Ethanol and aqueous extracts characterization from guava (Psidium guajava L.) and avocado (Persea americana Mill.) residues

Now a days there is a functional food tendency, which composition containing bioactive compounds is obtained from plant secondary metabolites as a mechanism defense from anabolism and catabolism from primary metabolites. Their constantly consumption offer favorable health effects (Callejas & Pablo, 2002). These substances are a chemical compounds heterogeneous family of and their presence is related to the species, families or morphological plant part studied (Shahidi & Ambigaipalan, 2015).


Introduction
Now a days there is a functional food tendency, which composition containing bioactive compounds is obtained from plant secondary metabolites as a mechanism defense from anabolism and catabolism from primary metabolites. Their constantly consumption offer favorable health effects (Callejas & Pablo, 2002). These substances are a chemical compounds heterogeneous family of and their presence is related to the species, families or morphological plant part studied (Shahidi & Ambigaipalan, 2015).
Mexican avocado and guava productions occupies the first and fifth, correspondent places in worldwide production (México, 2017;Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria, 2017). Commonly, these crops during harvest, industrial processing, use or consumption generate agronomic residues (endocarp seeds, epicarp and leaves) that could be very important as bioactive compounds in foods by their metabolites presented (Food and Agriculture Organization, 2014).
Water as universal solvent and ethanol low toxicity are the most important solvents that have been found (Xavier et al., 2015). Their polarity is linked not only to the type of interatomic junctions (ionic or covalent type), but also to the presence of polar functional groups (hydroxyl, amino) and because of the form hydrogen bridge ability (Ringuelet & Viña, 2013). Therefore, the objective of the present research was to characterize the bioactive compounds with antioxidant properties in guava (Psidium guajava L.) and avocado (Persea americana Mill.) residues (pulp or epicarp and leaves) at different ethanol concentrations (T1 100%, T2 75%, T3 50% and T4 25%, v/v) and in aqueous extracts (T5).

Materials and methods
Experimental part was carried out at the Agricultural Sciences Faculty, Autonomous University of Mexico State, Toluca, Mexico. Leaves, fruit or fruit parts (epicarp or mesocarp) were obtained from young trees randomly compound mixtures from a rural production area during 2018 winter. Guava (Indium guajava L.) variety was Calvillo and avocado (Persea americana Mill) variety

Ethanol and aqueous extracts characterization from guava (Psidium guajava L.) and avocado (Persea americana Mill.) residues
was Hass that were sample from Benito Juarez and Uruapan, Michoacan State, respectably.
Plant material was disinfected with 1% sodium hypochlorite and air forced dried in an oven (Felisa mod. F313A). Leaves and epicarp were dried at 50 °C, 48 h and avocado pulp for 96 h. Finally, dried material was ground until a less 1 mm size was reached (Salem et al., 2011).

Obtaining extracts by maceration
Ethanol and aqueous extracts were made following Salem et al. (2011) methodology with modifications. Ethanol extracts were performed using it at different concentrations (T1 100%, T2 75%, T3 50% and T4 25%, v/v) and aqueous (T5) extracts were done. Plant extracts were prepared at 125 mg/mL and macerated in darkness for 72 h. Finally, they were placed in a 39 °C water bath, 30min in order to facilitate their filtration, which was carried out in No. 41 (Quantitative 240 nm) Filter Paper and placed in amber bottles. Samples were in refrigeration for 24h.

Total phenol determination total (TP)
Folin Ciocalteu spectrophotometric determination followed the Arizmendi et al. (2015) and Spizzirri et al. (2009) methodology. 100 uL of each sample were mix with Folin-Ciocalteu reagent 47.8 uL and incubated for 15 min. 0.1% Na 2 CO 3 300 uL were added to 1080 uL distilled water followed of 2 h darkness incubation. Subsequently, 760 nm absorbance was measured in a spectrometer (GENESYS UV-VIS) and expressed in acid gallic (EAG) per g dry matter (dm) equivalence.

Saponins by solvent separation method determination (Salem et al., 2011).
Saponins quantification started with a secondary metabolites separation using a phase separation funnel. 10mL sample extract were weighed are poured into a funnel with 20mL ethyl acetate (99.7/100, analytical grade, Fermont  ) for 30 min. Phase separation was performed with phenols upper phase part that were removed. Lower part (various compounds) were returned to the funnel and 20 mL of n-butanol (99.9/100, analytical grade, Fermont  , Monterrey, Mexico) were added to separate saponins (SP) phase. Subsequently, quantitation was performed by solvent evaporation and results were registered as dried matter (dm) mg g -1 (Makkar et al., 1998;Salem et al., 2011).

Antioxidant Capacity (AC) (ABTS + ) determination
Antioxidant capacity was quantified using the ABTS method [2.20-azinobis-(3-ethylbenzothiazoline-6-acid)] by Mehta et al. (2014) and Archundia et al. (2019). Radical formation was performed by 7 mM ABTS solution and 140 mM potassium persulfate reaction dark incubated at 25 °C, 16 h. Fresh radical solution was diluted in ethanol analytical grade to achieve a 0.7 ± 0.02 absorbance at 734 nm. 10 mL plant extract was diluted in ethanol 100 mL; due to, 30 mL of this plant extract solution was mixed with ABTS radical solution 3 mL. Absorbance was measured at 734 nm after 6 min of reaction and results were expressed in antioxidant capacity of (TEAC) mmol/g dry matter (dm) Trolox equivalence.

pH determination
pH determination was carried out following Ramírez et al. (2013) methodology. Potential hydrogen (pH) measure was done with a potentiometer (Thermo Scientific Orion STAR A215).

Total Phenol determination
Total phenol (TP) avocado and guava results (Table 1) by solvent concentration or solvent type had shown significant differences between treatments (P ≤ 0.05) for both species. Highest values concentration was observed for 50% (v/v) ethanol extracts (26. 40 mg of EAG g -1 for avocado and 32.08 mg of EAG g -1 dm for guava). Similar results were reported by Monroy-Vázquez et al. (2007) with higher concentrations (2578 mg/mL) from Mexican chile ancho (Capsicum annuum L. grossum sendt) 50% ethanol extracts. Differences were suggested due to solvent polarity. Ethanol is a medium polarity as water is high polarity solvent; in addition to, their allowed to combine and as a result a higher attracting phenols effectiveness were achieved (Archundia et al., 2019).
For avocado vegetative part results (Table 1), epicarp avocado extracts presented the highest total phenol (44.00 EAG g -1 dm). These results are similar to Wang et al. (2010) (12.60 EAG g -1 dm) for Hass variety; besides, it was concluded that avocado seeds and epicarp as a bioactive compounds such as chlorophylls, carotenoids and phenolic compounds (B and A procyanidins) source. TP for guava extracts presented the highest values (29.57 mg of EAG g -1 dm) especially in leaves extracts (Table 1). Guava leaves have been reported with polyphenols (flavonoids, especially quercetin) content (Vargas-Alvarez et al., 2006).
For solvent type or concentration-avocado vegetative part interaction (Table 2). Epicarp 50% ethanol extracts presented the highest TP quantification (56.17 mg of EAG g -1 dm). Similar results were reported by Salmerón (2014) (53.67 mg GAE g -1 dm) from Hass epicarp 80% methanol extracts; nevertheless, methanol extracts are not allowed as food additives. Highest TP guava quantification (45.13 mg of EAG g -1 dm) was found in 50% ethanol-leaves extract interaction (Table 3). Pérez et al. (2014) found lower TP values (9.071 mg of GAE g -1 dm) in young leaves 80% methanol extracts from the same guava variety. Then, total phenol polarity could suggested as higher for the present solvent and guava variety studied (Archundia et al., 2019).

Saponins by solvent separation method determination (Salem et al., 2011)
Saponins results for both species (avocado and guava) show significant differences between solvents treatments (P ≤ 0.05) ( Table 1). Highest saponins concentration was found in 25% ethanol extracts (v/v) (47.18 mg g -1 and 44.54 mg g -1 , avocado and guava, respectably). Koomson et al. (2018) presented similar saponins results in Solanum torvum (53.50 mg g -1 ) 20% ethanol extracts. Saponins are glycosides chemical compounds with a steroidal or triterpenoid type skeleton, where water solubility is facilitated by its high molecular weight, monosaccharide residues presence and aglycone polar groups. Lower saponins solubility has reported from an 80% ethanol solutions concentration; rather than, lower ethanol concentrations performed higher saponins solubility (Donald et al., 2017). Avocado vegetative part extracts with the highest saponins content (46.22 mg g -1 dm) were leaves extracts; likewise, guava leaves extracts presented the highest saponins results (29.14 mg g -1 dm) (Table 1). It has been suggested that this is due to leaves saponins synthesis emerged as plant defense mechanism for their survival against predators (Acamovic & Brooker, 2005).

Antioxidant Capacity (AC)
Avocado and guava 25% ethanol extracts presented the highest AC values (196.62 TEAC/g dm avocado and 314.57 TEAC/g dm for guava) (Table 1). These results agree with Alvis et al. (2012) in AC Curcuma (Curcuma longa) 75% ethanol extracts (2649 mg of Trolox/L). For vegetative part results; further, AC in avocado extracts was found (315.95 TEAC/g dm) in epicarp. It has been suggested because of avocado antioxidants reported such as C, E, B 2 and B 6 , vitamins, pantothenic acid, potassium, and dietary fiber (Kagawa, 2001) specifically in epicarp (epicatechin) (Nose & Fujino, 1982) and catechin (Terasawa et al., 2006), high antioxidant capacity chemical compounds. AC 314.32 TEAC/g dm guava leaves extracts (Table 1) were the highest values found. It has been suggested due to its vitamin E, C, carotenes, monounsaturated fatty acids, sterols and polyphenols, phenolic acids, such as ferulic acid content; hence, they were attributed as antioxidant fruit capacity causes (Chen & Yen, 2007;Wang et al., 2010;Gutiérrez et al., 2006).   Note: Different letters in the columns indicated significant differences between means (P ≤ 0.05) and the equal letters indicated that there were no significant differences between means (P ≤ 0.05). X -= Medium. EAG g-1 dm = Equivalent of acid Gallic per g in dm, dm = dry matter. TEAC g -1 of dm equivalent of antioxidant capacity in Trolox. ----= Samples not analysed.

pH
pH results (Table 1) for avocado and guava presented significant differences (P ≤ 0.05). Avocado range extracts was 5.61 to 6.41, while guava extracts remained in a range of 4.20 to 5.07. Both species presented the same behavior for ethanol or water extracts obtaining pH acids; comparable, ethanol at different concentrations extracts became more basic pH. The most basic pH was 50% ethanol concentration. It was suggested due to the 7.0 water and 6.0 ethanol initial pH; together with, samples pH (around 6.0), maceration hours and temperatures used for extracts isolation. Escribano-Bailón & Santos-Buelga (2003) mentioned that pH determines the phenol solubility degree in the extraction solvent, because it influences the extraction of the compounds that are potentially water soluble; as a result, absolute ethanol or aqueous extracts exposed to the conditions described above presented lower phenols solubility than ethanol at different concentrations extracts and higher phenolic compounds were found in 50% ethanol solutions as a polarity balance consequence suggestion.
For the vegetative or fruit part factor for both species significant difference (P ≤ 0.05) were observed. Avocado extracts were from 5.80 to 6.25 pH. Leaves extracts presented more acidic pH than pulp extracts (Table 1). Guava extracts shown 4.12 to 5.52 pH values, which the most acidic pH was epicarp extract (Table 1). Guava leaves extracts presented higher pH values similar to 6.0 pH. It could be suggested because of  (a, b and c) in the columns indicated significant differences between vegetative part-solvent type or concentration interaction and different letters (v, w, x, y and z) indicated significant differences between treatments or ethanol concentration (T1 100%, T2 75%, T3 50% and T4 25%, v/v) and in aqueous extracts (T5). X -= Medium. DS = Standard deviation. EAG g-1 dm = Equivalent of acid Gallic per g in dm, dm = dry matter. TEAC g -1 of dm equivalent of antioxidant capacity in Trolox. ----= Samples not analysed. different conditions agroclimatic and natural agents during the plant growth until harvesting or sampling time influence; therefore, H + ions concentration in the vacuole from substrates such as sucrose and glucose, causing a putative decrease with slight pH changes and acidity reduction (García et al., 2015;Sánchez et al., 2014).
Vegetative part or fruit-type and solvent type or concentration interaction for avocado extracts included 5.43-6.71 pH values (Table 2). 50% ethanol extract-pulp extracts interaction were the most alkaline pH (6.71). Guava extracts resulted from 3.35 to 5.84 pH values, where the most alkaline pH value obtained was 50% ethanol-leaves extracts interaction. pH value has a significant effect over phenol extraction as more basic (even approaching 8.0) is it as higher phenol extraction concentration is presented (Sepúlveda et al., 2016). Therefore, it pH solvent extraction and samples, whether vegetative part or fruit used, is suggested relevant for bioactive compounds extraction. Note: p ≤ 0.05. Different letters (a, b and c) in the columns indicated significant differences between vegetative part-solvent type or concentration interaction and different letters (v, w, x, y and z) indicated significant differences between treatments or ethanol concentration (T1 100%, T2 75%, T3 50% and T4 25%, v/v) and in aqueous extracts (T5). X -= Medium. DS = Standard deviation. EAG g-1 dm = Equivalent of acid Gallic per g in dm, dm = dry matter. TEAC g -1 of dm equivalent of antioxidant capacity in Trolox. ----= Samples not analysed.

Conclusion
Bioactive compounds with antioxidant quantification were achieved in guava (Psidium guajava L.) and avocado (Persea americana Mill.) residues (pulp or epicarp and leaves) at different ethanol concentrations, where the best concentrations were 50 and 25% ethanol extracts for total phenol and saponins determinations with AC, respectably. Avocado epicarp with guava leaves and both species leaves under the analyzed conditions presented the highest bioactive compounds studied (total phenol and saponins, respectably). Antioxidant capacity was proportional to total phenols amount obtained. Avocado epicarp or guava leaves-25% ethanol interaction presented the best AC. Finally, according to previous reports avocado epicarp and guava leaves 25 or 50% ethanol extracts are putative potential options for human and animal food supplements food; in addition, present extracts studied are suggested as agro-industrial, pharmaceutical and chemical products active ingredients.