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Soxhlet extraction of phenolic compounds from Vernonia cinerea leaves and its antioxidant activity
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
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Soxhlet extraction of phenolic compounds from Vernonia cinerea leaves and its antioxidant activity ⁎
Oluwaseun R. Alaraa, , Nour H. Abdurahmana, Chinonso I. Ukaegbub a b
Centre of Excellence for Advanced Research in Fluid Flow (CARIFF), Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300, Gambang, Pahang, Malaysia
A R T I C LE I N FO
A B S T R A C T
Keywords: Vernonia cinerea Soxhlet extraction Total phenolic content Total flavonoid content Antioxidant
Recently, discovering natural antioxidants have gained more interest due to the fact that most infectious ailment, namely cardiovascular disorder, diabetes and cancer are associated with free radical cells. Thus, the extraction of phenolic compounds from Vernonia cinerea leaves through Soxhlet extraction method was studied. The effects of extraction time (1–4 h), feed-to-solvent (1:10–1:25 g/mL) and ethanol concentration (20–80% v/v) on the yield of extract, total phenolic content (TPC) and total flavonoid content (TFC) were examined. Moreover, the phenolic compounds and functional groups in the extract at maximum conditions were identified using Liquid Chromatography-Mass Spectrometry Quadrupole Time of Flight (LC–Q–TOF–MS) and Fourier Transform Infrared Spectrometry (FTIR), respectively. The antioxidant activity of the extract was as well investigated. The experimental results showed that the highest yield of extract (10.01 ± 0.85% w/w), TPC (53.96 ± 1.45 mg GAE/g d.w.) and TFC (30.09 ± 0.44 mg QE/g d.w.) were achieved using extraction time of 2 h, feed-to-solvent of 1:20 g/mL and ethanol concentration of 60% v/v. However, the extract reflected good antioxidant activity.
1. Introduction Naturally, the human body produces reactive oxygen species that are associated with diabetes, cancer, chronic, and cardiovascular diseases (Wickramaratne et al., 2016). World Health Organization reported that prevention is the most effective strategy against any disease than treatment (Javier David Vega et al., 2017). Therefore, regular consumption of vegetable and phytonutrient-rich fruit have been suggested to reduce the risk of these ailments. Moreover, antioxidants from natural products in terms of phenolic compounds are important components that can eliminate reactive oxygen species (Xu et al., 2016). Phenolic compounds are secondary metabolites abundantly found in vegetables and fruits, common dietary phenolic compounds are phenolic acids and flavonoids (Mojzer et al., 2016; Tuberoso and Orrù, 2008). Vernonia cinerea (Purple fleabane) is an annual herb that belongs to family Asteraceae. It spreads over Africa, tropical Asia and Australia, and can grow up to 1 m tall. The whole part of this plant possesses several ethnomedicinal uses. The whole plant had been reported to cure filariasis, intermittent fever, vaginal discharges, haematological disorders, tridosha, and as a blood tonic, purifier, astringent, and stomachic (Khare, 2007). Traditionally, the leaves of V. cinerea are being used in treating wounds, painful urination, rheumatoid arthritis, ⁎
ingestion, elephantiasis, asthma, malaria, hepatitis, erectile dysfunction, diabetes, and sexual impotence (Dogra and Kumar, 2015). In view of these usages, V. cinerea leaves had been reported to have different pharmacological activities, namely: anti-inflammatory, renoprotective, anti-tumour, antioxidant, anti-microbial, and anti-diabetic activities. In addition, the ethanolic extract inhibited 53.13% and 62.43% haemolysis of red blood cells in heat-induced and hypotonic solution conditions, respectively as compared to acetylsalicylic acid (Pratheeshkumar and Kuttan, 2011). Extraction is an important process in the recovery of phenolic compounds from plant matrix. It can either be a conventional or unconventional method. Conventional methods such as maceration, boiling, soaking, hydrodistillation, and Soxhlet have been used for several years. However, Soxhlet extraction is the most commonly used method for extracting phenolic compounds due to its lower processing cost, the simplicity of operation, suitability for both initial and bulk extraction, good for total recovery of extracts, consume lesser time, and solvent as compare to other conventional methods such as maceration or percolation (Seidel, 2012). More so, Soxhlet extraction has been a well-established technique which possesses a great performance as compared to other conventional extraction methods except that it is well applied to the extraction of thermolabile (Luque de Castro and García-Ayuso, 1998). Likewise, when comparing this method to other
Corresponding author. E-mail address: [email protected] (O.R. Alara).
https://doi.org/10.1016/j.jarmap.2018.07.003 Received 11 June 2018; Accepted 31 July 2018 2214-7861/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Alara, O.R., Journal of Applied Research on Medicinal and Aromatic Plants, https://doi.org/10.1016/j.jarmap.2018.07.003
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conventional methods, the larger yields can be extracted with a much smaller quantity of solvent (Handa et al., 2008). Therefore, this study focused on the Soxhlet extraction of phenolic compounds from V. cinerea leaves. Moreover, the phenolic profiling and functional groups in the extract at maximum Soxhlet extraction condition was evaluated using Liquid Chromatography-Mass Spectrometry Quadrupole Time of Flight (LC–Q–TOF–MS) and Fourier Transform Infrared Spectrometry (FTIR). The antioxidant activities of the extract were as well examined.
TPC =
(2)
where c is the concentration of TPC from the calibration curve (mg/L), V is volume (L) of solvent used in the extraction, and m represents the weight (g) of the dried sample used. 2.4. Total flavonoid content in the extracts The quantitative determination of TFC in the extract of V. cinerea leaves was determined using the methodology described in the previous reports (Alara et al., 2018a,b). An aliquot of 100 μL of extract solution (1 g/L) was thoroughly mixed with 100 μL of 2% AlCl3 solution and allowed to stand for 60 min at room temperature. The absorbance in the supernatant was measured at 420 nm using a UV-vis Spectrophotometer (Hitachi U-1800, Japan). Then, the concentration of TFC in the extract was calculated from the quercetin acid standard calibration curve (ranging from 50 to 500 mg/L) with equation of line y = 0.112x + 0.178, R2 = 0.9945 (where y is the absorbance at 420 nm and x is the concentration of TFC from the calibration curve), TFC was determined using Eq. (3) and results were reported as quercetin equivalent in milligram per gram of dried extract (mg QE/g d.w.). The analysis was repeated thrice and mean ± standard deviation was recorded.
2. Materials and methods 2.1. Sample collection and chemicals procurement Fresh samples of V. cinerea leaves were sourced from Universiti Malaysia Pahang, Gambang environment. The leaves were manually removed from the stem, washed in tap water and dried at room temperature for two weeks. Then, the dried samples were crushed using a blender, sieved to an average particle size of 105 μm and stored in an airtight dark container prior to the extraction process. The following chemicals and reagents were bought from Sigma Aldrich (M) Sdn Bhd, Selangor: Ethanol (99.5% purity), gallic acid, quercetin, Folin-Ciocalteu reagent, sodium carbonate anhydrous, methanol (99.9% purity), and aluminium chloride salt. The distilled water used was obtained from Faculty of Chemical and Natural Resources Engineering laboratory, Universiti Malaysia Pahang.
TFC =
c *V m
(3)
where c is the concentration of TFC from the calibration curve (mg/L), V is volume (L) of solvent used in the extraction, and m represents the weight (g) of the dried sample used.
2.2. Extraction of plant samples using Soxhlet technique
2.5. Fourier transform infrared spectrometry analysis
The plant sample of V. cinerea leaves (10 g) was weighed into a Soxhlet extractor thimble and placed in the extraction apparatus. Aqueous ethanol varied as 20, 40, 60, and 80% v/v was measured into a 250-mL conical flask depending on the feed-to-solvent ratio (1:10, 1:15, 1:20, and 1:25 g/mL). A heating mantle was used to reflux the mixture for varied extraction time between 1 and 4 h. After the extraction time has been reached, the extract solution was allowed to cool to room temperature. Then, filtered through a cone of filter paper (Whatman no 1) and concentrated to dryness using a rotary evaporator. Then, the extraction yield, TPC and TFC in the extract were evaluated. Eq. (1) was used to evaluate the percentage of extraction yield. The experimental procedure was replicated thrice and the mean ± standard deviation was recorded.
% Yield of extracts =
c *V m
Fourier transform infrared was employed to identify different characteristic functional groups in the extracts from V. cinerea leaves using Soxhlet extraction technique. The IR spectra were obtained using an FTIR (Nicolet iS5 iD7 ATR; Thermo Scientific, Germany) equipped with OMNIC software. The obtained IR spectra were scanned from wave number ranging from 4000 to 500 cm−1 with a resolution of 4 cm−1 (Alara et al., 2018a,b). The spectra obtained for the extracts were interpreted with a chart for characteristics infrared absorption frequencies of functional groups. 2.6. Identification of phenolic compounds using LC–Q–TOF–MS analysis
Weight of extracts from plant sample (w ) * 100% Weight of dried plant sample (w )
Phenolic compounds in the extracts at the highest Soxhlet extraction conditions were tentatively identified through LC–Q–TOF–MS analysis equipped with PDA detector (Waters Vion IMS, USA) and symmetry C18 column of 100 mm × 2.1 mm, 1.8 μm particle size (Waters Acquity UPLC HSS T3, USA). The separation conditions are as follows: The mobile phase comprised of water (A) and 100% acetonitrile (B) using a gradient elution of was 90% A and 10% B at 0–1.25 min, 45% A and 55% B at 1.25–4.17 min, 10% A and 90% B at 4.17–6.25 min, 90% A and 10% B at 6.25–8.34 min; the elution flow rate was set at 0.5 mL/ min and injection volume of 20 μL; the analysis was performed using SYNAPT mass spectrometer (Waters) coupled with an electrospray ionization operated in negative and positive ion modes, mass range of 100–1000 m/z, spray voltage 4 keV, column temperature of 40 °C, sample temperature of 15 °C, gas flow of 0.5 mL/min, desolvation temperature of 550 °C, and desolvation gas flow rate of 800 L/h. Phenolic compounds in the V. cinerea leaf extract were characterized by MS/MS fragmentation pattern reference standards.
(1)
2.3. Total phenolic content in the extracts The total phenolic content in the extracts of V. cinerea leaves was determined using Folin-Ciocalteu reagent method (Alara et al., 2018a,b). The extracts (1 mL) at a concentration of 5 g/L was mixed with 200 μL of Folin-Ciocalteu reagent. Then, 0.6 mL of 0.2 mM Na2CO3 solution was thoroughly mixed with the mixture after 5 min. The mixture was left for 120 min and absorbance was measured at 765 nm using a UV–vis Spectrophotometer (Hitachi U-1800, Japan). Thereafter, the concentration of TPC in the plant extract was calculated from the gallic acid standard calibration curve (ranging from 50 to 500 mg/L) with equation of line y = 0.0006x + 0.0169, R2 = 0.9903 (where y is the absorbance at 765 nm and x is the sample concentration from the calibration curve), TPC was determined using Eq. (2) and results were reported as gallic acid equivalent in milligram per gram of dried extract (mg GAE/g d.w.). The analysis was repeated thrice and mean ± standard deviation was recorded.
2.7. In vitro antioxidant activities of the extract 2.7.1. DPPH assay The hydrogen atom or electron donating ability of extracts from V. 2
3
± ± ± ± 24.44 28.44 30.09 27.01 0.87a 1.00b 1.45c 1.01b ± ± ± ± 49.05 51.14 53.96 50.05 7.04 ± 0.10a 8.88 ± 0.88b 10.01 ± 0.85c 8.03 ± 0.90b
Results are expressed as means ± standard deviation. Different letters along the column indicate a significant difference (p < 0.05).
20 40 60 80 0.87a 0.45b 0.22c 0.59a ± ± ± ± 23.04 25.88 28.39 24.43 1.45a 1.01b 0.99c 0.50a ± ± ± ± 46.22 48.18 51.13 45.55 0.09a 0.76b 0.22c 0.72a ± ± ± ± 6.99 8.01 9.22 7.17 1:10 1:15 1:20 1:25 0.21a 0.87b 0.22a 0.12a ± ± ± ± 22.11 26.22 23.55 21.22
F:S (g/mL) TFC (mg QE/g d.w.)
3.1.1. Effect of extraction time on the recoveries of extract, TPC and TFC Extraction time is imperative in reducing energy and cost of the extraction process. It is one of the most important factors that alter the recovery of phenolic compounds from plant matrix. This is due to the fact that over-exposure of plant sample to localize heating tends to degrade phenolic compounds (Mojzer et al., 2016). Thus, it is essential to determine the appropriate extraction time for higher recovery. The effects of extraction time variation on the yields of extracts, TPC and TFC using a Soxhlet apparatus were examined. The extraction time was varied as 1, 2, 3, and 4 h at a constant feed-to-solvent ratio of 1:10 g/mL and ethanol concentration of 20% v/v. The maxima extraction yields, TPC and TFC were achieved at 2 h of extraction time (Table 1). Further increase in the extraction time decline the recovery of phenolic compounds. This might be due to the degradation of phenolic compounds resulted from excessive heating of the plant samples (Dahmoune et al., 2014). Moreover, the obtained results followed the Fick’s second law of diffusion, whereby at certain time of extraction, final equilibrium will be reached between the extraction solvent and plant sample. Hence, the yields tends to decline beyond final equilibrium (Alara et al., 2018c).
TPC (mg GAE/g d.w.)
The effects of extraction factors which include extraction time, feedto-solvent ratio and ethanol concentration using Soxhlet extraction technique were examined on the recoveries of extracts, TPC and TFC from V. cinerea leaves. Table 1 vividly explains the effects of individual factors on the yields. Effect of an individual factor is discussed as follows.
Extraction time (h)
3.1. Effects of Soxhlet extraction factors on the yields
Effect of feed-to-solvent ratio
3. Results and discussion
Effect of extraction time
Table 1 Effects of Soxhlet extraction factors on the recoveries of extracts, TPC and TFC from V. cinerea leaf.
The experimental procedure and analysis were carried out in triplicate. The results were statistically analyzed using ANOVA and the significance was taken at a level of p < 0.05.
0.86a 1.02b 0.11a 0.27c
2.8. Statistical analysis
± ± ± ±
(5)
45.55 48.32 44.95 42.11
* 100%
0.10a 0.55b 0.14a 0.20c
Acontrol
± ± ± ±
Acontrol − Asample
TPC (mg GAE/g d.w.)
Percentage of ABTS+ • inhibition =
TFC (mg QE/g d.w.)
2.7.2. ABTS assay The ABTS scavenging ability of V. cinerea leaf extracts (or ascorbic acid) was assayed using a method described in previous the report (Alara et al., 2018a,b). A 150 μL of V. cinerea leaf extract at different concentrations (100–500 μg/mL) was mixed with 285 μL of ABTS solution (2.45 mM potassium persulfate solution and 7 mM ABTS). The reaction mixture was incubated at room temperature for 2 h and then the light absorption was recorded spectrophotometrically at 734 nm. The IC50 value was calculated. This assay was carried out in triplicate and mean ± SD was computed. The ability of the extract to scavenge ABTS radical was calculated as follows:
6.49 7.28 6.22 5.34
Effect of ethanol concentration
where Acontrol represents absorbance measure for the mixture of methanol and DPPH solution; and Asample represents absorbance of the mixture of extract from V. cinerea leaves and DPPH solution.
1 2 3 4
TPC (mg GAE/g d.w.)
(4)
Yield (% w/w)
* 100%
Ethanol concentration (% v/v)
Acontrol
Yield (% w/w)
Acontrol − Asample
Yield (% w/w)
Percentage of DPPH inhibition capacity =
TFC (mg QE/g d.w.)
cinerea leaves was verified using DPPH assay as described in the previous reports (Alara et al., 2018a,b). A 2000 μL of 0.1 mM DPPH was added to 200 μL of the extract (or ascorbic acid) at different concentrations (100–500 μg/mL), an absorbance of the mixture was recorded at 517 nm after incubation for 30 min in the dark at room temperature. Then, the IC50 value was calculated. This assay was carried out in triplicate and mean ± SD was computed. The ability of the extract to scavenge DPPH radical was calculated as follows:
0.74a 0.22b 0.44c 0.75b
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Fig. 1. The infrared spectra of extract from V. cinerea leaves using Soxhlet extraction. Table 2 Phenolic compounds in the extract from V. cinerea leaf using Soxhlet extraction technique. S/N
Observed RT (min)
Component name
Chemical formula
Observed m/z
Adducts
Total fragment found
1 2
0.68 1.31
C13H16O10 C25H24O12
331.0671 515.1199
−H −H
7 10
3
2.07
C8H8O2
135.0452
−H
0
4 5 6 7 8 9
3.79 3.95 4.11 4.49 0.96 1.09
1-Galloyl-β-D-glucose 3,5-O-Dicaffeoylquinic acid 4Hydroxyacetophenone Kukoamine A 6-Gingerol Caesalpins P Kakuol Quercetin-7-O-rutinoside Kaempferol
C28H42N4O6 C17H26O4 C16H12O6 C10H10O4 C27H30O16 C15H10O6
529.3048 293.1762 299.0561 193.0507 611.1605 287.0553
−H −H −H −H +H +H
6 0 1 3 2 1
Table 3 In vitro antioxidant activities of extract from V. cinerea leaf using Soxhlet extraction technique.
Extract Ascorbic acid
TPC (mg GAE/g d.w.)
TFC (mg GAE/g d.w.)
IC50 value of DPPH (μg/mL)
IC50 value of ABTS+• (μg/mL)
53.96 ± 1.45 –
30.09 ± 0.44 –
301.86 ± 2.44a 130.30 ± 1.20b
269.27 ± 1.11a 73.11 ± 1.00b
Results are expressed as means ± standard deviation. Different letters along the column indicate a significant difference (p < 0.05).
This finding is similar to the results obtained in the extraction of Centella asiatica, where the maximum yield of TPC was achieved using extraction time of 120 min (Chew et al., 2011). Thus, the extraction time of 2 h was selected as optimum for the next factor (feed-to-solvent ratio).
extraction yields in conventional extraction methods (Mojzer et al., 2016). The effects of feed-to-solvent ratio were examined on the recovery yields from V. cinerea leaves. At constant extraction time of 2 h and ethanol concentration of 20% v/v, the feed-to-solvent ratio was varied as 1:10, 1:15, 1:20, and 1:25 g/mL. It can be clearly seen in Table 1 that the recoveries of extracts and phenolic compounds tend to increase as the feed/solvent increases from 1:10 g/mL through 1:20 g/ mL. Beyond 1:20 g/mL of feed/solvent, the yields declined. The maximum recovery yields of extracts, TPC and TFC were obtained at 1:20 g/ mL and further increase reduced the yields. The solvent-to-feed ratio between 10:1 (mL/g) and 20:1 (mL/g) had been reported to give optimal yields (Veggi et al., 2013). Therefore, the feed-to-solvent ratio of 1:20 g/mL was considered for the next factor (ethanol concentration).
3.1.2. Effect of feed-to-solvent on the recoveries of extract, TPC and TFC The volume of extracting solvent is another important factor, a large volume of solvent requires more energy and time to condense extraction solution in the purification process. Plant matrix needs to be totally immersed in the solvent for higher recovery during the extraction process. In general, higher volume of solvent will increase the
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antioxidant activity of the extract (Shah et al., 2018). Kukoamine A which is a spermine conjugate that possess dihydrocaffeic acid had been examined having strong antioxidant activity against 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical in a range of 5–97.5% (Wang et al., 2016). Moreover, the anti-diabetic activity of extract from V. cinerea leaves can be attributed to the presence of 3,5-O-dicaffeoylquinic acid (Alara et al., 2018c; Chen et al., 2014). The anticancer property of this extract can be attributed to the presence of 6-gingerol (Kumara et al., 2017). Thus, the identified phenolic compounds in the extract show the antioxidant activity of V. cinerea leaves.
3.1.3. Effect of ethanol concentration on the recoveries of extract, TPC and TFC Finally, extraction time and feed-to-solvent ratio were fixed at 2 h and 1:20 g/mL while the ethanol concentration was varied between 20 and 80% v/v for the extraction of yields, TPC and TFC from V. cinerea leaves. It can be observed that the yields of extracts, TPC and TFC increased gradually from 20 to 60% v/v of ethanol concentration, beyond this ethanol concentration, the yields decline steadily. The optimal recovery yields of extracts (10.01 ± 0.85% w/w), TPC (53.96 ± 1.45 mg GAE/g d.w.) and TFC (30.09 ± 0.44 mg QE/g d.w.) were obtained at 2 h of extraction time, feed-to-solvent of 1:20 g/mL and ethanol concentration of 60% v/v for V. cinerea leaves. In the similar studies on the extraction of V. amygdalina leaves using Soxhlet extraction method, the TPC yields were reported to be 63.044 mg GAE/ g d.w., 97.0 ± 0.01 mg GAE/g d.w. and 38.834 mg GAE/g d.w. using ethanol, methanol, and ethyl acetate, respectively as the extracting solvents (Oriakhi et al., 2013). This can be explained by the solubility of phenolic compounds which depends on the nature of chemical composition in the plant sample and polarity of the solvent used (Wong et al., 2014). Hence, the use of binary solvent has been suggested to be better than mono-solvent in the extraction of phenolic compounds from plant matrix. The presence of water in extraction process ease the leaching of hydrophilic antioxidants.
3.4. In vitro antioxidant activity of the extracts The extract of V. cinerea leaves at the maximum conditions of Soxhlet extraction method was examined for its antioxidant activity using DPPH and ABTS radical scavengers. The results obtained was compared with that of ascorbic acid as presented in Table 3. Several studies had reported that lowest IC50 value indicates strong antioxidant activity (Alara et al., 2018a,b; Karabegovic et al., 2014). Although, the antioxidant activity of ascorbic acid was higher than the extract, however, the results show that the extract possessed antioxidant activity. This might be due to the effect of localized heating using Soxhlet extraction method which might have degraded the antioxidant in the extract (Karabegovic et al., 2014). The different scavenging activities observed using DPPH and ABTS might be due to the different mechanisms involved in the assays. It has been reported that high molecular weight phenolics possess the capacity to scavenge ABTS+• which depends on the molecular weight, nature of hydroxyl group substitution and number of aromatic rings (Khan et al., 2012). In addition, the presence of kukoamine A, a spermine conjugate with dihydrocaffeic acid has been investigated to potentially possess a strong antioxidant activity (Shah et al., 2018; Wang et al., 2016).
3.2. Identified functional groups in the extract Fourier transform infrared (FTIR) is one of the widely used characterizations for the identification and elucidation of functional groups in the plant extracts. This characterization was used to identify functional groups in the extract from V. cinerea leaves at the maximum conditions of Soxhlet extraction based on the peak value in the region of infrared radiation. The characteristic absorption peaks are presented in Fig. 1. The broad peak at 3328.79 cm−1 confirmed the presence of phenolic compounds. However, the presence of lipid-carbohydrate (mainly vas(CH2) and vs(CH2) stretching) can be attributed to the peaks at 2123.24 cm−1, 2977.79 cm−1 and 2901.36 cm−1. The peaks at 1643.14, 1452.99, 1383.43, and 1274.26 cm−1 indicates the presence of C]O stretching, NeH bending, bending of carboxylic acid (presence of tannins, flavonoids, glycosides, and saponins), and nucleic acid (vas (> P = O) stretching of phosphodiesters), respectively (Dilek et al., 2012). The band at 1274.26 cm−1 shows the presence of C–O groups of polyols such as hydroxyflavonoids (Oliveira et al., 2016). The presence of ester group and/or secondary alcohols could be associated with the presence of sharp peaks at 1085.30 cm−1 and 1043.52 cm−1. In addition, band peak at 877.39 cm−1 could be attributed to aromatic ring vibration. Thus, the observed characteristic fingerprints in the extract from V. cinerea leaves using Soxhlet extraction method reflected the presence functional characteristics associated with polyphenols and flavonoids.
4. Conclusion This study investigated the effects of Soxhlet extraction factors, namely: extraction time, feed-to-solvent ratio and ethanol concentration on the recoveries of extract, TPC, TFC from V. cinerea leaves. The highest yields were obtained using extraction time of 2 h, feed/solvent of 1:20 g/mL and ethanol concentration of 60% v/v. The LC–Q–TOF–MS and FTIR showed the presence of phenolic compounds in the extract. More so, the extract from V. cinerea leaves possesses potent antioxidant activity with reference to ascorbic acid. Thus, the extract from V. cinerea leaves can potentially be used as the natural antioxidant. Conflict of interest We declare no conflict of interest.
3.3. Identified phenolic compounds in the extracts
Acknowledgement
The identification of phenolic compounds in the extract from V. cinerea leaves at maximum conditions of Soxhlet extraction was carried out using LC–Q–TOF–MS. A total number of 9 phenolic compounds were tentatively identified by comparing spectral data with reference compounds. The identified phenolic compounds are presented in Table 2. The compounds are 1-galloyl-β-D-glucose (MS ion at m/z 331.0671), 3,5-O-dicaffeoylquinic acid (MS ion at m/z 515.1199), 4hydroxyacetophenone (MS ion at m/z 135.0452), kukoamine A (MS ion at m/z 529.3048), 6-gingerol (MS ion at m/z 293.1762), caesalpins P (MS ion at m/z 299.0561), and kakuol (MS ion at m/z 193.0507) were identified through negative ionization. However, only two flavonoids, namely quercetin-7-O-rutinoside and kaempferol were identified through positive ionization. The presence of 1-galloyl-β-D-glucose, kukoamine A, quercetin-7-O-rutinoside, and kaempferol indicate the
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Journal of Applied Research on Medicinal and Aromatic Plants journal homepage: www.elsevier.com/locate/jarmap
Soxhlet extraction of phenolic compounds from Vernonia cinerea leaves and its antioxidant activity ⁎
Oluwaseun R. Alaraa, , Nour H. Abdurahmana, Chinonso I. Ukaegbub a b
Centre of Excellence for Advanced Research in Fluid Flow (CARIFF), Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300, Gambang, Pahang, Malaysia
A R T I C LE I N FO
A B S T R A C T
Keywords: Vernonia cinerea Soxhlet extraction Total phenolic content Total flavonoid content Antioxidant
Recently, discovering natural antioxidants have gained more interest due to the fact that most infectious ailment, namely cardiovascular disorder, diabetes and cancer are associated with free radical cells. Thus, the extraction of phenolic compounds from Vernonia cinerea leaves through Soxhlet extraction method was studied. The effects of extraction time (1–4 h), feed-to-solvent (1:10–1:25 g/mL) and ethanol concentration (20–80% v/v) on the yield of extract, total phenolic content (TPC) and total flavonoid content (TFC) were examined. Moreover, the phenolic compounds and functional groups in the extract at maximum conditions were identified using Liquid Chromatography-Mass Spectrometry Quadrupole Time of Flight (LC–Q–TOF–MS) and Fourier Transform Infrared Spectrometry (FTIR), respectively. The antioxidant activity of the extract was as well investigated. The experimental results showed that the highest yield of extract (10.01 ± 0.85% w/w), TPC (53.96 ± 1.45 mg GAE/g d.w.) and TFC (30.09 ± 0.44 mg QE/g d.w.) were achieved using extraction time of 2 h, feed-to-solvent of 1:20 g/mL and ethanol concentration of 60% v/v. However, the extract reflected good antioxidant activity.
1. Introduction Naturally, the human body produces reactive oxygen species that are associated with diabetes, cancer, chronic, and cardiovascular diseases (Wickramaratne et al., 2016). World Health Organization reported that prevention is the most effective strategy against any disease than treatment (Javier David Vega et al., 2017). Therefore, regular consumption of vegetable and phytonutrient-rich fruit have been suggested to reduce the risk of these ailments. Moreover, antioxidants from natural products in terms of phenolic compounds are important components that can eliminate reactive oxygen species (Xu et al., 2016). Phenolic compounds are secondary metabolites abundantly found in vegetables and fruits, common dietary phenolic compounds are phenolic acids and flavonoids (Mojzer et al., 2016; Tuberoso and Orrù, 2008). Vernonia cinerea (Purple fleabane) is an annual herb that belongs to family Asteraceae. It spreads over Africa, tropical Asia and Australia, and can grow up to 1 m tall. The whole part of this plant possesses several ethnomedicinal uses. The whole plant had been reported to cure filariasis, intermittent fever, vaginal discharges, haematological disorders, tridosha, and as a blood tonic, purifier, astringent, and stomachic (Khare, 2007). Traditionally, the leaves of V. cinerea are being used in treating wounds, painful urination, rheumatoid arthritis, ⁎
ingestion, elephantiasis, asthma, malaria, hepatitis, erectile dysfunction, diabetes, and sexual impotence (Dogra and Kumar, 2015). In view of these usages, V. cinerea leaves had been reported to have different pharmacological activities, namely: anti-inflammatory, renoprotective, anti-tumour, antioxidant, anti-microbial, and anti-diabetic activities. In addition, the ethanolic extract inhibited 53.13% and 62.43% haemolysis of red blood cells in heat-induced and hypotonic solution conditions, respectively as compared to acetylsalicylic acid (Pratheeshkumar and Kuttan, 2011). Extraction is an important process in the recovery of phenolic compounds from plant matrix. It can either be a conventional or unconventional method. Conventional methods such as maceration, boiling, soaking, hydrodistillation, and Soxhlet have been used for several years. However, Soxhlet extraction is the most commonly used method for extracting phenolic compounds due to its lower processing cost, the simplicity of operation, suitability for both initial and bulk extraction, good for total recovery of extracts, consume lesser time, and solvent as compare to other conventional methods such as maceration or percolation (Seidel, 2012). More so, Soxhlet extraction has been a well-established technique which possesses a great performance as compared to other conventional extraction methods except that it is well applied to the extraction of thermolabile (Luque de Castro and García-Ayuso, 1998). Likewise, when comparing this method to other
Corresponding author. E-mail address: [email protected] (O.R. Alara).
https://doi.org/10.1016/j.jarmap.2018.07.003 Received 11 June 2018; Accepted 31 July 2018 2214-7861/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Alara, O.R., Journal of Applied Research on Medicinal and Aromatic Plants, https://doi.org/10.1016/j.jarmap.2018.07.003
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conventional methods, the larger yields can be extracted with a much smaller quantity of solvent (Handa et al., 2008). Therefore, this study focused on the Soxhlet extraction of phenolic compounds from V. cinerea leaves. Moreover, the phenolic profiling and functional groups in the extract at maximum Soxhlet extraction condition was evaluated using Liquid Chromatography-Mass Spectrometry Quadrupole Time of Flight (LC–Q–TOF–MS) and Fourier Transform Infrared Spectrometry (FTIR). The antioxidant activities of the extract were as well examined.
TPC =
(2)
where c is the concentration of TPC from the calibration curve (mg/L), V is volume (L) of solvent used in the extraction, and m represents the weight (g) of the dried sample used. 2.4. Total flavonoid content in the extracts The quantitative determination of TFC in the extract of V. cinerea leaves was determined using the methodology described in the previous reports (Alara et al., 2018a,b). An aliquot of 100 μL of extract solution (1 g/L) was thoroughly mixed with 100 μL of 2% AlCl3 solution and allowed to stand for 60 min at room temperature. The absorbance in the supernatant was measured at 420 nm using a UV-vis Spectrophotometer (Hitachi U-1800, Japan). Then, the concentration of TFC in the extract was calculated from the quercetin acid standard calibration curve (ranging from 50 to 500 mg/L) with equation of line y = 0.112x + 0.178, R2 = 0.9945 (where y is the absorbance at 420 nm and x is the concentration of TFC from the calibration curve), TFC was determined using Eq. (3) and results were reported as quercetin equivalent in milligram per gram of dried extract (mg QE/g d.w.). The analysis was repeated thrice and mean ± standard deviation was recorded.
2. Materials and methods 2.1. Sample collection and chemicals procurement Fresh samples of V. cinerea leaves were sourced from Universiti Malaysia Pahang, Gambang environment. The leaves were manually removed from the stem, washed in tap water and dried at room temperature for two weeks. Then, the dried samples were crushed using a blender, sieved to an average particle size of 105 μm and stored in an airtight dark container prior to the extraction process. The following chemicals and reagents were bought from Sigma Aldrich (M) Sdn Bhd, Selangor: Ethanol (99.5% purity), gallic acid, quercetin, Folin-Ciocalteu reagent, sodium carbonate anhydrous, methanol (99.9% purity), and aluminium chloride salt. The distilled water used was obtained from Faculty of Chemical and Natural Resources Engineering laboratory, Universiti Malaysia Pahang.
TFC =
c *V m
(3)
where c is the concentration of TFC from the calibration curve (mg/L), V is volume (L) of solvent used in the extraction, and m represents the weight (g) of the dried sample used.
2.2. Extraction of plant samples using Soxhlet technique
2.5. Fourier transform infrared spectrometry analysis
The plant sample of V. cinerea leaves (10 g) was weighed into a Soxhlet extractor thimble and placed in the extraction apparatus. Aqueous ethanol varied as 20, 40, 60, and 80% v/v was measured into a 250-mL conical flask depending on the feed-to-solvent ratio (1:10, 1:15, 1:20, and 1:25 g/mL). A heating mantle was used to reflux the mixture for varied extraction time between 1 and 4 h. After the extraction time has been reached, the extract solution was allowed to cool to room temperature. Then, filtered through a cone of filter paper (Whatman no 1) and concentrated to dryness using a rotary evaporator. Then, the extraction yield, TPC and TFC in the extract were evaluated. Eq. (1) was used to evaluate the percentage of extraction yield. The experimental procedure was replicated thrice and the mean ± standard deviation was recorded.
% Yield of extracts =
c *V m
Fourier transform infrared was employed to identify different characteristic functional groups in the extracts from V. cinerea leaves using Soxhlet extraction technique. The IR spectra were obtained using an FTIR (Nicolet iS5 iD7 ATR; Thermo Scientific, Germany) equipped with OMNIC software. The obtained IR spectra were scanned from wave number ranging from 4000 to 500 cm−1 with a resolution of 4 cm−1 (Alara et al., 2018a,b). The spectra obtained for the extracts were interpreted with a chart for characteristics infrared absorption frequencies of functional groups. 2.6. Identification of phenolic compounds using LC–Q–TOF–MS analysis
Weight of extracts from plant sample (w ) * 100% Weight of dried plant sample (w )
Phenolic compounds in the extracts at the highest Soxhlet extraction conditions were tentatively identified through LC–Q–TOF–MS analysis equipped with PDA detector (Waters Vion IMS, USA) and symmetry C18 column of 100 mm × 2.1 mm, 1.8 μm particle size (Waters Acquity UPLC HSS T3, USA). The separation conditions are as follows: The mobile phase comprised of water (A) and 100% acetonitrile (B) using a gradient elution of was 90% A and 10% B at 0–1.25 min, 45% A and 55% B at 1.25–4.17 min, 10% A and 90% B at 4.17–6.25 min, 90% A and 10% B at 6.25–8.34 min; the elution flow rate was set at 0.5 mL/ min and injection volume of 20 μL; the analysis was performed using SYNAPT mass spectrometer (Waters) coupled with an electrospray ionization operated in negative and positive ion modes, mass range of 100–1000 m/z, spray voltage 4 keV, column temperature of 40 °C, sample temperature of 15 °C, gas flow of 0.5 mL/min, desolvation temperature of 550 °C, and desolvation gas flow rate of 800 L/h. Phenolic compounds in the V. cinerea leaf extract were characterized by MS/MS fragmentation pattern reference standards.
(1)
2.3. Total phenolic content in the extracts The total phenolic content in the extracts of V. cinerea leaves was determined using Folin-Ciocalteu reagent method (Alara et al., 2018a,b). The extracts (1 mL) at a concentration of 5 g/L was mixed with 200 μL of Folin-Ciocalteu reagent. Then, 0.6 mL of 0.2 mM Na2CO3 solution was thoroughly mixed with the mixture after 5 min. The mixture was left for 120 min and absorbance was measured at 765 nm using a UV–vis Spectrophotometer (Hitachi U-1800, Japan). Thereafter, the concentration of TPC in the plant extract was calculated from the gallic acid standard calibration curve (ranging from 50 to 500 mg/L) with equation of line y = 0.0006x + 0.0169, R2 = 0.9903 (where y is the absorbance at 765 nm and x is the sample concentration from the calibration curve), TPC was determined using Eq. (2) and results were reported as gallic acid equivalent in milligram per gram of dried extract (mg GAE/g d.w.). The analysis was repeated thrice and mean ± standard deviation was recorded.
2.7. In vitro antioxidant activities of the extract 2.7.1. DPPH assay The hydrogen atom or electron donating ability of extracts from V. 2
3
± ± ± ± 24.44 28.44 30.09 27.01 0.87a 1.00b 1.45c 1.01b ± ± ± ± 49.05 51.14 53.96 50.05 7.04 ± 0.10a 8.88 ± 0.88b 10.01 ± 0.85c 8.03 ± 0.90b
Results are expressed as means ± standard deviation. Different letters along the column indicate a significant difference (p < 0.05).
20 40 60 80 0.87a 0.45b 0.22c 0.59a ± ± ± ± 23.04 25.88 28.39 24.43 1.45a 1.01b 0.99c 0.50a ± ± ± ± 46.22 48.18 51.13 45.55 0.09a 0.76b 0.22c 0.72a ± ± ± ± 6.99 8.01 9.22 7.17 1:10 1:15 1:20 1:25 0.21a 0.87b 0.22a 0.12a ± ± ± ± 22.11 26.22 23.55 21.22
F:S (g/mL) TFC (mg QE/g d.w.)
3.1.1. Effect of extraction time on the recoveries of extract, TPC and TFC Extraction time is imperative in reducing energy and cost of the extraction process. It is one of the most important factors that alter the recovery of phenolic compounds from plant matrix. This is due to the fact that over-exposure of plant sample to localize heating tends to degrade phenolic compounds (Mojzer et al., 2016). Thus, it is essential to determine the appropriate extraction time for higher recovery. The effects of extraction time variation on the yields of extracts, TPC and TFC using a Soxhlet apparatus were examined. The extraction time was varied as 1, 2, 3, and 4 h at a constant feed-to-solvent ratio of 1:10 g/mL and ethanol concentration of 20% v/v. The maxima extraction yields, TPC and TFC were achieved at 2 h of extraction time (Table 1). Further increase in the extraction time decline the recovery of phenolic compounds. This might be due to the degradation of phenolic compounds resulted from excessive heating of the plant samples (Dahmoune et al., 2014). Moreover, the obtained results followed the Fick’s second law of diffusion, whereby at certain time of extraction, final equilibrium will be reached between the extraction solvent and plant sample. Hence, the yields tends to decline beyond final equilibrium (Alara et al., 2018c).
TPC (mg GAE/g d.w.)
The effects of extraction factors which include extraction time, feedto-solvent ratio and ethanol concentration using Soxhlet extraction technique were examined on the recoveries of extracts, TPC and TFC from V. cinerea leaves. Table 1 vividly explains the effects of individual factors on the yields. Effect of an individual factor is discussed as follows.
Extraction time (h)
3.1. Effects of Soxhlet extraction factors on the yields
Effect of feed-to-solvent ratio
3. Results and discussion
Effect of extraction time
Table 1 Effects of Soxhlet extraction factors on the recoveries of extracts, TPC and TFC from V. cinerea leaf.
The experimental procedure and analysis were carried out in triplicate. The results were statistically analyzed using ANOVA and the significance was taken at a level of p < 0.05.
0.86a 1.02b 0.11a 0.27c
2.8. Statistical analysis
± ± ± ±
(5)
45.55 48.32 44.95 42.11
* 100%
0.10a 0.55b 0.14a 0.20c
Acontrol
± ± ± ±
Acontrol − Asample
TPC (mg GAE/g d.w.)
Percentage of ABTS+ • inhibition =
TFC (mg QE/g d.w.)
2.7.2. ABTS assay The ABTS scavenging ability of V. cinerea leaf extracts (or ascorbic acid) was assayed using a method described in previous the report (Alara et al., 2018a,b). A 150 μL of V. cinerea leaf extract at different concentrations (100–500 μg/mL) was mixed with 285 μL of ABTS solution (2.45 mM potassium persulfate solution and 7 mM ABTS). The reaction mixture was incubated at room temperature for 2 h and then the light absorption was recorded spectrophotometrically at 734 nm. The IC50 value was calculated. This assay was carried out in triplicate and mean ± SD was computed. The ability of the extract to scavenge ABTS radical was calculated as follows:
6.49 7.28 6.22 5.34
Effect of ethanol concentration
where Acontrol represents absorbance measure for the mixture of methanol and DPPH solution; and Asample represents absorbance of the mixture of extract from V. cinerea leaves and DPPH solution.
1 2 3 4
TPC (mg GAE/g d.w.)
(4)
Yield (% w/w)
* 100%
Ethanol concentration (% v/v)
Acontrol
Yield (% w/w)
Acontrol − Asample
Yield (% w/w)
Percentage of DPPH inhibition capacity =
TFC (mg QE/g d.w.)
cinerea leaves was verified using DPPH assay as described in the previous reports (Alara et al., 2018a,b). A 2000 μL of 0.1 mM DPPH was added to 200 μL of the extract (or ascorbic acid) at different concentrations (100–500 μg/mL), an absorbance of the mixture was recorded at 517 nm after incubation for 30 min in the dark at room temperature. Then, the IC50 value was calculated. This assay was carried out in triplicate and mean ± SD was computed. The ability of the extract to scavenge DPPH radical was calculated as follows:
0.74a 0.22b 0.44c 0.75b
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Fig. 1. The infrared spectra of extract from V. cinerea leaves using Soxhlet extraction. Table 2 Phenolic compounds in the extract from V. cinerea leaf using Soxhlet extraction technique. S/N
Observed RT (min)
Component name
Chemical formula
Observed m/z
Adducts
Total fragment found
1 2
0.68 1.31
C13H16O10 C25H24O12
331.0671 515.1199
−H −H
7 10
3
2.07
C8H8O2
135.0452
−H
0
4 5 6 7 8 9
3.79 3.95 4.11 4.49 0.96 1.09
1-Galloyl-β-D-glucose 3,5-O-Dicaffeoylquinic acid 4Hydroxyacetophenone Kukoamine A 6-Gingerol Caesalpins P Kakuol Quercetin-7-O-rutinoside Kaempferol
C28H42N4O6 C17H26O4 C16H12O6 C10H10O4 C27H30O16 C15H10O6
529.3048 293.1762 299.0561 193.0507 611.1605 287.0553
−H −H −H −H +H +H
6 0 1 3 2 1
Table 3 In vitro antioxidant activities of extract from V. cinerea leaf using Soxhlet extraction technique.
Extract Ascorbic acid
TPC (mg GAE/g d.w.)
TFC (mg GAE/g d.w.)
IC50 value of DPPH (μg/mL)
IC50 value of ABTS+• (μg/mL)
53.96 ± 1.45 –
30.09 ± 0.44 –
301.86 ± 2.44a 130.30 ± 1.20b
269.27 ± 1.11a 73.11 ± 1.00b
Results are expressed as means ± standard deviation. Different letters along the column indicate a significant difference (p < 0.05).
This finding is similar to the results obtained in the extraction of Centella asiatica, where the maximum yield of TPC was achieved using extraction time of 120 min (Chew et al., 2011). Thus, the extraction time of 2 h was selected as optimum for the next factor (feed-to-solvent ratio).
extraction yields in conventional extraction methods (Mojzer et al., 2016). The effects of feed-to-solvent ratio were examined on the recovery yields from V. cinerea leaves. At constant extraction time of 2 h and ethanol concentration of 20% v/v, the feed-to-solvent ratio was varied as 1:10, 1:15, 1:20, and 1:25 g/mL. It can be clearly seen in Table 1 that the recoveries of extracts and phenolic compounds tend to increase as the feed/solvent increases from 1:10 g/mL through 1:20 g/ mL. Beyond 1:20 g/mL of feed/solvent, the yields declined. The maximum recovery yields of extracts, TPC and TFC were obtained at 1:20 g/ mL and further increase reduced the yields. The solvent-to-feed ratio between 10:1 (mL/g) and 20:1 (mL/g) had been reported to give optimal yields (Veggi et al., 2013). Therefore, the feed-to-solvent ratio of 1:20 g/mL was considered for the next factor (ethanol concentration).
3.1.2. Effect of feed-to-solvent on the recoveries of extract, TPC and TFC The volume of extracting solvent is another important factor, a large volume of solvent requires more energy and time to condense extraction solution in the purification process. Plant matrix needs to be totally immersed in the solvent for higher recovery during the extraction process. In general, higher volume of solvent will increase the
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antioxidant activity of the extract (Shah et al., 2018). Kukoamine A which is a spermine conjugate that possess dihydrocaffeic acid had been examined having strong antioxidant activity against 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical in a range of 5–97.5% (Wang et al., 2016). Moreover, the anti-diabetic activity of extract from V. cinerea leaves can be attributed to the presence of 3,5-O-dicaffeoylquinic acid (Alara et al., 2018c; Chen et al., 2014). The anticancer property of this extract can be attributed to the presence of 6-gingerol (Kumara et al., 2017). Thus, the identified phenolic compounds in the extract show the antioxidant activity of V. cinerea leaves.
3.1.3. Effect of ethanol concentration on the recoveries of extract, TPC and TFC Finally, extraction time and feed-to-solvent ratio were fixed at 2 h and 1:20 g/mL while the ethanol concentration was varied between 20 and 80% v/v for the extraction of yields, TPC and TFC from V. cinerea leaves. It can be observed that the yields of extracts, TPC and TFC increased gradually from 20 to 60% v/v of ethanol concentration, beyond this ethanol concentration, the yields decline steadily. The optimal recovery yields of extracts (10.01 ± 0.85% w/w), TPC (53.96 ± 1.45 mg GAE/g d.w.) and TFC (30.09 ± 0.44 mg QE/g d.w.) were obtained at 2 h of extraction time, feed-to-solvent of 1:20 g/mL and ethanol concentration of 60% v/v for V. cinerea leaves. In the similar studies on the extraction of V. amygdalina leaves using Soxhlet extraction method, the TPC yields were reported to be 63.044 mg GAE/ g d.w., 97.0 ± 0.01 mg GAE/g d.w. and 38.834 mg GAE/g d.w. using ethanol, methanol, and ethyl acetate, respectively as the extracting solvents (Oriakhi et al., 2013). This can be explained by the solubility of phenolic compounds which depends on the nature of chemical composition in the plant sample and polarity of the solvent used (Wong et al., 2014). Hence, the use of binary solvent has been suggested to be better than mono-solvent in the extraction of phenolic compounds from plant matrix. The presence of water in extraction process ease the leaching of hydrophilic antioxidants.
3.4. In vitro antioxidant activity of the extracts The extract of V. cinerea leaves at the maximum conditions of Soxhlet extraction method was examined for its antioxidant activity using DPPH and ABTS radical scavengers. The results obtained was compared with that of ascorbic acid as presented in Table 3. Several studies had reported that lowest IC50 value indicates strong antioxidant activity (Alara et al., 2018a,b; Karabegovic et al., 2014). Although, the antioxidant activity of ascorbic acid was higher than the extract, however, the results show that the extract possessed antioxidant activity. This might be due to the effect of localized heating using Soxhlet extraction method which might have degraded the antioxidant in the extract (Karabegovic et al., 2014). The different scavenging activities observed using DPPH and ABTS might be due to the different mechanisms involved in the assays. It has been reported that high molecular weight phenolics possess the capacity to scavenge ABTS+• which depends on the molecular weight, nature of hydroxyl group substitution and number of aromatic rings (Khan et al., 2012). In addition, the presence of kukoamine A, a spermine conjugate with dihydrocaffeic acid has been investigated to potentially possess a strong antioxidant activity (Shah et al., 2018; Wang et al., 2016).
3.2. Identified functional groups in the extract Fourier transform infrared (FTIR) is one of the widely used characterizations for the identification and elucidation of functional groups in the plant extracts. This characterization was used to identify functional groups in the extract from V. cinerea leaves at the maximum conditions of Soxhlet extraction based on the peak value in the region of infrared radiation. The characteristic absorption peaks are presented in Fig. 1. The broad peak at 3328.79 cm−1 confirmed the presence of phenolic compounds. However, the presence of lipid-carbohydrate (mainly vas(CH2) and vs(CH2) stretching) can be attributed to the peaks at 2123.24 cm−1, 2977.79 cm−1 and 2901.36 cm−1. The peaks at 1643.14, 1452.99, 1383.43, and 1274.26 cm−1 indicates the presence of C]O stretching, NeH bending, bending of carboxylic acid (presence of tannins, flavonoids, glycosides, and saponins), and nucleic acid (vas (> P = O) stretching of phosphodiesters), respectively (Dilek et al., 2012). The band at 1274.26 cm−1 shows the presence of C–O groups of polyols such as hydroxyflavonoids (Oliveira et al., 2016). The presence of ester group and/or secondary alcohols could be associated with the presence of sharp peaks at 1085.30 cm−1 and 1043.52 cm−1. In addition, band peak at 877.39 cm−1 could be attributed to aromatic ring vibration. Thus, the observed characteristic fingerprints in the extract from V. cinerea leaves using Soxhlet extraction method reflected the presence functional characteristics associated with polyphenols and flavonoids.
4. Conclusion This study investigated the effects of Soxhlet extraction factors, namely: extraction time, feed-to-solvent ratio and ethanol concentration on the recoveries of extract, TPC, TFC from V. cinerea leaves. The highest yields were obtained using extraction time of 2 h, feed/solvent of 1:20 g/mL and ethanol concentration of 60% v/v. The LC–Q–TOF–MS and FTIR showed the presence of phenolic compounds in the extract. More so, the extract from V. cinerea leaves possesses potent antioxidant activity with reference to ascorbic acid. Thus, the extract from V. cinerea leaves can potentially be used as the natural antioxidant. Conflict of interest We declare no conflict of interest.
3.3. Identified phenolic compounds in the extracts
Acknowledgement
The identification of phenolic compounds in the extract from V. cinerea leaves at maximum conditions of Soxhlet extraction was carried out using LC–Q–TOF–MS. A total number of 9 phenolic compounds were tentatively identified by comparing spectral data with reference compounds. The identified phenolic compounds are presented in Table 2. The compounds are 1-galloyl-β-D-glucose (MS ion at m/z 331.0671), 3,5-O-dicaffeoylquinic acid (MS ion at m/z 515.1199), 4hydroxyacetophenone (MS ion at m/z 135.0452), kukoamine A (MS ion at m/z 529.3048), 6-gingerol (MS ion at m/z 293.1762), caesalpins P (MS ion at m/z 299.0561), and kakuol (MS ion at m/z 193.0507) were identified through negative ionization. However, only two flavonoids, namely quercetin-7-O-rutinoside and kaempferol were identified through positive ionization. The presence of 1-galloyl-β-D-glucose, kukoamine A, quercetin-7-O-rutinoside, and kaempferol indicate the
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