Piperine was used in this study in its raw form, and different steps, such as amide hydrolysis and amidation, were used to synthesize piperine derivatives containing a phenolic hydroxyl group. DPPH and ABTS free radical scavenging assays were used to assess piperine derivative antioxidant activities. We constructed an AAPH oxidative stress erythrocyte model to study the effect of piperine derivatives on the hemolysis rate of oxidatively damaged erythrocytes as well as the hemoglobin oxidation rate. This AAPH model was also used to determine piperine derivative effects on antioxidant enzyme activity and malondialdehyde (MDA) content. Results showed that spectroscopic methods could synthesize and identify piperine derivatives containing phenolic hydroxyl groups (H-1∼H-3). Moreover, DPPH and ABTS assay results showed that piperine derivative free radical clearance rates were higher compared with the parent compound. Additionally, piperine derivatives (H-1∼H-3) were found to provide protection to AAPH oxidatively damaged erythrocytes in their ability to inhibit AAPH-induced erythrocyte lysis, while hemoglobin oxidation was higher compared with the parent compound. Piperine derivatives may protect intracellular glutathione peroxidase (GSH-Px) antioxidant enzyme system activities, safeguarding against oxidative damage. This study synthesized novel piperine derivatives for use as potential antioxidant agent candidates.
Free radicals refer to any molecule with one or more unpaired electrons that is a product of normal body metabolism [
Piperine is an active alkaloid that is extracted from peppers, long peppers, and other plants. It produces a variety of physiological and pharmacological effects to occur, such as antioxidative [
Oxalyl chloride (98%) was purchased from Saan Chemical Technology Co., Ltd (Shanghai, China). TCI Development Co., Ltd (Shanghai, China), supplied 3-aminophenol (98.5%). J&K Scientific Co., Ltd, supplied 4-aminophenol (97%) and 2-aminophenol (99%). Piperine was purchased from Shaanxi Ciyuan Biotechnology Co., Ltd. Dichloromethane (DCM), petroleum ether (PE), methanol, and ethyl acetate (EA; analytical grade) were all purchased from Tianjin Fuyu Fine Chemical Co., Ltd. The methemoglobin (Hb) reagent kit, catalase (CAT) visible light reagent kit, total protein quantitation (BCA assay) reagent kit, malondialdehyde (MDA) reagent kit, glutathione peroxidase (GSH-Px) reagent kit, and total superoxide dismutase (T-SOD) reagent kit were all purchased from the Nanjing Jiancheng Bioengineering Institute. AAPH (2, 2′-azobis (2-amidinopropane) dihydrochloride) was purchased from Saan Chemical Technology Co., Ltd.
Male Sprague Dawley laboratory rats (280 ± 10 g) were obtained from Chengdu Dossy Experimental Animals Co., Ltd. All experimental animals underwent routine feeding and housing as well as
Piperine was used as a raw material to synthesize three structurally modified piperine derivatives based on the pathways shown in Figure
Schematic used in the preparation of piperine derivatives.
Piperine (10.69 g, 37.51 mmol) was dispersed in a potassium hydroxide (KOH) (237 g, 4.22 mol)/methanol solution (300 mL) and refluxed at 75°C. After 24 h, the solution was cooled and subjected to suction filtration to obtain a white solid. The solid was dispersed in small amounts of methanol, and 6 mol/L hydrochloric acid was used to adjust the pH level to one. Following this, suction filtration was conducted, after which the substance was dried to obtain compound 2. The product was a yellow powder with a mass of 7.61 g and a yield of 93.1%. 1H NMR (400 MHz, DMSO-d6):
Compound 2 (6.91 g, 31.7 mmol) was dispersed in 15 mL of anhydrous DCM. An oxalyl chloride-DCM solution (12 mol/L, 2.70 mL) was added and stirred for 2 h at room temperature to obtain an orange liquid. Vacuum distillation was used to remove oxalyl chloride and DCM to obtain an orange-red amide. It is important to note that this amide must be prepared fresh before use.
As shown in Figure
The H-1 crude product was then purified using column chromatography on a silica gel (mixture eluent: EA : PE = 2 : 1) to obtain an 84 mg product (yield: 58%) as a pale-yellow solid. 1H NMR (400 MHz, DMSO-d6):
The crude H-2 product was then purified using column chromatography on a silica gel (mixture eluent: EA : PE = 2 : 1) to obtain a 102 mg product (4a) (yield: 71%) as a pale-yellow solid. 1H NMR (400 MHz, DMSO-d6):
Finally, the H-3 crude product was purified using column chromatography on a silica gel (mixture eluent: PE : AE : HAc = 2 : 1 : 0.05) to obtain a 95 mg product (4a) (yield: 66%) as a pale-yellow solid. 1H NMR (400 MHz, DMSO-d6):
DPPH (2, 2-diphenyl-1-picrylhydrazyl) is regarded as a generator of free radicals and is widely used to quantify the antioxidative capacity of biological samples and foodstuff. In this study, a microplate reader was used to measure the
The ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay was conducted according to a modified method by Re et al. [
Centrifugation (5 min at 3500 rpm/min) was used to separate erythrocytes and plasma with the addition of an anticoagulant at 4°C. The erythrocyte pellet was collected and washed four times with phosphate-buffered saline (PBS) (pH 7.4) at 4°C, followed by centrifugation at 3500 rpm/min for 5 min. A suitable amount of PBS was then added to evenly mix and dilute the pellet to obtain a 10% erythrocyte suspension.
The erythrocyte suspension (100
The erythrocyte suspension (100
The erythrocyte suspension (100
The experiments were carried out in parallel triplicate. All data was expressed as the mean ± standard deviation (
DPPH and ABTS are regarded as free radical generators and widely used to quantify antioxidative capacities of biological samples and foodstuff. In this study, a microplate reader was used to measure the
As shown in Figure
DPPH and ABTS free radical clearance curves of piperine and its derivatives. (a) The DPPH free radical clearance rate (IP) of piperine and its derivatives at different concentrations. (b) The ABTS free radical clearance rate (IP) of piperine and its derivatives at different concentrations.
IC50 of the DPPH and ABTS free radical inhibition rates of the different piperine derivatives.
Compound | IC50 of DPPH radical oxidation inhibition rate (mM) | IC50 of ABTS radical oxidation inhibition rate ( |
---|---|---|
Piperine | — | — |
H-1 | 0.21 ± 0.43 | 8.70 ± 0.092 |
H-2 | 1.25 ± 0.14 | 6.15 ± 0.066 |
H-3 | 7.82 ± 2.52 | 6.04 ± 0.34 |
0.00097 ± 0.00078 | 9.80 ± 1.46 |
Figure
AAPH is the most common free radical inducer used in the construction of erythrocyte oxidative damage models. Studies have shown that AAPH has a strong ability to induce oxidative damage in erythrocytes, resulting in hemolysis. For this study, we constructed an erythrocyte oxidative damage model. Firstly, we assessed the protective abilities of piperine and its derivatives on AAPH-induced erythrocyte oxidative hemolysis, while further examining the effects of piperine derivatives on hemoglobin oxidation rates in erythrocytes. Erythrocytes are the best cellular model to study oxidative damage [
As shown in Figure
Protective effect duration of piperine derivatives on AAPH-induced erythrocyte hemolysis and time curves. (a) Time curves of the protective effects of piperine derivatives on AAPH-induced erythrocyte hemolysis at a concentration of 50
Erythrocyte oxidative damage causes hemoglobin oxidation and formation of methemoglobin. Therefore, quantifying methemoglobin levels in erythrocytes can reflect the level of erythrocyte oxidative damage. As shown in Figure
Duration of the protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation in erythrocytes and time curves. (a) Time curves of the protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation at a concentration of 50
Free radicals (i.e., ROS and reactive nitrogen species (RNS)) possess strong chemical reactivity and have the potential to cause oxidative damage to biological macromolecules. Under normal circumstances, oxidative and antioxidative systems in the body maintain a certain balance to protect tissues and organs from being attacked by oxidizing agents. Among the body’s enzymatic and nonenzymatic antioxidative systems, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) are major antioxidant enzyme systems [
Figure
Effects of piperine derivatives on AAPH-induced GSH-Px levels, SOD activity, and CAT activity in rat erythrocytes.
When 50
Figure
Figure
A free radical imbalance in the body causes unsaturated fatty acid oxidation to occur, subsequently resulting in the formation of lipid peroxides, of which malondialdehyde (MDA) is the most classic reaction end-product. MDA causes cytotoxicity as it can disrupt the integrity of the phospholipid membrane and induce cell death [
Effects of piperine derivatives on AAPH-induced MDA levels in rat erythrocytes.
In this study, naturally extracted piperine was used as a raw material to synthesize piperine derivatives containing a phenolic hydroxyl group. We demonstrated that the free radical clearance rates of piperine derivatives were higher compared with the parent compound. Piperine derivatives exhibited a potent antioxidant ability
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflicts of interest regarding the writing and publication of this study.
This study was financially supported by scientific research funds provided by Xi’an Medical University (2016YXXK09), Xi’an and Weiyang District Science and Technology Fund (201930), and Xi’an Science and Technology Bureau Project (2020KJRC0135).