Introduction

Reactive oxygen species (ROS), which include free radicals such as superoxide anion radicals, hydroxyl radicals and nonfree-radical species such as H2O2 and singled oxygen, are various forms of activated oxygen [1]. These molecules are exacerbating factors in cellular injury and aging process [2]. Iron, an essential metal for normal cellular physiology, can result in cell injury when in excess. This is because it plays a catalytic role in the initiation of free radical reactions. Fe (II) can react with hydrogen peroxide (H2O2) to produce the hydroxyl radical (OH•) via the Fenton reaction, whereas superoxide can react with iron (III) to regenerates iron (II) that can participate in the Fenton reaction [3].

Sodium nitroprusside, an anti-hypertensive drug, acts by relaxation of vascular smooth muscle; consequently, it dilates peripheral arteries and veins. However, sodium nitroprusside (SNP) can cause cytotoxicity through the release of cyanide and/or nitric oxide (NO) [4]. NO is a free radical with short half-life (<30 s). Although NO acts independently, it may also cause neuronal damage in cooperation with other reactive oxygen species (ROS) [5]. While quinolinic acid (QA), a neuroactive metabolite of the tryptophan–kinurenine pathway, produced by macrophages and microglia [6], is present in human and rat brain [7] it is involved in the pathogenesis of a variety of human neurological diseases [8]. Pharmacologically, QA is an endogenous glutamate agonist with a relative selectivity for the NMDA receptor in the brain [9]. Since it is not readily metabolized in the synaptic cleft, it stimulates the NMDA receptor for prolonged periods. This sustained stimulation results in opening of calcium channels causing Ca2+ influx followed by Ca2+-dependent enhancement of free radical production leading to molecular damage and often to cell death [10].

The brain and nervous system are particularly vulnerable to oxidative stress due to limited antioxidant capacity [9]. The brain is about 2% of person’s mass, but it utilizes 20% of the metabolic oxygen, and the vast majority of this energy is used by the neurons [11]. In addition, neurons cannot synthesize glutathione, a fundamental component of aerobic cell antioxidant machinery, but instead rely on surrounding astrocyte cells to provide useable glutathione precursors [11]. Because the brain has limited access to the bulk of antioxidants produced by the body, neurons are the first cells to be affected by a shortage of antioxidants, and are most susceptible to oxidative stress [11].

Pepper is an important agricultural crop because of its economic importance and the nutritional value of its fruits as an excellent source of natural colors and antioxidant compounds [12, 13]. A wide spectrum of antioxidant vitamins and phenolic compounds are present in pepper fruits such as vitamin C and polyphenols. Phenol compounds retard or inhibit lipid autoxidation by acting as radical scavengers [14]; this activity is principally based on the redox properties of their hydroxy groups and the structural relationships between different parts of their chemical structure [15, 16]. Consequently, they are essential antioxidants that protect against propagation of the oxidative chain. Whereas, vitamin C can chelates heavy metal ions [14], react with singlet oxygen and other free radicals, and suppresses peroxidation [15], reducing the risk of arteriosclerosis, cardiovascular diseases and some forms of cancer [3].

Capsicum pubescens, commonly known as tree pepper, has a distinctive thick-flesh, which is popularly used as vegetable condiment or made into a sauce [16, 17]. As a hot pungent flavor, it is mainly used as flavoring in cooked foods [16]. The fruit has antihemorrhoidal, antirheumatic, antiseptic, diaphoretic, digestive, irritant, rubefacients, sialagogue and tonic when taken in small amount [18]. Externally, it is used in the treatment of sprains, unbroken chilblains, neuralgia, pleurisy, etc. [17]. However, there is limited information on the antioxidant activity of C. pubescens as a dietary source of antioxidant. The present study therefore tend to assess the potentials of this pepper as a dietary source of antioxidant and to determine the state (ripe or unripe) in which the antioxidants are more abundant.

Materials and methods

Materials

Fresh samples of ripe and unripe C. pubescens were collected from a vegetable garden in Camobi, Santa Maria RS, Brazil. The authentication of the pepper was carried out in Departamento de Biologia, Universidade Federal de Santa Maria, Santa Maria RS, Brazil. All the chemicals used were analytical grade, while the water was glass distilled. The handling and the use of the animal were in accordance with NIH Guide for the care and use of laboratory animals. In the experiments, Wister strain albino rats weighing 200–230 g were collected from the breeding colony of Departamento de Biologia, Universidade Federal de Santa Maria, Santa Maria RS, Brazil. They were maintained at 25°C, on a 12 h light/12 h dark cycle, with free access to food and water.

Aqueous extract preparation

The inedible portion of ten samples of ripe (red) and unripe (green) pepper fruits were removed from the edible portion respectively, the edible portion was subsequently washed in distilled water and chopped into small pieces by table knife. The aqueous extract of the pepper was subsequently prepared by homogenizing the pepper in water (1:20 w/v); the homogenates were centrifuged at 2,000 rpm for 10 min. The supernatant was used for the assay.

Total phenol determination

The total phenol content was determined by adding 0.5 ml of the aqueous extract of the pepper to 2.5 ml, 10% Folin–Cioalteu’s reagent (v/v) and 2.0 ml, 7.5% sodium carbonate was subsequently added. The reaction mixture was incubated at 45°C for 40 min, and the absorbance was measured at 765 nm in the spectrophotometer. Gallic acid was used as standard phenol [19].

Vitamin C determination

Vitamin C was determined using the method of Benderitter et al. [20]. Briefly, 75 μl of DNPH (2 g of dinitrophenyl hydrazine, 230 mg thiourea and 270 mg in 100 ml, 5 M H2SO4) was added to 500 μl reaction mixture (300 μl of the pepper extracts with 100 μl, 13.3% trichloroacetic acid and water, respectively). The reaction mixture was subsequently incubated for 3 h at 37°C, then 0.5 ml H2SO4 65% (v/v) was added to the medium, and the absorbance was measured at 520 nm, and the vitamin C content of the sample was subsequently calculated, using vitamin C standard curve.

Fe2+ chelation assay

The ability of the aqueous extract to chelate Fe2+ was determined using a modified method of Minotti and Aust [21] with a slight modification by Puntel et al. [22]. Briefly, 150 μl of freshly prepared 500 μM FeSO4 was added to a reaction mixture containing 168 μl of 0.1 M Tris–HCl (pH 7.4), 218 μl saline and the aqueous extract of the pepper (0–25 μl). The reaction mixture was incubated for 5 min, before the addition of 13 μl of 0.25% 1, 10-Phenanthroline (w/v). The absorbance was subsequently measured at 510 nm in the spectrophotometer

Degradation of deoxyribose (Fenton’s reaction)

The ability of the aqueous extract of the pepper to prevent Fe2+/H2O2 induced decomposition of deoxyribose was carried out using the method of Halliwell and Gutteridge [5]. Briefly, freshly prepared aqueous extract (0–100 μl) was added to a reaction mixture containing 120 μl, 20 mM deoxyribose, 400 μl, 0.1 M phosphate buffer, 40 μl, 20 mM hydrogen peroxide and 40 μl, 500 μM FeSO4, and the volume made upto 800 μl with distilled water. The reaction mixture was incubated at 37°C for 30 min, and the reaction was stopped by the addition of 0.5 ml of 2.8% TCA (Trichloroacetic acid), this was followed by the addition of 0.4 ml of 0.6% TBA solution. The tubes were subsequently incubated in boiling water for 20 min. The absorbance was measured at 532 nm in spectrophotometer.

Reducing property

The reducing property of the pepper extracts was determined by assessing the ability of the extract to reduce FeCl3 solution as described by Pulido et al. [23]. Briefly, 2.5 ml aliquot was mixed with 2.5 ml, 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min, thereafter 2.5 ml, 10% trichloroacetic acid was added, and subsequently centrifuged at 650 rpm for 10 min, 5 ml of the supernatant was mixed with equal volume of water and 1 ml of 0.1% ferric chloride, the absorbance was later measured at 700 nm, a higher absorbance indicates a higher reducing power.

Preparation of brain homogenates

The rats were decapitated under mild diethyl ether anesthesia and the cerebral tissue (whole brain) was rapidly dissected and placed on ice and weighed. This tissue was subsequently homogenized in cold saline (1/10 w/v) with about 10-up-and-down strokes at approximately 1,200 rev/min in a Teflon–glass homogenizer. The homogenate was centrifuged for 10 min at 3,000g to yield a pellet that was discarded and a low-speed supernatant (S1) containing mainly water, proteins and lipids (cholesterol, galactolipid, individual phospholipids, gangliosides); DNA and RNA was kept for lipid peroxidation assay [8].

Lipid peroxidation and thiobarbibutric acid reactions

The lipid peroxidation assay was carried out using the modified method of Ohkawa et al. [24]. Briefly, 100 μl S1 fraction was mixed with a reaction mixture containing 30 μl of 0.1 M pH 7.4 Tris–HCl buffer, pepper extract (0–100 μl) and 30 μl of the pro-oxidant solution (250 μM freshly prepared FeSO4, 70 μM sodium nitroprusside and 1 mM quinolinic acid). The volume was made up to 300 μl by water before incubation at 37°C for 1 h. The color reaction was developed by adding 300 μl of 8.1% SDS (Sodium doudecyl sulphate) to the reaction mixture containing S1, this was subsequently followed by the addition of 600 μl of acetic acid/HCl (pH 3.4) mixture and 600 μl of 0.8% TBA (Thiobarbituric acid). This mixture was incubated at 100°C for 1 h. TBARS (Thiobarbituric acid reactive species) produced were measured at 532 nm and the absorbance was compared with that of standard curve using MDA (Malondialdehyde).

Analysis of data

The results of the triplicate were pooled and expressed as mean ± standard error (S.E.) [25]; student t test was carried out. Significance was accepted at P ≤ 0.05. The EC50 (extract concentration that will cause 50% inhibition of lipid peroxidation in the rat’s brain) of the pepper extracts were also determined.

Results and discussion

The results of the vitamin C content of C. pubescens are presented in Table 1. The results revealed that no significant difference (P > 0.05) exist between vitamin C content of the ripe and unripe C. pubescens [ripe (231.5 μg/g), unripe (224.4 μg/g)]. However, the vitamin C content of the pepper (ripe and unripe) was lower than that of Capsicum annuum, Tepin [ripe (332.5 μg/g) and unripe (250.2 μg/g)] [26], C. annuum L cv. Vergasa [27] and what Chu et al. [28] reported for red pepper, but higher than that of Capsicum chinese, Habanero [ripe (152.5 μg/g) and unripe (103.6 μg/g)] [26]. However, ripe and unripe C. pubescens had higher vitamin C content than that of lettuce, cucumber, celery, cabbage, onion, carrot, pineapple, banana, apple and some tropical green leafy vegetables [29, 30], but lower than that of orange, lemon and strawberry [30].

Table 1 Total phenol content and vitamin C content of Capsicum pubescens
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The results of the total phenol content of ripe and unripe C. pubescens are also presented in Table 1. The results revealed that the total phenol content of the ripe pepper (117.7 mg/100 g) was significantly (P < 0.05) higher than that of the unripe pepper (70.5 mg/100 g). The basis for the significant difference (P < 0.05) in the total phenol content cannot be categorically stated; however, the physiological changes that accompanied ripening that brings about changes in pigments may have caused a significant increase (P < 0.05) in the total phenol content of C. pubescens [26, 27]. However, the total phenol content of this pepper (ripe and unripe) is below that of C. annuum, Tepin, but within the same range with that of C. chinese, Habanero [26]. Furthermore, the trend in the phenol content with ripening agrees with the trend in C. chinese, Habanero [26], in that ripe pepper had higher total phenol content than the unripe pepper [26], but the same argument will not hold for C. annuum, Tepin [26].

As presented in Fig. 1 and Table 2, aqueous extracts of the ripe and unripe C. pubescens (1.7–8.3 mg/ml) caused a significant decrease (P < 0.05) in the lipid peroxidation in the rat’s brain homogenates in a dose-dependent manner. This inhibition in lipid peroxidation in the rat’s brain by the aqueous extract could be ascribed to the presence of vitamins and polyphenol compounds in the pepper (Table 1). Nevertheless, the aqueous extracts of the ripe pepper (EC50 = 3.5 mg/ml) significantly (P < 0.05) inhibited MDA production in the brain tissues than the unripe pepper (EC50 = 7.2 mg/ml). This difference in the inhibition of lipid peroxidation by the ripe and unripe pepper could be attributed to the difference in the total phenol content (Table 1).

Fig. 1
figure 1

Capsicum pubescens inhibits lipid peroxidation in rat’s brain. The percentage inhibition of MDA (Malondialdehyde) production was expressed with respect to basal brain tissue (without extract and pro-oxidant) that was taken as 100%

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Table 2 EC50 of aqueous extract of Capsicum pubescens
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However, it is worth noting that the ripening process that led to an increase in the total phenol content of C. pubescens would enhance the ability of the pepper to prevent MDA production in the brain tissue homogenates. Earlier reports have shown that phenolic compounds are responsible for most of the antioxidant activity in plants [26]. Phenolic compounds do this by removing free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce α-tocopherol radicals and inhibit oxidases [31–33]. Furthermore, the EC50 of pepper was higher than that of C. annuum, Tepin and C. chinese, Habanero that are considered the hottest pepper [26, 34].

Figure 2 shows the interaction (inhibition) of the pepper extracts with Fe (II)-induced lipid peroxidation in rat’s brain. The results revealed that incubation of the brain tissues in the presence of 25 μM Fe (II) caused 260% increase in the MDA content of the brain homogenates when compared with that of the basal (100%). This increase in lipid peroxidation in the presence of Fe2+ could be attributed to the ability of Fe2+ to catalyze one-electron transfer reactions that generate reactive oxygen species, such as the reactive OH*, which is formed from H2O2 through the Fenton reaction. Iron also decomposes lipid peroxides, thus generating peroxyl and alkoxyl radicals, which favors the propagation of lipid oxidation [35].

Fig. 2
figure 2

Capsicum pubescens inhibits Fe (II) induced lipid peroxidation in rat’s brain. The percentage inhibition of MDA (Malondialdehyde) production was expressed with respect to basal brain tissue (without extract and pro-oxidant) that was taken as 100%

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However, aqueous extracts of C. pubescens significantly inhibited (P < 0.05) Fe (II) induced lipid peroxidation in the rat’s brain in a dose-dependent manner (Fig. 2). Moreover, the ripe pepper significantly (P < 0.05) inhibited MDA production in the rat’s brain tissue homogenates than the unripe pepper. The decrease in the Fe2+ induced lipid peroxidation in the rat’s brain homogenates in the presence of the extracts could be as result of the ability of the extracts to chelate Fe2+ and/or scavenge free radicals produced by the Fe2+-catalyzed production of reactive oxygen species (ROS) in the rat’s brain [26]. However, the higher ability of the ripe pepper to protect the rat’s brain could be because of its higher phenol content (Table 1).

The results of the Fe (II) chelating ability of the pepper extracts are presented in Fig. 3. The results revealed that both extracts chelate Fe (II) in a dose-dependent manner, and there was no significant (P > 0.05) difference between the Fe (II) chelating ability of the ripe and unripe pepper. Likewise, the extracts did not significantly (P > 0.05) inhibit OH radical [Fe (II)/H2O2]-induced decomposition of deoxyribose as shown in Fig. 4. The reason for the inability of the extracts to prevent deoxyribose decomposition cannot be categorically stated. However, it is likely that the complex formed between the pepper phytochemicals and Fe would not initiate lipid peroxidation, but is active in Fenton reaction.

Fig. 3
figure 3

Fe (II) chelation ability of C. pubescens

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Fig. 4
figure 4

Inhibition of deoxyribose decomposition by C. pubescens

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Furthermore, extracts of the ripe and unripe C. pubescens significantly (P < 0.05) reduce Fe (III) to Fe (II) in a dose-dependent manner. However, ripe pepper extracts had a significantly higher (P < 0.05) reducing power than the unripe pepper (Fig. 5). This result agrees with the result of the total phenol content presented in Table 1 because the ripe pepper with higher phenol content had higher reducing power. Therefore, phenol and vitamin C are actively involved in the protection of brain tissue from Fe (II) induced lipid peroxidation, the mechanism through which they possibly do this, is by their Fe (II) chelating ability and reducing power; and not through hydroxyl radical scavenging ability.

Fig. 5
figure 5

Reducing power of C. pubescens

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The interaction of the aqueous extract of C. pubescens with sodium nitroprusside and quinolinic acid induced lipid peroxidation in rat’s brain are presented in Figs. 6 and 7, respectively. The results of the study revealed that sodium nitroprusside (178%) and quinolinic acid (222%) caused a significant increase in the MDA content of the brain. However, both extracts inhibited MDA production in the brain in a dose-dependent manner. Nevertheless, the ripe pepper extracts had a significantly higher (P < 0.05) inhibitory effect on the lipid peroxidation induced by both pro-oxidants than the unripe pepper. The higher inhibitory effect of the ripe pepper on lipid peroxidation in the rat’s brain is in line with the total phenol content and reducing power of the pepper.

Fig. 6
figure 6

Capsicum pubescens inhibits Sodium nitroprusside induced lipid peroxidation in rat’s brain. The percentage inhibition of MDA (Malondialdehyde) production was expressed with respect to basal brain tissue (without extract and pro-oxidant) that was taken as 100%

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Fig. 7
figure 7

Capsicum pubescens inhibits quinolinic acid induced lipid peroxidation in rat’s brain. The percentage inhibition of MDA (Malondialdehyde) production was expressed with respect to basal brain tissue (without extract and pro-oxidant) that was taken as 100%

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The protective properties of the pepper against sodium nitroprusside induced lipid peroxidation in the brain could be because of the ability of the antioxidant phytochemicals present in the aqueous extract to quench-scavenge the nitrous radical and Fe produced from the decomposition of sodium nitroprusside. The mechanism through which the extracts inhibited quinolinic acid induced lipid peroxidation in the brain tissues cannot be categorically stated; however, it could be attributed to the free radical scavenging ability and inhibition of the over-stimulation of NMDA receptor by the extracts.

Conclusion

Capsicum pubescens could prevent Fe (II), sodium nitroprusside and quinolinic acid induced lipid peroxidation in brain, this they do through the antioxidant activity of their phytochemicals (phenols and vitamin C). However, ripening, which brings about changes in the pigment of pepper, caused a significant increase (P < 0.05) in the total phenol content and consequently causes an increase in the protective ability of the C. pubescens on some pro-oxidant (Fe (II), sodium nitroprusside, quinolinic acid) induced lipid peroxidation in brain by enhancing the Fe (II) chelating and reducing power of the pepper.