Antioxidant and immunostimulatory activities In Vitro of Polysaccharides from Pomegrante Peels.
Summary: In the present study, the crude polysaccharides from pomegranate peels(CPP) were prepared by water-extraction technology. In vitro antioxidant assay, CPP showed strong inhibition of lipid peroxidation and reducing ability, moderate 1,1-diphenyl-2-picryldydrazyl(DPPH) radical scavenging activity. The immunostimulatory activity of CPP was also evaluated by using in vitro cell models. The results demonstrated that CPP could promote the splenocyte proliferation, increase the activity of acid phosphatase in peritoneal macrophages and strengthen peritoneal macrophages to devour neutral red in vitro.
Keywords: Pomegranate peels; Polysaccharide; Antioxidant activity; Immunostimulatory activity; In vitro
Polysaccharides, distributed widely in animals, plants and microorganisms, have been demonstrated to play an important role as free radical scavenger in the prevention of oxidative damage in living organism [1-3], and possess potential and potent capabilities of immunostimulatory activity by stimulating immune system and strengthening the specific and non-specific immune response in mice [4-6]. Therefore gradually growing attention has been paid to polysaccharides characterized as antioxidant and immunomodulatory natural products.
Pomegranate, a member of the Punicacea family, is a shrub or a small tree, originating from Asia and now widely cultivated in China. For centuries, the peel of the plant has been used as traditional chinese medicines. Over the last decade, studies indicate that the pomegranate peel possesses a number of biological activities such as antioxidant, immunoregulatory, antibacterial, antiviral, antitumor and anti-inflammatory activities [7-11]. Modern pharmacological experiments have proved that polyphenols is the most active ingredients. Most recent interests have been focused on the polyphenols to explore the pharmacological mechanisms of pomegranate peel. Whereas it is difficult to make significant progress to elucidate the underlying mechanism by which pomegranate take effect only by polyphenols .
Pomegranate peel contains multiple constituents, such as polyphenols, flavonoids and polysaccharides. Polyphenolic compounds, including flavonoids (anthocyanins, catechins and other complex flavonoids) and hydrolyzable tannins
To whom all correspondence should be addressed. (punicalin, pedunculagin, punicalagin, gallagic acid and ellagic acid esters of glucose) . As we know, pomegranate peel contains and more and more attentions have been cast on polyphenols by medical scientists and nutriationists due to their various important biological activities [9, 13]. However, a detailed and comprehensive extraction technology and biological activities of the crude polysaccharides from pomegranate peels(CPP) have not been reported so far. Therefore, evaluation of CPP activity will be important for the elucidation of function and utilization of the polymers. Herein, we report in detail the antioxidant and biological activities of CPP, by using different extracorporeal antioxidant methods and in vitro cell models.
Reagents and Materials
1,1-diphenyl-2-picryl-hy-drazyl (DPPH) was purchased from Sigma Chemical Co. (St. Louis, USA). Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Shanghai Sangon Biological Engineering Technology and Services Co., China. The male Kunming mice were purchased from the Experiment Animal Center of Academy of Military Medical Sciences (Beijing, China). All other chemicals used were ultra pure or analytic grade.
Extraction of Polysaccharides
Ripened pomegranates were obtained from Huaiyuan, Anhui Province of China. The peels were separated manually. The crude CPP was prepared according to our previous method . Briefly, the fresh peels collected were cut into pieces, dried by oven at 60 C and pulverized into powder. A 60-mesh sift was used to screen the powder, then extracted with hot water under the following conditions: extraction time 3h, extraction temperature 80C, solvent/material ratio 15(mL/g) and times of extraction, two times. The extract was filtered through a Whatman No. 1 filter paper. Then, more anhydrate ethanol was added to the filtrate to a final concentration of 80% (v/v). It was kept overnight at 4C to precipitate CPP.
Determination of Antioxidant Activity of CPP in vitro
Assay of Reducing Power
The reducing power of CPP was determined according to the method reported by Oyaizu  with slight modification. One milliliter of CPP was mixed with 1.0 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 1.0 mL of 1% potassium ferricyanide (K3Fe(CN6)). The reaction mixture was incubated in a temperature-controlled water bath at 50C for 20 min, followed by addition of 1.0 mL of 10% trichloroacetic acid. The mixture was then centrifuged at 750g using a centrifuge for 5 min at 25C. The supernatant obtained was treated with 0.2 mL of 1% FeCl3. The absorbance of the reaction mixture was measured as 700 nm. Increase in absorbance was used as the measure of reducing power.
Assay of DPPH Radicals Scavenging Activity
The DPPH radical scavenging activity was measured by using the method reported by Qiao  with slight modification. Briefly, 0.2 ml DPPH radicals(DPPH) solution (400 mol/L in dehydrated alcohol) was added to 1.0 ml of CPP solution, and then 2.0 mL of water was added. The mixture was shaken and allowed to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm against a blank (water instead of sample and DPPH solution). Lower absorbance of the reaction mixture indicates higher free-radical scavenging activity. The scavenging percentage was calculated by the following equation: scavenging percentage activity (%) = [1 -(A1 A2)/ A0]A-100, where A0 is the absorbance of the control (water instead of sample solution), A1 is the absorbance of the sample and A2 is the absorbance of the sample under identical conditions as A1 with water instead of DPPH solution.
Determination of Protective Activity against Fe-Induced Lipid Peroxidation
The inhibition of lipid peroxidation of CPP was determined by quantification of the lipid peroxide decomposition product MDA based on reaction with thiobarbituric acid using egg yolk as oxydable substrate . Briefly, 0.5 mL egg homogenate (10% in 0.2 M PBS (pH7.4), v/v) and 1.0 mL test sample were mixed, then 0.10 mL FeSO4 (0.025 M) was added to initiate lipid peroxidation. After incubation in 37C water bath for 120 min, 1.5 mL of 20% (w/v) acetic acid and 0.10 mL TCA were added to the mixture, then it was shaken vigorously and left at room temperature for 10 min. After that, 1.5 mL of 0.8% (w/v) thiobarbituric acid and 1.1% SDS (w/v) were added to quench the reaction. The mixture was shaken and heated at 95C for 15 min, and then was centrifuged at 10000 rpm for 5 min. The absorbance of the upper layer was measured at 532 nm. The inhibition of lipid peroxidation was calculated as follows: inhibition rate (%) = [1 -( A1 A2)/ A0]A-100,
In which A0 is the absorbance of the control group in the hydroxyl radicals generation system, A1 is the absorbance of the test group and A2 is the absorbance of the samples only.
Determination of Immunostimulatory Activity of CPP in vitro Assay of Splenocyte Proliferation
The assay of splenocyte proliferation was done according to the MTT-based colorimetric method with slight modification . Briefly, male Kunming mice were killed by cervical dislocation and the spleens were removed aseptically. A single spleen cells suspension was prepared by homogenization in 5.0mL RPMI-1640 medium. The suspension was centrifuged to afford cell pellet. The erythrocytes in cell pellet were lysed with Tris-NH4Cl lysing buffer (0.15M NH4Cl and 20mM Tris) for 3 min. The lysed solution was then centrifuged, washed twice with RPMI-1640 medium and adjusted to a density of 1A-107 cells/mL in the RPMI-1640 medium supplemented with FBS (10%), penicillin (100 unit/mL) and streptomycin (100g/mL). The spleen cell suspension was pipetted into 96-well flat-bottom plate (50L/well). Then, group I (normal control group) was treated with RPMI-1640 medium (50L/well). Group II (lower dose), Group III (low dose),
roup VI (medium dose) and group V (high dose) were treated with CPP (50L/well, fial concentration was 12.5, 25, 50, and 100g/mL, respectively). Group VI (positive control group) was treated with ConA (50L/well, final concentration 2.5g/mL). After incubated at 37C in a humidified 5% CO2 incubator for 72 h, MTT solution (10L/well, 5.0 mg/mL) was added and the plate was further incubated for 4 h. Then, 100L of 10% SDS in 0.01N HCl was added to each well and the plate was kept overnight for the dissolution of formazan crystals. The absorbance of each well at 570nm was measured by an ELISA plate reader (TECAN Infinite F200, Switzerland).
Assay of Acid Phosphatase Activity in Peritoneal Macrophages
The activity of acid phosphatase in peritoneal macrophages was determined according to the reported method with minor modification . In brief, sterile 3% thioglycollate medium (50 mL/kg body weight) was injected intraperitoneally into male Kunming mice as a stimulant to elicit peritoneal macrophages. Three days later, peritoneal exudates cells were harvested by a lavage of the peritoneal cavity with 5mL of ice-cold RPMI-1640 medium. The resulting cell suspension was centrifuged (1700rpm for 5min), washed twice with RPMI-1640 medium and adjusted to a density of 2A-106 cell/mL in the RPMI-1640 medium supplemented with FBS (10%), penicillin (100 unit/mL) and streptomycin (100g/mL). The cell suspension was added into a 96-well flat-bottom plate (100L/well) and the cells were allowed to adhere to the bottom of the plate at 37 C in a humidified 5% CO2 incubator for 3 h. Non-adherent cells were removed by washing three times with RPMI-1640 medium.
Then, fresh medium (50L/well, control group) or test sample (50L/well, CPP at a final concentration 12.5, 25, 50, and 100g/mL as lower dose, low dose, medium dose and high dose, respectively) was added to each well and the plate was incubated with macrophages at 37 C for 24 h. The culture medium was removed by rapid inversion and flicking of the plate. The macrophage monolayer in each well was solubilized by addition of 1% Triton X-100 (25L). Thereafter, 150L freshly prepared p-nitrophenyl phosphate (1 mg/mL) in 0.1Mcitrate buffer (pH 5.0) was added as a substrate for acid phosphatase, and the plate was incubated at 37 C for 1 h. The reaction was stopped by addition of 50L of 3.0M NaOH solution, and the absorbance (Abs) of the culture well was measured at 405nm using an ELISA plate reader. The stimulating index of acid phosphatase activity was calculated by the following equation:
Acid phosphatase activity =(Abssample - Abscontrol)/Abscontrol
Assay of Phagocytosis of Peritoneal Macrophages
Phagocytosis of peritoneal macrophages was measured according to the reported method with modification . Briefly, the peritoneal macrophages were prepared, seeded in a 96-well flat-bottom plate and allowed to adhere as done as previously described. Non-adherent cells were removed by washing three times with RPMI-1640 medium. Then, fresh medium (50L/well, control group) or test sample (50L/well, CPP at a final concentration 12.5, 25, 50, and 100g/mL as lower dose, low dose, medium dose and high dose, respectively) was added to each well and the plate was incubated at 37 C in a 5% CO2 incubator for 48 h. Then, neutral red solution (1%, 100L/well) was added to the cell plate. After further incubation for 60 min, the cells were washed three times with RPMI-1640 medium to remove excess neutral red. A mixture of glacial acetic acid (1%) and ethanol (1:1) was added to each well (100L/well), and the cell plate was incubated again at 37 C for 15 h.
Finally, the Abs of each well was measured at 540nm by an ELISA plate reader.
Analysis of the experimental data and one-way ANOVA were performed using the SPSS 16.0 for windows (SPSS, Chicago, IL, USA). Multiple comparisons of means were done by the least significance difference (LSD) test. p-Values of less than 0.05 were regarded as significant.
Results and Discussion
The Antioxidant Activity of CPP in vitro Reductive Potential of CPP
Various mechanisms, including reducing capacity, prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging have been reported to explain the antioxidant activities of polysaccharides . The reductive capacity of a compound may serves as a signicant indicator of its potential antioxidant activity . Fig. 1A shows that the reductive potential of capsule polysaccharides exhibited a dose-dependent activity within the test concentration range of 0-1250 g/mL. And the reductive powers of CPP increased significantly (p less than 0.01) with the increase of sample concentration.
DPPH Radicals Scavenging Activity of CPP
The DPPH free radical is a stable free radical, which has been widely accepted as a tool for estimating the free radical-scavenging activities of antioxidants . DPPH radical scavenging activity of the CPP was evident at all of the tested concentrations, but lower than that of ascorbic acid (Fig. 1B). The scavenging effect increased with the increase of concentration up to 1250 g/mL. The highest DPPH radical scavenging activities were 35.91% and 75.02% for CPP and ascorbic acid respectively at the concentration of 1250 g/mL. CPP showed weaker DPPH radical scavenging activity than ascorbic acid at the range of 0-1250 g/ml (pless than 0.05).
Protective Activity Against Fe-induced Lipid Peroxidation of CPP
Lipid peroxidation is a common consequence of free radical-mediated chain reactions and some of its end-products such as lipid hydroperoxides and various species have unpaired electrons or the ability to attract electrons from other molecules. All of the end-products mentioned above can damage DNA directly or indirectly . The effect of CPP on non-enzymatic peroxidation was shown in Fig. 1C. In the egg yolk lipid peroxidation system, CPP and ascorbic acid inhibited lipid peroxidation in a concentration-dependent manner. The inhibitory rates of lipid peroxidation were 77.12% and 87.86% for CPP and ascorbic acid respectively at a concentration of 1120 g/mL, indicating that both CPP possessed high lipid peroxidation inhibiting ability.
The Immunostimulatory Activity of CPP In Vitro Effect of CPP on Splenocyte Proliferation
The lymphocyte-mediated immunity plays an important role in the cellular and humoral immune responses. The capacity to elicit an effective T- and B-lymphocyte immunity can be shown by the stimulation of lymphocyte proliferation response . Therefore, we investigated the effect of CPP on splenocyte proliferation. The stimulating indexes of CPP were all exceed 0.9, indicating that CPP sample could promote the proliferation of splenocyte (Fig. 2A). In addition, the promoting effects significantly increased (p less than 0.05) with the increase of sample concentration ranging from 12.5 to 100g/mL. At a concentration of 50g/mL and 100g/mL, the stimulating index of CPP was 1.57 and 1.48, respectively. And they were higher than the stimulating index of positive control group(1.45) treated with Con A, which is commonly used as T cells mitogen.
Spleen is one of the major immune organs. There are T-lymphocyte(40%) and B-lymphocyte(60%) in spleen, and splenocyte proliferation is related to immunity improvement of T-lymphocyte or B- lymphocyte, especially the T-lymphocyte. The present results indicated that CPP had directly T cells and B cells mitogen effect on mouse splenocytes and could stimulate proliferation of spleen T lymphocyte and B lymphocyte, strengthen immunological response and improve immunity. And CPP tend to stimulate the immune suppression of B cell mediated immune response.
Effect of CPP on Acid Phosphatase Activity in Peritoneal Macrophages
Acid phosphatase, a marker enzyme of lysosome, is signal enzyme ofmacrophage activation. The activity of acid phosphatase in macrophages increases with the activation of macrophages and decreases with the inhibition of macrophages. Therefore, the immune function of macrophages can be represented by the activity of acid phosphatase . In the present study, we found that the activities of acid phosphatase in macrophages increased significantly by CPP in a dose-dependent manner (12.550 g/mL, Fig. 2B), while high dose of CPP (100g/mL) had the weaker stimulating activity than medium dose of CPP (50g/mL).The results indicated that CPP sample possessed the ability of stimulating or immunosuppressive acid phosphatase in peritoneal macrophages in a dose-dependent manner.
Macrophages, one of the major immunocyte, possess many immunefunctions, such as phagocytizing extraneous material, processing antigen, secreting cytokine and so on. Macrophages are known to be the first defense line in preventing foreign invasion . Usually, macrophages are in a state of dormancy and its phagocytosis is weaker. But macrophages may be activated by many immunoenhancers. After activation, macrophages can inhibit the growth and transferring of tumor cells and may have stronger immune functions of endocellular sterilization, exocellular oncolysis, phagocytosis and pinocytosis. The present results suggested that CPP could have the function of activating macrophages due to its activation on acid phosphatase in peritoneal macrophages. Therefore, CPP might practice improving the immunity by activating macrophages and strengthening acid phosphatase activity.
Effect of CPP on Devouring Neutral red of Peritoneal Macrophages
One of the immune functions of macrophage is devouring extraneous material. Macrophages phagocytize extraneous materials to produce phagosome and then scavenge those extraneous matters by using some enzymes including acid phosphatase. Thus, the immune activity of macrophages can be represented indirectly by its devouring percentage. The phagocytosis indices of CPP on devouring neutral red of peritoneal macrophages were all less than 1.0 (Fig. 2C), indicating that all the CPP samples had the moderate activities of promoting peritoneal macrophages to devour neutral red. Furthermore, the promoting effects increased with the increase of sample concentration ranging from 12.5 to 50g/mL. Notably, CPP (50g/mL) presented the strongest promoting effect, while high dose of CPP (100g/mL) had the weaker stimulating activity than medium dose of CPP (50g/mL).
The results suggested that CPP might practice improving immunity by strengthening macrophages phagocytosis and CPP also might be involved in activating both the adaptive and innate immunities to different extent . Therefore, the results mentioned above that CPP had much stronger immuno-enhancing activity in vitro.
In the present study, the antioxidant properties of crude polysaccharides from pomegranate peels(CPP) were demonstrated by using a variety of testing systems in vitro, CPP showed strong inhibition of lipid peroxidation and reducing ability, moderate 1,1-diphenyl-2-picryldydrazyl radical scavenging activity. Furthermore, the immunostimulatory activity of CPP was also evaluated by using in vitro cell models.
The results demonstrated that CPP could promote the splenocyte proliferation, increase the activity of acid phosphatase in peritoneal macrophages and strengthen peritoneal macrophages to devour neutral red in vitro. The results indicate that CPP may be a source of natural antioxidants and immunomodulator. Further works on the structure and biological activity in vivo of CPP is in progress.
This work was supported by Anhui Provincial Natural Science Foundation (1408085MH209) and Universities Natural Science Research Project of Anhui Province(KJ2012B094). The authors declare that there are no conflicts of interest.
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|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Feb 28, 2015|
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