Bioactive constituents from Flacourtia vogelii Hook.f. (Flacourtiaceae)

Abstract The chemical investigation of the methanolic root extract of Flacourtia vogelii led to the isolation of a new arylbenzoate derivative, vogelinal (1), together with thirteen known compounds (2–14). The structures of the isolates were elucidated by extensive spectroscopic and spectrometric analyses (1D and 2D NMR, ESI-MS) and by comparison with previously reported data. All the compounds were tested for their antioxidant, antifungal and antibacterial activities. Compound 7 exhibited the highest antioxidant potential, with RSa 50 of 11.80 ± 2.13 µg/mL, RSa 50 of 42.60 ± 6.32 µg/mL and RC50 of 51.60 ± 7.71 µg/mL for the DPPH, ABTS and FRAP assay, respectively. Compound 13 displayed weak antifungal effect with MIC value of 125 μg/mL against Candida parapsilosis. Compound 8 showed weak antibacterial effect with MIC value of 125 μg/mL, against Shigella dysenteria. The present study, conclude that this species could be a promising source of antioxidant and antibacterial constituents. Graphical Abstract


Introduction
Plants have been used as a source of medicine by many populations all over the world to meet their primary health care.Traditional treatment by medicinal plants is often dangerous due to the poor knowledge of the toxicity and dosage.Besides that, there is a global consensus on the benefits of Phyto pharmacy and at present medicinal plants occupy a key position in plant research and medicine.The importance of plants in medicine remains even of greater relevance with the current global shift to obtain drugs from plants sources, as a result of which attention has been given to the medicinal value of herbal remedies for safety, efficacy and economy (Aliyu et al. 2011).Plants constitute an important source of active ingredients which differ widely in terms of structure and therapeutic properties.Among the plants often used in traditional medicine, Flacourtia vogelii Hook.f. belonging to the Flacourtia genus, is known as a shrub or small tree, of at least 4 meters high, trunk with 10 cm of diameter and with globose pale green fruit containing 5 to 7 seeds; it is mainly found in tropical forest (Burkill 1994;Breteler 1989).The genus Flacourtia (Flacourtiaceae) which the name was giving by Etienne De Flacourt (1607-1660), contains about 20 species of small trees and shrubs native to tropical Africa and Asia.Several species of this genus are cultivated for their edible fruits and as ornamentals (Everett 1981).They are used in the traditional medicine of several countries for the treatment of ailments such as rheumatoid arthritis, gout, snake bite, fever, malaria, nephritic colic, diarrhea, dysentery, high blood pressure, toothache, diabetes, asthma, pre-and post-natal purification, anemia, skin eruption, piles, cough, liver disorder, weakness of limbs, bronchitis and as digestive (Elfita et al. 2019;Sasi et al. 2018;Sanjeeb et al. 2013;Agassounon et al. 2012;Yusuf et al. 2007).Previous phytochemical investigations of Flacourtia species resulted in the isolation of flavonoids, triterpenoids, fatty acids, steroids, alkaloids, tannins, saponins, anthocyanin, glycosides and phenolic compounds (Eramma and Devaraja 2021;Elfita et al. 2019;Alakolanga et al. 2015;Sanjeeb et al. 2013;Thai et al. 2022;Jayasinghe et al. 2012;Afifi et al. 2022;Sarkar et al. 2020), which exhibited several activities such as antibacterial, antifungal, antioxidant, anti-proliferative, hepatoprotective, antimalarial, antidiabetic, anti-inflamatoire and anti-asthmatic activity (Eramma and Devaraja 2021;Elfita et al. 2019;Sasi et al. 2018;Sanjeeb et al. 2013;Agassounon et al. 2012;Tyagi et al. 2010).
To the best of our knowledge, neither phytochemical nor pharmacological study associated with Flacourtia vogelii have been reported.In continuity of our search for bioactive constituents from Cameroonian medicinal plants, the methanolic root extract of F. vogelii was investigated.Herein, we report the isolation and structure elucidation of a new compound along with thirteen known ones.

Structure characterization of isolated compounds
The chemical investigation of the root extract of Flacourtia vogelii led to the isolation of a new arylbenzoate derivative, trivially named as vogelinal (1), together with thirteen known compounds (Figure 1).

Antibacterial activity
The antibacterial activities of the methanolic root extract and compounds 1 -14 were evaluated against gram positive bacteria strains and clinical bacteria isolates obtained from the Centre Pasteur du Cameroon (CPC) Pseudomonas aeruginosa ATCC10145, Staphylococcus aureus ATCC25922, Escherishia coli ATCC10536, Klessiella pneumonae ATCC13883, Shigella flexineri NR518, Shigella dysenteria CPC, Salmonella enteritidis ATCC13076, Salmonella typhimurium CPC and Salomonella typhi ATCC6539 using the microdilution broth susceptibility assay as previously described (Newton et al. 2002).The results (Table S2) showed that compound 8 displayed weak antibacterial activities against Shigella dysenteria with MIC values of 125 µg/mL, and bactericidal effect with MBC/MIC ratio of 2. Compound 1 showed weak antibacterial activity against Salmonella enteritidis with MIC value of 250 µg/mL, whilst compounds 4 and 13 displayed weak activities against Pseudomonas aeruginosa and Shigella flexineri, respectively, with MIC value of 250 µg/mL.The weak activities may be due to the fact that almost all the isolates are lipophilic compounds, which makes it difficult to enter the hydrophilic periplasmic space of bacteria strains or isolates.The methanolic root extract displayed moderate antibacterial activity against Escherishia coli and Salmonella typhimurium with MIC value of 250 µg/mL.This last result could justify the use of the plant in traditional medicine for the treatment of infectious diseases.

Antifungal activity
The antifungal activities of the root extract and compounds 1 -14 were evaluated against clinical fungal isolates obtained from the Centre Pasteur du Cameroon (CPC) Candida albicans CPC, Candida krusei CPC, Candida parapsilosis CPC, Cryptococcus neoformans CPC, Trichophyton mentagrophytes CPC, Microsporium audouinii CPC and Epidermophyton flocosum CPC.The results (Table S3) showed that compounds 13 exhibited weak antifungal activity against Candida parapsilosis with MIC value of 125 µg/mL, while compound 1 showed weak antifungal activity against Candida parapsilosis with MIC value of 250 µg/mL.The other compounds were found to be inactive against several fungal isolates.The weak activities may be due to the weak solubility of the isolates in polar solvents.No activity was observed with the root extract, implying that the synergetic effect is not benefit in the crude extract.

Antioxidant activity
The root extract and compounds 1-14 were also evaluated for their antioxidant capacity using DPPH, ABTS and FRAP assays (Table S4).Compound 7 exhibited the highest but moderate antioxidant potential, with RSa 50 of 11.80 ± 2.13 µg/mL, RSa 50 of 42.60 ± 6.32 µg/mL and RC 50 of 51.60 ± 7.71 µg/mL for the DPPH, ABTS and FRAP assay respectively.The other compounds were found to be either weakly active or inactive compare to the reference Vitamin C, which showed RSa 50 of 7.80 ± 1.24 µg/mL, RSa 50 of 11.20 ± 1.68 µg/mL and RC 50 of 18.00 ± 2.00 µg/mL using DPPH, ABTS and FRAP assays, respectively.Almost all the isolates are weak proton donor.Thus, the weak antioxidant potential may be due to the low availability of acid protons.

General experimental procedures
The 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 500 MHz NMR spectrometer (Rheinstetten, Germany) equipped with a 5 mm cryogenic DCH (H/13C) probe.Chemical shifts(δ) are reported in parts per million (ppm).Coupling constants (J) are reported in Hz.HRMS and MS were obtained on a Q Exactive Plus Hybrid quadrupole -orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, uSA) using electro-spray ionization in the positive-ion mode.The spray voltage was set at 3.5 kV; the sheath gas flow rate (N2) at 50 units; the capillary temperature at 320 °C; the S lens RF level at 50; and the probe heater temperature at 425 °C.Column chromatography was carried out on silica gel (70-230 mesh, Merck) and flash silica gel (230-400 mesh, Merck).TLC was performed on Merck precoated silica gel 60 F 254 aluminium foil, using ceric sulphate spray reagent for visualization.All reagents used were of analytical grade.

Plant materials
The roots of F. vogelii were collected on July 2016, in Yaoundé, Central region of Cameroon, more precisely at Kala hill, located between latitude 3° 52′ 0.001" N and longitude 11° 31′ 0.001" E, altitude above sea level 726 m.The plant was identified by Mr. Victor Nana of the National Herbarium of Cameroon, Yaoundé, where a voucher specimen (N° 30054/HNC) has been deposited.

Antibacterial test
The broth microdilution method was used for susceptibility testing of bacteria species in 96 well-microtiter sterile plates as described by Newton et al. (Newton et al. 2002).
For this, the samples were dissolved in 5% dimethylsulfoxide (DMSo) solution and diluted with Mueller Hinton broth to obtain a stock concentration of 500 µg/ml.Serial two-fold dilutions of the test substances were made with Mueller Hinton broth to yield a volume of 100 µl/well.This gave a concentration range of 250 to 0.97 µg/ml respectively.one hundred microliters of each bacterial suspension (containing about 1.5 × 10 6 CFu/mL) was added to respective wells containing the test samples and mixed thoroughly to give final concentrations ranging from 500 to 0.48 µg/ml.The solvent control, 5% DMSo did not show inhibitory effects on the growth of the bacteria.
Ciprofloxacin® (Bayer, Germany) at concentration of 64 to 0.5 µg/ml were used as the standard reference.The assay microtitre plates were incubated at 37 °C for 24 hrs.Inhibitory concentrations of the extracts were detected after addition of 50 µl of 0.2 mg/ ml p-iodonitrotetrazolium chloride (INT) (Sigma-Aldrich, South Africa) and incubated at 37 °C for 30 min (Mativandlela et al. 2006).Viable bacteria change the yellow dye of p-iodonitrotetrazolium chloride to a pink color.The assay is based on living cells' capacity to transform the INT dye tetrazolium ring into a pink-coloured formazan structure due to the action of mitochondrial and other dehydrogenases inside the cell.
The lowest concentration at which no visible colour changes was observed was considered as the MIC (Minimum Inhibitory Concentration).The bactericidal concentrations were determined by adding 50 µl aliquots of the preparations (without INT), which did not show any visible colour change after subculture from MIC assays, into 150 µl of extract-free Mueller Hinton broth.These preparations were further incubated at 37 °C for 48 hrs and bacterial growth was revealed by the addition of INT as above.
The smallest concentration at which no colour changes was observed was considered as the MBC.The tests were performed in triplicates.The ratio MBC/MIC was calculated to determine the bactericidal (MBC/MIC ≤ 4) and bacteriostatic (MBC/MIC > 4) effects.

Antifungal test 3.5.2.1. Preparation of yeast inocula.
The inocula of yeasts were prepared from 48h old cultures by picking numerous colonies and suspending them in sterile saline (NaCl) solution (0.9%).Absorbance was read at 530 nm and adjusted with the saline solution to match that of a 0.5 McFarland standard solution, corresponding to about 10 6 yeast cells/ml (CLSI.2008).

Determination of minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs).
Minimum Inhibitory Concentration (MIC) of each sample was determined by using broth microdilution techniques according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards, NCCLS) for yeasts (M27-A2), (CLSI. 2008).Stock solutions of the samples were prepared in 5% aqueous dimethylsulfoxide (DMSo) solution and diluted with Sabouraud Dextrose Broth (SDB) to give a concentration of 1 mg/ml.This was serially diluted two-fold to obtain a concentration range 500 to 0.24 μg/ml.The final concentration of DMSo in the well was less than 1% (preliminary analysis with 1% DMSo did not inhibit the growth of the test organisms).Negative control well consisted of 195 µl of SDB and 5 µl (containing about 10 6 cells/ml for yeast and 1 x 10 4 spores/ml for filamentous fungi) of the inoculum.The plates were covered with a sterile lid, and incubated on the shaker at 37 °C for 48 h (for yeasts).MICs were assessed visually after the corresponding incubation period and were taken as the lowest product concentration at which there was no growth or virtually no growth.The assay was repeated three times.For the MFCs determination, 5 µl aliquots from each well that showed no growth of microorganism were replicated into 195 µl of SDB and incubated at 37 °C for 48 h (yeasts).The lowest concentration that yielded no growth after the subculturing was taken as the MFC.

Antioxidant tests 3.5.3.1. DPPH assay.
The free radical scavenging activities of the sample were evaluated using the DPPH analysis as described by Mensor et al. (2001) and Noghogne et al. (2015).The radical scavenging activities of crude extract were evaluated through spectrophotometer using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical.When DPPH reacts with an antioxidant sample, which can donate hydrogen, it is reduced.The changes in colour were measured at wave length 517 nm under uV/Visible light spectrophotometer (Infinite M200, TECAN, Swiss).The extract (1000 µg/mL) was two-fold serially diluted with methanol.Fifty microliters of the diluted extract (1000 µg/mL) in methanol were mixed with 150 µL of 0.02% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) methanol solution, giving a final extract concentration range from 250 to 1.9531 µg/mL (250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9062 and 1.9531 µg/mL).After 30 min of incubation in the dark at room temperature, the optical density was measured.Ascorbic acid (Vitamin C) was used as positive control.Each assay was done in triplicate and the results, recorded as the mean ± standard deviation (SD).The radical scavenging activity (RSA, %) was calculated as follow:

ABTS assay.
The radical scavenging activities of the samples were evaluated spectrophotometrically using the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) free radical.When ABTS reacts with an antioxidant compound, which can donate hydrogen, it is reduced.The changes in colour were measured at 734 nm under uV/Visible light spectrophotometer (Infinite M200 (TECAN, Swiss)).
Pure methanol was used to calibrate the counter.The extract (1000 µg/mL) was twofold serially diluted with methanol.Twenty-five microliters of the diluted extract were mixed with seventy-five µL of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) methanol solution, to give a final extract concentration range of 250 − 1.9531 µg/mL (250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9062 and 1.9531 µg/mL).After 30 min of incubation in the dark at room temperature, the optical densities were measured at 734 nm.Ascorbic acid (Vitamin C) was used as control.Each assay was done in triplicate and the results, recorded as the mean ± standard deviation (SD) of the three findings, were presented in tabular form.The radical scavenging activity (RSA, in %) was calculated as follows:

RSA Absorbance of ABTS Absorbance of sample
Absorbance of ABTS % * *100

FRAP assay.
The ferric reduction potential (conversion potential of Fe 3+ to Fe 2+ ) of the samples was determined according to the method described by Padmaja et al. (2011).Briefly, the samples were first dissolved as for the DPPH assay.
25 µL from each dilution was introduced into a new microplate and 25 µL of 1.2 mg/ mL Fe 3+ solution added.The plates were pre-incubated for 15 mins at ambient temperature.After this time, 50 µL of 0.2% ortho-phenanthroline was added to obtain final extract concentrations of 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625 and 1.95325 µg/mL.The reaction mixtures were further incubated for 15 min at ambient temperature after which the absorbance was measured at 505 nm under uV/Visible light spectrophotometer (Infinite M200 TECAN, Swiss) against the blank (made of 25 µL methanol + 25 µL Fe3+ + 50 µL ortho-phenanthroline).Ascorbic acid (Vitamin C) was used as positive control.The assay was performed in triplicate.
From the obtained oD (optical Density), Reducing percentages were calculated for each concentration and used to determine the RC 50 from Dose-response curves.

Conclusion
overall, this research reports the isolation of a new arylbenzoate derivative, namely vogelinal (1) together with thirteen known compounds, including three friedelane type triterpenes (2-4) which confirmed the chemotaxonomic membership of Flacourtia vogelii to the Flacourtia genus.Compound 8 showed weak antibacterial activity with MIC of 125 μg/mL, against Shigella dysenteria and compound 13 displayed weak activity with MIC of 125 μg/mL, against Candida parapsilosis.In addition, compound 7 exhibited the highest antioxidant activity among all the isolates, with RSa 50 of 11,80 ± 2,13, RSa 50 of 42,60 ± 6,32 and RC 50 of 51,60 ± 7,71 µg/mL for the DPPH, ABTS, FRAP assay respectively.This result confirms the used of this plant in traditional medicine for the treatment of infectious diseases.