Phytochemical Characterizations of Maranthes polyandra (Benth.) Prance

Two new ursane-type triterpenoids, named Polyanside A (1) and B (2), along with eleven known compounds (3–13), were isolated and elucidated from Maranthes polyandra (Benth.) Prance. The structures of these compounds were elucidated based on chemical evidence and multiple spectroscopic data. Isolated compounds were evaluated for anti-cancer, anti-inflammatory activities, and cytotoxicity on a normal human cell line (BJ). None of them showed activity and cytotoxicity. The hexane fraction was analyzed by GC-MS, resulting in the identification of forty-one compounds. This is the first comprehensive study on the phytochemistry of M. polyandra.


Introduction
The history of medicinal plants is as old as the history of human beings. Natural products have played a vital role in drug discovery. The use of natural components from folk medicines requires a clear understanding of chemistry, efficacy, and safety. Now, there has been a surge in interest in valorizing the biological importance of medicinal plants [1,2]. It is a pressing priority to obtain potent phytoconstituent from different medicinal plants and to explore their promising benefits [3,4].
Thus, the current study aimed to explore the phytochemical constituent from M. polyandra through isolation and GC-MS analysis. Finally, two new triterpenoids (1 and 2), and eleven known compounds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) were isolated. In addition, GC-MS analysis of the hexane fraction also led to the identification of 41 compounds. This is the first comprehensive phytochemistry study of this species. Anti-cancer and anti-inflammatory activities and cytotoxicity of compounds 1, 2, 5, 6, 8, 11, and 13 were evaluated. None of them were active. Based on traditional uses, anti-inflammatory components might exist in this plant. Further study may be required to discover potent anti-inflammatory molecules from this species.

Structure Elucidation of Isolated Compounds
First, 80% MeOH extract of the stem bark of M. polyandra was fractioned by n-hexane for GC-MS analysis. The remaining residue was then isolated using chromatographic techniques, such as silica gel column chromatography (CC), C18 CC, Sephadex LH 20 CC, and HPLC. Thirteen compounds (1-13) were obtained, including two new compounds (1 and 2) and eleven known compounds (3-13) ( Figure 1). The structures of 1 and 2 were elucidated mainly through NMR techniques, primarily based on 1D NMR ( 1 H and 13 C NMR), 2D NMR (COSY, HSQC, HMBC, and NOESY), and MS techniques including EI-MS and HR-EI-MS. and antioxidant effects [14]. However, phytochemistry investigation of this species is extremely limited, except for a few studies [15][16][17][18]. Until now, only three compounds (xanthoxylin, β-eudesmol, luteolin) have been isolated from this plant [15]. The composition of seed oil has been analyzed by GC-MS [17,18]. In addition, GC-MS analysis on the extract and fractions of M. polyandra stem bark just confirmed the presence of some fatty acids [16]. Thus, the current study aimed to explore the phytochemical constituent from M. polyandra through isolation and GC-MS analysis. Finally, two new triterpenoids (1 and 2), and eleven known compounds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) were isolated. In addition, GC-MS analysis of the hexane fraction also led to the identification of 41 compounds. This is the first comprehensive phytochemistry study of this species. Anti-cancer and anti-inflammatory activities and cytotoxicity of compounds 1, 2, 5, 6, 8, 11, and 13 were evaluated. None of them were active. Based on traditional uses, anti-inflammatory components might exist in this plant. Further study may be required to discover potent anti-inflammatory molecules from this species.

Structure Elucidation of Isolated Compounds
First, 80% MeOH extract of the stem bark of M. polyandra was fractioned by n-hexane for GC-MS analysis. The remaining residue was then isolated using chromatographic techniques, such as silica gel column chromatography (CC), C18 CC, Sephadex LH 20 CC, and HPLC. Thirteen compounds (1-13) were obtained, including two new compounds (1 and 2) and eleven known compounds (3-13) ( Figure 1). The structures of 1 and 2 were elucidated mainly through NMR techniques, primarily based on 1D NMR ( 1 H and 13 C NMR), 2D NMR (COSY, HSQC, HMBC, and NOESY), and MS techniques including EI-MS and HR-EI-MS.  were suggestive of a urs-12-en-3-one skeleton. All NMR data showed great similarity with α-amyrone except an extra oxymethine signal at δ H 4.49 (br s) that is correlated with δ C 69.3 in HSQC [19,20]. The presence of a hydroxyl was confirmed at C-6 through COSY correlations between H-5 (δ H 1. 22 1.22) and H-6 (δ H 4.49) was observed. Thus, the structure of compound 1 was elucidated as shown in Figure 1 and named Polyanside A. Key 1 H-1 H COSY, HMBC, and NOESY correlations are shown in Figure 2 ( Figures S14-S21).

Phytochemical Investigation of Hexane Fraction by GC-MS
GC-MS analysis of the hexane fraction revealed the presence of different phytochem icals, which are shown in Figure 3 and listed in Table 2.
By deciphering the results obtained from the GC-MS analysis, it was observed th M. polyandra contained various phytochemicals that are known for their different medi nal and economical importance. These results were acquired firstly through gas chrom togram, in which area of the peaks indicated the relative concentration of the phytoco stituent present in hexane fraction, and their structures were identified through NIS online database for mass spectrometry. The obtained phytochemicals have been report to possess different biological activities, including antimicrobial, antioxidant, anti-inflam matory, and anti-cancer effects. These results provided new knowledge about the no polar components from M. polyandra.  (Table 1, Figures S27-S33) of 2 is in good agreement with 1, except one more oxymethine proton at δ H 3.14 (dd, J = 10.0, 5.0 Hz) and the absence of a carbonyl signal. The location of δ H 3.14 was confirmed at C-3 through HMBC correlations of δ H 3.14 with C-2 (δ C 27.4), C-5 (δ C 55.5), C-23 (δ C 17.0), and C-24 (δ C 28.05), along with COSY correlations between δ H 3.14 and H-2 (δ H 1.61 and 1.63). In addition, H-3 exhibited correlations with H-5 (δ H 0.74, d, 2.0 Hz), implying a β-orientation of the hydroxyl at C-3. Therefore, the structure of compound 2 was elucidated as shown in Figure 1 and named Polyanside B. Key COSY, HMBC, and NOESY correlations are shown in Figure 2 ( Figures S34-S41).
Compounds 1, 2, 5, 6, 8, 11, and 13 were performed for anti-cancer activity against MCF-7 cell (breast cancer), NCI-H460 (lung cancer), Hela (cervical cancer), and cytotoxicity against normal human cell line BJ, which were obtained from a cell culture biobank (PCMD, ICCBS) of American Type Culture Collection (ATCC), MTT assay was used for this activity (S3.5) [32]. All of them were observed to be inactive and nontoxic with inhibition < 50% at 50 µM. Compounds 1, 2, 5, 6, 8, 11, and 13 were also screened for nitric oxide (NO) inhibitory activity by a previously described method (S3.6) [33]. Unfortunately, all tested compounds displayed <50% inhibition at 25 µg/mL. The methanol extract and hexane fraction were tested for the same assays. However, they were inactive. Compound 6 was reported to possess a good antinociceptive effect conferring to hot-plate and tail-flick assays [34]. Compounds 6 and 11 have been claimed to be the responsible components of an active extract to inhibit the growth of A549 cells (lung carcinoma epithelial cells) by analyzing the extract by LC-MS-MS [35]. However, in the current study they were inactive against NCI-H460 (lung cancer). Sari et al. evaluated the antimicrobial potential of 6 and 8. It was observed that 6 inhibited S. aureus with MIC of 9.4 µg/mL. Meanwhile, 8 inhibited S. enterica with MIC of 37.5 µg/mL [36]. To the best of our knowledge, it is the first time to test compounds 1, 2, 5, 6, 8, 11, and 13 for their anti-cancer potential (against MCF-7, HeLa, and H460) as pure compounds.
By deciphering the results obtained from the GC-MS analysis, it was observed that M. polyandra contained various phytochemicals that are known for their different medicinal and economical importance. These results were acquired firstly through gas chromatogram, in which area of the peaks indicated the relative concentration of the phytoconstituent present in hexane fraction, and their structures were identified through NIST online database for mass spectrometry. The obtained phytochemicals have been reported to possess different biological activities, including antimicrobial, antioxidant, anti-inflammatory, and anticancer effects. These results provided new knowledge about the non-polar components from M. polyandra.

General Experimental Procedures
Low-resolution mass spectra EI-MS were chronicled on a JEOL MS route JMS 600H instrument, and HR-EI-MS was analyzed on Thermo Finnigan MAT 95XP linked with X-Calibur. The 1 H and 13 C NMR spectra were recorded on a Bruker Avance NEO-500, 400 NMR spectrometer in CDCl 3 at 500, 400, and 125 MHz, respectively. The UV was checked on the Evolution TM 300 Spectrophotometer, and FT-IR spectra were recorded on a Bruker Vector 22 spectrophotometer. Optical rotations were determined on a JASCO 2000 Polarimeter. The purity of the compounds was verified on TLC (Silica gel, Merck F254, 0.25 mm thickness). Melting points were determined in glass capillary tubes using the Buchi melting point apparatus. For the TLC plate's visualization, vanillin and ceric sulfate staining reagents were used. All experiments were performed at room temperature using solvents acquired commercially and used without further purification.

Collection of Plant Material
The stem bark of Maranthes polyandra (Benth.) Prance was collected by Mr. Kayode Muritala Salawu, a Senior Lecturer in the Department of Pharmacognosy and Drug Development, University of Ilorin, Kwara State, Nigeria, in August 2018 in the main campus of the University of Ilorin. The plant was identified and authenticated at the Herbarium Unit of the Department of Plant Biology, University of Ilorin, where the voucher specimen was deposited by the synonym Parinari polyandra Benth and voucher number (UILH/001/582/2021) was issued.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The hexane fraction was analyzed through Agilent 7000 GC/MS triple Quad, and Agilent 7890A GC system. The Agilent 7890A GC detector was used to accomplish the analysis. OPTIMA SN 23102-72 OPTIMA-5 was used to give temperature the maximum temperature during the analysis was 325 • C (30 m × 250 µm × 0.25 µm) and the phytocomponent were separated using helium as a carrier gas at a constant flow of 1.129 mL/min. A 2 µL volume of sample was injected, then analyzed by the Agilent 7000 triple quad mass detector. Initially, the temperature was maintained at 50 • C for 3 min, then it increased with 7 • C/min till 200 • C in 20 min, and then 7 • C/min till 300 • C in 25 min. Total runtime was 83.71 min. During this process, the injector temperature was maintained at 250 • C. Agilent 6890 gas chromatograph equipped with ZB-5MS (30 m × 0.32 ID and 0.25 µm film thickness) was combined with a Jeol, JMS-600H mass spectrometer operating in EI mode with ion source at 250 • C, and electron energy at 70 eV. Carrier gas volume was adjusted between 1.0 and 5.0 µL depending upon the detector response. The library used to identify the constituents was NIST Mass Spectral Search Program and Kovat's retention indices.

Conclusions
Two undescribed ursane-type triterpenoids, named Polyanside A (1) and B (2), along with eleven known compounds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13), were isolated and elucidated from Maranthes polyandra (Benth.) Prance. The structures of these compounds were elucidated based on chemical evidence and multiple spectroscopic data. The hexane faction was analyzed by GC-MS, resulting the identification of forty-one compounds. The results contributed new knowledge to the phytochemistry of M. polyandra. Unfortunately, the tested compounds 1, 2, 5, 6, 8, 11, and 13 were found to be inactive on the anti-cancer and inflammatory assay. In addition, other compounds were not able to be employed for activity evaluation due to the poor quantity. The limited quantity of initial material presented difficulty in isolating more pure components or a greater quantity of obtained compounds. Further study with a sufficient quantity of initial material is required to discover potent molecules from this plant.