Free radical scavenging, antioxidant activity And phenolic content of Salvadora oleoides Decne Leaves.

 

Akash Garg1*, Sanjeev Kumar Mittal2

1Department of Pharmacognosy, PDM School of Pharmacy, Karsindhu-126115  India

*Research Scholar at School of Pharmaceutical Sciences, RIMT University, Mandi Gobindgarh-147301   India

2 School of Pharmaceutical Sciences, RIMT University, Mandi Gobindgarh-147301   India

*Corresponding Author E-mail: akashvj2004@gmail.com

 

ABSTRACT:

Background: Salvadora oleoides Decne (Salvadoraceae) is facultative halophyte tree found in dry and arid regions of India. Traditionally, it is used in the treatment of various ailments like piles, tumors, bronchitis, asthma, rheumatism, fever, conjunctivitis, digestive disorders, opacity of cornea, as carminative and alexipharmic etc. The plant is reported to possess antihypoglycemic, hypolipidemic, analgesic and antimicrobial activities. The purpose of current research study was to evaluate the in vitro antioxidant potential of Salvadora oleoides Decne leaves.

Method: Dried powdered leaves of Salvadora oleoides Decne were extracted with different solvents (Petroleum ether, chloroform and methanol) using soxhlet extraction. To evaluate free radical scavenging and antioxidant abilities seven in vitro test systems were employed i.e. DPPH scavenging, ABTS scavenging, superoxide anion scavenging, nitric oxide scavenging, hydroxyl radical  scavenging ,  Total antioxidant potential  in a linoleic acid emulsion and FRAP assay.

Results: The extracts were proved to have significant antioxidant potential in models like DPPH scavenging, ABTS scavenging, total antioxidant potential in linoleic acid emulsion. In all these studies, methanol extract exhibited higher antioxidant potential as compared to chloroform extract and petroleum ether extract respectively. In antioxidant potential study models viz. superoxide anion scavenging, hydroxyl radical scavenging, FRAP assay and  NO scavenging assay, none of extracts exhibited much significant activity as compared to respective standards.

Conclusion: The antioxidant potentials and total phenolic content of different extracts followed the same order, i.e., there is a good correlation between antioxidant potential and total phenolic content. Results showed that these extracts especially methanol extract, could be considered as natural antioxidants and may be useful for curing diseases arising from oxidative degradation.

 

KEYWORDS: Salvadora oleoides Decne, DPPH, ABTS, Total phenolic content.

 

 


INTRODUCTION:

Herbal drugs constitute the major part of most indigenous medicine systems including the ancient Indian ayurvedic system of medicine. In the development of human knowledge of therapeutic use, scientists endeavored to isolate different chemical constituents from plants, subjected them to biological and pharmacological tests and then used them to prepare modern medicines. Reactive oxygen species (ROS) are highly reactive ions and “free radicals” (chemicals containing atoms with an unpaired electron in its outer orbit) involving oxygen molecules. They are byproduct of cellular respiration or synthesized by enzyme systems – phagocytic cells, neutrophils and macrophages (NADPH oxidase, myeloperoxidases).1 Normally in controlled homeostatic environment in body ROS are necessary for production of some hormones (thyroxine), to kill some types of bacteria and engulfed pathogens, normal cell functioning and cell signaling. Excessive production of ROS by self-perpetuating chain reactions has been implicated as mediator of numerous diseases including cancer, atherosclerosis, heart and neurodegenerative diseases. At normal physiological levels, ROS functions as “Redox messengers” in intracellular signaling and regulation, where as excessive ROS damage the cellular macromolecules such as lipids, proteins and nucleic acids through inducing oxidative modification and promote cell death.2,3 Regular supplements of exogenous antioxidants limit the deleterious effect caused by excessive ROS. Many naturally occurring antioxidant compounds of plant origin (vegetables, fruits, leaves, oil seeds, cereals crops, tree barks, roots, spices and herbs) have been identified as potential ROS scavengers.4

 

The chemical constituents (polyphenols, flavonoids etc.) of existing flora are potential antioxidants which is a largely unutilized asset provided by nature. A vast number of plants have been investigated in recent past for their pharmacological actions and free radical scavenging properties. It is the diverse chemical composition of different herbs which makes naturally occurring molecules a vital nut in all medicinal and therapeutic systems. Salvadora oleoides Decne (Salvadoraceae) is an oil-yielding medicinal and multipurpose tree. It is commonly known in India as meetha jal.5 It is facultative halophyte found in dry and arid regions of India (Rajasthan, Haryana, Punjab, Maharashtra and Gujarat).6 Traditionally, it is used in the treatment of various ailments like piles, tumors, bronchitis, asthma, rheumatism, fever, conjunctivitis, digestive disorders, opacity of cornea, as carminative and alexipharmic etc.7-9 The plant is reported to possess antihypoglycemic, hypolipidemic, analgesic and antimicrobial activities.10-12 The major chemical constituents of the plant are sterols like β-sitosterol and their glucosides, flavonoids, dihydroisocoumarin, terpenoids like methoxy-4-vinylphenol and cis-3-hexenyl benzoate. In addition aerial parts also contain phytol, n-hexadecanoic acid, trans-s-damascenone, octacosane, nonacosane, 1-octadecene, heptacosane, hexacosane, pentacosane and squalene. Two compounds octadecanoyl heptanoate and δ-lactone of 3-isobutyl-5-hydroxy-19-oxononadeconic acid were isolated from stem bark. A new dimeric dihydroisocoumarin, salvadorin was isolated from the chloroform fraction of Salvadora oleoides Decne. The chemical structure was established as 8-benzyl-6-[6-(6-ethyl-7-methyl-5,8-dihydro-2-napthalenyl)-1-oxo-3,4-dihydro-1H isochromen-8yl]-3,4-dihydro-1H-isochromen-1-one through spectroscopic techniques and chemical analysis.13-15

 

To the best of our knowledge, no work has been performed on the comparative antioxidant potential of various extracts of Salvadora oleoides leaves. Therefore, the present study was undertaken to investigate the in vitro antioxidant potential of various extracts of current plant drug.

 

EXPERIMENTAL:

The S. oleoides Decne leaves were collected from local flora of district Jind, Haryana (India) in November, 2012 and identified by Dr. H.B. Singh, Chief Scientist and Head, Raw Material Herbarium and Museum, National Institute of Science Communication and Information Resources, New Delhi. A Voucher Specimen vide reference number NISCAIR/RHMD/Consult/2012-13/2155/161 has been deposited in the same institute.

 

Chemicals and standards

Preparation of Extracts

The Salvadora oleoides Decne leaves were air dried at room temperature, then ground to a powder and passed through a 20-mesh sieve. The extraction of powdered plant material was performed using continuous hot percolation method (soxhlet extraction). Three samples (10 gram each) were extracted exhaustively using petroleum ether, chloroform and methanol individually. The extracts were then filtered through Whatman No. 1 filter paper. The extracts were evaporated in vacuum rotary evaporator and dry extracts were stored at -20şC until use.

 

Determination of total phenolic content (TPC)

Total phenolic (TP) content in the extracts was determined using Folin–Ciocalteu reagent (FCR) according to the method of Kumar et al. with slight modification.16 Gallic acid was used as a standard. Briefly, the solution of each extract (0.5 mL, 1 mg mL-1) was diluted to 10 mL with distilled water in a volumetric flask. FCR (1 mL) was added and mixed thoroughly, and then sodium carbonate solution (3 mL, 2 %) was added. The absorbance at 760 nm was measured after 2 h. The total phenolic content was determined by comparison with the standard calibration curve of gallic acid, and results are presented as milligrams of gallic acid equivalents (mg of GAE) per gram dry weight (g DW). All tests were conducted in triplicate.

 

DPPH radical scavenging assay

The free radical scavenging activity (antioxidant capacity) of the plant extracts on the stable radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was evaluated by the method reported by Shirwaikar et al.17 In this assay, a volume of 1.5 mL of methanol solution of the extracts at different concentrations (50, 100, 150, 200 and 250 µg mL-1) was mixed with 0.5 mL of the methanol solution of DPPH (0.1 mM). A solution containing all reagents except extract solution (blank methanol) served as a control. After 30 min of reaction at room temperature in the dark, the absorbance was measured at 517 nm against blank using a UV-VIS spectrophotometer. The experiment was performed in triplicate and the percent free radical scavenging activity was calculated according to the following equation:

 

% DPPH radical scavenging activity = [(Ac-As) / Ac] × 100 where Ac = absorbance of blank control and As =absorbance of test sample.18

 

L-ascorbic acid was used as the reference standard antioxidant. The inhibitory concentration needed to scavenge DPPH free radical by 50% (IC50) was calculated by regression analysis of the dose response curve plotted between percentage inhibition versus concentration of the test samples and the standard.

 

ABTS free radical scavenging assay

Total antioxidant status was measured using 2,2´-azinobis-[3-ethylbenzthiazoline]-6-sulphonic acid (ABTS) assay as described by Re et al.19 ABTS was dissolved in deionized water to 7 mM concentration and potassium persulphate added to a concentration of 2.45 mM. The reaction mixture was left to stand at room temperature overnight (12-16 h) in the dark before use. The resultant intensely-coloured ABTS•+ radical cation was diluted with 0.01 M PBS (phosphate buffered saline), pH 7.4, to give an absorbance value of approximately 0.70 at 734 nm. The sample extracts were diluted 100 times with the ABTS solution to a total volume of 1 mL. Absorbance was measured at time intervals of 30 min after addition of each extract. The assay was performed at least in triplicate. Solution containing 990 μL of PBS, to replace ABTS, was used as control. The assay relies on the antioxidant capability of the samples to inhibit the oxidation of ABTS to ABTS•+ radical cation. L-ascorbic acid was used as the reference standard antioxidant. The percentage free radical scavenging activity was calculated according to the following equation:

% ABTS scavenging activity = [(Ac-As) / Ac] × 100 where Ac = absorbance of control and As =absorbance of sample.

 

Total antioxidant activity determination in a linoleic acid emulsion by the ferric thiocyanate method

The methods  of Mitsuda et al. and Osawa and Namiki  were  slightly  modified.20,21  A mixture  of  4 mL  of  a weighed sample in  99.5% ethanol, 4.1 mL  of  2.51%  linoleic  acid  in  99.5%  ethanol, 8  mL  of  0.05M  phosphate  buffer  (pH 7.0)  and 3.9 mL  of water was placed in  a vial   with  a screw cap and then placed in  an oven at 40° C in  the  dark. To 0.1 mL of this solution was added 9.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocyanate. Precisely 3  min  after  addition of  0.1  mL  of    0.02M  ferrous chloride in  3.5%  hydrochloric acid to the reaction mixture,  the absorbance  of  red color was  measured  at 500 nm each  24 hr until  one day after absorbance  of the  control reached  maximum.22 During the linoleic acid oxidation, peroxides formed and these compounds oxidize Fe+2 to Fe+3. The latter Fe+3 ions form complex with SCN and this complex has maximum absorbance at 500 nm. The solutions without added extract used as blank samples. All data about total antioxidant activity are the average of triplicate analyses. In this test L-ascorbic acid was used as standard. The percentage inhibition of lipid peroxidation was calculated by following equation:

 

% Inhibition of lipid peroxidation = [(Ac-As) / Ac] × 100 where Ac = absorbance of control and As =absorbance of sample.

 

Superoxide anion radical scavenging activity

Superoxide anion radical scavenging of the extracts of S. oleoides Decne was measured by the method of Yen et al. with slight modifications.23,24 The reaction mixture, which contained 1 mL of different concentration extracts (50,100,150,200, and 250µg/mL) in distilled water, 1 mL of Phenozine methosulphate (PMS) (60 µM) in phosphate buffer (0.1 M, pH  7.4), 1 mL of Nicotinamide adenine dinucleotide (NADH) (468 µM) in phosphate buffer, and 1 mL  of Nitrobluetetrazolium chloride (NBT) (150 µM) in phosphate buffer, was incubated at  ambient temperature for 5 min, and the color was read at 560  nm against blank samples. L-ascorbic acid was used as the reference standard antioxidant. The percentage free radical scavenging activity was calculated according to the following equation:

 

% superoxide anon scavenging activity = [(Ac-As) / Ac] × 100 where Ac = absorbance of control and As =absorbance of sample

 

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging ability was determined according to the method of Klein, Cohen and Cederbaum with slight modification.25 1 ml solutions of different concentration (50, 100, 150, 200 and 250 µg/mL) of each extract was transferred to test tubes and dried to complete dryness.  1 mL of an iron EDTA solution (0.13% ferrous ammonium sulphate and 0.26% EDTA), 0.5 mL of EDTA (0.018%) and 1 mL of DMSO (0.85% v/v in 0.1 M phosphate buer, pH 7.4) were added to the test tubes and the reaction was initiated by adding 0.5 mL of 0.22% L-ascorbic acid. Test tubes were capped tightly and heated on a water bath at 80–90 şC for 15 min. The reaction was terminated by the addition of 1 mL of ice-cold trichloroacetic acid (17.5% w/v). 3 mL of Nash reagent (75.0 g of ammonium acetate, 3 mL glacial acetic acid and 2 mL of acetyl acetone were mixed and made up to 1L with distilled water) was added to all of the tubes and left at room temperature for 15 min for colour development. The intensity of the yellow colour formed was measured by UV-VIS spectrophotometer at 412 nm against reagent blank. Gallic acid was used as the reference standard antioxidant. The percentage hydroxyl radical scavenging is calculated by the formula.

 

Hydroxyl radical scavenging activity (%) = [(Ac-As) / Ac] × 100 where Ac = absorbance of control and As =absorbance of sample

 

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was performed according to method described by Benzie and Strain with some modifications.26 The stock solutions included 300 mM acetate buffer (pH 3.6), 10 mM Tri-2-pyridyl-s-triazine (TPTZ) solution in 40 mM hydrochloric acid, and 20 mM ferric chloride hexahydrate solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL ferric chloride hexahydrate solution, which was then warmed to 37°C before use. The sample solutions of plant extracts and standard L-L-ascorbic acid were prepared in methanol. 10 μL of each of sample solution was taken in separate test tubes and 2990 μL of FRAP solution was added to each to make a total volume of 3 mL. The sample solutions were allowed to react with the FRAP solution in the dark for 30 min. The absorbance of the coloured product (ferrous tripyridyltriazine complex) was checked at 593 nm. The FRAP values are expressed as micromoles of L-ascorbic acid equivalents (ASAE) per mg of the sample using the standard curve constructed for different concentrations of L-ascorbic acid. The results are expressed in % μmol ASAE mL-1..

 

Nitric oxide generation and assay of nitric oxide scavenging

Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide.27,28  Sodium nitroprusside (5 mM) in phosphate-buffered saline was mixed with different concentrations of the extracts and standard were dissolved in the suitable solvent systems and incubated at 25°C for 150 min. The samples from the above were reacted with Greiss reagent (1% sulphanilamide, 2% H3PO4 and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamme was read at 546 nm and referred to the absorbance of standard solutions of potassium nitrite treated in the same way with Griess reagent.29 L-ascorbic acid was used as the reference standard antioxidant. The percentage scavenging of NO for S. oleoides Decne and the standard antioxidant was calculated according to equation given below.

 

% Nitric oxide scavenging activity = [(Ac-As) / Ac] × 100 where Ac = absorbance of control and As =absorbance of sample.

 

Statistical analysis

All data are presented as means ± SEM. Differences at p < 0.05 to p < 0.001 were considered statistically significant. All the statistical analyses were performed with Prism Graphpad software or Excel 2003. Regression analyses were carried out using Microsoft Excel. Calibration curves of the standards were considered as linear if R2 > 0.99. IC50 values were calculated from either linear or logarithmic dose response curves where R2 > 0.90. (n = 3)

 

RESULTS AND DISCUSSION:

Determination of total phenolic content (TPC)

Phenolic compounds contain hydroxyl groups which contribute to the free radical scavenging and act as primary antioxidants. As depicted in the Table I the methanol extract found to have more TPC as compared to other two extracts. The highest quantity (mg) of gallic acid equivalents/gram dry weight was found in methanol extract.

 

TABLE 1. Total phenolic content of various extracts of S. oleoides Decne.

Sample

mg  GAE/ g DW*

(Mean ± SEM)**

Petroleum ether extract

33.20 ± 0.82

Chloroform extract

37.21 ± 0.74

Methanol extract

46.37 ± 1.17

*Milligram of gallic acid equivalent per gram dry weight of the sample

**All results are presented as mean ± standard mean error of three assays (n = 3).

 

DPPH radical scavenging assay

The DPPH assay method is based on the reduction of DPPH, a stable free radical. The free radical DPPH with an odd electron gives a maximum absorption at 517 nm (purple colour). Antioxidants donate one hydrogen atom to DPPH radical and thereby cause reduction of this free radical to stable diamagnetic molecule DPPHH and the absorbance is decreased and gives a yellow colour17.

 


 

Figure  1. The dose response curves for percentage scavenging of DPPH by extracts of S. oleoides in comparison with L-Ascorbic acid. The lines represent linear or logarithmic (as stated in legends) regression analysis results.

 

 

 


The DPPH radical scavenging effect of S. oleoides Decne extracts compared with L-ascorbic acid is presented in Fig. 1. The results depicted a significantly (p < 0.05) higher radical scavenging activity for the standard agent ascorbic acid in comparison to PEE and ME. At 200 µg/mL, the ME has significantly (p < 0.01) more free radical scavenging activity as compared to CE. The L-ascorbic acid depicted significantly (p < 0.01) more activity than CE at 200 µg/mL. Similarly both the ASA and ME  have significantly (p < 0.001) more activity than PEE at 200 µg/ml. ASA, CE and ME; all the three  possess markedly high (p < 0.001) free radical scavenging activity as compared to PEE at 250 µg/mL. ME is more (p < 0.01) potent DPPH radical scavenger than CE at 250 µg/mL. The dose response curves for percentage scavenging of DPPH by extracts of S. oleoides Decne along with L-ascorbic acid was drawn using regression analysis  and calculated IC50 values as  257.24, 169.94, 161.65, 152.27  µg/mL  for PEE, CE, ME and ASA respectively. 

 

ABTS free radical scavenging assay

The ABTS free radical scavenging effect of S. oleoides Decne extracts compared with ASA is presented in Fig. 2. Except for PEE, all other extracts have significantly higher ABTS radical scavenging activity relative to control at concentration 50 µg/mL (CE, p < 0.01; ME and L-ascorbic acid, p < 0.001). As expected L-ascorbic acid possess markedly (p < 0.001) higher free radical scavenging activity relative to PEE, CE and ME. At 50 µg/mL concentration CE has lower radical scavenging activity as compared to ME (p < 0.05). Importantly, ME was proved to be more efficient ABTS scavenger relative to CE at 200 and 250 µg/mL concentrations (p < 0.01 and p < 0.001 respectively). The CE and ME were found to have significantly (p < 0.01 and p < 0.001 respectively) greater scavenging activity in comparison to the PEE of S. oleoides Decne, which increased with the dose of extracts. As shown in Fig. 2, The dose response curves for percentage scavenging of ABTS by extracts of S. oleoides Decne along with L-ascorbic acid was drawn using regression analysis  and calculated IC50 values as  378.27, 260.75, 217.45, 129.80 µg/mL  for PEE, CE, ME and ASA respectively.

 

 


Fig. 2. The dose response curves for percentage scavenging of ABTS by extracts  of S. oleoides in comparison with L-ascorbic acid.

 


Total antioxidant activity determination in a linoleic acid emulsion by the ferric thiocyanate method

As depicted by results in Fig., 50 µg/ml concentration of L-ascorbic acid has significantly improved total antioxidant activity as compared to PEE (p < 0.001) and CE (p < 0.01). 50 µg/mL conc. of CE and ME showed significantly (p < 0.05, p < 0.01 respectively) more antioxidant activity relative to PEE fraction. All the extracts depicted marked (p < 0.001) antioxidant activity as compared to blank. The 100 µg/mL conc. of ASA, CE and ME possess significantly (p < 0.001, p < 0.05, p < 0.05 respectively) more antioxidant activity relative to PEE. Further, as expected the standard antioxidant has higher reducing capability as compared to CE and ME (p < 0.01). The 150 µg/mL concentration of L-ascorbic acid , CE and ME have more antioxidant activity (p < 0.001, p < 0.01, p < 0.001 respectively) as compared to PEE fraction. Importantly, the ME fraction has more (p < 0.05) antioxidant activity relative to CE fraction and as expected the L-ascorbic acid depicted profound (p < 0.001) antioxidant activity relative to CE. At conc. 250 µg/mL the standard antioxidant depicted significantly more reducing capability relative to PEE and CE fractions (p < 0.01). As presented in Fig. 3, The dose response curves for total antioxidant activity of linoleic acid emulsion by the ferric thiocyanate method by extracts of S. oleoides Decne along with L-ascorbic acid was drawn using regression analysis  and calculated IC50 values as  178.89, 145.81, 108.85, 104.31µg/mL  for PEE, CE, ME and ASA respectively.


 

Fig. 3. The dose response curves for percentage antioxidant / lipid peroxidation inhibition activity by extracts of S. oleoides in comparison with L-ascorbic acid.

 


Superoxide anion radical scavenging activity

As depicted by Table II, At 50 and 100 µg/mL neither of the extracts was effective in superoxide radical scavenging except for the standard L-ascorbic acid (p < 0.001). At 150 µg/mL the extracts depicted significant radical scavenging activity as presented in Table IV. Both CE and ME are compared to the PEE at higher concentrations. Further, the ME was found to be significantly (p < 0.05) more active relative to the CE at 250 µg/mL concentration. As expected the standard drug depicted significantly (p < 0.001) high free radical scavenging activity relative to all other three extracts. As found in results the CE and ME have markedly (p < 0.001) higher radical scavenging activity relative to the PEE. Further, ME at concentration 250 µg/mL was found to have significantly (p < 0.05) elevated radical scavenging activity in comparison to CE.

 

TABLE II. Superoxide anion radical scavenging assay by the various extracts of S. oleoides Decne.

Sample

Concentration

(µg/mL)

Superoxide anion radical scavenging, %

(Mean ± SEM)*

Petroleum ether extract (PEE)

50

0.81 ± 0.40

 

100

1.73 ± 0.87

 

150

2.96 ± 0.69a

 

200

8.84 ± 1.57b

 

250

9.88 ± 1.60b

Chloroform extract (CE)

50

1.46 ± 0.57

 

100

1.56 ± 0.15

 

150

6.62 ± 0.88c

 

200

13.27±1.72c

 

250

21.65 ± 0.53c

Methanol extract (ME)

50

1.40 ± 0.27

 

100

1.54 ± 0.22

 

150

8.06 ± 0.32c

 

200

16.99 ± 1.31c

 

250

28.38 ± 1.55c

L-ascorbic acid (ASA)

50

13.50 ± 2.01c

 

100

20.27 ± 0.53c

 

150

34.37 ± 0.62c

 

200

42.44 ± 1.49c

 

250

65.26 ± 1.97c

*All results are presented as mean ± standard mean error of three assays (n = 3); a p < 0.05, b p < 0.01, c p < 0.001 when compared with the negative control, i.e., blank/solvent.

 

Hydroxyl radical scavenging ability

Hydroxyl radical is an extremely reactive free radical formed in biological systems which can react and damage biomolecules found in living cells. Hydroxyl radicals can initiate lipid peroxidation of the cell membrane. Iron catalyzed OH radical formation degrades deoxyribose to thiobarbituric acid reactive substances (TBARS) which generates a pink chromogen on heating with thiobarbituric acid (TBA)25

TABLE III. Hydroxyl anion radical scavenging assay by the various extracts of S. oleoides Decne.

Sample

Concentration

(µg /mL)

Hydroxyl anion radical scavenging activity, %

(Mean ± SEM)*

Petroleum ether extract (PEE)

50

0.99 ±0.17

 

100

1.99 ± 0.29

 

150

5.6 ± 0.41a

 

200

6.33 ±1.68b

 

250

8.93 ± 0.31

Chloroform extract (CE)

50

1.67 ± 0.25

 

100

1.99 ± 0.18

 

150

3.88 ± 0.90

 

200

6.55 ± 0.73c

 

250

7.78 ± 0.35b

Methanol extract (ME)

50

1.84 ± 0.40

 

100

3.07 ± 0.46

 

150

11.73 ± 1.43c

 

200

12.32 ± 1.63c

 

250

12.51 ± 1.37c

Gallic acid (GA)

50

19.96 ± 0.94c

 

100

24.41 ± 0.55c

 

150

43.37 ± 1.74c

 

200

60.22 ±2.35c

 

250

72.92 ± 1.80c

*All results are presented as mean ± standard mean error of three assays (n = 3); a p < 0.05, b p < 0.01, c p < 0.001 when compared with the negative control, i.e., blank/solvent.

 

As depicted by results in Table III, the standard drug gallic acid has shown highest hydroxyl radical scavenging activity.  At 150 µg/mL, the extracts depicted significant radical scavenging activity. At this concentration there is significant difference between the hydroxyl scavenging activity of ME and PEE (p < 0.05), and ME and CE (p < 0.01). At 200 µg/mL, the results were almost same as that of 150 µg/mL concentration, the only difference being no significant difference between hydroxyl radical scavenging activity of ME and CE. Highest antioxidant activity of all extracts was observed at 250 µg/mL concentration.

 

Ferric reducing antioxidant power (FRAP) assay

Ferric ion reducing power assay measures the electron donating capacity of an antioxidant. The presence of reducing agents (i.e. antioxidants) causes the reduction of the Fe3+/ferricyanide complex to the ferrous form. The absorbance measured at 700 nm of the resultant blue-green coloured solution is proportional to the amount of Fe2+ in the system. Therefore an increased absorbance is indicative of higher reducing power26.

 

TABLE IV. Ferric reducing antioxidant power (FRAP) of various extracts of S. oleoides Decne.

Sample

Concentration

(µg mL-1)

 μmol ASAE/mL, %*

(Mean ± SEM)**

Petroleum ether extract (PEE)

50

0.96 ± 0.08

 

100

0.85 ± 0.26

 

150

0.94 ± 0.06

 

200

1.90 ± 0.51

 

250

2.82 ± 0.37a

Chloroform extract(CE)

50

1.22 ± 0.27a

 

100

1.41 ± 0.17a

 

150

1.95 ± 0.14

 

200

3.16 ± 0.32a

 

250

7.50 ± 0.29c

Methanol extract(ME)

50

1.08 ± 0.07

 

100

2.69 ± 0.26c

 

150

4.04 ± 0.12b

 

200

5.38 ± 0.56c

 

250

7.50 ± 0.29c

L-ascorbic acid(ME)

50

9.44 ± 0.46

 

100

16.74 ±0.39

 

150

25.77 ± 1.27

 

200

37.21 ±0.96

 

250

54.99 ±1.01

*Percentage  micromoles of L- ascorbic acid equivalent per milliliter of sample

**All results are presented as mean ± standard mean error of three assays (n = 3); a p < 0.05,  bp < 0.01, c p < 0.001 when compared with the negative control, i.e., blank/solvent.

 

As depicted by results in Table IV, the standard drug L-ascorbic acid has shown highest hydroxyl radical scavenging activity. At 50 µg/mL concentration, only CE showed significant FRAP relative to control (p < 0.05). ME showed markedly significant FRAP relative to PEE at all concentrations. At 100 µg/mL concentration ME possessed significantly (p < 0.05) more antioxidant activity relative to CE fraction. Only ME depicted consistent antioxidant activity dose-dependently.

 

Nitric oxide generation and assay of nitric oxide scavenging

Formation of NO is elevated in infections and inflammation, and in the aerobic environment NO reacts with oxygen to produce strong oxidants such as peroxynitrite anion (ONOO−). Although NO does not interact with biological macromolecules directly, peroxynitrite anion (ONOO−) will give rise to adverse effects such as DNA fragmentation, cell damage and neuronal cell death. Nitric oxide generated spontaneously from sodium nitroprusside (SNP) in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions causing diazotization of sulphanilamide which in turn undergoes coupling with naphthylethylenediamine dichloride forming an azo-dye. Scavengers of nitric oxide compete with oxygen which leads to reduced production of nitrite ions28.

 

TABLE V. Nitric oxide scavenging by various extracts of S. oleoides Decne.

Sample

Concentration

(µg mL-1)

NO scavenging, %

(Mean ± SEM)*

Petroleum ether extract (PEE)

50

1.01 ± 0.00

 

100

1.03 ± 0.02

 

150

1.10 ± 0.06

 

200

2.99 ± 0.06b

 

250

3.48 ± 0.29b

Chloroform extract (CE)

50

1.06 ± 0.03

 

100

1.33 ± 0.25

 

150

1.45 ± 0.28

 

200

1.67 ± 0.34

 

250

3.47 ± 0.26b

Methanol extract (ME)

50

1.04 ± 0.03

 

100

1.70 ± 0.22

 

150

2.75 ± 0.31a

 

200

3.99 ± 0.55b

 

250

5.97 ± 0.16b

L-ascorbic acid (ASA)

50

13.66 ± 0.37c

 

100

21.37 ±1.07c

 

150

32.72 ± 1.11c

 

200

44.54 ±0.78c

 

250

52.65 ±0.95c

*All results are presented as mean ± standard mean error of three assays (n = 3); a p <    0.05, b p < 0.01, c p < 0.001 when compared with the negative control, i.e., blank/solvent.

 

As shown in table V, all the extracts depicted insignificant NO scavenging activity at 50 µg/mL concentration. However, PEE and ME produced slightly higher activity as compared to CE. Further ME depicted significantly more NO scavenging activity as compared to CE and PEE  (p < 0.05) at 250 µg/mL concentration and also at 200 µg/mL concentration for CE (p < 0.05) alone. L-ascorbic acid has more marked NO scavenging activity as compared to various extracts of S. oleoides Decne.

 

CONCLUSION:

In the present study, antioxidant (Free radical scavenging) potential of various extracts (Petroleum ether, chloroform and methanol) of Salvadora oleoides Decne was studied utilizing various in vitro models. The extracts were proved to have significant antioxidant potential in various models like DPPH scavenging, ABTS scavenging, total antioxidant potential in linoleic acid emulsion. In all these studies, methanol extracts exhibited higher antioxidant potential as compared to chloroform extract and petroleum ether extract respectively. In antioxidant potential study models viz. superoxide anion scavenging, hydroxyl radical scavenging, FRAP assay and  NO scavenging assay, none of extracts exhibited much significant activity as compared to respective standards. The higher antioxidant potential of Methanol extract can be attributed to higher phenolic content (Table I) as compared to other extracts. It may be concluded that S. oleoides Decne can prove an effective and potential antioxidant agent. Further In vivo animal studies are indeed required to validate antioxidant potential and thereby use in treatment of diseases related to oxidative degradation.

 

ACKNOWLEDGEMENT:

The authors thank RIMT University, Mandi Gobindgarh (India) for the valuable support in carrying out the current research work.

 

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Received on 16.11.2017          Modified on 30.11.2017

Accepted on 20.01.2018       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(1): 27-35.

DOI: 10.5958/0975-4385.2018.00005.5