Immunomodulatory Потенциал Bibenzil-diгидрофенантрен дериватив изолируется от Calanthe cardioglossa.

The Orchidaceae family is the
second largest among angiosperms, encompassing approximately 850 genera
and 20,000 species. This family plays a significant role in the commercial
cut flower industry, and many of its species have documented uses
as remedies for various diseases. 1 The
genus Calanthe has a rich history of use in traditional
medicine across China (Traditional Chinese Medicine, TCM), Thailand,
India (Ayurveda), Indonesia, and Nepal for treating a range of ailments.
In TCM, around 14 species of Calanthe are utilized
in phytotherapy to address health issues such as traumatic injuries,
edema, and pain relief for conditions such as joint pain, toothaches,
and abdominal discomfort. 2 − 4 Moreover, various bioactive compounds
isolated from Calanthe species including phenanthrenes,
phenanthrenequinones, calanthoside, and 6′- O -β-D-apiofuranosylindican have demonstrated significant biological
activities, including antiplatelet aggregation, anticancer effects,
and hair restoration. 5 − 9 Calanthe cardioglossa Schltr. is a terrestrial
orchid distributed throughout Thailand, Laos, and Vietnam. This plant
has been used as a tonic in Thai ethnomedicine 2 , 10 and
has not yet been reported in any phytochemical studies. Therefore,
investigating the compounds present in this plant for their biological
activities is particularly intriguing.

Inflammation is a natural
process that helps the body heal and
defend itself from harm. A dynamic balance between pro-inflammatory
and anti-inflammatory processes is essential for controlling disease
and limiting pathology. However, when inflammation persists and becomes
chronic, it can be detrimental. Chronic inflammation is a key factor
in many diseases, and prolonged periods of covert or subclinical inflammation
may precede organ damage, leading to clinical manifestations, such
as obesity, cardiovascular disease (CVD), type 2 diabetes (T2D), and
neurological diseases. Strengthening the human immune system is a
potential strategy for addressing disease pathogenesis and reducing
mortality. Immunomodulatory treatments that utilize phytochemicals
derived from plants are gaining attention as promising approaches
to combat various diseases such as cancer, 11 neuroinflammation 12 and infections. 13 Medicinal plants are excellent sources of bioactive
compounds; they are affordable, widely accessible, and generally associated
with fewer side effects, making them ideal candidates for drug development.
Notable immune-active bioactive molecules from plants include bibenzyls, 11 , 14 phenanthrenes, 12 alkaloids, 15 , 16 and terpenoids. 17 Additionally, active
compounds derived from orchids have been reported to exhibit immunomodulatory
effects. 11 , 12 , 14 This study
aimed to isolate compounds from C. cardioglossa and
to evaluate their immunomodulatory activity in neuroinflammation,
i.e., multiple sclerosis (MS), an autoimmune inflammatory disease
of the central nervous system (CNS) that is a leading cause of chronic
neurological disability. 18 , 19

Results and Discussion

Compound 1 was
obtained as a brown, amorphous solid.
The molecular formula was determined to be C 30 H 26 O 6 based on its [M–H] − ion at m / z 481.1637 (calcd 481.1651 for C 30 H 25 O 6 ) in the HR-ESI-MS. The IR spectrum
showed the absorption bands at 3338 (hydroxyl groups), 1551 and 1465
(aromatic rings) cm –1 , and UV maxima at 217, 279,
and 298 nm, which corresponded to dihydrophenanthrene skeleton. 20 , 21 The 1 H NMR spectrum presented characteristics of dimeric
dihydrophenanthrene with signals of four methylene groups at δ H 2.52 (4H, m, H 2 -9 and H 2 -10) and δ H 2.75 (4H, m, H 2 -9′ and H 2 -10′).

For the first dihydrophenanthrene moiety (rings A and B), 1 H NMR showed signals for a singlet aromatic proton at δ H 6.59 (1H, s, H-3), the ABX spin system at δ H 6.66 (1H, br s, H-8), δ H 6.68 (1H, dd, J = 8.4, 2.8 Hz, H-6), δ H 8.06 (1H, d, J = 8.4 Hz, H-5), a methoxy group at δ H 3.87 (3H, s, MeO-4) and a hydroxyl proton at δ H 8.17 (1H, s, HO-7) ( Table 1 ). The locations of H-3 and H-5 were assigned by their HMBC
connection with C-4a (δ C 116.9). The HMBC correlation
between H-8 and C-9 (δ C 30.5) was also observed ( Figure 1 ). The methoxy group
was attached at C-4 (δ C 157.8) confirmed by its NOESY
cross-peak with H-3 and H-5 ( Figure 2 ). Therefore, the first phenanthrene moiety was 2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene
with C-1 substitution.

Table 1 1 H (400 M Hz) and 13 C NMR (100 MHz) Spectral Data of Compound 1 in
Acetone- d 6 (δ in ppm, J in Hz)   1 Position δ H ( J in Hz) δ C , type 1   117.3, C 2   154.9, C 3 6.59, s 99.0, CH 4   157.8, C 4a   116.9, C 4b   126.0, C 5 8.06, d (8.4) 130.1, CH 6 6.68, dd (8.4, 2.8) 113.3, CH 7   156.0, C 8 6.66, br s 114.6, CH 8a   140.2, C 9 2.52, m 30.5, CH 2 10 2.52, m 28.5, CH 2 10a   140.8, C 1′ 6.39, d (2.8) 106.0, CH 2′   159.4, C 3′ 6.40, d (2.8) 101.5,
CH 4′   155.9, C 4a′   115.7, C 4b′   126.2, C 5′ 8.15, s 132.6, CH 6′   121.3, C 7′   153.9, C 8′ 6.84, s 115.6, CH 8a′   139.3, C 9′ 2.75, m 30.4,
CH 2 10′ 2.75, m 31.5, CH 2 10a   141.4, C MeO-4 3.87, s 55.7, CH 3 MeO-2′ 3.74, s 55.3, CH 3 HO-7 8.17, s   HO-4′ 8.56, s

1 H (400 M Hz) and 13 C NMR (100 MHz) Spectral Data of Compound 1 in
Acetone- d 6 (δ in ppm, J in Hz)

Key HMBC (arrow) and COSY (bold) correlations of compounds 1 – 3 .

Key NOESY correlations of compounds 1 – 3 .

For the second dihydrophenanthrene moiety (rings
A′ and
B′), the 1 H NMR spectrum displayed two meta -coupled aromatic protons at δ H 6.39 (1H, d, J = 2.8 Hz, H-1′) and δ H 6.40 (1H,
d, J = 2.8 Hz, H-3′), two singlet aromatic
protons at δ H 6.84 (1H, s, H-8′) and δ H 8.15 (1H, s, H-5′), a methoxy group at δ H 3.74 (3H, s, MeO-2′) and one hydroxyl proton at δ H 8.56 (1H, s, HO-4′) ( Table 1 ). The assignments of H-1′ and H-8′
were deduced from their HMBC correlations to C-10′ (δ C 31.5) and C-9′ (δ C 30.4), respectively.
The assignments of H-3′ and H-5′ were confirmed by their
HMBC correlations with C-4a′ (δ C 115.7) ( Figure 1 ). The location of
the methoxy group at C-2′ was supported by its NOESY interactions
with H-1′ and H-3′ ( Figure 2 ). So, the second moiety of dihydrophenanthrene
was 4′,7′-dihydroxy-2′-methoxy-9′,10′-dihydrophenanthrene
with C-6′ substitution.

The connection between the two
moieties of dihydrophenanthrene
at C-1 (δ C 117.3) and C-6′ (δ C 121.3) was deduced by the HMBC correlations from H-5′ to
C-1. The experimental ECD spectrum of compound 1 exhibited
negative Cotton effects at 248 and 314 nm, and a positive Cotton effect
at 271 nm ( Figure 3 ). This spectral feature is closely matched with the calculated ECD
spectrum of the blestrianol B from Bletilla striata , 22 therefore the absolute configuration
of compound 1 was assigned as (a R ,a′ R ,a″ S )-configuration. From the evidence
mentioned above, it leads to the structure compound 1 as shown and is named calancardin A.

Experimental ECD spectra
in MeOH and enantiomer structure of compounds 1 – 3 .

Compound 2 was isolated as a brown
amorphous solid.
The formula was determined to be C 31 H 30 O 6 based on its [M–H] − ion at m / z 497.1914 (calcd 497.1964 for C 31 H 29 O 6 ) in the HR-ESI-MS. The IR spectrum
exhibited the absorption bands at 3337 (hydroxyl groups), 1576 and
1444 (aromatic rings) cm –1 . The UV spectrum showed
maximum absorption at 203, 279, and 298 nm, suggesting a bibenzyl-dihydrophenanthrene
skeleton. 20 , 22 This was confirmed by 1 H, 13 C NMR, and HSQC spectra. Two methylene groups at δ H 2.76 (4H, m, H 2 -9 and H 2 -10) with HSQC
correlations to the carbon atom at δ C 29.6 (C-9)
and δ C 30.6 (C-10) and two methylene groups at δ H 2.63 (2H, m, H 2 -α′) and 2.69 (2H,
m, H 2 -α″) with HSQC correlations to the carbon
at δ C 37.0 (C-α′) and δ C 37.3 (C-α″) exhibited characteristics of dihydrophenanthrene
and bibenzyl, respectively. 23

For
the dihydrophenanthrene moiety (rings A and B), the 1 H
and 13 C NMR spectra ( Table 2 ) closely resembled that of the second dihydrophenanthrene
moiety of compound 1 . The 1 H NMR spectrum
displayed two doublet aromatic protons at δ H 6.39
(1H, d, J = 2.8 Hz, H-1) and δ H 6.41
(1H, d, J = 2.8 Hz, H-3), two singlet aromatic protons
at δ H 6.85 (1H, s, H-8) and δ H 8.24
(1H, s, H-5), one methoxy group at δ H 3.74 (3H, s,
MeO-2) and two hydroxyl protons at δ H 7.59 (1H, s,
HO-7) and δ H 8.58 (1H, s, HO-4). A NOESY interaction
of MeO-2 with H-1 and H-3 places the methoxy group at C-2 ( Figure 2 ). From the above
evidence, the first moiety of compound 2 was 4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene
with a C-6 substitution.

Table 2 1 H (400 M Hz) and 13 C NMR (100 MHz) Spectral Data of Compounds 2 – 3 in Acetone- d 6 (δ
in ppm)   2   3   Position δ H ( J in Hz) δ C , type δ H ( J in Hz) δ C , type 1 6.39, d (2.8) 105.2,
CH   117.3, C 2   158.5, C   154.5, C 3 6.41, d (2.8) 100.6,
CH 6.58, s 99.0, CH 4   155.1, C   157.7, C 4a   114.9, C   116.8, C 4b   125.3, C   126.0, C 5 8.24, s 132.0, CH 8.06,
d (8.8) 130.1, CH 6   120.2, C 6.69, dd (8.8, 2.0) 113.3, CH 7   153.1, C   156.0, C 8 6.85, s 114.7, CH 6.68, br s 114.6, CH 8a   138.5, C   140.2, C 9 2.76, m 29.6, CH 2 2.52, t (7.2) 30.6, CH 2 10 2.76, m 30.6, CH 2 2.39, m 28.3, CH 2 10a   140.5, C   140.3, C 1′   143.4, C   145.1, C 2′   117.5, C 6.32, br t (2.0) 108.9, CH 3′   156.1, C   159.2, C 4′ 6.37, d (2.4) 99.1, CH 6.24, t (2.0) 99.7, CH 5′   159.9, C   161.8, C 6′ 6.44, d (2.4) 106.4, CH 6.31, br
t (2.0) 106.3, CH 1″   144.1, C   133.4,
C 2″ 6.49, t (2.0) 113.5, CH 6.87, d (2.0) 111.7,
CH 3″   159.7, C   148.3, C 4″ 6.59, ddd (8.0, 2.8, 0.8) 111.3, CH   143.6, C 5″ 6.57, t (8.0) 128.9,
CH   123.5, C 6″ 6.55, br d (8.0) 120.4, CH 6.54, d (2.0) 124.8, CH α′ 2.63, m 37.0, CH 2 2.84, br s 39.0, CH 2 α″ 2.69, m 37.3, CH 2 2.84, br s 38.1, CH 2 MeO-2 3.74, s 54.4, CH 3     MeO-4     3.87, s 55.7, CH 3 MeO-5′ 3.76, s 54.4, CH 3 3.71, s 55.2, CH 3 MeO-3″ 3.31, s 54.2, CH 3 3.89, s 56.2, CH 3 HO-4 8.58, s       HO-7 7.59, s   8.16, s   HO-3′ 7.31, s   8.21, s

1 H (400 M Hz) and 13 C NMR (100 MHz) Spectral Data of Compounds 2 – 3 in Acetone- d 6 (δ
in ppm)

For bibenzyl moiety (rings A′ and B′),
the 1 H NMR signals revealed two meta -coupled
aromatic
protons at δ H 6.37 (1H, d, J = 2.4
Hz, H-4′) and δ H 6.44 (1H, d, J = 2.4 Hz, H-6′), a set of aromatic proton for meta -substituted benzene at δ H 6.49 (1H, t, J = 2.0 Hz, H-2″), δ H 6.55 (1H,
br d, J = 8.0 Hz, H-6″), δ H 6.57 (1H, t, J = 8.0 Hz, H-5″), and δ H 6.59 (1H, ddd, J = 8.0, 2.8, 0.8 Hz, H-4″),
two methoxy groups at δ H 3.31 (3H, s, MeO-3″)
and δ H 3.76 (3H, s, MeO-5′) and one hydroxyl
proton (δ H 7.31, 1H, s, HO-3′) ( Table 2 ). The assignment of H-6′
was referenced from the HMBC correlation to C-α′ and
C-2′ (δ C 117.5). The position of H-2″
and H-6″ was confirmed by three-bond correlation to C-α″
in the HMBC spectrum. In addition, the 1 H– 1 H COSY of H-5″ with H-4″ and H-6″ was observed
( Figure 1 ). The location
of MeO-5′ at C-5′ (δ C = 159.9) was
confirmed by its NOESY correlations with H-4′ and H-6′.
The attachment of MeO at C-3″ (δ C 159.7) was
inferred by its NOESY correlations with H-2″ and H-4″
( Figure 2 ). Thus, this
bibenzyl moiety was 3′-hydroxy-5′,3″-dimethoxybibenzyl
with C-2′ substitution.

Compound 2 was derived
from dihydrophenanthrene and
bibenzyl moieties which connected at C-6 (δ C 120.2)
and C-2′ by the HBMC correlation of H-5 with C-2′. The
experimental ECD spectrum of this compound showed a negative Cotton
effect at 254 nm and a positive Cotton effect at 282 nm ( Figure 3 ). The absolute configuration
of compound 2 was assessed as (a S ,a′ S )-configuration when compared to (a S ,a′ S )-blestanol H from previous reported. 22 We named compound 2 calancardin B.

Compound 3 was obtained as a brown amorphous solid.
The molecular formula was C 31 H 30 O 7 based on its [M–H] − ion at m / z 513.1859 (calcd 513.1913 for C 31 H 29 O 7 ) in the HR-ESI-MS. The IR spectrum exhibited
the absorption bands at 3305 cm –1 (hydroxyl groups),
2952 and 1638 cm –1 (aromatic rings). This compound
was proposed to be a bibenzyl-dihydrophenanthrene skeleton based on
the maximum UV absorption peaks at 204 and 281 nm, 23 along with NMR spectra of dihydrophenanthrene characteristic
[two signals of methylene protons at δ H 2.39 (2H,
m, H 2 -10) and at δ H 2.52 (2H, t, J = 7.2 Hz, H 2 -9) with two methylene carbon signals
at δ C 28.3 (C-10) and at δ C 30.6
(C-9)] and bibenzyl characteristic [four methylene proton signals
at δ H 2.84 (4H, br s, H 2 -α′
and H 2 -α″) with two methylene carbon signals
at δ C 38.1 (C-α″) and at δ C 39.0 (C-α′)]. The 1 H and 13 C NMR data ( Table 2 ) of dihydrophenanthrene moiety in compound 3 (rings
A and B) was strikingly similar to the first dihydrophenanthrene moiety
of compound 1 . The 1 H NMR exhibited the following
signals: a singlet aromatic proton at δ H 6.58 (1H,
s, H-3), an ABX system proton at δ H 6.68 (1H, br
s, H-8), δ H 6.69 (1H, dd, J = 8.8,
2.0 Hz, H-6), δ H 8.06 (1H, d, J =
8.8 Hz, H-5), a methoxy group at δ H 3.87 (3H, s,
MeO-4) and a hydroxyl proton (δ H 8.16, 1H, s, HO-7).
The positions of H-3 and H-5 were confirmed by their HMBC correlations
with C-4a (δ C 116.8). H-8 was assigned by its HMBC
correlation with C-9. The attachment of the methoxy group to C-4 (δ C 157.7) was referenced from its NOESY correlation with H-3
and H-5 ( Figure 2 ).
Consequently, the structure of the dihydrophenanthrene moiety of compound 3 was 2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene with
C-1 substitution.

In addition, the 1 H NMR spectrum
displayed three meta -coupled aromatic protons at
δ H 6.24
(1H, t, J = 2.0 Hz, H-4′), δ H 6.31 (1H, br t, J = 2.0 Hz, H-6′), and δ H 6.32 (1H, br t, J = 2.0 Hz, H-2′),
a pair of meta -coupled aromatic proton at δ H 6.54 (1H, d, J = 2.0 Hz, H-6″) and
δ H 6.87 (1H, d, J = 2.0 Hz, H-2″),
together with three singlet protons at δ H 3.71 (3H,
s, MeO-5′), δ H 3.89 (3H, s, MeO-3″),
and δ H 8.21 (1H, s, HO-3′) ( Table 2 ). The position of H-2′
and H-6′ was assigned by the HMBC correlation to C-α′
and C-4′ (δ C 99.7). The assignment of H-2″
and H-6″ was referenced from the HMBC cross-peak to C-α″
and C-4″ (δ C 143.6) ( Figure 1 ). Moreover, two methoxy groups were placed
at C-5′ (δ C 161.8) and C-3″ (δ C 148.3) based on their NOESY correlation from MeO-5′
to H-4′ and H-6′ and MeO-3″ to H-2″ ( Figure 2 ). Therefore, the
second moiety of compound 3 was 3′,4″-dihydroxy-5′,3″-dimethoxybibenzyl
with C-5″ substitution.

The connection at C-1 (δ C 117.3) and C-5″
(δ C 123.5) of the two moieties was deduced by the
HMBC correlation from H-6″ to C-1. Furthermore, the experimental
ECD spectrum of compound 3 displayed a positive Cotton
effect at 254 nm and a negative Cotton effect at 282 nm ( Figure 3 ), for which the
absolute configuration of compound 3 was assessed as
(a R ,a′ R )-configuration by
comparison with (a R ,a′ R )-blestanol
F from literature data. 22 From the aforementioned
evidence, the structure of compound 3 was finally determined
and named calancardin C.

Calancardin A ( 1 ) is a
dimer of dihydrophenanthrene.
Calancardins B ( 2 ) and C ( 3 ) were dimers
of dihydrophenanthrene and bibenzyl, which were less common than dimers
formed between dihydrophenanthrene and dihydrophenanthrene/phenanthrene. 24 Bibenzyl-dihydrophenanthrene derivatives have
mostly been reported to be found in orchids including Bletilla
formosana , 25 Bletilla
striata , 22 , 26 Dendrobium parishii , 27 Dendrobium signatum , 28 Pholidota chinensis , 23 Pholidota yunnanensis , 20 and Pleione bulbocodioides . 29 , 30 Additionally, 12 known compounds were isolated
from the whole plant of C. cardioglossa : 3-(2-hydroxyphenyl)-4(3 H )-quinazolinone ( 4 ), 31 batatasin III ( 5 ), 32 gigantol
( 6 ), 33 coelonin ( 7 ), 34 lusianthridin ( 8 ), 20 ephemeranthoquinone ( 9 ), 35 gymconopin C ( 10 ), 36 blestriarene A ( 11 ), 37 phochinenin G ( 12 ), 23 bletillatin
A ( 13 ), 26 (2,3- trans )-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-10-methoxy-2,3,4,5-tetrahydrophenanthro[2,1-]
furan-7-ol ( 14 ), 38 and shanciol
G ( 15 ). 39 Their structures
were identified by comparing physicochemical data with the literature
data.

To investigate the immunomodulatory effects, all isolated
compounds
from C. cardioglossa , except calancardin C ( 3 ) and phochinenin G ( 12 ) due to insufficient
quantities, were tested for their activity in THP-1 cells, a human
monocytic cell line. Toxicity tests in THP-1 cells ( Figure 4 A) indicated that most compounds
(compounds 1 , 2 , 4 , 5 , 6 , 7 , 8 , 10 , 13 , 15 ) were not cytotoxic at a concentration
of 10 μM. In our previous study, 14 a concentration at 10 μM of bibenzyls has been shown to have
good immunomodulatory effects in an in vitro model
of LPS-induced inflammation in human primary peripheral blood mononuclear
cells (PBMCs). In this study, we also used the concentration of 10
μM to further evaluate the immunomodulatory effects under LPS
stimulation of the purified compounds in the THP-1 cell line as well
as in human PBMCs. Utilizing the THP-1 cell line, five compounds including
calancardin A ( 1 ), calancardin B ( 2) , batatasin
III ( 5 ), gigantol ( 6 ), and lusianthridin
( 8 ) demonstrated a significant reduction in LPS-induced
TNF-α production compared to the control group ( Figure 4 B). Among these five active
compounds, calancardin B ( 2 ) exhibited the most pronounced
activity in reducing the level of TNF-α production following
LPS stimulation. Consequently, we further investigated its immunomodulatory
effects on primary human PBMCs. To achieve this, we conducted in vitro assessments using PBMCs isolated from five patients
with MS (pwMS).

(A) Cell viability of isolated compounds at 10, 25, 50,
and 100
μM in LPS-stimulated THP-1 monocytes. (B) TNF-α level
of isolated compounds at 10 μM in LPS-stimulated THP-1 monocytes.
The results are shown as mean ± SD. One-way ANOVA followed the
correction of multiple comparisons (Tukey test), *** P < 0.001, ** P < 0.01, * P <
0.05.

In brief, PBMCs were stimulated with LPS following
a previously
described method. 12 , 14 Four hours after LPS stimulation,
we determined the expression of cytokines IFN-γ, IL-2, and TNF-α
in multiple cell types, including CD3 + T cells, CD14 + monocytes, CD19 – HLADR + and CD19 – HLADR – myeloid cells, as well as
CD19 + B cells using flow cytometry ( Figure 5 ). Unstimulated PBMCs served as negative
controls. No changes in IFN-γ and IL-2 could be observed in
all cell populations after LPS stimulation. As expected, we observed
an increased expression of TNF-α in CD14 + monocytes
but not in other immune cell population ( Figure 5 ). This specificity of monocytes in responding
to LPS is attributed to the binding of LPS to TLR4 receptors, which
facilitates TNF-α expression with the assistance of CD14, a
marker unique to monocytes. 40

Dot plots and
histograms display a flow cytometric gating strategy
in LPS-induced inflammatory cytokine (IFN-γ, IL-2, and TNF-α)
expression in human PBMCs of multiple sclerosis obtaining CD3 + T cells (G5), CD14 + monocytes (G6), and B cells
(G9). CD19 – HLADR + (G8) and B cells (G9).

PBMCs from pwMS that were coincubated with calancardin
B ( 2 ) (PBMCs + LPS + calancardin B) demonstrated a significant
reduction in LPS-induced TNF-α production in CD14 + monocytes, as compared with LPS-stimulated PBMCs without calancardin
B treatment (i.e., PBMCs + LPS) ( Figure 6 ). It is well established that pwMS present
both functional and compositional alterations in their monocyte populations,
particularly an increased level of pro-inflammatory cytokines such
as TNF-α during the active phases of the disease, which contribute
to neuroinflammation. 41 , 42 Our findings suggest a correlation
between monocyte activation and disease activity, underscoring the
central role of monocytes in MS pathology. Consequently, modulating
monocyte activity presents a promising therapeutic strategy for MS.
Approaches aimed at enhancing the anti-inflammatory functions of monocytes
or inhibiting their pro-inflammatory actions could lead to novel treatment
options for this condition.

Determination of the immune modulatory effects. Bar graphs
show
the percentage of frequency of TNF-α expression in the immune
cells of MS PBMCs (five biological replicates) after 4 h treatment
with 10 μM of active compound from C. cardioglossa with or without LPS stimulation. One-way ANOVA followed the correction
of multiple comparisons (Tukey test), **** P <
0.0001, *** P < 0.001, ** P <
0.01, * P < 0.05; ns, not significant.

The most potent compound, calancardin
B ( 2 ), is a
dimer formed from 9,10-dihydrophenanthrene (monomer: lusianthridin, 8 ) and bibenzyl (monomer: 3′-O-methylbatatasin III).
It has demonstrated the ability to inhibit LPS-induced TNF-α
expression in both THP-1 monocytes and PBMCs from pwMS. The effects
of calancardin B may be enhanced by the synergistic action of its
two monomers, both of which are capable of reducing TNF-α production
following LPS stimulation in THP-1 monocytes and PBMCs. In examining
the structure–activity relationships (SAR) of five active compounds
(compounds 1 , 2 , 5 , 6 , 8 ) within the THP-1 monocyte model, it was observed
that two bibenzyl compounds (batatasin III ( 5 ) and gigantol
( 6 )) and calancardin B ( 2 ), a bibenzyl-dihydrophenanthrene
derivative (a dimer of lusianthridin ( 8 ) and 3′-O-methyl-batatasin
III) exhibited low activity in inhibiting TNF-α production after
LPS stimulation. These compounds share a core structure featuring
a methoxy group at C-5 and a hydroxy group at C-3 within the bibenzyl
moiety. Furthermore, it is plausible to suggest that a methoxy group
at C-3′, combined with the absence of substitution at C-4′,
would yield higher activity compared to that of having a hydroxy group
at C-3′. This aligns with our previous findings, which indicated
that bibenzyl compounds associated with the inhibition of LPS-induced
TNF expression in PBMCs possess three methoxy groups located at C-3,
C-5, and C-3′. 14 For dihydrophenanthrene
derivatives, calancardin A ( 1 ) (a dimer of lusianthridin
( 8 ) and coelonin ( 7 )) and lusianthridin
( 8 ) resulted in a slight decrease in TNF-α levels
in THP-1 monocytes stimulated with LPS, while coelonin ( 7 ) did not demonstrate any inhibitory effect in these cells. The distinction
between these 9,10-dihydrophenanthrene compounds lies in their structural
features: lusianthridin ( 8 ) contains a methoxy group
at C-2 and a hydroxy group at C-4, whereas calancardin A ( 1 ), which is derived from lusianthridin ( 8 ) and coelonin
( 7 ), along with coelonin ( 7 ) itself, possesses
a hydroxy group at C-2 and a methoxy group at C-4.

In summary,
three newly elucidated dimers composed of dihydrophenanthrene
and bibenzyl derivatives, alongside 12 known compounds, were isolated
from C. cardioglossa . Notably, calancardin B ( 2 ), a dimer of dihydrophenanthrene and bibenzyl, displayed
a substantial reduction in TNF-α levels in both THP-1 cells
and monocytes isolated from pwMS. This finding indicates that calancardin
B may exert beneficial immunomodulatory effects, thereby offering
promising prospects for the development of treatments targeting immune-related
inflammatory diseases.

Experimental Section

General Experimental Procedures

Optical rotations were
measured on a Jasco P-2000 polarimeter (Hachioji, Tokyo, Japan). UV
spectra were recorded using a Shimadzu UV-1800 spectrophotometer (Kyoto,
Japan). The IR spectra were obtained by using a PerkinElmer FT-IR
spectrophotometer (Waltham, MA, USA). NMR spectra were recorded on
a Bruker Avance DPX-400 FT-NMR spectrometer (Billerica, MA, USA).
High-resolution electrospray ionization mass spectra (HRESIMS) were
recorded on an Agilent 6530 accurate-mass Q-TOF LC/MS instrument (Santa
Clara, CA, USA). CD spectra were measured with a Jasco J-815 spectrophotometer
(Hachioji, Tokyo, Japan). Semipreparative HPLC was performed on a
Shimadzu SPD-20A UV detector with a Shimadzu LC-20AD pumping system
(Kyoto, Japan) and VDSpher PUR 100 SIL (250 mm × 10 mm) column
(Berlin, Germany). Dry column vacuum chromatography (DCVC) and column
chromatography (CC) isolations were carried out on silica gel 60 (Merck,
Darmstadt, Germany) with particle sizes of 40–63 and 63–200
μm. Sephadex LH-20 (Merck, Darmstadt, Germany) was used for
separation by size exclusion chromatography. Thin layer chromatography
(TLC) was run on aluminum sheets of silica gel 60 F 254 (Merck,
NJ, USA) and detected under UV light. Human monocytes (THP-1 cells)
and RPMI-1640 medium were purchased from the American Type Culture
Collection (ATCC; Manassas, VA, USA). 2-Mercaptoethanol, dimethyl
sulfoxide (DMSO), lipopolysaccharides (LPS), and Human Tumor Necrosis
Factor (TNF-α) ELISA kit were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Fetal bovine serum (FBS) and penicillin/streptomycin
were obtained from Gibco (Gaithersburg, MA, USA). PrestoBlue cell
viability reagent was purchased from Thermo Fisher Scientific Inc.
(Rockford, IL, USA).

Plant Material

Plant material was purchased from the
Chatuchak market in April 2020. Plant identification was performed
by Yanyong Punpreuk, Department of Agriculture, Bangkok, Thailand.
A voucher specimen (BS-CC-012563) was deposited in the Department
of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical
Sciences, Chulalongkorn University.

Extraction and Isolation

The dried whole plants of C. cardioglossa (2 kg) were extracted with MeOH (3 ×
10 L) at room temperature. The MeOH extract (74.6 g) was subjected
to dry column vacuum chromatography (DCVC) using silica gel with a
hexane-acetone gradient to give 7 fractions (Fr.A-G). Fraction E (2.6
g) was isolated with column chromatography (CC) on silica gel (MeOH–CH 2 Cl 2 gradient) to give 7 fractions (EI-EVII). Fraction
EV (112.6 mg) was separated on Sephadex LH-20 (acetone) and then CC
(silica gel, acetone/hexane 1:9) to yield gigantol ( 6 ) (22.1 mg) and ceolonin ( 7 ) (23.1 mg). Faction EVII
(1.2 g) was isolated using Sephadex LH-20 (acetone) and then CC (silica
gel, EtOAc-hexane 1:3) to obtain batatasin III ( 5 ) (509.1
mg) and 3-(2-hydroxyphenyl)-4(3 H )-quinazolinone ( 4 ) (3.3 mg). Fraction EVI (151.0 mg) was purified by Sephadex
LH-20 (acetone) and then CC (silica gel, MeOH–CH 2 Cl 2 1.5:98.5) to yield lusianthridin ( 8 )
(5.0 mg). Fraction F (4.3 g) was isolated using CC on silica gel (MeOH–CH 2 Cl 2 gradient) to obtain 7 fractions (FI–FVII).
Faction FIII (60.3 mg) was purified using HPLC (silica gel, MeOH–CH 2 Cl 2 1.5:98.5) to yield ephemeranthoquinone ( 9 ) (4.3 mg), gymconopin C ( 10 ) (2.0 mg), and
blestriarene A ( 11 ) (7.7 mg). Fraction FIV (23.5 mg)
was isolated with Sephadex LH-20 (MeOH) and then HPLC (silica gel,
MeOH–CH 2 Cl 2 1.5:98.5) to give compound 1 (3.5 mg), phochinenin G ( 12 ) (1.7 mg), bletillatin
A ( 13 ) (2.1 mg), and (2,3- trans )-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-10-methoxy-2,3,4,5-tetrahydro-phenan
( 14 ) (5.0 mg). Fraction FVI (32.7 mg) was separated with
Sephadex LH-20 (MeOH) and then HPLC (silica gel, EtOAc-hexane 1:3)
to obtain compound 2 (3.3 mg), compound 3 (1.3 mg), and shanciol G ( 15 ) (2.2 mg).

Calancardin A ( 1 )

Brown amorphous solid;
α D 20 –
7.1 ( c 0.10, MeOH). UV (MeOH) λ max (log ε) 217 (2.98), 279 (1.91), 298 (1.51) nm; ECD (MeOH)
λ max (Δε) 224 (+4.57), 248 (−2.82),
271 (+0.39), 314 (−1.20) nm; IR (ATR) ν max 3338, 2935, 2853, 1647, 1617, 1551, 1465, 1247, 1211 cm –1 ; 1 H and 13 C NMR data, see Table 1 . HRESIMS m / z 481.1637 [M–H] − (calcd
481.1651 for C 30 H 25 O 6 ).

Calancardin B ( 2 )

Brown amorphous solid;
α D 20 +
5.8 ( c 0.10, MeOH). UV (MeOH) λ max (log ε) 203 (2.87), 279 (0.96), 298 (0.69) nm; ECD (MeOH)
λ max (Δε) 224 (+4.65), 254 (−6.20),
282 (+0.80), 309 (−5.62) nm; IR (ATR) ν max 3337, 2941, 2853, 1620, 1576, 1444, 1258 cm –1 ; 1 H and 13 C NMR data, see Table 2 . HRESIMS m / z 497.1914 [M–H] − (calcd 497.1964 for C 31 H 29 O 6 ).

Calancardin C ( 3 )

Brown amorphous solid;
α D 20 –
4.0 ( c 0.10, MeOH). UV (MeOH) λ max (log ε) 204 (3.46), 281 (1.03) nm; ECD (MeOH) λ max (Δε) 231 (−6.50), 254 (+0.25), 282 (−11.01)
nm; IR (ATR) ν max 3305, 2952, 2848, 1638, 1521, 1456,
1206, 1063 cm –1 ; 1 H and 13 C NMR data, see Table 2 . HRESIMS m / z 513.1859 [M–H] − (calcd 513.1913 for C 31 H 29 O 7 ).

Cell Viability and TNF-α Screening

THP-1 cells
were maintained in RPMI-1640 supplemented with 0.05 mM 2-mercaptoethanol,
10% FBS and 1% Pen/Strep at 5% CO 2 and 37 °C. For
cell viability testing, the PrestoBlue cell viability reagent was
used as in the previous study with slight modifications. 43 Cells were seeded at 1 × 10 6 cells/mL into 96-well plate (Corning, Jiangsu, Chaina) and treated
with various concentrations of compounds from C. cardioglossa for 4 h, then PrestoBlue was added, and the cells were incubated
for 30 min in dark conditions at 37 °C. After that, the absorbance
was measured at a fluorescence excitation 560 nm and an emission 590
nm using a microplate reader (CLARIOstar, BMG Labtech, Germany). For
TNF-α screening, the same method with previous study 44 was performed with some modifications. Briefly,
THP-1 cells at 5 × 10 5 cells/mL were seeded into 24-well
plate (Corning, ME, USA), then 100 μM of LPS and 10 μM
of each compound were put into well. After 4 h, the medium was collected
to measure the TNF-α with an ELISA kit.

Ethics and Human Peripheral Blood Mononuclear Cells (PBMCs)

The Ethics for this study was approved by the Ethics Committee
of Charite Universitätsmedizin Berlin (EA1/187/17). Buffy coats
were obtained from five patients with multiple sclerosis. Human MS
PBMCs were isolated and then cryopreserved in liquid nitrogen tank
as described in a prior study. 12

LPS Stimulation in PBMCs

Frozen human MS PBMCs were
thawed and resuspended with RPMI 1640 medium with 10% FBS. Cells were
seeded in an ultralow-attachment 96-well plate (Corning, New York,
USA) at a density of 2 × 10 5 cells per well. Then,
calancardin B ( 2 ) (10 μM) was added to the corresponding
well. After that, cells were stimulated for inflammatory condition
using LPS (100 ng/mL) and further incubated at 37 °C for 2 h.
Subsequently, brefeldin A (10 μg/mL) was added to the wells
and then incubated for 2 h. Eventually, all cells were harvested and
washed with PBS then analyzed using flow cytometry as described below.

Cytokines in LPS-Treated MS PBMCs Measurement Using Flow Cytometry

LPS-treated MS PBMCs samples were thawed and washed twice with
staining buffer (0.5% BSA in PBS with 2 mM EDTA). Cells were resuspended
in FcR-blocking buffer (1:100; Miltenyi Biotec, Bergisch Gladbach,
Germany) and then incubated at 4 °C for 10 min to block unspecific
antibodies. After incubation with blocking buffer, cells were stained
at 4 °C for 20 min with extracellular immunofluorescent antibodies
including CD3 (APC, HIT3a, Biolegend), CD14 (FITC, RMO52, Beckman
Coulter), CD19 (PE, HIB19, Biolegend) and HLA-DR-(APC/Cy7, L243, Biolegend)
antibodies diluted in staining buffer. Subsequently, 2% methanol-free
FA was added to fix the cells at 4 °C for 30 min. Cells were
washed with staining buffer and incubated at 4 °C for 30 min
with intracellular immunofluorescent antibodies including TNF (BV,
MAb11, Biolegend), IL-2 (PerCP/Cy5.5, MQ1–17H12, Biolegend)
and IFN-γ (PE/Cy7, 4S.B3, Biolegend) diluted in permeabilization
buffer (eBioscience, California, USA). After that, stained cells were
washed with staining buffer and centrifuged at 300 g at 12 °C for 10 min. Finally, the supernatants were discarded,
and pellets were resuspended in staining buffer. For evaluation of
extracellular antibodies and intracellular cytokines, pellets were
measured using BD CANTO II flow cytometer (BD Biosciences, New Jersey,
USA) with software BD DIVA version 8.1. Gating strategy and data analysis
were performed by FlowJo software, version 10.1 (Ashland, OR, USA).

Statistical Analysis

Data are indicated as mean ±
standard deviation (SD). Statistical analysis was conducted using
a one-way analysis of variance (ANOVA) followed by Tukey’s
test for comparisons between groups ( P < 0.05).
The analysis was performed with GraphPad Prism software version 8.0
(San Diego, CA, USA).

Acknowledgments

This research is supported by the 90th Anniversary
of Chulalongkorn University, Rachadapisek Sompote Fund (GCUGR1125672001D).
C.P. under the supervision of B.S. was funded by the Second Century
Fund (C2F) for PhD Scholarship, Chulalongkorn University. We are grateful
to Mr. Yanyong Punpreuk, Department of Agriculture, Bangkok, Thailand,
for plant identification.

Data Availability Statement

The NMR data
for Calancardins A–C ( 1 – 3 )
have been deposited in nmrXiv at https://nmrxiv.org/P89 and can be found at Calancardin A (#NMRXIV:S741),
Calancardin B (#NMRXIV:S743) and Calancardin C (#NMRXIV:S742).

Supporting Information Available

The Supporting Information
is
available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.4c01394 .

Spectrum data (UV, IR, HR-ESI-MS, and NMR (1D and 2D))
of three new compounds ( 1 – 3 ) ( PDF )

Author Contributions

B.S. and C.B.
conceived and designed the project. C.P. performed extraction and
isolation. C.P. and B.S. performed structure elucidation. W.C. performed
spectroscopy data. C.P. performed in vitro experiment
in THP-1 monocytes. A.D. performed in vitro experiment
in MS PBMCs. V.K. and C.B. analyzed flow cytometry data. C.B. designed
the antibody panels for mass cytometry. C.C. and P.R. performed in
data curation. C.P., V.K., C.B., and B.S. wrote the manuscript.

The
authors
declare no competing financial interest.

Supplementary Material

np4c01394_si_001.pdf

References

Associated Data

Supplementary Materials

np4c01394_si_001.pdf

Data Availability Statement

The NMR data
for Calancardins A–C ( 1 – 3 )
have been deposited in nmrXiv at https://nmrxiv.org/P89 and can be found at Calancardin A (#NMRXIV:S741),
Calancardin B (#NMRXIV:S743) and Calancardin C (#NMRXIV:S742).