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).
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).
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.
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.
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.
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.
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.
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
|
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%.
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%.
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%.
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%.
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%.
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.
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.
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.
