Chemotaxonomy and Antibacterial Activity of the Extracts and Chemical Constituents of Psychotria succulenta Hiern. (Rubiaceae)

The use of natural products for medicinal purposes is becoming more and more common nowadays, as evidenced by the presence in plants of secondary metabolites with different potentials such as antioxidant and antibacterial properties. We evaluated in this work the antimicrobial activities of the extracts and some isolated compounds from the seeds of Psychotria succulenta Hiern. (Rubiaceae), a Cameroonian medicinal plant traditionally used to cure microbial infections. The ethanol extract was prepared by maceration and extracted with ethyl acetate and n-butanol. The EtOAc (m = 168 g) and n-BuOH (m = 20 g) extracts were further fractionated by silica gel column chromatography to isolation of compounds. Their structures were elucidated by spectroscopic analysis and by comparison with published data. The antibacterial activity of extracts and compounds was assessed by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against pathogenic bacteria. Thirteen compounds including four alkaloids (veprisine (1), naucleofficine III (2), vepridimerine B (3), and vepridimerine C (4)), three triterpenes (barbinervic acid (5), 3-O-α-L-rhamnopyranosyl quinovic acid (6), and oleanolic acid (7)), one steroid (β-sitosterol-3-O-β-D-glucopyranoside (8)), four phenolic compounds (scopoletin (9), gallic acid (10), quercetin-3-O-β-D-glucopyranoside (11), and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12)), and one iridoid (borreriagenin (13)) were isolated from the EtOAc and n-BuOH extracts. These compounds were identified by 1D and 2D NMR combined analysis as well as by melting point comparison. The EtOH, EtOAc, and n-BuOH extracts exhibited significant antibacterial activities (MIC = 32‐128 μg/mL; MBC = 64‐256 μg/mL) against Staphylococcus aureus (Gram-positive bacterium), Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia (Gram-negative bacteria). Among the isolated compounds, scopoletin (9) showed a moderate activity against Klebsiella pneumoniae with MIC and MBC values of 16 μg/mL and 32 μg/mL, respectively. It appears that, chemotaxonomically, some of the isolated compounds have already been obtained from the genus Psychotria but to the best of our knowledge, this is the first report on the phytochemical investigation of P. succulenta. Although many other studies need to be achieved, our results support the use of P. succulenta in traditional medicine to cure infectious diseases particularly those caused by the tested bacteria.


Introduction
Infectious diseases caused by bacteria, viruses, fungi, and other parasites continue to cause enormous damage in the world. Gram-negative or Gram-positive bacteria are able to acquire resistance mechanisms to face environmental aggression (natural environment, competing bacteria, host defense, or antibiotics) either by the modification of sites of action of anti-infective molecules or by production of degradative enzymes. It is therefore important to develop new drugs using natural plants to fight antibiotic resistance and to limit undesirable side effects. Psychotria succulenta Hiern. (Rubiaceae) is a shrub of varying size between 1 and 2 m with yellow fruits of ripeness commonly found in most tropical regions [1]. Plants of the genus Psychotria (leaves, roots, barks, and rhizomes) are commonly used in traditional medicine for treating bronchial and gastrointestinal disorders [2]. They are also used for curing infections of female reproductive system [3].
To the best of our knowledge, no phytochemical nor pharmacological works have been achieved on P. succulenta. Previous works carried out on Psychotria species have shown that the different extracts (petroleum ether, chloroform, ethyl acetate, dichloromethane, ethanol, and methanol extracts), the fractions, and some isolated compounds exhibited interesting biological activities such as antibacterial, cytotoxic, antioxidant, antimycobacterial, and antimutagenic properties [4][5][6][7]. Plants of this genus are characterized as an abundant source of indole, monoterpene indole, quinoline, and isoquinoline alkaloids as well as flavonoids [8]. In the course of our search of bioactive compounds from some medicinal plants growing in Cameroon [9,10], we undertook the phytochemical study of P. succulenta, leading to the isolation and structure elucidation of thirteen compounds. Furthermore, the crude EtOH extract, the EtOAc and n-BuOH extracts as well as some of the isolated secondary metabolites were evaluated for their antibacterial activity, and the results are also presented.

Extraction
Procedure. The seeds of P. succulenta were dried at room temperature and then crushed in fine powder to give 2.8 kg. Two kilograms (2 kg) of this powder was extracted with ethanol (3 × 12 L) for 72 hours to yield 287.2 g of crude ethanol extract after evaporation of the solvent under reduced pressure. A part of this crude extract (250 g) was suspended in distilled water (600 mL), then extracted with EtOAc and n-BuOH, respectively. After evaporation of each solvent under reduced pressure, 168 g and 20 g of EtOAc and n-BuOH extracts were obtained, respectively.
Veprisine (1), vepridimerine B (3), and vepridimerine C (4) are quinolone-terpene alkaloids occurring mainly in plants of the Rutaceae family [12,14,32,33]. To the best of our knowledge, this is the first report of their isolation from Rubiaceae. Nevertheless, the cooccurrence of indole and quinoline alkaloids in the same plant species is well documented. These two classes of alkaloids were obtained from Araliopsis soyauxii (Rutaceae) [33], Melodinus yunnanensis (Apocynaceae) [34], Alstonia scholaris (Apocynaceae) [35], and Clausena lansium (Rutaceae) [36]. This further confirmed the fact that biosynthetically, quinoline alkaloids may be derived from ring expansion of indole alkaloids [35]. This seems to be the first report on the isolation of the flavonoid glycosides quercetin-3-O-β-glucopyranoside (11) and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-Lrhamnopyranoside (12) from a plant of the Psychotria genus although these secondary metabolites have already been found in the family Rubiaceae, precisely in Hedyotis diffusa and Hedyotis verticillata, respectively [31]. The iridoid borreriagenin (13) obtained during this work has already been isolated from some plants of the Rubiaceae family including Borreria verticillata [21] and Morinda longifolia [37], but to the best of our knowledge, this is the first report of its isolation from a plant of the genus Psychotria. The findings of the present study showed differences between the antibacterial activities of extracts from P. succulenta seeds. This suggests that P. succulenta contains several active principles with different polarities as shown by the nature of the isolated compounds. Indeed, the antibacterial activities of medicinal plants are correlated with the presence in their extracts of one or more bioactive secondary metabolites [38]. The n-BuOH extract was the most active following in decreasing order by the EtOAc extract and MeOH extract. This result reinforces the concept that P. succulenta contains also polar antibacterial compounds. These differences in antibacterial activities from different solvents had also been observed [9,11]. Hence, the n-BuOH extract was expected to produce significant active principles in this research. However, the results showed that ethyl acetate was the better solvent compared to the n-BuOH to isolate the phytochemicals (compounds 2, 5, and 9) that are most active toward the tested bacteria from P. succulenta. The ethyl acetate is a semipolar solvent and could effectively extract active principles with semipolar properties such as alkaloids, sterols, terpenoids, flavonoids, and glycosides from the plant [39]. Different parts (leaves, roots, barks, and rhizomes) of plants of the genus Psychotria are commonly used in traditional medicines for treating bronchial and gastrointestinal disorders such as cough, bronchitis, ulcer, and stomachache [2,40]. They are also used to cure infections of the female reproductive system [3]. Previous pharmacological works carried out on other Psychotria species like P. microlabastra (leaves, stem, and roots bark), P. gardineri (branches and leaves), and P. nigra (branches and leaves) have shown that methanol, dichloromethane, and hexane extracts exhibit antibacterial activities [41]. Our results allow us not only to validate the use of P. succulenta in traditional medicine but also to approve the literature data.
The findings of the present study showed that the MBC values are in general fourfold lesser than the MIC values on the corresponding bacteria; suggesting that the extracts and some isolated compounds from P. succulenta seeds have a bactericidal effect on the sensitive bacteria [42].
The results of the antibacterial activity of some isolated compound from P. succulenta seeds are in agreement with those of the literature. Indeed, veprisine isolated from the root wood of Teclea maniensis (Rutaceae) exhibited moderate to higher antimycobacterial activity against two mycobacterial strains, namely, Mycobacterium madagascariense DSM 44641 and Mycobacterium indicus pranii DSM 45239 with the MIC values of 657.9 μM and 2:63 × 10 3 μM, respectively [12]. Oleanolic acid isolated from Miconia species displayed antibacterial effect with MIC values ranging from 30 μg/mL to 70 μg/mL [43]. A phenolic coumarin scopoletin (7-hydroxy-6-methoxycoumarin) from Lasianthus lucidus Blume (Rubiaceae) proved to be effective against Pseudomonas aeruginosa ATCC 27853 (AmpC β-lactamase producing strain) and P. aeruginosa DMSC 37166 [44]. It was found that gallic acid had antimicrobial activity against P. aeruginosa, E. coli, S. aureus, and Lysteria monocytogenes through hydrophobicity changes, decrease of negative surface charge, and occurrence of local rupture or pore formation in the cell membranes with consequent leakage of essential intracellular constituents [45]. Finally, 10-acetyl borreriagenin isolated from the aerial parts of Hedyotis pilulifera (Rubiaceae) showed antibacterial activity against Staphylococcus aureus, with an MIC value of 100 μg/mL [46]. To the best of our knowledge, this is the first report on the antibacterial activities of the extracts, naucleofficine III, vepridimerine B, vepridimerine C, barbinervic acid, 3-O-α-Lrhamnopyranosyl quinovic acid, quercetin-3-O-β-D-glucopyranoside, kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside, and borreriagenin from P. succulenta seeds. The overall study emphasizes the potential of P. succulenta seeds as a sustainable source of broad spectrum antibacterial agents.