Gnetum africanum and Gnetum buchholzianum are found in the wild in the humid tropical forests of Nigeria, Cameroon, Central African Republic, Gabon, Democratic Republic of Congo and Angola [1] . In Cameroon, Gnetum africanum and Gnetum buchholzianum are found mainly in the Center, Littoral, South and South-West regions and are widely used for food needs [2] . Gnetum spp is known for its medicinal properties. In Nigeria, for example, the leaves known locally as “afang” are used for the treatment of spleen, angina and cathartic. In the Democratic Republic of Congo where they are known under the name of “fumbwa”, they are used to fight against nausea and an antidote against certain poisons, the leaves are used to fight against warts, hemorrhoids and boils. In Mozambique, this medicinal plant is used to fight against constipation because of its high fiber content [3] . It also softens the pain of contractions and therefore facilitates childbirth [4] . An antidiarheic activity of extracts from these leaves has been reported [5] . In Cameroon, the leaves are known as “okok” or “ekoke” or “eru”. The predominant use of which is food, and they are associated with peanuts in “okok” or with Talinum triangulare leaves for cooking “eru”. In south-eastern Nigeria and in the far north of Cameroon, they are eaten as salad [3] . Although, traditional medicine has reported some therapeutic uses of Gnetum spp. scientific evidence on their therapeutic effects and optimal conditions for extracting their bioactive substances is yet to be produced [6] . A study conducted showed that the methanol extracts of the leaves of Gnetum africanum and Gnetum buchholzanium are richer in secondary metabolites than hexane and ethyl acetate extracts after qualitative chemical screening [7] . Results from this work have shown that the therapeutic dose which restored the glycemia in the rats rendered diabetic was 200 mg/ml. This dose was found to have no toxic effect on the rats. This same dose was administered to the rats and rendered diarrhea thus restoring their digestive system. Several researchers have evaluated the chemical composition of Gnetum ssp. Which grow in different geographic areas and various species of the genus Gnetum. The isolation and elucidation of the structure of three phenolic compounds, namely 3,4-dimethoxychlorogenic acid (1), resveratrol (2) and 3-methoxyresveratrol (3) from the stem bark of Melinjo (Gnetum gnemon) have been made. Isolation of these compounds was performed by chromatography and structural elucidation was performed by interpretation of spectroscopic data including UV, IR, 1H and 13C 1D and 2D NMR, and FABMS. The results of this study also showed the activity of each compound as a radical hydroxyl scavenger of 3,4-dimethoxychlorogenic acid (3), resveratrol (2) and 3-methoxyresveratrol (3), with a IC50 of 523.7; 45.17; and 60.12; g/ml respectively [8] . From what have been mentioned above, Gnetum africanum and Gnetum buchholzianum, two species of the genius Gnetum generally encountered in Central Africa including Cameroon have not been the subjects of profound phytochemical studies. From which arose the aim of our research.

The analysis of the chromatographic profiles of the hydroethanolic extracts and fractions obtained with solvents of increasing polarity and as well as the quantification of phenolic compounds made it possible to highlight 15 chemical compounds and the statistically significant proportions of these compounds. It emerges from this analysis that the stilbenes are the family of the majority compounds (7/15) followed by the flavonoids (4/15). Phenols (3/15) and a known compound catechin (epigallocatchechin). The stilbenes are the most famous family of compounds of the genus Gnetum. These results obtained corroborate with those found by Fadi et al. (2011) [3] on cultivars of Gnetum gnemon revealed 15 compounds of the stilbenes family. Ali et al. (2011) [3] also obtained 9 stilbenes in the spice africanum. All the compounds detected by HPLC-MS in the hydroethanolic extract, as well as the fractions obtained were not identified. This is explained by the identification methods taken from the literature which have made it possible to identify compounds of the stibene family such as Gnetupendin D (1), Isorhaponcitin (4), Gnetuhaidin P (1'), Gnetupendin D (3'), Gnetuhainin C (4') [11] compared to usual spectroscopic methods (NMR, COZY, DEPT…). There is a low structural homogeneity between the chemical compounds of the two hydroethanolic extracts and between the different fractions obtained on the other hand. Common compounds are Gnetupendin D (1) and Homovanillyl alcohol (6) for both species and Gnetupendin D (3') for FAb. This homogeneity can be explained by the fact that these plants are from the same vegetable kingdom. The difference on the other hand, could be explained by the fact that these plants were harvested in two different agro-ecological areas and each compound is eluted in the solvent where it has the affinity. The contents of total flavonoids and polyphenols are respectively 29.870 ± 2.485 - 220.200 ± 35.500 (mg EQ/g extract) for the ethanolic extract and dicholoromethane fraction of Gnetum africanum welv. (EBa and DFa) and 2.233 ± 0.251 - 335.300 ± 57.810 (mg EQ/g extract) for the hydroethanolic extract and dicholoromethane fraction of Gnetum buchholzianum Engl. (EBb and DFb). On the other hand, the total polyphenol contents are between 6.993 ± 0.046 - 14.520 ± 0.557 (mg EQ/g extract) for the hydroethanolic extract and methanol fraction of Gnetum africanum Welv. (EBa and MFa) and 4.560 ± 0.010 - 16.250 ± 1.704 (mg EQ/g extract) for the hydroethanolic extract and dicholoromethan fraction of Gnetum buchholzianum engl. (EBb and DFb). The work carried out by Beack et al., [13] showed the high levels of total polyphenols in the fresh leaves of G. africanum and G. buchholzianum respectively of 507.17 ± 21.55 and 406.37 ± 3.09 (mg/100g DM). This would show that the levels of phenolic compounds decrease during the preparation of the alcoholic extracts. This difference in concentration could also be explained by the fact that various solvents of varying polarity were used. Thus, secondary metabolites are eluted differently depending on the solvent for which they have more affinity, which influences their concentration. In summary, the results of this study revealed that these two spices content phenolic compounds, flavonoids, and stilbenes and demonstrated the quantification of the phenolic compounds. These could be used to fight against multi-resistant infectious agents. They can also be used as natural sources of antioxidants.

At the end of our investigation, which focused on the qualitative and quantitative evaluation of phenolic compounds of hydroethanolic extracts and fractions obtained from the leaves of Gnetum africanum and Gnetum buchholzianum (Gnetaceae), we can conclude that 15 phenolic compounds divided into three families such as flavonoids, stilbenes and phenols have been characterized. The dichloromethane fractions showed significantly the total flavonoid contents removed from both extracts. In contrast, the total polyphenols content was high in the hydroethanol extract of Gnetum buchholzianum. These bioactive compounds identified and total phenolics compounds quantified can be exploited in pharmacological and pharmacokinetical activities to fight against multi-resistant infectious agents and as well as oxidative stress and antiinflammatory diseases.

The authors thank the phytochemistry and natural pharmaceutical substances laboratory of the Faculty of Medicine and Pharmaceutical Science of the University of Douala for the providing equipment for obtaining hydroethanolic extracts, the chemistry research unit of the Faculty of Sciences of the same University for the fractionation of hydroethanolic extracts and the Higher Normal School of the University of Yaounde I, for the analytical study of hydroethanolic extracts and fractions obtained using an HPLC/MS type device of the Ultimate 3000 brand ( Thermo Fisher, USA). The first author is grateful to the Ministry of Higher Education for the mobility fellowship.

The data are available upon request

The authors declared that there are no conflicts of interest.

Bertin Sone Enone, Sandrine Sikadeu and Celestine Magne carried out the experiments. Lahngong Methodius Shinyuy and Wilfred Ndogmo Tekapi involved in chemicals analysis. Valdi Steve Djova involved quantitive phenolic compounds analysis. Jean-Pierre Ngene, Charles Christian Ngoule, Jean Baptiste Hzounda, Juliette Koube and Hans Denis Bamal performed methodology and chromatographic profils analysis, Simone Véronique Fannang, Moïse Henri Julien Nko’o for chemical structure designed. Loé Gisèle Etame, Josiane Etang and Albert Mouelle Sone designed the study. All the authors were involved in the draft and review of the manuscript.

Enone, B.S., Etame-Loe, G.M.-M., Ngoule, C.C., Ngene, J.-P., Koube, J., Shinyuy, L.M., Fannang, S.V., Nko’o, M.H.J., Fokou, J.B.H., Bamal, H.D., Magne, C., Djova, V.S., Tekapi, W.N., Sikadeu, S., Etang, J. and Sone, A.M. (2022) Characterization and Quantification of Phenolic Compounds of Hydroethanolic Extracts and Fractions of Leaves Gnetum africanum (Welv.) and Gnetum buchholzianum (Engl.) (Gnetaceae). Open Journal of Applied Sciences, 12, 1304-1318. https://doi.org/10.4236/ojapps.2022.127090

1) Chemicals structures of components identified in EBa

1: Gnetupendin D; 2: Dimethoxygnetulin; 3: Methoxyparvifolol D; 4: Isorhaponcitin;

5: Dimethoxydihydropyrane riodictyol; 6: Alcool Homovanillyl; 7: Dimethoxyeriodictyol

2) Chemicals structures of components identified in EBb

1': Gnetuhaidin P; 2': Dimethoxydihydropyraneriodictyol; 3': Gnetupendin D; 4': Gnetuhainin C; 5': Trimethoxyluteolin; 6': Epigallocathechin; 7': Alcool Erythro guaiacylglycerol-β-O-4’-coniferyl; 8': Alcool Homovanillyl.