Two new triterpenoid saponins from Centella asiatica

Article abstract of DOI:10.1016/j.phytol.2021.06.012

Two undescribed ursane-type triterpene saponins, named asiaticoside H (1) and I (2), were isolated from the whole plants of Centella asiatica. The chemical structures of 1 and 2 were mainly characterized by extensive analysis of their 1D and 2D NMR and HR

Full text of DOI:10.1016/j.phytol.2021.06.012

Phytochemistry Letters 44 (2021) 102–105
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Two new triterpenoid saponins from Centella asiatica
Bo Ren, Wei Luo, Meng-jun Xie, Mei Zhang*
Key Laboratory of Standardization of Chinese Herbal Medicine, Ministry of Education, State Key Laboratory of Southwestern Chinese Medicine Resources, Chengdu
University of Traditional Chinese Medicine, Chengdu, 611137, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two undescribed ursane-type triterpene saponins, named asiaticoside H (1) and I (2), were isolated from the
whole plants of Centella asiatica. The chemical structures of 1 and 2 were mainly characterized by extensive
analysis of their 1D and 2D NMR and HRESIMS spectroscopic data and chemical derivation.
Centella asiatica
Triterpenoid saponins
Structural elucidation
ꢀ
–
1. Introduction
[M H] ion peak at m/z 1119.5604 (calc. for 1119.5593) in its negative
HRESIMS spectrum. The 1H-NMR spectrum of 1 displayed several
characteristic proton singals for six methyl proton signals at δH 0.90 ꢀ
1.15, an olefinic proton signal at δH 5.41 (brs) and four anomeric
methines at δH 6.14 (1H, d, J =8.16 Hz), 4.97 (1H, d, J =7.86 Hz), 5.30
(1H, d, J =3.54 Hz) and 5.79 (1H, brs) (Table 1). In the 13C-NMR spectra
Centella asiatica (L.) Urban, belonging to the family Apiaceae, is
widely distributed throughout the world, especially in the moist and
tropical regions of Asia, Africa and Oceania (James and Dubery, 2009).
In some countries of the world, C. asiatica has been used for treating skin
diseases and healing skin wounds for thousands of years (Brinkhaus
et al., 2000). In this plant, the triterpene saponins are main character-
istic constituents, which have been confirmed to be responsible for the
above medicinal benefits (Shen et al., 2019; Kwon et al., 2014).
C. asiatica has been traditionally used to improve brain function, boost
memory and prevent cognitive deficits (Shinomol and Muralidhara,
2011), which was proved to associate with the triterpene saponins and
caffeoylquinic acids in the plant that exhibit neuroprotective effects in
several in vitro models (Wu et al., 2020; Viswanathan et al., 2019;
Subaraja and Vanisree, 2019). Recently, C. asiatica extracts were
confirmed to have antibacterial activity (Sieberi et al., 2020). Asiatico-
side, a main component in C. asiatica, showed strong antibacterial ac-
tivity against some antibiotic-resistant bacterial strains, including
methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus
epidermidis (Zhang et al., 2006). In order to fully understand the
chemical compositions of C. asiatica and search for novel antibacterial
ingredients from this plant, the present study was focused on the
chemical investigation of the minor bioactive compounds. As a result,
two new triterpene saponins (Fig. 1) were obtained and identified.
of 1, four signals at δ 95.9, 105.0, 101.2 and 102.8 were readily
C
assigned to the anomeric carbons for three glucoses and one L-rhamnose
with the aid of HSQC spectrum (Table 1). The sugar residues were
further identified as D-glucose and L-rhamnose based on the methods
previously reported (Li et al., 2017). The 1H-NMR and 13C-NMR spec-
troscopic data were very similar to those of asiaticoside (Sung et al.,
1992), a major component in C. asiatica, except for the presence of one
more glucose moiety in 1. The double methyl proton signal assigned to
H-29 at δH 0.92 (3H, d, J =3.18 Hz) showed an HMBC correlation with
C-18 (δ 53.5), while another double methyl proton signal assigned to
H-30 atCδH 0.90 (3H, d, J =2.94 Hz) exhibited HMBC correlations with
C-19 (δ 39.5) and C-21 (δ 31.0), suggesting the ursane-type aglycone
C
C
of 1 (Fig. 2). The double bond was located between C-12 and C-13 in
view of the HMBC correlations of H-18 (δH 2.48) with C-12 (δ 126.1)
C
and C-13 (δC 138.9). The chemical shifts of C-2 and C-3 at δC 69.8 and
78.2 suggested the equatorial position of the hydroxyl groups at C-2 and
C-3 (Uddin Ahmad et al., 1986; Kojima et al., 1987). In the NOESY
spetrum of 1, the obvious cross signals of H-2 (δH 4.20)/H-25 (δH 1.06),
H-2 (δH 4.20)/H-24 (δH 1.03), H-5 (δH 1.77)/H-23 (δH 3.33) and H-3 (δH
4.02)/H-5 (δ 1.77) further confirmed the stereochemistries of H-2, H-3
H
2. Results and discussion
and the presence of an oxygen atom connected to C-23 (Fig. 2). A
glucose residue was confirmed to be connected to C-23 of the aglycone
due to the HMBC correlation between the anomeric proton at δH 5.30
Compound 1 was isolated as a white amorphous powder. The mo-
lecular formula of 1 was determined to be C54H88O24according to the
(1H, d, J =3.54 Hz) and the methylene signal at δ 73.6 (C-23).
C
* Corresponding author.
E-mail address: zhangmei63@cdutcm.edu.cn (M. Zhang).
https://doi.org/10.1016/j.phytol.2021.06.012
Received 7 April 2021; Received in revised form 25 May 2021; Accepted 8 June 2021
Available online 15 June 2021
1874-3900/© 2021 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

B. Ren et al.
Phytochemistry Letters 44 (2021) 102–105
Fig. 1. Chemical structures of the compounds 1-2.
Moreover, the chemical shift of the oxygenated methylene CH2-23 in 1
shifted to lower field by around 7 ppm than that of CH2-23 in asiatico-
side, also suggesting a sugar residue was located at C-23 in 1. The sugar
chain at C-28 of the aglycone was verified by the following important
HMBC correlations from H-1 (δH 6.14) of Glc-1 to C-28 (δC 176.7), H-1
3. Experimental section
3.1. General experimental procedures
Silica gel (100–200 mesh) and TLC plates were obtained from
Qingdao Haiyang Chemical Group Co. Ltd. (Qingdao, China). All sol-
vents were analytical grade and bought from KeLong (Chengdu, China).
Standard sugars were bought from Sigma-Aldrich (St. Louis, MO, USA).
Optical rotations were recorded on a PerkinElmer 341 polarimeter. NMR
spectra were performed on Bruker Avance-400 and Bruker Avance-600
spectrometers in C5D5N with TMS as an internal standard, respectively.
HR-ESI-MS data were acquired on a LTQ Orbitrap XL mass spectrometer.
The sugar derivatives were analyzed via a Shimadzu LC-20A series HPLC
instrument equipped with a SinoChrom ODS-BP C18 column. Semi-
preparative HPLC separation was carried out on a CXTH apparatus
with an UV3000 detector equipped with a Unisil-10ꢀ 120-C18 (250 × 30
(δH 4.97) of Glc-2 to C-6 (δ 69.5) of Glc-1, and H-1 (δH 5.79) of Rha to
C
C-4 (δ 78.6) of Glc-2 (Fig. 2). Finally, compound 1 was etablished as
3-O-β-CD-glucopyranosyl
2α,3β,23-trihydroxy-urs-12-ene-28-oic acid
28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl
(1→6)-β-D-glucopyranoside by detailed analyses of HSQC, HMBC,
NOESY and TOCSY spectra, and named asiaticoside H.
Compound 2 was isolated as a white amorphous powder. The mo-
ꢀ
–
lecular foumula of 2 was defined as C48H78O20based on the [M H] ion
peak at m/z 973.5018 (calc. for 973.5014) in the HRESIMS spectrum.
Comparison of the 1H-NMR and 13C-NMR data of 2 with those of
madecassoside, another major component in C. asiatica, suggested the
chemical structures of these two compounds were only differed in the
location of the double bonds. The configurations of the hydroxyl groups
mm, 10 μm) column.
at C-2, C-3 and C-6 were further verified by the cross signals of H-2 (δ
H
3.2. Plant materials
4.49)/H-25 (δH 1.74), H-3 (δH 4.01)/H-5 (δH 1.82), H-5 (δH 1.82)/H-23
(δH 4.33) and H-5 (δH 1.82)/H-6 (δH 5.06) in the NOESY spetrum of 2
(Fig. 2). The appearance of a methylene carbon signal at δC 112.7 ppm in
the 13C-NMR spectrum of 2 indicated the presence of an exocyclic
The whole plant material of C. asiatica was collected in Sichuan
Province, China, in July 2018. The plant was identified by Professor
Yuntong Ma of Chengdu University of TCM. A voucher specimen (NO.
ZZZYK20200803001ꢀ 30) was deposited in the authors′ lab.
double bond. Detailed analysis of the 13C-NMR data of 2 and 2
α,3β-
dihydroxy-urs-20(30)-en-28-oic acid (Qiao et al., 2014) demonstrated
they might share the same E ring. This was further confirmed by the
3.3. Extraction and isolation
HMBC cross-peaks from H3-29 (δ 1.13) to C-19, C-20 and H2-30 (δ
H
5.00) to C-20, C-21. The sugar chHain at C-28 of the aglycone was sup-
ported by the HMBC correlations from H-1 (δ 6.13) of Glc-1 to C-28 (δ
175.4), H-1 (δH 4.95) of Glc-2 to C-6 (δC 69.7)Hof Glc-1, and H-1 (δH 5.85C)
of Rha to C-4 (δ 78.6) of Glc-2 (Fig. 2). Thus, compound 2 was eluci-
The air-dried whole plants of C. asiatica (10.0 kg) were smashed and
extracted with 80 % MeOH (20 L) for 3 times (24 h each time) at 60 ◦C.
The combined extract was evaporated under reduced pressure to yield a
dark brown residue, which was suspended in water and then partitioned
successively with CH2Cl2 and n-BuOH. The resultant n-BuOH extract was
dried under reduced pressure and then separated over a silica gel col-
umn (30 × 100 cm) eluted with a gradient solvent system of H2O
saturated CH2Cl2/MeOH (10:1, 8:1, 6:1, 4:1, 2:1, 1:1) to yield eleven
fractions (Fr. 1 - Fr. 11) based on TLC analysis. Fr. 8 was subject to HPLC
dated
28-O-
as
2α
C ,3β,6β,23-tetrahydroxy-urs-20(30)-ene-28-oic
acid
α
-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl
(1→6)-β-D-glucopyranoside, and named asiaticoside I.
on a Unisil-10ꢀ 120-C18 (10 μm, 250 × 30 mm, 20 mL/min) column
103

B. Ren et al.
Phytochemistry Letters 44 (2021) 102–105
Table 1
1H and 13C NMR spectroscopic data of compounds 1 and 2 (δ, J in Hz, C5D5N).
1
2
position
δC
δH (J in Hz)
δC
δH (J in Hz)
1
47.9
68.9
78.8
43.6
48.4
19.1
33.2
40.4
48.5
38.4
24.0
126.1
138.9
42.8
28.8
24.8
48.7
53.5
39.5
39.3
31.0
37.0
73.6
14.4
17.7
18.1
24.0
176.7
17.6
21.6
1.32 (m), 2.27 (m)
4.20 (m)
50.1
69.4
78.3
44.5
49.5
67.9
42.0
42.2
51.9
40.9
22.2
26.1
38.5
42.0
28.3
27.2
48.0
49.9
41.3
150.5
34.4
35.3
66.7
15.6
17.1
19.9
15.3
175.4
25.0
112.7
1.41 (m), 2.47 (m)
4.49 (m)
2
3
4.02 (m)
4.01 (m)
4
5
1.77 (m)
1.82 (m)
6
1.37 (m), 1.53 (m)
1.37 (m), 1.85 (m)
5.06 (br s)
7
1.78 (m), 1.90 (m)
8
9
1.75 (m)
1.70 (m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Glc-1
1
2.05 (m)
1.42 (m), 1.70 (m)
2.39 (m), 2.45 (m)
3.01 (m)
5.41 (br s)
1.15 (m), 1.25 (m)
1.96 (m)
1.11 (m), 2.03 (m)
0.98 (m), 1.76 (m)
2.48 (d, 11.16)
1.33 (m)
1.14 (m)
2.75 (m)
0.87 (m)
1.25 (m), 1.37 (m)
1.78 (m), 1.92 (m)
3.33 (d, 9.48), 4.35 (m)
1.03 (s)
1.42 (m), 2.10 (m)
1.35 (m), 2.53 (m)
4.07 (m), 4.33 (m)
1.69 (s)
1.06 (s)
1.74 (s)
1.15 (s)
1.60 (s)
1.14 (s)
0.86 (s)
0.92 (d, 2.94)
0.90 (d, 3.18)
1.13 (d, 5.2)
5.00 (br s)
95.9
73.9
79.0
71.1
78.1
69.5
6.14 (d, 8.16)
4.32 (m)
95.5
73.9
79.0
71.5
78.0
69.7
6.13 (d, 7.48)
4.31 (m)
2
3
4.04 (m)
4.03 (m)
4
4.24 (m)
4.20 (m)
5
4.06 (m)
4.07 (m)
6
4.23 (m), 4.64 (m)
4.23 (m), 4.64 (m)
Glc-2
1
105.0
75.5
76.7
78.6
77.3
61.5
4.97 (d, 7.86)
3.93 (m)
104.9
75.5
76.6
78.8
77.2
61.5
4.95 (d, 7.52)
3.92 (m)
2
3
4.12 (m)
4.12 (m)
4
4.36 (m)
4.36 (m)
5
3.65 (m)
3.65 (m)
6
4.08 (m), 4.20 (m)
4.08 (m), 4.22 (m)
Glc-3
1
101.2
70.5
72.9
71.1
72.0
62.8
5.30 (d, 3.54)
4.38 (m)
2
3
4.65 (m)
4
4.48 (m)
5
4.38 (m)
6
4.35 (m), 4.40 (m)
Rha
1
102.8
72.7
72.9
74.1
70.5
18.7
5.79 (br s)
4.66 (m)
102.3
72.7
72.8
74.3
70.3
18.6
5.85 (br s)
4.62 (m)
2
3
4.53 (m)
4.52 (m)
4
4.12 (m)
4.14 (m)
5
4.89 (m)
4.95 (m)
6
1.67 (d, 6.12)
1.67 (d, 5.4)
1
with MeOH-H2O (57:43, v/v) as an eluent system to give six sub-
fractions (Fr. 8.1 - Fr. 8.6). Fr. 8.5 was further purified by the same
HPLC using CH3CN-H2O (25:75, v/v) as an eluant to afford compound 2
(61.6 mg). Fr. 10 was submitted to HPLC on a Unisil-10ꢀ 120-C18 (10
MeOH); H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N)
data see Table 1; HRESIMS: m/z 973.5018 [Mꢀ H]ꢀ (calcd for
C
48H77Oꢀ20, 973.5014).
μ
m, 250 × 30 mm, 20 mL/min) column with MeOH-H2O (57:43, v/v) as
3.4. Acid hydrolysis and determination of absolute configurations of
monosaccharides
an eluent system to give pure compound 1 (32.7 mg).
20
Asiaticoside H (1): White amorphous powder; [
α]
+11.7 (c 0.10,
D
1
MeOH); H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N)
Compounds 1-2 (each 2.0 mg) were mixed and dissolved in 2%
H2SO4 solution (2 mL) and heated at 100 ◦C for 12 h. The resultant
solution was cooled to room temperature and then extracted with EtOAc
three times (each 3 mL) to remove the aglycones. The remaining H2O
layer was neutralized to pH = 7 with aqueous solution of Ba(OH)2,
data see Table 1; HRESIMS: m/z 1119.5604 [Mꢀ H]ꢀ (calcd for
C
54H87Oꢀ24, 1119.5593).
Asiaticoside I (2): White amorphous powder; [
20
α
]
-4.0 (c 0.15,
D
104

B. Ren et al.
Phytochemistry Letters 44 (2021) 102–105
Fig. 2. Key HMBC and NOE correlations of compounds 1-2.
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water solution of compounds 1 and 2 was dried under reduced pressure
to give a residue (2.2 mg), which was dissolved in pyridine (0.3 mL)
containing L-cysteine methyl ester hydrochloride (2.5 mg) and heated at
Kojima, H., Tominaga, H., Sato, S., Ogura, H., 1987. Pentacyclic triterpenoids from
Prunella vulgaris. Phytochemistry 26, 1107–1111.
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component of Centella asiatica, inhibits melanogenesis in B16F10 mouse melanoma.
Mol. Med. Rep. 10, 503–507.
60 ◦C for 1 h. Then, phenylisothiocyanate (2.5
μL) was added and the
mixture was heated at 60 ◦C for another 1 h. The reaction mixture was
directly analyzed by reversed-phase HPLC performed on a Shimadzu LC-
20A series HPLC instrument equipped with a SinoChrom ODS-BP C18
column and CH3CN-H2O (25:75, v/v) containing 50 mM H3PO4 as the
mobile phase. From the hydrolysate of compounds 1 and 2, D-glucose
and L-rhamnose were identified by comparison of the retention times of
their derivatives with those of the authentic sugars derivatized in the
same way, which showed retention times of 19.88 and 24.17 min,
respectively.
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Liu, H.W., Wang, M.K., Wang, F., 2017. Flavonoid glycosides isolated from
Epimedium brevicornum and their estrogen biosynthesis-promoting effects. Sci. Rep.
7, 7760.
Qiao, A.M., Wang, Y.H., Xiang, L.M., Zhang, Z.X., He, X.J., 2014. Triterpenoids of sour
jujube show pronounced inhibitory effect on human tumor cells and antioxidant
activity. Fitoterapia 98, 137–142.
Shen, X.Q., Guo, M.M., Yu, H.Y., Liu, D., Lu, Z., Lu, Y.H., 2019. Propionibacterium acnes
related anti-inflammation and skin hydration activities of madecassoside, a
pentacyclic triterpene saponin from Centella asiatica. Biosci. Biotechnol. Biochem.
83, 561–568.
Shinomol, G., Muralidhara, M.M.B., 2011. Exploring the role of ‘“Brahmi”’ (Bocopa
monnieri and Centella asiatica) in brain function and therapy. Recent Pat. Endocr.
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Declaration of Competing Interest
No potential conflict of interest was reported by the authors.
Acknowledgements
Sieberi, B.M., Omwenga, G.I., Wambua, R.K., Samoei, J.C., Ngugi, M.P., Al-Bakri, A.G.,
2020. Screening of the dichloromethane: methanolic extract of Centella asiatica for
antibacterial activities against Salmonella typhi, Escherichia coli, Shigella sonnei,
Bacillus subtilis, and Staphylococcus aureus. Sci. World J., 6378712, 2020.
Subaraja, M., Vanisree, A.J., 2019. The novel phytocomponent asiaticoside-D isolated
from Centella asiatica exhibits monoamine oxidase-B inhibiting potential in the
rotenone degenerated cerebral ganglions of Lumbricus terrestris. Phytomedicine 58,
152833.
This work was financially supported by the Applied and Basic
Research Program of Sichuan Province (2018JY0186)
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glycosides from the bark of Schefflera octophylla. Phytochemistry 31, 227–231.
Uddin Ahmad, V., Bano, S., Bano, N., 1986. A triterpene acid from Nepeta hindostana.
Phytochemistry 25, 1487–1488.
Appendix A. Supplementary data
Viswanathan, G., Dan, V.M., Radhakrishnan, N., Nair, A.S., Nair, A.P.R., Baby, S., 2019.
Protection of mouse brain from paracetamol-induced stress by Centella asiatica
methanol extract. J. Ethnopharmacol. 236, 474–483.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2021.06.012.
Wu, Z.W., Li, W.B., Zhou, J., Liu, X., Wang, L., Chen, B., Wang, M.K., Ji, L.L., Hu, W.C.,
Li, F., 2020. Oleanane- and ursane-type triterpene saponins from Centella asiatica
exhibit neuroprotective effects. J. Agri. Food Chem. 68, 6977–6986.
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