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Drug Discoveries & Therapeutics. 2012; 6(1):18-23.
18
Identification and evaluation of agents isolated from traditionally
used herbs against Ophiophagus hannah venom
Rima Salama1, Jintana Sattayasai2, Arun Kumar Gande1, Nison Sattayasai3, Mike Davis1,
Eric Lattmann1,*
1 Division of Pharmacy, School of Life and Health Sciences, Aston University, Birmingham, England;
2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand;
3 Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.
*Address correspondence to:
Dr. Eric Lattmann, Division of Pharmacy, School of
Life and Health Sciences, Aston University, Aston
Triangle, Birmingham B4 7ET, England.
E-mail: e.lattmann@aston.ac.uk
ABSTRACT: The aim of this study was firstly to
identify active molecules in herbs, that are traditionally
used for the treatment of snake bite, such as Curcuma
antinaia, Curcuma contravenenum, Andrographis
paniculata, and Tanacetum parthenium; secondly to
test similar structurally related molecules and finally
to prepare and evaluate an efficient formulation
against Ophiophagus hannah venom intoxification.
Three labdane based compounds, including labdane
dialdehyde, labdane lactone, and labdane trialdehyde
and two lactones including 14-deoxy-11,12-didehydro-
andrographolide and parthenolide were isolated by
column chromatography and characterised. Using
the isolated rat phrenic nerve-hemidiaphragm
preparation, the antagonistic effect of crude extracts,
isolated compounds and prepared formulations
were measured in vitro on the inhibition of the
neuromuscular transmission. Inhibition on muscle
contraction, produced by the 5 μg/mL venom, was
reversed by test agents in organ bath preparations. A
labdane trialdehyde, isolated from C. contravenenum,
was identified as the best antagonising agent in the
low micromolar range. Tests on formulations of the
most potent C. contravenenum extract showed, that
the suppository with witepsol H15 was an effective
medicine against O. hannah venom. This study
elucidated the active compounds, accounting for the
antivenin activity of traditionally used herbs and
suggested the most suitable formulation, which may
help to develop potent medicines for the treatment of
snake bite in the future.
Keywords: Antivenin activity, rat nerve-hemidiapragm,
labdane dialdehyde, labdane trialdehyde, parthenolide,
desoxy-andrographolide
1. Introduction
The actual incidence and the severity of snake poisoning
are currently highly undervalued (1). The importance
of snake bites is considered a major occupational
disease causing both disabilities and mortalities. This
disease is causing devastation to individuals, who are
involved in agricultural work in the tropical regions
worldwide. Inaccurate epidemiological data resulted
in the underestimation of this international problem,
which requires both high attention and sincere efforts to
alleviate its burden (2). Based on these facts, the World
Health Organization (WHO) is calling for new, proven
and affordable treatments. Traditional medicine or
herbal medicine has long been used for the treatment of
snake bite worldwide for its affirmed effectiveness, easy
availability, and fine economic affordability. However,
the active ingredients contained in these herbs and most
effective formulations are still needed to be elucidated.
We have previously reported a labdane dialdehyde
structure (Figure 1), which was isolated from a
novel Curcuma zedoaroides species and exhibited
well antivenin activity (3). Species in the genus
Curcuma including Curcuma antinaia and Curcuma
contravenenum were in regular use in Thailand against
cobra intoxication, but they are very hard to find on local
markets in Isarn today. Besides of those herbs, another
two medical plants including Andrographis paniculata
and Tanacetum parthenium are widely applied for the
treatment of snake bite in India and China. Andrographis
paniculata is in use in India as a snake venom antidote.
DOI: 10.5582/ddt.2012.v6.1.18
Brief Report
Figure 1. Chemical structure of labdane dialdehyde.
H
O
O
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Drug Discoveries & Therapeutics. 2012; 6(1):18-23.
The leaves of A. paniculata, locally known as Nilavembu,
were grinded into a paste and applied topically at the site
of the snake's bite (4). Tanacetum parthenium, a member
of the Asteraceae family, which is also known as the
Compositae family, represents a herb well known for its
medicinal properties. Since ancient times this herb was
used by the Greeks and the Egyptians as well as the early
Europeans for the treatment of a number of illnesses,
such as headaches, stomach ache, menstrual pain, joint
pain, fever. The Chinese used this herb due to its healing
properties against insects and snake bites (5). In order to
further elucidate the ingredients, that account for antivenin
activity of these herbs and develop formulations, that are
effective against snake venom, we prepared and tested the
in vitro antivenin activity of the crude extracts, purified
compounds and certain formulations against Ophiophagus
hannah venom in the present study.
2. Materials and Methods
2.1. Materials
Lyophilized O. hannah venom was obtained from the
Queen Suavabha Memorial Institute, Bangkok, Thailand.
The venom was dissolved in normal saline, aliquoted
and kept at –20°C as stock solution. Herbs including
C. zedoaroides, C. antinaia, C. contravenenum, A.
paniculata, and T. parthenium were purchased from the
King Cobra Village, Khon Kaen Province, Thailand. The
chemicals and solvents were purchased from Aldrich
(Gillingham, UK) and Lancaster Synthesis (Lancaster,
UK). Mass spectra were obtained by Atmospheric
Pressure Chemical Ionisation (APCI), using a Hewlett-
Packard 5989b quadrupole instrument (Vienna, Austria).
Both proton and carbon NMR spectra were obtained on
a Brucker AC 250 instrument (Follanden, Switzerland),
calibrated with the solvent reference peak. Infra-red
spectra were plotted from KBr discs on a Mattson 300
FTIR spectrophotometer (Coventry, UK).
Laboratory and HPLC grade dichloromethane,
petroleum ether 60-80ºC, ethyl acetate, chloroform-d
and methanol were purchased from Fisher-Scientific
(Waltham, MA, USA). The Soxhlet extraction system
was the Quickfit (C5/23) model with (24/29) joint from
BÜCHI, Switzerland. The heating mantle used was
the Heidolph EKT 3001 from Sigma-Aldrich, UK.
The rotary evaporator used was the BÜCHI Rotavapor
Model R-144, Switzerland. 1H-NMR and 13C-NMR were
recorded in Bruker advance 400 in chloroform-d. Column
chromatography was carried out on silica gel and thin-
layer chromatography (TLC) on TLC-silica gel 60 F254.
2.2. Preparation of crude extracts and isolation of the
tested compounds
The rhizomes of C. zedoaroides, C. antinaia, and C.
contravenenum (30 g of the dried, powdered rhizomes)
were extracted with dichloromethane using a Soxhlet
extraction apparatus, respectively. Once the Soxhlet
extractions were completed the solvent was removed under
reduced pressure from the round bottom flasks using a
rotary evaporator. The extract content of C. zedoaroides,
C. antinaia, and C. contravenenum were gathered and the
entry code Kae1, Mia3, and Rat7 was assigned. Column
chromoatography of the crude extract was performed using
petroleum ether:ethyl acetate (60:40) as solvent system and
the compounds were detected with UV light, permanganate,
and 2,4-dinitrophenylhydrazine (DNPH). The Kae1
extract finally provided labdane dialdehyde; Mia3 extract
finally provided labdane dialdehyde and labdane lactone;
Rat7 extract finally provided labdane dialdehyde, labdane
lactone, and labdane trialdehyde. The spectra data of these
three compounds are shown in the Appendix.
Fifty grams of dried and ground leaves of A.
paniculata were extracted with 500 mL of methanol
using to a Soxhlet extractor. The solvent was evaporated
off solvent using rotary evaporation. TLC analysis was
performed with a dichloromethane:methanol (95:5) solvent
mixture, and both andrographolide and 14-deoxy-11,12-
didehydroandrographolide, were purified by column
chromatography. The spectra data of 14-deoxy-11,12-
didehydroandrographolide are shown in the Appendix.
Fifty grams of grinded plant material T. parthenium
was extracted with 500 mL of chloroform using a Soxhlet
extractor. The extraction was monitored by TLC with
ethyl acetate:petroleum ether 60-80°C (70:30) and the TLC
plate was developed with vanillin reagent and heated at
400°C for 1 min. Two point five gram of crude extract was
dissolved in a minimum volume of ethyl acetate in the heat
and 1/3 of the volume of petrol ether was added and cooled
on ice. The yellowish sample was recrystallised from ethyl
acetate:petroleum ether (70:30) to give parthenolide, 130 mg,
as an off white powder. The spectra data of this compound
are shown in the Appendix.
2.3. Preparation of C. contravenemum formulations
Ethanolic solution (entry code: OHRat7): For the pre-
paration of the ethanolic solution, 0.5 g of the concentrated
extract of C. contravenemum was dissolved in 5 mL of
ethanol and divided into two portions of 2.5 mL each. To one
of the samples OHRat7 3 drops of trifluoroacetic acid were
added. Both samples were incubated at 36°C f or a period of 1
week, then tested.
Water for injection (entry code: Aq Rat7 ): For the pre-
paration of a solution for the injection, 2.5 equivalents of an
aqueous solution of 10% Na2S2O5 to 0.2 g of the extract in
0.1 mL DMSO were mixed until homogenous and water was
added until a final volume of 4 mL.
Suppositories (entry code: Rat7S): Suppositories (3
g) containing 30% active ingredients were prepared by the
fusion method as previously described (6). Sixty six grams
of C. contravenemum rhizomes, giving 1.98 g crude extract,
were extracted with 300 mL of acetone as described. The
19
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Drug Discoveries & Therapeutics. 2012; 6(1):18-23.
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3. Results and Discussion
3.1. In vitro antivenin activity of crude extracts from the
genus Curcuma
The protecting effects of crude extracts Kae1, Mia3,
and Rat7 from C. zedoaroides, C. antinaia, and C.
contravenenum against O. hannah venom on the
neuromuscular transmission of the rat phrenic nerve-
hemidiaphragm are shown in Table 1. The Kae1 extract
was obtained in the highest yield (7%) and at a 100
μg/mL dose the response was still above 60%. The best
protection was determined for Rat7 extract (yield 3.0%),
giving for the 100 μg/mL organ bath concentration a more
than 80% of the original contraction of the diaphragm.
Mia3 extract was obtained in the lowest yield (2.6%)
and demonstrated the lowest antivenom activity. The
muscle contract response was determined as 56.5% when
expososed to 100 μg/mL of this extract.
3.2. Analysis of the ingredients in the crude extracts
and evaluation of their antivenin activity
The crude extracts from the five medicinal herbs, used
in the present study, were further purified and generated
the test compounds (Figure 2). The antivenin activities of
solvent was evaporated off to about a third of the volume
and 6 g witepsol H15, a synthetic fat was added and the
complete solvent was then evaporated off in vacuum. The
homogeneous melt was poured into the suppository moulds
and after cooling the suppositories were obtained. The
melting point of the prepared suppositories was detected at
32ºC.
2.4. In vitro antivenin activity assay
Spraque Dawley rats (200-250 g) were obtained from
the Animal House, Faculty of Medicine, Khon Kaen
University. The treatment procedures, according to
current UK legislation, were approved by the bioethics
committee, Faculty of Medicine, Khon Kaen University
(HO 2434-76). Animals had free access to fresh water and
food pellets. They were exposed to automated 12 h light
cycles.
Rat phrenic nerve-hemidiaphragms were prepared
according to the staff of the Department of Pharmacology,
University of Edinburgh (1970) and the contractile
responses were studied. The entire nerve-muscle
preparation was submerged in 50 mL Kreb's solution with
carbogen and the temperature was maintained at 37°C.
The phrenic nerve was stimulated with a rectangular-
wave pulse of 0.5 msec/0.5 Hz through a bipolar platinum
electrode, using a Grass Model S-48 stimulator. Muscle
contraction was recorded with a force transducer and
Grass Polygraph recorder. The indicated doses of crude
extract (Kae1, Mia3, and Rat7), test solutions of the
isolated compounds in DMSO, and prepared formulations
(OHRat7, AqRat7, and Rat7S) were added, respectively,
to a circulating water bath at 37°C for 30 min and then
venom was added and 30 min later the muscle contraction
was accessed. Two phrenic nerve-hemidiaphragm
preparations were obtained from one animal. Control,
DMSO, was set to 100%, 5 μg/mL venom to 0%. The %
test response (muscle contraction) was measured at 30
min in presence of the test compounds and the venom.
In the control group, 5 μg/mL venom alone gradually
and completely inhibited the indirectly-evoked twitches
within 30 min (0% response). In presence of the antidote
the contraction remained at a certain percentage of the
full contraction. This effect was used here to screen the 3
Curcuma plant extracts, the isolated compounds and the
formulations of the best plant.
Entry Code
Kae1
Mia3
Rat7
Table 1. Antivenin activity of crude extracts from Curcuma species
Source
C. zedoaroides
C. antinaia
C. contravenenum
Yield
7.0%
2.6%
3.0%
Composition
Labdane dialdehyde, 79%
Labdane dialdehyde, 45%
Labdane lactone, 39%
Labdane dialdehyde, 32%
Labdane lactone, 37%
Labdane trialdehyde, 8%
Muscle contraction response
32.1% (50 μg/mL); 63.1% (100 μg/mL)
26.4% (50 μg/mL); 56.5% (100 μg/mL)
53.5% (50 μg/mL); 82.6% (100 μg/mL)
Measurement time is 30 min; full contraction 100%, venom 0% at 5 μg/mL.
Figure 2. Chemical structures of the isolated compounds.
H
O
O
H
O
HO
O
H
O
O
O
H
HO
OH
O
O
OH
HO
HO
OO
O
Labdane dialdehyde Labdane lactone Labdane trialdeh yde
Labdane diol
14-Deoxy-11,12-
didehydroandrographolide Pa rth en olide
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Drug Discoveries & Therapeutics. 2012; 6(1):18-23.
these agents were examined and the results are shown in
Table 2. The crude extract Kae1 from C. zedoaroides is
a very good source of the labdane dialdehyde, which was
obtained from this species in 79% yield and excellent
purity, by simple column chromatography. A 10 μg/mL
concentration of this compound showed 83% protection
against the snake venom.
The crude extract Mia3 from C. antinaia provided
in addition to labdane dialdehyde, a second labdane
derivative, identified as labdane lactone (Figure 2),
which showed a bioactivity about 60%. Its formation can
be understand if we assume that the labdane dialdehyde
is oxidised to the corresponding carboxylic acid and the
second aldehyde is able to form the hydroxyl-lactone
structure of labdane lactone. The proportions of labdane
dialdehyde and labdane lactone in Mia3 were detected
as 45% and 39%, respectively. Labdane lactone was
previously isolated from members of the Zingiberaceae
family, which are traditionally used as a medicine
against inflammatory diseases. This antiinflammatory
agent regulates NF-κB-regulated cellular responses in
particular it inhibited NF-κB activation, suppressed
phosphorylation, p65 nuclear translocation and reporter
gene transcription (11).
From C. contravenenum extract Rat7, in addition to
labdane dialdehyde and labdane lactone, a third molecule
labdane trialdehyde (Figure 2) was finally isolated. The
percentages of labdane dialdehyde, labdane lactone, and
labdane trialdehyde in this extract were determined as
32%, 37%, and 8%, respectively. Labdane trialdehyde
21
maintained nearly a full diaphragm contraction and
with 99.5% protection it is considered the best venom
antidote, reported to date. Comparing the activities of
these three molecules, the labdane trialdehyde is the most
potent antivenin agent, followed by labdane dialdehyde
and labdane lactone. Through analyzing the relationship
between activities and structures of the above isolated
compounds, the succindialdehyde structure may be
essential and if the dialdehyde moiety was reduced to
a diol the activity was lost (labdane diol, Figure 2).
The labdane trialdehyde was previously isolated from
myoga extracts on the search for inhibitors of human
platelet aggregation and human 5-lipoxygenase. This
compound was found to be a potent inhibitor of human
platelet aggregation and human 5-lipoxygenase (7). The
percentage, in which it is present in this curcuma species,
is with 8% a good new source of this agent and therefore,
it can be evaluated for further therapeutic applications.
Guided by previous enzyme-linked immunosorbent
assay (ELISA) study (8) T. parthenium (Feverfew) and
A. paniculata were also investigated in addition to the
Curcuma plant. From A. paniculata, andrographolide
and 14-deoxy-11,12-didehydroandrographolide were
isolated and tested. The desoxy-derivative displayed a
weak inhibition of 32% at the 10 μM test concentration
while andrographolide was found inactive as antidote. As
14-deoxy-11,12-didehydroandrographolide is only present
in less than 0.01% in the traditional formulation, known as
Nilavembu, it cannot be recommended. The concentration
and resulting of this the active amount of 14-deoxy-11,12-
didehydroandrographolide is simply too low to have any
medicinal effect. T. parthenium is a herb well known for
its medicinal properties. The Chinese used this herb due
to its healing properties against insects and snake bites.
Overall parthenolide isolated from this herb had reasonable
antivenom activity of about 54% and it can easily be
isolated in good quantities by crystallisation. It is the only
European herb, which is readily available in a commercial
formulation as a tablet from e.g. simply supplements (9).
3.3. In vitro antivenin activity of C. contravenenum
formulations
C. contravenenum is the easiest plant in terms of plant
production and has the biggest rhizome based on the
total weight of the entire plant. It gave a 3.0% yield of
the crude extract (Rat7) which exhibited most potent
antivenin activity. For these reasons, three formulations of
Rat7 extract were prepared and tested on their antivenin
activity (Table 3). The ethanolic extract of Rat7 was
Entry Code
OHRat7
AqRat7
Rat7S
Table 3. Antivenin activity of crude extracts from Curcuma species
Formulation
10% in ethanol
Hydrogensulfite adduct; 5% in water
Suppository 30%
Muscle contraction response
1.1% (500 μg/mL); 0.2% (1,000 μg/mL)
5.1% (1,000 μg/mL); 6.3% (5,000 μg/mL)
46.1% (200 μg/mL); 77.4% (400 μg/mL)
Measurement time is 30 min; full contraction 100%, venom 0% at 5 μg/mL.
Compound
Labdane dialdehyde
1 μM
10 μM
Labdane diol
1 μM
10 μM
Labdane lactone
1 μM
10 μM
Labdane trialdehyde
1 μM
10 μM
14-Deoxy-11,12-di-
dehydroandrographolide
1 μM
10 μM
Parthenolide
1 μM
10 μM
Muscle contraction response
66.1%
82.6%
0
2.1%
45.6%
61.9%
83.5%
99.5%
12.2%
32.4%
29.4%
54.0%
Table 2. Antivenin activity of isolated compounds
Measurement time is 30 min; full contraction 100%, venom 0% at 5
μg/mL.
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Drug Discoveries & Therapeutics. 2012; 6(1):18-23.
22
found inactive, and therefore formulations in Thai whisky
cannot be recommended. Chemically, the aldehyde
functionality has been converted into an ethoxy semi-
acetale. Thus, a protected, chemically unreactive structure
was formed without any bioactivity. The formulation
as hydrogen sulfite adduct was supposed to work as an
injection in water, but again with the loss of the chemical
reactivity the bioactivity was also lost, at least in the in
vitro (ex vivo) experiment. The formulation of the extract
Rat7 as a suppository worked well; in vitro 77% response
remained for the 400 μg/mL dose, compared with the
original extract of Rat7 at a 100 μg/mL dose, which
gave 83% venom protection. Based on a good in vitro-in
vivo correlation, we obtained from a previous study, 1-2
suppositories (3 g) should translate into a working anti-
venom medication.
Three curcuma species, i.e. C. zedoaroides, C.
antinaia, and C. contravenenum, and two other traditional
medicines A. paniculata, and T. parthenium were
confirmed active against O. hannah venom in our ex-
vivo assay. In addition to the labdane dialdehyde, which
was discovered in C. zedoaroides in our previous study,
labdane lactone and labdane trialdehyde, isolated from
curcuma species C. antinaia and C. contravenenum
were found effective now against the venom. Labdane
trialdehyde is the best anti-neurotoxic agent known
to date. However, the isolated labdane trialdehyde is
unstable. It is supposed, that the natural plant formulation
is stabilising labdane trialdehyde with the labdane
dialdehyde. Feverfew extract at high doses may be used
for snake venom intoxication as a common European
alternative. It should be noted, that the only efficient
formulation is a suppository, in addition to the preparation
of the freshly grinded root of the fresh rhizome. These
results provided evidences about the usefulness of some
traditional medicines as antidotes and gave clues on
the drug development in the future. Further studies are
ongoing to replace the in vitro antivenin assay, used in this
study, by an in vitro method, in which chicken intestine is
used and not laboratory animal tissue.
Acknowledgements
This study was partly supported by the Khon Kaen
University Research Fund. We are grateful for the
technical assistance of Wanchai Airarat.
References
1. Warrell DA. Snake bite. Lancet. 2010; 375:77-88.
2. Cruz LS, Vargas R, Lopes AA. Snakebite envenomation
and death in the developing world. Ethn Dis. 2009; 19(1
Suppl 1):S1-42-46.
3. Chavaeerach A, Sudmoon R, Tanee T, Mokkamul P,
Sattayasai N, Sattayasai J. Two new species of Curcuma
(Zingiberaceae) used as cobra-bite antidotes. J Syst
Evolut. 2008; 46:80-88.
4. Rajadurai M, Vidhya VG, Ramya M, Bhaskar A. Ethno-
medicinal plants used by the traditional healers of
pachamalai hills, Tamilnadu, India. Ethno-Med. 2009;
3:39-41.
5. Lee KH, Wang HK, Itokawa H, Morris-Natschke SL.
Current perspectives on Chinese medicines and dietary
supplements in China, Japan and United States. Yao Wu
Shi Pin Fen Xi. 2000; 8:219-228.
6. Adegboye TA, Itiola OA. Physical and release properties
of metronidazole suppositories. Trop J Pharm Res. 2008;
7:887-896.
7. Suebsasana S, Pongnaratorn P, Sattayasai J, Arkaravichien
T, Tiamkao S, Aromdee C. Analgesic, antipyretic, anti-
inflammatory and toxic effects of andrographolide
derivatives in experimental animals. Arch Pharm Res.
2009; 32:1191-1200.
8. Daduang S, Sattayasai N, Sattayasai J, Tophrom P,
Thammathaworn A, Chaveerach A, Konkchaiyaphum
M. Screening of plants containing Naja naja siamensis
cobra venom inhibitory activity using modified ELISA
technique. Anal Biochem. 2005; 341:316-325.
9. Kemper K. Seven herbs every paediatrician should
know. Contemporary Paediatrics. 1996; 13:79-90.
10. Lattmann E, Sattayasai J, Sattayasai N, Staaf A,
Phimmasone S, Schwalbe CH, Chaveerach A. In-vitro
and in-vivo antivenin activity of 2-[2-(5,5,8a-trimethyl-
2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-
succinaldehyde against Ophiophagus hannah venom. J
Pharm Pharmacol. 2010; 62:257-262.
11. Kunnumakkara AB, Ichikawa H, Anand P, Mohankumar
CJ, Hema PS, Nair MS, Aggarwal BB. Coronarin D,
a labdane diterpene, inhibits both constitutive and
inducible nuclear factor-κB pathway activation, leading
to potentiation of apoptosis, inhibition of invasion, and
suppression of osteoclastogenesis. Mol Cancer Ther.
2008; 7:3306-3317.
12. Tiuman TS, Ueda-Nakamura T, Garcia Cortez DA, Dias
Filho BP, Morgado-Diaz JA, de Souza W, Nakamura CV.
Antileishmanial activity of parthenolide, a sesquiterpene
lactone isolated from Tanacetum parthenium. Antimicrob
Agents Chemother. 2005; 49:176-182.
(Received January 23, 2012; Revised February 1, 2012;
Accepted February 3, 2012)
Appendix
Labdane dialdehyde. 2-[2-(5,5,8a-Trimethyl-2-
methylene-decahydro-naphthalen-1-yl)-ethylidene]-
succinaldehyde. The 1H-NMR and 13C-NMR data are
identical with Lattmann et al. (10).
1H-NMR (CDCl3, 300 MHz): δ: 9.7 + 9.5 (1H, 1H,
CHO), 6.75 (1H, t, C12H), 4.90 + 4.40 (1H, 1H, s,
C17H), 3.40 (2H, d, C14H), 2.40 (3H, m, C11H,
C6H), 2.0-1.0 (10H, m), 0.9 + 0.8 + 0.7 (9H, s, 3×Me);
13C-NMR: δ: 196.38, 192.60 CHO, C15 + C16; 146.96,
133.78 C8, C13, 159.05 C12, 106.33 C17, 55.20, 54.18
C6, C7, 32.22, 39.34 C1, C5, 34.15, 23.83,14.23 Me,
40.54, 38.30, 38.15, 36.78, 23.55, 23.52, 18.23 C14,
C11, C9, C10, C2, C3, C4.
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Drug Discoveries & Therapeutics. 2012; 6(1):18-23. 23
Labdane lactone. 5-Hydroxy-3-[2-(5,5,8a-trimethyl-
2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-
dihydro-furan-2-one. The 1H-NMR and 13C-NMR data
are identical with Kunnumakkara et al. (11).
1H-NMR (CDCl3, 300 MHz): δ: 6.69 (br s, 1H), 5.94 (br
s, 1H), 4.81 (s, 1H), 4.36 (br s, 1H), 2.99 (m, 1H), 2.71
(m, 1H), 2.40-2.36 (m, 2H), 2.20 (m, 1H), 2.05-1.08 (m,
12H), 0.89 (s, 3H), 0.82 (s, 3H), 0.71 (s, 3H); 13C-NMR
(75 MHz): δ: 170.65, 148.01, 143.45, 124.48, 107.52,
96.46, 56.02, 55.21, 41.90, 39.94, 39.12, 37.68, 33.57,
33.45, 25.58, 23.99, 21.62, 19.21, 14.23.
Labdane trialdehyde. 2-[2-(2-Formyl-5,5,8a-
trimethyl-decahydro-naphthalen-1-yl)-ethylidene]-
succinaldehyde. The 1H-NMR and 13C-NMR data are
identical with Suebsasana et al. (7).
1H-NMR (CDCl3, 300 MHz): δ: 9.96 (s, 1H, H-17), 9.62
(s, 1H, H-15), 9.50 (s, 1H, H-16), 7.10 (dd, 1H, J = 6.0,
8.8, H-12), 3.54 (d, 1H, J = 17, H-14a), 3.48 (d, 1H, J =
17, H-14b), 2.85 (m, 1H, J = 8.8,12, 15.2, H-11a), 2.65
(m, 1H, J = 4, 6, 15.2, H-11b), 2.50 (m, 1H, H-8), 2.35 (m,
1H, H-7b), 1.92 (m, 1H, H-5), 1.75 (m, 1H, H-1b), 1.60
(m, 2H, H-6), 1.42 (m, 1H, H-3b), 1.40 (m, 2H, H-2), 1.40
(m, 1H, H-7a), 1.22 (m, 1H, H-3a), 1.15 (m, 1H, H-1a),
1.05 (m, 1H, H-9), 0.88 (s, 2H, H-18), 0.82 (s, 3H, H-19),
0.80 (s, 3H, H-20); 13C-NMR (75 MHz): δ: 205.37 (C-17),
198.84 (C-15), 194.80 (C-16), 158.72 (C-12), 138.35
(C-13), 56.87 (C-9), 55.03 (C-5), 49.23 (C-8), 43.16
(C-3), 40.29 (C-14), 39.88 (C-10), 39.88 (C-1), 34.25
(C-4), 34.25 (C-18), 25.65 (C-7), 27.14 (C-11), 22.31
(C-19), 20.11 (C-6), 19.82 (C-2), 16.16 (C-20).
14-Deoxy-11,12-didehydro-andrographolide. The
1H-NMR and 13C-NMR data are identical with
Suebsasana et al. (7).
1H-NMR (CDCl3, 300 MHz): δ: 7.43 (1H, t, J = 1.76
Hz, H-14), 6.85 (1H, dd, J = 10.1 and 15.8 Hz, H-12),
6.15 (1H, d, J = 15.8 Hz, H-11), 4.86 (2H, d, J = 1.3 Hz,
H-15), 4.75 (1H, d, J = 1.8 Hz, 17a), 4.49 (1H, d, J = 1.8
Hz, 17b), 4.12 (1H, d, J = 11.0 Hz, H-19a), 3.39 (1H, t,
J = 5.3 Hz, H-3), 3.38 (1H, d, J = 11.4 Hz, H-19b), 1.22
(3H, s, H-18), 0.83 (3H, s, H-20); 13C-NMR: δ: 172.2
(C=O, C-16), 148.0 (C, C-8), 142.8 (CH, C-12), 136.0
(CH, C-11), 129.2 (C, C-13), 121.1 (CH, C-14), 109.2
(CH2, C-17), 80.8 (CH, C-3), 69.5 (CH2, C-15), 64.2
(CH2, C-19), 61.7 (CH, C-9), 54.7 (CH, C-5), 43.0 (C,
C-4), 38.5 (C, C-10), 38.2 (CH2, C-1), 36.6 (CH2, C-7),
28.1 (CH2, C-2), 22.9 (CH2, C-6), 22.6 (CH3, C-18),
15.9 (CH3, C-20).
Parthenolide. The 1H-NMR and 13C-NMR data are
identical with Tiuman et al. (12).
1H-NMR (CDCl3, 300 MHz): δ: 6.34 (d, J = 3.6 Hz,
H-13α), 5.62 (d, J = 3.0 Hz, H-13β), 5.21 (dd, J = 2.7,
12.0 Hz, H-1), 3.86 (t, J = 8.4 Hz, H-6), 2.79 (d, J = 9.0
Hz, H-5), 2.74 to 2.82 (m, H-7), 2.32 to 2.44 (m, H-9β),
2.32 to 2.49 (m, H-2β), 2.11 to 2.21 (m, H-2α, H-3β,
H-8α, H-9α), 1.72 (s, H-14), 1.70 to 1.77 (m, H-8β),
1.31 (s, H-15), 1.20 to 1.28 (m, H-3α). 13C-NMR (CDCl3,
75.5 MHz): δ: 169.3 (C-12), 139.2 (C-11), 134.6 (C-10);
125.3 (C-1), 121.3 (C-13), 82.4 (C-6), 66.4 (C-5), 61.5
(C-4), 47.7 (C-7), 41.2 (C-9), 36.3 (C-3), 30.6 (C-8),
24.1 (C-2), 17.3 (C-15), 16.9 (C-14).