Validation, Optimization and Hepatoprotective Effects of Boeravinone B and Caffeic Acid Compounds from Boerhavia diffusa Linn

Abstract

Boerhavia diffusa, also known as Punarnava, is a plant of the Nyctaginaceae family that has been utilized in traditional medicine to cure a variety of ailments. The goal of this study was to use response surface methodology (RSM) to optimize the maximum percentage yield of boeravinone B and caffeic acid from Boerhavia diffusa roots, and simultaneous determination of boeravinone B and caffeic acid in newly developed single solvent system and demonstrate the hepatoprotective benefits of boeravinone B and caffeic acid. The extraction process examined extraction time, extraction temperature 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 successfully 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 and caffeic 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 the absorbance mode at 254 nm. The maximum percentage yield of caffeic acid and boeravinone B from Boerhavia diffusa require appropriate extraction parameters such as temperature, time, organic solvents and 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 extraction of boeravinone B. The boeravinone B (200 µg/mL) and caffeic acid (200 µg/mL) showed the most significant hepatoprotective activity compared with standard sylimarin in HepG2 cell induced with galactosamine 40 mM toxicity. The findings supported B. diffusa’s traditional use as a functional food forhuman health benefits.

1. Introduction

Boerhavia diffusa has been traditionally used by Indian and Brazilian indigenous and tribal people. Ayurvedic medicine traditionally uses the plant’s root and leaves to treat a wide range of ailments, such as viral jaundice and liver dysfunction. This plant isreported to have behavioral and neuroendocrine effect and anti-depressant activity [1], anti-angiogenic activity [2], anti-convulsant and anti-epileptic activity [3], anti-inflammatory activity [4], inhibitory effect of prostatic hyperplasia [5], anti-genotoxic effect [6], thrombolytic, cytotoxic and anti-microbial activities [7], arsenic trioxide-induced cardiotoxicity [8], anti-urolithic activity [9], anti-hyperglycaemic and reno-protective effect [10], acetaminophen-induced liver toxicity [11], anti-proliferative and anti-estrogenic effects [12], immunomodulatory and anti-metastatic activity [13,14], radio-protective activity [15], potent anti-breast cancer activity [16], intestinal activity [17], immunosuppressive activity [18], anti-diabetic activity [19], Ca²⁺ channel antagonistic activity [20], chemo-preventive action [21], glycol-induced urolithiasis [22], and inhibition of human cervical cancer cells [23]. Main Rotenoids area type isoflavone compound (known as boeravinones) that are isolated from the roots of this plant; they showed potent anti-cancer [24,25], anti-inflammtory [26], cardioprotective [27] powerful anti-oxidant and geno-protective properties [28], intestinal motility and spasmolytic activity [29]. Caffeic acid (3,4-dihydroxycinnamic acid) is one of the major polyphenolic compounds found in many plant species, showing anti-viral activity [30], immunostimulatory activity [31], cardioprotective activity [32], hepatoprotective activity [33], anti-cancer activity [34] and anti-hepatocellular carcinoma activity [35]. For detecting phenolic compounds in medicinal plants, several analytical techniques such as liquid chromatography–mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), high-performance thin layer chromatography (HPTLC)and ultra-performance liquid chromatography (UPLC). Boeravinone B and caffeic acid are rarely applied by use of their similar analytical techniques. Ilyas et al. in 2013 reported only identification and quantification of caffeic acid and boeravinone in single-solvent system toluene: ethyl acetate: formic acid: methanol (3:3:0.8:0.2 v/v) but tailing peak and separation and resolution of band of caffeic acid and boeravinone was very poor [36]. Here, a validated high-performance thin layer chromatography (HPTLC) method has been newly developed based on single solvent system (toluene: ethyl acetate: formic acid (5:4:1 v/v) for simultaneous validation, quantification and optimization of boeravinone B and caffeic acid in the plants. The selected HPTLC method is accurate, precise, simple, specific, less time-consuming, cost-effective and can separate boeravinone B and caffeic acid compounds from 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 phenolic acids. The comparison of the entire chromatograms allows for the detection of minor similarities and differences between the plants under investigation. The objective of the study was to simultaneously quantify, validate, optimize and to evaluate hepatoprotective activity of boeravinone B and caffeic acid in hydroalcoholic extracts of B. diffusa.

2. Materials and Methods

2.1. 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 used as developing systems for HPTLC analysis. All samples and standards used for the analysis were filtered through a membrane filter, 0.22 μm pore size (HIMEDIA, Mumbai, India). Extraction method using a Soxhlet apparatus (Omega, Mumbai, India) and Rotary evaporator (Buchi R-114, Switzerland). CAMAG HPTLC system (Muttenz, Switzerland) equipped with a Linomat IV sample applicator was used for quantification and optimization. HepG2 cell line (National Centre for Cell Science (NCCS), Pune, India), Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS) and MTT assay kit, trypsin EDTA, 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, eppendorf tube, 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).

2.2. Collection and Extraction of Plant Materials

In February 2011, we collected the roots of B. diffusa in Maruthmallai, Kanyakumari district, Tamilnadu, India. It was identified and authenticated by Dr. V. Chelladurai, Research Officer, 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 extraction by using 95% methanol and 50% (v/v) hydro-alcohol for 6 h at 37 °C, respectively. The methanol and hydro-alcoholic extracts were evaporated and dried at 45 °C with help of rotary 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-alcoholic alcohol yielded 9.9% weight-for-weight.

2.3. HPTLC Instrumentation

2.3.1. Preparation of Sample Solution

A total of 100 mg of extracts were dissolved in 10 mL of methanol and sonicated the solution for 15 min and then makeup with 10 mL HPLC grade methanol, filtered through a membrane 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 of 4 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 bands was 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 the development volume was20 mL. In a 10 × 10 cm twin-trough glass chamber saturated with the mobile phase, linear ascending development was performed. At room temperature, the optimal chamber saturation time for the mobile phase was 20 min. The chromatogram run was 8.5 cm long, and the TLC plates were dried in an oven at 50 °C for 5 min after development. CAMAG TLC Scanner 3 in absorbance-reflectance mode at 254 nm, operated by Win-CATS software, was used for densitometric scanning. The deuterium lamp was used as a source of radiation. The peak area response was compared to the amount of medication in a linear regression.

2.3.5. Preparation of Standards

The standard solution of bioactive compounds was prepared by dissolving 1.0 mg of boeravinone B and caffeic acid in 10 mL of methanol as stock solution and stored at 2 to 8 °C. These boeravinone B and caffeic acid were further diluted as per the requirement to plot a final concentration for quantification.

2.3.6. Analytical Method Evaluation

The lowest diluted solutions of the two reference compounds in the calibration curves were further diluted to a series of concentrations with HPLC grade methanol for the determinations of limits of detection (LOD) and quantification (LOQ). The LOD and LOQ under 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 and LOQ 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 calibration solutions by using peak area against concentration. To evaluate the accuracy, the pre- analyzed samples were spiked with standard at three different known concentration levels i.e., 50, 100 and 150% and the mixtures were re-analyzed by the proposed method. Precision of 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 caffeic acid were carried out at different levels of concentrations 100, 200, 400 ng/band. The identification of bands was carried out in triplicate. The R.S.D. was taken as a measure of precision. The resolution between the peaks of caffeic acid and Boeravinone B found satisfactory (2.1 ± 0.2). The developed method resolution found to be good enough to avoid any possible merging between the peaks. Satisfactory recovery values suggest that there 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 (Minneapolis, MN, USA) was used (Minneapolis, Stat-Ease Inc., MN, and USA). This style is suitable for investigating constructing second-order polynomial models and quadratic response 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 confirmed the region of interest. The dependent and independent variables are recorded in

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

Factors Independent Variables	Levels Used
	Low (−1)	Medium	High (+1)
B1 = Time in min	30	60	90
B2 = Temperature (°C)	30	45	60
B3 = Solvent concentration (v/v)	40	60	80
Dependent variables
C1 = Boeravinone B	Maximized percentage yield
C2 = 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 + A₈B₂² + A₉B₃²

(1)

where A₀ is the intercept, A₁ to A₉ are the regression coefficients, R is the dependent variable and B_1, B₂ and B₃ are the independent variables.

The data of the maximum percentage yield of boeravinone B and caffeic acid in the hydro-alcoholic extract of plant materials were recorded through arithmetical improvement by using Design-Expert software system. The Box–Behnken Design style spreadsheet is shown in

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

Run	Factor-1 (A1): Time (Min)	Factor-2(A2): Temperature (°C)	Factor-3 (A3): Solvent Concentration		% Yield Boeravinone B (C1)	% Yield Caffeic Acid(C2)
			Methanol (%)	Water Content (%)
01	30	30	60	40	0.044	0.011
02	90	30	60	40	0.048	0.018
03	30	60	60	40	0.045	0.017
04	90	60	60	40	0.039	0.017
05	30	45	40	60	0.050	0.036
06	90	45	40	60	0.027	0.048
07	30	45	80	20	0.011	0.046
08	90	45	80	20	0.032	0.0384
09	60	30	40	60	0.036	0.041
10	60	60	40	60	0.047	0.068
11	60	30	60	40	0.035	0.067
12	60	60	80	20	0.015	0.043
13	60	45	60	40	0.0086	0.066
14	60	45	60	40	0.0085	0.065
15	60	45	60	40	0.0086	0.066
16	60	45	60	40	0.0085	0.065
17	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 and incubated overnight. After 24 h, the media was flicked off, the cells were treated with boeravinone B and caffeic acid (100 and 200 µg/mL) in separate wells of a 24-well plate and incubated for 2 h. Silymarin (250, 500 µg/mL) was used as a reference standard. After incubation, the cell was treated with D-galactosamine at a concentration of 40 mM and was allowed to incubate for 2 h. After incubation, the cells were washed and incubated for 1 h with MTT (20 μL of 5 mg/mL of MTT in PBS) was added to each well. Formation of formazan crystal was observed under a microscope after 2 h. In case crystal, formation was not proper, incubation was continued for an hour more. Media was removed and the remaining formazan crystals were dissolved in 200 μLof DMSO in each well. The cell culture plate was kept on a shaker for 15 min; the absorbance was recorded by ELISA reader at 540 nm.

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

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

3. Results

3.1. Development of Mobile Phase

Separation of standard markers studies were carried out on the working standard solution of boeravinone B and caffeic acid in HPLC-grade methanol. Originally, many trials 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 and optimization of boeravinone B and caffeic acid gave a well-defined and sharp peak. We obtained sharp bands upon saturating the twin trough plate development chamber with the developing system at room temperature for 30 min. The band of boeravinone B and caffeic acid was recorded on HPTLC plate scanned at 254 nm (Figure 1).

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 retention factor of caffeic acid (R_f = 0.58), boeravinone B (R_f = 0.71), respectively. The calibration curve’s linear regression data revealed a good linear relationship throughout a concentration 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, respectively (

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

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

). 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 (Table 3) showed that the repeatability of method. The developed HPTLC method’s outstanding 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 establish that the total system operated successfully, system suitability criteria such as peak symmetry, resolution (Rs), capacity factor (K), and selectivity was tested. As demonstrated in Table 3, the obtained values were within acceptable ranges.

3.3. Box–Behnken Design-Experiment

A three-level factorial, Box–Behnken Design-Expert applied statistical modeling experimental style was performed by applying 17 investigational trial runs, as recorded in Table 2. Standard boeravinone B and caffeic acid determined that the preferred model was the squared model and also the equivalent values of standard deviation and percentage coefficient of variation for the various planned models is listed in

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

together with the regression equations typically for ultimately elite responses. Only statically significant (p < 0.0001) for boeravinone B and caffeic acid respectively coefficient is enclosed in the equations. A positive value indicated the optimization of boeravinone B and caffeic acid in hydroalcoholic extracts, while a negative value represents an inverse relationship between the response and the factor. It is clear from the equations that the factor time (B₁), as well as temperature (B₂) has an uncooperative effect and 40:60:80 v/v solvent ratios (B₃) have a productive on the responses (C₁ and C_2,). It also indicates that the correlation between factors and response is not always linear. When more than one factor is replaced at the same 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 result in the case of the square root of various factors does not repeat history, as shown by its performance. In the case of the square root of various factors, B²₂ (extraction temperature) and B²₃ (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 the extractions were established based on percentage yield of boeravinone B and caffeic acid.

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 B₁² + 0.021 B₂² + 0.0054 B₃²….

(2)

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

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

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

3.5. Responseof Independent Factors on Caffeic Acid (C₂)

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

Caffeic acid = 0.066 + 0.0014 B₁ + 0.00093 B₂ + 0.000013 B₃−0.0016 B₁ B₂−0.005 B₁ B₃−0.013 B₂ B₃−0.031 B₁²−0.019 B₂² + 0.0074 B₃²

(3)

Factor B₂ is perceived to have an increased profound impact on the proportion of caffeic acid percentage yield compared to issue B₁ and B₂. Because the factor B (temperature level) inflated, the sharp percentage yield of caffeic acid conjointly inflated slowly as a result of thermostable compound. Figure 2E shows the impact of temperature levels on caffeic acid proportion yield.

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

.

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 therapeutic significance of natural extracts or their constituents, as well as the standardization of crude pharmaceuticals, necessitate phytochemical study [37]. The literature review supports the fact that many natural products exhibiting hepatoprotective activity need to be standardized and optimized due to degradation of polyphenolic compounds during extraction 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 propagated as a medicinal plant. The current study aims to develop and validate high performance 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 time consuming, cost-effective, and can separate boeravinone B and caffeic acid compounds from their 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 comparatively elevated amount of boeravinone B and caffeic acid compared to alcoholic and aqueous extracts. This may be because medium polar retinoid and flavonoid glycosides present in 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 metabolites. Under the same time and temperature conditions, the solvent used and the chemical property of the sample are the most important factors. In the present investigation, the application of the Box–Behnken Design for the optimization of boeravinone B and caffeic acid based on temperature of extraction (°C), time of extraction (min) and solvent concentration ratio (%v/v).

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

Caffeic acid and boeravinone B have been of interest for theirhealth benefits, and the present 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 report on the validation, quantification, and optimization of boeravinone B and caffeic acid in Boerhavia diffusa (Linn).The proposed HPTLC method was performed according to the guidelines of the International Conference on Harmonization (ICH) for the quantification 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 caffeic acid and boeravinone B from Boerhavia diffusa require appropriate extraction parameters such as temperature, time, organic solvents, and water content, which can be achieved using the Box–Behnken statistical design, providing a time: temperature: solvent ratio (30:45:40 v/v) for the extraction of caffeic acid and 60:60:40 v/v for the extraction of boeravinone B. The hepatoprotection of standard silymarin showed ranging from 78.7 percent at 100 µg/mL to 84.34 percent 200 µg/mL, caffeic acid varied from 46.17 percent at 100 µg/mL to 52.34 percent at 200 µg/mL and boeravinone B ranged from 40.89 percent at 100 µg/mL to 62.21 percent at 200 µg/mL. The boeravinone B and caffeic acid showed the most significant hepatoprotective activity compared with standard silymarin in HepG2 cell induced with galactosamine 40 mM toxicity. The findings supported Boerhavia diffusa’s traditional use as a functional food for human health benefits.