comprehensive metabolomics analysis based on uplc-q/tof
TRANSCRIPT
RSC Advances
PAPER
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.
View Article OnlineView Journal | View Issue
Comprehensive m
aSchool of Pharmaceutical Sciences, Jilin
130021, Jilin, China. E-mail: liuj
+86-431-85619803bResearch Center of Natural Drug, Jilin Univ
Cite this: RSC Adv., 2020, 10, 8396
Received 28th November 2019Accepted 18th February 2020
DOI: 10.1039/c9ra09965d
rsc.li/rsc-advances
8396 | RSC Adv., 2020, 10, 8396–84
etabolomics analysis based onUPLC-Q/TOF-MSE and the anti-COPD effect ofdifferent parts of Celastrus orbiculatus Thunb.
Na Yang,a Han Wang,a Hongqiang Lin,a Junli Liu,a Baisong Zhou,a Xiaoling Chen,a
Cuizhu Wang,ab Jinping Liu *ab and Pingya Li*a
The root, stem and leaf of Celastrus orbiculatus Thunb. (COT) have all been used as Chinese folk medicine.
Aiming at revealing the secondary metabolites and screening the anti-COPD effect of COT, the
comprehensive phytochemical and bioassay studies were performed. Based on the ultra-high
performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry
(UPLC-Q/TOF-MSE), the screening analysis of components in COT was conducted with the UNIFI
platform, the metabolomics of the three parts were analyzed with multivariate statistical analysis.
Cigarette smoke extract (CSE)-stimulated inflammatory model in A549 cells was used to investigate the
biological effect of the three parts. A total of 120 compounds were identified or tentatively characterized
from COT. Metabolomics analysis showed that the three parts of COT were differentiated, and there
were 13, 8 and 5 potential chemical markers discovered from root, stem and leaf, respectively. Five
robust chemical markers with high responses could be used for further quality control in different parts
of COT. The root, stem and leaf of COT could evidently reduce the levels of pro-inflammatory factors in
a dose-dependent way within a certain concentration range. The stem part had a stronger anti-COPD
effect than root and leaf parts. This study clarified the structural diversity of secondary metabolites and
the various patterns in different parts of COT, and provided a theoretical basis for further utilization and
development of COT.
1. Introduction
Celastrus orbiculatus Thunb. (COT), belonging to the familyCelastraceae and the genus Celastrus L., is widely distributedthroughout China.1 The parts including root, stem and leaf allcould been used as Chinese folk medicine to treat rheumatoidarthritis, vomiting, abdominal pain, and snakebites.2,3 The rootof COT was reported to possess anti-tumor,4 antiviral,5 bacte-riostatic6 and lipid-lowering1 activities, and about 50compounds, including triterpenes, sesquiterpenoids, steroidsand organic acids, were isolated from the root or root bark.3,7–9
The stem was also reported to have anti-inammation,10 anti-cancer11 and fatty liver amelioration12 effects, and nearly 100compounds including triterpenes, diterpenoids, steroids,avonoids, phenolics and benzoquinone were isolated.13–16 Forthe leaf part, previous studies have found that the extract of ithad insecticidal effect and hypoglycemic effect,17 while onlya few avonoids were isolated.18 It was revealed that there weresignicant variation for the contents of celastrol or total
University, Fujin Road 126, Changchun
[email protected]; [email protected]; Tel:
ersity, Changchun 130021, Jilin, China
20
alkaloids in different parts of COT.19,20 Along with continuousexpansion of the folk and clinical application of COT, an in-depth study on the chemical constituents in different parts ofCOT has attracted more and more attention. However, thecomprehensive comparative study on the chemical compositionbetween root, stem and leaf parts of COT has not been reportedso far.
Recently, the UPLC-Q/TOF-MS method has been innovativelyused for screening and identifying chemical components inherbal medicines and traditional Chinese medicine. And theglobal proling of various metabolites were reported. As part ofthese research works, we reported this method to detect somenatural products including Platycodon grandiorum andGinseng root.21,22 Our research results showed that this methodis high throughput, comprehensive, simple and efficient. As faras we know, the UPLC-Q/TOF-MSmethod has not been reportedto identify the components in COT. So, the study in this papercomparatively analyzes the phytochemicals of root, stem andleaf parts of COT by using the UPLC-Q/TOF-MS method for therst time and nds out the similarities and differences betweenthem.
Chronic Obstructive Pulmonary Disease (COPD), predictedto rank as the third leading cause of death in the world,23 ismainly caused by signicant exposure to harmful gases or
This journal is © The Royal Society of Chemistry 2020
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
particles.24 Cigarette smoking was the leading environmentalrisk factor for COPD around the world, and cigarette smokerswere more likely to develop respiratory symptoms and hada higher COPD mortality rate. Along with the progressive lunginammation, some pro-inammatory mediators such as IL-1b,IL-6 and TNF-a participated in the occurrence and developmentof COPD.25 Although the COT had been used in treating variousinammatory diseases, the effect on the cigarette smoke extract(CSE)-induced inammatory reaction has not been reported sofar.
In the present study, the main medicinal parts of COT (root,stem and leaf) were chosen as the test sample. On one hand, thesimilarities and differences of phytochemicals in three partswere analyzed by using UNIFI platform and untargeted metab-olomics based on UPLC-Q/TOF-MSE. The components andpotential chemical markers to prole diverse classications ofmetabolites of three parts were investigated. On the other hand,the effects on CSE-induced inammatory reaction of these threeparts were explored in A549 cells. The anti-COPD activity ofdifferent parts was preliminarily discussed. This comprehensivestudy could reveal the structural diversity of secondary metab-olites and the different patterns of main medicinal parts ofCOT, and provide the data for further clinical application inanti-COPD. The study on the phytochemistry and the pharma-cological activity of various parts were both signicantly valu-able to the research and development of COT.
2. Experiment2.1. Materials and reagents
A total of 10 batches of fresh COT were collected from differentgrowth areas in China (Table 1). All herbs were authenticated bythe authors according to Hunan Province Local Standard forTraditional Chinese Medicine (2009 edition) for “Celastrusorbiculatus Thunb.”. The corresponding specimens had beendeposited in the Research Center of Natural Drug, JilinUniversity, China.
Methanol and acetonitrile were of LC/MS grade purchasedfrom Fisher Chemical Company. Formic acid was bought fromSigma-Aldrich Company, St. Louis, MO, USA. Deionized waterwas puried by Millipore water purication system (Millipore,Billerica, MA, USA). All other chemicals were analytically pure.Cigarettes for bioassay analysis were Xiongshi cigarette (China
Table 1 The detail information of the collected COT samples
No. Source
COT 01 Yichun city, Heilongjiang provinceCOT 02 Changchun city, Jilin province, ChCOT 03 Huhhot city, Inner Mongolia autonCOT 04 Lanzhou city, Gansu province, ChCOT 05 Taian city, Shandong province, ChCOT 06 Xi'an city, Shanxi province, ChinaCOT 07 Zhengzhou city, Henan province,COT 08 Chengdu city, Sichuan province, CCOT 09 Changsha city, Hunan province, CCOT 10 Jinhua city, Zhejiang province, Ch
This journal is © The Royal Society of Chemistry 2020
Tobacco Zhejiang Industrial Co., Ltd, Hangzhou, China), eachcigarette contained 11 mg of tar, 0.7 mg of nicotine, and 13 mgof carbon monoxide. Human lung carcinoma A549 cells wereobtained from the Department of Pathogen Biology, BasicMedical College, Jilin University. ELISA kits were bought fromNanjing Jiancheng Bio-engineering Institute.
2.2. Sample preparation of three parts of COT
Took the whole fresh COT, and separated the root, stem and leafpart respectively to get 30 test samples including root part (R1–R10) samples, stem part (S1–S10) samples and leaf part (L1–L10)samples. The aforementioned parts were air-dried, grinded andsieved with Chinese National Standard Sieve No. 3, R40/3 seriesto obtain the homogeneous powder respectively. Each powderwas weighted (2.0 g) accurately and extracted thrice (3 hours pertime) with 100 mL of 80% methanol at 80 �C, cooled, ltered,collected and combined the ltrate of each sample, concen-trated and evaporated to dryness.
For metabonomics analysis, each residue (all approximately2.0 mg) was dissolved in 1.0 mL of 80% methanol respectively,aer being ltered with a syringe lter (0.22 mm), 30 test solu-tions (RM1–RM10, SM1–SM10 and LM1–LM10) were obtained, whichwas injected into the UPLC system directly. Furthermore, toensure the suitability consistency and the stability of MS anal-ysis, a sample for quality control (QC) was prepared by pooling20 mL from every test solution, namely containing all of theconstituents in this analysis.
For screening analysis, the test solutions of root part (RS),stem part (SS) and leaf part (LS) were prepared by pooling 100 mLfrom RM1–RM10, SM1–SM10 and LM1–LM10 solutions, respectively.
For bioassay analysis, the test samples (Rbio, Sbio and Lbio) ofroot part, stem part and leaf part were prepared by combiningeach residue of R1–R10, S1–S10 and L1–L10, respectively. Then,Rbio, Sbio and Lbio were dissolved in water at the concentration of3.2 mg mL�1 to get the stock solutions stored in 4 �C.
2.3. Ultra-high performance liquid chromatographycombined with quadrupole time-of-ight tandem massspectrometry (UPLC-Q/TOF-MSE)
The separation and detection of components were performedon the UPLC system combined with Xevo G2-XS Q/TOF mass
Collection time
, China 12th August, 2017ina 3rd August, 2017omous region, China 10th August, 2017ina 13th August, 2017ina 5th July, 2017
14th July, 2017China 15th July, 2017hina 7th July, 2017hina 27th July, 2017ina 2nd July, 2017
RSC Adv., 2020, 10, 8396–8420 | 8397
Tab
le2
Compoundsidentifiedfrom
theroot,stem
andleaf
ofCOTbyUPLC
-QTOF-MSE
a
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
10.59
C29H
28O
850
4.18
2850
4.18
43�2
.754
9.18
10[M
+H
�COO]�,3
82.140
0[M
�H
�C7H
5O2]�,3
11.085
7[M
�H
�C11H
12O
3]�
Interiotherin
AR
33
2#1.23
C19H
20O
634
4.12
5634
4.12
60�1
.136
7.14
86[M
+Na]
+,3
27.118
5[M
+H
�H
2O]+,
315.07
07[M
+H
�2�
CH
3]+,2
38.059
1[M
+H
�C7H
7O]+,2
24.034
1[M
+H
�C8H
9O]+
5,7-Dihyd
roxy-6,8-
dimethyl-3(S)-3-(3-
methoxy-40-hyd
roxybe
nzyl)
chroman
-4-one
S—
32.43
C9H
12O
418
4.07
2818
4.07
36�4
.418
5.07
91[M
+H]+,1
67.058
4[M
+H
�H
2O]+,
136.05
93[M
+H
�H
2O
�CH
2OH]+
Euc
ommidiol
R,S
34
43.60
C16H
18N
2O3
286.13
2828
6.13
173.5
309.12
20[M
+Na]
+,2
42.084
1[M
+H
�3�
CH
3]+,
225.09
89[M
+H
�2�
OCH
3]+
PicrasidineB
S,L
a
53.64
C22H
28O
11
468.16
1646
8.16
32�3
.446
9.16
60[M
+H]+,2
90.103
6[M
+H-Glu]+,2
32.069
1[M
+H
�C3H
7O-Glu]+
Cim
icifug
aglycoside
R35
63.66
C19H
30O
838
6.19
2538
6.19
41�4
.240
9.18
11[M
+Na]
+,2
26.012
4[M
+H
�C6H
9O5]+,
178.08
37[M
+H
�2�
CH
3-Glu]+,1
90.125
5[M
+H
�H
2O-Glu]+
Roseo
side
R,S
,L36
73.97
C25H
28O
642
4.19
0142
4.18
863.6
447.18
12[M
+Na]
+,4
07.122
6[M
+H
�H
2O]+,
303.05
39[M
+H
�C9H
14]+,1
76.053
3[M
+H
�H
2O
�C9H
14�
C6H
5O2]+
Norku
rarinon
eR
37
84.17
C19H
20O
634
4.12
4434
4.12
60�4
.734
5.13
16[M
+H]+,2
22.053
0[M
+H
�CH
3�
C7H
7O]+,1
53.051
1[M
+H
�C11H
12O
3]+
2,3-Dihyd
ro-5,7-dihyd
roxy-
8-methoxy-3-[(4-
methoxyp
hen
yl)m
ethyl]-6
-methyl-4H-1-ben
zopy
ran-4-
one
S38
94.44
C15H
18O
832
6.09
9332
6.10
02�2
.932
5.08
70[M
�H]�,1
28.042
6[M
�H
�H
2O-Glu]�,
100.04
09[M
�H
�HCOOH-Glu]�,7
5.05
70[M
�H
�C3H
3O2-Glu]�
Glucosido
-p-cou
maric
acid
Rs
104.60
C20H
20O
534
0.12
9534
0.13
11�4
.534
1.13
55[M
+H]+,2
56.064
1[M
+H
�C5H
9O]+,
238.06
92[M
+H�H
2O�C5H
9O]+,1
93.071
3[M
+H
�C9H
7O2]+
PsorachalconeA
S—
11※
5.02
C32H
34O
12
610.20
4861
0.20
50�0
.460
9.14
46[M
�H]�,5
41.201
1[M
�H
�O
�C2H
3O]�,3
71.189
8[M
�H
�O
�2�
C5H
3O3]�
Orbiculin
IL
—
125.28
C27H
36O
13
568.21
3956
8.21
56�2
.859
1.20
31[M
+Na]
+,3
59.145
9[M
+H
�CH
2OH-
Glu]+,3
45.125
1[M
+H
�CH
3�
C11H
13O4]+
CitrusinB
S,L
s
135.39
C15H
10O
730
2.04
1530
2.04
27�4
.130
3.04
84[M
+H]+,1
78.032
0[M
+H
�C6H
5O3]+,
108.02
86[M
+H
�H
2O
�C9H
5O4]+
Isoe
tin
Ss
14※
5.53
C27H
30O
16
610.15
3961
0.15
340.9
611.16
13[M
+H]+,4
32.104
0[M
+H-Glu]+,2
53.038
8[M
+H
�2�
Glu]+
Luteolin
7,40-diglucoside
L39
155.65
C24H
32O
10
480.20
0748
0.19
962.3
503.18
99[M
+Na]
+,3
86.151
7[M
+H
�2�
H2O
�COOCH
3]+,3
60.142
6[M
+H
�C8H
9O]+,1
91.071
5[M
+H
�C8H
9O
�C9H
13O3]+
Ilexin
L3S
—
165.66
C20H
24O
737
6.15
0537
6.15
22�4
.539
9.13
88[M
+Na]
+,3
16.121
7[M
+H
�2�
CH
3�
CH
2OH]+,3
10.132
0[M
+H
�2�
H2O
�CH
3O]+,
138.05
97[M
+H
�C12H
15O
5]+
Vladinol
CR
40
175.74
C15H
10O
730
2.04
1830
2.04
27�2
.930
3.04
91[M
+H]+,2
85.036
3[M
+H
�H
2O]+,
110.02
81[M
+H
�C9H
5O5]+
Que
rcetin
Sb
8398 | RSC Adv., 2020, 10, 8396–8420 This journal is © The Royal Society of Chemistry 2020
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Tab
le2
(Contd.)
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
185.87
C19H
18O
431
0.11
9331
0.12
05�3
.830
9.12
66[M
�H]�,2
79.082
1[M
�H
�2�
CH
3]�,
278.09
77[M
�H
�OCH
3]�,1
73.053
2[M
�H
�OCH
3�
C8H
9]�,1
04.070
3[M
�H
�C11H
9O4]�
6,7-Dim
ethoxy-2-
phen
ethylch
romon
eS
s
195.96
C10H
10O
317
8.06
2317
8.06
30�3
.917
9.06
87[M
+H]+,1
64.037
4[M
+H
�CH
3]+,
120.04
91[M
+H
�COOCH
3]+,8
6.03
07[M
+H
�C6H
5O]+
Methyl-p-cou
marate
R41
206.75
C17H
14O
834
6.06
8834
6.06
89�0
.134
7.07
61[M
+H]+,3
29.062
9[M
+H
�H
2O]+,
298.06
88[M
+H
�H
2O�
OCH
3]+,2
19.046
0[M
+H
�H
2O
�C6H
5O2]+
Aksilarin
Rs
217.22
C14H
16O
424
8.10
4024
8.10
49�3
.524
9.11
05[M
+H]+,2
06.087
7[M
+H
�CH
3CO]+,
193.10
07[M
+H
�C3H
4O]+
Evodinnol
R42
227.58
C21H
22O
637
0.14
1037
0.14
16�1
.737
1.14
83[M
+H]+,220
[M+H�C9H
11O2]+,152
[M+
H�
C12H
11O
4]+
(+)-7,8-
Dideh
ydroarctigen
inR
43
237.73
C17H
20O
428
8.13
6028
8.13
62�0
.531
1.12
52[M
+Na]
+,2
74.109
0[M
+H
�CH
3]+,
238.08
42[M
+H
�2�
H2O
�CH
3]+
(+)-Celap
han
olA
R,S
s
248.47
C34H
44O
14
676.27
3867
6.27
311.0
677.28
11[M
+H]+,5
85.239
0[M
+H
�H
2O�CH
3�
C2H
3O2]+,5
56.265
0[M
+H�C7H
5O2]+,4
41.199
7[M
+H
�4�
C2H
3O2]+
Celan
gulinIV
S,L
—
258.57
C22H
24O
740
0.14
0740
0.15
22�3
.840
1.15
59[M
+H]+,3
83.062
3[M
+H
�H
2O]+,
365.21
18[M
+H
�2�
H2O
]+,2
34.076
3[M
+H
�C9H
11O3]+
Aschan
tin
R,S
44
268.83
C20H
20O
534
0.12
9534
0.13
11�4
.534
1.13
55[M
+H]+,3
26.064
1[M
+H
�CH
3]+,
299.05
62[M
+H
�C3H
7]+,1
50.056
2[M
+H
�C4H
7
�C8H
7O2]+
Corylifol
BS
45
278.94
C20H
18O
533
8.11
4333
8.11
54�3
.333
9.12
09[M
+H]+,2
18.094
5[M
+H
�C8H
7O]+,
176.07
35[M
+H
�H
2O
�C9H
7O2]+
Dem
ethoxycurcu
min
R46
288.95
C20H
20O
635
6.12
4735
6.12
60�3
.635
7.13
20[M
+H]+,2
45.082
0[M
+H
�CH
3�
C6H
5O2]+,9
6.03
46[M
+H
�H
2O
�C14H
11O
4]+
Leachianon
eG
Rs
298.96
C18H
20O
531
6.13
1731
6.13
111.8
339.12
09[M
+Na]
+,2
51.062
4[M
+H
�2�
H2O�
2�
CH
3]+,1
74.090
0[M
+H
�2�
H2O
�C7H
7O]+
(3R-cis)-3,4-Dihyd
ro-3,4-
diol-7-m
ethoxy-3-[(4-
methoxyp
hen
yl)m
ethyl]-
2H-1-ben
zopy
ran
R,S
—
30#
8.98
C22H
28O
842
0.17
9942
0.17
843.4
443.16
96[M
+Na]
+ ,20
5.08
43[M
+H
�2�
CH
2OH
�C8H
9O3]+,1
67.066
4[M
+H
�C13H
18O
5]+
(+)-Lyon
iresinol
S47
319.81
C24H
26O
844
2.16
1144
2.16
28�3
.844
3.16
75[M
+H]+,3
84.146
6[M
+H
�C2H
3O2]+,
381.12
68[M
+H
�2�
CH
3O]+,3
07.073
1[M
+H
�CH
3�
2�
CH
3O
�C2H
3O2]+
Interiorin
CR,S
—
3210
.35
C20H
18O
432
2.12
1832
2.12
054.0
323.12
91[M
+H]+,2
68.100
4[M
+H
�C4H
7]+,
254.08
34[M
+H
�C5H
9]+
Neo
bavaiso
avon
eR
48
3311
.39
C18H
34O
533
0.23
9233
0.24
06�4
.035
3.22
84[M
+Na]
+,2
77.212
9[M
+H
�3�
H2O]+,
150.11
00[M
+H
�2�
H2O
�C8H
17O2]+,8
1.05
69[M
+H
�H
2O
�C4H
7O2�
C8H
17O2]+
9-Octad
ecen
oicacid
R,S
s
3413
.75
C18H
20O
430
0.13
4830
0.13
62�4
.332
3.12
40[M
+Na]
+,1
38.055
6[M
+H
�C10H
11O2]+,
107.04
65[M
+H
�CH
3O
�C10H
11O2]+
tran
s-3,30,5,5
0 -Tetramethoxystilben
eS
—
35*
14.06
C30H
48O
650
4.34
4850
4.34
51�0
.6Virga
ureagenin
GR
49
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 8396–8420 | 8399
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Tab
le2
(Contd.)
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
549.34
17[M
+HCOO]�,2
63.342
3[M
�H
�C14H
24O
3]�,2
39.323
9[M
�H
�C16H
24O3]�
3614
.33
C28H
42O
545
8.30
5345
8.30
324.6
457.29
81[M
�H]�,3
44.332
1[M
�H
�2�
H2O
�HCOOH
�CH
2OH]�,2
33.295
7[M
�H
�C13H
20O
3]�,
3b,4b,23-Trihyd
roxy-24,30
-dinorolean-12,20
(29)-dien-
28-oic
acid
R50
3715
.19
C28H
46O
546
2.33
4946
2.33
450.8
461.32
76[M
�H]�,4
25.327
0[M
�H
�2�
H2O]�,
193.23
96[M
�H
�C17H
32O
2]�
Polypo
rusteron
eF
R51
3815
.26
C39H
54O
661
8.39
2661
8.39
201.0
641.38
57[M
+Na]
+,5
33.334
4[M
+H
�C3H
4�
HCOOH]+,3
98.248
6[M
�2�
CH
3�
C3H
4�
C8H
7O3]+
Lup-20
(29)-en-28-oic-3b
-yl
caffeate
Rs
39*
15.47
C30H
40O
446
4.29
3446
4.29
271.5
509.25
51[M
+HCOO]�,3
86.269
3[M
�H
�H
2O�
C2H
3O2]�,1
99.222
8[M
�H
�C17H
28O
2]�
Pristimerin
R52
40※
15.84
C33H
38O
957
8.25
3557
8.25
163.3
577.26
83[M
�H]�,5
61.268
0[M
�H
�O]�,
547.21
87[M
�H
�2�
CH
3]�,5
34.217
6[M
�H
�C2H
3O]�
Orbiculin
AR,L
s
4116
.28
C30H
46O
447
0.33
9947
0.33
960.6
469.33
26[M
�H]�,4
54.315
7[M
�H
�CH
3]�,
451.33
03[M
�H
�H
2O]�,3
42.352
2[M
�H
�C8H
15O]�
11-Oxoka
nsenon
olR
53
4216
.52
C30H
46O
650
2.33
1550
2.32
944.1
547.33
26[M
+HCOO]�,4
55.323
6[M
�H
�HCOOH]�,4
01.350
5[M
�H
�3�
H2O
�HCOOH]�
Esculen
ticacid
Rs
4316
.66
C31H
44O
651
2.31
1851
2.31
38�3
.951
1.30
45[M
�H]�,4
95.292
9[M
�H
�O]�,
467.24
12[M
�H
�CO2]�,2
33.253
5[M
�H
�C17H
26O
3]�
Paeo
nen
olideG
R,L
54
4416
.79
C32H
40O
855
2.27
1755
2.27
23�1
.159
7.26
99[M
+HCOO]�,4
87.225
0[M
�H
�H
2O�
CH
3�
CH
3O]�,1
93.106
5[M
�H
�C21H
26O
5]�
Sauc
erneo
lA
R,S
,L55
4516
.80
C27H
44O
849
6.30
5549
6.30
363.8
541.30
50[M
+HCOO]�,4
61.302
6[M
�H
�2�
H2O]�,3
78.287
6[M
�H
�C6H
13O2]�
Turke
steron
eR,L
56
4616
.81
C30H
50O
244
2.39
0744
2.38
11�0
.944
1.37
13[M
�H]�,4
23.328
6[M
�H
�H
2O]�,
233.34
56[M
�H
�C14H
24O
]�Olean
olic
alcohol
S,L
57
4717
.06
C20H
34O
433
8.24
6133
8.24
571.1
361.23
53[M
+Na]
+,2
67.223
9[M
+H
�4�
H2O]+,
262.16
73[M
+H
�CH
3�
2�
CH
2OH]+,2
78.012
4[M
+H
�C3H
5O2]+
Hallol
Ra
4817
.22
C18H
28O
227
6.20
8627
6.20
89�1
.027
7.21
41[M
+H]+,2
48.219
7[M
+H
�C2H
5]+,
222.15
20[M
+H�C4H
7]+,6
7.05
79[M
+H�C2H
5�
C11H
17O
2]+
6,9,12
,15-
Octad
ecatetraen
oicacid
R58
4917
.23
C16H
30O
225
4.22
4925
4.22
461.2
277.21
41[M
+Na]
+,1
25.116
0[M
+H
�C4H
9�
C3H
5O2]+,1
12.080
4[M
+H
�H
2O
�C9H
17]+
Oleop
almitic
acid
R,S
,L59
50*
17.25
C30H
44O
446
8.32
2546
8.32
40�3
.246
7.24
41[M
�H]�,4
34.290
5[M
�H
�H
2O
�CH
3]�,4
23.332
0[M
�H
�CO2]�
3b-H
ydroxy-2-oxoolean-12-
ene-22
,29-actone
R—
51*
17.30
C27H
40O
646
0.28
3346
0.28
251.7
483.27
51[M
+Na]
+,3
61.235
5[M
+H
�C6H
12O
]+,
417.22
82[M
+H
�CO2]+,3
19.248
0[M
+H
�C8H
14O2]+
Lucide
nic
acid
NR
60
52#
17.38
C27H
44O
443
2.32
2343
2.32
40�3
.8Chlorogenin
R,S
,La
8400 | RSC Adv., 2020, 10, 8396–8420 This journal is © The Royal Society of Chemistry 2020
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Tab
le2
(Contd.)
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
431.22
17[M
�H]�,3
97.342
8[M
�H
�O�
H2O]�,
395.32
58[M
�H
�2�
H2O]�,2
49.101
9[M
�H
�C11H
18O
2]+
5317
.56
C20H
22O
432
6.15
1532
6.15
18�0
.832
7.15
88[M
+H]+,3
12.129
5[M
+H
�CH
3]+,
256.10
67[M
+H
�2�CH
3�C3H
5]+,2
03.223
7[M
+H
�C7H
8O2]+
Deh
ydrodiisoe
ugen
olR
61
54※
17.62
C30H
48O
548
8.35
2348
8.35
024.4
487.29
17[M
�H]�,4
69.324
0[M
�H
�H
2O]�,
433.32
97[M
�H�3�H
2O]�,4
11.103
3[M
�H
�2
�CH
3�
HCOOH]+,4
05.302
9[M
�H
�2�
H2O�
HCOOH]�
Orthosph
enic
acid
R,S
,La
5517
.80
C35H
40O
757
2.27
8857
2.27
742.4
571.27
38[M
�H]�,5
55.282
6[M
�H
�O]�,
331.23
34[M
�H
�O
�C6H
5�
C9H
7O2]�
CelastrineB
R,S
,L—
5617
.84
C30H
46O
345
4.34
4545
4.34
47�0
.545
5.34
92[M
+H]+,408
.343
5[M
+H�H
2O�CHO]+,
325.22
91[M
+H
�CH
3�
C6H
11O
]+(13a
,14b
,17a
,20S
,24Z
)-La
nosta-26-hyd
roxy-3-oxo-
7,24
-dien-21-al
R,S
a
57*
18.02
C27H
44O
748
0.31
0648
0.30
874.0
479.24
39[M
�H]�,4
64.290
3[M
�H
�CH
3]�,
461.29
34[M
�H
�H
2O]�,4
14.277
9[M
�H
�H
2O
�C2H
7O]�,2
02.023
9[M
�H
�C11H
22O3]+
Viticosterone
R,S
,L62
5818
.11
C30H
46O
345
4.34
6445
4.34
473.8
453.34
14[M
�H]�,4
35.360
8[M
�H
�H
2O]�,
409.33
19[M
�H
�CO2]�,3
76.371
6[M
�H
�H
2O
�CH
3�
CO2]�,2
23.210
0[M
�H
�C15H
18O
2]�
WilforlideA
Rs
5918
.20
C28H
48O
241
6.36
3641
6.36
54�4
.146
1.36
18[M
+HCOO]�,2
65.259
1[M
�H
�C9H
10O2]�,1
49.230
9[M
�H
�C19H
38]�
g-Tocop
herol
R63
6018
.24
C30H
38O
11
574.24
2357
4.24
141.5
597.23
15[M
+Na]
+,3
95.189
7[M
+H
�C2H
3O2�
C7H
5O2]+,3
39.172
4[M
+H
�4�
C2H
3O2]+
Ejap3
R,L
a
61#
18.38
C16H
32O
225
6.23
9325
6.24
02�3
.727
9.23
03[M
+Na]
+,1
99.178
7[M
+H
�C4H
9]+,
69.055
3[M
+H
�2�
CH
3�
C9H
17O2]+
Methyl
(12S
)-12
-methyltetrad
ecan
oate
Sa
6218
.41
C32H
44O
855
6.30
1655
6.30
36�3
.655
5.29
10[M
�H]�,4
63.260
4[M
�H
�H
2O�
CH
3
�C2H
3O2]�,3
13.228
5[M
�H
�H
2O�
C7H
13O2�
C5H
3O2]�
14,15-Epo
xy-14H
-cyclop
enta
[a]
phen
anthrene,
bufa-20,22
-dien
olidede
riv.
R—
63*
18.46
C30H
42O
751
4.29
3151
4.29
310.1
515.30
17[M
+H]+,4
69.285
9[M
+H
�HCOOH]+,
417.26
08[M
+H
�C6H
10O]+,3
77.229
7[M
+H
�C9H
14O]+
Gan
oderen
icacid
AR
60
64#
18.67
C30H
48O
345
6.35
8345
6.36
04�4
.745
7.36
75[M
+H]+,4
39.352
0[M
+H
�H
2O]+,
424.26
88[M
+H
�H
2O�CH
3]+,3
30.246
5[M
+H
�C8H
15O]+
Kan
senon
olS
64
6518
.69
C16H
32O
225
6.23
9425
6.24
02�2
.730
1.23
76[M
+HCOO]�,9
7.09
91[M
�H
�C6H
13�
C3H
5O2]�,6
8.05
54[M
�H
�CH
3�
C8H
17�
C2H
3O2]�
4,8,12
-Trimethyltridecan
oicacid
S65
6618
.69
C18H
30O
227
8.22
3327
8.22
46�4
.527
9.22
92[M
+H]+,2
61.235
3[M
+H
�H
2O]+,
72.055
4[M
+H
�C11H
19�
C2H
3O2]+
Gam
olen
icacid
R,S
66
6718
.74
C30H
46O
447
0.33
9947
0.33
960.6
469.33
26[M
�H]�,4
54.315
7[M
�H
�CH
3]�,
451.33
03[M
�H
�H
2O]�,4
07.372
6[M
�H
�H
2O
�CO2]�,2
61.410
2[M
�H
�C14H
24O
]�
3b-H
ydroxy-11a
,12a
-epo
xy-
13b,28-ursolide
Ra
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 8396–8420 | 8401
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Tab
le2
(Contd.)
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
6818
.83
C28H
36O
748
4.24
5848
4.24
61�0
.646
9.25
97[M
�OH]+,3
63.235
0[M
+H
�C7H
6O2]+,
345.18
52[M
+H
�H
2O�
C7H
6O2]+,2
18.170
8[M
+H
�2�
H2O
�C7H
6O2�
C6H
5O2]+
ScutelloneI
R67
6918
.94
C30H
46O
447
0.33
9947
0.33
960.6
471.33
26[M
+H]+,4
25.315
7[M
+H
�HCOOH]+,
289.32
41[M
+H
�C11H
18O
2]+,2
49.338
3[M
+H
�C14H
22O
2]+
Hed
erag
onic
acid
R68
7018
.97
C30H
48O
345
6.36
0645
6.36
040.5
457.36
78[M
+H]+,4
39.223
1[M
+H
�H
2O]+,
249.34
37[M
+H
�C14H
24O
]+,2
03.165
3[M
+H
�C14H
24O
�HCOOH]+
Ursolic
acid
R,S
,L69
7119
.00
C18H
30O
329
4.21
8429
4.21
95�3
.631
7.20
60[M
+Na]
+,2
77.211
5[M
+H
�H
2O]+,
172.08
63[M
+H
�C9H
15]+,1
24.108
1[M
+H
�C9H
15O3]+
9-Oxooctade
ca-10,12
-dien
oicacid
R,S
70
7219
.13
C30H
42O
751
4.29
3151
4.29
310.1
515.30
04[M
+H]+,4
54.266
0[M
+H
�CH
3�
HCOOH]+,4
15.267
6[M
+H
�C6H
12O]+,3
21.239
3[M
+H
�C11H
14O
3]+
Gan
oderen
icacid
BR
60
73*
19.30
C11H
14O
217
8.10
0117
8.09
944.2
201.08
86[M
+Na]
+,164
.073
9[M
+H
�CH
3]+,
107.05
89[M
+H
�CH
3O�
C3H
5]+,7
6.05
07[M
+H
�2�
CH
3O
�C3H
5]+
Isoh
omog
enol
R71
7419
.35
C29H
48O
242
8.36
7542
8.36
544.7
451.35
68[M
+Na]
+,4
11.244
9[M
+H
�H
2O]+,
274.22
05[M
+H
�C10H
19O
]+,2
56.226
0[M
+H
�H
2O
�C10H
19O]+
Seringo
sterol
R,L
72
7519
.46
C30H
44O
751
6.30
8051
6.30
87�1
.351
5.30
07[M
�H]�,5
00.286
5[M
�H
�CH
3]�,
497.33
83[M
�H
�H
2O]�,3
22.176
7[M
�H
�2�
H2O
�C8H
13O
3]�
Gan
oderic
acid
AR
73
7619
.72
C22H
30O
435
8.21
3235
8.21
44�3
.338
1.20
24[M
+Na]
+,3
16.206
3[M
+H
�C2H
3O]+,
273.16
48[M
+H
�C3H
7�
C2H
3O2]+
(3S,4a
S,10
aS)-3-(Acetyloxy)-
2,3,4,4a
,10,10
a-hexah
ydro-
6-hyd
roxy-1,1,4a-trim
ethyl-
7-(1-m
ethylethyl)-9(1H
)-ph
enan
threnon
e
Rs
7719
.73
C30H
50O
650
6.36
3250
6.36
074.9
505.35
59[M
�H]�,4
3.39
09[M
�H
�4�
H2O]�,
431.28
34[M
�H
�CH
3�
C3H
7O]�
13b,17b
-Epo
xyalisol
AR
s
7819
.89
C30H
46O
345
4.34
3845
4.34
47�2
.045
5.34
92[M
+H]+,4
09.343
5[M
+H
�HCOOH]+,
391.15
17[M
+H
�H
2O�
HCOOH]+,2
49.331
7[M
+H
�C14H
22O
]+
Olean
onic
acid
R,S
74
7920
.05
C30H
46O
548
6.33
3448
6.33
45�2
.448
7.34
06[M
+H]+,4
41.334
4[M
+H
�HCOOH]+,
413.34
36[M
+H
�H
2O
�CH
3�
HCOOH]+,
395.32
78[M
+H
�2�
HCOOH]+,2
49.535
8[M
+H
�C14H
22O3]+
Ceanothic
acid
Ra
80*
20.24
C28H
42O
647
4.29
8647
4.29
811.0
497.29
08[M
+Na]
+,4
57.234
2[M
+H
�H
2O]+,
411.23
24[M
+H
�H
2O�
CH
3�
CH
3O]+,2
85.039
9[M
+H
�H
2O
�C9H
16O3]+
Methyl
lucide
nateQ
R75
8120
.36
C29H
34O
851
0.22
5651
0.22
540.3
533.21
48[M
+Na]
+,4
10.176
0[M
+H
�C2H
3O
�C3H
6O]+,2
68.145
5[M
+H
�C2H
3O
�C7H
5O
�C5H
3O2]+
Orbiculin
BR
—
8220
.36
C30H
48O
345
6.36
1745
6.36
043.0
Olean
olic
acid
R,S
,L74
8402 | RSC Adv., 2020, 10, 8396–8420 This journal is © The Royal Society of Chemistry 2020
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Tab
le2
(Contd.)
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
455.35
44[M
�H]�,3
91.344
0[M
�H
�H
2O
�HCOOH]�,1
49.370
7[M
�H
�H
2O
�C18H
27O2]�
8321
.14
C19H
38O
433
0.27
7033
0.27
700.1
353.26
32[M
+Na]
+,3
13.216
0[M
+H
�H
2O]+,
300.04
86[M
+H
�CH
3O]+,2
40.067
9[M
+H
�C3H
7O3]+
b-M
onop
almitin
S,L
76
84#
21.46
C39H
54O
661
8.39
4961
8.39
204.6
619.40
02[M
+H]+,5
65.412
6[M
+H
�3�
H2O]+,
472.35
57[M
+H
�C9H
7O2]+,4
56.344
3[M
+H
�C9H
7O3]+,4
11.135
6[M
+H
�C14H
24O]+
27-p-E-
Cou
maroyloxyursolicacid
S77
85#
21.49
C30H
46O
243
8.34
8543
8.34
98�2
.943
9.35
59[M
+H]+,4
21.343
2[M
+H
�H
2O]+,
390.11
26[M
+H
�H
2O�
CH
3O]+,2
50.344
3[M
+H
�C13H
17O]+
5-Deh
ydroka
roun
idiol
S—
86*
21.72
C13H
12O
220
0.08
3420
0.08
31�3
.320
1.08
86[M
+H]+,1
86.059
7[M
+H
�CH
3]+,
165.09
96[M
+H
�2�
H2O
]+,1
40.053
3[M
+H
�C2H
5O2]+
Safynol
R78
87*
22.03
C12H
16O
219
2.11
5719
2.11
503.7
215.10
45[M
+Na]
+,1
63.049
0[M
+H
�2�
CH
3]+,
150.17
39[M
+H�C3H
7]+,135
.217
3[M
+H�CH
3�
C3H
7]+,1
19.069
5[M
+H�CH
3�C2H
3O2]+,9
1.05
56[M
+H
�C3H
7�
C2H
3O2]+
Carvacryl
acetate
Ra
88*
22.04
C29H
38O
445
0.27
6645
0.27
70�0
.945
1.28
44[M
+H]+,4
33.259
1[M
+H
�H
2O]+,
405.27
71[M
+H
�HCOOH]+,2
15.362
1[M
+H
�C15H
24O
2]+,2
00.091
7[M
+H
�CH
3�
C15H
24O
2]+
Celastrol
R,S
,L79
8922
.05
C9H
10O
134.07
3713
4.07
323.4
157.06
32[M
+Na]
+,1
20.117
3[M
+H
�CH
3]+,
106.07
04[M
+H�C2H
5]+,7
7.04
77[M
+H�C2H
5�
CHO]+
4-Ethylbe
nzaldeh
yde
R80
90*
22.22
C29H
36O
444
8.26
0944
8.26
14�1
.044
9.26
79[M
+H]+,4
34.254
8[M
+H
�CH
3]+,
403.26
06[M
+H
�HCOOH]+,2
67.331
5[M
+H
�C12H
6O2]+
25-(9/
8)Abe
o-24
-nor-
friede
lan-2,3-dioxo-
1(10
),4,6,9(11)-tetraen
-29-
oicacid
R—
9122
.46
C38H
44N
2O6
624.32
0062
4.32
203.2
647.31
13[M
+Na]
+,5
03[M
+H�C8H
9O]+,3
14.139
5[M
+H
�CH
3�
C18H
16N
O2]+,2
97[M
+H
�C7H
7O
�C11H
17O2N]+,2
66.079
0[M
+H
�CH
3�
C8H
9O�
C13H
18N
O2]+
Neferin
R81
9222
.78
C32H
48O
551
2.34
8651
2.35
02�3
.151
1.34
13[M
�H]�,4
93.243
6[M
�H
�H
2O]�,
465.10
23[M
�H
�HCOOH]+,4
34.237
9[M
�H
�H
2O
�C2H
3O2]�,3
70.249
1[M
�H
�C8H
13O2]�
Gan
oderic
acid
XR
82
9322
.80
C12H
16O
219
2.11
5919
2.11
504.2
215.10
52[M
+Na]
+,1
78.089
3[M
+H
�CH
3]+,
107.51
47[M
�C5H
11O]+,7
8.05
71[M
+H
�C6H
11O2]+
Amyl
benzoate
Rs
9422
.81
C30H
52O
346
0.39
2046
0.39
170.8
483.38
13[M
+Na]
+,4
07.359
3[M
+H
�H
2O]+,
368.30
12[M
+H
�H
2O�
C3H
7O2]+,2
53.015
4[M
+H
�C14H
24O
]+
Oliba
num
olH
S,L
83
9522
.82
C30H
48O
447
2.35
4347
2.35
53�2
.147
3.36
15[M
+H]+,4
55.348
3[M
+H
�H
2O]+,
427.35
61[M
+H
�HCOOH]+,3
33.152
7[M
+H
�C9H
16O]+,2
90.014
6[M
+H
�CH
3�
C11H
20O]+
Ech
inocysticacid
R,S
84
9622
.83
C16H
32O
225
6.23
9025
6.24
02�4
.5Methyl
pentade
canoa
teR,S
,Ls
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 8396–8420 | 8403
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Tab
le2
(Contd.)
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
279.22
76[M
+Na]
+,1
55.123
0[M
+H
�C5H
11�
CH
3O]+,8
5.04
88[M
+H
�C10H
21�
OCH
3]+
9723
.02
C22H
36O
436
4.26
0136
4.26
14�3
.736
5.26
65[M
+H]+,2
66.219
0[M
+H
�C6H
11O]+,
219.17
49[M
+H
�H
2O
�C2H
3O2�
C5H
9O]+
VitetrifolinE
S85
9824
.29
C12H
16O
219
2.11
5919
2.11
504.2
215.10
52[M
+Na]
+,1
64.089
3[M
+H
�CH
3]+,
136.05
87[M
+H
�C4H
9]+,7
8.05
71[M
+H
�C6H
11O2]+
Isop
entylbe
nzoate
Rs
9924
.31
C11H
14O
217
8.10
0317
8.09
944.7
201.08
95[M
+Na]
+,1
47.044
1[M
+H
�CH
3]+,
136.04
91[M
+H
�C2H
3O]+,1
22.047
7[M
+H
�C3H
5O]+,7
7.01
46[M
+H
�CH
3O
�C4H
7O]+
Anisylaceton
eR
86
100#
24.51
C30H
52O
346
0.38
9846
0.39
17�4
.148
3.36
82[M
+Na]
+,4
25.332
5[M
+H
�2�
H2O]+,
336.24
43[M
+H
�C9H
17]+,2
95.035
7[M
+H
�C12H
22]+
Neo
panaxad
iol
S87
101
24.71
C30H
48O
244
0.36
5644
0.36
540.3
441.37
29[M
+H]+,4
23.361
1[M
+H
�H
2O]+,
259.16
87[M
+H
�C11H
18O
2]+,2
19.031
2[M
+H
�C14H
22O
2]+
Daturan
olon
eR,S
88
102
24.94
C30H
48O
424.37
0142
4.37
05�1
.042
5.37
74[M
+H]+,4
07.363
3[M
+H
�H
2O]+,
392.34
59[M
+H
�H
2O�CH
3]+,2
18.192
6[M
+H
�C14H
23O
]+
Olean
-9(11),12-dien
-3b-ol
R,S
s
103*
25.48
C21H
24O
434
0.16
7234
0.16
75�0
.836
3.15
73[M
+Na]
+,1
89.071
1[M
+H
�CH
3�
C8H
9O2]+,1
73.093
9[M
+H
�CH
3O
�C8H
9O2]+,
107.04
94[M
+H
�CH
3O
�C13H
15O2]+
Deh
ydrodiisoe
ugen
olmethyl
ether
R89
104
25.67
C30H
46O
447
0.33
7247
0.33
96�4
.147
1.34
45[M
+H]+,4
56.201
6[M
+H
�CH
3]+,
398.51
07[M
+H�C3H
5O2]+,3
79.031
7[M
+H�2�
HCOOH]+,3
12.237
0[M
+H
�H
2O�
C8H
13O
2]+,
234.45
81[M
+H
�CH
3�
C3H
5O2�
C8H
13O
2]+
Nigranoicacid
Ra
105
25.70
C37H
42O
13
694.28
0169
4.27
783.6
695.28
73[M
+H]+,5
15.220
1[M
+H
�3�C2H
4O2]+,
393.22
28[M
+H
�3�
C2H
4O2�
C7H
6O2]+,
331.01
67[M
+H
�2�
C2H
4O2�
C7H
6O2�
C7H
5O2]+
Celah
inD
S,L
s
106
25.91
C28H
48O
400.36
9840
0.37
05�1
.642
3.35
91[M
+Na]
+,3
86.263
8[M
+H
�CH
3]+,
326.28
23[M
+H
�H
2O
�CH
3�
C3H
7]+,2
54.226
6[M
+H
�H
2O�
C8H
17O]+,2
13.225
2[M
+H
�H
2O
�C11H
22O]+
Fungisterol
R,S
a
107
25.93
C27H
44O
384.33
9038
4.33
92�0
.638
5.34
63[M
+H]+,3
67.041
7[M
+H
�H
2O]+,
256.25
47[M
+H
�H
2O�
C8H
15]+,2
15.241
5[M
+H
�H
2O
�C11H
20]+
(E)-22
-Deh
ydroch
olesterol
R90
108
25.96
C29H
48O
412.36
8641
2.37
05�4
.641
3.37
46[M
+H]+,2
60.220
4[M
+H
�CH
3�
C10H
19]+,2
57.215
1[M
+H
�H
2O
�C10H
19]+
28-Isofucosterol
R,S
a
109
26.22
C30H
48O
345
6.35
8845
6.36
04�3
.545
7.36
51[M
+H]+,4
39.214
2[M
+H
�H
2O]+,
411.35
84[M
+H
�HCOOH]+,2
65.181
2[M
+H
�C13H
20O
]+
Maytenoicacid
Rs
110
26.28
C29H
48O
412.37
0241
2.37
05�0
.841
3.37
75[M
+H]+,3
95.367
6[M
+H
�H
2O]+,
256.22
75[M
+H�H
2O�C10H
19]+,2
18.721
8[M
+H
�CH
3�
C13H
24]+
Chon
dryllasterol
R91
8404 | RSC Adv., 2020, 10, 8396–8420 This journal is © The Royal Society of Chemistry 2020
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Tab
le2
(Contd.)
No.
t R(m
in)
Form
ula
Calcu
lated
mass(D
a)Theo
retical
mass(D
a)Masserror
(ppm
)MSE
frag
men
tation
Iden
tication
Source
Ref.
111
26.31
C18H
36O
228
4.27
2428
4.27
152.7
307.26
16[M
+Na]
+,1
40.131
2[M
+H
�2�
CH
3�
C6H
11O2]+,7
0.05
83[M
+H
�2�
CH
3�
C11H
21O2]+
Hexad
ecan
oicacid
Rs
112
26.62
C30H
48O
424.36
8642
4.37
05�4
.442
5.37
47[M
+H]+,4
10.358
4[M
+H
�CH
3]+,
367.30
99[M
+H�CH
3�C3H
7]+,273
.312
0[M
+H�
C11H
20]+
Fernen
one
R,L
92
113
26.83
C30H
48O
244
0.36
5644
0.36
540.3
441.37
29[M
+H]+,4
23.361
1[M
+H
�H
2O]+,
412.01
47[M
+H
�CHO]+,2
33.189
0[M
+H
�C14H
24O
]+,2
04.316
7[M
+H
�CHO
�C14H
24O]+
Olean
olic
alde
hyd
eR,S
,La
114
27.08
C28H
48O
241
6.36
6741
6.36
542.9
439.35
59[M
+Na]
+,1
92.121
2[M
+H
�C16H
33]+,
151.07
31[M
+H
�C19H
38]+
Cum
otocop
herol
R93
115
27.25
C24H
38O
439
0.27
5239
0.27
70�4
.341
3.26
44[M
+Na]
+,1
79.080
9[M
+H�C2H
5�C4H
9
�C8H
17O
]+,1
49.146
9[M
+H
�C8H
17�
C8H
17O
]+,
77.021
6[M
+H
�2�
C9H
17O2]+
Flexim
elR,S
,Lb
116
27.52
C30H
48O
424.37
0942
4.37
050.9
425.37
82[M
+H]+,4
10.357
2[M
+H
�CH
3]+,
259.21
41[M
+H
�C11H
18O
]+,2
21.193
9[M
+H
�C14H
20O
]+
b-Amyron
R,S
,L94
117※
27.84
C36H
44O
962
0.30
0362
0.29
852.9
621.30
83[M
+H]+,5
62.285
1[M
+H
�C2H
3O2]+,
461.23
03[M
+H�C2H
3O2�C5H
9O2]+,2
91.208
8[M
+H
�C2H
5�
C2H
3O2�
2�
C7H
5O2]+
Celafolin
D-3
S,L
s
118
27.88
C30H
48O
244
0.36
5644
0.36
540.3
441.37
29[M
+H]+,4
23.361
1[M
+H
�H
2O]+,
398.53
59[M
+H
�C3H
7]+,2
88.228
8[M
+H
�C11H
21]+,2
47.011
7[M
+H
�C14H
26]+
Polasterol
AR,S
,La
119
28.10
C29H
46O
410.25
3541
0.25
49�3
.441
1.35
99[M
+H]+,3
93.369
6[M
+H
�H
2O]+,
272.22
61[M
+H
�C10H
19]+,2
31.208
4[M
+H
�C13H
24]+
Corbisterin
R,L
95
120
28.49
C30H
50O
244
2.38
1344
2.38
110.5
465.37
05[M
+Na]
+,4
25.364
7[M
+H
�H
2O]+,
291.35
80[M
+H
�C10H
16O
]+,2
51.513
3[M
+H
�C13H
20O
]+
3-Oxo-11b
-hyd
roxyfriede
lane
S96
a*Characteristiccompo
nen
tinroot;#
characteristiccompo
nen
tinstem
;※ch
aracteristiccompo
nen
tinleaf;a:com
paredwithsp
ectralda
taob
tained
from
Wiley
Subs
cription
Services,Inc.(U
SA);
b:compa
redwithNISTChem
istryWeb
Boo
k;s:
compa
redwiththereference
compo
unds
;R:theroot
ofCelastrus
orbiculatusThun
b.;S:
thestem
ofCelastrus
orbiculatusThun
b.;L:
theleaf
ofCelastrus
orbiculatusThun
b.
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 8396–8420 | 8405
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
spectrometer (Waters Co., Milford, MA, USA) with an electro-spray ionization (ESI) interface.
The ACQUITY UPLC BEH C18 column (100 mm � 2.1 mm,1.7 mm) was bought from Waters Corporation (Milford, MA,USA). The moving phrase was consisted of eluent A (0.1%methanoic acid in water, v/v) and eluent B (0.1%methanoic acidin acetonitrile, v/v) in a liner gradient program (0–2 min, 10% B;2–26 min, 10 / 90% B; 26–28 min, 90% B; 28–28.1 min, 90 /
10% B; 28.1–40 min, 10% B) with a ow rate of 0.4 mL min�1.Set the temperature of column and the sample manager at 30 �Cand 15 �C, respectively. 10% and 90% acetonitrile in aqueoussolution were used as weak and strong wash solventsrespectively.
The optimized MS parameters were as follows: sourcetemperature (150 �C), desolvation temperature (400 �C), conevoltage (40 V), capillary voltage at 2.6 kV (ESI+) and 2.2 kV (ESI�),cone gas ow (50 L h�1) and desolvation gas ow (800 L h�1).MSE mode was chosen with low energy of 6 V and high energy of20–40 V.26,27 The mass spectrometer was calibrated with sodiumformate in the range of 100 to 1200 Da in order to ensure themass reproducibility and accuracy. Leucine enkephalin (m/z556.2771 in ESI+ and 554.2615 in ESI�) was used as externalreference for Lock Spray™ injected at a constant ow of 10mL min�1. The QC sample was injected randomly 4 timesthroughout the whole work list. All of the volume injection ofthe samples and QC was 5 mL per run. During data acquisition,the data for screening analysis was performed in MSE
continuum mode, the data for metabolomics analysis was per-formed in MSE centroid mode. Data recording was performedon MassLynx V4.1 workstation (Waters, Manchester, UK).
2.4. Screening analysis of components in three parts of COTby UNIFI platform
UNIFI 1.7.0 soware (Waters, Manchester, UK) was used fordata analysis.28,29
Firstly, in addition to the internal Traditional MedicineLibrary on UNIFI platform, the chemical constituent investiga-tion was conducted. As the result, a self-built database ofchemical compounds isolated from the genus of Celastrus L.was established by searching the online databases includingWeb of Science, Medline, PubMed, ChemSpider and ChinaNational Knowledge Infrastructure (CNKI). The compoundname, molecular formula and chemical structure of compo-nents were obtained in the database.
Secondly, the raw data obtained from Masslynx workstationwere compressed by Waters Compression and Archival Toolv1.10, then were imported into the UNIFI soware.
Thirdly, the compressed data were processed by thestreamlined work ow of UNIFI soware in order to quicklyidentify the chemical compounds which were matched thecriteria with Traditional Medicine Library and self-built data-base. The main parameters of processed method were as follow:2D peak detection was set to 200 as the minimum peak area. Inthe 3D peak detection, the peak intensity of high energy and lowenergy was taken more than 200 and 1000 times as theparameter respectively. Selected +H and +Na as positive adducts
8406 | RSC Adv., 2020, 10, 8396–8420
and +COOH and –H as negative adducts. Leucine enkephalinwas used as reference compound in order to get exact massaccuracy, with [M + H]+ 556.2766 for positive ion and [M � H]�
554.2620 for negative ion. As a result, the comprehensivechemical constituents screening list was accomplished.
Finally, a lter was set to rene the results, with the masserror between �5 and 5 ppm and response value over 5000.Each compound was veried by compared with the character-istic MS fragmentation patterns reported in literature or theretention time of the reference substances.
2.5. Metabolomics analysis of three parts of COT
The raw data acquired by Masslynx workstation was processedon MakerLynx XS V4.1 soware (Waters, Milford, CT, USA).Firstly, the raw data were processed with alignment, decon-volution, and data reduction, etc. The main parameters of theprocess method were as follows: retention time (0–28 min),retention time window (0.20), mass (100–1200 Da), masstolerance (0.10), mass window (0.10), minimum intensity(5%), marker intensity threshold (2000 counts) and noiseelimination (level 6). As a result, the list with mass andretention time corresponded to the responses based on all thedetected peaks from each data le were shown in ExtendedStatistics (XS) Viewer. Secondly, multivariate statistical anal-ysis, both principle component analysis (PCA) and orthogonalprojections to latent structures discriminant analysis (OPLS-DA), were performed on the MakerLynx soware to analyzethe resulting data.
PCA, a classical unsupervised low dimensional patternrecognition model, was used to show pattern recognition andmaximum variation, and the overview and classication wereobtained. OPLS-DA was used to obtain the maximum separationbetween two different groups. S-plots, which could providevisualization of the OPLS-DA predictive results, were created toexplore the potential chemical markers which contributed tothe differences.
Meanwhile, metabolites with VIP value > 4.0 and p-value <0.001 were considered as potential chemical markers.30,31
Futhermore, permutation test was also performed to providea reference distribution with the R2/Q2 values to indicatestatistical signicance. Finally, the analysis results were shownin Simca 15.0 soware (Umetrics, Malmo, Sweden).
2.6. Bioassay analysis of three parts of COT
2.6.1 Preparation of cigarette smoke extract. The prepara-tion of the cigarette smoke extract (CSE) was basically the sameas previous reports.32 Put the smoke from one cigarette into20 mL culture medium (300 s per cigarette). The CSE solutionwas incubated at 37 �C for 30 min aer being ltered with a 0.22mm sterile lter. The CSE solution was prepared freshly and wasused within 30 min. This prepared CSE solution was consideredto have the highest concentration (100%).
2.6.2 Cell viability assay. The nal concentrations (20.0,40.0, 80.0, 160.0, 320.0 mg mL�1) of each test samples (Rbio, Sbioand Lbio) were acquired by diluting the stock solutions withDulbecco's Modied Eagle Medium (DMEM). A549 cells were
This journal is © The Royal Society of Chemistry 2020
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
cultured in 96-well plates at a density of 5 � 105 cells per welltreated with CSE (0%, 5%, 10%, 20%, 30% and 40%) for 18 h, ortreated with Rbio, Sbio and Lbio solutions (0.0, 20.0, 40.0, 80.0,
Fig. 1 The base peak intensity (BPI) chromatograms in root, stem and le
This journal is © The Royal Society of Chemistry 2020
160.0, 320.0 mg mL�1) for 24 h. The growth-inhibition effect ofCSE and the effect of the drugs on viability of A549 cells wereevaluated by MTT assay.
af of COT in ESI+ and ESI�.
RSC Adv., 2020, 10, 8396–8420 | 8407
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
2.6.3 Drug treatment. For all groups, A549 cells werecultured in 96-well plates at a density of 5 � 105 cells per mL for18 h. In CSE group, the cells were treated with a certain dose of
Fig. 2 Chemical structures of compounds identified in the root, stem a
8408 | RSC Adv., 2020, 10, 8396–8420
CSE without drug intervened. In drug groups, the cells weretreated with both CSE and drugs (Rbio, Sbio or Lbio). In controlgroup, A549 cells were cultured normally without the CSE or
nd leaf of COT.
This journal is © The Royal Society of Chemistry 2020
Fig. 2 (contd.)
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 8396–8420 | 8409
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Fig. 2 (contd.)
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
drugs. In positive group, the cells were treated with both CSEand dexamethasone (5 mg mL�1).
2.6.4 Enzyme-linked immunosorbent assay. The contentsof IL-1b, IL-6 and TNF-a in the cell culture supernatant weredetermined with ELISA kits. All procedures were performedaccording to the manufacturer's instructions.
2.6.5 Statistical analysis. Statistical analysis was performedon Graphpad Prism 6.0 soware (CA, USA). The results wereexpressed as mean� SD. Two tailed test or a one-way analysis ofvariance (ANOVA) was used to calculate statistical signicantdifference (p < 0.05).
8410 | RSC Adv., 2020, 10, 8396–8420
3. Results and discussion3.1. Screening analysis of components of three parts of COT
A total of 120 compounds, including 91 in ESI+ mode and 29 inESI� mode, were identied or tentatively characterized fromthree parts of COT (Table 2). The base peak intensity (BPI)chromatograms were shown in Fig. 1. The chemical structureswere shown in Fig. 2, the results showed that COT was rich innatural components with various structural patterns. On onehand, according to the reference, there were nearly 50, 100, 10compounds were reported from the root, stem, leaf parts of COT,
This journal is © The Royal Society of Chemistry 2020
Fig. 2 (contd.)
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
respectively. While in this study, there were 92, 56 and 32components were identied or tentatively characterized fromroot part, stem part and leaf part of COT, respectively. And mostof the components were identied from COT for the rst time.Various kinds of structures, including triterpeniods,
This journal is © The Royal Society of Chemistry 2020
sesquiterpenoids, steroids, avonoids, organic acid and organicacid esters, phenylpropanoids, diterpeniods, monoterpenoids,alkaloid and others, were contained in each part of COT. Thenumbers (% of the total identied components in each part) andstructural types of compounds identied from root, stem and leaf
RSC Adv., 2020, 10, 8396–8420 | 8411
Fig. 4 The PCA of root (R), stem (S), leaf (L) groups in ESI+ and ESI�. QC: quality control.
Fig. 3 The numbers (% of the total identified components in each part) and structural types of compounds identified from root, stem and leaf ofCOT. R: the root of Celastrus orbiculatus Thunb.; S: the stem of Celastrus orbiculatus Thunb.; L: the leaf of Celastrus orbiculatus Thunb.
Fig. 5 OPLS-DA plots/permutation tests/S-plots between R and S&L. R: the root of Celastrus orbiculatus Thunb.; S: the stem of Celastrusorbiculatus Thunb.; L: the leaf of Celastrus orbiculatus Thunb.
8412 | RSC Adv., 2020, 10, 8396–8420 This journal is © The Royal Society of Chemistry 2020
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
Fig. 6 OPLS-DA plots/permutation tests/S-plots between S and R&L. R: the root of Celastrus orbiculatus Thunb.; S: the stem of Celastrusorbiculatus Thunb.; L: the leaf of Celastrus orbiculatus Thunb.
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
of COT were shown in Fig. 3. There were 34, 18 and 10 triterpe-niods identied from root, stem and leaf, respectively, accountedfor 37%, 32% and 31% of the total components in each part. So,
Fig. 7 OPLS-DA plots/permutation tests/S-plots between L and R&S.orbiculatus Thunb.; L: the leaf of Celastrus orbiculatus Thunb.
This journal is © The Royal Society of Chemistry 2020
it was concluded that triterpeniods were the major constituentsin three parts of COT. Moreover, according to each percentage,the root part of COT was also rich in organic acid and organic
R: the root of Celastrus orbiculatus Thunb.; S: the stem of Celastrus
RSC Adv., 2020, 10, 8396–8420 | 8413
Fig. 8 Heat-map visualizing the intensities of the potential chemicalmarkers.
Fig. 9 The cytotoxicity effects of different concentrations of cigarettesmoke extract (CSE) on A549 cells. **p < 0.01, compared with 0% CSEgroup.
Fig. 10 The cell viability effects of different concentrations of Rbio, Sbiogroup.
8414 | RSC Adv., 2020, 10, 8396–8420
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
acid esters, steroids and phenylpropanoids. The stem part wasalso rich in organic acid and organic acid esters, and avonoids.The leaf part was also rich in steroids, and sesquiterpenoids. Thepercentage of avonoids in the total identied components instem was higher than the percentages in root part or in leaf part.The percentages of sesquiterpeniods and steroids in the totalidentied components in leaf was higher than the percentages inroot part or in stem part. On the other hand, the sharedcomponents (30 for root and stem, 22 for root and leaf, 23 forstem and leaf, 15 for root, stem and leaf) were also found in ourstudy. As shown in Fig. 3, the structures of shared componentswere various, while triterpeniods held themajority. Celastrol, oneof the triterpeniods, was shown to distribute in root, stem andleaf, which was consistent with the ref. 19. So our research workcould provide the scientic data to clarify the chemical compo-sition of COT, particularly for the root and the leaf parts.
Although the study provided evidences to elucidate thechemical composition of COT, there were still some unresolvedissues. For example, as shown in BPI chromatograms, therewere some unidentied components. Further research shouldbe carried on the identication of these unknown compounds.
3.2. Metabolomics analysis of three parts of COT
PCA score 2D plots in both ESI+ and ESI� were established asshown in Fig. 4. The QC samples were clustered tightly and were inthemiddle of the three groups in PCA, which indicated the systemhad satisfactory stability. The samples from root of COT wereclearly gathered together, which indicated there was a goodsimilarity among them, and this phenomenon was also observedin stem and leaf of COT. Meanwhile, the root, stem and leafgroups were easily divided into three clusters, indicating that thesethree parts of COT could be differentiated in both ESI+ and ESI�.
In order to further distinguish one part from the other twoparts, OPLS-DA plots, S-plots, permutation tests, and VIP valueswere obtained to see which variables were responsible forsample separation97 (Fig. 5–7). In OPLS-DA plots, each spotrepresented a sample. From the perspective of OPLS-DA, onepart was clearly separated from the other two parts. Theparameters such as R2 and Q2 indicated the model had good
and Lbio solution on A549 cells. **p < 0.01, compared with 0 mg mL�1
This journal is © The Royal Society of Chemistry 2020
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
ability of prediction and reliability in both ESI+ and ESI�modes.The permutation plots showed the original point on the rightwas clearly higher than all Q2-values (blue) on the le, whichindicated the original models were valid. To identify themetabolites contributing to the discrimination, S-plots weregenerated under OPLS-DA model. Each spot in S-plots repre-sented a variable. The variables with VIP > 4 and p < 0.001 wereconsidered as potential chemical markers. The possiblemolecular formula of the markers were calculated by high-accuracy quasi-molecular ion with mass error between�5 ppm. A total of 26 robust known chemical markers (markedin Table 2) enabling the differentiation between one part withthe other two parts were identied and marked in S-plots.
Fig. 11 The effects of inflammatory cytokine IL-1b, IL-6 and TNF-a on rcells. ##p < 0.01, compared with control group; *p < 0.05, compared w
This journal is © The Royal Society of Chemistry 2020
According to the reference, it was revealed that there wassignicant variation for the contents of celastrol or total alkaloidsin different parts of COT. While in this study, there were 13, 8 and5 potential chemical markers including celastrol discovered fromroot, stem and leaf, respectively. The markers in root including 8triterpenoids (35, 39, 50, 51, 63, 80, 88, 90), 1 steroids (57), 1organic acid esters (87), 1 phenylpropanoids (103) and 2 othercompounds (73, 86). The markers in stem including 4 triterpe-noids (64, 84, 85, 100), 1 avonoids (2), 1 phenylpropanoids (30), 1steroids (52) and 1 organic acid esters (61). The markers in leafincluding 3 sesquiterpenoids (11, 40, 117), 1 avonoids (14) and 1triterpenoids (54). Additionally, among these potential chemicalmarkers, the contents of 57 and 88 in root, 52 in stem, 40, 54 and117 in leaf were much higher than in the other two parts (p <
oot, stem and leaf (20–160 mg mL�1) of COT in CSE-stimulated A549ith CSE group; **p < 0.01, compared with CSE group.
RSC Adv., 2020, 10, 8396–8420 | 8415
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
0.001). While components 35, 39, 50, 51, 63, 73, 80, 86, 87, 90 and103 were detected only in root, components 2, 30, 61, 64, 84, 85and 100 were detected only in stem, components 11 and 14 weredetected only in leaf part under the detect condition.
The detected result of 88 (celastrol) in our study, with muchhigher contents in root than in stem and leaf, was consistentwith the ref. 19. While there were a few differences between ourresults and the references. In the present study, compound 39(pristimerin) was only detected in root, and 11 (orbiculin I) wasonly detected in leaf. According to the reports, 39 was onceisolated from stem14 though mainly from root,98,99 and 11 iso-lated from root.3 The reason was the concentrations of themwere lower than the lowest detection limits. It was worthmentioning that some chemical markers with high responses inUPLC-MS, two triterpenoids (39 and 88) in root, one avonoids(2) in stem and two sesquiterpenoids (11 and 40) in leaf, couldbe used for further quality control of three parts of COTrespectively.
In order to systematically evaluate the chemical markers,a heat-map was generated. The hierarchical clustering heatmap, intuitively visualizing the difference level of potentialchemical markers in different parts, was shown in Fig. 8. Thehigher values were indicated by red squares, the lower valueswere indicated by green squares.
3.3. Bioactivity evaluation
3.3.1 Cytotoxicity of CSE and the three parts of COT on theviability of A549 cells. The results of MTT showed that theviability of A549 cells was obviously affected (p < 0.01) by 30% or40% CSE (Fig. 9). Therefore, 20% CSE was chosen as stimulus inthe following experiments. Additionally, as shown in Fig. 10, theviability of A549 cells were not signicantly affected by the Rbio,Sbio and Lbio solutions at 20–160 mg mL�1. So we evaluated theeffects of Rbio, Sbio and Lbio solutions at 20–160 mg mL�1 on CSE-stimulated A549 cells.
3.3.2 Effect of root, stem and leaf of COT on CSE-stimulated pro-inammatory cytokine levels in A549 cells.The inammatory development was characterized by the releaseof pro-inammatory mediators such as interleukin-1b (IL-1b),interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a).Whether the root, stem and leaf of COT could inhibit the releaseof IL-1b, IL-6 and TNF-a in CSE-stimulated lung epithelial cellswas investigated in this paper. As shown in Fig. 11, theproduction of IL-1b, IL-6 and TNF-a in A549 cells was obviouslyincreased aer treated with CSE (p < 0.01). However, treatedwith the Rbio, Sbio and Lbio solutions could evidently decreasethe levels of pro-inammatory factors in a good dose-dependentway with a certain range of 20–160 mg mL�1 in CSE-stimulatedcells. The Rbio solution could signicantly decrease the levelsof IL-1b, TNF-a (80 mg mL�1, p < 0.05; 160 mg mL�1, p < 0.01) andIL-6 (40 and 80 mg mL�1, p < 0.05; 160 mg mL�1, p < 0.01). TheSbio solution could signicantly decrease the levels of IL-1b,TNF-a (40 mg mL�1, p < 0.05; 80 and 160 mg mL�1, p < 0.01) andIL-6 (20 and 40 mg mL�1, p < 0.05; 80 and 160 mg mL�1, p < 0.01).The Lbio solution could signicantly decrease the levels of IL-6(80 mg mL�1, p < 0.05; 160 mg mL�1, p < 0.01) and TNF-a (80
8416 | RSC Adv., 2020, 10, 8396–8420
and 160 mgmL�1, p < 0.05), but showed no signicantly effect onIL-1b. The above results showed that the Sbio solution hada stronger anti-inammation effect than Rbio and Lbio. It issuggested that to explore the anti-COPD effect of the COT stemin vivo is meaningful in further research. The different activitiesof root, stem and leaf of COT might be caused by the variousphytochemicals in these three parts of COT. The phytochemicalstudy showed that three parts of COT were differentiated. Thebioassay study showed that three parts of COT could reduce thelevels of pro-inammatory factors to varying degrees. And thestem part had a stronger anti-COPD effect than root and leafparts. As we all know, the material basis of different pharma-cological activities is the different chemical composition. Theresults of the two parts of our study showed that the differentactivities of root, stem and leaf of COT might be caused by thevarious phytochemicals in these three parts of COT.
4. Conclusions
In conclusion, for screening analysis, a total of 120 compounds(15 shared components), including 92 from root, 56 from stemand 32 from leaf, were identied or tentatively characterizedfrom COT. Each part of COT was rich in various kinds ofstructures, especially triterpeniods held the majority. Formetabolomic analysis, the root, stem and leaf of COT weredifferentiated in both ESI+ and ESI� modes. There were 13, 8and 5 potential chemical markers identied from root, stemand leaf, respectively. Among the above robust markers, 5robust chemical markers with high responses in UPLC-MS, 2triterpenoids (pristimerin and celastrol) in root, 1 avonoids{5,7-dihydroxy-6,8-dimethyl-3(S)-3-(3-methoxy-40-hydrox-ybenzyl) chroman-4-one} in stem and 2 sesquiterpenoids(orbiculin I and orbiculin A) in leaf, could be used for furtherquality control in three parts of COT respectively. For bioassayanalysis, the root, stem and leaf of COT could evidently reducethe levels of pro-inammatory factors in a dose-dependent waywithin a certain range of 20–160 mg mL�1 in CSE-induced A549cells. The results showed that the stem part had a stronger anti-COPD effect than root and leaf parts. The different activitiesmight be caused by the various phytochemicals in these threeparts of COT. This comprehensive phytochemical studyrevealed both the structural diversity of secondary metabolitesand the different distributions in different parts of COT. Itcould provide a theoretical basis for further utilization anddevelopment of COT. And the identication of anti-COPDcomponents from COT will be explored deeply in the futurebased on the current results of this study.
Conflicts of interest
The authors declare no conicts of interests.
References
1 Y. Zhang, Y. H. Si, L. Zhai, S. D. Guo, J. L. Zhao, H. Sang,X. F. Pang, X. Zhang, A. B. Chen and S. C. Qin, Celastrusorbiculatus Thunb. reduces lipid accumulation by
This journal is © The Royal Society of Chemistry 2020
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
promoting reverse cholesterol transport in hyperlipidemicmice, Lipids, 2016, 51, 677–692.
2 M. R. Wang, Research progress of a Chinese herb Celastruson anti-tumor effect, J. Chin. Med., 2010, 6, 1055–1057.
3 H. Z. Jin, B. Y. Hwang, H. S. Kim, J. H. Lee, Y. H. Kim andJ. J. Lee, Antiinammatory constituents of Celastrusorbiculatus inhibit the NF-kappaB activation and NOproduction, J. Nat. Prod., 2002, 65, 89–91.
4 D. Q. Hou, N. Bai, Z. C Wei, B. Li, G. X. Tang and Y. F. Gao,Celastrus orbiculatus Celastraceae Thunb extracts inhibitproliferation and migration of oral squamous cellcarcinoma cells by blocking NF-kB pathway, Trop. J. Pharm.Res., 2019, 18, 1259–1264.
5 Z. Q. Liu, R. X. Zhou and M. Q. Wu, Study on antiviralcomponents of Celastrus orbiculatus Thunb., Acta Acad.Med. Jiangxi, 1983, 1–5.
6 X. G. Tao, Bacteriostatic effect of Celastrus root skin extractionuid on Staphylococcus aureus, Hunan Agric. Univ., 2012.
7 H. Y. Ni, Z. H. Zhang, W. J. Song and L. F. Guo, Chemicalconstituents of root bark of Celastrus orbiculatus Thunb.,Chin. Pharmaceut. J., 2014, 49, 21.
8 Y. Jiang, H. Li and S. Q. Luo, Study on the chemicalconstituents of Celastrus orbiculatus Thunb., Chin. Tradit.Herb. Drugs, 1996, 27, 73–89.
9 J. Wu, 1. Preparation and biological activity of cucurbitacinderivatives. 2. Study on the chemical constituents andbiological activities of the root bark of Celastrus orbiculatusThunb., Dissertation, University of Chinese Academy ofSciences, 2011.
10 Y. D. Zhu, L. Liu, L. Hu, W. Q. Dong, M. Zhang, Y. Q. Liu andP. Li, Effect of Celastrus orbiculatus in inhibitingHelicobacter pylori induced inammatory response byregulating epithelial mesenchymal transition and targetingmiR-21/PDCD4 signaling pathway in gastric epithelial cells,BMC Complementary Altern. Med., 2019, 19, 91.
11 H. B. Wang, L. D. Tao, T. Y. Ni, H. Gu, F. Jin, X. J. Dai, J. Feng,Y. B. Ding, W. M. Xiao, S. Y. Guo, T. D. S. Hisamitsud,Y. Y. Qian and Y. Q. Liu, Anticancer efficacy of the ethylacetate extract from the traditional Chinese medicine herbCelastrus orbiculatus against human gastric cancer, J.Ethnopharmacol., 2017, 205, 147–157.
12 Y. Zhang, Y. H. Si, L. Zhai, N. N. Yang, S. T. Yao, H. Sang,D. D. Zu, X. Xu, S. C. Qin and J. H. Wang, Celastrusorbiculatus Thunb. ameliorates high-fat diet-induced non-alcoholic fatty liver disease in guinea pigs, Pharmazie,2013, 68, 850–854.
13 B. Y. Hwang, H. S. Kim, J. H. Lee, Y. S. Hong, J. S. Ro, K. S. Leeand J. J. Lee, Antioxidant benzoylated avan-3-ol glycosidefrom Celastrus orbiculatus, J. Nat. Prod., 2001, 64, 82–84.
14 J. Li, Discovery of natural products with lipid-loweringactivity and their mechanism of action, Dissertation,University of Chinese Academy of Sciences, 2018.
15 J. J. Li, J. Yang, F. Lu, Y. T. Qi, Y. Q. Liu, Y. Sun and Q. Wang,Chemical constituents from the stems of Celastrusorbiculatus, Chin. J. Nat. Med., 2012, 10, 279–283.
16 X. Q. Chen, K. Zan and Q. Wang, Olean-type triterpenes ofCelastrus orbiculatus, Biochem. Syst. Ecol., 2012, 44, 338–340.
This journal is © The Royal Society of Chemistry 2020
17 Y. W. Liu, Efficacy test of crude ethanol extracts of Celastrusorbiculatus Thunb. and Ginkgo biloba leaves on Agrolimaxagrestis Linnaeus, J. Anhui Agric. Sci., 2011, 39, 19176–19185.
18 X. X. Yu, T. T. Zhang and D. Y. Wang, Chemical constituentsin hypoglycemic active fraction of Celastrus orbiculatus leaf,J. Chin. Med. Mater., 2014, 6, 998–1000.
19 Y. Zhang, Y. J. Cui, S. D. Guo, N. N. Yang, Y. H. Si andS. C. Qin, Using response surface methodology foroptimization of extraction process followed by capillaryzone electrophoresis for determination of celastrol inCelastrus orbiculatus Thunb, Chin. J. Hosp. Pharm., 2014,34, 704–709.
20 Y. H. Zhang, L. H. Yang and C. M. Li, Determination ofgeneral alkaloids from Celastrus orbiculatus Thunb. inGuilin area, Acad. J. Second Mil. Med. Univ., 2004, 25, 1012.
21 C. Z. Wang, N. Q. Zhang, Z. Z. Wang, Z. Qi, B. Z. Zheng,P. Y. Li and J. P. Liu, Rapid characterization of chemicalconstituents of Platycodon grandiorum and its adulterantAdenophora stricta by UPLC-QTOF-MS/MS, J. MassSpectrom., 2017, 52, 643–656.
22 H. Q. Lin, H. L. Zhu, J. Tan, C. Z. Wang, Q. H. Dong, F. L. Wu,H. Wang, J. L. Liu, P. Y. Li and J. P. Liu, Comprehensiveinvestigation on metabolites of wild-simulated americanGinseng root based on ultra-high performance liquidchromatography-quadrupole time-of-ight massspectrometry, J. Agric. Food Chem., 2019, 67, 5801–5819.
23 W. S. Tang, H. Y. Peh, W. P. Liao, C. H. Pang, T. K. Chan,S. H. Lau, V. T. Chow and W. S. Wong, Cigarette smoke-induced lung disease predisposes to more severe infectionwith nontypeable haemophilus inuenzae: protectiveeffects of andrographolide, J. Nat. Prod., 2016, 79, 1308–1315.
24 L. M. Fabbri, F. Luppi, B. Beghe and K. F. Rabe, Update inchronic obstructive pulmonary disease 2005, Am. J. Respir.Cell Mol. Biol., 2006, 175, 1056–1065.
25 X. Liang, J. Wang, R. J. Guan, L. Zhao, D. F. Li, Z. Long,Q. Yang, J. Y. Xu, Z. Y. Wang, J. K. Xie and W. J. Lu, Limaxextract ameliorates cigarette smoke-induced chronicobstructive pulmonary disease in mice, Int. J.Immunopharmacol., 2018, 54, 210–220.
26 H. Q. Lin, H. L. Zhu, J. Tan, H. Wang, Q. H. Dong, F. L. Wu,Y. H. Liu, P. Y. Li and J. P. Liu, Non-targeted metabolomicanalysis of methanolic extracts of wild-simulated and eld-grown American Ginseng, Molecules, 2019, 24, 1053.
27 Y. R. Wang, C. Z. Wang, H. Q. Lin, Y. H. Liu, Y. M. Li, Y. Zhao,P. Y. Li and J. P. Liu, Discovery of the potential biomarkersfor discrimination between Hedyotis diffusa and Hedyotiscorymbosa by UPLC-QTOF/MS metabolome analysis,Molecules, 2018, 23, 1525.
28 H. L. Zhu, H. Q. Lin, J. Tan, C. Z. Wang, H. Wang, F. L. Wu,Q. H. Dong, Y. H. Liu, P. Y. Li and J. P. Liu, UPLC-QTOF/MS-based nontargeted metabolomic analysis of mountain- andgarden-cultivated Ginseng of different ages in northeastChina, Molecules, 2019, 24, 33.
29 H. Q. Lin, H. L. Zhu, J. Tan, H. Wang, Z. Y. Wang, P. Y. Li,C. F. Zhao and J. P. Liu, Comparative analysis of chemicalconstituents of Moringa oleifera leaves from China and
RSC Adv., 2020, 10, 8396–8420 | 8417
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
India by ultra-performance liquid chromatography coupledwith quadrupole-time-of-ight mass spectrometry,Molecules, 2019, 24, 942.
30 C. Z. Wang, N. Q. Zhang, Z. Z. Wang, Z. Qi, H. L. Zhu,B. Z. Zheng, P. Y. Li and J. P. Liu, Nontargetedmetabolomic analysis of four different parts of Platycodongrandiorum grown in northeast China, Molecules, 2017,22, 1280.
31 Z. J. Zou, Z. H. Liu, M. J. Gong, B. Han, S. M. Wang andS. W. Liang, Intervention effects of puerarin on bloodstasis in rats revealed by a 1H-NMR-based metabonomicapproach, Phytomedicine, 2015, 22, 333–343.
32 L. Chen, Q. Ge, G. Tjin, H. Alkhouri, L. H. Deng,C. A. Brandsma, I. Adcock, W. Timens, D. Postma,J. K. Burgess, J. L. Black and B. G. G. Oliver, Effects ofcigarette smoke extract on human airway smooth musclecells in COPD, Eur. Respir. J., 2014, 44, 634–646.
33 R. S. Coleman, S. R. Gurrala, S. Mitra and A. Raao,Asymmetric total synthesis of dibenzocyclooctadienelignan natural products, J. Org. Chem., 2005, 70, 8932.
34 H. M. Hua, H. Q. Yin, B. Q. Li and Y. H. Pei, Study on theconstituents of the bark of Eucommia ulmoides, Mol. PlantBreed., 2003, 1, 801–803.
35 Z. G. Zheng, R. S. Wang, H. Q. Cheng, T. T. Duan, B. He,D. Tang, F. Gu and Q. Zhu, Isolated perfused lungextraction and HPLC-ESI-MS(n) analysis for predictingbioactive components of Saposhnikoviae Radix, J. Pharm.Biomed. Anal., 2011, 54, 614–618.
36 C. Masuoka, M. Ono, Y. Ito and T. Nohara, Antioxidative,antihyaluronidase and antityrosinase activities of someconstituents from the aerial part of Piper elongatum VAHL,Food Sci. Technol. Res., 2003, 9, 197–201.
37 S. Sato, J. Takeo, C. Aoyama and H. Kawahara, Na+-glucosecotransporter (SGLT) inhibitory avonoids from the rootsof Sophora avescens, Bioorg. Med. Chem., 2007, 15, 3445–3449.
38 Y. N. Lin, D. N. Zhu, J. Qi, M. J. Qin and B. Y. Yu,Characterization of homoisoavonoids in differentcultivation regions of Ophiopogon japonicus and relatedantioxidant activity, J. Pharm. Biomed. Anal., 2010, 52, 757–762.
39 S. Schwaiger, C. Seger, B. Wiesbauer, P. Schneider,E. P. Ellmerer, S. Sturm and H. Stuppner, Development ofan HPLC-PAD-MS assay for the identication andquantication of major phenolic edelweiss (Leontopodiumalpium Cass.) constituents, Phytochem. Anal., 2006, 17, 291–298.
40 M. J. Chang, T. M. Hung, B. S. Min, J. C. Kim, M. H. Woo,J. S. Choi, H. K. Lee and K. Bae, Lignans from the fruits ofForsythia suspensa (Thunb.) Vahl protect high-densitylipoprotein during oxidative stress, J. Agric. Chem. Soc. Jpn.,2008, 72, 2750–2755.
41 S. I. Falcao, M. Vilas-Boas, L. M. Estevinho, C. Barros,M. Domingues and S. M. Cardoso, Phenoliccharacterization of northeast Portuguese propolis: usualand unusual compounds, Anal. Bioanal. Chem., 2010, 396,887–897.
8418 | RSC Adv., 2020, 10, 8396–8420
42 V. Kumar, V. Karunaratne, M. R. Meegalle and K. Sanath, 1-[20,40-dihydroxy-30,50-di-(300-methylbut-200-enyl)-60-methoxy]phenylethanone from Acronychia pedunculata, root bark,Phytochemistry, 1989, 28, 1278–1279.
43 T. Matsumoto, K. Hosono-Nishiyama and H. Yamada,Antiproliferative and apoptotic effects of butyrolactonelignans from Arctium lappa on leukemic cells, Planta Med.,2005, 72, 276–278.
44 T. J. Schmidt, S. Hemmati, E. Fuss and A. W. Alfermann, Acombined HPLC-UV and HPLC-MS method for theidentication of lignans and its application to the lignansof Linum usitatissimum L. and L. bienne Mill, Phytochem.Anal., 2006, 17, 299–311.
45 S. Yin, C. Q. Fan, Y. Wang, L. Dong and J. M. Yue,Antibacterial prenylavone derivatives from Psoraleacorylifolia, and their structure-activity relationship study,Bioorg. Med. Chem., 2004, 12, 4387–4392.
46 H. L. Jiang, A. Somogyi, N. E. Jacobsen, B. N. Timmermannand D. R. Gang, Analysis of curcuminoids by positive andnegative electrospray ionization and tandem massspectrometry, Rapid Commun. Mass Spectrom., 2006, 20,1001–1012.
47 Q. H. Ye, W. M. Zhao and G. W. Qin, Lignans fromDendrobium chrysanthum, J. Asian Nat. Prod. Res., 2004, 6,39–43.
48 X. Y. Guan, H. F. Li, W. Z. Yang, C. H. Lin, C. Sun, B. R. Wang,D. A. Guo and M. Ye, HPLC-DAD-MSn analysis and HPLCquantitation of chemical constituents in Xian-ling-gu-baocapsules, J. Pharm. Biomed. Anal., 2011, 55, 923.
49 H. C. Beatriz, C. P. Miriam, V. C. Claudia, L. O. Jesus F,A. G. Agustin and J. N. Pedro, New saponins from Sechiummexicanum, Magn. Reson. Chem., 2009, 11, 994–1003.
50 S. J. Xu, L. Yang, X. Zeng, M. Zhang and Z. T. Wang,Characterization of compounds in the Chinese herbal drugMu-Dan-Pi by liquid chromatography coupled toelectrospray ionization mass spectrometry, Rapid Commun.Mass Spectrom., 2010, 20, 3275–3288.
51 Y. Y. Zhao, X. L. Cheng, Y. M. Zhang, Y. Zhao, R. C. Lin andW. J. Sun, Simultaneous determination of eight majorsteroids from Polyporus umbellatus by high-performanceliquid chromatography coupled with mass spectrometrydetections, Biomed. Chromatogr., 2010, 24, 222–230.
52 C. Niampoka, R. Suttisri, R. Bavovada, H. Takayama andN. Aimi, Potentially cytotoxic triterpenoids from the rootbark of Siphonodon celastrineus Griff, Arch. Pharmacal Res.,2005, 28, 546–549.
53 L. Y. Wang, N. L. Wang, X. S. Yao, S. Miyata and S. Kitanaka,Euphane and tirucallane triterpenes from the roots ofEuphorbia kansui and their in vitro effects on the celldivision of Xenopus, J. Nat. Prod., 2003, 66, 630–633.
54 X. Li, S. H. Li, J. X. Pu, S. X. Huang and H. D. Sun, Chemicalconstituents from Paeonia anomala subsp.veitchii(Paeoniaceae), Acta Bot. Yunnanica, 2007, 29, 259–262.
55 C. F. Hossain, Y. P. Kim, S. R. Baerson, L. Zhang,R. K. Bruick, K. A. Mohammed, A. K. Agarwal, D. G. Nagleand Y. D Zhou, Saururus cernuus lignans-potent small
This journal is © The Royal Society of Chemistry 2020
Paper RSC Advances
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
molecule inhibitors of hypoxia-inducible factor-1, Biochem.Biophys. Res. Commun., 2005, 333, 1026–1033.
56 F. M. Darwish and M. G. Reinecke, Ecdysteroids and otherconstituents from Sida spinosa L., Phytochemistry, 2003, 62,1179–1184.
57 N. Sanchez-Avila, F. Priego-Capote, J. Ruiz-Jimenez andM. D. Luque de Castro, Fast and selective determination oftriterpenic compounds in olive leaves by liquidchromatography-tandem mass spectrometry with multiplereaction monitoring aer microwave-assisted extraction,Talanta, 2009, 78, 0–48.
58 M. A. Alamsjah, S. Hirao, F. Ishibashi and Y. Fujita, Isolationand structure determination of algicidal compounds fromUlva fasciata, J. Agric. Chem. Soc. Jpn., 2005, 69, 2186–2192.
59 T. Sugiura, S. Nakane, S. Kishimoto, K. Waku, Y. Yoshiokaand A. Tokumura, Lysophosphatidic acid, a growth factor-like lipid, in the saliva, J. Lipid Res., 2002, 43, 2049–2055.
60 C. R. Cheng, M. Yang, Z. Y. Wu, Y. Wang, F. Zeng, W. Y. Wu,S. H. Guan and D. A. Guo, Fragmentation pathways ofoxygenated tetracyclic triterpenoids and their applicationin the qualitative analysis of Ganoderma lucidum bymultistage tandem mass spectrometry, Rapid Commun.Mass Spectrom., 2011, 25, 1323–1335.
61 R. Bortolomeazzi, G. Verardo, A. Liessi and A. Callea,Formation of dehydrodiisoeugenol and dehydrodieugenolfrom the reaction of isoeugenol and eugenol with DPPHradical and their role in the radical scavenging activity,Food Chem., 2009, 118, 256–265.
62 M. B. Gallo, W. C. Rocha, U. S. da Cunha, F. A. Diogo, F. C. daSilva, P. C. Vieira, J. D. Vendramim, J. B. Fernandes, M. F. daSilva and L. G. Batista-Pereira, Bioactivity of extracts andisolated compounds from Vitex polygama (Verbenaceae)and Siphoneugena densiora (Myrtaceae) against Spodopterafrugiperda (Lepidoptera: Noctuidae), Pest Manage. Sci.,2006, 62, 1072–1081.
63 K. Nagy, C. Courtet, C. Marie, B. Holst and M. Kussmann,Comprehensive analysis of vitamin E constituents inhuman plasma by liquid chromatography-massspectrometry, Anal. Chem., 2007, 79, 7087–7096.
64 L. Y. Wang, N. L. Wang, X. S. Yao, S. Miyata and S. Kitanaka,Euphane and Tirucallane Triterpenes from the Roots ofEuphorbia kansui and their in vitro Effects on the CellDivision of Xenopus, J. Nat. Prod., 2003, 66, 630–633.
65 H. Simone and V. Walter, Exploring the fatty acids of vernixcaseosa in form of their methyl esters by off-line coupling ofnon-aqueous reversed phase high performance liquidchromatography and gas chromatography coupled to massspectrometry, J. Chromatogr. A, 2010, 52, 8270–8278.
66 C. Pettinella, S. H. Lee, F. Cipollone and I. A. Blair, Targetedquantitative analysis of fatty acids in atherosclerotic plaquesby high sensitivity liquid chromatography/tandem massspectrometry, J. Chromatogr. B: Anal. Technol. Biomed. LifeSci., 2007, 850, 168–176.
67 Y. L. Lin and Y. H. Kuo, Four new neoclerodane-typediterpenoids, scutellones B, G, H, and I, from aerial partsof Scutellaria rivularis, Chem. Pharm. Bull., 1989, 37, 582–585.
This journal is © The Royal Society of Chemistry 2020
68 X. A. Wen, J. Liu, L. Y. Zhang, P. Z. Ni and H. B. Sun,Synthesis and biological evaluation of arjunolic Acid,bayogenin, hederagonic acid and 4-epi-hederagonic acid asglycogen phosphorylase inhibitors, Chin. J. Nat. Med.,2010, 8, 441–448.
69 R. Tundis, B. Deguin, F. Menichini and F. Tillequin, Iridoidsfrom Putoria calabrica, Biochem. Syst. Ecol., 2002, 30, 689–691.
70 X. Li, P. Gao, B. Gjetvaj, N. Westcott and M. Y. Gruber,Analysis of the metabolome and transcriptome of Brassicacarinata seedlings aer lithium chloride exposure, PlantSci., 2009, 177, 68–80.
71 K. Kuroda and A. Nakagawaizumi, Analytical pyrolysis oflignin: products stemming from b-5 substructures, Org.Geochem., 2006, 37, 665–673.
72 S. E. Ayyad, S. Z. Sowellim, M. S. El-Hosini and A. Abo-Atia,The structural determination of a new steroidal metabolitefrom the brown alga Sargassum asperifolium, Z.Naturforsch., C: J. Biosci., 2003, 58, 333–336.
73 H. W. Seo, T. M. Hung, M. K. Na, H. J. Jung, J. C. Kim,J. S. Choi, J. H. Kim, H. K. Lee, I. S. Lee, K. H. Bae,M. S. Hattori and B. S. Min, Steroids and triterpenes fromthe fruit bodies of Ganoderma lucidum and their anti-complement activity, Arch. Pharmacal Res., 2009, 32, 1573–1579.
74 T. H. Lee, J. L. Chiou, C. K. Lee and Y. H. Kuo, Separation anddetermination of chemical constituents in the roots of RhusJavanica L. Var. Roxburghiana, J. Chin. Chem. Soc., 2005, 52,833–841.
75 K. Iwatsuki, T. Akihisa, H. Tokuda, M. Ukiya, M. Oshikubo,Y. Kimura, T. Asano, A. Nomura and H. Nishino, Lucidenicacids P and Q, methyl lucidenate P, and othertriterpenoids from the fungus Ganoderma lucidum andtheir inhibitory effects on Epstein-Barr virus activation, J.Nat. Prod., 2003, 66, 1582.
76 C. A. Luo, W. N. Zhang, C. Q. Sheng, C. J. Zheng, J. Z. Yao andZ. Y. Miao, Chemical composition and antidiabetic activityof Opuntia Milpa Alta extracts, Chem. Biodiversity, 2010, 7,2869–2879.
77 B. S. Siddiqui, U. Ghani, S. T. Ali, S. B. Usmani and S. Begum,Triterpenoidal constituents of the leaves of Carissa carandas,Nat. Prod. Lett., 2003, 17, 153.
78 L. Liu, Y. Cheng and H. Zhang, Phytochemical Analysis ofAnti-atherogenic Constituents of Xue-Fu-Zhu-Yu-Tangusing HPLC-DAD-ESI-MS, Chem. Pharm. Bull., 2004, 52,1295–1301.
79 D. Q. Luo, H. Wang, X. Tian, H. J. Shao and J. K. Liu,Antifungal properties of pristimerin and celastrol isolatedfrom Celastrus hypoleucus, Pest Manage. Sci., 2005, 61, 85–90.
80 M. Skoumal, R. M. Rodriguez, P. L. Cabot, F. Centellas,J. A. Garrido, C. Arias and E. Brillas, Electro-Fenton, UVAphotoelectro-Fenton and solar photoelectro-Fentondegradation of the drug ibuprofen in acid aqueousmedium using platinum and boron-doped diamondanodes, Electrochim. Acta, 2009, 54, 2077–2085.
RSC Adv., 2020, 10, 8396–8420 | 8419
RSC Advances Paper
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 2
6 Fe
brua
ry 2
020.
Dow
nloa
ded
on 4
/29/
2022
10:
51:1
3 A
M.
Thi
s ar
ticle
is li
cens
ed u
nder
a C
reat
ive
Com
mon
s A
ttrib
utio
n 3.
0 U
npor
ted
Lic
ence
.View Article Online
81 J. Yang and K. L. Zhou, NMR spectroscopic analysis ofneferine and isoliensinine, Magn. Reson. Chem., 2004, 42,994–997.
82 C. H. Li, P. Y. Chen, U. M. Chang, L. S. Kan, W. H. Fang,K. S. Tsai and S. B. Lin, Ganoderic acid X, a lanostanoidtriterpene, inhibits topoisomerases and induces apoptosisof cancer cells, Life Sci., 2005, 77, 0–265.
83 M. Yoshikawa, T. Morikawa, H. Oominami and H. Matsuda,Absolute Stereostructures of olibanumols A, B, C, H, I, and Jfrom olibanum, gum-resin of Boswellia carterii, andinhibitors of nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages, Cheminform,2009, 57, 957.
84 E. H. Joh and D. H. Kim, A sensitive liquid chromatography-electrospray tandem mass spectrometric method forlancemaside A and its metabolites in plasma anda pharmacokinetic study in mice, J. Chromatogr. B: Anal.Technol. Biomed. Life Sci., 2010, 878, 1875–1880.
85 G. Alam, I. G. Gandjar, L. Hakim, H. Timmerman,R. Verpoorte and S. Wahyuono, Tracheospasmolyticactivity of vitetrifolin-E isolated from the leaves of Vitextrifolia L, Maj. Farm. Indones., 2003, 14, 188–194.
86 M. Meier, B. Kohlenberg and N. Braun, Isolation of anisylacetone from Agarwood Oil, J. Essent. Oil Res., 2003, 15,54–56.
87 L. N. Tao, Q. Y. Meng, Y. Jian, R. Xing and H. R. Guo, A newpanaxadiol from the acid hydrolysate of Panax ginseng,Chin. Chem. Lett., 2009, 20, 687–689.
88 K. Wang, H. Sun, B. Wu and Y. Pan, Two Novel Oleantriterpenoids from Celastrus hypoleucus, Helv. Chim. Acta,2010, 88, 990–995.
89 G. Li, C. S. Lee, M. H. Woo, S. H. Lee, H. W. Chang andJ. K. Son, Lignans from the bark of Machilus thunbergii andtheir DNA topoisomerases I and II inhibition andcytotoxicity, Biol. Pharm. Bull., 2004, 27, 1147–1150.
90 L. P. Ponomarenko, I. V. Stonik, N. A. Aizdaicher,T. Y. Orlova, G. I. Popovskaya, G. V. Pomazkina and
8420 | RSC Adv., 2020, 10, 8396–8420
V. A. Stonik, Sterols of marine microalgae Pyramimonascf.cordata (Prasinophyta), Attheya ussurensis sp. nov.(Bacillariophyta) and a spring diatom bloom from LakeBaikal, Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol.,2004, 138, 65–70.
91 J. Wandji, F. Tillequin, D. A. Mulholland, J. C. Shirri,N. Tsabang, E. Seguin, P. Verite, F. Libot and Z. T. Fomum,Pentacyclic triterpenoid and saponins from Gambeyaboukokoensis, Phytochemistry, 2003, 64, 845–849.
92 F. Wu, K. Koike, T. Nikaido, K. Ishii, T. Ohmoto and K. Ikeda,Terpenoids and avonoids from Arenaria kansuensis, Chem.Pharm. Bull., 1990, 38, 2281–2282.
93 C. R. Chen, L. H. Chao, M. H. Pan, Y. W. Liao andC. I. Chang, Tocopherols and Triterpenoids from Sidaacuta, J. Chin. Chem. Soc., 2007, 54, 41–45.
94 C. M. Sandison, R. Alexander, R. I. Kagi and C. J. Boreham,Early diagenetic transformation of organic matter ina marine-inuenced lignite, Org. Geochem., 2003, 34, 1081–1102.
95 C. Johannes and R. L. Lorenz, Preparation and massspectrometry of 14 pure and 18O2-labeled oxidationproducts from the phytosterols b-sitosterol andstigmasterol, Anal. Biochem., 2004, 325, 107–116.
96 M. X. Chen, D. Y. Wang and J. Guo, ChemInform abstract: 3-Oxo-11b-hydroxyfriedelane from the roots of Celastrusmonospermus, Cheminform, 2010, 34, 114–117.
97 X. W. Chang, B. R. Wang, T. Wang, D. K. Li, Y. Zhao,Y. L. Zhang, D. Z. Zhou and Z. L. Ye, Plant metabolomicsapproach for age discrimination of mountain cultivatedginseng using UPLC-Q-TOF/MS, China J. Chin. Mater. Med.,2016, 41, 3609–3614.
98 J. Wu, Y. Zhou, L. Y. Wang, J. P. Zuo and W. M. Zhao,Terpenoids from root bark of Celastrus orbiculatus,Phytochemistry, 2012, 7, 159–168.
99 L. Zhang, Z. J. Xu, Y. J. Feng and D. Y. Wang, Chemicalconstituents of roots of Celastrus orbiculatus, J. Chin. Med.Mater., 2013, 36, 569.
This journal is © The Royal Society of Chemistry 2020