Phenolic Antioxidants from Casimiroa edulis. Leaves

Abstract

In the present current, quercetin and its 3-O.-rutinoside, the ethyl acetate and ethanol extracts, and two new flavones from Casimiroa edulis. Llave et Lex (Rotaceae) were tested for their antioxidant activity via scavenging by the ABTS^˙+ [2,2′-azinobis(3-ethylbenzothiazolone-6-sulfonic acid)]free radical. Among all the tested fractions and compounds, the ethanol extract exhibited the strongest antioxidant activity followed by EtOAc extract, giving 842 and 712 µM Trolox equivalents/g dry weight, respectively. Moderate activities were detected for the other tested materials. The two new flavones were identified as 5-methoxy-6-hydroxyflavone and its 6-O.-β.-D-glucoside. The structure elucidation was based on UV, electrospray ionization mass spectrometry (ESIMS), ¹H and ¹³C NMR, proton-proton correlation spectroscopy (¹H-¹H COSY), distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlations spectrum (HMBC).

Keywords:

• Antioxidants
• Casimiroa edulis
• flavonoids
• free radicals
• new flavones
• Rotaceae

Introduction

Plants are sources of natural antioxidants, and some of the compounds have significant antioxidative properties and health benefits (Exarchou et al., 2002). The antioxidative effect is mainly due to phenolic compounds such as flavonoids, phenolic acids, tannins, and phenolic diterpenes (Shahidi et al.,1992; Chung et al., 1998; Pietta, 2000). Casimiroa edulis. Llave et Lex (Rotaceae) is a species indigenous to temperate zones of Mexico and Central America, and it is popularly called “zapote blanco,” which has been known since pre-Hispanic times for its interesting sedative-like effects and its use as a sleep inducer (Romero et al., 1983). The tree is cultivated in Egypt for its edible fruits. In folk medicine, a concoction of leaves and occasionally of seeds is administered for sedative action. The aqueous and alcohol extracts of leaves and seeds, respectively, showed anticonvulsant, hypnotic, antihypertension, diuretic, anti-inflammatory (Petit-Play et al., 1982), and sedative activities (Magos & Vidrio, 1991; Magos et al., 1995). Other pharmacological activities have been studied for the plant in Mexico and in the United States. (Aiko et al., 1998; Magos et al., 1999). Secondary metabolites of various plant parts of Casimiroa edulis. that have been previously reported include imidazole (Romero et al., 1983), quinolinone derivatives (Toube et al., 1967; Dreyer, 1968), zapotidine alkaloids (Kincl et al., 1958), furanoquinoline alkaloids together with 2-quinolone and 4-quinolones, edulein, edulitin, edulinine, casmiroin (Dryer, 1968; Enriques et al., 1984) (viz., casmiroedine, casimiroine, skimmianine, 1-methyl-2-phenyl-4-quinolone, eduline) (Power & Callan, 1911; Aebi, 1956; Raman et al., 1962), coumarins, furanocoumarins and scopoletin methyl ether (Dreyer, 1968; Kincl et al., 1958), isoimpinellin, casmiroin, skimmianine, edulein, the histamine derivatives such as N.,N.-dimethylhistamine (Dreyer, 1968),casmidine and casimiroedin (Enriques et al., 1984), as compounds of marked hypotensive activity. Flavonoids including 5,6,2′,6′-tetramethoxyflavone (known as zapotin) (Dreye & Bertelli, 1976; Garratt et al., 1967) and limonoids (Sondheimer et al., 1959; Murphy et al., 1968) all have been previously isolated from leaves. No previous work on fruits of C. edulis. has been reported except for the study of its provitamin content among the fruits of 33 different species in the U.S. market (Aebi, 1956) and the 5,6-dihydroxyflavone from the plant bark (Iriarte et al., 1956). In the current study, the leaves of C. edulis. were studied for their antioxidant activity and for determination of the active compounds.

Materials and Methods

Apparatus

Melting points were determined on a Kofler hot-stage apparatus (UK) and are uncorrected; mass spectra (electrospray negative ion) sample was dissolved in acetonitrile on a Micromass Quattro spectrometer (Germany). ¹H and ¹³C NMR spectra, using external electronic referencing through the deuterium resonance frequency of the solvent, were determined at 600.17 or 150.91 MHz, respectively, with a JEOL ECA 600 spectrometer fitted with an auto 5 mm X/H probe. Carbon atom types were established in the ¹³C NMR spectrum by employing a combination of broad- and proton-decoupled and distortionless enhancement by polarization transfer (DEPT) experiments with 64 K data points over a spectrum width of 17,605 · 6 Hz. [¹J._C–H] and ²J._C–H and ³J._C–H]. ¹H-¹³C correlations were established by using Heteronuclear multiple quantum correlation (HMQC) and Heteronuclear multiple bound correlation (HMBC) pulse sequences, respectively. ¹H-¹H correlations were by double quantum filtered correlation spectroscopy (COSY).

The antioxidant assay kit was purchased from Randox Laboratories (Crumlin, Co. Antrim, United Kingdom), and the assay was performed using a Boehringer Mannheim Hitachi 717 automatic analyzer (Japan). Aluminum sheets 20 × 20 cm Silica gel G 60 F254 Merck KGaA 64271 (Darmstadt, Germany), was used for thin-layer chromatography. Silica gel 60 particle size (70–230 mesh) was used for column chromatography. Solvent systems (a) chloroform:ethanol (9:1), (b) ethyl acetate:methanol:water (30:5:4), (c) ethyl acetate:methanol:acetic acid:water (65:15:10:10) were used for developing the chromatoplates. Visualization of chromatograms was achieved under UV before and after exposure to ammonia vapor or by spraying with aluminum chloride (Stahl, 1969). All solvents used were of analytical grade.

Plant material

The leaves of Casimiroa edulis. Llave et Lex (Rotaceae) were collected from Dakahlia governorate, Egypt, in 2001. The plant identification was verified by the late Prof. N. El-Hadidi, Professor of Botany, Botany Department, Faculty of Science, Cairo University, and a voucher specimen has been deposited in the herbarium of the Desert Research Center, Cairo, Egypt. The plant sample was air-dried in the shade and then reduced to a fine powder that was packed in tightly closed containers and stored for phytochemical and biological studies.

Preparation of the plant extracts

The leaves (2 kg) of C. edulis. were exhaustively extracted with 95% ethanol (EtOH), 3 × 5 l; the extract was filtered under vacuum at a temperature not exceeding 35°C, and concentrated in a rotary evaporator to yield a brownish syrup residue (150 g), which was partitioned between 10 l ethyl acetate (EtOAc) and 1 l H₂O. The ethyl acetate fraction, after removal of the solvent and drying over Na₂SO₄, afforded 30 g of extracts that were subjected to column chromatography on silica gel (500 g) using CHCl₃:MeOH (99:1 v/v). Fractions of 80 cm³ were collected; the combined fractions were evaporated to give 600, 400, and 1000 mg, respectively. The first fraction was discarded (chlorophyll and fatty matter), and the other two fractions were reapplied on a silica gel column using ethyl acetate with gradually increased amounts of methanol; purification involved preparative TLC. Final purification was done on Sephadex LH-20 using MeOH/H₂O as eluent. Crystallization from chloroform yielded compounds 1–4 (Figure 1).

Figure 1 The compounds isolated from Casimiroa edulis.

Toxicological studies

LD₅₀ of the tested extracts were determined (Finney, 1964), and for this purpose, albino mice (20–22 g) were divided into groups of five animals each. Preliminary experiments were done to determine the minimal dose that kills all mice and the maximal dose that fails to kill any animal. Several doses at equal intervals were administrated orally to a group of five mice. Animals were kept under observation for 24 h during which time symptoms of toxicity and rate of mortality in each group were recorded from which the LD₅₀ therapeutic dose was calculated.

Antioxidant measurements

The alcohol and ethyl acetate extracts were centrifuged (2000 × g., 10 min) to remove insoluble material and the supernatants retained. Antioxidant activity in the supernatants in addition to the isolated compounds 1–4 were determined in vitro. (Table 1) via scavenging of the ABTS^˙+ [(2,2′ -azinobis(3-ethylbenzothiazolone-6-sulfonic acid)] radical, which was generated by a metmyoglobin/hydrogenperoxide system (Millere & Rice-Evans, 1997). Each plant extract (10 µl) was added separately to a 1-cm pathlength spectrophotometer cuvette (1 ml capacity) containing 20 mM phosphate-buffered saline, pH 7.4, 2.5 µM myoglobin, and 150 µM ABTS. The reaction was initiated by addition of 75 µM hydrogen peroxide, and the absorbance change at 734 nm was monitored at 30°C. A quantitative relationship exists between the absorbance at 734 nm measured after 6 min and the antioxidant property of the plant extract, as determined relative to Trolox (a water-soluble vitamin E analogue) antioxidant standards, and expressed in terms of mM Trolox equivalents (mMTE). Samples of the plant extracts for antioxidant activity were dried to constant weight (80°C for 20 h), and the final antioxidant activity was expressed in terms of mMTE/gram dried tissue. A two-point calibration was used. In the first stage of the assay, myoglobin was reacted with hydrogen peroxide to produce the ferrimyoglobin free radical, which was then incubated with a chromagen, 2,2-amino-di-(3-ethylbenzthiazole sophonate) to produce ATBSR+, a radical cation with a blue-green color measured at 600 nm. Antioxidants in the added plant aqueous extracts suppressed the blue-green color to a degree that was proportional to their concentration and antioxidant property. The absorbance of the resulting oxidized solution was compared with that of the calibrated standard, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Results were expressed as µmol Trolox equivalents per gram plant dry weight.

Table 1 Antioxidant activity of different extracts and isolated compounds of Casimiroa edulis. Llave et Lex

Extracts and isolated compounds	Antioxidant capacity (µmol Trolox equivalents/g dry weight)
Alcohol extract	842
Ethyl acetate extract	712
6-Hydroxy 5-methoxyflavone	452
5-Methoxyflavone 6-O.-β.-D-glucoside	389
Quercetin	772
Quercetin 3-O.-rutinoside	640

6-Hydroxy 5-methoxyflavone(1)

(50 mg) yellow needle crystals, R_f = 0.78 (system a), m.p. 322°C. UV (MeOH), λ_max, nm: 265, 310; (AlCl₃): 266, 312; (AlCl₃/HCl) 266, 310; (NaOAc) 267, 313; (NaOAc/H₃BO₃) 267, 313; (NaOMe) 308 and 329, ¹³C NMR (methanol-d.₆ 600 MHz): δ. ppm 62.46 (OCH₃), 108.19 (C-3), 149.10 (C-5), 119.48 (C-10), 132.49 (C-1′), 127.39 (C-2′ and 6′), 130.26 (C-3′ and 5′), 148.57 (C-6), 133.1 (C-4′), 115.28 (C-8), 125.63 (C-7), 154.19 (C-9), 164.18 (C-2). 180. 29 (C-4).

5-Methoxyflavone 6-O-β-D-glucoside (2)

(100 mg) yellow needle crystals, R_f = 0.65 (system b), m.p. 290°C. UV (MeOH), λ_max, nm: 267, 312; (AlCl₃) 267, 313; (AlCl₃/HCl) 268, 313; (NaOAc) 267, 313; (NaOAc/H₃BO₃) 267, 314; (NaOMe) 266, 311: ¹H NMR (Methanol-d.₆ 600 MHz): δ ppm 7.98 (d, J. = 8.24 Hz, H-2′-6′), 7.72 (d, J. = 9.54 Hz, H-7), 7.54 (m, 3H, H-3′-5′-4′), 7.45 (d, J. = 9.27 Hz, H-8) 4.96 (d, J. = 7.56 anomeric glucoside proton), 3.9 (S, 3H, OCH₃), 3–3.9 (m, rest of sugar protons) ¹³C NMR (Methanol-d.₆ 600 MHz): δ ppm 62.46 (OCH₃), 108.19 (C-3), 115.28 (C-8), 125.63 (C-7), 119.48 (C-10), 132.49 (C-1′), 130.28 (C-3′ and 5′), 127.46 (C-2′ and 6′), 133.04 (C-4′), 149.10 (C-5), 149.28 (C-6), 154.19 (C-9), 164.18 (C-2), 180. 29 (C-4), 103.38 (C-1″) 62.5 (OCH₃), 62.74 (C-6″), 78.4 (C-5″), 78.11 (C-″), 75.05 (C-2″), 71.34 (C-4″).

Results and Discussion

The known compounds 3–4 quercetin and its 3-O.-rutinoside were identified through their color reactions, UV spectra in methanol and with different shift reagents, ESIMS, ¹H NMR with authentic samples, and published data (Mabry et al., 1970; Markham, 1982). The LD₅₀ were found to be over 6 g/kg±0.4. This result revealed the safe use of the plant.

Compound 2 gave 1 and glucose on acid hydrolysis, and 1 and 2 showed yellow color under UV that did not change upon exposure to ammonia vapors. The UV in methanol exhibited two weak bands at λ 310 and 265 nm, respectively, with a weak bathochromic shift in both bands upon addition of sodium methoxide (NaOCH₃) for compound 1. The other flavonoid diagnostic reagents gave no shifts for both compounds, indicating the weakness of phenolic hydroxyls in both the new compounds 1 and 2. Both compounds display a distinguishing ¹H NMR pattern, as it showed a group of signals around δ. ppm 7.55–7.53 (m, 3H), which was assigned to H-3′,5′, and 4′; however, owing to the small chemical shift differences, it was difficult to distinguish between these latter resonances on the basis of the ¹H-¹H COSY spectrum; in addition, the HMSQ experment (heteronuclear single quantum coherence) clearly demonstrated that the H-4′ resonance appeared downfield of the H-3′, 5′ resonance (Dagfinn et al., 1996). The 2′, 6′ protons were located as doublet, J. 8.24 Hz at δ 7.89. Two ortho. coupled aromatic protons were further located at δ. 7.7 and 7.45 J. 9.54 Hz and 9.27 Hz assigned for H-7 and 7–8, respectively (Dagfinn et al., 1996). H-3 gave a strong singlet at δ 6.75; a sharp singlet at δ 3.9 was also detected for both compounds, assigned for a methoxy group OCH₃. In addition to all the mentioned signals, 2 also displays one signal for an anomeric protonat δ 4.9 (d, J. 7.5 Hz) indicating a β.-glucosyl moiety; furthermore, the remainder of the sugar protons were detected between δ 3–3.9. The ¹³C NMR of 2 along with the distortionless enhancement by polarization transfer (DEPT) experment allowed the identification of seven quaternary, one methine, one methyl, and thirteen methylene carbons. Electrospray ionozation mass spectrometry (ESIMS) in its negative mode exhibits m./z. 429.3 (M − 1), its positive mode gave 431.2 (M + 1) and 453.2 (M + Na). Thus, all these data established for 2 the molecular formula of C₂₂H₂₂0₉. HMSQ proved the suggested linkage of the β.-glucosyl moiety to C-6. HMBC (heteronuclear multiple bond correlations spectrum) allowed the confirmation of all the assigned protons and carbons as it showed long range coupling of H-8 with carbon 10, 6, and 9, also H-7 with carbon 6 and 9 and the OCH₃with C-5. The structure of compound 1 was similarly confirmed.

Antioxidant activity (Rice-evans & Miller, 1994)

This study has revealed that the total antioxidant content ranged from 389 to 842 µmol Trolox equivalents/g dry weight. Alcohol and ethyl acetate extracts exhibited 842 and 712 µmol Trolox equivalents/g dry weight; compounds 3 and 4 were found to have the highest activity (640 and 772 µmol/g, respectively).

Acknowledgment

All spectra were recorded in the Chemistry and Forensic Medicine Department, School of Life Science, Bradford University, Bradford, United Kingdom. The research was supported at the University of Texas at Austin by the Robert A. Welch Foundation (grant F-130).