Plant material collection and identification

Fresh leaves of C. ambrosioides were collected in Bukavu city, located in the eastern part of the Democratic Republic of Congo, between April and October 2019. Plant materials were identified and authenticated by the Department of Biology of “Centre de Recherche en Sciences Naturelles CRSN/Lwiro”, and voucher specimens deposed under number LWI563359346.

Preparation of leaf the methanol extract

The leaves were air-dried at room temperature and then manually grounded to fine powders (Mowla, Alauddin, Rahman, Ahmed, & K, 2009; Tafesse, Hymete, Mekonnen, & Tadesse, 2017). According to this protocol, the leaf powder (1.144 kg) was repeatedly extracted with the methanol in an Erlenmeyer flask by occasional shaking and stirring. The different obtained extracts were concentrated on a rotary evaporator (at 40-50°C) to obtain the crude quote (232.99 gr: yield 20.4%).

Fractionation of C ambrosioides methanol extract using Vacuum Liquid Chromatography

The methanol extract was subjected to vacuum liquid chromatography (VLC) on silica gel using the n-hexane, n-hexane-dichloromethane (1:1), dichloromethane, dichloromethane-methanol (1:1), methanol, and methanol-water (9:1) as the mobile phases, respectively. These sub-fractions were freed of solvents on rotavapor and further dried in the fuming hood for one week before submitting pharmacological studies.

Identification of phytochemicals by GC-FID and GC-MS

Gas Chromatography equipped with flame ionization detector (FID), capillary column SPB-5 was used. The experimental mass spectra of the volatile compounds were compared with the electronic mass spectral data reported in the literature (NIST database) for the identification of compounds (Khan et al., 2016; Wang et al., 2018). ChemDraw Ultra 8.0 software was used for drawing materials' structures.

Alpha-glucosidase Inhibition Assay

The enzyme inhibition assay is based on the breakdown of the substrate to produce a colored product, followed by measuring the absorbance (Kurihara, Sasaki, & Hatano, 1994).

Determination of DPPH Radical Scavenging Activity

The free radical scavenging activity was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH) using the method described by Gulcin et al. (Gülçin, Alici, & Cesur, 2005).

Figure 1

Typical chromatogram of chemical compounds present in the n-hexane fraction of C.ambrosioides

Phytochemical identification

Figure 1 indicates a typical chromatogram of chemical compounds present in the n-hexane fraction of C. ambrosioides. However, Figure 4; Figure 3; Figure 2 shows the structures of all compounds identified in the plant. A total of 58 phytoconstituents were identified by GC and GC–MS analysis (Table 1).

Figure 2

Phytoconstituents (1-20) identified in the n-hexane fraction from C. ambrosioides methanol extract

Identified compounds were grouped in 11 classes of substances, including aliphatic hydrocarbons (35.54%), diterpenes (20.94%), esters (15.17%), triterpenes (11.07%), bromine-containing (7.05%), diverse functional groups (3.76%), aromatic hydrocarbons (2.45%), sesquiterpenes (2.15%), alcohols (1.31%), ketones (0.27%), monoterpenes (0.15%), and fatty acids (0.13%). The main compounds were heptacosane (30.48%), phytol (20.94), and squalene (11.07%).

Cyclohexanol, 2,4-dimethyl- (1), (Z)-7-hexadecene (2), tetradecane (3), caryophyllene (4), (Z)-β-farnesene (5), α-caryophyllene (6), pentadecane (7); 3,4,4-trimethyl-3-(3-oxo-but-1-enyl)-bicyclo[4.1.0]heptan-2-one (8), germacrene D (9), cadina-1(10),4-diene (10), benzene, (1-propylheptadecyl)- (11), dihydroactinidiolide (12), benzene, (1-ethyloctyl)- (13), (E)-5-octadecene (14), nonadecane (15), benzene, (1-methylnonadecyl)- (16), benzene, (1-pentylhexyl)- (17), benzene, (1-butylheptyl)- (18), benzene, (1-propyloctyl)- (19), benzene, (1-ethylnonyl)- (20), 1-decanol, 2-hexyl- (21), heptadecane (22), tetradecane, 2,6,10-trimethyl- (23), benzene, (1-methyldecyl)- (24), methyl tetradecanoate (25), cyclohexane, 1,1,3-trimethyl-2-(3-methylpentyl)- (26), benzene, (1-pentylheptyl)- (27), β-Guaiene (26), benzene, (1-propylnonyl)- (29), benzene, (1-ethyldecyl)- (30), 1-nonadecene (31), dodecane, 2-phenyl- (32), isopropyl myristate (33), benzene, (1-pentyloctyl)- (34), phytol (35), 2-pentadecanone, 6,10,14-trimethyl- (36), (7a-isopropenyl-4,5-dimethyloctahydroinden-4-yl)methanol (37), (Z)-7-hexadecenoic acid, methyl ester (38), hexadecanoic acid, methyl ester (39), hexadecanoic acid, ethyl ester (40), heneicosane (41), 9,12-octadecadienoic acid, methyl ester (42), (Z)-9-octadecenoic acid, methyl ester (43), octadecanoic acid, methyl ester (44), octadecanoic acid, ethyl ester (45), heptacosane (46) ,

Figure 3

Phytoconstituents (21-41) identified in the n-hexane fraction from C. ambrosioides methanol extract

eicosanoic acid, methyl ester (47), 7-methyl-Z-tetradecen-1-ol acetate (48), trans-13-Octadecenoic acid (49), 17-octadecynoic acid, methyl ester (50), (12-Methyl-E,E-2,13-octadecadien-1-ol (51), oleic acid (52), ethyl iso-allocholate (53), oleic acid, 3-(octadecyloxy)propyl ester (54), squalene (55), ethanol, 2-(octadecyloxy)- (56), Z-(13,14-epoxy)tetradec-11-en-1-ol acetate (57), and ethanol, 2-(9-octadecenyloxy)-, (Z)- (58).

Previous studies have reported some compounds extracted from the leaves, mainly in pentane and essential oil. In this present study, we report the chemical composition of the n-hexane fraction of methanolic extract of leaves, showing 58 phytochemicals belonging to 11 classes of substances. Those compounds include α-guaiene (Sagrero-Nieves & Bartley, 1995), α-caryophyllene and caryophyllene (Gbolade, Tira-Picos, & Nogueria, 2010; Gillij, Gleiser, & Zygadlo, 2008; Jaramillo, Duarte, & Delgado, 2012), squalene (Reyes-Becerril, Angulo, Sanchez, Vázquez-Martínez, & López, 2019), phytol (Jaramillo et al., 2012), dihydroactinidiolide (Reyes-Becerril et al., 2019); 3,7,11,15-tetramethyl-2-hexadecen-1-ol and 1-nonadecene (Mostafa et al., 2016); and 9,12-octadecadienoic acid, methyl ester (Reyes-Becerril et al., 2019). Essential oils are a complex mixture of volatile plant compounds composed of terpenoids (mainly monoterpenes and sesquiterpenes) and phenolic compounds. The essential oil's chemical composition is highly variable from plant to plant, even in the same species, related to different factors (abiotic, biotic, methods of extraction, conservation, and postharvest conditions) (Mkaddem et al., 2022). Although the n-hexane fraction of the methanolic extract is far from essential oil, these results show the presence of a good number of terpene compounds.

Figure 4

Phytoconstituents (42-58) identified in the n-hexane fraction from C. ambrosioides methanol extract

On the other hand, our study showed a few phytoconstituents close to those identified by other authors. For example, germacrene D, hexadecanoic, and octadecanoic acids were identified in our sample with their methyl and ethyl esters. Germacrene D-4-ol (Gillij et al., 2008), hexadecanoic acid (Pino, Marbot, & Real, 2003), and octadecanoic acid (Shah & Khan, 2017) without their esters were identified in essential and the methanol (ethyl acetate) extract. In the line of our results, tetradecane, caryophyllene oxide, hexadecanoic acid, caryophyllene, germacrene D, 9, 12-octadecadienoic acid, methyl ester, oleic acid, phytol, tetradecane, squalene, heneicosane, and methyl derivatives have been identified by GC-MS analysis in the n-hexane fraction/extract of different plant species (Godwin, Akinpelu, Makinde, Aderogba, & Oyedapo, 2015; Govindarajan et al., 2016; Ivanov et al., 2018; Nadaf, Nasrabadi, & Halimi, 2012). It has been observed that in the n-hexane fraction or extract of different plants, there is a remarkable variability of compounds, particularly the methyl esters. According to the literature, the methyl esters are possible artifacts due to the extraction with methanol (Venditti, 2018).

Forty-six out of sixty-one phytoconstituents are reported for the first time by the plant. Based on literature data, approximately 330 compounds (including their isomers) have been identified in different extracts, fractions of C. ambrosioides, and the majority (59.54%) mainly in essential oil (Kasali et al., 2021). However, contrary to our results, a chemical investigation of the n-hexane extract from Brazilian C. ambrosioides showed the presence of seven monoterpenes, include α-terpinene, p-cymene, benzyl alcohol (Z)-ascaridole, carvacrol, and (E)-ascaridole (Jardim, Jham, Dhingra, & Freire, 2010).

In vitro pharmacological evaluations

Table 2 reports the in vitro antidiabetic (α-glucosidase) and antioxidant investigations of the leaf methanol extract and its fractions.

The methanol extract and fractions demonstrated in vitro antidiabetic property by inhibiting α-glucosidase activity. According to their IC₅₀ values, dichloromethane-methanol bit was the most effective (20.4± 0.72 µM), followed by the methanol fraction (22.2± 0.93 µM), the dichloromethane fraction (23.7± 0.84 µM), and the n-hexane-dichloromethane fraction (25.4± 0.82 µM).

On the other vein, fractions F5 (methanol) and F6 (methanol-water) showed the best antioxidant potential than crude extract and different fractions. Their IC₅₀ values of 44.9 ± 0.07 and 48.8 ± 0.04 (µM), respectively, were close to the IC₅₀ value of the standard drug (BHA).

According to our results (Table 2), all compounds showed antidiabetic potential, and according to the classification of the sample based on IC₅₀ or CC₅₀ (Indrayanto, Putra, & Suhud, 2021), they possess moderate activity. Nevertheless, the most potent fractions are located in the polarity range between the methanol and dichloromethane fractions. Several phytoconstituents can exist in that range of polarity, including steroids, glycosides, alkaloids, anthraquinones, tannins, flavonoids, phenolic acids, peptides, polysaccharides, etc. However, as natural α-glucosidase inhibitors, flavonoids, alkaloids, terpenoids, steroids, quinines, phenylpropanoids, anthocyanins, tannins, phenolics, curcuminoids, miscellaneous, are the most found (Kumar, Narwal, Kumar, & Prakash, 2011; Yin, Zhang, Feng, Zhang, & Kang, 2014). Moreover, previous studies reported the inhibition effect of either the dichloromethane extract or fraction on α-glucosidase (Ferulago bracteata, Croton bonplandianum, Rhizophora apiculata, etc.). With IC₅₀ of 3.9 ± 0.71 (µM), 1-deoxynojirimycin presented enzyme inhibition 9.2 times greater than the methanol extract and 5.2 times methanol-dichloromethane fraction. For example, similar to our results, the methanol extract of Ceiba pentandra inhibited 87.79% of α-glucosidase. However, acarbose (Drug standard) inhibited 10 times potent than that of the methanol extract (Nguelefack et al., 2020).

lso reported the intense antioxidant activity of the methanol and the methanol-water fractions of C. ambrosioides close to the standard drug (BHA). There is a high probability of finding flavonoids and their glucosides in these fractions. It is known that the polyphenolic compounds include flavonoids, are suitably extracted in hydroalcoholic solutions (Luna, Ramírez-Garza, & Saldívar, 2020). The best-described pharmacological potential of flavonoids is their antioxidant capacity, depending on functional groups' arrangement about the nuclear structure. Scavenging reactive oxygen species, upregulation or protection of antioxidant defenses, and suppressing their formation through enzyme inhibition and chelation of trace elements involved in a free radical generation are the primary antioxidant mechanisms of natural flavonoids (Kumar & Pandey, 2013).