Effect of Air-Drying Temperature on Phenol Compound and Essential Oils Content in Lemon Leaves

Biological Material

The leaves of Citrus Limon were collected in March 2017 from the governorate of Gabes (Tunisia), located in the arid bioclimatic stage characterized by annual precipitation between 88 and 230 mm/year and an average temperature of 18.5°C.

• Essential oil Extraction: Lemon leaves used to extract essential oil were cut into small pieces and then dried at different drying temperatures.

Drying Process

Fresh lemon leaves were cut manually with stainless steel knife into rectangular samples.

Drying experiments were performed in a laboratory scale, convective and horizontal (designed and constructed in the Applied Thermodynamics laboratory, National Engineering School of Gabes). This dryer is worked in closed loop and is equipped with a programmable controlling system for drying air parameters.

The convective drying kinetics was measured at four temperature levels (30, 40, 50 and 60°C). The choice of these drying temperatures is justified by the fact that it is proven in the literature that plant products cannot withstand sufficiently high drying temperatures. Indeed, a temperature above 60°C can lead to the loss of the quality of our product such as the loss of volatile compounds (essential oils and aromas) [15].

The air velocity was set at 1m/s as measured by digital anemometer. The loss of weight of the sample was recorded automatically every 2 or 5 minutes. Finally, the dry solid was placed in an oven at 105°C for 24 hours to obtain its dry mass.

Drying Kinetics Expressed in Terms of Empirical Models

Five mathematical models (Table 1) were applied to establish a drying law characteristic of the treated product. These models provide the expression of the dimensionless variable moisture ratio, Xr, as a function of the drying time.

The parameters were identified directly on the raw data using a nonlinear regression program (“lsqcurvefit”) with MATLAB software.

The plotting of various curves appearing in this manuscript was carried out using SigmaPlot 12.5 software.

The correlation coefficient, R2,is one of the testimonies to analyze model precision which describes the drying curves which expression is:

n ∑

2 exp 2 1 ( ) 1 ( )

X X R X X − = − −

ipre i i n = ∑

2 exp 1 i i =

In addition to R2, the RMSE mean square error is used to study the smoothing precision. This static parameter is calculated as follows:

2

1 n ( ) i pre i i RMSE X X n = = − ∑

exp, , 1

Estimation of Diffusion Coefficient

The water diffusion coefficient D is currently identified from experimental drying kinetics by fitting the experimental curves to the simplified solution of the Fick’s differential equation. The simplest and most often used solution is the one corresponding to a slab which surface water content is supposed equal to the equilibrium product water content (Xeq) forgiven air parameters [16]. It is important to note that this assumption is valid when the drying rate is fully controlled by internal water transfer. In this case, the Fick’s equation solution averaged over the sample thickness L2 and truncated to the first term (meaningful for largetimes) is:

2 X X e X X

2 4 2 8 Dt eq L

− − = − Where D is the water diffusivity (m2/s) and L is the thickness of the product (m).

π π cr eq

Hydro Distillation (HD)

Several factors may affect essential oils’ productivity and chemical formula; among them we find the drying temperature and extraction technique.

Efficiency Improvement Simplex method: To reduce the number of experiments demonstrating how the factors affecting the experiments influenced the results of the experiments alone or in combination with other factors, Simplex method was employed.

This optimization of extraction efficiency was done with via factors:

• Drying temperature [30, 60] (°C);
• Water / plant matter ratio [4, 10];
• The extraction time [90, 180].

The hydro-distillation is the most widely used process in the extraction of active ingredients from the aromatic plants in spite of its high-energy cost and its negative impact on the environment.

In this extraction process, the product to be treated is fully immersed in water, which is then boiled. Starting  up of the heater  is carried out with an optimal adjustment of the heating power to allow a stability of the extraction at a constant speed. The water and water vapors pass through the plant material and are mixed with the volatile essential oil molecules which then are recovered after condensation. The installations used for the management of this process are very simple to build. In fact, the need to heat large amounts of water and consequently increased energy extraction cost are the major disadvantages of this process.

The cohobating can be present in this process to send all aromatic waters back to the boiler, which is separated from the essential oil by simple settling [17].

Chemical Composition of EOs of Lemon Leaves (GC- MS): Chromatographic analyses were performed on a gas chromatograph electronically controlled of pressure type Agilent (series HP 6890) fitted with a capillary column HP-5 (5% diphenyl-dimethyl siloxane); (30 m x 0.25 mm, thickness: 0.25 µm).

Detection was assured by a  flame ionization  detector (FID) at 250°C, supplied by a mixture of  gases (H2/Air). Nitrogen was used as a carrier gas with a flow of 1.7 ml/min. This unit is equipped with a split less type PTV injector with a split injection mode (split ratio: 1/50, flow: 66 ml / min, injected volume: 1 µl). The programming of the temperature ranged from 50 to 200°C, during 5 min, with a gradient of 4°C/min.

The essential oil components are identified by comparing the mass spectrum of each peak separated by CG with those reported as references in specialized mass spectrometry libraries.

Reagents

The reagents used in this research are:

• Distilled water;
• Folin-Ciocalteu reagent (2M);
• Sodium carbonates(7%);
• Sodium nitrite (5%);
• Aluminum chloride (AlCl3, 6H2O);
• Sodium hydroxide (1M).

Experimental Techniques

Extraction of essential oil is accomplished by hydro distillation technique (equipment designed and constructed in the Applied Thermodynamics laboratory, National Engineering School of Gabes). The different dispensing of polyphenols and flavonoids were performed using a UV- visible spectrophotometer (brand Genesys 10S UV.VIS).

Total Phenol Quantification

Harvested plant was dried at 30, 40, 50 and 60°C. Total phenol was extracted by mixing 3 g of the dry leaf powder in 30 ml of water. After 12h, the solution was centrifuged at 5000 rpm for 15 minutes. The upper phase was recuperated for later use.

Absorption in the visible domain is common and results in staining of the sample [18].

Total Phenolic Content Determination

A volume of 125 μl of extract prepared in distilled water was addedto 125 μl of Folin-Ciocalteu reagent (diluted 10 times) [19]. After 3 minutes, a volume of 1250 μl of Na2CO3 (70 mg/ml) is added to the solution, and then the total is adjusted to 3 ml with distilled water. After 90 minutes of incubation, the total phenolic content (TPC) was analyzed spectrophotometrically using the Folin_Ciocalteu method at awavelength of 760 nm. The TPC of lemon leaves was expressed as equivalents of Gallic acid/g of dried leaves (mg- GAE/g-DL). Flavonoid Content Quantification A volume of 250 µl of extract is combined with 75 µl of NaNO2 (5%). 6 minutes later, 150 μl of (AlCl3, 6H2O) are added to the solution. After 5 minutes, 500 μl NaOH (1 M) will be added and the total will be adjusted to 2500 μlbyH2O. The total flavonoid content (TFC) of the extracts was determined at 510 mns. The amount of TFC was reported based on mg- cateching equivalent per g of dried leaves (mg-CE/g-DL) [20].

Error Estimation

Using basic error propagation rules, the errors on the measured and calculated parameters can be calculated in the following way:

The relative error of the water content measurement: ΔX/X= Δm/m where m is the sample mass and Δm/m is the relative error of mass on line and in situ measurement. This relative error is strongly dependent on the balance precision and weighing conditions (perturbations induced by the air flow). In the case of this study, the value of 0.06 will be used.

• The relative error of the drying rate determination: Δ(dX/dt)/(dX/dt) = 2 ΔX/X. This error will thus equal 0.12.
• The relative error of the water diffusivity calculation: ΔD/D = 3 ΔX/X. This error will thus equal 0.18.

Drying Curves

The initial moisture content of lemon leaves is about 1.8 ± 0.1 g water /g dry matter. Figure 1 shows the results of drying experiments on thin layers in a laboratory dryer with continuous measurement of the sample mass. The drying temperature varies within a range of 30 to 60° C (V = 1 m / s, RH = 60%).

As shown in Figure 1, at constant relative humidity and air velocity, increasing the temperature will decrease the drying time as expected.

Click to enlarge

The temperature showed a very clear influence. Indeed, by increasing the temperature, the drying time has decreased exponentially (the drying time is reduced from about 10 h at 30°C to 3 h at 60°C). Reducing the drying time under the effect of the air temperature is desired in practice, since the capacity of a dryer will be increased and will allow a considerable reduction in drying costs. The response of the drying rate to the temperature is a characteristic property of medicinal plant species. In similar experiments with Chamomilla recutita flowers, the drying time was reduced from 52 h at 30°C to 1.6 h at 60°C [21]. For the roots of Echinacea angustifolia, the relationship was 56 h at 30°C to 6.5 h at 60°C.

The absence of the initial phase and the constant rate phase completely conforms the literature. Indeed, Sghaier, et al. [22] pointed out that when the product is in the form of thin leaves the heating up and the constant rate period are not visible.

Modelling of Drying Curves

The drying kinetics data obtained for the four temperatures was fitted to five empirical kinetic models (Table 1).

It was noted that the R2 values (Table 1) ranged from 0.9604 to 0.9996. The temperature dependence of the drying kinetics can be drawn from the fact that the value of the parameter “k” (Table 1) increased for all models with the increase in the drying temperature.

The results given in Table 1 show that the Logarithmic model has the highest values of R2 as well as the lowest values of RMSE compared to the other models for all temperature levels.

Estimation of Diffusion Coefficient

The diffusion coefficient De was derived from the drying curves recorded during hot air drying trials in the pilot dryer by fitting method. The influence of temperature was deduced from these data (Table 2).

Effect of Drying on Essential Oils Extraction

Essential Oils Yield: Essential oil extraction yield is defined as follows:

in (%) 100 Mass of essential oil theorganic phase Initial mass of dry plant material η = The operating conditions for essential oil extraction from lemon leaves by hydro distillation technique are optimized by simplex method in order to minimize the extraction time and the water/plant material ratio and increase the yield.

The 3-dimensional simplex for lemon leaves is given in Table 3.

The analysis of this experimental  design shows that the essential oil yield varies between 0.3158% and 0.598%, which confirms that the factors studied have reduced the essential oil yield.

Optimum conditions giving a better yield of essential oil of lemon leaves (0.6%) are an air-drying temperature of 24°C, a handling time of 125 min and a water /vegetable matter ratio of 3.357.This indicated that significant loss or degradation of EOs occurred during the drying process [23]. Dorneles, et al. [24] also demonstrated that drying air temperature resulted in the decreasing the content of essential oil from leaves. However, according to Xing, et al. [25], oven drying showed the highest EO yield of Purple Perilla Leaves.

Chemical Composition of EOs from Citrus Limon: The chemical composition of EOs from Citrus limon at different air-drying temperatures is observed and showed in Figure 2.

The essential oil of lemon leaves includes 12 major components with a total of 89.26%, 77.08%, 79.19%, 72.86% respectively for the drying temperatures of 23.7, 30, 45 and 60°C.

Figure 2 shows the chromatogram obtained by GC / MS

giving the retention time as a function of absorbance.

Chemical formula of EOs of citrus limon is influenced by the heat treatment of the plant material. Indeed, for a temperature of 23.7˚C, the majority peak corresponds to the compound manoyloxid (44.05%), then limonene (18.97%) and beta-pinene (11.15%). While at 30˚C, the maximum level corresponds to beta-caryophyllene (46.06%) with the disappearance of limonene and the remarkable decrease in beta-pinene content (2.9%). The increase of the drying temperature up to 45˚C induces a chemical composition rich mainly in nerol (26.3%), neryl acetate (20.2%) and beta-caryophyllene (15.31%) with a further appearance of limonene. Finally, the qualitative analysis of the essential oils of the product dried at 60°C gives a majority beta- caryophyllene content which reaches 21.29% with a presence of limonene and beta-pinene. These show that there is a significant effect of air-drying temperature on the perfection of EOs of_citrus limon_.

Rocha, et al. [26] have shown that raising the drying temperature above 40°C reduces the fractions of the main compounds of Ocimum Selloi Benth essential oil (elimicine, trans-caryophyllene, germacrene-D, cyclobermacrene).

Essential oils of lemon leaves are valued mainly in the aromatherapy, perfumery and cosmetics markets. They are sought after for their fragrant or therapeutic properties. The main consumer markets are the developed countries (Europe, Japan, North America), which represent 80% of the world’s markets.

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Effect of Heat Treatment on Phenolic Concentration in Citrus Limon

Table 4 shows that raising the drying temperature induces the increase in the concentration of phenolic compounds. In fact, it goes from 33.83 mg of mg-GAE/g-DL at 30˚C to 51.97 mg of mg-GAE/g-DL at 60˚C.

This can be explained by the fact that in the case of this plant, the heat treatment promotes evacuation of phenolic compounds from the solid form. These results agree with those found by Boudhioua, et al. [27] who studied the impact of heat treatment process on total phenolic rate of four varieties of olive leaves.

Impact of Drying on Flavonoid Rate

The method using AlCl3 allows the estimation of flavonoid contents in extracts of the plant studied, by measuring their absorbance by means of UV / visible spectrophotometry (Table 4).

For the lemon leaves, the flavonoid content reaches its optimum at 40˚C (it goes from 0.655 mg of mg-CE/g-DLto 1.041 mg of mg-CE/g-DL at 40˚C to decrease again until 0.63 mg mg-CE/g-DLat 60˚C). These results show that the drying temperature could alter the active constituents of plant products, such as flavonoids. Hence the recommendations that require the drying of plants should not be at high temperatures to avoid the loss of active constituents.

Zhishen, et al. [28] quantified flavonoids in mulberry leaves by applying different modes of drying. They found that the fresh leaves contain more flavonoids than those dried in the air or in the oven.

Chen, et al. [29] found that the concentration of flavonoids in orange peel extracts declined with a drying temperature below 80°C and improved at anutmost drying temperature. Ho, et al. [30] reported that the flavonoid content of lemon leaves increased with drying time.