Analysis of the Influence of Different Processing Methods on the Main Effective Components in Oviductus Ranae by High-Performance Liquid Chromatography with Diode Array Detector

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Introduction
Oviductus Ranae (OR) is one of the "Treasures of Changbai Mountain" in China, which is also known as "the new three treasures of northeast China" together with ginseng and velvet antler.It was frst recorded in the "Compendium of Materia Medica."It is not only used as a nourishing and beautifying food in the folk [1] but also used as a medicine with the functions of tonifying the kidney, benefting essence, nourishing yin, and moistening the lungs.In addition, OR can be used for treatment of infrmity after illness, fatigued spirit, lack of strength, palpitation and insomnia, perspire during sleep, cough, and hemoptysis [2][3][4].Terefore, the OR industry has gradually become one of the pillar industries in northeast China.However, OR is derived from the oviduct of Rana temporaria chensinensis David.It is common knowledge that OR needs to be processed, whether used as food or medicine.Trough years of research studies on this medicine [5,6], our research group found that the processing and cooking methods of OR are often determined by the appearances and the tastes of fnished products.Te processing methods are relatively rough and have no scientifc guidance basis.
High-performance liquid chromatography is an important method to analyze the content of active components in traditional Chinese medicine or Chinese patent medicine [15][16][17].Te optimum processing technology was confrmed by the analysis of the infuence of diferent processing and cooking methods on the main efective components in OR by high-performance liquid chromatography with diode array detector (HPLC-DAD).Te study in this paper has guiding signifcance for the edible and processing of OR.

Preparation of Standard Solutions.
Appropriate amounts of 1-methyl hydantoin and estradiol were weighted precisely and dissolved with methanol via ultrasound to obtain stock standard solution (I) with the concentration of 1-methyl hydantoin 0.341 mg•mL −1 and estradiol 0.354 mg•mL −1 , respectively.Appropriate amount of 4-cholesten-3-one was weighted precisely and dissolved with methanol via ultrasound to obtain stock standard solution (II) with the concentration of 4-cholesten-3-one 1.71 mg•mL −1 .Cholesterol (42.00 mg) and 7-keto-cholesterol (4.58 mg) were weighted and placed in a 10 mL volumetric fask.Subsequently, 1 mL stock standard solution (I) and 1 mL stock standard solution (II) were added into the volumetric fask above and dissolved with methanol (fnal adjusted volume: 10 mL) to obtain the stock standard solution (III) with the concentration of 1methyl hydantoin 34.1 μg•mL −1 , estradiol 35.4 μg•mL −1 , cholesterol 4200 μg•mL −1 , 7-keto-cholesterol 458 μg•mL −1 , and 4-cholesten-3-one 171 μg•mL −1 .Finally, 1 mL of stock standard solution (III) was dissolved with methanol (fnal adjusted volume: 10 mL) and shaken well to obtain mix standard solutions.
2.4.Preparation of Test Solutions.About 5.0 g of OR power was weighed accurately and placed in a round-bottom fask.Ten, 100 mL of methanol was added into the round-bottom fask and was refux extracted for 30 min.Te sample was extracted twice in total.Ten, the methanol extracts were combined and evaporated to dryness via roller evaporator.Finally, the residue was redissolved with methanol (fnal adjusted volume: 5 mL) to obtain test solution.Te processed products of OR were prepared as per the method above.

Processing Methods of OR. Ten processing methods were applied in this study:
A. Boiling: Take an appropriate amount of OR and crush it, add 8 times water, boil for 30 min, and dry by airing B. Stewing: Take an appropriate amount of OR and crush it, add 20 times water, stew until water is absorbed completely, and cool and dry by airing C. Wine frying: Take an appropriate amount of OR and crush it, add 1/10 times yellow wine, mix thoroughly, heat gently until ustulate, and cool by airing D. Charcoal frying: Take an appropriate amount of OR and crush it, place in a hot pot, heat with high heat until burnt black, and cool by airing E. Frying: Take an appropriate amount of OR and crush it, heat gently until brown, and cool by airing F. Drying at 100 °C: Take an appropriate amount of OR and crush it, heat at 100 °C for 4 h, and cool and dry by airing G. Drying at 60 °C: Take an appropriate amount of OR and crush it, heat at 60 °C for 4 h, and cool and dry by airing H. Drying at 40 °C: Take an appropriate amount of OR and crush it, heat at 40 °C for 4 h, and cool and dry by airing 2 Journal of Chemistry

Calibration Curves, Limits of Detection, and Quantifcation.
Te stock standard solution (precise 0.2 mL, 0.4 mL, 1.0 mL, 2.0 mL, and 4.0 mL) was placed in a 10 mL volumetric fask and was adjusted to scale with methanol, respectively, to obtain the standard serial working solutions.Ten, the above standard working solutions were injected into HPLC for analysis, respectively.In addition, the standard solution in Section 3.3 was diluted gradually with methanol.Te limits of detection (LOD) and the limits of quantifcation (LOQ) were determined by three times and ten times of the signalnoise ratio, respectively.

Sample Content Determination.
Tree batches of OR and its processed products were prepared into test solutions based on the method in Section 2.4 and injected into HPLC for analysis according to the chromatographic conditions in Section 2.5, followed by the calculation of the contents of fve various analytes via the external standard method.

Calibration Curves, Limits of Detection, and
Quantifcation.Te standard curve was drawn by using the chromatographic peak area (Y) as the vertical axis and the concentration of the standard solution (X) as abscissa.Besides, the limit of detection (LOD) and limit of quantitation (LOQ) were calculated, respectively.Te results are displayed in Table 2, which indicated that fve components presented good linear relationships within their determination ranges as well as good sensitivity under the proposed chromatographic conditions.
Te contents of fve components were measured and the recovery rates were calculated, respectively.Te average recoveries of those fve components were in the range of  3, which suggested that the developed method was of good accuracy.

Sample Content Determination.
Te proposed HPLC-DAD method was applied to the simultaneous determination of fve main efective components in OR and its processing products.Te determination results are displayed and summarized in Figures 1 and 2 and in Table 4. Contents of measured components were in the range of 0.0003 to 3.9907 mg/g.Te concentration changes of fve chemical constituents in OR and OR processing products are shown in Figure 3.

Contents Determination Results.
OR not only has good efcacy but also tastes great after proper cooking.It has the reputation of "the head of Eight Treasures" as early as the Qing Dynasty [18,19].In the process of its cooking and processing, methods (including stewing, boiling, wine frying, frying, drying, and so forth) were usually applied.Moreover, changes of contents of fve components (1-methyl hydantoin, estradiol, cholesterol, 7-keto-cholesterol, and 4-cholesten-3-one) in OR processed by diferent methods were compared.Te results indicated that the contents of each analyte were diferent in OR processed via diferent methods.Te contents of fve components in OR processed by drying, wine frying, frying, and charcoal frying all decreased in some degree.Contents of 4-cholesten-3-one in OR processed by stewing and frying increased slightly, while the contents of 1-methyl hydantoin, estradiol, cholesterol, and 7-ketocholesterol were all decreased.Te results showed that OR unprocessed or processed by drying at a low temperature was recommended for use of eating and treating.
Drying is an indispensable processing method for food and traditional Chinese medicine, which is also suitable for the processing of OR [20].It was found that the contents of 1-methyl hydantoin, 4-cholesten-3-one, estradiol, cholesterol, and 7-keto-cholesterol in OR all decreased when dried at the temperature of 100 °C, 60 °C, and 40 °C.Te content of estradiol decreased signifcantly when OR was dried at the temperature of 100 °C, while less contents reduction were discovered when OR was dried at other temperatures.Estradiol has good pharmacodynamic efects.It can not only supplement estrogen and regulate hormone balance in vivo but also prevent osteoporosis.Estradiol can be used to treat leukopenia, breast cancer, and prostate cancer [21].However, estradiol is a double-edged sword for some people.It may lead to hormone abnormality, breast pain, weight gain, hypertension, gallstone, and liver function abnormalities [22].Terefore, OR should be dried according to treatment and tonic needs.
Frying method is usually applied when OR is processed.Te fried OR tastes delicious.And sometimes some yellow rice wine can be added properly in the process of frying, which can reduce the fshy smell and make OR tastes more delicious.In the pursuit of delicious food, people often ignore the content changes of efective ingredients in OR.Te local temperature of the medicinal materials is often too high in the process of frying, which may make some heatsensitive efective ingredients deteriorate.Te results displayed that contents of fve components in OR processed by wine frying, frying, and charcoal frying decreased signifcantly.Tus, OR should not be fried at high temperature so as not to afect its efects.
Stewed OR, fried OR, and original medicine materials are the most common way when people eat OR used as food or medicine.Te results also showed that contents of 1methyl hydantoin, estradiol, cholesterol, and 7-ketocholesterol in OR processed by stewing and frying decreased compared to those of original medicine materials, while content of 4-cholesten-3-one increased slightly.It was found that stewing has a little infuence on the content of each analyte, while frying has a great infuence on the content of 1-methyl hydantoin and estradiol.OR contains bacteria and microorganisms since it is a kind of animal product.

Journal of Chemistry
Terefore, the stewing method can kill bacteria and microorganisms under the premise of ensuring the efcacy of OR, which can guarantee the safety of OR as well.Journal of Chemistry were better using Alltima ™ C 18 column and methanol-0.1% phosphoric acid solution system.In addition, based on the previous study of our research group, the detective wavelengths were confrmed as 215 nm (1-methyl hydantoin, estradiol, and cholesterol) and 240 nm (7-keto-cholesterol and 4-cholesten-3-one), at which each component could be determined at the maximum absorption.

Conclusion
In this paper, a HPLC-DAD method was developed and verifed for the simultaneous determination of fve main efective components (1-methyl hydantoin, estradiol, cholesterol, 7-ketocholesterol, and 4-cholesten-3-one) in OR.In addition, the proposed method was applied to investigate the changes of fve constituents in OR prepared by diferent processing methods.
Te results indicated that OR unprocessed or processed by drying at a low temperature was recommended for use of eating and treating.Tis study provides guidance for the edible and processing technology of OR, which also lays a research foundation for product development and quality research.
Reagents.Oviductus Ranae (batch number: 20180301, 20180302, and 20180303) was gathered from Jingyu County of Baishan City in Jilin Province and identifed by professor Jiang Dacheng of Changchun University of Chinese Medicine.Te samples were in accordance with the relevant requirements of "Pharmacopoeia of People's Republic of China (2015 version, volume I)."

Table 2 :
Linear relationships of various constituents.
Methods.First of all, prepared methods of test samples were investigated in this paper.Te infuence of diferent extraction solvents (water, methanol, ethanol, and anhydrous ethanol) on the transfer rates of the test sample and the separation efects of each analyte were studied.

Table 4 :
Results of content determination of various components (mg/g, n � 3).
C 18 , Angilent TC-C 18 and Kromasil 100-5 C 18 ) and diferent mobile phase systems (methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid solution, and methanol-0.2%phosphoric acid solution) were compared, respectively.Te results exhibited that the baseline was basically separated and the peak shapes of the chromatographic peak of each analyte