Isolation, identification and quantification of brazilein content in sappan wood (Caesalpinia sappan) traditional drink using LC HRMS and HPLC method

Abstract

For decades, the demand for attractive colors in foods and beverages has been steadily increasing. Brazilein, a red pigment extracted from the heartwood of Caesalpinia sappan L. (CS), is commonly used in various traditional drinks, including the Indonesian herbal beverage wedang uwuh. However, brazilein's red color is notably unstable due to its sensitivity to heat, light, oxygen, and pH fluctuations, making it challenging to analyze. This study aimed to determine the brazilein content in both CS heartwood (a single-source material) and wedang uwuh (a mixed-source product). Before this, the samples underwent blanching and drying at various temperatures before extraction. The brazilein was extracted using the maceration method employing 96% ethanol for 72 hours. The resulting extracts were analyzed using High-Performance Liquid Chromatography (HPLC). The result showed that the measurement was validated and exhibited a Correlation Coefficient (R2) of 0.9997, Limit of Detection (LOD) of 0.0095 (µg/mL), and Limit of Quantification (LOQ) of 0.0287 (µg/mL). The brazilein concentrations varied with drying temperature, ranging from 2.5383±0.01% to 3.0155±0.01% (w/w) in CS and from 1.6335±0.01% to 3.0168±0.01% (w/w) in wedang uwuh. Notably, the lower brazilein content in wedang uwuh reflects its complex composition and the additional processing steps it undergoes, which likely contribute to pigment degradation.

Keywords:
Wedang uwuh; Red pigment; Chromatography; UPLC-MS/MS, HPLC

Highlights

Using advanced chromatographic techniques, the study isolates brazilein from sappan wood extract and mixed sample (wedang uwuh traditional drink)

The analysis confirms significant content of brazilein in sappan wood extract and mixed sample (wedang uwuh traditional drink

1 Introduction

Brazilein is one of the most prevalent chemicals found in the heartwood of Caesalpinia sappan L. (CS), as well as in the heartwood of other tropical species like brazilwood (Paubrasilia echinata) and Mexican logwood (Haematoxylum brasiletto H. Karst.) (Ngamwonglumlert et al., 2020). Due to its numerous beneficial functional properties, CS is now cultivated in diverse regions worldwide, including Indonesia, Malaysia, Thailand, Hawaii, Northern India, Papua New Guinea, the Solomon Islands, Sri Lanka, Taiwan, and the Philippines (Dapson & Bain, 2015).

Brazilein provides several pharmacological benefits, including antioxidant and anti-inflammatory effects (Hariyati et al., 2023), antibacterial activity against Shigella flexneri (Qi et al., 2021), and cytotoxic effects against several cancer cell lines, such as colorectal, breast, lung, and cervical cancers, suggesting its potential as a chemo-preventive agent (Raptania et al., 2024). CS heartwood, a traditional medicinal plant, is produced in regions such as Taiwan, China, India, Myanmar, Vietnam, Sri Lanka, and the Malay Peninsula. Dried CS heartwood has been used for centuries in traditional oriental medicine, typically prepared as an aqueous decoction to invigorate the blood, promote menstruation, and alleviate pain and swelling. Additionally, it has been valued for its astringent and diuretic properties and has been recommended for treating certain skin diseases (Liang et al., 2013).

Aside from its potential health benefits, brazilein is widely recognized as a traditional food colorant. CS heartwood has long been used as a colorant in several Asian cuisines and beverages, not only to enhance aesthetic appeal but also to add functional benefits. It serves as the sole coloring ingredient in traditional drinks like wedang secang and wedang uwuh (Indonesia) and pathimukham (India). The importance of brazilein analysis in food and colorant applications cannot be overstated. As consumers increasingly prioritize health and environmental sustainability, the demand for natural additives in food products is rising. Brazilein, with its red color pigment and antioxidant properties, presents a promising alternative to synthetic dyes and colorants, which are often associated with adverse health effects. However, to ensure the safety, efficacy, and quality of brazilein as a colorant, rigorous analytical methods are essential.

Changes in the molecular structure of brazilein during food processing can lead to undesirable color shifts (Ngamwonglumlert et al., 2020), underscoring the need for precise measurement of brazilein content both in its pure form and in processed or mixed food products. While several studies have focused on the identification and quantification of brazilein in pure CS heartwood extracts, limited research has examined its presence in complex food matrices or in mixtures containing CS heartwood and other ingredients.

Given that CS heartwood is typically consumed as part of complex food products mixed with other ingredients, it is important to develop reliable analytical methods for the accurate quantification of brazilein in such matrices. Due to its significant role in health and functionality, developing a reliable method for brazilein determination is essential. Although previous studies have focused on measuring brazilein in its pure form without any treatment, limited information is available on analytical approaches for determining this compound in real food samples. This study aims to develop a quantification method of brazilein in a complex food matrix, such as mixed herbal drinks, as well as samples subjected to heat treatments like blanching and drying.

Natural pigments, including brazilein, are generally unstable once extracted from their natural sources. They are highly sensitive to external factors like heat, light, oxygen, and pH changes during food processing and cooking. For phenolic pigments like brazilein, stability is particularly influenced by pH. Besides pH, temperature also affects the stability of brazilein. It has been reported that increasing the pH, heating temperature, and exposure time can significantly reduce brazilein content (Ngamwonglumlert et al., 2020).

Brazilein exhibits a distinct color shift depending on the pH of its environment, transitioning from yellow to orange and red as the pH increases from 3 to 7 to 9, respectively. These color changes were associated with structural alterations caused by deprotonation. At higher pH levels, brazilein adopts a fully deprotonated form, while at lower pH, it reverts to a neutral, zero net-charge form. Notably, brazilein solutions at pH 9 show the lowest thermal stability, as the deprotonated molecules tend to convert into Quinone intermediates, which are prone to oxidative degradation. In contrast, at pH 3, brazilein maintains a more stable color and molecular structure during heating, suggesting greater resistance to thermal breakdown. Both the deprotonation and degradation processes of brazilein at elevated pH levels could be accelerated by heat, resulting in significant changes in color, molecular structure, and the breakdown of brazilein at higher temperatures (Ngamwonglumlert et al., 2020).

Analyzing brazilein content in food and colorant applications requires a comprehensive approach involving extraction, purification, and quantification techniques. Chromatographic methods, particularly High-Performance Liquid Chromatography (HPLC), are widely recognized for their precision and reliability in quantifying brazilein. Moreover, spectrophotometric analysis serves as a valuable tool for assessing the pigment's stability and its interaction with various food matrices.

In practical applications, CS heartwood, the primary source of brazilein, is often combined with other ingredients. In Indonesia, for example, CS heartwood is used as a key ingredient in traditional drinks such as wedang secang, wedang uwuh, bir plethok, liang tea, and sarabba. In this study, the brazilein content in wedang uwuh (a mixed beverage) was measured and compared with that in pure CS heartwood. Wedang uwuh is a traditional herbal drink from Yogyakarta, Indonesia, made from a blend of CS heartwood, ginger, cinnamon leaves, nutmeg leaves, clove leaves, and clove stalks, typically in a ratio of 9:5:1:1:1:1.

This study aims to develop a reliable method for brazilein determination in complex mixtures and to apply this method to wedang uwuh as a real-world sample. However, challenges remain in standardizing the preparation of wedang uwuh ingredients, including CS heartwood, which is non-standard in the existing production process. In conventional practice, fresh ingredients of wedang uwuh are typically sun-dried without standardized procedures, resulting in significant variations in quality. For instance, sun-drying causes the CS heartwood to develop a reddish color, but this process occurs under uncontrolled conditions, making it difficult to ensure color stability in food applications. To address this variability, this study also examines the effects of controlled heat treatments, including blanching and drying, on brazilein content. This approach aims to identify the differences in brazilein concentration between pure CS heartwood and mixed samples, providing a clearer understanding of how these preparatory steps impact pigment retention and quality.

2 Materials and methods

2.1 Materials

Fresh CS heartwood samples were obtained from Kemuning Forest in District Gunung Kidul of Central Yogyakarta. Other ingredients, including ginger, cinnamon leaves, nutmeg leaves, clove leaves, and clove stalks, were sourced directly from farmers in Samigaluh, Kulon Progo, Yogyakarta. The freshly harvested CS heartwood was cut into smaller pieces, approximately 300mm × 100 mm × 100 mm. Fresh ginger was sliced transversely across its diameter, following a cross-fiber orientation, into pieces roughly 300 mm × 100 mm × 50 mm, while the fresh leaves were used whole without size reduction.

The prepared herbs were then processed into chopped form, and selected portions were blanched before undergoing drying, as outlined in Table 1. For comparison, sun-drying without blanching was also performed on some samples, which were designated as the control (C) group. The chemicals and reagents used in this study included adsorbents (silica gel 60 PF254 containing gypsum), ethanol, ethyl acetate, Milli Q (deionized) water, and spraying reagent (H2SO4), all of which were purchased from Merck.

Table 1
Experimental design and selection of conditions for sample preparation.

Type of processing	Type of drying	Drying condition		Processing condition
		Temperature (°C)	Time (h)	Temperature (°C)	Time (min)
Non blanching	Sun drying (C)	40	48	27	1
	Cabinet drying (NBCD)	55	12	27	1
Water Blanching	Sun drying (BSD)	40	48	85	1
	Cabinet drying (BCD)	55	12	85	1
Steam Blanching	Sun drying (SSD)	40	48	100	1
	Cabinet drying (SCD)	55	12	100	1

2.1 Chromatographic method

Chromatographic separation was performed using a Reliant® T3 C18 reverse-phase column (150 x 4.6 mm, 5 µm) with an optimized mobile phase consisting of water (solvent A) and acetonitrile (solvent B). The column was eluted using a linear gradient program, starting at 15% B, ramping to 100% B for 5 min, and then returning to 15% B. Methanol was used to dilute the brazilein stock solution.

2.2 Preparation of sample

The samples were divided into two categories: single-source CS heartwood and a mixed sample, wedang uwuh, a traditional drink model composed of CS heartwood, ginger, cinnamon leaves, nutmeg leaves, clove leaves, and clove stalks in specific ratios (9:5:1:1:1:1). The CS Heartwood was initially cut into blocks and subjected to treatment as described in Table 1. Similarly, each component of the mixed sample was processed as in Table 1. Post-treatment, all samples, including both the single and mixed groups, were ground into a fine powder for analysis.

2.3 Extraction, fractionation, and purification

A total of 300 grams of fresh plant material was collected in bulk, washed, dried, ground, and extracted with 96% ethanol for 72 hours using the maceration method, yielding 18 grams of crude extract. The crude extract was then fractionated using vacuum liquid chromatography (VLC), eluted with a gradient of increasing polarity (EtOAc: MeOH: H₂O). Fractions exhibiting similar profiles on thin-layer chromatography (TLC) were combined, resulting in three major fractions (I-IV).

Fraction IV (1.5 g) was subsequently purified using Radial Chromatography (RC) on a 2 mm-thick silica gel plate, eluted with a mixture of 90% EtOAc, 8% MeOH, and 2% H₂O, with 5% incremental increases in polarity. This process yielded 7 mg of pure brazilein. The chromatographic profiles were initially examined using smaller TLC plates, visualized under ultraviolet (UV) light (254 nm) and stained with H₂SO₄ spraying reagent, followed by heat treatment. The spectra of brazilein were confirmed using Mass Spectrometry (MS) and MS/MS, as shown in Figure 1.

Figure 1
MS and MS/MS spectra of Brazilein.

2.4 HPLC quantitative analysis of brazilein contents

2.4.1 Finding the detection wavelength

Individual methanolic solutions of brazilein were scanned over the wavelength range of 210-600 nm using a photo diode array (PDA) detector (Waters). The detection wavelength for the simultaneous analysis of brazilein and related compounds was selected based on the crossing point of the brazilein spectra of the spectral overlay.

2.4.2 HPLC instrument and conditions

The HPLC analyses were conducted using a Waters LC system (Milford, Massachusetts, USA) equipped with a quaternary pump, on-line degasser, auto-sampler, and a photodiode array detector (model 2998). The system was controlled, and the data were acquired and processed using Empower software. Chromatographic separation was carried out with a Reliant® T3 C18 reverse-phase column (150 x 4.6 mm, 5 µm) with an optimized mobile phase comprising water (solvent A) and acetonitrile (solvent B). The column was eluted using a linear gradient program as follows: 85% A and 15% B for 10 min (0-10 min), 100% B for 5 min (10-15 min), and then 85% A and 15%B for the final 5 min (15-20 min). The mobile phase was filtered through a 0.22 µm filter and degassed using ultrasonication before use. The injection volume was set at 5 µL, and the column temperature was maintained at 30 °C throughout the chromatographic run.

2.4.3 Preparation of stock and calibration solution

Stock standard solutions of brazilein, with a final concentration of 1 mg/mL, were prepared by dissolving an appropriate amount of pure brazilein in methanol as an individual solution. All solutions were stored in light-protected vials to prevent degradation and ensure compound stability.

2.5 Method validation

2.5.1 Specificity and selectivity

To assess specificity, chromatograms of blank samples (prepared using the solvent extraction system) were compared with those of fractions containing brazilein. Selectivity, defined as the method’s ability to clearly separate different peaks, was evaluated using the resolution (Rs) parameter. A value of Resolution (Rs) > 1.5 is required to confirm that the method is sufficiently selective. The Rs value is calculated using the following formula (Equation 1):

$Rs=\frac{t{R}_2-t{R}_1}{\frac{1}{2}\left(W_1+W_2\right)}$ (1)

tR1, tR2: retention time for each peak (tR1 < tR2)

W1, W2: width of each peak

2.5.2. Preparation of calibration solution of brazilein and linearity

Calibration solutions were prepared by diluting the brazilein stock solution in methanol at same concentration to achieve a series of concentrations ranging from 3.95 to 62.5 µg/mL. The linearity of the method was assessed by plotting the concentration of brazilein against the corresponding peak area, with the regression line calculated using the least squares method. Linearity was confirmed if the correlation determinant (R²)> 0.99, indicating a strong linear relationship. The Limit of Detection (LOD) and quantification were calculated using the standard deviation approach, based on the standard deviation (SD) of the y-intercept and the slope of the calibration curve. These parameters were determined by which the LOD and Limit of Quantification (LOQ) were calculated based on Equation 2 and Equation 3 below, respectively.

$\mathrm{L}\mathrm{O}\mathrm{D}\mathrm{}=\mathrm{}3.3\mathrm{}\mathrm{x}\mathrm{}\mathrm{S}\mathrm{D}/\mathrm{S}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}$ (2)

$\mathrm{L}\mathrm{O}\mathrm{Q}\mathrm{}=\mathrm{}10\mathrm{}\mathrm{x}\mathrm{}\mathrm{S}\mathrm{D}/\mathrm{}\mathrm{S}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}$ (3)

2.6 Data analysis

The calibration curve was analyzed using least squares regression, performed in Microsoft Excel (Microsoft Inc., USA). Mean and standard deviation values were also calculated using Excel.

3 Results and discussion

The Caesalpiniaceae family is a significant source of pink and bright reddish-purple dyes, widely cultivated in Southeast Asia for the red dye extracted from its heartwood. Ethnobotanically, in Indonesia, CS heartwood has long been used as a functional food, particularly in traditional herbal drinks. CS heartwood is known for its properties. When brewed, CS heartwood produces a distinctive reddish-orange solution, making it a popular ingredient in beverages like wedang uwuh and wedang secang from the Yogyakarta region. As the popularity of CS heartwood-based beverages grows, there have been efforts to produce instant versions of wedang uwuh in convenient forms such as powders, sachets, and syrups. However, research by Ngamwonglumlert et al. (2020) indicated that the brazilein, the primary colorant and active compound in CS heartwood, is highly sensitive to changes in pH and temperature, complicating its analysis in complex food matrices.

This study aimed to develop a reliable method for analyzing brazilein content in both pure CS heartwood and mixed herbal drinks. As part of this investigation, the effects of various heat treatments, including water blanching, steam blanching, sun drying, and cabinet drying, were examined. Non-blanched, sun-dried samples were included as a control, as this is the most common method currently used in the traditional process of making wedang secang and wedang uwuh. The findings from this study are expected to provide practical recommendations for preparing high-quality herbal drink ingredients based on their brazilein content, thereby preserving both the color and functional properties of the final product.

3.1 Isolation and identification of brazilien

The first step in this study was the isolation of brazilein compound from the CS heartwood. The chemical structure of brazilein is presented in Figure 2. The purification process was performed using various chromatography methods, yielding brazilein in colors ranging from yellow to red. This isolated compound was then characterized using Ultra Performance Liquid Chromatography (UPLC)-MS/MS method with ES (-) mode, producing a molecular ion peak ESI-MS at m/z 284.94 (C16H12O5) with an exact mass of 284.07 for brazilein. These fragmentation values and m/z ratios aligned with previously reported data [9]. The resulting chromatograms from this analysis are presented in Figure 3. This isolated compound will be used as a reference standard for the quantitative analysis of brazilein content in both single-source CS heartwood samples and mixed herbal drink samples, employing the HPLC method.

Figure 2
Molecular structure of brazilein.

Figure 3
Brazilein chromatogram.

3.2 Determination of brazilein contents in the food matrix

The samples of the food matrix included wedang secang and wedang uwuh, which represent single-source and mixed herbal drinks made from CS heartwood, respectively. The fresh ingredients were first chopped, then subjected to steam or water blanching, followed by sun drying or cabinet drying. A control sample, prepared without blanching and dried using the sun, was also included to reflect traditional processing methods. After drying, all samples were ground into powder form for brazielin analysis.

3.2.1 Finding the detection wavelength

Spectral analysis of methanolic solutions containing brazilein reveals certain absorption characteristics, as shown in Figure 4. Specifically, brazilein dissolved in methanol exhibits a maximum absorption at 286 nm. This wavelength is particularly advantageous for HPLC analysis for several reasons. First, it allows for the simultaneous quantification of multiple compounds in complex, typically alcoholic solutions, which is critical because most real-world samples contain a mixture of substances that must be analyzed together. By selecting a wavelength where the target compounds have distinct absorbance profiles, it is possible to differentiate between them without the need for separate tests, enhancing both efficiency and accuracy. Additionally, this approach reduces analysis time and complexity, while also improving the precision and reliability of the results. Importantly, detecting a peak at this specific wavelength facilitates accurate strain separation, ensuring the effective isolation of the target compound within complex formulations (Starecki, 2008).

Figure 4
The spectra of brazilein in methanolic solution.

3.2.2 Method validation

• Specificity and selectivity

The method specificity is demonstrated in Figure 5, where no peaks are observed at the retention time corresponding to the brazilein peak in the blank solvent (Figure 5a), indicating the absence of interference from other substances. In contrast, the fraction sample (Figure 5b) displayed a clear brazilein peak at a retention time of 8.58 minutes, confirming the method's ability to accurately identify brazilein. Additionally, the method's selectivity was assessed based on baseline separation parameters, with Rs values for brazilein exceeding 1.5 (Figure 5b), ensuring clear and distinct separation from other compounds. These results confirm that the method is both specific and selective for the accurate detection and quantification of brazilein.

Figure 5
Chromatographic separation for specificity and selectivity assessment. (a) blank and (b) sample extract from water blanching and cabinet drying (BCD).

• Linearity, range, and sensitivity

The regression parameters obtained from the least-squares method are presented in Table 1. As shown in Table 2, the method exhibited strong linearity for brazilein within the concentration range of 2.26–72.5 µg/mL, with correlation determinant (R²) exceeding 0.999 within this range, demonstrating excellent linearity and reliability. Linearity was further validated by calculating the % Relative Standard Deviation (RSD) of the slope values, all of which were below 2%, ensuring the method's reliability and precision (Hollands et al., 2017; Surana et al., 2021; Bhaskaran et al., 2021)

Table 2
Validation parameters for brazilein (n=3).

Parameter	Brazilein standard
Linearity range (µg/mL)	2.26 – 72.5
Linear equation	y= 10226x + 72019
Determinant coefficient of (R2)	0.9997
Limit of Detection (µg/mL)	0.0095
Limit of Quantification (µg/mL)	0.0287

The sensitivity of the method was evaluated through the determination of the LOD and LOQ. The calculated LOD for brazilein was 0.009 µg/mL, while the LOQ was 0.02 µg/mL. These results confirm the method’s high sensitivity, making it capable of detecting and quantifying very low concentrations of the compounds. This level of sensitivity is crucial for accurately analyzing samples with minimal amounts of these compounds, thereby enhancing the method's applicability in various analytical contexts (Shrivastava & Gupta, 2011).

• Method confirmation by FTIR and LC-HRMS testing

The Fourier Transform Infrared Spectroscopy (FTIR) analysis of the CS heartwood sample revealed characteristic absorption peaks at 3337 cm ^-1 for (-OH group), 1613 cm ^-1 for (C = O / C = C group), 1508 cm ^-1 for (C = C group), 1446 cm ^-1 for (C = C group), 1380 cm ^-1 for (C-O group), 1250 cm ^-1 for (C-O group), 1180 cm ^-1 for (C-O group), 1115 cm ^-1 for (C-O group), and 1036 cm ^-1 for (C-O group) confirming the presence of brazilein compounds (Figure 6). (Nandiyanto et al., 2019). Additionally, the LC High Resolution Mass Spectrometry (HRMS) analysis detected brazilein compounds in both the pure CS heartwood sample and the mixed sample (wedang uwuh), as shown in Figure 7. Based on these FTIR and LC-HRMS results, HPLC analysis was subsequently performed to quantify the brazilein content in both the single CS heartwood samples and the mixed (wedang uwuh) samples.

Figure 6
FTIR absorption spectrum of CS heartwood extract.

Figure 7
LC-HRMS chromatogram of CS heartwood extract.

3.3 Application of the developed method

The red color of brazilein pigment in CS heartwood contributes significantly to the quality attributes of food and beverages. In addition to its vibrant color, brazilein exhibits antioxidant properties, making it essential to accurately quantify its content in complex food matrices. The method developed in this study successfully determined brazilein in its pure form and was subsequently applied to real samples, specifically wedang uwuh — a traditional herbal drink made from a mixture of CS heartwood, ginger, cinnamon leaves, nutmeg leaves, clove leaves, and clove stalk. This study also aimed to investigate the impact of different preparation treatments, including blanching and drying, on the brazilein content of these samples. These processes are commonly used in the preparation of ready-to-drink herbal beverages, but involve heat exposure, which can affect the stability of brazilein. The chromatogram and brazilein content of wedang uwuh are presented in Figure 8 and Table 3, respectively.

Figure 8
HPLC chromatogram of CS heartwood. extract (a), formation of wedang uwuh (b) with brazilein as standard.

Table 3
Brazilein content in C. sappan extract and mixed herbal drink.

No.	Treatment	Brazilein content (%) *
		CS heartwood	Mixed samples (wedang uwuh)
1	Non-Blanching-Sun Drying	2.5383 ± 0.01	1.8988 ± 0.01
	(NBSD/Control)
2	Non-Blanching-Cabinet Drying	2.5383 ± 0.01	2.6729 ± 0.01
	(NBCD)
3	Water Blanching-Sun Drying	2.5857 ± 0.01	2.6729 ± 0.01
	(BSD)
4	Water Blanching-Cabinet Drying	3.0155 ± 0.01	3.0168 ± 0.01
	(BCD)
5	Steam Blanching-Sun Drying	2.1573 ± 0.01	1.6335 ± 0.01
	(SSD)
6	Steam Blanching-Cabinet Drying (SCD)	1.8200 ± 0.01	1.8069 ± 0.01

The developed method successfully detected brazilein in wedang uwuh, despite the presence of other ingredients such as cinnamon, ginger, and clove leaves, which can potentially interfere with the analysis. This highlights the method's high sensitivity and specificity, allowing for accurate isolation and identification of brazilein within a complex matrix. This capability is essential, as brazilein is the primary compound responsible for the distinctive red color of wedang uwuh, ensuring product quality and safety.

Food colorants such as anthocyanin, β‐carotene, riboflavin, red beet powder, chlorophyll powder, and cochineal are generally less stable when exposed to high temperatures, light and fluctuations in pH levels (Wijesekara & Xu, 2024). Similarly, brazilein, the primary pigment in CS heartwood, is also sensitive to changes in pH and heat, which can significantly impact its color. In addition to pH and temperature, factors like oxidation and light exposure can also lead to color alterations in dyes (Ngamwonglumlert & Devahastin, 2023). These elements can result in modifications or breakdown of compounds, leading to fading or total loss of color (Ngamwonglumlert et al., 2017).

Given these challenges, selecting the appropriate physical form for natural pigments is critical. For instance, carbonated beverages containing anthocyanin-based colorants can undergo color changes during storage with light exposure control (Montibeller et al., 2018). Therefore, liquid forms are not recommended for natural colorants as they are prone to degradation, alteration, and color loss. In contrast, instant powder forms are generally more stable, as the reduced water content minimizes the risk of fermentation and pH shifts, preserving the colorant's stability over time.

In this study, CS heartwood was utilized to produce a functional herbal drink or mixed herbal drink known as wedang uwuh, which was prepared in dry powder form. The raw material, primarily CS heartwood, underwent multiple heating processes during blanching and drying. Blanching is a crucial step in food processing, especially for fruits and vegetables, as it helps inactivate enzymes, preserve color and flavor, and remove trapped air (Dorantes-Alvarez et al., 2017).

The findings indicate that the sample preparation method significantly influenced the brazilein content. The highest brazilein yield was obtained using water blanching at 85 °C for 1 minute, followed by cabinet drying at 55 °C. This treatment produced 3.0155±0.01% (w/w) brazilein for single samples and 3.0168±0.01% (w/w) brazilein for mixed samples.

Water blanching at 85 °C for 1 minute is particularly effective in preserving brazilein, the red pigment in CS heartwood, while enhancing its antioxidant activity. Brazilein, being water-soluble, partially dissolves into the blanching water, allowing for the release of antioxidant compounds without significant pigment loss, thus maintaining the vivid red color. However, at excessively high temperatures or prolonged exposure, brazilein can degrade, leading to color fading and reduced antioxidant properties. Therefore, 85 °C is considered the optimal blanching temperature to preserve both the bright red hue and the functional properties of brazilein. The findings of this study indicate that the conventional processing method, which typically involves sun drying without blanching, should be replaced with a combination of water blanching and cabinet drying. This approach is recommended for its ability to enhance brazilein content, thereby improving the overall quality and functional properties of the final product.

4 Conclusion

The distinctive red color of the brazilein compound in CS heartwood is a key factor in its appeal for use in food and beverage products. In addition to its vibrant color, brazilein also acts as a potent antioxidant, making its identification and quantification in real samples crucial. In this study, the HPLC measurement technique for brazilein was validated with a correlation coefficient (r) of 0.9997, a LOD of 0.0095 µg/mL, and a LOQ of 0.0287 µg/mL, demonstrating the method's high precision and sensitivity. The optimized sample preparation method, which involved blanching with water at 85 °C for 1 minute followed by cabinet drying at 55 °C, resulted in the highest brazilein yield for both pure and mixed samples. The developed method was successfully implemented to measure brazilein in pure CS heartwood as well as in the real samples, such as wedang uwuh with and without treatment. These findings highlight the practical application of this method, recommending blanching and cabinet drying as effective approaches for retaining brazilein content in processed herbal products. The presence of brazilein in the samples was confirmed by FTIR and LC HRMS analysis.

Acknowledgements

We express our gratitude to Universitas Ahmad Dahlan Yogyakarta Indonesia for financial support through the Hibah Invitasi Program 2024 with the Grant Number PD-093/SP3/LPPM-UAD/IX/2024) for the research and Universitas Gadjah Mada Yogyakarta Indonesia through Recognisi Tugas Akhir (RTA) Program with the Grant Number 5075/UN1.P.II/Dit-Lit/PT.01.01/2023 for the publication.

Data Availability Statement

The data supporting this study are not publicly available due to agreements, but can be requested from the corresponding author upon reasonable request.

• Cite as:
  Septiyani, R., Wikandari, R., Santoso, U., & Raharjo, N. C. (2025). Isolation, identification and quantification of brazilein content in sappan wood (Caesalpinia sappan) traditional drink using LC HRMS and HPLC method. Brazilian Journal of Food Technology, 28, e2024130. https://doi.org/10.1590/1981-6723.13024.
• Funding:
  Universitas Ahmad Dahlan (Grant Number PD-093/SP3/LPPM-UAD/IX/2024) and Universitas Gadjah Mada (Grant Number 5075/UN1.P.II/Dit-Lit/PT.01.01/2023).

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Edited by

• Associate Editor:
  Juliano Lemos Bicas.

Publication Dates

• Publication in this collection
  03 Nov 2025
• Date of issue
  2025

History

• Received
  21 Jan 2025
• Accepted
  09 June 2025