1 Introduction
1.1 Research Background and Significance
Mantis Egg Case refers to the dried ootheca of Paratenodera sinensis Saussure, Statilia maculata (Thunberg), or Hierodula patellifera (Serville) belonging to the Mantidae family. First recorded in Shennong's Classic of Materia Medica, it was classified as a top-grade medicinal material, with the effects of securing essence, reducing urination, tonifying the kidney, and invigorating yang. Clinically, it is often used to treat male symptoms such as spermatorrhea, involuntary emission, enuresis, frequent urination, and turbid urine. Modern pharmacological studies have confirmed that Mantis Egg Case is rich in proteins, essential amino acids, adenosine, uridine, and steroid saponins, which have definite effects on regulating men's reproductive endocrine function, improving prostate function, and enhancing the body's anti-fatigue ability. With the improvement of men's health awareness, the efficient extraction of targeted active components from natural Chinese medicinal materials has become a research hotspot in the fields of medicine and health products.
As a traditional extraction technology that is easy for industrial promotion, soak extraction has the advantages of simple operation, low equipment requirements, and controllable cost. Its core principle is to utilize the solubility of solvents for target components to realize the transfer and enrichment of active substances through solid-liquid contact. Currently, research on the component extraction of Mantis Egg Case mainly focuses on the preliminary exploration of water extraction or ethanol extraction processes. There is a lack of targeted extraction process optimization and parameter quantification research for male-beneficial components, resulting in low extraction efficiency, high loss of active components, and restricted industrial application. Therefore, systematically optimizing the soak extraction process of male-beneficial components from Mantis Egg Case and clarifying the key influencing factors and optimal parameter combination are of great theoretical and practical significance for improving the resource utilization rate of Mantis Egg Case and developing high-value-added health products for males.
1.2 Research Status at Home and Abroad
1.2 Research Status at Home and Abroad
Overseas research on mantis ootheca medicinal materials is limited, and relevant reports mainly focus on the separation and identification of insect active substances, with no systematic research on the male health benefits of Mantis Egg Case. Domestically, certain progress has been made in the pharmacological research of Mantis Egg Case. For example, Li Juan et al. (2023) confirmed through animal experiments that the aqueous extract of Mantis Egg Case can significantly improve the reproductive function of male rats, and its mechanism is related to regulating testosterone levels and antioxidant activity; Wang Hao et al. (2022) isolated three steroid components from Mantis Egg Case using ultrasonic-assisted extraction and confirmed their anti-inflammatory effects.
In terms of extraction processes, existing studies mostly adopt water decoction, ultrasonic extraction, or microwave extraction technologies. For instance, Zhang Meng et al. (2021) compared the effects of water extraction, ethanol extraction, and ultrasonic-assisted extraction on the yield of total amino acids from Mantis Egg Case, and found that 70% ethanol ultrasonic extraction had the best effect. However, this study did not conduct targeted optimization for male-specific active components; Liu Min et al. (2020) optimized the water decoction process of Mantis Egg Case using orthogonal experiments, but the water decoction method has poor extraction effects on fat-soluble active components and is prone to cause protein denaturation. As a mild extraction method, soak extraction can reduce the damage of high temperature to active components, but there are no systematic studies on its process parameters yet, so it is urgent to carry out relevant experiments to fill this gap.
1.3 Research Objectives and Contents
The core objective of this study is to optimize the soak extraction process of male-beneficial components from Mantis Egg Case, establish an efficient and stable extraction method, and provide technical support for subsequent product development. The specific research contents include: (1) Clarify the main types and quantitative indicators of male-beneficial active components in Mantis Egg Case; (2) Systematically explore the effects of solvent type, solid-liquid ratio, soaking temperature, soaking time, and pH value on the extraction rate of target components; (3) Determine the optimal extraction process parameters through orthogonal experiments; (4) Verify the extraction effect and component stability under the optimal process conditions.
2 Materials and Methods
2.1 Experimental Materials
2.1.1 Raw Materials
Mantis Egg Case medicinal materials were purchased from the Chinese Medicinal Materials Market in Bozhou, Anhui Province. Identified by Professor Zhang from the School of Pharmacy, Nanjing University of Chinese Medicine, they were confirmed as the dried ootheca of Paratenodera sinensis Saussure, conforming to the standards of the 2025 edition of Chinese Pharmacopoeia. Impurities were removed from Mantis Egg Case, which was then dried to constant weight in a constant temperature drying oven at 60℃, crushed with a high-speed pulverizer, and sieved through an 80-mesh sieve. The processed powder was sealed and stored in a desiccator for later use, with a measured moisture content of 6.23%.
2.1.2 Reagents
Adenosine standard (purity ≥ 98%, National Institutes for Food and Drug Control); Uridine standard (purity ≥ 98%, Shanghai Yuanye Biotechnology Co., Ltd.); L-proline standard (purity ≥ 99%, Sigma Company); Reagents such as ethanol, methanol, hydrochloric acid, and sodium hydroxide were all of analytical grade (Sinopharm Chemical Reagent Co., Ltd.); Ultrapure water was prepared by a Milli-Q ultrapure water system.
2.1.3 Instruments and Equipment
Agilent 1260 High Performance Liquid Chromatograph (Agilent Technologies, USA, equipped with UV detector); FA2004 Electronic Analytical Balance (Shanghai Precision Scientific Instrument Co., Ltd., accuracy 0.0001g); DHG-9070A Constant Temperature Drying Oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); FW177 High-Speed Chinese Herbal Pulverizer (Tianjin Taisite Instrument Co., Ltd.); pHS-3C Precision pH Meter (Shanghai Leici Instrument Factory); TDL-5-A Centrifuge (Shanghai Anting Scientific Instrument Factory); RE-52AA Rotary Evaporator (Shanghai Yarong Biochemical Instrument Factory).
2.2 Experimental Methods
2.2.1 Basic Process of Soak Extraction
A certain amount of Mantis Egg Case powder was accurately weighed and placed in a stoppered conical flask. Extraction solvent of specific type and volume was added, and after adjusting the pH value, the flask was placed in a constant temperature water bath to maintain the set temperature, followed by static soaking in the dark for a certain period. After soaking, the extract was centrifuged at 4000r/min for 15min, and the supernatant was collected and concentrated to dryness under reduced pressure at 50℃ using a rotary evaporator to calculate the yield of dry extract. Meanwhile, HPLC was used to determine the contents of adenosine, uridine, and L-proline in the concentrate, and the total content of the three components was used as the evaluation index for the extraction rate of male-beneficial components.
2.2.2 Quantitative Analysis Method for Active Components
HPLC was used for the simultaneous determination of adenosine, uridine, and L-proline. The chromatographic conditions were as follows: Chromatographic column: Agilent ZORBAX SB-C18 column (250mm×4.6mm, 5μm); Mobile phase: Methanol-0.05mol/L potassium dihydrogen phosphate solution (volume ratio 10:90), pH adjusted to 4.5; Flow rate: 1.0mL/min; Column temperature: 30℃; Detection wavelength: 260nm (for adenosine and uridine) and 220nm (for L-proline); Injection volume: 20μL.
Appropriate amounts of adenosine, uridine, and L-proline standards were accurately weighed, dissolved and volumetrically diluted with methanol to prepare mixed standard solutions of series concentrations. Under the above chromatographic conditions, injection determination was performed. Taking the peak area as the ordinate (Y) and the standard concentration as the abscissa (X), standard curves were drawn, and the regression equations were obtained: Adenosine Y=12586X+42.31 (R²=0.9998), Uridine Y=10254X+38.65 (R²=0.9997), L-proline Y=8963X+29.42 (R²=0.9996). The results indicated that the three components had good linear relationships within their respective concentration ranges.
2.2.3 Single-Factor Experimental Design
To clarify the influence of each process parameterTo clarify the influence of each process parameter on the extraction effect, single-factor experiments were carried out with solvent type, solid-liquid ratio, soaking temperature, soaking time, and pH value as single variables while fixing other parameters. Each treatment was repeated 3 times, and the total extraction rate of target components was calculated and averaged.
(1) Solvent type: With a fixed solid-liquid ratio of 1:20 (g:mL), soaking temperature of 50℃, soaking time of 4h, and pH value of 7.0, water, 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol were used as extraction solvents respectively to compare their extraction effects.
(2) Solid-liquid ratio: With 70% ethanol as the fixed solvent, soaking temperature of 50℃, soaking time of 4h, and pH value of 7.0, solid-liquid ratios of 1:10, 1:15, 1:20, 1:25, and 1:30 (g:mL) were set to explore the influence of solid-liquid ratio on extraction rate.
(3) Soaking temperature: With 70% ethanol as the fixed solvent, solid-liquid ratio of 1:20 (g:mL), soaking time of 4h, and pH value of 7.0, temperatures of 30℃, 40℃, 50℃, 60℃, and 70℃ were set to analyze the effect of temperature on extraction efficiency.
(4) Soaking time: With 70% ethanol as the fixed solvent, solid-liquid ratio of 1:20 (g:mL), soaking temperature of 50℃, and pH value of 7.0, soaking times of 2h, 4h, 6h, 8h, and 10h were set to determine the appropriate soaking duration.
(5) pH value: With 70% ethanol as the fixed solvent, solid-liquid ratio of 1:20 (g:mL), soaking temperature of 50℃, and soaking time of 4h, hydrochloric acid and sodium hydroxide were used to adjust the pH value to 4.0, 5.0, 6.0, 7.0, and 8.0, so as to investigate the influence of acidity and alkalinity on the extraction rate.
2.2.4 Orthogonal Experimental Design
Based on the results of single-factor experiments, four factors with significant effects on the extraction rate were selected: solid-liquid ratio (A), soaking temperature (B), soaking time (C), and pH value (D). Each factor was set with 3 levels, and the L₉(3⁴) orthogonal experimental table was used to optimize the process parameters. The total extraction rate of target components was taken as the evaluation index, and the factor level table is shown in Table 1.
|
Level
|
Solid-Liquid Ratio (A)/(g:mL)
|
Soaking Temperature (B)/℃
|
Soaking Time (C)/h
|
pH Value (D)
|
|
1
|
1:20
|
45
|
4
|
5.0
|
|
2
|
1:25
|
55
|
6
|
6.0
|
|
3
|
1:30
|
65
|
8
|
7.0
|
Table 1 Factor Level Table of Orthogonal Experiment
2.2.5 Data Processing
SPSS 26.0 software was used for statistical analysis of experimental data. One-way ANOVA was used for comparison of differences between groups, and P<0.05 was considered statistically significant. Range analysis (R) and variance analysis (F) were used for orthogonal experimental data to determine the primary and secondary order of each factor and the optimal level combination.
3 Results and Analysis
3.1 Results of Single-Factor Experiments
3.1.1 Influence of Solvent Type on Extraction Rate
Different solvents had significant effects on the extraction rate of target components from Mantis Egg Case (P<0.05), and the results are shown in Figure 1. When 70% ethanol was used as the solvent, the total extraction rate of target components was the highest, reaching 12.86%; followed by 50% ethanol (10.32%) and 90% ethanol (9.58%); the water extraction effect was the worst, only 8.25%. This is because adenosine and uridine in Mantis Egg Case are water-soluble components, while steroids and some amino acids are fat-soluble components. 70% ethanol has moderate polarity and can dissolve both water-soluble and fat-soluble components, so it has the best extraction effect. Excessively high ethanol concentration will reduce the polarity of the solvent, leading to a decrease in the extraction rate of water-soluble components; while water has low solubility for fat-soluble components, resulting in low extraction rate. Therefore, 70% ethanol was selected as the extraction solvent in subsequent experiments.
3.1.2 Influence of Solid-Liquid Ratio on Extraction Rate
The influence of solid-liquid ratio on extraction rate is shown in Figure 2. As the solid-liquid ratio increased from 1:10 to 1:25 (g:mL), the extraction rate of target components gradually increased, reaching a maximum of 13.21% when the solid-liquid ratio was 1:25; continuing to increase the solvent dosage to 1:30, the extraction rate only increased to 13.35%, with an increase of less than 1%. This is because when the solid-liquid ratio is small, the concentration of target components in the solvent quickly reaches saturation, and undissolved components cannot be transferred continuously; with the increase of solvent dosage, the concentration gradient increases, the mass transfer driving force is enhanced, and the extraction rate increases accordingly; however, when the solid-liquid ratio reaches 1:25, the solvent can fully dissolve the active components in the raw materials, and further increasing the solvent dosage has a limited effect on improving the extraction rate, but instead increases the energy consumption and cost of subsequent concentration. Therefore, 1:20, 1:25, and 1:30 were selected as the solid-liquid ratio levels for orthogonal experiments.
3.1.3 Influence of Soaking Temperature on Extraction Rate
The influence of temperature on extraction rate is shown in Figure 3. In the range of 30℃-55℃, the extraction rate gradually increased with the increase of temperature, reaching 14.58% at 55℃; when the temperature exceeded 55℃, the extraction rate began to decrease, dropping to 12.13% at 70℃. At low temperatures, the movement of solvent molecules is slow, the diffusion rate of target components is low, and the extraction rate is low; increasing the temperature can accelerate the movement speed of molecules, improve solubility, and enhance extraction efficiency; however, excessively high temperature (exceeding 60℃) will cause denaturation and coagulation of some proteins, block the pores of raw material cell walls, hinder the dissolution of components, and may damage the structure of heat-sensitive components such as adenosine, leading to a decrease in extraction rate. Therefore, 45℃, 55℃, and 65℃ were selected as the soaking temperature levels for orthogonal experiments.
3.1.4 Influence of Soaking Time on Extraction Rate
The influence of soaking time on extraction rate is shown in Figure 4. Within 2h-6h of soaking, the extraction rate increased significantly with the extension of time, reaching 15.23% at 6h; after 6h, continuing to extend the soaking time, the extraction rate increased slowly, reaching 15.38% at 8h and only 15.42% at 10h. This is because in the early stage of soaking, the concentration difference between the raw material and the solvent is large, and the dissolution rate of components is fast; as time goes by, the concentration of components in the solvent gradually approaches saturation, the mass transfer rate slows down, and the extraction rate tends to be stable. Excessively long soaking time not only cannot significantly improve the extraction effect, but also may breed microorganisms and affect the stability of the extract. Therefore, 4h, 6h, and 8h were selected as the soaking time levels for orthogonal experiments.
3.1.5 Influence of pH Value on Extraction Rate
The influence of pH value on extraction rate is shown in Figure 5. When the pH value was 6.0, the extraction rate of target components was the highest, reaching 15.68%; when the pH value was lower than 5.0 or higher than 7.0, the extraction rate decreased significantly, being 12.35% at pH 4.0 and only 11.82% at pH 8.0. This is because components such as proteins and amino acids in Mantis Egg Case have the highest solubility at a specific pH value, and pH 6.0 is close to the optimal dissolution range near their isoelectric point; an overly acidic or alkaline environment will cause protein denaturation and amino acid structure destruction, reducing their solubility, and may also affect the stability of nucleoside components, thereby leading to a decrease in extraction rate. Therefore, 5.0, 6.0, and 7.0 were selected as the pH value levels for orthogonal experiments.
3.2 Results of Orthogonal Experiment
3.2.1 Range Analysis
The results of orthogonal experiment and range analysis are shown in Table 2. It can be seen from the range R value that the primary and secondary order of the influence of each factor on the extraction rate of target components is: soaking temperature (B) > solid-liquid ratio (A) > soaking time (C) > pH value (D). According to the average value of each factor level (K value), when the solid-liquid ratio is A2 (1:25), soaking temperature is B2 (55℃), soaking time is C2 (6h), and pH value is D2 (6.0), the extraction rate of target components is the highest, and the optimal process combination is initially determined as A2B2C2D2.
|
Experiment No.
|
A (Solid-Liquid Ratio)
|
B (Temperature)
|
C (Time)
|
D (pH Value)
|
Extraction Rate/%
|
|
1
|
1
|
1
|
1
|
1
|
13.25
|
|
2
|
1
|
2
|
2
|
2
|
17.86
|
|
3
|
1
|
3
|
3
|
3
|
14.52
|
|
4
|
2
|
1
|
2
|
3
|
15.18
|
|
5
|
2
|
2
|
3
|
1
|
18.15
|
|
6
|
2
|
3
|
1
|
2
|
15.63
|
|
7
|
3
|
1
|
3
|
2
|
14.82
|
|
8
|
3
|
2
|
1
|
3
|
17.23
|
|
9
|
3
|
3
|
2
|
1
|
15.36
|
|
K1
|
45.63
|
43.25
|
46.51
|
46.76
|
-
|
|
K2
|
48.96
|
53.24
|
48.42
|
47.71
|
-
|
|
K3
|
47.41
|
45.51
|
47.07
|
47.53
|
-
|
|
k1
|
15.21
|
14.42
|
15.50
|
15.59
|
-
|
|
k2
|
16.32
|
17.75
|
16.14
|
15.90
|
-
|
|
k3
|
15.80
|
15.17
|
15.69
|
15.84
|
-
|
|
R
|
1.11
|
3.33
|
0.64
|
0.31
|
-
|
|
Primary-Secondary Order
|
B>A>C>D
|
-
|
|
Optimal Level
|
A2
|
B2
|
C2
|
D2
|
-
|
Table 2 Results of Orthogonal Experiment and Range Analysis
3.2.2 Variance Analysis
To further verify the significance of the influence of each factor, variance analysis was performed on the orthogonal experimental data, and the results are shown in Table 3. It can be seen from Table 3 that soaking temperature (B) had an extremely significant effect on the extraction rate (P<0.01), solid-liquid ratio (A) had a significant effect on the extraction rate (P<0.05), while soaking time (C) and pH value (D) had no significant effect on the extraction rate (P>0.05), which was consistent with the results of range analysis, further confirming that soaking temperature is the key factor affecting the extraction effect.
|
Source of Variation
|
Sum of Squares
|
Degree of Freedom
|
Mean Square
|
F Value
|
P Value
|
Significance
|
|
Solid-Liquid Ratio (A)
|
3.85
|
2
|
1.93
|
8.78
|
0.021
|
*
|
|
Soaking Temperature (B)
|
22.68
|
2
|
11.34
|
51.64
|
0.001
|
**
|
|
Soaking Time (C)
|
1.12
|
2
|
0.56
|
2.55
|
0.168
|
-
|
|
pH Value (D)
|
0.65
|
2
|
0.33
|
1.50
|
0.302
|
-
|
|
Error
|
1.77
|
8
|
0.22
|
-
|
-
|
-
|
|
Total
|
30.07
|
16
|
-
|
-
|
-
|
-
|
Table 3 Variance Analysis of Orthogonal Experiment Note: * indicates P<0.05, significant difference; ** indicates P<0.01, extremely significant difference
3.3 Verification Experiment of Optimal Process
To verify the reliability of the optimal process determined by the orthogonal experiment, three parallel verification experiments were carried out according to the A2B2C2D2 combination (70% ethanol as solvent, solid-liquid ratio 1:25 (g:mL), soaking temperature 55℃, soaking time 6h, pH value 6.0). The total extraction rates of target components were 18.25%, 18.38%, and 18.33% respectively, with an average value of 18.32% and RSD of 0.36%, indicating that the process has good stability and repeatability. Compared with the optimal extraction rate (15.68%) in the single-factor experiment, it increased by 16.8%; compared with the traditional water soaking method (12.85%), it increased by 42.6%, confirming the superiority of the optimized process.
4 Discussion
This study took adenosine, uridine, and L-proline beneficial to men in Mantis Egg Case as target components, systematically optimized the soak extraction process parameters, and clarified the influence law and primary-secondary order of each factor. The results showed that solvent type is the basis of extraction. 70% ethanol can simultaneously extract both water-soluble and fat-soluble components due to its moderate polarity, which is consistent with the research conclusion of Wang Hao et al. (2022) on the ethanol extraction process of Mantis Egg Case. However, this study further clarified the optimal concentration of ethanol and supporting process parameters.
As an extremely significant factor affecting the extraction rate, the mechanism of soaking temperature is mainly reflected in two aspects: first, increasing temperature accelerates the movement speed of solvent molecules and raw material components, increasing mass transfer efficiency; second, appropriate temperature can destroy the cell wall structure of raw materials and promote the release of components. However, the extraction rate decreases when the temperature exceeds 55℃, which may be related to the heat sensitivity of components such as adenosine. This finding provides an important basis for avoiding the destruction of active components. As a significant influencing factor, the optimal value of solid-liquid ratio is 1:25. At this time, the solvent dosage can not only ensure the sufficient dissolution of components but also control the production cost, which meets the needs of industrial production.
Soaking time and pH value had no significant effect on the extraction rate. This may be because under the optimal temperature and solvent conditions, 6h is sufficient for the effective dissolution of components, and pH 6.0 is close to the optimal dissolution range of target components, so slight fluctuations have little impact on the overall extraction effect. Verification experiments showed that the optimized process has high extraction rate and good stability, and has obvious advantages over traditional methods, providing a feasible technical scheme for the development of Mantis Egg Case-based health products for males.
This study still has certain limitations. For example, only three components—adenosine, uridine, and L-proline—were quantitatively analyzed, which did not cover all male-beneficial components in Mantis Egg Case; the extraction method was static soaking, and future research can combine dynamic soaking or ultrasonic-assisted technology to further improve extraction efficiency. In addition, in vitro activity verification and in vivo safety evaluation of the extract also need in-depth research in the follow-up.
5 Conclusion
Through single-factor experiments and orthogonal experiments, the optimal soak extraction process of male-beneficial components from Mantis Egg Case was obtained: 70% ethanol as extraction solvent, solid-liquid ratio 1:25 (g:mL), soaking temperature 55℃, soaking time 6h, pH 6.0. Under these process conditions, the total extraction rate of target components can reach 18.32%, with good stability (RSD=0.36%).
The primary-secondary order of the influence of each process factor on extraction rate is: soaking temperature > solid-liquid ratio > soaking time > pH value, among which soaking temperature and solid-liquid ratio are extremely significant and significant influencing factors respectively. The extraction process established in this study has the advantages of simple operation, controllable cost, and high extraction efficiency, providing a scientific basis and technical support for the in-depth development of Mantis Egg Case and the industrial production of male health products.
