Study on Optimization of Ultrasonic Extraction Process for Benzoic Acid as a Harmful Component in Paeonia lactiflora Pall

Abstract

Objective

To optimize the ultrasonic extraction process for benzoic acid as a harmful substance in Paeonia lactiflora Pall. (P. lactiflora Pall.).

Methods

Methanol and ethanol solutions at different concentration gradients (25, 50, 75%) were prepared to investigate the effects of extraction solvents on the extraction efficiency of benzoic acid. The influences of ultrasonic frequency (35, 50 Hz), ultrasonic power (40, 60, 80, 100 W), ultrasonic time (10, 20, 30, 40, 50, 60 minutes), and ultrasonic temperature (20, 30, 40, 50°C) on the extraction efficiency were examined. Orthogonal experiments were conducted to analyze the effects of temperature, time, and ultrasonic power on the extraction efficiency and to screen the optimal ultrasonic extraction process.

Results

Various influencing factors had certain effects on the extraction efficiency of benzoic acid from P. lactiflora Pall. Single-factor analysis revealed that 25% methanol, ultrasonic frequency of 50 Hz, ultrasonic power of 40 W, ultrasonic time of 10 minutes, and ultrasonic temperature of 30°C yielded the highest extraction efficiency for benzoic acid. The order of influence of different factors on the extraction efficiency was temperature > time > power. The optimal conditions obtained from orthogonal experiments were: extraction solvent of 25% methanol, ultrasonic frequency of 50 Hz, ultrasonic time of 20 minutes, ultrasonic power of 40 W, and ultrasonic temperature of 30°C.

Conclusion

Under the conditions of 25% methanol as the extraction solvent, ultrasonic frequency of 50 Hz, ultrasonic time of 20 minutes, ultrasonic power of 40 W, and ultrasonic temperature of 30°C, the extraction efficiency of benzoic acid from P. lactiflora Pall. was the highest. This method offers advantages such as simple operation, small sample size requirement, and low solvent consumption, providing a reliable analytical approach for quality control and safety evaluation of P. lactiflora Pall.

 

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Keywords

Paeonia lactiflora Pall. - benzoic acid - ultrasonic extraction - process optimization

Paeonia lactiflora Pall. (P. lactiflora Pall.) is the dried root of the Ranunculaceae plant P. lactiflora Pall., also known as “parting grass” or “flower of prime minister.” While its flowers have high ornamental value, its roots are used medicinally. P. lactiflora Pall. is classified into Baishao (Paeoniae Radix Alba) and Chishao (Paeoniae Radix Rubra), both of which are commonly used as Chinese medicinal materials. According to the 2020 edition of the Chinese Pharmacopoeia, Baishao (Paeoniae Radix Alba) is the dried root of P. lactiflora Pall., processed by washing and boiling to remove the skin or by removing the skin before boiling.[1] It is primarily cultivated. Chishao (Paeoniae Radix Rubra), on the other hand, is the dried root of P. lactiflora Pall. or P. veitchii Lynch.[1] The chemical components of P. lactiflora Pall. mainly include paeoniflorin, benzoylpaeoniflorin, albiflorin, benzoic acid, tannins, volatile oils, gallic acid, and fatty oils, etc.[2] [3] [4] [5] Among these, paeoniflorin is the main component of P. lactiflora Pall., known for its sedative, anti-inflammatory, coronary vasodilating, and platelet aggregation inhibitory effects, making it the preferred chemical quality control indicator.

Benzoic acid is generally regarded as a harmful component in P. lactiflora Pall., as it increases the detoxification burden on the liver and produces certain toxic side effects.[6] It is classified as a harmful substance in food.[7] Benzoic acid is commonly used as a preservative to inhibit the growth of fungi, bacteria, and molds. It also has industrial value in the synthesis of fibers, resins, coatings, etc. Modern research indicates that benzoic acid is a major harmful substance in Baishao (Paeoniae Radix Alba) and is also found in Chishao (Paeoniae Radix Rubra).

Studies have found that the content of benzoic acid undergoes significant changes after Baishao (Paeoniae Radix Alba) is processed by boiling.[6] Therefore, determining the content of benzoic acid, a harmful component in P. lactiflora Pall., can serve as an important indicator for evaluating the quality of Baishao (Paeoniae Radix Alba) and Chishao (Paeoniae Radix Rubra) slices. Currently, there are various methods for determining benzoic acid content, such as thin-layer chromatography, gas chromatography, thin-layer chromatography–ultraviolet spectrophotometry, and enzyme-linked immunosorbent assay. Although these methods have their respective advantages and disadvantages in detecting benzoic acid content, traditional extraction methods consume more materials and require longer processing times. Thus, optimizing the extraction process for benzoic acid from P. lactiflora Pall. is of great significance. Based on previous research, this study employs response surface methodology to optimize the ultrasonic extraction process for benzoic acid from P. lactiflora Pall. and explores the optimal extraction conditions.[8] [9] [10] [11] [12] By optimizing the extraction process, not only can the extraction efficiency of benzoic acid be improved to ensure the quality of P. lactiflora Pall.-based medicines, but it also provides a theoretical basis for addressing the inappropriate medication use of medicinal materials in the market[13] and offers effective technical support for quality control of Chinese medicinal materials.[14]

Materials

Drugs and Reagents

Baishao (Paeoniae Radix Alba) slices (Daqing Fuyuan Slice Processing and Sales Co., Ltd., place of origin: Anhui, China; production license number: Hei Y20010278) were identified by Professor Donghua Wei of Harbin Medical University as derived from P. lactiflora Pall. Benzoic acid reference standard (National Institutes for Food and Drug Control, China; batch number: 110736-200732); methanol (Thermo Fisher Scientific, USA; chromatographic grade); phosphoric acid (Tianjin Yongda Chemical Reagent Co., Ltd., China; batch number: 20110625); ethanol (Shenyang Huadong Reagent Factory, China; analytical grade); water: Wahaha mineral water (China).

 

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Instruments

High-performance liquid chromatography (HPLC) system (model: 996-486, Waters, United States); electronic analytical balance (model: AL204, Mettler Toledo Instruments (Shanghai) Co., Ltd., China); drying oven (model: DF205, Beijing Xicheng District Second Medical Device Factory, China); ultrasonic instrument (model: SK250HP, Shanghai Kedao Ultrasonic Instrument Co., Ltd., China).

 

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Methods

Chromatographic Conditions

This study used HPLC to determine the benzoic acid content in ultrasonic extracts. The chromatographic conditions were as follows: column: LichroCART RP-C₁₈ (4 × 250 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid aqueous solution (40:60); flow rate: 1 mL/min; detection wavelength: 232 nm; column temperature: 25°C; injection volume: 10 μL.

 

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Preparation of Reference Solution

Accurately weigh 3.16 mg of benzoic acid reference standard, dissolve in methanol to a volume of 50 mL, and prepare a benzoic acid solution containing 63.2 μg/1 mL to obtain the benzoic acid reference solution.

 

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Preparation of Test Sample Solution

Grind the test medicinal material samples, pass through a 60-mesh sieve, and dry at 50°C to constant weight. Then, accurately weigh 2.00 g of the fine powder, place it in a conical flask, add 35 mL of extraction solvent, and ultrasonicate for 30 minutes. Cool to room temperature, filter, and pass the filtrate through a 0.45-μm organic filter membrane to obtain the test sample. The relative extraction amount is calculated by comparing the peak area with that of the reference solution to evaluate the effects of different extraction conditions.

 

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Effect of Different Extraction Solvents on Benzoic Acid Extraction Efficiency

Benzoic acid is easily soluble in organic solvents. According to the method described in Section 2.3, prepare test sample solutions to investigate the effects of different concentrations of methanol (25%, 50%, 75% MeOH) and ethanol (25%, 50%, 75% EtOH) on the extraction efficiency of benzoic acid.

 

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Effect of Different Ultrasonic Conditions on Benzoic Acid Extraction Efficiency

To achieve the optimal extraction efficiency for benzoic acid, test sample solutions were prepared according to the method described in Section 2.3. Single-factor experiments were conducted to investigate the effects of ultrasonic frequency (35 and 50 Hz), ultrasonic time (10, 20, 30, 40, 50, and 60 min), ultrasonic power (40, 60, 80, and 100 W), and ultrasonic temperature (20, 30, 40, and 50°C) on the extraction efficiency of benzoic acid.

 

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Orthogonal Experimental Design for Screening the Optimal Extraction Process of Benzoic Acid

To further optimize the extraction process of benzoic acid, an orthogonal experimental design was adopted. With the ultrasonic frequency set at 50 Hz, orthogonal experiments were conducted using ultrasonic time, ultrasonic power, and ultrasonic temperature as variables ([Table 1]) to determine the optimal extraction process.

Table 1 Orthogonal optimization experimental design table

Levels	Factors
	Ultrasonic time (t/min)	Ultrasonic power (P/%)	Ultrasonic temperature (t/℃)
1	10	40%	20
2	30	80%	30
3	50	100%	40

 

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Results

Effect of Different Extraction Solvents on Benzoic Acid Extraction Efficiency

Benzoic acid is easily soluble in organic solvents. The effects of different concentrations of methanol (25, 50, 75% MeOH) and ethanol (25, 50, 75% EtOH) on the extraction efficiency of benzoic acid were investigated, with ultrasonic times set at 20, 30, and 40 minutes, respectively. The results showed that methanol exhibited higher extraction efficiency than ethanol at all concentrations, with 25% methanol yielding the highest extraction efficiency. Thus, 25% MeOH was determined to be the optimal extraction solvent ([Table 2]).

Table 2 Effect of different extraction solvents on benzoic acid extraction efficiency

Extraction solvents	Ultrasonic time (t/min)	Content of benzoic acid (%)
25% EtOH	20 min	0.1084
	30 min	0.1107
	40 min	0.1387
50% EtOH	20 min	0.0503
	30 min	0.0494
	40 min	0.0554
75% EtOH	20 min	0.0634
	30 min	0.0565
	40 min	0.0633
25% MeOH	20 min	0.1627
	30 min	0.1235
	40 min	0.1267
50% MeOH	20 min	0.0637
	30 min	0.0684
	40 min	0.0861
75% MeOH	20 min	0.0771
	30 min	0.1010
	40 min	0.0960

Note: Data are presented as mean values of three replicates, with standard deviations within 2%.

 

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Effect of Different Ultrasonic Conditions on Benzoic Acid Extraction Efficiency

Effect of Ultrasonic Frequency on Benzoic Acid Extraction Efficiency

Ultrasonic frequencies of 35 and 50 Hz were set, and the extraction efficiency of benzoic acid at different frequencies was measured. The experimental results showed that the extraction efficiency at 50 Hz was higher than that at 35 Hz, with an approximately 2-fold increase. Thus, the optimal ultrasonic frequency was determined to be 50 Hz ([Table 3]).

Table 3 Effect of different ultrasonic frequencies on benzoic acid extraction efficiency

Ultrasonic frequency	Content of benzoic acid (%)
50 Hz	0.1427
35 Hz	0.0816

Note: Data are presented as mean values of three replicates, with standard deviations within 2%.

 

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Effect of Ultrasonic Power on Benzoic Acid Extraction Efficiency

Four different ultrasonic power levels—40, 60, 80, and 100 W—were tested, and the extraction efficiency of benzoic acid at each power level was measured. The results indicated that ultrasonic power had a minor effect on the extraction efficiency of benzoic acid, with no significant differences observed (p > 0.05). This suggests that within the studied power range, variations in power had little impact on the extraction of benzoic acid. Thus, the optimal ultrasonic power was determined to be 40 W ([Table 4]).

Table 4 Effect of different ultrasonic power on benzoic acid extraction efficiency

Power (P/W)	Content of benzoic acid (%)
100	0.1391
80	0.1399
60	0.1450
40	0.1720
P	0.085

Note: Data are presented as mean values of three replicates, with standard deviations within 2%.

 

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Effect of Ultrasonic Time on Benzoic Acid Extraction Efficiency

This study investigated the extraction efficiency at six different ultrasonic times: 10, 20, 30, 40, 50, and 60 minutes. The results showed that ultrasonic time had a certain effect on the extraction efficiency of benzoic acid. The lowest extraction efficiency was observed at 10 minutes, whereas the highest was at 60 minutes. However, no significant differences were found among the extraction efficiencies at 20, 30, 40, and 50 minutes, and there was no significant difference between 20 and 60 minutes (p > 0.05). This indicates that high extraction efficiency can be achieved within a short ultrasonic time, and extending the time did not significantly improve the extraction efficiency. The ultrasonic time ultimately selected for this study was 20 minutes ([Table 5]).

Table 5 Effect of different ultrasonic times on benzoic acid extraction efficiency

Ultrasonic time (min)	Content of benzoic acid (%)
10	0.1392
20	0.1520
30	0.1621
40	0.1580
50	0.1683
60	0.1819

Note: Data are presented as mean values of three replicates, with standard deviations within 2%.

 

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Effect of Ultrasonic Temperature on Benzoic Acid Extraction Efficiency

This study investigated the extraction efficiency of benzoic acid at four different ultrasonic temperatures: 20, 30, 40, and 50°C. The results showed that ultrasonic temperature had no significant effect on the extraction efficiency of benzoic acid (p > 0.05; [Table 6]). The highest extraction efficiency was observed at 30°C, which was determined to be the optimal extraction temperature.

Table 6 Effect of different ultrasonic temperatures on benzoic acid extraction efficiency

Temperature (℃)	Content of benzoic acid (%)
20	0.1391
30	0.1503
40	0.1452
50	0.1313
p	0.241

Note: Data are presented as mean values of three replicates, with standard deviations within 2%.

 

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Orthogonal Experimental Design Analysis of the Optimal Extraction Process for Benzoic Acid

To further optimize the extraction process of benzoic acid, an orthogonal experimental design was employed. With the ultrasonic frequency set at 50 Hz, orthogonal experiments were conducted using ultrasonic time, ultrasonic power, and ultrasonic temperature as variables ([Table 7]). Variance analysis of the experimental results using SPSS software revealed that ultrasonic temperature had the greatest impact on extraction efficiency (F = 1.343, p > 0.05), followed by ultrasonic time (F = 1.403, p > 0.05), and ultrasonic power had the least impact (F = 0.475, p > 0.05) ([Table 7]). Thus, the order of influence of factors on benzoic acid extraction efficiency was: temperature > time > power.

Table 7 Orthogonal optimization experimental results

Experiment number	Factors			Content of benzoic acid (%)
	A Time (min)	B Power (W)	°C Temperature (℃)
1	20	40%	20	0.1140
2	20	80%	30	0.1280
3	20	100%	40	0.1342
4	30	40%	30	0.1211
5	30	80%	40	0.1283
6	30	100%	20	0.1270
7	50	40%	40	0.1398
8	50	80%	20	0.1347
9	50	100%	30	0.1282
K1	0.1254	0.1250	0.1252
K2	0.1255	0.1303	0.1258
K3	0.1342	0.1298	0.1341
R	0.0088	0.0053	0.0089

Note: Data are presented as mean values of three replicates, with standard deviations within 2%.

Analysis of the K-values from the orthogonal experiments showed that for the time factor, the 50-minute level had the highest K-value; for the power factor (B), the B2 (80%) level had the highest K-value; and for the temperature factor (C), the C3 (40°C) level had the highest K-value. Although the effects of the factors did not reach statistical significance, based on the single-factor analysis results, the optimal extraction conditions were determined to be an ultrasonic time of 20 minutes, ultrasonic power of 40%, and ultrasonic temperature of 30°C.

 

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Discussion

Currently, various methods are available for extracting benzoic acid, such as organic solvent extraction and supercritical fluid extraction.[15] [16] [17] Hot water extraction uses high-temperature water as a solvent to dissolve active components from plants, but this method involves high temperatures, low extraction efficiency, and significant losses.[16] Organic solvent extraction improves extraction efficiency through the “like dissolves like” principle but still consumes considerable resources.[17] [18] Microwave extraction directly acts on water molecules, rapidly rupturing plant cell walls to quickly release active components, resulting in high extraction efficiency. However, it does not address the issue of high temperatures during extraction. Supercritical fluid extraction and ultra-high-pressure rapid extraction offer high efficiency but are not yet widely adopted

Ultrasonic extraction is an ideal method for extracting active components from Chinese medicines. By applying ultrasound to the extraction solution, plant cell walls are effectively disrupted, promoting the dissolution of active components. Compared with traditional heat reflux extraction, ultrasonic extraction offers advantages such as lower temperatures, shorter processing times, and higher efficiency, avoiding the destruction of biological activity in herbal medicines caused by high temperatures.[19] [20] [21] [22] Additionally, ultrasonic extraction requires less solvent, is simple to operate, and has high safety, aligning with the principles of green chemistry. This study experimentally verified the effects of different ultrasonic times, frequencies, power levels, and temperatures on the extraction efficiency of benzoic acid. The results demonstrated that the optimal extraction conditions were an ultrasonic frequency of 50 Hz, ultrasonic time of 20 minutes, ultrasonic power of 40 W, and ultrasonic temperature of 30°C.

Traditional Chinese medicine (TCM) has played a vital role in disease prevention and treatment since ancient times, representing a precious heritage of the Chinese nation. The quality of Chinese medicinal materials directly affects clinical efficacy, making the establishment of scientific and rational quality control standards crucial for promoting the high-quality development of TCM. Traditional quality control standards for Chinese medicines often use the content of active ingredients as indicators, with less emphasis on nonactive components, particularly harmful substances such as benzoic acid, for which there are no clear regulatory limits.

In P. lactiflora Pall., benzoic acid is generally regarded as a harmful component. Its presence increases the detoxification burden on the liver and may cause toxic side effects, such as liver function damage or other health issues, when consumed in certain quantities.[6] Therefore, determining the benzoic acid content in P. lactiflora Pall. cannot only serve as an important indicator for evaluating its quality but also ensure the safety and efficacy of the medicinal material. With the diverse varieties of P. lactiflora Pall. and the disorder in the market, improving the quality control of Chinese medicines and ensuring clinical application are essential steps for advancing TCM development. The experimental results of this study have identified the optimal ultrasonic extraction process for benzoic acid, providing technical support for quality control of benzoic acid in P. lactiflora Pall. Based on this, further establishing limits for benzoic acid content is of great significance for standardizing the quality of P. lactiflora Pall. in the market and ensuring the safety of clinical medication.

As a major harmful component in P. lactiflora Pall., benzoic acid is present in both Baishao (Paeoniae Radix Alba) and Chishao (Paeoniae Radix Rubra). Therefore, it is necessary to set limits on benzoic acid content to better evaluate the processing and quality of white and Chishao (Paeoniae Radix Rubra) from different origins. While controlling the content of paeoniflorin, how to regulate the standard for benzoic acid remains a topic worthy of further exploration.

The main difference between Baishao (Paeoniae Radix Alba) and Chishao (Paeoniae Radix Rubra) lies in their pharmacological effects and applications. Baishao (Paeoniae Radix Alba) is primarily used for nourishing blood and nourishing yin, whereas Chishao (Paeoniae Radix Rubra) is mainly used for promoting blood circulation and reducing swelling. Although both contain benzoic acid, their contents and impacts differ.[22] Thus, when establishing control standards for benzoic acid, the distinct characteristics of Baishao (Paeoniae Radix Alba) and Chishao (Paeoniae Radix Rubra) must be considered to ensure the scientificity and rationality of the standards. This study, by optimizing the ultrasonic extraction process, provides a reliable method for benzoic acid extraction and lays the foundation for further research on control standards for benzoic acid. Building on existing research achievements, different benzoic acid content standards can be considered to accommodate various clinical application needs. By establishing scientific and rational control standards for benzoic acid content, the healthy development of the TCM industry can be promoted.

 

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Conclusion

Under the conditions of 25% methanol as the extraction solvent, ultrasonic frequency of 50 Hz, ultrasonic time of 20 minutes, ultrasonic power of 40 W, and ultrasonic temperature of 30°C, the extraction efficiency of benzoic acid from P. lactiflora Pall. was the highest. This method offers advantages such as simple operation, small sample size requirement, and low solvent consumption, providing a reliable analytical approach for quality control and safety evaluation of P. lactiflora Pall.

 

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Conflict of Interest

The authors declare no conflict of interest.

CRediT Authorship Contribution Statement

Shangyue Chen: Conceptualization, and project administration. Guiming Guo and Gang Chen: Formal analysis. Yanxin Zhai: Data curation, and writing -original draft. Jingliang Xie, Xu Zhao, and Mingxue Cai: Investigation. Xuegang Zhou: Resources, and writing -original draft.

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Publication History

Received: 02 March 2025

Accepted: 14 June 2025

Article published online:
30 September 2025