Introduction

Medicinal plants are an important therapeutic aid for various ailments. Ethiopia and other developing nations in Asia and Africa continue to rely largely on medicinal plants in their daily lives. 80% of the population in poor nations gets their primary healthcare from traditional medicine, with around 80% of these medications being plant extracts, according to a World Health Organization (WHO) report1. It is commonly known that the majority of developing nations utilize traditional medicine and medicinal herbs as the normative basis for maintaining good health. In addition, modern civilizations’ usage of medicinal plants has centered on their extraction for use in cosmetics and the production of various medications, including chemotherapeutics from these plants as well as from traditionally utilized natural remedies in rural areas2,3.

Cordia is a genus of flowering plants that belongs to the family Boraginaceae. It is one of the most representative members of this family. Cordia is a tree or shrub, sometimes subscandent in the borage family. The 300 species belonging to the genus Cordia are found in a broad range across tropical and subtropical areas globally, making them an important and diverse group of plants4. The genus Cordia species are used as a febrifuge and to treat wounds, tumors, gout, ulcers, and boils. The plant parts, like fruits, leave, stem bark, seeds, and roots, of most species of this genus have been used in traditional medicine5.

Cordia africana, known as “Wanza” in Amharic, is a species of flowering tree, the evergreen small to medium sized tree that grows to a height of 4–15 m is native to tropical Africa6, and is one of the main sources of traditional medicines serving to treat different diseases of human beings. The different parts of C. africana are used for the treatment of various diseases in folk medicine. It is widely used for traditional management of malaria by local people in different parts of Ethiopia7 .The roots of C. africana are used for the treatment of migraine, broken body/hand, wounds, gastritis/constipation, and asthma8 and the stem bark is used as an antispasmodic and anti-inflammatory, while in East Africa, it is employed to treat wounds, skin infections, and gastritis5, particularly, the leaf part squeezed with a drop of water and drunk the concoction with a cup of coffee is claimed to be used for diarrhea in the Tigray and Afar regions of Ethiopia9.

Antimicrobial drugs derived from medicinal plants offer an attractive alternative from antibiotics, as they function differently and can be effectively utilized to treat diseases caused by resistant bacteria strains10. Consequently, there is increasing interest in medicinal plants with antimicrobial potentials because of their medicinal and health benefits.

Despite its wide use in traditional medicine, there are no exhaustive studies which means only a little work has been done on this species. For example, in vivo antidiarrheal activity screening of methanolic extract of the root bark of C. africana reported that it has antidiarrheal activity comparable to atropine11. Methanolic seed extract of C. africana have significant antiulcer activity12. Oleic acid isolated from root bark C. africana conducted in Kenya reported that has antibacterial activity with that of standard drug vancomycin13. The leaves, stem bark and fruit of C. africana also showed inhibitory effects against different bacteria strains14. But no study have been done on the phytochemical investigation, isolation of compound and antibacterial activity from the root parts C. africana.

Material and methods

Plant material collection

The roots of C. africana were gathered in February 2023 from Yirgalem, located in the Sidama Region with 6.7473°N, 38.4067°E. The plant was verified by botanist Mr. Reta Regasa from the Department of Biology at Hawassa College of Teachers Education and voucher specimens of the plant material were deposited at the National Herbarium, Ethiopia, for future reference with the voucher number designated as GT011/2023.The study comply with relevant institutional, national, and international guidelines and legislation.

Preparation of plant material

The roots of the plant collected were gently washed with tap water and chopped into small pieces, air-dried at room temperature for two months, and ground to ease the use of extraction processes and deposited in a dry plastic bag until used for analysis.

Extraction

The pulverized root (500 g) of C .africana was sequentially extracted with dichloromethane/methanol (1:1 v/v) and methanol using maceration techniques for 72 h with continuous shaking at 40 °C.The solution was filtered using Whatmann No. 1 filter paper, and the residual solvent in each gradient extract was removed using a rotary evaporator under reduced pressure. The marc from the former extraction procedure was extracted with methanol likewise. The mass of the raw extracts was measured using an analytical balance and stored in the hood for further examination.

Phytochemical screening test

The crude extracts were subjected to phytochemical screening tests or secondary metabolite identification using standard procedures in order to look into the presence of some secondary metabolites, such as alkaloids, anthraquinones, flavonoids, glycosides, phenols, saponins, steroids, and tannins15.

Antibacterial activity test

The crude extracts and isolated compounds were evaluated in vitro for their anti-bacterial activities against four bacteria strains: E. coli ATCC 35,218, S.aureus ATCC 25,923, S. pyogenes ATCC 19,615, and P. aeruginosa ATCC 27,853 using disc diffusion method16,17,18. The bacterial stock cultures were maintained on the nutrient agar slants. The test solutions were prepared by dissolving 250 \(\mu g/ml\) and 300 \(\mu\) g/ of the test samples to achieved final stock concentration of 250 \(\mu g/ml\) and 300 \(\mu\) g/ml in DMSO to achieve final stock concentrations. The freshly grown liquid culture of the test pathogen solution with similar turbidity and 0.5 McFarland was seeded over the Müeller-Hinton Agar medium plates. The plates were shaken gently to allow the evenly mixed bacterial cells and agar to mix. Sterile paper discs (6 mm in diameter, Whatmann No. 3) were separately soaked in the above stock solutions (samples and standards) and then applied over the seeded plates at equidistance. Control experiments were carried out under similar conditions using ampicillin for antibacterial activity tests as a standard drug. The plates were then inverted and incubated at 37 °C for 24 h.19.

After the incubation period, the plates were observed for a clearance zone around the disks. Clear inhibition zones formed around the discs indicated the presence of antibacterial activity and were measured in millimeters. The mean of the inhibition zone of each test sample was taken to evaluate the antibacterial activity20. The inhibition zones produced by the plant extracts were compared with the inhibition zones produced by commercial standard antibiotics ampicillin.

Compound isolation and characterization

The dichloromethane/methanol (1:1 v/v) crude extract was absorbed onto silica gel and then subjected to column chromatography filled with silica gel. The column was filled in such a way that (dry silica or alumina is added directly to a column, and solvent was allowed to trickle through in portions, then with pressure). Fractionation was performed using solvents system n-hexane and ethyl acetate with gradually increasing polarity. The fractions were concentrated under reduced pressure using Rotary evaporator. The fractions that show single spot were made to dry under reduced pressure. The isolated compounds were kept under refrigerator until spectral data was generated. The structures of the isolated compounds were elucidated by using analyzing the spectral data of IR, 1H-NMR, 13C-NMR, DEPT-135 and comparing the experimental spectral data with literature.

Result NAD discussion

Mass of the crude extracts

The extracts were weighted and the percentage yield was calculated. Relatively higher yield of extract was obtained with dichloromethane: methanol (1:1 v/v) (3.2%, 16 g) compared to methanol 1.1%, 5.5 g.

Phytochemical screening

In this study phytochemical screening tests were performed for preliminary examination of secondary metabolites of the crude extracts of DCM and MeOH (1:1 v/v) and MeOH extract. The result of the phytochemical screening revealed the presence of terpenoids, flavonoids, phenolics, glycosides, anthraquinones, tannins, and steroids as in (Table 1). Based on the phytochemical observations mentioned above C. africana is one of the plants that has a large number of secondary metabolites. These metabolites may be responsible for the plant’s traditional therapeutic applications, and more research on the plant could lead to the creation of new drugs.

Table 1 Phytochemical screening test results of the DCM and MeOH (1:1) and MeOH extracts of C. africana root.
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Isolation and characterization of compounds

Dried dichloromethane/methanol (1:1 v/v) crude extract (14 g) was adsorbed onto 20 g of silica gel (meshing size 60–120) and subsequently placed onto a column containing 160 g of silica gel (meshing size 60–120), employing n-hexane to establish a medium with the lowest polarity at the beginning of the elution process. Two compound (GT1 and GT2) were isolated in this study. The structure of the isolated compounds were elucidated based on the spectral data (IR, NMR) and similar report from the literature.

Compound GT1 was isolated in (19:1) n-hexane/ethyl acetate solvent system as yellow oil 25 mg with 0.6 Rf value. The FTIR spectrum (Appendix 1) of the compound GT1 indicated that it has carbonyl group stretching band at 1742 cm−1 suggested that the compound was most likely an ester. The weak band at 3003.10 cm−1 represents C-H stretch of an alkene whereas the bands at 2922 and 2855 cm-1 indicate C-H stretching of methyl groups and the bond (CH2) was represented by a band of rocking at 722 cm−1.

The 1H NMR Bruker (CDCl3, 400 MHz) spectrum of compound -GT1 (Appendix 2) the peak at δ H 0.89 (t, 3H, J = 6.6 Hz) indicated protons of methyl groups, the peaks at δ 4.27 (dd, 2H, J = 4.2, 12 Hz, glyceryl CH2O), 5.24 (m, glyceryl CHO) indicated glyceryl proton, 2.29 (t, 2H, J = 7.2 Hz) indicate protons of methylene bonded to a carbonyl group, the peaks at δH 5.33–5.40 (m, 1H) indicates the presence of olefinic proton, the peak at δ 2.77 ( t, 2H, J = 6.6 Hz ) indicated protons of a methylene group flanked by two C = C bonds or double allylic CH2, the peaks at 2.0–2.03 (m, 2H) indicates allylic protons, 1.23–1.35 (m, 2H) was assigned to the aliphatic methylene group protons while, the peak at δ 2.31 indicated protons of a methylene bonded to a carbonyl group (Table 2).

Table 2 Comparison of the 1H NMR (400 MHz, CDCl3) and 13C NMR (101 MHz, CDCl3) data of the compound GT1 (trilinolein) and reported data21,22.
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The 13C-NMR Bruker (CDCl3, 101 MHz) spectrum of compound -GT1 (Appendix 3) displayed four type of carbon being the carbonyls (C = O) with the signals at δ 173.3 (C-1), and 172.8 (C- 1’) ppm, the signals at 127.9–130.2 ppm were assigned to the olefinic carbons atoms at δ 130.0 (C-9), δ 129.7 (C-9’), 129.6 (C-10, 10’), 130.2 (C-13, 13’) and 127.9 (C-12, 12’), glycerol carbons with signals at δ 62.1 (C-1’’) and 68.9 (C-2’’) ppm and aliphatic carbons ranging from 14.1 (C-18) to 34.2 (C-2) ppm. The terminal methyl carbon (C-18) was detected at δ 14.1 ppm, while the methylene group was noted at 22.7 (C-17). Methylene carbons at δ 24.9 (C-3) were identified in the β-position relative to the carboxyl group; a peak at δ 25.6 (C-11) indicated a methylene group situated between olefinic carbons (Table -2).

The DEPT-135 Bruker (CDCl3, 101 MHz) spectrum of compound -GT1 (Appendix 4) further verified the existence of methyl carbon at δ14.1 (C-18),methylene carbon at δ 62.1 (C-1’), 34.1 (C-2), 34.2 (C-2’), 31.9 (C-16, C-16’), 29.7 (C-7), 29.6 (C-7’), 29.4 ( C-15 and C-15’), 29.3 (C-5 and C-5’), 29.2 (C-8), 29.1 (C-6), 29.2 (C-6’), 27.2 (C-8,C- 8’,C-14, and C- 14’), 27.1 (C-11), 24.9 (C-3), 24.9 (C-3’), and 22.7 (C-17), Oxygenated methine carbon at δ 68.9 (C-2’’), and olefinic methine carbons at δ 130.0 (C-9), δ 129.7 (C-9’), 129.6 both (C-10, 10’), 127.9 both (C-12, 12’) and 130.2 both (C-13, 13’) .The lack of peaks at δ 172.8 (C-1’) and 173.3 (C-1) in the DEPT-135 spectra, which were seen in the 13C-NMR spectra, further validated the presence of quaternary carbon atoms in the ester carbonyl group (Table 2).

Using thus spectroscopic data (IR, 1H-NMR, 13C-NMR, and DEPT-135) and a comparison with existing studies, the compound was identified as triglyceride trilinolein.21,23,24 (Fig. 1). Compound-GT1 was isolated from C. africana plant for the first time and was identified as trilinolein. Trilinolein is a natural triacylglycerol with linoleic acid as the fatty acid residue in all three esterified positions of glycerol. It possesses several pharmacologic activities, such as in vitro antioxidant, myocardial22, and cardiovascular protective effects and inflammatory25, which also confirms with this plants traditional use.

Fig. 1
figure 1

Proposed structure of compound GT1.

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Compound GT2 was obtained in (18:2) n-hexane/ethyl acetate solvent system as a crystalline solid with an Rf value of 0.55, melting point of 129–131 °C and mass 60 mg. The IR spectra (Appendix 5) shows the presence of OH functionality absorption at 3422 cm-1,aliphatic –C-H stretching at 2925 cm−1, C = C stretching at 1656 cm−1 and C-O stretching at 1374.5 cm−1.

The 1H NMR Bruker (CDCl3, 400 MHz) (Appendix 6) spectrum displayed three regions namely aliphatic, olefinic and hydroxylated on the spectrum signals. The appearance of the singlets at δ H 0.69 (3H, s) and 1.02 (3H, s) confirming the presence of two CH3 attached to quaternary carbons. A signal at δ 5.36 (1H, d, J = 7.5 Hz) suggested the presence of one olefinic proton. A multiplet centered at δ 3.54 (1H, m), characteristic of proton germinal to a hydroxyl group at C-3. A signals representing the methyl groups protons were observed at δ 1.02 (3H, s), 0.94 (3H, d, J = 8.5 Hz), and 0.81 (3H, d, J = 8.1 Hz). The appearance of the complex multiplet at δ 2.29 and 2.32 is revealed that the two CH2 adjacent to carbon attached to OH group (Table 3).

The 13C NMR Bruker (CDCl3, 101 MHz) (Appendix 7), showed recognizable signals at δ 140.7 (C-5) ppm and 121.7 (C-6) ppm which are typical of alkene double bonds, the peak at 19.4 (C-19) and 11.9 (C-18) ppm correspond to angular methyl carbon atoms, the signal at 71.8 (C-3) ppm was assignable to the beta hydroxyl group attached to the carbon (Table 3).

Table 3 The 1H NMR (400 MHz, CDCl3), and 13C NMR (101 MHz, CDCl3) spectral data of the compound GT2 and literature reported data of β –sitosterol26,27,28.
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The overall spectrum of the compound closely compared to that of β – sitosterol29 shown in Fig. 2. Compound-GT2 was isolated from C. africana root for the first time and was identified as β –sitosterol.

Fig. 2
figure 2

Proposed structure of compound GT2.

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However, β-sitosterol was previously isolated from the stem bark of C. africana30. β-sitosterol is a naturally occurring plant compound called a phytosterols which exhibits a variety of biological and medicinal properties such as analgesic, immunomodulatory, antimicrobial, anticancer, anti-inflammatory, wound healing effect, antioxidant and anti-diabetic activities26, which also agrees with this plant traditional use.

Antibacterial activities test result

The crude extracts of dichloromethane/methanol (1:1 v/v), methanol, and isolated compounds (compound GT1 and GT2) were tested for their antibacterial properties against four different bacterial strains, two gram positive S. pyogenes ATCC 19,615, S. aureus ATCC 25,923, and two gram negative, P. aeruginosa ATCC 2785 and E. coli ATCC 25,922 (Appendix 8). The disc diffusion method was used and the zones of growth inhibition of the crude extract and isolated compounds were measured in millimeter and compared to the standard positive control ampicillin. The zone of inhibition measured are reported in (Table 4).

Table 4 Antibacterial inhibition zones (mm) of crude extracts and isolated compounds of C. africana.
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The present result revealed that the inhibition zone of the methanol extract showed comparable antibacterial activities with that of the standard drug ampicillin (13.5 mm, 12 mm, 12.5 mm and 13 mm) against S. pyogenes ATCC 19,615, P. aeruginosa ATCC 27,853, E. coli ATCC 25,922, and S. aureus ATCC 25,923 (12.5 mm, 11.5 mm, 11 mm, and 10.5 mm) respectively at the concentration of 300 μg/ml. Dichloromethane/methanol (1:1v/v) extract showed that moderate activity with that of the standard drug (ampicillin) against E. coli ATCC 25,922 (9.5 mm) and S. pyogenes ATCC 19,615. (9 mm) at the concentration of 300 μg/ml. Compound-GT2 also showed significant antibacterial activity comparable to that of standard drug (ampicillin) against P. aeruginosa ATCC 27,853 (11 mm), S. pyogenes ATCC 19,615 (10 mm), E. coli ATCC 25,922 (9 mm), and S. aureus ATCC 25,923 (10.5 mm) at concentration of 300 μg/ml. Compound-GT1 showed moderate activity compared to that of standard drug (ampicillin 13 mm and 12 mm ) on the tested bacteria strains; S. pyogenes ATCC 19,615 (9 mm) and S. aureus ATCC 25,923 (8.5 mm) at concentration of 300 μg/ml. The methanol extract were showed comparable activities with Ampicillin (12 mm) against P. aeruginosa ATCC 27,853 (11.5 mm), E. coli ATCC 25,922 (11 mm) at concentration of 300 μg/ml.

Methanol extract and compound-GT2 showed significant activities compared to ampicillin than dichloromethane/methanol (1:1v/v) extract and compound-GT1. The findings of this study indicate that C. africana root extract and the isolated compounds show antibacterial properties against both gram-negative and gram-positive pathogens. Comparatively, methanol has revealed better antibacterial activities against four extract. These finding is consistent with previous literature14.

Conclusion

The present study provides insight into the phytochemical profile and antibacterial activities of C. africana against the selected bacterial strains. Phytochemical investigation of the root extract from C. africana showed the presence of, flavonoids, glycosides, terpenoids, tannins, phenolics, and steroids. However, alkaloids were not found in the crude extracts of the root of C. africana (DCM: MeOH (1:1 v/v) and MeOH). Two compounds GT1 and GT2 were isolated from the DCM: MeOH (1:1 v/v) extract of the root of C. africana. The structure elucidations of GT1, and GT2 were in good agreement with the spectroscopic data of trilinolein and β-sitosterol respectively. Both compounds are isolated for the first time from the root of C. africana. The isolated compounds and crude extracts exhibited considerable antibacterial activities that were comparable to that of standard drug ampicillin against all selected bacteria, with diameter zones of inhibition ranging from 9 to 12 mm. The findings of the antibacterial study agree with the results of previous work14. Therefore, the antibacterial activities of the crude extracts and isolated compounds support the traditional application of these plants in treating bacterial infections.