Optimization of ultrasonic-assisted extraction followed by capillary zone electrophoresis for determination of celastrol concentration in Celastrus orbiculatus Thunb Ying Zhang1,2, Xin-sheng Fang1, Ying-jie Cui2, Shou-dong Guo2, and Jian-hua Wang1* 1 State Key Laboratory of Crop Biology, Shandong Key laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Taian, Shandong, People’s Republic of China; 2. Institute for Atherosclerosis in Taishan Medical College, Taian, Shandong, People’s Republic of China. Corresponding author: Dr. Jianhua Wang, College of Agronomy, Shandong Agricultural University, Taian, Shandong 271018, P. R. China; E-mail: sdauwangjh@163.com; Fax: +86-538-8242226 Abbreviations: C. orticulatus Thunb (Celastrus orbiculatus Thunb.); UAE (ultrasonic-assisted extraction); CZE (capillary zone electrophoresis); RSM (Response Surface Methodology); TCMS (traditional Chinese medicines); BBD (Box-Behnken design) 1 Abstract Celastrol is the main active compound of the plant genus Celastrus L., and has multiple biological properties. Unfortunately, the methods for accurate analysis and extraction of the compound remain poorly investigated. In this study, optimized ultrasonic-assisted extraction (UAE) and efficient capillary zone electrophoresis (CZE) analysis methods were developed for rapid extraction and simultaneous determination of celastrol concentration in Celastrus orbiculatus Thunb. The UAE process was optimized using a central composite experimental design. The optimal conditions for celastrol were 80% methanol, a solvent to material ratio of 20 mL/g, and an extraction time of 50 minutes. The CZE analysis method was validated as having good linearity, precision, reproduction, and accuracy. Using optimized UAE and CZE methods, the concentration of celastrol in root, stem, bark, and leaf of C .orticulatus Thunb. was found to be 5.132, 1.513, 0.324, and 0.343 mg/g, respectively. UAE-CZE is a fast, convenient, and appropriate method for determination of celastrol concentration in C. orticulatus Thunb. These results could be valuable for breeding a new variety of Celastrus L. species, which may have application in the clinic, pharmacy, and in research. Keywords: Celastrus orbiculatus Thunb., ultrasonic-assisted extraction, capillary zone electrophoresis, celastrol, response surface methodology 2 1. Introduction Celastrus orbiculatus Thunb. (C. orticulatus Thunb.) is a perennial creeping plant, belonging to the Celastrus L. family, and is one of the traditional Chinese medicines (TCMs). It is distributed northeast, northwest, southwest, and north of China, as well as in Korea and Japan [1]. Its stem, root, leaf, fruit, and seed are all TCMs. Studies have shown that C. orticulatus Thunb. contains many compounds such as flavones, terpenes, alkaloids, and organic acids [2]. It has many properties such as sedative, hypnotic, pesticide, antitumor, antibacterial, anti-inflammatory, and antioxidation [3]. Its roots have been used as a traditional herbal medicine to treat fever, chills, joint pain, edema, rheumatoid arthritis, and bacterial infection in Chinese folk medicine [4]. Pharmaceutical studies and clinical practices have demonstrated that a number of sesquiterpenes and triterpenes, including celastrol, possess notable antibacterial, anti-tumor, insect antifeedant and cytotoxic activities [5]. Celastrol, a quinone methide triterpenoid isolated from the Celastraceae family, is the main triterpenoid component of C. orbiculatus Thunb. It has been used for years as a natural remedy for inflammatory conditions, and exhibits various biological properties, including chemopreventive, antioxidant, antitumor and neuroprotective effects [6]. Celastrol has been shown to inhibit IL-1 release from lipopolysaccharide (LPS)-stimulated human peripheral mononuclear cells [7,8], and has also been shown to induce suppression of TNF-induced NF-кB activation [9,10]. Celastrol also attenuates hypertension-induced inflammation and oxidative stress in vascular smooth muscle cells (VSMCs) via heme oxygenase (HO-1) induction; thus, it may serve as a novel drug for treating hypertension [11]. Recently, celastrol, regarded as a potent antioxidant and anti-inflammatory drug, was used as a hopeful treatment for Alzheimer’s disease (AD) [12]. In addition, celastrol showed the most potent inhibitory activity in a reporter gene 3 assay, with an IC50 of 0.27 µM [13]. In addition, it is a natural proteasome inhibitor that has great potential for cancer prevention and treatment [14]. In view of its widespread biological activities, the extraction of celastrol has been of much interest to pharmaceutical chemists. Conventional methods for extracting celastrol from herbal drugs are hot reflux extraction (HRE) and soxhlet extraction (SE), and conventional solvents used for celastrol extraction are ether, ethanol, chloroform, and methanol [15,16]. Much attention has been given to the application of ultrasonic-assisted extraction (UAE) in the life and environmental sciences [17]. Through cavitation, UAE disrupts the cell membranes of plants, increasing the spread of solvent into the cell and efficiently stripping it of its contents, thereby shortening extraction time and improving extraction efficiency [18,19]. Thus, UAE has been of interest for extracting active compounds including flavonoids [20], alkaloids [21], anthraquinones [22], total lignans [23], saponins [24], naphtha [25], and other components [26-28] from herbal medicines or TCMs. Classic optimization studies use a one-factor-at-a-time approach, in which only one factor is variable at a time, while all others are kept constant. However, this approach is time-consuming and expensive. Response surface methodology (RSM), a multivariate statistic technique, can overcome this problem [29], as it accounts for possible interaction effects between variables. Thus, optimizing the extraction process using RSM by establishing a mathematical model would not only give a clearer understanding of the effects of various factors on extraction, but would also help locate the optimal region of extraction. In addition, RSM has been successfully used to model and optimize the extraction process of active components in herbal drugs [30–32]. Previous methods for determination of celastrol concentration have been HPLC [33] and HPLC –MS [34-36]. Until now, no paper has been published on the extraction and determination 4 of celastrol concentration in TCMs or herbal drugs using UAE and capillary zone electrophoresis (CZE). The objective of this study was to develop rapid, reliable, and reproducible UAE for the rapid extraction and simultaneous CZE determination of celastrol concentration in C. orticulatus Thunb. To achieve this objective, the operational parameters of the UAE procedure (extraction time, solvent to material ratio, and solvent concentration) were optimized applying RSM. Furthermore, the celastrol concentration in different parts of C. orbiculatus Thunb. was determined with the developed UAE and CE methods. 2. Materials and Methods 2.1 Reagents C. orbiculatus Thunb. was collected from Taishan Mountain in China, and chipped to slices (40 mesh). All slices were dried at a normal temperature until constant mass was observed. Standard celastrol was obtained from Tianjin Yifang Science and Technology Co. (Tianjin, China); the purity of the standards was above 98% (w/w). Sodium borate (analytical grade) was purchased from Tianjin Bodi Chemical Co. and methanol (HPLC grade) was from Fisher Co. (USA). Double-distilled water was made with Milli-Q SYSTHESIS Ultrapure Water System (Millipore Co., France) in our laboratory. 2.2 CZE apparatus Celastrol was quantified using a Quartz Uncoated fused silica capillary column, 75 µm (id)× 50.2 cm (total length) and 40 cm (effective length, and a Beckman P/ACETMMDQ CZE system (Beckman Coulter Co., USA) equipped with a PDA detector. The range of voltage used was 0 to 30 kV. Data acquisition was carried out with 32 Karat TM software, version 5.0 (Beckman Coulter 5 Co. USA). Optimization conditions were 10% methanol, 70 mM sodium borate buffer solution (pH, 9), 22 kv separation voltage and a UV detection wavelength of 210 nm. Under these conditions, the compound was well separated within 15 minutes. The chromatogram of celastrol is shown in Figure 2. 2.3 Methods 2.3.1 Preparation of standard curve Celastrol (1.17 mg) was precisely weighed and dissolved in methanol in a 5.0 ml volumetric flask, to a final stock solution of 0.234 mg/ml. We then made serial dilutions with methanol to obtain different working concentrations. All solutions were stored under refrigeration (4℃). Absorbency was determined at room temperature and a standard curve was obtained. 2.3.2 Extraction methods Slices of C. orbiculatus Thunb. were crushed and sieved using a 40 mesh standard sieve. Ultrasonic-assisted extraction (UAE) was conducted in an ultrasonic bath (Shumei® KQ-600DB ultrasonic instrument, Kunshan, China). The effect of single factors including solvents, solvent to material ratio, and extraction time on celastrol yield was first evaluated. Based on the single factor experiments, Box-Behnken design (BBD) experiments were used to optimize the UAE extraction process. Sample solutions were centrifuged at 5000 rpm and filtered through a 0.22 µm membrane filter, (then was injected in 5s)-comment. Quantitative determination of the active compounds in the extracts was performed using external standards by means of a five-point calibration curve. 6 2.3.3 Box-Behnken design Based on the single factor experiment, a central composite design was used to design UAE tests to explore the variables that affect ultrasonic extraction. The variables considered in UAE optimization were solvent concentration, solvent to material ratio, and extraction time. Three levels of each variable were chosen for the trials, including one central point and two axial points [37], and each factor chosen were marked as +1, 0, or -1. Each factor was evaluated at the following intervals: 70–90% for solvent concentration, 10–20 mL/g for solvent to material ratio, and 40–60 minutes for extraction time. The experimental factors and levels are shown in Table 1. 2.3.4 Software The construction and analysis of the experimental design, and the response surface for reaching the optimal conditions, were carried out using the Statistical Analysis System Design-expert software, version 7.1.6 Trial. 3 Results and Discussion 3.1 Effect of solvents on extraction yield Three grams of stem powder (40 mesh) were put into a 100 mL glass iodine flask, and soaked for 20 minutes with 30 mL of different concentrations of methanol (30%, 50%, 70%, 80%, 90%, and 100% methanol), followed by extraction with UAE for 50 minutes at 30℃. The results (Fig. 3) indicated that celastrol yield slowly increased when methanol concentrations exceeded 80%, and there was no significant difference in yield between 80% and 90% methanol (p>0.05). Therefore, 80% methanol was chosen as the solvent. 7 3.2 Effect of solvent to material ratio on extraction yield Three grams of stem powder (40 mesh) were extracted with 80% methanol for 50 minutes with different solvent to material ratios (1:5, 1:10, 1:15 and 1:20) at 30℃. The results are shown in Figure 4. Celastrol yield clearly varied under different solvent to material ratios, and reached its maximal yield when the solvent to material ratio was 1:15. Therefore, 1:15 was chosen as the optimal solvent to material ratio. 3.3 Effect of time on extraction yield Three grams of stem powder (40 mesh) were extracted with 80% methanol for 10, 20, 30, 40, 50 and 60 minutes at 30 C, and at a solvent to material ratio of 1:15. The results are shown in Figure 5. Maxium celastrol yield was obtained at an extraction time of 50 minutes. There was no significant difference in celastrol yield (p>0.05) between 50 and 60 minutes. Therefore, 50 minutes was chosen as the optimal extraction time. 3.4 Optimization of UAE using RSM Fifteen experiments were carried out according to the conditions shown in Table 2.The extraction yield (mg/g) is also shown in Table 2. All experiments were performed randomly to minimize the effects of uncontrolled factors that could introduce bias into the measurements. Each analysis was performed in triplicate. This model was used to obtain a surface response fitting the data to a polynomial model, the evaluation of the effects of each factor and also the interaction effects between factors.-comment Equation 1 shows the obtained model for celastrol. YCL= 5.44295-0.25397 X1+0.100195 X2+0.042788 X1X3-0.00116 X2 X3+0.001968 X12-0.00557 X22-0.0004 X32 8 X3+0.002044 X1X2+0.000182 Analysis of variance (ANOVA) for the fitted quadratic polynomial model is presented in Table 3. The proposed mathematical model was significant (p<0.05), and lack of fit was insignificant with a coefficient of determination (R2) of 0.903. Therefore, the model was accepted and could be used for analyses and forecast for celastrol. The extraction solvent showed a largely significant positive effect on celastrol yield (p<0.005). The solvent to material ratio and extraction time showed insignificant (p>0.05) effects on celastrol yield. The fitted surfaces and contours are shown in Figures 6-8, and can be used to appraise the (mutual effects on the concentrations of celastrol about experimental factors).comment The shape of the contours can reflect the strength and firmness of the mutual effects. An oval shape represents strength, roundness, and firmness. In the case of roundness, the mutual effect can be ignored [38]. The three-dimensional plot (Fig. 6) of the interaction methanol concentration × solvent to material ratio shows that the extraction yield of celastrol increased with increasing methanol concentration. However, the solvent to material ratio did not have a significant effect. The same effect of methanol concentration and extraction time can be seen in Figure 7. The slope of the response surface of solvent to material ratio was bigger than the extraction time. However, in Figure 8, the maximum celastrol yield was obtained under an extraction time of about 50 minutes and a solvent to material ratio between 15-20 mL/g .By resolving the model, the optimization condition was methanol concentraction was 80%, the solvent to material ratio was 20 mL/g, and the extraction time was 50 minutes. The predicted value of celastrol yield of celastrol using this model was 1.4750 mg/g. 3.5 Validation of the analysis method This is the first time that UAE and CZE have been applied for the extraction and simultaneous 9 determination of celastrol concentration in C. orbiculatus Thunb. The CZE chromatograms of standard compounds and samples are shown in Figure 2. The chromatographic method presented good linearity in the concentration range considered. The regression equation of celastrol was YCL=543282 x+1967.2 (r=0.9920) with a linear range of 0.039~0.117 µg/µL. The limit of detection (LOD) was used to evaluate sensitivity of the analysis method. LOD is defined as the lowest concentration at which the signal is larger than three times the baseline noise (S/N=3). The measured LOD value for celastrol was 1.21 mg/mL. Intraday and interday variations were used to determine the precision of CZE method. Intraday variation was determined by analyzing in triplicate the same mixed standard solution five times within one day, while for the interday variability test, the standard solution was examined in triplicate for three consecutive days. The RSD of the retention time (TR) and peak area (AP) were taken as measures of precision. The RSDs of TR and AP for intraday were 1.15% and 2.09%, respectively, and for interday were 1.37% and 2.84%, respectively. Reproducibility of the novel extraction and CZE method was determined by analyzing six replicates under optimal extraction conditions in one day. The RSD of the compound was 1.92% (n=6). A recovery test was used to evaluate the accuracy of this method. The recovery of celastrol was 101.13%, with a RSD<4.00% (n=6). 3.6 Verification of UAE and application of the developed method To test the optimized conditions for celastrol, 3 g stem (40 mesh) were extracted under the optimized conditions. The celastrol concentration in the stem of C. orbiculatus Thunb. was 1.513 mg/g, compared to the predicted value 1.475 mg/g; the error was 2.56%. The regression equation may have reflected the effects of the three factors on celastrol extraction of celastrol. Celastrol 10 concentrations in different parts of C. orbiculatus Thunb. were also determined using the developed UAE and CZE methods. It could be seen that the concentrations of celastrol in the root, bark, and leaf of C. orbiculatus Thunb. were 5.132,1.513, 0.324, and 0.343 mg/g, respectively. The concentration of celastrol in the root was three times that in the stem, and sixteen times that in the bark and leaf. These results are valuable for breeding a new variety of Celastrus L. species, which may have application in the clinic, pharmacy, and in research. 4. Concluding Remarks UAE is a physical process of extraction [17], and CZE is an analytical technique with the advantage of a high resolution and the fact that it consumes little solvent [39]. An optimal UAE method and an efficient CZE method have been used for extraction and simultaneous determination of celastrol concentration in C. orbiculatus Thunb. The developed CZE analytical method was validated as having good linearity, precision, and accuracy. Thus, the combination of UAE–CE is a faster, convenient, and appropriate method for quantitative analysis of celastrol in C.orbiculatus Thunb. Furthermore, there is significant variability in the concentrations of celastrol in different parts of C.orbiculatus Thunb, which is valuable for breeding new varieties of Celastrus L. species, which may be applicable in the clinic, pharmacy, and in research. Acknowledgements We greatly acknowledge the financial support from the Taishan Scholar Fund of Shandong province (No. 67 [2008]) and Natural Science Foundation of Taishan Medical college (No. 0104 [2010]). Figure Legends Fig. 1 Chemical structure of Celastrol 11 Fig. 2 Chromatogram of Celastrol in standard and samples(A:standard B: stem ) Fig. 3 The effect of methanol volume fraction on yield Fig. 4 The effect of yield of solvent to material ratio on yield Fig. 5 The effect of extraction time on yield Fig. 6 Fitted surface and contour map for the yield of celastrol as a function of methanol concentration (X1) and solvent to material ratio (X2). (extraction time=50 min) Fig. 7 Fitted surface and contour map for the yield of celastrol as a function of methanol concentration (X1) and time (X3). (solvent to material ratio =15 mL/g.) Fig. 8 Fitted surface and contour map for the yield of celastrol as a function of solvent to material ratio(X2) and extraction time(X3) . 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