Chemovariation and antibacterial activity of extracts and isolated compounds from species of Ixora and Greenea (Ixoroideae, Rubiaceae)

Plant materials

For comparative phytochemical study, 25 specimens from eight species of Ixora and two species of Greenea were collected from northeastern and southern Thailand and identified by S. Vajrodaya and V. Chamchumroon (Table 1). Voucher specimens were deposited in the herbarium at the Department of Botany, Faculty of Science, Kasetsart University, Bangkok, Thailand. We collected leaves, stem bark, and root bark for each plant. With some species, we could only obtain certain parts from a few specimens due to the plants’ scattered distributions across the landscape. The genus Greenea was only represented by two species (G. corymbosa and G. montana) because the other species reported from Thailand are only available as herbarium specimens or are limited to areas not accessible for collecting (Tange, 2013).

Extraction

Air-dried parts (0.6–267 g leaf, stem bark, or root bark) of Ixora and Greenea species were chopped into small pieces, powdered separately using a blender, and then macerated with methanol (20–400 mL) for a week in the dark at room temperature. The methanolic extracts were filtered and concentrated using a rotary evaporator at 37 °C to obtain the semi-solid crude extract, which was subsequently partitioned into hydrophilic and lipophilic extracts using distilled water and chloroform, respectively. The lipophilic and hydrophilic crude extracts were then evaporated to dryness for further experiments. For phytochemical profiling, 10 mg of each extract was prepared in methanol (HPLC grade) and filtered through a 0.45 µm nylon filter prior to analysis by HPLC.

Identification of chemical profiles

High performance liquid chromatography analysis of lipophilic extracts (10 mg) was performed on an Agilent 1100 series with a UV photodiode array detector at wavelengths of 230, 254, and 280 nm. A reverse phase ChromSepher 5, C18 column (250 × 4.6 mm) was used for analytical separation. The eluent consisted of (A) an aqueous buffer containing 0.015M ortho-phosphoric acid at pH 3 and 0.0015M tetrabutylammonium hydroxide and (B) methanol. The mobile phase started at 60% B for 16 min, then increased to 90–100% B within 6 min, with 100% B continuing for 6 min at a flow rate of 1.0 mLmin⁻¹. HPLC analysis of the hydrophilic extract (10 mg) started with the mobile phase at 10% B, then increased to 70% B within 15 min, next to 80% B within 5 min, and finally increased to 100% B within 2 min and continued for 6 min at a flow rate of 0.5 mLmin⁻¹. The injection volume was 20 µL for all analyses.

Thin layer chromatography was used to analyze lipophilic extracts on pre-coated silica gel 60 F₂₅₄ plates (Merck, Kenilworth, NJ, USA) using a solvent mixture consisting of hexane and ethyl acetate (1:1, v/v). Hydrophilic extracts were analyzed on TLC plates using a solvent mixture of ethyl acetate/methanol/water (6:4:0.4, v/v/v). Compound detection was performed either under UV irradiation or derivatization with anisaldehyde reagent.

Isolation of compounds

Three compounds were isolated successfully from the following species:

Ixora javanica (RB041): The lipophilic extract (130 mg) of stem bark (dry weight 12 g) was separated using column chromatography with a silica gel 60 column (0.04–0.063 mm, Merck, Kenilworth, NJ, USA) with 30% ethyl acetate in hexane. For each fraction, 20 mL was collected. Fractions with blue fluorescence under UV light at 365 nm were combined to obtain compound 1 (10 mg).

Greenea montana (RB011): The lipophilic extract (570 mg) of stem bark (dry weight 48 g) was fractionated using a silica gel 60 column (0.2–0.5 mm, Merck, Kenilworth, NJ, USA) with 30% ethyl acetate in hexane. For each fraction, 20 mL was collected. Fractions with slightly dark blue fluorescence under UV light at 365 nm were combined to obtain a mixture of compounds 1 and 2 (2.8 mg).

Ixora cibdela (RB046-048): The hydrophilic extract (one g) of root bark (dry weight 26.89 g) was fractionated using Sephadex LH-20 with MeOH as eluent to get ten fractions of 20 mL each (labelled S06). Then, fraction S06-F1 (538 mg) was separated on a silica gel 60 column (0.04–0.063 mm, Merck, Kenilworth, NJ, USA) with increasing polarity of chloroform/methanol mixtures: methanol (at 2:1 v/v) to obtain seven fractions of 30 mL each (labelled SG04). From these, fraction SG04-F1 (12 mg) was subjected to preparative TLC using solvent mixtures of ethyl acetate/methanol/water (7:2:1) to get compound 3 (three mg).

The isolated compounds were identified using nuclear magnetic resonance (NMR) and then used as reference compounds for comparative analyses of different plant parts of Ixora and Greenea species from different habitats based on retention time and the UV spectra of HPLC profiles.

Structure elucidation

The structures of the isolated compounds were deduced from 1D- (¹H, ¹³C) and 2D- (COSY, HSQC, HMBC) NMR spectroscopic data. All NMR spectra were recorded on a Bruker Avance II 400 (resonance frequencies 400.13 MHz for ¹H and 100.63 MHz for ¹³C) equipped with a five mm observation broadband probe head (BBFO) with z-gradients at room temperature and using a standard Bruker pulse program. The samples were dissolved in 0.6 ml of CDCl₃ or MeOD (each 99.8% D, Euriso-top, Saint-Aubin Cedex, France). Chemical shifts in ppm were determined with reference to residual solvent signals (CDCl₃: 7.26 ppm for ¹H, 77.0 ppm for ¹³C, MeO-d4: 3.31 ppm for ¹H, and 49.0 ppm for ¹³C). The chemical shifts of ¹H and ¹³C NMR of scopoletin (1), isofraxidin (2), and geniposidic acid (3) are given in the Supplemental File.

Antibacterial activity

The bacterial strains used in this experiment were methicillin-resistant S. aureus (MRSA) ATCC 43300, S. aureus ATCC 25923, vancomycin-resistant Enterococcus faecium UCLA192, E. faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa DMST 37166, P. aeruginosa ATCC 27853, carbapenem-resistant (KPC-producing) Klebsiella pneumoniae ATCC-BAA 1705, K. pneumoniae ATCC-BAA 1706, extended-spectrum beta-lactamase (SHV-18 producing) K. pneumoniae ATCC 700603, Acinetobacter baumannii ATCC 19606, Stenotrophomonas maltophila DMST 19079, and Salmonella choleraesuis ATCC 10708. These strains were obtained from the Department of Medical Science, Ministry of Public Health, Bangkok, Thailand.

Antibacterial activities were only tested for lipophilic extracts from I. javanica, I. brunonis, I. nigricans, and G. montana, because these species had complete extract sets including all three different parts of each plant. In addition, isofraxidin could not be isolated as a single pure compound (see isolation of compounds section), and therefore only scopoletin and geniposidic acid were tested.

Disk diffusion assays were used for antibacterial testing of lipophilic extracts, scopoletin, and geniposidic acid. Bacterial strains were inoculated into Muller–Hinton broth (MHB-Difco, Sparks, MD, USA). Bacterial suspensions equivalent in density to 0.5 McFarland (comparable to 1.5 × 10⁸ CFU/mL) were then inoculated on Muller–Hinton agar (MHA-Difco, USA). Plant extracts (400 µg) were dissolved in 100 µL dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA), loaded into six mm diameter cylinder cups, and placed on media containing the test organism. Plates were subsequently incubated for 18–20 h at the optimal temperature for the bacterial strain. The resulting inhibition zones (diameter of clear zone; mm) were compared to those of standard antibiotics piperacillin/tazobactam (100/10 µg), amikacin (30 µg), ceftriaxone (30 μg), cefotaxime (30 µg), ceftazidime (30 μg), ciprofloxacin (five µg), nalidixic (30 μg), imipenem (10 μg), trimethoprim/sulfamethoxazole (1.25/23.75 µg), and vancomycin (30 µg) with control species K. pneumoniae ATCC 700603 and P. aeruginosa strain ATCC 27853, following the recommendation of the Clinical and Laboratory Standards Institute (CLSI, 2017) for quality control of antimicrobial testing (Table S2).

A broth microdilution test was also used to determine the minimum inhibitory concentrations (MICs) of various lipophilic extracts, scopoletin, and geniposidic acid. Serial dilutions of extracts in DMSO, ranging from 1,024 to 0.5 µg/mL, were prepared and placed into 96-well microplates. To these were added a standard inoculum of the tested bacteria in MHB, which was then incubated at 37 °C for 18 h. The MIC was determined from the lowest concentration of extract that showed inhibition of bacterial growth.

Occurrence pattern of coumarins and iridoid in the studied Ixora and Greenea species

Phytochemical examination and isolation of extracts from Ixora and Greenea species revealed three known compounds: two coumarins, scopoletin (1) and isofraxidin (2), and one iridoid glycoside, geniposidic acid (3). These compounds were identified using NMR spectroscopy. The occurrences of these compounds varied among extracts from different plant parts (leaves, stem bark, and root bark) in species of both genera collected from different provinces (Table 1). All three were detected in all parts of the studied Greenea samples. In contrast, these compounds occurred sporadically in different parts of Ixora plants. Among Ixora species, isofraxidin was found only in stem and root bark extracts of samples collected from northeastern Thailand. Meanwhile, extracts from I. pendula did not contain either scopoletin or isofraxidin, making it completely unlike other Ixora species.

Antibacterial activity

Lipophilic extracts from Ixora and Greenea species showed antibacterial activity against three of the tested microorganisms: the Gram-positive bacteria methicillin-resistant S. aureus ATCC 43300 and S. aureus ATCC 25923 and the Gram-negative bacteria S. maltophila DMST 19079. Other microorganisms did not show any susceptibility. The inhibition zone diameters and MIC values of studied extracts against susceptible bacteria strains are presented in Fig. 1. All extracts from I. nigricans showed potential antimicrobial activity against two strains of the Gram-positive bacterium S. aureus, with zones of inhibition measuring 10–18 mm. For I. javanica, only the root bark extract was active, affecting the Gram-positive bacterium S. aureus and the Gram-negative bacterium S. maltophila DMST 19079; the zones of inhibition measured 8–10 mm. Extracts from I. brunonis and G. montana showed antibacterial activity only in the microdilution assay.

In microdilution assays, lipophilic extracts from all parts of I. nigricans, I. brunonis, and G. montana and the root bark extract of I. javanica showed strong antibacterial activity against the Gram-positive, methicillin-resistant S. aureus ATCC 43300, with MIC values varying from 64 to 256 µg/mL. In addition, the lipophilic root bark extract of I. javanica also showed antibacterial activities against the Gram-positive S. aureus ATCC 25923 (MIC value 256 µg/mL) and against the Gram-negative S. maltophila DMST 19079 (MIC value 128 µg/mL).

The inhibition zone diameters and MIC values of isolated scopoletin and geniposidic acid are presented in Fig. 1. Scopoletin showed antibacterial activities against the Gram-positive bacteria S. aureus ATCC 43300 and E. faecium UCLA192 and the Gram-negative bacterium S. maltophila DMST 19079, with similar zones of inhibition at eight mm. In microdilution assays, scopoletin showed antibacterial activities against the Gram-positive bacteria S. aureus ATCC 43300 and E. faecium (MIC value 128 µg/mL), and also against Gram-negative S. maltophila strains (MIC value 256 µg/mL). Geniposidic acid did not show any antibacterial activity.

Detected compounds

Three known compounds (scopoletin, isofraxidin, and geniposidic acid) were detected and isolated from the extracts of Ixora and Greenea species. Scopoletin (1) is a widely distributed coumarin found in a number of plant families such as Rutaceae and Apiaceae (Jain & Joshi, 2012), Clusiaceae (Marcondes et al., 2015), Convolvulaceae (Sood, Pradhan & Biswasroy, 2014; Khan & Hossain, 2015; Ojha, Mishra & Mishra, 2016), Solanaceae (Valizadeh et al., 2014), Asteraceae (Adams, Efferth & Bauer, 2006), Burseraceae (Mogana, Teng-Jin & Wiart, 2013), Asclepiadaceae (El-Demerdash et al., 2009), and Rubiaceae (Swe, 2008; Mohammed et al., 2013; Chen, Zhang & Chen, 2016). Isofraxidin (2) is found in species of Araliaceae (Maruyama et al., 2008; Yamazaki & Tokiwa, 2010; Bai et al., 2011; Lee et al., 2013), Asteraceae (Kwak, Lee & Schmitz, 2001; Gliszczyńska & Brodelius, 2012; Yim et al., 2017), Apiaceae (Yim et al., 2017), Simaroubaceae (Hwang et al., 2012), Rutaceae (Brown et al., 1988), Chloranthaceae (Niu et al., 2012; Li et al., 2016), and Rubiaceae (De Araújo et al., 2012).

Iridoids (especially iridoid glycosides) are reported as one of the most common secondary metabolites in Ixoroideae (Inouye et al., 1988; Martins & Nunez, 2015). In this study, only geniposidic acid (3) was isolated from I. cibdela and detected in I. diversifolia, I. pendula, G. montana, and G. corymbosa collections. Geniposidic acid was reported for the first time in I. chinensis in 1975 by Takeda, Nishimura & Inouye, but has not been reported in other species of the genus. Our results reveal that iridoid glycosides also occur in other Ixora and Greenea species. Similarly, other iridoid compounds may yet be present in our tested species. For example, ixoroside and ixoside were reported in I. chinensis (Takeda, Nishimura & Inouye, 1975), asperuloside was reported from I. casei, I. japonica, and I. macrothyrsa (Inouye et al., 1988), and deacetylasperulosidic acid was reported in I. odorata (Inouye et al., 1988).

The occurrences of scopoletin, isofraxidin, and geniposidic acid in leaf extracts of the two studied Greenea species were entirely different from those in the leaves of the tested Ixora species. However, these compounds are not the best candidates for chemotaxonomic markers to distinguish these genera, as they were detected in other parts for specimens of both Greenea species collected from the south, as well as in I. cibdela collected from the northeast. The presence or absence of secondary metabolites may be caused by a number of factors such as soil, season of collection, habitat, plant organ, plant age, and genetic mutation (Martins & Nunez, 2015). Moreover, the availability of building blocks for the biosynthesis of secondary metabolites and infection by microorganisms also affect metabolite production (Wilart, 2013). Building blocks for secondary metabolites are synthesized from intermediates or end-products of primary metabolic pathways such as photosynthesis, glycolysis, and the Krebs cycle; catalyzing enzymes and cofactors in these pathways can yield various classes of natural products, depending on a sequence of reactions and rearrangement in the biosynthesis pathway (Dewick, 2002; Ribera & Zuñiga, 2012; Gokulan, Khare & Cerniglia, 2014). This suggests that chemical variation among different plant parts and collecting locations may result from different biotic or abiotic factors affecting the accumulation of secondary metabolites.

Antibacterial activities

The findings of this study were similar to previous work on methanolic leaf and stem extracts of I. coccinea that reported broad antibacterial activities, especially against the Gram-positive bacterium S. aureus (Annapurna et al., 2003; Mani et al., 2014). However, while extracts from all parts of I. brunonis and G. montana showed distinct antibacterial activity in MIC assays, no zone of inhibition was observed in disc diffusion assays for these extracts (Fig. 1). This discrepancy may have been caused by the diffusion of extracts into the agar medium of the disc diffusion assay, making their antibacterial activity less potent than when in the microdilution well of the MIC assay (Mani et al., 2014).

Iridoids have been reported to possess a number of biological and pharmacological activities such as neuroprotective, anti-inflammatory, immunomodulator, hepatoprotective, cardioprotective, anticancer, antioxidant, antimicrobic, hypoglycaemia, hypolipidemic, choleretic, antispasmodic, and purgative properties (Tundis et al., 2008). In particular, geniposidic acid has been reported to inhibit the mycelium development of the plant pathogenic fungus Colletotrichum gloeosporioides (Luciano, Lima & Silveira, 2010), but the present study did not find evidence of antibacterial activity at concentrations of 0.5–1,024 µg/mL. However, only moderate antibacterial activity has been reported for the related secoiridoid sweroside, with its MIC at the high concentration of 1.0 g/mL (Horn et al., 2001). Therefore, higher concentrations are required for conclusive evaluation of the antibacterial activity of the iridoid glucoside geniposidic acid.

The antibacterial effects of coumarins have been shown to depend on the substitution patterns in their structures. Scopoletin has previously been shown to have antifungal, antibacterial, antioxidant, and anti-inflammatory properties (Gnonlonfin, Sanni & Brimer, 2012; Balouiri, Sadiki & Ibnsouda, 2016). Scopoletin consists of disubstituted simple coumarins (O-CH₃ at C6 and OH at C7) or hydroxycoumarins, which exhibit greater antibacterial activity against Gram-positive bacteria than Gram-negative bacteria, perhaps due to exclusion by the antibiotic efflux pump mechanism of Gram-negative bacteria (Tegos et al., 2002; Yang et al., 2016). However, other hydroxycoumarins, especially esculetin (OH at C6 and C7) and daphnetin (OH at C7 and C8), have demonstrated enhanced antibacterial activity against Gram-negative bacteria (De Souza, Monache & Smania, 2005).

In our results, scopoletin was not the only active compound identified among the constituents of lipophilic extracts. There could be synergistic effects for compounds in crude extracts, making the combination more effective than any single isolated constituent (Rasoanaivo et al., 2011). Synergism of mixed compounds in lipophilic extracts may be responsible for the antibacterial activity of a crude extract through various mechanisms, such as prevention from degradation by enzymes, effects on cell signaling and transportation, and overcoming multi-drug resistance mechanisms (Gilbert & Alves, 2003). In particular, the I. javanica specimen RB041 of showed scopoletin throughout the plant, but its lipophilic extracts from parts other than the root did not exhibit antibacterial activity, while the leaf extract from I. brunonis specimen RB032 contained no scopoletin but did exhibit some activity. Therefore, the antibacterial activities of these plants can be ascribed to other groups of compounds such as terpenoids and steroids.