Antioxidant Activity of Extract and a New Isolated Dioxaspiran Derivative of Tripleurospermum disciforme.

ABSTRACT

Antioxidants are important substances with the ability to protect the body from damage caused by free radical–induced oxidative stress. A variety of free-radical scavenging antioxidants exist within the body, many of which are derived from dietary sources such as fruits, vegetables, and teas. In this study, various extracts of Tripleurospermum disciforme. (C. A. Mey) Schultz Bip. (Compositae) with different solvents and a new isolated compound from its chloroform extract were tested by linoleic acid peroxidation for antioxidant activity using 1,3-diethyl-2-thiobarbituric acid as reagent. The chloroform extract was found to be the most active one (IC₅₀ = 10.75 µg/ml), comparable to α-tocopherol (IC₅₀ = 14.75 µg/ml) as positive control. The isolated dioxaspiran derivative showed an IC₅₀ value of 185.50 µg/ml with the same condition. Since this is higher than the IC₅₀ value of the crude chloroform extract, other effective compounds may be responsible for such a difference.

Keywords:

• Antioxidant
• dioxaspiran derivative
• linoleic acid
• Tripleurospermum disciforme

Introduction

It is widely accepted that a variety of disorders such as heart disease, chronic renal failure, diabetes mellitus, cancer, immune dysfunction, and aging are closely related to the peroxidation reactions in living organisms (Maxwell, 1995; Halliwell, 2000; Young & Woodside, 2001; Heinecke, 2003). Recent studies show that free-radical scavengers, both endogenous and exogenous, whether synthetic or natural, can be effective for the treatment or prophylaxis of disorders attributed to free-radical damage (Maxwell, 1995; Halliwell, 2000; Cesquini et al., 2003). There is growing interest toward antioxidants of herbal resources such as vegetables, fruits, teas, and grain products (Larson, 1988; Cao et al., 1996; Gazzani et al., 1998; Velioglu, 1998).

Tripleurospermum disciforme. (C. A. Mey) Schultz Bip. (Compositae) (locally known as babuneh.) is a perenial or biennial plant 10–70 cm high with a wide distribution in most central and southern parts of Iran. Two synonyms, Chrysanthemum disciforme. C. A. Mey and Matricaria disciforme. (C. A. Mey) DC, are also presented for this species (Ghahreman, 1996). In the old medico-pharmaceutical books of Iran, several scientific names are introduced for babuneh including Anthemis nobilis. L., Anthemis wiedemanniana. Fisch & Mey, Matricaria chamomilla. L., and Tripleurospermum disciforme. (C. A. Mey) Schultz Bip (Hooper et al., 1937). Today, what is presented as babuneh in the market is mostly T. disciforme. with a botanical character that is slightly different from the famous species of Matricaria chamomilla. (Amin, 1991). Babuneh, with a warm temperament, is described in The Canon of Medicine. as neurotonic and antifever (Avicenna, 1985). Its flowers have also been used as a carminative, stimulant, and febrifuge (Hooper et al., 1937).

A literature survey showed a few phytochemical investigations on this species including isolation of sesquiterpenes and flavonoids (Harborne, 1970; Revazona, 1970). As part of our ongoing research on natural antioxidants, the current study was performed to evaluate the antioxidant activity of various extracts of T. disciforme. with different solvents and an isolated compound from its chloroform extract.

Materials and Methods

Plant material

The plant material was collected from Siah Bisheh (northern of Iran) in May 2002 and was authenticated by the Herbal Museum, Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences (voucher no. 6634-TEH). The plant flowers were separated, air-dried in the shade at room temperature, and kept in an air-tight, light-protected container.

Chemicals

Linoleic acid and 1,3-diethyl-2-thiobarbituric acid (DETBA) were obtained from Merck (Darmstadt, Germany) and Aldrich Chemical Co. (Milwaukee, WI, USA), respectively. α-Tocopherol, sodium dodecyl sulfate (SDS), and butylated hydroxytoluene (BHT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents were from Merck and were of analytical grade.

Instruments

¹H NMR (400\ MHz) spectrum was measured in CDCl₃ using Varian 400 Unity plus. spectrometer (CA, USA). Chemical shifts were in parts per million (ppm) from tetramethylsilane (TMS) as an internal standard. Mass spectra were recorded with a Finigan TSQ-MAT 70 mass spectrometer (CA, USA) at 70 eV. Fourier-transform infrared (FTIR) spectra were obtained with a Nicolet FTIR 550 spectrometer (WI, USA).

Extraction

The air-dried flowers were ground into fine powder, and 1500 g was soaked in 3 l of hexane at room temperature overnight. The solvent was filtered, and the residue was macerated 3 more days with the same solvent. The solvent was filtered and evaporated to dryness under reduced pressure at 40°C to give a hexane extract (55.4 g). Further extraction processes were carried out with chloroform, ethyl acetate, methanol, and water in order of increasing polarity to give 18.1, 4.8, 61.4, and 32.7 g of soluble fractions, respectively.

Measurement of antioxidant activity

The antioxidant activity of plant extracts against peroxidation of linoleic acid was determined based on a method reported by Furuta et al. (1997). α-Tocopherol was used as a reference compound. Three dilutions of each extract (0.02, 0.2, and 2 mg/ml) were prepared. For a typical assay, an aliquot of 20 µl of each dilution was mixed with 20 µl of 2 mg/ml linoleic acid in ethanol and incubated at 80°C for 60 min. Incubated samples were cooled in an ice bath followed by addition of 200 µl of 20 mM BHT, 200 µl of 8% SDS, and 400 µl of distilled water. After mixing, a solution of 3.2 ml of 1.25 mM DETBA in sodium phosphate buffer (0.125 M, pH = 3.0) warmed to 50°C was added, mixed, and heated at 95°C for 15 min, and cooled in an ice bath. Then, 4 ml of ethyl acetate was added to each tube, vortexed to extract the pink adduct from the aqueous phase, and centrifuged at 700 g. for 10 min (F₁). A control, containing linoleic acid and other additives without antioxidants, representing 100% lipid peroxidation was also prepared (F₂). The fluorescence intensities of ethyl acetate layer of sample (F₁) and control solution (F₂) were measured at an excitation wavelength of 515 nm and an emission wavelength of 555 nm in a spectrofluorimeter (Model RF-5000, Shimadzu, Kyoto, Japan) against their own blanks (F₃ and F₄, respectively) prepared as described above without linoleic acid. The antioxidant activity was calculated as the percent of peroxidation inhibition using the following equation (Okada & Okada, 1998):

All extracts and reference substance were assayed in triplicate, and the results were averaged. Percent inhibition versus log concentration curve was plotted, and the concentration of sample required for 50% inhibition was determined and expressed as IC₅₀ values.

Isolation of major constituents

By screening the antioxidant activity of various extracts, the chloroform extract showed the highest activity. Therefore, this extract was subjected to preparative TLC on silica gel 60 PF254 (Merck, Darmstadt, Germany) with the eluent system of chloroform-ethyl acetate (98.5:1.5). Two fractions were isolated with the following characteristics: fraction 1, yellow spot in UV light 366, R_f = 0.7; and fraction 2, brown spot in UV light 254, R_f = 0.9. These fractions were further purified by TLC on the same silica gel with a different eluent system (hexane-ethyl acetate, 80:20), and two compounds (1 and 2) were furnished. Purification of the compound 2 was not achieved due to minute quantity of this compound. Identification of compound 1 was carried out by comparison of its spectroscopic data (MS, IR, and ¹H NMR) with those reported in the literature for similar structures. The antioxidant activity of this compound was also determined as described above.

Results and Discussion

T. disciforme. is a plant traditionally used in Iran for various ailments. The antioxidant activity of different concentrations of the flower extracts of this species with various solvents was determined against linoleic acid peroxidation. The results are shown in Table 1. The antioxidant efficiency of these extracts increased in the following order: hexane (IC₅₀ = 189.75 µg/ml) < ethyl acetate (IC₅₀ = 124.75 µg/ml) < methanol (IC₅₀ = 56.00 µg/ml) < water (IC₅₀ = 44.25 µg/ml) < α.-tocopherol (IC₅₀ = 14.75 µg/ml) < chloroform(IC₅₀ = 10.75 µg/ml). The chloroform extract represented the highest activity, which was slightly more effective than α.-tocopherol.

Table 1.. Yield, % of peroxidation inhibition (mean ± SD) of different extracts of Tripleurospermum disciforme., and IC₅₀ against linoleic acid (1 mg/ml) in the reaction mixture.

		Peroxidation inhibition (%)
Plant extract	Yield (%)	0.01 mg/ml	0.1 mg/ml	1 mg/ml	IC50 (µg/ml)
Hexane	3.69	−10.72 ± 1.95	19.44 ± 2.91	84.13 ± 2.10	189.75
Chloroform	1.20	62.24 ± 2.02	80.82 ± 2.48	90.77 ± 0.60	10.75
Ethyl acetate	0.32	13.31 ± 0.75	61.27 ± 3.66	71.96 ± 1.85	124.75
Methanol	4.10	12.62 ± 1.70	64.88 ± 2.40	96.92 ± 0.80	56.00
Water	2.18	20.32 ± 1.79	62.80 ± 1.92	97.55 ± 1.36	44.25
α.-Tocopherol	—	—	—	—	14.75

The chloroform extract was subjected to silica gel, and compound 1 was isolated as described before. The yield of compound 1 was 5.31% of chloroform extract and 0.06% of dried flower powder. The spectral data of compound 1 were as follows:

IR bands were detected at ν (CHCl₃) 2177 (C=C), 1750 (COOR) and 1638, 1550 (C=C) cm⁻¹. ¹H -NMR (400 MHz) in CDCl₃ gave signals at δ 1.86 (3H, dd, J. = 7.2 Hz, J. = 2 Hz, H_L), 1.96 and 1.98 (6H, dd, J. = 1.6 Hz, H_E & H_F), 5.43 (1H, d, J. = 1.6 H_Z, H_D), 5.50 (1H, d, J. = 2.4 H_Z, H_I), 5.70–5.74 (m, 3H, H_A,H_B,H_J), 6.15–6.26 (m, 2H, H_G,H_K) and 6.30–6.40 (m, 2H, H_H, H_C); EI-MS spectrometry (EI, WI) showed the molecular formula of C₂₀H₁₈O₄. The spectral fragmentation was at m/e. 322 (10%), 292 (14%), 237 (29%), 160 (100%), 145 (16%), 131 (80%), 103 (57%), 54 (17%), and 39 (29%).

The structure of compound 1 was identified by comparison of its spectral data with those reported in the literature for some similar structures (Bohlmann et al., 1965; Bohlmann & Zdero, 1970; Bohlman & Bethke, 1971). The structure of compound 1 was confirmed to be an acetylenic derivative of dioxaspiran (Fig. 1). The identity of compound 1 was proved mainly on the basis of NMR spectrum and the fragment ions in mass spectrum. Isolation of this compound and its structure has not so far been reported according to our bibliography.

Figure 1 Chemical structure of compound 1.

The IC₅₀ value of compound 1 was 185.50 µg/ml; much higher than the crude chloroform extract, as shown in Table 1. This means that there might be some other effective compounds in the crude extract that affect the antioxidant activity of chloroform extract. Further investigation is needed to clarify this point.