Phytochemical analysis and antioxidant activity of chloroform extract of Cassis alata

 

J. Sujatha¹, S. Asokan², S. Rajeshkumar³

¹PG and Research Department of Microbiology, Marudupandiyar College, Vallam, Thanjavur, Tamil Nadu, India - 613 403

²Department of Microbiology, Annai college of Arts and Science, Kovilacheri, Kumbakonam-612 503

³School of Bio-sciences and Technology, VIT, Vellore – 632014, TN, India

 

ABSTRACT:

The present study was designed to evaluate the phytochemical analysis and anti-oxidant properties of chloroform extract of Cassia alata. The compounds were extracted using chloroform. The chemical bonds present in the extract were identified by FTIR and find out the various phytochemical constituents was characterized by gas chromatography-mass spectrometry (GC-MS) and High performance liquid chromatography analysis. The chloroform leaf extract of the plant was mainly found to contain ten compounds. The evaluation of the antioxidant activities of the extract was examined against DPPH free radical and also calculates the IC₅₀ concentration to scavenge the free radical.

 

 

INTRODUCTION:

Medicinal plants are the nature’s gift for human beings to treatment of various health related problems. These are the important resources to find a novel new drugs and new therapeutic agents ¹. This phytotherapy has fewer side effects compared to synthetic or chemical drugs. Free radicals such as peroxidise, super dioxide, nitric oxide, DPPH are generated by normal metabolic process. ROS (reactive oxygen species) are suppresses the activity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxides (GPx) in body ². The oxidant and antioxidant levels in tissues are occurred in balanced conditions. If the oxidants of ROS are present in elevated levels cause oxidative stress to the organism ^3,4.

 

They can disturb normal metabolic activity of body by repairing DNA, lipids, carbohydrate and protein and also they also are a major cause of heart disease, stroke, cancer and aging ⁵. Consequently, the exploration of new drug from plants that can reduce oxidative stress becomes very important.

 

The plant Cassia alata was belongs to the family Fabaceae and subfamily Caesalpinioideae. This species mainly considered as weed and easily growing shrub. It grows 3-4 m tall and has leaves with 50-80 cm long. The flowers are yellow in colour. C. alata is otherwise known as ringworm bush. This plant has very effective activity against ringworm and other fungal infections of the skin^6,7. It has several medicinal properties like laxative, purgative and antioxidant activity due to the presence of tannins and saponins in the leaves.  C. alata leaves helps to controlling cardiovascular disease and fat control. Apart from these properties C. alata leaves also possess antimicrobial, antitumor, wound healing, anti-inflammatory, antimutagenic and analgesic activities ^8-10. Different studies reported that the juice of C. alata leaves considered as a medicine for parasitic skin diseases and constipation^{11, 12}. The roots used for treating gastrointestinal disorders, asthma and UTIs.

In this study, the bioactive phytochemicals present in chloroform leaf extract of C.alata were screened and characterized by GC-MS, HPLC and FTIR techniques. Furthermore the antioxidant activity was analysed against DPPH free radical.  

 

MATERIALS AND METHODS:

Collection of plant materials:

The leaves of Cassia alata were collected from Wandiwash, South India.

 

Preparation of chloroform extract:

The leaves were shade dried in room temperature for 3-5 days and ground into fine powder.  10 g of fine powder was macerating for 24 hours with 100 ml of chloroform solvent. Then, the extract was filtered through what man No.1 filter paper and collects the filtrate. The collected filtrate was concentrated by vacuum rotary evaporator. The resulting colloidal extract was used in phytochemical screening and antioxidant activity studies.

 

Phytochemical screening of chloroform extracts ¹³:

Tests for Alkaloids (Mayer’s test):

In this test Mayer’s reagent was used which contains (Potassium mercuric Iodide). 1 ml of test sample was mixed with one or two drops of Mayer’s reagent and the formation of cream color precipitate indicate the presence of alkaloids.

 

Tests for glycosides:

To this test, 1ml of the test extract was mixed with 1ml of conc. H₂SO₄ and allowed to standby for 2 min. In the end a reddish color precipitate indicates the presence of glycosides.

 

Test for carbohydrate

Take1m of the filtrate with 5ml Benedict’s reagent and boil for 5 minutes. Changing of colour indicates the presence of carbohydrates.

 

Test for protein

1ml of filtrate with 5 to 6 drops of Millon’s reagent develops white precipitate which turns red on heating.

 

Test for anthraquinones:

1 ml of sample was mixed with equal volume of benzene followed by 10% ammonia and observes the formation of red color while adding ammonia indicates the presence of anthraquinones.

 

Test for Resins:

The positive result of presence of resins in extract was assayed by mixing 1 ml of the extracts, 3-4 ml of CuSO₄ solution in a test tube and tubes were shaken vigorously. Then the reaction mixture was allowed to stand and the resulting solution if shows green color precipitate indicate the presence of resins.

Test for Quinones:

In this test 1 ml of test sample extract was treated with KOH and the color change was appeared from red to blue indicates the presence of Quinones.

 

Test for Saponins:

The presence of saponin was tested by froth test. In a test tube 2 ml of extract is taken and shaken vigorously. After that it shows bubbles in the test tube indicate the presence of saponins.

 

Tests for Phenols:

Presence of phenol was identified by development of blue color while treating 2 ml of the testing extract with 1ml of ferric chloride solution.

 

Tannins:

To 1ml of extract was treated with few drops of ferric chloride solution and heated on water bath, dark green colour indicates for presence of tannins.

 

Flavanoids:

In a test tube, 2 ml of extract was mixed with few drops of diluted hydrochloric acid was and a small pinch of Zinc or magnesium turnings were added and the reaction mixture was boiled in water bath for few minutes. The solution turns into red or brown indicates presence of flavonoids.

 

Steroids:

For steroid test, 2 ml of acetic anhydride was treated with 1 ml of sample extract. The colour of the solution was turned from violet to bluish green after adding of 2 ml con. H₂SO₄ indicating the presence of steroids.

 

Terpenoids :

A few drops of chloroform and 2 ml of conc. H₂SO₄ were added with 2 ml of sample extract which may form layer. A reddish brown color was observed indicates presence of terpenoids.

 

Test for proteins and sugars:

Protein assay test was performed by using Biuret Reagent method and sugar test was assayed by using folin – phenol reagent.

 

Finally the phytochemicals of Cassia alata chloroform was analysed by FTIR analysis, GC-MS analysis and HPLC.

 

Antioxidant activity of chloroform extract of C. alata leaves:

Antioxidant activity of chloroform extract was performed by the method of DPPH scavenging assay. To this assay, 1 mM DPPH was prepared by dissolving in methanol and protect the solution from sunlight by covering aluminium foil. Different concentration of extract like 5, 20, 40, 60, 80 and 100 µg/ml were taken about 3 ml and mixed with 0.5 ml of DPPH solution and incubate in dark condition for 30 minutes. After incubation the intensity of the reaction mixture was measured in Spectrophotometer at 517 nm. Ascorbic acid was used a positive control. Methanol solution served as blank. Control is maintained without addition of leaf extract. The percentage of DPPH scavenging activity was calculated by following the equation:

 

 

The IC50 value was calculated from the data.

 

Statistical analysis:

All the experiments were performed thrice and delivered the data in the form of graphs were plotted by using MS Excel ® software 2010.

 

RESULTS AND DISCUSSION:

Screening of Phytochemicals:

The phytochemical analysis of chloroform extract of leaf Cassia alata was analysed for the compounds such as tannin, phenol, saponin, flavonoid, steroid, terpenoids, glycosides, alkaloids, and anthraquinones. Among them, the preliminary phytochemical analysis revealed the presence of seven compounds i.e. alkaloids, Glycosides, phenols, tannins, flavonoid, steroids, proteins and anthraquinones, resins, quinines, saponins terpenoid, and sugars were absence.

 

Table 1: Phytochemical screening of chloroform extract of C. alata

 

FTIR:

FTIR characterization technique was used to identify chemical bonds in compounds which are present in extract of leaf. C. alata leaf extract has six peaks were observed by FTIR spectrum shown in figure 1. There are six compounds present in chloroform extract of C.alata leaves (Table 2). The active functional groups are hydrogen bonded O-H stretching phenols and alcohols and C-H stretching alkanes were present at the peak 3391cm-1 and 2956 cm-1 respectively. The absorption band at 2845 cm -1 reveals that presence of O-H stretching carboxylic acids. The presence of C=O stretching ketones was observed at the peak 1675 cm-1. The peak at 1470 cm-1 indicates the presence of aromatic C-C stretched compounds. The narrow peak at 1023 cm-1 corresponding to C-N stretch aliphatic amines.

 

Figure 1: FTIR spectrum of chloroform extract of C. alata leaves

 

Table 2: FTIR spectrum of chloroform extract of C. alata leaves

S. No	Wave number (cm-1)	Functional groups
1	3391	Hydrogen bonded O-H stretching phenols and alcohols
2	2956	C-H stretching alkanes
3	2845	O-H stretching carboxylic acids
4	1675	C=O stretching ketones
5	1470	C-C stretched (in –ring) aromatics
6	1023	C-N stretch aliphatic amines

 

GC-MS analysis:

The characterization technique of GC-MS spectrum of the chloroform extract of C.alata leaves showed 10 peaks indicate the presence of 10 different phytochemical components (Figure 2). The identified chemical constituents are represented in Table 3. The results of GC-MS analysis revealed that the presence of 1-Decanol, 2-Ethyl-, Phytol, Sulfurous acid, Pentadecyl 2-propyl ester, Heptacosane, Octacosyl Trifluoroacetate, tetracosane, 16-Heptadecenal, Vitamin E, Octadecanal, 3-Butoxy-1,1,1,5,5,5-Hexamethyl-3-(trimethylsiloxy) trisiloxane and 3,50-Dioxa-2,51-Disiladopentacontane.

 

Figure 2: GC-MS analysis of chloroform extract of C. alata leaves

 

Table 3: GC-MS analysis of chloroform extract of C. alata

 

Figure 3: HPLC chromatogram characterization of chloroform extract of C. alata

 

HPLC analysis:

HPLC chromatograms showing two phyto-constituents of the chloroform extract of C. alata leaves is shown in Figure 3. It appears to contain predominantly one compound which was eluted at a retention time (Rt) of 2.409 min while the minor peak was observed at a Rt of 4.550 min.

 

Antioxidant activity of chloroform extract of C. alata:

In order to the antioxidant activity of chloroform leaf extract of C. alata was determined by DPPH activity.  The activity of leaf extract was compared standard ascorbic acid by measuring absorption intensity in the Spectrophotometer at the wavelength of 517 nm.  While increasing the concentration of plant extract the percentage of inhibition of DPPH inhibition also increased. However, the inhibition percentage is directly proportional to the concentration of leaf extract (Fig 4 and 5).

 

 

The IC50 value is defined as a substance that required inhibiting the 50 % of targeted materials. In this assay, the IC50 values of chloroform extract was found to be 19.5µg/ml (Table 4). The antioxidant activities of the plant may be due to the presence of flavonoids in the leaves. Tannins have astringent properties and biological activities like antioxidant, anti-oxidant, antimicrobial and anti-inflammatory properties¹⁴.

 

Our report revealed that the extract of C. alata shows excellent antioxidant activity due to the presence of phytochemicals like phytol, esters and fatty acids. Phytol has antioxidant activity was proved by Santos et al in 2003¹⁵. Similarly, Bhattacharya and Rana ¹⁶ were isolated phytol from Lagasea mollis and reported that it act as antimycobacterial agent. C. alata leaf extract has Octadecanal in highest which also actively involved in the pharmaceutical applications. Ara et al ¹⁷ reported that the compounds octadecanal has the antimicrobial effect against pathogenic microorganisms. Plants are the very major sources of pharmaceutically important bioactive compounds ^{18, 19, 20, 21, 22}. 

 

Figure 4: DPPH free radical scavenging activity of chloroform extract of C. alata

 

 

Figure 5: Linear regression analysis of DPPH free radical scavenging activity of chloroform extract of C. Alata

Table 4: DPPH free radical scavenging activity of chloroform extract of C. alata

Concentration of chloroform C. alata extract (µg/ml)	% Inhibition of DPPH free radical
	Plant extract	Standard
5	18.35±0.74	12.63±1.53
10	35.03±1.16	20.31±1.65
20	52.21±1.45	34.79±1.18
30	70.13±1.33	46.05±1.65
40	81.66±1.06	56.74±1.24
50	92.19±1.56	73.59±1.33

 

The plants are playing a major role in the antioxidant activity. Based on that only other applications of the plants are continued ^{23, 24, 25, 26, 27}. This study clearly indicates the antioxidant potential of the traditional herbal plant Cassia alata.

CONCLUSION:

The result obtained from the phytochemical screening carried out indicates that Cassia alataL has alkaloids, Glycosides, phenols, tannins, flavonoid, and steroids. FTIR shows that presence of carboxylic acids, alkanes and alcohol chemical bonds in the leaf extract. GC-MS spectrum confirmed presence of ten compounds like vitamin E, Phytol, Sulfurous acid, Heptacosane, Octacosyl Trifluoroacetate, tetracosane, 16-Heptadecenal, Vitamin E, Octadecanal etc. HPLC revealed that extract contain two major components. Moreover, these phyto-constituents were responsible DPPH free radical scavenging activity was examined. Therefore, plant extract has more effect of low or high concentration usage and considered as alternative resource for its therapeutic quality.

 

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Accepted on 24.09.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2):439-444.