Antibacterial and antioxidant activities, lethality assay and chemical profile in crude extract of Biancaea sappan (L.) Tod. for anti-Vibrio agent

2.1 Extraction of plant heartwood

Heartwood of B. sappan was collected from medicinal plants shops in Tha Sala sub-district, Nakhon Si Thammarat province. Heartwood powder (50 g) was soaked in 300 mL (1:6 w/v) of 95 % ethanol with shaking at 150 rpm for 5 days (Hemthanon and Ungcharoenwiwat, 2022), and the crude extract was separated by a double layer of white cloth and filter paper. The extract was concentrated using a rotary evaporator at 50 °C and stored in glass bottle covered with aluminum foil at −20 °C until use. The yield (%) was calculated by formula: Extract yield (%) = (crude extract (g) / plant powder (g)) × 100. The B. sappan extract (BSE) (1,250 mg) was prepared in DMSO (1 mL) and diluted with distilled water to 125 mg/mL (10 % DMSO) before filtering with a 0.22 μm membrane. The BSE stock (125 mg/mL) was stored at −20 °C until next study.

2.2 Bacterial strains and chemicals

The pathogenic bacteria causing foodborne illness, wound infection, and other general diseases including two Gram-positive bacteria (B. cereus TISTR747 and S. aureus TISTR2329) and three Gram-negative bacteria (E. coli TISTR527, P. aeruginosa TISTR357 and V. parahaemolyticus TISTR1596) were used for antibacterial activity assay. All bacterial strains were provided by TISTR Culture Collection (Bangkok MIRCEN, Bangkok, Thailand). Each strain was cultured on nutrient agar (NA) at 35 °C for 18–24 h. All chemicals were of analytical grade.

2.3 Antibacterial activity assay

2.4 Determination of total phenolic content

The BSE was qualitatively determined total phenolic content (TPC) by the Folin–Ciocalteu assay modified from Wong-Paz et al. (Wong-Paz et al., 2014). Briefly, 40 μL of BSE and 40 μL of Folin–Ciocalteu reagent were mixed at room temperature (RT, 28 ± 2 °C) for 5 min, and 40 μL of 0.01 M sodium carbonate was then added. The mixture was allowed to stand for 5 min in the dark, and then diluted with DI water. All tubes were measured the absorbance at 790 nm, and distilled water was used as a blank. The TPC was expressed as milligrams of gallic acid equivalents per gram of dried extract (mg GAE/g) in accordance with calibration curve of gallic acid.

2.5 Determination an antioxidant activity

BSE antioxidant activity was evaluated using 2,2–diphenyl–1–picrylhydrazyl (DPPH) radical scavenging activity with modified from Farasat et al. (Farasat et al., 2014). Briefly, BSE stock solutions were made to obtain concentrations ranging from 0.03 to 31.25 mg/mL. The samples (100 μL) were mixed with 1 mL of 100 µM DPPH in MeOH, and reactions were performed at RT in the dark for 30 min. The absorbance was measured at 515 nm, and 95 % ethanol and 95 % ethanol + DPPH solutions were used as controls, and all assays were tested in triplicate. The percentage of radical scavenging activity was calculated using the formula: $$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{({\mathrm{A}}_{515}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-{\mathrm{A}}_{515}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e})\times 100}{{\mathrm{A}}_{515}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$$

DPPH scavenging activity was recorded and plotted on a graph, and the half-maximal inhibitory concentration (IC₅₀) value was then calculated.

2.6 Thermal and solvent stabilities

2.7 Brine shrimp lethality bioassay

BSE toxicity was evaluated on the basis of its ability to kill brine shrimp (A. salina) as proposed by Meyer et al. (Meyer et al., 1982), with some modifications by Ramos et al (Ramos et al., 2009). For the experiment, 400 μL of BSE was prepared in 4.6 mL of 2.5 % artificial seawater to obtain various concentrations (0.12, 0.24, 0.49, 0.98, 1.96, 3.91, 7.81, 15.62, and 31.25 mg/mL) in vials that contained ten live brine shrimp nauplii. All treatments and controls were let to stand at RT for 24 h. The experiment was performed in triplicate. After incubation, the surviving nauplii in each vial were counted and calculated as % mortality. An acute toxicity was determined using the median lethal concentration test (LC₅₀), which was the concentration of BSE that killed brine shrimp (50 %) at an exposure time.

The toxicity evaluation with the LC₅₀ value was performed using Clarkson’s toxicity index (Clarkson et al., 2004) as follows: non-toxic (LC₅₀ > 1,000 µg/mL), low toxicity (LC₅₀ 500–1,000 µg/mL), medium toxicity (LC₅₀ 100–500 µg/mL), and high toxicity (LC₅₀ 0–100 µg/mL).

2.8 Chemical profiling of BSE

The chemical profiling of BSE was determined by LC-Q-TOF-MS/MS. The analysis was performed on an Agilent 1290 Infinity II LC system coupled to a G6545 quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, USA). The analysis was carried out with Zorbax Eclipse Plus C18 Rapid Resolution HD (150 mm length × 2.1 mm inner diameter, particle size 1.8 μm) (Agilent Technologies, USA) maintained at 25 °C. The mobile phase composed of 1 % acetic acid in water (solvent A) and acetonitrile (solvent B) applying the following gradient: 0–5 min (A: 95 %, B: 5 %), 5–10 min (A: 80 %, B: 20 %), 10–15 min (A: 70 %, B: 30 %), 15–20 min (A: 65 %, B: 35 %), 20–25 min (A: 55 %, B: 45 %), 25–30 min (A: 25 %, B: 75 %), 30–32 min (A: 5 %, B: 95 %) system with a flow rate 0.2 mL/min. The sample volume was 2 μL, and mass spectra were acquired in the negative ion mode with a mass range of 100–1,500 m/z for MS and MSMS in automatic mode. Mass spectral analysis was carried out on a MassHunter WorkStation-Qualitative Analysis, and Agilent personal Compound Database and Library (PCDL) version B.08.00 build 92 was employed to create the custom database.

2.9 Anti-Vibrio activity in seawater

The time–kill behavior at MIC and MBC values of BSE against V. parahaemolyticus TISTR1596 was determined. The bacterial strain was cultured and prepared to yield approximately 10⁸ CFU/mL. The BSE stock solution (125 mg/mL) was diluted to make 1MIC, 2MIC, 3MIC, and 4MIC in a test tube containing 10 mL of seawater, and 10 % DMSO was used as a control treatment. The tests were performed in triplicate. The bacterial suspension (100 µL) was added to each tube (10⁶ CFU/mL), and all treatments were incubated at 35 ± 2 °C for 0, 3, 6 and 12 h. Ten microliters of each treatment was then placed onto TCBS agar and incubated at 35 ± 2 °C for 24 h. The bacterial growth at each time point was determined and calculated in CFU/mL.

2.10 Statistical analysis

The results were expressed as the mean ± standard deviation (SD). All experiments were conducted in triplicate and analyzed using a one-way analysis of variance. Data were analyzed using SPSS (IBM Corp., Armonk, NY, USA). Values were considered statistically significant at p < 0.05.

3.1 Plant extraction yield

The ethanolic BSE was obtained using the maceration method with 95 % ethanol. The BSE had a red–brown color, a sticky paste texture, and a distinctive smell. Fifty grams of leaf powder was used, and 4.78 g of the crude extract was obtained, which was a BSE yield of 9.56 % (w/w). Most previous studies reported that the sappan heartwood contained a red dye component that contained brazilin. Brazilin was the major active ingredient in group of flavonoids that is a phenolic compound. It can be dissolved in high polarity solvents because of the hydroxyl group in its structure (Rondão et al., 2013; Nirmal et al., 2015). It was extracted along with the phytochemical substances in sappan heartwood, and the crude extract was a shiny brown powder with a 17.60 % yield (Bukke et al., 2015). Therefore, important factors affecting the extraction yield may be the type, amount, and form of phytochemical substances that could dissolve in the extraction solvents.

3.2 Determination of the minimum inhibitory concentration and minimum bactericidal concentration

Antibacterial activity against the five pathogenic bacteria was determined. The results are shown in Table 1. BSE showed highly efficient inhibitory effects on Gram-positive and Gram-negative bacteria, with a MIC range of 0.49–3.91 mg/mL. It had a low MIC value toward V. parahaemolyticus TISTR1596 (0.49 mg/mL), S. aureus TISTR2329, B. cereus TISTR747 (0.98 mg/mL), and E. coli TISTR527 (3.91 mg/mL). As a result, the sappan methanolic extract which inhibited both groups of bacteria and showed the highest inhibitory to E. coli MTCC443 and B. subtilis MTCC10619 (MIC = 2 mg/mL) (Bukke et al., 2015). In the same way, the MBC values of BSE were 0.49, 15.62, 31.25, and 62.50 mg/mL against V. parahaemolyticus, S. aureus, B. cereus TISTR747, and E. coli, respectively. Therefore, the BSE had the highest bioactivity against V. parahaemolyticus, which might play a role in the specificity of the main compounds in crude extract to the bacterial cell wall. The main compounds in the BSE were phenolic compounds that could interact with the peptidoglycans of Gram-positive bacteria and the outer membrane of Gram-negative bacteria. There are many modes of action in the BSE such as membrane disruption, enzyme inactivation, and adhesin binding (Tiwari et al., 2011) that could affect the inhibitory and bactericidal activity toward two groups of pathogenic bacteria (Gram-positive and Gram-negative bacteria). Additionally, the stability and toxicity of both crude extracts were then determined, and preliminary evaluated as anti-Vibrio substance in seawater for its application in shrimp farming.

3.3 Total phenolic content and antioxidant property

The TPC of the BSE was measured using the Folin–Ciocalteu assay. BSE had a high TPC (31.46 mg GAE/g). However, the aqueous extract of B. sappan in Thailand had been reported a TPC of 147.02 ± 0.63 mg GAE/g (Taokaenchan et al., 2017). The amount of TPC in plants depends on the source and type of plant as well as the type of extraction solvent. The antioxidant activity in the BSE was measured using the DPPH assay. The percentage of inhibition of BSE at different concentrations is shown in Table 2. The BSE exhibited the highest scavenging activity on the DPPH radical at 83.05 % for 3.91 mg/mL and had an IC₅₀ value of 0.288 mg/mL. The antioxidant effects accorded to the amount of the TPC resulted from antioxidants containing an aromatic ring and a hydroxyl group as free radical terminators (Abdel-Hameed, 2009), which can bind with any free radical. The BSE had effective DPPH radical scavenging activity and exhibited remarkable antioxidant activity at a low concentration of 0.98 mg/mL (81.67 %). Furthermore, Butkrup et al. (Butkhup and Samappito, 2011) found that the scavenging effect of BSE on the DPPH radical was 82.93 % at 2.00 mg/mL of crude extract.

3.4 Stability studies of the total phenolic content and antibacterial substances under various conditions

The phytochemical substances in BSE were determined at different temperatures (room temperature, pasteurization, and autoclave) and with different diluents (distilled water and seawater). The thermal stability of TPC is shown in Fig. 1. The TPC increased to over 100 % under both pasteurization and sterilization conditions (116.27 and 121.01 %), which indicates that it is resistant to heat degradation because of temperature kinetics that increased the TPC (Molaveisi et al., 2019). Generally, the temperature affected the amount of phenolic content, broke the phenolic structure, and caused instability. However, some phenolic compounds were heat tolerant such as quercetin in the Leguminosae family, which showed an increasing phenolic compound content at 120 °C (Sharma et al., 2015). Furthermore, Ghafoor et al. (Ghafoor et al., 2019) found that the difference in the TPC may be due to the temperature and plant structure. For the effect of diluent on TPC for 24 h, the remaining TPC in distilled water was over 90 %, while that in seawater was approximately 40 %–50 % at 12–24 h (Fig. 1B). Therefore, BSE had the stability of phytochemical substances in distilled water. The crude extracts were not stable in seawater, which may have been caused by the interaction between the ions in seawater and the hydroxyl group in the phenolic compounds (Atkins, 1922), which could lead to a decrease in the TPC. However, the BSE had low stability in seawater, but it still had high anti-Vibrio activity with antibacterial substances that were similarly active, and it showed the same lowest of MIC and MBC against this strain (Table 3).

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Fig. 1

Thermal stability of the TPC in BSE at room temperature, pasteurization (63 °C, 20 min) and autoclave (121 °C, 15 min) conditions (A). Stability of the TPC in BSE dissolved in distilled water and seawater at 0, 6, 12, and 24 h (B).

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3.5 Toxicity test using A. salina model

The BSE was tested using A. salina to evaluate the median lethal concentration (LC₅₀). A. salina larvae mortality in crude extracts after 24 h is shown in Table 4. A. salina mortality ranged from 0.12 to 31.25 mg/mL of crude extract, and the acute toxicity test of crude extracts within 24 h showed a LC₅₀ value > 1,000 µg/mL, which indicated a non-toxic substance (Clarkson et al., 2004). A LC₅₀ value at 7.374 mg/mL of BSE killed up to 50 % of the A. salina population. Fikriah et al. (Fikriah and Lestari, 2011) studied the acute toxicity test using brine shrimp and found that the ethanolic extracts from bark and stem of Sopang were toxic, with a LC₅₀ value below 1,000 µg/mL at 204.644 and 239.883 µg/mL. The main active component affecting A. salina larvae mortality was a flavonoid. The flavonoid mode of action was binding of the OH-group to integral cell membrane proteins, which interferes with active transport of Na⁺ and K⁺ ions. The active substance–integral protein complex disrupted Na⁺ ion transport into the cell, and the resulting high accumulation caused the cell membrane to rupture (Nurhayati et al., 2006). However, heartwood extract of B. sappan (250–5000 mg/kg) was reported to be safe with no acute or subacute toxicity in rats (Sireeratawong et al., 2010; Nirmal et al., 2015).

3.6 Chemical profiling of BSE

The total ion chromatograms (TIC) of BSE in the negative mode from LC-Q-TOF-MS/MS are shown in Fig. 2. Eighty-four retention times of peak were detected, and 45 proposed compounds were identified from fragmentation patterns in tandem MS compared with libraries and fragmentation profile. From TIC, 10 major peaks were demonstrated at R_t 2.275–14.534 min (Table 5). The highest area sum (24.47 %) at R_t 7.994 min was identified as 4,7-Dihydroxy-2H-1-benzopyran-2-one or 4,7 dihydroxycoumarin that belonged to the class of 7-hydroxycoumarins. It has not been reported in B.sappan, and several studies have reported its derivatives to antiproliferative activity against four different cancer cell lines (Govindaiah et al., 2019). One member, umbelliferone (UMB), showed B. cereus inhibition and antioxidant activity with non-toxic in acute or chronic oral gavage administration to laboratory animals (Cruz et al., 2020). Next, the peak of 7.772 min (9.45 % area sum) was assigned as Methyl 7-desoxypurpurogallin-7-carboxylate trimethyl ether that was cyclic ketone phenolic compound and methylation on purpurogallin carboxylic acid exhibiting many bioactivities (Jhoo, 2008). Brazilein was then found at R_t 10.410 min (8.38 % area sum) that was reported anti-inflammatory, anticancer, antibacterial and antioxidant properties (Liang et al., 2013; Krihariyani et al., 2020). Biochanin A, an O-methylated isoflavone in the class of 7-hydroxyisoflavone, was detected at R_t 13.174 (8.41 % area sum). It had been reported the high inhibitory against pathogenic bacteria as Xanthomonas axonopodis pv. glycines that DNA synthesis and flagella formation were altered as well as the composition of the bacterial membrane (Kai-Xuan et al., 2020). It had also been indicated their anti-inflammatory, anticancer, neuroprotective, antioxidant, anti-microbial, and hepatoprotective properties. Next, (2S,2″S,3S,3″R,4S)-3,4′,5,7-Tetrahydroxyflavan (2->7,4->8)-3,4′,5,7-tetra hydroxyflavan or geranin A (4.13 %) was an A-type proanthocyanin that was found anti-protozoa and antioxidant activities (Calzada et al., 2001; Maldonado et al., 2005). Finally, heliannone C (3.83 %) was identified with 285.07 m/z that had few reports of this bioactive flavonoid. As result and their activities, there is no report available on the synergistic activity of each substance, but the crude extract normally contained various active substances that was the combination form leading to be a possible synergism of biological activities.

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Fig. 2

LC-MS/MS chromatograms of ethanolic extract of B. sappan (BSE) in negative mode and MS zoomed spectrums of six major components.

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3.7 Determination of anti-Vibrio activity in seawater

The anti-Vibrio activity was tested in seawater at 1MIC–4MIC of crude extract for 0–12 h. The colony surviving in TCBS agar was measured by calculating the colony forming units (CFU/mL), and survival trends are shown in Fig. 3.

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Fig. 3

Morphology of brine shrimp (A. salina) at various BSE concentrations after 24 h.

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The control condition was tested in DMSO, and the bacterial number was 10⁵ to 10⁶ CFU/mL at a dilution of 10^-6 to 10^-1, which showed a slight increase from 0 h to 12 h. The bactericidal activity of BSE increased in a concentration-dependent manner. The 1MIC, 2MIC, and 3MIC values showed a gradual decreasing trend until there was less than 50 % of bacteria at 1 h. All BSE concentrations showed a high efficiency for killing V. parahaemolyticus within 6 h. The 3MIC and 4MIC values killed V. parahaemolyticus within the shortest time (2 h) followed by 2MIC (3 h) and 1MIC (6 h). The 4MIC value had the highest effective inhibition and killed V. parahaemolyticus within 1 h. The anti-Vibrio activity might be affected by the various types of phenolic compounds found in B. sappan heartwood including brazilin. Brazilin was a major active compound and had a variety of actions (Nirmal et al., 2015) including interfering with Na⁺ ions, enzyme inactivation, interfering with energy metabolism, and membrane disruption (Fikriah and Lestari, 2011; Tiwari et al., 2011). Cell membrane rupture may have caused cell death. The BSE showed a strong inhibitory effect and can be used in shrimp farming or other aquatic fields where V. parahaemolyticus is problematic (see Fig. 4).

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Fig. 4

Time–kill curve for V. parahaemolyticus in B. sappan extract at 1, 2, 3, and 4MIC for 12 h.

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