New dammarane-type saponins from gynostemma pentaphyllum

Article abstract of DOI:10.3390/molecules24071375

Six new dammarane-type saponins, gypenosides CP1-6 (1 6), along with 19 known compounds 7 25, were isolated and characterized from the aerial parts of Gynostemma pentaphyllum. Among these compounds, eight dammarane-type saponins, 2, 5, 6, 7, 11, 12, 13, and 15, exhibited the greatest antiproliferative effects against two human tumor cell lines (A549 and HepG2).

Full text of DOI:10.3390/molecules24071375

Article
New Dammarane-type Saponins from Gynostemma
pentaphyllum
Po-Yen Chen 1,2, , Chih-Chao Chang 1, , Hui-Chi Huang 3, , Li-Jie Zhang , Chia-Ching Liaw ,
†
†
†
1
4
Yu-Chi Lin , Nham-Linh Nguyen 1,5, Thanh-Hoa Vo 1,5, Yung-Yi Cheng 6,7
1
,
Susan L. Morris-Natschke , Kuo-Hsiung Lee 6,7,* and Yao-Haur Kuo 1,5,8,
6
*
1
Division of Chinese Materia Medica Development, National Research Institute of Chinese Medicine, Taipei
12, Taiwan; peik35@gmail.com (P.-Y.C.); changhcym@gmail.com (C.-C.C.);
1
lijizhang@hotmail.com (L.-J.Z.); m8952612@hotmail.com (Y.-C.L.)
2
3
4
5
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112,
Taiwan
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical
University, Taichung 404, Taiwan; hchuang@mail.cmu.edu.tw
Department of Research and Development, Starsci Biotech Co. Ltd., Taipei 112, Taiwan;
biogodas@hotmail.com
The Ph.D. program in Clinical Drug Development of Chinese Herbal Medicine, College of Pharmacy,
Taipei Medical University, Taipei 11031, Taiwan; nguyenlinhnham4201@gmail.com (N.-L.N.);
hoavo0808@gmail.com (T.H.V.)
6
Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599-7568, USA; yungyi@email.unc.edu (Y.Y.C.); susan_natschke@unc.edu (S.L.M.-N.);
khlee@unc.edu (K.-H.L.)
7
8
Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung
4
0402, Taiwan
Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University,
Taichung 404, Taiwan
*
Correspondence: yhlee@unc.edu (K.-H.L.); kuoyh@nricm.edu.tw (Y.-H.K.);
Tel.: +919-962-0066 (K.-H.L.); +886-2-2820-1999 (ext. 7061) (Y.-H.K.)
These authors contributed equally to this work.
†
Academic Editor: Francesco Epifano
Received: 23 February 2019; Accepted: 5 April 2019; Published: 8 April 2019
Abstract: Six new dammarane-type saponins, gypenosides CP1-6 (16), along with 19 known
compounds 7–25, were isolated and characterized from the aerial parts of Gynostemma pentaphyllum.
Among these compounds, eight dammarane-type saponins, 2, 5, 6, 7, 11, 12, 13, and 15, exhibited
the greatest antiproliferative effects against two human tumor cell lines (A549 and HepG2).
Keywords: Gynostemma pentaphyllum; Jiaogulan; dammarane-type saponins; gypenosides
antiproliferative activity
1
. Introduction
Gynostemma pentaphyllum (Thunb.) Makino (family Cucurbitaceae) is an ethnomedicine
frequently used in Asian countries as a functional food and tea [1–4]. Due to its various
pharmacological activities, including anti-inflammatory [5–7], antioxidative [8,9], anti-
hyperlipidemic [3,10], hypoglycemic [3,11,12], and antitumor effects [13–18], it is marketed in Asia in
dietary supplements, such as Jiaogulan tea and Jiaogulan concentrated juice [4,12,19]. Dammarane-
type triterpene saponins or gypenosides are the major components responsible for the plant’s
pharmacological activities [1–7,11,15,17,20–25]. To date, more than 210 compounds, including over
Molecules 2019, 24, 1375; doi:10.3390/molecules24071375
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1375
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180 gypenosides, along with flavonoids [9,15,17] and polysaccharides [8,16] have been isolated from
G. pentaphyllum. Moreover, gypenosides are structurally like the ginsenosides, which are well-known
pharmacologically active components of ginseng root (Panax ginseng) [26]. Thus, G. pentaphyllum is a
unique non-Panax plant rich in gypenosides [26,27].
In our present study, 25 components were isolated from an ethanol extract of the aerial parts of
G. pentaphyllum. Their structures were identified from NMR, IR and HRMS spectroscopic data.
Among them, six new gypenosides CP1-6 (1–6) (Figure 1) and 10 known dammarane-type saponins
7–16, seven known flavonoid glycosides 17–23, and two known sesquiterpene glycosides 24 and 25,
were obtained from the title plant. All isolated compounds were evaluated for antiproliferative
activities against human lung cancer (A549) and hepatoma (HepG2) cell lines.
Figure 1. Chemical structures of compounds 1–6 isolated from G. pentaphyllum.
2. Results and Discussion
Gypenoside CP1 (1), [α]26
D
+11.5 (c 0.2, MeOH), has the molecular formula C55
H92O24, as
+
established by NMR and HRESIMS (m/z 1159.5874 [M + Na] , calcd for C55
H92O24Na 1159.5876),
indicating ten degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl,
−
1
1
carbonyl, and olefinic groups at 3358, 1736, and 1638 cm , respectively. In the H-NMR spectrum
Tables 1 and 2), signals were observed for nine tertiary methyl groups [δ 0.86, 0.92, 0.97, 1.00, 1.10,
.36, 1.62, 1.68, and 2.04 (each 3H, s)] and an olefinic proton [δ 5.13 (1H, bt, J = 7.0 Hz)]. The C-NMR
Tables 1 and 2) and DEPT spectra showed resonances for 55 carbons, among which 30 were aglycone
carbons including 8 methyls [δ 16.3 (C-18), 17.4 (C-30), 17.7 (C-29), 17.8 (C-19), 18.0 (C-27), 22.4 (C-
1), 25.9 (C-26), 28.6 (C-28)], 4 oxygenated carbons [δ 68.2 (C-2), 71.5 (C-12), 84.9 (C-20), 96.5 (C-3)],
(
H
1
3
1
H
(
C
2
C
1
1
and a pair of olefinic carbons [δ 126.1 (C-24), 132.2 (C-25)]. The H- H COSY and HMBC correlations
C
fully established the planar structure of 1 (Figure 2). Furthermore, oxygenations at C-2, C-3, and C-
1
1
1
2
1
2 were corroborated by H- H COSY correlations between H
.95) and H-9 (δ 1.48)/H -11 (δ 1.28; 1.82)/H-12 (δ 3.74)/H-13 (δ
.89)/H -15 (δ 1.03; 1.57) as well as the HMBC long-range correlation of H-3 with carbon signals at
41.8 (C-4) and 57.2 (C-5). The molecular formula and 1D and 2D-NMR spectroscopic data of 1
suggested a dammarane-type saponin, a typical constituent of Gynostemma species, with the same
2
-1 (δ
H
2.09; 0.87)/H-2 (δ
H
3.72)/H-3 (δ
H
H
2
H
H
H
1.73)/H-17 (δ
H
2.28) /H
2
-16 (δ 1.33;
H
2
H
δ
C

Molecules 2019, 24, 1375
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aglycone as that of 2α,3β,12β,20(S)-tetrahydroxydammar-24-ene [28,29]. The aglycone accounted for
0 carbon signals, leaving 25 carbon signals assignable to four sugar moieties and one acetyl group
3
1
3
[
δ
C
20.9 (CH
.30 (d, J = 7.5 Hz)/δ
105.0. HMBC cross peaks of H-1′ to C-3, H-1′′ to C-2′, H-1′′′ to C-20, and H-1′′′′ to C-
′′′ determined the positions of the four sugars. Acid hydrolysis of 1 yielded -glucose (Glc) and -
D
3
) and 172.8 (C=O)] in the C NMR spectrum. Four anomeric signals were observed at δ
H
4
C
105.5, δ 4.42 (d, J = 7.5 Hz)/δ 104.7, δ 4.56 (d, J = 8.0 Hz)/δ 98.1, and δ 4.71
H
C
H
C
H
(
d, J = 7.5 Hz)/δ
C
6
D
xylose (Xyl) in a ratio of 3:1 based on HPLC analysis of the component monosaccharides compared
with the standard sugars [30]. A long-range correlation between proton and carbon signals at δ 4.15,
.31 (H-6′′) and δ 172.8 (C=O), respectively, was consistent with acetylation of the 6′′-OH. In the
NOESY spectrum of 1 (Figure 2), cross peaks were found between H-2 (δ 3.72)/Me-29 (δ 1.10), Me-
9 (δ 1.10)/Me-19 (δ 0.97), Me-19 (δ 0.97)/Me-18 (δ 1.00), and Me-18 (δ 1.00)/H-13 (δ 1.73),
indicating β-orientations of Me-29, Me-19, Me-18, and H-13. However, the NOESY correlations of H-
H
4
C
H
H
2
H
H
H
H
H
H
3
(δ
H
2.95)/Me-28 (δ
H
0.86), H-3 (δ
H
2.95)/H-5 (δ
H
0.84), H-5 (δ
H
0.84)/H-9 (δ
H
1.48), H-9 (δ
H
1.48)/Me-
30 (δ
H
0.92), and Me-30 (δ
H
0.92)/H-17 (δ
H
2.28) suggested α-orientations of H-5, H-9, H-17, Me-28,
and Me-30. The configuration of C-20 in 1 was determined to be S based on a comparison of the 13C-
NMR spectroscopic data of 1 and gypenoside XLVI [29]. The complete structure of 1 (gypenoside
CP1) was elucidated as 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-[(6-O-acetyl-β-
D
-
glucopyranosyl)-(1→2)-β- -glucopyranosyl]-20-O-[β- -xylopyranosyl-(1→6)-β- -glucopyranoside].
D
D
D
Figure 2. Key COSY, HMBC, and NOESY correlations of 1.

Molecules 2019, 24, 1375
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Table 1. H- and 13C-NMR spectroscopic data of the aglycone of gypenosides CP1-6 (1–6).
1
1
2
(mult, J in Hz)
2.07 (m)
3
4
(mult, J in Hz)
1.70 (m)
5
6
No.
δ
H
(mult, J in Hz)
.09 (dd, 5.0, 13.0)
.87 (m)
δ
C
δH
δ
C
δH
(mult, J in Hz)
δ
C
δH
δ
C
δH
(mult, J in Hz)
δ
C
δH
(mult, J in Hz)
δC
2
2.07 (dd, 4.8, 12.6)
0.88 (m)
2.03 (dd, 4.2, 12.6)
0.87 (m)
2.03 (dd, 4.2, 12.6)
0.87 (m)
47.7
1
47.8
47.8
47.8
40.2
47.7
0
0.91 (t, 8.0)
3.70 (m)
0.98 (m)
2
3
4
5
6
7
8
9
3.72 (m)
2.95 (d, 9.0)
-
68.2
96.5
41.8
57.2
19.3
35.7
41.0
51.0
38.8
31.0
71.5
49.7
52.4
31.5
27.3
52.9
16.3
17.8
84.9
22.4
36.8
23.8
126.1
132.2
25.9
18.0
28.6
17.7
17.4
68.2
96.5
41.8
57.2
19.4
35.7
41.0
51.0
38.8
31.1
71.8
49.8
52.5
31.6
27.2
53.1
16.2
17.8
84.9
22.8
36.7
24.2
125.9
132.3
25.9
17.9
28.5
17.8
17.2
3.69 (m)
68.2
96.5
41.8
57.2
19.4
35.7
41.0
51.0
38.8
31.0
71.5
49.8
52.4
31.5
27.3
52.9
16.3
17.8
84.9
22.4
36.7
23.8
126.1
132.2
25.9
18.0
28.5
17.79
17.3
1.68 (m); 1.95 (m)
3.11 (dd, 3.0, 9.0)
27.3
91.0
40.5
57.6
19.3
35.9
41.0
51.1
37.9
30.8
71.7
49.7
52.4
31.5
27.3
52.9
16.3
16.8
85.0
22.4
36.7
23.8
126.1
132.2
25.9
18.0
28.4
16.7
17.4
3.70 (m)
68.2
96.6
41.8
57.2
19.4
35.6
40.9
50.9
38.8
31.1
71.8
49.7
52.5
31.6
27.2
53.1
16.2
17.9
84.9
22.9
36.7
24.3
125.9
132.3
25.9
18.0
28.6
17.9
17.1
3.70 (m)
68.2
96.6
41.8
57.2
19.4
35.6
40.9
51.0
38.7
30.9
71.5
49.7
52.4
31.5
27.2
52.9
16.2
17.9
84.9
22.4
36.8
24.1
126.1
132.2
25.9
18.0
28.6
17.9
17.3
2.93 (d, 9.5)
-
2.93(d, 9.6)
2.92 (d, 9.6)
-
2.92 (d, 9.6)
-
0.84 (d, 11.5)
0.84 (d, 10.5)
0.82 (m)
1.46 (m); 1.53 (m)
1.57 (m); 1.28 (m)
-
0.74 (d, 11.4)
1.43 (m); 1.53 (m)
1.55 (m); 1.26 (m)
-
0.79 (d, 11.4)
1.37 (m); 1.50 (m)
1.47 (m); 1.19 (m)
0.79 (d, 11.4)
1.37 (m); 1.50 (m)
1.47 (m); 1.19 (m)
-
1.49 (m); 1.56 (m)
1.57 (m); 1.29, (m)
-
1.46 (m); 1.54 (m)
1.55 (m); 1.30 (m)
-
1.48 (m)
1.50 (m)
1.47 (m)
1.42 (dd, 3.0, 13.2)
1.45 (m)
-
1.45 (m)
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
-
-
-
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1.82 (m);1.28 (m)
3.74 (m)
1.86 (m); 1.28 (m)
3.67 (m)
1.82 (m); 1.28 (m)
3.73 (m)
1.78 (m); 1.22 (m)
3.71 (m)
1.81 (m); 1.22 (m)
3.66 (m)
1.70 (t, 10.8)
-
1.81 (m); 1.22, (m)
3.66 (m)
1.73 (d, 11.5)
-
1.74 (d, 10.5)
1.73 (d, 10.8)
-
1.72 (t, 10.8)
-
1.70 (t, 10.8)
-
1.57 (m); 1.03 (m)
1.89 (m); 1.33 (m)
2.28 (m)
1.58 (m); 1.06 (m)
1.92 (m); 1.38 (m)
2.27 (m)
1.00 (s)
1.58 (m); 1.04 (m)
1.89 (m); 1.33 (m)
2.28 (m)
1.57 (m); 1.03 (m)
1.95 (m), 1.32 (m)
2.28 (m)
1.55 (m); 1.03 (m)
1.91 (m); 1.38 (m)
2.28 (m)
0.89 (s)
1.55 (m); 1.03 (m)
1.91 (m); 1.38 (m)
2.28 (m)
1.00 (s)
0.99 (s)
0.99 (s)
0.89 (s)
0.97 (s)
0.94 (s)
0.93 (s)
0.88 (s)
0.84 (s)
0.84 (s)
-
-
-
-
-
-
1.36 (s)
1.34 (s)
1.35 (s)
1.35 (s)
1.33 (s)
1.33 (s)
1.79 (m); 1.53(m)
2.02 (m); 2.15 (m)
5.13 (bt, 7.0)
-
1.80 (m); 1.60 (m)
2.00 (m)
5.10 (brt, 7.0)
-
1.79 (m); 1.52 (m)
2.05 (m); 2.16(m)
5.11 (m)
1.80 (m); 1.52 (m)
2.05 (m); 2.14 (m)
5.12 (m)
1.80 (m); 1.61 (m)
2.06 (m)
5.11 (bt, 7.2)
-
1.80 (m); 1.61 (m)
2.06 (m)
5.11 (bt, 7.2)
-
-
1.68 (s)
1.68 (s)
1.68 (s)
1.62 (s)
0.82 (s)
1.07 (s)
0.91 (s)
1.68 (s)
1.69 (s)
1.69 (s)
1.62 (s)
1.62 (s)
1.62 (s)
1.62 (s)
1.62 (s)
0.86 (s)
0.82 (s)
0.77 (s)
0.83 (s)
0.83 (s)
1.10 (s)
1.08 (s)
1.02 (s)
1.07 (s)
1.07 (s)
0.92 (s)
0.92 (s)
0.91 (s)
0.89 (s)
0.89 (s)
Table 2. H- and 13C-NMR spectroscopic data of the sugar moieties of gypenosides CP1-6 (1–6).
1
No.
1
2
3
4
5
6

Molecules 2019, 24, 1375
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δ
H
δ
C
δH
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δH
δC
1
2
3
4
5
6
′
′
′
′
′
′
4.42 (d, 7.5)
3.56 (m)
104.7
82.4
78.7
70.9
78.0
62.3
4.42 (d, 7.5)
3.55 (m)
104.6
82.7
78.7
70.9
77.9
62.3
4.41 (d, 7.8)
3.56 (m)
104.6
82.7
78.7
70.9
78.0
62.3
4.40 (d, 7.8)
3.45 (m)
105.2
83.4
78.6
71.2
77.5
62.7
4.42 (d, 7.8)
3.54 (dd, 7.8, 9.0)
3.59 (m)
104.6
83.2
78.8
70.9
77.9
62.3
4.41 (d, 7.8)
3.55 (dd, 7.8, 9.0)
3.58 (m)
104.6
83.2
78.8
70.9
78.0
62.3
3.58 (m)
3.58 (m)
3.58 (m)
3.54 (t, 9.0)
3.37 (m)
3.36 (m)
3.36 (m)
3.32 (m)
3.36 (m)
3.36 (m)
3.34 (m)
3.19 (m)
3.34 (m)
3.23 (m)
3.20 (m)
3.35 (m)
3.85 (dd, 5.0, 12.0)
3.85 (dd, 5.0, 12.0)
3.66 (dd, 5.0, 12.0)
4.71 (d, 7.5)
3.24 (dd, 7.5, 9.0)
3.35 (m)
3.86 (dd, 1.8, 12.0)
3.66 (dd, 5.4, 12.0)
4.70 (d, 7.8)
3.24 (t, 8.4)
3.35 (m)
3.83 (dd, 1.8, 12.0)
3.65 (dd, 5.4, 12.0)
4.62 (d, 7.8)
3.23 (t, 8.4)
3.86 (dd, 1.8, 12.0)
3.66 (m)
3.86 (dd, 1.8, 12.0)
3.65 (dd, 5.4, 12.0)
4.71 (d, 7.8)
3.23 (t, 8.4)
3
.66 (dd, 5.0, 12.0)
4.71 (d, 7.5)
3.23 (m)
1
2
3
4
5
6
′′
′′
′′
′′
′′
′′
105.0
76.1
77.9
71.4
75.3
65.0
105.2
76.1
77.9
71.4
75.3
64.7
105.2
76.1
77.9
71.4
75.3
64.7
105.5
76.4
77.7
71.3
75.5
64.7
4.71 (d, 7.8)
3.24 (t, 8.4)
105.4
76.1
78.0
71.4
75.2
65.2
105.4
76.1
78.1
71.3
75.3
65.1
3.35 (m)
3.35 (m)
3.35 (m)
3.34 (m)
3.29 (m)
3.32 (m)
3.32 (m)
3.32 (m)
3.33 (m)
3.33 (m)
3.43 (m)
3.45 (m)
3.45 (m)
3.44 (m)
3.46 (dd, 1.8, 5.4)
4.20 (dd, 5.4, 12.0)
4.38 (dd, 1.8, 12.0)
3.46 (m)
4.15 (dd, 5.0, 12.0)
4.18 (dd, 5.0, 12.0)
4.37 (dd, 5.0, 12.0)
4.18 (dd, 4.8, 12.0)
4.36 (dd, 1.8, 12.0)
4.18 (dd, 4.8, 12.0)
4.37 (dd, 1.8, 12.0)
4.20 (dd, 4.8, 12.0)
4.38 (dd, 1.8, 12.0)
4
.31 (dd, 5.0, 12.0)
1
2
3
4
5
6
7
8
9
a
a
a
a
a
a
a
a
a
172.8
20.9
168.1
168.1
123.5
146.6
18.1
168.2
123.5
146.6
18.1
138.4
127.4
129.6
128.6
129.6
127.4
134.6
122.7
38.7
138.4
127.3
129.7
128.6
129.7
127.3
134.6
122.7
38.7
2.04 (s)
5.88 (dq, 1.5, 15.5)
7.00 (dq, 7.0, 15.5)
1.89 (dd, 1.5, 7.0)
123.6 5.88 (dq, 1.5, 15.6)
146.6 7.00 (dq, 7.2, 15.5)
5.89 (dq, 1.8, 15.6)
7.00 (dq, 7.2, 15.6)
1.89 (dd, 1.8, 7.2)
7.38 (d, 7.2)
7.29 (t, 7.2)
7.38 (d, 7.2)
7.29 (t, 7.2)
18.1
1.89 (dd, 1.8, 7.2)
7.20 (t, 7.2)
7.21 (t, 7.2)
7.29 (t, 7.2)
7.29 (t, 7.2)
7.38 (d, 7.2)
6.52 (d, 16.2)
6.33 (dd, 7.2, 16.2)
3.30 (m)
7.38 (d, 7.2)
6.52 (d, 16.2)
6.33 (dd, 7.2, 16.2)
3.30 (m)
1
0a
173.4
98.3
173.4
98.1
1
2
3
4
5
6
’’’
’’’
’’’
’’’
’’’
’’’
4.56 (d, 8.0)
3.12 (m)
3.33 (m)
3.31 (m)
3.32 (m)
98.1
75.3
78.6
71.4
76.7
70.1
4.60 (d, 8.0)
3.08 (m)
98.3
75.4
78.3
71.2
78.0
62.5
4.56 (d, 7.8)
3.11 (m)
98.1
75.3
78.6
71.4
76.7
70.1
4.56 (d, 8.4)
3.11 (m)
98.1
75.3
78.6
71.4
76.7
70.1
4.59 (d, 7.8)
3.07 (m)
4.56 (d, 7.8)
3.12 (m)
75.4
75.3
3.34 (m)
3.32 (m)
3.32 (m)
3.35 (m)
78.2
3.33 (m)
78.6
3.32 (m)
3.32 (m)
3.32 (m)
3.33 (m)
71.2
3.32 (m)
71.4
3.18 (m)
3.39 (m)
3.39 (m)
3.20 (m)
77.9
3.38 (m)
76.7
3.73 (dd, 5.5, 11.5)
3.64 (dd, 5.5, 11.5)
3.77 (dd, 2.0, 11.5)
3.72 (dd, 5.4, 11.4)
4.00 (dd, 2.4, 11.4)
4.29 (d, 7.2)
3.20 (m)
3.73 (m)
3.64 (m)
62.5
3.73 (dd, 5.4, 11.4)
4.00 (dd, 1.8, 11.4)
4.29 (d, 7.8)
3.20 (d, 7.2)
3.30 (m)
70.1
4
.00 (dd, 2.0, 11.5)
4.30 (d, 7.5)
3.20 (m)
4.00 (dd, 1.8, 11.4)
4.29 (d, 7.8)
3.19 (d, 7.2)
3.29 (m)
3.77 (dd, 2.4, 12.0)
1
2
3
4
5
’’’’
’’’’
’’’’
’’’’
’’’’
105.5
74.8
77.5
71.2
66.8
105.6
74.8
77.5
71.2
66.8
105.6
74.8
77.5
71.2
66.8
105.6
74.8
77.5
71.2
66.8
3.30 (m)
3.30 (m)
3.47 (m)
3.46 (m)
3.47 (m)
3.46 (m)
3.18 (m)
3.18 (m)
3.18 (m)
3.18 (m)
4
.00 (dd, 2.0, 11.5)
3.84 (dd, 5.4, 11.4)
3.84 (dd, 5.4, 11.4)
3.84 (dd, 5.4, 11.4)

Molecules 2019, 24, 1375
6 of 13
Gypenoside CP2 (2) was isolated as a white powder. Its molecular formula was determined to
be C52
H
86
O
20 from HRESIMS and 13C-NMR spectroscopic analysis. Comparison of the H and 13C-
1
NMR spectroscopic data of 1 and 2 (Tables 1 and 2) indicated that both compounds have the same
aglycone; however, compound 2 contains a but-2-enoyl unit (δ 7.00, 5.88, and 1.89/δ 168.1, 146.6,
23.6, and 18.1) but lacks the xylose and acetyl group found in 1. The location of the but-2-enoyl group
at Glc-C-6′′ was confirmed by the correlations observed in the HMBC between δ 4.18, 4.37 (H-6′′)
and δ 168.1 (C-1a, C=O) (Figure 3). The NMR spectra showed the presence of three β-glucopyranosyl
signals [δ 4.42 (d, J = 7.5 Hz)/δ 104.6, δ 4.71 (d, J = 7.5 Hz)/δ 105.2, δ 4.60 (d, J = 8.0 Hz)/δ 98.3],
which were confirmed to be from -glucose via acid hydrolysis. Long-range correlations were also
observed between δ 4.42 (H-1′) and δ 96.5 (C-3), δ 4.71 (H-1′′) and δ 82.7 (C-2′), and δ 4.60 (H-1′′′)
and δ 84.9 (C-20) indicating that the three sugars were attached to C-3, C-2′, and C-20, respectively.
Thus, the structure of gypenoside CP2 (2) was elucidated as 2α,3β,12β,20S-tetra-hydroxydammar-24-
H
C
1
H
C
H
C
H
C
H
C
D
H
C
H
C
H
C
ene-3-O-{[6-O-(E)-but-2-enoyl]-β- -glucopyranosyl-(1→2)-β- -glucoyranosyl}-20-O-β- -
D D D
glucopyranoside.
Figure 3. Key COSY and HMBC correlations of 2 and 5.
Gypenoside CP3 (3) was isolated as a white powder. Its molecular formula was determined as
1
3
1
13
C
57
H
94
O
24 from HRESIMS and C-NMR spectroscopic analysis. The H- and C-NMR spectra (Tables
and 2) of 3 showed signals assignable to a 3-O-{[6-O-(E)-but-2-enoyl]-β- -glucopyranosyl-(1→2)-β-
-xylopyranosyl-(1→6)-β- -glucopyranosyl] moiety, which
were virtually superimposable onto those of 2; however, carbon signals (δ 66.8, 71.2, 74.8, 77.5, and
05.6) consistent with an additional sugar moiety were also present. Four anomeric signals were
found at δ 4.29 (d, J = 7.2 Hz)/δ 105.6, δ 4.41 (d, J = 7.8 Hz)/δ 104.6, δ 4.56 (d, J = 7.8 Hz)/δ 98.1,
and δ 4.70 (d, J = 7.8 Hz)/δ 105.2. Acid hydrolysis of 3 yielded -glucose and -xylose (3:1). The
signals for CH -6′′′ (δ 3.72, 4.00/δ 70.1) in 3 were shifted downfield compared to those in 2 (δ 3.64,
.77/δ 62.5), indicative of the attachment of a -xylose at CH -6′′′ in 3 [29]. Moreover, long-range
correlations (HMBC) between δ 4.41 (H-1′) and δ 96.5 (C-3), δ 4.70 (H-1′′) and δ 82.7 (C-2′), δ 4.56
84.9 (C-20), and δ 4.29 (H-1′′′′) and δ 70.1 (C-6′′′) indicated the following sugar
-glucose at C-3, C-2′, and C-20, respectively and
1
D
D
-glucopyranosyl} moiety and a 20-O-[β-
D
D
C
1
H
C
H
C
H
C
H
C
D
D
2
H
C
H
3
C
D
2
H
C
H
C
H
(H-1′′′) and δ
C
H
C
locations,
D
D
-xylose at C-6′′′. Accordingly, compound
Molecules 2019, 24, 1375; doi:10.3390/molecules24071375
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1375
7 of 13
3
(gypenoside CP3) was determined as 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-{[6-O-(E)-
but-2-enoyl]-β- -glucopyranosyl-(1→2)-β- -glucopyranosyl}-20-O-[β- -xylopyranosyl-(1→6)-β-
glucopyranoside].
D
D
D
D
-
1
3
Gypenoside CP4 (4) was isolated as a white powder. The HRESIMS and C-NMR spectroscopic
23. Analysis of the H- and 13C-NMR spectra
1
data of 4 suggested its molecular formula to be C57
H94
O
(
Tables 1 and 2) gave 57 signals, of which 30 were assigned to the triterpene skeleton. The further
comparison of the 1D and 2D NMR data of 3 and 4 indicated the structural similarity in a 3β,12β,20S-
trihydroxydammar-24-ene with four sugar moieties, except for the replacement of an oxymethine (δ
8.2, C-2) by a methylene (δ 27.3, C-2) at aglycone in 4. Detailed checking the NMR data together
with the analysis of acid hydrolysis, the glycone moiety of 4 were composed 3 units of of -glucose
and one -xylose. Thus, gypenoside CP4 was determined as 3β,12β,20S -trihydroxydammar-24-ene-
-O-{[6-O-(E)-but-2-enoyl]-β- -glucopyranosyl-(1→2)-β- -glucopyranosyl}-20-O-[β-
xylopyranosyl-(1→6)-β- -glucopyranoside].
The HRESIMS of gypenoside CP5 (5) showed a quasimolecular ion at m/z 1129.5928 [M + Na]+
calcd. for C58 20Na 1129.5923), corresponding to the molecular formula C58 20. Like previous
C
6
C
D
D
3
D
D
D
-
D
(
H
90
O
H90O
isolates, compound 5 has a 2α,3β,12β,20 (S)-tetrahydroxydammar-24-ene skeleton, due to the
similarity of the H- and 13C-NMR spectroscopic data (Tables 1 and 2). Detailed analysis of the NMR
1
and HRESIMS data of 5 and 2, suggested that both compounds possess the same aglycone and
glucopyranosyl moieties, while compound 5 contains a phenyl moiety not found in 2. The cross peaks
between δ 4.20, 4.38 (H-6′′) and δ 173.4 (C-10a) in the HMBC spectrum of 5 (Figure 3) indicated that
the (E)-but-2-enoyl ester at Glc C-6′′ in 2 was replaced by a (E)-4-phenylbut-3-enoyl unit in 5.
Accordingly, the structure of (gypenoside CP5) was confirmed as 2α,3β,12β,20S-
tetrahydroxydammar-24-ene-3-O-{[6-O-(E)-4-phenyl-but-3-enoyl]-β- -glucopyranosyl-(1→2)-β-
glucopyranosyl}-20-O-β- -glucopyranoside.
The positive HRESIMS of compound 6 showed a molecular ion peak at m/z 1261.6340 [M + Na]+
calcd for C63 24Na, 1261.6346), which was 132 amu more than the molecular ion of 5, presumably
corresponding to a xylose group. The 1D and 2D NMR spectroscopic data of 6 showed similar signals
as those of 5 except for an additional unit in 6 characterized by a xylose signals (δ 105.6, 77.5, 74.8,
1.2, and 66.8). Acidic hydrolysis of 6 also furnished D-glucose and D-xylose. Based on the above
D
-
H
H
5
D
D
-
D
(
H98O
C
7
corroborations, the structure of
6 (gypenoside CP6) was determined as 2α,3β,12β,20S-
tetrahydroxydammar-24-ene-3-O-{[6-O-(E)-4-phenyl-but-3-enoyl]-β-D-glucopyranosyl-(1→2)-β-
D
-
glucopyranosyl}-20-O-[β- -xylopyranosyl-(1→6)-β- -glucopyranoside].
D
D
After the detailed spectroscopic analysis and chemical hydrolysis mentioned above, compounds
1-6 were proved as novel chemical structures and named as gypenosides CP1-CP6, respectively. The
remaining nineteen isolates were identified as 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-β-
glucopyranosyl-20-O-[β- -6-O-acetylglucopyranosyl-(1→2)-β- -glucopyranoside (7) [31],
gypenoside XLVI (8) [29], gypenoside LVI (9) [29], gypenoside LVII (10) [32], gypenoside LXXVII (11)
33], gypenoside L (12) [29], 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-β- -glucopyranosyl-20-
O-β- -glucopyranoside (13) [34], gypenoside XLII (14) [35], gypenoside Rd (15) [36] and 2α,3β,20S-
trihydroxydammar-24-ene-3-O-[β- -glucopyranosyl-(1→2)-β- -glucopyranosyl]-20-O-[β- -xylo-
pyranosyl-(1→6)-β- -glucopyranoside] (16) [37], together with seven flavonoids, quercetin-3-O-α-
rhamnopyranosyl(1→2)-β- -galactopyranoside (17) [38], quercetin-3-neohesperidoside (18) [39],
kaempferol-3-O-α- -rhamnopyranosyl-(1→2)-β- -galactopyranoside (19) [40], kaempferol-3-O-α-
rhamnopyranosyl-(1→2)-β- -glucopyranoside (20) [41], quercetin-7-O-β- -glucoside (21) [42],
kaempferol-7-O-β- -galactopyranoside (22) [43], and isorhamnetin-7-O-β- -glucopyranoside (23)
44], and two sesquiterpene glucosides, (6R,7E,9R)-9-hydroxy-megastigman-4,7-dien-3-one-9-O-β-
glucopyranoside (24) [45], and (E)-4-[3′-(β- -glucopyranosyloxy)butylidene]-3,5,5-trimethyl-2-
D
-
D
D
[
D
D
D
D
D
D
L-
D
L
D
L-
D
D
D
D
[
D-
D
cyclohexen-1-one (25) [46]. The structures of the known compounds were identified by comparing
their NMR data with published literature.
All isolates 125 were evaluated for antiproliferative activities against two human tumor cell
lines, adenocarcinoma (A549) and human liver carcinoma (HepG2) and the results are shown in Table
3. Although none of the isolates showed significant cytotoxcity against the two human cell lines,

Molecules 2019, 24, 1375
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certain dammarane-type triterpene saponins (2, 5, 6, 7, 11, 12, 13, and 15) were more potent than the
remaining compounds. The EC50 values of these eight compounds against the HepG2 cell line ranged
from 29.3 to 100.6 μM, while only four compounds (2, 11, 13 and 15) exhibited EC50 values of less than
1
00 μM (EC50 59.4~87.3 μM) against A549 cells. Among the six new gypenosides, compound 2 was
the most potent against A549 cells and compound 5 was among the most potent against HepG2 cells.
Table 3. Antiproliferative data for compounds 125 against cancer cell lines.
A549 Cell Line
Inhibition (%) a EC50 (μM) Inhibition (%) EC50 (μM)
HepG2 Cell Line
Cmpd.
Number of Sugars
1
2
3
4
5
6
7
8
9
16.4 ± 3.97
84.1 ± 7.97
23.3 ± 6.20
17.0 ± 2.00
42.7 ± 1.41
29.7 ± 5.34
37.1 ± 0.78
6.5 ± 5.16
(-) b
38.9 ± 3.82
83.0 ± 3.91
44.0 ± 2.28
46.4 ± 2.60
71.1 ± 0.60
55.5 ± 6.45
53.8 ± 3.43
30.7 ± 5.32
31.1 ± 7.76
44.1 ± 1.96
93.1 ± 0.99
60.4 ± 6.34
73.3 ± 1.81
24.5 ± 2.79
56.5 ± 1.55
37.3 ± 0.53
11.5 ± 1.17
9.4 ± 3.02
(-)
4
3
4
4
3
4
3
3
4
3
2
2
2
4
3
4
-
59.4 ± 2.51
60.4 ± 0.63
(-)
(-)
(-)
(-)
(-)
29.3 ± 0.26
(-)
54.2 ± 2.07
(-)
89.1 ± 3.75
(-)
(-)
14.5 ± 6.04
20.9 ± 2.62
94.4 ± 0.28
27.8 ± 11.35
65.1 ± 7.29
17.9 ± 2.72
43.2 ± 3.67
25.2 ± 1.35
13.1 ± 2.88
16.1 ± 5.32
19.3 ± 1.40
13.0 ± 3.40
15.5 ± 2.73
22.9 ± 12.78
21.7 ± 9.46
10.9 ± 6.06
5.6 ± 3.12
(-)
(-)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
(-)
(-)
70.1 ± 2.34
76.2 ± 2.10
(-)
100.7 ± 1.36
87.3 ± 3.39
68.4 ± 0.57
(-)
(-)
73.8 ± 2.86
75.4 ± 1.30
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
-
32.5 ± 3.83
18.0 ± 2.99
29.2 ± 2.45
26.6 ± 6.30
31.2 ± 3.45
25.2 ± 3.65
6.5 ± 3.17
-
-
-
-
-
-
-
a
Inhibition (%) of pure compounds against cell lines at 100 μg/mL; b (-): ED50 > 100 g/mL.
3. Materials and Methods
3.1. General Experimental Procedures
The optical rotations were determined using a JASCO P-2000 polarimeter (Jasco Co., Tokyo,
Japan). The infrared (IR) spectra were measured on a Mattson Genesis II spectrophotometer (Thermo
Fisher Scientific Inc., Waltham, MA, USA). Electrospray ionization mass spectrometry (ESIMS) data
were obtained on an LCQ mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
High-resolution electronic ionization mass spectrometry (HREIMS) data were measured on a
Finnigan MAT-95XL mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Nuclear
magnetic resonance (NMR) spectra were recorded using by Bruker AC-400 FT-NMR (Bruker BioSpin,
Rheinstetten, Germany), Varian unit Inova 500 MHz, and Varian VNMRS 600 MHz spectrometers
(
Aglient Technologies, Santa Clara, CA, USA). Diaion HP-20 (Mitsubishi Chemical Co., Tokyo,
Japan), Sephadex LH-20 (GH Healthcare, Uppsala, Sweden), and silica gel 60 (Merck 70–230 and 230–
00 mesh, Merck, Darmstadt, Germany) were used for column chromatography, and precoated silica
4
gel (Merck 60 F-254) plates were used for TLC. The spots on TLC were detected by spraying with an
anisaldehyde-sulfuric acid solution and heating at 100 °C. HPLC separations were performed on a
Shimadzu LC-6AD series instrument (Shimadzu Inc., Kyoto, Japan) with an SPD-10A UV detector

Molecules 2019, 24, 1375
9 of 13
and a 380-LC ELSD detector (Aglient Technologies, Santa Clara, CA, USA), which was equipped with
a Cosmosil 5C18 AR-II column (Nacalai Tesque, Inc., Kyoto, Japan).
3.2. Plant Material
The aerial parts of Gynostemma pentaphyllum (8.4 kg) were purchased from Zheng Yuen Tang
Biotech Co. Ltd. in Kaohsiung, Taiwan in August 2013. A voucher specimen (NRICM, No. 20130101)
has been deposited in the National Research Institute of Chinese Medicine, Taipei, Taiwan.
3.3. Extraction and Isolation
The dried aerial parts of G. pentaphyllum (8.4 kg) were extracted three times at 60 °C with 95%
ethanol (EtOH). The EtOH soluble portion was concentrated to give a crude extract (8 L). The
concentrated EtOH extract was partitioned with n-hexane and H O (1:1, v/v) to give a n-hexane
portion (847.2 g). The aqueous layer was further partitioned with EtOAc to give an EtOAc portion
279.5 g). Then, the H O portion was loaded onto a Diaion HP-20 column (11 × 72 cm), and
successively eluted with H O, 25% MeOH, 50% MeOH, 75% MeOH, 100% MeOH, and 100% EtOAc
2
(
2
2
to obtain five fractions (Fr-1 to 6). Fr-4 (298.7 g) was further chromatographed on a Sephadex LH-20
column with 60% MeOH as the eluent to give four fractions (Fr-4-1 to Fr-4-4). Fr-4-3 was further
purified by semi-preparative HPLC using 35% CH
3
CN in H
.0 mL/min to give compounds 1 (71.5 mg), 7 (30.3 mg), 8 (54.2 mg), 9 (32.1 mg), and 16 (30.4 mg). Fr-
was chromatographed on a LH-20 column with 60% MeOH as the eluent to yield eight fractions
2
O as the solvent system at a flow rate of
2
3
(
Fr-3-1 to Fr-3-8). Fr-3-2 and Fr-3-3 were further separated by semi-preparative HPLC with 35%, and
8% CH CN in H O as the solvent system, respectively. Compounds 24 (17.2 mg) and 25 (5.4 mg)
were obtained from Fr-3-3, and compound 14 (15.4 mg) was separated from Fr-3-2. Fr. 5 was further
fractioned with a step gradient elution of H O-MeOH (from 30:70 to 0:100, v/v) on a C18-gel flash
column to afford six fractions (Fr-5-1 to Fr-5-6). Fr-5-2, and Fr-5-3 were further purified by semi-
preparative HPLC using 38% CH CN in H O as the solvent system at a flow rate of 2.0 mL/min to
give compounds 2 (5.2 mg), 3 (3.1 mg), 4 (2.8 mg), and 10 (4.5 mg). Fr-5-4, was further purified by
semi-preparative HPLC using 48% CH CN in H O as the solvent system at a flow rate of 2.0 mL/min
to give compounds 5 (2.6 mg), 6 (2.1 mg), 12 (11.2 mg), and 13 (7.9 mg). The EtOAc portion was loaded
onto a LH-20 column eluting with CH Cl /MeOH (1:1, v/v) to afford 10 fractions (Fr-E-1 to Fr-E-10).
Fr-E-2 was further purified by semi-preparative HPLC using 80% CH CN in H O as the solvent
system at a flow rate of 2.0 mL/min to give compound 11 (8.7 mg). Fr-E-8 was further purified by
semi-preparative HPLC using 38% CH CN in H O as the solvent system at a flow rate of 2.0 mL/min
to give compounds 17 (13.7 mg), 18 (42.7 mg), 19 (30.2 mg), 20 (48.0 mg), 21 (7.0 mg), 22 (4.2 mg), and
1
3
2
2
3
2
3
2
2
2
3
2
3
2
23 (16.2 mg).
1
13
3
.4. Spectroscopic Data ( H- and C-NMR spectra of 1-6 were also provided by the supplementary materials)
Gypenoside CP1 (1), White amorphous powder; [α]26
D
+11.5 (c 0.2, MeOH); IR (KBr) νmax 3358, 2945,
−
1
1
) and 13C- (125 MHz, methanol-d
) NMR data,
2
876, 1736, 1638, 1082 cm ; H- (500 MHz, methanol-d
4
4
+
see Tables 1 and 2, respectively; HRESIMS m/z 1159.5874 [M + Na] (calcd for C55
H92O24Na, 1159.5876).
Gypenoside CP2 (2), White amorphous powder; [α]26
+15.7 (c 0.2, MeOH); IR (KBr) νmax 3362, 2941,
D
−
1
1
) and 13C- (125 MHz, methanol-d
) NMR data,
2
872, 1715, 1650, 1074 cm ; H- (500 MHz, methanol-d
4
4
+
see Tables 1 and 2, respectively; HRESIMS m/z 1053.5607 [M + Na] (calcd for C52
H86O20Na, 1053.5610).
Gypenoside CP3 (3), White amorphous powder; [α]26
+12.9 (c 0.2, MeOH); IR (KBr) νmax 3370, 2928,
D
−
1
1
) and 13C- (150 MHz, methanol-d
) NMR data,
2
876, 1715, 1650, 1078 cm ; H- (600 MHz, methanol-d
4
4
+
see Tables 1 and 2, respectively; HRESIMS m/z 1185.6030 [M + Na] (calcd for C57
H94O24Na, 1185.6033).
Gypenoside CP4 (4), White amorphous powder; [α]26
+6.6 (c 0.2, MeOH); IR (KBr) νmax 3350, 2937,
D
−
1
1
) and 13C- (150 MHz, methanol-d
) NMR data,
2
868, 1728, 1650, 1078 cm ; H- (600 MHz, methanol-d
4
4
+
see Tables 1 and 2, respectively; HRESIMS m/z 1169.6084 [M + Na] (calcd for C57
H94O23Na, 1169.6084).

Molecules 2019, 24, 1375
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Gypenoside CP5 (5), White amorphous powder; [α]26
D
+7.7 (c 0.2, MeOH); IR (KBr) νmax 3370, 2921,
−
1 1
) and 13C- (150 MHz, methanol-d
2
872, 1679, 1074 cm ; H- (600 MHz, methanol-d
4
4
) NMR data, see
+
Tables 1 and 2, respectively; HRESIMS m/z 11129.5928 [M + Na] (calcd for C58
H O
90 20Na, 1129.5923).
Gypenoside CP6 (6), White amorphous powder; [α]26
+12.9 (c 0.2, MeOH); IR (KBr) νmax 3370, 2921,
D
−
1
1
) and 13C- (150 MHz, methanol-d
2
864, 1687, 1070 cm ; H- (600 MHz, methanol-d
4
4
) NMR data, see
+
Tables 1 and 2, respectively; HRESIMS m/z 1261.6340 [M + Na] (calcd for C63
H O
98 24Na, 1261.6346).
3
.5. Acid Hydrolysis of Dammarane-Type Glycosides
Each isolated compound (1.0 mg) was treated with 2 N methanolic HCl (2 mL) under conditions
of reflux at 90 °C for 1 h. Each mixture was extracted with CH Cl to afford the aglycone portion, and
the aqueous layer was neutralized with Na CO and filtered. To the evaporated filtrate was added 1-
trimethylsilyl)imidazole and pyridine (0.2 mL), and the mixture was stirred at 60 °C for 5 min. After
the reaction mixture was dried under a stream of N , each residue was partitioned between CHCl
and H O. Each CH Cl fraction was subjected to gas chromatography (GC, column: Varian capillary
2
2
2
3
(
2
3
2
2
2
column CP-chirasil-L-val for optical isomers, 25 m × 0.25 mm, 0.12 μm; column temperature, 50–150
3
°
C, 30 °C/min, 150–180 °C, 0.8 °C /min; injector temperature, 200 °C; He carrier gas, 2.0 kg/cm ; mass
detector, Thermo, DSQ2; electron energy, 70 eV). Under these conditions, the sugars of each reactant
were identified by comparison with authentic standards ( -glucose and -xlyose).
D
D
3.6. Antiproliferation Assay
The isolates were tested for antiproliferative effects against HepG2 (human hepatocellular
carcinoma), A549 (human lung adenocarcinoma) tumor cell lines and the M10 (human mammary
epithelial) cell line in vitro using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] colorimetric method based on previously published procedures [47]. Two cell lines were
maintained optimal medium (Life Technologies) supplemented with 2 mM -glutamine and 10%
L
heat-inactivated fetal bovine serum (FBS) (Life Technologies) under standard culture conditions.
After treatment with serial dilutions of tested compounds for 48 h, the alamar blue assay (Biosource
International, Nivelles, Belgium) was used to obtain the half maximal inhibitory concentration (IC50).
Doxorubicin was used as a positive control. Plates were incubated at 37 °C for 6 h prior to measure
the absorbance at 570 nm and at 600 nm wavelengths using a spectrophotometric plate reader
(DYNEX Technologies, Chantilly, VA, USA). Experimental data were normalized to control values.
Mitomycin c was used as a positive control (A549 cell line: 0.1 ± 0.01 μg/mL; HepG2 cell line: 0.1 ±
0.01 μg/mL).
4. Conclusions
In this study, we isolated and characterized six new and ten known dammarane-type triterpene
saponins as well as eight known flavonoids and two known sesquiterpene glucosides from a 95%
EtOH extract of dried aerial parts of G. pentaphyllum. All the new chemical structures of compounds
1–6 were elucidated completely and tentatively named gypenosides CP1–CP6. These new isolates
were obtained from the titled plant for the first time, and not yet found in other natural resources or
synthesized molecules before. For the derived chemical structures of 5 and 6, (E)-4-phenylbut-3-enyl
unit is first time to be found in nature. This study adds to the present phytochemical and properties
information on this plant species, together with the studies performed and compiled by others [1],
could assist in future modification of dammarane-type compounds as anticancer or other therapeutic
agents.
Supplementary Materials: The H- and 13C-NMR spectra of compounds 1–6 are available online.
1
Author Contributions: Y.-H.K and K.-H.L. supervised the study. P.-Y.C., N.-L.N. and T.H-.V performed the
experiments. L.-J.Z, C.-C.L., and Y.-C.L analyzed the data. C.-C.C., H.-C.H., C.-C.L., Y.-C.L., Y.Y.C. and S.L.M.-
N. wrote the paper.

Molecules 2019, 24, 1375
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Funding: This work was supported by grants from the National Science Council (NSC098-2811-B-077-002 and
NSC98-2320-B-077-005-MY3) and Ministry of Science and Technology (MOST104-2320-B-077 -006 -MY3),
Taiwan, as well as from the Ministry of Health and Welfare (MM10601-0160), Taiwan.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–6 are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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