In Vitro Antioxidant and Antibacterial Screening of flowers Extract from Cassia auriculata Linn.

 

Rohit Kumar Bargah*, Anil Kushwaha, Arun Tirkey, Bimlesh Hariwanshi

Department of Chemistry, Govt. S.P.M. College, Sitapur, Surguja (C.G.) India , 497111

 

ABSTRACT:

Nature has been a powerful source of enormous medicines for thousands of years and number of modern drugs has been extracted and exploited from natural sources, which are used in traditional systems of medicines. The main aim of the present study was to evaluate. In- Vitro antioxidant, antibacterial activity and preliminary phytochemical screening of the ethanolic and aqueous extract of flowers of Cassia auriculata Linn. Belonging to the family Caesalpiniaceae. Phytochemicals screening of the crude extract revealed the presence of flavonoids, steroids, saponins, phenolic compound, terpenoids, carbohydrates, were present in all extract while alkaloids and tannins were absent only in ethanolic extract. The antioxidant activity of flowers of Cassia auriculata was assessed with the help of antioxidant activities like 2, 2-Diphenyl-1-picryl hydrazyl (DPPH) scavenging assay. Total phenol and flavonoids content were measured. Flowers show most antioxidant activity in both extracts due to higher phenolic and flavonoids content that indicated the direct correlation among antioxidant potential, total phenol and flavonoids. The presence of the bioactive constituents have been linked to the antimicrobial activity of the plant materials against gram positive and gram negative bacteria using agar disc diffusion method. Minimum inhibition concentration value ranged from 50, 75, 100 µg/ml.Ethanolicextract exhibited significantly good antibacterial activity in dose depended manner. Therefore, it is concluded that Cassia auriculata possess phenolic properties that has considerable antioxidant and antibacterial properties.

 

 

1. INTRODUCTION:

Medicinal plants represent a rich source of antibacterial and antioxidant agents. The use of medicinal herbs has become an important part of daily life despite. The progress in modern medical and pharmaceutical research^[1]. Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind.

 

Natural products, either as pure compound or as standardized plant extracts provide unlimited opportunities for new drugs leads because of the unmatched availability of chemical diversity^[2]. Free radicals are known to play an important role in the origin of life and biological evolution implicating their beneficial effect on the organism.

 

Antioxidants are radical scavengers which protect the human body against free radical which may cause pathological condition such as cancer, cataract coronary, heart diseases, rheumatoid, arthritis, diabetes, Alzheimers diseases and ageing process^[3]. Cassia auriculate [Linn.] is a common Indian medicinal plant belong to family Caesalpiniaceae and well known for its phytochemical compositions^[4,5], pharmaceutical application and Therapeutic potential^[6]. The shrub is especially famous for its attractive yellow flower which is used in the treatment of skin disorders and body odour^[7]. The leaves are mainly used to treat anthelmentic infections, ulcer, Leprosy and other skin diseases. Its bark is used as a astringent and fruits anthelminthic, seeds used to treat in eye trouble. The Flowers are used to treat urinary discharge, diabetes and throat irritation^[8,9]. The plant has been reported to possess antipyretic, hepatoprotective, antidiabetic and antihyperglyceamic activity^[10]. Medicinal plant possess an unlimited and untapped wealth of chemical compounds with high drug potential that make these plants useful as sources of medicines. Cassia species have been of interest in phytochemical due to their excellent medicinal value. Cassia auriculata are an important rich source of secondary metabolites, anthraquinones derivatives and medicine and phanmacopoeia^[11]. The Cassia auriculata plant containts preliminary phytochemical constituents such as alkaloids, phenols, glycosides, flavonoids, tannins, saponins, carbohydrates and anthraquinone derivatives health the pharmacological activity^[12]. This study will help to design the new potential drugs. The present investigation deal with evaluating the antioxidant and antibacterial activity of aqueous and ethanolic extract of flowers of Cassia auriculata. It is anticipated phytochemical with adequate antibacterial efficacy will be used for the treatment of bacterial infections. The potential developing antimicrobial from higher plants appears rewarding as it will lead to the development of a phytomedicine to act against microbes^[13].

 

2. METHOD AND MATERIALS:

2.1 Collection of plant materials:

Fresh flowers of Cassia auriculata were collected from Chalata village Sitapur, District Surguja Chhattisgarh India, during the month of March – April 2018 and Identified by Prof. Head of Botany, Dr. N.K. Singh, Govt. College Sargaon, Distt.- Mungeli (C.G.). The flowers were thoroughly washed, shade, dried, homogenized to fine powder and stored in airtight –bottles.

 

2.2 Extraction:

150gm of plant sample Flower were cut into small pieces and further grinded placed in the soxhlet extractor for the extraction of bioactive compound. The extracts were concentrated by solvent evaporated to dryness under reduced pressure using a rotary evaporator and the final crude was stored in a refrigerator at 4ºCS until further use. A portion of the powder was subjected to successive extraction with ethanol and aqueous for 24-48 Hrs. using soxhlet apparatus.

 

2.3 Preliminary phytochemical screening:

The extracts were subjected to preliminary phytochemical testing to detect the presence of different phytochemical constituents. The plant extract was carried out qualitatively for the presence of Alkaloids, Carbohydrate, Tannins, Flavonoids, Saponins, Terpenoids, and Steroids by using the stander method^[14].

 

Determination of total phenol content:

The amount of total phenol content was determined by Folin – Ciocalteu’s reagent method^[15]. 0.5ml of extract (1mg/ml) and 0.1 Folin –Ciocalteu’s reagent 0.5 N) was mixted and the mixture was incubated at room temperature for 15 minutes. Then 2.5ml saturated sodium carbonate solution was added and further incubated for 30minutes at room temperature and the absorbance was measured at 760nm. Gallic acid was used as a positive control. Total phenol values are expressed in term of Gallic acid equivalent (mg/g of extracted compound).

 

Determination of Flavonoid content:

The amount of Flavonoids content was determined by aluminum chloride colorimetric method^[16]. The reaction mixture of 3ml consisted of 1.0ml of sample (1mg/ml), 1.0ml of methanol, 0.5ml of Aluminum chloride (1.2%) and 0.5ml Potassium acetate (120mm). This was incubated at room temperature for 30minutes. The absorbance of all samples was measured at 415nm. Quercetin was used as a positive control. Flavonoid content is expressed in term of Quercetin equivalent (mg/g of extracted compound).

 

2.4 Micro-organism (Bacterial culture’s and growth condition):

The antibacterial activity was carried out on the extracts with 2000µg/ml against the gram positive bacteria: Bacillus subtilis, Staphylococcus aureus and gram negative bacteria: Escherichia coli, Pseudomonas aeruginosa. Nutrient agar medium was used for the maintenance of the tested bacterial organism. Mueller Hinton Agar (M.H.A) was used in all bioassays applying the disc diffusion method.

 

Disc diffusion method:

Sensitivity of the bacterial strains to the different – extract was determined by the Kirby – Bauer disc diffusion susceptibility test. A suspension of the tested bacteria was spread on the solid media plates ^[17]. Filter paper disc (4mm in diameter) were loaded and after staying at 4^oc for 2h, the plates were incubated at 37^oc for 24Hrs.^[18]. The diameter of the inhibition zones were measured in mm. The inhibition zones obtained were is compound with a positive control were set using standard antibiotic ciprofloxacin^[19].

 

2.5. Antioxidant activity:

2, 2-Diphenyl- picryl- hydrazyl (DPPH) Free Radical Scavenging Activity:

This assay was used in many studies for testing antioxidant activity. 2, 2 –Diphenyl -1-picryl hydrazyl stable radical (DPPH) evidently offers a convenient and accurate method for titrating the oxidizable groups of natural and synthetic antioxidants. This assay was based on the relation of a methanolic solution of the colored free radical (DPPH) by free radical scavenger^[20]. The degradation of DPPH was evaluation by composition with a control sample without hydrogen donating compounds. The decrease in absorbance of DPPH at its absorbance maximum of 517nm was proportional to be conformed of free radical Scavenger added to DPPH reagent solution. Lower absorbance of reaction mixture indicated higher antioxidant activity^[21].In this experiment methanolic solution of DPPH (100mm, 2.5ml), 0.05ml of each extracts dissolved in methanol was added different concentrations (50-250µg/ml) reaction mixture was shaken and after 30minut at room temperature, the absorbance value were measured 517nm and converted into percentage of antioxidant activity (%AA) Ascorbic acid was used as standard^[22]. The degree of discoloration indicator the scavenging efficacy of the extract was calculated by the following equation^[23].

 

                                      Abs (control)- Abs (sample)

%Scavenger of DPPH = –––––––––––––––––––––×100

                                              Abs (control)

 

 

3. RESULT AND DISCUSION:

Phytochemical analysis:

The phytochemical constituents of the different extracts of Cassia auriculata flower investigated are summarized in table [1.1]. This preliminary screening test revealed the presence of medicinally active constituents in the two extract of Cassia auriculata flower studied. The study indicated that Steroids, Saponin, Phenolic Compounds, Terpenoids, Flavonoids and Carbohydrate were present in all extracts while cardiac glycoside were absent in the both extracts. Alkaloids and tannin were absent only ethanolic extract. Flavonoids are hydrorylatedphenolic substance known to be synthesized by plant in response to microbial infection^[24]. Total Phenolic, Flavonoids are formed to be more in flower ethanoilc extract. Phenolic compounds are powerful chain breaking antioxidant^[25]. Herbal derived antioxidant especially poly phenols and Flavonoids have been recognized to have Anticancer, antidiabetic, antiaging, properties and preparation of cardio vascular disease^{[26 -27]}.

 

Table 1.1 : phytochemical investigation of Cassia auriculata flower on different extract.

S. No	Phytochemical constituents	Methanol	Aqueous
01	Alkaloids	-	+
02	Flavonoids	+	+
03	Steroids	+	+
04	Saponin	+	+
05	Tannin	-	+
06	Phenolic compound	+	+
07	Terpenoids	+	+
08	Cardiac Glycoside	-	-
09	Carbohydrates	+	+

 

Table 1.2: Free radical scavenging capacity of aqueousextract of Cassia auriculata Flowers

Values are mean ± SEM of three determinations

 

 

Table 1.3: Free radical scavenging capacity of ethanol extract of Cassia auriculata Flowers

Concentration (µg/ml)	DPPH Scavenging %
	Ethanol Extract	Ascorbic Acid
50	30.6±1.23	92.3±0.42
100	48.1±0.83	-
150	61.3±1.34	-
200	79.5±0.72	-
250	93.4±0.69	-
IC50	109.93	-

Values are mean ± SEM of three determinations

 

Antioxidant activity:

Antioxidant activity of the plant extract showed good result by increasing the concentration of the compound (50, 100, 150, 200, 250µg/ml) which is comparable to the IC₅₀ value for ascorbic acid. DPPH scavenging data depicted in table [1.2 and 1.3]. The IC₅₀ value for ethanol and aqueous extract were formed to be 109.93 and 133.67µg/ml. Antioxidant that block the reactive oxygen species may be involved in preventing oxidative disease like cardiovascular disease, neurovascular and autoimmune disease. The present plant extracts were also great value to prevent these disease as it has good antioxidant properties.

 

Antibacterial activity:

Study on the antibacterial activity of ethanol and aqueous extract of dry flowers of Cassia auriculata was coducted using agar disc diffusion method was shown in table [1.4 and 1.5]. The antibacterial activity of the microorganism: Bacillussubtilis, Staphylococcus aureus, Escheria coli, Pseudomonas aeruginosa among the various solvents tested. The maximum activity was observed against all organisms in both extract. The highest zone of inhibition was observed against staphylococcus aureus at 100µg/ml of ethanol and aqueous extract. It is used to detect the antibacterial activity by comparing the standard drugs Ciproflaxin (10µg/ml). The Cassia auriculata flowers are known to contain various active principle of therapeutic value and possess biological activity against various diseases. The antibacterial properties of Cassia auriculata extracts may be due to the presence of phenolic constituents.

 

 

Table:1.4: Antibacterial activity of ethanolic extract of Cassia auriculata flowers

 

Table:1.5: Antibacterial activity of aqueous extract of Cassia auriculata flowers

 

 

4. CONCLUSION:

The present study evaluated the presence of various biologically active metabolites in the ethanol and aqueous flower extract of Cassia auriculata. Cassia auriculata a rich source of phytoconsituents having immense antioxidant potential. The plant is rich in phenolic and flavonoids since extracts. Possess good free radical scavenging activity and antibacterial activity. It would be promising for then development as ingredients of functional food and pharmaceuticals drugs.

 

5. ACKNOWLEDGMENT:

The author are thank full to the principal Prof. Shashima Kujur Govt. S.P.M. College Sitapur, Surguja and Special thanks to the Principal Dr. Amit Roy and Dr. Ram Kumar Sahu, Department of Pharmacy Raipur Chhattisgarh, India providing their invaluable support and provide all the research facilities.

 

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 14.08.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6):2624-2628.