Normal-temperature efficient extraction method of cortisol in pig hair

Abstract

The invention provides a pig hair cortisol hormone extraction method, which comprises: 1, washing a hair sample with isopropanol, and drying for 3-5 h at a temperature of 50-70 DEG C to obtain washed hair; 2, the washed hair is ground, ground hair is obtained, and the grinding time is 10-20 min; 3, the ground hair makes contact with methyl alcohol for 8-24 h at the rotating speed of 0-1400 rpm, then centrifugation is conducted, and supernate is obtained. By utilizing the method provided by the invention, the volatilization of the incubation medium is effectively inhibited, the contact between the incubation liquid and the sample is increased, the extraction amount of the cortisol hormone in the pig hair is increased, the cost is effectively reduced, and the method can be widely applied to the field of animal science.

Description

Technical Field

[0001] The invention relates to the field of animal husbandry science, and in particular to a method for extracting cortisol hormone from pig hair. Background Art

[0002] Cortisol synthesis begins with cholesterol within the mitochondria of the adrenal cortex. Cholesterol is converted to pregnenolone under the catalysis of cytochrome P450scc. Pregnenolone is then converted to progesterone and 17-hydroxyprogesterone, followed by 11-dehydrocortisol. Finally, 11-dehydrocortisol is converted to cortisol via 11-hydroxylase. Cortisol, a glucocorticoid, primarily regulates the metabolism of sugars, fats, and proteins. Its counterpart, corticosterone, is a mineralocorticoid produced by the zona glomerulosa cells of the adrenal cortex and primarily functions to maintain water and electrolyte balance. Corticosterone and cortisol can be converted into each other under the action of 11-βHSD1 and 11-βHSD2.

[0003] Cortisol is primarily produced by the HPA (hypothalamic-pituitary-adrenal axis). The medial paramedian nucleus of the hypothalamus releases corticotropin-releasing factor (CRH). CRH acts on the anterior pituitary gland via the portal venous system, stimulating the secretion of adrenocorticotropic hormone (ACTH). After entering the bloodstream, ACTH acts on the adrenal cortex, stimulating the synthesis and secretion of cortisol. Cortisol secretion is regulated by a negative feedback mechanism. When cortisol levels rise, the secretion of CRH and ACTH is inhibited.

[0004] Cortisol plays an important role in the metabolism of sugars, lipids, and proteins, as well as in immune regulation and cardiovascular regulation. Specifically, cortisol stimulates gluconeogenesis in the liver while reducing peripheral tissue sensitivity to insulin and inhibiting its uptake, leading to elevated blood sugar levels. In the short term, cortisol promotes fat breakdown, while long-term high levels of cortisol can lead to lipogenesis. Cortisol can reduce inflammatory responses by inhibiting the function of immune cells. Cortisol can promote vasoconstriction and regulate sodium reabsorption, making it a potential cause of hypertension. In pig farming, cortisol levels are often an important reference for evaluating the pig's growth environment and stress status.

[0005] The vast majority of cortisol in the body (90%) is bound to the carrier protein cortisol-binding globulin (CBG). Free cortisol accounts for only 5%-10%. Cortisol levels in the body fluctuate significantly during the day and night. Currently, traditional methods for extracting cortisol include saliva, sweat, serum, and tissue fluid sampling. Serum and tissue fluid are invasive, while sweat and saliva are non-invasive. Serum sampling can calculate the body's total cortisol level (including free and bound cortisol); tissue fluid can be sampled during sleep at night and is primarily used to measure cortisol concentrations at specific times. However, these two methods are prone to causing harm to livestock and causing stress reactions during actual operation. Sweat is easy to sample, and the level of cortisol secretion over a period of time can be calculated by wearing wearable measuring devices, but the cortisol level is easily fluctuated while the subject is sweating; saliva sampling collects samples of free cortisol, but saliva contains cortisone, which will be converted into cortisol under the action of the above-mentioned 11-βHSD2 secreted by the salivary glands. Therefore, the free cortisol concentration measured in saliva will be higher than the actual value.

[0006] In recent years, methods for measuring free cortisol concentrations in hair samples have become increasingly mature. Free cortisol present in the capillaries of hair follicles can diffuse into the hair medulla and accumulate there. Measuring the cortisol content of a given length of hair can reveal the average level of cortisol in the body during that period of hair growth. This method is easy to use, minimally invasive to livestock, and can assess cortisol levels over a specific period of time. However, in practical application, this technology still faces numerous challenges that need to be addressed. A rapid, efficient, and practical method for detecting hair cortisol is urgently needed. Summary of the Invention

[0007] The present application discovered that existing methods for extracting cortisol from hair all have defects in the conditions for drying the hair after washing, the method for crushing the hair, and the conditions for incubating the crushed hair with methanol, which seriously affect the extraction efficiency of cortisol. However, this defect has not been noticed. Based on this, the present invention provides a room-temperature and efficient extraction technology for cortisol from pig hair to overcome this defect.

[0008] The present invention provides a room-temperature efficient extraction technology for cortisol from pig hair, the method comprising the following steps: Step 1: Wash the hair sample with isopropyl alcohol and dry it at 50-70°C for 3-5 hours to obtain washed hair; Step 2: Grinding the washed hair to obtain ground hair, preferably, the grinding time is 10-20 minutes; Step 3: The ground hair is exposed to methanol at a speed of 0-1400 rpm for 8-24 hours and then centrifuged to obtain a supernatant.

[0009] Preferably, in step 3, the temperature of the contact between the hair sample and methanol is 18°C-25°C, the time is 12h-20h, and the rotation speed is 600-1200rpm; Preferably, the time is 12-16 hours and the rotation speed is 800-1200 rpm; for example, the temperature is 20°C, the time is 16 hours, and the rotation speed is 800 rpm, or the temperature is 20°C, the time is 16-20 hours, and the rotation speed is 1200 rpm.

[0010] Preferably, in step three, the centrifugation condition is 10,000-12,000 rpm for 3-6 min.

[0011] Preferably, the ratio of ground hair to methanol is 40-60:1 mg / ml, preferably 45-55:1 mg / ml, for example 50:1 mg / ml, 0.2 ml of methanol is used for every 10 mg of hair, and 0.6 ml of methanol is used for every 30 mg of hair.

[0012] Preferably, in step 1, the drying temperature is 55-65° C. and the drying time is 3-5 hours, for example, the drying temperature is 60° C. and the drying time is 4 hours. Under these conditions, static electricity on the hair after drying can be reduced, thereby improving the efficiency of subsequent treatments.

[0013] Optionally, in step 1, the isopropyl alcohol washing time is 1.5-4 minutes, preferably, 2 minutes, which can wash away the pollution on the hair surface without affecting the cortisol inside the hair.

[0014] Optionally, in step 2, the rotor and steel balls required for grinding should be pre-cooled at -20°C.

[0015] Optionally, in step 2, the hair is cut into small segments and the grinding condition is 10-20 minutes at 30-35 Hz. The hair is considered to be fully ground when there are no more segments. Preferably, the grinding time is 15 minutes.

[0016] Optionally, the hair is taken from a mammal.

[0017] Optionally, the mammal is a cow, pig, sheep, dog, rabbit, etc. Preferably, the mammal is a pig.

[0018] Application of the extraction method of the present invention in detecting cortisol in animal hair.

[0019] Optionally, the mammal is a cow, pig, sheep, dog, rabbit, etc. Preferably, the mammal is a pig.

[0020] Beneficial technical effects: The present invention provides a method for efficiently extracting cortisol from pig hair at room temperature, effectively inhibiting the volatilization of the incubation medium and increasing the contact between the incubation solution and the sample. Under the incubation conditions of incubating methanol and the hair sample at room temperature at 800 rpm for 16 hours, the cortisol extraction efficiency reaches a peak of 18.722±2.027 pg / mg. The extraction process is stable and controllable, the extraction efficiency is significantly improved, and the cost is effectively reduced. This method can be widely used in the field of animal science. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the standard curve for cortisol detection.

[0022] Figure 2 Figure 3 shows the changes in hair cortisol content under different incubation conditions. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0026] Example 1: Extraction of cortisol from pig hair The hair cortisol hormone extraction method disclosed in Chinese Patent 201510570829.X (published on December 16, 2015) was used to extract cortisol from the hair of Landrace and Large White pigs. The steps are as follows: (1) Take about 60 mg of hair from three Large White pigs and three Landrace pigs, and store them in a dark place until the day of the experiment.

[0027] (2) Place the hair sample in a 15ml centrifuge tube, add 10ml of isopropanol to each tube and shake and wash twice, each wash for 3 minutes.

[0028] (3) The washed hair was placed in an oven and dried at 70°C for 4 hours to obtain washed and dried hair.

[0029] (4) Cut the hair into small pieces and transfer them into a 5 ml centrifuge tube. Grind the hair using a ball mill at 35 Hz for 5 min.

[0030] (5) Take 50 mg of hair from each sample and place it in a 1.5 ml centrifuge tube. Add 1 ml of methanol to each tube and incubate in a metal bath at 80°C for 16 h.

[0031] During the experiments, the inventors discovered that pig hair could not be completely ground into a powder. Furthermore, because methanol's boiling point of 64.7°C is lower than the temperature at which hair and methanol are incubated (80°C), this accelerated methanol volatilization. Using conventional incubation conditions, the inventors employed sealing measures such as sealing film and explosion-proof clamps, but these measures were unable to address the methanol volatilization issue. Ultimately, only two of the 18 samples contained any methanol liquid in the centrifuge tubes.

[0032] Example 2: Screening of incubation medium for extraction of cortisol hormone from pig hair To improve the extraction efficiency of cortisol from pig hair, the existing extraction method (published on December 16, 2015, in document 201510570829.X) was modified. Comparing samples revealed that the hair samples used in the existing method were taken from cows, which have a soft texture, while pig hair is harder. The current grinding time was not universally applicable and should be appropriately extended. Furthermore, the incubation medium was screened. Six Landrace pig hair samples were collected to create a mixed hair sample. Cortisol was extracted using methanol, ethanol, acetone, and isopropanol, respectively. Three parallel experiments were performed for each medium. The steps were as follows: (1) Take 500 mg of mixed hair samples without hair follicles from six pigs and store them in the dark until the day of the experiment.

[0033] (2) Weigh the mixed hair sample, take about 50 mg of hair from each parallel tube and place it in a 15 ml centrifuge tube. Add 10 ml of isopropanol to each tube and shake and wash it twice, each washing for 2 minutes.

[0034] (3) The washed hair was placed in an oven and dried at 60°C for 4 hours to obtain washed and dried hair.

[0035] (4) Cut the hair into small pieces and transfer them into a 5 ml centrifuge tube. Grind the hair using a ball mill at 35 Hz for 15 min. Use a 5 mm diameter steel ball. Grind the hair until there are no more hair segments. The rotor and steel balls should be pre-cooled at -20 °C.

[0036] (5) For each parallel sample, 30 mg of hair was placed in a 2 ml centrifuge tube. 0.6 ml of methanol, 0.6 ml of ethanol, 0.6 ml of acetone, and 0.6 ml of isopropanol were added to the tube, respectively. The tube was incubated at room temperature (18-25 degrees Celsius, preferably 20 degrees Celsius) for 24 hours.

[0037] (6) Place the incubated samples in a centrifuge and centrifuge at 12,000 rpm for 3 minutes. Pipette 0.3 ml of the supernatant from each sample into a 1.5 ml centrifuge tube and store at -20°C until the day of measurement.

[0038] (7) Use the salivary cortisol test kit and perform the experimental operation according to the instructions. The specific steps are as follows: Add 50µl of standard, control, and sample duplex assay to each well; add 50µl of enzyme conjugate to each well; incubate at room temperature on a shaker at 900 rpm for 60 minutes; dilute the 10x concentrated wash buffer 10-fold with distilled water and set aside; spin dry the plate and wash four times with 300µl of wash buffer per well. After washing, tap the wells with absorbent paper to remove any remaining droplets; add 200µl of substrate solution to each well; incubate at room temperature in the dark for 30 minutes without shaking; add 50µl of stop solution to each well to terminate the enzymatic reaction (the blue color immediately turns yellow); measure the absorbance (OD) of each well at 450nm within 15 minutes after adding the stop solution.

[0039] Table 1 Standard product data

[0040] Table 2 Cortisol content in pig hair extracted using different incubation media

[0041] According to the standard data in Table 1, the Figure 1 The standard curve shown in Figure 2 shows cortisol concentration on the horizontal axis and absorbance signal on the vertical axis. The cortisol concentration in the sample was calculated by substituting the sample absorbance into the standard curve. The cortisol content of samples treated with different incubation media is shown in Table 2. The above experiments show that under the same incubation conditions, increasing the grinding time and lowering the incubation temperature to room temperature can effectively improve the extraction efficiency of cortisol in the sample. Among them, methanol has the most significant extraction efficiency, which is 7.3 times that of acetone and 2.7 times that of ethanol. It also has the best reproducibility, with a standard deviation of 15.2% and the highest extraction efficiency.

[0042] Example 3 Effects of different incubation conditions and incubation times on cortisol extraction Based on Example 2, this method further optimized the conditions for extracting cortisol from hair. Three pig hair samples provided by a cooperative pig farm in Xinjiang were used for cortisol extraction. Three replicates were made for each sample, and an orthogonal experiment with different incubation conditions and times was performed. The steps are as follows: (1) Take about 1500 mg of hair from three Large White pigs and three Landrace pigs and store them in a dark place until the day of the experiment.

[0043] (2) Weigh the hair sample, take about 50 mg of hair from each parallel tube and place it in a 15 ml centrifuge tube. Add 10 ml of isopropanol to each tube and shake and wash it twice, each washing for 2 minutes.

[0044] (3) The washed hair was placed in an oven and dried at 60°C for 4 hours to obtain washed and dried hair.

[0045] (4) Cut the hair into small pieces and transfer them into a 5 ml centrifuge tube. Grind the hair using a ball mill at 35 Hz for 15 min. Use a 5 mm diameter steel ball. Grind the hair until there are no more hair segments. The rotor and steel balls should be pre-cooled at -20 °C.

[0046] (5) For each parallel experiment, 30 mg of hair was collected and placed in a 2 ml centrifuge tube. 0.6 ml of methanol was added to each tube. The cells were incubated at room temperature (20 degrees Celsius) using static, shaker (800 rpm), and shaker (1200 rpm). The incubation time was 12 h, 16 h, and 20 h under each condition, respectively. The experimental design matrix is ​​shown in Table 3.

[0047] (6) Place the incubated samples in a centrifuge and centrifuge at 12,000 rpm for 5 minutes. Pipette 0.3 ml of the supernatant from each sample into a 1.5 ml centrifuge tube and store at -20°C until the day of measurement.

[0048] (7) Use the salivary cortisol detection kit and perform the experimental operation according to the instructions. The specific operating steps are the same as those in Example 2.

[0049] Table 3 Experimental design matrix

[0050] Table 4 Cortisol content in pig hair extracted with different rotation speed and incubation time combinations

[0051] Changes in cortisol content in hair extracted under different incubation conditions Figure 2As shown, the horizontal axis represents different combinations of rotation speed and incubation time, expressed as rotation speed (rpm) - incubation time (h). For example, 800-16 represents a treatment combination of a rotation speed of 800 rpm and an incubation time of 16 h, 0-12 represents a treatment combination of a rotation speed of 0 (i.e., static state) and an incubation time of 12 h, and so on. Specifically, the cortisol content of pig hair extracted under different combinations of rotation speed and incubation time is shown in Table 4. Under the incubation conditions of 800 rpm-16 h, the cortisol extraction efficiency reached a peak of 18.722±2.027 pg / mg, which was 48.6% higher than that of the traditional static incubation method (the cortisol extraction efficiency of 0 rpm-16 h was 12.596±1.137 pg / mg) (p<0.01); when the rotation speed was increased to 1200 rpm, the cortisol extraction efficiency of the incubation time of 16 h (1200 rpm-16 h) was 16.413±3.593 pg / mg, which was 8.3% lower than that of 800 rpm-16 h; the extraction efficiency of 800 rpm-16 h was 14.8% higher than that of 800 rpm-12 h, and decreased by 17.9% when extended to 20 h (800 rpm-20 h). The intra-assay coefficient of variation was 6.43% and the inter-assay coefficient of variation was 10.53% after incubation at 800 rpm for 16 h.

[0052] In summary, increasing the grinding time in this application and incubating at 800 rpm for 16 hours at room temperature can effectively improve the extraction efficiency and concentration of cortisol in pig hair, and has higher stability and wide applicability.

[0053] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for extracting hair cortisol hormone, characterized in that: The method comprises the following steps: Step 1: Wash the hair sample with isopropyl alcohol and dry it at 50-70°C for 3-5 hours to obtain washed hair; Step 2: Grinding the washed hair to obtain ground hair, preferably, the grinding time is 10-20 minutes; Step 3: The ground hair is exposed to methanol at a speed of 0-1400 rpm for 8-24 hours and then centrifuged to obtain a supernatant.

2. The extraction method according to claim 1, wherein In step 3, the temperature of the hair sample in contact with methanol is 18°C-25°C, the time is 12h-20h, and the rotation speed is 600-1200rpm; Preferably, the time is 12-16 hours and the rotation speed is 800-1200 rpm; For example, the temperature is 20° C., the time is 16 h, and the rotation speed is 800 rpm; or, the temperature is 20° C., the time is 16-20 h, and the rotation speed is 1200 rpm.

3. The extraction method according to claim 2, characterized in that The centrifugation condition in step 3 is 10000-12000 rpm for 3-6 min.

4. The extraction method according to claim 2, characterized in that The ratio of the ground hair to methanol is 40-60:1 mg / ml, preferably 45-55:1 mg / ml, for example 50:1 mg / ml.

5. The extraction method according to claim 4, characterized in that In the step 1, the drying temperature is 55-65° C., and the drying time is 3-5 hours. For example, the drying temperature is 60° C., and the drying time is 4 hours.

6. The extraction method according to claim 5, characterized in that The isopropanol washing time in step 1 is 1.5-4 minutes.

7. The extraction method according to any one of claims 1 to 6, characterized in that The hair is obtained from a mammal.

8. The extraction method according to claim 6, characterized in that The mammal is a pig.

9. Use of the extraction method according to any one of claims 1 to 6 in the detection of cortisol in animal hair.

10. The use according to claim 9, characterized in that The animal is a pig.

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