Optimized microwave assisted extraction (MAE) of alkaloids and polyphenols from Berberis roots using multiple-component analysis

Berberis, one of the major sources of berberine and polyphenols, is widely accepted genus for its medicinal properties. The inclusion of these phytochemicals in different health formulations has widened its scope in pharmaceuticals and nutraceuticals. In the present study, multi-component analysis (MCA) has been used to extract these nutraceutical compounds from Berberis jaeschkeana roots under microwave-assisted extraction (MAE) conditions. To determine the optimum extraction condition, different factors, including, microwave power, sample to solvent ratio, irradiation time, solvent pH and solvent concentration were tested under 42 experiments. The MCA includes, Plackett-Burmen and Central Composite Design and analyzes model fitness, regression coefficient (β), analysis of variance (ANOVA) and 3D response curve. The results showed significant model fitness and involvement of linear, quadratic and interactive effect of different factors. Under optimized MAE condition, [i.e. 1 g of sample extracted through 70 mL of a solution (100% methanol pH 2.0), provided microwave power of 598 W for 2 min of irradiation time], the berberine and palmatine contents were recorded as 46.38 mg g−1 and 20.54 mg g−1 respectively. Under optimized condition, the yield of alkaloids were found closer to the models’ predicted value. Similarly, total phenolic content and antioxidant activities were also found closer to the models’ predicted value. To test the suitability of the optimized MAE condition for other species i.e., Berberis asiatica, extraction of alkaloids and polyphenolics was conducted and recorded higher yield to the previous records. Moreover, under optimum extraction condition, six and seven polyphenolic compounds from B. jaeschkeana and B. asiatica were quantified respectively. The proposed MAE optimization design using MCA contributes towards faster and greener extraction of alkaloids and polyphenolics with higher yield. Moreover these greener approaches could sustainably utilize species during extract preparation and harnessing its nutraceutical and pharmaceutical potential. This study design could also be replicated on other valuable species or compounds for effective extraction of nutraceutical components and sustainable utilization of natural products.

in Italy 16 . Similarly, 'Zyflamend Softgel Capsule' , which acts as antioxidant and anti-inflammatory dietary supplement to manage osteoarthritis and rheumatoid arthritis also contain berberine 17 .
To obtain the desired secondary metabolites and/or extract as a whole, plant materials undergoes various processes, including drying, extraction, separation and purification 21,29 . These bioprocessing conditions have direct effect on the extraction yield and extract quality, thus determine the final product cost. Traditionally, Berberis root extract was prepared by boiling, decoction and infusion, while, maceration and microwave assisted extraction (MAE) techniques are used during laboratory experiments 29,30 . Reports revealed that various compounds of nutraceutical and pharmaceutical interest have been extracted by MAE method 21,[31][32][33] . The MAE is reported as a green extraction method, as it provides both faster extraction and lesser or no solvent consumption 34,35 . The excess heat and pressure developed during the process increases mass transfer and helps in extracting plant components in lesser time with increased yield as compared to conventional extraction methods 31,[36][37][38][39][40][41] . Excessive heat during MAE was also reported to degrade the heat sensitive compounds 32,33,35,42 , thus the processing conditions (i.e., microwave power, irradiation time, type of solvents and composition) needs to be optimized for obtaining better quality of extract and compounds.
To determine the optimum processing conditions, various efforts have been made. As such, single factor analysis (univariate) determines only the effect of a single variable at a time over the responses and neglects the interactive effect between variables, thus leads to a larger number of experiments with futile results. On the other hand, a multiple-component analysis (MCA) not only determines the individual effect but also the interactive effects of variables 41 . Response surface methodology (RSM) is one such MCA, which needs lesser experimental runs and thus could be a choice for optimization of process conditions 43,44 . To date, the extraction of berberine and other alkaloids have successfully been optimized from stem part of Berberis amurensis 30 and rhizome of Coptis chinensis 45 using RSM. Although, the root part of Berberis spp. is reported to contain the higher amount of alkaloids [46][47][48] , however the optimum extraction condition has not yet been developed under advanced extraction technique. Thus, this study was designed to apply MCA, including Plackett-Burman design (PBD) and central composite design (CCD) for optimizing MAE conditions for alkaloids and phenolics from B. jaeschkeana C.K. Schneid roots. Also, the optimized MAE extraction conditions were tested for their suitability/reliability in Berberis asiatica roots.
Microwave assisted extraction and phytochemical analysis. Microwave assisted extraction (MAE). MAE was carried out using multiwave-3000 microwave reaction system (Anton-Paar, Germany, GmbH) consisted of 8 closed extraction vessels equipped with infrared temperature sensor, vessel mark sensor, controllers and a magnetic stirrer at the base. For extraction, 1 g of the powdered root sample was dissolved in different volumes and concentrations of methanol at different pH values (Supplementary Tables 1, 2). The mixture was placed in closed vessel extracting chamber inside the microwave reaction system. Microwave power at different levels and time periods (Supplementary Tables 1, 2) was applied according to the model design and the filtered extract was stored at −20 °C. All dependent variables (responses) were measured within two weeks from the storage time.
Analysis of total phenols (TP). The TP was measured by Folin-Ciocalteau's colorimetric method 49 . The quantification of TP content was done using a gallic acid standard curve and estimated as mg gallic acid equivalent/g dry sample (mg GAE/g dw). Briefly, 0.5 mL of Folin-ciocalteu solution was added to diluted 5 mL of extract. Thereafter, sodium bicarbonate (Na 2 CO 3 ; 7% w/v) was added to the mixture. After proper mixing, the mixture was kept for 90 minutes at room temperature in dark. The absorbance of resultant blue color was measured at 765 nm under spectrophotometer (Hitachi U-2001, Japan).
Determination of alkaloids and polyphenolic compounds. Alkaloids. Alkaloids (berberine and palmatine) were analyzed using high performance liquid chromatography (HPLC) (LC-10AT, Shimadzu Liquid Chromatography, Japan) equipped with the binary pump and diode-array detection (DAD-MZOA) unit 50 . Briefly, 10 µL of the (1 mg of dry extract dissolved in 1 mL of methanol) extract was injected and separated using C18 reverse phase column (250 mm × 4.6 mm i.d., 5 µm, Purosphere, Merck, Darmstadt, Germany) maintained at 25 ± 1 °C. The mobile phase comprises acetonitrile (A) and 0.13% potassium dihydrogen phosphate of pH 2.5 (B) at 50:50 ratio and flow at 1.0 mL/min for 20 min of total run time. Depending on the lambda max, detector wavelength was set at 345 nm and berberine and palmatine content was recorded as mg 100 g −1 dry weight. All experiments were conducted in triplicate (n = 3).
Polyphenolics. Moreover, polyphenolics were analyzed as per the method 20,51 , with minor modifications. Briefly, 10 µL of the (1 mg of dry extract dissolved in 1 mL of methanol) extract was injected and separated using C18 reverse phase column (250 mm × 4.6 mm i.d., 5 µm, Purosphere, Merck, Darmstadt, Germany) maintained at 25 ± 1 °C. The mobile phase consisted of a mixture of methanol and 0.1% (v/v) ortho-phosphoric acid at the ratio of 40:60 (v/v) and the flow rate was maintained at 0.8 mL/min for total run time of 40 min. The DAD wavelengths were selected from 254 to 330 nm. A total of fifteen polyphenolic standards were used and the final concentration of compounds was determined as mg 100 g −1 dry weight. All experiments were conducted in triplicate (n = 3).

Multiple-component analysis (McA).
The experiments were designed for determining optimum MAE conditions for maximizing alkaloid and polyphenolic contents. Factors such as microwave power, sample to solvent ratio, solvent pH, solvent concentration and irradiation time were selected as independent variables for alkaloids and polyphenolic extraction under MAE (Supplementary Tables 1 and 2).
For MCA, a systematic approach was followed. The independent variables were first screened based on the significant effect over the responses using Plackett-Burman design (PBD). PBD only measures linear effect of individual factors, hence PBD was conducted to determine the significant effect of microwave power, irradiation time, sample to solvent ratio, solvent pH and concentration at two levels over the response variables (i.e., berberine, palamatine and TP content) (Supplementary Table 1). The significant factors were then tested using central composite design (CCD) to determine the linear, quadratic and interactive effects over the responses (i.e., berberine, palamatine, TP content and antioxidant activity) (Supplementary Table 2). The second order polynomial equation was applied to determine the effects as: where, Y is the response variable, Xi and Xj are the independent variables, and k is the number of tested factors (k = 4). The regression coefficient is defined as β 0 for intercept, βi for linear, βii for quadratic and βij for the cross-product term. 3D graphs were generated using regression coefficient and analysis of variance (ANOVA) was conducted to find the significant (p < 0.05) effect of the model terms. optimum MAe condition and Validation of the model. Keeping all the responses as maximum and factors level within the range, the optimal MAE condition for alkaloids, polyphenols and antioxidant activity were determined and validated. The optimal conditions were generated by the model based on the response value and desirability was used to select the best optimal MAE condition. The experiment was further conducted (in triplicate) on the selected optimum extraction condition and coefficient of variation (CV) was determined to validate the model. To test the reliability of the optimized MAE condition, root samples of Berberis asiatica were also tested for alkaloids and polyphenolic antioxidants extraction.
Statistical analysis. The obtained results were subjected to analysis of variance (ANOVA) and all the experiments were conducted in triplicates. The statistical software used was SPSS V.17.0 (IBM Corporation).

Results and Discussion
Screening factors (plackett-Burman Design). Microwave power. Microwave power was used at two levels (100 and 300 W) for PBD and showed a significant variation in berberine, palmatine and TP content (Supplementary Table 1). All the responses increased with increasing microwave power, which might be due to increase in mass transfer by microwave 36,37 . Meanwhile, the excess heat also leads to degradation of compounds 35,42 , which depends on the irradiation time and physiochemical properties of compounds and has not been seen during the current study, even at higher microwave power.
Irradiation time. Irradiation time did not show any significant difference in the responses (Supplementary  Table 1), however, lower irradiation time was found equally effective as higher. Considering the energy loss and possibility of degradation of compounds at higher irradiation time, it was kept at a lower level for CCD model.

Solvent pH.
A significant effect of solvent pH was recorded for berberine and palmatine content (Supplementary Table 1). The pH levels for PBD model were set at acidic (pH 2.5) and near neutral (pH 6). Increasing the solvent pH from acidic to near neutral, a significant increase in the degradation of berberine and palmatine was observed.
Thus, for CCD model the solvent pH levels were kept at lower value.
Solvent concentration. For MAE condition, methanol was selected as solvent of choice because of the fact that it has higher solubility of compounds of interest and also having higher dissipation factor (tan δ), which provides greater microwave heat absorption and transfer by the solvent under MAE 31,37 . As such, increasing methanol concentration from 20 to 80%, berberine and palmatine concentration increases significantly (Supplementary  Table 1), thus further tested at higher levels in CCD model. Overall, among different tested independent variables under PBD, microwave power, sample to solvent ratio, solvent pH and concentration showed significant variations in the responses. Also, the model F-value for berberine, palmatine and TP was found significantly fit with good coefficient of determination (R 2 ) (Supplementary Table 1).

Multiple-component optimization (central composite design). Fitting the model. The CCD
showed a significant (p < 0.05) model fitness ( Table 1). The coefficient of determination (R 2 ) value of all the response variables were found to be higher and also lack of fit was found to be non-significant (

3D Response Surface Analysis for Alkaloids and Polyphenolic Antioxidants. Effect of microwave power (X1):
Microwave power showed significant linear, quadratic and interactive effect on all the responses (Table 1). Berberine concentration decreased significantly (p < 0.05) with increasing microwave power, while a significant (p < 0.001) positive linear effect was found on ABTS, FRAP and DPPH antioxidant activity (Table 1). No significant linear effect of microwave power has been recorded on TP extraction at lower level, however as the power increases above 400 W, a significant positive quadratic effect has been seen (Fig. 1h). For FRAP and ABTS antioxidant activity, a positive significant quadratic effect of microwave power (X 1 ) has been recorded at higher levels ( Fig. 2a-f).
Effect of sample to solvent ratio (X2): A significant linear positive effect of sample to solvent ratio has been observed for all responses except for FRAP antioxidant activity (Table 1). At higher ratio, above 1:50 gmL −1 , a significant (p < 0.001) decrease in FRAP activity was recorded as negative quadratic effect (Fig. 2g). However, berberine content was found to be increased with further increasing in solvent volume as a significant (p < 0.001) positive quadratic effect (Fig. 1a,d,e). Interestingly, a significant positive interaction between sample to solvent ratio and solvent concentration (X 1 X 4 ) was also recorded for all the responses (Figs. 1 and 2), except for palmatine and DPPH activity. Similarly, a significant positive interactive effect between sample to solvent ratio and microwave power (X 1 X 2 ) has been seen for some of the responses. As such, with increasing both X 1 and X 2 , a significant increase in berberine (p < 0.001) (Fig. 1a), palmatine (p < 0.05) (Fig. 1f), ABTS (p < 0.01) (Fig. 2a) and DPPH antioxidant activity (p < 0.01) (Fig. 2i) have been recorded.
Effect of solvent concentration (X4): For all the responses except TP content, with increasing methanol concentration (X 4 ), a significant (p < 0.001) linear increase in response values were recorded, while at higher concentration, a significant (p < 0.05) quadratic negative and significant (p < 0.001) quadratic positive effect was recorded www.nature.com/scientificreports www.nature.com/scientificreports/ for FRAP antioxidant activity and berberine content, respectively (Table 1, Figs. 1 and 2). With increasing methanol concentration along with microwave power (X 1 X 4 ), a significant decrease in berberine content (p < 0.01) was recorded (Fig. 1c), however for TP (p < 0.05) (Fig. 1k), ABTS (p < 0.05) (Fig. 2c) and FRAP antioxidant activity (p < 0.001) (Fig. 2f), an increase in response value was recorded. Interestingly, when both methanol concentration and sample to solvent ratio (X 2 X 4 ) acts together, a significant (p < 0.01) increase in all response values have been observed (Figs. 1, 2). However, significant (p < 0.001) positive interactive effect between methanol concentration and solvent pH (X 3 X 4 ) was only recorded for FRAP antioxidant activity (Fig. 2h). jaeschkeana alkaloids and polyphenolics extraction was selected based on the maximum desirability obtained for the model. Under optimum MAE condition, 1 g of root powder sample was mixed with 70 mL of absolute methanol having pH value of 2.0 and the mixture was kept inside the microwave system for 2 min ramp time at 598 W of microwave power. The response values were determined and found very close to the model predicted value, as CV ranges from 0.5 to 5.6% (Table 2). Under these condition, TP (21.27 mg GAE g −1 ), and ABTS (21.96 mM AAE g −1 ), FRAP (230.14 mM AAE g −1 ) and DPPH (53.73 mM AAE g −1 ) antioxidant activities were found to be well fitted with model predicted value (Table 2). Also, berberine and palmatine contents were found 46.38 and 20.54 mg g −1 , respectively (Table 3, Supplementary Fig. 1a).
Under optimum MAE conditions, Berberis asiatica root samples were extracted and recorded 88.71 mg g −1 of berberine and 18.68 mg g −1 of palmatine concentration along with TP (30.43 mg GAE g −1 ), and ABTS (26.47 mM AAE g −1 ), FRAP (247.37 mM AAE g −1 ) and DPPH (66.21 mM AAE g −1 ) antioxidant activity (Table 2). Moreover, these plant extracts under optimum MAE conditions showed significantly higher berberine and palmatine contents, as compared to earlier reports ( Table 4). The increased yield at lesser extraction time, coupled with simple and environment friendly MAE optimized condition has proven to be an effective green extraction method for these compounds.

HPLC-DAD analysis of polyphenolics under optimum MAE condition.
Out of fifteen screened polyphenolic compounds, the HPLC-DAD analysis detected and quantified a total of six polyphenolic compounds in B. jaeschkeana and seven in B. asiatica roots under optimal MAE condition ( Table 3). This study first time detected the presence of phloridzin, 3-hydroxybenzoic acid, chlorogenic acid, vanillic acid, catechin, gallic acid, caffeic acid and rutin in root part of Berberis species. The HPLC chromatograms of polyphenolics obtained from B. Jaeschkeana roots were presented in Supplementary Figure 1b. In comparison to B. jaeschkeana, B. asiatica contain polyphenolics in higher concentrations, except for 3-hydroxybenzoic acid. Also, phloridzin was not detected in B. asiatica, while caffeic acid and rutin could not detected in B. jaeschkeana (Table 3). These polyphenolics are well studied for their nutraceutical properties and found to play important role in combating against   24,25,27,52 . Moreover, these compounds along with berberine were found effective to treat a number of medical complications (Fig. 3).

conclusions
An improved microwave-assisted extraction method for pharmaceutically and nutraceutically important natural compounds from Berberis species has been developed and validated. Under optimum MAE condition, alkaloids viz. berberine (46.38 mg g −1 ) and palmatine (20.54 mg g −1 ) from B. jaeschkeana roots were found higher as compared to earlier reported concentrations. Similarly, optimum MAE condition was also found suitable for extraction of these compounds from Berberis asiatica roots, and obtained comparatively higher concentrations to the earlier reports. For the first time six polyphenolic compounds in B. jaeschkeana and seven in B. asiatica root part have been detected and quantified at optimal MAE condition. Comparison between the species reveals a higher concentration of berberine and polyphenolic antioxidant compounds in B. asiatica as compared to B. jaeschkeana. As berberine, palmatine and polyphenolic compounds are being commercially used in large number of pharmaceutical and nutraceutical products, the present study provides reliable, repeatable and economical MAE method, which showed improved extraction yield. Also, the multi-component analysis was found to be successful in designing MAE method for extracting valuable compounds of nutraceutical and pharmaceutical importance. Thus, the present study could be replicated in other high-value medicinal plants for their effective and sustainable utilization in nutraceuticals and pharmaceuticals.