Isolation and identification of bioactive compounds from Tinospora cordifolia stem extracts as antibacterial materials in seawater environments

2.1 Materials

Tinospora cordifolia plants were provided by the Indonesian Medical and Aromatic Crops Research Institute (IMACRI) - Bogor. The plant samples were identified in the Herbarium Bogoriense laboratory, Directorate of Scientific Collection Management of BRIN, Cibinong – Bogor (ID number: B-1810/II.6.2/DI.05.07/6/2022). Pseudomonas aeruginosa (ATCC 15,442 strain) was obtained from IPB University Culture Collection (IPBCC) – Department of Biology, IPB University.

The solvents for extraction, fractionation, and isolation were distilled methanol, n-hexane, ethyl acetate, and distilled water. TLC silica gel sheet 60 F₂₅₄ aluminium sheets (Merck, 1.05554.0001) were used for thin layer chromatography, and silica gel (0.063–0.200 mm, Merck 1.07734.1000) was used for column chromatography. The antibacterial test materials were Brain Heart Infusion (BHI, BD, Bacto 237500), Mueller Hinton Agar (MHA), and synthetic seawater media (Marine Art, SF-1, Japan). Both tetracycline and ampicillin (Sigma) were used for positive control.

2.2 Extract preparation

The stems of T. cordifolia plants were washed and dried before being cut into small pieces and pulverized in a blender. The powdered simplicia was stored in a closed container at room temperature. One thousand grams of powdered simplicia were extracted using the repeated maceration method with 50% methanol (v/v) in the macerator for three consecutive days. The filtrate was filtered and evaporated using a rotary vacuum evaporator after maceration. Furthermore, the extract was collected, weighed, and refrigerated until further processing.

2.3 Isolation of the active compounds

The flow chart in Fig. 1 shows the process of isolating the active compound from the T. cordifolia plant extract. The extract was fractionated using column chromatography and solvents containing n-hexane, ethyl acetate, and methanol. After preparation, 12.5 g of crude stem extract was slowly added to the column. In this study, column chromatography used a flow rate of 2 mL/min with 100 mg/mL of concentration and 125 mL of volume. After that, the eluent was streamed and collected. The eluent was gradually changed from nonpolar to polar. A rotary vacuum evaporator was used to collect and evaporate the results of the column chromatographic elution. The fraction concentrates were then visualized, and aggregation was performed using the same thin-layer chromatography (TLC) profile. The active fraction was then isolated using a two-stage column chromatography method. 94.5 mg of the active fraction was prepared with a volatile solvent, 90 mg of silica gel was added, and the mixture was loaded into the chromatography column. The column chromatography elution results were placed in a vial and dried in an oven. The isolates or subfractions were then separated using TLC to produce pure isolates and combinations based on the same TLC profile. The subfraction (isolate) was weighed and placed in a bottle, and its activity was determined. Furthermore, for pure isolates, the compound structure was analysed and identified.

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Fig. 1

Flowchart of the isolation of bioactive compounds from T. cordifolia plants consisting of crude extract maceration, including initial column chromatography, secondary column chromatography with the most active fraction, and identification of the isolated compounds.

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2.4 Phenolic and flavonoid content analyses

The total phenolic content (TPC) of the resulting extract and active fraction was analysed using the Folin-Ciocalteu method (Singleton, Orthofer, and Lamuela-Raventós, 1999) with gallic acid as a standard. Pipette 500 L of sample solution and reference solution (25, 50, 100, 150, and 200 µL) into a test tube and dilute with 250 µL of Folin-Ciocalteu reagent and distilled water to a volume of 4 mL. After 8 min, 750 µL of 20% Na₂CO₃ was added and shaken until homogeneous. The mixing solution remained at room temperature for 2 h. Absorption was measured at a wavelength of 765 nm. Each measurement was repeated twice to obtain the average phenol content. The total phenolic content was represented as gallic acid equivalents (mg/g extract) by using the calibration regression equation of the curve.

Meanwhile, the total flavonoid content was analysed by the colorimetry aluminium chloride (AlCl₃) method described by Chang et al. with slight modification (Chang et al., 2002). 4 mg of quercetin was dissolved in 4 mL methanol. The standard curve measurements used 10, 20, 30, 40, and 50 µg/mL concentrations with standard solution volumes of 50, 100, 150, 200, and 250 µL. Each test tube (standard and sample) received 2 mL of distilled water and 150 µL of NaNO₂ (5%). After 5 min, 150 µL of AlCl₃ (10%) was added. After 6 min, 2 mL of 1 M NaOH was added, and the volume was adjusted with distilled water to 5 mL. After homogenizing the solution mixture, the absorbance was measured using a UV–Vis spectrophotometer at 510 nm.

2.5 Antibacterial assay

Antibacterial activity was assessed by serial broth dilution methods following previous methods as described elsewhere (Priyanto et al., 2022). Before the antibacterial test, all instruments were cleaned and sterilized in a UV chamber for 24 h. The media and nutrients were prepared according to the manufacturer’s instructions and autoclaved for 15 min at 121 °C. The 96-well microplate well (Iwaki) was filled with 100 µL of BHI medium (diluted with artificial seawater), and well (A) was filled with 100 µL of the extract solution (diluted on methanol) before generating a series of dilution concentrations (serially dilution was set starting from 5000 to 50 μg/mL for fraction samples, and from 300 to 10 μg/mL for sub-fraction and isolate samples). Next, 100 µL of the 24-hour cultured bacterial suspension was added to each well plate and incubated at room temperature for 24 h. This study used the standard McFarland 0.5 of 24-hour bacterial culture, equivalent to 1 × 10⁸ CFU/mL. The turbidity and clarity of each incubated test medium were compared to the control media solution to determine the minimum inhibitory concentration (MIC). When the inhibition of bacterial growth is at a lower concentration, referred to as the MIC value, the test (Mueller Hinton Agar (MHA) media is cleaner. After incubation, the minimum bacterial concentration (MBC) was observed by searching for areas on the agar medium with no bacterial growth.

2.6 Analysis thin layer chromatography (TLC)

TLC analysis isolated or separated the active compound from the liquid solution. Aluminium plates and silica gel 60 F₂₅₄ as the stationary phase were used for TLC analysis. The mobile phase (eluent) was a mixture of n-hexane, ethyl acetate, and methanol. The sample (fraction/isolate) was dissolved in the appropriate solvent and shaken until completely dissolved. Furthermore, the solution was spotted on the bottom plate line (1 cm from the bottom), dried, and then eluted in a chamber containing eluent in a specific ratio. The plates were removed and dried after being completely adsorbed (up to the top line of the plates). The spots formed were observed at wavelengths of 254 and 366 nm.

2.7 Identification of bioactive compounds by LC-MS, FT-NMR, and FT-IR

The molecular weights of the compounds were determined using liquid chromatography-mass spectrometry (LC-MS) with a UNIFI chromatography instrument (Xevo G2-XS QToF) equipped with Mariner Biospectrometry software. The LCMS test was performed with methanol solvent, a 5 µL injection volume, and a C-18 column (15 mm × 2 mm) with a 0.2 mL/minute flow rate. Furthermore, Fourier transfer nuclear magnetic resonance (FT-NMR) JEOL JNM 500 MHz was used to identify pure compounds from isolated results, such as proton (¹H NMR), carbon (¹³C NMR), and 2D-NMR, which included HMQC and HMBC. Approximately 3 mg of the pure isolate was dissolved in a special deuterated solvent (CD₃OD) and then placed in a unique glass tube for NMR in the centre of the magnetic field tank to be measured by NMR. The functional groups (chemical bonds) of the compounds were detected using Fourier transform infrared spectrometry (FT-IR) (Bruker-Tension II). The scan frequency range was 4000 cm⁻¹ to 500 cm⁻¹, with a resolution of 4 cm⁻¹ for 45 s.

3.1 Yield extract

In this study, the maceration technique was used to obtain yield extracts because, compared to other techniques, maceration is a more straightforward process, requires less equipment and is widely used on a large scale (Vongsak et al., 2013). T. cordifolia stem extract with 50% methanol solvent yielded a 20.35% yield. This study calculated yield by weighing the dry extract (concentrated extract) and dividing it by the initial weight (powdered plant material). The extraction efficiency is influenced by extraction time, temperature, method, solvent, and phytochemical composition (Elboughdiri, 2018; Ida Madiha et al., 2017). Because methanol has a boiling point of 65 °C, it is commonly used as a solvent in maceration. In comparison, ethanol has a boiling point of 78 °C, and ethyl acetate has a boiling point of 77.1 °C (Nawaz et al., 2020). As a result, the temperature used to evaporate the resulting extract with methanol solvent is not too high, reducing the risk of overheating and destroying the secondary metabolite content of the extract. As a result, the time needed to evaporate the sample is relatively short.

3.2 Fractionation and antibacterial activity

Fractionation was accomplished through column chromatography with silica gel as the stationary phase and combined solution as the mobile phase. The mobile phases in the fractionation mixtures were hexane, ethyl acetate, and methanol solvents with increasing polarity. Table 1 shows the T. cordifolia stem extract fractionation results and the eluent ratio after combining based on similarities in TLC spots. Methanol produced the highest yield (21.94%), followed by ethyl acetate (10.40%). The type, composition, and activities of the extracted phytochemicals were influenced by the polarity of the solvent used in extraction and fractionation (Nawaz et al., 2020). Ethyl acetate is a semipolar solvent (index polarity of 4.4) that can dissolve alkaloids, flavonoids, sterols, and terpenoids. Methanol is polar (with an index polarity of 5.1). It can dissolve polar compounds, such as amino acids, flavones, flavonoids, glycosides, polyphenols, saponins, phenolic compounds, tannins, and terpenoids (Sri Widyawati et al., 2014). Furthermore, hexane is nonpolar but can dissolve lignin, lipids, terpenoids, and sterols (Sri Widyawati et al., 2014). This result demonstrates that T. cordifolia contains more phytochemicals with polar properties.

Additionally, the flow velocity influences the fractionation process, which is affected by gravity, the polarity of the eluent, and the density of the stationary phase (silica gel) (Ravisankar et al., 2019; Pirrung et al.; 2018). In addition, the resulting fractions were subjected to an antibacterial test to determine their antibacterial activity level and the fraction's minimal inhibitory concentration. Table 2 displays the antibacterial activity of T. cordifolia stem extract fractions against P. aeruginosa bacteria. According to the antibacterial activity results, the 1st and 2nd fractions exhibited the lowest minimum inhibitory concentration (MIC) values (140 and 156 µg/mL). In contrast, the minimum inhibitory concentration (MIC) of positive control (tetracycline and ampicillin) was 3.9 µg/mL. The MIC values of the 1st and 2nd fractions are close to those of the positive control, indicating that the 1st and 2nd fractions possess a potent inhibitory effect; consequently, they were isolated for the subsequent processes.

3.3 Total phenolic and flavonoid contents

The Folin-Ciocalteu and colorimetric aluminium chloride (AlCl₃) methods were used to determine the increase in the content of these two phytochemicals (phenolic and flavonoids) in the active fraction. The TPC and TFC values of the initial extract increased significantly, according to the results in Table 3.

The total phenolic content and bioactive properties of extracts significantly impact their antioxidant and antimicrobial activities (Sukiman et al., 2022). Studies have shown that an increase in the phenolic content of plant extracts leads to an increase in antioxidant and antibacterial activity (Kadir, 2021). Correlation studies of phytochemical content on biological activity were also conducted in the T. crispa stem extracts (Sukiman et al., 2022). Based on the TPC and TFC values, it is highly probable that the active fraction belongs to the phenol group and is responsible for antibacterial activities. Therefore, isolation and further analysis are needed to obtain the active compound and its molecular structure.

3.4 LCMS of active fractions

The analysis result of liquid chromatography-mass spectrophotometry (LCMS) for the active fraction is presented in Fig. 2.

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Fig. 2

Results of LCMS analysis for active fractions.

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The active fraction chromatogram results are shown in Fig. 2, and there is a peak with a high intensity at a retention time of 1.9 min. At 1.9 min of retention time, a dominant (high) spectrum, m/z 314.22 (M + H)⁺, indicates a molecular weight of 313.22. Previous researchers reported that the m/z spectrum of 312.1235 (M−H)⁺ indicated the compound N-trans-feruloyltyramine (Khan et al., 2021). Another study found an N-feruloyl tyramine compound in the 314 (M + H)⁺ spectrum (Sun et al., 2019). These findings support previous findings that the T. cordifolia plant contains the compound N-trans-feruloyltyramine, also known as moupinamide, with a molecular weight of 313.13141 (Royani et al., 2023).

3.5 Isolation of the active Fraction and antibacterial properties

The active fraction was isolated to obtain pure isolates with activity against P. aeruginosa bacteria. Table 4 shows the mass gain results and the eluent ratio used.

Based on the similarity of the TLC plate results, ten subfractions were obtained from the isolation process. The fifth subfraction showed the highest mass gain (40.2 mg) of approximately 42.5%. The 7th fraction exhibited the lowest mass gain (about 3 mg) and is indicated as a pure compound with a single TLC spot, as shown in Fig. 3.

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Fig. 3

Spot TLC isolate results under UV lamp 254 and 366 nm of T. cordifolia subfraction 7th.

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The subfractions were then subjected to antibacterial tests to determine their activity against bacterial inhibition. Table 5 summarizes the findings of the antibacterial activity test performed on each subfraction against bacteria capable of forming biofilms.

The results of the antibacterial activity revealed that the minimum inhibitory concentration (MIC) values ranged from 16 to 128 µg/mL with varying minimum bacterial concentration (MBC) values, as shown in Table 5. Antibacterial activity from the subfraction results tended to be much stronger than that of the fractions and extracts, which could occur because the secondary metabolite content in the subfraction was purer, which significantly impacted its activity. In addition to the bacterial test, pure isolates were analysed with FT-IR, ¹H NMR, and ¹³C NMR to determine their molecular structure.

3.6 Identification of bioactive compounds

The ¹H NMR and ¹³C NMR spectra of the pure isolate are shown in Fig. 4 and Table 6, respectively. The results of the ¹H NMR spectrum of the isolate indicate that there are two aromatic rings, namely, the substitution at 1–4 (A2B2) on the benzene ring, in which the two signals overlap because they are symmetrical, namely, at δ_H 6.72 (2H, d, 8 Hz) and δ_H 7.06 (2H, d, 8 Hz). The aromatic ring with the ABX system (trisubstituted at C 1, 3, and 4) at δ_H 6.79 (d, 8 Hz), 7.01 (dd, 1.5; 8 Hz), and 7.15 (d, 1.5 Hz). Trans double bonds were detected at δ_H 6.40 (d, 16 Hz) and 7.42 (d, 16 Hz), and CH₂-CH₂ groups were detected at δ_H 2.75 (2H, t, 7 Hz) and 3, 47. (2H, t, 7 Hz). In addition to a methoxy group (—OCH₃) at 3.87 (s), as summarized in the structure shown in Fig. 4. The ¹H NMR and ¹³C NMR (Table 6) spectral data are consistent with published data (Al-Taweel et al., 2012; Jiang et al., 2015; Marques et al., 2022).

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Fig. 4

Analysis of isolated bioactive compounds (N-(4-hydroxy-3-methoxy-E-cinnamoyl)-4-(2-aminoethyl) phenol) with HMQC.

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According to the specific results of ¹H NMR and ¹³C NMR spectral data, this isolate was identified as N-(4-hydroxy-3-methoxy-E-cinnamoyl)-4-(2-aminoethyl) phenol, the synonym of which is 4-(2-aminoethyl) N-trans-feruloyltyramine and moupinamide, with the structure shown in Fig. 4. This compound has never been reported in T. cordifolia previously. However, N-trans-Feruloyltyramine has been isolated from other species, including Smilax aristolochiifolia (Amaro et al., 2014), Polyalthia suberosa (Soi-ampornkul et al., 2022), Solanum procumbens (Solanaceae) (Tran et al., 2019), Datura metel (Xu et al., 2018), and Bassia indica (Othman et al., 2021). This assumption was supported by the results of the ¹³C NMR spectrum measurements (Table 6) and the H and C correlation measurements (HMQC), as shown in Fig. 4 and Table 6.

Then, as shown in Fig. 5, the results are supported by HMBC long-range correlation, which can determine the position of the chemical movement value in the molecular structure. The primary correlation of HMBC also reveals two aromatic rings on the benzene ring (at δ_H 6.72 (2H, d, 8 Hz) and 7.06 (2H, d, 8 Hz)) as well as aromatic rings at δ_H 6.79 (d, 8 Hz), 7.01 (dd, 1.5; 8 Hz), and 7.15 (dd, 1.5; 8 Hz) (d, 1.5 Hz). Furthermore, trans double bonds were discovered at δ_H 6.49 (d, 16 Hz) and 7.42 (d, 16 Hz), as well as the presence of CH₂-CH₂ groups at δ_H 2.75 (2H, t, 7 Hz) and 3.47 (d, 16 Hz) (2H, t, 7 Hz). The long-range correlation coupling in the HMBC spectrum between the signal (3H, 3.87, s) and C at 149.94 can be used to determine the position of the methoxy group at C-3.

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Fig. 5

Chemical shift value and summary HMBC correlation for an isolate from T. cordifolia.

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Fig. 6 shows the FT-IR spectrum of the T. cordifolia isolates, which confirms the presence of the Moupinamide compound. Significant absorption bands at 3350–3300 cm⁻¹ (N—H bending) and 1651 cm⁻¹ (C⚌O stretch) in the IR spectra of the isolate indicate the amide function. An aromatic group was present, as evidenced by absorption at 1588 cm⁻¹ and between 880 and 680 cm⁻¹. It was also identified that in this isolate, the aliphatic functional group (CH₂) at 3010 cm⁻¹, carbonyl (C⚌O) at 1700 cm⁻¹, and ester at 1210 cm⁻¹.

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Fig. 6

FT-IR spectra of isolate results from T. cordifolia.

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The results of the antibacterial test of pure isolates versus crude extracts and active fractions, as illustrated in Fig. 7, indicate that the isolates are promising against the biofilm bacterium P. aeruginosa. The antibacterial activity was relatively high against P. aeruginosa, with a minimum inhibitory concentration of 32 µg/mL, as demonstrated by the bacterial test data. Furthermore, the MIC results in Fig. 7 support the relationship between phenolic and flavonoid content and antibacterial activity. The antibacterial activity increases with increasing concentrations of TPC and TFC in the active fraction of the crude extract (Table 3).

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Fig. 7

Comparison of MIC values for crude extracts, active fractions, and isolates from T. cordifolia plants.

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Several studies have shown that moupinamide or N-trans feruloyltyramine (NTF) compounds isolated from plants show various bioactivities, such as Liriope muscari (Liliaceae), which exhibits antioxidant activity (Li et al., 2012), and Celtis africana, which possesses anti-inflammatory and antioxidant activity (Al-Taweel et al., 2012). Other studies have discovered that N-trans feruloyltyramine from the fruit of Lycium barbarum can potentially be a neuroprotective and neurogenic agent for age-related brain diseases (Khan et al., 2021). Sidastrum paniculatum was effective against Aedes aegypti L (Marques et al., 2022). Piper wallichii has been shown to exhibit antibacterial pathogenic activity (Nongmai et al., 2022). Based on the data and some literature, it appears that moupinamide is responsible for the antibacterial activity of the plant extracts and isolates investigated in this study.