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Carotenoids, pigments, phenolic composition and antioxidant activity of Oxalis corniculata leaves
Food Bioscience 32 (2019) 100472
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Food Bioscience journal homepage: www.elsevier.com/locate/fbio
Carotenoids, pigments, phenolic composition and antioxidant activity of Oxalis corniculata leaves
T
Alam Zeb∗, Muhammad Imran Department of Biochemistry, Faculty of Biological Sciences, University of Malakand, Chakdara, 18800, Khyber Pakhtunkhwa, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: Oxalis corniculata Phenolic compounds Carotenoids Chlorophyll Sleeping beauty
Carotenoids, pigments, phenolic composition and antioxidant activity of Oxalis corniculata leaves grown in dry, marshy and moist areas were evaluated. High-performance liquid chromatography analyses showed 13 phenolic compounds, which were higher in samples from dry areas, followed by those from marshy and moist areas. Kaempferol-3-sophorotrioside, quercetin-3-(caffeoyldiglucoside)-7-glucoside, isorhamnetin-3-caffeoyl-7-glucoside, and p-coumaric acid were the major compounds. High levels of all-E-violaxanthin, all-E-neoxanthin, all-Elutein, and 9-Z-lutein were present in samples from marshy and moist areas. Phytofluene, hydroxy-pheophytin a, and chlorophyll b were major pigments. Pigments were higher in samples from marshy areas, followed by moist and lowest in dry area samples. Total phenolic and flavonoids contents varied among the samples. Oxalis corniculata leaves showed high radical scavenging activity attributed to the high levels of carotenoids and phenolic compounds. Results showed that Oxalis corniculata leaves are a good source of bioactive substances such as carotenoids and phenolics for the uses in food fortification and as ingredients.
1. Introduction
(Tibuhwa, 2017). However, detail fingerprinting of these compounds was not reported. Similarly, the presence of important glycosides, including dihydroxy-5-methoxyflavon-8-glucopyranoside was reported in the Chinese Oxalis (Liu, Staerk, Nielsen, Nyberg, & Jäger, 2015). Bordoloi et al. (2016) reported the antioxidant activity, phenolic composition, mineral content and antifungal properties of Oxalis leaves along with other herbs. Quercetin, rutin and gallic acid were quantified in the leaves, while no other phenolic compounds were reported in Oxalis leaves. Another Indian variety of Oxalis was shown to contain phydroxybenzoic acid, ferulic acid, and rutin (Mukherjee, Pal, Chakraborty, Koley, & Dhar, 2018). In Pakistan, the plant is used for medicinal purposes, as a vegetable or as a salad, however, very little is known about its phytochemical constituents (Abbasi, Shah, & Khan, 2015). All these reported studies on Oxalis corniculata did not analyze phenolic compounds in detail. Carotenoids and pigments are important organic compounds, imparting color to plant leaves, foods and agriculture products. Carotenoids have a significant role in light harvesting and photoprotection in photosynthesis in plants (Damjanovic, Ritz, & Schulten, 1998). Pigments such as chlorophyll and their derivatives are used in the food industry and in medicine (Solymosi & Mysliwa-Kurdziel, 2017). However, these compounds have not been studied in Oxalis corniculata. This study was, therefore, done to evaluate carotenoids,
Oxalis corniculata L (sleeping beauty) is an important herb commonly grown in temperate zones in Europe, America and Asia. It is found in temperate zones around the world (Sharma & Kumari, 2014). The plant grows in semi-dry, moist, and wet spots such as yards, farms, and forest. The leaves are yellowish green, tri-hearted, alternate and delicate attached to the stem. Oxalis corniculata leaves are known to have several medicinal properties such as oxidative stress reduction in Parkinson disease and neurological disorders (Aruna, Rajeswari, & Sankar, 2016), anti-diabetic (Al-Qalhati, Waly, Al-Attabi, & Al-Subhi, 2016), reduction in chemical-induced pulmonary toxicity (Ahmad, Khan, & Shah, 2015), immunomodulatory effects (Shafi & Banerjee, 2018), anti-inflammatory (Dighe, Kuchekar, & Wankhede, 2016), antibacterial (Mukherjee et al., 2013), antifungal and insecticidal activities (Rehman, Rehman, & Ahmad, 2015). These effects were due to the presence of several important bioactive compounds. Zhang, Dong, and Cheng (2018) studied essential oils from the Chinese Oxalis corniculata. The authors identified 28 compounds, representing 85.2% of the total oil. The major compounds were geranyl acetate (13.3%), terpinolene (9.2%), hexadecanoic acid (7.7%), linalool oxide (7.4%), and geraniol (6.4%). In the Tanzanian variety of Oxalis, phytosterols, coumarins, terpenes, and flavonoids were identified
∗
Corresponding author. Department of Biochemistry, Faculty of Biological Sciences, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan. E-mail addresses: [email protected], [email protected] (A. Zeb).
https://doi.org/10.1016/j.fbio.2019.100472 Received 4 September 2018; Received in revised form 25 September 2019; Accepted 28 September 2019 Available online 02 October 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Identification and variations in phenolic composition of different Oxalis corniculata L. leaves samples. Peak
Retention time (min)
λ max (nm)
Compound
Amount (× 102 μg/g) Dry
1 1 2 2.7 3 3.3 4 4.7 5 5.6 6 7.6 7 7.8 8 8.8 9 10.5 10 10.8 11 13 12 13.5 13 17.3 Total Amount (μg/g)
p-Hydroxybenzoic acid p-Coumaroylhexose Sinapic acid p-Coumaric acid Caffeic acid Kaempferol-3-(p-coumaroyldiglucoside)-7-glucoside 3-O-Caffeoylquinic acid Kaempferol-3-O-glucoronide Vitexin-6-O-malonyl-2-O-xyloside Isorhamnetin-3-caffeoyl-7-glucoside Quercetin-3-(caffeoyldiglucoside)-7-glucoside Kaempferol-3-O-sophorotioside Ellagic acid deoxyhexoside
256 316 323, 311, 323, 320, 314, 336, 335, 337, 335, 346, 362,
Marshy a
240 233 296, 274, 232 271, 271, 271, 270, 320, 315,
232 232 236 235 236 237 272 244
0.28 ± 0.01 0.41 ± 0.02a 1.8 ± 0.1a 12.1 ± 0.1a 1.28 ± 0.04a 1.13 ± 0.02a 0.71 ± 0.04a 3.1 ± 0.1a 5.4 ± 0.1a 16.0 ± 0.1a 22.6 ± 0.1a 32.0 ± 0.2a 2.01 ± 0.04a 98.6a
Moist b
0.38 ± 0.02 0.093 ± 0.01b 0.79 ± 0.03b 6.1 ± 0.1b 0.71 ± 0.03b 1.24 ± 0.03b 0.54 ± 0.02b 2.03 ± 0.04b 3.5 ± 0.1b 9.5 ± 0.1b 10.2 ± 0.1b 13.4 ± 0.1b 4.06 ± 0.01b 52.4b
0.51 ± 0.02c 0.14 ± 0.01c 0.77 ± 0.02b 3.7 ± 0.1c 0.55 ± 0.03c 0.11 ± 0.01c 1.07 ± 0.04c 3.02 ± 0.01a 1.0 ± 0.1c 5.1 ± 0.1c 4.4 ± 0.1c 14.6 ± 0.1c 0.34 ± 0.02c 35.1c
Values are means with a standard deviation of the triplicate measurements. Different letters (a-c) in the same compound represent significance at p < 0.05 using post-hoc Tukey's test.
at 1790 × g for 15 min using an Eppendorf centrifuge (model 5702R, Eppendorf AG, Hamburg, Germany) at 25 °C. The above extraction was done three times and finally, the extracts were pooled and concentrated using solvent evaporation under vacuum. The residue was dissolved in high performance liquid chromatography (HPLC) solvent B, and 2 mL from each sample was filtered using a polytetrafluoroethylene (PTFE) syringe filter with 0.45 μm pore size (Agilent Technologies, Waldbronn, Germany) into HPLC vials (2 mL). The phenolic compounds were separated with the help of an Agilent Zorbax Eclipse C18 column (4.6 × 100 mm, 3.5 μm). The gradient chromatography was carried out with an Agilent HPLC model 1260 Infinity Better (Agilent Technologies) system consists of a quaternary pump, auto-sampler and diode array detector (DAD). The solvent system consisted of solvent A, which consisted of methanol-acetic aciddeionized water (10:2:88, v/v) and solvent B (methanol-acetic aciddeionized water, 90:2:8, v/v). The elution started with 100% A, reached 85% A in 5 min, at 20 min it was 50% A, and finally reached 30% A at 25 min (Zeb, 2015). The elution flow rate was 1 mL/min. The DAD chromatograms were obtained at 280, 320 and 360 nm and the wavelength used were selected by the operator. The absorption spectra for every peak were automatically scanned in the range of 200–600 nm. The identification of phenolic compounds was carried out using the retention time and absorption spectra of the available standard compounds (p-hydroxybenzoic acid, sinapic acid, 3-caffeoylquinic acid, caffeic acid, and p-coumaric acid). The compounds were quantified against the respective standard calibration curves. In the case where the standard compound was not available, the calibration curves of those compounds with a relative response factor (RRF) such as rutin and quercetin-3-glucoside were used. The RRF was calculated manually for each unknown compound as the ratio of the response factor of the individual related compound and response factor of the unknown. The amount of phenolic compounds were calculated and expressed as μg/g of fresh weight (fw).
pigments and the phenolic composition of Oxalis grown with the availability of different water resources. 2. Materials and methods 2.1. Reagents and chemicals Methanol, ethanol, all-E-violaxanthin, all-E-neoxanthin, all-E-lutein, chlorophyll a and b, p-hydroxybenzoic acid, p-coumaric acid, 3-caffeoylquinic acid, quercetin-3-rutinoside, and quercetin-3-glucoside were from Sigma-Aldrich (Sigma-Aldrich, Darmstadt, Germany). Caffeic acid was purchased from Tokyo Chemical Industries (TCI, Tokyo, Japan). Ultrapure deionized double distilled water was prepared using a Labtech Automatic Water-still (model LWD-3010D, Labtech, Namyangju, South Korea) coupled with an ELGA deionizer (model B114/B, ELGA LabWater, High Wycombe, UK). All chemicals and reagents used in this study were of HPLC or ACS grade and were obtained from Sigma-Aldrich unless otherwise mentioned. 2.2. Sample collection and preparation The fresh leaves of Oxalis corniculata were collected from different places of Lower Dir, Khyber Pakhtunkhwa (Pakistan), i.e., Chakdara (34.6666° N, 72.0290° E) and Talash (34.7415° N, 71.8720° E) which are popular habitats for the plant. The plant was kindly authenticated by Dr. Nasrullah Khan, Department of Botany, University of Malakand (Chakdara, Pakistan). Fresh plant leaves were collected from three different habitats, i.e., dry, marshy and moist areas. The samples from the moist area had been growing in fresh water; the marshy area was near the river, while the dry area was open with no shrubs or trees. The low to high temperatures were 13–22, 14–25 and 11–20 °C in the moist, dry and marshy areas, respectively. The air moisture levels of collection areas were 63, 47 and 65% in the moist, dry and marshy areas, respectively, while moisture contents of Oxalis corniculata leaves were 78.6% (moist), 71.2% (dry) and 76.5% (marshy). The leaves as harvested from the collected plant samples were separated and then washed with distilled water, and crushed to form a paste using a mortar and pestle. The samples for analysis were prepared from the paste materials.
2.4. Analysis of carotenoids and pigments One g of each sample of Oxalis leaves was extracted with ice-cold absolute ethanol (10 mL) and Vortex for 1 hr using a Witeg Wisd Vortex mixer (model VM-10, Witeg Labortechnik, Wertheim, Germany). After Vortexing, ice-cold absolute ethanol (10 mL) having 0.1% BHT (w/v) was added and again subjected to Vortexing for 30 min. The extractions were done in the presence of ambient light and repeated until the leaves were no longer green. Under vacuum conditions at 35 °C, the solvent was evaporated and final residue of each sample was dissolved in 2 mL 2-propanol and filtered with the PFTE syringe filters into HPLC vials
2.3. Analysis of phenolic compounds Each sample (1 g) was extracted with 10 mL of methanol-water (9:1, v/v) in 50 mL capped glass tubes. The tubes were placed in a shaking water bath at 35 °C for 1 h. After shaking, the samples were centrifuged 2
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Fig. 1. Representative HPLC-DAD chromatograms of phenolic compounds at 320 nm. (A) Dry samples, (B) marshy and (C) moist samples. Each peak in the chromatogram represents individual phenolic compound with details characteristics shown in Table 1.
every peak were measured from 200 to 750 nm. The chromatograms were obtained at 450 (for carotenoids), and 650 nm (for pigments) using an Agilent OpenLAB chromatography data system (CDS) ChemStation edition version C.01.05 software. The carotenoids were identified with the help of the available standards (all-E-violaxanthin, all-Eneoxanthin, all-E-lutein, chlorophyll a and b), or by comparing absorption spectra reported in earlier literature (Crupi, Milella, & Antonacci, 2010; Delgado-Pelayo, Gallardo-Guerrero, & HorneroMendez, 2014). In the case where the standard compound was not available, the calibration curves of those compounds with a relative response factor such as chlorophyll a, chlorophyll b, all-E-violaxanthin and all-E-lutein etc. were used. The quantitation of compounds was done using the peak areas (calculated after automatic integration by the software) using the respective standard calibration curves and expressed as μg/g of the fresh weight.
(2 mL). A reversed-phase HPLC system with the same specifications as previously was used for the separation and identification of carotenoids and pigments. The separations were done with the same C18 column with the same specifications but specifically maintained and equilibrated for carotenoids and pigments. The tertiary mobile phase system consisted of solvent A (methanol:deionized water, 92:8, v/v) with 10 mM ammonium acetate; deionized water containing 0.01 mM ammonium acetate as solvent B and solvent C was 100% methyl tertiary butyl ether (MTBE). The injection volume was 50 μL with the solvent flow rate of 1 mL/min. The gradient program was that of Zeb (2017). The gradient program was started with 80% A, 18% B and 2% C at 0 min. It changed to 80% A, 12% B and 8% C at 3 min, and at 25 min, it reached 65% A, 5% B with 30% C. The final gradient at 40 min was 60, 0, and 40% of A, B, and C, respectively. The initial solution was then run for 10 min to re-initialize the column. The absorption spectra of 3
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Fig. 2. Biplot of the principal component analysis (PCA) used for quantitative data of different samples of Oxalis corniculata. (A) PCA of phenolic compounds, and (B) PCA of carotenoids and pigments. The variables are phenolic compounds, pigments and carotenoids identified and quantified using HPLC.
4
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Fig. 3. Representative HPLC-DAD chromatograms of carotenoids and pigments at 450 nm. (A) Dry samples, (B) marshy and (C) moist samples. Each peak in the chromatogram represents individual compound with details characteristics shown in Table 2.
and Ullah (2017). Phenolic compounds were extracted in samples of the Oxalis corniculata leaves with ethanol in a ratio of 1:10, w/v (100%), methanol (100%) and methanol-water (9:1, v/v). The combined extract sample (0.5 mL) was mixed with 2.5 mL Folin Ciocalteau (FC) reagent and 2 mL 7.5% sodium carbonate and kept in the dark for 1 h. The absorbance of the sample was measured using a spectrophotometer (model PharmaSpec UV-1700, Shimadzu, Kyoto, Japan) at 765 nm. The TPC was calculated using a gallic acid standard curve and shown as mg of gallic acid equivalents (GAE)/g.
2.5. Principal component analysis Principal component analysis (PCA) was carried out using XLSTAT software (version 19.5.47062, 2017, Addinsoft, New York, NY, USA) macros for MS Excel (Microsoft Office 2010; Microsoft Austria GmbH, Vienna, Austria). All phenolic compounds, carotenoids, and pigments identified for each sample were included. The PCA biplot mapped the variables (individually identified compound) and samples (dry, marshy, and moist) using loadings and scores in respective dimensional spaces determined by the principal components (PC). The Spearman correlation coefficient and square cosine values were determined between the samples.
2.7. Total flavonoids contents Total flavonoids contents (TFC) were extracted with methanol, ethanol, and water as above. The combined extract solution (0.5 mL) was mixed with 2% aluminium trichloride (AlCl3) and kept for 1 h at 25 °C. The absorbance of the mixture was measured at 420 nm with the
2.6. Total phenolic contents Total phenolic content (TPC) was measured using the method of Zeb 5
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Table 2 Identification and characterization of carotenoids and pigments profile of different Oxalis corniculata L. leaves samples. Peak
Rt (min)
λ max (nm)
Identity
Amount (μg/g) Dry
1 1.1 2 4.4 3 5.7 4 6.2 5 7.2 6 8.4 7 8.8 8 9.1 9 9.9 10 14.5 11 15.8 12 19.1 13 20.4 14 22.3 15 22.9 16 25.4 Total Pigments Total Carotenoids
Phytofluene Peophorbide b All-E-violaxanthin Pheophytin b Hydroxy-pheophytin b All-E-neoxanthin Hydroxy-pheophytin a Hydroxy-pheophytin a' Pheophytin a All-E-lutein 9-Z-lutein Chlorophyll b Chlorophyll b epimer 13-Hydroxy-chlorophyll b Chlorophyll b' Chlorophyll a
366, 654, 470, 666, 652, 466, 666, 666, 666, 472, 468, 654, 648, 650, 648, 664,
347, 600, 440, 610, 598, 436, 610, 610, 608, 446, 440, 599, 602, 600, 600, 618,
328 438 416 410 522, 416 532, 534, 536, 422 418 436 468 462 464 432
Marshy a
433 502, 406 504, 408 506, 407
8.3 ± 0.3 0.21 ± 0.01a 1.3 ± 0.1a 0.92 ± 0.1a 1.7 ± 0.1a 2.6 ± 0.1a 8.1 ± 0.3a 5.9 ± 0.2a 3.8 ± 0.1a 128 ± 2a 57+1a 33 ± 1a 6.4 ± 0.5a 6.5 ± 0.2a 1.14 ± 0.02a 2.9 ± 0.1a 78.9 189
b
18 ± 1 1.01 ± 0.01b 8.8 ± 0.2b 5.4 ± 0.1b 11.5 ± 0.3b 14+1b 33+1b 21+1b 4.5 ± 0.1b 98+1b 106 ± 2b 54 ± 2b 6.9 ± 0.4a 13 ± 1b 2.1 ± 0.1b 4.9 ± 0.3b 176 227
Moist 52 ± 2c 1.1 ± 0.1b 7.7 ± 0.2c 5.1 ± 0.3b 8.8 ± 0.6c 39+1c 3.9 ± 0.1c 26.7 ± 0.2c 4.6 ± 0.1b 113 ± 1c 66+2c 33+3a 4.6 ± 0.5b 2.2 ± 0.1c 0.44 ± 0.01c 1.46 ± 0.02c 144 225
Values are means with a standard deviation of the triplicate measurements. Different letters (a-c) in the same compound represent significance at p < 0.05 using post-hoc Tukey's test.
samples from marshy areas, followed by dry and moist areas. Thus, the total amount of polyphenols determined using HPLC was 46 and 64% lower in the samples from marshy and moist areas, respectively, as compared to dry areas.
spectrophotometer against the blank (Mohdaly, Smetanska, Ramadan, Sarhan, & Mahmoud, 2011). The TFC was calculated using a quercetin standard curve and shown as mg of QE/g. 2.8. Antioxidant activity
3.2. Principal component analysis of phenolic compounds Different extracts of the Oxalis corniculata leaves were prepared, i.e., in methanol, ethanol, and methanol-water. The different extracts (5 μL) were incubated in the dark for 30 min with 0.1 mM of DPPH solution (1.95 μL). The absorbance of the samples was measured at 515 nm with the spectrophotometer against the DPPH solution according to the previously reported method (Zeb & Ullah, 2017). The radical scaven-
Fig. 2A shows the biplot of the loadings and scores in the respective dimensional spaces. The principal components accounted for 99.1% of the variance due to the sample origin. The F1 variance was higher (89.1%), followed by F2 (9.99%). The score plot also showed some homogeneity among certain variables. Samples from dry and marshy areas showed higher Spearman correlation with respect to vitexin-6-Omalonyl-2-O-xyloside and quercetin-3-(caffeoyldiglucoside)-7-glucoside. The moist area samples showed a correlation in terms of p-coumaric acid, kaempferol-3-O-sophorotioside, kaempferol-3-O-glucuronide and isorhamnetin-3-caffeoyl-7-glucoside. Other phenolic acids had relatively low correlations.
(Ac − As)
ging activity was calculated as RSA (%) = ⎡ Ac ⎤ × 100 , where Ac ⎣ ⎦ is absorbance of the control, and As is absorbance of the sample. 2.9. Data analyses Data were expressed as the mean ± standard deviation (SD) of 3 or more independent measurements. One-way analysis of variance (ANOVA) was done followed by the post hoc test using the Holm-Sidak method at α = 0.05 using the XLSTAT software macros for MS Excel.
3.3. Variations in carotenoids and pigments composition Carotenoids and pigments in Oxalis corniculata were effectively separated as shown in Fig. 3. The identified and quantified carotenoids and pigments are shown in Table 2. Five carotenoids and 11 pigments were identified. Table 2 shows the variation in carotenoids and pigments present in Oxalis corniculata samples. Phytofluene, all-E-neoxanthin and hydroxy-pheophytin a' levels were highest (p < 0.05) in the samples from the moist areas, followed by marshy areas. Significantly (p < 0.05) higher amounts of all-E-violaxanthin, hydroxypheophytin b, 9-Z-lutein, and chlorophyll b were found in the samples from the marshy areas, followed by the moist areas and then dry areas. A relatively low amount of pheophorbide b and chlorophyll b' were found in all samples. Pheophytin b was found in higher amounts in both moist and marshy area samples and lowest in dry area samples. Hydroxy-pheophytin a, 13-hydroxy-chlorophyll b, chlorophyll b', and chlorophyll a were found in significantly (p < 0.05) higher amounts in marshy area samples followed by dry and then moist area samples. Pheophytin a was significantly (p < 0.05) higher in moist and marshy areas samples compared to dry area samples. All-E-lutein was higher in the dry area samples, followed by the moist area and then marshy area samples. The total amounts of carotenoids were significantly higher
3. Results 3.1. Variations in phenolic compounds Table 1 shows the characteristics of phenolic compounds in Oxalis corniculata leaves of dry, moist and marshy origins. In total, 13 phenolic compounds were separated, identified and quantified as shown in Fig. 1. These phenolic compounds include hydroxybenzoic acids, cinnamic acids, flavonoids and their derivatives. Table 1 shows the variations in the phenolic composition of Oxalis corniculata leaves grown in different areas. The p-hydroxybenzoic acid and 3-caffeoylquinic acid was significantly higher in samples from moist areas, followed by marshy and dry areas. The amount of p-coumaroyl hexose, sinapic acid, p-coumaric acid, caffeic acid, vitexin-6-Omalonyl-2-O-xyloside, isorhamnetin-3-caffeoyl-7-glucoside, quercetin3-(caffeoyldiglucoside)-7-glucoside, kaempferol-3-O-glucuronide and kaempferol-3-O-sophorotrioside were significantly (p < 0.05) higher in samples from dry areas than moist and marshy areas. Kaempferol-3(p-coumaroyldiglucoside)-7-glucoside was significantly higher in 6
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3.5. Total phenolic contents The TPC of Oxalis corniculata are shown in Fig. 4. The TPC for extracts of dry, moist and marshy area samples differed with extraction solution used. The highest TPC was obtained using ethanol, followed by methanol, while methanol-water extracts had the lowest levels for all samples. In ethanolic extracts, marshy samples contain the highest TPC, followed by moist and lowest in dry area samples. The methanolic extracts of marshy samples contain the highest amount followed by dry and lowest in moist area samples. In methanol-water extracts, moist samples showed the highest TPC, followed by marshy and lowest in dry area samples. The net TPC was highest in marshy area samples. 3.6. Total flavonoid contents Different solvents were used to extract flavonoids (Fig. 4) from the Oxalis corniculata. In ethanolic extracts, the lowest total flavonoids contents (TFC) were measured in dry area samples, whereas marshy and moist area samples had similar amounts. A similar trend was also observed in the case of methanolic extracts. In the case of methanolwater extracts, the TFC was highest (p < 0.05) in the dry area followed by moist area samples. 3.7. Radical scavenging activity The radical scavenging activities (RSA) of Oxalis corniculata were studied in different extracts as shown in Fig. 4. The RSA values were significantly higher in the ethanolic extracts of all samples Oxalis corniculata as compared to methanol and methanol-water extracts. In the case of methanolic extracts, the marshy area samples showed significantly (p < 0.05) higher RSA values as compared to dry and moist area samples. The RSA of methanol-water was the lowest among all the extractions. 4. Discussion Thirteen phenolic compounds were identified and quantified in the leaves Oxalis corniculata grown with different moisture conditions (moist, marshy and dry). They were p-hydroxybenzoic acid, p-coumaroylhexose, sinapic acid, p-coumaric acid, caffeic acid, kaempferol3-(p-coumaroyldiglucoside)-7-glucoside, caffeoylquinic acid, kaempferol-3-O-glucuronide, vitexin-6-O-malonyl-2-O-xyloside, isorhamnetin3-caffeoyl-7-glucoside, quercetin-3-(caffeoyldiglucoside)-7-glucoside, kaempferol-3-O-sophorotioside, and ellagic acid deoxyhexoside. An earlier study reported only three phenolic compounds, i.e., rutin, gallic acid, and catechol using reference standards (Bordoloi et al., 2016). However, none of these compounds was found in the present study. Ibrahim et al. (2012) identified 12 phenolic compounds in the Oxalis corniculata from Karachi, Pakistan. However, no quantitative results had been reported. Another study reported a very specific glycoside called corniculatin (Ibrahim et al., 2013). Similarly, another study reported p-hydroxybenzoic acid, ferulic acid, and rutin in Oxalis corniculata leaves (Mukherjee et al., 2018). These compounds were not reported in the present study, which may be due to the difference in the area of collection of plant samples, or variety and analysis. This study confirms that there were significant differences in the phenolic composition of the selected samples from the different area. Higher amounts of phenolic compounds were reported in the samples from the dry area. This may be due to the expression of specific genes, which are responsible for the formation of enzymes which synthesize phenolic compounds. The nature of dryness of the sample collection areas in the present study may thus be contributing to the abiotic stress. During the abiotic stress, specific phenolic compounds are accumulated to cope with oxidative stress (Martinez et al., 2016). A high correlation of phenolic compounds in the PCA biplot also showed that dry and marshy areas were significantly correlated with respect to
Fig. 4. Total phenolic contents, total flavonoids contents and radical scavenging activity of Oxalis corniculata leaves collected from dry, marshy and moist areas. The extractions were done in 100% ethanol, 100% methanol and 90:10% methanol:water. Different letters (a–c) in the same extract represent significance differences using Tukey's test at p < 0.05 among the samples.
compared to pigments in all samples.
3.4. Principal component analysis of carotenoids and pigments The biplot shown in Fig. 2B represents loadings and scores in the respective dimensional spaces. The principal components showed 96.3% variance in dry, marshy and moist area samples. The F1 variance was higher (82.7%), followed by F2 (13.5%). This score plot also showed some homogeneity among certain variables. The samples from dry and marshy areas showed a higher Spearman correlation coefficient between hydroxy-pheophytin a and chlorophyll b. The moist area samples showed a higher correlation in terms of lutein and its isomers, phytofluene, hydroxy-pheophytin a', and neoxanthin. The squared cosine values of lutein was 0.988, 9-Z-lutein (0.999), phytofluene (0.892), hydroxyl-pheophytin a' (0.773) and neoxanthin were 0.843. All other pigments and all-E-violaxanthin had relatively low correlations. 7
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neoxanthin, all-E-lutein, and 9-Z-lutein contents were found at high levels as compared to pigments. Carotenoids were higher in marshy and moist area samples. Phytofluene, hydroxy-pheophytin a, and chlorophyll b were the major pigments. Pigments were significantly higher in the marshy area, followed by the moist area samples. Total phenolics and flavonoids varied in different solvents and among the samples. The results showed that Oxalis corniculata leaves had a high radical scavenging activity which may be due to the high levels of carotenoids and phenolics. This herb has the potential to be a source of functional food ingredients.
the location of Oxalis corniculata. The chromatographic separation of Oxalis corniculata leaves showed for the first time, a total of 16 carotenoids and pigments. The pigments included phytofluene, pheophorbide b, pheophytin b, hydroxy-pheophytin b, hydroxy-pheophytin a, hydroxy-pheophytin a', pheophytin a, chlorophyll b, chlorophyll b epimer, 13-hydroxy-chlorophyll b, chlorophyll b' and chlorophyll a. The carotenoids were all-E-violaxanthin, all-E-neoxanthin, all-E-lutein, and 9-Z-lutein. The carotenoid β-carotene was previously reported in Oxalis leaves (Rajyalakshmi et al., 2001). Since Oxalis corniculata is an edible and medicinal herb, the high amounts of carotenoids and pigments suggested that the plant is a potential source of commercial food-grade carotenoids and pigments (Mukherjee et al., 2018). The total amounts of carotenoids were significantly higher than pigments. The highest amounts of total phenolic compounds were found in the samples of marshy areas followed by moist areas. Similarly, marshy and moist area samples were shown to contain a higher amount of carotenoids while dry area samples had the lowest level. The lower values of carotenoids and pigments in dry area samples may be due to the higher synthesis of phenolic compounds. This was confirmed by the significantly high correlation coefficient (R2 = 0.9047) between total carotenoids and total phenolics. Similarly, a correlation coefficient (R2 = 0.6832) between total pigments and total phenolics were also observed. This also suggested that phenolic compounds are present in high amounts with relatively stressed conditions as compared to carotenoids. There was a high homogeneity among the marshy and dry area samples as confirmed using the PCA biplot (Fig. 2). Spectrophotometric studies of TPC showed relatively higher amounts in marshy area samples as compared to dry and moist area samples. However, TPC in all samples and extracts were lower than reported for Indian samples (Bordoloi et al., 2016; Mukherjee et al., 2018), and higher than samples from Bangladesh (Tukun et al., 2014). The difference may be due to variety, climate, soil conditions, and method of extractions and analysis. Similarly, TFC of the dry area samples were low in ethanol and methanol extractions compared to the methanol-water extract. Ethanol extraction resulted in low TFC in all samples. The TFC was relatively higher compared to the values reported previously (Mukherjee et al., 2018). The higher RSA values were observed in ethanol extracts in all samples. The higher RSA was strongly correlated with the amounts of TPC and TFC of the respective samples. The RSA values of the present samples were consistent with the RSA reported for Oxalis corniculata leaves (Bordoloi et al., 2016). TPC, TFC, and consequently RSA may vary with plant species, plant body part and variety. For example, TPC and TFC of Pistacia vera were higher in methanolic extracts as compared to aqueous extracts (Taghizadeh et al., 2018). An inverse was reported in Flammulina velutipes and Hypsizygus tessellatus, where TPC and TFC were higher in aqueous extracts as compared to methanolic extract (Shah, Ukaegbu, Hamid, & Alara, 2018). The use of different extraction solvents for the determination of phenolic compounds, flavonoids, and radical scavenging activity is advantageous (Silva, Costa, Calhau, Morais, & Pintado, 2017). These results showed that bioactive components solubilized in each solvent may be a good source for possible food supplementation or medicinal applications, suggesting that Oxalis corniculata leaves can be considered a good source of food ingredients or supplement.
Declaration of competing interest The authors confirmed that they have no conflict of interest with respect to the work described in this manuscript. Acknowledgement The authors are grateful to the Higher Education Commission (HEC) of Pakistan for providing financial assistance under the National Research Program for Universities (NRPU) funded project No 2344. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fbio.2019.100472. References Abbasi, A. M., Shah, M. H., & Khan, M. A. (2015). Wild edible vegetables of lesser Himalayas: Ethnobotanical and nutraceutical aspects. Cham, Switzerland: Springer International Publishing978-3-319-09542-4. Ahmad, B., Khan, M. R., & Shah, N. A. (2015). Amelioration of carbon tetrachlorideinduced pulmonary toxicity with Oxalis corniculata. Toxicology and Industrial Health, 31(12), 1243–1251. Al-Qalhati, I. R. S., Waly, M., Al-Attabi, Z., & Al-Subhi, L. K. (2016). Protective effect of Pteropyrum scoparium and Oxalis corniculata against streptozotocin-induced diabetes in rats. Federation of American Societies for Experimental Biology Journal, 30 1176.5. Aruna, K., Rajeswari, P. D. R., & Sankar, S. R. (2016). The effects of Oxalis corniculata l. extract against MPTP induced oxidative stress in mouse model of Parkinson's disease. Journal of Pharmaceutical Sciences and Research, 8(10), 1136. Bordoloi, M., Bordoloi, P. K., Dutta, P. P., Singh, V., Nath, S., Narzary, B., et al. (2016). Studies on some edible herbs: Antioxidant activity, phenolic content, mineral content and antifungal properties. Journal of Functional Foods, 23, 220–229. Crupi, P., Milella, R. A., & Antonacci, D. (2010). Simultaneous HPLC-DAD-MS (ESI+) determination of structural and geometrical isomers of carotenoids in mature grapes. Journal of Mass Spectrometry, 45(9), 971–980. Damjanovic, A., Ritz, T., & Schulten, K. (1998). Light-harvesting and photoprotection by carotenoids in photosynthesis. Biophysical Journal, 74(2), A76. Delgado-Pelayo, R., Gallardo-Guerrero, L., & Hornero-Mendez, D. (2014). Chlorophyll and carotenoid pigments in the peel and flesh of commercial apple fruit varieties. Food Research International, 65, 272–281. Dighe, S., Kuchekar, B., & Wankhede, S. (2016). Analgesic and anti-inflammatory activity of β-sitosterol isolated from leaves of Oxalis corniculata. International Journal of Pharmacological Research, 6, 109–113. Ibrahim, M., Hussain, I., Imran, M., Hussain, N., Hussain, A., & Mahboob, T. (2013). Corniculatin A, a new flavonoidal glucoside from Oxalis corniculata. Brazilian Journal of Pharmacognosy, 23(4), 630–634. Ibrahim, M., Imran, M., Ali, B., Malik, A., Afza, N., Aslam, M., et al. (2012). Phytochemical studies on Oxalis corniculata. Journal of the Chemical Society of Pakistan, 34(5), 1260–1265. Liu, Y., Staerk, D., Nielsen, M. N., Nyberg, N., & Jäger, A. K. (2015). High-resolution hyaluronidase inhibition profiling combined with HPLC–HRMS–SPE–NMR for identification of anti-necrosis constituents in Chinese plants used to treat snakebite. Phytochemistry, 119, 62–69. Martinez, V., Mestre, T. C., Rubio, F., Girones-Vilaplana, A., Moreno, D. A., Mittler, R., et al. (2016). Accumulation of flavonols over hydroxycinnamic acids favors oxidative damage protection under abiotic stress. Frontiers of Plant Science, 7, 838. Mohdaly, A. A. A., Smetanska, I., Ramadan, M. F., Sarhan, M. A., & Mahmoud, A. (2011). Antioxidant potential of sesame (Sesamum indicum) cake extract in stabilization of sunflower and soybean oils. Industrial Crops and Products, 34(1), 952–959. Mukherjee, S., Koley, H., Barman, S., Mitra, S., Datta, S., Ghosh, S., et al. (2013). Oxalis corniculata (Oxalidaceae) leaf extract exerts in vitro antimicrobial and in vivo anticolonizing activities against Shigella dysenteriae 1 (NT4907) and Shigella flexneri 2a (2457T) in induced diarrhea in suckling mice. Journal of Medicinal Food, 16(9), 801–809. Mukherjee, S., Pal, S., Chakraborty, R., Koley, H., & Dhar, P. (2018). Biochemical
5. Conclusion Carotenoids, pigments and phenolics of Oxalis corniculata leaves grown in different environments were measured using HPLC-DAD. Oxalis corniculata leaves were a good source of important bioactive compounds such as kaempferol-3-sophorotrioside, quercetin-3-(caffeoyldiglucoside)-7-glucoside, isorhamnetin-3-caffeoyl-7-glucoside, and p-coumaric acid. Phenolic compounds were higher in dry area samples, followed by marshy and moist area samples. All-E-violaxanthin, all-E8
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Characterization, 11(3), 1248–1253. Solymosi, K., & Mysliwa-Kurdziel, B. (2017). Chlorophylls and their derivatives used in food industry and medicine. Mini Reviews in Medicinal Chemistry, 17(13), 1194–1222. Taghizadeh, S. F., Rezaee, R., Davarynejad, G., Karimi, G., Nemati, S. H., & Asili, J. (2018). Phenolic profile and antioxidant activity of Pistacia vera var. Sarakhs hull and kernel extracts: The influence of different solvents. Journal of Food Measurement and Characterization, 12(3), 2138–2144. Tibuhwa, D. D. (2017). Antioxidant potentialities and antiradical activities of Oxalis corniculata Linn from Tanzania. Journal of Applied Biosciences, 116(1), 11590–11600. Tukun, A. B., Shaheen, N., Banu, C. P., Mohiduzzaman, M., Islam, S., & Begum, M. (2014). Antioxidant capacity and total phenolic contents in hydrophilic extracts of selected Bangladeshi medicinal plants. Asian Pacific Journal of Tropical Medicine, 7, S568–S573. Zeb, A. (2015). A reversed phase HPLC-DAD method for the determination of phenolic compounds in plant leaves. Analytical Methods, 7(18), 7753–7757. Zeb, A. (2017). A simple, sensitive HPLC-DAD method for simultaneous determination of carotenoids, chlorophylls and alpha-tocopherol in leafy vegetables. Journal of Food Measurement and Characterization, 11(3), 979–986. Zeb, A., & Ullah, F. (2017). Reversed phase HPLC-DAD profiling of carotenoids, chlorophylls and phenolic compounds in adiantum capillus-veneris leaves. Frontiers in Chemistry, 5, 29. https://doi.org/10.3389/fchem.2017.00029. Zhang, S., Dong, J., & Cheng, H. (2018). Essential oil composition of the leaves of Oxalis corniculata from China. Chemistry of Natural Compounds, 54(2), 380–381.
assessment of extract from Oxalis corniculata L.: Its role in food preservation, antimicrobial and antioxidative paradigms using in situ and in vitro models. Indian Journal of Experimental Biology, 56(4), 230–243. Rajyalakshmi, P., Venkatalaxmi, K., Venkatalakshmamma, K., Jyothsna, Y., Devi, K. B., & Suneetha, V. (2001). Total carotenoid and beta-carotene contents of forest green leafy vegetables consumed by tribals of south India. Plant Foods for Human Nutrition, 56(3), 225–238. Rehman, A., Rehman, A., & Ahmad, I. (2015). Antibacterial, antifungal, and insecticidal potentials of Oxalis corniculata and its isolated compounds. International Journal of Analytical Chemistry, 2015. 842468 https://doi.org/10.1155/2015/842468. Shafi, T., & Banerjee, S. (2018). An immunomodulatory profile of aqueous extract of Oxalis corniculata linn. in albino Wistar rats and Swiss albino mice. International Journal of Pharmaceutical Sciences and Research, 9(1), 208–213. Shah, S. R., Ukaegbu, C. I., Hamid, H. A., & Alara, O. R. (2018). Evaluation of antioxidant and antibacterial activities of the stems of Flammulina velutipes and Hypsizygus tessellatus (white and brown var.) extracted with different solvents. Journal of Food Measurement and Characterization, 12(3), 1947–1961. Sharma, R. A., & Kumari, A. (2014). Phytochemistry, pharmacology and therapeutic application of Oxalis corniculata Linn. – a review. International Journal of Pharmacy and Pharmaceutical Sciences, 3(6), 6–12. Silva, S., Costa, E. M., Calhau, C., Morais, R. M., & Pintado, M. M. E. (2017). Production of a food grade blueberry extract rich in anthocyanins: Selection of solvents, extraction conditions and purification method. Journal of Food Measurement and
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Contents lists available at ScienceDirect
Food Bioscience journal homepage: www.elsevier.com/locate/fbio
Carotenoids, pigments, phenolic composition and antioxidant activity of Oxalis corniculata leaves
T
Alam Zeb∗, Muhammad Imran Department of Biochemistry, Faculty of Biological Sciences, University of Malakand, Chakdara, 18800, Khyber Pakhtunkhwa, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: Oxalis corniculata Phenolic compounds Carotenoids Chlorophyll Sleeping beauty
Carotenoids, pigments, phenolic composition and antioxidant activity of Oxalis corniculata leaves grown in dry, marshy and moist areas were evaluated. High-performance liquid chromatography analyses showed 13 phenolic compounds, which were higher in samples from dry areas, followed by those from marshy and moist areas. Kaempferol-3-sophorotrioside, quercetin-3-(caffeoyldiglucoside)-7-glucoside, isorhamnetin-3-caffeoyl-7-glucoside, and p-coumaric acid were the major compounds. High levels of all-E-violaxanthin, all-E-neoxanthin, all-Elutein, and 9-Z-lutein were present in samples from marshy and moist areas. Phytofluene, hydroxy-pheophytin a, and chlorophyll b were major pigments. Pigments were higher in samples from marshy areas, followed by moist and lowest in dry area samples. Total phenolic and flavonoids contents varied among the samples. Oxalis corniculata leaves showed high radical scavenging activity attributed to the high levels of carotenoids and phenolic compounds. Results showed that Oxalis corniculata leaves are a good source of bioactive substances such as carotenoids and phenolics for the uses in food fortification and as ingredients.
1. Introduction
(Tibuhwa, 2017). However, detail fingerprinting of these compounds was not reported. Similarly, the presence of important glycosides, including dihydroxy-5-methoxyflavon-8-glucopyranoside was reported in the Chinese Oxalis (Liu, Staerk, Nielsen, Nyberg, & Jäger, 2015). Bordoloi et al. (2016) reported the antioxidant activity, phenolic composition, mineral content and antifungal properties of Oxalis leaves along with other herbs. Quercetin, rutin and gallic acid were quantified in the leaves, while no other phenolic compounds were reported in Oxalis leaves. Another Indian variety of Oxalis was shown to contain phydroxybenzoic acid, ferulic acid, and rutin (Mukherjee, Pal, Chakraborty, Koley, & Dhar, 2018). In Pakistan, the plant is used for medicinal purposes, as a vegetable or as a salad, however, very little is known about its phytochemical constituents (Abbasi, Shah, & Khan, 2015). All these reported studies on Oxalis corniculata did not analyze phenolic compounds in detail. Carotenoids and pigments are important organic compounds, imparting color to plant leaves, foods and agriculture products. Carotenoids have a significant role in light harvesting and photoprotection in photosynthesis in plants (Damjanovic, Ritz, & Schulten, 1998). Pigments such as chlorophyll and their derivatives are used in the food industry and in medicine (Solymosi & Mysliwa-Kurdziel, 2017). However, these compounds have not been studied in Oxalis corniculata. This study was, therefore, done to evaluate carotenoids,
Oxalis corniculata L (sleeping beauty) is an important herb commonly grown in temperate zones in Europe, America and Asia. It is found in temperate zones around the world (Sharma & Kumari, 2014). The plant grows in semi-dry, moist, and wet spots such as yards, farms, and forest. The leaves are yellowish green, tri-hearted, alternate and delicate attached to the stem. Oxalis corniculata leaves are known to have several medicinal properties such as oxidative stress reduction in Parkinson disease and neurological disorders (Aruna, Rajeswari, & Sankar, 2016), anti-diabetic (Al-Qalhati, Waly, Al-Attabi, & Al-Subhi, 2016), reduction in chemical-induced pulmonary toxicity (Ahmad, Khan, & Shah, 2015), immunomodulatory effects (Shafi & Banerjee, 2018), anti-inflammatory (Dighe, Kuchekar, & Wankhede, 2016), antibacterial (Mukherjee et al., 2013), antifungal and insecticidal activities (Rehman, Rehman, & Ahmad, 2015). These effects were due to the presence of several important bioactive compounds. Zhang, Dong, and Cheng (2018) studied essential oils from the Chinese Oxalis corniculata. The authors identified 28 compounds, representing 85.2% of the total oil. The major compounds were geranyl acetate (13.3%), terpinolene (9.2%), hexadecanoic acid (7.7%), linalool oxide (7.4%), and geraniol (6.4%). In the Tanzanian variety of Oxalis, phytosterols, coumarins, terpenes, and flavonoids were identified
∗
Corresponding author. Department of Biochemistry, Faculty of Biological Sciences, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan. E-mail addresses: [email protected], [email protected] (A. Zeb).
https://doi.org/10.1016/j.fbio.2019.100472 Received 4 September 2018; Received in revised form 25 September 2019; Accepted 28 September 2019 Available online 02 October 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Identification and variations in phenolic composition of different Oxalis corniculata L. leaves samples. Peak
Retention time (min)
λ max (nm)
Compound
Amount (× 102 μg/g) Dry
1 1 2 2.7 3 3.3 4 4.7 5 5.6 6 7.6 7 7.8 8 8.8 9 10.5 10 10.8 11 13 12 13.5 13 17.3 Total Amount (μg/g)
p-Hydroxybenzoic acid p-Coumaroylhexose Sinapic acid p-Coumaric acid Caffeic acid Kaempferol-3-(p-coumaroyldiglucoside)-7-glucoside 3-O-Caffeoylquinic acid Kaempferol-3-O-glucoronide Vitexin-6-O-malonyl-2-O-xyloside Isorhamnetin-3-caffeoyl-7-glucoside Quercetin-3-(caffeoyldiglucoside)-7-glucoside Kaempferol-3-O-sophorotioside Ellagic acid deoxyhexoside
256 316 323, 311, 323, 320, 314, 336, 335, 337, 335, 346, 362,
Marshy a
240 233 296, 274, 232 271, 271, 271, 270, 320, 315,
232 232 236 235 236 237 272 244
0.28 ± 0.01 0.41 ± 0.02a 1.8 ± 0.1a 12.1 ± 0.1a 1.28 ± 0.04a 1.13 ± 0.02a 0.71 ± 0.04a 3.1 ± 0.1a 5.4 ± 0.1a 16.0 ± 0.1a 22.6 ± 0.1a 32.0 ± 0.2a 2.01 ± 0.04a 98.6a
Moist b
0.38 ± 0.02 0.093 ± 0.01b 0.79 ± 0.03b 6.1 ± 0.1b 0.71 ± 0.03b 1.24 ± 0.03b 0.54 ± 0.02b 2.03 ± 0.04b 3.5 ± 0.1b 9.5 ± 0.1b 10.2 ± 0.1b 13.4 ± 0.1b 4.06 ± 0.01b 52.4b
0.51 ± 0.02c 0.14 ± 0.01c 0.77 ± 0.02b 3.7 ± 0.1c 0.55 ± 0.03c 0.11 ± 0.01c 1.07 ± 0.04c 3.02 ± 0.01a 1.0 ± 0.1c 5.1 ± 0.1c 4.4 ± 0.1c 14.6 ± 0.1c 0.34 ± 0.02c 35.1c
Values are means with a standard deviation of the triplicate measurements. Different letters (a-c) in the same compound represent significance at p < 0.05 using post-hoc Tukey's test.
at 1790 × g for 15 min using an Eppendorf centrifuge (model 5702R, Eppendorf AG, Hamburg, Germany) at 25 °C. The above extraction was done three times and finally, the extracts were pooled and concentrated using solvent evaporation under vacuum. The residue was dissolved in high performance liquid chromatography (HPLC) solvent B, and 2 mL from each sample was filtered using a polytetrafluoroethylene (PTFE) syringe filter with 0.45 μm pore size (Agilent Technologies, Waldbronn, Germany) into HPLC vials (2 mL). The phenolic compounds were separated with the help of an Agilent Zorbax Eclipse C18 column (4.6 × 100 mm, 3.5 μm). The gradient chromatography was carried out with an Agilent HPLC model 1260 Infinity Better (Agilent Technologies) system consists of a quaternary pump, auto-sampler and diode array detector (DAD). The solvent system consisted of solvent A, which consisted of methanol-acetic aciddeionized water (10:2:88, v/v) and solvent B (methanol-acetic aciddeionized water, 90:2:8, v/v). The elution started with 100% A, reached 85% A in 5 min, at 20 min it was 50% A, and finally reached 30% A at 25 min (Zeb, 2015). The elution flow rate was 1 mL/min. The DAD chromatograms were obtained at 280, 320 and 360 nm and the wavelength used were selected by the operator. The absorption spectra for every peak were automatically scanned in the range of 200–600 nm. The identification of phenolic compounds was carried out using the retention time and absorption spectra of the available standard compounds (p-hydroxybenzoic acid, sinapic acid, 3-caffeoylquinic acid, caffeic acid, and p-coumaric acid). The compounds were quantified against the respective standard calibration curves. In the case where the standard compound was not available, the calibration curves of those compounds with a relative response factor (RRF) such as rutin and quercetin-3-glucoside were used. The RRF was calculated manually for each unknown compound as the ratio of the response factor of the individual related compound and response factor of the unknown. The amount of phenolic compounds were calculated and expressed as μg/g of fresh weight (fw).
pigments and the phenolic composition of Oxalis grown with the availability of different water resources. 2. Materials and methods 2.1. Reagents and chemicals Methanol, ethanol, all-E-violaxanthin, all-E-neoxanthin, all-E-lutein, chlorophyll a and b, p-hydroxybenzoic acid, p-coumaric acid, 3-caffeoylquinic acid, quercetin-3-rutinoside, and quercetin-3-glucoside were from Sigma-Aldrich (Sigma-Aldrich, Darmstadt, Germany). Caffeic acid was purchased from Tokyo Chemical Industries (TCI, Tokyo, Japan). Ultrapure deionized double distilled water was prepared using a Labtech Automatic Water-still (model LWD-3010D, Labtech, Namyangju, South Korea) coupled with an ELGA deionizer (model B114/B, ELGA LabWater, High Wycombe, UK). All chemicals and reagents used in this study were of HPLC or ACS grade and were obtained from Sigma-Aldrich unless otherwise mentioned. 2.2. Sample collection and preparation The fresh leaves of Oxalis corniculata were collected from different places of Lower Dir, Khyber Pakhtunkhwa (Pakistan), i.e., Chakdara (34.6666° N, 72.0290° E) and Talash (34.7415° N, 71.8720° E) which are popular habitats for the plant. The plant was kindly authenticated by Dr. Nasrullah Khan, Department of Botany, University of Malakand (Chakdara, Pakistan). Fresh plant leaves were collected from three different habitats, i.e., dry, marshy and moist areas. The samples from the moist area had been growing in fresh water; the marshy area was near the river, while the dry area was open with no shrubs or trees. The low to high temperatures were 13–22, 14–25 and 11–20 °C in the moist, dry and marshy areas, respectively. The air moisture levels of collection areas were 63, 47 and 65% in the moist, dry and marshy areas, respectively, while moisture contents of Oxalis corniculata leaves were 78.6% (moist), 71.2% (dry) and 76.5% (marshy). The leaves as harvested from the collected plant samples were separated and then washed with distilled water, and crushed to form a paste using a mortar and pestle. The samples for analysis were prepared from the paste materials.
2.4. Analysis of carotenoids and pigments One g of each sample of Oxalis leaves was extracted with ice-cold absolute ethanol (10 mL) and Vortex for 1 hr using a Witeg Wisd Vortex mixer (model VM-10, Witeg Labortechnik, Wertheim, Germany). After Vortexing, ice-cold absolute ethanol (10 mL) having 0.1% BHT (w/v) was added and again subjected to Vortexing for 30 min. The extractions were done in the presence of ambient light and repeated until the leaves were no longer green. Under vacuum conditions at 35 °C, the solvent was evaporated and final residue of each sample was dissolved in 2 mL 2-propanol and filtered with the PFTE syringe filters into HPLC vials
2.3. Analysis of phenolic compounds Each sample (1 g) was extracted with 10 mL of methanol-water (9:1, v/v) in 50 mL capped glass tubes. The tubes were placed in a shaking water bath at 35 °C for 1 h. After shaking, the samples were centrifuged 2
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Fig. 1. Representative HPLC-DAD chromatograms of phenolic compounds at 320 nm. (A) Dry samples, (B) marshy and (C) moist samples. Each peak in the chromatogram represents individual phenolic compound with details characteristics shown in Table 1.
every peak were measured from 200 to 750 nm. The chromatograms were obtained at 450 (for carotenoids), and 650 nm (for pigments) using an Agilent OpenLAB chromatography data system (CDS) ChemStation edition version C.01.05 software. The carotenoids were identified with the help of the available standards (all-E-violaxanthin, all-Eneoxanthin, all-E-lutein, chlorophyll a and b), or by comparing absorption spectra reported in earlier literature (Crupi, Milella, & Antonacci, 2010; Delgado-Pelayo, Gallardo-Guerrero, & HorneroMendez, 2014). In the case where the standard compound was not available, the calibration curves of those compounds with a relative response factor such as chlorophyll a, chlorophyll b, all-E-violaxanthin and all-E-lutein etc. were used. The quantitation of compounds was done using the peak areas (calculated after automatic integration by the software) using the respective standard calibration curves and expressed as μg/g of the fresh weight.
(2 mL). A reversed-phase HPLC system with the same specifications as previously was used for the separation and identification of carotenoids and pigments. The separations were done with the same C18 column with the same specifications but specifically maintained and equilibrated for carotenoids and pigments. The tertiary mobile phase system consisted of solvent A (methanol:deionized water, 92:8, v/v) with 10 mM ammonium acetate; deionized water containing 0.01 mM ammonium acetate as solvent B and solvent C was 100% methyl tertiary butyl ether (MTBE). The injection volume was 50 μL with the solvent flow rate of 1 mL/min. The gradient program was that of Zeb (2017). The gradient program was started with 80% A, 18% B and 2% C at 0 min. It changed to 80% A, 12% B and 8% C at 3 min, and at 25 min, it reached 65% A, 5% B with 30% C. The final gradient at 40 min was 60, 0, and 40% of A, B, and C, respectively. The initial solution was then run for 10 min to re-initialize the column. The absorption spectra of 3
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Fig. 2. Biplot of the principal component analysis (PCA) used for quantitative data of different samples of Oxalis corniculata. (A) PCA of phenolic compounds, and (B) PCA of carotenoids and pigments. The variables are phenolic compounds, pigments and carotenoids identified and quantified using HPLC.
4
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Fig. 3. Representative HPLC-DAD chromatograms of carotenoids and pigments at 450 nm. (A) Dry samples, (B) marshy and (C) moist samples. Each peak in the chromatogram represents individual compound with details characteristics shown in Table 2.
and Ullah (2017). Phenolic compounds were extracted in samples of the Oxalis corniculata leaves with ethanol in a ratio of 1:10, w/v (100%), methanol (100%) and methanol-water (9:1, v/v). The combined extract sample (0.5 mL) was mixed with 2.5 mL Folin Ciocalteau (FC) reagent and 2 mL 7.5% sodium carbonate and kept in the dark for 1 h. The absorbance of the sample was measured using a spectrophotometer (model PharmaSpec UV-1700, Shimadzu, Kyoto, Japan) at 765 nm. The TPC was calculated using a gallic acid standard curve and shown as mg of gallic acid equivalents (GAE)/g.
2.5. Principal component analysis Principal component analysis (PCA) was carried out using XLSTAT software (version 19.5.47062, 2017, Addinsoft, New York, NY, USA) macros for MS Excel (Microsoft Office 2010; Microsoft Austria GmbH, Vienna, Austria). All phenolic compounds, carotenoids, and pigments identified for each sample were included. The PCA biplot mapped the variables (individually identified compound) and samples (dry, marshy, and moist) using loadings and scores in respective dimensional spaces determined by the principal components (PC). The Spearman correlation coefficient and square cosine values were determined between the samples.
2.7. Total flavonoids contents Total flavonoids contents (TFC) were extracted with methanol, ethanol, and water as above. The combined extract solution (0.5 mL) was mixed with 2% aluminium trichloride (AlCl3) and kept for 1 h at 25 °C. The absorbance of the mixture was measured at 420 nm with the
2.6. Total phenolic contents Total phenolic content (TPC) was measured using the method of Zeb 5
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Table 2 Identification and characterization of carotenoids and pigments profile of different Oxalis corniculata L. leaves samples. Peak
Rt (min)
λ max (nm)
Identity
Amount (μg/g) Dry
1 1.1 2 4.4 3 5.7 4 6.2 5 7.2 6 8.4 7 8.8 8 9.1 9 9.9 10 14.5 11 15.8 12 19.1 13 20.4 14 22.3 15 22.9 16 25.4 Total Pigments Total Carotenoids
Phytofluene Peophorbide b All-E-violaxanthin Pheophytin b Hydroxy-pheophytin b All-E-neoxanthin Hydroxy-pheophytin a Hydroxy-pheophytin a' Pheophytin a All-E-lutein 9-Z-lutein Chlorophyll b Chlorophyll b epimer 13-Hydroxy-chlorophyll b Chlorophyll b' Chlorophyll a
366, 654, 470, 666, 652, 466, 666, 666, 666, 472, 468, 654, 648, 650, 648, 664,
347, 600, 440, 610, 598, 436, 610, 610, 608, 446, 440, 599, 602, 600, 600, 618,
328 438 416 410 522, 416 532, 534, 536, 422 418 436 468 462 464 432
Marshy a
433 502, 406 504, 408 506, 407
8.3 ± 0.3 0.21 ± 0.01a 1.3 ± 0.1a 0.92 ± 0.1a 1.7 ± 0.1a 2.6 ± 0.1a 8.1 ± 0.3a 5.9 ± 0.2a 3.8 ± 0.1a 128 ± 2a 57+1a 33 ± 1a 6.4 ± 0.5a 6.5 ± 0.2a 1.14 ± 0.02a 2.9 ± 0.1a 78.9 189
b
18 ± 1 1.01 ± 0.01b 8.8 ± 0.2b 5.4 ± 0.1b 11.5 ± 0.3b 14+1b 33+1b 21+1b 4.5 ± 0.1b 98+1b 106 ± 2b 54 ± 2b 6.9 ± 0.4a 13 ± 1b 2.1 ± 0.1b 4.9 ± 0.3b 176 227
Moist 52 ± 2c 1.1 ± 0.1b 7.7 ± 0.2c 5.1 ± 0.3b 8.8 ± 0.6c 39+1c 3.9 ± 0.1c 26.7 ± 0.2c 4.6 ± 0.1b 113 ± 1c 66+2c 33+3a 4.6 ± 0.5b 2.2 ± 0.1c 0.44 ± 0.01c 1.46 ± 0.02c 144 225
Values are means with a standard deviation of the triplicate measurements. Different letters (a-c) in the same compound represent significance at p < 0.05 using post-hoc Tukey's test.
samples from marshy areas, followed by dry and moist areas. Thus, the total amount of polyphenols determined using HPLC was 46 and 64% lower in the samples from marshy and moist areas, respectively, as compared to dry areas.
spectrophotometer against the blank (Mohdaly, Smetanska, Ramadan, Sarhan, & Mahmoud, 2011). The TFC was calculated using a quercetin standard curve and shown as mg of QE/g. 2.8. Antioxidant activity
3.2. Principal component analysis of phenolic compounds Different extracts of the Oxalis corniculata leaves were prepared, i.e., in methanol, ethanol, and methanol-water. The different extracts (5 μL) were incubated in the dark for 30 min with 0.1 mM of DPPH solution (1.95 μL). The absorbance of the samples was measured at 515 nm with the spectrophotometer against the DPPH solution according to the previously reported method (Zeb & Ullah, 2017). The radical scaven-
Fig. 2A shows the biplot of the loadings and scores in the respective dimensional spaces. The principal components accounted for 99.1% of the variance due to the sample origin. The F1 variance was higher (89.1%), followed by F2 (9.99%). The score plot also showed some homogeneity among certain variables. Samples from dry and marshy areas showed higher Spearman correlation with respect to vitexin-6-Omalonyl-2-O-xyloside and quercetin-3-(caffeoyldiglucoside)-7-glucoside. The moist area samples showed a correlation in terms of p-coumaric acid, kaempferol-3-O-sophorotioside, kaempferol-3-O-glucuronide and isorhamnetin-3-caffeoyl-7-glucoside. Other phenolic acids had relatively low correlations.
(Ac − As)
ging activity was calculated as RSA (%) = ⎡ Ac ⎤ × 100 , where Ac ⎣ ⎦ is absorbance of the control, and As is absorbance of the sample. 2.9. Data analyses Data were expressed as the mean ± standard deviation (SD) of 3 or more independent measurements. One-way analysis of variance (ANOVA) was done followed by the post hoc test using the Holm-Sidak method at α = 0.05 using the XLSTAT software macros for MS Excel.
3.3. Variations in carotenoids and pigments composition Carotenoids and pigments in Oxalis corniculata were effectively separated as shown in Fig. 3. The identified and quantified carotenoids and pigments are shown in Table 2. Five carotenoids and 11 pigments were identified. Table 2 shows the variation in carotenoids and pigments present in Oxalis corniculata samples. Phytofluene, all-E-neoxanthin and hydroxy-pheophytin a' levels were highest (p < 0.05) in the samples from the moist areas, followed by marshy areas. Significantly (p < 0.05) higher amounts of all-E-violaxanthin, hydroxypheophytin b, 9-Z-lutein, and chlorophyll b were found in the samples from the marshy areas, followed by the moist areas and then dry areas. A relatively low amount of pheophorbide b and chlorophyll b' were found in all samples. Pheophytin b was found in higher amounts in both moist and marshy area samples and lowest in dry area samples. Hydroxy-pheophytin a, 13-hydroxy-chlorophyll b, chlorophyll b', and chlorophyll a were found in significantly (p < 0.05) higher amounts in marshy area samples followed by dry and then moist area samples. Pheophytin a was significantly (p < 0.05) higher in moist and marshy areas samples compared to dry area samples. All-E-lutein was higher in the dry area samples, followed by the moist area and then marshy area samples. The total amounts of carotenoids were significantly higher
3. Results 3.1. Variations in phenolic compounds Table 1 shows the characteristics of phenolic compounds in Oxalis corniculata leaves of dry, moist and marshy origins. In total, 13 phenolic compounds were separated, identified and quantified as shown in Fig. 1. These phenolic compounds include hydroxybenzoic acids, cinnamic acids, flavonoids and their derivatives. Table 1 shows the variations in the phenolic composition of Oxalis corniculata leaves grown in different areas. The p-hydroxybenzoic acid and 3-caffeoylquinic acid was significantly higher in samples from moist areas, followed by marshy and dry areas. The amount of p-coumaroyl hexose, sinapic acid, p-coumaric acid, caffeic acid, vitexin-6-Omalonyl-2-O-xyloside, isorhamnetin-3-caffeoyl-7-glucoside, quercetin3-(caffeoyldiglucoside)-7-glucoside, kaempferol-3-O-glucuronide and kaempferol-3-O-sophorotrioside were significantly (p < 0.05) higher in samples from dry areas than moist and marshy areas. Kaempferol-3(p-coumaroyldiglucoside)-7-glucoside was significantly higher in 6
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3.5. Total phenolic contents The TPC of Oxalis corniculata are shown in Fig. 4. The TPC for extracts of dry, moist and marshy area samples differed with extraction solution used. The highest TPC was obtained using ethanol, followed by methanol, while methanol-water extracts had the lowest levels for all samples. In ethanolic extracts, marshy samples contain the highest TPC, followed by moist and lowest in dry area samples. The methanolic extracts of marshy samples contain the highest amount followed by dry and lowest in moist area samples. In methanol-water extracts, moist samples showed the highest TPC, followed by marshy and lowest in dry area samples. The net TPC was highest in marshy area samples. 3.6. Total flavonoid contents Different solvents were used to extract flavonoids (Fig. 4) from the Oxalis corniculata. In ethanolic extracts, the lowest total flavonoids contents (TFC) were measured in dry area samples, whereas marshy and moist area samples had similar amounts. A similar trend was also observed in the case of methanolic extracts. In the case of methanolwater extracts, the TFC was highest (p < 0.05) in the dry area followed by moist area samples. 3.7. Radical scavenging activity The radical scavenging activities (RSA) of Oxalis corniculata were studied in different extracts as shown in Fig. 4. The RSA values were significantly higher in the ethanolic extracts of all samples Oxalis corniculata as compared to methanol and methanol-water extracts. In the case of methanolic extracts, the marshy area samples showed significantly (p < 0.05) higher RSA values as compared to dry and moist area samples. The RSA of methanol-water was the lowest among all the extractions. 4. Discussion Thirteen phenolic compounds were identified and quantified in the leaves Oxalis corniculata grown with different moisture conditions (moist, marshy and dry). They were p-hydroxybenzoic acid, p-coumaroylhexose, sinapic acid, p-coumaric acid, caffeic acid, kaempferol3-(p-coumaroyldiglucoside)-7-glucoside, caffeoylquinic acid, kaempferol-3-O-glucuronide, vitexin-6-O-malonyl-2-O-xyloside, isorhamnetin3-caffeoyl-7-glucoside, quercetin-3-(caffeoyldiglucoside)-7-glucoside, kaempferol-3-O-sophorotioside, and ellagic acid deoxyhexoside. An earlier study reported only three phenolic compounds, i.e., rutin, gallic acid, and catechol using reference standards (Bordoloi et al., 2016). However, none of these compounds was found in the present study. Ibrahim et al. (2012) identified 12 phenolic compounds in the Oxalis corniculata from Karachi, Pakistan. However, no quantitative results had been reported. Another study reported a very specific glycoside called corniculatin (Ibrahim et al., 2013). Similarly, another study reported p-hydroxybenzoic acid, ferulic acid, and rutin in Oxalis corniculata leaves (Mukherjee et al., 2018). These compounds were not reported in the present study, which may be due to the difference in the area of collection of plant samples, or variety and analysis. This study confirms that there were significant differences in the phenolic composition of the selected samples from the different area. Higher amounts of phenolic compounds were reported in the samples from the dry area. This may be due to the expression of specific genes, which are responsible for the formation of enzymes which synthesize phenolic compounds. The nature of dryness of the sample collection areas in the present study may thus be contributing to the abiotic stress. During the abiotic stress, specific phenolic compounds are accumulated to cope with oxidative stress (Martinez et al., 2016). A high correlation of phenolic compounds in the PCA biplot also showed that dry and marshy areas were significantly correlated with respect to
Fig. 4. Total phenolic contents, total flavonoids contents and radical scavenging activity of Oxalis corniculata leaves collected from dry, marshy and moist areas. The extractions were done in 100% ethanol, 100% methanol and 90:10% methanol:water. Different letters (a–c) in the same extract represent significance differences using Tukey's test at p < 0.05 among the samples.
compared to pigments in all samples.
3.4. Principal component analysis of carotenoids and pigments The biplot shown in Fig. 2B represents loadings and scores in the respective dimensional spaces. The principal components showed 96.3% variance in dry, marshy and moist area samples. The F1 variance was higher (82.7%), followed by F2 (13.5%). This score plot also showed some homogeneity among certain variables. The samples from dry and marshy areas showed a higher Spearman correlation coefficient between hydroxy-pheophytin a and chlorophyll b. The moist area samples showed a higher correlation in terms of lutein and its isomers, phytofluene, hydroxy-pheophytin a', and neoxanthin. The squared cosine values of lutein was 0.988, 9-Z-lutein (0.999), phytofluene (0.892), hydroxyl-pheophytin a' (0.773) and neoxanthin were 0.843. All other pigments and all-E-violaxanthin had relatively low correlations. 7
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neoxanthin, all-E-lutein, and 9-Z-lutein contents were found at high levels as compared to pigments. Carotenoids were higher in marshy and moist area samples. Phytofluene, hydroxy-pheophytin a, and chlorophyll b were the major pigments. Pigments were significantly higher in the marshy area, followed by the moist area samples. Total phenolics and flavonoids varied in different solvents and among the samples. The results showed that Oxalis corniculata leaves had a high radical scavenging activity which may be due to the high levels of carotenoids and phenolics. This herb has the potential to be a source of functional food ingredients.
the location of Oxalis corniculata. The chromatographic separation of Oxalis corniculata leaves showed for the first time, a total of 16 carotenoids and pigments. The pigments included phytofluene, pheophorbide b, pheophytin b, hydroxy-pheophytin b, hydroxy-pheophytin a, hydroxy-pheophytin a', pheophytin a, chlorophyll b, chlorophyll b epimer, 13-hydroxy-chlorophyll b, chlorophyll b' and chlorophyll a. The carotenoids were all-E-violaxanthin, all-E-neoxanthin, all-E-lutein, and 9-Z-lutein. The carotenoid β-carotene was previously reported in Oxalis leaves (Rajyalakshmi et al., 2001). Since Oxalis corniculata is an edible and medicinal herb, the high amounts of carotenoids and pigments suggested that the plant is a potential source of commercial food-grade carotenoids and pigments (Mukherjee et al., 2018). The total amounts of carotenoids were significantly higher than pigments. The highest amounts of total phenolic compounds were found in the samples of marshy areas followed by moist areas. Similarly, marshy and moist area samples were shown to contain a higher amount of carotenoids while dry area samples had the lowest level. The lower values of carotenoids and pigments in dry area samples may be due to the higher synthesis of phenolic compounds. This was confirmed by the significantly high correlation coefficient (R2 = 0.9047) between total carotenoids and total phenolics. Similarly, a correlation coefficient (R2 = 0.6832) between total pigments and total phenolics were also observed. This also suggested that phenolic compounds are present in high amounts with relatively stressed conditions as compared to carotenoids. There was a high homogeneity among the marshy and dry area samples as confirmed using the PCA biplot (Fig. 2). Spectrophotometric studies of TPC showed relatively higher amounts in marshy area samples as compared to dry and moist area samples. However, TPC in all samples and extracts were lower than reported for Indian samples (Bordoloi et al., 2016; Mukherjee et al., 2018), and higher than samples from Bangladesh (Tukun et al., 2014). The difference may be due to variety, climate, soil conditions, and method of extractions and analysis. Similarly, TFC of the dry area samples were low in ethanol and methanol extractions compared to the methanol-water extract. Ethanol extraction resulted in low TFC in all samples. The TFC was relatively higher compared to the values reported previously (Mukherjee et al., 2018). The higher RSA values were observed in ethanol extracts in all samples. The higher RSA was strongly correlated with the amounts of TPC and TFC of the respective samples. The RSA values of the present samples were consistent with the RSA reported for Oxalis corniculata leaves (Bordoloi et al., 2016). TPC, TFC, and consequently RSA may vary with plant species, plant body part and variety. For example, TPC and TFC of Pistacia vera were higher in methanolic extracts as compared to aqueous extracts (Taghizadeh et al., 2018). An inverse was reported in Flammulina velutipes and Hypsizygus tessellatus, where TPC and TFC were higher in aqueous extracts as compared to methanolic extract (Shah, Ukaegbu, Hamid, & Alara, 2018). The use of different extraction solvents for the determination of phenolic compounds, flavonoids, and radical scavenging activity is advantageous (Silva, Costa, Calhau, Morais, & Pintado, 2017). These results showed that bioactive components solubilized in each solvent may be a good source for possible food supplementation or medicinal applications, suggesting that Oxalis corniculata leaves can be considered a good source of food ingredients or supplement.
Declaration of competing interest The authors confirmed that they have no conflict of interest with respect to the work described in this manuscript. Acknowledgement The authors are grateful to the Higher Education Commission (HEC) of Pakistan for providing financial assistance under the National Research Program for Universities (NRPU) funded project No 2344. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fbio.2019.100472. References Abbasi, A. M., Shah, M. H., & Khan, M. A. (2015). Wild edible vegetables of lesser Himalayas: Ethnobotanical and nutraceutical aspects. Cham, Switzerland: Springer International Publishing978-3-319-09542-4. Ahmad, B., Khan, M. R., & Shah, N. A. (2015). Amelioration of carbon tetrachlorideinduced pulmonary toxicity with Oxalis corniculata. Toxicology and Industrial Health, 31(12), 1243–1251. Al-Qalhati, I. R. S., Waly, M., Al-Attabi, Z., & Al-Subhi, L. K. (2016). Protective effect of Pteropyrum scoparium and Oxalis corniculata against streptozotocin-induced diabetes in rats. Federation of American Societies for Experimental Biology Journal, 30 1176.5. Aruna, K., Rajeswari, P. D. R., & Sankar, S. R. (2016). The effects of Oxalis corniculata l. extract against MPTP induced oxidative stress in mouse model of Parkinson's disease. Journal of Pharmaceutical Sciences and Research, 8(10), 1136. Bordoloi, M., Bordoloi, P. K., Dutta, P. P., Singh, V., Nath, S., Narzary, B., et al. (2016). Studies on some edible herbs: Antioxidant activity, phenolic content, mineral content and antifungal properties. Journal of Functional Foods, 23, 220–229. Crupi, P., Milella, R. A., & Antonacci, D. (2010). Simultaneous HPLC-DAD-MS (ESI+) determination of structural and geometrical isomers of carotenoids in mature grapes. Journal of Mass Spectrometry, 45(9), 971–980. Damjanovic, A., Ritz, T., & Schulten, K. (1998). Light-harvesting and photoprotection by carotenoids in photosynthesis. Biophysical Journal, 74(2), A76. Delgado-Pelayo, R., Gallardo-Guerrero, L., & Hornero-Mendez, D. (2014). Chlorophyll and carotenoid pigments in the peel and flesh of commercial apple fruit varieties. Food Research International, 65, 272–281. Dighe, S., Kuchekar, B., & Wankhede, S. (2016). Analgesic and anti-inflammatory activity of β-sitosterol isolated from leaves of Oxalis corniculata. International Journal of Pharmacological Research, 6, 109–113. Ibrahim, M., Hussain, I., Imran, M., Hussain, N., Hussain, A., & Mahboob, T. (2013). Corniculatin A, a new flavonoidal glucoside from Oxalis corniculata. Brazilian Journal of Pharmacognosy, 23(4), 630–634. Ibrahim, M., Imran, M., Ali, B., Malik, A., Afza, N., Aslam, M., et al. (2012). Phytochemical studies on Oxalis corniculata. Journal of the Chemical Society of Pakistan, 34(5), 1260–1265. Liu, Y., Staerk, D., Nielsen, M. N., Nyberg, N., & Jäger, A. K. (2015). High-resolution hyaluronidase inhibition profiling combined with HPLC–HRMS–SPE–NMR for identification of anti-necrosis constituents in Chinese plants used to treat snakebite. Phytochemistry, 119, 62–69. Martinez, V., Mestre, T. C., Rubio, F., Girones-Vilaplana, A., Moreno, D. A., Mittler, R., et al. (2016). Accumulation of flavonols over hydroxycinnamic acids favors oxidative damage protection under abiotic stress. Frontiers of Plant Science, 7, 838. Mohdaly, A. A. A., Smetanska, I., Ramadan, M. F., Sarhan, M. A., & Mahmoud, A. (2011). Antioxidant potential of sesame (Sesamum indicum) cake extract in stabilization of sunflower and soybean oils. Industrial Crops and Products, 34(1), 952–959. Mukherjee, S., Koley, H., Barman, S., Mitra, S., Datta, S., Ghosh, S., et al. (2013). Oxalis corniculata (Oxalidaceae) leaf extract exerts in vitro antimicrobial and in vivo anticolonizing activities against Shigella dysenteriae 1 (NT4907) and Shigella flexneri 2a (2457T) in induced diarrhea in suckling mice. Journal of Medicinal Food, 16(9), 801–809. Mukherjee, S., Pal, S., Chakraborty, R., Koley, H., & Dhar, P. (2018). Biochemical
5. Conclusion Carotenoids, pigments and phenolics of Oxalis corniculata leaves grown in different environments were measured using HPLC-DAD. Oxalis corniculata leaves were a good source of important bioactive compounds such as kaempferol-3-sophorotrioside, quercetin-3-(caffeoyldiglucoside)-7-glucoside, isorhamnetin-3-caffeoyl-7-glucoside, and p-coumaric acid. Phenolic compounds were higher in dry area samples, followed by marshy and moist area samples. All-E-violaxanthin, all-E8
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