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Nobiletin antigen as well as preparation method and application thereof

A technology of nobiletin and antigen, which is applied in the field of nobiletin antigen and its preparation, which can solve the problems of high detection cost, inapplicability of rapid detection, and long time consumption, and achieve low synthesis cost, simple and clear synthesis steps, and good effect Effect

Active Publication Date: 2018-09-04
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nobiletin content analysis methods include high-performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), RP-HPLC method, HPLC-ESI-MS / MS method, HPLC-DAD, which have high sensitivity and good accuracy , but these detection methods require the use of expensive instruments and equipment, which are expensive and time-consuming, and are not suitable for on-site rapid detection

Method used

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  • Nobiletin antigen as well as preparation method and application thereof
  • Nobiletin antigen as well as preparation method and application thereof
  • Nobiletin antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the preparation of nobiletin-ovalbumin (nobiletin-OVA) antigen

[0045] Synthetic roadmap as figure 1 shown.

[0046] 1) Synthesis of compound shown in formula I

[0047] In a 50mL three-necked flask, add 0.3g nobiletin and 0.2g carboxymethoxylamine hemihydrochloride, then add 15mL pyridine, heat to 100°C, react for 24h and cool to room temperature, pour the reaction solution into 100mL water, Adjust the pH value to 3 with concentrated hydrochloric acid, extract with ethyl acetate (3×50 mL), dry over anhydrous sodium sulfate, spin precipitation, and column chromatography to obtain 0.2 g of the product with a yield of 56%.

[0048] 1 H NMR(600MHz,dmso)δ12.70(s,1H),7.48(d,J=8.3Hz,1H),7.36(s,1H),7.09(d,J=8.5Hz,1H),7.05(s ,1H),4.63(s,2H),3.93(s,3H),3.89(s,3H),3.84(s,3H),3.81(s,3H),3.79(s,3H),3.63(s, 3H).

[0049] 13 C NMR(151MHz,dmso)δ171.97,153.34,151.40,149.28,148.67,146.58,144.52,144.11,142.98,138.31,124.42,119.01,112.23,108.82,108.70,92.01,70.86,62....

Embodiment 2

[0061] Embodiment 2, the preparation of nobiletin-bovine serum albumin (nobiletin-BSA) antigen 1) the synthesis of nobiletin hapten shown in formula I and its activation are no different from those in Example 1, no longer repeat.

[0062] 2) Slowly drop the compound shown in the formula II obtained in step 2) in Example 1 into the carrier protein solution

[0063] (The carrier protein solution is 157.5mg BSA dissolved in 10mL phosphate buffered saline (PBS) with a pH value of 7.4), the molar ratio of the compound of formula II to the carrier protein is 15:1, and stirred overnight at 4°C.

[0064] 3) Dialysis: The reaction solution obtained in step 2) was dialyzed for three days with a PBS solution with a pH value of 7.4 and a concentration of 0.01mol / L, and the completely dialyzed reaction product solution (Nobiletin-BSA) was diluted to 1 mg / mL The solution was frozen at -40°C until use. The function of dialysis is to remove unreacted nobiletin haptens and other small molecu...

Embodiment 3

[0067] Embodiment 3, application of nobiletin-bovine serum albumin (nobiletin-BSA) antigen

[0068] 1. Preparation of Antibody Using Nobiletin-Bovine Serum Albumin (Nobiletin-BSA) Antigen

[0069] (1) Take 8-10 week old Bal b / c mice as experimental animals.

[0070] (2) Basic immunization: obtain the diluted nobiletin-BSA antigen solution (concentration is 1mg / mL) from the embodiment 2, add an equal volume of Freund's complete adjuvant after filtering through a sterile filter, and use a magnetic stirrer to Stir well to emulsify until dripping into water does not spread. Bal b / c mice were injected subcutaneously with emulsified complete antigen at multiple points in the abdominal cavity and back, and the injection dose was 0.1 mg emulsified antigen / mouse.

[0071] (3) Booster immunization: 2 weeks after the basic immunization, take 1 mL of the above-mentioned diluted nobiletin-BSA antigen solution, then add 1 mL of Freund's incomplete adjuvant, fully stir and emulsify with a ...

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Abstract

The invention discloses a nobiletin antigen as well as a preparation method and application thereof. The preparation method comprises the steps of reacting nobiletin and a compound B containing amidogen and carboxyl at the same time, so as to obtain a compound C; carrying out coupled reaction on the compound C and N-hydroxy succinimide under the existence of dicyclohexyl carbodiimide or 1-ethyl-(3-dimethylamino propyl)carbonyl diimine hydrochloride to obtain a compound D; carrying out coupled reaction on the compound D and carrier protein to obtain the nobiletin antigen. The nobiletin antigenis applied to preparing an enzyme linked immuno sorbent assay kit for detecting nobiletin in a sample, a luminescence immune kit of nobiletin, or an immunoaffinity chromatographic column. The preparation method provided by the invention has the advantages of convenience and quickness in obtaining the nobiletin antigen, concise synthesis steps, low synthesis cost and good effect. An antibody obtained through the immunization of the nobiletin antigen is good in specificity and low in minimum detection limit value.

Description

technical field [0001] The invention relates to the technical field of compound preparation and application, in particular to a nobiletin antigen and its preparation method and application. Background technique [0002] The common name of nobiletin is Nobiletin; the chemical name is: naringenin 7-rhamnoside; the trade name is: nobiletin, nobiletin, tangeretin; CAS registration number: 478-01-3; the molecular formula is: C21H22O8; relative molecular weight: 402.13. Its chemical structural formula is as follows: [0003] [0004] Nobiletin (Nobiletin) has antioxidant activity and can inhibit the growth of tumor cells, wherein Nobiletin has a good effect on Hela cells, THP-1 cells, Colon26 cells, S180 cells, human breast cancer cells MDA-MB-231 inhibition. The research on the mechanism of nobiletin's anti-cancer effect is gradually in-depth. It is found that nobiletin directly inhibits the activity of MEK, thereby inhibiting the phosphorylation of ERK and the expression o...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K1/107G01N33/531G01N33/53
CPCC07K1/1077C07K14/765C07K14/77C07K14/795C07K19/00G01N33/53G01N33/531
Inventor 崔永亮焦必宁赵静赵其阳张耀海陈爱华王成秋何悦
Owner SOUTHWEST UNIVERSITY
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