Compounds from Toddalia asiatica: Immunosuppressant Activity and Absolute Configurations

Article abstract of DOI:10.1021/acs.jnatprod.0c00564

In a screening of an extract library from plants used in Traditional Chinese Medicine the MeOH extract of Toddalia asiatica inhibited proliferation of human primary T cells with an IC50 of 25.8 μg/mL. Activity in the extract was tracked by HPLC activity profiling, and a total of 15 compounds were characterized. Three compounds, toddalic acid (6) and both enantiomers (7a and 7b) of toddanolic acid (7), were new natural products, and two recently published compounds, (2′R)-toddalolactone 3′-O-β-d-glucopyranoside (10) and (2′S)-toddalolactone 2′-O-β-d-glucopyranoside (11), were described in detail for the first time. The absolute configurations of compounds 8, 9, 10, 12, 13, and 15 were determined by comparison of experimental and calculated ECD spectra. For glucosides 9 and 10, ECD data and chiral-phase HPLC of the aglycones after enzymatic hydrolysis confirmed the results. Nitidine chloride (4) inhibited proliferation of primary human T cells with an IC50 of 0.4 μM.

Full text of DOI:10.1021/acs.jnatprod.0c00564

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Article
Compounds from Toddalia asiatica: Immunosuppressant Activity
and Absolute Configurations
̌
Jakob K. Reinhardt, Amy M. Zimmermann-Klemd, Ombeline Danton, Martin Smiesko,
̈
Carsten Grundemann, and Matthias Hamburger*
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ABSTRACT: In a screening of an extract library from plants used
in Traditional Chinese Medicine the MeOH extract of Toddalia
asiatica inhibited proliferation of human primary T cells with an
IC50 of 25.8 μg/mL. Activity in the extract was tracked by HPLC
activity profiling, and a total of 15 compounds were characterized.
Three compounds, toddalic acid (6) and both enantiomers (7a
and 7b) of toddanolic acid (7), were new natural products, and
two recently published compounds, (2′R)-toddalolactone 3′-O-β-
D-glucopyranoside (10) and (2′S)-toddalolactone 2′-O-β-D-
glucopyranoside (11), were described in detail for the first time.
The absolute configurations of compounds 8, 9, 10, 12, 13, and 15
were determined by comparison of experimental and calculated
ECD spectra. For glucosides 9 and 10, ECD data and chiral-phase HPLC of the aglycones after enzymatic hydrolysis confirmed the
results. Nitidine chloride (4) inhibited proliferation of primary human T cells with an IC50 of 0.4 μM.
nder normal conditions the immune system is able to
chromatogram and the activity of 1 min microfractions
collected is shown in Figure S2 (Supporting Information).
Inhibition of T cell proliferation was found in the micro-
fractions eluted at 14, 15, and 26 min. Compounds 3−10 in
the time windows of activity were subsequently isolated by a
combination of liquid−liquid partitioning, centrifugal counter-
current chromatography, preparative flash chromatography,
and HPLC on RP stationary phases. In addition, compounds 1,
2, and 11−15 were isolated from adjacent regions in the
HPLC chromatogram (Figure S2, Supporting Information).
The active fraction at 26 min contained a minor peak with the
same mass as nitidine (4). However, this peak was not found
after further fractionation of the extract.
1
Udifferentiate between self and nonself. However, auto-
immune diseases can develop if this function is impaired. In
rheumatoid arthritis, for example, autoreactive T cells attack
the synovial lining of the joints, resulting in tissue destruction
and inflammation.2 Autoimmune diseases are typically treated
with immunosuppressive drugs, but therapy may be accom-
panied by severe side effects.3 Thus, there is a need for better-
tolerated treatment options. To find new natural product leads
with immunosuppressive activity, a library of extracts from
plants used in Traditional Chinese Medicine (TCM) was
screened for their ability to inhibit T cell proliferation in vitro.
The MeOH extract from roots of Toddalia asiatica Lam.
(Rutaceae) was a promising hit, as it showed an IC50 value of
25.8 μg/mL without concomitant cytotoxicity (Figure S1,
Supporting Information). T. asiatica grows in most parts of
Asia and Africa.4 Its roots are only occasionally used in TCM
(Fei Long Zhang Xue),5 but more commonly as an
antimalarial drug in different African countries.6,7 The
phytochemistry of T. asiatica has been widely studied, and
numerous coumarins, alkaloids, and phenolic compounds have
been isolated from the roots of the plant.8−10 Most notably, a
structurally diverse set of coumarins have been identified,
including glucosides and prenylated derivatives.
Compound 1 was identified as syringic acid by analysis of
HRESIMS and NMR data (Table S1, Supporting Information)
and comparison with literature data.12
Compound 2 had a molecular formula of C21H23NO5
(HRESIMS data m/z 370.1646 [M + H]+, calcd for
+
C21H24NO5 , 370.1649). With the aid of 1D and 2D NMR
data recorded at 23 and 60 °C (Table S2) and comparison
Received: May 22, 2020
RESULTS AND DISCUSSION
■
The active compounds in the extract were tracked by HPLC-
based activity profiling.11 The overlay of an analytical HPLC
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Chart 1
Figure 1. Isolated mixture of compounds 3a and 3b and proposed reaction of compound 3b to yield 3c.
with literature data, compound 2 was identified as
allocryptopine.13
Compound 4 was identified as nitidine by analysis of its 1D
and 2D NMR spectra in DMSO-d6 with trifluoracetic acid
(Table S4, Supporting Information). Nitidine chloride was
Fagaridine (3b, identical to isofagaridine14) was obtained as
the major compound in a 4:1 mixture with dihydrofagaridine
(3a).15 Both were identified by comparison of their NMR data
(Table S3, Supporting Information) with the literature.14,15 To
prepare fagaridine (3b) as the respective chloride for biological
testing, the mixture was passed through an ion-exchange resin.
In the resulting compound a change was observed for the
chemical shifts of C-6 (δC 86.3) and H-6 (δH 5.53). The
presence of a methoxy group at C-6 was supported by an
HMBC correlation of H-6 to a carbon at δC 51.8. However, no
correlating protons were seen in the HSQC-DEPT spectrum.
This suggested the formation of the pseudobase of fagaridine
in the presence of methanol-d4 used for the NMR measure-
ments (Figure 1). Indeed, similar reactions have been
described before.16,17
successively prepared using an ion-exchange resin.18 The H
1
NMR shifts in DMSO-d6 (Table S4, Supporting Information)
and MS data (HRESIMS data m/z 348.1223 [M]+, calcd for
+
C21H18NO4 , 348.1230) were in good agreement with
published data.19
Compound 5 was identified as dihydronitidine (Table S4,
Supporting Information).20,21
For compound 6, a molecular formula of C12H10O6
(HRESIMS data m/z 251.0542 [M + H]+, calcd for
+
C12H11O6 , 251.0550) was determined, which, in combination
with 1D and 2D NMR data (Table 1), suggested a coumarin.
However, no signals for the proposed carboxylic acid moiety at
C-6 appeared in the HMBC and 13C NMR spectra. The
compound was methylated with trimethylsilyl diazomethane
B
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1
Table 1. H and 13C NMR Spectroscopic Data (500 MHz,
compared to ab initio calculated spectra of the enantiomers
(Figure S3(B), Supporting Information). The measured ECD
spectrum had a low signal-to-noise ratio and showed no
significant features of an expected π→π* transition at 220 nm
hinting at a scalemic mixture. The enantiomers were
subsequently separated by chiral-phase HPLC. ECD spectra
(Figure S3(B), Supporting Information) and the specific
rotations of the single enantiomers were recorded. The specific
rotations and ECD values for compound 7b were smaller than
could be expected. This was most likely due to an impurity not
observed in the NMR. Comparison to the ab initio calculated
ECD spectra identified 7a as (−)-(2′S)-toddanolic acid and 7b
as (+)-(2′R)-toddanolic acid.
Methanol-d4) for Compounds 6 and 6a
6
6a
a
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
2
3
4
4a
5
6
7
8
8a
11
12
13
14
163.2, C
113.2, CH
140.9, CH
108.2, C
155.2, C
118.7 , C
161.7, C
96.1, CH
162.2, C
6.24, d (9.8)
8.03, d (9.8)
113.5, CH
140.0, CH
108, C
6.28, d (9.8)
8.02, d (9.8)
156.3, C
115.1, C
161.6, C
96.3, CH
158.1, C
167.1, C
63.7, CH3
57.1, CH3
52.9, CH3
a
6.74, s
6.79, s
3.92, s
3.90, s
3.89, s
157.4, C
c
The 2D structure of compound 8 (Table S5, Supporting
Information) corresponded to that of hesperidin.23,24
63.4, CH3
57.1, CH3
3.99, s
3.90, s
Compounds 9 and 10 were identified by 1D and 2D NMR
data analysis as toddalolactones bearing a sugar moiety at C-3′.
Hydrolysis and subsequent GCMS analysis of the sugar
afforded D-glucose in both cases. Therefore, 9 and 10 were
epimers at C-2′, and this was corroborated by a difference in
13C chemical shifts of CH3-4′ (δC 21.9 in 9 and δC 23.0 in 10)
and CH3-5′ (δC 24.0 in 9 and δC 23.4 in 10). Compound 9
showed a specific rotation of [α]2D5 −45 and [α]D25 −2.5 for 10.
The (2′S)-epimer (9) was reported earlier by Lin et al. with a
specific rotation of [α]2D5 −44.25 In a recent publication,
however, the (2′R)-epimer 10 was reported as the
“enantiomer” of the (2′S)-epimer, with 13C NMR data virtually
identical to those of Lin’s (2′S)-epimer, but with exactly
opposite specific rotations.26 This was highly unexpected in
light of our data, and the absolute configuration of C-2′ in both
compounds was therefore examined in detail.
b
b
a
13C extracted from 1H−13C 2D inverse-detected experiments.
Overlapping signals. Signal not found.
b
c
(TMS-DAM) to obtain methyl ester 6a (Figure S17,
Supporting Information) with a molecular formula of
C13H12O6 (HRESIMS data m/z 265.0706 [M + H]+, calcd
+
for C13H13O6 , 265.0707), and the structure was corroborated
by NMR data as the methyl ester of 6. Thus, toddalic acid (6)
was confirmed as a new coumarin and only similar to buntasin,
another naturally occurring coumarin carboxylic acid found in
some Citrus species.22
The molecular formula of compound 7 was determined as
C16H22O7−(HRESIMS data m/z 327.1437 [M − H]−, calcd for
C16H23O7 , 327.1449). NMR data (Table 2) indicated an
The absolute configuration at C-2′ was determined by ECD.
The experimental spectra of 9 and 10 showed Cotton effects
(CEs) at 225, 253, and 330 nm with opposite signs. In the
calculated spectra (Figure 2) the sign for the CE at 330 nm
was negative for both epimers. Therefore, the CEs at 225 and
253 nm were used for the assignment. The two negative CEs
measured for 9 were in good agreement with the calculated
CEs for (2′S)-toddalolactone 3′-O-β-D-glucopyranoside. The
spectrum measured for 10 showed positive CEs at 225 and 253
nm. The observed CE at 225 nm matched with a positive CE
1
Table 2. H and 13C NMR Spectroscopic Data (500 MHz,
Methanol-d4) for Compound 7
position
δC, type
δH (J in Hz)
HMBC
1
2
3
4
5
6
7
8
1’
1’
2’
3′
4’
5′
1″
2″
3″
156.7, C
114.1, C
159.9, C
113.5, C
158.9, C
96.7, CH
62.2, CH3
56.1, CH3
27.2, CH2
6.30, s
1, 2, 4, 5
3
5
a
3.75, s
3.76, s
a
2.66, dd (13.7, 10.1)
2.86
3.56, dd (9.8, 2.8)
3, 4, 5, 2′, 3′
3, 4, 5, 2′, 3′
1′, 3′, 4
a
79.9, CH
74.2, C
a
25.4, CH3
25.8, CH3
20.9, CH2
35.3, CH2
178.1, C
1.23, s
2′, 3′, 5′
2′, 3′, 4’
2, 2″, 3″
1″, 3″
a
1.22, s
a
2.87
2.54, t (8.2, 8.2)
a
Overlapping signals.
aromatic ring bearing one hydroxy group, two methoxy groups
(CH3-7 and CH3-8; δH 3.75 and 3.76), a 2-methylbutan-2,3-
diol side chain, and a propionic acid residue (C-3″; δC 178.1).
The location of substituents on the aromatic ring were
determined with the aid of 13C NMR and HMBC data, leading
to the 2D structure of 7. To determine the absolute
configuration at C-2′, an ECD spectrum was recorded and
Figure 2. Comparison of experimental and calculated ECD spectra of
compounds 9 and 10 in MeOH (calcd spectrum shifted +5 nm).
C
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1
Table 3. H and 13C NMR Spectroscopic Data (500 MHz, Methanol-d4) for Compounds 9, 10, and 11
9
10
11
pos.
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
2
3
4
4a
5
6
7
8
8a
9
10
1′
1′
2′
3′
4′
5′
1″
2″
3″
4″
5″
6″
6″
163.5, C
112.7, CH
141.3, CH
108.5, C
157.7, C
120.3, C
163.7, C
96.4, CH
156.2, C
64.0, CH3
56.9, CH3
27.2, CH2
163.5, C
112.7, CH
141.4, CH
108.5, C
157.8, C
120.4, C
163.9, C
96.4, CH
156.3, C
64.0, CH3
56.9, CH3
27.2, CH2
163.7, C
112.6, CH
141.6, CH
108.5, C
157.9, C
120.0, C
164.1, C
96.3, CH
156.4, C
64.2, CH33
56.9, CH
27.0, CH2
6.22, d (9.8)
8.00, d (9.8)
6.20, d (9.6)
7.97, d (9.6)
6.21, d (9.5)
7.98, d (9.5)
6.72, s
6.69, s
6.71, s
a
a
a
3.90, s
3.91, s
3.88, s
3.88, s
3.90, s
a
a
a
3.90, s
2.90, dd (13.6, 10.1)
2.79, dd (13.6, 2.0)
2.89, dd (13.7, 9.8)
2.76, dd (13.7, 2.4)
2.98, dd (13.7, 10.0)
2.72, d (13.7)
a
a
a
77.6, CH
82.0, C
3.81
77.2, CH
81.6, C
3.79
88.1, CH
75.1, C
24.3, CH3
26.6, CH3
106.1, CH
76, CH
78.1, CH
71.7, CH
77.3, CH
62.9, CH2
3.87
a
a
21.9, CH3
24.0, CH3
98.7, CH
75.3, CH
78.1, CH
71.7, CH
77.8, CH
62.8, CH2
1.36, s
1.35, s
23.0, CH3
23.4, CH3
98.3, CH
75.5, CH
78.3, CH
71.8, CH
77.9, CH
62.9, CH2
1.34, s
1.35, s
1.30, s
1.25, s
4.20, d (7.0)
a
a
a
a
4.57, d (7.6)
4.55, d (7.6)
a
3.22, dd (8.4, 7.6)
3.4, dd (8.4, 8.4)
3.19, dd (9.5, 7.6)
3.38, dd (9.5, 7.9)
3.06
3.23
a
a
a
a
3.32
3.31
3.28
3.06
a
a
3.29
2.90, m
a
3.65, dd (11.7, 5.0)
3.66, dd (11.7, 4.7)
3.22
a
a
a
3.82, m
3.82
3.26
a
Overlapping signals.
in the calculated spectrum of (2′R)-toddalolactone 3′-O-β-D-
glucopyranoside, but no interpretable CE was seen in the
calculated spectrum at 253 nm. To corroborate the assign-
ments, compounds 9 and 10 were subjected to acid hydrolysis
with HCl.25 This led to an almost quantitative formation of
ketone 9a and, via a rearrangement, to aldehyde 9b (Figures
S18 and S81−S84, Supporting Information) and a small
amount of the aglycones. Using chiral-phase chromatography
on a Daicel Chiralpak IG column, the aglycone of 9 was
identified as (2′S)-toddalolactone, and that of 10 as (2′R)-
toddalolactone (Figure S128, Supporting Information).
Enzymatic hydrolysis of 9 and 10 with cellulase was used to
obtain the aglycones in sufficient amounts for ECD and
specific rotation (SR) analysis (Figures S123−S126, Support-
ing Information). ECD identified the aglycone of 9 as the
(2′S) enantiomer, and the one from 10 as the (2′R)-
enantiomer (Figure S8, Supporting Information). Collectively,
compound 9 was identified as (2′S)-toddalolactone 3′-O-β-D-
glucopyranoside and compound 10 as (2′R)-toddalolactone
3′-O-β-D-glucopyranoside. Coumarin 10 has been recently
reported, but the assignment of its absolute configuration was
solely based on OR data and a questionable correlation with
previously known glucoside 9.26
Compound 11 had a molecular formula of C22H30O11
(HRESIMS data m/z 493.1672 [M + Na]+, calcd for
C22H30O11Na+, 493.1680), and 1D and 2D NMR data
(Table 3) identified it as toddalolactone 2′-O-β-D-glucopyrano-
side.26 As the absolute configuration of 11 was hitherto only
based on SR data, an ECD spectrum was recorded and
compared to calculated spectra of both possible epimers
(Figure S9, Supporting Information). However, the calculated
spectra were highly similar; thus the analysis was inconclusive.
Acidic hydrolysis of 11 afforded the aglycone together with the
respective ketone and aldehyde (Figure S18, Supporting
Information). The aglycone was identified via chiral-phase
HPLC as (2′S)-toddalolactone (Figure S128, Supporting
Information), and compound 11 was conclusively identified
as (2′S)-toddalolactone 2′-O-β-D-glucopyranoside.
A molecular formula of C39H48O17 was determined for
compound 12 (HRESIMS data m/z 811.2758 [M + Na]+,
calcd for C39H48O17Na+, 811.2784). Using 1D and 2D NMR
data, compound 12 was identified as hazaleanin B (Table S7,
Supporting Information), which was previously reported from
Fagara rhetza (Rutaceae)27 and Zhuyeqing Liquor.28 ECD
analysis corroborated the published absolute configuration as
(2R,3R,4S)-hazaleanin B (Figure S11, Supporting Informa-
tion).
Compound 13 had a molecular formula of C39H50O17
(HRESIMS data m/z 813.2916 [M + Na]+, calcd for
C39H50O17Na+, 813.2940). By 1D and 2D NMR analysis 13
was identified as hazaleanin A.27 ECD data analysis was used to
independently confirm the absolute configuration. Owing to
the high conformational flexibility of 13, the conformational
analysis was performed not in bulk but in explicit MeOH by
means of molecular dynamics simulations. A comparison of the
experimental and calculated spectra (spectra in Figure S13,
selected conformers in Figures S19−S22, Supporting Informa-
tion) resulted in the assignment of 13 as (2S,3S)-hazaleanin in
accordance with the findings of Shibuya et al.27 This is the first
report of the presence of hazaleanins A and B in T. asiatica.
Compound 14 was identified by 1D and 2D NMR data as p-
coumaroyltyramine (Table S8, Supporting Information).29
Compound 15 had a molecular formula of C16H20O6
(HRESIMS data m/z 309.1329 [M + Na]+, calcd for
C16H20O6Na+, 309.1333), and 1D and 2D NMR data (Table
S9, Supporting Information) identified the compound as
D
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Figure 3. Inhibitory effects of compounds 1−15 on the proliferation of human T lymphocytes. Human PBMCs (2 × 105) were stained with CFSE
and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs, 100 ng/mL each). Unstimulated cells served as a negative control
(NC). Afterward, cells were incubated for 72 h in the presence of medium (PC), cyclosporin A (CsA; 4.16 μM), camptothecin (CPT; 300 μM), or
compounds 1−15 at concentrations between 0.3 and 30 μM. Cell division was analyzed by flow cytometry. The percentage of proliferating cells
was normalized to the stimulated control and depicted as mean standard deviation. n = 3. *p < 0.05.
toddalolactone.25,30 The negative sign of the specific rotation
suggested (2′S)-toddalolactone (15a).25 This was supported
by the ECD spectrum, which was compared to the calculated
spectra of both enantiomers 15a and 15b (Figure S15,
Supporting Information). The SR of 15 ([α]2D5 −10) compared
to (2′S)-toddalolactone (15a) obtained by enzymatic hydrol-
ysis of 9 ([α]2D5 −51) indicated the enantiomeric excess of 15a
as 20%. This analysis was corroborated by a 1H NMR
detected only at 10 and 30 μM concentrations for compound 4
and at 30 μM concentration for 5. Nitidine chloride (4) has
been previously found to modulate reactive microgliosis after
traumatic brain injury at 0.1 μM, and inhibition of the ERK
and NF-κB signaling pathways in microglia cells was
reported.35 Inhibition of T cell proliferation by nitidine
chloride (4) observed in our study could thus be caused by
inhibition of these pathways. Nitidine chloride reportedly also
induces apoptosis in HCT116 human colorectal cancer cells
via inhibition of ERK.36 In conclusion, nitidine chloride (4) is
a potential lead, with inhibition of T cell proliferation at sub-
micromolar concentrations and an apparent cytotoxicity only
at a 25-fold higher concentration. This possibility was also
corroborated by a patent (CN102008474B) filed for the use of
nitidine in the treatment of autoimmune diseases and resisting
transplant rejection.37 The related dihydronitidine (5) has
been reported to selectively induce apoptosis in various tumor
cell lines when tested at low micromolar concentrations.38
31
experiment in the presence of the shift reagent Eu(hfc)3
(29% enantiomeric excess of 15a) and chiral-phase HPLC on a
Daicel Chiralpak IG column (25% enantiomeric excess of
15a).
In conclusion, a total of 15 compounds were isolated from
the MeOH extract of T. asiatica, of which toddalic acid (6) and
toddanolic acid (7) were new natural products. Although
compound 10, the 2′-epimer of 9, was reported recently, we
provide here the first full structural assignment and
spectroscopic data. The co-occurrence of 9 and 10 indicates
that both enantiomers of toddalolactone are naturally present
in the plant.32 The carboxylic moiety at C-6 in coumarin 6 is
unusual from a biosynthetic perspective, and only one similar
naturally occurring coumarin has been reported so far.22
Toddanolic acid (7) is structurally related to toddalolactone
(15). An artifactual formation of 7 can be ruled out given that
an opening of the lactone ring and reduction of the double
bond would require conditions (e.g., basic pH, metal catalyst,
and NaBH4) far from the ones used during the isolation.33,34
Compounds 1−15 were tested for their ability to suppress
the proliferation of stimulated human primary T lymphocytes
in vitro. Compounds 1−3 and 6−15 showed slight inhibition
of T cell proliferation at the highest test concentration of 30
μM. Compounds 4 and 5 concentration-dependently inhibited
T cell proliferation with IC50’s of 0.4 μM (R2 = 0.7) and 6.7
μM (R2 = 0.9), respectively (Figure 3). Cytotoxic effects were
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured at concentrations between 0.4 and 1 mg/mL in MeOH
on a PerkinElmer 341 polarimeter with a 10 cm microcell. UV and
ECD spectra were recorded in MeOH (50−150 μg/mL) on a
Chirascan CD spectrometer using 1 mm path precision cells (110 QS,
Hellma Analytics). NMR data were recorded on a Bruker Avance III
NMR spectrometer operating at 500.13 MHz for 1H and 125.77 MHz
for 13C nuclei. 1H NMR, COSY, HSQC, HMBC, and NOESY spectra
were measured at 23 °C in a 1 mm TXI probe with a z-gradient. 13C
NMR/DEPTQ spectra were recorded at 23 °C in 3 mm tubes using a
1
5 mm BBI probe. For compound 2, H, COSY, HSQC, and HMBC
spectra were also recorded at 60 °C. All spectra were analyzed using
Bruker TopSpin 3.5 and ACDLabs NMR Workbook software suites.
NMR spectra were recorded in CDCl3 (Sigma-Aldrich), methanol-d4,
DMSO-d6, or TFA (all Armar Chemicals). HPLC-PDA-ELSD-ESIMS
E
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data were recorded in the positive and negative mode on a Shimadzu
LC-MS/MS 8030 triple quadrupole ESIMS system connected via a T-
splitter (1:10) to a Shimadzu HPLC system consisting of a degasser,
binary high-pressure mixing pump, autosampler, column oven, and
diode array detector, and via a T-split to an Alltech 3300 ELSD
detector. Data were acquired and processed with the LabSolution
software suite. Semipreparative HPLC separations were carried out
with an Agilent HP 1100 Series system consisting of a quaternary
pump, autosampler, column oven, and a diode array detector
(G1315B). Preparative HPLC separations were carried out using
Agilent 1290 Infinity II preparative binary pumps and an Agilent 1100
DAD detector in line with an Agilent 6120 Quadropole LC/MS
detector. The column effluent was collected via an active split (100:1)
between HPLC columns and detectors. Chemstation software was
used for data acquisition and processing. Waters SunFire C18 (3.5 μm,
3.0 × 150 mm i.d., equipped with a guard column 10 × 3.0 mm i.d.),
SunFire Prep C18 (5 μm, 10 × 150 mm i.d., equipped with a guard
column 10 × 10 mm i.d.), and SunFire Prep C18 OBD (5 μm, 30 ×
150 mm i.d., equipped with a guard column 10 × 20 mm i.d.)
columns were used for reverse-phase analytical, semipreparative, and
preparative separations, respectively. Chiral-phase HPLC separations
were performed at a flow rate of 1 mL/min on either a Daicel
Chiralpak IG column (5 μm, 250 × 4.6 mm) eluted with 20%
CH3CN in H2O for 10 min or a Daicel Chiralpak IF column (5 μm,
250 × 4.6 mm) eluted with 20% CH3CN in H2O, both containing
0.1% formic acid (FA), for 10 min. HPLC-grade MeOH, CH3CN
(Scharlau Chemie), and water from a Milli-Q water purification
system (Merck Millipore) were used for HPLC separations. The
mobile phase used for analytical HPLC contained 0.1% FA.
Technical-grade solvents purified by distillation were used for
extraction, open column chromatography, and CPC separations.
Silica gel (15−40 μm, Merck) and C18 modified silica (RP-18
LiChroprep 25−40 μm) were used for open column chromatography.
HRESIMS data were measured on an LQT XL Orbitrap mass
spectrometer (Thermo Scientific) coupled to a Shimadzu LC-MS
system equipped with a Waters SunFire C18 column (3.5 μm, 3.0 ×
150 mm i.d., equipped with a guard column 10 × 3.0 mm i.d.).
Trimethylsilyldiazomethane was purchased from Tokio Chemical
Industry, and Lewatit MonoPlus SP 112 (Na+ form), Eu(hfc)3, and
cellulase enzyme blend were from Sigma-Aldrich, Germany.
and a mixture of 1500 mL of CHCl3 and 500 mL of MeOH. The
organic layers were combined and dried in vacuo, yielding 17.3 g of an
enriched fraction. A precipitate at the interface of the aqueous and the
organic layers was collected separately, filtered, and washed with cold
CHCl3 and MeOH to give 548 mg of 8. Aliquots of the enriched
fraction (1.9 to 3.0 g per run) were subjected to centrifugal partition
chromatography (CPC) using CHCl3/MeOH/H2O (25:30:20) at a
flow rate of 5 mL/min in descending mode. Six fractions (A−F) were
collected, and a switch to ascending mode gave four additional
fractions (G−K). Fraction E (559 mg) was separated on a C18 column
(25−40 μm, 5 × 47 cm) connected to an Interchim Puriflash 4100
system [water + 0.1% FA (A), CH3CN + 0.1% FA (B); 27% B (0−5
min) 27 → 44% B (5−202 min), 44 → 100% (202−220 min), 100%
(220−245 min); flow rate 22 mL/min; sample dissolved in 1.3 mL of
MeOH], and fractions E1−E16 were obtained. E6 (6 mg) was purified
by semipreparative RP HPLC [CH3CN + 0.1% FA (A), H2O + 0.1%
FA (B); 18% B (0−20 min) 18 → 20% B (20−22 min), 20 → 100%
(22−26 min), 100% (26−31 min); flow rate 4 mL/min;
concentration 50 mg/mL in MeOH, injection volume 10−40 μL]
to yield dihydronitidine (5, 0.6 mg). The rest of fraction E (600 mg)
was chromatographed on a silica gel column (15−40 μm; 3.5 × 35
cm) connected to an Interchim Puriflash 4100 system [CHCl3 (A),
MeOH (B); 100% A (0−5 min) 0 → 2% B (5−10 min), 2 → 5%
(10−20 min), 5 → 30% (20−110 min), 30 → 50% (110−175 min),
50 → 55% (175−190 min), 55 → 57% (190−191 min), 57 → 100%
(191−205 min), 100% (205−260 min); flow rate 20 mL/min; solid
state introduction, with the sample adsorbed on 3 g of silica gel (63−
200 μm, Merck)], and 24 fractions (EA−EX) were obtained. Fraction
EL (36 mg) was separated by preparative RP HPLC [CH3CN + 0.1%
FA (A), H2O + 0.1% FA (B); 15% B (0−24 min) 15 → 25% B (24−
35 min), 25 → 35% B (35−40 min), 35 → 60% B (40−45 min), 60
→ 100% B (45−47 min), flow rate 25 mL/min; concentration 90
mg/mL in MeOH, injection volume 390 μL] to afford a mixture of 3a
and 3b (3.5 mg). Fraction EN (9.3 mg) was recrystallized from
MeOH/H2O (1:2) to obtain syringic acid (1, 4.5 mg). Fraction EP
(22 mg) was separated by preparative RP HPLC [CH3CN + 0.1% FA
(A), H2O + 0.1% FA (B); 15% B (0−3 min) 30 → 35% B (3−16
min), 35 → 100% (16−20 min); flow rate 20 mL/min; concentration
55 mg/mL in MeOH/H2O (1:3); injection volume 400 μL], and
fractions EP1−EP4 were collected. EP3 (5.8 mg) was purified by
semipreparative RP HPLC [CH3CN + 0.1% FA (A), H2O + 0.1% FA
(B); 24% B (0−20 min) 24 → 100% B (20−22 min), 100% B (22−
27 min), flow rate 4 mL/min; concentration 2.5 mg/mL MeOH/H2O
(1:1), 3 injections with injection volumes 40−70 μL] to afford 12
(3.6 mg) and 13 (1.5 mg). Fraction EQ (60 mg) was separated by
preparative RP HPLC [CH3CN + 0.1% FA (A), H2O + 0.1% FA (B);
23% B (0−30 min) 23 → 100% B (30−31 min), 100% B (31−36
min); flow rate 20 mL/min; concentration 83 mg/mL in MeOH/
H2O (1:3); 2 injections of 300−400 μL] to afford 9 (20 mg), 10 (11
mg), and 11 (5.1 mg). Fraction ET (55 mg) was dissolved in 700 μL
MeOH/H2O (3:1), and the supernatant was subjected to preparative
HPLC [CH3CN + 0.1% FA (A), H2O + 0.1% FA (B); 13% B (0−2
min), 13 → 25% B (2−32 min), 25 → 29% (32−42 min), 29 → 60%
(42−50 min), 60% (50−52 min), 60 → 100% B (52−55 min), flow
rate 20 mL/min; concentration 75 mg/mL in MeOH/H2O (3:1);
injection volume 700 μL] to yield 2 (1.6 mg). Fraction EV (22 mg)
was separated by preparative RP HPLC [CH3CN + 0.1% FA (A),
H2O + 0.1% FA (B); 21% B (0−3 min) 21 → 29% B (3−24 min), 29
→ 100% (24−30 min), flow rate 20 mL/min; concentration 55 mg/
mL in MeOH/H2O (1:1); injection volume 400 μL] to afford
fractions EV1−EV5. Fraction EV1 (5.8 mg) was purified by
semipreparative RP HPLC [CH3CN + 0.1% FA (A), H2O + 0.1%
FA (B); 11% B (0−30 min) 11 → 100% B (30−32 min), 100% B
(32−38 min), flow rate 4 mL/min; concentration 33 mg/mL in
MeOH/H2O (1:2), 2 injections of 60 μL], to afford 6 (1.0 mg) and 7
(1.5 mg). As compound 7 was obtained as a scalemic mixture of both
enantiomers, the enantiomers 7a and 7b were separated via chiral-
phase HPLC on a Daicel Chiralpak IF column [CH3CN + 0.1% FA
(A), H2O + 0.1% FA (B); 20% B (0−10 min), flow rate 1 mL/min;
concentration of 5 mg/mL; 8 injections with 10 μL]. Compound 7b
Plant Material. T. asiatica roots were purchased from Bozhou
Swanf Commerce and Trade Co., Ltd. (Bozhou, China) in January
2018. A voucher specimen (number 0 1026) has been deposited at
the Division of Pharmaceutical Biology, University of Basel,
Switzerland.
Microfractionation. Microfractionation of T. asiatica MeOH
extract was carried out by analytical RP-HPLC on an LC-MS 8030
system (Shimadzu) connected with an FC 204 fraction collector
(Gilson). A solution of 10 mg/mL extract in DMSO was prepared,
and three injections were performed: 2 × 30 μL for microfraction,
using only the PDA (0.6 mg of extract in total), and 1 × 10 μL with
PDA-ELSD-ESIMS detectors without microfractionation. Water
containing 0.1% FA (solvent A) and CH3CN with 0.1% FA (B)
were used as a mobile phase. A linear gradient from 5 to 60% B in 30
min was followed by 5 min at 100% B. Microfractions of 1 min each
were collected from the beginning of minute 2 to the end of minute
34. Corresponding microfractions of two successive injections were
collected into the same wells of a 96-deep-well plate. The plates were
dried in a Genevac EZ-2 evaporator.
Extraction and Isolation. T. asiatica roots were ground with a
Retsch ZM1 centrifugal mill. A 740 g amount of the powdered roots
was extracted with 4 × 2.2 L of DCM over 24 h. The remaining plant
material was extracted with 3 × 2.2 L of MeOH over 24 h. The
combined MeOH extracts were dried in vacuo and freeze-dried to
afford 38.7 g of dry residue. A 35.7 g amount of MeOH extract was
subjected to liquid−liquid partitioning using a two-phase system
containing 928 mL of CHCl3, 500 mL of MeOH, and 570 mL of
H2O. After removal of the organic phase, the aqueous phase was
successively extracted with a mixture of 800 mL of CHCl3 and 200
mL of MeOH, a mixture of 500 mL of CHCl3 and 500 mL of MeOH,
F
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Article
eluted at 7.3 min, and compound 7a at 8.4 min. Fraction EW (58 mg)
was dissolved in 580 μL MeOH and 10 μL of FA and subjected to
preparative RP HPLC [CH3CN (A), H2O (B); 16% B (0−48 min),
16 → 100% B (48−53 min); flow rate 25 mL/min; concentration of
90 mg/mL; injection volume 550 μL], to afford 4 (10.1 mg). The
hydrochloride of 4 was prepared via filtration over Lewatit MonoPlus
SP 112 resin.
Hazaleanin B (12): yellow solid; [α]2D5 +14 (c 0.09 g/100 mL,
MeOH); UV λmax (MeOH) (log ε) 205 (5.0), 328 (4.3) nm; ECD
(MeOH, c 0.13 mM, 0.1 cm); Δε +43.7 (203 nm), −18.5 (218 nm),
1
+14.5 (244 nm), +4.3 (272 nm), −1.9 (287 nm); H and 13C NMR,
see Table S7, Supporting Information; HRESIMS m/z 811.2758 [M
+ Na]+ (calcd for C39H48O17Na, 811.2784).
Hazaleanin A (13): white solid; [α]2D5 −9 (c 0.05 g/100 mL,
MeOH); UV λmax (MeOH) (log ε) 205 (4.9), 329 (4.2) nm; ECD
(MeOH, c 0.13 mM, 0.1 cm); Δε +20.5 (208 nm), −7.3 (217 nm),
+1.4 (239 nm); 1H and 13C NMR, see Table S7, Supporting
Information; HRESIMS m/z 813.2916 [M + Na]+ (calcd for
C39H50O17Na, 813.2940).
1
Syringic acid (1): white solid; H and 13C NMR, see Table S1,
Supporting Information; HRESIMS m/z 181.0489 [M + H − H2O]+
+
(calcd for C9H9O4 , 181.0495).
Allocryptopine (2): colorless solid; 1H and 13C NMR, see Table S2,
Supporting Information; HRESIMS m/z 370.1646 [M + H]+ (calcd
+
1
N-p-Coumaroyltyramine (14): yellowish solid; H and 13C NMR,
for C21H24NO5 , 370.1649).
Fagaridine and dihydrofagaridine (3b and 3a): red solid; 1H and
13C NMR, see Table S3, Supporting Information; HRESIMS m/z
see Table S8, Supporting Information; HRESIMS m/z 284.1274 [M
+ H]+ (calcd for C17H18NO3, 284.1281).
+
334.1059 [M]+ (calcd for C20H16NO4 , 334.1074).
Toddalolactone (15): white solid; [α]2D5 −10 (c 0.07 g/100 mL,
MeOH); UV λmax (MeOH) (log ε) 205 (4.5), 225 (4.1), 330 (4.0)
nm; ECD (MeOH, c 0.21 mM, 0.1 cm); Δε +3.1 (203 nm), −1.0
Compound 3b: 1H and 13C NMR, see Table S3, Supporting
Information.
Nitidine chloride (4): yellow solid; 1H and 13C NMR, see Table S4,
1
(229 nm); H and 13C NMR, see Table S9, Supporting Information;
Supporting Information; HRESIMS m/z 338.1223 [M]+ (calcd for
HRESIMS m/z 309.1329 [M + Na]+ (calcd for C16H20O6Na,
309.1333).
+
C21H18NO4 , 338.1230).
Dihydronitidine (5): light yellow solid; 1H and 13C NMR, see
Table S4, Supporting Information; HRESIMS m/z 348.1220 [M −
(2′S)-Toddalolactone (15a): colorless solid; [α]2D5 −51 (c 0.04 g/
100 mL, MeOH); UV λmax (MeOH) (log ε) 196 (4.8), 205 (4.7), 226
(4.4), 330 (4.1) nm; ECD (MeOH, c 0.21 mM, 0.1 cm); Δε +8.8
(203 nm), −6.3 (229 nm), −1.8 (256 nm), −1.7 (327 nm).
(2′R)-Toddalolactone (15b): colorless solid; [α]2D5 50 (c 0.06 g/
100 mL, MeOH); UV λmax (MeOH) (log ε) 197 (4.7), 205 (4.6), 225
(4.3), 330 (4.0) nm; ECD (MeOH, c 0.21 mM, 0.1 cm); Δε +5.8
(225 nm), +2.7 (253 nm), +1.0 (329 nm).
+
H]+ (calcd for C21H18NO4 , 348.1230) and m/z 334.1064 [M −
+
CH3]+ (calcd for C20H16NO4 , 334.1074).
Toddalic acid (6): colorless solid; UV λmax (MeOH) (log ε) 206
1
(4.4) nm, 323 (3.9) nm; H and 13C NMR, see Table 1; HRESIMS
+
m/z 251.0542 [M + H]+ (calcd for C12H11O6 , 251.0550).
1
Compound 6a: colorless solid; H and 13C NMR, see Table 1;
+
HRESIMS m/z 265.0706 [M + H]+ (calcd for C13H13O6 , 265.0707).
Ion Exchange. The resin (Lewatit MonoPlus SP 112 (Na+ form),
500 mg) was placed in a 3 mL glass vial with a magnetic stirrer. The
resin was washed twice with 10 mL of HPLC grade MeOH while
slowly stirring. Compound 2 or 4, respectively, was dissolved in
MeOH at a concentration of 1.7 mg/mL, added to the washed resin,
and stirred until the color of the supernatant had faded. The
supernatant was discarded, and the resin was washed twice with 10
mL of MeOH. Then, 1 mL of a 10% NaCl solution in H2O/MeOH
(1:1) was added while stirring, followed by 5 mL of MeOH. The
supernatant was collected, and the step was repeated once again. The
combined supernatants were dried under a flow of N2, suspended in
1.5 mL of HPLC grade water, and centrifuged at 13 200 rpm for 8
min. The supernatant was discarded, and the precipitate was washed
twice more with 1 mL of H2O, yielding the respective chloride.
Preparation of 6a. Compound 6 (0.1 mg, 0.4 μmol) was
dissolved in 75 μL of DCM and 25 μL of MeOH in a vial under an
argon atmosphere, and 5 μL of trimethylsilyl diazomethane (0.6 M in
n-hexane, 3 μmol) was added. The mixture was vortexed and allowed
to stand at room temperature for 1 h. The reaction was stopped by
the addition of 3.4 μL of 1.7 M acetic acid in H2O. The reaction
mixture was dried in a flow of N2 to yield 6a.
Enantiomeric Excess Determination of Toddalolactone
(15). Eu(hfc)3 (0.7 mg) was dissolved under argon in CDCl3 at a
concentration of 1.5 equiv of 15 per 120 μL, and the solution was
transferred under argon to the mixture of 15a and 15b. The resulting
solution was transferred into a 3 mm NMR tube under argon, and 1H
NMR spectra were recorded.
Acid Hydrolysis of 9, 10, and 11. Analogous to acid hydrolysis
for GCMS sugar analysis,24 approximately 1 mg of 9, 10, or 11,
respectively, was dissolved in 1 mL of 2 N aqueous HCl in a 4 mL
sealable vial. The vial was placed in an oven at 105 °C for 105 min.
After cooling, the mixture was extracted with EtOAc (3 × 1 mL). The
organic layers were combined and dried under N2. The aglycones 15a
and 15b, respectively, were identified as minor compounds via chiral-
phase HPLC on a Daicel Chiralpak IG column eluting with 20%
CH3CN. (2′R)-Toddalolactone (15b) eluted at 4.74 min, and (2′S)-
toddalolactone (15a) at 4.93 min, respectively (Figure S128,
Supporting Information). The major compounds recovered after
Toddanolic acid (7): colorless solid; [α]2D5 −12 (c 0.07 g/100 mL,
MeOH); UV λmax (MeOH) (log ε) 204 (4.1), 281 (3.0) nm; ECD
(MeOH, c 0.97 mM, 0.1 cm); Δε +1.49 (204 nm), −0.8 (230 nm);
1H and 13C NMR, see Table 2, Supporting Information; HRESIMS
−
m/z 327.1437 [M − H]− (calcd for C16H23O7 , 327.1449).
(−)-(2′S)-Toddanolic acid (7a): colorless solid; [α]2D5 −20 (c 0.03
g/100 mL, MeOH); UV λmax (MeOH) (log ε) 204 (4.5), 281 (3.3)
nm; ECD (MeOH, c 0.48 mM, 0.1 cm); Δε −3.4 (205 nm), −1.2
(231 nm), −0.5 (273 nm).
(+)-(2′R)-Toddanolic acid (7b): colorless solid; [α]2D5 32 (c 0.02 g/
100 mL, MeOH); UV λmax (MeOH) (log ε) 204 (4.5), 282 (3.4) nm;
ECD (MeOH, c 0.32 mM, 0.1 cm); Δε +8.0 (205 nm), +2.6 (232
nm), +1.1 (275 nm).
(2′S)-Hesperidin (8): pale yellow solid; UV λmax (MeOH) (log ε)
200 (4.8), 224 (4.5), 283 (4.3), 331 (3.6) nm; ECD (MeOH, c 0.32
mM, 0.1 cm); Δε +20.2(200 nm) +11.0 (223 nm), −10.4 (289 nm),
+2.5 (334 nm); 1H and 13C NMR, see Table S5, Supporting
Information; HRESIMS m/z 633.1778 [M + Na]+ (calcd for
C28H34NaO15+, 633.1790) (m/z 633.1778 [M + Na]+, calcd for
C28H34NaO15+, 633.1790).
(2′S)-Toddalolactone 3′-O-β-D-glycopyranoside (9): yellow solid;
[α]2D5 −45 (c 0.14 g/100 mL, MeOH); UV λmax (MeOH) (log ε) 205
(4.6), 226 (4.1), 330 (4.1) nm; ECD (MeOH, c 0.32 mM, 0.1 cm);
Δε +10.8 (203 nm), −4.6 (227 nm), −1.7 (248 nm), −1.1 (332 nm);
1H and 13C NMR, see Table 3; HRESIMS m/z 493.1669 [M + Na]+
(calcd for C22H30O11Na+, 493.1680).
Compounds 9a and 9b. 1H and 13C NMR, see Table S6,
Supporting Information.
(2′R)-Toddalolactone 3′-O-β-D-glycopyranoside (10): yellow
solid; [α]2D5 −2.5 (c 0.12 g/100 mL, MeOH); UV λmax (MeOH)
(log ε) 205 (4.6), 225 (4.2), 328 (4.0) nm; ECD (MeOH, c 0.21 mM,
0.1 cm); Δε −2.2 (202 nm), +2.6 (226 nm), +1.0 (251 nm), +0.9
1
(334 nm); H and 13C NMR, see Table 3; HRESIMS m/z 493.1670
[M + Na]+ (calcd for C22H30O11Na+, 493.1680).
(2′S)-Toddalolactone 2′-O-β-D-glycopyranoside (11): yellow
solid; [α]2D5 −46 (c 0.08 g/100 mL, MeOH); UV λmax (MeOH)
(log ε) 204 (4.5), 224 (4.1), 329 (3.9) nm; ECD (MeOH, c 0.32 mM,
0.1 cm); Δε +12.9 (204 nm), −5.2 (227 nm), −1.9 (248 nm), −1.5
1
(332 nm); H and 13C NMR, see Table 3; HRESIMS m/z 493.1672
hydrolysis of 9, 10
aldehyde 9b (Figure S18, Supporting Information).
̧, and 11, respectively, were the ketone 9a and the
[M + Na]+ (calcd for C22H30O11Na, 493.1680).
G
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Enzymatic Hydrolysis of 9, 10, and 11. Approximately 1 mg of
9, 10, or 11 was dissolved in 100 μL of cellulase blend (Sigma-
Aldrich, Cellulase, enzyme blend, SAE0020) diluted 1:1 with purified
water. The mixture was stirred at 37 °C for 22.5 h. Afterward, the
reaction mixture was diluted with 900 μL of H2O and extracted with
EtOAc (3 × 1 mL). The organic layers were combined and dried
under N2. Compounds 9 and 10 afforded their respective aglycone,
while 11 was recovered unreacted.
T Cell Proliferation Assay. Lymphocytes were isolated, washed
twice in cold PBS, and resuspended in PBS at a concentration of 5 ×
106 cells/mL. Cells were stained for 10 min at 37 °C with
carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 μM:
Sigma-Aldrich, St. Louis, MO, USA). The staining was stopped by
washing twice with complete medium. Stained lymphocytes (2 × 106
cells/mL) were stimulated with anti-human CD3 (clone OKT3) and
anti-human CD28 (clone 28.2) mAbs (each 100 ng/mL; eBioscience)
in the presence of either medium, CsA (4.16 μM; Novartis Pharma),
CPT (300 μM; Tocris), or plant extracts/single compounds
(concentration range 0.3−30 μM) and incubated for 72 h. The
negative control remained unstimulated. Flow cytometric analysis of
the cell division was performed using a FACSCalibur instrument (BD
Biosciences).
Determination of Apoptosis and Necrosis of T Cells.
Lymphocytes were isolated, washed twice in cold PBS, and
resuspended in medium at a concentration of 2 × 106 cells/mL.
Cells were stimulated with anti-human CD3 (clone OKT3) and anti-
human CD28 (clone 28.2) mAbs (each 100 ng/mL; eBioscience) in
the presence of either medium, CPT (300 μM; Tocris), 0.5% Triton-
X 100, or plant extracts and cultivated for 48 h. The negative control
remained unstimulated. Cultured cells were washed with PBS and
stained with annexin V-FITC using the apoptosis detection kit
(eBioscience) according to the manufacturer’s instructions. Propi-
dium iodide (eBioscience) was added and cells were stained for 15
min at room temperature in the dark. Apoptosis and necrosis rates
were determined by flow cytometric analysis using a FACSCalibur
instrument (BD Biosciences).
Testing of Microfractions. The dried microfractions in 96-deep-
well plates were dissolved in 25 μL of DMSO by sonication and
mixing with a pipet. Of these stock solutions, dilutions of 1/1, 1/3, 1/
10, and 1/30 were prepared and tested in duplicates for T lymphocyte
proliferation inhibition as described above. Assuming an equal
distribution of 200 ng substance in each of the microfractions,
theoretical IC50 values were calculated as a relative measure of activity.
They were normalized to 100%, with the highest value representing
100%.
Analysis of Data. For statistical analysis, data were processed with
Microsoft Excel and SPSS software (IBM, version 22.0, Armonk,
USA). Statistical significance was determined with the SPSS software.
Statistical significance was determined with the SPSS software by a
one-way ANOVA, followed by Dunnett’s post hoc pairwise
comparisons. Values are presented as mean SD for the indicated
number of independent experiments. The asterisks represent
significant differences from controls (*p < 0.05).
Computational Methods. Conformational analysis was per-
̈
̈
formed with Schrodinger MacroModel 11.0 (Schrodinger, LLC, New
York) employing the OPLS2005 (optimized potential for liquid
simulations) force field in H2O for geometrical optimization in two
steps. In the first step, a global minimum was searched using 20 000 or
30 000 steps, depending on the size of the molecule. In the second
step, the global minimum was used for a conformational search (4000
steps) choosing the five conformers to be subjected to geometrical
optimization and energy calculation applying DFT with Becke’s
nonlocal three-parameter exchange and correlation functional and the
Lee−Yang−Parr correlation functional level (B3LYP), using the
B3LYP/6-31G** basis set, the SCRF method, and the CPMC model
for solvation (MeOH for ECD calculations) with the Gaussian 09
program package.39 Excitation energy (denoted by wavelength in
nm), rotator strength (Rstr), dipole velocity (Rvel), and dipole length
(Rlen) were calculated in MeOH by TD-DFT/B3LYP/6-31G(d,p).
ECD curves were obtained on the basis of rotator strengths with a
half-band of 0.25 eV using SpecDis v1.71.40
Conformational Search for 13. The conformational flexibility
and relative structural complexity of 13 required a more sophisticated
conformational analysis. The 3D structures of all four stereoisomers
were built in the Maestro modeling environment.41 First, a
conformational search was performed to obtain a reasonable starting
conformation by sampling a total of 30 000 conformers using a mixed
torsional/low-mode sampling method, extended torsional sampling,
OPLS 2005 force field, and the implicit water solvent model in
MacroModel.42 The water solvent model was selected from the list of
available implicit models (water, CHCl3, octanol), because it offers
properties closest to MeOH. Next, a periodic boundary system was
created by placing the global minimum structure identified in the
conformational search into a cubic system (edge length 50 Å) of
preorganized MeOH molecules. The whole periodic boundary system
was fully optimized using the default relaxation protocol implemented
in Desmond,43−45 composed of seven stages allowing the solvent
molecules to optimally arrange with respect to the solute. The last
frame of the equilibration stage was used as input for the production
simulation in the total duration of 0.48 ms (NPT ensemble and
standard conditions T = 300 K, p = 101.325 kPa) with frames being
sampled every 480 ps (in total 1000 frames were saved per
simulation). All MD simulations were done using Desmond software.
The MD trajectories were postprocessed using the default clustering
analysis of Desmond. Representative solute structures of the 10 most
significant clusters were used as input for Gaussian 09 calculations as
described earlier.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00564.
Ethics Statement. Patients gave their written consent to donate
blood for scientific research. All experiments conducted on human
material were approved by the Ethics Committee of the University of
Freiburg (55/14; 11.02.2014), and all methods used were compliant
with the regulations of the Ethics Committee.
Preparation and Cultivation of Human Peripheral Lympho-
cytes. Peripheral blood mononuclear cells (PBMCs) were isolated
from the blood of healthy adult donors obtained from the Blood
Transfusion Centre (University Medical Center, Freiburg, Germany).
Venous blood was centrifuged on a LymphoPrep gradient (density:
1.077 g/cm3, 20 min, 500g, 20 °C; Progen). After centrifugation, cells
were washed twice with PBS and subsequently cultured in RPMI 1640
medium supplemented with 10% heat-inactivated fetal calf serum (GE
Healthcare Life Sciences), 2 mM L-glutamine, 100 U/mL penicillin,
and 100 U/mL streptomycin (all from Life Technologies). The cells
were cultured at 37 °C in a humidified incubator with a 5% CO2/
95% air atmosphere.
T lymphocyte proliferation data for T. asiatica MeOH
extract, 1H and 13C NMR data for compounds 1−5, 6a,
8, and 12−15; experimental and computed ECD spectra
of 7, 8, 11, and 15 as well as UV spectra for compounds
7−13 and 15; 1D and 2D NMR spectra and chiral-phase
HPLC data (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Matthias Hamburger − Pharmaceutical Biology, Pharmacenter,
University of Basel, 4056 Basel, Switzerland; orcid.org/
0000-0001-9331-273X; Phone: +41 61 207 14 25;
Email: matthias.hamburger@unibas.ch; Fax: +41 61 207 14
74
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https://dx.doi.org/10.1021/acs.jnatprod.0c00564
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
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Amy M. Zimmermann-Klemd − Center for Complementary
Medicine, Institute for Infection Prevention and Hospital
Epidemiology, Faculty of Medicine, University of Freiburg,
79106 Freiburg, Germany
Ombeline Danton − Pharmaceutical Biology, Pharmacenter,
University of Basel, 4056 Basel, Switzerland
Martin Smiesko − Pharmacenter, University of Basel, 4056
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00564
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
C.G. is supported by PRIAM-BS (Verein Stiftungsprofessur fu
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