SUSTAINABILITY ASPECTS OF CARAPA GUIANENSIS AUBL OIL FOR ANTIINFLAMMATORY USES

Objective: The objective of this study is to describe the lipidomic profile of andiroba oil and its socio-economic characteristics focused on the activities of limonoids as an alternative for the generation of nonsteroidal anti-inflammatory drugs in order to relate the generation of products that act on circular and external peripheral inflammation. Theoretical Reference: In this context, andiroba oil ( C. guianensis ) has a wealth of lipid substances with chemical and socio-economic potential, highlighting its importance for local populations and its potential for sustainable bioeconomics, as this species contains nonsteroidal antiinflammatory drugs from Amazonian oil plants. Method: The methodology adopted includes describing the lipidomic profile of andiroba oil analyzed by GC/MS, using a Zebrom 5HT capillary column specifically designed to analyze both the methyl esters of the various fatty acids and the limonoids and steroids. The bibliographic search was carried out in the CAPES periodicals on the Scopus database, using specific keywords such as andiroba, Carapa, limonoids and antiinflammatories. Three samples of andiroba oil collected in different states were used to compare the lipidomic profiles. Results and Discussion: The results obtained revealed the presence of limonoids 7-desacetyl-7-oxogedunin, 7-oxogedunin, deacetyl gedunin; steroids campesterol, stigmasterol, b-sitosterol and various medium and long chain fatty acids. These results showed valuable information about the presence of limonoids, which have anti-inflammatory properties related to the chemical composition, which can vary with the season, the place of collection and the differences in the extraction processes of this oil. However, there is no total absence of limonoids


INTRODUCTION
This paper describes the importance of andiroba oil, specifically the species C.
guianensi and C. procera in the Amazon bioeconomy and the role of riverside communities in sustainably using forest resources.The analysis of andiroba oil shows the richness of lipid compounds and describes the chemical composition of andiroba oil and its variations.4 In this study, the extraction of non-timber products, such as andiroba oil, stands out because it has little or no environmental impact and highlights forest functionality, making it an economically viable practice and one of the most effective ways of preserving the forest.
The aim is to provide new information on the use of to promote initiatives with greateroil in peripheral inflammatory applications and initiatives with greater and more significant scientific backing within the andiroba production chain.
Notably, andiroba oil is a natural source of fatty acids linked by esterification to form acylglycerols.The oil's lipid composition is predominantly comprised of oleic, palmitic, stearic and linoleic acids bound to glycerol, accounting for approximately 96 to 98% of its total lipids.
In comparison, the remaining 2 to 4% consists of unsaponifiable material, which includes saponins, limonoids, steroids and other lipids.
This work aims to deepen the theory of the use of andiroba oil on various inflammations based on the metabolic profile determined through GC/MS analysis for new economic perspectives.The study focuses on lipidomics, highlighting limonoids and the oil's potential for COX-1 inhibition, thus directing the investigation towards an alternative approach with prospects for application in circular peripheral anti-inflammatory uses and towards a socioeconomic view.

THEORETICAL FRAMEWORK
The Amazon rainforest, an essential source of natural resources, has been severely impacted by human activities, particularly by the unsustainable exploitation of inputs as raw materials for commercial and industrial purposes.One example is the andirobe tree species, which belong to the Meliaceae family and are distributed in the Carapa genus (Cui et al., 2023), (Conab, 2017).When located in the floodplain forests of the Amazon estuary, these species are relevant to riverine and non-riverine peoples, fishermen, small farmers, Indians and various groups of Amazonian population, including those in the metropolitan region.These species were used for three different purposes until two decades ago: the first was oil extraction, the second was timber extraction and the third was the use of bark, heartwood and flowers in folk medicine (Ferraz, Camargo, & Sampaio, 2002), (Busman, 2016).At the moment, Amazonian people have changed their behavior and are working in new directions with four different objectives.The first is the extraction of oil; the second is the collection of seeds, the third is the preservation of the andiroba tree and the fourth is the search for new perspectives for bioeconomy and sustainability.
Within this vision, the extraction of non-timber products, which have little or no environmental impact, and forestry functionality, an economically viable practice, are presented as practical means of maintaining harmony in the rainforest.(Nardi-Santos, 2013).The most significant observation is that local populations in the Amazon are driving initiatives to develop agro-industrial production chains (Christenhusz & Byng, 2016), often in collaboration with governmental and non-governmental institutions (Martiniano, Machado Junior, Silva, & Silva, 2023).In this case, it is seeking to develop green pharmacy models, specifically in the production of soaps, candles, creams, and lotions and, specifically, in the fractionation of andiroba oil and bark tincture, as well as in the collection and germination of seeds to generate seedlings, as this plant has great potential as a medicinal plant (Chia et al., 2018).
Historically, herbal medicines have played an essential role in medical care, with extensive use documented in pharmacopeias until the 19th century (Cook, 1946).In particular, medicinal plants remain vital sources of medicine, even with the significant advances in medicine since the mid-twentieth century.Plants have become crucial for identifying chemical compounds that can fight disease, as highlighted by (Leandro, Jardim, & Gavilanes, 2017).In the Amazon, a diverse range of plant species has been used as a source of medicines, with andirobeiras (Carapa spp) being notable examples, noting that the genus Carapa, belonging to the Meliaceae is taxonomically classified under the order Sapindales, class Magnoliopsida, phylum Tracheophyta and kingdom Plantae, as described by (Santos et al., 2020).
According to the World Health Organization, medicinal plants are widely used by most people in developing countries for their health needs (Lima Melro et al., 2020) A recent study on the lipid profile of andiroba oil in Pará and Amazonas states identified five distinct metabolites: limonoids, acylglycerides, free fatty acids, steroids and phospholipids (Reis & Santos, 2021).In addition, carotenoids and traces of hydrocarbons can occasionally be detected.
Several ethnopharmacological studies have been used as a basis for scientific investigations and application of andiroba oil, including its repellent and larvicidal properties, insecticides (Miranda Júnior, Dolabela, Da Silva, Póvoa, & Maia, 2012) carrapaticidal, anthelmintic, healing and antiinflammatory properties (Araujo-lima et al., 2018).In this context, for the first time, we are investigating the bioeconomic potential related to the antiinflammatory activity of limonoids, a type of tetranortriterpenoid found in andiroba oil, which can inhibit cyclooxygenases 1 and 2. Notably, andiroba oil is preferred for topical use, and COX-1 inhibition is a crucial research target, as it could lead to the development of therapeutic agents with improved safety profiles.COX-1, like COX-2, also promotes the production of thromboxane A2, which is a pro-aggregate substance, acting on platelet aggregation, controlled, has relevant importance for life, and is part of the first stage of the hemostasis process, controlling hemorrhagic processes (Budsberg, 2009).
Blocking mainly COX 1 in platelets and the vascular endothelium promotes the reduction of thromboxane A2, reducing the thrombogenic effect (Armstrong et al., 2011).
During this action, COX-2 present in the endothelium, if it is not inhibited or if it is little inhibited, will continue to produce PGI2, which is an anti-aggregant substance, extending this effect, similar to the effect caused by acetylsalicylic acid (Costa-Tort et al., 2021).Thus, it can be inferred that the action of the limonoids present in andiroba oil has the potential to inhibit COX-1.
Selective or highly potent COX-1 inhibitors (Cingolani et al., 2017)have the potential to create more effective or targeted treatments for various diseases, including peripheral circulation disorders such as phlebitis.Phlebitis usually occurs in superficial veins close to the skin, usually in the arms and legs, and is commonly associated with inflammation of the veins and arteries.Andiroba oil has a wide range of traditional uses, including treating muscle contusions, dislocations, tendonitis, dermatitis and sprains.It also relieves symptoms associated with fever, malaria, parasitic infections, insect bites, arthritis, rheumatism and pain (Leandro et al., 2017).

OBTAINING THE MOLECULES TO BE ANALYZED
The metabolic concentrate rich in limonoids was obtained from the oil of C. guianensis until they were processed.Sisgen registration: AF60078.

OBTAINING THE EXTRACTS WITH ULTRASOUND-ASSISTED SOLVENTS
The seeds (200g) were boiled for 20 minutes in water at 100°C in a closed 5L stainless steel container under a constant pressure system.After boiling, the seeds were dehulled and the homogenized and crushed grains were transferred to a 500 mL conical glass flask.The extraction was carried out in a UNIQUE ultrasonic bath, model USC -1800, with a frequency of 40 KHz and a power of 155 Watts RMS, maintained at a temperature of 50 °C for 30 minutes.
The solvent used was analytical grade n-hexane.A volume of 200 mL of the solvent was added to 60 g of biomass to carry out the hexane extraction (3x).The hexane extract was filtered and the remaining biomass was used for other purposes.After the evaporation of the solvents, the dry extract was quantified.

DERIVATIZATION OF THE SAMPLES
The samples were derivatized with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) to select the free molecules that had not been hydrolyzed and the others with free hydroxyls.Two samples were used, with 10 mg of oil added to each 2 ml conical tube, followed by 50 μL of the derivatizer.The tubes were kept in an ultrasonic bath for 20 minutes at 30 °C.The solvent was evaporated at 35° C / 5 psi / 20 min, and 500 μl of the mixture n-hexane: CH2 Cl2 in a ratio of 1: 1 (v / v) were added to the tubes respectively.They were shaken for 10 seconds, and the contents were transferred to 2 mL glass vials with caps and septum and submitted for GC/MS analysis.x retention time.The lipid concentration was calculated by normalizing the peak area and validating the retention times.

RESULTS AND DISCUSSIONS
Andiroba oil has a group of substances belonging to the triterpenes, specifically the tetranortripernoids, known as limonoids, present in the unsaponifiable fraction.(Roy & Saraf, 2006).This fraction also contains steroid structures and can be observed through the lipidomic profile, where the fatty acids are presented in the form of methyl esters and for this reason are eluted more quickly (green circle), before the tetranortripenoids and steroids (orange circle), Figure 1.11 circulatory inflammation (varicose veins and phlebitis) and strains (sprains, strains, insect bites and knocks).However, many uses are not limited to these symptoms but to inflammation in general, including infectious inflammation (Rodrigues et al., 2023).For this reason, andiroba oil is often mixed with copaiba oil, which has a high antimicrobial and parasitic potential (Dhorm Pimentel de Moraes, Tavares, Soares Rocha, de Paula, & Giorgio, 2018) But its use is little or never associated with the infectious aspect.In general, articles, theses and other scientific works don't focus on such specific points, describing general points, and it is for this reason that this work highlights such relevant points involving the potential of this oil, whether singular or associated.It can be seen that between 2004 and 2024 there is a growing trend in research into andiroba oil, mainly due to its anti-inflammatory properties, Figure 4.This demonstrates the demand for and interest in this oil due to its biological aspect in cosmetology, health, pharmacy, insect repellency and other activities that border on the economic aspect.

Figure 4
Publications on Andiroba oil The sources of natural products with antiinflammatory potential have a more significant impact because this type of symptom is associated with body pain.In this sense, the search for active ingredients in plants is based on the scientific aspects related to easily accessible plants and the high productivity of their active ingredients.It can be seen that the countries involved in the use of plants to combat diseases are spread across Europe, Asia, and the Americas, and Brazil stands out in terms of publications related to andiroba (Figure 5).

Highlighting scientific publications on andiroba oil
There are 154 documents published by Brazil The study of andiroba oil is different because the keywords used are intensely related to "andiroba", "Meliaceae" and "Carapa guianensis".This is divided into the three most commonly used keywords, more recently, as of 2018, research has been directed towards the search for unsaturated fatty acids, w-3, w-9 and w-9 and it is in this aspect that andiroba oil has been explored.However, "emulsification" has also been explored due to the oil's high emulsification potential and the search for "limonoids", the most important target substances from the point of view of antiinflammatory activity.Figure 6 shows the keywords described by the authors in a search of 360 scientific articles on the Scopus database.
The most studied species in Brazil is C. guianensis Aubl, found in the northern states of Pará, Amazonas, Acre, Roraima, Rondônia and Amapá.It also occurs in the states of Maranhão, Mato Grosso and Bahia.

Figure 6
Bibliometrics with keywords and trends It can be seen that on the occurrence map, the area delimited by the green line represents the northern states of Brazil, including the Amazon rainforest, with occurrence points.In the states outside the northern region, the occurrence points are represented by yellow dots Figure 7. 14 In this sense, the C. guianensis species has great potential for application and is strongly related to sustainable use by Amazonian peoples, including riverside communities.By hand, the oil is obtained by collecting the seeds, cleaning them, cooking them in water at 100º C until the kernel mass softens, resting them until they reach room temperature, between 28-32o C, removing the kernels manually with homemade utensils, and kneading the cooked kernels until they form a cake.This cake is left to rest in the shade or in the sun to release the oil, which is collected, processed and stored in jars with lids.(Santos & Salgado, 2020).In general, this oil is considered crude, or more modernly extra virgin oil.When the community or agro-family processes the seeds in large quantities, the excess oil is sent to oil marketing companies, cosmetics companies or fractionation companies for resale.This last aspect has a wide reach among Amazonian populations, as it is when it is purchased for external use to combat many inflammations.
In the industrial extraction of andiroba oil, factories use some more specific steps, such as cleaning the kernels, weighing, drying, pre-cooking and pressing, filtering or centrifuging, pumping the oil, packaging, storage and distribution.In some cases, the waste is also treated.
Some of the stages are replaced, and this depends on the size of the factory and its technological conditions.
When it comes to commercial oil, quality control is essential and chemical analysis must accurately express the composition of the oil, especially the substances related to the active ingredient.In this case, andiroba oil was subjected to lipidomic analysis using the GC/MS technique after derivatization to integrate all the lipid substances present in the oil.This analysis revealed the presence of a diverse range of fatty acids, steroids and limonoids.Identifying these compounds, particularly the limonoids, is very promising for the development of sustainable applications aimed at generating antiinflammatory products.The four limonoids identified in andiroba oil vary in their presence and concentration in the samples analyzed.It should be noted that gedunin is common in andiroba oil, although it can appear in a deacetylated form, as shown in Table 1.However, it can be detected, usually in residual concentrations, as observed in (Araujo-lima et al., 2018) e (Nascimento et al., 2019).
The search for natural alternatives to combat skin inflammation has been intensified in recent years in our research group, with studies carried out on Amazonian oilseeds and their effects on the enzymes Cyclooxygenases (COXs) and Lipoxynagenases (LOXs), which act in the biotransformation of arachidonic acid, an important cell regulator.(Wang et al., 2021).In this flow of the inflammatory chain, the mechanism of action of limonoids from andiroba, acting as NSAIDs, is presented as a candidate for inactivating these enzymes and making andiroba oil a promising source for generating products with high peripheral anti-inflammatory efficiency.

CONCLUSION
The study emphasizes the importance of andiroba oil in the bioeconomy and the role of riverside communities in using forest resources sustainably.The analysis of andiroba oil shows the wealth of valuable compounds and describes that the chemical composition of andiroba oil can vary with seasonality and the place of collection.Due to differences in the oil extraction process, which may also be associated with seasonality or the place of collection, there is always a variation in composition, which is reflected in the presence or absence of substances, 16 generally observed that the active antiinflammatory principles are found in the unsaponifiable fraction, and the application of in silico studies has emerged as an alternative to classify their effects on cyclooxygenase (COX) enzymes.
seeds.The Andirobeiras (C guianensis) seeds were sent from INPA (National Institute for Amazonian Research) Adolph Ducke Park Reserve, in Manaus -AM (OA1); Mancio Lima in Acre (OA2) and Forestry station in Manaus-Am (OA3).The seeds were kept frozen at -20 o C ANALYSIS OF TARGET SAMPLES BY GC-MSMass spectrometry analyses were carried out using a Thermo Scientific Trace 1300 gas chromatograph (GC) coupled to an AI 1310 MS-ISQ Single Quadrupole autosampler mass spectrometer equipped with a ZB-5HT capillary column (30m x 0.25mm x 0.1μm), using helium gas as a carrier at a flow rate of 1mL/min.Sample injection of 1.0 μL in Splitless mode.The injector ran at 220 °C and the oven temperature adjustment started at 50 °C to 200 °C (8 °C / min.),remaining for 1 min, rising to 300 °C (15 °C / min.),being maintained for 5 min, rising again to 350 °C (15 °C / min.)and remaining for a further 9 minutes.The MS-ISQ was operated with the interface at 280 °C, the ionization source at 280 °C, the mass range (40-1000 Da) with 1 scan/s and electron ionization at 70 eV.The substances were identified by comparing the mass spectra with those of the commercial libraries NIST2011, WILEY2009, FAMES2011

Figure 1
Figure 1Chromatogram of the lipid profile of andiroba oil

Figure 2
Figure 2Bound lipids present in vegetable oils

Figure 3
Figure 3Triterpenes linked to sugars, forming saponins