1. Introduction
Andrographis paniculata Nees, an annual herb in the Acanthaceae family, is one of the most famous herbal resources extensively used as a traditional medicine in China, India, Thailand, and Scandinavia for prevention and treatment of fever, dysentery, diarrhea, inflammation, sore throat, and snakebites [
1,
2]. Furthermore, it is a promising new way for the treatment of several serious diseases, including HIV [
3], AIDS [
4], and numerous symptoms associated with immune disorders [
1,
3]. The main active compounds in
A. paniculata are believed to be the diterpenes, and andrographolide (AP
1) and dehydroandrographolide (AP
3,
Figure 1) are the important and major diterpenoids in
A. paniculata with a wide range of pharmacological activities, such as antiviral [
3], anti-inflammatory [
5], hepatoprotective [
6], anticancer [
7], immunostimulant [
7], antiangiogenic [
8] and antihyperglycemic [
9].
Figure 1.
The chemical structure of andrographolide (AP1) and dehydroandrographolide (AP3).
Figure 1.
The chemical structure of andrographolide (AP1) and dehydroandrographolide (AP3).
Heat reflux extraction (HRE) is the most widely used conventional technique for the extraction of diterpenoids from
A. paniculata. However, HRE causes the consumption of large amounts of volatile and hazardous organic solvents, and needs long extraction times and consumes more energy. Besides, the extraction efficiency of diterpenoids by conventional extraction method is not satisfactory [
1,
10]. Novel methods for the extraction of diterpenoids including supercritical carbon dioxide extraction [
10], micellar extraction [
11] and microwave assisted extraction [
12] have drawn significant research attention in the past decade. However, complex equipment construction, high equipment expenditure and low material throughput have made them difficult in industrial scale-up applications [
13]. Therefore, it is important to improve conventional extraction technique and establish an efficient, simple and fast extract method for industrial scale-up applications.
Vacuum assisted extraction (VAE) technology is developed based on pressurized liquid extraction (PLE) except in the vacuum controlled region [
14]. A VAE device for the extraction of active compounds from traditional Chinese medicine has been designed in our research group [
15]. It is a cheap, simple, fast and efficient method which can be implemented by simply upgrading the conventional equipment with the addition of a vacuum controller device. Like PLE, VAE can accelerate the release of solutes from the plant matrix by the vacuum assisted breakdown of cell components, and facilitate the solid-liquid mass transfer between the extraction solvent and matrix. The working pressure is stable and adjustable from 0 to 1000 mbar. In the VAE system, the extraction process is carried out at a temperature below the boiling point of the solvent by adjusting the vacuum setting of the system. The boiling temperature of the solvent is relative with the saturated vapor pressure above the solvents, so boiling at low temperatures can be realized by adjusting the system vacuum. Moreover, under vacuum, small bubbles appear and ascend among the liquid-solid phase, resulting in the violent movement of solvent and further improving the liquid-liquid mass transfer.
Nowadays, there is some literature describing vacuum-assisted extraction technologies, including vacuum microwave assisted extraction [
16,
17] and vacuum ultrasoound-assisted extraction [
18], which could enhance the extraction efficiency and reduce the extraction time. Nevertheless, most of the studies are based on novel extraction techniques including ultrasonic-assisted and microwave-assisted extraction, which are difficult to use in industrial scaled-up applications due to the complex equipment construction [
13]. HRE is still the most widely used conventional technique for the extraction of bioactive components from Chinese herbs in industry production processes. To our knowledge, there are no studies of vacuum-assist extraction methods based on the heat reflux extraction technique. The objective of this study was to explore the feasibility of VAE for extraction of diterpenoids from
A. paniculata. The effects of boiling temperature, ethanol concentration, extraction time, extraction cycles, and ratio of liquid to solid were investigated. Response surface methodology (RSM) was used to build a model between the yield value and these independent variables, and to optimize the extraction conditions of diterpenoids from
A. paniculata in order to provide valuable information for industrial purposes.
3. Experimental Section
3.1. Materials
A. paniculata was obtained from a local drugstore in Nanchang (China). Reference samples of andrographolide (AP1) and dehydroandrographolide (AP3) were supplied by the National Institute for the Control of Pharmaceuticals and Biological Products (Beijing, China). Ethanol (analytical grade), acetonitrile (chromatographic grade), and formic acid (chromatographic grade) were obtained from local chemical suppliers.
3.2. Apparatus
An Agilent 1200 HPLC system equipped with a quaternary solvent delivery system, an autosampler and variable-wavelength ultraviolet detector (VWD) (Agilent Technologies, Santa Clara, CA, USA) was used for HPLC analysis. A Phenomenex reversed-phase Gemini C18 column (250 × 4.6 mm, 5 μm) and a Phenomenex C18 guard column (Phenomenex, Torrance, CA, USA) were used for all chromatographic analysis. VAE experiments were carried out with a V850 Vacuum Controller (Büchi Labortechnik AG, Flawil, Switzerland).
3.5. Response Surface Methodology (RSM)
RSM was applied in two stages, first to identify the significant factor for extraction efficiency of AP1 and AP3 and yield of extracta sicca using a Placket-Burman design and later the significant variables resulted from the Placket-Burman design were optimized by using a Box-Behnken design. All experiments aimed at optimizing the conditions of extraction for greatest efficiency. Each experiment was conducted in triplicate and the average yield value of AP1, AP3 and ES was used for statistical analysis.
The experiments were carried out in random order to avoid systematic errors. The statistical software package Design-expert 8.0.6 was used for regression analysis of the data and estimation of the regression equation coefficients. A second-order response function was applied to establish an empirical model that relates the response measured to the independent variables. This is shown in Equation (6):
where
Y is the measured response variable,
b0 is a constant,
bi is the linear coefficient,
bii is the quadratic coefficient,
bij is the two factors interaction coefficient, and
Xi and
Xj are independent variables of the system. The quality of the fitted polynomial model was expressed by the coefficient of determination (
R2), and its statistical significance was checked by
p-test and
F-test.
3.6. Optimization
A desirability function approach was used to optimize the three responses simultaneously. Each response can be assigned a significance degree relative to the other responses:
where
di is the partial desirability function of each response obtained from the transformation of the individual response of each experiment.
ri is the significance degree of each response.
4. Conclusions
In this study, an effective vacuum assisted extraction method was successfully used to extract diterpenoids from A. paniculata. RSM with a Placket-Burman design, Box-Behnken design and desirability function were employed to get the optimal extraction conditions for diterpenoids in a quick and economical way. Coefficient of determination of the three models suggested good fit. Compared with conventional HRE, bioactive components can be efficiently extracted from A. paniculata by the optimal VAE conditions (boiling temperature 65 °C, ethanol concentration 50%, extraction time 16 min, extraction cycles 1 and liquid-solid ratio 12:1) and the extraction yields of AP1 and AP3 was 137.7% and 122.1%, the yield of ES was 8.0%. The experimental results proved a good accordance with the predicted values. The optimized VAE not only accelerated the extraction rate and improved the efficiency of the extraction yield of bioactive components, but also shortened the extraction time and energy compared to conventional HRE, which shows great potential for becoming an alternative technique for industrial scale-up applications.