Margalit Bergman

The antioxidant activity of aqueous spinach extract: chemical identification of active fractions

In previous studies we have elucidated the presence of powerful, natural antioxidants (NAO) in water extracts of spinach leaves and demonstrated their biological activity in both in vitro and in vivo systems. In the present study, the chemical identity of several of these antioxidant components is presented. Spinach leaves were extracted with water and the 20,000 g supernatant which contained the antioxidant activity was extracted with a water:acetone (1:9) solution. The 20,000 g supernatant obtained was further purified on reverse phase HPLC using C-8 semi-preparative column. Elution with 0.1% TFA resulted in five hydrophilic peaks. Elution with acetonitrile in TFA resulted in seven additional hydrophobic peaks. All the peaks were detected at 250 nm. All the fractions obtained showed antioxidant activity when tested using three different assays. Based on 1H and 13C NMR spectroscopy four of the hydrophobic fractions were identified as glucuronic acid derivatives of flavonoids and three additional fractions as trans and cis isomers of p-coumaric acid and others as meso-tartarate derivatives of p-coumaric acid. The present study demonstrates for the first time the presence of both flavonoids and p-coumaric acid derivatives as antioxidant components of the aqueous extract of spinach leaves.

Antioxidant activity of the polysaccharide of the red microalga Porphyridium sp

The cells of the red microalga Porphyridium UTEX 637 are encapsulated within a sulfated polysaccharide whose external part (i.e., the soluble fraction) dissolves into the medium. It is thought that the main function of the polysaccharide is to protect the algal cells from the extreme environmental conditions, such as drought and high light, prevailing in their native sea-sand habitat. In this study, we evaluated the antioxidant properties of the water-soluble polysaccharide of Porphyridium sp. by determining the ability of a polysaccharide solution to inhibit: (1) autooxidation of linoleic acid, as determined by the standard thiobarbituric acid (TBA) and ferrous oxidation (FOX) assays; and (2) oxidative damage to 3T3 cells as determined by the dichlorofluorescein (DCFH) assay. In all three assays, the polysaccharide inhibited oxidative damage in a dose-dependent manner. Antioxidant activity was also exhibited by fractions of the polysaccharide obtained by sonication followed by separation on a reverse-phase HPLC with a C8 semi-preparative column. It is suggested that the antioxidant activity of the sulfated polysaccharide protects the alga against reactive oxygen species produced under high solar irradiation, possibly by scavenging the free radicals produced in the cell under stress conditions and transporting them from the cell to the medium.

The effect of natural antioxidants, NAO and apocynin, on oxidative stress in the rat heart following LPS challenge

Oxidative damage plays a key role in septic shock induced by lipopolysaccharide (LPS) which is known to enhance the formation of reactive oxygen species (ROS). In this study, biochemical parameters indicative of oxidative stress were tested in the rat heart following LPS challenge, with and without pretreatment with the antioxidants NAO (natural antioxidant) and apocynin. NAO is a natural antioxidant isolated and purified from spinach and its main components are flavonoids and coumaric acid derivatives. Treatment with LPS alone significantly (P<0.05) increased the malondialdehyde (MDA) level in heart, both in cytosolic and mitochondrial fractions by 1.5- and 2.4-fold, respectively, and in plasma (2.66 fold). In the heart homogenate, the level of hydroperoxides also increased significantly (P<0.05). In addition, LPS treatment significantly (P<0.05) increased NADPH oxidase activity in the heart microsomal fraction by approximately 10-fold compared to control. Pretreatment for 7 days with either apocynin or NAO prior to the LPS challenge significantly (P<0.05) improved rat survival, decreased MDA levels in both fractions and decreased microsomal NADPH-oxidase activity, compared to LPS alone. Catalase (CAT) activity slightly increased at 24 h post-LPS injection in LPS group and returned to the control level in the apocynin treated group. No meaningful changes were indicated for glutathione peroxidase activity among all the treatment groups. The activities of cytosolic and mitochondrial superoxide dismutase (SOD) enzymes significantly (P<0.05) increased approximately 20% in the LPS-treated group, compared to control. Apocynin significantly (P<0.05) decreased SOD level in the mitochondrial fraction with no effect on the cytosolic fraction; whereas, NAO had no important effect on SOD level in both fractions. The beneficial pretreatment effects of the antioxidants against oxidative stress in the rat heart presented in this study may suggest a potential chemopreventive effect of this compound in sepsis prevention.

Scavenging of reactive oxygen species by a novel glucurinated flavonoid antioxidant isolated and purified from spinach.

NAO is a natural water soluble antioxidant that was isolated and purified from spinach leaves. Using HPLC, NMR, and CMR spectroscopy, the main components were identified as flavonoids and p-coumaric acid derivatives. The NAO was found to be a very effective antioxidant in several in vivo and in vitro biological systems. In the present study, the antioxidant activity of the novel antioxidant glucurinated flavonoid (GF) isolated and characterized from NAO, is compared to well-known antioxidants. In addition, the direct free radical scavenging properties of the purified component GF were studied using the electron spin resonance (ESR) technique. GF and NAO were found to be superior to EGCG and NAC and to the Vitamin E homologue Trolox in inhibiting reactive oxygen species (ROS) formation in the autooxidation system of linoleic acid and in fibroblasts exposed to metal oxidation. GF and NAO were found to inhibit the ESR signal intensity of DMPO-O(2) radical formation during the riboflavin photodynamic reaction. 10 mM GF caused approximately 90% inhibition in the intensity of the ESR signal, while NAO at a concentration of 60 microg/ml caused an inhibition of about 50%. Using the Fenton reaction, GF and NAO were found to inhibit DMPO-OH radical formation. A concentration of 2 mM GF caused a 70% inhibition in the intensity of the DMPO-OH radical ESR signal, while propyl gallate at the same concentration caused only 50% inhibition. Furthermore, both GF and NAO also inhibited the (1)O(2) dependent TEMPO radical generated in the photoradiation TPPS4 system. About 80% inhibition was obtained by 4 mM GF. The results obtained indicate that the natural antioxidants derived from spinach may directly affect the scavenging of ROS and, as a consequence, may be considered as effective sources for combating oxidative damage.

Composition, Efficacy, and Safety of Spinach Extracts

Spinach leaves, containing several active components, including flavonoids, exhibit antioxidative, antiproliferative, and antiinflammatory properties in biological systems. Spinach extracts have been demonstrated to exert numerous beneficial effects, such as chemo- and central nervous system protection and anticancer and antiaging functions. In this review article, we present a compilation of data generated in our laboratories and those of other investigators describing the chemical composition of spinach, its beneficial effects, relative safety information, and its recommended inclusion in the human diet. A powerful, water-soluble, natural antioxidant mixture (NAO), which specifically inhibits the lipoxygenase enzyme, was isolated from spinach leaves. The antioxidative activity of NAO has been compared to that of other known antioxidants and found to be superior in vitro and in vivo to that of green tea,N-acetylcysteine (NAC), butylated hydroxytoluene (BHT), and vitamin E. NAO has been tested for safety and is well tolerated in several species, such as mouse, rat, and rabbit. NAO has been found to be nonmutagenic and has shown promising anticarcinogenic effects in a few experimental models, such as skin and prostate cancer; it has not shown any target-organ toxicity or side effects. The current review provides epidemiological and preclinical data supporting the efficacy of extracts of spinach and the safety of its consumption.

Slowing tumorigenic progression in TRAMP mice and prostatic carcinoma cell lines using natural anti-oxidant from spinach, NAO--a comparative study of three anti-oxidants.

The TRAMP model and human prostatic cancer (PCA) cell lines DU145 and PC3 are useful for chemopreventive studies. We compared the efficacy of 3 anti-oxidants [a water-soluble natural anti-oxidant, NAO (200 mg/kg), found in spinach leaves; epigallocatechin-3 gallate, EGCG (200 mg/kg), a major green tea polyphenol; and N-acetylcysteine, NAC (125 mg/kg)] plus vehicle in slowing spontaneous tumorigenic progression in TRAMP and wild-type male mice. Sacrifices occurred on weeks 5, 9, and 13. Prostatic histopathology and oxidative-stress blood markers were evaluated. Hyperplasias were ranked by a combination of severity grade and distribution (focal, multifocal, and diffuse). The effectivity of each tested compound in reducing the severity/focalness of hyperplasia varied from lobe to lobe. NAO exerted a significant effect on the dorsal and lateral lobes; NAC, on the anterior and ventral lobes, and EGCG, on the ventral lobe. When the most severe hyperplasia in all 4 lobes of TRAMPs was evaluated, only NAO reduced hyperplasia at weeks 9 and 13. Plasma peroxide levels in TRAMPs were reduced following oral administration of NAO or NAC for 13 weeks; EGCG only slightly reduced these levels. In NAO-treated DU145 and PC3 PCA cells, inhibition of cellular proliferation occurred in a dose-dependent manner, increasing numbers of G1 cells and reducing ROS levels. The anti-oxidative and antiproliferative properties of NAO may explain its efficacy in slowing the spontaneous prostatic carcinogenic process in the TRAMP and its effects in the cell lines.

Lipopolysaccharide-induced oxidative stress in the liver: comparison between rat and rabbit.

In the present study the effect of LPS on biochemical systems involved in radical formation and scavenging processes in tissues from rabbit (LPS-sensitive) and rat (LPS-resistant) was investigated. The results obtained show a significant enhancement in the endogenous antioxidative enzyme system in rats as a result of LPS injection. In rats, 24 h after LPS injection, glutathione peroxidase (G-POX) and superoxide dismutase (SOD) activities were increased by 60% and 120%, respectively, compared to the control. However, in rabbits the increase in these activities was relatively mild. Moreover, NADPH-oxidase activity, which produces superoxide radical, was increased about twofold in rabbit, 15 h following LPS injection. In rats, injection of LPS did not result in any significant changes in the activity of this enzyme. In rats, a decrease in malonaldehyde (MDA) levels appeared after injection of LPS, while in contradistinction, the peroxidative levels of lipids in the rabbits liver were increased about 3-fold. Injection of D-galactosamine (Gal-N) in combination with LPS significantly increased the sensitivity of rats to LPS characterized by a significant increase in NADPH-oxidase activity. This study indicates that one possible mechanism (among others) that may explain the relative sensitivity of rabbits compared to rat, may be related to the increase in the production of reactive oxygen substances (ROS) which is not accompanied by a concomitant increase of the protective antioxidative enzymes. Furthermore, the relative resistance of the rat was found to be related to an increase in the activity of the protective antioxidative systems following administration of LPS.

Effects of water-soluble antioxidant from spinach, NAO, on doxorubicin-induced heart injury

Doxorubicin (DOX) produces clinically restorative responsesinnumeroushumancancers,butitscardiotoxicity has limited its usefulness. Because reactive oxygen species may affect DOX-induced antitumor activity and cardiotoxicity, weevaluated the prophylactic effect ofspinachnatural antioxidant (NAO) on DOX-induced cardiotoxicity and oxidative stress in female Balb/c mice using histological, electron microscopical and biochemical parameters. Mice were treated with NAO for 7 days prior to and/or for 6 days after DOX administration. Pretreatment with NAO (cumulative dose: 130 mg/kg) did not hinder the effectiveness of DOX. Light and electron microscopy of DOX-treated heart revealed myocardial degeneration. When administered combined before and after DOX, NAO conferred the most significantcardiacprotection.TheeffectsofNAOonthelipid peroxidation product, malondialdehyde, and on H2O2 / hydroperoxides were examined on day 6 following DOX administration; levels of both were elevated in DOX-treated mice, compared to control. Pretreatment with NAO prevented these changes. Pretreatment with NAO before DOX administration decreased catalase and increased super oxide dismutase activities compared to the DOX group. Our results suggest usage of NAO in combination with DOX as a prophylactic strategy to protect heart muscle from DOX inducedcellulardamage.

Effects of Antioxidants Apocynin and the Natural Water-Soluble Antioxidant from Spinach on Cellular Damage Induced by Lipopolysaccaride in the Rat

Oxidative damage plays a key role in septic shock induced by the endotoxin lipopolysaccaride (LPS) by enhancing the formation of reactive oxygen species such as superoxide anion radicals, peroxides, and their secondary product, malondialdehyde, especially in the liver. In this study, histopathologic changes in several organs were compared among groups of male Wistar rats that had been injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group that had been injected with LPS without pretreatment with antioxidants, an untreated control group, and groups that had been injected with either of the 2 antioxidants only. The antioxidants used were a water-soluble natural antioxidant from spinach (NAO) and the NADPH oxidase inhibitor apocynin. Hematoxylin-and-eosin-stained slides were prepared, and lesions were semiquantitatively scored. Exposure to LPS alone was associated with multifocal hepatocellular necrosis and acute inflammation, thymic and splenic lymphoid necrosis, ocular retinal hemorrhage and acute endophthalmitis, adrenal medullary vacuolation and necrosis and acute inflammation, and decreased adrenal cortical cytoplasmic vacuolation (consistent with depletion of steroidal hormone contents). Results indicated that pretreatment with both antioxidants for 8 days reduced, in some organs, the necrotic and inflammatory changes associated with the LPS challenge. These findings suggest a potential therapeutic application for these antioxidants in clinical sepsis.

Protection of fibroblasts (NIH-3T3) against oxidative damage by cyanidin-3-rhamnoglucoside isolated from fig fruits (Ficus carica L.).

Anthocyanins, plant secondary metabolites, have been recognized for their health-promoting properties when consumed by humans. In this study, the antioxidant properties of a major anthocyanin in fresh fig fruits, cyanidin-3-rhamnoglucoside (C3R), were evaluated by various assays in vitro and correlated with the protection afforded by C3R to cultured NIH-3T3 fibroblast cells. C3R inhibited lipid peroxidation from producing peroxy radicals (ROO(*)) and MDA in a dose-dependent manner, and a high calculated stoichiometric coefficient [n] for peroxy radicals was demonstrated. In addition to its scavenging of reactive oxygen species (ROS), C3R showed a strong chelating activity toward the Fe(2+) ion. Finally, pretreatment with C3R inhibited proapoptotic processes that were initiated by the oxidation of lysosome membranes in fibroblast cells. The high antioxidant potential, with several modes of action of purified C3R, may contribute to health benefits gained by the consumption of fresh fig fruits.