Maxim, a shrub in the family Loganiaceae, is distributed widely in the southwest and central regions of China. It was first recorded in “Kai Bao Ben Cao
”, a one thousand year-old work on traditional Chinese medicines. Buddleja officinalis
is known as “Mi Meng Hua
” in Chinese and it is listed in the Chinese Pharmacopoeia. The flower buds and inflorescences of this medicinal plant are commonly used for the treatment of eye diseases, including conjunctival congestion and soreness, photophobia, delacrimation, and cloudy and blurred vision, as well as hepatic asthenia [1
]. Recent research has shown that the main components of B. officinalis
are flavonoids, phenethyl alcohol glycosides, and saponins [2
], although alkaloids and carotenoids are also present in the plant. Linarin is the major flavone and an important bioactive constituent of this plant, and it was used as the index component of B. officinalis
in the 2015 edition of the Chinese Pharmacopeia. A small number of other constituents (acteoside, luteolin, etc.) have been also considered for quality control for B. officinalis
in earlier reports [6
], but those studies had some weaknesses. As is well known, Chinese herbal medicines typically have a variety of chemical components and the ingredients may have synergistic or antagonistic effects. One or two chemical components, therefore, do not reflect the true efficacy of the herbal medicine and establishing a quality evaluation system for the multi-index constituents of B. officinalis
is thus very necessary. To date, a method for the simultaneous quantification of multiple major compounds of B. officinalis
, including flavonoids, phenethyl alcohol glycosides, alkaloids, and carotenoids has not been reported.
It is widely acknowledged that optimal extraction methods are very important in the establishment of a multi-index component determination system. Both conventional (cold soak, reflux and Soxhlet) extraction methods and non-conventional (microwave-assisted and ultrasound-assisted) extraction methods have been used to extract active constituents from natural products. The optimal extraction method is selected using factors such as the extraction efficiency and the time taken for the extraction process. Many extraction parameters (e.g., number of extractions, solvent composition, extraction time and liquid to solid ratio) significantly affect the extraction efficiency of crude medicines.
The response surface methodology (RSM) is a statistical method for optimizing multivariate data, which is used widely to optimize extraction process [8
]. The principle of RSM is to establish a multiple quadratic regression equation to fit the functional relationships between factors and response values, and to then find the optimal process parameters by analyzing the regression equation [9
]. The function of RSM and single factor tests is similar, but fewer experiments are needed for RSM, thereby reducing the usage of solvents and time is required, RSM can also provide sufficient information on interactions between factors, whereas single factor tests cannot [10
]. Optimization of the extraction conditions for B. officinalis
has been reported previously [11
] but these studies only described the optimum conditions for the extraction of the total flavonoids or yellow pigments, and they did not identify single components. In the present study, the extraction of 11 active ingredients of B. officinalis
was simultaneously optimized for the first time.
The quality of Chinese herbal medicine is also influenced by different plant development stages and harvest time. In general, the harvest time has a great influence on the quality of medicinal materials. As illustrated by the old Chinese saying, “In March, Yinchen is a herb but useless straw in April, and firewood in May” [13
], the active components in Yinchen change during different seasons. Therefore, in the present study we attempted to identify the best harvest time for B. officinalis
The objectives of the present study were to optimize the extraction process, establish an HPLC-DAD method for quantitative analysis, and determine the best harvest time for B. officinalis.
3. Materials and Methods
3.1. Plant Samples
Buddleja officinalis Maxim samples (flower buds and inflorescences) used for optimizing the extraction conditions was purchased from Bozhou (Anhui Province, China). Samples during different flowering stages (bud, early flower, full blossom, and withering stage) were collected from Guizhou Province between March and April in 2016, and dried in the sun (according to the result of pre-experiments) until the moisture content was <12% (according to the stipulation for flower medicines in the Chinese Pharmacopoeia). Moisture content was determined using a Sartorius MA 35 rapid moisture meter (Sartorius AG, Göttingen, Germany) at 105 °C, using the method established in our laboratory. The experimental samples were ground into fine powders, sieved through a 60-mesh sieve, and then stored in a drier over silica gel.
3.2. Chemicals and Reagents
Reference substances, echinacoside (Y1), apigenin-7-O-glucuronide (Y5) and acacetin (Y11), were purchased from Baoji Herbest Bio-Tech Co. Ltd. (Baoji, China). Luteolin-7-O-rutinoside (Y2), acteoside (Y3), luteolin-7-O-glucoside (Y4), neobudofficide (Y6), linarin (Y7), N1,N5,N10-(Z)-tri-p-coumaroylspermidine (Y8), crocin III (Y9) and apigenin (Y10) were separated and purified (>98% by HPLC analysis) in our own laboratory and their structures were confirmed using MS and 1H-NMR and 13C-NMR spectroscopy. HPLC grade methanol and acetonitrile were purchased from Hanbon Sci. & Tech Co. Ltd. (Huaian, China) and Merck (Darmstadt, Germany), respectively. Purified water was obtained from Wahaha Group Co. Ltd. (Hangzhou, China). All of the other chemicals and solvents were analytical grade and were purchased from Nanjing Chemical Regents Co. Ltd. (Nanjing, China).
3.3. Ultrasound-Assisted Extraction (UAE)
Powdered samples (0.5 g) of B. officinalis were mixed with aqueous methanol (0–100%) using varying ratios of solvent to solid (10–50 mL/g) and extracted for different periods of time (20–50 min) at ambient temperature in 50-mL centrifuge tubes, before they were exposed to ultrasound using a KH5200DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co. Ltd., Kunshan, China). The mixtures were centrifuged at 1400× g for 10 min and the insoluble sludge was re-extracted. The supernatants were combined and diluted to 50 mL with the extraction solvent. Samples (1 mL) of the extracts were then filtered through a 0.22-µm microfiltration membrane before further analysis.
3.4. HPLC Analysis
HPLC analyses were conducted using an Agilent Series 1260 LC instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler, column temperature controller, DAD, quaternary pump, and online degasser. The analytes were separated using a Hanbon Megres C18 column (4.6 mm × 250 mm, 5 µm, Hanbon Sci. & Tech Co. Ltd., Huaian, China). The injection volume was 10 µL, the flow rate was 1.0 mL/min, and the column temperature was 25 °C. The detection wavelengths were set at 330 nm and 440 nm (maxima for crocin III) based on the the results obtained by full wave scanning. The mobile phase comprised solvent A (0.1% aqueous formic acid, v/v) and solvent B (acetonitrile) with the following elution gradient: 0–5 min (8–10% B), 5–10 min (10–18% B), 10–20 min (18–18.5% B), 20–34 min (18.5–30% B), 34–38 min (30–30% B), 38–41 min (30–37% B), 41–50 min (37–95% B), and 50–55 min (95–95% B). The total run time was 55 min and the time of column equilibration was 5 min. The mixed standard stock solution of 11 reference compounds was prepared by dissolving echinacoside (0.154 mg/mL), luteolin-7-O-rutinoside (0.061 mg/mL), acteoside (0.834 mg/mL), luteolin-7-O-glucoside (0.0255 mg/mL), apigenin-7-O-glucuronide (0.058 mg/mL), neobudofficide (0.1615 mg/mL), linarin (0.39 mg/mL), N1,N5,N10-(E)-tri-p-coumaroylspermidine (0.250 mg/mL), crocin III (0.254 mg/mL), apigenin (0.0625 mg/mL), and acacetin (0.029 mg/mL) in 60% aqueous acetonitrile. The solution was used for calibration and to calculate the linear correlation coefficient of the curve. The results were expressed as mg/g dry weight.
3.5. Validation of the HPLC-DAD Method
3.5.1. Linearity, LOD and LOQ
The mixed standard stock solution containing the 11 reference compounds was prepared as described in Section 3.4
. Then the standard solution was diluted to seven concentration levels using 60% acetonitrile to construct the calibration curves, and the linear relation is measured by the calculation of R2
. The LOD and LOQ represent the lowest concentrations that can be detected at signal-to-noise ratios of 3 and 10, respectively.
The intra-day precision was evaluated by injecting the same standard solution six times in one day and the inter-day precision was evaluated by injecting the same standard solution twice each day for three consecutive days.
The repeatability was tested by analyzing five sample solutions in parallel, which were prepared using the method described in Section 3.3
The stability was determined by measuring the peak area of a fresh sample solution at different time points (0, 2, 4, 6, 8, 12, 24 and 48 h).
The accuracy of the HPLC method was assessed by measuring the recovery rates. A previously analyzed sample of powder (0.25 g, accurately weighed) was spiked with the 11 standard compounds at 1 × concentration level (n
= 5). Solutions were prepared as described in Section 3.3
and then analyzed.
3.6. Experimental Design
3.6.1. Preliminary Experiments
In the preliminary experiments, five factors (extraction method, number of extractions, methanol/ethanol concentration, extraction time and liquid to solid ratio) were chosen for evaluation. Extraction methods were heat reflux, UAE and cold soak extraction; number of extractions was 1, 2 and 3; methanol/ethanol concentrations were 0%, 20%, 40%, 60%, 80% and 100%; extraction times were 20, 30, 40 and 50 min; liquid to solid ratios were 10:1, 20:1, 30:1, 40:1 and 50:1 mL/g.
3.6.2. Response Surface Methodology
The conditions for the extraction of B. officinalis
were optimized using RSM. The Box-Behnken design (BBD) was used to evaluate the effects of three independent variables (methanol concentration/X1
, liquid-to-solid ratio/X2
, and extraction time/X3
) on the extraction yield of the 11 compounds from B. officinalis
. Appropriate ranges for the methanol concentration (60–100%), liquid to solid ratio (20–40 mL/g), and extraction time (20–40 min) were selected based on the results of single factor tests. The three levels for each variable were coded as +1, 0 and −1 to denote high, middle, and low values, respectively. In total, 17 experimental runs were conducted, which comprised 12 factorial and five central point experiments. The results were fitted to a quadratic polynomial regression model as follows:
where Y represents the predicted response and Xi
are the independent variables. The regression coefficients of the intercept, linear, quadratic, and interaction terms are defined as b0
, and bij
The data generated by the RSM experiments were analyzed using Design Expert version 8.06 (Stat-Ease Inc., Minneapolis, MN, USA). The adequacy of the model was assessed by analysis of variance (ANOVA). The BBD outputs also contained contour and three-dimensional surface response plots, which indicated the relationships between the responses and independent variables.
3.7. Validation of the Model
The validity of the model was demonstrated based on comparisons of the experimental values obtained under optimal conditions and the predicted values based on the coefficient of variation (CV, %).
3.8. Statistical Analysis
Assays were performed in triplicate and data are expressed as the mean value ± standard deviation. The data generated by the RSM experiments were analyzed using Design Expert version 8.06. The adequacy of the model was assessed by analysis of variance (ANOVA). The BBD outputs also contained contour and three-dimensional surface response plots, which indicated the relationships between the responses and independent variables.