Extraction and characterization of total phenolic and flavonoid contents from bark of Swietenia macrophylla and their antimicrobial and antioxidant properties

2.1 Plant material

The bark of S. macrophylla was collected from the botanical garden located in Manipal, Karnataka-India (13°22′11″ N, 74°47′6″ E). The bark was dried at room temperature in shades, milled and sieved (300-µm particle size); powdered plant materials were stored in an airtight container at room temperature and preserved under darkness to avoid oxidation of compounds (Rhazi et al., 2019; Muñiz-Márquez et al., 2013).

2.2 Reagents and standards

Ethanol, methanol, acetone, and distilled water were used as extraction solvents. These solvents were of analytical grade. Rhodanine, Bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), tri-ethanolamine, potassium hydroxide, sodium nitrite, aluminum chloride, tannic acid, sodium hydroxide, Rutin, sodium carbonate, Folin-Ciocalteau reagent, ferric chloride, and phosphate buffer solution were used as reagents in this study and all procured from Hi-media Laboratories Pvt. ltd., Mumbai-India and Sigma Aldrich Co. LLC, Mumbai-India.

2.3 Solvent extraction

The one gram of powdered bark was taken in four different tubes, to which 20 mL of 50 %v/v different solvents such as methanol, ethanol, and acetone were added individually, whereas in the fourth tube 20 mL of pure distilled water was added. Then the tubes were kept for extraction in the rotary shaker for 48 h at 32 °C and 170 rpm. All experiments were carried out independently to screen the best extraction solvent. Thereafter, the extraction mixture was centrifuged for 10 min at 4 °C and 4000 rpm. After centrifuge, the extract (supernatant) was separated and the solvent was evaporated in the water bath at temperatures equivalent to the solvent’s boiling point till the volume of extract was reduced to half. After evaporation, the extracts were collected in vials and preserved at 4 °C for further analysis of the contents of total phenolic compounds, gallic acid, hydrolyzable tannic acid, and flavonoids (Muñiz-Márquez et al., 2013; Naima et al., 2015; Makkar, 2003). All the experiments were carried out in triplicates and average values with standard deviations were reported.

3.1 Estimation of total phenolic compound

The total phenolic compound (TPC) of the S. macrophylla bark extracts was estimated by using the Folin-Ciocalteu method (Blainski et al., 2013). Briefly, 0.2 mL plant extract diluted to 1 mL with distilled water was mixed with 10 mL Folin-Ciocalteau reagent (10 %v/v). After 5 min, 8 mL of 7.5 %w/v of aqueous sodium carbonate was added to the mixture and incubated at room temperature for 2 h. After incubation, the reaction mixture was read at 710 nm using UV-spectrophotometer against a blank without extract. The amount of total phenolic content in extract was expressed as mg of gallic acid equivalents (GAE)/ gram dry weight of plant material (gdw).

3.2 Estimation of total flavonoid content

The total flavonoid content (TFC) of the S. macrophylla bark extracts was estimated by using the spectrophotometric method (Kim et al., 2003; Zhishen et al., 1999). Briefly, 0.2 mL plant extract diluted five times with distilled water was mixed with 2.4 mL of distilled water. To this, 0.3 mL of 5 % sodium nitrite was added, mixed well, and incubated for 5 min. Then, 0.3 mL of 10 % Aluminum chloride was added, mixed well, and incubated for 6 min. Finally, 2 mL of 1 M sodium hydroxide was added, mixed in the reaction mixture, and read the absorbance at 510 nm. using UV-spectrophotometer against a blank without extract. The amount of TFC in the extract was expressed as mg of rutin equivalents/gram dry weight of plant material (gdw) i.e. mg rutin/gdw.

3.3 Estimation of hydrolysable tannic acid

The hydrolyzable tannic acid content of the S. macrophylla bark extracts was estimated by using the protein precipitation method of Ann-Hagerman and Larry-Butler (Ann-Hagerman and Larry-Butler, 1978). Briefly, 0.2 mL extract was diluted to 1 mL with distilled water and mixed with 2 mL of BSA solution. Incubated for 15 min at 30 °C, then centrifuged at 4 °C and 7000 rpm for 15 min. After centrifuge, the supernatant was discarded and to the pellet 4 mL of SDS-tri ethanolamine solution was added to dissolve the pellet. Further, 1 mL of ferric chloride solution was added, mixed immediately, and incubated for 15 min at 30 °C. After incubation, the absorbance was read using UV-spectrophotometer at 510 nm against a blank without extract. A standard curve was attained by finding the absorbance of standard solutions of tannic acid with concentrations ranging from 0.1 to 1 mg/mL.

3.4 Estimation of gallic acid content

The gallic acid content of the S. macrophylla bark extracts was estimated by using the spectrophotometric method (Sharma et al., 2000). Briefly, 0.25 mL extract was mixed with 0.25 mL citrate buffer (0.05 M and pH 5.0) and 0.3 mL of methanolic rhodanine, mixed well, and incubated for 5 min at 30 °C. After 5 min, 0.2 mL of 0.5 N potassium hydroxide was added and incubated at 30 °C for a further 5 min. Thereafter, 4 mL of distilled water was added and incubated at 30 °C for 10 min. Finally, the absorbance was read using UV-spectrophotometer at 520 nm against a blank without extract (i.e. instead of extract, citrate buffer was added). A standard curve was attained by finding the absorbance of standard solutions of 0.05 mM gallic acid in citrate buffer and expressed in mg/mL.

5.1 Thin-layer chromatography (TLC)

In this study, the resolution of hydrolyzable tannins, gallic acid, total phenols, and flavonoids in the bark extract of S. macrophylla were analyzed using TLC as described elsewhere (Das et al., 2019; Banu and Nagarajan, 2014). The silica gel sheet of dimensions 6 cm × 15 cm and thickness of 175–225 µm (Merck, TLC Silica gel 60 F₂₅₄) was used to perform the TLC. The straight line parallel to a smaller dimension was drawn 1 cm from the end of the plate with the pencil on either side. The two spots were marked with a 2 cm distance on the straight line on one side only while keeping the margin of 1 cm between a wall of the plate and spots. These spots were useful to load the plant extracts and standard for analysis. The silica gel sheets were activated by soaking the sheet with methanol and then drying at 110 °C for 10 min. The shots of bark extract of S. macrophylla and specific standard were spotted individually on the activated silica gel plate with the help of a wing micropipette and placed in a chromatographic chamber comprising different solvent systems. The spotted plates are placed in such a way that the spots do not touch the solvent. The chamber was closed with the lid and allowed to stand for a sufficient amount of time to separate the phytochemicals. Once the solvent reaches the second line, plates were removed and air-dried at room temperature. The specific spraying reagents were sprayed, the colored spots developed were marked, and dried and their distance was measured from the center of the spot. The retention factor Rf of the sample was calculated using the formula shown below: $$Retentionfactor,R_f\frac{Dis\tan cebysolute}{Dis\tan cetravelledbysolvet}$$

The composition of the specific solvent system (i.e. mobile phase) and the type of spraying reagents used to separate the phytochemical constituents qualitatively of bark extracts of S. macrophylla is shown in Table 3 as described elsewhere (Banu and Nagarajan, 2014).

5.2 Fourier transform infrared spectroscopy (FTIR) spectroscopy

Structural characterization of plant extract was carried out by Fourier Transform Infrared spectroscopy (SHIMADZU). The IR peaks were analyzed to confirm the presence of functional groups.

6.1 Antimicrobial activity

The antimicrobial activity of bark extract of S. macrophylla was tested against two strains of Gram-positive bacteria (Bacillus species and S. aureus) and two strains of Gram-negative bacteria (E. coli and S. marcescens). These strains were procured from the culture collection of the Institute of Microbial Technology, Chandigarh, India. All the above-mentioned strains are pre-cultured overnight in nutrient broth media at 32 °C and 180 rpm.

Next, the agar well diffusion method was adopted to check the antimicrobial activity of plant extract as described elsewhere (Gonelimali et al., 2018; Mostafa et al., 2018). The 0.1 mL of overnight grown strains were spread plated on sterilized and solidified nutrient agar plates individually. The wells of size 0.25 cm in diameter were drilled using the sterile cork borer. Then, 0.1 mL of steam-sterilized plant extract (5 %w/v) was added to the respective wells and placed in the refrigerator for 30 min to diffuse well the plant extract into the agar. Later, the plates were incubated at 37 °C for 24 h. The antibacterial activity was calculated by measuring the clear zone formation around the well using a Vernier caliper. Tannin acid and gallic acid at a concentration of 0.1 %w/v were taken as a positive control.

6.2 Antioxidant property studies

Electrochemical analysis of antioxidant property of plant extract was executed with SP 150 Biologic potentiostat with a three-electrode system. Ag/AgCl and platinum electrodes were used as reference and counter electrodes respectively. A glassy carbon electrode of 3 mm diameter was used as the working electrode. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) studies were carried out to check the antioxidant property. Stainless steel was also used as the working electrode in CV. The extracts were analyzed using 0.1 M phosphate buffer at pH 7. The extracts were prepared by dissolving 5 mL of the plant extract in 25 mL of methanol and ultrasonication was done for 30 min (40 kHz frequency) at room temperature. Post ultrasonication, the extract was subjected to CV and DPV by adding 200 mg/mL of extract concentration to 20 mL of 0.1 M PBS buffer (pH 7.0). The concentration of the extract was increased from 23.5 µM to 235 µM. CV was performed to analyze electrochemical responses between 0.0 and 1.25 V potential window. Scan rates varied between 10 and 100 mV/s. DPV was conducted between − 0.50 V to 1.00 V potential window, with a pulse amplitude of 20 mV and pulse width of 50 ms.

7.1 Impact of different extraction solvents

The total phenolic contents of the S. macrophylla bark were extracted by using different commonly used solvents such as water, methanol, ethanol, and acetone at 50 % v/v and the results are shown in Fig. 1. The results specified that the use of different solvents resulted in different extraction yields of bioactive compounds. 50 % acetone extract showed a higher yield of all the bioactive compounds quantified, which could be due to the high solubility of these compounds in acetone. The highest concentrations of bioactive compounds such as TPC, flavonoids, hydrolyzable tannins, and gallic acid were found to be 26.537 ± 1.56 mg GAE/gdw, 48.584 ± 1.76 mg Rutin/gdw, 9.491 ± 1.03 mg/mL, and 41.11 ± 1.534 µg/mL respectively in 50 % acetone extract. The lowest concentrations of these compounds were observed in water extract (Fig. 1). The choice of significant solvents plays an important role in the extraction of bioactive compounds and their applications in food products and therapeutics (Lasano et al., 2019). Solvents and their polarities will influence the solubility of bioactive compounds in a sample. The solvent penetration into the plant materials and extraction of bioactive compounds from them is more efficient with solvents having similar polarities of compounds (Dhanani et al., 2017; Mohammedelnour et al., 2017). Hence, the effect of different solvents on extraction was carried out to select the most influential solvent to recover phenolic compounds. In this study, the order of high yield of bioactive compounds by using solvents was as follows:

Acetone > Ethanol > Methanol > Water.

Dirar et al (Dirar et al., 2019) reported that the acetone and ethanol solvents are efficient in extracting a higher amount of phenolic and flavonoid compounds from plant sources. Our results are in good agreement with the results reported (N. F. Lasano NF, N. S. Ramli, A. H. Hamid, R. Karim, M. S. Dek, R. Shukri, 2019; Dhanani et al., 2017).

7.2 Optimization of solvent extraction of a total phenolic compound by central composite design (CCD)

The five factors acetone concentration (A), the volume of acetone (B), agitation speed (C), extraction temperature (D), and extraction time (E) were selected based on our preliminary studies to study their interactions and also to find their optimal levels using CCD. The experimental design matrix for the extraction of TPC from S. macrophylla bark is depicted in Table 2. The CCD showed an enhanced TPC yield of 49.53 mg GAE/gdw at 50 % (v/v) acetone concentration, 20 mL volume of acetone, agitation speed of 170 rpm, 32 °C of extraction temperature, and extraction time of 24 h (Run 5, 9, 19, 23, 24, 28 in Table 2). Next, the results were fitted to a second-order regression equation (Eq. (2)).

Where Acetone concentration (A), %v/v; Volume of acetone (B), mL; Agitation speed (C), rpm; Extraction temperature (D), °C; and Extraction time (E), h.

The outcome of an analysis of variance (ANOVA) specifies that the regression model predicts and fits the experimental results very well presenting a high R² and F- values (Table 4). In this study, the linear (except C), quadratic, and interactive (except AD, BC, and BD) model terms were found to be statistically significant for the extraction of TPC from the bark of S. macrophylla as the P-value was < 0.05 (Table 4). The interaction effects among the factors were visualized with 3D- surface plots as shown in Fig. 2. The interaction between AB, AC, AE, BE, CD, CE, and DE have shown a positive effect on the extraction of TPC representing that the levels selected were converging towards the center point (Level 0). Whereas the interaction between AD, BC, and BD showed a negative effect could be due to the different combinations of factor levels, even though the factors A, B, and D are individually significant.

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Fig. 2

Surface plots showing the interaction effects of the significant factors on the extraction of the TPC process from the bark of S. macrophylla with the remaining factors held constant at the center level of the central composite design: (a) Acetone concentration (A) and Volume of acetone (B); (b) Acetone concentration (A) and Agitation speed (C); (c) Volume of acetone (B) and Extraction time (E); (d) Agitation speed (C) and Extraction temperature (D); (e) Agitation speed (C) and Extraction time (E); and (f) Extraction temperature (D) and Extraction time (E).

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The accuracy of the model is tested by using the normal probability plot (Fig. 3). It is the plot between the residuals and the predicted response that also helps to check the normality assumption (Sebayang et al., 2017). If the data points are scattered randomly in the plot shows an inadequate fit and gives meager results. Fig. 3 depicts that the normality assumption is satisfied as all data points are along a straight line indicating that the model provides an adequate estimate of the actual system. Hence, it can be established that the regression model equation (Eq.2) is very much satisfactory to explain the extraction of the TPC process using acetone solvent.

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Fig. 3

The normal probability plots.

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The regression model (Eq. (2)) was solved by using a response optimizer tool of MINITAB 17.0. The acquired optimal levels of individual factors for the improved TPC yield are as follows: Acetone concentration (A) = 50 %(v/v), Volume of acetone (B) = 20 mL, Agitation speed (C) = 170 rpm, Extraction temperature (D) = 32 °C, and Extraction time (E) = 24 h; all of them were found to be present in the experimental levels (Table 1). Under these optimal conditions, the predicted TPC yield was 52.085 mg GAE/gdw (Fig. 4).

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Fig. 4

The optimization plot for extraction of TPC from the bark of S. macrophylla.

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Lastly, the regression model equation (Eq. (2)) was validated by performing the experiments in triplicates at the above-mentioned optimal levels. It was found that the experimental and predicted TPC yield was 51. 24 ± 0.89 mg GAE/gdw and 52.085 mg GAE/gdw respectively (Table 5), which indicates the adequacy of the model. This optimization method exhibited a 2.0-fold increase in the extraction of TPC from the bark of S. macrophylla using acetone as a solvent when compared to unoptimized conditions (26.537 ± 1.56 mg GAE/gdw). Che Sulaiman et. al (Che Sulaiman et al., 2017) reported an increase of 1.3 times on the extraction of TPC from Clinacanthus nutans Lindau leaves with the use of RSM for optimization, indicating that CCD employed in optimization of TPC extraction from S. macrophylla correlated with better results as there was a 2.0-fold increase in TPC yield. This is the first report on the extraction of bioactive compounds from the bark of S. macrophylla, which represents a high yield of total phenolic, flavonoids, and hydrolyzable tannins with antimicrobial and antioxidant properties.

7.3 Characterization of extracted total phenolic compounds

7.3.1

7.3.1 Thin-layer chromatography (TLC)

The TLC system was used to separate and identify the contents of flavonoids, phenolic compounds, tannic acid, and gallic acid in acetone extracts of S. macrophylla bark. In this system, Precoated silica gel sheets were used as the stationary phase and the equivalent solvent system was used as the mobile phase as shown in Table 3. Gallic acid, Rutin, and Tannic acid were used as a standard to identify the phenolic compounds and gallic acid, flavonoids, and tannic acid respectively. These compounds were separated in the TLC system and further identified by spraying their respective coloring agents as depicted in Table 3. The colored spots were spotted on the system indicating the presence of phenolic compounds, flavonoids and tannic acids with R_f values of standards Gallic acid, Rutin and Tannic acid are 0.624, 0.958, and 0.978 respectively (Fig. 5a-c). However, the compound gallic acid was not separated and identified (Fig. 5d). This indicates the absence of the component, or presence in the least concentrations in the bark of S. macrophylla, and also showed the gallic acid content of 41.11 ± 1.534 µg/mL using a spectrophotometric method as shown in Fig. 1, whereas other components are in the terms of mg/mL. The values of R_f mentioned for the phenols, flavonoids and tannic acid are in good agreement with reports reported elsewhere (Dinakaran et al., 2019), which represents the presence of bioactive compounds.

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Fig. 5

The qualitative analysis of bioactive compounds in acetone extracts of S. macrophylla bark: Phenols (a), Flavonoids (b), Hydrolysable tannins (c), and Gallic acid (d) using thin layer chromatography with reference standards gallic acid (G), rutin (R), tannic acid (T), and gallic acid (G) respectively.

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7.4 Fourier transform infrared spectroscopy (FTIR) spectroscopy

The FTIR of the studied sample is displayed in Fig. 6. The band at 3309 cm⁻¹ is related to hydroxyl (OH wagging) of a phenolic group (Barud et al., 2013). A band was found at 1638 cm⁻¹ due to the C⚌O stretching vibration of flavonoids. A shoulder at 1700 cm⁻¹ may be due to stretching vibration of C⚌O group (Mot et al., 2011), 1509 cm⁻¹ related to aromatic ring deformations (Franca et al., 2014), 1375 cm^-1C-O stretching of acid groups or rocking vibration of aromatic CH, and 1236 cm⁻¹ due to vibration of C—O group of polyols of hydroxy-flavanoids (Silva et al., 2014). The results are in accord with Chukwuneke et al. (Chukwuneke, 2012), indicating that the functional groups identified are similar to the ones presented in this study.

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Fig. 6

FTIR spectra of bark extract of S. macrophylla.

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7.5 Application studies

7.5.1

7.5.1 Antimicrobial activity and minimum inhibitory concentration

The antimicrobial activity of the extract of S. macrophylla bark at 5 % (w/v) was tested against four pathogenic strains i.e. two-gram positive strains Bacillus sp, and S. aureus, and two-gram negative strains E. coli, and S. marcescens. In this study, the extract exhibited the area of inhibition (AOI) against the gram-positive strains tested whereas no AOI was found against gram-negative strains tested. Bacillus sp. and S. aureus showed an AOI of 181.4 mm² (21 mm clear zone of diameter with 5 mm standard disc) and 113.1 mm² (18 mm clear zone of diameter with 5 mm standard disc) respectively after 72 h of incubation at 37 °C (Fig. 7). The effectiveness of the extract was estimated by measuring the MICs against the strains with AOI. The extract of S. macrophylla bark showed the MIC value of 20 % (w/v) and 15 % (w/v) against Bacillus sp and S. aureus respectively. Our results showed a good antimicrobial effect against gram-positive bacteria, and also the results are in good agreement with Yuan et al (Yuan et al., 2021). This is the first report on the extraction of bioactive compounds from the bark of S. macrophylla, which represents a high yield of total phenolic, flavonoids, and hydrolyzable tannins with antimicrobial and antioxidant properties.

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Fig. 7

Results of antimicrobial activity and minimum inhibitory concentration.

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7.5.2

7.5.2 Electrochemical evaluation of antioxidant properties

The evaluation of the antioxidant properties of plant extract was carried out using CV and DPV. Fig. 8 depicts the redox behavior of compounds present in the plant extract with well-defined oxidation and reduction peaks; in which the CVs were recorded from − 0.2 to 1.4 V at various scan rates. The anodic peak potentials (E_pa) as shown in Fig. 8a, 0.17 V and 0.81 V were due to the presence of various antioxidants with varying oxidation potentials (Phenolic compounds and flavonoids). These results confirm that plant extract contains multiple antioxidants. The low and high oxidation potential values obtained from voltammograms indicate that the extract contains both compounds with good and intermediate antioxidant capacity.

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Fig. 8

CV of 141 µM plant extract in phosphate buffer. a) at 10 mV/s; b) at various scan rates using glassy carbon as the working electrode; c) at various scan rates using stainless steel as the working electrode.

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The CVs were analyzed separately in two different setups by taking glassy carbon and stainless steel(ss) as the working electrodes. The graphs obtained (Fig. 8b and c) confirm the presence of identical peak potentials. As can be seen from Fig. 8b and c that the intensities of the CV increase with an increase in the scan rate. Although a higher scan rate implies greater sensitivity, the charging current also rises. Since it is a diffusion-controlled process at higher scan rates, the graphs look slightly distorted.

Fig. 9 shows the DPV graph for the bark extract of S. macrophylla. This also confirms the presence of a lower oxidation potential peak in agreement with the CV results leading to the antioxidant property. As can be seen from Figure b there is an increase in peak current density with the increase in the concentration of plant extract. This shows the linear relation between the two parameters. The peak current density for this approach is determined by the electron transfer kinetics and the diffusion coefficient of the electroactive species, in addition to the concentration.

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Fig. 9

DPV plots for the bark extract of S. macrophylla. a) DPV of extract in phosphate buffer; and b) Variation of the peak current density, in DPV with a concentration of plant extract.

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