Manniosides G-J, New Ursane- and Lupane-Type Saponins from Schefflera mannii (Hook.f.) Harms

Four previously unreported triterpenoid saponins named 3β-hydroxy-23-oxours-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (mannioside G) (1), 23-O-acetyl-3β-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (mannioside H) (2), ursolic acid 28-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl] ester (mannioside I) (3), and 3β-hydroxy-23-oxolup-20(29)-en-28-oic acid 28-O-β-D-glucopyranosyl ester (mannioside J) (4) were isolated as minor constituents from the EtOAc soluble fraction of the MeOH extract of the leaves of Schefflera mannii along with the known compounds 23-hydroxyursolic acid 28-O-β-D-glucopyranosyl ester (5), ursolic acid 28-O-β-D-glucopyranosyl ester (6), pulsatimmoside B (7) betulinic acid 28-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl] ester (8), 23-hydroxy-3-oxo-urs-12-en-28-oic acid (9), hederagenin (10), ursolic acid (11), betulinic acid (12), and lupeol (13). Their structures were elucidated by a combination of 1D and 2D NMR analysis and mass spectrometry. The MeOH extract, the EtOAc and n-BuOH fractions, and some of the isolated compounds were evaluated for their antibacterial activity against four bacteria: Staphylococcus aureus ATCC1026, Staphylococcus epidermidis ATCC 35984, Escherichia coli ATCC10536, and Klepsiella pnemoniae ATCC13882. They were also screened for their antioxidant properties, but no significant results were obtained.


Introduction
Schefflera mannii (Hook.f.) Harms, also recognized as Astropanax mannii, is a member of the Araliaceae family, which contains 602 known species indigenous to Asia, Africa, and the southwest Pacific regions [1,2].Plants of the genus Schefflera are used in traditional medicine to treat various ailments, including inflammation, rheumatism, fever, pain, diarrhea, cancer, traumatic pain, liver diseases, wound healing, chronic cough, malaria, and bites from animals and as a general tonic [2].Although sesquiterpenes, phenylpropanoids, and lignans are obtained from some Schefflera species, the most common group of secondary metabolites extracted from this genus are triterpenoids and their glycosides [3][4][5][6].Triterpene glycosides, also called saponins, are reported to exhibit a large spectrum of biological activities, including anti-inflammatory [7,8], antibacterial [9,10], antiviral [11,12], cytotoxic [13], and antioxidant [14,15] activities.Recently, we described the isolation and structure elucidation of fifteen saponins, including five new derivatives from the MeOH extract of Schefflera mannii [3].In this paper, the EtOAc soluble fraction of the MeOH extract of this plant was investigated, leading to the discovery of thirteen secondary metabolites among with four previously unreported ursane and lupane glycosides.
The 1 H NMR spectrum of compound 1 exhibited six methyl signals (Tables 1 and 2), including four singlets at δ H 0.90 (CH 3 -24, CH 3 -25), 0.74 (CH 3 -26), and 1.07 (CH 3 -27) as well as two doublets at δ H 0.80 (J = 6.5 Hz, CH 3 -29) and 0.86 (J = 5.8 Hz, CH 3 -30).It also showed one olefinic proton signal at δ H 5.16 (brt, J = 3.8 Hz, H-12), one oxygen-bearing  [27] provided evidence that the residue was a glucopyranosyl unit.Furthermore, the coupling constant observed for the anomeric proton (J = 8.0 Hz) indicated that this glucopyranosyl unit was in the β configuration.The D configuration was assumed, as it is most commonly encountered in the plant kingdom [14,28].This was supported by the fact that more than 200 triterpenoid saponins have been isolated from plants of the genus Schefflera, and in their structures, all the glucopyranosyl units are of D configuration [5,[29][30][31][32].Furthermore, fifteen triterpene saponins were recently isolated from Schefflera mannii, and the configuration of their glucopyranosyl units was determined to be D by GC analysis [3].In the HMBC spectrum, the correlation depicted from the anomeric proton at δ H 5.24 (d, J = 8.0 Hz, H-1 ′ ) to the carbon at δ C 176.6 (C-28) revealed that the glucopyranosyl unit was linked at C-28.Based on the above corroboration, the structure of compound 1 was elucidated as 3β-hydroxy-23-oxours-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester, a previously unreported saponin to which the trivial name mannioside G was given.
Comparison of the aglycone portion of compound 2 with that of 1 (Tables 1 and 2) revealed that the formyl group present in compound 1 was replaced by an O-acetylated oxymethylene group.This was further supported by the methyl singlet observed at δ H 2.07 (CH 3 CO) and the HMBC correlations depicted from CH 3 -24 (δ H 0.76, s) to C-3 (δ C 71.3), C-4 (δ C 41.4), C-5 (δ C 47.5), and C-23 (δ C 65.6) and from H-23a (δ H 4.03, d, J = 11.3) and H-23b (δ H 3.90, d, J = 11.3) to CH 3 CO (δ C 171.4), C-3 (δ C 71.3), and C-5 (δ C 47.5).Full assignment of all the proton and carbon signals of the aglycone of compound 2 was accomplished by careful examination of the HSQC, HMBC, and 1 H-1 H COSY spectra, which was finally elucidated as 23-O-acetyl-3β-hydroxyurs-12-en-28-oic acid.In the ROESY spectrum, the correlation depicted between H-3 (δ H 3.55, dd, J = 11.4,5.1 Hz) and one of the hydroxymethylene protons at δ H 3.90 (d, J = 11,3 Hz, H-23b) supported the β orientation of the hydroxyl group at C-3.The sugar residue was shown to be constituted of a glucopyranosyl unit following careful examination of the 1D and 2D NMR data (Supplementary Materials) in comparison with those reported in the literature [27].Its β configuration was deduced from the large coupling constant observed between H-1 ′ and H-2 ′ (J = 8.1 Hz), while the D configuration was assumed to be the same as for compound 1.The HMBC correlation observed from H-1 ′ (δ H 5.36, d, J = 8.0 Hz) to the carbon at δ C 176.5 (C-28) supported the attachment of the sugar unit at C-28.Accordingly, the structure of compound 2 was determined to be 23-O-acetyl-3β-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester, a new ursane-type saponin to which the trivial name mannioside H was assigned.
The antibacterial activity of the crude extract, fractions, and some isolated compounds was evaluated using the broth microdilution method on two Gram-positive, namely Staphylococcus aureus ATCC1026 and Staphylococcus epidermidis ATCC35984, and two Gramnegative, namely Escherichia coli ATCC10536 and Klepsiella pnemoniae ATCC13882, bacterial strains.The results were discussed according to the antimicrobial cut-off points of plant extracts and pure compounds described by Kuete in 2010 [35].The extract and fractions displayed inhibitory potential ranging from weak to significant (64-1024 µg/mL) against the bacterial strains tested (Table 3).The extract and all the fractions tested had moderate activity against the two Gram-negative bacteria: Escherichia coli ATCC10536 and Klepsiella pnemoniae ATCC13882 (126 < MIC < 512 µg/mL).The n-BuOH fraction significantly inhibited the growth of Staphylococcus epidermidis ATCC 35984 with an MIC value of 64 µg/mL, while the EtOAc fraction exhibited a moderate activity against Staphylococcus aureus ATCC1026 with an MIC value of 512 µg/mL.Compounds 5, 9, and 10-13 were not active against all the tested bacterial strains.Our results agreed with the literature on the antibacterial activity of lupeol [36] and betulinic acid [37], which were not active against some bacterial strains.However, oleanolic acid was reported to exhibit good to moderate activity against Staphylococcus aureus and Escherichia coli [37].Since triterpenoids and their glycosides were reported to exhibit antioxidant activity [14,38], the extract, fractions, and some isolated compounds were screened for their antioxidant properties using the DPPH and FRAP methods, but no significant effect was observed when compared to L-ascorbic acid used as positive control.

General Experimental Procedures
IR spectra were obtained using a Perkin Elmer Spectrum 400 FT-IR/FT-NIR spectrometer (Bruker, Billerica, MA, USA), equipped with a universal ATR sampling accessory. 1H and 13 C NMR spectra were recorded in CD 3 OD on a Bruker Avance 400 (400 MHz for 1 H and 100 MHz for 13 C) (Bruker, Ettlingen, Germany).All chemical shifts (δ) are given in ppm with reference to the residual solvent signal, and coupling constants (J) are in Hz.HRESIMS (high-resolution electrospray ionization) was recorded at the Central Analytical Facility at Stellenbosch University using a Waters Synapt G2 spectrometer (Milford, MA, USA).The ionization source was an ESI + , with a cone voltage 15 V. Semi-preparative HPLC (Luna 10 µm C18 (2) column) was performed with an Agilent Technologies 1260 Infinity (G4286B, 1220 LC system) HPLC pump connected to a G1362A Agilent Refractive Index Detector.The flow rate was 3 mL/min, and 250 µL of solution was injected each time.Column chromatography was performed using silica gel 60 (Merck, Darmstadt/Germany) (0.063-0.200 mm and 0.04-0.063mm) and Sephadex LH-20.The following solvent systems were used: MeOH for Sephadex column chromatography and mixtures of hexane−EtOAc, EtOAc−MeOH, and EtOAc−MeOH−H 2 O for silica gel column chromatography.Thin-layer chromatography (TLC) was performed on Merck precoated silica gel 60 RP-18 F254S and silica gel 60 F254 aluminum foil.The plates were revealed using a UV lamp (254-365 nm) and 10% H 2 SO 4 reagent followed by heating.

Plant Material
The leaves of Schefflera mannii (Hook.f.) Harms were collected in Dschang (5 • 27 ′ 0 ′′ N and 10 • 4 ′ 0 ′′ E), West Region of Cameroon, in November 2017.The plant material was identified at the Cameroon National Herbarium in Yaoundé by Mr. Nana Victor in comparison with a voucher specimen deposited under the reference N • 35063/HNC.

Extraction and Isolation
The air-dried and pulverized leaves (3 kg) were macerated three times with MeOH (95%) (15 L) at room temperature (each time for 24 h).The filtrate obtained was concentrated under reduced pressure to give 405 g of extract (yield 13.5%).An amount of 369 g of the crude methanolic extract was suspended in water (1 L) and successively partitioned with EtOAc (3 × 1 L) and n-BuOH (3 × 1 L).The solutions were evaporated under reduced pressure to afford 201.8 and 36.6 g of EtOAc and n-BuOH fractions, respectively.A part of the EtOAc fraction 196 g was subjected to silica gel column chromatography eluted with hexane-EtOAc (from hexane-EtOAc 10% to EtOAc 100%) and then EtOAc-MeOH (from EtOAc 100% to EtOAc-MeOH 30%) with increasing polarity to give four sub-fractions (A-D).

The Antibacterial Activity
The antibacterial activity was conducted according to the technique described by Mbaveng et al. [39].The microorganisms comprised two Gram-positive, namely Staphylococcus aureus ATCC1026 and Staphylococcus epidermidis ATCC35984, and two Gram-negative, namely Escherichia coli ATCC10536 and Klepsiella pnemoniae ATCC13882, bacterial strains.They were obtained from the Research Unit of Bacteriology of "Centre Pasteur de Yaoundé" (Cameroon).They were kept in the laboratory in the mixture of glycerol and Mueller Hinton Broth (MHB) (1:1) at the temperature of −4 • C, and the activation was achieved using the streak technique on agar medium.Doxycycline was used as positive control.

The Antioxidant Activity
The assay was carried out using the method previously described by Mensor et al. [40].A volume of 20 µL methanol was poured in the last seven lines of a 96-well plate, and then, 20 µL of methanolic solution of samples at 2 mg/mL were poured into the first two wells of each row (four rows per sample).The dilution was conducted following a geometric series with the common ratio 2 in the other wells.After that, 180 µL of methanolic solution of DPPH (0.08 mg/mL) was put into each well of the first three rows, and the same volume of methanol was put into the well of the fourth row.The next step was the incubation of the plates for 30 min in obscurity at room temperature; and finally, the absorbance of each well was read using the spectrophotometer (FLUOstar Omega for microplate, Gainesville, FL, USA) at 517 nm and then converted into percentages of antioxidant activity.The positive control used was L-ascorbic acid.The experiment was conducted three times successively.The percentages of antioxidant activity were calculated by using the following formula.
% antioxidant activity = ( tive, namely Escherichia coli ATCC10536 and Klepsiella pnemoniae ATCC13882, bacterial strains.They were obtained from the Research Unit of Bacteriology of "Centre Pasteur de Yaoundé" (Cameroon).They were kept in the laboratory in the mixture of glycerol and Mueller Hinton Broth (MHB) (1:1) at the temperature of −4 °C, and the activation was achieved using the streak technique on agar medium.Doxycycline was used as positive control.

The Antioxidant Activity
The assay was carried out using the method previously described by Mensor et al. [40].A volume of 20 µL methanol was poured in the last seven lines of a 96-well plate, and then, 20 µL of methanolic solution of samples at 2 mg/mL were poured into the first two wells of each row (four rows per sample).The dilution was conducted following a geometric series with the common ratio 2 in the other wells.After that, 180 µL of methanolic solution of DPPH (0.08 mg/mL) was put into each well of the first three rows, and the same volume of methanol was put into the well of the fourth row.The next step was the incubation of the plates for 30 min in obscurity at room temperature; and finally, the absorbance of each well was read using the spectrophotometer (FLUOstar Omega for microplate) at 517 nm and then converted into percentages of antioxidant activity.The positive control used was L-ascorbic acid.The experiment was conducted three times successively.The percentages of antioxidant activity were calculated by using the following formula.The reducing power of the tested samples was determined following the method reported by Benzie and Strain [41].For the preparation of the FRAP reagent, a buffer solution of sodium acetate (300 mM, pH 3.6), a solution of 2,4,6-tris (2-pyridyl)-1,3,5-s-triazine TPTZ (10 mM), and a ferric chloride solution were mixed together at the ratio 10:1:1, respectively.A volume of 5 µL of each sample at 2 mg/mL was mixed with 95 µL of the FRAP reagent, and the mixture was incubated in the dark for 30 min at 37 °C.The optical density was read by using a spectrophotometer at 593 nm.L-ascorbic acid was used as positive control.The antioxidant potential of each sample was calculated from the calibration curve of ferrous sulfate solution and expressed in millimole equivalent of FeSO4 per gram of sample.

Conclusions
Phytochemical investigation of the EtOAc soluble fraction of the MeOH extract of the leaves of Schefflera mannii led to the discovery of four previously unreported triterpenoid saponins, namely manniosides G-J along, with nine known metabolites.Only very little amounts of the new compounds were obtained (less than 1.5 mg); consequently, they were not screened for their biological activities.Although compounds 5, 9, and 10-13 did not show antibacterial and antioxidant activities, the present work once again strengthens the chemotaxonomy of plants of the genus Schefflera since they are known to be an important Abs (DPPH) − (Abs (trial) − Abs (sample) tive, namely Escherichia coli ATCC10536 and Klepsiella pnemoniae ATCC13882, bacterial strains.They were obtained from the Research Unit of Bacteriology of "Centre Pasteur de Yaoundé" (Cameroon).They were kept in the laboratory in the mixture of glycerol and Mueller Hinton Broth (MHB) (1:1) at the temperature of −4 °C, and the activation was achieved using the streak technique on agar medium.Doxycycline was used as positive control.

The Antioxidant Activity
The assay was carried out using the method previously described by Mensor et al. [40].A volume of 20 µL methanol was poured in the last seven lines of a 96-well plate, and then, 20 µL of methanolic solution of samples at 2 mg/mL were poured into the first two wells of each row (four rows per sample).The dilution was conducted following a geometric series with the common ratio 2 in the other wells.After that, 180 µL of methanolic solution of DPPH (0.08 mg/mL) was put into each well of the first three rows, and the same volume of methanol was put into the well of the fourth row.The next step was the incubation of the plates for 30 min in obscurity at room temperature; and finally, the absorbance of each well was read using the spectrophotometer (FLUOstar Omega for microplate) at 517 nm and then converted into percentages of antioxidant activity.The positive control used was L-ascorbic acid.The experiment was conducted three times successively.The percentages of antioxidant activity were calculated by using the following formula.The reducing power of the tested samples was determined following the method reported by Benzie and Strain [41].For the preparation of the FRAP reagent, a buffer solution of sodium acetate (300 mM, pH 3.6), a solution of 2,4,6-tris (2-pyridyl)-1,3,5-s-triazine TPTZ (10 mM), and a ferric chloride solution were mixed together at the ratio 10:1:1, respectively.A volume of 5 µL of each sample at 2 mg/mL was mixed with 95 µL of the FRAP reagent, and the mixture was incubated in the dark for 30 min at 37 °C.The optical density was read by using a spectrophotometer at 593 nm.L-ascorbic acid was used as positive control.The antioxidant potential of each sample was calculated from the calibration curve of ferrous sulfate solution and expressed in millimole equivalent of FeSO4 per gram of sample.

Conclusions
Phytochemical investigation of the EtOAc soluble fraction of the MeOH extract of the leaves of Schefflera mannii led to the discovery of four previously unreported triterpenoid saponins, namely manniosides G-J along, with nine known metabolites.Only very little amounts of the new compounds were obtained (less than 1.5 mg); consequently, they were not screened for their biological activities.Although compounds 5, 9, and 10-13 did not show antibacterial and antioxidant activities, the present work once again strengthens the chemotaxonomy of plants of the genus Schefflera since they are known to be an important The reducing power of the tested samples was determined following the method reported by Benzie and Strain [41].For the preparation of the FRAP reagent, a buffer solution of sodium acetate (300 mM, pH 3.6), a solution of 2,4,6-tris (2-pyridyl)-1,3,5-striazine TPTZ (10 mM), and a ferric chloride solution were mixed together at the ratio 10:1:1, respectively.A volume of 5 µL of each sample at 2 mg/mL was mixed with 95 µL of the FRAP reagent, and the mixture was incubated in the dark for 30 min at 37 • C. The optical density was read by using a spectrophotometer at 593 nm.L-ascorbic acid was used as positive control.The antioxidant potential of each sample was calculated from the calibration curve of ferrous sulfate solution and expressed in millimole equivalent of FeSO 4 per gram of sample.

Conclusions
Phytochemical investigation of the EtOAc soluble fraction of the MeOH extract of the leaves of Schefflera mannii led to the discovery of four previously unreported triterpenoid saponins, namely manniosides G-J along, with nine known metabolites.Only very little amounts of the new compounds were obtained (less than 1.5 mg); consequently, they were not screened for their biological activities.Although compounds 5, 9, and 10-13 did not show antibacterial and antioxidant activities, the present work once again strengthens the chemotaxonomy of plants of the genus Schefflera since they are known to be an important source of triterpenoids and their glycosides (saponins).However, given that saponins are well known for their cytotoxic, antifungal, and anti-inflammatory properties, we intend to reisolate these compounds in large amounts in order to evaluate these biological activities during our future investigations.

Hz)
glucopyranosyl] ester, a new ursane-type glycoside named mannioside I. Compound 4 was obtained as a gum with a molecular formula of C 36 H 56 O 9 on the basis of the HRESIMS spectrum, which displayed the sodium adducts at m/z 655.3826 [M + Na] + (Calcd.for C 36 H 56 O 9 Na + : 655.3817) and 1287.7780[2M + Na] + (Calcd.for C 72 H 112 O 18 Na +

Table 3 .
Minimum inhibitory concentrations of the extract, fractions, and some compounds isolated from S. mannii.