An Antibacterial Isovaleronitrile Diglycoside From Detarium microcarpum Guill. Perr. ( Fabaceae )

Abstract

A new isovaleronitrile diglycoside, named microcarposide (1), together with 6 known compounds: lupeol (2), betulinic acid (3), β-sitosterol glucoside (4), methyl gallate (5), luteolin (6), and epicatechin (7), was isolated from the methanolic extract of the fruits of Detarium microcarpum Guill. Perr. The structures of the compounds were determined by extensive analysis of 1D- and 2D-¹H and ¹³C NMR spectroscopic data in conjunction with mass spectrometry and by comparison with data reported in the literature. Compound 1 was characterized as (2R)-2-[(6″-O-β-l-rhamnopyranosyl-β-d-glucopyranosyl)oxy]-3-methylbutanenitrile. Some of the isolated compounds were evaluated for their antibacterial activities against several microorganisms; only compound 1 was active against Salmonella typhi, Salmonella enteritidis, and Salmonella typhimurium with minimum inhibition concentration values of 153.4, 76.7, and 76.7 μM, respectively.

The genus Detarium is a member of the family Fabaceae, subdivision Caesalpinioideae, and tribe Detarieae. ¹ Only 2 species of this genus are reported in the literature. The first, Detarium senegalensis J.F. Gmel., grows in riparian and dry forests areas, while the second, D. microcarpum Guill. and Perr., is a fruit bearing tree growing to a height of about 10 m, which is distributed in dry savannah regions of some western and central Africa countries such as Benin, Burkina Faso, Guinea Bissau, Guinea, Niger, Nigeria, Senegal, Ghana, Togo, Cameroon, and Central African Republic. ^{2 -4} Different parts of this latter plant have been used in traditional medicine for the treatment of several illnesses such as stomach disorders, venereal diseases, and gastrointestinal ailments. The fruit pulp is rich in minerals, such as Ca, P, Fe, K, Na, and Mg, and essential vitamins, such as C, E, B2, and folic acid, which serves as a major food supplement during the dry season. ⁵ It is worth noting that fruits of plants of the Fabaceae family contain cyanogenic glycosides. ⁶ These are secondary plant metabolites that have been used as chemotaxonomic markers. They are present in more than 2500 plant species of which the most represented botanical families are Fabaceae, Rosaceae, Linaceae, and Asteraceae. ⁶

Previous phytochemical investigations of the fruit flour of D. microcarpum reported about 42% carbohydrates; 36% lipids; and 11% protein, terpenoids, and phenolic compounds, some of which exhibited a wide range of biological properties, including antimicrobial, antimalarial, and cytotoxic effects. ^{7 -9} The seed coat is also reported to possess antimicrobial activity, which could be used in the control of infectious diseases. ¹⁰ Although different parts of D. microcarpum are used for the treatment of microbial and parasitic diseases, we have found no information regarding their use in the treatment of typhoid fever. However, in Cameroon, the local population from where this plant was harvested uses its fruit pulp and root bark decoctions for the treatment of this disease. Thus, in order to either confirm or overturn the ethnobotanical uses of this plant in the treatment of typhoid fever, and as part of our ongoing search for anti-Salmonella extracts and secondary metabolites from Cameroonian medicinal plants, we have undertaken the chemical and pharmacological investigation of this plant.

In this paper, we report the structural elucidation of a new isovaleronitrile diglycoside, named microcarposide (1), along with 6 known compounds (2-7), as well as evaluating their antibacterial activity and especially their anti-Salmonella activity.

Results and Discussion

Silica gel column chromatography (CC) of the methanol extract of fruits of D. microcarpum led to the isolation of a new isovaleronitrile diglycoside, named microcarposide (1), along with 6 known compounds lupeol (2), ¹¹ betulinic acid (3), ¹² β-sitosterol glucoside (4), ¹³ methyl gallate (5), ¹⁴ luteolin (6), ¹⁵ and epicatechin (7). ¹⁶ The structures of the known compounds were determined based on the analysis of their spectroscopic data, which showed complete agreement with those reported in the literature (Figure 1).

Figure 1

Structures of compounds 1-7.

Compound 1 was obtained as a white powder from the EtOAc/MeOH (9:1) fraction. It reacted positively both with Molisch’s and the cyanogenic reagents, suggesting its sugar nature and the presence in its structure of a cyanide moiety. Its molecular formula, C₁₇H₂₉NO₁₀, implying 4 degrees of unsaturation, was established from its HR-ESI-TOF-MS (Supplemental Figure S1), which showed, in the positive mode, the protonated molecular ion peak [M+H]⁺ at m/z 408.1865 (calcd for C₁₇H₃₀NO₁₀ ⁺ : 408.1864). The presence of the cyanide group in this compound was confirmed by the stretching vibration band observed at υ 2356 cm⁻¹ in the IR spectrum. This spectrum also displayed vibration bands characteristic of a hydroxyl group (υ 3363 cm⁻¹) and C _sp3-H (υ 2920 cm⁻¹) of aliphatic carbons (Supplemental Figure S2). The 17 carbon atoms of the molecular formula were confirmed by the broad band proton decoupled ¹³C NMR spectrum (Table 1), which showed 17 signals. These were sorted by DEPT and HMQC techniques into 12 _sp3 methine carbon signals [among which were 11 oxymethine signals appearing at δ _C 101.9 (C-1′), 101.2 (C-1″), 76.9 (C-5′), 76.1 (C-3′), 73.4 (C-2′), 72.4 (C-4″), 71.9 (C-2), 68.8 (C-3″), 70.9 (C-2″), 70.4 (C-4′), 71.1 (C-5″); and 1 methine signal at δ _C 31.7 (C-3)]; 1 sp ³ oxymethylene signal at δ _C 67.0 (C-6′), and 3 methyl group signals at δ _C 18.4 (C-6″), 18.2 (C-4), and 17.6 (C-5). Thus, the compound contained 1 quaternary sp nitril carbon signal at δ _C 118.1 (C-1).

Table 1

¹H (500 MHz) and ¹³C (125 MHz) NMR Spectral Data and HMBC Correlations of Compound (1) in DMSO.

Position	1H NMR δ H (nH, m, J in Hz)	13C NMR δ C (m)	HMBC
1	-	118.1
2	4.51 (1H, d, 5.6)	71.9	C1′, C1, C3, C4
3	2.06 (1H, m)	31.7
4	0.97 (3H, d, 6.8)	18.2
5	1.11 (3H, d, 6.7)	17.6
1′	4.34 (1H, d, 7.8)	101.9	C2, C3′, C5′
2′	2.99 (1H, dd, 9.9, 6.5)	73.4
3′	3.32 (1H, s)	76.1
4′	3.05 (1H, dt, 8.9, 4.5)	70,4
5′	3.18 (1H, dt, 12.6, 6.5)	76.9
6a′	3.81 (1H, dd, 11.9, 4.3)	67.0	C1″
6b′	3.48 (1H, dd, 11.9, 2.2)		C1″
1″	4.59 (1H, d, 5.8)	101.2	C6′, C3″, C5″
2″	3.63 (1H, d, 9.0)	70.9
3″	3.43 (1H, d, 3.9)	68.8
4″	3.20 (1H, d, 4.2)	72.4
5″	3.42 (1H, d, 3.2)	71.1
6″	1.13 (3H, d, 6.2)	18.4

The combined analysis of the ¹H NMR (Table 1) and HMQC (Supplemental Figure S6) spectra of compound 1 showed a set of signals at δ _H 4.51 (1H, d, J = 5.6 Hz, H-2)/δ _C 71.9 (C-2), δ _H 2.06 (1H, m, H-3)/δ _C 31.7 (C-3), δ _H 0.97 (3H, d, J = 6.8 Hz, H-4)/δ _C 18.2 (C-4), and δ _H 1.11 (3H, d, J = 6.7 Hz, H-5)/δ _C 17.6 (C-5), which were assigned to an isovaleronitrile aglycone type moiety (C₅H₉NO). ¹⁷ The ¹H NMR spectrum of compound 1 also exhibited 2 doublets of 1 proton each at δ _H 4.34 (1H, d, J = 7.8 Hz, H-1′) and δ _H 4.59 (1H, d, J = 5.8 Hz, H-1″), which correlated in the HMQC spectrum with the corresponding carbons C-1′ at δ _C 101.9 and C-1″ at δ _C 101.2, indicative of the presence of 2 sugar moieties. Also observed in these spectra were 2 sets of signals. The first, corresponding to hydroxyl methine and methylene signals at δ _H/δ _C 4.34 (1H, d, J = 7.8 Hz, H-1′)/101.9 (C-1′), 2.99 (1H, dd, J = 9.9, 6.5 Hz, H-2′)/73.4 (C-2′), 3.32 (1H, s, H-3′)/76.1 (C-3′), 3.05 (1H, dt, J = 8.9, 4.5 Hz, H-4′)/70.4 (C-4′), 3.18 (1H, dt, J = 12.6, 6.5 Hz, H-5′)/76.9 (C-5′), 3.81 (1H, dd, J = 11.9, 4.3 Hz, H-6a′)/67.0 (C-6′), and 3.46 (1H, dd, J = 11.9, 2.2 Hz, H-6b′)/67.0 (C-6′), was a characteristic of d-glucopyranosyl moiety, ¹⁸ whereas the second set, including signals at δ _H/δ _C 4.59 (1H, d, J = 5.8 Hz, H-1″)/101.2 (C-1″), 3.63 (1H, d, J = 9.0, H-2″)/70.9 (C-2″), 3.43 (1H, d, J = 3.9, H-3″)/68.8 (C-3″), 3.20 (1H, d, J = 4.2, H-4″)/72.4 (C-4″), 3.42 (1H, d, J = 3.2, H-5″)/71.1 (C-5″), and 1.13 (3H, d, J = 6.2, H-6″)/18.4 (C-6″), was attributable to an l-rhamnopyranosyl moiety. ^18,19 Complete assignment of the protons and carbons of 2 sugar units was achieved by analysis of the COSY, HMQC, and HMBC spectra of this compound.

The linkage between the sugar units and the aglycone and that between 2 sugar units remained to be determined.

The fragment ion observed at m/z 309 (M⁺−99) in the HR-ESI-TOF-MS of compound 1, corresponding to the loss of the aglycone, confirmed that the aglycone was linked to the sugar moieties through an oxygen atom. Furthermore, the HMBC (Supplemental Figure S8) correlations observed between the anomeric proton H-1′ of the d-glucose unit at δ _H 4.34 (1H, d, J = 7.8 Hz) and carbons C-3ʹ (δ _C 76.1), C-5ʹ (δ _C 76.9), and C-2 (δ _C 71.9) of the aglycone, clearly confirmed its direct attachment to the aglycone (Figure 2). Concerning the linkage between 2 sugar units, the HMBC (Supplemental Figures S8 and S9) correlations were used once again. The HMBC correlation observed between the H-6ʹ protons of the d-glucose moiety at δ _H 3.81 (1H, dd, J = 11.9, 4.3 Hz, H-6a′) and at 3.48 (1H, dd, J = 11.9, 2.2 Hz, H-6b′) with the anomeric carbon C-1ʺ of the l-rhamnose unit at δ _C 101.2 and H-1ʺ anomeric proton of the l-rhamnose unit at δ _H 4.49 (1H, d, J = 5.8 Hz) and the C-6ʹ (δ _C 67.0) carbon of the d-glucose unit established the connectivity between 2 sugar units.

Figure 2

Key 2D NMR correlations of compound 1.

The relative stereochemistry of the anomeric protons of 2 sugar units was established to be β from J coupling constant values between H-1′and H-2′ (³ J _H1′-H2′ = 5.0 Hz), and H-1″ and H-2″ (³ J _H1″-H2″ = 5.0 Hz), respectively. ^18,19

The absolute configuration of the stereogenic center C-2 of the aglycone was established by comparing the chemical shifts and coupling constants of its proton with those of 2 closely related epimers, heterodendrin and epi-heterodendrin (Supplemental Figure S12). ²⁰ These 2 compounds are glucosides possessing in their structure the same aglycone part directly linked to the same sugar unit as in compound 1. The fact that the ¹H NMR data of our compound (chemical shifts and coupling constants) (Table 2) show very close similarities to those of epihetrodendrine let us assign the « R » configuration to carbon C-2 as in the epihetrodendrine epimer. ²¹ Thus, from the above data, compound 1, to which the trivial name microcarposide was attributed, was assigned to be (2 R ) 3-methyl-2-[β- l-rhamnopyranoside-1(1→6)-β- d-glucopyranosyl]butanenitrile or (2 R ) 2-[β- l-rhamnopyranoside-1(1→6)-β- d-glucopyranosyl]isovaleronitrile or 6ʹ-O-rhamnosyl-(R)-epiheterodendrine.

Table 2

Different Chemical Shift Values for Heterodendrin, Epiheterodendrin, ²¹ and Compound 1.

Position	(S)-Heterodendrine		(R)-Epiheterodendrine		Compound 1
	δ H (600 MHz, DMSO-d 6)	δ C (150 MHz, DMSO-d 6)	δ H (600 MHz, DMSO-d 6)	δ C (150 MHz, DMSO-d 6)	δ H (500 MHz, DMSO-d 6)	δ C (125 MHz, DMSO-d 6)
1	-	117.8	-	118.6	-	118.1
2	4.74 (J = 6.2 Hz)	70.7	4.55 (J = 5.4 Hz)	72.6	4.51 (J = 5.6 Hz)	71.9
1′	4.32 (J = 7.6 Hz)	100.9	4.29 (J = 7.7 Hz)	103.4	4.34 (J = 7.8 Hz)	101.9

Compounds 1 to 5 were assayed for their antibacterial potency against 3 Salmonella strains, Salmonella typhi (ST), Salmonella typhimurium (STM), and Salmonella enteritidis (SE). As depicted in Supplemental Table S1, only compound 1 exhibited a moderate activity against 3 strains, with minimum inhibition concentration (MIC) values of 153.4, 76.7, and 76.7 µM on ST, STM, and SE, respectively. However, as compared with the reference drug (RD), ciprofloxacine, compound 1 was much less active. However, the activity of this compound against the microbial strains could justify the use of the fruit pulp of this plant for the treatment of infectious diseases, including typhoid fever.

Experimental

General Experimental Procedures

Melting points were measured on a Buchii melting point apparatus. Optical rotations were recorded on a Perkin-Elmer-241 MC Polarimeter, IR spectra on a Bruker Fourier transform/infrared (ATR) spectrophotometer, mass spectra (ESI-MS) on a Thermo-Finnigan LCQ DECA mass spectrometer, and HR-ESI-MS with a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer. 1D- and 2D-NMR spectra were recorded in deuterated solvents on either a Bruker ARX 500 or an AVANCE DMX 600 NMR spectrometer (proton at 500 MHz and carbon ¹³C at 125 MHz). All chemical shifts (δ) were measured in parts per million (ppm) using a residual solvent signal as a secondary reference relative to tetramethylsilane as internal standard, while coupling constants (J) are given in Hz. Solvents were distilled prior to use. Analytical grade solvents were used for LCMS. Column chromatography was performed using Merck MN silica gel 60 M (0.04-0.063 nm), and thin layer chromatography (TLC) on aluminum silica gel 60 F₂₅₄ (Merck) precoated plates (0.2 mm layer thickness). Compounds were visualized on TLC either by the use of an UV lamp (254 and 366 nm) or by heating after spraying with 20% H₂SO₄ (v/v) solution. Different mixtures of n-hexane (hex), EtOAc, CH₂Cl₂, and MeOH were used as eluting solvents.

Plant Material

The fruits of D. microcarpum were harvested in Gamba savanna (Mvina division, Adamaoua region of Cameroon) in March 2018. Identification was made by M. Ngansop Eric, a botanist of the Cameroon National Herbarium, Yaoundé, where a voucher specimen has been deposited under the registration number HNC/57227.

Extraction and Isolation

Air-dried powdered fruit of D. microcarpum (1200 g) was extracted 3 times (3 × 10 L) by maceration at room temperature (about 25 °C) in methanol for 48 hours. After filtration, the resulting solution was concentrated under reduced pressure to give a dark crude extract (145 g).

Part of this extract (130 g) was fractionated using successively hex (4 × 500 mL) dichloromethane (DCM; 4 × 500 mL), ethyl acetate (EA; 4 × 500 mL), and n-butanol (n-BuOH; 4 × 500 mL) through flash chromatography over silica gel (200 g), to yield 4 fractions, namely fraction A (10 g), B (19 g), C (30 g), and D (57 g), respectively. Fraction D (57 g), resulting from n-butanol, was subjected to silica gel CC (column dimension: 4.0 × 60 cm), eluting with a gradient of DCM/MeOH (100:0, 1500 mL; 95:5, 1500 mL; 90:10, 1500 mL; 80:20, 1500 mL; 70:30, 1500 mL; 60:40, 1500 mL; and 50:50, 1500 mL) to yield lupeol (2) (4 mg), β-sitosterol glucoside (4) (3.5 mg), and microcarposide (1) (8.3 mg). Fraction B (19 g), resulting from dichloromethane, was subjected to silica gel CC (column dimension: 3.0 × 65 cm), eluting with a gradient of hex:EtOAc (100:0, 1500 mL; 90:10, 1500 mL; 80:20, 1500 mL; 70:30, 1500 mL; 60:40, 1500 mL; 50:50, 1500 mL; 40:60, 1500 mL; 30:70, 1500 mL; and 0:100, 1500 mL) to afford betulinic acid (3) (7 mg). In a similar manner, fraction C (30 g), resulting from EA, was subjected to silica gel CC (column dimension: 3.0 × 65 cm), eluting with a gradient of hex:EtOAc (100:0, 1500 mL; 90:10, 1500 mL; 80:20, 1500 mL; 70:30, 1500 mL; 60:40, 1500 mL; 50:50, 1500 mL; 40:60, 1500 mL; 30:70, 1500 mL; and 0:100, 1500 mL) to yield methyl gallate (5) (4.1 mg), luteolin (6) (5.3 mg), and epicatechin (7) (6.5 mg).

In Vitro Anti-Salmonella Assays

The antibacterial activity of compounds 1 to 5 was evaluated using the microdilution method. ²² For determination of MICs and minimal bactericidal concentrations (MBCs) of these compounds, 3 Salmonella strains were used: ST, STM, and SE (Supplemental Table S3).

The MIC of compounds 1 to 5 was determined through the broth microdilution method in 96-well microtiter plates. The 96-well plates were prepared by dispensing into each well 50 µL of Mueller Hinton broth. The test substances were initially prepared in DMSO in broth medium at 25 mg/mL. A volume of 100 µL of each test sample was added to the first wells of the microtiter plate. Serial 2-fold dilutions of these test samples were made and 50 µL of inoculum standardized at 10⁶ CFU/mL. The last wells (no. 12) served as sterility controls (contained broth only) or negative control (broth plus inoculum). This gave final concentration ranges from 306.80 to 0.29 μM (for compound) and 386.30 to 0.37 μM (for RD: Ciprofloxacin). The plates were incubated at 37 °C for 24 hours. The MICs of the test compounds were determined following the addition of 20 µL of resazurin (alamar blue TM Cell Viability Reagent) solution. Viable bacteria reduced the yellow due to a pink color. The MIC corresponded to the lowest well concentration where no color change was observed, indicating no growth of microorganism. The MBC was determined by adding 50 µL aliquots of the clear wells to 100 µL of freshly prepared broth medium and incubating at 37 °C for 24 hours. The MBC was regarded as the lowest concentration of test sample that did not produce a color change as above. All tests were performed in triplicate on 2 different occasions.

Microcarposide (1)

C₁₇H₂₉NO₁₀, white powder (8.3 mg); $-[\alpha {]}_{589}^{20}=-99$ (c = 0.5, DMSO); – IR ν_max cm⁻¹ (KBr): 3419(-OH), 2934(-C _sp3-H), 2201(-C≡N), 1422-812(-C _sp3-O), 665-417(-C _sp3-H); ¹H NMR (500 MHz, DMSO): δ _H 4.59 (1H, s, H-1″), 4.51 (1H, d, J = 5.6 Hz; H-2), 4.34 (1H, d, J = 7.8 Hz, H-1′), 3.81 (1H, dd, J = 11.9, 2.2 Hz, H-6b′), 3.63 (1H, d, J = 9.0 Hz, H-2″), 3.46 (1H, d, J = 11.9, 4.3 Hz, H-6a′), 3.43 (1H, d, J = 3.9 Hz, H-3″), 3.42 (1H, d, J = 3.2 Hz, H-5″), 3.32 (1H, s, H-3′), 3.20 (1H, d, J = 4.2 Hz, H-4″), 3.18 (1H, dt, J = 12.6, 6.5 Hz, H-5′), 3.05 (1H, dt, J = 8.9, 4.5 Hz, H-4′), 2.99 (1H, dd, J = 9.9, 6.5 Hz, H-2′), 2.06 (1H, dq, J = 13.3, 6.7 Hz, H-3), 1.13 (3H, d, J = 6.2 Hz, H-6″), 1.11 (3H, d, J = 6.7 Hz, H-5), 0.97 (3H, d, J = 6.8 Hz, H-4); ¹³C NMR (125 MHz, DMSO): δ _C 118.1 (C-1), 101.9 (C-1′), 101.2 (C-1″), 76.9 (C-5′), 76.1 (C-3′), 73.4 (C-2′), 72.4 (C-4″), 71.9 (C-2), 68.8 (C-3″), 70.9 (C-2″), 70,4 (C-4′), 71.1 (C-5″), 67.0 (C-6′), 31.7 (C-3), 18.4 (C-6″), 18.2 (C-4), 17.6 (C-5), see Supplemental Table S1; HR-ESI-TOF-MS: m/z 408.1865 (calcd. for C₁₇H₃₀NO₁₀ ⁺: 408.1864).

Supplemental Material

Supplementary Material 1 - Supplemental material for An Antibacterial Isovaleronitrile Diglycoside From Detarium microcarpum Guill. Perr. (Fabaceae)

Supplemental material, Supplementary Material 1, for An Antibacterial Isovaleronitrile Diglycoside From Detarium microcarpum Guill. Perr. (Fabaceae) by William F. Feudjou, Arnaud M. Mbock, Marlyse B. W. Ouahouo, Valérie T. Sielinou, Racéline K. Gounoue, Pierre Mkounga, Bruno N. Lenta, Théophile Dimo, Fabrice B. Fekam, Norbert Sewald and Augustin E. Nkengfack in Natural Product Communications