Cyclooxygenase inhibitory constituents from Houttuynia cordata

Phytomedicine Vol. 2 (4), pp. 305-308, 1996 © 1996 by Gustav Fischer Verlag, Stuttgart· Jena . New York

Cyclooxygenase inhibitory constituents from Houttuynia cordata R. BAUER1, A. PROBSTLE2, H. LOTIER2, W. WAGNER-REDECKER3, and U. MATIHIESEN4 Institut fur Pharmazeutische Biologie, Heinrich-Heine-Universitat Dusseldorf, Universitatsstr, 1, D-40225 Dusseldorf, Germany. 2 Institut fur Pharmazeutische Biologie, Ludwig-Maximilians-Universitat Miinchen, Karlstr. 29 , D-80333 Miinchen, Germany. 3 Finnigan MAT GmbH, Barkhausenstr. 2, D-28197 Bremen, Germany. 4 Spurenelementlabor der med. Einrichtung, Heinrich-Heine-Universitat Dusseldorf, Universitatsstr, 1, D-40225 Dusseldorf, Germany.

1

Summary The n-hexane extract of Houttuynia cordata was shown to inhibit prostaglandin synthase in vitro. Phytochemical examination led to the identification of five fatty acids (linolenic, linoleic, oleic, palmitic and stearic), cepharanone B, phytol and stigmast-4-ene-3,6-dione. The inhibitory effect of the extract on prostaglandin formation in vitro could be attributed mainly to linoleic and linolenic acid. Key Words: Houttuynia cordata, Saururaceae, fatty acids, phytol, stigmast-4-ene-3,6-dione, pyridine alkaloids, LC-MS, cyclooxygenase inhibition .

Introduction The aerial parts of Houttuynia cordata Thunb. (Yu Xing Cao) have been used in traditional Chinese medicine as a detoxicant and an antiinflammatory and antipyretic agent, especially for the respiratory tract and skin problems (Chang and But, 1986). Previous investigations of H. cordata showed it contained flavonoids and fatty acids (Takagi et aI., 1978), essential oil (Tutupalli and Chaubal, 1975), a benzamide (Nishiya et aI., 1988), aristolactams and 4,5dioxoaporphines (Probstle and Bauer, 1992; Jong and Jean, 1993 a) and pyridine derivatives (Jong and Jean, 1993 b; Probstle et aI., 1994). Recently, the steam distillate of H. cordata has been reported to have virucidal effects (Kyoko et aI., 1995). Based on our phytochemical and pharmacological investigations of H. cordata, we now report on further lipophilic constituents and their pharmacological activities.

Material and Methods The plant material was provided and identified by Prof. Lou Zhicen, Dept. of Pharmacognosy, BeijingMedical University, by Prof. Won S. Woo, Natural Products Research Institute, Seoul National University, Korea, and by the botanical garden of the University of Dusseldorf, Germany. Voucher specimens are on deposit at the Institute of Pharmaceutical Biology, Munich.

HPLC: Hibar LiChroCART 125-4 LiChrospher RP18, 5 Jl (for LC-MS: 100-4, 3 u) (Merck) with MeCNIH2 0 , 50-95% linearly in 30 min, 95-100% linearly in 15 min, flow: 1.0 mllmin (for LC-MS: 0.6 mllmin), detection 210 nm. GC: Column: DB Wax, 60 m x 0.25 mm ID, 25 urn film thickness (J+W Scientific); mobile phase: argon; inj. temp.:

306

R. Bauer et al.

250°C; det. temp. 250°C (FID); column temp.: 150220°C (5°C/min), 14-60 min isotherm at 220°C; retention times: palmitic acid 30.7 min, stearic acid 41.5 min, oleic acid 43.4 min, linoleic acid 47.5 min, linolenic acid 50.3 min.

Preparation of fatty acid methylesters: 2 mg of n-hexane extract were boiled with 2 ml of methanolic NaOH solution (5 min); this was followed by the addition of 4 ml of BF3-methanol (20%) via the condenser. After boiling 2 min, 3 ml of n-hexane were added and the boiling was continued for another 2 min. The n-hexane phase, containing the methylesters was removed from the mixture using a saturated NaCl solution and was dried over NaS04' The n-hexane phase was evaporated to dryness and the residue dissolved in 500/ll n-hexane (Van Wijngaarden, 1967).

Mass spectra of fatty acids (LC-MS): Palmiticacid EI-MS 256 [M]+, 213, 185, 129, 73. Stearic acid EI-MS: 284 [M]+, 241,185,129,73. Oleic acid ElMS: 282 [M]+, 264, 220,180,111,97,69. Linoleicacid ElMS: 280 [M]+, 123, 109, 95, 81, 67. Linolenic acid EI-MS: 278 [M]+, 163, 135, 108,93, 79, 67. Extraction: 750 g dried aerial parts of H. cordata were powdered and exhaustively extracted with n-hexane in a Soxhlet apparatus (yield 35 g).

Isolation: MPLC on silica gel (particle size < 63 urn, column: Biichi B-685 46 x 4.9 em) n-hexane/EtOAc gradient (nhexane 100%, 98%, 95%, 90%, 80%, 70%, 50%, 500ml each) 10ml/min, fractions 20ml; MPLC on RP18 (Multoprep, particle size 25/40/lm, CS, Eschweiler, column: 46 x 1,5 em) MeOH/H 20 gradient (75%-100% MeOH), 2.5 ml/min; fractions 10 ml; analysis of eluates by TLC (silica gel 60F254, thickness: 0.25 mm, Merck; nhexane/ EtOAc 5:3; detection: UV 365 nm, vanillinIH2S04 reagent).

Structure elucidation: IH-NMR spectra were recorded at 400 MHz in DMSOd6 , using TMS as internal standard. UV spectra were recorded online by a photodiode array detector (HP 1040 A), EI-MS and CI-MS by a Kratos MS 80 RFA, and LC-MS by a Finnigan MAT SSQ 710 instrument with particle beam interface. Cepharanone B (1): C17H 13N0 3 , EI-MS: 279 [M]+, 264,

236,221,193. UV "max (nm) 230, 260, 275, 285,315,385. IR V (cm-t) 3180 (NH), 1720 (CO), 1380. IH-NMR (DMSO-d6 ) 10.9 (s, NH), 9.1 (dd, H-5), 8.0 (dd, H-8), 7.6 (m, H-6, H-7), 7.6 (s, H-2), 7.2 (s, H-9), 4.1 (2 s, 20Me). Phytol (2): C20H400, EI-MS: 296 [M]+, 278, 196, 137, 123. UV "max (nm) 200. IR V (cm'") 3400,2900. IH-NMR (CDC1 3 ) 5.4 (t, H-3), 4.1 (d, H-1 a, b), 2.0 (t, H-4 a, b), 1.7 (s, CH 3 ) , 1.3 (m, CH 2), 0.8 (4xd, 4 CH 3 ). 13C-NMR (CDC1 3 ) 140.2, 123.1,59.4,39.9,39.3, 37.4, 36.6, 32.8, 32.7,29.7,28.0,25.1,24.8,24.5,22.7,22.6,19.7,16.1. Stigmast-4-ene-3,6-dione (3): C29H4602, EI-MS: 426 [M]+, 400, 280, 163, 137, 83. UV "max (nm) 200,243. IR V (crn') 2950,1680. IH-NMR (CDC1 3 ): 6.1 (s, H-4), 2.7 (dd, H-7), 2.5 (d, t, H-8, H-9), 2.1 (n, H-14), 2.0 (m, H-17), 1.9 (m, H-1, H-2), 1.6 (m, H-15, H-16), 1.4 (m, H-11, H-12), 1.3 (m, H-20, H-28), 1.2 (s, H-19), 1.0 (d, H-21), 0.9 (t, H29),0.8 (d, H-26, H-27), 0.8 (s, H-18). 13C-NMR (CDC1 3 ): 202.5 (C-3), 199.5 (C-6), 161.1 (C-5), 125.4 (C-4), 56.5 (C-14), 55.8 (C-17), 51.0 (C-9), 46.8 (C-1), 45.8 (C-24), 42.5 (C-13), 39.8 (C-12), 39.1 (C-10), 36.0 (C-20), 35.5 (C-8), 34.2 (C-7), 33.9 (C-22), 33.8 (C-2), 29.1 (C-25), 28.0 (C-16), 17.5 (C-21), 11.9 (C-21), 11.9 (C-29), 11.8 (C-18). X-ray structure analysis: Stigmast-4-ene-3,6-dione (3) crystallized from n-hexane as thin transparent yellow platelets. Crystal data: a = 7.780 (3), b = 12.580 (5), c = 27.260 (10) A, ~ = 90.42 (2)°, monoclinic space group C2, z = 4,1864 unique reflections were measured on a Siemens R 3 m diffractometer with Ni-filtered Cu K, radiation up to 2'\) = 114°. From these, 1305 were treated as observed with F 2: -tc (F). Absorption correction was measured and applied. Structure elucidation was performed by means of SHELXTL. Isotropic refinements converged at R = 13.2%. COX-assay: The test was performed with cyclooxygenase from sheep seminal vesicle microsomes. Cyclooxygenase activity was determined in a microtiter scale as previously described (Redl et al., 1994). The incubation mixture contained 1 ug enzyme preparation in 190 ul 0.1 M tris buffer (pH 8.0), 1 nM reduced glutathion, 1 mM epinephrinhydrogentartrate and 0.05 mM Na 2EDTA. 10/ll of test substance dissolved in EtOH p. a. were added and preincubated for 5 min. The reaction was started by addition of 10/ll of 4.5 /lM 1-14C-arachidonic acid (0.05/lCi) and incubated for 20 min at 3rc. The reaction was stopped by adding 10/ll of 10% formic acid. Arachidonic acid and its labelled metabolites were separated by reversed-phaseHPLC, detected by radioactivity monitoring and quantified via peak areas. Activity was calculated as % inhibition of PGE2 formation in comparison to a blank run (pure ethanol) without the inhibitor. Statistics: Control measurements were performed in duplicate. Results are means of three experiments (% inhibition). The maximum observed standard deviation (absolute) of the test was 7%. Positive control measurements

Cyclooxygenase inhibito ry constituents from Houttuynia cordata

, CH3 0

307

o N-H

8 13

, CH30

9 3

o Cepharanone B

Stlgmast-4-e ne-3,6-dlo ne

4 1il

OH

(min)

Fig. 1. HPLC separation of the n-hexane extract of H. cordata monitored by UV (210 nm). 1 = Cepharanone B, 2 = a-Linolenic acid, 3 = Linoleic acid, 4 =Oleic acid, 5 = Palmitic acid, 6 =Stearic acid, 7 = Phytol, 8 = 3,5-Didecanoyl-pyridine, 9 = 2-Nonyl-5decanoyl-pyridine, 10 =3,5-Didecanoyl-4-nonyl-l ,4-dihydropyridine, 11 = 3-Decanoyl-4-nonyl-5-dodecanoyl-1,4-dihydropyridine, 12 = 3,5-Didodecanoyl-4-nonyl-1,4-dihydropyridine, 13 = 1,3,5-Tri-decanoyl-benzene.

were performed with indomethacin at a concentration of its IC so value (1.2 11M ). ICso values were determined by regression analysis of the mean results at four different concentrations.

Results and Discussion Th e aerial parts of H. cordata were extracted with n-hexane and chloro phyll was removed by vacuum chromatography on RP18 with methanol as the eluent. The resulting extract exhibited stro ng inhibitory activity in the cyclooxygenase (COX) inhibiti on assay (IC so = 5.6Ilg/ml). In order to identify the active principles, the constituents of the nhexane extract were ana lyzed by HPLC (Fig. 1). Several compounds with very low UV absorbance were detected by using LC-MS with a particle beam interface. By comparing the on-line recorded El spectra with spectra from the library, the five main constituents were identified as ex-linolenic, linoleic, oleic, palmitic, and stearic acids. The results were confirm ed by GC of the fatty acid meth yl esters after meth ylating the n-hexane extract with boro n tr ifluoride methanol, and compar ing them with reference comp ounds. Quantitat ive analysis of fatty acids in the nhexan e extract of several samples of H. cordata showed that their total concentra tio ns varied from 8 to 32 % of the n-hexane extract, corresponding to 0.3-1.1 % in the plant materi al (Tab. 1). Fractionation and isolation of furth er constituents of the n-hexane extra ct was achieved by MPLC and semipreparative HPLC. This resulted in the identification of cepharanone B (1), phytol (2), and stigmast-4-ene-3,6-dione (3) (Fig. 2), as well

Phytol

Fig.2. Formulas of isolated compounds. as five pyridine derived alkaloids and a triacylbenzene derivat ive, which we have already reported (Probsrle et al., 1994 ). Cephara none B (1) has been previously isolated from the chloroform extrac t of H. cordata and was identified by NMR and MS (Probstle and Bauer, 1992). Phytol (2) could be identified by GC-MS and comparison with reference data. Stigmast-4-ene-3,6-dione (3) crystallized from n-hexane. The structure of 3 could be elucidated by X-ra y anal ysis and N MR. As the measurement had been carri ed out at room temperature, strong thermal movements of the side cha in at C-17 made localizati on of C-25- C-29 impossible. Therefore, the refinement was not anisotropically finished. However, the structure of the side chain could be determined by 13C-NM R, by comparison with literature data (Goad, 1991), and by DEPT experiments, since 3 had pre viously been isolated from Sam bucus ebulus (Tunmann and Grimm, 1974 ). Testing the isolated com pounds, ph ytol (ICso = 44 11M) and stigmast-4-ene-3,6-dione (IC so = 148 11M) showed medium to low inhibitor y activity in the COX assay. Cepharanonc B and the pyridine derived alkaloids exhib ited only weak activity. Linoleic (ICso = 0.5IlM ), oleic (ICso = 14.011 M ), palmitic (IC so = 20.0IlM ), and linolenic acids (IC so = 23.0 11M) showed the highest inhibitory activity of Table 1. Determination of fatty acids by GC in the n-hexane extracts of different samples of H. cordata (Extr. 1 = from Korea, Ext r, 2 = from China, Extr, 3 = from Germany) . content ['Yo]

Extr. 1

palmitic acid stearic acid oleic acid linoleic acid a-linolenic acid

2.1 2.2 1.9 1.4 1.0

5.7 2.4 3.7 2.9 1.9

5.0 2.0 5.5 4.4 13.0

total fatty acids total fatty acids in the plant material

8.6

16.6

29.9

0.6

1.1

0.3

Extr. 2

Extr. 3

308

R. Bauer et al.

Table2. Inhibition of cyclooxygenase by lipophilic compounds isolated from H. cordata. Inhibition [%] (cone. 200IlM/ml) cepharanone B palmitic acid stearic acid oleic acid linoleic acid linolenic acid phytol 3,5-decanoylpyridine 1,4-dihydrophyridine 1,3,5-tridecanoylbenzene ~-sitosterol

stigmast-4-ene-3,6-dione

6.2 79.0 50.5 83.2 100.0 85.0 83.0

IC so [1lM/ml] 20.0 14.0 0.5 23.0 55.7

1~.4

89.0 3.4 83.0 60.0

141.0 23.7 148.0

all the compounds isolated from H. cordata so far (Tab. 2). Therefore, the in vitro activity of the n-hexane extract is probably due to its content of unsaturated fatty acids. The activity of linoleic acid in vitro is even better than that of indomethacin (IC so = 1.2IlM). After extracting the dried aerial parts of H. cordata with hot water, we could detect cepharanone B but none of the other lipophilic compounds. This hot water extract exhibited almost no inhibitory activity on prostaglandin formation. Therefore, our results so far cannot explain the effectiveness of a tea preparation. In China, fresh leaves of H. cordata are often eaten as a health food or are applied topically for skin problems. The fatty acids might playa role as active principles in those cases. Polyunsaturated fatty acids are known to exhibit antiinflammatory, hormonal and immunomodulatory activities in vivo (Melnik 1993; Borelli et al., 1994; Chenoy et al., 1994). Oils from evening primrose and borage seeds, known to be rich in polyunsaturated fatty acids, have been used successfully against chronic diseases like psoriasis and rheumatoid arthritis (Kiehl et aI., 1994; Berth-Jones and Graham-Brown, 1993; Tolleson and Frithz, 1993). In vitro the exhibit activity in suppression of diverse T lymphocyte functions (proliferation, cytotoxcity, IL-2 production) and inhibition of prostaglandin and leukotriene B4 formation (Zurier 1993). Therefore, unsaturated fatty acids in general may playa role as anti-inflammatory and immunomodulatory agents in many drugs.

Linolensaure - Ergebnis einer Doppelblindstudie. Z. Hautkr. 69: 523-4, 1994. Chang, H., But, P.: In: Pharmacology and Applications of Chinese Materia Medica, pp. 827-833, World Scientific, Singapore, 1986. Chenoy, R., Hussain, S., Tayob, Y., O'Brien, P.M., Moss, M. Y., Morse, P.E: Effect of oral gammalinolenic acid from evening primrose oil on menopausal flushing. Brit. Med.]. 308: 501-3, 1994. Goad, J. L.: Phytosterols. In: Methods in Plant Biochemistry (Dey, P.M., Harbone, J. R., eds.) Vol. 7, pp. 369-429; Academic Press, London, New York, Tokyo, Toronto, Sydney, 1991. Jong, T.-T., Jean, M.-Y.: Constituents of Houttuynia cordata and the crystal structure of vomifoliol.]. Chin. Chem. Soc. (Taipeh) 40: 399-402, 1993 b. Jong, T.-T., Jean, M.-Y.: Alkaloids from Houttuynia cordata. ]. Chin. Chem. Soc. (Taipeh) 40: 301-3, 1993 a. Kiehl, R., Ionescu, G., Manuel, P., et al.: Klinische, immun- und lipidmodulatorische Effekte von einer Behandlung mit ungesattigten Fettsauren bei atopischem Ekzem. Z. Hautkr. 69: 42-8, 1994. Kyoko, H., Mioko, K., Toshimitsu, H.: Virucidal effects of the steam distillate from Houttuynia cordata and its components on HSV-1, influenza virus and HIV. Planta Med. 61: 237-41,1995. Melnik, B.: Prophylaxe und Therapie der Neurodermitis mit Gamrnalinolensaure. Allergologie 16: 454-8, 1993. Nishiya, H., Ishiwata, K., Komatsu, K., Nakata, 0., Kitamura, K., Fujii, S.: Platelet aggregation inhibitors from Jyu-yaku (Houttuyniae Herb). Chem. Pharm. Bull. 36: 1902-4, 1988. Probstle, A., Neszmelyi, A., Jerkovich, G., Wagner, H., Bauer, R.: Novel Pyridine and 1,4-Dihydropyridine Alkaloids from Houttuynia cordata. Nat. Prod. Lett. 4: 235-240, 1994. Probstle, A., Bauer, R.: Aristolactams and a 4,5-dioxoaporphine derivative from Houttuynia cordata. Planta Med. 58: 568-9, 1992. Redl, K., Breu, W., Davis, B., Bauer, R.: Antiinflammatory Active Polyacetylenes from Bidens campylotheca. Planta Med. 60: 58-62, 1994. Takagi, S., Yamaki, M., Masuda, K., Kubota, M.: On the constituents of the terrestrial part of Houttuynia cordata. Shoyakugaku Zasshi 32: 123-5, 1978. Tolleson, A., Frithz, B.: Borage oil, an effective new treatment for infantile seborrhoic dermatitis. Brit.]. Dermatol. 129: 95,1993. Tunmann, P., Grimm, H.J.: Uber ein Steroidketon in der Wurzel von Sambucus ebulus. Arch. Pharm. 307: 891-893, 1974. Tutupalli, L. V., Chaubal, M. G.: Saururaceae. V. Composition of essential oil from foliage of Houttuynia cordata and chemosystematics of Saururaceae. Lloydia 38: 92-6, 1975. Van Wijngaarden, D.: Modified rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Ana/. Chem. 39: 848-9, 1967. Zurier, R. B.: Fatty acids, inflammation and immune response. Prostaglandins, Leukotrienes and Essential Fatty Acids 48: 57-62, 1993.

References Berth-Jones, J., Graham-Brown, R. A.: Placebocontrolled trial of essential fatty acid supplementation in atopic dermatitis. Lancet 341: 1557-60, 1993. Broelli, S., Bresser, H., Belsan, L.: Externe Therapie mit gamma-

Address

R. Bauer, Institut fur Pharmazeutische Biologie, HeinrichHeine-Universitat Dusseldorf, Universitatsstr, 1, D-40225 Dusseldorf, Germany.