1. INTRODUCTION
Throughout history, medicinal plants have been used to manage infectious diseases, and their use has gained wide acceptance in many countries as an alternative approach to the treatment of specific ailments [1]. The growing use and acceptance of medicinal plants reflect their potential as a reservoir of bioactive compounds for drug discovery and development. Bougainvillea glabra, a hardy species of Bougainvillea, is distinguished by its colorful bracts. In Thailand, this species is popular as an ornamental plant and is recognized for its medicinal properties. B. glabra has been found to harbor phytoconstituents such as phenolic compounds, flavonoids, and alkaloids that impart a wide range of therapeutic properties [2]. B. glabra has generated significant interest in the diverse properties of its phytochemical composition. In addition to bioactive compounds, this plant contains various antioxidative molecules that counter oxidative activity, including the activities of free radicals that disrupt the stability of cellular molecules [3].
Certain phenolic compounds obtained through plant extraction exhibit pharmaceutical properties. Consequently, plants that produce large quantities of phenolics may potentially be sources of higher concentrations of antioxidants, antibiotics, anticancer agents, and antidiabetics [4]. The majority of natural phenolics are hydrophilic, while some are lipophilic. In the assessment of bioactive compounds, researchers frequently prioritize, as we do here, the determination of total phenolic compounds (TPC).
The surge in antibiotic overuse has raised concerns about antimicrobial resistance, as it has seriously narrowed the available healthcare choices for effective antimicrobial treatments [5]. The search for innovative antimicrobial agents has been an ongoing topic and the phytochemical composition of B. glabra holds possibilities for the discovery of new substances with natural antimicrobial properties. Recognized as an edible medicinal plant, B. glabra has emerged as a potential source of antimicrobial compounds, given the diverse phytochemical classes presented within its flowers [2].
Phytochemical compounds found in Bougainvillea have also demonstrated anticancer properties. Studies have revealed that specific compounds in the genus of Bougainvillea can impede the growth of various cancer cell lines [2,6]. For example, dichloromethane and methanol extracts of B. glabra inhibited a wide range of cancer cells, including MCF-7, MDA-MB-231, CaSki, DU-145, and SW-480 [7]. Another study showed that the new bioactive compound given the name bougainvinones, from the bark of B. spectabilis, displayed cytotoxic effects on KB, HeLa S-3, HT-29, MCF-7, and HepG2 cancer cells [6].
Numerous studies have explored the components and phytochemical properties of bioactive compounds in the purple flowers of B. glabra. However, our study contributes additional insights by providing information on antimicrobial resistance against previously unstudied stains of clinically infectious bacteria and anticancer properties against cell lines that have not been investigated before. These novel findings are based on the purple flowers of B. glabra collected in Songkhla Province, situated in the southern part of Thailand.
2. MATERIALS AND METHODS
2.1. Sample Collection and Preparation
Fresh purple flowers of Bougainvillea sp. had been collected from the Songkhla Province of Thailand and were then taxonomically identified and confirmed as B. glabra following the series of dichotomous keys of Flora of Thailand, Volume 5, Part 3 [8].
Fresh purple flowers from B. glabra were harvested and carefully rinsed with distilled water. The flowers were then dried in an oven at 60°C for 24 h. The dried flowers were ground into a fine powder that was sequentially extracted using the chosen analytical solvent grade: hexane, ethyl acetate, methanol, and water. The powdered flower was first extracted with hexane with shaking at 200 rpm for 16 h at a ratio of 1:10 w/v, with 100 g of flower in 1 L of hexane. The extract was then filtered using Whatman No.1 filter paper, and the clear extract was collected. The residue after hexane extraction then underwent subsequent extraction in the same condition using ethyl acetate, methanol, and water as solvents. The clear extracts obtained from hexane, ethyl acetate, and methanol were concentrated using a Buchi R-134 rotary evaporator and the clear extract obtained from water was dehydrated using a freezer dryer.
2.2. Total Phenolic Contents
To assess phenolic contents, a 100 mg extract sample was dissolved in 1 mL of 70% ethanol with regular shaking for 1 h at 4°C. The determination of TPC was carried out using a 96-well microplate format following a previous work [9] with some modifications. Each reaction included 20 µL of either (1) 70% ethanol as a blank, (2) various concentrations of gallic acid (Merck Co., Germany) as a standard, or (3) one of the extracts. Subsequently, 100 µL of 10% Folin–Ciocalteu reagent (Sisco Research Laboratories, India) and 80 µL of 7.5% Na2CO3 were added. The reaction was incubated at room temperature for 1 h. TPC was then determined by measuring absorbance at 765 nm and was reported as milligrams of gallic acid equivalent per gram of dry weight (mg GAE g−1 DW).
2.3. Antioxidant Activities
2.3.1. DPPH-free radical scavenging activity
To evaluate free radical scavenging activity, a 100 mg extract sample was dissolved in 1 mL of 70% ethanol with regular shaking at 4°C for 1 h. The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was conducted using a 96-well microplate format [10]. Each well contained 190 µL of DPPH and 10 µL of either (1) 70% ethanol as a blank, (2) various concentrations of Trolox (Merck Co., Germany) as a standard, or (3) one of the extracts. The total volume of the DPPH assay (200 µL) was incubated at room temperature for 1 h. The optical density, measured at an absorbance of 517 nm, was used to determine the percentage of inhibition in the DPPH assay. The % inhibition was calculated as follows:
The scavenging capacity of DPPH was expressed as the Trolox equivalent antioxidant capacity (TEAC) in milligrams Trolox per gram of DW.
2.3.2. ABTS-free radical scavenging activity
To investigate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity [11], a working solution of ABTS+ was prepared by combining 40 mM potassium persulfate (K2S2O8) with 7 mM ABTS. This working solution was stored at 4°C for 16 h before use. The ABTS+ working solution was diluted with 50 mM phosphate buffer (pH 7.4) until the absorbance at 734 nm coincided with an optical density (OD) of 0.7. Extract samples of 100 mg were dissolved in 1 mL of 50 mM phosphate buffer (pH 7.4) with regular shaking for 1 h. The ABTS+ assay was conducted using a 96-well microplate spectrophotometer. In each well, 180 µL of the working ABTS+ solution was mixed with 20 µL of either (1) 50 mM phosphate buffer (pH 7.4) as a blank, (2) various concentrations of Trolox as a standard, or (3) one of the extracts. All reactions were incubated at 30°C for 6 min. OD (A) for absorbance was then measured at a wavelength of 734 nm and % inhibition was calculated as follows:
The scavenging capacity of ABTS was expressed as the TEAC in milligrams Trolox per gram of DW.
2.3.3. Ferric-reducing antioxidant power
Free radical scavenging activity was also investigated by using the ferric-reducing antioxidant power (FRAP) assay [12]. The FRAP reagent was prepared by combining 10 mL of 300 mM acetate buffer at pH 3.6, 1 mL of 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) in 40 mM HCl, and 1 mL of 20 mM FeCl3. A volume of 150 µL of the FRAP reagent was added to a 96-well plate along with 20 µL of either (1) distilled water as a blank, (2) various concentrations of Trolox as a standard, or (3) one of the extracts. The reaction was incubated in darkness at 25°C for 10 min. Absorbance was then measured at 593 nm. Reducing power was calculated and reported in milligrams Trolox per gram of DW.
2.4. Antimicrobial Properties
2.4.1. Determination of antimicrobial activity
All the microbial strains in this study were obtained from the Department of Microbiology, School of Medicine, Walailak University, Thailand. The following Gram-positive bacteria were used to investigate antimicrobial screening properties of the extracts: Bacillus cereus ATCC11778, B. subtilis 7988 (clinical isolate), B. subtilis ATCC6051, Listeria monocytogenes (clinical isolates), Staphylococcus aureus ATCC25923, S. aureus ATCC29213, methicillin-resistant S. aureus (clinical isolate), S. epidermidis ATCC35984, and Streptococcus pyogenes ATCC19615. The following Gram-negative bacteria were used to screen the antimicrobial properties of the extracts: Acinetobacter baumannii MDR, Escherichia coli ATCC25922, E. coli O157:H7 (clinical isolates), Klebsiella pneumoniae ATCC70063, Pseudomonas aeruginosa ATCC27853, Salmonella enterica serotype Typhi (clinical isolates), Shigella enteritis (clinical isolates), and Vibrio cholerae (clinical isolates).
Gram-positive and -negative bacteria were maintained at 4°C on nutrient agar (Gibco; Thermo Fisher Scientific, Inc) and Luria–Bertani (LB) agar (Gibco; Thermo Fisher Scientific, Inc), respectively. The cultures were grown overnight in Mueller Hinton (MH) broth (Gibco; Thermo Fisher Scientific, Inc) at 37°C and then diluted to approximately 8 × 108 colony-forming units (CFU/mL) for all tests. For the positive control, ampicillin was employed specifically against Gram-positive bacteria, while gentamicin was utilized against Gram-negative bacteria.
2.4.2. Overlay spotted screening for antimicrobial activity
The protocol used to assess antimicrobial activity was slightly adjusted based on Wang et al. [13]. In brief, bacterial strains were cultured overnight at 37°C in MH broth, then diluted to a concentration of bacterial suspension at 107 CFU/mL, and swabbed on MH agar in three different directions. Extracts were individually prepared at 100 mg/mL in DMSO and sterilized through a 0.22 sterile hydrophilic PTFE filter. To screen extract sensitivity, 10 µL of each extract at 100 mg/mL were spotted on the swabbed MH agar plates and then incubated overnight at 37°C. Bacterial sensitivity to the extract was determined by observing clear spots on the bacterial lawn.
2.4.3. Microdilution technique for minimum inhibitory concentration and minimum bactericidal concentration
Microdilution technique for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in this study was adapted from Mordmuang et al. [14]. To determine the MIC, 100 µL of MH broth was transferred into each well of a 96-well plate. Extracts were subjected to twofold serial dilution. In brief, 100 µL of each sterile extract, initially diluted in MH broth at 32 mg/mL, underwent twofold dilutions in each well, resulting in final extract concentrations ranging from 16 mg/mL to 0.25 µg/mL. Additionally, 100 µL of each diluted bacterial cell suspension containing 106 CFU/mL was added to each well of the 96-well plate. For the growth control, 1% DMSO diluted in MHB was used instead of the extract. The entire plate was then incubated at 37°C. The MIC was determined as the lowest concentration of extract that rendered the solution in each well visibly clear.
For the investigation of MBC, 10 µL of each well, starting from the MIC concentration and onward, was plated onto MH agar and subsequently incubated at 37°C with 5% CO2. The MBC was defined as the concentration in the well that exhibited no bacterial growth on the agar plates.
2.5. Anticancer properties
2.5.1. Cancer cell culture
Four cancer cell lines were used in this study—A549 (representing lung cancer), HT-29 (representing colon cancer), MCF-7 (representing breast cancer), and KKU-213A (representing bile duct cancer). Additionally, two normal cell lines, human dermal fibroblast (HDF) and an immortalized human cholangiocyte cell line, MMNK-1, were included for comparative analysis. All cell lines were cultivated in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) and 1% antibiotic–antimycotic (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured and incubated under standard conditions at 37°C with 5% CO2.
2.5.2. Cytotoxicity assay
The cytotoxic effects of the studied extracts were assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 3 × 103 cells in 100 µL of medium were seeded into each well of a 96-well plate and incubated overnight. In the following day, the old media were replaced with media containing various concentrations of the extracts (62.5, 125, 250, 500, 750, and 1,000 µg/mL), and medium containing DMSO was used as a vehicle control (0 µg/mL). The cells were incubated for 72 h before being subjected to the MTT assay to assess the effect of each extract on cancer and normal cell lines. In the MTT assay, a final concentration of 0.5 mg/mL MTT solution was added to each well and incubated for 2 h. Excess MTT solution was removed, and formazan crystals were solubilized with 100 µL of DMSO. Absorbance was recorded at 540 nm using a microplate reader (Spark® multimode microplate reader, TECAN, Switzerland). Percent cell viability was calculated using the following formula:
2.6. GC-MS Analysis of Phytochemical Compounds
The four plant extracts were analyzed by gas chromatography (GC, Agilent 7890) coupled with triple quadrupole mass spectrometry (MS, Agilent 7000D) [15]. The less polar extracts, hexane and ethyl acetate, were separately chromatographed in an Agilent 19091-433 HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm; maximum temperature 340°C) while the more polar extracts, methanol and aqueous, were separated in an Agilent 19091N-113I HP-INNOWax capillary column (30 m × 0.25 mm × 0.25 μm; maximum temperature 340°C). Ultrahigh-purity helium, flowing at a constant rate of 1 mL/min, served as the carrier gas. The ionizing energy was set at 70 eV. The oven temperature was programmed from 60°C (held for 2 min) to 300°C at a rate of 3°C/min. Crude extract samples were diluted with ethanol in a 1:10 ratio (v/v). The resulting particle-free diluted crude extracts were injected via syringe using a spitless setting. Data were acquired by collecting full-scan mass spectra within the range of 35–550 amu. Constituent contents of the crude extracts were expressed as percentages calculated from peak areas. Peak identification of chemical compounds in the crude extracts relied on GC retention time. Individual peaks in the GC after mass spectral analyses were identified using the databases of Wiley10 and the National Institute Standard and Technology (NIST) version 17.
2.7. Statistical Analysis
Each experiment was conducted in five replicates unless otherwise specified. The normality of data distribution was assessed using the Shapiro–Wilk test, and the homogeneity of variance was examined using the Bartlett test. Comparative analysis was performed through an ANOVA with Tukey’s post-hoc test. Statistical significance was considered when the p-value was less than 0.05. The presentation of all data includes mean values accompanied by the standard deviation (SD).
3. RESULTS
3.1. Total Phenolic Contents
An understanding of the chemical composition of B. glabra is crucial to the optimal retrieval of phenolic and antioxidant contents through solid–liquid extraction. To capture both hydrophilic and lipophilic phenolics, the solvents used ranged from low to high polarity. The methanol extract exhibited the highest TPC with 26.97 ± 1.70 mg GAE g−1 DW, followed by the aqueous extract (15.82 ± 2.58 mg GAE g−1 DW), ethyl acetate extract (8.69 ± 0.61 mg GAE g−1 DW), and hexane extract (2.19 ± 0.16 mg GAE g−1 DW) [Figure 1A].
 | Figure 1: Total phenolic contents (TPC) (A) and in vitro antioxidant capacities of the dried flower of B. glabra extracted in different solvents were determined by DPPH (B), ABTS (C), and FRAP (D) assays and all data were reported as means ± SD from five replicate experiments. Different letters indicate a significant difference (p < 0.05) from the ANOVA test.
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3.2. In vitro Antioxidant Activities
To evaluate antioxidants present in the flower extracts of B. glabra, three colorimetric antioxidant assays were used: the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and FRAP assays. These in vitro assays are routinely employed to assess the antioxidant capacity of plant extracts.
3.2.1. DPPH-free radical scavenging activity
The fundamental principle of the DPPH assay is the conversion of stable DPPH radicals. When DPPH• receives a hydrogen atom from antioxidant molecules, a color change from purple to clear yellow occurs. The color change reduces absorbance at 517 nm when 70% ethanol is used as the extraction solvent. Therefore, the antioxidant capacity of the extract is determined by comparing the plant extract concentration used to reduce the DPPH molecule to the standard concentration of an antioxidant molecules such as Trolox or L-ascorbic acid. The methanol extract (1.03 ± 0.04 mg TEAC g−1 DW) demonstrated a significantly higher antioxidant capacity than the water extract (0.62 ± 0.02 mg TEAC g−1 DW), ethyl acetate extract (0.17 ± 0.01 mg TEAC g−1 DW), and hexane extract (0.08 ± 0.01 mg TEAC g−1 DW) [Figure 1B].
3.2.2. ABTS radical scavenging activity
The ABTS assay is based on the decolorization that occurs when the free radical molecule, ABTS•+, is reduced to ABTS by antioxidant molecules. The antioxidant capacity is determined by measuring the decolorization of ABTS•+. The advantage of the ABTS assay is that antioxidant capacity can be determined in various pH conditions. In this study, we used physiological pH (pH 7.4). The highest antioxidant capacity was found in the methanol extract (5.45 ± 0.19 mg TEAC g−1 DW), which demonstrated a significantly higher antioxidant capacity than the water (3.39 ± 0.23 mg TEAC g−1 DW), ethyl acetate (2.22 ± 0.21 mg TEAC g−1 DW), and hexane extracts (0.20 ± 0.02 mg TEAC g−1 DW) [Figure 1C]. Compared to the DPPH assay, the antioxidant capacity determined by the ABTS assay was nearly fivefold higher.
3.2.3. FRAP radical scavenging activity
Since the dried flowers of B. glabra are water-soluble at acidic pH (pH 3.6), the FRAP assay was also used to determine the antioxidant capacity of the flower extracts. FRAP assays are also colorimetric assays. Antioxidant activity is indicated by the blue color that appears when the ferric tripyridyltriazine complex (Fe3+-TPTZ) is reduced to a ferrous complex (Fe2+-TPTZ). The ethyl acetate extract (1.13 ± 0.03 mg TEAC g−1 DW) showed the greatest antioxidant capacity followed by methanol (0.30 ± 0.03 mg TEAC g−1 DW), hexane (0.20 ± 0.14 mg TEAC g−1 DW), and water extracts (0.15 ± 0.04 mg TEAC g−1 DW) [Figure 1D].
3.3. Antimicrobial properties
3.3.1. Antimicrobial screening against pathogenic
The antimicrobial screening test using 10 µL of 100 mg/mL (1 mg) of each extract revealed that the studied extracts produced a wide range of inhibitory growth effects on different bacterial lawns [Table 1]. The hexane and ethyl acetate extracts both similarly demonstrated inhibitory activity against B. cereus ATCC11778, B. subtilis ATCC6051, B. subtilis 7988 (clinical isolate), L. monocytogenes (clinical isolate), S. aureus ATCC29213, MRSA, A. baumannii MDR, E. coli O157:H7, and S. enteritis (clinical isolate). The ethyl acetate extract also inhibited S. epidermidis ATCC35984, E. coli ATCC25922, K. pneumoniae (clinical isolate), S. enterica serotype Typhi (clinical isolate), and V. cholerae (clinical isolate), but not P. aeruginosa ATCC27853, unlike the hexane extract. The methanolic extract displayed a narrower antimicrobial profile, specifically against B. cereus ATCC11778, B. subtilis ATCC6051, B. subtilis 7988 (clinical isolate), S. epidermidis ATCC35984, S. pyogenes ATCC19615, A. baumannii MDR, and S. enteritis (clinical isolate). The aqueous extract showed no inhibitory effects against any of the tested bacteria.
Table 1: Antimicrobial activity of four different extracts of B. glabra screened against various pathogenic bacteria.
| Strains | Extracts | Positive Control
(amp/gen) | Negative Control
(1% DMSO) |
|---|
| Hexane | Ethyl acetate | Methanol | Aqueous |
|---|
| Gram-positive bacteria |
| Bacillus cereus ATCC11778 | + | + | + | – | +* | – |
| Bacillus subtilis 7988 (clinical isolate) | + | + | + | – | +* | – |
| B. subtilis ATCC6051 | + | + | + | – | +* | – |
| Listeria monocytogenes (clinical isolates) | + | + | – | – | +* | – |
| Staphylococcus aureus ATCC25923 | – | – | – | – | +* | – |
| S. aureus ATCC29213 | + | + | – | – | +* | – |
| Methicillin-resistant S. aureus (clinical isolate) | + | + | – | – | –* | – |
| Staphylococcus epidermidis ATCC35984 | – | + | + | – | +* | – |
| Streptococcus pyogenes ATCC19615 | – | – | + | – | +* | – |
| Gram-negative bacteria |
| Acinetobacter baumannii MDR | + | + | + | – | +** | – |
| Escherichia coli ATCC25922 | – | + | – | – | +** | – |
| E. coli O157:H7 (clinical isolates) | + | + | – | – | +** | – |
| Klebsiella pneumoniae ATCC70063 | – | + | – | – | +** | – |
| Pseudomonas aeruginosa ATCC27853 | + | – | – | – | +** | – |
| Salmonella enterica serotype Typhi (clinical isolates) | – | + | – | – | +** | – |
| Shigella enteritis (clinical isolates) | + | + | + | – | +** | – |
| Vibrio cholerae (clinical isolates) | – | + | – | – | +** | – |
3.3.2. The MICs and MBC against pathogenic bacteria
The MIC and MBC varied across the organisms investigated [Table 2]. The hexane extract demonstrated strong inhibitory activity against Gram-positive Bacilli, specifically B. cereus ATCC11778, which presented relatively low concentrations to inhibit the growth of B. cereus ATCC11778 at the MIC and MBC of 256 and 512 µg/mL, respectively. The MIC and MBC against B. subtilis ATCC6051 were at 2 mg/mL and 2 mg/mL, respectively, whereas those values against B. subtilis 7988 were higher in a clinical isolate at 4 mg/mL and 8 mg/mL. The inhibition effect of ethyl acetate and methanol extracts against B. cereus ATCC11778 showed around twofold higher at MICs and MBCs where those values revealed quite similar to both MIC and MBC against B. subtilis isolates. When the extracts were administered at high concentrations (measured in mg/mL), the MICs demonstrated effectiveness against the bacteria including L. monocytogenes (clinical isolate), S. aureus ATCC29213, MRSA (clinical isolate), S. epidermidis ATCC35984, S. pyogenes ATCC19615, and Gram-negative bacteria including A. baumannii MDR, E. coli ATCC25922, E. coli O157:H7, K. pneumoniae (clinical isolate), P. aeruginosa ATCC27853, S. enterica serotype Typhi (clinical isolates), S. enteritis (clinical isolate), and V. cholerae (clinical isolate).
Table 2: Minimum inhibitory concentrations and minimum bactericidal concentrations of four different extracts of B. glabra against various pathogenic bacteria.
| Strains | Minimum Inhibitory Concentration (MIC)/Minimum Bactericidal Concentration (MBC) in µg/mL | Minimum Inhibitory Concentration (MIC) of Ampicillin/Gentamycin (µg/mL) | Negative Control
(1% DMSO) |
|---|
| Hexane | Ethyl acetate | Methanol | Water |
|---|
| Gram-positive bacteria |
| Bacillus cereus ATCC11778 | 256/512 | 512/1024 | 512/2048 | R | 0.5/0.25 | R |
| Bacillus subtilis 7988 (clinical isolate) | 4000/8000 | 4000/4000 | 4000/>8000 | R | 0.5/0.5 | R |
| B. subtilis ATCC6051 | 2048/2048 | 4000/8000 | 4000/>4000 | R | 0.125/0.125 | R |
| Listeria monocytogenes (clinical isolate) | >16000/NA | >8000/NA | R | R | 0.25/2 | R |
| Staphylococcus aureus ATCC25923 | R | R | R | R | 0.25/0.5 | R |
| S. aureus ATCC29213 | >16000/NA | >8000/NA | R | R | 0.25/0.125 | R |
| Methicillin-resistant S. aureus (clinical isolate) | >16000/NA | >8000/NA | R | R | NA/0.25 | R |
| Staphylococcus epidermidis ATCC35984 | R | >8000/NA | >16000/NA | R | 0.5/0.25 | R |
| Streptococcus pyogenes ATCC19615 | R | R | >16000/NA | R | 0.25/0.125 | R |
| Gram-negative bacteria |
| Acinetobacter baumannii MDR | >16000/NA | >8000/NA | R | R | NA/1 | R |
| Escherichia coli ATCC25922 | >16000/NA | >8000/NA | R | R | NA/0.031 | R |
| E. coli O157:H7 (clinical isolates) | >16000/NA | >8000/NA | R | R | NA/0.063 | R |
| Klebsiella pneumoniae (clinical isolate) | R | >8000/NA | R | R | NA/0.062 | R |
| Pseudomonas aeruginosa ATCC27853 | >16000/NA | R | R | R | NA/4 | R |
| Salmonella enterica serotype Typhi (clinical isolates) | R | >8000/NA | R | R | NA/2 | R |
| Shigella enteritis (clinical isolate) | >16000/NA | >8000/NA | >16000 | R | NA/1 | R |
| Vibrio cholerae (clinical isolate) | R | >8000/NA | R | R | NA/4 | R |
3.4. Anticancer Activity
The studied extracts exhibited anticancer activity against various human cancer cell lines. The anticancer activity of the extracts was assessed using the MTT assay. Results indicated that the hexane extract from B. glabra flowers exhibited the most potent cytotoxic effects against all four cancer cell lines (colon cancer cell line [HT-29], lung cancer [A549], breast cancer [MCF-7], and bile duct cancer [KKU-213A]) followed by the ethyl acetate and methanol extracts. The water extract had a mild to moderate effect on these cell lines [Figure 2]. Among the tested cancer cell lines, the colon cancer cell line HT-29 showed the highest sensitivity to the extracts, followed by lung cancer (A549), breast cancer (MCF-7), and bile duct cancer (KKU-213A) cell lines [Figure 2].
 | Figure 2: The anticancer activity of the dried flower of B. glabra extracted in different solvents was tested against four cancer cell lines: lung cancer (A549), colon cancer (HT-29), breast cancer (MCF-7), and bile duct cancer (KKU-213A) and two normal cell lines: HDF derived from human skin fibroblast cells and MMNK-1 derived from the epithelial lining of the bile duct. The charts show the results of cytotoxicity assays involving B. glabra flower extracted in hexane (A), ethyl acetate (B), methanol (C), and water (D). Results are means ± SD from five replicate experiments. Additional data can be found in Supplementary Data Tables 1–4.
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3.5. GC-MS Analysis of Phytochemical Compounds
In the hexane extract [Table 3], the major chemical constituents were triterpene (squalene) and one natural tocotrienol, or vitamin E (1-benzopyran-6-ol, 3,4-dihydro-2,5,7,8-tetramethyl-2-[4,8,12-trimethyltridecyl]). Several diterpenes were identified (Kaur-16-ene, phytol and phytol acetate). A group of esters was also detected, including 9,12,15-octadecatrienoic acid, methyl ester, 9,12-octadecadienoic acid, methyl ester and hexadecanoic acid, and methyl ester. The chemical components in the ethyl acetate extract [Table 4] were mostly 2-hydroxypropane-1,3-diyl diacetate, fatty acid (hexadecenoic acid), and triterpene (squalene). In the methanol extract [Table 5], most of the chemical constituents were classified into an ester group (hexadecanoic acid, methyl ester, 9,12,15-octadecatrienoic acid, methyl ester, and 9-octadecenoic acid, methyl ester). The aqueous extract [Table 6] contained cyclic hydrocarbons (2,3-dihydro-benzofuran, 1,2-cyclopentanedione and 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl).
Table 3: The major chemical components of the hexane extract of B. glabra flower were identified by GC-MS analysis.
| No. | Identification | Formula | Rt (min) | Percentage |
|---|
| 1 | 2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene | C30H50 | 67.43 | 14.04 |
| 2 | 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltrideca-3,7,11-trienyl)-3,4-dihydrochromen-6-ol | C29H50O2 | 73.89 | 9.92 |
| 3 | methyl octadeca-9,12,15-trienoate | C19H32O2 | 48.52 | 1.99 |
| 4 | 9,12-Octadecadienoic acid | C18H32O2 | 50.03 | 1.87 |
| 5 | 5,5,9-trimethyl-14-methylidenetetracyclohexadecane | C20H32 | 46.27 | 1.10 |
| 6 | 3,7,11,15-tetramethylhexadec-2-en-1-ol | C20H40O | 48.91 | 0.91 |
| 7 | 7,11,15-trimethyl-3-methylidenehexadec-1-ene | C20H38 | 40.33 | 0.90 |
| 8 | methyl octadeca-9,12-dienoate | C19H34O2 | 48.33 | 0.81 |
| 9 | methyl hexadecanoate | C17H34O2 | 43.18 | 0.26 |
| 10 | 3,7,11,15-tetramethylhexadec-2-enyl] acetate | C22H42O2 | 41.73 | 0.23 |
| 11 | (3,5-ditert-butylphenoxy)-trimethylsilane | C14H22O | 28.43 | 0.15 |
| 12 | Undecane | C11H24 | 11.31 | 0.15 |
Table 4: The major chemical components of the ethyl acetate extract of B. glabra flower were identified by GC-MS analysis.
| No. | Identification | Formula | Rt (min) | Percentage |
|---|
| 1 | (3-acetyloxy-2-hydroxypropyl) acetate | C.H12O5 | 17.36 | 5.61 |
| 2 | Hexadecanoic acid | C16H32O2 | 44.42 | 3.58 |
| 3 | 2,3-dihydroxypropyl acetate | C.H10O4 | 10.93 | 3.45 |
| 4 | 2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene | C30H50 | 67.42 | 3.16 |
| 5 | 7,11,15-trimethyl-3-methylidenehexadec-1-ene | C20H38 | 40.32 | 2.67 |
| 6 | 3,7,11,15-tetramethylhexadec-2-en-1-ol | C20H40O | 48.90 | 2.19 |
| 7 | 2,3-dihydro-1-benzofuran | C.H.O | 16.28 | 1.35 |
| 8 | (2-acetyloxy-3-hydroxypropyl) acetate | C.H12O5 | 17.56 | 1.24 |
| 9 | 3,7,11,15-tetramethylhexadec-2-enyl] acetate | C22H42O2 | 41.72 | 0.62 |
| 10 | 6-hydroxy-4,4,7a-trimethyl-6,7-dihydro-5H-1-benzofuran-2-one | C11H16O3 | 37.43 | 0.51 |
| 11 | methyl octadeca-9,12-dienoate | C19H34O2 | 48.33 | 0.40 |
Table 5: The major chemical components of the methanol extract of B. glabra flower were identified by GC-MS analysis.
| No. | Identification | Formula | Rt (min) | Percentage |
|---|
| 1 | (2-acetyloxy-3-hydroxypropyl) acetate | C3H8O3 | 21.42 | 6.20 |
| 2 | Methyl hexadecanoate | C17H34O2 | 19.99 | 5.34 |
| 3 | Methyl octadeca-9,12,15-trienoate | C19H32O2 | 26.55 | 4.56 |
| 4 | Methyl (Z)-octadec-9-enoate | C19H36O2 | 24.42 | 4.14 |
| 5 | Hexadecanoic acid | C16H32O2 | 32.82 | 3.63 |
| 6 | Methyl octadeca-9,12-dienoate | C19H34O2 | 25.29 | 3.28 |
| 7 | 4-Methoxy-4-oxobutanoic acid | C5H8O4 | 22.02 | 1.60 |
| 8 | 4-Ethenyl-2-methoxyphenol | C9H10O2 | 19.03 | 1.58 |
| 9 | Octadec-9-enoic acid | C18H34O2 | 36.62 | 1.48 |
| 10 | Furan-2-ylmethanol | C5H6O2 | 10.36 | 1.15 |
| 11 | 5-Methylfuran-2-carbaldehyde | C6H6O2 | 9.44 | 0.63 |
| 12 | Phenylmethanol | C7H8O | 13.39 | 0.32 |
Table 6: The major chemical components of the aqueous extract of B. glabra flower were identified by GC-MS analysis.
| No. | Identification | Formula | Rt (min) | Percentage |
|---|
| 1 | 2,3-Dihydro-1-benzofuran | C8H8O | 22.60 | 6.82 |
| 2 | Cyclopentane-1,2-dione | C5H6O2 | 11.73 | 2.72 |
| 3 | 3,5-Dihydroxy-6-methyl-2,3-dihydropyran-4-one | C6H8O4 | 20.07 | 2.37 |
| 4 | 4-Hydroxy-2,5-dimethylfuran-3-one | C6H8O3 | 15.90 | 2.18 |
| 5 | 4-Ethenyl-2-methoxyphenol | C9H10O2 | 18.85 | 2.07 |
| 6 | Methyl carbamate | C2H5NO2 | 9.90 | 1.93 |
| 7 | Furan-2-ylmethanol | C5H6O2 | 10.28 | 1.76 |
| 8 | 2,3,5-Trimethylcyclohexa-2,5-diene-1,4-dione | C12H10O4 | 34.10 | 1.66 |
| 9 | 2-Methoxyphenol | C7H8O2 | 13.02 | 1.52 |
| 10 | Hexadecanoic acid | C16H32O2 | 32.59 | 1.44 |
| 11 | Benzoic acid | C7H6O2 | 23.41 | 0.98 |
| 12 | Phenol | C6H6O | 15.36 | 0.80 |
4. DISCUSSION
Our results demonstrated that the majority of the phenolic compounds in the dried flower of B. glabra were hydrophilic, as indicated by the high TPC in the methanol and aqueous extracts. This finding is consistent with previous studies of B. glabra [7], which also reported the highest TPC found in methanolic extracts. While one may criticize the Folin–Ciocalteu method for its indirect nature and potentially unspecific results, it has been widely used and remains an acceptable means of quantifying TPC. More direct methods, such as HPLC and GC-MS, have been employed to detect novel phenolic compounds in B. alba [16] and can be used to directly quantify individual phenolic compounds. Intriguingly, we found that the TPC of the purple flowers of B. glabra in our study (26.97 ± 1.70 mg GAE g−1 DW) was comparable to the TPCs reported in studies from flowers of Bougainvillea x buttiana Holttum and Standl (var. Rose) in Mexico (21.14 mg GAE g−1 DW) [17] and from the aerial parts of B. glabra in Pakistan (24.01 mg GAE g−1 DW) [7]. This similarity suggests that plants from the genus Bougainvillea consistently produce similar TPCs, even when cultivated in different geographical areas.
Various phenolic compounds exhibit varying degrees of free radical scavenging activity, influenced by the number of hydroxyl groups they possess. As a result, many studies have observed a positive correlation between TPC and antioxidant activities [18]. In this study, the antioxidant activities detected in the DPPH and ABTS assays were consistent with the TPC of the different extracts. Meanwhile, the ethyl acetate extract exerted the highest antioxidant activity in the FRAP assay. While antioxidant activities indicated by the FRAP assay have usually shown a positive correlation with TPC, a study of antioxidants in certain fresh fruit juices [19] found, as we did, that the results of an FRAP assay did not show a correlation with TPC. The criteria for quantifying antioxidants and the individual chemical characteristics of bioactive compounds in plant extracts are the main factors influencing the degree of antioxidant activity.
B. glabra flower exhibits notable potential as an effective antimicrobial agent against a wide array of pathogenic bacterial infections in both ATCC strains and clinical isolates. In our investigation, the purple B. glabra flowers displayed distinctive chemical compositions and biological properties that contribute additional information to the phytochemical profiles identified in previous studies. The hexane and ethyl acetate extracts demonstrated inhibitory effects on a broader spectrum of clinically relevant bacteria, encompassing methicillin-resistant strains of S. aureus (clinical isolate) and S. epidermidis ATCC35984, clinical isolates of Bacillus spp. and L. monocytogenes (clinical isolate), and multidrug-resistant strain of A. baumannii. Furthermore, both hexane and ethyl acetate extracts inhibited the growth of the pathogenic Gram-negative bacteria E. coli O157:H7 (clinical isolates), S. enteritis (clinical isolate), and S. enterica serotype Typhi (clinical isolate) at high concentrations of the extracts in mg/mL unit. Ethanolic and methanolic flower extracts of different Bougainvillea species have presented antimicrobial properties against a broad spectrum of bacteria including S. aureus, E. coli, P. aeruginosa, and B. cereus [20,21]. Swamy et al. also investigated the antimicrobial activity of the Bougainvillea bract against K. pneumoniae, P. vulgaris, P. aeruginosa, B. subtilis, as well as fungi. While the bract is highlighted for its antimicrobial potential, leaf extracts of Bougainvillea are also considered promising antimicrobial agents as flowers are originally adapted from leaves [22]. In addition, Ravikumar and Pratheep revealed that methanolic extract of B. glabra flower inhibited the growth of S. aureus, B. subtilis, P. putida, E. coli, and Candida albicans [20]. While the microbial properties of Bougainvillea flowers have been tested in previous studies, this study critically contributes essential insights into the antimicrobial properties, particularly against untested bacterial strains, S. epidermidis ATCC35984, clinical isolates of Bacillus spp., L. monocytogenes, pathogenic Gram-negative bacteria E. coli O157:H7, and multidrug-resistant strain of A. baumannii.
Medicinal plants have been extensively exploited in the treatment of various ailments, and B. glabra is recognized as a promising candidate for investigation. Despite its acknowledged potential, the understanding of its phytochemical properties, antimicrobial activity, and anticancer effects is not yet complete. In this study, we have demonstrated that the purple flowers of B. glabra extracts possess anticancer activity testing on the colon cancer cell line HT-29, the lung cancer (A549), the breast cancer (MCF-7), and the bile duct cancer (KKU-213A), aligning with previous research findings that indicate their consistent efficacy in suppressing various cancer cell lines. Saleem et al. illustrated that both methanol and dichloromethane Bougainvillea extracts effectively inhibited the growth of breast (MCF-7 and MDA-MB-231), cervical (CaSki), brain (DU-145), and colon (SW-480) cancer cell lines [7]. Moreover, an ethanol extract of B. glabra leaf demonstrated cytotoxic effects on HT-29, AGS (gastric adenocarcinoma), and BL-13 (bladder cancer) cell lines [23].
The belief that remedies from natural sources are safer may not always be true. Therefore, safety assessments are mandatory in the search for new drugs derived from medicinal plants. The study of Krishna and Sundararajan conducted toxicity studies on a methanol extract of B. glabra using a Wistar albino rat model [24]. The study suggested that the extract was well-tolerated and did not show any observed toxicity in both acute and sub-chronic settings. Our study [Figure 2] showed that, against the normal skin fibroblast HDF cells, high concentrations of all our extracts exhibited only mild toxicity. When our extracts were tested on normal bile duct cells, MMNK-1, results showed that water [Figure 2D] and methanol [Figure 2C] extracts had only a mild effect. On the other hand, the ethyl acetate [Figure 2B] and hexane [Figure 2A] extracts significantly suppressed the growth of MMNK-1. The major difference between these two normal cell lines is that HDF is a normal slow-growing cell derived from human skin fibroblast cells, while MMNK-1 is derived from the epithelial lining of the bile duct, modified to be immortalized cells [25]. Therefore, they may not truly represent normal cells. Our findings add to the information regarding the safety of Bougainvillea extracts, suggesting that they exhibit a definite antiproliferative effect against malignant cells but show low to no effect on slow-dividing normal skin fibroblast cells.
In this study, certain chemical components detected in the purple flowers of B. glabra flowers in the Songkhla Province, Thailand, were consistent with those detected in previous studies [26,27]. In the present investigation, certain constituents were identified within the purple flowers of B. glabra for the first time. For example, 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one in the ethyl acetate extract and furan (2-Furancarboxaldehyde, 5-methyl-) in the methanol extract. GC-MS analysis identified myristic acid as a primary contributor to the reduced microbial growth rate, with palmitic acid also playing a partial role. Diterpenes, including phytol, were identified as potential antimicrobial agents. Palmitic acid and phytol were also detected in the GC-MS analysis of our hexane [Table 3], ethyl acetate [Table 4], and methanol [Table 5] extracts where they might potentially be the cause of the inhibition of Gram-positive and Gram-negative bacteria [28]. The differences in the number and quantity of terpenes and phenols identified in the various extracts of Bougainvillea flower may be key factors in explaining its high antioxidant and antimicrobial activities, as well as the promising suppression of certain cancer cell lines. As anticipated, both plant species and geographical location can impact metabolic production [29], particularly in the synthesis of secondary metabolite pathways.
Despite the high phenolic content in the methanol extract, it may not exhibit the anticipated levels of antimicrobial and anticancer activities, as demonstrated in a previous study on Rubus idaeus L. [30], where various types of antioxidant activity were observed, but antimicrobial activity against different strains was not detected. Furthermore, we cannot discount the possibility that certain microbial and anticancer agents present in hexane and ethyl acetate extracts may be present in lesser quantities but could potentially offer higher potency in terms of antimicrobial and anticancer activities.
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