Prenylated Flavonoids and C-15 Isoprenoid Analogues with Antibacterial Properties from the Whole Plant of Imperata cylindrica (L.) Raeusch (Gramineae)

The local botanical Imperata cylindrica in Cameroon was investigated for its antibacterial potency. The methanol extract afforded a total of seven compounds, including five hitherto unreported compounds comprising three flavonoids (1–3) and two C-15 isoprenoid analogues (4 and 5) together with known derivatives (6 and 7). The novelty of the flavonoids was related to the presence of both methyl and prenyl groups. The potential origin of the methyl in the flavonoids is discussed, as well as the chemophenetic significance of our findings. Isolation was performed over repeated silica gel and Sephadex LH-20 column chromatography and the structures were elucidated by (NMR and MS). The crude methanol extract and isolated compounds showed considerable antibacterial potency against a panel of multi-drug resistant (MDR) bacterial strains. The best MIC values were obtained with compound (2) against S. aureus ATCC 25923 (32 µg/mL) and MRSA1 (16 µg/mL).


Introduction
Infectious diseases cause 15 million deaths each year, accounting for approximately 27.12% of deaths worldwide [1]. The rapid emergence of multi-drug resistant bacteria has severely compromised the efficacy of antibiotics and, hence, the fight against bacterial infections worldwide [2]. The search for new compounds to tackle bacterial resistance has become imperative. African flora is a rich source of bioactive substances, and several medicinal plants have proven their ability to inhibit the growth of Gram-negative bacteria [3][4][5][6]. The present study focusses on Imperata cylindrica (Gramineae), an aggressive grass endemic to tropical and subtropical Asia, Australia, Africa, and southern Europe [7][8][9]. This plant species has different names in various languages around the World, such as "speargrass" in Nigeria, "alang-alang" in Asia, "cogongrass" in America, or "Ngnig-gah" in Western Cameroon, amongst others [7]. I. cylindrica can grow up to 1.2 m high with narrow and rigid leaves, bearded at the base and glabrous in their upper part [10]. It adversely affects agroforestry operations because it suppresses the growth of trees and crop plants and is a fire hazard [7,[11][12][13]. I. cylindrica constitutes one of the most reputed species of the genus narrow and rigid leaves, bearded at the base and glabrous in their upper part [10]. It adversely affects agroforestry operations because it suppresses the growth of trees and crop plants and is a fire hazard [7,[11][12][13]. I. cylindrica constitutes one of the most reputed species of the genus after some species previously thought to be members of this genus were reassigned to other groups of plants. It is reputed in traditional medicine for the treatment of hemorrhage, improvement of urination, and immunostimulation [14]. The rhizomes of this plant are advised by traditional healers as a diuretic and are used to treat inflammations and fever in Korean herbal medicine [15]. Investigations carried out on this plant species have revealed antibacterial and anticancer activities [16,17]. Furthermore, the root extract of I. cylindrica is non-toxic upon short and long-term oral administration [18]. The chemistry and pharmacology of I. cylindrica are diverse and rich. The plant contains coumarins and chromones [19], lignans [20], methoxylated flavonoids [19], phenolic acids [21], and steroids [22]. Crude extracts and isolated compounds from this plant showed a broad band of biological potency including cytotoxic, neuroprotective, and vasodilatory effects [20]. Within the frame of our ongoing research into the characterization of bioactive metabolites from Cameroonian folk medicine [23][24][25][26][27][28], we report herein the occurrence of five unprecedented compounds in nature with antibacterial properties.
Likewise, compound 3 was isolated in the form of a yellow powder and its molecular formula C21H20O6 was deduced from its HRESI-MS, which showed the sodium adduct [M + Na] + at m/z 391.1140 (calcd. m/z 391.1152 for C21H20O6Na + ). The IR spectrum showed absorption bands at 3355 (O-H), 2922 (C-H), 1642 (C=O), 1600 (C=C), and 1087 (C-O) cm −1 . The NMR spectra of 3 (Table 2) were similar to those of compound 2 except the lack of resonances attributable to characteristic aliphatic signals of flavanones and rather the appearance of a one-proton singlet at δH/δC 6.55 (1H, s, H-3)/113.3 (C-3) in compound 3. Moreover, each of the meta-coupled protons H-2′ and H-6′, as well as the olefinic proton H-3, showed HMBC cross peaks to an oxygenated olefinic carbon at δC 146.1 (C-2). These resonances identified compound 3 as a flavone [24,34].
Likewise, compound 3 was isolated in the form of a yellow powder and its molecular formula C 21 (Table 2) were similar to those of compound 2 except the lack of resonances attributable to characteristic aliphatic signals of flavanones and rather the appearance of a one-proton singlet at δ H /δ C 6.55 (1H, s, H-3)/113.3 (C-3) in compound 3. Moreover, each of the meta-coupled protons H-2 and H-6 , as well as the olefinic proton H-3, showed HMBC cross peaks to an oxygenated olefinic carbon at δ C 146.1 (C-2). These resonances identified compound 3 as a flavone [24,34]. Compounds 2 and 3 are unique in nature because of the upfield methyl group encountered at C-8 in both structures paired with the prenyl substituent at C-5 . Gancaonin E, a known compound related both to 2 and 3, is prenylated at the C-8 position [35]. The methyl group at C-8 in our derivatives might originate from the oxidation of the prenyl substituent in related analogues. Various flavonoids containing methyl groups in either position C-6 or C-8 have been isolated [33]. Thus, 2 and 3 are newly characterized and we proposed the trivial names cylindricines A (2) and B (3), respectively. The olefin was also adjacent to a hydroxylated quaternary carbon at δ C 71.9 (C-5), bearing a methyl group as evidenced by HMBC contacts from the methyl to nearby resonances C-4 (δ C 156.2), C-5 (δ C 71.9), and C-6 (δ C 49.6). The COSY spectrum evidenced square correlations between the methine H-6 (δ H 2.86, m) and the methylene protons H-7a (δ H 2.99, dd, J = 15.9, 6.1 Hz) and H-7b (δ H 2.88, dd, J = 15.9, 6.1 Hz). Both groups of protons also showed HMBC contacts to a second α, β-unsaturated carbonyl moiety at δ C 203.4 (C-8), 140.9 (C-10), and 137.5 (C-9), bearing a methyl at δ H /δ C 1.75 (s, H-15)/10.9 (C-15) and a hydroxymethylene at δ H /δ C 4.36 (d, J = 5.7 Hz, H-11)/58.9 (C-11) judged by HMBC contacts between carbons C-9, C-8, and C-10.
The relative stereochemistry of the stereocenters and olefins was established based on the coupling constants and ROESY cross peaks (Figure 3) of 4. H-4 and H-3 showed a cis arrangement with J = 10.3 Hz as further suggested by the absence of ROESY interaction between these protons. In addition, the ROESY spectrum showed interactions from the methyl H-14 to H-7a but to none of the geminal dimethyl at C-1, indicative of the equatorial position of the methyl C-14. The β-position of H-6 was confirmed by cross peaks visible from H-7b to the β-methyl H-13 and from H-6 to H-13 and H-7b. The interaction from H-7b to the olefin H-10 indicated a δ-transoid conformation of the α, β-unsaturated ketone at C-8 with a cis-relationship of the methyl and hydroxymethylene groups as illustrated by ROESY cross peaks from H-15 to H-11. Compound 4 was thus characterized as 3Z, 9E, 5S, 6R, similar to reported analogues in the literature [36,37]. Compound 4 was thus a new C-15 isoprenoid analogue related to abscisic acid, a well-known plant hormone involved in abscission [37]. We proposed the trivial name cylindracid A (4).
Molecules 2021, 26, x FOR PEER REVIEW 6 of 11 5S, 6R, similar to reported analogues in the literature [36,37]. Compound 4 was thus a new C-15 isoprenoid analogue related to abscisic acid, a well-known plant hormone involved in abscission [37]. We proposed the trivial name cylindracid A (4). Compound 5 was obtained as a brown oil and its molecular formula C11H16O4 was deduced from its HRESI-MS, which exhibited the pseudo-molecular ion [M−H] − at m/z 211.0985 (calcd. m/z 211.0975 for C11H15O4 − ). The NMR spectra profiles of 5 (Table 1) were similar to those of compound 4 except the lack of resonances of the prenyl group attached to C-7 and the appearance of a carboxylic group resonance at δC 177.5 (C-8) in compound 5. Compound 5 could be derived from its parent compound 4 by oxidation of the side prenyl chain at C-7. Its stereochemistry was found to be similar to that of compound 4, the angular methyl C-11, and the new carboxylic group, with both being equatorially oriented. Compound 5 was thus newly characterized and we proposed the trivial name cylindracid B. The corresponding IR, MS and NMR spectra of each of the compounds described here are available as supplementary material ( Figures S1-S38).
While compounds 4 and 5 were isolated in low amounts and thus some signals were missing in their respective 1 H-NMR and 13 C-NMR spectra, the signal assignment for both compounds could be completed by using the respective spectra of the mixture of 4 and 5.
The known compounds were identified as 3',4',5,5',7-pentahydroxyflavanone (6) [38] and mearnsetin (7) [39] by comparison of their NMR and MS data with those reported in the literature. A mixture of stigmasterol and β-sitosterol was also isolated and identified by comparison of the analytical TLC and IR spectra with a sample from our laboratory.
As shown by the results presented in Table 3, the crude methanol extract of I. cylindrica, as well as the isolated compounds, inhibited the growth of all the tested Gram-positive and Gram-negative microorganisms. Depending on the microorganism, the MIC values of the crude extract ranged from 256 to 1024 μg/mL, while the MIC values of the pure compounds ranged from 128 to 1024 μg/mL (1), 16 to 128 μg/mL (2), 64 to 256 μg/mL (3), 512 to 1024 μg/mL (4), 64 to 512 μg/mL (5), and 32 to 128 μg/mL (6).   (Table 1) were similar to those of compound 4 except the lack of resonances of the prenyl group attached to C-7 and the appearance of a carboxylic group resonance at δ C 177.5 (C-8) in compound 5. Compound 5 could be derived from its parent compound 4 by oxidation of the side prenyl chain at C-7. Its stereochemistry was found to be similar to that of compound 4, the angular methyl C-11, and the new carboxylic group, with both being equatorially oriented. Compound 5 was thus newly characterized and we proposed the trivial name cylindracid B. The corresponding IR, MS and NMR spectra of each of the compounds described here are available as supplementary material (Figures S1-S38).
While compounds 4 and 5 were isolated in low amounts and thus some signals were missing in their respective 1 H-NMR and 13 C-NMR spectra, the signal assignment for both compounds could be completed by using the respective spectra of the mixture of 4 and 5.
The known compounds were identified as 3',4',5,5',7-pentahydroxyflavanone (6) [38] and mearnsetin (7) [39] by comparison of their NMR and MS data with those reported in the literature. A mixture of stigmasterol and β-sitosterol was also isolated and identified by comparison of the analytical TLC and IR spectra with a sample from our laboratory.
As shown by the results presented in Table 3, the crude methanol extract of I. cylindrica, as well as the isolated compounds, inhibited the growth of all the tested Gram-positive and Gram-negative microorganisms. Depending on the microorganism, the MIC values of the crude extract ranged from 256 to 1024 µg/mL, while the MIC values of the pure compounds ranged from 128 to 1024 µg/mL (1), 16 to 128 µg/mL (2), 64 to 256 µg/mL (3), 512 to 1024 µg/mL (4), 64 to 512 µg/mL (5), and 32 to 128 µg/mL (6).

Discussion
This study reports on the characterization of seven compounds, including five flavonoids (1-3, 6 and 7) and two C-15 isoprenoid analogues (4 and 5). Compounds 1-3 are prenylated flavonoids and are identified and described for the first time in this study. This is the first report of prenylated flavonoids from the genus Imperata. Compounds 4 and 5 are also new and have been identified for the first time. Compounds 6 and 7 were readily known but are isolated and described for the first time from the family Gramineae. Nonetheless, nonprenylated flavonoids have already been reported from Imperata species [19,22,40]. Imperata cylindrica has a very high molecular diversity, scanning methoxylated flavonoids [19], coumarins and chromones [19], lignans [8,20], phenolic acids [21], and terpenoids [41,42]. The most encountered groups of flavonoids are non-prenylated and unmethylated at position 8. The prenylated flavonoids described in this work are isolated for the first time in the genus Imperata. The terpenoids most often found in Imperata cylindrica or in the genus Imperata are triterpenes. However, a sesquiterpene has already been isolated from Imperata [20]. Compounds 4 and 5 isolated and described herein enrich the chemotaxomia of the plant. Our results confirm the molecular diversity observed in the literature for the genus Imperata.
Following the definition by [4], the crude methanol extract of I. cylindrica showed moderate antibacterial activity against almost all the tested bacteria strains. Compounds 1 and 4 displayed weak activities toward all the tested microorganisms while compound 3 moderately inhibited the growth of E. aerogenes ATCC 13048 and S. aureus MRSA1. Compound 5 exerted moderate activity on E. aerogenes ATCC 13048, comparable to the activity obtained with compound 6 against E. coli AG102 and S. aureus ATCC 25923. The best activity of compound 2 was obtained against S. aureus ATCC 25923 and MRSA1. The known antimicrobial mechanisms associated with plant secondary metabolites [43] may explain the antibacterial effect of isolated compounds from I. cylindrica. Although the antimicrobial activity of the methanol extract of I. cylindrica was previously demonstrated against a panel of Gram-positive MDR bacteria [16], to the best of our knowledge, the chemical and antibacterial profiling of compounds from I. cylindrica against MDR microorganisms is being reported here for the first time. This study supports the antimicrobial traditional use of I. cylindrica.

General Experimental Procedures
Electrospray ionization (ESI) mass spectra were recorded on a 1200-series HPLCsystem or a 1260-series Infinity II HPLC-system (Agilent-Technologies, Santa Clara, CA, USA) with binary pump and integrated diode array detector coupled to an LC/MSD-Trap-XTC-mass spectrometer (Agilent-Technologies) or an LC/MSD Infinity lab LC/MSD (G6125B LC/MSD). High resolution mass spectra were recorded on a Micromass-Q-TOF-Ultima-3-mass spectrometer (Waters, Milford, MA, USA) with LockSpray-interface and a suitable external calibrant. Infrared (IR) spectra were recorded on a FTIR-spectrometer (Bruker Tensor 27, Billerica, MA, USA) equipped with a diamond ATR unit and are reported in terms of frequency of absorption in cm −1 . NMR spectra were recorded on a Bruker Avance III 500 HD ( 1 H: 500 MHz, 13 C: 125 MHz) or Avance 600 ( 1 H-NMR: 600 MHz and 13 C-NMR: 151.1 MHz). Chemical shifts δ (ppm) are reported relative to residual solvent signal. 2D spectra (COSY, HMQC, HMBC) and DEPT-135 spectra were used for signal assignment. Chromatographic purification of compounds was performed on silica gel (35-70 µm, Acros Organics, Fair Lawn, NJ, USA) and Sephadex LH-20. Thin-layer chromatography (TLC) was carried out on silica plates (TLC Silica 60 F 254 by Merck, Kenilworth, NJ, USA) and spots were detected by spraying with 20% H 2 SO 4 followed by charring at 100 • C.

Plant Material
The whole plant of Imperata cylindrica was collected at Dschang in the western Region of Cameroon in April 2020. The identification was performed at the Cameroon National Herbarium (Yaoundé, Cameroon) by comparison with the voucher specimen kept under the voucher number 30139/SRF-Cam.

Cylindraucine (1)
Orange amorphous powder; IR (ν max ) 3306, 2923, 1709, 1610, 1586, 1150, 1069 cm −1 ; 1 H-NMR (500 MHz, CDCl 3 -CD 3 OD) and 13 C-NMR (125 MHz, CDCl 3 -CD 3 OD): see Table 1 The bacterial growth inhibition capacity of the crude methanol extract of Imperata cylindrica and its isolated compounds was assessed on a panel of six multi-drug resistance (MDR) bacteria: E. coli (ATCC 8739, AG 102), Enterobacter aerogenes (ATCC 13048, EA27), and Staphylococcus aureus (ATCC 25923, MRSA1). The MIC and MBC determinations were performed using the rapid INT colorimetric assay as described by [44], with some modifications as previously described [45,46]. Briefly, 100 µL of Müller Hinton Broth (MHB) was introduced into each well of a 96-well microtiterplate, followed by introduction of 100 µL solution of samples (crude extract, isolated compounds or chloramphenicol as reference). DMSO was used to dissolve the samples, at a final concentration below 1%. Preliminary assays with DMSO 1% did not show any effect on the growth of the tested bacteria. Two-fold dilution series of samples were made and the tested bacterial concentration was 1.5 × 10 6 colony forming unit (CFU)/mL. Plates were sealed with parafilm and incubated at 37 • C for 18 h. The negative control consisted of wells containing only MHB and inoculum, while the positive control was made up of MHB, inoculum, and a reference antibiotic (chloramphenicol). After 18 h incubation at 37 • C, the MIC of each sample was determined as the lowest sample concentration at which no bacterial growth was observed following the addition of 40 µL INT (0.2%). MBC was detected by adding 50 µL aliquots of the preparations, which did not show any growth after incubation during MIC assays, to 150 µL of MHB. These preparations were further incubated at 37 • C for 48 h. All the assays were performed in triplicate and repeated three times.

Conclusions
This study afforded five compounds unprecedented in nature, flavonoids (1-3) and C-15 sesquiterpenoid analogues (4 and 5). These findings enrich the chemotaxonomy of the plant, genus, and even family studied. The isolated compounds display considerable antibacterial activity against MDR strains. This study supports the uses of the plant in folk medicine to relieve urinary infections and might lead to an eventual standardization of a phytodrug based on both the chemical constituents and the biological features highlighted here amongst others.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.