, Ali Jaber*
, Ghassan Ibrahim
, Edmomd Cheble
STORAGE EFFECT ON THE GC-MS PROFILING AND ANTIOXIDANT ACTIVITIES OF ESSENTIAL OILS FROM LEAVES OF
ANNONA SQUAMOSA L.
Sara Hammoud, Ali Jaber*, Ghassan Ibrahim, Edmomd Cheble
Laboratory for Research and Development of Medicines and Natural Products, RDMPN, Faculty of Pharmacy, Lebanese University, Beirut, Lebanon.
Aim and objective: Medicinal plants, their biological activities, and their phytochemical contents are important for finding safe and potent new compounds for therapeutic use. In order to investigate the chemical contents and to evaluate the storage effect on the antioxidant activity of Lebanese Annona squamosa (AS) leaf essential oil, the current study was undertaken.
Methods: Shade-dried leaves of AS were taken from Batroun (Lebanon), and the essential oil (EO) was extracted by hydrodistillation. The gas chromatography-mass spectrometry (GC-MS) technique was used to analyze the composition of the EO. Concerning the antioxidant activity, two different methods namely radical scavenging activity (DPPH test) and ferric reducing antioxidant power (FRAP) were used.
Results: A total of 21 compounds were identified. The majority of the identified compounds belong to sesquiterpenoids. β-elemene (11.39 -14.14 %) and β-carophyllene (10.15 – 15.56 %) were the most abundant components. On the other hand, the storage of the plant materials containing the EOs or the EOs themselves leads to a loss in the volatile compounds, which is reflected in the bioactivity as shown in the results of the antioxidants assays. The EOs demonstrated antioxidant activities with IC50 lower than 9 μg.mL-1. DPPH test and FRAP test exhibited a strong positive correlation (r=0.99).
Conclusion: The obtained results suggest that EO extracts from AS have an antioxidant to protect people. Thus, the EO of fresh samples of AS can have interesting applications in versatile areas such as the pharmaceutical and food industries.
Keywords: Annona squamosa, Antioxidant, DPPH, Essential oil, FRAP, GC-MS.
INTRODUCTION
Medicinal plants are part of the history of all continents. Through the centuries, knowledge about plants has been organized, documented, and passed down across generations1. Herbal medicine is now used daily as prevention rather than therapy to protect our health. They are resources of phytochemicals such as flavonoids, polyphenols, alkaloids, tannins, terpenoids, coumarins, and others. Annona squamosa (AS) edible fruit plants belonging to the Annonaceae family is commonly known as the sugar apple, custard apple, and sweetsop2. Research on this plant showed several medicinal properties such as cardiotonic, antimicrobial, insecticidal, and anti-cancerous activities3. Including, but not limited to, Chen et al., isolated new diterpenes and tested their cytotoxic activities4. One of the five diterpenes evaluated showed potent cytotoxicity with an IC50 value of under 20 µM.
AS spread in many countries among them Lebanon, due to its geographical location, and its Mediterranean climate where it adapts well. It is a small, well-branched tree that grows at altitudes of 0 to 2,000 m and does well in hot, dry climates. The plant has been reported to possess a wide variety of pharmacological activities5. Essential oils (EO), volatile compounds extracted from plants, are complex compounds with strong odors, made up of various plant metabolites6. EOs are believed to have many different biological activities7, it have been used for their positive effects on humans since ancient times, as attested by early writings8. Furthermore, the composition of EOs in the same plant species is affected by several parameters, such as harvest time, extraction method, and protection9-11. Numerous investigations were done to study the chemical components of A. squamosa Essential oils (ASEOs), terpenes and sesquiterpenes were the major reported classes2,12,13. ASEOs have shown a wide range of biological properties13-16. EO from AS bark showed significant antimicrobial activity against two gram-positive bacteria species namely Bacillus subtilis (non-pathogenic bacterium) and S. aureus (opportunistic pathogen)12.With IC50 values less than 20 g.mL-1, the ASEO showed strong trypanocidal and antimalarial effects. Furthermore, significant ultrastructural changes, particularly in the cell membrane and mitochondria, block the growth of amastigotes and ultimately lead to necrotic parasite death13. Furthermore, EO from AS pericarps exhibited significant anti-hepatoma activities with IC50 lower than 55 μg.mL-1 against SMMC-7721 hepatoma cell line14.
Earlier studies were devoted to studying the leaves and seeds of Lebanese AS5,17. However, there is no report concerning the essential oils. The present study aimed to extract and identified the volatile organic components from dry leaves of AS using hydrodistillation and GC/MS, to study the changes in EO composition during storage of leaves in paper bags or the storage of EO samples, and measure the effect on the antioxidant activity.
MATERIALS AND METHODS
Plant materials: Collection, Identification, and Preparation
The leaves of AS were collected from the producer in Batroun, in northern Lebanon (80-100 m AMSL). The plant has been identified and confirmations were done via the Flora of the presidency of Madras, by Gamble J.S.18. The voucher specimen (No. 1806) of the plant material is maintained in our laboratory.
AS leaves were collected in February and beginning of March 2019 from Annona trees. The leaves were shade dried for 3 weeks and then pulverized into fine powder. The powder was divided into two batches, the first being stored at room temperature in dark for further use in the next year, and the second part was undergone hydro-distillation extraction to yield the first EO sample (S1). The latter, in turn, was stored at -18°C, in stoppered glass vessels containing some air for one year yielding the second EO sample (S2) (March 2020). Finally, the third sample (S3) is the EO obtained after fresh extraction of the conserved leaves.
Extraction of essential oils of A. squamosa (ASEO)
Foremost, 50 g of AS powder were added into a 1000 mL round bottom flask containing 500 mL of distilled water. The hydro-distillation was performed in a Clevenger-type distillation apparatus designed according to British Pharmacopoeia specifications19. After 3 hours of distillation, the ASEO was collected in the receiver arm. For further use, the oils were sealed and maintained in amber glass vials at 4°C.
Analyses of volatile organic compounds
One microliter of ASEO sample was diluted (1:100) with hexane and injected into the gas chromatography‑ mass spectrometry (GC-MS) system. GC SHIMADZU QP2010 system was used to analyze the volatile compounds in the N. tabacum extract (without derivatization). DB-5MS (5% Diphenyl/95% Dimeth-ylpolysiloxan) capillary column having (30 m length, 0.25 i.d., film thickness 0.28 µm) and helium as carrier gas (1 mL/min, constant flow) was used for compound separation. The oven temperature was programmed from 65°C (2 min initial time) increased to 300°C at 10°C/min (isothermal for the final time). The actual temperature in the MS source reached 230ºC, and the MS was operated in the electron impact mode at 70EV ion source energy. The injector temperature was 250°C, while the injection volume was 1 µL and a total run of one hour is performed, with a mass detector scan range m/z=50–550. Data receipt and processing were performed using Shimadzu GC-MS solution software. The detected compounds were tentatively identified, by MS spectral correlations using NIST08 (National Institute of Standards and Technologies, Mass Spectra Libraries), as well as published data.
In vitro antioxidant activities
The antioxidant activity was measured using two methods namely DPPH free radical scavenging assay and reducing power assay.
DPPH assay
2, 2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) method for determining antioxidant activity isa spectrophotometric method based on the hydrogen atom transfer and single electron transfer mechanisms. These assays included radical scavenging activity, based on the antioxidant reduction of the violet DPPH radical via a hydrogen atom transfer mechanism, resulting in the color change (Sirivibulkovit et al.,). The violet DPPH radical is measured by a UV-Vis spectrophotometer at approximately 515 to 520 nm. Several solutions of increasing concentrations varying from 0.81 ng.mL-1 to 10.81 ng.mL-1 of ascorbic acid were prepared in test tubes. About 1 mL of the ASEO solution of different concentrations (5 to 25 µg.mL-1) was taken in test tubes, then 1 mL of the DPPH methanolic solution (81.15 μM) was added. Simultaneously, a control was generated by mixing 1 mL of DPPH solution with 1 mL methanol. After 30 min of incubation in dark at room temperature, the decrease in absorbance of each mixture (due to quenching of DPPH free radicals) was determined at 517 nm against a blank (methanol) using a UV-VIS spectrophotometer.
Based on graphic values of the percentage of DPPH inhibition vs EO concentrations, the half-maximal inhibitory concentration (IC50) (the concentration of the sample needed to inhibit 50% of the DPPH) of each sample was estimated. The antioxidant activities of all the samples were compared to the antioxidant activity of ascorbic acid, i.e., ascorbic acid was used as a reference standard.
Reducing power assay
The reducing power assay method is designed based on the reduction potential of the components by reacting with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+). The latter product mixed with ferric chloride forms a ferric–ferrous complex having an absorption maximum of 700 nm21.The reducing ability of ASEO was determined according to a method reported by Oyaizu (Oyaizu, 1986). The aliquots of different concentrations (10 to 100μg.mL-1) of the standard/test sample (methanolic solutions) were mixed with 2.5 mL of (pH 6.6) phosphate buffer + 2.5 mL of (1%) potassium ferricyanide. Subsequently to a cooling step, the mixture was placed in a water bath at 50°C for 20 minutes. About 2.5 mL (10%) trichloroacetic acid aliquots were added to the mixture, which was then centrifuged for 10 minutes at 3000 rpm. The upper layer of solution of 2.5 mL was mixed with 2.5 mL distilled water and a freshly prepared 0.5 mL of (0.1%) ferric chloride solution. The absorbance was measured at 700 nm in a UV spectrometer. The solutions were prepared on the day of the experiment and well protected from sunlight. Ascorbic acid at various concentrations (5 to 40μg.mL-1) was used as standard.
he sample was prepared using a similar procedure but by replacing the EO with an equal volume of methanol. The absorbance values were plotted against the concentration, and a linear regression analysis was carried out. The higher absorbance of the reaction mixture indicates a greater reducing power. All data were recorded as mean ± SD for three replicates.
Statistical analysis
The experimental runs and the analyses were carried out in triplicate. The experimental results derived in the study were expressed as the mean ± standard deviation (SD). The correlation coefficients were calculated with Pearson’s test using Excel 2013 (Microsoft Corporation, Redmond, WA, USA). Linear regression analysis was used to calculate the IC50 values. Independent samples t-tests were used to analyze the obtained data. Statistical significance was considered where p<0.05.
RESULTS AND DISCUSSION
Compound identification using GC-MS
The essential oil obtained by hydro-distillation from the leaves of A. squamosa was yellowish-green and the yield was 0.1% (v/w), based on dry weights. The chemical constituents of ASEO samples were analyzed by GC-MS (Figure 1). This led to the identification of different compounds that were determined by referring to previously published articles and referring to the suggestions of the NSIT Library23. The chemical composition of the ASEO is shown in Table 1.
The components are listed in order of their elution on the DB-5MS column. The results showed that the EO of the three samples was mainly composed of the sesquiterpenoids. By comparing samples, S2 to S3, some of the sesquiterpenoids that were present in S3 were not found in S1. Besides, by comparing sample S2 with the results from the previous year (S1), the compounds that were present in S1 disappeared from S2. A total of 19 (61.2%), 11 (51.07%), and 10 (43.33%) compounds were identified in the sample S1, S2, and S3 respectively. ASEO was predominantly composed of sesquiterpenes (50.78%), and the remainders are monoterpenes. Bicyclic sesquiterpenes comprised 19.66% of the sample. The three major constituents that were discovered were δ-elemene (11%), carophyllene (10.15%), and β-elemene (14.14%). While β-elemene is absent, the two other compounds are in agreement with the results of Al-Nemari et al.,24,. EOs from the leaves of numerous Annonaceae genera, including Annona, have been discovered to include spathulenol and caryophyllene oxide, which could be used as chemotaxonomic identifiers for these genera25,26. After storage, the percentage of α-humulene increases from 0.6 to 2.57% in agreement with the work of Mockutë10.
In contrast, although the amounts of compounds with caryophyllene (β‑caryophyllene + caryophyllene oxide) were nearly the same in fresh and stored EOs, caryophyllene oxide decreased from 8.15% (S1) and disappear in S2. On the other hand, the proportion of constituents with a low molecular weight (monoterpenes) significantly decrease, while the conditional percentage of larger molecular weight molecules having three isoprene units (sesquiterpenes) increased as a result of the above decrease. It's worth noting that the content in α-pinene, linalool, and thymol in S2 increases per the results of Baritaux et al.9. Other researchers have found lower amounts of monoterpenes and higher levels of certain sesquiterpenes in dill and ginger27,28.
EOs derived from fruit and seeds of annonacea species are mainly consti-tuted of monoterpene hydrocarbons29, while sesqui-terpenes predominate EOs of leaves as hydrocarbons forms and in bark and roots as oxygenated forms.
This profile variation of the samples can be attributed to the fact that EO components are known to readily transform into one another by oxidation, isomerization, cyclization, or dehydrogenation reactions induced either enzymatically or chemically, due to their structural link within the same chemical group30. There have been many reports about the composition of EOs from the different parts of A. Squamosa).
For instance, the chemical profile of EO from the leaves of A. Squamosa growing in Badagary (Nigeria) was mainly composed of (E)-caryophyllene (38.9%) and eugenol (30.2%)31. This research work aims to study the effect of the extraction factor on the volatile compounds occurring in AS. The GC-MS method highlighted the difference in the content of the EO whether it was extracted and conserved in the refrigerator or preserved in the leaves.
The chemical composition of EOs and plant secondary metabolites, in general, is affected by different abiotic
factors, namely climate, growing conditions, or harvest time are the most studied32. Sesquiterpenes isolated from EOs are among compounds with promising antimicrobial activity35,36. These encompass β-caryophyllene, a sesquiterpene extensively present in EOs, which possesses anti-inflammatory and anticarci-nogenic activities37. Its oxygenated form caryophyllene oxide, present at 8.15% in the obtained EOAS, owns high antimicrobial properties38. Furthermore, β-elemene presents good antitumor and anti-inflamm-atory activities without obvious cytotoxicity or clinical side effects39.
DPPH assay
In the antioxidant test of essential oils, much positive control can be used, such as quercetin, trolox, α-tocopherol, ascorbic acid, and many others herein ascorbic acid was used in the two tests. The three assessed EOs were able to reduce the stable, purple-colored radical DPPH· to yellow colored DPPH-H, thus samples S1, S2 and S3 had IC50 values of 6, 9, and 8 μg.mL‑1 respectively (Figure 2) and varied signify-cantly (p<0.05). The positive control (ascorbic acid) had an IC50 value of 3 μg.mL‑1. The highest antioxidant activity was obtained with the sample with the lowest IC50.
Among the samples, S1 revealed the lowest IC50, this means that sample S1 has more ability to inactivate free radicals leading to more antioxidant activity. Based on the previous GC-Ms results, this could be explained by the fact that the number of secondary metabolites (such as phenolic compounds, terpenoids) present in the ASEO was influenced by several factors including duration of conservation, and chemical variability. The obtained IC50 values are in good agreement with those obtained for a different part of A. squamosa40. The IC50 is lower than the 1.33 mg.mL‑1 reported for seed oil41. In addition, the results showed that it outperforms EO from the leaf of its peers A. muricata (244.8 μg.mL‑1)42.
FRAP method
The antioxidant potential of plant extracts or EOs may be determined by their reducing power43.The reducing power of ASEO was determined for samples S1, S2, and S3 at different concentrations (Figure 3). It was observed that the absorbance of all samples gradually increases with the increasing concentration of oil. Also, the capacity of the extracts to reduce Fe3+ to Fe2+is lower than that of ascorbic acid, and sample S1 had the greatest reducing power. The reducing power of the three ASEOs is found lesser than the positive control compound, and there was a significant difference at p<0.05. Current findings were, qualitatively, in agreement with the observation obtained with AS leaves extracts37,38, 44,45, where they found that the reducing power of ascorbic acid exceeds that of AS extracts. In other words, the reducing power of A. squamosa extracts referred to its electron transfer capacity in a redox reaction, leading to the neutralization of free radicals and forming stable products. It has been reported that the reducing power of extracts probably depends on the hydrogen-donating ability present in terpenoids and
phenolic compounds. As a result, antioxidants can be thought of as reductants, and the inactivation of oxidants.
Significant correlations were obtained between the ASEO and the antioxidant activities via two different assays. Table 3 shows the correlation between DPPH and FRAP assays of ASEOs obtained along different storage conditions. A positive correlation exists between the way of conservation and DPPH(r=0.88) and FRAP (r=0.81) assays. So, there is a high possibility of the same reasons, same mechanisms or same bioactive compounds influence the antioxidant activity ASEO with DPPH and FRAP assays. On the other side, it merits featuring that, a significant correlation was obtained between the two antioxidant assays, and various examinations have likewise revealed this relationship46,47.
Limitations
Besides the limited number of samples, the limitation of the study was the biological activities tests, restricted to antioxidant tests, which might cover other potential biological effects, such as anticancer and antimicrobial.
CONCLUSION
The phytochemical content and antioxidant activity of A. squamosa essential oils were influenced by the time of conservation. In this work, the phytochemical screening using GC-MS revealed that different oil of ASEO contain sesquiterpenoids. In addition, many compounds present in EO obtained from fresh samples disappeared with time even with conservation at -18°C. Same results were obtained with conserved plant materials. The results of antioxidant activity showed that freshly prepared EO samples from freshly dried leaves had exerted the best antioxidant activity. The results were confirmed by using two antioxidant assays namely DPPH and reducing power assay. Thus, EO of fresh samples can have an interesting application in versatile areas such as the pharmaceutical and food industries. Further studies can be performed to reveal the reactions responsible for the biological activities present in the ASEOs. Evaluation of the anticancer activity of sample S1 against cell lines can be done in the future since antioxidant is correlated to anticancer activity.
ACKNOWLEDGEMENTS
The authors are grateful to the Lebanese University (Faculty of Pharmacy and Faculty of Sciences) Lebanon for providing gall chemicals and products necessary to carry out this work. The GC-MS spectra were performed at the Lebanese Agricultural Research Institute Laboratory. The assistance of the staff is gratefully appreciated.
CONFLICT OF INTEREST
The authors declare that there is no real, potential, or perceived conflict of interest for this article.
AUTHOR CONTRIBUTIONS
Concept: A.J., G.I., E.C.; Design: A.J., G.I., E.C.; Control: A.J., E.C.; Materials: A.J.; Data Collection and/or processing: S H., A.J.; Analysis and/or interpretation: S.H., A.J., G.I., E.C.; Literature review: S.H., A.J.; Manuscript writing: S.H., A.J.
REFERENCES