Diarylheptanoids from Dioscorea villosa (Wild Yam)

Article abstract of DOI:10.1021/np300603z

A fractionation methodology aimed at the metabolomic mining of new phytoconstituents for the widely used botanical, wild yam (Dioscorea villosa), makes use of 1D qHNMR and 2D NMR profiles along the preparative fractionation pathway. This quantifiable and structural guidance led to the isolation of 14 diarylheptanoids (1-14), including five new compounds (1-5) with a tetrahydropyrano core skeleton. The structures, including the absolute configurations of both new and previously known diarylheptanoids, were assigned by a combination of HRESIMS, 1D and 2D NMR, ¹H iterative full spin analysis (HiFSA), and Mosher's ester method. The isolation yields were consistent with yields predicted by qHNMR, which confirms the (semi)quantifiable capabilities of NMR-based preparative metabolomic mining. The qHNMR-aided approach enabled the identification of new and potentially significant chemical entities from a small fraction of the plant extract and, thereby, facilitated the characterization of the residual complexity of the D. villosa secondary metabolome. LC-MS profiling of different D. villosa accessions further confirmed that the diarylheptanoids represent genuine secondary metabolites, which can serve as a new class of markers for botanical integrity analysis of D. villosa.

Full text of DOI:10.1021/np300603z

Article
pubs.acs.org/jnp
Diarylheptanoids from Dioscorea villosa (Wild Yam)†
Shi-Hui Dong, Dejan Nikolic, Charlotte Simmler, Feng Qiu, Richard B. van Breemen, Djaja D. Soejarto,
́
Guido F. Pauli, and Shao-Nong Chen*
UIC/NIH Center for Botanical Dietary Supplements Research, Department of Medicinal Chemistry and Pharmacognosy, College of
Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States
S
* Supporting Information
ABSTRACT: A fractionation methodology aimed at the metabolomic mining of new phytoconstituents for the widely used
botanical, wild yam (Dioscorea villosa), makes use of 1D qHNMR and 2D NMR profiles along the preparative fractionation
pathway. This quantifiable and structural guidance led to the isolation of 14 diarylheptanoids (1−14), including five new
compounds (1−5) with a tetrahydropyrano core skeleton. The structures, including the absolute configurations of both new and
1
previously known diarylheptanoids, were assigned by a combination of HRESIMS, 1D and 2D NMR, H iterative full spin
analysis (HiFSA), and Mosher’s ester method. The isolation yields were consistent with yields predicted by qHNMR, which
confirms the (semi)quantifiable capabilities of NMR-based preparative metabolomic mining. The qHNMR-aided approach
enabled the identification of new and potentially significant chemical entities from a small fraction of the plant extract and,
thereby, facilitated the characterization of the residual complexity of the D. villosa secondary metabolome. LC-MS profiling of
different D. villosa accessions further confirmed that the diarylheptanoids represent genuine secondary metabolites, which can
serve as a new class of markers for botanical integrity analysis of D. villosa.
chemically converted into the mammalian hormone progester-
one.4−6 Subsequently, diosgenin was used as starting material
for the synthesis of cortisone and norethindrone.7 The latter
became known as a highly potent and orally active progesta-
tional agent and was the key ingredient in the first birth control
pills in the 1960s.7
Today, dietary supplements containing wild yam extracts are
popular among women for the alleviation of menopausal
symptoms and are widely used as alternatives to hormone
replacement therapy, although no direct evidence exists for the
estrogenic activity of wild yam extracts.8,9 A comprehensive
literature survey reveals that phytochemical information on wild
yam is limited in chemical diversity: so far, only 12 steroidal
saponins and two flavan-3-ol glycosides having been reported as
he genus Dioscorea in the family Dioscoreaceae comprises
Tover 600 species, which are found throughout the tropical
and temperate regions of the world. Dioscorea species are
widely used as botanical dietary supplements. These plants are
well known for containing steroidal saponins, which have been
used as marker compounds for quality control of the botanical
products.1 The roots and rhizomes of Dioscorea villosa L. are
known as “wild yam”. This species, native to North America, is
a twining tuberous vine. Since the 18th century, herbalists have
been using wild yam to treat menstrual cramps and problems
related to childbirth, as well as for upset stomach and coughs.2
In 1940, the rhizomes of D. villosa were discovered to be an
important source of diosgenin,3 a phytoestrogen that acts on
the mammary epithelium of ovariectomized mice and can be
†Residual Complexity and Bioactivity, Part 16 (see S1,
Supporting Information).
Received: September 1, 2012
Published: December 17, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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1
Figure 1. Representation of the concept of H NMR-guided fractionation for the metabolomic mining of Dioscorea phytoconstituents. A 30 mg
aliquot of crude MeOH extract of wild yam was carefully dried in vacuo and dissolved in 600 μL of DMSO-d6, and a high S/N 1H NMR spectrum
was collected (400 MHz, 5 mm broadband probe with ATM, NS = 5 K). Under these conditions, signals in the aromatic region between δH 6.0 and
8.5 (2.47%) were readily detected, and some of them could even be recognized as diagnostic AA′XX′ resonances of the subsequently isolated
diarylheptanoids.
major secondary metabolites.1,3,10−12 From both chemo-
taxonomic and metabolomic perspectives, Dioscorea species
can also be considered underexplored and likely contain other
classes of secondary metabolites. The relatively narrow
spectrum of known phytoconstituents limits our current
understanding of the significance of wild yam as a dietary
supplement, in particular with regard to the potential relief of
menopausal symptoms. In addition to considering the relevance
of residual complexity (see ref 13 and S1, Supporting
Information) in the assessment of phytoconstituents from D.
villosa, and plant metabolomes in general, the present study also
took into account the role of alternative chromatography in the
discovery of new classes of secondary metabolites, as has been
shown for well-studied plants such as black cohosh (Actaea
racemosa (L.) Nutt., syn. Cimicifuga racemosa L.).14 As part of
the research in the UIC/NIH Botanical Center, aimed at the
metabolomic mining of new and potentially interesting
bioactive constituents, the present study explores preparative
fractionation methodology, which utilizes the combination of
both 1D and 2D NMR spectral profiles and diversified
chromatography. One starting point was the 1H NMR
spectrum of the MeOH extract of wild yam, which is dominated
by the signals of the steroidal saponins and saturated lipids in
the range between δ 0.5 and 6.0. Figure 1 shows that, under
higher sensitivity NMR conditions, numerous minor reso-
nances can be detected between δ 6.0 and 8.5, which cannot be
assigned to known Dioscorea constituents. Using qHNMR, an
enrichment efficiency parameter (see Results and Discussion)
was established to guide the fractionation workflow toward the
isolation of the putatively new aromatic metabolites.
members of this metabolite class from D. villosa.15 Structure
elucidation was performed to the level of absolute configuration
on the basis of 1D and 2D NMR data, H iterative full spin
system analysis (HiFSA), advanced Mosher’s ester analysis, and
chiral HPLC. Furthermore, LC-MS profiling confirmed that the
diarylheptanoids represent genuine secondary metabolites of
wild yam (S2, Supporting Information).
1
Primary fractionation of the crude MeOH extract used a
MeOH−H2O solvent gradient on a preparative C18 solid-phase
extraction (SPE) cartridge and effectively enriched the aromatic
components into three fractions, which were further purified by
VLC, MPLC, and HPLC. The resulting 14 aromatic
compounds include both new tetrahydropyrano (1−5) and
acyclic (6−14) diarylheptanoids, marking the first report of
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Figure 2. ΔδS−R values of MTPA esters of 1, 4, 6, and 8 used for the determination of absolute configuration.
The occurrence of diarylheptanoids in the genus Dioscorea
was first discovered in 2004,16 reporting three such compounds
from D. spongiosa, a species that had been previously
authenticated as D. septemloba.17 Since then, two additional
analogues have been reported from D. spongiosa18 and one from
D. bulbifera.19 Diarylheptanoids are well-known and major
secondary metabolites from the Zingiberaceae. They possess a
1,7-diphenylheptane skeleton and exhibit prominent pharma-
cological activities such as estrogenic, anticancer, antibacterial,
antioxidative, anti-inflammation, and antiosteoporotic proper-
ties.15 The first diarylheptanoid to be discovered was curcumin,
a yellow pigment from turmeric (Curcuma longa L.,
Zingiberaceae) first purified in 1815.15 Curcumin has been
studied extensively both in vitro and in vivo, including human
clinical trials for a variety of diseases, e.g., multiple myeloma,
pancreatic cancer, myelodysplastic syndromes, colorectal
cancer, psoriasis, and Alzheimer’s disease.15,20−22 Considering
the immense body of existing research evidence, the present
metabolomic mining of a variety of diarylheptanoids in wild
yam widens the biochemical profile of Dioscorea plants and
potentially offers new biochemical leads for the development of
wild yam and other Dioscorea botanical products. Herein, we
present the details of the isolation methodology and the
structure elucidation of these new diarylheptanoids.
compounds, thus, serving as the enrichment efficiency
parameter. The most concentrated aromatic fractions, 4−6,
were combined for further purification by VLC, MPLC, and
HPLC. The purification scheme eventually afforded 14
diarylheptanoids (1−14), five of which (1−5) were new
compounds containing a tetrahydropyrano ring in the heptane
portion of the molecule. The absolute configurations of 6 and 7
were assigned for the first time. The presence of enantiomeric
pairs in four of the purified products (2/3, 4/5, 6/7, and 8/9)
was recognized only during the effort to assign their individual
absolute configurations using the advanced Mosher’s ester
method. The generation of HiFSA data sets (see subsection
General Experimental Procedures in the Experimental Section)
for 1−7 (S4, Supporting Information) aided the unambiguous
assignments of the partially complex 1H NMR signals and, thus,
was instrumental in the interpretation of the chiral NMR
experiments. Moreover, the confirmation of δ and J values
(Table 1) by HiFSA to 0.1 ppb and 0.01 Hz precision,
respectively, provided valuable data for unambiguous future
structure dereplication of these compounds.
Structure Elucidation. Compound 1 was obtained as a
white, amorphous powder, and its molecular formula was
established as C19H20O4 on the basis of HRESIMS. Thus, the
carbon skeleton required 10 indices of hydrogen deficiency.
The 1H NMR spectrum of 1 (Table 1) exhibited the typical dt-
like resonances of two aromatic AA′XX′ spin systems at δH
7.228 and 6.714 (J = 8.5 and 2.6 Hz, each 2H), as well as 7.215
and 6.754 (J = 8.4 and 2.5 Hz, each 2H), respectively,
indicating the presence of two p-disubstituted benzene rings.
Accordingly, the 13C BB and DEPT NMR spectra of 1 (Table
1) exhibited four methine resonances at δC 128.80, 128.74,
116.09, and 116.39 (each 2C), as well as four quaternary carbon
resonances at δC 158.27, 157.90, 135.04, and 130.02. Two
proton resonances at δH 6.533 and 6.062 exhibiting a large
RESULTS AND DISCUSSION
■
Targeted Purification. The purification scheme that
yielded the diarylheptanoids started with the EtOAc partition
of the MeOH extract of D. villosa roots/rhizomes. A suspension
of this partition in MeOH−H2O (1:9, v/v) was loaded on a
vacuum SPE cartridge containing C18-RP silica gel as a packing
material. Eleven primary fractions were collected, from elution
with a gradient of a MeOH−H2O solvent system from 0:10 to
1
10:0 in 10% intervals. The H NMR spectra of all primary
1
coupling constant of 16.0 Hz were also observed in the H
fractions were examined for the abundance of aromatic
resonances. In order to establish a quantifiable measure for
the enrichment of the unknown aromatic metabolites, the
resonances of the steroidal methyl groups appearing between δ
0.5 and 1.3 were used as an internal reference and the integral
ratio of the unexplored aromatic region (δ 6.0 to 8.5) relative to
the lipophilic region (δ 0.5 to 1.3) was calculated (see
Extraction and Isolation in the Experimental Section as well as
S3, Supporting Information). This value was determined for
both the extract and the primary fractions and allowed
monitoring of the relative content of the aromatic target
NMR spectrum of 1, indicating the presence of an E double
bond. The 13C DEPT experiment demonstrated three oxy-
genated methines at δC 75.20, 74.76, and 65.47, along with two
methylene groups at δC 40.97 and 39.61. These diagnostic
NMR data clearly showed that 1 is a diarylheptanoid.15 The
1H−1H COSY NMR spectrum of 1 showed a continuous series
of correlations from H-1 to H-7, indirectly establishing the C−
C connectivity of the heptane moiety. The presence of an
oxygen bridge between C-1 and C-5 and, thus, the
tetrahydropyrano partial structure of the heptane moiety were
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Figure 3. Comparison of the 1H NMR spectra of the MTPA esters of the enantiomeric mixture of 2 and 3 (δH 4.5 to 5.8). The resonances of H-1
and H-3 could be readily assigned on the basis of their chemical shifts and splitting patterns. However, the four resonances of H-1 revealed a small
but significant difference in their integrals (47.6:52.4, and vice versa), providing evidence for the fact that the isolate was an enantiomeric mixture of
2 and 3 rather than a pure single enantiomer.
determined by the HMBC correlation from H-1 to C-5.
Similarly, HMBC correlations of the H,C pairs H-2′/C-1, H-6′/
C-1, H-1/C-1′, H-1/C-2′, and H-1/C-6′ as well as H-2″/C-7,
H-6″/C-7, and H-6/C-1″ linked the two benzene rings to their
respective C-1 and C-7 positions of the heptane unit.
Consequently, the two OH groups were assigned to C-4′ and
C-4″, which is supported by their chemical shifts and the
molecular formula. ROESY correlations observed between the
four pairs H-1/H-5, H-2α/H-4α, H-3/H2-2, and H-3/H2-4
indicated that the tetrahydropyrano ring assumes a chair
conformation. The large coupling constants between H-1 and
H-2α (J = 12.0 Hz) as well as H-5 and H-4α (J = 11.7 Hz)
indicated the trans-diaxial positions of these protons, leaving
equatorial positions for H-2β and H-4β in the tetrahydropyrano
ring. This was supported by the occurrence of a W-type long-
range coupling constant between H-2β and H-4β (4J = 2.1 Hz).
H-3 was also demonstrated to be in an equatorial position by its
quintet-like splitting pattern arising from only small coupling
constants (dddd, J = 3.0, 2.9, 2.8, and 2.7 Hz). Finally, the
advanced Mosher’s ester procedure23,24 was employed to
determine the absolute configuration of C-3, bearing a
secondary OH group. Treatment with (R)- and (S)-MTPA
chlorides led to esterification of the secondary C-3 OH group
to afford the (R)- and (S)-MTPA derivatives (1R and 1S),
respectively. By observing the 1H NMR chemical shift
difference values (ΔδS−R) of the heptane moiety, the absolute
configuration of C-3 was determined to be S (Figure 2). Hence,
the structure of 1 was elucidated as (1S,3S,5R,6E)-1,7-bis(4-
hydroxyphenyl)-1,5-epoxy-3-hydroxyhept-6-ene.
and showed the characteristic resonances for two p-
disubstituted benzene rings, three oxygenated methines, and
four methylene groups. Using the same approach as for 1,
utilizing 1H−1H COSY and HMBC spectra, the gross structures
of 2 and 3 were determined to be 6,7-dihydro derivatives of 1, a
finding consistent with the molecular weight and presence of
1
two additional mass units. Upon comparison of the two H
NMR data sets of 1 and 2/3, the observation of congruent
splitting patterns of the signals of H-1, H-3, and H-5 indicated
that the compounds have identical relative configurations,
which was supported by the ROESY correlations of the pairs H-
1/H-5, H-3/H2-2, and H-3/H2-4.
In order to determine the absolute configuration of C-3 in
the original isolate (eventually determined to be an
enantiomeric mixture of 2 and 3), the (R)- and (S)-MTPA
1
derivatives were prepared, which exhibited two very similar H
NMR spectra. The only noticeable difference was the reversed
integration ratio of two sets of resonances in a ratio of
52.4:47.6, indicating that this sample is indeed an enantiomeric
mixture of compounds, 2 and 3.25,26 In the 1H NMR spectra of
the two MTPA derivatives, the resonances of H-1 and H-3
could be clearly assigned on the basis of their chemical shifts
and splitting patterns (Figure 3). Owing to the MTPA
acylation, the H-3 resonances of the derivatives overlapped at
much lower field. The phenyl ring of the MTPA moiety
exhibited the smallest shielding/deshielding effect on H-3 and
did not produce diastereotopic dispersion. In contrast, the
esterification widened the diastereotopic dispersion of the two
H-1 resonances, yielding two separate signals with different
integrations, 52.4:47.6, which was used to determine the ratio
of two parent enantiomers (Figure 3). Subsequently, the
chemical shift difference of the two H-1 resonances was used to
assign the absolute configuration of C-3: as the ΔδS−R values of
the H-1 resonances in the major and minor diastereomers were
+0.221 and −0.221 ppm, respectively, the absolute config-
urations of C-3 of the two parent enantiomers had to be S and
R, respectively (Figure 3). Thus, the structures of 2 and 3 were
Compounds 2 and 3 were purified in the form of an
enantiomeric mixture by HPLC with an RP C18 column. As the
1H NMR spectrum of this mixture before MTPA derivatization
showed a purity of 94.8% assuming it was a single compound,
1D and 2D NMR spectra were recorded for the elucidation of
the gross structure and relative configurations. The HRESIMS
1
gave a molecular formula of C19H22O4. The H and 13C NMR
data (Table 1) revealed that 2 and 3 were also diarylheptanoids
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deduced as (1S,3S,5S)-1,7-bis(4-hydroxyphenyl)-1,5-epoxy-3-
hydroxyheptane and (1R,3R,5R)-1,7-bis(4-hydroxyphenyl)-1,5-
epoxy-3-hydroxyheptane, respectively. In further support of this
finding, the enantiomeric mixture of 2 and 3 was separated by
normal-phase HPLC on a chiral column (Chiralcel OJ; 10 μm,
250 × 4.6 mm) as shown in Figure 4. Using UV detection at
UV 216 nm were used to calculate their relative abundance
ratio as 83.7:16.3. The qHNMR evaluation of the (R)- and (S)-
MTPA derivatives resulted in a different ratio of 76.2:23.8. This
observed difference between UV and qHNMR results might
partially be due to UV-active impurities, but most likely resulted
from the quantification error of the qHNMR assay in this
particular case. As shown only recently,27 both the accuracy and
the precision of qHNMR quantification depend on the signal-
to-noise ratio (S/N), leading to an overestimation of content
and higher errors as the S/N drops below 150. Under the
chosen conditions (∼300 μg sample, 600 μL, 5 mm RT
broadband probe, 400 MHz), the qHNMR spectra of the
MTPA derivatives exhibited a S/N of 13 for the minor
diastereomer (∼20%) in an already mass-limited sample. The
conclusion that the qHNMR-based content is likely too high is
consistent with observations made in the recent qHNMR
validation study27 and supports the UV-based enantiomeric
ratio as being more reliable in this case.
Compounds 6 and 7 were also obtained as an enantiomeric
mixture and further separated by normal-phase HPLC using the
same chiral column as for 2 and 3 (Figure 4). While the peak
areas of HPLC at UV 213 nm showed an enantiomeric ratio of
1
57.2:42.8, overlap of H NMR resonances of the pairs of (R)-
and (S)-MTPA esters did not allow qHNMR quantification.
The molecular formula of 6 and 7 was determined to be
1
C19H20O4 using HRESIMS. Interpretation of the H and 13C
NMR spectra (Table 1) suggested identity with a previously
reported gross structure, 5-hydroxy-1,7-bis(4-hydroxyphenyl)-
1-hepten-3-one, for which no absolute configuration assign-
ment had been made.28 Two aliquots of the major enantiomer,
6, were treated with (R)- and (S)-MTPA chlorides to form
diastereomeric derivatives. Analysis of their H and H−1H
COSY NMR spectra indicated that 6 has the 5R configuration;
accordingly, the minor enantiomer possesses the 5S config-
uration (Figure 2). Similarly, the structures of 6 and 7 were
elucidated as (5R,1E)-1,7-bis(4-hydroxyphenyl)-5-hydroxy-
hept-1-en-3-one and (5S,1E)-1,7-bis(4hydroxyphenyl)-5-hy-
droxyhept-1-en-3-one, respectively.
1
1
Figure 4. Chiral separation of three pairs of enantiomers (2/3, 4/5,
and 6/7) by HPLC (2/3 and 6/7: Chiralcel OJ, 10 μm, 250 × 4.6
mm; 4/5: Chiralpak IA, 5 μm, 250 × 4.6 mm).
224 nm, the peak areas of the two enantiomers were calculated
to give an abundance ratio of 47.6:52.4, which was fully
consistent with the chiral qHNMR determination (compounds
3 to 2).
Seven previously reported diarylheptanoids (8−14) were
characterized from D. villosa for the first time. Compounds 8
and 9 were obtained as enantiomeric mixtures (64.5:35.5) and
determined to be (5S)-1,7-bis(4-hydroxyphenyl)5-hydroxyhep-
tan-3-one and (5R)-1,7-bis(4-hydroxyphenyl)-5-hydroxyhep-
tan-3-one, respectively,29,30 by analysis of their NMR and MS
data as well as by a Mosher’s ester analysis (Figure 2); the three
diarylheptanoids 10−12 were assigned as (4E,6E)-1,7-bis(4-
hydroxyphenyl)hepta-4,6-dien-3-one,31 (3R*,5S*)-1,7-bis-
(4hydroxyphenyl)-3,5-dihydroxyheptane,32 and (3R,5R)-1,7-
bis(4hydroxyphenyl)-3,5dihydroxyheptane,33 respectively, by
comparing their NMR and optical rotation data with those in
the literature; compounds 13 and 14 were obtained as a
mixture (separable, but interconverting) in the ratio 87.3:12.7,
as determined by qHNMR, and their structures were elucidated
as (4Z,6E)-1,7-bis(4-hydroxyphenyl)-5-hydroxyhepta-4,6-dien-
3-one and (1E)-1,7-bis(4-hydroxyphenyl)hept-1-ene-3,5-dione,
respectively,31,34 by 1D and 2D NMR as well as MS analysis.
Stereochemical and Full Spin Analysis. While the
advanced Mosher’s ester method was efficient for the
determination of the absolute configuration of diarylheptanoids
bearing secondary OH groups (1−9), the method was
unsuitable for the symmetric compounds 11 and 12, as the
proton resonances surrounding the OH group(s) in the
derivatives could not be unambiguously assigned. However,
Similarly, compounds 4 and 5 were also obtained as an
1
enantiomeric mixture, recognized upon H NMR analysis of
their MTPA derivatives. The isolates gave the same molecular
formula, C19H22O4, based on HRESIMS. The 1H and 13C NMR
spectra of 4/5 showed the diagnostic signals of a diary-
lheptanoid, in which resonances for two p-disubstituted
benzene rings, three oxygenated methines, and four methylene
groups were observed (Table 1). Analysis of the 1H−1H COSY
and HMBC spectra led to the same gross structure as that of 2
and 3. The coupling patterns of the tetrahydropyrano protons
H-1, H-3, and H-5 indicated that all three protons assume axial
positions in the chair-like conformer, which was further
supported by the NOESY correlations of the pairs H-1/H-3,
H-3/H-5, and H-5/H-1. Using the same method described for
1
2/3, H NMR analysis of the (R)- and (S)-MTPA derivatives,
the absolute configurations of C-3 in 4 and 5 were determined
to be R and S, respectively (Figure 2). Thus, the structures of 4
and 5 were determined as (1S,3R,5S)-1,7-bis(4-hydroxyphen-
yl)-1,5-epoxy-3-hydroxyheptane and (1R,3S,5R)-1,7-bis(4-hy-
droxyphenyl)-1,5-epoxy-3-hydroxyheptane, respectively. Shown
in Figure 4, the enantiomeric pair 4/5 could also be separated
by normal-phase HPLC on a chiral column (Chiralpak IA; 5
μm, 250 × 4.6 mm). The peak areas of the two enantiomers at
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the absolute configuration of enantiomers could be assigned in
analogy with that of 2/3, 4/5, and 8/9 by analyzing the H
series of mostly new diarylheptanoids from a widely used
botanical.
1
In addition to providing qualitative guidance about the partial
structure of unknown metabolites, the present approach is even
capable of predicting isolation yields through qHNMR-based
estimation of their contents.39 The major diarylheptanoid, 12,
was estimated to be present at a concentration of 0.045%,
which compares well with the isolation yield of 0.023% (230
ppm, 205.0 mg from 900 g of extract), especially when
considering unavoidable loss during the four-step isolation
procedure. Similarly, minor aromatic signals, such as the typical
2H pseudo-doublets of the AA′XX′ spin system, allowed the
prediction of isolation yields below 0.002% (<20 ppm) for
further diarylheptanoids, which again matches the achieved
yields of 0.8−15.2 mg for the other isolates, 1−11, 13, and 14.
These preliminary results indicate that the assumptions about
proton and molar ratios required for performing the qHNMR
calculations are valid for practical purposes and allow a
reasonably close prediction of both individual yields and
required scale for the isolation procedure.
Concluding Remarks. While the biological impact and
potential of the wild yam diarylheptanoids require further
study, which is ongoing in our laboratory, the present report
establishes a link between the ubiquitous residual complexity
(RC) of crude metabolomes, such as plant extracts, and the
approach of qHNMR-guided metabolomic mining. These
findings not only extend the utility of qHNMR applications,
but also complement previous conclusions about the relevance
of RC. For example, the RC of a clinical black cohosh extract
was uncovered only after using a pH-targeted approach (pH
zone refinement CPC), which led to the identification of N-
containing metabolites, including the serotonergic active
principle contained in the plant.14
Another link relates to the dereplication and targeted analysis
of individual metabolites in residually complex samples. As has
been shown for the structurally diverse triterpenes of black
cohosh, rapid dereplication can be achieved from standard
(q)HNMR spectra of (residually) complex mixtures, provided
that characteristic and coherent spectroscopic information is
available.13 By presenting the HiFSA profiles of compounds 1−
7, the present study facilitates future studies of Dioscorea
botanicals at the chemistry/biology interface by supplying
comprehensive information for dereplication. As shown
recently, this knowledge also enables the precise quantification
of multiple marker compounds by a combination of HiFSA and
qHNMR,35 thus laying the groundwork for the quantifiable
assessment of the RC of Dioscorea preparations.
NMR spectra of their mixed (R)- and (S)-MTPA esters. In
general, enantiomeric ratios can be determined by integration
1
of H resonances, provided the isolates exhibit some degree of
enantiomeric excess and the MTPA derivatives show
sufficiently disperse diastereotopic proton signals.
Finally, in order to facilitate the future structural
dereplication of congeneric diarylheptanoids from Dioscorea
species, as well as analogues from other genera such as Alpinia,
Zingiber, Curcuma, and Alnus, but also to support development
1
of qHNMR standardization protocols, the precise H NMR
profiles of the newly characterized botanical markers were
generated by means of HiFSA, using the PERCH software tool,
for compounds 1, 2/3, 4/5, and 6/7.35 Molecular structures of
the selected diarylheptanoids were used as starting points to
1
analyze each discrete spin system and predict the basic H
NMR parameters (δ and J). Then, the predicted NMR
parameters were optimized through iterative spin system
calculations using the PERCHit iterator, until the quantum-
1
mechanical simulations replicated the experimental H NMR
spectra (S4, Supporting Information). The final simulated
HiFSA spectra exhibited excellent agreement with the observed
spectra for all spectral lines and line intensities, with a total
root-mean-square deviation (“residual”) of less than 0.07%.
These results further validated the elucidated structures (S4,
Supporting Information). The simulated HiFSA spectra
represent highly precise fingerprints, which can be used to,
unambiguously, identify the marker compounds and distinguish
their resonances from those of impurities by comparison with
the experimental spectra. This enables qHNMR-based
determination of content,35 sample purity,36,37 and purity−
activity investigation.38 The digital HiFSA spectra of these
secondary metabolomic markers can also serve as references for
future metabolomic standardization of wild yam botanicals.35
Diarylheptanoids in Dioscorea villosa. While diary-
lheptanoids are mainly distributed in the roots, rhizomes, and
bark of Alpinia, Zingiber, Curcuma, and Alnus species,15 this is
the first report of this compound class from the rhizomes/roots
of D. villosa. Taking into account existing evidence for the
presence of diarylheptanoids in the genus Dioscorea, it was still
important to confirm that diarylheptanoids represent genuine
secondary metabolites of wild yam. The LC-MS profiling
performed in the present study detected the most abundant
diarylheptanoid, 12, in the crude extract of an authentic, in-
house-cultivated wild yam specimen (S2, Supporting Informa-
tion). Additional evidence for the genuine presence of
diarylheptanoids in wild yam came from characteristic
correlation patterns in the aromatic region of the 1H−1H
COSY NMR spectra of the crude extract, matching those of the
isolates (S56, Supporting Information).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Perkin-Elmer 241 polarimeter at 22 °C in MeOH.
UV spectra were acquired on a Molecular Devices Spectra Max Plus
384 spectrophotometer. IR spectra were acquired on a Perkin-Elmer
577 IR spectrometer. For this purpose, MeOH solutions of the
compounds were dripped onto the IR sample holder and evaporated
to form a thin sample film. NMR spectra were obtained on a Bruker
AV-400 (5 mm broadband probe with automatic tuning and matching
(ATM) capability) or a DPX-400 (5 mm 1H/13C/31P/19F QNP
With respect to the preparative mining of secondary
metabolites, the use of 1H NMR-based information about
structural fragments was shown to provide valuable guidance
for the development of purification protocols. Combined with
diverse chromatographic methodology, this approach facilitates
the discovery of previously unknown and/or potentially
interesting metabolites, even from relatively well-studied plants
or other complex biological matrices. Once specific proton
resonances have been identified and linked to a characteristic
structural class or partial structure, a tailored and more targeted
fractionation protocol can be developed, as is shown here for a
probe) NMR spectrometer (Bruker, Zurich, Switzerland) using
̈
methanol-d4 (for compound 1, by adding 10% CDCl3), pyridine-d5,
or DMSO-d6 as the solvent. The chemical shifts of the residual solvent
signals (δH 3.310 and δC 49.15 for methanol-d4; δH 8.740 for pyridine-
d5; δH 2.500 for DMSO-d6) were used as the chemical shift reference
and also as internal calibrants for qHNMR quantification. Offline
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NMR data processing was performed with MestReNova software
version 8.0.0-10524 for Windows OS (Mestrelab Research, Santiago
de Compostela, Spain). All NMR experiments were performed using
20 to afford the enantiomeric mixture of 8 and 9 (4.0 mg). The
secondary subfraction D was subjected to MPLC on HW-40 gel,
eluted with neat MeOH, to give four tertiary subfractions, D-SF-I to
D-SF-IV. Subfraction D-SF-II was further separated by silica gel VLC,
eluted with hexanes−EtOAc (3:1, v/v), to give five quaternary
subfractions, D-SF-II-1 to D-SF-II-5. D-SF-II-2 was purified by a
semipreparative HPLC using C18 reversed-phase and Chiralcel OJ
chiral columns, successively, to give compounds 6 (3.8 mg) and 7 (3.2
mg), respectively. Fraction D-SF-II-2 was also purified by semi-
preparative HPLC using reversed-phase C18 and Chiralpak IA
columns, successively, to yield compounds 4 (2.5 mg) and 5 (0.8
mg). The secondary subfraction E was purified by silica gel VLC, using
isocratic elution with hexanes−EtOAc (1:1, v/v), to give 11 tertiary
subfractions, E-SF-I to E-SF-XI. E-SF-VIII was determined to be a
pure compound, 12 (205.0 mg). E-SF-V was purified by semi-
preparative HPLC, using a C18 reversed-phase column, to give
compounds 1 (3.3 mg), 11 (3.0 mg), and the enantiomeric mixture of
2 and 3, which was further separated into 2 (2.1 mg) and 3 (1.9 mg)
by semipreparative HPLC with a Chiralcel OJ column. TLC fraction
monitoring as described above was used throughout the separation
process.
1
standard Bruker pulse sequences. The H NMR data were processed
with double zero-filling and Lorentz−Gauss resolution enhancement
(LB −1.8 Hz and GF 0.04 = GB 1.0 Hz) prior to Fourier
transformation. Calculations for the 1H NMR iterative full spin
analysis were performed with the PERCH software package v.2010.1
(PERCH Solutions Ltd., Kuopio, Finland). HRESIMS was carried out
on a Waters Q-TOF Synapt mass spectrometer using the negative
mode. LC-MS analysis for study of the genuine nature of
diarylheptanoid 12 was performed on an AB Sciex 4000 LIT
QTRAP equipped with a Shimadzu UFLC system using a YMC-
Pack ODS-AQ column (150 × 2.1 mm, 3 μm, 12 nm), with the ESI
ion source operating in the positive mode.
Semipreparative HPLC was carried out using a Waters 600
controller with a Waters 2996 photodiode array detector, using
YMC-Pack ODS-AQ (250 × 10 mm, S-5, 12 nm), Chiralcel OJ (250 ×
4.6 mm, 10 μm), and Chiralpak IA (250 × 4.6 mm, 5 μm) columns.
Silica gel (230−400 mesh, Macherey-Nagel), C18 reversed-phase silica
gel (Macherey-Nagel), Sephadex LH-20 (Sigma), and HW-40F gel
(Tosoh) were used for VLC and MPLC. General fraction monitoring
following preparative chromatographic separations was done by TLC
analysis with precoated glass TLC plates (250 μm thickness, K6F Si
gel 60, EM Science, Germany). The compounds were visualized by
spraying the dried plates with 5% H2SO4 in EtOH, followed by heating
at 120 °C for 10 min. All solvents used for LC were of analytical and
chromatographic grade, purchased from Sigma-Aldrich Co., St. Louis,
MO, USA. The (R)- and (S)-MTPA chlorides were purchased from
the same vendor.
(1S,3S,5R,6E)-1,7-Bis(4-hydroxyphenyl)-1,5-epoxy-3-hydroxy-
hept-6-ene (1): white, amorphous powder; [α]2D2 +14.6 (c 0.12,
MeOH); UV (MeOH) λmax (log ε) 263 (4.49) nm; IR (MeOH) νmax
1
3342, 1611, 1515, 1238, 1020, 828 cm−1; H and 13C NMR data, see
Table 1; HRESIMS negative mode m/z 311.1289 [M − H]− (calcd for
C19H19O4, 311.1283).
(1S,3S,5S)-1,7-Bis(4-hydroxyphenyl)-1,5-epoxy-3-hydroxyhep-
tane (2): white, amorphous powder; [α]2D2 +190.2 (c 0.02, MeOH);
UV (MeOH) λmax (log ε) 276 (3.90), 224 (4.65) nm; IR (MeOH)
1
νmax 3310, 2922, 1614, 1515, 1236, 1172, 1070, 1031, 827 cm−1; H
Plant Material. Wild-harvested rhizomes/roots of Dioscorea villosa
L. (4.5 kg) were purchased from Mountain Rose Herbs in August
2011. Authentic rhizomes/roots of in-house-cultivated D. villosa L.
were collected in the UIC Dorothy Bradley Atkins Medicinal Plant
Garden in October 2010. Both samples were authenticated by one of
the authors (D.D.S.). A voucher specimen of the commercial sample
(accession number: BC630) has been deposited at the Field Museum
of Natural History Herbarium, Chicago, IL.
and 13C NMR data, see Table 1; HRESIMS negative mode m/z
313.1446 [M − H]− (calcd for C19H21O4, 313.1440).
(1R,3R,5R)-1,7-Bis(4-hydroxyphenyl)-1,5-epoxy-3-hydroxyhep-
tane (3): white, amorphous powder; [α]2D2 −166.7 (c 0.01, MeOH);
UV (MeOH) λmax (log ε) 276 (3.90), 224 (4.65) nm; IR (MeOH)
1
νmax 3336, 2922, 1614, 1515, 1232, 1172, 1069, 1030, 827 cm−1; H
and 13C NMR data, see Table 1; HRESIMS negative mode m/z
313.1446 [M − H]− (calcd for C19H21O4, 313.1440).
Extraction and Isolation. Authentic, in-house-cultivated D. villosa
(BC 601, 5 g) was extracted with MeOH to give 980.8 mg of crude
extract, of which 128.5 mg was subjected to an SPE VLC using 6 g of
C18 reversed-phase silica gel as packing material. An 11-step gradient of
MeOH−H2O (0:10 to 10:0, v/v, 10% interval) was used as the mobile
(1S,3R,5S)-1,7-Bis(4-hydroxyphenyl)-1,5-epoxy-3-hydroxyhep-
tane (4): white, amorphous powder; [α]2D2 +32.9 (c 0.22, MeOH); UV
(MeOH) λmax (log ε) 276 (3.54), 216 (4.39) nm; IR (MeOH) νmax
1
3336, 2944, 1614, 1515, 1449, 1367, 1236, 1066, 831 cm−1; H and
13C NMR data, see Table 1; HRESIMS negative mode m/z 313.1448
1
phase, and the H NMR spectra of all 11 fractions were examined in
[M − H]− (calcd for C19H21O4, 313.1440).
order to systematically explore the content of aromatic compounds.
1
(1R,3S,5R)-1,7-Bis(4-hydroxyphenyl)-1,5-epoxy-3-hydroxyhep-
tane (5): white, amorphous powder; [α]2D2 −30.6 (c 0.09, MeOH); UV
(MeOH) λmax (log ε) 276 (3.54), 216 (4.39) nm; IR (MeOH) νmax
The integral ratio of two H NMR resonance regions, δH 6.0 to 8.5
and δH 0.5 to 1.3, was used as the parameter to measure the
enrichment efficiency of the target compounds. The ratio was 5.3:94.7
in the MeOH extract, and the ratios for the three primary fractions
with the highest abundance of aromatic resonances were 45.0:55.0 (fr.
4), 34.2:65.8 (fr. 5), and 40.7:59.3 (fr. 6). Thus, fractions 4−6 were
combined to give one enriched aromatic compounds fraction.
1
3336, 2944, 1614, 1515, 1449, 1367, 1236, 1066, 831 cm−1; H and
13C NMR data, see Table 1; HRESIMS negative mode m/z 313.1448
[M − H]− (calcd for C19H21O4, 313.1440).
(5R,1E)-1,7-Bis(4-hydroxyphenyl)-5-hydroxyhept-1-en-3-one (6):
yellow, amorphous powder; [α]2D2 +21.9 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 327 (4.37), 213 (4.38) nm; IR (MeOH) νmax
The dried and milled rhizomes/roots of D. villosa (BC 630, 4.5 kg)
were extracted with MeOH (3 × 6000 mL) to give 900 g of crude
extract. This was suspended in H2O−MeOH (9:1), then successively
partitioned at room temperature between hexanes, CHCl3, EtOAc, and
n-BuOH. Using the aforementioned enrichment strategy, the EtOAc-
soluble partition (43.0 g) was subjected to a C18 SPE VLC, affording
the enriched aromatic compounds fraction (2.4 g), which was
chromatographed on a silica gel VLC eluted with a CHCl3−MeOH
gradient (100:1 to 5:1, v/v) to give five secondary subfractions (A to
E). Subfraction B was further fractionated by MPLC on HW-40F gel,
eluted with MeOH, to afford six tertiary subfractions (B-SF-I to B-SF-
VI). Subfraction B-SF-V was subjected to a silica gel VLC, eluted with
an isocratic SS (hexanes−acetone, 2.5:1, v/v), to afford compound 10
(15.2 mg). Subfraction B-SF-VI was subjected to a silica gel VLC,
eluted with the isocratic SS of hexanes−EtOAc (1:1, v/v), to yield a
mixture of compounds 13 and 14 (2.1 mg). The secondary subfraction
C was chromatographed over silica gel by VLC, eluted with hexanes−
EtOAc (2:1, v/v), and was further purified by LPLC on Sephadex LH-
1
3309, 2943, 1600, 1582, 1514, 1242, 1170, 1021, 819 cm−1; H and
13C NMR data, see Table 1; HRESIMS negative mode m/z 311.1289
[M − H]− (calcd for C19H19O4, 311.1285).
(5R,1E)-1,7-Bis(4-hydroxyphenyl)-5-hydroxyhept-1-en-3-one (7):
yellow, amorphous powder; [α]2D2 −18.1 (c 0.11, MeOH); UV
(MeOH) λmax (log ε) 327 (4.37), 213 (4.38) nm; IR (MeOH) νmax
1
3310, 2940, 1598, 1581, 1514, 1204, 1169, 821 cm−1; H and 13C
NMR data, see Table 1; HRESIMS negative mode m/z 311.1289 [M
− H]− (calcd for C19H19O4, 311.1285).
Preparation of the (R)- and (S)-MTPA Ester Derivatives of 1.
Two aliquots of compound 1 (0.3 mg each in 50 μL) were transferred
into two NMR tubes and dried under vacuum overnight at room
temperature. Then, 6 μL of (R)- or (S)-MTPA chloride and 600 μL of
pyridine-d5 were successively added. The NMR reaction tubes were
immediately sealed, shaken vigorously to ensure even mixing, and
1
stored in a desiccator overnight until the reaction was complete. H
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Journal of Natural Products
Article
NMR spectra were used to monitor the reaction. The 1H NMR
spectra of the final (R)- and (S)-MTPA adducts were recorded directly
after each reaction, and the chemical shifts were assigned based on
1H−1H COSY NMR experiments. Ambiguous signals were excluded
1
spectra of compounds 8−14, and the HiFSA simulated H
NMR spectra of 1−7. This material is available free of charge
via the Internet at http://pubs.acs.org.
from the calculation of ΔδS−R values.23,24
1H NMR data of the (R)-MTPA ester of 1 (400 MHz, pyridine-d5)
(S38, Supporting Information): δ 6.814 (1H, d, J = 15.9 Hz, H-7),
6.449 (1H, dd, J = 15.9, 5.5 Hz, H-6), 5.757 (1H, br quintet, J = 2.6
Hz, H-3), 4.715 (1H, dd, J = 11.8, 1.1 Hz, H-1), 4.621 (1H, m, H-5),
2.228 (1H, m, H-2a), 2.194 (1H, m, H-4a), 1.953 (1H, m, H-4b),
1.922 (1H, m, H-2b).
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1 (312) 996-7253. Fax: +1 (312) 996-7107. E-mail:
sc4sa@uic.edu.
Notes
1H NMR data of the (S)-MTPA ester of 1 (400 MHz, pyridine-d5)
(S40, Supporting Information): δ 6.764 (1H, d, J = 16.3 Hz, H-7),
6.430 (1H, dd, J = 16.3, 5.5 Hz, H-6), 5.765 (1H, br quintet, J = 2.8
Hz, H-3), 4.952 (1H, dd, J = 12.0, 2.2 Hz, H-1), 4.469 (1H, m, H-5),
2.264 (1H, m, H-2a), 2.169 (1H, m, H-4a), 1.993 (1H, m, H-2b),
1.903 (1H, m, H-4b).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. J. G. Napolitano for his kind assistance
with HiFSA procedures. We are also thankful to Dr. J. B.
McAlpine for his valuable comments and suggestions during
the manuscript preparation. This research was supported by
grant P50AT000155 (UIC/NIH Botanical Center), co-funded
by the National Center for Complementary and Alternative
Medicine (NCCAM) and the Office of Dietary Supplements
(ODS), both of the National Institutes of Health.
Preparation of the (R)- and (S)-MTPA Ester Derivatives of 2
and 3. The (R)- and (S)-MTPA esters of the mixture of 2 and 3
obtained from the isolation scheme were produced by following the
general Mosher reaction procedure described for 1.
1H NMR data of the (R)-MTPA ester of 2 (400 MHz, pyridine-d5)
(S42, Supporting Information): δ 5.683 (1H, m, H-3), 4.603 (1H, dd,
J = 11.6, 1.6 Hz, H-1).
1H NMR data of the (S)-MTPA ester of 2 (400 MHz, pyridine-d5)
(S43, Supporting Information): δ 5.683 (1H, m, H-3), 4.824 (1H, dd,
J = 11.6, 1.6 Hz, H-1).
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1H NMR data of the(R)-MTPA ester of 3 (400 MHz, pyridine-d5)
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J = 11.6, 1.6 Hz, H-1).
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= 17.1, 4.4 Hz, H-4b), 2.816 (2H, m, H-7), 2.186 (2H, m, H-6).
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Figures S52 and S54 in the Supporting Information.
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* Supporting Information
The IR and 1D and 2D NMR spectra of compounds 1−7, the
1
1H NMR and H−1H COSY spectra of the (R)- and (S)-
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MTPA esters of compounds 1−6, 8, and 9, the H NMR
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