The Study on Extraction Process and Analysis of Components in Essential Oils of Black Pepper (Piper nigrum L.) Seeds Harvested in Gia Lai Province, Vietnam

Black pepper (Piper nigrum L.) is a tropical crop with extensive medicinal potential in ethnomedicine and nutraceutical applications. The essential oil of black pepper finds wide applications in inhabitation of respiratory infections and soothing of muscular pains due to its warming and energizing property. The pungent bioactive piperine is responsible for this function, and therefore, efficient technology is required for an optimal extraction process of this compound. In the present article, we have developed a procedure for extracting black pepper essential oil from Vietnam, optimizing conditions that affect the extraction process. The effect of process parameters, namely material size, preservation method, the concentration of sodium chloride, the concentration of soak time, the ratio of material to water, temperature extraction, time extraction on the extraction yield, and relative efficiency were investigated. Results demonstrated that 20 g of black pepper milled with a mesh size of 160 obtained 0.48 g of essential oil (2.4%) at a raw material to water ratio of 1/21 (g/mL) at 150 °C in a time of 5.2 h. GC-MS (Gas chromatography–mass spectrometry) spectra showed that 3-carene (29.21%), D-limonene (20.94%), caryophyllene (15.05%), and β-pinene (9.77%) were present as major components. These results suggested that the essential oil extracted from Vietnamese black pepper is applicable in the manufacturing processes of insecticides and air deodorizers.

again. The purpose of pre-cooling is to reduce the undesirable odor and volatility of the materials after milling, which is caused at elevated temperatures. Finally, sodium chloride (NaCl, Bach Khoa Ltd., Ho Chi Minh City, Vietnam) was dissolved in water and added to the flask containing the material, followed by gentle shaking of the brine/material suspension.

Extraction of Essential Oil
After being soaked with NaCl solution for the required time period, 20 g of black pepper was placed into a 1000 mL volume flask connected directly to the Clevenger apparatus (Bach Khoa Ltd., Ho Chi Minh City, Vietnam) and heated with the Heating Mantle User Manual heater (1000.EU.05, 300 W, Glassco Laboratory Equipment Pvt. Ltd., Ambala Cantt, India). The experimental set up for the extraction stage is illustrated in Figure 1. The extraction time is started when the first drop of condensed essential oil is condensed and dropped into the oil extraction system. After the extraction process is completed, the essential oil is recovered and dehydrated with sodium sulfate (Na2SO4, Sigma-Aldrich, St. Louis, MO, USA) and stored in a storage tank at 10 °C.
The yield of black pepper oil obtained (%) is calculated by the following formula:

Optimizing the Extraction of Essential Oils by Hydrodistillation
Factors affecting the extraction of black pepper essential oil, including material size, preservation conditions, concentration and soak time in the NaCl solution, and distillation conditions, were examined in this study. First of all, considered sizes of the material included whole seeds, mesh 40, and mesh 160, where the latter two correspond to 40 and 160 mesh size filtration after grinding. Following that, the influence of raw material preservation was also investigated by taking into account different conditions including the temperature (room temperature and cold storage of 10 °C), state of the lid (closed lid and open lid- Figure 2), and time of preservation (24-72 h). In addition, the concentration and soak time of the NaCl solution were investigated, ranging from 1-5% and from 1-5 h, respectively. Finally, the main conditions affecting the extraction process including the ratio of raw material to solvent (1/5-1/35 g/mL), extraction time (1-6 h), and extraction temperature (130-200 °C) were also surveyed. These experimental conditions were optimized through single-factor and the response surface methodology (RSM).

Determination of Constituents of the Essential Oil by GC-MS
Gas Chromatography-Mass Spectrometry (GC-MS, GC Agilent 6890 N, MS 5973 inert, Agilent Technologies, Santa Clara, CA, USA) was used to analyze the components contained in the essential oils of black pepper. Firstly, a 25 μL sample of essential oil was added in 1.0 mL n-hexane (Sigma -Aldrich, St. Louis, MO, USA) and then dehydrated with Na2SO4. For the HP5-MS column, head column pressure was 9.3 psi. GC-MS were obtained under the following conditions: Carrier gas He; flow rate 1.0 mL/min; split 1:100; injection volume 1.0 μL; injection temperature 250 °C; oven temperature progress included an initial hold at 50 °C for 2 min, a rise to 80 °C at 2 °C/min, a rise to 150 °C at 5 °C/min, a rise to 200 °C at 10 °C/min and a rise to 300 °C at 20 °C/min for 5 min.

The Influence of Material Size on the Yield Essential Oil
As the outer peels of the black pepper were thick and hard, the efficiency of the extraction process could be hindered without the peeling process. Therefore, it is necessary to perform treatment accordingly to the different sizes. Based on Figure 3, the amount of attained oil increased as the size of the black pepper decreased. The lowest yield at 0.25% is achieved without peeling, and significantly higher yields, at 1.6% and 1.8%, were obtained at mesh 40 and mesh 160, respectively. This is explained by the fact that when the black pepper is crushed, the cells containing oil were broken, facilitating the water penetration into the oil-containing bags. This, in turn, induces steam to transport the oil to the Clevenger apparatus. In addition, the powdered material for the attained oil has a natural odor and is light green compared to the material in seed size that has a light yellow color due to prolonged exposure to heat.  Figure 4 displayed the effect of the preservation method on the yield of black pepper extraction. Overall, lidded preservation gave higher oil content than the method of open storage. Cool preservation was also showed to be more efficient than storage at ambient temperature for maintaining the oil quantity. In terms of preservation period, longer storage time is also associated with lower oil content. Since essential oil is a mixture of numerous volatile compounds, the closed preservation and low temperature helped reduce the diffusion of essential oil into the air. Therefore, the best preservation conditions are selected at 10 °C with lidded preservation.

The Influence of the Concentration of Sodium Chloride Solution on the Yield of Essential Oil
The results from the investigation on the concentration of NaCl solution, used to immerse the raw material, were analyzed in Figure 5. In the extraction process, the mixture of essential oils, and water formed the emulsion. When adding salt to the extraction mixture, not only could the loss of essential oil in the form of emulsion be avoided, but the solubility of some non-polar components of essential oil into the water medium could also be reduced. In addition, the NaCl salt also plays an important role as an electrolyte that increases the density and polarization of water, which makes the separation of essential oil from water easier. As NaCl concentration reaches 2%, oil yield was found to be declining. This could be explained by the osmotic pressure difference exerted by the higher solute concentration in the external environment in comparison with that inside the oil-containing cells. This effectively causes water inside the cell to osmose out, hindering the separation of essential oils from the material. Figure 5 showed that in the same extraction conditions, the sample with a NaCl concentration of 2% gave the best results with the yield of 2%, and at a concentration of 4-5% the lowest results were observed at approximately 1.85%. Therefore, the optimal concentration of NaCl is selected as 2% for the best yield of the essential oil.

NaCl concentration (%)
From Figure 6, the soak time of the NaCl 2% solution prior to extraction evidently enhances the extract efficiency due to the time needed to induce the penetration of essential oil into the environment. On the other hands, prolonged exposure to salt also assists in breaking down oil bags, and high water emulsion could facilitate the transport of oil by steam. The results showed that oil yield was increasing with the soak period, reaching the maximum yield at 3 h. Afterward, increasing soak time reduces extraction efficiency. Here, a 3-h soak time was selected for the highest efficiency of 2.2%.

The Effect of the Material-to-Water Ratio on the Essential Oil Yield
In the steam extraction process, when heating the mixture of water and material, the water vapor permeates the epidermis, which contains essential oils, breaks down the essential oils, and attracts the oil by steam. If the amount of water is insufficient to dissolve the colloids and salt wrapping the pouch of the essential oil, the oil is unable to escape. Using more water for extraction will cause greater diffusion of oil into the water, leading to enhanced solubility and increased the yield of soluble components, as shown in Figure 7. On the contrary, excess water could dissolve or emulsify the oil, reducing the amount of oil yield and the economic efficiency of the distillation due to increased energy consumption and extraction duration. Based on Figure 7, although both ratios of 1/20 (g/mL) and 1/25 (g/mL) gave the highest yield oil (2.2%), the 1/20 (g/mL) ratio could save a significant water use and in turn brings high economic value with only a marginal tradeoff in yield. Therefore, the ratio of 1/20 (g/mL) was chosen for the next survey.  Material to water (g/mL)

The Influence of Temperature and Time Extraction on the Yield of Essential Oil
In Figures 8 and 9, it is shown that as the temperature or time of the extraction increases, the yield of black pepper essential oil also increased. However, to an optimum threshold, the yield of essential oil was no longer increased, as shown in Figure 8. Based on Figure 8, the highest yield of oils was obtained at 180 °C at 2.25% and decreased to 1.95% at 200 °C due to the decomposition of some components at high temperature. At both temperatures, the essential oil is light yellow due to a small amount of material being splashed into the flask and burned. The temperature of 150 °C is preferred to 180 °C due to the energy efficiency and the green color of the oil, indicating the absence of denaturation of some substances in the product. On the other hand, Figure 9 showed that the extraction yield was found to cease rising when prolonging time extraction past 5 h. Therefore, 5 h was chosen as the extraction time for subsequent surveys.

Optimization of the Experimental Conditions Using Response Surface Methodology (RSM)
After having obtained the conditions for extraction of the oil, three factors, including a ratio of water-to-material of 20:1, extraction time of 5 h, and an extraction temperature of 150 °C were selected for further optimization using RSM. The optimum conditions of the three variables, determined by aforementioned single factor investigations, were used to construct the experimental design for RSM. Accordingly, twenty experimental attempts were specified and performed. The Tables 1 and 2  Overall, the model suitability was determined through several indicators, including the model F-value, p-values of variables, F-value of lack of fit, and R 2 . To be specific, the model is less likely to feature noise due to the significant model F-value of 47.72. Regarding variable p-values, model results showed that most variables, except for AC and AB, are statistically significant. This suggests that model reduction is unnecessary and that the resulting model is adequate to explain the data. In addition, the high R 2 and the closeness of data points corresponding to actual and predicted data to the 45-degree line (Figure 10) suggested the relative accuracy of the quadratic model.  Figure 10. Plots of (a) actual data versus predicted data and (b) residuals calculated from the model.
Plotting the response variable (oil yield) with respect to two variables yielded three surfaces as follows.
Closer examination of the surface plots in Figure 11 revealed the hill-shaped relationship between the experimental factors and the response. Evidently, the plot indicated the presence of a maximum oil yield where the oil yield declines thereafter with pairwise increases of two conditions. Attempting to determine the maxima of the model yielded optimum conditions of A = 1/21, B= 5.2, and C = 151, corresponding to the predicted yield of 2.42%. To validate these parameters, we conducted three real experiments with similar parameters. The results were shown in Table 3. Reportedly, the actual yields in experiments conducted with optimal conditions were found to be approximate to the predicted yields. This suggests the suitability of the quadratic model for predicting the yield.  In comparison with previous studies, some main points regarding the conditions of extraction of the oil are summarized as follows (Table 4).

The Result of GC-MS
The results of the GC-MS analysis of the black pepper essential oil extracted at the optimized conditions were presented in Table 5. We have identified 26 compounds representing 99.86% of the total compounds in the black pepper essential oils. Amounts of other non-identified compounds present in the oils were negligible. Compounds were considered as traces when their amounts were less than 0.05%. In our study, we found that 3-carene (29.21%), D-limonene (20.94%), β-caryophyllene (15.05%), α-pinene (4.69%), and β-pinene (9.77%) are major constituents of black pepper essential oil ( Figure 12). In comparison with the results of previous studies, the contents of 3-carene and Dlimonene were significantly higher than most results of other black pepper cultivars. For βcaryophyllene, the compound was found in lower quantity in comparison with black pepper from India when extracted with the same conditions. It is also worth noting that unlike other black pepper cultivars, Vietnamese black pepper oil lacks a sabinene component.  The sheer abundance of 3-carene and D-limonene suggested that Vietnamese black pepper is a promising ingredient for the production of insect repellent [37] and air deodorizer. To elaborate, when making a comparison of the compositions of different essential oils, it is revealed that Dlimonene and camphor exhibit mosquito repellency [38]. Also, in another study, the combination of 3-carene, D-limonene, and α-pinene was shown to be prevalent in air fresheners and air deodorizers [40]. Other possible uses of the essential oil of the Vietnamese black pepper could include antioxidant, antimicrobial, or insecticidal agent applications [40][41][42].

Conclusions
In this study, the conditions for optimal hydrodistillation extraction of essential oil from Vietnamese black pepper (Piper nigrum L.) seeds were investigated. In addition, GC-MS also was used to reveal the chemical composition of the produced oil. Optimal conditions included a water-tomaterial ratio of 1:21 g/mL, the extraction temperature of 150 °C, and the extraction time of 5.2 h. In addition, the materials should be preserved at a temperature of 10 °C in lidded storage. These conditions corresponded to the optimum efficiency of 2.45%. Furthermore, twenty-six compounds of essential oil in black pepper have been identified using gas chromatography-mass spectrometry (GC-MS). The results showed that 3-carene (29.21%), D-limonene (20.94%), β-caryophyllene (15.05%), βpinene (9.77%), and α-pinene (4.69%) are the major constituents of the essential oil. From the analysis of the results obtained, it is possible to conclude that the essential oil extracted from Vietnamese black pepper is suitable for the production of insect repellant and air freshener.