Two new triterpenes from Commicarpus grandiflorus (A. Rich.) Standl. aerial parts exudate

Abstract The exudate of Commicarpus grandiflorus (A. Rich.) Standl. flowering aerial parts was investigated for its chemical composition. Nine compounds were isolated, five triterpenes and four methylated flavones, of which two were new natural triterpenes, 2α,3β,11α-olean-18-en-2,3,11-triol (1) and 2α,3β-olean-12-en-2,3-diol-11-one (2) that were named commicarpotriol and commicarpodiol, respectively. Structural characterization was carried out using 1D, 2D NMR, and MS techniques and the antimicrobial activity of all isolates was evaluated. GRAPHICAL ABSTRACT


Introduction
Commicarpus grandiflorus (A. Rich.) Standley (Nyctaginaceae) is one of five species wildly growing in Saudi Arabia. The genus Commicarpus is morphologically very close to the genus Boerhavia, thus originally considered as a section of Boerhavia. In 1909, Standley separated the genus Commicarpus from Boerhavia for reasons related to differences in habit and morphology (climbing plants in Commicarpus, diffuse in Boerhavia); in addition, Commicarpus fruits have viscid and mucilaginous glands, the perianth in Commicarpus is a funnel-shaped while in Boerhavia is bell-shaped (Struwig and Siebert 2013).
The aerial parts of C. grandiflorus produce abundant quantity of viscid and sticky exudates, which cause sometimes a stiff attachment of some insects to death. The water extract of the leaves is popularly used to kill head lice by washing the hair (Al-Fatimi 2019) and to treat lower extremity weakness and rheumatism (Kidane et al. 2014). A previous phytochemical study revealed the presence of flavonol 3-O-glucosides, betulinic acid, and b-sitosterol in the plant (Abou-Hussein et al. 2017). The petroleum ether subfraction of methanolic extract of C. grandiflorus showed a potent antiprotozoal effect against Plasmodium falciparum, Trypanosoma brucei, T. cruzi, and Leishmania infantum (Abdel-Sattar et al. 2010). The methanol extract displayed antimicrobial activity in agar diffusion method against Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus, Sarcina lutea, Mycobacterium phlei, Bacillus subtilis, Candida albicans, and Escherichia coli (Abdel-Sattar et al. 2008). Since plant exudates are considered as a protective factor from infections caused by bacteria, fungi and viruses, as well as insect repellent (Nagy et al. 2017;Morimoto 2019;Zhang et al. 2020), the aim of this study was to isolate the constituents of C. grandiflorus surface exudate and to evaluate their antimicrobial activity. This work is part of our ongoing research project on investigation of surface exudate of Saudi Arabia medicinal plants (Dal Piaz et al. 2018).

Results and discussion
The surface exudate of C. grandiflorus aerial parts was fractionated by Biotage Isolera column chromatography followed by RP-HPLC, leading to the isolation of two new triterpenes (1,2) and seven known compounds (3-9) ( Figure 1).
Compound 1 displayed the molecular formula C 30 H 50 O 3 as determined by HRESIMS and 13 C NMR spectra (sodiated molecular ion at m/z 481.3649 [M þ Na] þ ). Analysis of the 1 H and 13 C NMR spectra revealed the presence of a triterpene having an oleanane skeleton with three hydroxyl groups and one double bond (Mahato and Kundu 1994). Analysis of the 1D TOCSY spectra in combination with the COSY experiment revealed the presence of a -CH 2 -CH-OH-CH-OH spin system for the ring A. The irradiation of the signal at The ring B showed a -CH-CH 2 -CH 2 -spin system, the ring D a -CH 2 -CH 2 -spin system, while the ring E two separated spin systems constituted by a -CH-and a -CH 2 -CH 2 -moiety. Analysis of the HSQC-DEPT spectrum allowed to define the direct hydrogen-carbon correlations of all atoms in the molecule and to distinguish CH 2 signals from CH. Finally, the HMBC experiment established the exact position of the functional groups due to the unambiguous correlations between H-1-C-3 and H-1-C-5; H-9-C-1, H-9-C-10, H-9-C-11, and H-9-C-14; H-13-C-14 and H-13-C-18; H-19-C-17; Me-25-C-1, Me-25-C-5, Me-25-C-8, and Me-25-C-10; Me-28-C-17, Me-28-C-18, and Me-28-C-22. The stereochemistry of the hydroxyl group at C-11 was established by the 2D-ROESY experiment which showed the correlations between d 3.94 (H-11) and d 1.17 (Me-25) and d 2.46 (H-13) allowing to establish H-11 in b position; similarly, H-2 resulted in b position due to the correlation between d 3.61 (H-2) and d 0.84 (Me-24), while H-3 resulted in a position (Topçu and Ulubelen 1999). Thus, compound 1 was characterized as 2a,3b,11a-olean-18-en-2,3,11-triol, and named commicarpotriol, a new natural triterpene.
Compound 2 had molecular formula C 30 H 48 O 3 and molecular weight of 456 u, as inferred from the HRESIMS mass spectrum recorded in the positive ion mode showing the sodiated molecular ion [M þ Na] þ at m/z 479.3501 and the protonated molecular ion [M þ H] þ at m/z 457.3676. In the HRESIMS/MS fragmentation spectrum the peak at m/z 435.2619 [M þ Na-44] þ , due to the loss of 44 u was observed. The 1 H-NMR spectrum showed again the presence of signals typical of a triterpene having an oleanane skeleton (Braca et al. 2001). The COSY correlations, observed between H 2 -1-H-3, H-5-H 2 -7, H 2 -15-H 2 -16, H-18-H 2 -19 and H 2 -21-H 2 -22, let to obtain each spin system of the triterpene rings. Moreover, the HSQC spectra revealed the presence of five spin systems corresponding to the respective rings of the triterpene in which two hydroxymethynes were evident in ring A and an unsaturation conjugated to a carbonyl group in ring C. Finally, the HMBC spectrum allowed to determine the exact position of the functional groups by correlations between H-9-C-1, H-9-C-11, and H-9-C-26; H-12-C-18 and H-12-C-19; Me-25-C-1, Me-25-C-5, Me-25-C-9, and Me-25-C-10; Me-28-C-17, Me-28-C-18, and Me-28-C-22 and to identify compound 2 as 2a,3bolean-12-en-2,3-diol-11-one, named commicarpodiol, a new naturally occurring triterpene.
Finally, the metabolomic profile of C. grandiflorus aerial parts exudate was obtained through analytical investigation by HPLC-PDA-ESI-MS/MS operating in both negative and positive ion modes, resulting in two different chromatographic profiles. In particular, the LCMS profile obtained in the negative ion mode ( Figure S14) was characterized by three main peaks corresponding to compounds belonging to the class of methylated flavones. The identification of the compounds was performed by comparison of chromatographic data (retention times, t R ), UV and ESI-MS (full and fragmentation patterns) with data of pure compounds obtained from the isolation process and identified by 1D and 2D NMR experiments in the present work. The three compounds showed two bands of maximum absorption at k max ¼ 275-280 and k max ¼ 330-335, typical of flavones. In addition, the loss of -CH 3 ([M-H-15] -) in the ESI-MS/MS fragmentation experiments of precursor ions confirmed the presence of methyl groups on the core of the flavones. Therefore, compounds were identified as 6, 7, 8, and 9. The chromatogram recorded in the positive ion mode ( Figure S15) showed a different profile with several peaks observed in the apolar region fully identified as triterpenes, by comparison of chromatographic and MS data with the data of reference standards, isolated in the present work. The compounds were identified as 1, 2, 4, and 5.
Plant exudate composition is quite variable and includes high-molecular-weight molecules (sugar, lipids, proteins) and low-molecular-weight signalling molecules (organics and amino acids) (Badri and Vivanco 2009). These mixtures generally play a protective role for plants, exerting biological activities such as antimicrobial and antioxidant. Moreover, some compounds herein isolated from the exudate of C. grandiflorus aerial parts have been previously reported as antimicrobial agents; in particular, Brez ani et al. (2018)  Based on these evidences, and taking into account the difficulty of using the whole exudate to perform biological assays due to its complexity and viscosity, the isolated compounds were assayed for their potential antimicrobial activity. Compounds 1-9 were therefore tested against a collection of Gram-positive and Gram-negative bacteria (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus mutans, Citrobacter, Salmonella, Bacillus subtilis, Shigella, Bacillus clausii, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Listeria monocytogenes), at concentration from 10 to 200 lg/mL (0, 10, 25, 50, 100, 200 lg/mL). As reported in Table 1, among the analysed compounds, 1, 8, and 9 were active against S. mutans, whereas only compound 8 showed an antimicrobial activity against S. aureus (Table  1), in agreement with the data present in the literature. More interestingly, compound 1 showed a significant activity against S. mutans. A comparison between the structure of 1 and those of the other structurally related but inactive compounds (2, 4, and 5), suggested that the hydroxyl group at C-11 could play a key role in the bioactivity of this molecule.
S. mutans and S. aureus are considered the most relevant bacteria in the transition of non-pathogenic commensal oral microbiota which contribute to the dental caries process and are associated with progression of periodontal diseases (Struzycka 2014); these bacteria are etiological agents of the oral cavity pathologies and are involved in other diseases such as cardiovascular disease, pneumonia, rheumatoid arthritis, pancreatic cancer, and colorectal cancer (Frias-Lopez and Duran-Pinedo 2020). The anti-S. mutans activity measured for 1 could therefore suggest it as a possible lead for the development of new therapeutic agents against oral infections.

General experimental procedures
For NMR analyses, a Bruker DRX-400 spectrometer (Topspin software) was used operating at 400 and 100 MHz for 1 H and 13 C NMR spectra, respectively. All spectra were recorded at 300 K in CD 3 OD (Merck). Chemical shifts were reported in d (ppm) and referred to the solvent signals d H 3.31 and d C 49.0 . The standard pulse sequence and phase cycling were used for COSY, TOCSY, HSQC, HMBC, and ROESY experiments. TLC was performed on precoated Kieselgel 60 F254 plates (Merck); compounds were detected by spraying with Ce(SO 4 ) 2 /H 2 SO 4 (Sigma Aldrich). Column chromatography was performed over the BiotageV R Isolera TM Spektra flash purification instrument (Agilent) with silica gel (Merck); RP-HPLC separations were conducted on a Shimadzu LC-8A series pumping system equipped with a Shimadzu RID10A refractive index detector and a Shimadzu injector, using a C 18 m-Bondapak column (30 cm x 7.8 mm, 10 mm, Waters-Milford). HPLC-PDA-ESI-MS/MS was carried out using a Surveyor LC pump, a Surveyor autosampler, coupled with a Surveyor PDA detector and a LCQ Advantage ion trap and electrospray ionization source mass spectrometer (ThermoFinnigan), equipped with the Xcalibur 3.1 program. Analysis was performed using a Synergi Fusion-RP column (Phenomenex) 4.6 Â 150 mm size, 4 mm particles. A mixture consisting of methanol (solvent A) (Merck) and water acidified with formic acid (0.1%) (solvent B) (Merck) was used as the eluent, according to the following linear gradient: 0-65 min, 40-100% A, 65-80 min, 100% A.

Plant material
Flowering aerial parts of C. grandiflorus were collected at Wadi Thee Ghazal in the mountains of Taif region of Saudi Arabia in January 2015 and identified by one of the authors, Prof. A. Bader. A voucher specimen (number SA-IT/2015-2) has been deposited at the herbarium of the Laboratory of Pharmacognosy, Faculty of Pharmacy at Umm Al-Qura Univesrity, Makkah, Saudi Arabia.

Extraction and isolation
The aerial parts of C. grandiflorus (135 g) were subjected to extraction for 30 s in CHCl 3 and an exudate with semi-liquid consistency (10.6 g) was obtained. A portion of the exudate (2.5 g) was subjected to silica gel column chromatography using the BiotageV R Isolera TM Spektra flash purification method, eluting with solvent mixtures of increasing polarity, n-hexane, CHCl 3 , and MeOH. The sample was loaded into SNAP samplet cartridge of 340 g flash silica and the wavelengths considered during the analysis were k ¼ 254 and 320 nm. A total of 98 fractions were collected and grouped into 13 main fractions, A-L, due to the characteristics inferred from thin-layer chromatography (TLC) on 60 F254 silica gel plate using CHCl 3 and CHCl 3 -MeOH (95:5) as eluents and cerium sulfate as a spray reagent. Fractions B (20.4 mg), C (33.1 mg), D (44.7 mg), G (24.2 mg), H (54.5 mg), and L (38.1 mg) were injected into the RP-HPLC system with MeOH-H 2 O (95:5) as mobile phase at flow rate of 2 mL/min. Compound 6 (2.3 mg, t R 10 min) from fraction B, compounds 3 (2.8 mg, t R 13 min) and 4 (2.4 mg, t R 25 min) from fraction C, compounds 7 (2.9 mg, t R 9 min) and 8 (2.9 mg, t R 11 min) from fraction D, compound 5 (2.5 mg, t R 31 min) from fraction G were purified, respectively. Compound 1 (2.6 mg, t R 15 min) was obtained from fraction L and compounds 2 (2.1 mg, t R 14 min), 3 (29.6 mg, t R 28 min), and 5 (3.0 mg, t R 32 min) were isolated from fraction H. Finally, fraction F (58.5 mg) was subjected to RP-HPLC with MeOH-H 2 O (4:1) as a mobile phase at flow rate of 2 mL/min yielding compound 9 (2.3 mg, t R 9 min).

Spectroscopic data
Compound suspensions of microorganisms), a vehicle control (sterile culture medium with DMSO), and antimicrobial agent chlorhexidine gluconate (CHX) were used. The MIC was determined as the lowest drug concentration that inhibited visible bacterial growth. All determinations were done in triplicate, Student's t-test was used, and the data considered statistically significant at p 0.05.

Conclusions
The phytochemical study of C. grandiflorus flowering aerial parts exudate allowed the isolation and the identification of nine secondary metabolites (1-9), belonging to the classes of methylated flavones and triterpenes, of which two were new natural triterpenes. In the latest years, the need for new antibacterial constituents has become a gradually central issue to respond to pathogens resistance mechanisms, which are increasing at an alarming rate. Compound 1 showed anti-S. mutans activity, for this reason it could therefore be suggested as a possible lead for the development of new therapeutic agents against oral infections. Moreover, to further investigate the antibacterial activity of 1, the possible mechanism of action against bacteria such as efflux pump inhibition, biofilm inhibition will be studied.