TLC Detection of Acanthopanax Senticosus in Cistanche and Epimedium Black Tea

Abstract

The present application relates to a thin layer chromatography detection method of Acanthopanax senticosus in Cistanche Epimedium black tea, comprising the following steps: preparing a test solution of Cistanche Epimedium black tea to be tested, a reference solution of isofraxidin and Acanthopanax senticosus reference medicinal material solution; spotting the test solution of Cistanche Epimedium black tea to be tested, the reference solution of isofraxidin and Acanthopanax senticosus reference medicinal material solution on the same silica gel thin layer plate, developing them with a developing agent, taking them out, drying them, and then performing a color development treatment; wherein the developing agent is chloroform and a methanol solution; the methanol solution is methanol or a methanol-water solution. The thin layer chromatography detection method has good separation effect on Acanthopanax senticosus in Cistanche Epimedium black tea, high detection accuracy, simple operation, and low cost.

Description

Technical Field

[0001] The present application relates to the technical field of food detection, and in particular to a thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea. Background Art

[0002] Cistanche and Epimedium Black Tea is a health food that can relieve physical fatigue, made from Cistanche, jujube, epimedium, Acanthopanax, black tea, dextrin and other main raw and auxiliary materials, using Chinese medicine water extraction technology combined with modern technology. Acanthopanax is one of the important ingredients in this product, and accurate testing of it can ensure the quality and efficacy of the product.

[0003] Acanthopanax is a plant of the Araliaceae family that is used as medicine in its dried roots, rhizomes or stems. Modern research has found that Acanthopanax contains a variety of ingredients, including glycosides, polysaccharides, flavonoids, lignans, phenylpropanoids, coumarins, and trace elements and amino acids such as Na, Cu, Mg, Ca, Sn, and Mn. It has a variety of physiological functions, including immune regulation, anti-aging, anti-inflammatory, and anti-stress. The quality control of Acanthopanax medicinal materials is carried out in accordance with the thin layer chromatography method (General Rule 0502) in the "Chinese Pharmacopoeia 2020 Edition Part 1" to control the isofraquinone in it.

[0004] At present, there are problems such as complex operation, low accuracy and high cost in detecting the Acanthopanax senticosus components in Cistanche and Epimedium black tea. Summary of the invention

[0005] Based on this, the present application provides a thin layer chromatography detection method for Acanthopanax senticosus in Cistanche Epimedium black tea with good separation effect, high detection accuracy, simple operation and low cost.

[0006] The specific technical solutions are as follows:

[0007] A thin layer chromatography detection method for Acanthopanax senticosus in Cistanche and Epimedium black tea, comprising the following steps:

[0008] Prepare the test solution of Cistanche deserticola and Epimedium black tea, the reference solution of isofraxidin and the reference medicinal material solution of Acanthopanax senticosus;

[0009] The test solution of the Cistanche deserticola and Epimedium black tea to be tested, the isofraxidin reference solution and the Acanthopanax senticosus reference medicinal material solution are spotted on the same silica gel thin layer plate, developed with a developing agent, taken out, dried and then subjected to color development treatment;

[0010] Wherein, the developing agent is a solution of chloroform and methanol in a volume ratio of (10-19):(1-10);

[0011] The methanol solution is methanol or a methanol aqueous solution with a volume percentage of 20% to 30%.

[0012] In some of the embodiments, the developing agent is chloroform and methanol in a volume ratio of (18-19):(1-2).

[0013] In some of these embodiments, the color development process includes the step of observing under ultraviolet light.

[0014] In some embodiments, the wavelength of the ultraviolet lamp is 365nm.

[0015] In some embodiments, the test solution of Cistanche deserticola and Epimedium black tea comprises the following steps:

[0016] Adding a first solvent to the tested Cistanche deserticola and Epimedium black tea for extraction, evaporating the obtained extract to dryness to prepare a first solid;

[0017] After the first solid is dissolved in a second solvent, a third solvent is added for extraction, and the third solvent phase is collected and evaporated to dryness to prepare a second solid;

[0018] The second solid is added into the fourth solvent for dissolution to prepare the test solution of the Cistanche deserticola and Epimedium black tea to be tested;

[0019] And / or, preparing the Acanthopanax senticosus control medicinal material solution comprises the following steps:

[0020] Adding a first solvent to an Acanthopanax senticosus reference medicinal material for extraction, and evaporating the obtained extract to dryness to prepare a first solid;

[0021] After the first solid is dissolved in a second solvent, a third solvent is added for extraction, and the third solvent phase is collected and evaporated to dryness to prepare a second solid;

[0022] The second solid is added into the fourth solvent for dissolution to prepare the Acanthopanax senticosus control medicinal material solution.

[0023] In some embodiments, the first solvent is independently an ethanol aqueous solution with a volume concentration of 70% to 80%.

[0024] In some embodiments, the second solvent is each independently water, and the third solvent is each independently chloroform.

[0025] In some embodiments, the fourth solvent is each independently methanol.

[0026] In some embodiments, the first solvent is added for extraction, and the step of evaporating the obtained extract to dryness has one or more of the following characteristics:

[0027] (1) The extraction temperature is heated to reflux;

[0028] (2) The extraction time is 50 to 70 minutes;

[0029] (3) The evaporation temperature is 55℃~65℃.

[0030] In some embodiments, after adding the second solvent to dissolve, adding the third solvent to extract, and collecting the third solvent phase to dryness have one or more of the following characteristics:

[0031] (1) The volume ratio of the second solvent to the third solvent is (1-3):1;

[0032] (2) The number of extractions is 1 to 3 times;

[0033] (3) The evaporation temperature is 25℃~35℃.

[0034] The above-mentioned thin layer chromatography detection method of Acanthopanax senticosus in Cistanche Epimedium black tea can achieve effective separation of isofraquinone, a marker component of Acanthopanax senticosus in Cistanche Epimedium black tea, by adopting a suitable developing agent and a thin layer plate for detection, and then detect Acanthopanax senticosus with high accuracy and high sensitivity. The detection operation is simple, the detection time is short, and the cost is low. The corresponding thin layer chromatography spots are clear and free of interference, which improves the accuracy and comprehensiveness of the quality control of Cistanche Epimedium black tea, and is conducive to the establishment and overall evaluation of the quality standard of Cistanche Epimedium black tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the thin layer plate image of Example 1 using chloroform-methanol (19:1) as the developing solvent.

[0036] Figure 2 This is the thin layer plate image of Example 2 using chloroform-methanol (16:4) as the developing solvent.

[0037] Figure 3 This is the thin layer plate image of Example 3 using chloroform-methanol (13:7) as the developing solvent.

[0038] Figure 4 This is the thin layer plate image of Example 4 using chloroform-methanol (10:10) as the developing solvent.

[0039] Figure 5 This is the thin layer plate image of comparative example 1 using chloroform-methanol (7:13) as the developing solvent.

[0040] Figure 6 This is the thin layer plate image of comparative example 2 using chloroform-methanol (4:16) as the developing solvent.

[0041] Figure 7 This is the thin layer plate image of comparative example 3 using chloroform-methanol (7:13) as the developing solvent.

[0042] Figure 8 This is the thin layer plate image of comparative example 4 using chloroform-75% methanol (19:1) as the developing solvent.

[0043] Fig. 9 This is the thin layer plate image of comparative example 5 using chloroform-50% methanol (19:1) as the developing solvent.

[0044] Fig.10 This is the thin layer plate image of Example 5 using chloroform-25% methanol (19:1) as the developing solvent.

[0045] Fig.11 This is the thin layer plate image of comparative example 6 using chloroform-methanol-formic acid (19:1:0.3) as the developing solvent.

[0046] Fig.12 This is the thin layer plate image of comparative example 7 using chloroform-formic acid-water (6:2:1) as the developing solvent.

[0047] Fig.13 This is the thin layer plate image of comparative example 8 using toluene-ethyl acetate-formic acid (20:4:0.5) as the developing solvent.

[0048] Fig.14 This is a thin layer plate diagram of comparative example 9 using a polyamide plate as the thin layer plate.

[0049] Fig.15 This is a thin layer plate diagram of Example 6 using a silica gel H plate as the thin layer plate.

[0050] Fig.16 This is a thin layer plate diagram of Example 7 using a silica gel GF254 plate as the thin layer plate.

[0051] Among them, the samples spotted on each thin layer plate are, from left to right, the test solution, the isofraxidin reference solution, the Acanthopanax control medicinal material solution (the aforementioned solutions are spotted once) and the negative control medicinal material solution. DETAILED DESCRIPTION

[0052] The following is a further detailed description of the thin layer chromatography detection method of Acanthopanax senticosus in the Cistanche Epimedium black tea of ​​the present application in conjunction with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0054] The optional scope of the terms "and / or", "or / and", and "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, said any and all combinations include any combination of any two related listed items, any more related listed items, or all related listed items.

[0055] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0056] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0057] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0058] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0059] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0060] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.

[0061] The room temperature in the present application generally refers to 4°C to 30°C, preferably 20±5°C.

[0062] In some examples of the present application, a thin layer chromatography detection method of Acanthopanax senticosus in Cistanche Epimedium black tea is provided, comprising the following steps:

[0063] Prepare the test solution of Cistanche deserticola and Epimedium black tea, the reference solution of isofraxidin and the reference medicinal material solution of Acanthopanax senticosus;

[0064] The test solution of the Cistanche deserticola and Epimedium black tea to be tested, the isofraxidin reference solution and the Acanthopanax senticosus reference medicinal material solution are spotted on the same silica gel thin layer plate, developed with a developing agent, taken out, dried and then subjected to color development treatment;

[0065] Wherein, the developing agent is a solution of chloroform and methanol in a volume ratio of (10-19):(1-10);

[0066] The methanol solution is methanol or a methanol aqueous solution with a volume percentage of 20% to 30%.

[0067] It can be understood that after the color development treatment, it is possible to judge whether the sample contains isoflavone and preliminarily determine its content based on the spot position, color and other characteristics of the test sample and the reference substance on the thin layer plate.

[0068] Specifically, the volume percentage of the methanol aqueous solution includes, but is not limited to, 20%, 23%, 25%, 27%, 30% or a range between any two of the foregoing.

[0069] Specifically, the volume ratio of chloroform to methanol in the developing agent includes but is not limited to: 19:1, 16:4, 13:7, 10:10 or a range between any two of the foregoing. Further, the developing agent is chloroform and methanol in a volume ratio of (18-19):(1-2).

[0070] Without limitation, the silica gel thin layer plate may be, for example, a silica gel G plate, a silica gel H plate, or a silica gel GF254 plate, etc. In some examples, the silica gel thin layer plate is a silica gel G plate.

[0071] In some of the examples, the color development process includes a step of observing under an ultraviolet lamp. Further, the wavelength of the ultraviolet lamp is 365nm.

[0072] In addition, the accuracy and sensitivity of the detection can be further improved by effectively extracting the isofraxidin component in the Cistanche and Epimedium black tea to be tested.

[0073] In some of these examples, the test solution of Cistanche deserticola and Epimedium black tea includes the following steps:

[0074] Adding a first solvent to the tested Cistanche deserticola and Epimedium black tea for extraction, evaporating the obtained extract to dryness to prepare a first solid;

[0075] After the first solid is dissolved in a second solvent, a third solvent is added for extraction, and the third solvent phase is collected and evaporated to dryness to prepare a second solid;

[0076] The second solid is added into the fourth solvent for dissolution to prepare the test solution of the Cistanche deserticola and Epimedium black tea to be tested.

[0077] In some of the examples, preparing the Acanthopanax senticosus control medicinal material solution includes the following steps:

[0078] Adding a first solvent to an Acanthopanax senticosus reference medicinal material for extraction, and evaporating the obtained extract to dryness to prepare a first solid;

[0079] After the first solid is dissolved in a second solvent, a third solvent is added for extraction, and the third solvent phase is collected and evaporated to dryness to prepare a second solid;

[0080] The second solid is added into the fourth solvent for dissolution to prepare the Acanthopanax senticosus control medicinal material solution.

[0081] Further, in the step of adding a first solvent for extraction and evaporating the obtained extract to dryness:

[0082] In some examples, the first solvent is independently an ethanol aqueous solution with a volume concentration of 70% to 80%. Specifically, the volume concentration of the ethanol aqueous solution includes but is not limited to: 70%, 73%, 75%, 77%, 80% or a range between any two of the foregoing.

[0083] In some of these examples, the extraction temperature is heated to reflux.

[0084] In some examples, the extraction time is 50 min to 70 min. Specifically, the extraction time includes but is not limited to: 50 min, 55 min, 58 min, 60 min, 62 min, 65 min, 70 min or a range between any two of the foregoing.

[0085] In some examples, the evaporation temperature is 55° C. to 65° C. Specifically, the evaporation temperature includes, but is not limited to, 55° C., 57° C., 60° C., 63° C., 65° C., or a range between any two of the foregoing.

[0086] Further, after adding the second solvent to dissolve, adding the third solvent to extract, collecting the third solvent phase and evaporating to dryness:

[0087] In some examples, the second solvent is each independently water, and the third solvent is each independently chloroform.

[0088] In some examples, the volume ratio of the second solvent to the third solvent is (1-3): 1. Specifically, the volume ratio of the second solvent to the third solvent includes but is not limited to: 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1 or a range between any two of the foregoing.

[0089] In some examples, the number of extractions is 1 to 3. Specifically, the number of extractions includes, but is not limited to: 1, 2, 3, or a range between any two of the foregoing.

[0090] In some examples, the evaporation temperature is 25° C. to 35° C. Specifically, the evaporation temperature includes, but is not limited to, 25° C., 27° C., 30° C., 33° C., 35° C., or a range between any two of the foregoing.

[0091] Further, in the step of adding a fourth solvent for dissolving:

[0092] In some examples, the fourth solvent is each independently methanol.

[0093] For experimental parameters not specified in the following specific embodiments, reference is made to the instructions given in the present application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by manufacturers.

[0094] The raw materials and reagents involved in the following specific embodiments can be obtained from the market, or those skilled in the art can prepare them according to known means. The Cistanche Epimedium Black Tea used in the embodiment is from Xinjiang Huachun Pharmaceutical Co., Ltd., with the following proportions: Cistanche Tubulosa 1410g, jujube 990g, epimedium 990g, Acanthopanax Senticosus 990g, black tea 2010g, dextrin 99g, and this product is a 3.0g / bag bagged tea brewing agent.

[0095] Example 1

[0096] This embodiment is a thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea.

[0097] (1) Preparation of reference substance stock solution

[0098] Take the isoflurane reference substance and add methanol to make a solution containing 1 mg per 1 mL as the reference substance solution.

[0099] (2) Preparation of control medicinal material solution

[0100] Take 5 g of Acanthopanax senticosus control medicinal material, add 50 mL of 75% ethanol aqueous solution, heat and reflux for 1 hour, filter, evaporate the filtrate at 60°C, add 10 mL of water to the residue to dissolve it, shake and extract with chloroform twice, 5 mL each time, combine the chloroform solutions, evaporate at 30°C, add 1 mL of methanol to the residue to dissolve it, and use it as the control medicinal material solution.

[0101] (3) Preparation of test solution

[0102] Take 5g of Cistanche deserticola and Epimedium black tea powder, add 50mL of 75% ethanol aqueous solution, heat and reflux for 1 hour, filter, evaporate the filtrate at 60℃, dissolve the residue in 10mL of water, shake and extract with chloroform twice, 5mL each time, combine the chloroform liquid, evaporate at 30℃, dissolve the residue in 1mL of methanol, and use it as the test solution.

[0103] (4) Negative control medicinal materials

[0104] To prepare the negative control medicinal material of Cistanche tubulosa and epimedium black tea, the four medicinal materials of Cistanche tubulosa, jujube, epimedium and black tea were mixed and ground according to the compound ratio, and the negative control medicinal material solution was prepared in the same way as the test sample solution.

[0105] (5) Chromatographic conditions

[0106] According to the thin layer chromatography method (General Rule 0502), 10 μL of each of the above four solutions was taken and spotted on the same silica gel G thin layer plate, and chloroform-methanol (19:1) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 mn). The results were as follows: Figure 1 shown.

[0107] Examples 2 to 5 and Comparative Examples 1 to 8

[0108] This embodiment and comparative example are thin layer chromatography detection methods for Acanthopanax senticosus in Cistanche Epimedium Black Tea. The steps are the same as those in Example 1, and the main difference is that different developing agents are used. The specific developing agents are shown in Table 1 below. The detection results are shown in Table 1 below. Figures 2 to 13 As shown:

[0109] Table 1

[0110]

[0111] Combination Figures 1 to 13 It can be seen that the developing agents of Examples 1 to 5 can effectively separate the characteristic ingredient of Acanthopanax senticosus in Cistanche Epimedium Black Tea, and the spots are clear, the interference of miscellaneous peaks is small, and the detection results are accurate, among which Example 1 is the best. Comparative Examples 1 to 8 change the type of developing agent, and there are problems such as overlap with adjacent spots, unclear spots, and excessive development, which makes it difficult to detect Acanthopanax senticosus in Cistanche Epimedium Black Tea.

[0112] Examples 6-7 and Comparative Example 9

[0113] This embodiment and comparative example are thin layer chromatography detection methods for Acanthopanax senticosus in Cistanche Epimedium Black Tea. The steps are the same as those in Example 1, and the main difference is that different thin layer plates are used. The specific thin layer plates are shown in Table 2 below. The detection results are shown in Table 2 below. Figures 14 to 16 As shown:

[0114] Table 2

[0115]

[0116] Combination Figure 1 and 14From Figure 1 to 16, it can be seen that Examples 1, 6 to 7 use different types of silica gel plates, which can effectively separate the characteristic ingredient of Acanthopanax senticosus in Cistanche Epimedium Black Tea, and the spots are clear, the interference of miscellaneous peaks is small, and the detection results are accurate, among which Example 1 is the best. Comparative Example 9 uses a polyamide plate, and after development, the spots are blurred, the edges are unclear, and it presents an irregular diffuse morphology, which makes it difficult to accurately distinguish its specific shape, and it is difficult to detect Acanthopanax senticosus in Cistanche Epimedium Black Tea.

[0117] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0118] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A thin layer chromatography detection method for Acanthopanax senticosus in Cistanche and Epimedium black tea, characterized in that: The steps include: Prepare the test solution of Cistanche deserticola and Epimedium black tea, the reference solution of isofraxidin and the reference medicinal material solution of Acanthopanax senticosus; The test solution of the Cistanche deserticola and Epimedium black tea to be tested, the isofraxidin reference solution and the Acanthopanax senticosus reference medicinal material solution are spotted on the same silica gel thin layer plate, developed with a developing agent, taken out, dried and then subjected to color development treatment; Wherein, the developing agent is a solution of chloroform and methanol in a volume ratio of (10-19):(1-10); The methanol solution is methanol or a methanol aqueous solution with a volume percentage of 20% to 30%.

2. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 1, characterized in that: The developing agent is chloroform and methanol in a volume ratio of (18-19):(1-2).

3. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 1, characterized in that: The color development process includes a step of observation under ultraviolet light.

4. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 3, characterized in that: The wavelength of the ultraviolet lamp is 365nm.

5. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to any one of claims 1 to 4, characterized in that: The test solution of Cistanche deserticola and Epimedium black tea to be tested comprises the following steps: Adding a first solvent to the tested Cistanche deserticola and Epimedium black tea for extraction, evaporating the obtained extract to dryness to prepare a first solid; After the first solid is dissolved in a second solvent, a third solvent is added for extraction, and the third solvent phase is collected and evaporated to dryness to prepare a second solid; The second solid is added into the fourth solvent for dissolution to prepare the test solution of the Cistanche deserticola and Epimedium black tea to be tested; And / or, preparing the Acanthopanax senticosus control medicinal material solution comprises the following steps: Adding a first solvent to an Acanthopanax senticosus reference medicinal material for extraction, and evaporating the obtained extract to dryness to prepare a first solid; After the first solid is dissolved in a second solvent, a third solvent is added for extraction, and the third solvent phase is collected and evaporated to dryness to prepare a second solid; The second solid is added into the fourth solvent for dissolution to prepare the Acanthopanax senticosus control medicinal material solution.

6. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 5, characterized in that: The first solvents are each independently an ethanol aqueous solution with a volume concentration of 70% to 80%.

7. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 5, characterized in that: The second solvent is independently water, and the third solvent is independently chloroform.

8. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 5, characterized in that: The fourth solvent is each independently methanol.

9. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 5, characterized in that: The step of adding the first solvent for extraction and evaporating the obtained extract to dryness has one or more of the following characteristics: (1) The extraction temperature is heated to reflux; (2) The extraction time is 50 to 70 minutes; (3) The evaporation temperature is 55℃~65℃.

10. The thin layer chromatography detection method of Acanthopanax senticosus in Cistanche and Epimedium black tea according to claim 5, characterized in that: After adding the second solvent to dissolve, adding the third solvent to extract, and collecting the third solvent phase to dryness, the steps have one or more of the following characteristics: (1) The volume ratio of the second solvent to the third solvent is (1-3):1; (2) The number of extractions is 1 to 3 times; (3) The evaporation temperature is 25℃~35℃.

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