Simultaneous Estimation of Rhein and Aloe-Emodin in Traditional and Ultrasound-Based Extracts of Rheum palmatum L. (Rhubarb) Using Sustainable Reverse-Phase and Conventional Normal-Phase HPTLC Methods

: The greenness indices of literature analytical procedures for the simultaneous measurement of rhein and aloe-emodin have not been determined. As a consequence, the ﬁrst goal of this study was to design and validate a sensitive and sustainable reverse-phase high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of rhein and aloe-emodin in a traditional extract (TE) and ultrasound-based extract (UBE) of commercial Rhubarb and Rhubarb plant extracts in comparison to the conventional normal-phase HPTLC method. The second goal was to determine the greenness indices for both methods using the AGREE approach. For the sustainable reverse-phase HPTLC approach, the method was linear in the 50–1000 ng/spot range for rhein and 25–1000 ng/spot range for aloe-emodin. However, for the conventional normal-phase HPTLC approach, the method was linear in the 50–600 ng/spot range for rhein and 100–600 ng/spot range for aloe-emodin. The limit of detection (LOD) for rhein and aloe-emodin was 16.81 ng/spot and 8.49 ng/spot, respectively, using the sustainable analytical method. However, the LOD for rhein and aloe-emodin was 18.53 ng/spot and 39.42 ng/spot, respectively, using the conventional analytical method. For the simultaneous determination of rhein and aloe-emodin, the sustainable analytical method was more sensitive, accurate, precise, and robust than the conventional analytical method. The amount of rhein and aloe-emodin was higher in the UBE of commercial Rhubarb and Rhubarb plant extract over their TE. For the simultaneous quantiﬁcation of rhein and aloe-emodin in the TE and UBE of marketed Rhubarb and Rhubarb plant extract, the sustainable analytical method was superior to the conventional analytical method. The AGREE index for the sustainable reverse-phase and conventional normal-phase HPTLC methods was determined to be 0.78 and 0.49, respectively, indicating an excellent greenness proﬁle of the sustainable reverse-phase HPTLC method over the conventional normal-phase HPTLC approach. The sustainable analytical method was found to be superior to the conventional analytical method based on these results.


Introduction
Anthraquinones are the phenolic compounds which are present in small amounts in plants [1].These compounds are present in various plants, such as Rheum palmatum (Rhubarb, family: Polygonaceae), Rhamnus purshiana (cascara, family: Rhamnaceae), Aloe

Introduction
Anthraquinones are the phenolic compounds which are present in small amounts in plants [1].These compounds are present in various plants, such as Rheum palmatum (Rhubarb, family: Polygonaceae), Rhamnus purshiana (cascara, family: Rhamnaceae), Aloe barbadensis (aloe, family: Liliaceae), and Senna alata (candle bush, family: Fabaceae) [2,3].Various phytochemicals have been isolated from Rhubarb, however, the major phytochemicals are anthraquinones [4].The main anthraquinones present in Rhubarb are rhein, emodin, aloe-emodin, chrysophanol, and physcion [4,5].Rhein (Figure 1A) and aloeemodin (Figure 1B) have several pharmacological activities, such as laxative, purgative, anti-inflammatory, anti-aging, antibacterial, antioxidant, and anti-tumor activities [1][2][3][4][5][6][7].Because rhein and aloe-emodin can be found in a variety of plants and marketed formulations, their qualitative and quantitative standardization is required.Different pharmaceutical approaches for the simultaneous measurement of rhein and aloe-emodin in plant extracts, traditional Chinese medicine (TCM), marketed dosage forms, and physiological fluids were found in the literature.For the simultaneous quantification of rhein and aloe-emodin in plant extracts, marketed dosage forms and TCM, various high-performance liquid chromatography (HPLC) methods using ultraviolet (UV) detection [4,[8][9][10][11][12] or diode array (DAD) detection [5] have been reported.HPLC-DAD and HPLC-fluorescence (FLD) assays have also been reported for the simultaneous quantification of rhein and aloe-emodin in dog plasma samples and plasma, urine, and cerebrospinal fluids of rats [13,14].Rhein and aloe-emodin were also determined simultaneously in various plant extracts using some ultra-high-performance liquid chromatography (UHPLC)-DAD and UHPLC-mass spectrometry (MS) methods [15,16].A UHPLC-MS/MS method has also been reported for the simultaneous determination of rhein and aloeemodin in rat plasma samples and their pharmacokinetic assessment [17].For the simultaneous determination of rhein and aloe-emodin in rat plasma samples after oral administration of Rhubarb extract, a liquid-chromatography-mass spectrometry (LC-MS) method was also utilized [18].A high-performance thin-layer chromatography (HPTLC)-UV method has been documented for the simultaneous quantification of aloe-emodin and emodin in Rheum emodi, Cassia alata, and Aloes, using the toluene/ethyl acetate/formic acid solvent system [19].However, the linearity and greenness profile of this method was much inferior to the current sustainable HPTLC method [19].For the simultaneous quantification of rhein and aloe-emodin in Senna alata leaves and commercial products, a HPTLC-UV assay using an ethyl acetate/methanol/water solvent system has also been documented [20].However, the linearity, precision, and greenness profile of this method was much inferior to the current sustainable HPTLC method [20].Some other analytical assays, such as the colloidal gold immunochromatographic strip [21], capillary zone electrophoresis [5,22], synchronous fluorescence spectroscopy [23], near-infrared spectroscopy [24], and ultrasound emulsification ionic liquid microextraction [25] assays have also been reported for the simultaneous quantification of rhein and aloe-emodin in Rhubarb and other plant extracts.Literature on the simultaneous quantification of rhein and aloeemodin indicated different pharmaceutical assays of quantification.The greenness index Different pharmaceutical approaches for the simultaneous measurement of rhein and aloe-emodin in plant extracts, traditional Chinese medicine (TCM), marketed dosage forms, and physiological fluids were found in the literature.For the simultaneous quantification of rhein and aloe-emodin in plant extracts, marketed dosage forms and TCM, various high-performance liquid chromatography (HPLC) methods using ultraviolet (UV) detection [4,[8][9][10][11][12] or diode array (DAD) detection [5] have been reported.HPLC-DAD and HPLC-fluorescence (FLD) assays have also been reported for the simultaneous quantification of rhein and aloe-emodin in dog plasma samples and plasma, urine, and cerebrospinal fluids of rats [13,14].Rhein and aloe-emodin were also determined simultaneously in various plant extracts using some ultra-high-performance liquid chromatography (UHPLC)-DAD and UHPLC-mass spectrometry (MS) methods [15,16].A UHPLC-MS/MS method has also been reported for the simultaneous determination of rhein and aloe-emodin in rat plasma samples and their pharmacokinetic assessment [17].For the simultaneous determination of rhein and aloe-emodin in rat plasma samples after oral administration of Rhubarb extract, a liquid-chromatography-mass spectrometry (LC-MS) method was also utilized [18].A high-performance thin-layer chromatography (HPTLC)-UV method has been documented for the simultaneous quantification of aloe-emodin and emodin in Rheum emodi, Cassia alata, and Aloes, using the toluene/ethyl acetate/formic acid solvent system [19].However, the linearity and greenness profile of this method was much inferior to the current sustainable HPTLC method [19].For the simultaneous quantification of rhein and aloe-emodin in Senna alata leaves and commercial products, a HPTLC-UV assay using an ethyl acetate/methanol/water solvent system has also been documented [20].However, the linearity, precision, and greenness profile of this method was much inferior to the current sustainable HPTLC method [20].Some other analytical assays, such as the colloidal gold immunochromatographic strip [21], capillary zone electrophoresis [5,22], synchronous fluorescence spectroscopy [23], near-infrared spectroscopy [24], and ultrasound emulsification ionic liquid microextraction [25] assays have also been reported for the simultaneous quantification of rhein and aloe-emodin in Rhubarb and other plant extracts.Literature on the simultaneous quantification of rhein and aloe-emodin indicated different pharmaceutical assays of quantification.The greenness index of any of the stated analytical methods, on the other hand, was not determined.Furthermore, no sustainable HPTLC techniques for the simultaneous measurement of rhein and aloe-emodin have been described.For the evaluation of the greenness index, various methodologies have been reported [26][27][28][29][30].The development of green analytical methodologies is an important step in pharmaceutical and phytochemical analysis in order to reduce environmental toxicity [27,28].For the evaluation Agronomy 2022, 12, 1295 3 of 17 of the greenness index, only the Analytical GREENness (AGREE) methodology considers all twelve principles of green analytical chemistry (GAC) [28].Accordingly, the greenness index of the current sustainable and conventional HPTLC experiments was evaluated using the AGREE approach [28].Sustainable/greener HPTLC methods offer several advantages compared to conventional liquid chromatography methods [29,30].However, the HPTLC methods have certain limitations.The main limitation of the developed sustainable HPTLC is associated with its application in the simultaneous determination of rhein and aloe-emodin in biological samples.
The first goal of the current study was to design and validate a more accurate, precise, robust, sensitive, and sustainable reverse-phase HPTLC assay for the simultaneous determination of rhein and aloe-emodin in traditional extracts (TE) and ultrasound-based extracts (UBE) of marketed Rhubarb and Rhubarb plant extracts in comparison to the conventional normal-phase HPTLC assay.The second goal was to determine the greenness indices of both assays using the AGREE approach.Using the International Council for Harmonization (ICH) Q2-R1 guidelines, the sustainable and conventional analytical assays for the simultaneous estimation of rhein and aloe-emodin were validated [31].

Materials
Standard rhein and aloe-emodin and commercial Rhubarb powder extract were obtained from E-Merck (Darmstadt, Germany).HPLC-grade solvents, such as methanol, ethanol, and chloroform were obtained from Sigma Aldrich (St. Louis, MO, USA).The roots of Rhubarb (Rheum palmatum) were procured from Alexandria, Egypt, and authenticated by the Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-kharj, Saudi Arabia.The water (conductivity = < 1.0 µS/cm and resistivity = 18.2 MΩ•cm) was procured from the Milli-Q unit in the laboratory.All other solvents and reagents used were of analytical grades.

Instrumentation and Chromatography
The HPTLC CAMAG TLC system (CAMAG, Muttenz, Switzerland) was utilized for the simultaneous estimation of rhein and aloe-emodin in pure forms, TE, and UBE of marketed Rhubarb, and Rhubarb plant extract.The samples were applied as 6 mm bands using a CAMAG Automatic TLC Sampler 4 (ATS4) Sample Applicator (CAMAG, Geneva, Switzerland).The CAMAG microliter syringe (Hamilton, Bonaduz, Switzerland) was associated with the sample applicator.For the simultaneous estimation of rhein and aloe-emodin, the application rate was set to 150 nL/s.The TLC plates were established in a CAMAG automated developing chamber 2 (ADC2) (CAMAG, Muttenz, Switzerland) with a distance of 80 mm.The development chamber (dimensions: 21 × 21 × 9 cm) was saturated with vapors of respective mobile phases for 30 min at 22 • C. At a wavelength of 257 nm, rhein and aloe-emodin were identified.The UV lamp was used for the detection.Densitometry analysis was performed under the linear ascending reflectance-absorbance mode.The scanning rate and slit dimensions were set to 20 mm/s and 4 × 0.45 mm 2 , respectively.Each experiment was performed using three or six replicates.The software utilized was WinCATs (version 1.4.3.6336,CAMAG, Muttenz, Switzerland).
The most noteworthy differences between the normal-phase and reverse-phase HPTLC procedures were the HPTLC plates and mobile phase compositions.The HPTLC plates in the conventional analytical method were glass plates (plate size: 10 × 20 cm) pre-coated with normal-phase silica gel (particle size: 5 µm) 60F254S plates (E-Merck, Darmstadt, Germany), whereas the HPTLC plates in the sustainable analytical method were glass plates (plate size: 10 × 20 cm) pre-coated with reverse-phase silica gel (particle size: 5 µm) 60F254S plates (E-Merck, Darmstadt, Germany).The mobile phase in the conventional analytical method was chloroform/methanol (70:30, v/v); however, the sustainable analytical method used ethanol/water (60:40, v/v).

Calibration Plots and Quality Control (QC) Solutions for Rhein and Aloe-Emodin
Individual stock solutions of rhein and aloe-emodin were produced by dissolving 10 mg of both compounds in the stated amount of respective mobile phases, yielding a final stock solution of 100 µg/mL for both compounds (n = 6).For the conventional analytical method, concentrations in the 50-600 ng/spot range for rhein and 100-600 ng/spot range for aloe-emodin were obtained by an appropriate dilution.For the sustainable analytical method, concentrations in the 50-1000 ng/spot range for rhein and 25-1000 ng/spot for aloe-emodin were obtained by an appropriate dilution.For the conventional analytical method, 200 µL of each solution of rhein and aloe-emodin were applied to normal-phase HPTLC plates and reverse-phase HPTLC plates for the sustainable analytical method.Utilizing both methodologies, the peak areas of each solution of rhein and aloe-emodin were recorded.The calibration curves for rhein and aloe-emodin were created by graphing the peak areas of both compounds versus concentrations (n = 6).Three independent QC samples were acquired fresh for the examination of different validation parameters.

Sample Preparation for the Simultaneous Determination of Rhein and Aloe-Emodin in TE of Marketed Rhubarb and Rhubarb Plant Extract
The fresh roots of the Rhubarb plant were obtained and crushed to obtain the fine powder.The powder Rhubarb was extracted using a hot process method.Approximately 1.0 g of powdered samples from commercial and plant were soaked in 50 mL of ethanol in chloroform.The obtained mixtures were heated at 70 • C for one hour in a water bath.The extraction process was repeated thrice and centrifuged at 3000× g at 25 • C for 30 min.The supernatant was obtained and filtered via Whatman filter paper (No. 41).The filtrate was evaporated under reduced pressure at 40 • C using a rotary evaporator.The samples containing 10 mg/mL of commercial extract and process extracts were obtained.These samples were used for the simultaneous quantification of rhein and aloe-emodin in the TE of commercial Rhubarb and Rhubarb plants using the sustainable reverse-phase HPTLC and conventional normal-phase HPTLC assays.

Sample Preparation for the Simultaneous Quantification of Rhein and Aloe-Emodin in UBE of Commercial Rhubarb and Rhubarb Plant Extract
The UBE was carried out using ultrasonic vibrations utilizing the Ultrasonic processor-200 Ht, power 200 W (Darmstadt, Germany).Approximately 1.0 g of each powdered sample was soaked in 50 mL of ethanol in chloroform.The UBE was performed using the above apparatus for one hour.The temperature was fixed at 25 • C and the frequency was fixed at 26 kHz.The UBE process was repeated thrice and centrifuged at 3000× g at 25 • C for 30 min.The supernatant was obtained and filtered using Whatman filter paper (No. 41).A rotary vacuum evaporator was used to evaporate filtrate at 40 • C. The obtained samples were used for the simultaneous determination of rhein and aloe-emodin in the UBE of commercial Rhubarb and Rhubarb plants using the sustainable reverse-phase HPTLC and conventional normal-phase HPTLC assays.

Validation Parameters
Using the ICH-Q2-R1 guidelines, the conventional and sustainable analytical methods for the simultaneous estimation of rhein and aloe-emodin were validated for linearity, system suitability, accuracy, precision, robustness, specificity, and sensitivity [31].By plotting the peak area of both compounds against their concentrations, the linearity for both compounds was determined.The conventional analytical method's linearity for rhein and aloe-emodin was tested in the 50-600 ng/spot and 100-600 ng/spot ranges, respectively (n = 6).For the sustainable analytical assay, the linearity of rhein and aloe-emodin was tested in the 50-1000 ng/spot and 25-1000 ng/spot ranges, respectively (n = 6).
The determination of the retardation factor (R f ), asymmetry factor (As), and theoretical plates number per meter (N/m) was utilized to assess the system suitability parameters of both assays for the simultaneous estimation of rhein and aloe-emodin.For both assays, the R f , As, and N/m values were determined using their given equations [29].
For the simultaneous estimation of both compounds, the %recovery was utilized to determine the accuracy of both assays.The accuracy of the conventional analytical assay for both compounds was assessed at three QC levels: low QC (LQC; 100 ng/spot), medium QC (MQC; 400 ng/spot), and high QC (HQC; 600 ng/spot) for both compounds.For rhein, the accuracy of the sustainable analytical method was assessed at LQC (100 ng/spot), MQC (400 ng/spot), and HQC (1000 ng/spot).For aloe-emodin, the accuracy of the sustainable analytical method was tested at LQC (50 ng/spot), MQC (400 ng/spot), and HQC (1000 ng/spot).At each QC level, the %recovery for rhein and aloe-emodin (n = 6) was calculated using both assays.
Intra/inter-assay variation was determined for both assays of rhein and aloe-emodin.For both assays, the intra-assay precision was assessed by measuring freshly made rhein and aloe-emodin samples at LQC, MQC, and HQC on the same day (n = 6).For both assays (n = 6), the inter-assay precision for rhein and aloe-emodin was assessed by determining newly created samples at LQC, MQC, and HQC on three consecutive days.
The robustness of the rhein and aloe-emodin assays was determined by making small purposeful modifications in the mobile phase composition.For rhein and aloe-emodin, the sustainable mobile phase of ethanol/water (60:40, v/v) was changed to ethanol/water (62:18, v/v) and ethanol/water (58:42, v/v) for the sustainable reverse-phase HPTLC assay, and the differences in the HPTLC response and R f values were noted (n = 6).For rhein and aloe-emodin, the conventional mobile phase of chloroform/methanol (70:30, v/v) was changed to chloroform/methanol (72:28, v/v) and chloroform/methanol (68:32, v/v) for the conventional normal-phase HPTLC assay, and the differences in the HPTLC response and R f values were noted (n = 6).
Using a "standard deviation" methodology, the sensitivity of both assays for the simultaneous estimation of rhein and aloe-emodin was determined as "limit of detection (LOD) and limit of quantification (LOQ)".Rhein and aloe-emodin "LOD and LOQ" values were calculated with the help of their reported formulae for both assays (n = 6) [31].
To assess the specificity of both assays for simultaneous determination of rhein and aloe-emodin, the R f values and UV spectra of rhein and aloe-emodin in marketed Rhubarb and Rhubarb plant extracts were compared to those of standard rhein and aloe-emodin.

Application of Conventional and Sustainable Analytical Methods in the Simultaneous Estimation of Rhein and Aloe-Emodin in Commercial Rhubarb and Rhubarb Plant Extracts
For both assays, the obtained samples of commercial Rhubarb and Rhubarb plant extracts were spotted on normal-phase HPTLC plates for the conventional normal-phase HPTLC assay and reverse-phase HPTLC plates for the sustainable reverse-phase HPTLC assay.For both assays, the HPTLC responses were measured using the same experimental circumstances used for the simultaneous estimations of standard rhein and aloe-emodin (n = 3).For both assays, the amounts of rhein and aloe-emodin in marketed Rhubarb and Rhubarb plant extracts were determined using the calibration curves for rhein and aloe-emodin.

Greenness Assessment by AGREE Approach
The AGREE approach [28] was used to determine the greenness indices for both assays for the simultaneous estimation of rhein and aloe-emodin.The AGREE indices (0.0-1.0) for both assays were determined using AGREE's analytical greenness calculator (version 0.5, Gdansk University of Technology, Gdansk, Poland, 2020).

Method Development
For the establishment of a valid band for the simultaneous estimation of the rhein and aloe-emodin standards, and the TE and UBE of commercial Rhubarb and Rhubarb plant extract using the conventional normal-phase HPTLC assay, different chloroform/methanol concentrations within the 30-90% chloroform range were investigated as the conventional mobile phases.The results obtained suggested that the chloroform/methanol (70:30, v/v) solvent system presented well-separated and intact HPTLC peaks for rhein at R f = 0.53 ± 0.02 and of aloe-emodin at R f = 0.27 ± 0.01 (Figure 2).Rhein and aloe-emodin were both found to have As values of 1.12 and 1.07, which are both very reliable.As a result, the chloroform/methanol (70:30, v/v) was selected as the final solvent system for the simultaneous determination of the rhein and aloe-emodin standards, and the TE and UBE of commercial Rhubarb and Rhubarb plant extracts, using the conventional normal-phase HPTLC assay.

Method Development
For the establishment of a valid band for the simultaneous estimation of the rhein and aloe-emodin standards, and the TE and UBE of commercial Rhubarb and Rhubarb plant extract using the conventional normal-phase HPTLC assay, different chloroform/methanol concentrations within the 30-90% chloroform range were investigated as the conventional mobile phases.The results obtained suggested that the chloroform/methanol (70:30, v/v) solvent system presented well-separated and intact HPTLC peaks for rhein at Rf = 0.53 ± 0.02 and of aloe-emodin at Rf = 0.27 ± 0.01 (Figure 2).Rhein and aloeemodin were both found to have As values of 1.12 and 1.07, which are both very reliable.As a result, the chloroform/methanol (70:30, v/v) was selected as the final solvent system for the simultaneous determination of the rhein and aloe-emodin standards, and the TE and UBE of commercial Rhubarb and Rhubarb plant extracts, using the conventional normal-phase HPTLC assay.Different ethanol/water ratios within the 30-90% ethanol range were examined as sustainable solvent systems for the establishment of a valid band for the simultaneous quantification of rhein and aloe-emodin utilizing the sustainable reverse-phase HPTLC assay.As shown in Figure 3, all the planned solvent systems were created under chambersaturation conditions.The results showed that the ethanol/water (60:40, v/v) mobile phase presented well-resolved and intact HPTLC peaks for rhein at Rf = 0.20 ± 0.01 and of aloeemodin at Rf = 0.86 ± 0.02 (Figure 2).Rhein and aloe-emodin were both found to have As values of 1.10 and 1.05, which are both very reliable.As a result, the ethanol/water (60:40, v/v) was selected as the final solvent system for the simultaneous determination of rhein and aloe-emodin standards, and the TE and UBE of commercial Rhubarb and Rhubarb plant extracts, using the sustainable reverse-phase HPTLC assay.The scanning was performed in the wavelength range of 200-400 nm.The highest HPTLC response was predicted at a wavelength of 257 nm for rhein and aloe-emodin when the spectral bands for Different ethanol/water ratios within the 30-90% ethanol range were examined as sustainable solvent systems for the establishment of a valid band for the simultaneous quantification of rhein and aloe-emodin utilizing the sustainable reverse-phase HPTLC assay.As shown in Figure 3, all the planned solvent systems were created under chambersaturation conditions.The results showed that the ethanol/water (60:40, v/v) mobile phase presented well-resolved and intact HPTLC peaks for rhein at R f = 0.20 ± 0.01 and of aloe-emodin at R f = 0.86 ± 0.02 (Figure 2).Rhein and aloe-emodin were both found to have As values of 1.10 and 1.05, which are both very reliable.As a result, the ethanol/water (60:40, v/v) was selected as the final solvent system for the simultaneous determination of rhein and aloe-emodin standards, and the TE and UBE of commercial Rhubarb and Rhubarb plant extracts, using the sustainable reverse-phase HPTLC assay.The scanning was performed in the wavelength range of 200-400 nm.The highest HPTLC response was predicted at a wavelength of 257 nm for rhein and aloe-emodin when the spectral bands for rhein and aloe-emodin were obtained under densitometry mode.As a consequence, the entire simultaneous quantification of rhein and aloe-emodin took place at 257 nm.
rhein and aloe-emodin were obtained under densitometry mode.As a consequence, the entire simultaneous quantification of rhein and aloe-emodin took place at 257 nm.

Validation Parameters
The ICH-Q2-R1 guidelines were applied to evaluate linearity, system suitability, accuracy, precision, robustness, specificity, and sensitivity for the simultaneous determination of rhein and aloe-emodin [31].The outcomes for the linearity evaluation of rhein and aloe-emodin calibration plots using the sustainable and conventional analytical methods are included in Table 1.The calibration plots for rhein and aloe-emodin using the sustainable and conventional analytical methods are presented in Figure 4.For the sustainable analytical assay, rhein and aloe-emodin calibration plots were linear in the 50-1000 ng/spot and 25-1000 ng/spot ranges, respectively.Rhein and aloe-emodin's determination coefficients (R 2 ) were estimated to be 0.9972 and 0.9962, respectively.Rhein and aloeemodin's regression coefficients (R) were estimated to be 0.9985 and 0.9980, respectively.These data of R 2 and R were significant for rhein and aloe-emodin (p < 0.05).These outcomes showed a strong relation between the peak areas and concentrations of rhein and aloe-emodin.All these outcomes suggested the suitability of the sustainable analytical method for the simultaneous determination of rhein and aloe-emodin.

Validation Parameters
The ICH-Q2-R1 guidelines were applied to evaluate linearity, system suitability, accuracy, precision, robustness, specificity, and sensitivity for the simultaneous determination of rhein and aloe-emodin [31].The outcomes for the linearity evaluation of rhein and aloe-emodin calibration plots using the sustainable and conventional analytical methods are included in Table 1.The calibration plots for rhein and aloe-emodin using the sustainable and conventional analytical methods are presented in Figure 4.For the sustainable analytical assay, rhein and aloe-emodin calibration plots were linear in the 50-1000 ng/spot and 25-1000 ng/spot ranges, respectively.Rhein and aloe-emodin's determination coefficients (R 2 ) were estimated to be 0.9972 and 0.9962, respectively.Rhein and aloe-emodin's regression coefficients (R) were estimated to be 0.9985 and 0.9980, respectively.These data of R 2 and R were significant for rhein and aloe-emodin (p < 0.05).These outcomes showed a strong relation between the peak areas and concentrations of rhein and aloe-emodin.All these outcomes suggested the suitability of the sustainable analytical method for the simultaneous determination of rhein and aloe-emodin.
For the conventional analytical method, rhein and aloe-emodin calibration plots were linear in the 50-600 ng/spot and 100-600 ng/spot ranges, respectively.Rhein and aloe-emodin's R 2 were estimated to be 0.9952 and 0.9956, respectively.Rhein and aloeemodin's R were estimated to be 0.9975 and 0.9977, respectively.These data of R 2 and R were significant for rhein and aloe-emodin (p < 0.05).These results also indicate a good correlation between the peak areas and concentrations of rhein and aloe-emodin.All these outcomes suggested the suitability of the conventional analytical method for the simultaneous determination of rhein and aloe-emodin.However, the sustainable analytical method was more linear than the conventional analytical method.
The system suitability parameters for the sustainable and conventional analytical methods are included in Table 2.For the simultaneous determination of rhein and aloeemodin, the R f , As, and N/m for the sustainable and conventional analytical assays were estimated to be satisfactory.For the conventional analytical method, rhein and aloe-emodin calibration plots were linear in the 50-600 ng/spot and 100-600 ng/spot ranges, respectively.Rhein and aloe-emodin's R 2 were estimated to be 0.9952 and 0.9956, respectively.Rhein and aloe-emodin's R were estimated to be 0.9975 and 0.9977, respectively.These data of R 2 and R were significant for rhein and aloe-emodin (p < 0.05).These results also indicate a good correlation between the peak areas and concentrations of rhein and aloe-emodin.All these outcomes suggested the suitability of the conventional analytical method for the simultaneous determination of rhein and aloe-emodin.However, the sustainable analytical method was more linear than the conventional analytical method.For assessing rhein and aloe-emodin, the %recovery was used to determine the accuracy of both assays.The accuracy data for the sustainable and conventional analytical assays are listed in Table 3.Using the sustainable analytical method, the %recoveries of rhein and aloe-emodin at three distinct QC levels were estimated to be 99.12-101.30%and 98.86-101.15%,respectively.Using the conventional analytical method, the %recoveries of rhein and aloe-emodin at three distinct QC levels were estimated to be 96.12-102.74 and 95.72-103.83%,respectively.These data indicated that both assays were accurate for the simultaneous determination of rhein and aloe-emodin.For the simultaneous determination of rhein and aloe-emodin, however, the sustainable analytical method was highly accurate over the conventional analytical method.The precision of both assays was determined as intra/inter-day variations and expressed as the % of the coefficient of variance (%CV).Table 4 lists the data of intra/inter-day variations for the simultaneous quantification of rhein and aloe-emodin utilizing the sustainable and conventional analytical assays.For the sustainable analytical assay, the %CVs of rhein and aloe-emodin for the intra-day variance were determined to be 0.79-0.95%and 0.76-0.84%,respectively.The %CVs of rhein and aloe-emodin for the inter-day precision were determined to be 0.81-1.02%and 0.80-0.93%,respectively.For the conventional analytical assay, the %CVs of rhein and aloe-emodin for the intra-day variance were determined to be 2.32-3.27%and 2.27-3.94%,respectively.The %CVs of rhein and aloe-emodin for the inter-day variance were determined to be 2.41-3.36 and 2.59-4.07%,respectively.These outcomes indicated that both were precise for the simultaneous determination of rhein and aloe-emodin.However, the sustainable analytical assay was highly precise over the conventional analytical assay for the simultaneous quantification of rhein and aloe-emodin.By introducing some planned changes in the mobile phase compositions, the robustness of both assays was determined.Table 5 lists the outcomes of robustness analysis using the sustainable and conventional analytical assays.For the sustainable assay, the %CVs for rhein and aloe-emodin were determined to be 1.00-1.05%and 0.93-1.02%,respectively.Rhein and aloe-emodin R f values were also determined to be 0.19-0.21and 0.85-0.87,respectively.
For the conventional method, the %CVs for rhein and aloe-emodin were determined to be 2.63-2.77%and 2.91-2.99%,respectively.Rhein and aloe-emodin R f values were also determined to be 0.52-0.54and 0.26-0.28,respectively.These findings suggested that both assays were robust for the simultaneous determination of rhein and aloe-emodin.For the simultaneous determination of rhein and aloe-emodin, however, the sustainable analytical assay was highly robust over the conventional analytical assay.
The LOD and LOQ values were used to determine the sensitivity of both assays.The LOD and LOQ for rhein and aloe-emodin using the sustainable and conventional analytical assay are listed in Table 1.Utilizing the sustainable analytical assay, the LOD and LOQ for rhein were determined to be 16.81 ± 0.18 and 50.53 ± 0.54 ng/spot, respectively.Utilizing the sustainable analytical assay, the LOD and LOQ for aloe-emodin were determined to be 8.49 ± 0.12 and 25.47 ± 0.36 ng/spot, respectively.Using the conventional analytical assay, the LOD and LOQ for rhein were determined to be 18.53 ± 0.23 and 55.59 ± 0.69 ng/spot, respectively.Utilizing the conventional analytical assay, the LOD and LOQ for aloe-emodin were determined to be 39.42 ± 0.62 and 118.26 ± 1.86 ng/spot, respectively.These findings showed that both assays were sensitive to the simultaneous determination of rhein and aloe-emodin.For the simultaneous determination of rhein and aloe-emodin, however, the sustainable analytical assay was highly sensitive to the conventional analytical assay.The specificity of the analytical assay for the simultaneous quantification of rhein and aloe-emodin was assessed by comparing the R f values and overlay UV spectra of rhein and aloe-emodin in the TE and UBE of commercial Rhubarb and Rhubarb plant extracts with those of standard rhein and aloe-emodin.The superimposed UV spectra of the standard rhein and aloe-emodin, as well as rhein and aloe-emodin in the TE and UBE of commercial Rhubarb and Rhubarb plant extracts, are included in Figure 5.At the wavelength of 257 nm, the highest HPTLC response of the rhein and aloe-emodin standards and the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were recorded.The identical UV spectra, R f values, and wavelengths of rhein and aloe-emodin standards and the TE and UBE of marketed Rhubarb and Rhubarb plant extract demonstrated the specificity of the analytical technique for the simultaneous measurement of rhein and aloe-emodin.
of 257 nm, the highest HPTLC response of the rhein and aloe-emodin standards and the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were recorded.The identical UV spectra, Rf values, and wavelengths of rhein and aloe-emodin standards and the TE and UBE of marketed Rhubarb and Rhubarb plant extract demonstrated the specificity of the analytical technique for the simultaneous measurement of rhein and aloe-emodin.

Application of Conventional and Sustainable Analytical Methods in the Simultaneous Analysis of Rhein and Aloe-Emodin in TE and UBE of Marketed Rhubarb and Rhubarb Plant Extracts
The sustainable HPTLC approach can be considered an alternative method of regular chromatographic assays for the simultaneous quantification of both compounds in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts.For the sustainable analytical assay, the densitograms of rhein and aloe-emodin from the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were identified by comparing their HPTLC band at Rf = 0.20 ± 0.01 for rhein and Rf = 0.86 ± 0.02 for aloe-emodin with those of standard rhein and aloe-emodin.For the conventional analytical assay, the densitograms of rhein and aloe-emodin from the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were identified by comparing their HPTLC band at Rf = 0.53 ± 0.02 for rhein and Rf = 0.27 ± 0.01 for aloe-emodin with those of standard rhein and aloe-emodin.The supplementary Figure S1 (Figure S1) shows HPTLC densitograms of rhein and aloe-emodin in the TE of commercial Rhubarb and Rhubarb plant extracts employing the sustainable analytical assay, which revealed identical peaks of rhein and aloe-emodin to those of standard rhein and aloe-emodin.In addition, four additional peaks were also detected in the TE of commercial Rhubarb and Rhubarb plant extracts.Figure 6 shows HPTLC densitograms of rhein and aloe-emodin in the UBE of commercial Rhubarb and Rhubarb plant extracts using the sustainable analytical assay, which also revealed identical rhein and aloeemodin peaks to those of standard rhein and aloe-emodin.In addition, five and four additional peaks were also detected in the UBE of commercial Rhubarb and Rhubarb plant

Application of Conventional and Sustainable Analytical Methods in the Simultaneous Analysis of Rhein and Aloe-Emodin in TE and UBE of Marketed Rhubarb and Rhubarb Plant Extracts
The sustainable HPTLC approach can be considered an alternative method of regular chromatographic assays for the simultaneous quantification of both compounds in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts.For the sustainable analytical assay, the densitograms of rhein and aloe-emodin from the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were identified by comparing their HPTLC band at R f = 0.20 ± 0.01 for rhein and R f = 0.86 ± 0.02 for aloe-emodin with those of standard rhein and aloe-emodin.For the conventional analytical assay, the densitograms of rhein and aloe-emodin from the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were identified by comparing their HPTLC band at R f = 0.53 ± 0.02 for rhein and R f = 0.27 ± 0.01 for aloe-emodin with those of standard rhein and aloe-emodin.The supplementary Figure S1 shows HPTLC densitograms of rhein and aloe-emodin in the TE of commercial Rhubarb and Rhubarb plant extracts employing the sustainable analytical assay, which revealed identical peaks of rhein and aloe-emodin to those of standard rhein and aloe-emodin.In addition, four additional peaks were also detected in the TE of commercial Rhubarb and Rhubarb plant extracts.Figure 6 shows HPTLC densitograms of rhein and aloe-emodin in the UBE of commercial Rhubarb and Rhubarb plant extracts using the sustainable analytical assay, which also revealed identical rhein and aloe-emodin peaks to those of standard rhein and aloe-emodin.In addition, five and four additional peaks were also detected in the UBE of commercial Rhubarb and Rhubarb plant extracts, respectively.Figure S2 shows HPTLC densitograms of rhein and aloe-emodin in the TE of commercial Rhubarb and Rhubarb plant extracts using the conventional analytical assay, which also revealed identical peaks of rhein and aloe-emodin to those of the standard rhein and aloe-emodin.In addition, three and four additional peaks were also detected in the TE of commercial Rhubarb and Rhubarb plant extracts, respectively.Figure 7 shows HPTLC densitograms of rhein and aloe-emodin in the UBE of commercial Rhubarb and Rhubarb plant extracts using the conventional analytical assay, which also revealed identical peaks of rhein and aloe-emodin to those of standard rhein and aloe-emodin.In addition, four additional peaks were also detected in the UBE of commercial Rhubarb and Rhubarb plant extracts.
extracts, respectively.Figure S2 shows HPTLC densitograms of rhein and aloe-emodin in the TE of commercial Rhubarb and Rhubarb plant extracts using the conventional analytical assay, which also revealed identical peaks of rhein and aloe-emodin to those of the standard rhein and aloe-emodin.In addition, three and four additional peaks were also detected in the TE of commercial Rhubarb and Rhubarb plant extracts, respectively.Figure 7 shows HPTLC densitograms of rhein and aloe-emodin in the UBE of commercial Rhubarb and Rhubarb plant extracts using the conventional analytical assay, which also revealed identical peaks of rhein and aloe-emodin to those of standard rhein and aloeemodin.In addition, four additional peaks were also detected in the UBE of commercial Rhubarb and Rhubarb plant extracts.The detection of extra peaks in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts demonstrated that both analytical methods can be efficiently utilized for the simultaneous analysis of rhein and aloe-emodin in the presence of different phytochemicals/impurities. For the sustainable and conventional HPTLC assays, the amounts of rhein and aloe-emodin in the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were calculated from the calibration curves of rhein and aloe-emodin and the results are summarized in Table 6.extracts, respectively.Figure S2 shows HPTLC densitograms of rhein and aloe-emodin in the TE of commercial Rhubarb and Rhubarb plant extracts using the conventional analytical assay, which also revealed identical peaks of rhein and aloe-emodin to those of the standard rhein and aloe-emodin.In addition, three and four additional peaks were also detected in the TE of commercial Rhubarb and Rhubarb plant extracts, respectively.Figure 7 shows HPTLC densitograms of rhein and aloe-emodin in the UBE of commercial Rhubarb and Rhubarb plant extracts using the conventional analytical assay, which also revealed identical peaks of rhein and aloe-emodin to those of standard rhein and aloeemodin.In addition, four additional peaks were also detected in the UBE of commercial Rhubarb and Rhubarb plant extracts.The detection of extra peaks in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts demonstrated that both analytical methods can be efficiently utilized for the simultaneous analysis of rhein and aloe-emodin in the presence of different phytochemicals/impurities. For the sustainable and conventional HPTLC assays, the amounts of rhein and aloe-emodin in the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were calculated from the calibration curves of rhein and aloe-emodin and the results are summarized in Table 6.The detection of extra peaks in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts demonstrated that both analytical methods can be efficiently utilized for the simultaneous analysis of rhein and aloe-emodin in the presence of different phytochemicals/impurities. For the sustainable and conventional HPTLC assays, the amounts of rhein and aloe-emodin in the TE and UBE of commercial Rhubarb and Rhubarb plant extracts were calculated from the calibration curves of rhein and aloe-emodin and the results are summarized in Table 6.
For the sustainable reverse-phase HPTLC assay, the amounts of rhein in the TE of commercial Rhubarb and Rhubarb plant extracts were found to be 2.78 ± 0.102 mg/g and 2.51 ± 0.092 mg/g, respectively.However, the amounts of rhein in the UBE of commercial Rhubarb and Rhubarb plant extracts were determined to be 4.33 ± 0.110 mg/g and 3.81 ± 0.101 mg/g, respectively.The amounts of aloe-emodin in the TE of marketed Rhubarb and Rhubarb plant extracts were found to be 4.61 ± 0.112 mg/g and 3.84 ± 0.108 mg/g, respectively.However, the amounts of aloe-emodin in the UBE of marketed Rhubarb and Rhubarb plant extracts were determined to be 5.74 ± 0.121 mg/g and 4.71 ± 0.114 mg/g, respectively.For the conventional normal-phase HPTLC assay, the amounts of rhein in the TE of marketed Rhubarb and Rhubarb plant extracts were found to be 0.016 ± 0.001 mg/g and 0.008 ± 0.000 mg/g, respectively.However, the amounts of rhein in the UBE of marketed Rhubarb and Rhubarb plant extracts were determined to be 0.036 ± 0.003 mg/g and 0.026 ± 0.002 mg/g, respectively.The amounts of aloe-emodin in the TE of marketed Rhubarb and Rhubarb plant extracts were found to be 0.523 ± 0.005 mg/g and 0.461 ± 0.004 mg/g, respectively.However, the amounts of aloe-emodin in the UBE of marketed Rhubarb and Rhubarb plant extracts were determined to be 0.609 ± 0.08 mg/g and 0.504 ± 0.006 mg/g, respectively.Compared to the conventional normal-phase HPTLC assay, amounts of rhein and aloe-emodin in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts were found significantly higher using the sustainable reverse-phase HPTLC assay (p < 0.05).In addition, the UBE procedure showed significantly higher amounts of rhein and aloe-emodin compared to their TE procedure p < 0.05).As a consequence, the UBE procedure for the extraction of rhein and aloe-emodin is preferred superior to the TE procedure.Based on all these results and observations, the sustainable HPTLC assay has been considered superior to the conventional normal-phase HPLTC assay for the simultaneous analysis of both compounds in their TE and UBE.

Greenness Assessment Using AGREE
Different approaches have been documented for the greenness assessment of analytical methods [26][27][28][29][30].However, only AGREE uses all twelve principles of GAC for the greenness assessment [28].As a consequence, the greenness of both analytical methods was determined by the AGREE method.The representative pictograms for the AGREE indices of the sustainable reverse-phase and conventional normal-phase HPTLC assays are presented in Figure 8.The AGREE indices for both assays were predicted by assigning different scoring systems.Based on the current experiments, different scores were assigned to twelve different components, such as sample treatment, sample amount, device positioning, sample preparation stages, degree of automation, derivatization, waste, analysis throughput, energy consumption, sources of solvents/reagents, toxicity, and operator's safety.Finally, the average AGREE index was calculated for both methods.The AGREE indices were predicted to be 0.78 and 0.49 for the sustainable reverse-phase and conventional normal-phase HPTLC assays, respectively.These findings suggested the excellent greenness profile of the sustainable reverse-phase HPTLC assay compared to the conventional normal-phase HPTLC assay for the simultaneous analysis of both compounds.ysis throughput, energy consumption, sources of solvents/reagents, toxicity, and operator's safety.Finally, the average AGREE index was calculated for both methods.The AGREE indices were predicted to be 0.78 and 0.49 for the sustainable reverse-phase and conventional normal-phase HPTLC assays, respectively.These findings suggested the excellent greenness profile of the sustainable reverse-phase HPTLC assay compared to the conventional normal-phase HPTLC assay for the simultaneous analysis of both compounds.

Conclusions
In the literature, there are scarce of sustainable analytical assays for the simultaneous analysis of rhein and aloe-emodin.As a consequence, compared to the conventional normal-phase HPTLC assay, the present study was performed to design and validate an accurate, precise, robust, sensitive, and sustainable reversed-phase HPTLC assay for the simultaneous determination of rhein and aloe-emodin in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts.For the simultaneous analysis of both compounds, the sustainable analytical assay is more linear, accurate, precise, robust, and sensitive than the conventional analytical assay.The amounts of rhein and aloe-emodin in marketed Rhubarb and Rhubarb plant extracts were significantly higher using the sustainable analytical assay compared to the conventional analytical assay.The UBE procedure for the extraction of rhein and aloe-emodin has been considered superior to the TE procedure.The AGREE indices presented the excellent greenness nature of the sustainable analytical assay compared to the conventional analytical assay.For the simultaneous analysis of both compounds in commercial Rhubarb and Rhubarb plant extracts, the sustainable HPTLC assay has been found superior to the conventional HPTLC assay based on these results.In addition, because of the application of green solvent systems against the commonly used hazardous solvents for the simultaneous quantification of rhein and aloeemodin, the developed assay is also safe and sustainable compared to reported analytical methods.

Conclusions
In the literature, there are scarce of sustainable analytical assays for the simultaneous analysis of rhein and aloe-emodin.As a consequence, compared to the conventional normal-phase HPTLC assay, the present study was performed to design and validate an accurate, precise, robust, sensitive, and sustainable reversed-phase HPTLC assay for the simultaneous determination of rhein and aloe-emodin in the TE and UBE of marketed Rhubarb and Rhubarb plant extracts.For the simultaneous analysis of both compounds, the sustainable analytical assay is more linear, accurate, precise, robust, and sensitive than the conventional analytical assay.The amounts of rhein and aloe-emodin in marketed Rhubarb and Rhubarb plant extracts were significantly higher using the sustainable analytical assay compared to the conventional analytical assay.The UBE procedure for the extraction of rhein and aloe-emodin has been considered superior to the TE procedure.The AGREE indices presented the excellent greenness nature of the sustainable analytical assay compared to the conventional analytical assay.For the simultaneous analysis of both compounds in commercial Rhubarb and Rhubarb plant extracts, the sustainable HPTLC assay has been found superior to the conventional HPTLC assay based on these results.In addition, because of the application of green solvent systems against the commonly used hazardous solvents for the simultaneous quantification of rhein and aloe-emodin, the developed assay is also safe and sustainable compared to reported analytical methods.

Figure 3 .
Figure 3. Developed thin-layer chromatography (TLC) plate for standard rhein, standard aloeemodin, and Rhubarb plant extract samples developed using ethanol/water (60:40 v/v) as the sustainable mobile phase for the sustainable reverse-phase HPTLC assay.

Figure 3 .
Figure 3. Developed thin-layer chromatography (TLC) plate for standard rhein, standard aloeemodin, and Rhubarb plant extract samples developed using ethanol/water (60:40 v/v) as the sustainable mobile phase for the sustainable reverse-phase HPTLC assay.

Figure 4 .
Figure 4. Representative calibration curves of standard rhein and aloe-emodin obtained using (A) sustainable reverse-phase HPTLC and (B) conventional normal-phase HPTLC methods.

Figure 6 .
Figure 6.Sustainable reverse-phase HPTLC densitograms of rhein and aloe-emodin in (A) UBE of marketed Rhubarb extract and (B) UBE of Rhubarb plant extract.

Figure 7 .
Figure 7. Conventional normal-phase HPTLC densitograms of rhein and aloe-emodin in (A) UBE of marketed Rhubarb extract and (B) UBE of Rhubarb plant extract.

Figure 6 .
Figure 6.Sustainable reverse-phase HPTLC densitograms of rhein and aloe-emodin in (A) UBE of marketed Rhubarb extract and (B) UBE of Rhubarb plant extract.

Figure 6 .
Figure 6.Sustainable reverse-phase HPTLC densitograms of rhein and aloe-emodin in (A) UBE of marketed Rhubarb extract and (B) UBE of Rhubarb plant extract.

Figure 7 .
Figure 7. Conventional normal-phase HPTLC densitograms of rhein and aloe-emodin in (A) UBE of marketed Rhubarb extract and (B) UBE of Rhubarb plant extract.

Figure 7 .
Figure 7. Conventional normal-phase HPTLC densitograms of rhein and aloe-emodin in (A) UBE of marketed Rhubarb extract and (B) UBE of Rhubarb plant extract.

Table 1 .
Outcomes of linearity evaluation for the simultaneous quantification of rhein and aloeemodin utilizing the sustainable and conventional analytical assays (mean ± SD; n = 6).

Table 1 .
Outcomes of linearity evaluation for the simultaneous quantification of rhein and aloeemodin utilizing the sustainable and conventional analytical assays (mean ± SD; n = 6).

Table 2 .
System suitability parameters of rhein and aloe-emodin for the sustainable and conventional analytical methods (mean ± SD; n = 3).

Table 3 .
Accuracy data of rhein and aloe-emodin for the sustainable and conventional analytical assays (mean ± SD; n = 6).
CV: coefficient of variance.

Table 4 .
Intra/inter-day precision data of rhein and aloe-emodin for the sustainable and conventional analytical assays (mean ± SD; n = 6).
CV: coefficient of variance.

Table 5 .
Robustness data of rhein and aloe-emodin for the sustainable and conventional analytical assays (mean ± SD; n = 6).
CV: coefficient of variance; R f : retardation factor.

Table 6 .
Application of sustainable and conventional analytical methods in simultaneous analysis of rhein and aloe-emodin in traditional extract (TE) and ultrasound-based extract (UBE) of commercial Rhubarb and Rhubarb plant extract (mean ± SD; n = 3).