6,8-Di-C-methyl-flavonoids with neuroprotective activities from Rhododendron fortunei

Article abstract of DOI:10.1016/j.fitote.2016.06.008

Six 6,8-di-C-methyl-flavonoids, (2R,3R)-6,8-di-C-methyl-5,7,4′-trihydroxyflavanonol 7-O-β-D-gluco-pyranoside (1), (2R,3R)-6,8-di-C-methyl-5,7,4′-trihydroxyflavanonol 7-O-β-D-xylopyranosyl(1?→?6)-β-D-glucopyranoside (2), 6,8-di-C-methylkaempferol 7-O-β-D-glucopyranoside (3), (2R)-farrerol (4a), (2R/2S)-farrerol 7-O-β-D-glucopyranoside (5), and (2R/2S)-farrerol 7-O-β-D-xylopyranosyl(1?→?6)-β-D-glucopyranoside (6), and four known analogues, farrerol (4b), (2R,3R)-6,8-di-C-methyldihydrokae-mpferol (7), 6,8-di-C-methylkaempferol 7-O-β-D-glucopyranoside (8), and 6,8-di-C-methylkaempferol (9), were isolated from the twigs and leaves of Rhododendron fortunei. The structures of compounds 1–9 were determined by spectroscopic analyses (HRESIMS, 1D and 2D NMR, and CD) and chemical methods. Compounds 1–9 were evaluated for their neuroprotective effects on the human neuroblastoma SH-SY5Y cells apoptosis induced by hydrogen peroxide (H₂O₂) and amyloid β peptide (Aβ), respectively. Compounds 1–3 and 5–9 exhibited significant neuroprotective effects against H₂O₂-induced SH-SY5Y cell apoptosis, and compound 8 exhibited the strongest activity with a improvement of cell viability by about 30% at the concentration of 10?μM. Compounds 1–9 showed significant neuroprotective effects against Aβ-induced SH-SY5Y cell apoptosis.

Full text of DOI:10.1016/j.fitote.2016.06.008

6,8-Di-C-methyl-flavonoids with neuroprotective activities from Rhododen-
dron fortunei
Yongji Lai, Hong Zeng, Meijun He, Huiqin Qian, Zhaodi Wu, Zengwei
Luo, Yongbo Xue, Guangmin Yao, Yonghui Zhang
PII:
DOI:
Reference:
S0367-326X(16)30133-2
doi: 10.1016/j.fitote.2016.06.008
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17 June 2016
20 June 2016
Please cite this article as: Yongji Lai, Hong Zeng, Meijun He, Huiqin Qian, Zhaodi
Wu, Zengwei Luo, Yongbo Xue, Guangmin Yao, Yonghui Zhang, 6,8-Di-C-methyl-
flavonoids with neuroprotective activities from Rhododendron fortunei, Fitoterapia (2016),
doi: 10.1016/j.fitote.2016.06.008
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Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
6,8-Di-C-methyl-flavonoids with neuroprotective activities from
Rhododendron fortunei
Yongji Laia,b, Hong Zenga, Meijun Hec, Huiqin Qiana, Zhaodi Wua, Zengwei Luoa, Yongbo Xuea,
Guangmin Yaoa,*, Yonghui Zhanga,*
a Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan 430030, People’s Republic of China.
b Department of Pharmacy, the Central Hospital of Wuhan, Wuhan 430014, People’s Republic of China.
c Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi 445500, People’s Republic of China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Six 6,8-di-C-methyl-flavonoids, (2R,3R)-6,8-di-C-methyl-5,7,4′-trihydroxyflavanonol 7-O-β-D-gluco-
pyranoside (1), (2R,3R)-6,8-di-C-methyl-5,7,4′-trihydroxyflavanonol 7-O-β-D-xylopyranosyl(1→6)-β-
D-glucopyranoside (2), 6,8-di-C-methylkaempferol 7-O-β-D-glucopyranoside (3), (2R)-farrerol (4a),
(2R/2S)-farrerol 7-O-β-D-glucopyranoside (5), and (2R/2S)-farrerol 7-O-β-D-xylopyranosyl(1→6)-β-
D-glucopyranoside (6), and four known analogues, farrerol (4b), (2R,3R)-6,8-di-C-methyldihydrokae-
mpferol (7), 6,8-di-C-methylkaempferol 7-O-β-D-glucopyranoside (8), and 6,8-di-C-methylkaempferol
(9), were isolated from the twigs and leaves of Rhododendron fortunei. The structures of compounds
1–9 were determined by spectroscopic analyses (HRESIMS, 1D and 2D NMR, and CD) and chemical
methods. Compounds 1–9 were evaluated for their neuroprotective effects on the human
neuroblastoma SH-SY5Y cells apoptosis induced by hydrogen peroxide (H2O2) and amyloid β peptide
(Aβ), respectively. Compounds 1–3 and 5–9 exhibited significant neuroprotective effects against H2O2-
induced SH-SY5Y cell apoptosis, and compound 8 exhibited the strongest activity with a improvement
of cell viability by about 30% at the concentration of 10 μM. Compounds 1–9 showed significant
neuroprotective effects against Aβ-induced SH-SY5Y cell apoptosis.
Available online
Keywords:
C-methyl-flavonoids
Structure elucidation
Neuroprotective effects
Rhododendron fortunei
Ericaceae
2015 Elsevier B.V. All rights reserved.
1. Introduction
Alzheimer′s disease (AD) is a chronic neurodegenerative disease, and is the leading cause of dementia1. Oxidative stress-induced cell
damage and the deposition of β-amyloid (Aβ) peptide into senile plaques in the extracellular space are the manifest pathological process
of AD2,3. Natural products from plants, marine organisms, and bacteria have been reported to be an important source of Alzheimer’s
drug leads4,5. Flavonoids are widely distributed in plants, and are reported to possess neuroprotective effects by inhibiting the formation
of reactive oxygen species (ROS), as well as by preventing Aβ aggregation and Aβ-induced toxicity6,7. Although flavonoids are
abundant in nature, C-methyl-flavonoids having methylations on carbon are rare8. Literature survey revealed that C-methyl-flavonoids
16–18
were mainly reported from plants of Caesalpiniaceae9, Didieraceae10,11, Dryopteridaceae12, Ericaceae13,14, Fabaceae15, Myrtaceae
,
Onocleaceae19, Pinaceae8,20–23, and Ranunculaceae24, and dem- onstrated anticancer8, antifungal15, antiviral17, antiinflammatory9,
antibacterial9, antioxidant18, antimicrobial24 , neuroprotective7, peroxynitrite scavenging20, and aldose reductase inhibitory19 activities.
Rhododendron fortunei Lindil. (Ericaceae), an evergreen shrub or small tree, is native to China, and widely distributes in Anhui,
Fujian, Guangdong, Guangxi, Guizhou, Henan, Hubei, Hunan, Jiangxi, Shaanxi, Sichuan, Yunnan, and Zhejiang provinces of China25.
The leaves and flowers of R. fortunei are used as a folk medicine in China to treat skin ulcers, menstrual disorder, leucorrhea,
hemorrhage of digestive tract, and cough26. Literature survey revealed limited phytochemical studies on R. fortunei. Preliminary
phytochemical studies of our term on the twigs and leaves of R. fortunei led to the isolation of twelve known compounds, five
flavonoids including a C-methyl-flavonoids, (2R, 3R)-6,8-di-C-methyldi- hydrokaempferol (7, in the reference the structure was
determined as (2R, 3S)-6,8-di-C-methyldihydrokaempferol by mistake), a diterpenoid, and six phenylpropanoids27. Farrerol (4b), a
well-known 6,8-di-C-methyl-flavonoid in the genus of Rhododendron, has been reported to possess the neuroprotective activity7. In
order to discovery more 6,8-di-C-methyl-flavonoids with neuropective activity from R. fortunei, the chemical constituents of this plants
were further investigated. In the follow-up study, six 6,8-di-C-methyl-flavonoids (1–3, 4a, 5–6) and four known analogues (4b, 7–9)
(Figure 1) were obtained. Here, we described the isolation and structural determination of the ten 6,8-di-C-methyl-flavonoids, and their
neuroprotective effects on the human neuroblastoma SH-SY5Y cells apoptosis induced by hydrogen peroxide (H2O2) and amyloid-β
*
Corresponding authors. Tel./fax: +86 027 83692892
E-mails: zhangyh@mails.tjmu.edu.cn (Y Zhang);
gyap@hust.edu.cn (G. Yao).
1

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(Aβ) peptide, respectively.
2. Results and discussion
The dried twigs and leaves of R. fortunei were extracted by 95% EtOH. The extract was concentrated under reduced pressure to
remove the EtOH, and successively extracted by petroleum ether, CHCl3, EtOAc, and n-BuOH to yield four extraction fractions.
Repeated chromatographic purification of the EtOAc and n-BuOH fractions by silica gel, RP C-18, Sephadex LH-20, and HPLC
resulted in the isolation of ten C-methyl-flavonoids, of which six are new compounds. Comparing their NMR data with reported values,
we assigned the sructures of the four known C-methyl-flavonoids to be farrerol (4b)22, (2R,3R)-6,8-di-C-methyldi-hydrokaempferol
(7)21, 6,8-di-C-met-hylkaempferol 7-O-β-D-glucopyranoside (8)23, and 6,8-di-C-methyl-kaempferol (9)18.
Figure 1. Chemical structures of 6,8-di-C-methyl-flavonoids 1–9.
Compound 1 was obtained as a yellow amorphous solid. The molecular formula of 1 was determined to be C23H26O11 by the quasi-
molecular ion [M + Na]+ at m/z 501.1354 (calcd for C23H26O11Na, 501.1373) in the HRESIMS and the 13C NMR data. The IR spectrum
of 1 showed the presence of hydroxy (3381 cm–1) and conjugated carbonyl (1630 cm–1) groups. In addition, the strong absorption at 285
1
and 368 nm in the UV spectrum inferred 1 to be a flavonoid. The H NMR spectrum of 1 (Table 1) exhibited resonances for a typical
AA′BB′ coupling system, two oxymethines, a glucopyranose at 4.74 (d, J = 7.6 Hz, H-1′′), 3.77 (dd, J = 5.2, 11.8 Hz, H-6′′a), 3.66 (dd,
J = 2.2, 11.8 Hz, H-6′′b), 3.53 (t, J = 8.3 Hz, H-2′′), 3.44 (t, J = 8.8 Hz, H-3′′), 3.39 (t, J = 8.8 Hz, H-4′′), and 3.18 (m, H-5′′), and two
methyl singlets. The 13C NMR (Table 2) and DEPT spectra displayed resonances for a ketone carbonyl, eight substituted aromatic
carbons, four aromatic methines, a glucopyranose unit (δC 105.5, C-1′′; 78.3, C-5′′; 78.0, C-3′′; 75.9, C-2′′; 71.6, C-4′′; 62.8, C-6′′), two
oxymethines, and two methyls. The above NMR data suggested that compound 1 is a glycosylated derivatives of the known compound
1
721. The HSQC, H-1H COSY, and HMBC data were used to establish the planar structure of compound 1 (Figure 2). The HMBC
correlations of 6-CH3 (δH 2.15) with C-5 (δC 159.7), C-6 (δC 113.5), and C-7 (δC 163.2) and 8-CH3 (δH 2.08) with C-7, C-8 (δC 112.3),
C-9 (δC 159.1) indicated that these two methyl groups were located at C-6 and C-8. Moreover, the glucosidation position was deduced
to be at C-7 based on the HMBC correlation of the anomeric proton H-1′′ (δH 4.74) and C-7. To determine the absolute configuration of
the glucopyranose, 1 was hydrolyzed by 2 M CF3CO2H, and the trimethylsilylthiazolidine derivatives of the hydrolysate were prepared.
The absolute configuration of the glucose in 1 was determined to be D by comparing the GC retention time of the
trimethylsilylthiazolidine derivatives of the hydrolysate with those of standard D- and L-glucose. The β-glucosyl linkage was deduced
from the coupling constant J = 7.6 Hz of the anomeric proton H-1′′ (δH 4.74, d). The large coupling constant J = 11.8 Hz between H-2
and H-3 revealed their trans relationship, and the strong positive Cotton effect at 353 nm in the CD spectrum suggested the absolute
configuration of 1 was 2R,3R21,28. Thus, compound 1 was characterized as (2R,3R)-6,8-di-C-methyl-5,7,4′-trihydroxyflava-nonol 7-O-β-
D-glucopyranoside.
Figure 2. Key 1H-1H COSY and HMBC correlations of compound 1.
Table 1 1H NMR Data (400 MHz) for Compounds 1–4, J in Hz.
position
1a
2b
3c
4a
Aglycone
2

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5.29 dd
(2.9, 12.8)
2.71 dd
(2.9, 17.1)
3.05 dd
(12.8, 17.1)
2
4.98 d (11.8)
5.38 d (11.8)
3α
3β
4.56 d (11.8)
5.13 d (11.8)
2′ 6′
3′ 5′
Me-6
Me-8
Glc
1′′
7.37 d (8.5)
6.84 d (8.5)
2.15 s
7.87 d (8.3)
7.30 d (8.3)
2.68 s
8.10d (8.8)
6.96 d (8.8)
2.19 s
7.32 d (8.5)
6.83 d (8.5)
2.00 s
2.08 s
2.55 s
2.40 s
1.99 s
4.74 d (7.6)
3.53 t (8.3)
3.44 t (8.8)
3.39 t (8.8)
5.46 d (6.8)
4.34 overlap
4.33 overlap
4.29 m
4.65 d (7.7)
3.35 overlap
3.26 m
2′′
3′′
4′′
3.22 m
5′′
6′′
3.18 m
3.66 dd
(2.2, 11.8)
3.77 dd
(5.2, 11.8)
4.12 m
2.99 t (8.6)
3.53 dd
(4.0, 11.0)
3.85 d
4.30 m
4.75 d (10.8)
(11.0)
Xyl
1′′′
2′′′
3′′′
4′′′
5′′′
4.86 d (7.4)
3.93 t (8.1)
4.08 m
4.07 d (7.4)
2.88 d (8.1)
3.24 m
4.17 m
3.20 m
4.33 overlap
2.95 t (11.2)
3.63 dd
(5.2, 11.2)
3.65 t (10.6)
a Measured in methanol-d4; b Measured in pyridine-d6;
c Measured in DMSO-d6.
Compoud 2 was obtained as a yellow amorphous powder. The [M + Na]+ ion peak at m/z 633.1785 in the HRESIMS and 13C-NMR
data determined the molecular formula of 2 to be C28H34O15 (calcd for C28H34O15Na, 633.1795). The NMR data of 2 (Tables 1 and 2)
were similar to those of 1 , and the major differences were that there is an additional xylose moiety (δH 4.86, H-1′′′; 3.93, H-2′′′; 4.08,
H-3′′′; 4.17, H-4′′′; 4.33, 3.65, H-5′′′; δC 106.1, C-1′′′; 75.3, C-2′′′; 78.7, C-3′′′; 71.6, C-4′′′; 67.5 C-5′′′)29 in 2, and the H-6′′(δH 4.30, 4.75)
and C-6′′(δC 70.3) of the glucose unit in 2 were deshielded more than those in 1. Thus, this additional xylose moiety in 2 should be
connected to the C-6′′ of the glucose. The correlations from H-1′′′ (δH 4.86) of the xylose to C-6′′ (δC 70.3) of the glucose in the HMBC
spectrum verified the linkage between these two sugar moieties to be 7-O-xylopyranosyl(1→6)-glucopyranose. The absolute
configuration of the sugar moieties was identified as D-glucose and D-xylose by comparing the GC retention times of the trimethylsilyl-
thiazolidine derivative of the hydrolysate with those of standards, D- and L-glucose, and D- and L-xylose. The β-linkage for two sugar
units was deduced from the large coupling constants of the anomeric protons (J = 6.8 Hz for glucose and J = 7.4 Hz for xylose). The
trans-relationship of H-2 and H-3 in 2 was determined by the large coupling constant J = 11.8 Hz, and the 2R,3R absolute configuration
was suggested by the strong positive Cotton effect at 353 nm in the CD spectrum21,28, which is almost the same as that of 1. Thus,
compound
2
was designated as (2R,3R)-6,8-di-C-methyl-5,7,4′-trihydroxyflavano-nol 7-O-β-D-xylopyranosyl(1→6)-β-D-
glucopyranoside.
Table 1 1H NMR Data (400 MHz) for Compounds 1–4, J in Hz.
position
1a
2b
3c
4a
Aglycone
2
3
84.9
74.2
84.9
74.3
147.7
136.1
176.5
155.0
113.1
158.5
109.8
151.0
106.2
122.0
129.7
115.6
159.5
9.0
80.1
44.1
4
200.8
159.7
113.5
163.2
112.3
159.1
105.0
129.5
130.4
116.3
159.3
9.3
201.5
159.9
113.0
163.1
112.1
158.8
105.2
129.6
130.8
116.8
160.1
10.1
198.4
160.3
104.8
164.2
104.1
159.3
103.2
131.5
128.8
116.3
158.8
7.4
5
6
7
8
9
10
1′
2′ 6′
3′ 5′
4′
Me-6
Me-8
Glc
1′′
2′′
9.8
10.5
9.2
8.1
105.5
75.9
106.2
76.2
104.5
74.0
3

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3′′
4′′
78.0
71.6
78.3
62.8
78.8
71.9
78.2
70.3
76.2
69.7
76.5
68.5
5′′
6′′
Xyl
1′′′
2′′′
3′′′
4′′′
5′′′
106.1
75.3
78.7
71.6
67.5
103.7
73.2
75.7
69.5
65.5
a Measured in methanol-d4; b Measured in pyridine-d6;
c Measured in DMSO-d6.
Compound 3 was isolated as a yellow amorphous powder. In conjunction with the 13C NMR data, the HRESIMS data of 3 at m/z
631.1621 [M + Na]+ suggested a molecular formula of C28H32O15 (calcd for C28H32O15Na, 631.1639), two mass units less than that of
compound 2. The resonances in the 1H (Table 1) and 13C (Table 2) NMR spectra of 3 were similar to those of 2, except that two olifinic
quaternary carbons (δC 147.7, 136.1) in 3 replaced two oxymethines (δH 5.38, 5.13; δC 84.9, 74.3) in 2. So, 3 should be a 2,3-dehydro-
derivative of 2. The HMBC correlations from H-1′′′ (δH 4.07, d) of the xylose to C-6′′ (δC 68.5) of the glucose, and from H-1′′ (δH 4.65,
d) of the glucose to C-7 (δC 158.5) established the connections of these two sugar units to aglycone. The absolute configurations of
these two sugar moieties were determined to be D-glucose and D-xylose by the GC analysis of the trimethylsilylthiazolidine derivatives
of the hydrolysate and the standards, D- and L-glucose, and D- and L-xylose. The coupling constants of anomeric protons (δH 4.65, d, J =
7.7 Hz, H-1′′; 4.07, d, J = 7.4 Hz, H-1′′′) assgined the β-linkage of the D-glucose and D-xylose. Hence, compound 3 was identified as
6,8-di-C-methylkaempferol 7-O-β-D-xylopyranosyl (1→6)-β-D-glucopyranoside.
Compound 4 is a pair of racemate, while Compounds 5 and 6 are mixtures of two epimers, respectively. Before the isolation of
compounds 5 and 6, 4 was deemed to be an optically pure compound. The structural elucidation of compounds 5 and 6 hinted that 4
was likely to be a pair of racemate, which was confirmed by chiral separation and CD in the subsequent study.
Figure 3. Chiral HPLC analysis of compounds 4a (peak 1)
and 4b (peak 2).
Figure 4. CD spectra of compounds 4a and 4b.
The molecular formula of compound 4, C17H16O5, was determined from the 13C NMR data and HRESIMS data at m/z 301.1069 [M +
H]+ (calcd for C17H17O5, 301.1076). Compound 4 was isolated as a pair of enantiomers. After many attempts, compound 4 was separaed
to 4a (0.4 mg, tR = 18.5 min) and 4b (0.6 mg, tR = 20.2 min) by a chiral HPLC using a Chiralpack AD-RH column, eluting with
4

ACCEPTED MANUSCRIPT
CH3CN–H2O = 35:65; flow rate: 1.0 mL/min (Figure 3). Their CD spectra (Figure 4) are totally different, and are almost mirror images
of each other. The absolute configuration of C-2 in compound 4a was determined as R by a positive Cotton effect at 290 nm and a
negative Cotton effect at 334 nm in the CD spectrum22,28, while the 2S configuration in compound 4b was determined by the negative
Cotton effect at 290 nm and the positive Cotton effect at 335 nm in the CD spectrum. Thus, compound 4a was established as (2R)-
farrerol. In fact, natural farrerol, a mixture of R and S configuration with a ratio of 2:3, has been reported in the previous studies30. In
this study, enantiomers were separated from each other and their physicochemical properties were supplemented.
Compound 5 was obtained as a yellow amorphous solid. The quasi-molecular ion [M + Na]+ at m/z 485.1408 in the HRESIMS,
together with the 13C NMR data, suggested a molecular formula of C23H26O10 (calcd for C23H26O10Na, 485.1424). The NMR spectra
indicated that compound 5 was isolated as a mixture of two epimers (5a and 5b) at C-2, however, these two epimers could not be
seperated by RP C18 or Chiralpack AD-RH HPLC. The NMR spectra exhibited resonances for two 6,8-di-C-methyl-5,7,4′-trihydroxy-
flavanone moieties, and two glucopyranose units. The glucopyranose unit was located at C-7 on the basis of correlations of H-1′′ (δH
4.73, d, J = 7.6 Hz; 4.74 , d, J = 7.6 Hz) and C-7 (δC 162.4, 162.6) in the HMBC spectrum. In order to determine the absolute
configuration of the glucopyranose, compound 5 was hydrolyzed by 2 M CF3CO2H. The trimethyl-silylthiazolidine derivative of the
hydrolysate showed a peak at 16.56 min in the GC, which was consistent with that of standard D-glucose (Rt = 16.63 min), rather than
L-glucose (Rt = 17.29 min). Thus, the absolute configuration of the glucose was determined as D. The β-glucosyl linkage was deduced
from the coupling constant J = 7.6 Hz of the anomeric protons H-1′′. The aglycone, which was obtained from the cellulase hydrolysis of
5, was identified to be a mixture of 2R and 2S-farrerol (4a and 4b, respectively) by Chiralpack AD-RH HPLC and NMR analysis.
Therefore, compound 5 was elucidated as (2R/2S)-farrerol 7-O-β-D-glucopyranoside.
SciFinder search showed that (2R)-farrerol 7-O-β-D-glucopyranoside was reported from R. concinnum by Zhao and their
colleagues31. However, when we study the detailed structures in the paper we found that the authors incorrectly assgined the absolute
configuration of their compound as (2R)-farrerol 7-O-β-D-glucopyranoside based on a negative Cotton effect at 270 nm and a positive
Cotton effect at 306 nm in the CD spectrum. Actually, that CD stpectrum should suggest a 2S configuration of flavanonol according to
the rules of Slade22,28. In particular, the authors claimed that the compound was known, and the data for the compound was the same as
that of a compound in the reference12. In fact, there was a (2S)-farrerol 7-O-β-D-glucopyranoside, instead of (2R)-farrerol7-O-β-D-
gluco-pyranoside, in their cited reference12. Therefore, the real (2R)-farrerol 7-O-β-D-glucopyranoside is never reported. Thus, in
compund 5 the epimer (2R)-farrerol 7-O-β-D-glucopyranoside (5a) is new, while the other (5b) is known.
The HRESIMS data at m/z 617.1762 [M + Na]+ and the 13C NMR data determined the molecular formula of compound 6, a yellow
amorphous solid, to be C28H34O14 (calcd for C28H34O14Na, 617.1846). The NMR spectra of 6 revealed that there were two sets of
resonances for a 6,8-di-C-methyl-5,7,4′-trihydroxy-flavanone, a glucopyranose, and a xylopyranose. Similar to those of compound 5,
the NMR data suggested that compound 6 was also isolated as a mixture of two epimers (6a and 6b) at C-2, which could not be
seperated by an RP C18 or a Chiralpack AD-RH HPLC. HMBC correlations from the anomeric protons H-1′′ of the glucose to C-7, and
from the anomeric protons H-1′′′ of the xylose to C-6′′ of the glucose established the connection of the glucose unit to C-7, and the
xylose unit to C-6′′ of the glucose unit, respectively. The aglycone, obtained from the cellulase hydrolysis of 6, was identified to be a
mixture of 2R and 2S-farrerol (4a and 4b, respectively) by Chiralpack AD-RH HPLC analysis. To determine the absolute configuration
of the glucopyranose and the xylopyranose, compound 6 was hydrolized by 2M CF3CO2H, and the trimethylsilylthiazolidine derivatives
of the hydrolysate was prepared. Comparison the retention times of the trimethylsilylthiazolidine derivatives of hydrolysate with those
of standards D- and L-glucose, and D- and L-xylose, assigned the absolute configuration of the glucose and xylose in compound 6 to be
D. The β-linkage of the D-glucose and D-xylose in compound 6 were deduced from the coupling constants of anomeric protons of the
glucose (δH 4.78, 4.77, d, J = 7.6 Hz, H-1′′) and the xylose (δH 4.16, d, J = 7.0 Hz, H-1′′′). Hence, the structure of compound 6 was
eluciated as (2S/2R)-farrerol 7-O-β-D-xylopyranosyl(1→6)-β-D-glucopyranoside.
Since farrerol (4b) has been reported to possess the neuroprotective activity against Aβ-induced cell death7, compounds 1–9 were
evaluated for neuroprotective effects on human neuroblastoma SH-SY5Y cells using the MTT method. As shown in Table 3,
compounds 1–3 and 5–9 exhibited significant neuroprotective effects against H2O2-induced SH-SY5Y cell apoptosis. Among them, 6,8-
di-C-flavonols (3, 8, and
Table 3 Neuroprotective effects of compounds 1–9 against hydrogen peroxide (H2O2 300 μM) and amyloid-β-protein (Aβ 1.0 μM)-induced human
neuroblastoma SH-SY5Y cells apoptosis. Data (cell viability (%) assessed using MTT assay) are expressed as mean ± SEM based on three independent
experiments.
H2O2 (300 μM)
Aβ (1.0 μM)
Concentration of test compounds (μM)
Concentration of test compounds (μM)
0.1
1
10
Modela
0.1
1
10
Modela
Cell viability
(%)
Vitamin Eb
82.1 ± 1.4
83.7 ± 2.0
82.2 ± 1.3
103.7 ± 2.3
76.9 ± 2.1
73.9 ± 0.9
73.9 ± 0.9
73.9 ± 0.9
76.4 ± 1.2
71.0 ± 2.9
71.0 ± 2.9
71.0 ± 2.9
73.9 ± 0.9
76.4 ± 1.2
80.2 ± 1.0
94.2 ± 4.0
90.6 ± 2.5
90.4 ± 5.3
85.4 ± 1.4
88.4 ± 3.5
87.1 ± 0.7
88.2 ± 4.3
94.3 ± 3.6
73.3 ± 0.7
76.1 ± 1.3
76.1 ± 1.3
77.0 ± 1.6
73.3 ± 0.7
73.3 ± 0.7
73.3 ± 0.7
76.1 ± 1.3
77.0 ± 1.6
92.8 ± 2.4
84.8 ± 3.3
91.8 ± 2.1
79.9 ± 4.8
78.2 ± 1.8
80.9 ± 1.3
96.9 ± 2.2
92.8 ± 1.6
91.9 ± 2.4
86.1 ± 3.6
88.1 ± 1.2
83.2 ± 1.9
83.1 ± 3.7
84.4 ± 1.6
85.6 ± 0.6
88.2 ± 1.8
1
2
3
4
5
6
7
8
77.5 ± 2.0
79.8 ± 2.3
98.7 ± 1.4
74.6 ± 0.7
78.9 ± 1.8
75.6 ± 1.8
84.3 ± 0.9
98.7 ± 2.7
79.4 ± 2.1
82.2 ± 1.4
101.9 ± 1.9
77.3 ± 1.8
79.9 ± 0.9
79.6 ± 0.5
91.1 ± 3.4
104.8 ± 1.8
78.2 ± 1.5
80.0 ± 2.4
92.8 ± 1.6
107.8 ± 0.6
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9
85.0 ± 1.7
87.2 ± 3.5
89.0 ± 2.1
76.4 ± 1.2
81.5 ± 2.0
90.2 ± 2.4
93.8 ± 1.2
77.0 ± 1.6
a Model group was only treated with H2O2 or Aβ
b Positive group
 P < 0.05  P < 0.01 compared with the H2O2-treated model group.  P < 0.05  P < 0.01 compared with the Aβ-treated model group
6

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9) showed more potent neuroprotection than 6,8-di-C-flavanonols (1, 2, and 7) and 6,8-di-C-flavanones (5 and 6). Thus, a larger
conjugated system in the molecules of flavonones may be beneficial for the neuroprotective activity against H2O2-induced SH-SY5Y
cell apoptosis. Comparing the structures and activities it seems that the glycosidation at A ring of the 6,8-di-C-flavonoids does not
affect the activity. In addition, compounds 1–9 displayed more significant neuroprotective effects against Aβ-induced SH-SY5Y cell
apoptosis than the positive control, 10 μM vitamin E. Our bioassay data confirmed that 6,8-di-C-flavonoids possess potential
neuroprotective effects7.
3. Experimental section
3.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer PE-341 polarimeter (Perkin-Elmer, Waltham, MA, USA). UV spectra were
recorded on a Varian Cary 50 UV/VIS spectrophotometer (Varian, Salt Lake City, UT, USA). CD spectra were measured with a Jasco
J-810 spectrometer (Jasco, Easton, MD, USA). IR spectra were recorded on a Bruker Vertex 70 FT-IR spectrophotometer (Bruker,
Karlsruhe, Germany). NMR spectra were recorded on Bruker AM-400 spectrometers (Bruker, Karlsruhe, Germany). Chemical shifts
were given in ppm with reference to the residual methanol-d4 (δH 3.31/δC 49.0), pyridine-d5 (δH 8.74/δC 150.3), and DMSO-d6 (δH
2.50/δC 39.5) signals. HRESIMS data were acquired using a Thermo Fisher LTQ XL LC/MS (Thermo Fisher, Palo Alto, CA, USA).
Column chromatography was performed using silica gel (100−200 mesh and 200−300 mesh, Qingdao Marine Chemical Inc., Qingdao,
China), ODS (50 μm, YMC, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Semipreparative HPLC was
performed on an Agilent 1100 HPLC (Agilant, Santa Clara, CA, USA) with UV detector and an Ultimate XB-C18 (10 × 250 mm, 5 μm)
column. The chiral HPLC isolation was accomplished by a Daicel Chiralpak AD-RH (4.6 × 150 mm, 5 μm; Daicel Chemical Ltd,
Tokyo, Japan) on a Dionex Ultimate 3000 HPLC (Dionex, Sunnyvale, CA, USA). Solvents were distilled prior to use, and
spectroscopic grade solvents were used. TLC was carried out on precoated silica gel GF254 plates. GC analysis was carried out on an
Agilent 7820A gas chromatography system (Agilant, Santa Clara, CA, USA).
3.2. Plant materials
Leaves of R. fortunei were collected at about 1500 m altitude, latitude 30°10′39.47″ North and longitude 109°44′42.93″ East in
Changlinggang, Enshi, Hubei Province, People′s Republic of China, in June 2010, and authenticated by one of the authors (M He). A
voucher specimen (No. 20100613) was deposited in the herbarium of Hubei Key Laboratory of Natural Medicinal Chemistry and
Resource Evaluation, Tongji Medical College, Huazhong University of Technology and Science.
3.3. Extraction and isolation
The dried and powdered twigs and leaves of R. fortunei (6.0 kg) were extracted with 95% EtOH (30 L) at room temperature for four
times. After the solvent was removed under reduced pressure, the EtOH extract (800 g) was suspended in H2O and partitioned with
petroleum ether, CHCl3, EtOAc, and n-BuOH, successively. The EtOAc partition fraction (31.3 g) was separated on a silica gel CC
(200–300 mesh) eluted with CHCl3–MeOH (from 1:0 to 3:1) to obtain six fractions (EA–EF). Fraction EB was subjected to ODS CC
(50–100% MeOH) to obtain EB1–EB6. Subfraction EB3 eluted by 65% MeOH was purified on a silica gel CC, eluting with petroleum
ether–Me2CO (3.5:1) to obtain Compound 7 (18 mg). Subfraction EB4 eluted by 75% MeOH was fractionated on a silica gel CC,
eluting with CHCl3-MeOH (25:1) to give three fractions EB4.1, EB4.2, and EB4.3. Fraction EB4.1 was purified by Sephadex LH-20 and
silica gel CC to afford compound 4 (9.6 mg), which was further isolated on a Chiralpack AD-RH (CH3CN–H2O = 35:65; flow rate 1.0
mL/min) to obtain compounds 4a (0.4 mg, tR = 18.5 min) and 4b (0.6 mg, tR = 20.2 min). Compound 9 (13.7 mg) was obtained by
recrystallization from fraction EB4.1 in MeOH. Fractions EE was subjected to ODS CC eluted with MeOH–H2O (20:80→100:0) to
afford seven subfractions EE1–EE7. The subfraction EE3 was subjected to Sephadex LH-20 CC and then purified by semi-preparative
HPLC (MeOH–H2O = 41:59, flow rate: 2 mL/min) to obtain compound 1 (64 mg, tR = 24.4 min). Compound 5 (8.2 mg, tR = 23.6 min)
was isolated from the subfraction EE4 by repeated chromatography including silica gel CC, ODS, and Sephadex LH-20 and final semi-
preparative HPLC (MeOH–H2O = 54:46, flow rate 2 mL/min). The subfraction EE5 was first fractionated by silica gel CC (CHCl3–
MeOH = 8:1) and further purified by semi-preparative HPLC (MeOH–H2O = 62:38, flow rate: 2 mL/min) to afford compound 8 (15.4
mg, tR = 21.0 min).
The n-BuOH extract (68.5 g) was subjected to silica gel CC eluted with CHCl3–MeOH–H2O (10:1:0→2:1:0.01) to give five fractions
BA–BE. Fraction BD was separated by ODS CC eluted with MeOH–H2O (20:80, 40:60, 60:40, 80:20, and 100:0) to give five fractions
(BD1–BD5), and BD2 was fractionated by silica gel CC again to three subfractions BD2.1, BD2.2, and BD2.3. Compound 6 (110 mg, tR =
28 min) was purified by semi-preparative (MeOH-H2O = 43:57, flow rate: 2.5 mL/min) from subfraction BD2.2. Subfraction BD2.3 was
further purified by repeated silica gel and Sephadex LH-20 CC to give compound 2 (1 g). Fraction BD3 was subjected to repeated
chromatography including silica gel CC, ODS, and Sephadex LH-20, and finally purified by semi-preparative HPLC (MeOH–H2O =
60:40, flow rate: 2 mL/min) to obtain compound 3 (47 mg).
(2R,3R)-6,8-di-C-methyldihydrokaempferol 7-O-β-D-glucopyr-anoside (1): Yellowish amorphous solid, []20 D +30.8 (c 0.13,
MeOH); UV (MeOH) λmax (log ε) 285 (4.23), 368 (3.60) nm; CD (c 0.44 mM, MeOH) 299 (Δε –5.68), 353 (Δε +2.60) nm; IR (film)
vmax 3381, 2922, 1630, 1518, 1450, 1362, 1271, 1174, 1107, 1070, 1039 cm–1; 1H and 13C NMR data, see Tables 1 and 2, respectively;
(+)-HRESIMS: m/z 501.1354 [M+Na]+ (calcd for C23H26O11Na, 501.1373).
(2R,3R)-6,8-di-C-methyldihydrokaempferol 7-O-β-D-xylopyra-nosyl(1→6)-β-D-glucopyranoside (2): Yellowish amorphous solid,
[]20 D +0.4 (c 0.38, MeOH); UV (MeOH) λmax (log ε) 283 (4.19)， 361 (3.55) nm; CD (c 0.26 mM, MeOH) 295 (Δε –5.98), 353 (Δε
+2.88) nm; IR (film) vmax 3371, 2924, 1628, 1518, 1448, 1364, 1271, 1173, 1107, 1067 cm–1; 1H and 13C NMR data, see Tables 1 and 2,
respectively; (+)-HRESIMS: m/z 633.1785 [M+Na]+ (calcd for C28H34O15Na, 633.1795).
6,8-di-C-methylkaempferol 7-O-β-D-xylopyranosyl(1→6)-β-D-glucopyranoside (3): Yellow amorphous solid, []20 D –4.5 (c 0.20,
MeOH : DMSO = 4:1); UV (MeOH) λmax (log ε) 276 (4.31), 334 (4.24), 378 (4.23) nm; IR (KBr) vmax 3402, 2923, 1641, 1612, 1555,
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1
1511, 1478, 1419, 1381, 1339, 1299, 1281, 1174, 1120, 1069 cm–1; H and 13C NMR data, see Tables 1 and 2, respectively; (+)-
HRESIMS: m/z 631.1621 [M+Na]+ (calcd for C28H32O15Na, 631.1639).
(2R)-farrerol (4a): Yellowish amorphous solid; []20 D –21.8 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 297 (4.11), 347(3.53) nm;
CD (c 0.33 mM, MeOH) 290 (Δε +5.47), 334 (Δε –1.63) nm; IR (KBr) vmax 3400, 2921, 1637, 1587, 1516, 1444, 1362, 1229, 1174,
1
1117, 1066, 1031 cm–1; H and 13C NMR data, see Tables 1 and 2, respectively; (+)-HRESIMS: m/z 301.1069 [M+H]+ (calcd for
C17H17O5, 301.1076).
(2R/2S)-farrerol 7-O-β-D-glucopyranoside (5): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 283 (4.21), 363 (3.56) nm; IR
(KBr) vmax 3378, 2920, 1627, 1518, 1445, 1351, 1278, 1176, 1124, 1069, 1038 cm–1; 1H NMR (CD3OD, 400 MHz) δ 7.33(4H, d, J = 8.4
Hz, H-2′ and H-6′), 6.83 (4H, d, J = 8.4 Hz, H-3′ and 5′), 5.33-5.36 (2H, H-2), 4.74 and 4.73 (2H, d, J = 7.6 Hz, H-G1′′), 3.79-3.75 and
3.68-3.64 (4H, H-G6′′), 3.53 (2H, t, J = 8.3 Hz, H-G2′′), 3.44 and 3.43 (2H, t, J = 8.8 Hz, H-G3′′), 3.39 (2H, t, J = 8.8 Hz, H-G4′′),
3.19-3.14 (2H, H-G5′′), 3.15-3.08 and 2.82-2.76 (4H, H-3), 2.14 (6H, s, Me-6), 2.13 (6H, s, Me-8). 13C NMR (CD3OD, 100 MHz) δ
200.1 and 200.0 (C-4), 162.6 and 162.4 (C-7), 159.8 (C-5), 159.3 (C-9), 158.9 (C-4′), 131.3 (C-1′), 128.9 (C-2′ and C-6′), 116.4 (C-3′
and C-5′), 113.0 (C-6), 111.9 and 11.8 (C-8), 106.3 (C-10), 105.3 (C-G1′′), 80.1 (C-2), 78.1 (C-G5′′), 77.9 (C-G3′′), 75.7 (C-G2′′), 71.5
(C-G4′′), 62.7 (C-G6′′), 44.3 (C-3), 9.8 (Me-8), 9.1 (Me-6); (+)-HRESIMS: m/z 485.1408 [M+Na]+ (calcd for C23H26O10Na, 485.1424).
(2R/2S)-farrerol 7-O-β-D-xylopyranosyl(1→6)-β-D-glucopyra-noside (6): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 282
(4.23), 363 (3.59) nm; IR (KBr) vmax 3370, 2926, 1630, 1612, 1518, 1444, 1349, 1278, 1170, 1124, 1065, 1045 cm–1; 1H NMR (CD3OD,
400 MHz) δ 7.36 and 7.35 (4H, d, J = 8.5 Hz, H-2′ and H-6′), 6.83 (4H, d, J = 8.5 Hz, H-3′ and 5′), 5.44 and 5.37 (2H, dd, J = 2.7, 13.0
Hz, H-2), 4.78 and 4.77 (2H, d, J = 7.6 Hz, H-G1′′), 4.16 (2H, d, J = 7.0 Hz, H-X1′′′), 3.98-3.95 and 3.75-3.69 (4H, H- G6′′), 3.82-3.77
and 3.07-3.02 (4H, H-X5′′′), 3.55-3.50 (2H, H-G2′′), 3.46-3.39 (4H, H-G5′′ and H-X4′′′), 3.37-3.35 (4H, H-G3′′ and H-G4′′), 3.28-3.13
and 2.80-2.73 (4H, H-3), 2.13 (6H, s, Me-6), 2.11 and 2.10 (6H, s, Me-8). 13C NMR (CD3OD, 100 MHz) δ 200.1 and 200.0 (C-4), 162.6
and 162.4 (C-7), 159.8 (C-5), 159.3 (C-9), 158.9 (C-4′), 131.3 (C-1′), 129.0 (C-2′ and C-6′), 116.3 (C-3′ and C-5′), 112.9 (C-6), 112.0
and 111.8 (C-8), 106.4 (C-10), 104.8 (C-G1′′), 104.7 and 104.6 C-X1′′′), 80.4 and 80.0 (C-2), 78.1 and 77.9 (C-G3′′), 77.8 (C-G5′′),
77.6 and 77.5 (C-X3′′′), 75.8 (C-G2′′), 74.8 and 74.7 (C-X2′′′), 71.5 (C-G4′′), 71.1 (C-X4′′′), 69.4 and 69.2 (C-G6′′), 66.7 and 66.6 (C-
X5′′′), 44.4 and 44.2 (C-3), 9.8 (Me-8), 9.2 and 9.1 (Me-6); (+)-HRESIMS: m/z 617.1762 [M+Na]+ (calcd for C28H34O14Na, 617.1846).
3.4. Acid hydrolysis of compounds 1–3, 5, and 6 and determina-tion of the absolute configuration of monosaccharides
Compounds 1 (2 mg), 2 (4 mg), 3 (4 mg), 5 (4 mg), and 6 (3.5 mg) were each refluxed in 2M CF3CO2H (MeOH–H2O, 1:4, 2 mL) for
2 h. The reaction mixture was then evaporated under reduced pressure to dryness until neutral. The residue was diluted in H2O (2 mL)
and then extracted with EtOAc (2 mL × 3). The H2O layer including the sugar component was evaporated, and the part of sugar residue
was compared with the authentic sample by TCL (silica gel; CHCl3–MeOH–H2O, 6:4:1). The other residue and L-cysteine methyl ester
hydrochloride (3 mg) were dissolved in 2 mL re-distilled dry pyridine and heated at 60 °C for 2 h. The reaction mixture was
concentrated to dryness and then 400 μL trimethylsilyl imidazole was added to the residue, followed by incubation at 60 °C for 1 h32.
Finally, the mixture was partitioned between n-hexane (600 μl) and H2O (2 mL). The n-hexane fraction was detected by GC under the
following conditions: capillary column, OV-17 (30 m × 0.32 mm × 0.5 μm, Lanzhou Zhongke Antai Analysis Technology Co., Ltd.,
China); the column temperature was raised from 220 °C to 250 °C at the rate of 2 °C/min and then maintained for 10 min; injection
temperature, 250 °C; detector FID, detector temperature, 250 °C; carrier gas, N2; split ratio, 10:1; flow rate, 1.0 mL/min. The reference
D-xylose, and L-xylose, D-glucose, L-glucose derivatives were prepared by similar reaction, which showed retention times of 11.86,
12.51, 16.63, and 17.29 min, respectively. By comparing the retention times of the trimethylsilylthiazolidine derivatives of the
hydrolysate and the standards, the monosaccharides in compounds 1 and 5 was determined to be D-glucose, and the monosaccharides in
compounds 2, 3, and 6 were identified as D-xylose and D-glucose.
3.5. Cellulase hydrolysis of compounds 5 and 6
Compounds 5 (4 mg) and 6 (6 mg) were each hydrolyzed by cellulase (50 mg) in H2O (10 mL) at 50 °C for 24 h. The reaction
mixture was extracted by EtOAc (2 mL × 5). The aglycone in the EtOAc extraction was further analyzed by Chiralpack AD-RH HPLC.
3.6. Neuroprotective activities of compounds 1–9 against H2O2- or Aβ-induced SH-SY5Y cells apoptosis
The neuroprotective activities of compounds 1–9 against H2O2- or Aβ-induced apoptosis in human neuroblastoma SH-SY5Y cells
were evaluated using the MTT method. The SH-SY5Y cells (100μl) were seeded into 96-well plates at a density of 4 × 104 cells/well in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 100 unit/mL penicillin and 100 μg/mL
streptomycin in a humid atmosphere of 5% CO2 at 37 °C and incubated for 24 h3. Then the cells were pre-treated with test compounds
(10, 1, and 0.1 μM) and Vitamin E (10 μM, positive control) for 1 h before the addition of H2O2 or aggregated Aβ (3 days at 37 °C).
After 24 h exposure to H2O2 or Aβ, 10 μL of MTT (5 mg/mL in PBS) were added to each well and incubated for 4 h at 37 °C in the
dark. The formazan converted from MTT was dissolved in 100 μL DMSO in each well. The absorbance was measured at 570 nm in an
ELISA plate reader. The results were presented as the percentage of survival relative to the controls. Data were expressed as means ±
standard errors of the mean (SEM). Statistical analyses were performed using Dunnett-t test after analysis of variance.
Acknowledgements
We thank Professor Y. Chen at Hawaii Pacific University for editing the manuscript, and the Analytical and Testing Center at
Huazhong University of Science and Technology for assistance in conducting CD and IR analyses. We greatly acknowledge the
financial supports from the National Natural Science Foundation of China (81001368 and 31170323), the Natural Science Foundation
of Hubei Province of China (2010CDB03206), the Specialized Research Fund for the Doctoral Program of Higher Education
(20100142120069), the Scientific Research Foundation for the Returned Oversea Chinese Scholars (G. Yao), the State Education
Ministry of China, and the Fundamental Research Funds for the Central Universities (HUST: 2014 QN131).
Competing financial interests
8

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The authors declare no competing financial interests.
Appendix A. Supplementary data
Supplementary data associated with this article including HRESIMS, 1D and 2D NMR spectra, IR, UV, and CD spectra, and GC
analysis of sugar for compounds 1−6 are can be found online at http://
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Conflict of interest statement
The authors (Yongji Lai, Hong Zeng, Meijun He, Huiqin Qian, Zhaodi Wu, Zengwei
Luo, Yongbo Xue, Guangmin Yao, Yonghui Zhang) declared that they have no conflicts of
interest to this work (6,8-Di-C-methyl-flavonoids with neuroprotective activities from
Rhododendron fortunei).
We declare that we do not have any commercial or associative interest that represents
a conflict of interest in connection with the work submitted.
Yonghui Zhang
6.16.2016
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ACCEPTED MANUSCRIPT
6,8-Di-C-methyl-flavonoids with neuroprotective activities
from Rhododendron fortunei
Yongji Laia,b, Hong Zenga, Meijun Hec, Huiqin Qiana, Zhaodi Wua, Zengwei Luoa, Yongbo Xuea,
Guangmin Yaoa,*, Yonghui Zhanga,*
Six 6,8-di-C-methyl-flavonoids (1–3, 4a, 5–6) and four known compounds (4b, 7–9) were isolated. Their
neuroprotective activities were evaluated.
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