Antibacterial Activity of Three Medicinal Lasianthus (Rubiaceae) Extracts on Human Resistant Pathogenic Bacteria

Aim: To evaluate the activity of medicinal Lasianthus extracts on bacteria and to determine the chemical characters among three medicinal Lasianthus (Rubiaceae) species. Materials and Methods: The phytochemical investigation of L. cyanocarpus, L. hirsutus and L. lucidus were performed using thin layer chromatography and high performance liquid chromatography (HPLC) techniques. Four antibacterial resistant strains including Staphylococcus aureus ATCC 43300 (MRSA), Klebsiella pneumoniae ATCC BAA 1705 (carbapenemase; KPC-producing strain) and Pseudomonas aeruginosa ATCC 27853 (AmpC β-lactamase producing strain) were used. The phytochemical investigation of L. cyanocarpus, L. hirsutus and L. lucidus were performed using thin layer chromatography and high performance liquid chromatography (HPLC) techniques. We also detected the effects of extracts on bacteria by using a scanning electron microscope. Results: The lipophilic extracts from the three plants revealed the terpenoids, coumarin and iridoid. HPLC showed similarities in the chemical profiles of both leaf and stem bark extracts. L. lucidus lipophilic extracts revealed the greatest effect against S. aureus and P. aeruginosa at 400 and 100 μg/mL respectively, whereas L. cyanocarpus extracts prominently affected K. pneumoniae at 400 μg/mL. Cell lysis and leakage of bacteria treated with extracts were observed. Conclusion: Our findings surprisingly showed the potential of antibacterial effect among resistant pathogenic bacteria. We also revealed the comparable signals of the chemical characters from the three Lasianthus. These findings support the traditional use related infectious diseases and it might be possible to further develop the antibiotic agents.


Introduction
The World Health Organization (WHO) reports that there are globally high levels of antimicrobial resistance in common bacteria coupled with a lack of coordination and understanding of antimicrobial resistance [1,2]. A low estimate suggests that antibiotic resistance is at present causing 700,000 deaths worldwide annually and this figure is projected to reach 10 million by 2050 [3]. Pathogenic bacterial infections, especially infections from Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumannii infect several organs including the skin and soft tissue, respiratory tract, urinary tract, bloodstream, and intra-abdomen [4]. Thus, the infectious control of resistant strains and the use of appropriate available antibacterial agents is the best way to struggle infections in the resistant era. Nowadays, scientists must encounter new antimicrobial agents from natural sources which bring new hope for treating multidrug resistant bacteria [5].
Plants in the genus Lasianthus (Rubiaceae) make up a large group with more than 180 species, mostly in tropical Asia where approximately 160 species are found in primary forest [6,7]. Different species of the genus Lasianthus (Rubiaceae) have been used as traditional medicines in tropical Asia to treat a variety of diseases, among which fevers and infectious diseases stand out [8][9][10].
At present, several species of Lasianthus are considered by authors to possess antibacterial properties [11]. It has been suggested in other reports that medicinal species of Lasianthus have been studied phytochemically, but there are no previous reports of isolated compounds and the bioactivity of the many species investigated here, 9 medicinal Lasianthus species, and in particular of their effects against pathogenic bacteria.
This study aimed to investigate antibacterial activity against seven pathogenic bacteria of 3 medicinal Lasianthus species related to infectious diseases. We also evaluated secondary metabolite compounds and chemical profiles from these potentially medicinal Lasianthus species.

Ethnomedicinal plants review
We collected all the available information on Lasianthus spp. Firstly, we reviewed all authoritative botanical data including Flora [12], related journals and botanical texts assembling details on plant distribution, morphological characteristics, medicinal uses and modes of preparation. We then crossreferenced these data via online databases and compared them with herbaria records in Thailand, Aarhus University Herbarium, Denmark and The Kew Herbarium, The United Kingdom.

Plant material
Mature leaves and stem bark of the medicinal Lasianthus species were collected between July and December, 2015, from tropical forests in Thailand. Voucher specimens of all Lasianthus were deposited at the Department of Botany, Kasetsart University, Bangkok Forest Herbarium (BKF), and Aarhus University Herbarium (AAU). The plant samples were identified and compared with type specimens: L. cyanocarpus (syntype K! 000763940), L. hirsutus (syntype K!000777044) and L. lucidus (type K!000763966, (S.S. Larsen, T. Christian. and D. Sookchaloem 46739 (AAU)).

Preparation of plant extractions
Two-hundred gram of mature leaves and stem bark were dried under shade and powdered using an electric mill. The powder was macerated with methanol for seven days. Extracts were subsequently filtered and then concentrated using rotary evaporation at 37ºC, until the crude extracts were semi-solid. The concentrated crude extracts were partitioned into a hydrophilic extract in distilled water and lipophilic extract in chloroform. The lipophilic extracts were stored at a temperature below -45ºC.

Antibacterial screening
Screening of the lipophilic extracts for antibacterial effect was determined by diffusion method. The standard inoculums (0.5 McFarland) were spread evenly on Muller-Hinton agar. After drying, each disk paper containing 200 µg of extract was placed on Muller-Hinton agar with the tested organisms. The agar plate was incubated at 37ºC for 24 hours. E. coli ATCC 25922 and S. aureus ATCC 25923 strains were used as control species with standard antibiotic disks [13].
The leaf and stem bark extracts solutions in dimethyl sulfoxide (DMSO-Sigma-Aldrich, USA) were serially diluted with 2-fold concentrations ranged from 6.25 μg/mL to 3,200 μg/mL. A standard inoculum of test bacteria in Mueller-Hinton broth (MHB-Oxoid, Basingstoke, UK) was poured into each well of the 96-well plate. DMSO was used as a control. The organisms were cultured at 37ºC for 18 hours. The MIC was documented as the lowest concentration of extracts that inhibited visible growth.

Chromatography techniques
The lipophilic extracts were placed and investigated on Thin Layer Chromatography pre-coated silica gel 60 F254 plates (Merck) using a solvent system of hexane ethyl acetate (7:3 v/v) and detected under UV irradiation (365 nm and 254 nm). The Relative front (Rf) values of each fluoresced spot were determined as a TLC pattern. The TLC plates were sprayed with detecting reagent to screen major secondary metabolites. We used an anisaldehyde sulfuric acid reagent for terpenoid detection. Dragendorff's reagent, vanillin-sulfuric acid reagent, 10% NaOH in ethanol and Wieffering test were used for alkaloids, higher alcohols (including steroids, phenols, essential oils), coumarin and iridoids, respectively (Merck). Samples of 10 mg/mL of lipophilic extracts in methanol (Merck) were performed using HPLC with methanol gradient of 60-100% in an aqueous buffer (0.015 M ortho-phosphoric acid pH 3 and 0.015 M tetrabutyl ammonium hydroxide). The HPLC analysis was done on an Agilent 1100 series system and detected with a UV photodiode arrays detector with wavelengths of 230 nm.

Scanning electron microscopy (SEM)
For scanning electron microscopy (SEM), P. aeruginosa, S. aureus and K. pneumoniae (105 CFU/mL) in lipophilic extracts at the MIC of each such strain combined with MHB were incubated at 37ºC for six hours and were then filtered through a 0.22 µM filter paper. The bacteria on the filter paper were fixed by a 2.5% glutaraldehyde in 0.2 M sodium phosphate saline buffer (pH 7.2) for 12 hours at 4ºC. The fixed bacteria were washed three times with 0.2 M sodium phosphate saline buffer (pH 7.2) and were then post-fixed in 1% osmium tetroxide (OsO 4 ) in distilled water for one hour [14]. After that, the filter paper was cleaned up with three cycles of distilled water. The fixed samples were dehydrated in a series of acetone and then dried at the critical

Statistical Analysis
The assay was done in triplicate to get the statistical average of the result. Mean value for the zone of inhibition in mm and MIC values in μg/mL were calculated.

Medicinal plant species review
With respect to ethnomedicinal information, a total of 3 species for treating infected wounds which have been documented from tropical Asia have been listed in the present article. Fresh dosing of the 3 species, direct application and use of the leaves as poultice wounds, as well as the usage of leaves and stem bark, are the most frequently recorded ways of administration. Their uses and modes of remedy are reported in Table 1. Juice from leaves to stop bleeding, on wounds, and stem bark or roots boiled as decoction together, the liquid being drunk to reduce fever. [23]

Antibacterial activity
From the results of the zone diameters, the 3 plant extracts showed the highest antibacterial activity against P. aeruginosa, followed by S. aureus and K. pneumoniae, respectively ( Table 2). With the clear zone of lipophilic extract, the leaf and stem bark extracts of L. lucidus showed the best potential antibacterial activity against P. aeruginosa ATCC 27853 and S. aureus ATCC 43300(MRSA). Similarly, MIC of L. lucidus stem bark and leaf extracts were determined at 200 µg/ml and 100µg/ml for P. aeruginosa ATCC 27853 and 400 µg/ml for S. aureus ATCC 43300 (MRSA) respectively ( Table 3). The leaf and stem bark extracts of L. hirsutus inhibited P. aeruginosa ATCC 27853 at 800 µg/ml concentration.

Qualitative chromatography analyses
The results on the TLC plates appeared positive to terpenoids, alkaloids, coumarin and iridoids and presented the bands after spraying with vanillin sulfuric acid reagent for detection of the higher alcohols, phenols, steroids, and essential oils. The TLC profiles of the leaf extracts showed similarities and differences from the stem bark extracts when defined by Rf values and qualitative bands or spots from each reagent. The results of the qualitative tests performed on lipophilic extracts from leaves and stem bark are shown in Table 4. The HPLC profiles showed the three dominant peaks of the leaf extracts of the three medicinal Lasianthus, while only one dominant peak appeared in the stem bark extracts. The retention time and significant UV spectra of comparable signals of the lipophilic extracts of leaves and stem bark are shown in Figure 1. From these observations, the HPLC profiles of the leaf extracts showed similar chemical characteristics and profiles for each of the three species. The stem bark extracts of L. cyanocarpus and L. hirsutus showed similar profiles but these differed from L. lucidus when diagnosed with UV λmax at a signal wavelength of 230 nm.
Moreover, the prolonged shape of P. aeruginosa could be observed when compared with that untreated bacteria ( Figure  2).
Comparative HPLC chromatograms (Profiles) of leaf and stem bark lipophilic extracts.

Discussion
This study was the first to investigate the effects of leaves and stem bark from L. cyanocarpus, L. hirsutus and L. lucidus lipophilic extracts on S. aureus, P. aeruginosa, and K. pneumoniae. Similarly, Dinda et al. [15] reported iridoids from Rubiaceous plants demonstrated antimicrobial activity. However, our lipophilic extracts had promising inhibitory effects against P. aeruginosa even at concentrations of 100 µg/ml, and thus can be a nice source for antibacterial agents. Moreover, we first revealed the cellular morphological changes via an electron microscope. The prolonged cells and the leakage of content were originally thought to be the results of by cell wall detriment like β-lactam [16].
Qualitative information for specific constituents in leaf and stem bark extracts from Lasianthus were investigated using color detection with different reagents was evidence to support the classification of the three medicinal Lasianthus species. The HPLC analysis determined the retention time and UV spectra signals. The results from this study present the first report of the chemical characteristics of these plants demonstrated with chromatographic techniques. In the report of Lukasz and Monika [17] the authors used TLC as a tool for botanical material investigation, then focused on chemotaxonomy for analysis of plant extracts and plant classification. Moreover, our HPLC profile among three species also supported the TLC findings showing the same number of compounds which could be detected under UV wavelength. Thus, HPLC is used to separate and quantify chemical compounds that have been dissolved in solution and to determine a specific compound in solution [18]. Lasiantheae which differs from the alkaloid-rich genus, Psychotria, is a significant chemical characteristic. The genus Ronabea has been placed in the same tribe as Psychotria (tribe Psychotrieae), but molecular evidence supports the separation of these two genera into different tribes as well [19]. A previous report on the phytochemistry of Lasianthus by Takeda et al. [20] isolated the three new compounds lasianthionoside A, B and C from leaves of L. fordii together with iridoid glucosides and asperuloside, deacetyl asperuloside, methyl deacetyl asperulosidate and citroside A. Furthermore, Dallavalle et al. in 2004 [21] were able to isolate the glucosidic compound (stigmasterol 3-o-â-D-glucoside) from L. gardneri, a species growing in the mountain forests of Sri Lanka. This is the first report of the chemical profiles of these three medicinal Lasianthus which may lead to the future discovery of chemical markers in chemotaxonomy and bioactive compounds for uses of medicinal Lasianthus species in South East Asia. The three medicinal Lasianthus in this study have been reported as traditional remedies for treating infected wounds and stopping the bleeding as well as reducing fever possibly caused by infection. The results of the antibacterial activity and chromatographic trends of the extracts conform to the available information on traditional uses [22,23].
In the future, there is a need to clarify the antibacterial activity and the exact mechanism of action of the extracts as well as to undertake research to discover the chemical compounds from the plant resources, which is expected to lead to a novel discovery of drugs.

Conclusion
Our work is the first report on three medicinal Lasianthus extracts which supports the traditional uses related to treating infectious disease and showed antibacterial effects against bacterial pathogens which are serious health problems. Qualitative analyses of lipophilic leaf and stem bark extracts from the three Lasianthus were undertaken using chromatographic techniques (TLC and HPLC). Effective pure compounds should be isolated from these plants to obtain alternative chemicals for use in chemotaxonomy and medicinal plant work.