In vitro anti-inflammatory and anti-ulcer activities of aqueous extract of Emilia sonchifolia

Emilia sonchifolia , an annual herb belonging to the Asteraceae family, has long been recognized for its potential in treating cutaneous infections, inflammation, and wounds. This study aimed to assess the anti-inflammatory and anti-ulcer effects of Emilia sonchifolia using protein denaturation assay and Acid Neutralizing Capacity (ANC) methods. The acid neutralizing capacity method revealed that the extract significantly reduced acidity to 8.35 at a concentration of 1000 mg/ml, outperforming the standard Aluminum hydroxide + Magnesium hydroxide combination which achieved a value of 13.2 at 500 mg/ml. Furthermore, the results from the protein denaturation assay demonstrated a positive correlation between increased concentration and percentage inhibition of inflammation. The extract exhibited the highest inhibition percentage of 44.99 at a concentration of 200 µg/ml, and the standard diclofenac sodium achieved 84.120 at 100 µg/ml. These findings underscore the remarkable medicinal potential of aqueous extract of Emilia sonchifolia , particularly in its applications as an anti-inflammatory and anti-ulcer agent. In summary, the aqueous extract of Emilia sonchifolia demonstrated significant anti-inflammatory activity across various concentrations, when compared to the effectiveness of the standard drug diclofenac sodium. Additionally, the extract displayed anti-ulcer activity when compared to the standard Aluminum hydroxide + Magnesium hydroxide combination. Phytochemical analysis unveiled the presence of alkaloids, glycosides, saponins, flavonoids, terpenoids, phenolic compounds, steroids, and carbohydrates in Emilia sonchifolia . These phytochemical constituents likely contribute to the extract's protective properties against protein denaturation and its anti-ulcer effects.


Introduction
Plants have been extensively used to treat human diseases from the beginning of humanity. Medicinal plants produce a number of secondary metabolites that can be used for the creation of therapeutically advantageous powerful drugs. Pharmacological benefits associated with these metabolites can be proved by the assistance of evidence-based research and theories 1 .
According to World Health Organization (WHO), underdeveloped countries mainly rely on plant derived medications for treating ailments and globally more than 20,000 plants have been identified as therapeutically active. In this article a deliberative effort has been made to investigate anti-inflammatory and anti-ulcer properties of the plant 'Emilia sonchifolia' 2 .
Emilia sonchifolia, also known as lilac tasselflower, purple sow thistle or cupids shaving brush is an important edible medicinal plant coming under Asteraceae family. They are widely distributed throughout India as a weed in the cultivated fields, wasteland area, grassy fields, roadsides and teak forests. It is considered to be one among the "Ten Sacred Flowers' of Kerala, state in India, collectively known as 'Dasapushpam' (Dhasha: ten, pushpam: flower). In Malayalam, it is called "Muyalcheviyan." It is an annual herb with a branched taproot. Stems are weak, erect or often branched at the base, smooth or sparingly hairy, 10 to 60 cm tall. This species is recognized by the Sow thistle like leaves. Lower leaves are deeply and irregularly toothed, kidney-shaped, ovate, triangular-ovate or obovate, 4-16 cm long, 1-8 cm wide with narrowly winged stalks. Upper leaves are smaller, alternately arranged, usually entire, sometimes coarsely toothed, stalkless and somewhat clasping the main stem. Inflorescence is an involucrate flower head resembling a single flower, 1.2-1.4 cm long, 4-5 mm wide, urn-shaped, long-stalked, at the end of branches. Flowering branches usually dichotomously branched with 3-6 heads, each head or capitulum a composite of numerous florets. The cup of the flower-head is green, cylindrical, somewhat inflated below. Florets are 30-60 per head, purple, scarlet, red, pink, orange, white or lilac 3 .

Plant distribution
The native range of this species is Tropical & Subtropical old world.

Uses
 The leaves juice of Emilia sonchifolia is used for the treatment of eye inflammations, night blindness, cuts and wounds, sore ears, infantile tympanites and bowel complaints.  The leaves are also used against fever and dysentery.
 The flower heads are chewed and kept in the mouth for about 10 minutes to prevent tooth decay  This plant has astringent, depurative, diuretic, expectorant, febrifuge and sudorific properties.  Diverse biological activities like cytotoxic, antitumor, anti-inflammatory, analgesic, antinociceptive, antiulcer, modulatory effects, antiviral, erythropoietic and hepatoprotective of the Emilia sonchifolia have been reported 8,9 . 2. Material and methods

Preparation of extract
The whole plant was washed thoroughly with sufficient water and chopped into smaller pieces. It was dried under shade for 3 weeks and reduced to coarse powder using an electrical blender and kept in an airtight container with proper labelling for future use. The powdered plant material (72.5 g) was extracted by macerating in distilled water (500 ml) for 72 hours. The liquid extract was strained using muslin cloth, allowed to cool and filtered. The filtrate was concentrated using heating mantle at 35 ℃. The extract was stored in refrigerator at 4 ℃ until used for the experiment 10 .

Phytochemical screening
The aqueous plant extract of the whole plant of Emilia sonchifolia were subjected to chemical tests to identify the constituents present in the plant.

Detection of alkaloids
The extract was mixed with few drops of dil. HCl and was then filtered. Test for alkaloids was carried out in this filtrate.

Mayer's Test
One or two drops of Mayers reagent (mercuric chloride 1.36 g dissolved in 60ml distilled water and mixed in a solution of 5 g of potassium iodide in 10 ml distilled water) was added to the filtrate through the sides of the test tube, formation of white creamy precipitate indicates the presence of alkaloids.

Wagner's Test
Wagner's reagent (1.27 g of iodine and 2 g potassium iodide dissolved in 5 ml distilled H2O) was added to the filtrate. Formation of reddish-brown precipitate indicates the presence of alkaloids.

Detection of glycosides
Keller killiani test To 2 ml of extract, added glacial acetic acid, 1 drop of 5% FeCl3 and conc. H2SO4. Reddish brown color appears at the junction of the 2 liquid layers and upper layer appears bluish green.

Baljet's test
To a few ml of extract, added 1 ml of sodium picrate solution and observed for the color change from yellow to orange which reveals the presence of glycosides.

Detection of terpenoids
Salkowski test 5 ml of extract was mixed with 2 ml of chloroform and about 3 ml of conc.H2SO4 was carefully added. At the separation level of the two liquids, a reddish-brown ring forms, this indicates the presence of terpenoids.

Detection of carotenoids
About 0.02 g of plant extract was mixed with chloroform, mixed well and then the mixture was filtered. To the filtrate, conc. H2SO4 was added, formation of a blue color at the interface indicates the presence of carotenoids.

Detection of steroids
Libermann-Burchard test 1 ml of extract was treated with 0.5 ml of acetic anhydride and 1 ml of H2SO4 carefully. A color change from violet to blue or green indicates the presence of steroids.

Detection of saponin
Foam test About 0.5 gm of extract was mixed with 2 ml of distilled water and heated for few minutes and filtered. The filtrate was vigorously shaken. The persistent froth was observed for 10 minutes, this indicates the presence of saponins.

Detection of flavonoids
 The extract was shaken with 1 ml of dilute ammonia solution and conc.H2SO4. Formation of yellow color indicates the presence of flavonoids.

 Aqueous sodium hydroxide test
To a few ml of extract, aqueous NaOH was added. Formation of yellowish orange color indicates the presence of flavonoids.

Detection of phenolics
Ferric chloride test To the plant extract, a few drops of 1% aqueous or alcoholic ferric chloride were added. The formation of bluish-black color indicates the presence of phenol.

Lead acetate test
A small quantity of extract was dissolved in distilled water and 3 ml of 10% lead acetate solution was added to it. A bulky white precipitate indicates the presence of phenolic compounds.

Detection of quinine
One ml of the plant extract was mixed with 5 ml of con. HCl. The formation of yellow precipitate indicates the presence of quinone.

Detection of tannin
The sample was mixed with distilled water and boiled for 5 minutes. It was filtered and was used for the test. Two drops of 10% ferric chloride were added to 1 ml of the filtrate. Formation of bluish or greenish or brownish black color indicates the presence of tannins.

Detection of carbohydrates
Molisch test 2 ml of the prepared filtrate were mixed with 0.2 ml of alcoholic solution of α-naphthol 10% and 2 ml of sulphuric acid, a reddish violet zone is formed, this indicates the presence of carbohydrates or glycosides 11,12,13 .

Protein denaturation method using Bovine Serum Albumin (BSA)
Protein Denaturation is a process in which proteins lose their tertiary structure and secondary structure by application of external stress or compound, such as strong acid or base, a concentrated inorganic salt, an organic solvent or heat.
Most biological proteins lose their biological function when denatured. Denaturation of proteins is a well-documented cause of inflammation 14 .
Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed medications in the world because of their verified effectiveness in reducing pain and inflammation. NSAIDs has accounted for prevention of the protein denaturation, which acts as antigens and prompts autoimmune diseases. As part of the investigation on the mechanism of the anti-inflammation activity, ability of plant extract to inhibit protein denaturation was studied. It was effective in inhibiting heat induced albumin denaturation 15 .

Acid Neutralizing Capacity (ANC) importance
The acid-neutralizing capacity (ANC) is the amount of acid that can be neutralized by an antacid. The United States Pharmacopoeia (USP) describes the ANC test as a back-titration method using sodium hydroxide (0.5 N solution) to a set endpoint of pH 3.5 to determine the number of milliequivalents of acid (hydrochloric acid 1 N solution) neutralized by the minimum labeled dosage (MLD) of an antacid 20 .
Antacids are alkaline drugs that neutralize gastric acidity and exert a buffering effect to stabilize the pH of the gastric juice. They are divided into 2 categories: absorbable compounds such as sodium bicarbonate, calcium carbonate (CaCO3), and magnesium carbonate (MgCO3), and non-absorbable compounds such as aluminum phosphate (AlPO4), aluminum hydroxide (Al (OH)3), and magnesium hydroxide (Mg (OH)2). They are usually marketed as a combination of 2 or 3 compounds 21 .
To the 5 ml quantity of each extract individually, water was added and mixed well to make up the total volume up to 70 ml. Then 30 ml of 1 N HCl was added into standard and test preparation and stirred for 15 minutes, 2-3 drops of phenolphthalein solution was added and mixed. The excess HCl was immediately titrated with 0.5 N Sodium hydroxide solution drop wise until a pink color is appeared 23 .
The moles of acid neutralized is calculated by,

Results and discussion
The whole plant of Emilia sonchifolia was dried, powdered and extracted using water as solvent by cold maceration method. The percentage yield of aqueous extract of Emilia sonchifolia were calculated and depicted in the table 2.

Protein denaturation
Anti-inflammatory activity of Emilia sonchifolia by protein denaturation using Bovine Serum Albumin (BSA).
The In vitro anti-inflammatory activity of aqueous extract of Emilia sonchifolia at different concentration was performed by Protein denaturation assay and the results were shown in table 4. From the results it was observed that the percentage inhibition of inflammation increased with increased concentration. The higher percentage of inhibition was obtained at the concentration of 200 (µg/ml) found to be 44.99, it was comparable with diclofenac sodium standard 84.12 at 100 (µg/ml) 27,28 .

In-vitro anti -ulcer activity
The in-vitro acid neutralizing effects of aqueous extract of whole plant of Emilia sonchifolia in different concentrations (100 mg, 200 mg, 500 mg, and 1000 mg per ml) were compared with the standard antacid AHMH-500 mg/ml. The results showed concentration dependent reduction in acid neutralizing capacity per gram of antacid was found as 158.5, 62, 22.5 and 8.35 respectively. Similarly, ANC value of the standard AHMH (500 mg) was found to be 13.2 which is quite similar to the concentration of test drug. Whereas, test drug concentration 1000 mg was found to neutralize acid more significantly as compared to standard 29,30 . The results are tabulated in Table 5.

Conclusion
Emilia sonchifolia commonly known as 'lilac tassel flower ' or 'muyalcheviyan' in Malayalam is an annual herb of the family 'Asteraceae'. Due to its several ethnomedical uses, it has been investigated for a good number of pharmacological activities. The results of this study revealed that aqueous extracts of Emilia sonchifolia possess anti-inflammatory and anti-ulcer properties. These properties may be due to the strong occurrence of polyphenolic compounds such as alkaloids, flavonoids, tannins, carbohydrates, glycosides, saponins, steroids and phenols.
The extract fractions serve as free radical inhibitors or scavengers or acting possibly as primary oxidants and inhibited the protein denaturation. The increments in absorbances of test samples with respect to control indicated stabilization of protein i.e., inhibition of protein denaturation by Emilia sonchifolia extract and reference drug diclofenac sodium. It is a scientific validation of its traditional uses in the management of inflammation.
The acid neutralizing property of the plant was determined by back titration method which was found useful in neutralizing the HCl. Results from ANC indicated a concentration-dependent decrease in acid neutralizing capacity compared to each gram of antacid administered.
Therefore, this study gives an idea that the aqueous extracts of the plant Emilia sonchifolia can be used as lead compound for designing a potent anti-inflammatory or anti-ulcer drug. However further definitive studies are required via detailed experimentation to ascertain the mechanisms and constituents for its properties.