1 Introduction

The use of therapeutic plants is as old as human civilization. Man’s knowledge of his environment has greatly improved over time and hence, his living conditions as well. This folkloric knowledge is the foundation of modern ethnopharmacology that is transmitted from generation to generation. By the middle of the 19th century, three-quarter of all existing drugs were from plant origin (Raphael, 2011) and many pharmaceutical companies were already preparing crude therapeutic formulations (Estes, 1988). These therapeutic effects have been attributed to the many secondary metabolites synthetized by plants for multiple purposes including: protection, interaction with their environment and attraction of pollinators (Pagare et al., 2015). Secondary metabolites have increasingly attracted the interest of researchers, resulting in the invention of ultra-modern methods for isolating pure compounds from plants that can solve the health issues of humans (Salihu et al., 2020). To date, two-thirds of the world’s population rely on herbal medicine taken in diverse forms for therapeutic purposes. This is justified by their efficacy not only in providing lead molecules against multiple drug resistant (MDR) conditions, but also because they are less toxic and much more available compared to synthetic drugs (Nikaido, 2009). As part of our on-going search for new bioactive compounds that can counteract bacterial resistance, this study was aimed at investigating the antibacterial effect of the extract and isolates from the stem bark and sap of Staudtia kamerunensis Warb (Myristicaceae), a plant that grows in the Central region of Africa.

Staudtia kamerunensis Warb. (Myristicaceae), is an evergreen tree, medium sized to large, and can reach up to 40 m high. The stem has a diameter of 90–110 cm; it is branchless up to 25 m and cylindrical. The fruit is an ellipsoid drupe of 2–5 cm × 1.5–4.5 cm that usually occurs in clusters of up to 20. They are fleshy when ripe and possess a single ovoid seed which is dark brown with a red or pink aril as envelop and is lobed at the apex (Oyen & Louppe, 2012). These species are identified by many synonyms namely; Staudtia congensis, Staudtia niohue Pierre, Staudtia spititata and Staudtia kamerunensis that has one variant, Staudtia gabonensis, whose fruits are smaller than the later. It has been identified as the only species of the genus Staudtia (Oyen & Louppe, 2012). Different parts of the tree are believed to possess multiple curative properties; bark decoction is used to treat menstrual issues, diarrhoea, lung ailments and cough in Central African traditional medicine. In the Democratic Republic of Congo (DRC), bark decoction is used to treat coughs, skin problems, oedema, wounds and oral infections while bark sap is used to treat snakebites, irritated eyes, bleeding, wounds and diarrhoea. In Gabon, the sap is rubbed against the mouths of new-borns to help them with teething and the pulverized bark mixed with padouk wood powder (Pterocarpus soyauxii Taub.) is used to treat ulcers, especially yaws. Boiled wood chips are used in treating gonorrhoea and rheumatism while fresh twigs are crushed, salted, and eaten as an aphrodisiac in Congo (Oyen & Louppe, 2012). Previous chemical investigations carried out on this species and other species of the Myristicaceae family reported the isolation of lignans as one of the major components of the species. Noumbissie et al., 1992 isolated a diterpene known as staudtienic acid (Noumbissie et al., 1992) from the stem bark. The work of Yankep et al., 1999 on the seeds and the stem bark of the species yielded seven lignans and one glycerol trimyristate.

Microbial resistance remains a major concern to public health and search for new potent antimicrobial agent is crucial (Baker et al., 2022). The scarcity of biological investigations on this plant species, coupled to its usage for bacteria-related diseases in ethno-pharmacology prompted us to the chemical investigations of its stem bark and sap, to isolate the biologically active compounds that can serve as lead for new antimicrobial agents, and to justify its use in traditional pharmacopeia.

2 Material and methods

3 Instrumental

NMR analysis was carried out in CDCl₃ and DMSO‑d₆ solvents, on a magnet of 500 MHz coupled to an Avance III HD 500 MHz console. All spectra were referenced using the chemical shifts of the different solvents, both for ¹H and ¹³C. Tetramethylsilane (TMS) was used as the internal standard. A last generation of Quadrupole Time-of-Flight (Q-TOF) Mass Spectrometry system (Bruker Compact), was used for the analysis of the pure isolates, using HR-ESIMS positive mode as ionisation method.

4 Results and discussion

The phytochemical study of the ethyl acetate extract of the stem bark and sap of S. Kamerunensis, resulted in the isolation of six compounds namely; biochanin A (1), formononetin (2), 3-(1,3-Benzodioxol-5-yl)-5,6,7-trimethoxy-4H-1-benzopyran-4-one (3), and epicatechin (4) from the stem bark, (2α, 3β) Oleanan-12-ene-2,3-diol (5) and 2α,3β-dihydroxylup-20-ene (6) from the sap.

Compound 1, 2, 3, 4 have the characteristic C6-C3-C6 backbone and hence belong to the large class of flavonoids. Compounds belonging to the flavan-3-ol and other polyphenolic compounds are generally omnipresent in plant species, and are responsible for the colour observed in fruits, flowers, (Amalesh et al., 2011). They play an important role in the protection of plants against microbial aggression (Amalesh et al., 2011). To the best of our knowledge, this is the first report of flavonoids, from this plant species. Yankep and collaborators (1999) reported the isolation of lignans from the seed and stem bark of this species. From biosynthetic viewpoint, lignans and flavonoids both originates from the phenylpropanoid pathway, which helps us to understand the existence of both in the stem bark of this specie. In his review, Valderrama reports the isolation of many flavonoids among which (-) epicatechin from Myristica fragans seeds, biochanin A from the seed of Virola surinamensis (Martínez Valderrama, 2000). In this review, several isoflavonoids are reported with the methylene dioxy moiety attached to ring Zeng et al. (1994) isolated formononetin from the stem bark of Knema glomerata Merr. Compound 5 and 6 are two isomeric pentacyclic triterpenoids. The difference between the two compounds is on the fifth ring. In compound 6, the fifth ring is a five membered ring with an isopropenyl moiety attached at carbon C-19, while compound 5 has the normal six-membered ring of pentacyclic triterpenoids. These compounds were obtained from the same fraction as a mixture. A further purification of the mixture was carried out and compound 5 was obtained as pure entity. Compound 6 rather, was difficult to obtain pure. To the best of our knowledge, this is the first phytochemical investigation of the sap of species belonging to this genus and species of the family and the first report of this class of compounds from the genus. Fig. 1 depicts the chemical structures of the isolated compounds from the extract of the stem bark and sap of the tree.

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Fig. 1

Structures of compounds (1–6) isolated from the stem bark and sap of S. kamerunensis.

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Table 1 presents the MIC values of the crude extract and of the ethyl acetate partition (EASK), (-) epicatechin (4), oleanan-12-ene-2α,3β –diol, (5) and the mixture of oleanan-12-ene-2α,3β -diol and 2α,3β-dihydroxylup-20-ene (6), to study their synergistic effects. The crude extracts showed significant antibacterial activity against both Gram-positive and Gram-negative bacteria strains. The lowest MIC value of the crude extract was 15.625 µg/mL and was against Bacillus subtilis, Enterococcus faecalis among the Gram-positive, against Enterobacter cloacae, Proteus vulgaris, Klebsiella oxytoca, and Proteus mirabilis, among Gram-negative strains. This makes the crude extract 1.7 times more potent than Ampicillin against Bacillus subtilis, Enterococcus faecalis, Enterobacter cloacae, Klebsiella oxytoca, and Proteus mirabilis, 26.6 times more potent against Proteus vulgaris, and 4.1 times more potent against Pseudomonas aeruginosa. These results justify the uses of the stem bark of this plant species against many bacteria-related diseases (Cough, lung complaints, wounds etc.).

A comparative study was carried out between the structures of oleanan-12-ene-2α, 3β -diol and maslinic acid isolated by Kamdem et al., (2022). The only difference between the two structures is the presence of a carboxylic acid at position 17 of the maslinic acid. Maslinic acid displayed excellent and consistent antibacterial characteristic against the same strains that were used in this study. A structure activity study can lead us to conclude that the carboxylic acid enhanced the activity of maslinic acid. The activity of maslinic acid is 16 times enhanced against P. aeruginosa, P. mirabilis, E. coli, K. pneumonia among Gram-negative strains, against S. epidermidis, M. smegmatis among Gram-positive strains, in comparison with oleanan-12-ene-2α, 3β-diol. These findings gives us an insight in the mechanism of action of maslinic acid in killing bacteria: the reactive site being the carboxylic acid. Deeper studies are required to have a clear understanding of its way of action. Oleanan-12-ene-2α, 3β-diol showed better but almost similar activity to maslinic acid against Bacillus subtilis, Enterococcus faecalis and Klebsiella oxytoca. The difference may be attributed to the presence of the carboxylic acid, but further studies are needed for clarification.

The biological activity of compounds can improve when combined with others. This effect is known as synergistic activity. The synergistic effect of pentacyclic triterpenoids has been studied as a pathway to counteract the emergence of antibacterial resistance. Chung and collaborators studied the synergistic effect of combining three pentacyclic triterpenoids with existing antibacterial medicines. The study of the combination of α-amyrin and betulinic acid revealed the synergistic effect of these compounds against Staphyloccocus aureus with an FIC (fractional inhibitory concentration of individual compounds) of 0.5 or less. A combination of α-amyrin, betulinic acid and betulinaldehyde did not inhibit the growth of the bacterial cells at the tested concentrations thus suggesting lack of synergy (Chung et al., 2011). Oleanan-12-ene-2α, 3β -diol and 2α, 3β-dihydroxylup-20-ene, are very similar in their structure, except for their fifth ring. A synergistic activity was observed; the activity of Oleanan-12-ene-2α, 3β -diol was doubled especially against Gram-negative strains (P. vulgaris, P. mirabilis, E. coli, K. pneumonia) and against Mycobacterium smegmatis among Gram-positive. Its activity was increased 16 times against P. aerogenes. The activity is instead decreased against Bacillus subtilis. Further studies can be developed in the pathway of utilizing the combination of these tritepenoids from the sap of this species against microbial attacks, more specifically Gram-negative aggressions.

Epicatechin also portrayed excellent antimicrobial activity compared to the standard drugs (see Table 1). Masika et al. (2004) reported the antimicrobial activity of epicatechin against four strains namely (B. subtilis, S. aureus, E. coli, P. aeruginosa). The MIC reported for E. coli (62.5 µg/mL) was 4 times better than what is reported here. The compound presented weak activity compared to ours, against the other strains (B.S −250 µg/mL, S.A. – 250 µg/mL, P.A. 62.5 µg/mL). Esquezani and co-workers attributed the antimicrobial effect of fractions from coconut husks to the relatively high abundance of catechin and epicatechin against S. aureus bacteria strains and acyclovir- resistant Herpes simplex virus type 1 (HSV-1-ACVr) (Esquenazi et al., 2002). Cetin-Karaca and Newman tested epicathecin against three different strains of E. coli (FTJ, ATCC43895, ATCC 35150) at pH = 5.6, and found it active with MIC being more or less equal to 20.0, 20.0 and 15.0 (mg/L) respectively (Cetin-Karaca and Newman, 2015). Escandón and collaborators reported epicatechin active against Helicobacter pylori (Escandón et al., 2016). Epicatechin also demonstrated synergistic effects with theaflavin from black tea when tested against eight clinical isolates of Stenotrophomonas maltophilia and Acinetobacter baumannii (Betts et al., 2011). Theaflavin tested alone was very active (p $\le 0.05$ ), at concentration $\ge 3\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L}$ . When coupled with epicatechin at a ratio of 2:1 (Theaflavin: epicatechin), and a concentration level above 1 mg/mL, the effectiveness was significantly increased (p $\le 0.05$ ).

5 Conclusion

The chemical investigation of the stem bark and sap of S. kamerunensis led to the isolation of six compounds, three isoflavonoids and one flavonoid from the stem bark, and a mixture of two pentacyclic triterpenoids from the sap. This is the first report of the isolation of flavonoids and triterpernoids from the species as well as of the antimicrobial studies. The species is known to be a rich source of lignans as the other members of the Myristicaceae family. The ethyl acetate extract, epicatechin, Oleanan-12-ene-2α, 3β -diol and the mixture of triterpenoids have shown significant antibacterial activity against strains tested compared to standard drugs. This justifies the local use of this plant against bacteria-related infections, and can serve as a source for the development of potent antibacterial treatment.

Funding.

The Centre for Natural Product Research (CNPR), and Drug Discovery and Smart Molecules Research Laboratory are acknowledged for providing funding for this research project, as well as the National Research Foundation, South Africa (Grant numbers 116740).

CRediT authorship contribution statement

Jordan L. Tonga: Investigation, Conceptualization, Writing – original draft. Michael H.K. Kamdem: Investigation, Formal analysis, Writing – review & editing. Julio I. M. Pagna: Supervision, Formal analysis. Thierry Y. Fonkui: . Charlotte M. Tata: Supervision, Writing – review & editing. Marthe C.D. Fotsing: Formal analysis, Writing – review & editing. Ephrem A. Nkengfack: Project administration, Supervision, Writing – review & editing. Edwin M. Mmutlane: Supervision, Writing – review & editing. Derek T. Ndinteh: Supervision, Writing – review & editing.