97.99 045
101.98 23.0
100.81 9.73
98.23 6.08
82.25 13.04
77.20 4.79
88.49 2.0
100.78 28.77
92.80 18.98
98.6 19.37
3
4
5
6
7
8
Methanol extract (160 μg/ml)
Methanol extract (40 μg/ml)
Positive control (Arbutin)
Notes: the results are expressed as mean values sd (n = 3). Final concentration of the compounds and the positive control
(arbutin) was 20 μM.

NATURAL PRODUCT RESEARCH
5
equal to that obtained with the positive control arbutin at the same concentration. For com-
pound 1, it has potent melanin synthesis inhibition but with a relative little effect on cell viability
about 84% at a final concentration of 20 μM while for compounds 5 and 6, the melanin content
in melanoma cells decreased to about 75 and 72% at a concentration of 20 μM, reflect their
good activity in inhibition of melanogenesis without affecting the cell viability as shown in
(Table 2). Compounds 7 and 8 showed weak inhibition activity. All the compounds did not
affect the viability of melanocytes; reflect their safety in inhibition of melanogenesis.The meth-
anol extract showed about 60% inhibition of melanin at a final concentration of 160 μg/mL
and by decreasing the concentration to 40 μg/mL, the extract suppressed the melanin content
in the cells to about 45% without causing obvious cytotoxicity in both cases on melanocytes.
3. Conclusions
Two new flavonoid glycosides, along with six known ones, were isolated from the flowers
of Vicia faba. The results indicated that compound 2 showed 53 and 77% lipase inhibition
activity in concentrations of 400 and 800 μg/mL, respectively. Our study also showed that
the new compounds 1 and 2 showed about 38 and 42.5% inhibition of melanogenesis at a
final concentration of 20 μM. All the compounds did not affect the viability of melanocytes;
reflect their safety in inhibition of melanogenesis. These results suggest the potential use
of Vicia faba L, flowers as a lipase and a melanogenesis inhibitor and the possible use for it
as a skin whitening agent.
Data analyses
Mean melanin production (μg/mL) and the percentage of control of melanogenesis are
reported as the mean SD. Statistical significance was determined by Dunnettʼs multiple
test p < 0.05 was considered statistically significant.
Supplementary material
Additional supporting information including the experimental section and the NMR/MS data of the
new compounds can be found in the online version of this article at the publisher’s website.
Acknowledgements
We would like to thank Research and Education Support Center of the faculty of Agriculture, Kyushu
University for supporting facilities for HR-LC-TOF MS analysis.
Disclosure statement
No potential conflict of interest was reported by the authors.
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