INTRODUCTION:

Inflammation is a pathophysiological response of living tissues to injuries that leads to the local accumulation of plasmatic fluids and blood cells. Though it is a defense mechanism, the complex events and mediators are involved in the inflammatory reactions can induce, maintain and aggravate too many diseases¹.

Asthma is an inflammatory disease of the airways characterized by episodes of acute bronchoconstriction causing shortness of breath, cough, chest tightness, wheezing, and rapid respiration. Among several non-infectious respiratory disorders in humans, allergic asthma is the most common disease that affects breathing. Allergic asthma is a chronic inflammatory disease of the airways, characterized by bronchial obstructive reactions, airway inflammation and airway hyper reactivity to variety of stimuli such as allergens, histamine, methacholine.

Drugs, which are in use presently for the management of pain and inflammatory conditions, are narcotics, (e.g. opioids) or non-narcotics, (e.g. salicylates) and corticosteroids like hydrocortisone^3,4. However, these drugs are known to provide symptomatic relief, but do not abrogate the underlying causes or the disease. Furthermore, prolonged medication may cause adverse effects such as gastro intestinal ulcers and thrombosis leading to discontinuation of treatment⁵. Hence, there is a need to evaluate and standardize a drug or formulation, which is nontoxic and free of any associated adverse effects.

Current therapy in the management of asthma includes bronchodilators such as β₂-agonists and muscarinic receptor antagonistc⁶. Despite the beneficiary effects in relief of symptoms of asthma, the use of these bronchodilators is limited because of their adverse effect profile⁶. Therefore, there is a need to find alternative therapies that are more-effective and safe to treat asthmatic condition⁷. Based on the fact that many asthmatic drugs are of plant origin, herbal medicine is a promising approach for current efforts of finding improved activity. Ayurvedic formulations are found to be clinically useful remedies in numerous disorders with advantages of better patient compliance and less cost⁸. Syzygium cumini is one of the widely used medicinal plants in treating many diseases like diabetes, asthma, inflammation, etc. which is native to the tropics, particularly to tropical America and Australia. The plant bark is having tannins which are the important constituent of S. cumini bark by phytochemical screening. Tannins are known to inhibit the histamine response⁸and also to inhibit PG synthesis¹⁰. This dissertation investigated the anti-inflammatory activity and anti-asthmatic activity of the ethanolic extract of plant bark in classical animal models of inflammation namely carrageenan induced paw edema in rats and also asthma effect in histamine induced bronchospasm in guinea pigs.

MATERIALS AND METHODS:

Collection and extraction:

The plant Syzygium cumini (L.) tree bark was collected, identified and authenticated by Mr. V. Chelladurai, Research officer of botany, Central council for ayurveda and siddha, Government of India. A voucher specimen of the plant is being maintained in the herbarium of our laboratory for future reference. After collection of the plant, the bark was removed and was washed thoroughly in tap water and dried in shade for about 10 days under controlled temperature (25±2ºC). Then the raw material was powdered and passed through a 40 mesh sieve and stored in a well closed container for further use. Coarsely powered bark was successively soxhlated using petroleum ether, chloroform and ethanol for 72 h. The extracts were filtered and the solvents were evaporated to dryness under reduced pressure in an Eyela rotary evaporator at 40⁰C to 45⁰C. The preliminary phytochemical investigations of bark extract of Syzygium cumini (L.) were carried out by the standard methods¹¹.

Animals:

Wister albino rats weighing 150-200gm and Guinea pigs weighing 250-300gm were procured from animal house of MNR College of Pharmacy, Sangareddy, Telangana, India. The experimental protocol was approved and ethical clearance for the handling of experimental animals was obtained from Institutional Animal Ethics Committee as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and used for the studies. The animals were housed individually in polypropylene cages, under standard laboratory conditions (12:12 hour light and dark cycle; at an ambient temperature of 25±3⁰C; 35-60% of relative humidity). The animals were fed with standard rat pellet diet and water ad libitum during the experiment.

Study was conducted at Institutional Animal Ethics Committee, MNR College of Pharmacy, Sangareddy. (Reg No: 1434/PO/a/11/CPCSEA).

Phytochemical investigations:

The concentrated Syzygium cumini ethanolic extract (SEE) is used for the preliminary screening of various phytoconstituents viz. carbohydrates, alkaloids, steroids, sterols, flavonoids, gums and mucilage, saponins, terpenes, fixed oils, amino acids and tannins were detected by usual methods prescribed in standard tests.

Acute toxicity studies:

Acute oral toxicity study was performed as per OECD-423 guidelines (acute toxic class method). Wistar rats (n =6) of either sex selected by random sampling technique were used for the study. The animals were kept fasting for overnight providing only water, after which the extracts were administered orally at the dose level of 5 mg/kg body weight by intragastric tube and observed for 14 days. If mortality was observed in 2-3 animals, then the dose administered was assigned as toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher dose such as 50, 300 and 2000 mg/kg body weight¹².

Anti-inflammatory activity study:

Carrageenan induced rat paw oedema:

The anti-inflammatory reaction is readily produced in rats in the form of paw edema using carrageenan. The rats were divided into four groups, each consisting of six animals.

Group I: Control animals were received 10ml/kg p.o.

Group II: Animals were received SEE at the dose of 200 mg/kg p.o.

Group III: Animals were received SEE at the dose of 400 mg/kg p.o.

Group IV: Animals received standard Diclofenac sodium at the dose of 5mg/kg p.o.

After 30 min of extracts administration, 0.1ml of 1% w/v carrageenan was injected into the right hind paw subplantar region of each rat. The left paw served as reference (non-inflammatory paw) for comparison. The paw volumes of both paws of control and extract treated rats were measured at 30, 60, 90 and 120 min after carrageenen administration¹³. The percentage edema inhibition for each rat and each group was obtained by using the formula.

Anti –inflammatory activity (%) = [(Vc-Vt)/Vc] ×100

Where

Vc = inflammatory increase in paw volume in the control group.

Vt = increase in paw volume in drug- treated animals.

Anti-asthmatic activity:

In - vivo method:

The Guinea pigs (250-300gm) were fasted for 12 h. Six animals were taken in each group. The test compounds, was administered orally at a dose of 200mg/kg and 400 mg/kg and challenged with histamine aerosol (0.2% aqueous solution of histamine acid chloride 3ml) in a vaponephrin pocket nebulizer sprayed into a closed transparent cage. The respiratory status reflecting the increasing degree of bronchoconstriction was recorded. The time for onset of convulsions (preconvulsion) was recorded. Animals remaining stable for more than 6 min were considered protected against histamine-induced bronchospasm. An intraperitoneal injection of chlorpheniramine maleate at a dose of 25mg/kg was given for the recovery of the test animals. The mean preconvulsion time of animals, treated with the test compounds was compared to control and is expressed in terms of percentage protection¹⁴.

Percent protection = [1- (T₁/ T₂)] × 100

Where

T_2
-Preconvulsive time of test compound.

T₁- Preconvulsive time of control.

In-vitro Method:

Mast cell stabilization activity:

The overnight fasted male Wistar rats were sacrificed with excess dose of anesthetic ether and the abdomen was cut open to expose the intestine. Pieces of mesentery with connecting lobes of fat and blood vessels were rapidly dissected out and cut into small pieces and placed in a beaker containing Ringer Locke (mm concentrations of NaCl 154, KCl 5.6, CaCl₂ 2.2, NaHCO₃ 6.0 and dextrose 5.5) solution for 20 min. Different dilutions of test drug (250 and 500µg/ml) were prepared in Ringer Locke solution. Then the tissues were incubated with C 48/80 (0.8µg/ml) for a period of 20 min. The pieces of mesentery were then placed on a clean slide. Excess fatty layers and adhering tissues were carefully removed. The trimmed tissue was placed in 4 % formaldehyde solution containing 0.1% o-Toludine blue for 2.5 min and the tissue was then de-stained with acetone and xylene for 5 min. The stained mesentery pieces were examined under a digital light microscope100 X magnification and 100 mast cells were counted, starting from the left hand side of the field and then proceeding clockwise. The number of intact, fragmented or disrupted mast cells was counted. A mast cell was considered disrupted if four or five granules were observed around the mast cells. The percentage of fragmented or disrupted and intact mast cells was calculated. Three pieces of mesentery were used for each concentration of the test sample. Transfer into the slide removes the white outer layer of tissue and one drop of ringer lock solution is added and mounting the coverslip, to view under the microscope. Mast cell separate from the tissue and put it in ringer lock solution for 20 mins^{15, 16}

Statistical analysis:

The data of the experiments were expressed as mean ± S.D. The statistical significance was determined by using Dunnett’s test and One-Way Analysis of Variance (ANOVA). The level of significance was considered to be P < 0.05.

RESULTS AND DISCUSSION:

Phytochemical studies:

From the phytochemical study, it has evaluated the presence of carbohydrates, proteins, amino acids, sterols, flavonoids and tannins in Syzygium cumini plant bark ethanolic extract (Table: 1).

Table1: Preliminary phytochemical screening of the ethanolic extracts of the bark of Syzygium cumini

SI. No.	Compound	Result
1	Alkaloids	-
2	Carbohydrates	+
3	Steroids	-
4	Sterols	+
5	Flavonoids	+
6	Gums and Mucilage	-
7	Saponins	-
8	Terpenes	-
9	Fixed oils	-
10	Amino acids	+
11.	Tannins	+

+ means present and – means absent

Phytochemical screening of S. cumini showed the presence of tannins, carbohydrates, flavonoids, amino acids and sterols. Tannins are known to posses the anti-asthmatic⁹, anti-inflammatory, anti-diabetic activity etc. It is now well known that mast cells are extensively involved in the pathophysiology of bronchial asthma^17.

Acute toxicity study:

The ethanolic bark extract of Syzygium cumini did not exhibit any signs of toxicity and mortality up to the dose level of 2000 mg/kg body weight. So, the extract is considered safe for further pharmacological screening tests.

Figure 1: The percentage reduction of inflammation in carrageenan induced rats.

Anti inflammatory study:

Ethanolic extract of Syzygium cumini was evaluated for anti- inflammatory activity. In carragenan induced paw oedema, the intaperitoneally administration of leaves extract produced a significant anti-inflammatory activity in 200 and 400 mg/kg body wt. in the rats in 400 mg/kg dose was showed highest anti-inflammatory potential comparing with the standard anti-inflammatory agent (Figure 1).

Figure 2: The percentage of degradation of mast cells.

C 48/80 + Vehicle

C 48/80 + 250 µg/ml

C 48/80 + 500 µg/ml

Figure 3: The microscopic examination of mesentery tissue at 100X magnification highlighting the damage of infection.

The ethanolic extract of S.cumini 400mg/kg has shown good significant reduction in the paw volume when compared to the control groups. The activity of the drug has maintained even after the 120 min, there it as shown a slight decrease in the activity.

Anti –asthmatic activity:

Invitro: Effect of extract on histamine induced mast cell degradation:

The SEE significantly decrease in degradation of the mast cell at 500µg/ml but 250µg/ml did not cause a significant decrease in degradation of mast cells. The SEE extract (500µg/ml) as shown decrease percentage of degranulation of mast cells towards the histamine induced mast cells, but 250µg/ml did not cause any significant effect. (Fig: 2, 3).

In-vivo: Effect of extract on histamine induced bronchoconstriction:

The SEE extract (400mg/kg) as shown significantly delayed onset of preconvulsion when compared with the SEE extract (200mg/kg). SEE at 400mg/kg produced significant delayed onset of preconvulsion induced by histamine, but 200mg/kg did not cause any significant effect (Figure: 4).

Figure 4: The percentage protection of histamine induced bronchoconstriction

The ethanolic extract of the Syzygium cumini has shown significant effect at 400mg/kg when compared to the 200mg/kg. SEE as shown a delayed on set of convulsion when compared to the control.

Here in our study a significant protection of rat mesenteric mast cells from disruption caused by compound 48/80 was observed with ethanolic extract of S.cumini bark. Therefore, ethanolic extract of S.cumini bark may make mast cell very less permeable to extravascular ions as compared with control group and give protection to mast cell degranulation. The present study S.cumini offered significant protection against compound 48/80 induced mast cell degranulation by stabilizing it, which is responsible for the decreasing airway inflammation by preventing release of various inflammatory mediators¹⁷.

CONCLUSION:

The study concluded that ethanolic bark extract of Syzygium cumini (L) showed anti-inflammatory activity and anti-asthmatic activity in a dose dependent manner. The pharmacological activities of the extract can be attrinuted to the presence of bioactive principles tannins. Our results contribute towards validation of traditional plant with Telugu name Neredu Pandu and botanical name mentioned by many authors as Syzygium cumini (L) and Eugenia jumbolana Lann in the treatment of inflammation and asthma.

ACKNOWLEDGEMENTS:

Authors are very much thankful to the management of MNR Educational Trust for providing us the facilities to carry out the research work.

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Received on 02.11.2019 Modified on 13.01.2020

Research J. Pharm. and Tech. 2020; 13(11):5210-5214.

DOI: 10.5958/0974-360X.2020.00911.7