Background and overview
An alkaloid with molecular formula C17H19NO3. It exists in the fruits of many pepper plants and is one of its pungent components, but the pungent flavor of pepper mainly comes from its isomer jiaweialkali. In commercial black pepper and white pepper, the content of piperine is 5% ~ 9%, sometimes as high as 11%. Piperine is a colorless monoclinic prismatic crystal; Melting point 130 ~ 133 ℃; Soluble in acetic acid, benzene, ethanol and chloroform, slightly soluble in ether, almost insoluble in water and petroleum ether; The entrance is tasteless at last and spicy after; A salt that can form crystals with strong acids. The injection of piperine has a good anticonvulsant effect on the seizure of glutamate and anisodine in mice. Clinical trial also has curative effect on some types of epilepsy. There is an official prescription for the treatment of epilepsy in China, “white pepper and Radish”, and the structurally simplified piperine analogue 3 – (3,4-methylenedioxyphenyl) acryloylpiperidine (trade name Kangxianling) has been synthesized. Piperine is more toxic to flies than pyrethrum.
Chemical composition analysis [2]
In 1819, the yellow crystalline substance piperine was first extracted from pepper. This alkaloid is the secondary pungent component of pepper. In addition, alkaloids such as piperine B, piperine, piperonate A and piperine were also found in the extract of pepper, which also had pungent and comforting taste. Piperine has the highest content and activity among all alkaloids in pepper. Many researchers have stopped separating and identifying the five analogues of piperine. They are identified as piperetine, piperonine, piperylin a, piperolein B and pipericine. Their chemical structures are shown in the figure below.
In addition, six other alkaloids were identified in 1997. The molecular formula of piperine is c17h19o3n,. The melting point of piperine is 128 ~ 130 ℃, which is very stable. After the hydrolysis of HNO3 solution or alkali solution, volatile piperidine (azacyclohexane) (c5h11n) will occur, and its acid hydrolysis product is piperine acid (c17h19o4). Piperine is very sensitive to light. Isopiperine and isopiperine will occur in the ethanol solution of piperine. Piperine exists in the form of four isomers: piperine, isopiperine, chavicine and isochavicine. However, isopiperine, isocavicine and chavicine all give out very weak pungent and comforting flavor. Grewe et al. Completed the chemical synthesis of these isomers, and their structures are shown in the figure.
Pharmacological effect [2]
Pepper is the most fundamental ingredient in Indian medicine and is used to treat a variety of diseases. Pharmacological studies have proved many of their traditional effects, such as analgesic and antipyretic effects, antioxidant and antibacterial effects. Piperine is an effective component of pepper. The pharmacological action of piperine is relatively common. At present, it has been found to have antioxidant, immune regulation, antitumor and other effects.
1. Antitumor effect
There are many reports on the antitumor effect of piperine. It is found that piperine can inhibit the lung metastasis induced by B16F-10 melanoma, significantly increase the composition of tumor and extend the survival time of tumor bearing mice. This shows that piperine has the effect of anti-tumor metastasis. It was found that oral piperine can significantly reduce the degree of lipid peroxidation, nucleotide content and polyamine decomposition in lung cancer mice induced by benzopyrene. Its inhibitory mechanism is to maintain protein damage and inhibit cell proliferation. In melanoma cell model, piperine could effectively inhibit the gene expression of ATF-2, NF kappaB and inflammatory cytokines.
2. Antioxidant effect
Based on the attack system of DPPH free radical, the ability of piperine to eliminate this kind of free radical in anhydrous ethanol medium was studied. Its ability to eliminate free radical can represent the strength of its antioxidant ability. Studies have proved that piperine has certain antioxidant activity, its antioxidant ability is equivalent to 60.05% of vitamin C, and fluctuates in acidic medium.
3. Effect on central nervous system
At present, it has been proved that piperine has many effects, such as sedation, hypnosis, anticonvulsant, skeletal muscle relaxation and antidepressant. By studying the effect of piperine on plant sleep, it is found that piperine can significantly increase the autonomous activity of mice, extend the sleep time of mice induced by sodium pentobarbital, and significantly add the deep sleep of rabbits. The mechanism can be related to the content of serotonin in the brain.
4. Immunomodulatory effect
The immunotoxicological study of piperine showed that: piperine continued to be administered to mice for five days, there was no obvious toxic effect, and the liver component was added normally. When studying the effect of piperine on thymocytes, it was found that piperine can inhibit the occurrence of reactive oxygen species (ROS), add the content of glutathione (GSH), inhibit cell apoptosis and the phenotypic changes of thymus dependent lymphocytes.
5. Increase the onset of inflammation
Piperine can increase the onset of inflammation and pain. Oral administration of piperine at a dose of 50mg / kg can significantly increase the onset of inflammation. Piperine has anti-inflammatory effect. Piperine and its homologues can be absorbed through the skin, so it can act on subcutaneous tissues, nerves and blood vessels. In addition, pharmacological studies have shown that piperine can prevent atherosclerosis, reduce blood lipid, have anticonvulsant and anti ulcer activities, maintain the liver, and will not damage the function of testis. And whether in vivo or in vitro, piperine can also slow down intestinal peristalsis. The effect of piperine on nervous system and sexual organs shows that it has the characteristics of anticonvulsant and vasodilation.
Extraction separation [3]
1. Supercritical fluid extraction (SFE) SFE is especially suitable for the extraction and separation of
natural substances rich in biological activity. It uses supercritical fluid as the extraction medium, uses the solvation effect of supercritical fluid to dissolve the liquid or solid mixture to be separated, and then reduces its density through decompression or temperature adjustment, so as to reduce its solvent ability and make the extract separate. SFE has been widely used in the fields of food, medicine and chemical industry because of its complex separation process and elementary extraction efficiency. Among them, SFE technology with CO2 as solvent has become the most important research and application technology in SFE technology because of its strong solubility and good selectivity to organic matter, and CO2 is non-toxic, inert and easy to obtain. The supercritical CO2 method was used to extract piperine from pepper. The study showed that the optimum extraction conditions were as follows: the extraction temperature was 40 ℃, the pressure was 200 bar, the addition amount of absolute ethanol was 7.5% (V: V), and the constant solvent flow rate of CO2 was (8 ± 2) g / min. Piperine in pepper was extracted by supercritical CO2. The optimum conditions for extracting piperine from 20g pepper powder with uniform particle size of 0.42mm were as follows: extraction temperature of 60 ℃, pressure of 300bar, extraction time of 45min and CO2 flow rate of 2l / min. Grinevicius et al. [12] extracted antitumor active substances from black pepper by supercritical CO2 method. The study showed that the second important components were piperine and terpenoids. When the extraction temperature was 40 ℃, the pressure was 200bar and the CO2 flow rate was (8 ± 2) g / min, the antitumor activity of the extract was the highest, and the content of piperine in the extract was the highest (6.035 ± 0.014)%.
2. Enzyme assisted supercritical carbon dioxide extraction
Enzyme assisted supercritical carbon dioxide extraction technology is a relatively new experimental technique, which can more effectively improve the extraction yield of piperine. Using this method to extract piperine from black pepper, the enzyme used is α- Amylase (from Bacillus licheniformis) studied and compared the changes in the yield of piperine extract before and after enzyme addition, and stopped the optimization experiment in batch and continuous form. The study shows that the best enzyme ratio is: enzyme amount: Black Pepper = 1:5000, time is 2.25h, and CO2 flow rate is 2L / min. In the continuous form, the specific activity of the enzyme increased by 2.13 times, and the batch form was added by 1.25 times. At the same time, the yield of piperine extract increased significantly.
3. Enzymatic extraction
Enzymatic extraction of active substances from Chinese herbal medicine is a major development of extraction technology. In recent years, there have been many researches on the extraction of plant effective components by enzyme technology, among which cellulase is the most widely used and the effect is more obvious. The secondary components of plant cell wall are cellulose and pectin. The addition of cellulase can effectively destroy the cell wall, which is conducive to the dissolution of active components in cells. Ultrasonic assisted enzymatic method can further strengthen the enzymatic hydrolysis of cellulose and improve the extraction rate of target substances. Using cellulase to extract piperine from white pepper, the conditions were optimized on the basis of single factor experiment. The optimum process for extracting piperine from 3.0g white pepper powder was determined as follows: cellulase was 6mg, enzymatic hydrolysis temperature was 50 ℃, pH value of enzymatic hydrolysis solution was 7 and time was 6h. Under this condition, the content of piperine in the extract could reach 5.29%.
4. Organic solvent method
Piperine is almost insoluble in water, slightly soluble in acetic acid, soluble in benzene or acetic acid, and easily soluble in chloroform and ethanol, so it can be extracted by organic solvent method. The piperine in pepper was extracted with 85% ethanol. The optimum process conditions were as follows: reflux extraction for 2 times, ethanol (volume): Pepper (mass) 10:1, filtrate merging, dilution, static cooling crystallization, anhydrous ethanol repeated crystallization, and the purity of piperine extracted was more than 98%. Piperine in pepper was extracted by Soxhlet extraction technology with methanol as extraction solvent. The content of crude piperine was 3.80%.
5. Ultrasonic assisted inorganic solvent extraction
Ultrasonic assisted inorganic solvent extraction is an ancient separation method. Ultrasonic cavitation will produce great pressure, improve the permeability of cell wall and contribute to the rapid release of effective components in cells. At the same time, the vibration effect and thermal effect of ultrasound can add mass transfer coefficient, increase the contact area of solute, and then add the solubility of solute. This method has the advantages of short time, high efficiency and can be stopped at room temperature. It is widely used in the extraction and separation of natural materials. Piperine was extracted from pepper by ultrasonic assisted organic solvent method. Ethanol was used as the extraction solvent. The extraction time was 18min, the solid-liquid ratio was 1:10, the ultrasonic power was 125w, the ultrasonic frequency was 25kHz and the temperature was 50 ℃. The research shows that the extraction efficiency of piperine is higher than that of traditional organic solvent extraction. Piperine was extracted from pepper by ultrasonic assisted inorganic solvent extraction. The optimum technological conditions were as follows: the volume fraction of ethanol was 95%, the extraction time was 45 min, the extraction temperature was 70 ℃, the material liquid ratio was 1:30 (G: ML). Under this condition, the content of piperine in the extract can reach 6.12%. 1.6 pressurized liquid extraction (PLE) pressurized liquid extraction (PLE) stops extraction by continuous solvent flowing through a fixed extraction column containing solid particles. It is characterized by less solvent demand, high extraction efficiency and increased extraction time. Using ethanol as ple solvent, 20g ground black pepper powder was put into the extraction container, the pressure was set to 100bar, the temperature was 40 ℃, the flow rate of B was maintained at 3ml / min, and the extraction time was 60min.
6. Ultrasonic assisted extraction of ionic liquids (iluae)
Piperine was extracted from white pepper by ultrasonic assisted extraction (iluae) based on a series of 1-Alkyl-3-methylimidazole ionic liquids. The effects of alkyl chain length and anion varieties on the extraction efficiency of piperine were evaluated. The results showed that they had an obvious effect on the extraction efficiency of piperine.
7. Microwave assisted organic solvent extraction
The principle of microwave assisted organic solvent extraction method is based on the fact that the ions and molecules in the solution bear the energy of microwave radiation and heat up, so as to improve the ability of solute entering the solvent. This method has the advantages of low energy consumption, short time, high efficiency and saving the use of extractant. Piperine in pepper was extracted by microwave assisted organic solvent method. The optimum extraction process was determined as follows: ethanol concentration was 80%, microwave power was 500W, extraction time was 60min, extraction temperature was 55 ℃, solid-liquid ratio was 80:50. Under these conditions, the content of piperine in the extract could reach 4.12%.
8. Ultrasonic microwave synergistic extraction
The ultrasonic microwave synergistic extractor is equipped with condensation installation, which can greatly improve the yield of pepper oleoresin in piperine and increase the loss of its volatile components. The effects of hot reflux extraction, Soxhlet extraction and ultrasonic microwave assisted extraction on the yield of pepper oleoresin were studied, and the content of piperine was analyzed and determined by HPLC. The results showed that there were significant differences in the contents of pepper oleoresin and piperine obtained by different extraction methods, and the yield of pepper oleoresin lost by ultrasonic microwave synergistic extraction was the highest.
9. Acid hydrolysis
Piperine is not soluble in water at all. Acid can be added in water to convert piperine into corresponding piperine salt, which is dissolved in water and then extracted.] Piperine was extracted from black pepper with acetic acid as extraction solution. 15g black pepper was extracted with 500g solvent. The content of piperine in the extract could reach 3.0%.
Determination method
A 2000 Pharmacopoeia rule allows the determination of piperine by high performance liquid chromatography (HPLC). At present, many literatures have reported the determination of piperine in drugs by high performance liquid chromatography (HPLC). The method is simple, accurate and reproducible. The content of piperine in pepper was determined by HPLC with acetonitrile ammonia acetate buffer as the active phase. Using C18 column and methanol water as the active phase, the content of piperine in Xiaoer Zhixie plaster was determined by HPLC at the detection wavelength of 343 nm. The quantitative analysis and determination methods of piperine in Pepper include thin-layer UV scanning method, colorimetry and UV spectrophotometry.
The thin-layer ultraviolet scanning method is limited by the instrument conditions, and it is difficult to popularize in some grass-roots units; The operation of colorimetry is complicated and cumbersome; UV spectrophotometry is complex, fast and accurate. Piperine can be determined by UV spectrophotometer at the maximum absorption wavelength of 342 ~ 345nm.
