2003
Isola ion and cha ac e isa ion o i inans, and os ane- ype
wi hanolides om Physalis pe u iana L.
Annika S ein‡1, Da e Compe a‡1, Bianka Ka ge2, Ma k B öns up1,2 and Jakob F anke*1
Full Resea ch Pape Open Access
Add ess:
1Cen e o Biomolecula D ug Resea ch, Leibniz Uni e si y Hanno e ,
Schneide be g 38, 30167 Hanno e , Ge many and 2Helmhol z Cen e
o In ec ion Resea ch, Inho ens asse 7, 38124 B aunschweig,
Ge many
Email:
Jakob F anke* - [email p o ec ed]
* Co esponding au ho ‡ Equal con ibu o s
Keywo ds:
and os anes; Physalis pe u iana; s e oids; s uc u e elucida ion;
wi hanolides
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
doi:10.3762/bjoc.15.196
Recei ed: 31 May 2019
Accep ed: 07 Augus 2019
Published: 23 Augus 2019
This a icle is pa o he hema ic issue "Te penes".
Gues Edi o : J. S. Dickscha
© 2019 S ein e al.; licensee Beils ein-Ins i u .
License and e ms: see end o documen .
Abs ac
Wi hanolides a e s e oidal lac ones widesp ead in Nigh shade plan s wi h o en po en an ip oli e a i e ac i i ies. Addi ionally, he
s uc u al di e si y o his compound class holds much po en ial o he disco e y o no el biological ac i i y. He e, we epo wo
newly cha ac e ised wi hanolides, named i inans, om Physalis pe u iana wi h highly unusual unca ed backbones ha esemble
mammalian and os ane sex ho mones. Based on biomime ic chemical eac ions, we p opose a model ha links hese compounds o
wi hanolide biosyn hesis. I inans ha e po en an ip oli e a i e ac i i ies, ha a e howe e lowe han hose o 4ß-hyd oxywi hano-
lide E. Ou wo k es ablishes and owi hanolides as a new subclass o wi hanolides.
2003
In oduc ion
T adi ional medicine has long been a sou ce o inspi a ion o
mode n d ug esea ch. An impo an example is Wi hania
somni e a, also known as ashwaghanda o Indian ginseng,
which has been used in Ayu edic medicine o ea a la ge
a ie y o ailmen s [1]. Ex ensi e s udies e ealed wi hanolides,
a class o s e oidal lac ones, o be p ima ily esponsible o he
medicinal e ec s [1,2]. A la ge ange o pha macological p op-
e ies has been assigned o wi hanolides, wi h an ip oli e a i e
ac i i ies being he mos po en ones [1]. Wi hanolides ha e
been also disco e ed in nume ous gene a o he han Wi hania,
o example Da u a, Dunalis, Ioch oma, Jabo osa, Lycium and
Physalis [3], esul ing in mo e han 300 known ep esen a i es
[3]. Physalis pe u iana is a wi hanolide p oduce o pa icula
ele ance as i is widely cul i a ed o i s edible be ies [4]. So
a , se e al wi hanolides ha e been epo ed om P. pe u iana
and o he Physalis species, mos p ominen ly physalins, pe u-
lac ones and 4ß-hyd oxywi hanolide E (1) [5-16]. As pa o ou
ongoing p og amme ocussed on he biochemis y o wi hano-
lides, ou aim was o gain u he insigh s in o he wi hanolide
p o ile o P. pe u iana. He e we epo i inans A (2) and B (3),
wo unusual unca ed wi hanolides wi h and os ane backbones.
We show ha oxida i e, bu no acidic o basic condi ions
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2004
Figu e 1: Wi hanolides om Physalis pe u iana. A) S uc u es o he newly cha ac e ised unca ed wi hanolides i inan A (2) and B (3) wi h an
and os ane backbone, oge he wi h he known wi hanolides 1 and 4–6 isola ed om P. pe u iana. B) P. pe u iana plan .
enable con e sion o he pu a i e p ecu so 4ß-hyd oxywi h-
anolide E (1) o i inan A (2). Based on his in insic eac i i y
we p opose a biosyn he ic model ha will se e as u he guid-
ance o elucida ing he enzyma ic basis o and os ane o ma-
ion in plan s in he u u e.
Resul s and Discussion
To isola e wi hanolides om P. pe u iana, we used a pu i ica-
ion s a egy based on p e ious epo s [17-19]. Nine weeks old
whole P. pe u iana plan s (140 g) we e ex ac ed wi h
H2O/MeOH (3:1) and di ided in o ac ions soluble in pe o-
leum e he , chlo o o m, and n-bu anol, espec i ely. The
chlo o o m ac ion was u he sepa a ed by lash ch oma og a-
phy on a C18 s a iona y phase, esul ing in h ee majo sub ac-
ions F1–F3. Final pu i ica ion by p epa a i e HPLC ollowed
by NMR analysis e ealed 4β-hyd oxywi hanolide E (1) as he
majo compound (50 mg) as well as he known me aboli es
wi hanolide E (4), wi hanolide F (5) and pe ulac one H (6) by
compa ison o li e a u e da a (Figu e 1) [9,20].
Two addi ional compounds a ac ed ou a en ion based on
hei unusual 1H NMR spec a (Table 1). Bo h showed wo
mul iple s in he ole inic egion, which a e highly cha ac e is ic
o wi hanolides wi h A- ing Michael accep o s. Howe e , com-
pa ed o o he wi hanolides, se e al signals we e missing. Typi-
cally, wi hanolides show i e single s o me hyl g oups in he
alipha ic egion, as well as he H-22 oxyme hine p o on o he
lac one moie y. Su p isingly, bo h compounds showed only wo
pu a i e me hyl signals, and no signal which migh co espond
o H-22. Thus, we easoned ha bo h unknown compounds
migh be unca ed wi hanolide-like compounds.
HRESIMS sugges ed a sum o mula o C19H24O5 o he i s
compound, which was suppo ed by he 13C spec um (Table 1).
By compa ing he spec um o NMR da a o o he wi hanolides,
we quickly iden i ied he Michael sys em in ing A based on
wo ole inic p o ons (δH 6.94 and 6.22 ppm), a seconda y
alcohol a C-4 (δH 3.79 ppm), a 5,6-epoxide (δH 3.37 (H-6)),
and a e ia y alcohol a C-14 (δC 80.9 ppm). COSY co ela-
ions suppo ed by HMBC analysis (Figu e 2A) e ealed an
in ac ABCD ing sys em wi h a subs i u ion pa e n iden ical o
4β-hyd oxywi hanolide E (1). Only a single, s iking di e ence
was no ed: C-17 was shi ed om 87.8 o 218.0 ppm, s ongly
sugges ing he p esence o a ke one ins ead o an alcohol. In
ag eemen wi h he p edic ed sum o mula and he absence o
all side chain ca bons, his comple ed he s uc u e o he
i s unknown compound, which we named i inan A (2,
Figu e 1).
The second unknown compound had a sum o mula o
C19H24O3 based on HRESIMS and 13C NMR (Table 1). In
con as o he i s compound, no epoxide and no seconda y
alcohol a C-4 was p esen , in ag eemen wi h he di e en
elemen al composi ion. Ins ead, 13C NMR indica ed a double
bond a C5–C6 (δC 135.7 and 124.3 ppm). O he wise, all spin
sys ems and co ela ions indica ed a ypical wi hanolide ABCD
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2005
Table 1: 13C and 1H NMR da a (CDCl3, 500 MHz, 298 K) o i inans A (2) and B (3) in compa ison o he known compound 4β-hyd oxywi hanolide E
(1, CDCl3, 400 MHz, 298 K, δ in ppm, J in Hz). Fo ca bon numbe ing see Figu e 1 and Figu e 2.
13C1H
posi ion 1 2 3 1 2 3
1 201.9 202.0 203.8 – – –
2 133.2 132.3 128.1 6.22 (1H, d, 9.9) 6.22 (1H, d, 10.0) 5.91 (1H, ddd, 10.0, 3.1, 1.2)
3 141.4 142.1 145.3 6.92 (1H, dd, 9.9, 6.1) 6.94 (1H, dd, 10.0, 5.8) 6.79 (1H, ddd, 10.0, 5.0, 2.6)
4 70.4 69.9 33.6 3.74 (1H, d, 6.1) 3.79 (1H, dd, 5.8, 2.4) 3.31 (1H, dddddd, 21.3, 2.8,
2.8, 2.8, 2.8, 2.8)a
2.88 (1H, dd, 21.2, 4.9)
5 64.2 63.9 135.7 – – –
6 63.1 63.1 124.3 3.28 (1H, b s) 3.37 (1H, m) 5.64 (1H, d , 5.7, 2.0)
7 26.0 24.9 24.1 2.03 (2H, m) 2.11 (1H, d , 14.2, 3.1)
1.84 (1H, ddd, 14.1, 11.7,
1.4)
2.08 (1H, m)
1.95 (1H, m)
8 34.3 32.6 35.5 1.83 (1H, m) 1.90 (1H, m) 1.88 (1H, m)
9 36.7 38.1 37.1 1.69 (1H, m) 1.51 (1H, m) 2.10 (1H, m)
10 47.9 47.8 50.9 – – –
11 21.5 20.4 21.6 1.72 (1H, m)
1.56 (1H, m) 1.91 (1H, m)
1.46 (1H, m) 2.34 (1H, m)
1.52 (1H, m)
12 29.8 24.3 25.0 2.25 (1H, m)
1.28 (1H, m) 1.66 (1H, d, 13.2)
1.55 (1H, m) 1.86 (1H, m)
1.63 (1H, m)
13 54.6 52.6 52.5 – – –
14 81.9 80.9 81.0 – – –
15 32.5 30.0 29.9 1.66 (1H, m)
1.59 (1H, m) 1.92 (2H, m) 1.96–1.85 (2H, m)
16 38.0 33.1 33.1 2.72 (1H, m)
1.45 (1H, m) 2.44 (1H, ddd, 18.9, 7.6, 4.1)
2.33 (1H, d , 18.8, 8.8) 2.35–2.46 (2H, m)
17 87.8 218.0 218.5 – – –
18 20.4 17.9 18.1 1.07 (3H, s) 1.01 (3H, s) 1.05 (3H, s)
19 16.9 17.8 19.2 1.42 (3H, s) 1.45 (3H, s) 1.27 (3H, s)
20 79.2 – – – – –
21 19.8 – – 1.42 (3H, s) – –
22 79.7 – – 4.88 (1H, dd, 11.8, 5.3) – –
23 34.4 – – 2.51 (2H, m) – –
24 150.8 – – – – –
25 121.6 – – – – –
26 166.0 – – – – –
27 12.5 – – 1.88 (3H, s) – –
28 20.8 – – 1.94 (3H, s) – –
14-OH n.d. 1.41 (1H, b s) 1.41 (1H, b s)
4-OH n.d. 2.57 (1H, d, 2.50) -
aAppa en dsex . See Figu e S19 (Suppo ing In o ma ion File 1) o de ails. n.d. no de ec ed.
ing sys em. Again, a ca bon wi h a dis inc down ield shi o
218.4 ppm was ound, demons a ing he p esence o a ke one
a C-17. Highly unusually, H-4β appea ed as a double o
sex e s (1:5:10:10:5:1) (dsex ) in he 1H NMR spec um. This
mul iple was explained as “dddddd” by a o al o six COSY
co ela ions (Figu e S19, Suppo ing In o ma ion File 1). The
esul ing compound was named i inan B (3, Figu e 1). A com-
pound o pu a i ely iden ical s uc u e was isola ed om
P. pe u iana be o e, bu only agmen a y physicochemical
da a has been epo ed so a [14].
To elucida e he ela i e s e eochemis y o i inans A (2) and B
(3), we analysed NOESY da a (Figu e 2). In he case o i inan
A (2), he β con igu a ion o OH-4 was deduced by he NOESY
co ela ion OH-4/CH3-19. OH-14 was assigned as α based on
he co ela ions OH-14/H-12α and H-9/H-12α. The 5,6-epoxide
was de e mined as β by a co ela ion om H-6 o H-3. These
assignmen s a e in comple e ag eemen wi h he ela i e s e eo-
chemis y o 4ß-hyd oxywi hanolide E (1). In i inan B, he con-
igu a ion o OH-14 could no be unambiguously in e ed om
NOE da a due o he signal o e lap o H-15 wi h H-12 and
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2006
Figu e 2: Key NMR co ela ions. (A) COSY and HMBC co ela ions o i inan A (2). (B) COSY and HMBC co ela ions o i inan B (3). (C) Key
NOESY co ela ions o i inan A (2). (D) Key NOESY co ela ions o i inan B (3).
o he p o ons. As an al e na i e, OH-14 α con igu a ion was
deduced om he chemical shi s o C-12 and C-9, which expe-
ience a s ong shielding γ-gauche e ec o OH-14α con igu a-
ions [21]. These da a indica e a ela i e s e eochemis y o
i inan B ma ching wi hanolide F (5).
I inans ep esen highly unusual wi hanolide de i a i es, as
hey lack he side-chain lac one ing ha is a common s uc-
u al ea u e o i ually all known wi hanolides [3], bu pos-
sess an and os ane backbone ins ead. While and os anes such as
and os e one (7) a e well-known human sex ho mones
(Figu e 3A) [22], hei occu ence in plan s is a e [23-26].
Only a single wi hanolide and os ane has been ully cha ac-
e ised be o e, cinedione (8), isola ed om Physalis cine as-
cens (Figu e 3A) [23]. We p opose he name and owi hano-
lides o his wi hanolide subclass, which so a appea s o be
cha ac e is ic o Physalis species.
The biosyn hesis o and os anes in mammals equi es h ee
enzyma ic s eps s a ing om choles e ol (9, Figu e 3B) [27].
Choles e ol (9) is con e ed o p egnenolone (10) by he
cy och ome P450 choles e ol side-chain clea age enzyme
(P450scc), which clea es he C20–C22 bond [27]. Then, he
bi unc ional P450c17 ac s as a 17α-hyd oxylase and 17,20-lyase
o gi e ise o and os anes [27]. Rela ed enzymes ha e no been
epo ed om plan s. We sea ched ansc ip ome da a o
P. pe u iana o pu a i e homologues o hese enzymes [28].
The bes hi s only had amino acid sequence iden i ies o
22–28%, indica ing ha no P450 enzymes o hese clans exis in
P. pe u iana. Al hough enzymes wi h simila ca aly ic ac i i y
migh ha e e ol ed con e gen ly in plan s, he di e en subs i-
u ion pa e n in he side chain sugges s ha a side-chain
clea age mechanism dis inc om mammals is in ol ed. While
he o de o oxida i e s eps in wi hanolide biosyn hesis is s ill
comple ely elusi e [29], we p opose ha his agmen a ion
occu s a a la e s age, when mos ypical wi hanolide unc ional-
isa ions ha e al eady been in oduced. Indeed, i inan A (2),
i inan B (3) and cinedione (8) can be di ec ly linked o he
known wi hanolides 4ß-hyd oxywi hanolide E (1), wi hanolide
F (5) and wi hanolide S [23], espec i ely (Figu e S20, Suppo -
ing In o ma ion File 1). I he agmen a ion occu ed ea ly in
he biosyn hesis, his would imply ha se e al biosyn he ic en-
zymes ha e o ole a e subs a es wi hou he lac one side chain.
We he e o e p opose ha he side-chain clea age enzyme in
wi hanolide biosyn hesis ac s a a la e s age, using common
pa hway end p oduc s such as 4ß-hyd oxywi hanolide E (1) as
i s subs a es. Two mechanisms a e concei able o his ans-
o ma ion (Figu e 3C): A non-oxida i e G ob agmen a ion
could make use o a push–pull mechanism be ween C-17 and
C-22, building on acid–base ca alysis. Al e na i ely, an en-
zyme could clea e he C17–C20 diol oxida i ely. Se e al P450
enzymes ha e been epo ed o be capable o clea ing diols,
p esumably ia a e ic pe oxo in e media e (Figu e 3C)
[30,31].
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2007
Figu e 3: S uc u es and biosyn hesis o and os anes. (A) And os ane backbone and and os e one (7) as a ypical mammalian sex ho mone. Cine-
dione (8) is he only o he ully cha ac e ised and owi hanolide known. (B) Biosyn hesis o and os anes in mammals. (C) Possible clea age mecha-
nisms in ol ed in and owi hanolide biosyn hesis in plan s.
To gain u he insigh s in o he biosyn he ic ou e and o
exclude ha and owi hanolides a e isola ion a e ac s [32], we
exposed 4ß-hyd oxywi hanolide E (1) as he likely p ecu so o
i inan A (2) o a ious chemical condi ions (Figu e 4). In
gene al, 1 was s able in all sol en s es ed, namely chlo o o m,
me hanol, DMSO and ace oni ile (da a no shown). T ea men
wi h acid a pH 3 caused no eac ion a all when hea ing up o
70 °C (Figu e 4A). A pH 0, se e al uniden i ied compounds
appea ed, bu no i inan A (2). In basic condi ions, only a single
uniden i ied p oduc was o med a pH 11 and 70 °C. Nex , we
es ed whe he 1 could be oxida i ely clea ed [33]. Incuba ion
o 1 wi h NaIO4 a oom empe a u e did no esul in any eac-
ion (da a no shown). Howe e , al hough i has been epo ed
ha pe ioda es a e no capable o clea ing di e ia y glycols

Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2008
Figu e 4: In insic eac i i y o 4ß-hyd oxywi hanolide E (1) unde acidic/basic and oxida i e condi ions, espec i ely. (A) LC–MS ch oma og ams (ELS
de ec ion) o 1 incuba ed a di e en pH alues. (B) LC–MS ch oma og ams (UV de ec ion a 200–400 nm) o 1 ea ed wi h di e en oxida i e
eagen s. The o ma ion o 2 in he NaIO4 eac ion was con i med by NMR analysis. See also Figu e S21 in Suppo ing In o ma ion File 1 o ex ac ed
ion ch oma og ams (EICs).
Table 2: An ip oli e a i e ac i i ies in di e en cell lines. Da a indica e EC50 alues ± SD in µM. A549 = human lung ca cinoma; L929 = mouse ib o-
blas ; KB-3-1 = human ce ix ca cinoma; MCF-7 = human b eas cance cell line.
Compound A549 L929 KB-3-1 MCF-7
4ß-hyd oxywi hanolide E (1) 3.74 ± 0.50 0.27 ± 0.30 1.11 ± 0.98 10.65 ± 6.18
i inan A (2)a5.01 ± 5.27 2.29 ± 0.88 4.62 ± 5.76 17.88 ± 7.27
i inan B (3)b3.45 ± 1.91 1.68 ± 1.78 2.40 ± 2.32 13.56 ± 9.18
s au ospo ine (posi i e con ol) 1.19 ± 0.99 <0.003 0.04 ± 0.01 0.16 ± 0.02
au ano in (posi i e con ol) >7.03 2.35 ± 0.83 1.59 ± 0.37 2.06 ± 0.60
aEs ima ed 90% pu i y based on 1H NMR. bEs ima ed 80% pu i y based on 1H NMR.
[33,34], we no ed o ma ion o small quan i ies o i inan A (2)
when pe o ming he eac ion a 70 °C (Figu e 4B and Figu e
S21 in Suppo ing In o ma ion File 1). The iden i y o i inan A
(2) was e i ied by isola ion o he co esponding compound by
p epa a i e HPLC (4% yield) ollowed by NMR analysis. This
esul con i ms ou NMR-based s e eochemical assignmen and
unambiguously links i inan A (2) o 4ß-hyd oxywi hanolide E
(1). We also pe o med an oxida i e clea age eac ion wi h ca -
aly ic amoun s o MoO2(acac)2 in DMSO as desc ibed by
Ga cía e al. [34], which also led o he o ma ion o ace
amoun s o i inan A (2). Ou expe imen s sugges ha i inan A
(2) and mos likely all and owi hanolides a e no isola ion a e-
ac s bu ue na u al p oduc s, which equi e an oxida i e en-
zyme o acili a e he C–C bond clea age. Fu u e s udies will
shed ligh on he enzyma ic basis o and owi hanolide o ma-
ion.
Conside ing he po en bioac i i ies o and os anes as well as
wi hanolides, we wonde ed whe he he loss o he side-chain
lac one would nega i ely impac he an ip oli e a i e ac i i y.
I inan A (2) and B (3) oge he wi h 4ß-hyd oxywi hanolide E
(1) as a posi i e con ol we e e alua ed agains a panel o ou
cell lines (Table 2). In ou assays we obse ed dec easing ac i -
i ies du ing he hi d and ou h eplica es, esul ing in la ge
s anda d de ia ions and po en ially indica ing limi ed s abili y
o hese compounds. None heless, EC50 alues o 4ß-hyd oxy-
wi hanolide E (1) we e in good ag eemen wi h p e iously
published alues [14,35,36]. I inans A (2) and B (3) we e 1.3 o
10- old less ac i e han 4ß-hyd oxywi hanolide E (1), wi h he
excep ion o i inan B (3) in A549 cells, which was equipo en .
Howe e , i inan A (2) and B (3) samples had a pu i y o 90%
and 80%, espec i ely. We he e o e canno exclude ha
uniden i ied impu i ies, which could no be emo ed by
epea ed p epa a i e HPLC, obscu e he ue EC50 alues o
i inans. We conclude ha i inans possess po en an ip oli e a-
i e ac i i y, ha is howe e educed compa ed o 4ß-hyd oxy-
wi hanolide E (1). Ou esul s demons a e he impo ance o
he lac one side chain o bioac i i y.
Conclusion
We ha e disco e ed and cha ac e ised i inans A and B,
wo new wi hanolides om P. pe u iana wi h unca ed back-
bones. They esemble mammalian sex ho mones o he
and os ane class. The ela i e s e eochemis y was elucida ed
based on NOESY analysis. Chemical s udies suppo a model
ha hese compounds a e o med by an oxida i e p ocess. We
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2009
p opose he name and owi hanolides o his wi hanolide
subclass.
Expe imen al
Gene al expe imen al p ocedu es
Seeds o Physalis pe u iana we e ob ained om Flo aSel ,
Spe li and Quedlinbu ge Saa gu . Plan s we e ini ially g own in
seed s a e soil (Kölle’s Bes e Anzuch e de) and la e ans-
e ed o po ing soil (Kölle’s Bes e P lanze de). Plan s we e
g own unde LED illumina ion (SANligh S2W) a
350 µmol s−1 m−2 PPFD wi h a 12 h pho ope iod and a
18–25 °C wi hou empe a u e and humidi y con ol. Plan s
we e wa e ed wice pe week wi h ap wa e as needed.
NMR spec a we e eco ded using B uke AscendTM 400 o
DRX 500 MHz spec ome e s ope a ing a 400 and 500 MHz
o 1H NMR and a 100 and 125 MHz o 13C NMR whe e
CDCl3 was used as sol en . Chemical shi s we e e e enced
ela i e o he esidual sol en signal o CDCl3 (δH = 7.26 ppm,
δC = 77.16 ppm) and exp essed in δ alues (ppm), wi h cou-
pling cons an s epo ed in Hz. Analysis was conduc ed wi h
TopSpin (Ve sion 4.0.6, B uke ). ATR-IR analysis was pe -
o med o he ange o 400–4000 cm−1 using a Shimadzu
IRA ini y 1S spec ome e wi h samples dissol ed in chlo o-
o m. Op ical o a ions we e measu ed wi h a Pe kin Elme 341
pola ime e . Using me hanol as sol en , he wa eleng h o
maximum abso p ion was de e mined on a Jasco V-630 spec-
opho ome e . Flash pu i ica ion was pe o med on a Bio age
Isole a One using columns desc ibed below. HRMS measu e-
men s we e ca ied ou on a Wa e s Alliance 2695 HPLC
coupled o a Mic omass LCT P emie mass spec ome e .
Fo analy ical and p epa a i e LC–MS a Wa e s ins umen was
used consis ing o a Wa e s 2767 au osample , Wa e s 2545
pump sys em, Wa e s 2998 diode a ay de ec o , Wa e s 2424
ELS de ec o , and a Wa e s SQ De ec o 2 o mass spec ome-
y in ESI+ and ESI– modes be ween m/z 150 and 1000. In ana-
ly ical mode, a Phenomenex Kine ex column (2.6 µm, C18,
100 Å, 4.6 × 100 mm) was used wi h a g adien o [sol en A:
H2O + 0.05% o mic acid; sol en B: ace oni ile + 0.045%
o mic acid; g adien : 10% o 90% B o e 10 min, 1 mL/min].
Samples we e dissol ed o a concen a ion o 10 mg/mL in
MeOH and 20 µL injec ed. In p epa a i e mode, a Phenomenex
Kine ex Axia column (5 µm, C18, 100 Å, 21.2 × 250 mm)
equipped wi h a Phenomenex Secu i y Gua d p ecolumn (Luna,
C5, 300 Å) was used in combina ion wi h he sepa a ion
g adien desc ibed below.
Ex ac ion and isola ion o wi hanolides
140 g o 9 weeks old, whole Physalis pe u iana plan s we e
ozen in liquid ni ogen and g ound o a ine powde . The
powde was ex ac ed wi h 500 mL H2O/MeOH (3:1) a oom
empe a u e o 3 h. A e il a ion and e apo a ion o he sol-
en unde educed p essu e, he c ude ex ac was esuspended
in 300 mL H2O and de a ed wi h 300 mL pe oleum e he . The
emaining aqueous laye was u he ex ac ed wi h 2 × 300 mL
CHCl3 ollowed by 2 × 300 mL n-BuOH. This esul ed in a
660 mg pe oleum e he ac ion, 386 mg CHCl3 ac ion and
1174 mg n-BuOH ac ion.
The CHCl3 ac ion was sepa a ed ia e e sed-phase lash
ch oma og aphy (Bio age SNAP KP-C18-HS 30 g column)
wi h a H2O/MeOH g adien . Samples we e adso bed on o Celi e
unde educed p essu e o d y loading. A g adien om 30% o
95% MeOH was used. F ac ions we e pooled guided by UV
maximum abso bance o o m main ac ion F1 (sub ac ions
1–21, 102 mg), F2 (sub ac ions 22–31, 12 mg) and F3
(sub ac ions 32–45, 48 mg). No wi hanolides we e de ec ed in
F2 based on LC–MS analysis and he e o e disca ded.
F ac ion F1 was u he sepa a ed by p epa a i e LC–MS.
The sample was dissol ed in MeOH o a concen a ion o
15 mg/mL. 100 µL was injec ed pe un. A sepa a ion g adien
was used [sol en A: H2O + 0.05% o mic acid; sol en B:
ace oni ile + 0.045% o mic acid; g adien : 10% o 90% B o e
10 min, 20 mL/min]. The pos -column low was spli
(100:1) and he mino i y low made up o 1 mL/min wi h
MeOH + 0.045% o mic acid o in-line analysis by UV, ELSD
and MS. The majo i y low was collec ed. The ollowing peaks
we e collec ed and iden i ied by NMR: R = 5.8–6.0 min (i inan
A (2), 6 mg); 7.2–8.0 min (4β-hyd oxywi hanolide E (1),
49 mg); 9.0–9.2 min (i inan B (3), 1 mg). The collec ed ac-
ions we e e apo a ed unde educed p essu e using a Ch is
RVC 2-25 CDplus o a ional acuum concen a o .
Main ac ion F3 was also sepa a ed by p epa a i e LC–MS
as desc ibed abo e, yielding he known compounds wi hano-
lide E (4) ( R = 7.3–7.6 min, 6 mg), pe ulac one H (6)
( R = 7.6–7.8 min, 9 mg) and wi hanolide F (5)
( R = 7.8–8.5 min, 9 mg) which we e iden i ied by NMR [20].
Analy ical da a
4β-Hyd oxywi hanolide E (1) was isola ed as a whi e c ys-
alline powde . NMR da a o 1 is lis ed in Table 1. All spec os-
copic p ope ies ma ched li e a u e da a [20].
I inan A (2): whi e c ys alline powde ; [α]D20 +10.48 (β = 0.62;
MeOH); UV (MeOH) λmax (log ε) 239 nm (3.93); IR (ATR,
CHCl3) νmax: 3460, 2967, 2930, 1734, 1674, 1454, 1373, 1092,
1036, 986, 922, 754 cm−1; o 1H and 13C da a see Table 1;
HRESIMS m/z: [M + Na]+ calcd o C19H24O5Na+, 355.1516;
ound, 355.1519.
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2010
I inan B (3): whi e c ys alline powde ; [α]D20 –10.00 (β = 0.06;
MeOH); UV (MeOH) λmax (log ε) 251 nm (3.94); IR (ATR,
CHCl3) νmax: 3402, 2955, 2930, 1682, 1383, 1259, 1215, 1136,
1088, 1016, 966, 806, 748 cm−1; o 1H and 13C da a see
Table 1; HRESIMS m/z: [M + Na]+ calcd o C19H24O3Na+,
323.1618; ound, 323.1626
BLAST sea ch o known and os ane
biosyn hesis enzymes
The known and os ane biosyn hesis enzymes Homo sapiens
P450scc (UniP o KB accession P05108) and Homo sapiens
P450c17 (P05093) we e used o sea ch epo ed Physalis pe u-
iana ansc ip ome da a [28] ia he BLASTn algo i hm. Bo h
enzymes yielded se e al ull-leng h hi s wi h amino acid se-
quence iden i ies o 22–28%.
Oxida i e clea age o 4β-hyd oxywi hanolide
E (1) o i inan A (2) by NaIO4
57.8 mg o NaIO4 (270.2 µmol, 7.0 equi ) in 400 µL ho H2O
was added o 19.4 mg 4ß-hyd oxywi hanolide E (1, 38.6 µmol,
1.0 equi ) in 1 mL MeOH. The eac ion was incuba ed a 70 °C
o 72 h in a hea block wi h shaking a 1000 pm. A e ha
ime a peak wi h m/z 315 co esponding o [M + H − H2O]+
wi h a e en ion ime o 3.5 min was obse ed by LC–MS,
co-elu ing wi h au hen ic i inan A (2). The eac ion mix u e was
sepa a ed by p epa a i e LC–MS as desc ibed abo e o gi e a
whi e c ys alline powde (0.5 mg, 4%), which was con i med o
be i inan A (2) by 1H NMR spec oscopy.
Oxida i e clea age o 4β-hyd oxywi hanolide
E (1) o i inan A (2) by MoO2(acac)2
2 µL o a MoO2(acac)2 s ock solu ion in DMSO (100 µg/µL,
0.6 µmol, 0.02 equi ) was added o 14.6 mg hyd oxywi hano-
lide E (1, 29.0 µmol, 1.0 equi ) in 100 µL DMSO. The eac ion
was incuba ed a 130 °C o 3 h in an oil ba h wi h s i ing a
400 pm. A e ha ime a peak wi h m/z 315 co esponding o
[M + H − H2O]+ wi h a e en ion ime o 3.5 min was obse ed,
co-elu ing wi h au hen ic i inan A (2). H2O (5 mL) and CHCl3
(5 mL) we e added o he eac ion mix u e. The laye s we e
sepa a ed and he aqueous phase was ex ac ed wi h chlo o o m
(3 × 5 mL). Combined o ganic laye s we e washed wi h
wa e (5 mL), d ied o e MgSO4, il e ed and he sol en was
e apo a ed unde educed p essu e. The esul ing c ude eac-
ion p oduc was hen analysed by analy ical HPLC as de-
sc ibed abo e.
An ip oli e a i e assays
The e ec o compounds on cell iabili y was p obed wi h a
WST-1 es using he p ocedu e o Ishiyama e al. [37] as modi-
ied by Sasse e al. [38]. The ollowing cell lines we e used:
mouse ib oblas cell line L929 (DSM ACC 2), human ce ix
ca cinoma cell line KB-3-1 (DSM ACC 158), he human lung
ca cinoma cell line A549 (DSMZ ACC 107) and human b eas
cance cell line MCF-7 (DSM ACC 115). The subcon luen
cells we e b ie ly washed wi h Ea le’s Balanced Sal Solu ion
(Gibco) wi hou Ca and Mg, ypsinized and e-suspended in
Dulbecco’s modi ied eagle’s medium ha con ained 5% e al
bo ine se um (FBS; L929, KB-3-1, A549) o Roswell Pa k
Memo ial Ins i u e medium ha con ained 5% FBS,
0.5% Minimum Essen ial Medium Non-Essen ial Amino Acids,
Gibco (MEM NEAA), 0.5% Glu aMAX (Gibco) and insulin a
5 μg/mL (MCF-7). 25 µL o se ial dilu ions o he es com-
pounds (64–0.06 µg/mL, ha we e made wi h a pipe ing obo
(epMo ion, Eppendo , Hambu g, Ge many), we e added o
25 μL aliquo s o a cell suspension (1500 cells o KB-3-1,
L929 and A549, 3000 cells o MCF-7) in 384 well mic o i e
pla es. Blank and sol en con ols we e incuba ed unde iden-
ical condi ions. A e an incuba ion pe iod o 5 days, 3 μL
WST-1 ( eady o use solu ion by Roche) was added. The incu-
ba ion ime o he pla es a 37 °C a ied be ween he cell lines
om 20 min o KB-3-1 and A549, L929 o 30 min, and 2 h
o MCF-7 be o e measu ing abso bance a 450 nm ( e e ence
600 nm) wi h an In ini e 200 PRO pla e eade (Tecan,
Männedo , Swi ze land). As posi i e con ol compounds,
Au ano in and S au ospo in we e applied. The abso bance o
he sol en con ol was se o 100%. The EC50 alues we e de-
e mined wi h Sigma Plo . All da a a e a e age alues om ou
biological eplica es.
Suppo ing In o ma ion
Suppo ing In o ma ion File 1
NMR, MS, UV and IR spec a o i inan A (2) and i inan B
(3). NMR da a o wi hanolide E (4), wi hanolide F (5) and
pe ulac one H (6).
[h ps://www.beils ein-jou nals.o g/bjoc/con en /
supplemen a y/1860-5397-15-196-S1.pd ]
Acknowledgemen s
This wo k has been ca ied ou wi hin he amewo k o
he SMART BIOTECS alliance be ween he Technische
Uni e si ä B aunschweig and he Leibniz Uni e si ä
Hanno e . This ini ia i e is suppo ed by he Minis y o
Science and Cul u e (MWK) o Lowe Saxony, Ge many.
LC–MS (INST 187/621) and NMR (INST 187/686-1) ins u-
men s unded by he Deu sche Fo schungsgemeinscha (DFG)
we e used. We hank P o . Russell Cox o his suppo and
help ul discussions, and Ma cel A nd o p elimina y wo k.
We hank D . Jö g Foh e and colleagues o suppo wi h NMR
measu emen s and Ka ja Kö ne and colleagues o excellen
lab suppo .
Beils ein J. O g. Chem. 2019, 15, 2003–2012.
2011
ORCID® iDs
Ma k B öns up - h ps://o cid.o g/0000-0002-8971-7045
Jakob F anke - h ps://o cid.o g/0000-0002-7603-6232
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