CITRUS WASTE COMPOSITION AND BIOACTIVE COMPOUNDS: A

REVIEW

Degree project

by

DANIEL ALFREDO SALAZAR DÍAZ

Presented to the engineering faculty of Universidad de los Andes In the partial fulfillment of the requirements for the degree of CHEMICAL ENGINEERING

December 2020

Chemical Engineering department

i

Citrus waste composition and bioactive compounds: a review

Copyright © 2018 Daniel Alfredo Salazar Díaz.

ii

CITRUS WASTE COMPOSITION AND BIOACTIVE COMPOUNDS: A

REVIEW

Degree project

by

DANIEL ALFREDO SALAZAR DÍAZ

Presented to the engineering faculty of Universidad de los Andes In the partial fulfillment of the requirements for the degree of CHEMICAL ENGINEERING

Approved by:

Advisor, Rocio Sierra Ramirez, PhD. Co-advisor, Daniel David Durán Aranguren, M.Eng and M.Sc. Jury, Niyireth Alicia Porras Holguin, PhD. Department director, Andrés Gonzalez Barrios, PhD.

December 2020

Chemical Engineering department

iii

ABSTRACT

Citrus fruits are used in many industries around the world, obtaining normally peels, seeds and pulps as residues, but these have properties and characteristics that can be used, then it was made a systematic literature review with the objective to identified composition, bioactive compounds and the effectiveness of the extraction methods to removes them.

The literature composition identification starts with a general composition description, where is identified the percentage presence of total carbohydrates (pectin, cellulose and hemicellulose), lignin, protein, ashes and extractives in the residue, being total carbohydrates and extractives the components with the major presence found. After, it was made a description by ultimate analysis and proximate analysis. The ultimate analysis give information of the chemical composition, in this was identified that citrus residues are principally composed by carbon and oxygen. The proximate analysis give information about the compounds behavior in a thermochemical process and in this was found that citrus residues are mainly composed by volatile matter and fixed carbon. Finally, it is made a description of the chemical composition of the ashes, finding the high presence of calcium and potassium.

The second part of the review is focused on the bioactive compounds and the extraction methods, dividing the analysis in essential oils and phenolic content. For essential oils is determined the yields and individual compounds composition reached from different residues from literature information, being limonene the main component of them and the solid-liquid extraction with acetone or ethanol as solvents the technique recommended.

For phenolic content is identified the total content and the individual phenolic compounds

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from different citrus residues from literature information, finding hesperidin, naringin and narirutin as the main compounds by presence and the use of ultrasound assisted and solid- liquid extraction as the techniques recommended to increase this content in the extracts.

Finally, it is evaluated the antioxidant activity of the extracts, where are found similar results for grapefruit peel and orange peel and were recommended the same techniques as in phenolic content, corroborating a correlation between them.

Keywords: citrus residues, bioactive compounds, antioxidant activity, citrus composition.

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TABLE OF CONTENTS

ABSTRACT ...... iv Keywords: ...... v LIST OF TABLES ...... 2 1. INTRODUCTION...... 4 2. OBJECTIVES ...... 8 2.1 General ...... 8 2.2 Specifics...... 8 3. RESULTS AND DISCUSION ...... 9 3.1 Research methodology ...... 9 3.2 Composition ...... 10 3.2.1 General citrus residue composition ...... 11 3.2.2 Ultimate analysis ...... 13 3.2.3 Proximate analysis...... 14 3.2.4 Ash’s chemical composition ...... 15 3.3 Extraction of citrus waste bioactive compounds ...... 16 3.3.1 Essential oils extraction ...... 21 3.3.2 Essential oils composition...... 23 3.3.3 Phenolic content of citrus residues...... 29 3.3.4 Composition of individual phenolic compounds...... 34 3.3.4 Antioxidant activity of citrus residues ...... 38 3.3.5 IC50 Values ...... 41 3. CONCLUSIONS ...... 43 4. REFERENCES ...... 45

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LIST OF TABLES

Table 1. Citrus waste composition ...... 13 Table 2. Ultimate analysis ...... 14 Table 3. Proximate analysis ...... 15 Table 4. Ash's chemical composition ...... 16

Table A1. Essential oil yields ...... 55 Table A2. Essential oils composition...... 56 Table A3. Total phenolic content (TPC) and total flavonoid content (TFC)...... 60 Table A4. Individual phenolic compounds...... 62 Table A5. Antioxidant activity of citrus residues...... 66 Table A6. IC50 values ...... 67

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LIST OF FIGURES

Figure 1. Research procedure ...... 10

Figure 2. Comparison of yields of extraction methods for essential oils ...... 22

Figure 3. Limonene percentage…………………………………………………………24

Figure 4. Comparison of extraction ...... 24

Figure 5. Percentage presence of high………………………………………………….26

Figure 6. Percentage presence of low ...... 26

Figure 7. Percentage of high presence………………………………………………….27

Figure 8. Presence of low presence ...... 27

Figure 9. Percentage of sesquiterpenes………………………………………………....28

Figure 10. Percentage of oxygenated ...... 28

Figure 11. Percentage of other oxygenated……………………………………………..29

Figure 12. Percentage of other compounds ...... 29

Figure 13. Comparison of phenolic content in different residues ...... 31

Figure 14. Comparison of extraction techniques for phenolic compounds in orange peels

...... 32

Figure 15. Presence found of individual flavanones in citrus residues ...... 34

Figure 16. Presence of flavanones……………………………………………………...37

Figure 17. Presence of other flavonoids ...... 37

Figure 18. Presence found of other phenolic compounds in citrus residues ...... 38

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INTRODUCTION

Citrus is the most widely produced fruit, including a group of several species and growing in more than 80 countries. Citrus fruits have an important role worldwide in providing nutrients and are used in medicine. Into this fruit family, oranges, mandarins, grapefruits

, pummelos, lemons, acid limes, among others can be found (Ladaniya, 2008). There are different varieties of the main citrus fruits like ponkan which is a mandarin variety. Also, there are citrus fruits that come from a specific of a region like Yuzu that is a citrus of eastern Asia. Additionally, clementine and mandora are hybrids between mandarin and orange.

At world level, the citrus production is about of 120 million tonnes, taking as residue the peel, seeds and pulp. These residues generally are composed by water, sugars, pectin, fiber, organic acids, amino acids, minerals, essential oils, flavonoids and vitamins.

Through which the residues can have an added value, due to the potential to elaborate commercial products valued in industries like the pharmaceutic, cosmetic, and food industry (Peñaranda et al., 2017). In its composition, bioactive compounds found in the extractives can be extracted using polar solvents (ethanol-water) and non-polar solvents

(Ortiz-Sanchez et al., 2020). Between them, valuable components like essential oils and phenolic compounds can be found.

Essential oils are a combination of terpenes that are generated from plants as secondary metabolites, then those molecules are not critical to their functioning. In orange, essential oils are located between the crust and a white section named albedo. The composition of

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the essential oil depends of factors such as weather, season and agricultural land (Ayala et al., 2017). The uses of essential oils are very varied, those include the uses in food industry, medicine, and cosmetic industry, among others. Some of the uses are: as flavouring in food industry, as diuretic in medicine, as essence for perfume, as pathogen inhibitor, as insect controller. The essential oils have different properties and effects depending to the fruit used. For instance, it has been observed that lemon essential oils could decrease the stress-induced behavioural acting on the sleep disorder. The difference in these uses and properties is due to a difference in the essential oils composition (Palazzolo et al., 2013).

The major chemical compound in the essential oils is limonene, which is a monoterpene with high presence in citrus peels, contributing to their smell (Kumar Joshi et al., 2019).

Polyphenols are defined as secondary plant metabolites contributing to fruit organoleptic and nutritive quality in terms of aroma, taste, flavor and color. Phenolic compounds include a wide variety of molecules that contain one or more hydroxyl groups in its aromatic rings. These compounds have been widely detected in the citrus residues, particularly the peels have been described as a great source of natural flavonoids

(Ferrentino et al., 2016). One of the most common polyphenols are flavonoids. These compounds can have healthy related properties with anti-cancer, antiviral and, anti- inflammatory activities, reduce capillary fragility and restrict human platelet aggregation

(Rafiq et al., 2018). The residue extracts, especially by its phenolic content, can be valorized due to its antioxidant activity or capacity. This refers to a substance capacity to inhibit the oxidative degradation (Londoño, n.d.). An antioxidant is any substance at low

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concentrations compared to an oxidizable substrate that significantly delays or prevents the oxidation of that substrate (Sehwag and Das, 2013).

To extract the essential oils and phenolic compounds, there are different methods that can be used. These methods use different principles like a solvent-sample interaction, distillation, enzymatic hydrolysis, among others. There are some variations to the extraction methods and aiming to extract the highest quantities of the target compounds.

The reuse and valorisation of citrus residues is of interest in Colombia due to the citrus production in the country. Colombia can be considered as citrus producer due to FAO reports, showing between 2008-2015 that the total production was higher than 190 thousand tonnes each year, exporting in average 8.7 thousand tonnes per year in that period, indicating the importance of these fruits for the country, being orange the most important citrus in terms of production and exportation, which leaves behind a considerable amount of residues. In Colombia citrus fruits are also imported. Between

2008-2015 the citrus imports were in average 15.17 thousand tonnes, where tangerines were the highest citrus imported in average 7.26 thousand tonnes per year.(FAO, 2016.)

For that reason, this work intends to review the compositional analysis and the bioactive compounds that can be extracted from citrus waste, with the objective to deepen the knowledge about this waste and the ways to valorise it by the extractives, then it is of interest to identify the characteristics of the residues, analysing the variability in the composition. In the same way is of interest to quantify and discriminate the extractive compounds, because each one has properties and characteristics that can be used in other

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industries and products, then adding value to the residue. Also is important to analyse the methods and conditions to make the extraction, because can give information about where must be directed the efforts to obtain these. A pertinent method to effectuate the research is through a systematic review methodology, because for this method it is necessary to have a clear question or objective, which was expressed previously and due to the research needs a search strategy, defining parameters and filters to select the information to use.

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1. OBJECTIVES

2.1 General

Develop a systematic literature review on citrus residues to identify main components, bioactive compounds and most effective extraction techniques.

2.2 Specifics

1. Determine the main components in citrus residues, as reported in the literature and

if there are important differences in a residue in comparison to another.

2. Identify among the extraction methods reported in the literature those that are most

effective in the removal of extractives compounds.

3. Determine into the extractives, which bioactive compounds are present.

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3. RESULTS AND DISCUSION

3.1 Research methodology

The objective of this review is to recollect information about citrus waste composition and valorization by extraction of bioactive compounds. The information was gathered using databases such as Science Direct, Springer link, Scopus, Wiley, Taylor and Francis and

ACS. These databases were selected due to their focus in engineering and science topics.

Articles that include valuable data about citrus residues and its valorization were considered. For that, the following keywords were used: ("citrus" OR "orange) AND

("waste" OR "residues") AND ("valorization" OR "bio-refineries").

The first filter was used to select research articles (which excluded books, conferences, presentations and reviews) that were published between 2005-2021. The second filter was done using the titles of the articles to identify information that could be directly related with the topic of this review. After that duplicate articles were removed. The third filter was done by screening the information provided by the abstracts. After that, half of the documents selected in the previous filter were dismissed. The procedure followed is based on is based on the PRISMA network for systematic reviews and meta-analysis (Mardani et al., 2019; Teigiserova et al., 2019) and is shown in Figure 1.

After this selection process, articles were classified into those that give information about citrus residues composition and in the other hand, those that give information about extraction of bioactive compounds, including the antioxidant activity of the extracts.

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Figure 1. Research procedure 3.2 Composition

The composition of citrus waste has been reported by several authors as way to identify possible valorization routes. In Table 1 a summary of the data reported in the literature is presented. In this table it can be noticed that the two main quantification methods employed are those proposed by NREL (National Renewable Energy Laboratory) and

AOAC (The Association of Official Analytical Chemists). It must be noted that both methods employ a different approach on how composition is measured. For example,

AOAC methods are based on digestible fiber measurements, and total carbohydrates are

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usually calculated by difference from the other compounds measured (which could depend on the values reported for each author) which makes this method semi quantitative. In the case of NREL and other NC (Non-Conventional) methods, measurements are focused on quantifying the specific structural polysaccharides for their use in biomass valorization.

Additionally, these methods differ in terms on their approach to the measurement of extractives. Whilst in the NREL water and ethanol are used as solvents, in the AOAC methods only non-polar solvents (e. g. petroleum ether).

3.2.1 General citrus residue composition

The results presented in Table 1 make evident that citrus wastes present considerable amounts of cellulose, hemicellulose, pectin, and extractives, which have been usually employed as the main fractions for waste valorization. Cellulose is found that the range for orange peel is of 9.2-30.17% of the composition. An outlier of 30.17% of cellulose is reported by (Ortiz-Sanchez et al., 2020), which can be attributed to the semi-quantitative measurement of holocellulose performed in that work using a combination of TAPPI and

NREL methods. For the lemon waste results reported (Masmoudi et al., 2008) the content of cellulose (11.97%) is lower than all the results reported for this measure for orange peel, except by the result obtained of 9.2% (Tsouko et al., 2020), but this result may be due that the free sugars content are taken as the only one extractive. The results from lemon waste reported (Pectin, protein and ash) are similar to the results reported for orange peels. For lignin, the results give a range of 1%-7%. For that, the data presented by

(Jiménez-Castro et al., 2020) of 14.77% is an outlier, that could be due to the report includes soluble and insoluble lignin, then there are possible variations during the

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quantification of those. Pectin is one of the most valuable components present in these residues. This is studied in different investigations and has an especial value for food industry. The composition of this component is in a range of 18%-23%. There is an atypical value of 11.18% (Ortiz-Sanchez et al., 2020) that is the lowest amount found and is out of the range determined, where a possible cause of that difference is the variation in the methods used to quantify the pectin, because these methods are not standardized. The ash content is the substance present in the lowest amounts with a range of 3.19-3.99%, that is a short range, indicating a similarity between the residues and is important due to the macro-elements presented (calcium, potassium, etc.) that will be analyzed later.

Finally, Extractive compounds refer to the substances that extracted through a sequential extraction with water and ethanol. The extractive content in orange peel is a range of 20.94 to 50% of the solid weight of the sample. It can be noticed that the inclusion of pulp and seeds to the evaluation of orange waste was reported only by (de la Torre et al., 2017) which is not too different to the composition of orange peel alone. In the data collected for orange peel in Table 1 an atypical value of 50.0±.01% of extractives is present

(Tsouko et al., 2020) (the highest value found), that could be attributed to the addition of free sugars into the value of total extractives. Another atypical value of extractives content was reported by (Jiménez-Castro et al., 2020), this value is of 5.56% that is by far the lowest measure for extractives, that can be explained because the article did not report a

13.41% of the samples composition.

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Table 1. Citrus waste composition

Total Residue Cellulose Hemicellulose Lignin Pectin Extractives Protein Ash Total Method Reference carbohydrates OP 57.42* 14.01±0.07 21.310±0.04 1.03±0.01 22.10±0.02 31,36 6.25±0.03 3.94±0.01 100 NC (Li et al., 2014) (de la Torre et al., OW** 51.5* 18.6±0.1 14.3±0.2 6.5±0.6 18.6±1.9 38.0±0.5 NR 3.7±0.1 99.7 NREL 2017) (Ortiz-Sanchez et al., OP 50.7* 30.17±0.50 9.35±4.36 5.07±2.14 11.18±0.16 30.55±1.07 4.86±0.20 3.61±0.20 94.79 NREL/TAPPI 2020) OP 51.5* 18.6±0.1 14.3±0.2 6.5±0.6 18.6±1.9 38.0±0.5 NR 3.7±0.1 99.7 NREL (Senit et al., 2019) (Lachos-Perez et al., OP 19.2* NR NR 16.8 19.62 20,94 6.85±0.00 3.99±0.04 67.78 NREL 2020) OP 36.6* 9.2±0.21 5.4±0.19 1.2±0.05 22.0±0.02 50.0±2.01 6.6±0.30 3.3±0.20 97.7 NREL (Tsouko et al., 2020) (Barrales et al., OP 82.8 NR NR NR NR NR 7.53±0.01 3.90±0.03 94.23 AOAC 2018) (Barrales et al., OP 84.2 NR NR NR NR NR 7.65±0.01 3.96±0.03 95.81 AOAC 2018) (Jiménez-Castro et OP 56.33* 21.23±1.20 12.08±0.42 14.77±1.75 23.02±1.40 5.56±0.37 6.74±0.20 3.19±0.25 86.59 NREL al., 2020) OP 52.79* 18.02* 16.17* 6.47 18.6 NR 3.72 62.98 NREL (Velasco et al., 2017) (Masmoudi et al., LW** 53.77+-0.03 11.97±0.90 NR NR 21.56±0.94 NR 7.88±0.70 3.24±0.07 98.42 AOAC 2008)

*Calculated from available data **Includes peel, pulp and the seeds OP, OW and LW as abbreviations of orange peel, orange waste and lemon waste respectively

3.2.2 Ultimate analysis

Table 2 shows the ultimate analysis of orange peel, lemon peel and ponkan peel. The information collected shows that oxygen, carbon and hydrogen are the citrus wastes main chemical components, these components represent about a 97% of the sample. A nitrogen composition is also reported in all the articles selected, but this quantity is near to the 1%, meaning that the nitrogen content is not abundant. Even though all authors indicate the presence of sulfur, this amount of this element is not higher than 0.586% (Kwon et al.,

2019). In the cases were Sulphur contents were not reported the amount of sulfur was too low to be detected. The elemental composition obtained for orange peel is similar to the data reported to lemon peel (Volpe et al., 2015). The results of ponkan peel (da Silva et al., 2019) differ from the other citrus by the carbon’s content, due to this content in the other residues was found in a range of 42.7-50.64%, while in ponkan peel was 39.72%, then is noticed a considerable difference.

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Table 2. Ultimate analysis

Residue Carbon Hydrogen Nitrogen Oxygen Sulfur Reference OP 42.7 6.4 1.0 47.6 NR (Alvarez et al., 2018) OP 44.53 6.243 0.498 48.143 0.586 (Kwon et al., 2019) OP 42 6 1 51 NR (Lachos-Perez et al., 2020) OP 44.22±0.27 5.90±0.06 0.76±0.06 46.33±0.41 0.11±0.01 (Siles et al., 2016) OP 50.64 5.57 1.11 42.59 0.09 (Volpe et al., 2015) LP 50.22 5.56 1.31 42.84 0.08 (Volpe et al., 2015) PP 39.72±0.01 5.89±0.01 1.64±0.06 50.55±0.06 NR (da Silva et al., 2019) OP, PP and LP as abbreviations of orange peel, ponkan peel and lemon peel respectively

3.2.3 Proximate analysis.

The proximate analysis (Table 3) is used to understand the behaviour of gaseous, liquid and solid components that can be obtained in a thermochemical process (da Silva et al.,

2019). It is a measure of the gross composition of biomass. Information was gathered about orange waste (OW), ponkan peel (PP), orange peel (OP) and lemon peel (LP). The results of volatile matter are similar in all the residues, with the lowest value being 74.1% (Alvarez et al., 2018) for orange waste and the highest 80.87% for ponkan peel (da Silva et al., 2019).

Also, there is not an important difference in the values reported for fixed carbon in the different peel residues. Comparing these data for the peels, fixed carbon content in orange peel is found in a range of 16.82-21.41% and the results found for lemon peel (19.93%) and ponkan peel (16.93) are into this range. The highest result for fixed carbon is obtained from orange waste. This residue includes peel, pulp and the pips, then the pulp and pips can give to the residue an increase on the amount of fixed carbon. Another interesting point is that the value reported of moisture for ponkan peel 7.80% (da Silva et al., 2019) is the highest among the other results.

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Some papers reported the high heating value of citrus waste, specifically for orange waste

(19.4 MJ kg−1) (Alvarez et al., 2018) and for orange peel (18.28 MJ kg−1) (da Silva et al.,

2019), with the highest values for orange waste including pulp and seeds. This is supported by (Volpe et al., 2015) that attributes an increasing in the HHV by the presence of the seed. The calculation for this parameter could be also done through the information obtained of fixed carbon and volatile matter (Ortiz-Sanchez et al., 2020) since the HHV is important to identify the available energy per unit mass. In comparison, citrus waste has a HHV higher than the values that have been previously reported for another residues or wastes like rice husk (12.8-14.9 MJ kg−1), corn stalks (17.82 MJ kg−1), sugar beet leaves

(17.72 MJ kg−1), hardwood waste (17.0 MJ kg−1) and paper waste (16.2 MJ kg−1). Also, it was found that the HHV of citrus waste is lower than the reported for polyethylene

(32.0-45.9 MJ kg−1) and tires (29.0-36.5 MJ kg−1) and present similar to the reported for coffee waste (19.8 MJ kg−1) (Boumanchar et al., 2019; Seitarides et al., 2008).

Table 3. Proximate analysis

Residue Volatile matter Fixed carbon Ash Moisture HHV (MJ kg-1) Reference OW** 74.1 23.6 2.3 1.5 19.4 (Alvarez et al., 2018) PP 80.87 16.93 2.20 7.80 NR (da Silva et al., 2019) OP 80.49±0.5 16.82±0.5 2.69±0.01 NR 18.28 (Ortiz-Sanchez et al., 2020) OP 75.90 21.47 2.63 3.06 NR (Volpe et al., 2015) LP 77.22 19.93 2.85 2.67 NR (Volpe et al., 2015) **Includes peel, pulp and the seeds

OP, PP, LP and OW as abbreviations of orange peel, ponkan peel lemon peel and orange waste respectively.

3.2.4 Ash’s chemical composition

Table 4 presents the results for the chemical composition of the ash. The information collected shows results for ponkan peel (PP), orange peel (OP) and lemon peel (LP). It

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can be observed that the main chemicals are potassium and calcium, in a range of 24.35-

33-15% and 22.13-29.47%, except for the data reported by (Volpe et al., 2015) that does not mention the calcium content. The presence of magnesium is evidenced in all the articles.

There are other elements reported, but their contents are in extremely low concentrations.

Some of these are Si, Al, Fe and Ti, having a presence lower than 1% of the ash. Also, it has been reported a low presence of Ba, Sr, Rb and Ce (da Silva et al., 2019) those are uncommon elements for this type of waste, especially Sr and Ce due to the radioactive isotopes that these elements have.

Table 4. Ash's chemical composition

Residue K Ca Mg P Na Reference PP* 33.1546 27.2609 6.4689 3.0071 1.3008 (da Silva et al., 2019) OP 30.9 29.47 4.78 8.34 1.98 (Alvarez et al., 2018) LP 24.35 22.13 3.60 NR 1.26 (Volpe et al., 2015) OP 25.86 NR 2.97 NR 1.04 (Volpe et al., 2015) OP, PP and LP as abbreviations of orange peel, ponkan peel and lemon peel respectively

3.3 Extraction of citrus waste bioactive compounds

Different methods were found to effectuate the extraction bioactive compounds of citrus waste. These methods are solid-liquid (SLE), microwave assisted (MAE), ultrasound assisted (UAE), enzymatic assisted (EAE), hydro-distillation (HD), steam distillation

(SD), solvent free microwave (SF), cold pressing (CP), supercritical carbon dioxide(SC-

CO2), subcritical water (SW), microwave assisted hydro-distillation (MAHD), pressurized liquid (PLE), accelerated assisted (ASE) , Soxhlet (SXE) and Microwave centrifuge pilot extraction (MW/C). To improve extraction yield, generally a sample pre- treatment is done. This pre-treatment usually consists of drying and grinding as a mean to reduce particle size and moisture. There are also other pretreatment methods, but these are

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too specific or use uncommon equipment. For instance (Peiró et al., 2019) studied polyphenol extraction by pulsed electric fields that use a PEF equipment to apply the electricity needed, the objective of the process is to increase the yields of a pressurized liquid extraction by conditioning the sample by electric pulses. Citrus bioactive compounds have also been retrieved as by-product of the process to recover pectic hydrocolloids (Cameron et al., 2016), where the essential oils extraction is part of the process as a mean to assure high quality pectin.

Solid-liquid extraction (SLE), also known as conventional solvent extraction is a method where a sample, usually pre-treated, is placed in a vessel with a solvent to extract the desired components. The solvent, due to its properties and affinity with the components to extract, removes them from the sample carrying out an extraction process. There are different solvents to effectuate the extraction and are divided by their molecular composition as polar and non-polar. The solvents found for this method in the related published research are mainly water, ethanol and methanol. This method is also enhanced or modified by adding heat or stirring which improves extraction time and yield.

Ultrasound-assisted extraction (UAE) and microwave assisted extraction (MAE) are variations of the conventional solid-liquid extraction, where the procedure is assisted using an ultrasonic bath or microwave radiation, respectively. This methods try to accelerate the extraction process, particularly for phenolic compounds (Barrales et al.,

2018; Dahmoune et al., 2013). The solid-liquid extraction at high pressures is another option. In this extraction, the viscosity of the solvent is reduced which enhances extraction time. There are two similar methods that use this principle for the extraction: Accelerated

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Assisted Extraction (ASE) and Pressurized Liquid Extraction (PLE). ASE uses an ASE extractor that is an automated system for extracting organic compounds, in the machine is placed the sample in cells adding the solvent, in which the relation of pressure and temperature maintain helps to maintain the solvent in liquid state during the extraction

(Nayak et al., 2015). PLE uses the injection of pressurized liquid in an extraction column, where the extraction is done in a system that includes pumps, valves, and heat exchangers

(Barrales et al., 2018) working at high pressures and temperatures.

Another variation is made with the Soxhlet extraction method. The system works by evaporating a solvent in a boiling flash which is condensed and let pass into a thimble where the extraction is made by contact with the sample. The setup counts with a recirculation system, where the extracts fall again into the boiling flask and then the process could be repeated in various cycles, reaching a higher yield in the extraction

(Battista et al., 2020).

The solid-liquid extraction can be also improved using other pre-treatments. One of those is the instant controlled pressure drop (DIC) procedure. The pre-treatment is based on a cycle divided in different stages, starting with a short high pressure and temperature stage where a saturated dry steam is injected. The next step has a thermo-mechanical effect issued from an abrupt pressure drop towards a vacuum of an absolute pressure of 5 kPa.

For this procedure, a DIC equipment is used with the objective to intensify the solid-liquid interactions through a higher porosity of the solid matrix, increasing the solvent transfer

(Louati et al., 2019).

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In the Hydro-distillation (HD) method, a mixture of water and the pre-treated sample is heated in a vessel to reach the boiling point of the mixture. Water works as solvent extracting the components. The steam produced flows throw a condenser and then it is deposited in a decanter. The essential oils are separated from water by the density difference, where the water remains at the bottom and the oils in the top (Ayala et al.,

2017). There are variations to this procedure, using different containers to do the heating process or adding stirring, trying to reduce the separation time, and improving the yield.

Another method using water is the steam distillation (SD) that uses steam at 90°C to effectuate the extraction (Ortiz-Sanchez et al., 2020). The steam flows through the sample extracting the compounds with higher affinity.

Some extraction methods work near critical conditions like the subcritical water (SW) extraction or above critical condition like supercritical carbon dioxide (SC-CO2) extraction. Subcritical water is water at temperatures above the boiling point and below its critical point. This water is pressurized to keep the liquid state. The extraction is conducted in a semi-continuous flow reactor, where the pressurized water is introduced

(Lachos-Perez et al., 2020). In SC-CO2 a high-pressure vessel is used where carbon dioxide flows through the residue. In supercritical state, carbon dioxide is held above or in its critical pressure and temperature resulting in a nonaggressive solvent to the organic material. It is a fluid with poor solubility for ionic and high-molecular-weight organic compounds, further it has green chemical properties like non-flammability and friendly environmentally (Kitto et al., 2019). It also can be used in mixtures with ethanol and carbon dioxide (Ndayishimiye et al., 2018).

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The use of enzymes for the extraction process takes advantage of the catalytic properties of enzymes to optimize the yields and times. In this method, a mixture of solvent with the enzyme is created to treat the residue powder by hydrolysis. After the hydrolysis the enzyme is inactivated keeping the samples in water bath at 90°C for 5 min. The hydrolysed slurry is centrifuged and the resultant supernatant is collected. The solvent used in the articles is 0.20 M sodium acetate buffer (pH 4.8) containing the enzyme Viscozyme L

(Nishad et al., 2019a, 2019b).

The use of solvents can be expensive. There is also a method that doesn’t need any solvent to extract the desired components that is solvent free microwave extraction (SF). The method consists in placing the sample in a microwave reactor, without any addition of solvent or water. The process causes the release of essential oils that are evaporated with the water present in the residue. Afterwards, a cooling system outside the microwave oven condenses the distillate (Aboudaou et al., 2019).

Another method uses cold pressing (CP) to created areas of compression in the peel by a laceration process using a needle, in these areas the oil flows to the exterior. After that, a cold pressing machine is used to extract the components. This is a mechanic method without heat treatment, avoiding oxidation reactions occurring along a temperature treatment, obtaining a high quality extract (Çakaloğlu et al., 2018).

There are another methods that combine the procedures described previously, one of them is the microwave assisted hydro distillation (MAHD), that is the conventional hydro distillation using microwave energy in the heating process (Bustamante et al., 2016).

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Microwave centrifuge pilot extraction (MW/C) combine centrifugal force with microwave heating, where is injected water steam to promote the extraction (Angoy et al., 2020). All the methods mentioned here have been explored in relationship with the extraction of bioactive compounds of orange waste, which will be discussed in detail in the next sections.

3.3.1 Essential oils extraction

Essential oils represent a high portion of the extracts from citrus fruits, these compounds are usually the first part of the extracts to be isolated. The method to retrieve essential oils could vary, but it is normally done through solid-liquid extraction taking advantage of the insolubility of certain molecules in water and ethanol. Table A1 presents the essential oils yields reached by different methods and conditions. For orange peel, including the pulps is noticed that using SLE achieves the highest yields, in range of 26.24-58.96% using acetone as solvent (Senit et al., 2019). Also, it was found that reducing the stirring ratio and increasing the pH of the solution increases the yields. These results could be improved using ethanol as solvent instead of acetone, due to the low solubility between the oils and that solvent. This can be supported with the results reported by (Battista et al., 2020) for orange peel. In that work, SXE was used comparing different solvents including acetone.

The results showed that using methanol caused the highest yields and using ethyl acetate or chloroform resulted in the lowest values. It was also found that using HD, MAHD and

SD the yields are the lowest with a considerable difference with SLE, as it is showed in the Figure 2. These methods have in common the use of high temperature water, and the

21

low yields can be explained, due to the fact that the composition of essential oils is mainly attributed to limonene that is insoluble in water (Kumar Joshi et al., 2019). HD achieves yields in a range of 0.019-4.48% and MAHD 0.88-1.822%, then the radiation assistance could decrease the extraction yield.

Extraction methods for essential oils in orange peels

SD

HD

d

o h

t SLE

e M MAHD

SXE

0 20 40 60 80 % p/p

Figure 2. Comparison of yields of extraction methods for essential oils

Another citrus residues that have been explored are yuzu which was submitted to SC-CO2 extraction (Ndayishimiye et al., 2018). In that study, a comparison of the yields between seeds, peels and the mixture (peels and seeds) were done and it was determined that the highest yields were from seeds and the lowest were from the peels and the mixture. Higher yields were also achieved without ethanol and using high pressure, which is a selective condition for non-polar compounds.

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3.3.2 Essential oils composition.

The essential oil’s composition could vary depending of the residue and the extraction method used, which can change their applicability. In Table A2 a comparison is shown of the essential oil’s composition obtained by different extraction methods. The essential oils found can be divided into: Monoterpenes, oxygenated monoterpenes, sesquiterpenes, oxygenated sesquiterpenes, oxygenated compounds and others.

Monoterpenes are the most abundant between the essential oils. Limonene belongs to this category, and has a presence in the essential oils of around 94-97%, then is the most important essential oil by percentage. The lowest value found during the extraction of limonene (74.38%) was obtained using HD with mandarin waste (Maria Patsalou et al.,

2020). This value is an outlier in comparison to other reports, which have a limonene percentage higher than 85%, including those results presented for citrus household residues and for whole orange as samples. This result for mandarin waste could be explained by the nature of residue used, because at the same conditions and using the same method of extraction, a higher quantity of limonene is found for orange peels (85.50%), ground orange peels (96.36%), non-ground orange peels (96.70%) and mandora peel

(Maria Patsalou et al., 2020), then in a residue comparison orange peel (OP) and Mandora peel (MDP) could be a high source of limonene compared to mandarin peel (MD) as is showed in the Figure 3. To make a selective extraction for limonene, it can be used different solvents, where an interesting alternative was proposed by (Ozturk et al., 2019), that evaluate the use of green solvents to replace the use of hexane for limonene extraction

23

with cyclopentyl methyl ether and 2-methyl-tetrahydrofuran obtaining similar results, then could be an alternative to be used in the methods described previously.

Limonene Limonene in orange peel 100

MAHD

s l

i SD o

90

l d

a SLE

i

o

t

h n

t MAE/C

e

e

s M

s 80 CP E

% SF HD 70 OP MP MDP 70 80 90 100 Residue %Essential oils

Figure 3. Limonene percentage Figure 4. Comparison of extraction presence in essential oils methods for limonene

In table A2 it is evident that when using HD an extremely low value of 74.43% of limonene is obtained using orange peel as a residue. This result is the most remarkable, because there were found another six papers using the same method and residue with a percentage of limonene in a range of 85.50-96.75%. The highest value of limonene content in the essential oils was 97.38% which was obtained by using MAHD process with orange peel as a residue. The same work presents a result of 96.75% of limonene by HD, resulting in values of recovery which are practically the same (~97%) (Bustamante et al., 2016),

24

then in a techniques comparison is noticed that these methods obtains the highest value, although the difference between the methods is not considerable (Figure 4).

Other monoterpenes with a considerable presence are 훼-pinene (0.27-1.45%), 훽-pinene

(0.02-3.26%), 훽-myrcene (0.06-0.27%), 훼-terpinene (0.01-4.27%) and 훾-terpinolene

(4.96%) and their proportion are shown in Figure 5. The rest of the monoterpenes reported have a percentage presence lower than 1%, as can be seen in Figure 6. Analysing the results found for the major presence monoterpenes, it is noticed that 훾-terpinolene is reported only paper using steam distillation as extraction method for orange peel (Ortiz-

Sanchez et al., 2020). It is possible that there could be a relationship between the use of steam distillation and the recovery of this component. For 훼-pinene the highest value

(1.45%) was found using hydro-distillation for orange peel (Ayala et al., 2017) and it’s an outstanding value, since this compound is reported in almost all the articles selected with a percentage in a range of 0.27-0.53%. 훽-pinene and 훼-terpinene have the same behaviour when hydro-distillation is used (Ayala et al., 2017) reaching a value of 3.26% and 4.27%

, respectively, These results differ from the range obtained in the rest of the data gathered with 0.02-0.56% of 훽-pinene and 0.01-0.305% of 훼-terpinene, respectively. For 훽- myrcene the highest value (2.28%) is obtained using steam distillation (Ortiz-Sanchez et al., 2020), and the lowest value was found for mandarin peels using hydro-distillation

(Maria Patsalou et al., 2020). Then, for the extraction of this compound could be better to use a method like steam distillation. Additionally, the low contents of monoterpenes found in mandarin (M. Patsalou et al., 2020) could be explained by the particular composition of the residue.

25

Monoterpenes (Low presence) Monoterpenes(High presence) 1.5 6

5 s

l

i

s

l

o i

1.0

l o

4

a

l

i

t

a

i

n

t e

n 3

s

e s

s 0.5 s

2 E

E

% % 1 0.0 0 e e e e e e e e e e e e e e e n n n n n n n n n n n n n n n e e le re e e le e e e e e e e e h in o d ra m o m in n n n c n l p p n y n i p i i i r i o n r i n -C i c r p p b y p in a e p la 3 -C p o e - - a M r C T r l p r - -t a ß S - te rp - e e e ß γ ß - e γ T h -T a -t -p a γ a

Figure 5. Percentage presence of high Figure 6. Percentage presence of low

presence monoterpenes in essential oils presence monoterpenes in essential oils

Regarding oxygenated monoterpenes, the molecule whit major presence is linalool in a range of 0.05-3.51%, also was found the presence in a high portion of linalool oxide and citronellol (Figure 7) . For linalool the lowest yield obtained for this substance (0.05%) was obtained in two different methods, using hydro-distillation and microwave assisted hydro-distillation (Bustamante et al., 2016). Even though in this case lo contents were obtained, in other works considerable amounts of linalool of 1.54% and 1.30+-0.035% have been obtained using the same method in orange peel and citrus household waste. The highest yield (3.51%) was found by steam distillation for orange peel (Ortiz-Sanchez et al., 2020), which could be recommended in comparison to other methods. Also, there are

26

other oxygenated monoterpenes reported, but they are present in percentages lower than the 1% of the composition of the essential oils. These molecules are terpin-4-ol, eucalyptol, citronellal, nerol, neral, geranial, genariol and alpha terpineol and their presence is showed in the Figure 8.

Oxygenated monoterpenes (High presence) Oxygenated monoterpenes

4 (Low presence) s

l 1.0

i 3

o

l a

i 0.8

s

t

l i

n 2

o

e

l

s a

s 0.6

i

t

E

1 n

e %

s 0.4

s E

0 % 0.2 l l e o o id ll x lo e o a n 0.0 l in o o L r o it l l l l l l l l l l C o o o a o a a o o a - e t ll r r i ri e in -4 n p e e e n a n L n i ly n N N ra n i i rp a o e e rp rp e c tr G e e T u i G -t T E C a

Figure 7. Percentage of high presence Figure 8. Presence of low presence oxygenated monoterpenes in essential oils oxygenated monoterpenes in essential oils

In Table A2, it can be observed that sesquiterpenes represent less than 1% of the essential oils. The most abundant of them is 훽-caryophyllene in a range of 0.01-0.89%. with it highest yield when obtained by the hydro-distillation of orange peel (Ayala et al., 2017).

Other sesquiterpenes present are: 훼-copaene, 훽-cubebene, 훽-elemene, valencene, copaene, 훼-humulene and germacrene (Figure 9). Regarding oxygenated sesquiterpenes, the presence of caryophellene oxide, 훽-sinensal, 훼-sisnensal, nootkatone and elemol, was

27

observed in percentage lower than 0.5% (Figure 10). Other oxygenated compounds are decanal, 훼-terpenyl acetate, citronellyl acetate, nonanal and octanal (Figure 11), with a composition lower than 1%. Between the oxygenated compounds, decanal stands out in a range of 0.16-0.87%. Other essential oils detected were z-carveol, trans-훼-bergamotene,

1,8-cineole, and perilla aldehyde (Figure 12). Z- carveol has the major presence with a value of 1.12% in one paper being this the only one report that has detected for this component during the hydro-distillation of orange peel.

Oxigenated sesquiterpenes

Sesquiterpenes 0.4

1.0

s l

i 0.3 o

s 0.8

l

i

l

o

a

i

l t

a 0.6 i

n 0.2

t

e

n

s

e s

s 0.4

s

E E

0.1 %

% 0.2

0.0 0.0 e e e e e e e e e l l e l n n n n n n n n n a a n o e e e e e e e e e s s o m ll c a l r a b l n n t y n p u c p e m y e e a le h e o m a o b le h n n k E p l u m c u e p i i t o a C r - - o -s -s o y V -h e a -C ß ß a o r a G ß ry n a a -C C ß

Figure 9. Percentage of sesquiterpenes Figure 10. Percentage of oxygenated

in essential oils sesquiterpenes in essential oils

28

Other Other oxygenated compounds 1.5

1.0

s

l

i o

s 0.8

l 1.0

l

i

a

o

i

t

l n

a 0.6

i

e

t

s

n s

e 0.5

E

s 0.4

s

%

E

% 0.2 0.0 l o e le e 0.0 e n o d v te e y r o n h l l l e e a i e a a a t t -C m -C ld n n n ta ta Z a 8 a a a ta e e rg , a n c c c c e 1 ll o e a a b ri N D O l l - e y y a P ll n h e e lp n p a o r s tr te n i - ra C a T

Figure 11. Percentage of other oxygenated Figure 12. Percentage of other compounds

monoterpenes in essential oils in essential oils

3.3.3 Phenolic content of citrus residues.

Two common methods are used to express the respective content of phenolic compounds.

Total phenolic content (TPC) is expressed in mg of gallic acid equivalent (GAE) per gram of dried residue and the total flavonoid content (TFC) in mg of quercetin equivalent (QE) per gram of dried residue. Flavonoids are a type of phenolic compounds and are important to the analysis by the high presence in this type of residue and the possibilities to value the waste by the different uses of those. The results of TPC and TFC for the different citrus residues are shown in Table A3. For phenolic content the highest value found was

71.0±8.5 mg GAE/gdb using UAE with ethanol 50% as solvent for grapefruit solid waste

29

(Garcia-Castello et al., 2015). The lowest result was 1.4±0.1 mg GAE/gdb using PLE with ethanol 99.5%.for orange peel (Barrales et al., 2018).

In order to valorise citrus waste by its phenolic content, peels acquire a high importance, because is the main residue obtained from these fruits. For orange peel (OP) a TPC was found in a range of 1.4-33.87 mg GAE/gdb, this is a wide range that means a wide variability in the results. Through this result it can be assume that the phenolic content in orange peel is higher than the content in lemon peel (LP) (5.9-15.92 mg GAE/gdb) and clementine peel (CP) (5.5 mg GAE/gdb) and lower than the content of yuzu peel (YP)

(12.17-43.64 mg GAE/gdb) and grapefruit peel (GP) (15.28-71.0 mg GAE/gdb) and this comparison is found in the Figure 13. Comparing the results of citrus peels with another fruit peels in TPC is found that in general have higher results than banana peel (3.8 mg

GAE/g),(Babbar et al., 2011) and mango peel (6.0-13.82 mg GAE/g db), except for clementine peel that has similar results (Guandalini et al., 2019).

30

Phenolic content

80 70

60

g

/ 50 E

A 40

G

g 30 m 20 10 0 OP GP CP YP LP Residue

Figure 13. Comparison of phenolic content in different residues

To extract the TPC in orange peel the EAE is the method that obtains the highest quantity as is shown in Figure 14, having a range of 31.704-33.87 mg GAE/gdb. This result is arguable and the method can’t be recommended totally, because the nature of the method is the enzymatic hydrolysis of the residue that releases a mixture of compounds, including sugars of the hydrolysed cell wall. As a result, the separation and quantification of phenolic compounds during EAE could be challenging. On the other hand, the use of SLE is recommended because it was found that a considerable content of 3.9-31.62 mg

GAE/gdb can be obtained using methanol as solvent. In contrast, it is not recommended to use SW, since this method results in the lowest TPC with 3.40-5.53 mg GAE/gdb. In lemon peel, MAE and UAE, report the highest values with 12.49-15 mg GAE/gdb and

11.09-15.92 mg GAE /gdb, respectively. Using grapefruit, UAE duplicates the quantity

31

obtained using solid liquid extraction. And in the case of yuzu only SC-CO2 was proved under different conditions with the best result being 43.64 mg GAE/gdb which was obtained mixing the carbon dioxide with ethanol at 45°C, 200 bar and 2 hours

(Ndayishimiye et al., 2018). In the case of clementine, only one result was reported and as consequence, it is not comparable to other extraction methods.

Extraction methods for phenolic content in orange peels

ASE MAE

d PLE

o h

t SW e

M EAE UAE SLE

0 10 20 30 40 mg GAE/ g

Figure 14. Comparison of extraction techniques for phenolic compounds in orange peels

Comparing the peels with other residues it was found a TPC of 15.76-17.24 mg GAE/gdb using UAE in residual lemon pomace from the juice industry. This shows that lemon pomace has a higher phenolic content than lemon peels. Also for yuzu using SC-CO2 a

32

comparison was made for phenolic and flavonoid content between peel, seeds and mixture

(peel and seed) (Ndayishimiye et al., 2018). In this comparison it was found that the phenolic content is higher in the extraction using the peel and the flavonoid content is higher using the seeds as sample. The mixture (peel and seeds) obtains intermediate results and do not improve the content obtained.

For flavonoid content the highest value found is 17.6 ±0.6 mg QE/gdb using SLE for orange peel assisted by heating and stirring with a mixture ethanol-water 40:60 (v/v) as solvent (Gómez-Mejía et al., 2019). The lowest result was 0.29±0.005 mg QE/gdb using

SLE for orange peel assisted by stirring with a mixture of methanol-water 80:20 (v/v) as solvent (Lagha-Benamrouche and Madani, 2013).

Lemon peel is the most attractive one to be valorised in terms of TFC with 18 mg QE/gdb

(Gómez-Mejía et al., 2019) which is the highest value, however the results in orange peel

(0.29-17.6 mg QE/gdb) and clementine peel (16.5 mg QE/gdb) were very close to that result, but are higher than yuzu peel (2.2-6.75 mg QE/gdb). TFC was not reported for grapefruit peel. Trying to obtain the highest content possible of TFC, using SLE with a mixture ethanol-water (40:60 v/v) at 90°C as extraction method could be the best option, because obtain the highest results for orange peel (17.6 mg QE/gdb), lemon peel (18 mg

QE/gdb) and clementine peel (16.5 mg QE/gdb) (Gómez-Mejía et al., 2019).The influence of the solvent and temperature is noticed, because the quantity extracted by SLE decrease considerably when is used a mixture of methanol-water at room temperature, obtaining a range of 0.29-1.29 mg QE/gdb (Lagha-Benamrouche and Madani, 2013) for orange peel varieties. Also the mixture proportion of the solvent is relevant, due to (Nishad et al.,

33

2019a, 2019b) use SLE with ethanol 70% at 60°C obtaining lower results (0.933-1.397 mg QE/gdb) compared to the analysed previously. For yuzu peel it was only found the use of SC in different conditions to effectuate the extraction, and because of that these results cannot be compared to other extraction methods.

3.3.4 Composition of individual phenolic compounds

Table A4 shows the results for individual phenolic compounds, where the most important components by major presence are naringin, hesperidin and narirutin, being the components to direct the valorisation efforts. The results are presented in mg of compound per g of dry basis and are shown in the Figure 15.

Flavanones (High presence)

Naringin

Narirutin

Hesperidin

0 20 40 60 80 mg/g db

Figure 15. Presence found of individual flavanones in citrus residues

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Naringin is a glycosylated flavonoid and it’s known for its antioxidant and scavenging activity (Nishad et al., 2019b). This compound was found for orange peel in a range of

10.796-49.03 mg/gdb that is a wide range, where the outlier results were those reported by (Barrales et al., 2018), because are lower by a high difference in comparison with the other results, being in a range of (0.062-0.4 mg/gdb), this article use PLE varying the ethanol concentration and the extraction temperature, and its observed that increasing those parameters increase the quantity extracted. Another particular outlier by the method used is with EAE that reaches the lowest quantity extracted for this residue, obtaining a range of 4.79-6.97 mg/gdb. Apart of the outlier values the naringin range from orange peel is of 10.79-49.03 mg/gdb. To extract naringin from citrus residues the use of SLE is recommended since this method resulted in the highest concentrations (49.03mg/gdb)

(Nishad et al., 2019a). Also is found a considerable amount by UAE, but in lower quantities than SLE at the same conditions. Grapefruit solid waste is another residue in which naringin can be extracted and is obtained in a range of 22-28 mg/gdb, where the ultrasound assistance can increase the quantity extracted.

Hesperidin is the most abundant flavonone glucoside present in citrus peels and its consumption may be associated with health benefits and in the prevention of many diseases, exhibiting biological as analgesic, anti-inflammatory, antyhypercholesterolemic, antihypertensive, diuretic, neuroprotective, cytotoxic against HEP-G2 cancer cells, among others (Victor et al., 2020). Those properties make hesperidin a compound of interest for its extraction. In orange peels it is found in a range of 7.1-55 mg/gdb, where the highest

35

concentration was found using PLE with methanol as solvent pressurized at 10 Mpa. The lowest result was found using UAE with a mixture water-ethanol 50% (v/v) at 30°C,

200rpm and 15 min of extraction time. These results could indicate that the ultrasound assistance is not effective to extract this compound in orange peel and which is supported by the range found using UAE that is of 7.1-7.7 mg/gdb and lower than the one reported for Soxhlet extraction (18), SCW (9.82-22.99) and PLE (12-58). Also, the concentration found for orange residues (peel, pulp and seeds) using a DIC pre-treatment with solid- liquid extraction reached a value of 65.01 mg/gdb, resulting in an increase of 537% in comparison to the results obtained without a pre-treatment (12.10 mg/gdb) (Louati et al.,

2019). The pre-treatment uses high pressure and temperature, similar to the extraction conditions used in PLE, then in order to valorise the orange residues extracting hesperidin those conditions (high pressure and temperature) could improve the results. For grapefruit solid waste (pulp and peel) hesperidin was found in a lower range 0.47-0.93 mg/gdb than the found for orange peel (7.1-55 mg/gdb). In these results, it was observed that ultrasound assistance could improve the recovery of hesperidin in grapefruit residues in comparison to conventional solid-liquid extraction.

Narirutin is another flavonone-glycoside, with high antioxidant capacity and applicability as anti-inflammatory agent common in the citrus peels. In orange peel is found in a range of 0.99-14 mg/g db, reaching the lowest value when SW is used (water ratio of 20 ml/min at 150°C and 4.92 min of residence time). To extract narirutin by this method, residence time and water ratio are crucial factors that increment the quantity extracted (Lachos-Perez et al., 2020). The highest value obtained for narirutin were reached using PLE with ethanol

36

75% at 65°C, 10MPa and 40 min of extraction time and PLE with ethanol 99.5% at 45°C,

10MPa and 40 min of extraction time (Barrales et al., 2018). For this extraction method the temperature and ethanol’s concentration are critical factors, which also increase the amount of narirutin obtained. This compound has been also extracted from grapefruit peel, but its content is very low with 0.43-0.83 mg/gdb (Garcia-Castello et al., 2015) in comparison to orange peel (0.99-14 mg/g db). In this residue the use of ultrasound waves duplicates the quantity extracted comparing to the use of solid-liquid with the same solvent, also the UAE uses less time.

Another flavanones found are neoeritrocin, naringenin, hesperetin and neohesperidin, but with low presence (Figure 16), also was found the presence of other flavonoids, where tangeritin could be remarkable (Figure 17) and other phenolic compounds (Figure 18), where gallic acid has the highest presence.

Flavanones (Low presence) Other flavonoids

1.5

Neoeritrocin

1.0

b d

Naringenin

g

/ g

Hesperetin m 0.5

Neohesperidin 0.0

n n n n ti i i ti ri h h e 0.0 0.5 1.0 1.5 e c c c g te te r n a a e a C ic u T p Q mg/g db E

Figure 16. Presence of flavanones Figure 17. Presence of other flavonoids

in citrus residues in citrus residues

37

Other phenolic compounds

4

3

b

d

g

/ 2

g m 1

0

id id id id id te c c c c c a a a a a a r c c c c c d i li li li i y e l il r ih ff ru a a d a e G n m C F a u a V o in z C ri lo F

Figure 18. Presence found of other phenolic compounds in citrus residues

3.3.4 Antioxidant activity of citrus residues

Different techniques and assays have been used to measure the antioxidant activity in citrus residues. In this research, the following methods were identified: α, α − diphenyl −

β − picrylhydrazy (DPPH), trolox Equivalent Antioxidant Capacity (TEAC), ferric reducing-antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and cupric ion reducing antioxidant capacity (CUPRAC) assays.

Methods used for measuring antioxidant activity

DDPH Assay

DPPH assay uses the stable free radical α, α − diphenyl − β − picrylhydrazyl (DPPH C18H12N5O6 M = 394.33). The delocalization of electron gives

38

it an intense violet colour. When the DPPH solution is mixed with the substrate that can donate a hydrogen atom, then leads to the reduced form with the loss of this violet colour.

In order to evaluate the antioxidant potential through free radical scavenging by the test samples, the change in optical density of DDPH is monitored. To effectuate the measurement the absorbance is evaluated at 517 nm.

TEAC

In the method of Trolox Equivalent Antioxidant Capacity (TEAC) the loss of colour is measured when an antioxidant is added to the blue-green chomophore ABTS+ (2,2-azino- bis(3-ethylbenz-thiazoline-6-sulfunic acid)). The antioxidant reduces ABTS+ to ABTS and decolorizes it.

FRAP

In the ferric reducing-antioxidant power (FRAP) essay the ability of antioxidants to reduce ferric ion is measured. This method is based on the reduction of the complex ferric iron and 2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) to the ferrous form at low pH. The reduction is observed by evaluation the change in absorption at 593 nm.

ORAC

In oxygen radical absorbance capacity (ORAC) method is measured the ability to scavenge oxygen radical (peroxy radicals). Oxygen radicals are generated using AAPH

39

(2,2-azobis 2-amidopropane dihydrochloride) and the method measure the decrease in fluorescence in the presence of free radicalscavengers.

CUPRAC

In cupric ion reducing antioxidant capacity (CUPRAC) method, the oxidizing reagent bis(neocuproine)copper(II) chloride (CU(II)-Nc reacts with polyphenols, where the reactive groups of polyphenols are oxidized to the corresponding quinones and and Cu

(II)-Nc is reduced to the highly colored Cu (I)-Nc chelate showing maximum absorption at 450 nm (Alam et al., 2013; Sehwag and Das, 2013).

Antioxidant activity

Table A5 shows the antioxidant activity measured for the different citrus residues. In general, for orange peel the highest antioxidant activity was observed using EAE, and the lowest results were found using SW. However, as in the TPC analysis this method not is recommended, then is better to use UAE or SLE with ethanol as solvent. For SW (Lachos-

Perez et al., 2020) reports an increment in the antioxidant activity by reducing the water ratio and having more residence time. These results corroborate the existence of a correlation between the TPC and the antioxidant activity. There are unusual results in the values reported by (Papoutsis et al., 2018), due to using CUPRAC values shows a high antioxidant activity compared to the other articles, in contrast using DPPH assay were obtained the lowest results in the same comparison. This is the unique report that has such as difference between the methods and also in this it’s observed that having a low particle size and high extraction temperatures the antioxidant activity increase. The results

40

reported by (Nishad et al., 2019b) and (Nishad et al., 2019a) compare the antioxidant activity in similar conditions and using the same methods (SLE, UAE, EAE) for orange peel and grapefruit respectively, giving that this residues have similar results. The comparison shows similar results for antioxidant activity using SLE and UAE and higher results for grapefruit peel using EAE.

3.3.5 퐈퐂ퟓퟎ Values

The antioxidant values also can be expressed by IC50 index. This value is the sample’s concentration necessary to inhibit 50% of the radical (Bechlin et al., 2020). A lower value of IC50 indicates higher antioxidant activity. In this research the use of IC50 index was found for the DPPH and ABTS radicals.

Table A6 shows the IC50 results for citrus residues. For the IC50values, it was found that the use of SLE and HD for orange peel as residue inhibits the DPPH radical. SLE showed more antioxidant activity in a range of 0.0258-0.901mg/ml, obtaining the best result ethylene glycol as solvent with temperature and stirring assistance (Ozturk et al., 2018), and the highest result (lowest in terms of antioxidant activity) using a mixture methanol- water (80:20, v/v) as solvent at room temperature, using magnetic blender and 22 hours of extraction time (Lagha-Benamrouche and Madani, 2013). Then, it is noticed that the effect of the temperature increases the antioxidant activity in the extracts. About HD, the antioxidant activity reached with this method is the lowest, with IC50 values in a range of

4.09-5.70 mg/ml. (Bechlin et al., 2020) presents that before the HD the sample is treated by an ozone flow, the results show that using a low ozone ratio and high extraction

41

temperatures was reached more antioxidant activity. A mixture of lemon ichang and mandarin was also analysed by its antioxidant activity, using SC-CO2. For this mixture were evaluated the results using the peels and a mixture (peels and seeds). It was obtained that including the seeds in the mixture, the antioxidant activity increases. That conclusion is supported by the use of the same method but measuring the antioxidant activity through the inhibition of ABTS radical. It is also important the evaluation of the solvents to use, then in a solvent comparison, trying to increase the antioxidant activity, (Ozturk et al.,

2018) compared different solvents at the same conditions. The results determined that using ethylene glycol the antioxidant activity is the highest followed by ethanol and water as the lowest.

There are also indicators similar to IC50. One of those is EC50. This parameter indicates the sample concentration needed to reach 50% of the maximum potency. for instance is used in the report by (Gómez-Mejía et al., 2019) that evaluate the antioxidant activity between orange, lemon and clementine peel, found using TEAC and DPPH assays expressed in the EC50 values that the antioxidant activity is highest for clementine and lowest for lemon.

42

3 CONCLUSIONS

Considering the results of different authors, it was found that the citrus residues are composed mainly by total carbohydrates (pectin, cellulose and hemicellulose), followed by extractive bioactive compounds, being of interest by the facilities to extract and by the properties and characteristics of those that can be used in different industries and products.

In this composition were found similar results among the residues. By the chemical composition (ultimate analysis), residues are composed principally by Carbon and

Oxygen, where ponkan peel is the residue with the lowest carbon percentage and the highest oxygen percentage compared with orange peel and lemon peel. In the behaviour of gaseous, liquid and solid components that can be obtained in a thermochemical process

(proximate analysis), it was obtained that fixed carbon and volatile matter have a considerable percentage presence in citrus residues and a high heating value also considerable compared to other materials, then indicating the possible utilities in thermochemical process as a source of energy. In this analysis were found differences between orange waste and the other residues (lemon peel, ponkan peel and orange peel), having the lowest volatile matter and the highest presence of fixed carbon, also was reported a HHV higher than the reported by orange peel. The residues also have an ash presence, where is found that are main composed by calcium and potassium, where ponkan peel has the highest presence of potassium and lemon peel has the lowest of potassium and calcium compared to ponkan peel and orange peel.

Among the extraction methods, for essential oils extraction, the use of SLE is the best option by yields and components found, obtaining the highest results using acetone as

43

solvent, however other studies identified that can be improved by the use of ethanol. For phenolic compounds the EAE obtains the highest value, however, by the nature of the extraction and the problems to separate the extracts is not recommended, then it is better use SLE, this by the values obtained and by the facilities to effectuate the extraction and to separate the compounds. In this category the highest results were found with ethanol as solvent. In the antioxidant activity, including the analysis of IC50 values, UAE and SLE are the methods recommended, where can be used ethanol and ethylene glycol as solvents.

In general for essential oils orange peel is the residue that has major presence. Into those was identified the presence of monoterpenes, oxygenated monoterpenes, sesquiterpenes, oxygenated sesquiterpenes and other oxygenated compounds, being mainly the presence of monoterpenes with limonene as principal compound by percentage presence. In the phenolic content grapefruit peel has the major presence. In individual phenolic compounds it was found mainly the presence of naringin, hesperidin and narirutin.

44

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5. ANNEX

Annex 1.

Table A1. Essential oil yields

Method Descripción EO yield (%) Reference SXE Orange peel 50 g, 2mm, 300 mL n-Hexane- Ethanol (50:50 v/v) 21.03 SXE Orange peel 50 g, 2mm,300 mL n-Hexane- Acetone (50:50 v/v) 10.86 SXE Orange peel 50 g, 2mm,300 mL n-Hexane 1.31 SXE Orange peel 50 g, 2mm,300 Ml Ethyl acetate 0.57 (Battista et al., 2020) SXE Orange peel 50 g, 2mm,300 mL Ethanol 24.18 SXE Orange peel 50 g, 2mm,300 mL Diethyl ether 0.77 SXE Orange peel 50 g, 2mm,300 Ml Chloroform 0.57 SXE Orange peel 50 g, 2mm,300 mL Acetone 12.99 Orange peel, <5 mm particles, Milestone RotoSYNTH MAHD 1.822 microwave, 800 W, 400 mbar, 45 min, 1:1.5 WOP:water ratio Orange peel, <5 mm particles, Milestone RotoSYNTH MAHD 0.140 microwave, 400 W, 100 mbar, 19 min, 1:2.5 WOP:water ratio (Bustamante et al., 2016) Orange peel, <5 mm particles, Milestone RotoSYNTH microwave, Step1: 1200 W-800 mbar-5 MAHD 0.88 min,Step2: 400 W-800 mbar-5 min Orange peel, <5 mm particles, Milestone RotoSYNTH microwave, Step1: 982 W-500 mbar-5 MAHD 1.87 min,Step2: 250 W-500 mbar-30 min Orange peel waste (peel and pulp), 0.5 mm particle size, 4.5 pH, 600 N (min^-1) stirring rate, SLE 26.24±1.25 4 L/S(L/kg), 1 hour (dry basis) Orange peel waste (peel and pulp), 1 mm particle size, 4.5 pH, 400 N(min^-1) stirring rate, 6 SLE 54.27±1.05 L/S (L/kg), 1 hour (dry basis) (Senit et al., 2019) Orange peel waste (peel and pulp), 1 mm particle size, 1.5 pH, 600 N(min^-1) stirring rate, 3 SLE 27.84±0.95 L acetone/kg residue (L/kg), 6 hour (dry basis) Orange peel waste (peel and pulp), 1 mm particle size, 4.5 pH, 400 N(min^-1) stirring rate, 6 SLE 58.96±1.05 L acetone/kg residue (L/kg), 6 hour (dry basis) Orange peel 500 g- 63% moisture-ratio 1:5 peels water- electrical resistance-particle size HD 1.05±0.011 between 4 and 5 nm-Extraction time 190 min (Hilali et al., 2019) Orange peel 500 g- 63% moisture-ratio 1:5 peels water-10 m^2 solar reflector coupled to a Solar HD 1.03±0.015 hydrodestilation unit-particle size between 4 and 5 nm- extraction time 120 min 100 g orange peel, Clevenger apparatus, 500 mL distilled water, 3 hour, 40 microg/L ozone HD 3.37±0.05 pretreatment, 40°C drying temperature (Bechlin et al., 2020) 100 g orange peel, Clevenger apparatus, 500 mL distilled water, 3 hour, 40 microg/L ozone HD 4.48±0.07 pretreatment, 60°C drying temperature Orange peel 64g-gridding in 500 cm^3-distillate water-50 min separation time, 2 min griding HD 4,40 (Ayala et al., 2017) time citrus peels of C. sinensis Osbeck-moisture content of 71–73% and were chopped in small HD 0,019 (Magare et al., 2020) size of 1.0–2.0 cm- 80 °C for 180 min, 1:1 solid/liquid ratio SD Orange peel-10%moisture-0.4 mm particle diameter-steam 90°C-water 0.84±0.01 (Ortiz-Sanchez et al., 2020) SC-CO2 Yuzu seed-CO2-45°C-200 bar-2h- CO2 rate 27g/min 15.45 SC-CO2 Yuzu peel-CO2-45°C-200 bar-2h- CO2 rate 27g/min 1.57 SC-CO2 Yuzu mixture (peel and seed)-CO2-45°C-200 bar-2h- CO2 rate 27g/min 8.22 SC-CO2 Yuzu seed-CO2-45°C-300 bar-2h- CO2 rate 27g/min 22.12 SC-CO2 Yuzu peel-CO2-45°C-300 bar-2h- CO2 rate 27g/min 1.87 SC-CO2 Yuzu mixture (peel and seed)-CO2-45°C-300 bar-2h- CO2 rate 27g/min 11.97 (Ndayishimiye et al., 2018) SC-CO2 Yuzu seed-CO2-ethanol-45°C-200 bar-2h- CO2 rate 27g/min 15.67 SC-CO2 Yuzu peel-CO2-ethanol-45°C-200 bar-2h- CO2 rate 27g/min 1.66 SC-CO2 Yuzu mixture (peel and seed)-CO2-ethanol-45°C-200 bar-2h- CO2 rate 27g/min 8.39 SC-CO2 Yuzu seed-CO2-ethanol-45°C-300 bar-2h- CO2 rate 27g/min 23.04 SC-CO2 Yuzu peel-CO2-ethanol-45°C-300 bar-2h- CO2 rate 27g/min 1.91 SC-CO2 Yuzu mixture (peel and seed)-CO2-ethanol-45°C-300 bar-2h- CO2 rate 27g/min 12.73

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Annex 2.

Table A2. Essential oils composition.

Reference (Ayala et al., 2017) (Aboudaou et al., 2019) (Angoy et al., 2020) Method HD SF HD CP HD MW/C Orange peel 500 g Orange peel Orange peel Orange peel defrosted and drined 1 kg whole Orange peel 64g- 200g-"DryDist" 200 g- 250 g- -a modified orange- gridding in 500 cm^3- microwave Clevenger Florentin semiindustrial pilot automated Description distillate water-50 min oven-200 W- type flask- combining cold pressing separation time, 2 min 10 min-100°C- apparatus- distillate centrifugal force and machine-1 griding time atmosferic water- water-2h- microwave heating- hour pressure 3hours 100°C water vapor-2.4 W/g 196 G 훼-Pinene 1.45 0.43 0.53 0.51 0.40±0.04 0.34±0.01 Camphene 0.99 훽-pinene 3.26 0.53±0.03 0.56±0.05 Sabinene 0.54 0.49 0.54 0.24±0.03 0.58±0.02 훽-Myrcene 1.54 1.64 1.87 1.82 1.82±0.05 1.73±0.02 Limoenene 74.43 94.64 95.48 95.06 95.0±0.2 94.7±0.1 훾-Terpinene 0.92 0.05 0.03 0.01 0.5±0.1 0.41±0.06 Terpinolene 0.01 0.02 0.01 0.14±0.01 0.11±0.01 훼-phellandrene 0.15 0.17 0.36 Monoterpenes 3-Craene 훼-terpinene 4.27 p-Cymene 0.27 훼-Terpinolene 훽-ocimene 0.02 0.02 0,02 훾-terpinene 0.05 0.03 0.01 terpinolene 0.01 0.02 0.01 훾-terpinolene Linalool oxide 0.82 Linalool 1.54 0.62 0.30 0.30 0.15±0.01 0.14±0.03 terpin-4-ol 0.03 0.06 0.02 0.10±0.01 0.02±0.01 Terpineol Eucalyptol Citronellal 0.87 0.06 0.03 0.04 Oxygenated Nerol 0.10 0.03 0.08 Monoterpenes Neral 0.05 0.03 0.06 Geranial 0.09 0.06 0.11 0.04±0.01 0.05±0.01 Genariol 0.04 0.01 훼 terpineol 0.10 0.06 0.05 Citronellol 0.78 훼- copaene 훽-Cubebene 0.64 훽-elemene 훽-Caryophyllene 0.89 0.02 0.02 0.01 Sesquiterpenes Valencene 0.02 0.05 0.02 Copaene 0.26 훼-humulene 0.16 0.01 0.03 0.03 Germacrene 0.12 Caryophellene oxide 훽-sinensal Oxygenated 훼-sinensal Sesquiterpenes nootkatone Elemol 0.36 0.01 Decanal 0.31 0.19 0.27 0.35±0.01 0.31±0.01 Other 훼-terpenyl acetate Oxygenated citronellyl acetate Compounds Octanal 0.02±0.04 0.06±0.06 Z-Carveol 1.12 Trans alpha-bergamotene Others 1,8-Cineole 0.16 Nonanal

56

Perilla aldehyde

57

Reference (Hilali et al., 2019) (Magare et al., 2020) (Ortiz-Sanchez et al., 2020) Method HD Solar HD SLE SLE SLE SD Orange peel Orange peel Orange Orange 500 g- 63% 500 g- 63% peel- peel- Orange moisture-ratio moisture- moisture moisture peel,moisture 1:5 peels ratio 1:5 content of content of content of water-10 m^2 peels water- 71–73%, 71–73%, 71–73%, 1.0– solar reflector Orange peel-10%moisture- electrical 1.0–2.0 cm 1.0–2.0 cm 2.0 cm Description coupled to a 0.4 mm particle diameter- resistance- particle particle particle size, hydrodestilati steam 90°C-water particle size size- 80 °C, size, 80 °C, 80 °C, 180 on unit- between 4 180 min, 180 min, , min, , 1:3 particle size 4- and 5 nm- 1:1 1:2 solid/liquid 5 nm- Extraction solid/liquid solid/liquid ratio extraction time 190 min ratio ratio time 120 min 훼-Pinene 0.39 0.37 0.434 0.515 0.453 Camphene 0.01 0.01 훽-pinene 0.02 0.02 Sabinene 0.19 0.16 0.273 0.301 0.281 훽-Myrcene 1.73 1.7 1.80 1.867 1.792 2.28 Limoenene 95.24 95.96 95.482 94.036 94.589 88.39 훾-Terpinene 0.03 0.02 Terpinolene 훼-phellandrene 0.08 0.05 Monoterpenes 3-Craene 0.14 0.08 훼-terpinene 0.02 0.01 0.234 0.255 0.305 p-Cymene 훼-Terpinolene 훽-ocimene 훾-terpinene terpinolene 훾-terpinolene 4.96 Linalool oxide Linalool 0.3 0.23 0.482 0.390 0.645 3.51 terpin-4-ol Terpineol Eucalyptol Citronellal 0.01 0.01 Oxygenated Nerol 0.01 0.01 Monoterpenes Neral 0.01 0.01 Geranial 0.01 0.01 Genariol 0.01 0.02 훼 terpineol Citronellol 훼- copaene 0.02 0.02 훽-Cubebene 0.01 0.01 훽-elemene 0.02 0.02 훽-Caryophyllene 0.02 0.02 Sesquiterpenes Valencene 0.11 0.13 Copaene 훼-humulene Germacrene Caryophellene oxide 0.02 0.01 훽-sinensal 0.01 0.02 Oxygenated 훼-sinensal 0.01 0.01 Sesquiterpenes nootkatone 0.01 0.02 Elemol Decanal 0.16 0.17 0.87 Other 훼-terpenyl acetate 0.02 0.02 Oxygenated citronellyl acetate 0.01 0.01 Compounds Octanal Z-Carveol Trans alpha-bergamotene 1,8-Cineole Others Nonanal Perilla aldehyde

58

Reference (Bustamante et al., 2016) (M. Patsalou et al., 2020) Method HD MAHD HD HD HD HD HD citrus Orange Mandarin Orange Orange peel ground non-ground household peel 100 peel 15-20 peel 15-20 100 g, 3 min mandora peel mandora peel kitchen citrus g-240 min g, 10 and g, 10 and at 2500 rpm 15-20 g, 10 15-20 g, 10 waste 15-20 100°C-3 25 mm, 25 mm, Description (<5mm and 25 mm, and 25 mm, g, 10 and 25 min at for 3 h for 3 h particles), for 3 h using for 3 h using mm, for 3 h 2500 rpm using using microwave Clevenger Clevenger using (<5mm Clevenger Clevenger oven apparatus apparatus Clevenger particles) apparatus apparatus apparatus 훼-Pinene 0.32 0.39 0.27 0.30 0.31±0.012 0.34±0.008 0.28±0.016 Camphene 훽-pinene 0.05 0.06 Sabinene 0.49 0.50 0.42 0.43 0.10±0.006 0.13±0.001 0.26±0.013 훽-Myrcene 0.74 0.79 0.73 0.92 1.35±0.022 1.53±0.017 1.32±0.042 Limoenene 96.75 97.38 74.38 85.50 96.36±0.085 96.70±0.021 94.41±0.198 훾-Terpinene Terpinolene 훼-phellandrene Monoterpenes 3-Craene 0.01±0.009 훼-terpinene p-Cymene 0.06±0.005 훼-terpinolene 0.20 0.18 훽-ocimene 훾-terpinene Terpinolene 훾-terpinolene Linalool oxide Linalool 0.05 0.05 0.54 0.47 0.53±0.014 0.18±0.005 1.30±0.035 Terpin-4-ol 0.01 0.01 0.04±0.002 0.07±0.004 Terpineol 0.01 0.01 Eucalyptol 0.07 0.06 Citronellal 0.08 0.08 0.05±0.003 0.01±0.001 Oxygenated Nerol 0.02±0.001 0.02±0.001 0.08±0.005 Monoterpenes Neral 0.07 0.04±0.001 0.05±0.002 0.17±0.006 Geranial Genariol 훼 terpineol 0.05±0.004 0.02±0.002 0.16±0.005 Citronellol 훼- copaene 훽-Cubebene 훽-elemene 훽-Caryophyllene Sesquiterpenes Valencene 0.09 0.34 0.07±0.003 0.34±0.015 0.06±0.003 Copaene 훼-humulene Germacrene Caryophellene oxide 훽-sinensal Oxygenated 훼-sinensal Sesquiterpenes Nootkatone Elemol Decanal 0.41 0.43 0.58±0.011 0.16±0.007 0.39±0.010 Other 훼-terpenyl acetate Oxygenated Citronellyl acetate Compounds Octanal 0.22 0.27 0.48±0.013 0.30±0.002 Z-Carveol Trans alpha-bergamotene na 0.01 1,8-Cineole Others Nonanal 0.07 0.06±0.005 0.09±0.009 Perilla aldehyde 0.24 0.08 0.02±0.004

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Annex 3.

Table A3. Total phenolic content (TPC) and total flavonoid content (TFC).

TPC (mg Method Descrption TFC (mg QE/g) Reference GAE/g) 0.5g Citrus sinensis cv. Malta peel-20 mL of 70% (v/v) ethanol-shaking water SLE 13.04 0.933 bath110 strokes per minute-60°C-2 hour Citrus sinensis cv. Malta peel-ultrasonicator with work‐ ing frequency of 20 kHz- (Nishad et al., UAE 15.90 1.050 room temperature 2019b) 0.5 g Citrus sintesis cv.Malta peel-20 mL sodium acetate buffer (0.2 M, pH 4.8) EAE 33.87 2.512 containing Viscozyme L-60°C SLE 0.5 g peel powder- Ethanol 70%- 2h-60°C-110 strokes per minute 15.28 1.397 Citrus paradisi l peels- ultrasonicator with working frequency fixed at 20 kHz-0.5 UAE 21.167 2.765 (Nishad et al., g 2019a) 0.5 g Citrus paradisi l peels-20 mL sodium acetate buffer (0.2 M, pH 4.8) EAE 31.704 3.299 containing Viscozyme L-60°C SC-CO2 Yuzu seed-CO2-45°C-200 bar-2h- CO2 rate 27g/min 15.32±0.02 5.17±0.01 SC-CO2 Yuzu peel-CO2-45°C-200 bar-2h- CO2 rate 27g/min 27.94±0.1 2.71±0.01 SC-CO2 Yuzu mixture (peel and seed)-CO2-45°C-200 bar-2h- CO2 rate 27g/min 24.1±0.1 4.95±0.2 SC-CO2 Yuzu seed-CO2-45°C-300 bar-2h- CO2 rate 27g/min 12.17±0.3 6.75±0.05 SC-CO2 Yuzu peel-CO2-45°C-300 bar-2h- CO2 rate 27g/min 22.4±0.04 2.2±0.02 SC-CO2 Yuzu mixture (peel and seed)-CO2-45°C-300 bar-2h- CO2 rate 27g/min 20.16±0.02 5.14±0.1 (Ndayishimiye et SC-CO2 Yuzu seed-CO2-ethanol-45°C-200 bar-2h- CO2 rate 27g/min 29.18±0.17 5.65±0.02 al., 2018) SC-CO2 Yuzu j peel-CO2-ethanol-45°C-200 bar-2h- CO2 rate 27g/min 43.64±0.5 3.32±0.01 SC-CO2 Yuzu mixture (peel and seed)-CO2-ethanol-45°C-200 bar-2h- CO2 rate 27g/min 40.94±0.3 5.06±0.4 SC-CO2 Yuzu seed-CO2-ethanol-45°C-300 bar-2h- CO2 rate 27g/min 27.31±0.2 4.46±0.06 SC-CO2 Yuzu peel-CO2-ethanol-45°C-300 bar-2h- CO2 rate 27g/min 40.71±1 3.67±0.1 SC-CO2 Yuzu mixture (peel and seed)-CO2-ethanol-45°C-300 bar-2h- CO2 rate 27g/min 37.28±0.8 4.01±0.01 5 g Orange peel-water 10ml/min-9.83 min resident time-semi-continuous flow SW 5.53±0.43 reactor- 150°C 5 g Orange peel-water 20ml/min-4.92 min resident time-semi-continuous flow (Lachos-Perez et SW 3.40±0.73 reactor- 150 °C al., 2020) 5 g Orange peel-water 30ml/min-3.28 min resident time-semi-continuous flow SW 4.31±0.44 reactor-150°C Orange Washington navel peel-5 g dried powder-50 ml methanol,water SLE 9.61±0.65 1.29±0.01 (800:200, v/v)- room temperature-22h-Mgnetic blender Orange Thomson navel peel-5 g dried powder-50 ml methanol,water (800:200, SLE 25.60±0.23 1.28±0.03 v/v)- room temperature-22h-Mgnetic blender Orange Sanguinelli peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- SLE 14.95±0.99 0.91±0.02 room temperature-22h-Mgnetic blender (Lagha- Orange Double fine peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- Benamrouche SLE 12.28±0.17 0.71±0.02 room temperature-22h-Mgnetic blender and Madani, Orange Portugaise peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- 2013) SLE 14.94±0.33 0.29±0.005 room temperature-22h-Mgnetic blender Orange Jaffa peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- room SLE 14.31±0.23 0.56±0.05 temperature-22h-Mgnetic blender Orange Bigarade peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- SLE 31.62±0.88 1.17±0.01 room temperature-22h-Mgnetic blender Orange peel,magnetic stirring 2 rpm-90°C-10 min- 50 mL of a mixture EtOH-H2O SLE 3.9±0.2 17.6±0.6 with 40:60 v/v Lemon peel,magnetic stirring 2 rpm-90°C-15 min- 50 mL of a mixture EtOH-H2O (Gómez-Mejía et SLE 5.9±0.4 18±2 with 40% v ethanol al., 2019) Clementine peel, magnetic stirring 2 rpm-90°C-15 min- 50 mL of a mixture EtOH- SLE 5.5±1.7 16.5±1.2 H2O with 20% v ethanol SLE Grapefruit solid waste- 50% Ethanol-48°C-270 min 36.5±4.2 (Garcia-Castello UAE Grapefruit solid waste- 50% Ethanol-48°C-32 min 71.0±8.5 et al., 2015) Orange peel waste (peel and pulp), 1 mm particle size, 1.2 pH, 600 N (min^-1), 6 SLE 4.4±0.018 L/S(L/kg), 1 hour Orange peel waste (peel and pulp), 1 mm particle size, 4.5 pH, 400 N(min^-1), 6 SLE 15.1±0.022 L/S (L/kg), 1 hour (Senit et al., Orange peel waste (peel and pulp), 1 mm particle size, 1.5 pH, 600 N(min^-1), 3 2019) SLE 6.6±0.033 L acetone/kg residue (L/kg), 6 hour Orange peel waste (peel and pulp), 0.5 mm particle size, 1.2 pH, 400 N(min^-1), SLE 12.3±0.059 3 L acetone/kg residue (L/kg), 6 hour

60

TFC (mg Method Descrption TPC (mg GAE/g) Reference QE/g) 2 g Orange peel lote 1-113 ml ethanol 50% (v/v)-Ultrasonic bath-30°C-15 min-after UAE 5.5±0.1 shaker 200 rpm 15 min 2 g Orange peel lote 2-113 ml ethanol 50% (v/v)-Ultrasonic bath-30°C-15 min-after UAE 6.0±0.1 shaker 200 rpm 15 min SXE lot 1 5 g orange peel-Soxhlet apparatus- 6 hours- absolute ethanol 5.8±0.1 Ethanol 99.5%- 45°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 1.6±0.3 47 kg solvent/kg sample Ethanol 99.5%- 65°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 4.6t±0.2 47 kg solvent/kg sample Ethanol 75%- 45°C-2 g Lote1 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- 47 PLE 11.2±0.1 kg solvent/kg sample Ethanol 75%- 65°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 14.9±0.7 47 kg solvent/kg sample Ethanol 50%- 45°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 9.6±0.9 (Barrales et al., 47 kg solvent/kg sample 2018) Ethanol 50%- 65°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 10.3±0.5 47 kg solvent/kg sample Ethanol 99.5%- 45°C-2 g Lote 2 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 1.4±0.1 47 kg solvent/kg sample Ethanol 99.5%- 65°C-2 g Lote 2 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 4.0±0.1 47 kg solvent/kg sample Ethanol 75%- 45°C-2 g Lote 2Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- 47 PLE 11.1±0.6 kg solvent/kg sample Ethanol 75%- 65°C-2 g Lote 2 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 15.9±0.2 47 kg solvent/kg sample Ethanol 50%- 45°C-2 g Lote 2 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 10.0±0.2 47 kg solvent/kg sample Ethanol 50%- 65°C-2 g Lote 2 Orange peel- 10 Mpa-40 min- flow rate 2.37 g/ min- PLE 10.3±0.5 47 kg solvent/kg sample RUN 1 Limon peels 1 g-50% Ethanol concentration-120s-400W-25 mL/g-A domestic MAE 15.00±0.83 microwave oven (NN-S674MF, Samsung, Malaysia) 2450 kHz working frequency RUN 5 Limon peels 1 g- 60% ethanol concentration- 120s-300W-25 mL/g-A MAE domestic microwave oven (NN-S674MF, Samsung, Malaysia) 2450 kHz working 12.49±0.99 frequency RUN 2 Limon peels 1 g-15 min- 30 amplitude- 30% ethanol solvent- An ultrasonic (Dahmoune et al., apparatus (SONICS Vibra cell, VCX 130 PB, Stepped UAE 11.09±0.32 2013) microtips and probes, No. 630-0422) was used for UAE with working frequency fixed at 20 kHz. RUN 13 - Limon peels 1g -10 min- 50 amplitude-50% ethanol solvent-An ultrasonic apparatus (SONICS Vibra cell, VCX 130 PB, Stepped UAE 15.92±1.23 microtips and probes, No. 630-0422) was used for UAE with working frequency fixed at 20 kHz. Particle size 1.40mm-30°C-20 min-150W- ultrasonic bath (Soniclean Pty Ltd., UAE Thebarton, Australia)- 15.76±0.18 4.65±0.14 operating at a frequency of 43 ± 2 kHz-1g/100mL solvent Extraction time 40 min-30°C-150W-1.40mm-ultrasonic bath (Soniclean Pty Ltd., UAE Thebarton, Australia)- 16.59±0.39 4.81±0.15 operating at a frequency of 43 ± 2 kHz-1g/100mL solvent (Papoutsis et al., Extraction temperature 50°C-20 min-150W-1.4mm-ultrasonic bath (Soniclean Pty 2018) UAE Ltd., Thebarton, Australia)- 17.24±0.15 4.84±0.08 operating at a frequency of 43 ± 2 kHz-1g/100mL solvent ultrasonic power 250 W-30°C-20 min-1.4mm-ultrasonic bath (Soniclean Pty Ltd., UAE Thebarton, Australia)- 15.77±0.21 4.66±0.09 operating at a frequency of 43 ± 2 kHz-1g/100mL solvent 1 g C. sinensis peel powders, acetone, Microwave oven n (Samsung model: NN- S674MF, MAE 12.09±0.06 Kuala Lumpur, Malaysia) frequency 2.45GHz, solvent to solid ratio 15-30mL/g, <80°C, 122 s, 500W 1 g orange peel powder(<125 microm), Ultrasonic system frequency fixed at 20kHz UAE (SONICS Vibra cell), 50 mL of 75,79% acetone, 8.33 min extraction time, 65.94% 10.35±0.04 (Nayak et al., 2015) extraction amplitude, 27+-2°C 1 g orange peel powder(<125 microm), diatomaceus earth, 50% acetone, 1500 psi, ASE 6.26±0.23 120°C, 21 min. 1 g orange peel powder(<125 microm), 50 mL of 50% aqueous acetone (v/v), SLE 10.21±0.01 thermostatic water bath, 60°C, 2 hours, 110 strokes per minute

61

Annex 4.

Table A4. Individual phenolic compounds.

Phlorid Feru Trimet Que Vanil Chloro Naringi zin Caffeic Catech Epicat lic hoxyb Couma rciti lic Metho metric Refere Solvent n dihydr acid in echin acid ezoic ric acid n acid d acid nce (mg/g) ate (mg/g) (mg/g) (mg/g) (mg acid (mg)g) (mg/ (mg/ (mg/g) (mg/g) /g) (mg/g) g) g) 0.5g Citrus sinensis cv. Malta peel-20 mL of 70% (v/v) 18.472 0.0342 0.0420 0.0081 SLE ethanol-shaking water 1.807* 0.127* * * * 7* bath110 strokes per minute- 60°C-2 hour (Nisha Citrus sinensis cv. Malta d et peel-ultrasonicator with 10.796 0.0471 al., UAE 0.935* 0.238* work‐ ing frequency of 20 * * 2019b kHz-room temperature ) 0.5 g Citrus sintesis cv.Malta peel-20 mL sodium acetate 0.0354 0.19 EAE 4.799* 0.208* 2.260* 0.455* buffer (0.2 M, pH 4.8) * 0* containing Viscozyme L-60°C 0.5 g peel powder- Ethanol 0.11 SLE 70%- 2h-60°C-110 strokes 49.03* 0.308* 2* per minute Citrus paradisi l peels- (Nisha ultrasonicator with working 42.038 0.12 UAE 0.086* d et frequency fixed at 20 kHz- * 1* al., 0.5 g 2019a) 0.5 g Citrus paradisi l peels- 20 mL sodium acetate buffer 0.0795 0.27 0.11 EAE 6.97* 0.145* 0.198* 3.052* (0.2 M, pH 4.8) containing * 8* 4* Viscozyme L-60°C Grapefruit solid waste- 50% SLE 22±2 Ethanol-48°C-270 min (Garci aet al., Grapefruit solid waste- 50% UAE 28±3 2015) Ethanol-48°C-32 min

Ethanol 75%- 45°C-2 g Lote1 Orange peel- 10 Mpa-40 0.10± PLE min- flow rate 2.37 g/ min- 0.03 47 kg solvent/kg sample Ethanol 75%- 65°C-2 g Lote 1 Orange peel- 10 Mpa-40 0.4±0. PLE min- flow rate 2.37 g/ min- 3 47 kg solvent/kg sample Ethanol 50%- 45°C-2 g Lote 1 0.062 Orange peel- 10 Mpa-40 PLE ±0.00 min- flow rate 2.37 g/ min- 9 47 kg solvent/kg sample Ethanol 50%- 65°C-2 g Lote 1 0.073 Orange peel- 10 Mpa-40 (Barral PLE ±0.00 min- flow rate 2.37 g/ min- es et 8 47 kg solvent/kg sample al., Ethanol 75%- 45°C-2 g Lote 2018) 2Orange peel- 10 Mpa-40 0.22± PLE min- flow rate 2.37 g/ min- 0.04 47 kg solvent/kg sample Ethanol 75%- 65°C-2 g Lote 2 0.226 Orange peel- 10 Mpa-40 PLE ±0.00 min- flow rate 2.37 g/ min- 5 47 kg solvent/kg sample Ethanol 50%- 45°C-2 g Lote 2 0.113 Orange peel- 10 Mpa-40 PLE ±0.00 min- flow rate 2.37 g/ min- 5 47 kg solvent/kg sample Ethanol 50%- 65°C-2 g Lote 2 0.13± PLE Orange peel- 10 Mpa-40 0.04

62

min- flow rate 2.37 g/ min- 47 kg solvent/kg sample

63

Neohesp Neoeritro Tanger Gallic Naring Hesper Hesperidin Narirutin Referen Method Solvent eridin cin itin acid enin itin (mg/g) (mg/g) ce (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) 5 g Orange peel-water 10ml/min- 1.95±0.2 SW 9.83 min resident time-semi- 22.99±0.66 2 continuous flow reactor- 150°C (Lacho 5 g Orange peel-water 20ml/min- 0.99±0.2 s-Perez SW 4.92 min resident time-semi- 9.82±<0.01 2 et al., continuous flow reactor- 150 °C 2020) 5 g Orange peel-water 30ml/min- 1.22±0.2 SW 3.28 min resident time-semi- 12.21±0.04 3 continuous flow reactor-150°C 0.011 Grapefruit solid waste- 50% 0.43±0.0 0.050±0. 0.09±0.0 (Garcia SLE 0.47±0.07 ±0.00 Ethanol-48°C-270 min 5 008 01 - 1 Castell 0.015 Grapefruit solid waste- 50% 0.83±0.0 0.11±0.0 0.16±0.0 o et al., UAE 0.93±0.03 ±0.00 Ethanol-48°C-32 min 4 1 1 2015) 4 2 g Orange peel Lote 1-113 ml 0.153 0.249 ethanol 50% (v/v)-Ultrasonic bath- 0.26± 0.24± UAE 7.1±0.7 5.0±0.4 ±0.00 ±0.00 30°C-15 min-after shaker 200 rpm 0.03 0.02 4 1 15 min 2 g Orange peel Lote2-113 ml 0.154 0.250 ethanol 50% (v/v)-Ultrasonic bath- 0.24± 0.24± UAE 7.7±0.8 5.5±0.1 ±0.00 ±0.00 30°C-15 min-after shaker 200 rpm 0.03 0.02 1 6 15 min lot 1 5 g orange peel-Soxhlet 1.1±0. 1.3±0. 1.38± 1.37± SXE 18±1 11±1 apparatus- 6 hours- absolute ethanol 1 2 0.08 0.06 Ethanol 99.5%- 45°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow 0.3±0. 0.5±0. 0.3±0. PLE 17±3 4±3 rate 2.37 g/ min- 47 kg solvent/kg 2 3 1 sample Ethanol 99.5%- 65°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow 0.3±0. 0.4±0. 0.3±0. PLE 41±6 6±2 rate 2.37 g/ min- 47 kg solvent/kg 1 2 1 sample Ethanol 75%- 45°C-2 g Lote1 Orange peel- 10 Mpa-40 min- flow 0.17± 0.26± 0.12± PLE 26±5 5±1 rate 2.37 g/ min- 47 kg solvent/kg 0.02 0.03 0.02 sample Ethanol 75%- 65°C-2 g Lote 1 Orange peel- 10 Mpa-40 min- flow 0.32± 0.48± 0.27± PLE 58±3 9±1 rate 2.37 g/ min- 47 kg solvent/kg 0.01 0.02 0.02 sample Ethanol 50%- 45°C-2 g Lote 1 (Barral 0.092 0.118 0.123 Orange peel- 10 Mpa-40 min- flow es et PLE 12±1 3.1±0.7 ±0.00 ±0.00 ±0.00 rate 2.37 g/ min- 47 kg solvent/kg al., 1 3 1 sample 2018) Ethanol 50%- 65°C-2 g Lote 1 0.154 0.193 Orange peel- 10 Mpa-40 min- flow 0.34± 0.12± PLE 34±1 2.4±0.3 ±0.00 ±0.00 rate 2.37 g/ min- 47 kg solvent/kg 0.02 0.02 1 6 sample Ethanol 99.5%- 45°C-2 g Lote 2 Orange peel- 10 Mpa-40 min- flow 1.09± 0.91± 0.85± PLE 42±1 14±2 rate 2.37 g/ min- 47 kg solvent/kg 0.03 0.02 0.06 sample Ethanol 99.5%- 65°C-2 g Lote 2 Orange peel- 10 Mpa-40 min- flow 0.5±0. 0.4±0. 0.32± PLE 55±2 7.1±0.9 rate 2.37 g/ min- 47 kg solvent/kg 2 1 0.07 sample Ethanol 75%- 45°C-2 g Lote 2Orange peel- 10 Mpa-40 min- flow 0.78± 0.37± 0.7±0. 0.64± PLE 37±1 5.4±0.3 rate 2.37 g/ min- 47 kg solvent/kg 0.09 0.05 1 0.06 sample Ethanol 75%- 65°C-2 g Lote 2 0.260 Orange peel- 10 Mpa-40 min- flow 0.54± 0.42± 0.54± PLE 54±1 14±2 ±0.00 rate 2.37 g/ min- 47 kg solvent/kg 0.03 0.03 0.09 2 sample Ethanol 50%- 45°C-2 g Lote 2 0.171 0.215 0.189 Orange peel- 10 Mpa-40 min- flow PLE 26±1 2.9±0.3 ±0.00 ±0.00 ±0.00 rate 2.37 g/ min- 47 kg solvent/kg 5 1 1 sample Ethanol 50%- 65°C-2 g Lote 2 0.36± 0.40± 0.35± PLE 34±1 4.2±0.1 Orange peel- 10 Mpa-40 min- flow 0.04 0.07 0.06

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rate 2.37 g/ min- 47 kg solvent/kg sample 30 g dry orange peel, 190 mL SLE 9.93 methanol, 3 days 13.4 g dry orange peel, 60 mL (Victor SLE 25.18 methanol, 55°C, 3 ours et al., 55.0 g fresh orange peel, 330mL 2020) SLE methanol, 55°C, 3 hours- second 28.45 cycle: 100 mL, 55°C, 3 hours. 500 mg Orange by product (peel, SLE pulp and seeds), 5 mL distillate 12.10 water, 4500 rpm stirring (Louati 500 mg Orange by product (peel, et al., pulp and seeds), 5 mL distillate SLE- 2019) water, 4500 rpm stirring.Pre- 65.01 DIC treatment: dry steam 0.60 Mpa, 4 cycles, 2 min thermal treatment time *Calculated from different units reported.

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Annex 5.

Table A5. Antioxidant activity of citrus residues.

CUPRAC FRAP (mg TEAC (mg Method Description DDPH (mg TE/g) Reference (mg TE/g) TE/g) TE/g) 0.5g Orange peel cv. Malta peel-20 mL of 70% (v/v) SLE ethanol-shaking water bath110 strokes per minute- 15.571* 8.235* 24.241* 7.491* 60°C-2 hour (Nishad et Orange cv. Malta peel-ultrasonicator with work‐ ing UAE 18.819* 11.218* 24.581* 10.044* al., 2019b) frequency of 20 kHz-room temperature 0.5 g Orange cv.Malta peel-20 mL sodium acetate buffer EAE 31.406* 20.056* 50.601* 17.718* (0.2 M, pH 4.8) containing Viscozyme L-60°C 0.5 g grapefruit peel powder- Ethanol 70%- 2h-60°C-110 SLE 13.093* 7.344* 20.784* 8.525* strokes per minute Grapefruit peel , ultrasonicator with working frequency (Nishad et UAE 17.721* 13.723* 23.760* 11.671* fixed at 20 kHz-0.5 g al., 2019a) 0.5 g grapefruit peel,20 mL sodium acetate buffer (0.2 EAE 34.480* 24.681* 53.995* 19.858* M, pH 4.8) containing Viscozyme L-60°C 5 g Orange peel-water 10ml/min-9.83 min resident 3.126±0.47 SW 1.131±0.168 time-semi-continuous flow reactor- 150°C 6 (Lachos- 5 g Orange peel-water 20ml/min-4.92 min resident 2.753±0.57 SW 1.442±0.028 Perez et al., time-semi-continuous flow reactor- 150 °C 3 2020) 5 g Orange peel-water 30ml/min-3.28 min resident 1.902±0.09 SW 0.846±0.088 time-semi-continuous flow reactor-150°C 0 Lemon pomace, Particle size 1.40mm-30°C-20 min- 150W- ultrasonic bath- 32.91±1.4 UAE 0.129±0.002 operating at a frequency of 43 ± 2 kHz-1g/100mL 4 solvent Lemon pomace, Extraction time 40 min-30°C-150W- 1.40mm-ultrasonic bath- 33.47±0.7 UAE 0.111±0.010 operating at a frequency of 43 ± 2 kHz-1g/100mL 4 solvent (Papoutsis Lemon pomace, Extraction temperature 50°C-20 min- et al., 2018) 150W-1.4mm-ultrasonic bath- 36.29±0.6 UAE 0.128±0.002 operating at a frequency of 43 ± 2 kHz-1g/100mL 7 solvent Lemon pomace, ultrasonic power 250 W-30°C-20 min- 1.4mm-ultrasonic bath- 33.61±0.4 UAE 0.117±0.007 operating at a frequency of 43 ± 2 kHz-1g/100mL 2 solvent *Calculated from different units reported.

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Annex 6.

Table A6. IC50 values

Method Description IC50 (mg/ml) Reference 0.5 g dry orange peel powder, ethanol, 1:10 solid/liquid ratio, 100 min, 40°C, 2800 rpm. SLE 0.0352±0.0004 DPPH 0.5 g dry orange peel powder, choline chloride:glycerol 1:2, 1:10 solid/liquid ratio, 100 SLE 0.0448±0.0019 min, 40°C, 2800 rpm. DPPH 0.5 g dry orange peel powder, choline chloride: ethylene glycol 1:4, 1:10 solid/liquid ratio, SLE 0.0306±0.0012 (Ozturk et al., 2018) 100 min, 40°C, 2800 rpm. DPPH 0.5 g dry orange peel powder, ethylene glycol, 1:10 solid/liquid ratio, 100 min, 40°C, 2800 SLE 0.0258±0.0006 rpm. DPPH 0.5 g dry orange peel powder, water, 1:10 solid/liquid ratio, 100 min, 40°C, 2800 rpm. SLE 0.0372±0.0003 DPPH Orange Washington navel peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- SLE 0.901±0.416 room temperature-22h-Mgnetic blender. DPPH Orange Thomson navel peel-5 g dried powder-50 ml methanol:water (800:200, v/v)- room SLE 0.612±0.652 temperature-22h-Mgnetic blender. DPPH Orange Sanguinelli peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- room SLE 0.696±0.326 temperature-22h-Mgnetic blender. DPPH (Lagha- Orange Double fine peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- room SLE 0.796±0.211 Benamrouche and temperature-22h-Mgnetic blender. DPPH Madani, 2013) Orange Portugaise peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- room SLE 0.743±0.144 temperature-22h-Mgnetic blender. DPPH Orange Jaffa peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- room SLE 0.759±0.138 temperature-22h-Mgnetic blender. DPPH Orange Bigarade peel-5 g dried powder-50 ml methanol,water (800:200, v/v)- room SLE 0.568±0.182 temperature-22h-Mgnetic blender. DPPH 100 g orange peel, Clevenger apparatus, 500 mL distilled water, 3 hour, 4 microg/L ozone HD 4.09±0.12 pretreatment, 60°C drying temperature . DPPH (Bechlin et al., 100 g orange peel, Clevenger apparatus, 500 mL distilled water, 3 hour, 40 microg/L ozone 2020) HD 5.70±0.04 pretreatment, 40°C drying temperature. DPPH dried powder of Limon ichang (citrus ichangensis) and mandarin mixture (peels and SC-CO2 0.75±0.06 seeds), CO2 27 g/min, 2 hours. DPPH dried powder of Limon ichang (citrus ichangensis) and mandarin peels, CO2 27 g/min, 2 SC-CO2 0.98±0.01 hours. DPPH (Ndayishimiye and dried powder of Limon ichang (citrus ichangensis) and mandarin mixture (peels and Chun, 2017) SC-CO2 1.26±0.08 seeds), CO2 27 g/min, 2 hours. ABTS dried powder of Limon ichang (citrus ichangensis) and mandarin peels, CO2 27 g/min, 2 SC-CO2 1.51±0.1 hours. ABTS

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