Plant Biology ISSN 1435-8603

RESEARCH PAPER

Morphological and chemical variation of Stemona tuberosa from

southern China – Evidence for heterogeneity of this medicinal
plant species
G. Chen1, L. Brecker2, S. Felsinger2, X.-H. Cai3, S. Kongkiatpaiboon4 & J. Schinnerl5
1 Kunming Botanical Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
2 University of Vienna, Faculty of Chemisty Institute of Organic Chemistry, W€ahringerstrasse 38, A-1090 Vienna, Austria
3 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
4 Drug Discovery and Development Center, Thammasat University, PathumThani, Thailand
5 Chemodiversity Research Group, Department of Botany and Biodiversity Research, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

Keywords ABSTRACT
Bai Bu; chemotaxonomy; plant secondary
metabolites; Stemona alkaloids; Stemona • The occurrence of bioactive alkaloids and tocopherols was studied in 15 different
tuberosa. provenances of Stemona tuberosa Lour. collected in southern China, to examine chem-
ical variation of individuals that show notable differences in flower characteristics.
Correspondence Morphological variations stimulated examination of chemical characteristics of these
J. Schinnerl, Chemodiversity Research Group, individuals.
Division of Systematic and Evolutionary • Methanolic root extracts of 15 individuals of S. tuberosa were comparatively assessed
Botany, University of Vienna, Rennweg 14, with HPLC-UV-DAD/ELSD. Five of seven compounds were co-chromatographically
A-1030 Vienna, Austria. identified. Two compounds were isolated and their structure elucidated using NMR
E-mail: johann.schinnerl@univie.ac.at and MS. Amounts of alkaloids and tocopherols were determined using HPLC-UV-
DAD/ELSD with the external standard method.
Editor
H. P. Mock
• Five alkaloids, tuberostemonine (1), tuberostemonine A (2), neotuberostemonine (3),
tuberostemonine N (4), stemoninine (5) and two 3,4-dehydrotocopherol derivatives
were identified. Within S. tuberosa alkaloid accumulation tends either towards
Received: 25 April 2017; Accepted: 30 May tuberostemonine (1) or stemoninine (5). All individuals show a notable co-occurrence
2017
of compounds 1 or 5 and 3,4-dehydro-d-tocopherol (6). These results coincide with
differences in flower morphology of S. tuberosa.
doi:10.1111/plb.12587
• Stemona tuberosa, as defined in the Flora of China, shows a remarkable variation in
flower morphology and additionally in the accumulation of alkaloids. The obtained
data show the need for future species delimitation to either species or subspecies level.

INTRODUCTION
Fleshy roots of several Stemona Lour. species (Stemonaceae–
Pandanales), also known as ‘Bai Bu’, are widely used in the tra-
ditional South-East Asian medicine as an antitussive agent
(Inta et al. 2008; Lee et al. 2008). Some species, in particular
S. tuberosa Lour., S. sessilifolia (Miq.) Miq. and S. japonica
(Blume) Miq. within this small monocot family (three genera
and ca. 33 species) have been well investigated and are known
to accumulate bioactive and structurally unique Stemona alka-
loids. Using guinea pigs, it was shown that the accumulated
alkaloids in roots of S. tuberosa, i.e. tuberostemonine and its
derivatives as well as stemoninine (Lin et al. 2006, 2008), are
responsible for the antitussive effects (Xu et al. 2006, 2010).
Furthermore, extracts obtained from S. tuberosa are used in
treatment of enteric helminth worms and various ectoparasites
in humans and livestock (Terada et al. 1982; Xu 2000).
The bioactivity and unique structures of the Stemona alka-
loids are still a focus of phytochemical research. Recently, sev-
eral studies of these alkaloids isolated from different species of
Stemona Lour. and Stichoneuron Hook.f. have been published
(Kongkiatpaiboon et al. 2011; Ramli et al. 2013; Gao et al.
2014). To date more than 150 Stemona alkaloids are known
(Greger 2006; Pilli et al. 2010; Wang & Chen 2014). These are
structurally characterised by a pyrrolo- or pyrido [1,2-a]-aze-
pine core. Greger (2006) classified them into protostemonine,
croomine and stichoneurine groups according to their core
skeleton (Fig. 1).
Tocopherols are present in all species and organs; these have
radical scavenging and several other activities (Munne-Bosch &
Alegre 2002; Falk & Munne-Bosch 2010). Tocopherols belong
to the vitamin E group and are structurally characterised by a
chromanol core and a phytol side chain. The substitution pat-
tern of the chromanol ring varies in C-methylation, forming
well-defined a, b, c and d tocopherol derivatives (IUPAC-IUB
Joint Commission on Biochemical Nomenclature (JCBN)
1982). The structurally closely related dehydrotocopherols pos-
sess a double bond between C3 and C4. Biosynthetically, they
are derived from shikimate and 1-deoxy-D-xylulose-5-phos-
phate pathways (Lichtenthaler 1999; Munne-Bosch & Alegre
2002; Lushchak & Semchuk 2012). Brem et al. (2004) first
described several dehydrotocopherol derivatives with antioxi-
dant properties as characteristic chemicals of various Stemona
species, mainly collected in Thailand. Recently, two optical iso-
mers of 3,4-d-dehydrotocopherol from roots of S. tuberosa
were published (Kil et al. 2015).

Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands 1
Morphological and chemical heterogeneity in S. tuberosa
From a morphological viewpoint, S. tuberosa individuals col-
lected in China show notable variation in flower characters
such as colour, size and shape. These might have been over-
looked in the past due to the ethnobotanical importance of the
roots. This apparent divergence suggests taxonomic hetero-
geneity (Fig. 2), which has already been noted in the Flora of
Thailand (Duyfjes & Inthachub 2011). Given our aim to reveal
the chemical variations within this species, we focus on both
composition of the alkaloids and tocopherol derivatives within
morphologically different individuals. In particular, we focus
on the phytochemical co-occurrences of alkaloids and toco-
pherols, since both are known chemical markers for S. tuberosa
and S. phyllantha Gagnep., respectively (Brem et al. 2004;
Schinnerl et al. 2007; Kongkiatpaiboon et al. 2011). We have
examined 15 accessions collected in southwest China in terms
of their phytochemistry and compared them with two reference
samples of S. tuberosa and S. phyllantha collected in Thailand
(data not shown).
MATERIAL AND METHODS
General experimental procedures
Thin layer chromatography (TLC) analyses on silica gel 60 F254
plates, 0.20-mm thick (tinfoil; Merck, Darmstadt, Germany)
were developed with CH2Cl2/EtOAc/MeOH/NH4OH (50:45:
5.5:0.5) and sprayed with Dragendorff or anisaldehyde reagent.
For preparative TLC, silica gel 60 F254 glass plates 0.25-mm
thick (Merck) were used. HPLC analyses were performed on
Agilent 1100 series (Agilent, Santa Clara, CA, USA) with simul-
taneous UV-diode array detection at 220/254/280/310 nm and
evaporative light scattering detection (ELSD; N2: 3.6 bar;
40 °C). HPLC employed a Hypersil BDS-C18 (250 9 4.6 mm,
5 lm particle size) column with methanol (MeOH) (B) and an
aqueous solution containing 10 mM ammonium acetate (A)
from 55% to 90% B in A within 19 min, 90–100% B in A
within 1 min, 100% B retained for 15 min and an injection vol-
ume of 10 ll at a flow rate of 1.0 ml
min 1.
Plant material
Plant material (Table 1) was collected in 2015 and identified by
G. Chen according to the Flora of China (Ji & Dufjes 1997).
2 Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands
Chen, Brecker, Felsinger, Cai, Kongkiatpaiboon & Schinnerl
Fig. 1. Main structural types of Stemona alkaloids
according to Greger (2006).
Herbarium specimens were deposited at the Herbarium of
Kunming Institute of Botany (KUN; http://www.kun.ac.cn).
The collected roots were sliced and dried at 35–40 °C before
the phytochemical examination.
Sample preparation for HPLC and TLC analyses
For HPLC measurements sliced roots were ground using a cof-
fee mill and an aliquot of the obtained powder (50.0 mg) was
accurately weighed and extracted with 0.7 ml methanol in an
Eppendorf cup under sonication for 15 min. After centrifuga-
tion at 14,000 g for 20 min, 0.5 ml of the supernatant was
directly subjected to HPLC. For TLC, the obtained supernatant
was evaporated and dissolved in 100 ll acetone.
Quantitative evaluation of the samples
An aliquot of 50 mg of each powdered sample was weighed
using an analytical balance and extracted twice with 500 ll ace-
tone. The combined extracts were adjusted to 2 mg
ml 1 with
acetone and subjected to HPLC. Tuberostemonine (1) and its
isomers 3 and 4 were quantified using ELS detection, while com-
pounds 5, 6 and 7 were quantified with UV detection at 220 nm
since these compounds possess a chromophore. For calibration
curves, tuberostemonine (1), stemoninine (5) and 3,4-dehydro-
d-tocopherol (6) were used as external standards. For 1, stan-
dards of 1000, 2000, 3000 and 4000 lg
ml 1; for 5, standards of
125, 250, 500, 750, 1000 and 1250 lg
ml 1; and for 6 standards
of 12.5, 125, 250 and 500 lg
ml 1 were prepared. Each standard
was subjected to HPLC three times. The calibration curves were
obtained by plotting the peak area versus amounts of the stan-
dards. The calculated results are shown in Table 3.
Identification of natural products
All compounds were identified by comparison of their analyti-
cal data with those of known reference compounds or with
data reported earlier. In particular, retention time in HPLC
measurements and UV spectra were used to compare sub-
stances with reference material. Co-chromatographically iden-
tified compounds were assigned by comparison of retention
times and UV spectra using authentic samples isolated in previ-
ous studies (Schinnerl et al. 2007; Kongkiatpaiboon et al.
Chen, Brecker, Felsinger, Cai, Kongkiatpaiboon & Schinnerl
Fig. 2. Flowers of S. tuberosa collected in southwest China. Individuals with flowers of type I accumulate tuberostemonine (1) and its derivatives; individuals
with flowers of type II accumulate stemoninine (5). Exceptions are samples collected in Chuxiong, with accumulation of 5, and Lincang with accumulation of 1
and 2 (Table 1). Photographs: G. Chen.
2011). Compounds 3 and 5 when isolated contained uncertain-
ties in the assignment of peaks in the obtained chromatograms.
Extraction and isolation of chromatographically
unidentifiable compounds
Chongzuo (CHEN20141106)
An amount of 8.64 g of sliced and dried roots was ground and
extracted twice for 2 days with 50 ml acetone and sonication.
The filtered extracts were combined and the solvent evapo-
rated. The resulting brownish residue (82 mg) was subjected to
column chromatography using Sephadex LH 20 (GE Health-
care, Little Chalfont, UK) and eluted with 30% acetone in
methanol. Fractions showing a positive spot with Dragendorff
reagent on TLC were pooled (10 mg) and finally purified by
preparative TLC, which produced 5.1 mg of compound 5.
Gulinqing (CHEN20131126)
An amount of 110 mg partly purified fractions was obtained
from 8.15 g powdered roots using acetone extraction and chro-
matography as described above. The final purification with
preparative TLC of Dragendorff-positive fractions (18 mg)
yielded 4.1 mg of pure compound 3.
Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands
Morphological and chemical heterogeneity in S. tuberosa
Structural elucidation
Identification of the two isolated compounds was made via a
detailed structural analysis using NMR and MS. For NMR
spectroscopic measurements each compound was dissolved in
acetone-d6 (~5.0 mg in 0.7 ml) and transferred into 5-mm high
precision NMR sample tubes. Measurements were performed
on a Bruker DRX-600 (Bruker, Billerica, MA, USA) at
600.13 MHz (1H) and 150.91 MHz (13C) using the Topspin 3.1
software. Measurement temperature was 298  0.05 K. Resid-
ual acetone-d5 was used as internal standard in 1H spectra (dH
2.05) and CD3OD in 13C spectra (dC 30.60 (CD3)). One-
dimension spectra were recorded with 14 ppm (1H) and
240 ppm (13C). To determine the two-dimension COSY,
TOCSY, NOESY, HMQC and HMBC spectra, 128 experiments
with 2048 data points each were recorded and Fourier-trans-
formed. Mass spectra were measured on a high-resolution
time-of-flight (HR-TOF) mass spectrometer (maXis; Bruker
Daltonics) by direct infusion electrospray ionisation (ESI) in
positive ionisation mode (mass accuracy 5 ppm). TOF MS
measurements were performed within the selected mass range
of m/z 100–2500. ESI used a capillary voltage of 4 kV to main-
tain a (capillary) current between 30 and 50 nA. All analytical
3
Morphological and chemical heterogeneity in S. tuberosa Chen, Brecker, Felsinger, Cai, Kongkiatpaiboon & Schinnerl

data were evaluated by structure determination as well as spec-

tral identities and similarities. In particular, data from NMR
measurements were evaluated using the combined CSEARCH
and SPECINFO database systems (Sch€ utz et al. 1997; Robien
2009). All data were further compared with the original pub-
lished data.

RESULTS AND DISCUSSION

Flower morphology
The flowers of S. tuberosa collected in China show distinct vari-
ations in size and shape (Fig. 2). For example, flowers of sam-
ples collected in Guilin, Yichang, Malipo, Chen zhou, Chahe,
Chuxiong, Fangchenggang and Mengzi are quite similar in size,
shape and colour. The dark parallel veins are clearly visible and
contrast with the light greenish colour of the tepals. Greenish
appendices form a strong contrast to the stamens and concur
in colour with the dorsal side of the tepals, which are approxi-
mately 7 cm long. These characters concur with those of
S. phyllantha from Thailand (Inthachub 2008; Inthachub et al.
2010), which is not listed for China in the Flora of China (Ji &
Dufjes 1997). Flowers of individuals collected in Tianlin, Jingxi,
Lincang and Chongzuo differ from flowers of the above
described flower type in length (ca. 3.5 cm), lanceolate form
and colour of the tepals. The ventral sides of the tepals are
strongly coloured and veins are nearly invisible. The appen-
dices of anthers form a strong contrast to the anthers and
tepals. Flower size of this type is approximately half that of the
former type. These described floral characters coincide with the
description of S. tuberosa from Thailand (Inthachub 2008;
Inthachub et al. 2010). Due to a lack of flowers, reliable state- Fig. 3. Structural formulae of alkaloids (1–5) and tocopherols (6 and 7)
ments on individuals collected in Fuligong, Gulinqing and detected in investigated individuals of S. tuberosa of both chemotypes.
Daxingzhai are not possible. Tuberostemonine (1), tuberostemonine A (2), neotuberostemonine (3),
tuberostemonine N (4), stemoninine (5), 3,4-dehydro-d-tocopherol (6), 3,4-
dehydro-c-tocopherol (7). Stereochemistry of 6 and 7 was not determined.
Distribution of alkaloids and tocopherols
The distribution pattern of alkaloids in roots of the investi-
gated individuals was determined by HPLC analyses combined collected in Fuligong. From the sample collected in Gulinqing,
with ELSD detection (Table 1; Fig. 4). This detection method neotuberostemonine (3) was isolated as main component.
was earlier applied to Stemona alkaloids lacking a chromophore In the samples collected in Daxingzhai, Jingxi, Chuxiong
group (Li et al. 2007; Kongkiatpaiboon et al. 2011). Two iden- and Chongzuo compounds 1–4 could not be detected. Results
tified compounds were further isolated and structurally verified shown in Table 1 indicate a preponderance of stemoninine (5)
using NMR and MS analyses. Structural formulae of the in these samples. Remarkably, the sample collected in Chux-
detected alkaloids are shown in Figs 3 and 4. From samples iong shows flower characters of the tuberostemonine chemo-
A–N (Table 1) the accumulation pattern of the alkaloids was type. The individual collected in Tianlin shows co-occurrence
revealed. Identification of alkaloids failed for the sample col- of stemoninine (5) together with neotuberostemonine (3). A
lected in Mengzi (O) due to the low alkaloid content and poor possible reason for the divergence between these two latter
condition of the available plant material. samples might be the occurrence of various phaeno- and/or
The alkaloid tuberostemonine and its derivatives (1–4) are genotypes in this area. Studies of the genome and/or the pro-
dominant in this species (Figs 3 and 4; Table 1). In samples teome may clarify these deviations. In general, the results are in
collected in Guilin, Yichang, Malipo, Lincang and Chen zhou accordance with previously published data (Jiang et al. 2006).
the compounds tuberostemonine (1) and tuberostemonine A In this study, similar results with the occurrence of compounds
(2) were detected. Identical patterns were earlier reported from 1, 3 and 5 in roots of S. tuberosa collected in China are
S. phyllantha, mainly collected in Thailand (Schinnerl et al. reported. Interestingly, croomine or its derivatives could not be
2007; Kongkiatpaiboon et al. 2011). In the sample collected in detected in the examined samples.
Fangchenggang, compounds (1) and (2) are, however, accom- The tocopherol derivatives 3,4-dehydro-d-tocopherol (6)
panied by their isomers neotuberostemonine (3) and and 3,4-dehydro-c-tocopherol (7) (Fig. 3) were detected as
tuberostemonine N (4), whilst in the sample from Chahe com- major and minor constituents, respectively, in 15 geographi-
pounds 1 and 4 are detectable. Tuberostemonine N (4) is the cally different provenances of S. tuberosa. Only in the individ-
only Stemona alkaloid that could be detected in the sample ual collected in Chahe was compound 7 not detectable

4 Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands
Chen, Brecker, Felsinger, Cai, Kongkiatpaiboon & Schinnerl Morphological and chemical heterogeneity in S. tuberosa

Fig. 4. HPLC profiles and UV spectra of 5, 6 and 7 of

S. tuberosa samples collected in Guilin, Fuligong,
Gulinqing and Chongzuo, representing the different
accumulation tendencies. a: HPLC-ELSD profiles, b:
HPLC-UV profiles at 220 nm. Tuberostemonine (1),
tuberostemonine A (2), Neotuberostemonine (3),
tuberostemonine N (4) and stemoninine (5) 3,4-
dehydro-d-tocopherol (6), 3,4-dehydro-c-tocopherol (7).

(Table 1). Despite such wide geographic distribution, this spe-
cies shows a conserved pattern of the chromenol moiety con-
taining tocopherols, while tocopherols containing a chromanol
moiety were not detected. Earlier reports from individuals of
S. tuberosa and S. phyllantha collected in different parts of
Thailand, Vietnam and Indonesia fit this pattern well (Table 2),
and concomitant investigated individuals of S. collinsiae Craib
and S. curtisii Hook.f. possess a similar pattern (Brem et al.
2004).
The biological role of tocopherol derivatives in this plant
species is still unknown. A feasible explanation is the protection
of other compounds from oxidation. In the case of S. tuberosa,
the main compound tuberostemonine (1) seems to be sensitive
against oxidation since numerous derivatives of this alkaloid

Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands 5
Morphological and chemical heterogeneity in S. tuberosa Chen, Brecker, Felsinger, Cai, Kongkiatpaiboon & Schinnerl

Table 1. Collection data and qualitative screening results of alkaloids and tocopherols from the roots of S. tuberosa.

Alkaloids Tocopherols

Origin Herbarium specimen 1 2 3 4 5 6 7

Guilin (A) CHEN20141017 ■ h ● ○

Yichang (B) CHEN20140813 ■ h ● ○
Malipo (C) CHEN20111002 ■ h ● ○
Lincang (D) CHEN20090324 ■ h ● ○
Chen zhou (E) CHEN20140622 ■ h ● ○
Fangchenggang (F) CHEN20141023 ■ h h h ● ○
Chahe (G) CHEN20160623 ■ h ●
Fuligong (H) CHEN20130928 ■ ● ○
Gulinqing (I) CHEN20131126 ■ ● ○
Tianlin (J) CHEN20150723 □ ■ ● ○
Daxingzhai (K) CHEN20140425 ■ ● ○
Jingxi (L) CHEN20130618 ■ ● ○
Chuxiong (M) CHEN20150629 ■ ● ○
Chongzuo (N) CHEN20141106 ■ ● ○
Mengzi (O) CHEN20061105 ● ○

Tuberostemonine (1), tuberostemonine A (2), neotuberostemonine (3), tuberostemonine N (4), stemoninine (5), 3,4-dehydro-d-tocopherol (6), 3,4-dehydro-c-
tocopherol (7) (Fig. 3). Squares (■, □) and circles (●, ○) are used for alkaloids and dehydro-tocopherols, respectively. The filled and empty symbols are used to
distinguish between major and minor amounts within each class of compound, respectively. A–O describes the origin of the samples with respect to Fig. 5.

are suspected to be artefacts (Greger 2006). Evaluation of the
plant material used in previous reports indicates regular
co-occurrence of 3,4-dehydro-d-tocopherol (6) with tuberoste-
monine (1) and its derivatives, or with croomine-type
compounds (Brem et al. 2004; Schinnerl et al. 2007; Kongkiat-
paiboon et al. 2011; Table 2). Such constant co-occurrence of 6
and 1, as well as the sensitivity against oxidation of 1 and the
antioxidative potential of 6, imply a protective function against
oxidation of the alkaloid. However, further studies on the dis-
tribution of 1 and 6 within root tissue and direct redox interac-
tion between the two molecules are necessary to confirm this
assumption (Table 2).
Quantities of tuberostemonine (1), stemoninine (5) and
3,4-dehydro-tocopherols (6 and 7)
Quantification of the above compounds confirmed the qualita-
tive screening results (Table 1). In general, samples containing
tuberostemonine (1) or its isomers 3 and 4 accumulate much
higher amounts of the respective alkaloids than the
Table 2. Published alkaloids and tocopherols of S. tuberosa and S. phyllan-
tha from previous studies. Numbering of compounds refers to Fig. 3.
stemoninine (5) accumulating samples (Table 3). All samples
belonging to the tuberostemonine chemotype showed distinct
variability in the accumulated amounts of 1. The samples from
Lincang and Fangchenggang accumulated only around 50% of
the calculated amounts of the other samples. Samples of the
stemoninine chemotype had rather similar amounts of 5, with
the exception of the sample collected in Chongzuo (0.55%;
Table 3), in which the concentration of 5 was much higher
than in the other four samples. These results differ from the
previously published data of Li et al. 2007 and Kongkiatpai-
boon et al. 2013. Li et al. (2007) employed HPLC coupled with
DAD and ELS detection, while Kongkiatpaiboon et al. (2013)
used TLC image analyses for quantification of these alkaloids.
In both studies, lower amounts of 1 and 5 were reported. Here
quantities of tocopherols were determined for the first time;
therefore, no comparable data are available for this plant
group. Generally, the amount of the determined tocopherols 6
and 7 was between 0.03% (Fangchenggang) and 0.18% (Chux-
iong). In general, the alkaloid/tocopherol ratio was much lower
in the tuberostemonine chemotype than in the stemoninine
chemotype. Since all the samples were collected from the wild,
the obtained results cannot be connected either to the age of
the plants or to biotic/abiotic factors that might influence the
content of these secondary metabolites.

Herbarium specimen/origin Alkaloids Tocopherols Country

Chemotaxonomic significance of tuberostemonine (1) and
HG 851 1, 2a,b
6c
Thailand stemoninine (5)
HG 919 1, 2a,b 6c Thailand
Drawing on studies with more comprehensive sampling, the
HG 918 Croomine deriv.a,b 6c Indonesia
tuberostemonine chemotype seems to be the major type found
HG 890 Croomine deriv.a,b 6c Thailand
throughout South-East Asia (Schinnerl et al. 2007; Kongkiat-
HG 879 Croomine deriv.a,b 6c Vietnam
paiboon et al. 2011). The newly identified stemoninine chemo-
HG 851 and HG 919 relate to S. phyllantha while HG 918, HG 890 and HG type forms a subgroup providing an interesting variation
879 are assigned to S. tuberosa. aKongkiatpaiboon et al. (2011) and bSchin- within this species. Obviously, the biosynthetic pathway of the
nerl et al. (2007), both deal with the occurrences of alkaloids. cdenotes Stemona alkaloids in this subgroup differs from the tuberoste-
occurrence of tocopherols (Brem et al. 2004). monine chemotype.

6 Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands
Chen, Brecker, Felsinger, Cai, Kongkiatpaiboon & Schinnerl Morphological and chemical heterogeneity in S. tuberosa

Table 3. Content of tuberostemonine (1), neotuberostemonine (3), lack of information on exact collection sites and incomplete
tuberostemonine N (4), stemoninine (5) and dehydrotocopherols (6 and 7). data on the occurrence of these alkaloids, no further geo-
Values are shown as percentage weight (% w/w). graphic segregation can be deduced. The occurrence of 1 and 2
Alkaloids Tocopherols
with some variation towards 3 and 4 coincides with results
reported earlier for S. phyllantha collected in Thailand and
Origin 1 3 4 5 6+7 China (Li et al. 2007; Kongkiatpaiboon et al. 2011). This varia-
tion is now evidenced in samples collected in Fangchenggang,
Guilin (A) 3.07 0.05
Chahe, Fuligong and Gulinqing (F–I; Table 1). The sample
Yichang (B) 3.08 0.07
collected in Tianlin (J) represents a special case due to the
Malipo (C) 3.16 0.09
co-occurrence of neotuberostemonine (3) and stemoninine
Lincang (D) 1.41 0.11
(5). Such a variation representing an intermediate between the
Chen zhou (E) 3.71 0.07
Fangchenggang (F) 1.41 0.03
two chemotypes was already known (Li et al. 2007). Taking
Chahe (G) 2.47 0.10
flower morphology of the examined individuals into account
Fuligong (H) 3.60 0.10 (Fig. 2), the subgroup of tuberostemonine (1) accumulating
Gulinqing (I) 3.60 0.11 individuals is generally similar in flower characters to S. phyl-
Tianlin (J) 0.22 0.12 lantha from Thailand (Inthachub 2008; Inthachub et al. 2010;
Daxingzhai (K) 0.23 0.07 Fig. 2). This differentiation in floral characters suggests species
Jingxi (L) 0.32 0.10 delimitation either at species or sub-species level, for which
Chuxiong (M) 0.26 0.18 DNA sequencing data will be necessary.
Chongzuo (N) 0.55 0.13 To summarise the obtained data, roots of S. tuberosa are
either characterised by accumulation of tuberostemonine and
Linear calibration parameters for tuberostemonine (1): y = 0.0077x 7.0933, its derivatives or accumulation of stemoninine; in this species
(r² = 0.9949); y = 2.7799x + 76.033, (r² = 0.9998) for stemoninine (5),
group, no protostemonine-type alkaloids could be detected.
y = 15.958x + 189.79 (r² = 0.9942) for tocopherols. Quantities of 3 and 4
Both types of compound show a clear segregation within this
were calculated using the calibration parameters for 1.
species that is additionally underlined by some differences in
floral characters. According to these results, individuals A–I
(Table 1) could tentatively be classified as S. phyllantha, while
the stemoninine (5) accumulating samples (K–N; Table 1)
should be retained as S. tuberosa. Thus, the present study also
constitutes a new floristic record for the flora of China. This
species also shows a clear trend in accumulation of the radical
scavenger 3,4-dehydro-d-tocopherol. The co-occurrence of
tuberostemonine or stemoninine together with this radical
scavenger may influence the stability of the respective alkaloids.
Overall, the obtained results are in accordance with previous
studies. In further research, identification of a phylogenetic
backbone and its comparison with studies of morphology and
chemodiversity in this genus would facilitate integration of
achieved results.

Fig. 5. Collection sites of the studied accessions of S. tuberosa. Abbrevia-
tions for countries are: CN, China; MM, Myamar; LAO, Laos; TH, Thailand.
A–O denote collection sites (Table 1). Tuberostemonine (1) accumulating
samples – filled circle (●); samples accumulating stemoninine (5) – asterisks
(*). x refers to sample in which alkaloids could not be detected.
The occurrence of tuberostemonine and stemoninine
chemotypes in samples collected in this study follows no clear
geographic segregation, indicating no clear regional separation
between the subgroups. Only a small area in the Chinese
mountain region close to Vietnam can be deduced from map-
ping the geographic position of the collection sites for occur-
rence of the stemoninine chemotype (Fig. 5). Due to both a
Isolated alkaloids
Neotuberostemonine (3) – amorphous powder; HRMS (ESI)
m/z: 376.2479 [C22H34NO4]+ (calcd. 376.2482); 1H NMR (ace-
tone-d6, 600 MHz, dH): 4.60 (dd, J = 3.3 & 3.0 Hz, 1H, H-11),
4.43 (ddd, J = 11.5, 7.7 & 5.4 Hz, 1H, H-18), 3.30 (ddd,
J = 14.0, 7.7 & 1.2 Hz, 2H, H-3), 3.23 (dd, J = 4.9 & 4.8 Hz,
1H, H-9a), 3.12 (m, 1H, H-5a), 3.01 (dq, J = 6.7 & 7.1 Hz, 1H,
H-13), 2.99 (m, 1H, H-5b), 2.64 (ddq, J = 12.1 5,4 & 7.0 Hz,
1H, H-20), 2.45 (m, 1H, H-19a), 2.18 (m, 1H, H-12), 1.91 (m,
1H, H-8a), 1.79 (m, 1H, H-9), 1.75 (m, 1H, H-1), 1.72 (m, 1H,
H-10), 1.71 (m, 2H, H-2a,b), 1.70 (m, 2H, H-6a,b), 1.69 (m, 1H,
H-16b), 1.62 (m, 1H, H-7a), 1.59 (m, 1H, H-8b), 1.50 (m, 1H,
H-19b), 1.49 (m, 1H, H-7b), 1.28 (m, 1H, H-16a), 1.17 (d,
J = 7.0 Hz, 3H, H-22), 1.16 (d, J = 7.0 Hz, 3H, H-15), 0.99 (t,
J = 7.2 Hz, 3H, H-17); 13C NMR (acetone-d6, 150 MHz, dH):
180.4 (s, C-14), 180.0 (d, C-21), 82.3 (d, C-11), 79.9 (d, C-18),
68.5 (d, C-3), 68.1 (d, C-9a), 51.6 (t, C-5), 43.7 (d, C-13), 43.0
(d, C-12), 39.0 (d, C-1), 38.0 (d, C-9), 36.9 (d, C-10), 36.1
(d, C-20), 35.9 (t, C-19), 34.7 (t, C-2), 31.1 (t, C-6), 30.9
(t, C-8), 24.8 (t, C-7), 22.7 (t, C-16), 15.9 (q, C-22), 12.3

Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands 7
Morphological and chemical heterogeneity in S. tuberosa Chen, Brecker, Felsinger, Cai, Kongkiatpaiboon & Schinnerl

(q, C-17), 11.3 (q, C-15). All data are in agreement with data
reported for this compound in Ye et al. (1994).
Stemoninine (5) – amorphous powder; UVMeOH 214 nm,
HRMS (ESI) m/z: 390.2272 [C22H32NO5]+ (calcd. 390.2275);
1
H NMR (acetone-d6, 600 MHz, dH): 6.90 (d, J = 1.6, 1H,
H-13), 4.22 (ddd, J = 9.4, 7.2 & 5.4 Hz, 1H, H-21), 4.01 (ddd,
J = 10.8, 9.3 & 3.2 Hz, 1H, H-7), 3.74 (dt, J = 9.7 & 5.4 Hz, 1H,
H-9), 3.46 (m, 1H, H-4a), 3.37 (ddd, J = 10.2, 7.0 & 5.4 Hz,
1H, H-2), 2.95 (m, 1H, H-4b), 2.64 (ddq, J = 11.4, 7.1 &
5.4 Hz, 1H, H-23), 2.43 (m, 1H, H-22a), 2.42 (m, 1H, H-8),
2.15 (ddd, J = 12.1, 6.8 & 5.5 Hz, 1H, H-11), 2.01 (m, 1H,
H-6a), 1.93 (m, 1H, H-10b), 1.86 (d, J = 1.6, 3H, H-18), 1.82
(m, 1H, H-19b), 1.72 (m, 1H, H-1a), 1.71 (m, 1H, H-5a), 1.57
(m, 1H, H-22b), 1.51 (m, 1H, H-10a), 1.47 (m, 1H, H-19a),
1.41 (m, 1H, H-6b), 1.39 (m, 1H, H-1b), 1.30 (m, 1H, H-5b),
1.16 (d, J = 7.1, 1H, H-23), 0.85 (t, J = 7.3, 3H, H-20); 13C
NMR (acetone-d6, 150 MHz, dH): 180.4 (s, C-24), 172.8 (s,
C-15), 115.4 (s, C-12), 147.2 (d, C-13), 134.1 (s, C-14), 84.7 (d,
C-21), 82.7 (d, C-7), 65.0 (d, C-2), 60.1 (d, C-9), 54.6 (d, C-8),
52.3 (d, C-11), 46.8 (t, C-4), 37.3 (t, C-6), 36.2 (d, C-23), 35.7
(t, C-22),28.0 (t, C-1), 27.9 (t, C-10), 21.8 (t, C-5), 21.7 (t,
C-19), 16.1 (q, C-26), 14.0 (q, C-20), 11.2 (q, C-18). All data
are in agreement with data reported for this compound in
Cheng et al. (1988).
ACKNOWLEDGEMENTS
We gratefully acknowledge Peter Unteregger (Institute of
Organic Chemistry, University of Vienna) for recording the
mass spectra, and Andreas Berger for critical reading of the
manuscript. Support for this study was provided by grants
from the Science and Technology Research Program of Kun-
ming Institute of Botany, the Chinese Academy of Sciences
(Grant No. KIB2017006) and the Young Academic and Techni-
cal Leader Raising Foundation of Yunnan Province to G. Chen
(2015HB091). The authors acknowledge aseanup.com for pro-
viding the map (http://www.aseanup.com/free-maps-asean-
southeast-asia/(2016/10/27).

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8 Plant Biology © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands