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Evaluation of antioxidant, anti-hemolytic and anticancer activity of various solvent extracts of Acacia hydaspica R. Parker aerial parts
© The Author(s). 2016
Received: 4 December 2015
Accepted: 23 July 2016
Published: 29 July 2016
Acacia hydaspica R. Parker, family leguminosae, is a medicinally important plant. Different plant parts are used in various ailments in folk medicine. The current study aimed at investigating the in vitro antioxidant, anti-hemolytic and anticancer activity of A. hydaspica.
Antioxidant potential was assessed using DPPH, ABTS and •OH, scavenging of H2O2, inhibition of lipid peroxidation and β-carotene bleaching inhibition assays. Anti-hemolytic activity was assessed using H2O2 induced hemolysis of RBCs. Anticancer potential was assessed using MTT assay. Spectrometric methods and HPLC-DAD analysis was performed for phytochemical screening.
EC50 values based on reduction of DPPH, ABTS and •OH, scavenging of H2O2, inhibition of lipid peroxidation and β-carotene bleaching for AHB, AHE and AHM were generally lower manifesting potential antiradical capacities. The fractions also exhibited significant (P <0.001) anti-hemolytic potential. Regarding IC50 values for anticancer activity against HCC-38 and MDA-MB-361 cancer cell lines; AHB, AHE and AHM exhibited significant (P <0.001) cyto-selection indices. Plant extracts showed no cytotoxicity against normal Vero cells (IC50 > 250 μg/ml). While significant (P <0.001) cytotoxicity was elicited by these extract/fractions against cancer cell lines. AHE was the most effective and IC50 was found to be 29.9 ± 0.909 μg/ml (SI = 9.83) and 39.5 ± 0.872 μg/ml (SI = 7.44) against MDA-MB-361 and HCC-38 cancer cells respectively. Higher amounts of TPC and TFC were exhibited by AHE and AHB as compared to other fractions. Gallic acid, catechin and myricetin were identified in AHE whereas gallic acid and catechin were identified in AHB by HPLC.
The presence of bioactive constituents in AHE and AHB might be responsible for antioxidant, anti-hemolytic and anticancer activities.
Cancer is a prominent reason of death in many developed and developing countries. Although the etiologies of cancer are varied, oxidative stress plays a major role for the pathophysiological developments. Oxidative stress forced by free radicals such as singlet oxygen species, superoxide, hydroxyl and ferrous may cause lipid peroxidation, protein damages, inflammation, autoimmune pathologies, DNA damage, altering cell-signaling pathways and modulating gene expression, induction and promotion of tumor [1, 2]. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used in the food industry because they are effective and less expensive than natural antioxidants. Synthetic antioxidants gained safety concerns and have been restricted due to their DNA damaging and other toxic effects . Complementary and alternative medicine is one of the emerging fields in health care today, especially as supportive medicine in treating diseases like cancer . Plant secondary metabolites such as flavonoids, terpenes, alkaloids, α-tocopherol and carotenoids have received considerable attention in recent years due to their diverse pharmacological properties, including cytotoxic and chemo-preventive effects. The possible health benefits of polyphenol consumption have been suggested to derive from their antioxidant properties [4, 5]. Therefore it is interesting to identify selectivity of plant extracts possessing antioxidant potential against cancer and normal cells. In fact, literature has verified an association between intake of diet rich in fruits and vegetables with a decline in oxidative stress induced disorders .
Acacia hydaspica R. Parker synonym A. eburnea, commonly known as ‘kikar’ family leguminosae is an economically important plant. It is a slender deciduous shrub, 1.2–1.8 m tall, twigs glabrous slightly zigzag; bark smooth, dark grey and 1.2–2.5 cm long stipular spines, seeds 1–8, areole not well marked. The bark and seeds are the source of tannin. Leaves serves as fodder for goats [7, 8]. The bark and seeds are the source of tannins. The plant is locally used as antiseptic. The traditional healers of India use various parts of the plant for the treatment of diarrhea; the leaves and the bark are useful in arresting secretion or bleeding. The pods are helpful in removing catarrhal matter and phlegm from the bronchial tubes. The gum dispels irascibility of the skin and soothes the inflamed membranes of the pharynx, alimentary canal and genito-urinary organs (http://trade.indiamart.com/details.mp?offer=6763150691). Gallic acid, catechin, rutin and caffeic acid have been identified in A. hydaspica by HPLC-DAD screening of crude methanol extract, while 7-O-galloyl catechin, +catechin and methyl gallate have been isolated from ethyl acetate fraction of A. hydaspica (AHE). The A. hydaspica possess anti-inflammatory, antipyretic and analgesic potentials . Polyphenolic compounds isolated from A. hydaspica induce apoptosis and inhibit cancer cell growth in vitro in breast and prostate cancer cells by modulating various signal transduction pathways . Various species of Acacia were investigated for their antioxidant and anticancer potentials in various animal models . The extracts from the bark and heartwood of Acacia confusa showed significant antioxidant activity in various antioxidant assays, including free radical and superoxide radical scavenging assays and lipid peroxidation assay as well as hydroxyl radical-induced DNA strand scission assay . A. mangium and A. auriculiformis heartwood extracts showed excellent quenching ability against DPPH free radicles . The antioxidant activities of bark extract of Acacia confusa and some of the isolated constituents from its ethyl acetate (EtOAc) fraction in various in vitro systems together with authentic antioxidant standards revealed that EtOAc fraction showed strong superoxide radical scavenging activity, reducing power, and ferrous ion-chelating ability. Results obtained indicated that the bark extracts from A. confusa have a great potential to prevent disease caused by the overproduction of radicals and also it might be used as a potential source of natural antioxidant agent . Heartwood extract of Acacia catechu induces apoptosis in human breast cancer cell . However to the best of our knowledge there is no single scientific report demonstrating the antioxidant and cyto-selective anticancer potential of A. hydaspica.
In this study qualitative phytochemical screening, total phenolic content (TPC), total flavonoid content (TFC), antioxidant and anti-hemolytic activities of extract/fractions were evaluated. Extracts with potent antioxidant activity were tested against normal (VERO) and cancer cell lines (HCC-38 and MDA-MB-361) in order to evaluate the cyto-selective potential against cancer and normal cells. Furthermore polyphenol constituents in active fractions were determined by HPLC-DAD chromatography using standard reference compounds.
The plant was collected from Kirpa village Islamabad, Pakistan. After identification with the help of relevant literature a voucher specimen was deposited (0642531) at the Herbarium of Pakistan Museum of Natural History, Islamabad.
Preparation of extract/fractions
The aerial parts (twigs and leaves) of the plant were dried in an aerated but shaded area. Dried material was ground by an electrical grinder to obtain 60 μm powder. The methanol extract was obtained by allowing 3 kg of powder to macerate three times in 95 % methanol (3 × 4000 ml) for five consecutive days. The supernatants were mixed and filtered. Solvent was evaporated by rotary vacuum evaporator (Buchi, R114, Switzerland). The residue was taken to dryness to obtain a viscous mass as the crude methanol extract (AHM). An amount of 12 g of AHM was suspended in water (250 ml) with continuous stirring then successively added (3 × 200 ml) following solvents; n-hexane, ethyl acetate, chloroform and n-butanol respectively, shake well and each layer was allowed to separate for 3 h in a separating funnel and at last water soluble fraction was obtained (AHA). Each of the fractions obtained were dried using a rotary evaporator. AHM and its five subsequent fractions: AHH, AHE, AHC, AHB and AHA were weighed and expressed in terms of percentage of air dried weight of plant material.
Ascorbic acid, aluminum chloride, 2,2-azino-bis-(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric chloride (FeCl3), Tween 80, β-carotene, (+)-catechin, gallic acid, rutin, quercitin, potassium persulphate, Folin-Ciocalteu’s reagent, ferrozine, gallic acid, rutin, linoleic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitro blue tetrazolium (NBT), linoleic acid, phenazine methosulphate (PMS), thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were purchased from Sigma Aldrich (Germany). Deoxyribose, riboflavin, sodium carbonate (Na2CO3), sodium hydroxide (NaOH), disodium hydrogen phosphate (Na2HPO4) and hydrogen peroxide (H2O2) were obtained from Wako Co. (Osaka, Japan). Potassium ferricyanide (K3Fe (CN) 6), triflouroacetic acid, sodium dihydrogen phosphate (NaH2PO4) and all solvents used were of analytical grade and were purchased from Sigma Aldrich (Germany).
Preliminary phytochemical screening
The methanol extract and its soluble fractions were subjected to phytochemical analysis by using the methods described previously for the detection of terpenoids, alkaloids , saponins, tannins, flavonoids , cardiac glycoside , reducing sugars , pholobatannins, coumarins and anthraquinones  by qualitative methods.
Estimation of total phenolic content (TPC)
The total phenols of AHM and its derived fractions were quantified by previously described spectrophotometric method . TPC was calculated from the calibration curve of gallic acid. Estimation of TPC was recorded in triplicate and expressed as mg of gallic acid equivalent/g of dry sample.
Estimation of total flavonoid content (TFC)
Aluminium chloride colorimetric technique with slight modifications was used for the estimation of TFC . Quantity of TFC was recorded in triplicate from the calibration curve of rutin and expressed as mg of rutin equivalent/g of dry sample.
Each sample was dissolved in 95 % methanol at a concentration of 1 mg/ml and diluted to prepare the serialized dilutions (10–500 μg/ml) for various antioxidant assays. Reference standard chemicals were used for comparison in all assays.
Antioxidant activity assessment assays
DPPH radical scavenging activity assay
Where Ad is the DPPH solution absorbance, Asd is the absorbance of solution containing test sample and DPPH solution, and Asa is the absorbance sample solution without DPPH. Each sample was analyzed in thrice.
As a standard reference compound ascorbic acid was employed.
Superoxide anion radical quenching assay
Gallic acid was used as a standard compound.
Hydroxyl radical quenching activity
Gallic acid was employed as a reference standard.
Hydrogen peroxide radical quenching assay
Hydrogen peroxide solution (200 mM) was prepared in phosphate buffer (50 mM, pH 7.4). 100 μl of test sample (0.1–0.5 mg/ml) mixed with 400 μl of 50 mM phosphate buffer (pH 7.4), then add 600 μl hydrogen peroxide solution and vortex the sample tubes. Note the absorbance of the solution at 230 nm after 10 min against a blank .
Ascorbic acid was used as a standard reference.
ABTS radical scavenging activity
Ascorbic acid was used as a standard control.
Iron chelating power
Catechin was used as a reference compound.
β- carotene bleaching test
At0 is the initial absorbance
At120 is the absorbance of solution after 120 min
Catechin and BHT were employed as a standard reference.
Anti-lipid peroxidation analysis
This test was performed in accord with scheme described earlier . The extract/fractions were dissolved in methanol to prepare varying concentrations of sample solutions (50–1000 μg/ml). An aliquot of 300 μl of CuCl2 solution (0.05 mM) was added to each test tube before adding sample (50 μl) and linoleic acid (100 μl). Mixture was vortexes for 5 s and kept for 20 h for incubation in shaking water bath set at 37 °C. Twenty microliter of BHT (prepared in 10 mM in ethanol) was poured to each test tube to stop the reaction. Solution of TBA was prepared by dissolving 0.67 % TBA in 0.1 M HCl by sonication and momentary heating. Afterward, 3 ml of this freshly prepared solution of TBA (thiobarbituric acid) was added to each sample tube and mixture was vortexed for 5 s. The sample tubes were kept in hot water bath for 10 min. After cooling the sample tubes, the pink aqueous layer was transferred to new test tubes containing 2.5 ml of 100 % n-butanol. Mixture was vortexed for 5 s and allowed to settle. Absorbance of pink solution was noticed at 532 nm using spectrophotometer.
BHA was used as a reference standard.
Total antioxidant capacity (TAC) (Phosphomolybdate assay)
Phosphomolybdate method was used to determine the antioxidant capacity of compounds . One thousand microliter of assay mixture comprising H2SO4 (0.6 M), sodium phosphate (0.028 M) and ammonium molybdate (0.004 M) was poured to the sample tubes containing 100 μl of test sample. Incubation of mixture was done for 90 min in hot water bath set at 95 °C. The absorbance of reaction mixture was noted at 765 nm after the samples were cooled.
Ascorbic acid employed as reference standard.
Reducing power assay
The protocol of Kumaran was followed for assessing the reducing ability of the extract/fractions . An amount of 0.5 ml of phosphate buffer (0.2 M, pH 6.6) and 0.5 ml of potassium ferricyanide was mixed with 0.5 ml of the extract/fractions (50–250 μg/ml) and incubated at 50 °C for duration of 20 min. 10 % TCA solution (0.5 ml) was added to the reaction mixture in order to stop the reaction. Afterwards, 0.5 ml solution was pipetted out from each reaction mixture tube and permitted to mix with ferric chloride (100 μl) and of distilled water (0.5 ml). Optical density of the chromogen made was note down at 700 nm after incubation of sample for 10 min. Higher absorbance values were proportionated to higher reducing potency.
Values obtained for gallic acid were used as reference standard.
Anti-hemolytic potential of extract/fractions was inspected by spectrophotometric procedure as described previously . Five milliliter of blood from a healthy person was collected in EDTA vials and centrifuged for 5 min at 1000 × g. Supernatant was removed and pellet was washed thrice with PBS (0.2 M, pH 7.4) before re-suspending in saline solution (0.5 %). 0.5 ml of the extract/fractions (100–1000 μg/ml in PBS) was dispensed to 1 ml of erythrocyte suspension and incubated at room temperature for 20 min. Next add 0.5 ml of H2O2 solution made in buffered saline to the reaction mixture for provoking oxidative degradation of the membrane lipids. Subsequently, the samples were centrifuged at 1000 × g for 10 min and the absorbance of supernatant was noted spectrophotometrically at 540 nm. The relative hemolysis was assessed in comparison with the hemolysis in the H2O2 treated (negative control), which was set as 100 %. For positive control phosphate buffer saline was used. Each set of experiments was performed in triplicate and inhibitory activity of different fractions was calculated and expressed as percent inhibition of hemolysis. Quercetin (100–500 μg/ml) treated in the similar manner was employed as a reference compound. The study protocol was in agreement with Helsinki Declaration. Study approval (Bch#0256) was obtained from the Ethical Review Committee, Quaid-i-Azam University. Islamabad. Informed consent was obtained from persons who participated in the study.
Cell lines and cell culture
HCC-38 (CRL-2314™, homosapien, mammary carcinoma epithelial cells, estrogen receptor negative), MDA-MB-361 (HTB-27™, homosapien, mammary gland/breast; derived from metastatic site: brain) and Vero (CCL-81™, normal kidney cells) cell lines were obtained from ATCC (Manassas, VA, USA). MDA-MB-361 and HCC-38 cells were routinely cultured in DMEM/F12, whereas Vero cells were grown in MEM media (Invitrogen), supplemented with 10 % FBS (Invitrogen 16000–044) and 1 % Penicillin/Streptomycin (Invitrogen 15140–122). The cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % oxygen at all times. HCC38 and Vero cells were seeded in 96-well microtiter plates at density 5 × 104 cells/well, whereas MDA361 cells were plated at 1.25 × 104 cells/well and incubated overnight with respective medium described above to obtain a 70 % confluent layer. The monolayer was treated with different concentrations (3.125–25 μg/ml) of the plant extract/fractions and incubated for 48 h at 37 °C. In all experiments a negative control and a positive control were maintained. Negative control contained only growth media while the positive control contained 50 % DMSO.
The principle of MTT is based on cellular reduction of soluble yellow MTT tetrazolium salt (3, 4, 5-(dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) to its purple color formazan product by the mitochondrial dehydrogenase in viable cells. MTT assay was used to determine cytotoxicity of A. hydaspica crude extract/fractions. After the end of treatment as described above the culture medium was replaced with fresh medium and MTT assay was performed . Absorbance was recorded using a plate reader (Spe 5 M) on 570 nm, with reference wavelength at 690 nm.
Estimation of cytotoxicity and IC50
Cell cytotoxicity was calculated as a percentage of corresponding control value (non-treated cells) obtained in a minimum of three independent experiments. The half-maximal inhibitory concentration values (IC50), defined as the concentration that inhibits 50 % of cell growth, were calculated from concentration-response curves.
IC50 values were calculated using Graph pad prism 5.
Preparation of standard for HPLC-DAD
Stock solutions of rutin, kaempherol, myricetin, gallic acid, catechin, caffeic acid and quercetin were prepared in methanol at concentration of 1 mg/ml and diluted with methanol to get 10, 20, 50, 100 and 200 μg/ml for the standard calibration curve. Calibration curves for standard analytes at 10, 20, 50, 100 and 200 μg/ml concentrations were found to be linear.
Preparation of samples for HPLC-DAD
Various analytes and plant extract/fractions stock solutions were prepared in methanol, at a concentration of 10–100 μg/ml. Samples were filtered through 0.45 μm membrane filter (Sortolon polymide; Sortorious). All samples were prepared freshly and used immediately for analysis or stored at 4 °C if not analyzed for more than 1 h.
SC is for standard compound
The concentration of standard compound in each fraction was expressed as μg/100 of dry plant powder.
All assays were performed in triplicates and results are expressed as mean ± SEM. Data of in vitro antioxidant and anticancer assays was analyzed with help of computerized Graph pad prism software to determine the EC50 and IC50 values. For analyzing the differences among EC50 values of different fractions in different antioxidant assays, a Completely Randomized AOV followed by Tukey HSD All-Pairwise Comparison Test was used, alpha set at 0.05 as a level of significance using Statistix 8.1 software. Correlation analysis was performed to determine the correlation between EC50 of various antioxidant assays and total phenolic and total flavonoid content.
Results and discussion
Extraction yield, total phenolic and flavonoid content in A. hydaspica methanol extract and its soluble fractions
Extraction yield (%)
TPC (mg gallic acid equivalent/g dry sample)
TFC (mg rutin equivalent/g dry sample)
87.6 ± 1.23c
127 ± 0.52c
57.7 ± 1.17e
71 ± 0.86a
120.3 ± 1.15b
129 ± 1.32b
36.0 ± 0.95f
34.5 ± 1.13d
129 ± 2.98a
139 ± 1.04a
73.3 ± 1.53d
95 ± 0.05c
Qualitative phytochemical screening
Phytochemical constituents of A. hydaspica methanol extract and its fractions
Total phenolic and flavonoid content
The profile of TPC and TFC in AHM and its derived fractions was determined from the standard calibration curve of gallic acid (R2 = 0.92) and rutin (R2 = 0.91) respectively. TPC varied widely, ranging from 36.0 ± 0.95 to 139 ± 1.04 mg gallic acid equivalent/g dry sample in the extract/fractions of A. hydaspica. However TFC varied from 34.5 ± 1.13 to 129.0 ± 2.98 mg rutin equivalent/g of dry sample. AHB showed the highest concentration (P <0.05) of TPC and TFC, followed by AHE > AHM > AHA > AHH > AHC (Table 2). So it is evident from the present data that AHB and AHE are the best solvents for fractioning polyphenol constituents, due to their polarity index and the best solubility for the type of metabolites in A. hydaspica. The results obtained in this study were in line to those of Sultana et al. , where the highest concentration of TPC had been determined in the bark of Acacia nilotica.
Antioxidant activity assessment
EC50 values of different antioxidant activities of extract and derived fractions of A. hydaspica
Hydrogen peroxide radical
Iron chelating power
β-carotene bleaching inhibition
Total antioxidant index at 250 μg/ml
Lipid peroxidation inhibition
19.5 ± 0.43a
43.0 ± 0.37b
39.0 ± 0.44a
40.3 ± 0.26a
193 ± 0.26d
41.7 ± 0.34a
86.4 ± 0.62d
1.6 ± 0.69b
156.1 ± 0.76d
46.5 ± 0.55b
59.3 ± 0.36c
42.3 ± 0.29b
80.6 ± 0.43c
401 ± 0.23e
91.7 ± 0.17b
173.5 ± 0.85d
1.2 ± 0.35c
562.3 ± 0.55g
18.0 ± 0.45a
38.3 ± 0.40a
35.7 ± 0.40a
37.2 ± 0.36a
173 ± 0.11c
40.3 ± 0.21a
48.4 ± 0.55b
1.7 ± 0.61b
61.3 ± 0.52c
65.0 ± 0.36c
90.8 ± 0.55d
63.0 ± 0.09c
115.6 ± 1.51d
600 ± 0.26e
130.1 ± 0.17d
260.6 ± 1.05f
0.7 ± 0.73e
467.3 ± 0.60f
16.7 ± 0.43a
49.0 ± 0.41b
33.0 ± 0.16a
42.0 ± 0.11a
98.0 ± 0.12a
39.0 ± 0.26a
66.2 ± 0.72c
1.7 ± 0.41a
50.0 ± 0.61b
39.5 ± 0.51b
46.3 ± 0.52b
47.0 ± 0.12b
68.1 ± 0.36b
478 ± 0.12e
103.0 ± 0.12c
131.6 ± 1.15e
1.3 ± 0.14e
401.1 ± 0.12e
15.0 ± 0.41a
35.0 ± 0.21a
61.0 ± 0.21b
1.8 ± 0.24b
32.3 ± 0.28a
29.0 ± 0.16a
43.5 ± 0.86a
37.7 ± 0.3a
40.4 ± 1.01a
59.3 ± 0.98c
50.11 ± 0.59b
DPPH radical scavenging activity
DPPH free radical quenching test is one among the most widely employed procedures to assess antioxidant potency of plant and biological samples . In the current testing; AHM, AHB and AHE depict appealingly greater DPPH quenching efficacy as compared to all other tested fractions and displayed lower EC50 values, which were non-significantly different from one another. The EC50 values in DPPH assay range from 16.7 ± 0.43–65 ± 0.36 μg/ml. AHB and AHE showed the lowest EC50 values (16.7 ± 0.43 and 18 ± 0.45 μg/ml), which were comparable to EC50 of reference compound (Ascorbic acid). DPPH radical scavenging activity of A. hydaspica extract and its various fractions showed good correlation with TPC (R2 = 0.9879) and TFC (R2 = 0.8477). Results of present investigation imply that A. hydaspica contain phyto-constituents that are proficient of donating hydrogen to a free radical in order to rescue the potential impairment. Most specifically, phenolic and flavonoid exhibit antioxidant activity due to the hydroxyl group attach to the aromatic ring which is capable donating electron and stabilizing the free radicals. The research conducted by Sultana et al.  in A. nilotica and Singh et al.  on Acacia auriculiformis revealed similar results.
Superoxide radical scavenging activity
Although superoxide anion is a weak oxidant, but it lead to the generation of powerful and hazardous hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative stress. Therefore, it is very important to study the scavenging of superoxide anion . The EC50 values in superoxide scavenging activities were in the order of AHE < AHM < AHB < AHA < AHH < AHC. When compared to ascorbic acid, the superoxide scavenging activity of the AHE was found to be statistically (P >0.05) similar. The potent electron scavenging ability of the methanol extract and its various fractions might be due its bioactive phytoconstituents like that are able to minimize the oxidation of biological macromolecules [26, 37]. A significant correlation was detected with TPC (R2 = 0.7909, P <0.05) while nonsignificant correlation (R 2 = 0.641, P >0.05) was observed with TFC. This strong superoxide radical neutralizing capacity of A. hydaspica might be functional therapeutically against oxidative stress induced ailments.
Hydroxyl radical scavenging activity
The evidence of OH radical scavenging activity by A. hydaspica extract and its fractions was determined by measuring the inhibition of 2-deoxyribose degradation by the free radicals generated during Fenton reaction. Crude methanol extract and derived fractions markedly scavenged OH radicals and prevented the degradation of 2-deoxyribose. A dose dependent mode was observed for hydroxyl radical scavenging activity. The lowest EC50 values were shown by AHB (36.0 ± 0.16 μg/ml) followed by AHE and AHM (37.7 ± 0.40 and 40.0 ± 0.44 μg/ml respectively). However EC50 values were significantly different from standard gallic acid. A significant correlation was observed with TPC (R2 = 0.844, P <0.01) and TFC (R2 = 0.776, P <0.05). The strong antioxidant activity of AHB and AHE might be utilized as a source of natural antioxidant in oxidative stress for minimizing the detrimental effects of hydroxyl radical in the body. A high scavenging activity of AHE for OH radical was reported in one of previous studies done in our lab.
Hydrogen peroxide radical scavenging activity
In the body, H2O2 is rapidly decomposed into oxygen and water and this may produce hydroxyl radicals (•OH) that can initiate lipid peroxidation and cause DNA damage . Therefore, the ability of plant extracts to scavenge hydrogen peroxide was also determined in order to get the idea that whether samples have same pattern of activity as OH radical reducing ability. Methanol extract/fractions of A. hydaspica possess significant ability to quench the hydrogen peroxide radicals, demonstrating the antioxidant potential of the plant. AHE proved to be efficient fraction against hydrogen peroxide (EC50 = 37.2 ± 0.36 μg/ml). EC50 values showed significant correlation with both TPC (R 2 = 0.844, P <0.01) and TFC (R2 = 0.776, P <0.05), attributing the activity to the occurrence of polyphenolic compounds that give electrons to hydrogen peroxide, thus neutralizing it into water. These findings are in line with the previous study.
ABTS radical scavenging assay
In this assay, the reaction of ABTS with potassium persulfate in the existence of hydrogen-donating antioxidants outcomes in the formation of ABTS+ blue/green chromophores, this reduction reaction is noted spectrophotometrically at an absorbance of 745 nm. This scheme of determination of antioxidant action is equally pertinent to hydrophilic and lipophilic classes of antioxidants like; flavonoids, hydroxycinnamates, carotenoids and antioxidants in the plasma . The result obtained indicated that AHM and its derived fractions scavenge the ABTS radicals in a dose dependent pattern. Among the extract/fractions lowest EC50 values for ABTS radical scavenging were determined for AHB (98.0 ± 0.1 μg/ml) while highest EC50 values were recorded for AHC (>500 ± 0.26 μg/ml) as shown in Table 1. However EC50 values of AHB were significantly lower than ascorbic acid (61 ± 0.2 μg/ml). The ABTS scavenging activity of the present study suggests that the phyto-constituents within the extract and various fractions of A. hydaspica might donate electron/hydrogen while minimizing the oxidative stress. Furthermore correlation analysis indicated significant correlation between ABTS radical scavenging activity and TPC (R2 = 0.881, P <0.001) as well as TFC (R2 = 0.857, P <0.001). The results are in line with the study of khan et al. .
Chelating activity on Fe2+
An imperative mechanism of antioxidant activity is the ability to chelate/counteract transition metals, which have the ability to demolish hydro peroxides and Fenton-type reactions. Therefore it was considered important to screen the iron (II) chelating ability of extract/fractions. The sequence for chelating power was AHB ~ AHE ≥ AHM > AHH > AHA > AHC. The EC50 values of AHB, AHE and AHM were close to the EC50 value of standard catechin. Correlation analysis suggested that the iron (II) chelating possessions of A. hydaspica may be accredited to its endogenous chelating agents like polyphenolic compounds . As some phenolic compounds have properly oriented functional groups, which can chelate metal ions to protect against oxidative damage . Chelating activity was correlated well with the phenolic (R2 = 0.8971, P <0.001) and flavonoid content (R2 = 0.8177, p <0.05) Our results are in good agreement with previous report where ethyl acetate fraction possessing polyphenolic constituents of A. auriculiformis showed highest metal chelating power .
β-carotene bleaching inhibition
In this test, antioxidant competence was evaluated by quantifying the inhibition of the conjugated diene-hydroperoxides and the volatile organic compounds creation as an outcome of linoleic acid oxidation. Hence, the antioxidant occurrence can impede the magnitude of β-carotene bleaching by counterbalancing the linoleate and other free radicals formed in the process. The color of reaction solution was retained for a long time in the presence of an antioxidant compound while a rapid decrease in absorbance was noticed in the absence of antioxidant . The antioxidant activity of A. hydaspica extract/fractions with regard to the β-carotene bleaching method could be ranked as AHE > AHB > AHM > AHA > AHH > AHC. The EC50 values of AHE (48.4 ± 0.55 μg/ml) were significantly different yet comparable with standard catechin (EC50 = 40.4 ± 1.01 μg/ml). However AHE showed better efficacy as compared to standard BHT. The differential efficacy of A. hydaspica extract/fractions to inhibit oxidation of linoleic acid emulsion is an indication of the complexity of the extract/fractions as well as potential interaction between the extract and emulsion components. Correlation analysis indicate significant correlation with phenolic (R2 = 0.9670, P <0.0001) and flavonoid (R2 = 0.8831, P <0.001) content. The correlation of phenolic and flavonoids with β-carotene bleaching inhibition potential was reported by other researchers as well [26, 50].
Anti-lipid peroxidation potential
Lipid peroxidation is involved in a number of pathological conditions so evaluation of antioxidant potential of natural and synthetic compounds requires an assay in lipid system too. The EC50 values in lipid peroxidation inhibition were in the order of AHB < AHE < AHM < AHA < AHC < AHH. EC50 values showed by AHB (50 ± 0.61 μg/ml) were significantly lower as compared to BHA values (EC50 = 59.3 ± 0.98 μg/ml). In this study the in vitro ability of plant extract/fractions to prevent the production of TBARS depicts the potential of samples to inhibit oxidation in lipid system. Significant correlation was observed with TPC (R 2 = 0.779, P <0.05) and TFC (R 2 = 0.836, P <0.05). Phenolic compounds are very important plant constituents because they exhibit antioxidant activities by inactivating lipid free radicals, or by preventing the decomposition of hydro-peroxides into free radicals. Results of lipid peroxidation were in line with the previous study of Singh et al. .
Total antioxidant capacity assay
The total antioxidant capacity of the extract/fractions was measured by phosphomolybdenum method based on the reduction of molybnedum (VI) to molybnedum (V) by the antioxidant action of extract and the subsequent formation of a green phosphate Mo (V) complex at acid pH of the medium with maximum absorbance at 695 nm . Total antioxidant capacity of the extract/fractions recorded at the highest dose of 250 μg/ml was in order of AHB (1.7 ± 0.015) ~ AHE (1.7 ± 0.08) > AHM (1.5 ± 0.016) > AHA (1.3 ± 0.05) > AHH (1.2 ± 0.02) > AHC (0.738 ± 0.012). The current analysis reveals that AHB and AHE displayed the uppermost antioxidant capacity. Latest researches proved that flavonoids and related polyphenols contributes substantially to the phosphomolybdate quenching capability of medicinal plants [46, 52]. AHB and AHE exhibited the highest antioxidant index comparable with ascorbic acid. Phosphomolybdenum assay in general detects antioxidants such as carotenoids, ascorbic acid, \( \alpha \)-tocopherol, and some phenolic, cysteine, and aromatic amines due to hydrogen and electron donating ability. The antioxidant capacities of the extract/fractions have a strong relationship with the solvent employed, mainly due to the different antioxidant potential of compounds with different polarities. Phytochemical analysis reveals the presence of various boiactive phytochemicals that might be attributed to the antioxidant capacity of A. hydaspica. Our result correlate well with the research of Tung at al. reporting gallic acid, catechin, myricetin along with other polyphenols in A. confusa leaves extract were responsible for the significant antioxidant potential .
Anti-hemolytic activity of A. hydaspica methanol extract and its soluble fractions against H2O2 induced hemolysis
Positive control (A)
Negative control (B)
Optical densityC at 560 nm concentration (μg/ml)
% Inhibition of hemolysis at 1000 (μg/ml)
1.45 ± 0.02
0.11 ± 0.05
0.69 ± 0.01
0.85 ± 0.05
0.96 ± 0.05
1.15 ± 0.01
1.45 ± 0.03
0.11 ± 0.05
0.65 ± 0.05
0.71 ± 0.05
0.88 ± 0.05
0.98 ± 0.05
1.45 ± 0.05
0.11 ± 0.05
0.51 ± 0.05
0.63 ± 0.03
0.71 ± 0.05
0.87 ± 0.02
1.45 ± 0.06
0.11 ± 0.05
0.72 ± 0.04
0.89 ± 0.09
1.14 ± 0.01
1.42 ± 0.02
1.45 ± 0.04
0.11 ± 0.05
0.67 ± 0.05
0.81 ± 0.05
0.92 ± 0.05
1.29 ± 0.03
1.45 ± 0.07
0.11 ± 0.05
0.49 ± 0.05
0.59 ± 0.05
0.64 ± 0.05
0.79 ± 0.05
1.45 ± 0.08
0.11 ± 0.05
0.72 ± 0.04
0.89 ± 0.09
1.14 ± 0.01
1.43 ± 0.02
The outcome of the current experiment presents the occurrence of primary antioxidants, which possess anti-hemolytic effect.
Assessment of anticancer potential by MTT assay
Cytotoxic effect of A. hydaspica methanol extract and its derived fractions on MDA-MB 361, HCC-38 and Vero cells of green monkey after 48 h of treatment
MDA361 cell line
HCC38 cell line
Vero cell line
46.9 ± 1.31*
75.9 ± 1.32*
254 ± 1.81
29.9 ± 0.91*
39.5 ± 0.87*
294 ± 1.55
37.1 ± 1.01*
56.1 ± 0.93*
258 ± 1.68
Retention time, optimized signal wavelength, and regression analysis of reference flavonoids determined by HPLC-DAD analysis
Retention time (min)
y = 3.0565x + 8.01
y = 1.7773x−48.49
y = 1.1764x + 7.96
y = 12.569x + 22.66
y = 12.950x−52.12
y = 2.560x−15.09
y = 10.967x + 125.59
y = 4.8624x + 35.01
HPLC-DAD profile of A. hydaspica ethyl acetate and n-butanol fractions
Quantity (μg/100 mg dry powder)
The standards were selected on the basis of their reported medicinal properties, for instance; catechin is an important phenolic compound with diverse beneficial health effects and its metabolites have shown therapeutic potential as antioxidant, anti-apoptotic, inhibit proliferation of breast cancer cells, block carcinogenesis and its effect is more pronounced in cancer cells as compared to normal cells . Gallic acid (GA) possesses potent antitumoral and antioxidant properties. The research conducted by Ali et al. also illustrate that gallic acid and polyphenols in the acetone extract of A. nilotica, are responsible for cytotoxic activity . Myricetin is also able to induce apoptosis of pancreatic cancer cells, human bladder carcinoma cell line, trigger apoptosis, regression of tumor growth, decrease metastasis and it increase bioavailability of tamoxifen, a drug used to treat breast cancer .
Our result correlate well with the research of Tung at al., reporting gallic acid, catechin, myricetin along with other polyphenols in ethyl acetate fraction of A. confusa leaves extract were responsible for the significant antioxidant and anticancer potential . This calls for further studies on the active components for proper assessment of their chemotherapeutic properties as well as their possible development as promising anticancer drugs.
The present study demonstrates the phytochemical profiling, in vitro antioxidant, anti-hemolytic and cyto-selective anticancer activity of A. hydaspica aerial parts extracts. Extracts with higher antioxidant capacity also had higher polyphenol content. It can be concluded that the extract obtained using higher polarity solvents were more effective radical scavengers then those obtained using less polar solvents. Ethyl acetate and n-butanol showed better characteristics as solvent for phenolic compounds. Furthermore these fractions tended to possess superior activity in lipid peroxidation inhibition and β-carotene bleaching assay as compared to BHA and BHT. Therefore, they might be used as preservative ingredients in the food and/or pharmaceutical industry. Moreover safety profile and chemotherapeutic potential of active fractions and methanol extract was determined by assessing the anti-hemolytic activity and in vitro testing against both cancer and normal cell lines. Bioactive compounds present in A. hydaspica active fractions might work synergistically and specifically in inhibiting proliferation of breast cancer cells with high SI value, suggesting that they might be used as a natural additive in human diets for cancer chemoprevention. However the evaluation and the discovery of new anticancer agents is long-term process that encompasses many steps by step approaches with the screening for anticancer properties, followed by the isolation and identification of bioactive compounds and finally in vivo anticancer activity testing in order to verify the aptitude of the compounds. Therefore further research would be required before such uses could be proposed with confidence.
ABTS, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid; AHA, Acacia hydaspica residual aqueous fraction of methanol extract of aerial parts; AHB, Acacia hydaspica n-butanol fraction of methanol extract of aerial parts; AHC, Acacia hydaspica chloroform fraction of methanol extract of aerial parts; AHE, Acacia hydaspica ethyl acetate fraction of methanol extract of aerial parts; AHH, Acacia hydaspica n-hexane fraction of methanol extract of aerial parts; AHM, Acacia hydaspica methanol extract of aerial parts; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DMEM/F12, Dulbecco’s modified eagle medium: nutrient mixture F-12; DPPH, 2,2-Diphenyl-1-Picrylhydrazyl; EDTA, ethylene diamine tetra acetic acid; PBS, phosphate buffer saline; PMS, phenazine methosulphate; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TFC, total flavonoid content; TPC, total phenolic content
We acknowledge Higher Education Commission (HEC) of Pakistan for awarding indigenous scholarship and IRSP scholarship for University of Minnesota, USA to the first author. Furthermore we acknowledge Deanship of Scientific Research at King Saud University for its technical assistant in this research project.
The project was partially funded by the Higher Education Commission (HEC) of Pakistan by awarding indigenous scholarship to the first author. We are grateful to the Deanship of Scientific Research at King Saud University for its funding of this research through Research Group Project number 193.
Availability of data and materials
All the data is contained in the manuscript.
TA made significant contributions to conception, design, experimentation, acquisition and interpretation of data and writing of manuscript. SR, MRK, SM, AA and MS made substantial contribution in interpretation of data and revising the manuscript for intellectual content. IUH made a contribution to the HPLC experimentation and analysis. All authors read and approved the final manuscript.
TA did PhD in Biochemistry/Pharmacological biology from Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan. SR is PhD Scholar from Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad and Researcher at the Department of Community Health Sciences, College of Applied Medical Sciences, Clinical nutrition program, King Saud University, Riyadh KSA. MRK did his Diploma in Unani Medicine and Surgery (DUMS) and is a registered practitioner of the National Council for Tibb of Pakistan. He is working as Associate Professor at the Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan. SM did MPhil in biochemistry/Pharmacological biology from Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan. AA is associate professor at Department of Community Health Sciences, College of Applied Medical Sciences, Clinical nutrition program, King Saud University, Riyadh KSA. MS did PhD in Biochemistry/Pharmacological biology from Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan. IUH is assistant professor at the Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study makes use of rats and human blood, and the experimental protocol for the use of animal and human blood was approved (Bch#0256) by the ethical board of Quaid-i-Azam University, Islamabad Pakistan.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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