A new benzophenone glycoside from the leaves of

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A new benzophenone glycoside from the leaves of Mitracarpus villosus Kenneth G. Ngwoke, Njideka Orame, Shuai Liu, Festus B. C. Okoye, Georgios Daletos & Peter Proksch To cite this article: Kenneth G. Ngwoke, Njideka Orame, Shuai Liu, Festus B. C. Okoye, Georgios Daletos & Peter Proksch (2017): A new benzophenone glycoside from the leaves of Mitracarpus villosus, Natural Product Research To link to this article: http://dx.doi.org/10.1080/14786419.2017.1306701

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Date: 22 March 2017, At: 06:52

Natural Product Research, 2017 http://dx.doi.org/10.1080/14786419.2017.1306701

A new benzophenone glycoside from the leaves of Mitracarpus villosus Kenneth G. Ngwokea,b, Njideka Orameb, Shuai Liua, Festus B. C. Okoyeb, Georgios Daletosa and Peter Prokscha a

Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, Duesseldorf, Germany; Faculty of Pharmaceutical Sciences, Department of Pharmaceutical and Medicinal Chemistry, Nnamadi Azikiwe University, Awka, Nigeria b

ABSTRACT

A new benzophenone glycoside, mitraphenone A (1), together with three known compounds (2–4) were isolated from the leaves of the traditionally used medicinal plant Mitracarpus villosus (Rubiaceae) collected in Nigeria. A combination of one- and two-dimensional NMR spectroscopic and mass spectrometric measurements were carried out to identify the structure of 1. All isolated compounds (1–4) were screened for their antibacterial activity against several Gram-positive and Gram-negative bacteria. Compound 1 exhibited moderate activity against Enterococcus faecium (strains ATCC 35667 and ATCC 700221) and Staphylococcus aureus ATCC 25923 with MIC values ranging from 25 to 50 μM.

ARTICLE HISTORY

Received 9 December 2016 Accepted 5 March 2017 KEYWORDS

Mitracarpus villosus; benzophenone glycoside; natural products; antibacterial activity

1. Introduction Natural products have been described as privileged structures that as a result of co-evolution are able to interact with various receptors in macro- and microorganisms, e.g via the activation/inhibition of regulatory enzyme(s) (Welsch et al. 2010; Ngwoke et al. 2014). Thus, natural products play an eminent role as lead compounds for drug discovery (Wätjen et al. 2009; Okoye et al. 2016). This impact can be exemplified by the fact that 49% of medicines, approved from 1940s to 2014, are derived or inspired from natural products (Newman & Cragg 2016). Historically, plants have been proved to be an important source of clinically CONTACT Georgios Daletos [email protected]; Peter Proksch [email protected] Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2017.1306701. © 2017 Informa UK Limited, trading as Taylor & Francis Group

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used pharmaceutical agents, including the anti-cancer drugs vincristine, vinblastine (vinca alkaloids) and etoposide (derivative of podophyllotoxin) (Chakravarthi et al. 2013; Greenwell & Rahman 2015), as well as the antimalarial agents, quinine and artemisinin (Martins & Nunez 2015). The extract of Mitracarpus villosus (Sw.) DC (Rubiaceae) has been used for the treatment of various diseases in the traditional setting in Nigeria. Particularly, it is a popular remedy for the treatment of ringworm and eczema infections (Iwu 2014). The antibacterial and antifungal properties of the crude extracts from M. villosus were also reported (Irobi & Daramola 1993; Oforkansi et al. 2011); however, no phytochemical studies on natural products from this plant have been carried out so far. During our ongoing search for new bioactive compounds from plants, we analysed the constituents of fresh leaves of M. villosus collected in Nigeria. Chemical investigation of the crude extract led to the isolation of four natural products, including a new benzophenone glycoside (1) and three known metabolites (2–4), which have been isolated from this plant for the first time. The results of the antibacterial screening of the isolated compounds (1–4) against several Gram-positive and Gram-negative bacteria are likewise discussed.

2. Results and discussion The crude extract (MeOH:CH2Cl2 (2:1)) of fresh leaves of M. villosus was subjected to liquid– liquid fractionation to afford n-hexane, EtOAc, n-BuOH and aqueous fractions. The n-BuOH and EtOAc fractions were further chromatographed on silica gel and Sephadex LH-20 followed by semi-preparative HPLC to obtain a new benzophenone glycoside (1), in addition to three known compounds (2–4) (Figure 1).

Figure 1. Structures of isolated compounds 1–4.

NATURAL PRODUCT RESEARCH

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Compound 1 was isolated as a brown solid and its molecular formula was established as C21H22O10 on the basis of the prominent ion peak at m/z 457.1105 [M + Na]+ in the HRESIMS spectrum. The UV spectrum of 1 showed absorptions at λmax 266 and 349 nm. The 1H and COSY NMR spectra recorded in MeOH-d4 revealed signals of an aromatic ABCD spin system (H-3 to H-6), as well as two singlets at δH 6.73 (H-3′) and 6.94 (H-6′), indicating the presence of 1,2-disubstituted (A) and 1,2,4,5-tetrasubstituted (B) benzene rings, respectively (Figure 1). This suggestion was further corroborated by analysis of the HMBC spectrum, in which the correlations from H-3 to C-1 (δc 139.6) and C-5 (δc 130.4), from H-6 to C-4 (δc 130.7) and C-2 (δc 140.8), from H-6′ to C-2′, C-4′ (δc 139.3) and C-5′, as well as from H-3′ to C-1′ (120.0), C-2′ and C-5′ were discerned (Figure S1). The deshielded resonances of C-2′ and C-5′ in B-ring (δc 158.5 and 149.5, respectively) suggested their oxygenated nature. Moreover, in the HMBC spectrum, the cross-peak observed from H-6 to the ester carbonyl carbon at δc 173.6 (1-COOH) established the linkage of a carboxylic function at C-1 (A ring). This was also evident from the fragment ion peak at m/z 389 [M−44−H]− in the MS spectrum, corresponding to the loss of a carboxyl moiety. Further HMBC correlations from H3-4′-CH3 (δH 2.25) to C-3′ (δc 119.8), C-4′ and C-5′, together with the NOE correlation between H3-4′-CH3 and H-3′ confirmed the attachment of the methyl group at C-4′ (B-ring) (Figure S1). Finally, the connection of A- and B-rings through a ketone carbonyl group (δc 205.6, CO) was established based on the HMBC correlations from both H-3 and H-6′ to the respective carbon (Figure S1). The remaining resonances in the 1H NMR spectrum of 1, including five oxymethine (H-1″ – H-5″) and a pair of non-equivalent oxymethylene protons (H2-6″), in the region between 3.01 and 4.53 ppm, were indicative of the presence of a sugar moiety, as confirmed by the observed MS fragment ion peak at m/z 273 [M−162+H]+ resulting from the loss of an hexose unit. A further detailed analysis of the chemical shifts and coupling constants of the respective protons (H-1″ – H2-6″) allowed the assignment of the sugar moiety as glucose. The β configuration of the anomeric carbon was established based on the large coupling constant between H-1″ and H-2″ (3J1″,2″ = 7.2 Hz) and the D configuration for glucose was proposed, as commonly encountered in nature (Brito-Arias 2007; Okoye et al. 2016). The 5′-O-glycosidic linkage of the sugar moiety to B-ring was corroborated based on the observed HMBC correlation from the anomeric proton H-1″ to C-5′, as well as from the NOE cross-peaks between H-1″ and both H-6′ and H3-4′-CH3 (Figure S1). Hence, compound 1 was identified as a new natural product and the name mitraphenone A was proposed. Compound 2 was isolated as a colourless solid, which formed needle-like crystals in MeOH. NMR, UV and MS spectra of 2 were identical to those reported for the common plant coumarin constituent, scopoletin, which was firstly isolated in 1884 (Moore 1911) and its antitumour and anticholinesterase properties have been reported thereafter (Rollinger et al. 2004; Liu et al. 2012). Likewise, compounds 3 and 4 were identified as anthraquinone-2-carboxylic acid (Burnett & Thomson 1967) and benz[g]isoquinoline-5,10-dione, respectively (Figure 1). The latter was previously isolated from Mitracarpus scarber and Psychotria camponutans (Rubiaceae) (Solis et al. 1995; Okunade et al. 1999) and its antibacterial, antifungal, antiplasmodial and anticancer properties have been described (Clark et al. 1984; Solis et al. 1995). However, to the best of our knowledge, this is the first report of the isolation of these compounds (2–4) from M. villosus. In the present study, all isolated compounds (1–4) were screened for their antibacterial activity towards a panel of Gram-positive and Gram-negative bacteria, including

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Staphylococcus aureus (ATCC 25923 and ATCC 700699), Enterococcus faecalis (ATCC 51299 and ATCC 29212), Enterococcus faecium (ATCC 35667 and ATCC 700221) and Acinetobacter baumanni (ATCC BAA 1605). Among tested compounds, 1 exhibited the strongest activity against E. faecium (ATCC 35667 and ATCC 700221) and S. aureus ATCC 25923 with MIC values ranging from 25 to 50 μM. Compounds 1–4 were also evaluated against Mycobacterium tuberculosis; however, none of them exhibited any significant activity (MIC > 50 μM). Likewise, the isolated compounds showed no inhibitory effect on the growth of L5178Y mouse lymphoma cells using the MTT assay (IC50 > 100 μM). Benzophenone O-glycosides are an intriguing class of compounds with several of them possessing diverse pharmacological activities (Elya et al. 2006; Nedialkov et al. 2009; Shu et al. 2012; Venditti & Ukwueze 2016). In accordance with our results, the antibacterial activity of guajaphenone A, isolated from the leaves of Psidium guajava (Ukwueze et al. 2015), as well as the radical scavenging and antioxidant properties of other associated benzophenone O-glycosides (Zheleva-Dimitrova et al. 2012) have been reported, thus, suggesting the high potential of this class of compounds for the development of new bioactive agents.

3. Experimental section 3.1. General experimental procedure Optical rotations were measured with a Jasco P-2000 polarimeter. 1D and 2D NMR spectra were recorded in MeOH-d4 on a Bruker AVANCE DMX 600 MHz NMR spectrometer (Bruker, Rheinstetten, Germany). HPLC/ESIMS data were recorded on a Thermo-Finnigan LCQ-Deca mass spectrometer (Thermoquest, Bremen, Germany) with an electrospray interface (ESI) coupled to a UV detector, and HRESIMS were recorded with a UHR-QTOF maXis 4G (Bruker Daltonics) mass spectrometer. HPLC–UV–DAD analysis was carried out on a HP 1100 system equipped with a photodiode array detector (Agilent technologies, Palo Alto, CA) with a Eurosphere C-18 column (5 mm, 125 X 4.6 mm i.d.; Knauer, Berlin, Germany). The ﬂow rate was set at 1 mL/min and the absorbance was detected at 254 nm with capillary temperature of 28°C and drift voltage of 20 eV. Analytical HPLC was performed with a Dionex P580A LPG pump equipped with a UV detector (UVD340S) using a Eurosphere C-18 column (5 mm, 125 × 4 mm i.d.; Knauer, Germany). Semi-preparative HPLC was performed with a Merck/ Hitachi L-7100 pump, coupled to a Merck/Hitachi UV detector (UV-L7400), using a Eurosphere C-18 column (10 mm, 300 × 8 mm i.d.; Knauer). The separation was carried out using a linear gradient of HPLC grade MeOH and nanopure water, monitored at 254 nm. Column chromatography was carried out using Merck MN silica gel 60 M (0.04–0.063 mm) or Sephadex LH-20 and the whole set-up was connected to a fraction collector (Retriever II, ISCO, Germany) and adjusted to a ﬂow rate of 2–3 mL/min. TLC plates precoated with Silica gel 60 F254 (layer thickness 0.2 mm, E. Merck, Darmstadt, Germany) were used with either CH2Cl2:MeOH (9:1) for semi-polar compounds or n-Hexane:EtOAc (8:2) for non-polar compounds as a mobile phase. The isolated compounds were detected by their UV absorption at 254 and 366 nm or by spraying the TLC plates with anisaldehyde reagent followed by heating at 110°C.

3.2. Plant material M. villosus leaves were collected in December 2015, from Agulu in Anambra State, Nigeria. The sample was identified by Dr Anthonia Emezia (Department of Pharmacognosy, UNIZIK,


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Awka, Nigeria) and a voucher specimen (No. PCG00556) was deposited at the Faculty of Pharmaceutical Sciences’ Herbarium.

3.3. Extraction and isolation Fresh leaves of M. villosus (900 g) were extracted over 48 h with 3 L of MeOH:CH2Cl2 (2:1) as solvent system. The solution was filtered using Buchner funnel and concentrated to dryness under vacuum at 40°C. The crude MeOH:CH2Cl2 (2:1) (52 g) was dispersed in 250 mL of distilled water and subjected to liquid–liquid fractionation using 250 mL each of n-hexane, EtOAc and n-BuOH in increasing order of polarities. The EtOAc fraction was concentrated under vacuum and subjected to vacuum liquid chromatography (VLC). Fraction 9 (412.3 mg, n-Hexane:EtOAc (1:1)) was further chromatographed over Sephadex LH-20 using MeOH as a mobile phase. Compound 4 (12 mg) was obtained as the fourth fraction. Fraction 10 was chromatographed over Sephadex LH-20 using MeOH as the eluting solvent and further subjected to semi-preparative HPLC purification to yield 2 (2.5 mg). In a similar manner, VLC fractions 1 and 4 of the n-BuOH phase were chromatographed over Sephadex LH-20, using CH2Cl2:MeOH (1:1) for elution, followed by semi-preparative HPLC to afford 1 (10 mg) and 3 (1.4 mg). Mitraphenone A (1): brown solid; UV (PDA): λmax 266 and 349 nm; [𝛼]20 D − 18 (c 0.10, MeOH); 1 H NMR (600 MHz, MeOH-d4) δ 7.94 (1H, m, H-6), 7.52 (1H, overlapped, H-4), 7.52 (1H, overlapped, H-5), 7.28 (1H, m, H-3), 6.94 (1H, m, H-6′), 6.73 (1H, s, H-3′), 4.53 (1H, d, J = 7.2 Hz, H-1″), 3.57 (1H, dd, J = 12.2, 3.6 Hz, H-6b″), 3.40 (1H, br t, J = 9.2 Hz, H-4″), 3.38 (1H, dd, J = 12.2, 2.5 Hz, H-6a″), 3.35 (1H, overlapped, H-3″), 3.34 (1H, overlapped, H-2″), 3.01 (1H, ddd, J = 9.2, 3.6, 2.5 Hz, H-5″), 2.25 (3H, s, 4′-CH3). 13C NMR (151 MHz, MeOH-d4) δ 205.6 (CO), 173.6 (1-COOH), 158.5 (C-2′), 149.5 (C-5′), 140.8 (C-2), 139.6 (C-1), 139.3 (C-4′), 130.7 (C-4), 130.4 (C-5), 130.2 (C-6), 127.8 (C-3), 120.0 (C-1′), 120.0 (C-6′), 119.8 (C-3′), 103.6 (C-1″), 78.0 (C-3″), 77.4 (C-5″), 74.7 (C-2″), 70.9 (C-4″), 61.7 (C-6″), 16.9 (4′-CH3); HRESIMS: m/z 457.1105 [M + Na]+ (Calcd for 457.1110, C21H22NaO10).

3.4. Antibacterial screening Antibacterial testing was carried out using broth microdilution method as described by Clinical and Laboratory Standards Institute (CLSI 2005). S. aureus (ATCC 25923 and ATCC 700699), E. faecalis (strains ATCC 51299 and ATCC 29212), E. faecium (strains ATCC 35667 and ATCC 700221), A. baumanni (strain ATCC BAA 1605) and M. tuberculosis were investigated. Moxifloxacin was used as a positive control for the Gram-positive strains, whereas rifampicin was used as a positive control for A. baumanni and M. tuberculosis.

3.5. Cytotoxicity assay Cytotoxicity against the mouse lymphoma cell line L5178Y was tested using the microculture tetrazolium (MTT) assay, as previously described (Müller et al. 1983). Experiments were carried out in triplicate and repeated three times. As negative controls, media with 0.1% EGMME/ DMSO were included in the experiments. The depsipeptide kahalalide F was used as a positive control.

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4. Conclusions In summary, one new benzophenone glycoside, mitraphenone A (1) in addition to three known compounds, scopoletin (2), anthraquinone-2-carboxylic acid (3) and benz[g]isoquinoline-5,10-dione (4) were isolated from fresh leaves of the medicinal plant M. villosus, collected in Nigeria. Interestingly, the new natural product mitraphenone A (1) showed considerable activity against E. faecium strains ATCC 35667 and ATCC 700221, as well as against S. aureus ATCC 25923 with MIC values in the range between 25 and 50 μM. However, compound 1 exhibited no cytotoxicity towards the mouse lymphoma cell line L5178Y, highlighting its selective antibacterial activity.

Acknowledgements The authors gratefully acknowledge TWAS-DFG for cooperative fellowship award that enabled this research. We are indebted to Prof. R. Kalscheuer (Heinrich Heine University, Duesseldorf, Germany) for antibacterial assays and to Prof. W. E. G. Müller (Johannes Gutenberg University, Mainz, Germany) for cytotoxicity assays.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding This work was supported by the TWAS-DFG.

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