INTRODUCTION:

Inflammation is a complex biological response of the immune system to various harmful stimuli, such as bacteria, viruses, trauma, damaged cells or chemical irritants¹. The main signs of inflammation include pain, swelling, heat, redness and loss of function². Although inflammation is a defensive mechanism that eliminates the effect of injurious agents, uncontrolled inflammation can lead to tissue damage, organ failure and chronic diseases including autoimmune, cardiovascular and neurodegenerative diseases and cancer^3,4. Different pro-inflammatory cytokines such as interleukin-1 beta (IL-1β), interleukin 6 (IL-6) and tumor necrosis alpha (TNF-α) are produced by some types of immune cells especially neutrophils and macrophages during the inflammatory process⁵.

During inflammation, lysosomes existed in the activated neutrophils release their components such as protease and bactericidal enzymes that produce various orders⁶. The extra cellular activity of these enzymes causes further tissue damage and contributes in acute and chronic inflammation^7,8.

Therefore, the stabilization of lysosomal membrane can limit the inflammatory response⁹. As RBC membrane is similar to the lysosomal membrane components, RBC membrane stabilization method is usually used to estimate the anti-inflammatory activity in-vitro^10,11.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most- frequently used drugs in inflammatory diseases¹². However, NSAIDs are known to possess variety of adverse effects including gastrointestinal toxicity, hypertension and renal failure, in addition to prolongation of bleeding time¹³. Use of NSAIDs is also found to be associated with an increased risk of acute myocardial infarction¹⁴. Thus, there is demand for development of new anti-inflammatory agents with fewer adverse effects and better safety profile¹⁵. Of the natural sources, phenolic compounds produced in plants as secondary metabolites and normally consumed in the diet were suggested to possess anti-inflammatory effect^16,17. More than 8000 plant- derived Phenolic compounds have been reported¹⁸. There are different families of phenolic compounds such as flavonoids, phenolic compounds, tannins and stilbenes¹⁹. Phenolic compounds promote immune system and described to have various pharmacological properties including anti-oxidant²⁰, anti-inflammatory²¹, antimicrobial²², anti-viral²³ and anti-cancer²⁴ activities.

Fruit juices are considered as a rich source of phenolic compounds²⁵. Literature about the anti- inflammatory role of dietary phenolics is lacking²⁶. Hence, this study is meant to evaluate the anti-inflammatory activity of phenolic compounds in their natural form. Black mulberry, pomegranate and orange juices were selected to study their anti-inflammatory activity based on their total phenolic content.

MATERIALS AND METHODS:

Instrument:

Centrifuge (Labofuge 200 Heraeus), Spectrophotometer (Jasco V-530 UV), Laboratory oven (Carbolite), Water bath (K and H Industries), Analytical balance (RADWAG, AS 220/C/2).

Chemicals:

Folin-Ciocalteu reagent was purchased from Sigma- Aldrich, Switzerland. Gallic acid was purchased from Biotech LTD, India. Sodium Carbonate was obtained from BDH, England. Sodium mono and di phosphate were purchased from Labocheme, India.

Samples preparation:

Fresh pomegranate, orange and black mulberry fruits were obtained from the local countryside. Juice samples were extracted according to Dussossoy et al. (2009)²⁷. The fruit was washed and squeezed and the resulted juice was then filtered and stored at -20°C until analysis.

Total Phenolic compounds determination:

The total phenolic content of each juice sample was determined using the Folin-Ciocalteu Method described by Vermerris and Nicholson, (2006)²⁸. Juice samples were diluted properly in distilled water prior to analysis. 2ml of freshly prepared 2% W/V Sodium Carbonate was added to 0.1ml of the diluted juice and mixed well. After 5 minutes, 0.1ml of a 1:1 dilution of Folin-Ciocalteu reagent was added. The reaction mixtures were allowed to stand for 30 minutes at room temperature before the absorbance was measured at 750nm. Distilled water served as a blank and gallic acid was used as a standard phenolic compound. A standard curve of gallic acid solutions in concentrations between (0.1-0.6g/l) was used to calculate the total phenolic content in each juice. The results were expressed as gallic acid equivalents (g_GAE/l). The method was performed in triplicate for each juice.

Red Blood cells (RBCs) membrane stabilization method:

The anti-inflammatory activity of each juice sample was evaluated depending on the method of sakat et al. (2009)²⁹ with minor modifications.

Preparation of RBCs suspension:

Fresh whole blood (5ml) was collected intravenously from healthy human volunteer who had not taken any of the NSAIDs for two weeks prior to the experiment and transferred to the heparinized tubes to prevent coagulation. The tubes were centrifuged at 3000rpm for 10 minutes and RBCs pellet were washed three times with equal volume of normal saline (0.9% NaCl). After that, 5% V/V RBCs suspension was made using normal saline.

Heat induced haemolysis:

Each juice was diluted with phosphate buffer (pH=7.4) prior to the experiment. The reaction mixture (2 ml) consisted of 1ml diluted juice sample of various phenolic compounds concentrations (10, 20, 30, 40, 50, and 60 µg/ml) and 1ml of 5% RBCs suspension. The control test tube contained normal saline (0.9% NaCl) instead of the sample. Ibuprofen was used as a reference drug and was prepared at the same concentrations of the sample using phosphate buffer (pH=7.4). All the tubes were incubated in water bath at 56°C for 30 minutes. The tubes were cooled under running tap water at the end of the incubation. The reaction mixture was centrifuged at 2500rpm for 5 minutes and the absorbance of the supernatant (hemoglobin) was measured at 560nm. The experiment was performed in triplicate for each juice and the percentage inhibition of Haemolysis was calculated by using the following formula:

(Abs control –Abs sample) X 100/ Abs control

Statistical analysis:

All the results were presented as mean ± standard deviation. The differences between juice samples and standard drug were analyzed using Student’s t-test. Differences were considered to be significant at p value < 0.05. All statistical analyses were performed using the Microsoft Excel 2016 Software.

RESULTS AND DISCUSSION:

Total phenolic compounds determination:

Total phenolic contents of black mulberry, pomegranate and orange juices are presented in table 1. The highest total phenolic content of 3.02 g_GAE/l was found in black mulberry juice. Al Asaad and Al Diab, (2017)²⁵ reported that black mulberry juice exhibited the highest phenolic content (ranged from 8.6 g_GAE/l to 11.2 g_GAE/l) among twelve types of fruit juices available in Syria.

Pomegranate juice content of phenolic compounds was 1.83 g_GAE/l. The resulted concentration was slightly higher than that reported by Gözlekçi et al. (2011)³⁰ which varied between (0.78-1.55 g_GAE/l). However, higher result was obtained by Anahita et al. (2015)³¹ which was 2.5 g_GAE/l.

Orange juice showed the lowest phenolic content among the studied juices as the concentration was 0.76 g_GAE/l. This finding was in accordance with Rapisarda et al. (1999)³² which ranged from 0.36 g_GAE/l to 1.14 g_GAE/l.

The differences in total phenolic content between our results and those in the literature can be attributed to several factors including cultivar, maturity stage, analytical method and environmental conditions³³.

Table 1: Total phenolic content in the studied juices

Juice type	Phenolic content (g_GAE/l)
Black mulberry juice	3.02 ± 0.08
Pomegranate juice	1.83 ± 0.001
Orange juice	0.76 ± 0.04

Results are expressed as mean ± SD, n=3

Effect of juice samples on heat induced haemolysis of RBCs:

Percentage inhibition of haemolysis values are shown in table 2. All juice samples showed good results in inhibiting the membrane lysis at different concentrations of phenolic compounds.

Black mulberry juice exhibited higher percentage inhibition of haemolysis at all concentrations than the other two juices which varied between (27.41- 43.19%) and this may be due to its higher phenolic content. Chen et al. (2016)³⁴ confirmed the anti-inflammatory activity of total flavonoid found in black mulberry fruits as the levels of some pro- inflammatory cytokines were significantly inhibited by the fruit extracts.

Pomegranate juice showed 24.06% and 39.06% as minimum and maximum percentage inhibition respectively. Similarly, Manukumar et al. (2014)³⁵ found that the pomegranate juice inhibited the heat induced haemolysis of RBCs (29.65 %) at concentration of 1000 µg/ml and the stabilizing activity of the juice was attributed to its polyphenolic content.

Orange juice protected RBC membrane against lysis where percentage inhibition ranged from 10.12% to 22.54%. Ghanim et al. (2007)³⁶ indicated that flavonoids in orange juice reduced inflammatory response in vitro while ascorbic acid did not cause any change.

Table 2: Percentage inhibition % of RBCs haemolysis by juice samples and Ibuprofen

Concentrations (µg/ml)	Samples
Concentrations (µg/ml)	Black mulberry juice	Pomegranate juice	Orange juice	Ibuprofen
10	27.41 0.45*	24.06 ± 0.76*	10.12 ± 0.53*	47.85 ± 0.18
20	40.57 ± 0.21*	39.06 ± 0.38*	22.54 ± 0.67*	62.29 ± 0.14
30	43.19 ± 0.42*	34.77 ± 0.77*	18.29 ± 0.41*	61.84 ± 0.08
40	32.62 ± 0.22*	29.40 ± 0.5*	13.15 ± 0.56*	52.15 ± 0.04
50	37.00 ± 0.34*	33.41 ± 0.2*	18.05 ± 0.49*	49.53 ± 0.08
60	35.12 ± 0.32*	30.12 ± 0.1*	15.3 ± 0.2*	51.33 ± 0.15

Each value represents the mean ± SD of triplicate. * p < 0.05 considered as significant

It is observed that all juice samples had higher percentage inhibition at low phenolic concentrations of 20µg/ml and 30µg/ml. This finding was coinciding with the statement of Oyekachukwu et al. (2017)³⁷, that the extracts of Annona muricata leaves inhibited heat induced haemolysis of RBCs at concentrations of 0.2 mg/ml and 0.4mg/ml better than at higher concentrations. Rajurkar et al. (2009)³⁸ also reported an increased anti-inflammatory activity of Abutilon indicum (L.) leaves extracts at low concentrations.

The potential mechanism for the stabilizing activity of juice samples could be an increase in the surface area/Volume ratio of RBC cells³⁹ which could be caused by the phenolic compounds as several studies reported that phenolic compounds can enhance RBC integrity against lysis^40,41.

The results indicate that the anti-inflammatory activity of the studied juices may be due to their total phenolic content. Dussossoy et al. (2009)²⁷ arrived at similar conclusion that the anti- inflammatory activity of Noni juice was probably attributed to the presence of phenolic compounds.

CONCLUSION:

Black mulberry juice had the higher anti-inflammatory activity than pomegranate juice and orange juice. It may be due to its higher phenolic content, which highlights the importance of dietary phenolics as promising anti-inflammatory agents. Therefore, the study recommends the consumption of rich-phenolics fruit juices as a part of healthy diet.

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Received on 26.07.2020 Modified on 20.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3685-3688.

DOI: 10.52711/0974-360X.2021.00637