Abstract
Phytochemical investigations on the n-BuOH-soluble fraction of the whole plant of Buddleja davidii led to the isolation of the phenylpropanoid glycosides 1-10. Their structures were determined by 1D and 2D NMR spectroscopic techniques. All the compounds showed potent antioxidative activity in three different tests, with IC50 values in the range 4.15-9.47 μM in the hydroxyl radical (˙OH) inhibitory activity test, 40.32-81.15 μM in the total ROS (reactive oxygen species) inhibitory activity test, and 2.26-7.79 μM in the peroxynitrite (ONOO−) scavenging activity test. Calceolarioside A (1) displayed the strongest scavenging potential with IC50 values of (4.15 ± 0.07, 40.32 ± 0.09, 2.26 ± 0.03μM) for ˙OH, total ROS and scavenging of ONOO−, respectively.
Introduction
Antioxidants, which scavenge active oxygen species (free radicals), were found in a variety of foodstuffs and are commonly referred to as scavengers [Citation1, Citation2]. Many oxidants are plant based and play an important role in protecting plants that are exposed to sunlight and live under severe oxygen stress. Antioxidants also play an important role in human health because the biologic defense mechanism cannot operate under severe oxygen stress. According to recent research, activated oxygen is thought to be major factor in aging, hardening of the arteries, diabetes, cancer and tissue in injury skin [Citation3, Citation4]. Indeed approximately 90% of age related diseases are linked to activated oxygen.
The genus Buddleja (synonym Buddleia), belonging to the family Buddlejaceae, comprises of 100 species mainly distributed in East Asia, America and South Africa. In Pakistan it is represented by four species [Citation5]. The genus Buddleja was previously placed in the family Loganiaceae [Citation6] while Hegnauer and Kooiman classify the families Buddlejaceae and Loganiaceae together in family Scrophlariaceae [Citation7].
Various species of the genus Buddleja are used for the treatment of a variety of ailments such as ulcer, conjunctival congestion, clustered nebulae and skin disorders. Different parts of B. asiatica are used as antiinflamatory, abortifaciant, antifungal and also for the treatment of skin diseases [Citation8, Citation9]. The leaves and flowers of B. globasa are used for washing wounds and treatment of ulcer [Citation9].The flowers of B. officinalis are used in Chinese medicine for the treatment of conjunctival congestion and clustered nebulae [Citation9].
Previous studies on the genus Buddleja have resulted in the isolation of various compounds including glycosides of phenylpropanoids, triterpenes, iridoids and flavonoids, sterols, aryl esters, phenolic fatty acid esters, alkaloid, lignans, neolignans, diterpenes and sesquiterpenes [Citation10, Citation11].
Buddleja davidii is deciduous shrub, up to 5 m tall with tap root system. Stem is erect and branched. Leaves are sub-sessile, 7-25 cm long, 1-7 cm broad. Stipules are interpetiolar and 2-lobed. Flowers are sub-sessile, bractcite, fragrant, forming a terminal 6-30 cm long inflorescence panicle. Capsules (fruits) are glabrous, up to 1 cm long. It is distributed in Pakistan, China, India and Afghanistan [Citation5].
As a part of our ongoing search for antioxidative constituents from the genus Buddleja [Citation12], we investigated the antioxidative activities of the solvent-partitioned fractions from the EtOH extract of the whole plant of Buddleja davidii (). The n-BuOH-soluble fraction of the plant showed stronger antioxidative activity than that of the other fractions 7.39±0.04 μg/mL, IC50 in hydroxyl radical inhibitory activity test) which prompted us to carryout phytochemical studies on this plant. As a result, from the n-BuOH-soluble fraction, we isolated and characterized ten known phenylpropanoid glycosides, Calceolarioside A (1) [Citation9], Isomartynoside (2) [Citation10], Martynoside (3) [Citation8], 2``-Acetylmartynoside (4) [Citation10], Jionoside D (5) [Citation10], Plantainoside C (6) [Citation10], Campneoside II (7) [Citation9], Angoroside C (8) [Citation10], Forsythoside B (9) [Citation9], Poliumoside (10) [Citation9] (). Their structures were elucidated by 1D and 2D NMR spectroscopic analyses.
Figure 1. Structures of Compounds 1–10.
Table 1. Antioxidative activity of the solvent fractions.
In the current study we have described the antioxidant activities of the phenylpropanoid glucosides (1-10), among which compounds (1, 7, 9 and 10) were isolated for the first time from the title plant species.
Materials and methods
Extraction and isolation
The shade-dried whole plant (6 kg) were chopped and extracted with EtOH (3 X 20 L) at room temperature for 96 h. The ethanolic extract was evaporated in vacuo to give a dark greenish residue (250 g), which was partitioned between CHCl3, EtOAc, n-BuOH and water to afford CHCl3- soluble fraction (80 g), EtOAc-soluble fraction (45 g) and n-BuOH-soluble fraction (30 g), respectively. The n-BuOH soluble fraction was subjected to vacuum liquid chromatography (VLC) over silica gel with a step gradient solvent system of MeOH:CHCl3 (1:9, 3:7, 5:5), and 100% MeOH as eluants to obtain four sub-fractions (1-4). Fractions 1 to 2 were combined and were subjected to silica gel column chromatography (CC) using MeOH:CHCl3 (2:8, 3:7) followed by CC over Sephadex LH-20 with pure water to get semi pure compounds 1-5 which were finally purified on recycling HPLC (LC 908 W) [ODS-M80 semi preparative column, MeOH:H2O (1:1), flow rate 3 ml/min), detection (UV and Refractive Index (RI) detectors). Fraction 3 and 4 were subjected to Sephadex LH-20 CC and then purified by recycling HPLC (MeOH:H2O, 6:4) to obtain compounds 6,7 and 8-10, respectively.
Evaluation of antioxidative activity
Measurement of the inhibition of total ROS generation
Rat kidney homogenates prepared from the kidneys of freshly killed male Wistar rats, weighing 130–180 g, were mixed with or without the suspension of extracts or compounds, which were dissolved in 10% EtOH (final concentration: 0.4%). The mixtures were then incubated with 12.5 mM 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA, Molecular Probes Inc., Eugene, Oregon), which was dissolved in 100% EtOH (final concentration: 0.2%), at 37 °C for 30 min. A 50 mM phosphate buffer (Wako Pure Chemical Industries, Osaka, Japan) solution at pH 7.4 was also used. DCHF-DA is a stable compound, which is hydrolyzed by intracellular esterase to yield a reduced, nonfluorescent compound, 2′,7′-dichlorodihydrofluorescein (DCHF). The ROS produced by the homogenates oxidizes the DCHF to highly fluorescent 2′,7′-dichlorofluorescein (DCF). The fluorescence intensity of the oxidized DCF was monitored using a microplate fluorescence spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT) with excitation and emission wavelengths of 460 and 530 nm, respectively [Citation13]. Trolox being an effective standard oxidant was used as a positive control.
Measurement of the inhibition of hydroxyl radical generation
Extracts or compounds that were dissolved in 10% EtOH (final concentration: 0.4%) were added to 1 mM H2O2 and 0.2 mM FeSO4 (Fisher Scientific, Fair Lawn, N.J.) and incubated at 37 °C for 5 min. Esterase-treated 2 M DCHF-DA (Molecular Probes Inc., Eugene, Oregon) in 100% EtOH was then added, and the changes in fluorescence were monitored on a microplate fluorescence spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT), with excitation and emission wavelengths of 460 and 530 nm, respectively, for 30 min [Citation14]. Trolox being an effective standard oxidant was used as a positive control.
Measurement of ONOO− scavenging activity
The ONOO− scavenging activity was measured by monitoring the oxidation of dihydrorhodamine 123 (DHR 123, Molecular Probes Inc., Eugene, Oregon) using a slight modification of the method reported by Kooy et al [Citation15]. DHR 123 (5 mM) in DMF, which was purged with N2, was stored as a stock solution at 80oC. This solution was then placed on ice and kept in the dark prior to the study. The buffer consisted of 90 mM NaCl, 50 mM Na3PO4, 5 mM KCl at pH 7.4, and 100 M diethylenetriaminepentaacetic acid (DTPA), each of which was prepared with high-quality deionized H2O and purged with N2. The final concentration of DHR 123 was 5 M. The background and final fluorescent intensities were measured 5 min after treatment with and without the authentic ONOO−. DHR 123 was oxidized rapidly by the authentic ONOO−, and the final fluorescent intensity of the oxidized DHR 123 was measured using a FL 500 microplate fluorescence reader (Bio-Tek Instruments Inc., Winooski, VT) at excitation and emission wavelengths of 480 and 530 nm, respectively. The results are expressed as the mean ± standard error (n = 3) for the final fluorescence intensity minus background fluorescence. The effects are expressed as the percent inhibition of DHR 123 oxidation, and the standard oxidant, DL-penicillamine was used as a positive control. The IC50s was defined as the concentrations of sample showing 50% scavenging activity and were calculated from a triplicate experiment in all of the three scavenging tests.
Results and discussion
Free radicals and ROS or RNS (reactive nitrogen species), including H2O2, ˙O2−, ˙OH, NO˙, and ONOO−, play an important role in the etiology of a variety of human degenerative diseases. These reactive species are formed in the body as a consequence of aerobic metabolism and damage all intracellular components, such as nucleic acids, proteins, and lipids. ROS are also implicated in both aging and various degenerative disorders [Citation16].
We investigated the general antioxidative effects of the compounds to inhibit ˙OH and total ROS and to scavenge authentic ONOO− for the crude fractions and isolated compounds 1–10. The n-BuOH-soluble fraction of the plant showed stronger antioxidative activity than that of the other fractions in all the three scavenging tests (). The bioassay-directed isolation from this fraction provided compounds 1-10. The compounds with phenolic hydroxyl groups in 1,4-orientation in their structures, showed strong antioxidative activity in those three tests (). Compounds 1 and 7, with 3,4-dihydroxyphenylpropanoids and 3,4-dihydroxyphenylethanoids moieties, showed stronger activity with the IC50 values (4.15 ± 0.07, 40.32 ± 0.09, 2.26 ± 0.03 and 4.72 ± 0.01, 43.71 ± 0.09, 2.71 ± 0.07) for ˙OH, total ROS and to scavenge ONOO−, respectively. In all the scavenging activity tests, Martynoside (3) and Jionoside D (5), with a 4-methoxyphenylethanoids moieties, showed lower activity than 1 and 7. Isomartynoside (2), which has a 1,4-methoxyphenylpropanoid as well 1,4-methoxyphenylethanoid moieties in structure, further showed less strong activity (IC50 values 7.10 ± 0.01, 67.12 ± 0.02, 4.91 ± 0.06) than all the tested compounds except compounds 8. Angoroside C (8) with a 4-methoxyphenylethanoids and three glycoside moieties was observed with lowest activities among the tested compounds with IC50 values 9.47 ± 0.03, 81.15 ± 0.07, 7.79 ± 0.04 for ˙OH, total ROS and to scavenge ONOO−, respectively, but still significant activities in all tests.
Table 2. Antioxidative activity of Compounds 1–10.
The antioxidative activity was also examined in term of the chemical structures including those of functional radical and its orientation. Hydroxy and methoxy groups in the 1,4- and 1,3-orientation (glycoside ring) are mainly involved in the scavenging of phenyl propanoids/phenylethanoids (1–10). This was observed in particular for Calceolarioside A (1) and Campneoside II (7) containing the hydroxyl group in the 1,4-orientation. We also found that the scavenging activity tended to decrease for increasing number of glycosidation in the series of the compounds as was observed in compounds 1, 7, 9 and 10. Furthermore, the development of scavenging activity was found low comparatively for compounds 3, 5, in which the methoxy group is in 1,4-orientation. Changing of hydroxyl group to methoxy group at 1,4-orientation also decrease the scavenging activity. The ortho substitution with electron donor methoxy group in 1,4-oriennted phenyl hydroxyl compounds slightly increase the scavenging activity. This was observed in compounds 3 and 2. Based on these results, a phenyl ring where the hydroxyl radical is in 1,4-orientation allows the oxygen atom to share a positive charge, thereby causing stabilization through delocalization. Because of the electron donating effect of the methoxy group in 1,3-orientation, it helps to stabilize positive charge and this is thought to influence the scavenging ability. The substitution radical of 1,2- or 1,4-orientation generally donate an electron to the aromatic ring to activate it, either by the resonance effect or inductive effect. This tendency was also found in the all scavenging tests against the phenylpropanoids (1–10). To the best of our knowledge this is a first report of such phenylpropanoids glycosides in these particular antioxidant activity tests.
The compounds 1–10 could lead to compounds for the treatment of oxidative stress-related human diseases. However, a further in vivo study would help in exploring the pharmacological properties of these compounds.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Related Research Data
References
- Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA (1990); 87: 1620–1624.
- Bohme GA, Bon C, Lemaire M, Reibaud M, Piot O, Stutzmann JM, Doble A, Blanchard JC Apparent synaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats. Proc Natl Acad Sci USA (1993); 90: 9191–9194.
- Ito N, Hirose M Apparent-carcinogenic and chemopreventive properties. Proc Cancer Res (1989); 53: 247–302.
- Beckman JS, Ye YZ, Anderson PJ, Chen J, Accavitti MA, Tarpey MM, White CR Apparent nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Proc Chem Hoppe-Seyler (1994); 375: 81–88.
- Abdullah P “Flora of West Pakistan” (1974); 56: p 1.
- Kapoor VK, Chawla AS, Gupta YC, Passannanti S, Paternostro MP Apparent of Buddleia species leaves. Proc (1982); 52: 235.
- Hegnauer R, Kooiman P Apparent taxonomic significance of iridoids of Tubiflorae sensu Wettstein. Proc Med (1978); 33: 1–33.
- Ahmad M, Sticher O Apparent of Acetacin-7-O-Rutenoside and Martynoside from Buddleja davidii. Proc Chem Soc Pak (1988); 10: 117–123.
- Liao Y-H, Houghton PJ, Hoult JRS Novel and known constituents from Buddleja species and their activity against Leukocyte Eicosanoid generation. J Nat Prod (1999); 62: 1241–1245.
- Yamamoto A, Nitta S, Miyase T, Ueno A, Wu L-J Phenylethanoid and lignan-iridoid complex glycosides from roots of Buddleja davidii. Phytochemistry (1993); 32: 421–425.
- Ahmad I, Malik A, Afza N, Fatima I, Tareen RB, Nawaz SA, Choudhary MI Iridoid glucoside and aryl ester from Buddleja crispa. Proc J Chem (2006); 80: 1483–1491.
- Ahmad I, Chen S, Peng Y, Chen S, Xu L Lipoxygenase inhibiting and antioxidant Iridoids from Buddleja crispa. Proc Enz Inhib Med Chem (2008); 23: 140–143.
- Label CP, Bondy SC Apparent and rapid quantitation of oxygen reactive species formation in rat synaptosomes. Neurochem Int (1990); 17: 435–440.
- Nagao A, Seki M, Kobayashi H Inhibition of xanthine oxidase by flavonoids. Proc Biotechnol Biochem (1999); 63: 1787–1790.
- Kooy NW, Royall JA, Ischiropoulos H, Beckman JS Apparent-mediated oxidation of dihydrorhodamine 123. Free Radical Biol Med (1994); 16: 149–156.
- Sgar S, Kallo IJ, Kaul N, Ganguly NK, Sharma BK Apparent free radicals in essential hypertension. Proc Cell Biochem (1992); 111: 103–108.