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Journal of Drug Delivery and Therapeutics

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Open Access  Full Text Article                                                                                                                Research Article 

Polyphenols contents and antioxidant potential of Nauclea latifolia Smith (Rubiaceae) acetonic fractions from Burkina Faso

Konate Almamy ¹*, Meda Roland Nâg-Tiéro ^2,3, Dicko Amadou ¹, Kam Eric Sami ², Koama Benjamin Kouliga ^2,3,4, Belem Hadidjatou ², Kabore Adama ¹, Traore Amadou ¹ And Tamboura Hamidou Hamadou ¹

¹ Laboratoire de Biologie et Santé Animales, Institut de l’Environnement et de recherches agricoles (INERA), 04 BP 8645 Ouagadougou 04, Burkina Faso.

² Laboratoire De Recherche Et d’Enseignement En Santé Et Biotechnologies Animales, Unité De Formation Et De Recherche En Sciences Et Techniques, Université Nazi Boni, 01 BP 1091 Bobo-Dioulasso 01, Burkina Faso.

³ Unité de Formation et de Recherche en Sciences et Techniques, Université Nazi Boni, 01 BP 1091 Bobo-Dioulasso 01, Burkina Faso.

⁴ Laboratoire De Médicine Et Pharmacopée Traditionnelle, Institut De Recherche En Sciences De La Santé (IRSS), 01 BP 2779 Bobo-Dioulasso, Direction Bobo-Dioulasso, Burkina Faso.

INTRODUCTION

Infectious diseases of livestock are a major threat to animal and human health in the world and their effective control is crucial ^1,2. Increases in the emergence or re-emergence of zoonotic infections pose significant additional threats to human health ^3,4. In Burkina Faso, the sanitary situation is characterised by a predominance of many epidemic and endemic diseases, together with a lack of qualified health worlkers. The best way to control of those infectious diseases is a holistic approach, which integrates robust diagnostic practices and vaccination. However, in many development countries, this approach is difficult to apply ⁵. Traditional herbal remedies are widely using by many people from Africa for their primary health care and the animal care for several reasons ^6,7. The traditional medicine is considered to be effective methods for curbing infectious diseases, reducing its intensity, shortening its course, or even preventing its recurrences ^6,7. The effectiveness of traditional medicine is mainly due to the choice of products, which optimize the functioning of the immune system and strengthen its reactivity. In addition to offering better and efficacious treatment options, the medicinal plants are also available, easily accessible and with minimal side effects relative to the conventional therapies ⁸. 

Nauclea latifolia Smith (syn. Sarcocephalus latifolius, Rubiaceae), is a plant widely used in West Africa for the treatment of several diseases ^9-11. In Burkina Faso, different extracts are used in the management of fevers, malaria, febrile states, dry colic, intestinal parasites, gastroenteritis, dysentery, vomiting, gonorrhea, syphilis, bilharzia and nervous attacks. Phytochemical analysis of extracts of the plant showed the presence of saponins, tannins alkaloids, flavonoids and phytate ^12-15. More recently, some researchers have discovery from a bio-guided purification of the roots of Nauclea latifolia, an active compound natural phytochemical as tramadol. This drug is available as a synthetic analgesic since the 1970s. It is also known to have a strong antibacterial property ^{16, 18}. Accordingly, it could be interesting to continue the investigating of this plant to ascertain and validate its medicinal value with a view to discovering new leads for better and improved management of infectious disease conditions. 

This study was conducted to investigate the antioxidant potential of the fraction n-hexane, dichloromethane, ethyl acetate and n-butanol of different parts of Nauclea latifolia. These could be the contributing factors to their health beneficial effects

MATERIAL AND METHODS

Plants Materials

Nauclea latifolia Smith (Rubiaceae) was collected at periphery of Ouagadougou (Burkina Faso) in September 2020. The botanical identification was done by Dr GANABA S. botanist at the National Herbarium of Burkina Faso where voucher specimens were kept. The part of plants collected were washed, dried in the shade at ambient temperature under the fan, crushed, and then sieved to obtain the fines powders.

Preparation of Extracts

The powdered plant samples (25 g) of each part were extracted with 250 ml of acetone (80 %) for 24 hours using an electric mixer. After filtration, acetone was removed using a rotary evaporator at 60^oC. The aqueous extract was then fractionated by successive liquid-liquid partitioning with an equal volume of n-hexane, dichloromethane, ethyl acetate and n-butanol. Extracts were stored at 4°C until being used.

Chemicals and reagents-

Chemicals include Folin-Ciocalteu, sodium carbonate, gallic acid, aluminium chloride (AlCl₃), sodium acetate, rutin, vanillin, tannic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS (2,2′-azinobis-(3-éthylbenzothiazoline-6-sulfonique)), potassium ferricyanide ((K3Fe(CN)6), trichloroacetic acid, iron III chloride, ascorbic acid, potassium persulfate, quercetin, hexane, ethyl acetate, methanol, n butanol. All chemicals and solvents used were of analytical grade and were purchased from Sigma Aldrich, and local suppliers.

Total Phenolics Content

Total phenolic content of each extract was evaluated according to the spectrophotometric method using the Folin-Ciocalteu reagent described by Meda et al., (2010) ¹⁹. A volume of 125 μl (0.1 mg/ml) of each extract was mixed with 625 μl of Folin-Ciocalteu reagent (0.2 N). After an incubation in the dark for 5 minutes, 500 μl of sodium carbonate (7.5% Na₂CO₃) was added to the mixture. After 2 hours’ incubation in the dark again, absorbance was read at 760 nm against a standard gallic acid curve (y = 4668e-3 *x-0.034, r² = 0.9991). The mean of three readings was used and the results were expressed in mg of gallic acid equivalent per 100 milligrams of dry extract (mg GAE/100mg). 

Total Flavonoids Content

The total flavonoids content in the extracts was determined by the aluminum trichloride
method described by Meda et al., (2010) ¹⁹. To 625 μl (0.1 mg/ml) of each extract solution were added 625 μl of AlCl₃ ( 2%), the whole was mixed and incubated in dark. After 10 minutes, the absorbances were read at 415 nm against a quercetin calibration curve (Y = 1.259e-02 * x; r2 = 0.9990). For each extract the average of 3 measurements was calculated and the amount of flavonoids was expressed in milligrams of quercetin equivalent per 100 milligrams of dry extract (mg QE/100mg).

Antioxidant Activity

DPPH radical method

The antioxidant power was carried out using the DPPH method according to the spectrophotometric method described by Meda et al., (2013).²⁰ In a test tube containing 0.375 ml of the sample (0.1 mg/ml) of each extract, 0.75 ml of the DPPH solution (20 mg /l) was introduced. After 15 minutes’ post-incubation of the mixture at room temperature in the dark, the absorbances were immediately read at 517 nm with a spectrophotometer (GENESYS 30). The radical scavenging activity (RSA) of each extract was measured using a reference curve of ascorbic acid. The antioxidant activity was expressed in µmol equivalent ascorbic acid per 1 gram of dry extract (µmol EAA/g).

 ABTS radical cation decolorization assay

The ABTS cation radical (ABTS^●+) scavenging capacity of antioxidants was determined as described by Meda et al., (2010). ABTS^●+ was regenerated by adding an aqueous solution of ABTS (7 mM) to 2.5 mM potassium persulfate solution, and the mixture is kept in the dark at room temperature for 12 hours before use. The mixture solution was then diluted with ethanol and the absorbance was adjusted to 0.700 (± 0.02) at 734 nm using spectrophotometer. 10 μl of each sample was mixed with 990 μl of the ABTS^•+ solution and incubated in the dark. After 15 minutes the absorbances were measured at 734 nm against a standard curve of ascorbic acid. 3 measures were carried out for each extract and the results were expressed in µmol equivalent ascorbic acid per 1 gram of dry extract (µmol EAA/g). 

Iron (III) to iron (II) reduction activity (FRAP) 

The FRAP assay was conducted following the method described by Meda et al., (2010). Briefly, 0.5 ml of extract (100 μg ml^-1) was added to 1.25 ml of phosphate buffer (0.2 M, pH 6.6) and 1.25 ml of potassium ferricyanide solution (1%). The resulting mix was then incubated at 50°C in a water bath. After 30 minutes 1.25 ml of trichloroacetic acid (10%) was added and the mixtures were centrifuged for 10 min at 3000 rpm. The supernatants of solutions (0.625 ml) were added to distilled water (0.625 ml) and to freshly prepared FeCl₃ solution (0.125 ml; 0.1%). The absorbances were read at 700 nm against a standard curve of ascorbic acid. The tests were performed in triplicate and expressed in µmol equivalent ascorbic acid per 1 gram of dry extract (µmol EAA/g). 

Statistical Analysis

The results were expressed as mean ± standard error of the mean (SEM). 2 way ANOVA multiple comparisons was applied for the statistical analysis, followed by Tukey's multiple comparisons test for post-hoc analysis. Statistical analyses were performed using GraphPad prism 9.12 and a p-value <0.05 was considered to be statistically significant.

RESULTS

Total Polyphenols Contents 

As a basis, total phenolics content was measured using the Folin–Ciocalteu reagent in each fraction. The results were derived from a calibration curve (y = 4668e-3 *x-0.034, r² = 0.9991) of gallic acid and expressed in gallic acid equivalents (GAE) per 100 milligrams dry extract weight. The content of phenolics in different fractions ranged from 2.05 to 58.16 mg GAE/100 mg. n-butanol fraction of bark, dichloromethane fraction of barks, ethyl acetate fraction of leaves and n-butanol fraction of leaves had the greatest phenolics contents (58.16 ± 0,76; 51.13 ± 0.99; 26.14 ± 0.61and 25.92 ± 0.91mg GAE/100 mg, respectively), while the smallest phenolics contents were found in root barks fractions of n-hexane fraction, root barks fractions of ethyl acetate fraction and root barks fractions of n-hexane fraction (2.05 ± 0.02; 2.21 ± 0.04 and 5.26 ± 0.16 mg GAE/100mg, respectively). 

As a basis, total flavonoids content was measured using aluminum trichloride reagent. The results were derived from a calibration curve (Y = 1.259e-02 * x; r2 = 0.9990) of quercetin equivalent and expressed in milligrams of quercetin equivalent per 100 milligrams of dry extract (mg QE/100mg). The total of flavonoids in different fractions ranged from 0.07 ± 0.01 to 4.85 ± 0.14mg QE/100mg. The leaves fractions have presented the greatest flavonoids contents and the smallest flavonoids contents were found in dichloromethane fraction of stem and root barks.

Table 1:  Phenolic content (TPC) and flavonoid contain (TFC) in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of roots, stem barks and leaves of Nauclea latifolia. 

Values are the mean of three replicates (TPC) ±SEM.

In vitro antioxidant activity determination

Trolox Equivalence Antioxidant Capacity in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia.

The results also showed a higher antioxidant content to capture the free radical (ABTS) in the different fractions leaves followed respectivily by the fractions of stem barks and he different fractions of root barks.

Figure 1: Antioxidant activity by ascorbic acid equivalent antioxidant capacity (ABTS) method in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia. Values are the mean of three replicates (ABTS) ±SEM

Table 2: Tukey's multiple comparisons test of Antioxidant activity by ascorbic acid equivalent antioxidant capacity (ABTS) method in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia

DPPH Radical Scavenging Activity in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia.

In this study, n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia were able to show free radical scavenging abilities (Figure 2). It was observed that the different fractions leaves had higher DPPH activity followed respectively by the fractions of stem barks and he different fractions of root barks.

Figure 2: Antioxidant activity by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity method in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia. Values are the mean of three replicates (DPPH) ±SEM

Table 3: Tukey's multiple comparisons test of Antioxidant activity by DPPH radical scavenging capacity method in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia

Ferric Reducing Antioxidant Power in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia.

The results also showed n-butanol fraction and DCM fraction of the stem barks had higher FRAP abilities to reduce ferric ions, followed by the all fractions of leaves and the ethyl acetate fraction of the stem barks. Root barks fractios showed lower FRAP abilities to reduce ferric ions. 

Figure 3: Antioxidant activity by ferric reducing (FRAP) method in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia. Values are the mean of three replicates (FRAP) ±SEM 

Table 4: Tukey's multiple comparisons test of Antioxidant activity ferric reducing (FRAP) in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of different parts of Nauclea latifolia

DISCUSSION

Polyphenol contain and antioxidant activity in n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of root barks, stem barks and leaves of Nauclea latifolia from Burkina Faso were evaluated. Polyphenols are known as potent antioxidants and contribute to some beneficial properties such as anti-viral and anti-microbial, anti-allergic, anti-inflammatory and immunomodulatory properties ^21-26. The polyphenols can be quantified by various methods, in this study, the total polyphenolic contents of the different fractions part of root barks, stem barks and leaves is determined using the diluted Folin-Ciocalteu reagent ²⁷. The total phenol and flavonoid content of the different fractions of the plant are presented in Table 1. The n-butanol fraction of root barks, dichloromethane fraction of root barks, ethyl acetate fraction of leaves and n-butanol fraction of leaves had the greatest phenolic contents. This variation could be explaining to various reasons such as solubility and polarity, environmental factors and origin of N. latifolia samples. Several analytical methods have been developed to assess the antioxidant properties of the medicinal plant extracts ²⁸.

In this study, the quantification of antioxidant activities of the different fractions have been focused on the DPPH, FRAP and ABTS methods of root bark, stem barks and leaves of Nauclea latifolia. The assays of antioxidants by ABTS and DPPH tests are based on the mechanism of the suppression of oxidative stress by the ability to scavenge free radicals. The assays of antioxidants by FRAP method is focused on the measure the ability of antioxidants to reduce ferric (Fe3+) ions. Antioxidants are broadly classified in 4 groups because of the complex nature of the redox-antioxidant system in vivo as: free radical scavengers, inhibitors of free radical formation, cellular and tissue damage repairers, and signaling messengers ^29,30. The results generated from this study demonstrated that different fractions of leaves followed by the fractions of stem barks and the different fractions of root barks respectively possessed good free radical scavenging activity.²⁹. The inhibition of free radical formation could protect against oxidative damage by suppressing the formation of active ROS/RNS ^29,30. These fractions could act as antioxidants, and serving possibility as scavengers or free radical inhibitors. Furthermore, antioxidants might have contributed to the different medicinal properties of this plant as reported ³¹.

Those results could be explained by the fact the antioxidant activity is greatly determined by the chemical structure and electron donation/reception capability of those polyphenols. Moreover, polyphenols due to the presence of a hydroxyl group are more soluble in polar organic solvents; therefore the extraction procedures and solvents could explain our results. 

CONCLUSION

In this study, the assessment of antioxidant activity has indicated that all parts of this plant had phenolic and flavonoid contents to varying degrees. This plant could be a source of natural antioxidants against several animal’s diseases. The quantification of antioxidant activity could serve as a indicators for the use of these parts of Nauclea latifolia against many human and animals diseases. Further works are necessary to better understand their ability to control animals infectious diseases.

ACKNOWLEDGMENT 

The authors acknowledge the support of Feed the future through the ENHANCE project which provided the PhD grant and financed this work. 

COMPETING INTERESTS 

The authors declare that they have no competing interests.

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