Neolignane and Monoterpene Glycosides Isolated from D. Don and Their Cytotoxicity Evaluation in HepG2 Cell Lines.

1
Introduction

Cupressus
torulosa D. Don ex Lamb.
(Cupressaceae), commonly known as the Himalayan cypress, Bhutan cypress,
or Surai, is a nonflowering, coniferous,
evergreen, and aromatic tree of the genus Cupressus. The genus Cupressus comprises more than 12 species
worldwide that have been used in traditional medicine to treat infections,
cough, inflammation, and for hepatoprotective activity.
Cupressus torulosa is an indigenous species to India and is widely distributed in the
foothills of the Himalayas (mainly the Northern Himalayas) at altitudes
of 1800–3300 m on limestone terrain in the western Himalayas,
extending to the eastern Himalayas across northeast India, including
Nepal, Bhutan, Tibet, northern Myanmar, and Vietnam. The needles (leaves) of C. torulosa have been used for their antiinflammatory, anticonvulsant, and wound-healing
properties. The chemistry of the nonvolatile
composition of the needle (leaves) extracts has not been examined
much; only the chemistry of volatile (essential oil) compositions
extracted from needles or leaves was explored in detail. The leaves
of the plant have a specific smell or aroma due to essential oil (cypress
oil) rich in monoterpenoids and diterpenoids, and the nonvolatile
leaf extract contains biflavanones, namely cupressuflavone, amentoflavone,
hinokiflavone, and flavone apigenin, and
some diterpenoids. The main chemotaxonomic
chemical compounds of the genus Cupressus are the
bioflavonoids,
,
and the presence of cupressuflavone
is a major biflavone in many species (C. sempervirens, C. glauca, C. goveniana, C. funebris, C. arizonica, and C. torulosa) of the genus Cupressus.

,
The biflavonoids isolated from
these species are very potent biologically active molecules and exhibit
various biological activities, including antibacterial, antiinflammatory,
antidiabetic, antimicrobial, antiviral, antioxidant, hypoglycemic,
anticancer, hepatoprotective, peripheral vasodilation, and lipid peroxidation
inhibition, etc.
−

Few species of this genus, such as C. sempervirens, have been studied intensively for their chemical constituents and
biological activities, and C. torulosa has rarely been investigated in this manner. Only a few reports
were indicate that the aqueous methanolic extract of C. torulosa needles exhibits antiinflammatory activity,
thereby supporting their traditional use in treating inflammatory
disorders. Other organic extracts (hexane,
ethyl acetate, and methanol) of C. torulosa leaves have also been reported for antibacterial properties. Previous reports on the antioxidant activities
of organic extracts, hexane, and chloroform showed weak antioxidant
activity, while polar methanolic extracts exhibited maximum antioxidant
properties, and the aqueous extract showed moderate antioxidant properties. The essential oil extracted from the leaves
of C. torulosa exhibited potent antimicrobial,
antioxidant, and antiinflammatory properties.
,
However, the detailed phytochemical investigation of nonvolatile
compositions and biological activities of this plant has not been
conducted. Therefore, as part of our continuing studies on the phytochemical
investigation of medicinal plants, we isolated three new compounds:
one previously undescribed neolignane glycoside (16)
and two undescribed monoterpene glycosides, 21 and 22, together with 26 known compounds. Among the known compounds,
12 compounds, 11–15, 17–20, 23–24, and 28, were also isolated
for the first time from this plant species. The isolated compounds
were evaluated for cytotoxicity activity against human liver cancer
HepG2 cell lines, and the IC50 values of the tested compounds
ranged from 37.54 μM to 271.13 μM. Among the evaluated
compounds, 13, 15, 18, 19, and 23 showed significant to moderate antiproliferative
properties in a concentration-dependent manner against HepG2 cancer
cell lines. Hence, compound 15 was selected for a detailed
investigation of the antiproliferative mechanism in HepG2 cell lines.

2
Results and Discussion
2.1
Structural Elucidation
LC/MS- and
HPLC-based phytochemical investigation of the acetone extract of C. torulosa was performed using sequential column
chromatography over normal-phase silica gel columns, dianion resin
HP-20, RP-C18 silica column, RP-C18 VLC, Sephadex
LH-20 column, along with preparative TLC. This resulted in the purification
of one new neolignane glycoside (16) and two previously
undescribed monoterpene glycosides (21 and 22), together with twenty-six known compounds (1–15, 17–20 and 23–29). The isolated
compounds were identified using chemical and spectroscopic data, including
1D and 2D NMR, IR, HR-ESI-MS, and circular dichroism (CD) spectral
analysis.
Compound 16 was isolated as a colorless
amorphous powder, with the optical rotation –30.12
(c, 0.0026
g/mL in MeOH). The molecular formula was determined as C30H40O11 by high-resolution electrospray ionization
mass spectrometry (HR-ESI-MS) analysis in positive-ion mode and displayed
an [M + H]+ ion peak at m/z 577.2245 (calcd. for C30H41O11,
577.2643), indicating 11 degrees of unsaturation. The IR spectrum
of the compound showed the presence of hydroxyl (3337 cm–1), carbonyl of ester group (1651 cm–1), and aromatic
ring (1507, 1454, and 1419 cm–1). The 1H NMR (Supporting Information, Figure S1) and 1H–1H COSY spectra (Supporting Information, Figure S5) revealed
the presence of a 1,3,4-trisubstituted benzene ring (ring A) with
δH 7.03 (1H, d, J = 1.9 Hz, H-2),
δH 7.09 (1H, d, J = 8.3 Hz, H-5),
and δH 6.91 (1H, dd, J = 8.3, 2.0
Hz, H-6), as well as a 1′,3′,4′,5′-tetrasubstituted
benzene ring (ring B) with δH 6.72 (1H, brs, H-2’)
and δH 6.71 (1H, brs, H-6’). Additionally,
an ethoxypropyl group was observed with δH 2.61 (t, J = 7.5 Hz, Ph–CH2, H-7’), δH 1.83 (m, CH2, H-8’), δH 3.58 (t, J = 6.5 Hz, OCH2, H-9’),
and -OEt with δH 4.11 (m, ovl, -OCH2,
H-1a) and δH 1.22 (3H, t, J = 6.0
Hz, H-1b). DEPT-135 (Supporting Information, Figure S3) displayed three negative
signals at δC 32.84 (C-7’), δC 35.70 (C-8’), and δC 61.49 (C-9’).
Furthermore, a sequence of methine–methine–methylene
[CH­(O)–CH­(Ph)–CH2OAc] was successively coupled
in this order: δH 5.57 (d, J = 6.0
Hz, H-7), δH 3.49 (m–ovl, H-8), and δH 3.84 (2H, m–ovl, H-9), in addition to an acetate group
-OAc at 2.02 (s, OCOCH
3, H-2b’).
Moreover, the 1H and 13C NMR (Table
) spectral data exhibited the
presence of 30 carbon peaks distributed between δC 172.9 and 14.4 ppm for eight quaternaries, 12 methines, five methylenes,
and methyls. The presence of an anomeric proton δH 5.37 (d, J = 1.8 Hz, H-1″), δC 101.5 (C-1″), and a methyl signal corresponding to
the attached sugar unit indicates the presence of a β-rhamnopyranosyl
in the molecule. The analysis of the cross-peaks of the 1H–1H COSY correlation suggested the presence of
a spin system between H-5 and H-6, establishing the connectivity and
substitution in ring A of the molecule. Furthermore, the analysis
of COSY, NOESY, and HMBC spectra led to the establishment of the side
chain of ring B (Figure
). Long-range correlations through 2
J and 3
J in the HMBC correlations (Figure
) were observed between
(H-1”/C-4) and (H-1''/C-2”), indicating the
connectivity
of the rhamnose unit. 1H–13C HMBC (Supporting Information, Figure S7) correlations between (H-2, H-6/C-7) and (H-2’, H-6’/C-7’)
suggested that compound 16 has two phenylpropanoid units.
A significant HMBC correlation was also identified between (H-7/C-4’,
H-7/C-8) and (H-7/C-5′). Thus, it can be concluded that the
two phenylpropanoids form a dihydrobenzofuran neolignan skeleton.
The NOESY correlation (H-3/OMe to H-2 and H-3′/OMe to H-2’),
HSQC correlation at [δH 3.80 (3H, s, OMe to C-3/OMe
at δC 56.49), and 3.87 (3H, s, OMe’ to C-3’/OMe’
at δC 56.81)], and HMBC correlations from (H-3/OMe
to C-3 and H-3′/OMe to C-3′), as shown in Figure
, suggest the positions of
methoxy groups at the C-3 and C-3′ carbons. Further, the linkage
of the acetate group at the C-9 position and the ethoxy group at the
C-9’ position was confirmed by the HMBC correlation, which
showed correlations from H-9 to C-1a’ of the acetate group
and from H-1a to C-2b of the ethoxy group. The coupling constant between
H-7 and H-8 (J
7 and 8 = 6.0
Hz) suggested that the preferred configuration of the two protons
was trans, further supported by the NOESY correlation
of H-7 with H-9b. The CD spectrum (Figure
) was used to determine the absolute configuration
of 16. The negative Cotton effects at 248 (−12.2)
and 283 (−7.3) nm, and a positive Cotton effect at 236 (+3.6)
nm, indicated that its absolute configuration was assigned as (7R, 8S), similar to that of Icariside E4
(15). Hence, this newly
isolated compound was fully characterized and is named Toruloside
A.

Compound 21 was isolated as a colorless
sticky substance.
The molecule exhibited +30.55 (c 0.003 g/mL in MeOH), and the molecular
formula C16H26O7 was deduced from
the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS)
in positive ion mode at m/z 331.17513
[M + H]+ (calcd. for C16H27O7 331.1751), along with the NMR data (Table
). Hence, the molecular formula indicates
that the molecule has four degrees of unsaturation. The IR spectrum
of compound 21 showed characteristic absorption bands
for the hydroxy group at 3362 cm–1 and the carbonyl
group at 1702 cm–1. The 1H NMR spectrum
of 21 (Table
) revealed the presence of signals for three methyl groups
at δH 0.93 (3H, d, J = 7.1 Hz, H-8),
δH 1.05 (3H, d, J = 6.8 Hz, H-9),
and δH 1.49 (3H, s, H-10); methylene, including oxygenated
methylene signals at δH 2.37 (1H, d, J = 17.0 Hz, H-3), δH 1.15 (1H, d, J = 7.1 Hz, H-3), δH 0.97 (1H, t, J = 5.0 Hz, H-6a), δH 1.21 (1H, dd, J = 7.9, 5.4 Hz, H-6b), and oxygenated signals at δH 3.84 (1H, dd, J = 11.8, 2.5 Hz, H-6′a) and
δH 3.68 (1H, dd, J = 11.9, 5.1 Hz,
H-6′b); seven methines at 2.33 (1H, m, H-5) and 2.08 (1H, q, J = 6.8 Hz, H-7), including an anomeric methine signal at
δH 4.58 (1H, d, J = 7.8 Hz, H-1′)
with four other oxygenated methine signals at δH 3.17–3.36
corresponding to the glucose units. The 13C NMR data (Table
), assigned with the
help of the HSQC and HMBC spectra, displayed 16 carbon resonances,
including a carboxy group at δC 216.02 (C-2), one
oxygenated methylene at δC 62.56, five oxygenated
methines, including an anomeric methine signal at δC 98.90, two quaternary carbon signals at δC 44.88
(C-1) and 81.71 (C-4), and three methyl groups at δC 19.39 (C-8), 20.43 (C-9), and 23.52 (C-10). The proton at H-5 showed 1H–1H COSY correlation with Ha-6/Hb-6, and
the HMBC correlation of H-3/C-1, C-2, and C-4; H-5/C-4 and C-6; H-7/C-1,
C-8, and C-9; and H-6/C-1, C-4, and C-5 allowed the dihydro-umbellulone-type
skeleton of 21 to be confirmed. The sugar unit was confirmed
as being linked at C-4 by the HMBC correlation of the anomeric proton
(H-1′) with C-4 (δC = 81.71) (Figure
). The absolute configuration
of C-1, C-4, and C-5 was determined by the CD spectrum (Figure
) and NOESY correlations of
H-10 to Ha-3, H-5, and Hb-6 (Figure
). The CD spectral data depicted two positive Cotton
effect at 213 (+76.2) , and 327 (+19.7) nm, and a negative Cotton
effect at 271 (−43.8) nm, which indicated the absolute configuration
of C-1 as R, C-4 as S, and C-5 as R. Hence, the structure of 21 was assigned as (1R, 4S, 5R) 3,4-dihydro-umbellulone-4-O-β-d-glucopyranose.
Compound 22 was isolated as a yellowish
sticky substance.
The molecule exhibited –149.00 (c 0.001
g/mL in MeOH), and the molecular formula C16H24O7 was determined by negative ion-mode HR-ESI-MS, which
showed a molecular ion peak at m/z 327.1449 [M–H]− (calcd. for C16H23O7, 327.1449), indicating five degrees of
unsaturation. The IR spectrum of compound 22 displayed
characteristic absorption bands of the hydroxy group at 3288 cm–1 and an unsaturated conjugated carbonyl group at 1724
cm–1. Analysis of the 1H NMR data of 22 (Table
) revealed one methyl singlet at δH 2.14 (3H, s,
H-10), one secondary methyl group at δH 1.12 (3H,
d, J = 7.0 Hz, H-9), and one olefinic proton at δH 5.3 (1H, s, H-3). The 13C NMR data (Table
), in combination with the HSQC
spectrum of 22, displayed 16 carbon resonances, characterized
as two methyls at δC 15.49 (C-9) and 18.16 (C-10);
three methylenes, including two oxygenated carbons at δC 73.26 (C-8) and 62.87 (C-6’); eight methines, including
a double bond at δC 124.14 (C-3) and an anomeric
signal at δC 104.43; and three nonprotonated carbons,
including a quaternary carbon at δC 39.26, one carbonyl
at δC 211.00 (C-2), and an olefinic signal at δC 182.06 (C-4). These data suggested that compound 22 was most likely an Umbellulone glycoside. 1H–1H COSY correlations identified a spin system between H-8/H-7
and H-7/H-9, indicating the connectivity of the isopropyl unit of
Umbellulone. Furthermore, the structure of compound 22 was established through comprehensive analysis of its 2D NMR spectra.
The 1H–13C-HMBC correlations (Figure
) from H-3 to C-2,
H-7 to C-2, C-6 to C-4, and H-10 to C-3 suggested that the 1a glycoside
system was located at C-8. The HMBC and 1H–13C-HSQC correlation of the anomeric proton (H-1′) with
C-8 (δC 73.26) (Figure
), along with a doublet signal at δH 4.23 (1H, d, J = 7.8 Hz) of the anomeric
proton, suggest the β-confirmation of the glucose unit. This
was further confirmed by acid hydrolysis of 22, and the
results of HPLC and GC-MS indicated that the sugar unit was β-D-pyranose.
The relative configuration of 22 was determined by extensive
analysis of the NOESY correlations (Figure
) of H-5/H-6b, indicating that they were
cofacial and assigned to be β-oriented. Other NOESY correlations
between H-8/H-9 confirmed the substitution position of the sugar moiety.
Its absolute configuration was further confirmed by circular dichroism
(CD) spectra (Figure
). The CD spectrum indicated two positive
Cotton effects at 235 (+52.4) and 324 (+53.2) nm and a negative Cotton
effect at 265 (−148.1) nm, which showed the absolute configuration
of C-1 and C-5 as S, and C-7 as R. Hence, the structure of compound 22 was elucidated as (1S, 5S, 7R)-Umbellulone 8-O-β-d-glucopyranoside.
The GC-FID
and GC-MS analysis of the nonpolar fraction
revealed
the presence of Umbellulone as a major component, accounting for approximately
19.8% (RT: 18.23 min; RI: 1171) of the total content (Table
) (Supporting Information, Figure S87).
In addition, the structures of 26 known compounds
were identified
by comparing the spectroscopic data (mainly 1H, 13C NMR, and HR-EI-MS analysis) with those reported in the literature.
The known compounds were identified as trans-communic
acid (1),
cis-communic acid (2), manool (3), sandaracopimeric acid (4),
,
sugiol (5),
,
15-acetoxyimbricatolic acid (6), imbricatolic acid (7),
,
cupressuflavone (8),
−

robustaflavone (9),
,
amentoflavone (10), (+)-volkensiflavone (11), 5,4′-dimethoxy-7-O-β-glucopyranosylflavone (12), rosin (13),
,
rosarin (14), icariside E4
(15)

,
juniperoside A (17), (1,4-hydroxy-3-methoxyphenyl)-2-[4-(3-rhamnoxyphenoxy]-1,3-propanediol) (18),
,
cupressoside A (19), α-hydroxyacetone glucoside (20), blumenol-C-glucoside (23), roseoside (24), β-sitosterol (25), sitosterol-3-O-β-d-glucoside (26), apigenin (27), β-catechin (28), and dodecan-1-ol (29) (Figure
). Among
the isolates, compounds 11–15, 17–20, 23, 24, and 28 were obtained from C. torulosa for the first time.

2.2
Biological Assay
Lignans are naturally
occurring compounds with a C6C3C6 core skeleton and are found in several
medicinal plants. They have been demonstrated promising anticancer
activity in various preclinical studies. These compounds can inhibit
cancer cell growth, induce apoptosis (programmed cell death), and
prevent metastasis and angiogenesis (formation of new blood vessels).
They achieve this through diverse molecular mechanisms, including
modulation of signaling pathways, regulation of cell cycle progression,
and modulation of inflammatory responses. Arctiin and magnolin are
a few examples of this class of compounds that have already proven
their efficacy against various cancers, including cervical, myeloma,
prostate, and colon cancers. Additionally, Phyllanthus sp.-derived lignans like phyllanthine have also proven their clinical
efficacy for hepatocellular malignancies, including NAFLDs. Keeping
in mind the wide application of lignans in hepatic diseases, we evaluated
the isolated lignans and other compounds for hepatocellular carcinoma
activity.

2.3
Cytotoxicity Assay of the Isolated Compounds
against HepG2 Cell Lines
The cytotoxic activity of isolated
compounds 1, 4–10, 13–16, 18, 19, and 21–24 was evaluated against the human liver cancer cell line HepG2 using
the MTT assay (Figure
) at using four different concentrations: 10, 25, 50, and 100 μM.
The IC50 values of the isolated compounds were calculated
from the MTT assay data by interpolating the concentration corresponding
to 50% cell viability within the linear dose–response range,
and the results were summarized in Table
. All of the tested compounds exhibited a
growth-inhibitory effect on hepatocellular carcinoma (HepG2) and showed
potent cytotoxicity, with IC50 values ranging from 37.54
μM to 271.13 μM. The compounds 13, 15, 18, 19, and 23 exhibited
significant inhibitory activity. In particular, compound 15 showed more potent antiproliferative activity in a concentration-dependent
manner (Figure
).
The IC50 of compound 15 was observed at 37.54
μM. Further, compound 15 was selected for a detailed
investigation of the antiproliferative mechanism in HepG2 cell lines.

2.4
Suppressed HepG2 Cell Migration with Treatment
of Compound 15
In the wound healing assay, cell
migration was assessed in response to mechanical scratch wounds in
the presence and absence of treatment. We observed a decrease in the
migratory capacity of the treated HepG2 cells with increasing concentrations
of compound 15 compared with the LPS-induced cells. To
quantify the effect of migration, the percentage of wound closure
after 24 h was determined. The data clearly showed that treatment
with different concentrations of compound 15 (27 μM,
37 μM, and 47 μM) caused a significant inhibition of cell
migration in a concentration-dependent manner (Figure
).

2.5
Mitochondrial ROS Production with Treatment
of Compound 15
Reactive oxygen species (ROS)
are a group of highly reactive peroxide molecules, ions, and free
radical species that evolve as regulators of several important signaling
pathways. It is now well accepted that a moderate increase in ROS
contributes to several pathological conditions, including the promotion
and progression of tumors due to their involvement in different signaling
pathways and induction of DNA mutations. ROS can also able to trigger
programmed cell death. Hence, we evaluated the production of ROS levels,
which have been observed to increase by nearly 64.78%, 68.49%, and
72.40% with increasing concentrations of 27 μM, 37 μM,
and 47 μM, respectively, compared to LPS-induced cells (Figure
).

2.6
Mitochondrial
Membrane Potential (MMP) Hyperpolarization
Assay
The mitochondrial membrane potential (MMP) is a crucial
indicator of mitochondrial health and cell survival. Its collapse
signals apoptosis; as pores open in the mitochondrial membrane, cytochrome
c leaks into the cell and triggers a chain reaction, leading to cell
death. Therefore, an increase in MMP serves as a warning sign of programmed
cell death and the production of ROS. Increasing concentrations of
compound 15 (27 μM, 37 μM, and 47 μM)
led to a significant increase in the level of MMP in HepG2 cells compared
to the LPS-induced cells (Figure
). LPS-induced cells showed a substantial increase
in the percentage level of MMP compared with the untreated cells.

2.7
Cell Cycle Regulation with the Treatment of
Compound 15
The figure shows the effect of compound 15 treatment on HepG2 cells after 24 h of exposure. Flow cytometry
results showed that 27 μM, 37 μM, and 47 μM-treated
cells significantly exhibited increased cell cycle arrest in the S-phase
(16.19%, 16.25%, and 16.50%) as compared to the LPS-induced cells
(14.14%) (Figure
).

2.8
Morphological Evaluation of Apoptosis
Chromatin
condensation and nuclear fragmentation in the nucleus were
observed in LPS-treated cells, providing specific evidence of apoptosis,
visualized through a fluorescence microscope. Apoptotic cells exhibiting
nuclear changes were visualized using the DNA-binding dye, Hoechst.
A significant increase in the number of apoptotic cells with altered
morphology was observed with an increasing concentration of compound 15 (Figure
). Interestingly, changes in the shape, size, and structure of HepG2
cells were observed, with the shrinkage of cells and the fragmentation
of membrane-bound apoptotic bodies. An increase in the level of chromatin
condensation and fragmentation of the nucleus indicates the occurrence
of cell death in HepG2 cells. Both Hoechst and propidium iodide stains
bind to DNA: Hoechst stains nuclear DNA in live cells, enabling total
cell population analysis suitable for live-cell imaging, while PI
penetrates only membrane-compromised cells, marking dead cells detected
with microscopy and flow cytometry.
,

3
Conclusion
In this study, we have reported
the extraction, isolation process,
and characterization of three previously undescribed compounds: a
neolignane glycoside (16) and two monoterpene glycosides (21 and 22), along with twenty-six known compounds, 1–15, 17–20, and 23–29,
from the acetone extract of C. torulosa. Compounds 11, 12, 13, 14, 15, 17, 18, 19, 20, 23, 24, and 28 were also isolated for the first time from this plant species.
The isolated compounds, 1, 4–10, 13–16, 18, 19, and 21–24, were evaluated for cytotoxic activity against the hepatocellular
carcinoma (HepG2) cell line and exhibited significant to moderate
activity, with IC50 values ranging from 37.54 μM
to 271.13 μM. Among the screened molecules, compound 15 showed a significant decrease in cell viability with increasing
concentration, with 50% cell viability at 37.54 μM. This study
provides a preliminary basis for the development and utilization of
the resources of C. torulosa. Compound 15 exhibited potent anticancer activity against HepG2 cells
in a concentration-dependent manner. Treatment with increasing concentrations
of 27 μM, 37 μM, and 47 μM significantly reduced
cell viability. It also inhibited cell migration, elevated reactive
oxygen species levels, and caused an increase in mitochondrial membrane
potential compared to LPS-induced cells, indicating oxidative stress
and mitochondrial involvement in cell death.
Furthermore, flow
cytometry analysis demonstrated S-phase cell
cycle arrest, suggesting disruption of DNA synthesis and cell cycle
progression. Morphological features, such as cell shrinkage, apoptotic
body formation, chromatin condensation, and nuclear fragmentation,
confirm the induction of apoptosis in HepG2 cells. Collectively, these
results suggest that compound 15 exerts antitumor effects
by inducing oxidative stress, inhibiting proliferation and migration,
arresting the cell cycle in the S-phase, and promoting apoptosis,
highlighting its potential as a therapeutic candidate against hepatocellular
carcinoma.

4
Experimental Section
4.1
General
Experimental Procedures
The
chemicals and reagents used for the whole experiment were fresh. All
of the solvents used in the extraction, isolation, and recrystallization
processes were fresh, double-distilled and of analytical grade. Different
pore sizes of silica gel (60–120, 100–200, and 230–400
mesh) (purchased from Avra Laboratories Pvt. Ltd.) and RP-C18 silica gel (Sigma-Aldrich) were used for the column chromatography,
while Sephadex LH-20 and dianion (resin) (Sigma-Aldrich) were used
in gel filtration chromatography. Preparative thin-layer chromatography
(PTLC) was used for analytical separations or purifications and was
performed on different-sized (0.5–2 mm) precoated silica gel
G/UV254 glass plates (Merck). Thin-layer chromatography
(normal and reverse phase) (TLC) analysis was performed on aluminum
TLC plates, which were silica gel-coated with fluorescent indicator
60 F254, 0.2 mm plates (Merck). The TLC plates were visualized
under short-wavelength (254 nm) and long-wavelength (365 nm) UV lamps
and detected by spraying with common TLC visualizing reagents: p-Anisaldehyde (Vanillin), phosphomolybdic acid (PMA), and
10% H2SO4 in EtOH (v/v). Optical rotations ([α]D) were recorded on a Horiba SEPA–300 High-Sensitive
Polarimeter. The IR spectra of compounds were recorded with JASCO
FT/IR-6X and Bruker/OPUS 75.18 spectrometers using a potassium bromide
(KBr) pellet or on ATR. The high-resolution electrospray ionization
mass spectrometry (HR-ESI-MS) was recorded on a 6545 Q-TOF LC/MS (Agilent
Technologies) or Orbitrap Exploris 240 mass spectrometer (Thermo Fisher
Scientific). The experimental circular dichroism (CD) spectra were
recorded on a Jasco J-1500 spectrometer. The 1D (1H, 13C, DEPT, 45, 90, and 135), and 2D-NMR (COSY, HSQC, HMBC,
and NOESY) spectra for this work were recorded on a Bruker AVANCE-300/500
MHz and 75/126 MHz NMR spectrometer, respectively, with or without
tetramethylsilane (TMS) as the internal standard. The chemical shift
was referenced to the solvent signal: deuterated methanol, DMSO-d
6, CDCl3, acetone-d
6, and D2O. All of the solvents used for NMR
experiments were deuterated, NMR grade, and purchased from Sigma-Aldrich.
The volatile composition of the nonpolar fraction was analyzed by
using Gas Chromatography (GC-FID, Agilent-8890), and Gas ChromatographyMass
Spectrometry (GC-MS) was recorded on a Clarus 680 GC interfaced with
a Clarus SQ 8C Quadrupole mass spectrometer of PerkinElmer. The HPLC
analysis of crude extract, pure compounds, and acid-hydrolyzed fractions
was performed by Waters Alliance 2695 with a 2998 PDA detector using
a Phenomenex Luna C18 (250 mm × 4.6 mm × 5 μm)
column.

4.2
Chemical Reagents
MTT {3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide} was supplied by MP Biomedicals. LPS and phosphorus iodide
were obtained from Sigma-Aldrich. JC-1 dye was purchased from G-Biosciences.
H2DCFDA was purchased from Invitrogen. Hoechst 33258 (bis-benzimide)
was procured from MP Biomedicals.

4.3
Plant
Material
Fresh plant material
was collected in December 2020 from Nainital, Uttarakhand. The plant
sample was identified as C. torulosa by the botanist at the CSIR–Central Institute of Medicinal
and Aromatic Plants, Lucknow-226015, India. A voucher specimen (Voucher
No. 8343) of C. torulosa was preserved
in the National Herbarium of the CSIR-Central Institute of Medicinal
and Aromatic Plants (CIMAP) for future reference.

4.4
Extraction and Isolation
The air-dried
leaves (1.2 kg) were crushed and extracted with hot acetone by the
heat reflux method (5 L, 24 h × 6) at 60–80 °C until
the extract was almost colorless.
,−

The filtrate was concentrated under reduced pressure in a rotary
evaporator at 40–45 °C, and the combined extract (184
g, 15.3%) was collected. The acetone extract was further fractionated
by column chromatography on silica gel SiO2 (2.7 kg, 120–200
mesh silica gel, 7 × 110 cm) and eluted successively with CHCl3: MeOH (100:00 to 00:100; 8 L for each gradient elution) to
afford ten fractions (A–J). Further, each fraction
was purified by different chromatographic techniques, including flash
chromatography, normal-phase column chromatography, vacuum liquid
chromatography (VLC), C18–VLC, RP-C18, Sephadex LH-20 column, and dianion HP-20 for polar sugar-containing
fractions. Fraction B (16.2 g) was subjected to normal-phase silica
gel column chromatography and eluted with acetone: hexane (100:00
to 00:100) to obtain subfractions B1–B8. Further, these subfractions were separately purified by flash and
small gravity columns, yielding compounds 1 (21 mg, 0.0018%), 2 (18 mg, 0.0015%), 29 (23.6 mg, 0.0019%), and 3 (9 mg, 0.0007%). Whereas fraction C (17.5 g) was subjected
to RCC (repeated column chromatography) and eluted with the same solvent
system with gradient polarity to obtain compounds 4 (117
mg, 0.0097%), 25 (143 mg, 0.0119%), and 5 (2.7 mg, 0.0002%). Similarly, fraction D (17 g) was subjected to
RCC and eluted with the same solvent system with polarity (20–60)
% to obtain compounds 6 (19 mg, 0.0016%) and 7 (115.5 mg, 0.0096%). Fraction E (13 g) was subjected to RCC and
eluted with 8–20% methanol/chloroform polarity to obtain compounds 11 (12 mg, 0.001%), 27 (4.5 mg, 0.0004%), 28 (21.8 mg, 0.0018%), and 26 (157 mg, 0.0131%).
Fraction F (18.6 g) was subjected to RCC and eluted with methanol/chloroform
polarity 8–25% to obtain compounds 8 (2.63 g,
0.2192%), 9 (147 mg, 0.0122%), and 10 (196
mg, 0.0164%). Fraction G (7 g) was first fractionated by normal-phase
column chromatography, eluted with a mobile phase solvent system of
methanol/chloroform at 8–25% polarity, and collected 14 subfractions
(G1–14). Further, each subfraction was purified by RP-C18 (reverse-phase column chromatography) or C18–VLC
eluted with a water/methanol polarity of 40–60% to obtain compound 13 (27 mg, 0.0023%). Fractions H (15 g), I (17 g), and J (37
g) were subjected to CC through dianion resin and eluted with gradient
polarity of water/methanol (100–00 to 00–100), yielding
subfractions H1–9, I1–12, and
J1–16. Further, each subfraction was purified by
RP-C18 and eluted with gradient polarity of water/methanol
(100–00 to 00–100) to obtain compounds 20 (37 mg, 0.0031%), 22 (54.7 mg, 0.0046%) and 23 (5 mg, 0.0004%) from subfraction H; compounds 15 (319
mg, 0.0269%), 16 (539 mg, 0.0449%), 19 (17
mg, 0.0014%), 21 (57.3 mg, 0.0048%), and 24 (6.8 mg, 0.0012%) from subfraction I; and compounds 12 (3.7 mg, 0.0003%), 14 (56.6 mg, 0.0047%), 17 (6.3 mg, 0.0005%), 18 (11 mg, 0.0009%) and 24 (7.9 mg, 0.0012%) from subfraction J.
4.4.1
Compound
(16)
Colorless,
amorphous powder; –30.12 (c 0.0026
g/mL in MeOH); UV (MeOH) λmax (log ε) 203,
232, 280 nm; CD (MeOH) λmax (Δε) 236
(+3.6), 248 (−12.2), 283 (−7.3) nm; IR (ATR) νmax 3289, 2947, 2829, 1672, 1582, 1534, 1466, 1406, 1302, 1237,
1166, 1112, 1017, 928, 699, and 631 cm–1; 1H (300 MHz) and 13C (75 MHz) NMR data, see Table
; HR-ESI-MS (positive-ion mode) m/z 577.2245 [M + H]+ (calcd.
for C30H41O11 577.2643).

4.4.2
Compound (21)
Colorless,
sticky; +30.55 (c 0.003 g/mL in
MeOH); UV (MeOH) λmax (log ε) 220, 266 nm;
CD (MeOH) λmax (Δε) 213 (+76.2), 271
(−43.8), 327 (+19.7) nm; IR (ATR) νmax 3362,
2961, 2926, 2880, 1702, 1456, 1380, 1213, 1074, 1034, 970, and 631
cm–1; 1H (500 MHz) and 13C
(126 MHz) NMR data, see Table
; HR-ESI-MS (positive-ion mode) m/z 331.17513 [M + H]+ (calcd. for C16H27O7 331.1751).

4.4.3
Compound
(22)
Yellowish,
sticky; –149.00 (c 0.001
g/mL in MeOH); UV (MeOH) λmax (log ε) 199,
220, 275 nm; CD (MeOH) λmax (Δε) 235
(+52.4), 265 (−148.1), 324 (+53.2), nm; IR (ATR) νmax 3288, 2931, 2829, 2878, 1724, 1649, 1600, 1455, 1412, 1366,
1153, 1072, 1024, and 629 cm–1; 1H (500
MHz) and 13C (126 MHz) NMR data, see Table
; HR-ESI-MS (negative-ion mode) m/z 327.1449 [M–H]− (calcd.
for C16H23O7 327.1449).

4.5
GC and GC-MS Analysis
The GC and
GC-MS analysis of the nonpolar fraction was performed according to
a previously reported method in Singh et al., 2023.

4.6
Acid Hydrolysis
Compounds 16, 21, and 22 (6.0
mg) were hydrolyzed in
2 M trifluoroacetic acid (TFA) (2.0 mL) at 80 °C for 24 h, separately.
After extraction with EtOAc (4 × 3.0 mL), the aqueous layer and
organic layer were evaporated under reduced pressure at 45 °C,
yielding the sugars and hydrolyzed compounds. The water-soluble sugar
part was analyzed directly by HPLC at 280 nm. The HPLC analysis was
performed on a Luna C18 column at 25 °C using 0.1%
TFA in CH3CN:0.1% TFA in H2O (25:75). L­(+)-Rhamnose
and β-d-glucose were confirmed as the sugar units.

4.7
MTT Assay
The cytotoxicity of the
isolated compounds was assessed by using the MTT test against HepG2
cells. After being seeded in 96-well plates (1× 104), the cells were exposed to varying concentrations of compounds
at 10, 25, 50, and 100 μM. After 24 h, the cells were incubated
for an additional 4 h with 10 μL of MTT. Following this, 100
μL of DMSO was added and incubated for 30 min at 37 °C.
An ELISA plate reader (model 680XR, Bio-Rad, Hercules, CA, USA) was
used to measure absorbance at a wavelength of 570 nm. The graph of
optical densities was used to determine the IC50.

4.8
Cell Migration Assay
HepG2 cells
were seeded at a density of 3 × 105 cells/well in
6-well plates. A sterile 10 μL pipette tip was used to create
a wound. After 24 h, cells were washed with PBS. A Nikon Eclipse E200
microscope was used to capture images of wound closure at 0 and 24
h following treatment with various concentrations of compound 15.
The equations for the calculation of the percentage
(%) of wound closure are given below:

4.9
ROS Assay
Dichlorofluorescein diacetate
(H2DCFDA) was employed to investigate intracellular ROS formation.
3 × 105 Cells were seeded in 6-well plates, stimulated
with LPS for 6 h, and then treated with compound 15 at concentrations
of 27 μM, 37 μM, and 47 μM for 24 h. The cells were
then exposed to 10 μM H2DCFDA for 30 min at 37 °C. The
cells were harvested, washed with PBS, and DCF fluorescence was measured
using the BD FACS Lyric flow cytometry system.

4.10
MMP Assay
About 1 × 105 cells per well
were seeded in 12-well plates. After 6 h of
LPS stimulation (1 μg/mL), the cells were exposed to different
concentrations of compound 15 (27, 37, and 47 μM)
for 24 h. The cells were then scraped, collected in a FACS tube, washed
with PBS, and analyzed for fluorescence intensities using the BD FACS
Lyric flow cytometry system to determine the level of mitochondrial
membrane potential (MMP) in the HepG2 cell line.

4.11
Cell Cycle Analysis
Approximately
3 × 105 HepG2 cells were seeded in six-well plates
and stimulated with LPS for 6 h. They were then treated with 27 μM,
37 μM, and 47 μM of compound 15 for 24 h. After harvesting
and fixing with 70% ethanol at 4 °C for 1 h, the cells were given
a PBS wash and stained with propidium iodide (PI). Cell cycle distribution
was analyzed using BD FACS Lyric flow cytometry.

4.12
Chromatin Condensation Assay
To
evaluate morphological changes in HepG2 cells treated with varying
concentrations of compound 15, 6-well plates were seeded with 3 ×
105 cells. After a 6 h stimulation with lipopolysaccharide
(LPS), the cells were treated for 24 h, rinsed with PBS, and fixed
in 4% paraformaldehyde for 10 min. The cells were rinsed again with
PBS and stained with Hoechst dye for 10 min at room temperature. The
morphology of the treated cells was photographed using an inverted
fluorescence microscope (Leica, DMI6000B, USA) equipped with Leica
software.

4.13
Statistical Analysis
All of the
results are presented as mean ± SD (standard deviation). The
data were analyzed by one-way ANOVA. GraphPad Prism, Version 8.0.2,
was used for statistical analysis.

Supplementary Material