Introduction                                                                                     

The discoveries of crude drugs are sometimes based on the alternative medicine developed by indigenous people. Faloak is a stem bark from the faloak tree (Sterculia quadrifida R.Br) used by people in East Nusa Tenggara Indonesia, as traditional medicine (Figure 1). Locals people believe faloak stems bark (FSB) can cure liver dysfunction, cancer, gastroenteritis, anti-diabetic, and fatigue recovery¹. Extract of faloak stems bark (FSB) contains bioactive compounds of alkaloids, flavonoids, phenols, and terpenoids ¹.  Exploration of FSB is necessary to know the bioactive compounds and study the extract’s biological activity.

Figure 1: Faloak (Sterculia quadrifida R.Br) stem bark Click here to view Figure

Selecting a suitable extraction method is crucial in isolating bioactive compounds in plant materials. Conventional extractions were applied for years to obtain a plant bioactive compound extract.  However, some conventional extractions are inefficient methods.³ Nowadays, microwave-assisted extraction, a green technology extraction, offers many advantages, especially thermo-sensitive compound extraction.²  Isolating of bioactive compounds from plant matrix using MAE is related to some parameters such as the chemical nature of the bioactive compounds and the plant matrix, time, ratio of ethanol and water, and temperature.⁴  Ethanol, a green solvent with low toxicity compared to methanol, evaporates quickly and is commonly used in pharmaceutical industries. The ratio of water and ethanol needs to be optimized to obtain maximum bioactive compounds in the plant.⁵ Water and ethanol are suitable for plant extraction using MAE, which has proved efficient for the phenolic compounds’ extraction from plants.^6-10 In extraction involving a microwave, water is essential since entrapped water in the plant matrix can interact with the microwave, causing cell rupture and releasing targeted compounds into the solvent.¹¹ The temperature of extraction is necessary to be optimized. High temperature can reduce the viscosity and surface tension which solvent penetrates better into the plant matrix cell.⁵ However, the degradation of bioactive compounds occurs at high temperatures. Extraction yields can be increased by extending the extraction time. However, a longer extraction time can lead to the degradation of heat-sensitive compounds.¹²   The extraction time using MAE is generally less than 30 min.¹³

Phenolic compounds contain many hydroxyl groups, which can be conjugated with sugars, acids, or alkyl groups. Therefore, one plant can have very diverse phenolics. Developing one extraction condition to obtain all the phenolic compounds is difficult. In this study, the optimal temperature and time of MAE for isolating the bioactive compound present in the FSB should be determined as to how those factors affect the phenol and flavonoid content of the extract. Moreover, a powerful analytical technique, HRMS, was used to profile the phytochemicals of the FSB extract.

Materials and Methods

Materials

Stems bark of faloak (Sterculia quadrifida R. Br ) was obtained from Kupang province of East Nusa Tenggara, Indonesia. All the chemical reagents were pro-analyze quality. Ethanol (Mallinkrodt), methanol (Merck), Gallic acid, Na₂CO_3, folin ciocalteau, quercetin, NaNO₂, AlCl₃, NaOH dan DPPH (2,2- diphenyl-1-picrylhydrazyl) (Sigma-Aldrich) were bought from local chemical reagent distributors.

Methods

Extraction of Faloak Stem Bark (FSB)

The steps of extraction using MAE are referred to Martati and Simamora¹⁴ . The air-dried and powdered FSB (4 g) was added with 40 mL of ethanol. The mixture was extracted using MAE (Anton Paar) at a combination of different temperatures (50, 60, and 70 °C) and lengths of extraction (5, 15, and 25 min).  The extract of FSB  was brought to room temperature. Impurities were separated through fine filter paper. The extracts were pooled in a small dark glass bottle and kept at -20°C until further measurements.

Quantification of total phenol (TPC)

Quantification of the TPC of FSB extract was measured by the Folin Ciocalteu (FC) assay according to Martati et al.¹⁴   Diluted FSB extract of 0.5 mL is reacted with 2.5 mL FC 10%, vortexed and then kept for 5 min. Then, the mixture was reacted with 2 mL of 7.5%  Na₂CO_3.  Keep the mixtures for 30 min at room temperature in a dark place. The absorbance of the FSB sample was read at 750 nm. Gallic acid (GA) was treated the same as the extract of FSB to obtain a calibration curve and used to quantify TPC as mg GAE/g.

Quantification of total flavonoid (TF)

Quantification of Total Flavonoid Content (TF) in the FSB extract was referred to Martati et al.¹⁵ with modification. A sample of FSB extract 1 mL is mixed with  4 mL of distilled water and reacted with a volume of 0.3 mL of NaNO₂  5%. After incubation for 5 min, 0.3 mL of  AlCl₃ 10% was combined and kept for 6 min. After 6 min, NaOH 1 M of  2 mL was added, and the total volume was 10 mL with distilled water. The mixture was mixed thoroughly. For quantification, the absorbance of the sample was read at 510 nm. A standard compound of quercetin was treated in the same steps for the sample to make a calibration curve. TF was quantified as mg QE/g sample. 

Radical scavenging activity (DPPH)

The antioxidant action of the extract FSB on free radicals was analyzed as in the method written by Martati et al.¹⁵ with small changes. DPPH was diluted in methanol to make a concentration of 0.3 mM. A serial dilutions of the FSB extract in methanol were prepared. A diluted extract of 3 mL was added with 1 mL of DPPH 0.3 mM. The sample mixture was kept in a dark room for 30 min. The sample’s absorbance value was measured at 517 nm. The radical scavenging activity was calculated using below equation:

where A_b is the absorbance of the methanol and DPPH solution (a blank), and A_e is the absorbance of the diluted FSB extract and DPPH solution. 

Analysis of LC-HRMS

Identification of the bioactive compounds in the extract of FSB was performed on Thermo Scientific Dionex Ultimate 3000 RSLCnano. The analytical column was Hypersil GOLD aQ 50 x 1 mm x 1.9 mm particle size. The column oven was 30° C.  The mobile phase was formic acid 0.1% (phase A) in water and acetonitrile containing formic acid 0.1% (phase B). A linear gradient mobile phase with a 40 mL/min flow rate was applied. Mobile phase A was applied from 100% to 95% in 2 min. Then, it decreased to 40% in 13 min, then to 5% in 7 min and was held for 3 min. Finally, It was increased to 95% and kept for 5 min. The total running time until finished was 30 min. High-Resolution Mass Spectrometer (Thermo Scientific Q Exactive) was set as follows: full scan settings were 70,000; the orbitrap resolution was 17,500. The software for processing data was a Compound Discoverer with mzCloud MS/MS Library. 

Design experiment and statistical analysis

The research applied the Randomized Block Design (RBD) method consisting of two factors extraction time (5, 15, and 25 min) and temperature variation (50, 60, and 70°C). From the combination of these two factors with three replications of each treatment, 27 experimental units were obtained. The measured parameter data were assessed using Analysis of Variance (ANOVA). Followed by a test to explore the difference between means (95% confidence interval). The data obtained were written as mean and standard deviation. The data were statistically analyzed using SPSS.

Results

TPC

The TPC of FSB extract was obtained from the extraction using MAE at a combination of temperature and time range from 49.07-81.17 mg GAE/g. Figure 2 shows the trend of TPC of FSB extract at a different combination of temperature and extraction time. It indicates that the yield of TPC increased as the temperature rose from 50 °C to 60 °C. At 70 °C, the TPC decreased. The maximum amount of  TPC was obtained at 60 °C for 25 min, amounting to 81.17 mgGAE/g. While the lowest TPC was extracted at 70 °C for 25 min, amounting to 49.07 GAE/g. Therefore, the yield of TPC was driven by the combination of temperature and extraction time.

Figure 2: TPC of FSB extract obtained at variation combination of temperature and time of extraction using MAE method.  Click here to view Figure

Total Flavonoid (TF)

The total flavonoid of FSB extract obtained from the extraction using MAE, at different temperatures and duration of extraction combinations ranged from 40.67 to 70.30 mg QE/g. The highest flavonoid was obtained by extraction at 60°C for 25 min.

Figure 3 shows the highest TF obtained at a temperature of 60 °C for 25 min amounting to 70.30 mgQE/g. While the lowest TF value was obtained at a temperature treatment of 70°C with an extraction time of 25 min, amounting to 40.67 mgQE/g.

Figure 3: TF of FSB extract obtained at various combinations of temperature and time of extraction using the MAE method.  Click here to view Figure

Radical scavenging activity (DPPH)

The radical scavenging activity of the FSB extract ranged from 47.96 to 73.40% (Figure 4). There is an increase in the radical scavenging activity of FSB extract obtained from extraction at 50 to 60 °C. Extraction of FSB at 70 °C resulted in a lower radical scavenging activity.

Figure 4: Radical scavenging activity of FSB extract obtained at variation combination of temperature and time of extraction using MAE method.  Click here to view Figure

Identification of bioactive compound in FSB extract

Figure 5 presents the chromatogram of the LC-HRMS analysis of FSB. The identified compounds are phenol, flavonoids, alkaloids, fatty acids, and their derivatives. Figure 6 presents several fragmentation patterns of compounds extracted from FSB. Some of the identified compounds were methyl cinnamate, vanillin, apocynin, scopoletin, L(-)-pipecolinic acid, arecoline, δ-valerolactam, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzaldehyde. A group of flavonoids was epicatechin and rutin. Fatty acids and their derivatives were eicosapentaenoic acid 9-oxo-10,12-octadecadienoic acid, 2,4-dihydroxy-heptadec-16-en-1-yl acetate, pinolenic acid, oleamide, α-eleostearic acid, 12-oxo phytodienoic acid, α-linolenoyl ethanolamide, palmitic acid, oleyl anilide dan 1-linoleoyl glycerol. The full list of compounds is attached in the supporting material (Table S1). There are some compounds identified as the same compounds, but they have different retention times, e.g. L(-)-Pipecolinic acid (1.01 and 0.87 min), (-)-Epicatechin (5.44 and 5.60 min) and Scopoletin (0.85 and 6.87 min). It might be that those compounds are isomers. Further analysis is needed to confirm this.

Figure 5: LC-HRMS chromatogram of FSB extract  Click here to view Figure

Figure 6: LC-HRMS fragmentation patterns of some compounds extracted from FSB Click here to view Figure

Discussion

The yield of TPC and TF increased as the extraction temperature rose from 50 to 60 °C. Extraction at 70 °C resulted in a lower product of TPC and TF. At temperatures up to 60 °C, the heat from microwave energy can improve mass transfer, a better diffusion rate, and reduce viscosity and surface tension, resulting in higher yields of flavonoids.¹⁶   Irradiation of microwave speeds up cell rupture by rapidly increasing temperature and internal pressure inside the cells as a booster for the destruction of the sample surface. It turns the release of the target compounds to the surrounding solvent.¹⁷  Energy generated from the heat and microwave interact with polar solvent and allows the solvent to enter the matrix of plants resulting in more extracted bioactive compounds.¹⁸  However,  extraction time had a very low negative significant impact on the TF of citrus peel extraction, indicating that increasing extraction time could decrease the TF slightly. ¹⁹ Temperature gave a positive effect up to 60 °C meaning that increasing the extraction temperature resulted in a good impact on radical scavenging activity, which was in line with the increase of TF and TPC as the extraction temperature was higher. The longer the duration of extraction and temperature would increase the degradation of some heat-labile antioxidative compounds.

There is an increase in the antioxidant activity of FSB extract obtained from extraction at 50 to 60 °C. Extraction at 70 °C resulted in lower antioxidant activity. The microwave energy causes plant tissue degradation, cellulose dehydration, and weakening of the microstructure. Therefore, the solvent penetrates easily into the cellular channels to release the solute into the solvent¹².  At higher temperatures, the MAE will destroy the cell matrix’s cell wall and the cells’ solute can be released into the surrounding solvent.^20,21 In this study, the increase in the total phenol can be accredited to the heating effect. An increasing the solvent temperature was caused by the dipole rotation of the solvent in the microwave field, which was furthermore increasing the phenolic compounds’ solubility. The combination of temperature and time could increase the solubility of phenolic compounds and lower the solvent’s viscosity, therefore, accelerating the release and dissolution of these compounds. However, degradation of the phenolic compounds can occur at a higher temperature²². A lower extraction temperature may produce a more stable extract but may result in a lower extraction yield. The heat stability of a plant during extraction refers to its ability to retain its chemical and physical when subjected to high temperatures during extraction.  The temperature significantly affected the extraction of flavonoids from citrus peel. Until 75 °C, the yield of flavonoids still increased, indicating that the extraction at 75 °C has not shown degradation of the flavonoids¹⁹.

Conclusion

This research showed an interaction between temperature and time of microwave-assisted extraction of FSB on TPC, TFC, and radical scavenging activity. The yield of TPC and TF has been higher as the rising temperature from 50 to 60 °C. Extraction at 70 °C decreased the yield TPC and TF. The newly identified specific phenolics and flavonoids were successfully profiled. Future research for testing the biological activity of the extract or the newly identified of phenol or flavonoid is necessary to study. 

Acknowledgment

The authors sincerely thank Midia Lestari Wahyu Handayani (Laboratory of Life Sciences Universitas Brawijaya Indonesia) for assisting the HRMS analysis.

Conflict of interest

The authors have no conflicts of interest to declare.

Funding Sources

This research received funding from The Ministry of Education, Culture, Research, and Technology Directorate General Higher Education Research and Technology Research through the Research of Thesis Magister 2022 with the contract number: 1071.20/UN10.C10/TU/2022. 

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