Antioxidant capacity and antibacterial activity from Annona cherimola phytochemicals by ultrasound-assisted extraction and its comparison to conventional methods

2.1 Reagent and plant materials

All reagents were used as received and deionized water was used through all experimental procedures. Free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), _L-ascorbic acid (ACS Reagent, ≥99%), methanol (MetOH, anhydrous, 99.8%), copper (II) chloride dihydrate (CuCl₂*2H₂O, ACS reagent, ≥99.0%), neocuproine (DMPHEN, 98%), gallic acid (GA, ACS reagent, ≥98%), sodium nitrate (NaNO₃), aluminum chloride (AlCl₃), Folin-Ciocalteu phenol reagent (2 M), ammonium acetate (CH₃CO₂NH₄, >99.9%), quercetin hydrate (≥95%), (±)-6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid (Trolox, 97%), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ, for spectroscopy det. (of Fe), ≥98%) and iron (III) chloride hexahydrate (FeCl₃*6H₂O, ACS Reagent, 97%) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Hydrochloric acid (HCl, 37%), sodium carbonate (Na₂CO₃, anhydrous, ACS Reagent, >99.5%), sodium hydroxide (pellets, ACS Reagent) and ethanol absolute (99.9%) were provided from J.T. Baker. All solutions were prepared using distilled water.

The leaves from the Annonaceae: Annona cherimola Mill and Annona muricata L. were obtained at the local market “José Ortiz de Domínguez – La Cruz” in Santiago de Querétaro, Querétaro, México. Samples were identified according to the guidelines published from Centro de Investigación Científica de Yucatán (CICY) herbarium (Romero-Soler and Cetzal-Ix, 2015). All used leaves were washed using tap water to eliminate dirt and impurities, then were dried using a conventional oven at 40 °C overnight and finally grounded and kept without humidity for later use.

For antibacterial tests, gram-positive Staphylococcus aureus #6538 and Gram-negative Pseudomonas aeruginosa #13338 were purchased from American Type Cell Culture (ATCC). Microorganism was grown in Luria Bertani media (LB) purchased by BD Bioxon and used as received.

2.2 Extraction methods for the Annonaceae family samples

Soxhlet extraction was used to enhance efficiency in comparison to other commonly used methods such as infusion or decoction as reported elsewhere (Daud et al., 2015; Gavamukulya et al., 2014; George et al., 2015; Mancini et al., 2018; Nawwar et al., 2012) and macerated as another classic extraction approach. For custard apple leaves (Annona cherimola Mill), the three extraction techniques: maceration, Soxhlet extraction and ultrasonic assisted extraction (UAE), were used. For the extraction of leaves from soursop (Annona Muricata L.), Soxhlet was the only extraction technique performed for the samples.

For maceration method, two-extractions per set of leaves were performed using water and ethanol as solvents. 10 g of dried leaves of soursop or custard apple were placed in a sealed baker alongside 150 mL of water or ethanol, which was left to stand for 7 days in the dark at room temperature. After this time, a light-brown extract for water samples and a bright-green extract for ethanol samples were obtained.

For Soxhlet extraction, two different solvents were used for extracting each set of leaves samples. The first extraction only used deionized water and the second extraction was performed using only ethanol. For either of the extractions, ten grams of selected dried leaves were added to 150 mL of the selected solvent (1:15 w/v). The extraction was made at each solvent boiling point for 48 h, to ensure optimal compound extraction, until the sample was run out. In both leaves’ samples, the extract color was dark-brown when using water as solvent while when using ethanol, the color was bright-green.

UAE was used in the search for a faster and more effective extraction of custard apple compounds. Variation of time and power was used to determine the best conditions for compound extraction using a sample:solvent ratio of 1:15 w/v. UAE was performed using a handheld ultrasonic homogenizer UP200Ht (Hielscher, Teltow, Germany). As observed in SX extraction, an extract with dark-brown color was obtained when water was used and while using ethanol, the extract was bright green. The color intensity observed from UAE was similar to SX results, but it was slightly more intense when the time of extraction was also increased.

For energy variation in the UAE, the relative amplitude of the maximum energy produced by the equipment, was varied from 50% to 100%. And the time was 5, 10 or 15 min. Finally, as performed in SX extraction, deionized water and ethanol were used for each of the described conditions.

After every extraction experiment, Whatman # 41 filter paper was used to separate the liquid phase and the samples were stored in sealed amber glass vials at 4° C until further use. Table 1 summarizes experiments performed.

2.3 Spectroscopy analysis of Annona muricata and Annona cherimola: Infrared spectroscopy and Ultraviolet–Visible characterization of the extracts

Spectroscopy characterization of the extract by an experimental FT-IR spectrum was performed and compared to a FT-IR calculated by density functional theory (DFT)spectrum.

Prior to the experimental measurement, samples from both water Soxhlet extracts (GW-SX and CW-SX) were dried using a vacuum desiccator for 48 h to obtain a paste. A Perkin Elmer Spectrum Two (Perkin Elmer,Waltham, MA, United States), was used for Attenuated Total Reflection (ATR) from 4000 to 400 cm⁻¹, to obtain characteristic vibrational modes of the main functional groups present in the paste formed from Annonaceae sp. extracts (Agu et al., 2018; Chen et al., 2013). The results were compared to the theoretical data obtained before.

For the theoretical frequency calculations of the two main annonacin components present in both extracts, bullatacin and aromin (Chen et al., 1999; Gavamukulya et al., 2017), were performed using Gaussian 16 (Frisch et al., 2016). The geometry was fully optimized assuming an ultrafine numeric integration cell, no symmetry restrictions and a temperature set at 278 K; using the hybrid functional Becke 3-Lee-Yang-Par (B3LYP) and with the 6-311g(d,p) basis sets. Simulation conditions were maintained in every step of re-optimization, while convergence in force/displacement parameters in numeric and analytical hessian, ensure a local energy minimum in the final structures. Additionally, no imaginary frequencies were obtained. The simulated IR spectrums were plotted, and the vibrational modes were analyzed using GaussianView program (Nielsen and Holder, 2003).

Then, an experimental UV–Vis spectrum, was obtained from a diluted 1:500 v/v sample from Annona cherimola Soxhlet extract in water on a 1 cm² quartz cuvette and measured from 800 to 200 nm in a VWR 1600-PC spectrophotometer (Delaware, PA, USA) and for comparison a theoretical calculation using Gaussian 16 [34] for aromin the major annonacin component of Annona cherimola Mill [32, 33] in presence of water, by time dependent density functional theory (TD-DFT) using the previously optimized structure was performed in order to obtain a theoretical UV–Vis spectrum to be compared with experimental results.

2.4 HPLC preliminary exploration of acetogenins

To gather evidence of the presence of acetogenins in the extracts a chromatographic analysis was performed using the protocol established by Yang et al (Yang et al., 2010). In short, chromatography was carried out using an HPLC Waters® 600S system (Waters Corporation, Milford, USA), equipped with a RP-C18 reversed-phase column (250 mm × 4.6 mm, 5 µm), coupled to a diode-array detector. For sample analysis 1 mg of dried extract was reconstituted in 1 mL of a methanol:water 85:15 (v/v) solution, and filtered with a 0.45 µm nylon syringe Acrodisc®. Mobile phase consisted of phase A (methanol), and phase B (water) with the following linear gradient conditions: 85% phase A over 40 min, followed by 85–95% phase A for 20 min. The flow rate was set at 1 mL/min and wavelength detection at 220 nm.

2.5 UV–Vis spectroscopy analysis and yield determination of Annona muricata and Annona cherimola extracts

Finally, the characteristic UV–Vis spectra of each sample were measured to 1) observe difference in absorbance when using different solvents for extraction, 2) determine the best parameters for the most efficient UAE and 3) to compare the results between samples and results of the other extraction methods (maceration and Soxhlet). For the UV–Vis characterization a diluted sample of 1:200 v/v in deionized water under a 1 cm² quartz cuvette was measured in the VWR 1600-PC spectrophotometer.

For yield determination of the extracts (Onoja et al., 2014) an overnight dried (at 90 °C) and empty baker was used. First, the beaker was weighed and then the extract was poured on to it. Then, the beaker was weighed after the extract has been concentrated at a constant weigh and finally the yield was calculated as follows:

The results of the extraction yields obtained for each of the performed extractions can be observed in Table 1.

2.6 Determination of total phenolics and total flavonoid of Annona muricata and Annona cherimola extracts

2.7 Determination of antioxidant capacity of Annona muricata and Annona cherimola extracts

Antioxidant capacity was determined by spectroscopy characterization using a VWR 1600-PC UV–Vis spectrophotometer. Experiments were performed using a 1 cm² quartz cuvette and _L-ascorbic acid was used as the standard. The calibration curves of all the assays were obtained using dilutions of an initial 1000 µM of ascorbic acid solution at 100 µM, 200 µM, 400 µM, 600 µM, and 800 µM. Results are expressed as μM of ascorbic acid equivalents (AE) per gram of dry Annona muricata or Annona cherimola leaves used.

2.8 Comparison of antibacterial activity of Annona cherimola extracts using different extraction methods against S. aureus

Antibacterial assays of Annona cherimola acetogenins dry water extracts (maceration, Soxhlet and UAE methods) were performed by the tube macrodilution method, in three independent experiments conducted in duplicate. Each extract was suspended in water at different concentrations: 2000, 1000, and 200 µg/mL, respectively. These concentrations were selected as a reference, in agreement with the previously reported antibacterial activity for other Annonaceous acetogenins (Bento et al., 2013; Trindade et al., 2020). In brief, an inoculum of S. aureus growth at 37 °C in LB media for 16 h, was diluted to a final concentration of 2 × 10⁵ CFU/mL. Extract suspensions were previously sonicated for 2 min, at 70% amplitude to homogenize. Each sample was placed in sterile Eppendorf tubes (1 mL) and mixed with 1 mL of bacterial suspension and incubated at 37 °C for 1 and 2 h respectively. An aliquot of 50 µL of each extract/bacteria interaction was plated in LB agar and incubated at 37 °C for 16 h for surviving bacteria count. Sterile buffer phosphate media (PBS) was used as a positive control. Antibacterial activity in percentage was calculated according to equation (2), where Co is the number of survival colonies in PBS control and C is the survival colonies in each extract sample:

3.1 Annona cherimola and Annona muricata FT-IR spectroscopy characterization: Experimental vs DFT

As observed from the FT-IR spectra of soursop and custard apple extracts from Fig. 1.a, both leaves samples practically have the same absorption bands with some difference in width and intensity, which indicates a similar chemical composition. This should be expected since both samples are from the same family species.

Close

Fig. 1

a) Experimental FT-IR spectra from the Annonaceae sp. extracts. b) FT-IR vibration spectra obtained from the optimized structures calculated by B3LYP from both acetogenin structures proposed. c) Bullatacin and d) aromin molecular structure after geometric optimization by B3LYP hybrid functional.

Export to PPT

From Fig. 1.a, the main characteristic vibrational modes that have been reported from Annona muricata L. are present in both extracts (Akintelu and Folorunso, 2019; Folorunso et al., 2019; Najihah et al., 2016; Santhosh et al., 2015; Sharmila and Sathya, 2017); broad band located at 3340 cm⁻¹ for O—H stretching vibration, at 2936 and 2846 cm⁻¹ assigned to alkane C—H asymmetric and symmetric vibration respectively. The band at 1735 cm⁻¹ is assigned to C⚌O stretching vibration. The band at 1068 cm⁻¹ can also be attributed to an alkene stretching vibration C⚌C and the band at 1040 cm⁻¹ is attributed to anhydrides C—O stretching vibration. Also, the band located at 826 cm⁻¹ is referred to substituted benzene structures. A more extensive description of the bands can be observed in Supplementary Information.

All the functional groups described, especially from O—H and C⚌O vibrations from aromatic rings are generally considered as part of flavonoids, triterpenoids, and phenolic compounds (Akintelu and Folorunso, 2019; Folorunso et al., 2019; Najihah et al., 2016; Santhosh et al., 2015; Sharmila and Sathya, 2017). Acetogenins are also considered, since all described vibrational modes from the functional groups has the potential to be fragments attached to the backbone of their structure.

To further confirm the presence of acetogenins from FT-IR spectra, a theoretical simulation using DFT with a hybrid functional such as the B3LYP, from two different acetogenin compounds, bullatacin for Annona muricata L. and aromin for Annona cherimola Mill (Fig. 1.c and d), is performed. These compounds are considered the most predominant in each extract (Chen et al., 1999; Yang et al., 2010).

As observed from Fig. 1.b, a similar band location, against experimental FT-IR from extracts; regarding the main functional groups, related to O—H, C⚌O, C⚌C, CH₃ and CH₂ molecular vibration is observed and can be further seen in Table S2 and Table S3.

From these theoretical results, it can be observed that a major component besides phenolic compounds in the extracts are acetogenins which confer parts of the antioxidant capacity of the extract as other interesting properties such as high antimicrobial activity which will be studied later.

3.2 Annona cherimola UV–Vis spectroscopy characterization: Experimental vs TD-DFT

In Fig. 2 is observed a comparison between the experimental UV–Vis results of the Annona cherimola Mill water extract from Soxhlet (Fig. 2.a) and the UV–Vis spectra of aromin theoretical calculations predicted by TD-DFT calculation in presence of water (Fig. 2.b).

Close

Fig. 2

a) Experimental UV–Vis spectra from a 1:500 v/v diluted sample of Annona cherimola Mill SX water extraction.and b) Theoretical UV–Vis spectra from aromin TD-DFT calculation.

Export to PPT

From experimental spectra from Fig. 2.a two bands are observed, one intense band located at 205 nm and another weak band at 265 nm meanwhile in the theoretical UV–Vis spectra in Fig. 2.b is observed a band centered at 257 nm.

As it has been previously reported (Hidalgo et al., 2019, 2020) the UV–Vis spectra calculations by B3LYP in methanol have a wavelength shift to lower energies for different Annonaceous acetogenin. These findings are in concordance with our results since in the calculated spectra the experimental appearing at 205 nm is shifted to higher wavelengths (257 nm). The difference is due to the solvent used for calculations and to intermolecular interaction present in the extract between aromin and the rest of the compounds, which are not considered for theoretical calculations and could also mask it.

The experimental and theoretical bands of 205 and 257 nm, respectively, are associated to π → π* and n → π* transitions from the C⚌C and C⚌O of the γ-lactone ring present in the acetogenin aromin (Afonso et al., 2017; Hidalgo et al., 2019, 2020; Rodrigues et al., 2019). On the other hand, the second band present in the experimental spectra at 265 nm, is associated with the presence of other OH rich compounds such as phenolic compounds which typically have a band associated in the same wavelength (Victor Talrose, 2011).

3.3 Preliminary analysis of actegenins presence in Soxhlet samples

Chromatograms from the water and ethanolic Soxhlet samples (CW-SX and CE-SX) can be observed in Fig. 3.

Close

Fig. 3

Chromatographic analysis of a) Soxhlet water extract and b) Soxhlet ethanolic extract.

Export to PPT

The chromatographs from extracts exhibited a comparable elution profile within 5–13 min, which could be attributed to the presence of 12,15-cis-squamostatin-A, and annoglaxin.

Given the scarce evidence of phytochemical profiles of chirimoya extracts, we can only assume that compounds on the co-eluting fractions within 5–13 min on water and ethanolic extracts show similar polarities and results are restrained to the acetogenins identified by Yang et al (Yang et al., 2010), and therefore it is possible that there are other acetogenins (e.g. bullatacin) still to be explored but its presence is confirmed by this extraction method supporting the FTIR and UV–Vis data obtain experimentally and theoretically.

3.4 Analysis of Annona cherimola extraction treatment by UV–Vis spectroscopy and yield of extraction

3.4.2

3.4.2 UAE parameters optimization for Annona cherimola extraction

For the UAE parameters optimization, the UV–Vis spectra of the experiments and yield can be observed in Fig. 4 (yields can also be observed from Table 1). Data can be directly compared because measurements were recorded using the same dilution factor. Spectra are presented from 200 to 400 nm to enhance visualization of the two principal bands to be compared (205 and 266 nm).

Close

Fig. 4

UV–Vis spectra from 200 to 400 nm, of the used parameters for Annona cherimola Mill water and ethanolic extract by UAE: a) 5 min UAE, b) 10 min UAE and c) 15 min UAE. Extraction yield (%) for samples from Annona cherimolla Mill: d) UAE from samples extracted using water and e) UAE ethanolic samples.

Export to PPT

From a general survey of all the UV–Vis spectra obtained using UAE presented in Fig. 4; suggests that to compare water or ethanolic samples only one of the two absorbance peaks obtained is needed; 266 nm for UAE-water samples and 205 nm for UAE-ethanolic samples.

By using either of the extraction solvent in UAE, it is seen that the parameters proposed affects similarly the components obtained. It is observed that when comparing samples at the same time, energy variation has a positive effect because when it is at maximum relative amplitude, the highest absorption peaks can be obtained, and extraction yield is increased.

In contrast, time does not affect the extraction positively in all the cases. When using 50% and 75% of the maximum energy input and time is increased, the maximum absorbance of the compounds (205/266 nm) and yield is also increased, but in contrast; when the maximum energy input is used and time is increased, the maximum absorbance at 205/266 nm and yield is also reduced.

These effects can be related to the physical phenomena of acoustic cavitation (Fig. S5) which consist in the production of microscopic bubbles due to high-intensity ultrasonic waves during rarefaction of the liquid. When the bubbles reach a volume at which they can no longer absorb energy, they collapse violently during a high-pressure cycle. During the implosion very high pressures (approx. 2000 atm) and temperatures (approx. 5000 K which is higher than the 60 °C in which it has been reported some acetogenins degradation (Gutiérrez et al., 2020)) are reached locally and the cavitation bubble results in a liquid jet up to 280 m/s velocity and the resulting shear forces break the cell wall mechanically and improve material transfer. Ultrasound can have either destructive or constructive effects to cells and thus in the extraction yields (absorbance intensity) depending on the sonication parameters employed (Kadkhodaee and Hemmati-Kakhki, 2007; Kim and Zayas, 1989; Technology).

The best extraction parameters (amplitude and time) for both solvent extractions in UAE, are obtained within a 5-minute extraction under the highest amplitude (CW-US13 and CE-US13) and these two samples are used for further discussions.

3.4.3

3.4.3 Comparison of UAE against conventional extraction methods

The data comparing the UV–Vis spectra and the extraction yields of different extraction methods can be observed in Fig. 5 (also for yields observe Table 1).

Close

Fig. 5

Comparison between the water and ethanol UV–Vis spectra obtained from different extraction methods used for Annona cherimola. a) Maceration, b) Soxhlet extraction and c) Ultrasound Assisted Extraction. d) Comparison between the extraction yields obtained from the different extraction used for Annona cherimola.

Export to PPT

For the different extraction methods where water was used as solvent, the absorbance at 266 nm (phenolics) can be ordered from the highest to the lowest as CW-SX > CW-US13 > CW-MC and the same trend could be also observed in the yield extraction. The maceration extraction technique despite to being the one with the longest time of compounds extraction (7 days) is the one with the lowest absorbance intensities and the lowest yield; even the band located at 206 nm can be clearly observed unlike CW-US13 and Soxhlet extractions, in which the higher concentration of acetogenins saturate the equipment resolution and does not allows a clear observation of the band.

If the ratio of extraction time/absorbance of the compounds at 266 nm and the ratio of extraction time/yield is compared; it is seen that MC is the less effective method because after 7 days, it has the lowest absorbance and the lowest yield (8.7%) and CW-US13 is the most effective method even though it does not have the highest absorption or the highest yield (12.7%) like Soxhlet (28.4%), it has the fastest extraction times and yield is higher to maceration which have the longest time of extraction. CW-US13 is 99.82% times faster than CW-SX and the difference of absorbance is only 1 [a.u] apart and its yield is ca. 45% from Soxhlet which could be a negligible difference considering the time and the energy required for Soxhlet extraction process.

Then, when ethanol is used as an extraction solvent the highest absorbance of compounds extraction (205 nm) and its extraction yield can be related to those of water as solvent: CE-SX > CE-US13 > CE-MC.

These six samples (CW-MC, CE-MC, CW-SX, CE-SX, CW-US13 and CE-US13), which are compared through their UV–Vis spectra and extraction yield, are also compared through their antioxidant capacity and their total phenolic and flavonoid content to observe more differences between extraction methods.

3.5 Preliminary analysis of acetogenins presence in UAE samples

The chromatograms from water and ethanolic UAE samples at five minutes under the maximum energy amplitude (CW-US13 and CE-US13) are observed in Fig. 6. All elution analysis were compared with that reported by Yang et al (Yang et al., 2009; Yang et al., 2010)

Close

Fig. 6

Chromatographic analysis of a) UAE water extract and b) UAE ethanolic extract.

Export to PPT

As observed in Soxhlet samples there is a comparable elution profile in early times (5–13 min), which are attributed to the presence of the acetogenins squamostatin-A, and annoglaxin. Among these chromatograms, water UAE (Fig. 6.a) and Soxhlet water extract (Fig. 3.a) show a very similar eluting profile and only the ultrasound-assisted ethanolic extract showed eluting fractions within 11–17, and 30–54 min (Fig. 6.b) that may refer the presence of the following acetogenins: annoglaxin, squamostatin-A, isodesacetyluvaricin, asiminecin, murisolin, and desacetyluvaricin.

The complete HPLC analysis (Fig. 3 and Fig. 6) indicates that such extraction methods would not be suited for acetogenins’ obtention using water as solvent. Contrarily, elution profile comparison between UAE and Soxhlet ethanol extracts clearly reveals an acetogenin-specific extraction of the latter technique (Fig. 6.b a vs Fig. 3.b), and therefore it was the most effective technique to obtain acetogenins from Annona cherimola extracts.

On the other hand, these results are restrained to the acetogenins identified previously (Yang et al., 2009; Yang et al., 2010), and therefore it is possible that there are other acetogenins still to be explored in which to precise identification additional techniques such as nuclear magnetic resonance spectroscopy (NMR) are required.

3.6 Total phenolic and total flavonoid content of the extract

The results for the total phenolic and total flavonoids from both spectroscopy (TP and TF) and electrochemical analysis (e-TP and e-TF) are showed in Table 2. The analysis from the data presented in Table 2 is detailed as follows.

3.7 Antioxidant capacity analysis of the extracts

The results for the different spectroscopy techniques used for Annona muricata and Annona cherimola can be seen in Table 3.

As an analysis from the spectroscopy methods used in Table 3, it is observed that FRAP has the highest values of μM ascorbic acid equivalents per gram of dry leave and contrary to DPPH and CUPRAC test, ethanolic samples have higher values than water samples.

These results give an insight into the antioxidant mechanism of the extracts tested. All samples react in the three performed tests, but because samples react in the DPPH assay, it could be inferred that there is a mixed electron atom transfer mechanism and hydrogen atom transfer mechanism. The difference between the reactivities in FRAP and CUPRAC assays can be explained due to a higher oxidation potential in FRAP (0.7 V) than CRUPRAC (0.6 V), which provokes that the phenolic compounds oxidize more rapidly by this technique (Apak et al., 2016; Gupta, 2015; Prior et al., 2005).

This explains why FRAP has the highest antioxidant capacity values of all the methods tested in the samples. Water samples have more compounds that follow the hydrogen atom transfer mechanism and ethanol extracts have more compounds that follow the electron atom transfer mechanism for free radical scavenging. This proves differences in type of the phenolics compounds and in Annonaceous acetogenins obtained in each solvent sample.

Also, it is important to highlight that Annona cherimola has higher values for DPPH and CUPRAC methodologies than Annona muricata, while the latest has higher values for FRAP than Annona cherimola. This reinforce the idea that different antioxidant mechanism is due to the different types of phenolics obtained in samples and even higher content of Annonaceos acetogenins since it has been reported that DPPH activity is related to the lactone ring present in the structure (Lima et al., 2010).

When compared our results from Annona cherimola to those in literature for other species and different zones of extraction (Adefegha et al., 2015; Daud et al., 2015; Gavamukulya et al., 2014; George et al., 2015; Loizzo et al., 2012; Md Roduan et al., 2019; Nam et al., 2017; Sanchez-Gonzales et al., 2019; SIQUEIRA et al., 2015), is observed for DPPH and FRAP values (there are no reported values for CUPRAC) that the data obtain is in the same magnitude order but is at least half of the reported values, indicating Soxhlet is not the most efficient method for compound extraction.

Regarding extractions methods, it is observed that Soxhlet samples have the highest values for antioxidant capacity from all methods performed for both Annonaceae species and both extraction solvents. The same trend as observed for absorbance, yield and total phenolics from Annona cherimola (Soxhlet > UAE > MC) is also observed in antioxidant capacity, indicating consistency in the results, indicating a correlation between the presence of phenolic content and antioxidant capacity (Coria-Téllez et al., 2018) and it should be expected since phenolic compounds are considered the major phytochemicals responsible for antioxidant capacity. That is why at higher concentration of total phenolics, there is higher antioxidant capacity.

Maceration even though is still highly used as in-house base protocol for the initial examination of components (Gori et al., 2021), is highly inefficient (specifically in time, but also as solvent consuming) comparing even with other classical methods, for the extraction of antioxidant compounds for it use of a possible specific application. Finally, even though UAE has antioxidant capacity values that are at least 50% lower to those obtained by Soxhlet extraction for all the tests (as also observed by UV–Vis and extraction yield), when considering is 99.82% faster it becomes an attractive alternative.

3.8 Antibacterial activity assay of the extracts

The extract obtained with all the bioactive metabolites from Annona cherimola Mill, have gained attention due to their wide range of biological features, produced by the potential antioxidant capacity (Ríos et al., 2003). In this sense, antibacterial activity assays were determined to compare the capacity of different extraction technologies from Annona cherimola leaves in water (CW-MC, CW-SX and CW-US13) against Gram-positive and Gram-negative bacteria. It is important to note that antibacterial activity against Gram-negative P. aeruginosa bacteria was not observed in any extracts evaluated, suggesting that specific cell structure composition in Gram-positive S. aureus plays a key role in the interaction with the extracted compounds.

Fig. 7 shows the antibacterial activity from Annona cherimola Mill water extracts obtained by different methods, against S. aureus. It can be seen in all methods; antibacterial activity is directly proportional to the extract concentration. For maceration method (Fig. 7a) antibacterial activity is ca. 80–90% at 2000 µg/mL, while for 1000 µg/mL is 60–80% at 1–2 h, respectively. At lower concentration (200 µg/mL), antibacterial activity is lower than 30%. Similar behavior is observed in Soxhlet extraction (Fig. 7b), where the higher antibacterial activity presented is at 200 µg/mL at 2 h. In both cases (CW-MC and CW-SX) antibacterial activity slightly increases after 2 h in contact with bacteria. However, by ultrasound assisted extraction (CW-US31 and CW-US33) the antibacterial activity increases, with an almost complete growth inhibition at 2000 µg/mL (Fig. 7c and d), while for 1000 µg/mL AA is above to ca. 90%.

Close

Fig. 7

Antibacterial activity percentage (%) of custard apple water extracts obtained by different extraction methods in water: a) maceration (7 days), b) Soxhlet extraction (48 h), c) ultrasonic assisted extraction (UAE, 5 min) at 50%, and d) UAE at 100%. AA assays were performed against S. aureus at 1 × 105 CFU/mL in LB media for 1 and 2 h. Different letters above the bars represent statistically significant differences at P > 0.05 according to Tukey comparison test (for more details of the statistical analysis see Table S4 of Supporting Information).

Export to PPT

The use of innovative ultrasound assisted methods to compounds extractions allows to obtain significant quantities of bioactive metabolites at lower time, in comparison with commonly used methods (maceration or Soxhlet extraction). Antibacterial activity behavior presented in the water extracts of Annona cherimola Mill has been associated with their capacity to inhibit enzymatic processes, which can interfere in the cellular mitochondrial respiration (Carmen Zafra-Polo et al., 1998; Hasmila et al., 2019). This is produced by the hydrogen atom transfer process, capable of modulating the formation of reactive oxygen species (Hasmila et al., 2019). Specifically, antibacterial activity of the water extract of custard apple against S. aureus suggest that interaction with the peptidoglycan presented in the outer membrane allows the complexes formation, which can promote the destabilization of bacteria permeability (Bento et al., 2013; Pinto et al., 2017).

It has been reported that Annona muricata L. extracts show antibacterial activity against S. aureus (Bento et al., 2013; Viera et al., 2010). In particular, Ríos et al. (Ríos et al., 2003) reports the antibacterial activity of the major constituents of the essential oil from Annona cherimola leaves, obtained by distillation process. It is clear that the extraction process is key in the final concentration of aromatic compounds, such as flavonoids, terpenoids and acetogenins (Hasmila et al., 2019). Our results demonstrate that by UAE method at the lowest time (CW-US13) compared with other processes (maceration at 7 days and Soxhlet at 48 h), it is possible to obtain different compounds including Annonaceous acetogenins with potential biological properties.