Optimization for ultra high pressure extraction of Scutellaria barbata by central composite design-response surface methodology

1 Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China. 2 Department of Pharmacy, Cang zhou Hospital of Integrated Traditional Chinese and Western Medicine of Hebei Province, Cangzhou,061001,China. 3 College of Life Science and Biopharmacology, Guangdong Pharmaceutical University, Guangzhou, 510006, China. 4 Department of Pharmacy, Cancer Hospital, Guangzhou Medical University, Guangzhou, 510095, China.


INTRODUCTION
Scutellaria barbata D. Don (Ban Zhi Lian, BZL), is a traditional Chinese medicine for clearing heats, relieving toxicity, reducing swelling, curing sores and abscesses (Dharmananda, 2004).Its anti-cancer property has recently been reported and the clinical trial of its extract for advanced breast cancer treatment was conducted in US (Fong et al., 2008).Previous investigations showed that this plant had over 30 flavonoids, more than 10 neoclerodane-type diterpenoids, triterpenoids and sterol glucosides, some of which exhibited interesting biological activities against tumor and viruses, immune adjustment, *Corresponding author.E-mail: linjm1231@21cn.com.#These authors contributed equally to this paper.Wang et al., 2010).There are various of methods for etc. (Yu et al., 2004;Hanna et al., 2010;Dai et al., 2010; extracting salidroside such as reflux extraction, ultrasonic extraction and Soxhlet extraction (Zhang et al., 2007).Besides long extracting time, most of these methods employ heating which could easily decompose some thermo-sensitive ingredients or transformation.
Ultra high pressures (UHP) are widely used in the ceramics, casting industry, pharmaceutics, metallurgy, plastics making and civil engineering.Literatures reported that UHP technique could shorten processing time and reach high extraction yields, without any adverse side effects on the activity and structure of the bioactive components.
This research used the UHP extraction technique and CCD-RSM to extract scutellarin, luteolin and apiolin from S. barbata, and also compared UHP with the extracting method of circulation reflux, ultrasonic and microwave.

Extraction of active ingredient from S. barbata by UHP
The S. barbata was crushed using a 24 mesh screen.About 2.0 g plant material was set in a sealed bag and solvent was added, the bubbles were sealed, the pressure was increased, keeping pressure for 90 s, then the pressure was removed and extracts were obtained, filtered using a 0.22 µm filter membrane, and then stored at 4°C.

High performance liquid chromatography analysis
Hypersil ODS C18 column (4.6 × 250 mm, 5 µm Dalian Yi Lite); a mobile phase of methanol (A) -acetic acid water (B pH 2.4), elution gradients of 0 to 60 min, A 22 to 64%; 0 to 60 min, B 78 to 36%; detection wavelength of 335 nm, flow rate of 1 ml•min -1 , and column temperature of 30°C were used.In this chromatography conditions, the chromatogram of reference substance and S. barbata samples are shown in Figure 1.

Preparation of standard curve
0.01378 g scutellarin, 0.00463 g luteolin and 0.00596 g of apigenin were added into a 50 ml flask, accurately, and appropriate amount of methanol was added to the mark to dissolve.0.05, 0.1, 0.5, 1.0, 2.0 and 5.0 ml of mixture control solution were transferred into a 10

B
Table 1.The level of form factors (r = 1.732).

Determination of extraction sample
Precise amount of sample filtration (30 µl) was analyzed with the earlier mentioned chromatographic conditions and measured three times for each sample.The average peak area value of scutellarin, luteolin and apigenin was gotten, respectively.The data were taken into the standard curve and the content of the sample was calculated.

Optimization of the UHP process of S. barbata using the CCD-RSM
Literature (Zhang et al., 2007;Xi et al., 2011;Corrales et al., 2008) showed that the main factors affecting the extraction efficiency are pressure, concentration solvent and solid-liquid ratio.Extraction pressure (X1), ethanol concentration (X2) and solid-liquid ratio (X3) were therefore selected as the main factors according to the CCD-RSM principles in this study to optimize the combination.The levels of the independent variables were -r, -1, 0, 1 and r, the experimental factors and levels of coding are shown in Table 1.The experiment design is shown in Table 2.The Design-Expert 8.0.4 software was used to for RSA analysis.

RESULTS
The results which were analyzed by Design-Expert 8.0.4 software are shown in Table 3.A multiple regression of three major factors (extraction pressure (X 1 ), ethanol concentration (X 2 ) and solid-liquid ratio (X 3 )) in 20 trials, Responses of experimental and composite scores in the regression equation predicted that the determination coefficient was 0.9648 (R 2 = 0.9648), showing that the model fitted well.Analysis of variance showed that the F value of the overall model test revealed a significant response (P <0.05) and that the effect of ethanol concentration on extraction rate of the three major components of S. barbata was also significant (P <0.05).
The determination coefficient of the model (R 2 = 0.9246) showed that the model could explain 92.46% of the response value of the change, and that the model fitted better, confirming that the analysis could permit a forecast of the main components of S. barbata.The CCD-RSM was adopted with the extraction rate of scutellarin (a), luteolin (b) and apigenin (c) of integrate scores as evaluation index, optimizing the ultra high pressure extraction process.The results are shown in Table 3 and the ANOVA results are in Table 4.The integrated score = 0.5X 1 +0.3X 2 +0.2X 3 .
The results of regression equation coefficient are shown in Table 4, which showed that X 2 was very significant and X 1 , X 3 were not significant in this model, from the F values, it could be seen that the single factor of the order: X 2 > X 1 > X 3 , the ethanol concentration > extraction pressure > solid-liquid ratio; X 2 2 was significantly quadratic, the other factors were not significant.X 1 , X 2 , X 3 and their interaction effects on the response are shown in Figures 2 to 4, which directly reflected the interaction of various factors on the response values.Graphics showed that the solid-liquid ratio and ethanol concentration affected most significantly, the extraction yield of S. barbata, showing a steep curve; while extraction pressure had the lowest significant impact.With the increasing or decreasing of the extraction pressure, the response value of corresponding curves did not change significantly.

Verification test
The optimal extraction conditions were obtained by Design-Expert 8.0.4 software which was: extraction pressure conditions of 204.72 MPa, ethanol concentration of 61.44% and solid-liquid ratio of 1:78.79, then the integrate score of 7.15.2.0 g of the three samples of S. barbata were set in a sealed bag, and 157.6 ml of 61.44% ethanol was added accurately, sealed, pressurized to 204.72 MPa, for 90 s, treated according to the earlier mentioned methods, then with 0.22 µm filter membrane, chromatographic conditions were determined as earlier mentioned, the extraction efficiency of scutellarin, luteolin and apigenin were 12.38, 1.76 and 2.31 mg g -1 , respectively, and with integrated score of     Literature (Zhang et al., 2004) suggested that in the extraction process of some herbs whose cell walls are easy to rupture, 100 MPa pressure was enough to break the cell wall for most of the herbs.For maximum extraction, solvent is the most important factor, followed by the liquid -solid ratio, and lastly, the extraction time and pressure.When compared with the conventional methods, ultra-high-pressure extraction time is short, only 2 min, much lesser than conventional methods, and has the advantages of higher extraction rate, simple operation, low energy consumption etc.The active ingredient of S. barbata extract can prevent heat loss and pharmacological effects or activity loss.In the next step, the pharmacodynamic comparison validation by different extraction methods will be studied.This effective extraction technologies and low-cost raw materials represent an environmental and economical alternative to conventional extraction methods where large amounts of organic solvents and long extraction times are required.
The use of extraction technologies will reduce raw materials, processing wastes and facilitate the production of natural valuable products.Meanwhile, the crude extract should be subjected to further separation and purification.The composition and the pharmacological properties of flavonoids need to be further studied.
Furthermore, the UHP technique could be used in combination with other techniques such as ultrasonic extraction and enzymatic extraction to improve the extracting rate and efficiency.

Figure 2 .
Figure 2. Effects of alcohol concentration and extraction pressure on extraction yield.

Figure 3 .
Figure 3. Effects of solid-liquid ratio and extraction pressure on extraction yield.

Figure 4 .
Figure 4. Effects of solid-liquid ratio and ethanol concentration on extraction yield.

Table 3 .
Results of design/response surface methodology.

Table 4 .
Response surface regression analysis results.

Table 5 .
Comparison results for different extraction methods.