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In this study, we explore the potential health benefits of Capsaicin, the active compound responsible for the spiciness in hot peppers. Scientists have been conducting experiments to investigate various methods of applying Capsaicin for preventive purposes against common illnesses, diseases, and cancer. This research delves into the extraction and distillation of Capsaicin, with implications for both oral and topical applications.
Scientists are currently investigating the multifaceted benefits of hot peppers and exploring diverse methods of utilizing them for disease prevention.
Research on chili peppers has already yielded an "optimized and validated method for simultaneous extraction, identification, and quantification of flavonoids and capsaicin..." (Marincas 255). The pungency of hot peppers is primarily attributed to the capsaicinoid Capsaicin, with hotter peppers containing higher levels of this compound. The quest to determine the Scoville unit ceiling has sparked global interest, driven in part by Ed Currie, the creator of the Carolina Reaper, the world's hottest recorded pepper. In the extraction process, freeze-dried peppers were combined with various solvents and subjected to extraction under different incubation times.
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The separation and quantification of capsaicin were achieved using a UHPLC system. Recent research has explored both oral and topical administration on animals and human cancer cells, demonstrating known health benefits. Further experimentation promises to expand our understanding and enhance human well-being.
In the extraction, identification, and quantification of capsaicin, fifty-three hot pepper samples were sourced from five different countries, as reported in the Food Chemistry Journal in 2018.
For thermal drying, the samples were stored in a laboratory dryer at 55°C for 48 hours.
Freeze-drying was conducted at -75°C for 48 hours. Each 0.5g portion of lyophilized pepper sample and thermal dried sample was separately mixed with 4ml of methanol and extracted for 12 hours in an ultrasound bath. Subsequently, the samples were centrifuged, and the supernatant of each sample was collected and injected.
Freeze-dried hot pepper samples (0.5g) were mixed with 4ml of different extraction solvents or a mixture of solvents to assess extraction efficiency. All samples were extracted in duplicate. The homogenized mixtures were agitated on a horizontal shaker for 4 hours at room temperature. Following extraction, the samples underwent centrifugation, and the supernatant was collected for analysis using the UHPLC system.
The extraction time optimization experiment for flavonoids and capsaicin was conducted in two approaches. In the first approach, 0.5g of lyophilized pepper samples were extracted with 4ml of MeOH (purchased from LGC Standards GmbH, Wesel, Germany) for 2, 4, 6, 12, and 24 hours. In the second approach, the same amount of lyophilized pepper samples was extracted with 4ml of MeOH for the same durations, assisted by ultrasound. Both sets of samples were extracted in duplicate. The mixtures from the first approach were agitated on a horizontal shaker at room temperature for the specified incubation times, while the samples from the second approach were placed in an ultrasonic bath at room temperature. After the designated time, the samples were centrifuged at 4000g for 15 minutes, and the supernatant was collected and injected into the UHPLC system. These approaches were evaluated to determine the optimal extraction conditions (Marincas 255-262).
Regarding the sample-to-solvent ratio, in accordance with The International Journal of Pharmaceutics, different proportions were used: 1:4, 1:6, and 1:8. Freeze-dried hot pepper samples (0.5g) were mixed with 2, 3, and 4ml of methanol and extracted for 12 hours in an ultrasound bath. Subsequently, the samples were centrifuged and injected into the UHPLC system.
In testing Capsaicin for oral and topical relief, rats were administered Capsaicin-infused candy to treat oral ulcers.
Male Sprague-Dawley rats (180–220 g) were procured from SPF Co., Ltd. (Beijing, China). All animal handling and surgical procedures strictly adhered to the NIH Guidelines for the Care and Use of Laboratory Animals. The rats were housed under standard laboratory conditions with free access to food pellets and water. Anesthesia was administered via intraperitoneal injection of chloral hydrate.
Candy mold models were designed using Solidworks 2013 (Dassault Systemes, S.A., USA). Each mold consisted of two symmetric or asymmetric parts with plane concaves that could be optimally designed for various shapes. The model files were saved in STL format, which were then processed using ANYCUBIC Photon Slicer software (2017, ANYCUBIC Co., Ltd., China) to create photon format files. The corresponding caps for the concaves were also designed. The programs were executed by connecting a flash disk to the SLA printer (Shenzhen Vertical Dimension Cube Technology Co., Ltd., China). LCD-type light-sensitive resins from GODSAID Science and Technology Co., Ltd., China were used to prepare the candy molds. Any residual resins on the 3D printed molds were removed by washing with ethanol and allowed to dry at room temperature.
Capsaicin, purchased from Wuhan Dongkangyuan Reagent Co., P188, and xylitol from Aladdin, were mixed and melted in a 120°C bath to create a transparent viscous liquid. The composition was 0.05%, 10%, and 89.95% (w/w) respectively. The liquid was poured quickly into the candy molds, filling them up to 4g, and then a corresponding cap with the same shape as the mold concaves was applied to press the filled viscous liquid until the surface was flat. The hot liquid in the mold was allowed to cool for 6 hours at room temperature until it solidified into candy. The two parts of the mold were separated, and the cap candy was unmolded. Additionally, pigments such as edible carmine, indigo, and water-soluble citric yellows were experimented with for candy coloration.
Capsaicin, blank candy powders, and capsaicin candy powders (100-meshed) were dried at 25°C under vacuum for 3 hours. Differential scanning calorimetry (DSC) was conducted using a Q2000 DSC instrument (TA Instruments, Waters, USA). Approximately 5–10mg of dry samples were heated from 30 to 200°C at a heating rate of 10°C/min. Nitrogen served as the purge gas at a flow rate of 50mL/min during the experiment.
X-ray powder diffraction (XRD) measurements were carried out using an X-ray powder diffractometer (D8 Advance, Bruker, Germany). The three types of dry samples mentioned earlier (about 5–10mg) were placed into the diffractometer. The parameters used included Cu as the target, an X-ray wavelength (λ) of 1.5418 Å, an electric voltage of 60kV, an electric intensity of 80mA, scanning angles (2θ) ranging from 3° to 60° with a step size of 0.02° at a rate of 5°/min.
The hardness of capsaicin candies was assessed using a YPD-3000 tablet hardness tester (Shanghai Huanghai Pharmaceutical Inspection Instrument Co., Ltd., China). A candy was horizontally inserted into the tester's cavity. Gradual compression of the barriers caused the candy to rupture, and its hardness was recorded. The experiments were conducted in triplicate for accuracy.
Capsaicin content in the candies was determined using high-performance liquid chromatography (HPLC) on the Agilent 1260 system (USA) (Shi and Zu, 2010). A Diamonsil C18 column (250mm × 4.6mm, 5μm) was maintained at 30°C. The injected volume was 10μL, and the mobile phase consisted of methanol/water (70:30, v/v) at a flow rate of 1mL/min, with UV detection at 280nm. Ruptured capsaicin candies were dissolved in the mobile phase and filtered through 0.45-μm membranes before analysis by HPLC.
A capsaicin candy was immersed in phosphate-buffered solutions (PBS, pH 6.8, 20mL) within a sealed 50-mL centrifuge tube, following a literature reference (Scoutaris et al., 2018). The tube was oscillated in a THZ-D Thermostat Oscillator (Taicang, China) at 37°C and 100rpm. The PBS solution was refreshed every 5 minutes until the candies had completely dissolved. The withdrawn solution was filtered through 0.45-μm membranes, and capsaicin content in the filtrate was determined by HPLC. Cumulative release rates of capsaicin were calculated.
The oral stimulation of capsaicin candies was assessed by two co-authors, H.J. and X.Y. (one female and one male), who were volunteers without oral diseases or smoking habits. An aliquot (0.1g) cut from capsaicin candies, containing 0.025%, 0.05%, or 0.10% capsaicin, was tasted in their mouths. The candies quickly dissolved, and they evaluated the subjective pungent levels induced by capsaicin stimulation until the pungency disappeared.
Rat oral ulcer models were established following the method reported by Hong et al. in 2018. In brief, rats were anesthetized using 10% chloral hydrate aqueous solutions at a dose of 4 mL/kg and immobilized. The rats' mouths were exposed, and their tongues were gently pulled out using tweezers. A melted phenol liquid (3.5 μL, approximately 60°C) was applied to a piece of round filter paper with a diameter of 5 mm. The paper was then placed on the tongue for 50 seconds and subsequently removed. The tongue was washed with saline to eliminate residual phenol. The successful establishment of the oral ulcer model was confirmed after 24 hours, characterized by pseudo-membranes on the tongue.
Twenty-four rats were equally divided into four groups. The control group received no treatment. Candies and Dexamethasone Acetate Adhesive Tablets were ground into 100-meshed powders, mixed with water to form pastes, and applied to the oral ulcers for 3 minutes. The dexamethasone group received a dose of 0.15 mg per rat, the blank candy group received a dose of 0.8 g per rat, and the capsaicin candy group received a dose of 0.4 mg of capsaicin in a paste of 0.8 g per rat. All treatments were administered once daily for 7 days. The ulcer was observed, photographed, and the ulcer area was analyzed using image analysis software (Image Pro Plus 6.0, Media Cybernetics, USA). The rate of ulcer healing was calculated using Eq. (1):
(1)Ulcer Healing Rate (%) = (A1 - An) / A1 × 100%, where An represents the ulcer area on a given day post-ulcer and A1 represents the ulcer area on the first day (Day 1).
On Day 7, the rats were sacrificed, and their tongues were excised for histopathological and immunohistochemical analysis.
The tongues were fixed in 10% formalin solutions and embedded in paraffin. Sections of 5 μm thickness were obtained and stained with H&E and Masson's trichrome, followed by observation under a microscope.
The tongue tissue slides were dewaxed, rehydrated, and washed with PBS. Endogenous peroxidase was inactivated with 3% hydrogen peroxide (H2O2) for 15 minutes at room temperature. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in the tongue tissues were determined using kits from Wuhan Servicebio Technology Co., Ltd., China, following the provided instructions. The slides were observed under a microscope.
All data were expressed as means ± standard deviations (SDs). Student's t-test was performed for all statistical analyses using Excel (Yiguan Jin Vol 568).
In the development of hot peppers like the Carolina Reaper, Ed Currie employs a simple yet effective cross-breeding technique by manually transferring pollen from one desired plant to another using a paintbrush. Holding the Guinness World Record for the hottest pepper, his passion and expertise in elevating capsaicin levels and spiciness in peppers exemplify the central role of capsaicin in the pursuit of extreme heat. While his work garners public interest, it also caters to those with a slightly masochistic
Below are the extracted flavonoids and capsaicinoids, their respective extraction times on a horizontal shaker and with ultrasound, along with their concentration readings:
| Compound | Extraction time on Horizontal Shaker | Concentration (µg/g)* | Extraction time with Ultrasound | Concentration (µg/g)* |
|---|---|---|---|---|
| Vitexin | 2 h | 12.71 ± 1.61 | 2 h | 17.58 ± 2.79 |
| 4 h | 20.93 ± 0.49 | 4 h | 18.19 ± 2.56 | |
| 6 h | 19.71 ± 1.01 | 6 h | 22.13 ± 3.64 | |
| 12 h | 14.63 ± 2.23 | 12 h | 24.38 ± 0.82 | |
| 24 h | 17.55 ± 2.05 | 24 h | 17.98 ± 0.47 | |
| Isoquercetin | 2 h | 5.36 ± 1.14 | 2 h | 5.63 ± 0.21 |
| 4 h | 8.22 ± 0.44 | 4 h | 11.34 ± 0.09 | |
| 6 h | 7.48 ± 4.16 | 6 h | 13.38 ± 1.71 | |
| 12 h | 3.26 ± 0.86 | 12 h | 32.28 ± 3.05 | |
| 24 h | 5.41 ± 0.38 | 24 h | 17.42 ± 0.55 | |
| Kaempferol-3-Glucoside | 2 h | 7.39 ± 3.23 | 2 h | 23.41 ± 4.24 |
| 4 h | 16.19 ± 0.28 | 4 h | 29.08 ± 0.42 | |
| 6 h | 22.52 ± 2.39 | 6 h | 29.25 ± 5.70 | |
| 12 h | 16.10 ± 0.62 | 12 h | 32.55 ± 2.83 | |
| 24 h | 16.94 ± 1.33 | 24 h | 34.79 ± 0.20 | |
| Myricetin | 2 h | 8.41 ± 1.58 | 2 h | 18.27 ± 0.31 |
| 4 h | 12.75 ± 2.36 | 4 h | 20.76 ± 1.18 | |
| 6 h | 18.10 ± 1.60 | 6 h | 22.87 ± 0.45 | |
| 12 h | 16.01 ± 1.96 | 12 h | 25.09 ± 0.76 | |
| 24 h | 16.34 ± 0.72 | 24 h | 23.88 ± 0.60 | |
| Capsaicin | 2 h | 662.87 ± 106 | 2 h | 627.59 ± 28.95 |
| 4 h | 705.50 ± 32 | 4 h | 637.02 ± 21.90 | |
| 6 h | 716.49 ± 16.85 | 6 h | 826.01 ± 23.78 | |
| 12 h | 608.38 ± 13.11 | 12 h | 846.97 ± 19.50 | |
| 24 h | 749.52 ± 16.45 | 24 h | 762.75 ± 103 |
* Concentrations are presented as mean ± standard deviation.
The world's hottest new craze is the growth and consumption of spicy peppers, with a preference for hotter varieties. Peppers have been used by humans for centuries and may have been one of the first spices ever created. Recent studies have revealed numerous health benefits associated with chili peppers. The active component responsible for the spiciness in hot peppers is Capsaicin. Capsaicin, along with flavonoids and other phytochemicals, shows promise in preventing various health conditions, including coronary heart disease, stroke, diabetes, cancer, and many other ailments. While scientists have already begun researching the beneficial attributes of capsaicin, the desire for innovative uses and additional health benefits continues to grow. This has inspired many, including myself, to advance our understanding of how spicy peppers can become and how substantial their health benefits can be.
Hot peppers, with their high capsaicin content, offer a promising avenue for improving health and preventing various diseases. This research has demonstrated efficient methods for extracting capsaicin and flavonoids from hot peppers, providing valuable insights into their concentrations over different extraction times and techniques. The potential applications of capsaicin in oral and topical treatments for conditions like oral ulcers further emphasize its importance in medicine.
Hot Pepper Benefits & Capsaicin Extraction and Distillation. (2024, Jan 17). Retrieved from https://studymoose.com/document/hot-pepper-benefits-capsaicin-extraction-and-distillation
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