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) . (80% methanol concentration)
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