Extraction and chemical characterization of neem seed oil ( Azadirachta indica )

, 1,2,3-trimethylbenzene and buticyclohexane. In addition to the volatiles in the oil, 7 fatty acids were found; oleic acid, linoleic acid, palmitic acid and stearic acid, with the oil extracted from the seeds having the highest concentration of oleic acid and linoleic acid in commercial neem oil. Given the presence of these chemical compounds in neem oil, it is suggested, for subsequent investigations, the isolation and application of organosulfur compounds as insecticides. The extraction of oil from flowers and neem seeds with solvents that are less harmful to the environment, together with its application, makes it possible to evaluate its pesticide potential. In addition, it is essential to evaluate the influence of neem on the bee population, analyzing cases of mortality and seeking solutions to avoid environmental damage. Additionally, the viability of neem oil as an industrial and hospital cleaning product is highlighted, exploring antimicrobial, antibacterial and biodegradable properties.


INTRODUCTION
The neem or neem tree (Azadirachta indica A. Juss) originates from Myanmar and the Indian subcontinent region (Islas et al., 2020;Sarkar et al., 2021).In the botanical classification, it belongs to the Meliaceae family, genus Azadirachta and species Indica (Sarkar et al., 2021).The tree has been used for over 2,000 years in India as an agricultural pesticide, in the production of drugs, cosmetics, fertilizers and disinfectants (Perera et al., 2021;Baby et al., 2022;Rana e Saxena, 2023 andAlba et al., 2023).It also has medicinal uses in the treatment of cancer (Jeelani et al., 2023), in the fight against the malaria vector (Ayinde et al., 2020) and is anti-inflammatory and antiviral (Loganathan et al., 2021).
In animals in the control of parasites, such as canine leishmaniasis (Zatelli et al., 2022).Among the plants with pesticidal activity, Azadirachta indica has several studies that demonstrate different uses against insects such as insecticide, larvicide (Jeelani et al., 2023), anti-food, reproduction inhibitor, development inhibitor (Kilani-Morakchi et al., 2021) and repellent (Ayinde et al., 2020;Ghosh et al., 2021).This is due to the large amount of phytochemicals, among which the substance azadirachtin is known as the most efficient for insecticide use, being the main one in biopesticide formulations (Perera et al., 2021;Rana e Saxena, 2023).Azadirachtin is present in varying concentrations in all parts of the plant, but the seeds have the highest concentration of azadirachtin, being more used in the production of oils for insecticide use (Chaudhary et al., 2021;Loganathan et al., 2021;Rana e Saxena, 2023).In view of this, oil extraction is considered the best way to take advantage of the benefits of the plant (Loganathan et al., 2021;Kumar et al., 2022;Rana e Saxena, 2023).
The methods used to extract neem oil are mechanical pressing, soxhlet, supercritical fluid, microwave and ultrasound (Chaudhary et al., 2021;Saha Tchinda et al., 2021).Soxhlet extraction is the most used to obtain bioactive molecules from natural sources (Fagbemi et al., 2021), its advantages are repetition in cycles of the sample solvent in the extraction and not requiring the filtration step after extraction (López-Bascón e Luque de Castro, 2020).On the other hand, the disadvantages are: high extraction time; degradation of thermolabile substances, due to the temperature used being close to the boiling point of the solvent (extractor); the need for another stage of evaporation of the solvent contained in the sample after extraction (Hirondart et al., 2020).In the extraction of neem oil via the Soxhlet system, the solvents that provide the most satisfactory yields are hexane (Chaudhary et al., 2021), as well as mixtures such as hexane/methanol and hexane/ethanol (Barbosa et al., 2023).However, other factors influence the choice of solvent.Selection based on polarity makes it possible to isolate related substances (Alba et al., 2023).In addition, the environmental issue is relevant, as hexane is a pollutant (Kumar et al., 2022).Green solvents such as acetone, isopropanol and ethanol also appear as options, useful for separating compounds and fatty acids of importance in the pharmaceutical and food industry (Saha Tchinda et al., 2021).
Neem seed oil has in its chemical constitution a diversity of volatile organic compounds (VOCs) belonging to different chemical classes such as: alcohols, acids, esters, phenols, flavonoids, sulphates, aldehydes, hydrocarbons and terpenes (Guchhait et al., 2022;Tulashie et al., 2021), which are responsible for the characteristic aroma of olive oil.In addition to the aroma, volatiles have antibiotic, antioxidant, antiviral, antitumor, antibacterial, antifungal properties and are used in the prevention and treatment of various diseases such as diabetes and cancer in humans (Rana e Saxena, 2023;Jeelani et al., 2023).Along with volatiles, neem oil has fatty acids in its composition, which are very important for the normal functioning of the human organism (Mulla et al., 2020;Saha tchinda et al., 2021).These fatty acids are used in pharmaceutical products (Zheng et al., 2021).
Thus, given the importance of the presence and use of these compounds, the general objective in the present study was the extraction of oil from the neem seeds through the soxhlet system, the characterization of the profile of volatile compounds by microextraction in solid phase headspace mode, followed by analysis by gas chromatography coupled with mass spectrometry HS-SPME/GC-MS, and fatty acids by gas chromatography with flame ionization detector GC-FID.

Collection of the neem seeds
The seeds of neem were collected in the months of November and December 2021, at the Federal University of Northern Tocantins (UFNT), Cimba unit (-7.181285262491843, -48.19801163546441) in Araguaína-Tocantins.The ripe seeds have been pulped using a common nylon sieve, and then dried, under the sun, for about 18 days.Although, the unripe seeds have been separately reserved, for one week, until they became fully ripe to be pulped.After all the seeds have been pulped, they were taken to a drying oven with air circulation of 55°C for 72 hours.
Figura 1 -Dry seeds after passing through the drying oven with air circulation

Oil extraction
After drying, the seeds were peeled and macerated in sufficient quantity for extraction, using a mortar (gral) with a porcelain pistil.The mass of the macerated sample was measured, about 11.0 g, on an analytical scale (Model AUY220, Shimadzu brand), then transferred to the paper filter cartridge, and then inserted into the soxhlet extraction system.The solvent used to extract the oil was the n-hexane analytical grade P.A. (quimex brand), where 8 extraction cycles were performed.
At the end of the extraction, the sample was taken to the rotary evaporator (brand Lucadema, model Luca-EV01) with a heating bath (brand Fisatom, and model 550).After evaporation of the solvent hexane, the oil was transferred to a beaker and anhydrous sodium sulfate (dynamic brand) was added to remove water remnants in the sample.Then, the sample containing the salt was filtered on filter paper, the residual mass was weighed on an analytical scale and the oil output was calculated, the measured value was 2.7g.

Microextraction solid phase headspace and chromatographic analyses
For the preparation of the sample by HS-SPME, approximately 1.26 g of the neem oil was weighed and transferred to a vial for headspace of 10 mL of transparent glass with a silicone cover.Subsequently, the vial was placed on the magnetic stirrer (Model, IKA C-mag HS 4) for 15 minutes at an approximate temperature of 45°C monitored by an infrared thermometer (model ST-600, brand Incoterm).Then, the needle of the holder was inserted into the vial, exposing the fiber CAR/PDMS (carboxen/polydimethylsiloxane film) to the headspace of the vial for 15 minutes to capture the volatiles.After that, the fiber was collected, and the holder was removed from the bottle and transferred to the GC-MS for analysis of the volatiles.
For the separation of volatile substances, it was used the gas chromatograph coupled to the mass spectrometer (GC-MS Agilent Technologies 7890B CG system and MSD 5977B).Before performing the chromatographic race, the fiber was conditioned according to the recommended conditions of the manual.Injector, 250 °C; initial oven temperature 40°C, with an increasing rate of 4 °C/min up to 220°C and after 20°C/min ending at 280 °C, the race had a total time 50 minutes; the temperature of the ionization source was 230 °C and quadrupole 150 °C.The carrier gas was helium (99.99 % purity), injection mode manual split 1:50, the column was the capillary type HP-5MS (5 % phenylmethylpolysiloxane) with dimensions 30 m x 25.0 μm x 0.25 μm, ionization energy of 70 eV.
For the identification of the compounds, it was used the NIST 2014 library present in the program provided by Agilent and the NIST Chemistry Book on the Web, the confirmation occurred by calculation and confirmation by reference to the Kovats index.
For analysis of the oil in the automatic injection mode with solvent, the oil is diluted at the ratio 1/100 in hexane (V/V) using a micropipette 10-100 μL and 100-1000 μL.The injection occurred in 1:50 split mode, and about 1 μL of the sample was injected automatically using a 10 μL glass syringe.The employed chromatographic conditions have been optimized according to the work of (Hossain et al., 2013).The initial temperature of the oven was 45 °C for 3 minutes, with a heating rate of 3 °C / minute, until reaching 150 °C with a variation of 20 °C / minutes ending at 250 °C, the analysis time lasting 48 minutes.Injector temperature, 155 °C and ionization source 230 °C, and quadrupole 150 °C.

Kovats index determination
A mixture of n-alkane pattern (C7-C30) has been used to determine the Kovats index of each compound detected in the oils analysis, through the retention times of n-alkanes, compounds and comparing values in the literature that have the same stationary phase of the capillary column (NIST Chemistry Book on the Web).The parameters of the chromatographic runs used have been the same as the oil analyses, except for the injection of fiber, which occurred in automatic mode.

Gas chromatograph with flame ionization detector (GC-FID)
For chromatographic analysis, 5,0 g of neem oil, 10 mL of methanol, and 50 mg of catalyst were added to a Teflon cup.The cup was introduced into a stainless-steel autoclave which was hermetically sealed and placed in a shaking heater with a temperature of 150 °C for a 4 hour period.After this time, the material was removed from the cup and centrifuged to separate the catalyst from the reaction medium.The excess methanol was evaporated using a rotary evaporator, thus obtaining the methyl esters of fatty acids.
It was weighed 114 mg of the sample and 1 mL of chloroform added.Subsequently, the oil was esterified, and a Gas Chromatograph was used GC -Shimadzu 2010 plus, with Rtx-Wax capillary column, coupled with flame ionization detector (FID), injecting (1 μL) of sample and with a programming column temperature of 210 °C for 50 minutes, the FID temperature of 250 °C and the flows of H2, air and carrier gas (N2) 40, 400 and 1.6 mL min-1, respectively.A seed oil pattern was used to identify fatty acids.A commercial sample of the neem oil, acquired in an agricultural products store located in the city of Teresina-PI, was analyzed under the same conditions for comparison with the oil profile extracted from the seeds.

RESULTS AND DISCUSSION
The average oil output was 23.73 % ± 0.07.In the literature, using the same technique and hexane solvent, it has been found results of 38.8 % (Yami et al., 2020), 40.35 % (Tesfaye et al., 2018), 52.3 % (Saha tchinda et al., 2021) and lower results, 21.51 % (Kaura et al., 1998) and13.33 % (Ungo-Kore et al., 2019).According to (Keneni e Marchetti, 2017) the grain oil output was 25-45 %, which is within the range presented in the literature.Oil output is related to tree development environment conditions, climate, rainfall, soil characteristics; genotype; conditions for the collection; storage of plant material; particle size (Fernandes et al., 2019;Neves e Carpanezzi, 2008;Saha Tchinda et al., 2021;Beyecha Hundi̇e et al., 2022).The oil yield can even be affected by the amount of sample used in the extraction.This is evidenced in articles where oil yields were cited.Consequently, these variables impacted the result of oil extraction output.
The abovementioned volatile and 2-methyl-2pentenal were identified in neem seeds oil (Mubarak e Kulatilleke, 1990) and in onion samples by SPME (Choi et al., 2017).The 2-methyl-2-pentenal had proven antifungal activity against Fusarium oxysporum f. sp.Cubense stipe 4 (Zhang et al., 2013).Performed extraction of yellow onion oil by steam carrier, some volatiles found were 3,4-dimethyliophene and (E)propenilpropiltrissulfide, one of the compounds with the highest concentration, being one of the main volatiles that collaborates with aroma and flavor of the onion oil (Cantrell et al., 2020).In our work, some sulfur compounds found in the neem seed oil were found in onions (Balandrin et al., 1988;Breu, 1996).This indicates the existence of similar metabolic processes in both species (Balandrin et al., 1988), although having this similar volatile profile, the aroma of the neem does not cause tear effect and loses intensity (Koul, 2004).
Conducted a study of the volatiles of Australian wild rice, identified an aroma similar to plastic, and found that one of the compounds that contribute to this odor is 2-Miristinoil pantethine (Zhao et al., 2022) that was also detected in our study.Toluene is present in the essential oil of the the neem leaf (Ebrahimi et al., 2013), inhibiting the presence of bacteria R. solanacearuam.Other compounds such as ethylbenzene and 1,3-dimethylbenzene obtained excellent antibacterial and antifungal activity (Mohamadpoor et al., 2022;Raza et al., 2016).
The volatile undecane was detected in buchanania lanzan seed oil (Desai et al., 2022) and dodecane compound in leaf oil of Strychnos nuxvomica (Suganthy e Gajendra, 2020), but no articles were found that report these volatiles in the neem oil.Detected dodecane and undecane in Pseudomonas spp.evaluated the effects of volatiles in promoting the growth of Vigna radiata seedlings, the benefits of these two substances in the seedling is to contribute to plant development, induced systemic resistance and antimicrobial action (Jishma et al., 2017).
Hydrocarbons, decane, dodecane, undecane, 2methyldecane, 3-methyldecane, 2-methylundecane and 4-methylundecane are present in plant extracts with antimicrobial properties (Bukvicki et al., 2013;Kumar Tyagi et al., 2013;Winnett et al., 2017).Thus, it is possible that the volatiles mentioned contribute synergistically to antimicrobial activity with other compounds of the neem oil.The oil extracted in this work was applied in hydroponic lettuce cultivation in another project.

Analysis of the neem oil by GC-FID
It observed by the analysis of the oil extracted from the seeds and the commercial oil of neem, the presence of the same fatty acids: palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid.The fatty acids of the oil extracted from the seeds have a higher percentage area, except linolenic, indicating higher concentration in relation to commercial oil.Moreover, a greater number of fatty acids was identified in the fixed oil extracted from the seeds.Figures 3 and 4 show the chromatograms of oil extracted from the seeds and commercial of neem, with the percentage areas of each compound.
The major fatty acids with their respective values of percentage areas found in seed oil were oleic acid 43.49%; linoleic acid 19.06 %; palmitic acid 18.71 % and stearic acid 16.67 %.While in commercial oil, the data for the same compounds were linoleic acid 40.76 %; oleic acid 33.38 %; palmitic acid 15.93 % and stearic acid 6.30 %.In the literature, all the compounds mentioned are among the most abundant in the neem oil analyzed in the GC-FID, with the highest concentration of oleic acid and linoleic acid (Mulla et al., 2020;Saha tchinda et al., 2021;Ismaila et al., 2022).
In other oils, it has been detected the presence of most of the fatty acids mentioned above, for example, in the oil of Moringa oleifera seeds, oleic acid is more abundant, with a percentage area of 70.2%; followed by palmitic acid 7.8%; stearic acid 7.5% and linoleic acid 3.1% (Zhao et al., 2019).In soybean oil fatty acid analysis studies, the major compounds are linoleic acid, oleic acid, palmitic acid, linolenic acid and stearic acid (Kostik et al., 2013;Zambiazi et al., 2007).As presented, soybean oils and Moringa Oleifera contain a strong presence of oleic acid and linoleic acid, as well as in our work with neem oil extracted from seeds and commercial oil.Some fatty acids found in our work have pesticide activity.The mixture of oleic, linoleic, palmitic and stearic and oleic effects with stearic acids showed ovicidal effects of egg positioning and death of larvae of C. maculatus (Lienard et al., 1993).In another study the fight against C. maculatus was also observed an insecticide property, with the use of oleic and linoleic acid that had greater efficiency and with the mixture with the four fatty acids mentioned above (Aider et al., 2016).Other properties of oleic acidic fatty acids, linoleic, palmitic and stearic are antibacterial activity (Casillas-Vargas et al., 2021), linoleic is related to decreased risks of cardiovascular diseases (Marangoni et al., 2020) and the high intake of oleic acid can help improve cases of hypertension, obesity, insulin resistance and inflammations (Granado-Casas e Mauricio, 2019).

CONCLUSIONS
The efficient extraction of neem seeds oil was performed by the soxhlet system using the hexane solvent, obtaining a good output equal to 23.73% ± 0.07.It was possible to detect 35 volatile compounds in the neem seeds oil, belonging to several chemical classes, which are responsible for the characteristic aroma of the oil.These same compounds are known for their antioxidant, biological, antibactericidal, antifungal, antimicrobial, insecticide, and anticancer activities.
Seed and commercial oils are rich in fatty acids and have practically the same compounds identified by GC-FID.However, the concentration of fatty acids is higher in the oil extracted from the seeds, and the oleic and linoleic acids are the compounds with the highest concentration.Fatty acids as well as volatile acids have biological activities essential to the functioning of the human organism and tree development.Thus, it's considered that all these chemical constituents present in the neem oil contribute synergistically to the various known benefits in using neem.
Given the presence of these chemical compounds in neem oil, it is suggested, for future research, to isolate and apply organosulfur compounds as insecticides.The extraction of oil from neem flowers and seeds, with solvents that are less harmful to the environment, makes it possible to assess its pesticide potential.Evaluating the impact of neem on bees, analyzing mortality and seeking solutions against environmental damage, is crucial.Additionally, the potential of neem oil as an industrial and hospital cleaning product is highlighted, exploring its antimicrobial, antibacterial and biodegradable properties.