All chemicals and reagents used in the experiments had analytical grade purity. Methanol, acetonitrile, ethanol, chloroform, acetone, and ethyl acetate were purchased from Fisher (USA). Ammonium acetate, formic acid, phosphoric acid, and ammonia solution (25%), magnesium sulfate anhydrous (MgSO₄), sodium acetate (NaOAc), and sodium chloride (NaCl) were obtained from Fisher (USA). Primary Secondary amine and Graphitized Carbon Black were purchased from Sigma-Aldrich (USA). Physalin B and D reference standards (assigned purity 98%) were supplied by Wuhan ChemFaces Biochemical Company.

Fresh P. angulata were collected in the Can Tho City, Kien Giang, Ca Mau, Hau Giang provinces, Mekong delta and Binh Dinh province, Central Southern coastal region, Vietnam. The plants were identified at the Department of Biology, Can Tho University (scientific name as Physalis angulata L.). The leaf samples were collected separately, dried at room temperature, pulverized, and analyzed individually. All samples were stored in black glass containers and kept at room temperature. Dried samples were collected from traditional drugstores located in these provinces.

Individual standard stock solutions of Physalin B and D (1000 μg/mL) in methanol were prepared and were stable for approximately 12 months. Working standard solutions were prepared daily by diluting the stock solutions with methanol to provide different concentrations. The standard stock and working solutions were protected from light and stored at 4 °C.

Method development, quantification, and validation studies were performed on UFLC Shimadzu (LC-20AD), detector DAD SPD-M20A. Chromatography separation was performed on an Agilent C₁₈ column (250 mm × 4,6 mm; 5 μm) and monitored at 225 nm with the column temperature at 30 °C. The gradient elution program was shown in Table 1. The mobile phase consisted of acetonitrile (A), methanol (B), and water (D) and pumped at a flow rate of 1.0 mL/min.

The proposed method was validated for selectivity, linearity, the limit of detection (LOD), the limit of quantification (LOQ), precision, and accuracy according to the Association of Official Analytical Chemists (AOAC) and ICH guidelines.

Extraction method

When employed as a source of natural chemicals, ultrasound-assisted extraction (UAE) is an intriguing approach for obtaining highly valuable compounds that might contribute to the increased value of some food by-products. The major advantages will be more efficient extraction, which will save energy, and the use of moderate temperatures, which will assist heat-sensitive chemicals. To successfully use the UAE, it is required to examine the impact of numerous process factors, including the most important ones are the applied ultrasonic power, frequency, extraction temperature, reactor parameters, and solvent-sample interaction.

The UAE procedure was optimized about the solvent (chloroform, ethyl acetate, acetone, methanol, ethanol), solid-liquid ratio (1:5, 1:10, 1:15, 1:20, 1:25 g/mL), sonication time (5, 10, 15, 20 and 25 min), and extraction times (once, twice, third, fourth, and fifth) under investigation. The sum of the Physalin B and D peak areas was used to evaluate the extraction efficiency.

The results of the optimization of the sample preparation procedure are shown in Fig. 2. Based on the solubility of the analytes and referring to some related references, five extraction solvents were selected: methanol, ethanol, acetone, chloroform, and ethyl acetate. The survey results showed that the extraction efficiency of Physalin from the sample matrix was highest with methanol solvent. Therefore, methanol was selected as the extraction solvent.

Figure 2. 

Results of the optimization of the sample preparation procedure.

A higher volume of extraction solvent can dissolve analytes more effectively. Thus, the liquid-to-solid ratio is also an important factor during extraction. To evaluate the effect of this factor on the extraction yields, we examined different ratios ranging from 1:5 to 1:25 g/mL. It was found that the Physalin B and D contents increased with an increase in the solid-liquid ratio from 1:5 to 1:10 g/mL. From ratio 1:15 to 1:25 g/mL, the contents of analytes decreased.

For the ultrasonic-assisted extraction method, the ultrasonic time factor addition to having a great influence on the extraction efficiency. Specifically, when increasing the ultrasonic time from 5 minutes to 10 minutes, the extraction efficiency increased significantly before stabilizing at 15 minutes. However, when the ultrasonic time was selected at 10 minutes, the extraction times were investigated. After 3 times of extraction, the extract contained active ingredients. It is recommended to choose an ultrasonic time of 15 minutes for follow-up surveys. The data shown in Fig. 1 illustrate that the ideal number of extractions was three in order to extract more than 99% of the analytes from the P. angulata matrices.

QuEChERS method was a sample preparation approach developed by Anastassiades et al. as a simple, rapid, effective, yet inexpensive way to extract analytes from the matrix, followed by a dispersive solid-phase extraction (d-SPE) cleanup of the extract (Bruzzoniti et al. 2014). QuEChERS method involves an acetonitrile salting-out extraction of a solid sample in an aqueous environment followed by dispersive solid phase extraction (d-SPE) to remove a majority of the remaining matrix interferences.

For the matrices of herbal, the removing impurities step in the sample preparation is really necessary. Because this step avoids a decrease in column efficiency when injecting samples into the chromatography system. The extraction solvent of choice was methanol - a strong soluble solvent, dissolving the majority of polar metabolites along with intermediate and low polar compounds. Therefore, when extracted with methanol, these substances, especially polar impurities, are dissolved simultaneously. This poses a requirement that a suitable impurities removal method be developed that can reduce impurities while still retaining the majority of the analyte. Among the extraction methods that can be used to extract organic compounds from medicinal and plant samples, the QuEChERS method is becoming increasingly popular (Malysheva et al. 2020; Xu et al. 2017). This method is based on the decrease in solubility of organic substances dissolved in water as the salt concentration in the aqueous solution increases.

Acetonitrile (MeCN) is evaluated as a solvent that can extract many active compounds with higher efficiency and selectivity than acetone or ethyl acetate. Furthermore, proper miscibility with water allows good penetration into the aqueous fraction of the sample, also allowing relatively easy separation of the different phases by the addition of salts. Commonly used salt-forming agents are inorganic salts such as anhydrous MgSO₄, NaCl, and NaOAc to create separation between the water and MeCN layer and remove the residual water layer in the sample, these salts promote the transfer of compounds. MgSO₄ completely separates the liquid-liquid phase and is better able to bind large amounts of water, while NaCl can reduce the co-extraction of other components in the matrix and thus produce good chromatographic peaks.

Therefore, the combined use of MgSO₄ and NaCl has been studied for better results than using each salt separately. A ratio of MgSO₄: NaCl (4:1) has been shown to be the most effective in terms of selectivity and separation of aqueous and organic phases, maintaining high recovery. Table 2 shows the ingredients for surveying the process of removing polar impurities. To evaluate the purification efficiency of each procedure, the total peak of impurities and Physalin of the tested samples were compared. The result in Table 2 illustrated that using 1.2 g MgSO₄ and 0.3 g NaCl then cleaned with 50 mg PSA and 7.5 mg GCB gave the highest Physalin content.

The impurity peak on the chromatogram after applying the QuEChERS method was significantly reduced. The results are shown in the area of ​​the impurity peak and the Physalin peak (Fig. 3).

Figure 3. 

The results of removing polar impurities and clean-up samples.

Most of the studies focused on the quantification of Physalin B and Physalin D in herbals of the same genus Physalis such as Physalis alkekengi L., Physalis franchetii (Zheng et al. 2012; Laczkó et al. 2017) with different matrices, so the chromatographic conditions are also different. After referring to previous research, it has been shown that common mobile phase solvents for the analysis of physalins include Physalin B and Physalin D in P. angulata by liquid chromatography method and based on the nature of physalin chemistry and solubility of two analytes, choose to investigate mobile phase with the following components: Acetonitrile, methanol, and water.

In the studies of Arruda et al. (Arruda et al. 2021) and Manuela Oliveira de Souza et al. (De Souza et al. 2013), the optimal chromatographic condition was a gradient elution program with acetonitrile composition and 0.05% TFA/water. However, our research used water and acetonitrile as mobile phases, the peak parameters were still satisfactory. Our studies used an aqueous unbuffered mobile phase, which is both friendly to the chromatographic system and to the environment. Because of the problem of unstable baselines and overlapping peaks, a gradient program incorporating 3 solvent channels should be investigated. As a result, the mobile phase used consisted of acetonitrile (A), methanol (B), and water (D). The gradient elution program was shown in Table 1.

The best detection wavelength is when the analytes give the highest absorption and must be different from the absorption wavelength of the solvent to avoid baseline noise. According to studies analyzing Physalin B and Physalin D by HPLC/PDA method (De Souza et al. 2013; Arruda et al. 2021) chose to investigate wavelengths from 225 nm and 310 nm. Scanning the spectrum in the UV 190 - 400 nm region, the results showed that at 225 nm, the analytes gave good signals. So that 225 nm was selected as the detection wavelength of the analyte process.