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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 2 | Page : 167-177
Extraction, purification, and anti-Inflammatory activity of steroid fraction from Physalis Alkekengi L. Var. Franchetii (Mast.) makino
Tian-Yi Xia1, Yi Wang2, Yan-Ni Yang1, Wu-Jing Wang1, Zi-He Ding1, Ren-Xing Zhong1, Ying Chen1, Wei Li1, Ming-Ming Peng1, Chuan-Qiu Li1, Li-Feng Shang3, Bing Liu4, Zhen-Yue Wang5, Chong-Rong Shi6, Zun-Peng Shu1
1 Guangdong Standardized Processing Engineering Technology Research Center of Traditional Chinese Medicine; Department of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, P.R. China
2 Guangdong Standardized Processing Engineering Technology Research Center of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, P.R. China
3 Guangdong Andao Medical Equipment Co. Ltd, Guangzhou, P.R. China
4 Department of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, P.R. China
5 Department of Pharmacy, Heilongjiang University of Chinese Medicine, Heilongjiang, P.R. China
6 Macau Association of Chinese Medicine (AIPPMCM), Macau SAR, P.R. China
Correspondence Address:
Dr. Zun-Peng Shu
Guangzhou Higher Education Mega Center, Guangdong Pharmaceutical University, No. 280, Guangzhou 510006, Guangdong Province
P.R. China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.372143
Objective: As a traditional medicinal plant listed in the Chinese Pharmacopeia, Physalis alkekengi L. var. franchetii (Mast.) Makino (PAF) has a long medicinal history and high economic value. PAF has immunomodulatory properties and can be used to treat acute lung injury and eczema. The aim of this study is to solve the problems of extraction and purification of active components from PAF. Materials and Methods: The solvent to be used for extraction and its concentration, the solid-to-liquid ratio, and extraction duration were investigated using a single-factor experiment. An orthogonal design (L9[34]) was used to determine the optimum extraction conditions. After optimization, the sample's concentrations and flow velocity, the eluents and their velocity, adsorption time, and the removed water volume were measured. The content of the five steroids in the sample was determined by high-performance liquid chromatography (HPLC). We also investigated the anti-inflammatory property of PAF calyxes before and after purification. Results: The optimum extraction and purification processes were determined by single-factor analysis. AB-8 was identified as the best macroporous adsorption resin for enrichment. After optimization, the average total steroid content was 71.83%, and the average recovery was 90% after purification. Among the five steroid components detected by HPLC, physalin F showed the highest content. Furthermore, the sample obtained after purification could significantly inhibit paw edema by egg whites induced. Conclusions: An environmentally-sustainable, efficient, and stable process was first optimized for enriching and purifying total steroids from PAF. The process has the potential for further development and utilization in the pharmaceutical industry.
Keywords: Anti-inflammatory activity, extraction and optimization process, physalins, Physalis alkekengi L. var. franchetii (Mast.) Makino, steroids
Physalis alkekengi L. var. franchetii (ast.) Makino (PAF) is a plant of the Physalis genus of the Solanaceae family known as “Jin-Deng-Long” (锦灯笼) in China, also popularly named “Gua-Jin-Deng” or “Hong-Gu-Niang” due to its similar appearance to a red lantern. As an edible medicinal homologous plant, PAF gives a ripe fruit with a slightly sour and bitter taste that contains a variety of Vitamins. The fruit can be eaten raw or processed into food or health products as fruit pulp. PAF, which is widely used in Chinese medicine, is recorded as Calyx Seu Fructus Physalis in the Chinese Pharmacopeia, 2015 edition for the treatment of throat pain and dysphonia, phlegmy cough, pemphigus, and eczema, among other ailments.[1] Moreover, PAF calyxes can be used as a medicine to clear heat, detoxify the body, and relieve pharyngitis.[2] Several pharmacological studies have also shown that PAF has anti-inflammatory properties, reduces acute lung injury, and boosts immune activity.[3],[4],[5]
To date, the chemical components known to be isolated from different parts of PAF include steroids, flavonoids, sterols, and alkaloids.[6] Steroid components, including steroid hormones, steroid alkaloids, cardiac glycosides, and steroid saponins,[7],[8] are widely used in the treatment of rheumatism, cardiovascular diseases, collagen diseases, lymphatic leukemia, organ transplantation, tumors, bacterial encephalitis, skin diseases, and endocrine disorders, among other diseases. Different steroid components are widely present in various plants.[9] In our previous work, we found that the steroidal component of PAF, physalin, has good antibacterial and anti-inflammatory properties.[10]
The extraction and purification of active ingredients are essential preliminary steps for the study of medicinal plants. Macroporous resins are organic polymers that are insoluble in acids, alkalis, and various organic solvents and have high adsorption properties. Compared with other traditional separation and purification techniques, macroporous resins offer many technical advantages such as simple operating procedures, environmental friendly processes, excellent selectivity, low production cost, and convenient regeneration.[11] Recently, macroporous resins have been successfully used for the separation and enrichment of bioactive compounds from a large number of natural products.[12],[13],[14] However, to date, there have been no reports on the separation and enrichment of total steroids from PAF using macroporous adsorption resins. Therefore, the purpose of this work was to develop a simple, environmentally friendly, and cost-effective purification method for the separation and purification of steroids from PAF by macroporous adsorption resin column chromatography. Furthermore, the anti-inflammatory properties of PAF samples were studied before and after purification. This study provides proof of concept for a novel process that could potentially be used for standard industrial production of PAF steroids and paves the way for their clinical application.
Materials and MethodsChemicals and apparatus
Hydrocortisone (Tianjin Jinyao Amino Acid co. LTD, Tianjin China), 1.5% sodium carboxymethyl cellulose (CMC-Na) solution, Chemicals and reagents were analytical grades. Physalin J, Physalin B, Physalin O, Physalin F, and Physalin P were prepared by our project team, and the purity reaches more than 98%. Methanol and acetonitrile were high-performance liquid chromatography (HPLC) grades (Dima Technology Co. Ltd, Beijing, China). Phosphoric acid (Hongxing Chemical Plant, Beijing, China). UV-756 spectrophotometer (Shanghai spectrum instruments co. LTD, Shanghai, China). HPLC-LC-2010A (Shimadzu Corporation, Shanghai, China), C18 column (5 μm, 150×4.6 mm, Shimadzu, Shanghai, China).
Plant material and adsorbents
In the present research, the calyxes of PAF were mainly collected from Heilongjiang province. All of these were identified by Prof. Li Shuyuan (Guangdong Pharmaceutical University, Guangzhou, China). Plants were stored for a maximum of 25 weeks.
Macroporous resins including HPD-100A, HPD-200B, HPD-700, DM130, HPD-500 (Baoen chemical co., ltd, Cangzhou, China), D-101 (Huida Chemical Co., Ltd, Tianjin, China), AB-8 (Chemical Plant of Nankai University, Tianjin, China). The chemical and physical properties of the tested resins according to the manufacturer are tabulated in [Table 1]. The resins were leached with 95% ethanol for 24 h. Then, the resins were washed with ethanol until an equal amount of distilled water was added to the effluent, and no white turbidity was observed. Finally, the resins were washed with deionized water until the outflow liquid did not smell like ethanol.
Table 1: Physical property of seven kinds of macroporous adsorption resinAnimals
All animal experimental protocols were approved by the Animal Care Ethics Committee of Guangdong Pharmaceutical University (certificate no.SCXK 2018–0022). One-hundred and two healthy adult standard deviation (SD) rats weight (180±20) g, both male and female animals were housed under standard laboratory conditions: humidity at 50% ± 5%, the temperature at 22°C ± 2°C, alternating 12 h light and dark cycles. All animals were given standard rodent chow, and allowed free access to water. After experiments, all rats were killed by cervical dislocation.
Optimization of the extraction process
Selection of extraction method
Took 15.0 g of dried PAF and divide it into three groups. Added 200 mL of 70% ethanol in each group. Afterward, the first group of PAF was extracted with ultrasonic extraction two times, 20 min each time. The second group was extracted with reflux extraction two times, 2 h each time. The last group was extracted twice by cold soaking, 48 h each time. The three groups were separately filtered and the filtrates were combined. The solvent was extracted and recovered by vacuum to obtain extracts. Calculated contracts yield and total steroid content in the extracts to determine the best method.
Single-factor experiments
To study the effect of ethanol concentration on extraction efficiency, 5.0 g of pretreated samples, extracted them with 200 mL of water and different concentration of ethanol (30%, 50%, 70%, and 90%) twice by heat reflux extraction, sustained for 2 h each time. To choose the optimum solvent amount (solid-to-liquid ratio), different solvent amount (20, 30, 40, 50, 60 multiples) were tested under the condition of extracted by 70% ethanol twice and each time for 2 h. To select the best extraction duration, different extraction duration (0.5, 1, 1.5, 2, 2.5, and 3 h) were tested with 200 mL of 70% ethanol, and extracted twice. Finally, different extraction times (1, 2, 3, and 4 times) on extraction efficiency were evaluated under conditions of 200 mL of 70% ethanol and extracted for 2 h. After each experiment, filtered and combined the filtrates. The solvent was extracted and recovered by vacuum to obtain extracts.
Orthogonal experiment
Based on the results of each single-factor experiment, respectively, selected three levels with favorable extraction efficiency from the four influencing factors: ethanol concentration, solvent amount (solid-to-liquid ratio), extraction duration, and extraction times. The L9 (34) orthogonal table was used for the orthogonal experiments. The experimental conditions of orthogonal experiments were listed in [Table 2], and weighed 5 g of material each time for experiment. Performed two parallel experiments. Through intuitive analysis, analysis of variance, and significance test of the results, the optimal extraction process of the total steroids of PAF was elected.
Measurement of total steroids content
9.66 mg of physalin F reference substance, added a small amount of methanol to dissolve it, and then, methanol was added up to 100 mL to obtain a reference solution with a concentration of 96.6 μg/mL. Measured 1.0, 2.0, 3.0, 4.0, and 5.0 mL of this solution, placed them in 10 mL test tubes with a stopper, and dried the methanol in a water bath. Subsequently, 0.2 mL of 5% vanillin-glacial acetic acid, 0.8 mL of perchloric acid was added, and water bath was at 70°C for 15 min. After taking out, the ice bath for 5 min, added 5 mL of glacial acetic acid. Through preliminary research, there is a maximum wavelength of 522 nm, so we used methanol as the blank, measured the absorbance at a wavelength of 522 nm. Besides, 6.6 mg of steroid extract was dissolved in water at a constant volume of 10 ml to obtain the sample solution with a concentration of 0.66 mg/mL. Using the above method, the maximum absorption was also found at 522 nm. Linear regression was conducted with concentration as abscissa and absorbance as ordinate. The regression equation was expressed as y = 13.333 × −0.0206 (R2 = 0.9998). The linear range was 9.66–48.3 μg/mL.
Screening of resin
Static adsorption and desorption test
Precisely weighed 5 g of each of the seven pretreated macroporous resins HPD-100A, HPD-200B, HPD-700, D-101, AB-8, DM130, HPD-500 (after removing water). Each of them was placed into a 250 mL Erlenmeyer flask with a stopper and added exactly 100 mL of total steroids aqueous solution, which known total content of 0.033 mg/mL. Then, the flask was shaken at room temperature with an oscillator for 4 h. After sufficient adsorption, filtered and determined the content of total steroids in the filtrate and the adsorption amount and adsorption rate of each resin at room temperature were calculated. When the resin was fully adsorbed, 100 mL 95% ethanol was added to the resin, soaked for 10 h at room temperature, shaked in an oscillator for 4 h, and filtered. Finally, determined the content of total steroids in the filtrate and calculated the adsorption/desorption amount and desorption rate based on the adsorption amount. The adsorption capacity, desorption capacity, and desorption ratio were calculated using the following equations:
Adsorption capacity:
Desorption capacity:
Desorption ratio:
where Qe is the adsorption capacity at adsorption equilibrium (mg/g dry resin); Dd is the desorption ratio (%); Qd is the desorption capacity after adsorption equilibrium (mg/g dry resin); C0 and Ce are the initial and absorption equilibrium concentrations of steroid in the solutions, respectively (mg/mL); V0 and Ve is the volume of the initial and absorption equilibrium sample solution (mL); M is the ratio of the water content; W is the weight of the hydrated resin (g); Cd is the concentration of steroid in the desorption solution (mg/mL); Vd is the volume of the desorption solution (mL).
Dynamic adsorption and desorption test
After the pretreatment, 5 g of each of the seven nonmoisture macroporous resins were accurately weighed and the column was wet-loaded with 95% ethanol. The resin bed volume was 100 mL, soaked for 24 h, treated with 95% ethanol at four times volume of resin bed (BV), a controlled flow rate of 2.0 BV/h, and then washed with plenty of distilled water until no alcohol taste. The sample solution with a total steroids concentration of C0 = 0.033 mg/mL was precisely absorbed, with a loading volume of V0 = 30 mL. The solution was passed through the resin bed at the same flow rate, and the outflow was collected. The resin column was washed with distilled water until the water washing solution became colorless. The aqueous solution was combined with the outflow solution, volume was V1 = 150 mL. Finally, the total steroid concentration was determined. Then, the resin was eluted with 70% ethanol at the same flow rate, and the eluent was collected in batches, one bottle per 10 mL, and the total steroid content in each bottle was determined. Combined all eluates and determined the concentration of total steroids. The adsorption capacity, desorption capacity, and desorption ratio were calculated using the equations in 2.6.1.
Optimization of adsorption conditions
Effect of sample factors
To investigate the influence of sample concentration on adsorption, 100 mL of sample solution (6.670 mg/mL) was taken and concentrated to 40 mL, divided it into four equal parts, each 10 mL one part, in which added different counts of water (0, 10, 20, and 30 mL), respectively, and the concentration was, respectively, diluted to 16.675, 8.338, 5.558, and 4.196 mg/mL. The solution with different concentrations was passed through a chromatographic column that filled with 5 g macroporous resin, and the flow rate was adjusted to 1 BV/h. Collected the liquid, 5 mL for one part. To study the effect of sample flow rate on adsorption, four samples solution with a known total steroids content of 6.670 mg/mL were taken, each 30 mL, and passed through four chromatographic columns filled with 5 g macroporous resin and controlled the flow to different rate (1, 2, 3, and 4 BV/h). Collected the liquid, 5 mL for one part. To determine the maximum loading volume, the sample solution (total steroids content of 13.340 mg/mL) was taken and passed through the chromatographic column containing 5 g macroporous resin at a flow rate of 1 BV/h for dynamic adsorption. The effluent was collected in sections, each one for every 2 mL, and collected 14 in total. After each liquid was collected, measured the absorbance value at 522 nm, when the absorption value increased suddenly, it is considered that adsorption saturation. Measured and recorded adsorption amount and absorbency were measured and recorded for further analysis.
Effect of elution factors
The commonly used eluents for macroporous adsorption resin include water, ethanol of different concentrations, methanol, acetone, etc. Considering safety and other reasons, water and different concentrations of ethanol were most used as eluents in the experiment. To select the optimum eluent concentration, five steroid sample solutions with a known content of 13.34 mg/mL (20 mL) were taken and passed through a chromatography column filled with 5 g of macroporous resin at a flow rate of 1 BV/h. After that, gradient elution with different eluents (water, 30%, 50%, 70%, and 90% ethanol) at a flow rate of 1 BV/h, respectively. Collected the eluates of each concentration and calculated the desorption of total steroids in each concentration of ethanol solution. To research the effect of elution rate on desorption ability, Four 20 mL solutions (known to have a total steroid content of 13.340 mg/mL) were passed through a column filled with 5 g macroporous resin at a flow rate of 1 BV/h respectively. Then, samples were eluted with 50% ethanol at different flow rates (1, 2, 3, 4 BV/h). Each eluate was collected to calculate the total steroid recovery. To ascertain the elution range, 20 mL of sample solution (total steroids content of 13.340 mg/mL) was taken and passed through a chromatography column filled with 5 g of macroporous resin at a flow rate of 1 BV/h. The resin was eluted with 50% ethanol at a flow rate of 2 BV/h, and the eluate was collected in sections, one for each 0.1 BV. Collected the eluate and measured absorbance at 522nm. After all the experiments, we recorded all data for further analysis.
Investigation of adsorption time and water consumption for removing impurities
To choose the best duration for adsorption, 20 mL, four portions of steroid sample solutions with total steroids content of 13.340 mg/mL were taken and respectively passed through a column filled with 5 g macroporous resin at a flow rate of 1 BV/h, and adsorbed in different duration (0, 1, 3, and 6 h). After that, the resin was eluted with 50% ethanol at a flow rate of 2 BV/h. Collected each eluent and recorded the volume of the eluent.
The sample solution of the same concentration was taken and adsorbed for 1 h at a flow rate of 1 BV/h through a chromatographic column containing 5 g macroporous resin, then washed with distilled water at a rate of 2 BV/h until tested negative by Molish reaction. Subsequently, the resin was eluted with 50% ethanol at a flow rate of 1 BV/h. The alcohol washing liquid was collected and concentrated under reduced pressure conditions to obtain total steroids extracts and measured the total steroids content. The amount of water that needed for final impurity removal is obtained according to the content variation.
High-performance liquid chromatography analysis
Conditions for high-performance liquid chromatography
The chromatographic separation was performed on a symmetry C18 column (5 μm, 150 mm × 4.6 mm) using acetonitrile (A) and 0.05% phosphoric acid solution (B) as the mobile phase at a flow rate of 1.0 mL/min. The gradient program was set as follows: 0–15 min, 5%–8% A; 15–25 min, 8%–18% A; 25–60 min, 18%–35% A. Detection was carried out at the wavelength of 230 nm. The column temperature was maintained at 35°C and the injection volume of each sample and the standard solution was 10 μL.
Sample and standard solution preparation
According to the method of extraction and purification, the total steroids were prepared. After that, the total steroids extract of 89.25 mg was precisely weighed and added with methanol into a 100 mL measuring bottle with a constant volume to obtain sample solutions with a concentration of 0.8925 mg/mL. Finally, the samples were filtrated through a 0.45 μm membrane filter before HPLC analysis.
Precision weighed the appropriate amount of Physalin J, Physalin B, Physalin O, Physalin F, Physalin P, added methanol to prepare solutions with concentrations of 0.108, 1.6, 1.1, 0.902, and 1.3 mg/mL to obtain the reference solutions.
Anti-inflammatory evaluation
Effects of Physalis alkekengi L. var. franchetii (Mast.) Makino on edema of rat paw caused by egg whites
According to body weight, the rats were randomly divided into six groups, each containing ten rats: (1) the model group, (2) the high-dose group (200.00 mg/kg), (3) the medium-dose group (100.00 mg/kg), (4) the low-dose group (50.00 mg/kg), (5) the positive control group (hydrocortisone injection 0.25 mg/kg), and (6) the total extract group (200.00 mg/kg). The positive control group was administered by intraperitoneal injection, the other groups were administered by gavage for seven consecutive days. The model group was injected with 10% fresh egg white for inflammatory modeling.
The rats were only given water for the first 18 h of the experiment. On the 7th day, the right hind limb of the rats was fixed, and the ankle joint was marked with a marker. Measured the normal paw volume of the right hind leg, and the average value was obtained continuously. Then, 1 h after intragastric administration, 0.1 mL of 10% fresh egg white was injected subcutaneously in the middle of the toes of the right hind limb. Paw volume was measured at 1, 2, 3, and 4 h after injection.
Effects of Physalis alkekengi L. var. franchetii (Mast.) Makino on peritoneal leukocyte migration in mice induced by sodium carboxymethyl cellulose
Randomly divided male and female healthy mice into six groups, 10 mice in each group: (1) the model group, (2) the positive control group (hydrocortisone injection 0.25 mg/kg), (3) the high-dose group (200.00 mg/kg), (4) the medium-dose group (100.00 mg/kg), (5) the low-dose group (50.00 mg/kg), and (6) the total extract group (200.00 mg/kg). Similarly, except for the model group, the other groups were continuously administered for 7 days. The positive control group was administered by intraperitoneal injection, and the other groups were given by gavage.
The mice were only given water for the first 18 h of the experiment. Forty minutes after the last dose, each mouse was intraperitoneally injected with 0.5 mL of 1.5% CMC-Na solution. The animals were sacrificed after 40 min. Each mouse was injected intraperitoneally with 5 mL of normal saline. Gently kneaded the abdomen of the mouse, extracted the peritoneal fluid to drop on the white blood cell counting plate, and counted.
Statistical analysis
All experiments were repeated three times per replicate. The data were expressed as the mean ± SD. The analysis of one-way analysis of variance, followed by Student's t-test for pairwise comparisons was used for the intergroup analysis; P < 0.05 was considered statistically significant.
Results and DiscussionDetermination of extraction method
Cold-soak, heat-reflux, and ultrasonic extraction are traditional methods with different advantages for extracting a variety of medicinal compounds.[15],[16][Figure 1]a shows that cold-soak and ultrasonic extractions were similar in their outcomes, both offering a low extract yield and total steroid content. Comparatively, the heat-reflux method was most effective, providing the highest extract yield and larger total steroid content. In addition, the heat-reflux method can reduce time, solvent consumption, and experimental costs. For this purpose, we chose the heat-reflux method as the optimal extraction method in our study.
Figure 1: Optimization of the extraction process of crude extractions: (a) ultrasound, cold-soak and heat-reflux methods, (b) ethanol concentration (30%, 50%, 70%, 90%), (c) solid-to-liquid ratio (1:20, 1:30, 1:40, 1:50, 1:60), (d) extraction duration in hours, (e) extraction timesSingle-factor effect on extraction
Selecting suitable solvents according to the polarity of the compound to be extracted is the first step in plant extraction.[17][Figure 1]b shows that comparing to 0% and 90%, 30%, 50%, and 70% ethanol solutions have higher extraction efficiencies. The extract yield for the 90% ethanol solution was limited, but the total steroid content was higher than that for the other ethanol solutions. Therefore, 50%, 70%, and 90% ethanol solutions were used as the factors for the orthogonal experiments. Next, an appropriate material–liquid ratio can improve extraction efficiency while avoiding excessive solvent waste.[18] As shown in [Figure 1]c, at 20, 30, and 40 multiples, the total steroid content was relatively high. When the solvent concentration continued to increase, the total steroid content decreased with an increase in the extract yield. The reason may be that as more solvent was added, the content of other extracted substances increased, so the extract yield increased, but the total steroid content decreased. Accordingly, we chose 20, 30, and 40 multiples as the factors for the orthogonal experiment. Moreover, extraction time is an indicator that can be used to achieve the highest efficiency and minimum energy cost.[19] As shown in [Figure 1]d, between 0.5 and 2 h, the total steroid content showed a linear upward trend, but after more than 2 h, the total steroid content began to decline. It may be that the amount of impurities was increased during prolonged extraction or that some steroidal components were destroyed by heating. Compared with 2 h, the total steroid content decreased at 2.5 h, but remained high. Moreover, the extraction yield was the highest at 2.5 h. Based on these findings, 1.5, 2, and 2.5 h were used as factor levels. In addition, frequency is an essential factor for extraction. [Figure 1]e shows that after performing heating reflux three times, the extract yield and total steroid content decreased. If the heat reflux continued, more impurities were generated. Therefore, the extraction time numbers of 1, 2, and 3 were used as the factors of the orthogonal experiment to further investigate the influence of the extraction frequency on the extraction efficiency.
Orthogonal results analysis
The orthogonal experiment is a common method for studying multiple factors and multiple levels, resulting in high efficiency, speed, and economy.[20],[21] Using the principle of orthogonality, some representative levels were selected from single-factor experiments for testing. The orthogonal experimental results were based on two experimental repetitions performed in parallel [Table 2] and [Table 3]. Intuitive analysis, variance analysis, and significance testing showed that the extraction duration had an extremely significant effect, and that the ethanol concentration had a significant effect, on the extraction results. The amount of extraction solvent and the number of extractions had no significant effect on the extraction results. The effects of various factors on the yield followed the order A > B > D > C. The best process was A2B3C2D2. However, considering that in the actual production process, the use of 90% ethanol will increase the production cost and high concentrations of ethanol increase the extraction of fat-soluble impurities (such as pigment, etc.). Concurrently, the single-factor experimental results showed that the extract yield for 90% ethanol was insufficient. In contrast, the extract yield and total steroid content were high for 70% ethanol. Ultimately, we determined A2B2C2D2 as the optimized extraction process, extracted by 30 multiples of 70% ethanol for 2 h each time.
Verification of extraction process
Three process verifications were carried out according to the extraction process: 5 g of dried PAF and 30 times of 70% ethanol for 2 h each time. The filtrates were filtered and combined. The solvent was extracted and recovered under vacuum to obtain the extracts. This process was repeated three times. The steroid contents of the three batches were 39.27%, 37.57%, and 38.78%, respectively. The average total steroid content was 38.21%. With this method of extraction, there were no significant changes in the extract weight and total steroid content among the three experimental results. Furthermore, this method is easy to operate, only requires simple equipment, uses a less polluting solvent, and has a lower production cost. Consequently, this method is stable, feasible, and suitable for industrial production, and can be used for the extraction of total steroids from PAF.
Determination of resin
First, the static adsorption and desorption capabilities of the resin were evaluated. The adsorption properties of different resins correlate with the chemical features of the resins and target compounds. When the polarity of the resins is similar to that of the adsorbed substance, adsorption effects are more likely to occur, according to the so-called similar miscibility principle.[22] It can be seen in [Figure 2]a that the adsorption effect of the macroporous resin with weak polarity was better than that of the macroporous resin with strong polarity. This is due to the weak polarity of steroid compounds, with a weaker polarity of the resin resulting in better binding. In addition, the adsorption and desorption capabilities were also dependent on the resins' physical features, including the surface area and average pore diameter. In low- and nonpolarity resins, the adsorption and desorption capacities of the resins with large particle sizes and large average pore sizes were dominant. In medium- and strong-polar resins, the resin with a large aperture also showed strong adsorption and desorption capabilities. However, a larger pore size also produced the opposite effect. Compared with HPD-700, the adsorption and desorption capacities of HPD-100A with a larger pore size were suboptimal even when the other conditions were the same. Overall, we observed that the static adsorption capacities of the AB-8 and HPD-200B resins were considerably higher than those of the other resins, but HPD-200B possessed a strong affinity for compounds so that the desorption capacity of the steroid was not outstanding.
Figure 2: (a) Static and (b) Dynamic adsorption/desorption capacity and desorption ratio of total steroids on seven resinsNext, to further verify the performance of the resins and better simulate their performance in actual production conditions, a dynamic adsorption experiment was performed, focusing on AB-8 and HPD-200B. In a dynamic experiment, the capacity of each resin is not only related to the physical and chemical properties of the resin and the adsorbate, but it is also affected by various external factors. HPD-100A performed better in the dynamic than in the static experiments, but its adsorption capacity remains insufficient [Figure 2]b. Medium- and high-polarity resins were still unsatisfactory. AB-8 maintained stable and efficient adsorption and desorption performance. Although HPD-200B had a strong adsorption capacity, its desorption capacity was not high, consistently with what was seen in the static experiments. At the same time, the AB-8 macroporous adsorption resin was relatively cheap, reducing the experimental cost. Taking the above factors into consideration, the AB-8 resin emerged as a suitable resin for further research.
Determination of adsorption conditions
Effect of sample concentration, flow rate, and leakage curve
In order to evaluate the effect of sample concentration on the macroporous adsorption resin, the adsorption process was carried out at various sample concentrations [Figure 3]a. Initially, the amount of resin adsorption increased quickly with the increasing sample concentration. Then, the increase in efficiency slowed down, with adsorption reaching the maximum efficiency at a sample concentration of 8.338 mg/mL. At relatively low values, the relationship between the concentration of the sample solution and the adsorption amount was in accordance with the classic Freundlich and Langmuir adsorption formulas. In addition, the concentration of the sample solution after pretreatment should not be greater than 16.675 mg/mL; otherwise, a large amount of precipitate will be generated. Taken together, the results pointed to an optimal concentration of between 8.338 and 16.675 mg/mL.
Figure 3: (a) Effect of loading concentration, and (b) Effect of loading velocity on the adsorptive capacity of resin, and (c) leakage curveThe sample flow rate also influences resin adsorption. If the flow rate is too high, the target compound leaks early without being fully adsorbed by the resin, leading to a reduced amount of resin adsorbed. However, if the adsorption flow rate is too low, the adsorption time will increase accordingly. As shown in [Figure 3]b, the effect of the flow rate on the adsorption capacity was assessed by loading the sample at a velocity of 1–4 BV/h. The resin adsorption amount was the highest at a loading flow rate of 1 BV/h. When the flow rate was >2 BV/h, the resin adsorption amount did not change significantly. At <1 BV/h, the flow rate could not be accurately controlled, causing great experimental difficulty. Therefore, the flow rate was controlled at 1 BV/h, which was judged to be most beneficial for the adsorption of the total steroids by industrial macroporous resins.
A leakage curve was used to determine the maximum sample volume. The leakage curve on the AB-8 resin was obtained based on the volume of the effluent liquid and the absorbance. As shown in [Figure 3]c, the leakage curve of the macroporous resin for the total steroids was S-shaped. When the volume of the effluent was 10 mL, the absorbance value of the effluent suddenly increased, indicating a significant leakage of the total steroids. When the volume of the effluent was 20 mL, the AB-8 macroporous resin reached adsorption saturation, and the total steroid leakage in the effluent reached its highest value. In general, a 10% ratio of the concentration in the effluent to the original concentration was defined as the leaking point. After the adsorption reached the leaking point, the adsorption affinity decreased and even disappeared, and the solutes leaked from the resins (i.e., the adsorption presumably reached saturation). Therefore, 20 mL of sample solution on the AB-8 resin (i.e., 4.0 mL/g resin) was determined as the sample volume for saturated adsorption.
Effect of eluents, elution velocity, and elution curve
The elution solvent is known to affect the removal of impurities on the resin and the efficiency of desorption. At the same time, the industry needs to consider the safety of the eluent, including whether it brings toxic substances into the extract. Therefore, the choice of the elution solvent is very important. In general, resins that can be deionized using deionized water or low-concentration ethanol and different concentrations of ethanol are used for desorption. In our study, different concentrations of ethanol and different elution times had a significant impact on the content of the separated components. As shown in [Figure 4]a, water could only elute a small number of steroids, so it could be used to remove impurities first. A large amount of steroids could be eluted using a 30% ethanol solution. A 50% ethanol solution could elute more steroids. The eluates for the 70% and 90% ethanol solutions contained only a small amount of steroids. In a previous study, we found that the amounts of PAF components with strong anti-inflammatory and antibacterial activities were high in 50% ethanol-eluting ingredients.[10] In this experiment, 50% ethanol also showed good desorption capability, essentially fully eluting the steroid components. Therefore, a 50% ethanol solution was used as the eluent.
Figure 4: Effects of different factors on the enrichment and purification of total steroids in PAF. (a) Elution results of different concentration of ethanol, (b) effect of eluting velocity on the recovery of total physalins, (c) elution curve of 50% alcohol, (d) effect of adsorbing time on the recovery of total physalins. PAF: Physalis alkekengi L. var. franchetii (Mast.) MakinoElution efficiency is also affected by the flow rate of the eluent. If the flow rate is too fast, the eluent and the adsorbed compounds will not sufficiently enter in contact with each other, and the elution efficiency will be poor. However, if the elution flow rate is too low, the production cycle will be prolonged, resulting in increased production costs. As shown in [Figure 4]b, when the elution flow rate was 2.0 BV/h, the recovery rate of the total steroids was the highest. As the elution flow rate increased, the total steroid recovery rate decreased. Consequently, 2.0 BV/h was selected as the optimal elution velocity.
To achieve the best elution process and save industrial costs, the volume of eluent also needs to be determined. When the elution volume is too small, the adsorbate cannot be eluted. However, when the elution volume is too high, the elution reagent is wasted and the experimental cost increases. As shown in [Figure 4]c, the initial absorbance value for 0–0.3 BV was low, indicating the presence of fewer steroid components. When 0.8 BV was collected, the absorbance decreased significantly, indicating that steroid components had basically been eluted. Therefore, it is best to collect an eluent volume between 0.3 and 0.8 BV.
Determination of adsorption time and water volume
An appropriate adsorption time is conducive to maximizing the enrichment of components while improving elution efficiency. If the adsorption time is too short, the steroid components cannot be fully absorbed. Conversely, if the adsorption time is too long, the components will tend to be locked onto the resin and will not be easily eluted. As shown in [Figure 4]d, the recovery rate was the highest at 1 h, and then dropped sharply as the adsorption time increased. Therefore, the adsorption time was best at 1 h, when less eluent was used and the recovery rate was high. Then, the Molish reaction was used to control the amount of water required to remove impurities. When the Molish reaction was negative, elution was stopped. The water consumption was determined to be approximately 100 mL, i.e., 2 BV.
Validation of purification process
The purification process was verified under the best conditions in each single-factor experiment. A 20-mL volume of PAF extract at a concentration of 13.34 mg/mL was slowly passed through a pretreated AB-8 macroporous resin column at a flow rate of 1 BV/h, adsorbed for 1 h, rinsed with 2 BV of distilled water at a flow rate of 2 BV/h, and tested by Molish reaction. When the reaction was negative, the mixture was eluted with 50% ethanol at a flow rate of 2 BV/h. An eluent volume ranging from 0.3 to 0.8 BV was concentrated to dryness under reduced pressure. This process was repeated three times. The steroid contents of the three batches were 70.17%, 72.21%, and 73.12%, respectively. The average total steroid content was 71.83%. The recovery rates were 90.28%, 91.18%, and 88.54%, respectively. The average recovery rate was 90.00%. The purification process using the macroporous adsorption resin method was stable and showed a high yield, representing a possible purification process for the total steroids in the plant.
Determination of index component content
The pharmacological activity of PAF depends on its chemical constituents and content. Therefore, the steroid components in the PAF extracts were analyzed using HPLC. In our previous research,[10] we identified five index components as follows: Physalin J, Physalin F, Physalin O, Physalin B, and Physalin P. The first step for content determination was to establish the linear regression equation of the five components [Table 4]. By HPLC quantitative analysis, the contents of the five steroids were determined to be the following: Physalin F (0.884%, Relative Standard Deviation (RSD) = 0.22%, n = 3), Physalin P (0.241%, RSD = 0.41%, n = 3), Physalin B (0.424%, RSD = 0.54%, n = 3), Physalin O (0.119%, RSD = 0.84%, n = 3), and Physalin J (0.343%, RSD = 0.33%, n = 3). The structural formula of the above compounds is shown in [Figure 5]. Previous studies have shown that Physalin F inhibits cancer cells.[23],[24] and that Physalin B has anti-cancer and anti-infection properties.[25],[26] The other three active ingredients need to be explored further. Based on the component analysis, animal experiments were further conducted to determine the pharmacological activity of PAF.
Figure 5: The structural formula of (a) Physalin F, (b) Physalin B, (c) Physalin J, (d) Physalin P, (e) Physalin OAnti-inflammatory evaluation
Inhibition of paw edema by egg whites
Steroid compounds have strong anti-inflammatory properties and are widely used in the clinic. Physalins, which are steroidal compounds found in PAF, have been proven to be effective anti-inflammatory agents.[10] The anti-inflammatory activity of steroids was verified in this study. [Figure 6] shows that there was no significant difference in the normal paw volume between the groups before the administration of the inflammatory agent (P > 0.05). After the administration of the inflammatory agent, each administration group was compared with the control group at different time points. The hydrocortisone group showed significant inhibition of paw edema in rats. The difference was statistically significant within 2 h of administration (P < 0.01), but the inhibitory effect decreased over time. The improvement in the high-dose and medium-dose groups was also significantly different from that of the control group within 2 h after modeling (P < 0.05). In contrast, the low-dose and unpurified total extracts did not significantly improve paw edema in rats (P > 0.05). In summary, purified PAF exerted an anti-inflammatory activity, which was dose dependent. The high-dose group showed effective anti-inflammatory activity, which was almost equivalent to that of hydrocortisone.
Figure 6: Effects of total steroids from PAF on edema of rat hind paw induced by fresh egg white. **P < 0.01, versus model, *P < 0.05. PAF: Physalis alkekengi L. var. franchetii (Mast.) MakinoInhibition of leukocyte migration
CMC-Na solution is often used to study the effects of drugs on inflammation in mice. In this experiment, the number of leukocytes in the abdominal cavity was counted as the inflammatory index value to determine the effect of drugs on inflammation in each group. As shown in [Figure 7], compared to the control group, the hydrocortisone and high-dose groups showed significant inhibition of the leukocyte migration in the abdominal cavity caused by CMC-Na (P < 0.01). Compared with the control group, the medium-dose and unpurified extract groups showed significant improvement in leukocyte migration (P < 0.05). The high-dose group showed stronger inhibition than the medium-dose group. In contrast, the low-dose group did not show significant improvement of leukocyte migration in the abdominal cavity caused by CMC-Na (P > 0.05). It was thus observed that PAF had an anti-inflammatory activity, which increased with increasing doses. These results suggest that the obtained PAF extract may potentially be applied in the treatment of inflammatory diseases.
Figure 7: Effect of total steroids from PAF on mice peritoneal leukocyte migration induced by sodium carboxymethyl cellulose solution. **P < 0.01, versus model, *P < 0.05. PAF: Physalis alkekengi L. var. franchetii (Mast.) Makino Conclusions Today, PAF Makino is not only consumed as a dietary supplement and fruit juice but can also be found in health products, medicines, and cosmetics. In this study, the extraction method, macroporous resin, and purification method have been optimized into a simple, green, and efficient macroporous adsorption resin AB-8 chromatography method for the separation and enrichment of total steroids from PAF. The results showed that the total steroid content after purification was 1.88 times higher than that before purification, and the recovery rate of the total steroids reached above 88%, meeting the new drug requirements in China (>50%). Furthermore, PAF was analyzed by HPLC and proved to contain a variety of anti-inflammatory active ingredients. Animal experiments further demonstrated that the enriched extract had a stronger anti-inflammatory effect, which could significantly reduce the swelling of rat feet caused by egg whites and reduced the migration of leukocytes in the abdominal cavity of mice caused by CMC-Na. Collectively, this study explored the best extract and enrichment process for the steroidal components of PAF, suggesting a new enrichment and purification process with profound significance for future studies and applications of PAF.
Acknowledgments
This study was supported by the Guangzhou Science and Technology Program in 2020– Basic and Applied Basic Research Project (Grant No. 202002030226), the National Natural Science Foundation of China (Grant No. 82174043), the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2021A1515011697). Thanks to professor Zun-Peng Shu for his careful teaching and thanks to the members of the research team for their great support and help.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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