Inflammatory diseases are probably the most common diseases in the century. Inflammation is the crucial first step in fighting off infection and healing wounds, which is a defence mechanism of the body. When inflammation persists immune system is always activated. This is known as chronic inflammation and can lead to chronic diseases. In some diseases the body’s defence system (immune system) inappropriately triggers an inflammatory response even when there are no foreign body’s to fight off. These diseases are called as autoimmune diseases. This can cause myocarditis, asthma attack and nephritis resulting in high blood pressure or kidney failure and colitis. Many diseases are associated with inflammation like Alzheimer’s diseases, heart diseases, diabetes, cancer and arthritis. Thus it becomes essential to prevent inflammation.

Platelet function is connected with inflammatory process and various free radicals are associated with diseases that alter the functions of platelets and bring inflammation. Platelets localize with leukocytes at sites of hemorrhage, within atherosclerotic and post angioplasty restenosis lesions and on areas of ischemic reperfusion injury. This heterotypic interaction between platelets and leukocytes links haemostatic/thrombotic and inflammatory responses. Thus we can regard anti inflammatory therapies as potentially antithrombotic. A vast amount of circumstantial evidence implicates oxygen derived free radicals, especially ROS and NO as mediators of inflammation and/or tissue destruction in inflammation and arthritic disorders. Massive burst of ROS during ischemia/reperfusion in turn lead to tissue injury causing serious complications in organ transplantation, stroke and myocardial infarction.

Though there are many synthetic drugs available for the treatment of inflammation and prevent platelet aggregation they all have side effects associated with their uses. Herbal formulations are considered to be less toxic and also free from their side effects than synthetic ones.  Numerous plants are claimed to possess anti-inflammatory, anti-platelet and anti-oxidant phyto constituents in folk medicine, however one among them is Cassia fistula Linn leaves. Since there are no specific scientific reports regarding its use as anti-inflammatory, anti-platelet and antioxidant activities, the plant was selected for this particular study with the aim to bring scientific evidence for its therapeutic uses. The main objective of the present study is to evaluate the anti-inflammatory, anti-platelet and antioxidant activities of Cassia fistula Linn leaves using various in vitro models. The scope of the study is attributed in exploring the potentials of the bioactive compounds from the medicinal trees and in revealing its safety & efficacy, there by realizing the promising ethno botanical herbs, towards the development of phyto medicine.

1. PLANT PROFILE:

• Plant name                   : Cassia fistula Linn
• Synonyms                    : Cassia exelsa , C. rhombifolia
• Family                          : Caesalpiniaceae
• Biological source         : It consist of dried leaves of Cassia fistula linn.
• Parts used                     : leaves, flowers, seed, fruits and root.   
  1. Vernacular Names:
• Sanskrit          :   Amaha
• Hindi              :   Amaltas
• English           :   Golden shower
• Malayalam     :    Konna
• Portugese       :    Canna fistula
• Spanish          :    Cana fistula

2.2 Description:

The tree is 6-9 m high; trunk straight; bark smooth and pale green when young, rough and dark brown when old; branches spreading slender. Leaves 23-30 cm long; main rhachis pubescent; stipule minute, linear-oblong, obtuse, pubescent. Leaflets 4-8 pairs ovate-oblong, bright green and glabrous above, paler and silvery-pubescent beneath. When young the midrib densely pubescent on the under side, base cuneate. Flowers in lax racemes 30-50 cm long; pedicals 3.8-5.7 cm long, slender, pubescent or glabrous. Calyx 1 cm long, divided to the base, pubescent; segments oblong, obtuse. Corolla 3.8 cm across, yellow; petals 5, subequal, obovate, shortly clawed, veined. Stamens all antheriferous, the 3 lowest the longest with very long curved filaments and oblong anthers dehiscing longitudinally, the 4 lateral with short straight filaments and versatile anthers opening by pores at the base, the remaining 3 much smaller, erect with indehiscent.

2.3 Chemical Constituent:

The Dried leaves of Cassia fistula Linn consists of tannins like Epicatechin, Total Phenolics like Proycanidin B₂, Bi-flavonoids, Tri-flavonoids, and Glycosides like Rhein glycosides, Sennoiside A and B, Chrysophanol, Physcion.

2.4 Medicinal Uses:

• The root is useful in skin diseases, leprosy, Tuberculous glands and syphilis; cures burning sensation.
• The leaves are laxative, and anti-periodic; heal ulcers; used in rheumatism; juice given in erysipelas.
• The buds improve taste; laxative; antipyretic; cure “kapha”, biliousness, skin diseases, leprosy.
• The flowers have flavour, with a bitter acrid taste; cooling, astringent; cure “kapha” and biliousness; cause flatulence.
• The fruit has flavour; digestible, cooling, purgative, antipyretic; cures leprosy, diseases of the heart, and abdominal pain.
• The seeds are sweetish, oily, laxative, carminative; improve the appetite.
• The leaves lessen inflammation.
• The flowers are purgative.
• The seeds are emetic.

Fig: 1 Cassia Fistula Linn

MATERIALS AND METHODS

3.1 Drugs and Chemicals:

Lipoxidase Enzyme, Linoleic acid, Tris-HCL Buffer, Ibuprofen, Sodium Citrate, ADP (adenosine-5-diphosphate-dicyclohexyl ammonium salt), DPPH (Diphenyl Picryl Hydrazine), Hydrogen Peroxide, Indomethacin, EDTA, Nitro Blue Tetrazolium (NBT), Dextrose, Citric acid, Ascorbic acid, 2-deoxy2-ribose, Hypoxanthine, Xanthine Oxidase, Butylated Hydroxyl Toluene, Sodium nitroprusside, Curcumin, Quercetin, Ethanol, Ferric Chloride, Potassium Ferricyanide, Sulphanilamide, Phosphoric acid, Ferric chloride, and Thio barbituric acid (TBA).

3.2 Collection and Authentication:         

The leaves of Cassis fistula was collected in the Rantham areas, identified and authenticated.  The aerial parts of the plant were thoroughly washed with water, in order to remove the earthy materials sticking to it. It was then dried under shade and powdered with a mechanical grinder and sieved through No.20 mesh sieve. The finely powdered leaves were kept in an airtight container until the time of use.

3.3 Preparation Of The Methanol Extract Of Cassia Fistula Linn Leaves:

The methanol extract of the leaves of Cassia fistula Linn was prepared by extraction using cold maceration process. 15g of finely powdered leaves were taken in mortar and triturated with small volume of methanol. A total volume of 150ml of methanol was added and stirred continuously in a mechanical shaker for 4 hours. It was then kept aside for 24 hours. It was again stirred in mechanical shaker for 4 hours kept aside for 12 hours. The contents were taken, filtered through muslin cloth; the filtrate was decanted and evaporated to dryness.

3.4 Preliminary Phytochemical Screening:

• Test for Alkaloids
• Test for Carbohydrates
• Test for Flavonoids
• Test for Saponins
• Test for Tannins
• Test for Steroids and Tri-terpenoids
• Test for Amino acid
• Test for Glycosides

3.5 In Vitro Anti-Inflammatory Methods:

• 5-Lipoxygenase inhibition activity
• 12-Lipoxygenase inhibition assay
• Human Red Blood Cells (HRBC) membrane stabilisation method using methanolic extract of Cassia fistula Linn leaves.

3.6 In Vitro Anti-Platelet Activity In Whole Blood:

3.7 In Vitro Antioxidant Studies:

• DPPH Assay
• Deoxyribose Degradation Assay (Scavenging Of Hydroxyl Radical)
• Superoxide Anion Scavenging Activity (NBT Reduction Assay)
• Reducing Power Ability
• Nitric Oxide Assay

3.8 Statistical Analysis:

All determinations are carried out in triplicate and the values are expressed as mean ± SEM.

RESULTS AND DISCUSSION

1. 2. Preparation Of Plant Extract:

Extraction of Cassia fistula Linn leaves was carried using methanol as solvent. The percentage yield of the methanol extract of the leaves of Cassia fistula Linn was found to be 15.9% w/w.  

4.2 Phytochemical Screening:

Preliminary phytochemical screening of Cassia fistula Linn leaves revealed the presence of flavonoids, glycosides, tannins and phenolic as shown in Table 1.

Table: 1 Preliminary Phytochemical Screening

1. 3. Pharmacological Activity:
      1. 5-Lipoxygenase Inhibition Assay Of Methanol Extract Of Cassia Fistula Linn:

Table: 2 5-Lipoxygenase Inhibition Assay Of Methanol Extract Of Cassia Fistula Linn

1. 1. 2. 12-Lipoxygenase Inhibition Assay Of Methanolic Extract Of Cassia Fistula Linn:

Table: 3  12-Lipoxygenase Inhibition Assay Of Methanol Extract Of Cassia Fistula Linn

1. 1. 3. Human Red Blood Cells (HRBC) Membrane Stabilisation Method Using

Methanolic Extract Of Cassia Fistula Linn:

Table: 4 Human Red Blood Cells (HRBC) Membrane Stabilisation Method Using Methanolic Extract Of Cassia Fistula Linn

1. 1. 4. DPPH Radical Scavenging Activity Of Cassia Fistula Linn Methanol Extract Of Leaves:

Table: 5 DPPH Radical Scavenging Activity Of Cassia Fistula Linn Methanol Extract Of Leaves

1. 1. 5. Hydroxyl Radical Scavenging Activity Of Cassia Fistula Linn Leaves Methanol Extract:

Table: 6 Hydroxyl Radical Scavenging Activity Of Cassia Fistula Linn Leaves Methanol Extract

1. 1. 6. Superoxide Radical Scavenging Assay Using Cassia Fistula Linn Leaves Methanol Extract:

Table: 7 Superoxide Radical Scavenging Assay Using Cassia Fistula Linn Leaves Methanol Extract

1. 1. 7. Reducing Power Assay Of Cassia Fistula Linn Leaves Methanol Extract:

Table: 8 Reducing Power Assay Of Cassia Fistula Linn Leaves Methanol Extract

1. 1. 8. Nitric Oxide Radical Scavenging Assay Using Methanol Extract Of Cassia Fistula Linn Leaves:

Table: 9 Nitric Oxide Radical Scavenging Assay Using Methanol Extract Of Cassia Fistula Linn Leaves