HEMI-SYNTHESIS AND UV-VISIBLE SPECTROPHOTOMETRIC CHARACTERIZATION OF 2,4-DINITROPHENYLHYDRAZONES DERIVED FROM CITRAL AND CITRONELLAL ESSENTIAL OILS OF TWO AROMATIC PLANTS ACCLIMATIZED IN CONGO-BRAZZAVILLE

respective Analysis

The orange-yellow and colorless essential oils with respective yields of 1.54% and 3.59% were extracted from the dry leaves of Cymbopogoncitratus (DC.) Stapf and Eucalyptus citriodora Hook. collected south of Brazzaville. Analysis by gas chromatography (GC) and by gas chromatography coupled with mass spectrometry (GC/MS) allowed the identification of fifteen (15) and eight (8) constituents representing (96.25%) and (98.46%) of total essential oils respectively. Cymbopogoncitratus oil consists mainly of geranial (51.99%) and neral (32.94%), two geometric isomers constituting citral which occupies a rate of 84.93%. While citronellal with a high level of (80.72%) and citronellol (10.48%) are the major compounds of the essential oil of Eucalyptus citriodora. Geranial (citral a) and citronellal 2,4dinitrophenylhydrazones were hemi-synthesized by a simple, easy method, respectively from essential oils of Cymbopogoncitratus and Eucalyptus citriodora with respective conversion rate (yields) of 20%. and 37%, in a short time (three to five minutes). Analysis of geranial and citronellal 2,4-dinitrophenylhydrazones by UV-visible spectrophotometry showed maximum absorption wavelengths of 390 nm and 370 nm respectively. The UV-visible spectrophotometric method employed for the determination of these hydrazones is convenient, fast and simple. The hemi-synthesized hydrazones could be useful in the pharmaceutical industry, in perfumery, cosmetic and in biomedicine.

…………………………………………………………………………………………………….... Introduction:-
Among the organic compounds contained in the essential oils of certain aromatic plants are carbonyl compounds (aldehydes and ketones) with high proportions reaching 85%. This is the case of citral (geranial and neral) and citronellal in the essential oils of Cymbopogoncitratus and Eucalyptus citriodora respectively (Ndzeli et al., 2019a; Ndzeli et al., 2019b). These are chemical compounds which have a remarkable interest, intervening in a wide field of application (cosmetics, perfumery, pharmaceuticals, food industry, chemical industry). They are endowed with various chemical properties due to the presence of the C=O double bond giving rise to several nucleophilic and electrophilic addition reactions (Vollhard, 1995). However, the main disadvantage or disadvantage of these Very few studies on the hemi-synthesis of hydrazones derived from aldehydes and ketones of essential oils are described in the literature. We can cite the hemi-synthesized hydrazones of essential oils with an aldehyde chemotype (cuminaldehyde and perillalaldehyde) recently carried out in Algeria (Bouchachia and Ameur, 2019).
To our knowledge, the hemi-synthesis of 2,4-dinitrophenylhydrazones from the aldehydes (citral and citronellal) of essential oils is not known.  (Jianrong et al., 2007) are used for the determination of 2,4-dinitrophenylhydrazones. However, these analytical methods involve long analysis time, tedious sample pretreatment, and high product cost.
The aim of this present work is to extract essential oils, to hemi-synthesize and to characterize 2,4dinitrophenylhydrazones derived from citral and citronellal by UV-Visible spectrophotometry, a simple and rapid method.

Extraction of essential oils:
After eight (8) days of drying at room temperature, in a ventilated room, the samples of Cymbopogoncitratus and Eucalyptus citriodora made up of the leaves are subjected to hydrodistillation for four (4) hours using an extractor. Clevenger type (Clavenger, 1928), fitted with a two (2) liter balloon. The condensate loaded with essential oil and hydrolate is collected. The essential oil is separated from the hydrolate by decantation. The extraction with diethyl ether is carried out to isolate the aqueous phase of the essential oil followed by drying of the ether phase with anhydrous sodium sulfate. Twenty-four (24) hours after evaporation of the diethyl ether in air, the essential oil is recovered. These operating conditions are summarized in Table I. The yield R in essential oil is calculated according to the following formula.
Tableau I :-Operating conditions for hydrodistillation of leaves of Cymbopogoncitratus (DC.) Stapf and Eucalyptus citriodora Hook.

Analysis of essential oils: Analysis by gas chromatography:
The quantification of the constituents was carried out using a Hewlett Packard HP 5890 type chromatograph equipped with a flame ionization detector equipped with HP ChemStation data acquisition software. The different constituents are separated using a DB5 capillary column (30 mx 0.25 mm), (film thickness 0.25 µm) under the following operating conditions: helium carrier gas (1 mL.min -1 ), temperature injector temperature: 280 °C, detector temperature: 280 °C. The oven is programmed at 50 °C for 5 minutes with a gradient of 5 ° C.min -1 from 50 to 300 °C, 5 minutes at 300 °C with a split mode injection of 1-20.

Analysis by gas chromatography-mass spectrometry:
Analysis by gas chromatography-mass spectrometry was carried out using a Hewlett Packard HP 6890 brand chromatograph coupled to an HP 5973 mass spectrometer. 584 gas: (helium: 1 mL.min -1 ), ionization energy (70 eV), temperature of injector (280 °C), detector temperature (280 °C). The oven is programmed at 50 °C for 5 minutes with a gradient of 5 °C. min -1 from 50 to 300 °C, 5 min at 300 °C with a 1-10 split mode injection.

Identification of constituents:
The various constituents of essential oil have been identified on the basis of their retention indices and their mass spectra by comparison with data from the literature (Adams, 2001;Joulain et al., 1998;Davies, 1990).

Hemi-synthesis of 2,4-dinitrophenylhydrazones:
The 2,4-dinitrophenylhydrazine reagent and essential oils from the species Cymbopogoncitratus and Eucalyptus citriodora are used for the preparation of hydrazones.
The method used has been reported by (Leclercq, 2008). It consists of dissolving 0.25 g of 2,4dinitrophenylhydrazine in 5 mL of methanol, followed by addition of 0.5 mL of concentrated sulfuric acid, then filtration of the lukewarm solution. To this solution are added 0.2 g of essential oil previously solubilized in a small volume of methanol. After about three (3) to five (5) minutes, the solid formed is filtered off and washed in a little methanol. If there is no solid, the solution is acidified with sulfuric acid. The precipitate is then recrystallized from ethanol and then dried. These conditions are summarized in (Table II).

Characterization of 2,4-dinitrophenylhydrazones: Determination of melting points:
The measurement of the melting temperatures of the prepared hydrazones is carried out using the kofler bench. The method consists of calibrating the device with benzoic acid which has a melting point of 122.35 °C. The carriage is moved horizontally until the cursor is at the border between solid and liquid. Then the movable index is moved vertically until it indicates the melting point of the standard. The hydrazone's melting point is then taken by depositing it at the cold end of the kofler bench and moving it towards the hot zone until the first drops of liquid appear. The carriage is then moved horizontally until the cursor is at the border between solid and liquid. The moving index then indicates the melting point. Three tests are carried out.

UV-visible spectrophotometric analysis:
The analysis of 2,4-dinitrophenylhydrazones is done by a UV-visible WPA Lightwawe II spectrophotometer, connected to an HP computer.

Essential oil solution:
The essential oil (10 to 20 mg) is placed in a 10 mL vial in which methanol is added to the mark.
Spectral scan: 2.4-dinitrophenylhydrazones scan: 0.5 mL of the 2,4-dinitrophenylhydrazine solution is added to 0.5 mL of the essential oil solution. An orange or brown precipitate forms depending on the essential oil used. The precipitate is left to stand for 10 minutes at room temperature and 5 ml of methanol are added to it: This is the solution of 2,4-dinitrophenylhydrazone.
We put in the reference tank • 1 mL of the 30% (V/V) water/ethanol solvent and in the measuring tank: Results and discussion:-Extraction and yield of essential oils: Hydrodistillation extraction from the dry leaves of Cymbopogoncitratus from Eucalyptus citriodora provides orange-yellow and colorless essential oils with yields of 1.54% and 3.59% respectively (Table III). The yield of Eucalyptus citriodora essential oil (3.59%) is higher than that of Cymbopogoncitratus essential oil (1.54%).
Eucalyptus citriodoraessential oil can be produced in high yields of up to 4-6% (Mapola, 2004). The result of the extraction yield of the essential oil of Eucalyptus citriodoraagrees with that described in Congo indicating a yield of 3.57% (Silou, et al., 2013), while that of the oil of Cymbopogoncitratus (DC.) Stapf (1.54%) is similar to that of essential oil of Togolese origin, which has a value of 1.60% (Koba et al., 2009).
Tableau III :-Extraction yield of essential oils from the leaves of Cymbopogoncitratus (DC.) Stapf and Eucalyptus citrodora Hook.

Chemical composition of essential oils: Cymbopogoncitratus:
The results of the chemical analysis of the essential oil extracted from the leaves of Cymbopogoncitratus are shown in (table VI). In total, fifteen (15) constituents were identified representing (96.25%) of the chemical composition of the total essential oil. The essential oil is rich in monoterpenes (95.87%) with a dominance of oxygenates (90.24%) of which the most remarkable are géranial (51.99%) and neral (32.94%), two geometric isomers constituting the citral which occupies a rate of 84.93%. Hydrocarbon monoterpenes represent a rate of 5.63% with mainly myrcene (5.52%) and (Z) -ocimene (0.11%) as components.
In the group of sesquiterpenes, the hydrocarbon compounds are represented by a single constituent, β caryophyllene, which occupies a very low level of (0.07%). We note the absence of oxygenates.   Eucalyptus citriodora: Table V collates the results of the chemical analysis of the essential oil extracted from the leaves of Eucalyptus citriodora. In total, eight (8) constituents were identified representing (98.46%) of the chemical composition of the total essential oil. The essential oil is characterized by a large amount of monoterpenes (97.96%) dominated by oxygenated monoterpenes (97.20%) of which the major compounds are citronellal with a high rate of (80.72%), citronellol (10.48%), lisopulegol (3.91%). We note the presence of a single hydrocarbon compound, β-pinene, which occupies a low level of (0.76%). The sesquiterpenes group remains marked by a single hydrocarbon compound, E caryophyllene, the level of which is low, amounting to (0.13%). Aliphatic compounds are characterized by ethylenic aldehyde 2,6 dimethyl hept-5-en-1-al in such a low proportion (0.37%).

Hemisynthesis of 2,4-dinitrophenylhydrazones:
Physical characterization: Table VI shows the results of the hemi-synthesis of 2,4-dinitrophenylhydrazones and their measured melting points. The reaction of 2,4-dinitrophenylhydrazine in the presence of sulfuric acid on the essential oils of Cymbopbogoncitratus and Eucalyptus citriodora gives rise to precipitates of orange and brown colors respectively. These colors are characteristic of 2,4-dinitrophenylhydrazone derivatives.
The essential oils of Cymbobogoncitratus and Eucalyptus citriodora contain a varied range of constituents, 2,4dinitrophenylhydrazones are formed after three (3) to five(5) minutes, the kinetics are slow so the extraction is also slow. Moreover, the yields of the hemi-synthesis of 2,4-dinitrophenylhydrazones obtained from these essential oils are low and respectively 20% and 37%. These low yields can be explained by the influence of the different constituents of essential oils during the reaction. In fact, the presence of the different constituents in essential oils changes the speed of extraction, resulting in slow kinetics. This slowing down means that there is competition or hindrance between the compound to be extracted and the other constituents of the mixture. This Competition is linked to steric and electronic effects.
Furthermore, the melting points of the hydrazones of the essential oils of Cymbopogoncitratus and Eucayptuscitriodora are 114 °C and 79 °C, respectively. These melting points are approximate to those in the literature (Rappoport, 1967) and correspond respectively to those of geranial and citronellal.

Cymbopogoncitratus(Géranial 2,4-dinitrophénylhydrazone):
The UV-visible absorption spectrum of geranial 2,4-dinitrophenylhydrazone from the essential oil of Cymbopogoncitratus plotted in (Figure 6) shows three main bands: 1. A low intensity band at 247 nm. This band is attributable to the π→π* transition of the C=C group of the ethylenic group; 2. A low intensity band at 298 nm. This band corresponds to the transition→σ * relating to the aniline aromatic system; 3. Another not very intense band at 390 nm relating to the n→π* transition characteristic of the C=N group of hydrazone.

Conclusion:-
Geranial (citral a) and citronellal 2,4-dinitrophenylhydrazones were hemi-synthesized by a simple, easy method from the essential oils of Cymbopogoncitratus (DC.) Stapf and Eucalyptus citriodora (Hook.) Respectively with conversion rate (yields) of 20% and 37 % respectively, in a short time (three to five minutes). Analysis of geranial and citronellal 2,4-dinitrophenylhydrazones by UV-visible spectrophotometry showed maximum absorption wavelengths of 390 nm and 370, respectively. The UV-visible spectrophotometric method employed for the determination of these hydrazones is convenient, fast and simple. The hemi-synthesized hydrazones could be useful in the pharmaceutical, perfume and cosmetic industries. Indeed, citral and citronellal hydrazones are known for their olfactory functions (Kaushik et al., 2016) which orient their uses in cosmetics and perfumery as hydrogels and in biomedicine (Kölmel and Kool, 2017; Xu and Liu, 2019).