OPTIMIZATION OF ULTRASOUND−ASSISTED EXTRACTION OF PHENOLIC COMPOUNDS FROM WOOD APPLE PULP: IDENTIFICATION OF PHYTOCHEMICALS USING GC−MS

Abstract

U ovom radu korištena je ultrazvučna ekstrakcija (UAE) za dobijanje fenoinih jedinjenja iz pulpe drvene jabuke. Za optimizaciju procesnih usiova korišćen je Box-Behnken dizajn. Rezultati su pokazaii da UAE imaju značajno više ukupnih fenoinih jedinjenja (TPC) (17,41±0,13 mg GAE/g suve mase) i TFC (6,73±0,19 mg RE/g suve mase) u pored strok signenju sa ekstrakcijom rastvarača koja je dala značajno niži TPC (12,01±0,17 mg GAE/g suve mase) ) i TFC = 4,47±0,09 mg RE/g suve mase). Optimalno dobijeni ekstrakti drvene pulpe jabuke su podvrgnuti fitokemijskom skriningu i nad strok signeno je da su bicikio[2.2.1]heptan, 2-(1-buten-3-ii)-servrogenin acetat, 4H-piran-4-on, 2,3-dihidro-3,5-dihidroksi-6-metii-, L-(+)-askorbinska kiselina 2,6-diheksadekanoat, cis-vakcenska kiselina, tiopen, 2-propii-, fenol, 2,4-bis(1,1-dimetHetii) i oktanoična kiselina bili prisutni samo u ekstraktima UAE. Pošto je prisustvo bioaktivnih komponenti u UAE značajno veće u pored strok signenju sa drugim tehnikama, izgleda da je ona bolja metoda za ekstrakciju

Alternate abstract:

In the present work, ultrasound-assisted extraction (UAE) was employed to extract the phenolic compounds from wood apple pulp. A Box-Behnken design was used to optimize the process variables. The results revealed that UAE have significantly higher TPC (17.41±0.13 mg GAE/g dW) and TFC (6.73±0.19 mg RE/g dW) in comparison with solvent extraction that gave noticeably lower TPC (12.01±0.17mg GAE/g dW) and TFC = 4.47±0.09 mg RE/g dW). Opiimaiiy obtained extracts of wood apple pulp were subjected to phytochemicai screening and it was found that bicycio[2.2.1]heptane, 2-(1-buten-3-yi)-serverogenin acetate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyi-, l-(+)-ascorbic acid 2,6-dihexadecanoate, cis-vaccenic acid, thiopene, 2-propyi-phenoi, 2,4-bis(1,1-dimethyiethyi) and octanoic acid were present only in UAE extracts. Since the bioactive component presence is remarkably higher in UAE compared to other techniques, UAE seems to be a better method for the extraction of phenolic compounds from wood apple pulp.

Headnote

Article Highlights

* Response surface methodology was applied for optimization of solvent extraction and ultrasound-assisted extraction

* UAE is an effective method to obtain maximum yield with minimum time and solvent consumption

* GC-MS analysis was carried out to identify the bioactive compounds present in the extract obtained from various extraction techniques

Abstract

In the present work, ultrasound-assisted extraction (UAE) was employed to extract the phenolic compounds from wood apple pulp. A Box-Behnken design was used to optimize the process variables. The results revealed that UAE have significantly higher TPC (17.41±0.13 mg GAE/g dW) and TFC (6.73±0.19 mg RE/g dW) in comparison with solvent extraction that gave noticeably lower TPC (12.01±0.17mg GAE/g dW) and TFC = 4.47±0.09 mg RE/g dW). Opiimaiiy obtained extracts of wood apple pulp were subjected to phytochemicai screening and it was found that bicycio[2.2.1]heptane, 2-(1-buten-3-yi)-serverogenin acetate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyi-, l-(+)-ascorbic acid 2,6-dihexadecanoate, cis-vaccenic acid, thiopene, 2-propyi-phenoi, 2,4-bis(1,1-dimethyiethyi) and octanoic acid were present only in UAE extracts. Since the bioactive component presence is remarkably higher in UAE compared to other techniques, UAE seems to be a better method for the extraction of phenolic compounds from wood apple pulp.

Keywords: wood apple pulp, ultrasound-assisted extraction, phytochemicais, solvent-assisted extraction.

U ovom radu korištena je ultrazvučna ekstrakcija (UAE) za dobijanje fenoinih jedinjenja iz pulpe drvene jabuke. Za optimizaciju procesnih usiova korišćen je Box-Behnken dizajn. Rezultati su pokazaii da UAE imaju značajno više ukupnih fenoinih jedinjenja (TPC) (17,41±0,13 mg GAE/g suve mase) i TFC (6,73±0,19 mg RE/g suve mase) u pored strok signenju sa ekstrakcijom rastvarača koja je dala značajno niži TPC (12,01±0,17 mg GAE/g suve mase) ) i TFC = 4,47±0,09 mg RE/g suve mase). Optimalno dobijeni ekstrakti drvene pulpe jabuke su podvrgnuti fitokemijskom skriningu i nad strok signeno je da su bicikio[2.2.1]heptan, 2-(1-buten-3-ii)-servrogenin acetat, 4H-piran-4-on, 2,3-dihidro-3,5-dihidroksi-6-metii-, L-(+)-askorbinska kiselina 2,6-diheksadekanoat, cis-vakcenska kiselina, tiopen, 2-propii-, fenol, 2,4-bis(1,1-dimetHetii) i oktanoična kiselina bili prisutni samo u ekstraktima UAE. Pošto je prisustvo bioaktivnih komponenti u UAE značajno veće u pored strok signenju sa drugim tehnikama, izgleda da je ona bolja metoda za ekstrakciju

fenoinih jedinjenja iz pulpe drvene jabuke. Ključne reči: pulpa drvene

(ProQuest: ... denotes formulae omitted.)

Wood apple is an underutilized fruit which is highly rich in antioxidants [1]. Hence, various parts of the plant possess curative properties [2,3]. In addition, the fruit pulp is said to have enormous amounts of phytochemicals like polyphenols, tannins, vitamins, amino acids and flavonoids [4,5]. These phytochemicals promote health benefits such as the reduction in the incidence of some degenerative diseases like cancer and diabetes [6], and reduction in risk factors of cardiovascular diseases [7]. Great attention has been paid to the replacement of synthetic additives by natural ones for the past few decades. Isolation, identification and characterization of bioactive compounds play a vital role. Extraction is one such process for the isolation and identification of these compounds [8]. Different extraction methods are used for the extraction of valuable compounds (i.e., Soxhlet, maceration, aqueous enzymatic extraction, etc.) [9] and the major drawback of these conventional extraction methods are longer duration, large amount of solvent consumption and high temperatures [10]. Due to its advantages, i.e., simplicity, inexpensive equipment, solvent consumption, minimum temperature and time, ultrasound-assisted extraction (UAE) has been used to overcome these drawbacks [11]. UAE is mainly attributed to behavior of cavitation bubbles upon pro pagation of acoustic waves which result in the disruption of the material matrix, facilitating the release of extractable compounds and enhancing mass transfer of solvent into the sample [12].

Optimization of the experimental conditions is a critical step in developing a successful extraction process due to the effect of various process variables on the extraction yield [13]. Response surface methodology (RSM), a statistical method used in mathematical modeling, has been successfully applied in optimizing extraction condition of a wide range of polyphenols, antioxidants and other metabolites in plants [14,15]. The present study was undertaken to evaluate the effect of extraction parameters, viz. extraction time, temperature and feed-solvent ratio on the extraction of bioactive compounds from wood apple fruit using methanol and to further optimize the conditions for obtaining maximum yield of polyphenols using RSM. The objective was to evaluate the suitability and efficiency of ultrasound-assisted extraction (UAE) as compared to conventional extraction methods (i.e., Soxhlet extraction and solvent extraction (SE). The screening of phytochemicals present in extracts was carried out using gas chromatography-mass spectroscopy (GC-MS).

MATERIALS AND METHODS

Pre-treatment

Fully matured wood apple fruits were purchased from a local market and the pulp was separated manually. The fruit pulp was air-dried for a week and then crushed and ground into powder using a blender. The coarse powder was then stored in an airtight container in a cool, dark and dry place. The sample was soaked in methanol for an hour before extraction [16].

Extraction methods

Soxhlet extraction

For the Soxhlet extraction, the powdered sample (10 g) and methanol (250 mL) were taken and the extraction process was executed at 50 °C for 6 h. After the extraction process, the extract was recovered which was used for further analysis [11].

Solvent extraction (SE)

For solvent extraction (SE) the sample was mixed with methanol in the ratio of 1:15, 1:20 and 1:25 g/mL in a conical flask covered with aluminum foil. With different time intervals of 4, 6 and 8 h; temperature: 35, 40 and 45 °C. All bottles were shaken by a temperature-controllable orbital shaker at 100 rpm [17].

Ultrasound-assisted extraction (UAE)

The powdered sample (1 g) was mixed with 15, 20 and 25 ml of methanol and was extracted for 5, 15 and 25 min in a bath-type ultrasonic extractor at 35, 40 and 45 °C temperature. After each extraction, the sample was cooled at room temperature. The samples were filtered using Whatman No. 1 filter paper before measuring the yield of the extract. It was stored for further analysis [18].

Analysis - total phenolic content (TPC)

Total phenolic content (TPC) was determined using the Folin-Ciocalteu method [19]. The sample extract (1 mL) was mixed with Folin-Ciocalteau reagent (2.5mL) and a few minutes later, 7.5% sodium carbonate (3 mL) was added. The mixture was kept in dark for half an hour and the absorbance was noted using a UV-Vis spectrophotometer. A standard curve was prepared using gallic acid diluted to different concentrations (10 mg/L to 500 mg/L). The results are expressed in mg of gallic acid equivalent (GAE)/g dry weight (dW).

Analysis - total flavonoid content (TFC)

Total flavonoid content (TFC) was determined using the aluminum chloride method [20]. The sample extract (1 mL) was mixed with aluminum chloride (1 mL), potassium acetate (1 mL) and sodium hydroxide (3 mL). The mixture was allowed to stand at room temperature and the absorbance was measured at 510 nm with a UV-Vis spectrophotometer. Rutin was used as the standard and the results were expressed in mg of rutin equivalent (RE) /g dry weight (dW).

Gas chromatography-mass spectroscopy (GC-MS)

The bioactive compounds were determined by subjecting the methanolic extract to gas chromatography and mass spectroscopy. GC-MS analysis was carried out using a Bruker 45X- GC-44 (GC) and SC-ION (MS) capillary column, the length of which was 15 m. Helium was used as carrier gas, the flow rate was set at 1 ml/min and the injection volume of the sample was 1 |iL. Temperature was set at 70 °C with a hold of 5 min. Total running time of GC was 45 min. Mass spectra were taken at 70 eV. Identification of components of GC-MS were conducted using the National Institute of Standards and Technology (NIST).

Response surface methodology (RSM)

Response surface methodology (RSM) is an effective technique employed for multiple regression analysis using quantitative data [21] and for optimizing complex processes. In the present study, RSM was used to optimize the process variables such as temperature, time and feed solvent ratio on TPC and TFC. In addition to that, their individual and interaction effects on responses (TPC and TFC) were also studied. Coding of the variables was done according to the following equation [19]:

... (1)

The experimental runs were established based on a Box-Behnken design (BBD). To calculate the number of experiments (N) required for the development of BBD the following equation was used:

... (2)

where k is the number of factors and C0 is the number of central points [19].

Model fitting

An empirical model with individual and interaction terms of independent variables was developed using a second-order polynomial equation [19]. The generalized form of the equation is given below.

... (3)

Y- predicted response, ß - predicted model intercept, ß, ß, ß - linear coefficients, ß11, ß2, ß3 - quadratic coefficients, ß2, ß13, ß3 - interaction coefficients and X, X2, X3 - independent variables.

RESULTS AND DISCUSSION

From the results (Table 1, a), it is observed that the yield of phenolic content ranged from 3.73 to 11.34 (mg GAE/g) and flavonoid content from 1.59 to 4.39 (mg RE/g). The TPC and TFC yield is found to be higher (11.34 mg of GAE/g and 4.39 mg of RE/g) at 40 °C, 6 h and 1:20 g/mL whereas the yield is lower (3.73 mg of GAE/g) at 35 °C, 6 h and 1:15 g/mL and (1.59 mg of RE/g) at 40 °C, 4 h and 1:15 g/mL. It is observed that when extraction time is increased, the phenolic content and flavonoid content of wood apple pulp extracts increased gradually, as shown in Table 1 (a). The value started to decline after 6 h. Further increase in extraction time increases the chance of degradation of phenolic compounds [22]. From the results, it is also observed that at higher temperatures there is a significant increase in the extraction of TPC and TFC. This may be due to the fact that at elevated temperatures mass transfer and penetration of solvent into the sample matrix increases [13,23]. Also, when increasing the temperature beyond 40 °C, it is observed that there are slight changes in the total phenolics. This may be due to degradation of bioactive components. Hence, temperature cannot be increased beyond a certain level even though it has positive effects [24]. Similarly, the increase in feedsolvent ratio increased TPC and TFC to 11.34 mg GAE/g and 4.39 mg RE/g, respectively. This may be due to the reason that when more solvent enters the cell walls, more phenolic compounds could infuse into the solvent if the feed-solvent ratio is higher.

Analysis of variance (ANOVA) was employed for testing the adequacy and fitness of the developed models. The regression coefficient and second order polynomial equation of the predicted model in coded variables are given in Table 2. Radj values (0.9996 for TPC and 0.9999 for TFC) were found to be equal to R2. An excellent correlation between the experimental and predicted response values were exhibited through the very high value of the correlation coefficient. The lack of fit and its associated p-value was significant which showed the fitness of the proposed statistical models (Table 3) [19].

Results (ultrasound-assisted extraction) showed that the yield of phenolic content ranged from 2.32 to 15.74 (mg GAE/g) and flavonoid content 0.45 to 6.25 (mg RE/g). From Table 1, the TPC and TFC yield is found to be higher (15.74 mg of GAE/g and 6.25 mg of RE/g) at 15 min, 40 °C and 1:20 g/mL whereas the yield is lower (2.32 mg of GAE/g) at 15 min, 35 °C and 1:15 g/mL and (0.45 mg of RE/g) at 15 min, 35 °C and 1:15 g/mL. It is observed that when extraction time is increased from 5 to 15 min, the phenolic content and flavonoid content of wood apple pulp extracts increased gradually from 2.32 to 15.74 mg GAE/g dW and 0.45 to 6.25 mg RE/g dW, respectively. The value started to decline to a minimum of 6.56 mg GAE/gdW and 2.19 mg RE/g dW at 25 min. This could be explained by Fick's second law of diffusion [23]. Further increase in extraction time increases the chance of degradation of phenolic compounds [22]. From the results, a significant increase in the extraction of TPC and TFC at elevated temperatures is also observed. This is due to enhanced mass transfer and penetration of solvent into the sample matrix [13,23]. When increasing the temperature beyond 40 °C, it is observed that there are slight changes in the total phenolics. This may be due to degradation of bioactive components. Hence, temperature cannot be increased beyond a certain level even though it has positive effects [24]. In addition, when the feed-solvent ratio increased from 1:15 to 1:20, the total phenolic content and flavonoid content also increased to 15.74 mg GAE/g and 6.25 mg RE/g, respectively. This may be due to the fact that more phenolic compounds could infuse into the solvent if the feed-solvent ratio is higher.

From Table 2 it is observed that the regression coefficient and second order polynomial equation of the predicted model in coded variables shows a significant contribution of each coefficient determined by p-value of ftest (p < 0.05). The model is generally considered to be fit based on a lack-of-fit test (p > > 0.05). The p-value obtained for the TPC and TFC indicated the suitability of the model to accurately predict the variations (Table 2 (b)). The quality of the fit model can be determined based on the coefficient of determination (R2) [19].

Optimization and validation

The numerical optimization of extraction parameters was carried out using design-expert statistical software based on the initial experimental results. The experiment was conducted according to the predicted and optimal conditions of 42 °C, 19 min and 1:23 g/mL feed-solvent ratio, the TPC and TFC values were found to be 17.41 mg GAE/g dW and 6.73 mg RE/g dW, respectively for UAE. Also, at 40 °C, 5.5 h, 1:24 g/mL, the TPC and TFC were 12.01 and 4.47 mg/g for SE. Similarly, at 50 °C, 6 h and 1:25 g/mL, maximum results were obtained as TPC = 20.53 mg/g and TFC = 8.62 mg/g for Soxhlet extraction.

Comparison of Soxhlet and solvent extraction methods with ultrasound-assisted extraction

The results disclosed ultrasound-assisted extraction with a maximum TPC (17.41±0.13 mg GAE/g dW) and TFC (6.73±0.19 mg RE/g dW) in comparison with solvent-assisted extraction that gave noticeably lower TPC (12.01±0.17 mg GAE/g dW) and TFC (4.47±0.09 mg RE/g dW). Whereas the Soxhlet extraction results TPC (20.53±0.11 mg GAE/g dW) and TFC (8.62±0.15 mgRE/gdW) reveal closer values as that of UAE, as shown in Table 4. This may be due to the fact that at higher temperature and longer extraction time, the polyphenol yield increases, whereas higher temperatures and longer duration can degrade certain bioactive compounds. Based on the results, the noteworthy improvement of UAE could be attributed to the acoustic cavitation of ultrasound and its mechanical effects that resulted in better recovery. Cavitation occurring near the surface of the plant's cell results in surface peeling, cell breakdown and erosion that further accentuates the recovery process [12]. The effect arising from acoustic cavitation ultimately leads to enhanced recovery of the desired compounds.

Phytochemical screening of components

The compound 3H-pyrazole, 4,5-dihydro-4-(2,2,3,3-tetrafluorocyclobutyl)possesses anticancer, antiviral, antioxidants and antimicrobial properties [25]. Anti-inflammatory compounds [26] like hexadecanoic acid, oleic acid, eicosanoic acid, octadecanoic acid, tetradecanoic acid, pentadecanoic acid and flavoring agents like 2-chloroethyl linoleate were observed (Tables 5-7).

Table 7 shows that eight additional phytoconstituents like bicyclo[2.2.1]heptane, 2-(1-buten-3-yl)-serverogenin acetate, 4Hpyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-, L-(+)-ascorbic acid 2,6-dihexadecanoate, c/s-vaccenic acid, thiopene, 2-propyl-phenol, 2,4-bis(1,1-dimethylethyl), octanoic acid, 2-hexyl- were present in UAE extracts only.

The biological activities of each of the identified phytocomponents contribute to its remarkably higher (97.715%) antioxidant, antimicrobial and anti-inflammatory activities. Whereas two components such as octanoic acid, 2-hexyl- and thiopene, 2-propyl- act as fragrance and flavoring agents (8.915%). Phytochemicals like pentadecanoic acid and nhexadecanoic acid (49.198%) identified in UAE are comparatively higher than SE (19.076%) and Soxhlet extraction (15.658%). Similarly, octadecanoic acid, pentadecanoic acid, which possess anti-inflammatory and cytotoxicity activities [30] are found to be 6.830% in Soxhlet extraction, 15.005% in solvent extraction, whereas they are much higher in UAE (16.963 and 49.198%).

CONCLUSION

The effect of extraction conditions on TPC and TFC were studied by RSM. The results showed that under the optimal conditions of 42 °C, 19 min and 1:23 g/mL, the TPC and TFC values were found to be 17.41±0.13 mg GAE/g dW and 6.73±0.19 mg RE/g dW, respectively for UAE. At 40 °C, 5.5 h, 1:24 g/mL, the TPC and TFC were 12.01±0.17 and 4.47±0.09 mg/g for SE. Similarly, at 50 °C, 6 h and 1:25 g/mL, maximum results were obtained as TPC = 20.53±0.11 mg/g and TFC = 8.62±0.15 mg/g for Soxhlet extraction. Further analysis of the optimized UAE, Soxhlet and SE extract through GC-MS reveals the presence of eight additional compounds: bicyclo[2.2.1]heptane, 2-(1-buten-3-yl)-, serverogenin acetate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-, L-(+)-ascorbic acid 2,6-dihexadecanoate, c/s-vaccenic acid, thiopene, 2-propyl-, phenol, 2,4-bis(1,1-dimethylethyl), octanoic acid, 2-hexyl-, identified in UAE extracts only, which confirmed the extraction of phenolic compounds using UAE was significantly higher than conventional methods. High TPC and TFC solidify UAE as an efficient extraction method for the recovery of natural antioxidants. Therefore, it can be said with certainty that UAE is comparatively a superior extraction method for the recovery of natural antioxidants from wood apple pulp.

Nomenclature

UAE - ultrasound-assisted extraction

SE - solvent extraction

TPC - total phenolic content

TFC - total flavonoid content

GAE - gallic acid equivalent

RE - rutin equivalent

dW - dry weight

BBD - Box-Behnken design

RSM - response surface methodology

GC-MS - gas chromatography-mass spectroscopy

Sidebar

Correspondence: S. Venkatachalam, Food Process Engineering Lab, Department of Chemical Engineering, A C Tech, Anna University, Chennai - 25, India.

E-mail: [email protected]

Paper received: 28 August, 2018

Paper revised: 28 March, 2019

Paper accepted: 8 April, 2019

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