INTRODUCTION:

For a long period of time, plants have been a valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies. About 80% of individuals from developed countries use traditional medicine, which has compounds derived from medicinal plants. Therefore, such plants should be investigated to better understand their properties, safety and efficiency¹. The cost of drugs in use today is too expensive for the majority of the population in the third world countries and therefore the search for some cheap sources of antimicrobial substances in nature become inevitable. Plants are good source for new safe, biodegradable and renewable drugs. The use of plants as therapeutic agents in addition to being used as food is age long.

So, there is a great awareness in the use and significance of these medicinal floras by the World Health Organization in several resource- poor nations². This has led to intensified efforts on the documentation of medicinal plants³.The plant Cassia auriculata Linn (Family: Caesalpiniaceae) commonly known as Tanners Senna, is distributed throughout hot deciduous forests of India and holds a very prestigious position in Ayurveda and Siddha systems of medicine⁴.

It is a leguminoustree in the subfamily Caesalpinioideae. It is commonly known by its local names matura tea tree, ranawara or avaram, (Tamil: ஆவாரைāvārai) or the English version avaramsenna. It is the State flower of Telangana⁵it occurs in the dry regions of India. Avaramsenna is a much-branched shrub with smooth cinnamon brown bark and closely pubescent branchlets. The plant has been reported to possess antipyretic, hepatoprotective, anti-diabetic, anti-peroxidative and antihyperglyceamic and microbial activity⁶. The flowers are used to treat urinary discharges, nocturnal emissions, diabetes and throat irritation⁷.

Reactive oxygen species (ROS) such as O₂, H₂O₂ and OH• are highly toxic to cells. Cellular antioxidant enzymes and the free radical scavengers normally protect a cell from toxic effects of the ROS. When generation of the ROS overtakes the antioxidant defense of the cells, oxidative damage of the cellular macromolecules (lipids, proteins and nucleic acid) occurs, leading finally to various pathological. Reactive nitrogen species (RNS) are the products of normal cellular metabolism. NO• is a small molecule that contains one unpaired electron on the antibonding (2π*y) orbital and is, therefore, a radical. NO• is generated in biological tissues by specific nitric oxide synthases (NOSs). Overproduction of reactive nitrogen species is called nitrosative stress. This may occur when the generation of reactive nitrogen species in a system exceeds the system’s ability to neutralise and eliminate them. Nitrosative stress may lead to nitrosylation reactions that can alter the structure of proteins and so inhibit their normal function⁸.

Free radicals are known to play an important role in origin of life and biological evolution implicating their beneficial effects on the organism. The cytotoxic effect of free radicals is deleterious to mammalian cells and mediates the pathogenesis of many chronic diseases, but it is responsible for killing of pathogens by activated macrophages in the immune system. Antioxidants fight against free radicals by protecting us from various diseases and scavenge of reactive oxygen radicals or protect the antioxidant defense mechanism. Reactive oxygen species (ROS) are capable of damaging biological macromolecules such as DNA, carbohydrates and proteins. ROS is a collective term, which includes not only oxygen radicals (O₂ and OH.) but also some non-radical derivatives of oxygen like H₂O₂, HOCl, and ozone (O₃). If human disease is believed to be due to the imbalance between oxidative stress and antioxidant defense, it is possible to limit oxidative tissue damage and hence prevent disease progression by antioxidant defense supplements. In addition, antioxidant activity may be regarded as a fundamental property important for life⁹.

In the present investigation we have reported the isolation of bioactive compounds from ether, ethanol and ethyl acetate extract of the plant and also seen their phytochemical analysis, from those isolated compounds in-vitro antioxidant analysis were carried out by different methods.

MATERIAL AND METHODS:

Preparation of powder material:

After authentication, the fresh healthy aerial parts of plant of Cassia auriculata dried properly in shade for 3 weeks, segregated, pulverized by a mechanical grinder and passed through a 40 mesh sieve. The powdered plant materials were stored in an airtight container, and used for further studies.

Preparation of extracts¹⁰:

About 1 kg of air-dried aerial parts of plant Cassia auriculata was extracted in soxhlet assembly successively with ether, ethyl acetate and ethanol (order of increasing polarity). Each time before extracting with the next solvent, the powdered material was dried at room temperature. Each extract was concentrated by using rotary vacuum evaporator. The extract obtained with each solvent was weighed and the percentage yield was calculated in terms of dried weight of the plant material. The colour and consistency of the extract were also noted. All the solvents used for this entire work were of analytical reagent grade (Merck, Mumbai).

Calculation of percentage yield:

The percentage yield was calculated for the extracts and major compounds with reference to the crude material taken using the formula given below.

Preliminary phytochemical screening of the crude extracts¹¹:

The extract was subjected to preliminary phytochemical screening for the detection of various plant constituents present. The term qualitative analysis refers to the establishing and providing the identity of a substance. The pharmacological actions of crude drugs are determined by the nature of their constituents. The phyto-constituents are responsible for the desired therapeutic properties. To obtain these pharmacological effects, the plant materials itself or extract in a suitable solvent or isolated active constituent may be used.

The various extracts were subjected to the following chemical tests separately for the identification of various active constituents (Kokate, 1999 and Harborne, 1998).

1. Tests for Alkaloids:

a. Dragondroff's Test: 1ml of the extract, 1ml of Dragondroff's reagent was added, formation of orange red precipitate indicated the presence of alkaloids.

b. Wagner's Test: 1ml of the extract, 2ml of Wagner's reagent was added, formation of reddish-brown precipitate indicated the presence of alkaloids.

c. Mayer's Test: 1ml of the extract, 3ml of Mayer's reagent was added, formation of full white precipitate confirmed the presence of alkaloids.

d. Hager's Test: 1ml of the extract, 3ml of Hager's reagent was added, formation of yellow precipitate confirmed the presence of alkaloids.

2. Test for Carbohydrates:

a. Molisch Test: 2ml of the extract, 1ml of 〈-naphthol solution was added, and concentrated sulphuric acid through the sides of test tube. Purple or reddish violet colour at the junction of the two liquids revealed the presence of carbohydrates.

b. Fehling's Test: 1ml of the extract, equal quantities of Fehling's solution A and B were added, upon heating formation of a brick red precipitate indicated the presence of carbohydrates.

c. Benedict’s test: 5ml of Benedict’s reagent, I ml of extract solution was added and boiled for 2 minutes and cooled. Formation of a red precipitate showed the presence of carbohydrates.

3. Tests for Proteins and Amino Acids:

a. Biuret Test: 1ml of the extract add 1 ml of 40% sodium hydroxide solution was added followed by 2 drops of 1% copper sulphate solution. Formation of a violet colour showed the presence of proteins.

b. Xanthoprotein Test: 1ml of the extract 1 ml of concentrated nitric acid was added. A white precipitate was formed, it was boiled and cooled. 20% of sodium hydroxide or ammonia was subsequently added, orange colour indicated the presence of aromatic amino acids.

c. Lead Acetate Test: 1ml of the extract, 1 ml of lead acetate solution is added. Formation of a white precipitate indicated the presence of proteins.

d. Ninhydrin Test: Two drops of freshly prepared 0.2% ninhydrin reagent were added to the extract solution and it was then heated. Development of blue colour revealed the presence of proteins, peptides or amino acids.

4. Tests for Phytosterol:

a. Libermann Burchard Test: The extract was dissolved in 2ml of chloroform in a dry test tube. 10 drops of acetic anhydride and 2 drops of concentrated sulphuric acid were added. The solution became red, then blue and finally bluish green, indicated the presence of steroids.

b. Salkowski Test: Dissolve the extract in chloroform and equal volume of concentrate sulphuric acid was added. Formation of bluish red to cherry red colour in chloroform layer and green fluorescence in the acid layer represented the steroid components in the tested extract.

5. Tests for Glycosides:

a. Legal Test: The extract was dissolved in pyridine and sodium nitroprusside solution was added to make it alkaline. Colour changes from pink to red colour showed the presence of glycosides.

b. Baljet Test: 1ml of the test extract, 1ml sodium picrate solution was added. Colour changes from yellow to orange colour revealed the presence of glycosides.

c. Borntrager’s Test: A few ml of dilute HCl was added to 1ml of the extract solution. It was then boiled, filtered and the filtrate was extracted with chloroform. The chloroform layer was then treated with 1ml of ammonia. The formation of red colour showed the presence of anthraquinone glycosides.

d. Keller Killiani Test: The extract was dissolved in acetic acid containing traces of ferric chloride and it was then transferred to a test tube containing sulphuric acid. At the junction, formation of a reddish brown colour, which gradually became blue, confirmed the presence of glycosides.

6. Test for Saponins:

a. About 1ml of methanol extract was diluted separately with distilled water to 20ml, and shaken in a graduated cylinder for 15 minutes. A1% 1cm layer of foam indicated the presence of saponins.

7. Test for Flavonoids:

a. Shinoda Test: 1ml of the extract, magnesium turnings were added followed by 1-2 drops of concentrated hydrochloric acid. Formation of red colour showed the presence of flavanoids.

8. Test for Tannins and Phenolic compounds:

a. 1ml of the extract, ferric chloride was added, formation of a dark blue or greenish black colour product showed the presence of tannins.

b. 1ml of the extract, potassium dichromate solution was added, formation of a precipitate showed the presence of tannins and phenolic compounds.

9. Test for Triterpenoids:

a. Two or three granules of tin metal in 2ml thionyl chloride solution were dissolved. 1ml of the extract was then added into the test tube. The formation of a pink colour indicated the presence of triterpenoids.

10. Test for Fixed Oils:

a. Spot Test: A small quantity of extract was pressed between two filter papers. Oil stains on paper indicated the presence of fixed oils.

b. Saponification Test: 1ml of the extract few drops of 0.5 N alcoholic potassium hydroxide was added along with a drop of phenolphthalein. The mixture was heated on a water bath for 1-2 hours. The formation of soap or partial neutralization indicated the presence of fixed oils.

Estimation of Total Phenol and Flavonoids content in various extracts of plant¹¹:

Determination of total phenol:

0.25g of sample, mixed with 2.5ml of ethanol and centrifuged at 2^oC for 10 minutes and the supernatant was preserved. Then, the sample was re-extracted with 2.5ml of ethanol 80% and centrifuged. The pooled supernatant was evaporated to dryness. Then, added 3ml of water to the dried supernatant. To this added 2 ml of sodium carbonate (20%) and 0.5ml of Folins phenol reagent. The reaction mixture was kept in boiling water bath for 1 min. The absorbance was measured at 650 nm in a spectrophotometer (Mallick, 1980).

Total flavonoids¹⁶:

Measurement of total flavanoids was performed based on the method described by Cameron et al., (1943). 0.5 ml of extract, 4ml of the vanillin reagent (1% vanillin in 70% conc. H₂SO₄) was added and kept in a boiling water bath for 15 minutes. The absorbance was read at 360nm. A standard was run by using catechol (110 µg/ml).

INVITRO ANTIOXIDANT ACTIVITY:

The in-vitro antioxidant activities of the various extracts of plant studies were determined by following methods namely,

1. DPPH assay

2. Superoxide anion scavenging activity

3. Nitric Oxide free radical scavenging capacity.

4. Hydroxyl free radical scavenging activity

5. Estimation of Total Phenol and Flavonoids content in various extracts of plant

DPPH photometric assay¹²:

The antioxidant reacts with stable free radical, DPPH and converts it to 1, 1-Diphenyl -2- picryl hydrazine. A coloured complex is formed which can be measured colorimetrically at 518nm.

The effect of extract on DPPH radical was assayed using the method of Mensor et al., (2001). A methanolic solution of 0.5ml of DPPH (0.4mM) was added to 1 ml of different concentrations of Cassia auriculata extract and allowed to react at room temperature for 30 minutes. Methanol served as the blank and DPPH in methanol without the extracts served as the positive control. After 30 min, the absorbance was measured at 518nm and converted into percentage radical scavenging activity as follows.

A₅₁₈ control - A₅₁₈ Sample

scavenging activity (%) = -------------------------- X 100

A₅₁₈ control

Where A₅₁₈ control is the absorbance of DPPH radical+ methanol; A₅₁₈ sample is the absorbance of DPPH radical+ sample extract/ standard.

Superoxide scavenging activity (NBT dye reduction method)¹³:

In this method, the superoxide is produced by riboflavin. The superoxide anions are subsequently made to reduce nitro blue tetrazolium which yield a chromogenic product, which is measured at 560nm.Measurement of superoxide anion scavenging activity was performed based on the method described by Winterbourne et al., (1975). The assay mixture contained sample with 0.1ml of Nitro blue tetrazolium (1.5 mM NBT) solution, 0.2ml of EDTA (0.1M EDTA), 0.05 ml riboflavin (0.12 mM) and 2.55ml of phosphate buffer (0.067 M phosphate buffer). The control tubes were also set up wherein DMSO was added instead of sample. The reaction mixture was illuminated for 30 min and the absorbance at 560nm was measured against the control samples. Ascorbate was used as the reference compound. All the tests were performed in triplicate and the results averaged. The percentage inhibition was calculated by comparing the results of control and test samples.

Nitric oxide scavenging activity¹⁴

Nitric oxide is a very unstable species under aerobic conditions. It reacts with O₂ to produce the stable product nitrates and nitrite through intermediates through NO₂ and N₃O₄. It is estimated by using Garrat method (Garrat, 1964). In the presence of the test compound, which is a scavenger, the amount of nitrous acid will decrease. The extent of decrease will reflect the extent of scavenging, which is measured at 540nm.

Preparation of Garrat’ reagent¹⁴:

Three ml of reaction solution containing 2ml of sodium nitroprusside (10mM) and 0.5ml phosphate buffer saline (1M) were incubated at 25ºC for 2.5hours. After incubation, 0.5mL of the reaction mixture containing nitrite was pipetted and mixed with 1 mL of sulphanilic acid reagent (0.33%) and allowed to stand for 5min for completing diazotization. After that 1ml of naphthalene diamine dihydrochloride (1% NEDA) was added, mixed and allowed to stand for half an hour. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generate nitric oxide, which interacts with oxygen to produce nitrite ions, which can be estimated by the use of Griess Illosvery reaction at 540nm.

Hydroxyl free radical scavenging activity¹⁵:

The basic of this assay is quantification of degradation product of 2-deoxy ribose by condensation with thiobarbituric acid (TBA). The radical was generated by the Fe³⁺ ascorbate-EDTA-H₂O₂system (Fenton reaction).This was performed by the method mentioned in Elizabeth. K., et al (1990). The reaction mixture contained 0.1ml deoxyribose (2.8mM), 0.1 ml EDTA (0.1mM), 0.1ml H₂O₂ (1mM), 0.1ml Ascorbate (0.1mM), 0.1ml KH₂PO₄-KOH buffer, pH 7.4 (20mM) and various concentrations of plant extract in a final volume of 1ml. The reaction mixture was incubated for 1 hour at 37⁰C. Deoxyribose degradation was measured as TBARS and the percentage inhibition was calculated.

RESULTS AND DISCUSSION:

Phytochemical Screening:

In the Preliminary phytochemical analysis of the Cassia auriculata, was revealed that Ethanol (1.57%w/w) aqueous (1.71%w/w) and Ethyl acetate extract (2.48%w/w) of plant was found to indicates the higher percentage yield of an extract. Various constituents such as Alkaloids, Carbohydrates, Glycosides, Flavonoids, steroids, Amino acids, Saponins, Proteins, Phenols and Tannins were present in the above said solvent extract, where Alkaloids also present in Chloroform extract. The ethanolic extract of Cassia auriculata was found to have higher content of phenolic and flavonoid components than ethyl acetate extract of plant are given in table 1. Phenolic compounds are known as powerful chain breaking antioxidants. Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl groups. Flavonoids are potent antioxidants present in foods of plants. Most beneficial effects of flavonoids are attributed to their antioxidant and chelating abilities.

Table 1: The total Phenolic content and flavonoids content various extracts of Cassia auriculata:

Extract	Total phenol content (mg/g of catechol) (±SEM)	Total flavonoids content (mg/g) (±SEM)*
Ethyl acetate extract	0.32 ± 0.27	1.72 ± 0.20
Ethanol extract	1.35 ±0.18	3.67 ± 0.45

*All values are expressed as mean ± SEM for three determinations

Anti-oxidant activity:

Antioxidants that block the reactive oxygen species may be involved in preventing oxidative diseases like cardiovascular diseases, neurovascular diseases and autoimmune diseases. The present plant extract was also great value to prevent these diseases as it has good antioxidant property.

The in-vitro antioxidant activities of the various extracts of Cassia auriculata were determined by above methods shows that the ethanolic extract of plant was found to be more effective than ethyl acetate extract. The DPPH radical scavenging activity of the extract increases with increasing concentration are shown in table 2. It shows the IC₅₀ of the ethanolic extract of Cassia auriculata and Rutin were found to be 260µg/ml and 490µg/ml. The IC₅₀ values of ethanolic extract of Cassia auriculata were found to have strong superoxide radical scavenging activity are displays in table 3; whereas, ethyl acetate showed weak activity when compared to that of standard Ascorbate. The IC₅₀ of the ethanolic extract of plant and Ascorbate were found to be 410µg/ml and 80µg/ml. The methanolic extract of Plant was found to be most effective in scavenging nitric oxide radical activity than that of ethyl acetate extract. But when compared to both extracts with Ascorbate (standard), the methanolic extract of the Plant showed significant results are given in table.4. The IC₅₀ of the ethanolic extract of Plant and Ascorbate were found to be 250µg/ml and 410µg/ml. The ethanolic extract of plant was capable of reducing DNA damage at all concentrations (IC₅₀ = 410 μg/ml). Ascorbic acid, used as a standard, IC₅₀ = 390 μg/ml. The ethanolic extract of Cassia auriculata was found to be more effective than ethyl acetate extract. The IC₅₀ of the ethanolic extract of plant and Ascorbate were found to be 410µg/ml and 390µg/ml were displays in table.5.

Antioxidant activity of Ethanol and Ethyl acetate extract of plant:

Table 2: Effect of various extracts of Cassia auriculata on DPPH assay:

Concentration (µg/ml)	% of activity (±SEM)*
Concentration (µg/ml)	Ethyl acetate Extract	Ethanol Extract	Standard (Rutin)
100	17.35 ± 0.13	37.90 ±0.15	19.58 ±0.16
200	22.67 ± 0.02	49.75 ±0.13	23.22 ±0.45
400	36.42 ± 0.8	66.66 ±0.23	54.13 ±0.80
800	37.63 ± 0.15	71.16 ±0.22	72.33 ±0.42
	IC₅₀=1080 µg/ml	IC₅₀=260 µg/ml	IC₅₀=490 µg/ml

*All values are expressed as mean ± SEM for three determinations

Table 3: Effect of various extracts of Cassia auriculata on Superoxide anion scavenging activity by NBT dye reduction method:

Concentration (µg/ml)	% of activity (± SEM)*
Concentration (µg/ml)	Ethyl acetate Extract	Ethanol extract	Standard (Ascorbate)
100	14.55 ± 0.33	31.53 ± 0.60	74.38 ±0.10
200	21.42 ± 0.70	35.33 ± 0.53	90.16 ±0.12
400	30.56 ± 0.20	61.61 ± 0.68	93.18 ± 0.26
800	42.35 ± 0.15	76.23 ± 0.80	98.52 ± 0.21
	IC50=1090 µg/ml	IC50= 410 µg/ml	IC50 = 80 µg/ml

*All values are expressed as mean ± SEM for three determinations

Table 4: Effect of various extracts of Cassia auriculata on Nitric oxide scavenging activity:

Concentration (µg/ml)	% of activity (±SEM)*
Concentration (µg/ml)	Ethyl acetate Extract	Ethanol Extract	Standard (Ascorbate)
100	18.60±0.65	41.58±0.62	26.13±0.08
200	23.10±0.80	50.12±0.30	30.30±0.05
400	31.16±0.20	58.30±0.25	60.60±0.30
800	38.60±0.28	65.85±0.64	55.54±0.21
	IC₅₀=1060 µg/ml	IC₅₀=250 µg/ml	IC₅₀=410 µg/ml

*All values are expressed as mean ± SEM for three determinations.

Table 5: Effect of various extracts of Cassia auriculata on Hydroxyl radical scavenging activity:

Concentration (µg/ml)	% of activity (±SEM)*
Concentration (µg/ml)	Ethyl acetate Extract	Ethanol Extract	Standard (Ascorbate)
100	20.32 ± 0.71	22.32 ± 0.76	25.83 ±0.17
200	42.79 ± 0.38	37.22 ± 0.41	29.31 ±0.15
400	48.80 ± 0.58	56.81 ± 0.65	62.62 ± 0.20
800	61.70 ± 0.40	48.69 ± 0.40	54.20 ± 0.10
	IC₅₀=200 µg/ml	IC₅₀=410 µg/ml	IC₅₀= 390 µg/ml

*All values are expressed as mean ± SEM for three determinations.

Antioxidant activity of the Cassia auriculata extract revealed that good results by increasing the concentration of the compounds at (100, 200, 400, and 800) various concentrations in different methods. Normally ascorbic acid is used as standard; it showed highest antioxidant activity at all concentrations. Secondary metabolites such as phenols and flavonoids mainly associated with this antioxidant property of the compound.

CONCLUSION:

This present study evaluated the presence of various biologically active metabolites in the various extract of Cassia auriculata and the ethanolic extract of Cassia auriculata has shown effective antioxidant activity in all the above assay techniques. The ethanolic extract of plant metabolites like phenols and flavonoids were accompanying with an antioxidant activity. The results shows in the present study indicate that the plant Cassia auriculata is a potential source of natural antioxidants.

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Received on 10.12.2019 Modified on 07.02.2020

Research J. Pharm. and Tech. 2020; 13(12):6150-6155.

DOI: 10.5958/0974-360X.2020.01073.2