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

Background A large number of secondary metabolites can be obtained from plants used for traditional medicine in two related genera (Ixora and Greenea) in the subfamily Ixoroideae (Rubiaceae), but there are only a few detailed studies on their bioactivities. Therefore, the main goals of this study were to determine the antibacterial activities of lipophilic extracts from plants of some Ixora and Greenea species native to Thailand, and to isolate some pure compounds from those extracts. Moreover, we compared the occurrence of compounds in different plant parts of samples from different habitats to better understand their variation. Methods A total of 56 lipophilic extracts were obtained from the leaves, stem bark, and root bark of eight Ixora and two Greenea species collected at various locations in Thailand. Isolated compounds were identified using nuclear magnetic resonance. Antimicrobial activities were evaluated against four Gram-positive and nine Gram-negative human pathogenic bacterial strains. Results Extracts from I. javanica, I. nigricans, I. brunonis, and G. montana, along with isolated scopoletin, exhibited antibacterial activities against Gram-positive methicillin-resistant Staphylococcus aureus ATCC 43300, with minimum inhibitory concentration values ranging from 64 to 256 µg/mL. The occurrence of scopoletin, isofraxidin, and geniposidic acid in lipophilic extracts showed some variation among different plant parts and species. Conclusions Lipophilic extracts of Ixora and Greenea species have the potential to be developed as anti-Gram-positive agents, in particular to counter infections of methicillin-resistant S. aureus strains. The chemical profiles showed differences between floristic regions but similarity within the same plant parts.


INTRODUCTION
As the resistance of microorganisms to available antibiotics increases, plant sources have become an attractive alternative for new drug discovery (Cowan, 1999). The World Health Organization (World Health Organization (WHO), 2017) has recently published a list of antibiotic-resistant bacteria, of which methicillin-resistant Staphylococcus aureus is considered a high-priority pathogen that urgently requires new antibiotic targeting. A number of studies have identified antibacterial activity against S. aureus strains in extracts from Ixora species (Annapurna et al., 2003;Mani et al., 2014). Furthermore, a large number of species in the genus Ixora are used as traditional treatments for many ailments. In folk medicine, species such as I. brunonis, I. cibdela, I. javanica, and I. nigricans are used to treat fevers, ophthalmic diseases, ear infections, wounds infections, dysentery, and diarrhea (Table S1). These diseases are mostly caused by bacteria and viruses; therefore, Ixora species are potential natural sources for antimicrobial drug development. Moreover, plants in the genus Greenea, native to Southeast Asia (Puff, Chayamarit & Chamchumroon, 2005;Tange, 2013), are closely related to Ixora according to phylogenetic analysis (Razafimandimbison et al., 2011;Kainulainen, Razafimandimbison & Bremer, 2013). Given this phylogenetic affinity, it is possible that extracts from Greenea could have bioactivities similar to those from Ixora.
In traditional medicine, different plant parts of Ixora species are used to treat different symptoms (Table S1). Thus, each plant part might possess different bioactive compounds that allow for different applications. To date, a few phytochemical studies of Ixora species have reported the presence of alkaloids, anthraquinones, phenolics, peptides, terpenoids, sterols, and iridoid glycosides (Chen, Zhang & Chen, 2016;Usha, Reginald & Immanuel, 2016). However, while there are several reports on the biological activities of extracts from Ixora species, only a handful of studies have identified their bioactive compounds. Similarly, only one phytochemical study of Greenea has been reported, which identified saponins, triterpenes and steroids in the leaves of G. corymbosa (Rahmani et al., 1985). Isolation and identification of bioactive compounds is needed for further determination of the therapeutic potential of these medicinal plants. We hypothesized that extracts and compounds from unstudied Ixora and Greenea species may have antibacterial effects, and that different plant parts and the localities from which the plants are obtained may affect the occurrence of these compounds.
The main goals of this study were to determine the antibacterial activities of lipophilic extracts from selected Ixora and Greenea species against selected human pathogenic bacteria strains, and to isolate and identify potentially active compounds in these species. A comparative phytochemical study was also conducted on 56 lipophilic extracts from the leaves, stem bark, and root bark of eight Ixora and two Greenea species collected from various locations in Thailand, using thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and, in some cases, by isolation. Our study is the first to report the composition and antibacterial activities of extracts from G. montana.

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). Table 1 Distribution of scopoletin (1), isofraxidin (2), and geniposidic acid (3) in leaves, stem bark and root bark extracts from examined Ixora and Greenea species collected from northeastern and southern Thailand.

Species
Collector No. Origin Leaves Stem bark Root bark

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 mm 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 Â 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 mL for all analyses. Thin layer chromatography was used to analyze lipophilic extracts on pre-coated silica gel 60 F 254 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).
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-( 1 H, 13 C) 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 1 H and 100.63 MHz for 13 C) 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 CDCl 3 or MeOD (each 99.8% D, Euriso-top, Saint-Aubin Cedex, France). Chemical shifts in ppm were determined with reference to residual solvent signals (CDCl 3 : 7.26 ppm for 1 H, 77.0 ppm for 13 C, MeO-d4: 3.31 ppm for 1 H, and 49.0 ppm for 13 C). The chemical shifts of 1 H and 13 C 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 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.
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 mg/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 mg/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 mg/mL) and against the Gram-negative S. maltophila DMST 19079 (MIC value 128 mg/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 mg/mL), and also against Gram-negative S. maltophila strains (MIC value 256 mg/mL). Geniposidic acid did not show any antibacterial activity.
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 mg/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.
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.

CONCLUSIONS
Our findings show that the lipophilic extracts of I. javanica, I. nigricans, I. brunonis, and G. montana from Thailand have potential to be used as antimicrobial agents against Gram-positive methicillin-resistant S. aureus strains. Detailed phytochemical investigations found scopoletin, isofraxidin, and geniposidic acid to be prevalent among the studied species. Isolated scopoletin showed anti-bacterial activity, as did several extracts without detected scopoletin, suggesting that scopoletin is not the only active compound in the lipophilic extracts. Further study is required to reveal other possible bioactive ingredients. The occurrence patterns of these compounds among the samples showed some distinction among the genera and species. Further analyses of more specimens from a broader set of species and from different floristic regions are necessary to obtain a more detailed picture of their chemical profiles.