Phytochemical characterization of aqueous and ethanol extracts from lime and orange rind in Guerrero, Mexico

doi:10.21203/rs.3.rs-4877550/v1

Background The phytochemicals that include both primary and secondary metabolites of the plants are of great interest in a variety of sectors, including the agricultural, pharmaceutical and cosmetic. Recently, it has been recognized that SMs could be used as a basis to develop biopesticides. The objective of this research was to identify and quantify compounds present in aqueous and ethanolic extracts from orange and lime rind from the state of Guerrero, Mexico.

Methods The objective of this study was to characterize aqueous and ethanol extracts of lime and orange rind. Lime and orange peel were collected and dried in the sun at room temperature. For characterization of plant extracts was it was done through gas chromatography-mass spectrometry (GC/MS) in an Agilent Technologies 7820A equipment, with mass selective detector (MSD, Agilent Technologies 5975), operated in the mode of complete radiofrequencies scan (full scan) in splitless mode, with an injection volume of 1 µL of sample.

Results The results from this study broaden the existing knowledge about the abundant phytochemical composition of aqueous and ethanol extracts of orange and lime. The aqueous extract of orange presented a dark brown color and the presence of 10 chemical compounds, of which D-limonene stood out as one of the predominant ones with a concentration of 95.66%, with a retention time of 4.1 min, followed by citric acid with 1.11%.

Conclusions This study represents a significant advance in the characterization and comprehension of orange and lime extracts, providing a solid base for future studies and practical applications in various scientific and commercial fields. The outstanding compounds in the extracts are D-limonene and citric acid. The continuous exploration of these compounds and their interactions promises continuing to drive discoveries that benefit both agriculture and public health globally.

Extracts

Phytochemical

Metabolites

Plants

Conventional agriculture (CA) is essential for food production globally since it provides most of the foods that people consume daily. This type of agriculture is characterized by the intensive use of chemical products, such as pesticides and fertilizers, as well as by the use of high-yield varieties, monocrops, genetically modified organisms, heavy machinery, intensive mechanization, and irrigation systems (Willer and Lernoud 2017; Le Campion et al. 2020). Although this model is efficient in terms of production, it presents important sustainability and environmental impact problems, among which some that stand out are air, soil and water pollution, due to the use of chemical pesticides that can affect local biodiversity, generate human health problems, contribute to climate change, and resistance problems; for example, for the case of weeds, in the year 2022, 350 cases of weed resistance to glyphosate were reported, and in addition multiple resistance was found in 23 species of weeds in 17 countries around the world (Arispe-Vázquez et al. 2023). For the case of insects, in the year 2021 it was informed that Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae) was already resistant to 33 active ingredients in different parts of the world (Cerna-Chávez et al. 2022). There are also monocrops, which in many cases increase the vulnerability of crops in the presence of pests and diseases, with the tendency to generate a continuous dependency on pesticides and fertilizers.

From a perspective of sustainability, CA suggests worries due to its intensive use of non-renewable resources, such as fossil fuels and energy for fertilizer production, while water consumption in irrigation systems can be unsustainable in regions prone to drought. These problems have led to a growing interest in more sustainable alternatives such as organic agriculture, agroecology, and other approaches that promote agricultural practices that are less dependent on chemical pesticides and more respectful with the environment and human health in the long term.

In the 2023 cycle, the surfaces sown in Mexico of lime (Citrus × limon L. Burm. f.) and orange (Citrus × sinensis (L.) Osbeck) were 222,643.40 and 353,609.46 ha, with a production of 3,239,914.70 and 4,942,658.65 million t, respectively. The value of lime and orange production was $MX 50,901,593.8. During that same cycle, the state of Guerrero had a production of Mexican lime and orange of 71,560.04 and 5,987.43 t, respectively (SIAP 2024). Concerning the state of Guerrero during the same cycle, the specific production was 71,560.04 t of Mexican lime, and these data highlight the significant contribution of the state of Guerrero to the national production of Mexican lime, although its orange production is relatively lower in comparison to other producing states in Mexico.

The phytochemicals that include both primary and secondary metabolites of the plants are of great interest in a variety of sectors, including the agricultural, pharmaceutical and cosmetic (Elshafie et al. 2023; Reshi et al. 2023). Primary metabolites are essential for the growth and development of plants, while secondary metabolites (SMs), also known as specialized metabolites, carry out crucial roles in the adaptation of plants to environmental stress and in their defense against predators and pathogens (Kroymann 2011; Fernie and Pichersky 2015; Yang et al. 2018; Isah, 2019; Pang et al. 2021; Fazili et al. 2022; Ochatt et al. 2022; Jeyasri et al. 2023; Salam et al. 2023; Sugiyama 2023); these SMs are produced in small amounts yet they are extremely important due to their bioactive properties.

The SMs can be classified into several categories, such as: phenols, terpenes, steroids, alkaloids and flavonoids, each with specific properties that can benefit both agriculture and other industries (Guerriero et al. 2018; Kessler and Kalske 2018). The current knowledge about how to obtain plants with high concentrations of these metabolites is vital to advance in agricultural and horticultural techniques that use economic and sustainable biomass (Selwal et al. 2023). In addition to their potential as biopesticides, SMs are also applied in agrochemicals, food additives, and the fragrance industry (Patil 2020). The production of SMs is influenced by a variety of factors both biotic and abiotic; among the biotic factors there are interactions with other pathogenic organisms, and on the other hand, among abiotic factors there is availability of water, light and temperature.

The type of fertilization applied and general agronomic management can also affect the production of SMs, and some studies have proven that certain fertilizers and agronomic practices can alter the concentration and composition of these compounds in plants. As a whole, understanding these factors and their adequate manipulation is essential to improve the production and quality of SMs, opening new opportunities for innovation in various fields. Therefore, the objective of this study was to characterize aqueous and ethanol extracts of lime and orange rind.

Study area and collection

The study was conducted in the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP)-Campo Experimental Iguala (CEIGUA) in Iguala de la Independencia, Guerreo, Mexico. Mexican lime Citrus aurantifolia Christm Swingle, was collected from an experimental plot of 2 ha in CEIGUA, which is located on geographic coordinates 18.337614 of latitude and − 99.511906 of longitude (Figs. 1 and 2). The orange rinds were obtained from juice shops in the central zone of the municipality of Iguala de la Independencia.

Figure 1. Geographic location of the study area. A = Mexico, B = State of Guerrero, C = Municipality of Iguala de la Independencia, D = Mexican lime plot

Processing of samples

The orange and lime rinds were sundried at room temperature, previous to their processing and analysis in the laboratory (Fig. 3). The rinds were ground until obtaining a fine powder in a blender and then the extraction of the compounds was conducted using 20 L jugs as containers. In each jug, 20 g of ground rind was placed and 1 L of tap water or ethanol alcohol was added, depending on the type of extraction. The jugs were placed outdoors at environmental temperature, taking advantage of the direct exposure to intense sunlight during a prolonged period; for the extraction with water, the jugs were left out for 53 days, while for the extraction with ethanol they were left only for 15 days for the extraction. During the entire time of extraction, regular monitoring was conducted to ensure that the conditions remained constant, and periodic observations were made of the level of liquid in the jugs (Toledo-Aguilar et al. 2024).

Filtration of the solutions was carried out at the end of the periods specified for each method of extraction in the Laboratory of Molecular Entomology and Alternatives for Pest Control of the Universidad Autónoma Agraria Antonio Narro. For the case of the extraction of phytochemical compounds through water, high-quality filters were used (Whatman No.1), which separated the rind residues from the liquid solution that contained the bioactive compounds. This step was repeated several times to ensure that no solid particles remained in the final solutions, which could interfere with later analyses. For the extraction through ethanol, a vacuum pump was used (Ulvac model DTC-22A) with filters (Whatman No.1), to eliminate any residual particle. Then, the ethanol extract was subjected to an evaporation process through a rotary evaporator, which operated at 150 rpm and 90°C, thus concentrating the compounds extracted. This procedure allowed obtaining a pure and concentrated extract, eliminating the ethanol alcohol from the final product. The use of the rotary evaporator (Yamato model VR300) allowed eliminating the solvent, preserving the integrity of the bioactive compounds extracted and avoiding their degradation from prolonged exposure. The extracts of lime and orange rinds were placed in amber glass containers to protect them from light and were stored in refrigeration at 5°C.

Figure 3. Peel placed outdoors at room temperature. A = Orange peel, B = Lime peel

Qualitative chemical analysis

The characterization of plant extracts was carried out in the Laboratory of Forest and Agricultural Health of INIFAP-Campo Experimental Pabellón (CEPAB). It was done through gas chromatography-mass spectrometry (GC/MS) in an Agilent Technologies 7820A equipment, with mass selective detector (MSD, Agilent Technologies 5975), operated in the mode of complete radiofrequencies scan (full scan) in splitless mode, with an injection volume of 1 µL of sample. The initial temperature slope was 50°C, followed by an increase of 10°C min^− 1 until 300°C, where it was kept for 1 min; then a post-run of 3 min at 300°C was added. The injection port was kept at 250°C and the source and quadruple were at 230°C and 150°C, respectively. The column used was HP-5MS 5% phenyl methyl silox: 30 m x 250 µm x 0.25 µm. High-purity helium was used ss carrying gas, with a flux of 1 mL min^− 1 with electron impact in ionization mode 70 eV. For the analysis of the aqueous extracts, the SPME-ID technique reported by Rivera-Dávila et al. (2022) was used. For the ethanol extracts, they were previously concentrated in a rotary evaporator at 50°C to a third of their volume; next, the ethanol extract was passed through micropore filters of 0.22 µm. After filtering, 1 µL of sample was injected for its analysis. The extractions were carried out by triplicate. The compounds were identified based on their retention indices and mass spectrums, using the W9N11.L database and a similarity index higher than 90%.

The results from this study broaden the existing knowledge about the abundant phytochemical composition of aqueous and ethanol extracts of orange and lime. The aqueous extract of orange presented a dark brown color and the presence of 10 chemical compounds, of which D-limonene stood out as one of the predominant ones with a concentration of 95.66%, with a retention time of 4.1 min, followed by citric acid with 1.11%. In this regard. It is important to highlight that in the aqueous extract of orange, there were other compounds such as: alpha-terpinene, linalool, palmitic acid, 4-terpineol, oleic acid, beta-pinene, 2-methoxy-4-vinylphenol and alpha-terpinolene at concentrations of ≤ 0.74%, which are classified within organic acids, terpenoid compounds, terpenic alcohols and phenols. Their concentrations and respective retention times and abundance are presented in Table 1, although the terpenes correspond to the largest compounds among SMs and have been widely studied due to their potential as antimicrobial, insecticide and weed control agents (Ninkuu). The ethanol extract of orange presented a dark yellow color (Fig. 4C, 5D) and 14 chemical compounds were observed, of which 6 were equal to those obtained from the aqueous extract of orange, although in higher concentrations, with the exception of D-limonene which reduced its concentration in 60.7% in the ethanol extract compared to the aqueous extract. In the ethanol extract of orange, 8 new compounds were found among which hexanedioic acid and caryophyllene stand out, which were observed at concentrations of 11.18% and 1.8%, respectively; the remaining 6 new compounds observed were present at concentrations of ≤ 0.80% (Table 1).

The aqueous extract of lime presented a reddish brown color and the presence of 11 chemical compounds was observed, their concentration being between 1.75 and 17.77%. The compounds that presented the highest concentrations were D-limonene and palmitic acid with 16.77 and 13.20%, respectively (Table 1, Fig. 5B). Meanwhile, the remaining 8 compounds showed a concentration of ≤ 9.51%. The ethanol extract of lime presented a dark orange color, and it was the extract where the highest number of chemical compounds was observed, with 25 from which only 7 were the same as those obtained in the aqueous extract of lime. Its concentration was between 0.15 and 36.94%. The compounds that presented the highest concentrations were citric acid with 36.94%, followed by oleic acid with 32.53% (Table 1, Fig. 5D). Likewise, it was seen that in the ethanol extract of lime the concentration of oleic acid, citric acid and caryophyllene increased, and the concentration of D-limonene, beta-bisabolen, 4-terpineol, alpha-farnesene, and caryophyllene decreased, compared to the aqueous extract.

Table 1

Components and concentration of the extracts under study
NE	NC	Compound**	Retention Time (min)	Concentration (%)
1	1	D-Limonene	4.147	95.66
	2	Citric acid	3.056	1.11
	3	Alpha-Terpinene	6.98	0.74
	4	Linalool	5.32	0.44
	5	Palmitic acid	16.36	0.42
	6	4-Terpineol	6.72	0.41
	7	Oleic acid	18.02	0.20
	8	Beta-Pinene	2.75	0.19
	9	2-methoxy-4-vinyl phenol	8.90	0.13
	10	Alpha-Terpinolene	5.15	0.12
2	1	D-Limonene	6.59	16.77
	2	Palmitic acid	23.39	13.20
	3	Camphene	12.70	9.51
	4	Oleic acid	25.05	9.19
	5	Beta-Bisaboleno	18.24	8.28
	6	Citric acid	1.53	5.77
	7	4-Terpineol	12.31	3.44
	8	Alpha-Farnesene	17.24	3.12
	9	Caryophyllene	17.06	2.99
	10	Tetradecanoic Acid	23.56	2.38
	11	9-Octadecanoic Acid-(z)-methyl E ester	24.61	1.75
3	1	D-Limonene	7.20	57.49
	2	Hexanedioic Acid	27.23	11.18
	3	Oleic acid	25.22	6.24
	4	Citric acid	3.97	5.82
	5	Palmitic acid	23.43	3.21
	6	Caryophyllene	18.24	1.80
	7	Alpha-Terpinolene	9.95	1.20
	8	β-Pinene	5.54	0.82
	9	β- Copaene	17.06	0.80
	10	α-Copaene	16.84	0.58
	11	9-Octadecanoic acid	21.25	0.51
	12	Delta-Cadinene	18.50	0.47
	13	Linoleic Acid	26.36	0.32
	14	β -Farnesene	17.54	0.25
4	1	Citric acid	4.50	36.94
	2	D-Limonene	5.10	5.16
	3	Gamma-Terpinene	5.45	1.35
	4	p-Cymene	5.89	0.83
	5	4-Terpineol	7.15	1.10
	6	Alpha-Terpineol	7.33	1.45
	7	Alpha-Myrcene	9.99	0.41
	8	Caryophyllene	10.43	3.69
	9	Alpha-Farnesene	10.86	0.84
	10	Beta-Bisabolene	11.34	4.06
	11	Lauric acid	12.04	1.13
	12	(+)-Aromadendrene	12.13	0.28
	13	Myristic acid	14.31	0.71
	14	Methyl-Palmitate	15.93	0.15
	15	Palmitic acid	16.54	10.16
	16	Limetin	16.84	1.54
	17	is-13-ac. Octadecanoic, methyl ester	17.63	0.19
	18	Linoleic acid	18.20	14.70
	19	Oleic acid	18.44	32.53
	20	Isopimpinellin	19.20	0.18
	21	Hexanedioic acid	20.25	3.57
	22	6-Octadecenoic acid	20.73	0.12
	23	Pentacosane	20.99	0.09
	24	Eicosane	22.47	0.15
	25	1,5,8-p-Mentatriene	22.90	0.64

NE = Extract number, 1 = aqueous orange extract, 2 = aqueous lime extract, 3 = ethanolic orange extract, 4 = ethanolic lime extract. NC = Compound number. **Predominant compound in the 4 extracts.

Masakazu et al. (1999) mention that D-limonene can be present at a concentration close to 95% in orange rinds, which agrees with what was seen in this study .The predominant chemical compounds were organic acids and terpenes, which are very common in natural extracts and essential oils; similarly, some authors mention factors that affect the percentage of active ingredients, such as geographic distribution, environmental conditions, temperature, rainfall, altitude, and sunlight hours (Hossain et al. 2014). Other authors mention that citruses have mainly phenolic compounds (flavonoids, phenolic acids and coumarins), terpenoids (limonoids and carotenoids), and pectins (Lado et al. 2018; Cebadera-Miranda et al. 2019; Lu et al. 2021).

Diverse studies have shown that D-limonene is a natural antioxidant and anti-inflammatory compound, and it has been studied for its potential not just as herbicide and organic insecticide (repellent), due to its chemical properties against weeds and insects. In this regard, Davidowski and DiMarco (2009) mention that orange oil has a considerable amount of limonene and has numerous applications, including as fuel in motors, a strong degreaser in cleaning applications, and a natural pesticide. Its biodegradable and less toxic properties compared to chemical herbicides make D-limonene an attractive and sustainable alternative. For example, studies have documented its ability to act as a natural repellent against mosquitos and other flying insects, highlighting its potential in pest control in a more ecological way (Michaelakis et al. 2009; Maia and Moore 2011; Pohlit et al. 2011; Giatropoulos et al. 2012).

D-limonene is recognized for its antioxidant potential, anti-inflammatory properties and anti-cancer properties and many health-promoting effects. (Akhavan-Mahdavi et al. 2022). These findings highlight the potential of orange and lime extracts as promising sources of bioactive compounds with applications in different industries such as health and agriculture. For example, the ethanol extract of orange presented great similarity in terms of color and viscosity to a commercial bioherbicide (Fig. 6) based on mullein, coconut oil, pine resin, puccinia fungus, and papain (as shown in the product’s label), so it is interesting to continue with the research of these extracts as biopesticides, among them as organic herbicides. Commercial and organic limonene products have been tested, applied in the field for weed management, attaining low but visible control effects on wide and narrow leaves (Fig. 7).

Figure 7. Effects of the application of commercial limonene products applied to weeds (ABC)

This study represents a significant advance in the characterization and comprehension of orange and lime extracts, providing a solid base for future studies and practical applications in various scientific and commercial fields. The outstanding compounds in the extracts are D-limonene and citric acid. The continuous exploration of these compounds and their interactions promises continuing to drive discoveries that benefit both agriculture and public health globally.

INIFAP Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias

CEIGUA Campo Experimental Iguala

CEPAB Campo Experimental Pabellón

CA Conventional agriculture

SMs Secondary metabolites

T Tons

ha Hectare

µL Microliter

L Liter

G Grams

°C Degrees Celsius

GC/MS Chromatography-mass spectrometry

NE Extract number

NC Component number

min Minutes

% Percentage

** Predominant compound in the 4 extracts

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

Data are available under reasoning demand.

Competing interests

The authors declare that they have no competing interests.

Funding

Fiscal funds - INIFAP project 1063336025.

Author contributions

Investigation JLAV, RTA, DHNC, MFV, LAFH; methodology JLAV, KVLR, LAFH, JFDN, SAS, JTS, MRV, AHJ, JMH, writing—original draft preparation JLAV, DACZ, JFDN, MRSV, JTS, writing—review and editing SAS, JMH, KVLR. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

The authors thank the staff involved in field activities.

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Phytochemical characterization of aqueous and ethanol extracts from lime and orange rind in Guerrero, Mexico

Status:

Version 1

Abstract

Figures

Background

Methods

Study area and collection

Processing of samples

Qualitative chemical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Version 1