Multi-Method Determination of Antioxidant Capacity, Phytochemical and Biological Investigation of Four Different Solvent Extractives of Leucophyllum frutescens (cenizo)

The four solvent extractives obtained from aerial parts of Leucophyllum frutescens were evaluated for their Total Antioxidant Activity (TAA) by ammonium molybdate method, scavenging potential by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox-Equivalent Antioxidant Capacity (TEAC) assays, metal-reducing potential by Cupric Reducing Antioxidant Capacity (CUPRAC) and Ferric Reducing Antioxidant Power (FRAP) assays, Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and their biological activities. The study concluded that BULE exhibited total antioxidant activity (226.235±1.222 mg AA.Eq.gm-1 DE±S.D) by molybdate method, CHLE exhibited more scavenging potential (DPPH 209.589±8.500 mg trolox Eq.gm-1 DE±S.D and TEAC 210.166±7.954 mg trolox Eq.gm-1 DE±S.D) and reducing potential (CUPRAC 646.889±16.889 mg trolox Eq.gm-1 DE±S.D & FRAP 472.981±15.625 mg trolox Eq.gm-1 DE±S.D). Phytochemical quantification concluded high TPC by BULE (189.369±1.393 mg GA.Eq.gm-1 DE±S.D) and high TFC by CHLE (232.458±1.589 mg Qu.Eq.gm-1 DE±S.D). Strong inhibition of α-glucosidase and urease enzymes was observed by HELE (IC50 0.3321±0.007 mg.ml-1±SD) and BULE (IC50 4.09±0.357 mg.ml-1±SD) extractives, respectively. The hemolytic effect shown by hexane extract (HELE) was higher with HA50 25.545±0.927 ug.ml-1±SD whereas methanol (MELE), chloroform (CHLE), and butanol (BULE) exhibited hemolytic effects at higher concentration with HA50 400.067±1.364, 321.394±1.332, and 332.957±0.465 µg.ml-1±SD, respectively. GC-MS profiling of HELE of L. frutescens was performed for qualitative analysis. The principal phytochemicals tentatively identified by GC-MS analysis of HELE accounts for fatty acids (60.221%), lignans (17.687%), ketones (3.358%), phenols (2.584%), sesquiterpenes (1.265%), and aldehydes (0.345%).


INTRODUCTION
sealed containers in the refrigerator for further evaluation.

Determination of Plant extracts Yield
The percent yield of methanol extract was calculated as: % Percent yield =

Multi-method Estimation of antioxidant Activity
The antioxidant potential was estimated by phosphomolybdenum, DPPH, TEAC, CUPRAC, and FRAP methods.

Total Antioxidant activity by phosphomolybdenum method
Total antioxidant activity was determined by adopting the method described in the literature with some modifications [8]. 200 µl of the extract solution (1 mg.ml -1 ) was added to 2 ml eppendorf tube and mixed with 1.8 ml reagent [0.6M H2SO4 (sulphuric acid), 28 mM NaH2PO4 (sodium phosphate), 4mM (NH4)6MO7O24 (ammonium molybdate)], the tube was capped and incubated in the water bath at temperature 95 o C for about 90 minutes. The absorbance of the resulting solution was measured at λ 695 nm using the IRMECO U2020 UV-visible spectrophotometer. A calibration curve was drawn between 20-200 µg.ml -1 , and the total antioxidant activity of extracts was expressed as mg ascorbic acid equivalent per gram of dry weight of extract (mg AA.Eq.gm -1 DE).

SCAVENGING POTENTIAL
Scavenging potential was determined by DPPH and TEAC assay (expressed in mg. trolox equivalent per gram of dried extract). Experimental procedure for DPPH [9] and TEAC [10] adopted from literature but with some modification are described below.

2,2-diphenyl-1-picrylhydrazyl (DPPH)
0.3127 mg.ml -1 extract solution was prepared in methanol. 150 µl of 200 mM DPPH solution was added to the microtiter plate well followed by the addition of 50 µl extract solution. The mixture was incubated at room temperature for 30 minutes. A similar procedure was adopted for the positive control (trolox). For blank instead of extract or trolox solution, 50 µl of methanol was used. Absorbance was measured at 517nm by Biotek-Synergy HT. A calibration curve of trolox was plotted between 5-100 µg.ml -1 for estimation of scavenging potential as mg trolox equivalent per gram of dried extract (mg trolox Eq.gm -1 DE).

Trolox Equivalent Antioxidant Capacity (TEAC)
1 ml extract solution (0.3127 mg.ml -1 ) was added to a test tube containing 2 ml mixture of an equal volume of 2.5 mM 2,2-azinobis(3-ethylbenothiazoline) 6-sulfonic acid and 2.45 mM potassium persulfate, incubating test tube in the dark for 30 minutes, and measured absorbance at 734 nm.
The calibration curve of trolox was plotted between 5-80 µg.ml -1 . Results were expressed in mg trolox equivalent per gram of dried extract (mg trolox Eq.gm -1 DE)

Reducing Antioxidant Potential
Reducing antioxidant potential was determined by CUPRAC (expressed mg. trolox equivalent per gram of dried extract) and FRAP (expressed mg. trolox equivalent per gram of dried extract). The procedures adopted are described below with minor modifications [10].

Cupric Reducing Antioxidant Capacity (CUPRAC)
0.5 ml of extract solution (0.3127 mg.ml -1 ) was mixed with a 3 ml reaction mixture of equal volumes of 10 mM CuCl2, 7.5 mM neocuprion, 1M ammonium acetate buffer pH 7 incubated for 30 minutes at room temperature and measured absorbance at 450nm. A blank was prepared with methanol instead of the extract solution. The calibration curve of trolox was plotted between 2.5-100 µg.ml -1 . Results were expressed as mg trolox equivalent per gram of dried extract (mg.trolox Eq.gm -1 of DE).

Total Phenolic Content (TPC)
Total phenolic content (TPC) of four extracts was estimated using Folin-ciocalteu (F-C) method described in the literature [11], with minor modification. Dried extract dissolved in methanol to get a stock solution 1 mg.ml -1 and gallic acid dissolved in methanol to get its aliquots 10, 20, 40, 60, 80, 100, 200 ug.ml -1 and calibration curve was drawn. 200 µl of extract solution or calibrators was added to 2 ml Eppendorf, 200 µl of Folin-ciocalteu reagent was added to it. The mixture was subjected to a vortex mixer followed by the addition of 0.8 ml of 700 µM sodium carbonate (Na2CO3) solution incubating at ambient temperature for 2 hours. Transferred 200µl of each assay mixture in 96 microtiter plate well and measured the absorbance at λ 765nm using Biotek-Synergy HT. Total phenolic content (TPC) of MELE, CHLE, and BULE was expressed as mg gallic acid equivalent per gram of dried extract (mg GA.Eq.gm -1 DE).
Note: F-C reagent should be added before the addition of 700 µM sodium carbonate solution to avoid air oxidation of phenols.

Total Flavonoid Content (TFC)
The total flavonoid content of MELE, HELE, CHLE, and BULE was determined by adopting a method available in the literature with minor modification [12]. A mixture of 1ml extract solution (1mg.ml -1 ), 4 ml deionized water, 300 µl of 5% sodium nitrite solution, and 300 µl of 10% AlCl3 solution added to glass test tube followed by the addition of 2 ml 1M sodium hydroxide solution.
The mixture was incubated for 6 minutes. 2.4 ml deionized water was added to the final mixture and measured the absorbance at λ 510 nm using IRMECO U2020 UV-visible spectrophotometer.
The calibration curve was plotted between 50-1000 µg.ml -1 . Total flavonoid content (TFC) of dried plant extract was expressed as milligram quercetin equivalent per gram of dried extract (mg Qu.Eq.gm -1 DE)

Alpha-glucosidase inhibition assay
α-glucosidase inhibition assay was performed according to method described in the literature with some modification [13]. A mixture of 10 µl of enzyme solution (1U/ml), 50µl of 50 mM phosphate buffer pH 6.8, and 20 µl of aliquots of the extract was added to 96 microtiter plate well and incubated for 15 minutes at 37 o C. The absorbance of the mixture was measured at λ 405 nm (preread). 20 µl of 0.5 mM solution of p-nitro-α-D-glucopyranoside was added to the mixture as substrate followed by incubation for 30 minutes at 37˚C. The absorbance was measured again at 405 nm (after-read). The same procedure was adopted for the positive control (quercetin) and negative control (methanol). The percent inhibition of α-glucosidase was calculated by using the following formula. (net absorbance = after readpre-read) Percent Alpha-glucosidase inhibition = (1− . ) 100 Where Abs stands for absorbance recorded

Urease inhibition assay (UIA)
The anti-urease activity of different solvent extractives was estimated by the method described in the literature [14]. A mixture of 20 µl of 0.025 % urease solution prepared in 1M phosphate buffer pH 7.0 and 20 µl of extract aliquot was added to the microtiter well followed by incubation for 15 minutes at 37 C. 60µl of 2.25 % aqueous urea added to the reaction mixture and kept in an incubator for 15 minutes at 37 ˚C . Measured the absorbance at λ 630 nm (pre-read). 60 µl of phenol reagent followed by 100 µl of sodium hypochlorite solution in alkali added to the above reaction mixture, again incubated for half an hour at 37 ˚C and measured the absorbance at λ 630nm (after-read). The same procedure was adopted for all aliquots of each extractive as well as hydroxyurea as a positive control. Note (net absorbance = after readpre-read). Percent enzyme inhibition was estimated by the following equation Hemolytic activity of MELE, HELE, CHLE, and BULE was estimated by the literature method [15]. The blood was withdrawn from a healthy volunteer and added to ethylenediaminetetraacetic acid (EDTA) tube. The EDTA tube containing blood was centrifuged. The plasma portion was discarded and the red portion was subjected to three times washing with phosphate buffer pH 7. Where Abs stands for absorbance recorded.

Instrument and chromatographic conditions
Molecular metabolomics study of HELE was performed by GC-MS analysis using the J&amp; W HP-5MS GC capillary column with dimension 30 m x 0.25 mm with particle size 0.25 µm. Helium was used as the carrier gas in constant flow mode with a flow rate of 1 ml.min -1 . The inlet temperature was set to 280 °C. The oven temperature ramped to 280 °C at a rate of 5 °C.min -1 starting from 60°C and then isothermal at 280 °C for 20 min. One micro-liter of HELE extract solution was injected and the split ratio was 20:1. The MS was set to scan mode from 40-550 amu and the EI voltage was 70 eV. The transfer line temperature kept at 280 °C and the ion source and quadrupole temperature were kept at 230°C and 150 °C, respectively.

Identification of components
The mass spectrum of each separated phytochemical on specific retention time was compared with the mass spectrum stored in the database of the National Institute Standard and Technology (NIST14). The name of each phytochemical with its retention time, molecular weight, and relative percent peak area was tabulated (Table 4).

Statistical analysis of data
For each experimental assay, three replicate values were expressed as the mean±standard deviation

Multi-Method Antioxidant activities
Reactive oxygen species such as OH • , HO2 • -, and OONO -, but not O2 • are normally produced during metabolic processes, and excessive accumulation of these ROS badly affects fatty acids, DNA, and proteins causing tissue injury and inflammation [16]. Therefore to enhance the defense system, these ROS need to get detoxified or scavenged by consuming antioxidants. To the best of our knowledge, the scientific literature review does not report the antioxidant activity of different solvent extracts of L. frutescens from Pakistan. A Mexican study reported the antioxidant activity of ethanolic extract on red blood cells using oxidant 2,2-azo-bis-(2-amidinopropane) dihydrochloride (AAPH). According to this study ethanol sub-fraction obtained from methanol extract of roots of L. frutescens (HF4) showed antioxidant activity at 100 µg.ml -1 [17]. In another study, the scavenging potential of the methanolic extract by DPPH and TEAC method was reported 280.43±4.97 µM QE/g FW and 266.20 ±5.63 µM TE/g FW) respectively.
In this study, the antioxidant potential has been estimated by total antioxidant activity using phosphomolybdenum, scavenging activity (DPPH and TEAC), and reducing potentials (CUPRAC and FRAP) methods ( Figure 2). The antioxidant activity of each extract has been different from each other which is found in accordance with TPC and TFC.
Antioxidant activity by all the above-mentioned methods has been presented in  [18]. The results of reducing potential observed by CUPRAC and FRAP for each extract was not significantly different (p > 0.05) from each other but found significantly different (p < 0.05) between the extracts. It has been observed that scavenging and reducing potential observed by any of the extracts is directly proportional to polyphenolic content [19].
Hence the author concludes that for achieving maximum benefits CHLE should be utilized alternative to methanolic or ethanolic leave extract.

Poly-phenolic contents
Polyphenols are biologically active compounds comprising chlorogenic acids, tannins, hydrolyzable tannins, and flavonoids mostly found in conjugation with sugar moieties.
Polyphenols are considered as nutraceuticals having a variety of pharmacological effects on the body after their consumption e.g. antibacterial, antiviral, antiparasitic, antidiabetic, anticancer, and antioxidant [20]. frutescens from Pakistan and rest of the Asian countries. In the present study, the results of TPC revealed that high polar solvents have more potential to extract phenolic compounds from L.
frutescens compared to chloroform, hexane, and methanol [21].   Hemolytic effects of these polar extracts might be due to the presence of high amount of phytotoxic lignans, such as diayangambin and epiashantin [6]. GC-MS analysis of HELE validated presence of hemolytic compounds in high proportion such as methyl stearate and di-isooctyl phthalate [22].

Alpha-glucosidase inhibition
Diabetes is a chronic metabolic disorder both in low and high-income countries and is one of the leading causes of death. Among the two types of diabetes, Diabetes II is most commonly found due to insulin resistance or less amount of insulin secretion.   Figure 4). The alpha-glucosidase inhibition activity of HELE is correlated with the presence of phytol, 9-octadecenoic acid, methyl ester, (E)-, eicosanoic acid, methyl ester, and gamma-sitosterol. The presence of these phytochemicals is validated by GC-MS analysis of HELE (Table 4).

Urease inhibition assay
In the US approximately 4.6 million people suffer peptic ulcers with 10% evidence of patients suffering from duodenal ulcers. About 70-90% of peptic ulcer and 90% duodenal ulcer is caused by helicobacter pylori. The helicobacter pylori can secrete urease extracellular, producing toxic effects on the epithelial lining of the gastric mucosa [23]. Urease converts transuded urea into ammonia which increases the pH of the surrounding environment resulting in a feasible environment for helicobacter pylori to grow and colonize. Four extractives obtained from the aerial part of L. frutescens were subjected to evaluation of anti-urease activity and it was observed that BULE exhibited maximum anti-urease activity with IC50 4.709 mg.ml -1 . The hydroxy urea was used as positive control and its IC50 value was calculated as 0.960 mg.ml -1 (Table 3).
frutescens. Alpha-amyrin and beta-amyrin exhibited an anti-inflammatory effect [26].  Table 5.  Conclusively, the results obtained provide the rationale for further extensive studies on biochemical analysis as well as the pivotal therapeutic potential of L. frutescens through in-vivo studies essentially focusing on isolation of antioxidants, antidiabetic, antibacterial, and phytotoxic compounds.