Antioxidant Activities of Methanol Extracts of Thirteen Cameroonian Antibacterial Dietary Plants

This study falls within the search for alternative solutions to problems related to diseases associated with oxidative stress. It involved the evaluation of antioxidant activities extracts from thirteen antibacterial Cameroonian food plants, namely, P. nigrum, A. cruentus, L. sativa, S. edule, S. nigrum, V. amygdalina, A. hybridus, V. hymenolepis, L. capensis, M. esculenta, C. melo, T. occidentalis, and T. triangulare. The thirteen plant extracts with a broad spectrum of antibacterial activity all showed total reducing powers ranging between 2.41 and 27.81 AAE (mg ascorbic acid equivalents per gram of dried extract) and total phenol contents between 2.65 and 35.03 GAE (mg of gallic acid equivalents per gram of dried extract) of dry extract. Except for extracts of L. capensis, the other 12 extracts showed flavonoid contents ranging between 0.29 and 5.99 RE (rutin equivalents per gram of dried extract). All 13 plant extracts also showed free radical scavenging activity against DPPH· with IC50 ranging between 1.65 and 310.52 μg/ml, while 12 of these extracts exhibited inhibitory activity against NO· radical (IC50 ranging from 19.77 to 157.72 μg/ml). Statistically insignificant positive correlations (
 
 p
 >
 0.5
 
 ) were found between antibacterial activities of these plants and their antioxidant activities. The different results of this study provide scientific evidence for the use of these antibacterial food plants in the control of different conditions associated with oxidative stress.


Introduction
Oxidative stress is a condition which arises when reactive oxygen species (ROS) production becomes higher than antioxidant defense of the organism. If antioxidant capacities of the organism decrease or there is an increase in ROS production, the systemic antioxidant/oxidative dysfunction can lead to the accumulation of oxidative damage, which in turn can lead to modification of biomolecules with the occurrence of reactions that lead to protein additions, DNA oxidation, and lipid peroxidation with cellular functional ability reduction and risk of disease increase. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels can be generated through signal transduction pathways and regulation of physiological events such as cell cycle, programmed cell death, and inflammation [1]. It has been established that infectious diseases trigger the production of ROS and RNS which are at the origin of an increase of susceptibility to secondary infections via a mechanism of tissue injury as well as impairment of epithelial barrier functions [2]. e body contains natural cleansing systems against free radicals and other reactive species. However, ROS production sometimes exceeds internal antioxidant capacity leading to oxidative stress. erefore, external antioxidants have an important function in human health. ese bioactive compounds act preventively and neutralize the formation of new reactive species and free radicals. e exogenous antioxidants are mainly derived from food and medicinal plants including fruits, vegetables, and spices which in recent years gained great interest in their antioxidant activity. ese plants contain natural antioxidants mainly made of phenolic compounds. Phenolic compounds include phenolic acids, polyphenols, and flavonoids that protect plants, fruits, and vegetables from oxidative damage [3].
In two previous research papers, we highlighted antibacterial activities of the thirteen Cameroonian antibacterial plants used in this study, namely, Piper nigrum, Amaranthus cruentus, Lactuca sativa, Sechium edule, Solanum nigrum, Vernonia amygdalina, Amaranthus hybridus, Vernonia hymenolepis, Lactuca capensis, Manihot esculenta, Cucurbita melo, Telfairia occidentalis, and the fruits of Piper nigrum [4,5]. ese edible plants are used for many purposes in African traditional medicine (Table 1). In our previous studies, we demonstrated that the methanol extracts of these 13 plants inhibit the growth of Gram-negative multidrug-resistant bacteria with MIC values ranging between 32 and 1024 μg/ml and can then be used in the control of bacterial infections including MDR phenotypes [4,5]. e present study aims to determine the contents in total phenols and total flavonoids and antioxidant activities of methanol extracts of thirteen plants.  Table 1).

Material and Methods
Each sample was air-dried at room temperature (RT), and the resulting powder was extracted for 48 h at RT using methanol as solvent. Afterward, the extracts were filtered using Whatman No.1 paper and the crude extracts were obtained after reduced pressure concentration.

Determination of Total Reducing Power.
e total reducing power of plant extracts was determined as previously described with some modifications. Briefly, the reaction mixture was prepared in phosphate buffer sodium (PBS) (0.2 M, pH 6.6) with 100 μg/ml of each extract, 440 μL of potassium ferrocyanide ([K 3 Fe(CN) 6 ] 1%), and 440 μL of 10% trichloroacetic acid. e samples were then centrifuged for 10 min at 3000 rpm, and 680 μL of supernatant from each tube was collected and introduced into a new tube containing FeCl 3 (140 μL) and demineralized water (680 μL). e absorbance of these new solutions was read at 700 nm and converted into milligrams of ascorbic acid equivalents (AAE) per gram of dried extract using a calibration curve (prepared using different concentrations of ascorbic acid used in the same manner as extracts).

Determination of Total Phenol Content.
e determination of total phenol content of plant extracts was carried out using the Folin-Ciocalteu technique [98]. Briefly, a stock solution of Folin-Ciocalteu reagent was diluted 10 times. en 500 μL of this solution was added to 100 μL of extract (10 mg/ml) dissolved in 10% DMSO. After 4 min of incubation at room temperature (RT), 400 μL of 7.5% of Na 2 CO 3 solution was added to the mixture and the absorbance of the final solution was read at 765 nm. e absorbance was further converted into milligrams of gallic acid equivalents (GAE) per gram of dried extract using a calibration curve prepared using different concentrations of gallic acid ( Figure 1) used in the same manner as extracts.

Determination of Total Flavonoid Contents.
e determination of total flavonoid content of each plant extract was carried out using the spectrophotometric method of aluminum chloride (AlCl 3 ) using rutin ( Figure 1) as a standard flavonoid. Briefly, 100 μL of each plant extract (10 mg/ml) was mixed with 400 μL of MeOH, 20 μL of AlCl 3 10%, 20 μL of acetic acid (CH 3 COOK, 1 M), and 560 μL of distilled water. e reaction mixture was then incubated for 4 min at RT. After the incubation period, the absorbance was measured at 415 nm and converted into micrograms of rutin equivalents (RE) per gram of dried extract using a calibration curve (OD vs. rutin concentration).
(2) e probit table was then used to convert %RSA into probits which were plotted against the logarithmic values of the concentrations, and a linear regression curve was established to calculate the RSa 50 , which are the amounts of sample necessary to decrease by 50% the free radical NO. e experiment was carried out in triplicate and the percentages of NO scavenged by test samples were compared to that of rutin.

Statistical Analysis.
Statistical Package for Social Science (SPSS, version 18.0) was used to carry out statistical analysis. Data from each experience were expressed as mean-± standard deviation (SD). One-way analysis of variance (ANOVA) followed by the Waller-Duncan post hoc test was used for group comparisons. Differences were considered significant at a p value ≥0.05.

Results
e antioxidant properties of extracts were assessed using five different methods. ese experiments allowed the obtention of variable results according to the extracts and tests used.
Compared to the total reducing power and content of total phenols, P. nigrum stood out clearly with the largest respective values of 30.12 AAE (Table 2) and 14.01 GAE (Table 3), while the other 12 extracts showed reducing power between 1.62 and 8.41 AAE (Table 2), and total phenols comprised between 2.34 and 7.71 GAE (Table 3) and a strong correlation was also observed between the results of these two methods with a coefficient of correlation of 0.868 (Table 4), reflecting the strong involvement of phenols in the reducing power of the extracts of this study. However, higher levels of flavonoids were not obtained with extracts showing the greatest levels of total phenols. Actually, among the 4 extracts showing the highest flavonoid content (Table 5), we noticed the presence of M. esculenta (the only one of the 4 extracts with one of the highest phenolic contents) and especially that of A. cruentus which showed the lowest phenolic content. is lack of correlation between flavonoid contents and phenolic contents and between flavonoid content and total reducing power of the extracts on the other hand was further confirmed through correlation factors (Table 4).

Journal of Chemistry
Correlation analysis was also made between the antibacterial activities of extracts (Supplementary data available here) and the antioxidant activities obtained with each of the five methods used in this work. e results presented in the last row of Table 4 showed that there are statistically insignificant positive correlations (p > 0.5) between the antibacterial activities of these plants and the antioxidant activities obtained with each of the five methods. e strongest correlation was observed between antibacterial activities and total phenols content, followed by free radical scavenging activity against nitric oxide. e weakest correlation was observed between antibacterial activities and total flavonoid content, followed by free radical scavenging activity against DPPH· and total reducing power.
is correlation analysis could suggest that although the correlation between the antibacterial and the antioxidant activities of the extracts in this study is not very high, the phenols they contain may exert both antibacterial and antioxidant activities. e radical scavenging activities of extracts expressed here as RSa 50 varied according to radicals and extracts. us, with the NO· radical, the greatest activity was observed with A. hybridus (RSa 50 � 19.77 μg/ml), greater than that of rutin used as reference antioxidant (RSa 50 � 157.72 g/ml). Four other extracts (T. occidentalis, V. amygdalina, L. capensis, and M. esculenta) also showed activities higher than that of rutin. Extracts of V. hymenolepis and S. nigrum exhibited free radical scavenging activity statistically similar to that of rutin. e remaining six samples exhibited activities, which, although significantly lower than that of rutin, is still important in most cases (Table 6).
Against the DPPH· free radical, all the 13 extracts exerted scavenging activities with RSa 50 ranging from 1.65 (P. nigrum) to 109.22 mg/ml (C. melo). Apart from the extract of P. nigrum which showed best radical scavenging activities against DPPH·, seven other extracts showed RSa 50 values which were lower than 50 mg/ml. ese extracts include S. edule, M. esculenta, L. capensis, V. hymenolepis, A. cruentus, and V. amygdalina (Table 7).
Negative correlation was observed between free radical scavenging activity of extracts against NO and phenol contents, while there were insignificant correlations between scavenging activity, reducing power, and total flavonoid content, suggesting that these elements do not play a significant role in the trapping of NO. A significant correlation was observed between the scavenging activity vis-à-vis the DPPH and total reducing power, showing that these two activities are linked to the same compounds, while nonsignificant correlations were observed between DPPH and the contents of flavonoids and total phenols.
Antioxidant activities undertaken in this study corroborate the results of other studies that reported the benefits of fruits and vegetables and give information about the subject. Fruits and vegetables are known for their ability to protect organisms through antioxidant properties. ey are very rich in antioxidants, and several publications were already made on different plants of this study (Table 1). Some highlights concern the wealth of some of these fruits and vegetables antioxidant reference such as vitamin C, vitamin E, and quercetin in A. hybridus, M. esculenta, P. vulgaris, and S. nigrum (Table 1).
Previous studies showed the plants of the genus Amaranthus used in this study, namely, A. hybridus and A. cruentus presented phenols in varying degrees depending on the extraction solvent [101]. Without determining the RSa 50 , the same study showed that A. cruentus and A. hybridus exerted antiradical activity against different radicals including NO°. Moreover, it has been proven that the extract of A. cruentus helps to fight against oxidative stress in rats by increasing the activity of different antioxidant enzymes of plasma [102].
A study has shown that the ethanolic, aqueous, and acetone extracts pomace of roots of B. vulgaris contains phenols (316.30 to 564.50 GAE) and flavonoids (200.50 to 253.50 RE) [103]. Much higher levels than those of the methanolic extract of the whole root were found in this study (9.28 GAE and 0.25 RE, respectively, for phenols and flavonoids). However, with an RSa 50 of 175.49 μg/ml, the extract presented an antiradical activity against DPPH°c omparable to the one found in a previous study (RSa 50 between 133 and 275 mg/ml) [103]. It was also demonstrated that the methanol extract inhibits lipid peroxidation, preserves the activity of plasma antioxidant enzymes, and prevents excessive fluidity of plasma membranes in rats that received CCl 4 [104].
It was previously shown that C. melo may be used as a source of natural antioxidant due to its contents in phenols of 26.4 ± 0.3 GAE and flavonoids of 69.7 ± 3.37 RE [105]. It is also capable of reducing the production of superoxide, nitrite, and peroxynitrite. ese antioxidant activities would be partly explained by its richness in antioxidants such as glutathione, carotenoids, or vitamin E [106]. C. pepo also contains phenols including flavonoids and a scavenging activity against DPPH°variables with content specifically fertilizer and soil composition in general [107].
It was also reported that ethanol extracts of leaves and seeds of aqueous extracts of S. edule possess antioxidant activities performed through the inhibition of lipid peroxidation, the activity of free radical scavenging against the β-carotene and DPPH°, with an RSa 50 of 2 mg/ml. ese previous studies showed that the aqueous and ethanolic extracts possess higher activity than that obtained (11.39 mg/ml) with the methanol extract by sheets which S. edule still presented one of the most reducing powers against DPPH° [38].
In addition to phenols and other phytochemicals (alkaloids, steroids, and saponins) highlighted in this work, the antioxidant activities of M. esculenta could also be explained by its high anthocyanins and vitamin C [43]. Indeed, it has also highlighted antiradical activities (against DPPH°and   Journal of Chemistry 7 superoxide radical) of methanol extracts and acetone from the said plant and their metal chelator and antioxidant against deoxyribose degradation effect. All these activities only expressed in percentages were variable depending on the test sample and in particular with a reduction percentage of DPPH°of 15.2% with 0.1 mM methanolic extract [43]. e methanol extract of S. nigrum has already made radical scavenging activity against DPPH°up to 92% (a much lower activity with the aqueous extract) with a correlation with its phenolic content. Isolated from this plant glycoproteins could also justify highlighted in this study activity because they exert concentration-dependent antiradical activity against DPPH°and superoxide anion, as well as activity against the oxidation of 2-deoxyribose [108].
It has been shown that three flavones (luteolin, luteolin 7-O-β-glucuronide, and luteolin 7-O-β-glucoside) isolated from V. amygdalina have protective effects against the oxidation of β-carotene and oleic acid, lutein being more active than BHT [109]. It was also shown that three daily administrations of the aqueous extract of the plant at doses of 50 and 100 mg/kg can prevent the induction (acetaminophen) in oxidative stress and lipid peroxidation in rats [110].
Several parameters must be considered to maximize the biological effects of different vegetables, fruits, and spices of this study. e oxidation is an essential biological process for the production of energy in many living organisms. However, certain pathological conditions such as microbial attacks cause an excessive production of reactive oxygen species (ROS) [111]. is excess of ROS in turn can cause many physiological disorders [112]. e extract of P. nigrum has both the highest antimicrobial activity and the highest antioxidant activity. Although the correlation between antioxidant and antibacterial activity is difficult to establish with other plant extracts of this study, the combination of these two activities observed in all these could be of great importance in the treatment of infectious diseases, as in addition to the elimination of the causative agent or the inhibition of growth, they may inhibit the oxidative processes that often aggravate the symptoms of these diseases.
Different plant extracts studied in this work showed antiradical activity against DPPH radicals and NO°. e antiradical force varies greatly between antioxidants and radical to another. According to the proposed scale [113], antioxidants activities are considered significant or high if the IC 50 < 50 mg/ml, moderate if 50 mg/ml < IC 50 < 100 mcg/ml, and low if IC 50 > 100 mcg/ml. According to this scale, extracts of P. nigrum, S. edule, M. esculenta, L. capensis, V. amygdalina, V. hymenolepis, A. cruentus, T. occidentalis, and S. nigrum can be classified as extracts with high activity against DPPH°free radical scavenging activity and extract of T. triangulare can be classified as extract with moderate scavenging activity against this radical. According to the same scale, although significant differences were not observed between the activity of many extracts (M. esculenta, L. capensis V. hymenolepis C. melo, S. nigrum A. hybridus, T. occidentalis, and V. amygdalina) and that of rutin (reference), only extracts of M. esculenta presented high activity against the NO°free radical, while moderate activities were observed with extracts of S. edule and V. hymenolepis using the same scale [113]. e radical scavenging activity of chemical compounds against DPPH radical is related to their ability to provide electrons and/or protons. Sodium nitroprusside, at physiological pH, decomposes in solution by producing NO°r adical. In the presence of oxygen, radical leads to the formation of nitrite from nitrate. e chromogen formed during diazotization with nitrite ions with Griess reagent has a maximum absorbance at a wavelength of 546 nm [100]. By complexing with the NO°radical, compounds contained in extracts of plants thus inhibited the formation of the chromogen. e elevated reducing powers of plant extracts of this study could be explained by high levels of total phenolics and flavonoids [114]. Reducing power is very often linked to the effect of the compounds that are electron donors [115]. e extract of P. nigrum would be the richest in reductones extracted followed by extracts T. triangulare, A. hybridus, M. esculenta, and V. amygdalina.
As demonstrated with the strong correlation between the phenol content and total reducing power, the action of phenols partly explains the reducing power and antiradical activity against DPPH plant extracts. e presence of phenols in all extracts is not a surprising fact, since they are present in all vascular plants [116]. Phenolic compounds from plants are powerful antioxidants capable of preventing oxidative damage to biomolecules such as DNA, lipids, and proteins that play a role in chronic diseases such as cancer and cardiovascular diseases [117,118]. It has also been shown that there is a linear relationship between the antioxidant power plant and the concentration of phenolic compounds [56] acting through their hydroxyl groups [119]. e subset of the most abundant polyphenols in our diet, flavonoids are known primarily for their antioxidant activity [119,120]. ey are a group of phenols particularly clever and quick to transfer an electron. is electron stabilizing free radicals explains the scavenging activity of various flavonoids. However, no significant correlation between total phenolic content and flavonoid content could explain the lack of a significant correlation between reducing power and total flavonoid content, which also presented a negative correlation with NO.

Conclusion
is study was aimed at evaluating the antioxidant activity of thirteen Cameroonian plants which previously displayed antibacterial activities. e thirteen plant extracts exhibited high antioxidant activities correlated to their reducing power. ey possess total reducing powers (from 2.41 to 27.81 μg ascorbic acid equivalents/g of dried extract), total phenol contents (2.65 and 35.03 mg of gallic acid equivalents/g of dried extract), total flavonoid contents (0.15 and 5.99 μg of rutin equivalents/g of dried extract), and free radical scavenging power against DPPH (IC50 varying between 1.65 and 375.43 μg/ml) and NO (IC50 varying from 19.77 to 157.72 μg/ml). Statistically insignificant positive correlations (p > 0.5) exist between the antibacterial 8 Journal of Chemistry activities of these plants and the antioxidant activities with the strongest correlations observed between antibacterial activities and total phenols content, followed by free radical scavenging activity against nitric oxide. ese important antioxidant activities demonstrated in the present study provide enough evidence to support the fact that the thirteen Cameroonian antibacterial plants can be used as a source of natural antioxidants and help combat oxidative stress which is sometimes associated with infectious conditions.

Data Availability
e experimental data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
DED VK, JAKN, JRK, and MM designed the experiments, carried out the study, and wrote the manuscript; all authors read and approved the final manuscript.