Phytochemical Analysis and Antimicrobial Activity Cassia auriculata

Shashikala M.*, B. Poojitha R. Shiva Ram Prasad, S. Palak, B. Kiran Teja

Department of Pharmacognosy, Gokaraju Rangaraju College of Pharmacy

 

*Correspondence: shashikala8052@grcp.ac.in

DOI: https://doi.org/10.71431/IJRPAS.2026.5603      

Article Information		Abstract
Research Article Received: 04/06/2026 Revised:   09/06/2026 Accepted: 09/06/2026 Published:30/06/2026   Keywords Cassia auriculata; Flower extract; Phytochemical screening; Total phenolic content; Total flavonoid content; Antimicrobial activity		Cassia auriculata widely used traditional herb in the diabetes and skin diseases and commonly known as Tanner’s Senna. In the present study phytochemical analysis and antimicrobial activity of the flowers of the plant were investigated.  The collected flowers were extracted with 80%of alcohol and tested for the presence of various phytoconstituents. The physicochemical parameters and total phenolic and total flavonoid content were determined. Thereafter the antimicrobial activity of the extract was performed. The phytochemical screening showed the presence of flavonoids, phenolics, tannins, alkaloids, glycosides, saponins, and terpenoids. TLC o analysis for flavonoids showed 2 spots with Rf value of 0.259, 0.53 and steroids showed 4 spots with Rf value of 0.228, 0.354, 0.6, 0.859. Total phenolic content was found to be 23.83µg, gallic acid equivalents and total flavonoid was 17.76µg, quercetin equivalents. The extract found to be effective against gram +ve and gram –ve bacteria with MIC against Staphylococcus aureus 6mm and and Bacillus subtilis 5mm and pseudomonas aeruginosa 8mm with 200mg concentration. Overall, the findings support the traditional use of Cassia auriculata flowers and suggest that the extract possesses significant phytochemical and antimicrobial potential.

 

INTRODUCTION

Since very old times, medicinal plants are being used by people for curing different diseases. Even now also, many people prefer plants because they are easy to get and mostly safe to use. These plants contain some useful substances like polyphenols, flavonoids, tannins and alkaloids. Such compounds help in showing antioxidant, antimicrobial and antidiabetic actions. Because of this reason, interest in plant based medicines is increasing day by day [1].

 

 

Cassia auriculata Linn. (Syn.: Senna auriculata (L.) Roxb.), Family Fabaceae (Caesalpiniaceae) (Figure 1), is described in the Herbal Pharmacopoeia of India as an important medicinal plant. The flowers are officially recognized as a crude drug and are traditionally used in the treatment of diabetes mellitus, UTI disorders. It is known by names like Avaram, Tanner’s Cassia and Tanner’s Senna. This plant mainly grows in India and Sri Lanka and it is also seen in some other regions like Indo Malaysia and Myanmar [2]. Flowers found to contain flavonoids, phenolic acids, alkaloids, tannins, and terpenoids. These polyphenols provide strong antioxidant properties beneficial for managing diabetes and metabolic disorders, [3]. In Ayurveda and Siddha system of medicine, this plant is used to treat diabetes, skin diseases, fever and urinary infections [4]. Many traditional healers consider this plant as very useful, especially the flowers. There are few reports regarding the phytochemical study of flowers and its antimicrobial activity. Thus in the present study the flowers of the Cassia auriculata were selected for phytochemical analysis and its antimicrobial activity.

Figure 1. Plant of Cassia auriculata

MATERIALS AND METHODS

The plant material was identified and authenticated at BSI (Botanical survey of India), Hyderabad. The flowers of Cassia auriculata were collected from the surroundings of Gokaraju Rangaraju College of Pharmacy, Bachupally, Telangana, India. The collected flowers were washed thoroughly under running tap water followed by rinsing, to remove adhering dirt and other extraneous matter. The flower material was then shade-dried at room temperature for 7–10 days in a well- ventilated area ((Figure 2). After complete drying, the flowers were coarsely powdered using an electric grinder, passed through a suitable sieve, and

stored in airtight containers until further use.

Figure 2. Dried flowers of Cassia auriculata

Preparation of extract and phytochemical study

50gm of coarsely powdered drug was placed in a stoppered container with the sufficient amount of 80% Methanol. It was allowed to stand for a period of at least 7 days (Figure 3), with frequent agitation and then strained through filter paper, the marc was pressed and the combined, was decanted after standing. The methanolic extract of Cassia auriculata (MECAL) was concentrated and stored in desiccator for further study.

                               Figure 3: Maceration Cassia auriculata powder

The phytochemical study is necessary to understand the basic component of drug which may help to construct the hypothetical action of the trail drug in the disease. The extract was tested for the presence of carbohydrates, proteins and amino acids, alkaloids, Phytosterol, saponins, Flavonoids, tannins and phenolic compounds [3,4].

Determination of physicochemical parameters [5] 

The coarse powder was subjected to the determination of physicochemical parameters total ash, acid-insoluble, water soluble ash value, alcohol-soluble extractives and water soluble extractive values

Thin layer chromatography study (TLC) [6,7]

Thin slurry of silica gel G was prepared in water and uniformly spread on the TLC plate and air dried. Then activated in hot air oven at 110⁰ C and used for application of the sample. The plates were developed in mobile phases, Hexane: Ethyl acetate: Methanol (7:2:1) and Toulene: Ethyl acetate: Formic acid (5:4:1) and Toluene: Methanol (8:2) for flavonoids and steroids respectively. The developed plates were kept in the iodine chamber and observed under UV chamber to locate the developed spots and Rf values were calculated.

Rf = Distance travelled by the sample / Distance travelled by the solvent

 

Determination of total phenols content [8]

Preparation of standard solution

Total phenolic content of the sample was determined using Folin-Ciocalteu method. The calibration curve of standard was prepared using various concentration of gallic acid. The concentrations of 10, 20, 30, 40 and 50 μg/ml were prepared from the stock solution of gallic acid (100 μg/ml). 1 ml solution of each concentration was diluted to 3 ml with distilled water in 10 ml of volumetric flask. Then 0.5 ml of Folin Ciocalteu’s reagent and 2 ml of Na₂CO₃ were added in each volumetric flask. Final volume was adjusted to 10 ml with distilled water.

Preparation of sample solution

100mg of the extract was dissolved in 50 ml of methanol (50%) and filtered. One ml filtrate was taken in a volumetric flask and proceed with the similar steps as mentioned in the preparation of standard solution. Estimation of total phenols was done on the basis of calibration curve of Gallic acid standard. Absorbance of each concentration was measured using UV/VIS spectrophotometer at 765 nm. Distilled water was used a blank.

Determination of total flavonoid content

Preparation of standard solution

Total flavonoid content of the sample was determined using aluminium chloride method. The calibration curve of standard was prepared using various concentration of quercetin. The various concentrations of 10, 20, 30, 40 and 50 μg/ml were prepared form the stock solution (100 μg/ml). 1 ml from each of the solution was pipetted in 10 ml volumetric flask and 1.5 ml of 95% methanol, 0.1 ml of 10% aluminium chloride and 0.1 ml of 1 M potassium acetate were added and mixed well. Final volume was adjusted to 10 ml with distilled water and incubated at room temperature for 30 min.

Preparation of sample solution

1 ml of methanol extract was pipette out in 10 ml of volumetric flask and similar steps were applied as mentioned in preparation of standard solution. Absorbance of each concentration was measured using UV/VIS spectrophotometer at 410 nm. On the basis of calibration curve of quercetin, total flavonoid content was determined.

Antimicrobial activity [9,10]

The antimicrobial activity of the plant extracts was determined by using disc diffusion method. The selected organisms were sub-cultured on Mueller Hinton Agar medium, incubated at 37°C for 24 h and stored at 4°C in the refrigerator to maintain stock culture. Petri plates were prepared with 20 ml of sterile Nutrient Medium Broth (NMB) was prepared by using Peptone, Beef extract, Yeast extract, NaCl. The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 min. The tests were conducted at two different concentrations at 100 and 200 µg /ml respectively of the crude extract. The discs were loaded with test solutions and left for 30 min at room temperature for diffusion of solution. Negative control was prepared using respective solvent. Amoxicillin (50µg/ml) was used as positive control. The plates were incubated for 24 h at 37°C. The diameter of the zones of inhibition was measured by measuring scale in millimeter (mm).

RESULT AND DISCUSSION

Preparation of the extract and preliminary phytochemical screening

MECAL was prepared using maceration method and % yield was found to be 7.2 w/w. The phytochemical study play important role to identify the constituents present in drug.  In this study, preliminary phytochemical screening of MECAL showed the presence of flavonoids, phenolics, tannins, alkaloids, glycosides, saponins and terpenoids (Figure 4). These results simply show that the flower extract contains many secondary metabolites, which are usually connected with medicinal uses of plants.

Figure 4. Phytochemical screening of methanolic extract of MECAL

Determination of physicochemical parameters

Physicochemical standards very important for the evaluation of crude drugs. It indicates identity, purity, and quality of crude drugs [5]. Ash values represents the inorganic content of the crude drug which may be deliberately added or naturally present. Extractive values approximately measures the amount of chemical constituents. Table 1 and figure 5 and 6 reveals the values of different physicochemical parameters and extractive values.

Thin layer chromatographic study

TLC studies gives the finger print of chemical constituents present in the extract [7]. TLC analysis of MECAL (Figure 7, a and b) showed the presence of 2 spots of flavonoids with Rf value of 0.259, 0.53 and steroids showed 4 spots with Rf value of 0.228, 0.354, 0.6, 0.859.

Table 1: Physicochemical parameters of Cassia auriculata

Physicochemical parameters	Values (%)
Total ash value	4.6
Acid insoluble ash value	1.1
Water soluble ash value	3.75
Alcohol soluble extractive value	18
Water soluble extractive value	12

 

Figure 5: Determination of ash values Figure 6: Determination of extractive values

a                                              b Figure 7 : TLC of a) flavonoids and b) steroids under UV and in iodine chamber

 

Determination of total phenolic and flavonoid content

Total phenolic and flavonoid of MECAL was checked by Folin–Ciocalteu and aluminium chloride method respectively. The values were written as gallic acid/quercetin equivalents per gram of extract. The results are given in the table 2 and 3 and and calibration curve are given in figure 8 and 9. The flavonoid content found to be 23.83 µg/ml / 4gm and phenolic content was found to be 17.76 µg/ml / 4gm. From this observation. The phenolic and flavonoid content was found to be present in good quantity. it can be said that these contents of the extract support antioxidant and antimicrobial activities.

Figure 8: Calibration curve of Gallic acid        Figure .9: Calibration curve of Quercetin

Antimicrobial Activity

The antimicrobial activity was assessed by the agar well diffusion method against selected Gram-positive bacteria, Gram-negative bacteria. The MECAL (Figure 9, Table 3) showed varying degrees of inhibition depending on the microbial strain and concentration used. Generally, a concentration-dependent increase in the zone of inhibition was observed. Gram-positive bacteria showed more sensitivity when compared to Gram-negative bacteria. This difference may be because Gram-negative bacteria have an extra outer membrane.

Figure  9: Antimicrobial  activity  of  MECAL extract 1-Blank, 2-100 mg 3- 200 mg 4-Antibiotic,  a)Bacillus b) Staphylococcus c) Pseudomonas species

 

 

Table 2: Antimicrobial activity of MECAL

Microorganism	Active Wells	Zone of inhibition (mm)
Bacillus	2, 3 &  4	3, 5 & 7
Staphylococcus	2, 3 & 4	4, 6, & 6
Pseudomonas	2, 3 & 4	5, 8 & 10
Bacillus	2, 3 &  4	3, 5 & 7
Staphylococcus	2, 3 & 4	4, 6, & 6

 

Overall, the results obtained in this laboratory study suggest that Cassia auriculata flower extract contains biologically active compounds with potential medicinal importance.

CONCLUSION

The study confirmed the presence of active compounds in Cassia auriculata flower extract using phytochemical tests and physicochemical analysis indicates the presence of ash values within the limits. TLC results showed flavonoids and steroids with clear Rf values, supporting the presence of key constituents. The extract also showed high flavonoid and phenolic content along with noticeable antimicrobial activity. These findings suggest that the plant extract has useful bioactive compounds and may offer potential therapeutic benefits when studied further in future laboratory and pharmacological research studies on plants. Overall, the observations made in this work show that Cassia auriculata contains useful compounds and the results look supportive for basic academic understanding. Further work needs to focus on the extraction of specific constituents and perform their antimicrobial activity

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

ACKNOWLEDGEMENT

We would like to acknowledge the management of Gokaraju Rangaraju College of Pharmacy for their invaluable support.

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