Pergamon

0031-9422(93)EO203-Q

Phymchniury.
Vol. M. No. 2, pp. 485479, 1994
Copyright 0 1994 Ekvier Science Ltd

Printed
InGmlBnItin.All
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0031~9422/94
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LIGNANS AND OTHER COMPOUNDS FROM THE MIXE INDIAN

MEDICINAL PLANT HYPTZS VERTICILLATA
MICHAELA
Institut

fiir Pharmazeutische

KUHNT,HORST
Biologie,

RIMPLER

and MICHAEL

Albert-Ludwig-Universitfit,

HEINRICH+

Schlnzlestr.

1, 79104 Freiburg,

F.R.G.

(Receivedin revised&m 23 November 1993)

Key Word Index-Hyptis
verticillata; Labiatae; lignans; (4R)-1,4dihydro-3-hydroxymethyl-5,6methylenedioxy-4-(3,4,5-trimethoxyphenyl)-naphthalene-2~ar~xylic
lactone (hyptinin); podorhizol; epipodorhizol; dehydropodophyllotoxin; 4-demethylpodophyllotoxin; podophyllotoxin; dehydrodesoxypodophyllotoxin;
sideritoflavone; rosmarinic acid; Mixe Indians (Oaxaca, Mexico).

Abstract-One

new and six known lignans, as well as sideritoflavone and rosmarinic acid were isolated from Hyptis
The new lignan, hyptinin, was fully characterized by spectroscopic methods (UV, H, CNMR, MS, IR,
CD). The known lignans were identified as dehydropodophyllotoxin.
dehydrodesoxypodophyllotoxin,
4demethyldesoxypodophyllotoxin, podophyllotoxin, podorhizol and epipodorhizol.
I;erticillata.

INTRODUCIION

[C,H,O,]+, which is a typical fragment for lactones. The

[M] + signal indicated a molecular formula of CZ2H,,,0,.
Hence, it seemed very likely that 1 was a lignan with the
typical substitution pattern consisting of a methylendioxy
function and a 3,4,5,-trimethoxyphenyl moiety. Further
indications for this substitution pattern were provided by
the IR peaks at 992 (methylendioxy) and 2850 cm- *
(methoxyl), and the H NMR data (Table 1). The IR peak
at 1755 cm- was typical for a five-membered lactone
ring with a double bond in conjugation to the carbonyl
group. On the basis of these results, six possible structures, which differ in the position of the lactone carbonyl
and the position of the double bond in ring B, were
possible. The UV spectroscopic data of the known aapopicropodophyllin (,&,_ 3 14 nm) and y-apopicropodophyllin (;,,,., 354 nm) [6] differ significantly from the UV
data of 1 (i.,, 289 nm). The distinct UV data of 1
indicated that the double bond was not conjugated to one
of the aromatic rings A or D, thus excluding a- and yapopicropodophyllin and their 9-0~0 isomers as possible
structures. The UV, CD and H NMR data of fl-apopicropodophyllin (la) [7] were similar but not identical
with the data of 1. Thus, structure 1, the 9-0~0 isomer of
la, remains the only alternative. This structure is supported by the chemical shifts of H-7, H-7x,/? and H-9a,/l
(Table 1), which are similar to those of the 9-0~0 lignan
konyanin lb [S]. Conclusive proof that the lactone
carbonyl is bonded to C-8 was obtained by NOE experiments: irradiation at H-7a (63.63) or H-7/3 (63.87)
only enhanced the H-7/3 or H-7x signals and the H-2
singlet (66.2). The lack of enhancement of the H-9a or H9/I signals is only compatible with structure 1. Furthermore, irradiation at H-9/? (64.87) gave a positive NOE
for H-7 (64.78) also confirming structure 1. The configuration at C-7 was proven by circular dichroism. The CD

Hyptis verticillata Jacq. is used medicinally by the Mixe

Indians of Oaxaca (Mexico) to treat gastrointestinal
disorders [l] and skin infections [2]. Similar uses for the
plant are reported from other parts of Central America
and the Caribbean [3]. While several members of the
genus have been investigated phytochemically in greater
detail, there is only one report on the chemical constituents of this species. /I-Peltatin and 4-demethyldesoxypodophyllotoxin have been isolated from the aerial parts
by German [4]. Therefore, we decided to investigate this
plant and especially the ether fraction. This phytochemical study is part of a larger project on the ethnobotany of
the Mixe, on the selected biological and pharmacological
effects of these plants and on bioactive compounds they
contain [2, 51. A detailed phytochemical investigation of
H. verticillata also is of interest, since ref. [4] is one of the
very few reports on the presence of aryltetralin lignans in
members of the Labiatae.
RESULTS

AND

DISCUSSION

The aerial parts of H. verficillata were percolated with

acetone-water mixtures followed by extraction of the
aqueous extract with petrol, ether and ethyl acetate. The
ether extract was then further purified by CC, LPLC and
HPLC, and yielded several lignans, sideritoflavone and
rosmarinic acid.
The new compound, 1, showed prominent peaks
in the high resolution MS at m/z 396, 181, 168, 135 and
73 due to [M] + [3,4,5-trimethoxybenzyl] + [3,4,5trimethoxyphenyl] +, [3,4-methylendioxybenzyl] + and

*Author

to whom correspondence

should

be addressed.
485

486

M. KLJHNTet al.

OH
OH

HO

curve of 1 was similar to that of la [6] suggesting an

~-arylsubstituent at C-7, but there is a lack of CD data
for closely related compounds.
Only for 7,8,8,7tetrahydronaphthalene
lignans have rules for the infiuence of the substituents at C-8 and C-8 on the signs of CD
spectra been established [9]. Hydrogenation of 1 at the
84 double bond using pattadium on charcoal gave one
main product (1~).The CD curve of tc showed a positive
sign for the first couplet at 286 nm, which indicates r-

configuration of the aryl substituent at C-7 [9]. The

substjtuents at C-8 and C-8 of fc are probably on the Iface, because under the conditions used hydrogen should
add at C-8 and C-8 on the face opposite to the 3,4,5trimethoxyphenyl ring at C-7 [lo]. The homogeneity of
lc was proven by the H NMR data (Table 1) and TLC.
This result is in accordance with the configuration predicted from the CD of 1. Obviously, the position of the oxogroup in the lactone ring does not affect the sign of the

Lignans from Hyptis wrricillnra

Table 1. HNMR spectral data of 1 and

6.7 s

._
6.62 s

8
9
2
3
3-OMe
4
4-OMe
5
5-OMe
6
7
8
9
@CH,#

3.63 dd (J = 22.5, 4.1)

3.87 dd (J = 22.5, 4.5)
-

6.36 s
3.76 s
3.78 s
3.76 s
6.36 s
4.79 In
4.79 dd (J = 17.2, 2.3)
4.87 d (J = 17.2)
5.93 d (J = 1.5)
5.94 d (J = 1.5)

lc,

487

and CNMR

123.9
107.8
147.3
147.1
109.6
123.7
29.3
138.3
172.3
129.7
105.8
157.9
56.3
153.3
60.8
157.9
56.3
105.8
42.8
128.2
71.1
101.4

spectral data of 1

6.54 s

6.46 s
3.58 dd (J = 11.9, 6)
3.68 dd (J = 11.9, 3.8)
2.88 m

6.41 s
3.83 s
3.93 s
3.83 s
6.41 s
4.6 d (5=6)
233 m
4.14dd(J=11.9,6)
4.19 dd (J= 11.9, 4.5)
6.07 s

CDCI,, chemical shifts (a, and S,) in ppm relative lo dcoc,, (7.24 ppm). coupling
constants (J) in Hz. H and %NMR spectra were recorded al 400 and 100 MHz,
resmtivelv. The assignment of rxotons and C-atoms of 1 was confirmed by H-H
C&Y and %-H COSY data.
at 270-290 nm of the CD curve. Thus, the
structure and absolute configuration of 1 is established.
We propose the name hyptinin for this new compound.
Compounds 2 and 3 could not be separated by chromatographic methods. The mixture was identified as the
diastereomeric compounds podorhizol(2) and epipodorhizol (3). The spectroscopic data (UV, IR, MS) were in
accordance with the data given in the literature [ 111. The
HNMR spectrum showed two sets of signals corresponding to structures 2 and 3. Most signals could be
unequivocally assigned to the H NMR data given in ref.
[ 111. The only problems were the overlapping signals of
the H-8 of 2 and of 3 and of the H-7a of 2 and H-8 of 3.
Therefore, it was not possible to see the coupling pattern
of both H-8 and the exact position of the H-7a of 2 and
the H-8 of 3. This problem was overcome by the utilization of NOE. Irradiation at H-7 of 3 gave positive
NOES only for H-8 and H-8 of 3. The H-8 signal now
appeared isolated as a double doublet at 2.62 ppm with
J ,,,*, = 8.25 Hz and J,.,, = 9.0 Hz, and the H-8 signal as
a complex multiplet at 2.46 ppm. Irradiation at H-7b of 2
gave a positive NOE only for H-7a (2.45 ppm, dd, J7.,78
= 13.5 Hz, J7z,g = 7.5 Hz) of 2 and irradiation at H-7 of 2
gave a positive NOE for H-8 (2.62 ppm, dd, J8..,,
=6.0 Hz, J7,,8, = 3.0 Hz) of 2.
In addition to 1-3, the lipophilic fractions of the ether
extract contained
dehydropodophyllotoxin
(4), 4demethylpodophyllotoxin
(S), podophyllotoxin (6) and
first couplet

dehydrodesoxypodophyllotoxin
(7). They were identified
by comparison with literature data [ 12, 133 and with
authentic samples. B-Peltatin and 4demethyldesoxypodophyllotoxin, which were isolated by V. F. German
from H. oerticillata grown in Jamaica [4], were not found.
Sideritoflavone (8) and rosmarinic acid (9), the latter of
which is widely distributed in the Lamiaceae, were isolated in larger amounts. These compounds were identified
by comparison with literature data 114, IS] and with
authentic samples. Since no high resolution NMR data of
8 and 9 are available in the scientific literature [14, 151,
the HNMR and 13CNMR data are included in the
Experimental section.
None of the above compounds have be-en reported
previously from H. uerticillata.
EXPERIMENTAL

General. NMR: 4OOMHz (H) or 100 MHz (C),

CDCI,: 1-8, CD,OD:9, chemical shifts are shown in 6values (ppm); EIMS: direct inlet system; IR: KBr, UV:
MeOH; OR: CHCI, at 25; CD: MeOH.
Plant material. Hyptis verticillata Jacq. was grown in
the botanical garden of Freiburg (F.R.G.) and identified
by one of the authors (M.H.). Voucher specimens (Heinrich and Antonio: GUI14 and No. 1277) are deposited at
the Institute for Pharmaceutical Biology, Albert-LudwigsUniversity of Freiburg (F.R.G.).

488

M.

KUHNT et al.

Extraction and isolation. Dried and powdered plant

material
(890 g aerial parts)
was percolated
with
Me&O-H,0
(4: 1) at room temp. to furnish the crude
extract (187 g). This was treated with H,O and then
extracted with petrol (48.3), Et,0 (21.7 g) and EtOAc
(20.43 g); leaving the H,O fr. (96.3 g). The Et20 extract
was
chromatographed
with
CH,CI,-MeOH-H,O
(70: 30: 3) on silica gel 60, frs of similar composition
were
combined after TLC-monitoring
and collected as follows:
fr. 46220=1,
fr. 365555O=II. Fr. I was then chromatographed with toluene-EtOAc-MeOH
(35: l5:3) on silica
gel 60 and yielded 2 main frs, 20044 = III and 45-90 = IV.
Fr. III was further purified using HPLC with MeOHMeCN-H,O
(20: I : 5) on a RP18 column (25 x 8 mm)
and yielded a mixt. of 2 and 3 (0.07% of the dried plant
material), 1(0.033%) and 4 (0.02%). Fr. IV was chromatographed with toluene-Me&O
(1: 1) on silica gel 60 and
yielded 2 frs, II-54=V
and 55-126=VI.
Fr. VI was
identified as 8 (0.071%). Fr. V was further purified using
LPLC with increasing concns of MeOH (50-80%) in
H,O on a RP18-column
yielding 5 (0.046%), 6 (0.25%)
and 7 (0.014%). Fr. II was chromatographed
with
EtOH-H,O
(1:4) on Sephadex
LH20 and yielded 9
(0.4 1%).
Hyptinin (1). UV ni:FH nm (log E): 320sh (5.52). 289
(5.94), 261 (6.24), 207 (6.96); [a]; +77.0; IR vkfi cm-:
2934,1752,1693,1588,1505,1483,1461,1420,1232,1128,
1036; EIMS 70eV m/z (rel. int.): 396 (0.25) [Ml,
312
(2.0), 269 (6.54). 195 (1.58), 181 (3.0). 168 (6.37), 157 (7.46),
147 (11.96), 135 (9.12) 97 (10.08) 73 (100); CD (MeOH;
c 1) AE: + 16.2 (239), + 12.4 (226), - 16.5 (205); H and
i3C NMR: Table 1.
8,8_Dihydrohyptinin (1~). Compound
1 (1.7 mg) was
hydrogenated
for 0.5 hr using Pd/C 10% and yielded
0.7 mg 1~; CD (0.5 mM, MeOH) AE: +0.22 (286);
H NMR: Table 1.
Mixture of podorhizol (2) and epipodorhizol (3). UV
ii::
nm (log E): 288 (5.65), 273 (5.63), 260sh (5.57), 209
(6.63); IR vkt: cm.- I: 3466,2916, 1760, 1589, 1506, 1499,
1488, 1459, 1419, 1330, 1240, 1036,926; EIMS 70eV m/z
(rel. int.): 416 (15.15) [Ml, 399 (9.16) 283 (11.14), 221
(6.28), 197(65.13), I81 (15.22), 169(61.51), l54(26.20), 135
(IOO), 77 (62.42); H NMR:86.64 (IH, d, J=8.3 Hz, 3: H5), 6.64 (2H, s, 3: H-2, H-6). 6.59 (IH, d, J=7.5 Hz, 2: H5),6.47(2H,s, 2: H-2. H-6),6.34(1H,dd,J=8.3,
I.5 Hz,3:
H-6),6.32(1H,d,J=1.5Hz,3:H-2),6.29(1H,dd,J=7.8,
1.5 HZ, 2: H-6), 6.22 (lH, d, .I= 1.8 HI 2: H-2). 5.91 and
5.93 (1H each, d, J = 1.5 Hz, 3: -OCH,-0-),
5.90 and
5.94(1H each, d, J= 1.5 Hz, 2: -0-CH,-O-j,
5.25 (lH,d,
J=2.5 Hz, 2: H-7), 4.81 (lH, d, J=8.3 HI 3: H-7), 4.35
(lH, dd, J=9, 7.5 Hz, 2: H-9a), 4.16 (lH, dd, 5=9.2,
7.5 Hz, 3: H-9a), 3.97 (lH, dd, J=9, 5.9 HI 2: H-9/I), 3.88
(6H,s,3:3,5-OMe),3.86(1H,dd,J=9.2,8.4
Hz,3:H-9j?),
3.83 (3H, s, 3: 4-OMe), 3.82 (6H, s, 2: 3,5-OMe), 3.8 (3H,
~,2:4-OMe),2.8(1H,m,2:H-8),2.62(1H,dd,J=6,3
HZ,
2: H-8), 2.62(lH,dd,J=9,8.3
Hz, 3: H-8), 2.46(1H, m,3:
H-8),2.45(1H,dd,J=13.5,7.5H~2:H-7a),2.26(lH,dd,
J=l3.5,
8.3 Hz, 2: H-7/.?), 2.23 (lH, dd, 5=13.5, 9Hz, 3:
H-7a), 2.16 (lH, dd, J= 13.5, 5.3 Hz, 3: H-7P).

Dehydropodophyllotoxin (4). UV J.k$ nm (log E):

340sh (5.47). 321 (5.8) 311 (5.78). 262 (6.42) 204 (6.65); IR
v~~~crn-: 2923,1763,1624,1582,1463,1413,1357,1239,
1122, 1035, 947; EIMS 70 eV m/z (rel. int.): 410 (23.92)
[M] +, 395 (9.81) 370 (14.72) 263 (8.58) 195 (19.01). 163
(17.79), 135 (65.03), 126 (39.65) 69 (87.73). 55 (100);
HNMR:67.47(1H,s,H-2),7.06(1H,.~,H-5),6.51(2H,s,
H-2, H-6), 6.06 and 6.04 (1H each, d, J= 1.8 Hz,
-O-CH,-O-),
5.33 (2H, br s, H-9a$), 3.93 (3H, s, 4OMe), 3.82 (6H, s, 3,5-OMe); 'C NM R: K-l (I 24.9) C2 (97.5), C-3 (149.7). C-4 (148.9) C-5 (102.0). C-6 (130.6),
C-7 (144.2), C-8 (119.2) C-9 (66.2) C-l (132.1), C-2
(107.7). C-3 (153. I), C-4 (I 33.4) C-5 (153. I), C-6 (107.9).
C-7 (130.2), C-8 (122.9), C-9 (170.0). -OCH,-O(101.9) 3-OMe (56.3), 4-OMe (61.1) 5-OMe (56.4).
4-Demethylpodophyllotoxin (5) and podophyllotoxin (6).
Identified by HPLC and TLC comparison with authentic
samples; HPLC: RP18 Nucleosil7 pm (25 x 4 mm), detection at 210 nm, MeOH-H,O
(4: I), 1.5 mlmin-,
R,
10.89 min: 6, 9.54 min: 5; TLC silica gel 60 F,,, Merck
CHCl,-MeOH
(25: l), R, 0.2: 5, 0.25: 6.
Dehydrodesoxypodophyllotoxin (7). UV ~.~.;nm (log
E): 339sh (5.31) 320sh (5.54). 263 (6.22). 219 (6.16); EIMS
70eV m/z (rel. int.): 394 (19.7) [M] +, 379 (10.9) 265 (4.61)
197(4.07), I81 (5.44), 163(9.74), 150(10.01), 118(19.9),93
(70.69),44(100); HNMR:67.68(1H.s,
H-7). 7.19(1H,s,
H-2), 7.11 (I H, s, H-5), 6.53 (2H, s, H-2, H-6). 6.07 (2H, s,
-OCH,-0-),
5.36 (2H, d, J = 1.3 Hz, H-9a$), 3.94 (3H, s,
4-OMe), 3.81 (6H, s, 3,5-OMe).
Sideritojiauone (8). UV ii:
nm (log E): 348 (5.89), 279
(5.82) 258 (5.77), 208 (6.08), (MeOH + AICI,) 433 (5.84),
364 (5.78) 308 (5.86), 276 (5.9), 238 (6.07) (MeOH
+ AICI,-HCI) 364 (5.99), 307 (5.89), 262 (5.84). 238 (6.03),
215 (6.02); IR vii: cm: 2930, 1690. 1655, 1600, 1560,
1380, 1285, 1120, 1072, 1030, 830; EIMS 70eV m/z (rel.
int.): 360(23.8) [Ml, 345(100), 211 (16.4), I83 (20.1). 137
(l8.6), 134 (23.7), 69 (38.5); HNMR:67.49
(IH, dd, J
=8.3, 2.3 Hz, H-6), 7.42 (IH, d, J=2.3 Hz, H-2), 6.99
(lH, d, 5=8.3 Hz, H-5), 6.55 (lH, s, H-3), 4.05, 3.95, 3.85
(3H each, s, 6,7,8-OMe); C NMR: 6 C-2 (165.4), C-3
(103.8). C-4 (183.8), C-4a (107.5), C-5 (146.5). C-6 (137.4)
C-7 (153.9) C-8 (132.0), C-8a (150.3). C-l (123.7). C-2
(114.1). C-3 (150.4) C-4 (150.4). C-5,(1 16.8) C-6 (120.3)
6,7,8-OMe (61.0, 61.9, 62.4).
Rosmarinic acid (9). H NMR: 6 7.5 (1 H, d, J = 15.8 Hz,
H-8), 7.03 (I H, d, J = 2.3 Hz, H-2), 6.92 (1 H, dd, ./ = 2.3,
8.3 Hz, H-6), 6.76 (IH, d, 5=8.3 Hz, H-5), 6.77 (lH, d, J
=2.3 Hz,H-2),6.67(lH,d,J=8.3
Hz,H-5),6.62(1H,dd,
J=2.3, 8.3 Hz, H-6), 6.25 (IH, d, 5=15.8 Hz, H-7), 5.09
(lH, dd, 5=3, 9.8 Hz, H-8) 3.10(lH, dd, 5=3, 14.3 Hz,
H-7x),
2.93 (lH,
dd, J=9.8,
14.3 Hz,
H-7,9);
3CNMR:GC-1
(127.9), C-2 (115.6), C-3 (149.4). c-4
(146.7),C-5(116.5),C-6(l22.9),C-7(146.7),C-8(115.6),C9 (l69.1), C-l (131.1). C-2 (117.5), C-3 (145.9), C-4
(144.8) C-5 (116.2) C-6 (121.8) C-7 (38.79), C-8 (77.6).
C-9 (I 77.9).
Acknowledgements-We
would like to thank Dr D.
Hunkler (Institute for Organic Chemistry, Freiburg) for

Lignans from Hyptis wrticillofo

performing the NMR experiments, Prof. R. Brossmer

(Institute for Biochemistry II, Heidelberg) and Prof.
A. W. Frahm (Institute for Pharmaceutical Chemistry,
Freiburg) for the CD data, and Prof. A. San Feliciano
(Fact&ad de Farmacia, Salamanca, Spain) for a generous
gift of the authentic sample of 5. The ethnobotanical
information is based on the knowledge of the inhabitants
of San Juan Guichicovi, Oaxaca, Mexica.

489

Phillipson, D. P., Schandelmaier, A. and Warhurst,

D. C. (1992) J. Ethnopharmucol. 36, 8 1.
Schrecker, A. W. and Hartwell, J. L. (1952) J. Am.
Chem. Sot. 74, 5676.

Ayres, D. C., Harris, J. A. and Hulbert, P. B. (1971)

J. Chem. Sot. (C) 11 I 1.
Gdzler, T., Gozler, B., Patra, A., Leet, J. E., Freyer,
A. J. and Shamma, M. (1984) Tetrahedron 40, 1145.
9. Hulbert, P. B., Klyne, W. and Scopes, P. M. (1981)
J. Chem. Res. (S) 27.

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