Citation: Thajudeen, K.Y.; Alsayari,

A.; Najib Ullah, S.N.M.; Salam, S.;

Elayadeth-Meethal, M.; Uoorakkottil,

I. Validation, Optimization and

Hepatoprotective Effects of

Boeravinone B and Caffeic Acid

Compounds from Boerhavia diffusa

Linn. Separations 2022, 9, 177. https://

doi.org/10.3390/separations9070177

Academic Editor: Liangliang Liu

Received: 24 May 2022

Accepted: 26 June 2022

Published: 18 July 2022

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separations

Article

Validation, Optimization and Hepatoprotective Effects ofBoeravinone B and Caffeic Acid Compounds fromBoerhavia diffusa LinnKamal Y. Thajudeen 1, Abdulrhman Alsayari 1, Shehla Nasar Mir Najib Ullah 1, Shahana Salam 2,Muhammed Elayadeth-Meethal 3 and Ilyas Uoorakkottil 4,*

1 Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha 62529, Saudi Arabia;kthajudeen@kku.edu.sa (K.Y.T.); alsayarikku@gmail.com (A.A.); sajibullah@kku.edu.sa (S.N.M.N.U.)

2 Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University,Al-kharj 11942, Saudi Arabia; s.salam@psau.edu.sa

3 Department of Animal Breeding and Genetics, College of Veterinary and Animal Sciences,Kerala Veterinary and Animal Sciences University, Wayanad 673576, Kerala, India;dremmuhammed@gmail.com

4 Department of Pharmacognosy and Phytochemistry, Moulana College of Pharmacy,Perinthalmanna 679321, Kerala, India

* Correspondence: dr.ilyasmcp@gmail.com

Abstract: Boerhavia diffusa, also known as Punarnava, is a plant of the Nyctaginaceae family that hasbeen utilized in traditional medicine to cure a variety of ailments. The goal of this study was to useresponse surface methodology (RSM) to optimize the maximum percentage yield of boeravinone Band caffeic acid from Boerhavia diffusa roots, and simultaneous determination of boeravinone B andcaffeic acid in newly developed single solvent system and demonstrate the hepatoprotective benefitsof boeravinone B and caffeic acid. The extraction process examined extraction time, extraction tem-perature and solvent concentration, which were optimized via Box–Behnken experimental design.The proposed HPTLC method for the quantification of boeravinone B and caffeic acid were success-fully validated and developed. The method was validated in term of linearity and detection limit,quantification limit, range, precision, specificity and accuracy. The separation of boeravinone B andcaffeic acid bands was achieved on HPTLC plate using formic acid: ethyl acetate: toluene (1:3:5 v/v)as developing system. Densitometric analyses of boeravinone B and caffeic acid was carried out in theabsorbance mode at 254 nm. The maximum percentage yield of caffeic acid and boeravinone B fromBoerhavia diffusa require appropriate extraction parameters such as temperature, time, organic solventsand water content, which can be achieved using the Box-Behnken statistical design provide time:temperature: solvent ratio (30:45:40 v/v) for extraction of caffeic acid and 60:60:40 v/v for extractionof boeravinone B. The boeravinone B (200 µg/mL) and caffeic acid (200 µg/mL) showed the mostsignificant hepatoprotective activity compared with standard sylimarin in HepG2 cell induced withgalactosamine 40 mM toxicity. The findings supported B. diffusa’s traditional use as a functional foodforhuman health benefits.

Keywords: Boerhavia diffusa; boeravinone B; caffeic acid; optimization; hepatoprotective activity;HPTLC

1. Introduction

Boerhavia diffusa has been traditionally used by Indian and Brazilian indigenous andtribal people. Ayurvedic medicine traditionally uses the plant’s root and leaves to treat awide range of ailments, such as viral jaundice and liver dysfunction. This plant isreported tohave behavioral and neuroendocrine effect and anti-depressant activity [1], anti-angiogenicactivity [2], anti-convulsant and anti-epileptic activity [3], anti-inflammatory activity [4], in-hibitory effect of prostatic hyperplasia [5], anti-genotoxic effect [6], thrombolytic, cytotoxic

Separations 2022, 9, 177. https://doi.org/10.3390/separations9070177 https://www.mdpi.com/journal/separations

Separations 2022, 9, 177 2 of 12

and anti-microbial activities [7], arsenic trioxide-induced cardiotoxicity [8], anti-urolithicactivity [9], anti-hyperglycaemic and reno-protective effect [10], acetaminophen-inducedliver toxicity [11], anti-proliferative and anti-estrogenic effects [12], immunomodulatoryand anti-metastatic activity [13,14], radio-protective activity [15], potent anti-breast can-cer activity [16], intestinal activity [17], immunosuppressive activity [18], anti-diabeticactivity [19], Ca2+ channel antagonistic activity [20], chemo-preventive action [21], glycol-induced urolithiasis [22], and inhibition of human cervical cancer cells [23]. Main Rotenoidsarea type isoflavone compound (known as boeravinones) that are isolated from the rootsof this plant; they showed potent anti-cancer [24,25], anti-inflammtory [26], cardioprotec-tive [27] powerful anti-oxidant and geno-protective properties [28], intestinal motility andspasmolytic activity [29]. Caffeic acid (3,4-dihydroxycinnamic acid) is one of the majorpolyphenolic compounds found in many plant species, showing anti-viral activity [30], im-munostimulatory activity [31], cardioprotective activity [32], hepatoprotective activity [33],anti-cancer activity [34] and anti-hepatocellular carcinoma activity [35]. For detectingphenolic compounds in medicinal plants, several analytical techniques such as liquidchromatography–mass spectrometry (LCMS), high-performance liquid chromatography(HPLC), high-performance thin layer chromatography (HPTLC)and ultra-performanceliquid chromatography (UPLC). Boeravinone B and caffeic acid are rarely applied by useof their similar analytical techniques. Ilyas et al. in 2013 reported only identificationand quantification of caffeic acid and boeravinone in single-solvent system toluene: ethylacetate: formic acid: methanol (3:3:0.8:0.2 v/v) but tailing peak and separation and res-olution of band of caffeic acid and boeravinone was very poor [36]. Here, a validatedhigh-performance thin layer chromatography (HPTLC) method has been newly devel-oped based on single solvent system (toluene: ethyl acetate: formic acid (5:4:1 v/v) forsimultaneous validation, quantification and optimization of boeravinone B and caffeicacid in the plants. The selected HPTLC method is accurate, precise, simple, specific, lesstime-consuming, cost-effective and can separate boeravinone B and caffeic acid compoundsfrom their other constituents. Because of advantages such as ease of sample preparation,optimization of specific chemicals and comparison of several samples on a single plate,comparable chromatographic techniques are frequently used to evaluate retinoid and phe-nolic acids. The comparison of the entire chromatograms allows for the detection of minorsimilarities and differences between the plants under investigation. The objective of thestudy was to simultaneously quantify, validate, optimize and to evaluate hepatoprotectiveactivity of boeravinone B and caffeic acid in hydroalcoholic extracts of B. diffusa.

2. Materials and Methods2.1. Chemicals and Instruments

We purchased standard boeravinone B and caffeic acid from NRPL, Bangalore, India.Ethyl acetate, toluene, methanol and formic acid (CDH Labs, Mumbai, India) were usedas developing systems for HPTLC analysis. All samples and standards used for the anal-ysis were filtered through a membrane filter, 0.22 µm pore size (HIMEDIA, Mumbai,India). Extraction method using a Soxhlet apparatus (Omega, Mumbai, India) and Rotaryevaporator (Buchi R-114, Switzerland). CAMAG HPTLC system (Muttenz, Switzerland)equipped with a Linomat IV sample applicator was used for quantification and optimiza-tion. HepG2 cell line (National Centre for Cell Science (NCCS), Pune, India), Dulbecco’smodified eagle medium (DMEM), fetal bovine serum (FBS) and MTT assay kit, trypsinEDTA, penicillin and streptomycin and DMSO (Sigma-Aldrich Co. LLC, St. Louis, MO,USA), Trypan blue solution and, galactosamine and absolute ethanol (HimediaLab Pvt.Ltd., Mumbai, India).Tissue culture flasks, 96- and 24-well microculture plates, eppendorftube, inverted microscope, serological pipette, heamocytometer (HimediaLab Pvt. Ltd.,Mumbai, India), laminar flow hoods (Khera instrument, New Delhi, India), CO2 incubator(NuAire, Plymouth, MN, USA), water bath, Deepfreezer (−20 ◦C).

Separations 2022, 9, 177 3 of 12

2.2. Collection and Extraction of Plant Materials

In February 2011, we collected the roots of B. diffusa in Maruthmallai, Kanyakumari dis-trict, Tamilnadu, India. It was identified and authenticated by Dr. V. Chelladurai, ResearchOfficer, Central Council for Research in Ayurveda and Siddha (Govt. of India), Tirunelveli,Tamil Nadu. The dried roots were grinded into a coarse powder and subjected to extractionby using 95% methanol and 50% (v/v) hydro-alcohol for 6 h at 37 ◦C, respectively. Themethanol and hydro-alcoholic extracts were evaporated and dried at 45 ◦C with help ofrotary evaporator under reduced pressure. The yield of the extract prepared with 95%methanol was 5.29% weight-for-weight, while the extract prepared with hydro-alcoholicalcohol yielded 9.9% weight-for-weight.

2.3. HPTLC Instrumentation2.3.1. Preparation of Sample Solution

A total of 100 mg of extracts were dissolved in 10 mL of methanol and sonicated thesolution for 15 min and then makeup with 10 mL HPLC grade methanol, filtered through amembrane filter, 0.22 µm pore size before injecting into the HPTLC system.

2.3.2. Preparation of Plate

Prior to usage, pre-coated silica gel 60 F254 aluminum TLC plates (Merck, Germany)were washed in methanol and dried. The samples were applied in the form of bands of4 mm width using Linomat V applicator (Muttenz, Switzerland) with a 100 µL syringe.The application rate was kept constant at 200 nL s−1, and the space between the two bandswas 9 mm.

2.3.3. Slit and Scanning Speed

The slit size was fixed at 5 0.45 mm, and the scanning speed was set to 5 mm s–1.

2.3.4. Chromatographic Conditions

The mobile phase was 1:3:5 v/v/v formic acid, ethyl acetate, and toluene and thedevelopment volume was20 mL. In a 10 × 10 cm twin-trough glass chamber saturated withthe mobile phase, linear ascending development was performed. At room temperature,the optimal chamber saturation time for the mobile phase was 20 min. The chromatogramrun was 8.5 cm long, and the TLC plates were dried in an oven at 50 ◦C for 5 min afterdevelopment. CAMAG TLC Scanner 3 in absorbance-reflectance mode at 254 nm, operatedby Win-CATS software, was used for densitometric scanning. The deuterium lamp wasused as a source of radiation. The peak area response was compared to the amount ofmedication in a linear regression.

2.3.5. Preparation of Standards

The standard solution of bioactive compounds was prepared by dissolving 1.0 mg ofboeravinone B and caffeic acid in 10 mL of methanol as stock solution and stored at 2 to8 ◦C. These boeravinone B and caffeic acid were further diluted as per the requirement toplot a final concentration for quantification.

2.3.6. Analytical Method Evaluation

The lowest diluted solutions of the two reference compounds in the calibration curveswere further diluted to a series of concentrations with HPLC grade methanol for thedeterminations of limits of detection (LOD) and quantification (LOQ). The LOD and LOQunder the present chromatographic conditions were determined at a signal-to-noise (S/N)ratio of 3 and 10, respectively. Calibration curves, regression equation along with LOD andLOQ for each compound have been validated as per the ICH guidelines Q2 (R1) (2005).Linearity was assessed with the aid of serially diluted calibration solutions of all standards.Calibration graphs were plotted on the basis of triplicate analysis of each calibrationsolutions by using peak area against concentration. To evaluate the accuracy, the pre-

Separations 2022, 9, 177 4 of 12

analyzed samples were spiked with standard at three different known concentration levelsi.e., 50, 100 and 150% and the mixtures were re-analyzed by the proposed method. Precisionof the method was determined by carrying out the intra-day and inter-day variation tests.The inter-day and intra-day variations for the determination of boeravinone B and caffeicacid were carried out at different levels of concentrations 100, 200, 400 ng/band. Theidentification of bands was carried out in triplicate. The R.S.D. was taken as a measureof precision. The resolution between the peaks of caffeic acid and Boeravinone B foundsatisfactory (2.1 ± 0.2). The developed method resolution found to be good enough toavoid any possible merging between the peaks. Satisfactory recovery values suggest thatthere was no interference happening between the standard peaks.

2.4. Optimization of Boeravinone B and Caffeic Acidin Hydroalcoholic Extracts

Using the Box-Behnken statistical design, three factors and levels were considered.There were seventeen runs of it. To optimize the design, the Stat-Ease V6 software (Min-neapolis, MN, USA) was used (Minneapolis, Stat-Ease Inc., MN, and USA). This style issuitable for investigating constructing second-order polynomial models and quadraticresponse surfaces. The designed style contains the replicated center point of the four-dimensional cube and a group of purposes lying at the center of every edge that confirmedthe region of interest. The dependent and independent variables are recorded in Table 1.

Table 1. Independent and dependent variables selected in Box–Behnken design.

Factors Independent Variables Levels UsedLow (−1) Medium High (+1)

B1 = Time in min 30 60 90B2 = Temperature (◦C) 30 45 60B3 = Solvent concentration (v/v) 40 60 80Dependent variables

C1 = Boeravinone B Maximized percentage yieldC2 = Caffeic acid Maximized percentage yield

The quadratic equation created by the Box–Behnken Design-Expert equation

R = A0 + A1 B1 + A2 B2 + A3 B3 + A4 B1 B2 + A5 B1 B3 + A6 B2 B3 + A7B12 + A8B2

2 + A9B32 (1)

where A0 is the intercept, A1 to A9 are the regression coefficients, R is the dependentvariable and B1, B2 and B3 are the independent variables.

The data of the maximum percentage yield of boeravinone B and caffeic acid in thehydro-alcoholic extract of plant materials were recorded through arithmetical improvementby using Design-Expert software system. The Box–Behnken Design style spreadsheet isshown in Table 2.

Table 2. Mentioned responses in Box–ehnken design experiment for 17 analytical trails.

RunFactor-1 (A1):Time (Min)

Factor-2(A2):Temperature (◦C)

Factor-3 (A3): Solvent Concentration % YieldBoeravinone B (C1)

% Yield CaffeicAcid(C2)Methanol (%) Water Content (%)

01 30 30 60 40 0.044 0.01102 90 30 60 40 0.048 0.01803 30 60 60 40 0.045 0.01704 90 60 60 40 0.039 0.01705 30 45 40 60 0.050 0.03606 90 45 40 60 0.027 0.04807 30 45 80 20 0.011 0.04608 90 45 80 20 0.032 0.038409 60 30 40 60 0.036 0.041

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Table 2. Cont.

RunFactor-1 (A1):Time (Min)

Factor-2(A2):Temperature (◦C)

Factor-3 (A3): Solvent Concentration % YieldBoeravinone B (C1)

% Yield CaffeicAcid(C2)Methanol (%) Water Content (%)

10 60 60 40 60 0.047 0.06811 60 30 60 40 0.035 0.06712 60 60 80 20 0.015 0.04313 60 45 60 40 0.0086 0.06614 60 45 60 40 0.0085 0.06515 60 45 60 40 0.0086 0.06616 60 45 60 40 0.0085 0.06517 60 45 60 40 0.0085 0.066

2.5. The Hepatoprotective Activity of Caffeic Acid and Boeravinone B

Cells were seeded at a density of 1 × 105 cells/ in each well of the 24-well plates andincubated overnight. After 24 h, the media was flicked off, the cells were treated withboeravinone B and caffeic acid (100 and 200 µg/mL) in separate wells of a 24-well plateand incubated for 2 h. Silymarin (250, 500 µg/mL) was used as a reference standard. Afterincubation, the cell was treated with D-galactosamine at a concentration of 40 mM andwas allowed to incubate for 2 h. After incubation, the cells were washed and incubatedfor 1 h with MTT (20 µL of 5 mg/mL of MTT in PBS) was added to each well. Formationof formazan crystal was observed under a microscope after 2 h. In case crystal, formationwas not proper, incubation was continued for an hour more. Media was removed andthe remaining formazan crystals were dissolved in 200 µLof DMSO in each well. The cellculture plate was kept on a shaker for 15 min; the absorbance was recorded by ELISAreader at 540 nm.

The % hepatoprotection of boeravinone B, caffeic acid and silymarin was obtained bythe following formula:

% Hepatoprotection = (Optical Density of Test sample / Optical Density of Control) × 100

3. Results3.1. Development of Mobile Phase

Separation of standard markers studies were carried out on the working standardsolution of boeravinone B and caffeic acid in HPLC-grade methanol. Originally, manytrials were established with different developing systems such as ethyl acetate: chloroform(5:5), ethyl acetate: chloroform (7:3), ethyl acetate: toluene (7:3), ethyl acetate: toluene (5:5).Finally, toluene: ethyl acetate: formic acid (5:4:1 v/v) for simultaneous quantification andoptimization of boeravinone B and caffeic acid gave a well-defined and sharp peak. Weobtained sharp bands upon saturating the twin trough plate development chamber withthe developing system at room temperature for 30 min. The band of boeravinone B andcaffeic acid was recorded on HPTLC plate scanned at 254 nm (Figure 1).

Separations 2022, 9, x FOR PEER REVIEW 6 of 13

Figure 1. HPTLC densitogram of caffeic acid (Rf = 0.58), boeravinone B (Rf = 0.71) in formic acid: ethyl acetate: toluene (1:3:5 v/v) presented on HPTLC plate scaned at 254 nm.

3.2. Method of Validation Figure 1 shows a representative chromatogram of boeravinone b and caffeic acid in

the established HPTLC technique. The HPTLC chromatogram in Figure 1 shows a reten-tion factor of caffeic acid (Rf = 0.58), boeravinone B (Rf = 0.71), respectively. The calibration curve’s linear regression data revealed a good linear relationship throughout a concentra-tion range of 60 to 240 µg mL−1 with a correlation coefficient (R2) value of 0.992 indicated acceptable linearity for caffeic acid and 50 to 250 µg mL−1 with a correlation coefficient (R2) value of 0.98.91 indicated acceptable linearity for boeravinone B (Table 2). Correlation coefficient is a statistical tool used to measure the strength or degree of this relationship, and, here, a high correlation coefficient value (a value extremely close to 1.0) indicates a high level of linear relationship between the peak area and concentration of caffeic acid and boeravinone B. The LOD and LOQ were calculated using the ICH Guidelines Q2 (R1) (2005) and were 50 ng, 200 ng for boeravinone B and 40 ng, 200 ng for caffeic acid, respec-tively (Table 3). The intraday and interday assay was used to determine the repeatability of the proposed HPTLC method, and the results was expressed in the term of %RSD (Ta-ble 3) showed that the repeatability of method. The developed HPTLC method’s outstand-ing precision was suggested by the low-percent RSD for repeatability and intermediate precision. The specificity of the developed HPTLC method for the analysis of boeravinone B and caffeic acid in the hydroalcoholic extract was recorded by comparing the spectra obtained in the standards and sample analyses. These spectra’s peak start, peak apex, and peak end positions were identical. The recovery trials were done to see how sensitive the method for estimating caffeic acid and boeravinone B. The standard addition approach was used to increase the concentration of caffeic acid and boeravinone B in the sample extract by 50%, 100%, and 150%. The percentage recoveries of the three concentrations ranged from 98.91% to 101.11% for boeravinone B and 99.91% to 100.71% for caffeic acid, indicating a high level of precision. The system suitability parameters of HPLC to estab-lish that the total system operated successfully, system suitability criteria such as peak symmetry, resolution (Rs), capacity factor (K), and selectivity was tested. As demon-strated in Table 3, the obtained values were within acceptable ranges.

Figure 1. HPTLC densitogram of caffeic acid (Rf = 0.58), boeravinone B (Rf = 0.71) in formic acid:ethyl acetate: toluene (1:3:5 v/v) presented on HPTLC plate scaned at 254 nm.

Separations 2022, 9, 177 6 of 12

3.2. Method of Validation

Figure 1 shows a representative chromatogram of boeravinone b and caffeic acidin the established HPTLC technique. The HPTLC chromatogram in Figure 1 shows aretention factor of caffeic acid (Rf = 0.58), boeravinone B (Rf = 0.71), respectively. Thecalibration curve’s linear regression data revealed a good linear relationship throughouta concentration range of 60 to 240 µg mL−1 with a correlation coefficient (R2) value of0.992 indicated acceptable linearity for caffeic acid and 50 to 250 µg mL−1 with a correlationcoefficient (R2) value of 0.98.91 indicated acceptable linearity for boeravinone B (Table 2).Correlation coefficient is a statistical tool used to measure the strength or degree of thisrelationship, and, here, a high correlation coefficient value (a value extremely close to 1.0)indicates a high level of linear relationship between the peak area and concentrationof caffeic acid and boeravinone B. The LOD and LOQ were calculated using the ICHGuidelines Q2 (R1) (2005) and were 50 ng, 200 ng for boeravinone B and 40 ng, 200 ng forcaffeic acid, respectively (Table 3). The intraday and interday assay was used to determinethe repeatability of the proposed HPTLC method, and the results was expressed in theterm of %RSD (Table 3) showed that the repeatability of method. The developed HPTLCmethod’s outstanding precision was suggested by the low-percent RSD for repeatabilityand intermediate precision. The specificity of the developed HPTLC method for the analysisof boeravinone B and caffeic acid in the hydroalcoholic extract was recorded by comparingthe spectra obtained in the standards and sample analyses. These spectra’s peak start,peak apex, and peak end positions were identical. The recovery trials were done to seehow sensitive the method for estimating caffeic acid and boeravinone B. The standardaddition approach was used to increase the concentration of caffeic acid and boeravinone Bin the sample extract by 50%, 100%, and 150%. The percentage recoveries of the threeconcentrations ranged from 98.91% to 101.11% for boeravinone B and 99.91% to 100.71%for caffeic acid, indicating a high level of precision. The system suitability parameters ofHPLC to establish that the total system operated successfully, system suitability criteriasuch as peak symmetry, resolution (Rs), capacity factor (K), and selectivity was tested. Asdemonstrated in Table 3, the obtained values were within acceptable ranges.

Table 3. Method validation parameters for boeravinon b and caffeic acid in B. diffusa.

Parameters Boeravinone B Caffeic Acid

LOD (ng) 50 40LOQ (ng) 200 200Resolution (Rs) 2.11 ± 0.74 1.93 ± 1.2Capacity factor (k’) 0.31 0.24Selectivity (α) 1.1 1.17Tailing factor 1 1Specificity Specific SpecificInterdayprecision (%RSD) 0.93 0.76Intraday precision (%RSD) 0.99 0.79Regression equation Y = −204.4 + 8.6X Y = −484.7X + 11.2Average recovery % 98.91 ± 1.2 99.91 ± 0.81Rf values 0.76 0.63

3.3. Box–Behnken Design-Experiment

A three-level factorial, Box–Behnken Design-Expert applied statistical modeling ex-perimental style was performed by applying 17 investigational trial runs, as recorded inTable 2. Standard boeravinone B and caffeic acid determined that the preferred model wasthe squared model and also the equivalent values of standard deviation and percentagecoefficient of variation for the various planned models is listed in Table 4 together withthe regression equations typically for ultimately elite responses. Only statically significant(p < 0.0001) for boeravinone B and caffeic acid respectively coefficient is enclosed in theequations. A positive value indicated the optimization of boeravinone B and caffeic acid in

Separations 2022, 9, 177 7 of 12

hydroalcoholic extracts, while a negative value represents an inverse relationship betweenthe response and the factor. It is clear from the equations that the factor time (B1), as wellas temperature (B2) has an uncooperative effect and 40:60:80 v/v solvent ratios (B3) havea productive on the responses (C1 and C2,). It also indicates that the correlation betweenfactors and response is not always linear. When more than one factor is replaced at thesame time, a factor can represent a various degree of response. Relationships of B1 and B2,as well as B1 and B3, induce uncooperative impacts on the response. However, the resultin the case of the square root of various factors does not repeat history, as shown by itsperformance. In the case of the square root of various factors, B2

2 (extraction temperature)and B2

3 (solvent concentration) produced positive results, and B21 (time of extraction)

showed the negative impact on the responses in Table 4. The last mixture ratios of theextractions were established based on percentage yield of boeravinone B and caffeic acid.

Table 4. Summary of results of regression analysis for models and responses (Y1-Y2) regressionequations for Quadratic models.

Models F Value R2 Adjusted R2 Predicted R2 SD C.V.%

Boeravinone B (Y1)

Linear 0.1218 −0.0808 −0.4134 0.018 -2F1 0.2745 −0.1609 −0.9616 0.018 -Cubic 0.9899 0.9598 - 0.0034 -Quadratic 0.9899 0.9769 0.9836 0.0026 9.48

Caffeic acid (Y2)

Linear 0.0033 −0.2267 −0.8001 0.023 -2F1 0.1187 −0.4101 −2.3379 0.024 -Cubic 0.9999 0.9995 - 0.00045 -Quadratic 0.9995 0.9989 0.9938 0.00069 1.52

3.4. Response of Independent Factors on Boeravinone B (C1)

Regression equation of the fitted quadratic models:

Boeravinone B = 0.00733 + 0.000075 B1−0.0023 B2 0.00812 B3 − 0.0029 B1 B2 +0.011 B1 B3 + 0.0078 B2 B3 + 0.017 B1

2 + 0.021 B22 + 0.0054 B3

2 . . . .(2)

Factor B1, which is the time (min) for extraction of plant materials used indicated thepercentage yield of boeravinone B in productive direction to that produced with factor B2.The productive coefficient composition of factor A produced the increase in percentageyield of boeravinone B, which improves in factor B1. It was confirmed that, when animproved time of extraction was applied, a higher concentration of the percentage ofboeravinone B was obtained (Figure 2A) shows the results of time in min of extraction onboeravinone B percentage yield.

Factor B2 (extraction temperature) was found to have an uncooperative result on theboeravinone B percentage yield compared to factor B3 (solvent concentration). As the factorB2 (temperature of extraction) level improved, demonstrated a positive impact on the per-centage yield of boeravinone B (Figure 2B), showing that the extraction temperature shouldbe increased to 60 degrees Celsius to get the highest percentage yield of boeravinone B.

In contrast to Factor B3 (extraction temperature), which indicated the maximumpercentage yield of boeravinone B, factor B3 (solvent concentration (water: methanol))produced a favourable impact on the percentage yield of boeravinone B. Boeravinone Bconcentration increased when a higher solvent concentration ratio was used (Figure 2C),demonstrating the influence of solvent ratio levels on boeravinone B percentage yield.

Separations 2022, 9, 177 8 of 12Separations 2022, 9, x FOR PEER REVIEW 9 of 13

Figure 2. Response surface plots of factor B2 vs. B1 against boeravinone B (C1) and caffeic acid (C2) respectively. (A) when an improved time of extraction was applied, higher concentrations of per-centage of boeravinone B were obtained; (B) as the factor temperature level improved, the boeravi-none B percentage yield also showed an uncooperative automatic impact; (C) when an elevated level of solvent ratio was applied, boeravinone B concentration also produced uncooperative im-pact; (D) when an increase time of extraction was applied, an elevated level of percentage yield of caffeic acid was investigated due to elevated level of penetration of solvent into plant extracts; (E) when the level of temperature increases, the sharp percentage yield of caffeic acid conjointly inflated slowly as a result of thermostable compound; (F) once an elevated level of solvent magnitude rela-tion was applied, the percentage yield of caffeic acid was minimized as a result of the medium polar compound.

3.6. Hepatoprotective Activity of Bioactive Compounds on HpeG2 Cell Induced with Galactosamine

In the study, the above-mentioned fractions were screened for hepatoprotective ac-tivity against GalN-induced cytotoxicity, by pre-incubating the cells with or without the extracts or sylimarin. A significant decrease in cell viability was observed upon treatment of HepG2 cells with GalN (40 Mm). Results are expressed as mean ± standard error mean (S.E.M). The percentage protection is represented in Figure 3 and Table 5.

Figure 2. Response surface plots of factor B2 vs. B1 against boeravinone B (C1) and caffeic acid(C2) respectively. (A) when an improved time of extraction was applied, higher concentrationsof percentage of boeravinone B were obtained; (B) as the factor temperature level improved, theboeravinone B percentage yield also showed an uncooperative automatic impact; (C) when anelevated level of solvent ratio was applied, boeravinone B concentration also produced uncooperativeimpact; (D) when an increase time of extraction was applied, an elevated level of percentage yieldof caffeic acid was investigated due to elevated level of penetration of solvent into plant extracts;(E) when the level of temperature increases, the sharp percentage yield of caffeic acid conjointlyinflated slowly as a result of thermostable compound; (F) once an elevated level of solvent magnituderelation was applied, the percentage yield of caffeic acid was minimized as a result of the mediumpolar compound.

3.5. Responseof Independent Factors on Caffeic Acid (C2)

As the equation shows that B1 has a productive impact on the caffeic acid percentageyield, When factor B (time of extraction) was increased, a productive level of caffeic acidpercentage yield was obtained due to solvent concentration penetrating into the plantmaterials, as shown in figure. This is because the equation shows that B1 has a productiveimpact on the caffeic acid percentage yield (Figure 2D).

Caffeic acid = 0.066 + 0.0014 B1 + 0.00093 B2 + 0.000013 B3−0.0016 B1 B2−0.005B1 B3−0.013 B2 B3−0.031 B1

2−0.019 B22 + 0.0074 B3

2 (3)

Factor B2 is perceived to have an increased profound impact on the proportion ofcaffeic acid percentage yield compared to issue B1 and B2. Because the factor B (temperaturelevel) inflated, the sharp percentage yield of caffeic acid conjointly inflated slowly as a

Separations 2022, 9, 177 9 of 12

result of thermostable compound. Figure 2E shows the impact of temperature levels oncaffeic acid proportion yield.

Factor B3 (40:60:80 v/v solvent ratio) indicated a distinguished lower caffeic acidpercentage yield as compared to that discovered on the issue B2, which showed a broadernegative impact. It had been discovered that, once an elevated level of solvent magnituderelation was applied, the percentage yield of caffeic acid was minimized as a result of themedium polar compound. Figure 2F indicates the impact of solvent magnitude affinitylevels on percentage yield of caffeic acid.

3.6. Hepatoprotective Activity of Bioactive Compounds on HpeG2 Cell Induced with Galactosamine

In the study, the above-mentioned fractions were screened for hepatoprotective activityagainst GalN-induced cytotoxicity, by pre-incubating the cells with or without the extractsor sylimarin. A significant decrease in cell viability was observed upon treatment of HepG2cells with GalN (40 Mm). Results are expressed as mean ± standard error mean (S.E.M).The percentage protection is represented in Figure 3 and Table 5.

Separations 2022, 9, x FOR PEER REVIEW 10 of 13

Table 5. In-vitro hepatoprotective activity of potent compounds present in B. diffusa.

Dose Treatment Control GLN(40 mM) Silymarin Boeravinone B Caffeic Acid 100 µg/mL 98.3 ± 1.1 15 ± 0.6 ## 78.7 ± 2.6 ** 40.89 ± 4.7 * 46.17 ± 4.4 *

Absorbance at 540 nm

3.5 0.459 2.757 1.431 1.616

200 µg/mL 84.35 ± 1.9 ** 66.21 ± 2.6 ** 52.34 ± 1.7 ** Absorbance at 540

nm 2.952 2.318 1.832

ns: non-significant, n = 3, data ± S.E.M. Groups I-4 were compared against group II using Dunnett’s posthoc test. ## Galactosamine toxic group significant, ns p > 0.05, ** p < 0.01, * p < 0.05.

Figure 3. Effect of the caffeic acid and boeravinone B on GlaN-induced toxicity in HepG2 cells. HepG2 cells were incubated in the presence/ absence of various compounds for 2h, prior to treat-ment with GlaN (40mm) for 2h. Thereafter, the cells were processed for the MTT assay. The results are expressed as mean ± SEM. ## Galactosamine toxic group significant, ns p > 0.05, ** p < 0.01, * p < 0.05 compared to the GalN (40 mM) control.

4. Discussion A significant number of medicinal plants are being employed for healing and for the

development of new pharmaceuticals all around the world. Understanding the therapeu-tic significance of natural extracts or their constituents, as well as the standardization of crude pharmaceuticals, necessitate phytochemical study [37]. The literature review sup-ports the fact that many natural products exhibiting hepatoprotective activity need to be standardized and optimized due to degradation of polyphenolic compounds during ex-traction time.

Several plants that are traditionally used are characterized for the presence of active ingredients using diverse methods [38,39]. Recently, B. diffusa has been increasingly prop-agated as a medicinal plant. The current study aims to develop and validate high perfor-mance thin layer chromatography (HPTLC) method for the simultaneous validation, quantification of boeravinone B and caffeic acid in the hydro alcoholic extract of B. diffusa in single solvent system. The HPTLC method is accurate, precise, simple, specific, less

Figure 3. Effect of the caffeic acid and boeravinone B on GlaN-induced toxicity in HepG2 cells.HepG2 cells were incubated in the presence/ absence of various compounds for 2h, prior to treatmentwith GlaN (40mm) for 2h. Thereafter, the cells were processed for the MTT assay. The results areexpressed as mean ± SEM. ## Galactosamine toxic group significant, ns p > 0.05, ** p < 0.01, * p < 0.05compared to the GalN (40 mM) control.

Table 5. In-vitro hepatoprotective activity of potent compounds present in B. diffusa.

Dose Treatment Control GLN(40 mM) Silymarin Boeravinone B Caffeic Acid

100 µg/mL 98.3 ± 1.1 15 ± 0.6 ## 78.7 ± 2.6 ** 40.89 ± 4.7 * 46.17 ± 4.4 *Absorbance at 540 nm 3.5 0.459 2.757 1.431 1.616

200 µg/mL 84.35 ± 1.9 ** 66.21 ± 2.6 ** 52.34 ± 1.7 **Absorbance at 540 nm 2.952 2.318 1.832

ns: non-significant, n = 3, data ± S.E.M. Groups I-4 were compared against group II using Dunnett’s posthoc test.## Galactosamine toxic group significant, ns p > 0.05, ** p < 0.01, * p < 0.05.

Separations 2022, 9, 177 10 of 12

4. Discussion

A significant number of medicinal plants are being employed for healing and for thedevelopment of new pharmaceuticals all around the world. Understanding the therapeuticsignificance of natural extracts or their constituents, as well as the standardization of crudepharmaceuticals, necessitate phytochemical study [37]. The literature review supports thefact that many natural products exhibiting hepatoprotective activity need to be standardizedand optimized due to degradation of polyphenolic compounds during extraction time.

Several plants that are traditionally used are characterized for the presence of ac-tive ingredients using diverse methods [38,39]. Recently, B. diffusa has been increasinglypropagated as a medicinal plant. The current study aims to develop and validate highperformance thin layer chromatography (HPTLC) method for the simultaneous validation,quantification of boeravinone B and caffeic acid in the hydro alcoholic extract of B. diffusain single solvent system. The HPTLC method is accurate, precise, simple, specific, less timeconsuming, cost-effective, and can separate boeravinone B and caffeic acid compounds fromtheir other constituents such as boeravinone A, C, D, E, F, G and flavonoid glycosides, etc.

In this method, the hydroalcoholic extract was found to contain a comparativelyelevated amount of boeravinone B and caffeic acid compared to alcoholic and aqueousextracts. This may be because medium polar retinoid and flavonoid glycosides presentin hydroalcoholic extract. The percentage yield of extracts depends on the temperature,time, and solvents of the extraction as well as the chemical moiety of secondary metabo-lites. Under the same time and temperature conditions, the solvent used and the chemicalproperty of the sample are the most important factors. In the present investigation, the ap-plication of the Box–Behnken Design for the optimization of boeravinone B and caffeic acidbased on temperature of extraction (◦C), time of extraction (min) and solvent concentrationratio (%v/v).

Shimma Ibrahim et al., in 2017, reported that natural plants have garnered great in-terest in the last few decades as therapeutic agents, since they produce inexpensive andless toxic products than synthetic drugs. To date, there is only one protective naturaldrug (Silymarin) that too is not curative and also has limitations in protecting the liverfrom viral attacks. Karimi, G. et al., in 2011, established that silymarin is useful forthetreatment of liver cirrhosis and hepatitis, and displaysimmumodulatory activity [40]. How-ever, silymarin’s structure containsa flavonoid moiety. Phenolic compounds (coumarins,flavonoids, xanthones, retinoid and tannins) are widely distributed in plants with potentanti-oxidant and anti-inflammatoryeffects, regulating immunity by interfering with im-mune cell regulation, proflammatory cytokines, and synthesis and gene expression andinhibitingphsophatidylinostide -3- kinase/ protein kinase [41].In the present study, isolatedconstituents caffeic acid (100 microgram/mL) and boeravinone B (200 µg/mL) showedsignificant hepatoprotective activity y compared with standard sylimarin in HepG2 cellinduced with galactosamine 40 mM toxicity.

Caffeic acid and boeravinone B have been of interest for theirhealth benefits, and thepresent HPTLC analytical study could be a future application to purify, identify, quantify,and optimize boeravinone B and caffeic acid in Boerhaviadiffusa.

5. Conclusions

The proposed method is innovative, since it is the first analytical method to reporton the validation, quantification, and optimization of boeravinone B and caffeic acid inBoerhavia diffusa (Linn).The proposed HPTLC method was performed according to theguidelines of the International Conference on Harmonization (ICH) for the quantifica-tion of boeravinone B and caffeic acid, which were successfully validated and developed.The method was validated in term of linearity and detection limit, quantification limit,range, precision, specificity, and accuracy. The maximum percentage yield of caffeicacid and boeravinone B from Boerhavia diffusa require appropriate extraction parameterssuch as temperature, time, organic solvents, and water content, which can be achievedusing the Box–Behnken statistical design, providing a time: temperature: solvent ratio

Separations 2022, 9, 177 11 of 12

(30:45:40 v/v) for the extraction of caffeic acid and 60:60:40 v/v for the extraction of boeravi-none B. The hepatoprotection of standard silymarin showed ranging from 78.7 percent at100 µg/mL to 84.34 percent 200 µg/mL, caffeic acid varied from 46.17 percent at 100 µg/mLto 52.34 percent at 200 µg/mL and boeravinone B ranged from 40.89 percent at 100 µg/mLto 62.21 percent at 200 µg/mL. The boeravinone B and caffeic acid showed the most signifi-cant hepatoprotective activity compared with standard silymarin in HepG2 cell inducedwith galactosamine 40 mM toxicity. The findings supported Boerhavia diffusa’s traditionaluse as a functional food for human health benefits.

Author Contributions: The contribution of the authors is as follows: conceptualization, I.U. andK.Y.T.; methodology, I.U.; investigation, I.U.; data curation, I.U., S.S., M.E.-M., S.N.M.N.U. and A.A.;writing—original draft preparation, I.U., K.Y.T. and M.E.-M.; writing—review and editing, I.U. andM.E.-M.; visualization, I.U.; supervision, K.Y.T.; funding acquisition, K.Y.T., S.N.M.N.U., A.A. and S.S.All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: All participants in the study provided their informed permission.

Data Availability Statement: The data that supports the finding of this study are also available fromthe corresponding author upon request.

Acknowledgments: The whole authors extend their appreciation to the Deanship of Scientific Re-search at King Khalid University for funding this work through General Research Project underGrant No: GRP/ 105/43.

Conflicts of Interest: The authors declare no conflict of interest.

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