Constituents of the leaves and twigs of Elaeagnus umbellata and their proliferative effects on human keratinocyte HaCaT cells

Article abstract of DOI:10.1016/j.fitote.2019.104374

Bioassay-guided fractionation of an extract of leaves and twigs of Elaeagnus umbellata led to the isolation of a serotonin derivative, N-[2-(5-hydroxyl-1H-indol-3-yl)ethyl]-butanamide (1), along with six flavonoid glycosides, kaempferol-3-O-β-D-xylopyranosyl(1 → 2)-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside (2), kaempferol-3-O-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside (3), kaempferol-3-O-α-L-rhamnopyranosyl(1 → 6)-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside (4), kaempferol-3-O-β-D-xylopyranosyl(1 → 2)-β-D-galactopyranoside (5), kaempferol-3-O-rutinoside (6), and kaempferol-3-O-β-D-glucopyranosyl(1 → 2)-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside (7). Their structures were elucidated using 1D/2D nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1–6 were evaluated for their proliferative effects on HaCaT keratinocytes; 1–5 promoted keratinocyte proliferation dose dependently. Compounds 3 and 4 showed potent activities. These results suggest that the leaves and twigs of E. umbellata have wound healing and skin cell regeneration potentials.

Full text of DOI:10.1016/j.fitote.2019.104374

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Constituents of the leaves and twigs of Elaeagnus umbellata and
their proliferative effects on human keratinocyte HaCaT cells
Sunil Babu Paudel, Jiyoung Park, Nam Hee Kim, Hyukjae Choi,
Eun-Kyoung Seo, Hyun Ae Woo, Joo-Won Nam
PII:
S0367-326X(19)31861-1
DOI:
https://doi.org/10.1016/j.fitote.2019.104374
FITOTE 104374
Reference:
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
16 September 2019
10 October 2019
12 October 2019
Please cite this article as: S.B. Paudel, J. Park, N.H. Kim, et al., Constituents of the leaves
and twigs of Elaeagnus umbellata and their proliferative effects on human keratinocyte
HaCaT cells, Fitoterapia (2018), https://doi.org/10.1016/j.fitote.2019.104374
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Constituents of the leaves and twigs of Elaeagnus
umbellata and their proliferative effects on human
keratinocyteHaCaTcells
Sunil Babu Paudela, Jiyoung Parkb, Nam Hee Kimb, Hyukjae Choia, Eun-Kyoung
Seob, Hyun Ae Woob,* and Joo-WonNama,*
a College of Pharmacy, YeungnamUniversity, Gyeongsan-si, Gyeongsangbukdo 38541, Korea
b College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul
120-750, Republic of Korea
*Corresponding authors.
E-mail addresses:hawoo@ewha.ac.kr (H.A. Woo) and jwnam@yu.ac.kr (J.-W. Nam)

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ABSTRACT
Bioassay-guided fractionation of an extract of leaves and twigs of Elaeagnus umbellata led to the
isolation of a serotonin derivative, N-[2-(5-hydroxyl-1H-indol-3-yl)ethyl]-butanamide (1), along
with six flavonoid glycosides, kaempferol-3-O-β-D-xylopyranosyl(1→2)-β-D-galactopyranoside-
7-O-α-L-rhamnopyranoside
(2),
kaempferol-3-O-β-D-galactopyranoside-7-O-α-L-
rhamnopyranoside (3), kaempferol-3-O-α-L-rhamnopyranosyl(1→6)-β-D-galactopyranoside-7-
O-α-L-rhamnopyranoside (4), kaempferol-3-O-β-D-xylopyranosyl(1→2)-β-D-galactopyranoside
(5), kaempferol-3-O-rutinoside (6), and kaempferol-3-O-β-D-glucopyranosyl(1→2)-β-D-
galactopyranoside-7-O-α-L-rhamnopyranoside (7). Their structures were elucidated using 1D/2D
nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1–6 were
evaluated for their proliferative effects on HaCaT keratinocytes; 1–5 promoted keratinocyte
proliferation dose dependently. Compounds 3 and 4 showed potent activities. These results
suggest that the leaves and twigs of E. umbellata have wound healing and skin cell regeneration
potentials.
Keywords:
Elaeagnus umbellata; Elaeagnaceae; serotonin derivative; flavonoid glycoside; cell proliferation;
HaCaT keratinocyte

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1. Introduction
The genus Elaeagnus (Elaeagnaceae) consists of approximately 90 species, which can be
found at altitudes ranging from 1,200 to 2,100 m and are mostly distributed in subtropical
regions of Asia [1, 2]. Various species of Elaeagnus, such as E. umbellata, E. pungens, E.
angustifolia, and E. multiflora, have been used as medicinal plants [1]. E. umbellata Thunb. is
well known for the lycopene contents in its berries, with antioxidant and anti-inflammatory
activities [2, 3]. In this study, we investigated the phytochemistry of the twigs and leaves, waste
products of this plant. E. umbellata has shown antiplasmodial [4], antioxidant [5, 6], and
antihypertensive [7] activities. Several secondary metabolites including β-carboline alkaloids [8],
coumarins [4], anthraquinone glycosides [9], sterols [4], and flavonoids [10] have been reported
for this species. In the present study to uncover skin-protective components through bioassay-
guided fractionation, a serotonin derivative and six flavonoid glycosides were isolated from the
leaves and twigs of E. umbellata and identified as N-[2-(5-hydroxyl-1H-indol-3-yl)ethyl]-
butanamide (1), kaempferol-3-O-β-D-xylopyranosyl(1→2)-β-D-galactopyranoside-7-O-α-L-
rhamnopyranoside (2) [11], kaempferol-3-O-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside
(3)
[12],
kaempferol-3-O-α-L-rhamnopyranosyl(1→6)-β-D-galactopyranoside-7-O-α-L-
rhamnopyranoside (4) [13], kaempferol-3-O-β-D-xylopyranosyl(1→2)-β-D-galactopyranoside (5)
[14], kaempferol-3-O-rutinoside (6) [15], and kaempferol-3-O-β-glucopyranosyl(1→2)-β-
galactopyranoside-7-O-α-rhamnopyranoside (7) [16] through spectroscopic data analysis
involving 1D and 2D nuclear magnetic resonance (NMR) (Fig. 1) and by comparing their data
with reported values. Various bioactivities have been reported for these isolated compounds,
such as the melatonin-inducing activity of 1, antioxidant activities of 2 and 4, melanogenesis
inhibitory activities of 3 and 6, and antimalarial activity of 5 [17-21].

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Skin is a self-renewing organ that defends the body against external threats such as heat,
infection, and ultraviolet radiation [22]. It also protects against microbes, light, and injury [23].
Skin cell proliferation is important for proper wound healing after skin injury. Skin wound
healing is a complex process involving the re-epithelialization of missing cellular structures and
the tissue layer through three phases: inflammation, proliferation, and remodeling [24]. The
smooth progression of these events leads to successful wound healing and restoration of the
disrupted anatomical and functional state of the skin [25]. Of the many cell types required during
wound healing, keratinocytes are important for epithelialization in the proliferative phase as they
are the predominant cells of the outermost layer of the skin [26]. Skin wound healing can be
facilitated by natural products with medicinal properties. Many studies on the wound healing
properties of natural products with anti-inflammatory, antioxidant, antibacterial, and pro-
collagen synthesis actions have been conducted [27]. For instance, it has been found that
saponins can enhance the synthesis of pro-collagen, while flavonoids have antiseptic and
antibacterial activities. These natural compounds can regulate one or more phases of skin wound
healing. Natural products and their phytochemicals have important roles in skin wound healing
and are used in the design of new synthetic compounds for this purpose [28]. To the best of our
knowledge, this is the first report on the isolation of these compounds from this species. Selected
compounds 1–6 were examined for their cell proliferation activities in HaCaT cells.
2. Experimental section
2.1. General experimental procedures
The NMR spectra were acquired on a 600 MHz Varian spectrometer (Varian, Billerica,
MA, U.S.A.) and a 250 MHz Bruker AVANCE DPX spectrometer (Bruker, Billerica, MA, USA).

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HR-MS was performed on a Waters ACQUITY UPLC system (Milford, MA, USA) coupled to a
Micromass Q-Tof Micro mass spectrometer and Agilent 6220 Accurate-Mass TOF LC/MS
system. Column chromatography was performed with silica gel 60 (230–400 mesh, Merck
KGaA, Darmstadt, Germany) and YMC-GEL ODS-A (12 nm, S-150 µm, YMC, Kyoto). TLC
was carried out on silica gel 60 F254 and RP-18 F254S aluminum plates (0.2-mm thickness, Merck
KGaA, Darmstadt, Germany) by visualization under UV light at 254 nm/365 nm and 10% (v/v)
sulfuric acid in EtOH followed by heating. Isolation was performed by HPLC (Waters
Corporation. U.S.A.) equipped with YMC-Pack ODS-AQ (10.0 × 250 mm: S-5 µm, 12 nm,
YMC, Kyoto) and J’Sphere ODS-M80 (10.0 × 250 mm: S-4 µm, 8 nm) columns.
2.2 Plant material
The twigs and leaves of E. umbellata were purchased from the Gongryongnara farm in
Goseong-gun, Gyeongsangnam-do, Korea and identified by J.-W. Nam, one of the authors. A
voucher specimen (no. YNPC004) has been deposited at the Natural Product Chemistry
Laboratory, College of Pharmacy, Yeungnam University.
2.3 Extraction and isolation
The dried twigs and leaves of E. umbellata (1.9 Kg) were extracted with 70% acetone (3
× 10 L) for 72 hr. at room temperature. The solvent was evaporated under reduced pressure to
yield the concentrated 70% acetone extract (31.0 g). This extract was suspended in deionized
H2O (600 mL) and sequentially fractionated with hexanes (5 × 600 mL), MC (3 × 600 mL),
EtOAc (2×600 mL), and saturated n-BuOH (2 × 600 mL). A portion of the n-BuOH extract (4.0
g) was separated through NP open column by using MC-MeOH (100:0 to 0:100, a step gradient
solvent system) to afford 18 fractions (Frc.1-Frc.18). Compound 1 was isolated from Frc. 6 (10.2

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mg) by RP HPLC (YMC-Pack ODS-AQ column, S-5 μm, 12 nm, 10.0 × 250 mm, 35% MeOH
in 65% acidified H2O with 0.1% FA, 2 mL/min). A small portion (3.5 mg) of compound 1 was
obtained from Frc. 4 (7.9 mg) and Frc. 5 (9.2 mg) with the same procedure. Compounds 2-5
were isolated from Frc. 15 by RP HPLC (YMC-Pack ODS-AQ column, S-5 μm, 12 nm, 10.0 ×
250 mm, 38% CH3CN in 62% acidified H2O with 0.1% FA, 2 mL/min). Compound 6 was
isolated from Frc. 14 by RP HPLC (J’Sphere ODS-M80 column, S-4 μm, 8 nm, 10.0 × 250 mm,
38% CH3CN in 62% acidified H2O with 0.1% FA, 2 mL/min). For compound 7, another batch of
E. umbellata twigs and leaves was extracted with 100% MeOH (3 × 48 L) for 3 days at room
temperature. This extract was evaporated under reduced pressure to give 400 g of concentrated
methanol extract. The methanol extract was separated by liquid-liquid extraction with deionized
H2O (1.5 L) and hexanes (1.5 L× 9), and the water layer was sequentially separated with MC
(1.5 L × 2), EtOAc (1.5 L× 2), and saturated n-BuOH (1.5 L× 3) to yield 39 g of n-BuOH
extract. The resulting n-BuOH extract was further fractionated through NP open column
chromatography using MC-MeOH (100:0 to 0:100) to yield 23 subfractions. From these 23
fractions, fractions 14-16 were combined for the next step NP-open column chromatography
using MC-MeOH (100:0 to 0:100) to give 14 fractions. Among the resulting 14 fractions,
fractions 9-11 were combined and subjected to reverse phase open column chromatography by
using MeOH- H2O (20:80 to 100:0) to yield 22 fractions. From these 22 fractions, fraction 7 was
injected in RP HPLC (YMC-Pack ODS-AQ column, S-5 μm, 10.0 × 250 mm, 22% CH3CN in
H2O, 2 mL/min) to give compound 7 (0.8 mg).
2.3.1. Data for compound 1
Brownish powder; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see
Table 1; HR-ESIMS: m/z 247.1439 [M+H]+ (calcd. 247.1441 for C14H19N2O2).

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2.3.2. Data for compound 2
1
Yellowish powder; H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see
Table 1; HR-ESIMS: m/z 727.2079 [M+H]+ (calcd. 727.2080 for C32H39O19).
2.4. Acid hydrolysis
To determine the absolute configurations of sugars in compound 2, a portion (0.5 mg) of
compound 2 was hydrolyzed with 1 N HCl (200 µL) at 80°C for 2 hr. The hydrolysates were
washed with EtOAc (200 µL × 3) to remove the aglycone. The resulting aqueous layer was dried
and treated with p-isothiocyanate to prepare arylthiocarbamoyl thiazolidine derivatives. The
reaction mixture was analyzed with RP HPLC system (Agilent Eclipse plus C18 column, 5 μm,
4.6 × 250 mm, 25% CH3CN in H2O, 0.8 mL/min, monitoring at 250 mm). Retention times of
detected arylthiocarbamoyl thiazolidine derivatives were compared with those of authentic D-
glucose, L-glucose, D-galactose, L-galactose, D-xylose, L-xylose and L-rhamnose (Sigma-
Aldrich, St. louis, MO, USA). The retention times of hydrolysates and authentic sugar are as
follows: D-galactose derivative (13.6 min), D-xylose derivative (18.3 min) and L-rhamnose
derivative (28.1 min).
2.5. Cell culture
HaCaT human keratinocytes were from cell Lines Service Gmbh (Eppelheim, Germany).
The HaCaT cells were cultured in Dulbecco’s modified Eagles’s medium (Hyclone)
supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/ streptomycin
(HyClone), and maintained at 37°C with 5% CO2 in a humidified atmosphere.
2.6. Evaluation of cell viability by CCK assay

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Cell viability was measured by cell counting kit-8 (CCK-8; Dojindo Laboratories,
Kumamoto, Japan) assay. CCK-8 allows very convenient assays by utilizing Dojindo’s highly
water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-soluble formazan dye upon
reduction in the presence of an electron mediator. CCK-8, being nonradioactive, allows sensitive
colorimetric assays for the determination of the number of viable cells in cell proliferation and
cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells to give an orange colored
product (formazan), which is soluble in the tissue culture medium. The amount of the formazan
dye generated by dehydrogenases in cells is directly proportional to the number of living cells.
The detection sensitivity using CCK-8 is higher than assays using other tetrazolium salts such as
MTT, XTT, MTS or WST-1. HaCaT cells were seeded in 96-well plates at a density of 0.2 × 104
cell/well, incubated for 24 h, and subsequently treated with various concentrations of agents for 3
days. The solution was removed from each well and replaced with CCK-8, was added to each
well and incubated at 37°C for 1 h. The absorbance wavelength was then measured at 450 nm
using spectrophotometer.
2.7. Statistical Analysis
All results were expressed as mean ± s.d. Student’s t‐test was used for comparisons
involving two groups.
3. Results and Discussion
Compound 1 was obtained as an amorphous brownish powder and showed a molecular ion
peak at m/z 247.1439 [M + H]+ (calcd. 247.1441) in the high-resolution electrospray ionization
mass spectrometry (HR-ESIMS) spectrum, consistent with an elemental formula of C14H18N2O2.

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The 1H NMR spectrum (Table 1) of 1 showed resonances at δH 6.65 (dd, J = 8.7, 2.2 Hz, 1H, H-
6), 6.93 (d, J = 2.2 Hz, 1H, H-4), and 7.15 (d, J = 8.7 Hz, 1H, H-7) indicating the presence of a
trisubstituted benzene ring, which was determined to be part of a 3,5-disubstituted indole moiety
based on heteronuclear multiple bond correlation (HMBC) correlations of H-2 (δH 7.00, s, 1H)
with C-8 and C-9 (Fig. 2). Moreover, two methylene signals at δH 2.86 (t, J = 7.4 Hz, 2H, H-10)
and 3.45 (t, J = 7.4 Hz, 2H, H-11) were observed. These were attributed to a 5-
hydroxytryptamine (serotonin) moiety, based on the correlated spectroscopy (COSY) cross-peak
1
of H-10/H-11 and the HMBC cross-peak of H-11 with C-3 (Fig. 2). In the H and 13C NMR
spectra (Table 1), additional signals for an n-propyl moiety at δH/δC 0.91 (t, J = 7.3 Hz, 3H, H-
16)/13.9 (C-16), 1.61 (h, J = 7.3 Hz, 2H, H-15)/20.4 (C-15), and 2.14 (t, J = 7.3 Hz, 2H, H-
14)/39.1 (C-14) were observed, and were further confirmed by the COSY correlations of H-15
with both H-14 and H-16. A resonance for a carbonyl group appeared at δC 176.1 (C-13) in the
13C NMR spectrum. Correlations of the signals at δH 3.45 (H-11) and 2.14 (H-14) with that at δC
176.1 in the HMBC spectrum revealed that the carbonyl group is located at C-13 to form a
butyramide group. Thus, 1 was determined to be a serotonin derivative, N-[2-(5-hydroxy-1H-
indol-3-yl)ethyl]-butanamide, which was isolated from a natural product for the first time in this
study. Serotonin derivatives are rarely found in plants. Since they were first identified in
safflower seeds, they have been found in various plant species and families. They not only act as
phytoalexin in plants but also have various biological activities such as antioxidant, anticancer
and skin whitening activities [29].
Compound 2 was isolated as an amorphous yellow powder and its molecular formula was
determined to be C32H38O19 based on the molecular ion peak [M + H]+ at m/z 727.2079 (calcd.
1
727.2080) in the HR-ESIMS spectrum. In the H NMR spectrum of 2, resonances for a typical

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AA'BB' spin system and an AB spin system were observed at δH 8.14 (d, J = 8.9 Hz, 2H, H-2',
6')/6.89 (d, J = 8.9 Hz, 2H, H-3', 5') and at δH 6.75 (d, J = 2.1 Hz, 1H, H-8)/6.45 (d, J = 2.1 Hz,
1
1H, H-6), respectively, suggesting that the aglycone moiety of 2 is a kaempferol [30]. The H
and 13C NMR spectra (Table 1) revealed the presence of three anomeric protons at δH 5.57 (d, J =
1.2 Hz, 1H, H-1'''')/δC 99.8 (C-1''''), 5.45 (d, J = 7.6 Hz, 1H, H-1'')/101.0 (C-1''), and 4.73 (d, J =
7.0 Hz, 1H, H-1''')/105.7 (C-1'''). Acid hydrolysis of 2 afforded D-galactose, D-xylose, and L-
rhamnose, which were identified by comparing their retention times in high-pressure liquid
chromatography (HPLC) analysis with those of standard sugars [31]. Furthermore, the anomeric
configurations of the sugar units were established to be β-D-galactose, β-D-xylose, and α-L-
rhamnose through their 3J coupling constants (Gal, d, J = 7.6 Hz; Xyl, d, J = 7.0 Hz; Rha, d, J =
1.2 Hz) [32, 33]. The linkage positions of the galactose and rhamnose moieties were confirmed
by HMBC cross-peaks between anomeric protons and aglycone carbons [δH 5.45 (H-1'') and δC
135.3 (C-3); δH 5.57 (H-1'''') and δC 163.4 (C-7)]. The 1→2 link between xylose and galactose
was confirmed by the HMBC cross-peaks of δH 4.73 (H-1''') and δC 80.6 (C-2''-galactose) (Fig. 2).
Thus, 2 was determined to be kaempferol-3-O-β-D-xylopyranosyl(1→2)-β-D-galactopyranoside-
7-O-α-L-rhamnopyranoside, which had been previously isolated from Trifolium lupinaster [11].
However, this is the first report of the absolute configurations of these sugars.
The cell proliferative effects of each compound on human keratinocyte HaCaT cells were
evaluated (Table 2) [34]. Because of its chemical similarity with the tested compounds,
kaempferol (Sigma-Aldrich) was used for comparison in the present biological study. Cells were
treated with the indicated concentrations of the selected compounds (1–6) or dimethyl sulfoxide
(DMSO) alone (vehicle) and cell viabilities were compared after three days of incubation using
cell counting kit-8 (CCK-8). Upon daily microscopic examination of the cells, no differences in

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cell attachment or morphology between the compound-treated and control cells during
incubation were observed. Therefore, compounds 1–5 promoted keratinocyte cell growth dose
dependently. Among the six compounds, 3 and 4 showed relatively high activities at
concentrations of 1, 5, and 10 µM and their proliferation activities were significantly greater than
that of kaempferol. In particular, incubation with low doses (1 and 5 µM) of compounds 3 and 4
promoted HaCaT cell proliferation. The viabilities of HaCaT cells following incubation with 3 at
concentrations of 1, 5, and 10 µM were 174.14%, 181.48%, and 193.94%, respectively. Similar
dose-dependent growth promotion by 4 was also observed; the proliferative effects increased to
181.82%, 180.47%, and 197.98%, respectively. Growth promotion rates in HaCaT cells caused
by compounds 1, 2, and 5 also showed significant increases at 10 µM (194.61%, 162.63%, and
194.70%, respectively).
In conclusion, compounds 1–5 showed proliferation activities for human keratinocyte HaCaT
cells; in particular, 3 and 4, at low doses, appeared to have the most potent proliferation activities
compared to those of kaempferol and the other compounds. Therefore, these isolated compounds
may be helpful in skin regeneration and wound healing in human skin and are also potential
cosmetic materials.
Declaration of Competing Interest
The authors declare no conflict of Interest.
Acknowledgements
This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education [NRF-

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2016R1D1A3B03933996]. The work was also funded in part by a 2018 Yeungnam University
Research Grant.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://.

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Figures and tables
Fig. 1. Chemical structures of compounds 1–7 (Rha = rhamnopyranoside; Gal =
galactopyranoside; Xyl = xylopyranoside; Glc = glucopyranoside).

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1
Fig. 2. Key 1H-1H COSY and H-13C HMBC correlations of compounds 1 and 2.

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Table 1. 1H (600 MHz) and 13C NMR (150 MHz) data of compounds 1 and 2.
Position
1
Position
2
δC
δH mult (J in Hz)
aglycone
δC
δH mult (J in Hz)
2
3
4
5
6
7
8
9
10
11
13
14
15
16
124.2
112.5
103.5
151.1
112.4
112.6
133.1
129.5
26.4
41.3
176.1
39.1
20.4
13.9
7.00 s
2
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
Gal
1''
2''
3''
4''
5''
158.7
135.3
179.9
162.9
100.5
163.4
95.4
157.9
107.4
122.6
132.5
116.3
161.7
116.3
132.5
6.93 d (2.2)
6.65 dd (8.7, 2.2)
7.15 d (8.7)
6.45 d (2.1)
6.75 d (2.1)
2.86 t (7.4)
3.45 t (7.4)
8.14 d (8.9)
6.89 d (8.9)
2.14 t (7.3)
1.61 h (7.3)
0.91 t (7.3)
6.89 d (8.9)
8.14 d (8.9)
101.0
80.6
75.2
70.3
77.1
5.45 d (7.6)
3.97 dd (9.7, 7.6)
3.72 dd (9.7, 3.4)
3.82 dd (3.4, 1.0)
3.45 td (6.0, 1.0)
3.61 dd (11.4, 6.0)
3.56 dd (11.4, 6.4)
6''
62.0
Xyl
1'''
2'''
3'''
4'''
105.7
75.1
77.3
71.1
4.73 d (7.0)
3.37 m
3.38 m
3.51 m
3.24 dd (11.5, 9.7)
/3.94 dd (11.5, 5.3)
5'''
66.8
Rha
1''''
2''''
3''''
4''''
5''''
99.8
71.7
72.1
73.6
71.3
5.57 d (1.2)
4.02 dd (3.3, 1.2)
3.84 dd (9.4, 3.3)
3.48 t (9.4)
3.59 dd (9.4, 6.1)
6''''
18.1
1.25 d (6.1)
Chemical shifts (δ) are expressed in ppm; J values are given in parentheses. Data were measured in CD3OD.

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Table 2. Cell proliferative effects of 1-6 on HaCaT cells.
Viable cells, %
DAY 0
DAY 3
Concentration of compound (µM)
Compound
vehicle
1
5
10
Kaempferola
158.25±1.07*
160.94±1.35*
153.87±0.84*
174.14±0.81*#
181.82±1.00*#
144.11±1.02*
146.46±1.67*
157.58±1.76*
161.62±1.67*
155.22±1.50*
181.48±0.51*#
180.47±1.17*#
145.79±1.35*
142.69±0.40*
193.27±2.36*
194.61±1.02*
162.63±0.67*
193.94±0.88*
197.98±2.19*#
194.70±0.79*
146.92±1.06*
1
2
3
100.00±1.20
155.22±1.35
4
5
6
a Positive control
* P<0.01 compared to 0 μM of each compound, # P<0.01 compared to kaempferol.

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Graphical abstract