Pooja Negi,Nishant Pandey,Jyoti,Tripti Mishra,Vivek Ahluwalia,Umesh Singh,Bhuwan B. Mishra
Isolation of Food Grade Dye from Flower Petals of Butea monosperma and Determination of Marker Compounds for Its Quantitative Analysis
Health concerns associated with synthetic dyes/colorants have fostered the use of natural coloring materials for food applications. This study has been carried out to extract a natural dye from the flower petals of Butea monosperma (family Fabaceae) under an eco-friendly and organic solvent-free approach. Hot aqueous extraction of dry B. monosperma flowers followed by lyophilization of the resulting extract furnished an orange-colored dye in ∼35% yield. Silica gel column chromatography of dye powder resulted in the isolation of three marker compounds, viz. iso-coreopsin (1), butrin (2), iso-butrin (3) which were characterized by spectral methods, e.g., ultra violet, Fourier-transform infrared spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry. The XRD analysis of isolated compounds established an amorphous nature for compounds 1 and 2 while compound 3 showed good crystallinity. The stability of dye powder and the isolated compounds 1–3 was determined by thermogravimetric analysis which showed excellent stability up to 200 °C. In trace metal analysis, the product B. monosperma dye powder exhibited low relative abundance <4% for Hg along with negligible concentrations of Pb, As, Cd, and Na. The detection and quantification of marker compounds 1–3 in the B. monosperma flower extracted dye powder were carried out by a highly selective UPLC/PDA method of analysis.
Isolation of Food Grade Dye from Flower Petals of Butea monosperma and Determination of Marker Compounds for Its Quantitative Analysis

Health concerns associated with synthetic dyes/colorants have fostered the use of natural coloring materials for food applications. This study has been carried out to extract a natural dye from the flower petals of Butea monosperma (family Fabaceae) under an eco-friendly and organic solvent-free approach. Hot aqueous extraction of dry B. monosperma flowers followed by lyophilization of the resulting extract furnished an orange-colored dye in ∼35% yield. Silica gel column chromatography of dye powder resulted in the isolation of three marker compounds, viz. iso-coreopsin (1), butrin (2), iso-butrin (3) which were characterized by spectral methods, e.g., ultra violet, Fourier-transform infrared spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry. The XRD analysis of isolated compounds established an amorphous nature for compounds 1 and 2 while compound 3 showed good crystallinity. The stability of dye powder and the isolated compounds 1–3 was determined by thermogravimetric analysis which showed excellent stability up to 200 °C. In trace metal analysis, the product B. monosperma dye powder exhibited low relative abundance <4% for Hg along with negligible concentrations of Pb, As, Cd, and Na. The detection and quantification of marker compounds 1–3 in the B. monosperma flower extracted dye powder were carried out by a highly selective UPLC/PDA method of analysis.

Colorants or color additives are the substances that impart characteristic colors to food or nonfood materials. Generally, artificial colorants, e.g., Red 40 (E129), Yellow 6 (E 110), Blue 2 (E 132), etc., are frequently used without interfering the taste or stability of food items. Notwithstanding the good coloring efficacy, several allied side effects, e.g., hyperactivity in children, allergenicity, toxicological, carcinogenicity, etc., have led to a global prohibition on use of synthetic colorants in the food matrix. This has encouraged a new tendency for application of natural colors/pigments in food items. In addition, a rising desire for labeling products as natural has resulted in a limited application of synthetic colorants in food. Thus, development of new natural colorants/dyes for application in foods and beverages is surging interest. Natural dyes/pigments are primarily derived from different parts of plants like roots, bark, leaves, fruits, and flowers. They have been considered nonallergic, nontoxic, noncarcinogenic, are also biodegradable in nature, and therefore pose no direct/indirect risk to the environment. The dye obtained from flowers petals of the plant Butea monosperma (Family Fabaceae), also known as Palash, has high potential for use as a colorant in a wide range of food and beverages, e.g., jam, chowmein, noodles, sausage, soft drinks, etc. The plant B. monosperma is native to the Indian subcontinent, Thailand, Germany, Japan, Vietnam, and China. Besides the coloring attributes of flower petals, the plant also contains abundant phytochemicals, e.g., alkaloids, tannin, polyphenols, glycosides, flavonoids, etc., which may be responsible for the medicinal value of the B. monosperma plant. Earlier, a diverse range of pharmacological activities, e.g., antidiabetic, antibacterial, antifungal, antiinflammatory, anticonvulsive, hepatoprotective, antifertility, wound healing, etc., have been prospected from different parts of the plant. Although a plethora of research is reported in the literature for the extraction and isolation of dye from B. monosperma flowers, the exact composition of the dye is not fully established. Most of the studies rely on the use of volatile organic solvents, e.g., n-hexane, ethyl acetate, ethanol, etc., for dye extraction from B. monosperma flowers. Despite the extraction efficacy, the residual impurities and other contaminants occurring in the organic solvents may pose health hazards on application in food prospective. In addition, the consistency of dye extracted by organic solvents also varies greatly; hence, many times it requires additional optimization studies before intended use. Majority of other reports are limited to the phytochemical investigation and bioassay of isolated compounds. The chromatographic methods reported on B. monosperma flower extract have their own shortcomings, e.g., low resolution, weak sensitivity, poor selectivity, use of exhaustive solvents, long analysis period, etc. To the best of our knowledge, there are no such reports on optimized and validated methods for detection and quantification of marker compounds in flower extract of B. monosperma. Therefore, the present research was performed with the explicit objectives of extracting natural dye powder from B. monosperma flowers via an organic solvent-free approach, isolation, and characterization of marker compounds in product dye, and development of an optimized and validated ultra-performance liquid chromatography (UPLC) method for quantification of marker compounds in the product dye. The developed UPLC/PDA method offers a high resolution and proper separation with minimum time of elution of each peak corresponding to the respective component present in the dye powder.

High-purity HPLC-grade chemicals and solvents were used in this work including orthophosphoric acid (H3PO4; AR grade; Merck, Mumbai, India), methanol, dichloromethane, and acetonitrile (all HPLC grades; Merck, Mumbai, India). All the experiments were carried out in Milli-Q ultra-pure water where the purification of water was done by the Milli-Q Integral 3 water purification system (Merck, Millipore). The dry B. monosperma flower petals were powdered by means of a universal grinding machine. In the absence of commercial analytical standards, the isolated compounds, viz., iso-coreopsin (1), butrin (2), and iso-butrin (3) were used as reference standards for quantitative analysis of B. monosperma dye powder. The purity and identity of reference standards were confirmed by chromatographic methods and spectral data analysis. Thin-layer chromatography analysis (TLC) was performed on 60 F254 silica gel precoated on aluminum plates, and visualization of spots was done either by spraying with 5% ethanol-KOH solution or under a UV lamp (λmax = 254 nm). Silica gel (200–400 mesh; Sigma Aldrich) column chromatography was done to isolate the compounds in high purity from dye powder.

UV spectra of isolated compounds were recorded on a Shimadzu UV–vis spectrophotometer (UV2600, Japan). IR spectra were recorded on an Agilent FT-IR spectrometer (Cary 660 series, USA). Melting points were measured on the Buchi melting point apparatus (M-565, Buchi, Switzerland). 1H NMR and 13C NMR were recorded on an Avance Neo 500 MHz FT-NMR Spectrometer (Bruker, Switzerland) at 500 and 125 MHz, respectively. Chemical shifts were recorded in δ (ppm) values relative to tetramethylsilane as an internal reference. Thermogravimetric analysis TGA analysis was done using a Perkin Elmer simultaneous thermal analyzer (STA 8000, USA) in the temperature range of 30 to 800 °C. High-resolution mass spectra (HR-MS) were recorded on Waters Micromass Q-Tof Micro, USA. Trace metal analysis was done on ICP-MS Agilent 7800 series (USA) using pure helium gas. XRD analysis was carried out on a Panalytical X’PERT PRO X-ray diffractometer at scattering angle (2θ) versus intensity, step size 0.001°, working voltage 45 kV with a Cu Kα Ni-filtered radiation (λ = 1.5406 Å). Elemental analysis was performed on an Elementar CHNOS Analyzer (Vario MACRO cube, Germany).

Chromatographic separation was performed on the Waters Acquity H-Class UPLC system with a quaternary solvent manager coupled with the Acquity λ PDA detector and column oven (Waters). The ZORBAX Eclipse Plus C18 column (100 × 4.6 mm; 3.5 μm) with isocratic elution was used to develop the optimized process. After running many trials, a solvent system consisting of degassed mobile phase (A) 0.01% ortho-phosphoric acid in water (pH = 4.7 ± 0.05) and (B) pure acetonitrile (HPLC grade) was used for elution. The optimized chromatographic conditions used are presented in Table S1.

The flower petals of B. monosperma were obtained from Vexcel Upkram Pvt. Ltd., Ranchi, Jharkhand (India). After verification of plant materials, it was shade dried up to moisture content <7 wt % followed by grinding with a commercial grinder to obtain a dry powder (∼0.5 mm, 30 mesh) of flower petals. A specimen sample (BM01-FL) of dry flower petals was preserved in our laboratory, Lab No. 2, Bioproduct Chemistry, Center of Innovative and Applied Bioprocessing (CIAB), SAS Nagar, Mohali-140306, Punjab, India.

A completely dried beaker was charged with dry powder of flower petals (20 g) in 500 mL of hot Milli-Q water in a manner that the plant material was fully immersed in the liquid. The extraction was carried out at five different temperatures (25, 40, 60, 80, and 100 °C) and at five different time intervals (45, 60, 90, 120, and 140 min). Absolute extraction of plant materials was achieved by stirring continuously for about 120 min at 1000 rpm with Milli-Q water temperature of 80 °C. After complete extraction (as monitored by a brix meter), residual materials were taken out from the liquor, and the extract was passed through a 0.4 μm filter to remove the fine colloidal particles. Filtration yielded a clear brown filtrate which was subjected to lyophilization giving a brown-colored dye as a product (7.0 g, yield ∼35%). B. monosperma dye powder (1.0 g) was chromatographed over silica gel (60–120 mesh) and eluted with mixtures of dichloromethane-methanol to give a total of six fractions (f1–6). The fraction f2 eluted with a mixture of dichloromethane-methanol (9:1) afforded iso-coreopsin (1, BM-O), recrystallization with dichloromethane-methanol (9:1) into orange-colored semisolid (30 mg, C21H22O10), Rf: 0.8; dichloromethane-methanol; silica gel (Merck). UV λmax: 215, 262, 348, 371 nm; FT-IR (KBr) vmax: 3397, 3327, 3098, 2363, 2028, 1709, 1610, 1439, 1183, 1072, 987, 729, 570, 475 cm–1; 1H NMR (500 MHz, DMSO-d6): δ 10.57 (br s, 1H), 8.75 (br s, 1H), 7.64 (d, J = 8.86 Hz, 1H), 7.27 (d, J = 1.90 Hz, 1H), 7.02 (dd, J = 1.90, 8.19 Hz, 1H), 6.84 (d, J = 8.13 Hz, 1H), 6.50 (dd, J = 2.19, 8.75 Hz, 1H), 6.34–6.33 (m, 1H), 5.43–5.39 (m, 1H), 5.08 (d, J = 3.71 Hz, 1H), 5.02–5.01 (m, 1H), 4.71 (d, J = 7.83 Hz, 1H), 4.57 (br s, 1H), 3.71 (br d, 1H), 3.50–3.44 (m, 4H), 3.17–3.08 (m, 3H), 2.64–2.63 (m, 1H); 13C NMR (125 MHz, DMSO-d6): δ 190.00 (C-4), 165.05 (C-7), 163.13 (C-8a), 147.02 (C-4′), 145.10 (C-3′), 129.92 (C-1′), 128.30 (C-5), 121.55 (C-6′), 115.71 (C-5′), 115.30 (C-2′), 113.28 (C-4a), 110.64 (C-6), 102.58 (C-8), 102.27 (C-1”), 78.96 (C-2), 77.15, 75.97, 73.30, 69.84, 60.67, 43.07 (C-3); ESI-MS: m/z [M + H]+ 435.1260 (calcd m/z 434.1213); Anal. Calcd. for C21H22O10: C, 58.06; H, 5.11; O, 36.83; Found: C, 58.01; H, 5.09; O, 36.77. Fraction f4 eluted with a mixture of dichloromethane-methanol (8:2) on crystallization afforded butrin (2, BM-W) as white amorphous solid (200 mg, mp = 250–255 °C, C27H32O15), Rf: 0.3; dichloromethane-methanol; silica gel (Merck). UV λmax: 217, 227, 272, 315 nm; FT-IR (KBr) vmax: 3733, 3308, 2880, 2194, 1967, 1663, 1606, 1437, 1246, 1183, 1073, 827, 595, 472 cm–1; 1H NMR (500 MHz, DMSO-d6): δ 8.78 (br s, 1H), 7.73 (d, J = 8.78 Hz, 1H), 7.29 (d, J = 1.95 Hz, 1H), 7.05 (dd, J = 1.83, 8.26 Hz, 1H), 6.85 (d, J = 8.19 Hz, 1H), 6.71 (dd, J = 2.26, 8.80 Hz, 1H), 6.67 (d, J = 2.27 Hz, 1H), 5.51–5.43 (m, 2H), 5.35 (d, J = 4.92 Hz, 1H), 5.09 (d, J = 4.34 Hz, 2H), 5.02–5.01 (d, J = 5.35 Hz, 2H), 4.99 (d, J = 7.39 Hz, 1H), 4.73 (d, J = 7.40 Hz, 1H), 4.58(t, J = 5.58 Hz, 1H), 4.54 (t, J = 5.58 Hz, 1H), 3.73–3.65 (m, 2H), 3.47–3.44 (m, 2H), 3.40–3.35 (m, 2H), 3.31–3.22 (m, 4H), 3.20–3.13 (m, 3H), 2.71 (dd, J = 2.79, 16.90 Hz, 1H); 13C NMR (125 MHz, DMSO-d6): δ 190.50 (C-4), 163.50 (C-7), 162.85 (C-8a), 147.11 (C-4′), 145.10 (C-3′), 129.71 (C-1′), 127.93 (C-5), 121.73 (C-6′), 115.70 (C-5′), 115.42 (C-2′), 115.31 (C-4a), 110.94 (C-6), 103.54 (C-8), 101.95 (C-1”), 99.73 (C-1″’), 79.30 (C-2), 77.22, 77.02, 76.40, 76.01, 73.31, 73.10, 69.91, 69.50, 60.74, 60.53, 43.0 (C-3); ESI-MS: m/z [M + H]+ 597.1808 (calcd m/z 596.1741); Anal. Calcd. for C27H32O15: C, 54.36; H, 5.41; O, 40.23; Found: C, 54.20; H, 5.22; O, 40.17. The mother liquor obtained after filtration of compound 2 (from fraction f4) was concentrated under the reduced pressure followed by recrystallization with dichloromethane-methanol afforded iso-butrin (3, BM-Y) as a yellow amorphous solid (500 mg; mp = 220–225 °C, C27H32O15), Rf: 0.5; dichloromethane-methanol; silica gel (Merck). UV λmax: 228, 269, 372 nm; FTIR (KBr) vmax: 3248, 2918, 2167, 1723, 1603, 1513, 1361, 1237, 1060, 811, 475 cm–1; 1H NMR (500 MHz, CD3OD): δ 8.11 (d, J = 9.12 Hz, 1H), 7.82 (d, J = 15.31 Hz, 1H), 7.73 (d, J = 1.95 Hz, 1H), 7.65 (d, J = 15.35, 1H), 7.33 (dd, J = 1.84, 8.22 Hz, 1H), 6.91 (d, J = 8.27 Hz, 1H), 6.71 (dd, J = 2.44, 9.09 Hz, 1H), 6.62 (d, J = 2.41 Hz, 1H), 5.02 (d, J = 7.21 Hz, 1H), 4.86 (d, J = 7.33 Hz, 1H), 3.98 (dd, J = 2.07, 12.0 Hz, 1H), 3.92 (dd, J = 2.12, 12.16 Hz, 1H), 3.74–3.70 (m, 2H), 3.54–3.47 (m, 6H), 3.43–3.41 (m, 1H), 3.39–3.35 (m, 1H); 13C NMR (125 MHz, DMSO-d6): δ 193.96 (C=O), 165.87 (C-2′), 165.13 (C-4′), 151.89 (C-4), 147.34 (C-3), 146.30 (C-beta), 133.20 (C-6′), 128.35 (C-1), 127.41 (C-6), 118.86 (C-alpha), 118.13 (C-2), 117.57 (C-5), 116.60 (C-1′), 109.40 (C-5′), 105.11 (C-3′), 104.28 (C-1″), 101.33 (C-1″’),78.71, 78.30, 77.88, 77.66, 74.94, 74.74, 71.70, 71.20, 62.75, 62.37; ESI-MS: m/z [M + H]+ 597.1813 (calcd m/z 596.1741); Anal. Calcd. for C27H32O15: C, 54.36; H, 5.41; O, 40.23; Found: 53.98; H, 5.36; O, 40.18.

The stock solution of extract P-1 (lyophilized dye powder) was prepared in a mixture of HPLC-grade methanol and water (1:1, v/v) to give the desired concentration of 1.0 mg/mL. Standard stock solutions of isolated reference compounds iso-coreopsin (1, BM-O) and iso-butrin (3, BM-Y) were prepared in HPLC grade methanol while compound butrin (2, BM-W) in purified Milli-Q water to give a desired concentration of 7.0 mg/mL. The stock solutions of each standard were diluted gradually to obtain seven different working concentrations. All samples were stored at 4 °C and were filtered through a 0.45 μm filter before UPLC analysis.

The developed UPLC/PDA method was validated according to the guidelines of the International Council for Harmonization (ICH). The method was tested with specificity, selectivity, linearity, precision, repeatability, limit of detection (LODs), limit of quantification (LOQs), recovery, and robustness.

In the quantification process, method specificity and selectivity are the vibrant parameters to evaluate the interference of different components present in the dye solution. Stock solutions of product dye and reference standards were prepared in their respective solvents. The chromatographic interferences were assessed by comparing chromatograms of blank solvents to chromatograms of samples prepared in each solvent. In addition, absorbance studies performed between 200 and 600 nm were examined for each component at six different dilutions.

The linearity of the proposed method was established by constructing the calibration curves over a concentration range from 35 to 7000 μg/mL. Seven different working concentrations were analyzed in triplicates to plot three independent calibration curves between peak areas of each standard to the concentration (μg/mL) of solution. The slope and correlation coefficient were determined by linear regression analysis of each calibration curve. Also, the standard deviation (SD) and coefficient of variation (% CV) were calculated in each case.

The LOD and LOQ values were calculated according to the ICH guidelines. LOD is defined as the smallest concentration of analyte that yields an accurate response but cannot be quantified, whereas, LOQ is the smallest quantity of analyte that yields a correct response. LOD and LOQ under proposed chromatographic conditions were calculated using the following formula:where σ, standard deviation of the response (y-intercept); S, slope of the calibration plot.

Precision is a measure of the closeness between a set of measurements from multiple identical samples. The intra- and inter-day precision test of the proposed method was calculated by using seven different concentration levels in triplicates in each case. For the intra-day precision test, 35, 70, 350, 700, 1400, 3500, and 7000 μg/mL of standard solutions were analyzed on the same day at different time intervals, while for the inter-day precision test, the same solutions were examined by replication for over three consecutive days. Additionally, the repeatability test of each standard solution was carried out to investigate the stability of the solution. Analysis was done by injecting the same sample solution for 0, 2, 4, 8, 12, and 24 h. Results of the precision and repeatability test were calculated in terms of standard deviation and coefficient of variation (% CV).

Recovery experiments were conducted to determine the accuracy of the proposed method. The recoveries of the quantification procedure were established by spiking 0.2 mL of standard solution at three different concentrations to 0.2 mL of aqueous extract. The analyses were done using the UPLC/PDA method described above and in triplicates at each concentration level. Recovery (%) of each component was calculated using the following equation:

Robustness was investigated to check the strength of the proposed UPLC/PDA method. Six different analytical parameters, viz., mobile flow rate, column temperature, mobile phase gradient, acetic acid concentration in the mobile phase, and detection wavelength, were studied individually by deliberately causing small variations under the optimum conditions. Each experiment was performed in triplicates and the respective influence of each variation on the content (%) and retention time (Rt) was evaluated with the help of UPLC analysis (Table 1).

The mode of the technique used to extract phytochemicals from plant material directly affects the extraction yield, hence considered as a key variable in the extraction procedure. The overall amount of these phytochemicals gets significantly affected by varying the operating parameters, such as the nature of the plant matrix and solvent used for extraction, types of methods followed, temperature conditions, and overall reaction time. Keeping this in view, each parameter was explored individually for the complete extraction of dye constituents from flower petals of B. monosperma. The present work deals with hot water extraction of dry B. monosperma flower petals and offers a completely organic solvent-free approach. A powder (∼0.5 mm, 30 mesh) of flower petals was used as a substrate for extraction due to the fact that smaller the particles shorter will be the path for the solvent to travel across the tissues, and consequently, lesser will be time taken in the extraction. In addition, the small particles exhibit a larger surface area and provide a greater mass transfer rate, thus may increase the overall extraction efficacy. The hot aqueous extraction approach was adopted over the extraction by using organic solvents due to the merits, e.g., environmental benign condition, requirement of less capital investment, safe operation, capability of discontinuous operation, no threat to product contamination by the solvent impurities, etc. Therefore, in a model experiment, dried powdered flower petals (20 g) were first extracted by stirring in Milli-Q water at ambient temperature for 1 h leading to extremely low recovery of dye powder (1.8 g) after lyophilization. In order to access the yield further, we optimized the extraction condition with respect to temperature and reaction time (Figure 1). The effect of temperature on extraction was studied at five different temperatures ranged between 25 and 100 °C. The total soluble contents in the resulting extract of each run were measured by a brix meter. The optimum reading in the brix meter was noticed when the extraction of plant material was carried out at 80 °C. A further increase in temperature of the aqueous medium, such as around 100 °C resulted in the development of smoky odor/smell in the reaction mixture due to a possible decomposition or charring of the dye components. Once the extraction temperature for the flower petals was optimized, we next studied the reaction time required for complete extraction. Here again, the brix of aqueous extract was compared when extraction time ranged between 45 and 140 min (a total of five different time intervals) at constant temperature (80 °C). The highest soluble contents in the brix meter were noticed when the extraction was carried out by stirring the flower petals for 2 h at 80 °C. The resulting extract on processing to lyophilization furnished a dye powder in good yield (7.0 g, ∼35% yield with respect to flower petals on a dry basis). Hence, it can be concluded that the extraction efficacy of dye powder from flower petals is highly dependable on the operating parameters used for extraction.

The dye powder obtained as the product from B. monosperma flower petals after aqueous extraction and processing to lyophilization was subjected further to column chromatographic purification for isolation of marker compounds 1–3 (Figure 2). The structural assessment of these compounds was affected by analyses, such as, NMR spectroscopy, mass spectrometry (HR-MS), and FT-IR spectroscopy (Figures S1–S9). The compounds were identified as iso-coreopsin (1, BM-O), butrin (2, BM-W), and iso-butrin (3, BM-Y). The stability of pure compounds was determined through TGA analysis under the N2 atmosphere. As evident from (Figure S10), the isolated compounds are generally stable up to 200 °C of the processing temperature. In the X-ray diffraction (XRD) analysis (Figure S15), broad peaks were observed in the case of compounds 1 and 2 which suggested the amorphous nature of isolated compounds, whereas sharp peaks observed in XRD spectrum for compound 3 indicated the crystalline nature. In the FT-IR spectrum (KBr), compound 1 displayed absorption bands characteristic of a carbonyl function (1709 cm–1), and a hydroxyl group (3396 cm–1). The UV spectrum exhibited the absorption bands characteristic of a flavone skeleton with absorption maxima at 262 nm (band II) and 371 nm (band I). Elemental analysis (Anal. Calcd.: C, 58.06%; H, 5.11%; O, 36.83%; Found: C, 58.01%; H, 5.09%; O, 36.77%) in combination with a molecular ion peak at m/z [M + H]+ 435.1260 established the molecular formula C21H22O10 for compound 1. In the 1H NMR spectrum (500 MHz, DMSO-d6), the two broad singlets that appeared at δ 10.57 and δ 8.75 were evidenced for two phenolic groups in compound 1. Six signals of aromatic protons were observed in the region δ 7.64 ppm (d, J = 8.86 Hz, H-5), δ 7.27 ppm (d, J = 1.90 Hz, H-2′), δ 7.02 ppm (dd, J = 1.90, 8.19 Hz, H-6′), δ 6.84 ppm (d, J = 8.13 Hz, H-5′), δ 6.50 ppm (dd, J = 2.19, 8.75 Hz, H-6), and δ 6.35–6.33 ppm (m, H-8). A glucose side chain in compound 1 was evidenced on the basis of an anomeric proton resonance observed at δ 4.71 ppm (linked at C-7, confirmed through HMBC correlation). In the 13C NMR spectrum (125 MHz, DMSO-d6), compound 1 exhibited a total of 21 carbon resonances. Nine signals corresponding to the carbons of ring A and C of the flavone moiety appeared at δ 78.96, 43.07, 190.0, 128.3, 110.64, 165.05, 102.58, 113.28, 163.13 ppm, and six carbon signals of ring B appeared at 129.92, 115.30, 145.10, 147.02, 115.71, 121.55 ppm. The anomeric carbon signal of the attached sugar unit was observed at δ 102.27 ppm and the position of attachment was confirmed by HMBC correlation as shown in Figure S3. Thus, the structure of compound 1 was identified as isocoreopsin, (2-(3,4-dihydroxyphenyl)-7-((3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) chroman-4-one). Compound 2 was obtained as a white amorphous solid (melting point 250–255 °C). Elemental analysis of compound 2 (Anal. Calcd.: C, 54.36%; H, 5.41%; O, 40.23%; Found: C, 54.20%; H, 5.22%; O, 40.17%) in combination with molecular ion peak at m/z [M + H]+ 597.1808 established the molecular formula C27H32O15. Similar to compound 1, the IR spectrum (KBr) of compound 2 revealed the presence of hydroxyl (3308 cm–1) and carbonyl (1663 cm–1) groups. The UV spectrum exhibited absorption maxima at 217 nm (band II) and 272 nm (band I), characteristic absorption bands of a flavone skeleton. In the 1H NMR spectrum (500 MHz, DMSO-d6) of compound 2, a broad singlet corresponding to the phenolic group was observed at δ 8.78, while the signals for six aromatic protons resonated in the region δ 7.73 (d, J = 8.78 Hz, H-5), δ 7.29 (d, J = 1.95 Hz, H-2′), δ 7.05 (dd, J = 1.83, 8.26 Hz, H-6′), δ 6.85 (d, J = 8.19 Hz, H-5′), δ 6.71 (dd, J = 2.26, 8.80 Hz, H-6), and δ 6.67 ppm (d, J = 2.27 Hz, H-8). The two anomeric proton resonances were observed at δ 4.71 (linked at C-3′, confirmed through HMBC correlation) and δ 4.97 (linked at C-7, confirmed through HMBC correlation). In the 13C NMR spectrum (125 MHz, DMSO-d6), compound 2 exhibited a total of 27 carbon resonances which confirmed the molecular formula to be C27H32O15. Nine signals observed were corresponding to the carbons of ring A and C of flavone moiety and resonated at δ 79.30, 43.00, 190.50, 127.93, 110.94, 163.50, 103.54, 115.31, and 162.80 ppm. A total of six carbon signals of ring B appeared at δ 129.71, 115.42, 145.10, 147.11, 115.70, and 121.73 ppm. The anomeric carbon signals of the two sugar moieties attached as the side chain were observed at δ 101.95 ppm and δ 99.73 ppm, and their respective attachments were confirmed by HMBC correlation as shown in Figure S6. Thus, the structure of compound 2 was identified as butrin, (2-(4-hydroxy-3-((3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl)-7-((3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl) oxy) chroman-4-one). Compound 3 was obtained as a yellow amorphous solid, melting point of 220–225 °C. The molecular formula of compound 3 was established as C27H32O15 based on elemental analysis (Anal. Calcd.: C, 54.36%; H, 5.41%; O, 40.23%; Found: C, 53.98%; H, 5.36%; O, 40.18%) in combination with molecular ion peak at m/z [M + H]+ 597.1813. FT-IR spectrum (KBr) revealed the presence of hydroxyl (3248 cm–1) and carbonyl (1723 cm–1) groups in compound 3. The UV spectrum exhibited absorption maxima at 228 nm (band II) and 372 nm (band I) that corresponded to a related α,β-unsaturated ketone skeleton. In the 1H NMR spectrum (500 MHz, CD3OD), the compound 3 exhibited a total of eight aromatic signals that appeared at δ 8.11 (d, J = 9.12 Hz, H-6′), δ 7.82 (d, J = 15.31 Hz, H-α), δ 7.73 (d, J = 1.95 Hz, H-2), δ 7.65 (d, J = 15.35, H-β), δ 7.33 (dd, J = 1.84, 8.22 Hz, H-6), δ 6.91 (d, J = 8.27 Hz, H-5), δ 6.71 (dd, J = 2.44, 9.09 Hz, H-3′), and δ 6.62 (d, J = 2.41 Hz, H-5′). The two anomeric protons of sugar residue were observed at δ 4.86 ppm (linked at C-3, confirmed through HMBC correlation) and at δ 5.02 ppm (linked at C-4′, confirmed through HMBC correlation). In the 13C NMR spectrum (125 MHz, DMSO-d6), compound 3 exhibited a total of 27 carbon resonances corresponding to the molecular formula to be C27H32O15. The six signals corresponding to the ring-A of the chalcone moiety were observed at δ 128.35, 118.13, 147.34, 151.89, 117.57, and 127.41 ppm. Likewise, six carbon signals of ring B resonated at δ 116.60, 165.87, 105.11, 165.15, 109.40, 133.20 ppm. The signals corresponding to the carbonyl carbon, C-α, and C-β were observed at δ 193.96, 118.86, and 146.30 ppm, respectively. The anomeric carbon signals of the two sugar moieties were observed at δ 104.28 ppm and δ 101.33 ppm and their respective attachments were confirmed by HMBC correlation as shown in Figure S9. On the basis of the above results, the structure of compound 3 was identified as isobutrin, (3-(4-hydroxy-3-((3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl)-1-(2-hydroxy-4-((3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) prop-2-en-1-one).

The development of an efficient and improved UPLC/PDA method was attained for the simultaneous evaluation of phytochemical constituents present in the B. monosperma extract. This developed method was achieved by undergoing a careful examination of the effect of different chromatographic parameters on the overall area, resolution, sharpness, and intensity of the peak. These parameters include the type of solvent system, nature of elution method, mobile phase ratio, type of organic acid modifiers, solvent flow rate, final column temperature and the wavelength used to detect specific peaks. We herein studied the binary solvent system comprising separate organic and aqueous phases in the isocratic mode in order to elute each component present in the extract. The organic phase was tested with solvents including methanol, MeCN, and the mixture of these two in a 1:1 ratio, while the aqueous phase was tried with organic acid modifiers such as formic acid, acetic acid, and ortho-phosphoric acid. Unsatisfactory results were obtained in the case of 0.01% formic acid and 0.01% acetic acid when used in the aqueous phase with either methanol or MeCN. However, quite satisfactory results were gained with varying ratios of MeCN and water consisting of 0.01% ortho-phosphoric acid in the aqueous phase. Optimal separation of respective components was ultimately achieved by possessing peaks with high resolution and intensity when the isocratic elution program was run for 30 min with 0.3 mL/min flow rate of pure MeCN and water with 0.01% ortho-phosphoric acid (pH = 3.5) in the specific ratio of 25:75 and the column temperature were maintained at 30 °C. The developed UPLC/PDA method was validated in particulars to selectivity, specificity, linearity, LOD, LOQ, precision, repeatability, recovery, and robustness in accordance with the ICH guidelines.

Selectivity is the ability of an HPLC method to separate analytes from each other. No interfering peaks at a similar retention time were observed in the chromatogram of isolated components. These reference components rapidly eluted with retention times of 20.317, 4.733, and 6.137 min as iso-coreopsin (1, BM-O), butrin (2, BM-W), and iso-butrin (3, BM-Y), respectively (Figure S11). The chromatographic peak purity for each component was found acceptable (>95%) by UPLC. The UPLC analysis (Figure S12) using the Hi-Plex column also established the presence of three nectar sugars, viz. sucrose, glucose, and xylose in the lyophilized B. monosperma dye powder (P-1). The quantitative determination of compounds occurring in dye powder is summarized in Table 2. Also, the absorbance studies were examined for each isolated component and found to be the same at chosen points in six different dilutions (Figure S13).

The linearity of the developed method was confirmed by plotting the calibration curve of each isolated component. The regression equation derived from the calibration curves showed a good linear relationship between the peak area and concentration of each component over the range of 35–7000 μg/mL for iso-coreopsin (1, BM-O), butrin (2, BM-W), and iso-butrin (3, BM-Y), respectively. Also, the correlation coefficient (r2) in each case was found to be more than 0.9989. The linearity plots with respective regression equations and correlation coefficient of each component are represented in Figure S14.

LOD is the least concentration of analyte that can be detected, while LOQ is the least concentration of analyte that can be quantified with acceptable precision. Based on the standard deviation of the response and the slope of the linear equation, the LOD and LOQ values of each component were calculated for the developed method. The limits of detection (signal-to-noise ratio of 3.3) of iso-coreopsin (1, BM-O), butrin (2, BM-W), and iso-butrin (3, BM-Y) were 81.47, 59.93 and 284.90 μg/mL, respectively, while their respective LOQ (signal-to-noise ratio of 10) was found to be 271.58, 199.76, and 949.66 μg/mL. The respective LOD, LOQ, and calibration curve parameters are summarized in Tables S2 and S3.

Precision is the measure of closeness between a set of measurements from multiple identical samples. It can be determined as intra- and inter-day precision. The assay values of each component on both occasions (intra- and inter-day) were found less than 5% which showed that the precision of the method was well acceptable. Also, the repeatability test of each sample solution was examined by replicate injections at 0, 2, 4, 8, 12, and 24 h respectively. Results showed satisfactory stability (% CV < 5) of the sample solution within 24 h at room temperature. The precision and repeatability data of optimized method of each component are presented in Tables S4 and S5.

To determine the accuracy of the method, recovery experiments of each component were performed by spiking the known amount of standard to the preanalyzed sample followed by the reanalysis. Three different concentration levels of the analytical standards were added to the samples in triplicates and mean recoveries of each were determined. The developed analytical method had shown good accuracy with overall recovery (%) in the range from 97.85 to 100.57 and acceptable % CV values i.e., less than 5 in each case (Table S6).

To investigate the robustness of the developed method, extract sample was analyzed by deliberately altering the five different experimental parameters. The variables evaluated in the study were flow rate, detection wavelength, ratio of mobile phase, concentration of acid modifier and column temperature which resulted in the change in the retention time and peak area response of major peak. This denotes that the procedure has been tested. The method was found highly robust regarding the parameters studied and their impact on content (%) and retention time are depicted in Table S7.

The dye powder obtained as a product from flower petals of B. monosperma was also investigated for the determination of trace metals using ICP-MS analysis. As summarized in Table S8, the dye powder displayed traces of mercury with significantly low relative abundance (RSD) <4%. Similarly, the presence of lead, arsenic, cadmium, and sodium could be detected only at negligible concentrations.

An accurate, sensitive, and repeatable UPLC/PDA method has been developed for the investigation of compounds present in dye/colorant powder obtained as the product from the flower extract of B. monosperma. The linearity, precision, stability, selectivity, sensitivity, accuracy, robustness, LOD, and LOQ of the devised method were all validated according to ICH recommendations. An organic solvent-free hot aqueous extraction approach for production of dye powder poses no threat to product contamination by the toxic impurities. In addition, the approach appears convenient, safe, economic, and highly scalable as the whole process of dye production from B. monosperma flower petals can be carried out with low-cost investments. Most of the dye constituents were isolated and characterized by analyses such as NMR spectroscopy, HR-MS, and FT-IR spectroscopy. Also, thermal studies and absorption studies of isolated constituents were performed. A near complete characterization of constituents occurring in B. monosperma dye may broaden its application for use as a coloring material in a variety of food products (e.g., sausage, jam, noodles, etc.), beverages (soft drinks), cosmetics, and pharmaceutical syrups.