Choushenosides A-C, three dimeric catechin glucosides from Codonopsis pilosula collected in Yunnan province, China

Article abstract of DOI:10.1016/j.phytochem.2018.05.012

Choushenosides A-C, three dimeric catechin glucosides, were isolated from the roots of Codonopsis pilosula cultivated at high elevations in Yunnan province of the People's Republic of China. The structures of these substances were determined by using spectroscopic and chemical methods. Biological evaluation showed that choushenoside C is a dose-dependent inhibitor of SIRT1.

Full text of DOI:10.1016/j.phytochem.2018.05.012

Phytochemistry153(2018)53–57
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Choushenosides A-C, three dimeric catechin glucosides from Codonopsis
pilosula collected in Yunnan province, China
Fu-Ying Qina,c, Li-Zhi Chenga,b, Yong-Ming Yanb, Bao-Hua Liub,∗∗, Yong-Xian Chenga,b,d,∗
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's
Republic of China
b
Guangdong Key Laboratory for Genome Stability & Disease Prevention, School of Pharmaceutical Sciences, School of Medicine, Shenzhen University Health Science
Center, Shenzhen 518060, People's Republic of China
c
University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450008, People's Republic of China
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Codonopsis pilosula (Campanulaceae)
Catechin
Dimers
Choushenosides A-C
SIRT1
Choushenosides A-C, three dimeric catechin glucosides, were isolated from the roots of Codonopsis pilosula
cultivated at high elevations in Yunnan province of the People's Republic of China. The structures of these
substances were determined by using spectroscopic and chemical methods. Biological evaluation showed that
choushenoside C is a dose-dependent inhibitor of SIRT1.
1. Introduction
frequent release of anal gases. This phenomenon is not associated with
the use of Dang Shen that is produced in northwest of China, which
Specialized metabolites are synthesized by metabolic pathways,
which in turn are regulated by congenital and acquired factors. A
consensus has been reached that the nature of specialized metabolites
in plants is dependent on the growing environment. Actually, this de-
pendence is also seen with microorganisms where it is widely observed
that one strain could produce many different compounds (OSMAC) in a
culture condition dependent manner (Jiang et al., 2016a,b). Codonopsis
pilosula (Franch.) Nannf. (Campanulaceae) is a perennial plant that
grows at altitudes of 1500–3000 m in the People's Republic of China.
Previous studies revealed that this plant contains phytosteroids, ses-
quiterpenes, triterpenes, alkaloids, alkylalcohol glycosides, phenylpro-
panoid glycosides, polyacetylene glycosides, neolignan, and poly-
saccharides (Yang et al., 2013). The roots of C. pilosula, known as Dang
Shen in Chinese, are a common traditional Chinese medicine used for
the treatment of Qi deficiency. The results of pharmacological in-
vestigations showed that the extract of C. pilosula regulates immunity,
improves learning and memory, and inhibits iNOS (Jiang et al.,
2016a,b; Jiang et al., 2015). In a recent study of traditional Chinese
medicines, we explored C. pilosula, a herb locally known as Chou Shen,
which is mainly found in Yunnan province. Local inhabitants of this
area have utilized the fresh roots of this plant as a vegetable for soup
making for many years even though its consumption causes the
belongs to the same species as does C. pilosula. This difference attracted
our attention and served as the basis of a study on Chou Shen aimed at
gaining insight into differences that exist between C. pilosula grown in
different regions and paving the way for developing Chou Shen as a
healthcare food. Below, we describe the results of this effort, which led
to the isolation, structural identification and biological evaluation of
three novel catechin dimers 1–3.
2. Results and discussion
2.1. Isolation and structure elucidation of 1–3
The EtOH extract of C. pilosula was partitioned between water and
EtOAc. The aqueous layer was concentrated in vacuo giving a residue
that was subjected to a chromatography protocol to produce 1–3.
Choushenoside A (1) was isolated as a brown yellow powder. The
13C NMR and DEPT spectra (Table 1) of 1 contain 22 carbon resonances
attributed to three methylene (one oxygenated), eleven methine (four
sp2, seven oxygenated sp3), and eight quaternary carbons (all sp2 in-
cluding five oxygenated). The 1H NMR spectrum of this substance
contains signals at δH 3.95 (d, J = 8.3 Hz, H-2), 3.68 (m, H-3), 2.86 (dd,
J = 16.0, 5.8 Hz, Ha-4), and 2.41 ppm (dd, J = 16.0, 9.4 Hz, Hb-4). In
∗
Corresponding author. 132 Lanhei Road, Kunming 650201, People's Republic of China.
Corresponding author.
∗∗
E-mail addresses: ppliew@szu.edu.cn (B.-H. Liu), yxcheng@szu.edu.cn (Y.-X. Cheng).
https://doi.org/10.1016/j.phytochem.2018.05.012
Received 7 October 2017; Received in revised form 12 May 2018; Accepted 17 May 2018
0031-9422/©2018ElsevierLtd.Allrightsreserved.

F.-Y. Qin et al.
Phytochemistry153(2018)53–57
addition, HMBC correlations of H-1′′/C-7 and CH2/C-5, C-6, C-7 in-
dicate the position of attachment of the sugar moiety.
Table 1
1H (600 MHz) and13C NMR (150 MHz) data of 1 in methanol-d4 (δ in ppm, J in
Hz).
At this point in the analysis, all NMR resonances can be fully as-
signed by using 2D NMR. However, characterization of the remaining
CH2 group at C-6 is ambiguous because the 1H NMR signal at 3.86 ppm
suggests that the methylene carbon is attached to oxygen whereas the
significantly upfield 18.1 ppm 13C NMR chemical shift of the carbon in
this group is not consistent with this conclusion. A solution to this
problem is found by assigning the structure of 1 to be a symmetric
dimer of a catechin derivative. To gain evidence for this possibility, the
HRMS of 1 was analyzed. The results show that the spectrum contains a
quasimolecular ion peak at m/z 939.2521 [M+Na]+ associated with
the molecular formula C43H48O22. This finding led to assignment of the
planar structure of 1. The trans relationship of H-2/H-3 in this sub-
stance was readily ascertained on the basis of 8.3 Hz coupling constant
in the doublet for H-2.
To assign the absolute configuration of the sugar moiety in 1, acid
hydrolysis was carried out. TLC and GC analysis showed that this
process generates D-glucose. Specifically, L-cysteine methyl ester hy-
drochloride derivatives of D- and L-glucose were prepared and com-
pared to that of the product formed by similar treatment of 1 by using
GC. The retention time for the L-cysteine methyl ester hydrochloride
derivative arising from 1 is 21.147 min, which is closer to that of D-
glucose (21.081 min) rather than that of L-glucose (21.615 min).
Choushenoside B (2), obtained as a brown yellow powder, has the
molecular formula C43H48O6 (20 degrees of unsaturation), based on
analysis of its HRESIMS, 13C NMR and DEPT spectra. The 13C NMR and
DEPT spectra show that this substance contains 43 carbons including
five methylene (two oxygenated), twenty-two methine (eight olefinic
and fourteen aliphatic), and sixteen quaternary carbons (sixteen ole-
finic including eight oxygenated). Data for 1 and the appearance of
Position δH
δC
Position δH
δC
2, 2‴
3, 3‴
4, 4‴
3.95, d (8.3)
3.68, m
2.86, dd (16.0,
5.8)
82.5 CH
69.1 CH
27.9 CH2 5′, 5″″
3′, 3″″
4′, 4″″
145.9 C
146.0 C
116.2 CH
6.70, d (8.1)
2.41, dd (16.0,
9.4)
5, 5‴
6, 6‴
7, 7‴
155.1 C
111.0 C
155.4 C
94.6 CH
154.3 C
103.5 C
132.5 C
6′, 6″″
6.70, br.d (8.1)
4.72, d (7.8)
3.56, t (8.1)
3.39, overlap
3.39, overlap
3.39, overlap
3.91, d (12.0)
3.74, dd (12.0,
4.4)
120.5 CH
101.7 CH
74.4 CH
77.9 CH
71.4 CH
77.8 CH
62.4 CH2
1″, 1″″'
2″, 2″″′
3″, 3″″′
4″, 4″″′
5″, 5″″′
6″, 6″″′
8, 8‴
6.19, s
9, 9‴
10, 10‴
1′, 1″″
2′, 2″″
6.66, d (1.6)
115.9 CH CH2
3.86, s
18.1 CH2
addition, an ABX spin pattern [δH 6.66 (d, J = 1.6 Hz, H-2′), 6.70 (d,
J = 8.1 Hz, H-5′), 6.70 (brd, J = 8.1 Hz, H-6′)] is observed in this
spectrum. Moreover, the spectrum contains resonances in the mid-field
region, which are characteristic of protons in a sugar moiety, along
other diagnostic signals that are similar to those of catechin-7-O-β-D-
glucoside (1a) (Foo and Karchesy, 1989). These findings suggest that 1
is a glucoside containing catechin. The major difference between the
spectra of 1a and 1 is that the resonance for H-6 of 1a is absent from the
spectrum of 1, being replaced by a methylene signal associated with a
group that is attached to C-6. This conclusion is demonstrated by ob-
servation of HMBC correlations (Fig. 2) of CH2/C-5, C-6, C-7. In
Fig. 1. The structures of catechin dimer glucosides 1–3 from Codonopsis pilosula.
54

F.-Y. Qin et al.
Phytochemistry153(2018)53–57
supported by HMBC correlations of H-1′′/C-5 and H-1′′′′′/C-7‴. The
coupling constants for H-2 (J = 6.2 Hz) and H-2‴ (J = 5.7 Hz) indicate
that two trans relationships are present in the pyran rings in 3. Finally,
evidence for the presence of the D-glucose moiety in 3 also comes from
analysis of the acid hydrolysis product formed in the manner described
for 1.
2.2. Biological evaluation
The nicotinamideadenosine dinucleotide (NAD)-dependent deace-
tylase SIRT1 regulates a wide range of cellular functions and is im-
plicated in many diseases such as those derived from aging, cancer,
neurodegeneration, metabolic and immune malfunctions (Mvunta
et al., 2017; Cho and Dai, 2016; Ma and Li, 2015; Luo et al., 2014; Cao
et al., 2016; Zhang et al., 2017). Considering the fact that traditional
applications of Chou Shen are associated with aging, the inhibitory
activities of 1–3 against SIRT1 were determined. The results show that
only 3 is an inhibitor of SIRT1. (Fig. 5), and that 1 and 2 are not active
even at concentrations as high as 200 μM. Owing to the differences
between the structure of 3 and those of 1 and 2, it can be proposed that
dimerization through rings A and B is important feature for SIRT1 in-
hibitory activity.
Fig. 2. Key 1H-1H COSY (
1.
), HMBC (
), and ROESY (
) correlations for
pairs of peaks in its NMR spectra led us to conclude that 2 is also a
dimer of a catechin derivative. Actually, the observation of 1H-1H COSY
correlations (Fig. 3, bold lines) of H-2/H-3/H-4, H-2‴/H-3‴/H-4‴ and
the presence of diagnostic signals for two ABX spin systems (H-2′, H-3′,
H-4′; H-2‴, H-3‴, H-4‴) confirmed this conclusion. Thus, the structure
of 2 is similar to that of 1 differing only in the connection of the two
catechin glycosides. Specifically, HMBC correlations of CH2/C-5‴, C-9,
H-6/C-5, C-10 show that C-8 in 2 is linked with C-6‴ via a methylene
carbon. In addition, the presence of HMBC correlations of H-1′′/C-7 and
H-1′′′′′/C-7‴ clearly indicates the locations of two sugar moieties in the
planar structure of 2 and, similarly, the JH-2,H-3 (7.3 Hz) and JH-2‴,H-3‴
(7.9 Hz) values indicate the presence of two trans relationships (Fig. 1).
Finally, evidence for the presence of a D-glucose residue in 2 comes
from analysis of the acid hydrolysis product carried out in the same
manner as described for 1.
3. Conclusions
To conclude, three undescribed catechin glucosides were isolated
from Codonopsis pilosula roots and structurally identified. Biological
evaluations revealed that choushenoside C is a SIRT1 inhibitor. This
study not only sets the foundation for developing C. pilosula as a
healthcare food for the prevention of SIRT1-associated disorders, but it
also suggests that choushenoside C might be a potent structural tem-
plate worth further optimization as a SIRT1 inhibitor.
4. Experimental
4.1. General experimental procedures
Choushenoside C (3), obtained as a brownish yellow powder, has
the molecular formula C42H46O6 (20 degrees of unsaturation), based on
analysis of its HRESIMS, 13C NMR and DEPT spectra. The 13C NMR and
DEPT spectra, along with the results of HSQC experiments, show that
this substance contains 42 carbons including four methylene (two
oxygenated), twenty-two methine (eight olefinic and fourteen ali-
phatic), and sixteen quaternary carbons (sixteen olefinic including eight
oxygenated). The 1H, 13C NMR resonances of 3 appear as pairs and
resemble those in the spectra of 2. The difference between the struc-
tures of 2 and 3 is that the two glycoside groups in the latter substance
are connected via C-8 and C-6′′′′ based on the observations of correla-
tions H-5′′′′/C-6′′′′, C-1′′′′, C-8, H-6/C-5, C-10, and H-3/C-10 (Fig. 4). In
addition, the existence of glycoside groups at C-5 and C-7‴ in 3 is
Column chromatography was undertaken on D101 macro-
porousresin (Tianjin Haiguang Chemical Co., Ltd., People's Republic of
China), RP-18 (40–60 μm; Daiso Co., Japan), and Sephadex LH-20
(Amersham Pharmacia, Sweden). Optical rotations were collected on a
Horiba SEPA-300 polarimeter. UV spectra were obtained on a Shimadzu
UV-2401PC spectrometer. CD spectra were measured on a Chirascan
instrument. GC analysis was performed using an Agilent 6890N gas
chromatography instrument. Semi-preparative or analytic HPLC was
carried out using an Agilent 1200 liquid chromatograph, the column
used was a 250 mm × 9.4 mm, i.d., 5 μm. NMR spectra were recorded
on a Bruker AV-400 or an AV-600 spectrometer with TMS as an internal
standard. ESIMS and HRESIMS were collected by an Agilent G6230TOF
MS spectrometer.
Fig. 3. Key 1H-1H COSY (
), HMBC (
), and ROESY (
) correlations for 2.
55

F.-Y. Qin et al.
Phytochemistry153(2018)53–57
Fig. 4. Key 1H-1H COSY (
), HMBC (
), and ROESY (
) correlations for 3.
(Fr.2.1.1–Fr.2.1.3). Of which, Fr.2.1.2 (3 g) was subjected to Sephadex
LH-20 (MeOH) followed by semi-preparative HPLC (MeOH/H2O, 20%,
flow rate: 3 mL/min) to yield 2 (4.5 mg, Rt = 16 min).
4.4. Compound characterization data
4.4.1. Choushenoside A (1)
Brown yellow powder; [α]D21.9–139.1 (c 0.24, MeOH); UV (MeOH)
λmax (logε) 286 (3.45), 229 (3.89), 208 (4.35) nm; CD (MeOH) Δε204
+74.10, Δε217 –79.09, Δε281 –3.80; ESIMS m/z 939 [M+Na]+
,
HRESIMS m/z 939.2521 [M+Na]+ (calcd for C43H48NaO22, 939.2535);
1H and 13C NMR data, see Table 1.
Fig. 5. Effects of compound 3 on SIRT1 activity. SIRT1 enzyme activity was
measured using a SIRT1 Fluorometric Drug Discovery Kit. The results are pre-
sented as the percentage of activity relative to the control in each group.
Statistical analysis was performed using one-way analysis of the variance
4.4.2. Choushenoside B (2)
Brown yellow powder; [α]D21.9 − 120.6 (c 0.23, MeOH); UV
(MeOH) λmax (logε) 289 (3.33), 231 (3.78), 207 (4.25) nm; CD
(MeOH) Δε200 +19.43, Δε218 –43.85, Δε282 –1.85; ESIMS m/z939 [M
(ANOVA) followed by Bonferroni's multiple comparison tests. **p
< 0.01,
+Na]+
,
HRESIMS
m/z
939.2525
[M+Na]+ (calcd
for
***p < 0.001 indicates significant differences between control and treatment
groups.
C43H48NaO22,939.2535); 1H and 13C NMR data, see Table 2.
Table 2
4.2. Plant material
1H (600 MHz) and13C NMR (150 MHz) data of 2 in methanol-d4 (δ in ppm, J in
Hz).
The roots of Codonopsis pilosula (Franch.) Nannf. (Campanulaceae)
were collected from Jinyuan of Xundian County (25°91′ N, 103°14′ E) in
Yunnan province, People's Republic of China, a cultivation base, in
November, 2015. The material cultivated at this site was previously
identified by Prof. De-Yuan Hong at Beijing Institute of Botany, Chinese
Academy of Sciences, People's Republic of China, and a voucher spe-
cimen (1016268) was deposited at the Herbarium of Kunming Institute
of Botany, Chinese Academy of Sciences, People's Republic of China.
Position δH
δC
Position δH
δC
2
3
4
4.77, d (7.3)
4.07, m
2.85, dd (16.4,
5.2)
83.1 CH
68.1 CH
28.2 CH2 4‴
2‴
3‴
4.44, d (7.9)
3.87, m
83.5 CH
68.8 CH
29.4 CH2
2.80, dd (16.3,
5.5)
2.41, dd (16.3,
8.6)
2.60, dd (16.4,
7.6)
5
155.1 C
96.1 CH
156.2 C
108.7 C
153.6 C
103.2 C
131.2 C
115.6 CH 2′′′′
146.1 C
146.2 C
116.0 CH 5′′′′
120.0 CH 6′′′′
102.6 CH 1′′′′′
74.5 CH
78.0 CH
71.1 CH
77.9 CH
5‴
6‴
7‴
155.6 C
110.5 C
156.2 C
95.7 CH
154.5 C
104.3 C
132.0 C
115.2 CH
146.3 C
146.4 C
116.2 CH
120.1 CH
102.2 CH
74.7 CH
78.1 CH
71.2 CH
78.0 CH
62.4 CH2
6
6.29, s
7
8
9
4.3. Extraction and isolation
8‴
6.19, s
9‴
10‴
1′′′′
10
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
The dried roots of C. pilosula (20 kg) were powdered and soaked in
85% aqueous EtOH (4 × 80 L × 24 h) to give a crude extract, which
was suspended in water followed by extraction with EtOAc to afford an
EtOAc soluble extract and an aqueous extract. The latter was divided
into three parts (Fr.1–Fr.3) by using a D101 macroporous resin column
eluted with an aqueous EtOH gradient (5%, 40%, and 100%). Fr.2
(30 g) was separated by using Sephadex LH-20 (MeOH) to yield two
fractions (Fr.2.1 and Fr.2.2). Fr.2.2 (19 g) was further separated by
using RP-18 column (MeOH/H2O, 10%–70%) to give eight fractions
(Fr.2.2.1–Fr.2.2.8). Of which, Fr.2.2.2 (2.3 g) was subjected to
Sephadex LH-20 (MeOH) followed by semi-preparative HPLC (MeOH/
H2O, 11%, flow rate: 3 mL/min) to yield 1 (5.5 mg, Rt = 24 min) and 3
(3.2 mg, Rt = 23 min). Fr.2.1 (11 g) was separated by using a RP-18
column (MeOH/H2O, 10%–70%) to generate three fractions
6.83, br. s
6.75, overlap
3′′′′
4′′′′
6.75, overlap
6.61, br. d (8.0)
4.79, d (7.8)
3.65, t (8.2)
3.43, overlap
3.43, overlap
3.43, overlap
3.84, overlap;
3.74, m
6.72, overlap
6.72, overlap
4.78, d (7.8)
3.61, t (8.3)
3.43, overlap
3.43, overlap
3.43, overlap
3.91, d (11.8);
3.74, m
2′′′′′
3′′′′′
4′′′′′
5′′′′′
62.2 CH2 6′′′′′
CH2
4.03, d (15.1)
3.86, overlap
17.6 CH2
56

F.-Y. Qin et al.
Phytochemistry153(2018)53–57
Table 3
containing 0.5U of SIRT1 enzyme, 1000 μM of NAD+ (Enzo Life
Sciences), 100 μM of SIRT1 peptide substrate (Enzo Life Sciences), and
the SIRT1 assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM
KCl, 1 mM MgCl2, 1 mg/mL BSA) along with the test compound at a
specific concentration. The plate was incubated at 37 °C for 30 min and
then subjected to Fluor de Lys developer solution (Enzo Life Sciences)
containing 2 mM nicotinamide. The plate was incubated at 37 °C for
another 30 min and the wells were read by using a fluorimeter with an
excitation wavelength of 360 nm and emission wavelength of 460 nm
(Karbasforooshana and Karimib, 2017).
1H (600 MHz) and13C NMR (150 MHz) data of 3 in DMSO‑d6 (δ in ppm, J in
Hz).
Position δH
δC
Position δH
δC
2
3
4
4.57, d (6.2)
3.81, m
80.5 CH
66.0 CH
26.8 CH2 4‴
2‴
3‴
4.64, d (5.7)
3.90, m
78.5 CH
64.2 CH
26.1 CH2
2.77, dd (16.4,
2.77, dd (16.3,
4.8)a
4.8)a
2.65, dd (16.4,
6.4)a
2.42, dd (16.3,
7.2)a
5
153.6 C
94.0 CH
154.9 C
108.0 C
152.1 C
101.3 C
129.1 C
113.9 CH 2′′′′
144.6 C
144.7 C
5‴
156.1 C
95.6 CH
156.6 C
94.5 CH
155.6 C
101.7 C
130.7 C
113.2 CH
143.5 C
143.8 C
119.2 CH
125.0 C
100.5 CH
73.0 CH
76.9 CH
69.4 CH
76.0 CH
60.4 CH2
6
6.27, s
6‴
7‴
8‴
6.05, d (1.6)
5.91, br. s
Acknowledgements
7
8
9
9‴
10‴
1′′′′
This study was supported by National Key Research and
Development Program of China (2017YFA0503900), the Major Project
on Biotechnology of Yunnan province (2014FC002), National Science
Fund for Distinguished Young Scholars (81525026).
10
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
6.62, br. s
6.59, s
6.47, s
3′′′′
4′′′′
6.64, d (8.4)
6.48, br. d (8.0)
4.70, d (7.6)
2.97, t (8.2)
3.23, overlap
3.17, overlap
3.23, overlap
3.70, overlap
3.53, m
115.0 CH 5′′′′
117.7 CH 6′′′′
Appendix A. Supplementary data
98.8 CH
73.2 CH
76.8 CH
69.5 CH
76.5 CH
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
4.70, d (7.6)
3.16, overlap
3.23, overlap
3.17, overlap
3.23, overlap
3.70, overlap
3.53, m
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.phytochem.2018.05.012.
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4.4.3. Choushenoside C (3)
Brown yellow powder; [α]D22.5–45.4(c 0.29, MeOH); UV (MeOH)
λmax (logε) 285 (3.34), 208 (4.22) nm; CD (MeOH) Δε203 +7.41,
Δε218 –18.60, Δε279 –2.95; ESIMS m/z 925 [M+Na]+, HRESIMS m/z
925.2379 [M+Na]+ (calcd for C42H46NaO22,925.2378); 1H and 13C
NMR data, see Table 3.
4.5. Acid hydrolysis
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Independent solutions of 1, 2 and 3 (1.0 mg) in 1 N HCl were stirred
at 70 °C for 5 h. After cooling, the mixtures were extracted with EtOAc.
In each case, the aqueous layer was neutralized with 1 N NaOH and
concentrated in vacuo giving a residue that was dissolved in anhydrous
pyridine (2 mL). To each of these solutions was added L-cysteine methyl
ester hydrochloride (2 mg) and the resulting mixtures were stirred at
60 °C for 1 h and concentrated in vacuo at 0 °C. Slow addition of 1-
(trimethylsiyl)imidazole to the mixtures was followed by stirring at
60 °C for 1 h. Aliquots (4 μL) of the supernatants were subjected to
chiral GC analysis to demonstrate that D-glucose is present in 1, 2 and 3
(Shi et al., 2014).
4.6. SIRT1 inhibition
To test for SIRT1 inhibition, each well of a plate was created
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