Anti-complementary components of Helicteres angustifolia

Article abstract of DOI:10.3390/molecules21111506

A first phenalenon derivative with an acetyl side chain at C-8, 8-acetyl-9-hydroxy-3-methoxy-7-methyl-1-phenalenon (compound 1), and a pair of new sesquilignan epimers at C-7″ of hedyotol C and hedyotol D analogs, hedyotol C 7″-O-β-D-glucopyranoside (compound 2) and hedyotol D 7″-O-β-D-glucopyranoside (compound 3) were isolated from the aerial parts of Helicteres angustifolia together with nine known compounds (4-12). Their structures were elucidated on the basis of spectroscopic methods, including mass spectroscopy, and 1D and 2D nuclear magnetic resonance. Eleven isolates exhibited anti-complementary activity. In particular, compounds 4 and 5 exhibited potent anti-complementary activities against the classical and alternative pathways with CH₅₀ values of 0.040 ± 0.009 and 0.009 ± 0.002 mM, and AP₅₀ values of 0.105 ± 0.015 and 0.021 ± 0.003 mM, respectively. The targets of compounds 4 and 5 in the complement activation cascade were also identified. In conclusion, the anti-complementary components of H. angustifolia possessed chemical diversity and consisted mostly of flavonoids and lignans in this study.

Full text of DOI:10.3390/molecules21111506

molecules
Article
Anti-Complementary Components of
Helicteres angustifolia
Xiang Yin 1, Yan Lu 2, Zhi-Hong Cheng 2 and Dao-Feng Chen 1,2,
*
1
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
yinxiang2zhy@163.com
Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China;
luyan@fudan.edu.cn (Y.L.); chengzhh@fudan.edu.cn (Z.-H.C.)
2
*
Correspondence: dfchen@shmu.edu.cn; Tel.: +86-21-5198-0135; Fax: +86-21-5198-0017
Academic Editor: Derek J. McPhee
Received: 11 October 2016; Accepted: 6 November 2016; Published: 10 November 2016
Abstract: A first phenalenon derivative with an acetyl side chain at C-8, 8-acetyl-9-hydroxy-
3-methoxy-7-methyl-1-phenalenon (compound
of hedyotol C and hedyotol D analogs, hedyotol C 7”-O-
hedyotol D 7”-O- -D-glucopyranoside (compound ) were isolated from the aerial parts of Helicteres
angustifolia together with nine known compounds ( 12). Their structures were elucidated on the
basis of spectroscopic methods, including mass spectroscopy, and 1D and 2D nuclear magnetic
resonance. Eleven isolates exhibited anti-complementary activity. In particular, compounds and
exhibited potent anti-complementary activities against the classical and alternative pathways
with CH50 values of 0.040 0.009 and 0.009 0.002 mM, and AP50 values of 0.105 0.015 and
and in the complement activation
1
), and a pair of new sesquilignan epimers at C-7”
β
-D-glucopyranoside (compound ) and
2
β
3
4
–
4
5
±
±
±
0.021 ± 0.003 mM, respectively. The targets of compounds
4
5
cascade were also identified. In conclusion, the anti-complementary components of H. angustifolia
possessed chemical diversity and consisted mostly of flavonoids and lignans in this study.
Keywords: Helicteres angustifolia; sterculiaceae; phenalenone; sesquilignan; anti-complement
1. Introduction
The complement system, as a chief component of innate immunity, plays a significant role in
host immune defense against infection and in the clearance of antigen-antibody complexes from
the bloodstream. It can be activated by a cascade mechanism through the classical pathway (CP)
,
alternative pathway (AP) or the lectin pathway [ ]. Generally, the normal activation of the
1
complement system leads to elimination of invading pathogens and optimal host response.
However, excessive activation of the complement system may induce tissue damage, inflammation
and a number of pathological situations such as systemic lupus erythematosus, rheumatoid arthritis
and acute respiratory distress syndrome [2,3]. Thus, inhibition of the complement system is
potentially therapeutic in diseases resulting from uncontrolled or overshooting complement activation.
The desirable properties of a valuable anti-complementary therapeutic agent are that it should be
inexpensive and highly specific, either having a long plasma half-life or being active orally [2].
However, none of the currently available agents meet all these criteria. A wide array of specific
complement inhibitors, for instance cobra venom factor (CVF), synthetic nafamastat mesilate (FUT-175),
a recombinant humanized antibody fragment, and a monoclonal antibody, Eculizumab, have been
developed to target various components of the complement cascade [4–8]. However, the clinical
development of this plethora of complement therapeutics has exhibited some side effects. Therefore,
there is an urgent need to search for naturally occurring and low-toxicity anti-complementary agents
from traditional Chinese medicines.
Molecules 2016, 21, 1506; doi:10.3390/molecules21111506
www.mdpi.com/journal/molecules

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The dried roots or whole plants of Helicteres angustifolia L. (Sterculiaceae) have been used
in Chinese folk medicine as anti-inflammatory, antidotal, analgesic, anti-bacterial and anti-cancer
agents [
penylpropanoids, quinones, lignans and flavonoids [10
complement system is a crucial trigger for inflammation [13
9
]. Previous phytochemical studies revealed that this plant mainly contains triterpenoids,
12]. It is commonly accepted that the
14]. Therefore, one may hypothesize
–
,
that complement inhibition intercepts the process of complement-dependent inflammation diseases.
In our effort to search for anti-complementary agents from Chinese herb medicines and to clarify their
anti-complementary constituents [
was found to show a potent anti-complementary activity, which encouraged us to investigate its
anti-complementary constituents systematically. In this study, one new skeleton compound ( ) and
two new compounds ( and ) were isolated from the aerial parts of this plant, together with nine
3,15,16], an ethanolic extract of the aerial parts of H. angustifolia
1
2
3
known compounds, on the basis of MS, 1D and 2D NMR, as well as comparison with the literature.
Herein, the isolation, characterization, and anti-complementary activity of these compounds are
reported. In addition, the target identification in the complement activation cascade has been
investigated for anti-complementary constituents.
2. Results and Discussion
In our search for anti-complementary activity compounds from plants, the 95% ethanolic extract
of H. angustifolia possessed a potent anti-complementary activity. Thus, the EtOAc-soluble fraction
was concentrated under reduced pressure to produce a residue that was subjected to multiple
chromatographies. From this active fraction, 12 compounds (
compound (1) and two new compounds (2 and 3) were isolated and identified.
Compound (Figure 1) was obtained as yellow crystals (CHCl3), and its molecular formula was
determined as C17H14O4 from HR-ESI-MS (m/z 283.0962 [M + H]+, calcd. for C17H15O4+, 283.0965).
The 1H-NMR spectrum (Table 1) exhibited signals for three aromatic protons (δH 8.43 (1H, d, J = 7.8 Hz
8.24 (1H, d, J = 7.8 Hz) and 7.58 (1H, t, J = 7.8 Hz)), an olefinic methine (δH 6.66 (1H, s)), two methyls
1–12) including one new skeleton
1
)
(
δH 2.72 and 2.58 (each 3H, s)), a methoxyl (δH 3.92 (3H, s)) and a peri-hydroxy (δH 17.36 (1H, s)),
as well as corresponding with 13C-NMR resonances at δC 127.9, 130.8, 123.7, 100.2, 32.1, 16.0, 56.4 and
174.2, respectively. Furthermore, the 13C-NMR spectrum (Table 1) showed two carbonyl carbon signals
(
δC 204.1 and 181.1) and eight quaternary carbon signals. The 1H-1H COSY spectrum revealed the
presence of a subunit C-4–C-5–C-6 (Figure 2). These NMR signals indicated that compound
1 had
a phenalenone skeleton as in the known compounds 7-methyl-1-phenalenon [17] and myeloconone
A2 [18]. The HMBC (Figure 2) correlations from 7-CH3 (δH 2.58) to C-7 (δC 144.1), C-8 (δC 136.3) and
C-6a (δC 125.4) indicated that the methyl was attached to C-7. Different from 7-methyl-1-phenalenon,
compound 1 possessed a methoxyl, an acetyl and a peri-hydroxy group in its structure. The HMBC
(Figure 2 and see Supplementary Materials) correlations from 3-OCH3 (δH 3.92) to C-3 (δC 166.6),
8-COCH3 (δH 2.72) to 8-COCH3 (δC 204.1) and C-8 (δC 136.3) further confirmed that the methoxyl was
connected to C-3, and the acetyl group was connected to C-8. Consequently, the structure of
1
was
deduced as 8-acetyl-9-hydroxy-3-methoxy-7-methyl-1-phenalenon. To our knowledge,
phenalenon derivative with an acetyl side chain connected to C-8.
1 is the first
1
Table 1. H- and 13C-NMR (600 and 150 MHz) data of compound 1 in C5D5N, δ in ppm, J in Hz.
No.
δ
δC
No.
δ
δC
H
H
1
2
181.1
100.2
166.6
120.8
127.1
127.9
123.7
130.8
125.4
7
144.1
136.3
6.66 s
8
9
3
174.2
3a
3b
4
9a
106.5
3-OCH3
7-CH3
8-COCH3
3.92 s
2.58 s
56.4
8.43 d (7.8)
7.58 t (7.8)
8.24 d (7.8)
16.0
5
204.1 (CO)
32.1 (CH3)
6
2.72 s
17.36 s
6a
9-OH

Molecules 2016, 21, 1506
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Figure 1. Structures of compounds 1–12.
Figure 2. Key HMBC and 1H-1H COSY correlations for compound 1.
Compound
2
(Figure 1) was obtained as white amorphous powder, and the molecular formula
+
was determined as C37H46O16 by HR-ESI-MS (m/z 769.2662 [M + Na]+, calcd. for C37H46NaO16
,
769.2678). The 1H- and 13C-NMR spectra (Table 2) showed characteristic signals for a sesquilignan
similar to hedyotol C [19], including eight aromatic protons (two sets of 1,2,4-trisubstituted phenyl
rings and a 1,2,3,5-tetrasubstituted phenyl ring), four methoxyl groups, two phenolic and five alcoholic
hydroxyl groups, one furan and one glycerol unit. In addition, an anomeric proton resonated
at δH 4.67 (1H, d, J = 7.8 Hz) and the large coupling constant indicated
Detailed inspection of the NMR data revealed that the structure of compound
hedyotol C 4”-O- -D-glucopyranoside [20], except that the glucosyl unit was connected to C-7” in
rather than to C-4”, as indicated by the HMBC correlations from H-1”0 (δH 4.67) to C-7” (δC 82.3) and
H-7” (δH 5.15) to C-1”0 (δC 104.4). The nature of sugar in compound
was further demonstrated to
be D-glucose by GC analysis of an acid-treated hydrolysate, as well as comparison of their 13C-NMR
data with that in the literature [20]. The relative configuration of compound was determined by
interpretation of the NOESY spectrum and the chemical shift ratio of C-7”:C-8” [21]. In the NOESY
β-glucosidic linkage.
2
was similar to
β
2
2
2

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spectrum of compound
2
(See Figure S13), the correlation of H-7” (δH 5.15) with H-8” (δH 4.42–4.44)
was observed, suggestive of the same
ratio of C-7”:C-8” (δC 82.3:86.6) to those of hedyotol C 4”-O-
configuration of compound was proposed as an erythro configuration. Therefore, compound
α
-orientation. Moreover, based on the similar chemical shift
β-D-glucopyranoside [20], the relative
2
2
was
established as hedyotol C 7”-O-β-D-glucopyranoside.
Table 2. 1H- and 13C-NMR (600 and 150 MHz) data for compounds
J in Hz.
2
and
3
in CD3OD,
δ
in ppm,
2
3
No.
δ
δC
δ
δC
H
H
1
2
3
4
5
6
7
8
133.7
111.0
149.1
147.4 a
116.1
120.1
87.5
133.7
111.0
149.1
6.97 d (1.8)
6.97 d (1.4)
147.4 a
116.1
120.1
87.5
6.79 d (8.1)
6.83 dd (8.1, 1.7)
6.79 d (8.1)
6.83 dd (8.0, 1.3)
4.73 d (3.6)
3.13–3.15 overlapped
4.27–4.31 overlapped,
3.88–3.91 overlapped
4.72 d (4.8)
55.3 b
55.3 b
72.9 c
3.14 ddd (11.3, 8.3, 4.9)
4.24–4.30 overlapped,
3.88–3.91 overlapped
9
72.9 c
0
1
138.9
104.2
154.3
136.2
87.2
138.9
104.1
154.5
136.4
87.3
0
0
0
2 ,6
6.64 s
6.66 s
0
4
7
8
3 ,5
0
0
4.75 d (4.6)
4.76 d (3.0)
0
55.7 b
72.8 c
55.7 b
72.7 c
3.14 ddd (11.3, 8.3, 4.9)
4.24–4.30 overlapped,
3.88–3.91 overlapped
3.13–3.15 overlapped
4.27–4.31 overlapped,
3.88–3.91 overlapped
0
9
00
00
00
00
00
00
00
00
1
2
3
4
5
6
7
8
132.4
112.7
148.4
147.1 a
115.3
121.9
82.3
130.8
113.1
148.7
147.1 a
115.5
122.0
77.7
7.03 d (1.7)
7.24 brs
6.72 d (8.1)
6.87 dd (8.1, 1.7)
5.15 d (6.2)
6.80 d (8.0)
6.92 dd (8.0, 1.3)
5.28 d (3.2)
4.42–4.44 m
86.6
4.26–4.27 m
86.8
4.05 dd (12.4, 4.0)
3.76 dd (12.4, 3.0)
3.88–3.91 overlapped
3.44 dd (11.4, 4.5)
00
9
61.5
61.4
β-D-glc
000
1
2
3
4
4.67 d (7.8)
3.27-3.29 m
3.38 d (9.0)
3.30–3.32 m
3.19 ddd (9.6, 5.4, 2.5)
3.73 dd (11.9, 2.3)
3.60 dd (11.8, 5.4)
3.87 s
104.4
75.7
78.1
71.5
77.8
4.21 d (7.5)
3.33–3.34 m
3.28–3.31 overlapped
3.28–3.31 overlapped
3.12–3.13 m
3.83–3.85 m
3.69 dd (11.9, 5.9)
3.88 s
101.0
75.2
77.7
71.9
77.8
000
000
000
000
5
000
6
62.7
62.7
56.41 d
56.6
56.37 d
56.5
3-OCH3
3 ,5 -OCH3
3 -OCH3
0
00
0
3.80 s
3.83 s
3.74 s
3.85 s
56.43 d
56.42 d
a−d
The assignments in each column may be interchanged.
Compound
compound
3
(Figure 1) was determined to have the same molecular formula C37H46O16 as
1
2
by HR-ESI-MS (m/z 769.2662 [M + Na]+). Their H- and 13C-NMR spectra closely
resembled each other (Table 2). The above evidence suggests that compounds
2 and 3 might be
stereoisomeric, which could be clarified in accordance with the NOESY spectrum and the chemical

Molecules 2016, 21, 1506
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shift ratio of C-7”:C-8” [21]. The differences between compounds
2
and
3
were the absence of NOESY
correlations between H-7” (δH 5.28) and H-8” (δH 4.26–4.27) (See Figure S20) and the similar chemical
shift ratio of C-7”:C-8” (δC 77.7:86.8) as compared to those of hedyotol D 4”-O- -D-glucopyranoside [20
in compound . This evidence confirmed compounds and were a pair of epimers at C-7”.
Accordingly, the relative configuration of compound was presumed as a threo configuration.
β
]
3
2
3
3
Consequently, compound 3 was identified as hedyotol D 7”-O-β-D-glucopyranoside.
Nine known compounds were identified as machicendonal (compound
4)
[22],
(7S,8R)-dihydrodehydrodiconiferyl alcohol (compound
(compound ) [24], potengriffioside A (compound
5,7,8,30-tetrahydroxy-40-methoxyflavone (compound
(compound 10) [26], hesperidin (compound 11) [27], and homoeriodictyol-7-O-
5
) [23], kaempferol-3-O-
) [25], kaempferol (compound
) [26], 5,7,8-trihydroxy-40-methoxyflavone
-D-glucopyranoside
β
-D-glucopyranoside
6
7
8) [24],
9
β
(compound 12) (Figure 1) [28] by comparing the spectroscopic data with those reported in the
literature. Compounds 4, 8–12 were isolated from the genus Helicteres for the first time.
All the isolated constituents (compounds
activity on CP and AP [16]. As shown in Figure 1 and Table 3, compounds
benzofuran lignans and possessed the most potent anti-complementary activity with CH50 values
of 0.040 0.009 and 0.009 0.002 mM and AP50 values of 0.105 0.015 and 0.021 0.003 mM,
respectively. Seven flavonoids (compounds 12) showed anti-complementary activity with CH50
and AP50 values of 0.143–1 mM and 0.311–1 mM, respectively. Compounds exhibited moderate
1–12) were evaluated for in vitro anti-complementary
4
and belonged to the
5
±
±
±
±
6–
1–3
activity on CP and weak activity on AP. It was found that flavonoids with 40-OH (compounds
12) showed stronger anti-complementary activity than those with a -OCH3 at C-40 (compounds
Thus, the 40-OH appears to be essential for the complementary activity of flavonoids.
8
9
and
11).
–
Table 3. Anti-complementary activity of compounds 1–12 against classical and alternative pathways.
Compound
CH50 (mM) a
AP50 (mM) a
1
2
3
4
5
6
7
8
0.744 ± 0.099
0.419 ± 0.043
0.249 ± 0.021
0.040 ± 0.009
0.009 ± 0.002 **,b
0.877 ± 0.081
0.143 ± 0.019
0.147 ± 0.022
0.232 ± 0.25
0.511 ± 0.043
>1
>1
>1
>1
0.105 ± 0.015
0.021 ± 0.003 **,b
>1
0.335 ± 0.040
0.311 ± 0.033
0.501 ± 0.065
0.984 ± 0.107
>1
9
10
11
12
0.351 ± 0.033
0.556 ± 0.061
Heparin c
0.026 ± 0.005
0.055 ± 0.008
a
Data were represented as mean
±
SD of three independent measurements (n = 3), and described as 50%
hemolytic inhibitory concentration (CH50c for classical pathway and AP50 for alternative pathway). **,b p < 0.01,
compared to the positive control group. Heparin was used as the positive control (mg/mL).
In order to illuminate the anti-complementary mechanism of compounds
of their targets in the complement activation cascade was conducted using complement-depleted
(C-depleted) sera [16]. As shown in Figure 3, compound regained the hemolytic capacity of
C5-depleted serum, and compound regained the hemolytic capacity of C4- and C5-depleted
sera. These findings suggested that compound probably acted on C1q, C2, C3, C4 and C9, while
compound interacted with the C1q, C2, C3 and C9 components of the complement. These results
indicate that different compounds can act on different targets in the complement activation cascade.
Thus, compounds and are promising candidates for development as anti-complementary agents
4 and 5, identification
4
5
4
5
4
5
from H. angustifolia. Moreover, the relevant pharmacology and toxicology of these compounds need
further investigations.

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Figure 3. Hemolytic assays of compounds
4 (A) and 5 (B) for individual components utilizing
C-depleted sera.
3. Materials and Methods
3.1. General Experimental Procedures
The 1D and 2D NMR spectra were recorded on a Bruker Ultrashield Plus 600 MHz spectrometer
(Bruker BioSpin Corporation, Billerica, MA, USA). Electrospray ionization mass spectrometry (ESI-MS)
spectra were recorded on an Agilent SL G1946D single quadrupole mass spectrometer (Agilent,
Foster, CA, USA). High-resolution electrospray ionization mass spectra (HR-ESI-MS) were determined
on a Bruker micro time of flight (TOF) spectrometer (Bruker Daltonics Corporation, Billerica, MA,
USA). The infrared (IR) spectra were measured on a Thermo Nicolet Avatar 360 spectrophotometer
(Thermo Nicolet, Madison, WI, USA). Ultraviolet (UV) spectra were obtained on a Lambda
25 spectrometer (PerkinElmer, Wellesley, MA, USA). The optical rotations were measured on a JASCO
P-1020 polarimeter (JASCO Corporation, Tokyo, Japan). The melting point was measured on a
micromelting point apparatus which was uncorrected (Yuhua Instruments Co., Ltd., Gongyi, China).
Gas chromatography (GC) was carried out on a Shimadzu GC-MS QP 2010 Ultra (Shimadzu
Corporation, Kyoto, Japan). Semipreparative high performance liquid chromatography (Semi-HPLC)
was run on an Agilent 1100 series (Agilent Technologies, Waldbronn, Germany), equipped
with a DAD detector and an ODS column (250
Medium pressure liquid chromatography (MPLC) was conducted on a MITSUBISHI GOT 1000
chromatographic instrument (Lisure Science (Suzhou) Co., Ltd., Suzhou, China) with a Santai ILOK
×
10.0 mm, 5 µm, Phenomenex Luna C18).
ODS column (390 mm
×
31.2 mm i.d.). Flash column chromatography was operated by use of
normal-phase silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and
gel Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden). Anti-complement 1q
((Anti-C1q), Human (Goat)), Anti-C2 (Human (Goat)) and Anti-C9 (Human (Goat)) were purchased
from Calbiochem (Merck KGaA, Darmstadt, Germany), and Anti-C3 (Human (Goat)), Anti-C4 (Human
(Goat)) and Anti-C5 (Human (Goat)) were obtained from Zhejiang Nanfang Reagent Factory, China.
Heparin sodium salt (
Ltd., China.
≥
150 IU/mg, dry basis) was purchased from Shanghai Aizite Biotech Co.
3.2. Plant Material
Dried aerial parts of H. angustifolia were purchased from Nanning, Guangxi Zhuang Autonomous
Region of China in March 2011, and authenticated by Prof. Dao-Feng Chen at Fudan University.
A voucher specimen (DFC-SZM20110812) has been deposited in the Herbarium of Materia Medica,
Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, China.
3.3. Extraction and Isolation
The extraction of the dried aerial parts of H. angustifolia with 95% ethanol was partitioned
successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc fraction was selected to further

Molecules 2016, 21, 1506
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purification due to its significant anti-complementary activity with CH50 value of 0.15
±
0.02 and
AP50 value of 0.29
(12 50 cm), eluted with CH2Cl2–MeOH (50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 0:1,
v/v). The CH2Cl2–MeOH (20:1) fraction (30 g) was subsequently chromatographed over a silica gel
column (10 40 cm), eluted with CH2Cl2–MeOH (30:1, 20:1, 10:1, 5:1, 1:1, and 1:2, v/v) to afford
±
0.03 mg/mL. The EtOAc extract (160 g) was subjected to a silica gel column
×
×
six fractions. Fraction 1 (3.2 g) was further separated by MPLC eluted with MeOH–H2O (10:90 to
90:10, v/v) at 25 mL/min to yield four subfractions (1-A to 1-D). Subfraction 1-B (180 mg) was purified
by semipreparative HPLC (MeOH–H2O, 42:58, v/v) at 3 mL/min to give compounds
28.35 min) and (28 mg, tR 35.65 min). Subfraction 1-C (330 mg) was applied to semipreparative
HPLC (MeOH–H2O, 53:47, v/v) at 3 mL/min to afford compounds 11 (25 mg, tR 27.34 min), (12 mg,
tR 38.73 min), and (17 mg, tR 45.45 min). Subfraction 1-D (550 mg) was separated on a Sephadex
LH-20 gel column (2.5 100 cm) using MeOH as the eluent to yield compounds 10 (7 mg) and
4 (6 mg, tR
6
1
8
×
9
(11 mg). Fraction 2 (8.2 g) was chromatographed by MPLC eluted with MeOH–H2O (20:80 to 80:20,
v/v) at 30 mL/min to afford six subfractions (2-A to 2-F). Subfraction 2-C (800 mg) was separated
by semipreparative HPLC, isocratically eluted with MeOH–H2O (30:70, v/v) at 3 mL/min to give
compound 12 (7 mg, tR 46.21 min). Subfraction 2-D (550 mg) was further subjected to a silica gel
column (3.5
yield five subfractions (2-D1 to 2-D5). Subfraction 2-D2 (290 mg) was applied to HPLC (MeOH–H2O,
40:60, v/v) to afford compounds (16 mg, tR 12.33 min) and (11 mg, tR 13.98 min). Subfraction 2-D3
×
30 cm) and eluted with a gradient of CH2Cl2–MeOH (20:1, 10:1, 5:1, 3:1, and 1:1, v/v) to
2
3
(90 mg) and subfraction 2-E (1.3 g) were respectively purified by Sephadex LH-20 (2.5
MeOH to give compounds 5 (18 mg) and 7 (0.9 g).
×
100 cm) with
3.4. Spectroscopic Data
8-Acetyl-9-hydroxy-3-methoxy-7-methyl-1-phenalenon (compound
1
): Yellow crystals (CHCl3); m.p.
(log ε): 243.3 (4.17), 342.5 (3.91) nm; IR (KBr) νmax: 3373 (OH), 2920,
max
325–330 ◦C; UV (CHCl3):
λ
1698 (C=O), 1623, 1574, 1337, 815 cm−1; 1H- and 13C-NMR (600 and 150 MHz, C5D5N) spectroscopic
data see Table 1; HR-ESI-MS: m/z 283.0962 [M + H]+ (calcd. for C17H15O4+; 283.0965).
25
Hedyotol C 7”-O-
(c 0.4, MeOH); UV (MeOH):
β
-D-glucopyranoside (compound
(log
2
): White amorphous powders; [
α
]
+14.25
D
λ
ε
): 218.0 (3.83), 279.7 (3.15) nm; IR (KBr)
ν
max: 3419 (OH),
max
2936, 1594, 1512, 1457, 1276, 1117 cm−1; 1H- and 13C-NMR (600 and 150 MHz, CD3OD) spectroscopic
data see Table 2; HR-ESI-MS: m/z 769.2662 [M + Na]+ (calcd. for C37H46NaO16+; 769.2678).
25
Hedyotol D 7”-O-
(c 0.3, MeOH); UV (MeOH):
β
-D-glucopyranoside (compound
(log
3
): White amorphous powders; [
α
]
–13.50
D
λ
ε
): 214.0 (3.93), 279.6 (3.22) nm; IR (KBr)
ν
max: 3419 (OH),
max
2926, 1600, 1506, 1463, 1123 cm−1; 1H- and 13C-NMR (600 and 150 MHz, CD3OD) spectroscopic data
see Table 2; HR-ESI-MS: m/z 769.2661 [M + Na]+ (calcd. for C37H46NaO16+; 769.2678).
3.5. Acid Hydrolysis of Compounds 2 and 3
The acid hydrolysis was performed according to the modified method of Song et al. [29].
◦
Compound
2
(2 mg) was dissolved in 2 mol/L HCl (1 mL), and heated at 95 C for 2 h. After hydrolysis
,
the reaction mixture was extracted with EtOAc and H2O, and the aqueous layer was neutralized with
silver carbonate (10 mg). Then the supernatant was concentrated to dryness and further evaporated
to dryness under N2 for 2 h. Subsequently, the residue was ◦stirred with L-cysteine methyl ester
hydrochloride (2 mg) in anhydrous pyridine (0.2 mL) at 60 C for 1 h. The dried reactant was
fractionalized with H2O and n-hexane (each 0.5 mL). The n-hexane layer was collected and subjected
to GC-MS. The acid hydrolysis of compound
The GC conditions were: column temperature 150 C, injector temperature 270 C, carrier gas N2.
D-glucose was detected from compounds and (tR 15.523 and 15.524 min) by comparison with
authentic samples: tRD-glucose 15.513 min, and L-glucose 15.808 min.
3 was conducted in the same procedure as compound 2.
◦
◦
2
3

Molecules 2016, 21, 1506
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3.6. Anti-Complementary Activity Assay Against CP and AP
The anti-complementary activities against the CP and AP were investigated with the method of
Xu et al. [16]. Heparin was used as positive control drug. The results of anti-complementary activity of
compounds 1–12 are displayed in Table 3. Anti-complement activity was determined as the mean of
triplicate measurements at each concentration and expressed as 50% inhibitory concentration (CH50
and AP50 values). All results were performed statistical analysis to compare the CH50 and AP50 values
between each compound and positive control with unpaired t test.
3.7. Identification of the Targets in the Complement Activation Cascade
Assay to identify the targets in the complement activation cascade was performed according to
the method of Di et al. [30]. The results of compounds
4 and 5 were displayed in Figure 3. Data were
expressed as mean ± SD of triplicate measurements.
4. Conclusions
A first phenalenon derivative (compound
new sesquilignan epimers (compounds and
known compounds were isolated from the aerial parts of H. angustifolia. Chemical structures of the
isolated compounds were identified on the basis of extensive spectroscopic data. Compounds 12
were isolated for the first time from the aerial parts of H. angustifolia. Eleven compounds exhibited
anti-complementary activity, and the targets of two compounds ( and ) in the complement activation
1) with an acetyl side chain at C-8 and a pair of
2
3
) with a glucosyl unit at C-7 together with nine
”
4, 8–
4
5
cascade were also identified. In conclusion, the anti-complementary constituents of H. angustifolia
possessed chemical diversity, and consisted mostly of flavonoids and lignans in this study.
Supplementary Materials: The 1D and 2D NMR spectra for compounds 1–3 are available as supporting data
online at http://www.mdpi.com/1420-3049/21/11/1506/s1.
Acknowledgments: This work was financially supported by the grant from the National Natural Science
Foundation of China (81330089 and 30925042). The authors are grateful to Wei-Liang Zhu and Ming-Yue Zheng at
the Shanghai Institute of Materia Medica for technical support.
Author Contributions: D.-F.C. conceived and designed the experiments; X.Y. performed the experiments; X.Y.,
Y.L. and Z.-H.C. analyzed the data; X.Y. wrote the paper; D.-F.C. edited and revised the paper. All authors read
and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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