Bioactivity-guided fractionation of Coronopus didymus: a free radical scavenging perspective.

Abstract

The whole plant aqueous extract of Coronopus didymus Linn, was fractionated on the basis of polarity and resulting fractions were evaluated for free radical scavenging ability. The most non-polar fraction (CDF1) was found to be more active than other fractions in scavenging DPPH, ABT[S.sup.[dot]-], nitric oxide and hydroxyl radicals in steady-state conditions. Stop-flow spectrometric studies showed 58.13% inhibition of 100 [micro]M DPPH at a concentration of 150[micro]g/ml of CDF1 in 1000s and 32.31% scavenging of 960 [micro]M ABT[S.sup.[dot]-] at a concentration of 300[micro]g/ml of CDF1 in 100s. The reaction of CDF1 with hydroxyl radicals produced by pulse radiolysis showed a transient spectrum with absorption peaks at 320, 390 and 400 nm, indicating the presence of flavonoids/related components. Competition kinetics with potassium thiocyanate against scavenging of hydroxyl radicals showed a reactivity of 0.1326 against thiocyanate. CDF1 also protected against Fenton reagent-induced calf thymus DNA damage at a concentration of 400 mg/ml indicating it to be the most potent fraction.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Coronopus didymus; Free radical scavenging; Stop-flow spectrometry; Pulse radiolysis; Competition kinetics

Introduction

Reactive oxygen species are cytotoxic due to the intermediate formed from univalent reduction of molecular oxygen, including the superoxide radical ([O.sub.2.sup.[dot]-]), hydrogen peroxide ([H.sub.2][O.sub.2]), and the hydroxyl radical (O[H.sup.dot]). These oxygen intermediates differ significantly in their interactions and can cause extensive cellular damage such as nucleic acid strand scission (Adelman et al., 1988), modification of polypeptides, lipid peroxidation, etc. (Pryor and Porter, 1990). The screening of the compounds, which scavenge the reactive oxygen species, could lead to promising radioprotectors. Most of the antioxidants used in therapy are derived from natural sources. About 28% of the drugs approved by the FDA between 1981 and 2002 are either natural products or chemicals derived from them (Weiss and Landauer, 2003; Clardy and Walsh, 2004). Therefore, exploration of phytochemicals as radioprotectors is a promising drug development strategy.

Coronopus didymus (CD) (Family: Brassicaceae) is being used as folk medicine to treat allergies and wounds in and around Chickballapur, Karnataka, India. CD has been reported to contain flavones, chrysoeriol and chrysoeriol-6"(OAc)-4'-[beta]-D-glucoside (Prabhakar, et al., 2002); benzyl cyanide (Inam ul Haque, 1989); and glucotrapaeolin, a glucoside of" benzyl isothiocyanate (McDowall et al., 1947). 1,8-dihyroxy anthraquinone has been reported at a concentration of 0.003% (De Ruiz et al., 1994). The flavonoids, chrysoeriol and its glycoside are reported to be good antioxidants and free radical scavengers (Mishra et al., 2003). Previously reported work from our labs include findings on the ability of the aqueous extract of CD in alleviating allergies, inflammation, hepatotoxicity, hyperglycemia, and promotion of wound healing (Prabhakar et al., 2002). The above findings stimulated us to study the bioactivity-guided fractionation of the aqueous extract through in vitro free radical scavenging studies.

Materials and methods

Extraction and fractionation

The aqueous extract of the plant was prepared by boiling about 200 g of the plant in water (11), concentrated and dried in a vacuum oven (CDW yield: 20 g). The aqueous extract (10 g) was treated with five successive 50 ml aliquots of methanol, centrifuged and pooled supernatant yielded 5.5 g methanol fraction. The methanol fraction was loaded on to a silica gel column built and eluted with chloroform followed by graded mixtures of chloroform-methanol. The following five fractions were collected separately and dried in vacuo: [1] Chloroform fraction (yield: 0.005 g), [2] 25% MeOH-CH[Cl.sub.3] fraction (CDF1 yield: 0.7 g), [3] 50% MeOH-CH[Cl.sub.3] fraction (CDF2 yield: 0.8 g), [4] 75% MeOH-CH[Cl.sub.3] fraction (CDF3 yield: 1.0 g) and [5] 100% MeOH fraction (CDF4 yield: 2.5 g).

Reaction with DPPH radical

The reaction of 100 [micro]M DPPH with CDW and CDF1 to CDF4 (5-500 [micro]g/ml) was followed by the steady-state method (Raj Sekhar Narla and Rao, 1995). Time-resolved study of CDF1 (125-500 [micro]g/ml) with 50 [micro]M DPPH was studied in a stop-flow spectrophotometer (Model SX 18 MV, Applied Photo Physics, UK).

Reaction with ABTS radical anion 

The reaction of 100 [micro]M ABT[S.sup.[dot]-] with CDW/CDF1 to CDF4 (2-200 [micro]g/ml) was also followed by the steady-state method (Raj Sekhar Narla and Rao, 1995). Time-resolved studies were carried out on solutions of CDF1 (25-300 [micro]g/ml) with aqueous solution of ABT[S.sup.[dot]-] (960 [micro]M) and the time-dependent absorbance changes were monitored at 645 nm in a stop-flow spectrophotometer.

Reaction with hydroxyl radical

Steady-state hydroxyl radical scavenging as measured by degradation of the deoxy-D-ribose method was performed on the solutions of CDW/CDF1 to CDF4 (3-300 [micro]g/ml) as described by Kunchandy and Rao (1989). The hydroxyl radical reaction of CDF1 was carried out using the pulse radiolysis technique, the detailed description of this technique is given in Priyadarsini et al. (1995). Radiation dosimetry was done using an aerated aqueous solution of 0.01 M potassium thiocyanate (KSCN). The dose per pulse was 18.5 Gy. Competition kinetics of hydroxyl radical scavenging by CDF1 against 1 mM KSCN at pH 7 was studied by monitoring (SCN)[.sub.2.sup.[dot]-] absorbance at 500 nm (Priyadarsini et al., 1995).

Nitric oxide scavenging studies

Different concentrations of CDW/CDF1 to CDF4 were incubated with sodium nitroprusside (1.66 mM) for 5h, then 0.5 ml Greiss reagent was added and absorbance was recorded at 546 nm (Sudheer Kumar et al., 2003).

Total antioxidant capacity

CDW/CDF1 to CDF4 were mixed with 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate), incubated at 95[degrees]C for 90min, cooled to room temperature, and absorbance measured at 695 nm. The antioxidant activity was expressed as the number of equivalents of ascorbic acid (ASC) (Prieto et al., 1999).

Calf thymus DNA protection studies

Fenton-induced calf thymus DNA protection studies of CDW/CDF1 to CDF4 were carried out (Lu et al., 2001). The final incubation mixture in a 1.5 ml Eppendorff tube contained 600 ng DNA, 20 mM phosphate buffer, 15 mM EDTA, 15 mM [H.sub.2][O.sub.2] and 8mM FeS[O.sub.4], incubated for 20min followed by the addition of loading buffer (30% glycerol in water). A volume of 20 [micro]l was loaded into wells and electrophoresis was carried out in TAE buffer (40 mM Tris base, 16mM acetic acid, 1 mM EDTA, pH 8.0) for 90min. Photographs were taken under UV (312 nm) transillumination to visualize DNA mobility.

Results

Reaction with DPPH radical

CDF1 was found to have maximum scavenging ability with an I[C.sub.50] of 69.083 [micro]g/ml. Standard ASC had an I[C.sub.50] of 9.76 [micro]g/ml. The sequence of increasing reactivity towards DPPH is ASC >CDF1 >CDF4 >CDF3 >CDF2 [approximately equal to] CDW (Table 1). The observed first-order decay rate constant ([k.sub.obs]) of CDF1 was linear with increasing concentrations of DPPH as studied by stop-flow spectrometry.

Reaction with ABT[S.sup.[dot]-]

The CDF1 was found to have an I[C.sub.50] of 3 [micro]g/ml (Fig 2). I[C.sub.50] of ASC was 1.64 [micro]g/ml. The sequence of reactivity towards ABT[S.sup.+] is ASC >CDF1 >CDW >CDF4 >CDF2 [approximately equal to] CDF3 (Table 1). The observed first-order decay rate constant for ABT[S.sup.[dot]-] was found to increase with increasing CDF1 concentrations in the stop-flow method.

Reaction with hydroxyl radical

The fractions CDF1, CDF2 and CDF3 exhibited an I[C.sub.50] of 11.42, 20.99 and 26.47 [micro]g/ml, respectively. However, the fraction CDF4 showed less scavenging ability with an I[C.sub.50] of 1093.5. Standard mannitol had an I[C.sub.50] of 2.95mg/ml. The transient spectrum of the reaction of CDF1 with O[H.sup.dot] is given in Fig. 1. The apparent reactivity of CDF1 against KSCN towards O[H.sup.dot] was determined and found to be 0.133 times that of KSCN (Spinks and Woods, 1990).

Reaction with nitric oxide

CDW/CDF1 to CDF4 exhibited equivalent ability to scavenge the nitric oxide and the I[C.sub.50] values are listed in Table 1.

Total antioxidant capacity

CDF1 and CDF4 were found to be most active and were equipotent. One milligram of CDF1/CDF4 was equivalent to 115.21 and 115.87 [micro]g of ASC, respectively (Table 2).

Calf thymus DNA protection studies

The fractions CDF1 to CDF4 did not damage the DNA by themselves as indicated in lanes 3, 5, 7 and 9. However, CDF1, CDF2 and CDF3 protected the hydroxyl radical-induced DNA damage as evident from lanes 4, 6 and 8 respectively. CDF4 did not protect the DNA damage (lane 10), which correlates well with the hydroxyl radical scavenging data (Fig. 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Discussion

Most of the natural products contain abundant potent antioxidants, screening them for identifying a radioprotective lead is an effective strategy. Our earlier studies on aqueous extract of CD revealed a good in vitro free radical scavenging activity, which encouraged us to look for its radioprotective ability in vivo (in-house data). In continuation with this work, we undertook the present study.

CDF1 was found to be better hydrogen and electron donors than the other fractions studied, as evidenced in DPPH and ABT[S.sup.[dot]-] assays, respectively. It also possesses 0.115 mg of ASC equivalent antioxidant capacity per mg of the fraction. Since, CDF1 showed better steady-state free radical scavenging and calf thymus DNA protection ability, we subjected it to nanosecond pulse radiolysis and stop flow spectrophotometric studies. The reaction with DPPH revealed initial fast decay followed by a slow decay in a concentration range of 125-500 [micro]g/ml. This is probably because two or more components react with DPPH at varying rates. The reaction of CDF1 with the hydroxyl radicals showed a transient spectrum with absorption peaks at 320, 390 and 400 nm, indicating the presence of flavonoids/related components (Mishra et al., 2003).

We had earlier isolated two flavonoids chrysoeriol and chrysoeriol-6"(OAc)-4'-[beta]-D-glucoside from the alcohol extract of CD, of which the latter was reported for the first time (Prabhakar et al., 2002). Mishra et al. reported the kinetic studies of these flavonoids and showed them to be active free radical scavengers. This suggests that the flavonoids present in the extract are probably one of the bioactive components responsible for the reactivity of CDF1. This is further supported by the fact that flavonoids like orientin and vicenin possess promising radioprotection abilities in vivo (Uma Devi et al., 2000). Isolation, characterization and testing the flavonoids/bioactive compounds from the promising fractions and subjecting them to in vivo radioprotection studies is a part of our future strategy.

Acknowledgements

This work is supported by a Grant Vide No. 2003/36/27/BRNS/2014 dated 13.02.2004 from the Board of Research and Nuclear Sciences, Department of Atomic Energy, Government of India, India.

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K.R. Prabhakar (a), V.P. Veeresh (a), K. Vipan (a), M. Sudheer (a), K.I. Priyadarsini (b), R.B.S.S. Satish (c), M.K. Unnikrishnan (a,*)

(a) Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal-576104, India

(b) Radiation Chemistry & Chemical Dynamics Division, Bhabha Atomic Research Center, Trombay, Mumbai--400 085, India

(c) Department of Radiobiology, Kasturba Medical College, Manipal--576104, India

Received 28 April 2005; accepted 4 July 2005

* Corresponding author. Tel: [phone]; fax: [phone].

E-mail address: [e-mail] (M.K. Unnikrishnan).

Table 1. Comparison of the I[C.sub.50] in [micro]g/ml of CDW and CDF1 to
CDF4 against various free radicals and their total antioxidant capacity

                                ABT[S.sup.+[dot]]  O[H.sup.dot]
               DPPH scavenging  scavenging         scavenging
Description    ([micro]g/ml)    ([micro]g/ml)      ([micro]g/ml)

CDW            473.00            14.80             Nil
CDF1            69.08             3.25               11.42
CDF2           470.21           142.07               20.99
CDF3           459.75           144.03               26.47
CDF4           235.07            84.04             1093.5
Ascorbic acid    9.76             1.64               --
Mannitol        --               --                2950.0

               NO scavenging  Ascorbic acid
Description    ([micro]g/ml)  equivalents/ mg ([micro]g)

CDW            276.54          86.71
CDF1           239.54         115.21
CDF2           230.39          88.85
CDF3           289.08          75.43
CDF4           227.61         115.87
Ascorbic acid   --             --
Mannitol        --             --

Table 2. Total antioxidant activity of the aqueous extract of CD and its
fractions

Description  Ascorbic acid equivalents per mg of the extract/fraction

CDW          0.08671
CDF1         0.11522
CDF2         0.08885
CDF3         0.07543
CDF4         0.11587