Planta Med. 57(1991) 515
Compounds Inhibiting Prostaglandin Synthesis Isolated from Ipomoea pes-caprae U. Pongprayoon3, P. Baeckström2. U. Jacobsson2, M. Lindström2. andL. Bohlin"4 1 2
Department of Pharmacognosy, Biomedical Center, Uppsala University, Box 579, S-75123 Uppsala, Sweden Department of Organic Chemistry, Royal Institute of Technology, S-10044 Stockholm, Sweden
Thailand Institute of Scientific and Technological Research. Bangkok 10900, Thailand Address for correspondence Received: December 18, 1990
their anti-inflammatory activity (4). Inflammation is a com-
The crude extract (IPA) of the plant Ipomoea pes-caprae (L.) R. Br. showed an inhibitory effect on prostaglandin synthesis in vitro. Bioassay-guided separation of the extract led to the isolation of four active compounds: 2-hydroxy-4,4,7-trimethyl- 1 (4H)-naphthalenone (1), (—)-mellein (2), eugenol (3), and 4-vinylguaiacol (4). Among the isolated compounds, 3 and 4 were the most active with IC50 values of 9.2 and 18 M, respectively. For I and 2 the IC50 values were 230 and 340 riM, respectively. The influence of 1, 2, 3, and 4 on
the formation of prostaglandins may partly explain a previously observed anti-inflammatory effect of the extract IPA.
Key words
plicated process, involving several different classes of mediators. Among the most important ones are the products of cyclooxygenase activity, prostaglandins. They possess direct inflammatory effects, as is the case for prostaglandin E2 and prostacyclin; both are potent vasodilators and hyperalgesic agents, and also synergise with other inflammatory mediators such as histamine and bradykinin (5).
This report describes the isolation and characterization of some compounds from the extract of!. pes-caprae, with inhibitory effects on the synthesis of prostaglandins.
Materials and Methods General experimental procedures Melting points were determined on an elec-
Ipomoea pes-caprae (L.) H. Br., 2-hy-
trothermal melting point apparatus and were uncorrected. Optical
droxy-4,4, 7-trimethyl-1(4R)-naphthalenone, (—)-mellein, eugenol, 4-vinylguaiacol, prostaglandin synthesis.
polarimeter. 1H- (400.13 MHz) and 13C-NMR (100.13 MHz) spectra
rotation in methanol solution was measured on a Perkin-Elmer
(Convolvulaceae) has been used in the traditional medicine of many tropical countries for the treatment of inflammatory disorders (1).
were recorded in deuteriochioroform on a Bruker AM 400 spectrometer. The sample used in the NOE experiment was degassed several times using the freeze-pump-thaw cycle. It was then kept under argon using a septum cap and the experiment was started within a couple of hours. The NOE difference technique was used as described by Hall and Sanders (6), and the spectra were recorded with the decoupler turned off during pulse and acquisition, preceded by 5s of irradiation either in or out of resonance. All coupling constants reported for the 1H-NMR spectra were measured after resolution enhancement of the data. Mass spectra were obtained on a Finnigan Model 4500 spectrometer connected to an
The plant extract obtained by petroleum
INCOS data system operating in the electron impact mode (70 eV). A DB 5 fused capillary column (30m, 0.32 mm i.d., 0.25gm film)
Introduction The plant Ipomoea pes-caprae (L.) R. Br.
ether extraction of the water distillate of the leaves was effective in a clinical study in patients with dermatitis caused by venomous jellyfish (2). It also exhibited a significant anti-
inflammatory activity in experimental animal models of acute inflammation when applied topically. The formation of prostaglandins in vitro was also inhibited (3).
Several classes of natural products have been shown to affect the inflammatory process through different modes of action. The greatest inhibitory effects on the
target enzymes cyclooxygenase and 5-lipoxygenase are found among phenolic compounds and arachidonic acid analogues. Furthermore, some triterpenoic acids, sesquiterpene lactones and polysaccharides exert immunomodulating activities by interfering with the complement system or T-lymphocyte populations, which may explain
was used with helium as a carrier gas. Absorption spectra were measured on a Varian Cary 19 spectrophotometer. The IR spectrum was recorded on a Polaris FT-IR spectrometer (Mattson Instruments) using KBr. Low pressure liquid chromatography was performed as described by Baeckström et al. (7). Silica gel (Merck 60, 0.040—0.063 mm) was dry packed in 15 or 25mm i.d. glass col-
umns and gradient elution was used. The eluent was delivered from a mixing chamber to the column with a metering pump at the rate of 30 mi/mm for 15mm i.d., and 60 ml/min for 25mm i.d. columns. The gradient was created with 200 ml hexane in the mixing chamber with consecutive additions of 50 ml portions of polar sol-
vent in increasing amounts (0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 100%) to a dropping funnel. TLC was performed on silica gel (Merck 60, percoated aluminium foil) eluted with 20% ethyl acetate
in hexane and visualized by spraying with vanillin and sulphuric acid in ethanol. Preparative GC was performed on a Pye Unicam 104 instrument with an FID detector connected to a computing integrator.
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Abstract
516 Planta Mcd. 57(1997)
U.PongprayoonetaL
The leaves of I. pes-caprac were collected along the seashores of Bangsaen, Thailand, during the period of May— August, 1987. Voucher specimens have been deposited in the Herbarium of the Department of Pharmaceuticals and Natural Products, Thailand Institute of Scientific and Technological Research, Bangkok, Thailand. The dried leaves were steam distilled and extracted with petroleum ether (b.p. 35—65 °C). The extract was then
treated with anhydrous magnesium sulphate and evaporated to dryness, producing an oil named IPA (0.05% yield).
Isolation, purification, and identWcation of active compounds IPA (5 g), which showed inhibitory activity on prostaglandin synthesis in vitro (3), was chromatographed on a 25mm i.d. column (60 g silica gel) and eluted with a gradient of increasing amounts of ethyl acetate in hexane to 100% ethyl acetate. The elution was continued by a gradient of increasing amounts of methanol in ethyl acetate. Thirty fractions (30 ml each) were collected and analyzed by TLC. Fractions with similar contents were combined to 6 fractions (A—F). The fractions were assayed for inhibition of prostaglandin synthesis, and two active fractions, C and D, were obtained as shown in Table 1. These fractions were indi-
vidually concentrated and redissolved in hexane and then partitioned against 1 N NaOH in aqueous solution. The aqueous phase was separated and neutralized with 1 N HC1 followed by extraction with hexane, which led to the isolation of the active acidic fractions. Further chromatography of these fractions on a 15mm i.d. column (10 g silica gel) and elution with a gradient of increasing amounts of dichloromethane in hexane [a small amount of MeOH (1 %) was added to the dichloromethane to minimize tailing] led to the isolation of compound 1 (15mg), compound 2 (14mg), and a mixture of compounds 3 and 4. The separation of 3 (50 mg) and 4(5mg) was performed on a preparative gas liquid chromatograph with a 6 m OV 101 column using a temperature programme from 60 to 180°C at a rate of 8 °C/min.
i/H-3, C-3/Me-1i, Me-12, C-4/Me-1i, Me-12, C-7/Me-13, C-8/ Me-i3 and C-iO/Me-11, Me-12. Compound I was identified as 2hydroxy-4,4,7-trimethyl-b(4H)-naphthalenone (8).
Compound 2: white needles; MS: rn/s 178 (Mi; —
66.3° (c 0.3, MeOH). Compound 2 was identified as (—)-mel-
lein (3,4-dihydro-8-hydroxy-3-methylisocoumarin) by comparing its 1H- and 13C-NMR data with those described in the literature (9).
Compound 3: colourless oil; MS: rn/s 164 (Mi. Compound 3 was identified as eugenol [2-methoxy-4-(2-propenyl)phenol] by comparison of its mass and 'H-NMR spectra with those of an authentic sample. Compound 4: colourless oil; MS: rn/s 150 (Mi; 1H-NMR: 63.92(3 H, s, OMe), 5.12 (1 H, dd, J= 0.9, 10.9 Hz, H-9), 5.59(1 H, dd, J= 0.9,17.6, H-95), 5.61(1 H, s, OH), 6.64(1 H, dd,J = 10.8, 17.5, H-8), 6.87(1 H, d, J= 8.0, H-6), 6.92(1 H, dd,J= 1.9, 8.0, H-5) and 6.94 (1 H, d, J = 1.9, H-3); 13C-NMR: 6 55.9 (OMe),
108.1 (C-3), 111.5 (C-9), 114.4 (C-6), 120.7 (C-5), 130.3 (C-4), 136.7 (C-8), 145.7 (C2a) and 146.6 (C-fl. Compound 4 was obtained in only very small amounts. Thus, the assignment of the 13CNMR spectrum was based on comparison with the assignments of the 13C-NMR spectrum of eugenol (10). On the basis of these data 4 was proposed to be ethenyl-2-methoxyphenol. The position of the ethenyl group was established in an NOB experiment. Irradiation
at 6 3.91 (OMe) resulted in enhancement of the signal at 6 6.94, while irradiation at 65.61 (OH) enhanced the signal at 66.87. Thus, 4 was identified as 4-ethenyl-2-methoxyphenol (4-vinylgnaiacol),
cf. (ii).
Prostaglandin synthesis assay The experiments were performed according to the method of White and Glassman (12). Bovine seminal vesicle microsomes (10pI, 20—30 pg protein) were pre-incubated with SOpl of cofactor solution (reduced glutathione and 1-adrenalin,
0.3mg/mi each in Tris buffer, pH 8.2) in an ice-water bath for
15mm. Vehicle or test solution (2Opl) and 2Opl of [14C]Table 1 Inhibition of prostaglandin formation by the L pes-caprae extract (IPA) and its fractions.
Fraction
% Vielda
Inhibitory Effect (%))
A
2Z0
B C
8.8 16.0 16.9 7.6 18.4
55±1 10±7 1±2
PA
D E
F aspirin
67±2 60±3
7±5 4±2 57 9
° Based on IPA. The same concentration (100 pglml( was used for all fractions. Mean SEM of two experiments, each experiment was performed in duplicate.
Compoundl: white needles; m.p. 118—118.5°C; IR (KBr); 3380, 1650cmH MS: m/z 202 (Mi, 187, 173, 159, 128, 115, 91,43; Amax (EtOH): 206 (e 22660), 258(10940), 298 nm (9020);
1H-NMR: 61.48(6 H, s, Me-Il, Me-12), 2.42(3 H, s, Me-13), 6.20(1 H, s, H-3), 6.41(1 H, s, OH), 7.43(1 H, dd,J= 2.1,8.2Hz, H-6), 7.47
(1 H, d, J 8.1, H-5), 8.01 (1 H, d, J 2.0, H-8); 13C-NMR; 621.0 (Me-13), 30.5 (Me-li, Me-12), 36.9 (C-4), 126.5 (C-5), 126.7 (C-3),
127.0 (C-8), 128.4 (C-9), 134.3 (C-6), 136.7 (C-7), 145.0 (C-2), 148.6 (C-b), 181.5 (C-i). The assignment of the 13C-NMR spectrum was based on the DEPT, H-C COSY, and the corresponding long-range experiments. Thus, long-range correlations were found between C-
arachidonic acid (16 Ci/ mol, 30 pM) were added and incubated at 37°C for 10 mm. A blank was kept in the ice-water bath. After incu-
bation, Spi of a carrier solution of unlabelled prostaglandins (0.2 mg/mi) were added and the reaction was terminated by adding 5pi of 2 N HCI. The unmetabolized arachidonic acid was sepa-
rated from the prostagiandin products by chromatography on a Bio-sii column and eiution with hexane/ dioxane/glacial acetic acid (70: 30: 1). Prostaglandin products were eluted with ethyl acetate/ methanol (85 : 15), and the activities of the samples were counted
in a Packard scintillation spectrometer. Regression analysis was used to calculate IC50 (concentration that gives 50% inhibition).
Chemicals The substances used were: reduced glutathione (Merck), 1-adrenalin (Apoteksboiaget, Sweden), [14C]-arachidonic acid (Amersham), arachidonic acid (Sigma), prostagiandins B2 and (Sigma), indomethacin (Sigma) and aspirin (Sigma).
Results and Discussion The crude extract (IPA) from the leaves of I. pes-caprae showed a significant inhibition ofprostaglandin formation. Fractionation of this extract, monitored by the prostaglandin synthesis inhibition bioassay, led to six fractions A—F, of which C and D exhibited inhibitory activity as shown in Table 1. By partition of the active fractions we succeeded in localizing the active constituents in the acidic interchangeable.
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Preparation of the plant extract
Planta Med. 57(1991) 517
Compounds Inhibiting Prostaglandin Synthesis Isolated from Ipomoea pes-caprae
OH 0
0
eugenol, was about half as potent as eugenol. Eugenol, a phenolic constituent of cloves (Syzygium aroma ticurn) arid nutmegs (Myristicafragrans), is a well-known inhibitor of
OH
H3
prostaglandin synthesis and also possesses anti-inflammatory activity in carrageenan-induced rat paw edema
H3C CH3 1
(13—15). Eugenol and guaiacol are also reported to inhibit leukocyte chemotaxis and prevent the production of oxygen
2
free-radicals by leukocytes (16). The potencies of 2-hy-
OH
droxy-4,4,7-trimethyl- 1 (4H)-naphthalenone (1) and (—)mellein (2) were in the same range as that of aspirin (IC50 =
(t...OCH3 H2
3
4
fractions as described above. Further purification led to the
isolation of four active compounds, 2-hydroxy-4,4,7trimethyl-1(4H)-naphthalenone (1), (—)-mellein (2), eugenol (3), and 4-vinylguaiacol (4).
This study confirms the anti-inflammatory
activity of I. pes-caprae shown in earlier studies (2, 3). Furthermore, four compounds responsible for a majority of the inhibitory effect exerted by the crude extract on prostaglandin synthesis have been identified. Further studies to
All compounds have been reported earlier, however, with varying quality of the spectroscopic data. Thus, full spectral assignments of 1 and 4 have been included based on one- and two-dimensional NMR spectroscopy as well as NOE measurements (see Materials and
identify compounds responsible for the previously observed in vivo anti-inflammatory activity are in progress.
Methods).
Acknowledgements
Compounds 1—4 inhibited prostaglandin synthesis in a concentration dependent manner (Figure 1). Among them, eugenol (3) was the most potent compound with an IC50 of 9.2 M, which was about ten times less potent than the reference drug indomethacin (IC50 = 0.7 pM). 4-Vinylguaiacol (4) (IC50 = 18 pM), which is an analogue of
One of the authors (U. P.) gratefully acknowledges the kind collaboration of Prof. T. Norm, Royal Institute of Technology, Stockholm, who provided the possibility of working in his laboratory when necessary. This research was financially supported by the Swedish Council for Planning and Coordination of Research (FRN).
Fig. 1 Inhibitory effect of 2-hydroxy.4,4,7.trimethyl.1(4H). naphthalenone (1), (—)-mellein (2), eugenol (3), and 4-vinylguaiacol (4) together with the two reference substances indomethacin and aspirin on prostaglandin synthesis in vitro.
1ot
Each point represents the mean SEM of three experiments, each experiment was performed in duplicate.
80
60
C
40
—.---..— Indomethacin
-....-.- Eugenol 20
— —
... — 4 -Vinylguaiacol
.• . — 2-hydroxy-4,4,74rimethyl-
—.----
--a--
1(411)-naplsthalenone (-)-Mellein Aspirin
00,1
10
Concentration (.Lg/mL)
100
1000
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230, 340 and 420pM, respectively). 2-Hydroxy-4,4,7trimethyl- 1 (4H)-naphthalenone was first reported as an oxidation product of 4,4, 7-trimethyl-3 ,4-dihydro-2(1H)naphthalenone (8). No pharmacological data have been reported for I in the literature. Mellein is known from Aspergums melleus and from other microbial sources (17, 18). It has been reported to possess anti-bacterial activity (19), but not anti-inflammatory activity.
518 Planta Med. 57(1991)
Sastni, B. N. (1965) in: The Wealth of India, Raw Materials 5, 251, 2
6
8
CSIR, New Delhi. Sunthonpalin, P., Wasuwat, S. (1985) Siniraj Hosp. Gaz. 37, 329.
Pongprayoon, U., Bohlin, L., Soonthornsaratune, P., Wasuwat, S. (1991) Phytotherapy Res. 5 (2), 63. Wagner, H. (1989) Planta Med. 55, 235. Salmon, J. A., Higgs, G. A. (1987) in: Bri. Med. Bull. 43(2), 285 , (Willoughby, D. A., ed.), Churchill 1.ivingstone, Edinburgh. Hall, L. D., Sanders, J. K. M. (1980) J. Am. Chem. Soc. 102, 5703. Baeckstrdm, P., Stridh, K., Li, L., Non T. (1987) Acta Chem. Scand. B41, 442. Davis, D. L., Stevens, K. L., Jurd, L. (1976) J. Agric. Food Chem. 24, 187. Mori, K., Gupta, A. K. (1985) Tetrahedron 41, 5295.
° Formacek, V., Desnoyer, L., Kellerhals, H. P., Keller, T., Clerc, J. T. in: 13C Data Bank, Vol. 1, Bruker Physik, Karlsruhe. " (1976) Shibata, S., Katsuyama. A., Noguchi, M. (1978) Agric. Biol. Chem. 42, 195. 12 White, H. L., Glassman, A. T. (1974) Prostaglandins 7, 123. 13 Wagner, H., Wierer, M., Bauer, R. (1986) Planta Med. 52, 184. 14 Bennett, A., Stamford, I. F., Tavares, I. A., Jacobs, S., Capasso, F., Mascolo, N., Autore, G., Romano, V.. Di Carlo, G. (1988) Phytotherapy Res. 2,124. Evans, W. C. (1989) in: Trease and Evans' Pharmacognosy, 13th ed., The Alden Press, Oxford. 16 Azuma, Y., Ozasa, Y., Takagi, N. (1986) J. Dent. Res. 65 (1), 53. 17 Nishikawa, H. (1933) J. Agric. Chem. Soc. Jpn. 772.
' 19
Aidridge, D. C., Gait, S., Giles, D., Turner, W. B. (1971) J. Chem. Soc. (C), 1623. Burton, H. 5. (1950) Nature, 165, 274.
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References
U. Pongprayoon et aL
Compounds Inhibiting Prostaglandin Synthesis Isolated from Ipomoea pes-caprae U. Pongprayoon3, P. Baeckström2. U. Jacobsson2, M. Lindström2. andL. Bohlin"4 1 2
Department of Pharmacognosy, Biomedical Center, Uppsala University, Box 579, S-75123 Uppsala, Sweden Department of Organic Chemistry, Royal Institute of Technology, S-10044 Stockholm, Sweden
Thailand Institute of Scientific and Technological Research. Bangkok 10900, Thailand Address for correspondence Received: December 18, 1990
their anti-inflammatory activity (4). Inflammation is a com-
The crude extract (IPA) of the plant Ipomoea pes-caprae (L.) R. Br. showed an inhibitory effect on prostaglandin synthesis in vitro. Bioassay-guided separation of the extract led to the isolation of four active compounds: 2-hydroxy-4,4,7-trimethyl- 1 (4H)-naphthalenone (1), (—)-mellein (2), eugenol (3), and 4-vinylguaiacol (4). Among the isolated compounds, 3 and 4 were the most active with IC50 values of 9.2 and 18 M, respectively. For I and 2 the IC50 values were 230 and 340 riM, respectively. The influence of 1, 2, 3, and 4 on
the formation of prostaglandins may partly explain a previously observed anti-inflammatory effect of the extract IPA.
Key words
plicated process, involving several different classes of mediators. Among the most important ones are the products of cyclooxygenase activity, prostaglandins. They possess direct inflammatory effects, as is the case for prostaglandin E2 and prostacyclin; both are potent vasodilators and hyperalgesic agents, and also synergise with other inflammatory mediators such as histamine and bradykinin (5).
This report describes the isolation and characterization of some compounds from the extract of!. pes-caprae, with inhibitory effects on the synthesis of prostaglandins.
Materials and Methods General experimental procedures Melting points were determined on an elec-
Ipomoea pes-caprae (L.) H. Br., 2-hy-
trothermal melting point apparatus and were uncorrected. Optical
droxy-4,4, 7-trimethyl-1(4R)-naphthalenone, (—)-mellein, eugenol, 4-vinylguaiacol, prostaglandin synthesis.
polarimeter. 1H- (400.13 MHz) and 13C-NMR (100.13 MHz) spectra
rotation in methanol solution was measured on a Perkin-Elmer
(Convolvulaceae) has been used in the traditional medicine of many tropical countries for the treatment of inflammatory disorders (1).
were recorded in deuteriochioroform on a Bruker AM 400 spectrometer. The sample used in the NOE experiment was degassed several times using the freeze-pump-thaw cycle. It was then kept under argon using a septum cap and the experiment was started within a couple of hours. The NOE difference technique was used as described by Hall and Sanders (6), and the spectra were recorded with the decoupler turned off during pulse and acquisition, preceded by 5s of irradiation either in or out of resonance. All coupling constants reported for the 1H-NMR spectra were measured after resolution enhancement of the data. Mass spectra were obtained on a Finnigan Model 4500 spectrometer connected to an
The plant extract obtained by petroleum
INCOS data system operating in the electron impact mode (70 eV). A DB 5 fused capillary column (30m, 0.32 mm i.d., 0.25gm film)
Introduction The plant Ipomoea pes-caprae (L.) R. Br.
ether extraction of the water distillate of the leaves was effective in a clinical study in patients with dermatitis caused by venomous jellyfish (2). It also exhibited a significant anti-
inflammatory activity in experimental animal models of acute inflammation when applied topically. The formation of prostaglandins in vitro was also inhibited (3).
Several classes of natural products have been shown to affect the inflammatory process through different modes of action. The greatest inhibitory effects on the
target enzymes cyclooxygenase and 5-lipoxygenase are found among phenolic compounds and arachidonic acid analogues. Furthermore, some triterpenoic acids, sesquiterpene lactones and polysaccharides exert immunomodulating activities by interfering with the complement system or T-lymphocyte populations, which may explain
was used with helium as a carrier gas. Absorption spectra were measured on a Varian Cary 19 spectrophotometer. The IR spectrum was recorded on a Polaris FT-IR spectrometer (Mattson Instruments) using KBr. Low pressure liquid chromatography was performed as described by Baeckström et al. (7). Silica gel (Merck 60, 0.040—0.063 mm) was dry packed in 15 or 25mm i.d. glass col-
umns and gradient elution was used. The eluent was delivered from a mixing chamber to the column with a metering pump at the rate of 30 mi/mm for 15mm i.d., and 60 ml/min for 25mm i.d. columns. The gradient was created with 200 ml hexane in the mixing chamber with consecutive additions of 50 ml portions of polar sol-
vent in increasing amounts (0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 100%) to a dropping funnel. TLC was performed on silica gel (Merck 60, percoated aluminium foil) eluted with 20% ethyl acetate
in hexane and visualized by spraying with vanillin and sulphuric acid in ethanol. Preparative GC was performed on a Pye Unicam 104 instrument with an FID detector connected to a computing integrator.
Downloaded by: University of Arizona Library. Copyrighted material.
Abstract
516 Planta Mcd. 57(1997)
U.PongprayoonetaL
The leaves of I. pes-caprac were collected along the seashores of Bangsaen, Thailand, during the period of May— August, 1987. Voucher specimens have been deposited in the Herbarium of the Department of Pharmaceuticals and Natural Products, Thailand Institute of Scientific and Technological Research, Bangkok, Thailand. The dried leaves were steam distilled and extracted with petroleum ether (b.p. 35—65 °C). The extract was then
treated with anhydrous magnesium sulphate and evaporated to dryness, producing an oil named IPA (0.05% yield).
Isolation, purification, and identWcation of active compounds IPA (5 g), which showed inhibitory activity on prostaglandin synthesis in vitro (3), was chromatographed on a 25mm i.d. column (60 g silica gel) and eluted with a gradient of increasing amounts of ethyl acetate in hexane to 100% ethyl acetate. The elution was continued by a gradient of increasing amounts of methanol in ethyl acetate. Thirty fractions (30 ml each) were collected and analyzed by TLC. Fractions with similar contents were combined to 6 fractions (A—F). The fractions were assayed for inhibition of prostaglandin synthesis, and two active fractions, C and D, were obtained as shown in Table 1. These fractions were indi-
vidually concentrated and redissolved in hexane and then partitioned against 1 N NaOH in aqueous solution. The aqueous phase was separated and neutralized with 1 N HC1 followed by extraction with hexane, which led to the isolation of the active acidic fractions. Further chromatography of these fractions on a 15mm i.d. column (10 g silica gel) and elution with a gradient of increasing amounts of dichloromethane in hexane [a small amount of MeOH (1 %) was added to the dichloromethane to minimize tailing] led to the isolation of compound 1 (15mg), compound 2 (14mg), and a mixture of compounds 3 and 4. The separation of 3 (50 mg) and 4(5mg) was performed on a preparative gas liquid chromatograph with a 6 m OV 101 column using a temperature programme from 60 to 180°C at a rate of 8 °C/min.
i/H-3, C-3/Me-1i, Me-12, C-4/Me-1i, Me-12, C-7/Me-13, C-8/ Me-i3 and C-iO/Me-11, Me-12. Compound I was identified as 2hydroxy-4,4,7-trimethyl-b(4H)-naphthalenone (8).
Compound 2: white needles; MS: rn/s 178 (Mi; —
66.3° (c 0.3, MeOH). Compound 2 was identified as (—)-mel-
lein (3,4-dihydro-8-hydroxy-3-methylisocoumarin) by comparing its 1H- and 13C-NMR data with those described in the literature (9).
Compound 3: colourless oil; MS: rn/s 164 (Mi. Compound 3 was identified as eugenol [2-methoxy-4-(2-propenyl)phenol] by comparison of its mass and 'H-NMR spectra with those of an authentic sample. Compound 4: colourless oil; MS: rn/s 150 (Mi; 1H-NMR: 63.92(3 H, s, OMe), 5.12 (1 H, dd, J= 0.9, 10.9 Hz, H-9), 5.59(1 H, dd, J= 0.9,17.6, H-95), 5.61(1 H, s, OH), 6.64(1 H, dd,J = 10.8, 17.5, H-8), 6.87(1 H, d, J= 8.0, H-6), 6.92(1 H, dd,J= 1.9, 8.0, H-5) and 6.94 (1 H, d, J = 1.9, H-3); 13C-NMR: 6 55.9 (OMe),
108.1 (C-3), 111.5 (C-9), 114.4 (C-6), 120.7 (C-5), 130.3 (C-4), 136.7 (C-8), 145.7 (C2a) and 146.6 (C-fl. Compound 4 was obtained in only very small amounts. Thus, the assignment of the 13CNMR spectrum was based on comparison with the assignments of the 13C-NMR spectrum of eugenol (10). On the basis of these data 4 was proposed to be ethenyl-2-methoxyphenol. The position of the ethenyl group was established in an NOB experiment. Irradiation
at 6 3.91 (OMe) resulted in enhancement of the signal at 6 6.94, while irradiation at 65.61 (OH) enhanced the signal at 66.87. Thus, 4 was identified as 4-ethenyl-2-methoxyphenol (4-vinylgnaiacol),
cf. (ii).
Prostaglandin synthesis assay The experiments were performed according to the method of White and Glassman (12). Bovine seminal vesicle microsomes (10pI, 20—30 pg protein) were pre-incubated with SOpl of cofactor solution (reduced glutathione and 1-adrenalin,
0.3mg/mi each in Tris buffer, pH 8.2) in an ice-water bath for
15mm. Vehicle or test solution (2Opl) and 2Opl of [14C]Table 1 Inhibition of prostaglandin formation by the L pes-caprae extract (IPA) and its fractions.
Fraction
% Vielda
Inhibitory Effect (%))
A
2Z0
B C
8.8 16.0 16.9 7.6 18.4
55±1 10±7 1±2
PA
D E
F aspirin
67±2 60±3
7±5 4±2 57 9
° Based on IPA. The same concentration (100 pglml( was used for all fractions. Mean SEM of two experiments, each experiment was performed in duplicate.
Compoundl: white needles; m.p. 118—118.5°C; IR (KBr); 3380, 1650cmH MS: m/z 202 (Mi, 187, 173, 159, 128, 115, 91,43; Amax (EtOH): 206 (e 22660), 258(10940), 298 nm (9020);
1H-NMR: 61.48(6 H, s, Me-Il, Me-12), 2.42(3 H, s, Me-13), 6.20(1 H, s, H-3), 6.41(1 H, s, OH), 7.43(1 H, dd,J= 2.1,8.2Hz, H-6), 7.47
(1 H, d, J 8.1, H-5), 8.01 (1 H, d, J 2.0, H-8); 13C-NMR; 621.0 (Me-13), 30.5 (Me-li, Me-12), 36.9 (C-4), 126.5 (C-5), 126.7 (C-3),
127.0 (C-8), 128.4 (C-9), 134.3 (C-6), 136.7 (C-7), 145.0 (C-2), 148.6 (C-b), 181.5 (C-i). The assignment of the 13C-NMR spectrum was based on the DEPT, H-C COSY, and the corresponding long-range experiments. Thus, long-range correlations were found between C-
arachidonic acid (16 Ci/ mol, 30 pM) were added and incubated at 37°C for 10 mm. A blank was kept in the ice-water bath. After incu-
bation, Spi of a carrier solution of unlabelled prostaglandins (0.2 mg/mi) were added and the reaction was terminated by adding 5pi of 2 N HCI. The unmetabolized arachidonic acid was sepa-
rated from the prostagiandin products by chromatography on a Bio-sii column and eiution with hexane/ dioxane/glacial acetic acid (70: 30: 1). Prostaglandin products were eluted with ethyl acetate/ methanol (85 : 15), and the activities of the samples were counted
in a Packard scintillation spectrometer. Regression analysis was used to calculate IC50 (concentration that gives 50% inhibition).
Chemicals The substances used were: reduced glutathione (Merck), 1-adrenalin (Apoteksboiaget, Sweden), [14C]-arachidonic acid (Amersham), arachidonic acid (Sigma), prostagiandins B2 and (Sigma), indomethacin (Sigma) and aspirin (Sigma).
Results and Discussion The crude extract (IPA) from the leaves of I. pes-caprae showed a significant inhibition ofprostaglandin formation. Fractionation of this extract, monitored by the prostaglandin synthesis inhibition bioassay, led to six fractions A—F, of which C and D exhibited inhibitory activity as shown in Table 1. By partition of the active fractions we succeeded in localizing the active constituents in the acidic interchangeable.
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Preparation of the plant extract
Planta Med. 57(1991) 517
Compounds Inhibiting Prostaglandin Synthesis Isolated from Ipomoea pes-caprae
OH 0
0
eugenol, was about half as potent as eugenol. Eugenol, a phenolic constituent of cloves (Syzygium aroma ticurn) arid nutmegs (Myristicafragrans), is a well-known inhibitor of
OH
H3
prostaglandin synthesis and also possesses anti-inflammatory activity in carrageenan-induced rat paw edema
H3C CH3 1
(13—15). Eugenol and guaiacol are also reported to inhibit leukocyte chemotaxis and prevent the production of oxygen
2
free-radicals by leukocytes (16). The potencies of 2-hy-
OH
droxy-4,4,7-trimethyl- 1 (4H)-naphthalenone (1) and (—)mellein (2) were in the same range as that of aspirin (IC50 =
(t...OCH3 H2
3
4
fractions as described above. Further purification led to the
isolation of four active compounds, 2-hydroxy-4,4,7trimethyl-1(4H)-naphthalenone (1), (—)-mellein (2), eugenol (3), and 4-vinylguaiacol (4).
This study confirms the anti-inflammatory
activity of I. pes-caprae shown in earlier studies (2, 3). Furthermore, four compounds responsible for a majority of the inhibitory effect exerted by the crude extract on prostaglandin synthesis have been identified. Further studies to
All compounds have been reported earlier, however, with varying quality of the spectroscopic data. Thus, full spectral assignments of 1 and 4 have been included based on one- and two-dimensional NMR spectroscopy as well as NOE measurements (see Materials and
identify compounds responsible for the previously observed in vivo anti-inflammatory activity are in progress.
Methods).
Acknowledgements
Compounds 1—4 inhibited prostaglandin synthesis in a concentration dependent manner (Figure 1). Among them, eugenol (3) was the most potent compound with an IC50 of 9.2 M, which was about ten times less potent than the reference drug indomethacin (IC50 = 0.7 pM). 4-Vinylguaiacol (4) (IC50 = 18 pM), which is an analogue of
One of the authors (U. P.) gratefully acknowledges the kind collaboration of Prof. T. Norm, Royal Institute of Technology, Stockholm, who provided the possibility of working in his laboratory when necessary. This research was financially supported by the Swedish Council for Planning and Coordination of Research (FRN).
Fig. 1 Inhibitory effect of 2-hydroxy.4,4,7.trimethyl.1(4H). naphthalenone (1), (—)-mellein (2), eugenol (3), and 4-vinylguaiacol (4) together with the two reference substances indomethacin and aspirin on prostaglandin synthesis in vitro.
1ot
Each point represents the mean SEM of three experiments, each experiment was performed in duplicate.
80
60
C
40
—.---..— Indomethacin
-....-.- Eugenol 20
— —
... — 4 -Vinylguaiacol
.• . — 2-hydroxy-4,4,74rimethyl-
—.----
--a--
1(411)-naplsthalenone (-)-Mellein Aspirin
00,1
10
Concentration (.Lg/mL)
100
1000
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230, 340 and 420pM, respectively). 2-Hydroxy-4,4,7trimethyl- 1 (4H)-naphthalenone was first reported as an oxidation product of 4,4, 7-trimethyl-3 ,4-dihydro-2(1H)naphthalenone (8). No pharmacological data have been reported for I in the literature. Mellein is known from Aspergums melleus and from other microbial sources (17, 18). It has been reported to possess anti-bacterial activity (19), but not anti-inflammatory activity.
518 Planta Med. 57(1991)
Sastni, B. N. (1965) in: The Wealth of India, Raw Materials 5, 251, 2
6
8
CSIR, New Delhi. Sunthonpalin, P., Wasuwat, S. (1985) Siniraj Hosp. Gaz. 37, 329.
Pongprayoon, U., Bohlin, L., Soonthornsaratune, P., Wasuwat, S. (1991) Phytotherapy Res. 5 (2), 63. Wagner, H. (1989) Planta Med. 55, 235. Salmon, J. A., Higgs, G. A. (1987) in: Bri. Med. Bull. 43(2), 285 , (Willoughby, D. A., ed.), Churchill 1.ivingstone, Edinburgh. Hall, L. D., Sanders, J. K. M. (1980) J. Am. Chem. Soc. 102, 5703. Baeckstrdm, P., Stridh, K., Li, L., Non T. (1987) Acta Chem. Scand. B41, 442. Davis, D. L., Stevens, K. L., Jurd, L. (1976) J. Agric. Food Chem. 24, 187. Mori, K., Gupta, A. K. (1985) Tetrahedron 41, 5295.
° Formacek, V., Desnoyer, L., Kellerhals, H. P., Keller, T., Clerc, J. T. in: 13C Data Bank, Vol. 1, Bruker Physik, Karlsruhe. " (1976) Shibata, S., Katsuyama. A., Noguchi, M. (1978) Agric. Biol. Chem. 42, 195. 12 White, H. L., Glassman, A. T. (1974) Prostaglandins 7, 123. 13 Wagner, H., Wierer, M., Bauer, R. (1986) Planta Med. 52, 184. 14 Bennett, A., Stamford, I. F., Tavares, I. A., Jacobs, S., Capasso, F., Mascolo, N., Autore, G., Romano, V.. Di Carlo, G. (1988) Phytotherapy Res. 2,124. Evans, W. C. (1989) in: Trease and Evans' Pharmacognosy, 13th ed., The Alden Press, Oxford. 16 Azuma, Y., Ozasa, Y., Takagi, N. (1986) J. Dent. Res. 65 (1), 53. 17 Nishikawa, H. (1933) J. Agric. Chem. Soc. Jpn. 772.
' 19
Aidridge, D. C., Gait, S., Giles, D., Turner, W. B. (1971) J. Chem. Soc. (C), 1623. Burton, H. 5. (1950) Nature, 165, 274.
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