Antioxidant and antiplasmodial activities of Razafimandimbisonia minor (Baill.) Kainul. &amp; B. Bremer leaves (Rubiaceae)

doi:10.21203/rs.3.rs-5332258/v1

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Antioxidant and antiplasmodial activities of Razafimandimbisonia minor (Baill.) Kainul. & B. Bremer leaves (Rubiaceae)

https://doi.org/10.21203/rs.3.rs-5332258/v1

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Background

Razafimandimbisonia minor (Baill.) Kainul. & B. Bremer (Rubiaceae), a specie endemic to Madagascar, is traditionally used in Malagasy medicine. A decoction of the leaves is employed to treat liver disorders, fatigue and fever. The aim of this study was to assess the plant's antioxidant activity, determine the content of total phenolic compounds and flavonoids, and analyze the antiplasmodial potential.

Methods

The leaves were collected, shade dried and ground. The methanol extract obtained by maceration was partitioned by liquid-liquid to produce hexane, ethyl acetate and aqueous fractions. Phytochemical screening was carried out using standard methods, followed by analysis of phenol and flavonoid contents, as well as antioxidant and antiplasmodial activity tests. Quantification of total polyphenols and flavonoids was carried out by UV spectrophotometry, using colorimetry with 10% Folin-Ciocalteu reagent and 2% aluminum chloride respectively. Antioxidant activity was measured by the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay. Antimalaria activity was assessed by measuring fluorescence with the SYBR Green dye on the chloroquine-resistant Plasmodium falciparum strain FCM29. Results were analyzed by ANOVA with Tukey's test.

Results

The methanol extract had a high polyphenol and flavonoid content, with concentrations of 280.8 ± 4.6 mg EGA/g and 50.4 ± 2.3 mg ER/g respectively. Biological tests revealed a better antioxidant activity of this extract, with a 50% inhibitory concentration of free radicals measured at 21.58 ± 0.05 µg/mL. But, the hexane and ethyl acetate extracts showed moderate antiplasmodial activity with concentrations of 21.32 and 24.42 µg/mL respectively, while the aqueous and methanol extracts showed weaker activity with concentrations of 41.5 µg/mL and 50.71 µg/mL, respectively.

Conclusion

These results display the phenolic and flavonoid content of the methanol extract of Razafimandimbisonia minor leaves, which corroborates with antioxidant activity, but the antimalarial activity is rather weak. They underline the value of using this plant for phytomedicinal properties, confirming the important role in malagasy traditional medicine.

Rubiaceae

Razafimandimbisonia minor

antioxidant

antimalarial

total polyphenol

total flavonoid

In Madagascar, a country renowned for exceptional biodiversity, with an endemicity rate of 80% [1], the use of medicinal plants is deeply rooted in local culture. In landlocked regions, traditional medicine remains a common practice [2]. Traditional practitioners use various plant parts, such as aerial parts, wood, bark, roots and seeds, which are recognized for their curative properties [3]. The phytochemical compound of these plants are of interest to researchers for their antibacterial, antiplasmodial and antioxidant potential [4].

Recent studies have shown that oxidative stress is linked to numerous diseases such as liver damage due to the oxidation of biomolecules as lipids [5]. This stress occurs when an imbalance exists between pro-oxidants and antioxidants, to the detriment of the latter [6]. Antioxidants, capable of neutralizing free radicals generated by cellular metabolism or external factors [7], inhibit free radical reactions and thus prevent cellular damage [8]. Among the various phytochemicals, polyphenols are well known for their ability to neutralize these free radicals, as are flavonoids, considered the most important class of polyphenols [9]. Antioxidant activity is often correlated with a high content of phenolic compound and flavonoids in extracts [10].

In this context, we focused on a plant endemic to Madagascar belonging to the Rubiaceae family, the fourth largest family of flowering plants with around 650 genera [11]. The genus Razafimandimbisonia, identified and described in 2009, belongs to the subfamily Ixoroideae and tribe Alberteae within the Rubiaceae [12]. This genus comprises five species: Razafimandimbisonia sambiranensis, Razafimandimbisonia humbotii, Razafimandimbisonia orientalis, Razafimandimbisonia regalis and Razafimandimbisonia minor [11] [12]. No chemical or biological studies have yet been reported on these species.

Razafimandimbisonia minor, known locally as “Hazomborondreo”, is commonly used in traditional pharmacopoeia to treat liver disorders, fatigue and fever [13]. Any fever in a patient is considered malaria until proven otherwise [14].

The aim of this study is to analyze the antioxidant activity of extracts from the leaves of this plant using the DPPH test, to quantify the polyphenol and flavonoid content, and to assess its antiplasmodial activity in order to validate its traditional use.

Plant material

The leaves of Razafimandimbisonia minor, (Baill.) Kainul. & B. Bremer (Rubiaceae) were collected in October 2014, in Andaingomadinika in the Moramanga district of the Alaotra Mangoro region. The plant was identified by Dr Stephan Richard RAKOTONANDRASANA, botanist at Centre National d'Application des Recherches Pharmaceutiques (CNARP). A voucher specimen, referenced ROL 734, was deposited in the herbarium of CNARP.

The harvested leaves were dried in a ventilated dryer at a temperature of 40°C for 2 weeks, then ground to obtain a powder.

Preparation of Razafimandimbisonia minor leaf extract

A total of (400 g) dried leaves were extracted by maceration with 1 L of absolute methanol for 72 h at room temperature, with repeated agitation and filtered. The filtrate was dried using rotary evaporator at temperature of 40 C. The methanol extract (40 g) was dissolved in 300 mL distilled water and partitioned gradiently into hexane and ethyl acetate (3 x 800 mL each) to obtain three fractions and dried.

Phytochemical screening

Phytochemical screening is a method for gathering information aimed at detecting principal families of chemicals found in plants, such as alkaloids, terpenoids, phenolic compounds, and polysaccharides. The presence or absence of major classes of compounds was characterized by color reactions and/or precipitations according to the methods described by Daira et al., 2016 and Norman, 1966 [15] [16].

Total polyphenol determination

The total polyphenol content of the methanol extract of R. minor was determined via UV spectrophotometry via the method of colorimetry with 10% (v/v) Folin-Ciocalteu via the methods of Nickavar and Esbati, (2012) and Stagos et al., (2012) [17] [18]. The calibration curve for gallic acid was prepared by mixing 1 mL of gallic acid solution in methanol at concentrations ranging from 200 to 25 mg/mL with 5 mL Folin Ciocalteu (10%) reagent. After 10 minutes, 4 mL of the sodium carbonate solution (75 mg/mL) was added, and the mixture was allowed to stand for 30 minutes in the dark. Then, the absorbance was measured at 765 nm, starting with the blank, followed by the least concentrated standard, and finally, the sample was assayed. The total polyphenol content of the R. minor extracts was expressed in mg of gallic acid equivalents per g of extract (mg EGA/g of extract).

Total flavonoid determination

The total flavonoid content of the aqueous extract of R. minor was determined via UV spectrophotometry via the method of colorimetry with 2% (w/v) aluminum trichloride (AlCl₃) via the methods of Nickavar and Esbati, (2012) and El Atki et al., (2019) [17] [19]. The calibration curve for rutin was prepared by mixing 3 mL of rutin solution in methanol at concentrations ranging from 0.1 to 0.006 mg/ml with 3 ml of AlCl₃ solution. Then, 3 ml of the sample (at concentrations ranging from 0.1 to 0.05 mg/ml) was mixed with 3 ml of the AlCl₃ solution. After 40 minutes of incubation in the dark, the absorbance was measured at 415 nm, starting with the blank, followed by the least concentrated standard, and finally, the sample was assayed. The total flavonoid content of the R. minor aqueous extract was expressed in mg of rutin equivalents per g of extract (mg ER/g of extract).

DPPH free radical scavenging activity assays

The DPPH^• scavenging capacity was evaluated as follows, with slight modifications according to the methods of Ahmad et al., (2010) and Awika et al., (2003) [20] [21]: 3800 µl of 4.5% DPPH prepared in methanol was combined with 200 µl of various concentrations of R. minor extract and ascorbic acid standards (0.003125–1 mg/ml). The mixtures were vortexed and incubated in the dark for 30 minutes. The absorbance was subsequently measured at 517 nm using a UV spectrophotometer. The antioxidant activity, which expresses the ability to scavenge free radicals, was estimated by the percentage of DPPH dye discoloration in solution in methanol. It is given by the following formula:

𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 (%)=(Abscontrol -Abstest)/Abscontrol ×100

The 50% inhibitory concentration of free radicals, or the IC₅₀, was subsequently obtained using the equation of the linear regression curve resulting from the percentage of inhibition as a function of concentration. Extracts with an IC₅₀ < 30 µg/ml were considered to have strong antioxidant activity, 30 µg/ml < IC₅₀ < 100 µg/ml were considered to have moderate activity, and an IC₅₀ > 100 µg/ml was considered to be inactive according to the scale of Ahmad et al., (2010).

Antimalaria activity

The antiplasmodial activity was evaluated by the technique described by Bennett et al. (2004) [22]. It is based on the measurement of the fluorescence emitted by the complex formed by the parasite's ds-DNA and the fluorophore SYBR Green I introduced into the culture medium of P. falciparum FCM29, a strain resistant to chloroquine. A mixture of 200 µL, composed of 100 µL of suspension of red blood cells infected with the FCM29 strain of P. falciparum (1% of parasitized red blood cells and 2% of healthy red blood cells in RPMI 1640 medium supplemented with 20% human serum), 50 µL of the product to be tested diluted in series and 50 µL of medium not enriched in human serum, is distributed in a 96-well microplate. The microplate is then incubated in a microaerophilic atmosphere at 37°C for 72 h. After incubation, a volume of 50 µL of SYBR Green I solution (Microprobes, Oregon, United States), diluted 20 times in RPMI 1640 medium supplemented with human serum, is added to each well. The resulting solutions are then mixed and left to stand for 60 min, protected from light at 37 ° C. Fluorescence is read at two wavelengths λ₁ = 485 nm, excitation wavelength and λ₂ = 518 nm, emission wavelength (Biotek - FLX800 microplate reader). The fluorescence rate is considered to be proportional to the amount of respective P. falciparum contained in each well. The IC₅₀ values, the concentrations of drugs which reduce the fluorescence rate by 50%, and therefore the growth, are then deduced by calculation.

According to Bero et al., (2010), the in vitro antiplasmodial activity of a compound is considered very high when its IC₅₀ < 1 µg/mL. For a 2 µg/mL ≤ IC₅₀ ≤ 11 µg/mL, the compound is active. When 11 µg/mL ≤ IC₅₀ ≤ 50 µg/mL, it is considered weakly active. Compounds with an IC₅₀ > 50 µg/mL are considered inactive [23].

Statistical analysis

All the experiments were performed in triplicate, and the results are presented as the means ± standard deviations. The data were analyzed using ANOVA. Significant differences between mean values were determined by Tukey's HSD test at a significance level of p < 0.05.

Phytochemical screening investigation

The results of the phytochemical screening of the leaves of R. minor are shown in Table 1.

Table 1

The results of phytochemical screening
Chemical families	Characterization reagents	Results
Phenolic compounds
Coumarins	NaOH, U.V. lamp λ 254 nm and 366 nm	+++
Flavonols	HCl, Mg, Alcool isoamylique	++
Anthocyanins	HCl, NH₄OH	-
Leucoanthocyanes	Hot HCl	++
Tannins	NaCl, Gelatin	++
Condensed tannins	FeCl₃	+++
Polyphenols	Gélatine 1%, NaCl	++
Terpenoids
triterpenoid	Acetic anhydride, H₂SO₄	++
unsaturated sterols	H₂SO₄	+++
Lactonic steroids	Acide picrique, soude	+++
Iridoids	HCl, Ethanol, Glycérol, CuSO₄	-
Saponins	Hauteur mousse	+++
Cardenolides	H₃PO₄, Acide trichloracétique, U.V. lamp	+++
Deoxy-2-sugar heterosides	FeCl₃, Acetic acid	+++
Alkaloids
Alkaloids	KI, I₂, HgCl₂, Bi(NO₃)₃, Tartaric acid	-

The secondary metabolites detected in the leaves of R. minor include phenolic compounds such as flavonoids, coumarins, and tannins; terpenoids such as triterpenoid, unsaturated sterols, lactonic steroids, cardenolides and deoxy-2-sugar heterosides and saponins.

Total polyphenol content

Antioxidant activity is often associated with high levels of phenolic compound. Polyphenol concentrations were determined on the basis of the gallic acid calibration curve according to the equation y = 0.0019x + 0.0306 with R² = 0.9996. The analysis revealed that the methanol extract possessed the highest concentration of phenolic compound with a concentration at 280.8 ± 4.6 mg EGA/g significantly compared to the hexane extract, ethyl acetate extract and aqueous extract with a concentration at 17.6 ± 2.6, 167.7 ± 8.7 and 145.1 ± 8.1 mg EGA/g, respectively (p˂0.05). Where, the hexane extract has the lowest polyphenol content.

Total flavonoid content

The antioxidant capacity of the plant's leaves is also supported by the presence of flavonoids. Flavonoid concentration was determined on the basis of the rutin calibration curve according to the equation y = 0.3341x + 0.0533 with R² = 0.9965. Total flavonoid content revealed that the methanol extract had the highest flavonoid content with a concentration of 50.4 ± 2.3 mg ER/g, although it was not significantly different from the ethyl acetate extract at a concentration of 43.0 ± 4.8 mg ER/g (p > 0.05). Furthermore, the hexane extract had the lowest flavonoid content at 1.4 ± 0.2 mg ER/g, compared with all other extracts. In short, the aqueous extract had modarete flavonoid content with a concentration at 31.5 ± 5.3 mg ER/g and significantly different compared to the ethyl acetate, methanol, and hexane extracts (p˂0.05). Data concerning total phenolic and flavonoid contents for each extract are presented in Table 2.

Antioxydant activity

Table 2 also shows that methanol and ethyl acetate extracts have a 50% DPPH radical scavenging capacity at concentrations of 21.58 ± 0.05 µg/mL and 22.55 ± 0.96 µg/mL respectively, statistically confirmed as no significant difference (p > 0.05). The hexane and aqueous extracts showed lower activity, with concentrations of 50.38 ± 0.63 µg/mL and 30.35 ± 3.58 µg/mL, respectively. However, vitamin E remained the most active with a concentration of 16.4 ± 0.04 µg/mL.

These results suggest that the extracts may contain significant quantities of free radical scavengers, due to their relatively high inhibitory concentrations.

The percentage of 50% inhibition of the DPPH• radical caused by the extracts from R. minor shown in Fig. 1.

Antiplamodial activity

Leaf extracts were evaluated for their in vitro antiplasmodial activity against strain FCM29 of P. falciparum. Table 2 shows that the hexane and ethyl acetate extracts had moderate activity, with concentrations of 21.30 ± 0.04 and 24.43 ± 0.03 µg/mL, respectively (p < 0.05). Aqueous and methanol extracts showed low activity, with concentrations of 41.60 ± 0.03 and 50.75 ± 0.01 µg/mL respectively. However, this activity remains very low compared with the reference, quinine, which had a concentration of 0.0012 ± 0.0030 µg/mL.

Table 2

Contents of total phenolic compounds, total flavonoids, and antioxidant and antiplasmodial activities of plant extracts.
Extracts	TPC (mg EG/g)	TFC (mg ER/g)	Antioxydant activity IC₅₀ (µg/mL)	Antiplasmodial activity IC₅₀ (µg/mL)
Aqueous	145.10 ± 8.10^a	31.50 ± 5.30^a	30.35 ± 3.58^a	41.60 ± 0.03^a
Hexane	17.60 ± 2.60^b	1.40 ± 0.20^d	50.38 ± 0.63^c	24.43 ± 0.03^b
EtOAc	167.70 ± 8.70^c	43.00 ± 4.80^b	22.55 ± 0,96^a	21.30 ± 0.04^c
MeOH	280.80 ± 4.60^d	50.40 ± 2.30^c,b	21.58 ± 0.05^d	50.75 ± 0.01^d
Vitamin E	-	-	16.46 ± 0.04^b	-
Quinine	-	-	-	0.0012 ± 0.0030^e

The tests were performed in triplicate, and the results are expressed as the mean values ± standard deviations. Values followed by different letters in the column are significantly different according to Tukey's post hoc test (p < 0.05).

The aim of this research was to evaluate the biological activity of the leaves of Razafimandimbisonia minor, a plant traditionally used in the sub-humid regions of Madagascar.

Phytochemical screening revealed the presence of various chemical classes in the leaf extract of this plant, including polyphenols, terpenoids and saponins. These results confirm the chemical compound richness of the Rubiaceae family, known for the production of terpenes, flavonoids, iridoids and coumarins [24] [25]. Polyphenols, in particular, have been highlighted for their key role in biological activities, as have flavonoids, considered the most important class of polyphenols [9].

Quantification of polyphenols and flavonoids in the hexane, ethyl acetate, aqueous and methanol extracts showed the following concentrations: methanol extract > ethyl acetate extract > aqueous extract > hexane extract. These observations confirm the results of Djeridane et al. (2006), who indicate that polyphenols are more concentrated in polar solvents, which also contain phenolic compounds such as flavonoids, tannins, anthocyanins and leucoanthocyanins [26]. In addition, the total flavonoid content of leaf extracts varies according to maturity and stage of development [27]. This concentration also depends on the polarity of the solvents used in extract preparation [28].

The results obtained are consistent with the antioxidant activity of the methanol extract, which showed 50% DPPH radical scavenging efficiency with a concentration of 21.58 ± 0.05 µg/mL. This was followed by the ethyl acetate and aqueous extracts, with concentrations of 22.55 ± 0.96 and 30.35 ± 3.58 µg/mL respectively. The hexane extract, showed lower activity, with a concentration of 50.38 ± 0.63 µg/mL. According to Guettaf et al. (2016), antioxidant activity is often correlated with a high content of phenolic compound in extracts [10]. The absence of phenolic compound in hexane extract could explain its low capacity to transfer electrons to DPPH free radicals [27] [29]. Hamia and colleagues have also demonstrated that extraction solvents influence the content of total phenols and their antioxidant power [30]. Several species of the Rubiaceae family have also demonstrated antioxidant activity [31]. Phenolic compound are known for their antioxidant capacities and their ability to regulate the activity of certain enzymes. They also have cardioprotective, antiallergic, anti-inflammatory, antiulcer, antidiabetic, antibacterial and antiviral properties. [32] [33] [34].

The results for antiplasmodial activity are inversely related to antioxidant activity. For antioxidant activity, the most polar extracts are the most active, while for antiplasmodial activity, less polar extracts, such as hexane and ethyl acetate extracts, show moderate activity. This variation in activity could be attributed to the presence of active lipophilic constituents, which do not extract in polar solvents such as water, with non-polar extracts being more active than polar ones. Ethyl acetate extract also showed moderate activity, probably due to the presence of secondary metabolites active against malaria parasites. This variation in activity could be due to the presence of active lipophilic constituents that do not extract into polar solvents such as water, with non-polar extracts being more active than polar extracts [35] [36]. Ethyl acetate extract also showed moderate activity due to the presence of secondary metabolites that are active against malaria parasites.

Outher studies have demonstrated that certain species of the Rubiaceae family are excellent sources of antimalarial agents [24]. For example, quinine, the first known antimalarial drug, has been used to treat malaria for centurie. This alkaloid was isolated from the bark of the Cinchona tree, belonging to the Rubiaceae family [37]. Several plants in this family are used for ornamental and medicinal purposes to treat a variety of conditions, such as coughs, constipation, abdominal disorders, anemia, arthritis, dermatitis, chickenpox and malaria [38].

In this study, the quantification of phenolic and flavonoid contents indicated that leaf extracts from this plant had varying levels of these compound. Determination of antioxidant activity in the extracts led to the conclusion that the recurrent use of this plant would be linked to their relative richness in polyphenolic constituents. In addition, Razafimandimbisonia minor leaves demonstrated moderate antimalarial activity against the P. falciparum strain, mainly in the hexane and ethyl acetate extracts.

These results highlight the potential of Razafimandimbisonia minor in malagasy traditional pharmacopoeia, due to its rich antioxidant compounds and moderate antimalarial activity, justifying its ethnobotanical use to treat liver disorders, fatigue and fever.

DPPH: 2,2-diphenyl-1-picrylhydrazyl

EGA : Gallic acid equvalents

ER: Rutin equivalents

IC_{50 :}50% inhibition concentration

TFC : Tolal flavonoids compound

TPC : Total phenolic compound

UV: Ultraviolet

Ethics approval and consent to participate

Not applicable.

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Not applicable.

Availability of data and materials

Not applicable

Competing interests

The authors declare no competing interests.

Funding

Not Funding

Authors’ contributions

H.R. conceptualized the investigations; V.R.E., J.R., and A.R. prepared the antioxidant and antiplasmodial technique; F.R. developed TFC analysis methods. L.R., V.R.E., R.T.S., and R.D.R.R. assisted in validating the paper; and D.A.R. and L.R. carried out the investigation during the studies. A.R. conducted the statistical tests throughout the biological tests. H.R. wrote the initial draft preparation, and V.R.E. supervised the article. All the authors reviewed and approved the published version of the text.

Acknowledgment

We thank the National Centre for Applied Pharmaceutical Research (CNARP) for the realization of this work and we also thank Njakarinala Ranarivelo for the Folin-Ciocalteu reagent for the TPC analysis.

Author details

^aDepartment of Chemistry, National Centre for Applied Pharmaceutical Research (CNARP), BP 702, Antananarivo 101-Madagascar. ^bDepartment of Pharmacodynamics, National Centre for Applied Pharmaceutical Research (CNARP), BP 702, Antananarivo 101- Madagascar.^cLaboratory of Organic Chemistry, Faculty of Science, University of Antananarivo- Madagascar.

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No competing interests reported.

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Antioxidant and antiplasmodial activities of Razafimandimbisonia minor (Baill.) Kainul. & B. Bremer leaves (Rubiaceae)

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Abstract

Figures

Background

Methods

Plant material

Phytochemical screening

Total polyphenol determination

Total flavonoid determination

DPPH free radical scavenging activity assays

Antimalaria activity

Statistical analysis

Results

Phytochemical screening investigation

Total polyphenol content

Total flavonoid content

Antioxydant activity

Antiplamodial activity

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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