A new antimutagen from Mentha cordifolia Opiz.

Mutation Research 515 (2002) 141–146

A new antimutagen from Mentha cordifolia Opiz. Irene M. Villaseñor∗ , Deborah E. Echegoyen, Jennifer S. Angelada Institute of Chemistry and Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines Received 25 September 2001; received in revised form 28 December 2001; accepted 28 December 2001

Abstract The CHCl3 extract of Mentha cordifolia Opiz. showed antimutagenicity against tetracycline. An antimutagen was purified by solvent partitioning and repeated normal phase-vacuum liquid chromatography (NP-VLC) using a micronucleus test-guided isolation and purification. Spectral analyses showed that the isolated antimutagen is possibly 6,7-bis-(2,2-dimethoxyethene)2,11-dimethoxy-2Z,4E,8E,10Z-dodecatetraendioic acid. It inhibited the mutagenicity of tetracycline by 68.7% at a dosage of 0.01 mg per 20 g mouse. Statistical analysis using Kruskal–Wallis one-way analysis of variance (ANOVA) by ranks showed that its variance differs from that of the solvent control group (tetracycline + dimethylsulfoxide (DMSO)) at α = 0.001. Moreover, the isolated antimutagen did not exhibit mutagenic activity at the same dosage. Statistical analysis showed that it is not mutagenic at 0.001 level of significance because its variance differs from that of tetracycline. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Mentha cordifolia Opiz.; Antimutagen; Micronucleus test

1. Introduction Mentha cordifolia Opiz. is commonly known as marsh mint, pepper mint, or mint [1] and is locally known in the Philippines as “yerba buena” or “herba bueba”. It was originally classified by Quisumbing [2] as M. arvensis Linn. (Family Labiatae). However, it was renamed M. cordifolia Opiz. (Family Lamiaceae) by Cantoria [3]. Cantoria described M. cordifolia as a hybrid of M. rotundifolia × M. spicata. The mint family is characterized by their volatile oils. Although, the type of volatile oil may vary, their uses are essentially the same as carminatives, expectorants, weak local anesthetics, as a skin wash for itchiness and insect bites, for pinworm (Enterobius) infections [4], for the treatment of dizzi∗ Corresponding author. E-mail addresses: [email protected], [email protected] (I.M. Villaseñor).

ness and arthritis [5], as an antispasmodic, sudorific, and emmenagogue, as a stimulant, stomachic, diuretic, and febrifuge [2,6]. It is also used for flatulence or gas pain, rheumatism (form of arthritis affecting the large joints of the extremities) or rheumatic joint pain, toothache [7], headache, muscle pain, dysmenorrhea, post-operative pain in secondary minor surgery, fainting, hysteria, and as a mouth wash [8]. M. cordifolia Opiz. is one of the priority plants under the Department of Science and Technology (DOST), Philippine Council for Health Research and Development (PCHRD), National Integrated Research Program on Medicinal Plants (NIRPROMP). The unextracted and unpurified leaves are produced in tablet form, including pediatric tablets. They have been proven to contain analgesic properties in clinical trial phases I, II, and III [9]. M. cordifolia Opiz. tablets were found to be antigenotoxic [10,11] against dimethylnitrosamine, N-nitrosopyrrolidine and tetracycline. They do not possess direct DNA damaging

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 0 0 6 - 2

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and chromosome-breaking effects before and after metabolic activation [10,11]. This study aims to isolate and characterize an antimutagenic constituent from M. cordifolia Opiz. leaves using a micronucleus test-guided isolation and purification. These bioactive constituents may serve as possible import substitute for therapeutics. It is said that the use of herbal medicines could save the Philippines over 700 million pesos worth of imports yearly.

2. Materials and methods 2.1. Extraction, isolation and purification The solvents used for extraction were single-distilled technical grade. M. cordifolia Opiz. leaves were purchased from the National Research Council of the Philippines (NRCP) in Bicutan, Taguig, MM. The leaves were homogenized and immersed in 95% ethanol. The ethanol extract was filtered and then concentrated under reduced pressure at 40 ◦ C. This fraction was then partitioned between hexane and water (6:1). The aqueous extract was further extracted with chloroform (CHCl3 ) and then with ethyl acetate (EtOAc). The hexane, CHCl3 , and EtOAc portions were concentrated in vacuo giving fractions FB, FC, and FD, respectively. Approximately 264 mg of the chloroform extract (FC) was adsorbed on silica gel 60 G (Merck) to form a sample powder. A 30 mm diameter column was dry packed using suction to make a 4–5 cm bed upon which the sample was added. The column was eluted with hexane, 5% gradient ratios of EtOAc in hexane, EtOAc, and 5% gradient ratios of ethanol in EtOAc into numbered test tubes. The column fractions were combined according to analytical thin layer chromatography (TLC) results into 11 sub-fractions, FC1– FC11. Normal phase-vacuum liquid chromatography (NPVLC) of FC6 using hexane, 5% gradient ratios of EtOAc in hexane, EtOAc, and 5% gradient ratios of ethanol in EtOAc resulted into four fractions, FC6A– FC6D. Further purification of FC6C was done by isocratic elution using 30% EtOAc–hexane as solvent. FC6C3.22 is a yellowish crystalline solid which degrades at 210.8 ◦ C: FT-IR in KBr (cm−1 ): 3527, 3291, 3100, 3017, 2967, 2850, 1673, 1606, 1515,

1426, 1388, 1105, 1031, 973, 951, 873, 848, 805; 1 H-NMR in CDCl in ppm (integration, multiplicity, 3 J values) [COSY] {HMBC}: 3.992 (1H, dd, J = 8.1 and 4.6 Hz) [7.421] {133.26,142.22}, 3.996 (6H, s), 4.144 (3H, d, J = 1 Hz) {142.223}, 6.611 (1H, d, J = 9.3 Hz), 7.031 (1H, dd, J = 20.2 and 8.5 Hz) [7.565], 7.421 (1H, dd, J = 5.1 and 2.1 Hz) [3.992] {120.790), 7.565 (1H, ddd, J = 19.0, 8.5, and 2.3 Hz) [7.031]; 13 C-NMR in ppm (DEPT) [HMQC]: 56.031, 56.089, 56.148 (–CH and CH3 ) [3.992,3.996], 61.497 (–CH3 ) [4.144], 62.132 (–CH) [3.992], 103.838 (–CH) [6.611], 108.348 (–CH) [7.421], 115.127 (–CH) [7.031], 120.790 (–CH) [7.565], 133.262 (–C), 142.878 (–C), 182.878 (–C); direct probe ESI-MS at 150 ◦ C m/e (percent relative abundance): 392 (1%), 374 (36%), 360 (70%), 345 (100%), 327 (10%), 302 (4%), 197 (28%), 169 (10%), 149 (8%), 84 (10%); ESI-MS [M+ ] at 454 amu. 2.2. Antimutagenicity bioassay: micronucleus test Swiss Webster albino mice, 7–12-week-old, were used. Five mice were used per dosage of the different isolates and five mice each for the positive, solvent, and spontaneous controls. Three slides were prepared per mouse. The required weights of the test isolates were dissolved in dimethylsulfoxide (DMSO) (Fluka-Garantie). The required weight (55 mg per kg mouse) of tetracycline (Upjohn) was dissolved in distilled water. Tetracycline was administered orally using a gavage while the test isolates were injected intraperitoneally to the test animals. The procedure is as described by Schmid [12]. The number of micronucleated polychromatic erythrocytes (MN-PCE) per 1000 PCE was counted using a high power microscope. 2.3. Statistical analysis The Kruskal–Wallis one-way ANOVA by ranks was used to determine whether or not the variance of the test group is statistically different from that of the control group. 2.4. Spectral analyses The FT-IR spectrum was obtained neat using a Bio-Rad FTS 40A: WIN IR spectrophotometer.

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The NMR spectra were obtained in CDCl3 using a JEOL LA 400 MHz. The EI-MS was acquired using a Finnegan MAT 95S trap mass spectrophotometer while the ESI-MS was obtained using a Finnegan MAT electrospray ionization mass spectrophotometer.

3. Results and discussion The results of the micronucleus test are summarized in Table 1. Bioassay of the crude extracts using a dosage of 2 mg per 20 g mouse showed that the chloroform extract (FC) decreased the MN-PCE induced by tetracycline by 61.7%. Fraction FC gave a percent yield of 0.14%. NP-VLC of FC resulted in fractions FC1–FC11. Subsequent bioassay of these fractions showed that FC2–FC4, FC6, and FC8 inhibited the mutagenic activity of tetracycline. These fractions were antimutagenic at a dosage of 0.1 mg per 20 g mouse. TLC results revealed that FC2–FC4 (Table 2) were mixtures of compounds with common Rf values. NP-VLC of FC2–FC4 followed by re-crystallization in ether–methanol solvent gave white needle-like crystals whose spectral data is similar to that reported for ␤-sitosterol (Villaseñor et al., accepted for publication) [13], which is the principal sterol in most higher plants.

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Further purification of FC6 yielded four fractions, FC6A–FC6D. The spots in FC6A and FC6B correspond to some of the spots found in FC3 and FC4 but are faint and very little in amount. Emphasis was given to the isolation of a brown spot in FC6C with an Rf 0.22 in 30% EtOAc in hexane. This is the same spot observed in FC8 at Rf 0.29 in 50% EtOAc in hexane (Table 2). Both FC6 and FC8 exhibited a 72.7% antimutagenic activity. Isocratic elution of FC6C by NP-VLC using 30% EtOAc–hexane as solvent was done until a semi-pure isolate, labeled FC6C3 was obtained. Subsequent bioassay showed that this mixture reduced the MN-PCE per 1000 PCE induced by tetracycline (10.17 ± 1.58) to 4.07 ± 0.89. Hence, FC6C3 reduced the mutagenic effect of tetracycline by 48.28% at a dosage of 0.06 mg per 20 g mouse. The results of the statistical analysis using Kruskal–Wallis ANOVA by ranks show that the variances of FC, FC2–FC4, FC6, FC8, and FC6C3 differs significantly from that of the solvent control at 0.001 level of significance. FC6C3 consists of two spots: Rf 0.40 in 30% EtOAc in hexane is UV-inactive and stains brown with I2 (v); and Rf 0.22 in 30% EtOAc in hexane is red under the UV lamp and also stains brown with I2 (v). A subsequent gas chromatography experiment showed that FC6C3 contained about 13% of FC6C3.40 and 81%

Table 1 Results of the antimutagenicity of M. cordifolia Opiz. extracts using the micronucleus test Test compound

Dosage (mg per 20 g mouse)

Spontaneous control Tetracycline + DMSO +FB +FC +FD +FC1 +FC2 +FC3 +FC4 +FC5 +FC6 +FC7 +FC8 +FC9 +FC10 +FC11

1.1 2.0 2.0 2.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Average no. of MN-PCE per 1000 PCE ± S.D. 1.67 10.17 6.5 3.89 7.42 5.28 2.89 2.07 1.75 5.21 2.78 4.67 2.78 4.33 5.83 4.27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.50 1.58 2.64 1.17 2.27 1.49 0.93 1.28 1.42 1.05 1.56 1.40 1.64 1.56 1.53 1.33

Inhibition (%)

36.1 61.8 27.0 48.1 71.6 79.6 82.8 48.8 72.7 54.1 72.7 57.4 42.7 58.0

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Table 2 Analytical TLC data for the CHCl3 fractions, FC1-FC11 Developing solvent EtOAc–hexane (25%) FC1

FC2

EtOAc–hexane (50%) FC3

FC4

FC5

FC6

FC7

FC7

FC8

FC9

0.70 0.66 0.56 0.44 0.40

0.67 0.55 0.44

0.56 0.43

0.56 0.41

0.41

0.32

0.32

0.35

0.35

0.52

0.23

0.21 0.12 0.07

0.21 0.15 0.08 0.03

0.23 0.12

0.36 0.28

0.36 0.29

0.29

0.03

0.20 0.12

0.19

FC10

FC11

0.94 0.79 0.70

0.27

0.07

0.12

0.14 0.12 0.09 0.04

Indicators: UV light, I2 vapors, vanillin–H2 SO4 heat, rf 0.56 in 25% EtOac–hexane was identified as ␤-sitosterol by spectral analysis.

of FC6C3.22. Recrystallization using ether–methanol gave FC6C3.22 as a yellowish solid. Confirmatory bioassay of FC6C3.22 (Table 3) showed that, at a dosage of 0.01 mg per 20 g mouse, isolate FC6C3.22 is antimutagenic as it was able to inhibit the mutagenic activity of tetracycline by 68.7%. Moreover, isolate FC6C3.22 did not exhibit mutagenic activity when administered alone. The number of MN-PCE it induces (2.42 ± 0.90) is very much less than that of the positive control (7.22 ± 1.09) and approximates that of the spontaneous control (1.78 ± 0.44). Statistical analysis showed that FC6C3.22 is antimutagenic at 0.001 level of significance. It is not mutagenic at 0.001 level of significance because its variance differs with that of tetracycline.

Spectral analysis of FC6C3.22 shows the presence of unsaturation and a conjugated carboxylic acid (–COOH) moiety. A signal at 2850 cm−1 is indicative of a CH3 –O stretch. The HMBC spectrum (Table 4) shows long range couplings (3 J) between carbon δ142.2 and protons δ4.00 and δ4.14 indicative of the presence of a =C–(OCH3 )2 moiety (fragment 1); while that between carbon δ133.3 and proton δ 4.00 is due to the presence of a =C–OCH3 moiety (fragment 2). The quaternary carbon at δ133.3 must have a C–COOH as its other substituent. The presence of a trans double bond is evident in a J value of ≈20 Hz between the protons at δ7.56 and 7.03. The COSY spectrum shows that the proton at δ7.42 is coupled to two protons at δ3.99 and 7.56. The HMBC spectrum

Table 3 Results of the antimutagenicity of M. cordifolia Opiz. isolate using the micronucleus test Test compound

Dosage (mg per 20 g mouse)

Spontaneous Tetracycline + DMSO Tetracycline + FC6C3.22 Tetracycline FC6C3.22

1.1 0.01 1.1 0.01

Average no. of MN-PCE per 1000 PCE ± S.D. 1.78 7.44 2.33 7.22 2.42

± ± ± ± ±

0.44 0.54 0.71 1.09 0.90

Inhibition (%)

68.7

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Table 4 NMR Spectral data for FC6C3.22 13 C-NMR

DEPT

HMQC (integration, multiplicity, J values)

COSY

56.0a

↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑

(–CH) (–CH3 )

3.99 (1H, dd, J = 8.1, 4.6 Hz) 4.00 (3H, s)

7.42

(–CH3 ) (–CH3 ) (–CH) (–CH) (–CH) (–CH)

4.14 4.00 6.61 7.42 7.03 7.56

56.1a 56.2a 61.5 62.1 103.8 108.3 115.1 120.8 133.3 142.2 182.9 a

(3H, (3H, (1H, (1H, (1H, (1H,

d, J = 1 Hz) s) d, J = 9.3 Hz) dd, J = 5.1, 2.1 Hz) dd, J = 20.2, 8.5 Hz) ddd, J = 19.0, 8.5, 2.1 Hz)

3.99, 7.56 7.56 7.03, 7.42

HMBC

7.42 4.00 4.00, 4.14

12.36

Interchangeable C signals.

shows that there is a long range coupling between proton δ7.42 and carbon δ120.8. Three fragments are therefore evident from the HMBC and HMQC spectra and the corresponding J values.

It is proposed that the carbon at δ133.3 is doubly bonded to the carbon at δ103.8 to have a more conjugated –COOH to account for the C=O stretch at 1673 cm−1 . It is further proposed that –COOH is cis to –H at δ6.61. These proposals are supported by additivity rules [14] with calculated values of δ107.8 (∆ 4.8 ppm) and δ6.45 (∆ 0.16 ppm), respectively. A structure assigned to FC6C3.22 is as follows, with a molecular formula of C11 H15 O5 and a molecular mass of 227 amu.

The ESI-MS shows a molecular ion peak at m/e 454. Hence, FC6C3.22 must have a symmetrical structure with a similar fragment at δ 56. An inspection

of the direct probe EI-MS taken at 150 ◦ C shows a base peak at m/e 345. The following m/e values would account for the following fragments: m/e 392 [2(227 − OCH3 )], 360 [2(227 − COOH)], 374 [392 − H2 O], 345 [374 − CO], 345 [345 − H2 O], and 197 [227 − OCH3 ]. Hence, FC6C3.22 is possibly 6,7-bis-(2,2-dimethoxyethene)-2,11-dimethoxy-2Z,4 E,8E,10Z-dodecatetraendioic acid. This preliminary structure is consistent with the structures of known antimutagens. Its sheer bulk may trap mutagens and hence, act as a blocking agent. Unsaturated compounds are also known to be free-radical scavengers [15–20] They may also react with alkylating agents through electrophilic addition [21–23]. Carboxylic acids, on the other hand, are generally regarded as the detoxification products of the mutagenic

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aldehydes [24,25]. Aldehyde dehydrogenase (ALDH) converts aldehydes to their corresponding carboxylic acids. References [1] C.H. Escueta, Herba Buena, Department of Agriculture and Food, Bureau of Plant Industry, Philippines, 1987. [2] E. Quisumbing, Medicinal Plants of the Philippines, Katha Publishing Co. Inc., 1978. [3] M.C. Cantoria, Pharmacognosy in Action. National Research Council of the Philippines, Bicutan, Tagig, MM, 1977. [4] M.L. Tan, Philippine Medicinal Plants in Common Use. Their Phytochemistry and Pharmacology, Luzon Secretariat of Social Action Q.C., 1978. [5] N.P.C. Maramba, J.D. Saludez, I.C. Sia, O.Y. Alegre, G.A. Solis-de Asis, L.B. Bagnaes, R.U. Macalagay, A.R. Bajao, Guidebook on the Proper Use of Medicinal Plants Katha Publishing Co. Inc., Quezon City, 1982. [6] L.S. de Padua, G.C. Lugod, J.V. Pancho, Handbook on Philippine Medicinal Plants, Vol. 1, Documentation and Information Section, Office of the Director for Research, UP Los Baños, 1977. [7] Ladion, Herminia Healing Wonders of Herbs. Philippine Publishing House, 1985. [8] N.P.C. Maramba, J.D. Saludez, I.C. Sia, O.Y. Alegre, G.A. Solis-de Asis, L.B. Bagnaes, R.U. Macalagay, A.R. Bajao, Guidebook on the Proper Use of Medicinal Plants. PCHRD, DOST, Bicutan, Tagig, MM, 1993. [9] N.P.C. Maramba, F.M. Dayrit, N.F. de Castro, H.R. Estrada, C.Y.L. Sylianco, A.L. Lingao, R.F. Quijano, E.G. Quintana, Selection and Scientific Validation of Medicinal Plants for Primary Health Care, Technical Report Series no. 12, Published by the PCHRD, DOST, 1991, pp. 42–43. [10] C.Y.L. Sylianco, J.A. Concha, A.P. Jocano, C.M. Lim, Antimutagenic effects of eighteen philippine medicinal plants, Phil. J. Sci. 115 (4) (1986) 293–298. [11] C.Y.L. Sylianco, F.R. Blanco, C.M. Lim, Mutagenicity, clastogenicity and antimutagenicity of medicinal plant tablets produced by the NSTA pilot plant IV, Yerba buena tablets, Phil. J. Sci. 115 (4) (1986) 299–305.

[12] W. Schmid, The micronucleus test, Mutat. Res. 31 (1) (1975) 9–15. [13] I.M. Villaseñor, J. Angelada, A.P. Canlas, D.E. Echegoyen, Bioactivity studies on ␤-sitosterol and its glucoside, Phytother. Res., in press. [14] C. Pretsch, S. Seibl, Spectral Data for Structure Determination of Organic Compounds, Springer, Berlin, 1989 (translated from the German by K. Biemann). [15] A. Aidoo, L.E. Lyncook, S. Lensing, M.E. Bishop, W. Wamer, In vivo antimutagenic activity of beta-carotene in rat spleen lymphocytes, Carcinogenesis. 16 (9) (1995) 2237–2241. [16] Y. Kuroda, N. Shima, K. Yazawa, K. Kaji, Desmutagenic and bio-antimutagenic activity of docosahexaenoic acid and eicosapentaenoic acid in cultured Chinese hamster v79 cells, Mutat. Res. Genet. Toxicol. Environ. Mutagen. 497 (1/2) (2001) 123–130. [17] Y. Nakamura, E. Suganuma, T. Matsuo, S. Okamoto, K. Sato, K. Ohtsuki, 2,4-Non-adienal and benzaldehyde bioantimutagens in Fushimi sweet pepper (Fushimi-togarashi), J. Agric. Food Chem. 47 (2) (1999) 544–549. [18] M.M. Mathews-Roth, Antitumor activity of beta-carotene, canthaxanthin and phytoene, Oncology 39 (1982) 33–37. [19] M.M. Mathews-Roth, Carotenoids quench evolution of excited species in epidermis exposed to UV-B (290–320 nm) light, Photochem. Photobiol. 43 (1986) 91–93. [20] M.M. Mathews-Roth, M. Pathak, Phytoene as a protective agent against sunburn (>280 nm) radiation in guinea pigs, Photochem. Photobiol. 21 (1975) 261–263. [21] M. Lahiri, S.V. Bhide, Effect of 4 plant phenols beta-carotene and alpha-tocopherol on 3(h)benzopyrene-DNA interaction invitro in the presence of rat and mouse liver post-mitochondrial fraction, Cancer Lett. 73 (1) (1993) 35–39. [22] I.M. Villaseñor, D.A. Edu, Antimutagen from the leaves of Carmona retusa (Vahl) Masam, Mutat. Res. 298 (1993) 215– 218. [23] I.M. Villaseñor, D.A. Edu, J.B. Bremner, Structure of an antimutagen from Carmona retusa (Vahl) Masam, Carcinogenesis 14 (1) (1993) 123–125. [24] T. Neudecker, D. Henschler, Allylisothiocyanate is mutagenic in Salmonella typhimurium, Mutat. Res. 156 (1985) 33–37. [25] I.M. Villaseñor, C.Y.L. Sylianco, F. Dayrit, Mutagens from the roasted seeds of M. oleifera., Mutat. Res. 224 (1989) 209–212.